Method for increasing KODA accumulation in plant, KODA production system, and method for producing plant with increased stress resistance

Abstract

To provide: a method for increasing the accumulation of 9,10-ketol-octadecadienoic acid (KODA) in plant individuals, tissues, or cells; a KODA production system; and a method for producing plants with increased stress resistance.SOLUTION: The present invention provides a method for increasing accumulation of KODA that includes a step of maintaining plant individuals, tissues, or cells in a medium under a phosphorus-deficient condition. The present invention also provides a system for producing KODA that includes plant individuals, tissues, or cells, including the 9-LOX gene and / or AOS gene, maintained in a medium with a dissolved phosphorus level of less than 1 mM. The present invention also provides a method for producing a plant with increased stress resistance that includes the steps of: introducing the 9-LOX gene and / or AOS gene to the plant and cultivating the plant in a medium under the phosphorus-deficient condition.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to methods and systems for increasing accumulation of 9,10-α-ketol linolenic acid (KODA) in plants. The present disclosure also relates to methods for producing plants with increased stress tolerance. [Background technology]

[0002] 9,10-α-ketol linolenic acid (also written as 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid or 9,10-ketol-octadecadienoic acid, abbreviated as KODA) is a physiologically active substance biosynthesized in plants, and is known to confer environmental stress resistance (to drought, disease, etc.) to plants and promote plant growth. Due to these remarkable properties, there are strong hopes for the practical application of KODA in the agricultural field. Non-Patent Document 1 reports a method for chemically synthesizing KODA (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Yokokawa et al., “Total Synthesis of α-Ketol Derivative of Linolenic Acid (KODA), a Flower-inducing Factor in Lemna paucicostata”, Chemistry Letters, Vol.32, No.9, pp.844-845. (2003) Summary of the Invention [Problem to be solved by the invention]

[0004] However, the amount of KODA biosynthesis (intracellular accumulation) in plants is generally very small, and no mass production system for KODA has been constructed using plants. The chemical synthesis method of Non-Patent Document 1 requires a large number of reaction steps, and the final yield is very low at 0.81%, so there is a problem in that the cost of KODA production is high. It is also possible to produce KODA in vitro using 9-LOX and AOS enzyme proteins extracted from genetically modified Escherichia coli, etc. However, there is a problem in that the cost is high because it is necessary to add expensive α-linolenic acid as a substrate raw material.

[0005] The present disclosure has been made to solve the problems described above, and its embodiments aim to provide a method for increasing the accumulation of KODA in a plant individual, tissue, or cell, a system for producing KODA, and a method for producing a plant with increased stress tolerance. [Means for solving the problem]

[0006] To achieve the above-mentioned objective, one aspect of the present disclosure is a method for increasing the accumulation of 9,10-α-ketol linolenic acid in a plant individual, tissue, or cell, the method comprising step (a) of maintaining the plant individual, tissue, or cell in a medium under phosphorus-deficient conditions.

[0007] One aspect of the present disclosure is a system for producing 9,10-α-ketol linolenic acid, comprising a plant individual, tissue, or cell containing a gene encoding 9-LOX and / or a gene encoding AOS, maintained in a medium having a dissolved phosphorus concentration of less than 1 mM.

[0008] One aspect of the present disclosure is a method for producing a plant with increased stress tolerance, the method comprising the steps of introducing at least one gene selected from a gene encoding 9-LOX and a gene encoding AOS into a plant, and cultivating the plant into which the gene has been introduced in a medium under phosphorus-deficient conditions. Effect of the Invention

[0009] The present disclosure provides methods for increasing the accumulation of KODA in plant individuals, tissues, or cells, a system for producing KODA, and a method for producing plants with increased stress tolerance. [Brief description of the drawings]

[0010] [Figure 1] The KODA content (μg / g fresh weight) of wild-type (WT), vector control (VC), and transformed strains grown in phosphate-sufficient (+Pi) or phosphate-deficient (-Pi) media is shown. For each strain, the graph on the left is +Pi and the graph on the right is -Pi. [Diagram 2] Photographs of the wild-type (WT), vector control (VC), and transformed strains grown in phosphate-sufficient (+Pi) or phosphate-deficient (-Pi) medium and exposed to high temperature stress (37°C, 4 or 5 days) are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Non-limiting embodiments of the present disclosure will be described below. The present disclosure is not limited to the examples in the following embodiments.

[0012] [Method for increasing KODA accumulation] In one embodiment, a method for increasing the accumulation of 9,10-α-ketol linolenic acid in a plant individual, tissue, or cell is provided, comprising the step (a) of maintaining the plant individual, tissue, or cell in a medium under phosphorus-deficient conditions.

[0013] In the embodiment, the plant is not particularly limited and may include terrestrial plants, aquatic plants, epiphytes, and rock plants. The plant of the embodiment includes, but is not limited to, plants of the Malvaceae, Chenopodiaceae, Rubiaceae, Brassicaceae, Poaceae, Cucurbitaceae, Asteraceae, Mulberry family, Pesamiaceae, Araceae, Solanaceae, Papaya family, Rosaceae, Fabaceae, Rutaceae, Oleaceae, and Liliaceae families. More specific examples of plants according to the embodiment include kiwi fruit (Actinidia deliciosa), celery (Apium graveolens var. dulce), Arabidopsis (Arabidopsis thaliana), beet (Beta vulgaris subsp. Vulgaris), cauliflower (Brassica oleracea var. botrytis), cabbage (Brassica oleracea var. capitata), broccoli (Brassica oleracea var. italica), rapeseed (Brassica rapa), and bok choy (Brassica rapa var.chinensis), tea plant (Camellia sinensis), bell pepper (Capsicum annuum), paprika (Capsicum annuum), safflower (Carthamus tinctorius), lime (Citrus aurantifolia), lemon (Citrus limon), orange (Citrus sinensis), grapefruit (Citrus x paradisi), coconut palm (Cocos nucifera), coffee plant (Coffea arabica), taro (Colocasia esculenta), cucumber (Cucumis sativus), pumpkin (Cucurbita maxima), persimmon (Diospyros kaki), buckwheat (Fagopyrum esculentum), barnyard grass (Echinochloa crus-galli), fig (Ficus carica), strawberry (Fragaria x ananassa), soybean (Glycine max), sunflower (Helianthus annuus), barley (Hordeum vulgare), lettuce (Lactuca sativa), duckweed (Lemna aoukikusa), apple (Malus domestica), banana (Musa spp.), tobacco (Nicotiana tabacum), basil (Ocimum basilicum), olive (Olea europaea), rice (Oryza sativa), avocado (Persea americana), parsley (Petroselinum crispum), beans (Phaseolus vulgaris), peas (Pisum sativum), pears (Pyrus pyrifolia), rosemary (Rosmarinus officinalis), sugarcane (Saccharum officinarum), tomato (Solanum lycopersicum), eggplant (Solanum melongena), potato (Solanum tuberosum), thyme (Thymus vulgaris), wheat (Triticum aestivum), grape (Vitis spp.), and Zea mays. Among these, Arabidopsis thaliana, duckweed, tomato, rapeseed, barnyard grass, or tobacco are more preferred. The Brassicaceae family is preferred, and Arabidopsis thaliana is more preferred.

[0014] In the method of the embodiment, a whole plant may be used, or a part of a whole plant, such as a tissue or a cell, may be used. As known to those skilled in the art, a plant tissue or cell may be maintained as a cultured tissue or a cultured cell.

[0015] The plant individual, tissue, or cell of the above embodiment can be maintained in a medium lacking phosphorus to increase the amount of KODA accumulated. The site where KODA accumulates is typically intracellular, but extracellular accumulation is not excluded. The site where KODA accumulates can be, but is not limited to, flowers, stems, roots, fruits, seeds, embryos, or combinations thereof (e.g., aboveground parts), or callus.

[0016] In an embodiment, 9,10-α-ketol linolenic acid refers to a compound also known as KODA, 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid, or 9,10-ketol-octadecadienoic acid. In this disclosure, 9,10-α-ketol linolenic acid may be referred to simply as "KODA." The structure of KODA is shown below.

[0017] [ka]

[0018] It will be understood by those skilled in the art that KODA may spontaneously exist in ionic or salt form depending on the equilibrium state in a particular aqueous environment. Therefore, KODA in this disclosure should be understood to encompass its ionic and salt forms. Salts of KODA can be, for example, sodium, potassium, lithium, ammonium, or other monovalent cation salts.

[0019] Maintaining a plant individual, tissue, or cell in a medium includes culturing the individual in the medium and culturing the tissue or cell in the medium, as is well known to those skilled in the art. Suitable media for maintaining a plant individual, tissue, or cell in a viable state are known to those skilled in the art, and may be, for example, soil, aqueous solution, agar medium, etc. Maintaining a plant individual, tissue, or cell in a medium lacking phosphorus may include changing the medium in which the plant individual, tissue, or cell was previously maintained to a medium in which the phosphorus concentration is reduced to such an extent that a significant increase in the amount of KODA accumulated in the plant individual, tissue, or cell occurs. The appropriate period for maintaining the plant individual, tissue, or cell in a medium lacking phosphorus may vary depending on the type of plant, tissue, or cell. The period may be, for example, at least 12 hours, at least 24 hours, at least 2 days, at least 3 days, at least 10 days, or at least 1 month. The period may also be, for example, one year or less, one month or less, 10 days or less, 3 days or less, 2 days or less, or 24 hours or less.

[0020] Phosphorus in the present disclosure may be present, for example, in the form of phosphate (or its ions). Thus, for example, the term "phosphorus concentration" does not necessarily mean that phosphorus is present as an elemental substance, but should be understood as the phosphorus atom equivalent concentration. For example, in the context of describing an experiment in which phosphorus is added to a medium only in the form of phosphate, such as in the Examples of the present application, the terms "phosphorus deficiency" and "phosphate deficiency" may be synonymous.

[0021] A medium under phosphorus-deficient conditions is a medium in which the phosphorus concentration has been reduced to a level at which a significant increase in the amount of KODA accumulated in the plant individual, tissue, or cell occurs. In the present disclosure, the phosphorus concentration of the medium can be expressed as the phosphorus atom equivalent concentration of dissolved phosphorus in the aqueous component of the medium. For example, if the medium is soil, the aqueous liquid can be separated from the solid matter in the soil by centrifugation, filtration, etc., and the dissolved phosphorus concentration of the aqueous liquid can be measured. If the medium is an agar medium, the dissolved phosphorus concentration of the aqueous solution before adding agar can be measured. The dissolved phosphorus concentration of the medium under phosphorus-deficient conditions can be specifically, for example, less than 1 mM, preferably 0.1 mM or less, more preferably 0.01 mM or less, and may be 0 mM.

[0022] Table 1 illustrates the composition of one of the media used for growing plants.

[0023] [Table 1]

[0024] For example, in the medium composition of Table 1, a medium in which the KH2PO4 concentration is changed to 0 mM is a phosphorus-deficient medium.

[0025] The method of the above embodiment has the surprising effect of increasing the amount of KODA accumulated in a plant body by simply changing the phosphate concentration in the medium, which is an inexpensive and easy procedure.

[0026] The plant individual, tissue or cell of the embodiment may contain a gene encoding 9-LOX and / or a gene encoding AOS, and more preferably contains both of these genes.These genes may be endogenous genes or exogenous genes.The exogenous gene may be artificially introduced into the plant individual, tissue or cell, for example, by transformation.

[0027] In the present disclosure, transformation includes increasing the expression of an exogenous gene compared to a corresponding non-transformed individual, tissue, or cell by introducing the gene. Transformation can be performed by various techniques known to those skilled in the art, including the Agrobacterium method, the gene gun (particle gun) method, the electroporation method, the PEG method, homologous recombination, and various genome editing techniques including the CRISPR / CAS9 method.

[0028] In the embodiment, the gene encoding 9-LOX (also referred to as 9-LOX gene) is a gene encoding 9-lipoxygenase (9-LOX). 9-LOX is an enzyme having an activity of introducing a hydroperoxy group to the 9-position of linolenic acid. Preferably, the 9-LOX encoded by the gene encoding 9-LOX is plant-derived 9-LOX, more preferably duckweed-derived 9-LOX. The 9-LOX encoded by the gene encoding 9-LOX may include one or more of amino acid substitutions, deletions, and additions, so long as it has the above-mentioned activity. More specifically, the 9-LOX may be an enzyme having 90% or more amino acid sequence identity to the 9-LOX derived from duckweed.

[0029] In the embodiment, the gene encoding AOS (also referred to as AOS gene) is a gene encoding allene oxide synthase (AOS). AOS is an enzyme having allene oxide synthase activity that synthesizes allene oxide from hydroperoxylated fatty acid. Preferably, the AOS encoded by the gene encoding AOS is plant-derived AOS, more preferably AOS derived from duckweed. The AOS encoded by the gene encoding AOS can include one or more of amino acid substitution, deletion, and addition, as long as it has the above activity. More specifically, AOS can be an enzyme having 90% or more amino acid sequence identity with AOS derived from duckweed.

[0030] In the embodiment, the genes encoding 9-LOX and AOS may be genes operably linked to a functional promoter. The functional promoter is not particularly limited as long as it can induce the expression of 9-LOX or AOS in a plant individual, tissue, or cell, and a suitable example includes the 35S promoter of cauliflower mosaic virus (CaMV) and its variants. The 9-LOX gene and / or AOS gene linked to a functional promoter may be included in any vector suitable for transformation. In addition, the 9-LOX and AOS genes may include untranslated regions (UTRs) in addition to the coding region.

[0031] In the present disclosure, a plant individual, tissue, or cell containing a gene may mean that the gene is present in the genome of the plant individual, tissue, or cell as an endogenous gene or is integrated as a foreign gene. Also, a plant individual, tissue, or cell containing a gene may mean that the gene is present extrachromosomally as an episome.

[0032] According to one embodiment, the method further comprises step (b) of extracting the accumulated KODA from the plant individual, tissue or cell after step (a) of maintaining the plant individual, tissue or cell in a phosphorus-deficient medium. The method of the present embodiment can also be considered as a method for producing KODA, since KODA is extracted after the amount of accumulated KODA is increased.

[0033] A specific procedure for extraction may include disrupting plant individuals, tissues, or cells. Disruption may be performed using tools and methods known to those skilled in the art, including, for example, a mortar and pestle, freezing, homogenizers, and bead mills. The KODA content in the sample may be increased by continuing the synthesis of KODA in the disrupted material. The KODA content may also be increased by adding alpha-linolenic acid to the disrupted material of the embodiment.

[0034] KODA can be extracted from disrupted or undisrupted plant samples, tissues, or cells using a variety of solvents, including water, acids such as formic acid, acetic acid, alcohols including methanol, ethanol, chloroform, other organic solvents, and mixtures thereof. Specific examples of preferred extraction solvents are water, 2% (v / v) formic acid / methanol, and mixtures thereof.

[0035] The extract obtained by the extraction process can be filtered to remove solid matter. Filtration can be performed by a method known to those skilled in the art, for example, using various spin columns and centrifuges. As known to those skilled in the art, the extract can be fractionated by chromatography to separate a fraction containing KODA.

[0036] [KODA manufacturing system] In one aspect, a system for producing KODA is provided. The system includes a plant, tissue, or cell that contains a gene encoding 9-LOX and / or a gene encoding AOS, and is maintained in a medium with a dissolved phosphorus concentration of less than 1 mM. It is understood that the system can be used to carry out one embodiment of the method for increasing the accumulation of KODA described above. The elements of the system (including the plant, gene, medium, and phosphorus concentration) can be applied to the explanations provided in relation to the above-mentioned method embodiment.

[0037] [Method for producing plants with increased stress tolerance] In one embodiment, a method for producing a plant (particularly a plant body) having increased stress tolerance is provided, which comprises the steps of introducing at least one gene selected from a gene encoding 9-LOX and a gene encoding AOS into a plant, and cultivating the plant into which the gene has been introduced in a medium under phosphorus-deficient conditions. It is preferable that both the 9-LOX gene and the AOS gene are introduced (e.g., transformed).

[0038] In an embodiment, stress tolerance may refer to various environmental stresses, including high temperature stress, drought stress, osmotic stress, starvation stress, and disease stress. KODA is known to be involved in plant tolerance to these various stresses. Since the KODA content in the plant is increased by cultivating the plant in a phosphorus-deficient medium, it is believed that the plant produced in this embodiment exhibits tolerance to these various stresses. In one embodiment, the stress tolerance is high temperature stress tolerance.

[0039] The elements of the method for producing the present embodiment (including the plant, medium, gene and its introduction, and phosphorus-deficient conditions) can be applied to the explanations provided in relation to the embodiment of the method for increasing the accumulation amount of KOD A. For example, the medium for phosphorus-deficient conditions can be a medium in which the dissolved phosphorus concentration is less than 1 mM. EXAMPLES

[0040] Examples of the present disclosure are described below, but the present disclosure is not limited to the examples described below.

[0041] Example 1. KODA accumulation in transformed and non-transformed plants under phosphorus deficiency conditions

[0042] Materials and Methods 1. Transformed Plants According to the method described in JP 2022-114271 A, a transformed strain was produced in which the 9-LOX gene and AOS gene of duckweed were expressed in the plant Arabidopsis thaliana. A vector control strain was also produced in the same manner, in which a transformation operation was performed using a control vector that does not have these genes.

[0043] Specifically, Arabidopsis was transformed by the inflorescence immersion method. That is, Arabidopsis grown until flower buds were formed was immersed in a suspension of Agrobacterium into which the target gene had been introduced for 3 minutes. The Arabidopsis inflorescence was then wrapped in plastic wrap and left to stand overnight under weak light, and the next day it was grown under normal conditions, and seeds were collected. The seeds were sown on MS medium containing drugs, and the desired transformants were obtained by selection using the drug resistance marker gene on the vector.

[0044] 2. Plant Growth Conditions Seeds of the wild type (WT), vector control type (VC), and transformed type were sterilized with 0.1% (v / v) Tween 20 and 20% (v / v) sodium hypochlorite solution and rinsed five times with sterilized ion-exchanged water. The sterilized seeds were sown individually on plates of MS medium (Table 2) containing 1% (w / v) sucrose and 0.8% (w / v) INA Agar as normal growth conditions, and then stored at 4°C in the dark for 2 to 4 days for vernalization. After growth for 10 days on normal growth medium (23°C, continuous light), the seeds were grown for 10 days on phosphorus-sufficient (+Pi) or phosphorus-deficient (-Pi) growth medium (Table 3) at 23°C under continuous light.

[0045] [Table 2]

[0046] [Table 3]

[0047] 3. Measurement of KODA content in crude extracts of plant leaves Samples of the aboveground parts of three plants frozen with liquid nitrogen were quickly crushed with a mortar and pestle. While cooling on ice, 250 μL of sterilized ion-exchanged water was added per 1 mg of fresh weight to suspend the crushed material, and the suspension was quickly centrifuged at 13,000 rpm for 2 minutes at 20 °C using a high-speed microcentrifuge, and 100 μL of the supernatant was collected. 100 μL of 2% (v / v) formic acid / methanol was added to this supernatant and stirred well. The mixture was dripped onto a titanium oxide and zirconium oxide bonded silica monolith spin column (MonoSpin (registered trademark) Phospholipid, GL Sciences Inc.), centrifuged at 5,000 rpm for 2 minutes at 20 °C using a high-speed microcentrifuge, and the filtrate was collected. The filtrate was further filtered through a 0.22 μm hydrophilic polyvinylidene fluoride membrane filter (4 mm Millex (registered trademark) Syringe Filters, Merck). The resulting solution was used as the KODA sample for LC-MS / MS analysis. The LC-MS / MS analysis device used was an LC-ESI-MS / MS (Shimadzu Corporation) that combines a high-performance liquid chromatograph LC-2040C 3D with a mass spectrometer LCMS-8050. The device was operated using the LabSolution that came with the device.

[0048] result Under phosphate-sufficient conditions, KODA was not detectable in samples from the wild-type and vector control plants. In contrast, KODA accumulation was observed in the transformed plants (Figure 1). Surprisingly, when these plants were grown in phosphate-deficient medium, both the wild-type and vector control plants accumulated small amounts of KODA. Furthermore, the amount of KODA produced by the transformed plants was significantly increased. That is, although the wild-type and vector control plants did not accumulate detectable KODA under phosphate-sufficient conditions, when they were transferred to phosphate-deficient conditions, detectable KODA accumulation occurred, and the transformed plants accumulated approximately 30-fold more KODA.

[0049] The above results suggest that the amount of KODA accumulated can be increased by maintaining not only the transformed strains but also the wild type and vector control strains in a medium lacking phosphorus. Conventionally, in order to increase the yield, the production of KODA required leaf crushing and subsequent incubation at room temperature, even for transformed strains. The results of this example show that the increase in the KODA concentration in the plant body can be achieved without transformation itself, and that both transformed and non-transformed strains can be achieved by simply maintaining them in a medium lacking phosphorus without the need for incubation of the leaf crush.

[0050] (Example 2) Improvement of high temperature stress tolerance under phosphorus deficiency conditions in plants transformed with 9-LOX gene and AOS gene

[0051] Materials and Methods 1. Transformed Plants The same wild-type strain, vector control strain, and transformed strain as used in Example 1 were used.

[0052] 2. Plant Growth Conditions The plants were grown in the same manner as in Example 1, except that the growth period in normal growth medium was 9 days, followed by a growth period in phosphorus-sufficient (+Pi) or phosphorus-deficient (-Pi) growth medium for 7 days, and then grown in normal growth medium (23°C, continuous light) for an additional 4 days. Then, the plants were switched to high temperature (37°C) conditions and grown under continuous light for 4, 5, or 6 days. After this high temperature stress treatment, the plants were further grown and observed under normal growth medium, normal growth temperature (23°C), and continuous light to observe recovery from high temperature stress, and stress tolerance was evaluated.

[0053] result The leaves that appear white in the photograph of Figure 2 are withered leaves. The wild-type strain and the vector control strain (as shown in Example 1, these strains showed only a modest increase in KODA amount under phosphorus-deficient conditions) began to wither during and died after stress treatment, regardless of the presence or absence of phosphorus. The transformed strains also withered after 5 days of high temperature stress treatment under phosphorus-sufficient conditions. In contrast, only the transformed strains maintained in a medium under phosphorus-deficient conditions were able to avoid withering and continue to grow under stress treatment, indicating that stress resistance had been conferred.

[0054] Although the present disclosure has been described with reference to the above several embodiments, the present disclosure is not limited to the above several embodiments. Various modifications can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0055] <Additional Notes> (Appendix 1) A method for increasing the accumulation of 9,10-α-ketol linolenic acid in a plant individual, tissue, or cell, comprising the step (a) of maintaining the plant individual, tissue, or cell in a medium under phosphorus-deficient conditions. (Appendix 2) The method according to claim 1, wherein the medium under phosphorus-deficient conditions has a dissolved phosphorus concentration of less than 1 mM. (Appendix 3) The method according to claim 1 or 2, wherein the plant individual, tissue, or cell is a plant individual, tissue, or cell that contains a gene encoding 9-LOX and / or a gene encoding AOS. (Appendix 4) The method according to any one of appendix 1 to 3, further comprising, after the maintaining step (a), a step (b) of extracting the accumulated 9,10-α-ketol linolenic acid from the plant individual, tissue, or cell. (Appendix 5) A system for producing 9,10-α-ketol linolenic acid, comprising a plant individual, tissue, or cell containing a gene encoding 9-LOX and / or a gene encoding AOS, maintained in a medium having a dissolved phosphorus concentration of less than 1 mM. (Appendix 6) A method for producing a plant with increased stress tolerance, comprising the steps of introducing at least one gene selected from a gene encoding 9-LOX and a gene encoding AOS into a plant, and cultivating the plant into which the gene has been introduced in a medium under phosphorus-deficient conditions. (Appendix 7) The method according to claim 6, wherein the medium under phosphorus-deficient conditions has a dissolved phosphorus concentration of less than 1 mM. (Appendix 8) 8. The method according to claim 6 or 7, wherein the stress resistance is high temperature resistance.

Claims

1. A method for increasing the accumulation of 9,10-α-ketol linolenic acid in a plant individual, tissue, or cell, comprising: The method comprises the step (a) of maintaining the plant individual, tissue or cell in a medium under phosphorus-deficient conditions.

2. The method according to claim 1 , wherein the phosphorus-deficient medium is a medium having a dissolved phosphorus concentration of less than 1 mM.

3. The method according to claim 1, wherein the plant individual, tissue, or cell is a plant individual, tissue, or cell that contains a gene encoding 9-LOX and / or a gene encoding AOS.

4. The method according to any one of claims 1 to 3, further comprising a step (b) of extracting the accumulated 9,10-α-ketol linolenic acid from the plant individual, tissue, or cell after the maintaining step (a).

5. A system for producing 9,10-α-ketol linolenic acid, comprising a plant individual, tissue, or cell containing a gene encoding 9-LOX and / or a gene encoding AOS, maintained in a medium having a dissolved phosphorus concentration of less than 1 mM.

6. A method for producing a plant with increased stress tolerance, comprising: introducing at least one gene selected from a gene encoding 9-LOX and a gene encoding AOS into a plant; and cultivating the plant into which the gene has been introduced in a medium under phosphorus-deficient conditions. method.

7. The method according to claim 6 , wherein the phosphorus-deficient medium is a medium having a dissolved phosphorus concentration of less than 1 mM.

8. The method according to claim 6 or 7, wherein the stress resistance is high temperature resistance.

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