Method for hydroponic cultivation of leguminous plant in high-concentration carbon dioxide environment

Abstract

The present invention provides a cultivation method for obtaining, in hydroponic cultivation in an environment with a high carbon dioxide concentration of 600-5,000 ppm, leguminous plant seeds having a high protein content, without lowering the protein content in the leguminous plant seeds. In the hydroponic cultivation of a leguminous plant in an environment with a high carbon dioxide concentration, the concentration of nitrogen dissolved in a nutrient solution is adjusted to 250-400 ppm to increase the protein content in seeds without causing reduction in yield. In addition, the cultivation period is shortened, and the amounts of free aspartic acid and free glutamic acid in the seeds are increased.

Description

Hydroponic Cultivation Method of Leguminous Plants under High-Concentration Carbon Dioxide Environment

[0001] The present invention relates to a hydroponic cultivation method of leguminous plants under a high-concentration carbon dioxide environment.

[0002] Generally, plant cultivation is carried out by soil cultivation in which soil is used as a medium and fertilizers and water are supplied as nutrients and moisture. On the other hand, a cultivation method using a nutrient solution in which liquid fertilizer components and water are mixed as a medium is called hydroponic cultivation.

[0003] In recent years, there has been a movement towards the practical application of so-called vegetable factories. Various vegetables are cultivated in the plant, and the core of which is hydroponic cultivation. As hydroponic cultivation methods for leguminous plants, Patent Document 1 and Non-Patent Document 1 are disclosed. Patent Document 1 aims to provide a cultivation method for leguminous plants capable of harvesting high-quality seeds, and discloses a method having a rhizosphere low-temperature step in which the roots of leguminous plants with enlarged seeds are kept in a cooled state. In Non-Patent Document 1, exploration of cultivation conditions suitable for hydroponic cultivation of edamame has been carried out, and cultivation density, sunshine hours, etc. have been studied.

[0004] In cultivation in vegetable factories, promoting the growth of crops is an important factor leading to improved productivity. Generally, when carbon dioxide is at a high concentration during crop cultivation, the growth of plants is promoted and productivity is increased. As an example of hydroponically cultivating leguminous plants under high-concentration carbon dioxide, Non-Patent Document 1 discloses the content of hydroponically cultivating edamame. In addition, Non-Patent Document 2 reports on the effects of high-concentration carbon dioxide on the growth of soybeans.

[0005] Japanese Unexamined Patent Application Publication No. 2021-78390

[0006] Kazuhiro Shoko et al., "Practical Application of Vegetable Factory (2) Hydroponic Cultivation Conditions of Edamame", Report of the Central Research Institute of Electric Power, Research Report: U91033, September 1991; Frontiers in Plant Science, (2018) Vol.9, Article 1413; Takashi Oyama, "Nitrate Absorption Metabolism and Nitrogen Fixation in Soybeans", Chemistry and Biology, Vol.29 (1991) p.433-443

[0007] In the cultivation of leguminous plants, a problem arises when the concentration of carbon dioxide in the cultivation environment is high, which leads to a decrease in the protein content of the seeds. Since leguminous seeds generally have high nutritional value as a source of protein, a decrease in protein content leads to a decline in seed quality. Non-patent document 1 describes the hydroponic cultivation of edamame under high-concentration carbon dioxide conditions, but does not mention the protein content of the seeds. Non-patent document 2 describes the decrease in protein content of soybeans due to increased carbon dioxide concentration, but does not disclose any solutions. Furthermore, non-patent document 2 suggests that the decrease in protein content due to increased carbon dioxide concentration cannot be solved by increasing the nitrogen supply. In addition, as described in non-patent document 3, it is known that attempting to apply large amounts of nitrogen fertilizer can lead to excessive growth of stems and leaves, lodging, poor fruit setting, and ultimately a reduction in yield. In vegetable factories, it is necessary to harvest high-quality crops in a short period of time, but for leguminous seeds, it is a difficult challenge to achieve both accelerated growth through carbon dioxide addition and the acquisition of high-quality seeds with high protein content.

[0008] The problem that this invention aims to solve is to provide a cultivation method for obtaining high-protein-content leguminous plant seeds without reducing the protein content in the seeds during hydroponic cultivation under a high-carbon dioxide environment of 600 to 5,000 ppm. In another embodiment, this invention aims to obtain novel leguminous plant seeds, specifically leguminous plant seeds with increased amounts of free aspartic acid and free glutamic acid, which are related to umami flavor.

[0009] The inventors diligently conducted research to address the above-mentioned problems. As a result, they discovered that, surprisingly, in hydroponic cultivation of leguminous plants under high-concentration carbon dioxide conditions, unlike cultivation under normal carbon dioxide conditions, deliberately increasing the dissolved nitrogen concentration in the nutrient solution to 250-400 ppm increases the protein content in the seeds without reducing yield, shortens the time to harvest, and further increases the amount of free aspartic acid and free glutamic acid in the seeds, thus solving the above-mentioned problems and completing the present invention.

[0010] In other words, the present invention relates to (1) a cultivation method for leguminous plants in which a nutrient solution containing 250 to 400 ppm of dissolved nitrogen is used in hydroponic cultivation of leguminous plants in which the carbon dioxide concentration in the cultivation environment during light irradiation is 600 to 5,000 ppm. (2) the cultivation method according to (1), wherein the nutrient solution cultivation method is hydroponics. (3) a method for producing seeds of leguminous plants, comprising cultivating leguminous plants in a nutrient solution containing 250 to 400 ppm of dissolved nitrogen in an environment where the carbon dioxide concentration in the environment during light irradiation is 600 to 5,000 ppm, and recovering seeds from the obtained plants. (4) the cultivation method according to (3), wherein the nutrient solution cultivation method is hydroponics. (5) a method for increasing the protein content in seeds of leguminous plants cultivated in which a carbon dioxide concentration in the environment during light irradiation is 600 to 5,000 ppm, using a nutrient solution containing 250 to 400 ppm of dissolved nitrogen. (6) A method for shortening the cultivation period of leguminous plants using a nutrient solution containing 250-400 ppm of dissolved nitrogen, with an ambient carbon dioxide concentration of 600-5,000 ppm during light irradiation. (7) A method for increasing free aspartic acid and free glutamic acid in the seeds of leguminous plants using a nutrient solution containing 250-400 ppm of dissolved nitrogen, with an ambient carbon dioxide concentration of 600-5,000 ppm during light irradiation. (8) Edamame with a total amount of free aspartic acid and free glutamic acid of 10-30 mg / g on a dry matter basis. (9) Edamame with a free glutamic acid content of 9.0-20 mg / g on a dry matter basis.

[0011] According to the present invention, in cultivation methods where the environment is controlled, such as in vegetable factories, high-protein-content leguminous seeds can be harvested in a short period of time under cultivation conditions with increased carbon dioxide concentration. In another embodiment, the present invention makes it possible to provide leguminous plant seeds with increased levels of free aspartic acid and free glutamic acid.

[0012] ■Nutrient-Based Cultivation In this specification, nutrient-based cultivation refers to a cultivation method that uses a nutrient solution in which nutrients necessary for plant growth are dissolved in a liquid, without using soil. Specific examples of nutrient-based cultivation include hydroponics, solid-substrate cultivation, and spray cultivation. In this specification, hydroponics refers to a cultivation method in which part or all of the roots of the plant to be cultivated are immersed in the nutrient solution. In this specification, solid-substrate cultivation refers to a cultivation method that uses various solid substrates instead of soil. In this specification, spray cultivation refers to a cultivation method in which part or all of the roots of the plant to be cultivated, which are exposed to the gas phase, are directly sprayed with nutrient solution for a certain period of time. The nutrient-based cultivation of the present invention refers to the above-mentioned hydroponics, solid-substrate cultivation, spray cultivation, etc., and the cultivation method of nutrient-based cultivation is not particularly limited, however, in a certain embodiment, the nutrient-based cultivation in the cultivation method of this embodiment is hydroponics.

[0013] ■ Leguminous Plants The leguminous plants used in the cultivation method of this embodiment are not particularly limited and include a wide range of plants classified as legumes. In a more specific embodiment, the leguminous plants are those that form root nodules when cultivated in soil. More specific examples of leguminous plants include adzuki beans, cowpeas, mung beans, black beans, wild beans, kidney beans, red beans, lima beans, broad beans, peas, chickpeas, lentils, soybeans, peanuts, pigeon peas, etc. In a more specific embodiment, the leguminous plant used in the cultivation method of this embodiment is the soybean. In this specification, the seeds of leguminous plants include both mature and immature seeds of the aforementioned plants.

[0014] ■Cultivation Equipment The cultivation method of this embodiment is typically carried out indoors. In a specific embodiment, the cultivation method of this embodiment is carried out in a facility known as a vegetable factory. As a specific example, the facility includes equipment for regulating temperature, equipment for irradiating light, equipment for circulating nutrient solution, equipment for regulating carbon dioxide concentration, etc. The indoors also includes facilities that allow sunlight to pass through, such as greenhouses and plastic greenhouses. In this case, some or all of the equipment for irradiating light, equipment for regulating temperature, etc., may be omitted.

[0015] ■Cultivation Temperature In one embodiment, the cultivation method of this embodiment is carried out with the temperature adjusted to 15 to 35°C, for example, 20 to 25°C. For example, in order to reproduce the temperature difference between day and night, the temperature may be adjusted to 25 to 30°C when light is being irradiated and to 15 to 20°C when light is not being irradiated.

[0016] ■Light Irradiation Device The light irradiation device used in the cultivation method of this embodiment is not particularly limited, and examples include fluorescent lamps and LED lighting, but LED lighting is preferred. In one embodiment, in the cultivation method of this embodiment, leguminous plants are irradiated with light at an illuminance of 5,000 to 20,000 lux, for example, 8,000 to 10,000 lux.

[0017] ■Carbon Dioxide Concentration In this embodiment, the carbon dioxide concentration during light irradiation in the cultivation environment refers to the carbon dioxide concentration within a radius of 20 cm from the plant body of a leguminous plant during light irradiation. Adjustment of the carbon dioxide concentration in the cultivation environment can be achieved by a device such as a carbon dioxide sensor that measures carbon dioxide concentration and a carbon dioxide supply device such as a carbon dioxide generator. In this invention, a high-concentration carbon dioxide environment refers to a concentration higher than the carbon dioxide concentration in the general atmosphere, specifically 600 ppm or higher. In this embodiment, the carbon dioxide concentration is 600 ppm or higher as an average concentration during light irradiation, preferably 700 ppm or higher, more preferably 800 ppm or higher, even more preferably 900 ppm or higher, and even more preferably 950 ppm or higher. Furthermore, from the viewpoint of the working environment when working in the facility, the average carbon dioxide concentration during light irradiation is 5,000 ppm or less, preferably 4,000 ppm or less, more preferably 3,000 ppm or less, even more preferably 2,000 ppm or less, and even more preferably 1,500 ppm or less. The range of carbon dioxide concentrations during light irradiation is 600–5,000 ppm, 700–4,000 ppm, 800–3,000 ppm, 900–2,000 ppm, and 950–1,500 ppm. In this specification, 400 ppm is referred to as the atmospheric carbon dioxide concentration condition. This concentration is based on the assumed atmospheric carbon dioxide concentration.

[0018] ■ Dissolved Nitrogen Concentration in the Nutrient Solution In this embodiment, the nutrient solution is a nutrient solution with a high concentration of dissolved nitrogen, specifically a nutrient solution with a dissolved nitrogen concentration of 250-400 ppm, 254-350 ppm, 258-300 ppm, 262-300 ppm, 262-280 ppm, or 262-270 ppm. Since a low nitrogen content in the nutrient solution leads to a decrease in the amount of protein in the seeds, the dissolved nitrogen concentration is 250 ppm or higher, preferably 254 ppm or higher, more preferably 258 ppm or higher, and even more preferably 262 ppm or higher. Furthermore, since excessively high nitrogen content leads to excessive growth of the above-ground parts, causing flower drop and reduced yield, the dissolved nitrogen concentration is 400 ppm or lower, preferably 350 ppm or lower, more preferably 320 ppm or lower, more preferably 300 ppm or lower, even more preferably 280 ppm or lower, and even more preferably 270 ppm or lower. Other components, such as phosphorus and potassium, can be appropriately adjusted by those skilled in the art depending on the leguminous plants being cultivated. For example, a nutrient solution containing various components (nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, iron, manganese, etc.) blended in proportions suitable for general plants can be used.

[0019] ■Measurement of Dissolved Nitrogen Concentration The dissolved nitrogen concentration in the nutrient solution can be measured as follows. The amount of nitrogen in the supernatant after centrifuging a homogenized nutrient solution at 8,000 × g for 5 minutes is analyzed by ion chromatography. For ion chromatography, a 940 Professional IC Vario (Metrohm) and an electrical conductivity meter are used as detectors. Cation analysis is performed as follows. An IC column Metroses C6-150 / 4.0 is used as the column, and a guard column Metroses C6 Guard / 4.0 is used, with the column oven set to 30°C. A 1.7 mM nitric acid - 1.7 mM dipicolinic acid mixed solution is used as the mobile phase, with the flow rate set to 0.9 mL / min, and 20 μl of sample is injected. Cation mixed standard solution I (mixed cation species Ca) is used as the standard. 2+ Mg 2+ na + ) (Fujifilm Wako Pure Chemical Corporation) and Cation Mixed Standard Solution II (Mixed Cation Species Na + NH4 + , K +Quantitative analysis is performed using (Fujifilm Wako Pure Chemical Industries, Ltd.). Anion analysis is performed as follows: An IC column Metrosep A Supp 5 250 / 4.0 is used as the column, and a guard column Metrosep A Supp 5 Guard / 4.0 is used, with the column oven set to 35°C. A 3.2 mM sodium nitrate - 1.0 mM sodium bicarbonate mixture is used as the mobile phase, with the flow rate set to 0.7 mL / min, and 20 μL of sample is injected. A CO2 suppressor is used, and 100 mM sulfuric acid is used as the regeneration solution. Sodium nitrate (Fujifilm Wako Pure Chemical Industries, Ltd.) and potassium dihydrogen phosphate (Fujifilm Wako Pure Chemical Industries, Ltd.) are used as standards for quantitative analysis. In this embodiment, the dissolved nitrogen concentration in the nutrient solution is the sum of the nitrogen concentrations contained in the ammonium ions and nitrate ions measured above.

[0020] ■Seed Yield In one embodiment, the seed yield per unit area of ​​leguminous plants in the cultivation method of this embodiment is, for example, 1,000g or more, 1,300g or more, and 1,600g or more per square meter. For example, it can be 1,000 to 5,000g, 1,300 to 4,000g, and 1,600 to 3,000g per square meter.

[0021] ■Immature Seeds and Mature Seeds In this specification, immature seeds of leguminous plants refer to seeds of leguminous plants that are immature but mature enough to be used for food. Specific examples include edamame for soybeans and green peas for peas. As a more specific example, in this specification, immature seeds of soybeans refer to seeds harvested between 30 and 40 days after flowering, and are called edamame. Mature seeds of soybeans refer to seeds harvested 50 days or more after flowering, and are called mature soybeans.

[0022] ■Cultivation Period In the cultivation method of this embodiment, cultivating leguminous plants in a nutrient solution with a high dissolved nitrogen concentration under a high carbon dioxide environment can shorten the cultivation period from sowing to seed harvest. For example, in the cultivation method of this embodiment, it is possible to shorten the cultivation period after sowing of mature soybeans by 5 days or more, 7 days or more, 9 days or more, and 11 days or more. For example, under conditions where the dissolved nitrogen concentration in the nutrient solution is 250 to 400 ppm and the carbon dioxide concentration during light irradiation is 600 to 5,000 ppm, harvesting can be exemplified within 80 days, 78 days, 76 days or within 74 days after sowing. Furthermore, the cultivation period after flowering can also be shortened by 5 days or more, 7 days or more, 9 days or more, and 11 days or more. For example, under conditions where the dissolved nitrogen concentration in the nutrient solution is 250 to 400 ppm and the carbon dioxide concentration during light irradiation is 600 to 5,000 ppm, harvesting can be exemplified within 50 days, 48 ​​days or within 46 days or within 44 days after flowering.

[0023] ■In an embodiment with a cultivation density, the cultivation density of leguminous plants in the cultivation method of this embodiment is 20 to 120 plants, 25 to 100 plants, 30 to 90 plants, and 40 to 80 plants per square meter. By setting the cultivation density within the above range, the height of the leguminous plants can be suppressed, which is advantageous for cultivation in plant factories, etc.

[0024] ■ Nutrient Solution Replacement In this cultivation method, the nutrient solution may be circulated. In this case, the nutrient solution may be replaced, for example, every three days, every five days, every week, every two weeks, or every three weeks. The timing of nutrient solution replacement can be determined from the remaining salt concentration. There are various methods for checking the salt concentration, but for example, checking the electrical conductivity is an easy method.

[0025] ■Measurement of Protein Content The protein content in seeds can be analyzed using the Dumas method. Specifically, the sample is freeze-dried into a powder and analyzed using rapid MAX N exceed (ELEMENTAR). The nitrogen-to-protein conversion factor used to calculate the protein amount is 6.25.

[0026] ■Protein content of mature seeds of leguminous plants cultivated under high-concentration carbon dioxide conditions Mature seeds of leguminous plants obtained by hydroponic cultivation according to this embodiment under high-concentration carbon dioxide conditions have a higher protein content compared to mature seeds obtained by conventional hydroponic cultivation. For example, mature soybeans grown using this cultivation method may have a protein content of 35-47% by mass, 36-45% by mass, and 37-43% by mass on a dry matter basis.

[0027] ■Protein content of immature seeds of leguminous plants cultivated under high-concentration carbon dioxide conditions Immature seeds of leguminous plants obtained by hydroponic cultivation according to this embodiment under high-concentration carbon dioxide conditions have a higher protein content compared to immature seeds obtained by conventional hydroponic cultivation. For example, edamame grown using this cultivation method may have a protein content of 31-40% by mass, 32-38% by mass, and 33-36% by mass on a dry matter basis.

[0028] ■Measurement of Free Amino Acid Amount The amount and ratio of free amino acids can be quantified as follows using liquid chromatography-mass spectrometry. A seed sample of a leguminous plant, such as a freeze-dried mature soybean or edamame powder sample, is mixed with 10 times the amount (v / w) of hexane, and the supernatant is removed. This is repeated three times to degrease and dry the sample, and then the sample is suspended in water at a concentration of 0.25% by mass or within the quantitative range of the amino acid mixed standard solution described later. The sample is then centrifuged (14,000 rpm for 5 minutes) to obtain the supernatant. The supernatant is passed through a cellulose acetate filter with a pore size of 0.2 μm and separated by liquid chromatography. A Discovery HS F5 HPLC column is used for liquid chromatography, and the column temperature is set to 40°C. Solvents A (0.1 vol% formic acid-water) and solvent B (0.1 vol% formic acid-acetonitrile) are used as solvents. The gradient conditions were as follows: from A100% to A75% / B25% for 5 minutes, from A75% / B25% to A65% / B35% for 6 minutes, from A65% / B35% to A5% / B95% for 4 minutes, from A5% / B95% to A95% / B5% for 4.5 minutes, and from A95% / B5% to A100% for 0.5 minutes, with a flow rate of 250 μL per minute. The sample was introduced into a mass spectrometer and analyzed in electrospray ionization positive mode. The analytical conditions for each amino acid are shown in Table 1, but quantification can be performed using amino acid mixed standard solution type H (Fujifilm Wako Pure Chemical Industries, Ltd.) as a standard.

[0029]

[0030] ■Amount of Free Aspartic Acid and Free Glutamic Acid in Immature Seeds In one embodiment, immature seeds of leguminous plants obtained by the cultivation method of this embodiment have increased amounts of free aspartic acid and free glutamic acid compared to immature seeds obtained by conventional hydroponic cultivation. In a more specific embodiment, edamame obtained by the cultivation method of this embodiment may have a free aspartic acid amount of 1.5 to 10 mg / g, 2.0 to 7.5 mg / g, or 2.5 to 5.0 mg / g on a dry matter basis, and a free glutamic acid amount of 9.0 to 20 mg / g, 9.3 to 16 mg / g, or 9.6 to 12 mg / g. In addition, the total amount of free glutamic acid and free aspartic acid may be 10 to 30 mg / g, 11 to 25 mg / g, or 12 to 20 mg / g. However, the above amounts of aspartic acid do not include asparagine, and the amount of glutamic acid does not include glutamine.

[0031] Therefore, in one embodiment, the present invention provides edamame having the above-mentioned amount of free amino acids.

[0032] ■Amount of Free Aspartic Acid and Free Glutamic Acid in Mature Seeds In one embodiment, mature seeds of leguminous plants obtained by the cultivation method of this embodiment have increased amounts of free aspartic acid and free glutamic acid compared to mature seeds obtained by conventional hydroponic cultivation. In a more specific embodiment, mature soybeans obtained by the cultivation method of this embodiment may have a free aspartic acid amount of 1.5 to 10 mg / g, 2.0 to 7.5 mg / g, or 2.5 to 5.0 mg / g on a dry matter basis, and a free glutamic acid amount of 1.5 to 9.0 mg / g, 2.0 to 7.0 mg / g, or 2.5 to 5.0 mg / g. In addition, the total amount of free glutamic acid and free aspartic acid may be 2.0 to 12 mg / g, 3.0 to 10 mg / g, or 4.0 to 8.0 mg / g. However, the above amounts of aspartic acid do not include asparagine, and the amount of glutamic acid does not include glutamine.

[0033] Therefore, in one aspect, the present invention provides mature soybeans having the above-mentioned amount of free amino acids.

[0034] ■ Increased Effect of Free Aspartic Acid and Free Glutamic Acid The above immature or mature seeds have increased levels of free aspartic acid and free glutamic acid compared to conventional immature or mature seeds, and an improvement in taste due to the increase in umami components can be expected.

[0035] In one embodiment, the present invention provides a method for producing seeds of leguminous plants, which includes recovering seeds from leguminous plants obtained by the above-described cultivation method. In another embodiment, the present invention provides progeny obtained from seeds obtained by the above-described production method.

[0036] In the above-described embodiments of immature seeds, mature seeds, manufacturing methods, and subsequent generations, all matters described in the above-described embodiments of cultivation methods apply to these embodiments.

[0037] The present invention will be specifically described below by the examples provided. Unless otherwise specified, percentages such as "%" in the examples are based on mass.

[0038] ■ Cultivation Example 1: Examination of nutrient solution with high nitrogen concentration under atmospheric carbon dioxide concentration conditions. The soybean variety Kamikaze Kaori (Snow Brand Seed Co., Ltd.) was used. The home hydroponics MASUCO (Kyowa Co., Ltd.) was used as the hydroponic cultivation device. The seeds of Kamikaze Kaori were germinated on a paper towel that had absorbed water, and when the hypocotyl had elongated to about 5 cm, they were transplanted into the hydroponic cultivation device. The nutrient solution with the composition described in Table 2 (Comparative Example 1: 230 ppm, Reference Example: 256 ppm) was used and replaced once a week. The hydroponic cultivation device was installed in an artificial weather chamber LPH-411PFDT-SPC (Nippon Ika Kikai Seisakusho Co., Ltd.), and LED lighting of about 10,000 lux was irradiated for 13 hours a day under the conditions of a plant flex bulb color (40W type), total luminous flux of 1,400 lm, and photosynthetic photon flux density of 280 μmol / m^2 / s. The temperature during irradiation was adjusted to 25 °C, and the temperature during non-irradiation was adjusted to 20 °C. The humidity was adjusted within the range of 60 - 70%. The cultivation density was 50 plants per square meter. When the electrical conductivity of the nutrient solution was measured using a compact electrical conductivity meter (Aquatint EC-33B, manufactured by HORIBA), it was 2.6 mS / cm immediately after nutrient solution replacement and 2.5 mS / cm after one week, and no significant decrease in salts was observed. Also, the carbon dioxide concentration was adjusted to 400 ppm. However, carbon dioxide was not supplied during non-irradiation of the LED lighting. The mature soybeans (51 days after flowering) were harvested respectively under the above conditions. The cultivation period was 82 days for all.

[0039]

[0040]

[0041] As shown in Table 3, when the nitrogen concentration in the nutrient solution was increased from 230 ppm (Comparative Example 1) to 256 ppm (Reference Example), a large number of flower drop was observed, and the number of seeds per plant decreased significantly. From this result, it was confirmed that increasing the nitrogen content in the nutrient solution would lead to a reduction in yield. This result was similar to the content described in Non-Patent Document 3.

[0042] ■ Cultivation Example 2: Examination of High-Nitrogen Concentration Nutrient Solution under High-Concentration Carbon Dioxide Conditions (Mature Soybeans) Using Kamikaze Fragrance (Snow Brand Seed Co., Ltd.) as the soybean variety, the test was conducted in the same manner as in Cultivation Example 1. However, the carbon dioxide concentration was adjusted to 400 ppm or 1,000 ppm, and the supply of carbon dioxide concentration when not irradiated with LED lighting was not carried out. Also, the nutrient solution with the composition described in Table 2 (Comparative Examples 1 and 2: 230 ppm, Example 1: 267 ppm) was used. Cultivation was carried out under the above conditions, and mature soybeans were harvested.

[0043]

[0044] As shown in Table 4, when the carbon dioxide concentration was increased from 400 ppm (Comparative Example 1) to 1,000 ppm (Comparative Example 2), the number of seeds and seed weight per plant increased, but the protein content of mature soybeans decreased significantly from 36.6% by mass to 33.6% by mass. On the other hand, even under the condition of a carbon dioxide concentration of 1,000 ppm, when the dissolved nitrogen concentration in the nutrient solution was deliberately increased from 230 ppm (Comparative Example 1) to 267 ppm (Example 1), as shown in Example 1, the number of seeds and seed weight per plant remained high, while the protein content improved significantly to 40.6% by mass. Furthermore, the protein content of Example 1 increased by 7% compared to Comparative Example 2. Despite the dissolved nitrogen concentration of 267 ppm in Example 1 being higher than the 256 ppm in the reference example shown in Table 3, no flower drop or yield reduction was observed, as seen in the reference example. In addition, the cultivation period from sowing to harvest in Example 1 was shortened to 73 days compared to 84 days in the control, Comparative Example 2. In particular, the number of days from flowering to harvest was 52 days and 54 days in Comparative Examples 1 and 2, respectively, while it was shortened to 43 days in Example 1. When the number of days required for one cultivation cycle is 84 days, the number of harvests per year is 4, but when it is 73 days, it becomes possible to harvest 5 times a year, and a significant improvement in productivity in vegetable factories can be expected. As shown in Table 3, at a carbon dioxide concentration of 400 ppm, increasing the dissolved nitrogen concentration in the nutrient solution (reference example) had adverse effects on cultivation such as flower drop and yield reduction. On the other hand, under the conditions of a carbon dioxide concentration of 1,000 ppm in this cultivation example (Example 1), it was found that deliberately increasing the dissolved nitrogen concentration in the nutrient solution did not cause a yield reduction, but rather increased the protein content of the seeds, resulting in improved seed quality compared to the control. In addition, it was found that the cultivation period was shortened, leading to improved productivity.

[0045] ■Cultivation Example 3: Investigation of High Nitrogen Concentration Nutrient Solution under High Carbon Dioxide Environment (Edamame) Using the soybean variety Kamikaze-ka (Snow Brand Seed Co., Ltd.), the experiment was conducted in the same manner as in Cultivation Example 2. In addition, Example 3 was conducted using nutrient solution C, which has a high proportion of ammoniacal nitrogen, and Example 4 was conducted using nutrient solution D, which has a high nitrogen concentration of 304 ppm. Edamame were harvested 31 days after flowering under the above conditions. The cultivation period for all cases was 62 days.

[0046]

[0047] Table 5 shows the protein content of edamame obtained under each condition. When the control nutrient solution was used, increasing the carbon dioxide concentration in the cultivation environment from 400 ppm (Comparative Example 3) to 1,000 ppm (Comparative Example 4) significantly decreased the protein content of edamame from 33.2% by mass to 29.8% by mass on a dry matter basis. However, by using nutrient solutions B and C, which have higher dissolved nitrogen concentrations, the protein content of edamame improved significantly to 34.5% by mass and 36.1% by mass, respectively (Examples 2 and 3). Compared to Comparative Example 4, the protein content of Example 2 increased by 4.7%, and that of Example 3 increased by 6.3%. Example 4, which used solution D with an even higher nitrogen concentration, showed a 3.3% increase. These results are similar to those for mature soybeans described above, and it is clear that increasing the dissolved nitrogen concentration in the nutrient solution improves the protein content of seeds even at the edamame stage.

[0048] ■Free amino acid composition of edamame and mature soybeans The free amino acids in edamame and mature soybeans were measured for Examples 1 and 2, and Comparative Examples 1, 2, 3, and 4.

[0049] Table 6 shows the analysis results for each free amino acid. In Examples 1 and 2, where the dissolved nitrogen concentration in the nutrient solution was increased to 267 ppm, both mature soybeans and edamame showed a tendency for increased free aspartic acid and free glutamic acid compared to the comparative examples where the dissolved nitrogen concentration was 230 ppm. Specifically, the free aspartic acid content was 3.0 mg / g or more by dry weight in both mature soybeans and edamame, which was a significant increase compared to the comparative examples. The aspartic acid content in Example 1 increased by 2.6 mg / g compared to the aspartic acid content of Comparative Example 1 and by 2.27 mg / g compared to the aspartic acid content of Comparative Example 2. Furthermore, the aspartic acid content in Example 2 increased by 1.9 mg / g compared to the aspartic acid content of Comparative Example 3 and by 2.42 mg / g compared to the aspartic acid content of Comparative Example 4. Regarding the free glutamic acid content, it was 9.96 mg / g in edamame and 2.96 mg / g in mature soybeans, showing an increase compared to each comparative example. The glutamic acid content of Example 1 increased by 1.97 mg / g compared to Comparative Example 1 and by 2.31 mg / g compared to Comparative Example 2. Furthermore, the glutamic acid content of Example 2 increased by 1.57 mg / g compared to Comparative Example 3 and by 2.96 mg / g compared to Comparative Example 4. In addition, the total amount of free glutamic acid and free aspartic acid, which are amino acids related to umami, was higher than in each comparative example, at 12.99 mg / g in edamame and 6.16 mg / g in mature soybeans, with a particularly significant increase in mature soybeans.

[0050] This invention provides a cultivation method for obtaining leguminous seeds with high protein content in a short period of time, even under high-carbon dioxide environments. Furthermore, this invention provides leguminous seeds with increased free aspartic acid and free glutamic acid. This invention is applicable to a wide range of fields, including agriculture and food production.

Claims

1. A cultivation method for hydroponic cultivation of leguminous plants where the carbon dioxide concentration during light irradiation in the cultivation environment is 600 to 5,000 ppm, using a nutrient solution containing 250 to 400 ppm of dissolved nitrogen.

2. The cultivation method according to claim 1, wherein the nutrient solution cultivation method is hydroponics.

3. A method for producing seeds of leguminous plants, comprising cultivating leguminous plants hydroponically using a nutrient solution containing 250 to 400 ppm of dissolved nitrogen, with an ambient carbon dioxide concentration of 600 to 5,000 ppm during light irradiation, and recovering seeds from the resulting plants.

4. The cultivation method according to claim 3, wherein the nutrient solution cultivation method is hydroponics.

5. A method for increasing the protein content in the seeds of leguminous plants by cultivating them using a nutrient solution containing 250–400 ppm of dissolved nitrogen, under environmental conditions where the carbon dioxide concentration during light irradiation is 600–5,000 ppm.

6. A method for shortening the cultivation period of leguminous plants, using a nutrient solution containing 250–400 ppm of dissolved nitrogen, while cultivating them at an ambient carbon dioxide concentration of 600–5,000 ppm during light irradiation.

7. A method for increasing free aspartic acid and free glutamic acid in the seeds of leguminous plants, using a nutrient solution containing 250–400 ppm of dissolved nitrogen, and cultivating them under environmental conditions where the carbon dioxide concentration during light irradiation is 600–5,000 ppm.

8. Edamame beans with a total amount of free aspartic acid and free glutamic acid of 10-30 mg / g on a dry matter basis.

9. Edamame with a free glutamic acid content of 9.0–20 mg / g on a dry matter basis.

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