Compositions and methods for producing tobacco plants and products with reduced or eliminated tobacco branches

Abstract

The present disclosure provides methods and compositions for controlling tobacco branch growth in tobacco by altering expression of different target genes.

Description

[0001] This application is a divisional application of an invention patent application with an application date of May 28, 2020, application number 202080048230.9, and invention name “Compositions and methods for producing tobacco plants and products with reduced or eliminated smoke branches”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 62 / 854,139, filed on May 29, 2019. The entirety of which is incorporated herein by reference. Technical Field

[0004] The present disclosure provides methods and compositions for improving the expression of nucleic acids and proteins useful for the reduction or elimination of tobacco shoots in plants.

[0005] Incorporation of Sequence Listing

[0006] This application contains a sequence listing submitted electronically in ASCII format and incorporated herein by reference in its entirety. The ASCII copy created on May 21, 2020 is named P34703WO00_SL.txt and is 380,869 bytes in size. Background Art

[0007] Tobacco is a plant species that shows particularly strong apical dominance. The molecular signal from the shoot apical meristem (SAM) mediates the hormone signal that effectively inhibits the growth of axillary buds. After removing the SAM (also referred to as "topping"), physiological and molecular changes occur, which enable new branches (or "tobacco forks") to grow from axillary meristems (buds). Tobacco forks growth causes the loss of yield and leaf quality. Tobacco forks are controlled by artificial removal and application of chemicals. Maleic hydrazide and fluoroformaldehyde are usually used for topping plants to inhibit axillary bud growth ("suckering"). However, the labor and chemical reagents for controlling tobacco forks are very expensive. Controlling the forking in tobacco by conventional breeding, mutation breeding and transgenic methods has become the main goal of decades, but so far, forking has not been successfully suppressed or eliminated by these methods. Recent molecular work has produced transgenic plants with tobacco forks reduced or eliminated, but the leaky expression of axillary bud degradation genes can cause the death of seeds and embryos and prevent the generation of continuous generations of transgenic plants. Therefore, the development of methods and compositions to prevent expression of axillary bud degradation genes in undesirable tissues and / or organs would result in reduced use of chemical agents and would reduce costs and labor associated with tobacco production. Brief description of the invention

[0009] In one aspect, the present disclosure provides a modified tobacco plant comprising a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein the mutation is not present in the endogenous gene of a control tobacco plant of the same variety, and wherein the modified tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping compared to the control tobacco plant when grown under comparable growth conditions.

[0010] In one aspect, the present disclosure provides a modified tobacco plant comprising a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein the mutation is not present in the endogenous gene of a control tobacco plant of the same variety.

[0011] In one aspect, the present disclosure provides a modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB and a WRKY transcription factor operably linked to a heterologous promoter, wherein the modified tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping compared to a control tobacco plant when grown under comparable growth conditions.

[0012] In one aspect, the present disclosure provides a modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor operably linked to a heterologous promoter.

[0013] In one aspect, the present disclosure provides a modified tobacco plant comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of reducing expression of an endogenous mRNA encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein the modified tobacco plant comprises no or reduced tobacco forks after topping compared to a control tobacco plant when grown under comparable growth conditions.

[0014] In one aspect, the present disclosure provides a modified tobacco plant comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of reducing the expression of an endogenous mRNA encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor.

[0015] In one aspect, the present disclosure provides a tobacco product comprising a cured tobacco material from a modified tobacco plant, the modified tobacco plant comprising a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein the mutation is not present in the endogenous gene of a control tobacco plant of the same variety, and wherein the modified tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping compared to the control tobacco plant when grown under comparable growth conditions.

[0016] In one aspect, the present disclosure provides a tobacco product comprising a cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein the mutation is not present in the endogenous gene of a control tobacco plant of the same variety.

[0017] In one aspect, the present disclosure provides a tobacco product comprising a cured tobacco material from a modified tobacco plant, the modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB and a WRKY transcription factor operably linked to a heterologous promoter, wherein the modified tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping compared to a control tobacco plant when grown under comparable growth conditions.

[0018] In one aspect, the present disclosure provides a tobacco product comprising a cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a polynucleotide sequence encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB and a WRKY transcription factor operably linked to a heterologous promoter.

[0019] In one aspect, the present disclosure provides a tobacco product comprising a cured tobacco material from a modified tobacco plant, the modified tobacco plant comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of reducing the expression of an endogenous mRNA encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein the modified tobacco plant comprises no or reduced tobacco forks after topping compared to a control tobacco plant when grown under comparable growth conditions.

[0020] In one aspect, the present disclosure provides a tobacco product comprising a cured tobacco material from a modified tobacco plant, the modified tobacco plant comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of reducing the expression of an endogenous mRNA encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor.

[0021] In one aspect, the present disclosure provides a method for producing a modified tobacco plant, comprising: (a) inducing a mutation in at least one tobacco cell at a genomic locus encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor; (b) selecting at least one tobacco cell comprising the mutation from step (a); and (c) regenerating a modified tobacco plant from the at least one tobacco cell selected in step (b), wherein when grown under comparable growth conditions, the modified tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping compared to a control tobacco plant lacking the mutation.

[0022] 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 a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor; (b) selecting at least one tobacco cell comprising the recombinant DNA construct; and (c) regenerating a modified tobacco plant from the at least one tobacco cell selected in step (b), wherein when grown under comparable growth conditions, the modified tobacco plant comprises no tobacco twigs or comprises reduced tobacco twigs after topping compared to a control tobacco plant lacking the recombinant DNA construct.

[0023] 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 small RNA molecule capable of reducing the expression of an endogenous mRNA encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor; (b) selecting at least one tobacco cell comprising the recombinant DNA construct; and (c) regenerating a modified tobacco plant from the at least one tobacco cell selected in step (b), wherein when grown under comparable growth conditions, the modified tobacco plant comprises no tobacco twigs or comprises reduced tobacco twigs after topping compared to a control tobacco plant lacking the recombinant DNA construct.

[0024] In one aspect, the present disclosure provides a method for preparing a tobacco product using cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein the mutation is not present in the endogenous gene of a control tobacco plant of the same variety, and wherein the modified tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping compared to the control tobacco plant when grown under comparable growth conditions.

[0025] In one aspect, the present disclosure provides a method for preparing a tobacco product using cured tobacco material from a modified tobacco plant, the modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB and a WRKY transcription factor operably linked to a heterologous promoter, and wherein the modified tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping compared to a control tobacco plant when grown under comparable growth conditions.

[0026] In one aspect, the present disclosure provides a method for preparing a tobacco product using cured tobacco material from a modified tobacco plant, the modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB and a WRKY transcription factor operably linked to a heterologous promoter, and wherein the modified tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping compared to a control tobacco plant when grown under comparable growth conditions.

[0027] In one aspect, the present disclosure provides a method for preparing a tobacco product using cured tobacco material from a modified tobacco plant, the modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB and a WRKY transcription factor operably linked to a heterologous promoter, wherein the modified tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping compared to a control tobacco plant when grown under comparable growth conditions.

[0028] In one aspect, the present disclosure provides a method for preparing a tobacco product using a cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a polynucleotide sequence encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB and a WRKY transcription factor operably linked to a heterologous promoter.

[0029] In one aspect, the present disclosure provides a method for preparing a tobacco product using cured tobacco material from a modified tobacco plant, the modified tobacco plant comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of reducing the expression of an endogenous mRNA encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein the modified tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping compared to a control tobacco plant when grown under comparable growth conditions.

[0030] In one aspect, the present disclosure provides a method for preparing a tobacco product using cured tobacco material from a modified tobacco plant, the modified tobacco plant comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of reducing the expression of an endogenous mRNA encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor.

[0031] In one aspect, the present disclosure provides a method comprising transforming a tobacco cell with a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor.

[0032] In one aspect, the present disclosure provides a method comprising transforming a tobacco cell with a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of reducing the expression of an endogenous mRNA encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor.

[0033] In one aspect, the present disclosure provides a method for producing a modified tobacco plant, comprising: (a) hybridizing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety, wherein the at least one tobacco plant of the first tobacco variety comprises a mutation in an endogenous gene encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein the mutation is not present in an endogenous gene of a control tobacco plant of the same variety, and wherein when grown under comparable growing conditions, the at least one tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping compared to the control tobacco plants; and (b) selecting offspring tobacco plants that comprise no tobacco forks or comprise reduced tobacco forks after topping compared to control tobacco plants of the same hybrid grown under comparable growing conditions.

[0034] In one aspect, the present disclosure provides a method for producing a modified tobacco plant, comprising: (a) hybridizing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein when grown under comparable growth conditions, the at least one tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping compared to a control tobacco plant; and (b) selecting an offspring tobacco plant that exhibits no tobacco forks or reduced tobacco forks after topping compared to a control tobacco plant of the same hybrid grown under comparable growth conditions.

[0035] In one aspect, the present disclosure provides a method for producing a modified tobacco plant, comprising: (a) hybridizing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of reducing expression of an endogenous mRNA encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein when grown under comparable growth conditions, the at least one tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping compared to a control tobacco plant when grown under comparable growth conditions; and (b) selecting an offspring tobacco plant that exhibits no tobacco forks or reduced tobacco forks after topping compared to a control tobacco plant of the same hybrid grown under comparable growth conditions.

[0036] Sequence Description

[0037] SEQ ID NOs: 1-44, 89-113 and 118-126 are nucleic acid sequences.

[0038] SEQ ID NOs: 45-88 are amino acid sequences of 114-117.

[0039] Further descriptions of the SEQ ID NOs provided herein can be found in Table 1 below.

[0040] Table 1. Description of sequences

[0041]

[0042]

[0043]

[0044] BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 is a Venn diagram depicting the overlap of differentially expressed genes in axillary buds between pairwise comparisons of water-treated and maleic hydrazide-treated tobacco plants 4, 24, and 72 hours after topping. MH4 refers to 4 hours after maleic hydrazide treatment; W4 refers to 4 hours after water treatment; MH24 refers to 24 hours after maleic hydrazide treatment; W24 refers to 24 hours after water treatment; MH72 refers to 72 hours after maleic hydrazide treatment; W72 refers to 72 hours after water treatment.

[0046] Figure 2 Depicted is the total mass of tobacco branches from a single P1_2.4::MYB plant one week after topping.

[0047] Figure 3 Depicted is the total mass of tobacco branches from a single P1_2.4::CDKI plant one week after topping.

[0048] Figure 4 Depicted is the total mass of tobacco forks from a single plant containing the amiRNA-1 construct one week after topping.

[0049] Figure 5 Depicted are the total mass of tobacco forks from individual plants containing the amiRNA-2 construct one week after topping.

[0050] Figure 6 Depicted is the total mass of tobacco forks from a single plant containing an amiRNA-3 construct one week after topping. DETAILED DESCRIPTION OF THE INVENTION

[0052] Unless otherwise defined, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art. Where a term is provided in the singular, the inventors also contemplate the aspects of the invention described by the plural form of the term. In the event of differences in terms and definitions used in references incorporated by reference, the terms used in this application shall have the definitions given herein. Other technical terms used have their ordinary meaning in the art in which they are used, as exemplified by various field-specific dictionaries, such as "The American Dictionary of Traditional Chinese Medicine," "The American Dictionary of Traditional Chinese Medicine," "The American Dictionary of Traditional Chinese Medicine," and "The American Dictionary of Traditional Chinese Medicine." Science Dictionary" (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), "McGraw-Hill Dictionary ofScientific 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).

[0053] Any references cited herein, including, for example, all patents, published patent applications, and non-patent publications, are hereby incorporated by reference in their entirety.

[0054] When a group of alternatives is presented, any and all combinations of the members that make up the group of alternatives are specifically contemplated. For example, if an item is selected from the group consisting of A, B, C, and D, the inventors expressly contemplate each individual alternative (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 a list of two or more items refers to any one of the listed items 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 one or both of A and B - i.e., A alone, B alone, or a combination of A and B. The expression "A, B, and / or C" is intended to mean A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0055] When numerical ranges are provided herein, the ranges should be understood to include the boundaries of the range and any numerical values ​​between the defined boundaries of the range. For example, "1-10" includes any number between 1 and 10, as well as the number 1 and the number 10.

[0056] As used herein, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0057] In one aspect, the modified tobacco plants provided herein comprise reduced or no tobacco forks after topping compared to control tobacco plants when grown under comparable growing conditions.

[0058] As used herein, "branching" refers to the development and / or growth of an axillary (or lateral) bud ("bush") from an axillary meristem that grows between a leaf and a stem. An axillary bud is a plumule that contains an axillary meristem, surrounding leaf tissue, and surrounding stem tissue. See, e.g., U.S. Patent Application Publication Nos. 2016 / 0281100; and 2017 / 0260535, which are incorporated herein by reference in their entirety.

[0059] As used herein, "topping" refers to the removal of the stem tip when the plant is close to maturity, including the apical meristem, flowers, and up to several adjacent leaves. Topping tobacco plants causes the loss of apical dominance. Before topping, the hormonal signal sent by the apical meristem makes the branching dormant to a great extent; topping removes the hormonal signal and allows the tobacco branch to grow ("topping-induced branching"). If the branching is fully controlled, topping increases yield, increases the value per acre, and produces the ideal improvement of the physical and chemical properties of tobacco leaves.

[0060] As used herein, "comparable growth conditions" refers to similar environmental conditions and / or agronomic practices used to grow two or more plant genotypes and to make meaningful comparisons between them, so that neither environmental conditions nor 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 nutrition (e.g., nitrogen and phosphorus). Agronomic practices include, for example, sowing, pruning, undercutting, transplanting, topping, and bifurcation. See Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford (1999), Chapters 4B and 4C of pp.70-103. It is understood in the art that "comparable growth conditions" do not require identical growth conditions.

[0061] As used herein, "modified" refers to plants, seeds, plant parts, plant cells and plant genomes that have been subjected to mutagenesis, genome editing, genetic transformation or a combination thereof.

[0062] In one aspect, the present disclosure provides a modified tobacco plant that does not contain a tobacco fork or contains a reduced tobacco fork after topping compared to a control tobacco plant when grown under comparable conditions. As used herein, a "reduction" in the number of tobacco forks, tobacco fork size, and / or the effect of tobacco forks on agronomic performance refers to a statistically significant reduction. As used herein, "statistically significant" refers to when an appropriate measure of statistical significance (e.g., a one-tailed two-sample t-test) is used, when the p-value is less than 0.05.

[0063] In one aspect, when grown under comparable conditions, the modified plants or methods provided herein require reduced management to control branching compared to control plants. As used herein, "management" refers to artificial removal of tobacco branches, application of chemicals (e.g., maleic hydrazide, flumetramid) to inhibit or remove tobacco branches, or both. In one aspect, when grown under comparable conditions, the modified plants or methods provided herein require reduced frequency of artificial tobacco branch removal, reduced frequency of chemical application, reduced amount of chemical application, or a combination thereof compared to control plants. See, for example, Fisher et al. "Topping, Managing Suckers, and Using Ethephon," pages 96-117 In: 2016 Flue-Cured Tobacco Information, North Carolin State University, which is incorporated herein by reference in its entirety.

[0064] In one aspect, the modified plants or methods provided herein require a reduced frequency of maleic hydrazide application to reduce or eliminate smoke branches. In one aspect, the modified plants or methods provided herein require a reduced amount of maleic hydrazide application to reduce or eliminate smoke branches. In one aspect, the modified plants or methods provided herein require a reduced frequency of flumetralin application to reduce or eliminate smoke branches. In one aspect, the modified plants or methods provided herein require a reduced amount of flumetralin application to reduce or eliminate smoke branches.

[0065] In one aspect, when grown under comparable conditions, the modified plant or method provided herein requires 1% of the control plant's need to manually remove the tobacco fork. In one aspect, when grown under comparable conditions, the modified plant or method provided herein requires 5% of the control plant's need to manually remove the tobacco fork. In one aspect, when grown under comparable conditions, the modified plant or method provided herein requires 10% of the control plant's need to manually remove the tobacco fork. In one aspect, when grown under comparable conditions, the modified plant or method provided herein requires 20% of the control plant's need to manually remove the tobacco fork. In one aspect, when grown under comparable conditions, the modified plant or method provided herein requires 30% of the control plant's need to manually remove the tobacco fork. In one aspect, when grown under comparable conditions, the modified plant or method provided herein requires 40% of the control plant's need to manually remove the tobacco fork. In one aspect, when grown under comparable conditions, the modified plant or method provided herein requires 50% of the control plant's need to manually remove the tobacco fork. In one aspect, the modified plants or methods provided herein require manual removal of cigarette forks at a frequency of 60% of that of control plants when grown under comparable conditions. In one aspect, the modified plants or methods provided herein require manual removal of cigarette forks at a frequency of 70% of that of control plants when grown under comparable conditions. In one aspect, the modified plants or methods provided herein require manual removal of cigarette forks at a frequency of 80% of that of control plants when grown under comparable conditions. In one aspect, the modified plants or methods provided herein require manual removal of cigarette forks at a frequency of 90% of that of control plants when grown under comparable conditions. In one aspect, the modified plants or methods provided herein require manual removal of cigarette forks at a frequency of 95% of that of control plants when grown under comparable conditions.

[0066] In one aspect, when grown under comparable conditions, the modified plants or methods provided herein require less than 1% of the control plants to manually remove the tobacco fork. In one aspect, when grown under comparable conditions, the modified plants or methods provided herein require less than 5% of the control plants to manually remove the tobacco fork. In one aspect, when grown under comparable conditions, the modified plants or methods provided herein require less than 10% of the control plants to manually remove the tobacco fork. In one aspect, when grown under comparable conditions, the modified plants or methods provided herein require less than 20% of the control plants to manually remove the tobacco fork. In one aspect, when grown under comparable conditions, the modified plants or methods provided herein require less than 30% of the control plants to manually remove the tobacco fork. In one aspect, when grown under comparable conditions, the modified plants or methods provided herein require less than 40% of the control plants to manually remove the tobacco fork. In one aspect, when grown under comparable conditions, the modified plants or methods provided herein require less than 50% of the control plants to manually remove the tobacco fork. In one aspect, the modified plants or methods provided herein require manual removal of cigarette forks less than 60% of the control plants when grown under comparable conditions. In one aspect, the modified plants or methods provided herein require manual removal of cigarette forks less than 70% of the control plants when grown under comparable conditions. In one aspect, the modified plants or methods provided herein require manual removal of cigarette forks less than 80% of the control plants when grown under comparable conditions. In one aspect, the modified plants or methods provided herein require manual removal of cigarette forks less than 90% of the control plants when grown under comparable conditions. In one aspect, the modified plants or methods provided herein require manual removal of cigarette forks less than 95% of the control plants when grown under comparable conditions.

[0067] In one aspect, when grown under comparable conditions, the modified plants provided herein require manual removal of tobacco branches at a frequency of 10%-95% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require manual removal of tobacco branches at a frequency of 20%-95% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require manual removal of tobacco branches at a frequency of 30%-95% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require manual removal of tobacco branches at a frequency of 40%-95% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require manual removal of tobacco branches at a frequency of 50%-95% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require manual removal of tobacco branches at a frequency of 60%-95% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require manual removal of tobacco branches at a frequency of 70%-95% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require 80%-95% of the control plants to manually remove the tobacco fork. In one aspect, when grown under comparable conditions, the modified plants provided herein require 90%-95% of the control plants to manually remove the tobacco fork. In one aspect, when grown under comparable conditions, the modified plants provided herein require 10%-75% of the control plants to manually remove the tobacco fork. In one aspect, when grown under comparable conditions, the modified plants provided herein require 10%-50% of the control plants to manually remove the tobacco fork. In one aspect, when grown under comparable conditions, the modified plants provided herein require 10%-25% of the control plants to manually remove the tobacco fork. In one aspect, when grown under comparable conditions, the modified plants provided herein require 50%-75% of the control plants to manually remove the tobacco fork. In one aspect, when grown under comparable conditions, the modified plants provided herein require 25%-75% of the control plants to manually remove the tobacco fork. In one aspect, the modified plants provided herein require manual removal of tobacco branches at a frequency of 25%-50% of that of control plants when grown under comparable conditions. In one aspect, the modified plants provided herein require manual removal of tobacco branches at a frequency of 1%-50% of that of control plants when grown under comparable conditions. In one aspect, the modified plants provided herein require manual removal of tobacco branches at a frequency of 1%-25% of that of control plants when grown under comparable conditions. In one aspect, the modified plants provided herein require manual removal of tobacco branches at a frequency of 1%-10% of that of control plants when grown under comparable conditions.

[0068] In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control the frequency of tobacco forks as 1% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control the frequency of tobacco forks as 5% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control the frequency of tobacco forks as 10% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control the frequency of tobacco forks as 20% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control the frequency of tobacco forks as 30% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control the frequency of tobacco forks as 40% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control the frequency of tobacco forks as 50% of the control plants. In one aspect, the modified plants provided herein require chemical applications to control tobacco branches at a frequency of 60% of that of control plants when grown under comparable conditions. In one aspect, the modified plants provided herein require chemical applications to control tobacco branches at a frequency of 70% of that of control plants when grown under comparable conditions. In one aspect, the modified plants provided herein require chemical applications to control tobacco branches at a frequency of 80% of that of control plants when grown under comparable conditions. In one aspect, the modified plants provided herein require chemical applications to control tobacco branches at a frequency of 90% of that of control plants when grown under comparable conditions. In one aspect, the modified plants provided herein require chemical applications to control tobacco branches at a frequency of 95% of that of control plants when grown under comparable conditions.

[0069] In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control the frequency of tobacco forks less than 1% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control the frequency of tobacco forks less than 5% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control the frequency of tobacco forks less than 10% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control the frequency of tobacco forks less than 20% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control the frequency of tobacco forks less than 30% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control the frequency of tobacco forks less than 40% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control the frequency of tobacco forks less than 50% of the control plants. In one aspect, the modified plants provided herein require chemical applications to control tobacco branches at a frequency less than 60% of that of control plants when grown under comparable conditions. In one aspect, the modified plants provided herein require chemical applications to control tobacco branches at a frequency less than 70% of that of control plants when grown under comparable conditions. In one aspect, the modified plants provided herein require chemical applications to control tobacco branches at a frequency less than 80% of that of control plants when grown under comparable conditions. In one aspect, the modified plants provided herein require chemical applications to control tobacco branches at a frequency less than 90% of that of control plants when grown under comparable conditions. In one aspect, the modified plants provided herein require chemical applications to control tobacco branches at a frequency less than 95% of that of control plants when grown under comparable conditions.

[0070] In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control tobacco branches at a frequency of 10%-95% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control tobacco branches at a frequency of 20%-95% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control tobacco branches at a frequency of 30%-95% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control tobacco branches at a frequency of 40%-95% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control tobacco branches at a frequency of 50%-95% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control tobacco branches at a frequency of 60%-95% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control tobacco branches at a frequency of 70%-95% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control tobacco branches at a frequency of 80%-95% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control tobacco branches at a frequency of 90%-95% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control tobacco branches at a frequency of 10%-75% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control tobacco branches at a frequency of 10%-50% of the control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require chemical applications to control tobacco branches at a frequency of 10%-25% of the control plants. In one aspect, the modified plants provided herein require chemical applications to control tobacco branches at a frequency of 50%-75% of that of control plants when grown under comparable conditions. In one aspect, the modified plants provided herein require chemical applications to control tobacco branches at a frequency of 25%-50% of that of control plants when grown under comparable conditions. In one aspect, the modified plants provided herein require chemical applications to control tobacco branches at a frequency of 25%-75% of that of control plants when grown under comparable conditions. In one aspect, the modified plants provided herein require chemical applications to control tobacco branches at a frequency of 1%-50% of that of control plants when grown under comparable conditions. In one aspect, the modified plants provided herein require chemical applications to control tobacco branches at a frequency of 1%-25% of that of control plants when grown under comparable conditions.In one aspect, the modified plants provided herein require chemical applications to control tobacco shoots at a frequency of 1% to 10% of that of control plants when grown under comparable conditions.

[0071] In one aspect, when grown under comparable conditions, the modified plants provided herein require a chemical spray volume of 1% of the volume of the control plant tobacco forks. In one aspect, when grown under comparable conditions, the modified plants provided herein require a chemical spray volume of 5% of the volume of the control plant tobacco forks. In one aspect, when grown under comparable conditions, the modified plants provided herein require a chemical spray volume of 10% of the volume of the control plant tobacco forks. In one aspect, when grown under comparable conditions, the modified plants provided herein require a chemical spray volume of 20% of the volume of the control plant tobacco forks. In one aspect, when grown under comparable conditions, the modified plants provided herein require a chemical spray volume of 30% of the volume of the control plant tobacco forks. In one aspect, when grown under comparable conditions, the modified plants provided herein require a chemical spray volume of 40% of the volume of the control plant tobacco forks. In one aspect, when grown under comparable conditions, the modified plants provided herein require a chemical spray volume of 50% of the volume of the control plant tobacco forks. In one aspect, when grown under comparable conditions, the modified plants provided herein require a chemical spray volume of 60% of the volume of the control plant tobacco forks. In one aspect, when grown under comparable conditions, the modified plants provided herein require a chemical spray volume of 70% of the volume of the control plant tobacco forks. In one aspect, when grown under comparable conditions, the modified plants provided herein require a chemical spray volume of 80% of the volume of the control plant tobacco forks. In one aspect, when grown under comparable conditions, the modified plants provided herein require a chemical spray volume of 90% of the volume of the control plant tobacco forks. In one aspect, when grown under comparable conditions, the modified plants provided herein require a chemical spray volume of 95% of the volume of the control plant tobacco forks.

[0072] In one aspect, when grown under comparable conditions, the modified plants provided herein require a chemical spray volume less than 1% of the volume of the control plant tobacco forks. In one aspect, when grown under comparable conditions, the modified plants provided herein require a chemical spray volume less than 5% of the volume of the control plant tobacco forks. In one aspect, when grown under comparable conditions, the modified plants provided herein require a chemical spray volume less than 10% of the volume of the control plant tobacco forks. In one aspect, when grown under comparable conditions, the modified plants provided herein require a chemical spray volume less than 20% of the volume of the control plant tobacco forks. In one aspect, when grown under comparable conditions, the modified plants provided herein require a chemical spray volume less than 30% of the volume of the control plant tobacco forks. In one aspect, when grown under comparable conditions, the modified plants provided herein require a chemical spray volume less than 40% of the volume of the control plant tobacco forks. In one aspect, when grown under comparable conditions, the modified plants provided herein require a chemical spray volume less than 50% of the volume of the control plant tobacco forks. In one aspect, when grown under comparable conditions, the modified plants provided herein require a chemical spray volume less than 60% of the volume of the control plant tobacco forks. In one aspect, when grown under comparable conditions, the modified plants provided herein require a chemical spray volume less than 70% of the volume of the control plant tobacco forks. In one aspect, when grown under comparable conditions, the modified plants provided herein require a chemical spray volume less than 80% of the volume of the control plant tobacco forks. In one aspect, when grown under comparable conditions, the modified plants provided herein require a chemical spray volume less than 90% of the volume of the control plant tobacco forks. In one aspect, when grown under comparable conditions, the modified plants provided herein require a chemical spray volume less than 95% of the volume of the control plant tobacco forks.

[0073] In one aspect, when grown under comparable conditions, the modified plants provided herein require 10%-95% less chemical spray volume than control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require 20%-95% less chemical spray volume than control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require 30%-95% less chemical spray volume than control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require 40%-95% less chemical spray volume than control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require 50%-95% less chemical spray volume than control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require 60%-95% less chemical spray volume than control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require 70%-95% less chemical spray volume than control plants. In one aspect, when grown under comparable conditions, the modified plant provided herein requires 80%-95% less chemical spray volume than control plants. In one aspect, when grown under comparable conditions, the modified plant provided herein requires 90%-95% less chemical spray volume than control plants. In one aspect, when grown under comparable conditions, the modified plant provided herein requires 10%-75% less chemical spray volume than control plants. In one aspect, when grown under comparable conditions, the modified plant provided herein requires 10%-50% less chemical spray volume than control plants. In one aspect, when grown under comparable conditions, the modified plant provided herein requires 10%-25% less chemical spray volume than control plants. In one aspect, when grown under comparable conditions, the modified plant provided herein requires 25%-50% less chemical spray volume than control plants. In one aspect, when grown under comparable conditions, the modified plant provided herein requires 25%-75% less chemical spray volume than control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require 50%-75% less chemical spray volume than control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require 1%-50% less chemical spray volume than control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require 1%-25% less chemical spray volume than control plants. In one aspect, when grown under comparable conditions, the modified plants provided herein require 1%-10% less chemical spray volume than control plants.

[0074] In one aspect, when grown under comparable conditions, the reduced tobacco forks comprise fewer total tobacco forks, smaller average tobacco fork sizes, or both, compared to control tobacco plants. In another aspect, when grown under comparable growth conditions, the smaller average tobacco fork size comprises a measure selected from a reduced average mass, a reduced average length, a reduced average diameter, or any combination thereof, compared to the tobacco forks of control tobacco plants. In another aspect, the smaller average tobacco fork size comprises a reduced average mass of tobacco forks. In another aspect, the smaller average tobacco fork size comprises a reduced average length of tobacco forks. In a further aspect, the smaller average tobacco fork size comprises a reduced average diameter of tobacco forks.

[0075] In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified tobacco plants comprise less total tobacco forks. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified tobacco plants comprise less average tobacco fork size. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified tobacco plants comprise shorter average tobacco fork length. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified tobacco plants comprise lower average tobacco fork quality. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified tobacco plants comprise shorter average tobacco fork diameter.

[0076] In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified tobacco plants comprise at least 1 tobacco fork less. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified tobacco plants comprise at least 2 tobacco forks less. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified tobacco plants comprise at least 3 tobacco forks less. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified tobacco plants comprise at least 4 tobacco forks less. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified tobacco plants comprise at least 5 tobacco forks less. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified tobacco plants comprise at least 7 tobacco forks less. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified tobacco plants comprise at least 10 tobacco forks less. In one aspect, when grown under comparable conditions, compared to a control tobacco plant, the modified tobacco plant comprises at least 15 tobacco forks few. In one aspect, when grown under comparable conditions, compared to a control tobacco plant, the modified tobacco plant comprises at least 20 tobacco forks few. In one aspect, when grown under comparable conditions, compared to a control tobacco plant, the modified tobacco plant comprises at least 25 tobacco forks few. In one aspect, when grown under comparable conditions, compared to a control tobacco plant, the modified tobacco plant comprises at least 30 tobacco forks few.

[0077] In one aspect, when grown under comparable conditions, compared with a control tobacco plant, the modified tobacco plant comprises at least 5% less tobacco forks. In one aspect, when grown under comparable conditions, compared with a control tobacco plant, the modified tobacco plant comprises at least 10% less tobacco forks. In one aspect, when grown under comparable conditions, compared with a control tobacco plant, the modified tobacco plant comprises at least 15% less tobacco forks. In one aspect, when grown under comparable conditions, compared with a control tobacco plant, the modified tobacco plant comprises at least 20% less tobacco forks. In one aspect, when grown under comparable conditions, compared with a control tobacco plant, the modified tobacco plant comprises at least 25% less tobacco forks. In one aspect, when grown under comparable conditions, compared with a control tobacco plant, the modified tobacco plant comprises at least 30% less tobacco forks. In one aspect, when grown under comparable conditions, compared with a control tobacco plant, the modified tobacco plant comprises at least 40% less tobacco forks. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, the modified tobacco plant comprises at least 50% less tobacco fork. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, the modified tobacco plant comprises at least 60% less tobacco fork. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, the modified tobacco plant comprises at least 70% less tobacco fork. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, the modified tobacco plant comprises at least 80% less tobacco fork. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, the modified tobacco plant comprises at least 90% less tobacco fork. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, the modified tobacco plant comprises at least 95% less tobacco fork.

[0078] In one aspect, fresh tobacco stick weight is used to measure average tobacco stick quality. In another aspect, dry tobacco stick weight is used to measure average tobacco stick quality.

[0079] In one aspect, when growing under comparable conditions, compared with the average cigarette fork quality of control tobacco plants, the average cigarette fork quality of modified tobacco plants is low by at least 5%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork quality of control tobacco plants, the average cigarette fork quality of modified tobacco plants is low by at least 10%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork quality of control tobacco plants, the average cigarette fork quality of modified tobacco plants is low by at least 15%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork quality of control tobacco plants, the average cigarette fork quality of modified tobacco plants is low by at least 20%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork quality of control tobacco plants, the average cigarette fork quality of modified tobacco plants is low by at least 25%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork quality of control tobacco plants, the average cigarette fork quality of modified tobacco plants is low by at least 30%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork quality of control tobacco plants, the average cigarette fork quality of modified tobacco plants is low at least 40%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork quality of control tobacco plants, the average cigarette fork quality of modified tobacco plants is low at least 50%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork quality of control tobacco plants, the average cigarette fork quality of modified tobacco plants is low at least 60%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork quality of control tobacco plants, the average cigarette fork quality of modified tobacco plants is low at least 70%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork quality of control tobacco plants, the average cigarette fork quality of modified tobacco plants is low at least 80%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork quality of control tobacco plants, the average cigarette fork quality of modified tobacco plants is low at least 90%. In one aspect, the average tobacco branch mass of the modified tobacco plants is at least 95% less than the average tobacco branch mass of a control tobacco plant when grown under comparable conditions.

[0080] In one aspect, the average fork length is measured at the base of the fork at the distal end thereof connecting the main tobacco stem to the fork stem.

[0081] In one aspect, when growing under comparable conditions, compared with the average cigarette fork length of control tobacco plants, the average cigarette fork length of modified tobacco plants is at least 1 centimetre short. In one aspect, when growing under comparable conditions, compared with the average cigarette fork length of control tobacco plants, the average cigarette fork length of modified tobacco plants is at least 2 centimetres short. In one aspect, when growing under comparable conditions, compared with the average cigarette fork length of control tobacco plants, the average cigarette fork length of modified tobacco plants is at least 3 centimetres short. In one aspect, when growing under comparable conditions, compared with the average cigarette fork length of control tobacco plants, the average cigarette fork length of modified tobacco plants is at least 4 centimetres short. In one aspect, when growing under comparable conditions, compared with the average cigarette fork length of control tobacco plants, the average cigarette fork length of modified tobacco plants is at least 5 centimetres short. In one aspect, when growing under comparable conditions, compared with the average cigarette fork length of control tobacco plants, the average cigarette fork length of modified tobacco plants is at least 10 centimetres short. In one aspect, the average tobacco fork length of the modified tobacco plant is at least 15 centimeters shorter than the average tobacco fork length of the control tobacco plant when grown under comparable conditions. In one aspect, the average tobacco fork length of the modified tobacco plant is at least 20 centimeters shorter than the average tobacco fork length of the control tobacco plant when grown under comparable conditions.

[0082] In one aspect, when grown under comparable conditions, compared with the average tobacco fork length of control tobacco plants, the average tobacco fork length of modified tobacco plants is short 0.5 cm-30 cm. In one aspect, when grown under comparable conditions, compared with the average tobacco fork length of control tobacco plants, the average tobacco fork length of modified tobacco plants is short 0.5 cm-25 cm. In one aspect, when grown under comparable conditions, compared with the average tobacco fork length of control tobacco plants, the average tobacco fork length of modified tobacco plants is short 0.5 cm-20 cm. In one aspect, when grown under comparable conditions, compared with the average tobacco fork length of control tobacco plants, the average tobacco fork length of modified tobacco plants is short 0.5 cm-15 cm. In one aspect, when grown under comparable conditions, compared with the average tobacco fork length of control tobacco plants, the average tobacco fork length of modified tobacco plants is short 0.5 cm-10 cm. In one aspect, when grown under comparable conditions, compared with the average tobacco fork length of control tobacco plants, the average tobacco fork length of modified tobacco plants is short 0.5 cm-5 cm. In one aspect, when grown under comparable conditions, the average tobacco fork length of the modified tobacco plant is 1 cm to 30 cm shorter than the average tobacco fork length of the control tobacco plant. In one aspect, when grown under comparable conditions, the average tobacco fork length of the modified tobacco plant is 1 cm to 20 cm shorter than the average tobacco fork length of the control tobacco plant. In one aspect, when grown under comparable conditions, the average tobacco fork length of the modified tobacco plant is 1 cm to 10 cm shorter than the average tobacco fork length of the control tobacco plant.

[0083] In one aspect, when growing under comparable conditions, compared with the average cigarette fork length of the control tobacco plant, the average cigarette fork length of the modified tobacco plant is short at least 5%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork length of the control tobacco plant, the average cigarette fork length of the modified tobacco plant is short at least 10%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork length of the control tobacco plant, the average cigarette fork length of the modified tobacco plant is short at least 15%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork length of the control tobacco plant, the average cigarette fork length of the modified tobacco plant is short at least 20%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork length of the control tobacco plant, the average cigarette fork length of the modified tobacco plant is short at least 25%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork length of the control tobacco plant, the average cigarette fork length of the modified tobacco plant is short at least 30%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork length of the control tobacco plant, the average cigarette fork length of the modified tobacco plant is short at least 40%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork length of the control tobacco plant, the average cigarette fork length of the modified tobacco plant is short at least 50%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork length of the control tobacco plant, the average cigarette fork length of the modified tobacco plant is short at least 60%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork length of the control tobacco plant, the average cigarette fork length of the modified tobacco plant is short at least 70%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork length of the control tobacco plant, the average cigarette fork length of the modified tobacco plant is short at least 80%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork length of the control tobacco plant, the average cigarette fork length of the modified tobacco plant is short at least 90%. In one aspect, the average tobacco twig length of the modified tobacco plants is at least 95% shorter than the average tobacco twig length of a control tobacco plant when grown under comparable conditions.

[0084] In one aspect, the fork diameter is measured at the base of the fork where it is adjacent to the main stem of the plant.

[0085] In one aspect, when growing under comparable conditions, compared with the average cigarette fork diameter of the control tobacco plant, the average cigarette fork diameter of the modified tobacco plant is short at least 5%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork diameter of the control tobacco plant, the average cigarette fork diameter of the modified tobacco plant is short at least 10%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork diameter of the control tobacco plant, the average cigarette fork diameter of the modified tobacco plant is short at least 15%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork diameter of the control tobacco plant, the average cigarette fork diameter of the modified tobacco plant is short at least 20%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork diameter of the control tobacco plant, the average cigarette fork diameter of the modified tobacco plant is short at least 25%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork diameter of the control tobacco plant, the average cigarette fork diameter of the modified tobacco plant is short at least 30%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork diameter of the control tobacco plant, the average cigarette fork diameter of the modified tobacco plant is short at least 40%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork diameter of the control tobacco plant, the average cigarette fork diameter of the modified tobacco plant is short at least 50%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork diameter of the control tobacco plant, the average cigarette fork diameter of the modified tobacco plant is short at least 60%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork diameter of the control tobacco plant, the average cigarette fork diameter of the modified tobacco plant is short at least 70%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork diameter of the control tobacco plant, the average cigarette fork diameter of the modified tobacco plant is short at least 80%. In one aspect, when growing under comparable conditions, compared with the average cigarette fork diameter of the control tobacco plant, the average cigarette fork diameter of the modified tobacco plant is short at least 90%. In one aspect, the average tobacco fork diameter of the modified tobacco plant is at least 95% shorter than the average tobacco fork diameter of a control tobacco plant when grown under comparable conditions.

[0086] In one aspect, when grown under comparable conditions, compared with the average tobacco branch diameter of the control tobacco plant, the average tobacco branch diameter of the modified tobacco plant is at least 1 millimeter short. In one aspect, when grown under comparable conditions, compared with the average tobacco branch diameter of the control tobacco plant, the average tobacco branch diameter of the modified tobacco plant is at least 2 millimeters short. In one aspect, when grown under comparable conditions, compared with the average tobacco branch diameter of the control tobacco plant, the average tobacco branch diameter of the modified tobacco plant is at least 3 millimeters short. In one aspect, when grown under comparable conditions, compared with the average tobacco branch diameter of the control tobacco plant, the average tobacco branch diameter of the modified tobacco plant is at least 4 millimeters short. In one aspect, when grown under comparable conditions, compared with the average tobacco branch diameter of the control tobacco plant, the average tobacco branch diameter of the modified tobacco plant is at least 5 millimeters short. In one aspect, when grown under comparable conditions, compared with the average tobacco branch diameter of the control tobacco plant, the average tobacco branch diameter of the modified tobacco plant is at least 6 millimeters short. In one aspect, when grown under comparable conditions, compared with the average tobacco branch diameter of the control tobacco plant, the average tobacco branch diameter of the modified tobacco plant is at least 7 millimeters short. In one aspect, when grown under comparable conditions, compared with the average tobacco branch diameter of the control tobacco plant, the average tobacco branch diameter of the modified tobacco plant is at least 8 millimeters short. In one aspect, when grown under comparable conditions, compared with the average tobacco branch diameter of the control tobacco plant, the average tobacco branch diameter of the modified tobacco plant is at least 9 millimeters short. In one aspect, when grown under comparable conditions, compared with the average tobacco branch diameter of the control tobacco plant, the average tobacco branch diameter of the modified tobacco plant is at least 1 centimetre short. In one aspect, when grown under comparable conditions, compared with the average tobacco branch diameter of the control tobacco plant, the average tobacco branch diameter of the modified tobacco plant is at least 1.5 centimetres short. In one aspect, when grown under comparable conditions, compared with the average tobacco branch diameter of the control tobacco plant, the average tobacco branch diameter of the modified tobacco plant is at least 2 centimetres short. In one aspect, when grown under comparable conditions, the average tobacco fork diameter of the modified tobacco plant is at least 3 centimeters shorter than the average tobacco fork diameter of the control tobacco plant. In one aspect, when grown under comparable conditions, the average tobacco fork diameter of the modified tobacco plant is at least 4 centimeters shorter than the average tobacco fork diameter of the control tobacco plant. In one aspect, when grown under comparable conditions, the average tobacco fork diameter of the modified tobacco plant is at least 5 centimeters shorter than the average tobacco fork diameter of the control tobacco plant.

[0087] Any nucleic acid molecule, protein or polypeptide that expection this paper provides is used together with any method that this paper provides.Any nucleic acid molecule, protein or polypeptide that expection this paper provides is used together with any plant, plant part, cell or seed that expection this paper provides.Any nucleic acid molecule, protein or polypeptide that expection this paper provides is used together with any tobacco plant, tobacco plant part, tobacco cell or tobacco seed that expection this paper provides.

[0088] The use of the term "polynucleotide" or "nucleic acid molecule" is not intended to limit the present disclosure to polynucleotides comprising deoxyribonucleic acid (DNA). For example, ribonucleic acid (RNA) molecules are also contemplated. One of ordinary skill in the art will recognize that polynucleotides and nucleic acid molecules may comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides both include naturally occurring molecules and synthetic analogs. The polynucleotides of the present disclosure also encompass all forms of sequences, including but not limited to single-stranded forms, double-stranded forms, hairpins, stem-loop structures, and the like. 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. The nucleic acid molecules may encode polypeptides or small RNAs.

[0089] The routine techniques of the art can be used to separate nucleic acids. For example, nucleic acids can be separated using any method including but not limited to recombinant nucleic acid technology and / or polymerase chain reaction (PCR). General PCR technology is described in, for example, PCR Primer: Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid technology includes, for example, restriction enzyme digestion and connection, which can be used to separate nucleic acids. Isolated nucleic acids can also be chemically synthesized 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. For example, polypeptides can also be purified by expressing nucleic acids in expression vectors. In addition, purified polypeptides can be obtained by chemical synthesis. The purity of the polypeptide can be measured using any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0090] In one aspect, the present disclosure provides methods for detecting recombinant nucleic acids and polypeptides in plant cells. Without limitation, hybridization detection nucleic acids can also be used. Hybridization between nucleic acids is discussed in detail in Sambrook et al. (1989, Molecular Cloning: Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0091] In one aspect, the endogenous gene provided herein comprises a polynucleotide sequence that is at least 80% identical to a sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the endogenous gene provided herein comprises a polynucleotide sequence that is at least 85% identical to a sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the endogenous gene provided herein comprises a polynucleotide sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the endogenous gene provided herein comprises a polynucleotide sequence that is at least 95% identical to a sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the endogenous gene provided herein comprises a polynucleotide sequence that is at least 96% identical to a sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the endogenous gene provided herein comprises a polynucleotide sequence that is at least 97% identical to a sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the endogenous gene provided herein comprises a polynucleotide sequence that is at least 98% identical to a sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the endogenous gene provided herein comprises a polynucleotide sequence that is at least 99% identical to a sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the endogenous gene provided herein comprises a polynucleotide sequence that is 100% identical to a sequence selected from SEQ ID NOs: 1-44 and 113.

[0092] As used herein, the term "polypeptide" refers to a chain of at least two covalently linked amino acids. A polypeptide can be encoded by a polynucleotide as provided herein. A protein as provided herein can be encoded by a nucleic acid molecule as provided herein. A protein can comprise a polypeptide as provided herein. As used herein, a "protein" refers to a chain of amino acid residues that can provide structure or enzymatic activity to a cell.

[0093] Antibodies can be used to detect polypeptides. Techniques for detecting polypeptides using antibodies include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. The antibodies provided herein can be polyclonal antibodies or monoclonal antibodies. Methods well known in the art can be used to prepare antibodies with specific binding affinity for the polypeptides provided herein. Methods known in the art can be used to attach the antibodies provided herein to solid supports, such as microtiter plates.

[0094] Detection (e.g., amplification products, hybridization complexes, polypeptides) can be performed using detectable labels. The term "label" is intended to encompass the use of direct labels as well as indirect labels. Detectable labels include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.

[0095] In one aspect, the endogenous gene provided herein encodes a polypeptide comprising an amino acid sequence that is at least 70% identical or similar to an amino acid sequence selected from SEQ ID NOs: 45-88 and 114. In one aspect, the endogenous gene provided herein encodes a polypeptide comprising an amino acid sequence that is at least 75% identical or similar to an amino acid sequence selected from SEQ ID NOs: 45-88 and 114. In one aspect, the endogenous gene provided herein encodes a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from SEQ ID NOs: 45-88 and 114. In one aspect, the endogenous gene provided herein encodes a polypeptide comprising an amino acid sequence that is at least 85% identical or similar to an amino acid sequence selected from SEQ ID NOs: 45-88 and 114. In one aspect, the endogenous gene provided herein encodes a polypeptide comprising an amino acid sequence that is at least 90% identical or similar to an amino acid sequence selected from SEQ ID NOs: 45-88 and 114. In one aspect, the endogenous gene provided herein encodes a polypeptide comprising an amino acid sequence that is at least 95% identical or similar to an amino acid sequence selected from SEQ ID NOs: 45-88 and 114. In one aspect, the endogenous gene provided herein encodes a polypeptide comprising an amino acid sequence that is at least 96% identical or similar to an amino acid sequence selected from SEQ ID NOs: 45-88 and 114. In one aspect, the endogenous gene provided herein encodes a polypeptide comprising an amino acid sequence that is at least 97% identical or similar to an amino acid sequence selected from SEQ ID NOs: 45-88 and 114. In one aspect, the endogenous gene provided herein encodes a polypeptide comprising an amino acid sequence that is at least 98% identical or similar to an amino acid sequence selected from SEQ ID NOs: 45-88 and 114. In one aspect, the endogenous gene provided herein encodes a polypeptide comprising an amino acid sequence that is at least 99% identical or similar to an amino acid sequence selected from SEQ ID NOs: 45-88 and 114. In one aspect, the endogenous gene provided herein encodes a polypeptide comprising an amino acid sequence that is 100% identical or similar to an amino acid sequence selected from SEQ ID NOs: 45-88 and 114.

[0096] As used herein, the term "percent identity" or "identical percent" with respect to two or more nucleotide or amino acid sequences is calculated by (i) comparing two optimally aligned sequences (nucleotides or amino acids) over a comparison window (one or more "comparable" regions), (ii) determining the number of positions at which the same nucleic acid base (for nucleotide sequences) or amino acid residue (for proteins and polypeptides) occurs in the two sequences to produce the number of matched positions, (iii) dividing the number of matched positions by the total number of positions in the comparison window, and then (iv) multiplying the quotient by 100% to produce the percent identity. If the "percent identity" is calculated relative to a reference sequence without specifying a particular comparison window, the percent identity is determined by dividing the number of matched positions over the comparison region by the total length of the reference sequence. Therefore, for the purposes of this application, when two sequences (query and subject) are optimally aligned (allowing gaps in their alignment), the "percent identity" of the query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions in the query sequence over its length (or comparison window), then multiplied by 100%.

[0097] When percent sequence identity is used with reference to amino acids, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, in which amino acid residues are substituted with other amino acid residues having similar chemical properties (e.g., charge or hydrophobicity), thereby not altering the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity."

[0098] In order to optimally align sequences to calculate their percent identity, various pairwise or multiple sequence alignment algorithms and programs are known in the art, such as ClustalW or SEQ ID NO: 1 which can be used to compare sequence identity or similarity between two or more nucleic acid or amino acid sequences. (BLASTTM) etc. Although other alignment and comparison methods are known in the art, the alignment and identity percentage (including the above-mentioned identity percentage range) between two sequences can be determined by 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 disclosure of which are incorporated herein by reference.

[0099] The term "complementarity percentage" or "complementarity percentage" used herein with respect to two nucleotide sequences is similar to the concept of identity percentage, but refers to the percentage of nucleotides of the query sequence that optimally base-pair or hybridize with nucleotides of the subject sequence when the query sequence and the subject sequence are arranged linearly and optimally base-paired without secondary folded structures (such as loops, stems or hairpins). This percentage of complementarity can be between two DNA chains, two RNA chains, or a DNA chain and an RNA chain. "Complementarity percentage" can be calculated by (i) optimally base-pairing or hybridizing two nucleotide sequences in a linear and fully extended arrangement (i.e., without folding or secondary structures) over a comparison window, (ii) determining the number of base pair positions between the two sequences over the comparison window to produce the number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions in the comparison window, and (iv) multiplying the quotient by 100% to obtain the complementarity percentage of the two sequences. The optimal base pairing of two sequences can be determined based on known pairings of nucleotide bases, such as GC, AT and AU, by hydrogen bonding. If the "complementarity percentage" is calculated relative to a reference sequence without specifying a particular comparison window, the percent identity is determined by dividing the number of complementary positions between the two linear sequences by the total length of the reference sequence. Thus, for the purposes of this application, when the two sequences (query and subject) are optimally base paired (allowing for mismatches or non-base paired nucleotides), the "complementarity percentage" of the query sequence is equal to the number of base paired positions between the two sequences divided by the total number of positions in the query sequence over its length, then multiplied by 100%.

[0100] In one aspect, the present disclosure provides a modified tobacco plant comprising a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein the mutation is not present in the endogenous gene of a control tobacco plant of the same variety, and wherein the modified tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping compared to the control tobacco plant when grown under comparable growth conditions.

[0101] In one aspect, the present disclosure provides a modified tobacco plant comprising a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein the mutation is not present in the endogenous gene of a control tobacco plant of the same variety.

[0102] In one aspect, the present disclosure provides a method for producing a modified tobacco plant, comprising: (a) inducing a mutation in at least one tobacco cell at a genomic locus encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor; (b) selecting at least one tobacco cell comprising a mutation from step (a); and (c) regenerating a modified tobacco plant from the at least one tobacco cell selected in step (b), wherein when grown under comparable growth conditions, the modified tobacco plant does not comprise a tobacco fork or comprises a reduced tobacco fork after topping compared to a control tobacco plant lacking the mutation. In one aspect, the method further comprises (d) growing the modified tobacco plant regenerated in step (c). On the other hand, the method further comprises (e) hybridizing the modified tobacco plant grown in step (d) with a second tobacco plant; and (f) obtaining at least one seed from the hybridization in step (e).

[0103] In one aspect, the modified tobacco plant, tobacco plant part, tobacco seed, tobacco cell or tobacco genome provided herein comprises one or more sudden changes in the endogenous gene encoding cyclin. In one aspect, the modified tobacco plant, tobacco plant part, tobacco seed, tobacco cell or tobacco genome provided herein comprises one or more sudden changes in the endogenous gene encoding cyclin dependent kinase. In one aspect, the modified tobacco plant, tobacco plant part, tobacco seed, tobacco cell or tobacco genome provided herein comprises one or more sudden changes in the endogenous gene encoding cyclin dependent kinase inhibitor. In one aspect, the modified tobacco plant, tobacco plant part, tobacco seed, tobacco cell or tobacco genome provided herein comprises one or more sudden changes in the endogenous gene encoding MYB. In one aspect, the modified tobacco plant, tobacco plant part, tobacco seed, tobacco cell or tobacco genome provided herein comprises one or more sudden changes in the endogenous gene encoding WRKY transcription factor.

[0104] Cyclin controls the progression of cells through the cell cycle. They usually play a role in the activation of CDK. Cyclin is characterized by having a conserved cyclin box domain. The cyclin box domain usually contains about 150 amino acids, which are organized into 5 α-helical regions. The cyclin box domain is important for binding other proteins such as CDK. See, for example, Noble et al., "The cyclin box fold: protein recognition in cell-cycle and transcription control," Trends Biochem. Sci. 22: 482-487 (1997); and Menges et al., "Genomic Organization and Evolutionary Conservation of Plant D-Type Cyclins," Plant Physiology, 145: 1558-1576 (2007), which are incorporated herein by reference in their entirety.

[0105] G1 (Gap1 phase) is the first part of the interphase of the cell cycle in eukaryotic cells. Cells synthesize mRNA and protein in the G1 phase, and the G1 phase ends with conversion to the S phase. The S phase (synthesis phase) is the second part of the interphase of the cell cycle, in which DNA is replicated. The S phase ends with conversion to the G2 phase. The G2 phase (Gap2 phase) typically involves rapid cell growth because cells are ready to transition to the M phase (mitotic phase). Cyclin and CDK are important regulators at the checkpoints between the transition from the G1 phase to the S phase and from the G2 phase to the M phase and during the G1 phase, the S phase, the G2 phase and the M phase.

[0106] There are two major groups of cyclins: the G1 / S cyclins required for controlling the cell cycle by the G1 / S transition, and the G2 / M cyclins required for controlling the cell cycle by the G2 / M transition. If the required cyclins or CDKs cannot function correctly, the cell cycle can stagnate at the G1 / S transition or the G2 / M transition and prevent the formation of daughter cells. On the one hand, the cyclins provided herein are G1 / S cyclins. On the other hand, the cyclins provided herein are G2 / M cyclins.

[0107] In one aspect, the disclosure provides polynucleotides encoding cell cycle proteins. In another aspect, the disclosure provides cell cycle proteins.

[0108] In one aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene, wherein the mutant polynucleotide encodes a mutant cyclin compared to a cyclin encoded by an endogenous gene lacking the mutation. In one aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene, wherein the mutant polynucleotide encodes a truncated cyclin compared to a cyclin encoded by an endogenous gene lacking the mutation. In another aspect, the present disclosure provides a non-naturally occurring truncated cyclin. In one aspect, the present disclosure provides a polynucleotide encoding a mutant mRNA of an endogenous gene comprising a premature stop codon, wherein the mutant mRNA encodes a truncated cyclin compared to an mRNA of an endogenous gene lacking the premature stop codon. .

[0109] In one aspect, the present disclosure provides polynucleotides comprising non-naturally occurring mutations in sequences encoding cyclin box domains. In one aspect, the present disclosure provides mutant polynucleotides comprising mutations in endogenous genes encoding cyclins, wherein the mutant polynucleotides encode cyclins lacking cyclin box domains. On the other hand, the present disclosure provides mutant polynucleotides comprising mutations in endogenous genes encoding cyclins, wherein the mutant polynucleotides encode cyclins comprising truncated cyclin box domains compared to cyclins encoded by endogenous genes lacking mutations. In one aspect, the present disclosure provides mutant polynucleotides comprising mutations in endogenous genes encoding cyclins, wherein the mutant polynucleotides encode cyclins that cannot bind to CDKs. In one aspect, the present disclosure provides mutant polynucleotides comprising mutations in endogenous genes encoding cyclins, wherein the mutant polynucleotides encode cyclins that exhibit reduced binding affinity to at least one CDK compared to cyclins encoded by endogenous genes lacking the mutations.

[0110] In one aspect, the mutated polynucleotide encodes a dominant negative allele of a cyclin. In another aspect, the mutated polynucleotide encodes a dominant positive allele of a cyclin. In another aspect, the mutated polynucleotide encodes a constitutively active cyclin. In another aspect, the mutated polynucleotide encodes an inactivated cyclin.

[0111] In one aspect, the endogenous gene encoding a cyclin comprises a polynucleotide sequence that is at least 80% identical to a sequence selected from SEQ ID NO: 6-32. In one aspect, the endogenous gene encoding a cyclin comprises a polynucleotide sequence that is at least 85% identical to a sequence selected from SEQ ID NO: 6-32. In one aspect, the endogenous gene encoding a cyclin comprises a polynucleotide sequence that is at least 90% identical to a sequence selected from SEQ ID NO: 6-32. In one aspect, the endogenous gene encoding a cyclin comprises a polynucleotide sequence that is at least 95% identical to a sequence selected from SEQ ID NO: 6-32. In one aspect, the endogenous gene encoding a cyclin comprises a polynucleotide sequence that is at least 96% identical to a sequence selected from SEQ ID NO: 6-32. In one aspect, the endogenous gene encoding a cyclin comprises a polynucleotide sequence that is at least 97% identical to a sequence selected from SEQ ID NO: 6-32. In one aspect, the endogenous gene encoding a cyclin comprises a polynucleotide sequence that is at least 98% identical to a sequence selected from SEQ ID NO: 6-32. In one aspect, the endogenous gene encoding a cyclin comprises a polynucleotide sequence that is at least 99% identical to a sequence selected from SEQ ID NOs: 6-32. In one aspect, the endogenous gene encoding a cyclin comprises a polynucleotide sequence that is 100% identical to a sequence selected from SEQ ID NOs: 6-32.

[0112] In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 70% identical to a sequence selected from SEQ ID NOs: 50-76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 75% identical to a sequence selected from SEQ ID NOs: 50-76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 80% identical to a sequence selected from SEQ ID NOs: 50-76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 85% identical to a sequence selected from SEQ ID NOs: 50-76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 50-76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 95% identical to a sequence selected from SEQ ID NOs: 50-76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 96% identical to a sequence selected from SEQ ID NOs: 50-76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 50-76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 50-76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 50-76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 50-76.

[0113] In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 70% similar to a sequence selected from SEQ ID NOs: 50-76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 75% similar to a sequence selected from SEQ ID NOs: 50-76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 80% similar to a sequence selected from SEQ ID NOs: 50-76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 85% similar to a sequence selected from SEQ ID NOs: 50-76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 90% similar to a sequence selected from SEQ ID NOs: 50-76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 95% similar to a sequence selected from SEQ ID NOs: 50-76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 96% similar to a sequence selected from SEQ ID NOs: 50-76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 97% similar to a sequence selected from SEQ ID NOs: 50-76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 98% similar to a sequence selected from SEQ ID NOs: 50-76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 99% similar to a sequence selected from SEQ ID NOs: 50-76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is 100% similar to a sequence selected from SEQ ID NOs: 50-76.

[0114] Cyclin-dependent kinases (CDKs) are a family of protein kinases involved in regulating the cell cycle, regulating transcription, and regulating mRNA processing. CDKs are considered to belong to the Enzyme Commission category 2.7.11.22. CDKs contain a kinase domain, and they interact or bind to cyclins. CDKs phosphorylate substrates on serine and threonine, so they can also be referred to as serine-threonine kinases. CDKs, together with cyclins, are necessary for cells to progress through the cell cycle. See, for example, Mironov et al., "Cyclin-Dependent Kinases and Cell Division in Plants─The Nexus," Plant Cell, 11:509-521 (1999), which is incorporated herein by reference in its entirety.

[0115] In one aspect, the disclosure provides a polynucleotide encoding a CDK. In another aspect, the disclosure provides a CDK.

[0116] In one aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene, wherein the mutant polynucleotide encodes a mutated CDK compared to a CDK encoded by an endogenous gene lacking the mutation. In one aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene, wherein the mutant polynucleotide encodes a truncated CDK compared to a CDK encoded by an endogenous gene lacking the mutation. In another aspect, the present disclosure provides a non-naturally occurring truncated CDK. In one aspect, the present disclosure provides a polynucleotide encoding a mutant mRNA of an endogenous gene comprising a premature stop codon, wherein the mutant mRNA encodes a truncated CDK compared to an mRNA of an endogenous gene lacking the premature stop codon.

[0117] In one aspect, the present disclosure provides polynucleotides comprising non-naturally occurring mutations in sequences encoding kinase domains. In one aspect, the present disclosure provides mutant polynucleotides comprising mutations in endogenous genes encoding CDKs, wherein the mutant polynucleotides encode CDKs lacking kinase domains. On the other hand, the present disclosure provides mutant polynucleotides comprising mutations in endogenous genes encoding CDKs, wherein the mutant polynucleotides encode CDKs comprising truncated kinase domains compared to CDKs encoded by endogenous genes lacking mutations. In one aspect, the present disclosure provides mutant polynucleotides comprising mutations in endogenous genes encoding CDKs, wherein the mutant polynucleotides encode CDKs that cannot bind to cell cycle proteins. In one aspect, the present disclosure provides mutant polynucleotides comprising mutations in endogenous genes encoding CDKs, wherein the mutant polynucleotides encode CDKs that cannot bind to cell cycle proteins. In one aspect, the present disclosure provides mutant polynucleotides comprising mutations in endogenous genes encoding CDKs, wherein the CDKs exhibit reduced binding affinity to at least one cell cycle protein compared to CDKs encoded by endogenous genes lacking the mutations.

[0118] In one aspect, the mutated polynucleotide encodes a dominant negative allele of a CDK. In another aspect, the mutated polynucleotide encodes a dominant positive allele of a CDK. In another aspect, the mutated polynucleotide encodes a constitutively active CDK. In another aspect, the mutated polynucleotide encodes an inactivated CDK.

[0119] In one aspect, the endogenous gene encoding CDK comprises a polynucleotide sequence that is at least 80% identical to a sequence selected from SEQ ID NOs: 1-4. In one aspect, the endogenous gene encoding CDK comprises a polynucleotide sequence that is at least 85% identical to a sequence selected from SEQ ID NOs: 1-4. In one aspect, the endogenous gene encoding CDK comprises a polynucleotide sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 1-4. In one aspect, the endogenous gene encoding CDK comprises a polynucleotide sequence that is at least 95% identical to a sequence selected from SEQ ID NOs: 1-4. In one aspect, the endogenous gene encoding CDK comprises a polynucleotide sequence that is at least 96% identical to a sequence selected from SEQ ID NOs: 1-4. In one aspect, the endogenous gene encoding CDK comprises a polynucleotide sequence that is at least 97% identical to a sequence selected from SEQ ID NOs: 1-4. In one aspect, the endogenous gene encoding CDK comprises a polynucleotide sequence that is at least 98% identical to a sequence selected from SEQ ID NOs: 1-4. In one aspect, the endogenous gene encoding CDK comprises a polynucleotide sequence that is at least 99% identical to a sequence selected from SEQ ID NOs: 1-4. In one aspect, the endogenous gene encoding CDK comprises a polynucleotide sequence that is 100% identical to a sequence selected from SEQ ID NOs: 1-4.

[0120] In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 70% identical to a sequence selected from SEQ ID NOs: 45-48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 75% identical to a sequence selected from SEQ ID NOs: 45-48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 80% identical to a sequence selected from SEQ ID NOs: 45-48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 85% identical to a sequence selected from SEQ ID NOs: 45-48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 45-48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 95% identical to a sequence selected from SEQ ID NOs: 45-48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 96% identical to a sequence selected from SEQ ID NOs: 45-48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 97% identical to a sequence selected from SEQ ID NOs: 45-48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 98% identical to a sequence selected from SEQ ID NOs: 45-48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 99% identical to a sequence selected from SEQ ID NOs: 45-48. In one aspect, the endogenous CDK comprises an amino acid sequence that is 100% identical to a sequence selected from SEQ ID NOs: 45-48.

[0121] In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 70% similar to a sequence selected from SEQ ID NOs: 45-48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 75% similar to a sequence selected from SEQ ID NOs: 45-48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 80% similar to a sequence selected from SEQ ID NOs: 45-48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 85% similar to a sequence selected from SEQ ID NOs: 45-48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 90% similar to a sequence selected from SEQ ID NOs: 45-48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 95% similar to a sequence selected from SEQ ID NOs: 45-48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 96% similar to a sequence selected from SEQ ID NOs: 45-48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 97% similar to a sequence selected from SEQ ID NOs: 45-48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 98% similar to a sequence selected from SEQ ID NOs: 45-48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 99% similar to a sequence selected from SEQ ID NOs: 45-48. In one aspect, the endogenous CDK comprises an amino acid sequence that is 100% similar to a sequence selected from SEQ ID NOs: 45-48.

[0122] Cyclin-dependent kinase inhibitors (CDKIs) are proteins that inhibit CDKs. CDKIs can bind to CDKs to prevent CDKs from binding to cyclins, thereby negatively regulating the cell cycle in eukaryotic organisms. In plants, CDKIs are divided into two families: KIP-related proteins (KRPs) and SIAMESE-related proteins (SMRs). See, for example, Kumar and Larkin, "Why do plants need so many cyclin-dependent kinase inhibitors?," Plant Signaling & Behavior, 12: e1282021 (2017), the entire contents of which are incorporated herein by reference.

[0123] In one aspect, the disclosure provides a polynucleotide encoding a CDKI. In another aspect, the disclosure provides a CDKI. In one aspect, the CDKI provided herein is a KRP. In another aspect, the CDKI provided herein is a SMR.

[0124] In one aspect, the present disclosure provides mutant polynucleotides comprising mutations in endogenous genes, wherein the mutant polynucleotides encode mutant CDKIs compared to CDKIs encoded by endogenous genes lacking the mutations. In one aspect, the present disclosure provides mutant polynucleotides comprising mutations in endogenous genes, wherein the mutant polynucleotides encode truncated CDKIs compared to CDKIs encoded by endogenous genes lacking the mutations. In another aspect, the present disclosure provides non-naturally occurring truncated CDKIs. In one aspect, the present disclosure provides polynucleotides encoding mutant mRNAs of endogenous genes comprising premature stop codons, wherein the mutant mRNAs encode truncated CDKIs compared to mRNAs of endogenous genes lacking the premature stop codons.

[0125] In one aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene encoding a CDKI, wherein the mutant polynucleotide encodes a CDKI that cannot bind to a CDK. In one aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene encoding a CDKI, wherein the mutant polynucleotide encodes a CDKI that exhibits reduced binding affinity for at least one CDK compared to a CDKI encoded by an endogenous gene lacking the mutation.

[0126] In one aspect, the mutated polynucleotide encodes a dominant negative allele of CDKI. In another aspect, the mutated polynucleotide encodes a dominant positive allele of CDKI. In a further aspect, the mutated polynucleotide encodes a constitutively active CDKI. In another aspect, the mutated polynucleotide encodes an inactivated CDKI.

[0127] In one aspect, the endogenous gene encoding CDKI comprises a polynucleotide sequence that is at least 80% identical to SEQ ID NO:5. In one aspect, the endogenous gene encoding CDKI comprises a polynucleotide sequence that is at least 85% identical to SEQ ID NO:5. In one aspect, the endogenous gene encoding CDKI comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO:5. In one aspect, the endogenous gene encoding CDKI comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:5. In one aspect, the endogenous gene encoding CDKI comprises a polynucleotide sequence that is at least 96% identical to SEQ ID NO:5. In one aspect, the endogenous gene encoding CDKI comprises a polynucleotide sequence that is at least 97% identical to SEQ ID NO:5. In one aspect, the endogenous gene encoding CDKI comprises a polynucleotide sequence that is at least 98% identical to SEQ ID NO:5. In one aspect, the endogenous gene encoding CDKI comprises a polynucleotide sequence that is at least 99% identical to SEQ ID NO:5. In one aspect, the endogenous gene encoding CDKI comprises a polynucleotide sequence that is 100% identical to SEQ ID NO:5.

[0128] In one aspect, the endogenous CDKI comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 49. In one aspect, the endogenous CDKI comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 49. In one aspect, the endogenous CDKI comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 49. In one aspect, the endogenous CDKI comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 49. In one aspect, the endogenous CDKI comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 49. In one aspect, the endogenous CDKI comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 49. In one aspect, the endogenous CDKI comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 49. In one aspect, the endogenous CDKI comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 49. In one aspect, the endogenous CDKI comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 49. In one aspect, the endogenous CDKI comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 49. In one aspect, the endogenous CDKI comprises an amino acid sequence that is 100% identical to SEQ ID NO:49.

[0129] In one aspect, the endogenous CDKI comprises an amino acid sequence that is at least 70% similar to SEQ ID NO:49. In one aspect, the endogenous CDKI comprises an amino acid sequence that is at least 75% similar to SEQ ID NO:49. In one aspect, the endogenous CDKI comprises an amino acid sequence that is at least 80% similar to SEQ ID NO:49. In one aspect, the endogenous CDKI comprises an amino acid sequence that is at least 85% similar to SEQ ID NO:49. In one aspect, the endogenous CDKI comprises an amino acid sequence that is at least 90% similar to SEQ ID NO:49. In one aspect, the endogenous CDKI comprises an amino acid sequence that is at least 95% similar to SEQ ID NO:49. In one aspect, the endogenous CDKI comprises an amino acid sequence that is at least 96% similar to SEQ ID NO:49. In one aspect, the endogenous CDKI comprises an amino acid sequence that is at least 97% similar to SEQ ID NO:49. In one aspect, the endogenous CDKI comprises an amino acid sequence that is at least 98% similar to SEQ ID NO:49. In one aspect, the endogenous CDKI comprises an amino acid sequence that is at least 99% similar to SEQ ID NO:49. In one aspect, the endogenous CDKI comprises an amino acid sequence that is 100% similar to SEQ ID NO:49.

[0130] MYB protein (MYB) is a part of the transcription factor family. In plants, MYB includes a conservative MYB DNA binding domain. The MYB DNA binding domain contains up to three incomplete repetitive sequences in a helix-turn-helix structure, each repetitive sequence is about 55 amino acids. These repetitive sequences are referred to as R1, R2 and R3, and it has been shown that the R2 / R3 repetitive sequence directly binds to the major groove of DNA. The subfamily of plant-specific MYB contains R2R3 type MYB domains and is referred to as R2R3 type MYB. Other subfamilies of MYB include R1 type, 3R type and 4R type MYB. For example, see Ambawat et al., "MYB transcriptionfactor genes as regulators for plant responses: an overview," Physiol. Mol. Biol. Plants, 19: 307-321 (2013), the entire contents of which are incorporated herein by reference.

[0131] In one aspect, the MYB provided herein is an R1 type MYB. On the other hand, the MYB provided herein is an R2R3 type MYB. On the other hand, the MYB provided herein is a 3R type MYB. On the other hand, the MYB provided herein is a 4R type MYB.

[0132] In one aspect, the disclosure provides a polynucleotide encoding MYB. In another aspect, the disclosure provides MYB.

[0133] In one aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene, wherein the mutant polynucleotide encodes a mutated MYB compared to the MYB encoded by the endogenous gene lacking the mutation. In one aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene, wherein the mutant polynucleotide encodes a truncated MYB compared to the MYB encoded by the endogenous gene lacking the mutation. On the other hand, the present disclosure provides a non-naturally occurring truncated MYB. In one aspect, the present disclosure provides a polynucleotide encoding a mutant mRNA of an endogenous gene comprising an early termination codon, wherein the mutant mRNA encodes a truncated MYB compared to the mRNA of the endogenous gene lacking the early termination codon.

[0134] In one aspect, the disclosure provides polynucleotides comprising non-naturally occurring mutations in sequences encoding MYB DNA binding domains. In one aspect, the disclosure provides mutation polynucleotides comprising mutations in endogenous genes encoding MYB, wherein the mutation polynucleotides encode MYB lacking MYB DNA binding domains. On the other hand, the disclosure provides mutation polynucleotides comprising mutations in endogenous genes encoding MYB, wherein the mutation polynucleotides encode MYB comprising truncated MYB DNA binding domains compared to MYB encoded by endogenous genes lacking mutations.

[0135] In one aspect, the disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene encoding MYB, wherein the mutant polynucleotide encodes a MYB that cannot bind to DNA. In one aspect, the disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene encoding MYB, wherein the mutant polynucleotide encodes a MYB that exhibits a reduced binding affinity to DNA compared to a MYB encoded by an endogenous gene lacking the mutation.

[0136] In another aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene encoding MYB, wherein the mutation polynucleotide encoding comprises a MYB comprising a truncated R2R3 type MYB domain compared to the MYB encoded by the endogenous gene lacking the mutation. On the other hand, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene encoding MYB, wherein the mutation polynucleotide encoding comprises a MYB comprising a truncated R1 type MYB domain compared to the MYB encoded by the endogenous gene lacking the mutation. On the other hand, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene encoding MYB, wherein the mutation polynucleotide encoding comprises a MYB comprising a truncated 3R type MYB domain compared to the MYB encoded by the endogenous gene lacking the mutation. On the other hand, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene encoding MYB, wherein the mutation polynucleotide encoding comprises a MYB comprising a truncated 4R type MYB domain compared to the MYB encoded by the endogenous gene lacking the mutation.

[0137] In one aspect, the polynucleotide encoding MYB is a dominant negative allele of the mutation. In another aspect, the polynucleotide encoding MYB is a dominant positive allele of the mutation. In another aspect, the polynucleotide encoding MYB is a constitutively active MYB. In another aspect, the polynucleotide encoding MYB is inactivated.

[0138] In one aspect, the endogenous gene encoding MYB comprises a polynucleotide sequence at least 80% identical to a sequence selected from SEQ ID NO: 33-44. In one aspect, the endogenous gene encoding MYB comprises a polynucleotide sequence at least 85% identical to a sequence selected from SEQ ID NO: 33-44. In one aspect, the endogenous gene encoding MYB comprises a polynucleotide sequence at least 90% identical to a sequence selected from SEQ ID NO: 33-44. In one aspect, the endogenous gene encoding MYB comprises a polynucleotide sequence at least 95% identical to a sequence selected from SEQ ID NO: 33-44. In one aspect, the endogenous gene encoding MYB comprises a polynucleotide sequence at least 96% identical to a sequence selected from SEQ ID NO: 33-44. In one aspect, the endogenous gene encoding MYB comprises a polynucleotide sequence at least 97% identical to a sequence selected from SEQ ID NO: 33-44. In one aspect, the endogenous gene encoding MYB comprises a polynucleotide sequence at least 98% identical to a sequence selected from SEQ ID NO: 33-44. In one aspect, the endogenous gene encoding MYB comprises a polynucleotide sequence at least 99% identical to a sequence selected from SEQ ID NOs: 33-44. In one aspect, the endogenous gene encoding MYB comprises a polynucleotide sequence 100% identical to a sequence selected from SEQ ID NOs: 33-44.

[0139] In one aspect, the endogenous MYB comprises an amino acid sequence at least 70% identical to a sequence selected from SEQ ID NOs: 77-88. In one aspect, the endogenous MYB comprises an amino acid sequence at least 75% identical to a sequence selected from SEQ ID NOs: 77-88. In one aspect, the endogenous MYB comprises an amino acid sequence at least 80% identical to a sequence selected from SEQ ID NOs: 77-88. In one aspect, the endogenous MYB comprises an amino acid sequence at least 85% identical to a sequence selected from SEQ ID NOs: 77-88. In one aspect, the endogenous MYB comprises an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOs: 77-88. In one aspect, the endogenous MYB comprises an amino acid sequence at least 95% identical to a sequence selected from SEQ ID NOs: 77-88. In one aspect, the endogenous MYB comprises an amino acid sequence at least 96% identical to a sequence selected from SEQ ID NOs: 77-88. In one aspect, the endogenous MYB comprises an amino acid sequence at least 97% identical to a sequence selected from SEQ ID NOs: 77-88. In one aspect, the endogenous MYB comprises an amino acid sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 77-88. In one aspect, the endogenous MYB comprises an amino acid sequence that is at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 77-88. In one aspect, the endogenous MYB comprises an amino acid sequence that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 77-88.

[0140] In one aspect, the endogenous MYB comprises an amino acid sequence at least 70% similar to a sequence selected from SEQ ID NO: 77-88. In one aspect, the endogenous MYB comprises an amino acid sequence at least 75% similar to a sequence selected from SEQ ID NO: 77-88. In one aspect, the endogenous MYB comprises an amino acid sequence at least 80% similar to a sequence selected from SEQ ID NO: 77-88. In one aspect, the endogenous MYB comprises an amino acid sequence at least 85% identical to a sequence selected from SEQ ID NO: 77-88. In one aspect, the endogenous MYB comprises an amino acid sequence at least 90% similar to a sequence selected from SEQ ID NO: 77-88. In one aspect, the endogenous MYB comprises an amino acid sequence at least 95% similar to a sequence selected from SEQ ID NO: 77-88. In one aspect, the endogenous MYB comprises an amino acid sequence at least 96% similar to a sequence selected from SEQ ID NO: 77-88. In one aspect, the endogenous MYB comprises an amino acid sequence at least 97% similar to a sequence selected from SEQ ID NO: 77-88. In one aspect, the endogenous MYB comprises an amino acid sequence at least 98% similar to a sequence selected from SEQ ID NOs: 77-88. In one aspect, the endogenous MYB comprises an amino acid sequence at least 99% similar to a sequence selected from SEQ ID NOs: 77-88. In one aspect, the endogenous MYB comprises an amino acid sequence 100% similar to a sequence selected from SEQ ID NOs: 77-88.

[0141] WRKY transcription factors are one of the largest families of transcriptional regulators known in plants. WRKY transcription factors are proteins containing a "WRKY domain". The WRKY domain contains about 60-70 amino acids and is involved in DNA binding. The WRKY domain contains a highly conserved "WRKY core domain" with an amino acid motif WRKYGQK (SEQ ID NO: 110). The conserved core of the WRKY domain can vary, and the amino acid motif WRKYGKK (SEQ ID NO: 111) is a commonly observed "WRKY core variant domain". The WRKY domain contains a globular shape consisting of 5 anti-effective parallel β chains, and a conserved core is found on the second β chain. The third β chain also contains a highly conserved amino acid motif PRSYY (SEQ ID NO: 112). In many WRKY transcription factors, the PR and SYY amino acids of the "PRSYY motif" (SEQ ID NO: 112) are encoded by different exons; the position of the inserted intron is highly conserved in the WRKY transcription factor. The WRKY domain also contains a "zinc finger region" comprising the amino acid motif CX4-5CX22-23HXH (SEQ ID NO: 115) or CX7CX23HXC (SEQ ID NO: 116), wherein X can be any amino acid, C is cysteine, and H is histidine. The WRKY domain also contains a "DWK salt bridge" motif, in which the conserved tryptophan (W) of the WRKY core (SEQ ID NO: 117) forms a triplet with aspartic acid (D) 4 amino acids upstream of W and lysine (K) 29 amino acids downstream of W. It is believed that the DWK salt bridge is important for stabilizing the WRKY domain. For example, see Rushton et al., "WRKY transcription factors," Trends in Plant Science, 15: 247-258 (2010), which is incorporated herein by reference in its entirety.

[0142] In one aspect, the disclosure provides polynucleotides encoding WRKY transcription factors. In another aspect, the disclosure provides WRKY transcription factors.

[0143] In one aspect, the disclosure provides polynucleotides comprising non-naturally occurring mutations, wherein the polynucleotides encode WRKY transcription factors. In one aspect, the disclosure provides polynucleotides encoding truncated WRKY transcription factors. In another aspect, the disclosure provides non-naturally occurring truncated WRKY transcription factors. In one aspect, the disclosure provides polynucleotides encoding mRNAs comprising premature stop codons, wherein the mRNAs encode truncated WRKY transcription factors.

[0144] In one aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene, wherein the mutant polynucleotide encodes a mutated WRKY transcription factor compared to a WRKY transcription factor encoded by an endogenous gene lacking the mutation. In one aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene, wherein the mutant polynucleotide encodes a truncated WRKY transcription factor compared to a WRKY transcription factor encoded by an endogenous gene lacking the mutation. In another aspect, the present disclosure provides a non-naturally occurring truncated WRKY transcription factor. In one aspect, the present disclosure provides a polynucleotide encoding a mutant mRNA of an endogenous gene comprising a premature stop codon, wherein the mutant mRNA encodes a truncated WRKY transcription factor compared to an mRNA of an endogenous gene lacking the premature stop codon.

[0145] In one aspect, the present disclosure provides polynucleotides comprising non-naturally occurring mutations in sequences encoding WRKY domains. In one aspect, the present disclosure provides mutant polynucleotides comprising mutations in endogenous genes encoding WRKY transcription factors, wherein the mutant polynucleotides encode WRKY transcription factors lacking WRKY domains. In another aspect, the present disclosure provides mutant polynucleotides comprising mutations in endogenous genes encoding WRKY transcription factors, wherein the mutant polynucleotides encode WRKY transcription factors comprising truncated WRKY domains compared to WRKY transcription factors encoded by endogenous genes lacking mutations.

[0146] In one aspect, the present disclosure provides polynucleotides comprising non-naturally occurring mutations in sequences encoding WRKY core domains. In one aspect, the present disclosure provides mutant polynucleotides comprising mutations in endogenous genes encoding WRKY transcription factors, wherein the mutant polynucleotides encode WRKY transcription factors lacking WRKY core domains. In another aspect, the present disclosure provides mutant polynucleotides comprising mutations in endogenous genes encoding WRKY transcription factors, wherein the mutant polynucleotides encode WRKY transcription factors comprising truncated WRKY core domains compared to WRKY transcription factors encoded by endogenous genes lacking mutations.

[0147] In one aspect, the present disclosure provides polynucleotides comprising non-naturally occurring mutations in sequences encoding WRKY core variant domains. In one aspect, the present disclosure provides mutant polynucleotides comprising mutations in endogenous genes encoding WRKY transcription factors, wherein the mutant polynucleotides encode WRKY transcription factors lacking WRKY core variant domains. In another aspect, the present disclosure provides mutant polynucleotides comprising mutations in endogenous genes encoding WRKY transcription factors, wherein the mutant polynucleotides encode WRKY transcription factors comprising truncated WRKY core variant domains compared to WRKY transcription factors encoded by endogenous genes lacking mutations.

[0148] In one aspect, the present disclosure provides polynucleotides comprising a non-naturally occurring mutation in a sequence encoding a PRSYY motif (SEQ ID NO: 112). In one aspect, the present disclosure provides mutant polynucleotides comprising a mutation in an endogenous gene encoding a WRKY transcription factor, wherein the mutant polynucleotide encodes a WRKY transcription factor lacking the PRSYY motif (SEQ ID NO: 112). In another aspect, the present disclosure provides mutant polynucleotides comprising a mutation in an endogenous gene encoding a WRKY transcription factor, wherein the mutant polynucleotide encodes a WRKY transcription factor comprising a truncated PRSYY motif (SEQ ID NO: 112) compared to a WRKY transcription factor encoded by an endogenous gene lacking the mutation.

[0149] In one aspect, the present disclosure provides polynucleotides comprising a non-naturally occurring mutation in a sequence encoding a zinc finger region. In one aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene encoding a WRKY transcription factor, wherein the mutant polynucleotide encodes a WRKY transcription factor lacking a zinc finger region. In another aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene encoding a WRKY transcription factor, wherein the mutant polynucleotide encodes a WRKY transcription factor comprising a truncated zinc finger region compared to a WRKY transcription factor encoded by an endogenous gene lacking the mutation.

[0150] In one aspect, the present disclosure provides polynucleotides comprising a non-naturally occurring mutation in a sequence encoding a DWK salt bridge motif. In one aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene encoding a WRKY transcription factor, wherein the mutant polynucleotide encodes a WRKY transcription factor lacking a DWK salt bridge motif. In another aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene encoding a WRKY transcription factor, wherein the mutant polynucleotide encodes a WRKY transcription factor comprising a truncated DWK salt bridge motif compared to a WRKY transcription factor encoded by an endogenous gene lacking the mutation.

[0151] In one aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene encoding a WRKY transcription factor, wherein the mutant polynucleotide encodes a WRKY transcription factor that is unable to bind to DNA. In one aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene encoding a WRKY transcription factor, wherein the mutant polynucleotide encodes a WRKY transcription factor that exhibits reduced binding affinity to DNA compared to a WRKY transcription factor encoded by an endogenous gene lacking the mutation.

[0152] In one aspect, the mutated polynucleotide encodes a dominant negative allele of a WRKY transcription factor. In another aspect, the mutated polynucleotide encodes a dominant positive allele of a WRKY transcription factor. In a further aspect, the mutated polynucleotide encodes a constitutively active WRKY transcription factor. In another aspect, the mutated polynucleotide encodes an inactivated WRKY transcription factor.

[0153] In one aspect, the endogenous gene encoding the WRKY transcription factor comprises a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 113. In one aspect, the endogenous gene encoding the WRKY transcription factor comprises a polynucleotide sequence that is at least 85% identical to SEQ ID NO: 113. In one aspect, the endogenous gene encoding the WRKY transcription factor comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 113. In one aspect, the endogenous gene encoding the WRKY transcription factor comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO: 113. In one aspect, the endogenous gene encoding the WRKY transcription factor comprises a polynucleotide sequence that is at least 96% identical to SEQ ID NO: 113. In one aspect, the endogenous gene encoding the WRKY transcription factor comprises a polynucleotide sequence that is at least 97% identical to SEQ ID NO: 113. In one aspect, the endogenous gene encoding the WRKY transcription factor comprises a polynucleotide sequence that is at least 98% identical to SEQ ID NO: 113. In one aspect, the endogenous gene encoding the WRKY transcription factor comprises a polynucleotide sequence that is at least 99% identical to SEQ ID NO: 113. In one aspect, the endogenous gene encoding a WRKY transcription factor comprises a polynucleotide sequence that is 100% identical to SEQ ID NO:113.

[0154] In one aspect, the endogenous WRKY transcription factor comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 114. In one aspect, the endogenous WRKY transcription factor comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 114. In one aspect, the endogenous WRKY transcription factor comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 114. In one aspect, the endogenous WRKY transcription factor comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 114. In one aspect, the endogenous WRKY transcription factor comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 114. In one aspect, the endogenous WRKY transcription factor comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 114. In one aspect, the endogenous WRKY transcription factor comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 114. In one aspect, the endogenous WRKY transcription factor comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 114. In one aspect, the endogenous WRKY transcription factor comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 114. In one aspect, the endogenous WRKY transcription factor comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 114. In one aspect, the endogenous WRKY transcription factor comprises an amino acid sequence that is 100% identical to SEQ ID NO:114.

[0155] In one aspect, the endogenous WRKY transcription factor comprises an amino acid sequence that is at least 70% similar to SEQ ID NO: 114. In one aspect, the endogenous WRKY transcription factor comprises an amino acid sequence that is at least 75% similar to SEQ ID NO: 114. In one aspect, the endogenous WRKY transcription factor comprises an amino acid sequence that is at least 80% similar to SEQ ID NO: 114. In one aspect, the endogenous WRKY transcription factor comprises an amino acid sequence that is at least 85% similar to SEQ ID NO: 114. In one aspect, the endogenous WRKY transcription factor comprises an amino acid sequence that is at least 90% similar to SEQ ID NO: 114. In one aspect, the endogenous WRKY transcription factor comprises an amino acid sequence that is at least 95% similar to SEQ ID NO: 114. In one aspect, the endogenous WRKY transcription factor comprises an amino acid sequence that is at least 96% similar to SEQ ID NO: 114. In one aspect, the endogenous WRKY transcription factor comprises an amino acid sequence that is at least 97% similar to SEQ ID NO: 114. In one aspect, the endogenous WRKY transcription factor comprises an amino acid sequence that is at least 98% similar to SEQ ID NO: 114. In one aspect, the endogenous WRKY transcription factor comprises an amino acid sequence that is at least 99% similar to SEQ ID NO: 114. In one aspect, the endogenous WRKY transcription factor comprises an amino acid sequence that is 100% similar to SEQ ID NO:114.

[0156] In one aspect, the modified tobacco plant, tobacco plant part, tobacco seed, tobacco cell or tobacco genome provided herein comprise one or more sudden changes.As used herein, " sudden change " refers to the change of the non-natural generation of DNA compared with the endogenous reference DNA sequence.Should be understood that when identifying sudden changes, the endogenous reference DNA sequence should be from identical tobacco varieties.For example, if the modified tobacco plant comprising sudden changes is from the TN90 kind, the endogenous reference sequence must be the endogenous TN90 sequence, rather than the homologous sequence from different tobacco varieties (for example K326).Similarly, if the modified tobacco cell comprising sudden changes is the TN90 cell, the endogenous reference sequence must be the endogenous TN90 sequence, rather than the homologous sequence from the tobacco cell of different tobacco varieties (for example K326).

[0157] In one aspect, the mutation provided herein produces the dominant allele of the mutation locus.Dominant allele is the allele that masks the contribution of the second allele at the same locus.Dominant allele can be "dominant negative allele" or "dominant positive allele". Dominant negative allele or anti-allele is the allele opposite to the normal allele function.Dominant negative allele typically functions abnormally and directly suppresses the activity of wild-type protein (for example, by dimerization) or suppresses the activity of the second protein required for the normal function of wild-type protein (for example, downstream components of activator or approach). For example, dominant negative allele removes or reduces the normal function of allele in heterozygous or homozygous state.Dominant positive allele can increase normal gene function (for example, super allele) or provide new function (for example, new allele) for gene.When the penetrance of linkage phenotype in allele heterozygous individual is lower than the penetrance observed in allele homozygous individual, semi-dominant allele occurs.

[0158] In one aspect, the mutations provided herein produce dominant negative alleles of the mutated locus. In another aspect, the mutations provided herein produce dominant positive alleles of the mutated locus.

[0159] As used herein, "induced" mutation refers to the generation of mutations in polynucleotide sequences by human intervention. Many suitable methods for inducing mutations in tobacco are known in the art. Non-limiting examples of these methods include the use of chemical mutagens, the use of radiation, and the use of nucleases. In one aspect, induced mutations include the use of a reagent selected from chemical mutagens, radiation, transposons, agrobacteria, and nucleases.

[0160] In one aspect, inducing mutations comprises using a chemical mutagen. In one aspect, the chemical mutagen comprises ethyl methanesulfonate (EMS).

[0161] In another aspect, inducing mutations comprises the use of radiation. In one aspect, the radiation comprises gamma rays, X-rays or ionizing radiation. In another aspect, the radiation comprises the use of fast neutrons.

[0162] In one aspect, inducing mutations comprises using a transposon. In another aspect, inducing mutations comprises using Agrobacterium.

[0163] In another aspect, inducing mutations comprises 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 mutations comprises the use of a CRISPR / Cas9 nuclease. In one aspect, inducing mutations comprises the use of a CRISPR / Cpf1 nuclease. In one aspect, inducing mutations comprises the use of a CRISPR / CasX nuclease. In one aspect, inducing mutations comprises the use of a CRISPR / CasY nuclease. In one aspect, inducing mutations comprises the use of a Csm1 nuclease.

[0164] Several types of mutations are known in the art. In one aspect, mutations include insertions. "Insertion" refers to the addition of one or more nucleotides or amino acids to a given polynucleotide or amino acid sequence, respectively, compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, mutations include deletions. "Deletion" refers to the removal of one or more nucleotides or amino acids of a given polynucleotide or amino acid sequence, respectively, compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, mutations include substitutions. "Substitution" refers to the replacement of one or more nucleotides or amino acids with a given polynucleotide or amino acid sequence, respectively, compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, mutations include inversions. "Inversion" refers to the end-to-end reversal of a fragment of a polynucleotide or amino acid sequence. In one aspect, mutations provided herein include mutations selected from insertions, deletions, substitutions, and inversions.

[0165] When the mutated messenger RNA (mRNA) is translated into a protein or polypeptide, a mutation in a gene coding region (e.g., an exon mutation) can produce a truncated protein or polypeptide. On the one hand, the present disclosure provides mutations that result in truncation of a protein or polypeptide. As used herein, a "truncated" protein or polypeptide comprises at least one less amino acid than an endogenous control protein or polypeptide. For example, if endogenous protein A comprises 100 amino acids, a truncated form of protein A may comprise 1-99 amino acids.

[0166] Not limited by any scientific theory, one way to cause protein or polypeptide truncation is by introducing premature termination codons in the mRNA transcripts of endogenous genes. In one aspect, the disclosure provides mutations that cause premature termination codons in the mRNA transcripts of endogenous genes. As used herein, "termination codons" refer to nucleotide triplet that sends protein translation termination signals in mRNA transcripts. "Premature termination codons" refer to termination codons located earlier (e.g., on the 5' side) than normal termination codon positions in endogenous mRNA transcripts. Unrestricted, several termination codons are known in the art, including "UAG", "UAA", "UGA", "TAG", "TAA" and "TGA".

[0167] In one aspect, the mutations provided herein include null mutations. As used herein, "null mutations" refer to mutations that confer complete loss of function to a protein encoded by a gene comprising the mutation, or mutations that confer complete loss of function to a small RNA encoded by a genomic locus. Null mutations can result in a lack of mRNA transcription products, a lack of small RNA transcription products, a lack of protein function, or a combination thereof.

[0168] The mutation provided herein can be located in any part of the endogenous gene. In one aspect, the mutation provided herein is located in the exon of the endogenous gene. In another aspect, the mutation provided herein is located in the intron of the endogenous gene. In a further aspect, the mutation provided herein is located in the 5'-untranslated region of the endogenous gene. In another aspect, the mutation provided herein is located in the 3'-untranslated region of the endogenous gene. In yet another aspect, the mutation provided herein is located in the promoter of the endogenous gene.

[0169] The screening of mutagenic tobacco plants and selection can be by any method known to those of ordinary skill in the art.The example of screening and selection method includes but is not limited to Southern analysis, PCR amplification, Northern blotting, RNase protection, primer extension, RT-PCR amplification, Sanger sequencing, next generation sequencing technology (for example Illumina, PacBio, Ion Torrent, 454) enzyme assay for detecting the enzyme or ribozyme activity of polypeptide and polynucleotide, for example PacBio, Ion Torrent, 454, and protein gel electrophoresis, Western blotting, immunoprecipitation and enzyme-linked immunosorbent assay for detecting polypeptide.Other techniques (such as in situ hybridization, enzyme staining and immunostaining) also can be used for detecting the existence or expression of polypeptide and / or polynucleotide.The method for implementing all reference techniques is known.

[0170] In one aspect, the mutation in the endogenous gene causes the expression level to decrease compared to the endogenous gene lacking the mutation. On the other hand, the mutation in the endogenous gene causes the expression level to increase compared to the endogenous gene lacking the mutation. In a further aspect, the mutation in the endogenous gene causes the activity level of the protein or polypeptide encoded by the endogenous gene with the mutation to decrease compared to the protein or polypeptide encoded by the endogenous gene lacking the mutation. In a further aspect, the mutation in the endogenous gene causes the activity level of the protein or polypeptide encoded by the endogenous gene with the mutation to increase compared to the protein or polypeptide encoded by the endogenous gene lacking the mutation.

[0171] In one aspect, compared to the genomic locus lacking a mutation, the mutation in the genomic locus causes a reduction in expression level. On the other hand, compared to the genomic locus lacking a mutation, the mutation in the genomic locus causes an increase in expression level. In a further aspect, compared to a protein or polypeptide encoded by a genomic locus lacking a mutation, the mutation in the genomic locus causes a reduction in the activity level of a protein or polypeptide encoded by the genomic locus with a mutation. In a further aspect, compared to a protein or polypeptide encoded by a genomic locus lacking a mutation, the mutation in the genomic locus causes an increase in the activity level of a protein or polypeptide encoded by the genomic locus with a mutation.

[0172] In the art, gene expression levels are routinely studied. As non-limiting examples, gene expression can be measured using quantitative reverse transcriptase PCR (qRT-PCR), RNA sequencing or Northern blotting. In one aspect, qRT-PCR is used to measure gene expression. On the other hand, Northern blotting is used to measure gene expression. On the other hand, RNA sequencing is used to measure gene expression.

[0173] In the art, protein activity levels are also routinely studied. For example, CDK activity can be measured using phosphorylation assays.

[0174] As used herein, the term "heterologous" refers to a combination of two or more DNA molecules or sequences (eg, promoters) and associated transcribable DNA sequences, coding sequences or genes when such a combination is man-made and not normally found in nature.

[0175] In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation is reduced by at least 1%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation is reduced by at least 5%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation is reduced by at least 10%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation is reduced by at least 15%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation is reduced by at least 20%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation is reduced by at least 25%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation is reduced by at least 50%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation is reduced by at least 75%. In one aspect, the expression level of an endogenous gene comprising a mutation is reduced by at least 90% compared to the expression level of an endogenous gene lacking the mutation. In one aspect, the expression level of an endogenous gene comprising a mutation is reduced by at least 95% compared to the expression level of an endogenous gene lacking the mutation.

[0176] In one aspect, compared to the expression level of an endogenous gene lacking a mutation, the expression level of an endogenous gene comprising a mutation is reduced by 1%-99%. In one aspect, compared to the expression level of an endogenous gene lacking a mutation, the expression level of an endogenous gene comprising a mutation is reduced by 1%-90%. In one aspect, compared to the expression level of an endogenous gene lacking a mutation, the expression level of an endogenous gene comprising a mutation is reduced by 1%-75%. In one aspect, compared to the expression level of an endogenous gene lacking a mutation, the expression level of an endogenous gene comprising a mutation is reduced by 1%-50%. In one aspect, compared to the expression level of an endogenous gene lacking a mutation, the expression level of an endogenous gene comprising a mutation is reduced by 1%-25%. In one aspect, compared to the expression level of an endogenous gene lacking a mutation, the expression level of an endogenous gene comprising a mutation is reduced by 1%-10%. In one aspect, compared to the expression level of an endogenous gene lacking a mutation, the expression level of an endogenous gene comprising a mutation is reduced by 75%-99%. In one aspect, the expression level of the endogenous gene comprising the mutation is reduced by 50%-99% compared to the expression level of the endogenous gene lacking the mutation. In one aspect, the expression level of the endogenous gene comprising the mutation is reduced by 25%-99% compared to the expression level of the endogenous gene lacking the mutation. In one aspect, the expression level of the endogenous gene comprising the mutation is reduced by 10%-99% compared to the expression level of the endogenous gene lacking the mutation. In one aspect, the expression level of the endogenous gene comprising the mutation is reduced by 50%-75% compared to the expression level of the endogenous gene lacking the mutation. In one aspect, the expression level of the endogenous gene comprising the mutation is reduced by 25%-75% compared to the expression level of the endogenous gene lacking the mutation. In one aspect, the expression level of the endogenous gene comprising the mutation is reduced by 10%-50% compared to the expression level of the endogenous gene lacking the mutation.

[0177] In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by at least 1%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by at least 5%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by at least 10%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by at least 15%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by at least 20%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by at least 25%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by at least 50%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by at least 75%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by at least 90%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by at least 95%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by at least 100%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by at least 150%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by at least 200%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by at least 250%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by at least 300%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by at least 400%. In one aspect, the expression level of an endogenous gene comprising a mutation increases by at least 500% compared to the expression level of an endogenous gene lacking a mutation. In one aspect, the expression level of an endogenous gene comprising a mutation increases by at least 750% compared to the expression level of an endogenous gene lacking a mutation. In one aspect, the expression level of an endogenous gene comprising a mutation increases by at least 1000% compared to the expression level of an endogenous gene lacking a mutation.

[0178] In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by 1%-1000%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by 1%-750%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by 1%-500%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by 1%-400%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by 1%-300%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by 1%-200%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by 1%-100%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by 1%-75%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by 1%-50%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by 1%-25%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by 1%-10%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by 100%-1000%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by 100%-750%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by 100%-500%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by 100%-250%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by 750%-1000%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by 500%-1000%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by 250%-1000%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by 1%-100%. In one aspect, compared with the expression level of the endogenous gene lacking mutation, the expression level of the endogenous gene comprising mutation increases by 50%-100%.

[0179] The term "endogenous gene" or "natural gene" used herein refers to the gene derived from the tobacco genome."endogenous gene" is a gene that has not been modified by human action before. On the one hand, the endogenous gene is a nuclear gene. On the other hand, the endogenous gene is a mitochondrial gene. On the other hand, the endogenous gene is a chloroplast gene.

[0180] As used herein, "gene" refers to a polynucleotide that can produce a functional unit (such as, but not limited to, a protein or a small RNA molecule). A gene may include a promoter, an enhancer sequence, a leader sequence, a transcription start site, a transcription stop site, a polyadenylation site, one or more exons, one or more introns, a 5'-untranslated region (UTR), a 3'-UTR, or any combination thereof. A "gene sequence" may include a polynucleotide sequence encoding a promoter, an enhancer sequence, a leader sequence, a transcription start site, a transcription stop site, a polyadenylation site, one or more exons, one or more introns, a 5'-UTR, a 3'-UTR, or any combination thereof. On the one hand, a gene encodes a small RNA molecule or a precursor thereof. On the other hand, a gene encodes a protein or a polypeptide.

[0181] As used herein, "genomic locus" refers to a fixed position on a chromosome. In one aspect, the genomic locus comprises a polynucleotide encoding a gene. In one aspect, the genomic locus comprises a polynucleotide encoding an endogenous gene. In another aspect, the genomic locus comprises a polynucleotide encoding a transgenic. In one aspect, the genomic locus can be transcribed from DNA into RNA. In one aspect, the genomic locus encodes messenger RNA. In another aspect, the genomic locus encodes a small RNA molecule.

[0182] As generally understood in the art, the term "promoter" refers to a DNA sequence that contains an RNA polymerase binding site, a transcription start site and / or a TATA box and assists or promotes the transcription and expression of an associated transcribable polynucleotide sequence and / or a gene (or transgene). The promoter can be synthesized, altered or derived from a known or naturally occurring promoter sequence or other promoter sequence. The promoter can also include a chimeric promoter containing a combination of two or more heterologous sequences. Therefore, the promoter of the present application can include a variant of a promoter sequence that is similar but not identical to other promoter sequences known or provided herein.

[0183] The promoter that drives expression in all or most tissues of a plant is called a "constitutive" promoter. The promoter that drives expression in certain periods or stages of development is called a "developmental" promoter. Relative to other tissues of an organism, the promoter that drives enhanced expression in certain tissues of an organism is called a "tissue preferred" promoter. Therefore, a "tissue preferred" promoter causes relatively high or preferred expression in a specific tissue of a plant, but has a lower expression level in other tissues of the plant. As a non-limiting example, a "axillary bud preferred promoter" causes relatively high or preferred expression in axillary bud tissue, but can have a lower expression level in other parts of the plant (e.g., roots, leaves, stems). A promoter that is expressed in a specific tissue of an organism and rarely expressed or not expressed in other tissues is called a "tissue specific" promoter. As a non-limiting example, an "axillary bud specific promoter" drives expression in axillary bud tissue, and rarely expressed or undetectable expression in other plant tissue types. An "inducible" promoter is a promoter that initiates transcription in response to environmental stimuli (e.g., heat, cold, drought, light) or other stimuli (e.g., wound or chemical application).

[0184] In one aspect, the promoter provided herein is an axillary bud specific promoter. In another aspect, the promoter provided herein is axillary bud preferred promoter. 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.

[0185] 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.

[0186] In one aspect, the heterologous promoter comprises an axillary bud-specific promoter. In another aspect, the heterologous promoter comprises an axillary bud-preferred promoter. In one aspect, the heterologous promoter comprises a constitutive promoter. In one aspect, the heterologous promoter comprises an inducible promoter. In one aspect, the heterologous promoter comprises a developmental promoter.

[0187] As used herein, "operably connected" refers to a functional connection between two or more elements. For example, an operable connection between a target polynucleotide and a regulatory sequence (e.g., a promoter) is a functional connection that allows the expression of the target polynucleotide. Operably connected elements can be continuous or non-continuous. In one aspect, a promoter provided herein is operably connected to a heterologous nucleic acid molecule.

[0188] In one aspect, the heterologous promoter comprises a polynucleotide sequence that is at least 90% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109. In one aspect, the heterologous promoter comprises a polynucleotide sequence that is at least 91% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109. In one aspect, the heterologous promoter comprises a polynucleotide sequence that is at least 92% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109. In one aspect, the heterologous promoter comprises a polynucleotide sequence that is at least 93% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109. In one aspect, the heterologous promoter comprises a polynucleotide sequence that is at least 94% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109. In one aspect, the heterologous promoter comprises a polynucleotide sequence that is at least 95% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109. In one aspect, the heterologous promoter comprises a polynucleotide sequence that is at least 96% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109. In one aspect, the heterologous promoter comprises a polynucleotide sequence that is at least 97% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109. In one aspect, the heterologous promoter comprises a polynucleotide sequence that is at least 98% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109. In one aspect, the heterologous promoter comprises a polynucleotide sequence that is at least 99% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109. In one aspect, the heterologous promoter comprises a polynucleotide sequence that is 100% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109.

[0189] In one aspect, the axillary bud preferred promoter comprises a polynucleotide sequence that is at least 90% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109. In one aspect, the axillary bud preferred promoter comprises a polynucleotide sequence that is at least 95% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109. In one aspect, the axillary bud preferred promoter comprises a polynucleotide sequence that is at least 96% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109. In one aspect, the axillary bud preferred promoter comprises a polynucleotide sequence that is at least 97% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109. In one aspect, the axillary bud preferred promoter comprises a polynucleotide sequence that is at least 98% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109. In one aspect, the axillary bud preferred promoter comprises a polynucleotide sequence that is at least 99% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109. In one aspect, the axillary bud preferred promoter comprises a polynucleotide sequence that is 100% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109.

[0190] In one aspect, the axillary bud-specific promoter comprises a polynucleotide sequence that is at least 90% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109. In one aspect, the axillary bud-specific promoter comprises a polynucleotide sequence that is at least 95% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109. In one aspect, the axillary bud-specific promoter comprises a polynucleotide sequence that is at least 96% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109. In one aspect, the axillary bud-specific promoter comprises a polynucleotide sequence that is at least 97% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109. In one aspect, the axillary bud-specific promoter comprises a polynucleotide sequence that is at least 98% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109. In one aspect, the axillary bud-specific promoter comprises a polynucleotide sequence that is at least 99% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109. In one aspect, the axillary bud-specific promoter comprises a polynucleotide sequence that is 100% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89-109.

[0191] In one aspect, the present disclosure provides a modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB and a WRKY transcription factor operably linked to a heterologous promoter, wherein the modified tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping compared to a control tobacco plant when grown under comparable growth conditions.

[0192] In another aspect, the present disclosure provides a modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB and a WRKY transcription factor operably linked to a heterologous promoter.

[0193] On the other hand, the 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 a polypeptide, the polypeptide being selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB and a WRKY transcription factor; (b) selecting at least one tobacco cell comprising the recombinant DNA construct; and (c) regenerating a modified tobacco plant from the at least one tobacco cell selected in step (b), wherein when grown under comparable growth conditions, the modified tobacco plant does not comprise a tobacco fork or comprises a reduced tobacco fork after topping compared to a control tobacco plant lacking the recombinant DNA construct. In one aspect, the method further comprises (d) growing the modified tobacco plant regenerated in step (c). On the other hand, the method further comprises (e) hybridizing the modified tobacco plant grown in step (d) with a second tobacco plant; and (f) obtaining at least one seed from the hybridization in step (e).

[0194] In one aspect, the disclosure provides a method comprising transforming a tobacco cell with a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor. In one aspect, the method further comprises regenerating a modified tobacco plant from the tobacco cell, wherein the modified tobacco plant comprises the recombinant DNA construct.

[0195] In one aspect, the disclosure provides a recombinant DNA construct comprising a polynucleotide provided herein. As used herein, the term "recombinant DNA construct" refers to a construct formed by a laboratory method (such as molecular cloning) of genetic recombination. In one aspect, the recombinant DNA construct is synthetically produced. On the other hand, the recombinant DNA construct comprises a promoter operably connected to a heterologous polynucleotide sequence. In one aspect, the recombinant DNA construct comprises a polynucleotide sequence encoding an amino acid sequence. On the other hand, the recombinant DNA construct comprises a polynucleotide sequence encoding a small RNA or its precursor. In one aspect, the recombinant DNA construct comprises a plasmid. On the other hand, the recombinant DNA construct comprises a vector.

[0196] As used herein, the terms "vector" or "plasmid" are used interchangeably and refer to a circular double-stranded DNA molecule physically separated from chromosomal DNA. In one aspect, the plasmid or vector used herein is capable of replicating in vivo. As used herein, a "transformation vector" is a plasmid capable of transforming plant cells. In one aspect, the plasmid provided herein is a bacterial plasmid. In another aspect, the plasmid provided herein is an Agrobacterium Ti plasmid or is derived from an Agrobacterium Ti plasmid.

[0197] In one aspect, the vector provided herein comprises a promoter. In one aspect, the vector provided herein comprises an axillary bud-specific promoter. In one aspect, the vector provided herein comprises an axillary bud-preferred promoter. In another aspect, the vector provided herein comprises a small RNA. In another aspect, the vector provided herein comprises a small RNA precursor. In one aspect, the vector provided herein comprises an artificial miRNA. In another aspect, the vector provided herein comprises an artificial miRNA precursor. In another aspect, the vector provided herein comprises a sequence at least 80% identical to a sequence selected from SEQ ID NO: 1-44 and 89-113, or a fragment thereof. In another aspect, the vector provided herein comprises a sequence at least 85% identical to a sequence selected from SEQ ID NO: 1-44 and 89-113, or a fragment thereof. In another aspect, the vector provided herein comprises a sequence at least 90% identical to a sequence selected from SEQ ID NO: 1-44 and 89-113, or a fragment thereof. In another aspect, the vector provided herein comprises a sequence at least 95% identical to a sequence selected from SEQ ID NO: 1-44 and 89-113, or a fragment thereof. In another aspect, the vectors provided herein comprise a sequence that is at least 96% identical to a sequence selected from SEQ ID NOs: 1-44 and 89-113, or a fragment thereof. In another aspect, the vectors provided herein comprise a sequence that is at least 97% identical to a sequence selected from SEQ ID NOs: 1-44 and 89-113, or a fragment thereof. In another aspect, the vectors provided herein comprise a sequence that is at least 98% identical to a sequence selected from SEQ ID NOs: 1-44 and 89-113, or a fragment thereof. In another aspect, the vectors provided herein comprise a sequence that is at least 99% identical to a sequence selected from SEQ ID NOs: 1-44 and 89-113, or a fragment thereof. In another aspect, the vectors provided herein comprise a sequence that is 100% identical to a sequence selected from SEQ ID NOs: 1-44 and 89-113, or a fragment thereof.

[0198] As used herein, a "fragment" of a nucleic acid sequence or amino acid sequence comprises a contiguous sequence segment that comprises 1%-99.99% of the total length of the reference sequence. For example, if the nucleic acid sequence comprises 1000 nucleotides, a fragment of the nucleic acid sequence may comprise any number between 10-999 contiguous nucleotides of the reference sequence. The present disclosure explicitly provides fragments of each of SEQ ID NOs: 1-114, where SEQ ID NOs: 1-114 are cited.

[0199] Many methods for introducing recombinant DNA constructs into plant cells are known in the art, which can be used according to the methods of the present application to produce transgenic plant cells and plants. According to the methods of the present invention, any suitable method or technique known in the art for transforming plant cells can be used. Effective methods for transforming plants include bacterial-mediated transformation, such as Agrobacterium-mediated or Rhizobium-mediated transformation and microparticle bombardment-mediated transformation. A variety of methods are known in the art to transform explants with transformation vectors through bacterial-mediated transformation or microparticle bombardment, and then cultivate these explants to regenerate or develop transgenic plants. Other methods of plant transformation, such as microinjection, electroporation, vacuum infiltration, pressure, ultrasonic treatment, silicon carbide fiber stirring, polyethylene glycol (PEG)-mediated transformation, etc., are also known in the art. Depending on the method and explant used, the transgenic plants produced by these transformation methods can be mosaic or non-mosaic for the transformation event.

[0200] The method of transforming plant cells is well known to those of ordinary skill in the art. For example, U.S. Patent Nos. 5,550,318; 5,538,880 6,160,208; 6,399,861; and 6,153,812 found specific instructions for transforming plant cells by microprojectile bombardment (e.g., gene gun transformation) with particles coated with recombinant DNA; and 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 describe Agrobacterium-mediated transformation, which are all incorporated herein by reference in their entirety. Other methods for transforming plants can be found in, for example, Compendium of Transgenic Crop Plants (2009) Blackwell Publishing. Any suitable method known to those skilled in the art can be used to transform tobacco cells with any nucleic acid molecule provided herein.

[0201] In one aspect, the method for providing nucleic acid molecules to tobacco cells comprises Agrobacterium-mediated conversion. On the other hand, the method for providing nucleic acid molecules to cells comprises PEG-mediated conversion. On the other hand, the method for providing nucleic acid molecules to cells comprises gene gun conversion. On the other hand, the method for providing nucleic acid molecules to cells comprises liposome-mediated transfection (lipofection). On the other hand, the method for providing nucleic acid molecules to cells comprises slow virus transfection.

[0202] Lipofection is described in, for example, U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam TM Lipofectin TM). Cationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides include those of WO91 / 17424 and WO91 / 16024. Delivery can be performed to cells (eg, in vitro or ex vivo administration) or target tissues (eg, in vivo administration).

[0203] Any tobacco cell that expection can regenerate fertile tobacco plant is to be used to put into practice useful receptor cell of the present disclosure.In one aspect, the recombinant DNA construct is introduced tobacco cell.In one aspect, the recombinant DNA construct is introduced tobacco protoplast cell.On the other hand, the recombinant DNA construct is introduced tobacco callus cell.In one aspect, the recombinant DNA construct is introduced tobacco cell, and said tobacco cell is selected from seed cell, fruit cell, leaf cell, cotyledon cell, hypocotyl cell, meristem cell, embryo cell, endosperm cell, root cell, bud cell, stem cell, flower cell, inflorescence cell, stem cell, stalk cell, style cell, stigma cell, torus cell, petal cell, sepal cell, pollen cell, anther cell, silk cell, ovary cell, ovule cell, pericarp cell and phloem cell.

[0204] Callus can be derived from various tissue sources, including but not limited to immature embryos or parts of embryos, seedling apical meristems, microspores, etc. Those cells that can be proliferated as callus can be used as recipient cells for transformation. Actual transformation methods and materials (e.g., various culture media and receptor target cells, transformation of immature embryos and subsequent regeneration of fertile transgenic plants) for preparing transgenic plants of the present disclosure are disclosed in, for example, U.S. Patents 6,194,636 and 6,232,526 and U.S. Patent Application Publication 2004 / 0216189, all of which are incorporated herein by reference.

[0205] In one aspect, the present disclosure provides a modified tobacco plant comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of reducing expression of an endogenous mRNA encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein the modified tobacco plant comprises no or reduced tobacco forks after topping compared to a control tobacco plant when grown under comparable growth conditions.

[0206] In another aspect, the present disclosure provides a modified tobacco plant comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of reducing expression of an endogenous mRNA encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor.

[0207] In a further aspect, the 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 a polypeptide, the polypeptide being selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor; (b) selecting at least one tobacco cell comprising the recombinant DNA construct; and (c) regenerating a modified tobacco plant from the at least one tobacco cell selected in step (b), wherein when grown under comparable growth conditions, the modified tobacco plant does not comprise a tobacco fork or comprises a reduced tobacco fork after topping compared to a control tobacco plant lacking the recombinant DNA construct. In one aspect, the method further comprises (d) growing the modified tobacco plant regenerated in step (c). On the other hand, the method further comprises (e) hybridizing the modified tobacco plant grown in step (d) with a second tobacco plant; and (f) obtaining at least one seed from the hybridization in step (e).

[0208] In another aspect, the disclosure provides a method comprising transforming a tobacco cell with a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably connected to a nucleic acid encoding at least one small RNA molecule, the small RNA molecule being capable of reducing the expression of an endogenous mRNA encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor. In one aspect, the method also comprises regenerating a modified tobacco plant from the tobacco cell, wherein the modified tobacco plant comprises the recombinant DNA construct.

[0209] As used herein, the term "small RNA molecule" refers to any non-coding RNA molecule. In addition to providing small RNA molecules, the present disclosure also provides small RNA precursor molecules for each small RNA molecule.

[0210] In one aspect, the small RNA molecule comprises a length of 18 nucleotides to 30 nucleotides. On the other hand, the small RNA molecule comprises a length of 18 nucleotides to 24 nucleotides. On the other hand, the small RNA molecule comprises a length of 18 nucleotides to 22 nucleotides.

[0211] In another aspect, the small RNA molecule has a length of 18 nucleotides. In another aspect, the small RNA molecule has a length of 19 nucleotides. In another aspect, the small RNA molecule has a length of 20 nucleotides. In another aspect, the small RNA molecule has a length of 21 nucleotides. In another aspect, the small RNA molecule has a length of 22 nucleotides. In another aspect, the small RNA molecule has a length of 23 nucleotides. In another aspect, the small RNA molecule has a length of 24 nucleotides. In another aspect, the small RNA molecule has a length of 25 nucleotides. In another aspect, the small RNA molecule has a length of 26 nucleotides. In another aspect, the small RNA molecule has a length of 27 nucleotides. In another aspect, the small RNA molecule has a length of 28 nucleotides.

[0212] In another aspect, the small RNA molecule has a length of at least 18 nucleotides. In another aspect, the small RNA molecule has a length of at least 19 nucleotides. In another aspect, the small RNA molecule has a length of at least 20 nucleotides. In another aspect, the small RNA molecule has a length of at least 21 nucleotides. In another aspect, the small RNA molecule has a length of at least 22 nucleotides. In another aspect, the small RNA molecule has a length of at least 23 nucleotides. In another aspect, the small RNA molecule has a length of at least 24 nucleotides. In another aspect, the small RNA molecule has a length of at least 25 nucleotides. In another aspect, the small RNA molecule has a length of at least 26 nucleotides. In another aspect, the small RNA molecule has a length of at least 27 nucleotides. In another aspect, the small RNA molecule has a length of at least 28 nucleotides.

[0213] In one aspect, the small RNA molecule comprises 18 nucleotides to 30 nucleotides. In another aspect, the small RNA molecule comprises 18 nucleotides to 24 nucleotides. In another aspect, the small RNA molecule comprises 18 nucleotides to 21 nucleotides. In another aspect, the small RNA molecule comprises 21 nucleotides to 24 nucleotides. In another aspect, the small RNA molecule comprises 21 nucleotides to 30 nucleotides.

[0214] In one aspect, the small RNA molecules provided herein are microRNA (miRNA). In one aspect, the small RNA molecules provided herein are artificial miRNA. In another aspect, the small RNA molecules provided herein are small interfering RNA (siRNA). In another aspect, the small RNA molecules provided herein are heterochromatic siRNA (hc-siRNA). In another aspect, the small RNA molecules provided herein are Piwi-interacting RNA (piRNA). In one aspect, the small RNA molecules provided herein are double-stranded RNA (dsRNA). In another aspect, the small RNA molecules provided herein are hairpin double-stranded RNA (hp-dsRNA). In another aspect, the small RNA molecules provided herein are trans-acting siRNA (ta-siRNA). In another aspect, the small RNA molecules provided herein are naturally occurring antisense siRNA (nat-siRNA). In another aspect, the small RNA molecules provided herein are Cas9-guide RNA (gRNA). In another aspect, the small RNA molecules provided herein are Cpf1-gRNA. In another aspect, the small RNA molecules provided herein are CasX-gRNA. In another aspect, the small RNA molecules provided herein are Csm1-gRNA.

[0215] In one aspect, the small RNA molecule is selected from dsRNA, siRNA, ta-siRNA and miRNA. In another aspect, the small RNA molecule is selected from miRNA, siRNA, hc-siRNA, piRNA, dsRNA, hp-dsRNA, ta-siRNA, nat-siRNA, Cas9-gRNA, Cpf1-gRNA, CasX-gRNA and Csm1-gRNA.

[0216] In one aspect, the small RNA molecules provided herein comprise polynucleotide sequences having at least 90% identity or complementarity with endogenous mRNA. In one aspect, the small RNA molecules provided herein comprise polynucleotide sequences having at least 91% identity or complementarity with endogenous mRNA. In one aspect, the small RNA molecules provided herein comprise polynucleotide sequences having at least 92% identity or complementarity with endogenous mRNA. In one aspect, the small RNA molecules provided herein comprise polynucleotide sequences having at least 93% identity or complementarity with endogenous mRNA. In one aspect, the small RNA molecules provided herein comprise polynucleotide sequences having at least 94% identity or complementarity with endogenous mRNA. In one aspect, the small RNA molecules provided herein comprise polynucleotide sequences having at least 95% identity or complementarity with endogenous mRNA. In one aspect, the small RNA molecules provided herein comprise polynucleotide sequences having at least 96% identity or complementarity with endogenous mRNA. In one aspect, the small RNA molecules provided herein comprise polynucleotide sequences having at least 97% identity or complementarity with endogenous mRNA. In one aspect, the small RNA molecules provided herein comprise a polynucleotide sequence having at least 98% identity or complementarity with an endogenous mRNA. In one aspect, the small RNA molecules provided herein comprise a polynucleotide sequence having at least 99% identity or complementarity with an endogenous mRNA. In one aspect, the small RNA molecules provided herein comprise a polynucleotide sequence having 100% identity or complementarity with an endogenous mRNA.

[0217] In one aspect, the small RNA molecule comprises a polynucleotide sequence having at least 85% identity or complementarity with a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence having at least 90% identity or complementarity with a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence having at least 91% identity or complementarity with a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence having at least 92% identity or complementarity with a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence having at least 93% identity or complementarity with a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence having at least 94% identity or complementarity with a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is at least 95% identical or complementary to a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is at least 96% identical or complementary to a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is at least 97% identical or complementary to a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is at least 98% identical or complementary to a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is at least 99% identical or complementary to a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is 100% identical or complementary to a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113.

[0218] In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical or complementary to at least 16 consecutive nucleotides of a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical or complementary to at least 17 consecutive nucleotides of a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical or complementary to at least 18 consecutive nucleotides of a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical or complementary to at least 19 consecutive nucleotides of a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical or complementary to at least 20 consecutive nucleotides of a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical or complementary to at least 21 consecutive nucleotides of a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical or complementary to at least 22 consecutive nucleotides of a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical or complementary to at least 23 consecutive nucleotides of a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical or complementary to at least 24 consecutive nucleotides of a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical or complementary to at least 25 consecutive nucleotides of a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical or complementary to at least 26 consecutive nucleotides of a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical or complementary to at least 27 consecutive nucleotides of a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical or complementary to at least 28 consecutive nucleotides of a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical or complementary to at least 29 consecutive nucleotides of a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical or complementary to at least 30 consecutive nucleotides of a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113.

[0219] In one aspect, the present disclosure provides small RNAs capable of reducing the expression of polynucleotides encoding cell cycle proteins. In one aspect, the present disclosure provides small RNAs capable of reducing the translation of polynucleotides encoding cell cycle proteins. In one aspect, the present disclosure provides small RNAs capable of reducing the expression of polynucleotides encoding CDK. In one aspect, the present disclosure provides small RNAs capable of reducing the translation of polynucleotides encoding CDK. In one aspect, the present disclosure provides small RNAs capable of reducing the expression of polynucleotides encoding CDKI. In one aspect, the present disclosure provides small RNAs capable of reducing the translation of polynucleotides encoding CDKI. In one aspect, the present disclosure provides small RNAs capable of reducing the expression of polynucleotides encoding MYB. In one aspect, the present disclosure provides small RNAs capable of reducing the translation of polynucleotides encoding MYB. In one aspect, the present disclosure provides small RNAs capable of reducing the expression of polynucleotides encoding WRKY transcription factors. In one aspect, the present disclosure provides small RNAs capable of reducing the translation of polynucleotides encoding WRKY transcription factors.

[0220] MicroRNA (miRNA) is a non-protein coding RNA, usually with about 19 to about 25 nucleotides (usually about 20-24 nucleotides in plants), which directs the trans-cleavage of target transcripts, negatively regulating the expression of genes involved in various regulatory and developmental pathways. In some cases, miRNA is used to direct the homologous processing of siRNA primary transcripts.

[0221] Many microRNA genes (MIR genes) have been identified and are publicly available in databases ("miRBase", available online at microrna(dot)sanger(dot)ac(dot)uk / sequences; see also Griffiths-Jones et al. (2003) Nucleic Acids Res., 31:439-441). MIR genes have been reported to be present in intergenic regions, isolated and in clusters in the genome, but may also be located completely or partially within introns of other genes (protein-coding and non-coding). In at least some cases, transcription of MIR genes can be under the facilitating control of the MIR gene's own promoter. The primary transcript, called "pri-miRNA", can be quite large (several thousand bases) and can be polycistronic, containing one or more pre-miRNAs (folded structures containing a stem-loop arrangement that is processed into a mature miRNA) as well as the usual 5' "cap" and polyadenylation tail of the mRNA.

[0222] Transgenic expression of miRNA (whether naturally occurring sequence or artificial sequence) can be used to regulate the expression of miRNA target genes. Inclusion of miRNA recognition sites in transgenic expressed transcripts can also be used to regulate the expression of transcripts. Recognition sites for miRNAs 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 may not necessarily affect inhibition.

[0223] Because miRNA is an important regulatory element in eukaryotic organisms, transgenic inhibition of miRNA can be used to manipulate biological pathways and reactions. Various uses of miRNA, their precursors, their recognition sites, and their promoters are described in detail in U.S. Patent Application Publication 2006 / 0200878A1, which is incorporated herein by reference. Non-limiting examples of these uses include: (1) expressing natural miRNA or miRNA precursor sequences to inhibit target genes; (2) expressing artificial miRNA or miRNA precursor sequences to inhibit target genes; (3) expressing transgenics with miRNA recognition sites, wherein the transgenics are inhibited when mature miRNA is expressed; (4) expressing transgenics driven by miRNA promoters.

[0224] As Zeng et al. (2002) Mol. Cell, 9: 1327-1333 demonstrated, for nucleotides in the miRNA stem region of a miRNA precursor, designing an artificial miRNA sequence can be as simple as replacing a sequence complementary to the expected target. A non-limiting example of a general method for determining nucleotide changes in a natural miRNA sequence to prepare an engineered miRNA precursor comprises the following steps: (a) selecting a unique target sequence of at least 18 nucleotides specific to a target gene, for example, by using a sequence alignment tool such as BLAST of a tobacco cDNA and genomic DNA database (see, for example, Altschul et al. (1990) J. Mol. Biol., 215: 403-410; Altschul et al. (1997) Nucleic Acids Res., 25:3389-3402) to identify target transcript orthologs and any potential matches to unrelated genes, thereby avoiding unintentional silencing of non-target sequences; (b) analyzing target genes for unwanted sequences (e.g., matches to sequences from non-target species) and scoring each potential 19-mer fragment for: GC content, Reynolds score (see Reynolds et al. (2004) Nature Biotechnol., 22:326-330), and functional asymmetry characterized by a negative difference in free energy (".DELTA..DELTA.G" or "ΔΔG") (see Khvorova et al. (2003) Cell, 114:209-216). Preferably, 19-mers having all or most of the following characteristics are selected: (1) Reynolds score>4, (2) GC content of about 40% to about 60%, (3) negative ΔΔG, (4) terminal adenosine, (5) lack of 4 or more consecutive identical nucleotides; (6) located near the 3' end of the target gene; (7) minimal difference from the miRNA precursor transcript. It is reported that the position of each third nucleotide in the siRNA is particularly important in affecting the RNAi efficacy, and the algorithm "siExplorer" is publicly available at rna.chem.tu-tokyo.ac.jp / siexplorer.htm (see Katoh and Suzuki (2007) Nucleic Acids Res., 10.1093 / nar / gkl1120); (c) determining the reverse complementary sequence of the selected 19-mer for preparing the modified mature miRNA. The other nucleotide at position 20 preferably matches the selected target sequence, and the nucleotide at position 21 is preferably selected not to be paired to prevent the spread of silencing on the target transcript, or is selected to pair with the target sequence to promote the spread of silencing on the target sequence; and (d) introducing the artificial miRNA into the plant.

[0225] In one aspect, the artificial miRNA provided herein is complementary to at least 18 consecutive nucleotides of a nucleic acid sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the artificial miRNA provided herein is complementary to at least 19 consecutive nucleotides of a nucleic acid sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the artificial miRNA provided herein is complementary to at least 20 consecutive nucleotides of a nucleic acid sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the artificial miRNA provided herein is complementary to at least 21 consecutive nucleotides of a nucleic acid sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the artificial miRNA provided herein is complementary to at least 22 consecutive nucleotides of a nucleic acid sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the artificial miRNA provided herein is complementary to at least 23 consecutive nucleotides of a nucleic acid sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the artificial miRNA provided herein is complementary to at least 24 consecutive nucleotides of a nucleic acid sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the artificial miRNA provided herein is complementary to at least 25 consecutive nucleotides of a nucleic acid sequence selected from SEQ ID NOs: 1-44 and 113. In one aspect, the artificial miRNA provided herein is complementary to at least 26 consecutive nucleotides of a nucleic acid sequence selected from SEQ ID NOs: 1-44 and 113.

[0226] In one aspect, the artificial miRNA provided herein is at least 75% complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the artificial miRNA provided herein is at least 80% complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the artificial miRNA provided herein is at least 85% complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the artificial miRNA provided herein is at least 90% complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the artificial miRNA provided herein is at least 91% complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the artificial miRNA provided herein is at least 92% complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the artificial miRNA provided herein is at least 93% complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the artificial miRNA provided herein is at least 94% complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the artificial miRNA provided herein is at least 95% complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the artificial miRNA provided herein is at least 96% complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the artificial miRNA provided herein is at least 97% complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the artificial miRNA provided herein is at least 98% complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the artificial miRNA provided herein is at least 99% complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the artificial miRNA provided herein is 100% complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113.

[0227] In one aspect, the artificial miRNA provided herein reduces or eliminates RNA transcription or protein translation of a target gene.

[0228] In one aspect, the artificial miRNA or its precursor is operably connected to an axillary bud specific promoter. On the other hand, the artificial miRNA or its precursor is operably connected to an axillary bud preferred promoter. On the other hand, the artificial miRNA or its precursor is operably connected to a promoter comprising a sequence selected from SEQ ID NO1:89-109 and a fragment thereof.

[0229] Tobacco is known in the art as a plant from the family Solanaceae. As used herein, the tobacco plant can be from any plant of the genus Nicotiana, including but not limited to 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 224063; Nicotiana glutinosa PI 555507; Nicotiana goodspeedii PI 241012; Nicotiana gossei PI 230953; Nicotiana hesperis PI 271991; Nicotiana knightiana PI 224063; Nicotiana glutinosa PI 555507; Nicotiana excelsior ... 555527; Nicotiana maritima PI 555535; Nicotiana megalosiphon PI 555536; Nicotiana nudicaulis PI 555540; Nicotiana paniculata PI 555545; Nicotiana plumbaginifolia PI 555548; Nicotiana arepanda PI 555552; ​​Nicotiana rustica; Nicotiana suaveolens PI 230960; Nicotiana sylvestris PI 555569; Nicotiana tomentosa PI 266379; Nicotiana tomentosiformis; and Nicotiana trigonophylla) PI555572. In one aspect, the tobacco plant described herein is a Nicotiana trigonophylla plant.

[0230] In one aspect, the modified tobacco plants, seeds, cells, hybrids, varieties or lines provided herein are derived essentially from or are in the genetic background of: BU 64, CC 101, CC 200, CC 13, CC 27, CC 33, CC 35, CC 37, CC 65, CC 67, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, CC 1063, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CU 263, DF911, GL 26H, GL338, GL 350, GL 395, GL 600, GL 737, GL 939, GL 973, GF 157, GF 318, RJR 901, HB 04P, K149, K 326, K 346, K 358, K394, K 399, K 730, NC 196, NC 37NF, NC 471, NC 55, NC 92, NC2326, NC 95, NC 925, PVH 1118, PVH 1452, PVH 2110, PVH 2254, PVH 2275, VA 116, VA119, KDH 959, KT 200, KT204LC, KY 10, KY 14, KY 160, KY 17, KY 171, KY 907, KY 907LC, KTY14 x L8 LC, Little Crittenden, McNair 373, McNair 944, male sterile KY 14x L8, Narrow Leaf Madole, MS KY171, Narrow Leaf Madole(phph), MS Narrow Leaf Madole, MSTND950, PD 7302LC, PD 7305LC, PD 7309LC, PD 7312LC, PD 7318LC, PD 7319LC, MSTKS2002, TKF 2002, TKF 6400, TKF 4028, TKF 4024, KT206LC, KT209LC, KT210LC, KT212LC, NC 100, NC102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH 129, NC 2002, Neal Smith Madole, OXFORD 207, ‘Perique’, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81, RG H51, RGH 4, RGH 51, RS1410, Speight 168, Speight 172, Speight179, Speight 210, Speight 220, Speight225, Speight 227, Speight 234, Speight G-28, Speight G-70, Speight H-6, SpeightH20, Speight NF3, TI1406, TI 1269, TN 86, TN86LC, TN 90, TN90LC, TN 97, TN97LC, TND94, TN D950, a TR (Tom Rosson) Madole, VA 309, VA 359, or any commercial tobacco variety according to standard tobacco breeding techniques known in the art.

[0231] In one aspect, the modified tobacco plant is selected from the group consisting of BU 64 plants, CC 101 plants, CC 200 plants, CC 13 plants, CC 27 plants, CC 33 plants, CC 35 plants, CC 37 plants, CC 65 plants, CC 67 ​​plants, CC 301 plants, CC 400 plants, CC 500 plants, CC 600 plants, CC 700 plants, CC 800 plants, CC 900 plants, CC 1063 plants, Coker 176 plants, Coker 319 plants, Coker 371 Gold plants, Coker 48 plants, CU 263 plants, DF911 plants, Galpao plants, GL 26H plants, GL 338 plants, GL 350 plants, GL 395 plants, GL 600 plants, GL 737 plants, GL 939 plants, GL 973 plants, GF 157 plants, GF 158 plants, GF 161 plants, GF 162 plants, GF 163 plants, GF 164 plants, GF 165 plants, GF 166 plants, GF 167 plants, GF 168 plants, GF 169 plants, GF 169 plants, GF 168 plants, GF 169 plants 318 plants, RJR 901 plants, HB 04P plants, K 149 plants, K 326 plants, K 346 plants, K 358 plants, K394 plants, K 399 plants, K 730 plants, NC196 plants, NC 37NF plants, NC 471 plants, NC 55 plants, NC 92 plants, NC2326 plants, NC 95 plants, NC925 plants, PVH 1118 plants, PVH 1452 plants, PVH 2110 plants, PVH 2254 plants, PVH 2275 plants, VA116 plants, VA 119 plants, KDH 959 plants, KT 200 plants, KT204LC plants, KY 10 plants, KY 14 plants, KY160 plants, KY 17 plants, KY 171 plants, KY 907 plants, KY 907LC plants, KTY14 x L8 LC plants, LittleCrittenden plants, McNair 373 plants, McNair 944 plants, male sterile KY 14 x L8 plants, NarrowLeaf Madole plants, MS KY171 plants, Narrow Leaf Madole (phph) plants, MS Narrow Leaf Madole plants, MS TND950 plants, PD 7302LC plants, PD 7305LC plants, PD 7309LC plants, PD 7312LC plants, PD 7318LC plants, PD 7319LC plants, MSTKS2002 plants, TKF 2002 plants, TKF 6400 plants, TKF4028 plants, TKF 4024 plants, KT206LC plants, KT209LC plants, KT210LC plants, KT212LC plants, NC 100 plants, NC 102 Plants, NC2000 plants, NC 291 plants, NC 297 plants, NC 299 plants, NC 3 plants, NC 4 plants, NC 5 plants, NC 6 plants, NC7 plants, NC 606 plants, NC 71 plants, NC 72 plants, NC 810 plants, NC BH129 plants, NC 2002 plants, Neal Smith Madole plants, OXFORD 207 plants, 'Perique' plants, PVH03 plants, PVH09 plants, PVH19 plants, PVH50 plants, PVH51 plants, R 610 plants, R 630 plants, R 7-11 plants, R7-12 plants, RG 17 plants, RG 81 plants, RG H51 plants, RGH 4 plants, RGH 51 plants, RS1410 plants, Speight 168 plants, Speight 172 plants, Speight 179 plants, Speight 210 plants, Speight 220 plants, Speight 225 plants, Speight 227 plants, Speight 234 plants, Speight G-28 plants, Speight G-70 plants, Speight H-6 plants, Speight H20 plants, Speight NF3 plants, TI 1406 plants, TI 1269 plants, TN 86 plants, TN86LC plants, TN 90 plants, TN90LC plants, TN 97 plants, TN97LC plants, TN D94 plants, TN D950 plants, TR (Tom Rosson) Madole plants, VA 309 plants, and VA 359 plants.

[0232] In another aspect, the modified tobacco cell is selected from BU 64 cells, CC 101 cells, CC 200 cells, CC 13 cells, CC 27 cells, CC 33 cells, CC 35 cells, CC 37 cells, CC 65 cells, CC 67 ​​cells, CC 301 cells, CC 400 cells, CC 500 cells, CC 600 cells, CC 700 cells, CC 800 cells, CC 900 cells, CC 1063 cells, Coker 176 cells, Coker 319 cells, Coker 371 Gold cells, Coker 48 cells, CU 263 cells, DF911 cells, Galpao cells, GL 26H cells, GL 338 cells, GL 350 cells, GL 395 cells, GL 600 cells, GL 737 cells, GL 939 cells, GL 973 cells, GF 157 ... 318 cells, RJR 901 cells, HB 04P cells, K 149 cells, K 326 cells, K 346 cells, K 358 cells, K394 cells, K 399 cells, K 730 cells, NC196 cells, NC 37NF cells, NC 471 cells, NC 55 cells, NC 92 cells, NC2326 cells, NC 95 cells, NC925 cells, PVH 1118 cells, PVH 1452 cells, PVH 2110 cells, PVH 2254 cells, PVH 2275 cells, VA116 cells, VA 119 cells, KDH 959 cells, KT 200 cells, KT204LC cells, KY 10 cells, KY 14 cells, KY160 cells, KY 17 cells, KY 171 cells, KY 907 cells, KY 907LC cells, KTY14 x L8 LC cells, LittleCrittenden cells, McNair 373 cells, McNair 944 cells, male sterile KY 14x L8 cells, NarrowLeaf Madole cells, MS KY171 cells, Narrow Leaf Madole (phph) cells, MS Narrow LeafMadole cells, MS TND950 cells, a PD 7302LC cell, PD 7305LC cell, PD 7309LC cell, PD7312LC cell, PD 7318LC cell, PD 7319LC cell, MSTKS2002 cells, TKF 2002 cells, TKF 6400 cells, TKF 4028 cells, TKF 4024 cells, KT206LC cell, KT209LC cell, KT210LC cell, a KT212LC cell, an NC100 cells, NC 102 cells, NC 2000 cells, NC 291 cells, NC 297 cells, NC 299 cells, NC3 cells, NC 4 cells, NC 5 cells, NC 6 cells, NC7 cells, NC 606 cells, NC 71 cells, NC 72 cells, NC810 cells, NC BH 129 cells, NC 2002 cells, Neal Smith Madole cells, OXFORD 207 cells, 'Perique' cells, PVH03 cells, PVH09 cells, PVH19 cells, PVH50 cells, PVH51 cells, R 610 cells, R630 cells, R 7-11 cells, R 7-12 cells, RG 17 cells, RG 81 cells, RG H51 cells, RGH 4 cells, RGH 51 cells, RS1410 cells, Speight 168 cells, Speight 172 cells, Speight 179 cells, Speight 210 cells, Speight 220 cells, Speight 225 cells, Speight 227 cells, Speight 234 cells, Speight G-28 cells, Speight G-70 cells, Speight H-6 cells, Speight H20 cells, Speight NF3 cells, TI1406 cells, TI 1269 cells, TN 86 cells, TN86LC cells, TN 90 cells, TN90LC cells, TN 97 cells, TN97LC cells, TN D94 cells, TN D950 cells, TR (Tom Rosson) Madole cells, VA 309 cells and VA 359 cells.

[0233] In another aspect, the modified tobacco seeds are selected from BU 64 seeds, CC 101 seeds, CC 200 seeds, CC 13 seeds, CC 27 seeds, CC 33 seeds, CC 35 seeds, CC 37 seeds, CC 65 seeds, CC 67 ​​seeds, CC 301 seeds, CC 400 seeds, CC 500 seeds, CC 600 seeds, CC 700 seeds, CC 800 seeds, CC 900 seeds, CC 1063 seeds, Coker 176 seeds, Coker 319 seeds, Coker 371 Gold seeds, Coker 48 seeds, CU 263 seeds, DF911 seeds, Galpao seeds, GL 26H seeds, GL 338 seeds, GL 350 seeds, GL 395 seeds, GL 600 seeds, GL 737 seeds, GL 939 seeds, GL 973 seeds, GF 157 seeds, GF 158 seeds, GF 161 seeds, GF 163 seeds, GF 164 seeds, GF 165 seeds, GF 166 seeds, GF 167 seeds, GF 168 seeds, GF 169 seeds, GF 170 seeds, GF 171 seeds, GF 172 seeds, GF 173 seeds, GF 174 seeds, GF 175 seeds, GF 176 seeds 318 seeds, RJR 901 seeds, HB 04P seeds, K 149 seeds, K 326 seeds, K 346 seeds, K 358 seeds, K394 seeds, K 399 seeds, K 730 seeds, NC196 seeds, NC 37NF seeds, NC 471 seeds, NC 55 seeds, NC 92 seeds, NC2326 seeds, NC 95 seeds, NC925 seeds, PVH 1118 seeds, PVH 1452 seeds, PVH 2110 seeds, PVH 2254 seeds, PVH 2275 seeds, VA116 seeds, VA 119 seeds, KDH 959 seeds, KT 200 seeds, KT204LC seeds, KY 10 seeds, KY 14 seeds, KY160 seeds, KY 17 seeds, KY 171 seeds, KY 907 seeds, KY 907LC seed, KTY14 x L8 LC seed, LittleCrittenden seed, McNair 373 seed, McNair 944 seed, male sterile KY 14x L8 seed, NarrowLeaf Madole seed, MS KY171 seed, Narrow Leaf Madole (phph) seed, MS Narrow LeafMadole seed, MS TND950 seed, a PD 7302LC seed, PD 7305LC seed, PD 7309LC seed, PD7312LC seed, PD 7318LC seed, PD 7319LC seed, MSTKS2002 seed, TKF 2002 seed, TKF 6400 seed, TKF 4028 seed, TKF 4024 seed, KT206LC seed, KT209LC seed, KT210LC seed, a KT212LCseed, an NC100 seeds, NC 102 seeds, NC 2000 seeds, NC 291 seeds, NC 297 seeds, NC 299 seeds, NC3 seeds, NC 4 seeds, NC 5 seeds, NC 6 seeds, NC7 seeds, NC 606 seeds, NC 71 seeds, NC 72 seeds, NC810 seeds, NC BH 129 seeds, NC 2002 seeds, Neal Smith Madole seeds, OXFORD 207 seeds, 'Perique' seeds, PVH03 seeds, PVH09 seeds, PVH19 seeds, PVH50 seeds, PVH51 seeds, R 610 seeds, R 630 seeds, R 7-11 seeds, R 7-12 seeds, RG 17 seeds, RG 81 seeds, RG H51 seeds, RGH 4 seeds, RGH 51 seeds, RS1410 seeds, Speight 168 seeds, Speight 172 seeds, Speight 179 seeds, Speight 210 seeds, Speight 220 seeds, Speight 225 seeds, Speight 227 seeds, Speight 234 seeds, Speight G-28 seeds, Speight G-70 seeds, Speight H-6 seeds, Speight H20 seeds, Speight NF3 seeds, TI1406 seeds, TI 1269 seeds, TN 86 seeds, TN86LC seeds, TN 90 seeds, TN90LC seeds, TN 97 seeds, TN97LC seeds, TN D94 seeds, TN D950 seeds, TR (Tom Rosson) Madole seeds, VA 309 seeds and VA 359 seeds.

[0234] As used herein, " tobacco plant " refers to whole tobacco plant.Tobacco cell or tobacco tissue culture derived from tobacco plant can comprise any tobacco plant part or tobacco plant organ (for example leaf, stem, root etc.), tobacco plant tissue, tobacco seed, tobacco plant cell and / or its offspring.Progeny plant can be from any filial generation, for example F1, F2, F3, F4, F5, F6, F7 etc.Tobacco plant cell is the biological cell of tobacco plant, takes from tobacco plant or derives from the cell of tobacco plant by cultivating.As used herein, " seedling " refers to be equal to or less than the tobacco plant of 14 days after sprouting.

[0235] In one aspect, plant component provided herein includes but is not limited to leaf, stem, root, seed, flower, pollen, anther, ovule, pedicel, fruit, meristem, cotyledon, hypocotyl, fruit pod, embryo, endosperm, explant, callus, tissue culture, tender shoot, cell and protoplast.In further aspect, the disclosure provides tobacco plant cell, tissue and the organ that are not propagation material and natural propagation of mediated plant.On the other hand, the disclosure also provides tobacco plant cell, tissue and the organ that are propagation material and natural propagation of mediated plant.On the other hand, the present invention provides tobacco plant cell, tissue and the organ that can not maintain self by photosynthesis.On the other hand, the disclosure provides somatic plant cell.Contrary to reproductive cell, somatic cell does not mediate plant propagation.

[0236] The tobacco cell, tobacco tissue and tobacco organ provided can be from seed, fruit, leaf, cotyledon, hypocotyl, meristem, embryo, endosperm, root, tender shoot, stem, fruit pod, flower, inflorescence, petiole, pedicel, style, stigma, receptacle, petal, sepal, pollen, anther, filament, ovary, ovule, pericarp, phloem and vascular tissue.On the other hand, the disclosure provides tobacco plant chloroplast.In further aspect, the disclosure provides epidermal cell, stomatal cell, trichome cell, root hair or storage root.

[0237] In one aspect, the disclosure provides tobacco protoplast cells. In another aspect, the disclosure provides tobacco callus cells. In another aspect, the disclosure provides tobacco seed cells. In another aspect, the disclosure provides tobacco fruit cells. In another aspect, the disclosure provides tobacco leaf cells. In another aspect, the disclosure provides tobacco cotyledon cells. In another aspect, the disclosure provides tobacco hypocotyl cells. In another aspect, the disclosure provides tobacco meristem cells. In another aspect, the disclosure provides tobacco embryo cells. In another aspect, the disclosure provides tobacco root cells. In another aspect, the disclosure provides tobacco stem cells. In another aspect, the disclosure provides tobacco flower cells. In another aspect, the disclosure provides tobacco inflorescence cells. In another aspect, the disclosure provides tobacco stem cells. In another aspect, the disclosure provides tobacco pedicel cells. In another aspect, the disclosure provides tobacco style cells. In another aspect, the disclosure provides tobacco stigma cells. In another aspect, the disclosure provides tobacco receptacle cells. In another aspect, the disclosure provides tobacco petal cells. In another aspect, the disclosure provides tobacco sepal cells. In another aspect, the disclosure provides tobacco pollen cells. In another aspect, the disclosure provides tobacco anther cells. In another aspect, the present disclosure provides tobacco filament cells. In another aspect, the present disclosure provides tobacco ovary cells. In another aspect, the present disclosure provides tobacco ovule cells. In another aspect, the present disclosure provides tobacco pericarp cells. In another aspect, the present disclosure provides tobacco phloem cells.

[0238] The present disclosure provides modified tobacco plants, modified tobacco plant parts, modified tobacco seeds and modified tobacco cells and preparation methods thereof. In one aspect, the present disclosure provides tobacco leaves of modified tobacco plants. In another aspect, the present disclosure provides tobacco seeds of modified tobacco plants. In another aspect, the present disclosure provides tobacco stems of modified tobacco plants. In another aspect, the present disclosure provides tobacco plant parts of modified tobacco plants. In another aspect, the present disclosure provides tobacco cells of modified tobacco plants. In another aspect, the present disclosure provides dried tobacco leaves of modified tobacco plants. In yet another aspect, the present disclosure provides cured tobacco leaves of modified tobacco plants. In another aspect, the present disclosure provides fermented tobacco leaves of modified tobacco plants.

[0239] On the other hand, the present disclosure provides alkaloids extracted from modified tobacco plants. On the other hand, the present disclosure provides nicotine extracted from modified tobacco plants. On the other hand, the present disclosure provides anatabine extracted from modified tobacco plants. On the other hand, the present disclosure provides anabasine extracted from modified tobacco plants. On the other hand, the present disclosure provides nornicotine extracted from modified tobacco plants.

[0240] In one aspect, modified tobacco plant, plant part, seed, cell or genome are cis-oriented.As used herein, " cis-oriented " refers to the genetic modification of plant, vegetable cell or plant genome, wherein all components (for example, promoter, donor nucleic acid, selection gene) only have plant origin (that is, do not use the component of non-plant origin).Transgenic plant, vegetable cell and plant genome provided herein can produce ready-to-use tobacco strain.On the other hand, modified tobacco plant provided herein does not comprise non-tobacco genetic material or sequence.

[0241] In one aspect, when grown under comparable conditions, the modified plant comprises increased leaf yield quality compared to a control tobacco plant.In one aspect, the leaf yield is selected from the group consisting of fresh leaf yield quality, dry leaf yield quality, and cured leaf yield quality.

[0242] In one aspect, when growing under comparable conditions, compared with a control tobacco plant, the modified plant comprises an increase of at least 0.5% leaf yield quality. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, the modified plant comprises an increase of at least 0.5% fresh leaf yield quality. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, the modified plant comprises an increase of at least 0.5% dry leaf yield quality. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, the modified plant comprises an increase of at least 0.5% cured leaf yield quality. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, the modified plant comprises an increase of at least 1% leaf yield quality. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, the modified plant comprises an increase of at least 1% fresh leaf yield quality. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, the modified plant comprises an increase of at least 1% dry leaf yield quality. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified plants comprise the quality of cured leaves output that increases by at least 1%. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified plants comprise the quality of leaf output that increases by at least 2%. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified plants comprise the quality of fresh leaves output that increases by at least 2%. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified plants comprise the quality of dry leaves output that increases by at least 2%. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified plants comprise the quality of cured leaves output that increases by at least 2%. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified plants comprise the quality of leaf output that increases by at least 3%. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified plants comprise the quality of fresh leaves output that increases by at least 3%. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, modified plant comprises a dry leaf yield quality that increases at least 3%. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, modified plant comprises a cured leaf yield quality that increases at least 3%. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, modified plant comprises a leaf yield quality that increases at least 4%. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, modified plant comprises a fresh leaf yield quality that increases at least 4%. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, modified plant comprises a dry leaf yield quality that increases at least 4%.In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified plants comprise the quality of cured leaves output that increases at least 4%. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified plants comprise the quality of leaf output that increases at least 5%. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified plants comprise the quality of fresh leaves output that increases at least 5%. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified plants comprise the quality of dry leaves output that increases at least 5%. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified plants comprise the quality of cured leaves output that increases at least 5%. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified plants comprise the quality of leaf output that increases at least 250%. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified plants comprise the quality of fresh leaves output that increases at least 250%. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified plants comprise the dry leaf yield quality that increases at least 250%. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified plants comprise the cured leaf yield quality that increases at least 250%. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified plants comprise the leaf yield quality that increases at least 25%. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified plants comprise the fresh leaf yield quality that increases at least 25%. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified plants comprise the dry leaf yield quality that increases at least 25%. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified plants comprise the cured leaf yield quality that increases at least 25%. In one aspect, when growing under comparable conditions, compared with control tobacco plants, modified plants comprise the leaf yield quality that increases at least 50%. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, modified plant comprises the fresh leaf yield quality that increases at least 50%. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, modified plant comprises the dry leaf yield quality that increases at least 50%. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, modified plant comprises the cured leaf yield quality that increases at least 50%. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, modified plant comprises the leaf yield quality that increases at least 75%. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, modified plant comprises the fresh leaf yield quality that increases at least 75%.In one aspect, when growing under comparable conditions, compared with a control tobacco plant, the modified plant comprises a dry leaf yield quality that increases at least 75%. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, the modified plant comprises a cured leaf yield quality that increases at least 75%. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, the modified plant comprises a leaf yield quality that increases at least 100%. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, the modified plant comprises a fresh leaf yield quality that increases at least 100%. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, the modified plant comprises a dry leaf yield quality that increases at least 100%. In one aspect, when growing under comparable conditions, compared with a control tobacco plant, the modified plant comprises a cured leaf yield quality that increases at least 100%.

[0243] "Curing" is a kind of aging process, which reduces moisture and causes the destruction of chlorophyll, so that tobacco leaves appear golden yellow, and starch is also converted into sugar. Therefore, compared with the green leaves of the harvest, cured tobacco has a higher reducing sugar content and a lower starch content. In one aspect, the tobacco plant or plant component provided herein can be cured using conventional methods, such as baking and curing, barn curing (barn-cured), open fire baking and curing, air-drying and curing or sun-curing and curing. For the description of various types of curing methods, see, for example (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). Cured tobacco is usually aged for several years (for example 2 to 5 years) in wooden barrels (for example vats) or cardboard boxes under compression conditions of 10% to about 25% moisture content. Referring to U.S. Patent numbers 4,516,590 and 5,372,149. Then, the tobacco of curing and aging can be further processed. Further processing includes curing the tobacco under vacuum with or without the introduction of steam at various temperatures, pasteurization, and fermentation.

[0244] In one aspect, the cured tobacco leaves provided herein are selected from air-cured tobacco leaves, open flame cured tobacco leaves, sun-cured tobacco leaves and cured tobacco leaves. On the other hand, the cured tobacco materials provided herein are selected from air-cured tobacco materials, open flame cured tobacco materials, sun-cured tobacco materials and cured tobacco materials. In one aspect, the cured tobacco leaves are from the tobacco varieties selected from the following: cured and cured varieties, golden varieties, burley varieties, Virginia varieties, Maryland varieties, dark varieties, oriental varieties and Turkish varieties. On the other hand, the cured tobacco materials are from the tobacco varieties selected from the following: cured and cured varieties, golden varieties, burley varieties, Virginia varieties, Maryland varieties, dark varieties, oriental varieties and Turkish varieties.

[0245] Typically, fermentation is characterized by high initial moisture content, heat generation and dry weight loss of 10 to 20%. 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). Cured, aged and fermented tobacco (e.g., cut, shred, puffed or mixed) can be further processed. See, for example, U.S. Patent Nos. 4,528,993; 4,660,577 and 4,987,907. In one aspect, the cured tobacco material of the present disclosure is cured by baking, cured by sun, cured by air or cured by open fire.

[0246] In one aspect, the tobacco plants, seeds, plant parts, plant cells and plant genomes provided herein are from a tobacco type selected from: flue-cured tobacco, sun-cured tobacco, air-cured tobacco, dark air-cured tobacco and dark open fire-cured tobacco. In another aspect, the tobacco plants, seeds, plant parts, plant cells and plant genomes provided herein are from a tobacco type selected from: burley tobacco, Maryland tobacco, golden tobacco, Virginia tobacco, Oriental tobacco, Turkish tobacco and tobacco.

[0247] In one aspect, the modified tobacco plant provided herein is a tobacco variety selected from the group consisting of a flue-cured variety, a golden variety, a burley variety, a Virginia variety, a Maryland variety, variety, dark variety, oriental variety and turkish variety.

[0248] In one aspect, the modified tobacco plant provided herein is selected from the group consisting of a flue-cured tobacco plant, a golden tobacco plant, a burley tobacco plant, a Virginia tobacco plant, a Maryland tobacco plant, Variety tobacco plants, dark variety tobacco plants, oriental variety tobacco plants and Turkish variety tobacco plants. In one aspect, the modified tobacco cells provided herein are selected from the group consisting of flue-cured variety tobacco cells, golden variety tobacco cells, burley variety tobacco cells, Virginia variety tobacco cells, Maryland variety tobacco cells, Variety tobacco cells, dark variety tobacco cells, oriental variety tobacco cells and Turkish variety tobacco cells. In one aspect, the modified tobacco plant parts provided herein are selected from the group consisting of flue-cured variety tobacco plant parts, golden variety tobacco plant parts, burley variety tobacco plant parts, Virginia variety tobacco plant parts, Maryland variety tobacco plant parts, Variety tobacco plant parts, dark variety tobacco plant parts, oriental variety tobacco plant parts and Turkish variety tobacco plant parts. In one aspect, the modified tobacco seeds provided herein are selected from the group consisting of cured variety tobacco seeds, golden variety tobacco seeds, burley variety tobacco seeds, Virginia variety tobacco seeds, Maryland variety tobacco seeds, variety tobacco seeds, dark variety tobacco seeds, oriental variety tobacco seeds and turkish variety tobacco seeds.

[0249] As used herein, by hybridizing two kinds of plants from different varieties or species, produce " hybrid ", make progeny comprise the hereditary material from each parent.Skilled technicians recognize that also can generate higher order hybrid.For example, can prepare the first hybrid by kind C and kind D hybridizing to produce C x D hybrid, and can prepare the second hybrid by kind E and kind F hybridizing to produce E x F hybrid.Can further hybridize the first and second hybrid to produce the high order hybrid (C x D) x (E x F) that comprises the hereditary information from all four parental varieties.In one aspect, the modified tobacco plant provided herein is a hybrid tobacco plant.On the other hand, the modified tobacco seed provided herein is a hybrid tobacco seed.

[0250] As used herein, the term "hybridization" refers to the intentional mating of two plants. On the one hand, hybridization comprises that the first tobacco plant is pollinated and / or fertilized by the second tobacco plant. The two tobacco plants that are hybridized can be distant relatives, close relatives or identical. In one aspect, the two tobacco plants that are hybridized are all modified tobacco plants. On the one hand, the two tobacco plants that are hybridized are identical tobacco varieties. In one aspect, the two tobacco plants that are hybridized are two different tobacco varieties. In one aspect, one of the two tobacco plants that are hybridized is male sterile. In one aspect, one of the two tobacco plants that are hybridized is female sterile. In one aspect, at least one of the two tobacco plants that are hybridized is a hybrid tobacco plant. In one aspect, at least one of the two tobacco plants that are hybridized is a modified tobacco plant.

[0251] In one aspect, the present disclosure provides a method for producing a modified tobacco plant, comprising: (a) hybridizing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety, wherein the at least one tobacco plant of the first tobacco variety comprises a mutation in an endogenous gene encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein the mutation is not present in an endogenous gene of a control tobacco plant of the same variety, and wherein when grown under comparable growing conditions, the at least one tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping compared to the control tobacco plants; and (b) selecting offspring tobacco plants that exhibit no tobacco forks or reduced tobacco forks after topping compared to control tobacco plants of the same hybrid grown under comparable growing conditions.

[0252] In another aspect, the present disclosure provides a method for producing a modified tobacco plant, comprising: (a) hybridizing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein when grown under comparable growth conditions, the at least one tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping compared to a control tobacco plant; and (b) selecting an offspring tobacco plant that exhibits no tobacco forks or reduced tobacco forks after topping compared to a control tobacco plant of the same hybrid grown under comparable growth conditions.

[0253] In one aspect, the present disclosure provides a method for producing a modified tobacco plant, comprising: (a) hybridizing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of reducing expression of an endogenous mRNA encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein when grown under comparable growth conditions, the at least one tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping compared to a control tobacco plant when grown under comparable growth conditions; and (b) selecting an offspring tobacco plant that exhibits no tobacco forks or reduced tobacco forks after topping compared to a control tobacco plant of the same hybrid grown under comparable growth conditions.

[0254] In one aspect, tobacco variety provided herein is male sterility. In another aspect, tobacco variety provided herein is cytoplasmic male sterility (CMS). In one aspect, modified tobacco plant provided herein is male sterility. In another aspect, modified tobacco plant provided herein is cytoplasmic male sterility. Male sterile tobacco plant can be produced by any method known in the art. The method for producing male sterile tobacco is described in Wernsman, EA, and Rufty, RC1987.Chapter Seventeen.Tobacco.Pages 669-698In:Cultivar Development.CropSpecies.WH Fehr (ed.), MacMill Publishing Go., Inc., New York, NY761pp.

[0255] In another aspect, the tobacco variety provided herein is female sterility. As a non-limiting example, female sterile plants can be prepared by mutating the STIG1 gene. See, for example, Goldman et al. 1994, EMBO Journal 13:2976-2984. In one aspect, the modified tobacco plant provided herein is female sterility.

[0256] Flue-cured tobacco (also called Virginia tobacco or golden tobacco) accounts for about 40% of the world's tobacco production. Because it reaches a golden to dark orange color during curing, flue-cured tobacco is often called "golden tobacco". Flue-cured tobacco has a light, bright aroma and taste. Flue-cured tobacco is usually high in sugar content and low in oil content. The main flue-cured tobacco growing countries are Argentina, Brazil, China, India, Tanzania and the United States. In one aspect, the modified tobacco plant or seed provided herein is a flue-cured tobacco variety selected from the group consisting of CC 13, CC 27, CC 33, CC35, CC 37, CC65, CC 67, CC 700, GF 318, GL 338, GL 368, GL 939, K 346, K 399, K326, NC 102, NC 196, NC 291, NC 297, NC 299, NC 471, NC 55, NC 606, NC 71, NC 72, NC 92, PVH 1118, PVH 1452, PVH2110, SPEIGHT 168, SPEIGHT 220, SPEIGHT 225, SPEIGHT 227, SPEIGHT 236, and any variety substantially derived from any one of the above varieties. In another aspect, the modified tobacco plant or seed provided herein is a flue-cured tobacco variety selected from the group consisting of Coker 48, Coker 176, Coker 371-Gold, Coker 319, Coker 347, GL 939, K 149, K326, K 340, K 346, K 358, K 394, K 399, K 730, NC 27NF, NC37NF, NC 55, NC 60, NC 71, NC 72, NC 82, NC 95, NC 297, NC 606, NC 729, NC 2326, McNair373, McNair 944, Ox207, Ox 414NF, Reams 126, Reams 713, Reams 744, RG 8, RG 11, RG Speight NF-3, Va116, Va 182, and any variety substantially derived from any of the above varieties. See WO2004 / 041006A1.In another aspect, the modified tobacco plant or seed provided herein is a flue-cured variety selected from K326, K346 and NC196.

[0257] Air-cured tobaccos include "burley", "Maryland" and "dark" tobaccos. The common factor associated with air-cured tobaccos is that they are cured primarily without artificial heat and humidity. Burley tobacco is light to dark brown in color, high in oil and low in sugar. Burley tobacco is typically air-cured in barns. The major burley growing countries are Argentina, Brazil, Italy, Malawi and the United States.

[0258] Maryland tobacco is extremely fluffy, has good burning properties, low nicotine and a neutral aroma. Major Maryland tobacco growing countries include the United States and Italy.

[0259] In one aspect, the modified tobacco plant or seed provided herein is a burley tobacco variety selected from the group consisting of Clay 402, Clay 403, Clay 502, Ky 14, Ky 907, Ky 910, Ky 8959, NC 2, NC 3, NC 4, NC 5, NC2000, Tn 86, Tn 90, Tn 97, R 610, R 630, R 711, R 712, NCBH 129, HB4488PLC, PD 7319LC, Bu 21×Ky 10, HB04P, Ky 14×L 8, Kt 200, Newton 98, Pedigo 561, Pf561, and Va 509. In another aspect, the modified tobacco plant or seed provided herein is a burley tobacco variety selected from the group consisting of TN90, KT209, KT206, KT212, and HB4488. In another aspect, the modified tobacco plant or seed provided herein is a Maryland tobacco variety selected from the group consisting of Md10, Md40, Md201, Md609, Md872, and Md341.

[0260] The main difference between dark air-cured tobacco and other types is the curing process, which gives the dark air-cured tobacco a brown to dark brown color and a unique aroma. Dark air-cured tobacco is mainly used to produce chewing tobacco and snuff. In one aspect, the modified tobacco plant or seed provided herein is a dark air-cured tobacco variety selected from the following: Sumatra, Jatim, Dominic Cubano, Besuki, One sucker, Green River, Virginia sun-cured and Paragu Passado.

[0261] Dark flame cured tobacco is usually cured with low burning wood fire on the closed curing room floor. Dark flame cured tobacco is typically used to make pipe tobacco, cigarette, chewing tobacco, snuff and strong cigar. The main planting area of ​​dark flame cured tobacco is Tennessee, Kentucky and Virginia in the U.S. In one aspect, the modified tobacco plant or seed provided herein is selected from following dark flame cured tobacco variety: Narrow Leaf Madole, Improved Madole, Tom Rosson Madole, Newton's VH Madole, Little Crittenden, Green Wood, Little Wood, Small Stalk Black Mammoth, DT 508, DT 518, DT 592, KY 171, DF 911, DF485, TN D94, TN D950, V309 and V359.

[0262] Oriental tobacco is also referred to as Greek aroma and Turkish tobacco, because they are typically planted in the eastern Mediterranean region, for example Turkey, Greece, Bulgaria, Macedonia, Syria, Lebanon, Italy and Romania.The little plant size of Oriental tobacco variety, little leaf size and unique aroma characteristics are that they adapt to the poor soil and the result of stress climate conditions in which they grow therein.In one aspect, modified tobacco plant or seed provided herein are selected from following Oriental tobacco variety: Izmir, Katerini, Samsun, Basma, Krumovgrad, Trabzon, Thesalian, Tasova, Sinop, Izmit, Hendek, Edirne, Semdinli, Adiyanman, Yayladag, Iskenderun, Duzce, Macedonian, Mavra, Prilep, Bafra, Bursa, Bucak, Bitlis, Balikesir, and basically derived from any one of any kind of above-mentioned variety.

[0263] Tobacco plants, plant parts and tobacco materials provided herein can be used for any tobacco products. As used herein, "tobacco product" is defined as any product prepared or derived from tobacco intended for human use or consumption. In one aspect, the tobacco product provided herein comprises a cured component from the tobacco plant provided herein. In another aspect, the tobacco product provided herein comprises a cured tobacco leaf from the tobacco plant provided herein. In one aspect, the disclosure provides a tobacco product comprising a cured tobacco material from any tobacco plant provided herein.

[0264] In one aspect, the present disclosure provides a tobacco product comprising a cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a mutation in an endogenous gene encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein the mutation is not present in an endogenous gene of a control tobacco plant of the same variety, and wherein when grown under comparable growth conditions, the modified tobacco plant does not comprise a tobacco fork after topping or comprises a reduced tobacco fork compared to a control tobacco plant. On the other hand, the present disclosure provides a tobacco product comprising a cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a mutation in an endogenous gene encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein the mutation is not present in an endogenous gene of a control tobacco plant of the same variety.

[0265] In one aspect, the present disclosure provides a method for preparing a tobacco product using a cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a mutation in an endogenous gene encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein the mutation is not present in an endogenous gene of a control tobacco plant of the same variety, and wherein when grown under comparable growth conditions, the modified tobacco plant does not comprise a tobacco fork after topping or comprises a reduced tobacco fork compared to a control tobacco plant. On the other hand, the present disclosure provides a method for preparing a tobacco product using a cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a polynucleotide sequence encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor operably linked to a heterologous promoter, and wherein when grown under comparable growth conditions, the modified tobacco plant does not comprise a tobacco fork after topping or comprises a reduced tobacco fork compared to a control tobacco plant.

[0266] In one aspect, the present disclosure provides a tobacco product comprising a cured tobacco material from a modified tobacco plant, the modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor operably linked to a heterologous promoter, and wherein when grown under comparable growth conditions, the modified tobacco plant does not contain a tobacco fork or contains a reduced tobacco fork after topping compared to a control tobacco plant. On the other hand, the present disclosure provides a tobacco product comprising a cured tobacco material from a modified tobacco plant, the modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor operably linked to a heterologous promoter.

[0267] In one aspect, the present disclosure provides a method for preparing a tobacco product using a cured tobacco material from a modified tobacco plant, the modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor operably linked to a heterologous promoter, wherein when grown under comparable growth conditions, the modified tobacco plant does not contain a tobacco fork or contains a reduced tobacco fork after topping compared to a control tobacco plant. On the other hand, the present disclosure provides a method for preparing a tobacco product using a cured tobacco material from a modified tobacco plant, the modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor operably linked to a heterologous promoter.

[0268] In one aspect, the present disclosure provides a tobacco product comprising a cured tobacco material from a modified tobacco plant, the modified tobacco plant comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule, the small RNA molecule being capable of reducing the expression of an endogenous mRNA encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein when grown under comparable growth conditions, the modified tobacco plant does not comprise a tobacco twig or comprises a reduced tobacco twig after topping compared to a control tobacco plant. On the other hand, the present disclosure provides a tobacco product comprising a cured tobacco material from a modified tobacco plant, the modified tobacco plant comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule, the small RNA molecule being capable of reducing the expression of an endogenous mRNA encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor.

[0269] In one aspect, the present disclosure provides a method for preparing a tobacco product using a cured tobacco material from a modified tobacco plant, the modified tobacco plant comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule, the small RNA molecule being capable of reducing the expression of an endogenous mRNA encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein when grown under comparable growth conditions, the modified tobacco plant does not contain a tobacco twig or contains reduced tobacco twigs after topping compared to a control tobacco plant. On the other hand, the present disclosure provides a method for preparing a tobacco product using a cured tobacco material from a modified tobacco plant, the modified tobacco plant comprising a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule, the small RNA molecule being capable of reducing the expression of an endogenous mRNA encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor.

[0270] Can extract or separate alkaloid compounds from any tobacco plant or plant part provided herein.In one aspect, tobacco product provided herein comprises alkaloid extracted from modified tobacco plant or its part.On the other hand, tobacco product provided herein comprises nicotine extracted from tobacco plant or tobacco plant part.On the other hand, tobacco product provided herein comprises anatabine extracted from tobacco plant or tobacco plant part.On the other hand, tobacco product provided herein comprises anabasine extracted from tobacco plant or tobacco plant part.In one aspect, tobacco product provided herein comprises nornicotine extracted from tobacco plant or plant part.In one aspect, tobacco product provided herein comprises alkaloid extracted from modified tobacco plant, wherein the alkaloid is selected from nicotine, nornicotine, anatabine and anabasine.

[0271] The alkaloid compounds extracted from tobacco plants or tobacco plant parts provided herein can be used to produce compositions suitable for non-combustible products. Exemplary non-combustible products include electronic cigarettes ("e-cigarettes"), electronic smoking products, e-steam products, atomized steam products, and heated tobacco products. In one aspect, non-combustible products provided herein include alkaloids extracted from tobacco plants or tobacco plant parts provided herein. In one aspect, non-combustible products provided herein include nicotine extracted from tobacco plants or tobacco plant parts provided herein. In one aspect, non-combustible products provided herein include anabasine extracted from tobacco plants or tobacco plant parts provided herein. In one aspect, non-combustible products provided herein include anatabine extracted from tobacco plants or tobacco plant parts provided herein. In one aspect, non-combustible products provided herein include nornicotine extracted from tobacco plants or tobacco plant parts provided herein.

[0272] In one aspect, the non-combustible product provided herein is an e-cigarette. In another aspect, the non-combustible product provided herein is an electronic smoking product. In another aspect, the non-combustible product provided herein is an atomized steam product. In another aspect, the non-combustible product provided herein is a heated tobacco product. In another aspect, the non-combustible product provided herein is an e-steam product.

[0273] Tobacco products provided herein include, but are not limited to, tobacco products (e.g., cigarettes, bidis, kreteks), cigar products (e.g., cigars, cigar wrappers, and cigarillos), pipe tobacco products, products derived from tobacco, nicotine products from tobacco, smokeless tobacco products (e.g., moist snuff, dry snuff, chewing tobacco, moist smokeless tobacco, fine cut chewing tobacco, long cut chewing tobacco, pouched chewing tobacco, snuff), films, chewables (e.g., chewing gum), lozenges, dissolving strips, labels, tablets, shaped portions, gels, consumer units, insoluble matrices, hollow shapes, reconstituted tobacco, expanded tobacco, etc. See, e.g., U.S. Patent Publication No. US 2006 / 0191548.

[0274] As used herein, "cigarette" refers to a tobacco product having a "rod" and a "filler". The cigarette "rod" includes the cigarette paper, the filter, the filter rod (for containing the filter material), the tipping paper that fixes the cigarette paper (including the filler) to the filter, and all the glue that fixes these parts together. "Filler" includes (1) all tobacco, including but not limited to reconstituted tobacco and expanded tobacco, (2) non-tobacco substitutes (including but not limited to herbs, non-tobacco plant materials and other flavors that may accompany tobacco in the cigarette paper), (3) casing, (4) flavoring, and (5) all other additives (mixed with tobacco and substitutes and rolled into cigarettes).

[0275] In one aspect, the present disclosure provides derivatized nicotine and methods of producing nicotine for use in products from the modified tobacco plants provided herein.

[0276] In one aspect, the methods provided herein include preparing a tobacco product using cured tobacco leaves from a modified tobacco plant provided herein.

[0277] As used herein, "reconstituted tobacco" refers to a portion of tobacco filler made of tobacco powder and other tobacco waste materials that is processed into sheets and cut into strips to simulate tobacco. In addition to saving costs, reconstituted tobacco is very important because it contributes to the contribution of cigarette taste by processing flavor development using the reaction between ammonia and sugar. In one aspect, the tobacco product provided herein comprises reconstituted tobacco derived from modified tobacco plants.

[0278] As used herein, "expanded tobacco" refers to a portion of tobacco filler that has been treated by expansion with a suitable gas so that the tobacco is "expanded," resulting in a decrease in density and an increase in filling power. It reduces the weight of the tobacco used in cigarettes.

[0279] Tobacco products derived from plants of the present disclosure also include cigarettes and other smoking products, particularly those smoking products including filter elements, wherein the smokable material rod includes cured tobacco in a tobacco blend. In one aspect, the tobacco product of the present disclosure is selected from cigarettes, kreteks, bidis, cigars, cigarillos, unventilated cigarettes, ventilated filter cigarettes, pipe tobacco, snuff, snus, chewing tobacco, moist smokeless tobacco, fine cut chewing tobacco, long cut chewing tobacco, and bagged chewing tobacco products. In another aspect, the tobacco product of the present disclosure is selected from chewing gum, tablets, lozenges, and dissolving strips.

[0280] In one aspect, the tobacco products provided herein comprise cured tobacco materials from modified tobacco plants. In another aspect, the tobacco products provided herein comprise cured tobacco leaf materials from modified tobacco plants. In another aspect, the tobacco products provided herein comprise cured tobacco stem materials from modified tobacco plants.

[0281] In another aspect, the tobacco product of the present disclosure is a smokeless tobacco product. Smokeless tobacco products do not burn, including but not limited to chewing tobacco, moist smokeless tobacco, snuff and dry snuff. Chewing tobacco is a roughly divided tobacco leaf, which is typically packaged in a large bag-shaped package and used for plugs or twists. Moist smokeless tobacco is a moist, more subdivided tobacco, provided in a loose form or in the form of a pouch, and is typically packaged in a round can and used as a pinch in or placed in a pouch between the cheeks and gums of an adult tobacco consumer. Snuff is heat-treated smokeless tobacco. Dry snuff is finely ground tobacco placed in the mouth or for the nasal cavity. In a further aspect, the tobacco product of the present disclosure is selected from loose leaf chewing tobacco, plug chewing tobacco, moist snuff and nasal snuff.

[0282] The disclosure also provides a method for manufacturing a tobacco product, and the tobacco product comprises a tobacco material from a tobacco plant provided herein. In one aspect, the method provided herein comprises modulating an aged tobacco material made from a tobacco plant provided herein, to increase its moisture content from about 12.5% ​​to about 13.5% to about 21%, mixing the modulated tobacco material to produce a desired mixture. In one aspect, the method for preparing a tobacco product provided herein further comprises carrying out casing or seasoning to the mixture. Typically, in the casing process, a casing or seasoning material is added to the mixture to improve their quality and develop certain desired flavor characteristics by balancing the chemical composition. Further details of the casing process can be found in Tobacco Production, Chemistry and Technology, Blackwell Science, 1999, edited by L.Davis and M.Nielsen.

[0283] The present disclosure provides tobacco materials from modified tobacco plants or parts thereof. The tobacco materials obtained from tobacco plants, cells, strains, varieties or hybrids of the present disclosure can be used to prepare tobacco products. In one aspect, tobacco materials include leaf materials. In one aspect, tobacco materials include stem materials. In one aspect, tobacco materials include fresh tobacco materials. In another aspect, tobacco raw materials include dry tobacco materials. In a further aspect, tobacco materials include cured tobacco materials. In another aspect, tobacco raw materials include fermented tobacco materials. In one aspect, tobacco materials provided herein can be used for any tobacco products provided herein. In one aspect, cured tobacco materials provided herein include air-cured tobacco materials. In another aspect, cured tobacco materials provided herein include open flame cured tobacco materials. In another aspect, cured tobacco materials provided herein include sun-cured tobacco materials. In another aspect, cured tobacco materials provided herein include cured tobacco materials. In another aspect, cured tobacco materials provided herein include cured tobacco materials. In another aspect, cured tobacco materials provided herein include cured tobacco materials. In another aspect, cured tobacco materials provided herein are selected from air-cured tobacco materials, open flame cured tobacco materials, sun-cured tobacco materials and cured tobacco materials.

[0284] Tobacco material provided herein can also be used but not limited to heat treatment (for example cooking, baking), seasoning, enzyme treatment, puffing and / or ripening method to process.Can use these technical processing fermentation and non-fermented tobacco.The example of suitable processed tobacco comprises dark air-cured, dark open fire cured, burley, cured and cigar filler or wrapper, and the product from whole leaf drying operation.In one aspect, tobacco fiber comprises based on fresh weight up to 70% dark tobacco.For example, as described in U.S. Publication No. 2004 / 0118422 or 2005 / 0178398, can modulate tobacco by heating, sweating and / or pasteurization step.

[0285] Tobacco material provided herein can be fermented.Typically, the feature of fermentation is that initial moisture content is high, produces heat and dry weight loss is 10 to 20%.See, for example, U.S. Patent No. 4,528,993; 4,660,577; 4,848,373 and 5,372,149.Except changing the fragrance of blade, fermentation can also change the color and texture of blade.Equally in fermentation process, evolution gas can be produced, oxygen can be absorbed, pH value can be changed, and the amount of water retained can be changed.See, for example, U.S. Publication No. 2005 / 0178398 and Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford).Before adding oral products, tobacco cured or cured and fermented can be further processed (for example, cutting, puffing, mixing, grinding or pulverizing) aging or aging and fermentation. In some cases, the tobacco is long cut fermented cured wet tobacco having an oven volatiles content of 48-50% by weight prior to mixing with the copolymer and optional flavorants and other additives.

[0286] In one aspect, tobacco material provided herein can be processed into required size.In some aspects, tobacco fiber can be processed into average fiber size less than 200 microns.In one aspect, tobacco fiber is 75 to 125 microns.In another aspect, tobacco fiber is processed into 75 microns or less size.In one aspect, tobacco fiber comprises long cut tobacco, and it can be cut or chopped into width is about 10 cuts / inch (cut / inch) until about 110 cuts / inch, and length is about 0.1 inch to about 1 inch.Double cut tobacco fiber can have the particle size of certain scope, makes about 70% double cut tobacco fiber fall between-20 mesh to 80 mesh size.

[0287] The tobacco material provided herein can be processed to have a total oven volatile content of about 10% by weight or more; about 20% by weight or more; about 40% by weight or more; about 15% by weight to about 25% by weight; about 20% by weight to about 30% by weight; about 30% by weight to about 50% by weight; about 45% by weight to about 65% by weight or about 50% by weight to about 60% by weight. It will be understood by those skilled in the art that "wet" tobacco typically refers to tobacco having an oven volatile content of about 40% by weight to about 60% by weight (e.g., about 45% by weight to about 55% by weight, or about 50% by weight). As used herein, "oven volatiles" are determined by calculating the weight loss percentage of the sample after drying the sample at 110° C. for 3.25 hours in a preheated forced draft oven. The total oven volatile content of an oral product may be different from the oven volatile content of the tobacco fiber used to prepare an oral product. The processing steps described herein can reduce or increase the oven volatile content.

[0288] The following exemplary, non-limiting embodiments are contemplated:

[0289] 1. A modified tobacco plant comprising a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein the mutation is not present in the endogenous gene of a control tobacco plant of the same variety, and wherein when grown under comparable growth conditions, the modified tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping compared to the control tobacco plant.

[0290] 2. A modified tobacco plant comprising a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB and a WRKY transcription factor, wherein the mutation is not present in the endogenous gene of a control tobacco plant of the same variety.

[0291] 3. A modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB and a WRKY transcription factor operably linked to a heterologous promoter, wherein when grown under comparable growth conditions, the modified tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping compared to a control tobacco plant.

[0292] 4. A modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB and a WRKY transcription factor operably linked to a heterologous promoter.

[0293] 5. A modified tobacco plant comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of reducing the expression of an endogenous mRNA encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein when grown under comparable growth conditions, the modified tobacco plant comprises no or reduced tobacco forks after topping compared to a control tobacco plant.

[0294] 6. A modified tobacco plant comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of reducing the expression of an endogenous mRNA encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, MYB and a WRKY transcription factor.

[0295] 7. The modified tobacco plant of embodiment 1 or 2, wherein the mutation comprises a mutation selected from the group consisting of an insertion, a deletion, a substitution, and an inversion.

[0296] 8. The modified tobacco plant of any one of embodiments 1, 2 or 7, wherein the mutation comprises a null mutation.

[0297] 9. The modified tobacco plant of any one of embodiments 1, 2, 7 or 8, wherein the mutation results in a premature stop codon in the mRNA transcript of the endogenous gene.

[0298] 10. The modified tobacco plant of any one of embodiments 1, 2, or 7-9, wherein the mutation results in truncation of the polypeptide.

[0299] 11. The modified tobacco plant of any one of embodiments 1, 2, or 7-10, wherein the mutation is located within an exon of the endogenous gene.

[0300] 12. The modified tobacco plant of any one of embodiments 1, 2, or 7-10, wherein the mutation is located within an intron of the endogenous gene.

[0301] 13. The modified tobacco plant of any one of embodiments 1, 2 or 7, wherein the mutation is located within the 5'-untranslated region (UTR) or the 3'-UTR of the endogenous gene.

[0302] 14. The modified tobacco plant of any one of embodiments 1, 2 or 7, wherein the mutation is located within the promoter of the endogenous gene.

[0303] 15. The modified tobacco plant of any one of embodiments 1, 2, or 7-14, wherein the endogenous gene comprises a polynucleotide sequence that is at least 80% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113.

[0304] 16. The modified tobacco plant of any one of embodiments 1, 2, or 7-15, wherein the endogenous gene encodes a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from SEQ ID NOs: 45-88 and 114.

[0305] 17. The modified tobacco plant of any one of embodiments 1, 2, or 7-16, wherein said mutation results in a decrease in the expression level of said endogenous gene compared to said control tobacco plant.

[0306] 18. The modified tobacco plant of any one of embodiments 1, 2, or 7-16, wherein said mutation results in an increased expression level of said endogenous gene compared to said control tobacco plant.

[0307] 19. The modified tobacco plant of any one of embodiments 1, 2, or 7-16, wherein said mutation results in a reduced level of activity of said polypeptide compared to said control tobacco plant.

[0308] 20. The modified tobacco plant of any one of embodiments 1, 2, or 7-16, wherein said mutation results in an increased level of activity of said polypeptide compared to said control tobacco plant.

[0309] 21. The modified tobacco plant of any one of embodiments 3-6, wherein the heterologous promoter comprises an axillary meristem-specific promoter.

[0310] 22. The modified tobacco plant of any one of embodiments 3-6, wherein the heterologous promoter comprises an axillary meristem-preferred promoter.

[0311] 23. The modified tobacco plant of any one of embodiments 3-6, wherein the heterologous promoter comprises a polynucleotide sequence that is at least 90% identical to a polynucleotide sequence selected from SEQ ID NOs: 89-109.

[0312] 24. The modified tobacco plant of any one of embodiments 3-6, wherein the small RNA molecule is selected from the group consisting of double-stranded RNA, small interfering RNA (siRNA), trans-acting siRNA and microRNA.

[0313] 25. The modified tobacco plant of embodiment 5 or 6, wherein the small RNA molecule comprises 18 nucleotides to 30 nucleotides.

[0314] 26. The modified tobacco plant of embodiment 5 or 6, wherein the small RNA molecule comprises a polynucleotide sequence that is at least 90% identical or complementary to the endogenous mRNA.

[0315] 27. The modified tobacco plant of embodiment 5 or 6, wherein the small RNA molecule comprises 18 nucleotides to 30 nucleotides.

[0316] 28. The modified tobacco plant of embodiment 5 or 6, wherein the small RNA molecule comprises a polynucleotide sequence having at least 90% identity or complementarity to a polynucleotide sequence selected from SEQ ID NO: 1-44 and 113.

[0317] 29. The modified tobacco plant of embodiment 5 or 6, wherein the small RNA molecule comprises a polynucleotide sequence that is identical or complementary to at least 18 consecutive nucleotides of a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113.

[0318] 30. The modified tobacco plant of any one of embodiments 1-29, wherein the polypeptide is a cell cycle protein.

[0319] 31. The modified tobacco plant of any one of embodiments 1-29, wherein the polypeptide is a CDK.

[0320] 32. The modified tobacco plant of any one of embodiments 1-29, wherein the polypeptide is a CDK inhibitor.

[0321] 33. The modified tobacco plant of any one of embodiments 1-29, wherein the polypeptide is MYB.

[0322] 34. The modified tobacco plant of any one of embodiments 1-29, wherein the polypeptide is a WRKY transcription factor.

[0323] 35. The modified tobacco plant of embodiment 30, wherein the cell cycle protein is encoded by a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 6-32.

[0324] 36. The modified tobacco plant of embodiment 30, wherein the cyclin comprises an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-76.

[0325] 37. The modified tobacco plant of embodiment 31, wherein the CDK is encoded by a polynucleotide sequence that is at least 80% identical to SEQ ID NOs: 1-4.

[0326] 38. The modified tobacco plant of embodiment 31, wherein the CDK comprises an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from SEQ ID NOs: 45-48.

[0327] 39. The modified tobacco plant of embodiment 32, wherein the CDK inhibitor is encoded by a polynucleotide sequence that is at least 80% identical to SEQ ID NO:5.

[0328] 40. The modified tobacco plant of embodiment 32, wherein the CDK inhibitor comprises an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from SEQ ID NO:49.

[0329] 41. The modified tobacco plant of embodiment 33, wherein the MYB is encoded by a polynucleotide sequence that is at least 80% identical to SEQ ID NOs: 33-44.

[0330] 42. The modified tobacco plant of embodiment 33, wherein the MYB comprises an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from SEQ ID NOs: 77-88.

[0331] 43. The modified tobacco plant of embodiment 34, wherein the WRKY transcription factor is encoded by a polynucleotide sequence that is at least 80% identical to SEQ ID NO:113.

[0332] 44. The modified tobacco plant of embodiment 34, wherein the WRKY transcription factor comprises an amino acid sequence that is at least 80% identical or similar to SEQ ID NO:114.

[0333] 45. The modified tobacco plant of any one of embodiments 1-44, wherein the modified tobacco plant is selected from the group consisting of a flue-cured variety, a golden variety, a burley variety, a Virginia variety, a Maryland variety, a dark variety, Tobacco varieties of the genus, Oriental and Turkish varieties.

[0334] 46. ​​The modified tobacco plant of any one of embodiments 1 to 45, wherein the modified tobacco plant is selected from the group consisting of BU 64 plants, CC 101 plants, CC 200 plants, CC 13 plants, CC 27 plants, CC 33 plants, CC 35 plants, CC 37 plants, CC 65 plants, CC 67 ​​plants, CC 301 plants, CC 400 plants, CC 500 plants, CC 600 plants, CC 700 plants, CC 800 plants, CC 900 plants, CC 1063 plants, Coker 176 plants, Coker 319 plants, Coker 371 Gold plants, Coker 48 plants, CU 263 plants, DF911 plants, Galpao plants, GL 26H plants, GL 338 plants, GL 350 plants, GL 395 plants, GL 600 plants, GL 737 plants, GL 939 plants, GL 973 plants, GF 157 plants, GF 318 plants, RJR 901 plants, HB 04P plants, K 149 plants, K 326 plants, K 346 plants, K 358 plants, K394 plants, K 399 plants, K 730 plants, NC 196 plants, NC 37NF plants, NC 471 plants, NC 55 plants, NC92 plants, NC2326 plants, NC 95 plants, NC 925 plants, PVH 1118 plants, PVH 1452 plants, PVH 2110 plants, PVH2254 plants, PVH 2275 plants, VA 116 plants, VA 119 plants, KDH 959 plants, KT 200 plants, KT204LC plants, KY 10 plants, KY 14 plants, KY 160 plants, KY 17 plants, KY 171 plants, KY 907 plants, KY907LC plants, KTY14 x L8 LC plants, Little Crittenden plants, McNair 373 plants, McNair 944 plants, male sterile KY 14 x L8 plants, Narrow Leaf Madole plants, MS KY171 plants, Narrow Leaf Madole (phph) plants, MS Narrow Leaf Madole plants, MS TND950 plants, PD 7302LC plants, PD7305LC plants, PD 7309LC plants, PD 7312LC plants, PD 7318LC plants, PD 7319LC plants, MSTKS2002 plants, TKF 2002 plants, TKF 6400 plants, TKF 4028 plants, TKF4024 plants, KT206LC plants, KT209LC plants, KT210LC plants, KT212LC plants, NC 100 plants, NC 102 plants, NC 2000 plants, NC 291 plants, NC297 plants, NC 299 plants, NC 3 plants, NC 4 plants, NC 5 plants, NC 6 plants, NC7 plants, NC 606 plants, NC71 plants, NC 72 plants, NC 810 plants, NC BH 129 plants, NC 2002 plants, Neal Smith Madole plants, OXFORD 207 plants, 'Perique' plants, PVH03 plants, PVH09 plants, PVH19 plants, PVH50 plants, PVH51 plants, R 610 plants, R 630 plants, R 7-11 plants, R 7-12 plants, RG 17 plants, RG 81 plants, RG H51 plants, RGH 4 plants, RGH 51 plants, RS1410 plants, Speight 168 plants, Speight 172 plants, Speight 179 plants, Speight 210 plants, Speight 220 plants, Speight 225 plants, Speight 227 plants, Speight234 plants, Speight G-28 plants, Speight G-70 plants, Speight H-6 plants, Speight H20 plants, Speight NF3 plants, TI 1406 plants, TI 1269 plants, TN 86 plants, TN86LC plants, TN 90 plants, TN90LC plants, TN 97 plants, TN97LC plants, TN D94 plants, TN D950 plants, TR (Tom Rosson) Madole plants, VA309 plants, and VA 359 plants.

[0335] 47. The modified tobacco plant of any one of embodiments 1-46, wherein said modified tobacco plant is a hybrid.

[0336] 48. The modified tobacco plant of any one of embodiments 1-47, wherein said modified tobacco plant is male sterile or cytoplasmic male sterile.

[0337] 49. The modified tobacco plant of any one of embodiments 1-47, wherein said modified tobacco plant is female sterile.

[0338] 50. The modified tobacco plant of any of embodiments 1, 3, or

[0009] , wherein the reduced tobacco branches comprise fewer total tobacco branches, a smaller average tobacco branch size, or both, compared to tobacco branches of the control tobacco plant when grown under comparable growing conditions.

[0339] 51. The modified tobacco plant of embodiment 50, wherein the smaller average tobacco twig size comprises a measurement selected from the group consisting of: reduced average mass, reduced average length, reduced average diameter, or any combination thereof, as compared to tobacco twigs of the control tobacco plant when grown under comparable growing conditions.

[0340] 52. The modified tobacco plant of any one of embodiments 1-51, wherein said modified plant has increased leaf yield quality compared to said control tobacco plant when grown under comparable growth conditions.

[0341] 53. The modified tobacco plant of embodiment 52, wherein the increased leaf yield quality comprises an increase of at least 0.5%.

[0342] 54. A tobacco leaf of the modified tobacco plant according to any one of embodiments 1-53.

[0343] 55. A tobacco seed of the modified tobacco plant of any one of embodiments 1-53.

[0344] 56. The tobacco leaf of embodiment 54, wherein the tobacco leaf is a cured tobacco leaf.

[0345] 57. The tobacco leaf according to embodiment 56, wherein the cured tobacco leaf is selected from the group consisting of air-cured tobacco leaves, flame-cured tobacco leaves, sun-cured tobacco leaves and flue-cured tobacco leaves.

[0346] 58. A tobacco product comprising an alkaloid extracted from the modified tobacco plant or part thereof of any one of embodiments 1-53.

[0347] 59. The tobacco product of embodiment 58, wherein the alkaloid is selected from nicotine, nornicotine, anatabine, and anabasine.

[0348] 60. The tobacco product of embodiment 58 or 59, wherein the tobacco product is selected from cigarettes, kreteks, bidis, cigars, cigarillos, unventilated cigarettes, ventilated filter cigarettes, pipe tobacco, snuff, snus, chewing tobacco, moist smokeless tobacco, fine cut chewing tobacco, long cut chewing tobacco, and pouched chewing tobacco products.

[0349] 61. The tobacco product of embodiment 58 or 59, wherein the tobacco product is selected from the group consisting of chewing gum, tablets, lozenges, and dissolving strips.

[0350] 62. The tobacco product of embodiment 58 or 59, wherein the tobacco product is a non-combustible product.

[0351] 63. The tobacco product of embodiment 62, wherein the non-combustible product is selected from the group consisting of an electronic cigarette, an electronic smoking product, an atomized vapor product, and a heated tobacco product.

[0352] 64. A tobacco product comprising cured tobacco material from a modified tobacco plant, the modified tobacco plant comprising a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein the mutation is not present in the endogenous gene of a control tobacco plant of the same variety, and wherein the modified tobacco plant comprises no or reduced tobacco forks after topping compared to a control tobacco plant when grown under comparable growth conditions.

[0353] 65. A tobacco product comprising cured tobacco material from a modified tobacco plant comprising a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein the mutation is not present in the endogenous gene of a control tobacco plant of the same variety.

[0354] 66. A tobacco product comprising cured tobacco material from a modified tobacco plant, the modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB and a WRKY transcription factor operably linked to a heterologous promoter, and wherein the modified tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping compared to a control tobacco plant when grown under comparable growth conditions.

[0355] 67. A tobacco product comprising cured tobacco material from a modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB and a WRKY transcription factor operably linked to a heterologous promoter.

[0356] 68. A tobacco product comprising cured tobacco material from a modified tobacco plant, the modified tobacco plant comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of reducing expression of an endogenous mRNA encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor, wherein the modified tobacco plant comprises no or reduced tobacco twigs after topping compared to a control tobacco plant when grown under comparable growth conditions.

[0357] 69. A tobacco product comprising cured tobacco material from a modified tobacco plant, the modified tobacco plant comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of reducing the expression of an endogenous mRNA encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor.

[0358] 70. The tobacco product of any one of embodiments 64-69, wherein the tobacco product is selected from cigarettes, kretek cigarettes, bidis, cigars, cigarillos, unventilated cigarettes, ventilated filter cigarettes, pipe tobacco, snuff, snus, chewing tobacco, moist smokeless tobacco, fine cut chewing tobacco, long cut chewing tobacco, and pouched chewing tobacco products.

[0359] 71. The tobacco product of any one of embodiments 64-69, wherein the cured tobacco material is from a tobacco variety selected from the group consisting of: flue-cured, golden, burley, Virginia, Maryland, dark, oriental, and Turkish.

[0360] 72. The tobacco product of any one of embodiments 64-69, wherein the cured tobacco material is selected from the group consisting of air-cured tobacco material, flame-cured tobacco material, sun-cured tobacco material, and flue-cured tobacco material.

[0361] 73. The tobacco product of any one of embodiments 64-69, wherein the cured tobacco material comprises cured tobacco leaf material.

[0362] 74. The tobacco product of any of embodiments 64-69, wherein the cured tobacco material comprises cured tobacco stem material.

[0363] 75. A method for producing a modified tobacco plant comprising:

[0364] (a) inducing a mutation in at least one tobacco cell at a genomic locus encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB and a WRKY transcription factor;

[0365] (b) selecting at least one tobacco cell comprising the mutation from step (a); and

[0366] (c) regenerating a modified tobacco plant from the at least one tobacco cell selected in step (b), wherein the modified tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping when grown under comparable growth conditions compared to a control tobacco plant lacking the mutation.

[0367] 76. A method for producing a modified tobacco plant, comprising:

[0368] (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 a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, MYB and a WRKY transcription factor;

[0369] (b) selecting at least one tobacco cell comprising the recombinant DNA construct; and

[0370] (c) regenerating a modified tobacco plant from the at least one tobacco cell selected in step (b), wherein the modified tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping when grown under comparable growth conditions, compared to a control tobacco plant lacking the recombinant DNA construct.

[0371] 77. A method for producing a modified tobacco plant comprising:

[0372] (a) introducing a recombinant DNA construct into at least one tobacco cell, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of reducing the expression of an endogenous mRNA encoding a polypeptide selected from the group consisting of a cyclin, a cyclin-dependent kinase (CDK), a CDK inhibitor, a MYB, and a WRKY transcription factor;

[0373] (b) selecting at least one tobacco cell comprising the recombinant DNA construct; and

[0374] (c) regenerating a modified tobacco plant from the at least one tobacco cell selected in step (b), wherein the modified tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping when grown under comparable growth conditions, compared to a control tobacco plant lacking the recombinant DNA construct.

[0375] 78. The method of embodiment 75, wherein the inducing comprises using an agent selected from the group consisting of a chemical mutagen, radiation, a transposon, Agrobacterium, and a nuclease.

[0376] 79. The method of embodiment 78, wherein the nuclease is selected from the group consisting of a meganuclease, a zinc finger nuclease, a transcription activator-like effector nuclease, a CRISPR / Cas9 nuclease, a CRISPR / Cpf1 nuclease, a CRISPR / CasX nuclease, a CRISPR / CasY nuclease, a Csm1 nuclease, or any combination thereof.

[0377] 80. The method of embodiment 78, wherein the chemical mutagen comprises ethyl methanesulfonate.

[0378] 81. The method of embodiment 78, wherein the radiation comprises gamma rays, X-rays, or ionizing radiation.

[0379] 82. The method of embodiment 77, wherein the small RNA molecule is selected from double-stranded RNA, small interfering RNA (siRNA), trans-acting siRNA and microRNA.

[0380] 83. The method of embodiment 77 or 82, wherein the small RNA molecule comprises at least 18 nucleotides.

[0381] 84. The method of any one of embodiments 77, 82 or 83, wherein the small RNA molecule comprises at least 90% identity or complementarity to the endogenous mRNA.

[0382] 85. The method of any one of embodiments 77, 82 or 84, wherein the small RNA molecule comprises 18 to 30 nucleotides.

[0383] 86. The method of any one of embodiments 77 or 82-85, wherein the small RNA molecule comprises at least 90% identity or complementarity to a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113.

[0384] 87. The method of any one of embodiments 77 or 82-86, wherein the endogenous mRNA comprises a sequence that is identical or complementary to at least 18 consecutive nucleotides of a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113.

[0385] 88. The method of any one of embodiments 75-87, wherein the at least one tobacco cell is a tobacco protoplast cell.

[0386] 89. The method of any one of embodiments 75-87, wherein the at least one tobacco cell is a tobacco callus cell.

[0387] 90. The method of any one of embodiments 75-87, wherein the at least one tobacco cell is selected from the group consisting of seed cells, fruit cells, leaf cells, cotyledon cells, hypocotyl cells, meristem cells, embryo cells, endosperm cells, root cells, bud cells, stem cells, flower cells, inflorescence cells, stem cells, peduncles cells, style cells, stigma cells, receptacle cells, petal cells, sepal cells, pollen cells, anther cells, silk cells, ovary cells, ovule cells, pericarp cells, and phloem cells.

[0388] 91. The method of any one of embodiments 75-77, wherein the method further comprises:

[0389] (d) growing the modified tobacco plant regenerated in step (c).

[0390] 92. The method of embodiment 91, wherein the method further comprises:

[0391] (e) crossing the modified tobacco plant grown in step (d) with a second tobacco plant; and

[0392] (f) obtaining at least one seed from the hybridization in step (e).

[0393] 93. The method of embodiment 76 or 77, wherein the heterologous promoter comprises an axillary meristem-specific promoter.

[0394] 94. The modified tobacco plant of embodiment 93, wherein the heterologous promoter comprises a polynucleotide sequence that is at least 90% identical to a polynucleotide sequence selected from SEQ ID NOs: 89-109.

[0395] 95. The method of embodiment 75, wherein the genomic locus comprises a polynucleotide sequence that is at least 80% identical to a polynucleotide sequence selected from SEQ ID NOs: 1-44 and 113.

[0396] 96. The method of embodiment 75, wherein the genomic locus encodes a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from SEQ ID NOs: 45-88 and 114.

[0397] 97. The method of embodiment 75, wherein the mutation results in reduced expression of the genomic locus compared to the control tobacco plant.

[0398] 98. The method of embodiment 75, wherein the mutation results in increased expression of the genomic locus compared to the control tobacco plant.

[0399] 99. The method of embodiment 75, wherein the mutation results in a decrease in the activity of the polypeptide compared to the control tobacco plant.

[0400] 100. The method of embodiment 75, wherein the mutation results in an increase in the activity of the polypeptide compared to the control tobacco plant.

[0401] 101. The method of any one of embodiments 75-100, wherein the polypeptide is a cell cycle protein.

[0402] 102. The method of any one of embodiments 75-100, wherein the polypeptide is a CDK.

[0403] 103. The method of any one of embodiments 75-100, wherein the polypeptide is a CDK inhibitor.

[0404] 104. The method of any one of embodiments 75-100, wherein the polypeptide is MYB.

[0405] 105. The method of any one of embodiments 75-100, wherein the polypeptide is a WRKY transcription factor.

[0406] 106. The method of embodiment 101, wherein the cyclin protein is encoded by a polynucleotide sequence that is at least 80% identical to SEQ ID NO:6-32.

[0407] 107. The method of embodiment 101, wherein the cyclin comprises an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from SEQ ID NOs: 50-76.

[0408] 108. The method of embodiment 102, wherein the CDK is encoded by a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 1-4.

[0409] 109. The method of embodiment 102, wherein the CDK comprises an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from SEQ ID NOs: 45-48.

[0410] 110. The method of embodiment 103, wherein the CDK inhibitor is encoded by a polynucleotide sequence that is at least 80% identical to SEQ ID NO:5.

[0411] 111. The method of embodiment 103, wherein the CDK inhibitor comprises an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from SEQ ID NO:49.

[0412] 112. The method of embodiment 104, wherein the MYB is encoded by a polynucleotide sequence that is at least 80% identical to SEQ ID NOs: 33-44.

[0413] 113. The method of embodiment 104, wherein the MYB comprises an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from SEQ ID NOs: 77-88.

[0414] 114. The method of embodiment 105, wherein the WRKY transcription factor is encoded by a polynucleotide sequence that is at least 80% identical to SEQ ID NO:113.

[0415] 115. The method of embodiment 105, wherein the WRKY transcription factor comprises an amino acid sequence that is at least 80% identical or similar to SEQ ID NO:114.

[0416] 116. The method of any one of embodiments 75-115, wherein the modified tobacco plant is a tobacco variety selected from the group consisting of a flue-cured variety, a golden variety, a burley variety, a Virginia variety, a Maryland variety, a dark variety, an oriental variety, and a Türkiye variety.

[0417] 117. The method of any one of embodiments 75-115, wherein the modified tobacco plant is selected from the group consisting of BU 64 plants, CC 101 plants, CC 200 plants, CC 13 plants, CC 27 plants, CC 33 plants, CC 35 plants, CC 37 plants, CC 65 plants, CC 67 ​​plants, CC 301 plants, CC 400 plants, CC 500 plants, CC 600 plants, CC 700 plants, CC 800 plants, CC 900 plants, CC 1063 plants, Coker 176 plants, Coker 319 plants, Coker 371 Gold plants, Coker 48 plants, CU 263 plants, DF911 plants, Galpao plants, GL 26H plants, GL 338 plants, GL 350 plants, GL 395 plants, GL 600 plants, GL 737 plants, GL 939 plants, GL 1064 plants, 973 plants, GF 157 plants, GF 318 plants, RJR 901 plants, HB 04P plants, K 149 plants, K 326 plants, K 346 plants, K 358 plants, K394 plants, K399 plants, K 730 plants, NC 196 plants, NC 37NF plants, NC 471 plants, NC 55 plants, NC 92 plants, NC2326 plants, NC 95 plants, NC 925 plants, PVH 1118 plants, PVH 1452 plants, PVH 2110 plants, PVH2254 plants, PVH 2275 plants, VA 116 plants, VA 119 plants, KDH 959 plants, KT 200 plants, KT204LC plants, KY 10 plants, KY 14 plants, KY 160 plants, KY 17 plants, KY 171 plants, KY 907 plants, KY 907LC plants, KTY14 x L8LC plants, Little Crittenden plants, McNair 373 plants, McNair 944 plants, male sterile KY 14 x L8 plants, Narrow Leaf Madole plants, MS KY171 plants, Narrow Leaf Madole (phph) plants, MS Narrow Leaf Madole plants, MS TND950 plants, PD 7302LC plants, PD 7305LC plants, PD 7309LC plants, PD 7312LC plants, PD 7318LC plants, PD 7319LC plants, MSTKS 2002 plants, TKF2002 plants, TKF 6400 plants, TKF 4028 plants, TKF4024 plants, KT206LC plants, KT209LC plants, KT210LC plants, KT212LC plants, NC 100 plants, NC 102 plants, NC 2000 plants, NC 291 plants, NC 297 plants, NC 299 plants, NC 3 plants, NC 4 plants, NC 5 plants, NC 6 plants, NC7 plants, NC 606 plants, NC 71 plants, NC 72 plants, NC 810 plants, NC BH 129 plants, NC 2002 plants, Neal Smith Madole plants, OXFORD 207 plants, 'Perique' plants, PVH03 plants, PVH09 plants, PVH19 plants, PVH50 plants, PVH51 plants, R 610 plants, R 630 p...

Claims

1. A cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a first polynucleotide sequence encoding a cyclin-dependent kinase (CDK) inhibitor polypeptide operably linked to a heterologous promoter, the CDK inhibitor polypeptide consisting of the amino acid sequence of SEQ ID NO: 80, the first polynucleotide sequence consisting of the nucleic acid sequence of SEQ ID NO: 36, wherein the heterologous promoter consists of a second polynucleotide sequence selected from the group consisting of SEQ ID NOs: 89-109, wherein when grown under comparable growth conditions, the modified tobacco plant comprises no tobacco twigs or comprises reduced tobacco twigs after topping compared to a control tobacco plant, wherein the cured tobacco material is cured leaf material, cured stem material, or both cured leaf material and cured stem material.

2. The cured tobacco material of claim 1, wherein the cured tobacco material is present in a tobacco product, and wherein the tobacco product is selected from the group consisting of cigarettes, cigars, pipe tobacco, snuff, and chewing tobacco.

3. The cured tobacco material of claim 2, wherein the cigarette is selected from the group consisting of kretek cigarettes, bidis, unventilated cigarettes and ventilated filter cigarettes.

4. The cured tobacco material of claim 2, wherein the cigar is a cigarillo.

5. The cured tobacco material of claim 1, wherein the cured tobacco material is present in a tobacco product, and wherein the tobacco product is a smokeless tobacco product.

6. The cured tobacco material of claim 5, wherein the smokeless tobacco product is selected from the group consisting of moist smokeless tobacco, fine cut chewing tobacco, long cut chewing tobacco, and pouched chewing tobacco.

7. The cured tobacco material of claim 5, wherein the smokeless tobacco product is selected from the group consisting of moist snuff and dry snuff.

8. The cured tobacco material of claim 1, wherein the cured tobacco material is from a tobacco variety selected from the group consisting of a flue-cured variety, a golden variety, a burley variety, a Virginia variety, a Maryland variety, a dark variety, an oriental variety, and a Turkish variety.

9. The cured tobacco material according to any one of claims 2-8, wherein the tobacco product further comprises reconstituted tobacco material from the modified tobacco plant.

10. 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 a cyclin-dependent kinase (CDK) inhibitor polypeptide, the nucleic acid encoding the CDK inhibitor consisting of the nucleic acid sequence of SEQ ID NO: 36, wherein the CDK inhibitor polypeptide consists of the amino acid sequence of SEQ ID NO: 80, and wherein the heterologous promoter consists of a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 89-109; (b) selecting at least one tobacco cell comprising the recombinant DNA construct; and (c) regenerating a modified tobacco plant from the at least one tobacco cell selected in step (b), wherein the modified tobacco plant comprises no tobacco forks or comprises reduced tobacco forks after topping when grown under comparable growth conditions, compared to a control tobacco plant lacking the recombinant DNA construct.

11. The method of claim 10, wherein the modified tobacco plant comprises (a) fewer total tobacco branches, (b) smaller average tobacco branch size, or (c) fewer total tobacco branches and smaller average tobacco branch size as compared to the control tobacco plant.

12. The method of claim 10, wherein the modified tobacco plant is from a tobacco type selected from the group consisting of Burley tobacco, Maryland tobacco, Blonde tobacco, Virginia tobacco, Oriental tobacco, and Turkish tobacco.

13. The method of claim 10, wherein said introducing comprises using bacteria-mediated transformation and microprojectile bombardment-mediated transformation.

14. The method of claim 10, wherein the introducing comprises using polyethylene glycol (PEG)-mediated transformation.

15. A method for preparing a tobacco product comprising using cured tobacco material from a modified tobacco plant, the modified tobacco plant comprising a first polynucleotide sequence encoding a cyclin-dependent kinase (CDK) inhibitor polypeptide operably linked to a heterologous promoter, the CDK inhibitor polypeptide consisting of the amino acid sequence of SEQ ID NO:80, the first polynucleotide sequence consisting of the nucleic acid sequence of SEQ ID NO:36, wherein the heterologous promoter consists of a second polynucleotide sequence selected from the group consisting of SEQ ID NOs:89-109, the modified tobacco plant comprising no tobacco twigs or comprising reduced tobacco twigs after topping compared to a control tobacco plant when grown under comparable growth conditions, and wherein the cured tobacco material is cured tobacco leaf material, cured tobacco stem material, or both cured tobacco leaf material and cured tobacco stem material.

16. The method of claim 15, wherein the tobacco product is selected from the group consisting of cigarettes, cigars, pipe tobacco, snuff, and chewing tobacco.

17. The method of claim 16, wherein the cigarette is selected from the group consisting of kretek, bidi, unventilated cigarette and ventilated filter cigarette.

18. The method of claim 16, wherein the cigar is a cigarillo.

19. The method of claim 15, wherein the tobacco product is a smokeless tobacco product.

20. The method of claim 19, wherein the smokeless tobacco product is selected from the group consisting of moist smokeless tobacco, fine cut chewing tobacco, long cut chewing tobacco, and pouched chewing tobacco.

21. The method of claim 19, wherein the smokeless tobacco product is selected from the group consisting of moist snuff and dry snuff.

22. The method of claim 15, wherein the cured tobacco material is from a tobacco variety selected from the group consisting of flue-cured, golden, burley, Virginia, Maryland, dark, oriental, and Turkish.

23. The method of claim 15, wherein the tobacco product further comprises reconstituted tobacco material from the modified tobacco plant.

24. The method of claim 15, wherein the method further comprises cooking, toasting, flavoring or enzymatically treating the cured tobacco material.

25. The method of claim 15, wherein the method further comprises fermenting the cured tobacco material.

26. The method of claim 15, wherein the method further comprises pasteurizing the cured tobacco material.

Images