Compositions and methods for producing tobacco plants, and products having altered alkaloid levels

Abstract

To identify genes that regulate tobacco nicotine levels, and to provide tobacco plants and products that contain altered nicotine levels while maintaining tobacco leaf quality.SOLUTION: The present disclosure provides tobacco Nic1b locus and associated genes (e.g., a group of ERF genes). Also, provided are tobacco plants with altered total alkaloid and nicotine levels and commercially acceptable leaf grade, their development via breeding or transgenic approaches, and production of tobacco products from these tobacco plants. Further, provided are compositions and methods for producing tobacco plants having novel mutations or alleles to reduce nicotine levels. Further, provided are sequence polymorphisms and molecular markers for breeding tobacco with reduced nicotine or alkaloids while maintaining tobacco leaf grade and tobacco product quality.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 616,959, filed January 12, 2018, U.S. Provisional Application No. 62 / 625,878, filed February 2, 2018, and U.S. Provisional Application No. 62 / 685,844, filed June 15, 2018, all of which are incorporated by reference in their entireties.

[0002] Incorporation of sequence listings The Sequence Listing contained in the file entitled "P34709WO00_SL.txt", created on January 11, 2019, which is 2,780,525 bytes (measured in MS-Windows®), is submitted electronically herewith and is incorporated by reference in its entirety.

[0003] Field The present disclosure provides the low alkaloid-associated chromosomal deletion region (LA-associated region), the tobacco Nic1b locus, and genes within or around the region or locus. Tobacco plants with altered total alkaloid and nicotine levels and commercially acceptable leaf grades, their development through breeding or transgenic approaches, and the production of tobacco products from these tobacco plants are also provided. [Background technology]

[0004] background Four major alkaloids are found in tobacco: nicotine, nornicotine, anabasine, and anatabine. Nicotine is the most abundant alkaloid, typically accounting for more than 90% of the total alkaloids in commercial tobacco cultivars. Nicotine biosynthesis occurs primarily in the tobacco roots. The tobacco plant then transports nicotine through the vascular bundles to the leaves, where it is then stored in vacuoles.

[0005] Various factors affect tobacco alkaloid levels, including genotype, environment, fertilization, and agronomic practices (e.g., nicotine production is stimulated by pinching, scarring, and herbivore damage). The low-alkaloid trait, first discovered in Cuban cigar tobacco cultivar strains, was introduced into cigarette cultivars through a series of backcrosses. Low-alkaloid tobacco germplasm was subsequently registered in the genetic background of cultivar Burley 21 (Legg et al., Crop Science, 10:212 (1970)). Genetic studies using the low-alkaloid Burley 21 (LA BU21) strain indicated that two unrelated loci contribute to nicotine levels in tobacco leaves. These two loci are designated Nic1 and Nic2. In LA BU21, the nic1 and nic2 (same as nicotine1 and nicotine2, respectively) mutations are semidominant. They exhibit a dose-dependent effect on nicotine levels, with the effect of nic1 being approximately 2.4 times stronger than that of nic2. Molecular characterization of the Nic2 locus has been reported. The nic2 mutation was shown to contain a deletion of a cluster of transcription factor genes from the ethylene response factor (ERF) family, such as ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168 (Shoji et al., Plant Cell, (10):3390-409 (2010) (Non-Patent Document 2)).

[0006] Reducing the total alkaloid content in tobacco can have many benefits. It can increase the value of tobacco as a biomass resource. Increasing nicotine alkaloids in tobacco plants can play an important role in protecting the plant from insects and herbivores.

[0007] Consistent with the role of alkaloids in insect defense, LA BU21 was reported to be extremely susceptible to insect damage (Legg et al., Crop Science, 10:212 (1970)). Further studies comparing flue-cured tobacco isogenic lines with low total alkaloid percentages (approximately 0.20%) to their "normal" recurring parents (total alkaloids 1.85-2.70%) reported that yield, grade index, total N, and reducing sugar content in the low-alkaloid lines were lower than in normal flue-cured cultivars (Chaplin and Weeks, Crop Science, 16(3):416-18 (1976)). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Legg et al., Crop Science, 10:212 (1970) [Non-patent document 2] Shoji et al., Plant Cell, (10):3390-409(2010) [Non-patent document 3] Chaplin and Weeks, Crop Science, 16(3):416-18 (1976) Summary of the Invention [Problem to be solved by the invention]

[0009] There is a need to identify genes that regulate tobacco nicotine levels and to develop tobacco plants and products that contain altered nicotine levels (e.g., reduced nicotine) while maintaining (if not superior) tobacco leaf quality. [Means for solving the problem]

[0010] overview In one aspect, the present disclosure provides a tobacco plant or part thereof comprising a mutation in the Nic1b_ERF locus, wherein the tobacco plant is capable of producing leaves having a USDA Grade Index value of 50 or greater when cured.

[0011] In another aspect, the disclosure provides a tobacco plant or part thereof comprising a mutation in the Nic1b_ERF locus and a mutation in the Nic2 locus, wherein the tobacco plant, when cured, is capable of producing leaves having a USDA Grade Index value equivalent to the USDA Grade Index value of a control plant when grown under similar growing conditions, wherein the control plant shares essentially the same genetic background as the tobacco plant except for the mutation.

[0012] In one aspect, the present disclosure further provides a non-transgenic tobacco plant or portion thereof comprising a nicotine level selected from the group consisting of less than 2.0%, wherein the tobacco plant, when cured, is capable of producing leaves having a USDA Grade Index value of 50 or greater.

[0013] In another aspect, the present disclosure also provides a tobacco plant or part thereof comprising a non-transgenic mutation in the Nic1b_ERF locus, wherein the non-transgenic mutation reduces the nicotine level of the tobacco plant to about 20% or less of the nicotine level of a control plant when grown under similar growing conditions, wherein the tobacco plant is capable of producing leaves, when cured, having a USDA grade index value equivalent to the USDA grade index value of the control plant, and wherein the control plant shares essentially the same genetic background as the tobacco plant except for the non-transgenic mutation.

[0014] In one aspect, the present disclosure provides a tobacco plant or part thereof comprising a mutation in the Nic1b_ERF locus, wherein the tobacco plant or part thereof comprises similar levels of one or more tobacco aroma compounds selected from the group consisting of 3-methylvaleric acid, valeric acid, isovaleric acid, labdenoids, cembrenoids, sugar esters, and reducing sugars compared to a control tobacco plant when grown under similar growing conditions.

[0015] In another aspect, the present disclosure provides a tobacco plant or portion thereof comprising a mutation in the Nic1b_ERF locus, wherein the mutation is not present in LA Burley 21. In one aspect, the tobacco plant provided herein comprises a shorter chromosomal introgression at the Nic1b locus compared to LA Burley 21. In another aspect, the tobacco plant provided herein does not comprise a deletion of an entire gene or an entire gene-coding sequence in the Nic1b_ERF locus.

[0016] In one aspect, the present disclosure provides a tobacco plant or portion thereof comprising one or more mutations in one or more genes comprising a sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 153, 154, 202, and 203, and fragments thereof. In one aspect, the tobacco plants provided herein comprise one or more non-naturally occurring mutant alleles at the Nic1b_ERF locus that reduce or eliminate one or more gene activities from the Nic1b_ERF locus. In one aspect, these mutant alleles result in lower nicotine levels.

[0017] In another aspect, the present disclosure provides a tobacco plant or portion thereof comprising one or more mutations in one or more genes comprising a coding sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 38, 48, 49, 52-54, 158, 159, 204, and 205, and fragments thereof.

[0018] In one aspect, the present disclosure provides a tobacco plant or portion thereof comprising one or more mutations in one or more genes encoding a polypeptide having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206, and 207, and fragments thereof.

[0019] In another aspect, the present disclosure provides a tobacco plant or portion thereof comprising a heterologous expression cassette comprising a Nic1b inhibitory sequence of a gene comprising a sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 153, 154, 202, and 203, and fragments thereof, wherein the inhibitory sequence is operably linked to a promoter functional in the plant cell, and the inhibitory sequence has at least 90% sequence identity to at least a 21-nucleotide fragment of the sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 153, 154, 202, and 203, and fragments thereof.

[0020] In another aspect, the present disclosure provides a recombinant DNA construct comprising a promoter operably linked to a polynucleotide encoding an RNA molecule that is functional in a tobacco cell and capable of binding to RNA encoding a polypeptide having an amino acid sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206, and 207, and fragments thereof, and wherein the RNA molecule represses expression of the polypeptide.

[0021] In one aspect, the present disclosure provides a recombinant DNA construct comprising a promoter functional in a tobacco cell and operably linked to a polynucleotide encoding a polypeptide having an amino acid sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206, and 207, and fragments thereof.

[0022] The present disclosure further provides flue-cured tobacco, tobacco blends, and tobacco products comprising plant material from the disclosed tobacco plants, strains, varieties, or hybrids.

[0023] The present disclosure also provides methods of breeding tobacco strains, cultivars, or varieties that contain desirable levels of total alkaloids or nicotine, e.g., low nicotine or no nicotine. In one aspect, the disclosure provides a method of introgressing a low-nicotine trait into a tobacco variety, the method comprising: (a) crossing a first tobacco variety that includes the low-nicotine trait with a second tobacco variety that does not have the low-nicotine trait to produce one or more progeny tobacco plants; (b) genotyping the one or more progeny tobacco plants for polymorphic markers associated with the low-nicotine trait, wherein the polymorphic markers are in a chromosomal interval flanked by any two SNP markers selected from the group consisting of SEQ ID NOs: 125-145 and 193-201, or by any two loci having sequences selected from the group consisting of SEQ ID NOs: 1, 3-37, 146, 149, 152-156, 202, 203, and 184-186; and (c) selecting the progeny tobacco plants that include the low-nicotine trait.

[0024] In one aspect, the disclosure provides a method of transferring a low-nicotine trait into a tobacco variety, the method comprising: (a) crossing a first tobacco variety including the low-nicotine trait with a second tobacco variety that does not have the low-nicotine trait to produce one or more progeny tobacco plants; (b) genotyping the one or more progeny tobacco plants for polymorphic markers associated with the low-nicotine trait, wherein the polymorphic markers are SNP markers selected from the group consisting of SEQ ID NOs: 125-145 and 193-201, or within 2 cM of any one of any loci having a sequence selected from the group consisting of SEQ ID NOs: 1, 3-37, 146, 149, 152-156, 202, 203, and 184-186; and (c) selecting the progeny tobacco plants that include the low-nicotine trait. In one aspect, the polymorphic markers are SNP markers selected from the group consisting of SEQ ID NOs: 125-145 and 193-201.

[0025] In another aspect, the disclosure provides a method for selecting tobacco plants having a low-nicotine trait, the method comprising: (a) isolating nucleic acids from a collection of tobacco germplasm; (b) assaying the nucleic acids for one or more markers closely linked to the Nic1b locus; and (c) selecting tobacco plants having the low-nicotine trait based on the marker assays. [The present invention 1001] 1. A tobacco plant or part thereof comprising a mutation in the Nic1b_ERF locus, wherein the tobacco plant, when cured, is capable of producing leaves having a USDA Grade Index value of 50 or greater. [The present invention 1002] 1001. The tobacco plant or part thereof of the present invention, wherein the tobacco plant is a Nicotiana tabacum plant. [The present invention 1003] 1002. The tobacco plant or part thereof of the present invention, wherein said tobacco plant further comprises a mutation in an ERF gene at the Nic2 locus. [The present invention 1004] The tobacco plant or part thereof of the present invention 1002, wherein the tobacco plant further comprises one or more mutations in two or more, three or more, four or more, five or more, six or more, or all seven genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168. [The present invention 1005] 1002. The tobacco plant or part thereof of the present invention, wherein said tobacco plant, when cured, is capable of producing leaves having a USDA Grade Index value of 70 or greater. [The present invention 1006] A tobacco plant or part thereof of the present invention 1002, wherein the tobacco plant, when cured, is capable of producing leaves having a USDA grade index value equivalent to the USDA grade index value of a control plant when grown and cured under similar conditions, and the control plant shares essentially the same genetic background as the tobacco plant except for the mutation. [The present invention 1007] 1002. A tobacco plant or part thereof of the present invention, wherein the tobacco plant contains a level of nicotine that is less than 40% of the nicotine level of a control plant when grown under similar growing conditions, and the control plant shares essentially the same genetic background as the tobacco plant except for the mutation. [The present invention 1008] 1002. The tobacco plant or part thereof of the present invention, wherein said tobacco plant comprises a nicotine level of less than 2.0%. [The present invention 1009] 1. A tobacco plant or part thereof comprising a non-transgenic mutation in the Nic1b_ERF locus, wherein the non-transgenic mutation reduces the nicotine level of the tobacco plant to less than 60% of the nicotine level of a control plant when grown under similar growing conditions, wherein the tobacco plant is capable of producing leaves, when cured, having a USDA grade index value equivalent to the USDA grade index value of the control plant, and wherein the control plant shares essentially the same genetic background as the tobacco plant except for the non-transgenic mutation, and wherein the tobacco plant is a Nicotiana tabacum plant. [The present invention 1010] A population of tobacco plants according to any one of 1001 to 1009 of the present invention. [The present invention 1011] A dried tobacco material from any one of the tobacco plants of the present inventions 1001 to 1009. [The present invention 1012] The cured tobacco material of the present invention, produced by a drying method selected from the group consisting of hot air drying, air drying, flame drying, and sun drying. [The present invention 1013] A tobacco blend comprising the dried tobacco material of the present invention. [The present invention 1014] A tobacco product comprising the dried tobacco material of the present invention. [The present invention 1015] The tobacco product of the present invention 1014 is selected from the group consisting of cigarettes, cigarillos, non-vented recess filter cigarettes, vented recess filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and cut tobacco. [The present invention 1016] The tobacco product of the present invention 1014, which is a smokeless tobacco product. [The present invention 1017] The tobacco product of the present invention, wherein the smokeless tobacco product is selected from the group consisting of loose-leaf chewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff. [The present invention 1018] A tobacco plant or part thereof comprising a mutation in the Nic1b_ERF locus, wherein said mutation is not present in LA Burley 21, LAFC53, and LN KY171. [The present invention 1019] The tobacco plant or part thereof of the present invention 1018, wherein said tobacco plant further comprises a mutation in an ERF gene at the Nic2 locus. [The present invention 1020] The tobacco plant or part thereof of the present invention 1018, wherein the mutation is located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, and 202-205, and fragments thereof. [The present invention 1021] The tobacco plant or part thereof of the present invention 1018, wherein the mutation is located in a gene encoding a polypeptide having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87, 88, 89, 180, 181, 206, and 207. [The present invention 1022] The tobacco plant or part thereof of the present invention 1018, wherein the tobacco plant is from a variety selected from the group consisting of flue-cured tobacco, air-cured tobacco, dark flue-cured tobacco, and Galpao tobacco, and Oriental tobacco. [The present invention 1023] The tobacco plant or part thereof of the present invention, wherein the tobacco plant is from a variety selected from the group consisting of Burley tobacco, Maryland tobacco, and dark air-cured tobacco. [The present invention 1024] A cured tobacco material from the tobacco plant of the present invention. [The present invention 1025] The cured tobacco material of the present invention 1024, wherein the tobacco plant contains nicotine at a level of 0.2% to 0.6%. [The present invention 1026] The cured tobacco material of the present invention 1024, wherein the tobacco plant contains nicotine at a level of 1.0% to 3.0%. [The present invention 1027] The cured tobacco material of the present invention 1024 produced by a drying method selected from the group consisting of hot air drying, air drying, flame drying, and sun drying. [The present invention 1028] A tobacco product comprising the dried tobacco material of the present invention. [The present invention 1029] The tobacco product of the present invention 1024 is selected from the group consisting of cigarettes, cigarillos, non-ventilated recess filter cigarettes, ventilated recess filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and cut tobacco. [The present invention 1030] The tobacco product of the present invention 1024, which is a smokeless tobacco product. [The present invention 1031] 1. A tobacco plant or part thereof comprising a heterologous expression cassette comprising a Nic1b_ERF inhibitory sequence of a gene comprising a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, and 202-205, and fragments thereof, wherein the inhibitory sequence is operably linked to a promoter functional in a plant cell, and the inhibitory sequence has at least 90% sequence identity to a fragment of at least 21 nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, and 202-205, and fragments thereof. [The present invention 1032] The tobacco plant or part thereof of the present invention 1031, wherein said Nic1b inhibitory sequence is capable of being transcribed as an inhibitory polynucleotide selected from the group consisting of a single-stranded RNA polynucleotide, a double-stranded RNA polynucleotide, and a combination thereof. [The present invention 1033] 1031. The tobacco plant or part thereof of the present invention, wherein the promoter is selected from the group consisting of a constitutive promoter, an inducible promoter, and a tissue-preferred promoter. [The present invention 1034] 1031. The tobacco plant or part thereof of the present invention, wherein the promoter is a root-specific promoter. [Brief explanation of the drawings]

[0026] [Figure 1] Breeding scheme to develop F5 lines carrying nic1 and nic2 deletion marker alleles (*(x) indicates self-fertilization). [Figure 2] Potential alternative splicing variants around locus g31431 and locus g31432. Dark boxes represent detected transcripts, while light boxes represent in silico generated CDS models. [Figure 3] Overexpression of ERF16, the LA-associated region deleted in LA BU21 (i.e., not containing an annotated gene, SEQ ID NO: 1, referred to as "Nic1bΔ"; see Example 2), or g32081 (a beta-glucosidase 18-like gene), but not ERF130, results in increased nicotine and total alkaloids in LA BU21. [Figure 4] Artificial microRNA-mediated suppression of individual Nic1b_ERFs (ERFnew, ERF210, ERF199, ERF29, and ERF91L2) reduces nicotine levels to approximately 50%–90% of control plants. Mean nicotine levels and standard errors are based on data from Table 7. DETAILED DESCRIPTION OF THE INVENTION

[0027] A brief description of arrays SEQ ID NO:1 shows the sequence of the LA-associated region identified from the TN90 x LA BU21 cross.

[0028] SEQ ID NO: 2 shows the sequence of the Nic1b region (including the LA-associated region).

[0029] SEQ ID NOs: 3-37 show the genomic coding sequences (gDNA that typically start with an ATG and end with a stop codon, but in some cases also contain untranslated region (UTR) sequences) of 35 annotated genes (NCGs) in the Nic1b region.

[0030] SEQ ID NOs: 38 to 72 show the cDNA sequences of 35 annotated genes (NCGs) in the Nic1b region.

[0031] SEQ ID NOs: 73 to 107 show the amino acid sequences encoded by 35 annotated genes (NCGs) in the Nic1b region.

[0032] SEQ ID NOs: 108 to 119 show exemplary mature artificial miRNA sequences (sense or antisense) for suppressing selected NCG genes.

[0033] SEQ ID NOs: 120 to 124 show exemplary guide RNA sequences for editing selected NCG genes.

[0034] SEQ ID NOs: 125-145 show the sequences of 21 SNP markers spanning or adjacent to the Nic1b region.

[0035] SEQ ID NOs:146 to 148 show the genomic coding, cDNA, and amino acid sequences of the g31432 locus, respectively.

[0036] SEQ ID NOs:149 to 151 show the genomic coding, cDNA, and amino acid sequences of the g31446 locus, respectively.

[0037] SEQ ID NOs: 152-156 show the genomic coding sequences of multiple protein-coding genes and non-coding RNAs from the Nic1b region. SEQ ID NOs: 157-161 show the corresponding cDNA sequences. SEQ ID NOs: 162-183 show the corresponding protein or non-coding RNA molecule sequences.

[0038] SEQ ID NOs: 184-186 show the genomic coding sequences of multiple transcription factor genes that exhibit differential expression between normal and reduced alkaloid tobacco strains. SEQ ID NOs: 187-189 show the corresponding cDNA sequences. SEQ ID NOs: 190-192 show the corresponding protein or non-coding RNA molecule sequences.

[0039] SEQ ID NOs: 193 to 201 show SNP marker sequences located immediately adjacent to multiple ERF genes from the Nic1b region or within genes encoding non-coding RNAs.

[0040] SEQ ID NOs:202 and 203 show the genomic coding sequences of two ERF genes associated with the Nic1b region. SEQ ID NOs:204 and 205 show the corresponding cDNA sequences. SEQ ID NOs:206 and 207 show the corresponding protein sequences.

[0041] SEQ ID NOs: 208 to 214 show the genomic coding sequences of the seven Nic2_ERF genes. SEQ ID NOs: 215 to 221 show the corresponding cDNA sequences. SEQ ID NOs: 222 to 228 show the corresponding protein sequences.

[0042] The various sequences include an "N" in a nucleotide sequence or an "X" in an amino acid sequence. "N" can be any nucleotide, e.g., A, T, G, C, or a deletion or insertion of one or more nucleotides. In some instances, a string of "N"s is shown. The number of "N"s does not necessarily correspond to the actual number of variable nucleotides at that position. The actual nucleotide sequence may be longer or shorter than the segment indicated by "N". Similarly, "X" can be any amino acid residue or a deletion or insertion of one or more amino acids. Again, the number of "X"s does not necessarily correspond to the actual number of variable amino acids at that position. The actual amino acid sequence may be longer or shorter than the segment indicated by "X".

[0043] Detailed Description Unless otherwise defined, scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art. Those skilled in the art will recognize that many methods can be used in the practice of the present disclosure. Indeed, the present disclosure is in no way limited to the methods and materials described. For purposes of this disclosure, the following terms are defined below.

[0044] Any references referred to herein, including, for example, all patents and publications, are incorporated by reference in their entirety.

[0045] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0046] The term "about" is used herein to mean approximately, roughly, in the region of, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth.

[0047] As used herein, "Nic1b locus" refers to any chromosomal position or location within or closely associated with the Nic1b region. "Nic1b region" refers to an approximately 1.5 million bp long chromosomal segment corresponding to SEQ ID NO:2 from the TN90 genome and bearing an allele associated with the low-alkaloid trait. "nic1b mutation" refers to a mutation in the Nic1b locus.

[0048] As used herein, Nic1b_ERF (or plural Nic1b_ERFs) refers to any one of the ERF genes or loci at or near the Nic1b locus, including, for example, ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210, and ERF91L2. See Table 11 and Kajikawa et al., Plant physiol. 2017, 174:999-1011. A "nic1b_erf mutation" refers to a mutation in the Nic1b_ERF gene. As used herein, all mutations or mutant alleles are shown in lowercase and italics. All names of genes, loci, or proteins are shown in or beginning with capital letters and may be italicized or not.

[0049] As used herein, Nic2_ERF (or plural Nic2_ERFs) refers to any one of the ERF genes or loci at or near the Nic2 locus, including, for example, ERF221, ERF115, ERF168, ERF17, ERF179, and ERF189. See Table 12; Shoji et al., Plant Cell, (10):3390-409 (2010); and Kajikawa et al., Plant physiol. 2017, 174:999-1011.

[0050] As used herein, a mutation refers to a heritable genetic modification introduced into a gene to change the expression or activity of the product encoded by the gene. Such a modification can occur in any sequence region of the gene, such as the promoter, 5'UTR, exon, intron, 3'UTR, or terminator region. In one embodiment, the mutation reduces, inhibits, or eliminates the expression or activity of the gene product. In another embodiment, the mutation increases, elevates, strengthens, or enhances the expression or activity of the gene product. In one embodiment, the mutation is not a natural polymorphism present in a particular tobacco variety or cultivar. As used herein, a "mutant allele" refers to an allele from a genetic locus, wherein the allele contains a mutation. As used herein, "mutagenesis" refers to generating a mutation without a transgene or without the mutation-related transgene remaining in the final mutant. In one embodiment, the mutagenesis is cisgenic. In another embodiment, the mutagenesis is via gene or genome editing. In a further embodiment, mutagenesis is via random mutagenesis, for example, chemical (eg, EMS) or physical (irradiation) mutagenesis.

[0051] As used herein, a tobacco plant may be any plant from the genus Nicotiana, including Nicotiana tabacum, Nicotiana amplexicaulis PI 271989, Nicotiana benthamiana PI 555478, Nicotiana bigelovii PI 555485, Nicotiana debneyi, Nicotiana excelsior PI 224063, Nicotiana glutinosa PI 555507, Nicotiana goodspeedii PI 241012, Nicotiana gossei PI 555509, ... 230953, Nicotiana hesperis PI 271991, Nicotiana knightiana PI 555527, Nicotiana maritima PI 555535, Nicotiana megalosiphon PI 555536, Nicotiana nudicaulis PI 555540, Nicotiana paniculata PI 555545, Nicotiana plumbaginifolia PI 555548, Nicotiana repanda PI 555552, Nicotiana rustica rustica), Nicotiana suaveolens PI 230960, Nicotiana sylvestris PI 555569, Nicotiana tomentosa PI 266379, Nicotiana tomentosiformistomentosiformis, and Nicotiana trigonophylla PI 555572. In one aspect, the tobacco plants described herein are Nicotiana tabacum plants.

[0052] In one aspect, the present disclosure provides a tobacco plant or part thereof comprising a mutation in the Nic1b_ERF locus, wherein the tobacco plant, when cured, is capable of producing leaves having a USDA grade index value of 50 or greater. In one aspect, the tobacco plant further comprises a mutation in an ERF gene at the Nic2 locus. In one aspect, the tobacco plant further comprises one or more mutations in two or more, three or more, four or more, five or more, six or more, or all seven genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168. In one aspect, the tobacco plant further comprises one or more mutations in ERF189, ERF115, or both. In one embodiment, the tobacco plant, when cured, is capable of producing leaves having a USDA grade index value selected from the group consisting of 55 or greater, 60 or greater, 65 or greater, 70 or greater, 75 or greater, 80 or greater, 85 or greater, 90 or greater, and 95 or greater. In another embodiment, the tobacco plant, when cured, is capable of producing leaves having a USDA grade index value equivalent to the USDA grade index value of a control plant when grown and cured in similar conditions, the control plant sharing essentially the same genetic background as the tobacco plant except for the mutation. In a further embodiment, the tobacco plant, when cured, is capable of producing leaves having a USDA grade index value that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% of the USDA grade index value of a control plant when grown in similar conditions, the control plant sharing essentially the same genetic background as the tobacco plant except for the mutation.In further embodiments, the tobacco plants, when dried, are capable of producing leaves having a USDA grade index value of 65% to 130%, 70% to 130%, 75% to 130%, 80% to 130%, 85% to 130%, 90% to 130%, 95% to 130%, 100% to 130%, 105% to 130%, 110% to 130%, 115% to 130%, or 120% to 130% of the USDA grade index value of the control plant. In further embodiments, the tobacco plants, when dried, are capable of producing leaves having a USDA grade index value of 70% to 125%, 75% to 120%, 80% to 115%, 85% to 110%, or 90% to 100% of the USDA grade index value of the control plant. In one embodiment, the tobacco plant contains a level of nicotine that is less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, or less than 80% of the nicotine level of a control plant when grown under similar growing conditions, wherein the control plant shares essentially the same genetic background as the tobacco plant except for the mutation. In another embodiment, the tobacco plant comprises a total alkaloid level selected from the group consisting of less than 3%, less than 2.75%, less than 2.5%, less than 2.25%, less than 2.0%, less than 1.75%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, and less than 0.05%. In another embodiment, the tobacco plant comprises a nicotine or total alkaloid level selected from the group consisting of less than 3%, less than 2.75%, less than 2.5%, less than 2.25%, less than 2.0%, less than 1.75%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, and less than 0.05%.In a further embodiment, the tobacco plant further comprises a transgene or mutation that directly suppresses the expression or activity of one or more genes encoding a product selected from the group consisting of PMT, MPO, QPT, BBL, A622, aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, ornithine decarboxylase, arginine decarboxylase, nicotine uptake permease (NUP), and a MATE transporter.

[0053] In one aspect, the present disclosure provides a tobacco plant or part thereof comprising a mutation in the Nic1b_ERF locus, wherein the tobacco plant, when cured, is capable of producing leaves having a USDA grade index value equivalent to that of a control plant when grown under similar conditions, and the control plant shares essentially the same genetic background as the tobacco plant except for the mutation. In one aspect, the tobacco plant further comprises a mutation in an ERF gene at the Nic2 locus. In one aspect, the tobacco plant further comprises one or more mutations in two or more, three or more, four or more, five or more, six or more, or all seven genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168. In one aspect, the tobacco plant further comprises one or more mutations in ERF189, ERF115, or both. In another embodiment, the tobacco plant is capable of producing leaves that, when cured, have a USDA Grade Index value selected from the group consisting of: 55 or greater, 60 or greater, 65 or greater, 70 or greater, 75 or greater, 80 or greater, 85 or greater, 90 or greater, and 95 or greater. In another embodiment, the tobacco plant is capable of producing leaves that, when cured, have a USDA Grade Index value selected from the group consisting of: 50-95, 55-95, 60-95, 65-95, 70-95, 75-95, 80-95, 85-95, 90-95, 55-90, 60-85, 65-80, 70-75, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, and 90-95. In further aspects, the tobacco plants are capable of producing leaves that, when dried, have a USDA grade index value that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% of the USDA grade index value of a control plant.In further embodiments, the tobacco plants, when dried, are capable of producing leaves having a USDA grade index value of 65% to 130%, 70% to 130%, 75% to 130%, 80% to 130%, 85% to 130%, 90% to 130%, 95% to 130%, 100% to 130%, 105% to 130%, 110% to 130%, 115% to 130%, or 120% to 130% of the USDA grade index value of the control plant. In further embodiments, the tobacco plants, when dried, are capable of producing leaves having a USDA grade index value of 70% to 125%, 75% to 120%, 80% to 115%, 85% to 110%, or 90% to 100% of the USDA grade index value of the control plant. In another embodiment, the tobacco plant contains a level of nicotine or total alkaloids that is less than 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of the nicotine or total alkaloid level of a control plant when grown under similar growing conditions. In another embodiment, the tobacco plant further contains a transgene or mutation that directly suppresses the expression or activity of one or more genes encoding a product selected from the group consisting of PMT, MPO, QPT, BBL, A622, aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, ornithine decarboxylase, arginine decarboxylase, nicotine uptake permease (NUP), and a MATE transporter.

[0054] In one embodiment, the Nic1b_ERF locus comprises one or more sequences selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, and 202-205. In one embodiment, the Nic1b_ERF locus comprises a sequence or chromosomal segment of less than 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, or 9000 nucleotides. In another embodiment, the Nic1b_ERF locus comprises a sequence or chromosomal segment of at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, or 9000 nucleotides. In further embodiments, the Nic1b_ERF locus comprises a sequence or chromosomal segment of between 100 and 300, 100 and 400, 100 and 500, 100 and 600, 100 and 700, 100 and 800, 100 and 900, 100 and 1000, 100 and 1500, 100 and 2000, 100 and 3000, 100 and 4000, 100 and 5000, 100 and 6000, 100 and 7000, 100 and 8000, or 100 and 9000 nucleotides. In one aspect, the Nic1b_ERF locus comprises a sequence or chromosomal segment of between 50 and 100, 100 and 200, 200 and 300, 300 and 400, 400 and 500, 500 and 600, 600 and 700, 700 and 800 and 900, 900 and 1000, 1000 and 1500, 1500 and 2000, 2000 and 3000, 3000 and 4000, 4000 and 5000, 5000 and 6000, 6000 and 7000, 7000 and 8000, or 8000 and 9000 nucleotides.

[0055] In one embodiment, the Nic1b_ERF locus comprises a sequence or chromosomal segment within 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 30000, 40000, 50000, 60000, or 70000 nucleotides of a SNP marker selected from the group consisting of SEQ ID NOs: 125-145 and 193-201. In another embodiment, the Nic1b_ERF locus comprises a sequence or chromosomal segment within 100 to 300, 100 to 400, 100 to 500, 100 to 600, 100 to 700, 100 to 800, 100 to 900, 100 to 1000, 100 to 1500, 100 to 2000, 100 to 3000, 100 to 4000, 100 to 5000, 100 to 6000, 100 to 7000, 100 to 8000, or 100 to 9000 nucleotides of a SNP marker selected from the group consisting of SEQ ID NOs: 125-145 and 193-201. In further embodiments, the Nic1b_ERF locus comprises a sequence or chromosomal segment within 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1500, 1500-2000, 2000-3000, 3000-4000, 4000-5000, 5000-6000, 6000-7000, 7000-8000, or 8000-9000 nucleotides of a SNP marker selected from the group consisting of SEQ ID NOs: 125-145 and 193-201.

[0056] In one embodiment, the Nic1b_ERF locus comprises a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, and 202-205, and fragments thereof. In another embodiment, the Nic1b_ERF locus comprises a sequence or chromosomal segment within 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 30000, 40000, 50000, 60000, or 70000 nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, and 202-205. In further embodiments, the Nic1b_ERF locus comprises a sequence or chromosomal segment within 100-300, 100-400, 100-500, 100-600, 100-700, 100-800, 100-900, 100-1000, 100-1500, 100-2000, 100-3000, 100-4000, 100-5000, 100-6000, 100-7000, 100-8000, or 100-9000 nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, and 202-205. In one embodiment, the Nic1b_ERF locus is selected from the group consisting of 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-2000, 1900-2100, 2100-2200, 2200-2300, 2300-2400, 2400-2500, 2500-2600, 2600-2700, 2700-2800, 2800-3000, 2900-3100, 3000-3200, 3100-3300, 3200-3400, 3300-3500, 3400-3500, 3500-3600, 3600-3700, 3700-3800, 3800-4000, 3900-4100, 41000-4200, 41000-4300, 4200-4400, 4300-4500, 4400-4500, 4500-4600, 4600-4700, 4 and sequences or chromosomal segments within 00-700, 700-800, 800-900, 900-1000, 1000-1500, 1500-2000, 2000-3000, 3000-4000, 4000-5000, 5000-6000, 6000-7000, 7000-8000, or 8000-9000 nucleotides.

[0057] In one aspect, the Nic1b_ERF locus comprises a sequence or chromosomal segment flanked by, but excluding, any two of the sequences selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, and 202-205, and fragments thereof. In one aspect, the Nic1b_ERF locus comprises a sequence or chromosomal segment flanked by, but excluding, any two of the SNP markers selected from the group consisting of SEQ ID NOs: 125-145 and 193-201. In one aspect, the Nic1b_ERF locus comprises a sequence or chromosomal segment flanked by, but excluding, any two of the sequences selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, and 202-205.

[0058] In one aspect, the present disclosure also provides a tobacco variety, cultivar, or line comprising a mutation selected from the group consisting of a nic1b_erf mutation, a nic2 mutation, and combinations thereof, wherein the tobacco variety, cultivar, or line has a leaf grade equivalent to the leaf grade of a control tobacco variety, cultivar, or line when grown under similar growing conditions, and the control tobacco variety shares essentially the same genetic background as the tobacco variety, cultivar, or line except for the mutation.

[0059]

[0013] In one aspect, the present disclosure further provides a non-transgenic tobacco plant or portion thereof comprising a nicotine or total alkaloid level selected from the group consisting of less than 3%, less than 2.75%, less than 2.5%, less than 2.25%, less than 2.0%, less than 1.75%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, and less than 0.05%, wherein the tobacco plant, when cured, is capable of producing leaves having a USDA Grade Index value of 50 or greater, 55 or greater, 60 or greater, 65 or greater, 70 or greater, 75 or greater, 80 or greater, 85 or greater, 90 or greater, and 95 or greater. In another embodiment, such non-transgenic tobacco plants are capable of producing leaves containing nicotine levels of less than 2.0% and, when cured, having a USDA Grade Index value of greater than or equal to 70. In a further embodiment, such non-transgenic tobacco plants are capable of producing leaves containing nicotine levels of less than 1.0% and, when cured, having a USDA Grade Index value of greater than or equal to 70.

[0060] In one aspect, the disclosure also provides a tobacco plant or portion thereof comprising a non-transgenic mutation, wherein the non-transgenic mutation reduces the nicotine or total alkaloid level of the tobacco plant to less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, or less than 80% of the nicotine level of a control plant when grown under similar growing conditions, wherein the tobacco plant, when cured, is capable of producing leaves having a USDA grade index value equivalent to the USDA grade index value of the control plant, and wherein the control plant shares essentially the same genetic background as the tobacco plant except for the non-transgenic mutation.

[0061] In one aspect, the present disclosure provides a tobacco plant or portion thereof comprising a mutation in the Nic1b_ERF locus, wherein the mutation is not present in LA Burley 21. In one aspect, the tobacco plant provided herein comprises a shorter chromosomal introgression at the Nic1b_ERF locus compared to LA Burley 21. In another aspect, the tobacco plant provided herein does not comprise a deletion of the entire gene or the entire gene coding sequence at the Nic1b_ERF locus. In one aspect, the tobacco plant provided herein is homozygous at the Nic1b_ERF locus. In another aspect, the tobacco plant provided herein is heterozygous at the Nic1b_ERF locus. In one aspect, the tobacco plant provided herein comprises a Nic1b_ERF mutation selected from the group consisting of a point mutation, a deletion, an insertion, a duplication, and an inversion. In one aspect, the Nic1b_ERF mutation in the tobacco plant provided herein is introduced by an approach selected from the group consisting of random mutagenesis and targeted mutagenesis. In another embodiment, the Nic1b_ERF mutation in the tobacco plants provided herein is introduced by a targeted mutagenesis approach selected from the group consisting of meganucleases, zinc finger nucleases, TALENs, and CRISPRs.

[0062] In one aspect, the tobacco plants provided herein comprise one or more mutations in one or more genes comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17-19, 153, 154, 202, 203, and 208-214, and fragments thereof. In one aspect, the one or more mutations reduce expression or activity of one or more genes comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17-19, 153, 154, 202, 203, and 208-214, and fragments thereof.

[0063] In one embodiment, the tobacco plants provided herein comprise one or more mutations in one or more genes comprising a coding sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 38, 48, 49, 52-54, 158, 159, 204, 205, and 215-221, and fragments thereof. In one embodiment, the one or more mutations reduce expression or activity of one or more genes comprising a coding sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 38, 48, 49, 52-54, 158, 159, 204, 205, and 215-221, and fragments thereof.

[0064] In one embodiment, the tobacco plants provided herein comprise one or more mutations in one or more genes encoding a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206, and 207, and 222-228, and fragments thereof. In one embodiment, the one or more mutations reduce the expression or activity of one or more genes encoding a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206, and 207, and 222-228, and fragments thereof.

[0065] In one aspect, the tobacco plants provided herein comprise one or more mutations in one or more genes comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 4, 14, 15, 17, 18, 19, 37, 39, 49, 50, 52, 53, 54, 202-205, and 72, and fragments thereof. In one embodiment, the one or more mutations reduce the expression or activity of one or more genes comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 4, 14, 15, 17, 18, 19, 37, 39, 49, 50, 52, 53, 54, 202-205, and 72, and fragments thereof.

[0066] In one embodiment, the tobacco plants provided herein comprise one or more mutations in one or more genes comprising a coding sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 49, 52, 53, 204, 205, and 54, and fragments thereof. In one embodiment, the one or more mutations reduce the expression or activity of one or more genes comprising a coding sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 49, 52, 53, 204, 205, and 54, and fragments thereof.

[0067] In one embodiment, the tobacco plants provided herein comprise one or more mutations in one or more genes encoding a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 84, 87, 88, and 89, and fragments thereof. In one embodiment, the one or more mutations reduce the expression or activity of one or more genes encoding a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 84, 87, 88, and 89, and fragments thereof.

[0068] LA Burley 21 (also referred to as LA BU21) is a low total alkaloid tobacco line produced by the incorporation of low alkaloid genes from Cuban cigar cultivars into Burley 21 through several backcrosses (Legg et al. 1970). It has approximately 0.2% total alkaloids (dry weight) compared to approximately 3.5% (dry weight) in its parent, Burley 21. LA BU21 has a leaf grade significantly lower than commercially acceptable standards. LA BU21 also exhibits other unfavorable leaf phenotypes characterized by lower yield, delayed maturation and senescence, higher susceptibility to insect herbivory, and poor end-product quality after drying (Chaplin and Weeks, Crop Sci. 16:416-418 (1976); Legg et al. Crop. Sci. 10:212 (1970); Chaplin and Burk, Crop Sci. 75:133-136 (1983)). LA BU21 leaves also exhibit traits such as higher polyamine content, higher chlorophyll content, and more mesophyll cells per unit leaf area.

[0069] Unless otherwise specified, measurements of alkaloid, polyamine, or nicotine levels (or other leaf chemistry or property characterization) or leaf grade index values ​​described herein for a tobacco plant, variety, cultivar, or strain refer to average measurements, including, for example, the average of multiple leaves from a single representative plant or the average measurement from a representative population of tobacco plants from a single variety, cultivar, or strain. Unless otherwise specified, nicotine, alkaloid, or polyamine levels (or other leaf chemistry or property characterization) of tobacco plants described herein are measured two weeks after topping in pooled leaf samples collected from leaf numbers 3, 4, and 5 after topping. In another embodiment, nicotine, alkaloid, or polyamine levels (or other leaf chemistry or property characterization) of tobacco plants are measured after topping in the leaves with the highest levels of nicotine, alkaloid, or polyamine (or other leaf chemistry or property characterization). In one embodiment, nicotine, alkaloid, or polyamine levels in a tobacco plant are measured at leaf numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 after topping. In another embodiment, nicotine, alkaloid, or polyamine levels (or another leaf chemistry or property characterization) of tobacco plants are measured after topping in pools of two or more leaves having consecutive leaf numbers selected from the group consisting of leaf numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. In another embodiment, nicotine, alkaloid, or polyamine levels (or another leaf chemistry or property characterization) of tobacco plants are measured after topping in leaves having leaf numbers selected from the group consisting of 1-5, 6-10, 11-15, 16-20, 21-25, and 26-30.In another embodiment, nicotine, alkaloid, or polyamine levels (or another leaf chemistry or property characterization) of tobacco plants are measured after topping in pools of two or more leaves having leaf numbers selected from the group consisting of 1-5, 6-10, 11-15, 16-20, 21-25, and 26-30. In another embodiment, nicotine, alkaloid, or polyamine levels (or another leaf chemistry or property characterization) of tobacco plants are measured after topping in pools of three or more leaves having leaf numbers selected from the group consisting of 1-5, 6-10, 11-15, 16-20, 21-25, and 26-30.

[0070] As used herein, leaf numbering is based on the leaf's position on the tobacco stem, with leaf number 1 being the youngest leaf (top) after topping and the highest leaf number being assigned to the oldest leaf (bottom).

[0071] The population of tobacco plants or collection of tobacco leaves for determining an average measurement (e.g., alkaloid or nicotine level or leaf grading) can be of any size, e.g., 5, 10, 15, 20, 25, 30, 35, 40, or 50. The average measurement or grad index value is determined according to standard industry-accepted protocols.

[0072] As used herein, "topping" refers to the removal of the top of the stem, including the SAM, flowers, and some adjacent leaves, when the tobacco plant is nearing vegetative maturity and at the beginning of reproductive growth. Typically, tobacco plants are topped at the bud stage (just after the flowers begin to appear). For example, greenhouse or field-grown tobacco plants can be topped when 50% of the plants have at least one open flower. Topping tobacco plants results in the loss of apical dominance and also induces increased alkaloid production.

[0073] Typically, the nicotine, alkaloid, or polyamine levels (or another leaf chemistry or property characterization) of the tobacco plant are measured about 2 weeks after topping. Other time points may also be used. In one embodiment, the nicotine, alkaloid, or polyamine levels (or another leaf chemistry or property characterization) of the tobacco plant are measured about 1, 2, 3, 4, or 5 weeks after topping. In another embodiment, the nicotine, alkaloid, or polyamine levels (or another leaf chemistry or property characterization) of the tobacco plant are measured about 3, 5, 7, 10, 12, 14, 17, 19, or 21 days after topping.

[0074] As used herein, "similar growing conditions" or "equivalent growing conditions" refers to similar environmental conditions and / or agronomic practices for growing and making meaningful comparisons between two or more plant genotypes, where neither the environmental conditions nor the agronomic practices contribute to and 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, sowing, clipping, undercutting, transplanting, pinching, and sucking. See Chapters 4B and 4C in Tobacco, Production, Chemistry and Technology, edited by Davis & Nielsen, Blackwell Publishing, Oxford (1999), pp. 70-103.

[0075] Alkaloids are complex nitrogen-containing compounds naturally occurring in plants that have pharmacological effects in humans and animals. Nicotine is the major natural alkaloid in commercial cigarette tobacco, accounting for approximately 90 percent of the alkaloid content in Nicotiana tabacum. Other major alkaloids in tobacco include cotinine, nornicotine, myosmine, nicotyrine, anabasine, and anatabine. Minor tobacco alkaloids include nicotine-n-oxide, N-methylanatabine, N-methylanabasine, pseudooxynicotine, 2,3-dipyridyl, and others.

[0076] In one embodiment, the tobacco plants provided herein comprise a lower level of total or individual alkaloids compared to a control tobacco plant lacking both the nic1b_erf mutation and the Nic1b_ERF-specific transgene when grown under similar growing conditions. In another embodiment, the tobacco plants provided herein comprise a lower level of one or more alkaloids selected from the group consisting of cotinine, nornicotine, myosmine, nicotyrine, anabasine, and anatabine compared to a control tobacco plant when grown under similar growing conditions. In one embodiment, a lower alkaloid or nicotine level refers to an alkaloid or nicotine level that is less than 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of the alkaloid or nicotine level of the control tobacco plant. In another embodiment, lower alkaloid or nicotine levels refer to alkaloid or nicotine levels of about 0.5% to 1%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 11% to 12%, 12% to 13%, 13% to 14%, 14% to 15%, 15% to 16%, 16% to 17%, 17% to 18%, 18% to 19%, 19% to 20%, 21% to 22%, 22% to 23%, 23% to 24%, 24% to 25%, 25% to 26%, 26% to 27%, 27% to 28%, 28% to 29%, or 29% to 30% of the alkaloid or nicotine levels of a control tobacco plant. In further embodiments, lower alkaloid or nicotine levels refer to alkaloid or nicotine levels that are about 0.5% to 5%, 5% to 10%, 10% to 20%, 20% to 30% of the alkaloid or nicotine levels of a control tobacco plant.

[0077] Alkaloid levels can be assayed by methods known in the art, such as gas-liquid chromatography, high-performance liquid chromatography, radioimmunoassay, and enzyme-linked immunosorbent assay-based quantification. For example, nicotine alkaloid levels can be measured by a GC-FID method based on CORESTA Recommended Method No. 7, 1987 and ISO standards (ISO TC 126N 394 E.; see also Hibi et al., Plant Physiology 100:826-35 (1992)) for methods using gas-liquid chromatography with a capillary column and an FID detector. Unless otherwise specified, all alkaloid levels described herein are measured using CORESTA Method No. 62, Determination of Nicotine in Tobacco and Tobacco Products by Gas Chromatographic Analysis, February 2005, and the methods defined in the Centers for Disease Control and Prevention's Protocol for Analysis of Nicotine, Total Moisture and pH in Smokeless Tobacco Products, published in Federal Register Vol. 64, No. 55, March 23, 1999 (and amended in Vol. 74, No. 4, January 7, 2009).

[0078] Alternatively, total alkaloids in tobacco can be measured using a segmented flow colorimetric method developed for the analysis of tobacco samples, as employed by Skalar Instrument Co. (West Chester, PA), and described by Collins et al., Tobacco Science 13:79-81 (1969). Briefly, tobacco samples are dried, crushed, and extracted prior to analysis of total alkaloids and reducing sugars. The method then uses acetic acid / methanol / water extraction and charcoal for decolorization. Total alkaloid determination is based on the reaction of cyanogen chloride with nicotine alkaloids in the presence of aromatic amines to form colored complexes, which are measured at 460 nm. Unless otherwise specified, total alkaloid or nicotine levels presented herein are based on dry weight (e.g., percent total alkaloids or percent nicotine).

[0079] In one embodiment, the tobacco plants provided herein comprise lower levels of nicotine compared to control tobacco plants lacking both the nic1b_erf mutation and the Nic1b_ERF-specific transgene when grown under similar growth conditions. In one embodiment, lower nicotine levels refer to average nicotine levels that are less than 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of the average nicotine levels of the control tobacco plants. In another embodiment, lower nicotine levels refer to average nicotine levels of about 0.5% to 1%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 11% to 12%, 12% to 13%, 13% to 14%, 14% to 15%, 15% to 16%, 16% to 17%, 17% to 18%, 18% to 19%, 19% to 20%, 21% to 22%, 22% to 23%, 23% to 24%, 24% to 25%, 25% to 26%, 26% to 27%, 27% to 28%, 28% to 29%, or 29% to 30% of the average nicotine levels of control tobacco plants. In further embodiments, lower nicotine levels refer to average nicotine levels that are about 0.5% to 5%, 5% to 10%, 10% to 20%, 20% to 30% of the average nicotine levels of control tobacco plants.

[0080] In one aspect, the tobacco plants provided herein have about 0.01%, 0.02%, 0.05%, 0.75%, 0.1%, 0.15%, 0.2%, 0.3%, 0.35%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.1% or more of a soluble fiber content of 100% or more of a soluble fiber content of 100% or more of a soluble fiber content of 100% or more of a soluble fiber content of %, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 5%, 6%, 7%, 8%, and 9%. In another aspect, the tobacco plants provided herein have a dry weight content of about 0.01% to 0.02%, 0.02% to 0.05%, 0.05% to 0.75%, 0.75% to 0.1%, 0.1% to 0.15%, 0.15% to 0.2%, 0.2% to 0.3%, 0.3% to 0.35%, 0.35% to 0.4%, 0.4% to 0.5%, 0.5% to 0.6%, 0.6% to 0.7%, 0.7% to 0.8%, 0.8% to 0.9%, 0.9% to 1%, 1% to 1.1%, 1.1% to 1.2%, 1.2% to 1.3%, 1.3% to 1.4, 1.4% to 1.5%, 1.5% to 1.6%, 1.6% to 1.7%, 1.7% to 1.8%, 1.8% to 1.9%, 1.9% to 2.0%, 2.0% to 2.1%, 2.0% to 2.2%, 2.1% to 2.3%, 2.2% to 2.4%, 2.3% to 2.5%, 2.4% to 2.5%, 2.5% to 2.6%, 2.6% to 2.7%, 2.7% to 2.8%, 2.8% to 2.9%, 2.9% to 3.0%, 2.0% to 3.1%, 2.1% to 3.2%, 2.2% to 3.3%, 2.3% to 3.4%, 2.4% to 3.5%, 2.5% to 3.6%, 2.5% to 3.7%, 2.6% to 3.8%, 2.7 and an average nicotine or total alkaloid level selected from the group consisting of 5%, 1.5% to 1.6%, 1.6% to 1.7%, 1.7% to 1.8%, 1.8% to 1.9%, 1.9% to 2%, 2% to 2.1%, 2.1% to 2.2%, 2.2% to 2.3%, 2.3% to 2.4%, 2.4% to 2.5%, 2.5% to 2.6%, 2.6% to 2.7%, 2.7% to 2.8%, 2.8% to 2.9%, 2.9% to 3%, 3% to 3.1%, 3.1% to 3.2%, 3.2% to 3.3%, 3.3% to 3.4%, 3.4% to 3.5%, and 3.5% to 3.6%. In further aspects, the tobacco plants provided herein comprise an average nicotine or total alkaloid level selected from the group consisting of about 0.01% to 0.1%, 0.02% to 0.2%, 0.03% to 0.3%, 0.04% to 0.4%, 0.05% to 0.5%, 0.75% to 1%, 0.1% to 1.5%, 0.15% to 2%, 0.2% to 3%, and 0.3% to 3.5% on a dry weight basis.

[0081] The present disclosure also provides tobacco plants with altered nicotine levels without negatively impacting other tobacco traits, such as the leaf grade index value. In one aspect, low-nicotine or nicotine-free tobacco varieties provide commercially acceptable grades of cured tobacco. Tobacco grade is assessed based on factors including, but not limited to, leaf position on the stem, leaf size, leaf color, leaf uniformity and integrity, maturity, feel, elasticity, gloss (relating to the intensity and depth of leaf coloration as well as brilliance), hygroscopicity (the tobacco leaf's ability to absorb and retain ambient moisture), and green color characteristics or hue. Leaf grade can be determined, for example, using the Official Standard Grade (7 U.S.C. § 511) published by the Agricultural Marketing Service of the U.S. Department of Agriculture.For example, the Official Standard Grades for Burley Tobacco (US Type 31 and Foreign Type 93) were effective November 5, 1990 (55 FR 40645); the Official Standard Grades for Flue-Cured Tobacco (US Types 11, 12, 13, 14, and Foreign Type 92) were effective March 27, 1989 (54 FR 7925); the Official Standard Grades for Pennsylvania Seedleaf Tobacco (US Type 41) were effective January 8, 1965 (29 FR 16854); the Official Standard Grades for Ohio Cigar-Leaf Tobacco (US Types 42, 43, and 44) ​​were effective December 8, 1963 (28 FR 11719 and 28 FR 11926); and the Official Standard Grades for Ohio Cigar-Leaf Tobacco (US Types 44, 45, and 46) were effective November 20, 1969 (34 See Official Standard Grades for Wisconsin Cigar-Binder Tobacco (US Types 54 and 55), effective November 20, 1969 (34 FR 17061); Official Standard Grades for Wisconsin Cigar-Binder Tobacco (US Types 54 and 55), effective November 20, 1969 (34 FR 17061); and Official Standard Grades for Georgia and Florida Shade-Grown Cigar-Wrapper Tobacco (US Type 62), effective April 1971. USDA Grade Index values ​​may be determined according to industry-recognized Grade Indexes.See, e.g., Bowman et al., Tobacco Science, 32:39-40 (1988); Legacy Tobacco Document Library (Bates Document #523267826-523267833, July 1, 1988, Memorandum on the Proposed Burley Tobacco Grade Index); and Miller et al., 1990, Tobacco Intern., 192:55-57 (all of the above references are incorporated by reference in their entirety). Unless otherwise specified, the USDA Grade Index for any plant described herein is a numerical representation of the received federal grade, from 0 to 100, weighted average of all stem locations. A higher Grade Index indicates higher quality. Alternatively, leaf grade can be determined via hyperspectral imaging. See, e.g., WO2011 / 027315 (published March 10, 2011; incorporated by reference in its entirety).

[0082] In one aspect, the tobacco plants provided herein comprise similar levels of one or more tobacco aroma compounds selected from the group consisting of 3-methylvaleric acid, valeric acid, isovaleric acid, labdanoids, cembranoids, sugar esters, and reducing sugars compared to a control tobacco plant when grown under similar growth conditions. In another aspect, the tobacco plants provided herein comprise a nic1b_erf mutation, a nic2 mutation, or a combination thereof that does not affect the levels of one or more tobacco aroma compounds selected from the group consisting of 3-methylvaleric acid, valeric acid, isovaleric acid, labdanoids, cembranoids, sugar esters, and reducing sugars.

[0083] As used herein, tobacco aroma compounds are compounds that are associated with the flavor and aroma of tobacco smoke.These compounds include but are not limited to 3-methylvaleric acid, valeric acid, isovaleric acid, cembranoid and labdanoid diterpenes, and sugar esters.The concentration of tobacco aroma compounds can be measured by any known metabolite profiling method in the art, including but not limited to gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance spectroscopy, and liquid chromatography-mass spectrometry.See The Handbook of Plant Metabolomics, edited by Weckwerth and Kahl (Wiley-Blackwell) (May 28, 2013).

[0084] As used herein, "reducing sugars" are any sugars (monosaccharides or polysaccharides) that contain free or potentially free aldehyde or ketone groups. Glucose and fructose act as nicotine buffers in cigarette smoke by reducing the pH of the smoke and effectively reducing the amount of "free" unprotonated nicotine. Reducing sugars balance the flavor of the smoke, for example, by modifying the sensory impact of nicotine and other tobacco alkaloids. An inverse relationship between sugar content and alkaloid content has been reported across tobacco varieties, within the same variety, and within the same plant strain, driven by growing conditions. Reducing sugar levels can be measured using a segmented flow colorimetric method developed for the analysis of tobacco samples, as employed by Skalar Instrument Co. (West Chester, PA), and described by Davis, Tobacco Science 20:139-144 (1976). For example, the sample is dialyzed against a sodium carbonate solution. Copper neocuproine is added to the sample and the solution is heated. The copper neocuproine chelate is reduced in the presence of sugar, resulting in a colored complex that is measured at 460 nm.

[0085] In one embodiment, the tobacco plants provided herein contain one or more non-naturally occurring mutant alleles at the Nic1b_ERF or Nic2 locus that reduce or eliminate one or more gene activities from the Nic1b_ERF or Nic2 locus. In one embodiment, these mutant alleles result in lower nicotine levels. Mutant Nic1b_ERF or Nic2 alleles can be introduced by any method known in the art, including random or targeted mutagenesis approaches.

[0086] Such mutagenesis methods include, but are not limited to, seed treatment with ethyl methyl sulfate (EMS) (Hildering and Verkerk, The use of induced mutations in plant breeding. Pergamon press, pp 317-320, 1965) or UV irradiation, X-rays, and fast neutron irradiation (see, e.g., Verkerk, Neth. J. Agric. Sci. 19:197-203, 1971; and Poehlman, Breeding Field Crops, Van Nostrand Reinhold, New York (3rd ed), 1987), transposon tagging (Fedoroff et al., 1984; U.S. Pat. Nos. 4,732,856 and 5,013,658), as well as T-DNA insertion methodologies (Hoekema et al., 1983; U.S. Pat. No. 5,149,645). EMS-induced mutagenesis involves chemically inducing random point mutations throughout the genome. Rapid neutron mutagenesis involves exposing seeds to neutron bombardment, which causes large deletions through double-stranded DNA breaks. Transposon tagging involves inserting a transposon into an endogenous gene to reduce or eliminate gene expression. Types of mutations that can occur in tobacco genes include, for example, point mutations, deletions, insertions, duplications, and inversions. Such mutations are preferably present in the coding region of a tobacco gene, but mutations in the promoter region, introns, or untranslated regions of a tobacco gene may also be desirable.

[0087] In addition, TILLING (Targeting Induced Local Lesions In Genomes), a rapid and automatable method of screening for chemically induced mutations using denaturing HPLC or selective endonuclease digestion of selected PCR products, is also applicable to the present disclosure. See McCallum et al. (2000) Nat. Biotechnol. 18:455-457. Mutations that affect gene expression or interfere with gene function can be determined using methods well known in the art. Insertional mutations in gene exons usually result in null mutants. Mutations in conserved residues can be particularly effective in inhibiting protein function. In one embodiment, the tobacco plant contains nonsense (e.g., stop codon) mutations in one or more of the NCG genes described herein.

[0088] In one aspect, the present disclosure also provides tobacco strains with altered nicotine levels while maintaining commercially acceptable leaf quality. These strains can be produced by introducing mutations into one or more genes at the Nic1b_ERF or Nic2 locus via precise genome engineering techniques, such as transcription activator-like effector nucleases (TALENs), meganucleases, zinc finger nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 systems, CRISPR / Cpf1 systems, CRISPR / Csm1 systems, and combinations thereof (see, e.g., U.S. Patent Application Publication No. 2017 / 0233756). See, e.g., Gaj et al., Trends in Biotechnology, 31(7):397-405 (2013).

[0089] Screening and selection of mutagenized tobacco plants can be performed by any methodology known to those skilled in the art. Examples of screening and selection methodologies include, but are not limited to, Southern analysis, PCR amplification for detecting polynucleotides, Northern blot, ribonuclease protection, primer extension, RT-PCR amplification for detecting RNA transcripts, Sanger sequencing, next-generation sequencing technology (e.g., Illumina, PacBio, Ion Torrent, 454), enzyme assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides, and protein gel electrophoresis, Western blot, immunoprecipitation, and enzyme-linked immunoassay for detecting polypeptides. Other techniques, such as in situ hybridization, enzyme staining, and immunostaining, can also be used to detect the presence or expression of polypeptides and / or polynucleotides. Methods for performing all of the techniques referenced are known.

[0090] In one aspect, a tobacco plant or plant genome provided herein is mutagenized or edited with a nuclease selected from the group consisting of a meganuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a CRISPR / Cas9 nuclease, a CRISPR / Cpf1 nuclease, or a CRISPR / Csm1 nuclease.

[0091] As used herein, "editing" or "genome editing" refers to the targeted mutagenesis of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides of an endogenous plant genomic nucleic acid sequence, or the removal or replacement of an endogenous plant genomic nucleic acid sequence. In one embodiment, the edited nucleic acid sequence provided has at least 99.9%, at least 99.5%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80%, or at least 75% sequence identity to the endogenous nucleic acid sequence. In one aspect, the edited nucleic acid sequences provided have at least 99.9%, at least 99.5%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80%, or at least 75% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, 202-205, and 208-221, and fragments thereof. In another embodiment, the edited nucleic acid sequences provided have at least 99.9%, at least 99.5%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80%, or at least 75% sequence identity to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, 202-205, and 208-221.

[0092] Meganucleases, ZFNs, TALENs, CRISPR / Cas9, CRISPR / Csm1, and CRISPR / Cpf1 induce double-stranded DNA breaks at target sites in genome sequences, which are then repaired by the natural process of homologous recombination (HR) or non-homologous end joining (NHEJ). Sequence modifications then occur at the cut site, which can include deletions or insertions resulting in gene disruption in the case of NHEJ, or the integration of donor nucleic acid sequences by HR. In one embodiment, a provided method includes editing a plant genome using a provided nuclease to mutate at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more nucleotides in the plant genome through HR using a donor polynucleotide. In one embodiment, the provided mutations are caused by genome editing using a nuclease. In another embodiment, the provided mutations are caused by non-homologous end joining or homologous recombination.

[0093] In one aspect, the mutations provided herein provide dominant mutants that activate the expression or activity of a gene of interest, e.g., a gene selected from the group consisting of a biosynthetic enzyme, a regulatory transcription factor, a transporter, a catabolic enzyme, or a combination thereof, for one or more antioxidants.

[0094] Meganucleases, typically identified in microorganisms, are unique enzymes with high activity and long recognition sequences (>14 bp) that result in site-specific digestion of target DNA. Engineered versions of naturally occurring meganucleases typically have extended DNA recognition sequences (e.g., 14-40 bp). Engineering meganucleases can be more challenging than engineering ZFNs and TALENs because the DNA recognition and cleavage functions of meganucleases are woven into a single domain. Specialized methods of mutagenesis and high-throughput screening have been used to generate novel meganuclease variants that recognize unique sequences and have improved nuclease activity.

[0095] ZFNs are synthetic proteins consisting of an engineered zinc finger DNA-binding domain fused to the cleavage domain of the FokI restriction endonuclease. ZFNs can be designed to cleave stretches of double-stranded DNA of almost any length due to the modification of the zinc finger DNA-binding domain. ZFNs form dimers from monomers composed of the nonspecific DNA cleavage domain of the FokI endonuclease fused to an engineered zinc finger array that binds to the target DNA sequence.

[0096] The DNA-binding domain of a ZFN typically consists of an array of three to four zinc fingers. The amino acids at positions -1, +2, +3, and +6 relative to the start of the zinc finger ∞-helix, which contribute to site-specific binding to the target DNA, can be modified and customized to accommodate specific target sequences. Other amino acids form a consensus backbone to generate ZFNs with different sequence specificities. Rules for selecting target sequences for ZFNs are known in the art.

[0097] The FokI nuclease domain requires dimerization to cleave DNA, so two ZFNs with C-terminal regions are required to bind to opposite DNA strands (separated by 5-7 bp) at the cleavage site. If the two ZF-binding sites are palindromic, the ZFN monomers can cleave the target site. The term ZFN, as used herein, is broad and includes monomeric ZFNs that can cleave double-stranded DNA without assistance from another ZFN. The term ZFN is also used to refer to one or both members of a pair of ZFNs engineered to work together to cleave DNA at the same site.

[0098] Without being limited by any scientific theory, because the DNA binding specificity of zinc finger domains can in principle be re-engineered using one of a variety of methods, customized ZFNs can theoretically be constructed to target almost any gene sequence. Published methods for engineering zinc finger domains include Context-dependent Assembly (CoDA), Oligomerized Pool Engineering (OPEN), and Modular Assembly.

[0099] TALEN is an artificial restriction enzyme that is generated by fusing transcription activator-like effector (TALE) DNA binding domain with FokI nuclease domain.When each member of a TALEN pair binds to the DNA site adjacent to the target site, the FokI monomer dimerizes and causes double-stranded DNA break at the target site.The term TALEN as used herein has a broad meaning, and includes monomeric TALEN that can cut double-stranded DNA without the assistance of another TALEN.The term TALEN is also used to refer to one or both members of a pair of TALENs that work together to cut DNA at the same site.

[0100] Transcription activator-like effectors (TALEs) can be engineered to bind virtually any DNA sequence. TALE proteins are DNA-binding domains derived from various plant bacterial pathogens in the genus Xanthomonas. Xanthomonas pathogens secrete TALEs into host plant cells during infection. TALEs translocate to the nucleus, where they recognize and bind to specific DNA sequences in the promoter regions of specific genes in the host genome. TALEs have a central DNA-binding domain composed of 13 to 28 repeat monomers of 33 to 34 amino acids. The amino acids in each monomer are highly conserved except for hypervariable amino acid residues at positions 12 and 13. The two variable amino acids are called repeat variable dinucleotides (RVDs). The amino acid pairs NI, NG, HD, and NN of the RVD preferentially recognize adenine, thymine, cytosine, and guanine / adenine, respectively, and modulation of the RVD allows recognition of consecutive DNA bases. This simple relationship between amino acid sequence and DNA recognition allowed the engineering of specific DNA-binding domains by selecting combinations of repeat segments containing appropriate RVDs.

[0101] In addition to wild-type FokI cleavage domains, mutant FokI cleavage domains with mutations have been designed to improve cleavage specificity and activity.FokI domains function as dimers and require two constructs with specific DNA binding domains for the target genome site with appropriate orientation and spatial arrangement.The number of amino acid residues between the TALEN DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites are both parameters for achieving high levels of activity.

[0102] The relationship between the amino acid sequence of the TALE binding domain and DNA recognition allows for designable proteins. TALE constructs can be designed using software programs such as DNA Works. Other methods for designing TALE constructs are known to those skilled in the art. See Doyle et al., Nucleic Acids Research (2012) 40:W117-122.; Cermak et al., Nucleic Acids Research (2011). 39:e82; and tale-nt.cac.cornell.edu / about.

[0103] The CRISPR / Cas9 system, CRISPR / Csm1, or CRISPR / Cpf1 system is an alternative to the FokI-based methods of ZFN and TALEN. The CRISPR system is based on an RNA-guided engineered nuclease that uses complementary base pairing to recognize DNA sequences at target sites.

[0104] The CRISPR / Cas9, CRISPR / Csm1, and CRISPR / Cpf1 systems are part of the adaptive immune system of bacteria and archaea, protecting them from invading nucleic acids such as viruses by cleaving foreign DNA in a sequence-dependent manner. Immunity is achieved through the integration of a short segment of invasive DNA, known as a spacer, between two adjacent repeats at the proximal end of the CRISPR locus. During subsequent encounters with the invasive DNA, the spacer-containing CRISPR array is transcribed and processed into approximately 40-nt long small interfering CRISPR RNA (crRNA), which, in combination with trans-activating CRISPR RNA (tracrRNA), activates and guides the Cas9 nuclease, which cleaves homologous double-stranded DNA sequences, known as protospacers, in the invasive DNA. A prerequisite for cleavage is the presence of a conserved protospacer-adjacent motif (PAM) downstream of the target DNA, which typically has the sequence 5-NGG-3, or less frequently NAG. Specificity is provided by a so-called "seed sequence" approximately 12 bases upstream of the PAM, which must match between the RNA and the target DNA. Cpf1 and Csm1 act in a manner similar to Cas9, but Cpf1 and Csm1 require tracrRNA.

[0105] In yet another embodiment, the provided tobacco plants further comprise one or more mutations in one or more genetic loci encoding nicotine demethylases (e.g., CYP82E4, CYP82E5, CYP82E10) that confer reduced amounts of nornicotine compared to control plants lacking one or more mutations in one or more genetic loci encoding nicotine demethylases (see U.S. Patent Nos. 8,319,011, 8,124,851, 9,187,759, 9,228,194, 9,228,195, and 9,247,706). In one embodiment, the modified tobacco plants described further comprise reduced nicotine demethylase activity compared to control plants when grown and cured under equivalent conditions.

[0106] The present disclosure also provides compositions and methods for inhibiting the expression or function of one or more polypeptides from the Nic1b_ERF locus in plants, particularly plants of the genus Nicotiana, including various commercial varieties of tobacco plants.

[0107] In one aspect, the disclosure provides a tobacco plant or portion thereof, comprising a heterologous expression cassette comprising a Nic1b_ERF inhibitory sequence of a gene comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, and 202-205, and fragments thereof, wherein the inhibitory sequence is operably linked to a promoter functional in a plant cell, and the inhibitory sequence is selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, and 202-205, and fragments thereof. 5, and fragments thereof. , 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotide fragments of a tobacco plant or portion thereof.In another aspect, the disclosure provides a tobacco plant or portion thereof, comprising a heterologous expression cassette comprising a Nic1b_ERF inhibitory sequence of a gene comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, and 202-205, and fragments thereof, wherein the inhibitory sequence is operably linked to a promoter functional in a plant cell, and the inhibitory sequence is selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, and 202-205, and fragments thereof. At least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of: 05, and fragments thereof. , 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotide fragments of a tobacco plant or portion thereof. In one embodiment, the Nic1b_ERF inhibitory sequence is capable of being transcribed as an inhibitory polynucleotide selected from the group consisting of single-stranded RNA polynucleotides, double-stranded RNA polynucleotides, and combinations thereof. In one embodiment, the Nic1b_ERF inhibitory sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 108-119.

[0108] As used herein, the terms "inhibit," "inhibition," and "inhibiting" are defined as any method known in the art or described herein that reduces the expression or function of a gene product of interest (e.g., a target gene product). "Inhibition" can be in the context of a comparison between two plants, e.g., a genetically altered plant and a wild-type plant. Alternatively, inhibition of target gene product expression or function can be in the context of a comparison between plant cells, organelles, organs, tissues, or plant parts within the same plant or between different plants, including comparisons between developmental or temporal stages within the same plant or plant part, or between plants or plant parts. "Inhibition" includes any relative decrease in the function or production of a gene product of interest, up to and including complete elimination of the function or production of the gene product. The term "inhibition" encompasses any method or composition that downregulates the translation and / or transcription of a target gene product or the functional activity of a target gene product. In one embodiment, the mRNA or protein level of one or more genes from the Nic1b locus in the modified plant is less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the mRNA or protein level of the same genes in a plant that is not mutant or has not been genetically modified to inhibit expression of those genes.

[0109] The term "inhibitory sequence" encompasses any polynucleotide or polypeptide sequence capable of inhibiting the expression or function of a gene involved in regulating nicotine biosynthesis from the Nic1b_ERF locus in a plant, such as a full-length polynucleotide or polypeptide sequence, a truncated polynucleotide or polypeptide sequence, a fragment of a polynucleotide or polypeptide sequence, a variant of a polynucleotide or polypeptide sequence, a nucleotide sequence in a sense orientation, a nucleotide sequence in an antisense orientation, a complement of a nucleotide sequence in a sense or antisense orientation, an inverted region of a nucleotide sequence, a hairpin of a nucleotide sequence, a double-stranded nucleotide sequence, a single-stranded nucleotide sequence, and combinations thereof. The term "polynucleotide sequence" includes sequences such as RNA, DNA, chemically modified nucleic acids, nucleic acid analogs, and combinations thereof.

[0110] Inhibitory sequences are designated by the name of the target gene product. Thus, a "Nic1b_ERF inhibitory sequence" refers to an inhibitory sequence that can inhibit the expression of a gene involved in nicotine biosynthesis regulation from the Nic1b_ERF locus in a plant, or inhibit the function of the gene product, for example, at the transcription and / or translation level. When the phrase "capable of inhibiting" is used in the context of a polynucleotide inhibitory sequence, it means that the inhibitory sequence itself exerts an inhibitory effect; or, if the inhibitory sequence encodes an inhibitory nucleotide molecule (e.g., a hairpin RNA, miRNA, or double-stranded RNA polynucleotide) or encodes an inhibitory polypeptide (e.g., a polypeptide that inhibits the expression or function of a target gene product), after its transcription (e.g., in the case of an inhibitory sequence encoding a hairpin RNA, miRNA, or double-stranded RNA polynucleotide) or its transcription and translation (e.g., in the case of an inhibitory sequence encoding an inhibitory polypeptide), the transcribed or translated product exerts an inhibitory effect on the target gene product (e.g., inhibits the expression or function of the target gene product), respectively.

[0111] The disclosed Nic1b_ERF inhibitory sequences can induce gene silencing via any silencing pathway or mechanism known in the art, including, but not limited to, sense suppression / co-suppression, antisense suppression, double-stranded RNA (dsRNA) interference, hairpin RNA interference and intron-containing hairpin RNA interference, amplicon-mediated interference, ribozymes, small interfering RNA, artificial or synthetic microRNA, and artificial trans-acting siRNA. Depending on the desired outcome, the Nic1b_ERF inhibitory sequence can range from at least about 20, 50, 70, 100, 150, 200, 250, 300, 350, or 400 nucleotides to the full-length polynucleotide encoding the disclosed protein. In one embodiment, the Nic1b_ERF inhibitory sequence can be a fragment having a length of about 50 to about 400 nucleotides, about 70 to about 350 nucleotides, about 90 to about 325 nucleotides, about 90 to about 300 nucleotides, about 90 to about 275 nucleotides, about 100 to about 400 nucleotides, about 100 to about 350 nucleotides, about 100 to about 325 nucleotides, about 100 to about 300 nucleotides, about 125 to about 300 nucleotides, or about 125 to about 275 nucleotides. In some embodiments, a fragment of a cytochrome P450 polynucleotide is about 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 400 nucleotides in length, and other such values ​​between about 70 and 400 nucleotides. In one aspect, a Nic1b_ERF inhibitory sequence may comprise about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0112] The use of the term "polynucleotide" is not intended to limit the present disclosure to polynucleotides made up of DNA. Those skilled in the art will recognize that polynucleotides can include ribonucleotides and combinations of ribonucleotides and deoxyribonucleosides. Such deoxyribonucleosides and ribonucleotides include both naturally occurring molecules and synthetic analogs. The polynucleotides of the present disclosure also encompass all forms of sequences, including, but not limited to, single-stranded forms, double-stranded forms, hairpins, and stem-and-loop structures.

[0113] In one aspect, the present disclosure provides a recombinant DNA construct comprising a promoter operably linked to a polynucleotide encoding an RNA molecule capable of binding to RNA encoding a polypeptide having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206, and 207, and 222-228, and fragments thereof, wherein the RNA molecule represses expression of the polypeptide. In one aspect, the RNA molecule is selected from the group consisting of microRNA, siRNA, and trans-acting siRNA. In another aspect, the recombinant DNA construct encodes a double-stranded RNA. Also provided are transgenic tobacco plants or parts thereof, cured tobacco materials, or tobacco products comprising these recombinant DNA constructs. In one aspect, these transgenic plants, cured tobacco materials, or tobacco products comprise lower levels of nicotine compared to control tobacco plants lacking the recombinant DNA construct. Further provided are methods for reducing nicotine levels in tobacco plants, comprising transforming tobacco plants with any of these recombinant DNA constructs.

[0114] As used herein, "operably linked" refers to a functional link between two or more elements. For example, an operably linked polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional link that allows expression of the polynucleotide of interest. Operably linked elements may be contiguous or non-contiguous.

[0115] As used herein and in reference to a sequence, "heterologous" refers to a sequence that originates from a foreign species or, if from the same species, has been substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. The term is also applicable to nucleic acid constructs, sometimes referred to herein as "polynucleotide constructs" or "nucleotide constructs." In this manner, a "heterologous" nucleic acid construct is intended to mean a construct that originates from a foreign species or, if from the same species, has been substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. Heterologous nucleic acid constructs include, but are not limited to, recombinant nucleotide constructs introduced into a plant or plant part thereof, for example, via transformation methods or subsequent breeding of a transgenic plant with another plant of interest. In one embodiment, the promoter used is heterologous to the sequence driven by the promoter. In another embodiment, the promoter used is heterologous to tobacco. In a further embodiment, the promoter used is native to tobacco.

[0116] In one embodiment, the modified tobacco plants described are cisgenic plants. As used herein, "cisgenesis" or "cisgenic" refers to the genetic modification of a plant, plant cell, or plant genome in which all components (e.g., promoter, donor nucleic acid, selection gene) originate exclusively from the plant (i.e., no components of non-plant origin are used). In one embodiment, the modified plants, plant cells, or plant genomes provided are cisgenic. The cisgenic plants, plant cells, and plant genomes provided can lead to ready-to-use tobacco lines. In another embodiment, the modified tobacco plants provided are free of any non-tobacco genetic material or sequences.

[0117] As used herein, "gene expression" refers to the biosynthesis or production of a gene product, including transcription and / or translation of the gene product.

[0118] Also provided herein are compositions and methods for overexpressing one or more polypeptides from the Nic1b_ERF locus in plants, particularly plants of the genus Nicotiana, including various commercial varieties of tobacco plants.

[0119] In one aspect, the present disclosure provides a recombinant DNA construct comprising a promoter functional in a tobacco cell and operably linked to a polynucleotide encoding a polypeptide having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206, and 207, and 222-228, and fragments thereof. Transgenic tobacco plants or parts thereof, cured tobacco materials, or tobacco products comprising these recombinant DNA constructs are also provided. In one aspect, these transgenic plants, cured tobacco materials, or tobacco products contain increased levels of nicotine compared to control tobacco plants not harboring the recombinant DNA construct. Further provided are methods for increasing nicotine levels in tobacco plants, comprising transforming a tobacco plant with any of these recombinant DNA constructs.

[0120] In one embodiment, for each transgenic or mutant strain carrying a Nic1b_ERF-related transgene or mutation, combinations of such transgenic or mutant strains with mutations or transgene events specific to one or more ERF genes at the Nic2 locus are also provided. Such one or more Nic2 ERF genes include, but are not limited to, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168 (Shoji et al., Plant Cell, (10):3390-409 (2010)). Plants having a combination of transgenic or mutagenic suppression of one or more, two or more, three or more, four or more, five or more, six or more, or seven or more Nic1b ERF-like genes (e.g., NCG1, NCG11, NCG12, NCG15, NCG16, NCG17, ERF101, ERF110, ERF16, ERF130) and one or more, two or more, three or more, four or more, or five or more Nic2 ERF genes (ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168) are of particular interest and provided herein. Plants having a combination of transgenic or mutagenic suppression of one or more, two or more, three or more, or all four Nic1b ERF-like genes (e.g., NCG12, NCG15, NCG16, NCG17, ERF101, ERF110, ERF16, ERF130) and transgenic or mutagenic suppression of ERF189, ERF115, or both are of further particular interest and provided herein.Further provided herein are plants that have the transgenesis or suppression of mutagenicity of one or more, two or more, three or more, or all four Nic1b ERF-like genes (e.g., NCG12, NCG15, NCG16, NCG17, ERF101, ERF110, ERF16, ERF130) in combination with the transgenesis or suppression of mutagenicity of ERF189. In another aspect, provided herein are plants that have the transgenesis or suppression of mutagenicity of one or more, two or more, three or more, or all four Nic1b ERF-like genes (e.g., NCG12, NCG15, NCG16, NCG17, ERF101, ERF110, ERF16, ERF130) in combination with the transgenesis or suppression of mutagenicity of ERF115. Plants having a transgenic or mutagenic suppression combination of one or more, two or more, three or more, or four or more Nic1b ERF-like genes (NCG1, NCG12, NCG15, NCG16, NCG17) and one or more, two or more, three or more, four or more, or five or more Nic2 ERF genes (ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168) are of particular interest and provided herein. Plants having a combination of transgenic or mutagenic suppression of one or more, two or more, three or more, or all four Nic1b ERF-like genes (NCG12, NCG15, NCG16, NCG17) with transgenic or mutagenic suppression of ERF189, ERF115, or both are of further particular interest and provided herein. Further provided herein are plants having a combination of transgenic or mutagenic suppression of one or more, two or more, three or more, or all four Nic1b ERF-like genes (NCG12, NCG15, NCG16, NCG17) with transgenic or mutagenic suppression of ERF189.In another aspect, provided herein are plants having the genetic introduction or suppression of mutagenicity of one or more, two or more, three or more, or all four Nic1b ERF-like genes (NCG12, NCG15, NCG16, NCG17) in combination with the genetic introduction or suppression of mutagenicity of ERF115.

[0121] In a further aspect, provided are tobacco plants having NCG12 introduced or suppressed mutagenicity and one or more, two or more, three or more, four or more, or five or more Nic2 ERF genes (ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168). In another aspect, provided are tobacco plants having NCG15 introduced or suppressed mutagenicity and one or more, two or more, three or more, four or more, or five or more Nic2 ERF genes (ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168). In another aspect, provided are tobacco plants having NCG16 introduced or suppressed mutagenicity and one or more, two or more, three or more, four or more, or five or more Nic2 ERF genes (ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168). In another aspect, provided are tobacco plants having NCG17 introduced or suppressed mutagenicity and one or more, two or more, three or more, four or more, or five or more Nic2 ERF genes (ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168). In another aspect, tobacco plants are provided that have transgenic or mutagenic suppression of ERF101 and transgenic or mutagenic suppression of one or more, two or more, three or more, four or more, or five or more Nic2 ERF genes (ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168).In another aspect, provided are tobacco plants having transgenic or mutagenic suppression of ERF110 and transgenic or mutagenic suppression of one or more, two or more, three or more, four or more, or five or more Nic2 ERF genes (ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168). In another aspect, provided are tobacco plants having transgenic or mutagenic suppression of ERF16 and transgenic or mutagenic suppression of one or more, two or more, three or more, four or more, or five or more Nic2 ERF genes (ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168). In another aspect, provided are tobacco plants having transgenic or mutagenic suppression of ERF130 and transgenic or mutagenic suppression of one or more, two or more, three or more, four or more, or five or more Nic2 ERF genes (ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168).

[0122] In one embodiment, tobacco plants are provided that have one or more Nic1b_ERF genes transgenic or mutagenic suppressed. In one embodiment, such plants contain lower nicotine or total alkaloid levels compared to control animals lacking the transgenic or mutagenic suppression under comparable conditions. In one embodiment, tobacco plants are provided that have one or more Nic1b_ERF genes transgenic or mutagenic suppressed and one or more, two or more, three or more, four or more, or five or more Nic2_ERF genes transgenic or mutagenic suppressed.

[0123] In one embodiment, tobacco plants are provided that have transgenic or mutagenic suppression of two or more Nic1b_ERF genes. In one embodiment, such plants contain lower nicotine or total alkaloid levels compared to control animals lacking transgenic or mutagenic suppression under comparable conditions. In one embodiment, tobacco plants are provided that have transgenic or mutagenic suppression of two or more Nic1b_ERF genes and transgenic or mutagenic suppression of one or more, two or more, three or more, four or more, or five or more Nic2_ERF genes.

[0124] In one embodiment, tobacco plants are provided that have transgenic or mutagenic suppression of three or more Nic1b_ERF genes. In one embodiment, such plants contain lower nicotine or total alkaloid levels compared to control animals lacking transgenic or mutagenic suppression under comparable conditions. In one embodiment, tobacco plants are provided that have transgenic or mutagenic suppression of three or more Nic1b_ERF genes and transgenic or mutagenic suppression of one or more, two or more, three or more, four or more, or five or more Nic2_ERF genes.

[0125] In one embodiment, tobacco plants are provided that have transgenic or mutagenic suppression of four or more Nic1b_ERF genes. In one embodiment, such plants contain lower nicotine or total alkaloid levels compared to control animals lacking transgenic or mutagenic suppression under comparable conditions. In one embodiment, tobacco plants are provided that have transgenic or mutagenic suppression of four or more Nic1b_ERF genes and transgenic or mutagenic suppression of one or more, two or more, three or more, four or more, or five or more Nic2_ERF genes.

[0126] In one embodiment, tobacco plants are provided that have five or more Nic1b_ERF genes introduced or mutagenicity suppressed. In one embodiment, such plants contain lower nicotine or total alkaloid levels compared to control animals lacking the introduced or mutagenicity suppressed under comparable conditions. In one embodiment, tobacco plants are provided that have five or more Nic1b_ERF genes introduced or mutagenicity suppressed and one or more, two or more, three or more, four or more, or five or more Nic2_ERF genes introduced or mutagenicity suppressed.

[0127] In one embodiment, tobacco plants are provided that have six or more Nic1b_ERF genes introduced or mutagenicity suppressed. In one embodiment, such plants contain lower nicotine or total alkaloid levels compared to control animals lacking the introduced or mutagenicity suppressed under comparable conditions. In one embodiment, tobacco plants are provided that have six or more Nic1b_ERF genes introduced or mutagenicity suppressed and one or more, two or more, three or more, four or more, or five or more Nic2_ERF genes introduced or mutagenicity suppressed.

[0128] In one embodiment, tobacco plants are provided that have transgenic or mutagenic suppression of fewer than two Nic1b_ERF genes. In one embodiment, such plants contain lower nicotine or total alkaloid levels compared to control animals lacking transgenic or mutagenic suppression under comparable conditions. In one embodiment, tobacco plants are provided that have transgenic or mutagenic suppression of fewer than two Nic1b_ERF genes and transgenic or mutagenic suppression of fewer than two, three, four, five, six, or seven Nic2_ERF genes.

[0129] In one embodiment, tobacco plants are provided that have transgenic or mutagenic suppression of fewer than three Nic1b_ERF genes. In one embodiment, such plants contain lower nicotine or total alkaloid levels compared to control animals lacking transgenic or mutagenic suppression under comparable conditions. In one embodiment, tobacco plants are provided that have transgenic or mutagenic suppression of fewer than three Nic1b_ERF genes and transgenic or mutagenic suppression of fewer than two, three, four, five, six, or seven Nic2_ERF genes.

[0130] In one embodiment, tobacco plants are provided that have transgenic or mutagenic suppression of fewer than four Nic1b_ERF genes. In one embodiment, such plants contain lower nicotine or total alkaloid levels compared to control animals lacking transgenic or mutagenic suppression under comparable conditions. In one embodiment, tobacco plants are provided that have transgenic or mutagenic suppression of fewer than four Nic1b_ERF genes and transgenic or mutagenic suppression of fewer than two, three, four, five, six, or seven Nic2_ERF genes.

[0131] In one embodiment, tobacco plants are provided that have transgenic or mutagenic suppression of fewer than five Nic1b_ERF genes. In one embodiment, such plants contain lower nicotine or total alkaloid levels compared to control animals lacking transgenic or mutagenic suppression under comparable conditions. In one embodiment, tobacco plants are provided that have transgenic or mutagenic suppression of fewer than five Nic1b_ERF genes and transgenic or mutagenic suppression of fewer than two, three, four, five, six, or seven Nic2_ERF genes.

[0132] In one embodiment, tobacco plants are provided that have transgenic or mutagenic suppression of fewer than six Nic1b_ERF genes. In one embodiment, such plants contain lower nicotine or total alkaloid levels compared to control animals lacking transgenic or mutagenic suppression under comparable conditions. In one embodiment, tobacco plants are provided that have transgenic or mutagenic suppression of fewer than six Nic1b_ERF genes and transgenic or mutagenic suppression of fewer than two, three, four, five, six, or seven Nic2_ERF genes.

[0133] In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of one or more Nic1b_ERF genes. In one embodiment, such plants contain higher nicotine or total alkaloid levels compared to control animals lacking the transgenic or mutagenic overexpression under comparable conditions. In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of one or more Nic1b_ERF genes and transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic2_ERF genes.

[0134] In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of two or more Nic1b_ERF genes. In one embodiment, such plants contain higher nicotine or total alkaloid levels compared to control animals lacking the transgenic or mutagenic overexpression under comparable conditions. In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of two or more Nic1b_ERF genes and transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic2_ERF genes.

[0135] In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of three or more Nic1b_ERF genes. In one embodiment, such plants contain higher nicotine or total alkaloid levels compared to control animals lacking the transgenic or mutagenic overexpression under comparable conditions. In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of three or more Nic1b_ERF genes and transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic2_ERF genes.

[0136] In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of four or more Nic1b_ERF genes. In one embodiment, such plants contain higher nicotine or total alkaloid levels compared to control animals lacking the transgenic or mutagenic overexpression under comparable conditions. In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of four or more Nic1b_ERF genes and transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic2_ERF genes.

[0137] In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of five or more Nic1b_ERF genes. In one embodiment, such plants contain higher nicotine or total alkaloid levels compared to control animals lacking the transgenic or mutagenic overexpression under comparable conditions. In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of five or more Nic1b_ERF genes and transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic2_ERF genes.

[0138] In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of six or more Nic1b_ERF genes. In one embodiment, such plants contain higher nicotine or total alkaloid levels compared to control animals lacking the transgenic or mutagenic overexpression under comparable conditions. In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of six or more Nic1b_ERF genes and transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic2_ERF genes.

[0139] In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of fewer than two Nic1b_ERF genes. In one embodiment, such plants contain higher nicotine or total alkaloid levels compared to control plants lacking transgenic or mutagenic overexpression under comparable conditions. In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of fewer than two Nic1b_ERF genes and transgenic or mutagenic overexpression of fewer than two, three, four, five, six, or seven Nic2_ERF genes.

[0140] In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of fewer than three Nic1b_ERF genes. In one embodiment, such plants contain higher nicotine or total alkaloid levels compared to control animals lacking transgenic or mutagenic overexpression under comparable conditions. In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of fewer than three Nic1b_ERF genes and transgenic or mutagenic overexpression of fewer than two, three, four, five, six, or seven Nic2_ERF genes.

[0141] In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of fewer than four Nic1b_ERF genes. In one embodiment, such plants contain higher nicotine or total alkaloid levels compared to control animals lacking transgenic or mutagenic overexpression under comparable conditions. In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of fewer than four Nic1b_ERF genes and transgenic or mutagenic overexpression of fewer than two, three, four, five, six, or seven Nic2_ERF genes.

[0142] In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of fewer than five Nic1b_ERF genes. In one embodiment, such plants contain higher nicotine or total alkaloid levels compared to control animals lacking transgenic or mutagenic overexpression under comparable conditions. In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of fewer than five Nic1b_ERF genes and transgenic or mutagenic overexpression of fewer than two, three, four, five, six, or seven Nic2_ERF genes.

[0143] In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of fewer than six Nic1b_ERF genes. In one embodiment, such plants contain higher nicotine or total alkaloid levels compared to control animals lacking transgenic or mutagenic overexpression under comparable conditions. In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of fewer than six Nic1b_ERF genes and transgenic or mutagenic overexpression of fewer than two, three, four, five, six, or seven Nic2_ERF genes.

[0144] In one embodiment, tobacco plants are provided that have one or more Nic2_ERF genes introduced or suppressed in mutagenesis and one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes introduced or suppressed in mutagenesis. In one embodiment, tobacco plants are provided that have two or more Nic2_ERF genes introduced or suppressed in mutagenesis and one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes introduced or suppressed in mutagenesis. In one embodiment, tobacco plants are provided that have three or more Nic2_ERF genes introduced or suppressed in mutagenesis and one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes introduced or suppressed in mutagenesis. In one embodiment, a tobacco plant is provided that has four or more Nic2_ERF genes introduced or suppressed mutagenicity and one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes introduced or suppressed mutagenicity. In one embodiment, a tobacco plant is provided that has five or more Nic2_ERF genes introduced or suppressed mutagenicity and one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes introduced or suppressed mutagenicity. In one embodiment, any of the above plants contains lower nicotine or total alkaloid levels compared to a control plant under equivalent conditions that does not have such Nic2_ERF or Nic1b_ERF genes introduced or suppressed mutagenicity.

[0145] In one embodiment, tobacco plants are provided that have the transgenesis or suppression of mutagenesis of fewer than two Nic2_ERF genes and the transgenesis or suppression of mutagenesis of fewer than two, fewer than three, fewer than four, fewer than five, or fewer than six Nic1b_ERF genes. In one embodiment, tobacco plants are provided that have the transgenesis or suppression of mutagenesis of two or more Nic2_ERF genes and the transgenesis or suppression of mutagenesis of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes. In one embodiment, tobacco plants are provided that have the transgenesis or suppression of mutagenesis of three or more Nic2_ERF genes and the transgenesis or suppression of mutagenesis of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes. In one embodiment, a tobacco plant is provided that has four or more Nic2_ERF genes introduced or suppressed mutagenicity and one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes introduced or suppressed mutagenicity. In one embodiment, a tobacco plant is provided that has five or more Nic2_ERF genes introduced or suppressed mutagenicity and one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes introduced or suppressed mutagenicity. In one embodiment, any of the above plants contains lower nicotine or total alkaloid levels compared to a control plant under equivalent conditions that does not have Nic2_ERF or Nic1b_ERF introduced or suppressed mutagenicity.

[0146] In one aspect, tobacco plants are provided that have transgenic or mutagenic overexpression of one or more Nic2_ERF genes and transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes. In one aspect, tobacco plants are provided that have transgenic or mutagenic overexpression of two or more Nic2_ERF genes and transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes. In one aspect, tobacco plants are provided that have transgenic or mutagenic overexpression of three or more Nic2_ERF genes and transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes. In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of four or more Nic2_ERF genes and transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes. In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of five or more Nic2_ERF genes and transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes. In one embodiment, such plants contain higher nicotine or total alkaloid levels compared to control plants under equivalent conditions that do not have transgenic or mutagenic overexpression of such Nic2_ERF or Nic1b_ERF genes.

[0147] In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of fewer than two Nic2_ERF genes and transgenic or mutagenic overexpression of fewer than two, fewer than three, fewer than four, fewer than five, or fewer than six Nic1b_ERF genes. In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of two or more Nic2_ERF genes and transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes. In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of three or more Nic2_ERF genes and transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes. In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of four or more Nic2_ERF genes and transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes. In one embodiment, tobacco plants are provided that have transgenic or mutagenic overexpression of five or more Nic2_ERF genes and transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes. In one embodiment, such plants contain higher nicotine or total alkaloid levels compared to control plants under equivalent conditions that do not have transgenic or mutagenic overexpression of Nic2_ERF or Nic1b_ERF.

[0148] In one aspect, a tobacco plant is provided that has one or two Nic2_ERF genes that are at least 90%, 95%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 208, 212, 215, and 219, and further has one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes that are at least 90%, 95%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, and 202-205, and that have been transgenic or have been mutagenic suppressed. In one embodiment, a tobacco plant is provided that has transgenic or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 and 226, and further has transgenic or mutagenic suppression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206, and 207. In one embodiment, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such transgenic or mutagenic suppression under equivalent conditions.

[0149] In one embodiment, a tobacco plant is provided that has one or two Nic2_ERF genes that contain a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 208, 212, 215, and 219, and further has one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes that contain a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, and 54, and the plant is further provided with one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes that contain a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, and 54. In one embodiment, a tobacco plant is provided that has transgenic or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 and 226, and further has transgenic or mutagenic suppression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, and 87-89. In one embodiment, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such transgenic or mutagenic suppression under equivalent conditions.

[0150] In one aspect, a tobacco plant is provided that has a transgenic or mutagenic suppression of one or two Nic2_ERF genes that comprise a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 208, 212, 215, and 219, and further has a transgenic or mutagenic suppression of one or more Nic1b_ERF genes that comprise a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, and 202-205. In one aspect, a tobacco plant is provided that has a genetically modified or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 and 226, and further has a genetically modified or mutagenic suppression of one or more Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206, and 207. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such genetic modification or mutagenic suppression under equivalent conditions.

[0151] In one aspect, a tobacco plant is provided that has a transgenic or mutagenic suppression of one or two Nic2_ERF genes that comprise a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 208, 212, 215, and 219, and further has a transgenic or mutagenic suppression of one or more Nic1b_ERF genes that comprise a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, and 54. In one aspect, a tobacco plant is provided that has a genetically modified or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 and 226, and further has a genetically modified or mutagenic suppression of one or more Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, and 87-89. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such genetically modified or mutagenic suppression under equivalent conditions.

[0152] In one aspect, a tobacco plant is provided that has a transgenic or mutagenic suppression of one or two Nic2_ERF genes that comprise a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 208, 212, 215, and 219, and further has a transgenic or mutagenic suppression of two or more Nic1b_ERF genes that comprise a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, and 202-205. In one aspect, a tobacco plant is provided that has a genetically modified or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 and 226, and further has a genetically modified or mutagenic suppression of two or more Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206, and 207. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such genetic modification or mutagenic suppression under equivalent conditions.

[0153] In one aspect, a tobacco plant is provided that has a transgenic or mutagenic suppression of one or two Nic2_ERF genes that comprise a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 208, 212, 215, and 219, and further has a transgenic or mutagenic suppression of two or more Nic1b_ERF genes that comprise a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, and 54. In one aspect, a tobacco plant is provided that has a genetically modified or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 and 226, and further has a genetically modified or mutagenic suppression of two or more Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, and 87-89. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such genetic modification or mutagenic suppression under equivalent conditions.

[0154] In one aspect, a tobacco plant is provided that has a transgenic or mutagenic suppression of one or two Nic2_ERF genes that comprise a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 208, 212, 215, and 219, and further has a transgenic or mutagenic suppression of three or more Nic1b_ERF genes that comprise a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, and 202-205. In one aspect, a tobacco plant is provided that has a genetically modified or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 and 226, and further has a genetically modified or mutagenic suppression of three or more Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206, and 207. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such genetic modification or mutagenic suppression under equivalent conditions.

[0155] In one aspect, a tobacco plant is provided that has a transgenic or mutagenic suppression of one or two Nic2_ERF genes that comprise a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 208, 212, 215, and 219, and further has a transgenic or mutagenic suppression of three or more Nic1b_ERF genes that comprise a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, and 54. In one aspect, a tobacco plant is provided that has a genetically modified or mutagenically suppressed one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 and 226, and further has a genetically modified or mutagenically suppressed three or more Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, and 87-89. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such genetically modified or mutagenic suppressed genes under equivalent conditions.

[0156] In one aspect, a tobacco plant is provided that has a transgenic or mutagenic suppression of one or two Nic2_ERF genes that comprise a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 208, 212, 215, and 219, and further has a transgenic or mutagenic suppression of fewer than two Nic1b_ERF genes that comprise a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, and 202-205. In one aspect, a tobacco plant is provided that has a genetically modified or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 and 226, and further has a genetically modified or mutagenic suppression of fewer than two Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206, and 207. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such genetic modification or mutagenic suppression under equivalent conditions.

[0157] In one aspect, a tobacco plant is provided that has a transgenic or mutagenic suppression of one or two Nic2_ERF genes that comprise a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 208, 212, 215, and 219, and further has a transgenic or mutagenic suppression of fewer than two Nic1b_ERF genes that comprise a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, and 54. In one aspect, a tobacco plant is provided that has a genetically modified or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 and 226, and further has a genetically modified or mutagenic suppression of fewer than two Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, and 87-89. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such genetic modification or mutagenic suppression under equivalent conditions.

[0158] In one aspect, a tobacco plant is provided that has a transgenic or mutagenic suppression of one or two Nic2_ERF genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 208, 212, 215, and 219, and further has a transgenic or mutagenic suppression of fewer than three Nic1b_ERF genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, and 202-205. In one aspect, a tobacco plant is provided that has a genetically modified or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 and 226, and further has a genetically modified or mutagenic suppression of fewer than three Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206, and 207. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such genetically modified or mutagenic suppression under equivalent conditions.

[0159] In one aspect, a tobacco plant is provided that has a transgenic or mutagenic suppression of one or two Nic2_ERF genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 208, 212, 215, and 219, and further has a transgenic or mutagenic suppression of fewer than three Nic1b_ERF genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, and 54. In one aspect, a tobacco plant is provided that has a genetically modified or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 and 226, and further has a genetically modified or mutagenic suppression of fewer than three Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, and 87-89. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such genetic modification or mutagenic suppression under equivalent conditions.

[0160] In one aspect, a tobacco plant is provided that has a transgenic or mutagenic suppression of one or two Nic2_ERF genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 208, 212, 215, and 219, and further has a transgenic or mutagenic suppression of fewer than four Nic1b_ERF genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, and 202-205. In one aspect, a tobacco plant is provided that has a genetically modified or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 and 226, and further has a genetically modified or mutagenic suppression of fewer than four Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206, and 207. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such genetically modified or mutagenic suppression under equivalent conditions.

[0161] In one aspect, a tobacco plant is provided that has a transgenic or mutagenic suppression of one or two Nic2_ERF genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 208, 212, 215, and 219, and further has a transgenic or mutagenic suppression of fewer than four Nic1b_ERF genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, and 54. In one aspect, a tobacco plant is provided that has a genetically modified or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 and 226, and further has a genetically modified or mutagenic suppression of fewer than four Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, and 87-89. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such genetically modified or mutagenic suppression under equivalent conditions.

[0162] In one aspect, a tobacco plant is provided that has a transgenic or mutagenic suppression of one or two Nic2_ERF genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 208, 212, 215, and 219, and further has a transgenic or mutagenic suppression of fewer than five Nic1b_ERF genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, and 202-205. In one aspect, a tobacco plant is provided that has a genetically modified or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 and 226, and further has a genetically modified or mutagenic suppression of fewer than five Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206, and 207. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such genetic modification or mutagenic suppression under equivalent conditions.

[0163] In one aspect, a tobacco plant is provided that has a transgenic or mutagenic suppression of one or two Nic2_ERF genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 208, 212, 215, and 219, and further has a transgenic or mutagenic suppression of fewer than five Nic1b_ERF genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, and 54. In one aspect, a tobacco plant is provided that has a genetically modified or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 and 226, and further has a genetically modified or mutagenic suppression of fewer than five Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, and 87-89. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such genetically modified or mutagenic suppression under equivalent conditions.

[0164] In one embodiment, mutagenic suppression of the Nic2_ERF gene does not involve a deletion of the entire Nic2_ERF gene or the entire Nic2_ERF coding region. In one embodiment, mutagenic suppression of the Nic2_ERF gene involves a deletion of the entire Nic2_ERF gene or the entire Nic2_ERF coding region. In one embodiment, mutagenic suppression of the Nic1b_ERF gene does not involve a deletion of the entire Nic1b_ERF gene or the entire Nic1b_ERF coding region. In one embodiment, mutagenic suppression of the Nic1b_ERF gene involves a deletion of the entire Nic1b_ERF gene or the entire Nic1b_ERF coding region.

[0165] In one aspect, a tobacco plant is provided that has transgenic or mutagenic overexpression of one or two Nic2_ERF genes comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 208, 212, 215, and 219, and further has transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, and 202-205. In one embodiment, a tobacco plant is provided that has transgenic or mutagenic overexpression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 and 226, and further has transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206, and 207. In one embodiment, the plant described in this paragraph comprises higher nicotine or total alkaloid levels compared to a control plant lacking such transgenic or mutagenic overexpression under equivalent conditions.

[0166] In one embodiment, a tobacco plant is provided that has transgenic or mutagenic overexpression of one or two Nic2_ERF genes comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 208, 212, 215, and 219, and further has transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, and 54. In one embodiment, a tobacco plant is provided that has transgenic or mutagenic overexpression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 and 226, and further has transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, and 87-89. In one embodiment, the plant described in this paragraph comprises higher nicotine or total alkaloid levels compared to a control plant lacking such transgenic or mutagenic overexpression under equivalent conditions.

[0167] In one aspect, lower nicotine or total alkaloid levels refer to a reduction of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, or 99% compared to a control plant under comparable conditions.

[0168] In one aspect, higher nicotine or total alkaloid levels refer to an increase of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, or 800% compared to a control plant under comparable conditions.

[0169] In one embodiment, the recombinant DNA construct or expression cassette may also contain a selectable marker gene for the selection of transgenic cells. Selectable marker genes include, but are not limited to, genes encoding antibiotic resistance, such as genes encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinone, and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markers include phenotypic markers such as β-galactosidase and fluorescent proteins, such as green fluorescent protein (GFP).

[0170] In one embodiment, the recombinant DNA construct or expression cassette comprises a promoter selected from the group consisting of a constitutive promoter, an inducible promoter, and a tissue-preferred promoter (e.g., a leaf-specific or root-specific promoter). Exemplary constitutive promoters include the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J 3:2723-2730); and the ALS promoter (U.S. Pat. No. 5,659,026). Exemplary chemical inducible promoters include the tobacco PR-1a promoter, which is activated by salicylic acid.Other chemical inducible promoters of interest include steroid-responsive promoters (see, for example, Schena et al. (1991) Proc.Natl.Acad.Sci.USA 88:10421-10425 and McNellis et al. (1998) Plant J.14(2):247-257 for glucocorticoid-inducible promoters) and tetracycline-inducible promoters (see, for example, Gatz et al. (1991) Mol.Gen.Genet.227:229-237, and U.S. Patent Nos. 5,814,618 and 5,789,156).Additional exemplary promoters that can be used include promoters that regulate thermoregulated gene expression, lightregulated gene expression (e.g., the pea rbcS-3A; maize rbcS promoter; the chlorophyll alb-binding protein gene found in pea; or the Arabssu promoter), hormoneregulated gene expression (e.g., the abscisic acid (ABA) responsive sequence from the Em gene of wheat; the ABA-inducible HVA1 and HVA22, and rd29A promoters of barley and Arabidopsis; etc.). and those involved in wound-inducible gene expression (e.g., wunl), organ-specific gene expression (e.g., tuber-specific storage protein genes; the 23 kDa zein gene from maize described by [R.]; or the β-phaseolin gene in common bean), or pathogen-inducible promoters (e.g., PR-1, prp-1, or β-1,3 glucanase promoter, the fungus-inducible wirla promoter in wheat, and nematode-inducible promoters TobRB7-5A and Hmg-1 in tobacco cured parsley, respectively).

[0171] In one embodiment, the provided tobacco plant further comprises increased or decreased expression of the activity of a gene involved in nicotine biosynthesis or transport. Genes involved in nicotine biosynthesis include, but are not limited to, arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinolinate phosphoribosyltransferase (QPT), and S-adenosyl-methionine synthetase (SAMS). The nicotine synthase that catalyzes the condensation step between nicotinic acid derivatives and methylpyrrolinium cations has not been elucidated, but two candidate genes (A622 and NBBl) have been proposed. See US2007 / 0240728A1 and US2008 / 0120737A1. A622 encodes an isoflavone reductase-like protein. In addition, several transporters may be involved in the transfer of nicotine. A transporter gene designated MATE has been cloned and characterized (Morita et al., PNAS 106:2447-52 (2009)).

[0172] In one embodiment, the provided tobacco plants further comprise increased or decreased levels of mRNA, protein, or both of one or more genes encoding a product selected from the group consisting of PMT, MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBBl, relative to a control tobacco plant. In another embodiment, the provided tobacco plants further comprise a transgene that directly suppresses expression of one or more genes encoding a product selected from the group consisting of PMT, MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBBl. In another embodiment, the provided tobacco plants further comprise a transgene or mutation that suppresses expression or activity of one or more genes encoding a product selected from the group consisting of PMT, MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBBl. In another aspect, the provided tobacco plants further comprise a transgene that overexpresses one or more genes encoding a product selected from the group consisting of PMT, MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBBl.

[0173] Also disclosed is the transformation of tobacco plants using the described recombinant constructs or expression cassettes using any suitable transformation method known in the art.Methods for introducing polynucleotide sequences into tobacco plants are known in the art, including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods."Stable transformation" refers to transformation in which the target nucleotide construct introduced into a plant is integrated into the plant's genome and can be inherited by its progeny."Transient transformation" is intended to mean that the sequence is introduced into a plant and is only expressed temporarily, or is only present in the plant temporarily.

[0174] Suitable methods for introducing polynucleotides into plant cells of the present disclosure include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Shillito et al. (1987) Meth. Enzymol. 153:313-336; Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (U.S. Patent Nos. 5,104,310, 5,149,645, 5,177,010, 5,231,019, 5,463,174, 5,464,763, 5,469,976, 4,762,785, 5,004,863, 5,159,135, 5,563,055, and 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, e.g., U.S. Pat. Nos. 4,945,050, 5,141,131, 5,886,244, 5,879,918, and 5,932,782; Tomes et al. (1995), Plant Cell, Tissue, and Organ Culture Fundamental Methods, edited by Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-926).Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio / Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); De Wet et al. (1985), The Experimental Manipulation of Ovule Tissues, eds. Chapman et al. (Longman, NY), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation).

[0175] In another embodiment, the recombinant construct or expression cassette may be introduced into a plant by contacting the plant with a virus or viral nucleic acid. Generally, such methods involve incorporating the expression cassette of the present disclosure into a viral DNA or RNA molecule. It is recognized that promoters for use in expression cassettes also encompass promoters utilized for transcription by viral RNA polymerase. Methods for introducing polynucleotides into plants and expressing the encoded proteins involving viral DNA or RNA molecules are known in the art. See, for example, U.S. Patent Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et al. (1996) Molecular Biotechnology 5:209-221.

[0176] Any plant tissue that can subsequently be propagated using clonal methods, whether by organogenesis or embryogenesis, may be transformed with a recombinant construct or expression cassette. By "organogenesis" is intended the process by which shoots and roots develop sequentially from the center of the meristem. By "embryogenesis" is intended the process by which shoots and roots develop together in a coordinated (not sequential) manner, whether from somatic cells or gametes. Exemplary tissues suitable for the various transformation protocols described include, but are not limited to, callus tissue, existing meristematic tissues (e.g., apical meristem, axillary bud, and root apical meristem) and inducing meristems (e.g., cotyledonary meristem and hypocotyl meristem), hypocotyl, cotyledon, leaf disc, pollen, and embryo.

[0177] In one aspect, the tobacco plant provided is from a tobacco species selected from the group consisting of flue-cured tobacco, air-cured tobacco, dark air-cured tobacco, dark flue-cured tobacco, Galpao tobacco, and Oriental tobacco. In another aspect, the tobacco plant provided is from a tobacco species selected from the group consisting of Burley tobacco, Maryland tobacco, and dark tobacco.

[0178] In one embodiment, the provided tobacco plant is of flue-cured tobacco background or exhibits one or more flue-cured tobacco characteristics described herein. Flue-cured tobacco (also known as Virginia or bright tobacco) accounts for approximately 40% of the world's tobacco production. Flue-cured tobacco is often also referred to as "bright tobacco" due to the golden yellow to deep orange color it achieves during curing. Flue-cured tobacco has a light, vibrant aroma and taste. Flue-cured tobacco is generally high in sugar and low in oil. Major flue-cured tobacco-growing countries are Argentina, Brazil, China, India, Tanzania, and the United States. In one aspect, provided low alkaloid or low nicotine tobacco plants or seeds include any of the following: CC 13, CC 27, CC 33, CC 37, CC 65, CC 67, CC 700, GF 318, GL 338, GL 368, GL 939, K 346, K 399, K326, NC 102, NC 196, NC 291, NC 297, NC 299, NC 471, NC 55, NC 606, NC 71, NC 72, NC 92, PVH 1118, PVH 1452, PVH 2110, SPEIGHT 168, SPEIGHT 220, SPEIGHT 225, SPEIGHT 227, SPEIGHT 230, SPEIGHT 232, SPEIGHT 234, SPEIGHT 236, SPEIGHT 238, SPEIGHT 239, SPEIGHT 240, SPEIGHT 241, SPEIGHT 242, SPEIGHT 243, SPEIGHT 244, SPEIGHT 245, SPEIGHT 246, SPEIGHT 247, SPEIGHT 248, SPEIGHT 249, SPEIGHT 250, SPEIGHT 251, SPEIGHT 252, SPEIGHT 253, SPEIGHT 254, SPEIGHT 255, SPEIGHT 256, SPEIGHT 257, SPEIGHT 258, SPEIGHT 259, SPEIGHT 260, SPEIGHT 261, SPEIGHT 262, SPEIGHT 263, SPEIGHT 264, SPEIGHT 265, SPEIGHT 266, SPEIGHT 267, S 236, and any variety essentially derived from any one of the foregoing varieties.In another aspect, provided low alkaloid or low nicotine tobacco plants or seeds include any of the following tobacco varieties: Coker 48, Coker 176, Coker 371-Gold, Coker 319, Coker 347, GL 939, K 149, K326, K 340, K 346, K 358, K 394, K 399, K 730, NC 27NF, NC 37NF, NC 55, NC 60, NC 71, NC 72, NC 82, NC 95, NC 297, NC 606, NC 729, NC 2326, McNair 373, McNair 944, Ox 207, Ox 414 NF, Reams 126, Reams 713, Reams 744, RG 8, RG 11, RG In a further embodiment, the low-alkaloid or low-nicotine tobacco plant, seed, hybrid, variety, or strain is a flue-cured tobacco background selected from the group consisting of K326, K346, and NC196.

[0179] In one embodiment, the provided tobacco plant is of an air-cured tobacco background or exhibits one or more characteristics of air-cured tobacco described herein. Air-cured tobaccos include burley, Maryland, and dark tobacco. A common factor is that the curing is primarily done without artificial sources of heat and humidity. Burley tobacco is light to dark brown in color, high in oil, and low in sugar. Burley tobacco is air-cured in barns. Major burley-growing countries are Argentina, Brazil, Italy, Malawi, and the United States. Maryland tobacco is very plump, has good burning characteristics, low nicotine, and a neutral aroma. Major Maryland-growing countries include the United States and Italy. In one embodiment, provided low alkaloid or low nicotine tobacco plants or seeds are of a burley tobacco background selected from the group consisting of Clay 402, Clay 403, Clay 502, Ky 14, Ky 907, Ky 910, Ky 8959, NC 2, NC 3, NC 4, NC 5, NC 2000, TN 86, TN 90, TN 97, R 610, R 630, R 711, R 712, NCBH 129, Bu 21 x Ky 10, HB04P, Ky 14 x L 8, Kt 200, Newton 98, Pedigo 561, Pf561, and Va 509. In a further embodiment, the low alkaloid or low nicotine tobacco plant, seed, hybrid, variety, or strain is in any burley background selected from the group consisting of TN 90, KT 209, KT 206, KT212, and HB 4488. In another embodiment, the provided low alkaloid or low nicotine tobacco plant or seed is in a Maryland tobacco background selected from the group consisting of Md 10, Md 40, Md 201, Md 609, Md 872, and Md 341.

[0180] In one embodiment, the provided tobacco plant is a dark air-cured tobacco background or exhibits one or more characteristics of dark air-cured tobacco described herein. Dark air-cured tobacco is distinguished from other varieties primarily by its curing process, which gives it its medium to dark brown color and distinctive aroma. Dark air-cured tobacco is primarily used in the production of chewing tobacco and snuff. In one embodiment, the provided low-alkaloid or low-nicotine tobacco plant or seed is a dark air-cured tobacco background selected from the group consisting of Sumatra, Jatim, Dominican Cubano, Besuki, One sucker, Green River, Virginia sun-cured, and Paraguan Passado.

[0181] In one embodiment, the provided tobacco plant is a dark flue-cured tobacco background or exhibits one or more characteristics of dark flue-cured tobacco described herein. Dark flue-cured tobacco is typically cured on the floor of a closed curing barn using a low-burning wood fire. The leaves have a low sugar content but a high nicotine content. Dark flue-cured tobacco is used to make pipe blends, cigarettes, chewing tobacco, snuff, and strong-flavored cigars. The primary growing regions for dark flue-cured tobacco are Tennessee, Kentucky, and Virginia in the United States. In one embodiment, the provided low alkaloid or low nicotine tobacco plants or seeds are on a dark flue-cured tobacco background 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, DT 508, DT 518, DT 592, KY 171, DF 911, DF 485, TN D94, TN D950, VA 309, and VA 359.

[0182] In one embodiment, the provided tobacco plant is of Oriental tobacco background or exhibits one or more of the characteristics of Oriental tobacco described herein. Oriental tobacco is also referred to as Greek, aromatic, and Turkish tobacco due to the fact that it is typically grown in the Eastern Mediterranean region, such as Turkey, Greece, Bulgaria, Macedonia, Syria, Lebanon, Italy, and Romania. In addition to the small plant and leaf size characteristic of today's Oriental varieties, their unique aromatic characteristics are the result of the plant's adaptation to the poor soil and harsh climatic conditions in which it grew over the past centuries. In one aspect, the provided low alkaloid or low nicotine tobacco plants or seeds are of an Oriental tobacco background selected from the group consisting of Izmir, Katerini, Samsun, Basma and Krumovgrad, Trabzon, Thesalian, Tasova, Sinop, Izmit, Hendek, Edirne, Semdinli, Adiyanman, Yayladag, Iskenderun, Duzce, Macedonian, Mavra, Prilep, Bafra, Bursa, Bucak, Bitlis, Balikesir, and any variety essentially derived from any one of the foregoing varieties.

[0183] In one aspect, the tobacco plants, seeds, hybrids, varieties, or strains described herein (which may be low alkaloid, low nicotine, high alkaloid, or high nicotine) include, but are not limited to, BU 64, CC 101, CC 200, CC 27, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CU 263, DF911, Galpao tobacco, GL 26H, GL 350, GL 600, GL 737, GL 939, GL 973, HB 04P, K 149, K 326, K 346, K 358, K394, K 399, K 730, KDH 959, KT 200, KT204LC, KY 10, KY 14, KY 160, KY 17, KY 171, KY 907, KY907LC, KTY14×L8 LC, Little Crittenden, McNair 373, McNair 944, msKY 14xL8, Narrow Leaf Madole, NC 100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH 129, NC 2002, Neal Smith Madole, OXFORD 207, "Perique" cigarette, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81, RG H51, RGH 4, RGH 51, RS 1410, Speight 168, Speight 172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI 1269, TN 86, TN86LC, TN 90, TN 97, TN97LC, TN D94, TN D950, TR (Tom Rosson) Madole, VA 309, or VA359, Maryland609, HB3307PLC, HB4488PLC, KT206LC, KT209LC, KT210LC, KT212LC, R610LC, PVH2310, NC196, KTD14LC, KTD6LC, KTD8LC, PD7302LC, PD7305LC, PD7309LC, PD7318LC, PD7319LC, PD7312LC, ShireyLC, or any commercial tobacco variety by standard tobacco breeding techniques known in the art, or in the genetic background thereof.

[0184] Any of the above specific varieties of dark air-cured, burley, Maryland, dark-fired, or Oriental are listed for illustrative purposes only. Any additional dark air-cured, burley, Maryland, dark-fired, or Oriental varieties are also contemplated in this application.

[0185] Populations of the described tobacco plants are also provided. In one embodiment, the population of tobacco plants has a plant density of about 5,000 to about 8,000, about 5,000 to about 7,600, about 5,000 to about 7,200, about 5,000 to about 6,800, about 5,000 to about 6,400, about 5,000 to about 6,000, about 5,000 to about 5,600, about 5,000 to about 5,200, about 5,200 to about 8,000, about 5,600 to about 8,000, about 6,000 to about 8,000, about 6,400 to about 8,000, about 6,800 to about 8,000, about 7,200 to about 8,000, or about 7,600 to about 8,000 plants per acre. In another embodiment, the population of tobacco plants is in a soil type having low to moderate fertility.

[0186] Also provided is a container of seeds from the tobacco plant described.The container of tobacco seeds of the present disclosure can contain any number, weight or volume of seeds.For example, the container can contain at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000 or more seeds, or more than about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000 or more seeds. Alternatively, the container may contain at least about 1 ounce, 5 ounces, 10 ounces, 1 pound, 2 pounds, 3 pounds, 4 pounds, 5 pounds or more of seeds, or more than about 1 ounce, 5 ounces, 10 ounces, 1 pound, 2 pounds, 3 pounds, 4 pounds, 5 pounds or more. The container of tobacco seeds may be any container available in the art. As non-limiting examples, the container may be a box, bag, packet, pouch, tape roll, tube, or bottle.

[0187] Cured tobacco materials made from the described low-alkaloid or low-nicotine tobacco plants are also provided. Cured tobacco materials made from the described tobacco plants having higher levels of total alkaloids or nicotine are further provided.

[0188] "Curing" is a ripening process that reduces moisture and results in the breakdown of chlorophyll, which gives tobacco leaves their golden color and converts starch to sugars. Cured tobacco, therefore, has a higher reducing sugar content and a lower starch content compared to harvested green leaf. In one embodiment, the provided green leaf tobacco can be dried using conventional means, such as hot air curing, barn drying, flame drying, air drying, or sun drying. See, for example, Tso (1999, Chapter 1, Tobacco, Production, Chemistry and Technology, edited by Davis & Nielsen, Blackwell Publishing, Oxford), for a description of different types of curing methods. Cured tobacco is typically aged in wooden drums (e.g., vats) or cardboard boxes under compressed conditions for several years (e.g., 2-5 years) at moisture contents ranging from 10% to about 25%. See U.S. Pat. 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 the introduction of steam at various temperatures, pasteurization, and fermentation. Fermentation is typically characterized by a high initial moisture content, heat production, and a loss of 10-20% of the dry weight. See, e.g., U.S. Pat. Nos. 4,528,993, 4,660,577, 4,848,373, and 5,372,149; U.S. Patent Application Publication No. 2005 / 0178398; and Tso (1999, Chapter 1, Tobacco, Production, Chemistry and Technology, edited by Davis & Nielsen, Blackwell Publishing, Oxford). Cured, aged, and fermented tobacco can be further processed (e.g., cut, shredded, expanded, or blended). See, e.g., U.S. Patent Nos. 4,528,993, 4,660,577, and 4,987,907. In one embodiment, the cured tobacco material of the present disclosure is sun-dried. In another embodiment, the cured tobacco material of the present disclosure is flue-dried, air-dried, or flame-dried.

[0189] Tobacco materials obtained from the tobacco strains, varieties, or hybrids of the present disclosure can be used to make tobacco products. As used herein, "tobacco product" is defined as any product made or derived from tobacco that is intended for human use or consumption.

[0190] Tobacco products provided include, but are not limited to, cigarette products (e.g., cigarettes and bidi cigarettes), cigar products (e.g., cigar wrapping tobacco and cigarillos), pipe tobacco products, tobacco-derived products, tobacco-derived nicotine products, smokeless tobacco products (e.g., moist snuff, dry snuff, and chewing tobacco), films, chewables, tabs, shaped portions, gels, consumable units, insoluble matrices, hollow shapes, reconstituted tobacco, and expanded tobacco. See, e.g., U.S. Patent Application Publication No. US2006 / 0191548.

[0191] As used herein, "cigarette" refers to a tobacco product having a "rod" and a "filler." The cigarette "rod" includes the cigarette paper, filter, plug wrap (used to contain the filter material), tipping paper that holds the cigarette paper (including the filler) to the filter, and any adhesives that hold these components together. "Filler" includes (1) all tobacco, including but not limited to reconstituted tobacco and expanded tobacco, (2) non-tobacco substitutes (including but not limited to herbs, non-tobacco plant materials, and other spices that may accompany the tobacco wrapped in the cigarette paper), (3) casing, (4) flavoring substances, and (5) all other additives (mixed with the tobacco and substitutes and wrapped into the cigarette).

[0192] As used herein, "reconstituted tobacco" refers to a portion of tobacco filler made from tobacco dust and other tobacco waste materials that has been processed into sheet form and cut into strips to resemble tobacco. In addition to cost savings, reconstituted tobacco is very important because of its contribution to cigarette taste from a flavor-developing process that uses ammonia and sugar reactions.

[0193] As used herein, "expanded tobacco" refers to a portion of tobacco filler that has been processed through the expansion of a suitable gas to "expand" the tobacco, resulting in a reduced density and greater filling capacity, which reduces the weight of the tobacco used in the cigarette.

[0194] Tobacco products derived from plants of the present disclosure also include cigarettes and other smoking articles, particularly those smoking articles containing a filter element in which a rod of smokable material contains dried tobacco within a tobacco blend. In one aspect, the tobacco product of the present disclosure is selected from the group consisting of cigarillos, non-ventilated recess filter cigarettes, vented recess filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, hookah, shredded tobacco, and cut tobacco. 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 smokeless tobacco, snus, and dry snuff. Chewing tobacco is coarsely divided tobacco leaves typically packaged in large pouches and used in plugs or twists. Moist smokeless tobacco is moist, more finely divided tobacco provided in loose or pouch form, typically packaged in round cans and used as pinches placed between the cheek and gum of adult tobacco consumers or in pouches. Snus is heat-treated smokeless tobacco. Dry snuff is finely ground tobacco placed in the mouth or used intranasally. 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. In yet another aspect, the tobacco product of the present disclosure is selected from the group consisting of electronically heated cigarettes, e-cigarettes, and electronic vaporization devices.

[0195] In one embodiment, the tobacco product of the present disclosure can be a blended tobacco product. In another embodiment, the tobacco product of the present disclosure can be a low-nicotine tobacco product. In a further embodiment, the tobacco product of the present disclosure can include nornicotine at a level of less than about 3 mg / g. For example, the nornicotine content in such products can be 3.0 mg / g, 2.5 mg / g, 2.0 mg / g, 1.5 mg / g, 1.0 mg / g, 750 μg / g, 500 pg / g, 250 pg / g, 100 pg / g, 75 pg / g, 50 pg / g, 25 pg / g, 10 pg / g, 7.0 pg / g, 5.0 pg / g, 4.0 pg / g, 2.0 pg / g, 1.0 pg / g, 0.5 pg / g, 0.4 pg / g, 0.2 pg / g, 0.1 pg / g, 0.05 pg / g, 0.01 pg / g, or undetectable.

[0196] In one aspect, the provided dried tobacco material or tobacco product has about 0.01%, 0.02%, 0.05%, 0.75%, 0.1%, 0.15%, 0.2%, 0.3%, 0.35%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.1% soluble tobacco. and a mean nicotine or total alkaloid level selected from the group consisting of 8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 5%, 6%, 7%, 8%, and 9%. In another embodiment, the provided dried tobacco material or tobacco product has a dry weight ratio of about 0.01% to 0.02%, 0.02% to 0.05%, 0.05% to 0.75%, 0.75% to 0.1%, 0.1% to 0.15%, 0.15% to 0.2%, 0.2% to 0.3%, 0.3% to 0.35%, 0.35% to 0.4%, 0.4% to 0.5%, 0.5% to 0.6%, 0.6% to 0.7%, 0.7% to 0.8%, 0.8% to 0.9%, 0.9% to 1%, 1% to 1.1%, 1.1% to 1.2%, 1.2% to 1.3%, 1.3% to 1.4%, 1.4% to 1.5%, 1.5% to 1.6%, 1.6% to 1.7%, 1.7% to 1.8%, 1.8% to 1.9%, 1.9% to 2.0%, 1.9% to 2.1%, 1.9% to 2.2%, 1.9% to 2.3%, 1.9% to 2.4%, 1.9% to 2.5%, 1.9% to 2.6%, 1.9% to 2.7%, 1.9% to 2.8%, 1.9% to 2.9%, 1.9% to 2.1%, 1.9% to 2.2%, 1.9% to 2.4%, 1.9% to 2.5%, 1.9% to 2.6%, 1.9% to 2.7%, 1.9% to 2.8%, 1.9% to 2.9%, 1.9% to 2.1%, 1.9% to 2.1%, and an average nicotine or total alkaloid level selected from the group consisting of 0.5%, 1.5% to 1.6%, 1.6% to 1.7%, 1.7% to 1.8%, 1.8% to 1.9%, 1.9% to 2%, 2% to 2.1%, 2.1% to 2.2%, 2.2% to 2.3%, 2.3% to 2.4%, 2.4% to 2.5%, 2.5% to 2.6%, 2.6% to 2.7%, 2.7% to 2.8%, 2.8% to 2.9%, 2.9% to 3%, 3% to 3.1%, 3.1% to 3.2%, 3.2% to 3.3%, 3.3% to 3.4%, 3.4% to 3.5%, and 3.5% to 3.6%. In further embodiments, the provided dried tobacco material or tobacco product comprises an average nicotine or total alkaloid level selected from the group consisting of about 0.01% to 0.1%, 0.02% to 0.2%, 0.03% to 0.3%, 0.04% to 0.4%, 0.05% to 0.5%, 0.75% to 1%, 0.1% to 1.5%, 0.15% to 2%, 0.2% to 3%, and 0.3% to 3.5% on a dry weight basis.

[0197] The present disclosure also provides methods for breeding tobacco strains, cultivars, or varieties containing desired levels of total alkaloids or nicotine, e.g., low nicotine or no nicotine. Breeding can be carried out via any known procedure. DNA fingerprinting, SNP mapping, haplotype mapping, or similar techniques may be used in marker-assisted selection (MAS) breeding programs to transfer or breed desired traits or alleles into tobacco plants. For example, breeders can use F1 hybrid plants or further cross F1 hybrid plants with other donor plants having agronomically desirable genotypes to create segregating populations in the F2 or backcross generation. Plants in the F2 or backcross generation can be screened for desired agronomic traits or chemical profiles using one of the techniques known in the art or listed herein. Depending on the expected inheritance pattern or the MAS technique used, self-pollination of selected plants can be performed before each backcross cycle to aid in identifying desired individual plants. Backcrossing or other breeding procedures can be repeated until the desired phenotype of the reverting parent is recovered. The reverting parent in this disclosure can be a flue-cured variety, a burley variety, a dark air-cured variety, a dark flue-cured variety, or an Oriental variety. Other breeding techniques can be found, for example, 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 Group, Inc., New York, NY (incorporated herein by reference in its entirety).

[0198] The results of plant breeding programs using the described tobacco plants include the useful strains, cultivars, varieties, progeny, selfed varieties, and hybrids of the present disclosure. As used herein, the term "variety" refers to a group of plants that share certain characteristics that separate them from other plants of the same species. Varieties are often, but not always, sold commercially. While possessing one or more robust traits, varieties are further characterized by very little overall variation between individuals within the variety. "Pure-stock" varieties may be produced by self-pollination and selection from a single parent using tissue or cell culture techniques, or by several generations of vegetative propagation. A variety may be essentially derived from another strain or variety. As defined by the International Convention for the Protection of New Varieties of Plants (December 2, 1961; revised at Geneva on November 10, 1972, October 23, 1978, and March 19, 1991), a variety is "essentially derived" from an earlier variety if it a) is derived primarily from the earlier variety, or from a variety derived primarily from the earlier variety while retaining the expression of essential characteristics resulting from the genotype or combination of genotypes of the earlier variety; b) is clearly distinguishable from the earlier variety; and c) matches the earlier variety in the expression of essential characteristics resulting from the genotype or combination of genotypes of the earlier variety, except for differences resulting from acts of derivation. Essentially derived varieties can be obtained, for example, by natural or induced mutants, somaclonal variants, selection of variant individuals from plants of the earlier variety, backcrossing, or transformation. A first tobacco variety and a second tobacco variety from which the first variety is essentially derived are considered to have essentially the same genetic background. A "strain," as distinguished from a variety, most often refers to a group of plants used non-commercially, for example, in botanical research. Strains typically exhibit little overall variation among individuals for one or more traits of interest, although there may be some variation among individuals for other traits.

[0199] In one aspect, the disclosure provides methods for transferring a low-nicotine trait into a tobacco variety, the methods comprising: (a) crossing a first tobacco variety that includes the low-nicotine trait with a second tobacco variety that does not have the low-nicotine trait to produce one or more progeny tobacco plants; (b) genotyping the one or more progeny tobacco plants for polymorphic markers associated with the low-nicotine trait, wherein the polymorphic markers are in a chromosomal interval flanked by any two SNP markers selected from the group consisting of SEQ ID NOs: 125-145 and 193-201; and (c) selecting a progeny tobacco plant that includes the low-nicotine trait. In another aspect, the methods further comprise backcrossing the selected progeny tobacco plant with the second tobacco variety. In a further aspect, the methods further comprise: (d) crossing the selected progeny plant with itself or with the second tobacco variety to produce one or more additional progeny tobacco plants; and (e) selecting an additional progeny tobacco plant that includes the low-nicotine trait. In one embodiment, the selecting step (e) comprises marker-assisted selection. In one embodiment, these methods result in a single-gene conversion comprising a low-nicotine trait. In one embodiment, these methods result in a single-gene conversion comprising a Nic1b_ERF introgression. In one embodiment, the second tobacco variety is an elite variety. In another embodiment, the genotyping step of these methods involves one or more molecular marker assays. In another embodiment, the polymorphic marker used in the method comprises a polymorphism selected from the group consisting of a single nucleotide polymorphism (SNP), an insertion or deletion (indel) in a DNA sequence, a simple sequence repeat (SSR) in a DNA sequence, a restriction fragment length polymorphism (RFLP), and a tag SNP. In another embodiment, the selected progeny tobacco plant comprises a shorter chromosomal introgression at the Nic1b_ERF locus compared to LA Burley 21.

[0200] In another aspect, the disclosure provides a method of transferring a low-nicotine trait into a tobacco variety, the method comprising: (a) crossing a first tobacco variety that includes the low-nicotine trait with a second tobacco variety that does not have the low-nicotine trait to produce one or more progeny tobacco plants; (b) genotyping the one or more progeny tobacco plants for polymorphic markers associated with the low-nicotine trait, wherein the polymorphic marker is within 20 cM, 10 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.5 cM, or less than 0.5 cM of any one of SNP markers selected from the group consisting of SEQ ID NOs: 125-145 and 193-201, or any locus having a sequence selected from the group consisting of SEQ ID NOs: 1, 3-37, 146, 149, 152-156, 202, 203, and 184-186; and (c) selecting the progeny tobacco plants that include the low-nicotine trait. In one embodiment, the method comprises simultaneously or in parallel selecting for one or more molecular markers associated with or closely linked to the Nic1b_ERF locus as well as one or more molecular markers associated with or closely linked to the Nic2 locus.

[0201] In one aspect, the disclosure provides a method for selecting tobacco plants having a low-nicotine trait, the method comprising: (a) isolating nucleic acids from a collection of tobacco germplasm; (b) assaying the nucleic acids for one or more markers closely linked to the Nic1b_ERF locus; and (c) selecting tobacco plants having the low-nicotine trait based on the marker assays. In one embodiment, the one or more markers assayed closely linked to the Nic1b_ERF locus are within about 20 cM, 10 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.5 cM, or less than 0.5 cM of any one of the SNP markers selected from the group consisting of SEQ ID NOs: 125-145 and 193-201, or any locus having a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, and 202-205. In another embodiment, the method further comprises assaying for one or more markers closely linked to the Nic2 locus. In one embodiment, the one or more markers assayed that are closely linked to the Nic2 locus are within about 20 cM, 10 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.5 cM, or less than 0.5 cM of any one of the polymorphic loci located in one of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168. In one embodiment, the method further comprises determining the nicotine level of the selected plant to confirm the low nicotine trait.

[0202] Also disclosed are methods for introgressing a low-nicotine trait into a tobacco variety, the methods comprising: (a) crossing a first tobacco variety including the low-nicotine trait with a second tobacco variety that does not have the low-nicotine trait to produce one or more progeny tobacco plants; (b) genotyping the one or more progeny tobacco plants for polymorphic markers associated with the low-nicotine trait, wherein the polymorphic markers are in a chromosomal interval flanked by any two SNP markers selected from the group consisting of SEQ ID NOs: 125-145 and 193-201; and (c) selecting the progeny tobacco plants including the low-nicotine trait. In one embodiment, these methods result in a single-gene conversion including the low-nicotine trait. In one embodiment, these methods result in a single-gene conversion including a Nic2 introgression. In one embodiment, the second tobacco variety is an elite variety. In another embodiment, the genotyping step of these methods involves one or more molecular marker assays. In another embodiment, the polymorphic marker used in the method comprises a polymorphism selected from the group consisting of single nucleotide polymorphisms (SNPs), insertions or deletions (indels) in DNA sequences, simple sequence repeats (SSRs) in DNA sequences, restriction fragment length polymorphisms (RFLPs), and tag SNPs. In another embodiment, the selected progeny tobacco plants comprise a shorter chromosomal deletion at the Nic2 locus compared to LA Burley 21.

[0203] As used herein, a "locus" is a chromosomal region where a polymorphic nucleic acid, trait determinant, gene, or marker is located. The loci of the present disclosure include one or more polymorphisms in a population, for example, alternative alleles exist in some individuals. As used herein, "allele" refers to alternative nucleic acid sequences at a particular locus. The length of an allele can be as short as one nucleotide base, but is typically longer. For example, a first allele may occur on one chromosome and a second allele may occur on a second homologous chromosome, such as occurs for different chromosomes in a heterozygous individual or between different homozygous or heterozygous individuals in a population. As used herein, the term "chromosomal interval" refers to a continuous linear span of genomic DNA on a single chromosome.

[0204] As used herein, centimorgan ("cM") is a unit of measure of recombination frequency. 1 cM is equal to a 1% chance that a marker at one locus is separated from a marker at a second locus due to crossover in a single generation. Genetic distances referred to herein can be calculated from recombination values ​​using the Kosambi function (Kosambi, The estimation of map distances from recombination values. Annals of Eugenics, 12:172-75 (1944)).

[0205] As used herein, "closely associated" or "associated" means that a marker or locus is within about 20 cM, 10 cM, 5 cM, 1 cM, 0.5 cM, or less than 0.5 cM of another marker or locus. For example, 20 cM refers to recombination between the marker and locus at a frequency equal to or less than about 20%.

[0206] As used herein, "introgression" or "introject" refers to the transfer of a desired allele of a genetic locus from one genetic background to another.

[0207] As used herein, "crossed" or "crossing" means producing offspring through fertilization (e.g., of a cell, seed, or plant), and includes crossing between plants (sexual) and self-fertilization or selfing.

[0208] As used herein, "backcrossing" and "backcrossing" refer to a method in which progeny plants are repeatedly crossed back to one of their parents. In a backcrossing scheme, the "donor" parent refers to the parent plant that has the desired gene or locus to be transferred. The "recipient" parent (used one or more times) or "recurrent" parent (used two or more times) refers to the parent plant into which the gene or locus is being transferred. The initial cross produces the F1 generation. The term "BC1" refers to the second use of the recurrent parent, "BC2" refers to the third use of the recurrent parent, and so on. In one aspect, backcrossing is performed repeatedly, with the progeny individuals of each successive backcross generation being themselves backcrossed to the same parent genotype.

[0209] As used herein, "single-gene converted" or "single-gene conversion" refers to a plant developed using the plant breeding technique known as backcrossing or through genetic engineering, in which essentially all of the desired morphological and physiological characteristics of the variety are restored in addition to a single gene that is transferred into the variety through the backcrossing technique or through genetic engineering.

[0210] As used herein, "elite variety" means any variety that has resulted from breeding and selection for superior agronomic performance.

[0211] As used herein, "selecting" or "selection" in the context of marker-assisted selection or breeding refers to the act of collecting or selecting desired individuals, usually from a population, based on certain predetermined criteria.

[0212] As used herein, the term "trait" refers to one or more detectable characteristics of a cell or organism that can be influenced by genotype. A phenotype can be observable with the naked eye or by any other means of evaluation known in the art, such as microscopy, biochemical analysis, genomic analysis, assays for specific disease resistance, etc. In some cases, a phenotype is directly controlled by a single gene or locus, e.g., a "single gene trait." In other cases, a phenotype is the result of several genes.

[0213] As used herein, "marker assay" refers to a method of detecting a polymorphism at a particular genetic locus using a particular method, such as measurement of at least one phenotype (e.g., seed color, flower color, or other visually detectable trait), restriction fragment length polymorphism (RFLP), single base extension, electrophoresis, sequence alignment, allele-specific oligonucleotide hybridization (ASO), random amplified polymorphic DNA (RAPD), microarray-based techniques, and nucleic acid sequencing techniques.

[0214] As used herein, "marker-assisted selection" (MAS) is a method in which a phenotype is selected based on a marker genotype. "Marker-assisted selection breeding" refers to a method of selecting for one or more desired traits in one or more plants by detecting one or more nucleic acids associated with the desired traits in the plants and then selecting plants or germplasm that have those one or more nucleic acids.

[0215] As used herein, "polymorphism" refers to the presence of one or more variations in a population. Polymorphisms may manifest as variations in the nucleotide sequence of a nucleic acid or variations in the amino acid sequence of a protein. Polymorphisms include the presence of one or more variations in a nucleic acid sequence or nucleic acid feature at one or more loci in a population of one or more individuals. Variations may include, but are not limited to, base changes of one or more nucleotides, insertions of one or more nucleotides, or deletions of one or more nucleotides. Polymorphisms may arise from random processes in nucleic acid replication, through mutagenesis, as a result of mobile genomic elements, from copy number variations, and during processes such as unequal crossing over, genome duplication, and chromosome breakage and fusion. Variations may be commonly found or may exist at low frequencies within a population; the former generally have greater utility in plant breeding, while the latter may be associated with rare but important phenotypic variations. Useful polymorphisms can include single nucleotide polymorphisms (SNPs), insertions or deletions (indels) in DNA sequences, simple sequence repeats (SSRs) in DNA sequences, restriction fragment length polymorphisms (RFLPs), and tag SNPs.Genetic markers, genes, DNA-derived sequences, RNA-derived sequences, promoters, 5' untranslated regions of genes, 3' untranslated regions of genes, microRNAs, siRNAs, tolerance loci, satellite markers, transgenes, mRNAs, ds mRNAs, transcriptional profiles, and methylation patterns can also comprise polymorphisms.In addition, the presence, absence, or copy number of the above-mentioned variations can also comprise polymorphisms.

[0216] As used herein, "SNP" or "single nucleotide polymorphism" refers to a sequence variation that occurs when a single nucleotide (A, T, C, or G) in a genomic sequence is altered or variable. A "SNP marker" exists when a SNP is mapped to a site on the genome.

[0217] As used herein, "marker" or "molecular marker" or "marker locus" is a term used to refer to a nucleic acid or amino acid sequence that is sufficiently unique to characterize a specific locus on the genome. Any detectable polymorphic trait can be used as a marker, so long as it is differentially inherited and exhibits linkage disequilibrium with the phenotypic trait of interest. Each marker is therefore an indicator of a specific segment of DNA with a unique nucleotide sequence. Map locations provide a measure of the relative location of specific markers relative to one another. When a trait is described as being associated with a given marker, it will be understood that the actual DNA segment whose sequence affects the trait generally cosegregates with the marker. More precise and specific localization of the trait can be obtained when markers are identified on both sides of the trait. By measuring the occurrence of markers in the progeny of a cross, the presence of the trait can be detected by a relatively simple molecular test without actually assessing the occurrence of the trait itself, which can be difficult and time-consuming because it requires growing plants to a stage and / or under environmental conditions where the trait can be expressed. In one embodiment, the markers used exhibit an LOD score of 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or 9 or more with the Nic1b_ERF or Nic2_ERF locus as measured using methods known in the art, such as Qgene version 2.23 (1996) and default parameters.

[0218] It is understood that any tobacco plant of the present disclosure can further comprise additional agronomically desirable traits, for example, by transformation with a genetic construct or transgene using techniques known in the art. Non-limiting examples of desirable traits include herbicide resistance, pest resistance, disease resistance; high yield; high grade index value; curability; drying quality; mechanical harvestability; holding capacity; leaf quality; height, plant maturity (e.g., early maturity, early-to-medium maturity, medium maturity, medium-to-late maturity, or late maturity); stem size (e.g., small, medium, or large stem); or leaf number per plant (e.g., low (e.g., 5-10 leaves), medium (e.g., 11-15 leaves), or many (e.g., 16-21) leaves), or any combination thereof. In one embodiment, the disclosed low-nicotine or nicotine-free tobacco plants or seeds contain one or more transgenes expressing one or more insecticidal proteins, such as the crystal protein of Bacillus thuringiensis or a nutritional insecticidal protein from Bacillus cereus, e.g., VIP3 (see, e.g., Estruch et al. (1997) Nat. Biotechnol. 15:137). In another embodiment, the tobacco plants further contain transferred traits that confer resistance to brown stem rot (U.S. Pat. No. 5,689,035) or cyst nematode resistance (U.S. Pat. No. 5,491,081).

[0219] The present disclosure also provides tobacco plants having yields equivalent to the yields of corresponding initial tobacco plants containing altered nicotine or total alkaloid levels but without such altered nicotine levels. In one aspect, the low-nicotine or nicotine-free tobacco varieties provide yields selected from the group consisting of about 1200-3500, 1300-3400, 1400-3300, 1500-3200, 1600-3100, 1700-3000, 1800-2900, 1900-2800, 2000-2700, 2100-2600, 2200-2500, and 2300-2400 lb / acre. In another embodiment, the low-nicotine or nicotine-free tobacco variety provides a yield selected from the group consisting of about 1200-3500, 1300-3500, 1400-3500, 1500-3500, 1600-3500, 1700-3500, 1800-3500, 1900-3500, 2000-3500, 2100-3500, 2200-3500, 2300-3500, 2400-3500, 2500-3500, 2600-3500, 2700-3500, 2800-3500, 2900-3500, 3000-3500, and 3100-3500 lb / acre. In further embodiments, the low-nicotine or nicotine-free tobacco plants provide a yield that is between 65% and 130%, between 70% and 130%, between 75% and 130%, between 80% and 130%, between 85% and 130%, between 90% and 130%, between 95% and 130%, between 100% and 130%, between 105% and 130%, between 110% and 130%, between 115% and 130%, or between 120% and 130% of the yield of a control plant having essentially the same genetic background except for the nic1b_erf mutation, the nic2 mutation, the Nic1b_ERF transgene, the Nic2 transgene, or a combination thereof. In further embodiments, the low-nicotine or nicotine-free tobacco plant provides a yield that is between 70% and 125%, 75% and 120%, 80% and 115%, 85% and 110%, or 90% and 100% of the yield of a control plant having essentially the same genetic background except for the nic1b_erf mutation, the nic2 mutation, the Nic1b_ERF transgene, the Nic2 transgene, or a combination thereof.

[0220] In one embodiment, the disclosed tobacco plants (e.g., low-nicotine, nicotine-free, or low-alkaloid tobacco varieties) do not exhibit one or more, two or more, three or more, or all of the LA BU21 traits selected from the group consisting of lower yield, delayed maturation and senescence, higher susceptibility to insect herbivory, increased polyamine content after topping, higher chlorophyll, more mesophyll cells per unit leaf area, and poor end-product quality after curing. In one embodiment, the disclosed tobacco plants (e.g., low-nicotine, nicotine-free, or low-alkaloid tobacco varieties) do not exhibit two or more of the LA BU21 traits selected from the group consisting of lower yield, delayed maturation and senescence, higher susceptibility to insect herbivory, increased polyamine content after topping, higher chlorophyll, more mesophyll cells per unit leaf area, and poor end-product quality after curing. In one embodiment, a disclosed tobacco plant (e.g., a low-nicotine, nicotine-free, or low-alkaloid tobacco variety) does not exhibit three or more of the LA BU21 traits selected from the group consisting of lower yield, delayed maturation and senescence, higher susceptibility to insect herbivory, increased polyamine content after topping, higher chlorophyll, more mesophyll cells per unit leaf area, and poor end-product quality after curing. In one embodiment, a disclosed tobacco plant (e.g., a low-nicotine, nicotine-free, or low-alkaloid tobacco variety) exhibits one or more, two or more, three or more, or all of the LA BU21 traits selected from the group consisting of lower yield, delayed maturation and senescence, higher susceptibility to insect herbivory, increased polyamine content after topping, higher chlorophyll, more mesophyll cells per unit leaf area, and poor end-product quality after curing, at a lower level compared to LA BU21, LAFC53, or LN KY171.In one aspect, the disclosed tobacco plants (e.g., low-nicotine, nicotine-free, or low-alkaloid tobacco varieties) exhibit two or more LA BU21 traits selected from the group consisting of lower yield, delayed maturation and senescence, higher susceptibility to insect herbivory, increased polyamine content after topping, higher chlorophyll, more mesophyll cells per unit leaf area, and poor end-product quality after curing, at lower levels compared to LA BU21, LAFC53, or LN KY171. In one aspect, the disclosed tobacco plants (e.g., low-nicotine, nicotine-free, or low-alkaloid tobacco varieties) exhibit three or more, or all of the following LA BU21 traits selected from the group consisting of lower yield, delayed maturation and senescence, higher susceptibility to insect herbivory, increased polyamine content after topping, higher chlorophyll, more mesophyll cells per unit leaf area, and poor end-product quality after curing, at lower levels compared to LA BU21, LAFC53, or LN KY171.

[0221] In one aspect, the disclosed modified tobacco plants (e.g., low-nicotine, nicotine-free, or low-alkaloid tobacco varieties) comprise modifications that confer a desired trait (e.g., low-nicotine, nicotine-free, or low-alkaloid) without substantially affecting a trait selected from the group consisting of yield, maturity and senescence, susceptibility to insect herbivory, polyamine content after topping, chlorophyll levels, mesophyll cell number per unit leaf area, and end-product quality after curing.

[0222] In one aspect, the disclosed modified tobacco plants comprise a modification that confers a desired trait (e.g., low nicotine, no nicotine, or low alkaloids) and further comprise traits substantially equivalent to an unmodified control plant, the traits being selected from the group consisting of yield, maturity and senescence, susceptibility to insect herbivory, polyamine content after topping, chlorophyll levels, mesophyll cell number per unit leaf area, and end-product quality after curing.

[0223] In one embodiment, the disclosed modified tobacco plants comprise a modification that confers a desired trait (e.g., low nicotine, no nicotine, or low alkaloids) and further comprise a yield of greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 100%, greater than 105%, greater than 110%, greater than 115%, greater than 120%, greater than 125%, greater than 130%, greater than 135%, or greater than 140% relative to the yield of an unmodified control plant. In one embodiment, the disclosed modified tobacco plants comprise a modification that confers a desired trait (e.g., low nicotine, no nicotine, or low alkaloids) and further comprise a yield of between 70% and 140%, 75% and 135%, 80% and 130%, 85% and 125%, 90% and 120%, 95% and 115%, or 100% and 110% relative to the yield of an unmodified control plant. In one aspect, the disclosed modified tobacco plants include modifications that confer a desired trait (e.g., low nicotine, no nicotine, or low alkaloids) and further include a yield of between 70% and 80%, 75% and 85%, 80% and 90%, 85% and 95%, 90% and 100%, 95% and 105%, 105% and 115%, 110% and 120%, 115% and 125%, 120% and 130%, 125 and 135%, or 130% and 140% relative to the yield of an unmodified control plant.

[0224] In one aspect, the disclosed modified tobacco plants comprise a modification that confers a desired trait (e.g., low nicotine, no nicotine, or low alkaloids) and further comprise a post-topping polyamine content that is greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 100%, greater than 105%, greater than 110%, greater than 115%, greater than 120%, greater than 125%, greater than 130%, greater than 135%, or greater than 140% relative to the post-topping polyamine content of an unmodified control plant. In one aspect, the disclosed modified tobacco plants comprise a modification that confers a desired trait (e.g., low nicotine, no nicotine, or low alkaloids) and further comprise a post-topping polyamine content of between 70% and 140%, between 75% and 135%, between 80% and 130%, between 85% and 125%, between 90% and 120%, between 95% and 115%, or between 100% and 110% relative to the post-topping polyamine content of an unmodified control plant. In one aspect, the disclosed modified tobacco plants comprise a modification that confers a desired trait (e.g., low nicotine, no nicotine, or low alkaloids) and further comprise a post-topping polyamine content of between 70% and 80%, 75% and 85%, 80% and 90%, 85% and 95%, 90% and 100%, 95% and 105%, 105% and 115%, 110% and 120%, 115% and 125%, 120% and 130%, 125 and 135%, or 130% and 140% relative to the post-topping polyamine content of an unmodified control plant.

[0225] In one embodiment, the disclosed modified tobacco plants comprise modifications that confer a desired trait (e.g., low nicotine, no nicotine, or low alkaloids) and further comprise chlorophyll levels that are greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 100%, greater than 105%, greater than 110%, greater than 115%, greater than 120%, greater than 125%, greater than 130%, greater than 135%, or greater than 140% relative to the chlorophyll level of an unmodified control plant. In one embodiment, the disclosed modified tobacco plants comprise a modification that confers a desired trait (e.g., low nicotine, no nicotine, or low alkaloids) and further comprise a chlorophyll level that is between 70% and 140%, 75% and 135%, 80% and 130%, 85% and 125%, 90% and 120%, 95% and 115%, or 100% and 110% relative to the chlorophyll level of an unmodified control plant. In one embodiment, the disclosed modified tobacco plants comprise a modification that confers a desired trait (e.g., low nicotine, no nicotine, or low alkaloids) and further comprise a chlorophyll level that is between 70% and 80%, 75% and 85%, 80% and 90%, 85% and 95%, 90% and 100%, 95% and 105%, 105% and 115%, 110% and 120%, 115% and 125%, 120% and 130%, 125 and 135%, or 130% and 140% relative to the chlorophyll level of an unmodified control plant.

[0226] In one aspect, the disclosed modified tobacco plants comprise a modification that confers a desired trait (e.g., low nicotine, no nicotine, or low alkaloids) and further comprise a number of mesophyll cells per unit leaf area that is 80% greater, 85% greater, 90% greater, 95% greater, 100% greater, 105% greater, 110% greater, 115% greater, 120% greater, 125% greater, 130% greater, 135% greater, or 140% greater than the number of mesophyll cells per unit leaf area of ​​an unmodified control plant. In one aspect, the disclosed modified tobacco plants comprise a modification that confers a desired trait (e.g., low nicotine, no nicotine, or low alkaloids) and further comprise a number of mesophyll cells per unit leaf area that is between 70% and 140%, 75% and 135%, 80% and 130%, 85% and 125%, 90% and 120%, 95% and 115%, or 100% and 110% relative to the number of mesophyll cells per unit leaf area of ​​an unmodified control plant. In one aspect, the disclosed modified tobacco plants comprise a modification that confers a desired trait (e.g., low nicotine, no nicotine, or low alkaloids) and further comprise a number of mesophyll cells per unit leaf area that is between 70% and 80%, 75% and 85%, 80% and 90%, 85% and 95%, 90% and 100%, 95% and 105%, 105% and 115%, 110% and 120%, 115% and 125%, 120% and 130%, 125 and 135%, or 130% and 140% greater than the number of mesophyll cells per unit leaf area of ​​an unmodified control plant.

[0227] In one aspect, the disclosed low-nicotine or nicotine-free tobacco varieties are adapted for mechanical harvesting. In another aspect, the disclosed low-nicotine or nicotine-free tobacco varieties are mechanically harvested.

[0228] In one embodiment, the provided tobacco plant is a hybrid plant. Hybrids can be produced by preventing self-pollination of a first variety of female parent plant (e.g., seed parent), allowing pollen from a second variety of male parent plant to fertilize the female parent plant, and allowing F1 hybrid seeds to form in the female plant. Self-pollination of female plants can be prevented by emasculating flowers at an early stage of flower development. Alternatively, pollen formation can be prevented in female parent plants using a form of male sterility. For example, male sterility can be caused by male sterility (MS), in which an introduced gene inhibits microspore formation and / or pollen formation, or transgenic male sterility, or self-incompatibility. Female parent plants containing MS are particularly useful. In embodiments where the female parent plant is MS, pollen may be harvested from the male fertile plant and manually applied to the stigma of the MS female parent plant, and the resulting F1 seeds are harvested.

[0229] The plant can be used to form a single-cross tobacco F1 hybrid. Pollen from the male parent plant is manually transferred to an emasculated or male-sterile female parent plant to form F1 seeds. Alternatively, a three-way cross can be performed in which a single-cross F1 hybrid is used as the female parent and crossed with a different male parent. As another alternative, a double-cross hybrid can be created by crossing the F1 progeny of two different single crosses with themselves. Self-incompatibility can be used for specific advantages to prevent self-pollination of the female parent when forming a double-cross hybrid.

[0230] In one embodiment, the low-nicotine or nicotine-free tobacco variety is male sterile. In another embodiment, the low-nicotine or nicotine-free tobacco variety is cytoplasmic male sterile. Male-sterile tobacco plants may be produced by any method known in the art. Methods for producing male-sterile tobacco are described in Wernsman, EA and Rufty, RC 1987. Chapter Seventeen. Tobacco. Pages 669-698 In: Cultivar Development. Crop Species. W.H. Fehr (ed.), MacMillan Publishing Company, Inc., New York, NY 761 pp.

[0231] In a further aspect, the tobacco parts provided include, but are not limited to, leaves, stems, roots, seeds, flowers, pollen, anthers, ovules, pedicels, fruits, meristems, cotyledons, hypocotyls, pods, embryos, endosperm, explants, callus, tissue cultures, shoots, cells, and protoplasts. In one aspect, the tobacco parts provided do not include seeds. In one aspect, the present disclosure provides tobacco plant cells, tissues, and organs that are not reproductive material and do not mediate the natural reproduction of plants. In another aspect, the present disclosure also provides tobacco plant cells, tissues, and organs that are reproductive material and mediate the natural reproduction of plants. In another aspect, the present disclosure provides tobacco plant cells, tissues, and organs that cannot be maintained through photosynthesis. In another aspect, the present disclosure provides somatic tobacco plant cells. Somatic cells, unlike germline cells, do not mediate plant reproduction.

[0232] The provided cells, tissues, and organs may be from seeds, fruits, leaves, cotyledons, hypocotyls, meristems, embryos, endosperms, roots, shoots, stems, pods, flowers, infloresences, stems, pedicels, styles, stigmas, receptacles, petals, sepals, pollen, anthers, filaments, ovaries, ovules, pericarp, phloem, or vascular tissue. In another aspect, the present disclosure provides tobacco plant chloroplasts. In a further aspect, the present disclosure provides epidermal cells, stomatal cells, leaf or root hairs, storage roots, or tubers. In another aspect, the present disclosure provides tobacco protoplasts.

[0233] Those skilled in the art will understand that tobacco plants naturally reproduce through seeds, rather than through asexual or vegetative propagation. In one aspect, the present disclosure provides tobacco endosperm. In another aspect, the present disclosure provides tobacco endosperm cells. In a further aspect, the present disclosure provides male or female sterile tobacco plants that cannot reproduce without human intervention.

[0234] In one aspect, the disclosure provides a nucleic acid molecule comprising at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 1-72, 147, 150, 157-161, 187-189, and 202-205, and fragments thereof. In one aspect, the disclosure provides a polypeptide or protein comprising at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73-107, 148, 151, 180-183, 206, 207, and 190-192. In another aspect, the disclosure provides a polypeptide or protein comprising at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73-107, 148, 151, 180-183, 206, 207, and 190-192. In another aspect, the disclosure provides a biologically active variant of a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 73-107, 148, 151, 180-183, 206, 207, and 190-192. Biologically active variants of proteins of the present disclosure may differ from the protein by as few as 1-15 amino acid residues, as few as 10, as few as 9, as few as 8, as few as 7, as few as 6, as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1 amino acid residue. Orthologous genes or proteins of genes or proteins from the Nic1b_ERF locus are also provided. "Orthologs" are genes that are derived from a common ancestral gene and are found in different species as a result of speciation.Orthologs may share at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity or similarity at the nucleotide and / or protein sequence level. The function of orthologs is often highly conserved between species.

[0235] As used herein, the term "sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refers to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When the percentage of sequence identity is used in relation to proteins, it is recognized that non-identical residue positions often differ by conservative amino acid substitutions, in which amino acid residues are replaced with other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity), thus not changing the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are considered to have "sequence similarity" or "similarity."

[0236] The provided nucleic acid molecules, polypeptides, or proteins can be isolated or substantially purified. An "isolated" or "purified" nucleic acid molecule, polypeptide, protein, or biologically active portion thereof is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. For example, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0237] The present disclosure further provides methods for producing tobacco products comprising tobacco material from the disclosed tobacco plants. In one embodiment, the method includes conditioning aged tobacco material from the tobacco plants to increase its moisture content from about 12.5% ​​to about 13.5% to about 21% and blending the conditioned tobacco material to produce a desired blend. In one embodiment, the method for producing a tobacco product further includes casing or flavoring the blend. Generally, during the casing process, casing or source materials are added to the blend to improve its quality by balancing its chemical composition and to develop certain desired flavor characteristics. Further details about casing processing can be found in Tobacco Production, Chemistry and Technology, edited by L. Davis and M. Nielsen, Blackwell Science, 1999.

[0238] The provided tobacco material may also be processed using methods including, but not limited to, heat treatment (e.g., cooking, toasting), flavoring, enzyme treatment, expansion, and / or curing. Both fermented and non-fermented tobaccos may be processed using these techniques. Examples of suitable processed tobaccos include dark air-cured, dark flue-cured, burley, hot-air-cured, and cigar filler or wrapper, as well as products from whole-leaf stemming operations. In one embodiment, the tobacco fiber comprises up to 70% dark tobacco on a fresh weight basis. For example, the tobacco may be conditioned by heating, steaming, and / or pasteurization steps, such as those described in U.S. Patent Application Publication Nos. 2004 / 0118422 or 2005 / 0178398.

[0239] The provided tobacco material can be subjected to fermentation. Fermentation is typically characterized by a high initial moisture content, heat production, and a loss of 10-20% of the dry weight. See, e.g., U.S. Patent Nos. 4,528,993, 4,660,577, 4,848,373, and 5,372,149. In addition to improving leaf aroma, fermentation can change either or both the color and texture of the leaf. Also, during the fermentation process, gases can be produced, oxygen can be taken up, pH can be altered, and the amount of water retained can be changed. See, e.g., U.S. Patent Application Publication No. 2005 / 0178398 and Tso (1999, Chapter 1, Tobacco, Production, Chemistry and Technology, edited by Davis & Nielsen, Blackwell Publishing, Oxford). The cured, or cured and fermented tobacco may be further processed (e.g., cut, expanded, blended, milled, or ground) before incorporation into an oral product. The tobacco is, in some cases, long-cut, fermented, cured, moist tobacco having an oven volatiles content of 48 to 50 percent by weight prior to mixing with the copolymer and, optionally, flavorings and other additives.

[0240] In one embodiment, the provided tobacco material can be processed to a desired size. In one embodiment, the tobacco fiber can be processed to have an average fiber size of less than 200 micrometers. In one embodiment, the tobacco fiber is 75 to 125 micrometers. In another embodiment, the tobacco fiber is processed to have a size of 75 micrometers or less. In one embodiment, the tobacco fiber comprises long-cut tobacco, which can be cut or shredded to a width of about 10 cuts per inch to about 110 cuts per inch and a length of about 0.1 inch to about 1 inch. The double-cut tobacco fiber can have a particle size range such that about 70% of the double-cut tobacco fiber falls between -20 mesh and 80 mesh.

[0241] The provided tobacco material can be processed to have a total oven volatile content of about 10% by weight or more, about 20% by weight or more, about 40% by weight or more, about 15% to about 25% by weight, about 20% to about 30% by weight, about 30% to about 50% by weight, about 45% to about 65% by weight, or about 50% to about 60% by weight. Those skilled in the art will appreciate that "moist" tobacco typically refers to tobacco having an oven volatile content of about 40% to about 60% by weight (e.g., about 45% to about 55% by weight, or about 50% by weight). As used herein, "oven volatile content" is determined by calculating the percentage weight loss of a sample after drying the sample for 3.25 hours in a forced air oven preheated to 110°C. Oral products may have an overall oven volatile content that differs from the oven volatile content of the tobacco fiber used to make the oral product. The processing steps described may reduce or increase the oven volatile content.

[0242] The following list provides a first set of exemplary embodiments: 1. A tobacco plant, or a tobacco genotype or tobacco part thereof, comprising a mutation in the Nic1b locus, wherein said tobacco plant is capable of producing leaves, when cured, having a USDA Grade Index value of 50 or greater. 2. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 1, wherein said tobacco plant further comprises a mutation in an ERF gene at the Nic2 locus. 3. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 1, wherein said tobacco plant further comprises one or more mutations in two or more, three or more, four or more, five or more, six or more, or all seven genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168. 4. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 1, wherein said tobacco plant further comprises one or more mutations in ERF189, ERF115, or both. 5. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 1, wherein said tobacco plant, when cured, is capable of producing leaves having a USDA Grade Index value selected from the group consisting of: a value of 55 or greater, a value of 60 or greater, a value of 65 or greater, a value of 70 or greater, a value of 75 or greater, a value of 80 or greater, a value of 85 or greater, a value of 90 or greater, and a value of 95 or greater. 6. The tobacco plant of embodiment 1, or a tobacco genotype or tobacco part thereof, wherein the tobacco plant, when cured, is capable of producing leaves having a USDA grade index value equivalent to the USDA grade index value of a control plant when grown and cured under similar conditions, and the control plant shares essentially the same genetic background as the tobacco plant except for the mutation. 7. The tobacco plant of embodiment 1, or a tobacco genotype or tobacco part thereof, wherein the tobacco plant, when cured, is capable of producing leaves having a USDA grade index value that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% of the USDA grade index value of a control plant when grown and cured under similar conditions, and the control plant shares essentially the same genetic background as the tobacco plant except for the mutation. 8. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 1, wherein said tobacco plant comprises a level of nicotine that is less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, or less than 80% of the nicotine level of a control plant when grown under similar growing conditions, and wherein said control plant shares essentially the same genetic background as said tobacco plant except for said mutation. 9. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 1, wherein said tobacco plant comprises a nicotine level selected from the group consisting of less than 3%, less than 2.75%, less than 2.5%, less than 2.25%, less than 2.0%, less than 1.75%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, and less than 0.05%. 10. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 1, wherein said tobacco plant further comprises a transgene or mutation that directly suppresses the expression or activity of one or more genes encoding a product selected from the group consisting of PMT, MPO, QPT, BBL, A622, aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, ornithine decarboxylase, arginine decarboxylase, nicotine uptake permease (NUP), and MATE transporter. 11. A tobacco plant, or a tobacco genotype or tobacco part thereof, comprising a mutation in the Nic1b locus, wherein said tobacco plant, when cured, is capable of producing leaves having a USDA grade index value equivalent to the USDA grade index value of a control plant when grown and cured under similar conditions, said control plant sharing essentially the same genetic background as said tobacco plant except for said mutation, or a tobacco genotype or tobacco part thereof. 12. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 11, wherein said tobacco plant further comprises a mutation in an ERF gene at the Nic2 locus. 13. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 11, wherein said tobacco plant further comprises one or more mutations in two or more, three or more, four or more, five or more, six or more, or all seven genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168. 14. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 11, wherein said tobacco plant further comprises one or more mutations in ERF189, ERF115, or both. 15. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 11, wherein said tobacco plant, when cured, is capable of producing leaves having a USDA Grade Index value selected from the group consisting of a value of 55 or greater, a value of 60 or greater, a value of 65 or greater, a value of 70 or greater, a value of 75 or greater, a value of 80 or greater, a value of 85 or greater, a value of 90 or greater, and a value of 95 or greater. 16. The tobacco plant of embodiment 11, or a tobacco genotype or tobacco part thereof, wherein the tobacco plant, when cured, is capable of producing leaves having a USDA grading index value of at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% of the grading index of the control plant. 17. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 11, wherein said tobacco plant comprises a level of nicotine that is less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, or less than 80% of the nicotine level of said control plant when grown under similar growing conditions. 18. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 11, wherein said tobacco plant further comprises a transgene or mutation that directly suppresses the expression or activity of one or more genes encoding a product selected from the group consisting of PMT, MPO, QPT, BBL, A622, aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, ornithine decarboxylase, arginine decarboxylase, nicotine uptake permease (NUP), and MATE transporter. 19. A plant or tobacco genotype of a tobacco variety containing a nic1b mutation, said tobacco variety having a leaf grading index equivalent to the leaf grading index of a control tobacco variety when grown under similar growing conditions, said control tobacco variety sharing essentially the same genetic background as said tobacco variety except for said mutation. 20. The plant or tobacco genotype of embodiment 19, wherein said tobacco variety further comprises a mutation in the ERF gene at the Nic2 locus. 21. The plant or tobacco genotype of embodiment 19, wherein said tobacco plant further comprises one or more mutations in two or more, three or more, four or more, five or more, six or more, or all seven genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168. 22. The plant or tobacco genotype of embodiment 19, wherein said tobacco plant further comprises one or more mutations in ERF189, ERF115, or both. 23. A non-transgenic tobacco plant, or tobacco genotype or tobacco part thereof, comprising a nicotine level selected from the group consisting of less than 3%, less than 2.75%, less than 2.5%, less than 2.25%, less than 2.0%, less than 1.75%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, and less than 0.05%, wherein the tobacco plant, when cured, is capable of producing leaves having a USDA Grade Index value of 50 or greater, 55 or greater, 60 or greater, 65 or greater, 70 or greater, 75 or greater, 80 or greater, 85 or greater, 90 or greater, and 95 or greater. 24. The non-transgenic tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 23, wherein said non-transgenic tobacco plant is capable of producing leaves containing a nicotine level of less than 2.0% and, when cured, having a USDA Grade Index value of 70 or greater. 25. The tobacco plant of non-transgenic embodiment 23, or a tobacco genotype or tobacco part thereof, wherein the non-transgenic tobacco plant is capable of producing leaves containing a nicotine level of less than 1.0% and, when cured, having a USDA Grade Index value of 70 or greater. 26. A tobacco plant, or tobacco genotype or tobacco part thereof, comprising a non-transgenic mutation in the Nic1b locus, wherein the non-transgenic mutation reduces the nicotine level of the tobacco plant to less than 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of the nicotine level of a control plant when grown under similar growing conditions, wherein the tobacco plant is capable of producing leaves, when cured, having a USDA grade index value equivalent to that of the control plant, and wherein the control plant shares essentially the same genetic background as the tobacco plant except for the non-transgenic mutation. 27. A population of tobacco plants of any one of embodiments 1-26. 28. A dried tobacco material from the tobacco plant of any one of embodiments 1-26. 29. The cured tobacco material of embodiment 28, produced by a curing method selected from the group consisting of hot air curing, air drying, flame drying, and sun drying. 30. A tobacco blend comprising the dried tobacco material of embodiment 28. 31. The tobacco blend of embodiment 30, wherein the dried tobacco material constitutes approximately at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% by weight of the dried tobacco in the tobacco blend. 32. The tobacco blend of embodiment 30, wherein the dried tobacco material constitutes approximately at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% by volume of the dried tobacco in the tobacco blend. 33. A tobacco product comprising the dried tobacco material of embodiment 28. 34. The tobacco product of embodiment 33, selected from the group consisting of cigarettes, cigarillos, non-vented recess filter cigarettes, vented recess filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and cut tobacco. 35. The tobacco product of embodiment 33, which is a smokeless tobacco product. 36. The tobacco product of embodiment 35, wherein said smokeless tobacco product is selected from the group consisting of loose-leaf chewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff. 37. Reconstituted tobacco comprising the dried tobacco material of embodiment 28. 38. A tobacco plant, or a tobacco genotype or part thereof, comprising a mutation in the Nic1b locus, said mutation not being present in the LA Burley 21 variety. 39. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein said tobacco plant comprises a shorter chromosomal introgression at the Nic1b locus compared to the LA Burley 21 variety. 40. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein said tobacco plant comprises a lower level of nicotine compared to a control tobacco plant not having said mutation when grown under similar growing conditions. 41. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein said tobacco plant comprises a level of nicotine that is less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, or less than 80% of the nicotine level in a control tobacco plant not having said mutation when grown under similar growing conditions. 42. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein said tobacco plant comprises a lower level of total alkaloids compared to a control tobacco plant not having said mutation when grown under similar growth conditions. 43. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein said tobacco plant comprises a lower level of one or more alkaloids selected from the group consisting of nicotine, nornicotine, anabasine, and anatabine compared to a control tobacco plant not having said mutation when grown under similar growth conditions. 44. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein said tobacco plant comprises a similar level of one or more compounds selected from the group consisting of 3-methylvaleric acid, valeric acid, isovaleric acid, labdanoids, cembranoids, sugar esters, and reducing sugars compared to a control tobacco plant not having said mutation when grown under similar growth conditions. 45. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein the mutation is homozygous. 46. ​​The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein the mutation is heterozygous. 47. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein said mutation is selected from the group consisting of a point mutation, a deletion, an insertion, a duplication, and an inversion. 48. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein said mutation is introduced by an approach selected from the group consisting of random mutagenesis and targeted mutagenesis. 49. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 48, wherein said targeted mutagenesis is mediated by meganucleases, zinc finger nucleases, TALENs, or CRISPRs. 50. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein said tobacco plant further comprises a mutation in an ERF gene at the Nic2 locus. 51. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein the mutation is located within a gene comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 3-37, 146, 149, 152-156, 202, 203, and 184-186, and fragments thereof. 52. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein said mutation reduces the expression or activity of said gene. 53. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein the mutation is located within a gene comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 4, 14, 15, 17, 18, 19, 37, 39, 49, 50, 52, 53, 54, 202-205, and 72, and fragments thereof. 54. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein the mutation is located within a gene comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 14, 17, 18, 19, 49, 52, 53, 202-205, and 54, and fragments thereof. 55. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein the mutation is located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 4, 14, 15, 17, 18, 19, 37, 39, 49, 50, 52, 53, 202-205, 54, and 72, and fragments thereof. 56. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein the mutation is located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 14, 17, 18, 19, 49, 52, 53, 202-205, and 54, and fragments thereof. 57. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein the mutation is located within a gene comprising a coding sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 49, 52, 53, 204, 205, and 54, and fragments thereof. 58. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein the mutation is located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 49, 52, 53, 204, 205, and 54, and fragments thereof. 59. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein the mutation is located in a gene encoding a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 84, 87, 88, and 89, and fragments thereof. 60. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein said plant further comprises reduced levels of mRNA, protein, or both of one or more genes encoding products selected from the group consisting of PMT, MPO, QPT, BBL, MATE, and A622, compared to a tobacco plant not having said mutation when grown under similar growth conditions. 61. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein said plant further comprises a transgene or mutation that suppresses the expression or activity of one or more genes encoding a product selected from the group consisting of PMT, MPO, QPT, BBL, A622, aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, ornithine decarboxylase, arginine decarboxylase, nicotine uptake permease (NUP), and MATE transporter. 62. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein said tobacco plant is a hybrid. 63. The tobacco plant of embodiment 38, or a tobacco genotype or tobacco part thereof, wherein said part is selected from the group consisting of leaves, stems, roots, seeds, flowers, pollen, anthers, ovules, pedicels, fruits, meristems, cotyledons, hypocotyls, pods, embryos, endosperms, explants, callus, tissue cultures, shoots, cells, and protoplasts. 64. The tobacco plant of embodiment 38, or a tobacco genotype or tobacco part thereof, wherein the tobacco plant is from a variety selected from the group consisting of flue-cured tobacco, air-cured tobacco, dark flue-cured tobacco, and Galpao tobacco, and Oriental tobacco. 65. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein said tobacco plant is from a variety selected from the group consisting of Burley tobacco, Maryland tobacco, and dark air-cured tobacco. 66. A population of tobacco plants according to embodiment 38. 67. A dried tobacco material from the tobacco plant of embodiment 38. 68. The cured tobacco material of embodiment 67, comprising a lower level of nicotine compared to a cured tobacco material from a control tobacco plant not carrying said mutation. 69. The dried tobacco material of embodiment 67, wherein the tobacco plant contains nicotine at a level of 0.2% to 0.6%. 70. The cured tobacco material of embodiment 67, wherein the tobacco plant comprises a nicotine level of 1.0% to 3.0%. 71. The cured tobacco material of embodiment 67, produced by a drying method selected from the group consisting of hot air drying, air drying, flame drying, and sun drying. 72. A tobacco blend comprising the dried tobacco material of embodiment 67. 73. A tobacco product comprising the dried tobacco material of embodiment 67. 74. The tobacco product of embodiment 73, selected from the group consisting of cigarettes, cigarillos, non-vented recess filter cigarettes, vented recess filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and cut tobacco. 75. The tobacco product of embodiment 73, which is a smokeless tobacco product. 76. The tobacco product of embodiment 75, wherein said smokeless tobacco product is selected from the group consisting of loose-leaf chewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff. 77. Reconstituted tobacco comprising the dried tobacco material of embodiment 67. 78. A recombinant DNA construct comprising a promoter operably linked to a polynucleotide that is functional in a tobacco cell and encodes a polypeptide having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73-107, 148, 151, 180-183, 206, 207, and 190-192, and fragments thereof. 79. A tobacco plant, or a tobacco genotype or tobacco part thereof, comprising the recombinant DNA construct of embodiment 78. 80. The tobacco plant of embodiment 79, or a tobacco genotype or tobacco part thereof, wherein said tobacco plant comprises a higher level of nicotine compared to a control tobacco plant not harboring said recombinant DNA construct. 81. A dried tobacco material from the tobacco plant of embodiment 79. 82. A tobacco product comprising the dried tobacco material of embodiment 81. 83. A method for increasing nicotine levels in a tobacco plant, comprising transforming the tobacco plant with the recombinant DNA construct of embodiment 78. 84. A recombinant DNA construct comprising a promoter operably linked to a polynucleotide encoding an RNA molecule capable of binding to RNA encoding a polypeptide having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73-107, 148, 151, 180-183, 206, 207, and 190-192, and fragments thereof, wherein the RNA molecule represses expression of the polypeptide. 85. A tobacco plant, or a tobacco genotype or tobacco part thereof, comprising the recombinant DNA construct of embodiment 84. 86. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 85, wherein the RNA molecule is selected from the group consisting of microRNA, siRNA, and trans-acting siRNA. 87. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 85, wherein said polynucleotide encodes a double-stranded RNA. 88. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 85, wherein said tobacco plant comprises a lower level of nicotine compared to a control tobacco plant not harboring said recombinant DNA construct. 89. A dried tobacco material from the tobacco plant of embodiment 85. 90. A tobacco product comprising the dried tobacco material of embodiment 89. 91. A method for reducing nicotine levels in a tobacco plant, comprising transforming the tobacco plant with the recombinant DNA construct of embodiment 84. 92. A tobacco plant, or a tobacco genotype or tobacco part thereof, comprising a heterologous expression cassette comprising a Nic1b inhibitory sequence of a gene comprising a sequence selected from the group consisting of SEQ ID NOs: 3-37, 146, 149, 152-156, 202, 203, and 184-186, and fragments thereof, wherein the inhibitory sequence is operably linked to a promoter functional in plant cells, and the inhibitory sequence is selected from the group consisting of SEQ ID NOs: 3-37, 146, 149, 152-156, 202, 203, and 184-186, and fragments thereof. 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotide fragment of the sequence. 93. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 92, wherein said Nic1b inhibitory sequence is capable of being transcribed as an inhibitory polynucleotide selected from the group consisting of single-stranded RNA polynucleotides, double-stranded RNA polynucleotides, and combinations thereof. 94. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 92, wherein said promoter is selected from the group consisting of a constitutive promoter, an inducible promoter, and a tissue-preferred promoter. 95. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 92, wherein the promoter is a root-specific promoter. 96. A tobacco plant, or a tobacco genotype or tobacco part thereof, comprising a heterologous expression cassette comprising a Nic1b inhibitory sequence of a gene comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 14, 15, 17, 18, 19, 37, 39, 49, 50, 52, 53, 54, 202-205, and 72, and fragments thereof, wherein the inhibitory sequence is operably linked to a promoter functional in plant cells, and the inhibitory sequence is selected from the group consisting of SEQ ID NOs: 4, 14, 15, 17, 18, 19, 37, 39, 49, 50, 52, 53, 54, 202-205, and 72, and fragments thereof. 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotide fragment of a sequence selected from the group consisting of: a tobacco plant, or a tobacco genotype or portion thereof, having at least 90% sequence identity to a fragment of at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotide fragment of a sequence selected from the group consisting of: 97. A method for transferring a low-nicotine trait to a tobacco variety, comprising: a. crossing a first tobacco variety that includes a low-nicotine trait with a second tobacco variety that does not have the low-nicotine trait to produce one or more progeny tobacco plants; b. genotyping the one or more progeny tobacco plants for polymorphic markers associated with the low nicotine trait, wherein the polymorphic markers are in a chromosomal interval flanked by any two SNP markers selected from the group consisting of SEQ ID NOs: 125-145 and 193-201; and c. selecting progeny tobacco plants containing the low nicotine trait. A method comprising: 98. The method of embodiment 97, further comprising backcrossing the selected progeny tobacco plant with the second tobacco variety. 99.d. crossing the selected progeny plant with itself or with the second tobacco variety to produce one or more additional progeny tobacco plants; and e. Selecting additional progeny tobacco plants containing the low nicotine trait. 98. The method of embodiment 97, further comprising: 100. The method of embodiment 99, wherein said selecting step (e) comprises marker-assisted selection. 101. The method of embodiment 97, wherein a single gene transformation comprising said low nicotine trait is produced. 102. The method of embodiment 97, wherein the second tobacco variety is an elite variety. 103. The method of embodiment 97, wherein said genotyping involves one or more molecular marker assays. 104. The method of embodiment 97, wherein the polymorphic marker comprises a polymorphism selected from the group consisting of a single nucleotide polymorphism (SNP), an insertion or deletion (indel) in a DNA sequence, a simple sequence repeat (SSR) in a DNA sequence, a restriction fragment length polymorphism (RFLP), and a tag SNP. 105. The method of embodiment 97, wherein said genotyping comprises assaying for a nucleic acid sequence located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 3-37, 146, 149, 152-156, 202, 203, and 184-186, and fragments thereof. 106. The method of embodiment 97, wherein said genotyping comprises assaying for a nucleic acid sequence located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 4, 14, 15, 17, 18, 19, 37, 39, 49, 50, 52, 53, 54, 202-205, and 72, and fragments thereof. 107. The method of embodiment 97, wherein said genotyping comprises assaying for a nucleic acid sequence located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 14, 17, 18, 19, 49, 52, 53, 202-205, and 54, and fragments thereof. 108. The method of embodiment 97, wherein said first tobacco variety is selected from the group consisting of LA Burley 21, LAFC53, and LN KY171. 109. The method of embodiment 97, wherein the selected progeny tobacco plants comprise a shorter chromosomal introgression at the Nic1b locus compared to LA Burley 21, LAFC53, and LN KY171. 110. A method for transferring a low-nicotine trait to a tobacco variety, comprising: a. crossing a first tobacco variety that includes a low-nicotine trait with a second tobacco variety that does not have the low-nicotine trait to produce one or more progeny tobacco plants; b. genotyping the one or more progeny tobacco plants for polymorphic markers associated with the low nicotine trait, wherein the polymorphic marker is within 20 cM of any one of the SNP markers selected from the group consisting of SEQ ID NOs: 125-145 and 193-201, or any locus having a sequence selected from the group consisting of SEQ ID NOs: 1, 3-37, 146, 149, 152-156, 202, 203, and 184-186; and c. selecting progeny tobacco plants containing the low nicotine trait. A method comprising: 111. The method of embodiment 110, wherein said genotyping comprises assaying for a nucleic acid sequence located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 3-37, 146, 149, 152-156, 202, 203, and 184-186, and fragments thereof. 112. The method of embodiment 110, wherein said genotyping comprises assaying for a nucleic acid sequence located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 4, 14, 15, 17, 18, 19, 37, 39, 49, 50, 52, 53, 54, 202-205, and 72, and fragments thereof. 113. The method of embodiment 110, wherein said genotyping comprises assaying for a nucleic acid sequence located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 14, 17, 18, 19, 49, 52, 53, 202-205, and 54, and fragments thereof. 114. A method for selecting tobacco plants having a low nicotine trait, comprising: a. isolating nucleic acids from a collection of tobacco germplasm; b. assaying the nucleic acid for one or more markers closely linked to the Nic1b locus; and c. Selecting tobacco plants having a low-nicotine trait based on said marker assay. A method comprising: 115. The method of embodiment 114, wherein the one or more markers are within about 20 cM, 10 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.5 cM, or less than 0.5 cM of any one of the SNP markers selected from the group consisting of SEQ ID NOs: 125-145 and 193-201, or any locus having a sequence selected from the group consisting of SEQ ID NOs: 1, 3-37, 146, 149, 152-156, 202, 203, and 184-186. 116. The method of embodiment 114, wherein said assaying comprises assaying for a nucleic acid sequence located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 3-37, 146, 149, 152-156, 202, 203, and 184-186, and fragments thereof. 117. The method of embodiment 114, wherein said assaying comprises assaying for a nucleic acid sequence located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 4, 14, 15, 17, 18, 19, 37, 39, 49, 50, 52, 53, 54, 202-205, and 72, and fragments thereof. 118. The method of embodiment 114, wherein said assaying comprises assaying for a nucleic acid sequence located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 14, 17, 18, 19, 49, 52, 53, 202-205, and 54, and fragments thereof. 119. The method of embodiment 114, further comprising determining the nicotine level of the selected plant to confirm the low nicotine trait. 120. The method of embodiment 114, wherein said collection of tobacco germplasm is a haploid breeding population. 121. A tobacco plant, or a tobacco genotype or tobacco part thereof, comprising a chromosomal introgression obtainable from any one of LA Burley 21, LAFC53, and LN KY171, wherein said chromosomal introgression is flanked by and does not include any two of SNP markers selected from the group consisting of SEQ ID NOs: 125-145 and 193-201, or is flanked by any two loci having sequences selected from the group consisting of SEQ ID NOs: 1, 3-37, 146, 149, 152-156, 202, 203, and 184-186, wherein said tobacco plant, when cured, is capable of producing leaves having a USDA Grade Index value of 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, or 95 or more. 122. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 121, wherein said tobacco plant comprises a nicotine level selected from the group consisting of less than 3%, less than 2.75%, less than 2.5%, less than 2.25%, less than 2.0%, less than 1.75%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, and less than 0.05%. 123. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 121, wherein the chromosomal introgression is flanked by and does not include any two SNP markers selected from the group consisting of SEQ ID NOs: 126-135. 124. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 121, wherein said chromosomal introgression is flanked by and does not include SNP markers SEQ ID NOs: 129-132. 125. A tobacco plant, or a tobacco genotype or tobacco part thereof, comprising a first chromosome introgression obtainable from any one of LA Burley 21, LAFC53, and LN KY171, wherein said first chromosome introgression is flanked by, but does not include, any two of Nic1b marker numbers 1 to 207, and said tobacco plant, when cured, is capable of producing leaves having a USDA grade index value equivalent to the USDA grade index value of a control plant when grown in similar growing conditions, and said control plant shares essentially the same genetic background as said tobacco plant except for said mutation, or a tobacco genotype or tobacco part thereof. 126. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 125, wherein said tobacco plant comprises a level of nicotine that is less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, or less than 80% of the nicotine level of said control plant when grown under similar growing conditions. 127. A population of tobacco plants according to any one of embodiments 121-126 and 136-141. 128. A dried tobacco material from the tobacco plant of any one of embodiments 121-126. 129. The cured tobacco material of embodiment 128, produced by a drying method selected from the group consisting of hot air drying, air drying, flame drying, and sun drying. 130. A tobacco blend comprising the dried tobacco material of embodiment 128. 131. The tobacco blend of embodiment 130, wherein the dried tobacco material constitutes approximately at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% by weight of the dried tobacco in the tobacco blend. 132. The tobacco blend of embodiment 130, wherein the dried tobacco material constitutes approximately at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% by volume of the dried tobacco in the tobacco blend. 133. A tobacco product comprising the dried tobacco material of embodiment 128. 134. The tobacco product of embodiment 133, selected from the group consisting of cigarettes, cigarillos, non-vented recess filter cigarettes, vented recess filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and cut tobacco. 135. The tobacco product of embodiment 133, selected from the group consisting of loose-leaf chewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff. 136. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein the mutation is located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 146, 153, 154, 17, 18, 19, 202, and 203, and fragments thereof. 137. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein the mutation is located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 153, 154, 17, 18, 19, 202, and 203, and fragments thereof. 138. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein the mutation is located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 153, 154, 17, 18, 19, and 203, and fragments thereof. 139. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein the mutation is located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 17, 18, and 19, and fragments thereof. 140. The tobacco plant or part thereof of embodiment 38, wherein the mutation is located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 3, 18, and 19, and fragments thereof. 141. The tobacco plant, or tobacco genotype or tobacco part thereof, of embodiment 38, wherein the mutation is located in a gene encoding a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87, 88, 89, 180, 181, 206, and 207, and fragments thereof.

[0243] Having now generally described the present disclosure, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limitations of the disclosure unless specified. [Example]

[0244] Example 1: Development of the TN90 strain with a low alkaloid trait and plants with a high intermediate alkaloid trait carrying the nic1 and nic2 deletion marker alleles A breeding population is developed from a cross between LA BU21 (Low Alkaloid Burley 21 with nic1 and nic2 mutations, donor parent) and the elite cultivar TN90 (recipient parent). The goal is to obtain low-alkaloid plants that also have improved leaf quality when compared to the parent LA BU21 strain, which has poor leaf characteristics. F2 plants with the desired characteristics (low alkaloid levels, improved leaf quality) are further self-pollinated to obtain the F3 and subsequently the F5 generations. Figure 1 shows the breeding method followed for the development of the self-pollinated TN90 strain with low-alkaloid traits.

[0245] As shown in Figure 1, 22 F3 plants are screened on the Axiome® array, which was developed based on approximately 170,000 polymorphic SNPs observed in several resequenced lines belonging to different tobacco species (hot wind, dark, oriental, and burley) in relation to the TN90 tobacco genome sequence database. Plant 22-6 showed approximately 40.6% restoration of the recipient parent genome and is selected for further development. The F3 generation also exhibits low alkaloid levels, which is consistent with those observed in LA BU21. The F4 generation is screened using Nic1 and Nic2 KASP assays (Nic1 and Nic2, respectively). KASP and Nic2 KASP(See SEQ ID NOS: 135 and 137 in US2016 / 0374387, which correspond to the markers.) All 192 screened F4 plants had two deletions, and three plants (ds1059-138, ds1059-143, and ds1059-192) were selected for further development. Self-pollinated seeds from these three plants were further collected and the F5 generation was evaluated. All 220 F5 plants were nic1 KASP Deletion alleles and nic2 KASP The F5 plants are homozygous for both deletion alleles. Alkaloid levels in the F5 plants are determined by measuring pooled leaf samples collected from the third, fourth, and fifth leaves from the top of the plant two weeks after topping. Not all plants exhibit the low-alkaloid trait; marked differences in leaf phenotypes are also observed, with some lines exhibiting good leaf quality consistent with that observed in commercial burley tobacco (Table 1).

[0246] Table 1. Alkaloid levels and leaf phenotypes of selected F5 and control plants. All F5 plants were nic1 KASP Deletion alleles and nic2 KASP Homozygous for both deletion alleles. TIFF2025157497000002.tif103162

[0247] Example 2: Identification of additional genomic regions associated with the low-alkaloid trait Homozygous nic1 KASP and nic2 KASP The F5 plants of Example 1, which have high alkaloid levels despite having the deletion allele, are nic1 KASP and nic2 KASP This suggests that the deletion markers do not exhibit 100% association with the low alkaloid trait. To investigate this finding, we investigated the nic1 gene in addition to LAFC53 (a flue-dried type with nic1 and nic2 introgressions), LN KY171 (a dark type with nic1 and nic2 introgressions), and KY171 (a dark variety without nic1 and nic2 introgressions). KASP and nic2KASP Two F5 strains (G61-36 and G61-39) homozygous for the deletion allele but exhibiting high alkaloid levels, as well as nic1 KASP and nic2 KASP Further resequencing was performed on two F5 strains (G61-31 and G61-37) that were homozygous for the deletion allele and exhibited low alkaloid levels. These seven strains, along with the previously resequenced strains BU21, HI BU21, LI BU21, and LA BU21, were mapped using the TN90 genome database.

[0248] Coverage analysis of the mapped reads led to the identification of a 2000-3000 bp region (SEQ ID NO: 1, hereafter referred to as the "LA-associated region") approximately 2 million bp downstream of the nic1 deletion segment NT1.0-Scaffold0002504 previously described in US2016 / 0374387. The LA-associated region was found in LA BU21, LI BU21, LN KY171, and LAFC53, as well as in the low-alkaloid homozygous nic1 KASP nic2 KASP In contrast, the LA-associated region is deleted in the F5 strains G61-31 and G61-37. In addition to wild-type (e.g., TN90, BU21, K326, NLM, Katerini), the high-alkaloid homozygous nic1 KASP nic2 KASP It is not deleted (i.e., present) in F5 strains G61-36 and G61-39. The LA-associated region does not contain any known genes. A serine-threonine protein phosphatase gene is found 4877 bp upstream of this region, and an ERF (homolog of ERF189) is found 59,945 bp downstream of this region.

[0249] Furthermore, SNP genotyping of F plants (22-6) revealed that the genomic region beginning 1,256,063 bp downstream of the nic1 deletion segment NT1.0-Scaffold0002504 and ending 2,766,118 bp downstream (approximately 1,510,055 bp long, SEQ ID NO: 2; referred to herein as the "Nic1b region") was heterozygous in F plants (including the LA-associated region). This suggests that the low-alkaloid allele in the Nic1b region may have originated in cigars (LA BU21 was developed from a cross between BU21 and a low-alkaloid Cuban cigar cultivar) and segregates in the resulting F and subsequent generations.

[0250] We also genotyped 47 F4 and 48 F5 plants using an Axiome array containing approximately 170,000 SNPs to further validate the correlation between alkaloid levels and the Nic1b region as well as the LA-associated region. Analysis of three progenitor F4 plants (ds1059-138, ds1059-143, and ds1059-192) showed the LA-associated and Nic1b regions as heterozygous or harboring the LA BU21-like (B) allele. Similarly, 48 F5 plants showed segregation in the LA-associated region and the Nic1b region, with individuals carrying the B allele (LA BU21-like) exhibiting low alkaloid levels (0.137%–0.417% dry weight), and individuals carrying the heterozygous state (H) or the wild-type allele (A) exhibiting moderate to high alkaloid levels (1.793%–5.419% dry weight).

[0251] Example 3: Identification of genes and markers in the Nic1b region Sequence analysis of the Nic1b region reveals at least 35 annotated genes from the region (Table 2). N ic1b C igar GeneThese NCG genes are referred to as "NCGs" ("NCGs") 1-35. Among these NCG genes, six ethylene-responsive transcription factor-like (ERF-like) genes were identified. RNA-seq data showed that 10 NCGs exhibited reduced expression in LA BU21 plants relative to BU21 from root samples collected 72 h after pinching. Seven NCGs, including four of the six ERF-like genes, were down-regulated in LA BU21 compared to BU21, while the other three NCGs were up-regulated.

[0252] Each of the 35 NCGs in LA BU21, TN90, and selected F5 plants is resequenced to identify specific natural mutations present in LA BU21 that may cause loss of gene function. Additional resequencing analysis is also performed to identify mutations in non-coding sequences (e.g., promoters) that may cause dysregulation of gene expression.

[0253] Additionally, polymorphisms within and adjacent to the Nic1b region will also be identified to develop SNP markers. 21 SNP markers will be developed for genotyping the Nic1b region (Table 3). Additional SNP markers are provided in Table 4. These markers are within the Nic1b region and immediately adjacent to various ERF genes. They possess alleles that can be used to distinguish normal-alkaloid strains from low-alkaloid strains.

[0254] (Table 2) Annotated genes in the Nic1b region. a g31431 and g31432 appear to exhibit alternative splicing (see Figure 2). b g31446 may represent an alternative splicing site. TIFF2025157497000003.tif236131TIFF2025157497000004.tif236149TIFF2025157497 000005.tif236149TIFF2025157497000006.tif236161TIFF2025157497000007.tif23660

[0255] (Table 3) Markers in and around the Nic1b region. (*) Genomic location is based on the TN90 genome, which is also used to represent NCG gene locations. TIFF2025157497000008.tif201137TIFF2025157497000009.tif201126

[0256] (Table 4) SNP markers closely adjacent to multiple ERF genes from the Nic1b region. (*) Genomic location is based on the reference TN90 genome. TIFF2025157497000010.tif201142TIFF2025157497000011.tif20144

[0257] Example 4: Transgenic approach to developing tobacco varieties with desirable nicotine levels Both overexpression and suppression approaches will be employed to investigate the function of NCGs and develop tobacco varieties with desirable nicotine levels. Two sets of transgenic plants will be generated: one overexpression approach using full-length coding sequences, and the other suppression approach using artificial microRNA or RNAi sequences. All 35 NCGs (NCG1-NCG35) that exhibit differential expression between TN90 and LA BU21 will be tested, focusing in particular on NCGs encoding ERF-like proteins. Some overexpression studies will be performed in a nic1 nic2 double mutant background (e.g., LA BU21) to test the ability of identified genes (individually or in combination) to complement or rescue the low-alkaloid mutant phenotype from the mutant nic1 allele.

[0258] For the expression of full-length coding sequences or artificial microRNA sequences, an expression vector is constructed to contain a cassette with a kanamycin selection marker (NPT II) under the direction of the actin 2 promoter and a NOS terminator, in addition to the CsVMV promoter and NOS terminator. Exemplary artificial microRNA sequences can be found in Table 2. Those skilled in the art will understand that other target sequences can be used in the construction of inhibitory constructs or other transgenic approaches for gene silencing (e.g., RNAi, trans-acting siRNA, etc.).

[0259] A nucleic acid construct carrying a transgene of interest is introduced into tobacco leaf discs using DNA bombardment or biolistic approaches. See, for example, Sanford et al., 1993, Methods Enzymol., 217:483-510; and Okuzaki and Tabei, 2012, Plant Biotechnology, 29:307-310. Briefly, plasmid DNA containing a transformation cassette is coated onto 1 μm gold particles (DNA / gold) as follows: The 1 μm gold particles are baked at 180°C for 12 hours to prepare a stock solution (40 mg / ml). To prepare a mixture for 10 shots, 100 μl of the stock solution is mixed with 40 μl of expression vector DNA (1 μg / μl), 100 μl of 2.5 M CaCl2, and 40 μl of 0.1 M spermidine in a 1.5 ml tube. The mixture is centrifuged at 13,000 × g for 30 seconds, and the pellet is washed with 500 μl of 100% ethanol. The DNA / gold mixture is suspended in 100 μl of water, and 10 μl is applied to the macrocarrier, allowed to dry, and then bombarded. Two shots per plate are bombarded using an 1,100-psi crushing disk under partial vacuum (711 mmHg) in a PDS-1000 / He system (Bio-Rad Laboratories, Hercules, CA, USA). Narrow Leaf Madole (NLM) and Tennessee 90 (TN90) tobacco leaf disks are used for transformation with RNAi constructs and full-length gene constructs. Whole tobacco leaves (approximately 45 × 30 mm long) are placed on MS medium overnight, and on the second day, the leaf disks are bombarded with the constructs. The leaves were then cut into small pieces (approximately 5 x 5 mm) and grown on TOM medium (MS medium containing 20 g sucrose / L; 1 mg / L IAA and 2.5 mg / L BAP) for 3-5 days at 27°C, after which they were transferred to TOM medium containing 300 mg / L kanamycin (TOM-Kan). The tissues were transferred to new TOM-Kan plates (27°C, 16 h light) every 2-3 weeks for 4-6 weeks. Kanamycin-resistant primary shoots were regenerated 4-6 weeks after bombardment. The shoots were then transferred to MS-kanamycin plates for root development.Leaves and / or roots from the T1 plants (and subsequent generations) are then evaluated to determine the amount of one or more alkaloids.

[0260] Example 5: Random mutagenesis to develop novel mutations in or around the Nic1b region that confer low-alkaloid traits Random mutagenesis of tobacco plants is performed using ethyl methanesulfonate (EMS) mutagenesis or fast neutron bombardment. EMS mutagenesis consists of chemically inducing random point mutations throughout the genome. Fast neutron mutagenesis consists of exposing seeds to neutron bombardment, which causes large deletions through double-stranded DNA breaks.

[0261] For EMS mutagenesis, 1 gram (approximately 10,000 seeds) of Tennessee 90 tobacco (TN90) seeds were washed in 0.1% Tween for 15 minutes and then soaked in 30 ml of ddH2O for 2 hours. 150 μl of 0.5% EMS (Sigma, catalog number M-0880) was then mixed with the seed / ddH2O solution and incubated under a hood at room temperature (RT; approximately 20°C) for 8-12 hours (rotating at 30 rpm). The liquid was then removed from the seeds and mixed overnight in 1 M NaOH for decontamination and discarded. The seeds were then washed twice with 100 ml of ddH2O for 2-4 hours. The washed seeds were then suspended in 0.1% agar solution.

[0262] The EMS-treated seeds in agar solution are evenly spread in flats onto water-soaked Carolina's Choice Tobacco Mix (Carolina Soil Company, Kinston, NC) at approximately 2,000 seeds / flat. The flats are then covered with plastic wrap and placed in a growth chamber. Once the seedlings emerge from the soil, the plastic wrap is punctured to gradually reduce humidity. The plastic wrap is completely removed after two weeks. The flats are moved to a greenhouse and treated with NPK fertilizer. The seedlings are packed into float trays and grown to transplanting size. The plants are then transplanted into the field. During growth, the plants self-pollinate to form M1 seeds. At maturity, five pods are harvested from each plant, and individual designations are given to the set of seeds from each plant. This forms the M1 population. A composite of M1 seeds from each M0 plant is grown, and leaves from the M1 plants are collected for DNA extraction. Target genes within, around, or adjacent to the Nic1b region will be amplified and sequenced to identify mutations in all 35 NCGs (NCG1-NCG35) that exhibit differential expression between TN90 and LA BU21, focusing in particular on NCGs encoding ERF-like proteins.

[0263] Example 6: Targeted mutagenesis to develop novel mutations in or around the Nic1b region that confer low-alkaloid traits We generate tobacco strains with low nicotine while maintaining high leaf quality by introducing mutations into and around the Nic1b locus (e.g., ERF101, ERF110, ERF16, ERF130, and NCGs) via precise genome engineering techniques, such as transcription activator-like effector nucleases (TALENs), meganucleases, zinc finger nucleases, and CRISPR (Cas9 system, Cpf1 system, or Csm1 system). We conduct genome modifications in commercial tobacco cultivars such as TN90, K326, and Narrow Leaf Madole. We edit all 35 NCGs (NCG1–NCG35) that exhibit differential expression between TN90 and LA BU21, focusing in particular on NCGs encoding ERF-like proteins.

[0264] For example, CRISPR guide RNAs are designed and synthesized to recognize specific target sequences from the NCG gene. Exemplary guide RNA sequences are provided in Table 2. The guide RNA and accompanying nucleic acid encoding the Cas9, Cpf1, or Csm1 protein (either in DNA plasmid or mRNA form) are then used to transform tobacco protoplasts. The CRISPR-Cas9 / Cpf1 / Csm1 ribonucleoprotein complex recognizes the specific NCG target sequence and introduces a double-strand break (DSB). The endogenous non-homologous end joining (NHEJ) DNA repair system repairs the DSB, which can introduce nucleotide deletions, insertions, or substitutions, resulting in potential loss-of-function mutations. Alternatively, a donor nucleic acid molecule with a desired sequence can be included in the protoplast transformation to serve as a template molecule to introduce the desired sequence at or near the CRISPR target site.

[0265] Tobacco protoplasts were isolated from TN90 tobacco leaves growing in magenta boxes in a growth chamber. Well-expanded leaves (5 cm) from 3- to 4-week-old plants were cut into 0.5- to 1-mm leaf strips from the center of the leaf. The leaf strips were transferred to a prepared enzyme solution (1% Cellulase R10, 0.25% Macerozyme R10, 0.4 M mannitol, 20 mM KCl, 20 mM MES (pH 5.7), 10 mM CaCl2, 0.1% BSA) by immersing both sides of the leaf strips. The leaf strips were vacuum infiltrated in the dark using a desiccator for 30 minutes, and digestion was continued in the dark at room temperature for 4 hours to overnight without agitation. The protoplasts were filtered through a 100 μm nylon filter and purified using 3 ml of Lymphoprep. Centrifuge the protoplasts, wash them with W5n solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES, 991 mg / L glucose, pH 5.7), and suspend them in W5n solution at a concentration of 5 x 105 / ml. Keep the protoplasts on ice for 30 minutes to allow them to settle to the bottom of the tube by gravity. Discard the W5n solution and resuspend the protoplasts in P2 solution at room temperature. Gently mix 50 µl of DNA (10-20 µg of plasmid), 500 µl of protoplasts (2 x 105 protoplasts), and 550 µl of PEG solution (40% v / v, 10 ml of 4 g of PEG4000, 0.2 M mannitol, 0.1 M CaCl2) in a 15 ml microcentrifuge tube and incubate the mixture at room temperature for 5 minutes.

[0266] Pellet the protoplasts and resuspend them in 1 ml of 2X 8EN1 (8EN1: NH4NO3-free MS salts, MS vitamins, 0.2% myo-inositol, 4 mM MES, 1 mg / L NAA, 1 mg / L IAA, 0.5 M mannitol, 0.5 mg / L BAP, 1.5% sucrose). Jelly the transformed protoplasts with an equal volume of low-melting agarose (LMA) and add 0.2 ml of protoplast-LAM dropwise to form beads. 10 ml of 8EN1 was added to the beads; on day 7, 5 ml of 8EN1 was removed and 5 ml of 8EN2 (8EN1 containing 0.25 M mannitol) was added; after another 7 days (day 14), 10 ml of 8EN2 was removed and 10 ml of 8EN2 was added; after another 7 days (day 21), 5 ml of 8EN2 was removed and 5 ml of 8EN3 (8EN1 containing 3% sucrose and no mannitol) was added; after another 7 days (day 28), 10 ml of 8EN3 was removed and 10 ml of 8EN3 was added. Protoplasts were maintained for 2 weeks for microcallus growth. When the callus reached approximately 5 mm (usually about 2 weeks), it was transferred to NCM solid medium. The callus was transferred to TOM-Kan solid medium for shoot growth, and transformed tobacco plants were regenerated using the methods described herein. The callus or regenerated plants are tested and selected for gene editing events that have the desired mutation in the NCG gene, generating both loss-of-function NCG alleles (e.g., premature stop codons or frameshifts) or other types of mutations (e.g., gain-of-function or new traits).

[0267] Similarly, genome editing techniques (e.g., Cas9, Cpf1, or Csm1-mediated CRISPR editing systems) are used to edit ERF genes from the Nic2 locus (ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168). Both loss-of-function ERF mutations (e.g., premature stop codons) and other types of mutations (e.g., gain-of-function or neo-traits) are generated.

[0268] Example 7: Use of a dominant repressor to repress ERF-like genes in the Nic1b region and produce low-alkaloid tobacco Develop dominant repressors for one or more ERF-like genes (e.g., NCG1, NCG11, NCG12, NCG15, NCG16, ERF101, ERF110, ERF16, ERF130, and NCG17) from the Nic1b region by attaching an ERF-binding amphipathic repression (EAR) motif to the C-terminus. When attached to a transcription factor, the EAR motif has been shown to actively repress the expression of the transcription factor's target genes (Hiratsu et al., Plant J. 34:733-739 (2003)).

[0269] Briefly, the ERF-EAR constructs are placed under the control of a constitutive promoter (e.g., the cauliflower mosaic virus (CaMV) 35S promoter) and used to transform wild-type tobacco by protoplast transformation or Agrobacterium, thereby generating transgenic lines for each construct as well as control lines transformed with an empty vector (VC). Expression of the transgene is verified. Both nicotine levels and the expression of nicotine biosynthesis-related enzymes (putrescine N-methyltransferase (PMT), N-methylputrescine oxidase (MPO), aspartate oxidase (AO), quinolinate synthase (QS), quinolinate phosphoribosyltransferase (QPT), and PIP family oxidoreductase A622) and the tonoplast-localized MATE family transporters MATE1 and MATE2 (MATE1 / 2) are examined, along with the expression levels of ornithine decarboxylase (ODC), arginine decarboxylase (ADC), spermidine synthase (SPDS), S-adenosylmethionine decarboxylase (SAMDC), and S-adenosylmethionine synthase (SAMS) genes.

[0270] Example 8: Breeding low-nicotine tobacco varieties The identified Nic1b region, the genes therein, and the associated molecular markers aid in breeding and producing low-alkaloid tobacco hybrids, varieties, and lines containing the nic1b_erf mutation (e.g., a complete or partial deletion of the Nic1b region) with commercially acceptable leaf quality. The NCG genes and markers are also used to screen for additional nic1b_erf alleles from various Nicotiana germplasm, e.g., different Nicotiana species or Nicotiana tabacum strains. A collection of 43 Nicotiana species, 49 Nicotiana rustica strains, and approximately 600 Nicotiana tabacum strains that can be screened is provided in Table 8 of U.S. Patent No. 7,700,834.

[0271] Germplasm identified as having a novel nic1b_erf allele can be used as source material for breeding with cultivated tobacco. Interspecific or intraspecific hybridization methods, combined with standard breeding methods such as backcrossing or pedigree methods, can be used to introgress the desired nic1b_erf or nic2 mutant allele from a donor source into cultivated tobacco. For example, a low-nicotine variety (e.g., a donor parent such as LA Burley 21) containing nic1, nic2, or both mutant alleles is crossed to an elite high-nicotine variety with a desirable genetic background and agronomically elite traits. F1 progeny plants from this cross are optionally assayed for one or more molecular markers, as exemplified in Table 3. The F1 progeny plants are then backcrossed to the parent elite high-nicotine variety (the regressing parent). Plants from the BC1 generation are genotyped using the molecular markers described and disclosed herein to select tobacco plants with smaller nic1b_erf or nic2 deletion segments. After multiple rounds of backcrossing (e.g., 5-7 generations), a new elite tobacco variety is obtained that contains both the low-alkaloid trait and other desirable traits from the recurrent parent elite strain. This new elite tobacco variety also does not contain any genetic disturbances associated with the low-alkaloid trait due to recombination events around the Nic1b and Nic2 loci. These recombination events unlink the nic1b_erf and nic2 mutations from any associated deleterious mutations, thus reducing or avoiding genetic disturbances. The above breeding and marker-assisted selection strategies can also be used to achieve pyramiding or stacking of the low-nicotine trait with other transgenes or natural alleles that reduce alkaloid or TSNA levels.

[0272] The low alkaloid tobacco hybrid, variety, or strain can be produced as a burley, dark, flue-cured, Maryland, or Oriental tobacco, or can be any of the following tobacco varieties: BU 64, CC 101, CC 200, CC 27, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CU 263, DF911, Galpao tobacco, GL 26H, GL 350, GL 600, GL 737, GL 939, GL 973, HB 04P, K 149, K 326, K 346, K 358, K394, K 399, K 730, KDH 959, KT 200, KT204LC, KY 10, KY 14, KY 160, KY 17, KY 171, KY 907, KY907LC, KTY14×L8 LC, Little Crittenden, McNair 373, McNair 944, msKY 14xL8, Narrow Leaf Madole, NC 100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH 129, NC 2002, Neal Smith Madole, OXFORD 207, "Perique" cigarette, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81, RG H51, RGH 4, RGH 51, RS 1410, Speight 168, Speight 172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI 1269, TN 86, TN86LC, TN 90, TN 97, TN97LC, TN D94, TN D950, TR (Tom Rosson) Madole, VA 309, or VA359, Maryland609, HB3307PLC, HB4488PLC, KT206LC, KT209LC, KT210LC, KT212LC, R610LC, PVH2310, NC196, KTD14LC, KTD6LC, KTD8LC, PD7302LC, PD7305LC, PD7309LC, PD7318LC, PD7319LC, PD7312LC, ShireyLC, or essentially any commercial tobacco variety by standard tobacco breeding techniques known in the art.

[0273] Example 9: Additional genes from the Nic1b region Several genes, including non-coding RNAs, were identified in the Nic1b region. They are listed in Table 5. With the exception of ERF16, all of these genes exhibit higher expression levels in TN90 and its high-alkaloid F5 sibling plants compared to LA BU21 and its low-alkaloid F5 sibling plants. ERF16 appears to be expressed at relatively similar levels in high-alkaloid and low-alkaloid plants. These additional genes will be further studied as outlined in Examples 4-7. For example, both overexpression and suppression approaches will be employed to investigate the function of these genes and develop tobacco varieties with desirable nicotine levels. Two sets of transgenic plants will be generated: one overexpression approach using full-length coding sequences and the other suppression approach using artificial microRNAs or RNAi sequences. Furthermore, mutational approaches (either random mutagenesis or targeted editing) will also be employed to generate mutant alleles of these genes.

[0274] Results from overexpression of selected genes or sequence segments from or near the Nic1b region are provided in Table 13 and Figure 3. Overexpression is driven using the 35S promoter. Individual TO transformants in LA BU21 were potted on the same day and contained control and overexpression constructs for ERF130, the LA-associated region deleted in LA BU21 (i.e., SEQ ID NO: 1; does not contain an annotated gene and is referred to as "Nic1bΔ"; see Example 2), g32081 (a beta-glucosidase 18-like gene), and ERF16. Plants were topped 12 weeks after potting. Alkaloid levels were determined by measuring pooled leaf blade samples collected from the third, fourth, fifth, and sixth leaves from the top of the plant two weeks after topping. Four or five independent TO plants were tested for each construct, and average levels of total or individual alkaloids were determined. As shown in Figure 3, this set of alkaloid data indicates that overexpression of ERF16 promotes nicotine production (a 76% increase over the control). Other minor alkaloids (including nornicotine, anabasine, and anatabine) also appear to be increased in ERF16-overexpressing plants, all of which contribute to the 76% increase in total alkaloids over the control. No obvious shift in nicotine or alkaloid levels is observed from overexpression of ERF130, the Nic1bΔ sequence, or g32081.

[0275] Example 10: Multiple transcription factors from chromosome 7 are differentially expressed between normal and reduced alkaloid tobacco lines As listed in Table 6, several additional transcription factors are identified from chromosome 7 that exhibit differential expression levels between BU21 and LA BU21. These additional genes will be further studied as outlined in Examples 4-7. For example, both overexpression and suppression approaches will be employed to investigate the function of these genes and develop tobacco varieties with desirable nicotine levels. Two sets of transgenic plants will be generated: one overexpression approach using full-length coding sequences, and the other suppression approach using artificial microRNA or RNAi sequences. Furthermore, mutational approaches (either random mutagenesis or targeted editing) will also be employed to generate mutant alleles of these genes.

[0276] Example 11: Various ERF genes from the Nic1b region are involved in nicotine biosynthesis As outlined in Example 4, artificial microRNA ("amiRNA") constructs are designed using the MIR6147 backbone to suppress various ERF genes from or near the Nic1b region (collectively referred to as "Nic1b_ERFs"). Some artificial microRNAs are designed to specifically suppress individual Nic1b_ERFs. Other microRNAs are designed to simultaneously target multiple Nic1b_ERFs. Some exemplary mature artificial microRNA sequences are provided in Table 2.

[0277] TO plants transformed with the Nic1b_ERF-specific amiRNA construct are potted on the same day as corresponding control plants carrying the amiRNA-GUS construct. Root tissue for gene expression studies is collected from plants while they are re-potted into larger pots. Alkaloid levels are determined by measuring pooled leaf blade samples collected from the third, fourth, fifth, and sixth leaves from the top of the plants two weeks after topping. Briefly, approximately 0.5 g of tobacco is extracted using liquid / liquid extraction into an organic solvent containing an internal standard and analyzed by gas chromatography (GC) with flame ionization detection (FID). Results can be reported as a weight percent (Wt%) or based on dry weight. Reporting data based on dry weight requires determination of oven volatiles (OV). Unless otherwise specified, total or individual alkaloid or nicotine levels shown herein are based on dry weight (e.g., percent total alkaloids or percent nicotine). A preliminary gene expression screen is performed using multiplex qPCR with actin as a control in a QuantStudio 5 or QS12K (ThermoFisher Scientific). A select subset of plants is also screened in a custom OpenArray using 18 genes in triplicate, including the actin control gene, in a QuantStudio QS 12K instrument.

[0278] Table 7 summarizes the alkaloid data from a set of independent T0 transformants that suppressed selected individual Nic1b_ERFs via artificial microRNAs. With some exceptions (e.g., 18GH2083), individualSuppression of Nic1b_ERFs reduces nicotine in the HI BU21 background (i.e., nic2 single mutant) but not in the wild-type background (e.g., t-NL Madole(PhPh)SRC) (Tables 7-9). This may suggest redundancy among Nic1b_ERFs and also indicates that the nic2 single mutant provides a more sensitive background for evaluating the role of each individual Nic1b_ERF in nicotine regulation. Suppression of individual Nic1b_ERFs in HI BU21 plants reduces nicotine levels to approximately 50%-90% of control plants (Table 7 and Figure 4). The nicotine reduction observed in amiRNA plants is consistent with reduced gene expression of multiple nicotine biosynthetic genes (Table 10).

[0279] T1 plants will be produced and further analyzed for kanamycin resistance segregation, alkaloid levels, and nicotine biosynthetic gene expression. Crosses will be performed to bring multiple amiRNAs into a single plant and simultaneously suppress multiple Nic1b_ERFs.

[0280] Example 12: Modulation of nicotine levels by manipulating ERF genes associated with the Nic1b and / or Nic2 loci To achieve desirable nicotine levels in tobacco, two groups of ERF genes are manipulated through overexpression or downregulation, and through either transgenic or mutagenesis approaches. Each group of ERFs is clustered around the Nic1b or Nic2 region. The first group of ERFs includes ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210, and ERF91L2, which are found herein to be clustered around the Nic1b region (individually Nic1b_ERF and collectively Nic1b_ERFs). See Table 11 and Kajikawa et al., Plant physiol. 2017, 174:999-1011. Core members of this first group of ERFs include ERFnew, ERF199, ERF19, ERF29, ERF210, and ERF91L2. This first group may also include JRE5L2. The second group of ERFs includes ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168 (individually Nic2_ERFs and collectively Nic2_ERFs), which appear to be deleted in HI BU21 (i.e., nic2 single mutants). See Shoji et al., Plant Cell, (10):3390-409 (2010). Core members of the second group include ERF189 and ERF115. This second group can also be expanded to include ERF163, ERF91L1, ERF104ΔC, ERF221, ERF115, ERF168, ERF17, ERF179, ERF17L1, ERF189, ERF17L2ΔC, JRE5L1, ERF104L1ΔC, and ERF168L1ΔC. See Table 12 and Kajikawa et al., Plant physiol. 2017, 174:999-1011.

[0281] Transgenic approaches include RNA interference-based gene suppression (e.g., antisense, co-suppression, hairpin, or inverted repeat-based RNAi, artificial microRNA, artificial trans-acting small interfering (ta-si)RNA) and dominant-negative suppressors (e.g., ERF-associated amphipathic suppression (EAR) motif). Transgene- or cis-gene-based suppression can be directed specifically at a single ERF gene or simultaneously at a subset of ERF genes. Dominant-negative suppressors can be made cisgenic, for example, by knocking in an EAR motif coding sequence in frame with the endogenous ERF coding sequence.

[0282] Mutagenesis approaches include random mutagenesis, either chemical (e.g., EMS-based TILLING) or physical (e.g., irradiation), and genome editing. Any genome editing technology described herein can be used, including, for example, TALEN, meganucleases, zinc finger nucleases, and CRISPR (Cas9 system, Cpf1 system, or Csm1 system).

[0283] Transgenic or mutagenesis approaches are used to generate mutant alleles of the first and second groups of ERFs, focusing on core members. Both loss-of-function (i.e., null) and weak mutant alleles are produced. Various combinations of Nic2_ERF mutant alleles are generated to suppress the level or activity of Nic2_ERF, potentially to a level or activity similar to that of the nic2 mutant allele found in HI BU21 or LA FC53. Various combinations of Nic1b_ERF mutant alleles are generated to suppress the level or activity of Nic1b_ERF, potentially to a level or activity similar to that of the nic1 mutant allele found in LI BU21 or LA FC53. Additional combinations of mutant alleles in various Nic2_ERF and Nic1b_ERF genes will be generated or introgressed into single tobacco plants to produce low-alkaloid tobacco strains with nicotine and total alkaloid levels similar to those found in LA BU21, LA FC53, or LN KY171.

[0284] (Table 5) Additional genes from or near the Nic1b region on chromosome 7. Start and stop genomic positions are provided based on the reference TN90 genome. Sequence identification numbers (SEQ ID NOs) are provided for each gene for the genomic coding DNA sequence (including introns, if applicable), cDNA sequence, and protein or other encoded RNA sequence. TIFF2025157497000012.tif23157

[0285] Table 6: Transcription factors from chromosome 7 that exhibit differential expression between normal and reduced alkaloid tobacco lines. Start and stop genomic positions are provided based on the reference TN90 genome. Sequence identification numbers (SEQ ID NOs) are provided for each gene for the genomic coding DNA sequence (including introns, if applicable), cDNA sequence, and protein or other encoded RNA sequence. TIFF2025157497000013.tif23131

[0286] Table 7. Set of T0 transformants mediated by artificial microRNAs individual We demonstrate that suppression of Nic1b_ERF reduces nicotine in the HI BU21 background (i.e., t-HI Burley 21, nic2 single mutant). The MIR6147 backbone is used for the artificial microRNA ("aMIR6147"). The "% Conv" column represents the percent conversion of nicotine to nornicotine. The average nicotine levels from multiple T0GUS control transformants are used to determine the relative nicotine levels in each individual Nic1b_ERF amiRNA T0 transformant. Plants are initially pinched 16 weeks after potting, and leaf samples are collected 2 weeks later for alkaloid analysis. TIFF2025157497000014.tif224128TIFF2025157497000015.tif224161TIFF2025157497000016.tif22445

[0287] Table 8: A second set of T0 plants transformed with artificial microRNAs targeting individual Nic1b_ERFs in the HI BU21 background (i.e., t-HI Burley 21, nic2 single mutant). This set of T0 plants was initially pinched 15 weeks after potting, and leaf samples were collected two weeks later for alkaloid analysis. Compared to the plants in Table 7, these plants were pinched one week earlier, which may result in a lack of consistency in the control plants (nicotine levels ranging from 1.52% to 2.78%); therefore, the amiRNA plants do not exhibit consistent nicotine reduction. The same set of amiRNA constructs as in Table 7 is used. The "% Conv" column represents the percent conversion of nicotine to nornicotine. The average nicotine levels from multiple T0GUS...

Claims

1. 1. A tobacco plant or part thereof comprising a mutation in the Nic1b_ERF locus, wherein said tobacco plant is capable of producing leaves having a USDA Grade Index value of 50 or greater when cured.

2. 2. The tobacco plant or part thereof of claim 1, wherein the tobacco plant is a Nicotiana tabacum plant.

3. 3. The tobacco plant or part thereof of claim 2, wherein the tobacco plant further comprises a mutation in the ERF gene at the Nic2 locus.

4. 3. The tobacco plant or part thereof of claim 2, wherein the tobacco plant further comprises one or more mutations in two or more, three or more, four or more, five or more, six or more, or all seven genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168.

5. 3. The tobacco plant or part thereof of claim 2, wherein the tobacco plant, when dried, is capable of producing leaves having a USDA Grade Index value of 70 or greater.

6. 3. The tobacco plant or part thereof of claim 2, wherein the tobacco plant, when cured, is capable of producing leaves having a USDA Grade Index value equivalent to the USDA Grade Index value of a control plant when grown and cured under similar conditions, and the control plant shares essentially the same genetic background as the tobacco plant except for the mutation.

7. 3. The tobacco plant or part thereof of claim 2, wherein the tobacco plant contains a level of nicotine that is less than 40% of the nicotine level of a control plant when grown under similar growing conditions, and the control plant shares essentially the same genetic background as the tobacco plant except for the mutation.

8. 3. The tobacco plant or part thereof of claim 2, wherein the tobacco plant comprises a nicotine level of less than 2.0%.

9. 1. A tobacco plant or part thereof comprising a non-transgenic mutation in the Nic1b_ERF locus, wherein the non-transgenic mutation reduces the nicotine level of the tobacco plant to less than 60% of the nicotine level of a control plant when grown under similar growing conditions, wherein the tobacco plant is capable of producing leaves, when cured, having a USDA Grade Index value equivalent to the USDA Grade Index value of the control plant, and wherein the control plant shares essentially the same genetic background as the tobacco plant except for the non-transgenic mutation, and wherein the tobacco plant is a Nicotiana tabacum plant.

10. A population of tobacco plants according to any one of claims 1 to 9.

11. A dried tobacco material from the tobacco plant according to any one of claims 1 to 9.

12. 12. The dried tobacco material of claim 11, produced by a drying method selected from the group consisting of hot air drying, air drying, flame drying, and sun drying.

13. A tobacco blend comprising the dry tobacco material of claim 11.

14. A tobacco product comprising the dried tobacco material of claim 11.

15. 15. The tobacco product of claim 14, wherein the tobacco product is selected from the group consisting of cigarettes, cigarillos, non-vented recess filter cigarettes, vented recess filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and cut tobacco.

16. 15. The tobacco product of claim 14, which is a smokeless tobacco product.

17. 17. The tobacco product of claim 16, wherein the smokeless tobacco product is selected from the group consisting of loose-leaf chewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff.

18. 1. A tobacco plant or part thereof comprising a mutation in the Nic1b_ERF locus, wherein said mutation is not present in LA Burley 21, LAFC53, and LN KY171.

19. 20. The tobacco plant or part thereof of claim 18, wherein the tobacco plant further comprises a mutation in the ERF gene at the Nic2 locus.

20. 19. The tobacco plant or part thereof of claim 18, wherein the mutation is located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, and 202-205, and fragments thereof.

21. 19. The tobacco plant or part thereof of claim 18, wherein the mutation is located in a gene encoding a polypeptide having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87, 88, 89, 180, 181, 206, and 207.

22. 19. The tobacco plant or part thereof of claim 18, wherein the tobacco plant is from a variety selected from the group consisting of flue-cured tobacco, air-cured tobacco, dark flue-cured tobacco, and Galpao tobacco, and Oriental tobacco.

23. 20. The tobacco plant or part thereof of claim 18, wherein the tobacco plant is from a variety selected from the group consisting of Burley tobacco, Maryland tobacco, and dark air-cured tobacco.

24. 20. A cured tobacco material from the tobacco plant of claim 18.

25. 25. The dried tobacco material of claim 24, wherein the tobacco plant contains a nicotine level of 0.2% to 0.6%.

26. 25. The dried tobacco material of claim 24, wherein the tobacco plant contains a nicotine level of 1.0% to 3.0%.

27. 25. The dried tobacco material of claim 24, produced by a drying method selected from the group consisting of hot air drying, air drying, flame drying, and sun drying.

28. A tobacco product comprising the dried tobacco material of claim 24.

29. 25. The tobacco product of claim 24, wherein the tobacco product is selected from the group consisting of cigarettes, cigarillos, non-vented recess filter cigarettes, vented recess filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and cut tobacco.

30. 25. The tobacco product of claim 24, which is a smokeless tobacco product.

31. 1. A tobacco plant or part thereof comprising a heterologous expression cassette comprising a Nic1b_ERF inhibitory sequence of a gene comprising a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, and 202-205, and fragments thereof, wherein the inhibitory sequence is operably linked to a promoter functional in a plant cell, and the inhibitory sequence has at least 90% sequence identity to a fragment of at least 21 nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, and 202-205, and fragments thereof.

32. 32. The tobacco plant or part thereof of claim 31 , wherein the Nic1b inhibitory sequence is capable of being transcribed as an inhibitory polynucleotide selected from the group consisting of a single-stranded RNA polynucleotide, a double-stranded RNA polynucleotide, and a combination thereof.

33. 32. The tobacco plant or part thereof of claim 31, wherein the promoter is selected from the group consisting of a constitutive promoter, an inducible promoter, and a tissue-preferred promoter.

34. 32. The tobacco plant or part thereof of claim 31, wherein the promoter is a root-specific promoter.

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