Methods and Compositions for Controlling Alkaloids in Tobacco

Abstract

The present disclosure relates to tobacco plants, plant parts, seeds, compositions, and methods for regulating the expression of decarboxylase genes in tobacco for controlling alkaloid levels.

Description

Technical Field

[0001] Field The present disclosure relates to tobacco plants, plant parts, seeds, compositions, and methods for regulating the expression of decarboxylase genes in tobacco for controlling alkaloid levels.

[0002] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 344,387, filed May 23, 2022, which is hereby incorporated by reference in its entirety.

[0003] Incorporation of Sequence Listing The sequence listing contained in the file "P34752WO00_SL.XML", created on May 2, 2023, and having 85,870 bytes (measured in MS - Windows®), is electronically submitted herewith and is hereby incorporated by reference in its entirety. A brief description of the nucleic acid and amino acid sequences is provided in Table 1.

[0004] (Table 1) Nucleic Acid and Amino Acid Sequences TIFF2025518037000002.tif255142

Background Art

[0005] Background Decarboxylase is an enzyme that catalyzes the decarboxylation of specific organic molecules. Decarboxylation is a chemical reaction that removes a carboxyl group and releases carbon dioxide. See Figure 1. The enzymes ornithine decarboxylase, arginine decarboxylase, and S - adenosylmethionine decarboxylase have previously been shown to be involved in the initial conversion of amino acids to putrescine in the pyrrolidine nicotine biosynthetic pathway. See Figure 2. In the pyridine nicotine biosynthetic pathway, no specific decarboxylase enzyme has been discovered. Herein, for the first time, the role of several decarboxylase genes in the pyridine nicotine biosynthetic pathway is elucidated.

Summary of the Invention

[0006] Summary In one aspect, the present disclosure provides a modified tobacco plant or a 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: 13 to 18.

[0007] In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule capable of binding to at least one endogenous nucleic acid sequence encoding a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18 and reducing its expression.

[0008] In one aspect, the present disclosure provides a modified tobacco plant or a 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 that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.

[0009] In one aspect, the present disclosure provides a method of producing a modified tobacco plant, comprising: (a) inducing at least one non-natural mutation in at least one tobacco cell in an endogenous nucleic acid sequence encoding a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18; (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).

[0010] In one aspect, the present disclosure provides a method for producing a modified tobacco plant, comprising: (a) introducing a recombinant DNA construct into at least one tobacco cell, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule capable of binding to at least one endogenous nucleic acid sequence encoding a polypeptide having at least 80% identity or similarity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18 and reducing its expression; (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).

[0011] In one aspect, the present disclosure provides a method for producing a modified tobacco plant, comprising: (a) introducing a recombinant DNA construct into at least one tobacco cell, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising 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: 13 to 18; (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).

[0012] In one aspect, the present disclosure provides a method comprising preparing a tobacco product using dried tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a non-natural mutation in an endogenous nucleic acid sequence, and the endogenous nucleic acid sequence encodes a polypeptide comprising 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: 13 to 18.

[0013] In one aspect, the present disclosure provides a method comprising preparing a tobacco product using dried tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding at least one low RNA molecule capable of binding to at least one 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: 13 to 18 and reducing its expression.

[0014] In one aspect, the present disclosure provides a method comprising preparing a tobacco product using dried tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.

[0015] 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 low RNA molecule capable of binding to at least one 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: 13 to 18 and reducing its expression.

[0016] 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 that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.

[0017] In one aspect, the present disclosure provides a method for producing a modified tobacco plant, 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 progeny tobacco seed, wherein 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 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: 13 to 18, and 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 progeny tobacco seed or a plant germinated therefrom, wherein at least one tobacco seed or a plant germinated therefrom comprises the non-natural mutation.

[0018] In one aspect, the present disclosure provides a method for producing a modified tobacco plant, 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 progeny tobacco seed, wherein at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding at least one low RNA molecule capable of binding to and reducing the expression of at least one endogenous nucleic acid sequence encoding a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18, and the recombinant DNA construct is not present in the endogenous nucleic acid sequence in a control tobacco plant of the same variety; and (b) selecting at least one progeny tobacco seed or a plant germinated therefrom, wherein at least one tobacco seed or a plant germinated therefrom comprises the recombinant DNA construct.

[0019] In one aspect, the present disclosure provides a method for creating a modified tobacco plant, 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 progeny tobacco seed, wherein at least one tobacco plant of the first tobacco variety contains a recombinant DNA construct, the recombinant DNA construct contains a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide containing 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: 13-18, and 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 progeny tobacco seed or a plant germinated therefrom, wherein at least one tobacco seed or a plant germinated therefrom contains the recombinant DNA construct.

[0020] In one aspect, the present disclosure provides a modified tobacco plant or a part thereof containing at least one unnatural mutation in an endogenous nucleic acid sequence that regulates gene expression or functional activity, wherein the gene encodes a polypeptide containing 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: 13-18.

[0021] In one aspect, the present disclosure provides a modified tobacco plant or a part thereof, comprising (a) a genetic modification in a gene, or (b) a genetic modification that targets a gene, wherein the genetic modification downregulates gene expression or activity, and 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: 1-12. BRIEF DESCRIPTION OF THE DRAWINGS

[0022]

Figure 1

Figure 2

Figure 3

Figure 4-1

Figure 4-2

Figure 4-3

Mode for Carrying Out the Invention

[0023] Detailed Description Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. When terms are provided in the singular, the inventors also contemplate aspects of this disclosure that are described by the plural of such terms. In the event of any discrepancy between 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 shall have their ordinary meaning in the technical fields in which they are used, as exemplified by various specialized dictionaries, such as "The American Heritage® Science Dictionary" (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), "McGraw-Hill Dictionary of Scientific and Technical Terms" (6th edition, 2002, McGraw-Hill, New York), or "Oxford Dictionary of Biology" (6th edition, 2008, Oxford University Press, Oxford and New York).

[0024] For example, any references cited herein, including all patents, published patent applications, and non-patent literature, are hereby incorporated by reference in their entirety.

[0025] When a grouping of alternatives is presented, any combination and all combinations of the members that make up the grouping of alternatives are specifically contemplated. For example, when an item is selected from a group consisting of A, B, C, and D, the inventors specifically contemplate each alternative individually (e.g., A alone, B alone, etc.), as well as combinations such as A, B, and D; A and C; B and C, etc. The term "and / or", when used in the listing of two or more items, means any one of the listed items, either alone or in combination with any one or more of the other listed items. For example, the expression "A and / or B" is intended to mean either 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.

[0026] When a numerical range is provided herein, the range is understood to include the endpoints of the range, as well as any number between the endpoints of the defined range. For example, "1 to 10" includes any number between 1 and 10, as well as the numbers 1 and 10.

[0027] When the term "about" is used with respect to a numerical value, it is understood to mean plus or minus 10%. For example, "about 100" includes 90 to 110.

[0028] As used herein, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, the terms "compound" or "at least one compound" may include a plurality of compounds including mixtures thereof.

[0029] Any tobacco plant or part thereof provided herein is specifically contemplated to be used with any method provided herein. Similarly, any modified tobacco plant or part thereof is specifically contemplated to be used with any method provided herein. Any nucleic acid sequence, amino acid sequence, or other composition provided herein is specifically contemplated to be used with any method provided herein.

[0030] Tobacco plants typically produce alkaloids at levels of 2% - 4% of their total dry weight. Nicotine is the major alkaloid compound in tobacco plants and often accounts for about 95% of the total alkaloid content of the plant. The remaining pool of alkaloids mainly includes other structurally related alkaloids such as anabasine, anatabine, and nornicotine.

[0031] Nicotine is synthesized in the roots via one of two major metabolic pathways, the pyridine nicotine biosynthetic pathway and the pyrrolidine nicotine biosynthetic pathway. See Figure 1.

[0032] In the pyridine nicotine biosynthetic pathway, quinolinate synthetase (QS) produces quinolinate, and the conversion of quinolinate to nicotinic acid mononucleotide (NAMN) is catalyzed by quinolinate phosphoribosyl-transferase (QPT). NAMN can be directly converted to nicotinic acid either by NAMN glycohydrolase or through a multi-step process involving the synthesis and breakdown of nicotinamide adenine dinucleotide (NAD). Further reduction of nicotinic acid subsequently forms 3,6-dihydro-nicotinic acid.

[0033] In the pyrrolidine nicotine biosynthetic pathway, decarboxylation of an amino acid (e.g., arginine) is necessary to form putrescine, and methylation of putrescine to N-methylputrescine is catalyzed by N-methyltransferase (PMT). Subsequently, N-methylputrescine is oxidized by diamine oxidase and spontaneously cyclizes to N-methyl-Δ 1 -pyrrolinium cation. This N-methyl-Δ 1 -pyrrolinium cation then 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.

[0034] Decarboxylase is an enzyme that catalyzes the removal (decarboxylation) of a carboxyl group from an organic molecule, releasing carbon dioxide. See Figure 2. Decarboxylases ornithine decarboxylase (ODC), arginine decarboxylase (ADC), and S-adenosylmethionine decarboxylase are known to be involved in the conversion of amino acids to putrescine in the pyrrolidine nicotine biosynthetic pathway. Provided herein are novel decarboxylases involved in the biosynthesis of nicotine or other alkaloids in tobacco.

[0035] In one aspect, the disclosure provides a modified tobacco plant or a 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: 13-18. In another aspect, the disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in an endogenous nucleic acid sequence that is at least 80% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-6. In another aspect, the disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in an endogenous nucleic acid sequence that is at least 80% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7-12.

[0036] In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in at least two different endogenous nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 12. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in at least three different endogenous nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 12. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in at least four different endogenous nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 12. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in at least five different endogenous nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 12. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in at least six different endogenous nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 12.

[0037] In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in at least two different endogenous nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 6. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in at least three different endogenous nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 6. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in at least four different endogenous nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 6. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in at least five different endogenous nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 6. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in each of SEQ ID NOs: 1 to 6.

[0038] In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in each of a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence, wherein the first endogenous nucleic acid sequence is at least 90% identical to SEQ ID NO: 1 and the second endogenous nucleic acid sequence is at least 90% identical to SEQ ID NO: 6. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in each of a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence, wherein the first endogenous nucleic acid sequence is at least 95% identical to SEQ ID NO: 1 and the second endogenous nucleic acid sequence is at least 95% identical to SEQ ID NO: 6. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in each of a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence, wherein the first endogenous nucleic acid sequence is at least 99% identical to SEQ ID NO: 1 and the second endogenous nucleic acid sequence is at least 99% identical to SEQ ID NO: 6. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in each of a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence, wherein the first endogenous nucleic acid sequence is 100% identical to SEQ ID NO: 1 and the second endogenous nucleic acid sequence is 100% identical to SEQ ID NO: 6.

[0039] In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in each of a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence, wherein the first endogenous nucleic acid sequence is at least 90% identical to SEQ ID NO: 2 and the second endogenous nucleic acid sequence is at least 90% identical to SEQ ID NO: 3. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in each of a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence, wherein the first endogenous nucleic acid sequence is at least 95% identical to SEQ ID NO: 2 and the second endogenous nucleic acid sequence is at least 95% identical to SEQ ID NO: 3. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in each of a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence, wherein the first endogenous nucleic acid sequence is at least 99% identical to SEQ ID NO: 2 and the second endogenous nucleic acid sequence is at least 99% identical to SEQ ID NO: 3. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in each of a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence, wherein the first endogenous nucleic acid sequence is 100% identical to SEQ ID NO: 2 and the second endogenous nucleic acid sequence is 100% identical to SEQ ID NO: 3.

[0040] In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7 to 12. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in at least two different endogenous nucleic acid sequences selected from the group consisting of SEQ ID NOs: 7 to 12. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in at least three different endogenous nucleic acid sequences selected from the group consisting of SEQ ID NOs: 7 to 12. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in at least four different endogenous nucleic acid sequences selected from the group consisting of SEQ ID NOs: 7 to 12. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in at least five different endogenous nucleic acid sequences selected from the group consisting of SEQ ID NOs: 7 to 12. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in each of SEQ ID NOs: 7 to 12.

[0041] In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in at least two endogenous nucleic acid sequences, wherein the at least two endogenous nucleic acid sequences encode different polypeptides comprising an amino acid sequence that is at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in at least three endogenous nucleic acid sequences, wherein the at least three endogenous nucleic acid sequences encode different polypeptides comprising an amino acid sequence that is at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in at least four endogenous nucleic acid sequences, wherein the at least four endogenous nucleic acid sequences encode different polypeptides comprising an amino acid sequence that is at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in at least five endogenous nucleic acid sequences, wherein the at least five endogenous nucleic acid sequences encode different polypeptides comprising an amino acid sequence that is at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in at least six endogenous nucleic acid sequences, wherein the at least six endogenous nucleic acid sequences encode different polypeptides comprising an amino acid sequence that is at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.

[0042] In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in at least two endogenous nucleic acid sequences, wherein the at least two endogenous nucleic acid sequences encode different polypeptides comprising an amino acid sequence that is 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in at least three endogenous nucleic acid sequences, wherein the at least three endogenous nucleic acid sequences encode different polypeptides comprising an amino acid sequence that is 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in at least four endogenous nucleic acid sequences, wherein the at least four endogenous nucleic acid sequences encode different polypeptides comprising an amino acid sequence that is 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in at least five endogenous nucleic acid sequences, wherein the at least five endogenous nucleic acid sequences encode different polypeptides comprising an amino acid sequence that is 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in at least six endogenous nucleic acid sequences, wherein the at least six endogenous nucleic acid sequences encode different polypeptides selected from the group consisting of SEQ ID NOs: 13-18.

[0043] In one aspect, the present disclosure provides a modified tobacco plant or a 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 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in an endogenous nucleic acid sequence that is at least 90% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 6. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in an endogenous nucleic acid sequence that is at least 90% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7 to 12.

[0044] In one aspect, the present disclosure provides a modified tobacco plant or a 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 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in an endogenous nucleic acid sequence that is at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 6. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in an endogenous nucleic acid sequence that is at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7 to 12.

[0045] In one aspect, the present disclosure provides a modified tobacco plant or a 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 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in an endogenous nucleic acid sequence that is at least 99% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-6. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in an endogenous nucleic acid sequence that is at least 99% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7-12.

[0046] In one aspect, the present disclosure provides a modified tobacco plant or a 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: 13-18, the modified tobacco plant comprises at least one non-natural mutation in a first endogenous nucleic acid sequence and at least one non-natural mutation in a second endogenous nucleic acid sequence, the first 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: 13-18, the second 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: 13-18, and the first and second endogenous nucleic acid sequences are not identical to each other.

[0047] In one aspect, the present disclosure provides a modified tobacco plant or a part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to at least one endogenous nucleic acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18 and can reduce its expression. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to at least one endogenous nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6 and can reduce its expression. In yet another aspect, the present disclosure provides a modified tobacco plant or a part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to at least one endogenous nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7 to 12 and can reduce its expression.

[0048] In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule capable of binding to at least one endogenous nucleic acid sequence that is at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18 and reducing its expression. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule capable of binding to at least one endogenous nucleic acid sequence that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6 and reducing its expression. In yet another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule capable of binding to at least one endogenous nucleic acid sequence that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7 to 12 and reducing its expression.

[0049] In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule capable of binding to at least one endogenous nucleic acid sequence that is at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18 and reducing its expression. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule capable of binding to at least one endogenous nucleic acid sequence that is at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6 and reducing its expression. In yet another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule capable of binding to at least one endogenous nucleic acid sequence that is at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7 to 12 and reducing its expression.

[0050] In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule capable of binding to at least one endogenous nucleic acid sequence that is at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18 and reducing its expression. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule capable of binding to at least one endogenous nucleic acid sequence that is at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6 and reducing its expression. In yet another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule capable of binding to at least one endogenous nucleic acid sequence that is at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7 to 12 and reducing its expression.

[0051] In one aspect, the present disclosure provides a modified tobacco plant or a part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule capable of binding to at least one endogenous nucleic acid sequence that is 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18 and reducing its expression. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule capable of binding to at least one endogenous nucleic acid sequence that is 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6 and reducing its expression. In yet another aspect, the present disclosure provides a modified tobacco plant or a part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule capable of binding to at least one endogenous nucleic acid sequence that is 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7 to 12 and reducing its expression.

[0052] In one aspect, the present disclosure provides a modified tobacco plant or a part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to and can reduce the expression of a first 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: 13 to 18, and a second 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: 13 to 18, wherein the first and second endogenous nucleic acid sequences are not identical to each other. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to and can reduce the expression of a first endogenous nucleic acid sequence encoding a polypeptide that is at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18, and a second endogenous nucleic acid sequence encoding a polypeptide that is at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18, wherein the first and second endogenous nucleic acid sequences are not identical to each other. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to and can reduce the expression of a first endogenous nucleic acid sequence encoding a polypeptide that is at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18, and a second endogenous nucleic acid sequence encoding a polypeptide that is at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18, wherein the first and second endogenous nucleic acid sequences are not identical to each other.In one aspect, the present disclosure provides a modified tobacco plant or a part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to and can reduce the expression of a first endogenous nucleic acid sequence encoding a polypeptide that is at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18, and a second endogenous nucleic acid sequence encoding a polypeptide that is at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18, wherein the first and second endogenous nucleic acid sequences are not identical to each other. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to and can reduce the expression of a first endogenous nucleic acid sequence encoding a polypeptide that is 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18, and a second endogenous nucleic acid sequence encoding a polypeptide that is 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18, wherein the first and second endogenous nucleic acid sequences are not identical to each other.

[0053] In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first and second endogenous nucleic acid sequences are each at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 6, and the first and second endogenous nucleic acid sequences are not identical to each other. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first and second endogenous nucleic acid sequences are each at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 7 to 12, and the first and second endogenous nucleic acid sequences are not identical to each other. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first and second endogenous nucleic acid sequences are each at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 6, and the first and second endogenous nucleic acid sequences are not identical to each other. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first and second endogenous nucleic acid sequences are each at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 7 to 12, and the first and second endogenous nucleic acid sequences are not identical to each other.In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first and second endogenous nucleic acid sequences are each at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-6, and the first and second endogenous nucleic acid sequences are not identical to each other. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first and second endogenous nucleic acid sequences are each at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 7-12, and the first and second endogenous nucleic acid sequences are not identical to each other. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first and second endogenous nucleic acid sequences are each at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-6, and the first and second endogenous nucleic acid sequences are not identical to each other. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first and second endogenous nucleic acid sequences are each at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 7-12, and the first and second endogenous nucleic acid sequences are not identical to each other.In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first and second endogenous nucleic acid sequences are each 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 6, and the first and second endogenous nucleic acid sequences are not identical to each other. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first and second endogenous nucleic acid sequences are each 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 7 to 12, and the first and second endogenous nucleic acid sequences are not identical to each other.

[0054] In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first endogenous nucleic acid sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 1, and the second endogenous nucleic acid sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 6. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first endogenous nucleic acid sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 1, and the second endogenous nucleic acid sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 6. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first endogenous nucleic acid sequence comprises a sequence that is at least 99% identical to SEQ ID NO: 1, and the second endogenous nucleic acid sequence comprises a sequence that is at least 99% identical to SEQ ID NO: 6. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first endogenous nucleic acid sequence comprises a sequence that is 100% identical to SEQ ID NO: 1, and the second endogenous nucleic acid sequence comprises a sequence that is 100% identical to SEQ ID NO: 6.

[0055] In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first endogenous nucleic acid sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 2, and the second endogenous nucleic acid sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 3. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first endogenous nucleic acid sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 2, and the second endogenous nucleic acid sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 3. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first endogenous nucleic acid sequence comprises a sequence that is at least 99% identical to SEQ ID NO: 2, and the second endogenous nucleic acid sequence comprises a sequence that is at least 99% identical to SEQ ID NO: 3. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first endogenous nucleic acid sequence comprises a sequence that is 100% identical to SEQ ID NO: 2, and the second endogenous nucleic acid sequence comprises a sequence that is 100% identical to SEQ ID NO: 3.

[0056] In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first endogenous nucleic acid sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 7, and the second endogenous nucleic acid sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 12. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first endogenous nucleic acid sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 7, and the second endogenous nucleic acid sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 12. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first endogenous nucleic acid sequence comprises a sequence that is at least 99% identical to SEQ ID NO: 7, and the second endogenous nucleic acid sequence comprises a sequence that is at least 99% identical to SEQ ID NO: 12. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first endogenous nucleic acid sequence comprises a sequence that is 100% identical to SEQ ID NO: 7, and the second endogenous nucleic acid sequence comprises a sequence that is 100% identical to SEQ ID NO: 12.

[0057] In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first endogenous nucleic acid sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 8, and the second endogenous nucleic acid sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 9. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first endogenous nucleic acid sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 8, and the second endogenous nucleic acid sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 9. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first endogenous nucleic acid sequence comprises a sequence that is at least 99% identical to SEQ ID NO: 8, and the second endogenous nucleic acid sequence comprises a sequence that is at least 99% identical to SEQ ID NO: 9. In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one low RNA molecule that binds to a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence and can reduce their expression, wherein the first endogenous nucleic acid sequence comprises a sequence that is 100% identical to SEQ ID NO: 8, and the second endogenous nucleic acid sequence comprises a sequence that is 100% identical to SEQ ID NO: 9.

[0058] In one aspect, a nucleic acid molecule encoding at least one low RNA molecule comprises a sequence that is at least 80% identical to one of SEQ ID NO: 32 and 33. In one aspect, a nucleic acid molecule encoding at least one low RNA molecule comprises a sequence that is at least 85% identical to one of SEQ ID NO: 32 and 33. In one aspect, a nucleic acid molecule encoding at least one low RNA molecule comprises a sequence that is at least 90% identical to one of SEQ ID NO: 32 and 33. In one aspect, a nucleic acid molecule encoding at least one low RNA molecule comprises a sequence that is at least 95% identical to one of SEQ ID NO: 32 and 33. In one aspect, a nucleic acid molecule encoding at least one low RNA molecule comprises a sequence that is at least 99% identical to one of SEQ ID NO: 32 and 33. In one aspect, a nucleic acid molecule encoding at least one low RNA molecule comprises a sequence that is 100% identical to one of SEQ ID NO: 32 and 33.

[0059] In one aspect, the present disclosure provides a modified tobacco plant or a 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 that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 - 18. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising 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: 1 - 6. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising a recombinant DNA construct comprising 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: 7 - 12.

[0060] In one aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in an endogenous nucleic acid sequence that regulates gene expression or activity, 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: 13 to 18. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof comprising at least one non-natural mutation in an endogenous nucleic acid sequence that regulates gene expression or activity, wherein the gene encodes an RNA sequence that is at least 80% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7 to 12. In one aspect, the endogenous nucleic acid sequence encodes a transcription factor that can bind to the nucleic acid sequence encoding the gene. As used herein, a "transcription factor" refers to a protein that controls the rate of transcription of a gene from DNA to messenger RNA by binding to a specific DNA sequence of the gene. Without being limited to any scientific theory, when a transcription factor binds to a gene, it turns on or enhances the transcription of the gene as compared to transcription in the absence of the transcription factor. In another aspect, the endogenous nucleic acid sequence encodes a repressor that can bind to the nucleic acid sequence encoding the gene. As used herein, a "repressor" refers to a protein that inhibits the transcription of a gene from DNA to messenger RNA by binding to a specific DNA sequence of the gene. Without being limited to any scientific theory, when a repressor binds to a gene, it turns off or reduces the transcription of the gene as compared to transcription in the absence of the repressor. In a further aspect, 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, quinolinate synthase produces quinic acid, which is a precursor required for the function of quinolate phosphoribosyltransferase. In one aspect, the gene comprises a nucleic acid sequence that is at least 80% identical or similar to SEQ ID NOs: 1 to 6.

[0061] In one aspect, the present disclosure provides a modified tobacco plant or a part thereof, comprising (a) a genetic modification in a gene, or (b) a genetic modification targeting a gene, wherein the genetic modification down-regulates the expression or activity of the gene, and the gene encodes an amino acid sequence having at least 80% identity or similarity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof, comprising (a) a genetic modification in a gene, or (b) a genetic modification targeting a gene, wherein the genetic modification down-regulates the expression or activity of the gene, and 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 6. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof, comprising (a) a genetic modification in a gene, or (b) a genetic modification targeting a gene, wherein the genetic modification down-regulates the expression or activity of the gene, and 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: 7 to 12. In another aspect, the present disclosure provides a modified tobacco plant or a part thereof, comprising (a) a genetic modification in a gene, or (b) a genetic modification targeting a gene, wherein the genetic modification down-regulates the expression or activity of the gene, and 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 12.

[0062] As used herein, "genetic modification" refers to a change in the genetic constitution of a plant or the plant genome. Genetic modifications can be introduced by methods including but not limited to mutagenesis, genome editing, genetic transformation, or combinations thereof. Genetic modifications include, for example, mutations in genes (e.g., non-natural mutations), or transgenes that target genes (e.g., an arginine decarboxylase (ADC) transgene that targets the ADC gene). As used herein, "targeting" refers to either directly upregulating or directly downregulating the expression or activity of a gene. As used herein, "direct" in the context of a transgene that affects the expression or activity of a gene refers to an effect exerted on the gene through physical contact or chemical interaction between the gene (e.g., promoter region or UTR region) or the product encoded therein (e.g., mRNA molecule or polypeptide) and the product encoded by the transgene (e.g., small RNA molecule or protein such as a transcription factor or dominant negative polypeptide variant). In one aspect, the transgene affects the expression or activity of the target gene without involving a transcription factor (e.g., the transgene does not encode a transcription factor and / or does not suppress the expression or activity of a transcription factor, and the transcription factor then controls the target gene).

[0063] mutation As used herein, "modification" in the context of a plant refers to a plant that contains a genetic change introduced for a particular purpose and beyond natural polymorphisms. Without limitation, a modified plant can contain non-natural mutations or recombinant DNA constructs. In one aspect, a modified tobacco plant contains non-natural mutations. In another aspect, a modified tobacco plant contains a recombinant DNA construct. In another aspect, a modified tobacco plant contains a genetic modification.

[0064] As used herein, "mutation" refers to a heritable genetic modification introduced into a gene to change the expression or activity of the product encoded by the gene's reference sequence. A mutation in a particular gene, for example, arginine decarboxylase (ADC), is referred to as an ADC variant. Such modifications can be in any sequence region of the gene, such as a promoter, 5' untranslated region (UTR), exon, intron, 3' UTR, or terminator region. In one aspect, the mutation reduces, inhibits, or eliminates the expression or activity of the gene product. In another aspect, the mutation increases, elevates, enhances, or augments the expression or activity of the gene product.

[0065] In one aspect, the mutation is not a natural polymorphism present in a particular tobacco variety or cultivar. In one aspect, the mutation is an "unnatural" or "non-naturally occurring" mutation. As used herein, "unnatural" or "non-naturally occurring" mutations refer to non-spontaneous mutations generated by human intervention and do not correspond to spontaneous mutations generated without human intervention. Non-limiting examples of human intervention include mutagenesis (e.g., chemical mutagenesis, ionizing radiation mutagenesis) and targeted genetic modification (e.g., CRISPR-based methods, TALEN-based methods, zinc finger-based methods). Unnatural mutations and non-naturally occurring mutations do not include spontaneous mutations that occur naturally (e.g., via aberrant DNA replication in the germline of a plant).

[0066] In one aspect, the mutation is not a natural polymorphism present in a particular tobacco variety or cultivar. It will be understood that when identifying a mutation, the reference DNA sequence should be from the same variety of tobacco. For example, if a modified tobacco plant containing the mutation is derived from the variety TN90, the endogenous reference sequence should be the endogenous TN90 sequence and not a homologous sequence from a different tobacco variety (e.g., K326). Similarly, if a modified tobacco cell containing the mutation is a TN90 cell, the endogenous reference sequence should be the endogenous TN90 sequence and not a homologous sequence from tobacco cells of a different tobacco variety (e.g., K326).

[0067] In one aspect, the tobacco plant or a part thereof is homozygous for at least one non-natural mutation. In another aspect, the tobacco plant or a part thereof is heterozygous for at least one non-natural mutation. In another aspect, the tobacco plant or a part thereof is homozygous for an introduced recombinant DNA construct. In another aspect, the tobacco plant or a part thereof is hemizygous for an introduced recombinant DNA construct. In a further aspect, the tobacco plant or a part thereof is heterozygous for an introduced recombinant DNA construct.

[0068] In one aspect, the mutations provided herein create a dominant allele at the mutated locus. A dominant allele is an allele that masks the contribution of a second allele at the same locus. A dominant allele can be a "dominant negative allele" or a "dominant positive allele". A dominant negative allele, or antimorph, is an allele that acts in opposition to normal allele function. A dominant negative allele typically either does not function normally and directly inhibits the activity of the wild-type protein (e.g., through dimerization) or inhibits the activity of a second protein (e.g., an activator or a downstream component of the pathway) that is required for the normal function of the wild-type protein. For example, a dominant negative allele suppresses or reduces the normal function of the allele in the heterozygous or homozygous state. A dominant positive allele can increase normal gene function (e.g., a hypermorph) or provide a new function to the gene (e.g., a neomorph). A semi-dominant allele occurs when the penetrance of the linked phenotype in an individual heterozygous for the allele is less than that observed in an individual homozygous for the allele.

[0069] In one aspect, the mutations provided herein create a dominant negative allele at the mutated locus. In another aspect, the mutations provided herein create a dominant positive allele at the mutated locus.

[0070] As used herein, "inducing a mutation" refers to generating a mutation in a polynucleotide sequence through 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 irradiation, the use of nucleases, the use of transposons, and the use of Agrobacterium. In one aspect, inducing a mutation includes the use of an agent selected from the group consisting of chemical mutagens, irradiation, transposons, Agrobacterium, and nucleases.

[0071] In one aspect, inducing a mutation includes the use of a chemical mutagen. In one aspect, the chemical mutagen includes ethyl methanesulfonate (EMS).

[0072] In another aspect, inducing a mutation includes the use of irradiation. In one aspect, the irradiation includes gamma rays, X-rays, ionizing radiation, or fast neutrons.

[0073] In one aspect, inducing a mutation includes the use of a transposon. In another aspect, inducing a mutation includes the use of Agrobacterium.

[0074] In a further aspect, inducing a mutation includes the use of a nuclease. 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 a mutation includes the use of a CRISPR / Cas9 nuclease. In one aspect, inducing a mutation includes the use of a CRISPR / Cpf1 nuclease. In one aspect, inducing a mutation includes the use of a CRISPR / CasX nuclease. In one aspect, inducing a mutation includes the use of a CRISPR / CasY nuclease. In one aspect, inducing a mutation includes the use of a Csm1 nuclease.

[0075] In one aspect, inducing a mutation includes the use of a base editor. As used herein, "base editor" refers to a catalytically impaired Cas nuclease fused to a nucleotide deaminase. In some aspects, the base editor further includes a DNA repair protein. In one aspect, the base editor is a cytosine base editor. The cytosine base editor enables the transition from C-G to T-A. In one aspect, the base editor is an adenine base editor. The adenine base editor enables the conversion from A-T to G-C. In one aspect, the base editor is a base editor from C to G.

[0076] In one aspect, inducing a mutation includes using a prime editor. As used herein, "prime editor" refers to a Cas nickase fused to an engineered reverse transcriptase. Prime editors can introduce all 12 transition and transversion mutations, as well as small insertions or deletions, and combinations thereof. Prime editors use a prime editing guide RNA (pegRNA) that specifies a target site for editing and encodes the desired edit. In one aspect, the pegRNA is provided to tobacco cells. In one aspect, the pegRNA includes a nucleic acid sequence that binds to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-9.

[0077] Additional information regarding base editors and prime editors, and their use in plants, can be found in Molla et al., Nature Plants, 7:1166-1187 (2021). See also Anzalone et al., Nature, 576:149-157 (2019), Komor et al., Nature, 533:420-424 (2016), and Gaudelli et al., Nature, 551:464-471 (2017).

[0078] Some types of mutations are known in the art. In one aspect, the mutation includes an insertion. An "insertion" refers to the addition of one or more nucleotides or amino acids to a given polynucleotide or amino acid sequence, respectively, as compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, the mutation includes a deletion. A "deletion" refers to the removal of one or more nucleotides or amino acids from a given polynucleotide or amino acid sequence, respectively, as compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, the mutation includes a substitution. A "substitution" refers to the replacement of one or more nucleotides or amino acids in a given polynucleotide or amino acid sequence, respectively, as compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, the mutation includes an inversion. An "inversion" refers to the case where a segment of a polynucleotide or amino acid sequence is inverted from end to end. "Duplication" refers to the case where a segment of a polynucleotide or amino acid sequence is repeated. The repeated segment can follow immediately after the original segment, or it can be separated from the original segment by one or more nucleotides or amino acids. In one aspect, the mutations provided herein include mutations selected from the group consisting of insertions, deletions, substitutions, duplications, and inversions.

[0079] 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 with respect to an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-12.

[0080] 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 with respect to an endogenous nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18.

[0081] In one aspect, the 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 mutation" refers to a mutation in a nucleic acid sequence that introduces a premature stop codon into the amino acid sequence. As used herein, "missense mutation" refers to a mutation in a nucleic acid sequence that causes a substitution within the amino acid sequence encoded by the nucleic acid sequence. As used herein, "frameshift mutation" refers to an insertion or deletion in a nucleic acid sequence that shifts the reading frame for translating the nucleic acid sequence into an amino acid sequence. "Splice site mutation" refers to a mutation in a nucleic acid sequence that retains an intron for protein translation or, alternatively, for an exon that is excluded from protein translation. Splice site mutations can cause nonsense, missense, or frameshift mutations.

[0082] Mutations in the coding region of a gene (e.g., exon mutations) can result in truncated proteins or polypeptides when the mutated messenger RNA (mRNA) is translated into a 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 contains at least one fewer amino acid compared to an endogenous control protein or polypeptide. For example, if the endogenous protein A contains 100 amino acids, a truncated version of protein A can contain from 1 to 99 amino acids. In one aspect, the non-natural mutations result in truncation of the polypeptide.

[0083] Without being limited to any scientific theory, one way to cause protein or polypeptide cleavage is by the introduction of a premature stop codon in the mRNA transcript of an endogenous gene. In one aspect, the present disclosure provides a mutation that results in a premature stop codon in the mRNA transcript of an endogenous gene. As used herein, a "stop codon" refers to a nucleotide triplet within an mRNA transcript that signals the termination of protein translation. A "premature stop codon" refers to a stop codon that is positioned earlier (e.g., 5'ward) than the normal stop codon position in an endogenous mRNA transcript. Without limitation, several stop codons are known in the art, including "UAG", "UAA", "UGA", "TAG", "TAA", and "TGA".

[0084] In one aspect, the mutations provided herein include null mutations. As used herein, a "null mutation" refers to a mutation that confers a complete loss of function for the protein encoded by the gene containing the mutation, or alternatively, a mutation that confers a complete loss of function for the small RNA encoded by the genomic locus. A null mutation can cause a lack of mRNA transcript production, a lack of small RNA transcript production, a lack of protein function, or a combination thereof.

[0085] The mutations provided herein can be located in any part of the endogenous gene. In one aspect, the mutations provided herein are located within the exons of the endogenous gene. In another aspect, the mutations provided herein are located within the introns of the endogenous gene. In a further aspect, the mutations provided herein are located within the 5'-UTR of the endogenous gene. In yet another aspect, the mutations provided herein are located within the 3'-UTR of the endogenous gene. In still another aspect, the mutations provided herein are located within the promoter of the endogenous gene. In yet another aspect, the mutations provided herein are located within the terminator of the endogenous gene. In one aspect, the non-natural mutations provided herein include mutations in sequence regions selected from the group consisting of promoters, 5'-UTRs, 3'-UTRs, exons, introns, and terminators.

[0086] Screening and selection of mutagenized tobacco plants can be carried out through any method known to those skilled in the art. Examples of screening and selection methods include, but are not limited to, Southern analysis, PCR amplification for detection of polynucleotides, Northern blot, RNase protection, primer extension, RT-PCR amplification for detection of RNA transcripts, Sanger sequencing, next-generation sequencing technologies (e.g., Illumina, PacBio, Ion Torrent, 454), enzyme assays for detecting the enzymatic or ribozyme activity of polypeptides and polynucleotides, as well as protein gel electrophoresis, Western blot, immunoprecipitation, and enzyme-linked immunosorbent assays for detecting polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining can also be used to detect the presence or expression of polypeptides and / or polynucleotides. Methods for performing the cited techniques are all known.

[0087] Nucleic Acids and Amino Acids As used herein, an "endogenous" nucleic acid sequence refers to a nucleic acid sequence that occurs naturally in the genome of an organism. Endogenous nucleic acid sequences do not include heterologous sequences inserted into the genome through intentional human intervention. Similarly, an endogenous amino acid sequence is a sequence that occurs naturally through the translation of an endogenous nucleic acid molecule. In one aspect, the nucleic acid sequences provided herein are endogenous nucleic acid sequences.

[0088] As used herein, "heterologous" refers to a sequence (nucleic acid or amino acid) that is of foreign origin or, in the case of being from the same species, has been substantially modified from its native form in composition and / or genomic locus by intentional human intervention. This term is also applicable to nucleic acid constructs, also referred to herein as "polynucleotide constructs" or "nucleotide constructs". Thus, a "heterologous" nucleic acid construct is intended to mean a construct that is of foreign origin or, in the case of being from the same species, has been substantially modified from its native form in composition and / or genomic locus by intentional human intervention. Examples of heterologous nucleic acid constructs include, but are not limited to, recombinant nucleotide constructs introduced into a plant or a plant part thereof through a transformation method or subsequent breeding of transgenic plants with another plant for a different purpose.

[0089] As used herein, "gene" refers to a polynucleotide capable of producing a functional unit (e.g., but not limited to, a protein or a non-coding RNA molecule). A gene can include a promoter, enhancer sequence, leader sequence, transcription start site, transcription termination site, 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, enhancer sequence, leader sequence, transcription start site, transcription termination site, 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 non-coding RNA molecule or its precursor. In another aspect, a gene encodes a protein.

[0090] As used herein with respect to two or more nucleotide or amino acid sequences, the terms "percent identity" or "percent identical" are calculated by: (i) comparing two optimally aligned sequences (nucleotide or amino acid) over a comparison window (the "alignable" region), (ii) determining the number of positions at which identical nucleic acid bases (in the case of nucleotide sequences) or amino acid residues (in the case of proteins and polypeptides) occur in both sequences to obtain 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 this quotient by 100% to obtain the percent identity. When "percent identity" is calculated in relation to a reference sequence without specifying a particular comparison window, the percent identity is determined by dividing the number of matched positions over the aligned region by the full length of the reference sequence. Thus, for the purposes of this application, when two sequences (query and target) are optimally aligned (taking into account 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), multiplied by 100%.

[0091] When percentage of sequence identity is used with respect to amino acids, it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions (where an amino acid residue is substituted for another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity) and thus does not change the functional properties of the molecule). When sequences differ by conservative substitutions, the percent sequence identity can 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".

[0092] Although not limited, two aliphatic (e.g., glycine, alanine, valine, leucine, isoleucine) amino acid residues can be substituted for each other in conservative substitutions, two hydroxyl (e.g., serine, cysteine, threonine, methionine) amino acid residues can be substituted for each other in conservative substitutions, two aromatic (e.g., phenylalanine, tyrosine, tryptophan) amino acid residues can be substituted for each other in conservative substitutions, two basic (e.g., histidine, lysine, arginine) amino acid residues can be substituted for each other in conservative substitutions, and two acidic (e.g., aspartic acid, glutamic acid, asparagine, glutamine) amino acid residues can be substituted for each other in conservative substitutions.

[0093] For optimal alignment of arrays for calculating percent identity, various pairwise or multiple sequence alignment algorithms and programs can be used to compare sequence identity or similarity between two or more nucleotide or amino acid sequences, such as ClustalW or Basic Local Alignment Search Tool (BLAST™). Other alignment and comparison methods are known in the art, but the alignment and percent identity between two sequences (including the ranges of percent identity noted above) can be determined by the ClustalW algorithm. See, for example, 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 hereby incorporated by reference.

[0094] As used herein with respect to two nucleotide sequences, the terms "percent complementarity" or "percent complementary" are similar to the concept of percent identity, but refer to the percentage of nucleotides in a query sequence that optimally base pair or hybridize to the nucleotides of a target sequence when the query and target sequences are linearly arranged and optimally base paired without secondary folding structures such as loops, stems, or hairpins. Such percent complementarity can be between two DNA strands, two RNA strands, or a DNA strand and an RNA strand. "Percent complementarity" can be calculated by (i) optimally base pairing or hybridizing two nucleotide sequences in a linearly and fully extended arrangement over a window of comparison (i.e., without folding or secondary structure), (ii) determining the number of positions of base pairs between the two sequences over the window of comparison to obtain the number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions in the window of comparison, and (iv) multiplying this quotient by 100% to obtain the percent complementarity of the two sequences. Optimal base pairing of two sequences can be determined based on known base pairing of nucleotide bases such as G-C, A-T, and A-U via hydrogen bonds. When "percent complementarity" is calculated in relation to a reference sequence without specifying a particular window of comparison, percent identity is determined by dividing the number of complementary positions between the two linear sequences by the full length of the reference sequence. Thus, for the purposes of this application, when two sequences (query and target) are optimally base formed (allowing for mismatches or unpaired nucleotides), the "percent complementarity" of the query sequence is equal to the number of positions of base pairing between the two sequences divided by the total number of positions in the query sequence over its length, multiplied by 100%.

[0095] The use of the terms "polynucleotide" or "nucleic acid molecule" is not intended to limit the present disclosure to polynucleotides that include deoxyribonucleic acid (DNA). For example, ribonucleic acid (RNA) molecules are also contemplated. One of ordinary skill in the art will recognize that polynucleotides and nucleic acid molecules can include ribonucleotides as well as combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. The polynucleotides of the present disclosure also include, but are not limited to, all forms of sequences including single-stranded forms, double-stranded forms, hairpins, stem and loop structures, etc. In one aspect, the nucleic acid molecules provided herein are DNA molecules. In another aspect, the nucleic acid molecules provided herein are RNA molecules. In one aspect, the nucleic acid molecules provided herein are single-stranded. In another aspect, the nucleic acid molecules provided herein are double-stranded. Nucleic acid molecules can encode polypeptides or small interfering RNAs.

[0096] As used herein, "recombinant nucleic acid" refers to nucleic acid molecules formed by laboratory methods of genetic recombination such as, but not limited to, molecular cloning. Similarly, "recombinant DNA construct" refers to DNA molecules formed by laboratory methods of genetic recombination.

[0097] Nucleic acids can be isolated using routine techniques in the art. For example, nucleic acids can be isolated using any method including, but not limited to, recombinant nucleic acid techniques and / or polymerase chain reaction (PCR). General PCR techniques are described, for example, in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation, which can be used to isolate nucleic acids. Isolated nucleic acids can also be chemically synthesized either as a single nucleic acid molecule or as a series of oligonucleotides. Polypeptides can be purified from natural sources (e.g., biological samples) by known methods such as DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. Polypeptides can also be purified, for example, by expressing nucleic acids in an expression vector. In addition, purified polypeptides can be obtained by chemical synthesis. The degree of purity of a polypeptide can be measured using any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0098] In one aspect, the disclosure provides methods for detecting recombinant nucleic acids and polypeptides in plant cells. Nucleic acids can also be detected using, but not limited to, hybridization. Hybridization between nucleic acids is discussed in detail in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0099] 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: 1-12 and 19-30. 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: 1-12 and 19-30. 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: 1-12 and 19-30. 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: 1-12 and 19-30. 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: 1-12 and 19-30. 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: 1-12 and 19-30. 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: 1-12 and 19-30. 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: 1-12 and 19-30. 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: 1-12 and 19-30. 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: 1-12 and 19-30. 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: 1-12 and 19-30. 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: 1-12 and 19-30. 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: 1-12 and 19-30.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: 1-12 and 19-30. 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: 1-12 and 19-30. 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: 1-12 and 19-30.

[0100] 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: 1-12. 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: 1-12. 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: 1-12. 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: 1-12. 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: 1-12. 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: 1-12. 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: 1-12. 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: 1-12. 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: 1-12. 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: 1-12. 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: 1-12. 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: 1-12. 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: 1-12. 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: 1-12.In another aspect, the endogenous nucleic acid sequences provided herein are at least 99% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-12. In another aspect, the endogenous nucleic acid sequences provided herein are 100% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-12.

[0101] 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 provided herein. A protein provided herein can be encoded by a nucleic acid molecule provided herein. A protein can include a polypeptide provided herein. As used herein, the term "protein" refers to a chain of amino acid residues that can provide structure or enzymatic activity to a cell.

[0102] A polypeptide can be detected using an antibody. Techniques for detecting a polypeptide using an antibody include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence. An antibody provided herein can be a polyclonal antibody or a monoclonal antibody. An antibody having specific binding affinity for a protein provided herein can be generated using methods well known in the art. An antibody provided herein can be attached to a solid support such as a microtiter plate using methods known in the art.

[0103] Detection (e.g., of an amplification product, of a hybridization complex, of a polypeptide) can be achieved using a detectable label. The term "label" is intended to encompass the use of both direct and indirect labels. Detectable labels include enzymes, hapten groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.

[0104] 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: 13 to 18. 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: 13 to 18. 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: 13 to 18. 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: 13 to 18. 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: 13 to 18. 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: 13 to 18. 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: 13 to 18. In another aspect, the amino acid sequences provided herein are at least 92% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In another aspect, the amino acid sequences provided herein are at least 93% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In another aspect, the amino acid sequences provided herein are at least 94% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In another aspect, the amino acid sequences provided herein are at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In another aspect, the amino acid sequences provided herein are at least 96% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In another aspect, the amino acid sequences provided herein are at least 97% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In another aspect, the amino acid sequences provided herein are at least 98% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.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: 13-18. 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: 13-18.

[0105] In one aspect, the nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 75% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the nucleic acid sequences provided herein encode 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: 13 to 18. In one aspect, the nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 85% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 88% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 91% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 92% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 93% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 94% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.In one aspect, the nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 96% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 97% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 98% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.

[0106] In one aspect, the endogenous nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the endogenous nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 75% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the endogenous nucleic acid sequences provided herein encode 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: 13 to 18. In one aspect, the endogenous nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 85% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the endogenous nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 88% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the endogenous nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the endogenous nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 91% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the endogenous nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 92% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the endogenous nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 93% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the endogenous nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 94% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In one aspect, the endogenous nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.In one aspect, the endogenous nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 96% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18. In one aspect, the endogenous nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 97% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18. In one aspect, the endogenous nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 98% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18. In one aspect, the endogenous nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18. In one aspect, the endogenous nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18.

[0107] Promoter As generally understood in the art, the term "promoter" includes an RNA polymerase binding site, a transcription start site, and / or a TATA box, and refers to a DNA sequence that aids or promotes the transcription and expression of an associated transcribable polynucleotide sequence and / or gene (or transgene). A promoter can be synthetically produced, altered, or derived from a known or naturally occurring promoter sequence or other promoter sequences. A promoter can also include a chimeric promoter that includes a combination of two or more heterologous sequences. Thus, the promoters of the present application can include variants of promoter sequences that are similar but not identical in composition to known or other promoter sequences (s) provided herein.

[0108] A promoter that drives expression in all or most tissues of a plant is referred to as a "constitutive" promoter. In one aspect, the constitutive promoter is selected from the group consisting of the cauliflower mosaic virus 35S promoter, the ubiquitin promoter, the actin promoter, the opine promoter, and the alcohol dehydrogenase promoter.

[0109] A promoter that drives expression during a specific period or stage of development is referred to as a "developmental" promoter.

[0110] A promoter that drives enhanced expression in a particular tissue of an organism relative to other tissues of the organism is referred to as a "tissue-preferred" promoter. Thus, a "tissue-preferred" promoter causes relatively high or preferential expression in a particular tissue(s) of a plant, but the level of expression in other tissue(s) of the plant is lower. As a non-limiting example, a root tissue-preferred promoter exhibits higher activity in root tissue, but may also exhibit activity, albeit at lower levels, in additional tissues such as stem, leaf, and flower tissues. A "tissue-specific" promoter causes expression only in a particular tissue. As a non-limiting example, a root tissue-specific promoter drives expression only in root tissue. In one aspect, the tissue-specific promoter is a root tissue-specific promoter. In another aspect, the tissue-specific promoter is a root tissue-preferred promoter.

[0111] In one aspect, the root tissue-preferred promoter is the cassava vein mosaic virus (CsVMV) promoter.

[0112] An "inducible" promoter is a promoter that initiates transcription in response to environmental stimuli such as heat, cold, drought, light, or other stimuli such as wounding or chemical application.

[0113] In one aspect, the promoter provided herein is a constitutive promoter. In another aspect, the promoter provided herein is an inducible promoter. In a further aspect, the promoter provided herein is a developmental promoter. In another aspect, the promoter is a tissue-preferential or tissue-specific promoter. In a further aspect, the promoter is selected from the group consisting of a constitutive promoter, a tissue-preferential promoter, a tissue-specific promoter, and an inducible promoter.

[0114] 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.

[0115] As used herein, "operably linked" refers to a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide of interest and a control sequence (e.g., a promoter) is a functional linkage that enables expression of the polynucleotide of interest. The elements operably linked can be contiguous or non-contiguous. In one aspect, the promoter provided herein is operably linked to a heterologous nucleic acid molecule.

[0116] low RNA molecule In one aspect, the nucleic acid molecule provided herein is a low RNA molecule. In another aspect, the nucleic acid molecule encodes a low RNA molecule.

[0117] As used herein, "low RNA molecule" refers to a non-coding RNA molecule that is 16 to 50 nucleotides in length. In one aspect, the low RNA molecule comprises 16 to 40 nucleotides. In another aspect, the low RNA molecule comprises 16 to 30 nucleotides. In another aspect, the low RNA molecule comprises 18 to 50 nucleotides. In another aspect, the low RNA molecule comprises 18 to 40 nucleotides. In another aspect, the low RNA molecule comprises 18 to 30 nucleotides. In another aspect, the low RNA molecule comprises 18 to 25 nucleotides. In another aspect, the low RNA molecule comprises 20 to 28 nucleotides. In another aspect, the low RNA molecule comprises 20 to 24 nucleotides. In another aspect, the low RNA molecule comprises 21 to 23 nucleotides. In another aspect, the low 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.

[0118] In one aspect, the low RNA molecule is selected from the group consisting of double-stranded RNA, small interfering RNA (siRNA), trans-acting siRNA, and microRNA (miRNA). In one aspect, the present disclosure also provides a guide RNA (gRNA) and a prime editing gRNA that bind to any one of SEQ ID NOs: 1 to 12.

[0119] miRNAs are generally about 19 to about 25 nucleotides (generally about 20 to 24 nucleotides in plants), induce cleavage in the trans of target transcripts, and negatively regulate the expression of genes involved in various regulatory and developmental pathways. In some cases, miRNAs function to induce co-phase processing of siRNA primary transcripts.

[0120] In plants, it is understood in the art that miRNAs and target nucleic acids often do not share perfect complementarity (although miRNAs and target nucleic acids can have perfect complementarity). miRNAs and their targets can have some mismatches between them, yet still allow 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).

[0121] In one aspect, the low RNA molecule comprises 100% complementarity with a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 12. In one aspect, the low 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 12. In one aspect, the low 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 12. In one aspect, the low 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 12. In one aspect, the low 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 12. In one aspect, the low 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 12. In one aspect, the low 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 12. In one aspect, the low 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 12. In one aspect, the low 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 12. In one aspect, the low 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 12.

[0122] Numerous microRNA genes (MIR genes) have been identified and are publicly available in databases (see "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 have been reported to occur both isolated within the genome and in clusters in intergenic regions, but can also be located wholly or in part within the 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. Transcription of MIR genes can, at least in some cases, be under the positive control of the promoters of the MIR genes themselves. The primary transcripts, termed "pri-miRNAs", can be very large (several kilobases), can be polycistronic, and contain one or more pre-miRNAs (folded structures containing the stem-loop arrangement that is processed into the mature miRNA), as well as the normal 5' "cap" and polyadenylation tail of an mRNA.

[0123] Maturation of mature miRNAs from their corresponding precursors (pri-miRNAs and pre-miRNAs) differs significantly between animals and plants. For example, in plant cells, most microRNA precursor molecules are thought to be processed entirely into mature miRNAs in the nucleus, whereas in animal cells, pri-miRNA transcripts are processed in the nucleus by the animal-specific enzyme Drosha, followed by transport of the pre-miRNA to the cytoplasm where it is further processed into the mature miRNA. Mature miRNAs in plants are typically 21 nucleotides in length.

[0124] Using transgenic expression of miRNA (whether a naturally occurring sequence or an artificial sequence), the expression of a target gene or genes of miRNA can be controlled. Inclusion of miRNA recognition sites in the transgenic expressed transcript is also useful for controlling the expression of the transcript. The recognition sites of miRNA have been verified in all regions of mRNA including the 5' untranslated region, the coding region, and the 3' untranslated region, indicating that the position of the miRNA target site relative to the coding sequence does not necessarily affect repression. Since miRNA is an important regulatory element in eukaryotes, transgenic repression of miRNA is useful for manipulating biological pathways and responses. Finally, the promoter of the MIR gene can have a very specific expression pattern (e.g., cell-specific, tissue-specific, time-specific, or inducible), and thus is useful in recombinant constructs for inducing such specific transcription of DNA sequences operably linked to the promoter. The various utilities of miRNA, their precursors, their recognition sites, and their promoters are described in detail in U.S. Patent Application Publication No. 2006 / 0200878A1, which is incorporated herein by reference. Non-limiting examples of these utilities include the following: (1) expression of a natural miRNA or miRNA precursor sequence for suppressing a target gene; (2) expression of an artificial miRNA or miRNA precursor sequence for suppressing a target gene; (3) expression of a transgene having a miRNA recognition site, where the transgene is suppressed when mature miRNA is expressed; (4) expression of a transgene driven by a miRNA promoter.

[0125] Designing artificial miRNA sequences can be as simple as replacing the nucleotides in the miRNA stem region of the miRNA precursor with sequences complementary to the intended target for the nucleotides, as shown by Zeng et al. (2002) Mol. Cell, 9: 1327-1333. One non-limiting example of a general method for determining nucleotide changes in a native miRNA sequence to produce an engineered miRNA precursor includes the following steps: (a) To identify any potential matches to orthologs of the target transcript and unrelated genes, for example, select a unique target sequence of at least 18 nucleotides specific to the target gene in both a tobacco cDNA and genomic DNA database, for example, by using a sequence alignment tool such as BLAST (see, for example, Altschul et al. (1990) J. Mol. Biol., 215: 403-410, Altschul et al. (1997) Nucleic Acids Res., 25: 3389-3402), thereby avoiding unintended silencing of non-target sequences; (b) Analyze the target gene for unwanted sequences (e.g., matches to sequences from non-target species), and score each potential 19-mer segment for functional asymmetry (".DELTA..DELTA.G" or "ΔΔG") characterized by GC content, Reynolds score (see Reynolds et al. (2004) Nature Biotechnol., 22: 326-330), and negative difference in free energy (see Khvorova et al. (2003) Cell, 115: 209-216). Preferably, a 19-mer having all or most of the following characteristics is selected: (1) Reynolds score > 4, (2) GC content of about 40% to about 60%, (3) negative ΔΔG, (4) terminal adenosine, (5) absence of consecutive runs of four or more of the same nucleotide, (6) position near the 3' end of the target gene, (7) minimal difference from the miRNA precursor transcript.The position of every third nucleotide in siRNA has been reported to be particularly important in affecting RNAi (RNA interference) efficacy, and the algorithm "siExplorer" is publicly available at rna.chem.t.u-tokyo.ac.jp / siexplorer.htm (see Katoh and Suzuki (2007) Nucleic Acids Res., 10.1093 / nar / gkl1120); (c) determining a 19-mer reverse complement selected for use in generating a modified mature miRNA. The additional nucleotide at position 20 preferably matches the selected target sequence, and the nucleotide at position 21 is selected such that it is either not base paired to prevent the spread of silencing on the target transcript or is base paired to the target sequence to promote the spread of silencing on the target transcript; and (d) transforming the artificial miRNA into a plant.

[0126] Without being limited to any scientific theory, it is understood in the art that both RNAi knockdown of a candidate gene (e.g., by use of an artificial miRNA or siRNA) and a mutation in the same candidate gene (e.g., a missense or nonsense mutation) can both cause reduced expression and / or reduced protein activity and can cause the same or similar phenotypes in plants. See, for example, Agrawal et al., Microbiology and Molecular Biology Reviews, 67:657-685 (2003).

[0127] In one aspect, the small molecule 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: 1-12. In one aspect, the small molecule 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: 1-12. In one aspect, the small molecule 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: 1-12. In one aspect, the small molecule 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: 1-12. In one aspect, the small molecule 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: 1-12. In one aspect, the small molecule 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: 1-12. In one aspect, the small molecule 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: 1-12. In one aspect, the small molecule 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: 1-12. In one aspect, the small molecule 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: 1-12. In one aspect, the small molecule 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: 1-12.

[0128] In one aspect, the small molecule 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: 7 to 12. In one aspect, the small molecule 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: 7 to 12. In one aspect, the small molecule 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: 7 to 12. In one aspect, the small molecule 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: 7 to 12. In one aspect, the small molecule 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: 7 to 12. In one aspect, the small molecule 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: 7 to 12. In one aspect, the small molecule 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: 7 to 12. In one aspect, the small molecule 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: 7 to 12. In one aspect, the small molecule 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: 7 to 12. In one aspect, the small molecule 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: 7 to 12.

[0129] In one aspect, the small molecule 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 NOs: 1-12. In another aspect, the small molecule 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 NOs: 1-12. In another aspect, the small molecule 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 NOs: 1-12. In another aspect, the small molecule 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 NOs: 1-12. In another aspect, the small molecule 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 NOs: 1-12. In another aspect, the small molecule 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: 1-12. In another aspect, the small molecule 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: 1-12.

[0130] In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence encoding a polypeptide that is at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18 and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence encoding a polypeptide that is at least 75% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18 and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a 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: 13 to 18 and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence encoding a polypeptide that is at least 85% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18 and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence encoding a polypeptide that is at least 88% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18 and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence encoding a polypeptide that is at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18 and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence encoding a polypeptide that is at least 91% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18 and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence encoding a polypeptide that is at least 92% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18 and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence encoding a polypeptide that is at least 93% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18 and reduce its expression.In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence encoding a polypeptide that is at least 94% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18 and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence encoding a polypeptide that is at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18 and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence encoding a polypeptide that is at least 96% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18 and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence encoding a polypeptide that is at least 97% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18 and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence encoding a polypeptide that is at least 98% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18 and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence encoding a polypeptide that is at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18 and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence encoding a polypeptide that is 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18 and reduce its expression.

[0131] In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence that is at least 70% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-12 or an RNA transcribed therefrom, and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence that is at least 75% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-12 or an RNA transcribed therefrom, and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-12 or an RNA transcribed therefrom, and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence that is at least 85% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-12 or an RNA transcribed therefrom, and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence that is at least 88% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-12 or an RNA transcribed therefrom, and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-12 or an RNA transcribed therefrom, and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence that is at least 91% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-12 or an RNA transcribed therefrom, and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence that is at least 92% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-12 or an RNA transcribed therefrom, and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence that is at least 93% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-12 or an RNA transcribed therefrom, and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence that is at least 94% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-12 or an RNA transcribed therefrom, and reduce its expression.In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence that is at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-12 or RNA transcribed therefrom, and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence that is at least 96% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-12 or RNA transcribed therefrom, and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence that is at least 97% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-12 or RNA transcribed therefrom, and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence that is at least 98% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-12 or RNA transcribed therefrom, and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence that is at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-12 or RNA transcribed therefrom, and reduce its expression. In one aspect, the low RNA molecules provided herein can bind to a nucleic acid sequence that is 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-12 or RNA transcribed therefrom, and reduce its expression.

[0132] As used herein, "capable of binding to" is synonymous with "capable of hybridizing to". In one aspect, a first nucleic acid molecule capable of binding to a second nucleic acid molecule binds to the second nucleic acid molecule. As used herein, a first nucleic acid molecule "hybridizes" to a second nucleic acid molecule via non-covalent interactions (e.g., Watson-Crick base pairing) in a sequence-specific antiparallel manner (i.e., the nucleic acid specifically binds to a complementary nucleic acid) under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. As is known in the art, standard Watson-Crick base pairing includes adenine pairing with thymine, adenine pairing with uracil, and guanine (G) pairing with cytosine (C) [DNA, RNA]. In addition, for hybridization between two RNA molecules (e.g., dsRNA), it is also known in the art that guanine bases can pair with uracil. For example, G / U base pairing is involved in part in the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anticodon base pairing with codons in mRNA. In the context of the present disclosure, the guanine of the protein-binding segment (dsRNA duplex) of the DNA-targeting RNA molecule of interest is considered to be complementary to uracil, and vice versa. As such, if G / U base pairing can be made at a given nucleotide position of the protein-binding segment (dsRNA duplex) of the DNA-targeting RNA molecule of interest, this position is not considered non-complementary, but rather complementary.

[0133] Hybridization and washing conditions are well known and are exemplified in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, 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, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). The temperature and ionic strength conditions determine the "stringency" of the hybridization.

[0134] Hybridization allows for base mismatches but requires that the two nucleic acids contain complementary sequences. The conditions appropriate for hybridization between two nucleic acids depend on variables well known in the art, such as the length and degree of complementarity of the nucleic acids. The greater the degree of complementarity between two nucleotide sequences, the higher the melting temperature (Tm) value for the hybrid of the nucleic acids having those sequences. For hybridization between nucleic acids having short stretches of complementarity (e.g., complementarity over 35 or fewer nucleotides), the position of the mismatch becomes important (see Sambrook et al.). Typically, the length of a hybridizable nucleic acid is at least about 10 nucleotides. Exemplary minimum lengths for hybridizable nucleic acids are at least about 15 nucleotides, at least about 20 nucleotides, at least about 22 nucleotides, at least about 25 nucleotides, and at least about 30 nucleotides). Further, one of ordinary skill in the art will recognize that the temperature and salt concentration of the wash solution can be adjusted as needed according to factors such as the length and degree of complementarity of the complementary region.

[0135] It is understood in the art that the sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid in order to be specifically hybridizable or hybridizable. Further, a polynucleotide can hybridize over one or more segments such that intervening or adjacent segments do not participate in a hybridization event (e.g., a loop structure or a hairpin structure). For example, an antisense nucleic acid in which 18 of 20 nucleotides are complementary to a target region and thus specifically hybridizes represents 90 percent complementarity. In this example, the remaining non-complementary nucleotides can be clustered or interspersed with the complementary nucleotides and need not be contiguous with each other or with the complementary nucleotides. The percent complementarity between specific stretches of nucleic acid sequences within a nucleic acid can be routinely determined using the BLAST® program (Basic Local Alignment Search Tool) and the PowerBLAST program known in the art (see Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), or using the default settings of the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

[0136] Reduced expression / activity In one aspect, a low RNA molecule reduces the expression of any nucleic acid sequence to which it can bind. In another aspect, a non-naturally occurring mutation provided herein reduces the expression of a mutated nucleic acid sequence as compared to the non-mutated nucleic acid sequence in a control plant grown under equivalent conditions.

[0137] Reduced expression of the endogenous nucleic acid sequence 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 blot (e.g., Northern blot), and RNA sequencing. The difference in expression can be described as absolute quantification or relative quantification. See, for example, Livak and Schmittgen, Methods, 25:402-408 (2001). When the endogenous nucleic acid sequence encodes a protein, the change in expression can be inferred by examining the accumulation of the encoded protein. Non-limiting examples of measuring protein accumulation include Western blot and enzyme-linked immunosorbent assay (ELISA).

[0138] In one aspect, the reduction in expression is measured using qRT-PCR. In another aspect, the reduction in expression is measured using an RNA blot. In another aspect, the reduction in expression is measured using RNA sequencing. In a further aspect, the reduction in expression is measured using a Western blot. In an even further aspect, the reduction in expression is measured using ELISA.

[0139] In one aspect, a non-natural mutation in a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-12 results in a reduced level of nucleic acid sequence expression compared to a nucleic acid sequence lacking the non-natural mutation in a control plant grown under equivalent 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: 13-18 results in a reduced level of nucleic acid sequence expression compared to a nucleic acid sequence lacking the non-natural mutation in a control plant grown under equivalent conditions.

[0140] In one aspect, the reduction in expression comprises at least a 1% reduction as compared to the expression in the same tissue of a control plant grown under equivalent conditions. In another aspect, the reduction in expression comprises at least a 5% reduction as compared to the expression in the same tissue of a control plant grown under equivalent conditions. In another aspect, the reduction in expression comprises at least a 10% reduction as compared to the expression in the same tissue of a control plant grown under equivalent conditions. In another aspect, the reduction in expression comprises at least a 25% reduction as compared to the expression in the same tissue of a control plant grown under equivalent conditions. In another aspect, the reduction in expression comprises at least a 50% reduction as compared to the expression in the same tissue of a control plant grown under equivalent conditions. In another aspect, the reduction in expression comprises at least a 75% reduction as compared to the expression in the same tissue of a control plant grown under equivalent conditions. In another aspect, the reduction in expression comprises at least a 90% reduction as compared to the expression in the same tissue of a control plant grown under equivalent conditions. In another aspect, the reduction in expression comprises at least a 95% reduction as compared to the expression in the same tissue of a control plant grown under equivalent conditions.

[0141] In one aspect, the reduction in expression includes a reduction of 1% to 99% compared to the expression in the same tissue of a control plant grown under equivalent conditions. In another aspect, the reduction in expression includes a reduction of 1% to 90% compared to the expression in the same tissue of a control plant grown under equivalent conditions. In another aspect, the reduction in expression includes a reduction of 1% to 75% compared to the expression in the same tissue of a control plant grown under equivalent conditions. In another aspect, the reduction in expression includes a reduction of 1% to 50% compared to the expression in the same tissue of a control plant grown under equivalent conditions. In another aspect, the reduction in expression includes a reduction of 1% to 25% compared to the expression in the same tissue of a control plant grown under equivalent conditions. In another aspect, the reduction in expression includes a reduction of 25% to 90% compared to the expression in the same tissue of a control plant grown under equivalent conditions. In another aspect, the reduction in expression includes a reduction of 50% to 90% compared to the expression in the same tissue of a control plant grown under equivalent conditions. In another aspect, the reduction in expression includes a reduction of 25% to 75% compared to the expression in the same tissue of a control plant grown under equivalent conditions.

[0142] In one aspect, the reduction in expression includes a statistically significant reduction compared to the expression in the same tissue of a control plant grown under equivalent conditions. One of ordinary skill in the art will recognize that any level of reduction is contemplated as long as the level of reduction has been determined to be statistically significant using an acceptable statistical hypothesis test. By way of non-limiting example, Student's t-test is one statistical hypothesis test that can be used to determine whether the reduction in expression between the modified plant and the control plant is statistically significant. As used herein, "statistically significant" refers to a p-value of 0.05 or less.

[0143] In one aspect, the non-natural mutation results in a reduced level of activity by a protein or polypeptide encoded by a nucleic acid sequence provided herein, as compared to the activity of a control plant grown under equivalent conditions. In another aspect, a non-natural mutation in an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-12 results in a reduced level of activity by a protein or polypeptide encoded by the nucleic acid sequence, as compared to the activity of a protein or polypeptide encoded by the endogenous nucleic acid sequence in a control tobacco plant when grown under equivalent conditions, and the nucleic acid sequence lacks the non-natural mutation in the control tobacco plant. In another aspect, a non-natural mutation in an endogenous nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18 results in a reduced level of activity by a protein or polypeptide encoded by the nucleic acid sequence, as compared to the activity of a protein or polypeptide encoded by the endogenous nucleic acid sequence in a control tobacco plant when grown under equivalent conditions, and the nucleic acid sequence lacks the non-natural mutation in the control tobacco plant.

[0144] As used herein, when referring to a protein or polypeptide, "activity" refers to the ability to perform an enzymatic function. By way of non-limiting example, the activity of a decarboxylase protein or polypeptide refers to the ability of the decarboxylase to remove a carboxyl group from an organic molecular substrate. Without being limited to any scientific theory, if a mutated decarboxylase protein has a reduced activity compared to a non-mutated control decarboxylase protein, the mutated decarboxylase may (a) be unable to remove a carboxyl group from any organic molecular substrate, (b) be able to remove a carboxyl group from an organic molecular substrate only at a reduced rate compared to the non-mutated decarboxylase protein, (c) be able to remove a carboxyl group only from a substrate that is not a suitable substrate for the non-mutated decarboxylase protein, or (d) be a combination of (b) and (c). Conversely, and without being limited to any scientific theory, if a mutated decarboxylase protein has an increased activity compared to a non-mutated control decarboxylase protein, the mutated decarboxylase may (a) be able to remove a carboxyl group from an organic molecular substrate at an increased rate compared to the non-mutated decarboxylase protein, (b) be able to remove a carboxyl group from a substrate that is not a suitable substrate for the non-mutated decarboxylase, or (c) be both (a) and (b). The activity of a decarboxylase can be measured using techniques standard in the art. See, for example, Yu et al., Enzyme Microb. Technol., 49:272-276 (2011), Seely and Pegg, J. Biol. Chem., 258:2496-2500 (1983), Mizusaki et al., Plant and Cell Physiology, 14:103-110 (1973).

[0145] Increased expression / activity In one aspect, the non-natural mutation results in increased expression of the nucleic acid sequence. In one aspect, the non-natural mutation results in an increased level of expression of the nucleic acid sequence when grown under equivalent conditions, compared to the expression of the nucleic acid sequence in the same tissue of a control tobacco plant, and the nucleic acid sequence lacks at least one non-natural mutation in the control tobacco plant.

[0146] In one aspect, the increased level of expression includes an increase of at least 5% compared to the expression in the same tissue of a control plant grown under equivalent conditions. In another aspect, the increased level of expression includes an increase of at least 10% compared to the expression in the same tissue of a control plant grown under equivalent conditions. In another aspect, the increased level of expression includes an increase of at least 25% compared to the expression in the same tissue of a control plant grown under equivalent conditions. In another aspect, the increased level of expression includes an increase of at least 50% compared to the expression in the same tissue of a control plant grown under equivalent conditions. In another aspect, the increased level of expression includes an increase of at least 75% compared to the expression in the same tissue of a control plant grown under equivalent conditions. In another aspect, the increased level of expression includes an increase of at least 100% compared to the expression in the same tissue of a control plant grown under equivalent conditions. In another aspect, the increased level of expression includes an increase of at least 200% compared to the expression in the same tissue of a control plant grown under equivalent conditions. In another aspect, the increased level of expression includes an increase of at least 500% compared to the expression in the same tissue of a control plant grown under equivalent conditions.

[0147] In one aspect, the non-natural mutation results in an increased level of activity of the protein or polypeptide encoded by the nucleic acid sequence when grown under equivalent conditions, compared to the activity of the protein or polypeptide encoded by the nucleic acid sequence in a control tobacco plant, and the nucleic acid sequence lacks at least one non-natural mutation in the control tobacco plant.

[0148] Alkaloid As used herein, "alkaloid" refers to complex nitrogen-containing compounds that occur naturally in tobacco and have pharmacological effects in humans or other animals. Non-limiting examples of alkaloids include nicotine, anabasine, anatabine, and nornicotine.

[0149] Nicotine is the major natural alkaloid in commercial cigarette tobacco, accounting for approximately 90% of the alkaloid content in Nicotiana tabacum. Other major alkaloids in tobacco include, but are not limited to, cotinine, nornicotine, myosmine, nicotyrine, anabasine, and anatabine. Trace amounts of tobacco alkaloids include, but are not limited to, nicotine - n - oxide, N - methylanatabine, N - methylanabasine, pseudooxynicotine, 2,3 - dipyridyl, and others.

[0150] In one aspect, the alkaloid is selected from the group consisting of anabasine, anatabine, nicotine, and nornicotine.

[0151] Alkaloid levels can be assayed 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, nicotine alkaloid levels can be measured by the CORESTA Recommended Method No. 7, 1987 and the ISO standard (GC - FID method based on ISO TC 126N 394 E). See also Hibi et al., Plant Physiology 100:826 - 35 (1992) for methods using gas - liquid chromatography equipped with a capillary column and an FID detector.

[0152] Alkaloid levels can be assayed from any plant tissue. Non-limiting examples include leaves and hairy root cultures. In addition, alkaloid levels can be assayed in dried plant material or in non-dried plant material.

[0153] Alternatively, the total alkaloids in tobacco can be measured using a segmented flow colorimetric method adapted by Skalar Instrument Co. (West Chester, PA) and developed for the analysis of tobacco samples as described by Collins et al., Tobacco Science 13:79-81 (1969). Briefly, tobacco samples are dried, ground, and extracted prior to the analysis of total alkaloids and reducing sugars. The method then uses acetic acid / methanol / water extraction and charcoal for decolorization. The determination of total alkaloids is based on the reaction of cyanide chloride with nicotine alkaloids in the presence of aromatic amines to form a colored complex that is measured at 460 nm.

[0154] In one aspect, the levels of individual alkaloids are measured using liquid chromatography with tandem mass spectrometry (LC / MS / MS) based on freeze-dried leaf samples. In one aspect, the levels of individual alkaloids are measured using liquid chromatography with tandem mass spectrometry (LC / MS / MS) based on freeze-dried hairy root culture samples.

[0155] Unless otherwise specified, tobacco plant nicotine or alkaloid levels (or other leaf chemical properties or trait evaluations) are measured after topping in pooled leaf samples collected from leaves 3, 4, and 5 after topping. As used herein, whenever a comparison between leaves from two plants (e.g., mutant plant vs. control plant) is referred to, it is always intended that the leaves be from the same or equivalent leaf position(s) and developmental stage(s) such that the comparison can demonstrate the effect due to genotype differences rather than other factors. By way of non-limiting illustration, leaf 3 of a control plant is intended as a reference point for comparison to leaf 3 of a modified plant containing a non-natural mutation or recombinant DNA construct.

[0156] As used herein, leaf numbering is based on the position of the leaves on the tobacco stem, with leaf number 1 after topping being the youngest leaf (upper) and the highest leaf number being assigned to the oldest leaf (lower).

[0157] As used herein, "topping" refers to the removal of the shoot apical meristem, flowers, and some of the largest adjacent leaves at the shoot apex when the tobacco plant is approaching vegetative maturity and at the onset of reproductive growth. Typically, tobacco plants are topped at the button stage (shortly after flowers begin to appear). For example, tobacco plants grown in a greenhouse or field can be topped when 50% of the plants have at least one flower. Topping a tobacco plant results in the loss of apical dominance and also induces increased alkaloid production.

[0158] Typically, the alkaloid level of a tobacco plant (or the characterization of another leaf chemistry or property; e.g., polyamine) is measured about two weeks after flower removal. Other time points can also be used. In one aspect, the alkaloid level of a tobacco plant (or the characterization of another leaf chemistry or property) is measured about 1, 2, 3, 4, or 5 weeks after flower removal. In another aspect, the nicotine, alkaloid, or polyamine level of a tobacco plant (or the characterization of another leaf chemistry or property; e.g., polyamine) is measured about 3, 5, 7, 10, 12, 14, 17, 19, or 21 days after flower removal.

[0159] In one aspect, a modified tobacco plant or a part thereof containing at least one non-natural mutation in an endogenous nucleic acid produces at least one leaf containing a reduced amount of at least one alkaloid as compared to the amount of alkaloid in a control tobacco plant lacking at least one non-natural mutation in the endogenous nucleic acid when grown under equivalent conditions.

[0160] In one aspect, a modified tobacco plant or a part thereof containing the recombinant DNA construct provided herein produces at least one leaf containing a reduced amount of at least one alkaloid as compared to the amount of alkaloid in a control tobacco plant lacking the recombinant DNA construct when grown under equivalent conditions.

[0161] In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 0.5% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 1% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 2% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 3% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 4% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 5% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 10% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 15% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 20% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 25% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 35% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 50% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 75% when grown under equivalent conditions as compared to a control tobacco plant.In one aspect, the reduced level of at least one alkaloid comprises at least a 90% reduction compared to control tobacco plants when grown under equivalent conditions.

[0162] In one aspect, at least one alkaloid at a reduced level includes a reduction of 1% to 99% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of 1% to 90% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of 1% to 80% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of 1% to 70% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of 1% to 60% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of 1% to 50% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of 1% to 40% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of 1% to 30% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of 1% to 20% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of 1% to 10% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of 1% to 5% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of 10% to 75% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of 10% to 50% when grown under equivalent conditions as compared to a control tobacco plant.In one aspect, the reduced level of at least one alkaloid comprises a 10% to 25% reduction compared to control tobacco plants when grown under equivalent conditions.

[0163] In one aspect, at least one alkaloid at a reduced level includes a reduction in nicotine of at least 0.5% when grown under equivalent conditions compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction in nicotine of at least 1% when grown under equivalent conditions compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction in nicotine of at least 2% when grown under equivalent conditions compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction in nicotine of at least 3% when grown under equivalent conditions compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction in nicotine of at least 4% when grown under equivalent conditions compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction in nicotine of at least 5% when grown under equivalent conditions compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction in nicotine of at least 10% when grown under equivalent conditions compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction in nicotine of at least 15% when grown under equivalent conditions compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction in nicotine of at least 20% when grown under equivalent conditions compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction in nicotine of at least 25% when grown under equivalent conditions compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction in nicotine of at least 35% when grown under equivalent conditions compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction in nicotine of at least 50% when grown under equivalent conditions compared to a control tobacco plant.In one aspect, the reduced level of at least one alkaloid includes a reduction of nicotine by at least 75% compared to a control tobacco plant when grown under equivalent conditions. In one aspect, the reduced level of at least one alkaloid includes a reduction of nicotine by at least 90% compared to a control tobacco plant when grown under equivalent conditions.

[0164] In one aspect, at least one alkaloid at a reduced level includes a reduction of nicotine from 1% to 99% when grown under equivalent conditions compared to control tobacco plants. In one aspect, at least one alkaloid at a reduced level includes a reduction of nicotine from 1% to 90% when grown under equivalent conditions compared to control tobacco plants. In one aspect, at least one alkaloid at a reduced level includes a reduction of nicotine from 1% to 80% when grown under equivalent conditions compared to control tobacco plants. In one aspect, at least one alkaloid at a reduced level includes a reduction of nicotine from 1% to 70% when grown under equivalent conditions compared to control tobacco plants. In one aspect, at least one alkaloid at a reduced level includes a reduction of nicotine from 1% to 60% when grown under equivalent conditions compared to control tobacco plants. In one aspect, at least one alkaloid at a reduced level includes a reduction of nicotine from 1% to 50% when grown under equivalent conditions compared to control tobacco plants. In one aspect, at least one alkaloid at a reduced level includes a reduction of nicotine from 1% to 40% when grown under equivalent conditions compared to control tobacco plants. In one aspect, at least one alkaloid at a reduced level includes a reduction of nicotine from 1% to 30% when grown under equivalent conditions compared to control tobacco plants. In one aspect, at least one alkaloid at a reduced level includes a reduction of nicotine from 1% to 20% when grown under equivalent conditions compared to control tobacco plants. In one aspect, at least one alkaloid at a reduced level includes a reduction of nicotine from 1% to 10% when grown under equivalent conditions compared to control tobacco plants. In one aspect, at least one alkaloid at a reduced level includes a reduction of nicotine from 1% to 5% when grown under equivalent conditions compared to control tobacco plants. In one aspect, at least one alkaloid at a reduced level includes a reduction of nicotine from 10% to 75% when grown under equivalent conditions compared to control tobacco plants.In one aspect, at least one alkaloid at a reduced level includes a reduction in nicotine of 10% to 50% when grown under equivalent conditions compared to control tobacco plants. In one aspect, at least one alkaloid at a reduced level includes a reduction in nicotine of 10% to 25% when grown under equivalent conditions compared to control tobacco plants.

[0165] In one aspect, the modified tobacco plant includes a nicotine level of 17 milligrams per gram (mg / g) or less of dry weight. In one aspect, the modified tobacco plant includes a nicotine level of 16 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nicotine level of 15 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nicotine level of 14 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nicotine level of 13 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nicotine level of 12 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nicotine level of 11 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nicotine level of 10 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nicotine level of 9 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nicotine level of 8 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nicotine level of 7 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nicotine level of 6 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nicotine level of 5 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nicotine level of 4 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nicotine level of 3 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nicotine level of 2 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nicotine level of 1.75 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nicotine level of 1.6 mg / g or less of dry weight.

[0166] In one aspect, at least one alkaloid at a reduced level includes a reduction of anatabine of at least 0.5% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of anatabine of at least 1% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of anatabine of at least 2% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of anatabine of at least 3% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of anatabine of at least 4% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of anatabine of at least 5% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of anatabine of at least 10% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of anatabine of at least 15% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of anatabine of at least 20% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of anatabine of at least 25% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of anatabine of at least 35% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of anatabine of at least 50% when grown under equivalent conditions as compared to a control tobacco plant.In one aspect, the reduced level of at least one alkaloid includes a reduction of anatabine by at least 75% compared to control tobacco plants when grown under equivalent conditions. In one aspect, the reduced level of at least one alkaloid includes a reduction of anatabine by at least 90% compared to control tobacco plants when grown under equivalent conditions.

[0167] In one aspect, at least one alkaloid at a reduced level includes a reduction in anatabine of from 1% to 99% when grown under equivalent conditions as compared to control tobacco plants. In one aspect, at least one alkaloid at a reduced level includes a reduction in anatabine of from 1% to 90% when grown under equivalent conditions as compared to control tobacco plants. In one aspect, at least one alkaloid at a reduced level includes a reduction in anatabine of from 1% to 80% when grown under equivalent conditions as compared to control tobacco plants. In one aspect, at least one alkaloid at a reduced level includes a reduction in anatabine of from 1% to 70% when grown under equivalent conditions as compared to control tobacco plants. In one aspect, at least one alkaloid at a reduced level includes a reduction in anatabine of from 1% to 60% when grown under equivalent conditions as compared to control tobacco plants. In one aspect, at least one alkaloid at a reduced level includes a reduction in anatabine of from 1% to 50% when grown under equivalent conditions as compared to control tobacco plants. In one aspect, at least one alkaloid at a reduced level includes a reduction in anatabine of from 1% to 40% when grown under equivalent conditions as compared to control tobacco plants. In one aspect, at least one alkaloid at a reduced level includes a reduction in anatabine of from 1% to 30% when grown under equivalent conditions as compared to control tobacco plants. In one aspect, at least one alkaloid at a reduced level includes a reduction in anatabine of from 1% to 20% when grown under equivalent conditions as compared to control tobacco plants. In one aspect, at least one alkaloid at a reduced level includes a reduction in anatabine of from 1% to 10% when grown under equivalent conditions as compared to control tobacco plants. In one aspect, at least one alkaloid at a reduced level includes a reduction in anatabine of from 1% to 5% when grown under equivalent conditions as compared to control tobacco plants. In one aspect, at least one alkaloid at a reduced level includes a reduction in anatabine of from 10% to 75% when grown under equivalent conditions as compared to control tobacco plants.In one aspect, the reduced level of at least one alkaloid includes a reduction of anatabine by 10% to 50% when grown under equivalent conditions as compared to control tobacco plants. In one aspect, the reduced level of at least one alkaloid includes a reduction of anatabine by 10% to 25% when grown under equivalent conditions as compared to control tobacco plants.

[0168] In one aspect, the modified tobacco plant includes an anatabine level of 1.5 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes an anatabine level of 1.4 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes an anatabine level of 1.3 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes an anatabine level of 1.2 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes an anatabine level of 1.1 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes an anatabine level of 1.0 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes an anatabine level of 0.9 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes an anatabine level of 0.8 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes an anatabine level of 0.7 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes an anatabine level of 0.6 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes an anatabine level of 0.5 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes an anatabine level of 0.41 mg / g or less of dry weight.

[0169] In one aspect, at least one alkaloid at a reduced level includes a reduction of anabasine by at least 0.5% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of anabasine by at least 1% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of anabasine by at least 2% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of anabasine by at least 3% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of anabasine by at least 4% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of anabasine by at least 5% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of anabasine by at least 10% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of anabasine by at least 15% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of anabasine by at least 20% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of anabasine by at least 25% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of anabasine by at least 35% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of anabasine by at least 50% when grown under equivalent conditions as compared to a control tobacco plant.In one aspect, the reduced level of at least one alkaloid includes a reduction of anabasine by at least 75% compared to a control tobacco plant when grown under equivalent conditions. In one aspect, the reduced level of at least one alkaloid includes a reduction of anabasine by at least 90% compared to a control tobacco plant when grown under equivalent conditions.

[0170] In one aspect, at least one alkaloid at a reduced level includes a reduction in anabasine of 1% to 99% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction in anabasine of 1% to 90% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction in anabasine of 1% to 80% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction in anabasine of 1% to 70% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction in anabasine of 1% to 60% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction in anabasine of 1% to 50% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction in anabasine of 1% to 40% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction in anabasine of 1% to 30% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction in anabasine of 1% to 20% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction in anabasine of 1% to 10% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction in anabasine of 1% to 5% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction in anabasine of 10% to 75% when grown under equivalent conditions as compared to a control tobacco plant.In one aspect, the reduced level of at least one alkaloid includes a reduction in anabasine of from 10% to 50% when grown under equivalent conditions compared to control tobacco plants. In one aspect, the reduced level of at least one alkaloid includes a reduction in anabasine of from 10% to 25% when grown under equivalent conditions compared to control tobacco plants.

[0171] In one aspect, the modified tobacco plant comprises an anabasine level of 0.6 mg / g or less on a dry weight basis. In one aspect, the modified tobacco plant comprises an anabasine level of 0.5 mg / g or less on a dry weight basis. In one aspect, the modified tobacco plant comprises an anabasine level of 0.4 mg / g or less on a dry weight basis. In one aspect, the modified tobacco plant comprises an anabasine level of 0.3 mg / g or less on a dry weight basis. In one aspect, the modified tobacco plant comprises an anabasine level of 0.25 mg / g or less on a dry weight basis. In one aspect, the modified tobacco plant comprises an anabasine level of 0.2 mg / g or less on a dry weight basis. In one aspect, the modified tobacco plant comprises an anabasine level of 0.19 mg / g or less on a dry weight basis. In one aspect, the modified tobacco plant comprises an anabasine level of 0.18 mg / g or less on a dry weight basis. In one aspect, the modified tobacco plant comprises an anabasine level of 0.17 mg / g or less on a dry weight basis. In one aspect, the modified tobacco plant comprises an anabasine level of 0.16 mg / g or less on a dry weight basis. In one aspect, the modified tobacco plant comprises an anabasine level of 0.15 mg / g or less on a dry weight basis. In one aspect, the modified tobacco plant comprises an anabasine level of 0.14 mg / g or less on a dry weight basis. In one aspect, the modified tobacco plant comprises an anabasine level of 0.13 mg / g or less on a dry weight basis. In one aspect, the modified tobacco plant comprises an anabasine level of 0.12 mg / g or less on a dry weight basis. In one aspect, the modified tobacco plant comprises an anabasine level of 0.11 mg / g or less on a dry weight basis. In one aspect, the modified tobacco plant comprises an anabasine level of 0.1 mg / g or less on a dry weight basis. In one aspect, the modified tobacco plant comprises an anabasine level of 0.09 mg / g or less on a dry weight basis. In one aspect, the modified tobacco plant comprises an anabasine level of 0.08 mg / g or less on a dry weight basis. In one aspect, the modified tobacco plant comprises an anabasine level of 0.07 mg / g or less on a dry weight basis. In one aspect, the modified tobacco plant comprises an anabasine level of 0.06 mg / g or less on a dry weight basis. In one aspect, the modified tobacco plant comprises an anabasine level of 0.05 mg / g or less on a dry weight basis. In one aspect, the modified tobacco plant comprises an anabasine level of 0.04 mg / g or less on a dry weight basis. In one aspect, the modified tobacco plant comprises an anabasine level of 0.035 mg / g or less on a dry weight basis.

[0172] In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 0.5% of nornicotine when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 1% of nornicotine when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 2% of nornicotine when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 3% of nornicotine when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 4% of nornicotine when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 5% of nornicotine when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 10% of nornicotine when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 15% of nornicotine when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 20% of nornicotine when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 25% of nornicotine when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 35% of nornicotine when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of at least 50% of nornicotine when grown under equivalent conditions as compared to a control tobacco plant.In one aspect, the reduced level of at least one alkaloid includes a reduction of nornicotine by at least 75% compared to control tobacco plants when grown under equivalent conditions. In one aspect, the reduced level of at least one alkaloid includes a reduction of nornicotine by at least 90% compared to control tobacco plants when grown under equivalent conditions.

[0173] In one aspect, at least one alkaloid at a reduced level includes a reduction of nornicotine from 1% to 99% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of nornicotine from 1% to 90% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of nornicotine from 1% to 80% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of nornicotine from 1% to 70% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of nornicotine from 1% to 60% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of nornicotine from 1% to 50% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of nornicotine from 1% to 40% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of nornicotine from 1% to 30% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of nornicotine from 1% to 20% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of nornicotine from 1% to 10% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of nornicotine from 1% to 5% when grown under equivalent conditions as compared to a control tobacco plant. In one aspect, at least one alkaloid at a reduced level includes a reduction of nornicotine from 10% to 75% when grown under equivalent conditions as compared to a control tobacco plant.In one aspect, the reduced level of at least one alkaloid includes a reduction of nornicotine by 10% to 50% when grown under equivalent conditions as compared to control tobacco plants. In one aspect, the reduced level of at least one alkaloid includes a reduction of nornicotine by 10% to 25% when grown under equivalent conditions as compared to control tobacco plants.

[0174] In one aspect, the modified tobacco plant includes a nornicotine level of 1.0 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nornicotine level of 0.9 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nornicotine level of 0.8 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nornicotine level of 0.7 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nornicotine level of 0.6 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nornicotine level of 0.5 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nornicotine level of 0.4 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nornicotine level of 0.3 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nornicotine level of 0.2 mg / g or less of dry weight. In one aspect, the modified tobacco plant includes a nornicotine level of 0.17 mg / g or less of dry weight.

[0175] plant As used herein, tobacco plants include Nicotiana tabacum, Nicotiana amplexicaulis PI 271989, Nicotiana benthamiana PI 555478, Nicotiana bigelovii PI 555485, Nicotiana debneyi, Nicotiana excelsior PI 224063, Nicotiana glutinosa PI 555507, Nicotiana goodspeedii PI 241012, Nicotiana gossei PI 230953, Nicotiana hesperis PI 271991, Nicotiana knightiana PI 555527, Nicotiana maritima PI 555535, Nicotiana megalosiphon PI 555536, Nicotiana nudicaulis PI 555540, Nicotiana paniculata PI 555545, Nicotiana plumbaginifolia PI 555548, Nicotiana repanda PI 555552, Nicotiana rustica, Nicotiana suaveolens PI 230960, Nicotiana sylvestris PI 555569, Nicotiana tomentosa PI 266379, Nicotiana tomentosiformis, and Nicotiana trigonophylla PIIt can be from any plant from the genus Nicotiana, including but not limited to 555572. In one aspect, the tobacco plants described herein are Nicotiana tabacum plants.

[0176] In one aspect, the tobacco parts provided include, but are not limited to, leaves, stems, roots, trichomes, seeds, flowers, pollen, anthers, ovules, pedicels, fruits, meristems, cotyledons, hypocotyls, sheaths, embryos, endosperms, explants, calli, tissue cultures, shoots, cells, and protoplasts. In one aspect, the tobacco parts provided do not include seeds. In one aspect, the present disclosure provides organs that do not mediate natural reproduction of the plant, rather than cells, tissues, and germplasm of the tobacco plant. In another aspect, the present disclosure also provides cells, tissues, and germplasm of the tobacco plant and organs that mediate natural reproduction of the plant. In another aspect, the present disclosure provides cells, tissues, and organs of a tobacco plant that cannot sustain itself through photosynthesis. In another aspect, the present disclosure provides somatic tobacco plant cells. Somatic cells, as opposed to germline cells, do not mediate plant reproduction.

[0177] The cells, tissues, and organs can be from seeds, fruits, leaves, cotyledons, hypocotyls, meristems, embryos, endosperms, roots, shoots, stems, trichomes, sheaths, flowers, inflorescences, stalks, pedicels, styles, stigmas, flower beds, petals, sepals, pollen, anthers, filaments, ovaries, ovules, pericarp, sieve parts, vascular tissues. In another aspect, the present disclosure provides chloroplasts of a tobacco plant. In a further aspect, the present disclosure provides epidermal cells, stoma cells, leaves or root hairs, storage roots, or tubers. In another aspect, the present disclosure provides protoplasts of tobacco.

[0178] One of ordinary skill in the art understands that tobacco plants reproduce naturally via seeds, rather than via asexual or vegetative propagation. In one aspect, the present disclosure provides endosperm of tobacco.

[0179] The present disclosure provides cells from the tobacco plants provided herein.

[0180] As used herein, "progeny tobacco plants" or "progeny tobacco seeds" can be from any hybrid generation, e.g., F 1 , F 2 , F 3 , F 4 , F 5 , F 6 , F 7 and so on.

[0181] In one aspect, the tobacco plant or a part thereof is of a tobacco variety selected from the group consisting of flue-cured varieties, bright varieties, burley varieties, Virginia varieties, Maryland varieties, dark varieties, Galpao varieties, Oriental varieties, and Turkish varieties. In certain aspects, the modified tobacco plant or a part thereof provided herein is of a tobacco variety selected from the group consisting of flue-cured varieties, bright varieties, burley varieties, Virginia varieties, Maryland varieties, dark varieties, Galpao varieties, Oriental varieties, and Turkish varieties.

[0182] In one aspect, the tobacco cells are of a tobacco variety selected from the group consisting of flue-cured varieties, bright varieties, burley varieties, Virginia varieties, Maryland varieties, dark varieties, Galpao varieties, Oriental varieties, and Turkish varieties. In one aspect, the modified tobacco cells are of a tobacco variety selected from the group consisting of flue-cured varieties, bright varieties, burley varieties, Virginia varieties, Maryland varieties, dark varieties, Galpao varieties, Oriental varieties, and Turkish varieties.

[0183] In one aspect, the tobacco leaves are of a tobacco variety selected from the group consisting of flue-cured varieties, bright varieties, burley varieties, Virginia varieties, Maryland varieties, dark varieties, Galpao varieties, Oriental varieties, and Turkish varieties.

[0184] In one aspect, the dried tobacco leaf or plant part is of a tobacco variety selected from the group consisting of flue-cured varieties, bright varieties, burley varieties, Virginia varieties, Maryland varieties, dark varieties, Galpao varieties, Oriental varieties, and Turkish varieties. One of ordinary skill in the art further understands that dried tobacco does not constitute a living body and cannot grow or reproduce.

[0185] Flue-cured tobacco (also called "Virginia" or "bright" tobacco) accounts for approximately 40% of the world's tobacco production. Flue-cured tobacco often turns from golden to deep orange during drying and is sometimes referred to as "bright tobacco". Flue-cured tobacco has a light and distinctive aroma and flavor. Flue-cured tobacco generally has a high sugar content and a low oil content. The major flue-cured tobacco cultivating countries are Argentina, Brazil, China, India, Tanzania, and the United States of America. In one aspect, the tobacco plants or seeds provided herein, or the modified tobacco plants or seeds, are of a flue-cured tobacco variety selected from the group consisting of the varieties listed in Table 2 and any variety essentially derived from any one of the aforementioned varieties. See WO2004 / 041006A1. In a further aspect, the modified tobacco plants or seeds provided herein are of a flue-cured variety selected from the group consisting of K326, K346, and NC196.

[0186] (Table 2) Flue-cured tobacco varieties TIFF2025518037000003.tif38159TIFF2025518037000004.tif255159

[0187] Air-cured tobacco includes "burley", "Maryland", and "dark" tobacco. A common factor associated with air-cured tobacco is that the drying is carried out mainly without using artificial heat sources and humidity sources. Burley tobacco ranges from light to dark brown, has a high oil content, and a low sugar content. Burley tobacco is typically air-dried in barns. The major burley cultivating countries include Argentina, Brazil, Italy, Malawi, and the United States of America.

[0188] Maryland tobacco is very fluffy and has good combustion characteristics, low nicotine, and a neutral aroma. The main Maryland tobacco-growing countries include the United States and Italy.

[0189] In one aspect, the tobacco plants or seeds, or modified tobacco plants or seeds provided herein are of a variety of burley tobacco selected from the group consisting of the tobacco varieties listed in Table 3 and any variety that is essentially derived from any one of the foregoing varieties. In a further aspect, the modified tobacco plants or seeds provided herein are of a burley variety selected from the group consisting of TN90, KT209, KT206, KT212, and HB4488.

[0190] (Table 3) Burley tobacco varieties TIFF2025518037000005.tif246167

[0191] In another aspect, the tobacco plants or seeds, or modified tobacco plants or seeds provided herein are of a variety of Maryland tobacco selected from the group consisting of the tobacco varieties listed in Table 4 and any variety that is essentially derived from any one of the foregoing varieties.

[0192] (Table 4) Maryland tobacco varieties TIFF2025518037000006.tif78128

[0193] Dark air-cured tobacco is mainly distinguished from other tobacco types by its drying process, and gives medium to dark brown color and a characteristic aroma to dark air-cured tobacco. Dark air-cured tobacco is mainly used for chewing tobacco and sniffing tobacco. In one aspect, the modified tobacco plant or seed provided herein is of a dark air-cured tobacco variety selected from the group consisting of Sumatra, Jatim, Dominican Cubano, Besuki, One sucker, Green River, sun-cured Virginia, and Paraguan Passado, and any variety that is essentially derived from any one of the foregoing varieties.

[0194] Dark fire-cured tobacco is generally dried by a low-burning fire on the floor of a closed drying shed. Dark fire-cured tobacco is typically used to make pipe blends, cigarettes, chewing tobacco, sniffing tobacco, and cigars with a strong flavor. The main cultivation areas for dark fire-cured tobacco are Tennessee, Kentucky, and Virginia in the United States of America. In one aspect, the tobacco plant or seed, or modified tobacco plant or seed provided herein is of a dark fire-cured tobacco variety selected from the group consisting of the tobacco varieties listed in Table 5 and any variety that is essentially derived from any one of the foregoing varieties.

[0195] (Table 5) Dark fire-cured tobacco varieties TIFF2025518037000007.tif193161

[0196] Oriental tobacco is also referred to as Greek tobacco, aroma tobacco, and Turkish tobacco, typically because it is cultivated in the Eastern Mediterranean region such as Turkey, Greece, Bulgaria, Macedonia, Syria, Lebanon, Italy, Romania, etc. The small plant size, small leaf size, and unique aromatic characteristics of Oriental tobacco varieties are the result of their adaptation to the poor soil and stressful climate conditions in which they are developed. In one aspect, the tobacco plants or seeds, or modified tobacco plants or seeds provided herein are of an Oriental tobacco variety selected from the group consisting of the tobacco varieties listed in Table 6 and any variety that is essentially derived from any one of the aforementioned varieties.

[0197] (Table 6) Oriental tobacco varieties TIFF2025518037000008.tif134153

[0198] In one aspect, the tobacco plants or seeds, or modified tobacco plants or seeds provided herein are of a cigar tobacco variety selected from the group consisting of the tobacco varieties listed in Table 7 and any variety that is essentially derived from any one of the aforementioned varieties.

[0199] (Table 7) Cigar tobacco varieties TIFF2025518037000009.tif79167

[0200] In one aspect, the tobacco plants or seeds, or modified tobacco plants or seeds provided herein are of a tobacco variety selected from the group consisting of the tobacco varieties listed in Table 8 and any variety that is essentially derived from any one of the aforementioned varieties.

[0201] (Table 8) Other tobacco varieties TIFF2025518037000010.tif43128

[0202] In one aspect, the tobacco plant or a part thereof is from a variety selected from the group consisting of the tobacco varieties listed in Tables 2, 3, 4, 5, 6, 7, and 8. In another aspect, the tobacco plant or a part thereof is from a variety listed in Table 2. In another aspect, the tobacco plant or a part thereof is from a variety listed in Table 3. In another aspect, the tobacco plant or a part thereof is from a variety listed in Table 4. In another aspect, the tobacco plant or a part thereof is from a variety listed in Table 5. In another aspect, the tobacco plant or a part thereof is from a variety listed in Table 6. In another aspect, the tobacco plant or a part thereof is from a variety listed in Table 7. In another aspect, the tobacco plant or a part thereof is from a variety listed in Table 8.

[0203] In one aspect, the modified tobacco plant or a part thereof is from a variety selected from the group consisting of the tobacco varieties listed in Tables 2, 3, 4, 5, 6, 7, and 8. In one aspect, the modified tobacco plant or a part thereof is from a variety listed in Table 2. In another aspect, the modified tobacco plant or a part thereof is from a variety listed in Table 3. In another aspect, the modified tobacco plant or a part thereof is from a variety listed in Table 4. In another aspect, the modified tobacco plant or a part thereof is from a variety listed in Table 5. In another aspect, the modified tobacco plant or a part thereof is from a variety listed in Table 6. In another aspect, the modified tobacco plant or a part thereof is from a variety listed in Table 7. In another aspect, the modified tobacco plant or a part thereof is from a variety listed in Table 8.

[0204] In one aspect, the tobacco seeds are from a variety selected from the group consisting of the tobacco varieties listed in Tables 2, 3, 4, 5, 6, 7, and 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.

[0205] In one aspect, the tobacco cells are from a variety selected from the group consisting of the tobacco varieties listed in Tables 2, 3, 4, 5, 6, 7, and 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.

[0206] All of the specific varieties mentioned above of hot air drying, dark air drying, burley, Maryland, dark flue-curing, cigar wrapper, or oriental type are listed for illustrative purposes only. In this application, any additional hot air drying, dark air drying, burley, Maryland, dark flue-curing, cigar wrapper, or oriental varieties are also contemplated.

[0207] In one aspect, the tobacco plants or varieties provided herein are inbred tobacco plants or varieties. As used herein, an "inbred" tobacco variety is a variety that has been bred for genetic homogeneity.

[0208] As used herein, a "hybrid" is produced by crossing two plants from different varieties or species such that the progeny contains genetic material from each parent. One of ordinary skill in the art will recognize that higher order hybrids can be produced as well. For example, a first hybrid can be made by crossing variety C with variety D to produce a CxD hybrid, and a second hybrid can be made by crossing variety F with variety E to produce an ExF hybrid. The first and second hybrids can be further crossed to produce a higher order hybrid (CxD)x(ExF) that contains genetic information from all four parent varieties. In one aspect, the modified tobacco plants provided herein are hybrid tobacco plants. In another aspect, the modified tobacco seeds provided herein are hybrid tobacco seeds. In one aspect, the tobacco plants or varieties provided herein are hybrid tobacco plants or varieties. In another aspect, the modified tobacco plants provided herein are hybrid tobacco plants.

[0209] In one aspect, the present disclosure provides a method for creating a modified tobacco plant, 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 progeny tobacco seed, wherein at least one tobacco plant of the first tobacco variety contains a non-natural mutation in an endogenous nucleic acid sequence, 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: 13 to 18, and the non-natural mutation does not exist in the endogenous nucleic acid sequence in a control tobacco plant of the first tobacco variety; and (b) selecting at least one progeny tobacco seed or a plant germinated therefrom, wherein at least one tobacco seed or a plant germinated therefrom contains the non-natural mutation. In another aspect, the present disclosure provides a method for creating a modified tobacco plant, 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 progeny tobacco seed, wherein at least one tobacco plant of the first tobacco variety contains a non-natural mutation in an endogenous nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6, and the non-natural mutation does not exist in the endogenous nucleic acid sequence in a control tobacco plant of the first tobacco variety; and (b) selecting at least one progeny tobacco seed or a plant germinated therefrom, wherein at least one tobacco seed or a plant germinated therefrom contains the non-natural mutation.In another aspect, the present disclosure provides a method for producing a modified tobacco plant, 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 progeny tobacco seed, wherein at least one tobacco plant of the first tobacco variety comprises a non-natural mutation in an endogenous nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7-12, and the non-natural mutation does not exist in the endogenous nucleic acid sequence of a control tobacco plant of the first tobacco variety; and (b) selecting at least one progeny tobacco seed or a plant germinated therefrom, wherein 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, 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 progeny tobacco seed, wherein 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 NOs: 1-12, and the non-natural mutation does not exist in the endogenous nucleic acid sequence of a control tobacco plant of the first tobacco variety; and (b) selecting at least one progeny tobacco seed or a plant germinated therefrom, wherein at least one tobacco seed or a plant germinated therefrom comprises the non-natural mutation. In one aspect, in any of the foregoing methods, the first tobacco variety and the second tobacco variety are the same tobacco variety. In another aspect, in any of the foregoing methods, the first tobacco variety and the second tobacco variety are two different tobacco varieties.

[0210] In one aspect, the present disclosure provides a method for producing a modified tobacco plant, 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 progeny tobacco seed, wherein at least one tobacco plant of the first tobacco variety contains a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding at least one low RNA molecule capable of binding to at least one endogenous nucleic acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18 and reducing its expression, and the recombinant DNA construct is absent in a control tobacco plant of the first tobacco variety; and (b) selecting at least one progeny tobacco seed or a plant germinated therefrom, wherein at least one progeny tobacco seed or a plant germinated therefrom contains the recombinant DNA construct. In another aspect, the present disclosure provides a method for producing a modified tobacco plant, 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 progeny tobacco seed, wherein at least one tobacco plant of the first tobacco variety contains a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding at least one low RNA molecule capable of binding to at least one endogenous nucleic acid sequence that is at least 80% identical or similar to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6 and reducing its expression, and the recombinant DNA construct is absent in a control tobacco plant of the first tobacco variety; and (b) selecting at least one progeny tobacco seed or a plant germinated therefrom, wherein at least one progeny tobacco seed or a plant germinated therefrom contains the recombinant DNA construct.In another aspect, the present disclosure provides a method for creating a modified tobacco plant, 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 progeny tobacco seed, wherein the at least one tobacco plant of the first tobacco variety contains a recombinant DNA construct, the recombinant DNA construct is operably linked to a heterologous promoter encoding at least one low RNA molecule capable of binding to at least one endogenous nucleic acid sequence that is at least 80% identical or similar to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7-12 and reducing its expression, and the recombinant DNA construct is not present in a control tobacco plant of the first tobacco variety; and (b) selecting at least one progeny tobacco seed or a plant germinated therefrom, wherein the at least one progeny tobacco seed or a plant germinated therefrom contains the recombinant DNA construct. In one aspect, in any of the aforementioned methods, the first tobacco variety and the second tobacco variety are the same tobacco variety. In another aspect, in any of the aforementioned methods, the first tobacco variety and the second tobacco variety are two different tobacco varieties.

[0211] In one aspect, the present disclosure provides a method for creating a modified tobacco plant, 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 progeny tobacco seed, wherein at least one tobacco plant of the first tobacco variety contains a recombinant DNA construct, the recombinant DNA construct contains a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide having 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: 13 to 18, and the recombinant DNA construct is not present in a control tobacco plant of the first tobacco variety; and (b) selecting at least one progeny tobacco seed or a plant germinated therefrom, wherein at least one progeny tobacco seed or a plant germinated therefrom contains the recombinant DNA construct. In another aspect, the present disclosure provides a method for creating a modified tobacco plant, 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 progeny tobacco seed, wherein at least one tobacco plant of the first tobacco variety contains a recombinant DNA construct, the recombinant DNA construct contains 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: 1 to 6, and the recombinant DNA construct is not present in a control tobacco plant of the first tobacco variety; and (b) selecting at least one progeny tobacco seed or a plant germinated therefrom, wherein at least one progeny tobacco seed or a plant germinated therefrom contains the recombinant DNA construct.In another aspect, the present disclosure provides a method for producing a modified tobacco plant, 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 progeny tobacco seed, wherein at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, 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: 7-12, and the recombinant DNA construct is not present in a control tobacco plant of the first tobacco variety; and (b) selecting at least one progeny tobacco seed or a plant germinated therefrom, wherein at least one progeny tobacco seed or a plant germinated therefrom comprises the recombinant DNA construct. In one aspect, in any of the methods described above, the first tobacco variety and the second tobacco variety are the same tobacco variety. In another aspect, in any of the methods described above, the first tobacco variety and the second tobacco variety are two different tobacco varieties.

[0212] As used herein, the term "crossing" refers to the intentional cross-breeding of two plants. In one aspect, crossing comprises pollination and / or fertilization of a first tobacco plant by a second tobacco plant. The two tobacco plants to be crossed can be distantly related, closely related, or the same. In one aspect, both of the two tobacco plants to be crossed are modified tobacco plants. In one aspect, the two tobacco plants to be crossed are of the same tobacco variety. In one aspect, the two tobacco plants to be crossed are of two different tobacco varieties. In one aspect, one of the two tobacco plants to be crossed is male sterile. In one aspect, one of the two tobacco plants to be crossed is female sterile. In one aspect, at least one of the two tobacco plants to be crossed is a hybrid tobacco plant. In one aspect, at least one of the two tobacco plants to be crossed is a modified tobacco plant.

[0213] In one aspect, the tobacco plants or varieties provided herein are male sterile. In another aspect, the tobacco plants or varieties provided herein are cytoplasmic male sterile (CMS). In one aspect, the modified tobacco plants or varieties provided herein are male sterile. In another aspect, the modified tobacco plants or varieties provided herein are cytoplasmic male sterile (CMS). Male sterile tobacco plants can be produced by any method known in the art. Methods for producing male sterile tobacco are described in Wernsman, E.A., and Rufty, R.C. 1987. Chapter Seventeen. Tobacco. Pages 669 - 698 In: Cultivar Development. Crop Species. W.H. Fehr (ed.), MacMillan Publishing Go., Inc., New York, N.Y. 761 pp.

[0214] In another aspect, the tobacco plants or varieties provided herein are female sterile. In another aspect, the modified tobacco plants or varieties provided herein are female sterile. As a non - limiting example, female sterile plants can be produced by mutating the STIG1 gene. See, for example, Goldman et al. 1994, EMBO Journal 13:2976 - 2984. In one aspect, the modified tobacco plants provided herein are female sterile.

[0215] Unless otherwise specified, all comparisons to a control plant require similar or equivalent growth conditions for the two plants being compared. As used herein, "grown under equivalent conditions", "similar growth conditions", or "equivalent growth conditions" refer to similar environmental conditions and / or agronomic practices for growing between two or more plant genotypes to make a meaningful comparison, such that neither the environmental conditions nor the agronomic practices contribute to or explain any differences observed between two or more plant genotypes. Environmental conditions include, for example, light, temperature, water (humidity), and nutrients (e.g., nitrogen and phosphorus). Agronomic practices include, for example, seeding, pruning, undercutting, transplanting, deflowering, and suckering. See Chapters 4B and 4C of Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford (1999), pp 70-103. As used herein, a "control plant" refers to a plant having the same or substantially the same genetic constitution as the modified plant being compared, except for the non-natural mutations or recombinant DNA constructs provided herein that have been introduced into the modified plant.

[0216] In one aspect, the modified tobacco plants described herein are low alkaloid varieties or low alkaloid plants. As a non-limiting example, LA Burley 21 (LA BU21) is a low alkaloid variety of tobacco. LA BU21 is created by incorporating low alkaloid gene(s) from the Cuban cigar variety into Burley21 through several backcrosses. It has a total alkaloid (dry weight) of about 0.2%, compared to about 3.5% (dry weight) of its parent, Burley21. LA BU21 has a leaf grade well below commercially acceptable standards. LA BU21 is also characterized by other undesirable leaf phenotypes, such as lower yields, delayed maturation and senescence, higher susceptibility to insect damage, and poor quality of the final product after drying. The leaves of LA BU21 further exhibit traits such as higher polyamine content, higher chlorophyll content, and more mesophyll cells per unit leaf area. For further characterization of the leaf phenotype of LA BU21, see US2019 / 0271000.

[0217] In one aspect, the present disclosure provides a tobacco plant or a part thereof that can produce leaves containing one or more polyamines at levels equivalent to those of equivalent leaves of a control plant that does not contain the low nicotine or low alkaloid conferring mutation or transgene (e.g., a genetic modification in or targeting one or more ADCs, AOs, or ODCs). In certain aspects, the levels of one or more polyamines that are equivalent are within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the levels in equivalent leaves of a control plant that does not contain the same mutation or transgene. In one aspect, the levels of one or more polyamines that are equivalent are 0.5% - 1%, 1% - 2%, 2% - 3%, 3% - 4%, 4% - 5%, 5% - 6%, 6% - 7%, 7% - 8%, 8% - 9%, 9% - 10%, 11% - 12%, 12% - 13%, 13% - 14%, 14% - 15%, 15% - 16%, 16% - 17%, 17% - 18%, 18% - 19%, or 19% - 20% of the levels in equivalent leaves of a control plant that does not contain the same mutation or transgene. In a further aspect, the levels of one or more polyamines that are equivalent are 0.5% - 5%, 5% - 10%, or 10% - 20% of the levels in equivalent leaves of a control plant that does not contain the same mutation or transgene.

[0218] In one aspect, the present disclosure provides an ADC mutant or transgenic tobacco plant or a part thereof, an AO mutant or transgenic tobacco plant or a part thereof, or an ODC mutant or transgenic tobacco plant or a part thereof that can produce leaves containing equivalent chlorophyll levels relative to equivalent leaves of a control plant that does not contain the same mutation or transgene. In certain aspects, the equivalent chlorophyll levels are within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the levels in equivalent leaves of a control plant that does not contain the same mutation or transgene. In one aspect, the equivalent chlorophyll levels are 0.5% - 1%, 1% - 2%, 2% - 3%, 3% - 4%, 4% - 5%, 5% - 6%, 6% - 7%, 7% - 8%, 8% - 9%, 9% - 10%, 11% - 12%, 12% - 13%, 13% - 14%, 14% - 15%, 15% - 16%, 16% - 17%, 17% - 18%, 18% - 19%, or 19% - 20% of the levels in equivalent leaves of a control plant that does not contain the same mutation or transgene. In a further aspect, the equivalent chlorophyll levels are 0.5% - 5%, 5% - 10%, or 10% - 20% of the levels in equivalent leaves of a control plant that does not contain the same mutation or transgene.

[0219] In one aspect, the present disclosure provides an ADC mutant or transgenic tobacco plant or a part thereof, an AO mutant or transgenic tobacco plant or a part thereof, or an ODC mutant or transgenic tobacco plant or a part thereof that can produce leaves containing mesophyll cells per unit of leaf area equivalent to those of equivalent leaves of a control plant that do not contain the same mutation or transgene. In certain aspects, the mesophyll cells per unit of leaf area equivalent are within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in equivalent leaves of a control plant that do not contain the same mutation or transgene. In one aspect, the mesophyll cells per unit of leaf area equivalent are between 0.5% and 1%, 1% and 2%, 2% and 3%, 3% and 4%, 4% and 5%, 5% and 6%, 6% and 7%, 7% and 8%, 8% and 9%, 9% and 10%, 11% and 12%, 12% and 13%, 13% and 14%, 14% and 15%, 15% and 16%, 16% and 17%, 17% and 18%, 18% and 19%, or 19% and 20% of the level in equivalent leaves of a control plant that do not contain the same mutation or transgene. In a further aspect, the mesophyll cells per unit of leaf area equivalent are between 0.5% and 5%, 5% and 10%, or 10% and 20% of the level in equivalent leaves of a control plant that do not contain the same mutation or transgene.

[0220] In one aspect, the present disclosure provides an ADC mutant or transgenic tobacco plant or a part thereof, an AO mutant or transgenic tobacco plant or a part thereof, or an ODC mutant or transgenic tobacco plant or a part thereof that can produce leaves having an equivalent epidermal cell size relative to equivalent leaves of a control plant that does not contain the same mutation or transgene. In certain aspects, the equivalent epidermal cell size is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in equivalent leaves of a control plant that does not contain the same mutation or transgene. In one aspect, the equivalent epidermal cell size is 0.5% to 1%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 11% to 12%, 12% to 13%, 13% to 14%, 14% to 15%, 15% to 16%, 16% to 17%, 17% to 18%, 18% to 19%, or 19% to 20% of the level in equivalent leaves of a control plant that does not contain the same mutation or transgene. In a further aspect, the equivalent epidermal cell size is 0.5% to 5%, 5% to 10%, or 10% to 20% of the level in equivalent leaves of a control plant that does not contain the same mutation or transgene.

[0221] In one aspect, the present disclosure provides an ADC mutant or transgenic tobacco plant or a part thereof, an AO mutant or transgenic tobacco plant or a part thereof, or an ODC mutant or transgenic tobacco plant or a part thereof that can produce leaves containing an equivalent leaf yield relative to equivalent leaves of a control plant that does not contain the same mutation or transgene. In certain aspects, the equivalent leaf yield is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in equivalent leaves of a control plant that does not contain the same mutation or transgene. In one aspect, the equivalent leaf yield is 0.5% to 1%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 11% to 12%, 12% to 13%, 13% to 14%, 14% to 15%, 15% to 16%, 16% to 17%, 17% to 18%, 18% to 19%, or 19% to 20% of the level in equivalent leaves of a control plant that does not contain the same mutation or transgene. In a further aspect, the equivalent leaf yield is 0.5% to 5%, 5% to 10%, or 10% to 20% of the level in equivalent leaves of a control plant that does not contain the same mutation or transgene.

[0222] In one aspect, the present disclosure provides an ADC mutant or transgenic tobacco plant or a part thereof, an AO mutant or transgenic tobacco plant or a part thereof, or an ODC mutant or transgenic tobacco plant or a part thereof that exhibits equivalent insect pest susceptibility relative to equivalent leaves of a control plant that does not contain the same mutation or transgene. In certain aspects, the equivalent insect pest susceptibility is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in equivalent leaves of a control plant that does not contain the same mutation or transgene. In one aspect, the equivalent insect pest susceptibility is between 0.5% and 1%, 1% and 2%, 2% and 3%, 3% and 4%, 4% and 5%, 5% and 6%, 6% and 7%, 7% and 8%, 8% and 9%, 9% and 10%, 11% and 12%, 12% and 13%, 13% and 14%, 14% and 15%, 15% and 16%, 16% and 17%, 17% and 18%, 18% and 19%, or 19% and 20% of the level in equivalent leaves of a control plant that does not contain the same mutation or transgene. In a further aspect, the equivalent insect pest susceptibility is between 0.5% and 5%, 5% and 10%, or 10% and 20% of the level in equivalent leaves of a control plant that does not contain the same mutation or transgene.

[0223] Any genetic modification provided herein can be introduced into any ADC mutant or transgenic tobacco plant or a part thereof, an AO mutant or transgenic tobacco plant or a part thereof, or an ODC mutant or transgenic tobacco plant or a part thereof.

[0224] The level of insect pest susceptibility can be assayed, for example, in an insect feeding assay, by methods known in the art. Briefly, a 1 / 4-inch layer of 0.7% agar in water is added to a 100 mm Petri dish and allowed to solidify. Leaf disks are cut from the lids of the Petri dishes, placed on the plates, and gently pressed into the agar. The leaf disks are harvested from the plants at the 4-5 leaf stage. The disks are harvested from the leaf blade only to exclude the major midrib. A single disk is harvested from each of the four largest leaves of the plant, generating four replicates per plant. Four plants are sampled for a total of 16 biological replicate test lines. One caterpillar at the second instar stage (e.g., Heliothis sp., Helicoverpa sp.) is added to the leaf and allowed to feed at ambient temperature for 48 hours. After 48 hours, the caterpillar larvae are weighed and the final larval weight is recorded.

[0225] In one aspect, a tobacco plant or a part thereof comprises a first genomic modification that provides a lower level of nicotine or total alkaloids relative to a control tobacco plant (e.g., in or targeting one or more ADC, AO, or ODC genes), and a second genomic modification that provides one or more traits at equivalent levels selected from the group consisting of total leaf polyamine level, total root polyamine level, total leaf chlorophyll level, number of mesophyll cells per unit leaf area, and leaf epidermal cell size, wherein the control plant does not have both the first and second genomic modifications. In certain aspects, a tobacco plant or a part thereof comprises a first genomic modification that provides a lower level of nicotine or total alkaloids relative to a control tobacco plant (e.g., in or targeting one or more ADC, AO, or ODC genes), and a second genomic modification that provides an equivalent level of total leaf polyamine level, wherein the control plant does not have both the first and second genomic modifications. In one aspect, a tobacco plant or a part thereof comprises a first genomic modification that provides a lower level of nicotine or total alkaloids relative to a control tobacco plant (e.g., in or targeting one or more ADC, AO, or ODC genes), and a second genomic modification that provides an equivalent level of total root polyamine level, wherein the control plant does not have both the first and second genomic modifications. In certain aspects, a tobacco plant or a part thereof comprises a first genomic modification that provides a lower level of nicotine or total alkaloids relative to a control tobacco plant (e.g., in or targeting one or more ADC, AO, or ODC genes), and a second genomic modification that provides an equivalent level of total leaf chlorophyll level, wherein the control plant does not have both the first and second genomic modifications. In one aspect, a tobacco plant or a part thereof comprises a first genomic modification that provides a lower level of nicotine or total alkaloids relative to a control tobacco plant (e.g., in or targeting one or more ADC, AO, or ODC genes), and a second genomic modification that provides an equivalent level of number of mesophyll cells per unit leaf area, wherein the control plant does not have both the first and second genomic modifications.In one aspect, a tobacco plant or a part thereof comprises 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 relative to a control tobacco plant, and a second genomic modification that provides an equivalent level of leaf epidermal cell size, wherein the control plant does not have both the first and second genomic modifications. In one aspect, the second genomic modification is in or targets an ADC, AO, or ODC gene.

[0226] In one aspect, the first genomic modification, the second genomic modification, or both comprise a transgene, a mutation, or both. In certain aspects, the genomic modification, the second genomic modification, or both comprise a transgene. In one aspect, the first genomic modification, the second genomic modification, or both comprise a mutation. In certain aspects, the first genomic modification, the second genomic modification, or both are not transgene-based. In one aspect, the first genomic modification, the second genomic modification, or both are not mutation-based.

[0227] In one aspect, the tobacco plants provided herein contain a reduced amount of total conjugated polyamines in the leaves relative to control tobacco plants. In certain aspects, the tobacco plants provided herein contain a reduced amount of total conjugated polyamines in the roots relative to control tobacco plants. Conjugated polyamines as used herein include soluble conjugated polyamines such as phenolamides containing a backbone consisting of free polyamines (e.g., putrescine, spermine, and / or spermidine) conjugated to one or more phenylpropanoids, such as ferulic acid, caffeic acid, and coumaric acid, but are not limited thereto. Conjugated polyamines also include insoluble conjugated polyamines incorporated into structural polymers such as lignin, but are not limited thereto. In one aspect, the tobacco plants provided herein contain a reduced amount of total free polyamines (e.g., putrescine, spermine, and spermidine) in the leaves relative to control tobacco plants. In certain aspects, the tobacco plants provided herein contain a reduced amount of total conjugated polyamines in the roots relative to control tobacco plants. In one aspect, the tobacco plants provided herein contain a reduced amount of the total conjugated form of one or more polyamines selected from the group consisting of putrescine, spermidine, and spermine in the leaves relative to control tobacco plants. In certain aspects, the tobacco plants provided herein contain a reduced amount of the total conjugated form of one or more polyamines selected from the group consisting of putrescine, spermidine, and spermine in the roots relative to control tobacco plants. In one aspect, the tobacco plants provided herein contain a reduced amount of the total free form of one or more polyamines selected from the group consisting of putrescine, spermidine, and spermine in the leaves relative to control tobacco plants. In certain aspects, the tobacco plants provided herein contain a reduced amount of the total conjugated form of one or more polyamines selected from the group consisting of putrescine, spermidine, and spermine in the roots relative to control tobacco plants.

[0228] In one aspect, the characteristics or traits of the tobacco plants described herein are measured at a time selected from the group consisting of immediately before flowering, at flower removal, 1 week post flower removal (WPT), 2 WPT, 3 WPT, 4 WPT, 5 WPT, 6 WPT, 7 WPT, 8 WPT, and at harvest. In certain aspects, the tobacco plants provided herein that include the first and second genomic modifications are capable of producing leaves having a leaf grade equivalent to that of the leaves from the control plants. In one aspect, the tobacco plants provided herein that include the first and second genomic modifications have a total leaf yield equivalent to that of the control plants.

[0229] In certain aspects, the tobacco plants of the present disclosure include the nic1 mutation, the nic2 mutation, or both.

[0230] In one aspect, the modified tobacco plants provided herein further include a transgene or mutation that directly suppresses the expression or activity of one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, or seventeen or more genes or loci encoding proteins selected from the group consisting of agmatine deiminase (AIC), arginase, diamine oxidase, methylputrescine oxidase (MPO), NADH dehydrogenase, phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinolinate phosphoribosyltransferase (QPT), S-adenosyl-methionine synthase (SAMS), A622, NBB1, berberine bridge enzyme-like (BBL), MYC2, Nic1_ERF, Nic2_ERF, ethylene response factor (ERF) transcription factors, nicotine uptake permease (NUP), and MATE transporter. See Dewey and Xie, Molecular genetics of alkaloid biosynthesis in Nicotiana tabacum, Phytochemistry 94(2013)10-27.

[0231] In one aspect, the modified tobacco plants provided herein further comprise a mutation in the ERF gene (Nic2_ERF) at the Nic2 locus. In one aspect, the modified tobacco plants provided herein further comprise 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 mutations in one or more of the 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 certain aspects, the modified tobacco plants provided herein further comprise one or more mutations in ERF189, ERF115, or both. In one aspect, the modified tobacco plants provided herein further comprise one or more transgenes that target and suppress 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 encoding the proteins selected from the group consisting of ERF32, ERF34, ERF39, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168.

[0232] In one aspect, the modified tobacco plants provided herein further comprise a mutation in the ERF gene at the Nic1 locus (Nic1_ERF) (or the Nic1b locus as in WO / 2019 / 140297). See also WO / 2018 / 237107. In one aspect, the modified tobacco plants provided herein further comprise one or more mutations in two or more, three or more, four or more, five or more, six or more, or seven or more genes selected from the group consisting of ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210, and ERF91L2. See WO / 2019 / 140297, and Kajikawa et al., Plant physiol. 2017, 174:999-1011. In one aspect, the modified tobacco plants provided herein further comprise one or more mutations in 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. In one aspect, the modified tobacco plants provided herein further comprise one or more transgenes that target and suppress one or more genes encoding one or more, two or more, three or more, four or more, five or more, six or more, or seven or more genes selected from the group consisting of ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210, and ERF91L2.

[0233] 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), quinolinate phosphoribosyltransferase (QPT), and S-adenosyl-methionine synthase (SAMS), A622, NBB1, BBL, MYC2, Nic1_ERF, Nic2_ERF, ethylene response factor (ERF) transcription factor, nicotine uptake permease (NUP), and MATE transporter, and further comprises a second genetic modification targeting one or more amino acid sequences that are at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18. In certain aspects, the modified tobacco plant provided herein comprises a first genetic modification and comprises a transgene targeting and suppressing 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), quinolinate phosphoribosyltransferase (QPT), and S-adenosyl-methionine synthase (SAMS), A622, NBB1, BBL, MYC2, Nic1, Nic2, ethylene response factor (ERF) transcription factor, nicotine uptake permease (NUP), and MATE transporter, and further comprises a second genetic modification 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: 13-18.

[0234] Dried tobacco / tobacco products In one aspect, the present disclosure provides a method comprising preparing a tobacco product using dried tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a non-natural mutation in an endogenous nucleic acid sequence encoding a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18. In another aspect, the present disclosure provides a method comprising preparing a tobacco product using dried tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a non-natural mutation in an endogenous nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6. In another aspect, the present disclosure provides a method comprising preparing a tobacco product using dried tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a non-natural mutation in an endogenous nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7-12.

[0235] In one aspect, the present disclosure provides a method comprising preparing a tobacco product using a dried tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding at least one low RNA molecule capable of binding to at least one endogenous nucleic acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18 and reducing its expression. In another aspect, the present disclosure provides a method comprising preparing a tobacco product using a dried tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding at least one low RNA molecule capable of binding to at least one endogenous nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6 and reducing its expression. In another aspect, the present disclosure provides a method comprising preparing a tobacco product using a dried tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding at least one low RNA molecule capable of binding to at least one endogenous nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7 to 12 and reducing its expression.

[0236] In one aspect, the present disclosure provides a method comprising preparing a tobacco plant using a dried tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, and the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In another aspect, the present disclosure provides a method comprising preparing a tobacco plant using a dried tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, and 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: 1 to 6. In another aspect, the present disclosure provides a method comprising preparing a tobacco plant using a dried tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, and 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: 7 to 12.

[0237] "Drying" is an aging process that reduces moisture, causes the destruction of chlorophyll, turns the tobacco leaves golden, and converts starch into sugar. Thus, dried tobacco has a higher reducing sugar content and a lower starch content compared to the harvested green leaves. In certain embodiments, the tobacco plants or plant components provided herein can be dried using conventional means such as hot air drying, barn drying, fire-cured drying, air drying, or sun drying. For an explanation of different types of drying methods, see, for example, Chapter 1 in Tso (1999, Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). Dried tobacco is typically aged under compression conditions for several years (e.g., 2 - 5 years) in wooden drums (e.g., hogsheads) or cardboard boxes, with a moisture content in the range of 10% to about 25%. See U.S. Patent Nos. 4,516,590 and 5,372,149. The dried and aged tobacco can then be further processed. Further processing includes conditioning the tobacco under vacuum, with or without the introduction of steam at various temperatures, pasteurization, and fermentation.

[0238] Information regarding the harvest of burley and dark tobacco varieties can be found in the 2019 - 2020 Burley and Dark Tobacco Production Guide (December 2018), issued by the University of Kentucky, the University of Tennessee, Virginia Tech, and North Carolina State University, which is hereby incorporated by reference in its entirety.

[0239] In one aspect, the disclosure provides dried tobacco material from any tobacco plant or part thereof provided herein. In one aspect, the disclosure provides dried tobacco material from any modified tobacco plant or part thereof provided herein.

[0240] In one aspect, the dried tobacco material includes a tobacco material selected from the group consisting of dried leaf material, dried stem material, dried bud material, dried flower material, and dried root material. In another aspect, the dried tobacco material includes dried leaf material, dried stem material, or both. In a further aspect, the dried tobacco material includes dried leaf material. In yet another aspect, the dried tobacco material includes dried stem material.

[0241] In one aspect, the dried tobacco material includes a hot air dried tobacco material. In another aspect, the dried tobacco material includes an air dried tobacco material. In another aspect, the dried tobacco material includes a direct fire dried tobacco material. In another aspect, the dried tobacco material includes a sun dried tobacco material. In another aspect, the dried tobacco material provided herein is selected from the group consisting of an air dried tobacco material, a direct fire dried tobacco material, a sun dried tobacco material, and a hot air dried tobacco material. In another aspect, the dried tobacco material is from a tobacco variety selected from the group consisting of a hot air dried variety, a bright variety, a burley variety, a virginia variety, a maryland variety, a dark variety, an oriental variety, and a turkish variety.

[0242] In one aspect, the dried tobacco leaves provided herein are selected from the group consisting of air dried tobacco leaves, direct fire dried tobacco leaves, sun dried tobacco leaves, and hot air dried tobacco leaves. In one aspect, the dried tobacco leaves are from a tobacco variety selected from the group consisting of a hot air dried variety, a bright variety, a burley variety, a virginia variety, a maryland variety, a dark variety, an oriental variety, and a turkish variety.

[0243] Fermentation typically begins with a high moisture content, generates heat, and is characterized by a 10-20% reduction in dry weight. See, for example, U.S. Patent Nos. 4,528,993, 4,660,577, 4,848,373, 5,372,149, U.S. Publication No. 2005 / 0178398, and Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). The dried, aged, and fermented tobacco can be further processed (e.g., cut, shredded, expanded, or blended). See, for example, U.S. Patent Nos. 4,528,993, 4,660,577, and 4,987,907. In one aspect, the present disclosure provides fermented tobacco material from any tobacco plant or part thereof provided herein. In another aspect, the present disclosure provides fermented tobacco material from any modified tobacco plant or part thereof provided herein.

[0244] Tobacco products can be made using tobacco materials obtained from the tobacco strains, varieties, or hybrids of the present disclosure. As used herein, "tobacco product" is defined as any product manufactured from or derived from tobacco intended for human use or consumption. In one aspect, the present disclosure provides a tobacco product comprising plant material from a tobacco plant provided herein. In another aspect, the present disclosure provides a tobacco product comprising plant material from a modified tobacco plant provided herein. In another aspect, the present disclosure provides a tobacco product comprising dried tobacco material. In another aspect, the present disclosure provides a tobacco product comprising fermented tobacco material. In another aspect, the present disclosure provides a tobacco product comprising a tobacco blend.

[0245] Examples of tobacco products include, but are not limited to, smoked tobacco products (e.g., cigarettes and bidis), cigar products (e.g., cigar wrapping tobacco and cigarillos), pipe tobacco products, products derived from tobacco, nicotine products derived from tobacco, smokeless tobacco products (e.g., moist snuff, dry snuff, and chewing tobacco), films, chewables, tabs, formed parts, gels, consumable units, insoluble matrices, hollow forms, reconstituted tobacco, expanded tobacco, etc. See, e.g., U.S. Patent Publication No. 2006 / 0191548.

[0246] As used herein, "cigarette" refers to a tobacco product having a "rod" and a "filler". The "rod" of a cigarette includes the cigarette paper, filter, plug wrap (used to contain the filter material), tip paper that holds the cigarette paper (containing the filler) to the filter, and any adhesives that hold these components together. The "filler" includes, but is not limited to, (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 spices that may accompany the tobacco wrapped within the cigarette paper), (3) casing, (4) flavorants, and (5) all other additives (mixed with the tobacco and substitutes and wrapped within the cigarette).

[0247] In one aspect, the tobacco product includes reconstituted tobacco. In another aspect, the present disclosure provides reconstituted tobacco comprising a dry tobacco material. As used herein, "reconstituted tobacco" refers to a portion of tobacco filler made from tobacco dust and other tobacco scrap materials, processed into sheet form, and cut into strips to resemble tobacco. In addition to cost reduction, reconstituted tobacco is very important because it contributes to the flavor of cigarettes from flavor generation processes using the reaction between ammonia and sugar.

[0248] In one aspect, the tobacco product includes expanded tobacco. As used herein, "expanded tobacco" refers to a portion of tobacco filler that is processed through the expansion of a suitable gas such that the tobacco is "filled", resulting in a reduction in density and an increase in fill volume. Thereby, the weight of the tobacco used in a cigarette is reduced.

[0249] Also, the plant-derived tobacco products of the present disclosure include cigarettes and other smoking articles, particularly smoking articles that include filter elements, and the rod of smokable material includes dried tobacco within a tobacco blend. In one aspect, the tobacco products of the present disclosure are selected from the group consisting of cigars, non-ventilated recess filter cigarettes, vented recess filter cigarettes, cigars, snuff, pipe tobacco, cigarillos, cigarette tobacco, chewing tobacco, leaf tobacco, hookah tobacco, shredded tobacco, and cut tobacco. In another aspect, the tobacco products of the present disclosure are selected from the group consisting of cigarettes, heated tobacco products, kreteks, bidis, cigars, cigarillos, non-ventilated cigarettes, vented recess filter cigarettes, pipe tobacco, snuff, snus, chewing tobacco, moist smokeless tobacco, fine cut chewing tobacco, long cut chewing tobacco, pouched chewing tobacco products, gums, tablets, lozenges, and soluble strips.

[0250] In one aspect, the tobacco product is a heated tobacco product. As used herein, a "heated tobacco product" is a tobacco product that is heated to a lower temperature (e.g., about 600 °C) than conventional cigarettes. When heated to an appropriate temperature, a heated tobacco product produces an aerosol or smoke that can be inhaled. A heated tobacco product is also referred to as a "non-combustion heated" tobacco product. Heated tobacco products are often used with an electronic device that uses a battery to heat the heated tobacco product, or an electronic device that uses a smoldering ember of ignited carbon to heat the heated tobacco product. In one aspect, the heated tobacco product is a film. In one aspect, the heated tobacco product does not burn when heated.

[0251] In one aspect, the tobacco product includes a reconstituted tobacco film. In one aspect, the tobacco product includes a humectant. In one aspect, the humectant is glycerin.

[0252] 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, nasal snuff, dry snuff, and sniff tobacco.

[0253] Smokeless tobacco products do not burn and include, but are not limited to, chewing tobacco, moist smokeless tobacco, snus, and dry snuff. Chewing tobacco is coarsely shredded tobacco leaves, typically packaged in a large pouch-like package and used for plugs or twists. Moist smokeless tobacco is moist, finely shredded tobacco, provided in loose or pouch form, typically packaged in a round can, and used by adult tobacco consumers as a pinch or pouch between the cheek and gum. Snus is heat-treated smokeless tobacco. Dry snuff is finely ground tobacco for oral or nasal use.

[0254] In yet another aspect, the tobacco product of the present disclosure is selected from the group consisting of electronic heated tobacco, electronic tobacco, and electronic vaporizers.

[0255] In one aspect, the tobacco product of the present disclosure can be a blended tobacco product.

[0256] In another aspect, the present disclosure provides a tobacco blend comprising a dried tobacco material. The tobacco blend can comprise any combination of dried tobacco, undried tobacco, fermented tobacco, unfermented tobacco, expanded tobacco, and reconstituted tobacco.

[0257] In one aspect, the tobacco blend comprises at least 5 wt% dried tobacco. In one aspect, the tobacco blend comprises at least 10 wt% dried tobacco. In one aspect, the tobacco blend comprises at least 15 wt% dried tobacco. In one aspect, the tobacco blend comprises at least 20 wt% dried tobacco. In one aspect, the tobacco blend comprises at least 25 wt% dried tobacco. In one aspect, the tobacco blend comprises at least 30 wt% dried tobacco. In one aspect, the tobacco blend comprises at least 35 wt% dried tobacco. In one aspect, the tobacco blend comprises at least 40 wt% dried tobacco. In one aspect, the tobacco blend comprises at least 45 wt% dried tobacco. In one aspect, the tobacco blend comprises at least 50 wt% dried tobacco. In one aspect, the tobacco blend comprises at least 55 wt% dried tobacco. In one aspect, the tobacco blend comprises at least 60 wt% dried tobacco. In one aspect, the tobacco blend comprises at least 65 wt% dried tobacco. In one aspect, the tobacco blend comprises at least 70 wt% dried tobacco. In one aspect, the tobacco blend comprises at least 75 wt% dried tobacco. In one aspect, the tobacco blend comprises at least 80 wt% dried tobacco. In one aspect, the tobacco blend comprises at least 85 wt% dried tobacco. In one aspect, the tobacco blend comprises at least 90 wt% dried tobacco. In one aspect, the tobacco blend comprises at least 95 wt% dried tobacco.

[0258] In one aspect, the tobacco blend comprises at least 5% by volume of dried tobacco. In one aspect, the tobacco blend comprises at least 10% by volume of dried tobacco. In one aspect, the tobacco blend comprises at least 15% by volume of dried tobacco. In one aspect, the tobacco blend comprises at least 20% by volume of dried tobacco. In one aspect, the tobacco blend comprises at least 25% by volume of dried tobacco. In one aspect, the tobacco blend comprises at least 30% by volume of dried tobacco. In one aspect, the tobacco blend comprises at least 35% by volume of dried tobacco. In one aspect, the tobacco blend comprises at least 40% by volume of dried tobacco. In one aspect, the tobacco blend comprises at least 45% by volume of dried tobacco. In one aspect, the tobacco blend comprises at least 50% by volume of dried tobacco. In one aspect, the tobacco blend comprises at least 55% by volume of dried tobacco. In one aspect, the tobacco blend comprises at least 60% by volume of dried tobacco. In one aspect, the tobacco blend comprises at least 65% by volume of dried tobacco. In one aspect, the tobacco blend comprises at least 70% by volume of dried tobacco. In one aspect, the tobacco blend comprises at least 75% by volume of dried tobacco. In one aspect, the tobacco blend comprises at least 80% by volume of dried tobacco. In one aspect, the tobacco blend comprises at least 85% by volume of dried tobacco. In one aspect, the tobacco blend comprises at least 90% by volume of dried tobacco. In one aspect, the tobacco blend comprises at least 95% by volume of dried tobacco.

[0259] In one aspect, the present disclosure provides a method of making a tobacco product using materials from any of the tobacco plants provided herein. In one aspect, the present disclosure provides a method of making a tobacco product using dried tobacco materials from any of the tobacco plants provided herein. In one aspect, the present disclosure provides a method of making a tobacco product, comprising: (a) providing a tobacco material from a tobacco plant provided herein; (b) drying the tobacco material to produce a dried tobacco material; and (c) producing a tobacco product, wherein the tobacco product comprises the dried tobacco material from step (b). In one aspect, the present disclosure provides a method of making a tobacco product, comprising: (a) providing a dried tobacco material from a tobacco plant provided herein; and (b) making a tobacco product, wherein the tobacco product comprises the dried tobacco material.

[0260] Transformation In one aspect, the present disclosure provides a method for producing a modified tobacco plant, comprising: (a) inducing a non-natural mutation in at least one tobacco cell in an endogenous nucleic acid sequence encoding a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18; (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, comprising: (a) inducing a non-natural mutation in at least one tobacco cell in an endogenous nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6; (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, comprising: (a) inducing a non-natural mutation in at least one tobacco cell in an endogenous nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7-12; (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, comprising: (a) inducing a non-natural mutation in at least one tobacco cell in an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-12; (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 one aspect, 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 cross in step (e).

[0261] In one aspect, the present disclosure provides a method for producing a modified tobacco plant, 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 low RNA molecule capable of binding to at least one endogenous nucleic acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18 and reducing its expression; (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, 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 low RNA molecule capable of binding to at least one endogenous nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6 and reducing its expression; (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, 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 low RNA molecule capable of binding to at least one endogenous nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7 to 12 and reducing its expression; (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 one aspect, any of the methods described above further includes (d) growing the modified tobacco plant regenerated in step (c). In another aspect, any of the methods described above further includes (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).

[0262] In one aspect, the present disclosure provides a method for producing a modified tobacco plant, 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 that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18; (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, 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 that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6; (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, 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 that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7-12; (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 one aspect, any of the aforementioned methods further comprises: (d) growing the modified tobacco plant regenerated in step (c). In another aspect, any of the aforementioned 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 cross in step (e).

[0263] In one aspect, the present disclosure provides a method comprising transforming tobacco cells with a recombinant DNA construct, wherein the recombinant DNA construct is operably linked to a heterologous promoter that is linked to a nucleic acid sequence encoding at least one low RNA molecule capable of binding to and reducing the expression of at least one endogenous nucleic acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18. In another aspect, the present disclosure provides a method comprising transforming tobacco cells with a recombinant DNA construct, wherein the recombinant DNA construct is operably linked to a heterologous promoter that is linked to a nucleic acid sequence encoding at least one low RNA molecule capable of binding to and reducing the expression of at least one endogenous nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6. In another aspect, the present disclosure provides a method comprising transforming tobacco cells with a recombinant DNA construct, wherein the recombinant DNA construct is operably linked to a heterologous promoter that is linked to a nucleic acid sequence encoding at least one low RNA molecule capable of binding to and reducing the expression of at least one endogenous nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7-12. In another aspect, the aforementioned method further comprises regenerating a modified tobacco plant from the transformed tobacco cells.

[0264] 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 that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. 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 that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6. 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 that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7 to 12. In another aspect, the aforementioned method further comprises regenerating a modified tobacco plant from the transformed tobacco cell.

[0265] Numerous methods for introducing recombinant DNA constructs into plant cells are known in the art and can be used to create transgenic plant cells and plants according to the methods of the present application. Any suitable method or technique for the transformation of plant cells known in the art can be used according to the present method. Effective methods for the transformation of plants include bacterial-mediated transformation such as Agrobacterium-mediated or Rhizobium-mediated transformation and particle bombardment-mediated transformation. Various methods for the regeneration or development of transgenic plants are known in the art, such as transforming explants with a transformation vector via bacterial-mediated transformation or particle bombardment and then culturing those explants subsequently. For example, other methods for plant transformation such as microinjection, electroporation, vacuum infiltration, pressure, sonication, silicon carbide fiber agitation, polyethylene glycol (PEG)-mediated transformation are also known in the art. Transgenic plants produced by these transformation methods can be chimeric or non-chimeric for the transformation event, depending on the method and explant used.

[0266] Methods for transforming plant cells are well known to those skilled in the art. For example, specific instructions for transforming plant cells with particles coated with recombinant DNA by microprojectile bombardment (e.g., biolistic transformation) can be found in U.S. Patent 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. Patent Nos. 5,159,135, 5,824,877, 5,591,616, 6,384,301, 5,750,871, 5,463,174, and 5,188,958, all of which are incorporated herein by reference. Additional methods for transforming plants can be found, for example, in Compendium of Transgenic Crop Plants (2009) Blackwell Publishing. Tobacco cells can be transformed with any of the nucleic acid molecules provided herein using any suitable method known to those skilled in the art.

[0267] In one aspect, a method for introducing a nucleic acid molecule into a tobacco cell comprises Agrobacterium-mediated transformation. In another aspect, a method for introducing a nucleic acid molecule into a tobacco cell comprises PEG-mediated transformation. In another aspect, a method for introducing a nucleic acid molecule into a tobacco cell comprises microprojectile transformation. In another aspect, a method for introducing a nucleic acid molecule into a tobacco cell comprises liposome-mediated transformation (lipofection). In another aspect, a method for introducing a nucleic acid molecule into a tobacco cell comprises lentiviral transfection.

[0268] Lipofection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides are those of WO91 / 17424 and WO91 / 16024. Delivery can be to cells (in vitro or ex vivo administration) or target tissues (e.g., in vivo administration).

[0269] Any tobacco cell capable of regenerating fertile tobacco plants is contemplated as a useful recipient cell for the practice of the present disclosure. In one aspect, a recombinant DNA construct is introduced into a tobacco cell. In one aspect, the recombinant DNA construct is introduced into a protoplast cell of tobacco. In another aspect, the recombinant DNA construct is introduced into a callus cell of tobacco. In one aspect, the recombinant DNA construct is introduced into a tobacco cell selected from the group consisting of seed cells, fruit cells, leaf cells, cotyledon cells, hypocotyl cells, meristematic cells, embryo cells, endosperm cells, root cells, shoot cells, stem cells, flower cells, inflorescence cells, stalk cells, pedicel cells, style cells, stigma cells, flower bed cells, petal cells, sepal cells, pollen cells, anther cells, filament cells, ovary cells, ovule cells, pericarp cells, and sieve element cells.

[0270] Callus can be initiated from a variety of tissue sources including, but not limited to, immature embryos or parts of embryos, shoot apical meristems of seedlings, microspores, etc. Those cells capable of growing as callus can function as recipient cells for transformation. Practical transformation methods and materials for making the transgenic plants of the present disclosure (e.g., various media and recipient target cells, transformation of immature embryos, and subsequent regeneration of fertile transgenic plants) are disclosed, for example, in U.S. Patent Nos. 6,194,636 and 6,232,526, and U.S. Patent Application Publication No. 2004 / 0216189, all of which are incorporated herein by reference.

[0271] Leaf grade As used herein, "USDA leaf grade criteria" refers to the subdivision of leaf types by group, quality, and color. In one aspect, the USDA grade quality score is quantified as a numerical representation of a grade from 0 to 100 determined by a certified tobacco leaf grade examiner and is a weighted average of all stem positions. The higher the grade criteria, the higher the quality. As used below, "points" refers to each integer numerical representation of the USDA leaf grade score. For example, the difference between a USDA leaf grade criteria score of 90 and a score of 85 is 5 points.

[0272] Alternatively, the grade of the leaf can be determined via hyperspectral imaging. See, for example, WO2011 / 027315, published Mar. 10, 2011, which is incorporated by reference in its entirety.

[0273] As used herein, "certified tobacco leaf grade examiner" refers to a person trained to grade tobacco leaves according to the USDA official standard grades published by the United States Department of Agriculture (USDA), 7 CFR §29, in Agricultural Marketing Systems. The USDA leaf grade criteria score can be assigned by an employee, a former employee, or a person otherwise trained to grade tobacco leaves according to the USDA official standard grades. Exemplary steps of standard operations for commercial inspections begin with the grower shipping the tobacco to market, after which the tobacco is placed flat in crates as lots. Each lot is weighed and then inspected by a certified tobacco leaf grade examiner. After the inspection, the grade examiner assigns a grade to each lot, which becomes a certificate of grade indicating group, quality, and color. The steps for grading experimental lots are similar, however, experimental tobacco is not put on the market or otherwise used for commercial purposes.

[0274] The grades of tobacco are evaluated based on factors including, but not limited to, leaf position, leaf size, leaf color, leaf uniformity and completeness, maturity, texture, elasticity, luster (related to leaf strength, color depth, and sheen), hygroscopicity (the ability of tobacco leaves to absorb and retain ambient moisture), and green color or color covering. The leaf grades can be determined, for example, using official standard grades published by the Agricultural Marketing Service of the United States Department of Agriculture (7 U.S.C. § 511). For example, refer to the official standard grades for Burley Tobacco (U.S. Type 31 and Foreign Type 93) (55 F.R. 40645) effective November 5, 1990, the official standard grades for Flue-Cured Tobacco (U.S. Types 11, 12, 13, 14, and Foreign Type 92) (54 F.R. 7925) effective March 27, 1989, the official standard grades for Pennsylvania Seedleaf Tobacco (U.S. Type 41) (29 F.R. 16854) effective January 8, 1965, the official standard grades for Ohio Cigar-Leaf Tobacco (U.S. Types 42, 43, and 44) (28 F.R. 11719 and 28 F.R. 11926) effective December 8, 1963, the official standard grades for Wisconsin Cigar-Binder Tobacco (U.S. Types 54 and 55) (34 F.R. 17061) effective November 20, 1969, the official standard grades for Wisconsin Cigar-Binder Tobacco (U.S. Types 54 and 55) (34 F.R. 17061) effective November 20, 1969, and the official standard grades for Georgia and Florida Shade-Grown Cigar-Wrapper Tobacco (U.S. Type 62) effective April 1971. The USDA grade indicator values can be determined according to industry-recognized grade indicators.For example, see Bowman et al, Tobacco Science, 32:39-40 (1988), Legacy Tobacco Document Library (Bates Document#523267826-523267833, July 1, 1988, Memorandum on the Proposed Burley Tobacco Grade Index), and Miller et al., 1990, Tobacco Intern., 192:55-57 (all of the foregoing references are incorporated by reference in their entirety).

[0275] Unless otherwise specified, for a tobacco plant, variety, cultivar, or strain, the measured values of leaf grade index values, alkaloids, or nicotine levels referred to herein refer to average measured values, for example, the average of multiple leaves of a single plant or the average measured value from a population of tobacco plants from a single variety, cultivar, or strain. The population of tobacco plants or collection of tobacco leaves for determining the average measured value (e.g., leaf grading or alkaloid or nicotine levels) can be of any size, for example, 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, or more. A population of at least five or more tobacco plants is used to determine the standard deviation. The average measured value or grade index value is determined according to standard protocols recognized in the industry.

[0276] As used herein, "USDA graded leaf group", "leaf group", or "group" is a type of division that covers closely related grades based on certain characteristics related to stem position, body, or general quality. The group is the first factor of the USDA grade. The determination of the group is part of the grading procedure and is assigned by a certified tobacco leaf grade examiner.

[0277] In one aspect, a modified tobacco plant comprising a non-natural mutation comprises an equivalent or higher USDA leaf grade index as compared to a control tobacco plant lacking the non-natural mutation when grown under equivalent conditions. In another aspect, a modified tobacco plant comprising a recombinant DNA construct comprises an equivalent or higher USDA leaf grade index as compared to a control tobacco plant lacking the recombinant DNA construct when grown under equivalent conditions.

[0278] As used herein, "equivalent" USDA leaf grade index refers to within 15%. For example, if a control plant has a USDA leaf grade index of 100, an equivalent USDA leaf grade index is 85 - 100.

[0279] In one aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 1% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 5% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 10% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 20% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 30% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 40% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 50% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 75% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 100% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions.

[0280] In one aspect, the modified tobacco plant comprises a USDA leaf grade index that is 1% to 100% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises a USDA leaf grade index that is 1% to 75% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises a USDA leaf grade index that is 1% to 50% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises a USDA leaf grade index that is 1% to 40% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises a USDA leaf grade index that is 1% to 30% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises a USDA leaf grade index that is 1% to 20% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises a USDA leaf grade index that is 1% to 10% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises a USDA leaf grade index that is 10% to 75% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises a USDA leaf grade index that is 10% to 50% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises a USDA leaf grade index that is 1% to 30% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions.

[0281] In one aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 1 point higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 2 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 3 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 4 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 5 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 6 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 7 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 8 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 9 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 10 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 11 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 12 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions.In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 13 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 14 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 15 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 16 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 17 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 18 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 19 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 20 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 25 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 30 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is at least 35 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent 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 equivalent 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 the control tobacco plant when grown under equivalent conditions.

[0282] In one aspect, the modified tobacco plant comprises a USDA leaf grade index that is 1 to 100 points higher than the USDA leaf grade index of the control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is 1 to 75 points higher than the USDA leaf grade index of the control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is 1 to 50 points higher than the USDA leaf grade index of the control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is 1 to 25 points higher than the USDA leaf grade index of the control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is 1 to 10 points higher than the USDA leaf grade index of the control tobacco plant when grown under equivalent conditions. In another aspect, the modified tobacco plant comprises a USDA leaf grade index that is 1 to 5 points higher than the USDA leaf grade index of the control tobacco plant when grown under equivalent conditions. 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 the control tobacco plant when grown u...

Claims

1. A modified tobacco plant or part thereof containing at least one non-natural mutation in an endogenous nucleic acid sequence, The modified tobacco plant or a portion thereof, wherein the endogenous nucleic acid sequence encodes a polypeptide containing an amino acid sequence that is at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 17, and 18.

2. The modified tobacco plant comprises at least one non-natural mutation in a first endogenous nucleic acid sequence and at least one non-natural mutation in a second endogenous nucleic acid sequence. The first endogenous nucleic acid sequence codes for a polypeptide that contains an amino acid sequence that is at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 17, and 18. The second endogenous nucleic acid sequence codes for a polypeptide that contains an amino acid sequence that is at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 17, and 18. The first endogenous nucleic acid sequence and the second endogenous nucleic acid sequence are not identical to each other. The modified tobacco plant or part thereof as described in claim 1.

3. The modified tobacco plant or portion thereof according to claim 2, wherein the first endogenous nucleic acid sequence includes a sequence that is at least 90% identical to sequence number 1, and the second endogenous nucleic acid sequence includes a sequence that is at least 90% identical to sequence number 6.

4. At least one small RNA molecule capable of binding to at least one endogenous nucleic acid sequence that encodes a polypeptide at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 17, and 18, and reducing its expression. A modified tobacco plant or part thereof, comprising a recombinant DNA construct containing a heterologous promoter operably linked to a nucleic acid encoding a certain.

5. The at least one low RNA molecule can bind to a first endogenous nucleic acid sequence encoding a polypeptide that is at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 17, and 18, and to a second endogenous nucleic acid sequence encoding a polypeptide that is at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 17, and 18, thereby reducing their expression. The first endogenous nucleic acid sequence and the second endogenous nucleic acid sequence are not identical to each other. The modified tobacco plant or part thereof as described in claim 4.

6. The modified tobacco plant or portion thereof according to claim 5, wherein the first endogenous nucleic acid sequence includes a sequence that is at least 90% identical to sequence number 1, and the second endogenous nucleic acid sequence includes a sequence that is at least 90% identical to sequence number 6.

7. The modified tobacco plant or part thereof according to claim 4 or 5, wherein the nucleic acid molecule encoding the at least one low RNA molecule comprises a nucleic acid sequence that is at least 90% identical to sequence number 32 or 33.

8. The modified tobacco plant or part thereof according to claim 1, wherein the tobacco plant, when grown under equivalent conditions, produces at least one leaf containing at least one of the alkaloids in a reduced amount 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 sequence.

9. The modified tobacco plant or portion thereof according to claim 4, wherein the tobacco plant, when grown under equivalent conditions, produces at least one leaf containing at least one of the alkaloids in a reduced amount compared to the amount of alkaloids in a control tobacco plant lacking the recombinant DNA construct.

10. The modified tobacco plant or part thereof according to claim 1 or 4, wherein the endogenous nucleic acid sequence includes a nucleic acid sequence that is at least 90% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, and 6.

11. The modified tobacco plant or part thereof according to claim 1 or 4, wherein the endogenous nucleic acid sequence includes a nucleic acid sequence that is at least 95% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, and 6.

12. The modified tobacco plant or part thereof according to claim 1 or 4, wherein the endogenous nucleic acid sequence includes a nucleic acid sequence that is 100% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, and 6.

13. The modified tobacco plant or part thereof according to claim 1 or 4, wherein the endogenous nucleic acid sequence includes a nucleic acid sequence that is at least 90% identical or complementary to a sequence selected from the group consisting of sequence numbers 7, 8, 9, 10, 11, and 12.

14. The modified tobacco plant or part thereof according to claim 1 or 4, wherein the endogenous nucleic acid sequence includes a nucleic acid sequence that is at least 95% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, 11, and 12.

15. The modified tobacco plant or part thereof according to claim 1 or 4, wherein the endogenous nucleic acid sequence includes a nucleic acid sequence that is 100% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, 11, and 12.

16. The modified tobacco plant or part thereof according to claim 1 or 4, wherein the polypeptide is at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 17, and 18.

17. The modified tobacco plant or part thereof according to claim 1 or 4, wherein the polypeptide is at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 17, and 18.

18. The modified tobacco plant or part thereof according to claim 1 or 4, wherein the polypeptide is 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 17, and 18.

19. The modified tobacco plant or portion thereof according to claim 1, wherein the tobacco plant is homozygous for at least one non-natural mutation.

20. The modified tobacco plant or portion thereof according to claim 1, wherein the tobacco plant is heterozygous for at least one non-natural mutation.

21. The modified tobacco plant or part thereof according to claim 8 or 9, wherein the at least one alkaloid is selected from the group consisting of anabasine, anatabine, nicotine, and nornicotine.

22. The modified tobacco plant or part thereof according to claim 8 or 9, wherein the reduced amount of at least one alkaloid comprises a reduction of at least 1%.

23. The modified tobacco plant or part thereof according to claim 1, wherein the at least one non-natural mutation comprises a mutation selected from the group consisting of insertions, deletions, substitutions, duplications, and inversions.

24. The modified tobacco plant or part thereof according to claim 1, wherein the at least one non-natural mutation comprises at least one mutation selected from the group consisting of nonsense mutations, missense mutations, frameshift mutations, and splice site mutations.

25. The modified tobacco plant or part thereof according to claim 1, wherein the at least one non-natural mutation includes a null mutation.

26. The modified tobacco plant or part thereof according to claim 1, wherein the at least one non-natural mutation results in the cleavage of the polypeptide.

27. The modified tobacco plant or part thereof according to claim 1, wherein the at least one non-natural mutation comprises a mutation in a sequence region selected from the group consisting of promoters, 5'-untranslated regions (UTRs), exons, introns, 3'-UTRs, and terminators.

28. The at least one non-natural mutation results in a reduced level of expression of the nucleic acid sequence compared to the expression of the nucleic acid sequence in the same tissue of a control tobacco plant when grown under equivalent conditions. The nucleic acid sequence lacks the at least one non-natural mutation in the control tobacco plant. The modified tobacco plant or part thereof as described in claim 1.

29. The at least one non-natural mutation results in a reduced level of activity of the protein or polypeptide encoded by the nucleic acid sequence, compared to the activity of the protein or polypeptide encoded by the nucleic acid sequence in a control tobacco plant when grown under equivalent conditions. The nucleic acid sequence lacks the at least one non-natural mutation in the control tobacco plant. The modified tobacco plant or part thereof as described in claim 1.

30. The modified tobacco plant or part thereof according to claim 4, 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.

31. The modified tobacco plant or part thereof according to claim 30, wherein the tissue-preferred promoter includes a root-preferred promoter.

32. The modified tobacco plant or part thereof according to claim 30, wherein the tissue-specific promoter includes a root-specific promoter.

33. The modified tobacco plant or part thereof according to claim 30, wherein the constitutive promoter is selected from the group consisting of the cauliflower mosaic virus (CaMV) 35S promoter, the ubiquitin promoter, the actin promoter, the opin promoter, and the alcohol dehydrogenase promoter.

34. The modified tobacco plant or part thereof according to claim 4, wherein the at least one low RNA molecule is selected from the group consisting of double-stranded RNA, small interfering RNA (siRNA), trans-acting siRNA, and microRNA.

35. The modified tobacco plant or part thereof according to claim 4, wherein the at least one low RNA molecule comprises 18 to 30 nucleotides.

36. The modified tobacco plant or portion thereof according to claim 4, wherein the at least one low 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: 7, 8, 9, 10, 11, and 12.

37. The modified tobacco plant or a portion thereof according to claim 1 or 4, wherein the modified tobacco plant is a tobacco variety selected from the group consisting of flue-cured varieties, light-colored varieties, Burley varieties, Virginia varieties, Maryland varieties, dark-colored varieties, Galpao varieties, Oriental varieties, and Turkish varieties.

38. The modified tobacco plant or a portion thereof according to claim 1 or 4, wherein the modified tobacco plant is a variety selected from the group consisting of tobacco varieties listed in Tables 2, 3, 4, 5, 6, 7, and 8.

39. The modified tobacco plant or a portion thereof according to claim 1 or 4, wherein the modified tobacco plant is a hybrid.

40. The modified tobacco plant or a part thereof according to claim 1 or 4, wherein the modified tobacco plant is male-sterile or cytoplasmically male-sterile.

41. The modified tobacco plant or a portion thereof according to claim 1 or 4, wherein the modified tobacco plant is female sterile.

42. A dried tobacco material derived from a modified tobacco plant or a part thereof as described in claim 1 or 4.

43. The dried tobacco material according to claim 42, comprising dried leaf material, dried stem material, or both.

44. The dried tobacco material according to claim 43, comprising hot-air dried tobacco material, air-dried tobacco material, fire-cured tobacco material, and sun-cured tobacco material.

45. A tobacco blend comprising the dried tobacco material described in claim 42.

46. The tobacco blend according to claim 45, comprising at least 10% by weight of dry tobacco.

47. The tobacco blend according to claim 46, comprising at least 10% by volume of dry tobacco.

48. A tobacco product comprising the tobacco blend described in claim 45.

49. A tobacco product comprising the dried tobacco material described in claim 42.

50. A tobacco product according to claim 48, selected from the group consisting of cigarettes, heated tobacco products, kretek, bidi cigarettes, cigars, cigarillos, non-ventilated cigarettes, vented recess filter cigarettes, pipe tobacco, snuff, snus, chewing tobacco, moist smokeless tobacco, fine-cut chewing tobacco, long-cut chewing tobacco, pouched chewing tobacco products, gum, tablets, lozenges, and soluble strips.

51. The tobacco product according to claim 48, which is a smokeless tobacco product.

52. The tobacco product according to claim 51, 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 snuff.

53. Reconstituted tobacco comprising the dried tobacco material described in claim 42.

54. A method comprising preparing a tobacco product using dried tobacco material from a modified tobacco plant, The modified tobacco plant contains non-natural mutations in the endogenous nucleic acid sequence, The endogenous nucleic acid sequence encodes a polypeptide that contains an amino acid sequence that is at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 17, and 18. The aforementioned method.

55. A method comprising preparing a tobacco product using dried tobacco material from a modified tobacco plant, The modified tobacco plant contains a recombinant DNA construct, The recombinant DNA construct is At least one small RNA molecule capable of binding to at least one endogenous nucleic acid sequence that encodes a polypeptide at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 17, and 18, and reducing its expression. Includes a heterologous promoter operably ligated to a nucleic acid sequence encoding, The aforementioned method.

56. The method according to claim 54 or 55, wherein the dried tobacco material comprises dried leaf material, dried stem material, or both.

57. The method according to claim 54 or 55, wherein the dried tobacco material includes hot air dried tobacco material, air dried tobacco material, direct flame dried tobacco material, and sun-dried tobacco material.

58. The method according to claim 54 or 55, wherein the tobacco product is selected from the group consisting of cigarettes, kreteks, bidi cigarettes, cigars, cigarillos, non-ventilated cigarettes, ventilated recess filter cigarettes, pipe tobacco, snuff, snus, chewing tobacco, moist smokeless tobacco, fine-cut chewing tobacco, long-cut chewing tobacco, pouch chewing tobacco products, gum, tablets, lozenges, and soluble strips.

59. The method according to claim 54 or 55, wherein the tobacco product is a smokeless tobacco product.

60. The method according to claim 59, 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 snuff.

61. The method according to claim 54 or 55, wherein the dried tobacco material is a tobacco variety selected from the group consisting of hot-air dried varieties, light-colored varieties, Burley varieties, Virginia varieties, Maryland varieties, dark-colored varieties, Galpao varieties, Oriental varieties, and Turkish varieties.

62. The method according to claim 54 or 55, wherein the endogenous nucleic acid sequence includes a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

Images