Method for Producing Eggplant with High Acetylcholine Content

Abstract

To provide a method for producing an eggplant with a high acetylcholine content.SOLUTION: A method for producing an eggplant plant with a high acetylcholine content includes the step for crossing a specific eggplant plant as a parent with another eggplant plant, where the specific eggplant plant has an acetylcholine content of 200 mg / kg or more per fresh fruit weight (FW) and / or 2500 mg / kg or more per dry fruit weight (DW) in fully ripened fruits. The fruits of the produced hybrid progeny feature a higher acetylcholine content compared to the fruits of the other eggplant plant.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing, for example, eggplant (Solanum melongena L.) with a high acetylcholine content, and to an eggplant with a high acetylcholine content obtained by said method. [Background technology]

[0002] Choline esters containing acetylcholine are known as functional components that suppress blood pressure rise (Non-Patent Document 1), and in recent years, they have been found to be specifically abundant in eggplants among fruits and vegetables (Non-Patent Documents 1 and 2 and Patent Documents 1 and 2), attracting attention. They have also been reported to have an effect on improving sleep (Patent Document 3). Since interest in hypertension and sleep improvement is high both at home and abroad, it is believed that there is a great social need for a method for producing eggplants with a high acetylcholine content that contributes to suppressing blood pressure rise and improving sleep.

[0003] On the other hand, when eating eggplant in Japan, it is common to eat unripe eggplants for fresh produce. Since the acetylcholine content in unripe eggplants for fresh produce is 3 to 8 mg / 100 g F.W. (Non-Patent Document 3), in order to ingest 2.3 mg of acetylcholine per day (Non-Patent Document 1), which is expected to have the function of lowering blood pressure, it is necessary to eat about 42 g of eggplants every day. In addition, in order to ingest 3.456 mg of acetylcholine per day (Patent Document 3), which is expected to have the effect of improving sleep, it is necessary to eat about 63 g of eggplants every day. However, the current situation in Japan is less than 20 g per person per day (Ministry of Internal Affairs and Communications Household Survey 2020), and it is not easy to meet the daily requirement with eggplants for fresh produce alone.

[0004] As mentioned above, in order to achieve the daily acetylcholine intake of 2.3mg or 3.456mg required for functional purposes, it would be necessary to eat more than twice as much eggplant as currently consumed, which is not realistic. In addition, as a way to solve this problem, it is possible to take it as an additive or supplement using dried powder, but the cost required to process ordinary eggplant into raw powder is a bottleneck and is preventing its widespread use.

Prior technical literature

Chartered Documents

[0005] [Patent Document 1] WO 2018 / 070545 [Patent Document 2] WO 2020 / 166494 [Patent Document 3] JP-2020-137462

Non-licensed documents

[0006]

Non-patent document 1

Non-patent document 2

Non-patent document 3

[0007] In view of the above-mentioned circumstances, an object of the present invention is to provide a method for producing eggplant with a high acetylcholine content. [Means for solving the problem]

[0008] As a result of intensive research to solve the above problems, the acetylcholine content of 100 eggplant core collections (Miyatake, K., Y. Shinmura, H. Matsunaga, H. Fukuoka and T. Saito, 2019. Construction of a core collection of eggplant (Solanum melongena L.) based on genome-wide SNP and SSR genotypes. Breeding Science, 69: 498-502.) was comprehensively investigated, and lines with high acetylcholine content were found. Self-breeding and selection were then performed to develop fixed lines AE-ACH03 and AE-ACH04 that have a stable high concentration of acetylcholine. It was discovered that by crossing these lines (AE-ACH03 and AE-ACH04) with existing eggplant varieties, it is possible to impart high acetylcholine content to existing eggplant varieties, and the present invention was completed.

[0009] That is, the present invention includes the following. [1] A method for producing an eggplant plant with a high acetylcholine content, comprising the steps of: crossing an eggplant plant having an acetylcholine content of 200 mg / kg or more fresh fruit weight (FW) and / or 2,500 mg / kg or more dry fruit weight (DW) in fully ripe fruits with another eggplant plant as a breeding parent; and producing progeny fruits having a higher acetylcholine content than the fruits of the other eggplant plants. [2] The method according to [1], wherein the parent eggplant plant is an eggplant plant identified by accession number FERM P-22468 (AE-ACH03) or an eggplant plant identified by accession number FERM P-22469 (AE-ACH04), or a progeny line thereof. [3] An eggplant plant with high acetylcholine content or a progeny thereof produced by the method according to [1] or [2]. [4] A part of the plant body of the eggplant plant with high acetylcholine content described in [3] or its progeny. Effect of the Invention

[0010] According to the present invention, it is possible to produce eggplants that contain acetylcholine at a concentration about 2 to 3 times higher in the fruit than common eggplant varieties for fresh produce by imparting high acetylcholine content to existing eggplant varieties that have properties suitable for processing. For example, by imparting high acetylcholine content to existing eggplant varieties that have properties suitable for processing, it is possible to significantly reduce the processing costs (raw material costs, transportation costs, storage costs, utility costs required for processing, labor costs, etc.) of dried powders that are assumed to be used as food additives or supplements. In addition, by imparting high acetylcholine content to existing eggplant varieties used for fresh produce, it is possible to expect blood pressure lowering effects and sleep improvement effects by cooking and eating eggplants for fresh produce, rather than using them as food additives or supplements. [Brief description of the drawings]

[0011] [Figure 1] This is a photograph showing the appearance of AE-ACH03 and 04. [Diagram 2]1 is a graph showing the acetylcholine content (per fresh fruit weight) of AE-ACH03 and 04 (Ach: acetylcholine, A03: AE-ACH03, A04: AE-ACH04). [Diagram 3] 1 is a graph showing the acetylcholine content (per dry weight of fruit) of AE-ACH03 and 04 (Ach: acetylcholine, A03: AE-ACH03, A04: AE-ACH04). [Figure 4] Photographs showing the appearance of eggplant parent lines and F1 lines. [Diagram 5] This is a graph showing the acetylcholine content (per fresh fruit weight) of the F1 lines between AE-ACH03, 04 and fresh eggplant lines (Ach: acetylcholine, A03: AE-ACH03, A04: AE-ACH04, 21: "AEP-PBFs21", 23: "AEP-PBFs23", mean ± SD). [Figure 6] This is a graph showing the acetylcholine content (per fruit dry weight) of the F1 lines between AE-ACH03, 04 and fresh eggplant lines (Ach: acetylcholine, A03: AE-ACH03, A04: AE-ACH04, 21: "AEP-PBFs21", 23: "AEP-PBFs23", mean ± SD). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention will be described in detail below.

[0013] The method for producing an eggplant plant with high acetylcholine content according to the present invention (hereinafter referred to as "the method") includes a step of crossing an eggplant plant with high acetylcholine content as a cross parent with another eggplant plant. The fruits of the hybrid progeny produced by the method have a higher acetylcholine content than the fruits of the other eggplant plants used in the cross.

[0014] The present invention is based on the finding that high acetylcholine content can be imparted to existing eggplant varieties by crossing the fixed lines AE-ACH03 or AE-ACH04, whose fruits contain high concentrations of acetylcholine, with the existing eggplant varieties.

[0015] Here, the eggplant plant with high acetylcholine content used as the breeding parent is, for example, an eggplant plant having an acetylcholine content of 200 mg / kg fruit fresh weight (FW) or more (preferably 250 mg / kg fruit fresh weight (FW) or more) and / or 2500 mg / kg fruit dry weight (DW) or more (preferably 3000 mg / kg fruit dry weight (DW) or more) in fully ripe fruit (fruits that have been in full bloom for about 2 to 3 months, when fruit enlargement has stopped and the fruit skin color has changed, and fruits with a yellow to brown skin in varieties and lines in which the fruit skin color has not changed). Examples of the eggplant plant as the breeding parent include the eggplant plant identified by the accession number FERM P-22468 (AE-ACH03) or the eggplant plant identified by the accession number FERM P-22469 (AE-ACH04), or their progeny lines.

[0016] AE-ACH03 and AE-ACH04 were deposited on February 24, 2023 at the National Institute of Technology and Evaluation, Patent Organism Depositary (NITE-IPOD) (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) under accession numbers FERM P-22468 and FERM P-22469, respectively.

[0017] Here, the progeny line of AE-ACH03 or AE-ACH04 means the next generation and subsequent generations of eggplants that inherit the property of having a high acetylcholine content in the fruits of the above-mentioned AE-ACH03 or AE-ACH04.

[0018] On the other hand, the "other eggplant plants" used for crossbreeding may be any eggplant varieties as long as they are crossbreedable, such as AEP-PBFs21, AEP-PBFs23 (all of which are parent lines (fixed varieties) of "Ano-kou No. 10," which is being applied for variety registration as an F1 variety of eggplant for fresh use that has parthenocarpy, resistance to bacterial wilt, and thornlessness), AE-P01, AE-P08, AE-P24 (parent lines (fixed varieties) of "Ano-minori" and "Ano-minori No. 2," which have parthenocarpy, "Senryo No. 2" (F1 variety), and "Chikuyo" (F1 variety).

[0019] In this method, an eggplant plant with a high acetylcholine content is used as a breeding parent (seed parent or pollen parent) and crossed with another eggplant plant (pollen or seeds) to produce a hybrid progeny (first generation hybrid (F1)). The eggplant plant with a high acetylcholine content may be either a seed parent or a pollen parent. The obtained hybrid progeny may also be backcrossed with the eggplant plant used to produce the hybrid progeny or another eggplant plant as a breeding parent (seed parent or pollen parent).

[0020] The fruits of the eggplant plant with high acetylcholine content produced by this method or its progeny have a higher acetylcholine content than the fruits of other eggplant plants used in the crossbreeding. When AEP-PBFs21 is used as the other eggplant plant, the fruits of the produced eggplant plant with high acetylcholine content or its progeny have an acetylcholine content of 118 mg / kg fresh fruit weight (FW) or more (preferably 148 mg / kg fresh fruit weight (FW) or more) and / or 1448 mg / kg dry fruit weight (DW) or more (preferably 1970 mg / kg dry fruit weight (DW) or more) in immature fruits (fruits just before the stop of enlargement and growth about one month after flowering). Furthermore, the fruits of the produced eggplant plant with high acetylcholine content or its progeny, for example, when AEP-PBFs23 is used as another eggplant plant, have an acetylcholine content of, for example, 189 mg / kg fresh fruit weight (FW) or more (preferably, 199 mg / kg fresh fruit weight (FW) or more) and / or 2381 mg / kg dry fruit weight (DW) or more (preferably, 2514 mg / kg dry fruit weight (DW) or more) in unripe fruits.

[0021] Here, the progeny line of the created eggplant plant with high acetylcholine content means eggplants of the next generation and subsequent generations that inherit the property of having a high acetylcholine content in the fruits of the above-mentioned created eggplant plant with high acetylcholine content compared to the fruits of the other eggplant plants used in the crossbreeding.

[0022] Furthermore, examples of parts of the plant body of the produced eggplant plant with high acetylcholine content or its progeny line include fruits, seeds, and the like. EXAMPLES

[0023] The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to these examples.

[0024] [Experimental Method] 1. Experimental Materials AE-ACH03 and 04 are fixed lines with stable and high concentrations of acetylcholine that were produced by selfing and selecting the mother line selected from the Nascore collection (Miyatake et al., 2019).

[0025] "Senryo No. 2" is a common eggplant variety used as fresh produce in Japan and is an F1 variety.

[0026] "AEP-PBFs21" and "AEP-PBFs23" are parent lines (fixed varieties) of "Ano-kou No. 10," which is being applied for registration as an F1 variety of eggplant for fresh use that has parthenocarpy, resistance to bacterial wilt, and thornlessness.

[0027] A03x21 and 21xA04 are F1 lines obtained by crossing AE-ACH03 (seed parent) with "AEP-PBFs21" (pollen parent) and F1 lines obtained by crossing AE-ACH04 (pollen parent) with "AEP-PBFs21" (seed parent), respectively.

[0028] A03x23 and 23xA04 are F1 lines obtained by crossing AE-ACH03 (seed parent) with "AEP-PBFs23" (pollen parent) and F1 lines obtained by crossing AE-ACH04 (pollen parent) with "AEP-PBFs23" (seed parent), respectively.

[0029] 2.Cultivation method AE-ACH03 and 04 and "Senryo 2" were sown in a greenhouse in March and planted in an open field in May. Fruits were harvested in August and used to measure acetylcholine content.

[0030] A03x21, 21xA04, A03x23, 23xA04, AEP-PBFs21, and AEP-PBFs23 were sown in a greenhouse in August and transplanted in a greenhouse in October. Fruits were harvested from November to January and used to measure acetylcholine content.

[0031] 3. Measurement of Acetylcholine Content The acetylcholine content was measured by the Ando method (Patent Application No. 2023-056633). The specific steps are shown below.

[0032] First, the eggplant hot water extract, to which triethylmethylammonium chloride (TEMA-Cl) (Tokyo Chemical Industry) was added as an internal standard, was filtered through a 0.45 μm pore size filter to remove insoluble residues, and then subjected to capillary electrophoresis (CE) analysis.

[0033] In the CE analysis, a polyimide resin-coated unmodified fused silica tube (inner diameter 75 μm, GL Sciences) was cut to 80 cm and used as a capillary. A CE system (Agilent 7100, Agilent) equipped with a photodiode array detector was used to detect major cations by indirect absorption, following a previous report (Yanai Rie et al., 2003). However, the analytical sample was pressure-injected at 50 mbar for 5 seconds with a final dilution ratio of 1 to 1000 times depending on the content of the target component. The electrophoretic solution consisted of 10 mM imidazole, 5 mM HIBA, 2 mM 18-crown-6-ether, and 0.2% (v / v) acetic acid, and 25 kV was applied for 10 to 12 minutes. Before each electrophoresis, the inside of the capillary tube was washed and equilibrated by running the electrophoretic solution for 5 minutes. The capillary tube was cooled to 25°C.

[0034] In detection, the reference wavelength was set to 215 nm and the detection wavelength was set to 310 nm, where there is no absorption, to use the indirect absorbance method in which the peak detected on the negative side at 215 nm is apparently inverted.

[0035] Acetylcholine (Tokyo Chemical Industry Co., Ltd.) and choline (Tokyo Chemical Industry Co., Ltd.) and major cationic components (ammonium ion (NH4), potassium ion (K), calcium ion (Ca), sodium ion (Na) and magnesium ion (Mg) mixed standard solution (Agilent)) were used as standards, and the components to be analyzed were quantified by the internal standard method.

[0036] [Results and Discussion] 1. Acetylcholine content AE-ACH03 and 04 are the progeny of a self-pollination and selection process of a mother line selected from the Nascore collection based on the criteria that it has high acetylcholine content and is similar in fruit shape and flesh quality to Japanese fresh eggplants (Figure 1).

[0037] AE-ACH03 and 04 were cultivated in an outdoor field, and the acetylcholine content of the fruit was measured. The results showed that the acetylcholine content of unripe AE-ACH03 was 238 mg / kg FW (2937 mg / kg DW), and that of AE-ACH04 was 169 mg / kg FW (2170 mg / kg DW), which was 2.5 to 3.5 times the acetylcholine content of the common eggplant variety "Senryo No. 2" (68 mg / kg FW (1061 mg / kg DW)) per fresh weight and 2 to 2.8 times the acetylcholine content per dry weight. In fully ripe fruits, the acetylcholine content of AE-ACH03 was 217 mg / kg FW (2867 mg / kg DW), and that of AE-ACH04 was 274 mg / kg FW (3111 mg / kg DW), which was about twice the acetylcholine content of "Senryo No. 2", a common eggplant variety, which was 127 mg / kg FW (1664 mg / kg DW) on both fresh and dry weight basis (Figures 2 and 3). The acetylcholine content was higher in fully ripe fruits than in unripe fruits, and thus allowing the fruit to fully ripen is an effective way to increase the acetylcholine content in eggplants.

[0038] 2. Acetylcholine content of F1 line Next, AE-ACH03 and 04 were crossed with the parent lines (AEP-PBFs21 and AEP-PBFs23) of "Ano-kou No. 10," a fresh eggplant variety that has been applied for registration as having resistance to bacterial wilt, parthenocarpy, and thornlessness, to produce an F1 line.

[0039] The acetylcholine content of the unripe fruit (Figure 4) of the F1 lines was measured. The acetylcholine content of A03x21 was 202 mg / kg FW (2702 mg / kg DW), and that of 21xA04 was 148 mg / kg FW (1970 mg / kg DW), which was 1.5 to 2.1 times the fresh weight and 1.7 to 2.3 times the dry weight of the "AEP-PBFs21" (97 mg / kg FW (1164 mg / kg DW)). On the other hand, the acetylcholine content of A03x23 was 199 mg / kg FW (2790 mg / kg DW), and that of 23xA04 was 212 mg / kg FW (2514 mg / kg DW), which was 1.1 to 1.2 times the content of "AEP-PBFs23" (177 mg / kg FW (1572 mg / kg DW)) on a fresh weight basis, but nearly twice that on a dry weight basis (Figures 5 and 6).

[0040] The reason why the acetylcholine content of A03x23 and 23xA04 was only slightly increased compared to "AEP-PBFs23" per fresh weight is because the dry matter percentage of "AEP-PBFs23" was 11.2%, which was higher than the dry matter percentages of A03x23 and 23xA04, which were 7.1% and 8.4%, respectively. Therefore, although the difference per fresh weight was 1.1 to 1.2 times, the difference per dry weight was nearly 2 times, which was a large difference.

[0041] As described above, eggplants with high acetylcholine content can be produced from existing varieties by crossing with AE-ACH03 or AE-ACH04.

[0042] In addition, because the acetylcholine content has doubled, the processing cost for the supplement raw material powder (raw material cost, transportation cost, storage cost, utility cost, labor cost, etc.) is estimated to be about half of that when using conventional eggplants for fresh produce. In addition, it is considered more desirable from the viewpoint of processing to create F1 lines with existing varieties, etc., rather than using AE-ACH03 and 04 as they are. For example, the appearance of A03x21, 21xA04, A03x23, and 23xA04 is closer to the appearance of eggplants commonly used in Japan than AE-ACH03 and 04 (Figures 1 and 4), so it is considered easier to apply the processing methods for existing varieties when processing these F1 lines.

[0043] Furthermore, simply cooking and eating the unripe fruit of the F1 lineage, rather than taking supplements, is expected to have the effect of lowering blood pressure and improving sleep. In this case, too, it is thought that the F1 lineage with existing varieties will be easier to eat in terms of texture and taste than using AE-ACH03 and 04 as is.

[0044] In terms of cultivation, these F1 lines may have advantages over AE-ACH03 and 04 in terms of bacterial wilt resistance, parthenocarpy, and yield.

[0045] Thus, the method according to the present invention is useful for producing an eggplant line with high acetylcholine content from an existing eggplant variety or the like having some desirable property.

[0046] References Miyatake, K., Y. Shinmura, H. Matsunaga, H. Fukuoka and T. Saito, 2019. Construction of a core collection of eggplant (Solanum melongena L.) based on genome-wide SNP and SSR genotypes. Breeding Science, 69: 498-502. Nishimura, M., M. Suzuki, R. Takahashi, S. Yamaguchi, K. Tsubaki, T. Fujita, J. Nishihira, and K. Nakamura. 2019. Daily ingestion of eggplant powder improves blood pressure and psychological state in stressed individuals: A randomized placebo-controlled study. Nutrients. 11:2797. Wang W, Yamaguchi S, Koyama M, Tian S, Ino A, Miyatake K, Nakamura K. 2020. LC-MS / MS Analysis of Choline Compounds in Japanese-Cultivated Vegetables and Fruits. Foods. 9:1029. Wang, W., S. Yamaguchi, A. Suzuki, N. Wagu, M. Koyama, A. Takahashi, R. Takada, K. Miyatake, and K. Nakamura. 2021. Investigation of the distribution and content of acetylcholine, a novel functional compound in eggplant. Foods. 10:81. Shinshu University. WO 2018 / 070545 A1. Composition containing choline ester for oral intake Shinshu University, ADEKA Co., Ltd. WO2020 / 166494. Eggplant-derived composition with blood pressure lowering effect Wellnas Co., Ltd. JP2020-137462A. Oral intake composition for improving sleep [Accession number]

[0047] FERM P-22468 FERM P-22469

Claims

1. A method for producing an eggplant plant with high acetylcholine content, comprising a step of crossing an eggplant plant specified by accession number FERM P-22468 or FERM P-22469, or a progeny thereof, as a breeding parent with another eggplant plant, the eggplant plant having an acetylcholine content of 200 mg / kg or more of fresh fruit (FW) and / or 2,500 mg / kg or more of dry fruit (DW) in fully ripe fruits, the eggplant plant being one of the eggplant plants specified by accession number FERM P-22468 or FERM P-22469, or a progeny thereof, the progeny fruits having a higher acetylcholine content than the fruits of the other eggplant plants.

2. An eggplant plant or its descendants having a high acetylcholine content produced by the method described in claim 1.

3. A part of the plant body of an eggplant plant with high acetylcholine content as described in claim 2 or its descendants.

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