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

Abstract

The present disclosure provides methods and compositions for regulating sucker growth in tobacco by altering the expression of different target genes.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 854,139, filed May 29, 2019. The entire content of this application is hereby incorporated by reference into this specification.

[0002] Field The present disclosure provides methods and compositions for improving the expression of nucleic acids and proteins useful for reducing or eliminating suckers in plants.

[0003] Incorporation of Sequence Listing This application includes a sequence listing that was electronically submitted in ASCII format, the entire of which is hereby incorporated by reference into this specification. The name of the ASCII copy created on May 21, 2020 is P34703WO00_SL.txt and the size is 380,869 bytes.

Background Art

[0004] Background Tobacco is a plant species that exhibits very strong apical dominance. Molecular signals from the shoot apical meristem (SAM) mediate hormonal signals that effectively inhibit the growth of axillary buds. Removal of the SAM (also known as "topping") causes physiological and molecular changes that enable the growth of new shoots (or "suckers") from axillary meristems (buds). The growth of suckers results in a decrease in yield and leaf quality. Suckers have been removed by manual removal and the application of chemicals. Maleic hydrazide and flumetralin are commonly used on topped plants to inhibit the growth of axillary buds ("sucker development"). However, the labor and chemicals for removing suckers are very expensive. Suppression of sucker development in tobacco through conventional breeding, mutagenesis breeding, and transgenic approaches has been a major goal for decades, but to date, no success in inhibiting or eliminating sucker development through these approaches has been achieved. Recent molecular studies have produced transgenic plants with reduced or absent suckers, but the leaky expression of axillary bud-degrading genes can lead to seed and embryo death and prevent the production of successive generations of transgenic plants. Therefore, the development of methods and compositions to prevent the expression of axillary bud-degrading genes in undesired tissues and / or organs will result in a reduction in the use of chemicals and reduce the costs and labor associated with tobacco production. SUMMARY OF THE INVENTION

[0005] Summary 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 cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, MYB, and WRKY transcription factors, wherein the mutation is not present in the endogenous gene in a control tobacco plant of the same variety, and wherein the modified tobacco plant, when cultivated under equivalent cultivation conditions, comprises no suckers or reduced suckers after topping compared to the control tobacco plant.

[0006] 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 cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, wherein the mutation is not present in the endogenous gene in a control tobacco plant of the same variety.

[0007] In one aspect, the present disclosure provides a modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, operably linked to a heterologous promoter, wherein the modified tobacco plant, when grown under equivalent cultivation conditions, comprises no or reduced suckers after topping as compared to a control tobacco plant.

[0008] In one aspect, the present disclosure provides a modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, operably linked to a heterologous promoter.

[0009] 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 cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, wherein the modified tobacco plant, when grown under equivalent cultivation conditions, comprises no or reduced suckers after topping as compared to a control tobacco plant.

[0010] 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 cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor.

[0011] In one aspect, the present disclosure provides a tobacco product comprising dried tobacco material from a modified tobacco plant comprising a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, wherein the mutation is not present in the endogenous gene in a control tobacco plant of the same variety, and wherein the modified tobacco plant, when grown under equivalent cultivation conditions, comprises no or reduced suckers after topping as compared to the control tobacco plant.

[0012] In one aspect, the present disclosure provides a tobacco product comprising dried tobacco material from a modified tobacco plant comprising a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, wherein the mutation is not present in the endogenous gene in a control tobacco plant of the same variety.

[0013] In one aspect, the present disclosure provides a tobacco product comprising dried tobacco material from a modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor operably linked to a heterologous promoter, wherein the modified tobacco plant, when grown under equivalent cultivation conditions, comprises no or reduced suckers after topping as compared to a control tobacco plant.

[0014] In one aspect, the present disclosure provides a tobacco product comprising dried 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, MYB, and a WRKY transcription factor, operably linked to a heterologous promoter.

[0015] In one aspect, the present disclosure provides a tobacco product comprising dried tobacco material from 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, wherein the modified tobacco plant, when grown under equivalent cultivation conditions, has no or reduced suckers after topping compared to a control tobacco plant.

[0016] In one aspect, the present disclosure provides a tobacco product comprising dried tobacco material from 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.

[0017] In one aspect, the present disclosure provides a method for producing a modified tobacco plant, comprising: (a) A step of inducing a mutation in at least one tobacco cell at a genomic locus encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor; (b) A step of selecting at least one tobacco cell containing the mutation from step (a); (c) A step of regenerating a modified tobacco plant from the at least one tobacco cell selected in step (b), wherein the modified tobacco plant, when cultivated under equivalent cultivation conditions, has no or reduced suckers after topping compared to a control tobacco plant lacking the mutation A method comprising the above is provided.

[0018] In one aspect, the present disclosure is a method for producing a modified tobacco plant, comprising: (a) A step of introducing a recombinant DNA construct into at least one tobacco cell, wherein the recombinant DNA construct comprises a heterologous promoter functionally linked to a nucleic acid encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor; (b) A step of selecting at least one tobacco cell containing the recombinant DNA construct; (c) A step of regenerating a modified tobacco plant from the at least one tobacco cell selected in step (b), wherein the modified tobacco plant, when cultivated under equivalent cultivation conditions, has no or reduced suckers after topping compared to a control tobacco plant lacking the recombinant DNA construct A method comprising the above is provided.

[0019] In one aspect, the present disclosure is a method for producing a modified tobacco plant, comprising: (a) A step of introducing a recombinant DNA construct into at least one tobacco cell, wherein the recombinant DNA construct comprises a heterologous promoter functionally 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 cyclin, cyclin-dependent kinase (CDK), CDK inhibitor, MYB, and WRKY transcription factor; (b) A step of selecting at least one tobacco cell containing the recombinant DNA construct; (c) A step of regenerating a modified tobacco plant from the at least one tobacco cell selected in step (b), wherein the modified tobacco plant, when cultivated under equivalent cultivation conditions, contains no or reduced suckers after topping as compared to a control tobacco plant lacking the recombinant DNA construct. Provided is a method comprising the above.

[0020] In one aspect, the present disclosure provides a method for preparing a tobacco product using dried tobacco material from a modified tobacco plant containing a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), CDK inhibitor, MYB, and WRKY transcription factor, wherein the mutation does not exist in the endogenous gene in a control tobacco plant of the same variety, and the modified tobacco plant, when cultivated under equivalent cultivation conditions, contains no or reduced suckers after topping as compared to the control tobacco plant.

[0021] In one aspect, the present disclosure provides a method for preparing a tobacco product using dried tobacco material from a modified tobacco plant containing a polynucleotide sequence encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), CDK inhibitor, MYB, and WRKY transcription factor, functionally linked to a heterologous promoter, wherein the modified tobacco plant, when cultivated under equivalent cultivation conditions, contains no or reduced suckers after topping as compared to the control tobacco plant.

[0022] In one aspect, the present disclosure provides a method comprising the step of preparing a tobacco product using dried tobacco material from a modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, operably linked to a heterologous promoter, wherein the modified tobacco plant, when cultivated under equivalent cultivation conditions, comprises no or reduced suckers after topping as compared to a control tobacco plant.

[0023] In one aspect, the present disclosure provides a method comprising the step of preparing a tobacco product using dried tobacco material from a modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, operably linked to a heterologous promoter, wherein the modified tobacco plant, when cultivated under equivalent cultivation conditions, comprises no or reduced suckers after topping as compared to a control tobacco plant.

[0024] In one aspect, the present disclosure provides a method comprising the step of preparing a tobacco product using dried tobacco material from a modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, operably linked to a heterologous promoter.

[0025] In one aspect, the present disclosure provides a method comprising the step of preparing a tobacco product using dried tobacco material from a modified tobacco plant comprising a recombinant DNA construct comprising a heterologous promoter functionally 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 cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, wherein the modified tobacco plant, when cultivated under equivalent cultivation conditions, has no or reduced suckers after topping as compared to a control tobacco plant.

[0026] In one aspect, the present disclosure provides a method comprising the step of preparing a tobacco product using dried tobacco material from a modified tobacco plant comprising a recombinant DNA construct comprising a heterologous promoter functionally 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 cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor.

[0027] In one aspect, the present disclosure provides a method comprising the step of transforming tobacco cells with a recombinant DNA construct comprising a heterologous promoter functionally linked to a nucleic acid encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor.

[0028] In one aspect, the present disclosure provides a method comprising the step of transforming tobacco cells with a recombinant DNA construct comprising a heterologous promoter functionally 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 cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor.

[0029] In one aspect, the present disclosure is a method for producing a modified tobacco plant, comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety, wherein the at least one tobacco plant of the first tobacco variety comprises a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, the mutation being absent in the endogenous gene in a control tobacco plant of the same variety, and the at least one tobacco plant, when cultivated under equivalent cultivation conditions, comprises no or reduced suckers after topping compared to the control tobacco plant; and (b) selecting a progeny tobacco plant that shows no or reduced suckers after topping compared to a control tobacco plant of the same hybrid cultivated under equivalent cultivation conditions; A method is provided that includes the above.

[0030] In one aspect, the present disclosure is a method for producing a modified tobacco plant, comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety, 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 cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, and the at least one tobacco plant, when cultivated under equivalent cultivation conditions, comprises no or reduced suckers after topping compared to a control tobacco plant; and (b) selecting a progeny tobacco plant that shows no or reduced suckers after topping compared to a control tobacco plant of the same hybrid cultivated under equivalent cultivation conditions; A method is provided that includes the above.

[0031] In one aspect, the present disclosure is a method for producing a modified tobacco plant, comprising: (a) A step of crossing 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 the expression of an endogenous genomic locus encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), CDK inhibitor, MYB, and WRKY transcription factor, and wherein the at least one tobacco plant, when cultivated under equivalent cultivation conditions, comprises no or reduced suckers after topping compared to a control tobacco plant; (b) A step of selecting progeny tobacco plants in which no or reduced suckers appear after topping compared to control tobacco plants of the same hybrid cultivated under equivalent cultivation conditions To provide a method comprising the same [The present invention 1001] A modified tobacco plant comprising a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, wherein the mutation does not exist in the endogenous gene in a control tobacco plant of the same variety, and the modified tobacco plant, when cultivated under equivalent cultivation conditions, contains no or reduced suckers after topping, as compared to the control tobacco plant. [The present invention 1002] A modified tobacco plant comprising a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, wherein the mutation does not exist in the endogenous gene in a control tobacco plant of the same variety. [The present invention 1003] A modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, functionally linked to a heterologous promoter, wherein the modified tobacco plant, when cultivated under equivalent cultivation conditions, contains no or reduced suckers after topping, as compared to a control tobacco plant. [The present invention 1004] A modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, functionally linked to a heterologous promoter. [The present invention 1005] A modified tobacco plant comprising a recombinant DNA construct comprising a heterologous promoter functionally 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 cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, wherein the modified tobacco plant, when cultivated under equivalent cultivation conditions, contains no or reduced suckers after topping, as compared to a control tobacco plant. [The present invention 1006] A recombinant DNA construct comprising a heterologous promoter functionally 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 cyclin, cyclin-dependent kinase (CDK), CDK inhibitor, MYB, and WRKY transcription factor A modified tobacco plant comprising the same. [Invention 1007] The modified tobacco plant according to Invention 1001 or 1002, 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 to 44 and 113. [Invention 1008] The modified tobacco plant according to Invention 1001 or 1002, 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 the group consisting of SEQ ID NOs: 45 to 88 and 114. [Invention 1009] The modified tobacco plant according to any one of Inventions 1003 to 1006, wherein the heterologous promoter comprises a polynucleotide sequence that is at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 89 to 109. [Invention 1010] The modified tobacco plant according to Invention 1005 or 1006, wherein the small RNA molecule comprises a polynucleotide sequence having at least 90% identity or complementarity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 44 and 113. [Invention 1011] Tobacco leaves of the modified tobacco plant according to any one of Inventions 1001 to 1006. [Invention 1012] Tobacco seeds of the modified tobacco plant according to any one of Inventions 1001 to 1006. [Invention 1013] The tobacco leaves according to Invention 1054, wherein the tobacco leaves are dried tobacco leaves. [Invention 1014] A tobacco product comprising an alkaloid extracted from the modified tobacco plant according to any one of Inventions 1001 to 1006 or a part thereof. [Invention 1015] A tobacco product comprising dried tobacco material from a modified tobacco plant containing a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), CDK inhibitor, MYB, and WRKY transcription factor, The mutation does not exist in the endogenous gene in the control tobacco plants of the same variety, and when the modified tobacco plant is cultivated under equivalent cultivation conditions, the tobacco product contains no or reduced suckers after topping compared to the control tobacco plant. [Invention 1016] A tobacco product comprising dried tobacco material from a modified tobacco plant containing a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), CDK inhibitor, MYB, and WRKY transcription factor, wherein the mutation does not exist in the endogenous gene in the control tobacco plants of the same variety. [Invention 1017] A tobacco product comprising dried tobacco material from a modified tobacco plant containing a polynucleotide sequence encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), CDK inhibitor, MYB, and WRKY transcription factor, functionally linked to a heterologous promoter, wherein when the modified tobacco plant is cultivated under equivalent cultivation conditions, it contains no or reduced suckers after topping compared to the control tobacco plant. [Invention 1018] A tobacco product comprising dried tobacco material from a modified tobacco plant containing a polynucleotide sequence encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), CDK inhibitor, MYB, and WRKY transcription factor, functionally linked to a heterologous promoter. [Invention 1019] A tobacco product comprising dried tobacco material from a modified tobacco plant containing a recombinant DNA construct comprising a heterologous promoter functionally 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 cyclin, cyclin-dependent kinase (CDK), CDK inhibitor, MYB, and WRKY transcription factor, wherein when the modified tobacco plant is cultivated under equivalent cultivation conditions, it contains no or reduced suckers after topping compared to the control tobacco plant. [Invention 1020] A tobacco product comprising dried tobacco material from 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 cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor. [Invention 1021] A method for producing a modified tobacco plant, the method comprising: (a) inducing a mutation in at least one tobacco cell at a genomic locus encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor; (b) selecting at least one tobacco cell comprising the mutation from step (a); (c) regenerating a modified tobacco plant from the at least one tobacco cell selected in step (b), wherein the modified tobacco plant, when grown under equivalent cultivation conditions, has no or reduced suckers after topping as compared to a control tobacco plant lacking the mutation. [Invention 1022] A method for producing a modified tobacco plant, the method comprising: (a) introducing a recombinant DNA construct into at least one tobacco cell, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor; (b) selecting at least one tobacco cell comprising the recombinant DNA construct; (c) regenerating a modified tobacco plant from the at least one tobacco cell selected in step (b), wherein the modified tobacco plant, when grown under equivalent cultivation conditions, has no or reduced suckers after topping as compared to a control tobacco plant lacking the recombinant DNA construct. [Invention 1023] A method for producing a modified tobacco plant, the method comprising: (a) A step of introducing a recombinant DNA construct into at least one tobacco cell, wherein the recombinant DNA construct comprises a heterologous promoter functionally 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 cyclin, cyclin-dependent kinase (CDK), CDK inhibitor, MYB, and WRKY transcription factor. (b) A step of selecting at least one tobacco cell containing the recombinant DNA construct. (c) A step of regenerating a modified tobacco plant from the at least one tobacco cell selected in step (b), wherein the modified tobacco plant, when cultivated under equivalent cultivation conditions, contains no or reduced suckers after topping as compared to a control tobacco plant lacking the recombinant DNA construct. [Inventive Concept 1024] A step of preparing a tobacco product using dried tobacco material from a modified tobacco plant containing a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), CDK inhibitor, MYB, and WRKY transcription factor. A method comprising: The mutation does not exist in the endogenous gene in a control tobacco plant of the same variety, and the modified tobacco plant, when cultivated under equivalent cultivation conditions, contains no or reduced suckers after topping as compared to the control tobacco plant. [Inventive Concept 1025] A step of preparing a tobacco product using dried tobacco material from a modified tobacco plant containing a polynucleotide sequence encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), CDK inhibitor, MYB, and WRKY transcription factor, functionally linked to a heterologous promoter. A method comprising: The modified tobacco plant, when cultivated under equivalent cultivation conditions, contains no or reduced suckers after topping as compared to the control tobacco plant. [Inventive Concept 1026] A step of preparing a tobacco product using dried tobacco material from a modified tobacco plant containing a polynucleotide sequence encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), CDK inhibitor, MYB, and WRKY transcription factor, functionally linked to a heterologous promoter. A method comprising: A method wherein the modified tobacco plant, when cultivated under equivalent cultivation conditions, contains no or reduced suckers after topping as compared to a control tobacco plant. [Inventive concept 1027] A step of preparing a tobacco product using dried tobacco material from a modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, functionally linked to a heterologous promoter comprising the method. [Inventive concept 1028] A step of preparing a tobacco product using dried tobacco material from a modified tobacco plant comprising a recombinant DNA construct comprising a heterologous promoter functionally 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 cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor comprising a method, wherein the modified tobacco plant, when cultivated under equivalent cultivation conditions, contains no or reduced suckers after topping as compared to a control tobacco plant. [Inventive concept 1029] A step of preparing a tobacco product using dried tobacco material from a modified tobacco plant comprising a recombinant DNA construct comprising a heterologous promoter functionally 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 cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor comprising the method. [Inventive concept 1030] A step of transforming tobacco cells with a recombinant DNA construct comprising a heterologous promoter functionally linked to a nucleic acid encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor comprising the method. [Inventive concept 1031] A step of transforming tobacco cells with a recombinant DNA construct comprising a heterologous promoter functionally 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 cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor A method comprising: [Invention 1032] A method for producing a modified tobacco plant, the method comprising: (a) Crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety, wherein the at least one tobacco plant of the first tobacco variety comprises a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, the mutation not being present in the endogenous gene in a control tobacco plant of the same variety, and the at least one tobacco plant, when cultivated under equivalent cultivation conditions, comprises no or reduced suckers after topping as compared to the control tobacco plant; (b) Selecting a progeny tobacco plant that shows no or reduced suckers after topping as compared to a control tobacco plant of the same hybrid cultivated under equivalent cultivation conditions. [Invention 1033] A method for producing a modified tobacco plant, the method comprising: (a) Crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety, 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 cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, and the at least one tobacco plant, when cultivated under equivalent cultivation conditions, comprises no or reduced suckers after topping as compared to a control tobacco plant; (b) Selecting a progeny tobacco plant that shows no or reduced suckers after topping as compared to a control tobacco plant of the same hybrid cultivated under equivalent cultivation conditions. [Invention 1034] A method for producing a modified tobacco plant, the method comprising: (a) A step of crossing 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 functionally linked to a nucleic acid encoding at least one small RNA molecule capable of reducing the expression of an endogenous genomic locus encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, and wherein the at least one tobacco plant, when cultivated under equivalent cultivation conditions, comprises no or reduced suckers after topping as compared to a control tobacco plant. (b) A step of selecting progeny tobacco plants in which no or reduced suckers appear after topping as compared to control tobacco plants of the same hybrid cultivated under equivalent cultivation conditions. [Invention 1035] The modified tobacco plant of Invention 1003, wherein the polynucleotide sequence comprises a polynucleotide sequence that is at least 80% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 44 and 113.

[0032] Brief description of the sequences Sequence numbers 1 to 44, 89 to 113, and 118 to 126 are nucleic acid sequences.

[0033] Sequence numbers 45 to 88 and 114 to 117 are amino acid sequences.

[0034] A further description of the sequence numbers provided in this specification can be found in Table 1 below.

[0035] (Table 1) Description of the sequences TIFF0007699549000001.tif177153TIFF0007699549000002.tif243153TIFF0007699549000003.tif242153

Brief description of the drawings

[0036] [Figure 1] A Venn diagram showing the overlap of genes differentially expressed in axillary buds between a pair of comparisons at 4 hours, 24 hours, and 72 hours after decapitation of water-treated and maleic hydrazide-treated tobacco plants. 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, and W72 refers to 72 hours after water treatment. [Figure 2] Shows the total mass of suckers from individual P1_2.4::MYB plants one week after decapitation. [Figure 3]Shows the total mass of the suckers from individual P1_2.4::CDKI plants one week after decapitation. [Figure 4] Shows the total mass of the suckers from individual plants containing the amiRNA-1 construct one week after decapitation. [Figure 5] Shows the total mass of the suckers from individual plants containing the amiRNA-2 construct one week after decapitation. [Figure 6] Shows the total mass of the suckers from individual plants containing the amiRNA-3 construct one week after decapitation.

Mode for Carrying Out the Invention

[0037] Detailed Description Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. When a term is provided in the singular, the inventors also contemplate aspects of the disclosure described by the plural of that term. In the event of a conflict between the terms and definitions used in the incorporated references, the terms used in this application shall have the definitions given herein. Other technical terms used shall have the ordinary meaning in the technical fields in which they are used, as exemplified by various technical field-specific dictionaries, such as "The American Heritage® Science Dictionary" (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), "McGraw-Hill Dictionary of Scientific and Technical Terms" (6th edition, 2002, McGraw-Hill, New York), or "Oxford Dictionary of Biology" (6th edition, 2008, Oxford University Press, Oxford and New York).

[0038] For example, any reference cited herein, including all patents, published patent applications, and non-patent publications, is hereby incorporated by reference in its entirety.

[0039] When a group of alternatives is presented, all combinations of the members that make up that group of alternatives are specifically contemplated. For example, when an item is selected from the group consisting of A, B, C, and D, the inventors specifically contemplate each alternative individually (e.g., A alone, B alone, etc.), as well as combinations such as A, B, and D; A and C; B and C, etc. The term "and / or" when used in a list of two or more items means any one of the listed items alone or any combination of one or more of the other listed items. For example, the expression "A and / or B" is intended to mean either or both of A and B, i.e., A alone, B alone, or the combination of A and B. The expression "A, B and / or C" is intended to mean A alone, B alone, C alone, the combination of A and B, the combination of A and C, the combination of B and C, or the combination of A, B and C.

[0040] When a numerical range is provided herein, that range is understood to include the endpoints of the range, as well as any number between the defined endpoints of the range. For example, "1 to 10" includes any number between 1 and 10, as well as the numbers 1 and 10.

[0041] As used herein, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, the terms "compound" or "at least one compound" can include a plurality of compounds including mixtures thereof.

[0042] In one aspect, the modified tobacco plants provided herein include reduced or no suckering growth after topping when grown under equivalent conditions compared to control tobacco plants.

[0043] As used herein, "sucker growth" refers to the development and / or growth of axillary (or lateral) buds ("suckers") from axillary meristems that grow between leaves and stalks. Axillary buds are embryonic shoots that include axillary meristems, surrounding leaf tissue, and surrounding stem tissue. See, for example, U.S. Patent Application Publication Nos. 2016 / 0281100 and 2017 / 0260535, which are hereby incorporated by reference in their entireties.

[0044] As used herein, "topping" refers to the removal of the shoot apex, including the shoot apical meristem, flowers, and some adjacent leaves, when the plant is approaching maturity. Topping of tobacco plants results in the loss of apical dominance. Prior to topping, sucker growth is largely kept dormant by hormonal signals emanating from the shoot apical meristem, and topping can remove the hormonal signals and allow sucker elongation ("topping-induced sucker growth"). If sucker growth is sufficiently controlled, topping can increase yield, increase value per acre, and result in desirable modifications to the physical and chemical properties of tobacco leaves.

[0045] As used herein, "equivalent growing conditions" refers to similar environmental conditions and / or agronomic practices for growing and making a meaningful comparison between two or more plant genotypes such that neither any differences in environmental conditions nor any agronomic practices contribute to or account for any differences observed between two or more plant genotypes. Environmental conditions include, for example, light, temperature, water, humidity, and nutrients (e.g., nitrogen and phosphorus). Agronomic practices include, for example, seeding, clipping, undercutting, transplanting, pinching, and sucker development. See Chapters 4B and 4C of Tobacco, Production, Chemistry and Technology, Davis & Nielsen eds., Blackwell Publishing, Oxford (1999), pp. 70-103. It is recognized in the art that "equivalent growing conditions" do not require identical growth conditions.

[0046] As used herein, "modified" refers to a plant, seed, plant part, plant cell, and plant genome that has been subjected to mutagenesis, genome editing, genetic transformation, or combinations thereof.

[0047] In one aspect, the disclosure provides a modified tobacco plant that, when grown under equivalent conditions, has no or reduced suckers after pinching compared to a control tobacco plant. As used herein, a "reduction" in the number of suckers, the size of the suckers, and / or the impact of the suckers on agronomic performance refers to a statistically significant reduction. As used herein, "statistically significant" refers to a p-value of less than 0.05 when using an appropriate measure of statistical significance (e.g., one-sided two-sample t-test).

[0048] In one aspect, the modified plants or methods provided herein require a reduction in management to control sucker growth when grown under equivalent conditions as compared to control plants. As used herein, "management" refers to manually removing suckers, applying chemicals (e.g., maleic hydrazide, flumetralin) to inhibit or remove suckers, or both. In one aspect, the modified plants or methods provided herein require a reduction in the frequency of manual sucker removal, a reduction in the frequency of chemical application, a reduction in the amount of chemical applied, or combinations thereof, as compared to control plants grown under equivalent conditions. See, for example, Fisher et al. "Topping, Managing Suckers, and Using Ethephon," pages 96-117 In: 2016 Flue-Cured Tobacco Information, North Carolina State University, which is incorporated herein by reference in its entirety.

[0049] In one aspect, the modified plants or methods provided herein require a reduced frequency of maleic hydrazide application to reduce or eliminate suckers. In one aspect, the modified plants or methods provided herein require a reduced amount of maleic hydrazide application to reduce or eliminate suckers. In one aspect, the modified plants or methods provided herein require a reduced frequency of flumetralin application to reduce or eliminate suckers. In one aspect, the modified plants or methods provided herein require a reduced amount of flumetralin application to reduce or eliminate suckers.

[0050] In one aspect, the modified plants or methods provided herein require manual removal of suckers at a frequency of 1% of the control plants when grown under equivalent conditions. In one aspect, the modified plants or methods provided herein require manual removal of suckers at a frequency of 5% of the control plants when grown under equivalent conditions. In one aspect, the modified plants or methods provided herein require manual removal of suckers at a frequency of 10% of the control plants when grown under equivalent conditions. In one aspect, the modified plants or methods provided herein require manual removal of suckers at a frequency of 20% of the control plants when grown under equivalent conditions. In one aspect, the modified plants or methods provided herein require manual removal of suckers at a frequency of 30% of the control plants when grown under equivalent conditions. In one aspect, the modified plants or methods provided herein require manual removal of suckers at a frequency of 40% of the control plants when grown under equivalent conditions. In one aspect, the modified plants or methods provided herein require manual removal of suckers at a frequency of 50% of the control plants when grown under equivalent conditions. In one aspect, the modified plants or methods provided herein require manual removal of suckers at a frequency of 60% of the control plants when grown under equivalent conditions. In one aspect, the modified plants or methods provided herein require manual removal of suckers at a frequency of 70% of the control plants when grown under equivalent conditions. In one aspect, the modified plants or methods provided herein require manual removal of suckers at a frequency of 80% of the control plants when grown under equivalent conditions. In one aspect, the modified plants or methods provided herein require manual removal of suckers at a frequency of 90% of the control plants when grown under equivalent conditions. In one aspect, the modified plants or methods provided herein require manual removal of suckers at a frequency of 95% of the control plants when grown under equivalent conditions.

[0051] In one aspect, the modified plants or methods provided herein require manual removal of suckers at a frequency of less than 1% of the control plants when grown under equivalent conditions. In one aspect, the modified plants or methods provided herein require manual removal of suckers at a frequency of less than 5% of the control plants when grown under equivalent conditions. In one aspect, the modified plants or methods provided herein require manual removal of suckers at a frequency of less than 10% of the control plants when grown under equivalent conditions. In one aspect, the modified plants or methods provided herein require manual removal of suckers at a frequency of less than 20% of the control plants when grown under equivalent conditions. In one aspect, the modified plants or methods provided herein require manual removal of suckers at a frequency of less than 30% of the control plants when grown under equivalent conditions. In one aspect, the modified plants or methods provided herein require manual removal of suckers at a frequency of less than 40% of the control plants when grown under equivalent conditions. In one aspect, the modified plants or methods provided herein require manual removal of suckers at a frequency of less than 50% of the control plants when grown under equivalent conditions. In one aspect, the modified plants or methods provided herein require manual removal of suckers at a frequency of less than 60% of the control plants when grown under equivalent conditions. In one aspect, the modified plants or methods provided herein require manual removal of suckers at a frequency of less than 70% of the control plants when grown under equivalent conditions. In one aspect, the modified plants or methods provided herein require manual removal of suckers at a frequency of less than 80% of the control plants when grown under equivalent conditions. In one aspect, the modified plants or methods provided herein require manual removal of suckers at a frequency of less than 90% of the control plants when grown under equivalent conditions. In one aspect, the modified plants or methods provided herein require manual removal of suckers at a frequency of less than 95% of the control plants when grown under equivalent conditions.

[0052] In one aspect, the modified plants provided herein require manual removal of suckers at a frequency of 10% to 95% of that of the control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require manual removal of suckers at a frequency of 20% to 95% of that of the control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require manual removal of suckers at a frequency of 30% to 95% of that of the control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require manual removal of suckers at a frequency of 40% to 95% of that of the control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require manual removal of suckers at a frequency of 50% to 95% of that of the control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require manual removal of suckers at a frequency of 60% to 95% of that of the control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require manual removal of suckers at a frequency of 70% to 95% of that of the control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require manual removal of suckers at a frequency of 80% to 95% of that of the control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require manual removal of suckers at a frequency of 90% to 95% of that of the control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require manual removal of suckers at a frequency of 10% to 75% of that of the control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require manual removal of suckers at a frequency of 10% to 50% of that of the control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require manual removal of suckers at a frequency of 10% to 25% of that of the control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require manual removal of suckers at a frequency of 50% to 75% of that of the control plants when cultivated under equivalent conditions.In one aspect, the modified plants provided herein require manual removal of suckers at a frequency of 25% to 75% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require manual removal of suckers at a frequency of 25% to 50% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require manual removal of suckers at a frequency of 1% to 50% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require manual removal of suckers at a frequency of 1% to 25% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require manual removal of suckers at a frequency of 1% to 10% of the control plants when grown under equivalent conditions.

[0053] In one aspect, the modified plants provided herein require application of a chemical substance to suppress sucker growth at a frequency of 1% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance to suppress sucker growth at a frequency of 5% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance to suppress sucker growth at a frequency of 10% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance to suppress sucker growth at a frequency of 20% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance to suppress sucker growth at a frequency of 30% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance to suppress sucker growth at a frequency of 40% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance to suppress sucker growth at a frequency of 50% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance to suppress sucker growth at a frequency of 60% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance to suppress sucker growth at a frequency of 70% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance to suppress sucker growth at a frequency of 80% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance to suppress sucker growth at a frequency of 90% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance to suppress sucker growth at a frequency of 95% of the control plants when grown under equivalent conditions.

[0054] In one aspect, the modified plants provided herein require chemical application to suppress sucker growth at a frequency of less than 1% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require chemical application to suppress sucker growth at a frequency of less than 5% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require chemical application to suppress sucker growth at a frequency of less than 10% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require chemical application to suppress sucker growth at a frequency of less than 20% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require chemical application to suppress sucker growth at a frequency of less than 30% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require chemical application to suppress sucker growth at a frequency of less than 40% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require chemical application to suppress sucker growth at a frequency of less than 50% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require chemical application to suppress sucker growth at a frequency of less than 60% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require chemical application to suppress sucker growth at a frequency of less than 70% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require chemical application to suppress sucker growth at a frequency of less than 80% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require chemical application to suppress sucker growth at a frequency of less than 90% of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require chemical application to suppress sucker growth at a frequency of less than 95% of the control plants when grown under equivalent conditions.

[0055] In one aspect, the modified plants provided herein require application of a chemical substance to suppress sucker growth at a frequency of 10% to 95% of the control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance to suppress sucker growth at a frequency of 20% to 95% of the control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance to suppress sucker growth at a frequency of 30% to 95% of the control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance to suppress sucker growth at a frequency of 40% to 95% of the control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance to suppress sucker growth at a frequency of 50% to 95% of the control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance to suppress sucker growth at a frequency of 60% to 95% of the control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance to suppress sucker growth at a frequency of 70% to 95% of the control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance to suppress sucker growth at a frequency of 80% to 95% of the control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance to suppress sucker growth at a frequency of 90% to 95% of the control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance to suppress sucker growth at a frequency of 10% to 75% of the control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance to suppress sucker growth at a frequency of 10% to 50% of the control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance to suppress sucker growth at a frequency of 10% to 25% of the control plants when cultivated under equivalent conditions.In one aspect, the modified plants provided herein require application of a chemical substance for suppressing sucker development at a frequency of 50% to 75% of that of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance for suppressing sucker development at a frequency of 25% to 50% of that of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance for suppressing sucker development at a frequency of 25% to 75% of that of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance for suppressing sucker development at a frequency of 1% to 50% of that of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance for suppressing sucker development at a frequency of 1% to 25% of that of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require application of a chemical substance for suppressing sucker development at a frequency of 1% to 10% of that of control plants when cultivated under equivalent conditions.

[0056] In one aspect, the modified plants provided herein require a chemical spraying volume that is 1% of the volume used to suppress the sucker growth of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume that is 5% of the volume used to suppress the sucker growth of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume that is 10% of the volume used to suppress the sucker growth of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume that is 20% of the volume used to suppress the sucker growth of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume that is 30% of the volume used to suppress the sucker growth of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume that is 40% of the volume used to suppress the sucker growth of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume that is 50% of the volume used to suppress the sucker growth of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume that is 60% of the volume used to suppress the sucker growth of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume that is 70% of the volume used to suppress the sucker growth of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume that is 80% of the volume used to suppress the sucker growth of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume that is 90% of the volume used to suppress the sucker growth of control plants when cultivated under equivalent conditions.In one aspect, the modified plants provided herein require a chemical spray volume that is 95% of the volume used to suppress sucker growth of control plants when grown under equivalent conditions.

[0057] In one aspect, the modified plants provided herein require a chemical spraying volume of less than 1% of the volume used to suppress the sucker growth of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume of less than 5% of the volume used to suppress the sucker growth of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume of less than 10% of the volume used to suppress the sucker growth of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume of less than 20% of the volume used to suppress the sucker growth of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume of less than 30% of the volume used to suppress the sucker growth of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume of less than 40% of the volume used to suppress the sucker growth of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume of less than 50% of the volume used to suppress the sucker growth of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume of less than 60% of the volume used to suppress the sucker growth of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume of less than 70% of the volume used to suppress the sucker growth of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume of less than 80% of the volume used to suppress the sucker growth of control plants when cultivated under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume of less than 90% of the volume used to suppress the sucker growth of control plants when cultivated under equivalent conditions.In one aspect, the modified plants provided herein require a chemical spray volume of less than 95% of the volume used to suppress sucker growth of control plants when grown under equivalent conditions.

[0058] In one aspect, the modified plants provided herein require a chemical spraying volume that is 10% to less than 95% of that of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume that is 20% to less than 95% of that of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume that is 30% to less than 95% of that of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume that is 40% to less than 95% of that of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume that is 50% to less than 95% of that of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume that is 60% to less than 95% of that of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume that is 70% to less than 95% of that of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume that is 80% to less than 95% of that of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume that is 90% to less than 95% of that of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume that is 10% to less than 75% of that of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume that is 10% to less than 50% of that of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume that is 10% to less than 25% of that of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume that is 25% to less than 50% of that of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require a chemical spraying volume that is 25% to less than 75% of that of the control plants when grown under equivalent conditions.In one aspect, the modified plants provided herein require 50% to less than 75% of the chemical spray volume of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require 1% to less than 50% of the chemical spray volume of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require 1% to less than 25% of the chemical spray volume of the control plants when grown under equivalent conditions. In one aspect, the modified plants provided herein require 1% to less than 10% of the chemical spray volume of the control plants when grown under equivalent conditions.

[0059] In one aspect, the reduced suckers include fewer total suckers, a smaller average sucker size, or both, compared to control tobacco plants when grown under equivalent conditions. In another aspect, the smaller average sucker size includes a measurement selected from the group consisting of a reduced average mass, a reduced average length, a reduced average diameter, or any combination thereof, compared to the suckers of control tobacco plants when grown under equivalent cultivation conditions. In another aspect, the smaller average sucker size includes a reduced average mass of the suckers. In a further aspect, the smaller average sucker size includes a reduced average length of the suckers. In a further aspect, the smaller average sucker size includes a reduced average diameter of the suckers.

[0060] In one aspect, the modified tobacco plants include fewer total suckers compared to control tobacco plants when grown under equivalent conditions. In one aspect, the modified tobacco plants include a smaller average sucker size compared to control tobacco plants when grown under equivalent conditions. In one aspect, the modified tobacco plants include a shorter average sucker length compared to control tobacco plants when grown under equivalent conditions. In one aspect, the modified tobacco plants include a lower average sucker mass compared to control tobacco plants when grown under equivalent conditions. In one aspect, the modified tobacco plants include a shorter average sucker diameter compared to control tobacco plants when grown under equivalent conditions.

[0061] In one aspect, the modified tobacco plant contains at least one fewer sucker when cultivated under equivalent conditions compared to the control tobacco plant. In one aspect, the modified tobacco plant contains at least two fewer suckers when cultivated under equivalent conditions compared to the control tobacco plant. In one aspect, the modified tobacco plant contains at least three fewer suckers when cultivated under equivalent conditions compared to the control tobacco plant. In one aspect, the modified tobacco plant contains at least four fewer suckers when cultivated under equivalent conditions compared to the control tobacco plant. In one aspect, the modified tobacco plant contains at least five fewer suckers when cultivated under equivalent conditions compared to the control tobacco plant. In one aspect, the modified tobacco plant contains at least seven fewer suckers when cultivated under equivalent conditions compared to the control tobacco plant. In one aspect, the modified tobacco plant contains at least ten fewer suckers when cultivated under equivalent conditions compared to the control tobacco plant. In one aspect, the modified tobacco plant contains at least fifteen fewer suckers when cultivated under equivalent conditions compared to the control tobacco plant. In one aspect, the modified tobacco plant contains at least twenty fewer suckers when cultivated under equivalent conditions compared to the control tobacco plant. In one aspect, the modified tobacco plant contains at least twenty-five fewer suckers when cultivated under equivalent conditions compared to the control tobacco plant. In one aspect, the modified tobacco plant contains at least thirty fewer suckers when cultivated under equivalent conditions compared to the control tobacco plant.

[0062] In one aspect, the modified tobacco plant contains at least 5% fewer suckers when cultivated under equivalent conditions compared to a control tobacco plant. In one aspect, the modified tobacco plant contains at least 10% fewer suckers when cultivated under equivalent conditions compared to a control tobacco plant. In one aspect, the modified tobacco plant contains at least 15% fewer suckers when cultivated under equivalent conditions compared to a control tobacco plant. In one aspect, the modified tobacco plant contains at least 20% fewer suckers when cultivated under equivalent conditions compared to a control tobacco plant. In one aspect, the modified tobacco plant contains at least 25% fewer suckers when cultivated under equivalent conditions compared to a control tobacco plant. In one aspect, the modified tobacco plant contains at least 30% fewer suckers when cultivated under equivalent conditions compared to a control tobacco plant. In one aspect, the modified tobacco plant contains at least 40% fewer suckers when cultivated under equivalent conditions compared to a control tobacco plant. In one aspect, the modified tobacco plant contains at least 50% fewer suckers when cultivated under equivalent conditions compared to a control tobacco plant. In one aspect, the modified tobacco plant contains at least 60% fewer suckers when cultivated under equivalent conditions compared to a control tobacco plant. In one aspect, the modified tobacco plant contains at least 70% fewer suckers when cultivated under equivalent conditions compared to a control tobacco plant. In one aspect, the modified tobacco plant contains at least 80% fewer suckers when cultivated under equivalent conditions compared to a control tobacco plant. In one aspect, the modified tobacco plant contains at least 90% fewer suckers when cultivated under equivalent conditions compared to a control tobacco plant. In one aspect, the modified tobacco plant contains at least 95% fewer suckers when cultivated under equivalent conditions compared to a control tobacco plant.

[0063] In one aspect, the average sucker mass is measured using the fresh sucker weight. In another aspect, the average sucker mass is measured using the dry sucker weight.

[0064] In one aspect, the modified tobacco plant comprises an average sucker mass that is at least 5% lower compared to the average sucker mass of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker mass that is at least 10% lower compared to the average sucker mass of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker mass that is at least 15% lower compared to the average sucker mass of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker mass that is at least 20% lower compared to the average sucker mass of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker mass that is at least 25% lower compared to the average sucker mass of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker mass that is at least 30% lower compared to the average sucker mass of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker mass that is at least 40% lower compared to the average sucker mass of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker mass that is at least 50% lower compared to the average sucker mass of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker mass that is at least 60% lower compared to the average sucker mass of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker mass that is at least 70% lower compared to the average sucker mass of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker mass that is at least 80% lower compared to the average sucker mass of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker mass that is at least 90% lower compared to the average sucker mass of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker mass that is at least 95% lower compared to the average sucker mass of a control tobacco plant when grown under equivalent conditions.

[0065] In one aspect, the average sucker length is measured from the base of the sucker, where the sucker joins the main tobacco stem, to the distal tip of the sucker stem.

[0066] In one aspect, the modified tobacco plant comprises an average sucker length that is at least 1 centimeter shorter, when grown under equivalent conditions, compared to the average sucker length of a control tobacco plant. In one aspect, the modified tobacco plant comprises an average sucker length that is at least 2 centimeters shorter, when grown under equivalent conditions, compared to the average sucker length of a control tobacco plant. In one aspect, the modified tobacco plant comprises an average sucker length that is at least 3 centimeters shorter, when grown under equivalent conditions, compared to the average sucker length of a control tobacco plant. In one aspect, the modified tobacco plant comprises an average sucker length that is at least 4 centimeters shorter, when grown under equivalent conditions, compared to the average sucker length of a control tobacco plant. In one aspect, the modified tobacco plant comprises an average sucker length that is at least 5 centimeters shorter, when grown under equivalent conditions, compared to the average sucker length of a control tobacco plant. In one aspect, the modified tobacco plant comprises an average sucker length that is at least 10 centimeters shorter, when grown under equivalent conditions, compared to the average sucker length of a control tobacco plant. In one aspect, the modified tobacco plant comprises an average sucker length that is at least 15 centimeters shorter, when grown under equivalent conditions, compared to the average sucker length of a control tobacco plant. In one aspect, the modified tobacco plant comprises an average sucker length that is at least 20 centimeters shorter, when grown under equivalent conditions, compared to the average sucker length of a control tobacco plant.

[0067] In one aspect, when grown under equivalent conditions, the modified tobacco plant has an average sucker length that is 0.5 centimeter to 30 centimeters shorter compared to the average sucker length of the control tobacco plant. In one aspect, when grown under equivalent conditions, the modified tobacco plant has an average sucker length that is 0.5 centimeter to 25 centimeters shorter compared to the average sucker length of the control tobacco plant. In one aspect, when grown under equivalent conditions, the modified tobacco plant has an average sucker length that is 0.5 centimeter to 20 centimeters shorter compared to the average sucker length of the control tobacco plant. In one aspect, when grown under equivalent conditions, the modified tobacco plant has an average sucker length that is 0.5 centimeter to 15 centimeters shorter compared to the average sucker length of the control tobacco plant. In one aspect, when grown under equivalent conditions, the modified tobacco plant has an average sucker length that is 0.5 centimeter to 10 centimeters shorter compared to the average sucker length of the control tobacco plant. In one aspect, when grown under equivalent conditions, the modified tobacco plant has an average sucker length that is 0.5 centimeter to 5 centimeters shorter compared to the average sucker length of the control tobacco plant. In one aspect, when grown under equivalent conditions, the modified tobacco plant has an average sucker length that is 1 centimeter to 30 centimeters shorter compared to the average sucker length of the control tobacco plant. In one aspect, when grown under equivalent conditions, the modified tobacco plant has an average sucker length that is 1 centimeter to 20 centimeters shorter compared to the average sucker length of the control tobacco plant. In one aspect, when grown under equivalent conditions, the modified tobacco plant has an average sucker length that is 1 centimeter to 10 centimeters shorter compared to the average sucker length of the control tobacco plant.

[0068] In one aspect, the modified tobacco plant comprises an average sucker length that is at least 5% shorter compared to the average sucker length of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker length that is at least 10% shorter compared to the average sucker length of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker length that is at least 15% shorter compared to the average sucker length of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker length that is at least 20% shorter compared to the average sucker length of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker length that is at least 25% shorter compared to the average sucker length of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker length that is at least 30% shorter compared to the average sucker length of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker length that is at least 40% shorter compared to the average sucker length of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker length that is at least 50% shorter compared to the average sucker length of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker length that is at least 60% shorter compared to the average sucker length of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker length that is at least 70% shorter compared to the average sucker length of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker length that is at least 80% shorter compared to the average sucker length of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker length that is at least 90% shorter compared to the average sucker length of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker length that is at least 95% shorter compared to the average sucker length of a control tobacco plant when grown under equivalent conditions.

[0069] In one aspect, the sucker diameter is measured at the base of the sucker, which is the location where the sucker joins the main stem of the plant.

[0070] In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 5% shorter compared to the average sucker diameter of a control tobacco plant when cultivated under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 10% shorter compared to the average sucker diameter of a control tobacco plant when cultivated under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 15% shorter compared to the average sucker diameter of a control tobacco plant when cultivated under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 20% shorter compared to the average sucker diameter of a control tobacco plant when cultivated under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 25% shorter compared to the average sucker diameter of a control tobacco plant when cultivated under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 30% shorter compared to the average sucker diameter of a control tobacco plant when cultivated under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 40% shorter compared to the average sucker diameter of a control tobacco plant when cultivated under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 50% shorter compared to the average sucker diameter of a control tobacco plant when cultivated under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 60% shorter compared to the average sucker diameter of a control tobacco plant when cultivated under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 70% shorter compared to the average sucker diameter of a control tobacco plant when cultivated under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 80% shorter compared to the average sucker diameter of a control tobacco plant when cultivated under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 90% shorter compared to the average sucker diameter of a control tobacco plant when cultivated under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 95% shorter compared to the average sucker diameter of a control tobacco plant when cultivated under equivalent conditions.

[0071] In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 1 millimeter shorter compared to the average sucker diameter of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 2 millimeters shorter compared to the average sucker diameter of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 3 millimeters shorter compared to the average sucker diameter of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 4 millimeters shorter compared to the average sucker diameter of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 5 millimeters shorter compared to the average sucker diameter of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 6 millimeters shorter compared to the average sucker diameter of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 7 millimeters shorter compared to the average sucker diameter of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 8 millimeters shorter compared to the average sucker diameter of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 9 millimeters shorter compared to the average sucker diameter of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 1 centimeter shorter compared to the average sucker diameter of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 1.5 centimeters shorter compared to the average sucker diameter of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 2 centimeters shorter compared to the average sucker diameter of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 3 centimeters shorter compared to the average sucker diameter of a control tobacco plant when grown under equivalent conditions.In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 4 centimeters shorter compared to the average sucker diameter of a control tobacco plant when grown under equivalent conditions. In one aspect, the modified tobacco plant comprises an average sucker diameter that is at least 5 centimeters shorter compared to the average sucker diameter of a control tobacco plant when grown under equivalent conditions.

[0072] Any nucleic acid molecule, protein or polypeptide provided herein is contemplated for use with any method provided herein. Any nucleic acid molecule, protein or polypeptide provided herein is contemplated for use with any plant, plant part, cell or seed provided herein. Any nucleic acid molecule, protein or polypeptide provided herein is contemplated for use with any tobacco plant, tobacco plant part, tobacco cell or tobacco seed provided herein.

[0073] The use of the terms "polynucleotide" or "nucleic acid molecule" is not intended to limit the present disclosure to polynucleotides containing deoxyribonucleic acid (DNA). For example, ribonucleic acid (RNA) molecules are also contemplated. One of ordinary skill in the art will recognize that polynucleotides and nucleic acid molecules can include ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. The polynucleotides of the present disclosure include all forms of sequences, including, but not limited to, single-stranded form, double-stranded form, hairpins, stem-loop structures, etc. In one aspect, the nucleic acid molecule provided herein is a DNA molecule. In another aspect, the nucleic acid molecule provided herein is an RNA molecule. In one aspect, the nucleic acid molecule provided herein is single-stranded. In another aspect, the nucleic acid molecule provided herein is double-stranded. The nucleic acid molecule can encode a polypeptide or small RNA.

[0074] Nucleic acids can be isolated using conventional techniques in the art. For example, nucleic acids can be isolated using any method including, but not limited to, recombinant nucleic acid techniques and / or polymerase chain reaction (PCR). General PCR techniques are described, for example, in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation that can be used to isolate 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. Polypeptides can also be purified, for example, by expressing nucleic acids in an expression vector. Furthermore, purified polypeptides can be obtained by chemical synthesis. The purity of a polypeptide can be measured using any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0075] In one aspect, the present disclosure provides a method for detecting recombinant nucleic acids and polypeptides in plant cells. Without limitation, nucleic acids can also be detected using hybridization. Hybridization between nucleic acids is discussed in detail by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0076] In one aspect, the endogenous gene provided herein comprises a polynucleotide sequence that is at least 80% identical to a sequence selected from the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of SEQ ID NOs: 1-44 and 113.

[0077] As used herein, the term "polypeptide" refers to a chain of at least two covalently linked amino acids. A polypeptide can be encoded by a polynucleotide provided herein. A protein provided herein can be encoded by a nucleic acid molecule provided herein. A protein can comprise a polypeptide provided herein. As used herein, "protein" refers to a chain of amino acid residues that can provide structure or enzymatic activity to a cell.

[0078] The polypeptide can be detected using an antibody. Techniques for detecting a polypeptide using an antibody include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence. The antibodies provided herein can be polyclonal or monoclonal antibodies. Antibodies having specific binding affinity for the polypeptides provided herein can be generated using methods well known in the art. The antibodies provided herein can be attached to a solid support such as a microtiter plate using methods known in the art.

[0079] (For example, of the amplification product, of the hybridization complex, of the polypeptide) Detection can be achieved using a detectable label. The term "label" is intended to encompass the use of indirect labels in addition to direct labels. Detectable labels include enzymes, hapten groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.

[0080] 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of SEQ ID NOs: 45-88 and 114.

[0081] As used herein, the term "percent identity" or "percent identical" 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 "alignable" regions); (ii) determining the number of positions at which identical nucleic bases (for nucleotide sequences) or identical amino acid residues (for proteins and polypeptides) occur in both sequences to obtain the number of matched positions; (iii) dividing the number of matched positions by the total number of positions in the comparison window; and then (iv) multiplying this quotient by 100% to obtain the percent identity. When "percent identity" is calculated with respect to a reference sequence without specifying a particular comparison window, the percent identity is determined by dividing the number of matched positions over the aligned regions by the full length of the reference sequence. Thus, in the present application, when two sequences (query and subject) are optimally aligned (allowing gaps during 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 the length of the query sequence (or the comparison window), and then multiplied by 100%.

[0082] When the percentage of sequence identity is used with respect to amino acids, positions of residues that are not identical often differ by conservative amino acid substitutions, where an amino acid residue is substituted by another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity), and it is recognized that the functional properties of the molecule are not changed. When sequences differ by conservative substitutions, the percent sequence identity can be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity."

[0083] Various pairwise or multiple sequence alignment algorithms and programs, such as ClustalW or Basic Local Alignment Search Tool (BLASTTM), can be used to compare sequence identity or similarity between two or more nucleotide or amino acid sequences in order to optimally align the sequences and calculate their percent identity. Other alignment and comparison methods are known in the art, but the alignment and percent identity (including the above percent identity ranges) between two sequences can be determined by the ClustalW algorithm. See, for example, Chenna et al., “Multiple sequence alignment with the Clustal series of programs,” Nucleic Acids Research 31:3497-3500 (2003), Thompson et al., “Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice,” Nucleic Acids Research 22:4673-4680 (1994); Larkin MA et al., “Clustal W and Clustal X version 2.0,” Bioinformatics 23:2947-48 (2007); and Altschul et al. “Basic local alignment search tool” J. Mol. Biol. 215:403-410 (1990), the entire contents and disclosures of which are incorporated herein by reference.

[0084] As used herein with respect to two nucleotide sequences, the terms "percent complementarity" or "percent complementary" are similar to the concept of percent identity, but refer to the percentage of nucleotides in a query sequence that optimally base pair or hybridize to the nucleotides of a subject sequence when the query and subject sequences are aligned linearly and optimally base pair without secondary folding structures such as loops, stems, or hairpins. Such percent complementarity can be between two DNA strands, two RNA strands, or a DNA strand and an RNA strand. "Percent complementarity" can be calculated by (i) optimally base pairing or hybridizing two nucleotide sequences in a linear and fully extended configuration (i.e., without folding or secondary structure) over a window of comparison, (ii) determining the number of positions that form base pairs between the two sequences over the window of comparison to obtain the number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions within the window of comparison, and (iv) multiplying this quotient by 100% to obtain the percent complementarity of the two sequences. Optimal base pairing of two sequences can be determined based on known base pairing of nucleotide bases such as G-C, A-T, and A-U via hydrogen bonding. When "percent complementarity" is calculated in relation to a reference sequence without specifying a particular window of comparison, percent identity is determined by dividing the number of complementary positions between the two linear sequences by the full length of the reference sequence. Thus, in this application, when two sequences (query and subject) are optimally base paired (allowing for mismatches or nucleotides that do not form base pairs), the "percent complementarity" of the query sequence is equal to the number of positions that form base pairs between the two sequences divided by the total number of positions in the query sequence over the length of the query sequence, and then multiplied by 100%.

[0085] 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 cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, wherein the mutation is not present in the endogenous gene in a control tobacco plant of the same variety, and wherein the modified tobacco plant, when grown under equivalent cultivation conditions, has no or reduced suckers after topping compared to the control tobacco plant.

[0086] In another 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 cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, wherein the mutation is not present in the endogenous gene in a control tobacco plant of the same variety.

[0087] In a further 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 the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, 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 the modified tobacco plant, when grown under equivalent cultivation conditions, has no or reduced suckers after topping compared to a control tobacco plant lacking the mutation. In one aspect, the method further comprises (d) cultivating the modified tobacco plant regenerated in step (c). In another aspect, the method further comprises (e) crossing the modified tobacco plant cultivated in step (d) with a second tobacco plant, and (f) obtaining at least one seed from the cross in step (e).

[0088] In one aspect, the modified tobacco plant, tobacco plant part, tobacco seed, tobacco cell or tobacco genome provided herein comprises one or more mutations in an endogenous gene encoding a cyclin. In one aspect, the modified tobacco plant, tobacco plant part, tobacco seed, tobacco cell or tobacco genome provided herein comprises one or more mutations in an endogenous gene encoding a 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 mutations in an endogenous gene encoding a 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 mutations in an endogenous gene encoding an MYB. In one aspect, the modified tobacco plant, tobacco plant part, tobacco seed, tobacco cell or tobacco genome provided herein comprises one or more mutations in an endogenous gene encoding a WRKY transcription factor.

[0089] Cyclins regulate the progression of cells through the cell cycle. Cyclins often play a role in the activation of CDKs. Cyclins are characterized by having a conserved cyclin box domain. The cyclin box domain typically contains about 150 amino acids, which are structured into five α-helical regions. The cyclin box domain is important for binding other proteins such as CDKs. 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.

[0090] G1 (Gap 1 phase) is the first part of interphase during the cell cycle in eukaryotic cells. Cells synthesize mRNA and proteins during G1 phase, and G1 phase ends with the transition to the S phase. The S phase (synthesis phase) is the second part of interphase during the cell cycle, during which DNA is replicated. The S phase ends with the transition to the G2 phase. G2 (Gap 2 phase) typically involves rapid cell growth as the cell prepares for the transition to the M phase (mitosis phase). Cyclins and CDKs are important regulators during the transitions from G1 to S phase and from G2 to M phase as well as at checkpoints during G1 phase, S phase, G2 phase, and M phase.

[0091] There are two major groups of cyclins: G1 / S cyclins required for the regulation of the cell cycle through the G1 / S transition, and G2 / M cyclins required for the regulation of the cell cycle through the G2 / M transition. If the required cyclin or CDK cannot function properly, the cell cycle arrests at the G1 / S transition or G2 / M transition, which can prevent the formation of daughter cells. In one aspect, the cyclin provided herein is a G1 / S cyclin. In another aspect, the cyclin provided herein is a G2 / M cyclin.

[0092] In one aspect, the present disclosure provides a polynucleotide encoding a cyclin. In another aspect, the present disclosure provides a cyclin.

[0093] 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 as compared to the cyclin 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 cyclin as compared to the cyclin encoded by the 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 as compared to the mRNA of the endogenous gene lacking the premature stop codon.

[0094] In one aspect, the present disclosure provides a polynucleotide comprising a mutation that does not naturally occur in the sequence encoding the cyclin box domain. In one aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene encoding a cyclin, wherein the mutated polynucleotide encodes a cyclin lacking the cyclin box domain. In another aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene encoding a cyclin, wherein the mutated polynucleotide encodes a cyclin comprising a truncated cyclin box domain compared to the cyclin encoded by the endogenous gene lacking the mutation. In one aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene encoding a cyclin, wherein the mutated polynucleotide encodes a cyclin that is unable to bind a CDK. In one aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene encoding a cyclin, wherein the mutated polynucleotide encodes a cyclin that exhibits a reduced binding affinity for at least one CDK compared to the cyclin encoded by the endogenous gene lacking the mutation.

[0095] 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 a further aspect, the mutated polynucleotide encodes a constitutively active cyclin. In another aspect, the mutated polynucleotide encodes an inactive cyclin.

[0096] In one aspect, the endogenous gene encoding cyclin comprises a polynucleotide sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 6 to 32. In one aspect, the endogenous gene encoding cyclin comprises a polynucleotide sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 6 to 32. In one aspect, the endogenous gene encoding cyclin comprises a polynucleotide sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 6 to 32. In one aspect, the endogenous gene encoding cyclin comprises a polynucleotide sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 6 to 32. In one aspect, the endogenous gene encoding cyclin comprises a polynucleotide sequence that is at least 96% identical to a sequence selected from the group consisting of SEQ ID NOs: 6 to 32. In one aspect, the endogenous gene encoding cyclin comprises a polynucleotide sequence that is at least 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 6 to 32. In one aspect, the endogenous gene encoding cyclin comprises a polynucleotide sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 6 to 32. In one aspect, the endogenous gene encoding cyclin comprises a polynucleotide sequence that is at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 6 to 32. In one aspect, the endogenous gene encoding cyclin comprises a polynucleotide sequence that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 6 to 32.

[0097] In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 70% identical to a sequence selected from the group consisting of SEQ ID NOs: 50 to 76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 50 to 76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 50 to 76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 50 to 76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 50 to 76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 50 to 76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 96% identical to a sequence selected from the group consisting of SEQ ID NOs: 50 to 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 to 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 to 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 to 76. In one aspect, the cyclin comprises an amino acid sequence that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 50 to 76.

[0098] In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 70% similar to a sequence selected from the group consisting of SEQ ID NOs: 50 to 76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 75% similar to a sequence selected from the group consisting of SEQ ID NOs: 50 to 76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 80% similar to a sequence selected from the group consisting of SEQ ID NOs: 50 to 76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 85% similar to a sequence selected from the group consisting of SEQ ID NOs: 50 to 76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 90% similar to a sequence selected from the group consisting of SEQ ID NOs: 50 to 76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 95% similar to a sequence selected from the group consisting of SEQ ID NOs: 50 to 76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 96% similar to a sequence selected from the group consisting of SEQ ID NOs: 50 to 76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 97% similar to a sequence selected from the group consisting of SEQ ID NOs: 50 to 76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 98% similar to a sequence selected from the group consisting of SEQ ID NOs: 50 to 76. In one aspect, the endogenous cyclin comprises an amino acid sequence that is at least 99% similar to a sequence selected from the group consisting of SEQ ID NOs: 50 to 76. In one aspect, the cyclin comprises an amino acid sequence that is 100% similar to a sequence selected from the group consisting of SEQ ID NOs: 50 to 76.

[0099] Cyclin-dependent kinases (CDKs) are a family of protein kinases involved in the regulation of the cell cycle, transcription, and mRNA processing. CDKs are recognized as belonging to Enzyme Commission class 2.7.11.22. CDKs contain a kinase domain and interact with or bind to cyclins. Since CDKs phosphorylate substrates on serine and threonine, they can also be called serine-threonine kinases. CDKs, together with cyclins, are required 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 hereby incorporated by reference in its entirety.

[0100] In one aspect, the present disclosure provides a polynucleotide encoding a CDK. In another aspect, the present disclosure provides a CDK.

[0101] In one aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene, wherein the mutated polynucleotide encodes a mutated CDK as compared to the CDK encoded by the endogenous gene lacking the mutation. In one aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene, wherein the mutated polynucleotide encodes a truncated CDK as compared to the CDK encoded by the 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 mutated mRNA of an endogenous gene comprising a premature stop codon, wherein the mutated mRNA encodes a truncated CDK as compared to the mRNA of the endogenous gene lacking the premature stop codon.

[0102] In one aspect, the present disclosure provides a polynucleotide comprising a mutation that does not naturally occur in the sequence encoding the kinase domain. In one aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene encoding a CDK, wherein the mutant polynucleotide encodes a CDK lacking a kinase domain. In another aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene encoding a CDK, wherein the mutant polynucleotide encodes a CDK comprising a truncated kinase domain compared to the CDK 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 encoding a CDK, wherein the mutant polynucleotide encodes a CDK that is unable to bind a cyclin. In one aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene encoding a CDK, which shows a reduced binding affinity for at least one cyclin compared to the CDK encoded by the endogenous gene lacking the mutation.

[0103] In one aspect, the mutant polynucleotide encodes a dominant negative allele of a CDK. In another aspect, the mutant polynucleotide encodes a dominant positive allele of a CDK. In a further aspect, the mutant polynucleotide encodes a constitutively active CDK. In another aspect, the mutant polynucleotide encodes an inactive CDK.

[0104] In one aspect, the endogenous gene encoding CDK comprises a polynucleotide sequence that is at least 80% identical to a sequence selected from the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of SEQ ID NOs: 1-4.

[0105] In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 70% identical to a sequence selected from the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of SEQ ID NOs: 45-48. In one aspect, the CDK comprises an amino acid sequence that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 45-48.

[0106] In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 70% similar to a sequence selected from the group consisting of SEQ ID NOs: 45 to 48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 75% similar to a sequence selected from the group consisting of SEQ ID NOs: 45 to 48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 80% similar to a sequence selected from the group consisting of SEQ ID NOs: 45 to 48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 85% similar to a sequence selected from the group consisting of SEQ ID NOs: 45 to 48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 90% similar to a sequence selected from the group consisting of SEQ ID NOs: 45 to 48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 95% similar to a sequence selected from the group consisting of SEQ ID NOs: 45 to 48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 96% similar to a sequence selected from the group consisting of SEQ ID NOs: 45 to 48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 97% similar to a sequence selected from the group consisting of SEQ ID NOs: 45 to 48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 98% similar to a sequence selected from the group consisting of SEQ ID NOs: 45 to 48. In one aspect, the endogenous CDK comprises an amino acid sequence that is at least 99% similar to a sequence selected from the group consisting of SEQ ID NOs: 45 to 48. In one aspect, the CDK comprises an amino acid sequence that is 100% similar to a sequence selected from the group consisting of SEQ ID NOs: 45 to 48.

[0107] Cyclin-dependent kinase inhibitors (CDKIs) are proteins that inhibit CDKs. CDKIs can bind to CDKs and prevent CDKs from binding to cyclins, thereby negatively regulating the cell cycle in eukaryotic cells. In plants, CDKIs are classified 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: el282021 (2017), which is incorporated herein by reference in its entirety.

[0108] 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 an SMR.

[0109] In one aspect, the disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene, wherein the mutated polynucleotide encodes a mutated CDKI compared to the CDKI encoded by the endogenous gene lacking the mutation. In one aspect, the disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene, wherein the mutated polynucleotide encodes a truncated CDKI compared to the CDKI encoded by the endogenous gene lacking the mutation. In another aspect, the disclosure provides a non-naturally occurring truncated CDKI. In one aspect, the disclosure provides a polynucleotide encoding a mutated mRNA of an endogenous gene comprising a premature stop codon, wherein the mutated mRNA encodes a truncated CDKI compared to the mRNA of the endogenous gene lacking the premature stop codon.

[0110] In one aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene encoding a CDKI, wherein the mutated polynucleotide encodes a CDKI that is unable to bind a CDK. In one aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene encoding a CDKI, wherein the mutated polynucleotide encodes a CDKI that exhibits a reduced binding affinity for at least one CDK as compared to the CDKI encoded by the endogenous gene lacking the mutation.

[0111] In one aspect, the mutated polynucleotide encodes a dominant negative allele of a CDKI. In another aspect, the mutated polynucleotide encodes a dominant positive allele of a CDKI. In a further aspect, the mutated polynucleotide encodes a constitutively active CDKI. In another aspect, the mutated polynucleotide encodes an inactive CDKI.

[0112] 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.

[0113] 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 CDKI comprises an amino acid sequence that is 100% identical to SEQ ID NO: 49.

[0114] 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 identical 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 CDKI comprises an amino acid sequence that is 100% similar to SEQ ID NO: 49.

[0115] The MYB protein (MYB) is part of a family of transcription factors. In plants, MYB contains a conserved MYB DNA binding domain. The MYB DNA binding domain contains up to three imperfect repeats of approximately 55 amino acids each in a helix-turn-helix structure. These repeats are called R1, R2, and R3, and the R2 / R3 repeat has been shown to bind directly to the major groove of DNA. A plant-specific subfamily of MYB contains the R2R3-type MYB domain and is called R2R3-type MYB. Other subfamilies of MYB include R1-type, 3R-type, and 4R-type MYB. See, for example, Ambawat et al., "MYB transcription factor genes as regulators for plant responses: an overview," Physiol. Mol. Biol. Plants, 19:307-321 (2013), which is incorporated herein by reference in its entirety.

[0116] In one aspect, the MYB provided herein is an R1-type MYB. In another aspect, the MYB provided herein is an R2R3-type MYB. In another aspect, the MYB provided herein is a 3R-type MYB. In another aspect, the MYB provided herein is a 4R-type MYB.

[0117] In one aspect, the disclosure provides a polynucleotide encoding MYB. In another aspect, the disclosure provides MYB.

[0118] In one aspect, the disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene, wherein the mutated polynucleotide encodes a mutated MYB as compared to the MYB encoded by the endogenous gene lacking the mutation. In one aspect, the disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene, wherein the mutated polynucleotide encodes a truncated MYB as compared to the MYB encoded by the endogenous gene lacking the mutation. In another aspect, the disclosure provides a non-naturally occurring truncated MYB. In one aspect, the disclosure provides a polynucleotide encoding a mutated mRNA of an endogenous gene comprising a premature stop codon, wherein the mutated mRNA encodes a truncated MYB as compared to the mRNA of the endogenous gene lacking the premature stop codon.

[0119] In one aspect, the present disclosure provides a polynucleotide comprising a mutation that is not naturally occurring in a sequence encoding a MYB DNA binding domain. In one aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene encoding MYB, wherein the mutated polynucleotide encodes a MYB lacking the MYB DNA binding domain. In another aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene encoding MYB, wherein the mutated polynucleotide encodes a MYB comprising a truncated MYB DNA binding domain as compared to the MYB encoded by the endogenous gene lacking the mutation.

[0120] In one aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene encoding MYB, wherein the mutated polynucleotide encodes a MYB that is unable to bind DNA. In one aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene encoding MYB, wherein the mutated polynucleotide encodes a MYB that exhibits a reduced binding affinity for DNA as compared to the MYB encoded by the endogenous gene lacking the mutation.

[0121] In another aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene encoding MYB, wherein the mutant polynucleotide encodes an MYB comprising an R2R3-type MYB domain truncated as compared to the MYB encoded by the endogenous gene lacking the mutation. In another aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene encoding MYB, wherein the mutant polynucleotide encodes an MYB comprising an R1-type MYB domain truncated as compared to the MYB encoded by the endogenous gene lacking the mutation. In another aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene encoding MYB, wherein the mutant polynucleotide encodes an MYB comprising a 3R-type MYB domain truncated as compared to the MYB encoded by the endogenous gene lacking the mutation. In another aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene encoding MYB, wherein the mutant polynucleotide encodes an MYB comprising a 4R-type MYB domain truncated as compared to the MYB encoded by the endogenous gene lacking the mutation.

[0122] In one aspect, the mutant polynucleotide encodes a dominant negative allele of MYB. In another aspect, the mutant polynucleotide encodes a dominant positive allele of MYB. In a further aspect, the mutant polynucleotide encodes a constitutively active MYB. In another aspect, the mutant polynucleotide encodes an inactive MYB.

[0123] In one aspect, the endogenous gene encoding MYB comprises a polynucleotide sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 33 to 44. In one aspect, the endogenous gene encoding MYB comprises a polynucleotide sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 33 to 44. In one aspect, the endogenous gene encoding MYB comprises a polynucleotide sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 33 to 44. In one aspect, the endogenous gene encoding MYB comprises a polynucleotide sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 33 to 44. In one aspect, the endogenous gene encoding MYB comprises a polynucleotide sequence that is at least 96% identical to a sequence selected from the group consisting of SEQ ID NOs: 33 to 44. In one aspect, the endogenous gene encoding MYB comprises a polynucleotide sequence that is at least 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 33 to 44. In one aspect, the endogenous gene encoding MYB comprises a polynucleotide sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 33 to 44. In one aspect, the endogenous gene encoding MYB comprises a polynucleotide sequence that is at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 33 to 44. In one aspect, the endogenous gene encoding MYB comprises a polynucleotide sequence that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 33 to 44.

[0124] In one aspect, the endogenous MYB comprises an amino acid sequence that is at least 70% 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 75% 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 80% 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 85% 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 90% 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 95% 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 96% 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 97% 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 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 MYB comprises an amino acid sequence that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 77-88.

[0125] In one aspect, the endogenous MYB comprises an amino acid sequence that is at least 70% similar 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 75% similar 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 80% similar 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 85% similar 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 90% similar 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 95% similar 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 96% similar 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 97% similar 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 98% similar 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% similar to a sequence selected from the group consisting of SEQ ID NOs: 77-88. In one aspect, the MYB comprises an amino acid sequence that is 100% similar to a sequence selected from the group consisting of SEQ ID NOs: 77-88.

[0126] WRKY transcription factors are one of the largest families of transcription regulators known in plants. WRKY transcription factors are proteins that contain a "WRKY domain." The WRKY domain contains approximately 60-70 amino acids and is involved in DNA binding. The WRKY domain contains a highly conserved "WRKY core domain" that has an amino acid motif of 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 composed of five antiparallel β-strands, and the conserved core is found on the second β-strand. The third β-strand also contains a highly conserved amino acid motif of 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, and the position of the intervening intron is highly conserved among WRKY transcription factors. The WRKY domain also contains a "zinc finger region" that includes an amino acid motif of CX4-5CX22-23HXH (SEQ ID NO: 115) or CX7CX23HXC (SEQ ID NO: 116), where X can be any amino acid, C is cysteine, and H is histidine. The WRKY domain also contains a "DWK salt bridge" motif, and the conserved tryptophan (W) of the WRKY core (SEQ ID NO: 117) forms a triplet with an aspartic acid (D) located 4 amino acids upstream of W and a lysine (K) located 29 amino acids downstream of W. The DWK salt bridge is thought to be important for stabilizing the WRKY domain. See, for example, Rushton et al., "WRKY transcription factors," Trends in Plant Science, 15:247-258 (2010), which is hereby incorporated by reference in its entirety.

[0127] In one aspect, the present disclosure provides a polynucleotide encoding a WRKY transcription factor. In another aspect, the present disclosure provides a WRKY transcription factor.

[0128] In one aspect, the present disclosure provides a polynucleotide comprising a non-naturally occurring mutation, wherein the polynucleotide encodes a WRKY transcription factor. In one aspect, the present disclosure provides a polynucleotide encoding a truncated WRKY transcription factor. 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 an mRNA comprising a premature stop codon, wherein the mRNA encodes a truncated WRKY transcription factor.

[0129] In one aspect, the present disclosure provides a mutant polynucleotide comprising a mutation in an endogenous gene, wherein the mutant polynucleotide encodes a mutant WRKY transcription factor that is mutated compared to the WRKY transcription factor 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 WRKY transcription factor that is truncated compared to the WRKY transcription factor encoded by the 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 that is truncated compared to the mRNA of the endogenous gene lacking the premature stop codon.

[0130] In one aspect, the present disclosure provides a polynucleotide comprising a mutation not naturally present in the sequence encoding the WRKY domain. In one aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene encoding a WRKY transcription factor, wherein the mutated polynucleotide encodes a WRKY transcription factor lacking the WRKY domain. In another aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene encoding a WRKY transcription factor, wherein the mutated polynucleotide encodes a WRKY transcription factor comprising a truncated WRKY domain as compared to the WRKY transcription factor encoded by the endogenous gene lacking the mutation.

[0131] In one aspect, the present disclosure provides a polynucleotide comprising a mutation not naturally present in the sequence encoding the WRKY core domain. In one aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene encoding a WRKY transcription factor, wherein the mutated polynucleotide encodes a WRKY transcription factor lacking the WRKY core domain. In another aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene encoding a WRKY transcription factor, wherein the mutated polynucleotide encodes a WRKY transcription factor comprising a truncated WRKY core domain as compared to the WRKY transcription factor encoded by the endogenous gene lacking the mutation.

[0132] In one aspect, the present disclosure provides a polynucleotide comprising a mutation that does not naturally occur in the sequence encoding the WRKY core variant domain. In one aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene encoding a WRKY transcription factor, wherein the mutated polynucleotide encodes a WRKY transcription factor lacking the WRKY core variant domain. In another aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene encoding a WRKY transcription factor, wherein the mutated polynucleotide encodes a WRKY transcription factor comprising a truncated WRKY core variant domain as compared to the WRKY transcription factor encoded by the endogenous gene lacking the mutation.

[0133] In one aspect, the present disclosure provides a polynucleotide comprising a mutation that does not naturally occur in the sequence encoding the PRSYY motif (SEQ ID NO: 112). In one aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene encoding a WRKY transcription factor, wherein the mutated polynucleotide encodes a WRKY transcription factor lacking the PRSYY motif (SEQ ID NO: 112). In another aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene encoding a WRKY transcription factor, wherein the mutated polynucleotide encodes a WRKY transcription factor comprising a truncated PRSYY motif (SEQ ID NO: 112) as compared to the WRKY transcription factor encoded by the endogenous gene lacking the mutation.

[0134] In one aspect, the present disclosure provides a polynucleotide comprising a mutation not naturally occurring in the sequence encoding the zinc finger region. In one aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene encoding a WRKY transcription factor, wherein the mutated polynucleotide encodes a WRKY transcription factor lacking the zinc finger region. In another aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene encoding a WRKY transcription factor, wherein the mutated polynucleotide encodes a WRKY transcription factor comprising a truncated zinc finger region as compared to the WRKY transcription factor encoded by the endogenous gene lacking the mutation.

[0135] In one aspect, the present disclosure provides a polynucleotide comprising a mutation not naturally occurring in the sequence encoding the DWK salt bridge motif. In one aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene encoding a WRKY transcription factor, wherein the mutated polynucleotide encodes a WRKY transcription factor lacking the DWK salt bridge motif. In another aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene encoding a WRKY transcription factor, wherein the mutated polynucleotide encodes a WRKY transcription factor comprising a truncated DWK salt bridge motif as compared to the WRKY transcription factor encoded by the endogenous gene lacking the mutation.

[0136] In one aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene encoding a WRKY transcription factor, wherein the mutated polynucleotide encodes a WRKY transcription factor that is unable to bind DNA. In one aspect, the present disclosure provides a mutated polynucleotide comprising a mutation in an endogenous gene encoding a WRKY transcription factor, wherein the mutated polynucleotide encodes a WRKY transcription factor that exhibits a reduced binding affinity for DNA as compared to the WRKY transcription factor encoded by the endogenous gene lacking the mutation.

[0137] In one aspect, the mutated polynucleotide encodes a dominant negative allele of the WRKY transcription factor. In another aspect, the mutated polynucleotide encodes a dominant positive allele of the 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 inactive WRKY transcription factor.

[0138] 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 the WRKY transcription factor comprises a polynucleotide sequence that is 100% identical to SEQ ID NO: 113.

[0139] 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 WRKY transcription factor comprises an amino acid sequence that is 100% identical to SEQ ID NO: 114.

[0140] 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 WRKY transcription factor comprises an amino acid sequence that is 100% similar to SEQ ID NO: 114.

[0141] In one aspect, the modified tobacco plants, tobacco plant parts, tobacco seeds, tobacco cells or tobacco genomes provided herein contain one or more mutations. As used herein, "mutation" refers to a non-naturally occurring change to the DNA as compared to an endogenous reference DNA sequence. It will be understood that when identifying a mutation, the endogenous reference DNA sequence should be from the same variety of tobacco. For example, if a modified tobacco plant containing a mutation is derived from the variety TN90, the endogenous reference sequence must be the endogenous TN90 sequence, not a homologous sequence from a different tobacco variety (e.g., K326). Similarly, if a modified tobacco cell containing a mutation is a TN90 cell, the endogenous reference sequence must be the endogenous TN90 sequence, not a homologous sequence from tobacco cells derived from a different tobacco variety (e.g., K326).

[0142] In one aspect, the mutations provided herein create a dominant negative allele at the mutated locus. A dominant allele is an allele that masks the contribution of a second allele at the same locus. The dominant allele can be a "dominant negative allele" or a "dominant positive allele". A dominant negative allele or antimorph is an allele that acts in opposition to the function of the normal allele. A dominant negative allele typically does not function normally and directly inhibits the activity of the wild-type protein (e.g., through dimerization) or inhibits the activity of a second protein required for the normal function of the wild-type protein (e.g., an activator or downstream component of the pathway). For example, a dominant negative allele suppresses or reduces the normal function of the allele in the heterozygous or homozygous state. A dominant positive allele can increase normal gene function (e.g., hypermorph) or provide a new function to the gene (e.g., neomorph). A semi-dominant allele occurs when the penetrance of the associated phenotype in an individual heterozygous for that allele is lower than the penetrance observed in an individual homozygous for the allele.

[0143] In one aspect, the mutations provided herein create a dominant negative allele at the mutated locus. In another aspect, the mutations provided herein create a dominant positive allele at the mutated locus.

[0144] As used herein, "inducing" a mutation refers to generating a mutation in a polynucleotide sequence by human intervention. Many suitable methods for inducing mutations in tobacco are known in the art. Non-limiting examples of such methods include the use of chemical mutagens, radiation, and nucleases. In one aspect, inducing a mutation includes the use of an agent selected from the group consisting of chemical mutagens, radiation, transposons, Agrobacterium, and nucleases.

[0145] In one aspect, inducing a mutation includes the use of chemical mutagens. In one aspect, the chemical mutagen includes ethyl methanesulfonate (EMS).

[0146] In another aspect, inducing a mutation includes the use of radiation. In one aspect, the radiation includes gamma rays, X-rays or ionizing radiation. In another aspect, the radiation includes the use of fast neutrons.

[0147] In one aspect, inducing a mutation includes the use of transposons. In another aspect, inducing a mutation includes the use of Agrobacterium.

[0148] In a further aspect, inducing a mutation includes the use of nucleases. In one aspect, the nuclease is selected from the group consisting of meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, CRISPR / Cas9 nuclease, CRISPR / Cpf1 nuclease, CRISPR / CasX nuclease, CRISPR / CasY nuclease and Csm1 nuclease. In one aspect, inducing a mutation includes the use of CRISPR / Cas9 nuclease. In one aspect, inducing a mutation includes the use of CRISPR / Cpf1 nuclease. In one aspect, inducing a mutation includes the use of CRISPR / CasX nuclease. In one aspect, inducing a mutation includes the use of CRISPR / CasY nuclease. In one aspect, inducing a mutation includes the use of Csm1 nuclease.

[0149] Several types of mutations are known in the art. In one aspect, a mutation includes an insertion. An "insertion" refers to the addition of one or more nucleotides or amino acids to a given polynucleotide or amino acid sequence, respectively, as compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, a mutation includes a deletion. A "deletion" refers to the removal of one or more nucleotides or amino acids from a given polynucleotide or amino acid sequence, respectively, as compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, a mutation includes a substitution. A "substitution" refers to the replacement of one or more nucleotides or amino acids in a given polynucleotide or amino acid sequence, respectively, as compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, a mutation includes an inversion. An "inversion" refers to the case where a segment of a polynucleotide or amino acid sequence is inverted between termini. In one aspect, the mutations provided herein include mutations selected from the group consisting of insertions, deletions, substitutions, and inversions.

[0150] Mutations in the coding region of a gene (e.g., mutations in exons) can result in truncated proteins or polypeptides when the mutated messenger RNA (mRNA) is translated into a protein or polypeptide. In one aspect, the present disclosure provides mutations that result in truncation of a protein or polypeptide. As used herein, a "truncated" protein or polypeptide contains at least one fewer amino acid as compared to an endogenous control protein or polypeptide. For example, if an endogenous Protein A contains 100 amino acids, a truncated form of Protein A can contain from 1 to 99 amino acids.

[0151] While not limited by any scientific theory, one way to cause cleavage of a protein or polypeptide is by introducing a premature stop codon into the mRNA transcript of an endogenous gene. In one aspect, the present disclosure provides a mutation that results in a premature stop codon in the mRNA transcript of an endogenous gene. As used herein, a "stop codon" refers to a nucleotide triplet within an mRNA transcript that signals the termination of protein translation. A "premature stop codon" refers to a stop codon that is located earlier (e.g., 5'ward) than the normal stop codon position in the endogenous mRNA transcript. Some stop codons, including but not limited to "UAG", "UAA", "UGA", "TAG", "TAA", and "TGA", are known in the art.

[0152] In one aspect, the mutations provided herein include null mutations. As used herein, a "null mutation" refers to a mutation that confers a complete loss of function to the protein encoded by the gene containing the mutation, or to a small RNA encoded by a genomic locus, or a mutation that confers a complete loss of function to a small RNA encoded by a genomic locus. A null mutation can cause a lack of mRNA transcript production, a lack of small RNA transcript production, a lack of protein function, or a combination thereof.

[0153] The mutations provided herein can be located in any portion of the endogenous gene. In one aspect, the mutations provided herein are located within an exon of the endogenous gene. In another aspect, the mutations provided herein are located within an intron of the endogenous gene. In a further aspect, the mutations provided herein are located within the 5'-untranslated region of the endogenous gene. In yet another aspect, the mutations provided herein are located within the 3'-untranslated region of the endogenous gene. In yet another aspect, the mutations provided herein are located within the promoter of the endogenous gene.

[0154] The screening and selection of the mutagenized tobacco plants can be by any method known to those skilled in the art. Examples of screening and selection methods include, but are not limited to, Southern analysis, PCR amplification for the detection of polynucleotides, Northern blot, RNase protection, primer extension, RT-PCR amplification for the detection of RNA transcripts, Sanger sequencing, next-generation sequencing technologies (e.g., Illumina, PacBio, Ion Torrent, 454) for the detection of the enzymatic or ribozyme activity of polypeptides and polynucleotides, enzyme assays, as well as protein gel electrophoresis, Western blot, immunoprecipitation, and enzyme-linked immunosorbent assay for the detection of polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining can also be used to detect the presence or expression of polypeptides and / or polynucleotides. Methods for performing all of the cited techniques are known.

[0155] In one aspect, a mutation in an endogenous gene results in a decreased level of expression as compared to the endogenous gene lacking the mutation. In another aspect, a mutation in an endogenous gene results in an increased level of expression as compared to the endogenous gene lacking the mutation. In a further aspect, a mutation in an endogenous gene results in a decreased level of activity by the protein or polypeptide encoded by the endogenous gene having the mutation as compared to the protein or polypeptide encoded by the endogenous gene lacking the mutation. In a further aspect, a mutation in an endogenous gene results in an increased level of activity by the protein or polypeptide encoded by the endogenous gene having the mutation as compared to the protein or polypeptide encoded by the endogenous gene lacking the mutation.

[0156] In one aspect, a mutation at a genomic locus results in a decreased level of expression compared to the genomic locus lacking the mutation. In another aspect, a mutation at a genomic locus results in an increased level of expression compared to the genomic locus lacking the mutation. In a further aspect, a mutation in a genomic locus results in a decreased level of activity by the protein or polypeptide encoded by the genomic locus having the mutation compared to the protein or polypeptide encoded by the genomic locus lacking the mutation. In a further aspect, a mutation in a genomic locus results in an increased level of activity by the protein or polypeptide encoded by the genomic locus having the mutation compared to the protein or polypeptide encoded by the genomic locus lacking the mutation.

[0157] Levels of gene expression are routinely investigated in the art. As non-limiting examples, gene expression can be measured using quantitative reverse transcriptase PCR (qRT-PCR), RNA sequencing or Northern blot. In one aspect, gene expression is measured using qRT-PCR. In another aspect, gene expression is measured using Northern blot. In another aspect, gene expression is measured using RNA sequencing.

[0158] Levels of protein activity are also routinely investigated in the art. For example, CDK activity can be measured using a phosphorylation assay.

[0159] As used herein, the term "heterologous" refers to a combination of two or more DNA molecules or sequences, such as a promoter and an associated transcribable DNA sequence, coding sequence or gene, where such a combination is artificial and not normally found in nature.

[0160] In one aspect, the level of expression of the endogenous gene containing the mutation is reduced by at least 1% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation is reduced by at least 5% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation is reduced by at least 10% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation is reduced by at least 15% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation is reduced by at least 20% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation is reduced by at least 25% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation is reduced by at least 50% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation is reduced by at least 75% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation is reduced by at least 90% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation is reduced by at least 95% compared to the level of expression of the endogenous gene lacking the mutation.

[0161] In one aspect, the expression level of the endogenous gene containing the mutation is reduced by 1% to 99% compared to the expression level of the endogenous gene lacking the mutation. In one aspect, the expression level of the endogenous gene containing the mutation is reduced by 1% to 90% compared to the expression level of the endogenous gene lacking the mutation. In one aspect, the expression level of the endogenous gene containing the mutation is reduced by 1% to 75% compared to the expression level of the endogenous gene lacking the mutation. In one aspect, the expression level of the endogenous gene containing the mutation is reduced by 1% to 50% compared to the expression level of the endogenous gene lacking the mutation. In one aspect, the expression level of the endogenous gene containing the mutation is reduced by 1% to 25% compared to the expression level of the endogenous gene lacking the mutation. In one aspect, the expression level of the endogenous gene containing the mutation is reduced by 1% to 10% compared to the expression level of the endogenous gene lacking the mutation. In one aspect, the expression level of the endogenous gene containing the mutation is reduced by 75% to 99% compared to the expression level of the endogenous gene lacking the mutation. In one aspect, the expression level of the endogenous gene containing the mutation is reduced by 50% to 99% compared to the expression level of the endogenous gene lacking the mutation. In one aspect, the expression level of the endogenous gene containing the mutation is reduced by 25% to 99% compared to the expression level of the endogenous gene lacking the mutation. In one aspect, the expression level of the endogenous gene containing the mutation is reduced by 10% to 99% compared to the expression level of the endogenous gene lacking the mutation. In one aspect, the expression level of the endogenous gene containing the mutation is reduced by 50% to 75% compared to the expression level of the endogenous gene lacking the mutation. In one aspect, the expression level of the endogenous gene containing the mutation is reduced by 25% to 75% compared to the expression level of the endogenous gene lacking the mutation. In one aspect, the expression level of the endogenous gene containing the mutation is reduced by 10% to 50% compared to the expression level of the endogenous gene lacking the mutation.

[0162] In one aspect, the level of expression of the endogenous gene containing the mutation increases by at least 1% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by at least 5% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by at least 10% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by at least 15% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by at least 20% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by at least 25% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by at least 50% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by at least 75% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by at least 90% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by at least 95% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by at least 100% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by at least 150% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by at least 200% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by at least 250% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by at least 300% compared to the level of expression of the endogenous gene lacking the mutation.In one aspect, the level of expression of the endogenous gene containing the mutation increases by at least 400% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by at least 500% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by at least 750% compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by at least 1000% compared to the level of expression of the endogenous gene lacking the mutation.

[0163] In one aspect, the level of expression of the endogenous gene containing the mutation increases by 1% to 1000% as compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by 1% to 750% as compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by 1% to 500% as compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by 1% to 400% as compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by 1% to 300% as compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by 1% to 200% as compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by 1% to 100% as compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by 1% to 75% as compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by 1% to 50% as compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by 1% to 25% as compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by 1% to 10% as compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by 100% to 1000% as compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by 100% to 750% as compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by 100% to 500% as compared to the level of expression of the endogenous gene lacking the mutation. In one aspect, the level of expression of the endogenous gene containing the mutation increases by 100% to 250% as compared to the level of expression of the endogenous gene lacking the mutation.In one aspect, the level of expression of an endogenous gene containing a mutation increases by 750% to 1000% compared to the level of expression of an endogenous gene lacking the mutation. In one aspect, the level of expression of an endogenous gene containing a mutation increases by 500% to 1000% compared to the level of expression of an endogenous gene lacking the mutation. In one aspect, the level of expression of an endogenous gene containing a mutation increases by 250% to 1000% compared to the level of expression of an endogenous gene lacking the mutation. In one aspect, the level of expression of an endogenous gene containing a mutation increases by 1% to 100% compared to the level of expression of an endogenous gene lacking the mutation. In one aspect, the level of expression of an endogenous gene containing a mutation increases by 50% to 100% compared to the level of expression of an endogenous gene lacking the mutation.

[0164] As used herein, the terms "endogenous gene" or "native gene" refer to a gene that occurs within the tobacco genome. An "endogenous gene" is a gene that has not been previously modified by human action. In one aspect, the endogenous gene is a nuclear gene. In another aspect, the endogenous gene is a mitochondrial gene. In a further aspect, the endogenous gene is a chloroplast gene.

[0165] As used herein, "gene" refers to a polynucleotide capable of producing a functional unit (e.g., but not limited to, for example, a protein or a small RNA molecule). A gene can include a promoter, an enhancer sequence, a leader sequence, a transcription start site, a transcription stop site, a polyadenylation site, one or more exons, one or more introns, a 5'-untranslated region (UTR), a 3'-UTR, or any combination thereof. A "gene sequence" can include a polynucleotide sequence encoding a promoter, an enhancer sequence, a leader sequence, a transcription start site, a transcription stop site, a polyadenylation site, one or more exons, one or more introns, a 5'-UTR, a 3'-UTR, or any combination thereof. In one aspect, the gene encodes a small RNA molecule or a precursor thereof. In another aspect, the gene encodes a protein or a polypeptide.

[0166] As used herein, "genomic locus" refers to a fixed position on a chromosome. In one aspect, a genomic locus includes a polynucleotide encoding a gene. In one aspect, a genomic locus includes a polynucleotide encoding an endogenous gene. In another aspect, a genomic locus includes a polynucleotide encoding a transgene. In one aspect, a genomic locus can be transcribed from DNA to RNA. In one aspect, a genomic locus encodes messenger RNA. In another aspect, a genomic locus encodes small RNA molecules.

[0167] As generally understood in the art, the term "promoter" contains an RNA polymerase binding site, a transcription start site and / or a TATA box, and refers to a DNA sequence that aids or promotes the transcription and expression of an associated transcribable polynucleotide sequence and / or gene (or transgene). A promoter can be synthetically produced, can be varied from a known or naturally occurring promoter sequence or other promoter sequence, or can be derived from a known or naturally occurring promoter sequence or other promoter sequence. A promoter can also include a chimeric promoter that includes a combination of two or more heterologous sequences. Thus, the promoters of the present application can include variants of promoter sequences that are similar but not identical in composition to known or other promoter sequences (one or more) provided herein.

[0168] A promoter that drives expression in all or most tissues of a plant is referred to as a "constitutive" promoter. A promoter that drives expression during a specific period or stage of development is called a "developmental" promoter. A promoter that drives enhanced expression in a specific tissue of an organism compared to other tissues of that organism is called a "tissue-preferred" promoter. Thus, a "tissue-preferred" promoter causes relatively higher or preferential expression in a specific tissue of a plant, but lower expression levels in other tissues of that plant. As a non-limiting example, an "axillary bud-preferred promoter" causes relatively higher or preferential expression in axillary bud tissue, but may have lower levels of expression in other parts of the plant (e.g., roots, leaves, stems). A promoter that is expressed within a specific tissue of an organism and is hardly or not at all 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 there is hardly or no detectable expression in other plant tissue types. An "inducible" promoter is a promoter that initiates transcription in response to environmental stimuli such as heat, cold, drought, light, or other stimuli such as wounding or chemical application.

[0169] In one aspect, the promoter provided herein is an axillary bud-specific promoter. In another aspect, the promoter provided herein is an 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.

[0170] 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.

[0171] In one aspect, the heterologous promoter includes an axillary bud-specific promoter. In another aspect, the heterologous promoter includes an axillary bud-preferred promoter. In one aspect, the heterologous promoter includes a constitutive promoter. In one aspect, the heterologous promoter includes an inducible promoter. In one aspect, the heterologous promoter includes a developmental promoter.

[0172] As used herein, "operably linked" refers to a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional linkage that enables expression of the polynucleotide of interest. Operably linked elements may be contiguous or non-contiguous. In one aspect, the promoter provided herein is operably linked to a heterologous nucleic acid molecule.

[0173] 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 to 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 to 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 to 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 to 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 to 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 to 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 to 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 to 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 to 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 to 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 to 109.

[0174] In one aspect, the axillary bud-preferential promoter comprises a polynucleotide sequence that is at least 90% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89 to 109. In one aspect, the axillary bud-preferential promoter comprises a polynucleotide sequence that is at least 95% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89 to 109. In one aspect, the axillary bud-preferential promoter comprises a polynucleotide sequence that is at least 96% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89 to 109. In one aspect, the axillary bud-preferential promoter comprises a polynucleotide sequence that is at least 97% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89 to 109. In one aspect, the axillary bud-preferential promoter comprises a polynucleotide sequence that is at least 98% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89 to 109. In one aspect, the axillary bud-preferential promoter comprises a polynucleotide sequence that is at least 99% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89 to 109. In one aspect, the axillary bud-preferential promoter comprises a polynucleotide sequence that is 100% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 89 to 109.

[0175] 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 to 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 to 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 to 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 to 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 to 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 to 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 to 109.

[0176] In one aspect, the present disclosure provides a modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), CDK inhibitor, MYB, and WRKY transcription factor, functionally linked to a heterologous promoter, wherein the modified tobacco plant, when cultivated under equivalent cultivation conditions, comprises no or reduced suckers after topping compared to a control tobacco plant.

[0177] In another aspect, the present disclosure provides a modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), CDK inhibitor, MYB, and WRKY transcription factor, functionally linked to a heterologous promoter.

[0178] In a further 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, said 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, an MYB, and a WRKY transcription factor; (b) selecting at least one tobacco cell comprising said recombinant DNA construct; and (c) regenerating a modified tobacco plant from said at least one tobacco cell selected in step (b), said modified tobacco plant having no or reduced suckers after topping as compared to a control tobacco plant lacking said recombinant DNA construct when grown under equivalent cultivation conditions. In one aspect, the method further comprises (d) cultivating the modified tobacco plant regenerated in step (c). In another aspect, the method further comprises (e) crossing the modified tobacco plant cultivated in step (d) with a second tobacco plant and (f) obtaining at least one seed from said crossing in step (e).

[0179] 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, an MYB, and a WRKY transcription factor. In one aspect, the method further comprises regenerating a modified tobacco plant from said tobacco cell, said modified tobacco plant comprising said recombinant DNA construct.

[0180] In one aspect, the present 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 laboratory methods of genetic recombination such as molecular cloning. In one aspect, the recombinant DNA construct is synthetically produced. In another aspect, the recombinant DNA construct comprises a promoter operably linked to a heterologous polynucleotide sequence. In one aspect, the recombinant DNA construct comprises a polynucleotide sequence encoding an amino acid sequence. In another aspect, the recombinant DNA construct comprises a polynucleotide sequence encoding a small RNA or a precursor thereof. In one aspect, the recombinant DNA construct comprises a plasmid. In another aspect, the recombinant DNA construct comprises a vector.

[0181] 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 can replicate in vivo. As used herein, a "transformation vector" is a plasmid capable of transforming a plant cell. In one aspect, the plasmid provided herein is a bacterial plasmid. In another aspect, the plasmid provided herein is, or is derived from, an Agrobacterium Ti plasmid.

[0182] In one aspect, the vectors provided herein include a promoter. In one aspect, the vectors provided herein include an axillary bud-specific promoter. In one aspect, the vectors provided herein include an axillary bud-preferred promoter. In another aspect, the vectors provided herein include a small RNA. In another aspect, the vectors provided herein include a small RNA precursor. In one aspect, the vectors provided herein include an artificial miRNA. In another aspect, the vectors provided herein include an artificial miRNA precursor. In another aspect, the vectors provided herein include a sequence selected from the group consisting of SEQ ID NOs: 1-44 and 89-113 or a sequence that is at least 80% identical to a fragment thereof. In another aspect, the vectors provided herein include a sequence selected from the group consisting of SEQ ID NOs: 1-44 and 89-113 or a sequence that is at least 85% identical to a fragment thereof. In another aspect, the vectors provided herein include a sequence selected from the group consisting of SEQ ID NOs: 1-44 and 89-113 or a sequence that is at least 90% identical to a fragment thereof. In another aspect, the vectors provided herein include a sequence selected from the group consisting of SEQ ID NOs: 1-44 and 89-113 or a sequence that is at least 95% identical to a fragment thereof. In another aspect, the vectors provided herein include a sequence selected from the group consisting of SEQ ID NOs: 1-44 and 89-113 or a sequence that is at least 96% identical to a fragment thereof. In another aspect, the vectors provided herein include a sequence selected from the group consisting of SEQ ID NOs: 1-44 and 89-113 or a sequence that is at least 97% identical to a fragment thereof. In another aspect, the vectors provided herein include a sequence selected from the group consisting of SEQ ID NOs: 1-44 and 89-113 or a sequence that is at least 98% identical to a fragment thereof. In another aspect, the vectors provided herein include a sequence selected from the group consisting of SEQ ID NOs: 1-44 and 89-113 or a sequence that is at least 99% identical to a fragment thereof. In another aspect, the vectors provided herein include a sequence selected from the group consisting of SEQ ID NOs: 1-44 and 89-113 or a sequence that is 100% identical to a fragment thereof.

[0183] As used herein, a "fragment" of a nucleic acid sequence or an amino acid sequence includes a contiguous segment of a sequence that comprises from 1% to 99.99% of the full length of the reference sequence. For example, if a nucleic acid sequence comprises 1000 nucleotides, a fragment of the nucleic acid sequence can comprise any number from 10 to 999 contiguous nucleotides of the reference sequence. Whenever SEQ ID NOs: 1-114 are referred to, this disclosure always explicitly provides each fragment of SEQ ID NOs: 1-114.

[0184] Numerous methods for introducing recombinant DNA constructs into plant cells are known in the art and can be used according to the methods of the present application to produce transgenic plant cells and plants. Any suitable method or technique known in the art for transforming plant cells can be used according to the methods of the present invention. Effective methods for plant transformation include bacterial-mediated transformation, such as Agrobacterium-mediated or Rhizobium-mediated transformation and particle gun-mediated transformation. Various methods are known in the art for transforming an explant with a transformation vector by bacterial-mediated transformation or particle gun and then culturing this explant, etc. subsequently to regenerate or develop a transgenic plant. Other methods for plant transformation, such as microinjection, electroporation, vacuum infiltration, pressure, sonication, silicon carbide fiber agitation, polyethylene glycol (PEG)-mediated transformation, etc. are also known in the art. Transgenic plants produced by these transformation methods can be chimeric or non-chimeric for the transformation event, depending on the method and explant used.

[0185] Methods for transforming plant cells are well known to those skilled in the art. For example, specific descriptions for transforming plant cells by particle bombardment with particles coated with recombinant DNA (e.g., particle gun transformation) can be found in U.S. Patent Nos. 5,550,318; 5,538,880; 6,160,208; 6,399,861; 6,153,812, and Agrobacterium-mediated transformation is described in U.S. Patent Nos. 5,159,135; 5,824,877; 5,591,616; 6,384,301; 5,750,871; 5,463,174; and 5,188,958, all of which are incorporated herein by reference. Further methods for transforming plants can be found, for example, in 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 of the nucleic acid molecules provided herein.

[0186] In one aspect, a method for providing a nucleic acid molecule to a tobacco cell includes Agrobacterium-mediated transformation. In another aspect, a method for providing a nucleic acid molecule to a cell includes PEG-mediated transformation. In another aspect, a method for providing a nucleic acid molecule to a cell includes particle gun transformation. In another aspect, a method for providing a nucleic acid molecule to a cell includes liposome-mediated transfection (lipofection). In another aspect, a method for providing a nucleic acid molecule to a cell includes lentiviral transfection.

[0187] Lipofection is described, for example, in U.S. Pat. Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides are included in International Publication Nos. WO 91 / 17424 and WO 91 / 16024. Delivery can be to cells (e.g., in vitro or ex vivo administration) or to a target tissue (e.g., in vivo administration).

[0188] Any tobacco cell from which a fertile tobacco plant can be regenerated is contemplated as a useful recipient cell for practicing the present disclosure. In one aspect, the recombinant DNA construct is introduced into a tobacco cell. In one aspect, the recombinant DNA construct is introduced into a tobacco protoplast cell. In another aspect, the recombinant DNA construct is introduced into a tobacco callus cell. In one aspect, the recombinant DNA construct is introduced into a tobacco cell selected from the group consisting of seed cells, fruit cells, leaf cells, cotyledon cells, hypocotyl cells, meristem cells, embryo cells, endosperm cells, root cells, shoot cells, stem cells, flower cells, inflorescence cells, stalk cells, pedicel cells, style cells, stigma cells, receptacle cells, petal cells, sepal cells, pollen cells, anther cells, filament cells, ovary cells, ovule cells, pericarp cells, and phloem cells.

[0189] Callus can be initiated from a variety of tissue sources including, but not limited to, immature embryos or parts of embryos, seedling apical meristems, microspores, etc. Cells that can grow as callus can serve as recipient cells for transformation. Practical transformation methods and materials for producing the transgenic plants of the present disclosure (e.g., various media and recipient target cells, transformation of immature embryos, and subsequent regeneration of fertile transgenic plants) are disclosed, for example, in U.S. Pat. Nos. 6,194,636 and 6,232,526 and U.S. Patent Application Publication No. 2004 / 0216189, all of which are incorporated herein by reference.

[0190] 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 cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, wherein the modified tobacco plant, when cultivated under equivalent cultivation conditions, has no or reduced suckers after pinching compared to a control tobacco plant.

[0191] 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 the expression of an endogenous mRNA encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor.

[0192] In a further 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 the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, 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 the modified tobacco plant, when cultivated under equivalent cultivation conditions, has no or reduced suckers after pinching compared to a control tobacco plant lacking the recombinant DNA construct. Provided is a method, which, in one aspect, further comprises the step of (d) cultivating the modified tobacco plant regenerated in step (c). In another aspect, the method further comprises the steps of: (e) crossing the modified tobacco plant cultivated in step (d) with a second tobacco plant; and (f) obtaining at least one seed from the crossing in step (e).

[0193] In another aspect, the present disclosure provides a method comprising the step of transforming tobacco cells 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 cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, MYB, and WRKY transcription factors. In one aspect, the method further comprises the step of regenerating a modified tobacco plant from the tobacco cells, wherein the modified tobacco plant comprises the recombinant DNA construct.

[0194] 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.

[0195] In one aspect, the small RNA molecule has a length of 18 to 30 nucleotides. In another aspect, the small RNA molecule has a length of 18 to 24 nucleotides. In another aspect, the small RNA molecule has a length of 18 to 22 nucleotides.

[0196] In another aspect, the small RNA molecule is 18 nucleotides in length. In another aspect, the small RNA molecule is 19 nucleotides in length. In another aspect, the small RNA molecule is 20 nucleotides in length. In another aspect, the small RNA molecule is 21 nucleotides in length. In another aspect, the small RNA molecule is 22 nucleotides in length. In another aspect, the small RNA molecule is 23 nucleotides in length. In another aspect, the small RNA molecule is 24 nucleotides in length. In another aspect, the small RNA molecule is 25 nucleotides in length. In another aspect, the small RNA molecule is 26 nucleotides in length. In another aspect, the small RNA molecule is 27 nucleotides in length. In another aspect, the small RNA molecule is 28 nucleotides in length.

[0197] In another aspect, the small RNA molecule is at least 18 nucleotides in length. In another aspect, the small RNA molecule is at least 19 nucleotides in length. In another aspect, the small RNA molecule is at least 20 nucleotides in length. In another aspect, the small RNA molecule is at least 21 nucleotides in length. In another aspect, the small RNA molecule is at least 22 nucleotides in length. In another aspect, the small RNA molecule is at least 23 nucleotides in length. In another aspect, the small RNA molecule is at least 24 nucleotides in length. In another aspect, the small RNA molecule is at least 25 nucleotides in length. In another aspect, the small RNA molecule is at least 26 nucleotides in length. In another aspect, the small RNA molecule is at least 27 nucleotides in length. In another aspect, the small RNA molecule is at least 28 nucleotides in length.

[0198] In one aspect, the small RNA molecule comprises from 18 to 30 nucleotides. In another aspect, the small RNA molecule comprises from 18 to 24 nucleotides. In another aspect, the small RNA molecule comprises from 18 to 21 nucleotides. In another aspect, the small RNA molecule comprises from 21 to 24 nucleotides. In another aspect, the small RNA molecule comprises from 21 to 30 nucleotides.

[0199] 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 heterochromatin 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.

[0200] In one aspect, the small RNA molecule is selected from the group consisting of dsRNA, siRNA, ta-siRNA, and miRNA. In another aspect, the small RNA molecule is selected from the group consisting of miRNA, siRNA, hc-siRNA, piRNA, dsRNA, hp-dsRNA, ta-siRNA, nat-siRNA, Cas9-gRNA, Cpf1-gRNA, CasX-gRNA, and Csm1-gRNA.

[0201] In one aspect, the small RNA molecules provided herein comprise a polynucleotide sequence having at least 90% identity or complementarity with an endogenous mRNA. In one aspect, the small RNA molecules provided herein comprise a polynucleotide sequence having at least 91% identity or complementarity with an endogenous mRNA. In one aspect, the small RNA molecules provided herein comprise a polynucleotide sequence having at least 92% identity or complementarity with an endogenous mRNA. In one aspect, the small RNA molecules provided herein comprise a polynucleotide sequence having at least 93% identity or complementarity with an endogenous mRNA. In one aspect, the small RNA molecules provided herein comprise a polynucleotide sequence having at least 94% identity or complementarity with an endogenous mRNA. In one aspect, the small RNA molecules provided herein comprise a polynucleotide sequence having at least 95% identity or complementarity with an endogenous mRNA. In one aspect, the small RNA molecules provided herein comprise a polynucleotide sequence having at least 96% identity or complementarity with an endogenous mRNA. In one aspect, the small RNA molecules provided herein comprise a polynucleotide sequence having at least 97% identity or complementarity with an 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.

[0202] In one aspect, the small RNA molecule comprises a polynucleotide sequence having at least 85% identity or complementarity with a polynucleotide sequence selected from the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence having at least 95% identity or complementarity with a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence having at least 96% identity or complementarity with a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence having at least 97% identity or complementarity with a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence having at least 98% identity or complementarity with a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence having at least 99% identity or complementarity with a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113.In one aspect, the small RNA molecule comprises a polynucleotide sequence having 100% identity or complementarity with a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113.

[0203] In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical to or complementary to at least 16 consecutive nucleotides of a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical to or complementary to at least 17 consecutive nucleotides of a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical to or complementary to at least 18 consecutive nucleotides of a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical to or complementary to at least 19 consecutive nucleotides of a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical to or complementary to at least 20 consecutive nucleotides of a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical to or complementary to at least 21 consecutive nucleotides of a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical to or complementary to at least 22 consecutive nucleotides of a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical to or complementary to at least 23 consecutive nucleotides of a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical to or complementary to at least 24 consecutive nucleotides of a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical to or complementary to at least 25 consecutive nucleotides of a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113.In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical to or complementary to at least 26 consecutive nucleotides of a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical to or complementary to at least 27 consecutive nucleotides of a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical to or complementary to at least 28 consecutive nucleotides of a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical to or complementary to at least 29 consecutive nucleotides of a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113. In one aspect, the small RNA molecule comprises a polynucleotide sequence that is identical to or complementary to at least 30 consecutive nucleotides of a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113.

[0204] In one aspect, the present disclosure provides a small RNA capable of reducing the expression of a polynucleotide encoding a cyclin. In one aspect, the present disclosure provides a small RNA capable of reducing the translation of a polynucleotide encoding a cyclin. In one aspect, the present disclosure provides a small RNA capable of reducing the expression of a polynucleotide encoding a CDK. In one aspect, the present disclosure provides a small RNA capable of reducing the translation of a polynucleotide encoding a CDK. In one aspect, the present disclosure provides a small RNA capable of reducing the expression of a polynucleotide encoding a CDKI. In one aspect, the present disclosure provides a small RNA capable of reducing the translation of a polynucleotide encoding a CDKI. In one aspect, the present disclosure provides a small RNA capable of reducing the expression of a polynucleotide encoding MYB. In one aspect, the present disclosure provides a small RNA capable of reducing the translation of a polynucleotide encoding MYB. In one aspect, the present disclosure provides a small RNA capable of reducing the expression of a polynucleotide encoding a WRKY transcription factor. In one aspect, the present disclosure provides a small RNA capable of reducing the translation of a polynucleotide encoding a WRKY transcription factor.

[0205] MicroRNA (miRNA) is generally a non-protein-coding RNA of about 19 to about 25 nucleotides (generally about 20 to 24 nucleotides in plants), guides the cleavage of target transcripts in trans, and negatively regulates the expression of genes involved in various regulatory and developmental pathways. In some cases, miRNA plays a role in guiding the in-phase processing of siRNA primary transcripts.

[0206] Numerous microRNA genes (MIR genes) have been identified and are publicly available in databases (see "miRBase," available online at microrna(dot)sanger(dot)ac(dot)uk / sequences; also see Griffiths-Jones et al. (2003) Nucleic Acids Res., 31:439-441). MIR genes have been reported to exist in isolated and intergenic regions within the genome, but can also be located entirely or partially within the introns of other genes (both protein-coding and non-protein-coding). Transcription of MIR genes can, in at least some cases, be under the positive control of the MIR gene's own promoter. The primary transcript, called "pri-miRNA," can be very large (several kilobases), can be polycistronic, and contains one or more pre-miRNAs (folded structures containing stem-loop arrangements that are processed into mature miRNAs), as well as the normal 5' "cap" and polyadenylation tail of mRNA.

[0207] Transgenic expression of miRNAs (whether naturally occurring sequences or artificial sequences) can be used to regulate the expression of one or more target genes of the miRNA. Inclusion of miRNA recognition sites in the transcripts expressed transgenically can also be useful in regulating the expression of those transcripts. miRNA recognition sites have been identified 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 repression.

[0208] Since miRNAs are important regulatory elements in eukaryotes, transgenic repression of miRNAs is useful for manipulating biological pathways and responses. The various utilities of miRNAs, miRNA precursors, miRNA recognition sites, and miRNA promoters are described in detail in U.S. Patent Application Publication No. 2006 / 0200878, which is incorporated herein by reference. Non-limiting examples of these utilities include: (1) expression of natural miRNA or miRNA precursor sequences to repress target genes; (2) expression of artificial miRNA or miRNA precursor sequences to repress target genes; (3) expression of a transgene having an miRNA recognition site (the transgene is repressed when mature miRNA is expressed); and (4) expression of a transgene driven by an miRNA promoter.

[0209] Designing artificial miRNA sequences can be as straightforward as replacing nucleotides in the miRNA stem region of the miRNA precursor with sequences complementary to the intended target, as demonstrated by Zeng et al. (2002) Mol. Cell, 9:1327-1333. One non-limiting example of a general method for determining nucleotide changes in a natural miRNA sequence to create an engineered miRNA precursor involves the following steps: (a) selecting a unique target sequence of at least 18 nucleotides specific to the target gene, for example, by using a sequence alignment tool such as BLAST (see, e.g., Altschul et al. (1990) J. Mol. biol., 215:403-410; Altschul et al. (1997) Nucleic Acids Res., 25:3389-3402) of both tobacco cDNA and genomic DNA databases to identify any potential matches with orthologs of the target transcript and unrelated genes, thereby avoiding unintended silencing of non-target sequences; (b) analyzing the target gene for undesirable sequences (e.g., matches with sequences from non-target species) and scoring each candidate 19-mer segment for functional asymmetry (".DELTA..DELTA.G" or "ΔΔG") characterized by GC content, Reynolds score (Reynolds et al. (2004) Nature Biotechnol., 22:326-330), and negative difference in free energy (see Khvorova et al. (2003) Cell, 114:209-216). Preferably, a 19-mer having all or most of the following characteristics is selected: (1) Reynolds score > 4, (2) GC content of about 40% to about 60%, (3) negative ΔΔG, (4) terminal adenosine, (5) no more than 4 consecutive identical nucleotides, (6) position near the 3' end of the target gene, and (7) minimal difference from the miRNA precursor transcript.The position of every third nucleotide in siRNA has been reported to be particularly important in affecting the efficacy of RNAi, and the algorithm "siExplorer" is publicly available at ma.chem.t.u-tokyo.ac.jp / siexplorer.htm (see Katoh and Suzuki (2007) Nucleic Acids Res., 10.1093 / nar / gkl1120); (c) determining the 19-mer reverse complement selected for use in generating a modified mature miRNA. The additional nucleotide at position 20 preferably matches the selected target sequence, and the nucleotide at position 21 is preferably selected such that it does not pair so as to prevent the spread of silencing on the target transcript, or pairs with the target sequence so as to promote the spread of silencing on the target transcript; and (d) transforming plants with the artificial miRNA.

[0210] In one aspect, the artificial miRNA provided herein is complementary to at least 18 consecutive nucleotides of 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 complementary to at least 19 consecutive nucleotides of 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 complementary to at least 20 consecutive nucleotides of 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 complementary to at least 21 consecutive nucleotides of 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 complementary to at least 22 consecutive nucleotides of 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 complementary to at least 23 consecutive nucleotides of 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 complementary to at least 24 consecutive nucleotides of 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 complementary to at least 25 consecutive nucleotides of 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 complementary to at least 26 consecutive nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-44 and 113.

[0211] 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.

[0212] In one aspect, the artificial miRNA provided herein reduces or abolishes the RNA transcription or protein translation of the target gene.

[0213] In one aspect, the artificial miRNA or its precursor is operably linked to an axillary bud-specific promoter. In another aspect, the artificial miRNA or its precursor is operably linked to an axillary bud-preferred promoter. In another aspect, the artificial miRNA or its precursor is operably linked to a promoter comprising a sequence selected from the group consisting of SEQ ID NOs: 89-109 and fragments thereof.

[0214] Tobacco is known in the art as a plant of the Solanaceae family. As used herein, tobacco plants include Nicotiana tabacum, Nicotiana amplexicaulis PI271989; Nicotiana benthamiana PI555478; Nicotiana bigelovii PI555485; Nicotiana debneyi; Nicotiana excelsior PI224063; Nicotiana glutinosa PI555507; Nicotiana goodspeedii PI241012; Nicotiana gossei PI230953; Nicotiana hesperis PI271991; Nicotiana knightiana PI555527, Nicotiana maritima PI555535; Nicotiana megalosiphon PI555536; Nicotiana nudicaulis PI555540; Nicotiana paniculata PI555545; Nicotiana plumbaginifolia PI555548; Nicotiana repanda PI555552; Nicotiana rustica; Nicotiana suaveolens PI230960; Nicotiana sylvestris PI555569; Nicotiana tomentosa PI266379, Nicotiana tomentosiformis;and can be from any plant of the genus Nicotiana including, but not limited to, Nicotiana trigonophylla PI555572. In one aspect, the tobacco plants described herein are Nicotiana tabacum plants.;

[0215] In one aspect, the modified tobacco plants, seeds, cells, hybrids, varieties or lines provided herein are essentially BU64, CC101, CC200, CC13, CC27, CC33, CC35, CC37, CC65, CC67, CC301, CC400, CC500, CC600, CC700, CC800, CC900, CC1063, Coker176, Coker319, Coker371Gold, Coker48, CU263, DF911, Galpao, GL26H, GL338, GL350, GL395, GL600, GL737, GL939, GL973, GF157, GF318, RJR901, HB04P, K149, K326, K346, K358, K394, K399, K730, NC196, NC37NF, NC471, NC55, NC92, NC2326, NC95, NC925, PVH1118, PVH1452, PVH2110, PVH2254, PVH2275, VA116, VA119, KDH959, KT200, KT204LC, KY10, KY14, KY160, KY17, KY171, KY907, KY907LC, KTY14xL8 LC, Little Crittenden, McNair373, McNair944, male sterile KY14xL8, Narrow Leaf Madole, MS KY171, Narrow Leaf Madole(phph), MS Narrow Leaf Madole, MS TND950, PD7302LC, PD7305LC, PD7309LC, PD7312LC, PD7318LC, PD7319LC, MSTKS2002, TKF2002, TKF6400, TKF4028, TKF4024, KT206LC, KT209LC, KT210LC, KT212LC, NC100, NC102, NC2000, NC291, NC297, NC299, NC3, NC4, NC5, NC6, NC7, NC606, NC71, NC72, NC810, NC BH 129, NC2002, Neal Smith Madole, OXFORD 207, "Perique", PVH03, PVH09, PVH19, PVH50, PVH51, R610, R630, R7-11, R7-12, RG17, RG81, RGDerived from H51, RGH4, RGH51, RS1410, Speight168, Speight172, Speight179, Speight210, Speight220, Speight225, Speight227, Speight234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI1406, TI1269, TN86, TN86LC, TN90, TN90LC, TN97, TN97LC, TN D94, TN D950, TR (Tom Rosson) Madole, VA309, VA359 or any commercial tobacco variety by standard tobacco breeding techniques known in the art, or having a genetic background of these.

[0216] In one aspect, the modified tobacco plants are BU64 plants, CC101 plants, CC200 plants, CC13 plants, CC27 plants, CC33 plants, CC35 plants, CC37 plants, CC65 plants, CC67 plants, CC301 plants, CC400 plants, CC500 plants, CC600 plants, CC700 plants, CC800 plants, CC900 plants, CC1063 plants, Coker176 plants, Coker319 plants, Coker371Gold plants, Coker48 plants, CU263 plants, DF911 plants, Galpao plants, GL26H plants, GL338 plants, GL350 plants, GL395 plants, GL600 plants, GL737 plants, GL939 plants, GL973 plants, GF157 plants, GF318 plants, RJR901 plants, HB04P plants, K149 plants, K326 plants, K346 plants, K358 plants, K394 plants, K399 plants, K730 plants, NC196 plants, NC37NF plants, NC471 plants, NC55 plants, NC92 plants, NC2326 plants, NC95 plants, NC925 plants, PVH1118 plants, PVH1452 plants, PVH2110 plants, PVH2254 plants, PVH2275 plants, VA116 plants, VA119 plants, KDH959 plants, KT200 plants, KT204LC plants, KY10 plants, KY14 plants, KY160 plants, KY17 plants, KY171 plants, KY907 plants, KY907LC plants, KTY14xL8 LC plants, Little Crittenden plants, McNair373 plants, McNair944 plants, male sterile KY14xL8 plants, Narrow Leaf Madole plants, MS KY171 plants, Narrow Leaf Madole(phph) plants, MS Narrow Leaf Madole plants, MSSelected from the group consisting of TND950 plants, PD7302LC plants, PD7305LC plants, PD7309LC plants, PD7312LC plants, PD7318LC plants, PD7319LC plants, MSTKS2002 plants, TKF2002 plants, TKF6400 plants, TKF4028 plants, TKF4024 plants, KT206LC plants, KT209LC plants, KT210LC plants, KT212LC plants, NC100 plants, NC102 plants, NC2000 plants, NC291 plants, NC297 plants, NC299 plants, NC3 plants, NC4 plants, NC5 plants, NC6 plants, NC7 plants, NC606 plants, NC71 plants, NC72 plants, NC810 plants, NC BH 129 plants, NC2002 plants, Neal Smith Madole plants, OXFORD 207 plants, "Perique" plants, PVH03 plants, PVH09 plants, PVH19 plants, PVH50 plants, PVH51 plants, R610 plants, R630 plants, R7-11 plants, R7-12 plants, RG17 plants, RG81 plants, RG H51 plants, RGH4 plants, RGH51 plants, RS1410 plants, Speight168 plants, Speight172 plants, Speight179 plants, Speight210 plants, Speight220 plants, Speight225 plants, Speight227 plants, Speight234 plants, SpeightG-28 plants, SpeightG-70 plants, SpeightH-6 plants, SpeightH20 plants, SpeightNF3 plants, TI1406 plants, TI1269 plants, TN86 plants, TN86LC plants, TN90 plants, TN90LC plants, TN97 plants, TN97LC plants, TN D94 plants, TN D950 plants, TR(Tom Rosson)Madole plants, VA309 plants and VA359 plants.

[0217] In another aspect, the modified tobacco cells are BU64 cells, CC101 cells, CC200 cells, CC13 cells, CC27 cells, CC33 cells, CC35 cells, CC37 cells, CC65 cells, CC67 cells, CC301 cells, CC400 cells, CC500 cells, CC600 cells, CC700 cells, CC800 cells, CC900 cells, CC1063 cells, Coker176 cells, Coker319 cells, Coker371Gold cells, Coker48 cells, CU263 cells, DF911 cells, Galpao cells, GL26H cells, GL338 cells, GL350 cells, GL395 cells, GL600 cells, GL737 cells, GL939 cells, GL973 cells, GF157 cells, GF318 cells, RJR901 cells, HB04P cells, K149 cells, K326 cells, K346 cells, K358 cells, K394 cells, K399 cells, K730 cells, NC196 cells, NC37NF cells, NC471 cells, NC55 cells, NC92 cells, NC2326 cells, NC95 cells, NC925 cells, PVH1118 cells, PVH1452 cells, PVH2110 cells, PVH2254 cells, PVH2275 cells, VA116 cells, VA119 cells, KDH959 cells, KT200 cells, KT204LC cells, KY10 cells, KY14 cells, KY160 cells, KY17 cells, KY171 cells, KY907 cells, KY907LC cells, KTY14xL8 LC cells, Little Crittenden cells, McNair373 cells, McNair944 cells, male sterile KY14xL8 cells, Narrow Leaf Madole cells, MS KY171 cells, Narrow Leaf Madole(phph) cells, MS Narrow Leaf Madole cells, MSSelected from the group consisting of TND950 cells, PD7302LC cells, PD7305LC cells, PD7309LC cells, PD7312LC cells, PD7318LC cells, PD7319LC cells, MSTKS2002 cells, TKF2002 cells, TKF6400 cells, TKF4028 cells, TKF4024 cells, KT206LC cells, KT209LC cells, KT210LC cells, KT212LC cells, NC100 cells, NC102 cells, NC2000 cells, NC291 cells, NC297 cells, NC299 cells, NC3 cells, NC4 cells, NC5 cells, NC6 cells, NC7 cells, NC606 cells, NC71 cells, NC72 cells, NC810 cells, NC BH 129 cells, NC2002 cells, Neal Smith Madole cells, OXFORD 207 cells, "Perique" cells, PVH03 cells, PVH09 cells, PVH19 cells, PVH50 cells, PVH51 cells, R610 cells, R630 cells, R7-11 cells, R7-12 cells, RG17 cells, RG81 cells, RG H51 cells, RGH4 cells, RGH51 cells, RS1410 cells, Speight168 cells, Speight172 cells, Speight179 cells, Speight210 cells, Speight220 cells, Speight225 cells, Speight227 cells, Speight234 cells, SpeightG-28 cells, SpeightG-70 cells, SpeightH-6 cells, SpeightH20 cells, SpeightNF3 cells, TI1406 cells, TI1269 cells, TN86 cells, TN86LC cells, TN90 cells, TN90LC cells, TN97 cells, TN97LC cells, TN D94 cells, TN D950 cells, TR(Tom Rosson)Madole cells, VA309 cells and VA359 cells.

[0218] In another aspect, the modified tobacco seeds are BU64 seeds, CC101 seeds, CC200 seeds, CC13 seeds, CC27 seeds, CC33 seeds, CC35 seeds, CC37 seeds, CC65 seeds, CC67 seeds, CC301 seeds, CC400 seeds, CC500 seeds, CC600 seeds, CC700 seeds, CC800 seeds, CC900 seeds, CC1063 seeds, Coker176 seeds, Coker319 seeds, Coker371Gold seeds, Coker48 seeds, CU263 seeds, DF911 seeds, Galpao seeds, GL26H seeds, GL338 seeds, GL350 seeds, GL395 seeds, GL600 seeds, GL737 seeds, GL939 seeds, GL973 seeds, GF157 seeds, GF318 seeds, RJR901 seeds, HB04P seeds, K149 seeds, K326 seeds, K346 seeds, K358 seeds, K394 seeds, K399 seeds, K730 seeds, NC196 seeds, NC37NF seeds, NC471 seeds, NC55 seeds, NC92 seeds, NC2326 seeds, NC95 seeds, NC925 seeds, PVH1118 seeds, PVH1452 seeds, PVH2110 seeds, PVH2254 seeds, PVH2275 seeds, VA116 seeds, VA119 seeds, KDH959 seeds, KT200 seeds, KT204LC seeds, KY10 seeds, KY14 seeds, KY160 seeds, KY17 seeds, KY171 seeds, KY907 seeds, KY907LC seeds, KTY14xL8 LC seeds, Little Crittenden seeds, McNair373 seeds, McNair944 seeds, male sterile KY14xL8 seeds, Narrow Leaf Madole seeds, MS KY171 seeds, Narrow Leaf Madole(phph) seeds, MS Narrow Leaf Madole seeds, MSSelected from the group consisting of TND950 seeds, PD7302LC seeds, PD7305LC seeds, PD7309LC seeds, PD7312LC seeds, PD7318LC seeds, PD7319LC seeds, MSTKS2002 seeds, TKF2002 seeds, TKF6400 seeds, TKF4028 seeds, TKF4024 seeds, KT206LC seeds, KT209LC seeds, KT210LC seeds, KT212LC seeds, NC100 seeds, NC102 seeds, NC2000 seeds, NC291 seeds, NC297 seeds, NC299 seeds, NC3 seeds, NC4 seeds, NC5 seeds, NC6 seeds, NC7 seeds, NC606 seeds, NC71 seeds, NC72 seeds, NC810 seeds, NC BH 129 seeds, NC2002 seeds, Neal Smith Madole seeds, OXFORD 207 seeds, "Perique" seeds, PVH03 seeds, PVH09 seeds, PVH19 seeds, PVH50 seeds, PVH51 seeds, R610 seeds, R630 seeds, R7-11 seeds, R7-12 seeds, RG17 seeds, RG81 seeds, RG H51 seeds, RGH4 seeds, RGH51 seeds, RS1410 seeds, Speight168 seeds, Speight172 seeds, Speight179 seeds, Speight210 seeds, Speight220 seeds, Speight225 seeds, Speight227 seeds, Speight234 seeds, SpeightG-28 seeds, SpeightG-70 seeds, SpeightH-6 seeds, SpeightH20 seeds, SpeightNF3 seeds, TI1406 seeds, TI1269 seeds, TN86 seeds, TN86LC seeds, TN90 seeds, TN90LC seeds, TN97 seeds, TN97LC seeds, TN D94 seeds, TN D950 seeds, TR(Tom Rosson)Madole seeds, VA309 seeds and VA359 seeds.

[0219] As used herein, "tobacco plant" refers to the whole tobacco plant. Tobacco cells or tobacco tissue cultures derived from a tobacco plant can include any tobacco plant part or tobacco plant organ (e.g., leaf, stem, root, etc.), tobacco plant tissue, tobacco seeds, tobacco plant cells and / or their progeny. Progeny plants can be derived from any hybrid generation, e.g., F1, F2, F3, F4, F5, F6, F7, etc. Tobacco plant cells are biological cells of a tobacco plant that are taken from a tobacco plant or obtained through culturing of cells taken from a tobacco plant. As used herein, "seedling" refers to a tobacco plant equal to or less than 14 days after germination.

[0220] In one aspect, tobacco plant parts provided herein include, but are not limited to, leaves, stems, roots, seeds, flowers, pollen, anthers, ovules, pedicels, fruits, meristems, cotyledons, hypocotyls, sheaths, embryos, endosperm, explants, callus, tissue cultures, shoots, cells and protoplasts. In a further aspect, the present disclosure provides tobacco plant cells, tissues and organs that are not germplasm and do not mediate the natural reproduction of the plant. In another aspect, the present disclosure also provides tobacco plant cells, tissues and organs that are germplasm and mediate the natural reproduction of the plant. In another aspect, the present disclosure provides tobacco plant cells, tissues and organs that cannot sustain themselves by photosynthesis. In another aspect, the present disclosure provides somatic plant cells. Somatic cells, unlike germline cells, do not mediate the reproduction of the plant.

[0221] The tobacco cells, tobacco tissues and tobacco organs provided can be derived from seeds, fruits, leaves, cotyledons, hypocotyls, meristems, embryos, endosperm, roots, shoots, stems, sheaths, flowers, inflorescences, stalks, pedicels, styles, stigmas, receptacles, petals, sepals, pollen, anthers, filaments, ovaries, ovules, pericarp, phloem and vascular tissue. In another aspect, the present disclosure provides tobacco plant chloroplasts. In a further aspect, the present disclosure provides epidermal cells, stoma cells, hair cells, root hairs or storage roots.

[0222] In one aspect, the present disclosure provides tobacco protoplast cells. In another aspect, the present disclosure provides tobacco callus cells. In another aspect, the present disclosure provides tobacco seed cells. In another aspect, the present disclosure provides tobacco fruit cells. In another aspect, the present disclosure provides tobacco leaf cells. In another aspect, the present disclosure provides tobacco cotyledon cells. In another aspect, the present disclosure provides tobacco hypocotyl cells. In another aspect, the present disclosure provides tobacco meristematic tissue cells. In another aspect, the present disclosure provides tobacco embryo cells. In another aspect, the present disclosure provides tobacco root cells. In another aspect, the present disclosure provides tobacco shoot cells. In another aspect, the present disclosure provides tobacco stem cells. In another aspect, the present disclosure provides tobacco flower cells. In another aspect, the present disclosure provides tobacco inflorescence cells. In another aspect, the present disclosure provides tobacco pedicel cells. In another aspect, the present disclosure provides tobacco pedicel cells of a floret. In another aspect, the present disclosure provides tobacco style cells. In another aspect, the present disclosure provides tobacco stigma cells. In another aspect, the present disclosure provides tobacco receptacle cells. In another aspect, the present disclosure provides tobacco petal cells. In another aspect, the present disclosure provides tobacco sepal cells. In another aspect, the present disclosure provides tobacco pollen cells. In another aspect, the present 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 teacher cells.

[0223] The present disclosure provides modified tobacco plants, modified tobacco plant parts, modified tobacco seeds and modified tobacco cells, as well as methods for producing them. In one aspect, the present disclosure provides tobacco leaves of a modified tobacco plant. In another aspect, the present disclosure provides tobacco seeds of a modified tobacco plant. In another aspect, the present disclosure provides tobacco stems of a modified tobacco plant. In a further aspect, the present disclosure provides tobacco plant parts of a modified tobacco plant. In another aspect, the present disclosure provides tobacco cells of a modified tobacco plant. In a further aspect, the present disclosure provides dried tobacco leaves of a modified tobacco plant. In still a further aspect, the present disclosure provides cured tobacco leaves of a modified tobacco plant. In yet another aspect, the present disclosure provides fermented tobacco leaves of a modified tobacco plant.

[0224] In another aspect, the present disclosure provides alkaloids extracted from a modified tobacco plant. In another aspect, the present disclosure provides nicotine extracted from a modified tobacco plant. In another aspect, the present disclosure provides anatabine extracted from a modified tobacco plant. In another aspect, the present disclosure provides anabasine extracted from a modified tobacco plant. In another aspect, the present disclosure provides nornicotine extracted from a modified tobacco plant.

[0225] In one aspect, the modified tobacco plant, plant part, seed, cell or genome is cisgenic. As used herein, "cisgenic" refers to the genetic modification of a plant, plant cell or plant genome in which all parts (e.g., promoter, donor nucleic acid, selectable gene) have only plant origin (i.e., parts of non-plant origin are not used). The cisgenic plants, plant cells and plant genomes provided herein can result in immediately usable tobacco lines. In another aspect, the modified tobacco plants provided herein do not contain non-tobacco genetic material or sequences.

[0226] In one aspect, the modified plant comprises an increased leaf yield quality when grown under equivalent conditions as 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.

[0227] In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 0.5% increased leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 0.5% increased fresh leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 0.5% increased dry leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 0.5% increased dried leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 1% increased leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 1% increased fresh leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 1% increased dry leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 1% increased dried leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 2% increased leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 2% increased fresh leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 2% increased dry leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 2% increased dried leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 3% increased leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 3% increased fresh leaf yield mass as compared to the control tobacco plant.In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 3% increased dry leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 3% increased dried leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 4% increased leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 4% increased fresh leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 4% increased dry leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 4% increased dried leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 5% increased leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 5% increased fresh leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 5% increased dry leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 5% increased dried leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 250% increased leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 250% increased fresh leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 250% increased dry leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 250% increased dried leaf yield mass as compared to the control tobacco plant.In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 25% increased leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 25% increased fresh leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 25% increased dry leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 25% increased dried leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 50% increased leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 50% increased fresh leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 50% increased dry leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 50% increased dried leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 75% increased leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 75% increased fresh leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 75% increased dry leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 75% increased dried leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 100% increased leaf yield mass as compared to the control tobacco plant. In one aspect, the modified plant, when cultivated under equivalent conditions, contains at least 100% increased fresh leaf yield mass as compared to the control tobacco plant.In one aspect, the modified plant comprises an increased cured leaf yield mass of at least 100% compared to a control tobacco plant when grown under equivalent conditions. In one aspect, the modified plant comprises an increased dried leaf yield mass of at least 100% compared to a control tobacco plant when grown under equivalent conditions.

[0228] "Curing" is an aging process that reduces moisture, causes destruction of chlorophyll, turns the tobacco leaf golden, and thereby converts starch to sugar. Thus, cured tobacco has a higher reducing sugar content and a lower starch content compared to the harvested green leaf. In one aspect, the tobacco plants or plant components provided herein can be cured using conventional means, such as hot air drying, barn-cured, fire curing, air drying, or sun drying. For an explanation of different types of curing methods, see, for example, Chapter 1 of Tso (1999, Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). Cured tobacco typically has a moisture content in the range of 10% to about 25% and is aged for several years (e.g., 2 - 5 years) in a compressed state in a wooden drum (e.g., a cask) or cardboard box. See U.S. Patent Nos. 4,516,590 and 5,372,149. The cured and aged tobacco can then be further processed. Further processing includes conditioning the tobacco under vacuum with or without introducing steam at various temperatures, pasteurization, and fermentation.

[0229] In one aspect, the dried tobacco leaf provided herein is selected from the group consisting of air-dried tobacco leaf, fire-dried tobacco leaf, sun-dried tobacco leaf, and yellow tobacco leaf. In another aspect, the dried tobacco material provided herein is selected from the group consisting of air-dried tobacco material, fire-dried tobacco material, sun-dried tobacco material, and yellow tobacco material. In one aspect, the dried tobacco leaf is from a tobacco variety selected from the group consisting of yellow variety, bright variety, burley variety, virginia variety, maryland variety, dark variety, oriental variety, and turkish variety. In another aspect, the dried tobacco material is from a tobacco variety selected from the group consisting of yellow variety, bright variety, burley variety, virginia variety, maryland variety, dark variety, oriental variety, and turkish variety.

[0230] Fermentation is typically characterized by a high initial moisture content, heat generation, and a 10 - 20% reduction in dry weight. See, e.g., U.S. Pat. Nos. 4,528,993; 4,660,577; 4,848,373; 5,372,149; U.S. Patent Application Publication No. 2005 / 0178398; and Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). The dried, aged, fermented tobacco can be further processed (e.g., cut, shredded, expanded, or blended). See, e.g., U.S. Pat. Nos. 4,528,993; 4,660,577; and 4,987,907. In one aspect, the dried tobacco material of the present disclosure is hot air dried, sun dried, air dried, or fire dried.

[0231] In one aspect, the tobacco plants, seeds, plant parts, plant cells, and plant genomes provided herein are from a type of tobacco selected from the group consisting of yellow tobacco, sun-cured tobacco, air-cured tobacco, dark air-cured tobacco, and dark fire-cured tobacco. In another aspect, the tobacco plants, seeds, plant parts, plant cells, and plant genomes provided herein are from a type of tobacco selected from the group consisting of burley tobacco, Maryland tobacco, bright tobacco, Virginia tobacco, Oriental tobacco, Turkish tobacco, and Galpao tobacco.

[0232] In one aspect, the modified tobacco plants provided herein are of a tobacco variety selected from the group consisting of yellow varieties, bright varieties, burley varieties, Virginia varieties, Maryland varieties, Galpao varieties, dark varieties, Oriental varieties, and Turkish varieties.

[0233] In one aspect, the modified tobacco plants provided herein are selected from the group consisting of yellow variety tobacco plants, bright variety tobacco plants, burley variety tobacco plants, virginia variety tobacco plants, maryland variety tobacco plants, galpao 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 yellow variety tobacco cells, bright variety tobacco cells, burley variety tobacco cells, virginia variety tobacco cells, maryland variety tobacco cells, galpao 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 yellow variety tobacco plant parts, bright variety tobacco plant parts, burley variety tobacco plant parts, virginia variety tobacco plant parts, maryland variety tobacco plant parts, galpao 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 yellow variety tobacco seeds, bright variety tobacco seeds, burley variety tobacco seeds, virginia variety tobacco seeds, maryland variety tobacco seeds, galpao variety tobacco seeds, dark variety tobacco seeds, oriental variety tobacco seeds, and turkish variety tobacco seeds.

[0234] As used herein, "hybrid" is produced by crossing two plants from different varieties or species such that the progeny contains genetic material from each parent. One of ordinary skill in the art will recognize that higher order hybrids can also be produced. For example, a first hybrid can be produced by crossing variety C with variety D to produce a C×D hybrid, and a second hybrid can be produced by crossing variety E with variety F to produce an E×F hybrid. The first and second hybrids can be further crossed to produce a higher order hybrid (C×D)×(E×F) that contains genetic information from all four parent varieties. In one aspect, the modified tobacco plants provided herein are hybrid tobacco plants. In another aspect, the modified tobacco seeds provided herein are hybrid tobacco seeds.

[0235] As used herein, the term "crossing" refers to the intentional mating of two plants. In one aspect, crossing includes pollination and / or fertilization of a first tobacco plant by a second tobacco plant. The two tobacco plants being crossed can be distantly related, closely related, or the same. In one aspect, both of the two tobacco plants being crossed are modified tobacco plants. In one aspect, the two tobacco plants being crossed are of the same tobacco variety. In one aspect, the two tobacco plants being crossed are of two different tobacco varieties. In one aspect, one of the two tobacco plants being crossed is male sterile. In one aspect, one of the two tobacco plants being crossed is female sterile. In one aspect, at least one of the two tobacco plants being crossed is a hybrid tobacco plant. In one aspect, at least one of the two tobacco plants being crossed is a modified tobacco plant.

[0236] In one aspect, the present disclosure is a method for producing a modified tobacco plant, comprising: (a) A step of crossing 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 contains a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), CDK inhibitor, MYB, and WRKY transcription factor, the mutation does not exist in the endogenous gene in control tobacco plants of the same variety, and when the at least one tobacco plant is cultivated under equivalent cultivation conditions, it contains no or reduced suckers after topping compared to the control tobacco plants; (b) A step of selecting progeny tobacco plants in which no or reduced suckers appear after topping compared to control tobacco plants of the same hybrid cultivated under equivalent cultivation conditions A method is provided that includes.

[0237] In another aspect, the present disclosure is a method for producing a modified tobacco plant, comprising: (a) A step of crossing 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 contains a recombinant DNA construct comprising a heterologous promoter functionally linked to a nucleic acid encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), CDK inhibitor, MYB, and WRKY transcription factor, and when the at least one tobacco plant is cultivated under equivalent cultivation conditions, it contains no or reduced suckers after topping compared to control tobacco plants; (b) A step of selecting progeny tobacco plants in which no or reduced suckers appear after topping compared to control tobacco plants of the same hybrid cultivated under equivalent cultivation conditions A method is provided that includes.

[0238] In a further aspect, the present disclosure is a method for producing a modified tobacco plant, comprising: (a) A step of crossing 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 functionally linked to a nucleic acid encoding at least one small RNA molecule capable of reducing the expression of an endogenous genomic locus encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), CDK inhibitor, MYB, and WRKY transcription factor, and the at least one tobacco plant, when cultivated under equivalent cultivation conditions, comprises no or reduced suckers after topping as compared to a control tobacco plant; (b) A step of selecting a progeny tobacco plant in which no or reduced suckers appear after topping as compared to a control tobacco plant of the same hybrid cultivated under equivalent cultivation conditions A method is provided that includes this.

[0239] In one aspect, the tobacco variety provided herein is male sterile. In another aspect, the tobacco variety provided herein is cytoplasmic male sterile (CMS). In one aspect, the modified tobacco plant provided herein is male sterile. In another aspect, the modified tobacco plant provided herein is cytoplasmic male sterile. Male sterile tobacco plants can be produced by any method known in the art. Methods for producing male sterile tobacco are described in Wernsman, E. A., and Rufty, R. C. 1987. Chapter Seventeen. Tobacco. Pages 669 - 698 In: Cultivar Development. Crop Species. W. H. Fehr (ed.), MacMillan Publishing Go., Inc., New York, N.Y. 761 pp.

[0240] In another aspect, the tobacco varieties provided herein are female sterile. As a non-limiting example, female sterile plants can be produced by mutating the STIG1 gene. See, for example, Goldman et al. 1994, EMBO Journal 13:2976-2984. In one aspect, the modified tobacco plants provided herein are female sterile.

[0241] Flue-cured tobacco (also called "Virginia" or "Bright" tobacco) accounts for approximately 40% of the world's tobacco production. Flue-cured tobacco is often also called "Bright tobacco" because of the bright yellow to dark orange color it reaches during curing. Flue-cured tobacco has a light, fresh aroma and taste. Flue-cured tobacco generally has a high sugar content and a low oil content. The main flue-cured tobacco cultivating countries are Argentina, Brazil, China, India, Tanzania, and the United States. In one aspect, the modified tobacco plants or seeds provided herein are of a flue-cured tobacco variety selected from the group consisting of CC13, CC27, CC33, CC35, CC37, CC65, CC67, CC700, GF318, GL338, GL368, GL939, K346, K399, K326, NC102, NC196, NC291, NC297, NC299, NC471, NC55, NC606, NC71, NC72, NC92, PVH1118, PVH1452, PVH2110, SPEIGHT168, SPEIGHT220, SPEIGHT225, SPEIGHT227, SPEIGHT236, and any variety that is essentially derived from any one of the foregoing varieties. In another aspect, the modified tobacco plants or seeds provided herein are of a flue-cured tobacco variety selected from the group consisting of Coker48, Coker176, Coker371-Gold, Coker319, Coker347, GL939, K149, K326, K340, K346, K358, K394, K399, K730, NC27NF, NC37NF, NC55, NC60, NC71, NC72, NC82, NC95, NC297, NC606, NC729, NC2326, McNair373, McNair944, Ox207, Ox414NF, Reams126, Reams713, Reams744, RG8, RG11, RG13, RG17, RG22, RG81, RG H4, RG H51, Speight H-20, Speight G-28, Speight G-58, SpeightG-70, Speight G-108, Speight G-l11, Speight G-l17, Speight 168, Speight 179, Speight NF-3, Va116, Va182, and any variety that is essentially derived from any one of the foregoing varieties.See International Publication No. WO 2004 / 041006. In a further aspect, the modified tobacco plants or seeds provided herein are yellow cultivars selected from the group consisting of K326, K346, and NC196.

[0242] Air-dried tobacco includes "Burley", "Maryland", and "Dark" tobaccos. The common factor linking air-dried tobaccos is that drying occurs mainly without artificial heat sources and moisture. Burley tobacco is light to dark brown in color, oily, and low in sugar. Burley tobacco is typically air-dried in barns. Major Burley-growing countries include Argentina, Brazil, Italy, Malawi, and the United States.

[0243] Maryland tobacco is very fluffy and has good burning characteristics, low nicotine, and a neutral aroma. Major Maryland-growing countries include the United States and Italy.

[0244] In one aspect, the modified tobacco plants or seeds provided herein are of Burley tobacco cultivars selected from the group consisting of Clay402, Clay403, Clay502, Ky14, Ky907, Ky910, Ky8959, NC2, NC3, NC4, NC5, NC2000, Tn86, Tn90, Tn97, R610, R630, R711, R712, NCBH129, HB4488PLC, PD7319LC, Bu21xKy10, HB04P, Ky14xL8, Kt200, Newton98, Pedigo561, Pf561, and Va509. In a further aspect, the modified tobacco plants or seeds provided herein are of Burley cultivars selected from the group consisting of TN90, KT209, KT206, KT212, and HB4488. In another aspect, the modified tobacco plants or seeds provided herein are of Maryland tobacco cultivars selected from the group consisting of Md10, Md40, Md201, Md609, Md872, and Md341.

[0245] Dark air-cured tobacco is mainly distinguished from other tobacco types by its drying method, which gives dark air-cured tobacco a medium to dark brown color and a unique aroma. Dark air-cured tobacco is mainly used in the production of chewing tobacco and snuff. In one aspect, the modified tobacco plants or seeds provided herein are of a dark air-cured tobacco variety selected from the group consisting of Sumatra, Jatim, Dominican Cubano, Besuki, One sucker, Green River, Virginia sun-cured, and Paraguan Passado.

[0246] Dark fire-cured tobacco is generally dried using smoldering firewood on the floor of a closed drying shed. Dark fire-cured tobacco is typically used to produce pipe blends, cigarettes, chewing tobacco, snuff, and full-bodied cigars. The main cultivation areas of dark fire-cured tobacco are the states of Tennessee, Kentucky, and Virginia in the United States. In one aspect, the modified tobacco plants or seeds provided herein are of a dark fire-cured tobacco variety selected from the group consisting of Narrow Leaf Madole, Improved Madole, Tom Rosson Madole, Newton’s VH Madole, Little Crittenden, Green Wood, Little Wood, Small Stalk Black Mammoth, DT508, DT518, DT592, KY171, DF911, DF485, TN D94, TN D950, VA309, and VA359.

[0247] Oriental tobacco is also called Greek, Aroma, and Turkish tobacco due to the fact that it is typically cultivated in the Eastern Mediterranean region such as Turkey, Greece, Bulgaria, Macedonia, Syria, Lebanon, Italy, and Romania. The small plant size, small leaf size, and unique aroma characteristics of Oriental tobacco varieties are the result of adaptation to the poor soils and stressful climate conditions in which this variety has been grown. In one aspect, the modified tobacco plants or seeds provided herein are of an Oriental tobacco variety selected from the group consisting of 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 any variety that is essentially derived from any one of the foregoing varieties.

[0248] The tobacco plants, plant parts, and tobacco materials provided herein can be used in any tobacco product. As used herein, "tobacco product" is defined as any product made from or derived from tobacco that is intended for human use or consumption. In one aspect, the tobacco products provided herein contain dried components from the tobacco plants provided herein. In another aspect, the tobacco products provided herein contain dried tobacco leaves from the tobacco plants provided herein. In one aspect, the disclosure provides a tobacco product comprising a dried tobacco material from any of the tobacco plants provided herein.

[0249] In one aspect, the present disclosure provides a tobacco product comprising dried tobacco material from a modified tobacco plant comprising a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, wherein the mutation is not present in the endogenous gene in a control tobacco plant of the same variety, and wherein the modified tobacco plant, when grown under equivalent cultivation conditions, comprises no or reduced suckers after topping as compared to the control tobacco plant. In another aspect, the present disclosure provides a tobacco product comprising dried tobacco material from a modified tobacco plant comprising a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, wherein the mutation is not present in the endogenous gene in a control tobacco plant of the same variety.

[0250] In one aspect, the present disclosure provides a method comprising the step of preparing a tobacco product using dried tobacco material from a modified tobacco plant comprising a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, wherein the mutation is not present in the endogenous gene in a control tobacco plant of the same variety, and wherein the modified tobacco plant, when grown under equivalent cultivation conditions, comprises no or reduced suckers after topping as compared to the control tobacco plant. In another aspect, the present disclosure provides a method comprising the step of preparing a tobacco product using dried tobacco material from a modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, the polynucleotide sequence being operably linked to a heterologous promoter, wherein the modified tobacco plant, when grown under equivalent cultivation conditions, comprises no or reduced suckers after topping as compared to the control tobacco plant.

[0251] In one aspect, the present disclosure provides a tobacco product comprising dried tobacco material from a modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, functionally linked to a heterologous promoter, wherein the modified tobacco plant, when cultivated under equivalent cultivation conditions, has no or reduced suckers after topping as compared to a control tobacco plant. In another aspect, the present disclosure provides a tobacco product comprising dried tobacco material from a modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, functionally linked to a heterologous promoter.

[0252] In one aspect, the present disclosure provides a method comprising the step of preparing a tobacco product using dried tobacco material from a modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, functionally linked to a heterologous promoter, wherein the modified tobacco plant, when cultivated under equivalent cultivation conditions, has no or reduced suckers after topping as compared to a control tobacco plant. In another aspect, the present disclosure provides a method comprising the step of preparing a tobacco product using dried tobacco material from a modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, functionally linked to a heterologous promoter.

[0253] In one aspect, the present disclosure provides a tobacco product comprising dried tobacco material from 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 cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, wherein the modified tobacco plant, when cultivated under equivalent cultivation conditions, has no or reduced suckers after topping as compared to a control tobacco plant. In another aspect, the present disclosure provides a tobacco product comprising dried tobacco material from 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 cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor.

[0254] In one aspect, the present disclosure provides a method comprising the step of preparing a tobacco product using dried tobacco material from 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 cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, wherein the modified tobacco plant, when cultivated under equivalent cultivation conditions, has no or reduced suckers after topping as compared to a control tobacco plant. In another aspect, the present disclosure provides a method comprising the step of preparing a tobacco product using dried tobacco material from 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 cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor.

[0255] Alkaloid compounds can be extracted or isolated from any tobacco plant or plant part provided herein. In one aspect, the tobacco products provided herein contain alkaloids extracted from a modified tobacco plant or a part thereof. In another aspect, the tobacco products provided herein contain nicotine extracted from a tobacco plant or a tobacco plant part. In another aspect, the tobacco products provided herein contain anatabine extracted from a tobacco plant or a tobacco plant part. In another aspect, the tobacco products provided herein contain anabasine extracted from a tobacco plant or a tobacco plant part. In one aspect, the tobacco products provided herein contain nornicotine extracted from a tobacco plant or a plant part. In one aspect, the tobacco products provided herein contain alkaloids extracted from a modified tobacco plant, and the alkaloids are selected from the group consisting of nicotine, nornicotine, anatabine, and anabasine.

[0256] The alkaloid compounds extracted from the tobacco plants or tobacco plant parts provided herein can be used to produce compositions suitable for use with non-combustible products. Exemplary non-combustible products include electronic cigarettes (“e-cigarettes”), electronic smoking articles, e-vapor products, aerosolized vapor products, and heated tobacco products. In one aspect, the non-combustible products provided herein contain alkaloids extracted from the tobacco plants or tobacco plant parts provided herein. In one aspect, the non-combustible products provided herein contain nicotine extracted from the tobacco plants or tobacco plant parts provided herein. In one aspect, the non-combustible products provided herein contain anabasine extracted from the tobacco plants or tobacco plant parts provided herein. In one aspect, the non-combustible products provided herein contain anatabine extracted from the tobacco plants or tobacco plant parts provided herein. In one aspect, the non-combustible products provided herein contain nornicotine extracted from the tobacco plants or tobacco plant parts provided herein.

[0257] 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 article. In another aspect, the non-combustible product provided herein is an aerosolized vapor 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-vapor product.

[0258] The tobacco products provided herein include, but are not limited to, cigarette products (e.g., cigarettes, bidi cigarettes, kreteks), cigar products (e.g., cigars, cigar wrapping tobacco, cigarillos), pipe tobacco products, tobacco-derived products, tobacco-derived nicotine products, 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, snus), films, chewables (e.g., gums), drops, dissolving strips, tabs, tablets, shaped parts, gels, consumable units, insoluble matrices, hollow shapes, reconstituted tobacco, expanded tobacco, etc. See, for example, U.S. Patent Application Publication No. 2006 / 0191548.

[0259] As used herein, "cigarette" refers to a tobacco product having a "rod" and a "filler". A cigarette "rod" includes cigarette paper, a filter, a plug wrap (used to contain the filter material), tipping paper that holds the cigarette paper (including the filler) to the filter, and all adhesives that hold these components together. "Filler" includes (1) all tobacco, including but not limited to reconstituted and expanded tobacco, (2) non-tobacco alternatives (including but not limited to herbs, non-tobacco plant materials, and other flavorings that may accompany the tobacco wrapped in cigarette paper), (3) casing, (4) flavorants, and (5) all other additives (mixed in the tobacco and alternatives and wrapped in the cigarette).

[0260] In one aspect, the present disclosure provides nicotine derived from a modified tobacco plant provided herein and a method of producing nicotine from a modified tobacco plant provided herein for use in a product.

[0261] In one aspect, the method provided herein includes preparing a tobacco product using dried tobacco leaves from a modified tobacco plant provided herein.

[0262] As used herein, "reconstituted tobacco" refers to a portion of tobacco filler made from tobacco dust and other tobacco scrap material, processed into sheet form, and cut into strips to resemble tobacco. In addition to cost reduction, reconstituted tobacco is very important for contributing to the flavor of cigarettes because it processes flavor generation using the reaction between ammonia and sugar. In one aspect, the tobacco product provided herein includes reconstituted tobacco derived from a modified tobacco plant.

[0263] As used herein, "expanded tobacco" refers to a portion of tobacco filler that has been processed through the expansion of a suitable gas such that the tobacco is "puffed" and its density is reduced and its filling capacity is increased. Expanded tobacco reduces the weight of the tobacco used in cigarettes.

[0264] Tobacco products derived from the plants of the present disclosure include cigarettes and other smoking articles, in particular smoking articles that include a filter element containing dried tobacco within a tobacco blend and a rod of smokable material. In one aspect, the tobacco products of the present disclosure are selected from the group consisting of cigarettes, kreteks, bidis, cigars, cigarillos, non-ventilated cigarettes, vented recess filter cigarettes, pipe tobacco, sniff tobacco, snus, chewing tobacco, moist snuff, finely cut chewing tobacco, long cut chewing tobacco, and pouch chewing tobacco products. In another aspect, the tobacco products of the present disclosure are selected from the group consisting of gums, tablets, drops, and dissolving strips.

[0265] In one aspect, the tobacco products provided herein include dried tobacco material from a modified tobacco plant. In another aspect, the tobacco products provided herein include dried tobacco leaf material from a modified tobacco plant. In another aspect, the tobacco products provided herein include dried tobacco stem material from a modified tobacco plant.

[0266] In another aspect, the tobacco product of the present disclosure is a smokeless tobacco product. Smokeless tobacco products are not combusted and include, but are not limited to, chewing tobacco, moist snuff, snus, and dry snuff. Chewing tobacco is typically coarsely cut tobacco leaves that are packaged in a large pouch-like package and used in a plug or twist form. Moist snuff is more finely cut moist tobacco that is provided in a loose form or a pouch form, typically packaged in a round can, and used as a pinch placed between the cheek and gum of an adult tobacco consumer or in a pouch. Snus is heat-treated smokeless tobacco. Dry snuff is finely ground tobacco that is placed in the mouth or used nasally. In a further aspect, the tobacco product of the present disclosure is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff.

[0267] The present disclosure further provides a method of manufacturing a tobacco product comprising tobacco material from a modified tobacco plant provided herein. In one aspect, the method provided herein conditions a cured tobacco material made from a modified tobacco plant provided herein to increase its moisture content from about 12.5% to about 13.5% to about 21%, and blends the conditioned tobacco material to produce a desired blend. In one aspect, the method of manufacturing a tobacco product provided herein further comprises casing or flavoring the blend. Generally, during the casing process, casing or source materials are added to the blend to balance the chemical composition to enhance the quality of the blend and to produce certain desired flavor characteristics. Further details of the casing process can be found in Tobacco Production, Chemistry and Technology, Edited by L. Davis and M. Nielsen, Blackwell Science, 1999.

[0268] The present disclosure provides tobacco materials from modified tobacco plants or parts thereof. The tobacco materials obtained from the tobacco plants, cells, lines, varieties or hybrids of the present disclosure can be used for manufacturing tobacco products. In one aspect, the tobacco material includes leaf material. In one aspect, the tobacco material includes stem material. In one aspect, the tobacco material includes fresh tobacco material. In another aspect, the tobacco material includes dried tobacco material. In a further aspect, the tobacco material includes cured tobacco material. In yet another aspect, the tobacco material includes fermented tobacco material. In one aspect, the tobacco materials provided herein can be used in any tobacco products provided herein. In one aspect, the dried tobacco materials provided herein include air-dried tobacco materials. In another aspect, the dried tobacco materials provided herein include fire-dried tobacco materials. In another aspect, the dried tobacco materials provided herein include sun-dried tobacco materials. In another aspect, the dried tobacco materials provided herein include yellow tobacco materials. In another aspect, the dried tobacco materials provided herein are selected from the group consisting of air-dried tobacco materials, fire-dried tobacco materials, sun-dried tobacco materials and yellow tobacco materials.

[0269] The tobacco materials provided herein can also be processed using methods including, but not limited to, heat treatment (e.g., cooking, toasting), flavoring, enzymatic treatment, expansion and / or curing. Both fermented and non-fermented tobacco can be processed using these techniques. Examples of suitable processed tobacco include dark air-cured, dark fire-cured, burley, yellow varieties, and cigar filler or wrapper, as well as products from a whole leaf stemming operation. In one aspect, the tobacco fiber comprises up to 70% dark tobacco on a fresh weight basis. For example, the tobacco can be conditioned by heating, sweating and / or pasteurization processes as described in U.S. Patent Application Publication No. 2004 / 0118422 or 2005 / 0178398.

[0270] The tobacco materials provided herein can be subjected to fermentation. Fermentation is typically characterized by a high initial moisture content, exotherm, and a 10-20% reduction in dry weight. See, for example, U.S. Patent Nos. 4,528,993; 4,660,577; 4,848,373; and 5,372,149. In addition to modifying the aroma of the leaf, fermentation can change either or both the color and texture of the leaf. During the fermentation process, evolved gases can be generated, oxygen can be taken up, the pH can change, and the amount of water retained can change. See, for example, U.S. Patent Application Publication No. 2005 / 0178398 and Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). The dried tobacco or dried and fermented tobacco can be further processed (e.g., cut, expanded, blended, comminuted or powdered) before incorporation into an oral product. The tobacco is, in some instances, a long-cut fermented dried moist tobacco having an oven volatile content of 48-50% by weight prior to mixing with a copolymer and, optionally, flavorants and other additives that may be included.

[0271] In one aspect, the tobacco materials provided herein can be processed to a desired size. In certain aspects, the tobacco fibers can be processed to have an average fiber size of less than 200 micrometers. In one aspect, the tobacco fibers are 75-125 micrometers. In another aspect, the tobacco fibers are processed to have a size of 75 micrometers or less. In one aspect, the tobacco fibers include long-cut tobacco that can be cut or shredded to a width of from about 10 cuts per inch to a maximum of about 110 cuts per inch and a length of from about 0.1 inch to a maximum of about 1 inch. The double-cut tobacco fibers can have a particle size range such that about 70% of the double-cut tobacco fibers fall between a mesh size of -20 mesh and 80 mesh.

[0272] The tobacco materials provided herein can be processed to have a total oven volatiles content of about 10 wt% or more; about 20 wt% or more; about 40 wt% or more; about 15 wt% to about 25 wt%; about 20 wt% to about 30 wt%; about 30 wt% to about 50 wt%; about 45 wt% to about 65 wt%; or about 50 wt% to about 60 wt%. One of ordinary skill in the art will understand that "wet" tobacco typically refers to tobacco having an oven volatiles content of about 40 wt% to about 60 wt% (e.g., about 45 wt% to about 55 wt%, or about 50 wt%). As used herein, "oven volatiles" is determined by calculating the percentage of weight loss of a sample after drying the sample for 3.25 hours in a preheated forced-air oven at 110°C. Oral products can have a total oven volatiles content different from that of the tobacco fibers used to produce the oral products. The processing steps described herein can decrease or increase the oven volatiles content.

[0273] The following exemplary, non-limiting embodiments are contemplated: 1. A modified tobacco plant comprising a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, wherein the mutation is not present in the endogenous gene in a control tobacco plant of the same variety, and wherein the modified tobacco plant, when grown under equivalent cultivation conditions, has no or reduced suckers after topping compared to the control tobacco plant. 2. A modified tobacco plant comprising a mutation in an endogenous gene encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), a CDK inhibitor, MYB, and a WRKY transcription factor, wherein the mutation is not present in the endogenous gene in a control tobacco plant of the same variety. 3. A modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), CDK inhibitor, MYB, and WRKY transcription factor, functionally linked to a heterologous promoter, wherein the modified tobacco plant, when cultivated under equivalent cultivation conditions, contains no or reduced suckers after topping, as compared to a control tobacco plant. 4. A modified tobacco plant comprising a polynucleotide sequence encoding a polypeptide selected from the group consisting of cyclin, cyclin-dependent kinase (CDK), CDK inhibitor, MYB, and WRKY transcription factor, functionally linked to a heterologous promoter. 5. A modified tobacco plant comprising a recombinant DNA construct comprising a heterologous promoter functionally 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 cyclin, cyclin-dependent kinase (CDK), CDK inhibitor, MYB, and WRKY transcription factor, wherein the modified tobacco plant, when cultivated under equivalent cultivation conditions, contains no or reduced suckers after topping, as compared to a control tobacco plant. 6. A modified tobacco plant comprising a recombinant DNA construct comprising a heterologous promoter functionally 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 cyclin, cyclin-dependent kinase (CDK), CDK inhibitor, MYB, and WRKY transcription factor. 7. The modified tobacco plant according to aspect 1 or 2, wherein the mutation comprises a mutation selected from the group consisting of insertion, deletion, substitution, and inversion. 8. The modified tobacco plant according to any one of aspects 1, 2, or 7, wherein the mutation comprises a non-expression mutation. 9. The modified tobacco plant according to any one of aspects 1, 2, 7, or 8, wherein the mutation results in a premature stop codon in the mRNA transcript of the endogenous gene. 10. The modified tobacco plant according to any one of aspects 1, 2, or 7 to 9, wherein the mutation results in shortening of the polypeptide. 11. The modified tobacco plant according to any one of aspects 1, 2, or 7 to 10, wherein the mutation is located within an exon of the endogenous gene. 12. The modified tobacco plant according to any one of aspects 1, 2, or 7 to 10, wherein the mutation is located within an intron of the endogenous gene. 13. The modified tobacco plant according to any one of aspects 1, 2, or 7, wherein the mutation is located within the 5'-untranslated region (UTR) or 3'-UTR of the endogenous gene. 14. The modified tobacco plant according to any one of aspects 1, 2, or 7, wherein the mutation is located within the promoter of the endogenous gene. 15. The modified tobacco plant according to any one of aspects 1, 2, or 7 to 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 to 44 and 113. 16. The modified tobacco plant according to any one of aspects 1, 2, or 7 to 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 the group consisting of SEQ ID NOs: 45 to 88 and 114. 17. The modified tobacco plant according to any one of aspects 1, 2, or 7 to 16, wherein the mutation results in a decreased level of expression of the endogenous gene as compared to the control tobacco plant. 18. The modified tobacco plant according to any one of aspects 1, 2, or 7 to 16, wherein the mutation results in an increased level of expression of the endogenous gene as compared to the control tobacco plant. 19. The modified tobacco plant according to any one of aspects 1, 2, or 7 to 16, wherein the mutation results in a decreased level of activity of the polypeptide as compared to the control tobacco plant. 20. The modified tobacco plant according to any one of aspects 1, 2, or 7 - 16, wherein the mutation results in an increased level of the activity of the polypeptide as compared to the control tobacco plant. 21. The modified tobacco plant according to any one of aspects 3 - 6, wherein the heterologous promoter comprises an axillary bud meristem - specific promoter. 22. The modified tobacco plant according to any one of aspects 3 - 6, wherein the heterologous promoter comprises an axillary bud meristem - preferential promoter. 23. The modified tobacco plant according to any one of aspects 3 - 6, wherein the heterologous promoter comprises a polynucleotide sequence that is at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 89 - 109. 24. The modified tobacco plant according to any one of aspects 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. 25. The modified tobacco plant according to aspect 5 or 6, wherein the small RNA molecule comprises 18 to 30 nucleotides. 26. The modified tobacco plant according to aspect 5 or 6, wherein the small RNA molecule comprises a polynucleotide sequence that has at least 90% identity or complementarity with the endogenous mRNA. 27. The modified tobacco plant according to aspect 5 or 6, wherein the small RNA molecule comprises 18 to 30 nucleotides. 28. The modified tobacco plant according to aspect 5 or 6, wherein the small RNA molecule comprises a polynucleotide sequence that has at least 90% identity or complementarity with a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1 - 44 and 113. 29. The modified tobacco plant according to aspect 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 the group consisting of SEQ ID NOs: 1 - 44 and 113. 30. The modified tobacco plant according to any one of aspects 1 - 29, wherein the polypeptide is a cyclin. 31. The modified tobacco plant according to any one of aspects 1 to 29, wherein the polypeptide is a CDK. 32. The modified tobacco plant according to any one of aspects 1 to 29, wherein the polypeptide is a CDK inhibitor. 33. The modified tobacco plant according to any one of aspects 1 to 29, wherein the polypeptide is an MYB. 34. The modified tobacco plant according to any one of aspects 1 to 29, wherein the polypeptide is a WRKY transcription factor. 35. The modified tobacco plant according to aspect 30, wherein the cyclin is encoded by a polynucleotide sequence that is at least 80% identical to SEQ ID NOs: 6 to 32. 36. The modified tobacco plant according to aspect 30, wherein the cyclin comprises an amino acid sequence...

Claims

1. A modified tobacco plant comprising a polynucleotide sequence encoding a cyclin-dependent kinase (CDK) inhibitor polypeptide functionally linked to a heterologous promoter, wherein the CDK inhibitor polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 49, and the heterologous promoter comprises a polynucleotide sequence that is at least 95% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 89-109.

2. The modified tobacco plant according to claim 1, wherein the polynucleotide sequence encoding the CDK inhibitor polypeptide is at least 95% identical to the polynucleotide sequence of SEQ ID NO:

5.

3. The modified tobacco plant according to claim 1, wherein the polynucleotide sequence encoding the CDK inhibitor polypeptide is identical to the polynucleotide sequence of SEQ ID NO:

5.

4. The modified tobacco plant according to claim 1, wherein the amino acid sequence is at least 99% identical to SEQ ID NO:

49.

5. The modified tobacco plant according to claim 1, wherein the amino acid sequence is identical to SEQ ID NO:

49.

6. The modified tobacco plant according to claim 1, wherein the heterologous promoter comprises a polynucleotide sequence that is at least 99% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 89-109.

7. The modified tobacco plant according to claim 1, wherein the heterologous promoter comprises a polynucleotide sequence that is identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 89-109.

8. The modified tobacco plant according to claim 1, which is of a tobacco variety selected from the group consisting of yellow varieties, bright varieties, burley varieties, virginia varieties, maryland varieties, dark varieties, Galpao varieties, oriental varieties, and Turkish varieties.

9. BU64 plant, CC101 plant, CC200 plant, CC13 plant, CC27 plant, CC33 plant, CC35 plant, CC37 plant, CC65 plant, CC67 plant, CC301 plant, CC400 plant, CC500 plant, CC600 plant, CC700 plant, CC800 plant, CC900 plant, CC1063 plant, Coker176 plant, Coker319 plant, Coker371Gold plant, Coker48 plant, CU263 plant, DF911 plant, Galpao plant, GL26H plant, GL338 plant, GL350 plant, GL395 plant, GL600 plant, GL737 plant, GL939 plant, GL973 plant, GF157 plant, GF318 plant, RJR901 plant, HB04P plant, K149 plant, K326 plant, K346 plant, K358 plant, K394 plant, K399 plant, K730 plant, NC196 plant, NC37NF plant, NC471 plant, NC55 plant, NC92 plant, NC2326 plant, NC95 plant, NC925 plant, PVH1118 plant, PVH1452 plant, PVH2110 plant, PVH2254 plant, PVH2275 plant, VA116 plant, VA119 plant, KDH959 plant, KT200 plant, KT204LC plant, KY10 plant, KY14 plant, KY160 plant, KY17 plant, KY171 plant, KY907 plant, KY907LC plant, KTY14xL8 LC plant, Little Crittenden plant, McNair373 plant, McNair944 plant, male sterile KY14xL8 plant, Narrow Leaf Madole plant, MS KY171 plant, Narrow Leaf Madole (phph) plant, MS Narrow Leaf Madole plant, MSThe modified tobacco plant according to claim 1, selected from the group consisting of TND950 plant, PD7302LC plant, PD7305LC plant, PD7309LC plant, PD7312LC plant, PD7318LC plant, PD7319LC plant, MSTKS2002 plant, TKF2002 plant, TKF6400 plant, TKF4028 plant, TKF4024 plant, KT206LC plant, KT209LC plant, KT210LC plant, KT212LC plant, NC100 plant, NC102 plant, NC2000 plant, NC291 plant, NC297 plant, NC299 plant, NC3 plant, NC4 plant, NC5 plant, NC6 plant, NC7 plant, NC606 plant, NC71 plant, NC72 plant, NC810 plant, NC BH 129 plant, NC2002 plant, Neal Smith Madole plant, OXFORD 207 plant, "Perique" plant, PVH03 plant, PVH09 plant, PVH19 plant, PVH50 plant, PVH51 plant, R610 plant, R630 plant, R7-11 plant, R7-12 plant, RG17 plant, RG81 plant, RG H51 plant, RGH4 plant, RGH51 plant, RS1410 plant, Speight168 plant, Speight172 plant, Speight179 plant, Speight210 plant, Speight220 plant, Speight225 plant, Speight227 plant, Speight234 plant, SpeightG-28 plant, SpeightG-70 plant, SpeightH-6 plant, SpeightH20 plant, SpeightNF3 plant, TI1406 plant, TI1269 plant, TN86 plant, TN86LC plant, TN90 plant, TN90LC plant, TN97 plant, TN97LC plant, TN D94 plant, TN D950 plant, TR (Tom Rosson) Madole plant, VA309 plant, and VA359 plant.

10. The modified tobacco plant according to claim 1, which is male sterile or cytoplasmic male sterile.

11. Tobacco leaves of the modified tobacco plant according to any one of claims 1-10.

12. Tobacco seeds of the modified tobacco plant according to any one of claims 1-10.

13. The tobacco leaf according to claim 11, wherein the tobacco leaf is a dried tobacco leaf.

14. A tobacco product comprising an alkaloid extracted from the modified tobacco plant according to any one of claims 1-10 or a part thereof.

15. A tobacco product comprising a dried tobacco material from a modified tobacco plant comprising a polynucleotide sequence encoding a cyclin-dependent kinase (CDK) inhibitor polypeptide functionally linked to a heterologous promoter, wherein the CDK inhibitor polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 49, and the heterologous promoter comprises a polynucleotide sequence that is at least 95% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 89-109.

16. The tobacco product according to claim 15, wherein the polynucleotide sequence encoding the CDK inhibitor polypeptide is at least 95% identical to the polynucleotide sequence of SEQ ID NO:

5.

17. The tobacco product according to claim 15, wherein the polynucleotide sequence encoding the CDK inhibitor polypeptide is identical to the polynucleotide sequence of SEQ ID NO:

5.

18. The tobacco product according to claim 15, wherein the amino acid sequence is at least 99% identical to SEQ ID NO:

49.

19. The tobacco product according to claim 15, wherein the amino acid sequence is identical to SEQ ID NO:

49.

20. The tobacco product according to claim 15, wherein the heterologous promoter comprises a polynucleotide sequence that is at least 99% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 89-109.

21. The tobacco product according to claim 15, wherein the heterologous promoter comprises a polynucleotide sequence that is identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 89-109.

22. The tobacco product according to any one of claims 15-21, selected from the group consisting of cigarettes, kreteks, bidis, cigars, cigarillos, non-ventilated cigarettes, vented recess filter cigarettes, pipe tobacco, snuff, chewing tobacco, moist smokeless tobacco, finely cut chewing tobacco, long cut chewing tobacco, and pouch chewing tobacco products.

23. The tobacco product according to any one of claims 15-22, wherein the dried tobacco material is selected from the group consisting of air-dried tobacco material, fire-dried tobacco material, sun-dried tobacco material, and hot air-dried tobacco material.

24. The tobacco product according to any one of claims 15 to 22, wherein the dried tobacco material comprises dried leaf material, dried stem material, or both dried leaf material and dried stem material.

25. 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 functionally linked to a polynucleotide sequence encoding a cyclin-dependent kinase (CDK) inhibitor polypeptide, the CDK inhibitor polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 49, and the heterologous promoter comprises a polynucleotide sequence that is at least 95% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 89 to 109; (b) selecting at least one tobacco cell comprising the recombinant DNA construct; (c) regenerating a modified tobacco plant from the at least one tobacco cell selected in step (b). The method as described above.

26. A method for producing a modified tobacco plant, comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct comprising a heterologous promoter functionally linked to a polynucleotide sequence encoding a cyclin-dependent kinase (CDK) inhibitor polypeptide, the heterologous promoter comprises a polynucleotide sequence that is at least 95% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 89 to 109, and the CDK inhibitor polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 49; (b) selecting a progeny tobacco plant comprising the recombinant DNA construct. The method as described above.

27. The method according to claim 25 or 26, wherein the heterologous promoter comprises a polynucleotide sequence that is at least 99% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 89 to 109.

28. The method according to claim 25 or 26, wherein the heterologous promoter comprises a polynucleotide sequence that is identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 89 to 109. Claim 29 The method according to claim 25 or 26, wherein the amino acid sequence is at least 99% identical to the amino acid sequence of SEQ ID NO:

49. Claim 30 The method according to claim 25 or 26, wherein the amino acid sequence is identical to the amino acid sequence of SEQ ID NO:

49. Claim 31 The method according to claim 25 or 26, wherein the polynucleotide sequence encoding the CDK inhibitor polypeptide is at least 95% identical to the polynucleotide sequence of SEQ ID NO:

5. Claim 32 The method according to claim 25 or 26, wherein the polynucleotide sequence encoding the CDK inhibitor polypeptide is identical to SEQ ID NO: 5.

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