Method for creating low-fertility new phytolacca americana germplasm through irradiation mutagenesis and application

Abstract

The invention discloses a method for creating low-fertility new phytolacca americana germplasm through irradiation mutagenesis and application. Belongs to the technical field of mutation breeding. According to the method disclosed by the invention, the < 60 > Co-gamma rays are used for carrying out irradiation treatment on the phytolacca americana seeds, so that a stable mutant group is successfully constructed. Through multi-generation screening, a low-breeding mutant strain with significantly improved biomass and limited reproductive capacity is obtained. Compared with wild phytolacca americana, the mutant strains show higher biomass and plant height in growth indexes, and show stronger growth vigor; the seed quantity and the pollen quantity are obviously lower than those of the wild type in breeding indexes, so that the ecological invasion risk is effectively reduced. According to the method, the heavy metal enrichment capacity of the phytolacca americana is reserved, a new thought and germplasm resources are provided for application of the phytolacca americana in heavy metal polluted land remediation, and the method has important ecological and environmental significance.

Description

Technical Field

[0001] The present invention relates to the technical field of mutagenesis breeding, and more specifically, to a method and application for creating a new low-fertility germplasm of Phytolacca americana through irradiation mutagenesis. Background Art

[0002] In recent years, with the increasingly serious problem of soil heavy metal pollution globally, the research on soil treatment using phytoremediation technology has become a hot topic. Phytolacca americana is a perennial herbaceous plant. It not only has a large biomass, grows rapidly, and has strong adaptability, but also has outstanding enrichment ability for various heavy metals such as manganese and cadmium. Moreover, with the increase of growth years, the root biomass and the ability to enrich heavy metals continuously increase, and it can continuously and efficiently remove heavy metal pollutants from the soil, showing great potential in the remediation of heavy metal contaminated soil and attracting much attention in the fields of ecological research and environmental remediation.

[0003] However, Phytolacca americana has a large number of seeds and strong reproductive ability, and it is extremely easy to reproduce in large numbers in the new environment, seriously threatening the biodiversity of the native ecosystem. Moreover, when its seeds are spread, the adsorbed heavy metals will return to the soil again, causing secondary pollution. These problems greatly limit the application of Phytolacca americana in the actual remediation of heavy metal pollution.

[0004] Irradiation mutagenesis technology is an important means for plant germplasm innovation and has been widely used in plant genetic improvement. For example, in the research on crops such as wheat and soybean, by 60 physically mutating seeds with Co-γ rays and other treatments, gene mutations have been successfully induced, and new varieties with excellent traits such as high yield and disease resistance have been cultivated. However, the method for creating a new low-fertility germplasm of Phytolacca americana through irradiation mutagenesis has not been reported yet.

[0005] In summary, how to provide a method and application for creating a new low-fertility germplasm of Phytolacca americana through irradiation mutagenesis is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a method and application for creating a new low-fertility germplasm of Phytolacca americana through irradiation mutagenesis.

[0007] The present invention uses irradiation mutagenesis technology to create a new low-fertility germplasm of Phytolacca americana, enabling it to retain advantages such as a large biomass and strong heavy metal enrichment ability while reducing the invasion risk, providing a safer and more effective solution for the remediation of heavy metal contaminated soil, and realizing the "turning waste into treasure" of Phytolacca americana in the remediation of heavy metal pollution, which is of great significance for improving the ecological environment and land remediation work.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] A method for creating new germplasm with low fertility of Phytolacca americana by irradiation mutagenesis, comprising the following steps:

[0010] (1) Select Phytolacca americana seeds with high plumpness and carry out 60 Co-γ ray irradiation treatment;

[0011] (2) Then treat the seeds with concentrated sulfuric acid and cultivate the first-generation mutants;

[0012] (3) Screen out plants with phenotypic mutations of high biomass or low reproduction, carry out bagging self-crossing treatment, and collect seeds for cultivating the second-generation mutants;

[0013] (4) Cultivate the seeds produced by self-crossing of the first-generation mutants, screen out plants with phenotypic mutations of high biomass and low reproduction, and obtain the second-generation mutant library;

[0014] (5) Select plants with large biomass and low seed setting rate from the second-generation mutant library as the new germplasm with low fertility of Phytolacca americana.

[0015] Further, the conditions of the irradiation treatment in step (1) are: the radiation intensity is 5 Gy / min, and the radiation dose is 300 Gy.

[0016] Further, the treatment with concentrated sulfuric acid in step (2) is to immerse the irradiated seeds in concentrated sulfuric acid for 8 minutes and then rinse them clean.

[0017] Further, the screening indexes in step (3) are:

[0018] The biomass observation indexes and standards include: ① The plant height is 15% or more higher than that of the wild-type plant; ② The leaf size is 15% or more larger than that of the wild-type plant;

[0019] The reproduction quantity observation indexes and standards include: ① The number of inflorescences is 60% or more less than that of the wild-type plant; ② The number of flowers is 50% or more less than that of the wild-type plant;

[0020] As long as the plant meets any one of the above biomass or reproduction quantity observation indexes and standards.

[0021] Further, the screening indexes in step (4) are:

[0022] The biomass observation indexes and standards include: ① The plant height is 90% or more higher than that of the wild-type plant; ② The leaf size is 80% or more larger than that of the wild-type plant;

[0023] The reproduction quantity observation indexes and standards include: ① The number of inflorescences is 70% or more less than that of the wild-type plant; ② The number of flowers is 70% or more less than that of the wild-type plant;

[0024] All of the above biomass and reproduction conditions must be met simultaneously.

[0025] Furthermore, the screening criteria in step (5) are as follows: select mutant plants with the largest biomass, the fewest inflorescence flowers, and the lowest seed setting rate.

[0026] Application of the new low-fertility germplasm of Phytolacca americana screened by the above method in heavy metal pollution remediation.

[0027] It can be seen from the above technical solutions that, compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0028] The method of the present invention utilizes 60 Co-γ rays to irradiate the seeds of Phytolacca americana, and successfully constructs a stable mutant population. Through multi-generation screening, low-fertility mutant lines with significantly increased biomass and limited reproductive ability are obtained. Compared with the wild-type Phytolacca americana, these mutants show higher biomass and plant height in growth indicators, indicating stronger growth potential; in reproductive indicators, the number of seeds and pollen is significantly lower than that of the wild-type, effectively reducing the risk of its ecological invasion. This method not only retains the heavy metal enrichment ability of Phytolacca americana, but also provides new ideas and germplasm resources for its application in the remediation of heavy metal polluted land, which has important ecological and environmental significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0030] Figure 1 It is a flow chart of creating a new low-fertility germplasm of Phytolacca americana by irradiation mutagenesis according to the present invention;

[0031] Figure 2 In step (5) of Embodiment 1 of the present invention, each inflorescence of Phytolacca americana after irradiation mutagenesis is bagged for self-crossing before flowering;

[0032] Figure 3 It is the first growth cycle of the mutant of Phytolacca americana after irradiation mutagenesis in Embodiment 1 of the present invention. Among them, (a) is the wild-type Phytolacca americana when the seedling grows to the 4-6 leaf stage; (b) is the trait after irradiation mutagenesis; (c) is the growth of the selected different mutant plants after bagging in step (5);

[0033] Figure 4This is the variation type of the leaves of Phytolacca americana mutants after irradiation mutagenesis in Example 1 of the present invention. Among them, (a) is the phenotype of the leaves of the control group; (b)-(e) are the leaf phenotypes of different mutants of the first generation of irradiated mutagenesis individuals.

[0034] Figure 5 This is the seed obtained after self-crossing of the first generation of Phytolacca americana mutants obtained by irradiation mutagenesis in step (5) of Example 1 of the present invention.

[0035] Figure 6 This is the phenotype of the Phytolacca americana irradiation mutagenesis mutant F2 in step (6) of Example 1 of the present invention. Among them, (a) is the phenotype of the whole etiolated individual; (b) is the phenotype of the inflorescence of the etiolated individual.

[0036] Figure 7 This is the flow chart of screening low-fertility mutants in the Phytolacca americana mutant library in step (7) of Example 1 of the present invention.

[0037] Figure 8 This is the comparison of the plant heights of the Phytolacca americana mutant strain and the wild-type control group in step (7) of Example 1 of the present invention. Among them, (a) is the wild-type control Phytolacca americana; (b) is the Phytolacca americana mutant strain FZ-100.

[0038] Figure 9 This is the comparison of the inflorescences of the wild-type control group and the mutant strain of Phytolacca americana in step (7) of Example 1 of the present invention. Among them, (a) is the inflorescence of the wild-type control Phytolacca americana; (b) is the inflorescence of the mutant strain FZ-100 Phytolacca americana.

[0039] Figure 10 This is the comparison of the immature infructescences of the wild-type control group and the mutant strain of Phytolacca americana in step (7) of Example 1 of the present invention. Among them, (a) is the performance of the wild-type control group after pollination and fruit setting of the inflorescence; (b) is the performance of the mutant strain FZ-100 after pollination and fruit setting of the inflorescence.

[0040] Figure 11 This is the comparison of the mature infructescences of the wild-type control group of Phytolacca americana and the mutant strain in step (7) of Example 1 of the present invention. The left side represents the infructescence of the wild-type Phytolacca americana, and the right side represents the infructescence of the FZ-100 mutant strain.

[0041] Figure 12 This is the fruit morphology and physiological performance of the seeds of the Phytolacca americana mutant strain and the wild-type control group in step (7) of Example 1 of the present invention. Among them, (a) and (b) are the comparisons of the fruits of the wild-type control group and the mutant Phytolacca americana at immature and mature stages, respectively. The left side of the figure is the wild-type control group, and the right side is the mutant Phytolacca americana; (c) is the cross-sectional view of the immature fruit. The left side of the figure is the wild-type control group, and the right side is the mutant Phytolacca americana. Detailed implementation mode

[0042] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying 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 belong to the scope of protection of the present invention.

[0043] The reagents required for the present invention are conventional experimental reagents, purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods, which will not be elaborated one by one here.

[0044] Example 1

[0045] (1) Seed preparation: Select Phytolacca americana seeds with high plumpness, and weigh 50 g as the irradiation mutagenesis treatment material according to the setting. Seeds with high plumpness have more abundant nutrients stored in themselves, which not only helps to improve the germination potential and germination rate of the seeds, but also enables the effect after irradiation treatment to remain relatively stable.

[0046] (2) Irradiation treatment: Use 60 Co-γ rays to irradiate the wild-type Phytolacca americana seeds in step (1). The intensity of the irradiation is 5 Gy / min, and the dose of the irradiation is 300 Gy. Since 60 Co is a radioactive isotope with a specific half-life, it is necessary to calculate the dose rate at the corresponding time and corresponding point according to its decay law to determine the specific irradiation time. During the irradiation process, the Phytolacca americana seeds are flipped at the set time t / 2 to ensure uniform distribution of the radiation dose.

[0047] (3) Seed pretreatment: Immerse the irradiated seeds in concentrated sulfuric acid for 8 minutes within 1 week after irradiation. Concentrated sulfuric acid has strong corrosiveness and can effectively damage the seed epidermis, causing its structure to rupture, creating favorable conditions for subsequent germination. After the treatment, the seeds need to be carefully taken out of the concentrated sulfuric acid and washed thoroughly to avoid the adverse effects of residual concentrated sulfuric acid on seed germination. The washed seeds are placed in a constant temperature incubator with the temperature set at 28°C to promote the smooth germination and growth of the seeds.

[0048] Table 1 Germination rate of irradiated mutagenic seeds

[0049]

[0050] As can be seen from Table 1, the germination rate of the irradiation mutagenesis experimental group was 51.24%, and the germination rates of the control groups were 93.75%, 84.38%, and 96.88% respectively, with an average germination rate of 91.67%. By comparing the data of the irradiation mutagenesis experimental group with that of the control groups, it can be concluded that the irradiation treatment achieved the expected semi-lethal effect. Irradiation treatment can induce gene mutations, but not all treated seeds will produce mutations. By setting the semi-lethal rate (LD50), the probability of gene mutations in the surviving seeds can be increased while ensuring the survival of a certain number of seeds. Under the semi-lethal rate condition, the surviving plants may carry a variety of different mutations, which increases genetic diversity and provides more options for subsequent screening of low-fertility plants.

[0051] (4) Seedling cultivation: Transplant the germinated seedlings into a seedling cultivation substrate (purchased from Xuanwei Miaoyuan Agricultural Science and Technology Co., Ltd.) and continue to cultivate them in a greenhouse until the seedlings grow to the 4-6 leaf stage. The conditions for greenhouse cultivation include: the cultivation temperature is 20-30 °C, and the relative air humidity for cultivation is 60-70%.

[0052] It takes 30-40 days to grow to the 4-6 leaf stage. In the first generation of mutants (f1), most of the phenotypes are the same as those of the wild type (the wild type is like Figure 3 (a)), and a small number show chlorosis, albinism, and abnormal shapes (such as Figure 3 (b), Figure 4 ).

[0053] (5) Construction of mutant population: Transplant the Phytolacca americana seedlings at the 4-6 leaf stage into flower pots for further cultivation, and continuously monitor the growth status of the seedlings during the cultivation process. The plants will flower after 4-5 months.

[0054] During the flowering period, observe the reproductive characteristics of the plants and screen out the mutant plants with obvious mutations in biomass or reproductive quantity. When screening the mutant plants, we make judgments based on the following observation indicators and standards. The observation indicators and standards for biomass include: ① The plant height is 15% or more higher than that of the wild type plants; ② The leaf size (single leaf area) is 15% or more larger than that of the wild type plants. The observation indicators and standards for reproductive quantity include: ① The number of inflorescences is 60% or less less than that of the wild type plants; ② The number of flowers is 50% or less less than that of the wild type plants. As long as the plant meets any one of the above observation indicators and standards for biomass or reproductive quantity, we will record it as a different plant.

[0055] Finally, 132 plants with significant differences from the wild type control group were screened out. These plants are significantly superior to the wild type control group in terms of biomass, or show low reproductive quantity compared to the wild type control group, such as Figure 3 (c).

[0056] The inflorescences of these mutant plants with high biomass or low fertility phenotypes were bagged for self-pollination,( Figure 2 ), and the seeds produced by self-pollination were collected. These seeds will be used as the starting materials for cultivating the second-generation mutants( Figure 5 ).

[0057] (6) Cultivation of the second-generation mutants: The seeds produced by self-pollination of the first-generation mutants were cultivated (the seeds were immersed in concentrated sulfuric acid for 8 minutes. After the treatment, the seeds were carefully taken out of the concentrated sulfuric acid and washed thoroughly to avoid the adverse effects of residual concentrated sulfuric acid on seed germination. The washed seeds were placed in a constant-temperature incubator at a temperature of 28 °C to promote the smooth germination and growth of the seeds. The germinated seedlings were transplanted into a seedling substrate and continued to be cultivated in a greenhouse until the seedlings grew to the 4-6 leaf stage. The greenhouse cultivation conditions included: the cultivation temperature was 20-30 °C, and the relative air humidity for cultivation was 60-70%). The purpose of this step was to further purify and confirm the low-fertility traits and ensure that these traits could be stably inherited to the offspring. During the flowering period, the reproductive characteristics of the plants were observed, and mutant plants with high biomass and low fertility were screened out. When screening the mutant plants, we made judgments based on the following observation indicators and standards. The biomass observation indicators and standards included: ① The plant height was 90% or more higher than that of the wild-type plants; ② The leaf size (single leaf area) was 80% or more larger than that of the wild-type plants. The fertility observation indicators and standards included: ① The number of inflorescences was 70% or more less than that of the wild-type plants; ② The number of flowers was 70% or more less than that of the wild-type plants. Only the plants that simultaneously met all the above biomass and fertility conditions were recorded as different plants, and the second-generation mutant library was obtained.

[0058] Finally, 12 plants with significant differences from the wild-type control group were screened out. The biomass of these plants was significantly higher than that of the wild-type control group, manifested as higher plant height and larger leaf area; at the same time, the fertility was significantly lower than that of the wild-type control group, manifested as fewer inflorescence numbers and flower numbers( Figure 6 ).

[0059] (7) Screening of low-fertility mutant plants: Select plants with high biomass and low seed-setting rate from the second-generation mutant library as new germplasms of Phytolacca americana with low fertility( Figure 7 ), and the screening criteria were: continuously observe and record the plant height, leaf size, number of inflorescences, number of flowers, and seed-setting rate of the mutants. Select the mutant plants with the largest biomass, the fewest inflorescence flowers, and the lowest seed-setting rate, that is, the ideal mutant materials with high biomass and low fertility.

[0060] During the continuous observation and recording of the biomass and reproductive capacity of mutants, it was found that the mutant FZ-100 exhibited abnormally tall plants, the largest leaf area, the fewest inflorescences, the fewest flowers, and shriveled and underdeveloped fruit clusters. The plant height, inflorescences, flowers, and seed quality of the mutant FZ-100 plants were measured and compared with the wild-type control group to evaluate the specific differences in growth and reproduction of the low-fertility mutant plants. For details, see Figures 8 to 12 , Table 2.

[0061] Table 2 Results of Biomass and Reproductive Capacity of Low-Fertility Mutant Plants

[0062]

[0063] The present invention discloses a method and application for creating a new low-fertility germplasm of Phytolacca americana by irradiation mutagenesis. This method not only retains the heavy metal enrichment ability of Phytolacca americana but also provides new ideas and germplasm resources for its application in the remediation of heavy metal contaminated land, which has important ecological and environmental significance.

[0064] In this specification, each embodiment is described in a progressive manner. The key point of each embodiment is to illustrate the differences from other embodiments. The same or similar parts among the embodiments can be referred to each other.

[0065] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for creating new low-fertility germplasm of Phytolacca americana through irradiation mutagenesis, characterized in that, It includes the following steps: (1) Select Phytolacca americana seeds with high plumpness and perform 60 Co-γ ray irradiation treatment; (2) Then, the seeds are treated with concentrated sulfuric acid to cultivate the first-generation mutants; (3) Select the plants with high biomass or low reproductive phenotype mutations, perform bagging and self-crossing treatment, and collect the seeds for cultivating the second-generation mutants; (4) Cultivate the seeds produced by self-crossing the first-generation mutants, and select the plants with high biomass and low reproductive phenotype mutations to obtain the second-generation mutant library; (5) Select the plants with large biomass and low seed-setting rate from the second-generation mutant library as the new low-fertility germplasm of Phytolacca americana.

2. The method according to claim 1, characterized in that, The conditions of the irradiation treatment in step (1) are: the radiation intensity is 5 Gy / min, and the radiation dose is 300 Gy.

3. The method according to claim 1, characterized in that The treatment with concentrated sulfuric acid in step (2) is to immerse the seeds after irradiation treatment in concentrated sulfuric acid for 8 minutes, and then rinse them clean.

4. The method according to claim 1, wherein The screening indexes in step (3) are: The biomass observation indexes and standards include: ① The plant height is 15% or more higher than that of the wild-type plants; ② The leaf size is 15% or more larger than that of the wild-type plants; The reproductive quantity observation indexes and standards include: ① The inflorescence quantity is 60% or more less than that of the wild-type plants; ② The flower quantity is 50% or more less than that of the wild-type plants; As long as the plant meets any one of the above biomass or reproductive quantity observation indexes and standards.

5. The method according to claim 1, wherein The screening indexes in step (4) are: The biomass observation indexes and standards include: ① The plant height is 90% or more higher than that of the wild-type plants; ② The leaf size is 80% or more larger than that of the wild-type plants; The reproductive quantity observation indexes and standards include: ① The inflorescence quantity is 70% or more less than that of the wild-type plants; ② The flower quantity is 70% or more less than that of the wild-type plants; It is required to meet all the above biomass and reproductive quantity conditions at the same time.

6. The method according to claim 1, wherein The screening index in step (5) is: select the mutant plants with the largest biomass, the fewest inflorescence flowers and the lowest seed-setting rate.

7. Application of the new low-fertility germplasm of Phytolacca americana screened by the method according to any one of claims 1 to 6 in heavy metal pollution remediation.

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