Method for chemically inducing male sterility and application thereof

Abstract

The invention relates to the technical field of chemically induced male sterility, in particular to a chemically induced male sterility method and application thereof. According to the method disclosed by the invention, a fixed sterile line, a maintainer line and a fixed restorer line are not provided, the non-hereditary male sterile line is generated only by carrying out chemical induced sterility on any parent inbred line in the excellent hybrid combination, and the hybrid can be prepared by taking the other parent inbred line as a pollination line; the chemically induced male sterile line (chemically killed line) can be used for preparing a heterozygous disease-resistant hybrid with a single dominant disease-resistant gene (containing a clubroot disease-resistant gene) and preparing a high-oil and high-yield hybrid with oil content, thousand seed weight, seed number and the like of rape with quantitative inheritable characters.

Description

Technical Field

[0001] The present invention relates to the technical field of chemically induced male sterility, and in particular to a method for chemically induced male sterility and its application. Background Art

[0002] For rapeseed hybrid seed production to obtain high-quality hybrid seeds, not only should there be a suitable isolation area around the seed production field, that is, a safe spatial isolation distance not contaminated by pollen outside the area, but also its parents must be pure and excellent, that is, the purity of the parents must reach more than 99%. This requires that the parent reproduction should have a more stringent isolation area than hybrid seed production, that is, a larger isolation distance. In rapeseed hybrid seed production in the Huanghuai region, Li Dianrong proposed that the spatial safety isolation distance around the seed production area must reach 1500m - 2000m, and the location of bees should also be 300m away from the edge of the seed production area. From the perspective of bee pollination, the isolation distance actually reaches 1800m - 2300m; while for parent reproduction, the isolation distance is required to be 4000m. Regarding the number of isolation areas for hybrid rapeseed seed production: cytoplasmic male sterility (CMS) three-line seed production requires four isolation areas, namely the reproduction of the parent sterile line, maintainer line, restorer line, and hybrid seed production. The hybrid soybean breeding team of the Jilin Academy of Agricultural Sciences believes that the cytoplasmic "three-line" method has high costs and complex processes; while the genic male sterility (GMS) two-line seed production requires three isolation areas, namely the reproduction of the parent male sterile two-line and restorer line and hybrid seed production.

[0003] In recent years, although chemically induced male sterility has attracted the general attention of rapeseed breeders, due to insufficient understanding and in-depth research and application, especially the key technologies have not been well applied, so the development of high-oil and high-yield rapeseed breeding has not been rapidly promoted. In our breeding nursery, every year, a large number of hybrid combinations are prepared with excellent parents, ranging from dozens to hundreds. These combinations are included in the combined test and plot test, and finally, excellent combinations meeting the breeding objectives are selected through two-level tests. High oil and high yield of rapeseed are the eternal goals of breeders, but it is not easy to achieve this goal.

[0004] In recent years, clubroot has developed rapidly in the Yangtze River Basin. It not only has a fast onset but also causes serious damage. The yield of lightly affected fields is reduced by 30% - 50%, and severely affected fields even result in a complete crop failure. Since this disease is a soil-borne disease, it is relatively difficult to control. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for chemically induced male sterility and its application. This method has neither a fixed sterile line nor a fixed maintainer line, nor a fixed restorer line. Only one of the parent inbred lines in an excellent hybrid combination needs to be chemically induced to male sterility to produce a non-heritable male sterile line (abbreviated as a male-killing line), and the other parent inbred line can be used as a pollination line to prepare hybrid seeds.

[0006] To solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A method for chemically inducing male sterility, comprising the following steps:

[0008] S1, selecting excellent variety A that meets the breeding goal as the female parent;

[0009] S2, selecting variety B as the male parent, crossing it with the female parent in S1, self-crossing the F1 generation, and obtaining three genotypes separated from the F2 generation, obtaining a total of six types of esterase isozyme bands of variety A, variety B, the hybrid, and the three genotypes separated from the F2;

[0010] S3, comparing the six enzyme bands obtained in step S2. If there is a new genotype among the three genotypes of the F2, then the cytoplasmic gene of this genotype is the cytoplasmic gene of the female parent, and the nuclear gene is the nuclear gene of the male parent;

[0011] S4, using the new genotype esterase band obtained in step S3 as the marker band of the new germplasm, performing plant selection on the F2 generation of the cross between variety A and variety B, and new germplasm can be obtained;

[0012] S5, if the new genotype esterase band obtained in step S3 is difficult to distinguish from the bands of any other germplasm in the segregation generation, then perform molecular marking on it to obtain the marker of the new germplasm, and then use the marker to screen.

[0013] The principle is as follows:

[0014]

[0015] Preferably, the cultivation method of the excellent variety in step S1 is to obtain plants through microspore culture, in vitro culture of the pollen of the first-generation hybrid to form haploid plants, and then doubling them into homozygous diploids.

[0016] Preferably, the specific method of the microspore culture includes pollen culture or anther culture.

[0017] An application of a method for chemically inducing male sterility, applied to chemically inducing male sterility of rapeseed and cruciferous vegetables, and also applicable to crops that can carry out the breeding approach of chemically inducing male sterility in cytoplasmic male sterility three-line and nuclear male sterility two-line systems.

[0018] An application of a method for chemically inducing male sterility, applied to breeding for resistance to clubroot disease.

[0019] The beneficial effects of the present invention:

[0020] The breeding method of the present invention uses a chemically induced male sterile line of rapeseed called "chemical emasculation line 1". For two parental inbred lines, they can induce male sterility in each other, and can be used for orthogonal or reciprocal crosses. The pairing is free and not affected by bad cytoplasm. Moreover, different from cytoplasmic and nuclear male sterile lines which are fixed and whose cytoplasm is not easy to change, the sterile trait of the induced male sterility is stable, overcoming the problem of micro-pollen self-pollination caused by the temperature sensitivity of cytoplasmic sterility, and reducing the seed production purity due to the emergence of sterile plants. In addition, the advantages of the F2 generation can also be utilized because the fertility of the offspring treated with the chemical hybridization agent does not segregate and no sterile plants will appear. From the genetic analysis, there are still some advantages.

[0021] The present invention has neither a fixed sterile line, maintainer line, nor a fixed restorer line. Only one of the parental inbred lines in an excellent hybrid combination needs to be chemically induced to be sterile to produce a non-genetic male sterile line "male-killing line", and the other parental inbred line can be used as the pollinator line to prepare hybrids. Moreover, the two parental lines in an excellent hybrid combination can also be used as male-killing lines or pollinator lines for each other. And they all belong to "male sterility" together with CMS and GMS, and are more convenient and labor-saving than CMS and GMS in application technology. The isolation area of the chemical emasculation line 1 of rapeseed is one less than that of cytoplasmic sterile hybrid seed production, and there is no such strict requirement for the reproduction of parental sterile lines; compared with the nuclear male sterile line (GMS) seed production, it does not need to remove 50% of the fertile plants, which is more labor-saving. This chemically induced male sterile line (chemical emasculation line 1) can be used to prepare hybrid disease-resistant varieties in combination with a single dominant disease-resistant gene (including the clubroot disease-resistant gene), and can be used to prepare high-oil and high-yield hybrid varieties in combination with the oil content, 1000-seed weight, number of seeds per pod, etc. of rapeseed with quantitative genetic traits.

[0022] The present invention can play a significant role in the breeding of clubroot disease resistance: Since the clubroot disease resistance gene of rapeseed is controlled by a few relatively independent major genes and mostly shows dominant single-gene inheritance, when a disease-resistant inbred line is used as one parent and another disease-susceptible inbred line is used as the other parent for hybridization, heterozygous resistance (disease resistance) genes can be obtained in the F1 generation. Therefore, if one of the combinations is cultivated into an excellent disease-resistant inbred line, and the "chemical emasculation line 1" approach is adopted in the selection of hybrid varieties, fast and good disease resistance effects can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic diagram of the new germplasm obtained by hybridizing with the chemical emasculation line 1 of the present invention. Among them, 208 is the esterase isozyme pattern of the female parent, 292 is the esterase isozyme pattern of the hybrid F1, and the new isozyme pattern indicates the obtained new germplasm;

[0024] Figure 2 It is a molecular marker diagram of the present invention's ♀432 (disease-susceptible rr) × ♂H5B (disease-resistant RR), F1;

[0025] Figure 3Example of the performance of the "432" strain that is not resistant to clubroot disease and the "H5B" strain that is resistant to clubroot disease in the field after hybridization, with the hybrid F1 showing "heterozygous resistance".

[0026] Figure 4 Molecular marker illustration of F1 from the cross of ♀797♀ (not resistant, rr) × ♂H5B (resistant, RR) in the present invention. Detailed implementation methods

[0027] For the convenience of those skilled in the art, the present invention will be further described below in conjunction with embodiments and drawings. The content mentioned in the implementation methods does not limit the present invention.

[0028] Each year, among dozens of selected hybridization combinations, the optimal combination of high combining ability parents is chosen. The better of the two parents in terms of traits is used as the female parent to induce male sterility (referred to as the male sterile line), and the other is used as the male parent for pollination (referred to as the pollination line) to prepare hybrid seeds. This technology is superior to the heterosis utilization methods of cytoplasmic male sterility three-line (CMS) and genic male sterility two-line (GMS), showing the characteristics of rapidity, safety, and high efficiency in technology. The male sterility that takes seven or eight generations to transfer in the cytoplasm can be completed within one year using the "chemical emasculation one-line" method, and no sterile plants appear in F2 to affect the yield. It can also play an important role in achieving high oil yield and breeding for clubroot disease resistance.

[0029] During the breeding process, the following points need to be noted:

[0030] 1. In the first breeding year, select parents with excellent traits in the breeding nursery, such as strains with more pod grains, high thousand-grain weight, high oil content, and strong stress resistance, and make dozens or more hybridization combinations. After harvesting, select 2 - 3 superior combinations.

[0031] 2. In the second year, induce male sterility (chemical emasculation) in the excellent female parent of the superior combination, and use the male parent as the pollination line to prepare chemical emasculation hybrids. The hybridization agent (emasculant) used for male sterility induction should be a rapeseed chemical hybridization agent with high efficiency, low toxicity, no residue, and no side effects. When the rapeseed is at the single nucleus stage and the stem height is about 20 cm, spray for the first time, and then spray for the second time 10 - 14 days after the first spray to maintain sterility until the end of flowering. Currently, SX-1 developed by the Shaanxi Hybrid Rapeseed Research Center can be selected as the chemical hybridization agent.

[0032] 4. To increase the seed production yield, the flowering periods of the male and female parents should coincide well. Since the female parent generally delays flowering by about 3 days after spraying the chemical emasculant, the flowering period of the male parent can be 3 - 5 days later than that of the female parent (let the female parent wait for the male parent to flower, not the other way around). To facilitate the female parent to better receive the pollen from the male parent for fruiting, the plant height of the male parent can be higher than or equal to that of the female parent. If the female parent is higher than the male parent, it is not conducive to pollination. For such combinations, pollination can be achieved by means of bees or wind.

[0033] 5. Given that high oil content and high yield are quantitative traits, for breeding high oil and high yield combinations, the oil content and yield factors of both parents in the combination should be high. For breeding disease-resistant combinations, one of the disease-resistant parents must be genetically dominant single-gene inheritance, so that the hybrid F1 will show heterozygous resistance. For example, for clubroot, most of the resistance genes found so far are dominant single genes, so the hybrid F1 will show the heterozygous resistance genes for disease resistance, which is a major issue in solving the losses caused by clubroot in current production.

[0034] 6. To improve the yield and quality of the combination, bees can be released in the seed production area, or at 10:00 am on windless sunny days and 2 - 3 pm, two people can pull ropes for pollination, or one person can use a rod to stir or use hands to spread the pollen, etc.

[0035] 7. To ensure the purity of the parents, microspore culture is adopted to purify and rejuvenate the parents or conduct multi-generation bagging and self-crossing for breeding.

[0036] Example 1

[0037] Create new germplasms using rapeseed excellent germplasm materials 208 and 159:

[0038] Use material 208 with a certain excellent trait as the female parent, and material 159 with another excellent trait as the male parent for hybridization. After hybridization, the hybrid F1292 is obtained. We made the esterase isozyme spectra of female parent 208 and hybrid F1292 in the laboratory; in the same year, the hybrid F1292 was planted in the breeding nursery, and in the second year, it was bagged and self-crossed during the flowering period to obtain the F2 generation, which can be separated into three genotypes: one is the female parent 208, one is the hybrid 292, and the other should be a new genotype. Make the esterase isozyme spectra of the three genotypes, and exclude the plants with the same isozyme spectra as the female parent 208 and hybrid F1292. Then the remaining plants should be new genotype germplasms with the cytoplasm of the female parent and the nucleus of the male parent. Since this is a genetic rule, there is no need to make esterase isozyme spectra for confirmation anymore. For example, Figure 1 It can be seen that the esterase isozyme spectra of female parent 208 and hybrid F1292 are the same (there are also differences), and the new enzyme spectrum on the right is undoubtedly the new germplasm.

[0039] If there is an enzyme spectrum that is neither the same as the female parent nor the F1 hybrid, then this enzyme spectrum should undoubtedly be a new germplasm. This selection method not only has a fast selection speed but also saves manpower, material resources, and costs. As for whether the germplasm is excellent, it can be identified by breeders or continue to create more excellent new germplasms.

[0040] Except for the above examples, any two rapeseed varieties (lines) with excellent traits are applicable to the method of the present invention for creating new hybrid germplasms.

[0041] Example 2

[0042] In the implementation of the "chemical emasculation line" breeding, we aimed to achieve high yields by having large and numerous grains. Considering the genetic stability of grain weight, we used large-grain varieties as female parents and multi-grain varieties as male parents for hybridization to obtain large-grain and multi-grain combinations. In April 2017, we selected 6 groups of germplasms of large-grain and small-grain varieties respectively for hybridization. We used the large-grain variety as the female parent "emasculation line", with an average 1000-grain weight of 4.97 g, and the small-grain variety as the male parent "pollination line", with an average 1000-grain weight of 3.95 g. In the autumn of 2017, their F1 was planted in the hybridization nursery. In the summer of 2018, their 1000-grain weight was harvested and examined. The result showed that the average 1000-grain weight of the F1 of the 6 combinations was 4.49 g, which was basically the same as the average 1000-grain weight of the two original parents of the combination, 4.47 g. (Table 1). On the contrary, we also selected 6 groups with different 1000-grain weights. We used the small-grain variety with an average 1000-grain weight of 3.67 g as the female parent "emasculation line" and the large-grain variety with an average 1000-grain weight of 4.90 g as the male parent "pollination line" for hybridization. Similarly, their F1 was planted in the hybridization nursery. In 2018, their F1 1000-grain weight was harvested and examined. The result showed that the average 1000-grain weight of the F1 was 4.29 g, which was 12.60% greater than the average 1000-grain weight of the original small-grain parent of the combination, 3.777 g, and 37.59% smaller than the average 1000-grain weight of the original large-grain parent of the combination (Table 2). The reciprocal crosses of large and small grains fully demonstrated that F1 belongs to quantitative inheritance in terms of grain weight and tends to the small-grain weight parent. If we want to increase the 1000-grain weight of F1, we should pay attention not to use germplasms with small 1000-grain weights in the hybridization parents. To increase the yield, it is best to use two germplasms with high 1000-grain weights. If this cannot be achieved, the germplasm with a low 1000-grain weight should not be too low, and try to use germplasms with a relatively high 1000-grain weight (Table 1, 2).

[0043] Table 1 Genetic performance of F1 of rapeseed large-grain variety × small-grain variety

[0044]

[0045]

[0046] Note:. The 1000-grain weight of F1 tends to the mean of the large and small grains of the combination

[0047] Table 2 Genetic performance of F1 of rapeseed small-grain variety × large-grain variety

[0048]

[0049]

[0050] Note: The 1000-grain weight of F1 tends to the average of the small-grain and large-grain combinations

[0051] Regarding the number of seeds per fruit, we selected three groups of germplasms and examined the performance of their F1 generations through hybridization. Among the three groups, the average number of seeds per fruit in the group with more seeds was 34.38, while the average number of seeds per fruit in the group with fewer seeds was 22.58. The average of the two was 28.48, and the average number of seeds per fruit in F1 was 28.13. Comparing F1 with the average number of seeds per fruit in the original germplasm (28.48), the two were extremely close. This also indicates that the number of seeds per fruit in F1 is close to the average of the groups with more and fewer seeds (Table 3).

[0052] Table 3 Genetic Performance of F1 from Crosses between Lines with More Seeds and Lines with Fewer Seeds in 2018

[0053]

[0054] From the above genetic research, it is known that to increase the number of seeds per pod in the hybrid F1, "male-sterilizing lines" must use germplasms with more seeds. It is not very difficult to reach the ideal level of large or numerous seeds based on the existing resources, so it is considered feasible to significantly increase the rapeseed yield.

[0055] To sum up, for the "chemical male-sterilization single-line" seed production method, only one line of the rapeseed hybrid combination needs to be used as the "inducing line, i.e., male-sterilizing line", and the other as the pollinating line. Moreover, the two lines can be interchanged, and there is no fixed "maintaining line" and "restoring line". Only "one line" is induced (chemically sterilized), and it is relatively flexible, so it is called "chemical male-sterilization single-line".

[0056] The isolation area for the chemical male-sterilization single-line in rapeseed is one less than that for cytoplasmic male-sterile hybrid seed production, and it is not as strict as the requirements for the reproduction of parental male-sterile lines; compared with the seed production of genic male-sterile lines (GMS), it does not require removing 50% of the fertile plants, thus saving more labor.

[0057] For the breeding of the chemical male-sterilization single-line in rapeseed, there is no need to backcross the male-sterile line for 6 - 7 generations or even more generations, nor to select the restoring line for 5 - 6 generations. Only one excellent hybrid combination needs to be selected, and its female parent is chemically sterilized. Generally, it can be completed in 1 - 2 generations; since there will be no male-sterile plants in seed production, it is very safe; at the same time, when selecting the cytoplasm, there is no need to use N cytoplasm as the maintaining line, S cytoplasm as the male-sterile line and restoring line, or the excellent 1:1 line in the selection of genic male sterility, etc., and continuous multi-generation selection is required to achieve the goal.

[0058] Example 3

[0059] In 2023, a combination resistant to clubroot was prepared using the chemical male-sterilization single-line method:

[0060] Chinese breeding experts have found multiple single-dominant resistance genes to clubroot disease in European turnips and Chinese cabbages. By using the method of "chemical hybridization one-line", a combination can be prepared by crossing the excellent non-disease-resistant germplasm of rapeseed with disease-resistant germplasm, and then chemical hybridization is carried out to obtain a clubroot disease-resistant hybrid. However, whether it is non-disease-resistant germplasm or disease-resistant germplasm, it should have excellent traits such as high oil content, high yield, high quality (the high-quality requirement is double-low, that is, erucic acid is less than 1%, and glucosinolate is less than 30 μmol / g cake), and resistance (tolerance) to Sclerotinia sclerotiorum.

[0061] Specific indicators: The oil content of high oil reaches more than 46%; The 1000-grain weight of high yield reaches more than 5 g; The number of grains per pod is about 30; The field incidence of Sclerotinia sclerotiorum < 5%. Only the hybrids prepared in this way have the traits of high oil, high yield and resistance to clubroot disease. Only then can they be registered and promoted.

[0062] The genetic and molecular identification of the above-mentioned clubroot disease-resistant cross-breeding is shown as follows:

[0063] Variety A (non-resistant) is represented by rr, and variety B (resistant) is represented by RR

[0064]

[0065] As Figure 2 shown, the clubroot disease-susceptible line 432 is represented by rr; the clubroot disease-resistant line H5B is represented by RR; the hybrid F1 is heterozygous resistant and is represented by Rr.

[0066] As Figure 4 shown, the clubroot disease-susceptible line Qinyou 797♀ is represented by rr; the clubroot disease-resistant line H5B is represented by RR; the hybrid F1 is heterozygous resistant and is represented by Rr.

[0067] As Figure 3 shown, left: The "432" line susceptible to clubroot disease has swollen root nodules, sparse fibrous roots, thinner stem segments, and poor growth and development; right: The hybrid F1 shows heterozygous resistance in the field, with normal roots, developed root systems, thick stem segments, and good growth and development.

[0068] By crossing the disease-susceptible hybrid F1 with the disease-resistant line, a disease-resistant variety can still be obtained. The genetic diagram is as follows:

[0069]

[0070] The mixed hybrids obtained from F1 not only have heterozygous resistance, but also have better adaptability and stable yield due to multiple genes.

[0071] To verify whether the above theory is correct, we planted six different combinations of a clubroot-resistant strain and a non-resistant strain in a disease nursery and conducted indoor inoculation tests. As a result, no combination was found to be infected with clubroot disease either indoors or outdoors, and the same result was shown in the field trials. Practice has fully proved the correctness of this theory. Based on this, it is believed that using the "chemical emasculation line" in breeding can quickly solve the problem of clubroot resistance in the short term and create real benefits for rapeseed in the disease area.

[0072] Example 4

[0073] In 2023, high-oil combinations were prepared using the chemical emasculation line:

[0074] The female parent (♀) 21Ld31-3, a high-oil strain with an oil content of 55%, was selected as the chemical emasculation line; the male parent (♂) 21Ld62-1, a high-oil strain with an oil content of 51.9%, was selected as the pollination line. Then, hybrid seeds were produced in the autumn of the same year using the "chemical emasculation line" method (or artificial seed production); the hybrids obtained in the summer harvest of the first year were planted in the combination test nursery in the autumn of the same year, and the hybrids were harvested in the summer of the second year. After measurement, the oil content of F1 reached 52.81%. The oil content is indeed relatively high. However, to become a variety that can be popularized and applied in production, not only the oil content should be high, but also the yield should be high. That is, the product of yield and oil content, the oil production, is the target requirement we pursue. Therefore, when selecting high-oil strains, the high-oil-content strains must also have high-yield traits. This requires selecting germplasms with a thousand-seed weight > 5g and about 30 seeds per pod, and also being resistant (tolerant) to the main disease, Sclerotinia sclerotiorum, in rapeseed production. The standard for selecting field Sclerotinia sclerotiorum-tolerant strains is that the incidence of Sclerotinia sclerotiorum < 5%. In terms of quality, it should meet the double-low standard, that is, the erucic acid is less than 1% and the glucosinolate is less than 30 μmol / g of cake. Therefore, in this case, selecting high-oil and high-yield parental strains is the key. It should also be noted here that the number of pods per plant, another factor affecting yield, is not included because it is too affected by environmental factors and has a high coefficient of variation (such as water, fertilizer, temperature, etc.). While the thousand-seed weight and the number of seeds per pod have a small coefficient of variation, high genetic transmissibility, and are relatively stable. Therefore, it is genetically based to put forward these two factors.

[0075] All technical features in this example can be modified in appearance according to actual needs.

[0076] The above embodiments are preferred implementation schemes of the present invention. In addition, the present invention can also be implemented in other ways. Any obvious replacement without departing from the concept of the technical solution of the present invention is within the protection scope of the present invention.

Claims

1. A method for chemically induced male sterility, characterized in that: It includes the following steps: S1. Select excellent variety A that meets the breeding goal as the female parent; S2. Select variety B as the male parent and cross it with the female parent in S1. The F1 generation is self-crossed, and three genotypes are separated in the obtained F2 generation. Six types of esterase isozyme bands of variety A, variety B, the hybrid, and the three genotypes separated in F2 are obtained; S3. Compare the six enzyme bands obtained in step S2. If there is a new genotype among the three genotypes in F2, the cytoplasmic gene of this genotype is the cytoplasmic gene of the female parent, while the nuclear gene is the nuclear gene of the male parent; S4. Use the new genotype esterase band obtained in step S3 as the marker band of the new germplasm, and conduct plant selection on the F2 generation of the cross between variety A and variety B to obtain a new germplasm; S5. If the new genotype esterase band obtained in step S3 is difficult to distinguish from the bands of any other germplasm in the segregation generation, perform molecular marking on it to obtain the marker of the new germplasm, and then use the marker to screen; 2. The method for chemically induced male sterility according to claim 1, characterized in that: The cultivation method of the excellent variety in step S1 is to obtain plants through microspore culture. The pollen of the first-generation hybrid is cultured in vitro to form haploid plants, and then doubled into homozygous diploids; 3. A method for chemically induced male sterility according to claim 2, characterized in that: The specific method of the microspore culture includes pollen culture or anther culture; 4. Use of a method for chemically induced male sterility according to any one of claims 1 - 3, characterized in that: It is applied to the chemical induction of male sterility in rapeseed and cruciferous vegetables, and is also applicable to crops that can adopt the breeding approach of chemical induction of male sterility in cytoplasmic male sterility three-line systems and genic male sterility two-line systems; 5. Use of a method for chemically induced male sterility according to any one of claims 1-3, characterized in that: It is applied to the breeding for resistance to clubroot disease.

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