CN111837957A - Culture medium for in-vitro directional screening of high-oil bodies of peanuts and screening method - Google Patents
Culture medium for in-vitro directional screening of high-oil bodies of peanuts and screening method Download PDFInfo
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Abstract
The invention discloses a culture medium for in vitro directional screening of high oil bodies of peanuts and a screening method, and belongs to the technical field of peanut breeding. The culture medium for in vitro directional screening of the peanut high-oil bodies comprises a somatic embryo germination and screening culture medium, a seedling forming and screening culture medium and a recovery culture medium; the somatic embryo germination and screening culture medium takes an MS culture medium as a basic culture medium, and 3-5 mmol/L HYP and 3-5 mg/L BAP are added; the seedling and screening culture medium takes an MS culture medium as a basic culture medium, and 7-9 mmol/L HYP and 1-3 mg/L BAP are added; the recovery medium takes an MS culture medium as a basic culture medium, and 1-3 mg/L BAP is added. The culture medium is adopted to screen the peanut high-oil bodies, so that the screening efficiency can be improved, and the hardening time of the screened plants in the later period can be effectively reduced.
Description
The application is a divisional application of a patent application of 'patent application No. 201810125227.7, application date 2018.02.08, application name of an in vitro directional screening method of peanut high oil bodies'.
Technical Field
The invention belongs to the technical field of peanut breeding, and particularly relates to a culture medium for in vitro directional screening of peanut high-oil bodies and a screening method.
Background
Peanuts are important oil crops in China and occupy an important position in national economy. More than 50% of peanuts produced in China are used for oil pressing, generally more than 50% of oil content is high-oil varieties or high-oil materials, and the pure profit can be increased by 7% when the oil content is increased by 1%, so that the cultivation of new high-oil peanut varieties becomes one of the main breeding targets of broad breeders. However, the peanut lacks high-oil-product resources, and high-oil progeny is difficult to obtain by crossbreeding, so the breeding of high-yield high-oil-product seeds is always a problem which is difficult to solve. The utilization of the mutagenesis technology can cause gene mutation or chromosome variation, but the mutation is non-directional, the conventional mutagenesis breeding can not directionally obtain the required high-oil mutant according to the breeding target, and the subsequent identification of the mutant requires a large amount of manpower, financial resources and material resources. The difficult problem can be solved by utilizing the combination of in vitro mutagenesis and tissue culture to carry out in vitro directional screening on the high oil body. Can save manpower, material resources and financial resources, is not limited by time and space, shortens breeding time, expands variation spectrum and improves variation rate. In vitro mutagenesis and directed screening of high oil bodies is an effective means for obtaining new high oil germplasm and cultivating new high oil varieties.
Radiation mutagenesis is one of important means for germplasm innovation and variety improvement, has the characteristics of high mutation frequency, large mutation range, stable and quick offspring, short breeding period and the like, and commonly used mutagenesis sources comprise gamma rays, X rays, fast neutrons, ion beams and the like. In China, the irradiation mutation breeding of peanuts starts in the 60 th 20 th century, and researchers have successively researched different mutation methods of peanuts in recent years for developing new breeding methods. Jiangdefeng et al (nuclear agriculture report, 2017, 31,1678-60Co-gamma ray irradiation (irradiation measurement 150Gy) is carried out on peanut variety Luhua No. 11 to obtain a new strain with pod character variation and crude fat content as high as 54.3 percent, and the result of the thesis shows that the induction can improve the oil content of the peanut, but the peanut cannot be directionally obtained and can only be randomly obtained, and some high-oil mutants are easy to eliminate due to low yield. Plum crown and the like (agricultural biotechnology, May 25,2017,33(5): 766-.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides the culture medium and the screening method for in vitro directional screening of the high oil bodies of the peanuts, which not only can improve the screening efficiency, but also can effectively reduce the seedling hardening time of the screened plants in the later period.
In order to solve the problems, the invention adopts the following technical scheme:
a culture medium for in vitro directional screening of high oil bodies of peanuts comprises a somatic embryo germination and screening culture medium, a seedling forming and screening culture medium and a growth recovery culture medium;
the somatic embryo germination and screening culture medium takes an MS culture medium as a basic culture medium, and 3-5 mmol/L HYP and 3-5 mg/L BAP are added;
the seedling and screening culture medium takes an MS culture medium as a basic culture medium, and 7-9 mmol/L HYP and 1-3 mg/L BAP are added;
the growth recovery culture medium takes an MS culture medium as a basic culture medium, and 1-3 mg/L BAP is added.
On the basis of the scheme, the concentration of HYP in the somatic embryo germination and screening culture medium is 4 mmol/L.
On the basis of the scheme, the concentration of HYP in the seedling and screening culture medium is 8 mmol/L.
An in vitro directional screening method of peanut high oil bodies uses the following culture medium for screening:
germination and screening culture medium of somatic embryo: taking an MS culture medium as a basic culture medium, and adding 3-5 mmol/L HYP and 3-5 mg/L BAP;
seedling formation and culture medium screening: taking an MS culture medium as a basic culture medium, and adding 7-9 mmol/L HYP and 1-3 mg/L BAP;
restoring the growth medium: an MS culture medium is used as a basic culture medium, and 1-3 mg/L BAP is added.
On the basis of the scheme, the in vitro directional screening method of the peanut high oil bodies comprises the following steps:
(1) transferring explants forming somatic embryos to a somatic embryo germination and screening medium for 4 weeks;
(2) transferring the explants with somatic embryos screened and alive by the step (1) to a seedling and screening culture medium for screening culture for 4 weeks;
(3) transferring the explants screened and cultured in the step (2) to a growth recovery culture medium for culture; after culturing for 4 weeks, the partially germinated somatic embryos resume growth;
(4) alternately culturing the somatic embryos recovered from the step (3) on a seedling forming and screening culture medium and a growth recovery culture medium until the regenerated plantlets grow to 1 cm; finally, the obtained seedlings are high-oil peanut seedlings or high-oil peanut bodies.
The culture conditions in the screening method are as follows: the culture temperature is 25-27 ℃, the illumination intensity is 2000-3000 Lx, and the daily illumination is 12-14 h.
On the basis of the above scheme, the explants in the step (1) are: selecting a seed embryo with plump seeds and a cotyledon removed, carrying out surface disinfection, taking the embryo lobule, inoculating the embryo lobule to a somatic embryo induction culture medium, and culturing under the conditions that the temperature of a culture room is 25-27 ℃, the illumination intensity is 2000-3000 Lx, and the daily illumination is 12-14 h, so as to induce the formation of the somatic embryo.
On the basis of the scheme, the somatic embryo induction culture medium takes an MS culture medium as a basic culture medium, and 4-12 mg/L2,4-D is added.
Compared with the prior art, the invention has the advantages and positive effects that:
1. the screening process is carried out in the culture medium, a large amount of culture materials without resistance are eliminated, a large amount of resources and cost can be saved, the culture and the directional screening can be carried out all the year round, and the operation is convenient. The method can create and screen new high-oil peanut varieties, broaden the genetic basis of peanuts, and overcome the difficulty that breeding of new high-yield high-oil varieties is difficult to break through due to the lack of high-oil variety resources in the peanuts.
2. The invention inoculates mature peanut seed embryo lobules on a somatic embryo induction culture medium to induce the formation of somatic embryos. Transferring the surviving explants to a somatic embryo germination culture medium containing 4m mol/L HYP and 3-5 mg/L BAP, and performing first high-oil directional screening; and transferring the seedlings to a seedling culture medium containing 8m mol/L HYP and 1-3 mg/L BAP after 4 weeks, performing secondary screening, recovering for 4 weeks after 4 weeks in the secondary screening process, and performing circulation, so that the screening efficiency can be improved, and the seedling hardening time of the screened plants in the later period can be effectively reduced.
3. The high oil body obtained by in vitro directional screening on the culture medium adopts a grafting method, overcomes the difficulties of difficult rooting of peanut tissue culture seedlings and low survival rate of domestication and transplantation, utilizes a seedling culture substrate as a germination accelerating substrate of a stock of a grafted seedling, directly transplants the high oil seedling as a scion into a field after aseptic grafting, and can greatly save the domestication and culture time in a domestication room. The small arched shed made of the mulching film after direct transplanting in the field has simple operation and good moisturizing effect, is beneficial to wound healing of the grafted seedlings, and can save manpower, material resources and financial resources.
Drawings
FIG. 1 shows phenotypic variation of high oil body progeny obtained by directional screening;
FIG. 2 is SSR electrophoretogram of progeny of Huayu No. 20 and high oil body in the invention; the left side is a primer pair PM 15; the right side is a primer pair of PM 297; CK: mutagenizing the parent floral development No. 20; others were high oil body M3 generation.
Detailed Description
The invention will be further described with reference to specific examples, to which, however, the scope of the invention is not limited. In addition, any modification of the present invention which is obvious to a person skilled in the relevant art and which does not depart from the gist of the invention or equivalent alternatives are intended to be within the scope of the present invention.
Example 1
The invention relates to an in vitro directional screening method of peanut high oil bodies, which comprises the following steps:
1. preparation of somatic embryo Induction Medium
(1) Preparing a somatic embryo induction culture medium, selecting an MS culture medium as a basic culture medium, and adding 2,4-D into the MS culture medium to form the somatic embryo induction culture medium. The addition amount of the 2,4-D is 2-12 mg/L. In the embodiment, the somatic embryo induction culture medium is MS + 5mg/L2,4-D +30g/L sucrose +8g/L agar; the pH of the somatic embryo induction medium was adjusted to 5.8.
2. Preparation of medium for somatic embryo germination and seedling formation and high oil body directional screening
(1) Preparing a somatic embryo germination and high-oil screening culture medium: an MS culture medium is selected as a basic culture medium, hydroxyproline and 6-Benzylaminopurine (BAP) are added into the MS culture medium to form a somatic embryo germination and high-oil directional screening culture medium, the addition amount of the hydroxyproline is 3-5 m mol/L, and the addition amount of the BAP is 3-5 mg/L. In the embodiment, the somatic embryo germination and screening medium is MS +4m mol/L hydroxyproline +4mg/L BAP +30g/L sucrose +8g/L agar, and the pH value of the somatic embryo germination and screening medium is adjusted to 5.8.
(2) Preparing a seedling and high-oil screening culture medium: an MS culture medium is selected as a basic culture medium, hydroxyproline and BAP are added into the MS culture medium to form a seedling and high-oil directional screening culture medium, the addition amount of the hydroxyproline is 7-9 m mol/L, and the addition amount of the BAP is 1-3 mg/L. In the embodiment, the seedling formation and screening culture medium is MS +8m mol/L hydroxyproline +2mg/L BAP +30g/L sucrose +8g/L agar, and the pH value of the somatic embryo germination and screening culture medium is adjusted to 5.8.
(3) Preparing a growth recovery culture medium: an MS culture medium is selected as a basic culture medium, BAP is added into the MS culture medium to form a growth recovery culture medium, and the addition amount of the BAP is 1-3 mg/L. In this example, the recovery medium was MS +2mg/L BAP +30g/L sucrose +8g/L agar, and the pH of the recovery growth medium was adjusted to 5.8. Culturing on a seedling and high-oil directional screening culture medium for 4 weeks, transferring to a growth recovery culture medium for culturing for 4 weeks, then transferring to a seedling and high-oil directional screening culture medium for culturing for 4 weeks, and alternately performing screening and recovery culture until the seedlings grown by the somatic embryos germinate grow to be more than 1 cm.
The invention uses proline analogue hydroxyproline as a screening agent, can tolerate hydroxyproline with a limit concentration, and surely causes mutation of related genes, so that the content of proline in a screened object is increased. Peanuts are oil crops, and proline degradation can be involved in fat synthesis. In the invention, hydroxyproline is added into the screening culture medium, so that high oil bodies can be screened. BAP is a synthetic purine plant growth regulator, can promote cell division, tissue differentiation, seed germination, lateral bud growth and the like, and is used for inducing somatic embryo germination and seedling formation.
3. Establishment of hydroxyproline screening concentration
Hydroxyproline is used as a screening pressure to be added into a culture medium for directional screening of high oil bodies. Culturing under the conditions of 25-27 ℃, illumination intensity of 2000-3000 Lx and daily illumination of 12-14 h
The explant material is a peanut seed flower culture No. 20 widely popularized and cultivated at present, and embryo with plump seeds and cotyledons removed is selected to be subjected to surface disinfection, and embryo leaflets are taken as test materials.
The tolerance of plants to stress varies in the various stages of growth and development. Similarly, the tolerance level to adversity stress at each stage under tissue culture conditions is also different. Hydroxyproline is used for simulating stress conditions, and is added into a culture medium for directional screening of high oil bodies, so that the optimum screening concentration of hydroxyproline in two different stages of germination and seedling formation of somatic embryos obtained by peanut embryo lobule tissue culture is researched.
(1) And (3) screening hydroxyproline in the somatic embryo germination stage, transferring explants forming somatic embryos after culturing for 4 weeks on a somatic embryo induction culture medium to a somatic embryo germination and screening culture medium for culturing, and adding 0, 2,4, 6 and 8mmol/L hydroxyproline in the somatic embryo germination and screening culture medium. After culturing for 4 weeks, on a somatic embryo germination culture medium without hydroxyproline, the somatic embryo germination is normal; on the somatic embryo germination and screening medium added with 4mmol/L hydroxyproline, although the somatic embryo is not completely lethal, the growth of the somatic embryo is severely inhibited; the embryos completely browned and died on the germination and screening medium of embryos added with 8m mol/L hydroxyproline. Therefore, 4mmol/L hydroxyproline was used as the high oil screening concentration for this incubation period.
(2) Hydroxyproline screening in the seedling stage, and normal culture is carried out without screening in the somatic embryo germination stage. Culturing the embryo lobular explant on an induction culture medium for 4 weeks to form a somatic embryo cluster, transferring the explant forming the somatic embryo cluster to a somatic embryo germination culture medium without hydroxyproline, and culturing for 4 weeks, wherein most somatic embryos germinate. Then transferring the germinated somatic embryo plexus to a seedling and screening culture medium for culture, and adding hydroxyproline of 0,4, 8 and 12m mol/L into the seedling and screening culture medium. 4w later, on a culture medium without hydroxyproline, the seedlings grown by somatic embryo germination grow normally; on a culture medium added with 4m mol/L hydroxyproline, the growth of seedlings grown by somatic embryo germination is inhibited, but the seedlings can also grow; the growth of the plantlets grown by somatic embryo germination on the seedling and screening medium supplemented with 8m mol/L hydroxyproline was severely inhibited. Therefore, 8m mol/L hydroxyproline was added as a high oil screening concentration for this incubation period.
4. Mutagenesis in combination with tissue culture
(1) Fast neutron irradiation and in vitro culture
The method is characterized in that mature and plump seeds of peanut variety No. 20 in flower cultivation are used as test materials to carry out fast neutron irradiation treatment (9-20 Gy), and 9.7Gy is adopted in the method. Removing cotyledons of the irradiated seeds, peeling off embryos, placing the seeds in a superclean workbench to be soaked in 75% alcohol for 20s, then soaking in 0.1% mercuric chloride for 12min, carrying out surface disinfection, rinsing with sterile water for 5 times, and then placing the seeds in a culture bottle filled with sterile water to be soaked for 8-12 h. Taking out the seed embryo which is disinfected and soaked on the surface, placing the seed embryo in a sterile culture dish, stripping embryo leaflets, then inoculating the seed embryo to a somatic embryo induction culture medium added with 5mg/L2,4-D, and culturing under the conditions that the temperature of a culture room is 25-27 ℃, the illumination intensity is 2000-3000 Lx, and the daily illumination is 12-14 h, so as to induce the formation of the somatic embryo. After 4 weeks of culture, about 40% of the explants formed somatic embryos, and other explants formed only callus or browned. While about 80% of the unirradiated seed embryo leaflet explants formed somatic embryos.
5. Directional screening of high oil bodies
After the embryonic lobules subjected to fast neutron irradiation treatment are cultured in vitro for 4 weeks, explants forming somatic embryos are transferred to a somatic embryo germination culture medium and a seedling culture medium in sequence to promote the germination and seedling of the somatic embryos, and high oil bodies are directionally screened. And (4) performing directional screening on the high oil body by using the determined hydroxyproline screening concentration. Firstly, 4m mol/L hydroxyproline is added into a somatic embryo germination culture medium, then 8m mol/L hydroxyproline is added into a seedling culture medium, and the culture is alternately carried out for 4 weeks by adopting screening pressure and non-screening pressure until the regenerated plantlet grows to be more than 1 cm.
The method specifically comprises the following steps: and (3) performing irradiation mutagenesis on the fast neutrons, culturing, transferring the fast neutrons to a somatic embryo germination and screening culture medium, culturing under the conditions that the temperature of a culture room is 25-27 ℃, the illumination intensity is 2000-3000 Lx, and the daily illumination is 12-14 h, and performing high-oil directional screening and somatic embryo germination and seedling formation simultaneously. Firstly, a screening culture medium added with 4m mol/L hydroxyproline is selected, most explants and somatic embryos die after 4 weeks of culture, and only a few explants survive. The explants with somatic embryos which are screened to live are transferred to a screening culture medium added with 8m mol/L hydroxyproline, and after 4 weeks of screening culture, partial explants and germinated somatic embryos are browned. After transferring to a growth recovery medium without hydroxyproline for 4 weeks, the partially germinated embryos recovered. Then, the medium added with 8m mol/L hydroxyproline is used for screening, and the steps are alternately carried out until seedlings are formed, and finally, the anther-bred No. 20 hydroxyproline resistant seedlings (high oil bodies) are obtained.
6. Grafting and transplanting of high oil bodies
(1) Preparation of grafting stock and aseptic grafting
Seedling growing medium is selected as germination accelerating medium, the seedling growing medium is placed in a culture bottle, and high-pressure sterilization is carried out after a proper amount of water is added.
And (2) selecting and cultivating seeds with No. 20 plump flowers and seeds for surface disinfection, soaking the seeds in sterile water for 4-6 hours, planting the seeds in the sterilized seedling substrate, cultivating the seeds under the conditions that the temperature of a culture room is 25-27 ℃, the illumination intensity is 2000-3000 Lx and the illumination is 12-14 hours per day, and after 11 days, germinating the stock seeds, extending the hypocotyl and grafting the stock seeds.
The plantlet which is obtained by the somatic embryo germination and growth obtained by the flower-growing No. 20 mutagenesis and directional screening is used as a scion, the plantlet is cut into a V shape, the flower-growing No. 20 seedling which is subjected to aseptic germination acceleration is used as a stock, the part above a cotyledon node is cut off, the middle part of a hypocotyl is longitudinally cut, then the scion is inserted into the stock, a grafting opening is sealed by a sealing film, the water loss is prevented, the opening is also in close contact, and a microtubule system of the stock and the scion is connected.
(2) Domestication and transplantation of grafted seedlings
And transplanting the grafted seedlings into a sterilized seedling raising substrate, and culturing for 4 days under the conditions that the temperature of a culture room is 25-27 ℃, the illumination intensity is 2000-3000 Lx, and the daily illumination is 12-14 h. And (3) opening a bottle cap for acclimatization for 2 days, directly transplanting the seedlings into the field, and carrying out ridging planting on the grafted seedlings at a ridge spacing of 50-60 cm and a plant spacing of 20-25 cm. After the grafted seedlings are transplanted into the field, small arched sheds are built by mulching films, 1 arched shed is built on each ridge, water is poured, a shading net is covered from 9 am to 4 pm in the early stage of transplanting 10-12 days, and the sun is prevented from directly drying. And (5) removing the small arched shed after transplanting for 3 weeks, performing field management according to a normal method, and enabling the grafted seedlings to grow normally, bloom and bear fruits.
7. High oil body progeny variation and segregation
The grafted high oil seedling (body) transplanted to the field is harvested according to a single plant, planted into a plant line in the next year, and the mutagenized parent flower-cultivated No. 20 is used as a control. The height of the No. 20 plant bred by the flower is 38-42 cm, the branches are thinned, the number of the branches is 9-11, the flower habit is continuous flowering, the pod is common, and the seed coat is pink. The high oil body progeny shows obvious variation and separation in various aspects of plant height, flowering habit, branch number, single plant fruiting number, pod shape, seed coat color and the like.
The number of the individual plants of the descendant of the high oil body No. b is increased, the number of branches is increased, and the number of branches of b-1 and b-2 is 16-17, as shown in a figure 1-b.
The branch number, the pod shape and the seed coat color of the high oil body descendant of No. d are obviously varied and separated, the branch number of d-1 is increased, the pod shape is suddenly separated into a bead shape, the seed coat color is suddenly changed into mauve, the branch number of d-2 is also increased, but the pod shape is a common shape, the seed coat color is pink, the branch number of d-3 is unchanged, but the pod shrinkage is extremely deep, and the shape is suddenly changed into a calabash shape, as shown in the figure 1-d.
The shape of the pod of the high oil body descendant of No. e and the number of the single plants are obviously varied and separated, the number of the single plants of e-2 is reduced, the pod is reduced, part of pod shrinkage is deep, the shape is mutated into a gourd shape, the number of the single plants of e-3 is increased, part of pod shrinkage is not obvious, the shape is a silkworm cocoon shape, the 20-blossom shrinkage is obvious, and the shape is a common shape, as shown in the figure 1-e.
The shape of the pod of the high oil body descendant of No. f and the number of the single plants are obviously varied and separated, the number of the single plants of f-1 is very little, the shrinkage of the f-2 single pod is deep, and the shape is changed into a gourd shape, as shown in a figure 1-f.
8. Detection of oil content of high oil body progeny
Peanut is one of oil crops, the oil content of a common variety is about 50%, and the high-oil variety or high-oil material is determined when the oil content reaches more than 55%. The net profit can be increased by 7 percent when the oil content is increased by 1 percent
After the high oil bodies obtained by combining in vitro mutagenesis and in vitro directional screening are subjected to selfing and homozygosis, the oil content of the seed kernels of the progeny strain is determined by adopting a near infrared method, and the oil content (50.01-60.03%) of all the progeny of the high oil bodies is higher than that (49.50%) of the mutagenized parent floriculture No. 20.
30 strains with high oil content are selected to be delivered to the oil and product quality supervision and inspection test center of the department of agriculture in 2014 and 2015 respectively for inspection, the mutagenic parent flower culture No. 20 is used as a control, the inspection results are shown in Table 1,
as can be seen from Table 1, the oil content of the mutagenized parent floral-bred No. 20 is 49.50%, and of 30 high-oil-body progeny strains detected, 28 high-oil-content lines reach the high-oil (over 55%) standard, wherein the oil content of 6 high-oil-body progeny strains is more than 59%, and the oil content of 10 high-oil-body progeny strains is 58-59% and is 59.63% at most.
TABLE 1 oil content of high oil bodies progeny
| Numbering | Oil content (%) |
| Flower cultivation 20 | 49.50 |
| 1 | 57.56 |
| 2 | 59.48 |
| 3 | 57.23 |
| 4 | 58.45 |
| 5 | 56.68 |
| 6 | 57.23 |
| 7 | 57.73 |
| 8 | 58.34 |
| 9 | 56.47 |
| 10 | 58.06 |
| 11 | 56.93 |
| 12 | 58.02 |
| 13 | 59.52 |
| 14 | 57.22 |
| 15 | 56.10 |
| 16 | 58.17 |
| 17 | 58.49 |
| 18 | 56.52 |
| 19 | 58.40 |
| 20 | 58.83 |
| 21 | 56.77 |
| 22 | 54.28 |
| 23 | 54.56 |
| 24 | 58.65 |
| 25 | 59.38 |
| 26 | 58.96 |
| 27 | 57.88 |
| 28 | 59.63 |
| 29 | 59.50 |
| 30 | 59.06 |
9. SSR detection of high oil bodies
And randomly selecting 13 high-oil-body offspring (from different high-oil bodies), extracting total DNA of the leaves, and detecting and analyzing DNA variation by using SSR primer pairs. The amplification products of 7 pairs of primers revealed polymorphisms between the mutant material and the mutagenized parent, florescence # 20, and 12 of the 13 tested materials presented more than 2 site mutations, as shown in table 2. As can be seen from Table 2, the SSR marker polymorphisms are expressed in a reduced number of amplified fragments (marked A) compared to the mutagenized parental floral No. 20; the number of amplified fragments increased (labeled B); the amplified fragments differ in length (labeled C). A. B, C the ratio of the three forms is 20.69%, 27.59% and 51.72%. The high oil mutation is shown to be caused by the mutation of the gene, and the plant height, the flowering habit, the branch number, the single plant fruiting number, the pod shape, the seed coat color and the like are all caused by the mutation of the gene. The oil content of the offspring of the same high oil body is separated because the high oil mutant gene is generated on one of the homologous chromosomes, and in the selfing process, the oil content of sister lines is different because the genes carried by the homologous chromosomes are separated and homozygous. FIG. 2 is an electrophoretogram of c-1, k-12, d-2, j-5 and l-12.
TABLE 2 SSR polymorphic analysis of high oil bodies and mutagenized parental floral No. 20
Note: a, the number of amplified fragments is reduced; b: the number of amplified fragments increases; c: difference in amplified fragment length
The specific primers are as follows:
according to the invention, fast seed irradiation is combined with tissue culture, hydroxyproline is used as indirect screening pressure for high oil screening, and the obtained high oil body is directly transplanted into a field through aseptic grafting, so that normal mature seeds are obtained. The phenotype of the high oil body progeny is obviously mutated and separated, the high oil body progeny is homozygous by selfing, the oil content is determined by adopting a near infrared method, the oil content of the high oil body progeny is higher than that of a mutagenic parent (the oil content is 49.50%), the oil content is detected by an agricultural department oil plant and product quality supervision test center, 28 oil content of 30 high oil body progeny strains to be detected reaches the high oil (over 55%), wherein the oil content of 6 high oil body progeny is more than 59%, the oil content of 10 high oil body progeny is 58-59%, and the highest oil content reaches 59.63%. DNA variation is detected and analyzed by an SSR method, more than 2 sites of 12 offspring (from different high oil bodies) of 13 high oil bodies to be detected are different from a mutagenic parent, which indicates that the oil content of the materials is improved due to gene mutation; in addition, phenotypic variation and yield increase are also caused by gene mutation.
Sequence listing
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<213> Artificial sequence (Arachis hypogaea Linn.)
<400>8
ggctcccttc gatgatgac 19
<210>9
<211>20
<212>DNA
<213> Artificial sequence (Arachis hypogaea Linn.)
<400>9
tgggcctaaa cccaacctat 20
<210>10
<211>20
<212>DNA
<213> Artificial sequence (Arachis hypogaea Linn.)
<400>10
ccacaaacag tgcagcaatc 20
<210>11
<211>20
<212>DNA
<213> Artificial sequence (Arachis hypogaea Linn.)
<400>11
ctttcttccc ccttgaacct 20
<210>12
<211>25
<212>DNA
<213> Artificial sequence (Arachis hypogaea Linn.)
<400>12
gatcaagtga aaatgttagt ataag 25
<210>13
<211>20
<212>DNA
<213> Artificial sequence (Arachis hypogaea Linn.)
<400>13
gggcttcact gcttttgatt 20
<210>14
<211>20
<212>DNA
<213> Artificial sequence (Arachis hypogaea Linn.)
<400>14
tgcgacttct gagaggacaa 20
Claims (8)
1. A culture medium for in vitro directional screening of high-oil peanut bodies is characterized by comprising a somatic embryo germination and screening culture medium, a seedling formation and screening culture medium and a recovery culture medium;
the somatic embryo germination and screening culture medium takes an MS culture medium as a basic culture medium, and 3-5 mmol/L HYP and 3-5 mg/L BAP are added;
the seedling and screening culture medium takes an MS culture medium as a basic culture medium, and 7-9 mmol/L HYP and 1-3 mg/LBAP are added;
the recovery medium takes an MS culture medium as a basic culture medium, and 1-3 mg/L BAP is added.
2. The culture medium for in vitro directional screening of peanut high oil bodies according to claim 1, wherein the concentration of HYP in the somatic embryo germination and screening culture medium is 4 mmol/L.
3. The culture medium for in vitro directional screening of high oil bodies of peanuts according to claim 1, wherein the concentration of HYP in the seedling and screening culture medium is 8 mmol/L.
4. An in vitro directional screening method for peanut high oil bodies, which is characterized in that the culture medium of any one of claims 1 to 3 is used for screening.
5. The in vitro directional screening method of the peanut high oil body according to claim 4, which comprises the following steps:
(1) transferring explants forming somatic embryos to a somatic embryo germination and screening medium for 4 weeks;
(2) transferring the explants with somatic embryos screened and alive by the step (1) to a seedling and screening culture medium for screening culture for 4 weeks;
(3) transferring the explants screened and cultured in the step (2) to a growth recovery culture medium for culture; after culturing for 4 weeks, the partially germinated somatic embryos resume growth;
(4) alternately culturing the somatic embryos recovered from the step (3) on a seedling forming and screening culture medium and a growth recovery culture medium until the regenerated plantlets grow to 1 cm; finally, the obtained seedlings are high-oil peanut seedlings or high-oil peanut bodies.
6. The in vitro directional screening method for peanut high oil bodies according to claim 5, wherein the culture conditions are as follows: the culture temperature is 25-27 ℃, the illumination intensity is 2000-3000 Lx, and the daily illumination is 12-14 h.
7. The method for directionally screening peanut high oil bodies in vitro according to claim 5 or 6, wherein the explants in the step (1) are: selecting a seed embryo with plump seeds and a cotyledon removed, carrying out surface disinfection, taking the embryo lobule, inoculating the embryo lobule to a somatic embryo induction culture medium, and culturing under the conditions that the temperature of a culture room is 25-27 ℃, the illumination intensity is 2000-3000 Lx, and the daily illumination is 12-14 h, so as to induce the formation of the somatic embryo.
8. The in vitro directional screening method for the peanut high oil bodies according to claim 7, wherein the somatic embryo induction culture medium takes an MS culture medium as a basic culture medium, and 4-12 mg/L2,4-D is added.
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| CN108353790A (en) * | 2018-02-09 | 2018-08-03 | 青岛农业大学 | A kind of breeding method of peanut high-oil kind |
| CN109156352A (en) * | 2018-10-09 | 2019-01-08 | 青岛农业大学 | One cultivate peanut mutant rapid screening method and application |
| CN109479721B (en) * | 2018-12-29 | 2019-06-07 | 青岛农业大学 | A kind of peanut plant regeneration method |
| CN117016385A (en) * | 2023-08-24 | 2023-11-10 | 安徽省农业科学院作物研究所 | Peanut transverse nitrogen ion beam implantation mutagenesis method |
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| CN102792892A (en) * | 2012-08-21 | 2012-11-28 | 青岛农业大学 | Method for directional screening of resistance body by peanut in-vitro mutation |
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| CN102792892A (en) * | 2012-08-21 | 2012-11-28 | 青岛农业大学 | Method for directional screening of resistance body by peanut in-vitro mutation |
Non-Patent Citations (3)
| Title |
|---|
| JIONGMING SUI ET AL.: "Generation of Peanut Drought Tolerant Plants by Pingyangmycin-Mediated In Vitro Mutagenesis and Hydroxyproline-Resistance Screening", 《PLOS ONE》 * |
| 李冠等: "利用离体诱变培育花生新品种宇花4 号", 《生物工程学报》 * |
| 赵明霞等: "离体筛选花生抗逆突变体及其后代特征特性研究", 《核农学报》 * |
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Address after: No. 700, Changcheng Road, Chengyang District, Qingdao City, Shandong Province, Shandong Applicant after: Qingdao Agricultural University Address before: College of chemistry and pharmacy, Qingdao Agricultural University, 700 Changcheng Road, Chengyang District, Qingdao City, Shandong Province 266000 Applicant before: Qingdao Agricultural University |
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Application publication date: 20201030 |