阅读说明：本技术 一种花生种质资源创制方法 (Peanut germplasm resource creating method ) 是由 王晶珊 刘斌 于 2021-09-14 设计创作，主要内容包括：本发明提供一种花生种质资源创制方法,属于花生新品种培育技术领域,经快中子辐照、航天诱变的花生种子,取出种胚进行表面消毒；经表面消毒的花生种胚在无菌水中浸泡然后放入灭菌的培养皿中,剥离胚小叶,随即接种于体细胞胚诱导培养基中进行培养；然后在体细胞胚萌发培养基上培养；以灭菌的沙子中无菌催芽的8～10日龄花生种子实生苗作为砧木,体细胞胚萌发长成的高1.5cm以上的小苗作为接穗,嫁接苗移栽田间前2个周注意保湿,之后按常规进行田间管理；成熟后按单株收获M2代种子,以诱变亲本作为对照。本发明能克服常规诱变技术突变体嵌和现象、大部分突变细胞不能遗传的瓶颈。(The invention provides a peanut germplasm resource creating method, which belongs to the technical field of new peanut variety cultivation, and is characterized in that peanut seeds subjected to fast neutron irradiation and space mutation are taken out, and the surface of the peanut seeds is disinfected; soaking the peanut embryo with the sterilized surface in sterile water, putting the soaked peanut embryo into a sterilized culture dish, stripping embryo leaflets, and then inoculating the peanut embryo into a somatic embryo induction culture medium for culture; then culturing on a somatic embryo germination culture medium; taking 8-10 days old peanut seed seedlings subjected to sterile germination acceleration in sterilized sand as stocks, taking small seedlings with the height of more than 1.5cm formed by somatic embryo germination as scions, paying attention to moisture preservation 2 weeks before transplanting the grafted seedlings into a field, and then carrying out field management according to a conventional method; after maturation, seeds of M2 generations were harvested from individual plants, and the mutagenized parents were used as controls. The invention can overcome the defects of mutant intercalation phenomenon and incapability of inheritance of most mutant cells in the conventional mutagenesis technology.)

1. A method for creating peanut germplasm resources is characterized by comprising the following steps: the method comprises the following steps:

(1) performing fast neutron irradiation and aerospace mutagenesis on peanut seeds, taking out a seed embryo, and performing surface disinfection;

(2) soaking the peanut embryos subjected to surface disinfection in sterile water for 8-12 hours;

(3) taking out the soaked embryo, placing into a culture dish subjected to autoclaving, stripping embryo leaflets, and immediately inoculating into a somatic embryo induction culture medium for culture;

(4) the induction medium of the somatic embryo cultured by inoculating embryo lobules is as follows: MS + 5-15 mg/L2, 4-D; adjusting the pH value of the culture medium and sterilizing;

(5) culturing on a somatic embryo induction culture medium, wherein the embryo lobular explant starts to form a somatic embryo, and then the somatic embryo is gradually formed; transferring the survival embryo leaflet explants forming somatic embryos to a somatic embryo germination culture medium for culture, and inducing the somatic embryos to germinate into seedlings;

(6) the somatic embryo germination culture medium comprises: MS + 4-6 mg/LBAP; adjusting the pH value of the culture medium and sterilizing;

(7) culturing on a somatic embryo germination culture medium, and subculturing once every 4 weeks to promote the somatic embryo to germinate into seedlings; when the plantlet grows to be more than 1.5cm, the plantlet can be used as a scion for grafting;

(8) taking 8-10 days old peanut seed seedlings subjected to sterile germination acceleration in sterilized sand as rootstocks, taking seedlings with the height of more than 1.5cm formed by somatic embryo germination as scions, performing sterile grafting in a super clean bench by adopting a grafting method, and wrapping a grafting opening by using a sealing film; culturing the grafted seedlings in sand for 3-5 days to promote the healing of grafting wounds, and then transplanting the grafted seedlings into the field;

(9) keeping moisture 2 weeks before transplanting the grafted seedlings into the field, and then performing field management according to the conventional method; after the seeds are matured, harvesting M2 seeds according to a single plant;

(10) the harvested M2 seeds were grown into rows per individual plant, and the mutagenized parents were used as controls.

2. The method for creating peanut germplasm resources according to claim 1, which is characterized by comprising the following steps: in the step (1), the disinfection method comprises the following steps: soaking the fabric in 70-75% alcohol for 20 seconds, soaking the fabric in 0.1% mercuric chloride solution for 8-10 minutes, and then washing the fabric with autoclaved sterile water for 3-5 times.

3. The peanut germplasm resource creation method of claim 2, characterized by comprising the following steps: in the step (1), the sterilization operation is carried out in a clean bench, and appliances for operation need to be sterilized under high pressure or burned by an alcohol lamp.

4. The method for creating peanut germplasm resources according to claim 1, which is characterized by comprising the following steps: in the step (3), each seed has 8 embryonic leaflets, namely a first pair of true leaves when the seed emerges, each true leaf has 4 leaflets, and a pair of true leaves has 8 leaflets, and the 8 leaflets are formed by the development of the 8 embryonic leaflets.

5. The peanut germplasm resource creation method of claim 4, wherein the method comprises the following steps: in the step (3), the reason for selecting the embryo lobule is as follows: compared with young leaves or mature leaves, the embryonic leaflets have low differentiation degree and strong regeneration capability.

6. The method for creating peanut germplasm resources according to claim 1, which is characterized by comprising the following steps: in the steps (4) and (6), the pH of the medium was adjusted to 5.8, and the medium was sterilized in an autoclave at 120 ℃ for 20 minutes.

7. The method for creating peanut germplasm resources according to claim 1, which is characterized by comprising the following steps: culturing for 2 weeks on a somatic embryo induction culture medium, wherein the embryo lobular explants start to form somatic embryos, and then the somatic embryos are gradually formed; due to the irradiation mutagenesis, part of the embryo leaflet explants are browned and die; generally, the appropriate mutagenesis dose is a dose at which half of the lethal dose or half of the regeneration rate is achieved, i.e., half of the explants cannot form somatic embryos; after culturing for 4 weeks, transferring the surviving embryo leaflet explants forming somatic embryos to a somatic embryo germination culture medium for culturing, and inducing the somatic embryos to germinate into seedlings.

8. The method for creating peanut germplasm resources according to claim 1, which is characterized by comprising the following steps: in the step (10), field observation is carried out in the growth period and the harvest period, and plant rows which are the same as parents and have no variation and separation are eliminated; the single plants in the same plant row are different in performance and obviously separated, and are different from the mutagenic parents, so that the embryonic cells of the regenerated plants are mutated cells, and the progeny of the regenerated plants are subjected to selfing, character variation and separation; selecting variant plants, selfing for 2-3 generations to enable the genes determining the variant characters to be homozygous, and creating new germplasm resources.

Technical Field

The invention belongs to the technical field of cultivation of new peanut varieties, and particularly relates to a method for creating peanut germplasm resources.

Background

Peanuts are important oil crops and economic crops and occupy an important position in national economy. The cultivation of a high-yield and high-quality special multi-resistance new peanut variety has important significance for improving the peanut yield and increasing the income of farmers. However, the problem of narrow genetic basis in current peanut cultivation is more and more prominent, the breakthrough progress of peanut breeding is limited to a great extent, the current northern peanut varieties generally have the relationship of peanuts in the summer, the southern peanuts generally have the relationship of lion head enterprises, so that the relationship of the peanut varieties is close, and the breakthrough is difficult to obtain when the conventional crossbreeding method is used for cultivating high-yield high-quality multi-resistance peanut varieties.

The use of mutagenesis techniques can create new individuals with new traits that do not exist in nature or are extremely rare. Fast neutron irradiation and space mutation are good mutation sources, but the space of irradiation treatment materials is limited, and peanut seeds are large, so that the number of the irradiation treated seeds can be only reduced. In order to increase the frequency of obtaining beneficial mutants, half lethal dose is generally used as an appropriate mutagenic dose, namely, about half of the irradiated seeds cannot normally germinate and grow, and only half of the dose which can germinate and grow into plants is used as an appropriate mutagenic dose. Thus, the number of irradiated seeds is small, and half of the irradiated seeds can not normally germinate into seedlings, so that the selective population of breeding can not be reached, because the mutagenic breeding usually needs a large enough population to obtain beneficial mutants.

Therefore, there is a great need for a method to obtain large numbers of plants from a limited number of irradiated seeds, thereby expanding the mutagenic selection population to obtain beneficial mutants.

Disclosure of Invention

In order to solve the technical problems of the background art, the invention provides a peanut germplasm resource creation method, which can overcome the bottlenecks that the conventional mutagenesis technology is used for realizing mutant intercalation phenomenon and most mutant cells cannot be inherited; more than 60 regeneration plants can be obtained from each irradiated seed, the breeding selection population is increased by more than 60 times, the selection population is greatly increased, the selection probability is improved, and the difficulties that 1 viable seed can only obtain 1 seedling in conventional mutation breeding, the mutation population is small and the breeding selection population cannot be reached can be solved.

In order to achieve the purpose, the invention adopts the following technical scheme: a method for creating peanut germplasm resources comprises the following steps:

(1) performing fast neutron irradiation and aerospace mutagenesis on peanut seeds, taking out a seed embryo, and performing surface disinfection;

(2) soaking the peanut embryos subjected to surface disinfection in sterile water for 8-12 hours;

(3) taking out the soaked embryo, placing into a culture dish subjected to autoclaving, stripping embryo leaflets, and immediately inoculating into a somatic embryo induction culture medium for culture;

(4) the induction medium of the somatic embryo cultured by inoculating embryo lobules is as follows: MS + 5-15 mg/L2, 4-D; adjusting the pH value of the culture medium and sterilizing;

(5) culturing on a somatic embryo induction culture medium, wherein the embryo lobular explant starts to form a somatic embryo, and then the somatic embryo is gradually formed; transferring the survival embryo leaflet explants forming somatic embryos to a somatic embryo germination culture medium for culture, and inducing the somatic embryos to germinate into seedlings;

(6) the somatic embryo germination culture medium comprises: MS + 4-6 mg/LBAP; adjusting the pH value of the culture medium and sterilizing;

(7) culturing on a somatic embryo germination culture medium, and subculturing once every 4 weeks to promote the somatic embryo to germinate into seedlings; when the plantlet grows to be more than 1.5cm, the plantlet can be used as a scion for grafting;

(8) taking 8-10 days old peanut seed seedlings subjected to sterile germination acceleration in sterilized sand as rootstocks, taking seedlings with the height of more than 1.5cm formed by somatic embryo germination as scions, performing sterile grafting in a super clean bench by adopting a grafting method, and wrapping a grafting opening by using a sealing film; culturing the grafted seedlings in sand for 3-5 days to promote the healing of grafting wounds, and then transplanting the grafted seedlings into the field;

(9) keeping moisture 2 weeks before transplanting the grafted seedlings into the field, and then performing field management according to the conventional method; after the seeds are matured, harvesting M2 seeds according to a single plant;

(10) the harvested M2 seeds were grown into rows per individual plant, and the mutagenized parents were used as controls.

Preferably, in step (1), the sterilization method comprises: soaking the fabric in 70-75% alcohol for 20 seconds, soaking the fabric in 0.1% mercuric chloride solution for 8-10 minutes, and then washing the fabric with autoclaved sterile water for 3-5 times.

Preferably, in step (1), the sterilization operation is performed in a clean bench, and the instruments used for the operation need to be autoclaved or burned by an alcohol lamp.

Preferably, in step (3), each seed has 8 embryonic leaflets, i.e., the first true leaves when the seed emerges, each true leaf has 4 leaflets, and a pair of true leaves has 8 leaflets, and the 8 leaflets are developed from the 8 embryonic leaflets.

Preferably, in step (3), the embryonic leaflets are selected for the following reasons: compared with young leaves or mature leaves, the embryonic leaflets have low differentiation degree and strong regeneration capability.

Preferably, in the steps (4) and (6), the pH of the medium is adjusted to 5.8, and the medium is sterilized in an autoclave at 120 ℃ for 20 minutes.

Preferably, after 2 weeks of culturing in step (5) on somatic embryo induction medium, the embryonic leaflet explants begin to form somatic embryos, followed by gradual somatic embryo formation; due to the irradiation mutagenesis, part of the embryo leaflet explants are browned and die; generally, the appropriate mutagenesis dose is a dose at which half of the lethal dose or half of the regeneration rate is achieved, i.e., half of the explants cannot form somatic embryos; after culturing for 4 weeks, transferring the surviving embryo leaflet explants forming somatic embryos to a somatic embryo germination culture medium for culturing, and inducing the somatic embryos to germinate into seedlings.

Preferably, in the step (10), the plant rows which are the same as the parents, have no variation and are separated are eliminated by field observation in the growth period and the harvest period; the single plants in the same plant row are different in performance and obviously separated, and are different from the mutagenic parents, so that the embryonic cells of the regenerated plants are mutated cells, and the progeny of the regenerated plants are subjected to selfing, character variation and separation; selecting variant plants, selfing for 2-3 generations to enable the genes determining the variant characters to be homozygous, and creating new germplasm resources.

The invention provides a method for creating peanut germplasm resources in large quantity by combining tissue culture with fast neutron irradiation, space mutation and the like. After the conventional mutagenesis treatment, only 1 seedling can grow from 1 seed. By using the method, the peanut seed embryo lobules after irradiation treatment are taken as explants to carry out tissue culture, each embryo lobule explant can regenerate more than 8 plantlets, each seed has 8 embryo lobules, and thus each seed can obtain more than 64 regenerated plantlets. The regenerated seedlings are transplanted to the field by grafting, more than 60 plants can be obtained from each seed, and the plants bloom and bear fruits. Secondly, conventional mutagenesis is carried out, only the meristematic cells at the top of the stem are mutated and can be inherited to the next generation, cells at other parts are differentiated cells, mutation cannot be inherited, and the phenomenon of mutant incorporation is serious. Because only meristematic cells have the capability of multidirectional differentiation, female and male cells can be differentiated to form female and male gametes through meiosis, and the female and male gametes are combined to generate the next generation. However, meristematic cells account for a very small proportion of the entire seed, and most of the cells are differentiated cells. The irradiated seeds are cultured by taking embryo leaflets, and plants are regenerated through an embryogenesis way, namely, cultured embryo leaflet explant cells have multiple differentiation potentials after dedifferentiation, each cell has the capacity of regenerating the plants, and the regeneration way (the embryogenesis way and the organogenesis way) can be regulated and controlled through the variety and the concentration of hormones added into a culture medium. The invention adds 2,4-D to regulate and control the embryogenesis path regeneration, namely, dedifferentiated cells form embryonic cells, the embryonic cells further develop into somatic embryos, and the somatic embryos germinate and regenerate plants. The embryonic cells originate from single cells, the mutation is also a single cell behavior, once the gene of the embryonic cells is mutated, all cells of somatic embryos formed by the embryonic cells and germinating and regenerating plants are mutant cells, so that the intercalation phenomenon of the mutant is overcome, and the characters controlled by the mutant genes can be transmitted to offspring.

The invention has the beneficial effects that:

the invention can overcome the bottlenecks that the conventional mutagenesis technology has the phenomenon of mutant intercalation and most of mutant cells can not be inherited; more than 60 regeneration plants can be obtained from each irradiated seed, the breeding selection population is increased by more than 60 times, the selection population is greatly increased, the selection probability is improved, and the difficulties that 1 viable seed can only obtain 1 seedling in conventional mutation breeding, the mutation population is small and the breeding selection population cannot be reached can be solved.

Detailed Description

The present application will be described in further detail with reference to examples. It is to be understood that the specific embodiments described herein are merely illustrative of the relevant invention and not restrictive of the invention. It should be noted that, for convenience of description, only the portions related to the present invention are shown below.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present application will be described in detail with reference to examples.

Embodiment 1, a method for creating peanut germplasm resources, comprising the following steps:

(1) performing fast neutron irradiation and aerospace mutagenesis on peanut seeds, taking out a seed embryo, and performing surface disinfection;

the disinfection method comprises the following steps: soaking in 70% ethanol for 20 s, soaking in 0.1% mercuric chloride solution for 8 min, and cleaning with sterilized water for 3 times;

the sterilization operation is carried out in a clean bench, and the instruments for operation need to be sterilized under high pressure or burned by an alcohol lamp.

(2) Soaking the peanut embryos subjected to surface disinfection in sterile water for 8 hours; the purpose of soaking makes the embryo lobule absorb water and stretch, and is easy to peel.

(3) Taking out the soaked embryo, placing into a culture dish subjected to autoclaving, stripping embryo leaflets, and immediately inoculating into a somatic embryo induction culture medium for culture; each seed has 8 embryonic lobules, namely a first pair of true leaves when the seed emerges, each true leaf has 4 lobules, a pair of true leaves has 8 lobules, and the 8 lobules are developed by the 8 embryonic lobules;

the reason for selecting embryo leaflets is as follows: compared with young leaves or mature leaves, the embryonic leaflets have low differentiation degree and strong regeneration capability. This is also the conclusion from trial and error of the earlier work of the present invention.

(4) The induction medium of the somatic embryo cultured by inoculating embryo lobules is as follows: MS +5mg/L2, 4-D; adjusting the pH of the culture medium to 5.8, and sterilizing in an autoclave at 120 ℃ for 20 minutes;

MS is an MS culture medium, comprises 16 elements necessary for plant growth, inorganic salt and organic components (vitamin, amino acid, 30g/L of cane sugar and 6g/L of agar);

the 2,4-D (dichlorophenoxyacetic acid) in the invention has the function of inducing the dedifferentiation of cultured embryonic lobular cells as a synthetic auxin and forming embryonic cells, and finally forming somatic embryos from the embryonic cells;

the sucrose has the functions of providing energy and substance synthesis carbon skeleton required by the growth of embryo lobules and adjusting the osmotic pressure of a culture medium;

the agar serves to solidify the medium and to change the liquid state to a solid state.

(5) After 2 weeks of culture on the somatic embryo induction medium, the embryo lobular explants start to form somatic embryos, and then the somatic embryos are gradually formed; due to the irradiation mutagenesis, part of the embryo leaflet explants are browned and die; generally, the appropriate mutagenesis dose is a dose at which half of the lethal dose or half of the regeneration rate is achieved, i.e., half of the explants cannot form somatic embryos; after culturing for 4 weeks, transferring the surviving embryo leaflet explants forming somatic embryos to a somatic embryo germination culture medium for culturing, and inducing the somatic embryos to germinate into seedlings.

(6) The somatic embryo germination culture medium comprises: MS +4 mg/LBAP; adjusting the pH of the culture medium to 5.8, and sterilizing in an autoclave at 120 ℃ for 20 minutes;

BAP (6-benzylaminopurine) is a cytokinin plant growth regulator, and has the function of inducing somatic embryos to germinate and grow into seedlings.

(7) Culturing on somatic embryo germination culture medium, subculturing once every 4 weeks, and promoting the somatic embryo to germinate into seedlings. When the plantlet grows to be more than 1.5cm, the plantlet can be used as a scion for grafting.

(8) Aseptic germination-accelerated 8-day-old peanut seed seedlings in sterilized sand are used as rootstocks, seedlings with the height of more than 1.5cm, which are germinated and grown by somatic embryos, are used as scions, aseptic grafting is carried out in a super clean workbench by adopting a grafting method, and a grafting opening is wrapped by a sealing film. And culturing the grafted seedling in sand for 3 days to promote the healing of the grafting wound, and then transplanting the grafted seedling into the field.

(9) And (4) paying attention to moisture preservation 2 weeks before transplanting the grafted seedlings into the field, and then performing field management according to the conventional method. After maturation, M2 seeds were harvested from each individual plant.

(10) The harvested M2 seeds were grown into rows per individual plant, and the mutagenized parents were used as controls. And (4) field observation in the growth period and the harvest period eliminates plant rows which are the same as the parents and have no variation and separation. The single plants in the same plant row have different performances and obvious separation, and are different from the mutagenic parents, so that the embryonic cells of the regenerated plants are mutated cells, and the progeny of the regenerated plants are subjected to selfing, character variation and separation. Selecting variant plants, selfing for 2 generations to make the genes determining the variant character homozygous, creating new germplasm resources.

Embodiment 2, a method for creating peanut germplasm resources, comprising the following steps:

(1) performing fast neutron irradiation and aerospace mutagenesis on peanut seeds, taking out a seed embryo, and performing surface disinfection;

the disinfection method comprises the following steps: soaking in 75% ethanol for 20 s, soaking in 0.1% mercuric chloride solution for 10 min, and washing with sterilized sterile water for 5 times;

the sterilization operation is carried out in a clean bench, and the instruments for operation need to be sterilized under high pressure or burned by an alcohol lamp.

(2) Soaking the peanut embryos subjected to surface disinfection in sterile water for 12 hours; the purpose of soaking makes the embryo lobule absorb water and stretch, and is easy to peel.

(3) Taking out the soaked embryo, placing into a culture dish subjected to autoclaving, stripping embryo leaflets, and immediately inoculating into a somatic embryo induction culture medium for culture; each seed has 8 embryonic lobules, namely a first pair of true leaves when the seed emerges, each true leaf has 4 lobules, a pair of true leaves has 8 lobules, and the 8 lobules are developed by the 8 embryonic lobules;

the reason for selecting embryo leaflets is as follows: compared with young leaves or mature leaves, the embryonic leaflets have low differentiation degree and strong regeneration capability. This is also the conclusion from trial and error of the earlier work of the present invention.

(5) The induction medium of the somatic embryo cultured by inoculating embryo lobules is as follows: MS +15mg/L2, 4-D; adjusting the pH of the culture medium to 5.8, and sterilizing in an autoclave at 120 ℃ for 20 minutes;

MS is an MS culture medium, comprises 16 elements necessary for plant growth, inorganic salt and organic components (vitamin, amino acid, 30g/L of cane sugar and 8g/L of agar);

the 2,4-D (dichlorophenoxyacetic acid) in the invention has the function of inducing the dedifferentiation of cultured embryonic lobular cells as a synthetic auxin and forming embryonic cells, and finally forming somatic embryos from the embryonic cells;

the sucrose has the functions of providing energy and substance synthesis carbon skeleton required by the growth of embryo lobules and adjusting the osmotic pressure of a culture medium;

the agar serves to solidify the medium and to change the liquid state to a solid state.

(5) After 2 weeks of culture on the somatic embryo induction medium, the embryo lobular explants start to form somatic embryos, and then the somatic embryos are gradually formed; due to the irradiation mutagenesis, part of the embryo leaflet explants are browned and die; generally, the appropriate mutagenesis dose is a dose at which half of the lethal dose or half of the regeneration rate is achieved, i.e., half of the explants cannot form somatic embryos; after culturing for 4 weeks, transferring the surviving embryo leaflet explants forming somatic embryos to a somatic embryo germination culture medium for culturing, and inducing the somatic embryos to germinate into seedlings.

(6) The somatic embryo germination culture medium comprises: MS +6 mg/LBAP; adjusting the pH of the culture medium to 5.8, and sterilizing in an autoclave at 120 ℃ for 20 minutes;

BAP (6-benzylaminopurine) is a cytokinin plant growth regulator, and has the function of inducing somatic embryos to germinate and grow into seedlings.

(7) Culturing on somatic embryo germination culture medium, subculturing once every 4 weeks, and promoting the somatic embryo to germinate into seedlings. When the plantlet grows to be more than 1.5cm, the plantlet can be used as a scion for grafting.

(8) Aseptic grafting is carried out on a super clean workbench by adopting a grafting method and a grafting opening is wrapped by a sealing film by taking a 10-day-old peanut seed seedling which is aseptically germinated in sterilized sand as a stock and a small seedling which is germinated and grown by a somatic embryo and is more than 1.5cm as a scion. And culturing the grafted seedlings in sand for 3-5 days to promote the healing of grafting wounds, and then transplanting the grafted seedlings into the field.

(9) And (4) paying attention to moisture preservation 2 weeks before transplanting the grafted seedlings into the field, and then performing field management according to the conventional method. After maturation, M2 seeds were harvested from each individual plant.

(10) The harvested M2 seeds were grown into rows per individual plant, and the mutagenized parents were used as controls. And (4) field observation in the growth period and the harvest period eliminates plant rows which are the same as the parents and have no variation and separation. The single plants in the same plant row have different performances and obvious separation, and are different from the mutagenic parents, so that the embryonic cells of the regenerated plants are mutated cells, and the progeny of the regenerated plants are subjected to selfing, character variation and separation. Selecting variant plants, selfing for 3 generations to make the genes determining the variant character homozygous, creating new germplasm resources.

Embodiment 3, a method for creating peanut germplasm resources, comprising the following steps:

(1) performing fast neutron irradiation and aerospace mutagenesis on peanut seeds, taking out a seed embryo, and performing surface disinfection;

the disinfection method comprises the following steps: soaking in 73% ethanol for 20 s, soaking in 0.1% mercuric chloride solution for 9 min, and cleaning with sterilized water for 4 times;

the sterilization operation is carried out in a clean bench, and the instruments for operation need to be sterilized under high pressure or burned by an alcohol lamp.

(2) Soaking the peanut embryos subjected to surface disinfection in sterile water for 10 hours; the purpose of soaking makes the embryo lobule absorb water and stretch, and is easy to peel.

(3) Taking out the soaked embryo, placing into a culture dish subjected to autoclaving, stripping embryo leaflets, and immediately inoculating into a somatic embryo induction culture medium for culture; each seed has 8 embryonic lobules, namely a first pair of true leaves when the seed emerges, each true leaf has 4 lobules, a pair of true leaves has 8 lobules, and the 8 lobules are developed by the 8 embryonic lobules;

the reason for selecting embryo leaflets is as follows: compared with young leaves or mature leaves, the embryonic leaflets have low differentiation degree and strong regeneration capability. This is also the conclusion from trial and error of the earlier work of the present invention.

(6) The induction medium of the somatic embryo cultured by inoculating embryo lobules is as follows: MS +10mg/L2, 4-D; adjusting the pH of the culture medium to 5.8, and sterilizing in an autoclave at 120 ℃ for 20 minutes;

MS is an MS culture medium, comprises 16 elements necessary for plant growth, inorganic salt and organic components (vitamin, amino acid, 30g/L of cane sugar and 7g/L of agar);

the 2,4-D (dichlorophenoxyacetic acid) in the invention has the function of inducing the dedifferentiation of cultured embryonic lobular cells as a synthetic auxin and forming embryonic cells, and finally forming somatic embryos from the embryonic cells;

the sucrose has the functions of providing energy and substance synthesis carbon skeleton required by the growth of embryo lobules and adjusting the osmotic pressure of a culture medium;

the agar serves to solidify the medium and to change the liquid state to a solid state.

(5) After 2 weeks of culture on the somatic embryo induction medium, the embryo lobular explants start to form somatic embryos, and then the somatic embryos are gradually formed; due to the irradiation mutagenesis, part of the embryo leaflet explants are browned and die; generally, the appropriate mutagenesis dose is a dose at which half of the lethal dose or half of the regeneration rate is achieved, i.e., half of the explants cannot form somatic embryos; after culturing for 4 weeks, transferring the surviving embryo leaflet explants forming somatic embryos to a somatic embryo germination culture medium for culturing, and inducing the somatic embryos to germinate into seedlings.

(6) The somatic embryo germination culture medium comprises: MS +5 mg/LBAP; adjusting the pH of the culture medium to 5.8, and sterilizing in an autoclave at 120 ℃ for 20 minutes;

BAP (6-benzylaminopurine) is a cytokinin plant growth regulator, and has the function of inducing somatic embryos to germinate and grow into seedlings.

(7) Culturing on somatic embryo germination culture medium, subculturing once every 4 weeks, and promoting the somatic embryo to germinate into seedlings. When the plantlet grows to be more than 1.5cm, the plantlet can be used as a scion for grafting.

(8) Sterile germination-accelerating 9-day-old peanut seed seedlings in sterilized sand are used as stocks, small seedlings with the height of more than 1.5cm, which are germinated and grown by somatic embryos, are used as scions, sterile grafting is carried out in a super clean bench by adopting a grafting method, and a grafting opening is wrapped by a sealing film. And culturing the grafted seedling in sand for 4 days to promote the healing of the grafting wound, and then transplanting the grafted seedling into the field.

(9) And (4) paying attention to moisture preservation 2 weeks before transplanting the grafted seedlings into the field, and then performing field management according to the conventional method. After maturation, M2 seeds were harvested from each individual plant.

(10) The harvested M2 seeds were grown into rows per individual plant, and the mutagenized parents were used as controls. And (4) field observation in the growth period and the harvest period eliminates plant rows which are the same as the parents and have no variation and separation. The single plants in the same plant row have different performances and obvious separation, and are different from the mutagenic parents, so that the embryonic cells of the regenerated plants are mutated cells, and the progeny of the regenerated plants are subjected to selfing, character variation and separation. Selecting variant plants, selfing for 2-3 generations to enable the genes determining the variant characters to be homozygous, and creating new germplasm resources.

The above description is only a preferred embodiment of the application and is illustrative of the principles of the technology employed. It will be appreciated by a person skilled in the art that the scope of the invention as referred to in the present application is not limited to the embodiments with a specific combination of the above-mentioned features, but also covers other embodiments with any combination of the above-mentioned features or their equivalents without departing from the inventive concept. For example, the above features may be replaced with (but not limited to) features having similar functions disclosed in the present application.

Other technical features than those described in the specification are known to those skilled in the art, and are not described herein in detail in order to highlight the innovative features of the present invention.