Method for cultivating leguminous plant

Abstract

Leguminous plants have had the problem that the plant height increases and therefore the plants have a risk of being incapable of effectively utilizing spaces and a risk of falling when the plants are densely packed in order to increase the yield of the plants in hydroponic cultivation under an environment where the extension of roots of plant bodies is not restricted. The present invention addresses the problem of providing a hydroponic cultivation method for leguminous plants, in which the plant length is maintained at a level suitable for hydroponic cultivation in dense hydroponic cultivation. It has been found that, in dense hydroponic cultivation of a leguminous plant at a density of 20-200 plants per square meter, the problem can be solved by performing the hydroponic cultivation of the leguminous plant using a nutrient solution having a high nitrate ion density.

Description

How to grow legumes

[0001] The present invention relates to a method for growing legumes by hydroponic culture and novel legume seeds.

[0002] Generally, plants are grown in soil using soil as a medium and then given fertilizer and water as nutrients and moisture. In contrast, a cultivation method using a nutrient solution made up of a mixture of liquid fertilizer and water as a medium is called hydroponics.

[0003] In recent years, there has been a movement toward the practical application of so-called vegetable factories, which cultivate various vegetables within plants. Non-Patent Documents 1, 2, and 3 disclose research on hydroponic soybean cultivation. In particular, Non-Patent Documents 1 and 3 describe a laboratory cultivation method for simultaneously cultivating multiple soybean varieties at high density. This method, in which individual plants are planted in test tubes and then arranged on trays for cultivation, is intended to experimentally obtain small amounts of seeds from each plant, and is not intended for practical use. Further testing has revealed that both the plant height and seed yield are significantly low.

[0004] Furthermore, Patent Document 1 discloses a hydroponic cultivation method for legumes. Patent Document 1 aims to provide a method for cultivating legumes that allows the harvest of high-quality seeds, and discloses a method that includes a rhizosphere low-temperature process that keeps the roots of legumes whose seeds have swelled in a cooled state.

[0005] Patent Publication No. 2021-78390

[0006] Masao Ishimoto et al., "Exploring Genetic Control Factors of Soybean Seed Components through Cultivation in a Fully Controlled Environment," Soy Protein Research, Vol. 19 (2016) pp. 24-29 Kazuhiro Shoji et al., "Practical Application of Vegetable Factories (2) Hydroponic Cultivation Conditions for Edamame," Report of the Central Research Institute of Electric Power Industry, Research Report: U91033, September 1991 Bioscience, Biotechnology, and Biochemistry, 2015, Vol. 79, No. 1, pp. 63-67

[0007] When legumes are grown in a nutrient solution in an environment that does not restrict root growth to increase yield, they grow taller (Non-Patent Document 2), which can lead to inefficient use of space and the risk of the plants falling over. The present invention aims to provide a method for growing legumes in a nutrient solution that maintains a height suitable for nutrient solution cultivation in a densely packed nutrient solution. In another aspect, the present invention aims to obtain novel legume seeds, specifically edamame (green soybeans) with improved free amino acid content.

[0008] The present inventors have conducted extensive research to solve the above problems, and as a result have found that the above problems can be solved by growing legumes in a nutrient solution with a high nitrate ion concentration in a densely packed hydroponic culture system with 20 to 200 plants per square meter, thereby completing the present invention.

[0009] That is, the present invention relates to: (1) a hydroponic cultivation method for legumes, in which legumes are cultivated at a cultivation density of 20 to 200 plants per square meter using a nutrient solution having a nitrate ion concentration of 10 to 30 mmol / L in an environment in which root elongation of the plant is not restricted; (2) the cultivation method according to (1) above, which is spray culture or hydroponic culture; (3) a method for producing seeds of legumes, which includes recovering seeds from plants obtained by the cultivation method according to (1) above; (4) edamame soybeans having a free asparagine content of 6 to 50 mg / g on a dry matter basis; (5) edamame soybeans having a free arginine content of 2.2 to 12 mg / g on a dry matter basis; (6) edamame soybeans having a free asparagine content of 6 to 50 mg / g on a dry matter basis and a free arginine content of 2.2 to 12 mg / g on a dry matter basis; (7) A method for controlling the height of legumes to 60 cm or less by cultivating them at a cultivation density of 20 to 200 plants per square meter using a nutrient solution with a nitrate ion concentration of 10 to 30 mmol / L; (8) A method for cultivating edamame at a cultivation density of 20 to 200 plants per square meter using a nutrient solution with a nitrate ion concentration of 10 to 30 mmol / L, and adjusting the amount of free asparagine in edamame to 6 to 50 mg / g on a dry matter basis; (9) A method for cultivating edamame at a cultivation density of 20 to 200 plants per square meter using a nutrient solution with a nitrate ion concentration of 10 to 30 mmol / L, and adjusting the amount of free arginine in edamame to 2.2 to 12 mg / g on a dry matter basis; (10) A method for cultivating edamame at a planting density of 20 to 200 plants per square meter using a nutrient solution with a nitrate ion concentration of 10 to 30 mmol / L, thereby setting the amount of free asparagine in edamame at a dry matter equivalent of 6 to 50 mg / g and the amount of free arginine at a dry matter equivalent of 2.2 to 12 mg / g; (11) A method for increasing the amount of free asparagine in edamame and mature soybeans, by cultivating edamame at a planting density of 20 to 200 plants per square meter using a nutrient solution with a nitrate ion concentration of 10 to 30 mmol / L.

[0010] According to the present invention, it is possible to provide a method for hydroponics of legumes, in which legumes are grown in a densely packed hydroponics environment without restricting root elongation, by using a nutrient solution with a high nitrate ion concentration, thereby maintaining a plant height suitable for hydroponics.In another aspect, it is possible to provide edamame soybeans having increased amounts of free amino acids, particularly free asparagine and free arginine, compared to those grown in soil.

[0011] Fig. 1 is a diagram showing the spray culture apparatus used in the examples. Fig. 2 is a diagram showing the plant height of soybeans cultivated in Cultivation Example 1. Fig. 3 is a diagram showing the plant height of soybeans cultivated in Cultivation Example 2. Fig. 4 is a diagram showing the yield per unit area of ​​mature soybeans cultivated in Cultivation Example 2. Fig. 5 is a diagram showing the plant height of soybeans cultivated in Cultivation Examples 3 and 4.

[0012] One aspect of the present invention provides a hydroponic culture method for legumes, in which legumes are grown in a densely packed nutrient solution at 20 to 200 plants per square meter in an environment that does not restrict root growth, using a nutrient solution with a nitrate ion concentration of 10 to 30 mmol / L.

[0013] ■ Cultivation Density In open-field cultivation, the appropriate cultivation density is generally about 10 to 20 plants per square meter, depending on the variety. On the other hand, when cultivating in a vegetable factory, a higher cultivation density is necessary for production efficiency. Therefore, the cultivation density of legumes in the cultivation method of this embodiment is preferably 20 or more plants per square meter, more preferably 25 or more plants, even more preferably 30 or more plants, even more preferably 35 or more plants, and even more preferably 40 or more plants. Increasing the cultivation density increases yield, but also increases plant height, which may require equipment such as supports to support the branches. Therefore, a density of 200 or less plants per square meter is preferred, more preferably 150 or less plants, even more preferably 120 or less plants, even more preferably 100 or less plants, and even more preferably 80 or less plants. 20 to 200 plants are preferred, more preferably 25 to 150 plants, even more preferably 30 to 120 plants, even more preferably 35 to 100 plants, and even more preferably 40 to 80 plants.

[0014] ■ Cultivation Form Legume plants are cultivated in an environment that does not restrict root elongation in this embodiment. That is, it is necessary to avoid restricting the liquid and / or gas phase space through which roots elongate, or to ensure a sufficiently large space. For example, it is preferable to ensure a space of 50 mL or more in which roots can grow, e.g., 70 mL or more, 100 mL or more, 200 mL or more, 1 L or more, 5 L or more, 10 L or more, or 20 L or more. There is no particular upper limit, but examples include 10,000 L or less, 5,000 L or less, 2,000 L or less, 1,000 L or less, 500 L or less, 100 L or less, and 50 L or less. Each of these spaces may contain a single individual plant, or multiple plants may share the space. It is important not to restrict root elongation; even if other plants coexist, root elongation is not restricted within the cultivation density range of this embodiment. In addition, in some embodiments, it is preferable to exclude methods of cultivating plants in test tubes, such as those described in Non-Patent Documents 1 and 3.

[0015] Hydroponics, as used herein, refers to a cultivation method that uses a nutrient solution containing nutrients necessary for plant growth dissolved in a liquid, without using soil. In contrast, soil culture is used herein to refer to a cultivation method that uses soil. Specific examples of hydroponics include hydroponics, solid medium culture, and spray culture. Hydroponics, as used herein, refers to a cultivation method in which some or all of the roots of the plant to be cultivated are submerged in a nutrient solution. Solid medium culture, as used herein, refers to a cultivation method that uses various solid media instead of soil. Spray culture, as used herein, refers to a cultivation method in which a nutrient solution is directly sprayed onto some or all of the roots of the plant to be cultivated, which are exposed to an air phase, for a certain period of time. The hydroponics of the present invention refer to the above-mentioned hydroponics, solid medium culture, spray culture, etc., and the hydroponics cultivation method is not particularly limited. However, in one embodiment, the hydroponics in this cultivation method is spray culture or hydroponics. Hydroponics, in which the roots are fully submerged in the nutrient solution and spraying is not required, is most preferred.

[0016] In one embodiment, spraying in aeroponics is carried out for 10 seconds to 1 minute, for example, at a frequency of 15 minutes to 2 hours, preferably 30 minutes to 1 hour.

[0017] Nitrate Ion Concentration in the Nutrient Solution The nutrient solution in this embodiment has a high nitrate ion concentration, specifically, a nitrate ion concentration of 10 to 30 mmol / L, for example, 10 to 25 mmol / L, or 15 to 20 mmol / L. Other components, such as phosphorus and potassium, can be adjusted appropriately by those skilled in the art depending on the legume being cultivated. For example, a nutrient solution containing various components (nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, iron, manganese, etc.) blended in proportions suitable for typical plants can be used. In a more specific embodiment, the nutrient solution in this embodiment is a 1 / 3 to 2 / 1 Hoagland nutrient solution. In an even more specific embodiment, the nutrient solution in this embodiment is a Hoagland nutrient solution concentrate. Hoagland nutrient solution is known to those skilled in the art, and an example of a specific method for preparing it is shown below.

[0018] Hoagland nutrient solution composition Stock solution H1 (diluted 200 times to make a 1 / 1 Hoagland solution) KNO3: 101 g / L (= 1M) Ca(NO3)2.4H2O: 236 g / L (= 1M) Stock solution H2 (diluted 500 times to make a 1 / 1 Hoagland solution) MgSO4.7H2O: 246 g / L (= 1M) Stock solution H3 (diluted 1,000 times to make a 1 / 1 Hoagland solution) NH4H2PO4: 115 g / L (= 1M) Stock solution H4 (diluted 10,000 times to make a 1 / 1 Hoagland solution) H3BO3: 1.85 g / L (= 0.03M) MnCl2.4H2O: 0.99 g / L (= 0.005M) (NH4)6Mo7O24·4H2O: 12.36g / L (=0.01M) ZnSO4·7H2O: 1.15g / L (=0.004M) Fe stock solution (dilute 10,000 times to make 1 / 1 Hoagland solution when used) Fe(III)-EDTA: 42.1g / 500mL (=0.2M) Store away from light with aluminum foil Cu stock solution (dilute 10,000 times to make 1 / 1 Hoagland solution when used) CuSO4·5H2O: 0.25g / 500mL (=0.002M) Store away from light with aluminum foil Mix the above stock nutrient solution in the following proportions, and adjust the total volume to 5L with water to obtain Hoagland stock nutrient solution (15mM as nitrate ions). H1: 25mL, H2: 10mL, H3: 5mL, H4: 500μL, Fe: 500μL, Cu: 500μL.

[0019] ■ Cultivation facility The cultivation method of this aspect is typically carried out indoors. In a specific embodiment, the cultivation method of this aspect is carried out in a facility known as a vegetable factory. As a specific example, the facility is equipped with equipment for adjusting temperature, equipment for irradiating light, equipment for circulating nutrient solution, and, if necessary, a device for adjusting carbon dioxide concentration. The indoor facility also includes equipment that transmits sunlight, such as a vinyl greenhouse or a greenhouse. In this case, some or all of the equipment for irradiating light, equipment for adjusting temperature, equipment for adjusting carbon dioxide concentration, etc. may be omitted.

[0020] ■ Spray Culture Method In one embodiment, in the cultivation method of this aspect, it is preferable that 50% or more, for example 80% or more, for example 100% of the roots of the legume plant are exposed to the gas phase. Here, "exposed to the gas phase" means a state in which the plant is not in direct contact with the nutrient solution, or is in temporary contact with the nutrient solution by spraying or the like, but is not in continuous contact. 50% of the roots refers to the portion of the tissue constituting the legume plant that corresponds to 50% by mass, assuming that the entire portion classified as roots is 100% by mass. Examples of methods for exposing 50% or more of the roots to the gas phase include a method in which sufficient space is secured between the lower end of the stem and the nutrient solution, and a method in which a screen such as a net is provided between the roots and the nutrient solution. For standard soybeans, the secured space can be, for example, 30 cm or more.

[0021] ■ Cultivation temperature In one embodiment, the cultivation method of this aspect is carried out by adjusting the temperature to 15 to 35° C., for example, 20 to 25° C. For example, to reproduce the temperature difference between daytime and nighttime, the temperature may be adjusted to 25 to 30° C. during light irradiation and 15 to 20° C. during non-irradiation.

[0022] Light Irradiation Device The light irradiation device used in the cultivation method of this embodiment is not particularly limited, and examples thereof include fluorescent lamps and LED lighting. LED lighting is preferred. In one embodiment, the cultivation method of this embodiment irradiates legume plants with light at an illuminance of 5,000 to 20,000 lux, for example, 8,000 to 10,000 lux.

[0023] ■ Nutrient Solution Replacement In the cultivation method of this embodiment, the nutrient solution may be circulated. In this case, the nutrient solution used may be replaced, for example, every 3 days, 5 days, 1 week, 2 weeks, or 3 weeks. The timing for replacing the nutrient solution can be determined from the remaining salt concentration. There are various methods for checking the salt concentration, but checking it using electrical conductivity is an easy method, for example.

[0024] ■ Plant Height In one embodiment, the plant height of legumes in the cultivation method of this embodiment is 60 cm or less, e.g., 40 cm or less, e.g., 15 to 60 cm, 20 to 50 cm, 25 to 40 cm, 25 to 50 cm, or 25 to 60 cm. A plant height of this level has advantages, such as reduced lodging resistance and the ability to prevent creeping legumes from entangling with each other or their surroundings. Furthermore, when cultivated in a limited space such as a vegetable factory, this level of plant height allows for vertical stacking of cultivation beds to increase yield, which is advantageous for reducing harvest costs. Furthermore, according to this embodiment, even short plants can produce sufficient yields for commercial cultivation. By cultivating legumes using a nutrient solution with a nitrate ion concentration of 10 to 30 mmol / L in an environment that does not restrict root elongation, the present invention enables both short plant height and high yield, even when cultivated at a planting density of 20 to 200 plants per square meter.

[0025] ■ Yield In one embodiment, the yield of mature seeds per unit area of ​​a legume plant in the cultivation method of this aspect can be, for example, 2,000 g or more, 3,000 g or more, or 4,000 g or more per square meter, for example, 2,000 to 6,000 g, 2,500 to 5,000 g, or 3,000 to 4,500 g per square meter.

[0026] ■ Legumes The legumes used in the cultivation method of this embodiment are not particularly limited and broadly include plants classified as legumes. In a more specific embodiment, the legumes are those that form nodules when cultivated in soil. More specific examples of legumes include adzuki beans, cowpeas, mung beans, black gram, rice beans, kidney beans, scarlet beans, lima beans, broad beans, peas, chickpeas, lentils, soybeans, peanuts, and pigeon peas. In a more specific embodiment, the legumes used in the cultivation method of this embodiment are soybeans. In this specification, "seeds of legumes" includes both mature and immature seeds of the aforementioned plants. Furthermore, "edamame" refers to immature soybean seeds. More specifically, "edamame" refers to soybeans harvested between 30 and 40 days after flowering, and "mature soybeans" refers to soybeans harvested 50 days or more after flowering.

[0027] Measurement of Free Amino Acid Content The amount and ratio of free amino acids can be quantified using liquid chromatography mass spectrometry as follows. A legume seed sample, such as freeze-dried powdered mature soybeans or green soybeans, is mixed with 10 volumes (v / w) of hexane and the supernatant is removed. This process is repeated three times to defatted and dried. The sample is then suspended in water at 0.25% by mass or a concentration within the quantification range of the amino acid standard solution described below, and centrifuged (14,000 rpm, 5 minutes) to obtain the supernatant. The supernatant is passed through a 0.2 μm pore size cellulose acetate filter and separated by liquid chromatography. Liquid chromatography is performed using a Discovery HS F5 HPLC column, with the column temperature set to 40°C. Solvents used are solvent A (0.1 vol% formic acid in water) and solvent B (0.1 vol% formic acid in acetonitrile). The gradient conditions were as follows: 100% A to 75% A / 25% B in 5 min, 75% A / 25% B to 65% A / 35% B in 6 min, 65% A / 35% B to 5% A / 95% B in 4 min, 5% A / 95% B to 95% A / 5% B in 4.5 min, and 95% A / 5% B to 100% A in 0.5 min. The flow rate was 250 μL per min. Samples were introduced into the mass spectrometer and analyzed in electrospray ionization positive mode. The analytical conditions for each amino acid are shown in Table 1. Quantitation can be performed using an amino acid mixture standard solution type H (Fujifilm Wako Pure Chemical Industries, Ltd.) as a standard.

[0028]

[0029] ■ Free Amino Acid Amount in Edamame In one embodiment, the edamame obtained by the cultivation method of this embodiment has increased free amino acids compared to edamame obtained by soil cultivation or conventional hydroponic cultivation. In a more specific embodiment, the edamame obtained by the cultivation method of this embodiment may have a free amino acid amount of 20 to 100 mg / g, e.g., 25 to 95 mg / g, or 30 to 90 mg / g, calculated on a dry matter basis. In a more specific embodiment, the edamame obtained by the cultivation method of this embodiment may have a free asparagine amount of 6.0 to 50 mg / g, e.g., 8.0 to 45 mg / g, or 10 to 40 mg / g, calculated on a dry matter basis. In a more specific embodiment, the edamame obtained by the cultivation method of this embodiment may have a free serine amount of 0.40 to 5.0 mg / g, e.g., 0.50 to 4.0 mg / g, or 0.60 to 3.0 mg / g, calculated on a dry matter basis. In a more specific embodiment, the edamame obtained by the cultivation method of this embodiment may have a free alanine content of 1.0 to 25 mg / g, e.g., 2.0 to 20 mg / g, or 3.0 to 15 mg / g, calculated on a dry matter basis. In a more specific embodiment, the edamame obtained by the cultivation method of this embodiment may have a free glutamine content of 0.25 to 10 mg / g, e.g., 0.40 to 8.0 mg / g, 0.60 to 7.0 mg / g, or 0.80 to 6.0 mg / g, calculated on a dry matter basis. In a more specific embodiment, the edamame obtained by the cultivation method of this embodiment may have a free histidine content of 1.2 to 12 mg / g, e.g., 1.5 to 10 mg / g, or 2.0 to 8.0 mg / g, calculated on a dry matter basis. In a more specific embodiment, the edamame obtained by the cultivation method of this embodiment may have a free arginine content of 2.2 to 12 mg / g, e.g., 2.7 to 10 mg / g, or 3.2 to 8.0 mg / g, calculated on a dry matter basis. In a more specific embodiment, the edamame obtained by the cultivation method of this embodiment may have a free threonine content of 0.45 to 4.0 mg / g, e.g., 0.55 to 3.0 mg / g, or 0.70 to 2.0 mg / g, calculated on a dry matter basis.

[0030] Therefore, in one aspect, the present invention provides edamame having the above-mentioned amount of free amino acids.

[0031] In some embodiments, mature soybeans obtained by the cultivation method of this embodiment have increased free amino acids compared to mature soybeans obtained by soil cultivation or conventional hydroponic cultivation. In more specific embodiments, the mature soybeans obtained by the cultivation method of this embodiment may have a free amino acid content of 3.0 to 20 mg / g, e.g., 3.5 to 15 mg / g, or 5.0 to 15 mg / g, calculated on a dry matter basis. In more specific embodiments, the mature soybeans obtained by the cultivation method of this embodiment may have a free asparagine content of 0.30 to 10 mg / g, 0.50 to 7.0 mg / g, or 0.80 to 5.0 mg / g, calculated on a dry matter basis.

[0032] Therefore, in one aspect, the present invention provides mature soybeans having the above-mentioned amounts of free amino acids.

[0033] ■ Effect of increasing free amino acids The above edamame has a higher amount of free asparagine than conventional edamame, and is expected to have effects such as improved endurance and fatigue recovery. The amount of free arginine is also increased, and similar effects such as fatigue recovery are expected. The amount of histidine is also increased, and is expected to have effects such as improving concentration and memory. The amount of threonine is also increased, and is expected to have an effect of improving gastritis, etc. The amount of glutamine is also increased, and is expected to have an effect such as accelerating recovery from muscle fatigue. The amount of alanine is also increased, and is expected to have effects such as fat burning. The amount of serine is also increased, and is expected to have a moisturizing effect on the skin, etc. Furthermore, the amount of free asparagine in the above mature soybeans is higher than in conventional mature soybeans, and is expected to have effects such as improved endurance and fatigue recovery. In a specific embodiment, the above edamame or mature soybeans can be used as raw materials for supplements, etc.

[0034] In one aspect, the present invention provides a method for producing seeds of a legume, which comprises collecting seeds from the legume obtained by the above-mentioned cultivation method aspect. In another aspect, the present invention provides progeny obtained from the seeds obtained by the above-mentioned production method.

[0035] In the above-mentioned immature seed embodiment, mature seed embodiment, production method embodiment, and progeny embodiment, all of the matters explained in the above-mentioned cultivation method embodiment are applied to these embodiments.

[0036] Hereinafter, the embodiments of the present invention will be described in more detail with reference to examples, etc. Unless otherwise specified, "%" and the like in the examples are based on mass.

[0037] Cultivation Example 1: Study of Soybean Spray Culture. The soybean varieties used were Kamifuka, Ajifuka, Sayamusume, and Yukine (all from Snow Brand Seed Co., Ltd.). The spray culture system used was a Sodatec Aero (Yakumo Trading Co., Ltd.). Figure 1 shows an overview of the spray culture system. Mature soybeans of each variety were germinated on absorbed paper towels and transplanted into the spray culture system when the hypocotyls reached approximately 5 cm. The chamber size was 60 cm x 40 cm x 18 cm, with a pump discharge pressure of 80 psi and a pump flow rate of 0.85 L / min. Hoagland nutrient solution concentrate (15 mM nitrate ion) was used as the nutrient solution. The planting density was 25 plants per square meter. The root space available for growth was approximately 43 L. The nutrient solution was sprayed directly onto the roots for 20 seconds every 30 minutes using the spray culture system. The nutrient solution was changed every week. The plants were illuminated with LED lighting at approximately 10,000 lux for 13 days per day, with the temperature adjusted to 25°C during illumination and 20°C during non-illumination. A net was placed 1 cm above the nutrient solution level at the bottom of the chamber to ensure that the majority of the roots, specifically at least 50%, were exposed to the air phase. The electrical conductivity of the nutrient solution was measured using a compact electrical conductivity meter (HORIBA LAQUAtwin EC-33B). It was 2.3 mS / cm immediately after the nutrient solution change and 2.2 mS / cm one week later, indicating no significant reduction in salt content. Furthermore, no nodule formation was observed on the large soybeans using this cultivation method. Each cultivar flowered 31–32 days after sowing. Edamame was ready for harvest approximately 30–40 days after flowering, and mature soybeans were ready for harvest approximately 60–70 days after flowering. Plant heights of all cultivars were within the target range of 30–40 cm (Figure 2). The taproot and most of the lateral roots remained in the air phase above the net. The yield per square meter was 2,346-2,644g.

[0038] Cultivation Example 2: Examination of Cultivation Density Under the same conditions as in Cultivation Example 1, the plant densities were adjusted to 25, 50, 75, and 100 plants per square meter. While there was a tendency for plant height to increase with increasing plant density, in this cultivation example, where a nutrient solution with a high nitrate ion concentration was used, plant heights were all below 60 cm, falling within the desired range. Furthermore, plant heights of 25, 50, and 75 plants per square meter were all below 40 cm (Figure 3).

[0039] The yield of mature soybeans per unit area for each variety is shown in Figure 4. There was no difference in yield between varieties, and as the planting density increased, yield increased, but the slope of the increase gradually became flatter.

[0040] Cultivation Example 3: Hydroponic Cultivation (Hoagland's Solution, 15 mM Nitrate) Soybeans were grown hydroponically under conditions that prevented root exposure to the gas phase. Eight plants (50 plants per square meter) were grown in a 40 cm x 39.6 cm x 26.3 cm Home Hyponica MUSUCO (Kyowa Co., Ltd.) hydroponic system. The nutrient solution and illumination conditions were the same as those in Cultivation Example 1. The root space available for growth was approximately 42 L. The nutrient solution was circulated in a 15 L liquid fertilizer tank and changed weekly. No root nodules were observed on the soybeans. Edamame were ready for harvest approximately 30–40 days after flowering, and mature soybeans were ready for harvest approximately 60–70 days after flowering. Plant heights for all cultivars ranged from 30–40 cm (Figure 5). Yields ranged from 2,766 to 4,093 g per square meter.

[0041] Cultivation Example 4: Hydroponic Cultivation (Standard Nutrient Solution, 7.5 mM Nitrate) Plants were grown densely at 25 plants per square meter in a standard nutrient solution containing 7.5 mM nitrate. The hydroponic cultivation equipment used was the same as in Cultivation Example 3. The root space allowed for growth was approximately 42 L. Cultivation was performed as in Cultivation Example 1, with the following nutrient solution: total nitrogen 115 ppm (same below), ammonia nitrogen 10 ppm, nitrate nitrogen 105 ppm, phosphoric acid (P2O5) 46.5 ppm, potassium (K2O) 188.5 ppm, lime (CaO) 109.5 ppm, magnesium (MgO) 40 ppm, manganese (MnO) 0.5 ppm, boron (B2O3) 0.5 ppm, iron (Fe) 1.45 ppm, copper (Cu) 0.01 ppm, zinc (Zn) 0.02 ppm, and molybdenum (Mo) 0.01 ppm. For all varieties, plant heights ranged from 57 to 74 cm (Figure 5). In this cultivation example, where nutrient solution with a normal nitrate ion concentration was used, some plants exceeded 60 cm in height. Plants exceeding 60 cm were at risk of lodging. Yields per square meter ranged from 811 to 1,966 g.

[0042] Cultivation Example 5: Ultra-High-Density Test Tube Cultivation A test was conducted with reference to Non-Patent Document 3. Two 12-hole test tube racks ("4WAY Flipper," manufactured by INA-OPTIKA) were connected together with the 17 cm x 5 cm top surface to form one set. Six 12 ml culture tubes (manufactured by INA-OPTIKA) with two holes drilled 1 cm and 2 cm from the bottom were inserted at intervals into the 24-hole test tube rack set described above, and then placed on a tray for cultivation. The soybean cultivar "Yukine" was used. Other conditions were the same as in Cultivation Example 1. The root space available for growth was approximately 12 mL. The cultivation density in this test was approximately 353 plants per square meter. Furthermore, the plant height averaged an extremely low 20.9 cm. While the fruit yield in other cultivation examples described herein was over 30 g per plant, this cultivation example only achieved 2.32 g per plant, demonstrating clearly insufficient growth.

[0043] Cultivation Example 6: High-Density Test Tube Cultivation Cultivation was performed in the same manner as Cultivation Example 5, with reference to Non-Patent Document 1. However, one 12 ml culture tube (manufactured by INA-OPTIKA) was inserted into the 24-well test tube rack set described above, which was then placed on a tray for cultivation. The soybean cultivar used was Kamifuka. Other conditions were the same as in Cultivation Example 1. The root space available for growth was approximately 12 ml. The cultivation density in this experiment was approximately 59 plants per square meter. Furthermore, the plant height was extremely low, averaging 21.5 cm. The fruit yield in this cultivation example was only 2.39 g / plant, and growth was clearly insufficient, as in Cultivation Example 5.

[0044] From Cultivation Examples 5 and 6, it was thought that regardless of the cultivation density, in cultivation methods in which the range of root growth is limited, such as in test tubes, the plant body cannot sufficiently absorb the nutrients necessary for growth, resulting in insufficient growth. As mentioned above, these cultivation methods are merely techniques for experimentally obtaining a small number of seeds, and even with the knowledge of these prior art technologies, it is impossible to conceive of any solution to the effect of nitrate ion concentration on plant height, let alone the problem of plant height increasing in high-density cultivation.

[0045] Cultivation Example 7: Investigation of free amino acid content The free amino acid content was measured in edamame harvested 31 to 36 days after flowering and in mature soybeans harvested 60 to 70 days after flowering for each variety. The results for Cultivation Example 1, which was cultivated using a nutrient solution spray culture, are shown in Table 2, and the results for Cultivation Example 3, which was cultivated hydroponically, are shown in Table 3. For comparison, the results for Cultivation Example 4, which was cultivated hydroponically using a normal nutrient solution, are shown in Table 4, and the free amino acid content in edamame and mature soybeans grown in outdoor soil culture is shown in Table 5.

[0046] ■Free amino acid content in edamame and mature soybeans (mature beans) harvested through hydroponic cultivation using a nutrient solution with a high nitrate ion concentration

[0047] ■ Free amino acid content in edamame and mature soybeans (mature beans) harvested through hydroponic cultivation using a nutrient solution with a high concentration of nitrate ions

[0048] ■ Free amino acid content in edamame and mature soybeans (mature beans) harvested through hydroponic cultivation in a conventional nutrient solution

[0049] ■ Free amino acid content in edamame and mature soybeans (mature beans) harvested through soil cultivation

[0050] Table 2, which shows nutrient solution spray culture, and Table 3, which shows hydroponic culture, show increased amounts of free amino acids in edamame and mature soybeans compared to the results in Table 4, which shows hydroponic culture in a normal nutrient solution. In edamame, the amounts of free asparagine, serine, alanine, glutamine, threonine, histidine, and arginine particularly increased. In mature soybeans, the amount of free asparagine particularly increased. Furthermore, Tables 2 and 3 show increased amounts of free amino acids in edamame and mature soybeans compared to Table 5, which was grown in soil. In edamame, the amounts of free asparagine, serine, alanine, glutamine, glutamic acid, histidine, and arginine particularly increased. In mature soybeans, the amount of free asparagine particularly increased.

[0051] Edamame grown in nutrient solution spray culture (Cultivation Example 1), hydroponics (Cultivation Example 3), normal nutrient solution hydroponics (Cultivation Example 4), and soil culture were tasted, and it was found that for all varieties, the spray culture and hydroponics had a richer flavor than the normal nutrient solution hydroponics and soil culture.

[0052] The present invention provides a method for hydroponics of legumes that maintains a plant height suitable for hydroponics even at high planting densities. The present invention also provides green soybeans with increased amounts of free asparagine and free arginine. The present invention can be used in a wide range of fields, including agriculture and the food production industry.

Claims

1. A hydroponic cultivation method for legumes in which plants are grown at a density of 20 to 200 plants per square meter, using a nutrient solution with a nitrate ion concentration of 10 to 30 mmol / L, in an environment that does not restrict the growth of the roots of the plants.

2. The cultivation method according to claim 1, which is spray culture or hydroponic culture.

3. A method for producing seeds of legumes, comprising recovering seeds from plants obtained by the cultivation method according to claim 1 or 2.

4. Edamame, which has a free asparagine content of 6 to 50 mg / g on a dry matter basis.

5. Edamame, which has a free arginine content of 2.2 to 12 mg / g on a dry matter basis.

6. Edamame, which has a free asparagine content of 6 to 50 mg / g on a dry matter basis and a free arginine content of 2.2 to 12 mg / g on a dry matter basis.

7. A method of controlling the height of legumes to less than 60 cm by cultivating them at a density of 20 to 200 plants per square meter using a nutrient solution with a nitrate ion concentration of 10 to 30 mmol / L.

8. A method of cultivating edamame at a density of 20 to 200 plants per square meter using a nutrient solution with a nitrate ion concentration of 10 to 30 mmol / L, which results in a free asparagine content of 6 to 50 mg / g on a dry matter basis.

9. A method of cultivating edamame at a planting density of 20 to 200 plants per square meter using a nutrient solution with a nitrate ion concentration of 10 to 30 mmol / L, resulting in a free arginine content of 2.2 to 12 mg / g on a dry matter basis.

10. A method of cultivating edamame at a planting density of 20 to 200 plants per square meter using a nutrient solution with a nitrate ion concentration of 10 to 30 mmol / L, which will result in a free asparagine content of 6 to 50 mg / g on a dry matter basis and a free arginine content of 2.2 to 12 mg / g on a dry matter basis.

11. A method for increasing the free asparagine content in edamame and mature soybeans by growing them at a density of 20 to 200 plants per square meter in a nutrient solution with a nitrate ion concentration of 10 to 30 mmol / L.

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