CN116250479A - Screening method of novel salt-tolerant selenium-rich wheat germplasm - Google Patents
Screening method of novel salt-tolerant selenium-rich wheat germplasm Download PDFInfo
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
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- A—HUMAN NECESSITIES
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- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
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- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/08—Immunising seed
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- A—HUMAN NECESSITIES
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Abstract
The invention provides a screening method of a novel salt-tolerant selenium-rich wheat germplasm, which specifically comprises the step of screening the novel high-salt-tolerant selenium-rich wheat germplasm by utilizing a method combining chemical mutagen mutagenesis, natural screening of saline-alkali soil, salt stress screening, selenium sensitivity screening and selenium content screening. The screening method not only solves the problem of low wheat yield caused by land salinization in China, but also can increase the selenium content of human body through grain supplement, and the screening follows the simple, convenient, quick and optimal screening principle, and provides an effective screening method for wheat nutritional element breeding by utilizing biological enhancement.
Description
Technical Field
The invention belongs to the technical field of crop breeding, and particularly relates to a screening method of a novel salt-tolerant selenium-enriched wheat germplasm.
Background
Selenium is a trace element necessary for human body, participates in various physiological and biochemical activities in the human body, has the functions of resisting cancer, resisting aging, enhancing immunity, protecting heart and the like, and is known as anticancer king, life fire and heart guard. Selenium deficiency of human body can cause keshan disease, bone joint disease and the like. More than 40 diseases such as diabetes, cataract, hepatitis and the like are reported to be associated with selenium deficiency. The important effects of selenium on the human body are mainly expressed in two aspects: on one hand, selenium-containing protein or selenium-containing enzyme such as glutathione peroxidase participates in a series of physiological activities of human body, such as thyroid hormone metabolism, redox reaction, immune reaction and the like, and has important significance on human health and immunity improvement; on the other hand, the selenium-containing compound can remove free oxygen of human body, thereby achieving the functions of resisting oxidation, resisting tumor and resisting aging. The world health organization recommends that the daily selenium intake of human bodies is 50-200 mug, the daily selenium intake recommended by the international selenium society is 50 mug, and according to investigation, the daily selenium intake of Chinese people is only 26-32 mug, which is far lower than the recommended intake. Therefore, the biological nutrition enhancement, namely, increasing the content of selenium in the grain crops through genetic breeding, agronomic practice and other modes, is a way for solving the problem of selenium deficiency and supplementing the safest, most economical, most simple and effective daily selenium intake.
Wheat is one of the important staple food crops, and about 1/3 of the population of population dependent wheat worldwide provides basic nutritional needs. Because the arable area of China is small, the salinization of the land is serious, and the yield and the nutritive value of the wheat are greatly limited. Therefore, the breeding of salt-tolerant selenium-rich wheat varieties is one of the important problems of food supply and nutrient element deficiency in China.
Disclosure of Invention
The invention aims to provide a screening method of a novel salt-tolerant selenium-enriched wheat germplasm. The screening method specifically comprises chemical mutagen mutagenesis, natural screening of saline-alkali soil, salt stress screening, selenium sensitivity screening and selenium content screening, is simple, and can provide a new idea for screening salt-tolerant selenium-enriched wheat.
In order to achieve the aim of the invention, the invention is realized by adopting the following technical scheme:
the invention provides a screening method of a novel salt-tolerant selenium-enriched wheat germplasm, which specifically comprises the following steps:
(1) Chemical mutagen mutagenesis: cleaning the surface of wheat seeds after disinfection, and carrying out mutagenesis treatment by using a chemical mutagen Ethyl Methylsulfonate (EMS) after seed soaking catalysis;
(2) And (3) natural screening of saline-alkali soil: sowing the mutagenized wheat seeds in saline-alkali soil, and harvesting wheat mutant seeds with strong salt tolerance and good growth vigor;
(3) Salt tolerance screening: selecting full wheat salt-tolerant mutant seeds and wild wheat seeds, carrying out surface disinfection and seed soaking catalysis, culturing in a culture dish containing high-concentration salt solution until rooting, and screening the wheat mutant seeds with root length obviously longer than that of the wild wheat seeds to obtain strong salt-tolerant wheat seeds;
(4) Selenium sensitivity screening: selecting strong salt-tolerant wheat seeds and wild wheat seeds with plump seeds, sterilizing the surfaces, soaking the seeds for catalysis, and then adding sodium selenite Na 2 SeO 3 Culturing in a culture dish of the solution until rooting, and screening wheat mutant seeds with root length obviously longer than that of wild wheat roots to obtain selenium-sensitive salt-tolerant wheat seeds;
(5) Selenium content screening: after surface disinfection, drying and grinding digestion are carried out on selenium-sensitive wheat salt-tolerant seeds and wild wheat seeds, the selenium content of the seeds is measured, and wheat mutant seeds with the selenium content remarkably higher than that of the wild wheat seeds are screened to obtain novel salt-tolerant selenium-rich wheat germplasm.
Further, the selenium-containing solution in the step (4) is a sodium selenite solution, and the concentration of the selenium-containing solution is 0.2mM-0.4mM.
Preferably, the selenium-containing solution is 0.3mM sodium selenite Na 2 SeO 3 A solution.
Further, the high-concentration salt solution in the step (3) is a sodium chloride solution, and the concentration of the salt solution is 120mM-180mM.
Preferably, the high concentration salt solution is 150mM sodium chloride solution.
Further, the salinity of the saline-alkali soil in the step (2) is 0.3% -0.7%.
Further, the surface disinfection step is as follows: the wheat seeds are treated by 1-3% sodium hypochlorite at 37 ℃ with shaking at 200rpm for 15min, and then washed 3 times with sterile water to complete sterilization.
Preferably, the concentration of sodium hypochlorite is 2%.
Further, the seed soaking catalysis comprises the following steps: the wheat seeds with the surface sterilized are soaked in the dark for 24 hours at 4 ℃.
Further, the chemical mutagen in the step (1) is ethyl methylsulfonate, and the concentration of the chemical mutagen is 0.3% -0.5%.
Preferably, the chemical mutagen is 0.4% EMS.
Further, the selenium content in the step (5) is determined by an ion body emission spectrometer.
Further, the sowing time in the step (2) is 220-250 days.
Compared with the prior art, the invention has the advantages that:
1. the invention establishes a composite screening method of 'wheat salt tolerance', 'selenium sensitivity of wheat seedlings' and 'selenium-enriched wheat new germplasm'. The new germplasm of wheat is derived from wheat mutagenesis mediated by chemical mutagen EMS. Screening a wheat salt-tolerant strain system mainly depends on the growth and yield of wheat plants under the natural condition of saline-alkali soil: and screening out new salt-tolerant high-yield wheat germplasm according to the wheat yield of the wheat in the unit area of the saline-alkali soil. And further screening a new strain with obviously changed sensitivity of seedlings to the externally added selenium from the salt-tolerant wheat mutant strain. And finally, measuring the selenium content of kernels in the new selenium-sensitive lines, and screening to obtain new selenium-enriched wheat germplasm, wherein the screening method is simple and has strong practicability.
2. The invention comprehensively utilizes the relevance of the selenium content of the wheat seeds and the sensitivity of the wheat seedlings to the externally added selenium and the relevance of the salt-tolerant wheat and the sensitivity of the selenium to the wheat seeds to carry out step-by-step screening, and the method is simple, convenient and quick and has clear evaluation and discrimination; the screening method of the novel wheat selenium-rich germplasm provides an effective screening method for wheat nutritional element breeding by utilizing biological enhancement.
Drawings
FIG. 1 shows the naturally screened salt tolerant wheat mutant induced by EMS using saline-alkali soil according to the present invention.
FIG. 2 shows the screening of novel wheat germplasm with strong salt tolerance by salt solution treatment according to the invention.
FIG. 3 shows the use of selenium solution in the present invention to screen selenium sensitive novel germplasm from strong salt tolerant wheat strains.
Detailed Description
The technical scheme of the invention is further described in detail by combining the following specific examples.
Example 1
A screening method of new selenium-enriched wheat germplasm specifically comprises the following steps:
1. ethyl Methylsulfonate (EMS) mutagenesis
(1) Surface disinfection: sterilizing the surface of wheat seeds by using 2% sodium hypochlorite and vibrating at 200rpm for 15min at 37 ℃, and then washing with sterile water for 3 times;
(2) Seed soaking catalysis: soaking the wheat seeds with the surfaces disinfected in the dark for 24 hours at the temperature of 4 ℃;
(3) Mutagenesis treatment: mutagenesis treatment is carried out on soaked wheat seeds by using 0.4% EMS for 24 hours;
(4) And (5) sowing in saline-alkali places: sowing the single wheat seeds after mutagenesis in saline-alkali soil with the salinity of 0.3% -0.7%;
(5) And (3) screening the saline-alkali soil: 8 months (240 days) after sowing, harvesting wheat mutant seeds with good growth vigor in saline-alkali soil by a single plant;
2. salt tolerance screening
(1) Surface disinfection: selecting full wheat mutant seeds obtained by screening from saline-alkali soil and wild wheat seeds of the same variety, sterilizing by using 2% sodium hypochlorite, vibrating at 37 ℃ and 200rpm for 15min, and flushing with sterile water for 3 times;
(2) Seed soaking catalysis: soaking the wheat seeds with the surface sterilized in the dark for 24 hours at the temperature of 4 ℃;
(3) Sodium chloride solution (NaCl) treatment: uniformly seeding all wheat seeds in a glass culture dish containing 150mM NaCl, paving two layers of filter paper in the culture dish, and culturing for 36 hours at room temperature; taking the root length of the wild wheat seedlings as a control, removing mutant strains with the root length shorter than or equal to the root length of the wild wheat seedlings, reserving mutant strains with the root length obviously longer than the root length of the wild wheat seedlings, and harvesting to obtain strong salt-tolerant wheat seeds after the conventional planting is continued;
3. selenium enrichment screening
(1) Surface disinfection: selecting strong salt-tolerant wheat seeds and wild wheat seeds with full seeds, sterilizing by using 2% sodium hypochlorite, vibrating at 37 ℃ and 200rpm for 15min, and flushing with sterile water for 3 times;
(2) Seed soaking catalysis: soaking the wheat seeds with the surface sterilized in the dark for 24 hours at the temperature of 4 ℃;
(3) Selenium sensitivity screening: all wheat seeds were evenly spot seeded with a seed containing 0.3mM sodium selenite (Na 2 SeO 3 ) In a culture dish of the solution, two layers of filter paper are paved in the culture dish, and the culture dish is cultivated for 5 days at room temperature; taking the root length of the wild wheat seedlings as a control, removing mutant strains with the root length shorter than or equal to the root length of the wild wheat seedlings, reserving mutant strains with the root length obviously longer than the root length of the wild wheat seedlings, and harvesting to obtain selenium-sensitive wheat seeds after the conventional planting is continued;
(4) Selenium content screening: the method comprises the steps of carrying out surface disinfection on harvested selenium-sensitive wheat seeds and wild wheat seeds by using 2% sodium hypochlorite, drying, grinding, heating and digesting, and measuring the selenium content of the seeds by using an ICP-OES inductively coupled plasma emission spectrometer; and screening seeds with the selenium content obviously higher than that of the wild seeds by taking the selenium content of the wild seeds as a control, namely the novel selenium-enriched and salt-tolerant wheat mutant germplasm.
Example 2: screening of novel selenium-enriched salt-tolerant wheat germplasm
According to the method in example 1, after EMS mutagenesis is carried out on wheat variety "Jimai 22" seeds, natural screening (shown in figure 1) of saline-alkali soil is carried out, the obtained selfed second generation mutant seeds are used as screening and identifying materials, meanwhile, the wheat variety "Jimai 22" seeds are used as wild type controls, and screening is carried out according to the following steps:
1. salt tolerance screening
(1) Surface disinfection: selecting 50 wheat mutant seeds with full seeds and wild seeds, placing the seeds and the seeds into a 50ml centrifuge tube simultaneously, adding 4ml of sterile water and 1ml of sodium hypochlorite, vibrating at 37 ℃ for 15min at 200rpm, and flushing the seeds with the sterile water for 3 times;
(2) Seed soaking catalysis: soaking the wheat seeds with the surfaces disinfected in the dark for 24 hours at the temperature of 4 ℃;
(3) Sodium chloride solution (NaCl) treatment: first, wheat seeds were inoculated into glass dishes containing sodium chloride at different concentrations of 0, 50, 100, 150 and 200mM, and after culturing for 24 hours, the change of root length after germination of the seeds at different concentrations was observed, and as the root length of wheat germination became shorter with increasing concentration of sodium chloride solution (NaCl), the influence of sodium chloride (NaCl) concentration on the root length of wheat was most pronounced at 150mM, so that 150mM sodium chloride (NaCl) solution was selected for screening. Uniformly and locally planting seeds in a glass culture dish paved with two layers of filter paper, adding a proper amount of 150mM NaCl, and placing the seeds in an illumination incubator for culturing for 48 hours;
(4) Salt tolerance identification: taking the root length of seedlings of 'Jimai 22' as a control, screening to find that the root length of seedlings of salt-tolerant wheat strains J31-1, J52-2 and J71-1 after salt treatment is obviously larger than that of wild type seedlings (shown in figure 2), thus obtaining 3 new germplasm of salt-tolerant wheat, and continuing further screening experiments;
2. selenium sensitivity screening
(1) Surface disinfection: selecting 50 wheat mutant seeds and Jimai 22 control seeds with full seeds respectively, putting the seeds and the seeds into a 50ml centrifuge tube, adding 40ml of sterile water and 10ml of sodium hypochlorite, vibrating at 37 ℃ for 15min at 200rpm, and flushing with the sterile water for 3 times;
(2) Seed soaking catalysis: soaking the wheat seeds with the surfaces disinfected in the dark for 24 hours at the temperature of 4 ℃;
(3) Sodium selenite solution (Na 2 SeO 3 ) And (3) treatment: first, na with different concentration is utilized 2 SeO 3 Treating wheat seeds with the solution (0.1-0.5 mM), observing and comparing the growth of root system after germination of seeds with Na 2 SeO 3 The sensitivity of the solution was found to decrease in root length with increasing selenate ion concentration, at Na 2 SeO 3 The root length variation was most pronounced at a solution concentration of 0.3mM, so that a suitable screening concentration of 0.3mM was chosen. The seeds were evenly planted in a culture dish with two layers of filter paper and added with a proper amount of 0.3mM Na 2 SeO 3 Placing the solution in an illumination incubator for culturing for 5d;
(4) Selenium sensitivity identification: screening and finding Na by using root length of seedlings of Jimai 22 as a control 2 SeO 3 Seedling root lengths of J31-1 and J52-2 in the solution treated mutants were significantly larger than that of the wild type (as shown in FIG. 3), thus obtaining 2 selenium-sensitive wheat seeds;
(5) Selenium element determination and identification: after the two selenium-sensitive wheat seeds and the wild wheat seeds are subjected to surface disinfection by using 2% sodium hypochlorite, the wheat seeds are dried, ground and heated for digestion, the selenium content of the seeds is measured by using an ICP-OES inductively coupled plasma emission spectrometer, and the results are shown in a table 1, wherein the selenium Se element contents of J31-1 and J52-2 are obviously higher than those of a control (Jimai 22), so that 2 new selenium-rich and salt-tolerant wheat mutant germplasm is obtained.
Table 1: new selenium-enriched wheat germplasm screening by selenium element determination
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (9)
1. The screening method of the novel salt-tolerant selenium-rich wheat germplasm is characterized by comprising the following steps of:
(1) Chemical mutagen mutagenesis: the wheat seed surface is disinfected and then cleaned, and after seed soaking catalysis, the chemical mutagen is used for mutagenesis treatment;
(2) And (3) screening the saline-alkali soil: sowing the mutagenized wheat seeds in saline-alkali soil, and harvesting mutant seeds with good growth vigor;
(3) Salt tolerance screening: selecting full mutant seeds and wild wheat seeds, carrying out surface disinfection and seed soaking catalysis, culturing until rooting by using a high-concentration salt solution, and screening mutant seeds with root length obviously longer than that of the wild wheat to obtain strong salt-tolerant wheat seeds;
(4) Selenium sensitivity screening: selecting strong salt-tolerant wheat seeds with full seeds and wild wheat seeds, carrying out surface disinfection and seed soaking catalysis, culturing until rooting by using a selenium-containing solution, and screening mutant seeds with root length obviously longer than that of the wild wheat to obtain selenium-sensitive wheat seeds;
(5) Selenium content screening: and (3) carrying out surface disinfection, drying, grinding and digestion on the selenium-sensitive wheat seeds and the wild wheat seeds, measuring the selenium content of the seeds, and screening mutant seeds with the selenium content obviously higher than that of the wild seeds to obtain new salt-tolerant selenium-rich wheat germplasm.
2. The method for screening a novel salt-tolerant selenium-enriched wheat germ plasm according to claim 1, wherein the selenium-containing solution in the step (4) is a sodium selenite solution with a concentration of 0.2mM-0.4mM.
3. The method for screening a novel salt-tolerant selenium-enriched wheat germplasm according to claim 1, characterized in that the high-concentration salt solution in the step (3) is a sodium chloride solution, and the concentration of the salt solution is 120mM-180mM.
4. The method for screening new germplasm of salt tolerant selenium enriched wheat according to claim 1, characterized in that the salinity of the saline-alkali soil in the step (2) is 0.3% -0.7%.
5. The method for screening new germplasm of salt tolerant selenium enriched wheat according to claim 1, characterized in that the step of surface disinfection is: the wheat seeds are treated by 1-3% sodium hypochlorite at 37 ℃ with shaking at 200rpm for 15min, and then washed 3 times with sterile water to complete sterilization.
6. The method for screening new germplasm of salt tolerant selenium enriched wheat according to claim 1, characterized in that the step of seed soaking catalysis is as follows: the wheat seeds with the surface sterilized are soaked in the dark for 24 hours at 4 ℃.
7. The method for screening new germplasm of salt tolerant selenium enriched wheat according to claim 1, characterized in that the chemical mutagen in the step (1) is ethyl methylsulfonate with a concentration of 0.3% -0.5%.
8. The method for screening new germplasm of salt tolerant selenium enriched wheat according to claim 1, characterized in that the selenium content in said step (5) is determined by an ion body emission spectrometer.
9. The method for screening a novel salt-tolerant selenium-enriched wheat germplasm according to claim 1, characterized in that the selenium element content in the novel salt-tolerant selenium-enriched wheat germplasm is not less than 125 μg/kg.
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