Method for inducing early flowering of gramineous plants

Abstract

To provide a technique for inducing early flowering of a gramineous plant.SOLUTION: The method for inducing early flowering comprises culturing a gramineous plant (e.g. rice variety'Kyo no Yume') having a functionally deficient mutation of a gene encoding gibberellin biosynthetic enzyme protein GA3ox2 under gibberellin treatment and a method for inducing early flowering of the gramineous plant comprising gibberellin is provided.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to a method for inducing early flowering in grass plants. [Background technology]

[0002] Gibberellins (GAs) are known as plant hormones that control plant growth and elongation. Gibberellins are a collective term for a very large group of compounds classified as diterpenes, with the basic structure of ent-gibberellane, and over 130 compounds have been identified. Gibberellins are widely used in agriculture to promote fruit growth and seed germination.

[0003] The GA biosynthesis pathway in plants has been elucidated. In the latter half of the GA biosynthesis pathway, GA20-oxidase (GA20ox) produces GA9 and GA 20 In the final step of GA biosynthesis, GA3-oxidase (GA3ox) converts GA9 to GA4 and GA 20 It is known that GA3ox converts to GA1. GA3ox1, GA3ox2, etc. are known as GA3ox.

[0004] The gene responsible for semi-dwarf rice varieties, which played a key role in the "Green Revolution" that dramatically increased grain production in the mid- to late 20th century, is now known to be SD1 (also known as OsGA20ox2), a GA biosynthetic enzyme gene (Non-Patent Document 1). Furthermore, the D18 (also known as OsGA3ox2) gene has been identified as one of the causative genes for dwarf rice mutants (Non-Patent Document 2). There are many other causative genes for GA-related dwarf mutants, and causative genes for not only GA-sensitive dwarf mutants but also GA-insensitive dwarf mutants have been identified (Non-Patent Document 3). Thus, even among GA-related dwarf mutants, the characteristics and causative genes for dwarfness are diverse.

[0005] The rice variety "Kyoto no Yume" is an extremely dwarf rice with a plant height of about 20 cm. Advantages of "Kyoto no Yume" include the fact that its extremely dwarf nature allows for multi-tiered cultivation, it is easy to cultivate in both hydroponics and soil, and the cultivation period required until harvest is relatively short. However, if the cultivation period until harvest of "Kyoto no Yume" could be further shortened, it would be possible to further improve production efficiency and increase annual yields. One possible way to shorten the cultivation period until harvest is to, for example, induce flowering earlier. However, no technology has been established to induce early flowering in rice. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Sasaki et al., Nature (2002) 416:701-702. [Non-patent document 2] Itoh et al., Proc Natl Acad Sci USA., (2001) 98(15): 8909-8914. [Non-patent document 3] Ueguchi-Tanaka M., et al., (2005) Nature 437, 693-698. Summary of the Invention [Problem to be solved by the invention]

[0007] An objective of the present invention is to provide a technique for inducing early flowering in grasses. [Means for solving the problem]

[0008] As a result of extensive research to solve the above problems, the inventors discovered that "Kyoto no Yume" has a functional deficiency mutation in the D18 gene, which encodes the gibberellin biosynthetic enzyme protein GA3ox2, and that early flowering can be induced by treating "Kyoto no Yume" with such a functional deficiency of the D18 gene with gibberellin, thereby completing the present invention.

[0009] That is, the present invention includes the following. [1] A method for inducing early flowering, comprising cultivating a grass plant having a loss-of-function mutation in the gene encoding the gibberellin biosynthetic enzyme protein GA3ox2 under gibberellin treatment. [2] The method according to [1] above, wherein the grass plant is rice. [3] The method according to [2] above, wherein the grass plant is the rice cultivar "Kyoto no Yume." [4] The method according to any one of the above [1] to [3], wherein the gibberellin is gibberellin A3. [5] The method according to any one of [1] to [4] above, wherein the cultivation under gibberellin treatment is carried out so that the roots of the grass family plant come into contact with gibberellin. [6] The method according to [5] above, wherein the cultivation of the grass family plant so that the roots of the grass family come into contact with gibberellin is carried out by cultivating the grass family plant in soil containing gibberellin. [7] An early flowering inducer in grasses that have a loss-of-function mutation in the gene encoding the gibberellin biosynthetic enzyme protein GA3ox2, which contains gibberellins. [8] The early flowering inducer according to [7] above, wherein the grass plant is rice. [9] The early flowering inducer according to [8] above, wherein the grass plant is the rice cultivar "Kyoto no Yume."

[10] The early flowering inducer according to any one of [7] to [9] above, wherein the gibberellin is gibberellin A3. [Effects of the Invention]

[0010] According to the present invention, early flowering of grasses can be effectively induced. [Brief explanation of the drawings]

[0011] [Figure 1] Figure 1 shows the coding sequence of the D18 gene of "Kyoto no Yume" and the mutations in the encoded deduced amino acid sequence. Arrows indicate the positions of sequence differences compared to the "Nipponbare" reference sequence of the D18 gene. [Figure 2] Figure 2 is a graph showing the change in plant height over time in "Kyoto no Yume" plants treated with GA3. Error bars indicate standard deviation. ** indicates a statistically significant difference (p<0.05) compared to the untreated GA3 plot (Dunnett test, n=10). [Figure 3] Figure 3 is a graph showing the effect of GA3 treatment on flowering. The vertical axis shows the number of days from sowing to flowering. Error bars indicate standard deviation, and asterisks indicate statistically significant differences compared to the GA3-untreated group (Dunnett's test, p<0.05, n=9 or 10). GA3 treatment was found to induce early flowering. [Figure 4] Figure 4 shows the yield status under different GA3 treatment conditions. The letters above each bar indicate statistically significant differences. A: Number of ears (number of ears per plant; Steel-Dwass test, p<0.05, n=10). B: Ear length (cm) (Tukey test, p<0.05, n=10). C: Fertility rate (%) (Tukey test, p<0.05, n=10). [Figure 5] Figure 5 is a graph showing the yield status under treatment conditions with different GA3 concentrations. The letters above each bar indicate statistically significant differences. A: Number of ripe seeds (seeds / plant) (Tukey's test, p<0.05, n=10). B: Seed yield (g / plant) (Tukey's test, p<0.05, n=10). C: Seed weight (mg / seed) (Tukey's test, p<0.05, n=10). DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in detail below. The present invention relates to a technique for inducing early flowering in a grass plant having a mutation in a gibberellin biosynthetic enzyme gene by treating the plant with gibberellin.

[0013] More specifically, the present invention relates to a method for inducing early flowering, which comprises cultivating a grass plant having a loss-of-function mutation in a gene encoding the gibberellin biosynthetic enzyme protein GA3ox2 under gibberellin treatment.

[0014] In the present invention, the grass plant may be any plant belonging to the family Poaceae, preferably a plant (rice) belonging to the genus Oryza, more preferably Oryza sativa L. or Oryza glaberrima.

[0015] The grass family plants that are the subject of the present invention are those that have a loss-of-function mutation in the gene encoding the gibberellin biosynthetic enzyme protein GA3ox2. Gramineae plants that have a loss-of-function mutation in the endogenous gene encoding the gibberellin biosynthetic enzyme protein GA3ox2 are typically dwarf plants.

[0016] The gibberellin biosynthetic enzyme protein GA3ox2 is a type of GA3-oxidase (GA3ox). The gene encoding the gibberellin biosynthetic enzyme protein GA3ox2 (also referred to as the GA3ox2 gene) can be the D18 gene. The gene encoding the gibberellin biosynthetic enzyme protein GA3ox2 is typically, but not limited to, an endogenous gene. The gene encoding the gibberellin biosynthetic enzyme protein GA3ox2 is typically a genomic gene.

[0017] An example of a gene encoding the gibberellin biosynthetic enzyme protein GA3ox2 is the D18 gene of the rice cultivar "Nipponbare." The D18 gene of the rice cultivar "Nipponbare" is a DNA encoding the amino acid sequence shown in SEQ ID NO: 2, and typically has the nucleotide sequence shown in SEQ ID NO: 1 as its coding sequence. In the present invention, the term "coding sequence" refers to a nucleotide sequence (usually including an initiation codon and a termination codon) that is translated into a protein, and is sometimes referred to as CDS. In another example, a gene encoding the gibberellin biosynthetic enzyme protein GA3ox2 is the following: (i) a gene that is a DNA comprising an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2, and that contains a base sequence that encodes a protein having oxidase activity; (ii) a gene that is a DNA comprising an amino acid sequence having an insertion, deletion, substitution, and / or addition of 1 to 10 (e.g., 1 to 2, 1 to 3, 1 to 5, or 1 to 7) amino acids in the amino acid sequence shown in SEQ ID NO: 2, and that contains a base sequence that encodes a protein having oxidase activity; (iii) a gene that is a DNA containing a base sequence that has 70% or more, preferably 80% or more, and more preferably 90% or more sequence identity to the base sequence shown in SEQ ID NO: 1 and encodes a protein having oxidase activity; and (iv) a gene that is a DNA containing a base sequence that has an insertion, deletion, substitution, and / or addition of 1 to 10 (e.g., 1 to 2, 1 to 3, 1 to 5, or 1 to 7) bases in the base sequence shown in SEQ ID NO: 1 and encodes a protein having oxidase activity; The gene may be selected from the group consisting of:

[0018] A loss-of-function mutation in a gene encoding the gibberellin biosynthetic enzyme protein GA3ox2 refers to a mutation in the nucleotide sequence of the gene that does not encode or is unable to express a functional (i.e., enzymatically active) gibberellin biosynthetic enzyme protein GA3ox2. A loss-of-function mutation in a gene encoding the gibberellin biosynthetic enzyme protein GA3ox2 may be due to, for example, a frameshift and / or the generation of a premature stop codon. In one embodiment, a loss-of-function mutation in a gene encoding the gibberellin biosynthetic enzyme protein GA3ox2 may be a mutation in the gene encoding the gibberellin biosynthetic enzyme protein GA3ox2 to a gene that is DNA containing the nucleotide sequence set forth in SEQ ID NO: 3, a gene that is DNA encoding the amino acid sequence set forth in SEQ ID NO: 6, or a gene that is DNA having the nucleotide sequence set forth in SEQ ID NO: 5 as its coding sequence. The present invention also provides a gene which is DNA comprising the nucleotide sequence set forth in SEQ ID NO: 3, a gene which is DNA encoding the amino acid sequence set forth in SEQ ID NO: 6, and a gene which is DNA having the nucleotide sequence set forth in SEQ ID NO: 5 as a coding sequence (or comprising the nucleotide sequence set forth in SEQ ID NO: 5), as well as the use of each of these genes in the production of a grass plant having a loss-of-function mutation in the gene encoding the gibberellin biosynthetic enzyme protein GA3ox2.

[0019] An example of rice having a mutation in the gene encoding the gibberellin biosynthetic enzyme protein GA3ox2, which is a gene that is DNA containing the nucleotide sequence set forth in SEQ ID NO: 3, a gene that is DNA encoding the amino acid sequence set forth in SEQ ID NO: 6, or a gene that is DNA having the nucleotide sequence set forth in SEQ ID NO: 5 as its coding sequence, is the rice cultivar "Kyoto no Yume." "Kyoto no Yume" is a preferred example of a grass plant having a loss-of-function mutation in the gene encoding the gibberellin biosynthetic enzyme protein GA3ox2.

[0020] Another example of a grass family plant having a loss-of-function mutation in the gene encoding the gibberellin biosynthetic enzyme protein GA3ox2 is a grass family plant (e.g., rice) into which the above-mentioned loss-of-function mutation, for example, a loss-of-function mutation derived from "Kyo no Yume," has been introduced into the gene encoding the gibberellin biosynthetic enzyme protein GA3ox2. Introduction of a loss-of-function mutation into the gene encoding the gibberellin biosynthetic enzyme protein GA3ox2 may be performed by techniques such as crossbreeding, mutagenesis, homologous recombination, or genome editing. A grass family plant having a loss-of-function mutation in the gene encoding the gibberellin biosynthetic enzyme protein GA3ox2 may be a hybrid (F1 species) derived from "Kyo no Yume" into which a loss-of-function mutation in the gene encoding the gibberellin biosynthetic enzyme protein GA3ox2 has been introduced, or a progeny thereof, or may be a mutant of "Kyo no Yume."

[0021] Gramineae plants having a loss-of-function mutation in the gene encoding the gibberellin biosynthetic enzyme protein GA3ox2 are sensitive to gibberellins. In the present invention, by treating a grass plant having a loss-of-function mutation in the gene encoding the gibberellin biosynthetic enzyme protein GA3ox2 with gibberellin, plant height can be increased and early flowering can be induced.

[0022] The gibberellin used in the gibberellin treatment is usually active gibberellin. Examples of active gibberellins include, but are not limited to, gibberellin A1 (GA1), gibberellin A3 (GA3), gibberellin A4 (GA4), and gibberellin A7 (GA7). The gibberellin used in the gibberellin treatment is more preferably GA3. GA3 is also called gibberellic acid.

[0023] In the present invention, a grass plant having a loss-of-function mutation in a gene encoding the gibberellin biosynthetic enzyme protein GA3ox2 is preferably cultivated under gibberellin treatment. The gibberellin treatment of a grass plant can be carried out by any method. For example, the gibberellin treatment can be carried out using gibberellin (gibberellin solution) at a concentration of 0.01 μM or more, preferably at a concentration of 0.1 μM to 100 μM, more preferably at a concentration of 0.2 μM to 50 μM, for example, 0.2 μM to 10 μM, or 1 μM to 5 μM.

[0024] In one embodiment, cultivation under gibberellin treatment is preferably carried out at least during a period including the time of transplanting seedlings of the grass family plant. In one embodiment, cultivation under gibberellin treatment may be carried out during a period including any time point between sowing and transplanting seedlings. In one embodiment, cultivation under gibberellin treatment may be carried out not only during a period including the time of transplanting seedlings, but also at a period including any time point from 2 weeks after transplanting seedlings, for example, between 2 weeks and 10 weeks, or between 3 weeks and 5 weeks after transplanting seedlings.

[0025] In one embodiment, cultivation under gibberellin treatment can be carried out so that the roots of the grass family plant come into contact with gibberellin. Cultivation so that the roots of the grass family plant come into contact with gibberellin can be carried out, for example, by cultivating the grass family plant in soil containing gibberellin (soil cultivation). Alternatively, cultivation so that the roots of the grass family plant come into contact with gibberellin can be carried out, for example, by hydroponically cultivating the grass family plant using a culture solution containing gibberellin. Cultivation so that the roots of the grass family plant come into contact with gibberellin allows the gibberellin to be absorbed by the roots of the grass family plant, thereby effectively exerting the functions of gibberellin, without having to spray gibberellin on the entire plant body or the above-ground parts of the grass family plant.

[0026] In the present invention, early flowering is induced in a grass plant having a loss-of-function mutation in the gene encoding the gibberellin biosynthetic enzyme protein GA3ox2 by cultivating the plant with gibberellin treatment. In the present invention, "early flowering" refers to an earlier flowering time in a gibberellin-treated grass plant compared to a control grass plant not treated with gibberellin. In a preferred embodiment, the "induction of early flowering" of the present invention results in an earlier flowering date in a gibberellin-treated grass plant than in a control grass plant not treated with gibberellin by at least 3 days, preferably at least 5 days, and more preferably at least 7 days, compared to a control grass plant not treated with gibberellin. In the present invention, the flowering date for each individual plant is defined as the date when the first panicle emerges and all spikelets on that panicle flower. Furthermore, whether or not "early flowering" is induced can be determined based on the average flowering date among individuals.

[0027] Gibberellin treatment of grasses carrying loss-of-function mutations in the gene encoding the gibberellin biosynthetic enzyme protein GA3ox2 has little adverse effect on seed quality or yield, making it suitable for use in cultivation for seed and grain production.

[0028] Furthermore, the present invention provides an early-flowering inducer for grasses that have a loss-of-function mutation in an endogenous gene encoding the gibberellin biosynthetic enzyme protein GA3ox2, which contains gibberellins. The gibberellins used in this early-flowering inducer and the grasses that have a loss-of-function mutation in an endogenous gene encoding the gibberellin biosynthetic enzyme protein GA3ox2, to which the early-flowering inducer is applied, are as described above.

[0029] The early-flowering inducer of the present invention may be a composition containing the above-mentioned gibberellin, for example, a liquid composition containing the above-mentioned gibberellin. The early-flowering inducer of the present invention may contain, in addition to gibberellin, additives acceptable for agricultural chemicals, such as an inert carrier (a solid carrier, a liquid carrier, etc.; for example, water), an excipient, a surfactant, a solubilizing agent, a suspending agent, a colorant, a flavoring agent, a preservative, a buffer, a pH adjuster, etc. The early-flowering inducer of the present invention preferably contains the above-mentioned gibberellin as an active ingredient, and preferably contains the above-mentioned gibberellin in an effective amount. The early-flowering inducer of the present invention may further contain other plant physiologically active ingredients.

[0030] The early flowering inducer according to the present invention can be suitably used, for example, to carry out gibberellin treatment in the above-mentioned early flowering induction method. [Example]

[0031] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0032] [Example 1] Analysis of the genomic DNA of "Kyoto no Yume" 1) Genomic DNA preparation A total of four green leaves (82 mg fresh weight) were collected from two soil-grown rice cultivars of "Kyo no Yume" (Oryza sativa L. cv Kyonoyume). These were frozen in liquid nitrogen, crushed with a mortar and pestle, and then analyzed using DNeasy. (R) Genomic DNA was purified using the Plant Mini kit (Qiagen). The resulting genomic DNA solution was quantified by absorbance measurement and subjected to agarose gel electrophoresis to confirm the quantity and quality of the DNA.

[0033] 2) Next-generation sequencing, mapping, and mutation extraction analysis Next-generation sequencing analysis and subsequent bioinformatics analysis were performed using 3.3 μg of the genomic DNA prepared above from "Kyoto no Yume."

[0034] First, the quality of the genomic DNA was confirmed by electrophoresis and fluorimetric quantification, and then a genomic shotgun library (insert size: 350 bp) was prepared. Paired-end short-read sequencing was performed using a NovaSeq6000 sequencer S4 flow cell with two 150 bp modules. The analysis specifications were a read length of 150 bp and a total data volume of 20 Gbase / total.

[0035] The resulting read data were cleaned using the program Trimmomatic (ver. 039) (Anthony et al. (2014) Bioinformatics, 30: 2114-2120. https: / / doi.org / 10.1093 / bioinformatics / btu170) to remove adapter sequences and low-quality reads. The cleaned reads were mapped to the reference genome sequence of the rice cultivar "Nipponbare" (Oryza sativa L. cv Nipponbare) using the mapping program BWA (ver. 0.7.17) (https: / / ftp.ncbi.nlm.nih.gov / genomes / all / GCF / 001 / 433 / 935 / GCF_001433935.1_IRGSP-1.0 / GCF_001433935.1_IRGSP-1.0_genomic.fna.gz). Next, duplicate reads were removed using Picard tools (ver. 1.111) (https: / / github.com / broadinstitute / picard). From the mapping results, bases that differed from the reference sequence were extracted using VCFtools (ver. 1.12) (Danecek et al., (2011) Bioinformatics, 27: 2156-2158. doi:10.1093 / bioinformatics / btr330), and a vcf file was generated.

[0036] 3) Identification of the mutation site in the D18 gene From the mutations identified in the vcf file obtained by the analysis in 2) above, mutations present within the gene region of the D18 gene (RAP-DB gene ID: Os01g0177400), which encodes GA3ox2, a gibberellin biosynthetic enzyme, were extracted. The coding sequence (nucleotide sequence) and corresponding amino acid sequence of the D18 gene of "Nipponbare" obtained from RAP-DB are shown in SEQ ID NOs: 1 and 2, respectively. The nucleotide sequence of the D18 gene of "Kyoto no Yume," which corresponds to the coding sequence of the D18 gene of "Nipponbare," is shown in SEQ ID NO: 3.

[0037] Analysis of the "Kyo-no-Yume" genomic DNA revealed two mutations in the D18 gene region compared with the reference sequence ("Nipponbare"). Both mutations were located in the coding sequence; one was an insertion deletion (InDel) and the other was a single nucleotide polymorphism (SNP) of G (guanine) to A (adenine) (Figure 1). Compared with the "Nipponbare" D18 gene sequence, the "Kyo-no-Yume" D18 gene sequence contained a mutation of 18 bp (CGACGCGGCGGCGCGGGC; SEQ ID NO: 4) corresponding to positions 165-182 of the "Nipponbare" reference sequence (SEQ ID NO: 1) to a 1 bp base (T) (a 17 bp deletion). This resulted in a frameshift, resulting in a mutation of amino acids 56 and beyond in the encoded GA3ox2 protein sequence, and a change of the 150th codon to a stop codon (Figure 1). This frameshift and the resulting generation of a stop codon changed the GA3ox2 protein from 370 amino acids in Nipponbare to 149 amino acids in Kyo no Yume, and the amino acid sequence from position 56 onwards became completely different from that of the Nipponbare GA3ox2 protein. Therefore, it is believed that the D18 gene in Kyo no Yume does not encode a functional GA3ox2 protein.

[0038] The results of this analysis showed that the D18 gene of "Kyoto Yume" has a loss-of-function mutation, which is a loss-of-function allele. "Kyoto Yume" can be said to lack the ability to synthesize GA3ox2 protein.

[0039] The coding sequence from the initiation codon to the termination codon of the D18 gene of "Kyoto Dream" (corresponding to positions 1 to 450 of SEQ ID NO: 3) and the corresponding deduced amino acid sequence are shown in SEQ ID NOs: 5 and 6, respectively.

[0040] [Example 2] Long-term cultivation test of "Kyoto no Yume" under GA treatment In Example 1, it was shown that "Kyoto Yume" has a functional loss mutation in the GA3ox2 gene and is unable to synthesize functional GA3ox2 protein. Based on this, the effect of gibberellin A3 (GA3) treatment on the cultivation of "Kyoto Yume" was investigated.

[0041] Fully ripened seeds of "Kyoto no Yume" were surface sterilized without removing the glume and sown on moist filter paper. An appropriate amount of 70% (v / v) ethanol was added to the fully ripened seeds and allowed to stand for 3 minutes. The ethanol was removed, and an appropriate amount of sodium hypochlorite solution (1% Cl) was added and stirred for 20 minutes to sterilize the fully ripened seeds. The sodium hypochlorite solution was then discarded, and the fully ripened seeds were washed five times with distilled water. The fully ripened seeds were sown in a petri dish (90 mm diameter) lined with 85 mm diameter filter paper, 5 mL of distilled water was added, and the seeds were incubated in a culture room at 28°C under a 16-hour photoperiod (light intensity 70 μmol / m 2 / s) and a dark period of 8 hours.

[0042] Eight days after sowing, seedlings were transplanted into a 1:1 mixture of granular rice seedling soil (Kumiai Granular Soil WD; National Federation of Agricultural Cooperative Associations) and vermiculite (Nittai). First, 6.3 kg of soil was placed in a 64 cm wide x 22 cm deep x 16 cm high (outer diameter) planter, and 4 L of GA3 solution (GA3 concentrations: 0 μM, 0.2 μM, or 1 μM) was added. Ten seedlings were transplanted per planter, with an average spacing of 10.6 cm (the day of transplanting = day 0 after transplanting). From this point onward, "Kyoto no Yume" varieties were grown in a glasshouse at 28°C under natural light. They were watered with tap water every 2–3 days, and on the 28th day after transplanting, 1 L of GA3 solution at the same concentration as at the time of transplanting was added per planter. No fertilization was performed until heading; the plants were grown on the fertilizer contained in the granular soil. At the heading stage, granular fertilizer NK-C6 (Central Chemical, NK Chemical C6) was applied as top dressing at 20 grains per planter (20 grains / 1300cm). 2 The fertilizer composition of the granular soil and granular fertilizer NK-C6 used was as follows: Kumiai Granular Fertilizer WD (per 3.0 kg): Nitrogen 0.6 g, Phosphorus 1.2 g, Potassium 1.0 g NK-C6: N 17%, P 0%, K 17%

[0043] The test groups treated with 0.2 μM or 1 μM GA3 solution on the day of transplantation and 28 days after transplantation are referred to as the 0.2 μM GA3 treatment group and the 1 μM GA3 treatment group, respectively, and the test group not treated with GA3 solution on the day of transplantation and 28 days after transplantation (GA3 concentration 0 μM) is referred to as the GA3 non-treatment group.

[0044] After transplanting, the plant height of each "Kyoto no Yume" plant was measured every two weeks for 70 days. The flowering date was also recorded at the heading stage. The flowering date for each plant was the day when the first panicle emerged and all of its spikelets flowered.

[0045] Watering was stopped approximately 5 weeks after the first flowering was observed during the cultivation test, and the seeds were then dried for 2 weeks before harvesting. At harvest, the number of panicles, panicle length, number of fertile seeds and sterile seeds per panicle, and weight of fertile seeds per kernel were measured, and the fertility rate and seed yield were calculated.

[0046] As shown in Figure 2, 14 days after transplanting, the plant height in the untreated GA3 plot was 8.1 cm, while in the 0.2 μM GA3 treatment plot it was 34.7 cm, and in the 1 μM GA3 treatment plot it was 46.4 cm, demonstrating a significant increase in plant height in the GA3 treatment plot. Furthermore, GA3 treatment consistently demonstrated a statistically significant increase in plant height up to 70 days after transplanting compared with the untreated GA3 plot. These results indicate that "Kyo no Yume" has a long-term response to GA3 treatment. Thus, it was demonstrated that treatment of "Kyo no Yume" with GA3 promotes aboveground shoot elongation.

[0047] Furthermore, when we investigated whether GA3 treatment had any effect on flowering, we found that the average number of days from sowing to flowering in the non-GA3 treated area was 67.8 days, whereas in the 0.2 μM GA3 treated area it was 60.7 days, and in the 1 μM GA3 treated area it was 59.5 days, which was significantly shorter (Figure 3). These results indicate that GA3 treatment accelerates (promotes) the flowering of "Kyoto no Yume."

[0048] At harvest, the average number of ears per plant was statistically significantly lower in the 1 μM GA3 treatment group than in the GA3-untreated and 0.2 μM GA3-treated groups (Fig. 4A). Furthermore, the average ear length (average of 10 plants) increased with GA3 treatment in a concentration-dependent manner (Fig. 4B). The fertility rate decreased in the 0.2 μM GA3 treatment group, but did not show any concentration-dependent changes (Fig. 4C). On the other hand, the number of fertile seeds per plant, seed yield, or fertile seed weight per kernel were not affected by GA3 treatment (Figs. 5A–C). These results suggest that GA treatment does not adversely affect the quality or yield of "Kyoto no Yume" rice.

Claims

1. A method for inducing early flowering comprises cultivating a grass plant having a loss-of-function mutation in a gene encoding the gibberellin biosynthetic enzyme protein GA3ox2 under gibberellin treatment.

2. The method according to claim 1 , wherein the grass plant is rice.

3. The method according to claim 2, wherein the grass plant is the rice cultivar "Kyoto no Yume."

4. Gibberellin is gibberellin A ３ The method according to any one of claims 1 to 3, wherein

5. 4. The method according to claim 1, wherein the cultivation under gibberellin treatment is carried out so that the roots of the grass family plant come into contact with the gibberellin.

6. 6. The method according to claim 5, wherein the cultivation of the grass plant so that the roots of the grass plant come into contact with gibberellin is carried out by cultivating the grass plant in soil containing gibberellin.

7. An agent for inducing early flowering in a grass plant having a loss-of-function mutation in a gene encoding the gibberellin biosynthetic enzyme protein GA3ox2, which contains gibberellin.

8. The early flowering inducer according to claim 7 , wherein the grass plant is rice.

9. The early flowering inducer according to claim 8, wherein the grass plant is the rice cultivar "Kyoto no Yume."

10. Gibberellin is gibberellin A ３ The early flowering inducer according to any one of claims 7 to 9,

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