Method for producing f1 hybrid seeds using partial male sterility genes

Abstract

The present invention relates to a method for producing a transgenic plant body in which a partial male sterile gene and an opaque endosperm marker gene are simultaneously induced to mutate using a CRISPR / Cas system, and producing seeds of a partial male sterile maintainer line and a high proportion of hybrid seeds by classifying the seeds through hybridization of the transgenic plant body with a wild type plant body. Flo5 / Os02g02840 The production method of hybrid seeds of the present invention can be used to induce partial male sterility in rice, and after simultaneously inducing mutation in an opaque endosperm marker gene, can be used as a maternal parent for hybrid seed production, making it possible to produce and classify seeds of a partial male sterile maintainer line and a high proportion of hybrid seeds, and thus can be variously utilized in the agricultural field.

Description

Technical Field

[0001] This invention relates to the following: a method for producing partially male-sterile maintainer line seeds and a method for producing high-proportion hybrid seeds: transgenic plants mutated simultaneously by a CRISPR / Cas system for a partially male-sterile gene and an opaque endosperm marker gene; and the transgenic plants are then hybridized with wild-type plants to induce mutations in the marker gene (…). Flo5 / Os02g02840 The classification of ) becomes possible. Background Technology

[0002] Rice ( Oryza sativa L. Rice is a staple crop used by more than half of the world's population. Considering the foreseeable growth in the world's population and current rice production levels, it is necessary to increase rice production to meet the growing food demand. However, rice cultivation faces numerous challenges, including a shrinking agricultural workforce, environmental impacts, and limitations in land and water resources. Therefore, developing diverse rice varieties is essential to address the increasing demand.

[0003] Heterosis is used in major crops to increase crop yields or acquire superior traits. Particularly in rice production, first-generation hybrid rice is known to show an approximately 30% increase in yield compared to the female parent through heterosis. However, hybrid rice production incurs high costs. To overcome this problem, hybrid rice production technology has been developing and utilizing male-sterile female parents to increase the probability of hybridization, moving towards economically viable hybrid seed production. This technology has now reached its third generation.

[0004] Male sterility is a core element of breeding systems. In rice, male sterility is mainly classified into two types: cytoplasmic male sterility (CMS) and genetic male sterility (GMS). Cytoplasmic male sterility is controlled by cytoplasmic factors, particularly genes found in mitochondria. Initially, a three-line hybrid breeding system based on CMS (first generation) was developed and widely used as a traditional method for producing hybrid rice. However, this system has drawbacks, requiring significant labor and cost to maintain the three lines and demanding precise field settings. To address this issue, genetic male sterility was utilized in hybrid rice production. A derived two-line system uses a photoperiod- or temperature-sensitive GMS system (collectively referred to as PTGMS, second generation), requiring no additional maintainer lines, thus simplifying the process. However, it suffers from the disadvantage of difficulty in constructing environmental conditions that regulate male sterility. Both first and second generations are limited by genetic diversity, exhibiting several fundamental limitations, including the production of potentially disease-vulnerable varieties, the instability of the CMS system, and the instability of the PTGMS system due to its excessive vulnerability to uncontrollable weather variations. Summary of the Invention

[0005] To facilitate the separation of hybrid seeds produced through hybridization and maintainer line seeds produced through self-fertilization, the inventors constructed a two-line hybridization system using the CRISPR / Cas system. This system utilizes a maternal line with simultaneous mutations in an opaque endosperm marker gene and a portion of a male sterility gene. This system can distinguish between self-pollinated and cross-pollinated seeds based on endosperm expression patterns. The inventors confirmed that the system can achieve partial maintenance of self-pollinated maternal lines and the production of hybrid seeds through hybridization with paternal lines, thus completing this invention.

[0006] Therefore, the object of the present invention is to provide transgenic rice that simultaneously suppresses the expression of some male sterility genes and opaque endosperm marker genes.

[0007] Another object of the present invention is a method for preparing transgenic rice that simultaneously inhibits the expression of some male sterility genes and opaque endosperm marker genes.

[0008] Another object of the present invention is to provide a first-generation hybrid seed produced by crossing the genetically modified rice with wild-type rice.

[0009] Another object of the present invention is to provide a method for producing first-generation hybrid seeds by crossing the genetically modified rice with wild-type rice.

[0010] To achieve the aforementioned objective, the present invention provides a transgenic rice that simultaneously suppresses a gene composed of the base sequence represented by SEQ ID NO: 1. GTrd5 ( Os11g20384 (or a gene consisting of the base sequence represented by SEQ ID NO: 2) GTrd9 ( Os03g51620 ) and the gene consisting of the base sequence represented by SEQ ID NO: 3. Flo5 ( Os02g02840 The expression of ).

[0011] To achieve the aforementioned further objective, the present invention provides a method for preparing transgenic rice, comprising the following steps: simultaneously inhibiting a gene composed of the base sequence represented by SEQ ID NO: 1. GTrd5 ( Os11g20384 (or a gene consisting of the base sequence represented by SEQ ID NO: 2) GTrd9 ( Os03g51620 ) and the gene consisting of the base sequence represented by SEQ ID NO: 3. Flo5 ( Os02g02840 The expression of ).

[0012] To achieve the other objective, the present invention provides a first-generation hybrid seed produced by crossing the transgenic rice as the female parent with wild-type rice.

[0013] To achieve the other objective, the present invention provides a method for producing first-generation hybrid seeds, characterized by comprising the following steps: hybridizing the transgenic rice as the female parent with wild-type rice.

[0014] The hybrid seed production method of the present invention can be used to induce partial male sterility in rice. After simultaneously inducing mutations in the hybrid seed and the opaque endosperm marker gene, it can be used as a maternal line for hybrid seed production to produce and classify maintainer line seeds and a high proportion of hybrid seeds, which can be used in a variety of ways in the agricultural field. Attached Figure Description

[0015] Figure 1 Show GTrd5 and GTrd9 Gene structure and phenotype of mutations.

[0016] Figure 2 Showing wild type and GTrd9 Cytological analysis results of the -1 mutant.

[0017] Figure 3 This illustrates the outline of the two-line hybrid rice breeding system of the present invention.

[0018] Figure 4 Show GTrd5Flo5 and GTrd9Flo5 Maintain the generation process of the system.

[0019] Figure 5 Showing the use GTrd5Flo5 and GTrd9Flo5 Production assessment of maternal hybrid seeds.

[0020] Figure 6 The heterosis observed in the F1 hybrid of the two-line hybrid rice breeding system of the present invention is shown. Detailed Implementation

[0021] The present invention will now be described in detail.

[0022] This invention provides a transgenic rice that simultaneously suppresses a gene composed of the base sequence represented by SEQ ID NO: 1. GTrd5 ( Os11g20384 (or a gene consisting of the base sequence represented by SEQ ID NO: 2) GTrd9 ( Os03g51620 ) and the gene consisting of the base sequence represented by SEQ ID NO: 3. Flo5 ( Os02g02840 The expression of ).

[0023] The scientific name of the term "rice" in this invention is Oryza sativa L.The rice is an annual herbaceous plant, and the rice in this invention can be any one of the following groups: Japonica type, Indica type and Javanica type.

[0024] In this invention, the term "male sterility" refers to the inability to achieve pollination, fertilization, and seed formation due to morphological or functional abnormalities of the male reproductive organs. This can manifest as temporary environmental variations or as a hereditary trait. Male sterility in this invention can be induced by a hereditary trait. Specifically, some forms of male sterility in this invention can be induced by inhibiting the activity of the cell nucleus involved in pollen tube elongation. GTrd5 or GTrd9 Gene expression is used to induce [something].

[0025] Furthermore, this invention provides a method for preparing transgenic rice, comprising the following steps: simultaneously inhibiting a gene composed of the base sequence represented by SEQ ID NO: 1. GTrd5 ( Os11g20384 (or a gene consisting of the base sequence represented by SEQ ID NO: 2) GTrd9 ( Os03g51620 ) and the gene consisting of the base sequence represented by SEQ ID NO: 3. Flo5 ( Os02g02840 The expression of ).

[0026] In this invention, the steps can be implemented by deleting or inserting a portion of the gene at a rate of 1 bp or more. For example, this can be achieved by deleting or inserting 10% of the full-length DNA, specifically by deleting or inserting at a rate of 0.01% or more. Furthermore, these steps can be implemented using various gene editing methods known in the art, preferably using CRISPR-Cas9 gene editing.

[0027] CRISPR-Cas9 is one of the gene editing technologies. Cas9 (CRISPR-associated protein 9) is an RNA-induced DNA cleaving enzyme that uses gRNA (guide RNA) to cut DNA sequences.

[0028] In this invention, the recombinant vector used in the transformation may also contain a DNA sequence encoding a gRNA capable of recognizing the target gene and a DNA sequence encoding a Cas protein.

[0029] Furthermore, in addition to exogenous genes such as T-DNA that can be inserted into the plant genome, the steps can also be performed using endogenous transposons such as TOS17 or by inducing mutations through X-ray or gamma-ray irradiation, or by using RNAi or antisense methods, but are not limited to these methods.

[0030] In this invention, the "T-DNA" refers to the transfer DNA within the tumor-inducing plasmid of Agrobacterium, a DNA fragment directed towards the nucleus of the host plant cell. The T-DNA has 25bp repeat sequences on both sides of its edges, and transfer begins at the left border and ends at the right border. The bacterial T-DNA is approximately 20,000bp in length, and its insertion causes insertional mutagenesis by disrupting the target gene. The inserted T-DNA sequence not only causes mutations but also marks the target gene.

[0031] The above-mentioned transformation can also be achieved using microinjection, electroporation, liposome-mediated transfection, calcium phosphate precipitation, etc. The preferred method of the present invention includes Agrobacterium-mediated transfection.

[0032] Furthermore, the present invention provides a first-generation hybrid seed produced by crossing the aforementioned transgenic rice as the female parent with wild-type rice.

[0033] To effectively transfer pollen, the hybridization method involves natural hybridization of gene-edited male-sterile rice with wild-type rice in cultivated land. Typically, it is recommended that the pollen parent be 6 to 10 times larger than the seed parent (male-sterile), but the planting pattern and ratio can be adjusted according to the environment.

[0034] Furthermore, the present invention provides a method for producing first-generation hybrid seeds, characterized by comprising the following steps: hybridizing transgenic rice as the parent plant with wild-type rice.

[0035] Preferably, one parent variety used in the hybridization is an aus-type or indica rice type, and the other is a japonica rice type, so as to maximize hybrid vigor. Representative pollen parent rice varieties with a wide range of affinity genes include N22, Dular, Moroberekan, Narin, Nekken1, etc., but are not limited to these.

[0036] Unless otherwise contradictory, the above-described content of this invention also applies, and any implementations by those skilled in the art with appropriate modifications are also included within the scope of this invention.

[0037] The present invention will be described in detail below through embodiments, but the scope of the present invention is not limited to the following embodiments.

[0038] Materials and Methods Plant materials and growth conditions It will be used as japonica rice ( Oryzasativassp.japonica Transformation systems based on the Dongjin (DJ) rice variety were cultured in a nursery at 28°C (16 hours light) and 22°C (8 hours dark) with a photoperiod of 80% humidity, or in a LMO (Legacy Modified Organism) isolation nursery at Kyung Hee University, and intracellular location analysis was performed.

[0039] Analysis of T-DNA insertion mutations Obtained from Kyung Hee University Seed Bank GTrd5 (2A-30498; cv. Huaying) and GTrd9 The T-DNA mutant of (2D-00110; cv. Dongjin) (Yi and An, 2013) was used. Heterochromatin (HZ) mutations were employed for isolation analysis and cross-hybridization. Genotyping was confirmed by polymerase chain reaction (PCR) in 50 μL of a mixture containing 20 ng of genomic DNA, 10X e-Taq buffer, 0.2 mM dNTPs, 0.5 U of e-Taq polymerase (Solgent, Daejeon, Korea), and 1 mM primers. The PCR protocol consisted of 35 cycles of 60 seconds at 94°C, 60 seconds at 60°C, and 120 seconds at 72°C. Primers used for genotyping analysis are shown in Table 1.

[0040] Table 1

[0041]

[0042] CRISPR / Cas-mediated mutations CRISPR / Cas9 constructs were generated by recognizing specific gRNAs using CRISPR direct (Naito et al., 2015). After oligomer synthesis, they were ligated into a BsaI-treated pRGEB32 binary vector (Addgene plasmid number: 63142). To generate double mutants, randomly arranged tRNA-gRNA structures were generated by PCR and then bound to the pRGEB32 binary vector using a speed cloning method (Moon and Jung, 2023). The resulting structures were then processed using Tojin rice (…). Oryzasativassp.japonicacv. As a wild-type (WT) rice, all transformation systems used Dongjin rice as the genetic background and utilized Agrobacterium tumefaciens (Agrobacterium) from callus tissue. AgrobacteriumtumefaciensPrepared by mediated co-culture.

[0043] Nucleic acid extraction and separation from mutant plants Genomic DNA was extracted from the samples using the cetyltrimethylammonium bromide (CTAB)-chloroform method. For T-DNA genotyping and CRISPR / Cas9 mutation sequencing, genomic DNA was amplified by PCR using gene-specific primers and analyzed at Macrogen (Seoul, South Korea) using the BigDye Terminator v3.1. Genotyping of T0 plants was performed using Sanger sequencing to screen for homozygous mutant systems.

[0044] Cytological analysis of pollen and pollen tubes Flowers and anthers were photographed using an Olympus SZX61 microscope. Before flowering, spikelets were collected, and pollen was extracted using tweezers. Pollen maturity was analyzed by staining with 1% I2-KI. The inner wall of the pollen was stained with 0.1% Calcofluor White and observed under UV light, while the outer wall was stained with 0.001% Auramine O and observed using the fluorescein isothiocyanate (FITC) channel on an Olympus BX61 microscope.

[0045] To observe pollen germination rate and pollen tube morphology in the laboratory, fresh pollen collected immediately after flowering was observed in solid or liquid pollen germination medium (PGM). Fresh PGM was prepared using 20% ​​(w / v) sucrose, 10% polyethylene glycol 4000, 3 mmol / L calcium nitrate, 40 mg / L boric acid, and 10 mg / L vitamin B1 (pH 6.8–7.0). PGM solidified with 1% agarose was covered with a coverslip and incubated for 20 minutes in a humid, dark chamber at 28°C. At least 150 pollen samples were used in each experiment, and germination rate was analyzed at a multiple of 3.

[0046] Callose staining of the pollen tubes (PT) of the pistil was performed using a modified method of Liu et al. (2016). Two hours after pollination, the pistils were cultured overnight in Carnoy's solution. The fixed pistils were washed and neutralized with 1M sodium hydroxide (NaOH), then stained with 0.05% aniline blue. The pistils fixed in 50% glycerol were observed under UV light using an Olympus BX61 fluorescence microscope.

[0047] Phenotypic analysis and field experiments To assess the seed set rate during cross-pollination, GTrd5Flo5 and GTrd9Flo5 Plants and homozygotes stPAC25The system was used for hybridization (Gold Rice; Zheng et al., 2017). Whole and incomplete kernels in the main cone were manually sorted, and the proportion of whole kernels was calculated based on seed color. To assess heterosis, [the following was done]... GTrd5Flo5 and GTrd9Flo5 After hybridization of plants with Dular rice varieties, the harvest yield of F1 hybrids was compared with that of the maternal line system.

[0048] Example 1. Genes required for male gamete transfer in rice in a two-line hybrid system. GTrd5 and GTrd9 Confirmation and Characterization The results of screening 107 T-DNA insertion systems of 628 genes preferentially expressed in pollen after the two-celled stage identified 20 systems that did not follow Mendelian segregation ratios, and these mutations were identified as gene transmission defects. gametictransferdefect,GTrd )mutation.

[0049] Defects were identified in these 20 systems that induced partial infertility. GTrd5 and GTrd9 Genes. (Related to...) GTrd5 and GTrd9 The T-DNA insertion systems for the gene (2A-30498 and 2D-00110) show a 1:1 ratio of wild-type (WT) to allotype binding (…). GTrd / GTrd The segregation ratio of the offspring was such that there were almost or no homozygous offspring (Table 2). To confirm whether the mutation caused a defect in the migration of male and female gametes, cross-hybridization was performed (Table 3). GTrd / GTrd When a plant is used as the paternal line to hybridize with a wild-type plant, only the following products are generated. GTrd / GTrd Descendants (WT), in contrast, use GTrd / GTrd When plants act as the maternal line, GTrd / GTrd and GTrd / GTrd The offspring ratio showed a normal 1:1. This result indicates that... GTrd5 and GTrd9 This is a gene essential for the transmission of male gametes. It is transmitted using the CRISPR / Cas9 system (…). Figure 1 Homozygous mutations obtained from (a) and (b) parts ( GTrd5-1 , GTrd5-2 , GTrd9-1 and GTrd9-2 The generation of ) occurs in two separate gene-editing systems ( Figure 1 Partial male infertility is shown in parts (c), (d), (f), and (g). In particular, GTrd5-1 and GTrd9-1 The mutations produced a considerable number of homozygous seeds that were not completely sterile, showing partial sterility, with productivity rates of at least 15% and 28%, respectively. Figure 1 (e) part, (h) part).

[0050] To investigate whether the observed partial sterility is due to pollen or pollen developmental defects, the morphology of the above structural elements was analyzed at the maturity stage. Figure 2 (a)-(c) Both the anthers and pollen of the wild-type and homozygous systems developed normally, and laboratory pollen germination tests also showed no significant difference in germination rate between the wild-type and the mutant. Figure 2 (d) and (f) parts). Therefore, it is anticipated that the partial male sterility of the above mutants is not due to maturation defects of the anthers or pollen or abnormal pollen development, but rather due to defects in pollen tube elongation occurring in a later stage. To confirm this, aniline blue staining was used to investigate the pollen tube elongation stage ( Figure 2 (e) and (g) steps). In the wild-type pistil, the pollen tube extends completely into the embryo sac, but GTrd5-1 and GTrd9-1 The pollen tubes of the mutant exhibit defects during elongation within the style, with only a very small fraction reaching the micropyle and completing fertilization. This result suggests... GTrd5-1 and GTrd9-1 Some of the sterile phenotypes observed in the mutants are caused by defects in the pollen tube elongation process.

[0051] Table 2

[0052] Table 3

[0053] Example 2. Constructing a maintenance system using opaque marker genes for two-line hybridization systems and some male sterility genes. GTrd5 or GTrd9 When mutations occur in genes, pollen tube elongation becomes defective, resulting in partial male sterility. Based on this characteristic, mutation and screening marker genes will be introduced into these genes to screen hybrid seeds, while simultaneously developing a two-line hybridization system that can maintain the system. Figure 3 Using substances that cause mutations in chalky white endosperm. FLO5 Genes serve as markers for distinguishing self-pollinated seeds (N et al., 2007). In the context of japonica rice, genes with mutations simultaneously in some male sterility genes and marker genes are used. GTrd5Flo5 and GTrd9Flo5 Mutation as maternal. FLO5 and GTrdBoth genes are inferior and can produce opaque seeds through self-pollination of the maintenance system, thus maintaining the system. However, when cross-pollinated with pollen from the donor system for hybridization breeding, hybrid seeds with a different appearance from those produced by self-pollination are produced.

[0054] Using CRISPR / Cas9-mediated genome editing FLO5 Preparation related GTrd5 and GTrd9 A double-mutation system. Analysis. GTrd5Flo5 The results of the base sequence analysis revealed single nucleotide deletions and insertions in the first exons of both genes, causing premature termination of the coding sequence. Similarly, in GTrd9Flo5 In the sequencing analysis, a 1bp insertion was found in both genes, leading to premature termination. Figure 4 (a) of the wild type and Flo5 Compared to single mutations, both pollen tube mutant alleles exhibited partial male sterility phenotypes without growth defects during both vegetative and reproductive growth stages. Figure 4 (part (b)). To evaluate its role as a marker for seed selection. FLO5 The function of genes was investigated, and seed phenotypes were examined. Figure 4 (part (c)). In from GTrd5Flo5 and GTrd9Flo5 Rice obtained from double mutant plants showed similarities to... Flo5 The single mutant exhibited a consistent phenotype of opaque, chalky endosperm. In contrast, the endosperm of rice obtained from the wild-type control group showed transparent and normal characteristics. Figure 4 (d) part). Flo5 Although the seed germination rate was not abnormal compared to the wild type, under normal nursery propagation conditions, the single mutant... GTrd5Flo5 and GTrd9Flo5 The seed germination rates in the double mutants were significantly reduced to approximately 11% and 23%, respectively. Figure 4 (e) of the seeds). Such a low seed germination rate is close to GTrd5 and GTrd9 The level observed in single mutants, thus confirming FLO5 Genes are effective screening markers that do not adversely affect seed production. Analysis of genes is used to identify genotypes. GTrd5Flo5 and GTrd9Flo5 The genotypes of the T1 progeny plants obtained from the system showed consistency with those of the T0 generation. Furthermore, to prevent the continuous expression of the Cas9 protein during reproduction and propagation, plants isolated from Cas9 were screened, and plants without the inserted gene in the T1 progeny were confirmed by PCR amplification targeting the hygromycin resistance gene. Figure 4 f).

[0055] Example 3. Evaluation passed GTrd5Flo5 and GTrd9Flo5 Two-line hybridization system of heteromorphic hybridization with golden rice variety To test the use GTrd5Flo5 and GTrd9Flo5 As a two-line hybridization system for maintaining the system, a golden rice variety that can be easily distinguished by seed color during cross-pollination is used as the pollen donor system. This allows for the determination of the production ratio of self-pollinated seeds to hybrid seeds during hybridization.

[0056] Without including certain male infertility genes, Flo5 Self-pollination accounted for 74.2% of all seeds (opaque seeds), while hybridization with the donor system accounted for 20.3% (golden seeds), which is about three times lower than the production rate of hybrid seeds. Figure 5 (Parts (a)-(c)). However, when using mutations that simultaneously carry part of the male sterility gene and the opacity marker gene... GTrd5Flo5 When used as the maternal line, the self-pollination rate decreased to 4.7% of all seeds (opaque seeds), while the cross-pollination rate with the donor system increased to 62.6%. Figure 5 (d) part - (f) part). In GTrd9Flo5 In this case, self-pollination accounts for 10.3% of all seeds (opaque seeds), while cross-pollination with the donor system increases to 64.3% of all seeds (golden seeds), resulting in a greater number of hybrid seeds formed through cross-pollination. Figure 5 (h)-(j)). This system enables the propagation of partially male-sterile seeds through self-pollination or cross-pollination, and when the partially male-sterile system is hybridized with a donor system, hybrid seed production can be increased, thereby easily distinguishing between preserved seeds and hybrid seeds based on the endosperm phenotype.

[0057] Example 4. Evaluation and Utilization GTrd5Flo5 Two-line hybridization system with Dular rice for hybrid rice breeding To evaluate the effectiveness of two-line hybrid systems in hybrid rice breeding, using japonica rice lines... GTrd5Flo5 The system used a maternal line and a Dular rice cultivar (Wang et al., 1998), a widely compatible indica rice line, as the paternal line for hybridization. F1 seeds generated through cross-pollination were separated into normal and opaque seeds, and then bred separately to confirm the retention of individual and hybrid characteristics. Individuals classified as opaque seeds are shown in the diagram. GTrd5Flo5 With the same genotype and partially male-sterile phenotype, individuals classified as having normal seeds, conversely, exhibited heterosis. Compared to the maternal line, F1 hybrids ( GTrd5Flo5 ×Dular) plant height increase ( Figure 6 (a) of the mature ears and seeds are increased in size. Figure 6 (parts (b) - (c)). Furthermore, the number at harvest was significantly higher than that of the maternal line, indicating significant heterosis. Figure 6 (part (d)). Such results show that a two-line hybrid system developed by combining partial male sterility genes with endosperm marker genes can significantly improve hybrid rice breeding systems.

[0058] sequence list An electronic document containing the sequence list is included.

Claims

1. A genetically modified rice, characterized in that, Simultaneously inhibit the gene composed of the base sequence represented by SEQ ID NO:

1. GTrd5 ( Os11g20384 (or a gene consisting of the base sequence represented by SEQ ID NO: 2) GTrd9 ( Os03g51620 ) and the gene consisting of the base sequence represented by SEQ ID NO:

3. Flo5 ( Os02g02840 The expression of ).

2. A method for preparing transgenic rice, characterized in that, The steps include: simultaneously inhibiting the gene composed of the base sequence represented by SEQ ID NO:

1. GTrd5 ( Os11g20384 (or a gene consisting of the base sequence represented by SEQ ID NO: 2) GTrd9 ( Os03g51620 ) and the gene consisting of the base sequence represented by SEQ ID NO:

3. Flo5 ( Os02g02840 The expression of ).

3. The method for preparing transgenic rice according to claim 2, characterized in that, The steps are achieved through gene editing using CRISPR-Cas9.

4. A method for producing first-generation hybrid seeds, characterized in that, The process includes the following steps: hybridizing the transgenic rice described in claim 1 with wild-type rice.

5. The method for producing first-generation hybrid seeds according to claim 4, characterized in that, The production method produces homozygous mutant seeds targeting the target gene, i.e., maintainer line seeds, and bialele mutant seeds, i.e., F1 hybrid seeds.

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