阅读说明：本技术 一种创制花生新种质与育种方法 (Novel created peanut germplasm and breeding method ) 是由 王晶珊 刘斌 于 2021-09-14 设计创作，主要内容包括：本发明提供一种创制花生新种质与育种方法,属于花生新品种培育技术领域,从花生种子的两个子叶瓣中间剥离种胚,以种胚作为诱变材料进行等离子体诱变消毒；经表面消毒的花生种胚在无菌水中浸泡,种胚取出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花生种子实生苗作为砧木,体细胞胚萌发长成的小苗作为接穗嫁接,成熟后按单株收获M2代种子,然后依次获得收获荚果M3代种子、单株荚果M4代种子,收获的M4代优良单株荚果种子按株行种植,选择优良株系。本发明利用等离子体诱变与组织培养结合的技术手段,克服花生种子大,无法利用等离子体诱变技术的瓶颈。(The invention provides a novel peanut variety creating and breeding method, belonging to the technical field of novel peanut variety cultivation, and the method comprises the steps of stripping a seed embryo from the middle of two cotyledons of a peanut seed, and carrying out plasma mutagenesis disinfection by taking the seed embryo as a mutagenesis material; soaking the peanut embryo with the sterilized surface in sterile water, taking out the embryo, putting the embryo into a sterilized culture dish, stripping embryo leaflets, and then inoculating the embryo into a somatic embryo induction culture medium for culture; then culturing on a somatic embryo germination culture medium; taking aseptic germination-accelerating peanut seed seedlings in sterilized sand as rootstocks, taking plantlets grown by somatic embryo germination as scions for grafting, harvesting seeds of M2 generation according to a single plant after maturation, then sequentially obtaining harvested pod seeds of M3 generation and seeds of M4 generation of the single plant, planting the harvested excellent single plant pod seeds of M4 generation according to plant rows, and selecting excellent plant lines. The invention utilizes the technical means of combining plasma mutagenesis and tissue culture to overcome the bottleneck that the peanut seeds are large and the plasma mutagenesis technology cannot be utilized.)

1. A novel peanut variety creating and breeding method is characterized in that: the method comprises the following steps:

(1) stripping the seed embryo from the middle of two cotyledon petals of the peanut seed, and carrying out plasma mutagenesis by taking the seed embryo as a mutagenesis material;

(2) performing surface disinfection on the peanut embryo subjected to plasma mutagenesis treatment;

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

(4) 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;

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

(6) 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;

(7) the somatic embryo germination culture medium comprises: MS + 4-6 mg/LBAP;

(8) 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;

(9) 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;

(10) 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;

(11) the harvested M2 generation seeds are planted into plants according to single plants, and the mutagenic parents are used as a control;

(12) selecting excellent single plants with multiple results, regular pods, 7-10 branches, lodging resistance and leaf spot resistance in plant rows with variant and separated traits of M2 generations, and harvesting seeds of M3 generations of pods independently;

(13) planting the harvested M3 excellent single-plant pod seeds according to plant rows, observing and marking single plants with early emergence, concentrated flowering, strong lodging resistance and upright plant shape in the growth period, selecting excellent single plants with more fruits, regular pods, 7-10 branches and leaf spot resistance in the harvest period, and harvesting M4 seeds of the single-plant pods independently;

(14) planting the harvested M4 excellent individual pod seeds according to plant rows, and mixing the excellent individual plants with consistent growth period and harvest period to form selected excellent plant lines;

(15) carrying out yield identification test on the selected excellent strains; the test is repeated for 3 times, and 1 ridge is sowed repeatedly each time; harvesting, drying in the sun, and weighing the yield; selecting strains with high yield to promote into strains;

(16) performing yield comparison test on the selected strains; the test is repeated for 3 times, and 3 ridges are sown repeatedly each time; taking a local variety regional test control variety or a local popularization variety as a control; harvesting, drying in the sun, and weighing the yield; and (4) selecting the strain with high yield, participating in provincial variety region tests, and reporting the registration of national non-main crop varieties after the tests are passed.

2. The method for creating new peanut germplasm and breeding according to claim 1, which is characterized in that: in the step (2), 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 method for creating new peanut germplasm and breeding according to claim 2, characterized by comprising the following steps: in the step (2), 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 new peanut germplasm and breeding according to claim 1, which is characterized in that: in the step (4), 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 developed from the 8 embryonic leaflets.

5. The method for creating new peanut germplasm and breeding according to claim 4, wherein the method comprises the following steps: in the step (4), 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 new peanut germplasm and breeding according to claim 1, which is characterized in that: in the steps (5) and (7), 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 new peanut germplasm and breeding according to claim 1, which is characterized in that: 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 new peanut germplasm and breeding according to claim 1, which is characterized in that: in the step (11), 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.

9. The method for creating new peanut germplasm and breeding according to claim 1, which is characterized in that: in the step (15), the sowing of the selected excellent strains in the yield identification test is double-grain sowing and ridging, the ridge distance is 80-90 cm, 2 rows are planted on each ridge, the small row distance on each ridge is 22-30 cm, the hole distance is 16-20cm, and 2 seeds are sown in each hole.

10. The method for creating new peanut germplasm and breeding according to claim 1, which is characterized in that: in the step (16), the sowing of the selected strain in the yield comparison test is double-grain sowing and ridging, the ridge distance is 80-90 cm, 2 rows are planted on each ridge, the small row distance on each ridge is 22-30 cm, the hole distance is 16-20cm, and 2 seeds are sown in each hole.

Technical Field

The invention belongs to the technical field of cultivation of new peanut varieties, and particularly relates to a novel created peanut germplasm and a breeding method.

Background

Peanuts, also known as peanuts, are the only crops which blossom on the ground and bear fruits underground, and are also called 'growing fruits' because of rich nutrition and important health-care function. The peanut kernels contain about 50% of fat, protein, multiple vitamins and multiple amino acids, and have the effects of enhancing memory, reducing cholesterol, preventing cardiovascular and cerebrovascular diseases, resisting oxidation, loosening bowel to relieve constipation and the like. The whole peanut is precious, and the peanut vine and the peanut shell are rich in protein and are good animal feed. And the peanut leaves have good hypnotic effect. Peanuts occupy an important position in national economy of China. The cultivation of new peanut varieties with high yield, high quality, special use and multiple resistance has important significance for improving the peanut yield and increasing the income of farmers. However, the genetic basis of peanuts is narrow and the diversity is poor, so that the peanut breeding becomes a bottleneck for breaking through, currently, northern peanut varieties generally have the relationship of peanuts, southern peanuts generally have the relationship of lion head enterprises, so that the relationship of the peanut varieties is close, and the novel high-yield, high-quality, special and multi-resistance peanut varieties cultured by the conventional hybrid breeding method are difficult to break through.

In order to break through the bottlenecks of narrow genetic basis and poor diversity of peanuts, peanut breeders try to create new germplasm by using a mutagenesis technology. The plasma mutagenic instrument is a novel instrument manufactured in recent years, has good mutagenic effect, is non-toxic, does not generate any pollution gas harmful to human bodies, and can carry out mutagenic treatment on microorganisms, animals and plants in a common laboratory. However, the plasma mutagenic apparatus has small mutagenic treatment space, short height and large peanut seeds, so that the plasma mutagenic apparatus cannot be used for mutagenic treatment, namely the plasma mutagenic technique cannot be used for peanut germplasm creation and breeding.

Disclosure of Invention

In order to solve the technical problems of the background art, the invention provides a novel peanut germplasm and a breeding method, which utilize a technical means of combining plasma mutagenesis and tissue culture, overcome the bottleneck that the peanut seed is large and can not utilize the plasma mutagenesis technology, create the novel peanut germplasm, provide materials for peanut breeding and open up a new way for peanut breeding; greatly increasing the selection population and improving the selection probability; overcomes the serious problem of mutant intercalation phenomenon.

In order to achieve the purpose, the invention adopts the following technical scheme: a novel peanut germplasm and a breeding method are created, and the method comprises the following steps:

(1) stripping the seed embryo from the middle of two cotyledon petals of the peanut seed, and carrying out plasma mutagenesis by taking the seed embryo as a mutagenesis material;

(2) performing surface disinfection on the peanut embryo subjected to plasma mutagenesis treatment;

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

(4) 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;

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

(6) 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;

(7) the somatic embryo germination culture medium comprises: MS + 4-6 mg/LBAP;

(8) 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;

(9) 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;

(10) 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;

(11) the harvested M2 generation seeds are planted into plants according to single plants, and the mutagenic parents are used as a control;

(12) selecting excellent single plants with multiple results, regular pods, 7-10 branches, lodging resistance and leaf spot resistance in plant rows with variant and separated traits of M2 generations, and harvesting seeds of M3 generations of pods independently;

(13) planting the harvested M3 excellent single-plant pod seeds according to plant rows, observing and marking single plants with early emergence, concentrated flowering, strong lodging resistance and upright plant shape in the growth period, selecting excellent single plants with more fruits, regular pods, 7-10 branches and leaf spot resistance in the harvest period, and harvesting M4 seeds of the single-plant pods independently;

(14) planting the harvested M4 excellent individual pod seeds according to plant rows, and mixing the excellent individual plants with consistent growth period and harvest period to form selected excellent plant lines;

(15) carrying out yield identification test on the selected excellent strains; the test is repeated for 3 times, and 1 ridge is sowed repeatedly each time; harvesting, drying in the sun, and weighing the yield; selecting strains with high yield to promote into strains;

(16) performing yield comparison test on the selected strains; the test is repeated for 3 times, and 3 ridges are sown repeatedly each time; taking a local variety regional test control variety or a local popularization variety as a control; harvesting, drying in the sun, and weighing the yield; and (4) selecting the strain with high yield, participating in provincial variety region tests, and reporting the registration of national non-main crop varieties after the tests are passed.

Preferably, in the step (2), 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 (2), 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 (4), 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 (4), 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 step (5) and step (7), 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 (6) 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 (11), 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.

Preferably, in the step (15), the sowing of the selected excellent strains in the yield identification test is double-grain sowing and ridging, the ridge distance is 80-90 cm, 2 rows are planted on each ridge, the small row distance on each ridge is 22-30 cm, the hole distance is 16-20cm, and 2 seeds are sown in each hole.

Preferably, in the step (16), the sowing of the selected strain in the yield comparison test is double-grain sowing and ridging, the ridge distance is 80-90 cm, 2 rows are planted on each ridge, the small row distance on each ridge is 22-30 cm, the hole distance is 16-20cm, and 2 seeds are sown in each hole.

The method uses the peanut seed embryo as the plasma mutagenic material, the main volume part of the peanut seed is two cotyledons, the seed embryo is very small, and the seed embryo as the plasma mutagenic material can not be limited by small mutagenic space. Secondly, after mutagenesis treatment, embryo lobules of the seed embryo are taken as explants to carry out tissue culture, each embryo lobule explant can regenerate more than 8 plantlets, and the seed embryo of each seed has 8 embryo lobules, so that 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. And thirdly, conventional mutagenesis is performed, 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 intercalation 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 method uses the seed embryo of the peanut seed to carry out mutagenesis treatment, then the embryo is taken out to be cultured, and the plant is regenerated through the embryogenesis way, namely, the cultured embryo leaflet explant cell has multiple differentiation potentials after dedifferentiation, each cell has the capacity of regenerating the plant, and the regeneration way (embryogenesis way and organogenesis way) can be regulated and controlled through the variety and concentration of the hormone added in the 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 stably inherited.

The invention has the beneficial effects that:

the invention utilizes the technical means of combining plasma mutagenesis and tissue culture, overcomes the bottleneck that the peanut seed is large and the plasma mutagenesis technology cannot be utilized, creates new peanut germplasm, provides materials for peanut breeding and opens up a new way for peanut breeding; greatly increasing the selection population and improving the selection probability; overcomes the serious problem of mutant intercalation phenomenon.

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 new peanut germplasm and breeding, comprising the following steps:

(1) and (3) stripping the seed embryo from the middle of two cotyledon petals of the peanut seed, and performing plasma mutagenesis by taking the seed embryo as a mutagenesis material.

(2) And (3) carrying out surface disinfection on the peanut embryo subjected to plasma mutagenesis treatment.

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 washing 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.

(3) The surface sterilized peanut embryos are soaked in sterile water for 8 hours. The purpose of soaking makes the embryo lobule absorb water and stretch, and is easy to peel.

(4) The soaked embryos are taken out, placed into an autoclaved culture dish, stripped of embryo leaflets, and then inoculated into a somatic embryo induction culture medium for culture. Each seed has 8 embryonic lobules, namely the first pair of true leaves when the seed emerges, each true leaf has 4 lobules, and 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 +5mg/L2, 4-D. The pH of the medium was adjusted to 5.8, and the medium was sterilized in an autoclave at 120 ℃ for 20 minutes.

MS is an MS culture medium, comprises 16 elements necessary for plant growth, and contains inorganic salt and organic components (vitamins, amino acid, 30g/L of sucrose and 6-8 g/L of agar).

The 2,4-D (dichlorophenoxyacetic acid) functions in the present invention as a synthetic auxin to induce de-differentiation of cultured embryonic lobular cells and formation of embryonic cells from which somatic embryos are ultimately formed.

The sucrose functions as a carbon skeleton for providing energy and material synthesis required for growth of embryo leaflets, and can regulate the osmotic pressure of the culture medium.

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

(6) After 2 weeks of culture on somatic embryo induction medium, the embryo leaflet explants began to form somatic embryos, after which somatic embryos 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 half lethal dose or half regeneration rate, i.e., half of the explants are unable to 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.

(7) The somatic embryo germination culture medium comprises: MS +4mg/LBAP +30g/L sucrose +6g/L agar. The pH of the medium was adjusted to 5.8, and the medium was sterilized 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.

(8) 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.

(9) 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.

(10) 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.

(11) 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.

(12) In the rows with variant and separated traits of M2 generation, selecting excellent single plants with multiple results, regular pods, 7 branches, lodging resistance and leaf spot resistance, and harvesting seeds of M3 generation of pods individually.

(13) The harvested M3 generation excellent single plant pod seeds are planted according to plant rows, single plants with early emergence, concentrated flowering, strong lodging resistance and upright plant shape are marked by observation in the growth period, the excellent single plants with more fruits, regular pods, 7 branches and leaf spot resistance are selected in the harvest period, and the M4 generation seeds of the single plant pod are harvested independently.

(14) The harvested M4 generation excellent individual pod seeds are planted according to plant rows, and excellent individual plants with consistent growth period and harvest period are mixed to become selected excellent plants.

(15) And carrying out yield identification test on the selected excellent strains. Double-grain sowing, ridging, wherein the ridge distance is 80cm, 2 rows are planted on each ridge, the small row distance on each ridge is 22cm, the hole distance is 16cm, and 2 seeds are sowed in each hole. The test was set up for 3 replicates, each replicate seeding 1 ridge. After harvesting, the seeds are dried in the sun and the yield is weighed. And selecting the strain with high yield to promote to the strain.

(16) The selected lines were subjected to yield comparison tests. Double-grain sowing, ridging, wherein the ridge distance is 80cm, 2 rows are planted on each ridge, the small row distance on each ridge is 22cm, the hole distance is 16cm, and 2 seeds are sowed in each hole. The experiment was set up for 3 replicates, each replicate seeding 3 ridges. Local variety regional test control varieties or local popularization varieties are used as controls. After being ripe and harvested, the seeds are dried in the sun and the yield is weighed. And (4) selecting the strain with high yield, participating in provincial variety region tests, and reporting the registration of national non-main crop varieties after the tests are passed.

Embodiment 2, a method for creating new peanut germplasm and breeding, comprising the following steps:

(1) and (3) stripping the seed embryo from the middle of two cotyledon petals of the peanut seed, and performing plasma mutagenesis by taking the seed embryo as a mutagenesis material.

(2) And (3) carrying out surface disinfection on the peanut embryo subjected to plasma mutagenesis treatment.

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 autoclaved 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.

(3) The surface sterilized peanut embryos are soaked in sterile water for 12 hours. The purpose of soaking makes the embryo lobule absorb water and stretch, and is easy to peel.

(4) The soaked embryos are taken out, placed into an autoclaved culture dish, stripped of embryo leaflets, and then inoculated into a somatic embryo induction culture medium for culture. Each seed has 8 embryonic lobules, namely the first pair of true leaves when the seed emerges, each true leaf has 4 lobules, and 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. The pH of the medium was adjusted to 5.8, and the medium was sterilized in an autoclave at 120 ℃ for 20 minutes.

MS is an MS culture medium, comprises 16 elements necessary for plant growth, and contains inorganic salt and organic components (vitamins, amino acid, 30g/L of sucrose and 6-8 g/L of agar).

The 2,4-D (dichlorophenoxyacetic acid) functions in the present invention as a synthetic auxin to induce de-differentiation of cultured embryonic lobular cells and formation of embryonic cells from which somatic embryos are ultimately formed.

The sucrose functions as a carbon skeleton for providing energy and material synthesis required for growth of embryo leaflets, and can regulate the osmotic pressure of the culture medium.

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

(6) After 2 weeks of culture on somatic embryo induction medium, the embryo leaflet explants began to form somatic embryos, after which somatic embryos 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 half lethal dose or half regeneration rate, i.e., half of the explants are unable to 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.

(7) The somatic embryo germination culture medium comprises: MS +6 mg/LBAP. The pH of the medium was adjusted to 5.8, and the medium was sterilized 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.

(8) 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.

(9) 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 seedling in sand for 5 days to promote the healing of the grafting wound, and then transplanting the grafted seedling into the field.

(10) 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.

(11) 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.

(12) In the rows with variant and separated traits of M2 generation, selecting excellent single plants with multiple results, regular pods, 10 branches, lodging resistance and leaf spot resistance, and harvesting seeds of M3 generation of pods individually.

(13) The harvested M3 generation excellent single plant pod seeds are planted according to plant rows, single plants with early emergence, concentrated flowering, strong lodging resistance and upright plant shape are marked by observation in the growth period, the excellent single plants with multiple fruits, regular pods, 10 branches and leaf spot resistance are selected in the harvest period, and the M4 generation seeds of the single plant pod seeds are harvested independently.

(14) The harvested M4 generation excellent individual pod seeds are planted according to plant rows, and excellent individual plants with consistent growth period and harvest period are mixed to become selected excellent plants.

(15) And carrying out yield identification test on the selected excellent strains. Double-grain sowing, ridging, wherein the ridge distance is 90cm, 2 rows are planted on each ridge, the small row distance on each ridge is 30cm, the hole distance is 20cm, and 2 seeds are sowed in each hole. The test was set up for 3 replicates, each replicate seeding 1 ridge. After harvesting, the seeds are dried in the sun and the yield is weighed. And selecting the strain with high yield to promote to the strain.

(16) The selected lines were subjected to yield comparison tests. Double-grain sowing, ridging, wherein the ridge distance is 90cm, 2 rows are planted on each ridge, the small row distance on each ridge is 30cm, the hole distance is 20cm, and 2 seeds are sowed in each hole. The experiment was set up for 3 replicates, each replicate seeding 3 ridges. Local variety regional test control varieties or local popularization varieties are used as controls. After being ripe and harvested, the seeds are dried in the sun and the yield is weighed. And (4) selecting the strain with high yield, participating in provincial variety region tests, and reporting the registration of national non-main crop varieties after the tests are passed.

Embodiment 3, a method for creating new peanut germplasm and breeding, comprising the following steps:

(1) and (3) stripping the seed embryo from the middle of two cotyledon petals of the peanut seed, and performing plasma mutagenesis by taking the seed embryo as a mutagenesis material.

(2) And (3) carrying out surface disinfection on the peanut embryo subjected to plasma mutagenesis treatment.

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 washing with autoclaved sterile 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.

(3) The surface sterilized peanut embryos are soaked in sterile water for 10 hours. The purpose of soaking makes the embryo lobule absorb water and stretch, and is easy to peel.

(4) The soaked embryos are taken out, placed into an autoclaved culture dish, stripped of embryo leaflets, and then inoculated into a somatic embryo induction culture medium for culture. Each seed has 8 embryonic lobules, namely the first pair of true leaves when the seed emerges, each true leaf has 4 lobules, and 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 +10mg/L2, 4-D. The pH of the medium was adjusted to 5.8, and the medium was sterilized in an autoclave at 120 ℃ for 20 minutes.

MS is an MS culture medium, comprises 16 elements necessary for plant growth, and contains inorganic salt and organic components (vitamins, amino acid, 30g/L of sucrose and 6-8 g/L of agar).

The 2,4-D (dichlorophenoxyacetic acid) functions in the present invention as a synthetic auxin to induce de-differentiation of cultured embryonic lobular cells and formation of embryonic cells from which somatic embryos are ultimately formed.

The sucrose functions as a carbon skeleton for providing energy and material synthesis required for growth of embryo leaflets, and can regulate the osmotic pressure of the culture medium.

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

(6) After 2 weeks of culture on somatic embryo induction medium, the embryo leaflet explants began to form somatic embryos, after which somatic embryos 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 half lethal dose or half regeneration rate, i.e., half of the explants are unable to 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.

(7) The somatic embryo germination culture medium comprises: MS +5mg/LBAP +30g/L sucrose +7g/L agar. The pH of the medium was adjusted to 5.8, and the medium was sterilized 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.

(8) 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.

(9) 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.

(10) 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.

(11) 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.

(12) In the rows with variant and separated traits of M2 generation, selecting excellent single plants with multiple results, regular pods, 8 branches, lodging resistance and leaf spot resistance, and harvesting seeds of M3 generation of pods individually.

(13) The harvested M3 generation excellent single plant pod seeds are planted according to plant rows, single plants with early emergence, concentrated flowering, strong lodging resistance and upright plant shape are marked by observation in the growth period, the excellent single plants with multiple fruits, regular pods, 8 branches and leaf spot resistance are selected in the harvest period, and the M4 generation seeds of the single plant pod are harvested independently.

(14) The harvested M4 generation excellent individual pod seeds are planted according to plant rows, and excellent individual plants with consistent growth period and harvest period are mixed to become selected excellent plants.

(15) And carrying out yield identification test on the selected excellent strains. Double-seed sowing, ridging, wherein the ridge distance is 85cm, 2 rows are planted on each ridge, the small row distance on each ridge is 26cm, the hole distance is 18cm, and 2 seeds are planted in each hole. The test was set up for 3 replicates, each replicate seeding 1 ridge. After harvesting, the seeds are dried in the sun and the yield is weighed. And selecting the strain with high yield to promote to the strain.

(16) The selected lines were subjected to yield comparison tests. Double-seed sowing, ridging, wherein the ridge distance is 85cm, 2 rows are planted on each ridge, the small row distance on each ridge is 26cm, the hole distance is 18cm, and 2 seeds are planted in each hole. The experiment was set up for 3 replicates, each replicate seeding 3 ridges. Local variety regional test control varieties or local popularization varieties are used as controls. After being ripe and harvested, the seeds are dried in the sun and the yield is weighed. And (4) selecting the strain with high yield, participating in provincial variety region tests, and reporting the registration of national non-main crop varieties after the tests are passed.

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.