JP2020130174A - Production method of plant whose weight is increased - Google Patents

Abstract

To provide a production method of a plant whose weight is increased.SOLUTION: A plant in which a function of phytochrome A gene is suppressed is cultured in at least 1.5 times of density of nutrient of density in a normal culture condition, for increasing weight of the plant relative to plant in which the function of phytochrome A is not suppressed.SELECTED DRAWING: None

Description

The present invention relates to a method for producing a plant having an increased weight. More specifically, a plant in which the function of the phytochrome A gene is suppressed is cultivated under high nutritional conditions to obtain a plant in which the function is not suppressed. It relates to a method of producing a plant having a relative weight increase.

According to the "World Population Forecast 2017 Revised Edition" released by the United Nations, the world's population of 7.6 billion is projected to reach 8.6 billion by 2030, and it is predicted that it will increase further thereafter, such as agricultural products that cover the increased population. There is an urgent need to secure it. Furthermore, biofuels are attracting attention as a measure against global warming, and the demand for agricultural products as raw materials as well as food is rapidly expanding.

However, since the farmland that can be cultivated is limited, it is necessary to increase the unit area or the yield (weight) per plant. In order to meet such needs, it is expected to develop technology that can further increase the productivity of agricultural products.

Takano M. Et al., The Plant Cell, 2001, Vol. 13, pp. 521-534 Takano M. Et al., The Plant Cell, 2005, Vol. 17, pp. 3311-325 Gurani MA. Et al., Biotechnol Adv. , January-February 2015, Vol. 33, No. 1, pp. 53-63 Supervised by Akira Hase, etc., Photosensing of plants-Reception and signal transduction of optical information (cell engineering separate volume-Plant cell engineering series), "1-1 Path of Phytochrome Research", Shujunsha, October 1, 2001 Published daily, page 37

The present invention has been made in view of the above problems, and an object of the present invention is to provide a method for producing a plant having an increased weight, which enables an increase in a unit area or a yield per individual plant.

The present inventors have conducted intensive studies to achieve the above object, and as a result of cultivating phyA-deficient rice (phyA-deficient rice) using a hydroponic solution under different nutritional conditions, phyA under high nutritional conditions. It was revealed that the weight of deficient rice was significantly increased compared to its parent variety. On the other hand, under undernutrition conditions (conditions in which the concentration of each nutrient (nitrogen, phosphorus, potassium, etc.) is 0.4 times that of the above-mentioned high nutritional conditions), no significant change is observed as compared with the parent varieties. I also found that.

Phytochrome (phy) is a major photoreceptor in plants and is known to play an important role in many physiological phenomena in plants. Rice has three phy (phyA, phyB, phyC) that play different roles, and a defective mutant (non-genetical recombination) of a gene encoding each phy has been isolated. However, it has been reported that phyA-deficient rice grown under normal cultivation conditions does not show any difference in expression traits as compared with the parent varieties (Non-Patent Documents 1 to 4), and under the above-mentioned high nutritional conditions. The weight increase of phyA-deficient rice was a surprising and unexpected effect from previous findings.

The present inventors further grow phyA-deficient rice plants and their parent varieties in open-field paddy fields by sparse planting (inter-strain 30 cm) or dense planting (inter-strain 15 cm, twice as high density as sparse planting). The nutritional conditions (fertilization amount) per individual were changed so that the nutrients of sparsely planted rice were doubled compared to those of densely planted rice. In normal paddy field cultivation, the distance between the plants is generally 15 to 20 cm, so that the nutrients of sparsely planted rice are 1.5 to 2 times that of the normal cultivation conditions.

As a result, it was clarified that in sparse planting, the dry weight of all plants and paddy was significantly increased in phyA-deficient rice as compared with the parent cultivar. On the other hand, in dense planting (normal cultivation conditions), no change was observed in the dry weight of all plants and paddy as compared with the parent varieties.

In addition, as a result of cultivating paddy fields with the amount of fertilizer applied twice as much as usual after unifying the planting density, the yield of phyA-deficient rice is increased as compared with the parent varieties under the high nutritional conditions as described above. Was also confirmed.

The present invention is based on the above findings, and more specifically, provides the following.
<1> A method for producing a plant with increased weight, in which a plant in which the function of the phytochrome A gene is suppressed is cultivated at a concentration at least 1.5 times higher than the concentration of nutrients under normal cultivation conditions. ,Method.
<2> The method according to <1>, wherein the nutrients are nitrogen, phosphoric acid and potassium.

By cultivating a plant in which the function of the phytochrome A gene is suppressed under high nutritional conditions (at least 1.5 times higher than the concentration of nutrients under normal cultivation conditions), the function is suppressed. Although it is not always clear why the weight is increased as compared with the non-plants, the present inventors speculate as follows.

That is, when the present inventors compared the amount of nutrients absorbed in phyA-deficient rice and its parent cultivar, the absorption capacity of many nutrients in phyA-deficient rice under high nutritional conditions was compared with that of the parent cultivar. It became clear that it greatly promoted. From this, it is presumed that the plant weight increased with the improvement of the nutrient absorption capacity under high nutritional conditions.

According to the present invention, it is possible to produce a plant with an increased weight, and thus it is possible to increase the yield per unit area or individual plant. Further, as described above, the plant in which the function of the phytochrome A gene used in the present invention is suppressed has a high absorption capacity of nutrients (fertilizers), so that the residual fertilizer is small and the environmental load is reduced.

In addition, there are various varieties of agricultural products having different characteristics such as suitability for cultivation environment, taste, and resistance, and producers select and cultivate varieties having characteristics suitable for the purpose. According to the present invention, in such varieties, by suppressing the function of the phytochrome A gene by genome editing or the like, the weight can be increased and the productivity can be improved without changing the characteristics of each cultivar. It will be possible.

In hydroponics of phyA-deficient rice (line A # 1) and its parent varieties, drying absorbed by 4, 24, and 48 hours after replacement with a new high-nutrient hydroponic solution or low-nutrition hydroponic solution. It is a graph which shows the result of having measured the absorption amount of various nutrients per weight (DW).

As shown in Examples described later, the present inventors have shown that phyA-deficient rice is significantly increased by cultivating it under high nutritional conditions as compared with its parent cultivar. On the other hand, it has also been found that no significant changes are observed in the cultivation under normal cultivation conditions and undernutrition conditions as compared with the parent varieties.

Therefore, the present invention is a method for producing a heavier plant in which a plant in which the function of the phytochrome A gene is suppressed is cultivated at a concentration at least 1.5 times higher than the concentration of nutrients under normal cultivation conditions. I will provide a.

(plant)
The "plant" to be weight-increased in the present invention is not particularly limited as long as the function of the phytochrome A gene is suppressed, and is, for example, a monocotic plant (for example, a gramineous plant such as rice, corn, sorghum, wheat). ) And angiosperms including dicotyledonous plants (eg, grasses such as tomatoes and potatoes), angiosperms, moss plants, fern plants, herbaceous plants, and woody plants. Further, it may be a genetically modified organism or a genome editor of these plants (for example, herbicide-tolerant crop, pest-tolerant crop, disease-tolerant crop, taste-enhancing crop, preservation-enhancing crop, yield-improving crop).

The "weight" increased by the present invention may be the weight of the whole plant or a part thereof. A part thereof is not particularly limited, and examples thereof include seeds, fruits, stems, leaves, roots, tubers, and flowers. Further, the weight may be a dry weight or a fresh weight (raw weight). In the present invention, "weight increase" means that the weight of the whole plant or at least a part thereof is significantly increased as compared with the plant in which the function of the phytochrome A gene is not suppressed. ..

(Phytochrome A gene)
In the present invention, the "phytochrome A gene" is a gene encoding phytochrome A (phyA), which is one of the photoreceptive proteins, and is, for example, a gene encoding a protein containing the amino acid sequence shown in the table below (see the table below). Examples thereof include genes encoding proteins containing the nucleotide sequences shown. In Table 1, "number of genes" indicates the number of phytochrome A genes (number of paralogus genes) possessed by each plant species.

In addition, it is possible that the nucleotide sequence is mutated in nature. And the amino acid encoded by it can be changed accordingly. Therefore, as long as the phytochrome A gene of the present invention encodes a protein having an activity of increasing the weight of a plant by suppressing its function, in the amino acid sequence shown in any of the ones shown in Table 1. Also included are genes encoding proteins consisting of amino acid sequences in which one or more amino acids have been substituted, deleted, added, and / or inserted. Here, the term "plurality" usually means 500 amino acids or less, preferably 400 amino acids or less, more preferably 300 amino acids or less, still more preferably 200 amino acids or less, more preferably 150 amino acids or less, still more preferably 100 amino acids or less, and more preferably. Within 50 amino acids (eg, within 40 amino acids, within 30 amino acids, within 20 amino acids), more preferably within 10 amino acids, particularly preferably within a few amino acids (eg, within 5 amino acids, within 4 amino acids, within 3 amino acids, 2 Within amino acids).

Further, at the present state of the art, those skilled in the art can use the nucleotide sequence information of a specific gene to identify the homologous gene from the same species or other plants. Methods for identifying homologous genes include, for example, hybridization techniques (Southern, EM, J. Mol. Biol., 98: 503, 1975) and polymerase chain reaction (PCR) techniques (Saiki, R. et al.). K., et al. Science, 230: 1350-1354, 1985, Saiki, RK et al. Science, 239: 487-491, 1988). To identify homologous genes, hybridization reactions are usually performed under stringent conditions. As the stringent hybridization conditions, 6M urea, 0.4% SDS, 0.5xSSC conditions or equivalent stringency hybridization conditions can be exemplified. Higher stringency conditions, such as 6M urea, 0.4% SDS, 0.1xSSC, can be used to expect the isolation of genes with higher homology. The phytochrome A gene of the present invention contains a DNA consisting of the nucleotide sequence shown in any of the ones shown in Table 1 as long as it encodes a protein having an activity of increasing the weight of a plant by suppressing its function. Contains genes containing DNA that hybridize with and stringent conditions.

The protein encoded by the identified homologous gene usually has high homology (high similarity), preferably high identity to that encoded by the particular gene. Here, "high" means at least 60% or more, preferably 70% or more, more preferably 80% or more, still more preferably 85% or more, more preferably 90% or more, still more preferably 95% or more (for example, 96). % Or more, 97% or more, 98% or more, 99% or more). The phytochrome A gene has 60% or more homology with the amino acid sequence described in any of the table 1 as long as it encodes a protein having an activity of increasing the weight of a plant by suppressing its function. Contains genes encoding amino acid sequences of sex (similarity) or identity.

Sequence homology can be determined using the BLAST program (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). The program is based on the algorithm BLAST (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993) by Karlin and Altschul. There is. For example, when analyzing the amino acid sequence by BLAST, the parameters are, for example, score = 50 and worldgth = 3. Further, when the amino acid sequence is analyzed by using the Gapped BLAST program, it can be performed as described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known.

Whether or not the protein encoded by the phytochrome A gene of the present invention described above has an activity of increasing the weight of a plant by suppressing its function is determined, for example, as shown in Examples described later. It can be determined by testing whether or not the weight of the plant cultivated under high nutritional conditions is increased by suppressing the function of.

(Plants in which the function of the phytochrome A gene is suppressed)
In the present invention, "suppression of the function of the phytochrome A gene" includes both complete suppression (inhibition) and partial suppression of the function. In addition, there are also plant species having a plurality of phytochrome A genes (paralogous genes such as phyA1 to phyA5) (for example, in the wheat shown in Table 1, the genes encoding the amino acid sequences shown by AEA40430, AEA40439 and AEA40449 are PhyA1 gene, phyA2 gene and phyA3 gene, respectively). In the case of such a plant species, it is sufficient that the function of at least one phytochrome A gene (for example, only the phyA1 gene in wheat) is suppressed, but all phytochrome A genes (for example, in wheat, the phyA1 gene, the phyA2 gene and the phyA3) need to be suppressed. It is preferable that the function of the gene) is suppressed. In addition, suppression of the function of the phytochrome A gene includes suppression of the expression of the phytochrome A gene and suppression of the activity of the protein encoded by the phytochrome A gene. Then, such suppression can be performed, for example, by introducing a mutation into a coding region, a non-coding region, a transcription control region (promoter region), or the like of the phytochrome A gene.

In the present invention, the mutation introduced into the phytochrome A gene is not particularly limited as long as the function of the gene is suppressed, and examples thereof include nucleotide substitution, deletion, addition, and / or insertion, but a nonsense mutation. , Frame shift mutations, null mutations are preferred. The number of mutations introduced into the phytochrome A gene is not particularly limited as long as the function of the gene is suppressed, and may be one, or a plurality (for example, two, three or less, five or less, etc.). 10 or less, 20 or less, 30 or less, 40 or less, 50 or less) may be used.

Further, as a mutation introduced into the phytochrome A gene, it is not necessary to lose the entire amino acid sequence of the protein encoded by the gene, and the mutation may be introduced into the gene so that a part thereof is lost or changed. ..

When a part of the protein expressed by a gene mutation is deleted, it is usually sufficient that 20% or more of the whole is deleted, preferably 30% or more, more preferably 40% or more, and further preferably. The deletion may be 50% or more, more preferably 60% or more, more preferably 70% or more, still more preferably 80% or more (for example, 85% or more, 90% or more, 95% or more).

In addition, in such a mutation that deletes a part, there is a mutation that deletes a region or a site that is considered to be important for exerting the activity of the protein encoded by the phytochrome A gene. Such regions can be grasped by those skilled in the art because, for example, in each sequence specified by the GenBank accession number shown in Table 1 above, information such as those regions is shown as "Region" and "Site". can do. Among these regions, all or part of them are deleted in the GAF domain because they are regions to which the photoreceptive chromophore, which is important for the activity of the protein encoded by the phytochrome A gene, binds. It is preferable that such a mutation is introduced. In addition, for example, in rice phytochrome A (consisting of the amino acid sequence described in XP_015630340), the GAF domain is a region consisting of amino acids at positions 220 to 412.

Introduction of a mutation into the phytochrome A gene can be achieved by a mutation introduction method known to those skilled in the art. Examples of such known methods include, but are not limited to, a genome editing method, a physical mutagenesis method, a method using a chemical mutagen, and a method of introducing a transposon or the like into genomic DNA.

Genome editing methods utilize site-specific nucleases (eg, zinc finger nucleases (ZFNs), transcriptional activation-like effector nucleases (TALENs), DNA double-strand break enzymes such as CRISPR-Cas9) to target genes. This is a method of modification. For example, ZFNs (US Patent No. 6265196, 8524500, 7888121, European Patent No. 1720995), TALENs (US Patent No. 8470973, US Patent No. 8586363), PPR (pentatric protein protein) (Nakamura et al.) In which a nuclease domain is fused. Fusion proteins such as Plant Cell Physiol 53: 1171-1179 (2012), CRISPR-Cas9 (US Pat. No. 8,695,359, International Publication No. 2013/176772), CRISPR-Cpf1 (Zetsche B. et al., Cell, 163) 3): 759-71, (2015)) and Target-AID (K. Nishida et al., Targeted nucleotide editing using hybrid prokaryotic and vertebrate advanced CRISPR. ), Etc., a method using a complex of a guide RNA and a protein can be mentioned.

Examples of the physical mutation introduction method include heavy ion beam (HIB) irradiation, fast neutron beam irradiation, gamma ray irradiation, and ultraviolet irradiation (Hayashi et al., Cyclotrons and Their Applications, 2007, 18th International Conference, 237). See pages 239 and Kazama et al., Plant Biotechnology, 2008, Vol. 25, pp. 113-117).

Examples of the method using a chemical mutant include a method of treating seeds and the like with a chemical mutant (see Zwar and Chandler, Planta, 1995, Vol. 197, pp. 39-48, etc.). The chemical mutant is not particularly limited, but is ethylmethanesulfoto (EMS), N-ethyl-N-nitrosourea (ENU), N-methyl-N-nitrosourea (MNU), sodium azide, sodium hydrogen sulfite. , Hydricylamine, N-methyl-N'-nitro-N-nitroguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), O-methylhydroxylamine, nitrite, formic acid and nucleotide analogs Can be mentioned.

As a method for introducing a transposon or the like into the genomic DNA, for _example, transposons such as _T OS 17, and a method of the T-DNA or the like is inserted into the genomic DNA of a plant (Kumar et al., Trends Plant Sci., 2001 year, 6 Vol. 3, No. 3, pp. 127-134, and Tamara et al., Trends in Plant Science, 1999, Vol. 4, No. 3, pp. 90-96).

For plants into which the mutation has been introduced by the above method, it can be confirmed that the mutation has been introduced into the phytochrome A gene by a known method. Examples of such known methods include DNA sequencing methods (next-generation sequencing methods and the like), PCR methods, analysis methods using microarrays, Southern blotting methods, and Northern blotting methods. According to such a method, whether or not a mutation has been introduced into the phytochrome A gene can be determined by comparing the sequence or length of the gene before and after the introduction of the mutation. In addition, by using Northern blotting, RT-PCR, Western blotting, ELISA, microarray analysis, etc., transcripts or translations of the phytochrome A gene in plants in which mutations have been introduced into the transcription control region, etc. If a decrease in the expression level of the product is observed, it can be confirmed that the plant is a plant in which a mutation has been introduced into the phytochrome A gene.

In addition, as another method for confirming that a mutation has been introduced into the phytochrome A gene, TILLING (Targeting Induced Local Lesions IN Genomes) can be mentioned (Srade et al., Transgenic Res., 2005). Year, Volume 14, pp. 109-115, and Comai et al., Plant J., 2004, Volume 37, pp. 778-786). In particular, when a non-selective mutation is introduced into the plant genome using the above-mentioned heavy ion beam irradiation or a chemical mutagen, the phytochrome A gene or a part thereof is amplified by PCR and then added to the amplification product. Individuals with mutations can be selected by the TILLING or the like. In this case as well, a plant having a null mutant of the phytochrome A gene can be obtained.

In addition, mutations introduced into genes other than the phytochrome A gene can be removed by crossing a plant into which a mutation has been introduced by the above method with a wild-type plant and performing backcrossing.

A plant in which the function of the gene is suppressed by introducing a mutation into the phytochrome A gene may be a heterozygote of the phytochrome A gene. In such a case, for example, by crossing F1 plants obtained by crossing the heterozygotes with a control plant to obtain F2 plants, phytochrome A into which the mutation is introduced from the F2 plants is obtained. Homozygotes carrying the gene can be selected. Further, from the viewpoint that the function of the phytochrome A gene is completely suppressed, a homozygote is preferable. In this case, the "plant that is a homozygous conjugate having the phytochrome A gene into which the mutation has been introduced" includes not only a plant having two phytochrome A alleles having mutations that are identical to each other, but also a first plant. A plant having a first phytochrome A allele encoding a protein having a mutation and suppressed activity and a second phytochrome A allele encoding a protein having a second mutation and suppressed activity. Is included.

In the present invention, as a method for suppressing the function of the phytochrome A gene, in addition to the above-mentioned mutagenesis, a method using a DNA encoding dsRNA (double-stranded RNA, for example, siRNA) complementary to the transcript of the phytochrome A gene, A method using a DNA encoding an antisense RNA complementary to a transcript of the phytochrome A gene (antisense DNA), a method using a DNA encoding an RNA having a ribozyme activity that specifically cleaves the transcript of the phytochrome A gene ( A method of targeting a transcript of the phytochrome A gene, such as the ribozyme method), can also be mentioned.

In the present invention, suppression of phytochrome A gene function can be performed on various plants, seeds or plant cells according to the above-mentioned methods and the like. Plant cells include cultured cells derived from plants as well as cells in plant bodies. In addition, various forms of plant-derived cells such as suspension-cultured cells, protoplasts, leaf sections, callus, immature embryos, pollen and the like are included.

Further, in the present invention, the above-mentioned site-specific nuclease, fusion protein or DNA encoding a guide RNA and protein complex, transposon-encoding DNA, double-stranded RNA-encoding DNA, and antisense RNA are encoded. DNA, DNA encoding RNA having ribozyme activity, or the like may be introduced into plant cells in the form of being inserted into a vector.

The vector into which the DNA for suppressing the function of the phytochrome A gene is inserted is not particularly limited as long as the inserted gene can be expressed in plant cells, but the DNA is constitutively or induced. May contain a promoter for expression. Examples of the promoter for constitutive expression include the 35S promoter of cauliflower mosaic virus, the actin promoter of rice, and the ubiquitin promoter of maize. In addition, as a promoter for inducible expression, it is known that it is expressed by external factors such as infection or invasion of filamentous fungi, bacteria, and viruses, low temperature, high temperature, drying, irradiation with ultraviolet rays, and spraying of a specific compound. Examples include promoters that have been used. Further, as a promoter for expressing a DNA encoding a short RNA such as siRNA as the DNA according to the present invention, a polIII-based promoter is preferably used.

Examples of a method for introducing the DNA or a vector into which the DNA is inserted into a plant cell include a particle gun method, an Agrobacterium-mediated method (Agrobacterium method), a polyethylene glycol method, and an electroporation method. Various methods known to those skilled in the art such as ration) can be used.

Even if it does not take the form of DNA, the above-mentioned site-specific nuclease, fusion protein, and transposon can be used as proteins, and the above-mentioned guide RNA, double-stranded RNA, antisense RNA, and RNA having ribozyme activity can be used as RNA. As a result, mutations can be introduced even when introduced into plant cells.

In addition, a plant with an increased weight can be obtained by regenerating a plant from a plant cell in which the function of a gene is artificially suppressed by the above-mentioned method or the like.

For example, in rice, a method for producing a transformed plant is a method in which a gene is introduced into a protoplast with polyethylene glycol to regenerate the plant (Datta, SK In Gene Transfer To Plants (Potrykus I and Spangenberg Eds). ) Pp66-74, 1995), a method of introducing a gene into a protoplast by an electric pulse to regenerate a plant (Toki et al. Plant Physiol. 100, 1503-1507, 1992), and a method of directly introducing a gene into a cell by a particle gun method. Then, a method for regenerating a plant (Christou et al. Bio / technology, 9: 957-962, 1991) and a method for regenerating a plant by introducing a gene via agrobacterium (Hiei et al. Plant J). .6: 271-282, 1994), some techniques have already been established and are widely used in the technical field of the present invention.

In addition, even for other plants, transformation and transformation using the method described in Tabei et al. (Edited by Yutaka Tabei, "Transformation Protocol [Plant Edition]", Kagaku-Dojin Co., Ltd., published on September 20, 2012) It can be regenerated into plants.

Further, once a plant in which the function of the phytochrome A gene is suppressed is obtained by the above-mentioned method or the like, it is possible to obtain offspring from the plant by sexual reproduction or asexual reproduction. Further, it is also possible to obtain breeding materials (for example, seeds, cutting ears, strains, callus, protoplasts, etc.) from the plant, its descendants or clones, and mass-produce the plant based on them. Therefore, the present invention includes offspring and clones of the increased weight of the plant, as well as their reproductive materials.

The preferred embodiment of the "plant in which the function of the phytochrome A gene is suppressed" according to the present invention has been described above, but the plant is not limited to the above-mentioned artificial mutagenesis and the like, and is natural. Plants in which the function of the phytochrome A gene is suppressed by mutation are also included.

In addition, there are various varieties of agricultural products having different characteristics such as suitability for cultivation environment, taste, and resistance, and the producer selects and cultivates varieties having characteristics suitable for the purpose. Therefore, in such an existing variety, if the function of the phytochrome A gene is suppressed by the above-mentioned method or the like, the weight can be increased (productivity is improved) without changing the characteristics of each variety.

(Plant cultivation method)
As shown in Examples described later, in the production method of the present invention, the above-mentioned plant in which the function of the phytochrome A gene is suppressed is at a concentration at least 1.5 times higher than the concentration of nutrients under normal cultivation conditions. By cultivating, a plant with increased weight can be obtained.

The "nutrient" according to the present invention may be any nutrient involved in plant growth, may be an essential nutrient, or may be a useful nutrient. Essential nutrients include, for example, a large amount of primary elements (nitrogen, phosphorus, potassium, carbon, hydrogen, oxygen), a large amount of secondary elements (magnesium, calcium, sulfur), and trace elements (boron, chlorine, manganese, iron, zinc, copper). , Molybdenum, nickel). Among these, nitrogen, phosphorus and potassium are preferable, and nitrogen, phosphorus, potassium and magnesium are more preferable as the nutrients according to the present invention.

In addition, the concentration of the above-mentioned nutrients under normal cultivation conditions can be understood by those skilled in the art as a standard fertilizer application amount set based on soil characteristics for each plant variety (for example, fertilizer application standard indicated by the Ministry of Agriculture, Forestry and Fisheries of Japan). can do. More specifically, the amount of fertilizer applied shown in Table 2 is given as the concentration of nutrients under normal cultivation conditions in each plant species.

In Table 2, the fertilization standard for rice cultivated in viscous soil is shown (however, the fertilization standard for diphosphorus pentoxide when cultivating Koshihikari etc. in viscous peat / sapric soil is shown. , 10 kg / 10a instead of 8 kg / 10a shown in Table 2).

In the present invention, the above-mentioned plant in which the function of the phytochrome A gene is suppressed may be cultivated at a concentration at least 1.5 times higher than the concentration of nutrients under the above-mentioned normal cultivation conditions, but 1.7. Cultivation at twice as high concentration is preferable, cultivation at twice as high concentration is more preferable, cultivation at 2.5 times higher concentration is more preferable, cultivation at three times higher concentration is more preferable, and cultivation at 3.5 times higher concentration is more preferable. Cultivation at a concentration four times higher is more preferable. In addition, the amount of nutrients can be at least 1 more than the normal cultivation conditions by adjusting the cultivation interval as shown in Example 2 described later, as well as adjusting the amount added to the soil or water. The concentration can be increased by 5 times.

The cultivation period in the production method of the present invention is not particularly limited, and may be cultivated from seeds or seedlings (seedlings, seedlings, etc.). The cultivation period is not particularly limited, and may include, for example, at least one period selected from the seedling period, the growing period, the overgrowth period, the maturity period, the flowering period, and the fruiting period. Further, the final stage of cultivation is not particularly limited, and examples thereof include a seedling stage, a growing stage, a prosperous stage, a maturity stage, a flowering stage, and a fruiting stage.

Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to the following Examples. In addition, this example was carried out using the materials and methods shown below.

(Rice)
PhyA-deficient rice (phyA-deficient rice) and its parent variety, Nihonbare, were subjected to the following experiments. The phyA-deficient rice is a rice selected from a mutant population (mutant panel) produced by the retrotransposon Tos17 produced by the former Agricultural and Biological Resources Research Institute (see Non-Patent Documents 1 and 2). In Examples 1 and 4 described later, phyA-deficient rice line A # 1 was used, and in Example 2, phyA-deficient rice line A # 2, which is a line independent of line A # 1, was used. In Example 3, the two systems A # 1 and A # 2 were used.

(Hydroponic cultivation)
Hydroponics was carried out using 6 rice plants per 330 mL of the following high-nutrition or low-nutrition hydroponic solution. The hydroponic solution was replaced with a new solution every two days, and the rice was grown until the five-leaf stage (approximately three weeks). The hydroponic solution was prepared by mixing various stock solutions as follows.

Stock liquid (I to V liquid)
Liquid I (500 mL stock)
KNO ₃ (potassium nitrate, MW = 101.1) 101.1g (2M)
NH ₄ H ₂ PO ₄ (Ammonium dihydrogen phosphate, MW = 115.03) 57.5 g (1 M)
Liquid II (200 mL stock)
MgSO ₄ -7H ₂ O (magnesium sulfate heptahydrate, MW = 246.48) 49.3g (1M )
Solution III (100 mL stock)
CaCl ₂ -2H ₂ O (calcium chloride dihydrate, MW = 147.01) 73.5g (5M )
IV solution (200 mL stock)
H ₃ BO ₃ (boric acid, MW = 61.83) 0.572 g (46.26 mM)
MnSO ₄ -5H ₂ O (Manganese (II) sulfate pentahydrate, MW = 241.08) 0.307g (6.37mM )
CuSO ₄ -5H ₂ O (copper (II) sulfate pentahydrate, MW = 249.7) 0.012g (0.24mM )
ZnSO ₄ -7H ₂ O (zinc (II) sulfate heptahydrate, MW = 287.56) 0.156g (2.71mM )
K ₂ MoO ₄ (potassium molybdate, MW = 238.13) 0.023 g (0.48 mM)
Liquid V (100 mL stock)
EDTA-Na-Fe (II) -H ₂ O (Fe-EDTA, MW = 385.06) 0.193 g (50 mM).

The highly nutritious hydroponic solution was prepared by diluting 500 μL of solution I, 500 μL of solution II, 100 μL of solution III, 500 μL of solution IV and 250 μL of solution V with ultrapure water (Mili-Q) to 1 L. On the other hand, the low-nutrition hydroponic solution was prepared by diluting the high-nutrition hydroponic solution with ultrapure water (Mili-Q) 2.5 times.

In hydroponics, rice was grown in a growth cabinet (manufactured by Sanyo Co., Ltd., product number: MLR-350) under light conditions of 16 hours light period / 8 hours dark period. The light source used was Biolux-A (manufactured by NEC), a fluorescent lamp for plant cultivation, and the temperature was set to 28 degrees and the cultivation was performed without setting the humidity.

(Paddy cultivation)
In Example 2 described later, rice seedlings (rice in the 4th to 5th leaf stage) were transplanted to paddy fields in the middle of May 2005. Fertilizers (basic fertilizer, spike fertilizer) were applied at normal fertilizer amounts. Specifically, the crops were cultivated at the amount of fertilizer applied based on the standard for cultivating ordinary crops of Nihonbare shown in Table 2. Rice seedlings were transplanted to paddy fields at intervals of 30 cm (sparse planting) or 15 cm (dense planting).

Further, in Example 3 described later, rice seedlings (rice in the 4th to 5th leaf stage) were transplanted to 2 plots of paddy fields at intervals of 15 cm in the middle of May 2019. One of the two paddy fields is under normal nutritional conditions (the amount of fertilizer applied based on the Nihonbare ordinary crop cultivation standards shown in Table 2), and the other is under high nutritional conditions (compared to the above-mentioned normal nutritional conditions). It was cultivated with twice the amount of fertilizer applied.

(Measurement of nutrient content)
The hydroponic solution sampled 4, 24, and 48 hours after the replacement of the new hydroponic solution was filtered with a 13AI 0.2 μm chromatodisc (manufactured by GL Sciences), and each nutrient content was subjected to an ion chromatograph (manufactured by Dionex). Measured using.

(Example 1)
As described above, phyA-deficient rice plants and parent varieties were cultivated under undernutrition conditions or high nutritional conditions (nutrients were 2.5 times higher than those under undernutrition conditions) using hydroponic solution. Then, each of the obtained seedlings was divided into an above-ground part and a root, and the dry weight was measured. The results obtained are shown in Table 3.

In Table 3, “A # 1” and “WT” indicate data of phyA-deficient rice (line A # 1) and parent varieties, respectively. The data are shown as mean ± standard error (SE) (n = 5). In addition, the values in phyA-deficient rice, which were significantly different from the values in the parent variety by the Student's t-test, are marked with an asterisk (* P <0.05).

As is clear from the results shown in Table 3, no significant change was observed between the phyA-deficient rice plant and the parent variety under undernutrition conditions. On the other hand, under high nutritional conditions, the weight of phyA-deficient rice was found to be significantly higher than that of the parent varieties.

(Example 2)
As described above, in paddy fields, phyA-deficient rice plants and parent varieties were cultivated by sparse planting (inter-strain: 30 cm, high nutritional conditions) or dense planting (inter-strains: 15 cm, normal nutritional conditions) until the paddy matured. Then, the dry weights of all the obtained plants and paddy were measured. The results obtained are shown in Table 4.

In normal paddy field cultivation, the distance between the strains is generally 15 to 20 cm (see http://www.tanbo-kubota.co.jp/foods/watching/15_2.html on the website of Kubota Co., Ltd.). ). Therefore, the sparsely planted rice has 1.5 to 2 times more nutrients than the normal cultivation conditions.

In Table 4, "A # 2" and "WT" indicate data of phyA-deficient rice (line A # 2) and parent varieties, respectively. Data are shown by mean ± standard error (SE) (total plant weight n = 20, paddy weight n = 16). In addition, the values in phyA-deficient rice, which were significantly different from the values in the parent cultivar by the Student's t-test, are marked with an asterisk (* P <0.05; ** P <0.01).

As is clear from the results shown in Table 4, it was clarified that in sparse planting, the dry weight of all plants and paddy increased 1.1 times in both phyA-deficient rice as compared with the parent cultivar. On the other hand, in dense planting (under normal nutritional conditions), no change was observed in the dry weight of all plants and paddy as compared with the parent varieties.

(Example 3)
As described above, in paddy fields, phyA-deficient rice plants (2 lines) and parent varieties were cultivated under normal nutritional conditions or high nutritional conditions (double the amount of fertilizer applied) until the paddy matured. Then, the dry weights of the obtained rice ears and the above-ground part (the part other than the ears) were measured. The results obtained are shown in Table 5.

In Table 5, "A # 1" and "A # 2" indicate the data of two independent lines of phyA-deficient rice, and "WT" indicates the data of the parent variety. Data are shown by mean ± standard error (SE) (cultivation under high nutritional conditions: n = 15, cultivation under normal nutritional conditions: n = 16). In addition, the values in phyA-deficient rice, which were significantly different from the values in the parent cultivar by the Student's t-test, are marked with an asterisk (*** P <0.001).

As is clear from the results shown in Table 5, in both of the two lines of phyA-deficient rice, a remarkable increase in yield was observed as compared with the parent cultivar when cultivated under high nutritional conditions.

(Example 4)
As shown in Examples 1 to 3, as a result of cultivating phyA-deficient rice under high nutritional conditions, its weight increased as compared with the parent variety. From this, it is expected that the nutrient absorption capacity of phyA-deficient rice is promoted under high nutritional conditions.

Therefore, in order to investigate the nutrient absorption capacity, as described above, hydroponics was carried out, and after exchanging with a new high-nutrition hydroponic solution or low-nutrition hydroponic solution, hydroponics sampled at 4, 24, and 48 hours. The content of each nutrient in the liquid was measured, and the amount of various nutrients absorbed per absorbed dry weight (DW) was calculated from the residual amount. Then, the absorption amounts of various nutrients were compared between the parent varieties and phyA-deficient rice. The obtained results are shown in FIG.

In FIG. 1, "A / WT_Low" indicates the ratio of phyA-deficient rice to the amount of absorption of various nutrients of the parent cultivar after exchanging the low-nutrition hydroponic solution, and "A / WT_High" is high-nutrition water. The ratio of phyA-deficient rice to the amount of absorption of various nutrients of the parent cultivar after exchanging the tillage solution is shown. Ammonium ion (NH 4 ^₊₎ and nitrate ion (NO 3 ^_-) "ND" in the not be calculated nutrient absorption ratio to parental cultivars and / or phyA deficient rice had exhausted absorbed after 24 hours Show that. On the other hand, "ND" in the fourth hour of the sulfate ion (SO 4 ^_2-) and calcium ions (Ca ²⁺⁾ are nutrient absorption ratio because there are more emissions out of the plants than the absorption quantity Indicates that it could not be calculated. The time nutrient absorption in rice is promoted different for each nutrient, nitrogen component of the ammonium ion (NH 4 ^₊₎ and nitrate ion (NO 3 ^_-) whereas the absorption at 4 hours become popular, Absorption of other nutrients is promoted after the 24th hour.

As is clear from the results shown in FIG. 1, no significant difference in the absorption amount of various nutrients was observed between the phyA-deficient rice and the parent cultivar under undernutrition conditions. On the other hand, under high nutritional conditions, it was revealed that the absorption amount of various nutrients in phyA-deficient rice was generally increased as compared with that of the parent varieties. From this result, it is suggested that the weight increase of phyA-deficient rice found in Examples 1 to 3 under high nutritional conditions is due to the increase in absorption capacity of various nutrients.

As described above, according to the present invention, it is possible to produce a plant having an increased weight, and by extension, it is possible to increase the unit area or the yield per plant. In addition, the plant in which the function of the phytochrome A gene used in the present invention is suppressed has a high absorption capacity of nutrients (fertilizers), so that the residual fertilizer is small and the environmental load is reduced. Therefore, the present invention is useful in the agricultural field.

Claims (2)

It is a method of producing plants with increased weight.
A method in which a plant in which the function of the phytochrome A gene is suppressed is cultivated at a concentration at least 1.5 times higher than the concentration of nutrients under normal cultivation conditions. The method of claim 1, wherein the nutrients are nitrogen, phosphoric acid and potassium.