Methods for increasing shoot and seed yields of long-day grasses

Abstract

To provide a method for increasing the yield of foliage and seeds of gramineous long-day plants.SOLUTION: The present invention discloses a method for increasing the yield of foliage and seeds of gramineous long-day plants by inhibiting the functions of PRR3 / 7 clade gene.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for increasing the shoot and seed yield of long-day grass plants. [Background technology]

[0002] In grasses, heading characteristics are one of the characteristics that are deeply related to environmental adaptability and growth stability. To achieve high yields, growth and development patterns must be adapted to seasonal changes during the cultivation period. Acceleration or delay of heading time is related to environmental factors such as temperature, but genetic factors such as low-temperature requirement, photoperiod responsiveness, and pure earliness are also known to be involved. For example, in barley, a long-day grass, the photoperiod-responsive gene Ppd-H1, a flowering control gene, is involved in the promotion of heading. It has been shown that knocking down Ppd-H1 in barley delays heading. In contrast, knocking out Ppd-H1 orthologues in rice and sorghum, short-day grasses, has been shown to accelerate heading (Non-Patent Documents 2 and 3).

[0003] Furthermore, it is known that natural mutations in wheat cultivars result in deletion of the Ppd-D1 promoter region, resulting in changes in the circadian rhythm of Ppd-D1 expression (Non-Patent Document 4). It has been shown that the heading date of these cultivars is earlier. Furthermore, GIGANTEA (GI) and FLOWERING LOCUS T (FT) are also known as flowering control genes. It has been shown that rice GI overexpression lines have a later heading date, while rice FT overexpression lines or an indica-type FT allele (Hd3a) have an earlier heading date (Non-Patent Document 6).

[0004] Increasing the yield of grasses and other plants is extremely important from the perspective of food security, and there is a need to identify genes involved in increased yield and to develop efficient production using these identified genes. [Prior art documents] [Patent documents]

[0005] [Non-Patent Document 1] Science 2005, 310, 1031-1034. [Non-patent document 2] Molecular Plant 2013, 6, 1877-1888. [Non-patent document 3] Proc. Natl. Acad. Sci. 2011, 108, 16469-16474. [Non-patent document 4] Therr. Appl. Genet. 2007, 115, 721-733. [Non-Patent Document 5] Nature 2003, 422, 719-722. [Non-patent document 6] Plant Cell Physiol. 2002, 43, 1096-1105. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a method for increasing the shoot and seed yield of a long-day grass plant. [Means for solving the problem]

[0007] In order to solve the above problems, the inventors conducted extensive research and found that suppressing the function of the PRR3 / 7 clade gene, which is a type of flowering control gene in long-day grasses, not only delays heading but also increases stem and leaf yield and seed yield.

[0008] The present invention has been completed based on these findings and includes the following broad aspects. [Section 1] A method for increasing shoot and seed yield in long-day grasses by suppressing the function of PRR3 / 7 clade genes. [Section 2] Item 2. The method according to Item 1, wherein the PRR3 / 7 clade gene is the PRR37 gene. [Section 3] Item 3. The method according to Item 1 or 2, wherein the function is suppressed by genome editing. [Section 4] Item 4. The method according to Item 3, wherein the genome editing is performed by direct introduction of a genome editing enzyme. [Section 5] Item 5. The method according to any one of Items 1 to 4, wherein the long-day plant of the Poaceae family is Triticeae. [Section 6] Item 6. The method according to any one of Items 1 to 5, wherein the long-day plant of the Poaceae family is a member of the genus Hordeum, tribe Triticum. [Effects of the Invention]

[0009] According to the present invention, the shoot and leaf yield and seed yield of long-day grass plants can be increased. [Brief explanation of the drawings]

[0010] [Figure 1] Ppd-H1 target sequence in a study using the barley cultivar "Nihonohoshi." The CRISPR / Cas9 target sequence was designed in the third exon of the flowering control gene Ppd-H1. The bold text indicates the PAM sequence, and the underlined text indicates the PAM sequence and target sequence. [Figure 2] TO screening in a study using the barley cultivar "Nihonohoshi." A: Electrophoresis results. B: DNA sequencing results. The sequences corresponding to positions 397 to 429 of Ppd-H1 are shown. NH1: SEQ ID NOs: 10 and 11. NH2: SEQ ID NOs: 12, 11, and 13. [Figure 3] Genotype of T1 individuals in a study using the barley cultivar "Nihonohoshi." A: Electrophoresis results. B: DNA sequencing results. The sequence corresponding to positions 394 to 432 of Ppd-H1 is shown. SEQ ID NO: 14. [Figure 4]Number of days to heading and grain yield of Ppd-H1 mutant strains in a study using the barley cultivar "Nihonohoshi." A: Appearance of wild-type strains and Ppd-H1 mutant strains (NH2.2-4) 62 days after germination. Heading is observed in the wild-type strain, but not in the mutant strain. B: Number of days to heading. Shows the average number of days to heading ± SE, assuming the day of germination is 0. Wild-type strain n=8, mutant strain n=8. ** indicates a significant difference from the wild-type strain (p<0.01) in a T-test. C: Number of tillers. D: Number of seeds. E: Grain yield. [Figure 5] Green leaf biomass of the Ppd-H1 mutant in a study using the barley cultivar "Nihonohoshi." A: Fresh weight. B: Dry weight. [Figure 6] Schematic diagram of the Ppd-H1 target sequence and construct used in a study using the barley cultivar "Toyonokaze." A: A CRISPR / Cas9 target sequence was designed in the third exon of the flowering control gene Ppd-H1. The PAM sequence is in bold, and the PAM and target sequences are underlined. B: Schematic diagram of the construct used to produce genome-edited individuals. pU6-gRNA encodes the Ppd-H1 target sequence. pUbi-Cas9 encodes spCas9 (=OsCas9) with codons modified for rice. ZmUbi: maize ubiquitin, TaU6: wheat ubiquitin. [Figure 7] Screening of particle-bombarded contemporary generations in a study using the barley cultivar "Toyonokaze." A: Screening overview. Particle-bombarded shoot apices were grown on culture medium, then potted and subjected to CAPS analysis using the fifth leaf. B: Gene sequences of mutant lines. The sequence corresponding to positions 398 to 428 of Ppd-H1 is shown. Gene sequences near the target sequence in the mutant lines (lines #1, #2, and #3) produced. Line #1: Insertion T (SEQ ID NO: 15), Deletion G (SEQ ID NO: 16). Line #2: Insertion C (SEQ ID NO: 17). Line #3: Insertion A (SEQ ID NO: 18), Insertion C (SEQ ID NO: 17). [Figure 8]Genotype analysis of next-generation individuals derived from #3 in a study using the barley cultivar "Toyonokaze." Five next-generation individuals were obtained from one ear of line #3. DNA was extracted from each individual and CAPS analysis was performed, revealing only uncleaved fragments in all individuals. The sequence of this fragment was then observed, revealing "Insertion A (SEQ ID NO: 18), Insertion C (SEQ ID NO: 17)" in #3-1 to #3-4, and "Insertion A (SEQ ID NO: 18)" in #3-5. [Figure 9] Number of days to heading for the Ppd-H1 mutant (#3 line) in a study using the barley cultivar "Toyonokaze." A: Appearance of the wild-type and Ppd-H1 mutant lines (#3-1 to #3-5 in Fig. 5) 63 days after germination. Heading is observed in the wild-type line, but not in the mutant line. B: Number of days to heading. Shows the average number of days to heading ± SE, with the germination day set to 0. Wild-type line n=6, mutant line n=5 (#3-1 to #3-5 in Fig. 5). ** indicates a significant difference from the wild-type line (p<0.01) in a t-test. [Figure 10] The number of tillers in the Ppd-H1 mutant was measured using the barley cultivar "Toyonokaze." The mean number of tillers ± SE is shown. Wild-type n = 6, mutant n = 5 (#3-1 to #3-5 in Fig. 5). ** indicates a significant difference from the wild-type (p<0.01) in the t-test. DETAILED DESCRIPTION OF THE INVENTION

[0011] According to one embodiment of the present invention, there is provided a method for increasing shoot and seed yield in long-day grass plants by suppressing the function of a flowering control gene such as a PRR3 / 7 clade gene.

[0012] Examples of long-day grass plants used in the present invention include Triticum triticum, Aetotrichum triticum, and Brachypodium triticum. Examples of Triticum triticum include Triticum, Hordeum, Secale, and Brachypodium spp. Examples of Aetotrichum triticum include Avena sativa. Examples of Brachypodium triticum include Orchardgrass and Brachypodium spp. Examples of Triticum triticum include Triticum aestivum and Brachypodium spp. Examples of Brachypodium triticum include Triticum aestivum (bread wheat) and Triticum durum (durum). Examples of Barley include Hordeum vulgare (barley). Examples of Rye include Secale cereale (cereale). Examples of Brachypodium spp. include Brachypodium distachyon (branch grass). Examples of plants of the genus Oat include oats (Avena sativa), etc. Examples of plants of the genus Orchardgrass include orchardgrass (Dactylis glomerata), etc. Examples of plants of the genus Lolium include perennial ryegrass (Lolium perenne), etc. Among these, barley, common wheat, and durum wheat are preferred.

[0013] As used herein, a flowering regulatory gene refers to a gene involved in the process of flower bud formation, such as the pseudo-response regulator (PRR) family, GI, FT, VRN1, and PhyC genes. Among these, the PRR family is preferred as a gene that increases shoot and seed yield by suppressing gene function. Examples of the PRR family include TOC1, AtPRR3, AtPRR5, AtPRR7, AtPRR9, and their orthologs. Among the PRR family, AtPRR3, AtPRR7, and their orthologs are preferred. Orthologs of AtPRR3 and AtPRR7 in long-day grasses are referred to as PRR3 / 7 clade genes. An example of an ortholog of AtPRR3 in long-day grasses is PRR37, such as Ppd-H1 (HvPRR37) in barley, Ppd-D1 (TaPRR37) in wheat, and BdPRR37 in wheatgrass. An ortholog of AtPRR7 in long-day grasses is PRR73, such as HvPRR73 in barley, TaPRR73 in wheat, and BdPRR73 in wheatgrass. Examples of such genes include a gene containing a polynucleotide having the nucleotide sequence of SEQ ID NO: 1, or a gene containing a polynucleotide with 55% or more sequence identity to a polynucleotide having the nucleotide sequence of SEQ ID NO: 1. SEQ ID NO: 1 is the cDNA sequence of Ppd-H1 from the barley cultivar "Nishinohoshi." Among these, genes encoding proteins involved in panicle formation and promotion of heading under long-day conditions, and in which suppression of gene function results in delayed heading, are preferred. As used herein, "delayed heading" refers to a delay of heading by typically 7 days or more, preferably 10 days or more, and more preferably 14 days or more, compared to a long-day grass plant (wild strain) in which the gene function is not suppressed and grown under the same conditions.

[0014] As used herein, a "polynucleotide having 55% or more sequence identity" refers to a polynucleotide in which one or more bases have been substituted, deleted, added, or inserted relative to the base sequence of SEQ ID NO: 1, or a polynucleotide consisting of a base sequence that has a homology of 55% or more, more preferably 70% or more, even more preferably 85% or more, particularly preferably 90% or more, and most preferably 95% or more relative to the base sequence of SEQ ID NO: 1.

[0015] Furthermore, the specific sequence of the gene is not limited as long as it encodes a functionally equivalent mutant, derivative, variant, allele, homolog, ortholog, or partial peptide of the protein encoded by the gene. Here, "functionally equivalent" means that the target protein has physiological or biochemical functions equivalent to those of the protein encoded by the gene. Examples of such proteins include a protein consisting of the amino acid sequence of SEQ ID NO: 2, or a protein consisting of an amino acid sequence substantially identical to that of SEQ ID NO: 2. SEQ ID NO: 2 is the Ppd-H1 amino acid sequence of the barley cultivar "Nishinohoshi." As used herein, "a protein consisting of a substantially identical amino acid sequence" includes a protein in which one or more amino acids are substituted, deleted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 2, or a protein consisting of an amino acid sequence that is 45% or more, more preferably 70% or more, even more preferably 85% or more, particularly preferably 90% or more, and most preferably 95% or more identical to the amino acid sequence of SEQ ID NO: 2.

[0016] In a preferred embodiment of the present invention, suppressing the function of the gene in a long-day grass plant not only delays heading but also increases stem and leaf yield and seed yield. It has been previously known that knocking down Ppd-H1 in barley delays heading, but it has not been known that it increases stem and leaf yield and seed yield. In the present invention, "stem and leaf yield" refers to the harvest weight of plant leaves and stems. "Seed yield" refers to the harvest weight of plant seeds. The weight may be dry weight or wet weight. "Increasing stem and leaf yield and seed yield" means increasing stem and leaf yield and seed yield compared to a long-day grass plant (wild strain) in which the function of the gene is not suppressed, grown under the same conditions. The stem and leaf yield is preferably increased by at least twofold, more preferably by at least threefold, and particularly preferably by at least fivefold, compared to a long-day grass plant (wild strain) in which the function of the gene is not suppressed, grown under the same conditions. The seed yield is preferably increased by 1.1 times or more, more preferably 1.3 times or more, and particularly preferably 1.5 times or more, compared to a long-day grass plant (wild strain) in which the gene function is not suppressed and grown under the same conditions. Regarding growth conditions, the growth temperature can be in the range of 15°C to 30°C, preferably 20°C to 25°C. The light-dark cycle is preferably 15 to 20 hours of light and 6 to 10 hours of darkness. The growth period is not particularly limited, but if the objective is to increase stem and leaf yield, it is 40 days or more, preferably 60 days or more, and 80 days or less. If the objective is to increase seed yield, it is 90 days or more, preferably 100 days or more, and 120 days or less. Furthermore, heading is delayed by 7 days or more, preferably 10 days or more, and more preferably 14 days or more, compared to a long-day grass plant (wild strain) in which the gene function is not suppressed.

[0017] Methods for suppressing gene function are not limited and known methods can be used, including, for example, methods for introducing mutations into genes, double-stranded RNA methods, antisense methods, and ribozyme methods.

[0018] Methods for introducing mutations into genes include, but are not limited to, chemical mutagenesis, physical mutagenesis, methods for introducing transposons or the like into genomic DNA, and methods using proteins that act on DNA or RNA (genome editing enzymes).

[0019] An example of a chemical mutagenesis method is treating seeds with a chemical mutagen. There are no particular limitations on the chemical mutagen, but examples include ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU), N-methyl-N-nitrosourea (NNU), sodium azide, sodium bisulfite, hydroxylamine, N-methyl-N'-nitro-N-nitroguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), O-methylhydroxylamine, nitrous acid, formic acid, and nucleotide analogs.

[0020] Examples of physical mutagenesis methods include fast neutron irradiation, gamma ray irradiation, heavy ion beam (HIB) irradiation, and ultraviolet irradiation.

[0021] Methods for introducing transposons or the like into genomic DNA include, for example, methods for inserting transposons such as TOS17, T-DNA, or the like into genomic DNA.

[0022] Methods using proteins (genome editing enzymes) that act on DNA or RNA include, for example, Crispr / Cas9, PRR motifs, endonucleases, zinc finger nucleases (ZFNs), TALENs (transcription activator-like effector nucleases), transposases, and site-specific recombinases.

[0023] The mutation is preferably introduced into the gene so that all or part of the amino acid sequence of the CCT motif of the gene is lost or altered. Furthermore, from the plant into which the mutation has been introduced, only desired mutants can be selected as needed. The double-stranded RNA method uses double-stranded RNA consisting of an oligo-RNA complementary to the mRNA of the gene and its complementary strand. When double-stranded RNA is introduced into cells, the mRNA complementary to that RNA is degraded, a phenomenon known as RNA interference, which has the ability to suppress the function of the target gene.

[0024] There is no limit to the length of the double-stranded RNA, as long as it can suppress the function of the target gene and is non-toxic. The double-stranded RNA may be, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs, and more preferably 19 to 27 base pairs. While it is desirable that the double-stranded RNA and the target sequence are identical, they may be substantially identical, i.e., homologous, to the extent that the above-mentioned RNA interference can be induced. Specifically, as long as the antisense strand sequence of the double-stranded RNA hybridizes with the target sequence, one or several (e.g., two or three) mismatches are acceptable. In other words, double-stranded RNAs include those in which one or several bases have been substituted, added, or deleted from the target sequence and are capable of inducing RNA interference, or those that have 90% or more, preferably 95% or more, and more preferably 98% or more homology with the target sequence and are capable of inducing RNA interference.

[0025] The antisense method uses DNA (antisense DNA) that encodes antisense RNA complementary to the transcription product of the gene. Antisense DNA inhibits transcription, splicing, or translation, thereby suppressing the function of the target gene.

[0026] To effectively inhibit the function of a target gene, the length of the antisense DNA is at least 15 bases, preferably 100 bases or more, and more preferably 500 bases or more. It is desirable that the antisense DNA and the target sequence are identical, but they may be substantially identical, i.e., homologous, to the extent that they can effectively inhibit gene function. Antisense DNA includes those that have 90% or more, preferably 95% or more, and more preferably 98% or more homology with the target sequence and can effectively inhibit gene function.

[0027] The ribozyme method uses DNA encoding RNA with ribozyme activity that specifically cleaves the transcription product of the gene. Examples of ribozymes include group I introns, RNase P, hammerhead ribozymes, and hairpin ribozymes.

[0028] Alternatively, the above-mentioned DNA encoding a transposon, DNA encoding a protein that acts on DNA or RNA, DNA encoding a double-stranded RNA, DNA encoding an antisense RNA, DNA encoding an RNA with ribozyme activity, etc. may be inserted into a vector and introduced into a plant cell. There are no particular limitations on the vector into which the DNA for suppressing the function of the gene is inserted, as long as it is capable of expressing the inserted gene in the cell.

[0029] The DNA, the vector containing the inserted DNA, the protein, etc. can be introduced into cells by known methods, such as the in planta particle bombardment (iPB) method, the Agrobacterium method, the particle gun method, the polyethylene glycol method, the electroporation method, the liposome method, the calcium phosphate precipitation method, the lipofection method, and the microinjection method.

[0030] As described above, various methods for suppressing gene function are known, but the method of introducing a mutation into a gene by genome editing is preferably used as the method of suppressing gene function in the present invention. By using genome editing, the targeted gene can be disrupted with high efficiency. In the case of gene disruption by genome editing, since only the targeted gene can be disrupted without leaving any trace of genetic recombination, some countries do not treat it as a modified plant. In the present invention, the method of introducing a mutation into a gene by the in planta particle bombardment (iPB) method is preferred because it allows genome editing of plants to be performed with good reproducibility. The iPB (in planta particle bombardment) method will be described below.

[0031] The in planta particle bombardment (iPB) method includes the steps of imbibing water into plant seeds, exposing the shoot apex of the seed embryo, and introducing nucleic acids and / or proteins into cells of the shoot apex. Note that, as used herein, the term "shoot apex" includes the growing point at the tip of the stem (shoot apical meristem) as well as tissue consisting of the growing point and several stem primordia arising from the growing point.

[0032] First, in the step of allowing plant seeds to absorb water, the absorption is carried out by soaking the seeds in water and incubating them. If necessary, a plant pesticide (e.g., PLANT PRESERVATIVE MIXTURE (registered trademark)) can be added as an additive to the water used for absorption. The absorption temperature can be, for example, in the range of 0 to 35°C, with 4 to 15°C being preferred. The absorption time depends on the dormancy state of the seeds, but is less than 48 hours after absorption, preferably 36 hours. This absorption step softens the seeds, making it easier to expose the shoot apex.

[0033] In the step of exposing the shoot apex of a seed embryo, for example, the shoot apex is exposed by removing the coleoptile and leaf primordia or the seed coat and cotyledons. Any exposing means may be used as long as it can remove the coleoptile and leaf primordia or the seed coat and cotyledons under a stereomicroscope, including, for example, a puncturing tool such as a needle with a diameter of about 0.2 mm, tweezers, a pipette, a syringe, and a cutting tool such as a scalpel or cutter. Next, the endosperm and excess scutellum are removed using a cutting tool such as a scalpel, and the embryo and scutellum containing the exposed shoot apex are placed on a medium with the shoot apex facing upward. Any known medium can be used as the medium, including, for example, MS medium, agar medium, etc.

[0034] In the step of introducing nucleic acid and / or protein into cells at the shoot apex, known genetic engineering techniques can be used as a method for introducing a target gene, such as a site-specific nuclease, into cells at the shoot apex, and are not particularly limited. Generally, a recombinant vector containing the target gene is prepared, and nucleic acid (e.g., recombinant vector) or protein can be introduced into the shoot apex by Agrobacterium, electroporation, particle gun, PEG-calcium phosphate, liposome, microinjection, whisker, plasma, laser injection, or other methods. From the perspective of efficiency of introduction into plants, particle gun method is preferred for introducing nucleic acid and / or protein into cells at the shoot apex. The particle gun method involves coating metal microparticles with nucleic acid and / or protein and then injecting them into cellular tissue.

[0035] The vector used in the present invention is not particularly limited, and known vectors such as pUC18, pGEM-3Zf, and pE(R3-R4)ZmUbi_OsCas9_Ver3 can be used. The vector used in the present invention can be prepared using known methods.

[0036] Proteins encoded by target genes, such as site-specific nucleases, include genome editing enzymes, such as CRISPR / Cas9, PRR motifs, endonucleases, zinc finger nucleases (ZFNs), TALENs (transcription activator-like effector nucleases), transposases, and site-specific recombinases.

[0037] When genome editing is performed by introducing a nuclease gene or protein (genome editing enzyme) into cells using particle gun technology, it is not necessary to integrate the gene into the genome to obtain a stable transformant. Genome editing can be performed by transiently expressing the nuclease gene. Genome editing can also be performed by directly introducing the genome editing enzyme (and guide RNA) into cells. Using these methods, the resulting genome-edited individual may not be a genetically modified organism.

[0038] In the present invention, examples of plant materials to be subjected to gene function inhibition include, depending on the method of function inhibition, plant tissues such as roots, stems, leaves, seeds, embryos, ovules, ovaries, shoot tips, and pollen, as well as slices thereof, cells, calluses, and protoplasts, but are not limited to these.

[0039] By regenerating plants from cells in which the gene function is suppressed, it is possible to obtain long-day plants of the grass family with increased green leaf mass and seed yield. Furthermore, once a long-day plant of the grass family in which the gene function is suppressed is obtained, it is possible to obtain progeny from the plant through sexual or asexual reproduction. Furthermore, mass production of plants is also possible using propagation materials such as seeds, cuttings, stems, callus, and protoplasts from plants, their progeny, or clones. Furthermore, the plants of the present invention include progeny plants such as the "T0 generation," which is the redifferentiated generation that has been subjected to transformation treatment, and the "T1 generation," which is the self-pollinated seed of a T0 generation plant, as well as hybrid plants and their progeny plants obtained by crossbreeding these plants as a single parent. [Example]

[0040] The present invention will be further explained below with reference to examples, but the present invention is not limited thereto.

[0041] In the following examples, unless otherwise specified, the experiments were carried out at room temperature and atmospheric pressure.

[0042] (1) Study using the barley cultivar "Nihonohoshi" (1-1) Determination of target sequence: Based on the entire Ppd-H1 sequence of the barley cultivar "Nishinohoshi," a 20-bp target sequence to be included in the guide RNA was searched for. CRISPRdirect (https: / / crispr.dbcls.jp / ) was used to search for the target sequence. The target sequence was 5'-CAGCTGCCTGTCAAATGGTG-3' (SEQ ID NO: 3) in the third exon of Ppd-H1, a sequence specific to the entire genome (Figure 1).

[0043] (1-2) In vitro transcription of guide RNA Guide RNA was prepared using the GeneArt Precision gRNA Synthesis Kit (Invitrogen). Oligo DNA containing the target sequence, 5'-TAATACGACTCACTATAGCACCATTTGACAGGCA-3' (SEQ ID NO: 4) and 5'-TTCTAGCTCTAAAACCAGCTGCCTGTCAAATGGT-3' (SEQ ID NO: 5), was heat-treated to form double-stranded DNA. This was annealed with the T7 promoter sequence and cr / tracrRNA sequence provided with the kit to construct template DNA. In vitro transcription was performed according to the manufacturer's instructions, and purified guide RNA was dissolved in nuclease-free water at 1 μg / μL.

[0044] (1-3) Preparation of barley shoot apex samples Dried seeds of the barley cultivar "Nishinohoshi" were sterilized by shaking in a 20% aqueous solution of sodium hypochlorite for 30 minutes. Sterilized seeds were sown on paper towels soaked in a 3% aqueous solution of plant preservative mixture (Nacalai Tesque) in a plastic petri dish. Sterilized seeds were imbibed in the dark at 4°C for 3 days to break dormancy and used for preparing shoot apical samples. The coleoptile, first, second, and third leaves were removed from the imbibed seeds' embryonic tissue using a fine needle to expose the tip of the shoot apical meristem. The embryonic tissue was then dissected from the endosperm and placed on MS medium [MS salt (Sigma) 4.3 g / L, maltose 30 g / L, MES 9.8 g / L, Phytagel 7 g / L]. 20–30 shoot apical samples were placed in the center of the medium to form a 1-cm-diameter donut-shaped circle.

[0045] (1-4) Preparation of RNP complexes Recombinant SpCas9 protein (2 μg / μL) was provided by the Advanced Analysis Center of the National Agriculture and Food Research Organization (NARO). A 1.5 mL tube was mixed with 10 μL of guide RNA (1 μg / μL), 5 μL of spCas9 solution, 2.5 μL of 10x CutSmart buffer, 0.5 μL of Recombinant RNase Inhibitor (TaKaRa), and 7 μL of nuclease-free HO, and the mixture was gently mixed. The mixture was allowed to stand for 10 minutes to allow the RNP complex to form.

[0046] (1-5) Preparation of RNP-bound gold particles Recombinant spCas9 protein (2 μg / μL) was added to a 1.5 mL tube with 10 μL of guide RNA (1 μg / μL), 5 μL of spCas9 solution, 2.5 μL of 10x CutSmart buffer, 0.5 μL of Recombinant RNase Inhibitor (TaKaRa), and 7 μL of nuclease-free water, and the mixture was gently mixed. The mixture was allowed to stand for 10 minutes to allow the formation of RNP complexes. Binding of the RNP complexes to 0.6 μm Gold microcarriers (Bio-Rad) was performed according to the method described in Sergei et al. (Svitashev, S., Schwartz, C., Lenderts, B., Young, J.K. & Mark Cigan, A. Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes. Nat. Commun. 7, 13274 (2016).).

[0047] (1-6) Introduction of RNP complexes into shoot apical samples Six microliters of the prepared RNP-bound gold particle suspension was spread onto a macrocarrier and allowed to dry at room temperature. This suspension was then fired four times onto shoot apex samples on MS medium using a particle gun. The particle gun used was a PDS-1000 / He Particle Delivery System (Bio-Rad), and the gold particle injection pressure was 1,350 psi. The RNP-bound gold particles were introduced into the 85 shoot apex samples by particle bombardment.

[0048] (1-7) Generation and selection of T0 individuals Plants transfected with the RNP complex were grown to the fifth leaf stage and genotyped using CAPS. DNA was extracted from the fifth leaf blade as described below, and the Ppd-H1 partial sequence was amplified using this as a template. The primers used for amplification were 5'-CGACCTTGTTCTCACAGAGG-3' (SEQ ID NO: 6) and 5'-TGGCACTTCCACTACCACTG-3' (SEQ ID NO: 7), and the PCR enzyme was KOD-One (Toyobo). PCR conditions consisted of 30 cycles of heat denaturation at 98°C for 10 seconds, annealing at 60°C for 5 seconds, and extension at 68°C for 5 seconds. Pvu II was added to the amplified product and digested at 37°C for at least 2 hours. The reaction products were separated by 2% agarose gel electrophoresis. Two plants contained undigested products (Figure 2A). These plants were designated NH1 and NH2. The resulting uncleaved product contained a single base insertion in the Ppd-H1 target sequence (Figure 2B). The genome editing efficiency (number of individuals containing the mutation relative to the number of individuals analyzed) in the T0 generation was 2.35% (Table 1).

[0049] [Table 1]

[0050] (DNA extraction) One hundred milligrams of plant tissue was flash-frozen in liquid nitrogen and disrupted using a multi-beads shocker. 400 μL of extraction solution (100 mM Tris-HCl (pH 9.0), 40 mM EDTA, 1.67% SDS) was added to the disrupted tissue and allowed to stand for 3 minutes. The extract was centrifuged at 5,000 G for 3 minutes, and 200 μL of the supernatant was collected. 500 μL of 99.5% EtOH and 20 μL of 3M sodium acetate were added, and the mixture was centrifuged at 12,000 G for 10 minutes to precipitate DNA. The precipitated DNA was washed with 70% EtOH and then dissolved in 100 μL of ultrapure water.

[0051] (1-8) Genotype analysis of T1 individuals The genotype of the T1 individuals obtained from the T0 selected individuals (NH1 and NH2) was examined by CAPS. The CAPS genetic analysis method was performed as described in (1-7). As a result, uncleaved products by CAPS were observed in the T1 individuals obtained from NH2. These products showed the same genetic mutation as the T0 individuals (Figure 3). In other words, the genetic mutation in NH2 was inherited to progeny. In this line, loss of function of Ppd-H1 due to the single-base insertion was predicted.

[0052] (1-9) Heading test and grain yield test Dried seeds of the wild-type strain and the Ppd-H1 homozygous mutant (T3 generation) isolated from the NH2.2-4 self-pollinated progeny were surface-sterilized by shaking in 20% sodium hypochlorite solution for 30 minutes. Sterilized seeds were sown on paper towels soaked in 3% PPM solution in plastic petri dishes. Sterilized seeds were allowed to absorb water in the dark at 4°C for 5 days to break dormancy. Germinated seeds (seeds with swollen roots) were planted 1 cm deep in approximately 2 L of potting soil and considered to be germination day 0. The wild-type strain and the Ppd-H1 mutant (NH2.2-4) were grown in a climate chamber set at 22°C under long-day conditions (16 h light / 8 h dark).

[0053] The day when the panicle elongated and the awn emerged from the tip of the leaf sheath was defined as the heading date. The harvested seeds were dried at 40°C for 7 days and then weighed. The heading date for the wild-type plant was 37.8 days after germination, while the heading date for the Ppd-H1 mutant was 98.3 days after germination. In other words, the Ppd-H1 mutant delayed heading by approximately 61 days (Fig. 4A, B). Furthermore, the Ppd-H1 mutant had increased tiller number, seed number, and grain yield compared to the wild-type plant (Fig. 4C-E).

[0054] (1-10) Green leaf biomass measurement We observed the green leaf biomass of the Ppd-H1 mutant during the vegetative growth stage. Wild-type and Ppd-H1 mutant lines (NH2.2-4) were grown at 22°C under long-day conditions (16 h light / 8 h dark). Each line was harvested just before or just after the panicle formation stage, and the fresh weight and dry weight were measured after drying at 80°C for 10 days. The green leaf biomass of the Ppd-H1 mutant was 9.5 times the fresh weight and 10.6 times the dry weight of the wild-type line (Figure 5). The Ppd-H1 mutant likely had an increased green leaf yield due to the longer vegetative growth period resulting from delayed heading.

[0055] (2) Study using the barley cultivar "Toyonokaze" (2-1) Determination of target sequence: Based on the entire Ppd-H1 sequence of the barley cultivar "Nishinohoshi," a 20-bp target sequence to be included in the guide RNA was searched for. CRISPRdirect (https: / / crispr.dbcls.jp / ) was used to search for the target sequence. The target sequence was 5'-CAGCTGCCTGTCAAATGGTG-3' (SEQ ID NO: 3) in the third exon of Ppd-H1, a sequence specific to the entire genome (Figure 6).

[0056] (2-2) Creating a construct Single-stranded oligomers containing the target sequences, 5'-GTTGCACCATTTGACAGGCAGCTG-3' (SEQ ID NO: 8) and 5'-AAACCAGCTGCCTGTCAAATGGTG-3' (SEQ ID NO: 9), were converted into double-stranded oligomers by heat treatment. These were then inserted into the BbsI site of the guide RNA expression vector pU6-gRNA. pUbi-Cas9 was used to express the codon-modified spCas9 (OsCas9).

[0057] (2-3) Plasmid purification The resulting construct was transformed into Escherichia coli DH5α. The recombinant Escherichia coli was cultured in LB medium containing 50 mg / L ampicillin for 16 hours, and the plasmid was extracted using the alkaline SDS method.

[0058] (2-4) Preparation of barley shoot apex samples The barley cultivar "Toyonokaze" was used. Dried seeds were surface-sterilized by shaking in a 20% sodium hypochlorite solution for 30 minutes. Sterilized seeds were sown on paper towels soaked in a 3% plant preservative mixture solution in a plastic petri dish. The sterilized seeds were imbibed in the dark at 4°C for 3 days to break dormancy and were used to prepare shoot apex samples. The coleoptile, first, second, and third leaves were removed from the imbibed seeds' embryonic tissue using a fine needle to expose the tip of the shoot apical meristem. The embryonic tissue was then dissected from the endosperm and placed on MS medium [MS salt (Sigma) 4.3 g / L, maltose 30 g / L, MES 9.8 g / L, Phytagel 7 g / L]. 20–30 shoot apex samples were placed in the center of the medium to form a 1-cm-diameter donut-shaped circle.

[0059] (2-5) Preparation of DNA-bound gold particles 0.6 μm Gold microcarriers (Bio-Rad) were suspended in nuclease-free water at 60 μg / μL to prepare a gold suspension. 2.5 μL each of pU6-gRNA (1 μg / μL) and pUbi-Cas9 (1.0 μg / μL) was placed in a 1.5 mL tube. 15 μL of the gold suspension, 25 μL of 2.5 M CaCl2, and 10 μL of 0.1 M spermidine were added and allowed to stand at room temperature for 5 minutes. The mixture was centrifuged at 9,000 G for 10 minutes to precipitate the DNA-bound gold particles. The supernatant was removed, 70 μL of 70% EtOH was added, and the mixture was centrifuged at 9,000 G for 10 seconds. The supernatant was removed again, and 70 μL of 99.5% EtOH was added, followed by centrifugation at 9,000 G for 10 seconds. The precipitated DNA-bound gold particles were resuspended in 25 μL of 99.5% EtOH.

[0060] (2-6) Introduction of DNA-bound gold particles into shoot apex samples Six microliters of the prepared DNA-bound gold particle suspension was spread onto a macrocarrier and allowed to dry at room temperature. This suspension was then fired four times onto shoot apex samples on MS medium using a particle gun. The particle gun used was a PDS-1000 / He Particle Delivery System (Bio-Rad), and the gold particle injection pressure was 1,350 psi. The DNA-bound gold particles were introduced into the 373 shoot apex samples by particle bombardment.

[0061] (2-7) Generation and selection of T0 individuals Plants transfected with DNA-bound gold particles were grown. Genotype analysis was performed on 204 plants grown to the fifth leaf stage using CAPS. DNA was extracted from the fifth leaf blade using the same method as in (1-7) and used as a template to amplify the Ppd-H1 partial sequence. The primers used for amplification were 5'-CGACCTTGTTCTCACAGAGG-3' (SEQ ID NO: 6) and 5'-TGGCACTTCCACTACCACTG-3' (SEQ ID NO: 7), and the PCR enzyme was KOD-One (Toyobo). PCR conditions consisted of 30 cycles of thermal denaturation at 98°C for 10 seconds, annealing at 60°C for 5 seconds, and extension at 68°C for 5 seconds. Pvu II was added to the amplified product and digested at 37°C for at least 2 hours. The reaction products were separated by 2% agarose gel electrophoresis. Observation of the reaction products revealed undigested products in three plants. These individuals were designated #1, #2, and #3. The resulting uncleaved products contained a single base insertion or deletion in the Ppd-H1 target sequence (Figure 7B). The genome editing efficiency (number of individuals containing mutations relative to the number of individuals analyzed) in the TO generation was 1.47% (Table 2).

[0062] [Table 2]

[0063] (2-8) Genotype analysis of T1 individuals The genotypes of T1 individuals obtained from the T0 selected individuals (#1, #2, and #3) were examined by CAPS. The CAPS genetic analysis method was performed as described in (2-7). As a result, no uncleaved products were observed in the T1 individuals obtained from #1 and #2, and the mutation was not inherited to the T1 individuals. On the other hand, uncleaved products were observed in five T1 individuals (#3-1 to #3-5) obtained from #3. These products showed the same genetic mutation as the T0 individuals (Figure 8). Therefore, the genetic mutation in #3 was inherited to progeny. In this line, loss of Ppd-H1 function due to the single-base insertion was predicted.

[0064] (2-9) Heading test Dried seeds of the wild-type strain and the Ppd-H1 mutant strain (#3 strain) were surface sterilized by shaking in a 20% sodium hypochlorite solution for 30 minutes. Sterilized seeds were sown on paper towels soaked in a 3% plant preservative mixture solution in a plastic petri dish. Sterilized seeds were allowed to absorb water for 5 days at 4°C in the dark to break dormancy. Germinated seeds (seeds with swollen roots) were planted 1 cm deep in approximately 1 L of potting soil and considered to be germination day 0. The wild-type strain and the Ppd-H1 mutant strain (#3 strain) were grown for 2 weeks in a climate chamber set at 22°C with a 16-hour light / 8-hour dark cycle, and then vernalized. Vernalization was performed by growing the seeds in a growth chamber set at 5°C with an 8-hour light / 16-hour dark cycle for 50 days. After vernalization, the plants were again grown in a climate chamber set at 22°C with a 16-hour light / 8-hour dark period.

[0065] The heading date was defined as the day when the panicle elongated and the awn emerged from the tip of the leaf sheath. The heading date for the wild-type plant was 57.8 days after germination, while the heading date for the Ppd-H1 mutant was 75.4 days after germination. In other words, the heading date was delayed by approximately 18 days in the Ppd-H1 mutant (Fig. 9). Furthermore, the number of tillers in the Ppd-H1 mutant was significantly increased compared to the wild-type plant (Fig. 10).

Claims

1. A method for increasing shoot and seed yield in long-day grasses by loss of function of the PRR37 gene.

2. The method of claim 1, wherein the functional suppression is achieved by genome editing.

3. The method described in claim 2, wherein the genome editing is performed by direct introduction of a genome editing enzyme.

4. The method according to any one of claims 1 to 3, wherein the long-day plant of the Poaceae family is Triticeae.

5. The method according to any one of claims 1 to 3, wherein the long-day plant of the Poaceae family is a member of the Triticeae family, Hordeum genus.

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