CN113287515B - Screening method of new salt-tolerant iron-rich wheat germplasm - Google Patents

Abstract

The invention provides a method for screening a new salt-tolerant iron-rich wheat germplasm, which specifically comprises the steps of screening the new high-salt-tolerant iron-rich wheat germplasm by a method combining chemical mutagen mutagenesis, saline-alkali soil natural screening, salt stress screening, iron sensitivity screening and iron 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 content of the iron element in the human body through grain supplement, follows the screening principle of simplicity, rapidness and excellent selection, and provides an effective screening method for breeding the nutritional elements of the wheat by utilizing biological enhancement.

Description

Screening method of new salt-tolerant iron-rich wheat germplasm

Technical Field

The invention belongs to the technical field of crop breeding, and particularly relates to a screening method of a new salt-tolerant iron-rich wheat germplasm.

Background

In China, the current situation of iron element deficiency is more serious, and the problem of malnutrition is more prominent especially for poor people. Iron is a necessary trace element for human body, and participates in various physiological and biochemical activities in human body. The lack of iron element in human body can reduce physical strength and damage intelligence, and serious patients can suffer from various diseases. A great deal of research points out that biological nutrition strengthening, namely, the content of the iron element in the grain crops is increased by means of genetic breeding, agricultural practice and the like, and the method is the safest, most economical, most convenient and most effective way for solving the problem of iron element deficiency.

Wheat is one of the important staple grain crops in China, but because the arable land area is small in China and the salinization of land is severe, the yield and the nutritive value of wheat are greatly limited. Therefore, screening of salt-tolerant and iron-tolerant wheat varieties is also one of the important problems in solving the shortage of grains in China.

Disclosure of Invention

The invention aims to provide a screening method of a new salt-tolerant iron-rich wheat germ plasm. The screening method specifically comprises the steps of chemical mutagen mutagenesis, saline-alkali soil natural screening, salt stress screening, iron sensitivity screening and iron content screening, is simple, and can provide a new idea for screening salt-tolerant and iron-rich wheat.

In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:

the invention provides a screening method of a new salt-tolerant iron-rich wheat germplasm, which specifically comprises the following steps:

(1) Chemical mutagen mutagenesis: the surface of the wheat seed is cleaned after being disinfected, and is subjected to mutagenesis treatment by using a chemical mutagen after being catalyzed by soaking;

(2) Naturally screening saline-alkali soil: sowing the mutagenized wheat seeds in saline-alkali soil, and harvesting the well-grown wheat mutant seeds;

(3) And (3) screening salt resistance: selecting wheat mutant seeds with plump seeds and wild type wheat seeds, performing surface disinfection and seed soaking catalysis, culturing in a culture dish containing high-concentration salt solution until the seeds take root, and screening the wheat mutant seeds with the root length longer than that of the wild type wheat seeds to obtain strong salt-tolerant wheat seeds;

(4) And (3) screening iron sensitivity: selecting strong salt-tolerant wheat seeds with plump seeds and wild type wheat seeds, performing surface disinfection and seed soaking catalysis, culturing in a culture dish containing an iron solution until the seeds take root, and screening wheat mutant seeds with root length longer than that of the wild type wheat seeds to obtain iron-insensitive wheat salt-tolerant seeds;

(5) Screening the iron content: and (3) after the salt-tolerant seeds of the iron-insensitive wheat and the wild type wheat seeds are subjected to surface disinfection, drying, grinding and digestion, measuring the iron element content of the seeds, and screening the wheat mutant seeds with the iron element content higher than that of the wild type seeds to obtain the new salt-tolerant iron-rich wheat germplasm.

Further, the high-concentration salt solution comprises a sodium chloride solution; the concentration is 120mM-180mM.

Preferably, the high-concentration salt solution is a 150mM sodium chloride solution.

Further, the iron-containing solution comprises an ethylene diamine tetraacetic acid iron sodium solution and a ferric nitrate solution; the concentration is 1.2mM-1.8mM.

Preferably, the iron-containing solution is a 1.5mM solution of sodium iron ethylenediaminetetraacetate EDTA-Fe.

Furthermore, the salinity of the saline-alkali soil is 0.3% -0.7%.

Further, the surface sterilization step is as follows: treating wheat seeds with 1-3% sodium hypochlorite at 37 deg.C under shaking at 200rpm for 15min, washing with sterile water for 3 times, and cleaning.

Preferably, the volume of sodium hypochlorite is 2%.

Further, the seed soaking catalysis step is as follows: the wheat seeds with sterilized surfaces are soaked in the dark at 4 ℃ for 24h.

Further, the chemical mutagen comprises EMS; the concentration is 0.3% -0.5%.

Preferably, the chemical mutagen is 0.4% EMS.

Further, the content of the iron element is determined by an ICP-OES inductively coupled plasma emission spectrometer.

Further, the sowing time in the step (2) is 220-250 days.

Further, the iron content in the new salt-tolerant iron-rich wheat germplasm obtained by screening is more than 2 times of that of wild wheat seeds.

Compared with the prior art, the invention has the advantages and beneficial effects that:

1. the invention establishes a composite screening method of 'wheat salt tolerance', 'iron sensitivity of wheat seedlings' and 'new iron-rich wheat germplasm'. The new wheat germplasm is derived from wheat mutagenesis mediated by a chemical mutagen EMS. The salt-tolerant wheat strain is screened mainly according to the growth and yield of wheat plants under the natural condition of saline-alkali soil: and screening out new salt-tolerant wheat germplasm according to the yield of the small wheat planted in the unit area of the saline-alkali soil. And further screening a new strain of which the sensitivity of seedlings to the added iron is obviously changed from the salt-tolerant wheat strain. And finally, determining the iron content of the grains in the new lines with changed iron sensitivity, and screening to obtain the new iron-rich wheat germplasm.

2. The invention comprehensively utilizes the relevance of the iron content of the wheat grains and the change of the iron sensitivity of the wheat seedlings to the added iron and the relevance of the salt-tolerant wheat and the iron sensitivity to carry out gradual screening, the method is simple, convenient and quick, and the evaluation and the discrimination are clear; and the NaCl solution and the EDTA-Fe solution used for screening are nontoxic and harmless to the seeds, and simultaneously, the dual safety of the self and the edible performance of the new germplasm obtained by screening is ensured. The screening method of the iron-rich new wheat germplasm provides an effective screening method for breeding the nutritional elements of the wheat by utilizing biological enhancement.

Drawings

FIG. 1 shows salt-tolerant wheat mutant strains mutagenized by EMS naturally screened by saline-alkali soil.

FIG. 2 shows that salt solution treatment is used to screen new wheat germplasm with strong salt tolerance.

FIG. 3 is a diagram of the present invention using iron solution to screen new iron insensitive germplasm from strong salt tolerant wheat lines.

Detailed Description

The technical solution of the present invention will be further described in detail with reference to the following specific examples.

Example 1

A method for screening new germplasm of iron-rich wheat specifically comprises the following steps:

1. EMS (Ethyl methylsulfonate) mutagenesis

(1) Surface disinfection: performing surface disinfection on the wheat seeds by using 2% sodium hypochlorite and shaking at the temperature of 37 ℃ and the rpm of 200 for 15min, and then washing the wheat seeds by using sterile water for 3 times;

(2) Seed soaking catalysis: soaking the wheat seeds with the sterilized surfaces in the dark at 4 ℃ for 24h;

(3) And (3) mutagenesis treatment: performing mutagenesis treatment on the soaked wheat seeds for 24 hours by using 0.4 percent EMS;

(4) Dibbling in saline and alkaline land: single-seed dibbling of the mutagenized wheat seeds into saline-alkali soil with the salinity of 0.3-0.7 percent;

(5) Screening saline-alkali soil: seeds of the wheat mutant which grow well in the saline-alkali soil are harvested individually 8 months (240 days) after sowing;

2. screening for salt tolerance

(1) Surface disinfection: selecting wheat mutant seeds obtained by screening from saline-alkali soil and full seeds and wild wheat seeds of the same variety, sterilizing by using 2% sodium hypochlorite, shaking at 37 ℃ and 200rpm for 15min, and washing with sterile water for 3 times;

(2) Seed soaking catalysis: soaking the wheat seeds with the sterilized surfaces in the dark at the temperature of 4 ℃ for 24 hours;

(3) Sodium chloride solution (NaCl) treatment: uniformly dibbling all wheat seeds in a glass culture dish containing 150mM NaCl, paving two layers of filter paper in the culture dish, and culturing for 36h at room temperature; taking the root length of the wild type wheat seedlings as a reference, rejecting mutant strains with the root length shorter than or equal to that of the wild type wheat seedlings, reserving the mutant strains with the root length obviously longer than that of the wild type wheat seedlings, and harvesting to obtain strong salt-tolerant wheat seeds after continuing conventional planting;

3. iron-rich screening

(1) Surface disinfection: selecting plump strong salt-tolerant wheat seeds and wild wheat seeds, sterilizing with 2% sodium hypochlorite, shaking at 37 deg.C and 200rpm for 15min, and washing with sterile water for 3 times;

(2) Soaking seed for catalysis: soaking the wheat seeds with the sterilized surfaces in the dark at the temperature of 4 ℃ for 24 hours;

(3) Iron sensitivity screening: uniformly dibbling all wheat seeds in a culture dish containing 150mM ethylene diamine tetraacetic acid iron sodium (EDTA-Fe) solution, paving two layers of filter paper in the culture dish, and culturing for 48h at room temperature; taking the root length of the wild type wheat seedling as a reference, rejecting a mutant strain with the root length shorter than or equal to that of the wild type wheat seedling, reserving the mutant strain with the root length obviously longer than that of the wild type wheat seedling, and harvesting to obtain the salt-tolerant seeds of the iron-insensitive wheat after continuing conventional planting;

(4) Screening the iron content: performing surface disinfection on the harvested salt-tolerant seeds of the iron-insensitive wheat and wild wheat seeds by using 2% sodium hypochlorite, drying, grinding, heating and digesting, and measuring the iron element content of the seeds by using an ICP-OES inductively coupled plasma emission spectrometer; and (3) screening seeds with the iron element content obviously higher than that of the wild seeds by taking the iron element content of the wild seeds as a reference, namely the novel wheat mutant germplasm rich in iron and resistant to salt.

Example 2: screening of new germplasm of Fe-rich salt-tolerant wheat

According to the method in the embodiment 1, after EMS mutagenesis is carried out on the wheat variety 'rufa 502' seeds, natural screening is carried out on saline-alkali soil (figure 1), the obtained selfing second generation mutant seeds are used as the material for screening and identification, meanwhile, the wheat variety 'rufa 502' seeds are used as wild type contrast, and screening is carried out according to the following steps:

1. screening for salt tolerance

(1) Surface disinfection: selecting 50 full wheat mutant seeds and wild seeds, simultaneously placing the seeds and the wild seeds in a 50ml centrifuge tube, adding 4ml of sterile water and 1ml of sodium hypochlorite, shaking at 37 ℃, vibrating at 200rpm for 15min, and washing with sterile water for 3 times;

(2) Seed soaking catalysis: soaking the wheat seeds with the sterilized surfaces in the dark at 4 ℃ for 24 hours;

(3) Sodium chloride solution (NaCl) treatment: firstly, wheat seeds are dibbled in glass culture dishes containing sodium chloride with different concentrations of 0, 50, 100, 150 and 200mM, after the seeds are cultured for 24 hours, the change of the root length of the germinated seeds under different concentrations is observed and compared, the root length of the germinated seeds is found to be shorter and shorter along with the increase of the concentration of the sodium chloride solution (NaCl), and the influence of the concentration of the sodium chloride (NaCl) on the root length of the wheat is most obvious when the concentration of the sodium chloride (NaCl) is 150mM, so the sodium chloride (NaCl) solution with 150mM is selected for screening. Uniformly dibbling the seeds in a glass culture dish paved with two layers of filter paper, adding a proper amount of 150mM NaCl, and putting the mixture in a lighting incubator for culturing for 36 hours;

(4) And (3) identifying salt tolerance: the seedling root length of the Rugen 502 is taken as a control, and the screening finds that the seedling root length of LY 1-98, LY 1-105, LY 1-119, LY1-29 and LY2-7 after salt treatment is obviously larger than that of a wild type (figure 2), so 5 new wheat germplasms with strong salt tolerance are obtained, and further screening experiments are carried out;

2. iron sensitivity screening

(1) Surface disinfection: selecting 50 full wheat mutant seeds and wild seeds in 50ml centrifuge tubes, adding 4ml sterile water and 1ml sodium hypochlorite, shaking at 37 deg.C and 200rpm for 15min, and washing with sterile water for 3 times;

(2) Seed soaking catalysis: soaking the wheat seeds with the surface disinfected in the dark at 4 ℃ for 24h;

(3) Treatment of ethylenediaminetetraacetic acid iron sodium solution (EDTA-Fe): firstly, wheat seeds are treated by EDTA-Fe (0.5-4 mM) with different concentrations, the sensitivity of the root growth of the germinated seeds to EDTA-Fe iron sodium solution is observed and compared, the length of the roots is firstly increased and then decreased along with the increase of the concentration of iron ions, and the roots reach the maximum length when the concentration of the EDTA-Fe solution is 1.5mM, so that 1.5mM is selected as the proper screening concentration. Uniformly dibbling the seeds in a culture dish paved with two layers of filter paper, adding a proper amount of 1.5mM EDTA-Fe solution, and putting the mixture into a light incubator for culturing for 48 hours;

(4) And (3) identifying iron sensitivity: taking the seedling root length of the Rugen 502 as a reference, screening finds that the seedling root length of LY1-29 and LY2-7 treated by EDTA-Fe solution is obviously greater than that of wild type, and also finds that the seedling root length of wheat mutant treated by EDTA-Fe solution is obviously greater than that of untreated wheat (figure 3), thus obtaining 2 iron-insensitive wheat salt-tolerant seeds;

(5) And (3) determination and identification of iron element: the two iron-insensitive wheat salt-tolerant seeds and wild type wheat seeds are subjected to surface disinfection by using 2% sodium hypochlorite, dried, ground and heated for digestion, and the iron element content of the seeds is measured by using an ICP-OES inductively coupled plasma emission spectrometer, so that the results are shown in table 1, the Fe element content of LY1-29 and LY2-7 is obviously higher than that of the wild type, and 2 iron-rich and salt-tolerant wheat mutant new germplasms are obtained.

Table 1: method for screening novel iron-rich wheat germplasm by utilizing iron element determination

Through the screening of the steps, the new variety of wheat which can resist high salt and is rich in iron is obtained for the first time, and the iron content of the new variety of the salt-resistant and iron-rich wheat obtained by screening is more than 2 times of that of the wild variety, so that the new variety of the wheat provides great advantages for the planting and breeding of the wheat, the wheat can be planted in saline-alkali soil, the growth environment of the wheat is expanded, and the increment and the yield are increased; meanwhile, the new wheat germplasm can provide more iron elements for a human body, and because the wheat is the essential food in diet, people can increase the intake of iron-rich food and reduce the occurrence of iron-deficiency diseases only in simple diet. In addition, the screening agent is a reagent which does not cause any harm to staple food crops, ensures the safety and universality of screening and the edible safety of wheat, has low cost and good screening effect, and completely meets the requirement of modern agricultural development.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described in the foregoing embodiments, or equivalents may be substituted for some of the features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding claims.

Claims (2)

1. The method for screening the new salt-tolerant iron-rich wheat germplasm is characterized by comprising the following steps:

(1) EMS mutagenesis:

(1) surface disinfection: performing surface disinfection on the wheat seeds by using 2% sodium hypochlorite and shaking at the temperature of 37 ℃ and the rpm of 200 for 15min, and then washing the wheat seeds by using sterile water for 3 times;

(2) seed soaking catalysis: soaking the wheat seeds with the sterilized surfaces in the dark at 4 ℃ for 24h;

(3) and (3) mutagenesis treatment: performing mutagenesis treatment on the soaked wheat seeds for 24 hours by using 0.4% EMS;

(4) dibbling the saline-alkali soil: single-seed dibbling of the mutagenized wheat seeds into saline-alkali soil with the salinity of 0.3-0.7 percent;

(5) screening saline-alkali soil: 8 months after sowing, harvesting seeds of the wheat mutant growing well in the saline-alkali soil by a single plant;

(2) And (3) screening salt resistance:

(1) surface disinfection: selecting wheat mutant seeds obtained by screening from saline-alkali soil and full seeds and wild wheat seeds of the same variety, sterilizing by using 2% sodium hypochlorite, shaking at 37 ℃ and 200rpm for 15min, and washing with sterile water for 3 times;

(2) seed soaking catalysis: soaking the wheat seeds with the sterilized surfaces in the dark at the temperature of 4 ℃ for 24 hours;

(3) and (3) treating with a sodium chloride solution: uniformly dibbling all wheat seeds in a glass culture dish containing 150mM NaCl, paving two layers of filter paper in the culture dish, and culturing for 36h at room temperature; taking the root length of the wild type wheat seedling as a reference, rejecting a mutant strain with the root length shorter than or equal to that of the wild type wheat seedling, reserving the mutant strain with the root length obviously longer than that of the wild type wheat seedling, and harvesting to obtain strong salt-tolerant wheat seeds after continuing conventional planting;

(3) Iron-rich screening:

(1) surface disinfection: selecting plump strong salt-tolerant wheat seeds and wild wheat seeds, sterilizing with 2% sodium hypochlorite, shaking at 37 deg.C and 200rpm for 15min, and washing with sterile water for 3 times;

(2) seed soaking catalysis: soaking the wheat seeds with the sterilized surfaces in the dark at 4 ℃ for 24 hours;

(3) and (3) screening iron sensitivity: uniformly dibbling all wheat seeds in a culture dish containing 150mM ethylene diamine tetraacetic acid ferric sodium solution, paving two layers of filter paper in the culture dish, and culturing for 48h at room temperature; taking the root length of the wild type wheat seedling as a reference, rejecting a mutant strain with the root length shorter than or equal to that of the wild type wheat seedling, reserving the mutant strain with the root length obviously longer than that of the wild type wheat seedling, and harvesting to obtain the salt-tolerant seeds of the iron-insensitive wheat after continuing conventional planting;

(4) screening the iron content: performing surface disinfection on the harvested salt-tolerant seeds of the iron-insensitive wheat and wild wheat seeds by using 2% sodium hypochlorite, drying, grinding, heating and digesting, and measuring the iron element content of the seeds by using an ICP-OES inductively coupled plasma emission spectrometer; and (3) screening seeds with the iron element content obviously higher than that of the wild seeds by taking the iron element content of the wild seeds as a reference, namely the novel wheat mutant germplasm rich in iron and resistant to salt.

2. The method for screening the new germplasm of the salt-tolerant iron-rich wheat according to claim 1, wherein the iron content in the screened new germplasm of the salt-tolerant iron-rich wheat is more than 2 times that in a wild type wheat seed.

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