Methods and compositions for modulating alkaloids in tobacco

Abstract

The present disclosure relates to tobacco plants, plant parts, seeds, compositions and methods related to modulating expression of novel genes in tobacco to modulate alkaloid levels.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 395,459, filed on August 5, 2022, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates to tobacco plants, plant parts, seeds, compositions and methods related to modulating expression of novel genes in tobacco to regulate alkaloid levels.

[0004] Incorporation of Sequence Listing

[0005] The sequence listing, which complies with the rules of WIPO Standard ST.26, is hereby incorporated by reference in its entirety. The sequence listing has been submitted as an electronic document via PatentCenter, encoded as XML in UTF-8 text. The electronic document is named "P34753WO00_SL.xml" and is 341,150 bytes in size (at Measured in ), and was created on July 26, 2023.

[0006] A listing of nucleic acid and amino acid sequences is provided in Table 1.

[0007] Table 1. Nucleotide and amino acid sequences

[0008]

[0009]

[0010]

[0011]

[0012] Background Art

[0013] Four major alkaloids are found in tobacco: nicotine, nornicotine, anabasine, and anatabine. Nicotine is the major alkaloid, typically accounting for more than 90% of the total alkaloids in commercial tobacco varieties. Nicotine biosynthesis occurs primarily in tobacco roots. Tobacco plants then transport nicotine via vascular bundles to leaves, where it is then stored in vacuoles. The accumulation level of nicotine and related alkaloids in tobacco leaves depends on biosynthesis and transport, both of which can be affected by environmental conditions, plant hormones, developmental state, and genetic regulators.

[0014] Provided herein are methods and compositions for modulating novel tobacco genes to modulate alkaloid levels in tobacco. Summary of the invention

[0015] In one aspect, the present disclosure provides a modified tobacco 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 NOs: 117 to 174.

[0016] In one aspect, the present disclosure provides a modified tobacco plant or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to an endogenous nucleic acid sequence and reducing its expression, the 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 NOs: 117 to 174.

[0017] In one aspect, the present disclosure provides a modified tobacco plant or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably 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: 117 to 174.

[0018] In one aspect, the present 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 a polypeptide in at least one tobacco cell, the 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: 117 to 174; (b) selecting at least one tobacco cell comprising the non-natural mutation from step (a); and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b).

[0019] In one aspect, the present 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 operably linked to a nucleic acid encoding at least one small RNA molecule, the at least one small RNA molecule being capable of binding to and reducing the expression of an endogenous nucleic acid sequence, the 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 NOs: 117 to 174; (b) selecting at least one tobacco cell comprising the recombinant DNA construct; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b).

[0020] In one aspect, the present 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 operably linked to a nucleic acid sequence encoding a polypeptide, the 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: 117 to 174; (b) selecting at least one tobacco cell comprising the recombinant DNA construct; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b).

[0021] In one aspect, the present disclosure provides a method comprising preparing a tobacco product using a cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a non-natural mutation in an endogenous nucleic acid sequence, wherein the endogenous nucleic acid sequence encodes 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: 117 to 174.

[0022] In one aspect, the present disclosure provides a method comprising preparing a tobacco product using a cured 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 operably linked to a nucleic acid sequence encoding at least one small RNA molecule, the at least one small RNA molecule being capable of binding to and reducing the expression of an endogenous nucleic acid sequence, the 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 NOs: 117 to 174.

[0023] In one aspect, the present disclosure provides a method comprising preparing a tobacco product using a cured 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 operably 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: 117 to 174.

[0024] In one aspect, the present disclosure provides a method comprising transforming a tobacco cell with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably 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 that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174.

[0025] In one aspect, the present disclosure provides a method comprising transforming a tobacco cell with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably 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: 117 to 174.

[0026] In one aspect, the present disclosure provides a method for producing a modified tobacco plant, 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 generation of tobacco seeds, wherein the at least one tobacco plant of the first tobacco variety comprises 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 NOs: 117 to 174, wherein the non-natural mutation is not present in the endogenous nucleic acid sequence in a control tobacco plant of the first tobacco variety; and (b) selecting at least one generation of tobacco seeds or a plant germinated therefrom, wherein the at least one tobacco seed or a plant germinated therefrom comprises the non-natural mutation.

[0027] In one aspect, the present disclosure provides a method for producing a modified tobacco plant, 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 first-generation 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 operably linked to a nucleic acid sequence encoding at least one small RNA molecule, the small RNA molecule being capable of binding to and reducing the expression of an endogenous nucleic acid sequence, the 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 NOs: 117 to 174, wherein the recombinant DNA construct is not present in an endogenous nucleic acid sequence in a control tobacco plant of the same variety; and (b) selecting at least one first-generation tobacco seed or a plant germinated therefrom, wherein the at least one tobacco seed or a plant germinated therefrom comprises the recombinant DNA construct.

[0028] In one aspect, the present disclosure provides a method for producing a modified tobacco plant, 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 generation 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 operably linked to a nucleic acid sequence encoding a polypeptide, the 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: 117 to 174, wherein the recombinant DNA construct is not present in the nucleic acid sequence in a control tobacco plant of the first tobacco variety; and (b) selecting at least one generation tobacco seed or a plant germinated therefrom, wherein the at least one tobacco seed or a plant germinated therefrom comprises the recombinant DNA construct.

[0029] In one aspect, the present disclosure provides a modified tobacco plant or part thereof, comprising at least one non-natural mutation in an endogenous nucleic acid sequence that modulates 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 NOs: 117 to 174. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Depicted are clusters of genes based on dissimilarity measured by topological overlap. The bars at the bottom of the graph represent different network modules.

[0031] Figure 2Depicted are subnetworks extracted from wgcna and visualized in Cytoscape. The genes (nodes) labeled at the center of each subnetwork are known nicotine biosynthesis genes. Abbreviations: AO3 (L-ascorbate oxidase 3); AO-2a (L-ascorbate oxidase 2a); BBL-3 (berberine bridge enzyme-like 3); ADC-1b (arginine decarboxylase 1b); BBL-4 (berberine bridge enzyme-like 4); QS1b (quinolinate synthase 1b); QS1a (quinolinate synthase 1a); ADC-1a (arginine decarboxylase 1a); PMT1b (putrescine methyltransferase 1b); BBL-5 (berberine bridge enzyme-like 4); QS1b (quinolinate synthase 1b); QS1a (quinolinate synthase 1a); ADC-1a (arginine decarboxylase 1a); PMT1b (putrescine methyltransferase 1b); BBL-6 (berberine bridge enzyme-like 4); QS1b (quinolinate synthase 1b); QS1a (quinolinate synthase 1a); ADC-1a (arginine decarboxylase 1a); PMT1b (putrescine methyltransferase 1b); BBL-7 (berberine bridge enzyme-like 7); BBL-8 (berberine bridge enzyme-like 8); QS1b (quinolinate synthase 1b); QS1a (quinolinate synthase 1a); ADC-1a (arginine decarboxylase 1a); PMT1b (putrescine methyltransferase 1b); BBL-9 (berberine bridge enzyme-like 4); L-1 (berberine bridge enzyme-like 1); PMT3 (putrescine methyltransferase 3); AO7 (L-ascorbate oxidase 7); ODC-1a (ornithine decarboxylase 1a); AO11 (L-ascorbate oxidase 11); ODC-1b (ornithine decarboxylase 1b); PMT1a (putrescine methyltransferase 1a); AIC-1b (agmatine deiminase 1b); A622-1 (isoflavone reductase homolog A622 1); PMT4 (putrescine methyltransferase 4); MPO1a (N-methylputrescine oxidase 1a); AO12 (L-ascorbate oxidase 12); ADC-1c (arginine decarboxylase 1c); LDC (lysine decarboxylase); AO4 (L-ascorbate oxidase 4); AO17 (L-ascorbate oxidase 17); A622-2 (isoflavone reductase homolog A622 2); A622-3 (isoflavone reductase homolog A622 3); BBL-2 (berberine bridge enzyme-like 2); and A622-4 (isoflavone reductase homolog A622 4).

[0032] Figure 3 Depicted are heat maps showing expression in terms of log2 fold change between before topping (BT), three days after topping (3D), and two weeks after topping (2W) in TN90 tobacco root samples.

[0033] Figures 4 to 16 Describe the relative expression of 14 novel tobacco genes in the root tissue of tobacco lines TN90 and CS118 (compared with tobacco eukaryotic translation elongation factor 1α (EF1α)).Expression was checked before topping (BT), after topping for three days (3d) and after topping for one week (1w).Error bars (if present) represent a standard deviation. Figure 4 The relative expression of g95400 (SEQ ID NO:58) is depicted. Figure 5 The relative expression of g82124 (SEQ ID NO:42) is depicted. Figure 6 The relative expression of g23499 (SEQ ID NO: 7) is depicted. Figure 7 The relative expression of g69182 (SEQ ID NO: 26) is depicted. Figure 8 The relative expression of g14663 (SEQ ID NO: 2) is depicted. Fig. 9 The relative expression of g71598 (SEQ ID NO: 28) is depicted. Fig.10 The relative expression of g74782 (SEQ ID NO: 30) is depicted. Fig.11 The relative expression of g92662 (SEQ ID NO:55) is depicted. Fig.12 The relative expression of g91334 (SEQ ID NO:51) is depicted. Fig.13 The relative expression of g93191 (SEQ ID NO:56) is depicted. Fig.14 The relative expression of g74738 (SEQ ID NO: 29) is depicted. Fig.15 The relative expression of g66301 (SEQ ID NO: 24) is depicted. Fig.16 The relative expression of g86407 (SEQ ID NO:47) is depicted.

[0034] Fig.17 Depicted are the relative expression of g22648 (compared to EF1α) in overexpression (OX) and knockdown (RNAi) lines compared to three vector control lines (VC-1, VC-2, and VC-3).

[0035] Fig.18 Depicted are the relative expression (compared to EF1α) of the putrescine N-methyltransferase (PMT) and quinolinate phosphoribosyltransferase (QPT) gene families in g22648 overexpression (OX) and knockdown (RNAi) tobacco lines compared to three vector control lines (VC-1, VC-2, and VC-3).

[0036] Fig.19 Depicted are nicotine levels in g22648 overexpression (OX) and knockdown (RNAi) tobacco lines compared to three vector control lines (VC).

[0037] Fig. 20 Depicted are nornicotine, anatabine, and anabasine levels in g22648 overexpression (OX) and knockdown (RNAi) tobacco lines compared to three vector control lines (VC).

[0038] Fig.21 Depicted are the relative expression of g95400 (compared to EF1α) in RNAi knockdown tobacco lines compared to vector control lines (VC).

[0039] Fig. 22 Depicted are the relative expression of the PMT and QPT gene families (compared to EF1α) in the g95400 RNAi knockdown tobacco lines compared to the vector control line (VC).

[0040] Fig.23 Depicted are nicotine levels in the g95400 RNAi knockdown lines compared to the vector control line (VC).

[0041] Fig.24 Depicted are nornicotine, anatabine, and anabasine levels in g95400 RNAi knockdown tobacco lines compared to vector control lines (VC).

[0042] Fig.25 Depicted are the relative expression of g74912 (compared to EF1α) in RNAi knockdown tobacco lines compared to three vector control lines (VC).

[0043] Fig.26 Depicted are the relative expression (compared to EF1α) of the PMT and QPT gene families in the g74912 RNAi knockdown tobacco lines compared to the two vector control lines (VC).

[0044] Fig. 27 Depicted are nicotine levels in g74912 RNAi knockdown tobacco lines compared to two vector control lines (VC).

[0045] Fig.28 Depicted are nornicotine, anatabine, and anabasine levels in g74912 RNAi knockdown tobacco lines compared to two vector control lines (VC).

[0046] Fig.29 Depicted are the relative expression of g12941 (compared to EF1α) in overexpression (OX) and knockdown (RNAi) lines compared to three vector control lines (VC-1, VC-2, and VC-3).

[0047] Fig.30 Depicted are the relative expression (compared to EF1α) of putrescine N-methyltransferase (PMT) and quinolinate phosphoribosyltransferase (QPT) in g12941 overexpression (OX) and knockdown (RNAi) tobacco lines compared to three vector control lines (VC-1, VC-2, and VC-3).

[0048] Fig.31 Depicted are nicotine levels in g12941 overexpression (OX) and knockdown (RNAi) tobacco lines compared to three vector control lines (VC-1, VC-2, and VC-3).

[0049] Fig.32 Depicted are nornicotine, anatabine, and anabasine levels in g12941 overexpression (OX) and knockdown (RNAi) tobacco lines compared to three vector control lines (VC-1, VC-2, and VC-3).

[0050] Fig.33 Depicted are the relative expression of g75446 (compared to EF1α) in RNAi knockdown tobacco lines compared to two vector control lines (VC).

[0051] Fig.34 Depicted are the relative expression (compared to EF1α) of the PMT and QPT gene families in the g75446 RNAi knockdown tobacco lines compared to the two vector control lines (VC).

[0052] Fig.35 Depicted are nicotine levels in g75446 RNAi knockdown tobacco lines compared to two vector control lines (VC).

[0053] Fig.36 Depicted are nornicotine, anatabine, and anabasine levels in g75446 RNAi knockdown tobacco lines compared to two vector control lines (VC).

[0054] Fig.37 Depicted are the relative expression of g31724 (compared to EF1α) in two RNAi knockdown tobacco lines compared to the vector control line (VC).

[0055] Fig.38 Depicted are the relative expression (compared to EF1α) of the PMT and QPT gene families in two g31724 RNAi knockdown tobacco lines compared to the vector control line (VC).

[0056] Fig.39 Depicted are nicotine levels in two g31724 RNAi knockdown tobacco lines compared to a vector control line (VC).

[0057] Fig.40 Depicted are nornicotine, anatabine, and anabasine levels in two g31724 RNAi knockdown tobacco lines compared to a vector control line (VC).

[0058] Fig.41 Depicted are the relative expression (compared to EF1α) of g14663 overexpression (OX) in two tobacco lines compared to the vector control line (VC).

[0059] Fig.42 Depicted are the relative expression (compared to EF1α) of the PMT and QPT gene families in two g14663 overexpressing (OX) tobacco lines compared to the vector control line (VC).

[0060] Fig.43 Depicted are nornicotine levels in two g14663 overexpressing (OX) tobacco lines compared to a vector control line (VC).

[0061] Fig.44Depicted are nornicotine, anatabine, and anabasine levels in two g14663 overexpressing (OX) tobacco lines compared to a vector control line (VC).

[0062] Fig.45 Depicted are the relative expression (compared to EF1α) of g71598 overexpression (OX) in two tobacco lines compared to the vector control line (VC).

[0063] Fig.46 Depicted are the relative expression (compared to EF1α) of the PMT and QPT gene families in two g71598 overexpressing (OX) tobacco lines compared to the vector control line (VC).

[0064] Fig.47 Depicted are nornicotine levels in two g71598 overexpressing (OX) tobacco lines compared to a vector control line (VC).

[0065] Fig.48 Depicted are nornicotine, anatabine, and anabasine levels in two g71598 overexpressing (OX) tobacco lines compared to a vector control line (VC).

[0066] Fig.49 Depicted are the relative expression of g82744 (compared to EF1α) in two RNAi knockout tobacco lines compared to two vector control lines (VC).

[0067] Fig.50 Depicted are the relative expression (compared to EF1α) of the PMT and QPT gene families in two g82744 RNAi knockout tobacco lines compared to two vector control lines (VC).

[0068] Fig.51 Depicted are nicotine levels in two g82744 RNAi knockout tobacco lines compared to two vector control lines (VC).

[0069] Fig.52 Depicted are nornicotine, anatabine, and anabasine levels in two g82744 RNAi knockout tobacco lines compared to two vector control lines (VC).

[0070] Fig.53 Depicted are the relative expression of g79774 (compared to EF1α) in three RNAi knockout tobacco lines compared to two vector control lines (VC).

[0071] Fig.54 Depicted are the relative expression (compared to EF1α) of the PMT and QPT gene families in three g79774 RNAi knockdown tobacco lines compared to two vector control lines (VC).

[0072] Fig.55Depicted are nicotine levels in three g79774 RNAi knockdown tobacco lines compared to two vector control lines (VC).

[0073] Fig.56 Depicted are nornicotine, anatabine, and anabasine levels in three g79774 RNAi knockdown tobacco lines compared to two vector control lines (VC).

[0074] Fig.57 Depicted are the relative expression of g82124 (compared to EF1α) in three RNAi knockdown tobacco lines compared to three vector control lines (VC).

[0075] Fig.58 Depicted are the relative expression (compared to EF1α) of the PMT and QPT gene families in three g82124 RNAi knockdown tobacco lines compared to three vector control lines (VC).

[0076] Fig.59 Depicted are nicotine levels in three g82124 RNAi knockdown tobacco lines compared to a vector control line (VC).

[0077] Fig.60 Depicted are nornicotine, anatabine, and anabasine levels in three g82124 RNAi knockdown tobacco lines compared to a vector control line (VC).

[0078] for Fig.17 , Fig.18 , Fig. 22 , Fig.25 , Fig.26 , Fig.29 , Fig.30 , Fig.33 , Fig.34 , Fig.37 , Fig.38 , Fig.41 , Fig.42 , Fig.45 , Fig.46 , Fig.49 , Fig.50 , Fig.53 , Fig.54 , Fig.57 and Fig.58 , error bars represent the range of observed data. DETAILED DESCRIPTION

[0079] Unless otherwise defined, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Where a term is provided in the singular, the inventors of the present invention also contemplate various aspects of the present disclosure described in the plural form of the term. In the event of differences in the terms and definitions used in the references incorporated by reference, the terms used in this application shall have the definitions given herein. Other technical terms used have their ordinary meaning in the field in which they are used, as exemplified by various field-specific dictionaries, such as "American Science Dictionary" (Editors of American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), "McGraw-Hill Dictionary of Scientific and Technical Terms" (6th ed., 2002, McGraw-Hill, New York), or "Oxford Dictionary of Biology" (6th ed., 2008, Oxford University Press, Oxford and New York).

[0080] Any references cited herein, including, for example, all patents, published patent applications, and non-patent publications, are hereby incorporated by reference in their entirety.

[0081] When a set of alternatives is presented, any and all combinations of the members that make up the set of alternatives are specifically contemplated. For example, if an item is selected from the group consisting of A, B, C, and D, the inventor will specifically contemplate each alternative individually (e.g., A alone, B alone, etc.) and combinations such as A, B, and D; A and C; B and C; etc. The term "and / or" when used in a list of two or more items refers to any one of the listed items by itself or in combination with any one or more 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.

[0082] When numerical ranges are provided herein, the ranges should be understood to include the edges of the range and any numbers between the defined edges of the range. For example, "between 1 and 10" includes any number between 1 and 10 as well as the number 1 and the number 10.

[0083] When the term "about" is used in reference to a number, it should be understood to mean plus or minus 10%. For example, "about 100" would include from 90 to 110.

[0084] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0085] Any tobacco plant that this paper provides or its part specifically contemplates and is used for using with any method that this paper provides.Similarly, any modified tobacco plant or its part specifically contemplates and is used for using with any method that this paper provides.Any nucleotide sequence, amino acid sequence or other compositions that this paper provides specifically contemplates and is used for using with any method that this paper provides.

[0086] Tobacco plants produce alkaloids usually at a level between 2% and 4% of their total dry weight. Nicotine is the main alkaloid compound in tobacco plants, and it accounts for about 95% of the total alkaloid content of plants usually. The remaining alkaloid pool mainly comprises other structurally related alkaloids, such as anabasine, anatabine and nornicotine.

[0087] Nicotine is synthesized in roots via one of two major metabolic pathways, the pyridine nicotine biosynthetic pathway and the pyrrolidine nicotine biosynthetic pathway.

[0088] In the pyridine nicotinoid biosynthetic pathway, quinolinic acid is produced by quinolinic acid synthase (QS), and quinolinic acid is converted to nicotinic acid mononucleotide (NAMN) catalyzed by quinolinic acid phosphoribosyltransferase (QPT). NAMN can be converted to nicotinic acid directly by NAMN glycohydrolase or by a multistep process involving the synthesis and degradation of nicotinamide adenine dinucleotide (NAD). Further reduction of nicotinic acid then forms 3,6-dihydronicotinic acid.

[0089] In the pyrrolidine nicotinoid biosynthetic pathway, an amino acid (e.g., arginine) is required to decarboxylate to form putrescine, and putrescine is methylated to N-methylputrescine catalyzed by N-methyltransferase (PMT). N-methylputrescine is then oxidized by diamine oxidase and spontaneously cyclized to N-methyl-Δ 1 -pyrrolinium cation. Then, the N-methyl-Δ 1 The -pyrrolinium cation couples with 3,6-dihydronicotinic acid or another nicotinic acid intermediate to form nicotine. However, the exact substrates and enzymes involved in these reactions remain unknown.

[0090] Here, novel genes involved in the biosynthesis of nicotine or other alkaloids in tobacco are provided.

[0091] In one aspect, the present disclosure provides a modified tobacco plant or part thereof, the modified tobacco 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 at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NO: 117 to 174. In another aspect, the present disclosure provides a modified tobacco plant or part thereof, the modified tobacco or part thereof comprising at least one non-natural mutation in an endogenous nucleic acid sequence, the endogenous nucleic acid sequence being at least 80% identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to 58. In another aspect, the present disclosure provides a modified tobacco plant or part thereof, the modified tobacco or part thereof comprising at least one non-natural mutation in an endogenous nucleic acid sequence, the endogenous nucleic acid sequence being at least 80% identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 59 to 116. In another aspect, the present disclosure provides a modified tobacco plant or part thereof, the modified tobacco or part thereof comprising at least one non-natural mutation in an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 116.

[0092] In one aspect, the present disclosure provides a modified tobacco plant or part thereof, the modified tobacco plant or part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule, the at least one small RNA molecule being capable of binding to and reducing the expression of an endogenous nucleic acid sequence, the endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In another aspect, the present disclosure provides a modified tobacco plant or part thereof, the modified tobacco or part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule, the at least one small RNA molecule being 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 58. In yet another aspect, the present disclosure provides a modified tobacco plant or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to an endogenous nucleic acid sequence and reducing its expression, the endogenous nucleic acid sequence being at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59 to 116.

[0093] In one aspect, the present disclosure provides a modified tobacco plant or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably 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: 117 to 174. In another aspect, the present disclosure provides a modified tobacco plant or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 58. In another aspect, the present disclosure provides a modified tobacco plant or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 59 to 116.

[0094] On the one hand, the present disclosure provides a kind of modified tobacco plant or its part, this modified tobacco plant or its part comprises at least one non-natural mutation in endogenous nucleic acid sequence, this endogenous nucleic acid sequence regulates the expression or activity of gene, wherein this gene encodes the polypeptide of amino acid sequence at least 80% identical or similar to the amino acid sequence of the group consisting of SEQ ID NO:117 to 174. On the other hand, the present disclosure provides a kind of modified tobacco plant or its part, this modified tobacco plant or its part comprises at least one non-natural mutation in endogenous nucleic acid sequence, this endogenous nucleic acid sequence regulates the expression or activity of gene, wherein this gene encodes the RNA sequence at least 80% identical or complementary to the nucleic acid sequence of the group consisting of SEQ ID NO:59 to 116. On the one hand, endogenous nucleic acid sequence encodes the transcription factor that can be combined with the nucleic acid sequence of coding gene. As used herein, "transcription factor" refers to the protein that controls the transcription rate of this gene from DNA to messenger RNA by combining with the specific DNA sequence of gene. Not limited by any scientific theory, if transcription factor is combined with gene, then compared with the transcription in the absence of transcription factor, this transcription factor opens or enhances the transcription of gene. On the other hand, the endogenous nucleic acid sequence encodes a repressor that can bind to the nucleic acid sequence encoding the gene. As used herein, "repressor" refers to a protein that inhibits the transcription of the gene from DNA to messenger RNA by binding to the specific DNA sequence of the gene. Without being limited by any scientific theory, if the repressor binds to the gene, the repressor turns off or reduces the transcription of the gene compared to the transcription in the absence of the repressor. In other aspects, the endogenous nucleic acid sequence encodes a protein that produces a precursor required for the polypeptide encoded by the gene to function. As a non-limiting example of a precursor, quinolinic acid synthetase produces quinolinic acid, which is a precursor required for quinolinic acid phosphoribosyl transferase to function. On the one hand, the gene comprises a nucleic acid sequence that is at least 80% identical or similar to SEQ ID NO: 1 to 58.

[0095] In one aspect, the present disclosure provides a modified tobacco plant or part thereof, comprising (a) a genetic modification in a gene; or (b) a genetic modification to the gene; wherein the genetic modification downregulates the expression or activity of the gene, wherein the gene encodes an amino acid sequence having at least 80% identity or similarity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In another aspect, the present disclosure provides a modified tobacco plant or part thereof, comprising (a) a genetic modification in a gene; or (b) a genetic modification to the gene; wherein the genetic modification downregulates the expression or activity of the gene, wherein the gene encodes a nucleic acid sequence having at least 80% identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 58. In another aspect, the present disclosure provides a modified tobacco plant or part thereof, comprising (a) a genetic modification in a gene; or (b) a genetic modification to the gene; wherein the genetic modification downregulates the expression or activity of the gene, wherein the gene encodes a nucleic acid sequence having at least 80% identity with a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 59 to 116. In another aspect, the present disclosure provides a modified tobacco plant or part thereof, comprising (a) a genetic modification in a gene; or (b) a genetic modification to the gene; wherein the genetic modification downregulates the expression or activity of the gene, wherein the gene encodes a nucleic acid sequence having at least 80% identity with a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 116.

[0096] As used herein, "genetic modification" refers to the change of the genetic composition of a plant or plant genome.Genetic modification can be introduced by methods including but not limited to mutagenesis, genome editing, genetic transformation, or a combination thereof.Genetic modification includes mutations (e.g., non-natural mutations) in, for example, a gene or a transgenic targeting gene (e.g., an arginine decarboxylase (ADC) transgenic targeting ADC gene).As used herein, "targeting" refers to directly up-regulating or directly down-regulating the expression or activity of a gene.As used herein, "directly" in the context of a transgenic affecting the expression or activity of a gene refers to physical contact or chemical interactions between products encoded via a gene (e.g., a promoter region or UTR region) or a product (e.g., an mRNA molecule or polypeptide) or a transgenic (e.g., a small RNA molecule or protein, such as a transcription factor or a dominant negative effect polypeptide variant) to exert influence on a gene.On the one hand, a transgenic affects the expression or activity of a target gene without involving a transcription factor (e.g., a transgenic does not encode a transcription factor and / or does not suppress the expression or activity of a transcription factor that in turn regulates a target gene).

[0097] mutation

[0098] As used herein, " modification " refers to the plant that comprises the genetic change that introduces and exceeds natural polymorphism for some purpose in the context of plant.Unrestricted, modified plant can comprise non-natural mutation or recombinant DNA construct.On the one hand, modified tobacco plant comprises non-natural mutation.On the other hand, modified tobacco plant comprises recombinant DNA construct.On the other hand, modified tobacco plant comprises genetic modification.

[0099] As used herein, "mutation" refers to a heritable genetic modification introduced into a gene to change the expression or activity of a product encoded by the reference sequence of the gene. A mutation of a gene, such as, for example, arginine decarboxylase (ADC) is referred to as an ADC mutant. Such modifications can be in any sequence region of a gene, for example, in a promoter, 5' untranslated region (UTR), exon, intron, 3'-UTR or terminator region. On the one hand, a mutation reduces, suppresses or eliminates the expression or activity of a gene product. On the other hand, a mutation increases, improves, strengthens or enhances the expression or activity of a gene product.

[0100] On the one hand, mutation is not the natural polymorphism present in specific tobacco varieties or cultivars.On the one hand, mutation is "non-natural" or "non-naturally occurring" mutation.As used herein, "non-natural" or "non-naturally occurring" mutation refers to the non-spontaneous mutation produced via human intervention, and does not correspond to the spontaneous mutation produced in the absence of human intervention.The limiting examples of human intervention include mutagenesis (for example, chemical mutagenesis, ionizing radiation mutagenesis) and targeted genetic modification (for example, based on the method of CRISPR, based on the method of TALEN, based on the method of zinc finger).Non-natural mutation and non-naturally occurring mutation do not include spontaneous mutation of abiogenesis (for example, via the abnormal DNA replication in plant germline).

[0101] On the one hand, sudden change is not the natural polymorphism that is present in specific tobacco variety or cultivar.Should be appreciated that when identifying sudden change, reference dna sequence should be from the tobacco of same kind.For example, if the modified tobacco plant that comprises sudden change is from kind TN90, then endogenous reference sequence must be endogenous TN90 sequence, rather than the homologous sequence from different tobacco varieties (for example, K326).Similarly, if the modified tobacco cell that comprises sudden change is the TN90 cell, then endogenous reference sequence must be endogenous TN90 sequence, rather than the homologous sequence from the tobacco cell of different tobacco varieties (for example, K326).

[0102] On the one hand, tobacco plant or its part are isozygous for at least one non-natural mutation.On the other hand, tobacco plant or its part are heterozygous for at least one non-natural mutation.On the other hand, tobacco plant or its part are isozygous for the recombinant DNA construction body of introducing.On the other hand, tobacco plant or its part are hemizygous for the recombinant DNA construction body of introducing.Aspect otherwise, tobacco plant or its part are heterozygous for the recombinant DNA construction body of introducing.

[0103] On the one hand, the mutation provided herein produces the dominant allele of the mutation locus.Dominant allele is the allele that covers the contribution of the second allele at the same locus.Dominant allele can be "dominant negative effect allele" or "dominant positive effect allele". Dominant negative effect allele or anti-allele are alleles opposite to the normal allele function.Dominant negative effect allele can not normally play a role usually, and either directly suppress the activity of wild-type protein (for example, by dimerization), or suppress the activity of the second protein required for the normal function of wild-type protein (for example, by the downstream component of activator or pathway).For example, dominant negative effect allele abolishes or reduces the normal function of allele in heterozygous or homozygous state.Dominant positive effect allele can increase normal gene function (for example, super allele) or provide new function (for example, new effect allele) for gene. A semidominant allele occurs when the penetrance of a linked phenotype in an individual heterozygous for the allele is less than the penetrance observed in an individual homozygous for the allele.

[0104] In one aspect, the mutations provided herein produce dominant negative alleles of the mutation locus. In another aspect, the mutations provided herein produce dominant positive alleles of the mutation locus.

[0105] As used herein, "induced" mutation refers to the generation of mutations in polynucleotide sequences via human 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 the one hand, induced mutations include the use of a reagent selected from the group consisting of chemical mutagens, radiation, transposons, Agrobacterium, and nucleases.

[0106] In one aspect, inducing mutations comprises using a chemical mutagen. In one aspect, the chemical mutagen comprises ethyl methanesulfonate (EMS).

[0107] In another aspect, inducing mutations comprises using radiation. In one aspect, the radiation comprises gamma rays, X-rays, ionizing radiation, or fast neutrons.

[0108] In one aspect, inducing mutations comprises using a transposon. In another aspect, inducing mutations comprises using Agrobacterium.

[0109] In a further aspect, inducing mutations comprises the use of nucleases. In one aspect, the nuclease is selected from the group consisting of a meganuclease, a zinc finger nuclease, a transcription activator-like effector nuclease, a CRISPR / Cas9 nuclease, a CRISPR / Cpf1 nuclease, a CRISPR / CasX nuclease, a CRISPR / CasY nuclease, and a Csm1 nuclease. In one aspect, inducing mutations comprises the use of a CRISPR / Cas9 nuclease. In one aspect, inducing mutations comprises the use of a CRISPR / Cpf1 nuclease. In one aspect, inducing mutations comprises the use of a CRISPR / CasX nuclease. In one aspect, inducing mutations comprises the use of a CRISPR / CasY nuclease. In one aspect, inducing mutations comprises the use of a Csm1 nuclease.

[0110] Several types of mutations are known in the art. On the one hand, mutations include insertions. "Insertion" refers to adding one or more nucleotides or amino acids to a given polynucleotide or amino acid sequence, respectively, compared to an endogenous reference polynucleotide or amino acid sequence. On the other hand, mutations include deletions. "Deletion" refers to removing one or more nucleotides or amino acids of a given polynucleotide or amino acid sequence, respectively, compared to an endogenous reference polynucleotide or amino acid sequence. On the other hand, mutations include substitutions. "Substitution" refers to replacing one or more nucleotides or amino acids with a given polynucleotide or amino acid sequence, respectively, compared to an endogenous reference polynucleotide or amino acid sequence. On the other hand, mutations include inversions. "Inversion" refers to when a fragment of a polynucleotide or amino acid sequence is reversed end to end. "Repetition" refers to when a fragment of a polynucleotide or amino acid sequence is repeated. The repeated fragment can be immediately after the original fragment, or it can be separated from the original fragment by one or more nucleotides or amino acids. On the one hand, mutations provided herein include mutations selected from the group consisting of insertions, deletions, substitutions, repetitions and inversions.

[0111] In one aspect, the non-natural mutation includes a mutation selected from the group consisting of substitution, deletion, insertion, duplication and inversion of one or more nucleotides relative to an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-116.

[0112] On the one hand, the non-natural mutation includes a mutation selected from the group consisting of substitution, deletion, insertion, duplication and inversion of one or more nucleotides relative to the endogenous nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 117 to 174.

[0113] 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, "nonsense mutations" refer to mutations in a nucleic acid sequence that introduce premature termination codons into an amino acid sequence by a nucleic acid sequence. As used herein, "missense mutations" refer to mutations in a nucleic acid sequence that result in replacements in an amino acid sequence encoded by a nucleic acid sequence. As used herein, "frameshift mutations" refer to insertions or deletions in a nucleic acid sequence that move frames to translate a nucleic acid sequence into an amino acid sequence. "Splice site mutations" refer to mutations in a nucleic acid sequence that result in introns being retained for protein translation or alternatively for exons being excluded from protein translation. Splice site mutations may result in nonsense mutations, missense mutations, or frameshift mutations.

[0114] When the mutant messenger RNA (mRNA) is translated into a protein or polypeptide, a mutation in the gene coding region (e.g., an exon mutation) may result in a truncated protein or polypeptide. In one aspect, the present disclosure provides mutations that result in truncation of a protein or polypeptide. As used herein, a "truncated" protein or polypeptide comprises at least one less amino acid compared to an endogenous control protein or polypeptide. For example, if endogenous protein A comprises 100 amino acids, a truncated form of protein A may comprise 1 to 99 amino acids. In one aspect, a non-natural mutation results in the truncation of a polypeptide.

[0115] Without being limited by any scientific theory, one method causing protein or polypeptide truncation is to introduce an early termination codon in the mRNA transcript of an endogenous gene. On the one hand, the present disclosure provides a mutation causing an early termination codon in the mRNA transcript of an endogenous gene. As used herein, "termination codon" refers to a nucleotide triplet that signals the termination of protein translation in an mRNA transcript. "Early termination codon" refers to a termination codon (e.g., on the 5' side) positioned earlier than the normal termination codon position in the endogenous mRNA transcript. Without limitation, several termination codons are known in the art, including "UAG", "UAA", "UGA", "TAG", "TAA" and "TGA".

[0116] On the one hand, the mutations provided herein include null mutations. As used herein, "null mutations" refer to mutations that confer a complete loss of function of a protein encoded by a gene comprising a mutation, or alternatively confer a complete loss of function of a small RNA protein 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.

[0117] The mutation provided herein can be located in any part of the endogenous gene. On the one hand, the mutation provided herein is located in the exon of the endogenous gene. On the other hand, the mutation provided herein is located in the intron of the endogenous gene. In a further aspect, the mutation provided herein is located in the 5'-UTR of the endogenous gene. On still another hand, the mutation provided herein is located in the 3'-UTR of the endogenous gene. On another hand, the mutation provided herein is located in the promoter of the endogenous gene. On another hand, the mutation provided herein is located in the terminator of the endogenous gene. On the one hand, the non-natural mutation provided herein includes a mutation in a sequence region selected from the group consisting of a promoter, a 5'-UTR, a 3'-UTR, an exon, an intron and a terminator.

[0118] The screening of mutagenic tobacco plants and selection can be carried out by any method known to those of ordinary skill in the art.The example of screening and selection method includes but is not limited to Southern analysis, PCR amplification, Northern blotting, RNase protection, primer extension, RT-PCR amplification, Sanger order-checking, next generation sequencing technology (for example, Illumina, PacBio, Ion Torrent, 454) for detecting the enzyme of polypeptide and polynucleotide or enzyme assay and protein gel electrophoresis, protein blotting, immunoprecipitation and the enzyme-linked immunosorbent assay for detecting polypeptide of ribozyme activity for detecting polynucleotide.Other technologies such as in situ hybridization, enzyme staining and immunostaining also can be used for detecting the existence or expression of polypeptide and / or polynucleotide.The method for carrying out all reference technologies is known.

[0119] Nucleic acids and amino acids

[0120] As used herein, an "endogenous" nucleic acid sequence refers to a nucleic acid sequence naturally present in the genome of an organism. An endogenous nucleic acid sequence does not include a heterologous sequence inserted into the genome via intentional human intervention. Similarly, an endogenous amino acid sequence is a sequence naturally present via translation of an endogenous nucleic acid molecule. In one aspect, the nucleic acid sequence provided herein is an endogenous nucleic acid sequence.

[0121] As used herein, "heterologous" refers to a sequence (nucleic acid or amino acid) derived from an alien species, or if from the same species, substantially modified from its native form in composition and / or genomic locus by intentional human intervention. The term also applies to nucleic acid constructs, also referred to herein as "polynucleotide constructs" or "nucleotide constructs". In this way, a "heterologous" nucleic acid construct means a construct derived from an alien species, or if from the same species, substantially modified from its native form in composition and / or genomic locus by 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 part, for example, via a transformation method or subsequent breeding of a transgenic plant with another plant of interest.

[0122] As used herein, "gene" refers to a polynucleotide that can produce a functional unit (e.g., without limitation, such as a protein or a small RNA molecule). A gene can include a promoter, an enhancer sequence, a leader sequence, a transcription start site, a transcription stop site, a polyadenylation site, one or more exons, one or more introns, 5'-UTR, 3'-UTR, or any combination thereof. A "gene sequence" can include a polynucleotide sequence encoding a promoter, an enhancer sequence, a leader sequence, a transcription start site, a transcription stop site, a polyadenylation site, one or more exons, one or more introns, 5'-UTR, 3'-UTR, or any combination thereof. In one aspect, a gene encodes a small RNA molecule or a precursor thereof. In another aspect, a gene encodes a protein.

[0123] As used herein, the term "percent identity" or "identical percent" with respect to two or more nucleotide or amino acid sequences is calculated by (i) comparing two optimally aligned sequences (nucleotides or amino acids) over a comparison window (one or more "comparable" regions), (ii) determining the number of positions at which the same nucleic acid base (for nucleotide sequences) or amino acid residue (for proteins and polypeptides) occurs in the two sequences to produce the number of matched positions, (iii) dividing the number of matched positions by the total number of positions in the comparison window, and then (iv) multiplying the quotient by 100% to produce the percent identity. If the "percent identity" is calculated relative to a reference sequence without specifying a particular comparison window, the percent identity is determined by dividing the number of matched positions over the comparison region by the total length of the reference sequence. Therefore, for the purposes of this application, when two sequences (query and subject) are optimally aligned (allowing gaps in their alignment), the "percent identity" of the query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions in the query sequence over its length (or comparison window), then multiplied by 100%.

[0124] When percent sequence identity is used with reference to amino acids, residue positions that are considered to be non-identical typically differ by conservative amino acid substitutions, in which an amino acid residue is substituted with another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity) and thus does not alter the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity."

[0125] In order to optimally align sequences to calculate their percent identity, various pairwise or multiple sequence alignment algorithms and programs are known in the art, such as ClustalW or Basic Local Alignment Search which can be used to compare sequence identity or similarity between two or more nucleotide or amino acid sequences. (BLAST TM ) etc. Although other alignment and comparison methods are known in the art, the alignment and identity percentage (including the above-mentioned identity percentage range) between two sequences can be determined by the ClustalW algorithm, see, e.g., Chenna et al., "Multiple sequence 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 X version 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 disclosures of which are incorporated herein by reference.

[0126] As used herein, the term "complementarity percentage" or "complementarity percentage" with respect to two nucleotide sequences is similar to the concept of identity percentage, but refers to the percentage of nucleotides of the query sequence that best base-pair or hybridize with nucleotides of the subject sequence when the query sequence and the subject sequence are arranged linearly and optimally base-paired without secondary folded structures (such as loops, stems, or hairpins). Such complementarity percentages can be between two DNA chains, two RNA chains, or a DNA chain and an RNA chain. "Complementarity percentage" can be calculated by (i) optimally base-pairing or hybridizing two nucleotide sequences in a linear and fully extended arrangement (i.e., without folding or secondary structures) over a comparison window, (ii) determining the number of base pair positions between the two sequences over a comparison window to produce a complementary number of positions, (iii) dividing the number of complementary positions by the total number of positions in the comparison window, and (iv) multiplying the quotient by 100% to obtain the complementarity percentage of the two sequences. The optimal base pairing of the two sequences can be determined by hydrogen bonding 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, the percent identity is determined by dividing the number of complementary positions between the two linear sequences by the total length of the reference sequence. Thus, for the purposes of this application, when the two sequences (query and subject) are optimally base paired (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 in the query sequence over its length, then multiplied by 100%.

[0127] The use of the terms "polynucleotide" or "nucleic acid molecule" is not intended to limit the present disclosure to polynucleotides comprising deoxyribonucleic acid (DNA). For example, ribonucleic acid (RNA) molecules are also contemplated. One of ordinary skill in the art will appreciate that polynucleotides and nucleic acid molecules can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. The polynucleotides of the present disclosure also encompass all forms of sequences, including but not limited to single-stranded forms, double-stranded forms, hairpins, stem-loop structures, and the like. On the one hand, the nucleic acid molecules provided herein are DNA molecules. On the other hand, the nucleic acid molecules provided herein are RNA molecules. On the one hand, the nucleic acid molecules provided herein are single-stranded. On the other hand, the nucleic acid molecules provided herein are double-stranded. The nucleic acid molecules can encode polypeptides or small RNAs.

[0128] As used herein, "recombinant nucleic acid" refers to a nucleic acid molecule formed by laboratory methods of genetic recombination (such as, but not limited to, molecular cloning). Similarly, a "recombinant DNA construct" refers to a DNA molecule formed by laboratory methods of genetic recombination.

[0129] The nucleic acid can be isolated using conventional techniques in the art. For example, any method can be used to isolate nucleic acid, including but not limited to recombinant nucleic acid technology and / or polymerase chain reaction (PCR). General PCR technology is described in, for example, PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, editor, Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid technology includes, for example, restriction enzyme digestion and connection, which can be used to isolate nucleic acid. The isolated nucleic acid can also be chemically synthesized as a single nucleic acid molecule or as a series of oligonucleotides. The polypeptide can be purified from a natural source (e.g., a biological sample) by known methods such as DEAE ion exchange, gel filtration and hydroxyapatite chromatography. It is also possible to purify the polypeptide, for example, by expressing the nucleic acid in an expression vector. In addition, the purified polypeptide can be obtained by chemical synthesis. The degree of purity of the polypeptide can be measured using any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis or HPLC analysis.

[0130] In one aspect, the present disclosure provides methods for detecting recombinant nucleic acids and polypeptides in plant cells. Without limitation, hybridization can also be used to detect nucleic acids. Hybridization between nucleic acids is discussed in detail in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0131] In one aspect, the nucleic acid sequences provided herein are at least 70% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the nucleic acid sequences provided herein are at least 75% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the nucleic acid sequences provided herein are at least 80% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the nucleic acid sequences provided herein are at least 85% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the nucleic acid sequences provided herein are at least 88% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the nucleic acid sequences provided herein are at least 90% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the nucleic acid sequences provided herein are at least 91% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the nucleic acid sequences provided herein are at least 92% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the nucleic acid sequences provided herein are at least 93% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the nucleic acid sequences provided herein are at least 94% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the nucleic acid sequences provided herein are at least 95% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the nucleic acid sequences provided herein are at least 96% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the nucleic acid sequences provided herein are at least 97% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the nucleic acid sequences provided herein are at least 98% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the nucleic acid sequences provided herein are at least 99% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the nucleic acid sequences provided herein are 100% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116.

[0132] In one aspect, the endogenous nucleic acid sequences provided herein are at least 70% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 75% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 80% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 85% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 88% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 90% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 91% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 92% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 93% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 94% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 95% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 96% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 97% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 98% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the endogenous nucleic acid sequence provided herein is at least 99% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the endogenous nucleic acid sequence provided herein is 100% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116.

[0133] As used herein, the term "polypeptide" refers to a chain of at least two covalently linked amino acids. A polypeptide can be encoded by a polynucleotide as provided herein. A protein as provided herein can be encoded by a nucleic acid molecule as provided herein. A protein can comprise a polypeptide as provided herein. As used herein, "protein" refers to a chain of amino acid residues that can provide structure or enzymatic activity to a cell.

[0134] Antibodies can be used to detect polypeptides. Techniques for using antibodies to detect polypeptides include enzyme-linked immunosorbent assay (ELISA), western blot, immunoprecipitation and immunofluorescence. Antibodies provided herein can be polyclonal antibodies or monoclonal antibodies. Methods well known in the art can be used to generate antibodies with specific binding affinity to polypeptides provided herein. Antibodies provided herein can be connected to solid supports (such as microtiter plates) using methods known in the art.

[0135] Detection (e.g., amplification products, hybridization complexes, polypeptides) can be accomplished using a detectable label. The term "label" is intended to encompass the use of direct labels as well as indirect labels. Detectable labels include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.

[0136] In one aspect, the amino acid sequences provided herein are at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In another aspect, the amino acid sequences provided herein are at least 75% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In another aspect, the amino acid sequences provided herein are at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In another aspect, the amino acid sequences provided herein are at least 85% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In another aspect, the amino acid sequences provided herein are at least 88% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In another aspect, the amino acid sequences provided herein are at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In another aspect, the amino acid sequences provided herein are at least 91% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In another aspect, the amino acid sequences provided herein are at least 92% identical or similar to the amino acid sequences selected from the group consisting of SEQ ID NOs: 117 to 174. In another aspect, the amino acid sequences provided herein are at least 93% identical or similar to the amino acid sequences selected from the group consisting of SEQ ID NOs: 117 to 174. In another aspect, the amino acid sequences provided herein are at least 94% identical or similar to the amino acid sequences selected from the group consisting of SEQ ID NOs: 117 to 174. In another aspect, the amino acid sequences provided herein are at least 95% identical or similar to the amino acid sequences selected from the group consisting of SEQ ID NOs: 117 to 174. In another aspect, the amino acid sequences provided herein are at least 96% identical or similar to the amino acid sequences selected from the group consisting of SEQ ID NOs: 117 to 174. In another aspect, the amino acid sequences provided herein are at least 97% identical or similar to the amino acid sequences selected from the group consisting of SEQ ID NOs: 117 to 174. In another aspect, the amino acid sequences provided herein are at least 98% identical or similar to the amino acid sequences selected from the group consisting of SEQ ID NOs: 117 to 174. In another aspect, the amino acid sequences provided herein are at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In another aspect, the amino acid sequences provided herein are 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174.

[0137] In one aspect, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 75% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the nucleic acid sequences provided herein encode polypeptides 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: 117 to 174. In one aspect, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 85% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 88% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 91% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 92% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 93% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 94% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 96% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 97% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 98% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174.In one aspect, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174.

[0138] In one aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 75% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the endogenous nucleic acid sequence provided herein encodes 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: 117 to 174. In one aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 85% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 88% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 91% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 92% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 93% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 94% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 96% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 97% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 98% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174.In one aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174.

[0139] Promoter

[0140] As generally understood in the art, the term "promoter" refers to a DNA sequence that contains an RNA polymerase binding site, a transcription start site and / or a TATA box and assists or promotes the transcription and expression of a related transcribable polynucleotide sequence and / or a gene (or transgenic). The promoter can be synthesized from, altered from, or derived from a known or naturally occurring promoter sequence or other promoter sequence. The promoter can also include a chimeric promoter comprising a combination of two or more heterologous sequences. Therefore, the promoter of the present application can include a variant of a promoter sequence that is similar but not identical to other promoter sequences known or provided herein.

[0141] Promoters that drive expression in all or most tissues of a plant are referred to as "constitutive" promoters. In one aspect, the constitutive promoter is selected from the group consisting of a cauliflower mosaic virus 35S promoter, an ubiquitin promoter, an actin promoter, an opine promoter, and an alcohol dehydrogenase promoter.

[0142] Promoters that drive expression during certain periods or stages of development are called "developmental" promoters.

[0143] A promoter that drives enhanced expression in certain tissues of an organism relative to other tissues of the organism is called a "tissue preferred" promoter. Therefore, a "tissue preferred" promoter causes relatively high or preferential expression in a specific tissue of a plant, but has a lower expression level in other tissues of the plant. As a non-limiting example, a root tissue preferred promoter shows higher activity in root tissue, but can also show activity in additional tissues such as stem, leaf and flower tissues, although at a lower level. A "tissue specific" promoter causes expression only in a specific tissue. As a non-limiting example, a root tissue specific promoter drives expression only in root tissue. On the one hand, a tissue specific promoter is a root tissue specific promoter. On the other hand, a tissue specific promoter is a root tissue preferred promoter.

[0144] In one aspect, the root tissue preferred promoter is the cassava vein mosaic virus (CsVMV) promoter.

[0145] An "inducible" promoter is one that initiates transcription in response to an environmental stimulus, such as heat, cold, drought, light, or other stimulus, such as wounding or chemical application.

[0146] In one aspect, the promoter provided herein is a constitutive promoter. In another aspect, the promoter provided herein is an inducible promoter. In another aspect, the promoter provided herein is a developmental promoter. In another aspect, the promoter is a tissue-preferred promoter or a tissue-specific promoter. In another aspect, the promoter is selected from the group consisting of a constitutive promoter, a tissue-preferred promoter, a tissue-specific promoter, and an inducible promoter.

[0147] In one aspect, the disclosure provides a heterologous promoter. In another aspect, the disclosure provides a promoter operably linked to a heterologous polynucleotide. In another aspect, the disclosure provides a polynucleotide sequence operably linked to a heterologous promoter.

[0148] As used herein, "operably linked" refers to a functional connection between two or more elements. For example, an operable connection between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional connection that allows expression of the polynucleotide of interest. Operably linked elements can be continuous or discontinuous. On the one hand, a promoter provided herein is operably linked to a heterologous nucleic acid molecule.

[0149] Small RNA molecules

[0150] In one aspect, the nucleic acid molecules provided herein are small RNA molecules. In another aspect, the nucleic acid molecules encode small RNA molecules.

[0151] As used herein, "small RNA molecule" refers to a non-coding RNA molecule with a length between 16 nucleotides and 50 nucleotides. On the one hand, the small RNA molecule comprises 16 nucleotides to 40 nucleotides. On the other hand, the small RNA molecule comprises 16 nucleotides to 30 nucleotides. On the other hand, the small RNA molecule comprises 18 nucleotides to 50 nucleotides. On the other hand, the small RNA molecule comprises 18 nucleotides to 40 nucleotides. On the other hand, the small RNA molecule comprises 18 nucleotides to 30 nucleotides. On the other hand, the small RNA molecule comprises 18 nucleotides to 25 nucleotides. On the other hand, the small RNA molecule comprises 20 nucleotides to 28 nucleotides. On the other hand, the small RNA molecule comprises 20 nucleotides to 24 nucleotides. On the other hand, the small RNA molecule comprises 21 nucleotides to 23 nucleotides. In another aspect, the small RNA molecule comprises 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, or 50 nucleotides.

[0152] In one aspect, the small RNA molecule is selected from the group consisting of double-stranded RNA, small interfering RNA (siRNA), trans-acting siRNA, and micro RNA (miRNA).

[0153] miRNAs typically have about 19 to about 25 nucleotides (usually about 20 to 24 nucleotides in plants) that direct the trans-cleavage of target transcripts, negatively regulating the expression of genes involved in various regulatory and developmental pathways. In some cases, miRNAs are used to direct the co-processing of siRNA primary transcripts.

[0154] It is understood in the art that in plants, miRNAs and targeting nucleic acids generally do not have perfect complementarity (although miRNAs and targeting nucleic acids can have perfect complementarity). There may be multiple mismatches between a miRNA and its target, but still enable the miRNA to reduce the expression and / or function of the target gene. See, for example, Liu et al., Plant Cell, 26:741-753 (2014) and Wang et al., Curr. Opin. Plant Biol., 27:118-124 (2015).

[0155] In one aspect, the small RNA molecule comprises 100% complementarity with a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 58. In one aspect, the small RNA molecule comprises at least 95% complementarity over 21 consecutive nucleotides of a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 58. In one aspect, the small RNA molecule comprises at least 90% complementarity over 21 consecutive nucleotides of a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 58. In one aspect, the small RNA molecule comprises at least 85% complementarity over 21 consecutive nucleotides of a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 58. In one aspect, the small RNA molecule comprises at least 95% complementarity over 20 consecutive nucleotides of a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 58. In one aspect, the small RNA molecule comprises at least 90% complementarity over 20 consecutive nucleotides of a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 58. In one aspect, the small RNA molecule comprises at least 85% complementarity over 20 consecutive nucleotides of a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 58. In one aspect, the small RNA molecule comprises at least 95% complementarity over 19 consecutive nucleotides of a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 58. In one aspect, the small RNA molecule comprises at least 90% complementarity over 19 consecutive nucleotides of a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 58. In one aspect, the small RNA molecule comprises at least 85% complementarity over 19 consecutive nucleotides of a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 58.

[0156] Many microRNA genes (MIR genes) have been identified and are publicly available in databases ("miRBase", available online at microrna[dot]sanger[dot]ac[dot]uk / sequences; see also, Griffiths-Jones et al. (2003) Nucleic Acids Res., 31:439-441). MIR genes are reported to occur in intergenic regions of the genome, either isolated or clustered, but may also be located completely 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. At least in some cases, transcription of MIR genes can be mediated by the MIR gene's own promoter. The primary transcript (called "pri-miRNA") can be very large (several kilobases) and can be polycistronic, containing one or more pre-miRNAs (folded structures containing a stem-loop arrangement that are processed into mature miRNAs) as well as the common 5' "cap" and polyadenylation tail of the mRNA.

[0157] The maturation of mature miRNAs from their corresponding precursors (pri-miRNA and pre-miRNA) differs significantly between animals and plants. For example, in plant cells, microRNA precursor molecules are thought to be processed largely completely in the nucleus into mature miRNAs, while in animal cells, pri-miRNA transcripts are processed in the nucleus by the animal-specific enzyme Drosha, followed by export of pre-miRNAs to the cytoplasm, where they are further processed into mature miRNAs. Mature miRNAs in plants are typically 21 nucleotides in length.

[0158] The transgenic expression of miRNA (whether naturally occurring sequence or artificial sequence) can be used to regulate the expression of one or more target genes of miRNA. Including miRNA recognition site in the transcript of transgenic expression is also useful for regulating the expression of transcript. The recognition site of miRNA has been verified in all regions of mRNA, including 5' untranslated region, coding region and 3' untranslated region, indicating that the position of miRNA target site relative to coding sequence may not necessarily affect inhibition. Since miRNA is an important regulatory element in eukaryotes, the transgenic inhibition of miRNA is useful for manipulating biological pathways and responses. Finally, the promoter of MIR gene can have a very specific expression pattern (for example, cell-specific, tissue-specific, time-specific or inducible), and therefore in the recombinant construct, it is useful to induce such specific transcription of the DNA sequence operably connected to them. The various uses of miRNA, its precursor, its recognition site and its promoter are described in detail in U.S. Patent Application Publication 2006 / 0200878A1, which is incorporated herein by reference. Non-limiting examples of these uses include: (1) expressing natural miRNA or miRNA precursor sequences to inhibit target genes; (2) expressing artificial miRNA or miRNA precursor sequences to inhibit target genes; (3) expressing transgenes with miRNA recognition sites, wherein the transgene is inhibited when the mature miRNA is expressed; (4) expressing transgenes driven by miRNA promoters.

[0159] Designing an artificial miRNA sequence can be as simple as substituting a sequence complementary to the intended target for nucleotides in the miRNA stem region of a miRNA precursor, as demonstrated by Zeng et al. (2002) Mol. Cell, 9: 1327-1333. A non-limiting example of a general method for determining nucleotide changes in a natural miRNA sequence to generate an engineered miRNA precursor includes the following steps: (a) selecting a unique target sequence of at least 18 nucleotides that is specific for a target gene (e.g., from both a tobacco cDNA database and a genomic DNA database), for example, by using a sequence alignment tool 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) to identify target transcript homologs and any potential matches to unrelated genes, thereby avoiding unintentional silencing of non-target sequences; (b) analyzing the target gene for unwanted sequences (e.g., matches to sequences from non-target species) and performing a GC content, Reynolds score (see, Reynolds et al. (2004) Nature 55:3389-3402) analysis of each potential 19-mer fragment. Biotechnol., 22:326-330) and functional asymmetry characterized by a 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 features 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) location close to the 3' end of the target gene; (7) minimal difference from the miRNA precursor transcript. It is reported that the position of every three nucleotides in siRNA is particularly important for affecting RNAi efficacy and algorithm, "siExplorer" is publicly available at rna[dot]chem[dot]t[dot]u-tokyo[dot]ac[dot]jp / siexplorer.htm (see, Katoh and Suzuki (2007) Nucleic Acids Res., 10.1093 / nar / gkl1120); (c) determining the reverse complement of the selected 19-mer for preparing modified mature miRNA. The additional nucleotide at position 20 is preferably matched to the selected target sequence, and the nucleotide at position 21 is preferably selected to be unpaired to prevent the spread of silencing on the target transcript or paired with the target sequence to promote the spread of silencing on the target transcript; and (d) transforming the artificial miRNA into plants.

[0160] Without being limited by any scientific theory, it is understood in the art that RNAi knockdown of a candidate gene (e.g., via the use of artificial miRNA or siRNA) and mutations in the same candidate gene (e.g., missense mutations or nonsense mutations) can result in reduced expression and / or reduced protein activity and can also result in the same or similar phenotype in plants. See, e.g., Agrawal et al., Microbiology and Molecular Biology Reviews, 67:657-685 (2003).

[0161] In one aspect, the small RNA provided herein comprises a nucleic acid sequence that is at least 75% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In one aspect, the small RNA provided herein comprises a nucleic acid sequence that is at least 80% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In one aspect, the small RNA provided herein comprises a nucleic acid sequence that is at least 85% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In one aspect, the small RNA provided herein comprises a nucleic acid sequence that is at least 90% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In one aspect, the small RNA provided herein comprises a nucleic acid sequence that is at least 95% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In one aspect, the small RNA provided herein comprises a nucleic acid sequence that is at least 96% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In one aspect, the small RNA provided herein comprises a nucleic acid sequence that is at least 97% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In one aspect, the small RNA provided herein comprises a nucleic acid sequence that is at least 98% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In one aspect, the small RNA provided herein comprises a nucleic acid sequence that is at least 99% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In one aspect, the small RNA provided herein comprises a nucleic acid sequence that is 100% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 116.

[0162] In one aspect, the small RNA provided herein comprises a nucleic acid sequence that is at least 75% complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59 to 116. In one aspect, the small RNA provided herein comprises a nucleic acid sequence that is at least 80% complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59 to 116. In one aspect, the small RNA provided herein comprises a nucleic acid sequence that is at least 85% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59 to 116. In one aspect, the small RNA provided herein comprises a nucleic acid sequence that is at least 90% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59 to 116. In one aspect, the small RNA provided herein comprises a nucleic acid sequence that is at least 95% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59 to 116. In one aspect, the small RNA provided herein comprises a nucleic acid sequence that is at least 96% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59 to 116. In one aspect, the small RNA provided herein comprises a nucleic acid sequence that is at least 97% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59 to 116. In one aspect, the small RNA provided herein comprises a nucleic acid sequence that is at least 98% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59 to 116. In one aspect, the small RNA provided herein comprises a nucleic acid sequence that is at least 99% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59 to 116. In one aspect, the small RNA provided herein comprises a nucleic acid sequence that is 100% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59 to 116.

[0163] On the one hand, the small RNA provided herein comprises a nucleic acid sequence that is at least 88.7% identical or complementary to at least 18 consecutive nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 116. On the other hand, the small RNA provided herein comprises a nucleic acid sequence that is at least 94.3% identical or complementary to at least 18 consecutive nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 116. On the other hand, the small RNA provided herein comprises a nucleic acid sequence that is 100% identical or complementary to at least 18 consecutive nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 116. On the other hand, the small RNA provided herein comprises a nucleic acid sequence that is at least 85% identical or complementary to at least 20 consecutive nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 116. On the other hand, the small RNA provided herein comprises a nucleic acid sequence that is at least 90% identical or complementary to at least 20 consecutive nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 116. In another aspect, the small RNA provided herein comprises a nucleic acid sequence that is at least 95% identical or complementary to at least 20 consecutive nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the small RNA provided herein comprises a nucleic acid sequence that is 100% identical or complementary to at least 20 consecutive nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 116.

[0164] In one aspect, the small RNA molecules provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the small RNA molecules provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 75% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the small RNA molecules provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the small RNA molecules provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 85% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the small RNA molecules provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 88% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the small RNA molecules provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the small RNA molecules provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 91% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the small RNA molecules provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 92% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the small RNA molecules provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 93% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the small RNA molecules provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 94% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the small RNA molecules provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the small RNA molecules provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 96% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174.In one aspect, the small RNA molecules provided herein are capable of binding to a nucleic acid sequence encoding a polypeptide at least 97% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174 and reducing its expression. In one aspect, the small RNA molecules provided herein are capable of binding to a nucleic acid sequence encoding a polypeptide at least 98% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174 and reducing its expression. In one aspect, the small RNA molecules provided herein are capable of binding to a nucleic acid sequence encoding a polypeptide at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174 and reducing its expression. In one aspect, the small RNA molecules provided herein are capable of binding to a nucleic acid sequence encoding a polypeptide 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174 and reducing its expression.

[0165] On the one hand, the small RNA molecules provided herein can bind to and reduce the expression of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 116 or a nucleic acid sequence at least 70% identical to the RNA transcribed therefrom. On the one hand, the small RNA molecules provided herein can bind to and reduce the expression of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 116 or a nucleic acid sequence at least 75% identical to the RNA transcribed therefrom. On the one hand, the small RNA molecules provided herein can bind to and reduce the expression of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 116 or a nucleic acid sequence at least 80% identical to the RNA transcribed therefrom. On the one hand, the small RNA molecules provided herein can bind to and reduce the expression of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 116 or a nucleic acid sequence at least 85% identical to the RNA transcribed therefrom. On the one hand, the small RNA molecules provided herein can bind to and reduce the expression of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 116 or a nucleic acid sequence at least 88% identical to the RNA transcribed therefrom. On the one hand, the small RNA molecules provided herein can bind to and reduce the expression of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 116 or a nucleic acid sequence at least 90% identical to the RNA transcribed therefrom. On the one hand, the small RNA molecules provided herein can bind to and reduce the expression of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 116 or a nucleic acid sequence at least 91% identical to the RNA transcribed therefrom. On the one hand, the small RNA molecules provided herein can bind to and reduce the expression of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 116 or a nucleic acid sequence at least 92% identical to the RNA transcribed therefrom. On the one hand, the small RNA molecules provided herein can bind to and reduce the expression of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 116 or a nucleic acid sequence at least 93% identical to the RNA transcribed therefrom. On the one hand, the small RNA molecules provided herein can bind to and reduce the expression of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 116 or a nucleic acid sequence at least 94% identical to the RNA transcribed therefrom. In one aspect, the small RNA molecules provided herein are capable of binding to and reducing the expression of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 116 or a nucleic acid sequence at least 95% identical to the RNA transcribed therefrom. In one aspect, the small RNA molecules provided herein are capable of binding to and reducing the expression of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 116 or a nucleic acid sequence at least 96% identical to the RNA transcribed therefrom. In one aspect, the small RNA molecules provided herein are capable of binding to and reducing the expression of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 116 or a nucleic acid sequence at least 97% identical to the RNA transcribed therefrom.In one aspect, the small RNA molecules provided herein are capable of binding to and reducing the expression of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 116 or a nucleic acid sequence at least 98% identical to the RNA transcribed therefrom. In one aspect, the small RNA molecules provided herein are capable of binding to and reducing the expression of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 116 or a nucleic acid sequence at least 99% identical to the RNA transcribed therefrom. In one aspect, the small RNA molecules provided herein are capable of binding to and reducing the expression of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 116 or a nucleic acid sequence 100% identical to the RNA transcribed therefrom.

[0166] As used herein, "capable of binding to" is synonymous with "capable of hybridizing to". On the one hand, a first nucleic acid molecule capable of binding to a second nucleic acid molecule binds to the second nucleic acid molecule. As used herein, under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength, a first nucleic acid molecule can "hybridize" with a second nucleic acid molecule (i.e., nucleic acid specifically binds to a complementary nucleic acid) in a sequence-specific, antiparallel manner via non-covalent interactions (e.g., Watson-Crick base pairing). As known in the art, standard Watson-Crick base pairing includes: adenine and thymine pairing, adenine and uracil pairing, and guanine (G) and cytosine (C) pairing [DNA, RNA]. In addition, it is also known in the art that for hybridization between two RNA molecules (e.g., dsRNA), guanine bases are paired with uracil. For example, in the context of tRNA anticodons and codon base pairing in mRNA, G / U base pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code. In the context of the present disclosure, a guanine of a protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule is considered complementary to a uracil, and vice versa. Thus, when a G / U base pair can be formed at a given nucleotide position of a protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule, that position is not considered non-complementary, but rather complementary.

[0167] Hybridization and washing conditions are well known and are described in 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 therein; and Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). Conditions of temperature and ionic strength determine the "stringency" of hybridization.

[0168] Hybridization requires that the two nucleic acids contain complementary sequences, but mismatches between bases are possible. The conditions suitable for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity (variables well known in the art). The greater the degree of complementarity between two nucleotide sequences, the greater the melting temperature (Tm) value of the nucleic acid hybrid with these sequences. For hybridization between nucleic acids with short fragment complementarity (for example, more than 35 or less nucleotides of complementarity), the position of mismatch becomes very important (see, Sambrook et al.). Generally, the length of hybridizable nucleic acids is at least about 10 nucleotides. The exemplary minimum length of hybridizable nucleic acids is: 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). In addition, the technician will recognize that the temperature and washing solution salt concentration can be adjusted as needed according to factors such as the length and degree of complementarity of the complementary region.

[0169] In the art, it is understood that the sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid to be specifically hybridizable or hybridizable. In addition, a polynucleotide may hybridize on one or more segments such that an intervening or adjacent segment does not participate in the hybridization event (e.g., a loop structure or a hairpin structure). For example, an antisense nucleic acid in which 18 of the 20 nucleotides of the antisense compound are complementary to the target region and will therefore specifically hybridize would represent 90% complementarity. In this example, the remaining non-complementary nucleotides may be clustered or interspersed with the complementary nucleotides and need not be adjacent to each other or to the complementary nucleotides. The percentage of complementarity between specific segments of nucleic acid sequences within a nucleic acid can be measured using 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 by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, using the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489) to determine conventionally.

[0170] Reduced expression / activity

[0171] In one aspect, the small RNA molecule reduces the expression of any nucleic acid sequence to which it is able to bind. In another aspect, the non-natural mutations provided herein reduce the expression of the mutant nucleic acid sequence compared to the non-mutated nucleic acid sequence in a control plant when grown under comparable conditions.

[0172] The expression reduction 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 (e.g., Northern blotting), RNA sequencing. Expression differences can be described as absolute quantification or relative quantification. See, e.g., Livak and Schmittgen, Methods, 25:402-408 (2001). If the endogenous nucleic acid sequence encodes a protein, the variation of expression can be inferred by checking the accumulation of the encoded protein. Non-limiting examples of measuring protein accumulation include Western blotting and enzyme-linked immunosorbent assay (ELISA).

[0173] In one aspect, qRT-PCR is used to measure the reduction in expression. In another aspect, Northern blot is used to measure the reduction in expression. In another aspect, RNA sequencing is used to measure the reduction in expression. In another aspect, Western blot is used to measure the reduction in expression. In yet another aspect, ELISA is used to measure the reduction in expression.

[0174] In one aspect, a non-natural mutation in a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 116 results in expression of a nucleic acid sequence that is reduced in level compared to a nucleic acid sequence lacking the non-natural mutation in a control plant when grown under comparable conditions. In one aspect, a non-natural mutation in a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174 results in expression of a nucleic acid sequence that is reduced in level compared to a nucleic acid sequence lacking the non-natural mutation in a control plant when grown under comparable conditions.

[0175] On the one hand, the reduction in expression comprises a reduction of at least 1% compared to expression in the same tissue of a control plant when grown under comparable conditions. On the other hand, the reduction in expression comprises a reduction of at least 5% compared to expression in the same tissue of a control plant when grown under comparable conditions. On the other hand, the reduction in expression comprises a reduction of at least 10% compared to expression in the same tissue of a control plant when grown under comparable conditions. On the other hand, the reduction in expression comprises a reduction of at least 25% compared to expression in the same tissue of a control plant when grown under comparable conditions. On the other hand, the reduction in expression comprises a reduction of at least 50% compared to expression in the same tissue of a control plant when grown under comparable conditions. On the other hand, the reduction in expression comprises a reduction of at least 75% compared to expression in the same tissue of a control plant when grown under comparable conditions. On the other hand, the reduction in expression comprises a reduction of at least 90% compared to expression in the same tissue of a control plant when grown under comparable conditions. On the other hand, the reduction in expression comprises a reduction of at least 95% compared to expression in the same tissue of a control plant when grown under comparable conditions.

[0176] On the one hand, the reduction in expression includes a reduction between 1% and 99% compared to the expression in the same tissue of the control plant when grown under comparable conditions. On the other hand, the reduction in expression includes a reduction between 1% and 90% compared to the expression in the same tissue of the control plant when grown under comparable conditions. On the other hand, the reduction in expression includes a reduction between 1% and 75% compared to the expression in the same tissue of the control plant when grown under comparable conditions. On the other hand, the reduction in expression includes a reduction between 1% and 50% compared to the expression in the same tissue of the control plant when grown under comparable conditions. On the other hand, the reduction in expression includes a reduction between 1% and 25% compared to the expression in the same tissue of the control plant when grown under comparable conditions. On the other hand, the reduction in expression includes a reduction between 25% and 90% compared to the expression in the same tissue of the control plant when grown under comparable conditions. On the other hand, the reduction in expression includes a reduction between 50% and 90% compared to the expression in the same tissue of the control plant when grown under comparable conditions. In another aspect, the reduction in expression comprises a reduction of between 25% and 75% compared to expression in the same tissue of a control plant when grown under comparable conditions.

[0177] In one aspect, the reduction in expression comprises a statistically significant reduction compared to the expression in the same tissue of a control plant when grown under comparable conditions. One of ordinary skill in the art will recognize that any reduction level is foreseeable, as long as the reduction level has been determined to be statistically significant using a recognized statistical hypothesis test. As a non-limiting example, a student's t-test is a statistical hypothesis test that can be used to determine whether the reduction in expression between a modified plant and a control plant is statistically significant. As used herein, "statistically significant" refers to a p-value less than or equal to 0.05.

[0178] On the one hand, non-natural mutation causes the activity of the protein or polypeptide encoded by the nucleic acid sequence provided herein compared with the activity of the control plant when growing under comparable conditions.On the other hand, the non-natural mutation in the endogenous nucleic acid sequence selected from the group consisting of SEQ ID NO:1 to 116 is compared with the activity of the protein or polypeptide encoded by the endogenous nucleic acid sequence in the control tobacco plant when growing under comparable conditions and has reduced the activity level of the protein or polypeptide encoded by the nucleic acid sequence, wherein this nucleic acid sequence lacks non-natural mutation in the control tobacco plant.On the other hand, the non-natural mutation in the endogenous nucleic acid sequence (wherein this endogenous nucleic acid sequence encodes the group amino acid sequence selected from SEQ ID NO:117 to 174) is compared with the activity of the protein or polypeptide encoded by the endogenous nucleic acid sequence in the control tobacco plant when growing under comparable conditions and has reduced the activity level of the protein or polypeptide encoded by the nucleic acid sequence, wherein this nucleic acid sequence lacks non-natural mutation in the control tobacco plant.

[0179] As used herein, "activity" when referring to a protein or polypeptide refers to the ability to perform an enzymatic function.

[0180] Increased expression / activity

[0181] On the one hand, non-natural mutation causes the expression of the increase of nucleotide sequence.On the one hand, non-natural mutation causes the expression of the described nucleotide sequence of increasing level compared with the expression of the described nucleotide sequence in the same tissue of the control tobacco plant when growing under comparable conditions, wherein said nucleotide sequence lacks at least one non-natural mutation in the described control tobacco plant.

[0182] In one aspect, the increased expression level comprises an increase of at least 5% compared to the expression in the same tissue of a control plant when grown under comparable conditions. In another aspect, the increased expression level comprises an increase of at least 10% compared to the expression in the same tissue of a control plant when grown under comparable conditions. In another aspect, the increased expression level comprises an increase of at least 25% compared to the expression in the same tissue of a control plant when grown under comparable conditions. In another aspect, the increased expression level comprises an increase of at least 50% compared to the expression in the same tissue of a control plant when grown under comparable conditions. In another aspect, the increased expression level comprises an increase of at least 75% compared to the expression in the same tissue of a control plant when grown under comparable conditions. In another aspect, the increased expression level comprises an increase of at least 100% compared to the expression in the same tissue of a control plant when grown under comparable conditions. In another aspect, the increased expression level comprises an increase of at least 200% compared to the expression in the same tissue of a control plant when grown under comparable conditions. In another aspect, increased expression levels comprise an increase of at least 500% compared to expression in the same tissue of a control plant when grown under comparable conditions.

[0183] In one aspect, the non-natural mutation results in an increased level of activity of a 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 when grown under comparable conditions, wherein the nucleic acid sequence lacks at least one non-natural mutation in the control tobacco plant.

[0184] Alkaloids

[0185] As used herein, "alkaloid" refers to a complex nitrogen-containing compound that occurs naturally in tobacco and has pharmacological effects on humans or other animals. Non-limiting examples of alkaloids include nicotine, anabasine, anatabine, and nornicotine.

[0186] Nicotine is the main natural alkaloid in commercial cigarette tobacco, and it accounts for about 90% of the alkaloid content in red tobacco. Without limitation, other major alkaloids in tobacco include cotinine, nornicotine, myosamine, dienicotine, anabasine and anatabine. Without limitation, minor tobacco alkaloids include n-oxynicotine, N-methyl anatabine, N-methyl anabasine, pseudooxynicotine, 2,3-bipyridine, etc.

[0187] In one aspect, the alkaloid is selected from the group consisting of anatabine, anatabine, nicotine and nornicotine.

[0188] Alkaloid levels can be measured by methods known in the art, for example, by quantification based on gas-liquid chromatography, high performance liquid chromatography, radioimmunoassay, enzyme-linked immunosorbent assay and mass spectrometry. For example, nicotinic alkaloid levels can be measured by GC-FID methods based on CORESTA recommended method No. 7, 1987 and ISO standard (ISO TC 126N 394E). Regarding the method of using gas-liquid chromatography equipped with a capillary column and a FID detector, see also, Hibi et al., Plant Physiology 100: 826-35 (1992).

[0189] Alkaloid levels can be determined from any plant tissue. Non-limiting examples include leaf and hairy root cultures. In addition, alkaloid levels can be determined in conditioned plant material or in plant material that has not yet been conditioned.

[0190] Alternatively, segmented flow colorimetry can be used to measure tobacco total alkaloids, which is developed for analyzing tobacco samples, as adapted by Skalar Instrument Co. (West Chester, PA) and by people such as Collins, Tobacco Science 13:79-81 (1969) described. In short, before analyzing total alkaloids and reducing sugars, tobacco samples are dried, ground and extracted. The method then adopts acetic acid / methanol / water extraction and charcoal decolorization. The mensuration of total alkaloids is based on the reaction of cyanogen chloride and nicotine alkaloids in the presence of aromatic amines to form colored complexes (it measures at 460nm).

[0191] In one aspect, liquid chromatography and tandem mass spectrometry (LC / MS / MS) are used to measure the levels of individual alkaloids based on freeze-dried modulated leaf samples. In one aspect, liquid chromatography and tandem mass spectrometry (LC / MS / MS) are used to measure the levels of individual alkaloids based on freeze-dried hairy root culture samples.

[0192] Unless otherwise stated, the nicotine or alkaloid level (or another leaf chemistry or property characterization) of tobacco plants is measured in mixed leaf samples collected from the 3rd, 4th and 5th leaves after topping after topping. As used herein, whenever a comparison between leaves from two plants (e.g., mutant plants and control plants) is mentioned, leaves from identical or comparable stem positions and developmental stages are meant so that the comparison can demonstrate the impact caused by genotype differences rather than other factors. As a non-limiting illustration, leaf 3 of the control plant is intended to be used as a reference point for comparison with leaf 3 of a modified plant comprising a non-natural mutation or a recombinant DNA construct.

[0193] As used herein, leaf numbering is based on the leaf position on the tobacco stem, with leaf number 1 being the youngest leaf (at the top) after topping, and the highest leaf number being assigned to the oldest leaf (at the bottom).

[0194] As used herein, " topping " refers to when tobacco plant approaches vegetative maturity and when reproductive growth begins left and right, removes the shoot apex, comprises shoot apex meristem, flower and several adjacent leaves at most.Usually, tobacco plant topping is carried out in button stage (soon after the flowers begin to appear).For example, when 50% of the plant had at least one open flower, the tobacco plant of greenhouse or field planting can be toppled.Topping can cause the loss of apical dominance to tobacco plant, and also can induce alkaloid production to increase.

[0195] In some embodiments, the alkaloid level of tobacco plant is measured in about 2 weeks after topping (or another leaf chemistry or characterization; For example, polyamines). Other time points can also be used. On the one hand, the alkaloid level of tobacco plant is measured in about 1,2,3,4 or 5 weeks after topping (or another leaf chemistry or characterization). On the other hand, the nicotine, alkaloid or polyamine level of tobacco plant is measured in about 3,5,7,10,12,14,17,19 or 21 days after topping (or another leaf chemistry or characterization; For example, polyamines).

[0196] In one aspect, a modified tobacco plant, or part thereof, comprising at least one non-natural mutation in an endogenous nucleic acid produces at least one leaf comprising a reduced amount of at least one alkaloid as compared to the amount of the alkaloid in a control tobacco plant lacking the at least one non-natural mutation in the endogenous nucleic acid when grown under comparable conditions.

[0197] In one aspect, a modified tobacco plant or part thereof comprising a recombinant DNA construct provided herein produces at least one leaf comprising a reduced amount of at least one alkaloid as compared to the amount of the alkaloid in a control tobacco plant lacking the recombinant DNA construct when grown under comparable conditions.

[0198] On the one hand, at least one alkaloid of the reduction level comprises at least 0.5% reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises at least 1% reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises at least 2% reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises at least 3% reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises at least 4% reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises at least 5% reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises at least 10% reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises at least 15% reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises and compares at least 20% reduction with the control tobacco plant when growing under comparable conditions.On the one hand, at least one alkaloid of reduction level comprises and compares at least 25% reduction with the control tobacco plant when growing under comparable conditions.On the one hand, at least one alkaloid of reduction level comprises and compares at least 35% reduction with the control tobacco plant when growing under comparable conditions.On the one hand, at least one alkaloid of reduction level comprises and compares at least 50% reduction with the control tobacco plant when growing under comparable conditions.On the one hand, at least one alkaloid of reduction level comprises and compares at least 75% reduction with the control tobacco plant when growing under comparable conditions.On the one hand, at least one alkaloid of reduction level comprises and compares at least 90% reduction with the control tobacco plant when growing under comparable conditions.

[0199] On the one hand, at least one alkaloid of the reduction level comprises a reduction of at least 1% to 99% compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises a reduction of at least 1% to 90% compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises a reduction of at least 1% to 80% compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises a reduction of at least 1% to 70% compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises a reduction of at least 1% to 60% compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises a reduction of at least 1% to 50% compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises a reduction of at least 1% to 40% compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises a reduction of at least 1% to 30% compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises a reduction of at least 1% to 20% compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises a reduction of at least 1% to 10% compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises a reduction of at least 1% to 5% compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises a reduction of at least 10% to 75% compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises a reduction of at least 10% to 50% compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises a reduction of at least 10% to 25% compared with the control tobacco plant when growing under comparable conditions.

[0200] On the one hand, at least one alkaloid of the reduction level comprises at least 0.5% nicotine reduction compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises at least 1% nicotine reduction compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises at least 2% nicotine reduction compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises at least 3% nicotine reduction compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises at least 4% nicotine reduction compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises at least 5% nicotine reduction compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises at least 10% nicotine reduction compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of the reduction level comprises at least 15% nicotine reduction compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises at least 20% nicotine reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises at least 25% nicotine reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises at least 35% nicotine reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises at least 50% nicotine reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises at least 75% nicotine reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises at least 90% nicotine reduction compared with the control tobacco plant when growing under comparable conditions.

[0201] On the one hand, at least one alkaloid of reduction level comprises that the nicotine reduction between 1% and 99% is compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises that the nicotine reduction between 1% and 90% is compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises that the nicotine reduction between 1% and 80% is compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises that the nicotine reduction between 1% and 70% is compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises that the nicotine reduction between 1% and 60% is compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises that the nicotine reduction between 1% and 50% is compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises that the nicotine reduction between 1% and 40% is compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises that the nicotine reduction between 1% and 30% is compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises that the nicotine reduction between 1% and 20% is compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises that the nicotine reduction between 1% and 10% is compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises that the nicotine reduction between 1% and 5% is compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises that the nicotine reduction between 10% and 75% is compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises that the nicotine reduction between 10% and 50% is compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises that the nicotine reduction between 10% and 25% is compared with the control tobacco plant when growing under comparable conditions.

[0202] On the one hand, the modified tobacco plant comprises a nicotine level less than or equal to 17 milligrams per gram (mg / g) dry weight. On the one hand, the modified tobacco plant comprises a nicotine level less than or equal to 16mg / g dry weight. On the one hand, the modified tobacco plant comprises a nicotine level less than or equal to 15mg / g dry weight. On the one hand, the modified tobacco plant comprises a nicotine level less than or equal to 14mg / g dry weight. On the one hand, the modified tobacco plant comprises a nicotine level less than or equal to 13mg / g dry weight. On the one hand, the modified tobacco plant comprises a nicotine level less than or equal to 12mg / g dry weight. On the one hand, the modified tobacco plant comprises a nicotine level less than or equal to 11mg / g dry weight. On the one hand, the modified tobacco plant comprises a nicotine level less than or equal to 10mg / g dry weight. On the one hand, the modified tobacco plant comprises a nicotine level less than or equal to 9mg / g dry weight. On the one hand, the modified tobacco plant comprises a nicotine level less than or equal to 8mg / g dry weight. On the one hand, the modified tobacco plant comprises a nicotine level less than or equal to 7mg / g dry weight. On the one hand, the modified tobacco plant comprises a nicotine level less than or equal to 6mg / g dry weight. On the one hand, the modified tobacco plant comprises a nicotine level less than or equal to 5mg / g dry weight. On the one hand, the modified tobacco plant comprises a nicotine level less than or equal to 4mg / g dry weight. On the one hand, the modified tobacco plant comprises a nicotine level less than or equal to 3mg / g dry weight. On the one hand, the modified tobacco plant comprises a nicotine level less than or equal to 2mg / g dry weight. On the one hand, the modified tobacco plant comprises a nicotine level less than or equal to 1.5mg / g dry weight. On the one hand, the modified tobacco plant comprises a nicotine level less than or equal to 1mg / g dry weight.

[0203] On the one hand, at least one alkaloid of reducing level comprises at least 0.5% anatabine reduction compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises at least 1% anatabine reduction compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises at least 2% anatabine reduction compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises at least 3% anatabine reduction compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises at least 4% anatabine reduction compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises at least 5% anatabine reduction compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises at least 10% anatabine reduction compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises at least 15% anatabine reduction compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises at least 20% anatabine reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises at least 25% anatabine reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises at least 35% anatabine reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises at least 50% anatabine reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises at least 75% anatabine reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises at least 90% anatabine reduction compared with the control tobacco plant when growing under comparable conditions.

[0204] On the one hand, at least one alkaloid of the reduction level includes anatabine reduction between 1% and 99% compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of the reduction level includes anatabine reduction between 1% and 90% compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of the reduction level includes anatabine reduction between 1% and 80% compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of the reduction level includes anatabine reduction between 1% and 70% compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of the reduction level includes anatabine reduction between 1% and 60% compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of the reduction level includes anatabine reduction between 1% and 50% compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of the reduction level includes anatabine reduction between 1% and 40% compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises anatabine reduction between 1% and 30% compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises anatabine reduction between 1% and 20% compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises anatabine reduction between 1% and 10% compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises anatabine reduction between 1% and 5% compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises anatabine reduction between 10% and 75% compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises anatabine reduction between 10% and 50% compared with the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises anatabine reduction between 10% and 25% compared with the control tobacco plant when grown under comparable conditions.

[0205] On the one hand, the modified tobacco plant comprises a anatabine level less than or equal to 1.5mg / g dry weight. On the one hand, the modified tobacco plant comprises a anatabine level less than or equal to 1.4mg / g dry weight. On the one hand, the modified tobacco plant comprises a anatabine level less than or equal to 1.3mg / g dry weight. On the one hand, the modified tobacco plant comprises a anatabine level less than or equal to 1.2mg / g dry weight. On the one hand, the modified tobacco plant comprises a anatabine level less than or equal to 1.1mg / g dry weight. On the one hand, the modified tobacco plant comprises a anatabine level less than or equal to 1.0mg / g dry weight. On the one hand, the modified tobacco plant comprises a anatabine level less than or equal to 0.9mg / g dry weight. On the one hand, the modified tobacco plant comprises a anatabine level less than or equal to 0.8mg / g dry weight. On the one hand, the modified tobacco plant comprises a anatabine level less than or equal to 0.7mg / g dry weight. On the one hand, the modified tobacco plant comprises a anatabine level less than or equal to 0.6mg / g dry weight. On the one hand, the modified tobacco plant comprises a anatabine level that is less than or equal to 0.5mg / g dry weight. On the one hand, the modified tobacco plant comprises a anatabine level that is less than or equal to 0.4mg / g dry weight. On the one hand, the modified tobacco plant comprises a anatabine level that is less than or equal to 0.3mg / g dry weight. On the one hand, the modified tobacco plant comprises a anatabine level that is less than or equal to 0.25mg / g dry weight. On the one hand, the modified tobacco plant comprises a anatabine level that is less than or equal to 0.2mg / g dry weight.

[0206] On the one hand, at least one alkaloid of the reduction level includes at least 0.5% of the neonicotinoid reduction compared to the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of the reduction level includes at least 1% of the neonicotinoid reduction compared to the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of the reduction level includes at least 2% of the neonicotinoid reduction compared to the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of the reduction level includes at least 3% of the neonicotinoid reduction compared to the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of the reduction level includes at least 4% of the neonicotinoid reduction compared to the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of the reduction level includes at least 5% of the neonicotinoid reduction compared to the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of the reduction level includes at least 10% of the neonicotinoid reduction compared to the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of the reduction level includes at least 15% of the neonicotinoid reduction compared to the control tobacco plant when grown under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises that compared with the control tobacco plant when growing under comparable conditions, at least 20% of neonicotinoid reduction is compared. On the one hand, at least one alkaloid of reducing level comprises that compared with the control tobacco plant when growing under comparable conditions, at least 25% of neonicotinoid reduction is compared. On the one hand, at least one alkaloid of reducing level comprises that compared with the control tobacco plant when growing under comparable conditions, at least 35% of neonicotinoid reduction is compared. On the one hand, at least one alkaloid of reducing level comprises that compared with the control tobacco plant when growing under comparable conditions, at least 50% of neonicotinoid reduction is compared. On the one hand, at least one alkaloid of reducing level comprises that compared with the control tobacco plant when growing under comparable conditions, at least 75% of neonicotinoid reduction is compared. On the one hand, at least one alkaloid of reducing level comprises that compared with the control tobacco plant when growing under comparable conditions, at least 90% of neonicotinoid reduction is compared.

[0207] On the one hand, at least one alkaloid of reducing level comprises that compared with the control tobacco plant when growing under comparable conditions, the neonicotinoid reduction between 1% and 99% is reduced. On the one hand, at least one alkaloid of reducing level comprises that compared with the control tobacco plant when growing under comparable conditions, the neonicotinoid reduction between 1% and 90% is reduced. On the one hand, at least one alkaloid of reducing level comprises that compared with the control tobacco plant when growing under comparable conditions, the neonicotinoid reduction between 1% and 80% is reduced. On the one hand, at least one alkaloid of reducing level comprises that compared with the control tobacco plant when growing under comparable conditions, the neonicotinoid reduction between 1% and 70% is reduced. On the one hand, at least one alkaloid of reducing level comprises that compared with the control tobacco plant when growing under comparable conditions, the neonicotinoid reduction between 1% and 60% is reduced. On the one hand, at least one alkaloid of reducing level comprises that compared with the control tobacco plant when growing under comparable conditions, the neonicotinoid reduction between 1% and 50% is reduced. On the one hand, at least one alkaloid of reducing level comprises that compared with the control tobacco plant when growing under comparable conditions, the neonicotinoid reduction between 1% and 40% is reduced. On the one hand, at least one alkaloid of reducing level comprises that compared with the control tobacco plant when growing under comparable conditions, 1% and 30% of neonicotinoids are reduced. On the one hand, at least one alkaloid of reducing level comprises that compared with the control tobacco plant when growing under comparable conditions, 1% and 20% of neonicotinoids are reduced. On the one hand, at least one alkaloid of reducing level comprises that compared with the control tobacco plant when growing under comparable conditions, 1% and 10% of neonicotinoids are reduced. On the one hand, at least one alkaloid of reducing level comprises that compared with the control tobacco plant when growing under comparable conditions, 1% and 5% of neonicotinoids are reduced. On the one hand, at least one alkaloid of reducing level comprises that compared with the control tobacco plant when growing under comparable conditions, 10% and 75% of neonicotinoids are reduced. On the one hand, at least one alkaloid of reducing level comprises that compared with the control tobacco plant when growing under comparable conditions, 10% and 50% of neonicotinoids are reduced. On the one hand, at least one alkaloid of reducing level comprises that compared with the control tobacco plant when growing under comparable conditions, 10% and 25% of neonicotinoids are reduced.

[0208] On the one hand, the modified tobacco plant comprises a neonicotinoid level that is less than or equal to 0.6mg / g dry weight. On the one hand, the modified tobacco plant comprises a neonicotinoid level that is less than or equal to 0.5mg / g dry weight. On the one hand, the modified tobacco plant comprises a neonicotinoid level that is less than or equal to 0.4mg / g dry weight. On the one hand, the modified tobacco plant comprises a neonicotinoid level that is less than or equal to 0.3mg / g dry weight. On the one hand, the modified tobacco plant comprises a neonicotinoid level that is less than or equal to 0.25mg / g dry weight. On the one hand, the modified tobacco plant comprises a neonicotinoid level that is less than or equal to 0.2mg / g dry weight. On the one hand, the modified tobacco plant comprises a neonicotinoid level that is less than or equal to 0.15mg / g dry weight. On the one hand, the modified tobacco plant comprises a neonicotinoid level that is less than or equal to 0.1mg / g dry weight.

[0209] On the one hand, at least one alkaloid of reducing level comprises at least 0.5% nornicotine reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises at least 1% nornicotine reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises at least 2% nornicotine reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises at least 3% nornicotine reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises at least 4% nornicotine reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises at least 5% nornicotine reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises at least 10% nornicotine reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reducing level comprises at least 15% nornicotine reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises at least 20% nornicotine reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises at least 25% nornicotine reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises at least 35% nornicotine reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises at least 50% nornicotine reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises at least 75% nornicotine reduction compared with the control tobacco plant when growing under comparable conditions. On the one hand, at least one alkaloid of reduction level comprises at least 90% nornicotine reduction compared with the control tobacco plant when growing under comparable conditions.

[0210] On the one hand, at least one alkaloid of reduction level comprises that compared with the control tobacco plant when growing under comparable conditions, 1% and 99% nornicotine reduction is compared. On the one hand, at least one alkaloid of reduction level comprises that compared with the control tobacco plant when growing under comparable conditions, 1% and 90% nornicotine reduction is compared. On the one hand, at least one alkaloid of reduction level comprises that compared with the control tobacco plant when growing under comparable conditions, 1% and 80% nornicotine reduction is compared. On the one hand, at least one alkaloid of reduction level comprises that compared with the control tobacco plant when growing under comparable conditions, 1% and 70% nornicotine reduction is compared. On the one hand, at least one alkaloid of reduction level comprises that compared with the control tobacco plant when growing under comparable conditions, 1% and 60% nornicotine reduction is compared. On the one hand, at least one alkaloid of reduction level comprises that compared with the control tobacco plant when growing under comparable conditions, 1% and 50% nornicotine reduction is compared. On the one hand, at least one alkaloid of reduction level comprises that compared with the control tobacco plant when growing under comparable conditions, 1% and 40% nornicotine reduction is compared. On the one hand, at least one alkaloid of reduction level comprises that compared with the control tobacco plant when growing under comparable conditions, 1% and 30% nornicotine reduction is compared. On the one hand, at least one alkaloid of reduction level comprises that compared with the control tobacco plant when growing under comparable conditions, 1% and 20% nornicotine reduction is compared. On the one hand, at least one alkaloid of reduction level comprises that compared with the control tobacco plant when growing under comparable conditions, 1% and 10% nornicotine reduction is compared. On the one hand, at least one alkaloid of reduction level comprises that compared with the control tobacco plant when growing under comparable conditions, 1% and 5% nornicotine reduction is compared. On the one hand, at least one alkaloid of reduction level comprises that compared with the control tobacco plant when growing under comparable conditions, 10% and 75% nornicotine reduction is compared. On the one hand, at least one alkaloid of reduction level comprises that compared with the control tobacco plant when growing under comparable conditions, 10% and 50% nornicotine reduction is compared. On the one hand, at least one alkaloid of reduction level comprises that compared with the control tobacco plant when growing under comparable conditions, 10% and 25% nornicotine reduction is compared.

[0211] On the one hand, the modified tobacco plant comprises a nornicotine level less than or equal to 1.0mg / g dry weight. On the one hand, the modified tobacco plant comprises a nornicotine level less than or equal to 0.9mg / g dry weight. On the one hand, the modified tobacco plant comprises a nornicotine level less than or equal to 0.8mg / g dry weight. On the one hand, the modified tobacco plant comprises a nornicotine level less than or equal to 0.7mg / g dry weight. On the one hand, the modified tobacco plant comprises a nornicotine level less than or equal to 0.6mg / g dry weight. On the one hand, the modified tobacco plant comprises a nornicotine level less than or equal to 0.5mg / g dry weight. On the one hand, the modified tobacco plant comprises a nornicotine level less than or equal to 0.4mg / g dry weight. On the one hand, the modified tobacco plant comprises a nornicotine level less than or equal to 0.3mg / g dry weight. On the one hand, the modified tobacco plant comprises a nornicotine level less than or equal to 0.2mg / g dry weight.

[0212] plant

[0213] As used herein, the tobacco plant can be from any plant of the genus Nicotiana, including but not limited to Nicotiana tabacum, Nicotiana claspingii PI 271989; Nicotiana benthamiana PI 555478; Nicotiana piciliana PI 555485; Nicotiana dibona; Nicotiana truncatum PI 224063; Nicotiana viscida PI 555507; Nicotiana guttata PI 241012; Nicotiana cossi PI 230953; Nicotiana occidentalis PI 271991; Nicotiana naitella PI 555527; Nicotiana seashore PI 555535; Nicotiana japonica PI 555536; Nicotiana nudica PI 555540; Nicotiana cone PI 555545; Nicotiana jasmine PI 555548; Nicotiana repens PI 555552; ​​Nicotiana calendula; Nicotiana spicata PI 230960; Nicotiana lindl PI 555569; Nicotiana velutipes PI 266379; Nicotiana tomentosa; and Nicotiana triangularis PI 555572. In one aspect, the tobacco plant described herein is a Nicotiana tabacum plant.

[0214] On the one hand, the tobacco parts provided include but not limited to leaf, stem, root, trichome, seed, flower, pollen, anther, ovule, pedicel, fruit, meristem, cotyledon, hypocotyl, pod, embryo, endosperm, explant, callus, tissue culture, bud, cell and protoplast.On the one hand, the tobacco parts provided do not comprise seed.On the one hand, the disclosure provides tobacco plant cells, tissue and the organ that are not propagation materials and the natural reproduction of mediated plants.On the other hand, the disclosure also provides tobacco plant cells, tissue and the organ as propagation materials and the natural reproduction of mediated plants.On the other hand, the disclosure provides tobacco plant cells, tissue and the organ that can not maintain self via photosynthesis.On the other hand, the disclosure provides tobacco plant cells.Contrary to germline cells, somatic cells do not mediate plant reproduction.

[0215] Cell, tissue and organ can be from seed, fruit, leaf, cotyledon, hypocotyl, meristem, embryo, endosperm, root, bud, stem, trichome, pod, flower, inflorescence, stem, pedicel, style, stigma, receptacle, petal, sepal, pollen, anther, filament, ovary, ovule, pericarp, phloem, vascular tissue.On the other hand, the disclosure provides tobacco plant chloroplast.Aspect otherwise, the disclosure provides epidermal cell, stomatal cell, leaf or root hair, storage root or tuber.On the other hand, the disclosure provides tobacco protoplast.

[0216] Skilled artisans know that tobacco plants reproduce naturally via seeds rather than via asexual or vegetative reproduction. In one aspect, the present disclosure provides tobacco endosperm.

[0217] The present disclosure provides cells from the tobacco plants provided herein.

[0218] As used herein, a "progeny tobacco plant" or "progeny tobacco seed" can be from any progeny generation, for example, F1, F2, F3, F4, F5, F6, F7, etc.

[0219] In one aspect, the tobacco plant or part thereof belongs to a tobacco variety selected from the group consisting of flue-cured varieties, brilliant varieties, burley varieties, Virginia varieties, Maryland varieties, black tobacco varieties, galpao varieties, oriental varieties and Turkish varieties. In one aspect, the modified tobacco plant provided herein or part thereof belongs to a tobacco variety selected from the group consisting of flue-cured varieties, brilliant varieties, burley varieties, Virginia varieties, Maryland varieties, black tobacco varieties, galpao varieties, oriental varieties and Turkish varieties.

[0220] In one aspect, the tobacco cell belongs to a tobacco variety selected from 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. In one aspect, the modified tobacco cell belongs to a tobacco variety selected from 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.

[0221] In one aspect, the tobacco leaves are of a tobacco variety selected from the group consisting of a flue-cured variety, a brilliant variety, a burley variety, a Virginia variety, a Maryland variety, a black tobacco variety, a garpao variety, an oriental variety, and a Turkish variety.

[0222] In one aspect, the curing tobacco leaf or plant portion is of a tobacco variety selected from the group consisting of flue-curing varieties, bright varieties, burley varieties, Virginia varieties, Maryland varieties, black tobacco varieties, garpao varieties, oriental varieties and Turkish varieties. Skilled artisans further understand that flue-curing tobacco does not constitute a living organism and cannot grow or reproduce.

[0223] Flue-cured tobacco (also referred to as "Virginia" or "bright" tobacco) accounts for about 40% of the world's tobacco production. Flue-cured tobacco is also commonly referred to as "bright tobacco" because it presents golden yellow to dark orange in the curing process. Flue-cured tobacco has a light, fresh aroma and taste. Flue-cured tobacco generally has a high sugar content and a low oil content. Main flue-cured tobacco growing countries are Argentina, Brazil, China, India, Tanzania and the U.S. In one aspect, tobacco plants or seeds or modified tobacco plants or seeds provided herein belong to the flue-cured tobacco variety selected from the group consisting of the variety listed in Table 2 and any variety of any one of the aforementioned varieties. Referring to, WO 2004 / 041006 A1. In other aspects, modified tobacco plants or seeds provided herein are flue-cured varieties selected from the group consisting of K326, K346 and NC196.

[0224] Table 2. Flue-cured tobacco varieties

[0225]

[0226]

[0227] Air-cured tobaccos include "burley", "maryland" and "dark" tobaccos. The common factor associated with air-cured tobaccos is that curing is done primarily without artificial heat and humidity. Burley tobacco is light to dark brown in color, high in oils and low in sugars. Burley tobacco is usually air-cured in barns. Major burley growing countries include Argentina, Brazil, Italy, Malawi and the United States.

[0228] Maryland tobacco is extremely fluffy, has good burning properties, low nicotine, and a neutral aroma. Major Maryland growing countries include the United States and Italy.

[0229] In one aspect, the tobacco plant or seed or modified tobacco plant or seed provided herein is a burley tobacco variety selected from the group consisting of the tobacco varieties listed in Table 3 and any variety substantially derived from any of the foregoing varieties. In a further aspect, the modified tobacco plant or seed provided herein is a burley variety selected from the group consisting of TN 90, KT 209, KT 206, KT212 and HB 4488.

[0230] Table 3. Burley tobacco varieties

[0231]

[0232]

[0233] In another aspect, the tobacco plant or seed or modified tobacco plant or seed provided herein is of a Maryland Nicotiana variety selected from the group consisting of the tobacco varieties listed in Table 4, and any variety derived substantially from any of the foregoing varieties.

[0234] Table 4. Maryland tobacco varieties

[0235]

[0236]

[0237] Dark air-cured tobacco is different from other tobacco types mainly in its curing process, and this process makes dark air-cured tobacco have medium brown to dark brown color and unique aroma.Dark air-cured tobacco is mainly used for producing chewing tobacco and snuff.In one aspect, modified tobacco plant or seed provided herein belong to the dark air-cured tobacco variety selected from the group consisting of Sumatra, Jatim, DominicanCubano, Besuki, One sucker, Green River, Virginia sun-cured and Paraguan Passado and any variety of any one variety basically derived from the aforementioned variety.

[0238] Dark flue-cured tobacco is usually prepared with low burning wood fire on the floor of closed preparation room.Dark flue-cured tobacco is generally used to make pipe mixture, cigarette, chewing tobacco, snuff and strong flavor cigar.The main growing area of ​​dark flue-cured tobacco is Tennessee, Kentucky and Virginia of the U.S. In one aspect, tobacco plant or seed provided herein or modified tobacco plant or seed belong to the dark flue-cured tobacco variety selected from the group of the tobacco variety composition listed in Table 5 and any variety of any variety basically derived from any variety in the aforementioned variety.

[0239] Table 5. Black tobacco varieties

[0240]

[0241]

[0242] Because Oriental tobacco is usually grown in this fact of the Eastern Mediterranean region (such as Turkey, Greece, Bulgaria, Macedonia, Syria, Lebanon, Italy and Romania), they are also referred to as Greek tobacco, aromatic tobacco and Turkish tobacco.The plantlet size, leaflet size and unique aroma characteristics of Oriental tobacco variety are the result of the poor soil and the tense climate conditions that they adapt to its growth.In one aspect, tobacco plant provided herein or seed or modified tobacco plant or seed belong to the Oriental tobacco variety selected from the group of the tobacco variety composition listed in Table 6 and any kind that is basically derived from any one in the aforementioned variety.

[0243] Table 6. Oriental tobacco varieties

[0244] Bafra (TI 1641) Edirne (TI 1671) Samsun (TC 536) Bahce (TI 1730) Ege(TI 1642) Samsun 959(TI 1570) Bahia(TI 1416) Ege-64(TI 1672) Samsun Evkaf (TI 1723) Bahia(TI 1455) Izmir (Akhisar) (TI 1729) Samsun Holmes NN(TC 540) Baiano(TI 128) Izmir (Gavurkoy) (TI 1727) Samsun Maden (TI 1647) Basma Izmir Ege 64 Samsun NO 15(TC 541) Basma (TI 1666) Izmir-Incekara (TI 1674) Samsun-BLK SHK Tol(TC 542) Basma Drama Izmir-Ozbas (TI 1675) Samsun-Canik (TI 1678) Basma Hybrid (PhPh) Jaka Dzebel (TI 1326) Samsun-Maden (TI 1679) Basma Zihna I Kaba-Kulak Saribaptar 407 - Izmir District Bitlis(TI 1667) Kagoshima Maruba(TI 158) Smyrna (TC 543) Bitlis(TI 1725) Katerini Smyrna No.23 (TC 545) Bubalovac (TI 1282) Katerini S53 Smyrna No.9(TC 544) Bursa(TI 1650) Krumovgrad 58 Smyrna-Blk Shk Tol(TC 546) Bursa (TI 1668) MS Basma Trabzon(TI 1649) Canik (TI 1644) MS Katerini S53 Trabzon(TI 1682) Djebel 174 (TI 1492) Nevrokop 1146 Trapezund 161 (TI 1407) Djebel 359 (TI 1493) Ozbas(TI 1645) Türkiye (TC 548) Djebel 81 Perustitza (TI 980) Turkish Angshit (TI 90) Dubec 566(TI 1409) Prilep(TI 1291) Turkish Samsum (TI 92) Dubec 7(TI 1410) Prilep(TI 1325) Turkish Tropizoid (TI 93) Dubek 566 (TI 1567) Prilep 12-2 / 1 Türkiye Varotic (TI 89) Duzce (TI 1670) Prilep 23 Xanthi (TI 1662)

[0245] In one aspect, the tobacco plant or seed or modified tobacco plant or seed provided herein is of a cigar tobacco variety selected from the group consisting of the tobacco varieties listed in Table 7 and any variety derived substantially from any of the foregoing varieties.

[0246] Table 7. Cigar tobacco varieties

[0247]

[0248] In one aspect, the tobacco plant or seed or modified tobacco plant or seed provided herein is of a tobacco variety selected from the group consisting of the tobacco varieties listed in Table 8 and any variety derived substantially from any of the foregoing varieties.

[0249] Table 8. Other tobacco varieties

[0250] Chocoa(TI 319) Hoja Parada (TI 1089) Hoja Parado (Galpoa) (TI 1068) Perique (St.James Parrish) Perique(TC 556) Perique(TI 1374) Sylvestris (TI 984) TI 179

[0251] In one aspect, the tobacco plant or part thereof is from a variety selected from the group consisting of the tobacco varieties listed in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8. In another aspect, the tobacco plant or part thereof is from a variety listed in Table 2. In another aspect, the tobacco plant or part thereof is from a variety listed in Table 3. In another aspect, the tobacco plant or part thereof is from a variety listed in Table 4. In another aspect, the tobacco plant or part thereof is from a variety listed in Table 5. In another aspect, the tobacco plant or part thereof is from a variety listed in Table 6. In another aspect, the tobacco plant or part thereof is from a variety listed in Table 7. In another aspect, the tobacco plant or part thereof is from a variety listed in Table 8.

[0252] In one aspect, the modified tobacco plant or part thereof is from a variety selected from the group consisting of the tobacco varieties listed in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8. In one aspect, the modified tobacco plant or part thereof is from a variety listed in Table 2. In another aspect, the modified tobacco plant or part thereof is from a variety listed in Table 3. In another aspect, the modified tobacco plant or part thereof is from a variety listed in Table 4. In another aspect, the modified tobacco plant or part thereof is from a variety listed in Table 5. In another aspect, the modified tobacco plant or part thereof is from a variety listed in Table 6. In another aspect, the modified tobacco plant or part thereof is from a variety listed in Table 7. In another aspect, the modified tobacco plant or part thereof is from a variety listed in Table 8.

[0253] In one aspect, the tobacco seeds are from a variety selected from the group consisting of the tobacco varieties listed in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8. In another aspect, the tobacco seeds are from a variety listed in Table 2. In another aspect, the tobacco seeds are from a variety listed in Table 3. In another aspect, the tobacco seeds are from a variety listed in Table 4. In another aspect, the tobacco seeds are from a variety listed in Table 5. In another aspect, the tobacco seeds are from a variety listed in Table 6. In another aspect, the tobacco seeds are from a variety listed in Table 7. In another aspect, the tobacco seeds are from a variety listed in Table 8.

[0254] In one aspect, the tobacco cells are from a variety selected from the group consisting of the tobacco varieties listed in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8. In another aspect, the tobacco cells are from a variety listed in Table 2. In another aspect, the tobacco cells are from a variety listed in Table 3. In another aspect, the tobacco cells are from a variety listed in Table 4. In another aspect, the tobacco cells are from a variety listed in Table 5. In another aspect, the tobacco cells are from a variety listed in Table 6. In another aspect, the tobacco cells are from a variety listed in Table 7. In another aspect, the tobacco cells are from a variety listed in Table 8.

[0255] All of the aforementioned specific varieties of flue-cured, dark-cured, burley, maryland, dark roasted, cigar, or oriental types are listed for exemplary purposes only. The present application also contemplates any additional flue-cured, dark-cured, burley, maryland, dark roasted, cigar, or oriental varieties.

[0256] In one aspect, the tobacco plant or variety provided herein is an inbred tobacco plant or variety. As used herein, an "inbred" tobacco variety is a variety that has been bred for genetic homogeneity.

[0257] As used herein, "hybrid" is produced by hybridizing two plants from different varieties or species so that progeny comprises the genetic material from each parent. Skilled craftsmen recognize that more advanced hybrids can also be produced.For example, the first hybrid can be hybridized with kind D to produce C x D hybrids by kind C, and the second hybrid can be hybridized with kind F to produce E x F hybrids by kind E. The first hybrid and the second hybrid can further hybridize to produce the advanced hybrid (C x D) x (E x F) comprising the genetic information from all four parental varieties.On the one hand, the modified tobacco plant provided herein is a hybrid tobacco plant.On the other hand, the modified tobacco seed provided herein is a hybrid tobacco seed.On the one hand, the tobacco plant or kind provided herein are hybrid tobacco plant or kind.On the other hand, the modified tobacco plant provided herein is a hybrid tobacco plant.

[0258] In one aspect, the present disclosure provides a method for producing a modified tobacco plant, 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 generation of tobacco seeds, wherein the at least one tobacco plant of the first tobacco variety comprises 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 NOs: 117 to 174, wherein the non-natural mutation is not present in the endogenous nucleic acid sequence in a control tobacco plant of the first tobacco variety; and (b) selecting at least one generation of tobacco seeds or a plant germinated therefrom, wherein the at least one tobacco seed or a plant germinated therefrom comprises the non-natural mutation. In another aspect, the present disclosure provides a method for producing a modified tobacco plant, 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 generation of tobacco seeds, wherein the at least one tobacco plant of the first tobacco variety comprises a non-natural mutation in 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 58, wherein the non-natural mutation is not present in the endogenous nucleic acid sequence in a control tobacco plant of the first tobacco variety; and (b) selecting at least one generation of tobacco seeds or a plant germinated therefrom, wherein the at least one tobacco seed or a plant germinated therefrom comprises the non-natural mutation. In another aspect, the present disclosure provides a method for producing a modified tobacco plant, 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 generation of tobacco seeds, wherein the at least one tobacco plant of the first tobacco variety comprises a non-natural mutation in 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: 59 to 116, wherein the non-natural mutation is not present in the endogenous nucleic acid sequence in a control tobacco plant of the first tobacco variety; and (b) selecting at least one generation of tobacco seeds or a plant germinated therefrom, wherein the at least one tobacco seed or a plant germinated therefrom comprises the non-natural mutation.On the other hand, the disclosure provides a method for producing a modified tobacco plant, 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 generation tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a non-natural mutation in an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 116, wherein the non-natural mutation is not present in the endogenous nucleic acid sequence in a control tobacco plant of the first tobacco variety; and (b) selecting at least one generation tobacco seed or a plant sprouted therefrom, wherein the at least one tobacco seed or a plant sprouted therefrom comprises the non-natural mutation. On the one hand, 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 foregoing methods, the first tobacco variety and the second tobacco variety are two different tobacco varieties.

[0259] In one aspect, the present disclosure provides a method for producing a modified tobacco plant, 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 first-generation 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 operably linked to a nucleic acid sequence encoding at least one small RNA molecule, the small RNA molecule being capable of binding to and reducing the expression of an endogenous nucleic acid sequence, the endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NO: 117 to 174, wherein the recombinant DNA construct is not present in a control tobacco plant of the first tobacco variety; and (b) selecting at least one first-generation tobacco seed or a plant germinated therefrom, wherein the at least one first-generation tobacco seed or a plant germinated therefrom comprises the recombinant DNA construct. In another aspect, the present disclosure provides a method for producing a modified tobacco plant, 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 generation 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 operably linked to a nucleic acid sequence encoding at least one small RNA molecule, the small RNA molecule being capable of binding to and reducing the expression of an endogenous nucleic acid sequence, the endogenous nucleic acid sequence being at least 80% identical or similar to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 58, wherein the recombinant DNA construct is not present in a control tobacco plant of the first variety; and (b) selecting at least one generation tobacco seed or a plant germinated therefrom, wherein the at least one generation tobacco seed or a plant germinated therefrom comprises the recombinant DNA construct. On the other hand, the present disclosure provides a method for producing a modified tobacco plant, 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 tobacco seed of the first tobacco variety, 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 operably linked to a nucleic acid sequence encoding at least one small RNA molecule, the small RNA molecule being capable of binding to and reducing the expression of an endogenous nucleic acid sequence, the endogenous nucleic acid sequence being at least 80% identical or similar to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 59 to 116, wherein the recombinant DNA construct is not present in a control tobacco plant of the first variety; and (b) selecting at least one tobacco seed of the first tobacco variety or a plant germinated therefrom, wherein the at least one tobacco seed of the first tobacco variety or a plant germinated therefrom comprises the recombinant DNA construct. On the one hand, 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.

[0260] In one aspect, the present disclosure provides a method for producing a modified tobacco plant, 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 generation 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 operably linked to a nucleic acid sequence encoding a polypeptide, the 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: 117 to 174, wherein the recombinant DNA construct is not present in a control tobacco plant of the first tobacco variety; and (b) selecting at least one generation tobacco seed or a plant germinated therefrom, wherein the at least one generation tobacco seed or a plant germinated therefrom comprises the recombinant DNA construct. In another aspect, the present disclosure provides a method for producing a modified tobacco plant, 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 generation 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 operably 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 58, wherein the recombinant DNA construct is not present in a control tobacco plant of the first tobacco variety; and (b) selecting at least one generation tobacco seed or a plant germinated therefrom, wherein the at least one generation tobacco seed or a plant germinated therefrom comprises the recombinant DNA construct. On the other hand, the disclosure provides a method for producing a modified tobacco plant, 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 tobacco seed of particle generation, 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 operably connected to a nucleic acid sequence, the nucleic acid sequence is at least 80% identical to the nucleic acid sequence of the group consisting of SEQ ID NO:59 to 116, wherein the recombinant DNA construct is not present in the control tobacco plant of the first tobacco variety; and (b) selecting at least one tobacco seed of particle generation or a plant sprouted therefrom, wherein the at least one tobacco seed of particle generation or a plant sprouted therefrom comprises the recombinant DNA construct. On the one hand, in any of the aforementioned methods, the first tobacco variety and the second tobacco variety are the same tobacco variety. On the other hand, in any of the aforementioned methods, the first tobacco variety and the second tobacco variety are two different tobacco varieties.

[0261] As used herein, term " hybridization " refers to the intentional mating of two plants.On the one hand, hybridization comprises pollination and / or fertilization of the second tobacco plant to the first tobacco plant.Two kinds of tobacco plants of hybridization can be distant relatives, close relatives or identical.On the one hand, two kinds of tobacco plants of hybridization are modified tobacco plants.On the one hand, two kinds of tobacco plants of hybridization belong to identical tobacco varieties.On the one hand, two kinds of tobacco plants of hybridization belong to two different tobacco varieties.On the one hand, one of two kinds of tobacco plants of hybridization is male sterile.On the one hand, one of two kinds of tobacco plants of hybridization is female sterile.On the one hand, at least one of two kinds of tobacco plants of hybridization is hybrid tobacco plant.On the one hand, at least one of two kinds of tobacco plants of hybridization is modified tobacco plant.

[0262] On the one hand, tobacco plant provided herein or kind are male sterile.On the other hand, tobacco plant provided herein or kind are cytoplasmic male sterile (CMS).On the one hand, modified tobacco plant provided herein or kind are male sterile.On the other hand, modified tobacco plant provided herein or kind are cytoplasmic male sterile (CMS).Male sterile tobacco plant can be produced by any method known in the art.The method for producing male sterile tobacco is at Wernsman, EA and Rufty, RC1987. Chapter 17. Tobacco. 669-698 pages, in: Cultivar Development. Crop Species. WH Fehr (editor), MacMillan Publishing Go., Inc., New York, NY, page 761, described.

[0263] On the other hand, tobacco plant or variety provided herein are female sterile. On the other hand, modified tobacco plant or variety provided herein are female sterile. As a non-limiting example, female sterile plants can be prepared by making the STIG1 gene mutation. See, for example, Goldman et al. 1994, EMBO Journal 13:2976-2984. On the one hand, modified tobacco plant provided herein is female sterile.

[0264] Unless otherwise stated, all comparisons with control plants require similar growth conditions or comparable growth conditions for the two plants compared. As used herein, "growing under comparable conditions", "similar growth conditions" or "comparable growth conditions" refer to similar environmental conditions and / or agronomic practices for growing and making meaningful comparisons between two or more plant genotypes, so that 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 nutrition (for example, nitrogen and phosphorus). Agronomic practices include, for example, sowing, pruning, undercutting, transplanting, topping and sprouting. See, Tobacco, Production, Chemistry and Technology, Chapter 4B and 4C, Davis & Nielsen, editors, Blackwell Publishing, Oxford (1999), pp. 70-103. As used herein, "control plants" refer to plants with the same or almost identical genetic composition as the modified plants compared, except for the non-natural mutations or recombinant DNA constructs provided herein that are introduced into the modified plants.

[0265] On the one hand, the modified tobacco plant described herein is a low alkaloid variety or a low alkaloid plant. As a non-limiting example, LABurley 21 (LABU21) is a low alkaloid variety of tobacco. LA BU21 is produced by incorporating the low alkaloid gene from Cuban cigar varieties into white rib 21 by multiple backcrossing. It has about 0.2% (dry weight) of total alkaloids (compared with about 3.5% (dry weight) of its parent white rib 21). LA BU21 has a leaf grade far below the commercially acceptable standard. LABU21 also shows other unfavorable leaf phenotypes, characterized in that yield is low, maturity and aging are delayed, susceptibility to insect herbivory is high, and the final product quality after modulation is poor. LABU21 leaves also show traits such as higher polyamine content, higher chlorophyll content, and more mesophyll cells per unit leaf area. For more characterization of LABU21 leaf phenotypes, see, US2019 / 0271000.

[0266] On the one hand, the disclosure provides tobacco plants or parts thereof, which contain mutations or transgenics conferring low nicotine or low alkaloids (e.g., in one or more ADCs, AOs or ODCs or targeted genetic modifications of one or more ADCs, AOs or ODCs) and can produce leaves containing comparable levels of one or more polyamines relative to comparable leaves of control plants not comprising the same mutation or transgenics. In one aspect, the comparable levels of one or more polyamines are within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5% or 1% of the levels in comparable leaves of control plants not comprising the same mutation or transgenics. In one aspect, comparable levels of one or more polyamines are between 0.5% and 1%, between 1% and 2%, between 2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%, between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and 10%, between 11% and 12%, between 12% and 13%, between 13% and 14%, between 14% and 15%, between 15% and 16%, between 16% and 17%, between 17% and 18%, between 18% and 19%, or between 19% and 20%. In additional aspects, comparable levels of one or more polyamines are between 0.5% and 5%, between 5% and 10%, or between 10% and 20% of the levels in comparable leaves of control plants that do not contain the same mutation or transgene.

[0267] In one aspect, the disclosure provides ADC mutant or transgenic tobacco plant or its part, AO mutant or transgenic tobacco plant or its part or ODC mutant or transgenic tobacco plant or its part, it can produce and comprise the comparable chlorophyll level with respect to the comparable leaves of control plant that does not comprise the same sudden change or transgene.In one aspect, the comparable chlorophyll level is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5% or 1% of the level in the comparable leaves of control plant that does not comprise the same sudden change or transgene. In one aspect, comparable chlorophyll levels are between 0.5% and 1%, between 1% and 2%, between 2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%, between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and 10%, between 11% and 12%, between 12% and 13%, between 13% and 14%, between 14% and 15%, between 15% and 16%, between 16% and 17%, between 17% and 18%, between 18% and 19%, or between 19% and 20%. In other aspects, comparable chlorophyll levels are between 0.5% and 5%, between 5% and 10%, or between 10% and 20% of the levels in comparable leaves of control plants that do not comprise the same mutation or transgene.

[0268] In one aspect, the disclosure provides ADC mutant or transgenic tobacco plant or part thereof, AO mutant or transgenic tobacco plant or part thereof, or ODC mutant or transgenic tobacco plant or part thereof, which can produce mesophyll cells containing a comparable number of mesophyll cells per unit leaf area relative to comparable leaves of control plants that do not contain the same mutation or transgene. In one aspect, the comparable number of mesophyll cells per unit leaf area 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 that do not contain the same mutation or transgene. In one aspect, the comparable number of mesophyll cells per unit leaf area is between 0.5% and 1%, between 1% and 2%, between 2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%, between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and 10%, between 11% and 12%, between 12% and 13%, between 13% and 14%, between 14% and 15%, between 15% and 16%, between 16% and 17%, between 17% and 18%, between 18% and 19%, or between 19% and 20%. In additional aspects, the comparable number of mesophyll cells per unit leaf area is between 0.5% and 5%, between 5% and 10%, or between 10% and 20% of the level in comparable leaves of control plants that do not contain the same mutation or transgene.

[0269] In one aspect, the disclosure provides ADC mutant or transgenic tobacco plant or its part, AO mutant or transgenic tobacco plant or its part or ODC mutant or transgenic tobacco plant or its part, it can produce the leaf of comparable epidermal cell size comprising the comparable leaf of control plant not comprising the same mutation or transgene.In one aspect, comparable epidermal cell size is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5% or 1% of the level in the comparable leaf of control plant not comprising the same mutation or transgene. In one aspect, comparable epidermal cell size is between 0.5% and 1%, between 1% and 2%, between 2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%, between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and 10%, between 11% and 12%, between 12% and 13%, between 13% and 14%, between 14% and 15%, between 15% and 16%, between 16% and 17%, between 17% and 18%, between 18% and 19%, or between 19% and 20%. In other aspects, comparable epidermal cell size is between 0.5% and 5%, between 5% and 10%, between 10% and 20% of the level in comparable leaves of control plants that do not comprise the same mutation or transgene.

[0270] On the one hand, the disclosure provides ADC mutant or transgenic tobacco plant or its part, AO mutant or transgenic tobacco plant or its part or ODC mutant or transgenic tobacco plant or its part, it can produce and comprise the comparable leaf yield with respect to the comparable leaf of control plant that does not comprise the same sudden change or transgene.In one aspect, the comparable leaf yield is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5% or 1% of the level in the comparable leaf of control plant that does not comprise the same sudden change or transgene. In one aspect, comparable leaf yield is between 0.5% and 1%, between 1% and 2%, between 2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%, between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and 10%, between 11% and 12%, between 12% and 13%, between 13% and 14%, between 14% and 15%, between 15% and 16%, between 16% and 17%, between 17% and 18%, between 18% and 19%, or between 19% and 20%. In other aspects, comparable leaf yield is between 0.5% and 5%, between 5% and 10%, or between 10% and 20% of the level in comparable leaves of control plants that do not comprise the same mutation or transgene.

[0271] In one aspect, the disclosure provides ADC mutants or transgenic tobacco plants or parts thereof, AO mutants or transgenic tobacco plants or parts thereof, or ODC mutants or transgenic tobacco plants or parts thereof, which exhibit comparable insect herbivory susceptibility relative to comparable leaves of control plants that do not comprise the same mutation or transgene. In one aspect, comparable insect herbivory susceptibility 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 that do not comprise the same mutation or transgene. In one aspect, the comparable insect herbivory susceptibility is between 0.5% and 1%, between 1% and 2%, between 2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%, between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and 10%, between 11% and 12%, between 12% and 13%, between 13% and 14%, between 14% and 15%, between 15% and 16%, between 16% and 17%, between 17% and 18%, between 18% and 19%, or between 19% and 20%. In other aspects, the comparable insect herbivory susceptibility is between 0.5% and 5%, between 5% and 10%, or between 10% and 20% of the level in comparable leaves of control plants that do not contain the same mutation or transgene.

[0272] Any of the genetic modifications provided herein can be introduced into any ADC mutant or transgenic tobacco plant or part thereof, AO mutant or transgenic tobacco plant or part thereof, or ODC mutant or transgenic tobacco plant or part thereof.

[0273] Insect herbivory susceptibility levels can be determined by methods known in the art (e.g., in insect feeding assays). In short, a quarter inch layer of 0.7% agar in water is added to a 100 mm petri dish and allowed to solidify. Leaf discs are cut from petri dish lids, placed in plates and gently pushed into the agar. Leaf discs are taken from plants at the 4-5 leaf stage. The discs are removed from the capsule discs just to exclude the main midrib. Single discs are taken from the four largest leaves of the plant, thereby producing 4 replicates per plant. Four plants were sampled for a total of 16 biological replicate test lines. A single budworm (e.g., Heliothis species, Helicoverpa species) in the second instar stage is added to the leaf and allowed to feed for 48 hours at ambient temperature. After 48 hours, the budworm larvae are weighed and the final larval weight is recorded.

[0274] In one aspect, a tobacco plant or part thereof comprises, relative to a control tobacco plant: a first genomic modification (e.g., in or targeting one or more 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 leaf area unit, and leaf epidermal cell size; and wherein the control plant does not have both the first genomic modification and the second genomic modification. In one aspect, a tobacco plant or part thereof comprises, relative to a control tobacco plant: a first genomic modification (e.g., in or targeting one or more 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 polyamine levels, wherein the control plant does not have both the first genomic modification and the second genomic modification. In one aspect, a tobacco plant or part thereof comprises, relative to a control tobacco plant: a first genome modification that provides a lower level of nicotine or total alkaloids (e.g., in one or more ADC, AO or ODC genes or targeting one or more ADC, AO or ODC genes); and a second genome modification that provides a comparable level of total root polyamine levels, wherein the control plant does not have both the first genome modification and the second genome modification. In one aspect, a tobacco plant or part thereof comprises, relative to a control tobacco plant: a first genome modification that provides a lower level of nicotine or total alkaloids (e.g., in one or more ADC, AO or ODC genes or targeting one or more ADC, AO or ODC genes); and a second genome modification that provides a comparable level of total leaf chlorophyll levels, wherein the control plant does not have both the first genome modification and the second genome modification. In one aspect, tobacco plants or parts thereof comprise, relative to control tobacco plants: a first genome modification (e.g., in one or more ADC, AO or ODC genes or targeting one or more ADC, AO or ODC genes), which provides a lower level of nicotine or total alkaloids; and a second genome modification, which provides a comparable level of mesophyll cell number per leaf area unit, wherein the control plant does not have both the first genome modification and the second genome modification. In one aspect, tobacco plants or parts thereof comprise, relative to control tobacco plants: a first genome modification, which provides a lower level of nicotine or total alkaloids (e.g., in one or more ADC, AO or ODC genes or targeting one or more ADC, AO or ODC genes); and a second genome modification, which provides a comparable level of leaf epidermal cell size, wherein the control plant does not have both the first genome modification and the second genome modification. In one aspect, the second genome modification is in or targeting ADC, AO or ODC genes, ADC, AO or ODC genes.

[0275] In one aspect, the first genome modification, the second genome modification, or both include a transgene, a mutation, or both. In one aspect, the genome modification, the second genome modification, or both include a transgene. In one aspect, the first genome modification, the second genome modification, or both include a mutation. In one aspect, the first genome modification, the second genome modification, or both are not based on a transgene. In one aspect, the first genome modification, the second genome modification, or both are not based on a mutation.

[0276] On the one hand, relative to the control tobacco plant, the tobacco plant provided herein comprises the total conjugated polyamines of reduction in leaf. On the one hand, relative to the control tobacco plant, the tobacco plant provided herein comprises the total conjugated polyamines of reduction in root. The conjugated polyamines used here include but are not limited to soluble conjugated polyamines, such as the phenolamides comprising the main chain of the free polyamines (for example, putrescine, spermine and / or spermidine) conjugated with one or more phenylpropanoids (such as ferulic acid, caffeic acid, coumaric acid) consisting of. Conjugated polyamines also include but are not limited to the insoluble conjugated polyamines incorporated into structural polymers (such as lignin). On the one hand, relative to the control tobacco plant, the tobacco plant provided herein comprises the total free polyamines (for example, putrescine, spermine and spermidine) of reduction in leaf. On the one hand, relative to the control tobacco plant, the tobacco plant provided herein comprises the total conjugated polyamines of reduction in root. On the one hand, relative to the control tobacco plant, the tobacco plant provided herein comprises the total conjugated form of reduction in leaf one or more polyamines selected from the group consisting of putrescine, spermidine and spermine. In one aspect, relative to the control tobacco plant, the tobacco plant provided herein comprises one or more polyamines selected from the group consisting of putrescine, spermidine and spermine in the total conjugated form of the reduction in the root. In one aspect, relative to the control tobacco plant, the tobacco plant provided herein comprises one or more polyamines selected from the group consisting of putrescine, spermidine and spermine in the total free form of the reduction in the leaf. In one aspect, relative to the control tobacco plant, the tobacco plant provided herein comprises one or more polyamines selected from the group consisting of putrescine, spermidine and spermine in the total conjugated form of the reduction in the root.

[0277] On the one hand, the characteristic of tobacco plant as described herein or proterties are measured at the time of the group being formed by the following items: before about to bloom, when topping, 1 week (WPT), 2WPT, 3WPT, 4WPT, 5WPT, 6WPT, 7WPT, 8WPT and results after topping.In one aspect, the tobacco plant comprising the first genome modification and the second genome modification provided herein can produce leaves with a leaf grade comparable to the leaf grade of the leaf from a control plant.On the one hand, the tobacco plant comprising the first genome modification and the second genome modification provided herein has a total leaf output comparable to a control plant.

[0278] In one aspect, the tobacco plants of the present disclosure comprise a nic1 mutation, a nic2 mutation, or both.

[0279] In one aspect, the modified tobacco plants provided herein further comprise a transgene or mutation that directly suppresses 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 a protein selected from the group consisting of: agmatine deoxyribonucleic acid (DABA) or DABA. Aminase (AIC), arginase, diamine oxidase, methylputrescine oxidase (MPO), NADH dehydrogenase, phosphoribosyl anthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinoline phosphoribosyltransferase (QPT), S-adenosylmethionine synthetase (SAMS), A622, NBB1, berberine bridge enzyme-like (BBL), MYC2, Nic1_ERF, Nic2_ERF, ethylene response factor (ERF) transcription factor, nicotine uptake permease (NUP) and MATE transporter. See, Dewey and Xie, Molecular genetics of alkaloid biosynthesis in Nicotiana tabacum, Phytochemistry 94 (2013) 10–27.

[0280] On the one hand, the modified tobacco plant provided herein further comprises the mutation in the ERF gene (Nic2_ERF) of Nic2 locus.On the one hand, the modified tobacco plant provided herein further 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 or all ten genes selected from the group consisting of ERF32, ERF34, ERF39, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17 and ERF168.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 plant provided herein further comprises one or more mutations in ERF189, ERF115 or both. In one aspect, the modified tobacco plants provided herein further comprise one or more transgenes targeting and suppressing a gene encoding 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 selected from the group consisting of ERF32, ERF34, ERF39, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17 and ERF168.

[0281] On the one hand, the modified tobacco plant provided herein further comprises a mutation in the ERF gene of the Nic1 locus (Nic1_ERF) (or the Nic1b locus as in WO / 2019 / 140297). See also, WO / 2018 / 237107. On the one hand, the modified tobacco plant provided herein further comprises two or more, three or more, four or more, five or more, six or more, seven or more genes selected from the group consisting of ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210 and ERF91L2. One or more mutations in the gene. See, WO / 2019 / 140297 and Kajikawa et al., Plant physiol.2017,174:999-1011. On the one hand, the modified tobacco plant provided herein further comprises one or more, two or more, three or more, four or more, five or more or all six genes selected from the group consisting of ERFnew, ERF199, ERF19, ERF29, ERF210 and ERF91L2.On the one hand, the modified tobacco plant provided herein further comprises one or more transgenics of targeting and inhibition genes, and the gene encoding is selected from the group consisting of ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210 and ERF91L2, two or more, three or more, four or more, five or more, six or more, seven or more genes.

[0282] 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, agmatine deiminase (AIC), arginase, diamine oxidase, arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinoline phosphoribosyltransferase (QPT) and S-adenosylmethionine synthetase (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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In one aspect, the modified tobacco plant provided herein comprises a first genetic modification comprising a transgene that targets and suppresses a gene or locus encoding a protein selected from the group consisting of aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinoline phosphoribosyltransferase (QPT) and S-adenosylmethionine synthetase (SAMS), A622, NBB1, BBL, MYC2, Nicl, 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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174.

[0283] Prepared Tobacco / Tobacco Products

[0284] In one aspect, the present disclosure provides a method comprising preparing a tobacco product using a modulated tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a non-natural mutation in an endogenous nucleic acid sequence, the endogenous 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: 117 to 174. In another aspect, the present disclosure provides a method comprising preparing a tobacco product using a modulated tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a non-natural mutation in an endogenous nucleic acid sequence, the endogenous nucleic acid sequence is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 58. In another aspect, the present disclosure provides a method comprising preparing a tobacco product using a modulated tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a non-natural mutation in an endogenous nucleic acid sequence, the endogenous nucleic acid sequence is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59 to 116.

[0285] In one aspect, the present disclosure provides a method comprising preparing a tobacco product using a cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA molecule, the at least one small RNA molecule being capable of binding to and reducing the expression of an endogenous nucleic acid sequence, the endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In another aspect, the present disclosure provides a method comprising preparing a tobacco product using a cured 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 operably linked to a nucleic acid sequence encoding at least one small RNA molecule, the at least one small RNA molecule being 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 58. In another aspect, the present disclosure provides a method comprising preparing a tobacco product using a cured 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 operably linked to a nucleic acid sequence encoding at least one small RNA molecule, the at least one small RNA molecule being capable of binding to and reducing 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: 59 to 116.

[0286] In one aspect, the present disclosure provides a method comprising preparing a tobacco plant using a cured 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 operably 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: 117 to 174. In another aspect, the present disclosure provides a method comprising preparing a tobacco product using a cured 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 operably 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 58. In another aspect, the present disclosure provides a method comprising preparing a tobacco product using a cured 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 operably 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: 59 to 116.

[0287] "Modulation" is to reduce moisture and destroy chlorophyll so that tobacco leaves are golden yellow and starch is converted into sugar by the aging process. Therefore, compared with the green leaves of the harvest, modulated tobacco has higher reducing sugar content and lower starch content. In one aspect, tobacco plants or plant components provided herein can be modulated using conventional means (for example, flue modulation, curing room modulation, baking, airing or sun baking). For description of different types of modulation methods, see, for example, Tso (1999, Tobacco, Production, Chapter 1 in Chemistry and Technology, Davis & Nielsen edited, Blackwell Publishing, Oxford). Modulated tobacco is usually aged for several years (for example, two to five years) under compression conditions in wooden barrels (for example, vats) or cardboard boxes, and the moisture content ranges from 10% to about 25%. See, U.S. Patent No. 4,516,590 and No. 5,372,149. Modulated and aged tobacco can then be further processed. Further processing includes conditioning of the tobacco under vacuum at various temperatures with or without the introduction of steam, pasteurization, and fermentation.

[0288] Information on harvesting Burley and Dark Tobacco varieties can be found in the 2019-2020 Burley and Dark Tobacco Production Guide (December 2018), published by the University of Kentucky, the University of Tennessee, Virginia Tech, and North Carolina State University, which is incorporated herein by reference in its entirety.

[0289] In one aspect, the present disclosure provides a cured tobacco material from any tobacco plant or part thereof provided herein. In one aspect, the present disclosure provides a cured tobacco material from any modified tobacco plant or part thereof provided herein.

[0290] On the one hand, the modulated tobacco material comprises the tobacco material selected from the group consisting of modulated leaf material, modulated stem material, modulated bud material, modulated flower material and modulated root material. On the other hand, the modulated tobacco material comprises modulated leaf material, modulated stem material or both. In other aspect, the modulated tobacco material comprises modulated leaf material. On the other hand again, the modulated tobacco material comprises modulated stem material.

[0291] On the one hand, the flue-cured tobacco material comprises a flue-cured tobacco material. On the other hand, the curing tobacco material comprises an air-cured tobacco material. On the other hand, the curing tobacco material comprises a sun-cured tobacco material. On the other hand, the curing tobacco material comprises a sun-cured tobacco material. On the other hand, the curing tobacco material provided herein is selected from the group consisting of an air-cured tobacco material, a flue-cured tobacco material, a sun-cured tobacco material and a flue-cured tobacco material. On the other hand, the curing tobacco material is from a tobacco variety selected from the group consisting of a flue-cured variety, a bright variety, a burley variety, a Virginia variety, a Maryland variety, a dark variety, an oriental variety and a Turkish variety.

[0292] On the one hand, the curing tobacco leaf provided herein is selected from the group consisting of air-cured tobacco leaf, flue-cured tobacco leaf, sun-cured tobacco leaf and flue-cured tobacco leaf. On the one hand, the curing tobacco leaf is from the tobacco variety selected from the group consisting of flue-cured variety, bright variety, burley variety, Virginia variety, Maryland variety, dark variety, oriental variety and Türkiye variety.

[0293] Generally, fermentation is characterized by high initial moisture content, heat generation and 10% to 20% dry weight loss.See, for example, U.S. Patent No. 4,528,993, No. 4,660,577, No. 4,848,373, No. 5,372,149; U.S. Publication No. 2005 / 0178398; and Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen edited, Blackwell Publishing, Oxford). Modulation, aging and fermented tobacco can be further processed (for example, cutting, chopping, puffing or mixing).See, for example, U.S. Patent No. 4,528,993; No. 4,660,577; and No. 4,987,907. On the one hand, the present disclosure provides a fermented tobacco material from any tobacco plant or part thereof provided herein. In another aspect, the present disclosure provides fermented tobacco material from any of the modified tobacco plants or parts thereof provided herein.

[0294] The tobacco material obtained from tobacco line, variety or hybrid of the present disclosure can be used for manufacturing tobacco products.As used herein, "tobacco product" is defined as any product intended for human use or consumption made from tobacco or derived from tobacco.On the one hand, the present disclosure provides tobacco products comprising the plant material from the tobacco plant provided herein.On the other hand, the present disclosure provides tobacco products comprising the plant material from the modified tobacco plant provided herein.On the other hand, the present disclosure provides tobacco products comprising modulated tobacco materials.On the other hand, the present disclosure provides tobacco products comprising fermented tobacco materials.On the other hand, the present disclosure provides tobacco products comprising tobacco blends.

[0295] Tobacco products include, but are not limited to, cigarette products (e.g., cigarettes and bidis), cigar products (e.g., cigar wrappers and cigarillos), pipe tobacco products, tobacco-derived products, tobacco-derived nicotine products, smokeless tobacco products (e.g., moist snuff, dry snuff, and chewing tobacco), films, chewable tablets, tablets, shaped portions, gels, consumables, insoluble matrices, hollow shapes, reconstituted tobacco, expanded tobacco, etc. See, e.g., U.S. Patent Publication No. US2006 / 0191548.

[0296] As used herein, "cigarette" refers to a tobacco product having a "rod" and a "filler". The "rod" of a cigarette includes the cigarette paper, the filter, the filter plug wrapper (for containing the filter material), the tipping paper that secures the cigarette paper (including the filler) to the filter, and all glue that secures these components together. "Filler" includes (1) all tobacco, including but not limited to reconstituted tobacco and expanded tobacco; (2) non-tobacco substitutes (including but not limited to herbs, non-tobacco plant materials, and other flavorings that may accompany the tobacco wrapped in the cigarette paper); (3) flavoring agents; (4) flavoring agents and (5) all other additives (mixed into the tobacco and substitutes and wrapped in the cigarette).

[0297] On the one hand, tobacco products include reconstituted tobacco. On the other hand, the present disclosure provides reconstituted tobacco comprising a curing tobacco material. As used herein, "reconstituted tobacco" refers to a portion of a tobacco filler made from tobacco dust and other tobacco waste materials, processed into a sheet form and cut into strips to resemble tobacco. In addition to saving costs, reconstituted tobacco is also very important because it contributes to the taste of cigarettes by using a reaction between ammonia and sugar to process flavor development.

[0298] In one aspect, the tobacco product includes expanded tobacco. As used herein, "expanded tobacco" refers to a portion of such tobacco filler that is processed by expansion of a suitable gas so that the tobacco is "expanded," resulting in reduced density and greater filling capacity. It reduces the weight of the tobacco used in cigarettes.

[0299] The tobacco product derived from the plant of the present disclosure also includes cigarettes and other smoking articles, particularly including those smoking articles of filter element, wherein the smokable material rod includes the modulation tobacco in the tobacco mixture.On the one hand, the tobacco product of the present disclosure is selected from the group consisting of cigarillos, non-ventilation groove filter cigarettes, ventilation groove filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, tobacco leaves, hookah tobacco, shredded tobacco and cut tobacco.On the other hand, the tobacco product of the present disclosure is selected from the group consisting of cigarettes, heated tobacco products, clove cigarettes, bidis, cigarillos, non-ventilation cigarettes, ventilation groove filter cigarettes, pipe tobacco, snuff, buccal tobacco, chewing tobacco, moist smokeless tobacco, finely cut chewing tobacco, long cut chewing tobacco, bagged chewing tobacco products, chewing gum, tablets, lozenges and dissolving strips.

[0300] In another aspect, the tobacco product of the present disclosure is a smokeless tobacco product.In one aspect, the smokeless tobacco product is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, snus, dry snuff, and oral tobacco.

[0301] Smokeless tobacco products do not burn, and include but are not limited to chewing tobacco, moist smokeless tobacco, snuff and dry snuff. Chewing tobacco is coarsely ground tobacco leaves, which are usually packaged in large bag-like packages and used in the form of plugs or twists. Moist smokeless tobacco is moist, more finely ground tobacco, which is provided in bulk or bagged form and is usually packaged in round cans and used as a clip or placed in a bag between the cheeks and gums of adult tobacco consumers. Snuff is heat-treated smokeless tobacco. Dry snuff is finely ground tobacco, which is placed in the mouth or used in the nasal cavity.

[0302] In yet another aspect, the tobacco product of the present disclosure is selected from the group consisting of an electrically heated cigarette, an electronic cigarette, an electronic vaporization device, and an inhaled nicotine product.

[0303] In one aspect, the tobacco product of the present disclosure can be a blended tobacco product.

[0304] In another aspect, the present disclosure provides a tobacco blend comprising a cured tobacco material. The tobacco blend can comprise any combination of cured tobacco, uncured tobacco, fermented tobacco, unfermented tobacco, expanded tobacco, and reconstituted tobacco.

[0305] In one aspect, a tobacco blend comprises at least 5% modulated tobacco by weight. In one aspect, a tobacco blend comprises at least 10% modulated tobacco by weight. In one aspect, a tobacco blend comprises at least 15% modulated tobacco by weight. In one aspect, a tobacco blend comprises at least 20% modulated tobacco by weight. In one aspect, a tobacco blend comprises at least 25% modulated tobacco by weight. In one aspect, a tobacco blend comprises at least 30% modulated tobacco by weight. In one aspect, a tobacco blend comprises at least 35% modulated tobacco by weight. In one aspect, a tobacco blend comprises at least 40% modulated tobacco by weight. In one aspect, a tobacco blend comprises at least 45% modulated tobacco by weight. In one aspect, a tobacco blend comprises at least 50% modulated tobacco by weight. In one aspect, a tobacco blend comprises at least 55% modulated tobacco by weight. In one aspect, a tobacco blend comprises at least 60% modulated tobacco by weight. In one aspect, a tobacco blend comprises at least 65% modulated tobacco by weight. In one aspect, a tobacco blend comprises at least 70% modulated tobacco by weight. In one aspect, a tobacco blend comprises at least 75% modulated tobacco by weight. In one aspect, the tobacco blend comprises at least 80% cured tobacco by weight. In one aspect, the tobacco blend comprises at least 85% cured tobacco by weight. In one aspect, the tobacco blend comprises at least 90% cured tobacco by weight. In one aspect, the tobacco blend comprises at least 95% cured tobacco by weight.

[0306] In one aspect, a tobacco blend comprises at least 5% modulated tobacco by volume. In one aspect, a tobacco blend comprises at least 10% modulated tobacco by volume. In one aspect, a tobacco blend comprises at least 15% modulated tobacco by volume. In one aspect, a tobacco blend comprises at least 20% modulated tobacco by volume. In one aspect, a tobacco blend comprises at least 25% modulated tobacco by volume. In one aspect, a tobacco blend comprises at least 30% modulated tobacco by volume. In one aspect, a tobacco blend comprises at least 35% modulated tobacco by volume. In one aspect, a tobacco blend comprises at least 40% modulated tobacco by volume. In one aspect, a tobacco blend comprises at least 45% modulated tobacco by volume. In one aspect, a tobacco blend comprises at least 50% modulated tobacco by volume. In one aspect, a tobacco blend comprises at least 55% modulated tobacco by volume. In one aspect, a tobacco blend comprises at least 60% modulated tobacco by volume. In one aspect, a tobacco blend comprises at least 65% modulated tobacco by volume. In one aspect, a tobacco blend comprises at least 70% modulated tobacco by volume. In one aspect, a tobacco blend comprises at least 75% modulated tobacco by volume. In one aspect, the tobacco blend comprises at least 80% cured tobacco by volume. In one aspect, the tobacco blend comprises at least 85% cured tobacco by volume. In one aspect, the tobacco blend comprises at least 90% cured tobacco by volume. In one aspect, the tobacco blend comprises at least 95% cured tobacco by volume.

[0307] change

[0308] In one aspect, the present 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 a polypeptide in at least one tobacco cell, the 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 NO: 117 to 174; (b) selecting at least one tobacco cell comprising the non-natural mutation from step (a); and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). In another aspect, the present 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 at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 58 in at least one tobacco cell; (b) selecting at least one tobacco cell comprising the non-natural mutation from step (a); and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). On the other hand, the present 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 at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 59 to 116 in at least one tobacco cell; (b) selecting at least one tobacco cell comprising the non-natural mutation from step (a); and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). On the other hand, the present 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 selected from the group consisting of SEQ ID NO: 1 to 116 in at least one tobacco cell; (b) selecting at least one tobacco cell comprising the non-natural mutation from step (a); and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). On the one hand, any of the aforementioned methods further comprises (d) growing the modified tobacco plant regenerated in step (c). In another aspect, any of the foregoing methods further comprises: (e) crossing the modified tobacco plant grown in step (d) with a second tobacco plant; and (f) obtaining at least one seed from the crossing in step (e).

[0309] In one aspect, the present 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 operably linked to a nucleic acid encoding at least one small RNA molecule, the at least one small RNA molecule being capable of binding to and reducing the expression of an endogenous nucleic acid sequence, the 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 NOs: 117 to 174; (b) selecting at least one tobacco cell comprising 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, the present 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 operably linked to a nucleic acid encoding at least one small RNA molecule, the at least one small RNA molecule being 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 58; (b) selecting at least one tobacco cell comprising the recombinant DNA construct; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). On the other hand, the present 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 operably connected to a nucleic acid encoding at least one small RNA molecule, the at least one small RNA molecule being 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:59 to 116; (b) selecting at least one tobacco cell comprising the recombinant DNA construct; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). On the one hand, any of the foregoing methods further comprises (d) growing the modified tobacco plant regenerated in step (c). On the other hand, any of the foregoing 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).

[0310] In one aspect, the present 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 operably linked to a nucleic acid sequence encoding a polypeptide, the 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 NO: 117 to 174; (b) selecting at least one tobacco cell comprising 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, the present 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 operably linked to a nucleic acid sequence, the nucleic acid sequence being at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 58; (b) selecting at least one tobacco cell comprising the recombinant DNA construct; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). On the other hand, the 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 operably linked to a nucleic acid sequence, the nucleic acid sequence being at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 59 to 116; (b) selecting at least one tobacco cell comprising the recombinant DNA construct; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). On the one hand, any of the foregoing methods further comprises (d) growing the modified tobacco plant regenerated in step (c). On the other hand, any of the foregoing 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).

[0311] In one aspect, the present disclosure provides a method comprising transforming tobacco cells with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA molecule, the at least one small RNA molecule being capable of binding to and reducing the expression of an endogenous nucleic acid sequence, the endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In another aspect, the present disclosure provides a method comprising transforming tobacco cells with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA molecule, the at least one small RNA molecule being 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 58. In another aspect, the present disclosure provides a method comprising transforming a tobacco cell with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA molecule, the at least one small RNA molecule being 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: 59 to 116. In another aspect, the aforementioned method further comprises regenerating a modified tobacco plant from the transformed tobacco cell.

[0312] In one aspect, the disclosure provides a method comprising transforming tobacco cells with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably 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: 117 to 174. In another aspect, the disclosure provides a method comprising transforming tobacco cells with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably 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 58. In another aspect, the disclosure provides a method comprising transforming tobacco cells with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59 to 116. In another aspect, the aforementioned method further comprises regenerating a modified tobacco plant from the transformed tobacco cell.

[0313] The various methods for introducing recombinant DNA constructs into plant cells are known in the art, and these methods can be used to produce transgenic plant cells and plants according to the methods of the present application. Any suitable method or technology known in the art for transforming plant cells can be used according to the method of the present invention. Effective methods for transforming plants include bacterial-mediated transformation, such as Agrobacterium-mediated or Rhizobium-mediated transformation and microparticle bombardment-mediated transformation. Various methods known in the art are used to transform explants using transformation vectors via bacterial-mediated transformation or microparticle bombardment, and then these explants are subsequently cultivated, etc. to regenerate or develop transgenic plants. Other methods for plant transformation, such as microinjection, electroporation, vacuum infiltration, pressure, ultrasonic treatment, silicon carbide fiber stirring, polyethylene glycol (PEG)-mediated transformation, etc. are also known in the art. The transgenic plants produced by these transformation methods can be mosaic or non-mosaic for the transformation event, depending on the method and explant used.

[0314] Methods for transforming plant cells are well known to those of ordinary skill in the art. For example, specific instructions for transforming plant cells by microprojectile bombardment using particles coated with recombinant DNA (e.g., gene gun transformation) are found in U.S. Pat. Nos. 5,550,318; 5,538,880, 6,160,208; 6,399,861; and 6,153,812, and Agrobacterium-mediated transformation is described in U.S. Pat. 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 Compendium of Transgenic Crop Plants (2009) Blackwell Publishing. Any suitable method known to those of skill in the art can be used to transform tobacco cells with any of the nucleic acid molecules provided herein.

[0315] On the one hand, the method that nucleic acid molecule is introduced into tobacco cell comprises agrobacterium-mediated conversion. On the other hand, the method that nucleic acid molecule is introduced into tobacco cell comprises PEG-mediated conversion. On the other hand, the method that nucleic acid molecule is introduced into tobacco cell comprises gene gun conversion. On the other hand, the method that nucleic acid molecule is introduced into tobacco cell comprises liposome-mediated transfection (lipofection). On the other hand, the method that nucleic acid molecule is introduced into tobacco cell comprises slow virus transfection.

[0316] Lipofection is described in, for example, U.S. Pat. Nos. 5,049,386; 4,946,787; and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam TM Lipofectin TM ). Cationic and neutral lipids suitable for effective receptor recognition lipofection of polynucleotides include those in WO 91 / 17424 and WO 91 / 16024. Can be delivered to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vitro administration).

[0317] Any tobacco cell that can therefrom regenerate fertile tobacco plant is considered to be useful receptor cell for implementing the present disclosure.On the one hand, the recombinant DNA construct is introduced into tobacco cell.On the one hand, the recombinant DNA construct is introduced into tobacco protoplast cell.On the other hand, the recombinant DNA construct is introduced into tobacco callus cell.On the one hand, the recombinant DNA construct is introduced into the tobacco cell of the group that is selected from the following item composition: seed cell, fruit cell, leaf cell, cotyledon cell, hypocotyl cell, meristem cell, embryo cell, endosperm cell, root cell, tender shoot cell, stem cell, flower cell, inflorescence cell, handle cell, pedicel cell, style cell, stigma cell, torus cell, petal cell, sepal cell, pollen cell, anther cell, filament cell, ovary cell, ovule cell, pericarp cell and phloem cell.

[0318] Callus can be started from various tissue sources, including but not limited to immature embryos or partial embryos, seedling apical meristems, microspores, etc. Those cells that can be used as callus proliferation can be used as the recipient cells of transformation. For preparing the practical transformation method and materials of transgenic plants disclosed herein (for example, various culture media and receptor target cells, the transformation of immature embryos and the regeneration of fertile transgenic plants subsequently) are disclosed in, for example, U.S. Patent No. 6,194,636 and No. 6,232,526 and U.S. Application Publication 2004 / 0216189, all of which are incorporated herein by reference. Those cells that can be used as callus proliferation can be used as the recipient cells of genetic transformation.

[0319] Leaf Level

[0320] As used herein, "USDA leaf grade index" refers to the classification of leaf types according to group, quality and color. In one aspect, the USDA grade quality score is quantified as a 0-100 digital representation of the grade determined by a certified tobacco grader, and is a weighted average of all stem positions. The higher the grade index, the higher the quality. As used below, "points" refers to each integer digital representation of the USDA leaf grade score. For example, the difference between a USDA leaf grade index score of 90 and a score of 85 is 5 points.

[0321] Alternatively, leaf grade may be determined via hyperspectral imaging. See, for example, WO 2011 / 027315 (published on March 10, 2011, and incorporated by reference in its entirety).

[0322] As used herein, "certified tobacco leaf grader" refers to personnel who are trained to grade tobacco leaves according to the USDA official standard grade defined by the United States Department of Agriculture (USDA) (agricultural marketing system as announced in 7CFR § 29). The USDA leaf grade index score can be assigned by an employee, a former employee, or a personnel trained otherwise to grade tobacco leaves according to the USDA official standard grade. The exemplary steps of the standard operation of commercial inspection services begin with growers transporting tobacco to the market, after which tobacco is arranged in batches on a flat basket. Each batch is weighed, and then inspected by a certified tobacco leaf grader. After the inspection, the grader assigns the grade to each batch, and the grade becomes a grade certificate indicating group, quality, and color. It is similar to be used for the step of grading the experimental batch; however, the experimental tobacco will not be marketed or otherwise used for commercial purposes.

[0323] Tobacco grade 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 ambient moisture), and subtle differences or shades of green. For example, the official standard grades published by the U.S. Department of Agriculture's Agricultural Marketing Service (7 U.S.C. §511) can be used to determine leaf grade. See, e.g., Official Standard Grades for Burley Tobacco (U.S. Type 31 and Foreign Type 93), effective November 5, 1990 (55 F.R. 40645); Official Standard Grades for Cured Tobacco (U.S. Types 11, 12, 13, 14 and Foreign Type 92), effective March 27, 1989 (54 F.R. 7925); Official Standard Grades for Pennsylvania Seed Leaf Tobacco (U.S. Type 41), effective January 8, 1965 (29 F.R. 16854); Official Standard Grades for Ohio Cigar Leaf Tobacco (U.S. Types 42, 43 and 44), effective December 8, 1963 (28 F.R. 11719 and 28 F.R. 11926); the official standard grade for cigar binder tobaccos in Wisconsin (U.S. Types 54 and 55), effective November 20, 1969 (34 F.R. 17061); the official standard grade for cigar binder tobaccos in Wisconsin (U.S. Types 54 and 55), effective November 20, 1969 (34 F.R. 17061); the official standard grade for shade-grown cigar wrapper tobaccos in Georgia and Florida (U.S. Type 62), effective April 1971. USDA grade index values ​​may be determined based on industry-accepted grade indices. See, e.g., Bowman et al., Tobacco Science, 32:39-40 (1988); Traditional 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 of the foregoing references are incorporated by reference in their entireties).

[0324] Unless otherwise noted, the measured value of the leaf grade index value, alkaloid or nicotine level of the tobacco plant, kind, cultivar or system mentioned herein refers to average measured value, comprises the mean value of the multi-leaf of for example individual plant or the average measured value of the colony of the tobacco plant from individual kind, cultivar or system.For example, the colony of the tobacco plant that is used to determine average measured value (, leaf classification or alkaloid or nicotine level) or the set of tobacco leaf can have any size, for example, 2,5,10,15,20,25,30,35,40,50 or more.Use the colony of at least 5 or more tobacco plants to determine standard deviation.Follow the standard protocol of industry recognition to determine average measured value or grade index value.

[0325] As used herein, "USDA grading group," "leaf group," or "group" is a classification covering types of closely related grades based on certain characteristics related to stem location, body, or general quality. Group is the first factor in USDA grade. Group determination is part of the grading process and is assigned by certified tobacco leaf graders.

[0326] On the one hand, the modified tobacco plant comprising non-natural mutations is compared with the control tobacco plant lacking non-natural mutations when growing under comparable conditions and comprises comparable or higher USDA leaf grade index.On the other hand, the modified tobacco plant comprising recombinant DNA constructs is compared with the control tobacco plant lacking recombinant DNA constructs when growing under comparable conditions and comprises comparable or higher USDA leaf grade index.

[0327] As used herein, "comparable" USDA Leaf Grade Index means within 15%. For example, if the control plant has a USDA Leaf Grade Index of 100, the comparable USDA Leaf Grade Index will be between 85 and 100.

[0328] On the one hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions is high at least 1% USDA leaf grade index.On the other hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions is high at least 5% USDA leaf grade index.On the other hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions is high at least 10% USDA leaf grade index.On the other hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions is high at least 20% USDA leaf grade index.On the other hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions is high at least 30% USDA leaf grade index.On the other hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions is high at least 40% USDA leaf grade index. On the other hand, modified tobacco plant comprises than the USDA leaf grade index of control tobacco plant when growing under comparable conditions is high at least 50% USDA leaf grade index.On the other hand, modified tobacco plant comprises than the USDA leaf grade index of control tobacco plant when growing under comparable conditions is high at least 75% USDA leaf grade index.On the other hand, modified tobacco plant comprises than the USDA leaf grade index of control tobacco plant when growing under comparable conditions is high at least 100% USDA leaf grade index.

[0329] On the one hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions is high 1% to high 100% USDA leaf grade index.On the one hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions is high 1% to high 75% USDA leaf grade index.On the one hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions is high 1% to high 50% USDA leaf grade index.On the one hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions is high 1% to high 40% USDA leaf grade index.On the one hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions is high 1% to high 30% USDA leaf grade index.On the one hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions is high 1% to high 20% USDA leaf grade index. On the one hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions is high 1% to high 10% USDA leaf grade index.On the one hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions is high 10% to high 75% USDA leaf grade index.On the one hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions is high 10% to high 50% USDA leaf grade index.On the one hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions is high 1% to high 30% USDA leaf grade index.

[0330] On the one hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions at least 1 point higher.On the other hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions at least 2 points higher.On the other hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions at least 3 points higher.On the other hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions at least 4 points higher.On the other hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions at least 5 points higher.On the other hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions at least 6 points higher. On the other hand, the modified tobacco plant comprises the USDA leaf grade index higher than at least 7 points of the USDA leaf grade index of the control tobacco plant when growing under comparable conditions.On the other hand, the modified tobacco plant comprises the USDA leaf grade index higher than at least 8 points of the USDA leaf grade index of the control tobacco plant when growing under comparable conditions.On the other hand, the modified tobacco plant comprises the USDA leaf grade index higher than at least 9 points of the USDA leaf grade index of the control tobacco plant when growing under comparable conditions.On the other hand, the modified tobacco plant comprises the USDA leaf grade index higher than at least 10 points of the USDA leaf grade index of the control tobacco plant when growing under comparable conditions.On the other hand, the modified tobacco plant comprises the USDA leaf grade index higher than at least 11 points of the USDA leaf grade index of the control tobacco plant when growing under comparable conditions.On the other hand, the modified tobacco plant comprises the USDA leaf grade index higher than at least 12 points of the USDA leaf grade index of the control tobacco plant when growing under comparable conditions. On the other hand, the modified tobacco plant comprises the USDA leaf grade index higher than at least 13 points of the USDA leaf grade index of the control tobacco plant when growing under comparable conditions.On the other hand, the modified tobacco plant comprises the USDA leaf grade index higher than at least 14 points of the USDA leaf grade index of the control tobacco plant when growing under comparable conditions.On the other hand, the modified tobacco plant comprises the USDA leaf grade index higher than at least 15 points of the USDA leaf grade index of the control tobacco plant when growing under comparable conditions.On the other hand, the modified tobacco plant comprises the USDA leaf grade index higher than at least 16 points of the USDA leaf grade index of the control tobacco plant when growing under comparable conditions.On the other hand, the modified tobacco plant comprises the USDA leaf grade index higher than at least 17 points of the USDA leaf grade index of the control tobacco plant when growing under comparable conditions.On the other hand, the modified tobacco plant comprises the USDA leaf grade index higher than at least 18 points of the USDA leaf grade index of the control tobacco plant when growing under comparable conditions.On the other hand, the modified tobacco plant comprises the USDA leaf grade index higher than at least 19 points of the USDA leaf grade index of the control tobacco plant when growing under comparable conditions.On the other hand, the modified tobacco plant comprises the USDA leaf grade index higher than at least 20 points of the USDA leaf grade index of the control tobacco plant when growing under comparable conditions.On the other hand, the modified tobacco plant comprises the USDA leaf grade index higher than at least 25 points of the USDA leaf grade index of the control tobacco plant when growing under comparable conditions.On the other hand, the modified tobacco plant comprises the USDA leaf grade index higher than at least 30 points of the USDA leaf grade index of the control tobacco plant when growing under comparable conditions.On the other hand, the modified tobacco plant comprises the USDA leaf grade index higher than at least 35 points of the USDA leaf grade index of the control tobacco plant when growing under comparable conditions. In another aspect, the modified tobacco plant comprises a USDA Leaf Grade Index that is at least 40 points higher than the USDA Leaf Grade Index of a control tobacco plant when grown under comparable conditions. In another aspect, the modified tobacco plant comprises a USDA Leaf Grade Index that is at least 50 points higher than the USDA Leaf Grade Index of a control tobacco plant when grown under comparable conditions.

[0331] On the one hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions is high 1 point to the USDA leaf grade index of high 100 points.On the other hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions is high 1 point to the USDA leaf grade index of high 75 points.On the other hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions is high 1 point to the USDA leaf grade index of high 50 points.On the other hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions is high 1 point to the USDA leaf grade index of high 25 points.On the other hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions is high 1 point to the USDA leaf grade index of high 10 points.On the other hand, modified tobacco plant comprises than the USDA leaf grade index of the control tobacco plant when growing under comparable conditions is high 1 point to the USDA leaf grade index of high 5 points. In another aspect, the modified tobacco plant comprises a USDA Leaf Grade Index that is 10 to 50 points higher than the USDA Leaf Grade Index of a control tobacco plant when grown under comparable conditions. In another aspect, the modified tobacco plant comprises a USDA Leaf Grade Index that is 10 to 25 points higher than the USDA Leaf Grade Index of a control tobacco plant when grown under comparable conditions.

[0332] On the one hand, the comparable USDA leaf grade index between the modified tobacco plant and the control tobacco plant when growing under comparable conditions is in 1 point.On the other hand, the comparable USDA leaf grade index between the modified tobacco plant and the control tobacco plant when growing under comparable conditions is in 2 points.On the other hand, the comparable USDA leaf grade index between the modified tobacco plant and the control tobacco plant when growing under comparable conditions is in 3 points.On the other hand, the comparable USDA leaf grade index between the modified tobacco plant and the control tobacco plant when growing under comparable conditions is in 4 points.On the other hand, the comparable USDA leaf grade index between the modified tobacco plant and the control tobacco plant when growing under comparable conditions is in 5 points.On the other hand, the comparable USDA leaf grade index between the modified tobacco plant and the control tobacco plant when growing under comparable conditions is in 6 points.On the other hand, the comparable USDA leaf grade index between the modified tobacco plant and the control tobacco plant when growing under comparable conditions is in 7 points.On the other hand, the comparable USDA leaf grade index between the modified tobacco plant and the control tobacco plant when growing under comparable conditions is in 8 points. On the other hand, the comparable USDA leaf grade index between the modified tobacco plant and the control tobacco plant when growing under comparable conditions is in 9 points.On the other hand, the comparable USDA leaf grade index between the modified tobacco plant and the control tobacco plant when growing under comparable conditions is in 10 points.On the other hand, the comparable USDA leaf grade index between the modified tobacco plant and the control tobacco plant when growing under comparable conditions is in 11 points.On the other hand, the comparable USDA leaf grade index between the modified tobacco plant and the control tobacco plant when growing under comparable conditions is in 12 points.On the other hand, the comparable USDA leaf grade index between the modified tobacco plant and the control tobacco plant when growing under comparable conditions is in 13 points.On the other hand, the comparable USDA leaf grade index between the modified tobacco plant and the control tobacco plant when growing under comparable conditions is in 14 points.On the other hand, the comparable USDA leaf grade index between the modified tobacco plant and the control tobacco plant when growing under comparable conditions is in 15 points.

[0333] Aroma / flavor

[0334] On the one hand, the modified tobacco plant provided herein comprises one or more tobacco aroma compounds of similar level compared to the control tobacco plant grown under comparable conditions, and these one or more tobacco aroma compounds are selected from the group consisting of 3-methylvaleric acid, valeric acid, isovaleric acid, diterpene, cembranoid diterpene, sugar ester and reducing sugar. As used herein, "similar" level refers to within 20%.

[0335] As used herein, " tobacco aroma compounds " are compounds relevant to the local flavor and aroma of tobacco smoke. These compounds include but are not limited to 3-methylvaleric acid, valeric acid, isovaleric acid, cembranoid diterpenes and labdenoid diterpenes and sugar esters. The concentration of tobacco aroma compounds can be measured by any known metabolite analysis method in the art, including but not limited to gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance spectroscopy, liquid chromatography-mass spectrometry. Referring to, The Handbook of Plant Metabolomics, edited by Weckwerth and Kahl, (Wiley-Blackwell) (May 28, 2013).

[0336] As used herein, "reducing sugar" is any sugar (monosaccharide or polysaccharide) with free or potential free aldehyde or ketone groups. Glucose and fructose act as nicotine buffers in cigarette smoke by reducing smoke pH and effectively reducing the amount of "free" non-protonated nicotine. Reducing sugar balances smoke flavor, for example, by changing the sensory impact of nicotine and other tobacco alkaloids. It is reported that there is a negative correlation between sugar content and alkaloid content in different tobacco varieties, the same variety and the same plant system, which is caused by planting conditions. Reducing sugar levels can be measured using segmented flow colorimetry, which is developed for analyzing tobacco samples, as adapted by Skalar Instrument Co. (West Chester, PA) and by Davis, Tobacco Science 20: 139-144 (1976). For example, the sample is dialyzed with sodium carbonate solution. Copper new cuprous reagent is added to the sample and the solution is heated. Copper new cuprous reagent chelate is reduced in the presence of sugar to produce a colored complex, which is measured at 460nm.

[0337] TSNA

[0338] In one aspect, the modified tobacco plants provided further comprise one or more mutations in one or more loci encoding nicotine demethylases (e.g., CYP82E4, CYP82E5, CYP82E10) that confer reduced amounts of nornicotine (see, U.S. Pat. Nos. 8,319,011; 8,124,851; 9,187,759; 9,228,194; 9,228,195; 9,247,706) compared to control tobacco plants lacking one or more mutations in one or more loci encoding nicotine demethylases when grown under comparable conditions. In one aspect, the modified tobacco plants further comprise reduced nicotine demethylase activity compared to control plants when grown and cured under comparable conditions. In another aspect, the tobacco plant provided further comprises one or more mutations or transgenes that provide elevated levels of one or more antioxidants (see, U.S. Patent Application Publication Nos. 2018 / 0119163 and WO 2018 / 067985). In another aspect, the tobacco plant provided further comprises one or more mutations or transgenes that provide reduced levels of one or more tobacco-specific nitrosamines (TSNAs). In one aspect, TSNA is selected from the group consisting of N'-nitrosonornicotine (NNN), 4-methylnitrosoamino-1-(3-pyridyl)-1-butanone (NNK), N'-nitrosoanatabine (NAT) and N'-nitrosoanatabine (NAB).

[0339] Example

[0340] The following non-limiting examples are contemplated:

[0341] 1. A modified tobacco 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 NOs: 117 to 174.

[0342] 2. A modified tobacco plant or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to an endogenous nucleic acid sequence and reducing its expression, wherein the endogenous nucleic acid sequence encodes a polypeptide that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NO: 117 to 174.

[0343] 3. A modified tobacco plant or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably 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: 117 to 174.

[0344] 4. The modified tobacco plant or part thereof of embodiment 1, wherein said tobacco plant produces at least one leaf comprising a reduced amount of at least one alkaloid as compared to the amount of said alkaloid in a control tobacco plant lacking said at least one non-natural mutation in said endogenous nucleic acid sequence when grown under comparable conditions.

[0345] 5. The modified tobacco plant or part thereof of embodiment 2 or 3, wherein said tobacco plant produces at least one leaf comprising a reduced amount of at least one alkaloid as compared to the amount of said alkaloid in a control tobacco plant lacking said recombinant DNA construct when grown under comparable conditions.

[0346] 6. The modified tobacco plant, or part thereof, of any one of embodiments 1, 2, or 4, wherein said endogenous nucleic acid sequence comprises a nucleic acid sequence that is at least 80% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-58.

[0347] 7. The modified tobacco plant or part thereof of embodiment 3, wherein the nucleic acid sequence comprises a nucleic acid sequence that is at least 80% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-58.

[0348] 8. The modified tobacco plant, or part thereof, of any one of embodiments 1, 2, or 4, wherein said endogenous nucleic acid sequence comprises a nucleic acid sequence that is at least 80% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 59-116.

[0349] 9. The modified tobacco plant or part thereof of embodiment 3, wherein the nucleic acid sequence comprises a nucleic acid sequence that is at least 80% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 59-116.

[0350] 10. The modified tobacco plant or part thereof of embodiment 1 or 4, wherein said tobacco plant is homozygous for said at least one non-natural mutation.

[0351] 11. The modified tobacco plant, or part thereof, of embodiment 1 or 4, wherein said tobacco plant is heterozygous for said at least one non-natural mutation.

[0352] 12. The modified tobacco plant, or part thereof, of any one of embodiments 4-6, 8, 10, or 11, wherein said at least one alkaloid is selected from the group consisting of anatabine, anatabine, nicotine, and nornicotine.

[0353] 13. The modified tobacco plant, or part thereof, of any of embodiments 4-to 6, 8, or 10-12, wherein said reduced amount of at least one alkaloid comprises a reduction of at least 1%.

[0354] 14. The modified tobacco plant, or part thereof, of any one of embodiments 1, 4, 10, or 11, wherein said at least one non-natural mutation comprises a mutation selected from the group consisting of an insertion, a deletion, a substitution, a duplication, and an inversion.

[0355] 15. The modified tobacco plant, or part thereof, of any one of embodiments 1, 4, 10, or 11, wherein said at least one non-natural mutation comprises at least one mutation selected from the group consisting of a nonsense mutation, a missense mutation, a frameshift mutation, and a splice site mutation.

[0356] 16. The modified tobacco plant, or part thereof, of any one of embodiments 1, 4, 10, 11, 14, or 15, wherein said at least one non-natural mutation comprises a null mutation.

[0357] 17. The modified tobacco plant or part thereof of any one of embodiments 1, 4, 10, 11, or 14-16, wherein said at least one non-natural mutation results in truncation of said polypeptide.

[0358] 18. The modified tobacco plant or part thereof of any one of embodiments 1, 4, 10, 11, or 14 to 17, wherein said at least one non-natural mutation comprises a mutation in a sequence region selected from the group consisting of a promoter, a 5'-untranslated region (UTR), an exon, an intron, a 3'-UTR, and a terminator.

[0359] 19. The modified tobacco plant or part thereof of any one of embodiments 1, 4, 10, 11, or 14 to 18, wherein said at least one non-natural mutation results in expression of said nucleic acid sequence at a reduced level compared to expression of said nucleic acid sequence in the same tissue of a control tobacco plant when grown under comparable conditions, wherein said nucleic acid sequence lacks said at least one non-natural mutation in said control tobacco plant.

[0360] 20. The modified tobacco plant or part thereof of any one of embodiments 1, 4, 10, 11, or 14 to 19, wherein said at least one non-natural mutation results in a reduced level of activity of a protein or polypeptide encoded by said nucleic acid sequence compared to the activity of the protein or polypeptide encoded by said nucleic acid sequence in a control tobacco plant when grown under comparable conditions, wherein

[0361] The nucleic acid sequence lacks the at least one non-natural mutation in the control tobacco plant.

[0362] 21. The modified tobacco plant or part thereof of any one of embodiments 2, 3, 5, 7 or 9, wherein said promoter comprises a promoter selected from the group consisting of a constitutive promoter, a tissue-preferred promoter, a tissue-specific promoter, and an inducible promoter.

[0363] 22. The modified tobacco plant or part thereof of embodiment 21, wherein said tissue-preferred promoter comprises a root-preferred promoter.

[0364] 23. The modified tobacco plant or part thereof of embodiment 21, wherein said tissue-specific promoter comprises a root-specific promoter.

[0365] 24. The modified tobacco plant or part thereof according to embodiment 21, wherein said constitutive promoter is selected from the group consisting of a cauliflower mosaic virus (CaMV) 35S promoter, an ubiquitin promoter, an actin promoter, an opine promoter, and an alcohol dehydrogenase promoter.

[0366] 25. The modified tobacco plant or part thereof according to embodiment 2, wherein the at least one small RNA molecule is selected from the group consisting of double-stranded RNA, small interfering RNA (siRNA), trans-acting siRNA, and microRNA.

[0367] 26. The modified tobacco plant or part thereof of embodiment 2, wherein said at least one small RNA molecule comprises between 18 nucleotides and 30 nucleotides.

[0368] 27. The modified tobacco plant or part thereof of embodiment 2, wherein said at least one small RNA molecule comprises a nucleic acid sequence that is at least 90% complementary to a sequence selected from the group consisting of SEQ ID NOs: 59-116.

[0369] 28. The modified tobacco plant or part thereof of any one of embodiments 1-27, wherein the modified tobacco plant is of a tobacco variety selected from the group consisting of flue-curing varieties, brilliant varieties, burley varieties, Virginia varieties, Maryland varieties, black tobacco varieties, galpao varieties, oriental varieties, and Turkish varieties.

[0370] 29. The modified tobacco plant, or part thereof, of any one of embodiments 1-116, wherein the modified tobacco plant is of a variety selected from the group consisting of the tobacco varieties listed in Tables 2-8.

[0371] 30. The modified tobacco plant, or part thereof, of any one of embodiments 1-29, wherein the modified tobacco plant is a hybrid.

[0372] 31. The modified tobacco plant, or part thereof, of any one of embodiments 1-30, wherein the modified tobacco plant is male sterile or cytoplasmic male sterile.

[0373] 32. The modified tobacco plant, or part thereof, of any one of embodiments 1-30, wherein the modified tobacco plant is female sterile.

[0374] 33. A cured tobacco material derived from the modified tobacco plant, or part thereof, of any one of embodiments 1-32.

[0375] 34. The cured tobacco material of embodiment 33, wherein the cured tobacco material comprises cured leaf material, cured stem material, or both.

[0376] 35. The cured tobacco material of embodiment 33 or 34, wherein the cured tobacco material comprises flue-cured tobacco material, air-cured tobacco material, flue-cured tobacco material, and sun-cured tobacco material.

[0377] 36. A tobacco blend comprising the cured tobacco material of any of embodiments 33-35.

[0378] 37. The tobacco blend of embodiment 36, wherein the tobacco blend comprises at least 10% by weight curing tobacco.

[0379] 38. The tobacco blend of embodiment 36, wherein the tobacco blend comprises at least 10% curing tobacco by volume.

[0380] 39. A tobacco product comprising the tobacco blend of any of embodiments 36-38.

[0381] 40. A tobacco product comprising the cured tobacco material of any of embodiments 33-35.

[0382] 41. The tobacco product of embodiment 39 or 40, wherein the tobacco product is selected from the group consisting of cigarettes, heated tobacco products, kretek cigarettes, bidis, cigars, cigarillos, unventilated cigarettes, ventilated groove filter cigarettes, pipe tobacco, snus, snus, chewing tobacco, moist smokeless tobacco, fine cut chewing tobacco, long cut chewing tobacco, pouched chewing tobacco products, chewing gum, tablets, lozenges, and dissolving strips.

[0383] 42. The tobacco product of embodiment 39 or 40, wherein the tobacco product is a smokeless tobacco product.

[0384] 43. The tobacco product of embodiment 42, wherein the smokeless tobacco product is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, nasal snuff, dry snuff, and oral tobacco.

[0385] 44. A reconstituted tobacco comprising the cured tobacco material of any of embodiments 33-35.

[0386] 45. A method of producing a modified tobacco plant, the method comprising:

[0387] (a) 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 NOs: 117 to 174;

[0388] (b) selecting at least one tobacco cell comprising the non-natural mutation from step (a); and

[0389] (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b).

[0390] 46. ​​A method of producing a modified tobacco plant, the method comprising:

[0391] (a) introducing a recombinant DNA construct into at least one tobacco cell, wherein the recombinant DNA construct comprises a heterologous promoter operably 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 NOs: 117 to 174;

[0392] (b) selecting at least one tobacco cell comprising the recombinant DNA construct; and

[0393] (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b).

[0394] 47. A method of producing a modified tobacco plant, the method comprising:

[0395] (a) introducing a recombinant DNA construct into at least one tobacco cell, wherein the recombinant DNA construct comprises a heterologous promoter operably 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: 117 to 174;

[0396] (b) selecting at least one tobacco cell comprising the recombinant DNA construct; and

[0397] (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b).

[0398] 48. The method of embodiment 45, wherein said at least one modified tobacco plant comprises a reduced amount of at least one alkaloid as compared to a control tobacco plant lacking said mutation when grown under comparable conditions.

[0399] 49. The method of embodiment 46 or 47, wherein said at least one modified tobacco plant comprises a reduced amount of at least one alkaloid as compared to a control tobacco plant lacking said recombinant DNA construct when grown under comparable conditions.

[0400] 50. The method of any one of embodiments 45, 46, 48 or 49, wherein the endogenous nucleic acid sequence is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 58.

[0401] 51. The method of embodiment 47 or 49, wherein the nucleic acid sequence is at least 80% identical to a sequence selected from the group consisting of SEQ ID NO: 1 to 58.

[0402] 52. The method of any one of embodiments 45, 46, 48 or 49, wherein the endogenous nucleic acid sequence is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 59 to 116.

[0403] 53. The method of embodiment 47 or 49, wherein the nucleic acid sequence is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 59 to 116.

[0404] 54. The method of any one of embodiments 48-53, wherein said at least one alkaloid is selected from the group consisting of anatabine, anatabine, nicotine, and nornicotine.

[0405] 55. The method of any one of embodiments 48-53, wherein said reduced amount of at least one alkaloid comprises at least a 1% reduction.

[0406] 56. A method according to any one of embodiments 45, 48, 50, 52, 54 or 55, wherein the non-natural mutation comprises a mutation selected from the group consisting of insertion, deletion, substitution, duplication and inversion.

[0407] 57. A method according to any one of embodiments 45, 48, 50, 52, or 54 to 56, wherein the non-natural mutation comprises a mutation selected from the group consisting of a nonsense mutation, a missense mutation, a frameshift mutation, and a splice site mutation.

[0408] 58. A method according to any one of embodiments 45, 48, 50, 52, or 54 to 57, wherein the non-natural mutation comprises a null mutation.

[0409] 59. The method of any one of embodiments 45, 48, 50, 52, or 54 to 58, wherein the non-natural mutation results in truncation of the polypeptide.

[0410] 60. The method of any one of embodiments 45, 48, 50, 52, or 54 to 59, wherein the non-natural mutation comprises a mutation in a sequence region selected from the group consisting of a promoter, a 5'-untranslated region (UTR), an exon, an intron, a 3'-UTR, and a terminator.

[0411] 61. The method of any one of embodiments 45, 48, 50, 52, or 54 to 60, wherein said inducing comprises using an agent selected from the group consisting of a chemical mutagen, radiation, a transposon, Agrobacterium, and a nuclease.

[0412] 62. The method of embodiment 61, wherein the nuclease is selected from the group consisting of a meganuclease, a zinc finger nuclease, a transcription activator-like effector nuclease, a CRISPR / Cas9 nuclease, a CRISPR / Cpf1 nuclease, a CRISPR / CasX nuclease, a CRISPR / CasY nuclease, a Csm1 nuclease, or any combination thereof.

[0413] 63. The method of embodiment 61, wherein the chemical mutagen comprises ethyl methanesulfonate.

[0414] 64. The method of embodiment 61, wherein the radiation comprises gamma rays, X-rays, ionizing radiation, or fast neutrons.

[0415] 65. The method of embodiment 46, wherein the small RNA molecule is selected from the group consisting of double-stranded RNA, small interfering RNA (siRNA), trans-acting siRNA, and microRNA.

[0416] 66. The method of embodiment 46, wherein the at least one small RNA molecule comprises 18 to 30 nucleotides.

[0417] 67. The method of embodiment 46, wherein the at least one small RNA molecule comprises a nucleic acid sequence that is at least 90% complementary to a sequence selected from the group consisting of SEQ ID NO: 59 to 116.

[0418] 68. The method of any one of embodiments 46, 47, or 49 to 53, wherein the promoter comprises a promoter selected from the group consisting of a constitutive promoter, a tissue-preferred promoter, a tissue-specific promoter, and an inducible promoter.

[0419] 69. The method of embodiment 68, wherein the tissue-preferred promoter comprises a root-preferred promoter.

[0420] 70. The method of embodiment 68, wherein the tissue-specific promoter comprises a root-specific promoter.

[0421] 71. The method of embodiment 68, wherein said constitutive promoter is selected from the group consisting of a cauliflower mosaic virus (CaMV) 35S promoter, an ubiquitin promoter, an actin promoter, an opine promoter, and an alcohol dehydrogenase promoter.

[0422] 72. The method of any one of embodiments 45-71, wherein said at least one tobacco cell is a tobacco protoplast cell.

[0423] 73. The method of any one of embodiments 45-71, wherein said at least one tobacco cell is a tobacco callus cell.

[0424] 74. The method of any one of embodiments 45 to 71, wherein said at least one tobacco cell is selected from the group consisting of seed cells, fruit cells, leaf cells, cotyledon cells, hypocotyl cells, meristem cells, embryo cells, endosperm cells, root cells, shoot 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.

[0425] 75. The method of any one of embodiments 45 to 47, further comprising:

[0426] (d) growing the modified tobacco plant regenerated in step (c).

[0427] 76. The method of embodiment 75, wherein the method further comprises:

[0428] (e) crossing the modified tobacco plant grown in step (d) with a second tobacco plant; and

[0429] (f) obtaining at least one seed from the hybridization in step (e).

[0430] 77. A method according to embodiment 45 or 48, wherein the at least one non-natural mutation results in expression of the nucleic acid sequence at a reduced level compared to expression of the nucleic acid sequence in the same tissue of a control tobacco plant when grown under comparable conditions, wherein the nucleic acid sequence lacks the at least one non-natural mutation in the control tobacco plant.

[0431] 78. The method of embodiment 77, wherein the reduced expression level comprises a reduction of at least 5%.

[0432] 79. A method according to embodiment 45 or 48, wherein the at least one non-natural mutation results in expression of the nucleic acid sequence at an increased level compared to expression of the nucleic acid sequence in the same tissue of a control tobacco plant when grown under comparable conditions, wherein the nucleic acid sequence lacks the at least one non-natural mutation in the control tobacco plant.

[0433] 80. The method of embodiment 79, wherein the increased level of expression comprises an increase of at least 5%.

[0434] 81. A method according to embodiment 45 or 48, wherein the at least one non-natural mutation results in a reduced level of activity of a 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 when grown under comparable conditions, wherein the nucleic acid sequence lacks the at least one non-natural mutation in the control tobacco plant.

[0435] 82. A method according to embodiment 45 or 48, wherein the at least one non-natural mutation results in an increased level of activity of a 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 when grown under comparable conditions, wherein the nucleic acid sequence lacks the at least one non-natural mutation in the control tobacco plant.

[0436] 83. The method of any one of embodiments 45-82, wherein said modified tobacco plant is of a tobacco variety selected from the group consisting of flue-curing varieties, brilliant varieties, burley varieties, Virginia varieties, Maryland varieties, black tobacco varieties, garpao varieties, oriental varieties, and turkey varieties.

[0437] 84. The method of any one of embodiments 45-83, wherein said modified tobacco plant is of a variety selected from the group consisting of the varieties listed in Tables 2-8.

[0438] 85. The method of any one of embodiments 45-84, wherein said modified tobacco plant is a hybrid.

[0439] 86. The method of any one of embodiments 45-85, wherein said modified tobacco plant is male sterile or cytoplasmic male sterile.

[0440] 87. The method of any one of embodiments 45-85, wherein said modified tobacco plant is female sterile.

[0441] 88. The method of embodiment 45 or 48, wherein said modified tobacco plant comprises a comparable or higher USDA Leaf Grade Index as compared to a control tobacco plant lacking said non-natural mutation when grown under comparable conditions.

[0442] 89. The method of embodiment 46 or 47, wherein said modified tobacco plant has a similar or higher USDA Leaf Grade Index as compared to a control tobacco plant lacking said recombinant DNA construct when grown under similar conditions.

[0443] 90. A method comprising preparing a tobacco product using a cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a non-natural mutation in an endogenous nucleic acid sequence, wherein the endogenous nucleic acid sequence encodes 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: 117 to 174.

[0444] 91. A method comprising preparing a tobacco product using a cured 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 operably linked to a nucleic acid sequence encoding at least one small RNA molecule, the at least one small RNA molecule being 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 NOs: 117 to 174.

[0445] 92. A method comprising preparing a tobacco product using a cured 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 operably 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: 117 to 174.

[0446] 93. The method of any one of embodiments 90-92, wherein the cured tobacco material comprises cured leaf material, cured stem material, or both.

[0447] 94. The method of any one of embodiments 90 to 92, wherein the cured tobacco material comprises flue-cured tobacco material, air-cured tobacco material, flue-cured tobacco material, and sun-cured tobacco material.

[0448] 95. The method of any one of embodiments 90-92, wherein the tobacco product is selected from the group consisting of cigarettes, kretek cigarettes, bidis, cigars, cigarillos, unventilated cigarettes, ventilated fluted filter cigarettes, pipe tobacco, snus, snus, chewing tobacco, moist smokeless tobacco, fine cut chewing tobacco, long cut chewing tobacco, pouched chewing tobacco products, chewing gum, tablets, lozenges, and dissolving strips.

[0449] 96. The method of any one of embodiments 90-92, wherein said tobacco product is a smokeless tobacco product.

[0450] 97. The method of embodiment 96, wherein the smokeless tobacco product is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, nasal snuff, dry snuff, and snus.

[0451] 98. The method of any one of embodiments 90-92, wherein the cured tobacco material is of a tobacco variety selected from 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.

[0452] 99. The method of any one of embodiments 90 to 98, wherein the endogenous nucleic acid sequence comprises a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116.

[0453] 100. The method of any one of embodiments 92 to 98, wherein the nucleic acid sequence comprises a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116.

[0454] 101. A method comprising transforming tobacco cells with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA molecule, the at least one small RNA molecule being capable of binding to and reducing the expression of an endogenous nucleic acid sequence, the 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: 117 to 174.

[0455] 102. A method comprising transforming tobacco cells with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably 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 NO: 117 to 174.

[0456] 103. A method for producing a modified tobacco plant, the method comprising:

[0457] (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 a...

Claims

1. A modified tobacco 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 NOs: 117 to 174.

2. A modified tobacco plant or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule, wherein the at least one small RNA molecule is capable of binding to an endogenous nucleic acid sequence and reducing its expression, wherein the endogenous nucleic acid sequence encodes a polypeptide that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NO: 117 to 174.

3. A modified tobacco plant or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide, wherein the polypeptide comprises 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: 117 to 174.

4. The modified tobacco plant or part thereof of claim 1, wherein said tobacco plant produces at least one leaf comprising a reduced amount of at least one alkaloid as compared to the amount of said alkaloid in a control tobacco plant lacking said at least one non-natural mutation in said endogenous nucleic acid sequence when grown under comparable conditions.

5. The modified tobacco plant or part thereof according to claim 2 or 3, wherein said tobacco plant produces at least one leaf comprising a reduced amount of at least one alkaloid as compared to the amount of said alkaloid in a control tobacco plant lacking said recombinant DNA construct when grown under comparable conditions.

6. A cured tobacco material derived from the modified tobacco plant or part thereof according to any one of claims 1 to 5.

7. The cured tobacco material of claim 6, wherein the cured tobacco material comprises cured leaf material, cured stem material, or both.

8. The cured tobacco material according to claim 6, wherein the cured tobacco material comprises flue-cured tobacco material, air-cured tobacco material, flue-cured tobacco material and sun-cured tobacco material.

9. A tobacco blend comprising the cured tobacco material according to any one of claims 6 to 8.

10. The tobacco blend of claim 9, wherein the tobacco blend comprises at least 10% by weight curing tobacco.

11. The tobacco blend of claim 9, wherein the tobacco blend comprises at least 10% curing tobacco by volume.

12. A tobacco product comprising the tobacco blend according to any one of claims 9 to 11.

13. A tobacco product comprising a cured tobacco material according to any one of claims 6 to 8.

14. The tobacco product of claim 12 or 13, wherein the tobacco product is selected from the group consisting of cigarettes, heated tobacco products, kretek cigarettes, bidis, cigars, cigarillos, unventilated cigarettes, ventilated groove filter cigarettes, pipe tobacco, snus, snus, chewing tobacco, moist smokeless tobacco, fine cut chewing tobacco, long cut chewing tobacco, pouched chewing tobacco products, chewing gum, tablets, lozenges, and dissolving strips.

15. The tobacco product according to claim 12 or 13, wherein the tobacco product is a smokeless tobacco product.

16. The tobacco product of claim 15, wherein the smokeless tobacco product is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, snus, dry snuff, and oral tobacco.

17. A reconstituted tobacco comprising the cured tobacco material according to any one of claims 6 to 8.

18. A method for producing a modified tobacco plant, the method comprising: (a) 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 NOs: 117 to 174; (b) selecting at least one tobacco cell comprising the non-natural mutation from step (a); and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b).

19. 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 operably linked to a nucleic acid encoding at least one small RNA molecule, wherein the at least one small RNA molecule is capable of binding to and reducing the expression of an endogenous nucleic acid sequence, wherein the endogenous nucleic acid sequence encodes a polypeptide that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174; (b) selecting at least one tobacco cell comprising the recombinant DNA construct; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b).

20. 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 operably 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: 117 to 174; (b) selecting at least one tobacco cell comprising the recombinant DNA construct; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b).

21. The method of claim 18, wherein the at least one modified tobacco plant comprises a reduced amount of at least one alkaloid compared to a control tobacco plant lacking the mutation when grown under comparable conditions.

22. The method of claim 19 or 20, wherein the at least one modified tobacco plant comprises a reduced amount of at least one alkaloid compared to a control tobacco plant lacking the recombinant DNA construct when grown under comparable conditions.

23. The method according to any one of claims 18 to 20, wherein the method further comprises: (e) growing the modified tobacco plant regenerated in step (c).

24. The method of claim 23, wherein the method further comprises: (g) crossing the modified tobacco plant grown in step (d) with a second tobacco plant; as well as (h) obtaining at least one seed from the hybridization in step (e).

25. according to the method for claim 18, wherein said at least one non-natural mutation causes the expression of the described nucleic acid sequence of reduction level compared with the expression of the described nucleic acid sequence in the same tissue of the control tobacco plant when growing under comparable conditions, wherein said nucleic acid sequence lacks the described at least one non-natural mutation in the described control tobacco plant.

26. A method comprising preparing a tobacco product using a cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a non-natural mutation in an endogenous nucleic acid sequence, wherein the endogenous nucleic acid sequence encodes 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: 117 to 174.

27. A method comprising preparing a tobacco product using a cured 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 operably linked to a nucleic acid sequence encoding at least one small RNA molecule, wherein the at least one small RNA molecule is capable of binding to and reducing the expression of an endogenous nucleic acid sequence, wherein the endogenous nucleic acid sequence encodes a polypeptide that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174.

28. A method comprising preparing a tobacco product using a cured 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 operably 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: 117 to 174.

29. The method of any one of claims 26 to 28, wherein the cured tobacco material comprises cured leaf material, cured stem material, or both.

30. The method of any one of claims 26 to 28, wherein the cured tobacco material comprises flue-cured tobacco material, air-cured tobacco material, flue-cured tobacco material, and sun-cured tobacco material.

31. The method of any one of claims 26 to 28, wherein the tobacco product is selected from the group consisting of cigarettes, kretek cigarettes, bidis, cigars, cigarillos, unventilated cigarettes, ventilated fluted filter cigarettes, pipe tobacco, snus, snus, chewing tobacco, moist smokeless tobacco, fine cut chewing tobacco, long cut chewing tobacco, pouched chewing tobacco products, chewing gum, tablets, lozenges, and dissolving strips.

32. The method of any one of claims 26 to 28, wherein the tobacco product is a smokeless tobacco product.

33. The method of claim 32, wherein the smokeless tobacco product is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, snus, dry snuff, and oral tobacco.

34. The method of any one of claims 26 to 28, wherein the cured tobacco material is of a tobacco variety selected from the group consisting of flue-cured varieties, bright varieties, burley varieties, Virginia varieties, Maryland varieties, black tobacco varieties, garpao varieties, oriental varieties and Turkish varieties.

35. The method of claim 26 or 27, wherein the endogenous nucleic acid sequence comprises a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116.

36. The method of claim 28, wherein the nucleic acid sequence comprises a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116.

37. A method comprising transforming tobacco cells with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA molecule, the at least one small RNA molecule being capable of binding to and reducing the expression of an endogenous nucleic acid sequence, the 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: 117 to 174.

38. A method comprising transforming tobacco cells with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably 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 NO: 117 to 174.

39. A method for producing a modified tobacco plant, 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 generation of tobacco seeds, wherein the at least one tobacco plant of the first tobacco variety comprises 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 NOs: 117 to 174, wherein the non-natural mutation is not present in the endogenous nucleic acid sequence in a control tobacco plant of the first tobacco variety; as well as (b) selecting at least one tobacco seed of the previous generation or a plant germinated therefrom, wherein the at least one tobacco seed or a plant germinated therefrom comprises the non-natural mutation.

40. A method for producing a modified tobacco plant, 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 generation of tobacco seeds, 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 operably linked to a nucleic acid sequence encoding at least one small RNA molecule, wherein the small RNA molecule is capable of binding to and reducing the expression of an endogenous nucleic acid sequence, wherein the endogenous nucleic acid sequence encodes a polypeptide that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174, wherein the recombinant DNA construct is not present in the endogenous nucleic acid sequence in a control tobacco plant of the same variety; as well as (b) selecting at least one tobacco seed of the first generation or a plant germinated therefrom, wherein the at least one tobacco seed or a plant germinated therefrom comprises the recombinant DNA construct.

41. A method for producing a modified tobacco plant, the method comprising: (a) crossing 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 generation of tobacco seeds, 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 operably linked to a nucleic acid sequence encoding a polypeptide, wherein the polypeptide comprises 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: 117 to 174, wherein the recombinant DNA construct is not present in the nucleic acid sequence in a control tobacco plant of the first tobacco variety; as well as (b) selecting at least one tobacco seed of the first generation or a plant germinated therefrom, wherein the at least one tobacco seed or a plant germinated therefrom comprises the recombinant DNA construct.

42. The method of any one of claims 39 to 41, wherein the plant germinated in step (b) comprises a reduced amount of at least one alkaloid compared to the control tobacco plant when grown under comparable conditions.

43. A modified tobacco plant or part thereof, comprising at least one non-natural mutation in an endogenous nucleic acid sequence that modulates 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 NOs: 117 to 174.

44. A cured tobacco material derived from the modified tobacco plant or part thereof according to claim 43.

45. A tobacco blend comprising the cured tobacco material according to claim 44.

46. ​​A tobacco product comprising the tobacco blend of claim 45.

47. A tobacco product comprising the cured tobacco material according to claim 44.

48. A reconstituted tobacco comprising the cured tobacco material according to claim 44.

49. A modified tobacco plant or part thereof, comprising: (a) a genetic modification in a gene; or (b) a genetic modification targeted to the gene; wherein the genetic modification downregulates the expression or activity of the gene, wherein the gene encodes a nucleic acid sequence having at least 80% identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 116.

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