Methods and compositions for modulating trichome density and flavor molecule secretion

Abstract

Glands are epidermal outgrowths in plants that are sites of synthesis and storage of metabolite compounds. Modulating the secretion of metabolite compounds in tobacco and tea can change the taste profile in products made from these plants. The present disclosure relates to methods for modulating glandular secretion, as well as compositions comprising altered glandular secretion of metabolites.

Description

Cross-references to related applications and incorporation of sequence listings

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 617,925, filed January 5, 2024, which is incorporated herein by reference in its entirety. A sequence list containing 90 SEQ ID NOs from SEQ ID NO:1 to SEQ ID NO:90, in a file named “P35070WO00_SL.xml”, measuring 263,609 bytes (on MS-Windows® OS), created on January 3, 2025, is filed electronically and incorporated herein by reference in its entirety. Technical Field

[0002] This disclosure relates to methods for regulating glandular trichome secretion, compositions comprising modified trichome secretion containing metabolites, and their use in plants including tobacco and tea. This disclosure also provides methods for regulating the metabolite profile from glandular trichome secretion, compositions comprising modified trichome secretion containing metabolites active as flavor molecules, and their use in plants including tobacco and tea. Background Technology

[0003] Glandular trichomes are epidermal organisms in plants that serve as sites for the synthesis and storage of metabolic compounds. They are found in stem, leaf, and floral tissues, providing protection against various biotic and abiotic stresses. Glandular trichomes also play a role in the biosynthesis, storage, and secretion of specialized or secondary metabolites. Other trichomes (such as non-glandular trichomes) protect plants from predators. For example (By providing a physical barrier) or protecting against ultraviolet radiation. Trichothecises are visible on the adaxial (upper) and abaxial (lower) leaf surfaces, although their density varies in some plant species.

[0004] Metabolites produced and secreted by glandular hairs are typically hydrophobic. For example (Fatty acid derivatives, flavonoids, terpenoids). Terpenoids constitute the largest and most diverse class of plant metabolites. The olefin backbone of terpenoids consists of multiples of five-carbon (C) isoprene units, mainly monoterpenes (10C), sesquiterpenes (15C), and diterpenes (20C). These terpenoids are most commonly produced by the condensation of five-carbon isoprene units (dimethylallyl diphosphate [DMAPP] and isopentenyl diphosphate [IPP]) through a continuous head-to-tail addition condensation of DMAPP and IPP.

[0005] The amount of secondary metabolites produced is usually closely related to the density of glandular trichomes present on the plant epidermis (Chalvin). et al. , Cell, 25:477-487 (2020)). One way to increase the amount of secondary metabolites produced in plants is to increase the density of trichomes present on the plant epidermis. Transcriptional regulation of trichome initiation has been shown to involve members of the MYB and C2H2 zinc finger transcription factor families. Studies have shown that Artemisia annua MYB1 ( AaMYB1 Overexpression of the transgene increased glandular trichome density and subsequently increased artemisinin production (Matias-Hernandez). et al. , Plant Journal , 90:520-534 (2017)).

[0006] Lipid transfer proteins (LTPs) play an important role in the transport of specialized metabolites in glandular trichomes. Studies have shown that overexpression of LTPs leads to an increase in secretions in plant glandular trichomes (Choi). et al. , Plant Journal , 70:480-491(2012)).

[0007] Given the crucial role of glandular trichomes in the biosynthesis and secretion of terpenoids, a deeper understanding of the genes, regulators, and signaling mechanisms involved in controlling trichome initiation and development in plants is needed. Understanding the patterns of secretion of these specialized metabolites into the trichome cuticle is also important. This disclosure provides candidate genes that can be used to modify trichome density and the metabolite secretion profile in plants. Modification of trichome density will also improve the transport of specialized metabolites in glandular trichomes. Altering the metabolite secretion profile will provide the ability to modulate the flavor profile of flavored tobacco and flavored tobacco products, with the aim of improving consumer satisfaction. Summary of the Invention

[0008] In one aspect, this disclosure provides a modified tobacco plant or a portion thereof, the modified tobacco plant or a portion thereof comprising at least one non-natural mutation in an endogenous nucleic acid sequence, wherein the endogenous nucleic acid sequence encodes a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40.

[0009] In one aspect, this disclosure provides a modified tobacco plant or a portion thereof comprising a recombinant DNA construct containing a heterologous promoter operatively linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of an endogenous nucleic acid sequence encoding a polypeptide that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40.

[0010] In one aspect, this disclosure provides a modified tobacco plant or a portion thereof comprising a recombinant DNA construct comprising a heterologous promoter operatively linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40.

[0011] In one aspect, this disclosure provides a method for producing a modified tobacco plant, the method comprising: inducing a non-natural mutation in an endogenous nucleic acid sequence encoding a polypeptide in at least one tobacco cell, the polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40; selecting at least one tobacco cell containing the non-natural mutation; and regenerating at least one modified tobacco plant from the selected at least one tobacco cell.

[0012] In one aspect, this disclosure provides a method for producing a modified tobacco plant, the method comprising: introducing a recombinant DNA construct into at least one tobacco cell, wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of an endogenous nucleic acid sequence encoding a polypeptide whose amino acid sequence is at least 80% identical or similar to that of the group consisting of SEQ ID NO: 21 to 40; selecting at least one tobacco cell containing the recombinant DNA construct; and regenerating at least one modified tobacco plant from the at least one selected tobacco cell.

[0013] In one aspect, this disclosure provides a method for producing a modified tobacco plant, the method comprising: introducing a recombinant DNA construct into at least one tobacco cell, wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence encoding a polypeptide, the polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40; selecting at least one tobacco cell containing the recombinant DNA construct; and regenerating at least one modified tobacco plant from the at least one selected tobacco cell.

[0014] In one aspect, this disclosure provides a method comprising preparing a tobacco product using a modified tobacco material derived from a modified tobacco plant, wherein the modified tobacco plant contains a non-natural mutation in an endogenous nucleic acid sequence, wherein the endogenous nucleic acid sequence encodes a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40.

[0015] In one aspect, this disclosure provides a method comprising preparing a tobacco product using modified tobacco material derived from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, and wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence encoding at least one small RNA molecule capable of binding to and reducing the expression of an endogenous nucleic acid sequence encoding a polypeptide that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40.

[0016] In one aspect, this disclosure provides a method comprising preparing a tobacco product using a modified tobacco material derived from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, and wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40.

[0017] In one aspect, this disclosure provides a method comprising transforming tobacco cells with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence encoding at least one small RNA molecule capable of binding to and reducing the expression of an endogenous nucleic acid sequence encoding a polypeptide that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40.

[0018] In one aspect, this disclosure provides a method comprising transforming tobacco cells with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence encoding a polypeptide, the polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40.

[0019] In one aspect, this disclosure provides a method for producing modified tobacco plants, the method comprising: hybridizing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one offspring tobacco seed, wherein the at least one tobacco plant of the first tobacco variety contains a non-natural mutation in an endogenous nucleic acid sequence, wherein the endogenous nucleic acid sequence encodes a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40, wherein the non-natural mutation is not present in the endogenous nucleic acid sequence of a control tobacco plant of the first tobacco variety; and selecting at least one offspring tobacco seed or a plant germinating therefrom, wherein the at least one tobacco seed or a plant germinating therefrom contains the non-natural mutation.

[0020] In one aspect, this disclosure provides a method for producing modified tobacco plants, the method comprising: hybridizing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one offspring tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence encoding at least one small RNA molecule capable of binding to and reducing the expression of an endogenous nucleic acid sequence, the endogenous nucleic acid sequence encoding a polypeptide whose amino acid sequence is at least 80% identical or similar to that of the group consisting of SEQ ID NO: 21 to 40, wherein the recombinant DNA construct is not present in the endogenous nucleic acid sequence in a control tobacco plant of the same variety; and selecting at least one offspring tobacco seed or plant germinating therefrom, wherein the at least one tobacco seed or plant germinating therefrom comprises the recombinant DNA construct.

[0021] In one aspect, this disclosure provides a method for producing modified tobacco plants, the method comprising: hybridizing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one offspring tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40, wherein the recombinant DNA construct is not present in the nucleic acid sequence of a control tobacco plant of the first tobacco variety; and selecting at least one offspring tobacco seed or a plant germinating therefrom, wherein the at least one tobacco seed or a plant germinating therefrom comprises the recombinant DNA construct.

[0022] In one aspect, this disclosure provides a modified tobacco plant or a portion thereof, the modified tobacco plant or a portion thereof comprising at least one non-natural mutation in an endogenous nucleic acid sequence that regulates the expression or functional activity of a gene, wherein the gene encodes a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40.

[0023] In one aspect, this disclosure provides a modified tobacco plant or a portion thereof comprising: (a) a genetic modification in a gene; or (b) a genetic modification targeting the gene; wherein the genetic modification downregulates the expression or activity of the gene, wherein the gene encodes a polynucleotide sequence having at least 80% identical nucleic acid sequence to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1 to 20.

[0024] On the one hand, this disclosure provides a modified Camellia The plant or part thereof, the modified camellia plant or part thereof comprising at least one non-natural mutation in an endogenous nucleic acid sequence, wherein the endogenous nucleic acid sequence encodes a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40.

[0025] On the one hand, this disclosure provides a modified Camellia The plant or a part thereof, the modified camellia plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operatively linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of an endogenous nucleic acid sequence encoding a polypeptide whose amino acid sequence is at least 80% identical or similar to that of the group consisting of SEQ ID NO: 21 to 40.

[0026] On the one hand, this disclosure provides a modified Camellia The plant or part thereof, the modified camellia plant or part thereof comprising a recombinant DNA construct comprising a heterologous promoter operatively linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40.

[0027] On one hand, this disclosure provides a method for generating modified... Camellia A plant-based method, comprising: in at least one CamelliaInducing a non-natural mutation in an endogenous nucleic acid sequence encoding a polypeptide in cells, the polypeptide containing at least 80% identical or similar amino acid sequences to those selected from the group consisting of SEQ ID NO: 21 to 40; selecting at least one containing said non-natural mutation. Camellia Cells; and from at least one of the selected cells Camellia Cell regeneration of at least one modified tobacco plant.

[0028] On one hand, this disclosure provides a method for generating modified... Camellia A plant-based method, comprising: introducing a recombinant DNA construct into at least one Camellia Cells, wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and downregulating the expression of an endogenous nucleic acid sequence encoding a polypeptide whose amino acid sequence is at least 80% identical or similar to that of the group consisting of SEQ ID NO: 21 to 40; at least one cell comprising the recombinant DNA construct is selected. Camellia Cells; and regeneration of at least one modified tobacco plant from at least one selected tobacco cell. Camellia plant.

[0029] On one hand, this disclosure provides a method for generating modified... Camellia A plant-based method, comprising: introducing a recombinant DNA construct into at least one Camellia Cells, wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence encoding a polypeptide comprising at least 80% identical or similar amino acid sequences to those selected from the group consisting of SEQ ID NO: 21 to 40; at least one cell comprising the recombinant DNA construct is selected. Camellia Cells; and regeneration of at least one modified tobacco plant from at least one selected tobacco cell. Camellia plant.

[0030] In one aspect, this disclosure provides a method for generating modified Camellia The plant method, which includes: taking the first Camellia at least one variety Camellia Plants and the Second Camellia at least one variety Camellia Plant hybridization to produce at least one offspring Camellia Seeds, wherein the first Camellia at least one of the varieties CamelliaThe plant contains a non-natural mutation in an endogenous nucleic acid sequence, wherein the endogenous nucleic acid sequence encodes a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40, wherein the non-natural mutation is not present in the first Camellia Variety comparison Camellia The endogenous nucleic acid sequence in the plant; and the selection of at least one generation Camellia Seeds or plants from which they germinate, wherein at least one of them is a seed. Camellia The seed or the plant from which it germinates contains the non-natural mutation.

[0031] In one aspect, this disclosure provides a method for generating modified Camellia The plant method, which includes: taking the first Camellia at least one variety Camellia Plants and the Second Camellia at least one variety Camellia Plant hybridization to produce at least one offspring Camellia Seeds, wherein the first Camellia at least one of the varieties Camellia The plant contains a recombinant DNA construct comprising a heterologous promoter operatively linked to a nucleic acid sequence encoding at least one small RNA molecule capable of binding to and downregulating the expression of an endogenous nucleic acid sequence encoding a polypeptide whose amino acid sequence is at least 80% identical or similar to that of the group consisting of SEQ ID NO: 21 to 40, and wherein the recombinant DNA construct is not present in a control of the same variety. Camellia In endogenous nucleic acid sequences in plants; and select at least one generation Camellia Seeds or plants from which they germinate, wherein at least one of them is a seed. Camellia The seed or the plant from which it germinates contains the recombinant DNA construct.

[0032] In one aspect, this disclosure provides a method for generating modified Camellia The plant method, which includes: taking the first Camellia at least one variety Camellia Plants and the Second Camellia at least one variety Camellia Plant hybridization to produce at least one offspring Camellia Seeds, wherein the first Camellia at least one of the varieties CamelliaThe plant contains a recombinant DNA construct comprising a heterologous promoter operatively linked to a nucleic acid sequence encoding a polypeptide comprising at least 80% identical or similar amino acid sequences to those selected from the group consisting of SEQ ID NO: 21 to 40, wherein the recombinant DNA construct does not contain the first Camellia The nucleic acid sequence in a control tobacco plant of a variety; and the selection of at least one generation of tobacco seed or plant germinating from it, wherein the at least one tobacco seed or plant germinating from it contains the recombinant DNA construct.

[0033] Sequence Description SEQ ID NO: 1 to 20 lists examples of nucleotide coding sequences for genes related to trichome density and flavor.

[0034] SEQ ID NO: 21 to 40 lists examples of amino acid sequences for genes related to trichome density and flavor.

[0035] SEQ ID NO: 41 to 60 lists examples of genomic DNA sequences for genes related to trichome density and flavor.

[0036] SEQ ID NO: 61 lists the sequence of the ALCS1 binary genetic construct for plant transformation. This construct contains the pCSVMV promoter, multiple cloning site, NOS terminator, ACTII promoter, NPTII resistance gene, and NOS terminator.

[0037] SEQ ID NO: 62 to 69 lists the nucleotide sequences of examples of promoters used for trichome-specific or preferred expression.

[0038] SEQ ID NO: 70 to 80 lists sequences of instances of promoters used for top-responsive root-specific or preferred expression.

[0039] SEQ ID NO: 81 to 90 list sequences of examples of promoters for topping-responsive leaf-specific or preferred expression. Attached Figure Description

[0040] Figure 1 An optical microscope photograph of a red-flowered tobacco leaf at 100x magnification is depicted, highlighting the structure of the glandular trichomes. Glandular trichome measurements were performed by measuring the width of the trichome glands and the length of the trichome stalks.

[0041] Figure 2 A comparison of selected trichome measurements from GR139 secretory (S) and GR139-non-secreting (NS) tobacco leaves is presented. Figure 2 A provides the total number of trichomes on both sides of the distal (lower) and proximal (upper) axis of the 5th leaf. Figure 2 B provides the average trichome length (in micrometers) on both the distal and proximal sides of the 5th leaf. Figure 2 C provides the average trichome width (in micrometers) on both the distal and adaxial sides of the 5th leaflet. Standard deviation is shown as an error bar.

[0042] Figure 3 Depicting Red Flos Tobacco An optical microscope image of a leaf at 100x magnification. The paraxial and abaxial surfaces are visible, with the paraxial surface at the top. Figure 3 A depicts the 5th leaf of GR139 secretory (S) red tobacco, and a depiction of the 5th leaf of GR139 non-secreting (NS) red tobacco. Figure 3 Compared to B.

[0043] Figure 4 The number of trichomes in tobacco varieties GR139 S, KDH-960, Hoja Parado, KDH-959, KDH-926 and AA-37-1 was quantitatively described. Figure 4 A shows the average number of paraxial trichomes. Figure 4 B shows the average number of distal trichomes. The standard deviation is shown as an error bar.

[0044] Figure 5 Depicting the GR139 S Red Flos Tobacco Background and in KDH959 Red Flos Tobacco Volcano plot of targeted RT-qPCR results for the top 10 candidate genes compared to the background. G82265, g58563, g58333, g58504, g58508, g82264, and g58509 were significantly upregulated. G82257 showed a flat expression pattern, and g82268 showed no significant change.

[0045] Figure 6 Depicting KDH926 Red Flos Tobacco Background and in KDH959 Red Flos Tobacco Volcano plot of targeted RT-qPCR results for the top 10 candidate genes compared to the background. G82265, g58563, g58333, g58504, g58508, g82264, and g58509 were significantly upregulated. G82257 was significantly downregulated, and g82268 showed no significant change.

[0046] Figure 7 Depicting KDH960 Red Flos Tobacco In the context of KDH959 Red Flos TobaccoVolcano plot of targeted RT-qPCR results for the top 10 candidate genes compared to the background. G82265, g58563, g58333, g58504, g58508, g82264, and g58509 were significantly upregulated. G82257 was significantly downregulated, and g82268 showed no significant change.

[0047] Figure 8 The results of targeted RT-qPCR for 16 candidate genes in GR139NS GIS-overexpressing tobacco, compared to the GR139NS control, are depicted. Actin was used as a control and set to zero. Fold changes relative to actin are shown along the Y-axis. Figure 8 A and Figure 8 B stands for independent experiment.

[0048] Figure 9 The results of targeted RT-qPCR for 16 candidate genes in GR139NS MYB86-overexpressing tobacco, compared to the GR139NS control, are depicted. Actin was used as a control and set to zero. Fold changes relative to actin are shown along the Y-axis. Figure 9 A and Figure 9 B stands for independent experiment.

[0049] Figure 10 The results of targeted RT-qPCR for 16 candidate genes in MYB86-overexpressing tobacco with galpao are depicted compared to the galpao control. Actin was used as a control and set to zero. Fold changes relative to actin are shown along the Y-axis. Figure 10 A, Figure 10 B and Figure 10 C stands for independent experiment.

[0050] Figure 11 The results of targeted RT-qPCR for 16 candidate genes in KDH960 MYB86-overexpressing tobacco, compared to the KDH960 control, are depicted. Actin was used as a control and set to zero. Fold changes relative to actin are shown along the Y-axis. Figure 11 A, Figure 11 B and Figure 11 C stands for independent experiment.

[0051] Figure 12 The results of targeted RT-qPCR for 16 candidate genes in TN90 GIS-overexpressing tobacco, compared to the TN90 control, are depicted. Actin was used as a control and set to zero. Fold changes relative to actin are shown along the Y-axis. Figure 12 A, Figure 12 B and Figure 12 C stands for independent experiment.

[0052] Figure 13The results of targeted RT-qPCR for 16 candidate genes in TN90 MYB86-overexpressing tobacco, compared to the TN90 control, are depicted. Actin was used as a control and set to zero. Fold changes relative to actin are shown along the Y-axis. Figure 13 A, Figure 13 B and Figure 13 C stands for independent experiment.

[0053] Figure 14 Principal component analysis (PCA) plots were created for 11 secretory and non-secretory tobacco varieties using only compounds associated with the following secondary metabolites: alkaloids and alkaloid derivatives, benzene rings, flavonoids, phenylpropanoids, and terpenoids. Detailed Implementation

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Where terms are provided in the singular, the inventors also contemplate describing aspects of this disclosure in the plural form of said terms. Where there are discrepancies between the terms and definitions used in the references incorporated herein by reference, the terms used herein shall have the definitions given herein. Other technical terms used have their general meaning in the field in which they are used, as exemplified in various field-specific dictionaries, such as “American Heritage® Dictionary of Science” (editors of American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), “McGraw-Hill Dictionary of Scientific and Technical Terms” (6th edition, 2002, McGraw-Hill, New York), or “Oxford Dictionary of Biology” (6th edition, 2008, Oxford University Press, Oxford and New York).

[0055] Any references cited herein, including, for example, all patents, published patent applications, and non-patent publications, are incorporated herein by reference in their entirety.

[0056] When presenting a set of alternatives, any and all combinations of the members constituting the set of alternatives are specifically envisioned. For example, if an item is a group selected from A, B, C, and D, the inventors specifically envision each alternative individually (e.g., A alone, B alone, etc.), as well as combinations such as A, B, and D; A and C; B and C, etc. When used in a list of two or more items, the term "and / or" means any one of the listed items on its own or in combination with any one or more of the other listed items. For example, the expression "A and / or B" is intended to mean one or both of A and B, i.e., A alone, B alone, or a combination of A and B. The expression "A, B, and / or C" is intended to mean A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0057] When a range of numbers is provided herein, the range should be understood to include any number between the edges of the range and the defining edges of the range. For example, "between 1 and 10" includes any number between 1 and 10, as well as the numbers 1 and 10.

[0058] When referring to numbers using the term "about," it should be understood as adding or subtracting 10%. For example, "about 100" would include numbers from 90 to 110.

[0059] As used herein, unless the context clearly indicates otherwise, the singular forms “an,” “a,” and “the” include plural indicators. For example, the terms “compound” or “at least one compound” can include multiple compounds, including mixtures thereof.

[0060] Any tobacco plant or portion thereof specifically conceived herein is intended for use with any methods provided herein. Similarly, any modified tobacco plant or portion thereof is specifically conceived for use with any methods provided herein. Any nucleic acid sequences, amino acid sequences, or other compositions specifically conceived herein are specifically conceived for use with any methods provided herein or with any tobacco plant (modified or unmodified).

[0061] This disclosure provides seeds obtained from any of the modified tobacco plants described herein. This disclosure also provides cells from any of the modified tobacco plants described herein.

[0062] Generally, trichomes are hair-like epidermal ectodermal organisms covering most above-ground plant tissues. Trichophyres tend to be multicellular, but unicellular trichomes are also known. Various types of trichomes can be found on a single plant, and they differ in shape, size, and cellular structure. Individual trichomes can be classified as glandular trichomes or non-glandular trichomes.

[0063] Glandular hairs are characterized by a head composed of cells that secrete or store large amounts of specialized metabolites. For example Unrestricted (terpenoids, phenylpropanoids, flavonoids, methyl ketones, acyl sugars). In the glandular trichomes, the trichomes can be further characterized as shield-shaped or head-shaped. Head-shaped glandular trichomes typically have a stalk whose length exceeds twice the height of the head, and the number of cells in the trichome is highly variable. Shield-shaped trichomes are short-stalked trichomes with a large head composed of four to eighteen cells arranged in one or two concentric circles.

[0064] Non-glandular trichomes typically provide plants with physical protection against biotic and abiotic stresses. For example, they can form protective trichomes against low humidity and high light intensity. For example Ultraviolet light), high or low temperatures, and insects or arachnids ( For example A physical barrier for mites to feed and / or lay eggs.

[0065] On one hand, trichomes are glandular trichomes. On the other hand, glandular trichomes are capitate glandular trichomes. On the other hand, glandular trichomes are shield-shaped glandular trichomes. On one hand, glandular trichomes are selected from the group consisting of capitate and shield-shaped glandular trichomes. On the other hand, trichomes are non-glandular trichomes.

[0066] In some cases, it may be desirable to increase the total number of trichomes on a plant or plant part. It may also be desirable to increase the density of trichomes on a plant or plant part. Without being limited by any scientific theory, plants containing an increased number of trichomes may produce an increased amount of one or more desired compounds compared to a control. See For example, U.S. Patent Application Publication No. 2022 / 0243214 A1; No. 2022 / 0243215 A1; and No. 2022 / 0243216 A1.

[0067] Infiltration As used in this article, "infiltration" refers to the expected alleles of a genotype locus being introduced from a genetic background ( For example (The genotype) is passed on to another genetic background.

[0068] As used in this article, “trilloid-associated single nucleotide polymorphism” refers to a polymorphism that is separate from trilloid-associated QTLs.

[0069] As used herein, “genotype” refers to the genetic makeup of a plant or cell. Two tobacco plants from different tobacco lines or varieties should be understood as having different genotypes. Alternatively, inbred lines typically contain the same genotype. However, without restriction, if a trichome-related QTL has been infiltrated into a single TN 90 plant, then a single TN 90 plant containing that trichome-related QTL will have a different genotype from all TN 90 plants lacking that trichome-related QTL.

[0070] As used herein, “genetic modification” refers to an alteration of the genetic composition of a plant or its genome. Genetic modifications can be introduced by methods including, but not limited to, mutagenesis, genome editing, genetic transformation, or combinations thereof. Genetic modifications include mutations (e.g., non-natural mutations) in, for example, genes or transgenes targeting genes (e.g., arginine decarboxylase (ADC) transgenes targeting the ADC gene). As used herein, “targeting” means directly upregulating or directly downregulating gene expression or activity. As used herein, “direct” in the context of transgenes affecting gene expression or activity means via the gene (… For example (startup subregion or UTR region) or the products encoded therein ( For example (mRNA molecules or polypeptides) and transgenic encoded products ( For example Transgenic molecules exert their influence on genes through physical contact or chemical interactions between small RNA molecules or proteins, such as transcription factors or dominant-negative effector peptide variants. On the one hand, transgenic molecules affect the expression or activity of target genes without involving transcription factors (…). For example Transgenic genes do not encode transcription factors and / or do not inhibit the expression or activity of transcription factors that instead regulate target genes.

[0071] mutation As used herein, “modification” in the context of plants refers to genetic alterations introduced for certain purposes that exceed natural polymorphism. Modified plants may contain non-natural mutations or recombinant DNA constructs. In one aspect, modified tobacco plants contain non-natural mutations. In another aspect, modified tobacco plants contain recombinant DNA constructs. In yet another aspect, modified tobacco plants contain genetic modifications. Modified plants, seeds, or plant parts may contain recombinant nucleic acid molecules. As used herein, “recombinant nucleic acid construct” refers to a nucleic acid molecule formed through laboratory methods of gene recombination (such as, without limitation, molecular cloning). In yet another aspect, modified plants, seeds, or plant parts contain genetic modifications. In yet another aspect, modified plants, seeds, or plant parts are transgenic plants, seeds, or plant parts.

[0072] As used herein, a “mutation” is a heritable genetic modification introduced into a gene to alter the expression or activity of the product encoded by the gene’s reference sequence. A mutation in a gene, such as arginine decarboxylase (ADC), is called an ADC mutant. Such modifications can be located in any sequence region of a gene, for example, in the promoter, 5ʹ-untranslated region (UTR), exon, intron, 3ʹ-UTR, or terminator region. On the one hand, mutations reduce, suppress, or eliminate the expression or activity of the gene product. On the other hand, mutations increase, enhance, strengthen, or strengthen the expression or activity of the gene product.

[0073] On the one hand, the mutation is not a naturally occurring polymorphism present in a particular tobacco variety or cultivar. On the other hand, the mutation is a “non-natural” or “non-naturally occurring” mutation. As used herein, a “non-natural” or “non-naturally occurring” mutation refers to a non-spontaneous mutation resulting from human intervention and does not correspond to a spontaneous mutation that occurs without human intervention. Non-limiting examples of human intervention include mutagenesis (e.g., chemical mutagenesis, ionizing radiation mutagenesis) and targeted genetic modifications (e.g., CRISPR-based methods, TALEN-based methods, zinc finger-based methods). Non-natural and non-naturally occurring mutations do not include spontaneously occurring mutations (e.g., via aberrant DNA replication in plant lines).

[0074] It should be understood that when identifying mutations, the reference DNA sequence should be derived from the same tobacco variety. For example, if the modified tobacco plant containing the mutation is from variety TN90, the endogenous reference sequence must be an endogenous TN90 sequence, not a homologous sequence from a different tobacco variety (e.g., K326). Similarly, if the modified tobacco cell containing the mutation is a TN90 cell, the endogenous reference sequence must be an endogenous TN90 sequence, not a homologous sequence from tobacco cells of a different tobacco variety (e.g., K326).

[0075] In one respect, the tobacco plant or a part thereof is homozygous for at least one non-natural mutation. In another respect, the tobacco plant or a part thereof is heterozygous for at least one non-natural mutation. In another respect, the tobacco plant or a part thereof is homozygous for the introduced recombinant DNA construct. In another respect, the tobacco plant or a part thereof is hemizygous for the introduced recombinant DNA construct. In yet another respect, the tobacco plant or a part thereof is heterozygous for the introduced recombinant DNA construct.

[0076] On one hand, this article presents the dominant alleles at the mutated locus. A dominant allele is an allele at the same locus that masks the contribution of a second allele. A dominant allele can be a "dominant negative-effect allele" or a "dominant positive-effect allele." A dominant negative-effect allele, or reversible allele, is an allele whose function is opposite to that of the normal allele. Dominant negative-effect alleles typically do not function properly and either directly inhibit the activity of wild-type proteins (…). For example (through dimerization), or inhibit the activity of the second protein required for the normal function of the wild-type protein (through dimerization). For example (Activators or downstream components of pathways). For example, dominant negative-effect alleles abolish or reduce the normal function of alleles in heterozygous or homozygous states. Dominant positive-effect alleles can increase the function of normal genes ( For example (superalle) or to provide new functions for genes ( For example(Novel alleles). A semi-dominant allele occurs when the penetrance of a linked phenotype in heterozygous individuals is less than the penetrance observed in homozygous individuals. On the other hand, the mutations presented in this paper produce dominant positive-effect alleles at the mutant loci.

[0077] As used herein, “induced” mutation refers to the generation of a mutation in a polynucleotide sequence through artificial intervention. Many suitable methods for inducing mutations in tobacco are known in the art. Non-limiting examples of such methods include the use of chemical mutagens, the use of radiation, the use of nucleases, the use of transposons, and the use of… Agrobacterium On one hand, induced mutations include the use of chemical mutagens, radiation, transposons, and... Agrobacterium Reagents consisting of a group of nucleases.

[0078] On one hand, mutagenesis can be induced by using chemical mutagens. One such chemical mutagen is ethyl methanesulfonate (EMS).

[0079] On the other hand, induced mutations involve the use of radiation. This radiation includes gamma rays, X-rays, ionizing radiation, or fast neutrons.

[0080] On the one hand, induced mutation involves the use of transposons. On the other hand, induced mutation involves the use of... Agrobacterium .

[0081] In another aspect, mutagenesis induction includes the use of nucleases. In one aspect, the nuclease is selected from the group consisting of a wide range of nucleases, zinc finger nucleases, transcription activator-like effector nucleases, CRISPR / Cas9 nucleases, CRISPR / Cpf1 nucleases, CRISPR / CasX nucleases, CRISPR / CasY nucleases, and Csm1 nucleases. In one aspect, mutagenesis induction includes the use of CRISPR / Cas9 nucleases. In one aspect, mutagenesis induction includes the use of CRISPR / Cpf1 nucleases. In one aspect, mutagenesis induction includes the use of CRISPR / CasX nucleases. In one aspect, mutagenesis induction includes the use of CRISPR / CasY nucleases. In one aspect, mutagenesis induction includes the use of Csm1 nucleases.

[0082] In one respect, induced mutations involve the use of base editors. As used herein, a "base editor" refers to a catalytically impaired Cas nuclease fused with a nucleotide deaminase. In some respects, base editors further include DNA repair proteins. In one respect, the base editor is a cytosine base editor. A cytosine base editor enables the conversion from CG to TA. In another respect, the base editor is an adenine base editor. An adenine base editor enables the conversion from AT to GC. In yet another respect, the base editor is a C to G base editor.

[0083] In one aspect, inducing mutations involves the use of a leader editor. As used herein, a "lead editor" refers to a Cas nickase fused to an engineered reverse transcriptase. The leader editor can introduce all 12 transition and transversion mutations, as well as small insertions or deletions, and combinations thereof. The leader editor uses a leader editing guide RNA (pegRNA), which specifies the target site for editing and encodes the desired edit. In one aspect, the pegRNA is provided to tobacco cells. In another aspect, the pegRNA contains a nucleic acid sequence that binds to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 4 to 9.

[0084] Additional information on base editors and lead editors and their uses in plants can be found in Molla et al., Nature Plants, 7:1166-1187 (2021). See also Anzalone et al., Nature, 576:149-157 (2019); Komor et al., Nature, 533:420-424 (2016); and Gaudelli et al., Nature, 551:464-471 (2017).

[0085] Several types of mutations are known in the art. In one aspect, mutations include insertions. An "insertion" refers to the addition of one or more nucleotides or amino acids, respectively, to a given polynucleotide or amino acid sequence compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, mutations include deletions. A "deletion" refers to the removal of one or more nucleotides or amino acids, respectively, from a given polynucleotide or amino acid sequence compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, mutations include substitutions. A "substitution" refers to the replacement of one or more nucleotides or amino acids, respectively, with a given polynucleotide or amino acid sequence compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, mutations include inversions. An "inversion" refers to the end-to-end reversal of a segment of a polynucleotide or amino acid sequence. A "repetition" refers to the duplication of a segment of a polynucleotide or amino acid sequence. The repeated segment may immediately follow the original segment, or it may be separated from the original segment by one or more nucleotides or amino acids. In one aspect, the mutations provided herein include those selected from the group consisting of insertions, deletions, substitutions, duplications, and inversions.

[0086] On the one hand, non-natural mutations include mutations of one or more nucleotides selected from the group consisting of substitutions, deletions, insertions, duplications, and inversions of endogenous nucleic acid sequences selected from the group consisting of SEQ ID NO: 1 to 20 and 41 to 60.

[0087] On the one hand, non-natural mutations include mutations consisting of substitutions, deletions, insertions, duplications, and inversions of one or more nucleotides relative to an endogenous nucleic acid sequence encoding an amino acid sequence consisting of the group consisting of SEQ ID NO: 21 to 40.

[0088] On the one hand, non-natural mutations include one or more mutation types selected from the group consisting of nonsense mutations, missense mutations, frameshift mutations, splice site mutations, and any combination thereof. As used herein, a “nonsense mutation” refers to a mutation in a nucleic acid sequence that introduces an early stop codon into an amino acid sequence. As used herein, a “missense mutation” refers to a mutation in a nucleic acid sequence that results in a substitution within the amino acid sequence encoded by the nucleic acid sequence. As used herein, a “frameshift mutation” refers to an insertion or deletion in a nucleic acid sequence that moves the frame to translate the nucleic acid sequence into an amino acid sequence. A “splice site mutation” refers to a mutation in a nucleic acid sequence that results in introns being retained for protein translation or, alternatively, exons being excluded from protein translation. Splice site mutations can lead to nonsense mutations, missense mutations, or frameshift mutations.

[0089] When mutated messenger RNA (mRNA) is translated into protein or polypeptide, the mutation in the gene coding region ( For example (Exon mutations) can lead to truncated proteins or peptides. In one aspect, this disclosure provides mutations that result in truncated proteins or peptides. As used herein, a “truncated” protein or peptide contains at least one fewer amino acid than an endogenous control protein or peptide. For example, if an endogenous protein A contains 100 amino acids, a truncated form of protein A can contain 1 to 99 amino acids. In another aspect, non-natural mutations result in truncated peptides.

[0090] Unrestricted by any scientific theory, one method to truncate proteins or peptides is to introduce premature stop codons into the mRNA transcripts of endogenous genes. In one aspect, this disclosure provides mutations that result in premature stop codons in the mRNA transcripts of endogenous genes. As used herein, a "stop codon" refers to a nucleotide triplet within the mRNA transcript that signals the termination of protein translation. A "premature stop codon" refers to a stop codon located earlier than the normal stop codon position in the endogenous mRNA transcript. For example (on the 5' side). Unrestricted, several stop codons are known in the art, including “UAG”, “UAA”, “UGA”, “TAG”, “TAA” and “TGA”.

[0091] In one respect, the mutations described herein include null mutations. As used herein, a “null mutation” is a mutation that confers complete loss of function to a protein encoded by a gene containing the mutation, or alternatively, a mutation that confers complete loss of function to a small RNA encoded by a genomic locus. Null mutations may result in a lack of mRNA transcript production, a lack of small RNA transcript production, a lack of protein function, or a combination thereof.

[0092] The mutations described herein can be located in any part of an endogenous gene. In one aspect, the mutations described herein are located within exons of an endogenous gene. In another aspect, the mutations described herein are located within introns of an endogenous gene. In yet another aspect, the mutations described herein are located within the 5ʹ-UTR of an endogenous gene. In yet another aspect, the mutations described herein are located within the 3ʹ-UTR of an endogenous gene. In yet another aspect, the mutations described herein are located within the promoter of an endogenous gene. In yet another aspect, the mutations described herein are located within the terminator of an endogenous gene. In one aspect, the non-natural mutations described herein include mutations selected from sequence regions consisting of the promoter, 5ʹ-UTR, 3ʹ-UTR, exons, introns, and terminators.

[0093] Screening and selection of mutagenic tobacco plants can be performed by any method known to those skilled in the art. Examples of screening and selection methods include, but are not limited to, Southern blotting, PCR amplification for detecting polynucleotides, Northern blotting, RNase protection, primer extension, RT-PCR amplification for detecting RNA transcripts, Sanger sequencing, and next-generation sequencing technologies. For example Enzymatic assays (such as those using Illumina, PacBio, Ion Torrent, and 454) to detect the activity of enzymes or ribozymes in peptides and polynucleotides, as well as protein gel electrophoresis, Western blotting, immunoprecipitation, and enzyme-linked immunosorbent assays (ELISA) for peptide detection. Other techniques include... In situ Hybridization, enzyme staining, and immunostaining can also be used to detect the presence or expression of peptides and / or polynucleotides. Methods for performing all reference techniques are known.

[0094] In one respect, compared to wild-type nucleic acid molecules, non-natural mutations include the insertion or deletion of at least one nucleotide in the nucleic acid molecule. In another respect, compared to wild-type nucleic acid molecules, non-natural mutations include the insertion or deletion of at least two nucleotides in the nucleic acid molecule. In another respect, compared to wild-type nucleic acid molecules, non-natural mutations include the insertion or deletion of at least three nucleotides in the nucleic acid molecule. In another respect, compared to wild-type nucleic acid molecules, non-natural mutations include the insertion or deletion of at least four nucleotides in the nucleic acid molecule. In another respect, compared to wild-type nucleic acid molecules, non-natural mutations include the insertion or deletion of at least five nucleotides in the nucleic acid molecule. In another respect, compared to wild-type nucleic acid molecules, non-natural mutations include the insertion or deletion of at least six nucleotides in the nucleic acid molecule. In another respect, compared to wild-type nucleic acid molecules, non-natural mutations include the insertion or deletion of at least seven nucleotides in the nucleic acid molecule. In another respect, compared to wild-type nucleic acid molecules, non-natural mutations include the insertion or deletion of at least eight nucleotides in the nucleic acid molecule. In another respect, compared to wild-type nucleic acid molecules, non-natural mutations include the insertion or deletion of at least nine nucleotides in the nucleic acid molecule. In one respect, compared to wild-type nucleic acid molecules, non-natural mutations include the insertion or deletion of at least 10 nucleotides in the nucleic acid molecule. In another respect, compared to wild-type nucleic acid molecules, non-natural mutations include the insertion or deletion of at least 15 nucleotides in the nucleic acid molecule. In another respect, compared to wild-type nucleic acid molecules, non-natural mutations include the insertion or deletion of at least 20 nucleotides in the nucleic acid molecule. In another respect, compared to wild-type nucleic acid molecules, non-natural mutations include the insertion or deletion of at least 30 nucleotides in the nucleic acid molecule. In another respect, compared to wild-type nucleic acid molecules, non-natural mutations include the insertion or deletion of at least 40 nucleotides in the nucleic acid molecule. In another respect, compared to wild-type nucleic acid molecules, non-natural mutations include the insertion or deletion of at least 50 nucleotides in the nucleic acid molecule. In another respect, compared to wild-type nucleic acid molecules, non-natural mutations include the insertion or deletion of at least 75 nucleotides in the nucleic acid molecule. In another respect, compared to wild-type nucleic acid molecules, non-natural mutations include the insertion or deletion of at least 100 nucleotides in the nucleic acid molecule. In another respect, compared to wild-type nucleic acid molecules, non-natural mutations include the insertion or deletion of at least 250 nucleotides in the nucleic acid molecule. On the one hand, compared to wild-type nucleic acid molecules, non-natural mutations include the insertion or deletion of at least 500 nucleotides in the nucleic acid molecule. On the other hand, compared to wild-type nucleic acid molecules, non-natural mutations include the insertion or deletion of at least 1000 nucleotides in the nucleic acid molecule.

[0095] In one respect, compared to wild-type nucleic acid molecules, non-natural mutations include insertions or deletions of 1 to 1000 nucleotides in the nucleic acid molecule. In another respect, compared to wild-type nucleic acid molecules, non-natural mutations include insertions or deletions of 1 to 750 nucleotides in the nucleic acid molecule. In another respect, compared to wild-type nucleic acid molecules, non-natural mutations include insertions or deletions of 1 to 500 nucleotides in the nucleic acid molecule. In another respect, compared to wild-type nucleic acid molecules, non-natural mutations include insertions or deletions of 1 to 250 nucleotides in the nucleic acid molecule. In another respect, compared to wild-type nucleic acid molecules, non-natural mutations include insertions or deletions of 1 to 100 nucleotides in the nucleic acid molecule. In another respect, compared to wild-type nucleic acid molecules, non-natural mutations include insertions or deletions of 1 to 75 nucleotides in the nucleic acid molecule. In another respect, compared to wild-type nucleic acid molecules, non-natural mutations include insertions or deletions of 1 to 50 nucleotides in the nucleic acid molecule. In another respect, compared to wild-type nucleic acid molecules, non-natural mutations include insertions or deletions of 1 to 25 nucleotides in the nucleic acid molecule. On the one hand, compared to wild-type nucleic acid molecules, non-natural mutations include the insertion or deletion of 1 to 10 nucleotides in the nucleic acid molecule. On the other hand, compared to wild-type nucleic acid molecules, non-natural mutations include the insertion or deletion of 1 to 5 nucleotides in the nucleic acid molecule.

[0096] In one aspect, compared to the wild-type amino acid sequence, the non-natural mutation includes the insertion or deletion of at least one amino acid residue in the amino acid sequence. In another aspect, compared to the wild-type amino acid sequence, the non-natural mutation includes the insertion or deletion of at least two amino acid residues in the amino acid sequence. In another aspect, compared to the wild-type amino acid sequence, the non-natural mutation includes the insertion or deletion of at least three amino acid residues in the amino acid sequence. In another aspect, compared to the wild-type amino acid sequence, the non-natural mutation includes the insertion or deletion of at least four amino acid residues in the amino acid sequence. In another aspect, compared to the wild-type amino acid sequence, the non-natural mutation includes the insertion or deletion of at least five amino acid residues in the amino acid sequence. In another aspect, compared to the wild-type amino acid sequence, the non-natural mutation includes the insertion or deletion of at least six amino acid residues in the amino acid sequence. In another aspect, compared to the wild-type amino acid sequence, the non-natural mutation includes the insertion or deletion of at least seven amino acid residues in the amino acid sequence. In another aspect, compared to the wild-type amino acid sequence, the non-natural mutation includes the insertion or deletion of at least eight amino acid residues in the amino acid sequence. In another aspect, compared to the wild-type amino acid sequence, the non-natural mutation includes the insertion or deletion of at least nine amino acid residues in the amino acid sequence. In one aspect, compared to the wild-type amino acid sequence, the non-natural mutation includes the insertion or deletion of at least 10 amino acid residues in the amino acid sequence. In another aspect, compared to the wild-type amino acid sequence, the non-natural mutation includes the insertion or deletion of at least 15 amino acid residues in the amino acid sequence. In another aspect, compared to the wild-type amino acid sequence, the non-natural mutation includes the insertion or deletion of at least 20 amino acid residues in the amino acid sequence. In another aspect, compared to the wild-type amino acid sequence, the non-natural mutation includes the insertion or deletion of at least 30 amino acid residues in the amino acid sequence. In another aspect, compared to the wild-type amino acid sequence, the non-natural mutation includes the insertion or deletion of at least 40 amino acid residues in the amino acid sequence. In another aspect, compared to the wild-type amino acid sequence, the non-natural mutation includes the insertion or deletion of at least 50 amino acid residues in the amino acid sequence. In another aspect, compared to the wild-type amino acid sequence, the non-natural mutation includes the insertion or deletion of at least 75 amino acid residues in the amino acid sequence. In another aspect, compared to the wild-type amino acid sequence, the non-natural mutation includes the insertion or deletion of at least 100 amino acid residues in the amino acid sequence. In one respect, non-natural mutations, compared to wild-type amino acid sequences, include the insertion or deletion of at least 250 amino acid residues in the amino acid sequence. In another respect, non-natural mutations, compared to wild-type amino acid sequences, include the insertion or deletion of at least 500 amino acid residues in the amino acid sequence. In yet another respect, non-natural mutations, compared to wild-type amino acid sequences, include the insertion or deletion of at least 1000 amino acid residues in the amino acid sequence.

[0097] In one aspect, compared to the wild-type amino acid sequence, non-natural mutations include insertions or deletions of 1 to 1000 amino acid residues in the amino acid sequence. In another aspect, compared to the wild-type amino acid sequence, non-natural mutations include insertions or deletions of 1 to 750 amino acid residues in the amino acid sequence. In another aspect, compared to the wild-type amino acid sequence, non-natural mutations include insertions or deletions of 1 to 500 amino acid residues in the amino acid sequence. In another aspect, compared to the wild-type amino acid sequence, non-natural mutations include insertions or deletions of 1 to 250 amino acid residues in the amino acid sequence. In another aspect, compared to the wild-type amino acid sequence, non-natural mutations include insertions or deletions of 1 to 100 amino acid residues in the amino acid sequence. In another aspect, compared to the wild-type amino acid sequence, non-natural mutations include insertions or deletions of 1 to 75 amino acid residues in the amino acid sequence. In another aspect, compared to the wild-type amino acid sequence, non-natural mutations include insertions or deletions of 1 to 50 amino acid residues in the amino acid sequence. In one respect, compared to the wild-type amino acid sequence, non-natural mutations include the insertion or deletion of 1 to 25 amino acid residues in the amino acid sequence. In another respect, compared to the wild-type amino acid sequence, non-natural mutations include the insertion or deletion of 1 to 10 amino acid residues in the amino acid sequence. In yet another respect, compared to the wild-type amino acid sequence, non-natural mutations include the insertion or deletion of 1 to 5 amino acid residues in the amino acid sequence.

[0098] Nucleic acids and amino acids The terms "coding nucleic acid sequence" and "coding sequence" are used interchangeably herein. The terms "genomic nucleic acid sequence" and "genomic sequence" are used interchangeably herein. The coding nucleic acid sequences provided herein include SEQ ID NO: 1 to 20. The endogenous nucleic acid sequences provided herein include SEQ ID NO: 41 to 60.

[0099] As used herein, "endogenous" nucleic acid sequences refer to nucleic acid sequences that naturally exist in the genome of an organism. Endogenous nucleic acid sequences do not include heterologous sequences that have been intentionally inserted into the genome through human intervention. Similarly, endogenous amino acid sequences are sequences that naturally exist through the translation of endogenous nucleic acid molecules. In this respect, the nucleic acid sequences provided herein are endogenous nucleic acid sequences.

[0100] As used herein, “heterologous” means a sequence (nucleic acid or amino acid) derived from a foreign species, or, if from the same species, substantially modified from its natural form at its composition and / or genomic loci through intentional human intervention. The term also applies to nucleic acid constructs, referred to herein as “polynucleotide constructs” or “nucleotide constructs.” In this way, a “heterologous” nucleic acid construct means a construct derived from a foreign species, or, if from the same species, substantially modified from its natural form at its composition and / or genomic loci through intentional human intervention. Heterologous nucleic acid constructs include, but are not limited to, recombinant nucleotide constructs that have been introduced into a plant or its plant parts, for example, via transformation methods or subsequent breeding of transgenic plants with another plant of interest.

[0101] As used in this article, "gene" refers to a gene that can produce functional units ( For example A gene can contain a polynucleotide sequence that encodes a promoter, enhancer sequence, leader sequence, transcription start site, transcription termination site, polyadenylation site, one or more exons, one or more introns, 5ʹ-UTR, 3ʹ-UTR, or any combination thereof. A "gene sequence" can contain a polynucleotide sequence encoding a promoter, enhancer sequence, leader sequence, transcription start site, transcription termination site, polyadenylation site, one or more exons, one or more introns, 5ʹ-UTR, 3ʹ-UTR, or any combination thereof. On one hand, a gene encodes a small RNA molecule or its precursor. On the other hand, a gene encodes a protein.

[0102] As used herein, the term "percentage of identity" or "percentage of similarity" relating to two or more nucleotide or amino acid sequences is calculated by: (i) comparing two best-aligned sequences (nucleotides or amino acids) over a comparison window (one or more "alignable" regions); (ii) determining the number of positions in both sequences where the same nucleic acid base (for nucleotide sequences) or amino acid residue (for proteins and peptides) occurs to produce a number of matching positions; (iii) dividing the number of matching positions by the total number of positions in the comparison window; and (iv) multiplying that quotient by 100% to produce the percentage of identity. If the "percentage of identity" is calculated relative to a reference sequence without specifying a particular comparison window, then the percentage of identity is determined by dividing the number of matching positions in the alignment region by the total length of the reference sequence. Therefore, for the purposes of this application, when two sequences (query and subject) are best-aligned (allowing gaps in their alignment), the "percentage of identity" of the query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions of the query sequence over its length (or comparison window), and then multiplied by 100%.

[0103] When referring to amino acids using a percentage of sequence identity, the positions of dissimilar residues are considered to differ primarily due to conserved amino acid substitutions, where the amino acid residues are considered to have similar chemical properties. For example The substitution of other amino acid residues (often due to charge or hydrophobicity) does not alter the functional properties of the molecule. When the conserved substitutions in a sequence differ, the percentage of sequence identity can be adjusted upwards to correct for the conservation of the substitution. Sequences that differ due to such conserved substitutions are said to have "sequence similarity" or "similarity".

[0104] Unrestricted, in conservative substitution, two aliphatic ( For example The amino acid residues (glycine, alanine, valine, leucine, isoleucine) can substitute for each other; the two hydroxyl groups ( For example Serine, cysteine, threonine, and methionine amino acid residues can substitute for each other; two aromatic ( For example Phenylalanine, tyrosine, and tryptophan amino acid residues can substitute for each other; two basic amino acids (PSA, phenylalanine, tyrosine, and tryptophan) can substitute for each other; For example Histidine, lysine, and arginine amino acid residues can substitute for each other; and two acidic ( For example The amino acid residues (aspartic acid, glutamic acid, asparagine, and glutamine) can substitute for each other.

[0105] To optimize sequence alignment and calculate their percentage of identity, various pairwise or multiple sequence alignment algorithms and programs are known in the art, such as ClustalW or Basic Local Alignment Search Tool® (BLAST), which can be used to compare sequence identity or similarity between two or more nucleotide or amino acid sequences. TM While other alignment and comparison methods are known in the art, the alignment and identity percentage (including the aforementioned range of identity percentages) between two sequences can be determined using the ClustalW algorithm. See, for example Chenna et al. , "Multiplesequence alignment with the Clustal series of programs," Nucleic Acids Research 31: 3497-3500 (2003); Thompson et al., “Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice,” Nucleic Acids Research 22: 4673-4680 (1994); Larkin MA et al. , "Clustal W and Clustal Xversion 2.0," Bioinformatics 23: 2947-48 (2007); and Altschul et al. , "Basic local alignment search tool". J. Mol. Biol. 215:403-410 (1990), the entire contents and publications of which are incorporated herein by reference.

[0106] As used herein, the term "complementarity percentage" or "complementary percentage" for two nucleotide sequences is similar to the concept of identity percentage, but refers to the percentage of nucleotides in the query sequence that have hybridized to the optimal base pair of the nucleotides in the subject sequence when the query and subject sequences are linearly aligned and have optimal base pairing without secondary folding structures (such as loops, stems, or hairpins). Such a complementarity percentage can be between two DNA strands, two RNA strands, or one DNA strand and one RNA strand. The "complementarity percentage" can be calculated by: (i) aligning the two nucleotide sequences in a linear and fully extended alignment (i.e., without folds or secondary structures) with optimal base pairing or hybridization on a comparison window; (ii) determining the number of base pair positions between the two sequences on the comparison window to produce the number of complementary positions; (iii) dividing the number of complementary positions by the total number of positions in the comparison window; and (iv) multiplying that quotient by 100% to obtain the complementarity percentage of the two sequences. Optimal base pairing of two sequences can be determined by hydrogen binding based on known pairings of nucleotide bases (such as GC, AT, and AU). If the "complementarity percentage" is calculated relative to a reference sequence without specifying a particular comparison window, then the identity percentage is determined by dividing the number of complementary positions between the two linear sequences by the total length of the reference sequence. Therefore, for the purposes of this application, when the two sequences (query and subject) are optimal base pairs (allowing for mismatches or non-base-paired nucleotides), the "complementarity percentage" of the query sequence is equal to the number of base-paired positions between the two sequences divided by the total number of positions of the query sequence over its length, then multiplied by 100%.

[0107] The use of the terms "polynucleotide" or "nucleic acid molecule" is not intended to limit this disclosure to polynucleotides containing deoxyribonucleic acid (DNA). Ribonucleic acid (RNA) molecules are also contemplated, for example. Those skilled in the art will recognize that polynucleotides and nucleic acid molecules can contain ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. The polynucleotides disclosed herein also encompass all forms of sequences, including but not limited to single-stranded, double-stranded, hairpin, stem-loop, and so on. In one aspect, the nucleic acid molecule provided herein is a DNA molecule. In another aspect, the nucleic acid molecule provided herein is an RNA molecule. In one aspect, the nucleic acid molecule provided herein is single-stranded. In another aspect, the nucleic acid molecule provided herein is double-stranded. Nucleic acid molecules can encode polypeptides or small RNAs.

[0108] As used herein, “recombinant nucleic acid” refers to a nucleic acid molecule formed through laboratory methods of gene recombination (such as, but not limited to, molecular cloning). Similarly, “recombinant DNA construct” refers to a DNA molecule formed through laboratory methods of gene recombination.

[0109] Nucleic acids can be isolated using techniques conventional in the art. For example, any method can be used to isolate nucleic acids, including but not limited to recombinant nucleic acid techniques and / or polymerase chain reaction (PCR). General PCR techniques are described, for example, in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, edited, Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation, which can be used to isolate nucleic acids. The isolated nucleic acids can also be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides. Nucleic acids can be isolated from natural sources using known methods such as DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. For example Peptides can be purified from biological samples. Alternatively, peptides can be purified, for example, by expressing nucleic acids in an expression vector. Furthermore, purified peptides can be obtained through chemical synthesis. The degree of purity of a peptide can be measured using any suitable method. For example Column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis were performed.

[0110] In one aspect, this disclosure provides methods for detecting recombinant nucleic acids and peptides in plant cells. Hybridization can also be used to detect nucleic acids, without limitation. Hybridization between nucleic acids is discussed in Sambrook. et al.This is discussed in detail in Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989.

[0111] On the one hand, oligonucleotide probes are TaqMan. TM Probe. TaqMan TM Probes are commonly used to improve the specificity of quantitative PCR. (TaqMan) TM probe depends Taq The polymerase's 5' to 3' exonuclease activity is used to cleave the double-labeled probe during hybridization with the complementary target sequence and to perform fluorophore-based detection. As in other quantitative PCR methods, the resulting fluorescence signal allows for quantitative measurement of product accumulation during the exponential phase of PCR; however, TaqMan... TM The probe significantly improves the specificity of detection.

[0112] In one aspect, the oligonucleotide probe contains at least 80% identical nucleic acid sequences to the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the oligonucleotide probe contains at least 85% identical nucleic acid sequences to the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the oligonucleotide probe contains at least 90% identical nucleic acid sequences to the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the oligonucleotide probe contains at least 91% identical nucleic acid sequences to the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the oligonucleotide probe contains at least 92% identical nucleic acid sequences to the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the oligonucleotide probe contains at least 93% identical nucleic acid sequences to the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In one aspect, the oligonucleotide probe contains at least 94% identical nucleic acid sequences to the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the oligonucleotide probe contains at least 95% identical nucleic acid sequences to the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the oligonucleotide probe contains at least 96% identical nucleic acid sequences to the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the oligonucleotide probe contains at least 97% identical nucleic acid sequences to the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the oligonucleotide probe contains at least 98% identical nucleic acid sequences to the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the oligonucleotide probe contains at least 99% identical nucleic acid sequences to the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. On the other hand, the oligonucleotide probe contains a nucleic acid sequence that is 100% identical to the nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 20 and 41 to 60.

[0113] On one hand, the oligonucleotide probe is adjacent to one or more polymorphic nucleotide positions at a marker locus. As used herein, "adjacent" means that the distance from the nearest end (3′ or 5′) of the oligonucleotide probe to the polymorphic nucleotide position is between 0 and 50 nucleotides. As used herein, "polymorphic nucleotide position" refers to the difference between two or more alleles at a given marker locus ( For example(Insertion, deletion, substitution). Polymorphic nucleotide positions can be identified by generating pairwise comparisons between allele sequences. For example, if the first allele contains the nucleotide sequence ATTTG and the second allele contains the nucleotide sequence TTTTG, then the first nucleotide will be the "polymorphic nucleotide position".

[0114] In one respect, the nucleic acid sequence provided herein is at least 70% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the nucleic acid sequence provided herein is at least 75% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the nucleic acid sequence provided herein is at least 80% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the nucleic acid sequence provided herein is at least 85% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the nucleic acid sequence provided herein is at least 88% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the nucleic acid sequence provided herein is at least 90% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the nucleic acid sequence provided herein is at least 91% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the nucleic acid sequence provided herein is at least 92% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the nucleic acid sequence provided herein is at least 93% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the nucleic acid sequence provided herein is at least 94% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the nucleic acid sequence provided herein is at least 95% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the nucleic acid sequence provided herein is at least 96% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. On the other hand, the nucleic acid sequence provided herein is at least 97% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. On the other hand, the nucleic acid sequence provided herein is at least 98% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. On the other hand, the nucleic acid sequence provided herein is at least 99% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. On the other hand, the nucleic acid sequence provided herein is 100% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60.

[0115] In one respect, the endogenous nucleic acid sequence provided herein is at least 70% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the endogenous nucleic acid sequence provided herein is at least 75% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the endogenous nucleic acid sequence provided herein is at least 80% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the endogenous nucleic acid sequence provided herein is at least 85% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the endogenous nucleic acid sequence provided herein is at least 88% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the endogenous nucleic acid sequence provided herein is at least 90% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the endogenous nucleic acid sequence provided herein is at least 91% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the endogenous nucleic acid sequence provided herein is at least 92% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the endogenous nucleic acid sequence provided herein is at least 93% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the endogenous nucleic acid sequence provided herein is at least 94% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the endogenous nucleic acid sequence provided herein is at least 95% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the endogenous nucleic acid sequence provided herein is at least 96% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. On the other hand, the endogenous nucleic acid sequence provided herein is at least 97% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. On the other hand, the endogenous nucleic acid sequence provided herein is at least 98% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. On the other hand, the endogenous nucleic acid sequence provided herein is at least 99% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. On the other hand, the endogenous nucleic acid sequence provided herein is 100% identical or complementary to the sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60.

[0116] As used herein, the term "peptide" refers to a chain of at least two covalently linked amino acids. Peptides may be encoded by polynucleotides provided herein. Proteins provided herein may be encoded by nucleic acid molecules provided herein. Proteins may comprise peptides provided herein. As used herein, "protein" refers to a chain of amino acid residues that provides structural or enzymatic activity to a cell.

[0117] Antibodies can be used to detect peptides. Techniques for detecting peptides using antibodies include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. The antibodies provided herein can be polyclonal or monoclonal antibodies. The antibodies provided herein that have specific binding affinity for peptides can be generated using methods well known in the art. The antibodies provided herein can be ligated to solid supports (such as microtiter plates) using methods known in the art.

[0118] Detection ( For example Amplification products, hybridization complexes, and peptides can be identified using detectable labels. The term "label" is intended to encompass both direct and indirect labeling. Detectable labels include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.

[0119] In one respect, the amino acid sequence provided herein is at least 70% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40. In another respect, the amino acid sequence provided herein is at least 75% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40. In another respect, the amino acid sequence provided herein is at least 80% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40. In another respect, the amino acid sequence provided herein is at least 85% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40. In another respect, the amino acid sequence provided herein is at least 88% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40. In another respect, the amino acid sequence provided herein is at least 90% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40. In another respect, the amino acid sequence provided herein is at least 91% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40. In another respect, the amino acid sequence provided herein is at least 92% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40. In another respect, the amino acid sequence provided herein is at least 93% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40. In another respect, the amino acid sequence provided herein is at least 94% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40. In another respect, the amino acid sequence provided herein is at least 95% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40. In another respect, the amino acid sequence provided herein is at least 96% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40. In another respect, the amino acid sequence provided herein is at least 97% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40. In another respect, the amino acid sequence provided herein is at least 98% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40. On the other hand, the amino acid sequences provided herein are at least 99% identical or similar to the amino acid sequences selected from the group consisting of SEQ ID NO: 21 to 40. On the other hand, the amino acid sequences provided herein are 100% identical or similar to the amino acid sequences selected from the group consisting of SEQ ID NO: 21 to 40.

[0120] In one aspect, the nucleic acid sequence provided herein encodes a polypeptide containing at least 70% identical or similar amino acid sequences to those selected from the group consisting of SEQ ID NO: 21 to 40. In another aspect, the nucleic acid sequence provided herein encodes a polypeptide containing at least 75% identical or similar amino acid sequences to those selected from the group consisting of SEQ ID NO: 21 to 40. In another aspect, the nucleic acid sequence provided herein encodes a polypeptide containing at least 80% identical or similar amino acid sequences to those selected from the group consisting of SEQ ID NO: 21 to 40. In another aspect, the nucleic acid sequence provided herein encodes a polypeptide containing at least 85% identical or similar amino acid sequences to those selected from the group consisting of SEQ ID NO: 21 to 40. In another aspect, the nucleic acid sequence provided herein encodes a polypeptide containing at least 88% identical or similar amino acid sequences to those selected from the group consisting of SEQ ID NO: 21 to 40. In another aspect, the nucleic acid sequence provided herein encodes a polypeptide containing at least 90% identical or similar amino acid sequences to those selected from the group consisting of SEQ ID NO: 21 to 40. In one aspect, the nucleic acid sequence provided herein encodes a polypeptide containing at least 91% identical or similar amino acid sequences to those selected from the group consisting of SEQ ID NO: 21 to 40. In another aspect, the nucleic acid sequence provided herein encodes a polypeptide containing at least 92% identical or similar amino acid sequences to those selected from the group consisting of SEQ ID NO: 21 to 40. In another aspect, the nucleic acid sequence provided herein encodes a polypeptide containing at least 93% identical or similar amino acid sequences to those selected from the group consisting of SEQ ID NO: 21 to 40. In another aspect, the nucleic acid sequence provided herein encodes a polypeptide containing at least 94% identical or similar amino acid sequences to those selected from the group consisting of SEQ ID NO: 21 to 40. In another aspect, the nucleic acid sequence provided herein encodes a polypeptide containing at least 95% identical or similar amino acid sequences to those selected from the group consisting of SEQ ID NO: 21 to 40. In another aspect, the nucleic acid sequence provided herein encodes a polypeptide containing at least 96% identical or similar amino acid sequences to those selected from the group consisting of SEQ ID NO: 21 to 40. In one aspect, the nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence that is at least 97% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40. In another aspect, the nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence that is at least 98% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40.In one aspect, the nucleic acid sequence provided herein encodes a polypeptide containing an amino acid sequence that is at least 99% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40. In another aspect, the nucleic acid sequence provided herein encodes a polypeptide containing an amino acid sequence that is 100% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40.

[0121] In one aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide containing at least 70% identical or similar amino acid sequences to those selected from the group consisting of SEQ ID NO: 21 to 40. In another aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide containing at least 75% identical or similar amino acid sequences to those selected from the group consisting of SEQ ID NO: 21 to 40. In another aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide containing at least 80% identical or similar amino acid sequences to those selected from the group consisting of SEQ ID NO: 21 to 40. In another aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide containing at least 85% identical or similar amino acid sequences to those selected from the group consisting of SEQ ID NO: 21 to 40. In another aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide containing at least 88% identical or similar amino acid sequences to those selected from the group consisting of SEQ ID NO: 21 to 40. In yet another aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide containing at least 90% identical or similar amino acid sequences to those selected from the group consisting of SEQ ID NO: 21 to 40. In one aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide containing an amino acid sequence that is at least 91% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40. In another aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide containing an amino acid sequence that is at least 92% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40. In another aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide containing an amino acid sequence that is at least 93% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40. In another aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide containing an amino acid sequence that is at least 94% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40. In another aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide containing an amino acid sequence that is at least 95% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40. In another aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide containing an amino acid sequence that is at least 96% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40. In one aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide containing an amino acid sequence that is at least 97% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40. In another aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide containing an amino acid sequence that is at least 98% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40.In one aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide containing an amino acid sequence that is at least 99% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40. In another aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide containing an amino acid sequence that is 100% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40.

[0122] promoter As commonly understood in the art, the term "promoter" refers to a DNA sequence containing an RNA polymerase binding site, a transcription start site, and / or a TATA box, and which assists or promotes the transcription and expression of an associated transcribed polynucleotide sequence and / or gene (or transgene). Promoters can be synthesized from, modified from, or derived from known or naturally occurring promoter sequences or other promoter sequences. Promoters can also include chimeric promoters comprising combinations of two or more heterologous sequences. Therefore, the promoters of this application can include variants of promoter sequences that are compositionally similar to but not identical to other known or provided promoter sequences.

[0123] Promoters that drive expression in all or most tissues of a plant are called "constitutive" promoters. Constitutive promoters are selected from a group consisting of the cauliflower mosaic virus 35S promoter, ubiquitin promoter, actin promoter, crown gall alkaloid promoter, and alcohol dehydrogenase promoter.

[0124] Promoters that drive expression at certain stages or phases of development are called "developmental" promoters.

[0125] Promoters that drive enhanced expression in certain tissues of an organism, relative to other tissues, are called "tissue-preferred" promoters. Therefore, "tissue-preferred" promoters elicit relatively high or preferential expression in specific tissues of a plant, but lower expression levels in other tissues. As a non-limiting example, root tissue-preferred promoters exhibit high activity in root tissues, but may also exhibit activity in additional tissues such as stems, leaves, and flowers, albeit at lower levels. "Tissue-specific" promoters elicit expression only in specific tissues. As a non-limiting example, root tissue-specific promoters drive expression only in root tissues. In one aspect, tissue-specific promoters are root tissue-specific promoters. In another aspect, tissue-specific promoters are root tissue-preferred promoters. In one aspect, tissue-specific promoters are leaf tissue-specific promoters. In another aspect, tissue-preferred promoters are leaf tissue-preferred promoters. In one aspect, tissue-specific promoters are trichome tissue-specific promoters. In another aspect, tissue-preferred promoters are trichome tissue-preferred promoters.

[0126] On the one hand, the root tissue preferred promoter is the cassava vein mosaic virus (CsVMV) promoter.

[0127] "Inducible" promoters are promoters that initiate transcription in response to environmental stimuli (such as heat, cold, drought, light, or other stimuli such as injury or chemical application).

[0128] In one respect, the promoters provided in this paper are constitutive promoters. In another respect, the promoters provided in this paper are inducible promoters. In yet another respect, the promoters provided in this paper are developmental promoters. In yet another respect, the promoters are tissue-preferred promoters or tissue-specific promoters. In yet another respect, the promoters are selected from the group consisting of constitutive promoters, tissue-preferred promoters, tissue-specific promoters, and inducible promoters.

[0129] In one aspect, this disclosure provides heterologous promoters. In another aspect, this disclosure provides promoters operatively linked to heterologous polynucleotides. In yet another aspect, this disclosure provides polynucleotide sequences operatively linked to heterologous promoters.

[0130] As used in this article, "operably linked" refers to a functional connection between two or more elements. For example, a polynucleotide of interest linked to a regulatory sequence (… For example An operatively linked promoter is a functional link that allows the expression of a polynucleotide of interest. The operatively linked elements can be continuous or discontinuous. In one respect, the promoters provided herein are operatively linked to heterologous nucleic acid molecules.

[0131] In one respect, an inducible promoter provides trichome-specific or preferred expression. In another respect, a trichome-specific or preferred inducible promoter comprises a sequence selected from the group consisting of SEQ ID NO: 62 to 69 and a functional fragment thereof. In another respect, a specific or preferred promoter can be an inducible promoter.

[0132] On one hand, inducible promoters provide root-specific or preferred expression. On the other hand, root-specific or preferred inducible promoters comprise sequences selected from the group consisting of SEQ ID NO: 70 to 80 and their functional fragments. Table 1 provides a comparison of estimated leaf and root-specific expression levels driven by SEQ ID NO: 70 to 80.

[0133] On one hand, inducible promoters provide leaf-specific or preferred expression. On the other hand, leaf-specific or preferred inducible promoters comprise sequences selected from the group consisting of SEQ ID NO: 81 to 90 and their functional fragments. Table 2 provides a comparison of estimated leaf and root-specific expression levels driven by SEQ ID NO: 81 to 90.

[0134] small RNA molecules On the one hand, the nucleic acid molecules presented in this article are small RNA molecules (also simply referred to as "small RNA"). On the other hand, the nucleic acid molecules encode small RNA molecules.

[0135] As used herein, a "small RNA molecule" refers to a non-coding RNA molecule with a length between 16 and 70 nucleotides. On one hand, a small RNA molecule contains 16 to 40 nucleotides. On another hand, a small RNA molecule contains 16 to 30 nucleotides. On another hand, a small RNA molecule contains 18 to 50 nucleotides. On another hand, a small RNA molecule contains 18 to 40 nucleotides. On another hand, a small RNA molecule contains 18 to 30 nucleotides. On another hand, a small RNA molecule contains 18 to 25 nucleotides. On another hand, a small RNA molecule contains 20 to 28 nucleotides. On another hand, a small RNA molecule contains 20 to 24 nucleotides. On another hand, a small RNA molecule contains 21 to 23 nucleotides. On the other hand, small RNA molecules contain 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 nucleotides.

[0136] On the one hand, small RNA molecules are selected from double-stranded RNA and small interfering RNA (siRNA). trans The group consisting of siRNA and microRNA (miRNA).

[0137] miRNAs typically have about 19 to 25 nucleotides (usually about 20 to 24 nucleotides in plants) and guide target transcripts. trans MiRNAs cleave and negatively regulate the expression of genes involved in various regulatory and developmental pathways. In some cases, miRNAs are used to guide the homologous processing of siRNA primary transcripts.

[0138] It should be understood in the art that in plants, miRNAs and target nucleic acids typically do not have perfect complementarity (although miRNAs and target nucleic acids can have perfect complementarity). Various mismatches may exist between miRNAs and their targets, but miRNAs can still reduce the expression and / or function of target genes. See For example, Liu et al. , Plant Cell , 26:741-753(2014) and Wang et al. , Curr . Opin.Plant Biol. , 27:118-124 (2015).

[0139] In one aspect, the small RNA molecule has 100% complementarity with a nucleic acid molecule containing a sequence selected from the group consisting of SEQ ID NO: 1 to 20 and 41 to 60. In another aspect, the small RNA molecule has at least 95% complementarity on 21 consecutive nucleotides of a nucleic acid molecule containing a sequence selected from the group consisting of SEQ ID NO: 1 to 20 and 41 to 60. In another aspect, the small RNA molecule has at least 90% complementarity on 21 consecutive nucleotides of a nucleic acid molecule containing a sequence selected from the group consisting of SEQ ID NO: 1 to 20 and 41 to 60. In another aspect, the small RNA molecule has at least 85% complementarity on 21 consecutive nucleotides of a nucleic acid molecule containing a sequence selected from the group consisting of SEQ ID NO: 1 to 20 and 41 to 60. In another aspect, the small RNA molecule has at least 95% complementarity on 20 consecutive nucleotides of a nucleic acid molecule containing a sequence selected from the group consisting of SEQ ID NO: 1 to 20 and 41 to 60. In one aspect, the small RNA molecule contains at least 90% complementarity on 20 consecutive nucleotides of a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the small RNA molecule contains at least 85% complementarity on 20 consecutive nucleotides of a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the small RNA molecule contains at least 95% complementarity on 19 consecutive nucleotides of a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In yet another aspect, the small RNA molecule contains at least 90% complementarity on 19 consecutive nucleotides of a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In yet another aspect, the small RNA molecule contains at least 85% complementarity on 19 consecutive nucleotides of a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60.

[0140] Many microRNA genes (MIR genes) have been identified and are publicly available in databases (“miRBase”, accessible online at micrornasangeracuk / sequences; see also Griffiths-Jones et al. (2003) Nucleic Acids Res., 31:439-441). MIR genes have been reported to occur in intergenic regions of the genome, either isolated or in clusters, but can also be located entirely or partially within introns of other genes (both protein-coding and non-protein-coding). For a review of miRNA biogenesis, see Kim (2005) Nature Rev. Mol. Cell. Biol., 6:376-385. In at least some cases, transcription of MIR genes can be promoted and controlled by the MIR gene's own promoter. Primary transcripts (called "pri-miRNAs") can be very large (several thousand bases) and can be polycistronic, containing one or more pre-miRNAs (containing stem-loop folded structures that are processed into mature miRNAs) as well as the common 5ʹ "cap" and polyadenylated tail of mRNAs.

[0141] The maturation of mature miRNAs from their corresponding precursors (pri-miRNAs and pre-miRNAs) differs significantly between animals and plants. For example, in plant cells, microRNA precursor molecules are thought to be processed into mature miRNAs largely entirely in the nucleus, while in animal cells, pri-miRNA transcripts are processed in the nucleus by the animal-specific enzyme Drosha, subsequently exporting pre-miRNAs to the cytoplasm, where they are further processed into mature miRNAs. Mature miRNAs in plants are typically 21 nucleotides in length.

[0142] Transgenic expression of miRNAs (whether naturally occurring or artificial sequences) can be used to regulate the expression of one or more target genes of the miRNA. The inclusion of miRNA recognition sites in the transgenic transcript is also useful for regulating transcript expression. MiRNA recognition sites have been validated in all regions of mRNA, including the 5ʹ untranslated region, coding region, and 3ʹ untranslated region, indicating that the position of the miRNA target site relative to the coding sequence does not necessarily affect repression. Since miRNAs are important regulatory elements in eukaryotes, transgenic repression of miRNAs is useful for manipulating biological pathways and responses. Finally, promoters of MIR genes can have highly specific expression patterns (e.g., cell-specific, tissue-specific, time-specific, or inducible), and are therefore useful in recombinant constructs for inducing such specific transcription of DNA sequences operatively linked to them. Various uses of miRNAs, their precursors, their recognition sites, and their promoters are described in detail in U.S. Patent Application Publication 2006 / 0200878 A1, which is incorporated herein by reference. Non-limiting examples of these uses include: (1) expressing natural miRNA or miRNA precursor sequences to repress target genes; (2) expressing artificial miRNA or miRNA precursor sequences to repress target genes; (3) expressing transgenes with miRNA recognition sites, wherein the transgene is repressed when the mature miRNA is expressed; and (4) expressing transgenes driven by miRNA promoters.

[0143] Designing artificial miRNA sequences can be as simple as replacing a sequence complementary to a nucleotide in the stem region of the miRNA precursor with a desired target, as demonstrated by Zeng et al. (2002) Mol. Cell, 9:1327-1333. A non-limiting example of a general method for determining nucleotide variations in a native miRNA sequence to produce an engineered miRNA precursor includes the following steps: (a) selecting a unique target sequence of at least 18 nucleotides specific to the target gene, for example, by using sequence alignment tools such as BLAST (e.g., see, Altschul et al. (1990) J. Mol. Biol., 215:403-410; Altschul et al. (1997) Nucleic Acids Res., 25:3389-3402), for example, against both tobacco cDNA and genomic DNA databases, to identify target transcript orthologs and any potential matches with unrelated genes, thereby avoiding unintentional silencing of non-target sequences; (b) analyzing undesirable sequences in the target gene ( For example(matching sequences of non-target species), and scoring each potential 19-mer segment with GC content, Reynolds score (see Reynolds et al. (2004) Nature Biotechnol., 22:326-330), and functional asymmetry characterized by negative free energy difference (“.DELTA..DELTA.G” or “ΔΔG”) (see Khvorova et al. (2003) Cell, 115:209-216). Preferably, 19-mers with all or most of the following characteristics are selected: (1) Reynolds score >4; (2) GC content between about 40% and about 60%; (3) negative ΔΔG; (4) terminal adenosine; (5) lack of consecutive runs of 4 or more identical nucleotides; (6) position close to the 3' end of the target gene; (7) minimal difference from miRNA precursor transcripts. It has been reported that the position of each three nucleotides in the siRNA is particularly important for affecting RNAi efficacy and algorithms. "siExplorer" is publicly available at rnachemtu-tokyoacjp / siexplorer.htm (see, Katoh and Suzuki (2007) Nucleic Acids Res., 10.1093 / nar / gkl1120); (c) determining the reverse complementary sequence of the selected 19-mer for the preparation of modified mature miRNA. The additional nucleotide at position 20 preferably matches the selected target sequence, and the nucleotide at position 21 is preferably chosen to be unpaired to prevent the spread of silencing on the target transcript or to pair with the target sequence to promote the spread of silencing on the target transcript; and (d) transforming the artificial miRNA into plants.

[0144] Without being bound by any scientific theory, it should be understood in this field that RNAi knockdown of candidate genes ( For example via the use of artificial miRNA or siRNA) and mutations in the same candidate gene ( For example Missense mutations (or nonsense mutations) can lead to reduced expression and / or reduced protein activity, or they can result in the same or similar phenotypes in plants. See For example, Agrawal et al. , Microbiology and Molecular Biology Reviews , 67:657-685 (2003).

[0145] In one aspect, inducing mutations involves the use of a leader editor. As used herein, a "lead editor" refers to a Cas nickase fused to an engineered reverse transcriptase. The leader editor can introduce all 12 transition and transversion mutations, as well as small insertions or deletions, and combinations thereof. The leader editor uses a leader editing guide RNA (pegRNA), which specifies the target site for editing and encodes the desired edit. In one aspect, the pegRNA is provided to tobacco cells. In another aspect, the pegRNA comprises a nucleic acid sequence that binds to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 20 or 41 to 60.

[0146] Additional information on base editors and leader editors and their uses in plants can be found in Molla. wait people , Nature Plants , 7:1166-1187(2021). See also Anzalone et al. , Nature , 576:149-157(2019); Komor et al. , Nature , 533:420-424 (2016); and Gaudelli et al. , Nature , 551:464-471(2017).

[0147] In one aspect, the small RNA provided herein contains a nucleic acid sequence that is at least 75% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the small RNA provided herein contains a nucleic acid sequence that is at least 80% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the small RNA provided herein contains a nucleic acid sequence that is at least 85% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the small RNA provided herein contains a nucleic acid sequence that is at least 90% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the small RNA provided herein contains a nucleic acid sequence that is at least 95% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the small RNA provided herein contains a nucleic acid sequence that is at least 96% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In one aspect, the small RNA provided herein contains a nucleic acid sequence that is at least 97% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the small RNA provided herein contains a nucleic acid sequence that is at least 98% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the small RNA provided herein contains a nucleic acid sequence that is at least 99% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In yet another aspect, the small RNA provided herein contains a nucleic acid sequence that is 100% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60.

[0148] In one aspect, the small RNA provided herein contains a nucleic acid sequence that is at least 75% complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the small RNA provided herein contains a nucleic acid sequence that is at least 80% complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the small RNA provided herein contains a nucleic acid sequence that is at least 85% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the small RNA provided herein contains a nucleic acid sequence that is at least 90% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the small RNA provided herein contains a nucleic acid sequence that is at least 95% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the small RNA provided herein contains a nucleic acid sequence that is at least 96% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In one aspect, the small RNA provided herein contains a nucleic acid sequence that is at least 97% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the small RNA provided herein contains a nucleic acid sequence that is at least 98% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the small RNA provided herein contains a nucleic acid sequence that is at least 99% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In yet another aspect, the small RNA provided herein contains a nucleic acid sequence that is 100% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60.

[0149] In one respect, the small RNA provided herein contains a nucleic acid sequence of at least 18 consecutive nucleotides that are at least 88.7% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the small RNA provided herein contains a nucleic acid sequence of at least 18 consecutive nucleotides that are at least 94.3% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the small RNA provided herein contains a nucleic acid sequence of at least 18 consecutive nucleotides that are 100% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the small RNA provided herein contains a nucleic acid sequence of at least 20 consecutive nucleotides that are at least 85% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another respect, the small RNA provided herein contains a nucleic acid sequence of at least 20 consecutive nucleotides that are at least 90% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. On the other hand, the small RNA provided herein comprises a nucleic acid sequence of at least 20 consecutive nucleotides that are at least 95% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. On the other hand, the small RNA provided herein comprises a nucleic acid sequence of at least 20 consecutive nucleotides that are 100% identical or complementary to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60.

[0150] In one aspect, the small RNA molecule provided herein can bind to and reduce the expression of nucleic acid sequences encoding polypeptides whose amino acid sequences are at least 70% identical or similar to those of the group consisting of SEQ ID NO: 21 to 40. In another aspect, the small RNA molecule provided herein can bind to and reduce the expression of nucleic acid sequences encoding polypeptides whose amino acid sequences are at least 75% identical or similar to those of the group consisting of SEQ ID NO: 21 to 40. In another aspect, the small RNA molecule provided herein can bind to and reduce the expression of nucleic acid sequences encoding polypeptides whose amino acid sequences are at least 80% identical or similar to those of the group consisting of SEQ ID NO: 21 to 40. In another aspect, the small RNA molecule provided herein can bind to and reduce the expression of nucleic acid sequences encoding polypeptides whose amino acid sequences are at least 85% identical or similar to those of the group consisting of SEQ ID NO: 21 to 40. In yet another aspect, the small RNA molecule provided herein can bind to and reduce the expression of nucleic acid sequences encoding polypeptides whose amino acid sequences are at least 88% identical or similar to those of the group consisting of SEQ ID NO: 21 to 40. In one aspect, the small RNA molecule provided herein can bind to and reduce the expression of nucleic acid sequences encoding polypeptides whose amino acid sequences are at least 90% identical or similar to those of the group consisting of SEQ ID NO: 21 to 40. In another aspect, the small RNA molecule provided herein can bind to and reduce the expression of nucleic acid sequences encoding polypeptides whose amino acid sequences are at least 91% identical or similar to those of the group consisting of SEQ ID NO: 21 to 40. In another aspect, the small RNA molecule provided herein can bind to and reduce the expression of nucleic acid sequences encoding polypeptides whose amino acid sequences are at least 92% identical or similar to those of the group consisting of SEQ ID NO: 21 to 40. In another aspect, the small RNA molecule provided herein can bind to and reduce the expression of nucleic acid sequences encoding polypeptides whose amino acid sequences are at least 93% identical or similar to those of the group consisting of SEQ ID NO: 21 to 40. In yet another aspect, the small RNA molecule provided herein can bind to and reduce the expression of nucleic acid sequences encoding polypeptides whose amino acid sequences are at least 94% identical or similar to those of the group consisting of SEQ ID NO: 21 to 40. On the one hand, the small RNA molecule provided herein can bind to and reduce the expression of nucleic acid sequences encoding polypeptides whose amino acid sequences are at least 95% identical or similar to those of the group consisting of SEQ ID NO: 21 to 40. On the other hand, the small RNA molecule provided herein can bind to and reduce the expression of nucleic acid sequences encoding polypeptides whose amino acid sequences are at least 96% identical or similar to those of the group consisting of SEQ ID NO: 21 to 40.In one respect, the small RNA molecule provided herein can bind to and reduce the expression of nucleic acid sequences encoding polypeptides whose amino acid sequences are at least 97% identical or similar to those of the group consisting of SEQ ID NO: 21 to 40. In another respect, the small RNA molecule provided herein can bind to and reduce the expression of nucleic acid sequences encoding polypeptides whose amino acid sequences are at least 98% identical or similar to those of the group consisting of SEQ ID NO: 21 to 40. In another respect, the small RNA molecule provided herein can bind to and reduce the expression of nucleic acid sequences encoding polypeptides whose amino acid sequences are at least 99% identical or similar to those of the group consisting of SEQ ID NO: 21 to 40. In yet another respect, the small RNA molecule provided herein can bind to and reduce the expression of nucleic acid sequences encoding polypeptides whose amino acid sequences are 100% identical or similar to those of the group consisting of SEQ ID NO: 21 to 40.

[0151] In one aspect, the small RNA molecules provided herein can bind to and reduce the expression of nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60, or nucleic acid sequences that are at least 70% identical to those transcribed from them. In another aspect, the small RNA molecules provided herein can bind to and reduce the expression of nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60, or nucleic acid sequences that are at least 75% identical to those transcribed from them. In another aspect, the small RNA molecules provided herein can bind to and reduce the expression of nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60, or nucleic acid sequences that are at least 80% identical to those transcribed from them. In yet another aspect, the small RNA molecules provided herein can bind to and reduce the expression of nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60, or nucleic acid sequences that are at least 85% identical to those transcribed from them. In one aspect, the small RNA molecules provided herein can bind to and reduce the expression of nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60, or the RNA transcribed from them, which are at least 88% identical. In another aspect, the small RNA molecules provided herein can bind to and reduce the expression of nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60, or the RNA transcribed from them, which are at least 90% identical. In yet another aspect, the small RNA molecules provided herein can bind to and reduce the expression of nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60, or the RNA transcribed from them, which are at least 91% identical. In yet another aspect, the small RNA molecules provided herein can bind to and reduce the expression of nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60, or the RNA transcribed from them, which are at least 92% identical. In one aspect, the small RNA molecules provided herein can bind to and reduce the expression of nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60, or the RNA transcribed from them, which are at least 93% identical. In another aspect, the small RNA molecules provided herein can bind to and reduce the expression of nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60, or the RNA transcribed from them, which are at least 94% identical. In yet another aspect, the small RNA molecules provided herein can bind to and reduce the expression of nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60, or the RNA transcribed from them, which are at least 95% identical. In yet another aspect, the small RNA molecules provided herein can bind to and reduce the expression of nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60, or the RNA transcribed from them, which are at least 96% identical.In one aspect, the small RNA molecules provided herein can bind to and reduce the expression of nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60, or nucleic acid sequences that are at least 97% identical to those transcribed from them. In another aspect, the small RNA molecules provided herein can bind to and reduce the expression of nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60, or nucleic acid sequences that are at least 98% identical to those transcribed from them. In another aspect, the small RNA molecules provided herein can bind to and reduce the expression of nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60, or nucleic acid sequences that are at least 99% identical to those transcribed from them. In yet another aspect, the small RNA molecules provided herein can bind to and reduce the expression of nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60, or nucleic acid sequences that are 100% identical to those transcribed from them.

[0152] As used herein, "capable of binding with" is synonymous with "capable of hybridizing with". In one sense, a first nucleic acid molecule capable of binding to a second nucleic acid molecule binds to that second nucleic acid molecule. As used herein, under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength, the first nucleic acid molecule can hybridize via non-covalent interactions (… For example Watson-Crick base pairing hybridizes with the second nucleic acid molecule in a sequence-specific, antiparallel manner. Right now (Specific binding of nucleic acids to complementary nucleic acids). As is known in the art, standard Watson-Crick base pairing includes: adenine to thymine, adenine to uracil, and guanine (G) to cytosine (C) [DNA, RNA]. Additionally, it is also known in the art that for two RNA molecules ( For example Hybridization between tRNA and dsRNA involves guanine bases pairing with uracil. For example, in the context of base pairing between the anticodon of tRNA and the codon in mRNA, the G / U base pairing portion is responsible for the degeneracy of the genetic code. Right now (Redundancy). In the context of this disclosure, guanine in the protein-binding fragment (dsRNA double strand) of the subject DNA targeting RNA molecule is considered complementary to uracil, and vice versa. Therefore, when a G / U base pair can be formed at a given nucleotide position in the protein-binding fragment (dsRNA double strand) of the subject DNA targeting RNA molecule, that position is not considered non-complementary, but rather complementary.

[0153] Hybridization and washing conditions are well known and illustrated in the following literature: Sambrook, J., Fritsch, EF, and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1; and Sambrook, J., and Russell, W., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). Temperature and ionic strength conditions determine the “rigidity” of the hybridization.

[0154] Hybridization requires two nucleic acids to contain complementary sequences, but base mismatches are possible. The suitable conditions for hybridization between two nucleic acids depend on the length and degree of complementarity (variables well known in the art). The greater the complementarity between the two nucleotide sequences, the higher the melting temperature (Tm) of the nucleic acid hybrid containing these sequences. For short-segment complementarity (… For example Hybridization between nucleic acids with complementarity of more than 35 or fewer nucleotides, the location of mismatches becomes important. See Sambrook et al. Typically, hybridizable nucleic acids are at least about 10 nucleotides in length. Exemplary minimum lengths of hybridizable nucleic acids are: at least about 15 nucleotides; at least about 20 nucleotides; at least about 22 nucleotides; at least about 25 nucleotides; and at least about 30 nucleotides. Furthermore, those skilled in the art will recognize that the temperature and the salt concentration of the washing solution can be adjusted as needed based on factors such as the length and degree of complementarity of the complementary regions.

[0155] In this art, it should be understood that the sequence of a polynucleotide does not need to be 100% complementary to the sequence of its target nucleic acid for specific hybridization or hybridization. Furthermore, polynucleotides can hybridize on one or more fragments such that inserted or adjacent fragments do not participate in the hybridization event. For example(Loop structure or hairpin structure). For example, an antisense nucleic acid in which 18 out of 20 nucleotides of the antisense compound are complementary to the target region and will therefore specifically hybridize will represent 90% complementarity. In this example, the remaining non-complementary nucleotides may cluster or be scattered with complementary nucleotides and do not need to be adjacent to each other or adjacent to complementary nucleotides. The percentage of complementarity between specific segments of a nucleic acid sequence can be determined using the BLAST® program (Basic Local Alignment Search Tool) and the PowerBLAST program known in the art (…). See Altschul et al. (J.Mol.Biol.,1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or routinely determined using the algorithm of Smith and Waterman (Adv. application Math., 1981, 2, 482-489) with default settings by using the Gap program (Wisconsin Sequence Analysis Package, version 8 for Unix, GeneticsComputer Group, University Research Park, Madison Wis.).

[0156] Decreased expression / activity On the one hand, small RNA molecules reduce the expression of any nucleic acid sequence to which they can bind. On the other hand, the non-natural mutations presented in this paper reduce the expression of mutant nucleic acid sequences compared to non-mutated nucleic acid sequences in control plants grown under similar conditions.

[0157] Unless otherwise stated, all comparisons with control plants require similar or comparable growth conditions for both plants being compared. As used herein, “grown under comparable conditions,” “similar growth conditions,” or “comparable growth conditions” refer to similar environmental conditions and / or agronomic practices used for growth and meaningful comparisons between two or more plant genotypes, such that the environmental conditions and / or agronomic practices will not contribute to or explain any differences observed between the two or more plant genotypes. Environmental conditions include, for example, light, temperature, water (humidity), and nutrients (…). For example (nitrogen and phosphorus). Agronomic practices include, for example, sowing, pruning, undercutting, transplanting, topping, and budding. SeeTobacco, Production, Chemistry and Technology, Chapters 4B and 4C, Davis & Nielsen, eds., Blackwell Publishing, Oxford (1999), pp. 70–103.

[0158] The reduction in expression of endogenous nucleic acid sequences can be measured using any suitable method known in the art. Non-limiting examples of measuring expression include quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), RNA blotting, etc. For example (Northern blotting) and RNA sequencing. Expression differences can be described as absolute or relative quantifications. See For example, Livak and Schmittgen, Methods , 25:402-408 (2001). If an endogenous nucleic acid sequence encodes a protein, changes in expression can be inferred by examining the accumulation of the encoded protein. Non-limiting examples of measuring protein accumulation include Western blotting and enzyme-linked immunosorbent assay (ELISA).

[0159] On one hand, qRT-PCR was used to measure the decrease in expression. On the other hand, RNA blotting was used to measure the decrease in expression. On yet another hand, RNA sequencing was used to measure the decrease in expression. In yet another aspect, Western blotting was used to measure the decrease in expression. And still another aspect, ELISA was used to measure the decrease in expression.

[0160] On one hand, non-natural mutations in nucleic acid sequences selected from the group consisting of SEQ ID NO: 1 to 20 and 41 to 60 result in reduced expression levels of nucleic acid sequences compared to those lacking the non-naturally mutated nucleic acid sequences in control plants grown under similar growth conditions. On the other hand, non-natural mutations in nucleic acid sequences encoding amino acid sequences selected from the group consisting of SEQ ID NO: 21 to 40 result in reduced expression levels of nucleic acid sequences compared to those lacking the non-naturally mutated nucleic acid sequences in control plants grown under similar growth conditions.

[0161] On one hand, non-natural mutations in nucleic acid sequences selected from the group consisting of SEQ ID NO: 1 to 20 and 41 to 60 result in reduced expression of the corresponding amino acid sequences (SEQ ID NO: 21 to 40) compared to those lacking the non-naturally mutated nucleic acid sequences in control plants grown under similar growth conditions. On the other hand, non-natural mutations in nucleic acid sequences encoding amino acid sequences selected from the group consisting of SEQ ID NO: 21 to 40 result in reduced expression of the corresponding amino acid sequences (SEQ ID NO: 21 to 40) compared to those lacking the non-naturally mutated nucleic acid sequences in control plants grown under similar growth conditions.

[0162] In one aspect, the reduction in expression includes a reduction of at least 1% compared to the expression in the same tissue of a control plant when grown under similar growth conditions. In another aspect, the reduction in expression includes a reduction of at least 5% compared to the expression in the same tissue of a control plant when grown under similar growth conditions. In another aspect, the reduction in expression includes a reduction of at least 10% compared to the expression in the same tissue of a control plant when grown under similar growth conditions. In another aspect, the reduction in expression includes a reduction of at least 25% compared to the expression in the same tissue of a control plant when grown under similar growth conditions. In another aspect, the reduction in expression includes a reduction of at least 50% compared to the expression in the same tissue of a control plant when grown under similar growth conditions. In another aspect, the reduction in expression includes a reduction of at least 75% compared to the expression in the same tissue of a control plant when grown under similar growth conditions. In another aspect, the reduction in expression includes a reduction of at least 90% compared to the expression in the same tissue of a control plant when grown under similar growth conditions. In another aspect, the reduction in expression includes a reduction of at least 95% compared to the expression in the same tissue of a control plant when grown under similar growth conditions.

[0163] As used in this article, when referring to proteins, “activity” means the ability to perform protein functions, such as binding nucleic acids, binding other proteins, or enzymes, without being restricted by any scientific theory.

[0164] In one aspect, the reduction in protein activity includes a reduction of at least 1% compared to the protein activity in the same tissue of a control plant when grown under similar growth conditions. In another aspect, the reduction in protein activity includes a reduction of at least 5% compared to the protein activity in the same tissue of a control plant when grown under similar growth conditions. In another aspect, the reduction in protein activity includes a reduction of at least 10% compared to the protein activity in the same tissue of a control plant when grown under similar growth conditions. In another aspect, the reduction in protein activity includes a reduction of at least 25% compared to the protein activity in the same tissue of a control plant when grown under similar growth conditions. In another aspect, the reduction in protein activity includes a reduction of at least 50% compared to the protein activity in the same tissue of a control plant when grown under similar growth conditions. In another aspect, the reduction in protein activity includes a reduction of at least 75% compared to the protein activity in the same tissue of a control plant when grown under similar growth conditions. In another aspect, the reduction in protein activity includes a reduction of at least 90% compared to the protein activity in the same tissue of a control plant when grown under similar growth conditions. On the other hand, the reduction in protein activity includes at least a 95% reduction compared to the protein activity in the same tissues of a control plant grown under similar growth conditions.

[0165] In one aspect, the reduction in expression includes a decrease of 1% to 99% compared to the expression in the same tissue of a control plant grown under similar growth conditions. In another aspect, the reduction in expression includes a decrease of 1% to 90% compared to the expression in the same tissue of a control plant grown under similar growth conditions. In another aspect, the reduction in expression includes a decrease of 1% to 75% compared to the expression in the same tissue of a control plant grown under similar growth conditions. In another aspect, the reduction in expression includes a decrease of 1% to 50% compared to the expression in the same tissue of a control plant grown under similar growth conditions. In another aspect, the reduction in expression includes a decrease of 1% to 25% compared to the expression in the same tissue of a control plant grown under similar growth conditions. In another aspect, the reduction in expression includes a decrease of 25% to 90% compared to the expression in the same tissue of a control plant grown under similar growth conditions. In another aspect, the reduction in expression includes a decrease of 50% to 90% compared to the expression in the same tissue of a control plant grown under similar growth conditions. On the other hand, the reduction in expression included a decrease of 25% to 75% compared to the expression in the same tissues of control plants grown under similar growth conditions.

[0166] In one aspect, the decrease in protein activity includes a reduction of 1% to 99% compared to the protein activity in the same tissue of a control plant grown under similar growth conditions. In another aspect, the decrease in protein activity includes a reduction of 1% to 90% compared to the protein activity in the same tissue of a control plant grown under similar growth conditions. In another aspect, the decrease in protein activity includes a reduction of 1% to 75% compared to the protein activity in the same tissue of a control plant grown under similar growth conditions. In another aspect, the decrease in protein activity includes a reduction of 1% to 50% compared to the protein activity in the same tissue of a control plant grown under similar growth conditions. In another aspect, the decrease in protein activity includes a reduction of 1% to 25% compared to the protein activity in the same tissue of a control plant grown under similar growth conditions. In another aspect, the decrease in protein activity includes a reduction of 25% to 90% compared to the protein activity in the same tissue of a control plant grown under similar growth conditions. On the other hand, the reduction in protein activity includes a decrease of 50% to 90% compared to the protein activity in the same tissue of a control plant grown under similar growth conditions. On the other hand, the reduction in protein activity includes a decrease of 25% to 75% compared to the protein activity in the same tissue of a control plant grown under similar growth conditions.

[0167] On one hand, the reduction in expression includes a statistically significant reduction compared to the expression in the same tissues of control plants grown under similar growth conditions. Those skilled in the art will recognize that any level of reduction is predictable, provided that the level of reduction has been determined to be statistically significant using accepted statistical hypothesis testing. As a non-limiting example, students... t The hypothesis test is a statistical hypothesis test used to determine whether the reduction in expression between modified and control plants is statistically significant. As used in this paper, "statistically significant" means a p-value less than or equal to 0.05.

[0168] On the one hand, non-natural mutations result in a reduced level of activity of proteins or peptides encoded by the nucleic acid sequences provided herein compared to the activity of control plants grown under similar growth conditions. On the other hand, non-natural mutations in endogenous nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60 reduce the activity level of proteins or peptides encoded by the nucleic acid sequences compared to the activity of proteins or peptides encoded by the endogenous nucleic acid sequences in control tobacco plants grown under similar growth conditions, wherein the nucleic acid sequences lack non-natural mutations in the control tobacco plants. On the other hand, non-natural mutations in endogenous nucleic acid sequences (wherein the endogenous nucleic acid sequence encodes histone sequences selected from the group consisting of SEQ ID NOs: 21 to 40) reduce the activity level of proteins or peptides encoded by the nucleic acid sequences compared to the activity of proteins or peptides encoded by the endogenous nucleic acid sequences in control tobacco plants grown under similar growth conditions, wherein the nucleic acid sequences lack non-natural mutations in the control tobacco plants.

[0169] As used in this article, when referring to proteins or polypeptides, "activity" means the ability to perform enzyme functions.

[0170] Increased expression / activity On one hand, the non-natural mutation leads to an increased expression of the nucleic acid sequence. On the other hand, the non-natural mutation leads to an increased level of expression of the nucleic acid sequence compared to the expression of the nucleic acid sequence in the same tissue of a control tobacco plant grown under similar growth conditions, wherein the nucleic acid sequence lacks at least one non-natural mutation in the control tobacco plant.

[0171] In one aspect, the increased expression level includes an increase of at least 5% compared to the expression in the same tissue of a control plant grown under similar growth conditions. In another aspect, the increased expression level includes an increase of at least 10% compared to the expression in the same tissue of a control plant grown under similar growth conditions. In another aspect, the increased expression level includes an increase of at least 25% compared to the expression in the same tissue of a control plant grown under similar growth conditions. In another aspect, the increased expression level includes an increase of at least 50% compared to the expression in the same tissue of a control plant grown under similar growth conditions. In another aspect, the increased expression level includes an increase of at least 75% compared to the expression in the same tissue of a control plant grown under similar growth conditions. In another aspect, the increased expression level includes an increase of at least 100% compared to the expression in the same tissue of a control plant grown under similar growth conditions. In another aspect, the increased expression level includes an increase of at least 200% compared to the expression in the same tissue of a control plant grown under similar growth conditions. On the other hand, the increased expression level includes an increase of at least 500% compared to the expression in the same tissues of control plants grown under similar growth conditions.

[0172] On one hand, the non-natural mutation results in an increased level of activity of the protein or polypeptide encoded by the nucleic acid sequence compared to the activity of the protein or polypeptide encoded by the nucleic acid sequence in a control tobacco plant grown under similar growth conditions, wherein the nucleic acid sequence lacks at least one non-natural mutation in the control tobacco plant.

[0173] plant As used herein, “control plant” refers to a plant with the same or nearly identical genetic composition as the modified plant being compared, except for the modifications described herein introduced into that modified plant. Right now In addition to non-natural mutations in endogenous genes, small RNAs, and recombinant nucleic acid constructs, control plants were grown under the same conditions and for the same period of time as the comparison plants.

[0174] As used in this article, tobacco plants can be derived from... tobacco Any plant of the genus, including but not limited to: Red Flos Tobacco (Nicotiana tabacum), Nicotiana amplexicaulis PI 271989; Ben's Tobacco (Nicotiana benthamiana) PI 555478; Nicotiana bigelovii (Tobacco of Bigelovii) PI 555485; De clan Tobacco (Nicotiana debneyi); High-quality tobacco (Nicotiana excelsior) PI 224063; Sticky tobacco (Nicotiana) glutinosa PI 555507 Nicotiana goodspeedii PI 241012; Gorbachev's tobacco (Nicotiana gossei) PI 230953; Western tobacco (Nicotiana hesperis) PI 271991; Tobacco Napier (Nicotiana knightiana) PI 555527; Seaside tobacco (Nicotiana maritima) PI 555535; Big pipe tobacco Grass (Nicotiana megalosiphon) PI 555536; Naked stem tobacco (Nicotiana nudicaulis) PI 555540; Conical tobacco (Nicotiana paniculata) PI 555545; Blue-leaved tobacco (Nicotiana plumbaginifolia) PI 555548; Nicotiana repanda (broadleaf tobacco) PI 555552; Yellow tobacco (Nicotiana rustica) ; cigarette Grass (Nicotiana suaveolens) PI 230960; Tobacco (Nicotiana sylvestris) PI 555569; fluff Tobacco (Nicotiana tomentosa) PI 266379. In one aspect, the tobacco plants described herein are Nicotiana tomentosiformis; and Triangular-leaved tobacco (Nicotiana trigonophylla) plants. red Tobacco Flower Other suitable plant species for the present disclosure may include members of the following genera:

[0175] Okra (Abelmoschus) The genera *Abies*, *Acer*, *Agrostis*, *Allium*, and *Alternanthera* (Alstroemeria), bromeliad (Ananas), andrographis, andropogon, wormwood Genus (Artemisia), Genus (Arundo), Genus (Atropa), Genus (Berberis), Genus (Beta), Red *Bixa*, *Brassica*, *Calendula*, *Camellia*, *Camptotheca* (Camptotheca), Capsicum, Carthamus, Catharanthus, Tripterygium The genera *Cephalotaxus*, *Chrysanthemum*, *Cinchona*, and *Citrullus*. The genera *Coffea*, *Colchicum*, *Coleus*, and *Cucumis* are all related to the same species. (Cucurbita), Cynodon, Datura, Dianthus, Foxglove (Digitalis), Dioscorea, Elaeis, Ephedra, Imperata (Erianthus), Eucalyptus, Festuca, Fragaria, Snowdrop (Galanthus), Glycine, Gossypium, Helianthus, Rubber Tree (Hevea), Hordeum, Hyoscyamus, Jatropha, Lettuce (Lactuca), Linum, Lolium, Lupinus, Tomato (Lycopersicon), Lycopodium, Manihot, Medicago, Mint (Mentha), Miscanthus, Musa, Nicotiana, Oryza, Millet (Panicum), Parthenium, Pennisetum, Petunia, and other genera. (Phalaris), Phleum, Pinus, Poa, Poinsettia, Poplar Genus: Populus, Rauwolfia, Ricinus, Rosa, Sugarcane (Saccharum), Salix, Sanguinaria, Scopolia, Rye (Secale), Solanum, Sorghum, Spartina, Spinacea, Artemisia Genus (Tanacetum), Genus (Taxus), Genus (Theobroma), Genus (Triticosecale), Genus (Wheat) Triticum, Uniola, Veratrum, Vinca, Vitis and The genus *Zea*. . Additional suitable species may include: Panicum species ( Panicum spp.), Sorghum species ( Sorghum spp.), Miscanthus species ( Miscanthus spp.), Saccharum species ( Saccharum spp.), Erianthus species ( Erianthus spp.), Populus species ( Populus spp.), big bluestem ( Andropogon gerardii ), elephant grass ( Pennisetum purpureum ), reed canary grass ( Phalaris arundinacea ), Bermuda grass (​ Cynodon dactylon ), tall fescue ( Festuca arundinacea ), grassland net grass ( Spartina pectinata ), alfalfa ( Medicago sativa ), Luzhu ( Arundo donax ),rye( Secale cereale ), species of the genus *Salix* ( Salix spp.), Eucalyptus Genus and species ( Eucalyptus spp.), triticale ( Triticosecale ), bamboo, sunflower ( Helianthus annuus ),safflower( Carthamus tinctorius ), Jatropha curcas ( Jatropha curcas ), castor bean ( Ricinus communis ), oil palm ( Elaeis guineensis ),flax( Linum usitatissimum ), Mustard greens (Brassica juncea), beets (Beta vulgaris) cassava ( Manihot esculenta ),tomato( Lycopersicon esculentum ),lettuce( Lactuca sativa ),banana( Musyclise alca ),potato( Solanum tuberosum ), cabbage ( Brassica oleracea ),Tea( Camellia sinensis ),strawberry( Fragaria ananassa ),cocoa( Theobroma cacao ),coffee( Coffeycliseca ),Grape( Vitis vinifera ),Pineapple( Ananas comosus ),chili( Capsicum annuum ),onion( Allium cepa ),melon( Cucumis melo ),cucumber( Cucumis sativus ), winter melon ( Cucurbita maxima ),pumpkin( Cucurbita moschata ),spinach( Spinacea oleracea ),watermelon( Citrullus lanatus Okra Abelmoschus esculentus ),eggplant( Solanum melongena ), species of the genus Rosa ( Rosa spp.), carnations ( Dianthus caryophyllus ), Petunia Genus and species ( Petunia spp.), poinsettia ( Poinsettia pulcherrimaa ), white lupine ( Lupinus albus ),oat( Uniola paniculata ), species of the genus *Gnaphalium* ( Agrostis spp.), aspen ( Populus tremuloides ), genus Pinus ( Pinus spp.), species of the genus Abies ( Abiesspp.), species of the genus Acer ( Acer spp.), barley ( Hordeum vulgare Kentucky bluegrass ( ) Poa pratensis ), species of ryegrass ( Lolium spp.) and cat's tail grass ( Phleum pratense ), willow branch millet ( Panicum virgatum ), sorghum ( Sorghum bicolor ), Miscanthus giganteus ( Miscanthus giganteus ), energy sugarcane ( Saccharum sp.), poplar ( Populus balsamifera ),corn( Zea mays ), soybeans Glycine max ),rape( Brassica napus ),wheat( Triticum aestivum ),cotton( Gossypium hirsutum ), rice ( Oryza sativa ),sunflower( Helianthus annuus ), alfalfa ( Medicago sativa ),beet( Beta vulgaris ) or pearl millet ( Pennisetum glaucum ).

[0176] In one respect, preferred plants for use in this disclosure include any plant or subspecies selected from the group consisting of: the genus *Nicotiana* (…). Nicotiana spp.), or Camellia genus ( Camellia (spp.). Appropriately, the tobacco plant is Nicotiana tabacum Tobacco plants, preferably, white-ribbed type Nicotiana tabacum Tobacco plants. Other plants applicable to this disclosure include, but are not limited to, camellias ( Camellia sinensis ).

[0177] In one aspect, the provided tobacco parts include, but are not limited to, leaves, stems, roots, trichomes, seeds, flowers, pollen, anthers, ovules, pedicels, fruits, meristems, cotyledons, hypocotyls, pods, embryos, endosperm, explants, callus, tissue cultures, buds, cells, and protoplasts. In another aspect, the provided tobacco parts do not include seeds. In one aspect, this disclosure provides tobacco plant cells, tissues, and organs that are not propagation material and do not mediate the natural reproduction of the plant. In another aspect, this disclosure also provides tobacco plant cells, tissues, and organs that serve as propagation material and mediate the natural reproduction of the plant. In another aspect, this disclosure provides tobacco plant cells, tissues, and organs that cannot sustain themselves through photosynthesis. In another aspect, this disclosure provides somatic tobacco plant cells. Unlike reproductive cells, somatic cells do not mediate plant reproduction.

[0178] Cells, tissues, and organs can originate from seeds, fruits, leaves, cotyledons, hypocotyls, meristems, embryos, endosperm, roots, buds, stems, trichomes, pods, flowers, inflorescences, stems, pedicels, styles, stigmas, receptacles, petals, sepals, pollen, anthers, filaments, ovaries, ovules, pericarps, phloem, and vascular tissues. In another aspect, this disclosure provides chloroplasts of tobacco plants. In yet another aspect, this disclosure provides epidermal cells, stomatal cells, leaf or root hairs, storage roots, or tubers. In yet another aspect, this disclosure provides tobacco protoplasts.

[0179] Skilled artisans know that tobacco plants reproduce naturally through seeds rather than through asexual or vegetative propagation. In this respect, the present disclosure provides tobacco endosperm.

[0180] This disclosure provides cells from the tobacco plant described herein.

[0181] As used in this article, "progeny tobacco plants" or "progeny tobacco seeds" can come from any progeny. for example , F1, F2, F3, F4, F5, F6, F7, etc.

[0182] On the one hand, tobacco plants or parts thereof belong to the group consisting of flue-cured varieties, bright varieties, burley varieties, Virginia varieties, Maryland varieties, black tobacco varieties, galpao varieties, oriental varieties, and Turkish varieties. On the other hand, the modified tobacco plants or parts thereof provided in this article belong to the group consisting of flue-cured varieties, bright varieties, burley varieties, Virginia varieties, Maryland varieties, black tobacco varieties, galpao varieties, oriental varieties, and Turkish varieties.

[0183] On one hand, tobacco cells belong to the group consisting of tobacco varieties selected from the following categories: flue-cured varieties, bright varieties, burley varieties, Virginia varieties, Maryland varieties, black tobacco varieties, galpao varieties, oriental varieties, and Turkish varieties. On the other hand, modified tobacco cells belong to the group consisting of tobacco varieties selected from the following categories: flue-cured varieties, bright varieties, burley varieties, Virginia varieties, Maryland varieties, black tobacco varieties, galpao varieties, oriental varieties, and Turkish varieties.

[0184] On the one hand, tobacco leaves belong to the group of tobacco varieties selected from the flue-cured varieties, bright varieties, burley varieties, Virginia varieties, Maryland varieties, black tobacco varieties, Galpao varieties, Oriental varieties, and Turkish varieties.

[0185] On the one hand, the leaves or plant parts of flavored tobacco belong to a group of tobacco varieties selected from the categories of flue-cured varieties, bright varieties, burley varieties, Virginia varieties, Maryland varieties, black tobacco varieties, Galpao varieties, Oriental varieties, and Turkish varieties. Skilled artisans further understand that flue-cured tobacco does not constitute a living organism and cannot grow or reproduce.

[0186] Smoked tobacco (also known as "Virginia" or "bright" tobacco) accounts for approximately 40% of the world's tobacco production. Smoked tobacco is often referred to as "bright" tobacco because it develops a golden to deep orange color during the smoking process. Smoked tobacco has a mild, fresh aroma and flavor. It is generally high in sugar and low in oil. The main countries growing smoked tobacco are Argentina, Brazil, China, India, Tanzania, and the United States. In this article, the tobacco plants or seeds, or modified tobacco plants or seeds, belong to the group consisting of the varieties listed in Table 3 and any varieties substantially derived from any of the aforementioned varieties. see WO 2004 / 041006 A1. In another respect, the modified tobacco plants or seeds provided herein are flue-cured varieties selected from the group consisting of K326, K346 and NC196.

[0187] Table 3. Tobacco varieties for flue gas conditioning.

[0188] Air-cured tobacco includes "Bulb," "Maryland," and "Dark" varieties. A common factor associated with air-cured tobacco is that the curing process takes place primarily in the absence of artificial heat and humidity. Burley tobacco ranges in color from light to dark brown, is high in oil, and low in sugar. Burley tobacco is typically air-cured in barns. Major Burley-growing countries include Argentina, Brazil, Italy, Malawi, and the United States.

[0189] Maryland tobacco is extremely fluffy, with good combustion properties, low nicotine content, and a neutral aroma. Major tobacco-growing countries in Maryland include the United States and Italy.

[0190] On the one hand, the tobacco plants or seeds or modified tobacco plants or seeds provided herein belong to the group consisting of the tobacco varieties listed in Table 4 and any varieties substantially derived from any of the aforementioned varieties. On the other hand, the modified tobacco plants or seeds provided herein are burley tobacco varieties selected from the group consisting of TN 90, KT 209, KT 206, KT212 and HB 4488.

[0191] Table 4. Burley tobacco varieties.

[0192] On the other hand, the tobacco plants or seeds or modified tobacco plants or seeds provided herein belong to the Maryland tobacco varieties selected from the groups of tobacco varieties listed in Table 5 and any varieties substantially derived from any of the aforementioned varieties.

[0193] Table 5. Maryland tobacco varieties.

[0194] The main difference between dark-cured tobacco and other tobacco types lies in its curing process, which gives it a medium to dark brown color and a distinctive aroma. Dark-cured tobacco is primarily used in the production of chewing tobacco and snuff. In this context, the modified tobacco plants or seeds described herein belong to any dark-cured tobacco variety selected from the group consisting of Sumatra, Jatim, Dominican Cuban, Besuki, One sucker, Green River, Virginia Sun-cured, and Paraguayan Passado, and any variety substantially derived from any of the aforementioned varieties.

[0195] Dark-cured tobacco is typically cured over a low-burning wood fire on the floor of an enclosed curating room. It is commonly used in the production of pipe blends, cigarettes, chewing tobacco, snuff, and strong cigars. The primary growing regions for dark-cured tobacco are Tennessee, Kentucky, and Virginia in the United States. In this context, the tobacco plants or seeds, or modified tobacco plants or seeds, described herein belong to any dark-cured tobacco variety selected from the groups of tobacco varieties listed in Table 6 and any variety substantially derived from any of the aforementioned varieties.

[0196] Table 6. Varieties of black tobacco.

[0197] Because Oriental tobacco typically grows in the eastern Mediterranean region (such as Turkey, Greece, Bulgaria, Macedonia, Syria, Lebanon, Italy, and Romania), it is also known as Greek tobacco, aromatic tobacco, and Turkish tobacco. The small plant size, leaf size, and distinctive aroma characteristics of Oriental tobacco varieties are a result of their adaptation to the poor soils and harsh climatic conditions in which they grow. In this article, the tobacco plants or seeds, or modified tobacco plants or seeds, provided are Oriental tobacco varieties belonging to the groups of tobacco varieties listed in Table 7 and any varieties substantially derived from any of the aforementioned varieties.

[0198] Table 7. Varieties of Oriental Tobacco.

[0199] On the one hand, the tobacco plants or seeds or modified tobacco plants or seeds provided herein belong to the cigar tobacco varieties selected from the groups of tobacco varieties listed in Table 8 and any varieties substantially derived from any of the aforementioned varieties.

[0200] Table 8. Cigar tobacco varieties.

[0201] On the one hand, the tobacco plants or seeds or modified tobacco plants or seeds provided herein belong to the tobacco varieties selected from the groups of tobacco varieties listed in Table 9 and any varieties substantially derived from any of the aforementioned varieties.

[0202] Table 9. Other tobacco varieties.

[0203] On the one hand, the tobacco plant or part thereof is selected from the group consisting of tobacco varieties listed in Tables 3 to 9. On the other hand, the tobacco plant or part thereof is selected from the varieties listed in Table 3. On the other hand, the tobacco plant or part thereof is selected from the varieties listed in Table 4. On the other hand, the tobacco plant or part thereof is selected from the varieties listed in Table 5. On the other hand, the tobacco plant or part thereof is selected from the varieties listed in Table 6. On the other hand, the tobacco plant or part thereof is selected from the varieties listed in Table 7. On the other hand, the tobacco plant or part thereof is selected from the varieties listed in Table 8. On the other hand, the tobacco plant or part thereof is selected from the varieties listed in Table 9.

[0204] On one hand, the modified tobacco plant or a portion thereof is selected from varieties grouped from the tobacco varieties listed in Tables 3 to 9. On one hand, the modified tobacco plant or a portion thereof is selected from the varieties listed in Table 3. On another hand, the modified tobacco plant or a portion thereof is selected from the varieties listed in Table 4. On another hand, the modified tobacco plant or a portion thereof is selected from the varieties listed in Table 5. On another hand, the modified tobacco plant or a portion thereof is selected from the varieties listed in Table 6. On another hand, the modified tobacco plant or a portion thereof is selected from the varieties listed in Table 7. On another hand, the modified tobacco plant or a portion thereof is selected from the varieties listed in Table 8. On another hand, the modified tobacco plant or a portion thereof is selected from the varieties listed in Table 9.

[0205] On the one hand, the tobacco seeds are selected from varieties grouped according to the tobacco varieties listed in Tables 3 to 9. On the other hand, the tobacco seeds are from the varieties listed in Table 3. On the other hand, the tobacco seeds are from the varieties listed in Table 4. On the other hand, the tobacco seeds are from the varieties listed in Table 5. On the other hand, the tobacco seeds are from the varieties listed in Table 6. On the other hand, the tobacco seeds are from the varieties listed in Table 7. On the other hand, the tobacco seeds are from the varieties listed in Table 8. On the other hand, the tobacco seeds are from the varieties listed in Table 9.

[0206] On one hand, the tobacco cells are from varieties selected from the groups consisting of tobacco varieties listed in Tables 3 to 9. On the other hand, the tobacco cells are from the varieties listed in Table 3. On the other hand, the tobacco cells are from the varieties listed in Table 4. On the other hand, the tobacco cells are from the varieties listed in Table 5. On the other hand, the tobacco cells are from the varieties listed in Table 6. On the other hand, the tobacco cells are from the varieties listed in Table 7. On the other hand, the tobacco cells are from the varieties listed in Table 8. On the other hand, the tobacco cells are from the varieties listed in Table 9.

[0207] All specific varieties of flue-cured, dark-cured, burley, Maryland, dark-roasted, cigar, or oriental types mentioned above are listed for illustrative purposes only. Any additional flue-cured, dark-cured, burley, Maryland, dark-roasted, cigar, or oriental varieties are also considered in this application.

[0208] As used in this article, “secretory type” [S] is a plant with a phenotype that includes at least secretory gonadal hairs.

[0209] As used in this article, “non-secretory type” [NS] refers to a plant with a phenotype that includes non-secretory glandular hairs.

[0210] On the one hand, the tobacco plants or varieties provided in this article are inbred tobacco plants or varieties. As used in this article, "inbred" tobacco varieties are varieties that have been bred for genetic homogeneity.

[0211] As used herein, a "hybrid" is produced by crossing two plants from different varieties or species, such that the offspring contain genetic material from each parent. Skilled artisans recognize that more advanced hybrids can also be produced. For example, a first hybrid can be produced by crossing variety C with variety D to produce a C x D hybrid, and a second hybrid can be produced by crossing variety E with variety F to produce an E x F hybrid. The first and second hybrids can be further crossed to produce higher-order hybrids (C x D) x (E x F) containing genetic information from all four parent varieties. In one respect, the modified tobacco plant provided herein is a hybrid tobacco plant. In another respect, the modified tobacco seeds provided herein are hybrid tobacco seeds. In one respect, the tobacco plant or variety provided herein is a hybrid tobacco plant or variety. In another respect, the modified tobacco plant provided herein is a hybrid tobacco plant.

[0212] In one aspect, this disclosure provides a method for producing modified tobacco plants, the method comprising: (a) hybridizing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one offspring tobacco seed, wherein the at least one tobacco plant of the first tobacco variety contains a non-natural mutation in its endogenous nucleic acid sequence, wherein the endogenous nucleic acid sequence encodes a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40, wherein the non-natural mutation is not present in the endogenous nucleic acid sequence of a control tobacco plant of the first tobacco variety; and (b) selecting at least one offspring tobacco seed or a plant germinating therefrom, wherein the at least one tobacco seed or a plant germinating therefrom contains the non-natural mutation. In another aspect, this disclosure provides a method for producing modified tobacco plants, the method comprising: (a) hybridizing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one offspring tobacco seed, wherein the at least one tobacco plant of the first tobacco variety contains a non-natural mutation in an endogenous nucleic acid sequence that is at least 80% identical to the nucleic acid sequence selected from the group consisting of SEQ ID NO: 41 to 60, wherein the non-natural mutation is not present in the endogenous nucleic acid sequence of a control tobacco plant of the first tobacco variety; and (b) selecting at least one offspring tobacco seed or a plant germinating therefrom, wherein the at least one tobacco seed or a plant germinating therefrom contains the non-natural mutation. In another aspect, this disclosure provides a method for producing modified tobacco plants, the method comprising: (a) hybridizing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one offspring tobacco seed, wherein the at least one tobacco plant of the first tobacco variety contains a non-natural mutation in an endogenous nucleic acid sequence that is at least 80% identical to the nucleic acid sequence selected from the group consisting of SEQ ID NO: 41 to 60, wherein the non-natural mutation is not present in the endogenous nucleic acid sequence of a control tobacco plant of the first tobacco variety; and (b) selecting at least one offspring tobacco seed or a plant germinating therefrom, wherein the at least one tobacco seed or a plant germinating therefrom contains the non-natural mutation.In another aspect, this disclosure provides a method for producing modified tobacco plants, the method comprising: (a) hybridizing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one offspring tobacco seed, wherein the at least one tobacco plant of the first tobacco variety contains a non-natural mutation in its endogenous nucleic acid sequence selected from the group consisting of SEQ ID NO: 41 to 60, wherein the non-natural mutation is not present in the endogenous nucleic acid sequence of a control tobacco plant of the first tobacco variety; and (b) selecting at least one offspring tobacco seed or a plant germinating therefrom, wherein the at least one tobacco seed or a plant germinating therefrom contains the non-natural mutation. In one aspect, in any of the foregoing methods, the first tobacco variety and the second tobacco variety are the same tobacco variety. In another aspect, in any of the foregoing methods, the first tobacco variety and the second tobacco variety are two different tobacco varieties.

[0213] In one aspect, this disclosure provides a method for producing modified tobacco plants, the method comprising: (a) hybridizing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one offspring tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence encoding at least one small RNA molecule capable of binding to and reducing the expression of an endogenous nucleic acid sequence encoding a polypeptide whose amino acid sequence is at least 80% identical or similar to that of a polypeptide selected from the group consisting of SEQ ID NO: 21 to 40, wherein the recombinant DNA construct is not present in a control tobacco plant of the first tobacco variety; and (b) selecting at least one offspring tobacco seed or a plant germinating therefrom, wherein the at least one offspring tobacco seed or a plant germinating therefrom comprises the recombinant DNA construct. In another aspect, this disclosure provides a method for producing modified tobacco plants, the method comprising: (a) hybridizing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one offspring tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence encoding at least one small RNA molecule capable of binding to and reducing the expression of an endogenous nucleic acid sequence that is at least 80% identical or similar to an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 20, wherein the recombinant DNA construct is not present in a control tobacco plant of the first tobacco variety; and (b) selecting at least one offspring tobacco seed or a plant germinating therefrom, wherein the at least one offspring tobacco seed or a plant germinating therefrom comprises the recombinant DNA construct. In another aspect, this disclosure provides a method for producing modified tobacco plants, the method comprising: (a) hybridizing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one offspring tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence encoding at least one small RNA molecule capable of binding to and reducing the expression of an endogenous nucleic acid sequence at least 80% identical or similar to an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NO: 41 to 60, wherein the recombinant DNA construct is not present in a control tobacco plant of the first tobacco variety; and (b) selecting at least one offspring tobacco seed or a plant germinating therefrom, wherein the at least one offspring tobacco seed or a plant germinating therefrom comprises the recombinant DNA construct. In one aspect, in any of the foregoing methods, the first tobacco variety and the second tobacco variety are the same tobacco variety.On the other hand, in any of the methods described above, the first tobacco variety and the second tobacco variety are two different tobacco varieties. In another aspect, the recombinant DNA construct of each of the above aspects comprises a promoter sequence selected from the group consisting of SEQ ID NO: 62 to 90.

[0214] In one aspect, this disclosure provides a method for producing modified tobacco plants, the method comprising: (a) hybridizing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one offspring tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40, wherein the recombinant DNA construct is not present in a control tobacco plant of the first tobacco variety; and (b) selecting at least one offspring tobacco seed or a plant germinating therefrom, wherein the at least one offspring tobacco seed or a plant germinating therefrom comprises the recombinant DNA construct. In another aspect, this disclosure provides a method for producing modified tobacco plants, the method comprising: (a) hybridizing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one offspring tobacco seed, wherein the at least one tobacco plant of the first tobacco variety contains a recombinant DNA construct, wherein the recombinant DNA construct contains a heterologous promoter operatively linked to a nucleic acid sequence at least 80% identical to the nucleic acid sequence of the group consisting of SEQ ID NO: 1 to 20, wherein the recombinant DNA construct is not present in a control tobacco plant of the first tobacco variety; and (b) selecting at least one offspring tobacco seed or a plant germinating therefrom, wherein the at least one offspring tobacco seed or a plant germinating therefrom contains the recombinant DNA construct. In another aspect, this disclosure provides a method for producing modified tobacco plants, the method comprising: (a) hybridizing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one offspring tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20, wherein the recombinant DNA construct is not present in a control tobacco plant of the first tobacco variety; and (b) selecting at least one offspring tobacco seed or a plant germinating therefrom, wherein the at least one offspring tobacco seed or a plant germinating therefrom comprises the recombinant DNA construct. In one aspect, in any of the foregoing methods, the first tobacco variety and the second tobacco variety are the same tobacco variety. In another aspect, in any of the foregoing methods, the first tobacco variety and the second tobacco variety are two different tobacco varieties. In yet another aspect, the recombinant DNA construct of each of the foregoing aspects comprises a promoter sequence selected from the group consisting of SEQ ID NOs: 62 to 90.

[0215] As used herein, the term "hybridization" refers to the intentional mating of two plants. In one aspect, hybridization includes pollination and / or fertilization of a first tobacco plant by a second tobacco plant. The two tobacco plants in a hybridization can be distantly related, closely related, or identical. In one aspect, both tobacco plants in a hybridization are modified tobacco plants. In one aspect, the two tobacco plants in a hybridization belong to the same tobacco variety. In one aspect, the two tobacco plants in a hybridization belong to two different tobacco varieties. In one aspect, one of the two tobacco plants in a hybridization is male-sterile. In one aspect, one of the two tobacco plants in a hybridization is female-sterile. In one aspect, at least one of the two tobacco plants in a hybridization is a hybrid tobacco plant. In one aspect, at least one of the two tobacco plants in a hybridization is a modified tobacco plant.

[0216] On the one hand, the tobacco plants or varieties provided herein are male-sterile. On the other hand, the tobacco plants or varieties provided herein are cytoplasmic male-sterile (CMS). On the one hand, the modified tobacco plants or varieties provided herein are male-sterile. On the other hand, the modified tobacco plants or varieties provided herein are cytoplasmic male-sterile (CMS). Male-sterile tobacco plants can be produced by any method known in the art. Methods for producing male-sterile tobacco are described in Wernsman, EA and Rufty, RC 1987. Chapter 17. Tobacco. pp. 669-698, in: Cultivar Development. Crop Species. WH Fehr (ed.), MacMillan Publishing Go., Inc., New York, NY 761 pp.

[0217] On the other hand, the tobacco plant or variety provided herein is female-sterile. On the other hand, the modified tobacco plant or variety provided herein is female-sterile. As a non-limiting example, it is possible to make... STIG1 Gene mutations are used to create female-sterile plants. see For example, Goldman et al. 1994, EMBO Journal 13:2976-2984. On the one hand, the modified tobacco plant presented in this paper is female-sterile.

[0218] In one respect, the modified tobacco plants described herein are low-alkaloid varieties or low-alkaloid plants. As a non-limiting example, LA Burley 21 (LA BU21) is a low-alkaloid tobacco variety. LA BU21 was produced by repeatedly backcrossing LA BU21 with low-alkaloid genes from Cuban cigar varieties incorporated into Burley 21. It has approximately 0.2% (dry weight) of total alkaloids (compared to approximately 3.5% (dry weight) of its parent, Burley 21). LA BU21 has leaf grades far below commercially acceptable standards. LA BU21 also exhibits other unfavorable leaf phenotypes, characterized by lower yields, delayed maturity and senescence, higher susceptibility to insect predation, and poorer final product quality after processing. LA BU21 leaves also exhibit traits such as higher polyamine content, higher chlorophyll content, and more mesophyll cells per unit leaf area. Further characterization of the LA BU21 leaf phenotype is available later. re fer to US2019 / 0271000.

[0219] In one aspect, this disclosure provides tobacco plants or portions thereof that exhibit comparable insect susceptibility to herbivorous growth relative to comparable leaves of control plants not containing the same mutation or transgene. In another aspect, the comparable insect susceptibility to herbivorous growth is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in comparable leaves of control plants not containing the same mutation or transgene. On the one hand, the susceptibility of comparable insects to herbivorous food was between 0.5% and 1%, 1% and 2%, 2% and 3%, 3% and 4%, 4% and 5%, 5% and 6%, 6% and 7%, 7% and 8%, 8% and 9%, 9% and 10%, 11% and 12%, 12% and 13%, 13% and 14%, 14% and 15%, 15% and 16%, 16% and 17%, 17% and 18%, 18% and 19%, or 19% and 20% in comparable leaves of control plants that did not contain the same mutations or transgenes. On the other hand, the susceptibility of comparable insects to herbivorous food was between 0.5% and 5%, 5% and 10%, or 10% and 20% in comparable leaves of control plants that did not contain the same mutations or transgenes.

[0220] The level of susceptibility to herbivorous insects can be determined using methods known in the art (e.g., in insect rearing assays). Briefly, a 0.7% agar aqueous solution of a quarter-inch layer is added to a 100 mm culture dish and allowed to solidify. Leaf discs are cut from the dish lid, placed in the dish, and gently pushed into the agar. Leaf discs are taken from plants at the 4-5 leaf stage. The discs are removed from the leaf discs only to exclude the main midrib. Single discs are taken from the four largest leaves of the plant, thus producing four replicas per plant. A total of 16 biological replication test lines were sampled from four plants. Single buds of the second instar stage ( ex ample , Heliothis Species , Helicoverpa (Species) was added to the leaves and allowed to feed for 48 hours at ambient temperature. After 48 hours, the budworm larvae were weighed and the final larval weight was recorded.

[0221] In one aspect, a tobacco plant or a portion thereof, relative to a control tobacco plant, comprises: a first genomic modification (e.g., in or targeting one or more NIC1, NIC2, QPT, PMT, ADC, AO, or ODC genes) that provides a lower level of nicotine or total alkaloids; and a second genomic modification that provides a comparable level of one or more traits selected from the group consisting of total leaf polyamine levels, total root polyamine levels, total leaf chlorophyll levels, number of mesophyll cells per unit leaf area, and leaf epidermal cell size; and wherein the control plant does not have both the first and second genomic modifications. In another aspect, a tobacco plant or a portion thereof, relative to a control tobacco plant, comprises: a first genomic modification (e.g., in or targeting one or more NIC1, NIC2, QPT, PMT, ADC, AO, or ODC genes) that provides a lower level of nicotine or total alkaloids; and a second genomic modification that provides a comparable level of total leaf polyamines, wherein the control plant does not have both the first and second genomic modifications. In one aspect, a tobacco plant or a portion thereof, relative to a control tobacco plant, comprises: a first genomic modification that provides lower levels of nicotine or total alkaloids (e.g., in or targeting one or more NIC1, NIC2, QPT, PMT, ADC, AO, or ODC genes); and a second genomic modification that provides comparable levels of total root polyamines, wherein the control plant does not have both the first and second genomic modifications. In another aspect, a tobacco plant or a portion thereof, relative to a control tobacco plant, comprises: a first genomic modification that provides lower levels of nicotine or total alkaloids (e.g., in or targeting one or more NIC1, NIC2, QPT, PMT, ADC, AO, or ODC genes); and a second genomic modification that provides comparable levels of total leaf chlorophyll, wherein the control plant does not have both the first and second genomic modifications. In one respect, the tobacco plant or a portion thereof, relative to the control tobacco plant, contains: a first genomic modification (e.g., in or targeting one or more NIC1, NIC2, QPT, PMT, ADC, AO, or ODC genes) that provides a lower level of nicotine or total alkaloids; and a second genomic modification that provides a comparable level of mesophyll cells per leaf area, wherein the control plant does not have both the first and second genomic modifications.In one aspect, the tobacco plant or a portion thereof, relative to the control tobacco plant, comprises: a first genomic modification that provides lower levels of nicotine or total alkaloids (e.g., in or targeting one or more NIC1, NIC2, QPT, PMT, ADC, AO, or ODC genes); and a second genomic modification that provides comparable levels of leaf epidermal cell size, wherein the control plant does not possess either the first or second genomic modification. In another aspect, the second genomic modification is in or targets one or more NIC1, NIC2, QPT, PMT, ADC, AO, or ODC genes.

[0222] On one hand, the first genomic modification, the second genomic modification, or both include transgenes, mutations, or both. On one hand, the genomic modification, the second genomic modification, or both include transgenes. On one hand, the first genomic modification, the second genomic modification, or both include mutations. On one hand, the first genomic modification, the second genomic modification, or both are not based on transgenes. On one hand, the first genomic modification, the second genomic modification, or both are not based on mutations.

[0223] In one aspect, the tobacco plants provided herein contain reduced amounts of total conjugated polyamines in their leaves compared to control tobacco plants. In another aspect, the tobacco plants provided herein contain reduced amounts of total conjugated polyamines in their roots compared to control tobacco plants. Conjugated polyamines as used herein include, but are not limited to, soluble conjugated polyamines, such as phenolamides comprising a backbone consisting of a free polyamine (e.g., putrescine, spermine, and / or spermidine) conjugated with one or more phenylpropanoids (e.g., ferulic acid, caffeic acid, coumaric acid). Conjugated polyamines also include, but are not limited to, insoluble conjugated polyamines incorporated into structural polymers (e.g., lignin). In one aspect, the tobacco plants provided herein contain reduced amounts of total free polyamines (e.g., putrescine, spermine, and spermidine) in their leaves compared to control tobacco plants. In one aspect, the tobacco plants provided herein contain reduced amounts of total conjugated polyamines in their roots compared to control tobacco plants. In one aspect, the tobacco plants provided herein contain reduced amounts of one or more polyamines in conjugated forms selected from the group consisting of putrescine, spermidine, and spermine in their leaves compared to control tobacco plants. In one respect, compared to the control tobacco plant, the tobacco plant provided herein contains reduced amounts of one or more polyamines selected from the group consisting of putrescine, spermidine, and spermine in its roots, in total conjugated forms. In another respect, compared to the control tobacco plant, the tobacco plant provided herein contains reduced amounts of one or more polyamines selected from the group consisting of putrescine, spermidine, and spermine in its leaves, in total free forms. In yet another respect, compared to the control tobacco plant, the tobacco plant provided herein contains reduced amounts of one or more polyamines selected from the group consisting of putrescine, spermidine, and spermine in its roots, in total conjugated forms.

[0224] In one aspect, the characteristics or traits of the tobacco plants described herein were measured at time points selected from the group consisting of: just before flowering, at topping, 1 week after topping (WPT), 2 WPT, 3 WPT, 4 WPT, 5 WPT, 6 WPT, 7 WPT, 8 WPT, and at harvest. In another aspect, the tobacco plants containing the first and second genomic modifications provided herein are capable of producing leaves with leaf grades comparable to those from control plants. In yet another aspect, the tobacco plants containing the first and second genomic modifications provided herein have a total leaf yield comparable to that of control plants.

[0225] In one respect, the tobacco plant disclosed herein includes nic1 mutation, nic2 Mutation or both.

[0226] On the one hand, the modified tobacco plant provided herein further comprises transgenes or mutations that directly inhibit one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, or seventeen or more genes or loci encoding proteins selected from the group consisting of: guanidine Deiminase (AIC), arginase, diamine oxidase, methyl putrescine oxidase (MPO), NADH dehydrogenase, phosphoribosyl-o-aminobenzoic acid isomerase (PRAI), putrescine N-methyltransferase (PMT), quinoline phosphoribosyltransferase (QPT), S-adenosylmethionine synthase (SAMS), A622, NBB1, berberine-bridged enzyme-like enzyme (BBL), MYC2, Nic1_ERF, Nic2_ERF, ethylene response factor (ERF) transcription factor, nicotine uptake permease (NUP), and MATE transporter. see , Dewey and Xie, Moleculargenetics of alkaloid biosynthesis in Nicotiana tabacum , Phytochemistry 94(2013) 10-27.

[0227] On the one hand, the modified tobacco plant provided in this paper further includes mutations in the ERF gene (Nic2_ERF) at the Nic2 locus. On the other hand, the modified tobacco plant provided in this paper further includes mutations selected from... ERF32 , ERF34 , ERF39 , ERF189 , ERF115 , ERF221 , ERF104 , ERF179 , ERF17 and ERF168 One or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all ten genes in the group. see Shoji et al., Plant Cell , (10):3390-409(2010); and Kajikawa et al. , Plant physiol. 2017, 174:999-1011. In one aspect, the modified tobacco plants provided in this paper further include... ERF189, ERF115Or one or more mutations of either. In one aspect, the modified tobacco plants provided herein further comprise one or more transgenes that target and repress genes, encoding selected... ERF32, ERF34, ERF39, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17 and ERF168 A group may consist of one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all ten proteins.

[0228] On the one hand, the modified tobacco plants provided in this article further include mutations in the ERF gene of the Nic1 locus (Nic1_ERF) (or the Nic1b locus as in WO / 2019 / 140297). See also WO / 2018 / 237107. In one aspect, the modified tobacco plants provided in this paper further include selected... ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210 and ERF91L2 One or more mutations in two or more, three or more, four or more, five or more, six or more, seven or more genes that make up the group. See , WO / 2019 / 140297 and Kajikawa et al. , Plant physiol 2017, 174:999-1011 。 On the one hand, the modified tobacco plants provided in this paper further include selected... ERFnew, ERF199, ERF19, ERF29, ERF210 and ERF91L2 The group comprises one or more, two or more, three or more, four or more, five or more, or all six genes, with mutations. In one aspect, the modified tobacco plant provided herein further includes one or more transgenes that target and repress genes encoding selected genes. ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210 and ERF91L2 A group consists of one or more, two or more, three or more, four or more, five or more, six or more, seven or more genes.

[0229] In one aspect, the modified tobacco plant provided herein further comprises a first genetic modification comprising a mutation in a gene or locus encoding a protein selected from the group consisting of aspartate oxidase, guanidine deiminase (AIC), arginase, diamine oxidase, arginine decarboxylase (ADC), methyl putrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosyl-2-aminobenzoic acid isomerase (PRAI), putrescine N-methyltransferase (PMT), quinoline phosphoribosyltransferase (QPT), and S-adenosylmethionine synthase (SAMS), A622, NBB1, BBL, MYC2, Nic1_ERF, Nic2_ERF, ethylene response factor (ERF) transcription factor, nicotine uptake permease (NUP), and MATE transporter; and further comprises a second genetic modification targeting one or more amino acid sequences that are at least 80% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 117 to 174. In one aspect, the modified tobacco plant provided herein comprises a first genetic modification comprising a transgene that targets and represses a gene or locus encoding a protein selected from the group consisting of aspartate oxidase, guanidine deiminase (AIC), arginase, diamine oxidase, arginine decarboxylase (ADC), methyl putrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosyl-2-aminobenzoic acid isomerase (PRAI), putrescine N-methyltransferase (PMT), quinoline phosphoribosyltransferase (QPT), and S-adenosylmethionine synthase (SAMS), A622, NBB1, BBL, MYC2, Nic1, Nic2, ethylene response factor (ERF) transcription factor, nicotine uptake permease (NUP), and MATE transporter; and further comprises a second genetic modification that targets one or more amino acid sequences that are at least 80% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 117 to 174.

[0230] Modified tobacco / tobacco products In one aspect, this disclosure provides a method comprising preparing a tobacco product using a modified tobacco material derived from a modified tobacco plant, wherein the modified tobacco plant contains a non-natural mutation in an endogenous nucleic acid sequence encoding a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NOs: 21 to 40. In another aspect, this disclosure provides a method comprising preparing a tobacco product using a modified tobacco material derived from a modified tobacco plant, wherein the modified tobacco plant contains a non-natural mutation in an endogenous nucleic acid sequence that is at least 80% identical to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 41 to 60. In yet another aspect, this disclosure provides a method comprising preparing a tobacco product using a modified tobacco material derived from a modified tobacco plant, wherein the modified tobacco plant contains a non-natural mutation in an endogenous nucleic acid sequence that is at least 80% identical to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 41 to 60.

[0231] In one aspect, this disclosure provides a method for preparing a tobacco product using a modified tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct containing a heterologous promoter operatively linked to a nucleic acid sequence encoding at least one small RNA molecule capable of binding to and reducing the expression of an endogenous nucleic acid sequence encoding a polypeptide whose amino acid sequence is at least 80% identical or similar to that of the group consisting of SEQ ID NOs: 21 to 40. In another aspect, this disclosure provides a method for preparing a tobacco product using a modified tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct containing a heterologous promoter operatively linked to a nucleic acid sequence encoding at least one small RNA molecule capable of binding to and reducing the expression of an endogenous nucleic acid sequence whose nucleic acid sequence is at least 80% identical to that of the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, this disclosure provides a method comprising preparing a tobacco product using modified tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, and wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence encoding at least one small RNA molecule capable of binding to and reducing the expression of an endogenous nucleic acid sequence, the endogenous nucleic acid sequence being at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 20 and 41 to 60.

[0232] In one aspect, this disclosure provides a method comprising preparing a tobacco plant using a modified tobacco material derived from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, and wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence encoding a polypeptide, the polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 to 40. In another aspect, this disclosure provides a method comprising preparing a tobacco plant using a modified tobacco material derived from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, and wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, this disclosure provides a method comprising preparing a tobacco product using a modified tobacco material derived from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, and wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 20 and 41 to 60.

[0233] "Cooking" is an aging process that reduces moisture and destroys chlorophyll, turning tobacco leaves golden yellow and converting starch into sugars. Therefore, cooked tobacco has a higher reducing sugar content and a lower starch content compared to harvested green leaves. On the one hand, the tobacco plant or plant components described herein can be processed using conventional methods (…). For example The process involves conditioning (such as flue conditioning, curing in the curing room, baking, drying, or sun-drying). Descriptions of different conditioning methods are provided below. See For example, Tso (1999, Tobacco, Production, Chemistry and Technology, Chapter 1, edited by Davis & Nielsen, Blackwell Publishing, Oxford). Modified tobacco is typically processed in wooden barrels ( For example Aged for several years under compressed conditions in large drums or cardboard boxes. For example (Two to five years), with a moisture content ranging from 10% to approximately 25%. See U.S. Patents 4,516,590 and 5,372,149. The conditioned and aged tobacco can then be further processed. This further processing includes conditioning the tobacco under vacuum with or without steam at various temperatures, pasteurization, and fermentation.

[0234] Information on harvesting Burley and Black tobacco varieties can be found in publications by the University of Kentucky, the University of Tennessee, Virginia Tech, and North Carolina State University. 2019-2020 Burley and Dark Tobacco Production Guide The article was found in December 2018 and its full text is incorporated into this paper by reference.

[0235] In one aspect, this disclosure provides modified tobacco materials derived from any tobacco plant or part thereof provided herein. In another aspect, this disclosure provides modified tobacco materials derived from any modified tobacco plant or part thereof provided herein.

[0236] On one hand, the processed tobacco material includes tobacco material selected from the group consisting of processed leaf material, processed stem material, processed bud material, processed flower material, and processed root material. On another hand, the processed tobacco material includes processed leaf material, processed stem material, or both. In yet another aspect, the processed tobacco material includes processed leaf material. And still another aspect, the processed tobacco material includes processed stem material.

[0237] On the one hand, modified tobacco materials include flue-modified tobacco materials. On the other hand, modified tobacco materials include air-cured tobacco materials. On another hand, modified tobacco materials include roasted tobacco materials. On yet another hand, modified tobacco materials include sun-dried tobacco materials. On yet another hand, the modified tobacco materials provided herein are selected from the group consisting of air-cured tobacco materials, roasted tobacco materials, sun-dried tobacco materials, and flue-modified tobacco materials. On yet another hand, the modified tobacco materials are selected from tobacco varieties selected from the group consisting of flue-modified varieties, light-colored varieties, Burley varieties, Virginia varieties, Maryland varieties, dark-colored varieties, Oriental varieties, and Turkish varieties.

[0238] On one hand, the processed tobacco leaves provided in this article are selected from the group consisting of air-dried tobacco leaves, flue-cured tobacco leaves, sun-dried tobacco leaves, and flue-processed tobacco leaves. On the other hand, the processed tobacco leaves are selected from the group consisting of flue-processed varieties, bright varieties, Burley varieties, Virginia varieties, Maryland varieties, dark varieties, Oriental varieties, and Turkish varieties.

[0239] Fermentation is typically characterized by high initial moisture content, heat generation, and a dry weight loss of 10% to 20%. See SeeFor example, U.S. Patents 4,528,993, 4,660,577, 4,848,373, and 5,372,149; U.S. Publication No. 2005 / 0178398; and Tso (1999, Chapter 1 of Tobacco, Production, Chemistry and Technology, edited by Davis & Nielsen, Blackwell Publishing, Oxford). Blended, aged, and fermented tobacco can be further processed (…). For example (cutting, chopping, puffing, or mixing). See For example, U.S. Patent Nos. 4,528,993, 4,660,577, and 4,987,907. In one aspect, this disclosure provides fermented tobacco material derived from any tobacco plant or part thereof provided herein. In another aspect, this disclosure provides fermented tobacco material derived from any modified tobacco plant or part thereof provided herein.

[0240] Tobacco materials obtained from the tobacco lines, varieties, or hybrids disclosed herein can be used to manufacture tobacco products. As used herein, "tobacco product" is defined as any product made from or derived from tobacco intended for human use or consumption. In one aspect, this disclosure provides tobacco products comprising plant material from the tobacco plants disclosed herein. In another aspect, this disclosure provides tobacco products comprising plant material from modified tobacco plants disclosed herein. In another aspect, this disclosure provides tobacco products comprising processed tobacco material. In another aspect, this disclosure provides tobacco products comprising fermented tobacco material. In another aspect, this disclosure provides tobacco products comprising tobacco blends.

[0241] Tobacco products include, but are not limited to, cigarette products ( For example Cigarettes and Biddi cigarettes), cigar products ( For example Cigar packaging tobacco and mini cigars), pipe tobacco products, tobacco-derived products, tobacco-derived nicotine products, smokeless tobacco products ( For example (including wet snuff, dry snuff, and chewing tobacco), films, chewing tablets, tablets, molded parts, gels, consumables, insoluble matrix, hollow shapes, reconstituted tobacco, puffed tobacco, etc. See, for example US Patent Publication No. US 2006 / 0191548.

[0242] As used herein, “cigarette” refers to a tobacco product having a “stem” and “fillings”. A cigarette “stem” includes cigarette paper, a filter tip, filter insert paper (for containing the filter material), a tipping paper that secures the cigarette paper (including the fillings) to the filter tip, and all adhesives that hold these parts together. “Fillings” include (1) all tobacco, including but not limited to reconstituted tobacco and puffed tobacco; (2) non-tobacco substitutes (including but not limited to herbs, non-tobacco plant materials, and other flavorings that may accompany the tobacco rolled into the cigarette paper); (3) flavorings; (4) seasonings; and (5) all other additives (mixed into the tobacco and substitutes and rolled into the cigarette).

[0243] On the one hand, tobacco products include reconstituted tobacco. On the other hand, this disclosure provides reconstituted tobacco comprising flavoring tobacco materials. As used herein, “reconstituted tobacco” refers to a portion of tobacco filler made from tobacco dust and other tobacco waste, processed into sheet form and cut into strips to resemble tobacco. In addition to cost savings, reconstituted tobacco is also important because it contributes to cigarette flavor by processing flavor development through the reaction between ammonia and sugar.

[0244] On one hand, tobacco products include expanded tobacco. As used herein, "expanded tobacco" refers to a type of tobacco filling that is processed by expanding a suitable gas, causing the tobacco to be "expanded," resulting in a lower density and a larger filling volume. This reduces the weight of the tobacco used in cigarettes.

[0245] Tobacco products derived from the plants disclosed herein also include cigarettes and other smoking articles, particularly those including filter elements, wherein the smoking material rod comprises modified tobacco within a tobacco mixture. In one aspect, the tobacco products of this disclosure are selected from the group consisting of cigarettes, non-ventilated grooved filter cigarettes, ventilated grooved filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, tobacco leaves, hookah tobacco, shredded tobacco, and cut tobacco. In another aspect, the tobacco products of this disclosure are selected from the group consisting of cigarettes, heated tobacco products, clove cigarettes, bidi cigarettes, cigars, cigarettes, non-ventilated cigarettes, ventilated grooved filter cigarettes, pipe tobacco, snuff, mouthwash, chewing tobacco, wet smokeless tobacco, finely chopped chewing tobacco, long-chopped chewing tobacco, bagged chewing tobacco products, chewing gum, tablets, lozenges, and dissolving strips.

[0246] On the other hand, the tobacco products disclosed herein are smokeless tobacco products. In one aspect, the smokeless tobacco products are selected from the group consisting of loose-leaf chewing tobacco, plugged chewing tobacco, wet snuff, nasal snuff, dry snuff, and oral tobacco.

[0247] Smokeless tobacco products do not burn and include, but are not limited to, chewing tobacco, wet smokeless tobacco, snuff, and dry snuff. Chewing tobacco consists of coarsely chopped tobacco leaves, typically packaged in large pouches and used as stoppers or wicks. Wet smokeless tobacco consists of moist, finer tobacco leaves, supplied in bulk or pouches and typically packaged in round cans and used as clips or placed in pouches between the cheeks and gums of adult tobacco consumers. Snuff is heat-treated smokeless tobacco. Dry snuff is finely ground tobacco, placed in the mouth or used in the nasal cavity.

[0248] On another aspect, the tobacco products disclosed herein are selected from the group consisting of electrically heated cigarettes, electronic cigarettes, and electronic vaporization devices.

[0249] On one hand, the tobacco product is a heated tobacco product. As used in this article, "heated tobacco product" refers to a product heated to a temperature lower than that of conventional cigarettes. For example Heated tobacco products (approximately 600°C) produce an inhalable aerosol or smoke when heated to a suitable temperature. Heated tobacco products are also known as "heat-not-burn" tobacco products. They are typically used in electronic devices that use batteries to heat the product, or in devices that use burning charcoal to heat it. In one aspect, heated tobacco products are thin films. In another aspect, heated tobacco products do not burn when heated.

[0250] On the one hand, tobacco products contain reconstituted tobacco film. On the other hand, tobacco products contain humectants. On yet another hand, the humectant is glycerin.

[0251] In one respect, the tobacco products disclosed herein may be blended tobacco products.

[0252] In one aspect, the tobacco product of this disclosure may comprise freeze-dried, unprocessed tobacco leaves or portions thereof. In another aspect, freeze-dried, unprocessed tobacco may be blended with processed tobacco as part of the tobacco product of this disclosure.

[0253] On the other hand, this disclosure provides a tobacco mixture comprising modified tobacco material. The tobacco mixture may comprise any combination of modified tobacco, unmodified tobacco, fermented tobacco, unfermented tobacco, expanded tobacco, and reconstituted tobacco.

[0254] In one aspect, the tobacco blend contains at least 5% modified tobacco by weight. In another aspect, the tobacco blend contains at least 10% modified tobacco by weight. In another aspect, the tobacco blend contains at least 15% modified tobacco by weight. In another aspect, the tobacco blend contains at least 20% modified tobacco by weight. In another aspect, the tobacco blend contains at least 25% modified tobacco by weight. In another aspect, the tobacco blend contains at least 30% modified tobacco by weight. In another aspect, the tobacco blend contains at least 35% modified tobacco by weight. In another aspect, the tobacco blend contains at least 40% modified tobacco by weight. In another aspect, the tobacco blend contains at least 45% modified tobacco by weight. In another aspect, the tobacco blend contains at least 50% modified tobacco by weight. In another aspect, the tobacco blend contains at least 55% modified tobacco by weight. In another aspect, the tobacco blend contains at least 60% modified tobacco by weight. In another aspect, the tobacco blend contains at least 65% modified tobacco by weight. In another aspect, the tobacco blend contains at least 70% modified tobacco by weight. In another aspect, the tobacco blend contains at least 75% modified tobacco by weight. In one aspect, the tobacco blend contains at least 80% modified tobacco by weight. In another aspect, the tobacco blend contains at least 85% modified tobacco by weight. In another aspect, the tobacco blend contains at least 90% modified tobacco by weight. In yet another aspect, the tobacco blend contains at least 95% modified tobacco by weight.

[0255] In one aspect, the tobacco blend contains at least 5% modified tobacco by volume. In another aspect, the tobacco blend contains at least 10% modified tobacco by volume. In another aspect, the tobacco blend contains at least 15% modified tobacco by volume. In another aspect, the tobacco blend contains at least 20% modified tobacco by volume. In another aspect, the tobacco blend contains at least 25% modified tobacco by volume. In another aspect, the tobacco blend contains at least 30% modified tobacco by volume. In another aspect, the tobacco blend contains at least 35% modified tobacco by volume. In another aspect, the tobacco blend contains at least 40% modified tobacco by volume. In another aspect, the tobacco blend contains at least 45% modified tobacco by volume. In another aspect, the tobacco blend contains at least 50% modified tobacco by volume. In another aspect, the tobacco blend contains at least 55% modified tobacco by volume. In another aspect, the tobacco blend contains at least 60% modified tobacco by volume. In another aspect, the tobacco blend contains at least 65% modified tobacco by volume. In another aspect, the tobacco blend contains at least 70% modified tobacco by volume. In another aspect, the tobacco blend contains at least 75% modified tobacco by volume. In one aspect, the tobacco mixture contains at least 80% modified tobacco by volume. In another aspect, the tobacco mixture contains at least 85% modified tobacco by volume. In another aspect, the tobacco mixture contains at least 90% modified tobacco by volume. In yet another aspect, the tobacco mixture contains at least 95% modified tobacco by volume.

[0256] change In one aspect, this disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) inducing a non-natural mutation in an endogenous nucleic acid sequence encoding an amino acid sequence that is at least 80% identical or similar to the amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40 in at least one tobacco cell; (b) selecting at least one tobacco cell from step (a) containing the non-natural mutation; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). In another aspect, this disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) inducing a non-natural mutation in an endogenous nucleic acid sequence in at least one tobacco cell that is at least 80% identical to the nucleic acid sequence selected from the group consisting of SEQ ID NO: 41 to 60; (b) selecting at least one tobacco cell from step (a) containing the non-natural mutation; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). In another aspect, this disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) inducing a non-natural mutation in at least one tobacco cell in an endogenous nucleic acid sequence that is at least 80% identical to an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60; (b) selecting at least one tobacco cell from step (a) containing the non-natural mutation; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). In another aspect, this disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) inducing a non-natural mutation in at least one tobacco cell in an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60; (b) selecting at least one tobacco cell from step (a) containing the non-natural mutation; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). In another aspect, any of the foregoing methods further comprises (d) growing the modified tobacco plant regenerated in step (c). In another aspect, any of the above methods further includes: (e) hybridizing the modified tobacco plant grown in step (d) with a second tobacco plant; and (f) obtaining at least one seed from the hybridization in step (e).

[0257] In one aspect, this disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) introducing a recombinant DNA construct into at least one tobacco cell, wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of an endogenous nucleic acid sequence encoding a polypeptide whose amino acid sequence is at least 80% identical or similar to that of a polypeptide selected from the group consisting of SEQ ID NO: 21 to 40; (b) selecting at least one tobacco cell containing the recombinant DNA construct; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). In another aspect, this disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) introducing a recombinant DNA construct into at least one tobacco cell, wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and downregulating an endogenous nucleic acid sequence that is at least 80% identical to an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60; (b) selecting at least one tobacco cell containing the recombinant DNA construct; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). In another aspect, this disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) introducing a recombinant DNA construct into at least one tobacco cell, wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and downregulating an endogenous nucleic acid sequence at least 80% identical to an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60; (b) selecting at least one tobacco cell containing the recombinant DNA construct; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). In another aspect, any of the foregoing methods further comprises (d) growing the modified tobacco plant regenerated in step (c). In another aspect, any of the above methods further comprises: (e) hybridizing the modified tobacco plant grown in step (d) with a second tobacco plant; and (f) obtaining at least one seed from the hybridization in step (e).

[0258] In one aspect, this disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) introducing a recombinant DNA construct into at least one tobacco cell, wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 to 40; (b) selecting at least one tobacco cell containing the recombinant DNA construct; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). In another aspect, this disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) introducing a recombinant DNA construct into at least one tobacco cell, wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60; (b) selecting at least one tobacco cell containing the recombinant DNA construct; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). In another aspect, this disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) introducing a recombinant DNA construct into at least one tobacco cell, wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence at least 80% identical to the nucleic acid sequence of the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60; (b) selecting at least one tobacco cell containing the recombinant DNA construct; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). In another aspect, any of the foregoing methods further comprises (d) growing the modified tobacco plant regenerated in step (c). In another aspect, any of the above methods further comprises: (e) hybridizing the modified tobacco plant grown in step (d) with a second tobacco plant; and (f) obtaining at least one seed from the hybridization in step (e).

[0259] In one aspect, this disclosure provides a method comprising transforming tobacco cells with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence encoding at least one small RNA molecule capable of binding to and reducing the expression of an endogenous nucleic acid sequence, the endogenous nucleic acid sequence encoding a polypeptide whose amino acid sequence is at least 80% identical or similar to that of the group consisting of SEQ ID NOs: 21 to 60. In another aspect, this disclosure provides a method comprising transforming tobacco cells with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence encoding at least one small RNA molecule capable of binding to and reducing the expression of an endogenous nucleic acid sequence, the endogenous nucleic acid sequence being at least 80% identical to that of the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, this disclosure provides a method comprising transforming tobacco cells with a recombinant DNA construct, wherein the recombinant DNA construct contains a heterologous promoter operatively linked to a nucleic acid sequence encoding at least one small RNA molecule capable of binding to and reducing the expression of an endogenous nucleic acid sequence, the endogenous nucleic acid sequence being at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In another aspect, the aforementioned method further comprises regenerating modified tobacco plants from transformed tobacco cells.

[0260] In one aspect, this disclosure provides a method comprising transforming tobacco cells with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence encoding a polypeptide, the polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 to 40. In another aspect, this disclosure provides a method comprising transforming tobacco cells with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In yet another aspect, this disclosure provides a method comprising transforming tobacco cells with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operatively linked to a nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and 41 to 60. In yet another aspect, the aforementioned method further comprises regenerating modified tobacco plants from the transformed tobacco cells.

[0261] On the one hand, plants suitable for genetic modification include monocots and dicots, as well as plant cell systems, including species from one of the following families: Acanthaceae, Allium family, Alstroemeriaceae, Amaryllidaceae, Apocynaceae, Arecaceae, Asteraceae, Berberidaceae, Bixaceae, Brassicaceae, Bromeliaceae, Caryophyllaceae, Cephalotaxaceae, Chenopodiaceae, Colchicaceae, Cucurbitaceae, and Dioscoreaceae. The genera *Ephedra*, *Euphorbiaceae*, *Fabaceae*, *Lamiaceae*, *Linaceae*, *Lycopodiaceae*, *Malvaceae*, *Melanthiaceae*, *Musaceae*, *Myrtaceae*, *Nyssaceae*, *Pinaceae*, *Plantaginaceae*, *Poaceae*, *Rosaceae*, *Rubiaceae*, *Salicaceae*, *Sapindaceae*, *Solanaceae*, *Taxaceae*, *Theaceae*, or *Vitaceae*. On the other hand, suitable species may include members of the following genera: Abelmoschus, Abies, Acer, Agrostis , Allium, Alstroemeria, Ananas, Andrographis, Andropogon, Artemisia, Arundo, Atropa, Berberis, Beta, Bixa , Brassica, Calendula, Camellia, Camptotheca, Capsicum, Carthamus, Catharanthus, Cephalotaxus, Chrysanthemum, Cinchona , Citrullus, Coffea, Colchicum, Coleus, Cucumis, Cucurbita, Cynodon, Datura, Dianthus, Digitalis, Dioscorea, Elaeis, Ephedra, Erianthus, Eucalyptus, Festuca, Fragaria, Galanthus, Glycine, Gossypium, Helianthus, Hevea, Hordeum, Hyoscyamus, Jatropha, Lactuca, Linum, Lolium, Lupinus, Lycopersicon , Lycopodium, Manihot, Medicago, Mentha, Miscanthus, Musa, Nicotiana, Oryza, Panicum, Parthenium, Pennisetum , Petunia, Phalaris, Phleum, Pinus, Poa, Poinsettia, Populus, Rauvolfia, Ricinus, Rosa , Saccharum, Salix, Sanguinaria, Scopolia, Secale, Solanum, Sorghum, Spartina, Spinacia, Tanacetum, Taxus , Theobroma, Triticale, Triticum, Arrhenatherum, Veratrum, Catharanthus, Vitis and Zea .

[0262] Various methods for introducing recombinant DNA constructs into plant cells are known in the art, and these methods can be used to generate transgenic plant cells and plants according to the methods of this application. Any suitable methods or techniques known in the art for transforming plant cells can be used according to this method. Effective methods for transforming plants include bacterial-mediated transformation, such as… Agrobacterium Mediated or Rhizobium Bacterial-mediated transformation and particle bombardment-mediated transformation. Various methods are known in the art for transforming explants using transformation vectors via bacterial-mediated transformation or particle bombardment, followed by culturing these explants to regenerate or develop transgenic plants. Other methods for plant transformation, such as microinjection, electroporation, vacuum permeation, pressure, sonication, silicon carbide fiber agitation, and polyethylene glycol (PEG)-mediated transformation, are also known in the art. Transgenic plants produced by these transformation methods can be chimeric or non-chimeric in response to the transformation event, depending on the method used and the explants.

[0263] Methods for transforming plant cells are well known to those skilled in the art. For example, this can be achieved by bombarding plant cells with particles coated with recombinant DNA. For example Specific details regarding the transformation of plant cells using a gene gun can be found in U.S. Patents 5,550,318, 5,538,880, 6,160,208, 6,399,861, and 6,153,812. Agro bacterium Mediated transformation is described in U.S. Patent Nos. 5,159,135; 5,824,877; 5,591,616; 6,384,301; 5,750,871; 5,463,174; and 5,188,958, all of which are incorporated herein by reference. Additional methods for transforming plants can be found, for example, in the Compendium of Transgenic Crop Plants (2009), Blackwell Publishing. Transforming tea plants ( Camellia Genus and species ( Camellia The method of ssp.) is also known in the art, for example in Mondal et al. , Plant Cell Rep .20:712-720 (2001). Any suitable method known to those skilled in the art can be used to transform tobacco cells using any of the nucleic acid molecules provided herein.

[0264] On the one hand, methods for introducing nucleic acid molecules into tobacco cells include AgrobacteriumMethods for introducing nucleic acid molecules into cells include PEG-mediated transformation, gene gun transformation, liposome-mediated transfection (liposome transfection), and lentiviral transfection.

[0265] lipid transfection For example As described in U.S. Patents 5,049,386, 4,946,787, and 4,897,355, and lipid transfection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Suitable receptors for effective recognition of polynucleotides in lipid transfection of cationic and neutral lipids include those in WO 91 / 17424 and WO 91 / 16024. Can be delivered to cells (…). For example, in vitro or ex vivo (application) or target tissue ( For example, in vivo (Application).

[0266] Any plant cell from which a fertile plant can be regenerated is considered a useful recipient cell for implementing this disclosure. Any tobacco cell from which a fertile tobacco plant can be regenerated is considered a useful recipient cell for implementing this disclosure. Camellia Any plant Camellia Cells are considered useful recipient cells for implementing this disclosure. In one aspect, a recombinant DNA construct is introduced into tobacco cells. In another aspect, a recombinant DNA construct is introduced into tobacco protoplast cells. In yet another aspect, a recombinant DNA construct is introduced into tobacco callus cells. In one aspect, a recombinant DNA construct is introduced into tobacco cells selected from the group consisting of: seed cells, fruit cells, leaf cells, cotyledon cells, hypocotyl cells, meristem cells, embryo cells, endosperm cells, root cells, bud cells, stem cells, flower cells, inflorescence cells, stalk cells, pedicel cells, style cells, stigma cells, receptacle cells, petal cells, sepal cells, pollen cells, anther cells, filament cells, ovary cells, ovule cells, pericarp cells, and phloem cells.

[0267] Callus can be initiated from various tissue sources, including but not limited to immature embryos or partial embryos, seedling apical meristems, microspores, etc. Cells capable of proliferating as callus can serve as recipient cells for transformation. Practical transformation methods and materials for preparing the transgenic plants disclosed herein (…) For exampleVarious culture media and recipient target cells, transformation of immature embryos and subsequent regeneration of fertile transgenic plants) are disclosed in, for example, U.S. Patent Nos. 6,194,636 and 6,232,526 and U.S. Application Publication 2004 / 0216189, all of which are incorporated herein by reference.

[0268] Leaf Level As used herein, the “USDA Leaf Grade Index” refers to the classification of leaf types based on group, quality, and color. On one hand, the USDA grade quality score is quantified as a 0-100 numerical representation of the grade, determined by a certified tobacco grader, and is a weighted average of all stem positions. A higher grade index indicates higher quality. As used below, “point” refers to each integer representation of the USDA leaf grade score. For example, the difference between a USDA leaf grade index score of 90 and 85 is 5 points.

[0269] Alternatively, leaf grade can be determined via hyperspectral imaging. See, for example, WO 2011 / 027315 (published on March 10, 2011, and incorporated herein by reference in its entirety).

[0270] As used herein, a “certified tobacco leaf grader” is a person trained to grade tobacco leaves according to USDA official standard grades as defined by the U.S. Department of Agriculture (USDA) (such as the Agricultural Marketing System as published in 7 CFR § 29). USDA leaf grade index scores can be assigned by employees, former employees, or individuals otherwise trained to grade tobacco leaves according to USDA official standard grades. An exemplary procedure for standard operating procedures in commercial inspection services begins with growers shipping tobacco to market, followed by arranging the tobacco in batches on flat baskets. Each batch is weighed and then inspected by a certified tobacco leaf grader. Following inspection, the grader assigns a grade to each batch, which becomes a certificate of grade indicating group, quality, and color. The procedures for grading experimental batches are similar; however, experimental tobacco is not brought to market or otherwise used for commercial purposes.

[0271] Tobacco grading is assessed based on factors including, but not limited to, petiole position, leaf size, leaf color, leaf uniformity and integrity, maturity, texture, elasticity, gloss (related to the intensity and depth of leaf coloration and brightness), hygroscopicity (the ability of tobacco leaves to absorb and retain moisture from the environment), and subtle differences in greenness or hue. For example, leaf grading can be determined using the official standard grading (7 USC §511) issued by the USDA Agricultural Marketing Service. See, for example,Official standard grades for Burley tobacco (U.S. Type 31 and Foreign Type 93), effective November 5, 1990 (55F.R.40645); Official standard grades for flue-cured tobacco (U.S. Types 11, 12, 13, 14 and Foreign Type 92), effective March 27, 1989 (54FR7925); Official standard grades for Pennsylvania seed tobacco (U.S. Type 41), effective January 8, 1965 (29FR16854); Official standard grades for Ohio cigar leaf tobacco (U.S. Types 42, 43, and 44), effective December 8, 1963 (28FR11719 and 28FR11719). FR11926); Official Standard Grading for Wisconsin Cigar Binder Tobacco (US Types 54 and 55), effective November 20, 1969 (34F.R.17061); Official Standard Grading for Wisconsin Cigar Binder Tobacco (US Types 54 and 55), effective November 20, 1969 (34 FR17061); Official Standard Grading for Georgia and Florida Shaded Cigar Wrapper Tobacco (US Type 62), effective April 1971. USDA grading index values ​​can be determined based on industry-accepted grading indices. See, for example Bowman et al., Tobacco Science , 32:39-40 (1988); Legacy Tobacco Document Library (Bates Document #523267826-523267833, July 1, 1988, Memorandum on the Proposed Burley Tobacco Grade Index); and Miller et al. 1990, Tobacco Intern. , 192:55-57 (All the aforementioned references are included in their full text by way of citation).

[0272] Unless otherwise stated, the leaf grade index values, alkaloid or nicotine levels of tobacco plants, varieties, cultivars or lines mentioned herein are average measurements, including, for example, the average of multiple leaves from a single plant or the average measurement from a population of tobacco plants of a single variety, cultivar or line. The determination of average measurements ( For example A population or collection of tobacco plants (or tobacco leaves, grading, alkaloid or nicotine levels) can be of any size, for example, 2, 5, 10, 15, 20, 25, 30, 35, 40, 50 or more. Use a population of at least 5 or more tobacco plants to determine the standard deviation. Follow industry-recognized standard protocols to determine the mean measurement or grading index value.

[0273] As used herein, “USDA grade leaf group,” “leaf group,” or “group” is a classification of types of grades based on certain characteristics related to stem location, body, or general quality. The group is the primary factor in USDA grading. Group determination is part of the grading process and is assigned by a certified tobacco leaf grader.

[0274] On the one hand, modified tobacco plants containing non-natural mutations have comparable or higher USDA leaf grade indices compared to control tobacco plants lacking non-natural mutations when grown under similar conditions. On the other hand, modified tobacco plants containing recombinant DNA constructs have comparable or higher USDA leaf grade indices compared to control tobacco plants lacking recombinant DNA constructs when grown under similar conditions.

[0275] As used in this article, a “comparable” USDA leaf grade index refers to the 15th percentile. For example, if a control plant has a USDA leaf grade index of 100, the comparable USDA leaf grade index will be between 85 and 100.

[0276] In one aspect, the modified tobacco plant contains a USDA leaf grade index that is at least 1% higher than that of the control tobacco plant when grown under similar growing conditions. In another aspect, the modified tobacco plant contains a USDA leaf grade index that is at least 5% higher than that of the control tobacco plant when grown under similar growing conditions. In another aspect, the modified tobacco plant contains a USDA leaf grade index that is at least 10% higher than that of the control tobacco plant when grown under similar growing conditions. In another aspect, the modified tobacco plant contains a USDA leaf grade index that is at least 20% higher than that of the control tobacco plant when grown under similar growing conditions. In another aspect, the modified tobacco plant contains a USDA leaf grade index that is at least 30% higher than that of the control tobacco plant when grown under similar growing conditions. In another aspect, the modified tobacco plant contains a USDA leaf grade index that is at least 40% higher than that of the control tobacco plant when grown under similar growing conditions. On the other hand, the modified tobacco plants contain a USDA leaf grade index that is at least 50% higher than that of the control tobacco plants when grown under similar growing conditions. On another hand, the modified tobacco plants contain a USDA leaf grade index that is at least 75% higher than that of the control tobacco plants when grown under similar growing conditions. On yet another hand, the modified tobacco plants contain a USDA leaf grade index that is at least 100% higher than that of the control tobacco plants when grown under similar growing conditions.

[0277] In one aspect, the modified tobacco plants contain a USDA leaf grade index that is 1% to 100% higher than that of the control tobacco plants grown under similar growing conditions. In another aspect, the modified tobacco plants contain a USDA leaf grade index that is 1% to 75% higher than that of the control tobacco plants grown under similar growing conditions. In another aspect, the modified tobacco plants contain a USDA leaf grade index that is 1% to 50% higher than that of the control tobacco plants grown under similar growing conditions. In another aspect, the modified tobacco plants contain a USDA leaf grade index that is 1% to 40% higher than that of the control tobacco plants grown under similar growing conditions. In another aspect, the modified tobacco plants contain a USDA leaf grade index that is 1% to 30% higher than that of the control tobacco plants grown under similar growing conditions. In another aspect, the modified tobacco plants contain a USDA leaf grade index that is 1% to 20% higher than that of the control tobacco plants grown under similar growing conditions. In one aspect, the modified tobacco plants contain a USDA leaf grade index that is 1% to 10% higher than that of the control tobacco plants grown under similar growing conditions. In another aspect, the modified tobacco plants contain a USDA leaf grade index that is 10% to 75% higher than that of the control tobacco plants grown under similar growing conditions. In another aspect, the modified tobacco plants contain a USDA leaf grade index that is 10% to 50% higher than that of the control tobacco plants grown under similar growing conditions. In yet another aspect, the modified tobacco plants contain a USDA leaf grade index that is 1% to 30% higher than that of the control tobacco plants grown under similar growing conditions.

[0278] In one aspect, the modified tobacco plant has a USDA leaf grade index that is at least 1 point higher than that of the control tobacco plant when grown under similar growing conditions. In another aspect, the modified tobacco plant has a USDA leaf grade index that is at least 2 points higher than that of the control tobacco plant when grown under similar growing conditions. In another aspect, the modified tobacco plant has a USDA leaf grade index that is at least 3 points higher than that of the control tobacco plant when grown under similar growing conditions. In another aspect, the modified tobacco plant has a USDA leaf grade index that is at least 4 points higher than that of the control tobacco plant when grown under similar growing conditions. In another aspect, the modified tobacco plant has a USDA leaf grade index that is at least 5 points higher than that of the control tobacco plant when grown under similar growing conditions. In another aspect, the modified tobacco plant has a USDA leaf grade index that is at least 6 points higher than that of the control tobacco plant when grown under similar growing conditions. On the other hand, the modified tobacco plants contain a USDA leaf grade index at least 7 points higher than that of the control tobacco plants grown under similar growing conditions. On the other hand, the modified tobacco plants contain a USDA leaf grade index at least 8 points higher than that of the control tobacco plants grown under similar growing conditions. On the other hand, the modified tobacco plants contain a USDA leaf grade index at least 9 points higher than that of the control tobacco plants grown under similar growing conditions. On the other hand, the modified tobacco plants contain a USDA leaf grade index at least 10 points higher than that of the control tobacco plants grown under similar growing conditions. On the other hand, the modified tobacco plants contain a USDA leaf grade index at least 11 points higher than that of the control tobacco plants grown under similar growing conditions. On the other hand, the modified tobacco plants contain a USDA leaf grade index at least 12 points higher than that of the control tobacco plants grown under similar growing conditions. On the other hand, the modified tobacco plants contain a USDA leaf grade index that is at least 13 points higher than that of the control tobacco plants when grown under similar growing conditions. On another hand, the modified tobacco plants contain a USDA leaf grade index that is at least 14 points higher than that of the control tobacco plants when grown under similar growing conditions. On another hand, the modified tobacco plants contain a USDA leaf grade index that is at least 15 points higher than that of the control tobacco plants when grown under similar growing conditions. On another hand, the modified tobacco plants contain a USDA leaf grade index that is at least 16 points higher than that of the control tobacco plants when grown under similar growing conditions.On the other hand, the modified tobacco plants contain a USDA leaf grade index that is at least 17 points higher than that of the control tobacco plants grown under similar growing conditions. On the other hand, the modified tobacco plants contain a USDA leaf grade index that is at least 18 points higher than that of the control tobacco plants grown under similar growing conditions. On the other hand, the modified tobacco plants contain a USDA leaf grade index that is at least 19 points higher than that of the control tobacco plants grown under similar growing conditions. On the other hand, the modified tobacco plants contain a USDA leaf grade index that is at least 20 points higher than that of the control tobacco plants grown under similar growing conditions. On the other hand, the modified tobacco plants contain a USDA leaf grade index that is at least 25 points higher than that of the control tobacco plants grown under similar growing conditions. On the other hand, the modified tobacco plants contain a USDA leaf grade index that is at least 30 points higher than that of the control tobacco plants grown under similar growing conditions. On the other hand, the modified tobacco plants contain a USDA leaf grade index that is at least 35 points higher than that of the control tobacco plants when grown under similar growing conditions. On another hand, the modified tobacco plants contain a USDA leaf grade index that is at least 40 points higher than that of the control tobacco plants when grown under similar growing conditions. On yet another hand, the modified tobacco plants contain a USDA leaf grade index that is at least 50 points higher than that of the control tobacco plants when grown under similar growing conditions.

[0279] On one hand, the modified tobacco plants have a USDA leaf grade index that is 1 to 100 points higher than that of the control tobacco plants grown under similar growing conditions. On the other hand, the modified tobacco plants have a USDA leaf grade index that is 1 to 75 points higher than that of the control tobacco plants grown under similar growing conditions. On another hand, the modified tobacco plants have a USDA leaf grade index that is 1 to 50 points higher than that of the control tobacco plants grown under similar growing conditions. On another hand, the modified tobacco plants have a USDA leaf grade index that is 1 to 25 points higher than that of the control tobacco plants grown under similar growing conditions. On another hand, the modified tobacco plants have a USDA leaf grade index that is 1 to 10 points higher than that of the control tobacco plants grown under similar growing conditions. On another hand, the modified tobacco plants have a USDA leaf grade index that is 1 to 5 points higher than that of the control tobacco plants grown under similar growing conditions. On the other hand, the modified tobacco plants have a USDA leaf grade index that is 10 to 50 points higher than that of the control tobacco plants when grown under similar growing conditions. On the other hand, the modified tobacco plants have a USDA leaf grade index that is 10 to 25 points higher than that of the control tobacco plants when grown under similar growing conditions.

[0280] On one hand, the comparable USDA leaf grade index between the modified tobacco plants and the control tobacco plants grown under similar growing conditions is within 1 point. On the other hand, the comparable USDA leaf grade index between the modified tobacco plants and the control tobacco plants grown under similar growing conditions is within 2 points. On the other hand, the comparable USDA leaf grade index between the modified tobacco plants and the control tobacco plants grown under similar growing conditions is within 3 points. On the other hand, the comparable USDA leaf grade index between the modified tobacco plants and the control tobacco plants grown under similar growing conditions is within 4 points. On the other hand, the comparable USDA leaf grade index between the modified tobacco plants and the control tobacco plants grown under similar growing conditions is within 5 points. On the other hand, the comparable USDA leaf grade index between the modified tobacco plants and the control tobacco plants grown under similar growing conditions is within 6 points. On the other hand, the comparable USDA leaf grade index between the modified tobacco plants and the control tobacco plants grown under similar growing conditions is within 7 points. On the other hand, the comparable USDA leaf grade index between the modified tobacco plants and the control tobacco plants grown under similar growing conditions is within 8 points. On the other hand, the comparable USDA leaf grade index between the modified tobacco plants and the control tobacco plants grown under similar growing conditions was within 9 points. On the other hand, the comparable USDA leaf grade index between the modified tobacco plants and the control tobacco plants grown under similar growing conditions was within 10 points. On the other hand, the comparable USDA leaf grade index between the modified tobacco plants and the control tobacco plants grown under similar growing conditions was within 11 points. On the other hand, the comparable USDA leaf grade index between the modified tobacco plants and the control tobacco plants grown under similar growing conditions was within 12 points. On the other hand, the comparable USDA leaf grade index between the modified tobacco plants and the control tobacco plants grown under similar growing conditions was within 13 points. On the other hand, the comparable USDA leaf grade index between the modified tobacco plants and the control tobacco plants grown under similar growing conditions was within 14 points. On the other hand, the comparable USDA leaf grade index between the modified tobacco plants and the control tobacco plants grown under similar growing conditions was within 15 points.

[0281] Aroma / Flavor Unless otherwise stated, when comparing the number of trichomes, trichome metabolite profiles, and / or trichome density between leaves from modified tobacco plants and leaves from control tobacco plants, it should be understood that the comparison must be made between leaves of the same age / growth stage. Leaves of any age or growth stage can be compared, provided they are identical. For example, V5 leaves from modified tobacco plants should be compared with V5 leaves from control tobacco plants. For clarification, it would be inappropriate, for the purposes of this disclosure, to compare the number and / or density of trichomes between newly emerging leaves and mature, senescent leaves.

[0282] Trichosome density can be measured by counting the number of trichomes present per unit area. Trichosome density can be determined using methods including, but not limited to, stereomicroscopes and image capture devices. For example Tools such as cameras and computer software are used. See For example, Mirnezami et al. , Appl Plant Sci. ,8(7):e11375(2020). Unless otherwise stated, the number of trichomes on a leaf can be measured by counting the total number of trichomes present on the entire leaf (including its petiole and blade (leaf)). The determination of the number of trichomes on a leaf can be performed using, but is not limited to, a stereomicroscope, an image capture device ( For example Tools such as cameras and computer software are used. See For example, Mirnezami et al. , Appl Plant Sci. ,8(7):e11375(2020).

[0283] In one respect, at least one leaf of the modified plant contains a greater average trichome density compared to the leaf of a control plant grown under comparable conditions. In another respect, the abaxial surface of at least one leaf of the modified plant contains a greater average trichome density compared to the abaxial surface of the leaf of a control plant grown under comparable conditions. In yet another respect, the adaxial surface of at least one leaf of the modified plant contains a greater average trichome density compared to the adaxial surface of the leaf of a control plant grown under comparable conditions.

[0284] In one respect, at least two leaves of the modified plant contain a greater average trichome density compared to two leaves of a control plant grown under comparable conditions. In another respect, most leaves of the modified plant contain a greater average trichome density compared to the same number of leaves of a control plant grown under comparable conditions. In yet another respect, all leaves of the modified plant contain a greater average trichome density compared to all leaves of a control plant grown under comparable conditions.

[0285] In one respect, at least one stem of the modified plant contains a greater average trichome density than the stem of a control plant grown under comparable conditions. In another respect, at least one flower of the modified plant contains a greater average trichome density than the flower of a control plant grown under comparable conditions. In another respect, at least one root of the modified plant contains a greater average trichome density than the root of a control plant grown under comparable conditions.

[0286] On the one hand, the modified plants had a higher average glandular trichome density compared to the control plants. On the other hand, the modified plants had a higher average non-glandular trichome density compared to the control plants.

[0287] In one respect, the modified tobacco plants presented herein contain similar levels of one or more tobacco aroma compounds compared to control tobacco plants grown under similar growth conditions. These one or more tobacco aroma compounds are selected from the group consisting of 3-methylvaleric acid, valeric acid, isovaleric acid, lysine, cephalosporin, glycol esters, and reducing sugars. As used herein, "similar" level means within 20%.

[0288] As used herein, “tobacco aroma compounds” are compounds associated with the flavor and aroma of tobacco smoke. These compounds include, but are not limited to, 3-methylvaleric acid, valeric acid, isovaleric acid, cephalosporin, and lisperanone diterpenes, as well as glycol esters. The concentration of tobacco aroma compounds can be measured by any metabolite analysis method known in the art, including but not limited to gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance spectroscopy, and liquid chromatography-mass spectrometry. See The Handbook of Plant Metabolomics, edited by Weckwerth and Kahl, (Wiley-Blackwell) (May 28, 2013).

[0289] As used herein, “reducing sugar” is any sugar (monosaccharide or polysaccharide) having a free or potentially free aldehyde or ketone group. Glucose and fructose act as nicotine buffers in cigarette smoke by lowering the smoke pH and effectively reducing the amount of “free” unprotonated nicotine. Reducing sugars balance tobacco flavor, for example, by altering the sensory effects of nicotine and other tobacco alkaloids. A negative correlation has been reported between sugar content and alkaloid content across different tobacco varieties, within the same variety, and within the same plant line, due to growing conditions. Reducing sugar levels can be measured using a segmented flow colorimetric assay, developed for the analysis of tobacco samples, adapted by Skalar Instrument Co. (West Chester, PA) and edited by Davis. Tobacco Science As described in 20:139-144 (1976). For example, the sample is dialyzed with sodium carbonate solution. A copper neocubic reagent is added to the sample and the solution is heated. The copper neocubic reagent chelate is reduced in the presence of sugar to produce a colored complex, which is measured at 460 nm.

[0290] Leaf surface biochemical substances: terpenes, alkaloids, alkaloid derivatives, benzene rings, flavonoids and phenylpropanoids Terpenes are a class of aromatic organic compounds produced by plants and some insects. Terpenes are members of the volatile unsaturated hydrocarbon group found in plant essential oils, based on the formula C2... 10 H 16 cyclic molecules; and related structures, such as, but not limited to, those having the formula C 15 H 24Terpenes are compounds of sesquiterpenes, or simple derivatives of such compounds. Terpenes are hydrocarbon molecules that plants often use to directly deter or attract herbivorous predators or parasites. Non-limiting examples of terpenes include citral, menthol, camphor, savelin A, and curcumin.

[0291] Pinene is a bicyclic monoterpene. Two naturally occurring structural isomers of pinene are known: α-pinene and β-pinene. Limonene is a monocyclic monoterpene and one of the two major compounds formed from pinene. Caryophyllene, especially β-caryophyllene, is a sesquiterpene common in many plants, including clove, cinnamon leaf, and black pepper. Linalool is an acyclic monoterpene, a key precursor in vitamin E formation, and has been isolated from many different plants. δ-3-carene is a bicyclic monoterpene. α-terpineol, terpinene-4-ol, and 4-terpineol are three closely related monoterpenes. Humulene is a sesquiterpene, also known as α-humulene.

[0292] On one hand, terpenes are terpenoids. Terpenoids (also called isoprene-like compounds) are modified terpenes containing additional functional groups, typically including oxygen. Terpenoids can be cyclic or acyclic, ranging in size from five-carbon hemiterpenes to long-chain complex molecules containing thousands of isoprene units. Terpenoids are formed by five-carbon isoprene units (… For example It is produced by the condensation of dimethylallyl diphosphate (DMAPP) and isoprenyl diphosphate (IPP), most commonly via a sequential head-to-tail addition of DMAPP to IPP. The initial cyclization process is catalyzed by different terpene synthases, and enzyme variation leads to variation in the monoterpene structure.

[0293] Terpenes are classified according to the number of isoprene units that make up the parent terpene. Hemiterpenes contain one isoprene unit. Monoterpenes contain two isoprene units. Sesquiterpenes contain three isoprene units. Diterpenes contain four isoprene units. Disesquiterpenes contain five isoprene units. Triterpenes contain six isoprene units. Tetraterpenes contain eight isoprene units. Polyterpenes contain more than eight isoprene units.

[0294] In one respect, polypeptides participate in the biosynthesis of at least one terpene. In another respect, polypeptides participate in the biosynthesis of at least one terpene compound. In another respect, polypeptides participate in the biosynthesis of at least one terpene compound selected from the group consisting of sesquiterpenes, monoterpenes, sesquiterpenes, diterpenes, disesquiterpenes, triterpenes, tetraterpenes, and polyterpenes. In yet another respect, polypeptides participate in the biosynthesis of at least one terpene selected from the group consisting of sesquiterpenes, monoterpenes, sesquiterpenes, diterpenes, disesquiterpenes, triterpenes, tetraterpenes, and polyterpenes.

[0295] In one aspect, the tobacco plant disclosed herein comprises, at increased levels, one or more terpenes selected from the group consisting of 3-hydroxy-3-methylglutaric acid, mevalonic acid, cis-trans-farnesol, carotene diol (1), carotene diol (2), carotene diol (3), β-cryptoxanthin, and geranylide. In another aspect, the tobacco plant disclosed herein comprises, at increased levels, one or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more terpenes selected from the group consisting of 3-hydroxy-3-methylglutaric acid, mevalonic acid, cis-trans-farnesol, carotene diol (1), carotene diol (2), carotene diol (3), β-cryptoxanthin, and geranylide. In one aspect, the tobacco plant disclosed herein comprises, at reduced levels, one or more terpenes selected from the group consisting of 3-hydroxy-3-methylglutaric acid, mevalonic acid, cis-trans-farnesol, carotene diol (1), carotene diol (2), carotene diol (3), β-cryptoxanthin, and geranylide. In another aspect, the tobacco plant disclosed herein comprises, at reduced levels, one or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more terpenes selected from the group consisting of 3-hydroxy-3-methylglutaric acid, mevalonic acid, cis-trans-farnesol, carotene diol (1), carotene diol (2), carotene diol (3), β-cryptoxanthin, and geranylide.

[0296] In one aspect, the tobacco plant disclosed herein contains one or more alkaloids selected from the group consisting of cotinine, 2-hydroxypyridine, nornicotinine, neonicotinine, nicotine, tyrosol, and rhodioloside at increased levels. In another aspect, the tobacco plant disclosed herein contains one or more, two or more, three or more, four or more, five or more, six or more, or seven or more alkaloids selected from the group consisting of cotinine, 2-hydroxypyridine, nornicotinine, neonicotinine, nicotine, tyrosol, and rhodioloside at increased levels. In another aspect, the tobacco plant disclosed herein contains one or more alkaloids selected from the group consisting of cotinine, 2-hydroxypyridine, nornicotinine, neonicotinine, nicotine, tyrosol, and rhodioloside at decreased levels. In yet another aspect, the tobacco plant disclosed herein contains one or more, two or more, three or more, four or more, five or more, six or more, or seven or more alkaloids selected from the group consisting of cotinine, 2-hydroxypyridine, nornicotinine, neonicotinine, nicotine, tyrosol, and rhodioloside at decreased levels.

[0297] In one aspect, the tobacco plant disclosed herein comprises one or more alkaloid derivatives selected from the group consisting of 3-pyridylacetic acid, nicotine-N-oxide, nocotinine, 6-hydroxynicotinine (1), and 6-hydroxynicotinine (2) at increased levels. In another aspect, the tobacco plant disclosed herein comprises one or more, two or more, three or more, four or more, or five or more alkaloid derivatives selected from the group consisting of 3-pyridylacetic acid, nicotine-N-oxide, nocotinine, 6-hydroxynicotinine (1), and 6-hydroxynicotinine (2) at increased levels. In another aspect, the tobacco plant disclosed herein comprises one or more alkaloid derivatives selected from the group consisting of 3-pyridylacetic acid, nicotine-N-oxide, nocotinine, 6-hydroxynicotinine (1), and 6-hydroxynicotinine (2) at decreased levels. In one aspect, the tobacco plant disclosed herein contains one or more, two or more, three or more, four or more or five or more alkaloid derivatives selected from the group consisting of 3-pyridylacetic acid, nicotine-N-oxide, norcotinine, 6-hydroxynicotine (1) and 6-hydroxynicotine (2) at reduced levels.

[0298] In one aspect, the tobacco plant disclosed herein comprises one or more benzene rings selected from the group consisting of 2,4,6-trihydroxybenzoic acid, 2,6-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, benzoic acid, gentianic acid-5-glucoside, hydroquinone β-glucopyranoside, protocatechuic acid-3-glucoside, salicylic acid, salicyluronic acid glucuronide, and salicylic acid-glucoside. In one aspect, the tobacco plant disclosed herein comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or eleven or more benzene rings selected from the group consisting of 2,4,6-trihydroxybenzoic acid, 2,6-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, benzoic acid, gentianic acid-5-glucoside, hydroquinone β-glucopyranoside, protocatechuic acid-3-glucoside, salicylic acid, salicyluronic acid glucuronide, and salicylic acid-glucoside. In one aspect, the tobacco plant disclosed herein comprises one or more benzene rings selected from the group consisting of 2,4,6-trihydroxybenzoic acid, 2,6-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, benzoic acid, gentianic acid-5-glucoside, hydroquinone β-glucopyranoside, protocatechuic acid-3-glucoside, salicylic acid, salicyluronic acid glucuronide, and salicylic acid-glucoside. In one aspect, the tobacco plant disclosed herein comprises, at reduced levels, one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or eleven or more benzene rings selected from the group consisting of 2,4,6-trihydroxybenzoic acid, 2,6-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, benzoic acid, gentianic acid-5-glucoside, hydroquinone β-glucopyranoside, protocatechuic acid-3-glucoside, salicylic acid, salicyluronic acid glucuronide, and salicylic acid-glucoside.

[0299] In one aspect, the tobacco plant disclosed herein contains one or more flavonoids selected from the group consisting of dihydrokaempferol, sennaol, dihydroquercetin, naringenin, quercetin, quercetin 3-glucoside, rutin (quercetin 3-rutin), 3-methoxyapigenin, kaempferol 3-O-glucoside / galactoside and isorhamnetin 3-rutin. In one aspect, the tobacco plant disclosed herein contains one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more flavonoids selected from the group consisting of dihydrokaempferol, sennaol, dihydroquercetin, naringenin, quercetin, quercetin 3-glucoside, rutin (quercetin 3-rutin), 3-methoxyapigenin, kaempferol 3-O-glucoside / galactoside and isorhamnetin 3-rutin. In one aspect, the tobacco plant disclosed herein contains reduced levels of one or more flavonoids selected from the group consisting of dihydrokaempferol, sennaol, dihydroquercetin, naringenin, quercetin, quercetin 3-glucoside, rutin (quercetin 3-rutin), 3-methoxyapigenin, kaempferol 3-O-glucoside / galactoside and isorhamnetin 3-rutin. In one aspect, the tobacco plant disclosed herein contains one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more flavonoids selected from the group consisting of dihydrokaempferol, sennaol, dihydroquercetin, naringenin, quercetin, quercetin 3-glucoside, rutin (quercetin 3-rutin), 3-methoxyapigenin, kaempferol 3-O-glucoside / galactoside and isorhamnetin 3-rutin.

[0300] As used in this article, the term "biosynthesis" refers to the production of complex molecules within plants or plant cells. For example (Unrestricted, terpenes or terpenoids). To "participate" in the biosynthesis of a compound, the polypeptide can directly interact with the substrate during the biosynthesis of that compound, or the polypeptide can affect (positively or negatively) the expression of polypeptides that directly interact with the substrate. For example (Transcription factors that promote the expression of enzymes that convert substrates into new forms or repressors that inhibit the expression of enzymes that convert substrates into new forms).

[0301] On one hand, peptides participate in the biosynthesis of hemiterpenes. On one hand, peptides participate in the biosynthesis of hemiterpenoids. On one hand, peptides participate in the biosynthesis of monoterpenes. On one hand, peptides participate in the biosynthesis of monoterpenoids. On one hand, peptides participate in the biosynthesis of sesquiterpenes. On one hand, peptides participate in the biosynthesis of sesquiterpenoids. On one hand, peptides participate in the biosynthesis of diterpenes. On one hand, peptides participate in the biosynthesis of diterpenoids. On one hand, peptides participate in the biosynthesis of disesquiterpenes. On one hand, peptides participate in the biosynthesis of disesquiterpenoids. On one hand, peptides participate in the biosynthesis of triterpenes. On one hand, peptides participate in the biosynthesis of triterpenoids. On one hand, peptides participate in the biosynthesis of tetraterpenes. On one hand, peptides participate in the biosynthesis of polyterpenoids. On one hand, peptides participate in the biosynthesis of monoterpenes. On one hand, peptides participate in the biosynthesis of polyterpenoids.

[0302] Terpene synthase (TPS) genes can be divided into seven evolutionary clades: TPS-a, TPS-b, TPS-c, TPS-d, TPS-e / f, TPS-g, and TPS-h. TPS-a, TPS-b, and TPS-g are limited to angiosperms, while TPS-d and TPS-h are found in gymnosperms and lycophytes. Selaginella moellendorffii ( Selaginalla moellendorffii This is unique to them. The TPS-a evolutionary branch mainly contains sesquiterpene synthases and diterpene synthases, while the TPS-b and TPS-g evolutionary branches mainly contain monoterpene synthases.

[0303] Terpenoid synthase-like genes In one aspect, the polypeptide involved in the biosynthesis of at least one terpene compound is a member of the TPS-a evolutionary branch. In another aspect, the polypeptide involved in the biosynthesis of at least one terpene compound is a member of the TPS-b evolutionary branch. In another aspect, the polypeptide involved in the biosynthesis of at least one terpene compound is a member of the TPS-c evolutionary branch. In another aspect, the polypeptide involved in the biosynthesis of at least one terpene compound is a member of the TPS-e / f evolutionary branch. In another aspect, the polypeptide involved in the biosynthesis of at least one terpene compound is a member of the evolutionary branch selected from the group consisting of TPS-a, TPS-b, TPS-c, TPS-e / f, and TPS-g.

[0304] On one hand, the terpene is menthol. On another hand, the terpene is menthol or a related compound. On another hand, the terpene is lysine diterpene. On another hand, the terpene is cepstrienyldiol. On another hand, the terpene is L-piperidine. On another hand, the terpene is neophytadiene. On another hand, the lysine diterpene is cis-fiberol. On another hand, the lysine diterpene is lysine diol. On another hand, the terpene is selected from the group consisting of menthol or a related compound, lysine diterpene, cepstrienyldiol, L-piperidine, and L-leucine. On another hand, the terpene is selected from the group consisting of menthol or a related compound, lysine diterpene, cepstrienyldiol, L-piperidine, L-leucine, and neophytadiene. On another hand, the terpene is selected from the group consisting of menthol or a related compound, lysine diterpene, cepstrienyldiol, L-piperidine, and L-leucine. On the one hand, the terpenes are selected from the group consisting of menthol, lysine diterpenes, cephalindiol, L-piperidine, L-leucine and neophytadiene.

[0305] On one hand, the terpene is myrcene. On another hand, the terpene is pinene. On another hand, the terpene is limonene. On another hand, the terpene is caryophyllene. On another hand, the terpene is linalool. On another hand, the terpene is terpinene. On another hand, the terpene is camphene. On another hand, the terpene is terpineol. On another hand, the terpene is phellandrene. On another hand, the terpene is carene. On another hand, the terpene is humulene. On another hand, the terpene is menthone. On another hand, the terpene is geraniol.

[0306] As used in this article, "menthol" refers to the substance with the chemical formula C. 10 H 20 The organic compound 5-methyl-2-(prop-2-yl)cyclohexane-1-ol, as designated by the International Union of Pure and Applied Chemistry (IUPAC). Menthol is also known as "(-)-menthol". Related compounds of menthol include, but are not limited to, (+)-menthol, (+)-isomenthol, (+)-neomenthol, (+)-neoisomenthol, (-)-isomenthol, (-)-neomenthol, and (-)-neoisomenthol. In one aspect, related compounds of menthol are selected from the group consisting of (+)-menthol, (+)-isomenthol, (+)-neomenthol, (+)-neoisomenthol, (-)-isomenthol, (-)-neomenthol, and (-)-neoisomenthol.

[0307] As used in this article, "neophydiene" refers to a substance with the chemical formula C 20 H 38 Organic compounds with the IUPAC name 7,11,15-trimethyl-3-methylenehexadecyl-1-ene.

[0308] As used in this article, "cemistrienediol" refers to a substance with the chemical formula C 20 H 34O2 and IUPAC names (1 R ,3 R 4 Z 8 Z ,12 S ,13 Z Cesartrienil is an organic compound of 1,5,9-trimethyl-12-propyl-2-ylcyclotetradecano-4,8,13-trien-1,3-diol. Cesartrienil is also known as "β-cesartrienil".

[0309] As used in this article, "L-pimolic acid" refers to a substance with the chemical formula C 20 H 30 O2 and IUPAC names (1 R 4 aR 4 bS 10 aR )-1,4 a -Dimethyl-7-propyl-2-yl-2,3,4,4 b ,5,9,10,10 a -Octahydrophenanthrene-1-carboxylic acid is an organic compound. L-piperidine is also known as "L-piperidine". In one aspect, L-piperidine is involved in the production of diterpene resin acids.

[0310] As used in this article, "L-leucine" refers to the substance with the chemical formula C6H4H2O. 12 NO2 and IUPAC names (2) S )-2-amino-4-methylvaleric acid is an amino acid.

[0311] As used in this article, "Lipastatine diterpenes" refers to terpenoid derivatives of the basic parent compound lipastatine diterpenes (a type of diterpenes). Lipastatine diterpenes have the chemical formula C1. 20 H 38 and IUPAC name (1 S ,2 S 4 aS 8 aR )-2,5,5,8 a -Tetramethyl-1-[(3 R [-3-methylpentyl]-1,2,3,4,4 a ,6,7,8-octahydronaphthalene.

[0312] A non-limiting example of a limonene diterpenoid is cis-haystol. As used herein, "cis-haystol" refers to a substance having the chemical formula C1. 20 H 34 O and IUPAC names (1 R ,2 R 4 aS 8 aS )-2,5,5,8 a -Tetramethyl-1-[(2Z )-3-methylpentan-2,4-dienyl]-3,4,4 a ,6,7,8-hexahydro-1 H 2-Naphthalene-2-ol is an organic compound.

[0313] As used in this article, "myrcene" refers to a substance with the chemical formula C 10 H 16 The compound with the IUPAC name 7-methyl-3-methylene-oct-1,6-diene. Myrcene, also known as β-myrcene or α-myrcene, belongs to the class of organic compounds called acyclic monoterpenes. Therefore, myrcene is considered an isoprene-like lipid molecule.

[0314] As used in this article, "pinene" refers to a substance with the chemical formula C 10 H 16 The compound with the IUPAC name (1S,5S)-2,6,6-trimethylbicyclo[3.1.1]hept-2-ene ((-)-α-pinene). (+)-α-pinene, also known as 2-pinene or acinte α, belongs to a class of organic compounds called bicyclic monoterpenes. These are monoterpenes containing exactly two rings that are fused together. α-pinene is a bicyclic [3.1.1]hept-2-ene in which methyl groups are substituted at positions 2, 6, and 6, respectively. It functions as a plant metabolite.

[0315] As used in this article, "limonene" refers to the compound with the chemical formula C. 10 H 16 The compound with the IUPAC name 1-methyl-4-(1-methylvinyl)-cyclohexene. (+ / -)-Limonene is a racemic mixture of limonene (a natural cyclic monoterpene).

[0316] As used in this article, "caryophyllene" or "β-caryophyllene" refers to caryophyllene with the chemical formula C 15 H 24 The organic compound with the IUPAC name (1R,4E,9S)-4,11,11-trimethyl-8-methylenebicyclo[7.2.0]undec-4-ene. β-Caryophyllene is classified as a sesquiterpene. (-)-β-Caryophyllene is a β-caryophyllene in which the stereocenter adjacent to the exocyclic double bond has an S configuration, while the remaining stereocenters have an R configuration. It is the most common form of β-caryophyllene and is found in many essential oils.

[0317] As used herein, "linalool" refers to compounds having the chemical formula C10H18O and the IUPAC name 3,7-dimethyloctyl-1,6-dien-3-ol. β-Linalool, also known as 3,7-dimethyl-1,6-octadien-3-ol or 2,6-dimethyloctyl-2,7-dien-6-ol, is classified as an acyclic monoterpene.

[0318] As used in this article, "terpinene" refers to a substance with the chemical formula C 10 H 16 Organic compounds with the IUPAC name 1-methyl-4-prop-2-ylcyclohexene. Terpinene, also known as α-terpinene or isoterpinene, is classified as a menthol monoterpene. These are monoterpenoid compounds with a structure based on an ortho-, meta-, or para-mentholane backbone.

[0319] As used in this article, "camphene" refers to the substance with the chemical formula C 10 H 16 Organic compounds with the IUPAC name 2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane. Camphene is classified as a bicyclic monoterpene, comprising two rings fused together. Camphene is a monoterpene with a bicyclic skeleton consisting of bicyclic [2.2.1]heptane with a geminal dimethyl substituted at position 2 and a methylene substituted at position 3.

[0320] As used herein, "terpineol" refers to monoterpene alcohols, which exist as four isomers: α-terpineol, β-terpineol, γ-terpineol, and terpinene-4-ol. β-terpineol and γ-terpineol differ only in the position of their double bonds. Terpineols are typically mixtures of these isomers, with α-terpineol being the predominant component. As used herein, "α-terpineol" refers to alcohols with the chemical formula C 10 H 18 O and IUPAC name 2-(4-methylcyclohexyl-3-en-1-yl)prop-2-ol is an organic compound.

[0321] As used herein, "phellandrene" refers to the cyclic monoterpene "α-phellandrene" or "β-phellandrene". As used herein, "α-phellandrene" refers to the cyclic monoterpene with the chemical formula C1. 10 H 16 α-Celphine is an organic compound with the IUPAC name 2-methyl-5-propyl-2-ylcyclohex-1,3-diene. α-Celphine is also known as α-phellandrene or dihydro-p-cymene. As used herein, "β-Celphine" refers to the compound with the chemical formula C2... 10 H 16 It is an organic compound with the IUPAC name 3-methylene-6-prop-2-ylcyclohexene. β-Celphine is also known as β-phellandrene or 2-peminadiene.

[0322] As used in this article, "carene" or "α-carene" refers to a substance with the chemical formula C 10 H 16 Organic compounds with the IUPAC name 3,7,7-trimethylbicyclo[4.1.0]hept-3-ene. α-carene, also known as 3-carene or δ-carene-3-ene, is classified as a bicyclic monoterpene.

[0323] As used in this article, "humulene" refers to a substance with the chemical formula C 15 H 24 Organic compounds with the IUPAC name (1E,4E,8E)-2,6,6,9-tetramethylcycloundec-1,4,8-triene.

[0324] As used in this article, "menthol" refers to a substance with the chemical formula C. 10 H 16 O and IUPAC name (5R)-5-methyl-2-prop-2-ylidenecyclohex-1-one. (+)-Menthone, also known as D-Menthone or cis-isomenthone, is classified as a menthane monoterpene.

[0325] As used in this article, "geraniol" refers to the substance with the chemical formula C 10 H 18 Geraniol is an organic compound with the IUPAC name (2E)-3,7-dimethyloctyl-2,6-dien-1-ol. Geraniol is an acyclic monoterpene compound, also known as 2E-geraniol, (e)-geraniol, or geraniyl alcohol.

[0326] On one hand, the polypeptide is geraniol-geraniol diphosphate synthase. On another hand, the polypeptide is 8-hydroxy-cobamic acid diphosphate synthase. On another hand, the polypeptide is cis-firol synthase. On another hand, the polypeptide is cephalotrinol synthase 2a. On another hand, the polypeptide is L-pinediene synthase. On another hand, the polypeptide is 2-isopropylmalate synthase. On another hand, the polypeptide is 2-oxoisovalerate dehydrogenase. On another hand, the polypeptide is geraniol diphosphate synthase. On another hand, the polypeptide is limonene synthase. On another hand, the polypeptide is limonene 3-hydroxylase. On another hand, the polypeptide is isopistenol dehydrogenase. On another hand, the polypeptide is menthone reductase. On another hand, the polypeptide is mentholatum synthase. On another hand, the polypeptide is neomenthol dehydrogenase. On another hand, the polypeptide is geraniol-geraniol pyrophosphate synthase. On another hand, the polypeptide is collavinyl diphosphate synthase. On another hand, the polypeptide is solanesyl diphosphate synthase. On another hand, the polypeptide is terpene synthase. On one hand, the polypeptide is 2-enal reductase. On another hand, the polypeptide is gemmaene synthase. On yet another hand, the polypeptide is cytochrome P450. On yet another hand, the polypeptide is carvacrol dehydrogenase. On yet another hand, the polypeptide is MYB61 transcription factor.

[0327] On one hand, the polypeptide was selected from the group consisting of geraniol diphosphate synthase (GDP), limonene synthase (LS), limonene 3-hydroxylase (L3OH), isopistinol dehydrogenase (IPD), menthol reductase, menthol furan synthase and GGPPS neomenthol dehydrogenase (NtNMD).

[0328] On one hand, the peptides were selected from the group consisting of geraniol geraniol diphosphate synthase (GGPPS2), 8-hydroxy-cobamic acid diphosphate synthase and cis-fiberol synthase.

[0329] On one hand, the polypeptides were selected from the group consisting of geraniol geraniol diphosphate synthase, 8-hydroxy-cobamic acid diphosphate synthase, cis-firol synthase, cephalothinol synthase 2a, L-pinediene synthase, 2-isopropylmalate synthase, 2-oxoisovalerate dehydrogenase, geraniol diphosphate synthase, limonene synthase, limonene 3-hydroxylase, isopistinol dehydrogenase, menthol reductase, menthol furan synthase, neomenthol dehydrogenase, leaf protein, and tofu spathiberene oxygenase.

[0330] On one hand, the polypeptide was selected from the group consisting of geraniol pyrophosphate synthase, collavinyl diphosphate synthase, solanesyl diphosphate synthase, terpene synthase, limonene synthase, isopirose enol dehydrogenase, 2-enal reductase, gemmaene synthase, 2-isopropylmalate synthase, and MYB61.

[0331] On one hand, the polypeptide was selected from the group consisting of geraniol geraniol diphosphate synthase, 8-hydroxy-cobamic acid diphosphate synthase, cis-firol synthase, cephalothinol synthase 2a, L-pinediene synthase, 2-isopropylmalate synthase, 2-oxoisovalerate dehydrogenase, geraniol diphosphate synthase, limonene synthase, limonene 3-hydroxylase, isopistinol dehydrogenase, menthol reductase, menthol furan synthase, neomenthol dehydrogenase, foliar protein, tofu scintillans diphosphate oxygenase, geraniol geraniol pyrophosphate synthase, collavinyl diphosphate synthase, solanesyl diphosphate synthase, terpene synthase, 2-enal reductase, gemmaene synthase, and MYB61.

[0332] On one hand, modified plants, seeds, or plant parts containing the recombinant nucleic acid provided herein contain an increased amount of at least one terpene compared to control plants, seeds, or plant parts lacking the recombinant nucleic acid molecule when grown under similar growth conditions. On the other hand, modified tobacco or... Camellia Plants, tobacco or Camellia Seeds or parts of tobacco plants compared with control tobacco plants lacking this recombinant nucleic acid molecule grown under similar conditions. Camellia Plants, tobacco or Camellia Seeds or tobacco or CamelliaThe plant portion contains an increased amount of at least one terpene compared to the plant portion.

[0333] In one aspect, the increased amount of at least one terpene comprises an increase of at least 0.5%. In one aspect, the increased amount of at least one terpene comprises an increase of at least 1%. In one aspect, the increased amount of at least one terpene comprises an increase of at least 2%. In one aspect, the increased amount of at least one terpene comprises an increase of at least 3%. In one aspect, the increased amount of at least one terpene comprises an increase of at least 4%. In one aspect, the increased amount of at least one terpene comprises an increase of at least 5%. In one aspect, the increased amount of at least one terpene comprises an increase of at least 10%. In one aspect, the increased amount of at least one terpene comprises an increase of at least 12.5%. In one aspect, the increased amount of at least one terpene comprises an increase of at least 15%. In one aspect, the increased amount of at least one terpene comprises an increase of at least 17.5%. In one aspect, the increased amount of at least one terpene comprises an increase of at least 20%. In one aspect, the increased amount of at least one terpene comprises an increase of at least 25%. In one aspect, the increased amount of at least one terpene comprises an increase of at least 30%. In one aspect, the increased amount of at least one terpene comprises an increase of at least 40%. In one aspect, the increased amount of at least one terpene comprises an increase of at least 50%. In one aspect, the increased amount of at least one terpene comprises an increase of at least 60%. In another aspect, the increased amount of at least one terpene comprises an increase of at least 70%. In another aspect, the increased amount of at least one terpene comprises an increase of at least 80%. In another aspect, the increased amount of at least one terpene comprises an increase of at least 90%. In another aspect, the increased amount of at least one terpene comprises an increase of at least 100%. In another aspect, the increased amount of at least one terpene comprises an increase of at least 150%. In another aspect, the increased amount of at least one terpene comprises an increase of at least 200%. In another aspect, the increased amount of at least one terpene comprises an increase of at least 250%. In another aspect, the increased amount of at least one terpene comprises an increase of at least 500%.

[0334] In one aspect, the increased amount of at least one terpene comprises an increase of 0.5% to 500%. In one aspect, the increased amount of at least one terpene comprises an increase of 0.5% to 250%. In one aspect, the increased amount of at least one terpene comprises an increase of 0.5% to 100%. In one aspect, the increased amount of at least one terpene comprises an increase of 0.5% to 75%. In one aspect, the increased amount of at least one terpene comprises an increase of 0.5% to 50%. In one aspect, the increased amount of at least one terpene comprises an increase of 0.5% to 25%. In one aspect, the increased amount of at least one terpene comprises an increase of 0.5% to 10%. In one aspect, the increased amount of at least one terpene comprises an increase of 0.5% to 5%. In one aspect, the increased amount of at least one terpene comprises an increase of 0.5% to 50%. In one aspect, the increased amount of at least one terpene comprises an increase of 0.5% to 500%. In one aspect, the increased amount of at least one terpene comprises an increase of 5% to 250%. In one aspect, the increased amount of at least one terpene comprises an increase of 5% to 100%. In one aspect, the increased amount of at least one terpene comprises an increase of 5% to 50%. In one aspect, the increased amount of at least one terpene comprises an increase of 25% to 500%. In another aspect, the increased amount of at least one terpene comprises an increase of 25% to 250%. In another aspect, the increased amount of at least one terpene comprises an increase of 50% to 100%. In another aspect, the increased amount of at least one terpene comprises an increase of 100% to 500%.

[0335] Terpenes can be extracted using any method known in the art, such as solvent extraction. See For example, Jiang et al. , Curr Protoc Plant Biol ., 1:345-358 (2016). Solvent-free extraction methods are also known in the art. See See For example, Yang and Xie, Science Direct , 162: 135-139 (2006).

[0336] The amount of terpenes in plants can be measured using any method known in the art, including, without limitation, gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance spectroscopy, and liquid chromatography-mass spectrometry. See The Handbook of Plant Metabolomics, edited by Weckwerth and Kahl, (Wiley-Blackwell) (May 28, 2013). In one respect, the amount of at least one terpene refers to the concentration of that terpene in the sampled tissue.

[0337] In one aspect, the modified tobacco plant provided herein further comprises a recombinant nucleic acid molecule containing a nucleic acid sequence encoding a polypeptide operatively linked to a heterologous promoter, wherein the polypeptide causes an increase in mean trichome density compared to control tobacco plants lacking the transgene or modification when grown under similar growth conditions. Non-limiting examples of genes that can be overexpressed to increase mean trichome density include... NtGIS , NtMYB86 , NbGIS .

[0338] TSNA On the one hand, the provided modified tobacco plant further contains, compared with control tobacco plants lacking one or more mutations in one or more loci encoding nicotine demethylase when grown under similar growth conditions, nicotine demethylase (…). For example, CYP82E4 , CYP82E5 , CYP82E10 One or more mutations at one or more loci of nicotinic acid (Nicotinic acid) confer a reduced amount of Nicotinic acid (Nicotinic acid). See (US Patent Nos. 8,319,011; 8,124,851; 9,187,759; 9,228,194; 9,228,195; 9,247,706). In one aspect, the modified tobacco plant further comprises reduced nicotine demethylase activity compared to control plants grown and processed under similar growth conditions. In another aspect, the provided tobacco plant further comprises one or more mutants or transgenics that provide elevated levels of one or more antioxidants. See (See U.S. Patent Application Publication Nos. 2018 / 0119163 and WO 2018 / 067985). In another aspect, the provided tobacco plant further comprises one or more mutants or transgenics providing reduced levels of one or more tobacco-specific nitrosamines (TSNAs). In one aspect, the TSNA is selected from the group consisting of N'-nitrosonicotinamide (NNN), 4-methylnitrosoamino-1-(3-pyridyl)-1-butanone (NNK), N'-nitrosoneonicotinamide (NAT), and N'-nitrosoneonicotinamide (NAB).

[0339] TSNA reduction Alkaloids are complex nitrogen-containing compounds that exist naturally in plants and have pharmacological effects in humans and animals. Nicotine is the main natural alkaloid in commercial cigarette tobacco and accounts for a significant portion of the total alkaloid content. Red Flower Smoke Approximately 90% of the alkaloids in tobacco are alkaloids. Other major alkaloids in tobacco include cotinine, nornicotine, mesmin, dienicotine, neonicotine, and neonicotinoid. Minor tobacco alkaloids include n-oxonicotine, N-methylneonicotine, N-methylneonicotine, pseudooxonicotine, and 2,3-bipyridine, etc.

[0340] Modified tobacco plants or portions thereof containing recombinant DNA constructs, wherein the levels of alkaloids operatively linked to a heterologous promoter can be determined by methods known in the art, such as quantification based on gas-liquid chromatography, high-performance liquid chromatography, radioimmunoassay, and enzyme-linked immunosorbent assay. For example, nicotine alkaloid levels can be measured using a GC-FID method based on CORESTA Recommendation No. 7, 1987 and ISO standard (ISO TC 126N 394 E). Regarding the use of gas-liquid chromatography equipped with a capillary column and an FID detector, See also , Hibi et al. , Plant Physiology 100: 826-35 (1992).

[0341] Alternatively, segmented flow colorimetry can be used to measure total alkaloids in tobacco. This segmented flow colorimetry was developed for the analysis of tobacco samples, as adapted by Skalar Instrument Co. (West Chester, PA) and developed by Collins. et al. , Tobacco Science As described in 13:79-81 (1969). In short, the tobacco sample was dried, ground, and extracted before the analysis of total alkaloids and reducing sugars. The method then employed acetic acid / methanol / water extraction and charcoal decolorization. The determination of total alkaloids was based on the reaction of cyanogen chloride with nicotine alkaloids in the presence of aromatic amines to form a colored complex (which was measured at 460 nm).

[0342] On the one hand, liquid chromatography with tandem mass spectrometry (LC / MS / MS) was used to measure the level of total TSNA or individual TSNA based on freeze-dried modulated leaf samples.

[0343] As used in this article, "marker assay" refers to a method that uses a single approach to detect markers at gene loci. For example Measure at least one phenotype (such as visually detectable traits, including red meat and precocity), restriction fragment length polymorphism (RFLP), single base extension, electrophoresis, sequence alignment, allele-specific oligonucleotide hybridization (ASO), random amplified polymorphic DNA (RAPD), microarray-based techniques, PCR-based techniques, and nucleic acid sequencing techniques.

[0344] As used herein, “polymorphism” refers to the presence of one or more variations in a population. Polymorphism can manifest as variations in the nucleotide sequence of nucleic acids or variations in the amino acid sequence of proteins. Polymorphism includes the presence of one or more nucleic acid sequence variations or nucleic acid features at one or more loci in a population of one or more individuals. This variation may include, but is not limited to, one or more nucleotide base changes, one or more nucleotide insertions, or one or more nucleotide deletions. Polymorphism can arise from random processes during nucleic acid replication, through mutagenesis, as a result of mobile genomic elements, from copy number variations, and during meiosis, such as unequal crossing over, genome replication, and chromosome breakage and fusion. This variation may be common or may be present at a low frequency within a population; the former has greater utility in general plant breeding, while the latter may be associated with rare but important phenotypic variations. Useful polymorphisms may include single nucleotide polymorphisms (SNPs), insertions or deletions (Indels) in DNA sequences, simple sequence repeats (SSRs) in DNA sequences, restriction fragment length polymorphisms (RFLPs), and tag SNPs. Genetic markers, genes, DNA-derived sequences, RNA-derived sequences, promoters, the 5' untranslated region of a gene, the 3' untranslated region of a gene, microRNAs, siRNAs, tolerance loci, satellite markers, transgenes, mRNA, ds mRNA, transcriptomic profiles, and methylation patterns can also contain polymorphisms. Furthermore, the presence, absence, or copy number variations of the aforementioned components can also contain polymorphisms.

[0345] As used in this article, "SNP" or "single nucleotide polymorphism" refers to a sequence variation that occurs when a single nucleotide (A, T, C, or G) in a genome sequence is altered or changed. When an SNP is located at a site on the genome, it is considered an "SNP marker".

[0346] As used herein, “germstone” refers to a living source of genetic material. Germstone can be a part of an organism or cell, or it can be separated from an organism or cell. Generally, germstone provides genetic material with a specific molecular structure that provides the physical basis for some or all of the genetic qualities of an organism or cell culture. As used herein, germstone includes cells, seeds, or tissues from which new plants can grow, or plant parts such as leaves, stems, pollen, ovules, or cells that can be cultured into complete plants.

[0347] As used in this paper, centimole (“cM”) is a unit of measurement for recombination frequency and genetic distance between two loci. 1 cM equals the 1% probability that a marker at one genetic locus will separate from a marker at a second locus due to crossover in a single generation. Genetic distance can be calculated from experimentally derived recombination values ​​using the Kosambi function (Kosambi, The estimation of map distances from recombination values). Annals of Eugenics , 12:172-75(1944)).

[0348] As used herein, “linked” or “genetically linked” means within approximately 20 cM, 19 cM, 18 cM, 17 cM, 16 cM, 15 cM, 14 cM, 13 cM, 12 cM, 11 cM, 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.5 cM, or less than 0.5 cM of another marker or locus on the same chromosome. For example, 10 cM means that recombination occurs between the marker and the locus at a highly predictable recombination frequency equal to or less than approximately 10%.

[0349] Genetic linkage of marker molecules can be established using gene mapping models, such as, but not limited to, flanking marker models reported by Lander and Botstein, Genetics, 121:185-199 (1989), and interval mapping based on the maximum likelihood method described by Lander and Botstein, Genetics, 121:185-199 (1989), implemented in the software package MAPMAKER / QTL (Lincoln and Lander, Mapping Genes Controlling QuantitativeTraits Using MAPMAKER / QTL, Whitehead Institute for Biomedical Research, Massachusetts, (1990)). Other software includes Qgene, version 2.23 (1996), Plant Breeding and Bioassays, Room 266, Emerson Building, Cornell University, Ithaca, NY, JoinMap (Kyazma BV, Wageningen, Netherlands), and mapQTL (Kyazma BV, Wageningen, Netherlands).

[0350] As used herein, the phrase “associated with” or “connected to” refers to an identifiable and / or measurable relationship between two entities. Therefore, a marker is “associated” with a trait when it is linked to or co-segregates with that trait, and when the presence of that marker is an indicator of whether and / or to what extent the desired trait or trait form will appear in plants / germplasm containing that marker. Similarly, a marker is “associated” or “linked” with an allele when it is linked to or co-segregates with that allele, and when the presence of that marker is an indicator of the presence of that allele in plants / germplasm containing that marker. For example, “a marker is linked to red meat” means that the marker is 10 cM or less from an allele co-segregating with red meat.

[0351] As used in this article, "hybrid" or "hybrid" means through fertilization ( For example The production of offspring (from cells, seeds, or plants) includes hybridization (sexual) between plants and self-fertilization (self-pollination).

[0352] As used in this article, "backcrossing" and "recursion" refer to the process in which offspring plants are repeatedly hybridized back to one of their parents. In a backcross scheme, the "do...

Claims

1. A modified tobacco plant or a portion thereof, said modified tobacco plant or a portion thereof comprising at least one non-natural mutation in an endogenous nucleic acid sequence, said endogenous nucleic acid sequence encoding a polypeptide comprising an amino acid sequence that is at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40.

2. The modified tobacco plant or a portion thereof according to claim 1, wherein the modified tobacco plant produces at least one leaf, the at least one leaf containing a reduced amount of at least one metabolite compared to the amount of at least one metabolite in a control tobacco plant lacking at least one non-natural mutation in the endogenous nucleic acid sequence when grown under similar growth conditions.

3. The modified tobacco plant or a portion thereof according to claim 1, wherein the modified tobacco plant belongs to the group consisting of flue-cured varieties, bright varieties, burley varieties, Virginia varieties, Maryland varieties, black tobacco varieties, Galpao varieties, Oriental varieties, and Turkish varieties.

4. A modified tobacco material, said modified tobacco material being derived from the modified tobacco plant or a portion thereof according to claim 1.

5. The modified tobacco material according to claim 4, wherein the modified tobacco material comprises flue-modified tobacco material, air-dried tobacco material, roasted tobacco material, or sun-dried tobacco material.

6. A tobacco product comprising the modified tobacco material according to claim 5.

7. The tobacco product according to claim 6, wherein the tobacco product is a smokeless tobacco product.

8. A modified tobacco plant or a portion thereof, said modified tobacco plant or a portion thereof comprising a recombinant DNA construct comprising a heterologous promoter operatively linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of an endogenous nucleic acid sequence, said endogenous nucleic acid sequence encoding a polypeptide whose amino acid sequence is at least 95% identical or similar to that of the group consisting of SEQ ID NO: 21 to 40.

9. The modified tobacco plant or a portion thereof according to claim 8, wherein the modified tobacco plant produces at least one leaf, the at least one leaf containing a reduced amount of at least one metabolite compared to the amount of at least one metabolite in a control tobacco plant lacking the recombinant DNA construct when grown under similar growth conditions.

10. The modified tobacco plant or a portion thereof according to claim 8, wherein the modified tobacco plant belongs to the group consisting of flue-cured varieties, bright varieties, burley varieties, Virginia varieties, Maryland varieties, black tobacco varieties, Galpao varieties, Oriental varieties and Turkish varieties.

11. A modified tobacco material, said modified tobacco material being derived from the modified tobacco plant or a portion thereof according to claim 8.

12. The modified tobacco material according to claim 11, wherein the modified tobacco material comprises flue-modified tobacco material, air-dried tobacco material, roasted tobacco material, or sun-dried tobacco material.

13. A tobacco product comprising the modified tobacco material according to claim 12.

14. The tobacco product according to claim 12, wherein the tobacco product is a smokeless tobacco product.

15. A modified tobacco plant or a portion thereof, the modified tobacco plant or a portion thereof comprising a recombinant DNA construct comprising a heterologous promoter operatively linked to a nucleic acid sequence encoding a polypeptide, the polypeptide comprising an amino acid sequence that is at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 to 40.

16. The modified tobacco plant or a portion thereof according to claim 15, wherein the modified tobacco plant produces at least one leaf, the at least one leaf containing an increased amount of at least one metabolite compared to the amount of at least one metabolite in a control tobacco plant lacking the recombinant DNA construct when grown under similar growth conditions.

17. The modified tobacco plant or a portion thereof according to claim 15, wherein the modified tobacco plant belongs to the group consisting of flue-cured varieties, bright varieties, burley varieties, Virginia varieties, Maryland varieties, black tobacco varieties, Galpao varieties, Oriental varieties and Turkish varieties.

18. A modified tobacco material, said modified tobacco material being derived from the modified tobacco plant or a portion thereof according to claim 15.

19. The modified tobacco material according to claim 18, wherein the modified tobacco material comprises flue-modified tobacco material, air-dried tobacco material, roasted tobacco material, or sun-dried tobacco material.

20. A tobacco product comprising the modified tobacco material according to claim 18.

21. The tobacco product according to claim 20, wherein the tobacco product is a smokeless tobacco product.

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