Tobacco material, tobacco product, and plant of genus nicotiana

Abstract

Provided is a tobacco product having improved fragrance and flavor. Specifically provided is a tobacco material including part of a plant of the genus Nicotiana, wherein: the plant of the genus Nicotiana has a mutation associated with the suppression of a function of an endogenous CAD gene; and the tobacco material contains 0.05 ppm or more of vanillin.

Description

Tobacco materials, tobacco products, and Nicotiana plants

[0001] The present invention relates to tobacco materials, tobacco products, and Nicotiana plants.

[0002] The flavor and aroma of tobacco products is a complex quality that appeals to human sensory organs and is thought to be based on the balance of various components contained in the raw material, tobacco leaves. Numerous flavor-contributing components have been investigated to improve the flavor and aroma of tobacco. Patent Document 1 describes the production of tobacco materials with increased content of flavor and aroma components (e.g., vanillin) by processing tobacco raw materials using industrial techniques.

[0003] Japanese Patent No. 6019216

[0004] Under these circumstances, there was room to consider methods that would have a lower environmental impact as a way to improve the flavor and aroma of tobacco products.

[0005] An object of one aspect of the present invention is to provide a tobacco product with an improved aroma and flavor.

[0006] In order to solve the above-mentioned problems, one embodiment of the present invention provides a tobacco material comprising a part of a Nicotiana plant, wherein the Nicotiana plant has a mutation resulting in functional inhibition of the endogenous cinnamyl alcohol dehydrogenase gene in at least one of: (a) an endogenous cinnamyl alcohol dehydrogenase gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1; and (b) an endogenous cinnamyl alcohol dehydrogenase gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2, and the tobacco material contains 0.05 ppm or more of vanillin.

[0007] In order to solve the above-mentioned problems, a Nicotiana plant according to one embodiment of the present invention has a mutation introduced into at least one of the following: (a) an endogenous cinnamyl alcohol dehydrogenase gene, the coding region of which is a polynucleotide encoding a polypeptide having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO:1; and (b) an endogenous cinnamyl alcohol dehydrogenase gene, the coding region of which is a polynucleotide encoding a polypeptide having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO:2; the mutation causes functional inhibition of the endogenous cinnamyl alcohol dehydrogenase gene; and the vanillin content of tobacco material obtained from the Nicotiana plant is higher than that of tobacco material obtained from an equivalent amount of a wild-type Nicotiana plant.

[0008] According to one aspect of the present invention, a tobacco product with improved flavor and taste can be realized.

[0009] 1 shows the vanillin content of Tsukuba No. 1 (wild-type Nicotiana plant) and a CAD mutant. 2 shows the appearance of the stem of CAD-A after the epidermis has been removed, and the positions of the upper and lower parts of the stem.

[0010] [Definition] "Nicotama plants" are a family of plants in the order Solanales, and belong to the Solanaceae family. In addition to recreational crops such as Nicotama, there are many other useful plants in the genera Capsicum, Solanum, and Tomato, which are often used as spices and food crops, and Petunia and Physalis, which are often used as ornamental plants. These species contain many useful plants with high commercial potential. As used herein, the term "Nicotama plants" encompasses entire plants (e.g., adults, seedlings, and seeds) and tissues (e.g., leaves, stems, flowers, roots, reproductive organs, embryos, and parts thereof). As used herein, the term "tobacco raw material" encompasses harvested products of these "Nicotama plants" and their dried products. For example, as used herein, harvested products of "Nicotama plants" include parts of the plant, such as leaves (tobacco leaves) and stems (e.g., stem remnants). Herein, when simply referring to "leaf," it refers to the integrated mesophyll (lamina) and midrib of the leaf of a Nicotama plant. When leaves are used as tobacco raw materials, the leaves themselves may be used as the tobacco raw material, or the mesophyll and midrib may be separated and either one may be used as the tobacco raw material. Separated mesophyll and midrib may also be combined in any ratio and used as the tobacco raw material. As used herein, "dried tobacco material" specifically refers to a dried product of a "tobacco raw material." Any known drying method may be used, such as yellow drying, hot air drying, or freeze drying. "Tobacco material" includes dried products of "tobacco raw materials," "dried tobacco materials," and any processed products thereof. Any known processing method may be used. "Tobacco material" is described in detail below. In this specification, the definition also includes the current generation into which a mutation has been introduced and the progeny obtained by crossbreeding this generation.

[0011] "Tobacco" refers to plants of the genus Nicotiana, most of which belong to the Solanaceae family, and the main commercially cultivated species are Nicotiana tabacum and Nicotiana rustica. In this specification, "nicotinoid plants" simply refers to plants of the genus Nicotiana, which belong to the Solanaceae family, and a typical example is Nicotiana tabacum. Details of Nicotiana plants will be explained in detail in Section 3. Nicotiana plants.

[0012] "Cinnamyl alcohol dehydrogenase (hereinafter also referred to as CAD)" is an enzyme responsible for the next conversion in the monolignol biosynthetic pathway (Chabannes et al. (2001) Plant J. 28(3):257-270.).

[0013] (i) Conversion of ρ-coumaraldehyde to ρ-coumaryl alcohol (ii) Conversion of caffeic aldehyde to caffeyl alcohol (iii) Conversion of coniferyl aldehyde to coniferyl alcohol (iv) Conversion of 5-hydroxyconiferaldehyde to 5-hydroxyconiferyl alcohol (v) Conversion of sinapaldehyde to sinapyl alcohol (vi) Conversion of cinnamaldehyde to cinnamyl alcohol The cinnamyl alcohol dehydrogenase gene of a Nicotiana plant is not particularly limited as long as it is an enzyme capable of any of the conversions (i) to (vi). Vanillin is not produced by the conversions (i) to (vi) in the monolignol biosynthetic pathway, in which CAD is directly involved.

[0014] For example, in plant species other than Nicotiana, it has been shown that in flax (Linum usitatissimum) plants with a silenced CAD gene, alkali treatment of heat-dried stems increases vanillin (Preisner et al. (2014) BMC Plant Biology 14:50). Furthermore, in pineapple (Ananas comosus), alkali treatment of stems of natural CAD mutants increases vanillin, ferulic acid, and coniferyl aldehyde (MacKay et al. (1997) PNAS 94 (15):8255-8260). In Nicotiana, alkali treatment of stems of antisense tobacco CAD transgenic plants also increases vanillin (Yahiaoui et al. (1998) Planta 204:8-15).

[0015] As a result of extensive research, the inventors have found that the vanillin content of tobacco materials obtained from CAD gene mutants of Nicotiana plants is increased compared to tobacco materials obtained from wild-type Nicotiana plants, and that tobacco materials containing these tobacco materials have an excellent aroma and flavor.

[0016] [1. Tobacco Material] A tobacco material according to one embodiment of the present invention comprises a part of a Nicotiana plant having a mutation that causes functional inhibition of an endogenous cinnamyl alcohol dehydrogenase gene. For example, the tobacco material comprises tobacco raw material harvested from a Nicotiana plant, and may be obtained by subjecting the tobacco raw material to any processing. Details of the Nicotiana plant are described in detail in Section [3. Nicotiana Plant].

[0017] The tobacco material contains at least 0.05 ppm of vanillin, which can improve the aroma and flavor of tobacco products. Vanillin is an organic compound having a vanilloid skeleton (also known as a vanillyl group). Vanillin may be either free vanillin or bound vanillin bound to a cell wall component, with free vanillin being preferred. For example, from the perspective of improving the aroma and flavor, it is particularly preferred that the desired content of vanillin, such as that represented by CAS number 121-33-5, is contained. The vanillin content may be 0.05 ppm or more, preferably 5 ppm or more, and more preferably 25 ppm. The vanillin content may also be 5000 ppm or less, 2000 ppm or less, 1000 ppm or less, or 250 ppm or less. Using such a tobacco material can further improve the aroma and flavor of tobacco products. The vanillin content may be measured by any method. For example, the vanillin content may be measured by conventionally known component analysis using GC-MS or LC-MS / MS. Furthermore, for example, in the component analysis, the vanillin content of a sample that has not been subjected to alkali treatment as pretreatment of the analytical sample may be measured. Furthermore, for example, for the purpose of specifically analyzing the vanillin content, the vanillin content of a sample that has been subjected to alkali treatment and high-temperature, high-pressure treatment as pretreatment of the analytical sample may be measured.

[0018] The tobacco material according to the present embodiment has a higher vanillin content than tobacco material produced using a wild-type Nicotiana plant. For example, the vanillin content of the tobacco material according to the present embodiment may be 1.3 times or more, 9 times or more, or 25 times or more the vanillin content of tobacco material produced using a wild-type Nicotiana plant.

[0019] As used herein, when comparing a Nicotiana plant according to the present embodiment or a material or product produced using said plant with a wild-type Nicotiana plant or a material or product produced using said plant, both may be produced under substantially the same conditions. As used herein, "substantially the same conditions" refers to conditions that can vary the vanillin content being substantially the same. Examples of conditions that can vary the vanillin content include plant growth conditions, harvest conditions, and tobacco raw material processing conditions. Hereinafter, when used herein, the expressions "increased vanillin content" or "high vanillin content" refer to a high vanillin content compared to a wild-type Nicotiana plant.

[0020] The tobacco material is harvested from a Nicotiana plant into which a mutation that causes functional suppression of the endogenous cinnamyl alcohol dehydrogenase gene has been introduced, and the tobacco material has a higher vanillin content than the same amount of tobacco material harvested from a wild-type Nicotiana plant.

[0021] The harvested material used for the tobacco raw material may have a vanillin content that is at least twice the vanillin content of the same amount of harvested material harvested from a wild-type Nicotiana plant. Furthermore, the vanillin content of the harvested material used for the tobacco raw material according to this embodiment may be at least 2.2 times, or at least 6.4 times, the vanillin content of the same amount of harvested material obtained from a wild-type Nicotiana plant.

[0022] The tobacco material according to this embodiment may include dried tobacco material as a tobacco raw material. The dried tobacco material is a dried product of tobacco raw material harvested from a Nicotiana plant into which a mutation that causes functional suppression of an endogenous cinnamyl alcohol dehydrogenase gene has been introduced. The dried tobacco material according to this embodiment has a higher vanillin content than dried tobacco material derived from a wild-type Nicotiana plant.

[0023] Cured tobacco materials are obtained by drying tobacco raw materials, which are harvested products of Nicotiana plants. Any drying method can be used, including, but not limited to, natural drying, warm air drying, and hot air drying.

[0024] The vanillin content of the dried tobacco material may be 1.3 times or more the vanillin content of the same amount of dried tobacco material derived from a wild-type Nicotiana plant. Furthermore, the vanillin content of the dried tobacco material according to this embodiment may be 9 times or more, 25 times or more, or even more than the vanillin content of the same amount of dried tobacco material derived from a wild-type Nicotiana plant.

[0025] Tobacco materials may be obtained by processing tobacco raw materials or dried tobacco materials in any manner. For example, tobacco raw materials or dried tobacco materials may be processed by drying, aging, extraction, harmonizing, flavoring, high-temperature and high-pressure treatment, distillation, pulverization, shredding, and the like. The obtained tobacco material may be in any of the following forms: cut filler, powder, particles, sheets, granules, and extract. These forms of tobacco materials are preferred from the viewpoint of applying the tobacco material to tobacco products.

[0026] The tobacco material may be in the form of cut filler. Hereinafter, tobacco material in the form of cut filler will be referred to as "raw material pieces." The particle size of the raw material pieces is preferably 0.5 to 1.18 mm. Such raw material pieces can be obtained, for example, by sieving in accordance with JIS Z 8815 using a stainless steel sieve conforming to JIS Z 8801. For example, 1) using a stainless steel sieve with 1.18 mm meshes, the raw material pieces are sieved by a dry mechanical shaking method for 20 minutes to obtain raw material pieces that pass through the stainless steel sieve with 1.18 mm meshes. 2) Subsequently, using a stainless steel sieve with 0.50 mm meshes, the raw material pieces are sieved by a dry mechanical shaking method for 20 minutes to remove the raw material pieces that pass through the stainless steel sieve with 0.50 mm meshes. In this way, raw material pieces can be prepared that pass through a stainless steel sieve (mesh opening = 1.18 mm) that defines the upper limit, but do not pass through a stainless steel sieve (mesh opening = 0.50 mm) that defines the lower limit.

[0027] The tobacco material may be in the form of particles or powder. Hereinafter, tobacco material in the form of particles or powder will be referred to as "tobacco particles." Tobacco particles include tobacco raw materials or dried tobacco materials that have been chopped into even smaller sizes than raw material pieces, as well as ground tobacco raw materials or dried tobacco materials. The particle size (D90) of the tobacco particles can be, for example, 10 to 1000 μm, and preferably 50 to 500 μm. The average particle size (D50) of the tobacco particles is preferably 20 to 1000 μm, and more preferably 50 to 500 μm. In this embodiment, the particle size (D90) and average particle size (D50) of the tobacco particles are determined by a laser diffraction / scattering method. Specifically, the particle size (D90) and average particle size (D50) of the tobacco particles are measured using a laser diffraction particle size distribution analyzer (e.g., HORIBA, Ltd. LA-950 (product name)). The amount of tobacco particles contained in 100% by weight of the non-tobacco plant material-containing composition can be 0 to 90% by weight, or may be 1 to 80% by weight, 5 to 80% by weight, 10 to 70% by weight, or 20 to 70% by weight.

[0028] The tobacco material may be in the form of a sheet. Hereinafter, tobacco material in the form of a sheet will be referred to as a "tobacco material-containing sheet." The tobacco material-containing sheet may be, for example, a sheet containing a single tobacco material, a sheet containing multiple tobacco materials, or a sheet containing tobacco material and a plant material other than a Nicotiana plant. Furthermore, the configuration of these sheets may be, for example, a single sheet, a stack of multiple sheets, or a crimped sheet. Furthermore, for example, the tobacco material-containing sheet may be composed of a layer containing tobacco material and a non-tobacco material layer containing a plant-derived material other than a Nicotiana plant.

[0029] As the plant other than Nicotiana plants, any known tobacco, i.e., a plant other than Nicotiana, can be used. For example, from the viewpoint of improving the aroma and taste, a plant having a known characteristic aroma may be used. Furthermore, as the plant material other than Nicotiana plants, the plant raw material may be used as it is, or may be subjected to any processing such as drying, crushing, extraction, or granulation.

[0030] The tobacco material-containing sheet may be manufactured by the following methods. The first method is to manufacture a paper-made sheet using a papermaking process. The second method is to mix an appropriate solvent such as water with a mixture containing dried tobacco material, a binder, an aerosol source, etc., to homogenize the mixture, and then cast the homogenized mixture thinly onto a metal plate or metal plate belt and dry it to manufacture a cast sheet. The third method is to mix an appropriate solvent such as water with dried tobacco material, homogenize the mixture, and extrude it into a sheet to manufacture a rolled sheet. Details of the types of homogenized sheets mentioned above are disclosed in "Tobacco Encyclopedia," Tobacco Research Center, March 31, 2009.

[0031] The tobacco material may be in the form of granules. Hereinafter, tobacco material in the form of granules will be referred to as "tobacco material-containing granules." The tobacco material-containing granules may be substantially spherical granules. The average particle size (D50) of the tobacco material-containing granules may be 1000 μm or less, 700 μm or less, 600 μm or less, 550 μm or less, or 400 μm or more. The average particle size (D50) of the non-tobacco plant material-containing granules refers to the average particle size (D50) based on a volume-based particle size distribution measured using a laser diffraction / scattering particle size distribution measurement method. Measurement of the average particle size using the laser diffraction / scattering particle size distribution measurement method can be performed in accordance with JIS Z8825:2013 (Particle size analysis - laser diffraction / scattering method). The average particle size (D50) can be measured, for example, using a laser diffraction / scattering particle size distribution measurement device (e.g., HORIBA, Ltd., LA-950).

[0032] The tobacco material may be in the form of an extract. Hereinafter, tobacco material in the form of an extract will be referred to as "tobacco extract." A tobacco extract is a substance or mixture that exhibits a flavor extracted from tobacco. Tobacco extracts can be prepared by known methods. Examples include: 1) a method in which tobacco raw materials or dried tobacco materials are subjected to extraction with an extraction medium to obtain a tobacco extract; 2) a method in which an extraction medium is added to tobacco raw materials or dried tobacco materials, followed by heating and collecting the generated vapor; and 3) a method in which the extraction medium is vaporized by heating and passed through the tobacco raw materials or dried tobacco materials, followed by collecting the vapor. Examples of extraction media include water or hydrophilic organic solvents such as alcohol. In method 1), water is preferably used as the extraction medium from the perspective of workability. In methods 2) and 3), alcohols such as propylene glycol, glycerin, or ethanol are preferably used as the extraction medium from the perspective of work efficiency. Acids or alkalis can also be used for extraction, as needed. The liquid obtained by extraction, containing the tobacco extract and extraction medium, is called a tobacco extract.

[0033] In one embodiment of the present invention, the dried tobacco material can be subjected to an alkali treatment (hereinafter also referred to as an alkali heating treatment). Flavor components can be generated through the alkali treatment, and these can be collected to prepare a tobacco extract liquid containing tobacco extract and water. In this case, it is preferable to extract the flavor components as a gas from the alkali-treated dried tobacco material, and then introduce the gas into water to transfer the flavor components to the water. Alternatively, the dried tobacco material can be prepared using the solid tobacco material after the alkali treatment. In this case, it is preferable to mix the tobacco extract liquid with the solid tobacco material to prepare the solid tobacco material. An example of alkali treatment involves adding an alkali substance to tobacco particles so that the solid-liquid ratio (w / v) is in the range of 1:5 to 1:20, heating the mixture at 50 to 200°C for 30 to 120 minutes under atmospheric pressure, and then cooling the resulting reaction mixture.

[0034] The alkaline substance used in the alkali treatment is preferably an alkaline liquid such as an aqueous sodium hydroxide solution or an aqueous potassium carbonate solution. The alkaline substance is supplied until the pH of the dried tobacco material reaches a specific range. This pH is preferably 8.0 or higher, more preferably 8.9 to 9.7. The pH of the dried tobacco material is the pH of water obtained by mixing the dried tobacco material with 10 times the amount of water. The tobacco material that has been subjected to the alkali treatment can also be neutralized by adding an acidic substance. The acidic substance used in the neutralization treatment is preferably an acidic liquid such as an aqueous phosphoric acid solution or an aqueous hydrochloric acid solution. The acidic substance is supplied until the pH of the dried tobacco material reaches a specific range. This pH is preferably 6.0 or higher, more preferably 6.5 to 8.0. The pH of the dried tobacco material is the pH of water obtained by mixing the dried tobacco material with 10 times the amount of water.

[0035] In one embodiment of the present invention, the dried tobacco material can be subjected to high-temperature, high-pressure treatment. Examples of high-temperature, high-pressure treatment include the method described in JP 10-66669 A. A solvent is added to tobacco particles so that the solid-liquid ratio (w / v) is in the range of 1:5 to 1:20, and the mixture is heat-treated at 100 to 250°C for 10 to 180 minutes while sealed in a pressure-controlled environment. The resulting reaction product is then returned to atmospheric pressure and cooled. Examples of the solvent include water and hydrophilic organic solvents such as alcohol.

[0036] In one embodiment of the present invention, the dried tobacco material can be steam distilled. Examples of steam distillation methods include those described in "Encyclopedia of Fragrances," p. 366 (published August 27, 1980, by Fujimaki Masao et al., Asakura Shoten). For example, water is placed in a still and brought to a boil, after which the dried tobacco material is placed in the still and distilled for several hours. The resulting steam-distilled water is collected, and acid is added to the water to adjust the pH to 3.0-4.0, followed by the addition of salt to a concentration of 20%. An extraction solvent is then added, and the mixture is subjected to liquid-liquid transfer using a separatory funnel. The solvent layer is collected, and an aqueous NaOH solution (pH 9-10) is added to remove the acid. The solvent layer is then concentrated using an evaporator to obtain a dry product. The water used for steam distillation may be filtered, deodorized, decolorized, etc.

[0037] In one embodiment of the present invention, the dried tobacco material may be subjected to ethanol extraction. For example, an ethanol extract may be obtained by adding 20 to 99.5% ethanol to the dried tobacco material so that the solid-liquid ratio (w / v) is 1:2 to 1:20, and then soaking the dried tobacco material at 4°C to 60°C for one hour to several months.

[0038] 2. Tobacco Products. A tobacco product according to one embodiment of the present invention comprises the tobacco material described above. Specific examples of tobacco products include cut tobacco products, cigars, pipe tobacco products, cigarettes (cigarettes), electronic cigarettes, smokeless tobacco products (including snus and snuff), and waterpipe tobacco products. In particular, the tobacco product may be any of electronic cigarettes, cigarettes, and smokeless tobacco products. Examples of electronic cigarette products include non-combustion high-temperature heated tobacco products, which use aerosol generated by heating a tobacco portion containing tobacco material as an aerosol source; non-combustion low-temperature heated tobacco products, which have an atomization section separate from the tobacco portion that atomizes the aerosol source, heat the atomization section, and inhale the tobacco flavor entrained in the aerosol as it passes through the tobacco portion; and non-heated tobacco products, which inhale the flavor without heating the tobacco portion. Herein, non-combustion high-temperature heated tobacco products and non-combustion low-temperature heated tobacco products are particularly referred to as "aerosol-producing products."

[0039] Preferred forms of tobacco material for use in aerosol products or cigarettes include cut filler, powder, particles, sheets, and granules. Furthermore, these forms of tobacco material may be prepared and mixed by any method to form a tobacco material-containing composition. By using at least one of such tobacco material itself and a tobacco material-containing composition, a flavor source for any flavor-generating article can be formed. The shape of the flavor source is not particularly limited, and may be, for example, granular, sheet, or block.

[0040] A "tobacco material-containing composition" includes tobacco material, and can also include, for example, a binder, an aerosol source, fibers, flavorings, etc. Each material that can be included in the tobacco raw material-containing composition will be described below.

[0041] The tobacco material-containing composition includes the tobacco material described above. The tobacco material contained in the tobacco material-containing composition may be in the form of tobacco particles or tobacco extract. The amount of tobacco particles contained in 100% by weight of the tobacco material-containing composition may be 0 to 90% by weight, 1 to 80% by weight, 10 to 70% by weight, or 20 to 70% by weight. The amount of tobacco extract contained in 100% by weight of the tobacco material-containing composition may be 0 to 90% by weight, 10 to 80% by weight, 10 to 70% by weight, 15 to 50% by weight, or 20 to 40% by weight.

[0042] The tobacco material-containing composition may contain any binder. Any binder component conventionally known in the art may be used as the binder. For example, cellulose derivatives, agar, guar gum, pectin, etc. may be used as binder components. When the tobacco material-containing composition contains a binder, the amount of binder contained in 100% by weight of the tobacco plant raw material-containing composition may be 1 to 30% by weight, preferably 5 to 10% by weight, and more preferably 5 to 8% by weight.

[0043] The tobacco material-containing composition may contain an aerosol source. Any aerosol source conventionally known in the art can be used as the aerosol source. When the tobacco material-containing composition contains an aerosol source, the amount of the aerosol source contained in 100% by weight of the tobacco plant raw material-containing composition is preferably 1 to 50% by weight, and more preferably 10 to 30% by weight.

[0044] The tobacco material-containing composition may contain fibers. Fibers conventionally known in the art can be used as appropriate. When the tobacco raw material-containing composition contains fibers, the amount of fibers contained in 100% by weight of the tobacco material-containing composition may be 2 to 15% by weight.

[0045] The tobacco material-containing composition may contain a flavoring. Any aerosol source conventionally known in the art may be used as the flavoring. The flavoring contained in the first flavor source and the flavoring contained in the second flavor source may be the same or different. When the tobacco material-containing composition contains a flavoring, the amount of flavoring contained in 100% by weight of the tobacco material-containing composition may be 5 to 20% by weight, and preferably 9 to 13% by weight.

[0046] The flavor source used in a tobacco product according to one embodiment of the present invention may be the tobacco material or tobacco material-containing composition in the form described above, or a mixture of an appropriate combination thereof. For example, it may be a mixture of a first flavor source containing the tobacco material in the form of tobacco particles or the tobacco material-containing composition described above, and a second flavor source containing the tobacco material described above. Furthermore, for example, the flavor source according to this embodiment may include a first flavor source containing cut filler as a tobacco material, and a second flavor source containing a tobacco material-containing sheet. Furthermore, the flavor source according to this embodiment may be a combination of tobacco materials or tobacco material-containing compositions in different forms. For example, the first flavor source may include cut filler or a tobacco material-containing sheet as a tobacco material, and the second flavor source containing tobacco particles may be attached to the surface of the first flavor source. Here, the "first flavor source" and the "second flavor source" each exist independently, and the flavor source is formed by physically mixing the two.

[0047] In the flavor source, the weight ratio of the first flavor source to the second flavor source may be 1:20 to 20:1, 1:10 to 10:1, or 1:5 to 5:1. In the flavor source, the weight of the first flavor source may be greater than the weight of the second flavor source. Also, in the flavor source, the weight of the first flavor source may be less than the weight of the second flavor source.

[0048] The flavor source may include a sheet-like first flavor source and a sheet-like second flavor source. Alternatively, a sheet of the first flavor source and a sheet of the second flavor source may be laminated. Alternatively, the flavor source may be a sheet of the first flavor source and a sheet of the second flavor source that are crimped together in a stacked state. The sheet-like flavor source can be produced by using the tobacco sheet described above.

[0049] The flavor source may include a first flavor source in a particulate or granular form and a second flavor source in a sheet form. Alternatively, the flavor source may include a first flavor source in a sheet form and a second flavor source in a particulate or granular form. In this case, one of the sheet-form first flavor source and the second flavor source may be folded, and the other of the first flavor source and the second flavor source in a particulate or granular form may be interposed between the gaps between the folded sheet-form first flavor source and the second flavor source. The particulate or granular flavor source can be produced by using the tobacco particles or tobacco material-containing granules described above.

[0050] The flavor source may include a first flavor source in a particulate or granular form and a second flavor source in a particulate or granular form. In this case, the average particle size of the first flavor source in a particulate or granular form and the average particle size of the second flavor source in a particulate or granular form may be different or substantially the same. Furthermore, the average particle size of the first flavor source may be larger than the average particle size of the second flavor source. Furthermore, the average particle size of the first flavor source may be smaller than the average particle size of the second flavor source.

[0051] 3. Nicotiana Plants A Nicotiana plant according to one embodiment of the present invention has a mutation introduced into it that causes functional suppression of an endogenous cinnamyl alcohol dehydrogenase gene. The vanillin content of the harvested product (leaves or stems) of the Nicotiana plant is higher than that of the harvested product (leaves or stems) of an equivalent amount of a wild-type Nicotiana plant. This configuration allows for the suppression of the function of a single gene to produce a plant with a high vanillin content in its tobacco leaves, cured leaves, and remaining stems. For example, the vanillin content of the harvested product obtained from the Nicotiana plant according to this embodiment may be at least twice the vanillin content of the harvested product obtained from the equivalent amount of a wild-type Nicotiana plant. Furthermore, the vanillin content of the harvested product obtained from the Nicotiana plant according to this embodiment may be at least 2.2 times, or at least 6.4 times, the vanillin content of the equivalent amount of a wild-type Nicotiana plant, or even more.

[0052] The vanillin content of the harvested product of the Nicotiana plant may be 0.05 ppm or more, preferably 5 ppm or more, and more preferably 25 ppm. Furthermore, the vanillin content of the harvested product of the Nicotiana plant may be 5000 ppm or less, 2000 ppm or less, 1000 ppm or less, or 250 ppm or less. Such a Nicotiana plant can further improve the flavor and aroma of tobacco products made therefrom.

[0053] The Nicotiana plant is not particularly limited as long as it belongs to the genus Nicotiana, and examples thereof include Nicotiana acaulis, Nicotiana acuminata, Nicotiana acuminata var. multzjlora, Nicotiana africana, Nicotiana alata, Nicotiana amplexicaulis, Nicotiana arentsii, Nicotiana attenuata, Nicotiana benavidesii, Nicotiana benthamiana, Nicotiana bigelovii, Nicotiana Nicotiana bigelovii, Nicotiana bonariensis, Nicotiana cavicola, Nicotiana clevelandii, Nicotiana cordifolia, Nicotiana corymbosa, Nicotiana debneyi, Nicotiana excelsior, Nicotiana forgetiana, Nicotiana fragrans, Nicotiana glauca, Nicotiana glutinosa, Nicotiana goodspeedii, Nicotiana gossei, Nicotiana ingrown ingulba), Nicotiana kawakamii, Nicotiana naitianaknightiana, Nicotiana langsdorfi, Nicotiana linearis, Nicotiana longiflora, Nicotiana maritima, Nicotiana megalosiphon, Nicotiana miersii, Nicotiana noctiflora, Nicotiana nudicaulis, Nicotiana obtusifolia, Nicotiana occidentalis, Nicotiana occidentalis subsp. hesperis, Nicotiana otophora otophora), Nicotiana paniculata, Nicotiana pauczjlora, Nicotiana petunioides, Nicotiana plumbaginifolia, Nicotiana quadrivalvis, Nicotiana raimondii, Nicotiana repanda, Nicotiana rosulata, Nicotiana rosulata subsp. Ingulba, Nicotiana rotundifolia, Nicotiana rustica Nicotiana rustica (Mulberry tobacco), Nicotiana setchellii, Nicotiana simulans, Nicotiana solanifoliasolanifolia, Nicotiana spegauinii, Nicotiana stocktonii, Nicotiana suaveolens, Nicotiana sylvestris, Nicotiana tabacum, Nicotiana thyrsiflora, Nicotiana tomentosa, Nicotiana tomentosiformis, Nicotiana trigonophylla, Nicotiana umbratica, Nicotiana undulata, Nicotiana velutina, Nicotiana wigandioides wigandioides), and hybrids of Nicotiana plants. Among these, Nicotiana tabacum and Nicotiana rustica, which are used as raw materials for tobacco leaf production, are particularly preferred. Nicotiana sylvestris can also be preferably used. The Nicotiana plant according to one embodiment of the present invention may particularly belong to Nicotiana tabacum or Nicotiana rustica.

[0054] Individuals resulting from the mutation or disruption of the above genes are referred to herein as mutants of Nicotiana plants (also simply referred to as mutants). Among Nicotiana plants, Nicotiana tabacum is an amphidiploid and possesses both a genome derived from its parent plant, Nicotiana sylvestris (also referred to as the "S genome") and a genome derived from Nicotiana tomentosiformis (also referred to as the "T genome"). In Nicotiana tabacum, genes with the same name are almost always present in both the S genome and the T genome. In the case of Nicotiana tabacum, the mutant may have the above mutation in either the S genome or the T genome. The mutant may also have the above mutation in both the S genome and the T genome. The mutation for eliminating a function may be a single mutation or multiple mutations in a single gene, and the type of mutation is not important. In the case of Nicotiana tabacum, any or all of the four alleles, two of which are present in each of the S genome and the T genome, may have mutations, and if mutations are present in multiple alleles, these mutations may be the same or different.

[0055] The functions of the CAD genes in both the S genome and the T genome may be specifically suppressed, or the function of the CAD gene in either the S genome or the T genome may be specifically suppressed. Specific suppression of the function of either the S genome or the T genome refers to suppressing the function of only the CAD gene in one of the S genome and the T genome without suppressing the function of the CAD gene in the other genome. To specifically suppress the function of only one of the CAD genes in the S genome or the T genome, it is preferable to introduce a change in the nucleotide sequence into only that one CAD gene.

[0056] In one embodiment of the present invention, a Nicotiana plant has suppressed function of an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 2. SEQ ID NO: 1 shows the amino acid sequence of an endogenous cinnamyl alcohol dehydrogenase (also referred to herein as "NtCAD-S") encoded by the S genome of Nicotiana tabacum, and SEQ ID NO: 2 shows the amino acid sequence of an endogenous cinnamyl alcohol dehydrogenase (also referred to herein as "NtCAD-T") encoded by the T genome of Nicotiana tabacum.

[0057] SEQ ID NO: 3 shows the CDS sequence of the NtCAD-S gene (encoding the amino acid shown in SEQ ID NO: 1). SEQ ID NO: 5 is the genomic DNA sequence of the NtCAD-S gene, which contains the nucleotide sequence shown in SEQ ID NO: 3 in its coding region. SEQ ID NO: 4 shows the CDS sequence of the NtCAD-T gene (encoding the amino acid shown in SEQ ID NO: 2). SEQ ID NO: 6 is the genomic DNA sequence of the NtCAD-T gene, which contains the nucleotide sequence shown in SEQ ID NO: 4 in its coding region.

[0058] An endogenous cinnamyl alcohol dehydrogenase gene of a Nicotiana plant according to one embodiment of the present invention may be (a) an endogenous cinnamyl alcohol dehydrogenase gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. Furthermore, an endogenous cinnamyl alcohol dehydrogenase gene of a Nicotiana plant according to one embodiment of the present invention may be (b) an endogenous cinnamyl alcohol dehydrogenase gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. This embodiment may satisfy one or more or all of the following conditions: The endogenous gene (a) contains a mutation that causes functional inhibition of the endogenous gene (a); The endogenous gene (a) does not contain a mutation that causes functional inhibition of the endogenous gene (b); and The endogenous gene (b) contains a mutation that causes functional inhibition of the endogenous gene (b). The endogenous gene of (b) does not contain a mutation that causes functional suppression of the endogenous gene of (a).

[0059] In a preferred embodiment, the Nicotiana plant has suppressed function of an endogenous gene comprising, as a coding region, a polynucleotide that has 90% or more (91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or 2 and encodes a polypeptide having CAD activity. When the Nicotiana plant has two endogenous genes, the coding region of one of the endogenous genes encodes a polypeptide having the sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or 2.

[0060] As used herein, the term "sequence identity (of an amino acid sequence)" refers to the percentage of identity between a reference (amino acid) sequence and a reference (amino acid) sequence, where the non-identical portions of the sequences are those in which substitutions, additions, deletions, or insertions (of amino acid residues) exist.

[0061] Here, the phrase "a polypeptide having 90% or more sequence identity to the amino acid sequence shown in ..." used to identify a polypeptide using an amino acid sequence listed in the Sequence Listing may refer to a polypeptide that is normally present in Nicotiana plants. As used herein, the terms "polypeptide" and "protein" have substantially the same meaning and may be used interchangeably.

[0062] Therefore, the above-mentioned specific polypeptides whose abundance is reduced in Nicotiana plants according to the present invention may be polypeptides that have a sequence identity of 90% or more with the respective amino acid sequences shown in the sequence listing, and it is preferable that the sequence identity is a higher percentage (e.g., 97% or more).

[0063] In one embodiment of the present invention, a Nicotiana plant has suppressed function of an endogenous gene comprising an amino acid sequence as set forth in SEQ ID NO: 1 or 2 in which one or more amino acids have been deleted, substituted, or added, and which comprises, as a coding region, a polynucleotide encoding a polypeptide having CAD activity. Here, the number of amino acids deleted, substituted, or added in each amino acid sequence is, for example, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1.

[0064] In one embodiment of the present invention, a Nicotiana plant has suppressed function of an endogenous gene comprising a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3 or 4 as a coding region.

[0065] In one embodiment of the present invention, a Nicotiana plant has suppressed function of an endogenous gene that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 3 or 4, and that contains as its coding region a polynucleotide that encodes a polypeptide having CAD activity.

[0066] A Nicotiana plant according to one embodiment of the present invention has a sequence identity of 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more to the nucleotide sequence set forth in SEQ ID NO: 3 or 4, and has suppressed function of an endogenous gene that includes, as a coding region, a polynucleotide that encodes a polypeptide having CAD activity.

[0067] Polynucleotides encoding the amino acid sequence shown in SEQ ID NO: 1 also encompass degenerate sequences of the nucleotide sequence shown in SEQ ID NO: 3 due to the degeneracy of the genetic code. Polynucleotides encoding the amino acid sequence shown in SEQ ID NO: 2 also encompass degenerate sequences of the nucleotide sequence shown in SEQ ID NO: 4 due to the degeneracy of the genetic code.

[0068] (Mutation) As used herein, "mutation" has the meaning commonly understood in the technical field to which the present application belongs, and refers to, for example, any change (e.g., substitution, deletion, insertion, addition, duplication, inversion, or translocation) of a base in a wild-type genome or an amino acid residue in a wild-type polypeptide. Therefore, "mutation of an endogenous gene" refers to a mutation of a gene that does not produce an original functional polypeptide (including a mutation that produces a polypeptide with reduced function or no function), a mutation of a gene that produces a polypeptide but reduces the amount produced, a mutation of a gene that produces a polypeptide but reduces the stability of the polypeptide, loss of a gene (a genomic DNA sequence including a coding region or an untranslated region), or a mutation that suppresses transcription from a gene (such as deletion of a transcriptional regulatory region or a transcription initiation region).

[0069] The mutation may be present in at least one of the promoter sequence (including a sequence located upstream (5') of the coding region), the terminator sequence (including a sequence located downstream (3') of the coding region), the 5' untranslated region, the 3' untranslated region, the conserved sequences at both ends of an intron (e.g., GT at the 5' end and AG at the 3' end), and the coding region. In particular, the mutation may be present in the coding region of an endogenous cinnamyl alcohol dehydrogenase gene. When a mutation is present in at least one of the promoter sequence, the terminator sequence, and the coding region, the mutation may be any one of substitution, deletion, insertion, addition, duplication, inversion, and translocation. In particular, the function of an endogenous gene may be suppressed, which comprises an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of a CAD polypeptide and which contains, as its coding region, a polynucleotide encoding a polypeptide having CAD activity. Here, the number of amino acids deleted, substituted or added in each amino acid sequence is, for example, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1.

[0070] Furthermore, a Nicotiana plant may have a mutation introduced outside the coding region of the endogenous cinnamyl alcohol dehydrogenase gene that causes functional suppression of the endogenous cinnamyl alcohol dehydrogenase gene. When the mutation is introduced outside the coding region of the endogenous cinnamyl alcohol dehydrogenase gene, the mutation may be an insertion of a polynucleotide that expresses an antisense RNA molecule, an RNAi molecule, or a co-suppression molecule that promotes degradation of mRNA transcribed from the endogenous cinnamyl alcohol dehydrogenase gene.

[0071] When a substitution results in loss of function, the substitution may occur in at least one of the promoter sequence, terminator sequence, 5' untranslated region and 3' untranslated region, conserved sequences at both ends of an intron, and coding region.

[0072] For example, substitutions in nucleotide sequences important for regulating gene expression, such as those in the promoter sequence, 5' untranslated region, and 3' untranslated region of a gene, can result in a decrease in the transcriptional activity of the gene or a decrease in the stability of the transcript from the gene. Both of these decreases can result in a decrease in the translation product due to a decrease in the transcript from the gene. Substitutions in the above-mentioned conserved sequences of introns (splice mutations) can cause abnormal splicing of mRNA, resulting in abnormal mRNAs with unnecessary introns added or inserted. Abnormal mRNAs can produce abnormal translation products, for example, due to frameshifts, or translation cannot be terminated.

[0073] If the nucleotide substitution in the coding region is a missense mutation (resulting in a reduced abundance of the wild-type polypeptide), the substitution will result in an amino acid different from the original amino acid, and may result in a polypeptide with reduced or no original function.

[0074] Substitutions in the coding region can also result in incomplete translation products or translation products that do not maintain their original function. Incomplete translation products arise due to the conversion of a codon encoding an amino acid to a stop codon (nonsense mutation). Incomplete translation products lack one or more consecutive amino acid residues, including the C-terminal amino acid residue, compared to the original translation product. The nonsense mutation occurs in any codon upstream of the original stop codon, preferably one or more codons upstream from the original stop codon. Therefore, translation products from genes containing nonsense mutations are incomplete. Translation products that lack their original function are generated by amino acid substitutions. In this case, the amount of transcript may be equivalent to that of wild-type plants. The translation product may exhibit a change in three-dimensional structure or a reduction in its function as a functional domain. One preferred embodiment of the mutations of the present invention is an amino acid substitution that results in a translation product that lacks its original function. Preferably, the amino acid substitution is a non-conservative substitution, which has a high potential to alter the function of the translation product. Non-conservative substitutions include substitutions of amino acids with different charge or hydrophobicity (e.g., substitutions of a basic amino acid with an acidic amino acid, a basic or acidic amino acid with a neutral amino acid, a neutral amino acid with a basic or acidic amino acid, or a polar amino acid with a non-polar amino acid), as well as substitutions of amino acids with side chains with different bulk (steric size).

[0075] As another example of a phenomenon caused by a nonsense mutation, when a nonsense mutation is present in the protein-coding region of a CAD gene, nonsense-mediated mRNA decay can occur (Brogna and Wen (2009) Nat. Structural Mol. Biol. 16: 107-113). Because nonsense-mediated mRNA decay causes transcript degradation, nonsense mutations can result in a decrease in transcript abundance. For nonsense-mediated mRNA decay to occur, it is preferable that the CAD gene contains at least one exon containing a nonsense mutation. In particular, it is more preferable that the exon containing the nonsense mutation is not the most downstream (3') exon constituting the CAD gene. The CAD gene in wild-type Nicotiana plants consists of five exons and four introns. Therefore, a preferred embodiment of a nonsense mutation that causes nonsense-mediated mRNA decay is one in which at least one nonsense mutation is present in exons 1 to 4 of the CAD gene.

[0076] Mutations other than substitutions (such as deletions and insertions) occurring in the promoter sequence, 5' untranslated region, and / or 3' untranslated region can result in a decrease in the amount of transcripts and polypeptides due to decreased transcriptional activity or stability, similar to substitutions. Mutations other than substitutions into conserved intron sequences can also result in the translation of polypeptides with amino acid sequences different from the original, similar to substitutions. Mutations other than substitutions into coding regions can also result in the translation of polypeptides with amino acid sequences different from the original due to deletions or insertions of amino acid residues (caused by deletions or insertions of multiples of three consecutive bases) or frameshifts. Furthermore, large deletions including the entire gene or insertions of large fragments into the gene can result in the loss of expression of the gene itself.

[0077] The mutation may be introduced by mutagen treatment, genome editing or gene knockout. In particular, the mutation may be introduced by mutagen treatment.

[0078] The mutagen treatment of the gene can be carried out by artificially applying the mutagen to a Nicotiana plant (and, if necessary, in combination with suppression of gene repair function). Examples of mutagens that can be used include chemical agents such as ethylmethanesulfonate (EMS), sodium azide, ethidium bromide, and nitrous acid, but are not limited to these as long as they cause mutations in the genomic DNA of Nicotiana plants. Examples of mutagens include gamma rays, heavy ion beams, X-rays, neutron rays, and UV rays, but are not limited to these as long as they are radiation that causes mutations in the genomic DNA of Nicotiana plants. EMS is preferred as a mutagen. These techniques are preferred because they do not require the addition of exogenous factors to the target plant. The gene recombination can be carried out by homologously recombining part or all of the target gene with a recombinant sequence according to known genetic engineering techniques. Genome editing of the gene can be performed by known techniques (e.g., zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and the CRISPR / Cas9 system). The gene knockout can be performed by inserting a known transposon (mobile genetic element) or T-DNA.

[0079] Nucleotide substitutions in the CAD gene by EMS treatment can result in, for example, (I) frameshift mutations, (II) truncation mutations (in which the N-terminal amino acid residue is essentially deleted), (III) splice mutations, or (IV) nonsense mutations. This is because EMS treatment tends to cause specific nucleotide changes in DNA (C → T substitutions and G → A substitutions). Substitutions that can occur in the CAD gene by EMS treatment include, for example, substitution of A for the third G in the ATG base sequence corresponding to the translation initiation codon methionine (also referred to as Met or M), which can result in (I) frameshift mutations or (II) truncation mutations. Furthermore, if the second G and / or the third G of TGG in the base sequence corresponding to tryptophan (also written as Trp or W) is substituted with A, or if the first C of CAA or CAG in the base sequence corresponding to glutamine (also written as Gln or Q) or of CGA corresponding to arginine (also written as Arg or R) is substituted with T, (IV) a nonsense mutation may occur. If G is substituted with A in the bases GT or AG located at both ends of the intron sequence, (III) a splice mutation may occur.

[0080] In the CRISPR / Cas9 system, if the guide RNA and the Cas9 protein are present in the target cell, and in the case of TALEN and ZFN, if the fusion protein (fused with a DNA binding domain and a nuclease) is present in the target cell, genome editing is possible. Therefore, the guide RNA and the Cas9 protein, as well as the fusion protein, can be directly introduced into the target cell. Methods for directly introducing them into the target cell include PEG method, electroporation method, and particle bombardment method. In addition, a vector into which a construct (including a polynucleotide encoding the guide RNA and the Cas9 protein, and any promoter and / or terminator) is inserted can be introduced into the target cell or tissue via Agrobacterium or the like.

[0081] In the CRISPR / Cas9 system, a complementary sequence of a nucleotide sequence immediately upstream of XGG on the genome forms a base pair with a part of the guide RNA, and the double-stranded genomic DNA is cleaved by Cas9 within the nucleotide sequence.

[0082] In TALEN, each of the pair of DNA-binding domains of the dimer-forming artificial nuclease binds to a nucleotide sequence present on both ends of the FokI cleavage domain via a 5- to 20-base spacer. The nucleotide sequences are present on one strand and the other strand of double-stranded genomic DNA, and therefore one of the pair of DNA-binding domains binds to one strand and the other to the other strand. The DNA-binding domain is composed of repeating units (modules) of 33 to 34 amino acid residues, with the number of modules corresponding to the number of bases to be bound.

[0083] In ZFNs, similar to TALENs, a pair of DNA-binding domains of a dimer-forming artificial nuclease bind to nucleotide sequences present on both ends of a FokI cleavage domain via a 5- to 20-base spacer. The DNA-binding domains are composed of multiple zinc finger modules.

[0084] As described above, artificially mutated Nicotiana plants have been described, but are not limited to these. For example, in Nicotiana plants, gene mutations or disruption may occur through spontaneous mutation. Spontaneous gene mutations are generally caused by replication errors and genetic damage. Such damage can be caused by exposure to naturally occurring known mutagens (e.g., radiation, ultraviolet light, etc.).

[0085] The various mutations described above can be easily introduced into Nicotiana plants by those skilled in the art. That is, based on this sequence information, regions in the genomes of various Nicotiana plants encompassed by the concept of the present invention into which mutations should be introduced can be appropriately determined.

[0086] Gene mutation or disruption can be determined by detecting the presence or absence of a mutation in the gene. Methods for detecting a mutation in a gene include: (1) a method in which a DNA sequence containing the mutation is amplified by PCR or the like, and then the DNA base sequence is directly decoded using a commercially available sequencer, (2) a method in which sequence differences are detected by differences in electrophoretic distance using the SSCP (Single Strand Conformation Polymorphism) method, (3) a method in which SNP (Single Nucleotide Polymorphism) is detected using the Cycle PCR method, (4) a method in which the presence or absence of a mutation is detected by cleaving mismatch sites using T7 Endonuclease I or the like, (5) a CAPS (Cleaved Amplified Polymorphic Sequence) method in which the presence or absence of a mutation can be determined based on the presence or absence of cleavage by restriction enzyme treatment, and (6) a dCAPS (Derived Amplified Polymorphic Sequence) method in which the presence or absence of a mutation can be determined based on the presence or absence of cleavage by restriction enzyme treatment using a primer set that intentionally contains mismatches. (7) A method for determining the presence or absence of a mutation by detecting whether or not a probe that specifically hybridizes to a mutant sequence has hybridized (PCR using a TaqMan probe); (8) A method for performing single-base extension using a primer adjacent to the mutation and detecting the presence or absence of a mutation based on the mass difference of the incorporated base (MassARRAY analysis); (9) In the case of deletions or insertions, a method for detecting mutations based on differences in electrophoretic mobility is available. However, any method that can determine the presence or absence of a mutation is sufficient. Alternatively, gene mutation or disruption can be determined by comparing the size and expression level of the protein resulting from the gene modification with those of the wild-type protein. Specifically, such a comparison can be performed, for example, by Western blotting.

[0087] Suppression of gene expression includes suppression of transcription from the gene to mRNA, suppression of translation from the gene to a polypeptide via mRNA (e.g., degradation of the mRNA), and suppression of the function of the translated polypeptide. mRNA degradation can result from the nonsense-mediated mRNA decay. Suppression of transcription can be achieved by inhibiting transcription factors that promote transcription from the gene and by inhibiting access of transcription initiation factors to the gene. Suppression of translation can be achieved using antisense RNA molecules, RNAi molecules, or co-suppression molecules. Suppression of polypeptide function can be achieved by molecules that inhibit the function of a functional polypeptide by binding to it (e.g., decoy nucleic acids, ribozymes, antibodies, and inhibitory peptides).

[0088] The vector used for transforming Nicotiana plants for the purpose of suppressing gene expression or introducing mutations into genes is not particularly limited, as long as it is capable of expressing a polynucleotide inserted therein in plant cells. Suitable vectors include, for example, pBI-, pPZP-, and pSMA-based vectors, which can introduce a polynucleotide of interest into plant cells via Agrobacterium. Binary vector-based plasmids (e.g., pBIG, pBIN19, pBI101, pBI121, and pPZP202) are particularly preferred.

[0089] When gene expression is suppressed by RNAi, a trigger sequence used to suppress the expression of a target gene by RNAi is inserted as a mutation into the vector. The trigger sequence is, for example, a polynucleotide (sense RNA portion) represented by a base sequence of at least 21 to 30 consecutive bases (e.g., 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, or 30 or more bases) that is part of a polynucleotide (which may have 0.1 to 1% substitutions) encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 or 2, and a polynucleotide (antisense RNA portion) represented by a base sequence complementary to the polynucleotide. The above-mentioned base sequence of "at least 21 to 30 consecutive bases" more specifically means a base sequence of 21 or more consecutive bases, 23 or more bases, 25 or more bases, 30 or more bases, 35 or more bases, 40 or more bases, 45 or more bases, 50 or more bases, 60 or more bases, 70 or more bases, 80 or more bases, 90 or more bases, or 100 or more bases.

[0090] The suppression (of transcription, translation, or polypeptide function) can be achieved, for example, by directly introducing a molecule for achieving the suppression into a plant, or by introducing a nucleic acid molecule encoding the molecule into a plant (transformation of the plant). Here, as a result of the transformation of the plant, the nucleic acid molecule is integrated into one or more arbitrary regions in the genome of the plant. In the case of Nicotiana tabacum, which is an amphidiploid plant, the nucleic acid molecule does not need to be integrated into both the S genome and the T genome as a result of the transformation of the plant, as long as the suppression is achieved.

[0091] (Function Suppression) In one embodiment of the present invention, a Nicotiana plant has suppressed function of an endogenous gene containing a polynucleotide consisting of the nucleotide sequence of a CAD gene as its coding region. As used herein, the term "endogenous gene function suppression" refers to a state in which a gene on the genome does not perform its original function. Therefore, "endogenous gene function suppression" encompasses "mutation of the endogenous gene," "disruption of the endogenous gene," and "suppression of endogenous gene expression" by a gene other than the endogenous gene (including a foreign gene). Furthermore, specifically suppressing function refers to suppressing only the function of the target gene without suppressing the functions of other genes. For example, it is desirable to avoid simultaneous suppression of the function of multiple genes under the control of the same transcription factor by suppressing the function of the cinnamyl alcohol dehydrogenase gene, which could lead to metabolic abnormalities.

[0092] In one embodiment of the present invention, the functional inhibition may be a reduction in the abundance of a native functional polypeptide translated from the coding region of the endogenous cinnamyl alcohol dehydrogenase gene, compared to a wild-type plant. A "reduced abundance" of a polypeptide refers to the presence of the polypeptide at 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less of the abundance of the wild-type polypeptide. The abundance of the polypeptide relative to the abundance of the wild-type polypeptide can be appropriately selected from the above values ​​so as to result in an increased vanillin content in the Nicotiana plant.

[0093] Preferably, the reduction in the abundance of the polypeptide in a Nicotiana plant according to the present invention is genetically and stably inherited in cultured cells, calli, protoplasts, seeds, and progeny obtained from the Nicotiana plant. Thus, the Nicotiana plant according to the present invention may be an individual generated from cultured cells, calli, protoplasts, seeds, and progeny produced through artificial manipulation, and these materials for obtaining the individual are included in the scope of the present invention.

[0094] The functional inhibition may be a reduction in translation of the native functional polypeptide compared to a wild-type plant. The translation of the polypeptide occurs due to a reduction in mRNA (due to the abundance of mRNA, such as instability of the mRNA itself, promotion of mRNA degradation, or suppression of mRNA transcription) or a reduction in the amount of translation from mRNA (due to a deficiency, inhibition of recruitment, or functional deficiency of translation components (tRNA and ribosomes)).

[0095] The functional inhibition may be a reduction in the abundance of mRNA transcribed from the endogenous cinnamyl alcohol dehydrogenase gene compared to a wild-type plant. The reduction in the amount of mRNA transcription occurs, for example, by suppressing transcription from the endogenous gene to mRNA. Transcription inhibition can be achieved by, for example, inhibiting access of transcription initiation factors to the endogenous gene as a result of introducing a mutation into the endogenous gene.

[0096] The functional inhibition may be the promotion of degradation of mRNA transcribed from the endogenous gene. mRNA degradation can be caused by the production of abnormal mRNA (causing nonsense-mediated mRNA decay), the presence of exogenous factors that degrade mRNA, the activation of endogenous components that degrade mRNA, or the presence of a degradation-promoting sequence in mRNA. In a Nicotiana plant, the promotion of degradation of mRNA transcribed from the endogenous gene results in a decrease in the amount of mRNA in the Nicotiana plant. That is, in the Nicotiana plant, the functional inhibition may be a decrease in the amount of mRNA transcribed from the endogenous gene compared to a wild-type plant. Here, "a decrease in the amount of mRNA transcribed from an endogenous gene" refers to the presence of 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less of the transcript, based on the amount of the transcript of the endogenous gene in a wild-type plant.

[0097] In the Nicotiana plant, the mutation may be insertion of a polynucleotide expressing a factor that promotes degradation of mRNA transcribed from the endogenous gene outside the region where the endogenous gene is located. The factor may be an antisense RNA molecule, an RNAi molecule, or a co-suppression molecule.

[0098] In one embodiment of the present invention, a Nicotiana plant has suppressed function of an endogenous gene, the endogenous gene containing a polynucleotide as its coding region that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to a polynucleotide consisting of a nucleotide sequence encoding an endogenous CAD gene and that encodes a polypeptide having CAD activity.

[0099] Stringent conditions refer to conditions under which a double-stranded polynucleotide specific to the nucleotide sequence is formed, but the formation of a non-specific double-stranded polynucleotide is significantly suppressed. In other words, they can be said to be conditions under which hybridization occurs between highly homologous nucleic acids, for example, at a temperature 15°C, preferably 10°C, and more preferably 5°C lower than the melting temperature (Tm value) of a double-stranded polynucleotide that perfectly matches the probe. For example, hybridization conditions include 68°C for 20 hours in a typical hybridization buffer. One example of such a condition is hybridization for 16 to 24 hours in a buffer solution consisting of 0.25 M Na2HPO4, pH 7.2, 7% SDS, 1 mM EDTA, and 1x Denhardt's solution at a temperature of 60 to 68°C, preferably 65°C, and more preferably 68°C, followed by two 15-minute washes in a buffer solution consisting of 20 mM Na2HPO4, pH 7.2, 1% SDS, and 1 mM EDTA at a temperature of 60 to 68°C, preferably 65°C, and more preferably 68°C. Another example is a hybridization solution containing 25% formamide, or, under more stringent conditions, 50% formamide, 4x SSC (sodium chloride / sodium citrate), 50 mM Hepes pH 7.0, 10x Denhardt's solution, and 20 μg / ml denatured salmon sperm DNA, where prehybridization is performed overnight at 42°C, followed by addition of a labeled probe and incubation at 42°C overnight. The washing solution and temperature conditions for subsequent washes can be approximately "1x SSC, 0.1% SDS, 37°C," or, for more stringent conditions, approximately "0.5x SSC, 0.1% SDS, 42°C," or, for even more stringent conditions, approximately "0.2x SSC, 0.1% SDS, 65°C." Thus, the more stringent the hybridization washing conditions, the more likely it is that DNA with high homology to the probe sequence will be isolated.However, the above combinations of SSC, SDS, and temperature conditions are merely examples, and those skilled in the art can achieve similar stringency by appropriately combining the above or other factors that determine hybridization stringency (e.g., probe concentration, probe length, hybridization reaction time, etc.). For example, those skilled in the art can easily obtain such genes by referring to Molecular Cloning (Sambrook, J. et al., Molecular Cloning: a Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, 10 Skyline Drive, Plainview, NY (1989)).

[0100] 4. Treatment of Tobacco Raw Material In one embodiment of the present invention, the tobacco raw material can be treated with alkali. Flavor components can be generated through the alkali treatment, and the flavor components can be collected to prepare a tobacco extract liquid containing tobacco extract and water. In this case, it is preferable to extract the flavor components as a gas from the alkali-treated tobacco raw material and introduce the gas into water to transfer the flavor components to the water.

[0101] The alkaline substance used in the alkaline treatment is preferably an alkaline liquid such as an aqueous sodium hydroxide solution or an aqueous potassium carbonate solution. In this case, the alkaline substance is supplied until the pH of the tobacco raw material falls within a specific range. This pH is preferably 8.0 or higher, more preferably 8.9 to 9.7. The pH of the tobacco raw material is the pH of water obtained by mixing the tobacco raw material with 10 times the amount of water.

[0102] In one embodiment of the present invention, tobacco raw materials can be subjected to high-temperature, high-pressure treatment. Examples of high-temperature, high-pressure treatment include the method described in JP-A-10-66669. A solvent is added to tobacco particles so that the solid-liquid ratio (w / v) is in the range of 1:5 to 1:20, and the mixture is heat-treated at 100 to 250°C for 10 to 180 minutes while sealed in a pressure-controlled environment. The resulting reaction product is then returned to atmospheric pressure and cooled. Examples of the solvent include water and hydrophilic organic solvents such as alcohol.

[0103] In one embodiment of the present invention, tobacco raw materials can be steam distilled. Examples of steam distillation methods include those described in "Encyclopedia of Fragrances," p. 366 (published August 27, 1980, by Fujimaki Masao et al., Asakura Shoten). For example, water is placed in a distillation still and brought to a boil, after which the tobacco raw materials are placed in the still and distilled for several hours. The resulting steam-distilled water is collected, and acid is added to the water to adjust the pH to 3.0-4.0, followed by the addition of salt to a concentration of 20%. An extraction solvent is then added, and the mixture is subjected to liquid-liquid inversion using a separatory funnel. The solvent layer is collected, and an aqueous NaOH solution (pH 9-10) is added to remove the acid. The solvent layer is then concentrated using an evaporator to obtain a dry product. The water used for steam distillation may be filtered, deodorized, decolorized, etc.

[0104] In one embodiment of the present invention, the tobacco material may be subjected to ethanol extraction. For example, an ethanol extract may be obtained by adding 20 to 99.5% ethanol to the tobacco material so that the solid-liquid ratio (w / v) is 1:2 to 1:20, and then soaking the tobacco material at 4°C to 60°C for one hour to several months.

[0105] [5. Method for producing a Nicotiana plant] One embodiment of the present invention comprises the step of introducing a mutation that specifically causes functional suppression of an endogenous cinnamyl alcohol dehydrogenase gene into the endogenous gene in the genome of a Nicotiana plant. Details of the mutation introduced into the Nicotiana plant are described in Section [3. Nicotiana plant].

[0106] The introducing step may include introducing the mutation into the coding region of the endogenous cinnamyl alcohol dehydrogenase gene. Further, the introducing step may be performed by mutagen treatment, genome editing, or gene knockout. In particular, the introducing step may be mutagen treatment.

[0107] The introducing step may also include introducing a mutation that specifically suppresses the function of the endogenous cinnamyl alcohol dehydrogenase gene into the genome of the Nicotiana plant outside the coding region of the endogenous cinnamyl alcohol dehydrogenase gene. The suppression of function is a reduction in the abundance of mRNA transcribed from the endogenous cinnamyl alcohol dehydrogenase gene compared to a wild-type plant. More preferably, the suppression of function may be promotion of degradation of the mRNA transcribed from the endogenous gene. Furthermore, the introduction step may include inserting a polynucleotide that expresses an antisense RNA molecule, an RNAi molecule, or a co-suppressor molecule that promotes degradation of the mRNA transcribed from the endogenous cinnamyl alcohol dehydrogenase gene.

[0108] In the above production method, an individual exhibiting a desired phenotype may be further selected from a mutant population of plants having mutations. As an example of selecting an individual, a procedure for selecting a desired individual from a mutant population (panel) obtained by treatment with a mutagen will be described.

[0109] One example of a method for producing Nicotiana plants with a mutation in the CAD gene is to treat Nicotiana plants with a mutagen such as EMS, as described above, to generate a panel of Nicotiana plants with mutations throughout the entire genome, and then extract genomic DNA. The CAD gene is amplified from each panel of genomic DNA or from a pool of these using gene-specific primers, the nucleotide sequence of the amplified product is determined, and lines with homozygous mutations are selected. In the case of Nicotiana plants, lines with homozygous mutations in the S genome and T genome are obtained, and these are crossed to produce F1. Furthermore, the resulting self-pollinated progeny (F2) are cultivated, and from these lines, lines with homozygous mutations in both the S genome and the T genome are obtained (a 1 / 16 probability due to bifactorial recessiveness).

[0110] Selection of individuals exhibiting a desired phenotype may be based on vanillin content. For example, individuals whose tobacco material or raw material contains an increased vanillin content compared to the vanillin content of wild-type tobacco material or raw material may be selected.

[0111] Therefore, the method of one embodiment may further include one or more steps: creating a population (panel) of Nicotiana plants in which mutations have been introduced throughout the genome of the Nicotiana plant; extracting genomic DNA from lines included in the panel; determining the base sequence of the CAD gene in the genomic DNA; selecting lines containing homozygous mutations from the panel; and confirming metabolites or CAD activity in the lines or tobacco materials or raw materials containing the lines.

[0112] The strain can be crossed with a non-mutagenized strain at any time before the step of confirming the measurement of metabolites or CAD activity is performed. Crossing allows for the elimination of mutations that may exist in genes other than the CAD gene. In certain embodiments, the strain with a mutation in the CAD gene can be backcrossed multiple times with a non-mutagenized strain (the original strain used to generate the panel).

[0113] Genomic DNA from a Nicotiana plant mutant may be extracted by a known method, or a commercially available extraction kit may be used. The genomic DNA may be crudely purified or may be a purified product that has undergone several purification steps.

[0114] Polynucleotide amplification can be carried out by, for example, PCR, but other known gene amplification methods such as LCR (ligase chain reaction) or LAMP (loop-mediated isothermal amplification) may also be used.

[0115] Primer sequences for amplifying each polynucleotide can be designed, for example, from the base sequence. For example, in the case of Nicotiana tabacum, S-type-specific and T-type-specific regions are first identified based on the results of a homology analysis between the base sequences of SEQ ID NO: 5 (the genomic sequence of the S-type CAD gene) and SEQ ID NO: 6 (the genomic sequence of the T-type CAD gene). Primers can be designed for these regions to specifically amplify the S-type and T-type CAD genes, respectively, from extracted genomic DNA (including S-type and T-type). The primers can be selected from S-type- or T-type-specific regions, preferably introns, 5'-untranslated regions, or 3'-untranslated regions. The primer length is preferably 15 to 30 bases, particularly preferably 17 to 25 bases. The primer sequence may be designed based on a region specific to the base sequence or a region common to both base sequences. Furthermore, as long as the primer functions as a primer for amplifying a sequence of a predetermined number of bases, including a mutation site, the primer sequence may contain one or more substitutions, deletions, and / or additions. Furthermore, the primer may be labeled with a fluorescent substance or a radioactive substance, if necessary.

[0116] The length of each polynucleotide to be amplified is not particularly limited as long as it is a length that allows use of various detection methods described below, but is, for example, 20 to 5,000 bases, more preferably 50 to 2,000 bases, even more preferably 100 to 700 bases, and still more preferably 100 to 500 bases.

[0117] Functionally deficient mutants of Nicotiana tabacum plants with mutations in two alleles (a total of four alleles, including alleles in both the T and S genomes, in the case of Nicotiana tabacum) can be obtained, for example, by the following method. As described above, Nicotiana tabacum plants are treated with a mutagen to create a mutant population (panel) with mutations throughout the genome, and genomic DNA is extracted. Using gene-specific primers, target genes (polynucleotides) are amplified from the genomic DNA of the panel, the nucleotide sequences of the products are determined, and lines with homozygous mutations are selected. For example, in the case of Nicotiana tabacum, lines (M2) with homozygous mutations in both the S and T genomes are first obtained, and then crossed to create F1. Furthermore, the resulting self-pollinated progeny (F2) are cultivated, and lines with homozygous mutations in both the S and T genomes are obtained from among them. To obtain functionally deficient mutants of Nicotiana tabacum plants with mutations in only one of the S and T genomes, it is sufficient to confirm that the genes in the non-target genome in the obtained M2 are not mutated.

[0118] [6. Breeding Progeny] One embodiment of the present invention may be a progeny of the above-described Nicotiana plant or a Nicotiana plant obtained by the above-described production method, or a breeding progeny obtained by crossing the Nicotiana plant. Mutant breeding has been conducted for many plant species, including rice, wheat, barley, and soybean. For example, mutants isolated from a mutant population treated with a mutagen contain numerous mutations in addition to the target gene. Therefore, backcrossing is generally performed to remove excess mutations. By crossing with a cultivar with superior traits, the traits of the mutant can be introduced into the cultivar, resulting in a cultivar with higher added value. Because the traits of mutants are derived from mutations, backcrossing requires the selection of individuals with mutations. In this case, the fewer mutations that result in the target trait (in this invention, vanillin content), the fewer mutations to focus on, reducing the effort required for backcrossing. Efficient backcrossing requires a simple method for detecting the presence or absence of mutations and whether the mutations are homozygous or heterozygous. This method can be performed using the mutation detection method described below. In addition, by performing Marker Assisted Selection (MAS) using background markers that show polymorphism between mutants and cultivars, lines with a high reversion rate to cultivars can be efficiently obtained with a small number of crosses. In the case of Nicotiana plants, known SNPs and SSRs (Simple Sequence Repeats) can be used as polymorphic markers. If necessary, new polymorphic markers can be obtained and used by decoding the genome sequence of the tobacco used and identifying differences in base sequence and number of repeat sequences.

[0119] [7. Other] One embodiment of the present invention provides a method for determining whether a Nicotiana plant has an increased vanillin content, comprising the steps of: obtaining a sample by harvesting a part of the Nicotiana plant; detecting a mutation in the genome contained in the sample that specifically causes functional suppression of the endogenous CAD gene; and determining that the Nicotiana plant in which the mutation is detected is a Nicotiana plant with an increased vanillin content.

[0120] Here, the suppression of the function results in an increase in the vanillin content in tobacco leaves or cured leaves of Nicotiana plants. That is, the above determination method is used in methods for producing Nicotiana plants, etc.

[0121] One embodiment of the present invention provides a method for breeding Nicotiana plants, comprising the step of crossing Nicotiana plants having a high vanillin content determined by the above-described determination method.

[0122] One embodiment of the present invention provides a progeny of the above-mentioned Nicotiana plant, a Nicotiana plant obtained by the above-mentioned production method, a Nicotiana plant determined by the above-mentioned determination method, or a Nicotiana plant obtained by the above-mentioned breeding method, or a breeding progeny obtained by crossing the Nicotiana plant. For example, when the Nicotiana plant is a Nicotiana plant and is Nicotiana tabacum, suppression of the function of two CAD genes (NtCAD-T and NtCAD-S) results in a high vanillin content. This enables breeding using a bifactorial recessive inheritance pattern, targeting mutations in the NtCAD-T gene and mutations in the NtCAD-S gene, thereby reducing the effort required for breeding compared to conventional methods.

[0123] Mutant breeding has been conducted for many plant species. For example, mutants isolated from a mutant population treated with a mutagen contain numerous mutations in addition to the target gene. Therefore, backcrossing is generally performed to remove excess mutations. In this crossing, a cultivar possessing superior traits is crossed with the mutant, allowing the desired traits of the mutant to be introduced into an existing cultivar. The resulting breeding progeny can be a variety that adds significant value to an existing cultivar. In this case, the fewer the number of target mutations that result in increased vanillin content, the fewer mutations that need to be focused on, reducing the effort required for backcrossing. The Nicotiana plant described above can be advantageously used from the perspective of breeding because it can increase vanillin content through a single gene mutation.

[0124] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0125] (Summary) By summarizing the above embodiments, the present invention can be summarized as follows.

[0126] (1) A tobacco material comprising a part of a Nicotiana plant, wherein the Nicotiana plant has a mutation resulting in functional inhibition of the endogenous cinnamyl alcohol dehydrogenase gene in at least one of: (a) an endogenous cinnamyl alcohol dehydrogenase gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1; and (b) an endogenous cinnamyl alcohol dehydrogenase gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2; and the tobacco material contains 0.05 ppm or more of vanillin.

[0127] (2) The tobacco material according to (1), wherein the tobacco material comprises dried tobacco material.

[0128] (3) The tobacco material according to (1) or (2), wherein the tobacco material is in the form of any one of cut filler, powder, particles, sheets, granules, and extracts.

[0129] (4) A tobacco product comprising the tobacco material according to any one of (1) to (3).

[0130] (5) The tobacco product according to (4), wherein the tobacco product is any one of an electronic tobacco product, a cigarette, and a smokeless tobacco product.

[0131] (6) A Nicotiana plant, wherein a mutation that causes functional inhibition of the endogenous cinnamyl alcohol dehydrogenase gene has been introduced into at least one of the following: (a) an endogenous cinnamyl alcohol dehydrogenase gene, the endogenous cinnamyl alcohol dehydrogenase gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1; and (b) an endogenous cinnamyl alcohol dehydrogenase gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2; and wherein the vanillin content of the product harvested from the Nicotiana plant is higher than that of the product harvested from an equivalent amount of a wild-type Nicotiana plant.

[0132] (7) The Nicotiana plant according to (6), which belongs to Nicotiana tabacum or Nicotiana rustica.

[0133] (8) The Nicotiana plant according to (6) or (7), wherein the vanillin content is at least twice the vanillin content of leaves harvested from the same amount of the wild-type Nicotiana plant.

[0134] (9) The Nicotiana plant according to any one of (6) to (8), wherein the mutation is introduced by mutagen treatment, genome editing, or gene knockout.

[0135] (10) The Nicotiana plant according to any one of (6) to (9), wherein the mutation is introduced into at least one coding region of the endogenous cinnamyl alcohol dehydrogenase gene.

[0136] An embodiment of the present invention will now be described.

[0137] Example 1: Identification of tobacco CAD mutants The genome sequence of the tobacco (Nicotiana tabacum) cultivar "Tsukuba No. 1" was analyzed, and a CAD (NtCAD-S, ID: nttv1s022g02367, SEQ ID NO: 5) thought to be derived from the S genome and a CAD (NtCAD-T, ID: nttv1s791g11064, SEQ ID NO: 6) thought to be derived from the T genome were identified. The CDS sequences of NtCAD-S (SEQ ID NO: 3) and NtCAD-T (SEQ ID NO: 4) were subjected to a homology search against the NCBI database, and it was confirmed that the genome sequences shown in SEQ ID NOs: 5 and 6, respectively, are sequences related to the CAD gene of tobacco (Nicotiana tabacum). The amino acid sequence of NtCAD-S (SEQ ID NO: 1) and the amino acid sequence of NtCAD-T (SEQ ID NO: 2) were also subjected to a similar search, and it was confirmed that the amino acid sequences shown in SEQ ID NOs: 1 and 2, respectively, are the amino acid sequences of CAD.

[0138] Tobacco mutants harboring mutations in CAD-S or CAD-T were isolated. The nucleotide sequences of the NtCAD-S and NtCAD-T gene regions of 2,000 tobacco mutant lines were analyzed to identify the mutations. Specifically, 2,000 tobacco mutants were generated by EMS treatment of seeds from the tobacco cultivar Tsukuba No. 1. Self-pollinated progeny seeds (M2 seeds) obtained from each M1 generation of 2,000 mutant individuals were sown. DNA extracted from eight seedlings per line was bulked (Tajima et al. (2011) Ann. Phytopathol. Soc. Jpn. 77: 258) and analyzed for nucleotide sequence. Three and two lines, respectively, harbored nonsense mutations in the coding region of the NtCAD-S gene (SEQ ID NO: 3) and the coding region of the NtCAD-T gene (SEQ ID NO: 4). Two mutants were selected for each gene. That is, for NtCAD-S, a line in which C at position 5' to 2237 of the genome sequence shown in SEQ ID NO: 5 was mutated to T, i.e., line NtCAD-S-1 (SEQ ID NO: 7) in which C at position 5' to 112 of the CDS sequence shown in SEQ ID NO: 3 was mutated to T, and a line in which C at position 5' to 2282 of the CDS sequence shown in SEQ ID NO: 3 was mutated to T, i.e., line NtCAD-S-2 (SEQ ID NO: 8) in which C at position 5' to 157 of the CDS sequence shown in SEQ ID NO: 3 was mutated to T. For NtCAD-T, a line in which C at position 5' to 3833 of the genome sequence shown in SEQ ID NO: 6 was mutated to T, i.e., line NtCAD-T-1 (SEQ ID NO: 9) in which C at position 5' to 394 of the CDS sequence shown in SEQ ID NO: 4 was mutated to T, and a line in which C at position 5' to 4725 of the CDS sequence shown in SEQ ID NO: 4 was mutated to T, i.e., line NtCAD-T-2 (SEQ ID NO: 10) in which C at position 5' to 715 of the CDS sequence shown in SEQ ID NO: 4 was mutated to T were selected. Seeds from these lines were sown and DNA was extracted at the seedling stage. PCR was performed using this DNA as a template and primers, and individuals carrying the mutation homozygously were selected. KOD One (registered trademark) PCR Master Mix (TOYOBO Corporation) was used for PCR. As a result, two lines carrying a mutation in the NtCAD-S gene and two lines carrying a mutation in the NtCAD-T gene were obtained. Table 1 shows the line names, the CAD sequences of the lines, and the primer sequences used to confirm the mutation or select the mutants.

[0139]

[0140] NtCAD-S-1 and NtCAD-T-1 were grown and crossed in a greenhouse to obtain the F1 generation (NtCAD-F1-1). NtCAD-S-2 and NtCAD-T-2 were grown and crossed in a greenhouse to obtain the F1 generation (NtCAD-F1-2). Each F1 generation was grown and selfed in a greenhouse to obtain two F2 generations derived from different mutant lines (NtCAD-F2-1 and NtCAD-F2-2). These two F2 generations were sown, DNA was extracted from the transplanted seedlings, and PCR was performed using the primers listed in Table 1 to amplify the surrounding sequence containing the mutation. PCR was then performed again to assign P7 and P5 sequences and individual barcode sequences to the amplified product for sequence analysis using iSeq 100 (Illumina), and sequence analysis was performed to confirm the genotype of each individual. As a result, we found two individuals, NtCAD-sstt-A (hereafter referred to as "CAD-A") and NtCAD-sstt-B (hereafter referred to as "CAD-B"), which are homozygous for mutations in both the NtCAD-S and NtCAD-T genes. We also found two individuals, NtCAD-SSTT-A (a sister line of NtCAD-sstt-A) and NtCAD-SSTT-B (a sister line of NtCAD-sstt-B), which do not have mutations in either gene. These individuals were selfed to obtain the F3 generation.

[0141] Example 2. Evaluation of Components Contributing to Flavor and Flavor (Samples) Eighteen F3 generations of CAD-A and CAD-B were grown in the field and pinned at the flowering stage. Three true leaves were harvested from each individual seven weeks after pinning. As a control, Tsukuba No. 1 was grown in the same manner, and three true leaves were harvested. Leaves were harvested from 15 individuals in good condition (12 individuals for CAD-A), and three individuals (a total of nine leaves) were mixed in equal proportions to form one sample, resulting in a total of five samples (four samples for CAD-A only). The harvested leaves were dried until yellow, and used as cured leaf samples. Three leaves were selected from each sample, the midribs were removed, and the leaves were freeze-dried to form mature leaf samples.

[0142] Leaf vein samples were obtained from leaves harvested from three Nicotiana plants of each line. The CAD-A and CAD-B mutant vein samples were mixed in equal proportions and then crushed. Hereinafter, the sample obtained by mixing and crushing multiple samples will be simply referred to as "CAD."

[0143] The stems of the CAD-A mutant and the control Tsukuba No. 1 were harvested after leaf collection at the flowering stage (immediately after topping) and then stripped of their epidermis to expose the cortex. These were then force-dried at 60°C for 48 hours to prepare the stem (xylem after topping) samples.

[0144] Seven weeks after stem stabbing, two stems were harvested from each of the CAD-A and CAD-B mutants and the control, Tsukuba 1, after leaf removal. The stems were then peeled to expose the cortex and forced-dried at 60°C for 48 h. These were used as xylem samples. The CAD-A and CAD-B mutant samples were mixed in equal proportions and then crushed.

[0145] (Results) Three of the five dry leaf samples, as well as one each of the mature leaf sample, the harvest-stem sample, the flowering-stem sample, and the midrib sample, were crushed and subjected to component analysis by GC-MS and LC-MS / MS. The results of the GC-MS and LC-MS / MS analyses are shown in Tables 2, 3, and 4. The tables show the mean values ​​(n = 3) for the dry leaf sample only, and the significant differences from the control, Tsukuba No. 1 (n = 3). Statistical analysis was performed using Dunnett's test (** indicates p < 0.01, * indicates p < 0.05). In both CAD mutants, CAD-A and CAD-B, vanillin content was significantly higher than in the control for the mature leaf sample, the dry leaf sample, the harvest-stem sample, the flowering-stem sample, and the midrib sample. In the CAD mutants, the content was 2.2 to 6.4 times higher in mature leaf samples (CAD-A, CAD-B), 1.3 to 4.3 times higher in dry leaf samples (CAD-A, CAD-B), 9 times higher in the harvest-stem sample (CAD), 25 times higher in the flowering-stem sample (CAD-A), and 4.4 times higher in the midrib sample (CAD) compared to the control.

[0146] Table 2 shows the semi-quantitative values ​​of vanillin and other components in the dry leaf sample, the harvest-stem sample, the flowering-stem sample, and the backbone sample.

[0147]

[0148] Table 3 shows the semi-quantitative values ​​of vanillin and other components in mature leaf samples.

[0149]

[0150] Table 4 shows the quantitative values ​​of vanillin in dry leaf samples, stem samples at the flowering stage, and midrib samples of the CAD mutant.

[0151]

[0152] Example 3 Preparation of Tobacco Materials and Evaluation of Flavor and Taste Tobacco materials were prepared using the flowering-stem CAD-A mutant and dried leaves of Tsukuba No. 1, and the tobacco materials were impregnated into cigarettes, followed by flavor and taste tests.

[0153] Tobacco material was prepared from the dried leaves of Tsukuba No. 1 after crushing. The dried leaves were then mixed with propylene glycol (PG) at a ratio of 1:3. Tobacco material was prepared from the flowering-stem CAD-A mutant by extracting the components with 95% food additive alcohol (Traceable 95) at a solid-liquid ratio of 1:9 for approximately 1 hour at 30-40°C. The ethanol fraction was then concentrated to dryness, and the residue was dried and crushed while being classified. Finally, the dried product and the residue powder were mixed with PG at a ratio of 1:4. Unflavored cigarettes were prepared, and the tobacco material prepared from the dried leaves of Tsukuba No. 1 and the flowering-stem CAD-A mutant was applied to the shreds.

[0154] Compared with the dried leaves of the control, Tsukuba No. 1, five out of six trained tasters rated the stems of the CAD-A mutant as having an increased sweetness and six out of six as having a vanilla-like aroma, demonstrating that the stems of the CAD mutant exhibit an enhanced aroma and flavor.

[0155] Example 4. Obtaining tobacco CAD single mutants and evaluating components contributing to flavor and aroma The F2 generation of the two lines obtained in Example 1 (NtCAD-F2-1 and NtCAD-F2-2) was subjected to sequence analysis to confirm the genotype of each individual, and individuals homozygous for a mutation in either the NtCAD-S or NtCAD-T gene, NtCAD-ssTT-A (hereinafter "CAD-A-sT"), NtCAD-SStt-A (hereinafter "CAD-A-St"), NtCAD-ssTT-B (hereinafter "CAD-B-sT"), and NtCAD-SStt-B (hereinafter "CAD-B-St"), were identified. These individuals were selfed to obtain the F3 generation.

[0156] Eighteen F3 plants each of CAD-A-sT, CAD-A-St, CAD-B-sT, and CAD-B-St were grown in the field and pinned at the flowering stage. Seven weeks after pinning, three true leaves were harvested from each plant. Tsukuba No. 1 was grown in the same manner as a control, and three true leaves were harvested. Leaves were harvested from 15 plants in good condition, and three plants (a total of nine leaves) were mixed in equal proportions to form one sample, for a total of five samples. The harvested leaves were dried to a yellow color and used as the cured leaf sample. Three leaves from each sample were selected, crushed, and subjected to component analysis by GC-MS and LC-MS / MS.

[0157] GC-MS analysis was performed as follows: 0.05 g of ground dried leaf sample was placed in a 15 mL screw-cap glass centrifuge tube, and 50 μL of the internal standard (20 ppm 6-methyl-quinoline) solution was added to each weighed sample. 10 mL of ethanol was then added to each sample, stirred using a Bordeaux valve, and the lid was tightly closed. Shaking extraction was performed using a medium-sized constant-temperature shaker, Bioshaker® BR-43FH MR (Taitec Co., Ltd., Saitama), at 200 rpm, 50°C, and 1 hour. The extracted solution was filtered through a 0.45 μm PTFE filter and subjected to GC-MS analysis. Other detailed analytical conditions are as follows. GC / MS: 7890B / 5977B (Agilent Technologies, Tokyo) Column: DB-WAX IU; 122-7032UI; 0.25 mm, 30 m, 0.25 μm (Agilent Technologies, Tokyo) Injection method: Splitless Injection port temperature: 250°C Heating program: 50°C for 2 min, 20°C / min to 120°C for 2 min, 10°C / min to 250°C for 24 min Measurement time: 44.5 min Post run: 5.0 mL / min; 30 min Measurement mode: SIM / SCAN

[0158] LC-MS / MS analysis was performed as follows. A 0.1 g sample of ground dried leaves was placed in a 15 mL screw-cap glass centrifuge tube, and 5.0 mL of a methanol solution containing the internal standard phlorizin at a concentration of 2.0 μg / mL was added. The tube was then sealed with a screw cap. Shaking extraction was performed using a medium-sized, thermostatically controlled shaker, Bioshaker® BR-43FH-MR (Taitec Co., Ltd., Saitama), at 250 rpm and 70°C for 1 hour. The extracted solution was filtered through a 0.45 μm pore size PTFE filter and subjected to analysis by liquid chromatography tandem mass spectrometry (LC-MS / MS). Other detailed analytical conditions are as follows.・High-performance liquid chromatograph: 1260 infinity (Agilent Technologies, Tokyo) ・Mass spectrometer: 6470 Triple Quad LC / MS (Agilent Technologies, Tokyo) ・Column: InfinityLab Poroshell 120 CS-C18, inner diameter 2.1 mm, length 150 mm, particle size 2.7 μm (Agilent Technologies, Tokyo) ・Column temperature: 35°C ・Eluent A: 20 mM ammonium formate-ultrapure water ・Eluent B: 20 ​​mM ammonium formate-acetonitrile:ultrapure water = 9:1 (v / v) ・Elution conditions: 1) 0 to 2 minutes: Eluent A:eluent B = 95:5, run for 2 minutes (eluent A 95% / eluent B 5%) 2) 2 to 10 minutes: Apply a linear gradient of eluent A:eluent B from 95:5 to 55:45. 3) 10 to 20 minutes: Apply a linear gradient of eluent A:eluent B from 55:45 to 0:100. 4) 20 to 25 minutes: Flow eluent B for 5 minutes (eluent A 0% / eluent B 100%). Flow rate: 0.2 mL / min. Injection volume: 2.0 μL. Ion source parameters: Ionization method: electrospray ionization (ESI). Nebulizer gas: nitrogen. Nebulizer gas temperature: 350°C. Nebulizer gas flow rate: 5 L / min. Nebulizer pressure: 35 psi. Sheath gas: 350°C. Sheath gas flow rate: 11 L / min. Capillary voltage: 3500 V. Mass spectrometer parameters: Ion polarity: negative. Collision gas: nitrogen. Measurement mode: selected reaction monitoring (SRM).

[0159] (Results) In all four CAD single mutant lines, CAD-A and CAD-B, there was no significant difference in vanillin content compared to the control, Tsukuba No. 1. Table 5 shows the semi-quantitative values ​​of vanillin and other components in dry leaf samples from CAD single mutant individuals, obtained as a result of component analysis by GC-MS and LC-MS / MS.

[0160] Example 5. Evaluation of components contributing to flavor and aroma in stem tissue (Analysis of vanillin and vanillin analogs in stem tissue) The F3 generation of Tsukuba No. 1, CAD-A, and CAD-B was grown in a field, and the stems were harvested at the flowering stage. Immediately after harvest, nine stems were separated into three tissues: epidermis, cortex, and pith, and each tissue was dried at 60°C for 48 hours. The dried tissues were pulverized using a Labomill LM-05 (Dalton Corporation) and subjected to component analysis by GC-MS and LC-MS / MS.

[0161] The results of GC-MS and LC-MS / MS analyses are shown in Table 6. In Table 6, the semiquantitative values ​​for each tissue (epidermis, cortex, and pith) of the control, Tsukuba No. 1, are shown relative to the semiquantitative values ​​of 1. In both CAD mutants, CAD-A and CAD-B, all analyzed components tended to increase, except for vanillylamine. In particular, in the cortex, vanillin increased 7.97-11.77-fold, and ferulic acid increased 11.17-24.46-fold in both CAD mutants, CAD-A and CAD-B, compared with the control, Tsukuba No. 1. Quantitative analysis of vanillin in each tissue of Tsukuba No. 1 and CAD-A revealed that vanillin in the cortex was 12.00 ppm in Tsukuba No. 1 and 108.90 ppm in CAD-A, accounting for 60-77.45% of the vanillin in the entire trunk. Furthermore, vanillin glucoside, which was not detected in the cortex of Tsukuba No. 1, was detected in the cortex of both CAD-A and CAD-B mutants, whereas vanillylamine was not detected in the pith of both CAD-A and CAD-B mutants.

[0162] (Analysis of vanillin content by trunk part and harvest time) CAD-A F3 was grown in a field, and trunks were harvested at both the flowering and harvest times. Immediately after harvest, the bark of each of nine trunks was removed, and the trunk was divided into two parts, the upper and lower parts, from the center of the trunk (see Figure 2 ). Each part was dried at 60°C for 48 hours. The resulting dried material from the nine trunks was divided into three parts, each consisting of a mixture of three trunks, and then finely pulverized using a Labo Mill LM-05 (Dalton Co., Ltd.) for component analysis by GC-MS.

[0163] The results of GC-MS analysis are shown in Table 7. In CAD-A-flowering stage-stem (without epidermis), vanillin was 6.85 times higher in the lower part than in the upper part, vanillic acid was 1.66 times higher, and vanillyl alcohol was 1.73 times higher. On the other hand, in CAD-A-harvest stage-stem (without epidermis), vanillin was 1.47 times higher in the lower part than in the upper part, vanillic acid was 1.24 times higher, and vanillyl alcohol was 0.73 times higher.

[0164] Example 6. Preparation of tobacco materials by fine grinding, high-temperature, high-pressure treatment, steam distillation, and ethanol extraction, and evaluation of components contributing to flavor and aroma Tobacco materials were prepared using the stems (without epidermis), dried leaves, and midribs at the flowering stage of CAD-A and Tsukuba No. 1, respectively, through various processes. The various CAD-A and Tsukuba No. 1 samples used were finely ground using a Labo Mill LM-05 fine grinder (manufactured by Dalton Co., Ltd.), and 50 mg of each tobacco material was taken after undergoing the various treatments A to D described below, or left as is (untreated), and the vanillin content of each tobacco material was quantitatively analyzed by GC-MS.

[0165] (A: Finely milled) Tobacco material was prepared by blending dried leaves of Tsukuba No. 1 and CAD-A varieties in a ratio of 1:3 with propylene glycol (PG). The same procedure was used to prepare the midrib, except that the dried leaves were replaced with midrib. For the flowering stem (without epidermis), components were first extracted with 99.5% ethanol at a solid-liquid ratio of 1:9 for approximately 1 hour at 30-40°C. The solid-liquid separation was then performed. The ethanol fraction was concentrated to dryness, and the residue was dried and pulverized. Finally, the dried material and the residue powder were blended in a ratio of 1:4 with PG, and finely milled again in a Silverson L5M-A laboratory mixer to prepare tobacco material.

[0166] (B: High-temperature, high-pressure treatment) Using Tsukuba No. 1 and CAD-A anthesis-stage stems (without epidermis), anthesis-stage stems (without epidermis):water ratio was adjusted to 1:7, and the mixture was subjected to high-temperature, high-pressure treatment with stirring in a TAS-01 reactor (manufactured by Taiatsu Glass Industries Co., Ltd.) at a temperature of 150°C and a pressure of 380 kPa for 3 hours to produce tobacco materials. The tobacco materials were produced using the same procedure as above, except that the anthesis-stage stems (without epidermis) were replaced with the anthesis-stage stems (without epidermis) for the midribs.

[0167] (C: Steam Distillation) Tobacco material was prepared using 400 g of flowering-stage stem (without epidermis) from Tsukuba No. 1 and CAD-A. 4 L of tap water was placed in a stockpot and brought to a boil at 100°C using an induction heater. After boiling, heating was stopped and 400 g of the flowering-stage stem (without epidermis) sample was placed in the stockpot. The lid of the stockpot was closed, heating was resumed, and distillation was continued for 2 hours. A tap was connected to the condenser, and tap water was passed through it to act as a chiller. Distillation was continued for 100 minutes, and 1.0 L of the resulting steam-distilled sample was collected and 20% (w / w) sodium chloride was added. 1 L of ethyl acetate was added to the steam-distilled sample, and the mixture was subjected to liquid-liquid transfer using a separatory funnel. The ethyl acetate layer was collected and concentrated under reduced pressure using a rotary evaporator to obtain a dry solid. The vanillin content of the obtained essential oils was approximately 465 mg / kg for CAD-A and approximately 335 mg / kg for Tsukuba No. 1. For quantitative analysis of vanillin content, the essential oils were diluted 100 times with 99.5% ethanol before use.

[0168] (D: Ethanol Extraction Treatment) Using the flowering stage-stem (without epidermis) of Tsukuba No. 1 and CAD-A, flowering stage-stem (without epidermis):99.5% ethanol was prepared at a ratio of 1:5 and allowed to stand for 24 hours at 25° C. After 24 hours, the supernatant was collected to prepare tobacco materials.

[0169] (Quantitative Analysis of Vanillin Content of Each Tobacco Material) 10.0 mL of an ethanol solution containing the internal standard 6-methylquinoline at a concentration of 0.1 μg / mL was added to each 15 mL screw-cap test tube, followed by 50 mg of either tobacco material prepared by the above-mentioned treatments A to D or untreated tobacco material. The tubes were then capped with screw caps, and shaking extraction was performed at 200 rpm, 50°C, and 1 hour using a medium-sized, thermostatically controlled shaker, Bioshaker® BR-43FH-MR (Taitec Co., Ltd., Saitama). The extracted solution was filtered through a 0.45 μm pore size PTFE filter and subjected to GC-MS analysis. Other detailed analytical conditions are as follows: Gas chromatograph: 7890B (Agilent Technologies, Tokyo) Column: DB-WAX UI, inner diameter 0.25 mm, length 30 m, particle size 0.25 μm (Agilent Technologies, Tokyo) Sample inlet temperature: 250°C Column oven heating conditions: 1) 0 to 2 min: Hold at 50°C 2) 2 to 7.5 min: Heat to 120°C at 20°C / min, then hold for 2 min 3) 7.5 to 50.5 min: Heat to 250°C at 10°C / min, then hold for 30 min Carrier gas: Helium Flow rate: 1.0 mL / min Sample injection volume: 1.0 μL Sample injection method: Pulsed splitless (20 psi, 2 min) Mass spectrometer: 5977A MSD (Agilent Technologies, Tokyo) Ionization method: Electron impact ionization (EI) Ion polarity: positive Electron impact energy: 70 eV Mass ion source temperature: 230°C Mass quadrupole temperature: 150°C

[0170] The quantitative ions and qualitative ions of the internal standard substance, 6-methylquinoline, were set as shown in Table 8.

[0171] The quantitative value of vanillin was calculated by the calibration curve method from the ratio of the area of ​​the ion chromatogram of vanillin at m / z = 151.0 to the area of ​​the ion chromatogram of the internal standard 6-methylquinoline at m / z = 143.0. Four calibration curve solutions (7.81, 62.5, 125, and 250 ng / mL) were analyzed in the vanillin standard solution, which contained 100 ng / mL of 6-methylquinoline as the internal standard, over the concentration range of 7.81 to 250 ng / mL. Quantitation was performed using a linear calibration curve with a correlation coefficient of 0.9999 for the area ratio of the ion chromatograms of 6-methylquinoline and vanillin.

[0172] (Results) The vanillin content (ppm) per 1 g of each sample is shown in Table 9. The vanillin content of the untreated control Tsukuba No. 1 was 9.67 ppm in the stem (without bark) at the flowering stage, 9.98 ppm in the dry leaves, and 5.17 ppm in the midrib. The vanillin content of the untreated CAD-A was 80.11 ppm in the stem (without bark) at the flowering stage, 21.32 ppm in the dry leaves, and 7.01 ppm in the midrib. The vanillin content of the finely ground CAD-A was 4.18 ppm in the stem (without bark) at the flowering stage, 9.98 ppm in the dry leaves, and 3.18 ppm in the midrib. The vanillin content of the finely ground CAD-A was 33.07 ppm in the stem (without bark) at the flowering stage, 4.77 ppm in the dry leaves, and 3.34 ppm in the midrib. The vanillin content of the control Tsukuba No. 1 flowering-stem (without bark) after high-temperature and high-pressure treatment was 14.27 ppm, while that of CAD-A flowering-stem (without bark) was 56.84 ppm and that of the midrib was 7.66 ppm. The vanillin content of the control Tsukuba No. 1 flowering-stem (without bark) essential oil concentrate after steam distillation was 30.53 ppm, while that of CAD-A flowering-stem (without bark) essential oil concentrate was 995.75 ppm. The vanillin content of the control Tsukuba No. 1 flowering-stem (without bark) after ethanol extraction was 2.93 ppm, while that of CAD-A flowering-stem (without bark) essential oil concentrate was 17.02 ppm. The ethanol extract of CAD-A flowering-stem (without bark) also had a vanilla-like aroma.

[0173] Example 7. Preparation of tobacco material subjected to alkali heating treatment, and evaluation of components contributing to flavor and flavor Tobacco materials were prepared using stems (without epidermis), dried leaves, and backbone at the flowering stage of CAD-A and Tsukuba No. 1. The various CAD-A and Tsukuba No. 1 samples used were finely pulverized using a Labo Mill LM-05 fine grinder and subjected to the processes described below. 50 mg of each tobacco material was then sampled, and the vanillin content in each tobacco material was quantitatively analyzed by GC-MS.

[0174] (Alkali Heating Treatment) The alkali heating treatment method was as follows. 1) 3.0 g of each finely ground sample was added to 40 mL of 1 M NaOH, and heat extraction was performed at 80°C for 2 hours under atmospheric pressure. 2) Neutralization was performed with a 1 M phosphoric acid solution. 3) The liquid phase was filtered, and ethyl acetate was mixed with the filtrate. The portion eluted in the ethyl acetate (ethyl acetate extract) was distilled to obtain a dry solid. The dry solid was redissolved in ethanol. 4) The residue was dried at 60°C for 2 hours. 5) The ethanol-redissolved product of the dry solid obtained in 3) was added back to the residue from 4), and the ethanol was air-dried to obtain an alkali heating treated tobacco material.

[0175] As a control, a tobacco material was prepared by replacing 40 mL of 1M NaOH with 40 mL of distilled water in step 1) above and omitting step 2), and this was used as a mock-treated tobacco material.

[0176] (Quantitative Analysis of Vanillin Content in Mock-Treated Tobacco Material) 10.0 mL of an ethanol solution containing the internal standard 6-methylquinoline at a concentration of 0.1 μg / mL was added to a 15 mL screw-cap test tube, followed by 50 mg of the tobacco material prepared by the above-mentioned mock treatment. The tube was then capped with a screw cap, and shaking extraction was performed at 200 rpm, 50°C, and 1 hour using a medium-sized constant-temperature shaking incubator, Bioshaker® BR-43FH-MR (manufactured by Taitec Corporation). The extracted solution was filtered through a 0.45 μm pore size PTFE filter and subjected to GC-MS analysis.

[0177] (Method for extracting vanillin using alkaline heating treatment) 30.0 μL of an acetone solution (50 μg / mL) of 1,3-dimethoxybenzene, an internal standard, was added to a 15 mL screw-cap test tube. 100 mg of ground tobacco material prepared by the alkaline heating treatment described above was added, and 3.0 mL of 1 M NaOH in MilliQ water was added. The tube was then sealed with a screw cap and heated at 80°C in a heat block for 1 hour. After heating, the tube was allowed to cool to room temperature, and 210 μL of 85% aqueous phosphoric acid solution was added to the test tube and neutralized by vortexing. After stirring, 3.0 mL of ethyl acetate was added and vortexed. Vanillin and 1,3-dimethoxybenzene were extracted into the organic layer. The tube was centrifuged at 2000 rpm for 3 minutes, and the upper (organic) layer was subjected to GC-MS analysis.

[0178] The detailed analytical conditions other than those mentioned above for the mock-treated and alkali-heat-treated GC-MS analyses are common to all of the following: Gas chromatograph: 7890B (Agilent Technologies, Tokyo) Column: DB-WAX UI, inner diameter 0.25 mm, length 30 m, particle size 0.25 μm (Agilent Technologies, Tokyo) Sample inlet temperature: 250°C Column oven heating conditions: 1) 0 to 2 min: Hold at 50°C 2) 2 to 7.5 min: Heat to 120°C at 20°C / min, then hold for 2 min 3) 7.5 to 50.5 min: Heat to 250°C at 10°C / min, then hold for 30 min Carrier gas: Helium Flow rate: 1.0 mL / min Sample injection volume: 1.0 μL Sample injection method: Pulsed splitless (20 psi, 2 min) Mass spectrometer: 5977A MSD (Agilent Technologies, Tokyo) Ionization method: Electron impact ionization (EI) Ion polarity: positive Electron impact energy: 70 eV Mass ion source temperature: 230°C Mass quadrupole temperature: 150°C

[0179] The quantitative ions and qualitative ions of the vanillin to be quantified, and 6-methylquinoline and 1,3-dimethoxybenzene, which are internal standard substances for quantification of vanillin in the mock treatment and alkali-heating treatment, respectively, were set as shown in Table 10.

[0180] The quantitative value of mock-treated vanillin was calculated by the calibration curve method using the ratio of the area of ​​the ion chromatogram of vanillin (m / z = 151.0) to the area of ​​the ion chromatogram of the internal standard 6-methylquinoline (m / z = 143.0). Four calibration curve solutions (7.81, 62.5, 125, and 250 ng / mL) were analyzed by GC-MS in the range of 7.81–250 ng / mL for vanillin standard solutions spiked with 100 ng / mL of 6-methylquinoline (internal standard). Quantitation was performed using a linear calibration curve with a correlation coefficient of 0.9999 for the area ratio of the ion chromatograms of 6-methylquinoline and vanillin.

[0181] The quantitative value of alkali-heated vanillin was calculated by the calibration curve method using the ratio of the area of ​​the ion chromatogram of vanillin (m / z = 151.0) to the area of ​​the ion chromatogram of the internal standard 1,3-dimethoxybenzene (m / z = 138.0). Six concentrations of vanillin standard solutions (3.13, 6.25, 12.5, 25.0, 50, and 100 μg / mL) were added to 50 μg / mL of the internal standard 1,3-dimethoxybenzene. Each 30 μL of the calibration curve solution was placed in a screw-cap test tube and analyzed using the procedure described in the Extraction Method for Cell Wall-Bound Vanillin. The resulting ethyl acetate solution was analyzed by GC-MS. Quantitation was performed using a linear calibration curve with a correlation coefficient of 0.9999 for the area ratio of the ion chromatograms of 1,3-dimethoxybenzene and vanillin.

[0182] (Vanillin Content Quantification Results) The vanillin content of the alkali-heat-treated tobacco material was 60.56 ppm at the anthesis-stem (without epidermis), 45.35 ppm at the anthesis-stem (with epidermis), and 15.69 ppm in the dried leaves of the control, Tsukuba No. 1. Meanwhile, the vanillin content of CAD-A was 1491.59 ppm at the anthesis-stem (without epidermis), 504.62 ppm at the anthesis-stem (with epidermis), and 74.27 ppm in the dried leaves. Furthermore, the vanillin content of the anthesis-stem (without epidermis) of CAD-B dried leaves after alkali-heat treatment was 79.94 ppm. Furthermore, the vanillin content of CAD-A at the anthesis-stem (without epidermis) was 24.6-fold higher than that of the control, Tsukuba No. 1.

[0183] (Evaluation of Tobacco Material Aroma and Taste) Tobacco materials prepared from untreated and alkali-heated stems of Tsukuba No. 1 and CAD-A varieties at the flowering stage were used to evaluate the aroma and taste of both cigarettes and heated tobacco products. For the evaluation of cigarette aroma and taste, the total weight of shredded tobacco packed into the cigarette was set at 715 mg, and each tobacco material was blended at 5% of the weight of the unflavored cigarette shredded tobacco. Specifically, 35 mg of tobacco material was blended per product to be evaluated. For the evaluation of the aroma and taste of heated tobacco products, reconstituted sheets were prepared using the tobacco materials using the method described below, and mixed with unflavored reconstituted tobacco shredded tobacco prepared by papermaking for evaluation. The total weight of the tobacco material packed into the heated portion of the heated tobacco product was set at 254 mg, and the reconstituted sheets prepared using each tobacco material sample were filled into the heated portion at a weight equivalent to 15% of the weight of the unflavored reconstituted tobacco shredded tobacco prepared by papermaking. Specifically, 54 mg of the reconstituted sheet was formulated per article to be evaluated.

[0184] (Preparation of reconstituted sheets) Reconstituted sheets for evaluating the flavor and taste of heated tobacco products were prepared according to the following procedure. 1) 3 g of tobacco material, 20 mL of water, and 1.8 g of carboxymethyl cellulose were mixed, and 1.8 g of glycerin was added to prepare a slurry. 2) Plastic wrap was wrapped around a horizontal base of a glass plate, and the slurry prepared in 1) was spread on the wrap. 3) The plastic wrap on which the slurry was spread in 2) was placed together with the base in an oven, and heated and dried at an internal temperature of 80°C for 3 hours to form the slurry into a sheet. 4) The heat-dried sheet in 3) was allowed to cool and cut into a size of 2 mm x 10 mm.

[0185] (Evaluation of cigarette aroma and flavor) Five trained panelists evaluated the cigarette aroma and flavor. When comparing cigarettes containing the alkali-heat-treated tobacco material of Tsukuba No. 1 with cigarettes containing the control untreated tobacco material of Tsukuba No. 1, one out of five panelists responded that the sweetness was improved compared to the control. On the other hand, when comparing cigarettes containing the untreated tobacco material of CAD-A, five out of five panelists responded that the sweetness was improved compared to the control. Furthermore, when comparing cigarettes containing the alkali-heat-treated tobacco material of CAD-A, five out of five panelists responded that the sweetness was more strongly improved compared to the control, and also responded that they perceived a vanilla-like aroma.

[0186] (Evaluation of Flavor and Aroma of Heated Tobacco) Five trained panelists evaluated the flavor and aroma of heated tobacco. When comparing the heated tobacco containing the alkali-heat-treated tobacco material of Tsukuba No. 1 with the control heated tobacco containing the untreated tobacco material of Tsukuba No. 1, one out of five panelists responded that the sweetness was improved compared to the control. On the other hand, when comparing the heated tobacco containing the untreated tobacco material of CAD-A, four out of five panelists responded that the sweetness was improved compared to the control. Furthermore, when comparing the heated tobacco containing the alkali-heat-treated tobacco material of CAD-A, four out of five panelists responded that the sweetness was improved compared to the control, and three out of five panelists responded that they perceived a vanilla-like aroma.

[0187] The present invention can be used in tobacco products.

Claims

1. A tobacco material containing a part of a tobacco plant, wherein the tobacco plant contains, as a coding region, an endogenous cinnamyl alcohol dehydrogenase gene encoding a polypeptide having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, and at least one of the endogenous cinnamyl alcohol dehydrogenase genes containing, as a coding region, a polynucleotide encoding a polypeptide having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2 has a mutation that causes suppression of the function of the endogenous cinnamyl alcohol dehydrogenase gene, and the tobacco material contains 0.05 ppm or more of vanillin.

2. The tobacco material according to claim 1, wherein the tobacco material includes a dried tobacco material.

3. The tobacco material according to claim 1 or 2, which is in any form of cut filler, powder, particles, sheet, granule, and extract.

4. A tobacco product containing the tobacco material according to any one of claims 1 to 3.

5. The tobacco product according to claim 4, wherein the tobacco product is any one of an e-cigarette product, a cigarette, and a smokeless tobacco product.

6. A tobacco plant, wherein at least one of the endogenous cinnamyl alcohol dehydrogenase genes containing, as a coding region, a polynucleotide encoding a polypeptide having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, and the endogenous cinnamyl alcohol dehydrogenase gene containing, as a coding region, a polynucleotide encoding a polypeptide having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2 has a mutation that causes suppression of the function of the endogenous cinnamyl alcohol dehydrogenase gene, and the vanillin content of the harvest obtained from the tobacco plant is higher than the vanillin content of the harvest obtained from the same amount of wild-type tobacco plants.

7. The tobacco plant according to claim 6, which belongs to Nicotiana tabacum or Nicotiana rustica.

8. The tobacco plant according to claim 6 or 7, wherein the vanillin content is at least twice the vanillin content of the harvest obtained from the same amount of the wild-type tobacco plant.

9. The tobacco plant according to any one of claims 6 to 8, wherein the mutation is introduced by mutagenesis treatment, genome editing, or gene knockout.

10. The tobacco plant according to any one of claims 6 to 9, wherein the mutation is introduced into at least one coding region of the endogenous cinnamyl alcohol dehydrogenase gene.

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