Method for mutagenizing wheat by high-energy carbon ions with different parameters

Abstract

The invention relates to the technical field of agricultural biology, in particular to a method for mutagenizing wheat through high-energy carbon ions with different parameters. The method comprises the following steps: selecting screened excellent seeds as a mutagenic material; the screened mutagenesis materials are treated in different seed placement modes; setting mutagenesis parameters of high-energy carbon ions; performing carbon ion beam radiation on the mutagenesis materials with different seed placement modes according to the set mutagenesis parameters, and storing wheat seeds with different mutagenesis parameters and placement mode combinations; the stored wheat seeds with different mutagenesis parameters and placement combinations are subjected to a water culture seedling test and planted in a field; and carrying out biological effect analysis on the wheat subjected to water culture seedling and the wheat planted in the field. According to the method, a plurality of parameters of carbon ion radiation are set, so that a proper mutagenesis parameter combination is obtained, and a more efficient and systematic technical means is provided for high-energy heavy ion mutagenesis breeding of wheat.

Description

Technical Field

[0001] The invention relates to the field of agricultural biotechnology, and in particular to a method for inducing wheat mutation with high-energy carbon ions of different parameters. Background Art

[0002] Wheat (Triticum aestivum L.) is one of the most important food crops in the world, with important agricultural, economic and social values. Its yield and quality are directly related to global food security and sustainable agricultural development. Traditional wheat breeding methods mainly rely on natural variation and hybrid breeding, but there are problems such as long breeding cycles and limited types of variation. In recent years, with the development of physical mutagenesis breeding technology, the use of physical factors such as high-energy ion beams, X-rays, and gamma rays to induce mutations in crops has become one of the important means to create excellent new germplasm of crops and cultivate new varieties (Stadler 1928; Yamaguchi et al. 2009; Du et al. 2022).

[0003] High-energy heavy ion (such as carbon ion) mutagenesis has a higher relative biological effect than conventional radiation (gamma rays, X-rays, etc.) (Tanaka et al. 1997; Hirano et al. 2022), causing changes in physiological and biochemical reactions in plants (Tanaka, Shikazono, and Hase 2010), triggering different types of mutations such as gene mutations and chromosome rearrangements (Hirano et al. 2015), causing changes in related traits, and beneficial mutations can be used to accelerate the screening of new materials and promote the breeding of new varieties (Shikazono et al. 2005; Yamaguchi et al. 2009; Arase et al. 2011; Wang et al. 2021). However, the efficiency of high-energy carbon ion mutagenesis is affected by many factors, and there is no relevant report on the appropriate parameters for high-energy carbon ion mutagenesis of wheat. Therefore, analyzing the biological effects of high-energy carbon ion mutagenesis on wheat through different parameter combinations and developing new methods for high-energy carbon ion mutagenesis of wheat will provide important support for the construction of a high-energy heavy ion efficient mutagenesis technology system and promote the improvement of crop mutagenesis breeding efficiency. Summary of the invention

[0004] The present invention solves the problem that there is no relevant report on the suitable parameters for high-energy carbon ion mutagenesis of wheat at present, and provides a method for high-energy carbon ion mutagenesis of wheat with different parameters. By setting multiple parameters of carbon ion radiation, a suitable combination of mutagenesis parameters is obtained, thereby providing a more efficient and systematic technical means for high-energy heavy ion mutagenesis breeding of wheat.

[0005] The technical solution for which the present invention seeks protection is as follows:

[0006] A method for inducing wheat mutagenesis using high-energy carbon ions with different parameters comprises the following steps:

[0007] S1: Select the selected high-quality seeds as mutagenic materials;

[0008] S2: The mutagenic materials screened in S1 are processed by different seed placement methods;

[0009] S3: setting the mutagenesis parameters of high-energy carbon ions, the parameters including: radiation energy, dose rate, energy transfer line density, radiation dose, and radiation time;

[0010] S4: subjecting the mutagenic materials of different seed placements in S2 to carbon ion beam radiation according to the mutagenic parameters set in S3, and saving the wheat seeds of different combinations of mutagenic parameters and placements;

[0011] S5: Part of the wheat seeds with different mutagenesis parameters and placement combinations stored in S4 were subjected to hydroponic seedling experiments, and the other part was planted in the field;

[0012] S6: Analyze the biological effects of wheat plants grown hydroponically in S5 and wheat plants planted in the field.

[0013] Preferably, the mutagenic material in S1 is an excellent individual plant of a wheat variety with a clear genetic background and robust growth, and wheat seeds with uniform size and full grains are selected after harvest.

[0014] In the above method, the wheat varieties are Wheat Jing 411 and Wheat Heyou 1.

[0015] Preferably, the placement method in S2 includes: fixing the embryos upward with double-sided tape, laying them flat, and placing them randomly.

[0016] Preferably, the mutagenic parameters of the high-energy carbon ions in S3 include radiation energy of 80 MeV / u, dose rate of 60 Gy / min, energy transfer line density of 35 KeV μm -1 and 70KeVμm -1 , irradiation dose is 20Gy-120Gy, and irradiation time is 0.33min-2.00min.

[0017] Preferably, the carbon ion beam radiation in S4 is uniformly irradiated according to the mutagenesis parameters set in S3.

[0018] In the above method, the storage temperature of the wheat seeds in S4 is -20°C.

[0019] Preferably, the hydroponic seedling test in S5 includes seed pre-germination treatment and disinfection, soaking, germination and determination of seedling physiological indicators.

[0020] Preferably, the biological effect analysis in S6 includes the growth of hydroponic seedlings and field M 1 The emergence of the first generation of plants was observed, and the reduction rate of seedling height, root length and field M were calculated. 1 Plant emergence rate.

[0021] Beneficial effects:

[0022] The present invention provides a method for mutagenizing wheat with high-energy carbon ions of different parameters. The method can accurately control the irradiation parameters of high-energy carbon ions, set different irradiation parameter combinations to irradiate the selected excellent seeds, and obtain the best radiation parameter combination when the seedling height is reduced by 50% and the emergence rate is 50%, thereby effectively realizing the directional improvement of wheat traits, and then realizing the precise mutagenization of required traits, solving the problem that there is no relevant report on the suitable parameters for mutagenizing wheat with high-energy carbon ions. These advantages make high-energy carbon ion mutagenization one of the important tools in modern wheat genetic improvement and breeding, especially in the context of severe challenges faced by global climate change and agricultural production, and have high application value and promotion prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The present invention shows an embodiment of an embodiment of the present invention wherein the seeds of wheat induced by high-energy carbon ion radiation are placed in a manner such that: the first row corresponds from left to right to three different seed placements of wheat variety Jing 411, namely, using double-sided tape to fix the seed embryo upward (U), seeds laid flat (T), and seeds randomly placed (R); the second row corresponds from left to right to three different seed placements of wheat variety Heyou 1, namely, using double-sided tape to fix the seed embryo upward (U), seeds laid flat (T), and seeds randomly placed (R).

[0024] Figure 2 The present invention is a flowchart of indoor wheat seedling germination by high-energy carbon ion radiation according to an embodiment of the present invention.

[0025] Figure 3 This is the biological effect of high-energy carbon ion irradiation on Xiaomaijing 411 in accordance with the embodiment of the present invention.

[0026] Figure 4 This is the biological effect of high-energy carbon ion irradiation on Wheat Kernel No. 1 in the embodiment of the present invention. DETAILED DESCRIPTION

[0027] The present invention will be further described in detail below in conjunction with specific examples. It should be understood that the following examples are only for explanation and illustration and do not limit the scope of the present invention in any way.

[0028] Sources of biological materials:

[0029] Wheat Jing 411: a known variety that has passed variety approval (purchased from Beijing Seed Company).

[0030] Wheat Heyou No. 1: a known variety that has passed variety approval (purchased from Beijing Seed Company).

[0031] The present invention provides a method for inducing wheat mutation using high-energy carbon ions with different parameters, which specifically comprises the following steps:

[0032] S1: Selecting screened high-quality seeds as mutagenic materials; in a specific embodiment of the present invention, high-quality individual plants of the wheat varieties Jing411 and Heyou1 with clear genetic background and robust growth are selected, and after harvesting, seeds with uniform size and full grains are selected as mutagenic materials.

[0033] S2: Place the seeds selected in S1 into a 60 mm culture dish, use double-sided tape to fix the seeds in three different arrangements: embryo facing up (U), seeds laid flat (T), and seeds randomly placed (R), and seal the dish with a sealing film;

[0034] S3: Setting the mutagenic parameters of high-energy carbon ions, the parameters include: radiation energy, dose rate, energy transfer line density, radiation dose, and radiation time; in a specific embodiment of the present invention, different carbon ion radiation parameters are set as follows:

[0035] Irradiation energy: The energy of carbon ion irradiation is set to 80MeV / u, which can effectively penetrate the seeds without causing excessive damage to the DNA of wheat seeds.

[0036] Irradiation dose rate: The carbon ion dose rate is 60Gy / min, which can balance the relationship between the biological effects induced by radiation and the cell repair ability, while ensuring the stability and repeatability of the experimental results.

[0037] Linear Energy Transfer Density (LET): Two LET values ​​are set, low LET value is 35KeVμm -1 and high LET value 70KeVμm -1 , providing an important reference for the optimization of radiation parameters.

[0038] Irradiation dose: The radiation dose of carbon ions is set as a gradient dose of 20Gy, 40Gy, 60Gy, 80Gy, 100Gy, and 120Gy to study the effects of different doses on organisms, explore the optimal dose range of carbon ion mutagenesis, improve the reliability and efficiency of the experiment, and provide a theoretical basis for carbon ion beam dose in practical applications.

[0039] Irradiation time: The irradiation time was set to 0.33min, 0.67min, 1.00min, 1.33min, 1.67min, and 2.00min according to the target dose to ensure the seeds were exposed to carbon ion radiation for a certain period of time, thereby increasing the probability of mutation.

[0040] S4: According to the mutagenesis parameters set in S3, the mutagenic materials with different seed placement methods in S2 are irradiated with carbon ion beams, and wheat seeds with different combinations of mutagenesis parameters and placement methods are saved; according to different seed placement methods, different radiation energy transfer linear densities and different radiation doses, different irradiation parameter combinations are set as shown in Table 1.

[0041] Table 1. Different parameter combinations for wheat induced by high-energy carbon ion mutagenesis

[0042] deal with <![CDATA[LET(KeVμm -1 )]]> Seed placement Radiation dose / Gy L35-U 35 Embryo facing up (U) 0、20、40、60、80、100、120 L70-U 70 Embryo facing up (U) 0、20、40、60、80、100、120 L35-T 35 Tile 0、20、40、60、80、100、120 L70-T 70 Tile 0、20、40、60、80、100、120 L35-R 35 Random (R) 0、20、40、60、80、100、120

[0043] According to the different parameter combinations in Table 1, the prepared wheat seeds were placed in the biological radiation terminal of the heavy ion research facility of the Institute of Modern Physics, Chinese Academy of Sciences (Lanzhou, Gansu) for irradiation. During the irradiation process, uniform irradiation was ensured, the irradiation parameters were controlled, and each parameter combination was repeated 3 times; the seeds after high-energy carbon ion irradiation were stored in a -20°C refrigerator for subsequent research.

[0044] S5: Part of the wheat seeds with different mutagenesis parameters and placement combinations stored in S4 were subjected to hydroponic seedling experiments, and the other part was planted in the field;

[0045] Indoor seedling test Figure 2 ): First disinfect some of the irradiated seeds with 1% sodium hypochlorite for 15 minutes, then rinse the seeds with an appropriate amount of distilled water for 3-4 times until the seeds have no pungent smell, then add an appropriate amount of sterile distilled water to the culture dish to soak the seeds, and let it stand for 12-18 hours until the wheat seeds germinate and turn white. After turning white, put them in a culture room (temperature 23℃, relative humidity 60%-80%, light 16 hours / dark 8 hours, light 2000lx) for hydroponic seedling test. When the buds are 2-3cm long, transfer them to Hoagland nutrient solution for cultivation and observe the growth of the seedlings. The unirradiated seeds serve as the control group.

[0046] The irradiated seeds were planted in the field to obtain M 1 Generation of plants.

[0047] S6: Analyze the biological effects of wheat plants grown hydroponically in S5 and wheat plants planted in the field.

[0048] The height and root length of the seedlings grown for 7 days were measured under different radiation parameters, and the percentage of reduction compared with the control group was calculated. The graphs were drawn using professional graphics software such as GraphPad Prism. 1 The emergence of the first generation plants in the field was investigated and the emergence rate was calculated.

[0049] Experimental Results

[0050] The invention uses high-energy carbon ion mutagenesis treatment of wheat by setting different parameter combinations to perform biological effect analysis, including physiological index determination of wheat indoor seedling test and field M 1 The statistical analysis of the plant emergence rate yielded the following conclusions:

[0051] like Figure 3 As shown in the figure, when wheat Jing 411 was irradiated with high-energy carbon ions, the greater the irradiation dose, the higher the reduction rate of seedling height and root length compared with the untreated control group, and the trends of the two were relatively consistent; no matter which seed placement method was used, at an LET value of 70KeVμm -1 The LET value is 35KeVμm -1 The sensitivity is higher. When the LET value is 35KeVμm -1 The half-life dose is 117-132Gy, and the LET value is 70KeVμm -1 The semi-dwarfing dose was 80-83 Gy. The irradiated wheat Jing 411 dry seeds were planted in the field. 1 The germination rate was investigated during the seedling stage and it was found that the germination rate gradually decreased with the increase of dosage. Statistical analysis showed that the LET value was 35KeVμm -1 The dose required to reduce the emergence rate by 50% is 114-117 Gy, and the LET value is 70 KeVμm -1 The dose required to reduce the emergence rate by 50% was 67-71 Gy.

[0052] like Figure 4 As shown in the figure, when wheat Heyou No. 1 was irradiated with high-energy carbon ions, the seedling height and root length gradually decreased with the increase of radiation dose; the sensitivity was also different under different LET value treatments. -1 The half-maximum dose is 106-126Gy, and the LET value is 70KeVμm -1 The half-maximum dose is 66-68 Gy. 1 The results showed that the germination rate decreased with the increase of dosage; the LET value was 35KeVμm -1 The dose required to reduce the emergence rate by 50% is 95-131 Gy, and the LET value is 70 KeV μm -1 The dose required to reduce the emergence rate by 50% was 47-62 Gy.

[0053] Based on the above, the LET value of wheat varieties Jing 411 and Heyou No. 1 is 70KeVμm -1 The LET value is 35KeVμm -1 The sensitivity is stronger, and Heyou No. 1 is more sensitive to carbon ion radiation than Jing 411. According to the biological effects of high-energy carbon ion mutation in wheat with different parameter combinations, it is preliminarily determined that the wheat variety Jing 411 is suitable for the LET value of 35KeVμm -1 , a dose of 110-130Gy for radiation treatment, and an LET value of 70KeVμm -1 , the dose is 70-80Gy for radiation treatment; Nuclear No. 1 is suitable for LET value of 35KeVμm -1 , a dose of 100-130Gy for radiation treatment, and a LET value of 70KeVμm -1 , radiation treatment is performed at a dose of 50-70Gy.

[0054] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A method for inducing wheat mutagenesis using high-energy carbon ions with different parameters, characterized in that: The steps include: S1: Select the selected high-quality seeds as mutagenic materials; S2: The mutagenic materials screened in S1 are processed by different seed placement methods; S3: setting the mutagenesis parameters of high-energy carbon ions, the parameters including: radiation energy, dose rate, energy transfer line density, radiation dose, and radiation time; S4: subjecting the mutagenic materials of different seed placements in S2 to carbon ion beam radiation according to the mutagenic parameters set in S3, and saving the wheat seeds of different combinations of mutagenic parameters and placements; S5: Part of the wheat seeds with different mutagenesis parameters and placement combinations stored in S4 were subjected to hydroponic seedling experiments, and the other part was planted in the field; S6: Analyze the biological effects of wheat plants grown hydroponically in S5 and wheat plants planted in the field.

2. The method for inducing wheat mutation with high-energy carbon ions of different parameters according to claim 1, characterized in that: The mutagenic material described in S1 is an excellent individual plant of a wheat variety with a clear genetic background and robust growth, and wheat seeds with uniform size and full grains are selected after harvest.

3. The method for inducing wheat mutagenesis using high-energy carbon ions with different parameters according to claim 1 or 2, characterized in that: The wheat varieties are Wheat Jing 411 and Wheat Heyou 1.

4. The method for inducing wheat mutation with high-energy carbon ions of different parameters according to claim 3, characterized in that: The placement methods described in S2 include: fixing the embryos upward with double-sided tape, laying them flat, and placing them randomly.

5. The method for inducing wheat mutation with high-energy carbon ions of different parameters according to claim 3, characterized in that: The mutagenesis parameters of high-energy carbon ions in S3 include radiation energy of 80 MeV / u, dose rate of 60 Gy / min, energy transfer line density of 35 KeV μm -1 and 70KeVμm -1 , irradiation dose is 20Gy-120Gy, and irradiation time is 0.33min-2.00min.

6. The method for inducing wheat mutation with high-energy carbon ions of different parameters according to claim 5, characterized in that: The carbon ion beam radiation in S4 is uniformly irradiated according to the mutagenesis parameters set in S3.

7. The method for inducing wheat mutation with high-energy carbon ions of different parameters according to claim 3, characterized in that: The storage temperature of wheat seeds in S4 was -20℃.

8. The method for inducing wheat mutation with high-energy carbon ions of different parameters according to claim 3, characterized in that: The hydroponic seedling test described in S5 includes seed pre-germination treatment and disinfection, soaking, germination and determination of seedling physiological indicators.

9. The method for inducing wheat mutation with high-energy carbon ions of different parameters according to claim 3, characterized in that: The biological effect analysis described in S6 includes observations on the growth of hydroponic seedlings and the emergence of M1 generation plants in the field, and calculations of the seedling height reduction rate, root length reduction rate, and field M1 plant emergence rate.

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