阅读说明：本技术 包含降低的烟碱和降低的烟草特异性亚硝胺的烟草植物 (Tobacco plants comprising reduced nicotine and reduced tobacco specific nitrosamines ) 是由 C·库迪蒂普迪 许冬梅 J·斯特里克兰德 M·F·德戈多伊鲁索 于 2020-01-23 设计创作，主要内容包括：本公开提供用于减少烟碱的方法。还提供了具有降低的烟草特异性亚硝胺(TSNA)的烟草植物。本公开还提供具有低烟碱和增加的抗氧化能力的经修饰的烟草植物。进一步提供了具有低烟碱和低TSNA的烟草植物或材料。还提供了本文提供的烟草植物的熟化烟草材料和包含该熟化烟草材料的烟草制品。(The present disclosure provides methods for reducing nicotine. Also provided are tobacco plants having reduced Tobacco Specific Nitrosamines (TSNA). The present disclosure also provides modified tobacco plants having low nicotine and increased antioxidant capacity. Further provided are tobacco plants or materials having low nicotine and low TSNA. Cured tobacco materials of the tobacco plants provided herein and tobacco products comprising the cured tobacco materials are also provided.)

1. A tobacco plant, or a part thereof, comprising a first genetic modification that inhibits one or more genes from the NIC1b locus, the NIC2 locus, or both, and further comprising a second genetic modification that increases the conversion of nicotine to nornicotine.

2. The tobacco plant, or part thereof, of claim 1, wherein said first genetic modification comprises a nic1b mutant allele.

3. The tobacco plant, or part thereof, according to claim 2, wherein said nic1b mutant allele is derived from the species cuba cigar, low alkaloid burley tobacco 21(LABU21), low intermediate burley tobacco 21(LIBU21), low alkaloid flue-cured tobacco 53(LAFC53), low nicotine KY171(LNKY171), or a species derived therefrom.

4. The tobacco plant, or part thereof, according to claim 1, wherein said first genetic modification comprises a transgene or mutation that reduces the expression or activity of one or more Ethylene Response Factor (ERF) genes from the NIC1b locus.

5. The tobacco plant, or part thereof, of claim 4, wherein said first genetic modification comprises a transgene targeting one or more genes selected from the group consisting of: ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210, and ERF91L 2.

6. The tobacco plant, or part thereof, of claim 1, wherein said first genetic modification comprises a nic2 mutant allele.

7. The tobacco plant, or part thereof, according to claim 1, wherein said first genetic modification comprises a transgene or mutation that reduces the expression or activity of one or more Ethylene Response Factor (ERF) genes from the NIC2 locus.

8. The tobacco plant, or part thereof, of claim 7, wherein said first genetic modification comprises a transgene targeting one or more genes selected from the group consisting of: ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF 168.

9. The tobacco plant, or portion thereof, of any one of claims 1-8, wherein said second genetic modification overexpresses nicotine demethylase.

10. The tobacco plant, or part thereof, of any one of claim 9, wherein said second genetic modification comprises a transgene encoding said nicotine demethylase.

11. The tobacco plant, or part thereof, of any one of claims 1-8, wherein said second genetic modification comprises a transgene encoding one or more of CYP82E4, CYP82E5 and CYP85E10 polypeptide.

12. The tobacco plant, or part thereof, of any one of claims 1-8, wherein said second genetic modification comprises genome editing that increases the expression or activity of one or more of CYP82E4, CYP82E5 and CYP85E10 polypeptides.

13. The tobacco plant, or part thereof, of claim 11 or 12, wherein said second genetic modification increases the expression or activity of CYP82E4v2, or comprises a transgene encoding CYP82E4v 2.

14. The tobacco plant, or part thereof, according to any one of claims 1-13, wherein said tobacco plant further comprises a third genetic modification that increases the content of one or more antioxidants as compared to a control plant lacking the third genetic modification.

15. The tobacco plant, or portion thereof, of claim 14, wherein said third genetic modification comprises a transgene encoding or targeting an antioxidant biosynthetic enzyme, a regulatory transcription factor for an antioxidant, an antioxidant transporter, an antioxidant metabolic enzyme, or a combination thereof.

16. The tobacco plant, or part thereof, of claim 14, wherein said third genetic modification comprises a transgene encoding one or more polypeptides selected from the group consisting of: AtPAP1, Ntmyb3A, Ntmyb3B, Ntmyb3C, NtJAF13, sta 1, NtAN1, and NtAN 2.

17. A tobacco plant, or part thereof, comprising a first genetic modification that increases the conversion of nicotine to nornicotine, and further comprising a second genetic modification that increases the content of one or more antioxidants.

18. The tobacco plant, or portion thereof, of claim 17, further comprising a third genetic modification that reduces nicotine in said tobacco plant.

19. Cured tobacco material from the tobacco plant of any one of claims 1-18.

20. A smoking article comprising cured tobacco material according to claim 19.

Technical Field

The present disclosure provides modified tobacco plants having low nicotine. The present disclosure also provides modified tobacco plants having low nicotine and increased antioxidant capacity. Also provided are tobacco plants comprising a reduced amount of a Tobacco Specific Nitrosamine (TSNA). Also provided are tobacco plants (which have been developed via breeding or transgenic approaches) having altered total alkaloid and nicotine content and commercially acceptable leaf grades, and the production of tobacco products from these tobacco plants.

Background

Four major alkaloids are found in tobacco: nicotine, nornicotine, anabasine and anatabine. Nicotine is the major alkaloid and typically accounts for over 90% of total alkaloids in commercial tobacco cultivars. Nicotine biosynthesis occurs primarily in the tobacco roots. The tobacco plant then transports the nicotine through the vascular bundle to the lamina where it is then stored in the vacuole.

A variety of factors affect the alkaloid content of tobacco, including genotype, environment, fertilization, and agronomic practices (e.g., tobacco production is stimulated by topping, injury, and herbivore injury). The low alkaloid trait originally found in the strain of the cuba cigar tobacco variety was introduced into the cigarette variety by a series of backcrosses. The low alkaloid tobacco germplasm was subsequently enrolled in the genetic background of the cultivar burley 21 (Legg et al, Crop Science,10:212 (1970)). Genetic studies using the low alkaloid burley 21(LA BU21) line showed that two unlinked loci promote nicotine content in tobacco leaves. These two loci are designated as Nic 1and Nic2 (also known as Nic 1and Nic2 (nicotine 1and nicotine 2, respectively.) the Nic 1and Nic2 mutations in LA BU21 are semi-dominant. they show a dose-dependent effect on nicotine levels, with the effect of Nic1 being about 2.4 times stronger than that of Nic 2. molecular characterization of the Nic2 locus has been reported. the Nic2 mutation is shown to contain a deletion in the transcription factor gene cluster from the Ethylene Response Factor (ERF) family.

There are many benefits to reducing the total alkaloid content in tobacco. It can improve the value of tobacco as biomass resource. Increasing the content of nicotinic alkaloids in tobacco plants can play an important role in protecting plants against insects and herbivores.

Cytochrome p450 catalyzes enzymatic reactions of a variety of chemically distinct substrates, including oxidative, peroxidative and reductive metabolism of endogenous and xenobiotic substrates. In plants, p450 is involved in biochemical pathways, including the synthesis of plant products such as phenylpropanoids, alkaloids, terpenoids, lipids, cyanogenic glycosides and glucosinolates (Chappel, Annu. Rev. plant Physiol. plant mol. biol. 198,49: 311-343). Cytochrome p450, also known as p450 heme-thiolate protein, generally acts as a terminal oxidase in multi-component electron transfer chains, known as a p 450-containing monooxygenase system. Specific reactions catalyzed include demethylation, hydroxylation, deamination and reduction of azo, nitro and N-oxide groups. Three cytochrome p450 s have been identified as nicotine demethylases (see U.S. Pat. Nos. 8,319,011; 8,124,851; 9,187,759; 9,228,194; 9,228,195; 9,247,706 as exemplary nicotine demethylase sequences).

Tobacco Specific Nitrosamines (TSNAs), such as N-nitrosonornicotine (NNN) and 4- (methylnitrosamino) -l- (3-pyridyl) -l-butanone (NNK), can be found in smokeless tobacco of cigarettes; mainstream smoke; and sidestream smoke. Air-cured and cured tobacco is reported to contain tobacco-specific nitrosamines. See "Effect of Air-Curing on the Chemical Composition of Tobacco", Wiernik et al, Recent adv. Tob. Sci, (1995),21, pp.39-80. According to Wiernik et al, TSNAs are not present in significant amounts in growing tobacco plants or fresh cut tobacco (green tobacco), but are formed during the curing process. It is stated that the bacterial population present on tobacco leaves to a large extent leads to the formation of nitrite from nitrate during the curing process and may influence the direct catalytic action of secondary amine nitrosation at physiological pH values. Affected secondary amines include tobacco alkaloids, which form TSNAs upon nitrosation.

There is a need to develop tobacco plants and products that contain altered nicotine levels (e.g., reduced nicotine) as well as reduced TSNAs while maintaining (if not producing better) tobacco leaf quality.

Disclosure of Invention

In one aspect, the present disclosure provides a tobacco plant, or a portion thereof, comprising a first genetic modification that inhibits one or more genes from the NIC1b locus, the NIC2 locus, or both, and further comprising a second genetic modification that increases the conversion of nicotine to nornicotine.

In another aspect, the present disclosure provides a tobacco plant, or a portion thereof, comprising a first genetic modification that inhibits one or more genes from the NIC1b locus, the NIC2 locus, or both, comprising a second genetic modification that increases the conversion of nicotine to nornicotine, and further comprising a third genetic modification that increases the content of one or more antioxidants as compared to a control plant lacking the third genetic modification.

In one aspect, the present disclosure provides a tobacco plant, or a portion thereof, comprising a first genetic modification that increases the conversion of nicotine to nornicotine, and further comprising a second genetic modification that increases the content of one or more antioxidants.

In one aspect, the present disclosure provides a tobacco plant, or part thereof, comprising a first genetic modification that provides mutagenesis or transgene suppression of one or more, two or more, three or more, four or more, five or more, six or more genes comprising a nucleotide sequence having at least 90% identity to SEQ ID Nos:10-19 and 30-36, and further comprising a second genetic modification that increases the conversion of nicotine to nornicotine. In one aspect, the present disclosure provides a tobacco plant, or part thereof, comprising a first genetic modification that provides mutagenesis or transgene suppression of one or more, two or more, three or more, four or more, five or more, six or more genes encoding nucleotide sequences having at least 90% identity to SEQ ID Nos:20-29 and 37-43, and further comprising a second genetic modification that increases the conversion of nicotine to nornicotine. In a further aspect, the tobacco plant further optionally comprises another genetic modification that increases the content of one or more antioxidants.

Brief description of the sequences

SEQ ID NOs: 1and 5 show exemplary coding and polypeptide sequences of the cytochrome P450 monooxygenase CYP82E4v2 gene from Nicotiana tabacum (Nicotiana tabacum).

Exemplary coding sequences and polypeptide sequences of the Myb3 gene of nicotiana tabacum are shown in SEQ ID NOs:2 and 6.

Exemplary coding sequences and polypeptide sequences of the Arabidopsis (Arabidopsis thaliana) PAP1 gene are shown in SEQ ID NOs:3 and 7.

Exemplary coding sequence and polypeptide sequence of Potato (Solanum tuberosum) AN1 are shown in SEQ ID NOs:4 and 8.

SEQ ID NO 9 shows the sequence of the Nic1b region.

10-19 show exemplary genomic sequences of the Nic1b _ ERF gene as listed in Table 8.

SEQ ID NOs 20-29 show exemplary genomic sequences of the Nic1b _ ERF gene as listed in Table 8.

30-36 show exemplary genomic sequences of the Nic2_ ERF gene as listed in Table 9.

37-43 show exemplary genomic sequences of the Nic2_ ERF gene as listed in Table 9.

An exemplary construct with two stacked expression cassettes for expression of NtMYB3 and NtCYP82E4 is shown in SEQ ID NO (CsVMV-NtMYB3-Nos-T-CsVMV-CYP82E 4-Nos-T).

SEQ ID NO 45 shows the amino acid sequence of the 6XHIS tag.

The various sequences may include an "N" in a nucleotide sequence or an "X" in an amino acid sequence. An "N" may be any nucleotide, such as A, T, G, C, or a deletion or insertion of one or more nucleotides. In some cases, a string of "N" s is shown. The number of "N" s does not necessarily correlate with the actual number of undetermined nucleotides at that position. The actual nucleotide sequence may be longer or shorter than the "N" segment shown. Similarly, "X" may be any amino acid residue or a deletion or insertion of one or more amino acids. Likewise, the number of "X" does not necessarily correlate with the actual number of undetermined amino acids at that position. The actual amino acid sequence may be longer or shorter than the "X" segment shown. Although A, T, G, C (as compared to A, U, G, C) is used in describing any SEQ ID in the sequence listing, SEQ ID may also refer to an RNA sequence depending on the context in which it is referenced.

Brief description of the drawings

FIG. 1: greenhouse grown T overexpressing nicotine demethylase CYP82E4v2₀Nicotine content in transgenic plants.

FIG. 2: greenhouse grown T overexpressing nicotine demethylase CYP82E4v2₀Reduced nicotine content in transgenic plants.

FIG. 3: greenhouse grown T overexpressing nicotine demethylase CYP82E4v2₀The content of anabasine in the transgenic plant.

FIG. 4: greenhouse grown T overexpressing nicotine demethylase CYP82E4v2₀Anatabine content in transgenic plants.

FIG. 5: t grown in the field overexpressing the nicotine demethylase CYP82E4v2₁Nicotine content in transgenic plants.

FIG. 6: t grown in the field overexpressing the nicotine demethylase CYP82E4v2₁Reduced nicotine content in transgenic plants.

FIG. 7: t grown in the field overexpressing the nicotine demethylase CYP82E4v2₁The content of anabasine in the transgenic plant.

FIG. 8: t grown in the field overexpressing the nicotine demethylase CYP82E4v2₁Anatabine content in transgenic plants.

FIG. 9: t overexpressing Myb3 in LATN90₀The transgenic plants exhibit increased antioxidant content as measured by the iron reduction antioxidant capacity (FRAP) assay.

FIG. 10: increased antioxidant content was observed in 3 generations of transgenic plants overexpressing Myb 3.

FIG. 11: t overexpressing Myb3 in LATN90₂The transgenic plants exhibit increased antioxidant content as measured by the iron reduction antioxidant capacity (FRAP) assay.

FIG. 12: greenhouse-grown T overexpressing nicotine demethylase CYP82E4v2 and Myb3₀Nicotine content in transgenic plants.

FIG. 13: greenhouse-grown T overexpressing nicotine demethylase CYP82E4v2 and Myb3₀Nicotine reduction in transgenic plants

FIG. 14: greenhouse-grown T overexpressing nicotine demethylase CYP82E4v2 and Myb3₀The content of anabasine in the transgenic plant.

FIG. 15: greenhouse-grown T overexpressing nicotine demethylase CYP82E4v2 and Myb3₀Anatabine content in transgenic plants.

FIG. 16: t overexpressing CYP82E4v2 and Myb3 in TN90₀The transgenic plants exhibit increased antioxidant content as measured by the iron reduction antioxidant capacity (FRAP) assay.

Detailed Description

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

Any reference cited herein, including, for example, all patents and publications, are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof. Where terms are provided in the singular, the inventors also contemplate aspects of the invention described by the plural of the terms, and vice versa. Where there are differences in terms and definitions used in references incorporated by reference, the terms used in this application shall have the definitions given herein. Other technical terms used have their ordinary meaning in The technical field used, as exemplified by various field-specific dictionaries, e.g. "The American"Science Dictionary "(edition of the American University Dictionary, 2011, Houghton Mifflin Harcourt, Boston and New York)," McGraw-Hill Dictionary of Scientific and Technical Terms "(6 th edition,2002, McGraw-Hill, New York) or" Oxford Dictionary of Biology "(6 th edition,2008, Oxford University Press, Oxford and New York).

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

When a range of values is provided herein, the range should be understood to include any value between the limits of the range and the limits of the range. For example, "1-10" includes any number between 1and 10, as well as the numbers 1and 10.

The term "about" as used herein means about, roughly, around, or within a range thereof (e.g., within 10%). 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.

As used herein, any adverb, preposition, or other modifier phrase (e.g., about, less than, greater than, at least, at most) is used in conjunction with a string or a set of alternatives (e.g., quantitative or qualitative descriptors) to modify the entire string or set including each listed member of such string or set, whether or not the adverb, preposition, or other modifier is replicated before each member.

As used herein, the "Nic 1b locus" or "Nic 1b locus" refers to any chromosomal location or site within the Nic1b region or closely linked to the Nic1b region. "Nic 1b region" refers to a chromosomal fragment of approximately 150 kilobases in length, corresponding to SEQ ID No.9 of the TN90 genome, and having alleles associated with low biobasicity. Nic1b region is described in U.S. application Nos.16/246,281 and 16/246,308, both filed on 11/1 month 2019; and PCT application nos. PCT/US2019/013345 and PCT/US2019/013363, both filed on 11/1/2019, all of which are incorporated herein by reference in their entirety. "Nic 1b mutation" refers to a mutation in the Nic1b locus.

As used herein, Nic1b _ ERF (or plurals, Nic1b _ ERFs) refers to any one of the ERF genes or loci at or near the Nic1b locus and includes, for example, ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210, and ERF91L 2. See Table 8and Kajikawa et al, Plant physiol.2017,174: 999-. "Nic 1b _ ERF mutation" refers to a mutation in the Nic1b _ ERF gene. As used herein, mutant or mutant alleles are shown in all lower case letters and italics. Gene, locus or protein names are shown with all or beginning with capital letters and can be italicized or non-italicized.

As used herein, Nic2_ ERF (or plurals, Nic2_ ERFs) refers to any of the ERF genes or loci at or near the Nic2 locus and includes, for example, ERF221, ERF115, ERF168, ERF17, ERF179, ERF 189. See table 9; shoji et al, Plant Cell, (10): 3390-; and Kajikawa et al, Plant physiol.2017,174: 999-.

Table 8: the ERF gene selected at or near the Nic1b region. The genomic coordinates of each ERF gene are shown as start and stop on chromosome 7. ERF nomenclature is based on Rushton et al, TOBFAC the database of tobaco translation factors, BMC biolnformatics 2008,9: 53. The DNA sequence of each Nic1b _ ERF was obtained from the TobFac dataset and used to probe the tobacco genome database to identify annotated gene models, transcripts and chromosomal coordinates. A sequence identification number (SEQ ID nos.) is provided for an exemplary genomic coding DNA sequence (including introns, if applicable) and polypeptide sequence for each gene.

Table 9: the ERF gene selected at or near the Nic2 region. The genomic coordinates of each ERF gene are shown as start and stop on chromosome 19. ERF nomenclature is based on Rushton et al, TOBFAC the database of tobaco translation factors, BMC Bioinformatics 2008,9: 53; shoji et al, Plant Cell, (10): 3390-; and Kajikawa et al, Plant physiol.2017,174: 999-. The DNA sequence of each Nic2_ ERF was obtained from the TobFac dataset or from the NCBI database and used to probe the tobacco genome database to identify annotated gene models, transcripts and chromosomal coordinates. A sequence identification number (SEQ ID nos.) is provided for an exemplary genomic coding DNA sequence (including introns, if applicable) and polypeptide sequence for each gene.

As used herein, "genetic modification" refers to an alteration in the genetic composition of a plant or plant genome. Genetic modifications can be introduced by methods including, but not limited to, mutagenesis, genome editing, genetic transformation, or a combination thereof. Genetic modifications include, for example, mutations in the gene (e.g., non-natural mutations) or transgenes targeting the gene. As used herein, "targeting" refers to directly up-regulating or directly down-regulating the expression or activity of a gene. As used herein, "directly" in the context of a transgene that affects gene expression or activity refers to the effect exerted on the gene by physical contact or chemical interaction between the gene (e.g., promoter region or UTR region) or a product encoded therein (e.g., an mRNA molecule or polypeptide) and a product encoded by the transgene (e.g., a non-coding small RNA molecule or protein, such as a transcription factor or dominant negative polypeptide variant). In one aspect, the transgene affects the expression or activity of the target gene without involving a transcription factor (e.g., the transgene does not encode a transcription factor and/or does not inhibit the expression or activity of a transcription factor that in turn regulates the target gene).

As used herein, a mutation refers to a heritable genetic modification introduced into a gene to alter the expression or activity of a product encoded by the gene. Such modifications may be in any sequence region of the gene, for example in the promoter, 5'UTR, exon, intron, 3' UTR or terminator regions. In one aspect, the mutation reduces, inhibits, or eliminates the expression or activity of the gene product. In another aspect, the mutation increases, enhances, potentiates or enhances the expression or activity of the gene product. In one aspect, the mutation is not a natural polymorphism present in a particular tobacco variety or cultivar. As used herein, "mutant allele" refers to an allele from a locus in which the allele comprises a mutation. As used herein, "mutagenesis" refers to the generation of mutations that do not involve a transgene or do not leave a mutation-associated transgene in the final mutant. In one aspect, the mutagen is a homologous transgene. In another aspect, the mutagenesis is via gene or genome editing. In another aspect, the mutagenesis is via random mutagenesis, such as chemical (e.g., EMS) or physical (r-irradiation) mutagenesis. It will be appreciated that when identifying mutations, the reference sequences should be from the same tobacco variety or background. For example, if the modified tobacco plant comprising the mutation is from the TN90 variety, the corresponding reference sequence should be an endogenous TN90 sequence, rather than a homologous sequence from a different tobacco variety (e.g., K326). In one aspect, the mutation is a "non-natural" or "non-naturally occurring" mutation. As used herein, "non-natural" or "non-natural" mutations refer to mutations that are not and do not correspond to spontaneous mutations that occur without human intervention. Non-limiting examples of human intervention include mutagenesis (e.g., chemical mutagenesis, ionizing radiation mutagenesis) and targeted genetic modification (e.g., CRISPR-based methods, TALEN-based methods, zinc finger-based methods). Non-natural mutations and non-naturally occurring mutations do not include naturally occurring spontaneous mutations (e.g., via abnormal DNA replication in the plant germline).

As used herein, a tobacco plant may be from any plant of the genus Nicotiana, including, but not limited to, Nicotiana tabacum (Nicotiana tabacum), Nicotiana claspiana (Nicotiana amplexicaulis) PI 271989; nicotiana benthamiana PI 555478; bikulao tobacco (Nicotiana bigelovii) PI 555485; tobacco di bonnie (Nicotiana debneyi); high tobacco (Nicotiana excelsior) PI 224063; tobacco mucosae (Nicotiana glutinosa) PI 555507; gutesbi tobacco (Nicotiana goodspedii) PI 241012; -Nicotiana tabacum (Nicotiana gossei) PI 230953; western tobacco (nicotiana heperis) PI 271991; nicotiana tabacum (Nicotiana knightiana) PI 555527; seashore tobacco (Nicotiana maritima) PI 555535; super large tube tobacco (Nicotiana megasiphon) PI 555536; naked stem tobacco (Nicotiana nudifloris) PI 555540; cone tobacco (Nicotiana paniculata) PI 555545; blue jasmine leaf tobacco (Nicotiana plumbaginifolia) PI 555548; tabacco remana (Nicotiana repanda) PI 555552; yellow flower tobacco (Nicotiana rustica); nicotiana suaveolens PI 230960; forest tobacco (Nicotiana sylvestris) PI 555569; nicotiana tomentosa PI 266379; tobacco fluff (Nicotiana tomentosa); and Nicotiana triangularis (Nicotiana trigenophylla) PI 555572. In one aspect, the tobacco plant described herein is a nicotiana tabacum plant.

In one aspect, the present disclosure provides a tobacco plant, or a portion thereof, comprising a first genetic modification that inhibits one or more genes from the NIC1b locus, the NIC2 locus, or both, and further comprising a second genetic modification that increases the conversion of nicotine to nornicotine. In one aspect, the tobacco plant or portion thereof comprises a mutation in the Nic1b locus. In one aspect, the Nic1b locus comprises a gene selected from the group consisting of: ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210, and ERF91L 2. In one aspect, the tobacco plant 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: ERFnew, ERF199, ERF16, ERF19, ERF29, ERF210, and ERF91L 2. In one aspect, the tobacco plant comprises one or more mutations in ERFnew, ERF16, or both. In one aspect, the first genetic modification comprises a transgene or mutation that reduces the expression or activity of one or more non-ERF genes from the NIC1b locus. In one aspect, the tobacco plant or portion thereof comprises a mutation in the Nic2 locus. In one aspect, the Nic2 locus comprises a gene selected from the group consisting of: ERF189, ERF115, ERF221, ERF104, ERF179, ERF17 and ERF 168. In one aspect, the tobacco plant 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 ERF 168. In one aspect, the tobacco plant comprises one or more mutations in ERF189, ERF115, or both. In another aspect, the tobacco plant comprises one or more transgenes encoding a nicotine demethylase (e.g., CYP82E4, CYP82E5, CYP82E 10). In another aspect, the tobacco plant comprises one or more transgenes capable of expressing a nicotine demethylase when in a plant cell (e.g., CYP82E4, CYP82E5, CYP82E 10). In another aspect, the tobacco plant comprises one or more transgenic CYP82E4v2 encoding a nicotine demethylase. In another aspect, the tobacco plant comprises one or more transgenic CYP82E4v2 capable of expressing nicotine demethylase.

In one aspect, the tobacco plant further comprises one or more transgenes encoding AN antioxidant regulatory protein (e.g., arabidopsis PAP1, potato AN1, nicotiana tabacum Myb 3). In one aspect, the tobacco plant further comprises one or more transgenes capable of expressing AN antioxidant regulatory protein (e.g., arabidopsis PAP1, potato AN1, nicotiana tabacum Myb 3).

In one aspect, the present disclosure provides a tobacco plant, or a portion thereof, comprising a first genetic modification that provides mutagenesis or transgene suppression of one or more, two or more, three or more, four or more, five or more, six or more genes comprising a nucleotide sequence having at least 85%, 90%, 95%, 97%, 99% identity to SEQ ID Nos:10-19 and 30-36, and further comprising a second genetic modification that increases the conversion of nicotine to nornicotine. In one aspect, the present disclosure provides a tobacco plant, or part thereof, comprising a first genetic modification that provides mutagenesis or transgene suppression of one or more, two or more, three or more, four or more, five or more, six or more genes encoding nucleotide sequences having at least 90% identity to SEQ ID Nos:20-29 and 37-43, and further comprising a second genetic modification that increases the conversion of nicotine to nornicotine. In a further aspect, the tobacco plant further optionally comprises another genetic modification that increases the content of one or more antioxidants.

In one aspect, a tobacco plant of the present disclosure comprises a mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes), one or more transgenes encoding a nicotine demethylase and one or more transgenes encoding an antioxidant regulatory protein. In one aspect, a tobacco plant of the present disclosure comprises a mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes), one or more transgenes capable of expressing a nicotine demethylase, and one or more transgenes capable of expressing an antioxidant regulatory protein. In one aspect, a tobacco plant of the present disclosure comprises a mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes), a transgene encoding arabidopsis PAP1, and a transgene encoding nicotine demethylase CYP82E4v 2. In one aspect, a tobacco plant of the present disclosure comprises a mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes), a transgene encoding potato AN1, and a transgene encoding nicotine demethylase CYP82E4v 2. In one aspect, a tobacco plant of the present disclosure comprises a mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes), a transgene encoding tobacco Myb3, and a transgene encoding nicotine demethylase CYP82E4v 2. In one aspect, a tobacco plant of the present disclosure comprises a mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes), a transgene encoding arabidopsis PAP1, and a transgene encoding nicotine demethylase CYP82E 5. In one aspect, a tobacco plant of the present disclosure comprises a mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes), a transgene encoding potato AN1, and a transgene encoding nicotine demethylase CYP82E 5. In one aspect, a tobacco plant of the present disclosure comprises a mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes), a transgene encoding nicotiana tabacum Myb3, and a transgene encoding nicotine demethylase CYP82E 5. In one aspect, a tobacco plant of the present disclosure comprises a mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes), a transgene encoding arabidopsis PAP1, and a transgene encoding nicotine demethylase CYP82E 410. In one aspect, a tobacco plant of the present disclosure comprises a mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes), a transgene encoding potato AN1, and a transgene encoding nicotine demethylase CYP82E 10. In one aspect, a tobacco plant of the present disclosure comprises a mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes), a transgene encoding nicotiana tabacum Myb3, and a transgene encoding nicotine demethylase CYP82E 10.

In one aspect, a tobacco plant of the present disclosure, or a portion thereof, comprises a first genetic modification that inhibits one or more genes from the Nic1b or Nic2 locus, and further comprises a second genetic modification that increases the conversion of nicotine to nornicotine. In another aspect, the first genetic modification comprises a nic1b or a nic2 mutant allele. In another aspect, the nic1b or nic2 mutant allele is derived from a cuba cigar tobacco variety, low alkaloid burley 21(LABU21), low intermediate burley 21(LIBU21), low alkaloid flue-cured tobacco 53(LAFC53), low nicotine KY171(LNKY171), or a variety derived therefrom. In another aspect, the nic1b or nic2 mutant allele is not derived from the cuba cigar variety, low alkaloid burley tobacco 21(LABU21), low intermediate burley tobacco 21(LIBU21), low alkaloid flue-cured tobacco 53(LAFC53), low nicotine KY171(LNKY171), or a variety derived therefrom. In another aspect, the first genetic modification comprises a transgene or mutation that reduces the expression or activity of one or more Ethylene Response Factor (ERF) genes from the Nic1b or Nic2 loci. In one aspect, the transgene targets one or more genes selected from the group consisting of: ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210, and ERF91L 2. In a further aspect, the transgene targets one or more genes selected from the group consisting of: ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF 168.

In one aspect, a tobacco plant of the present disclosure, or a portion thereof, comprises a genetic modification comprising a transgene encoding a nicotine demethylase. In another aspect, the transgene encodes a nicotine demethylase gene from a plant of Nicotiana species (Nicotiana). In another aspect, the nicotine demethylase comprises a non-natural, mutated or engineered amino acid sequence.

In a further aspect, the genetic modification comprising a transgene encoding a nicotine demethylase overexpresses one or more of CYP82E4, CYP82E5, and CYP85E10 polypeptides. In another aspect, the genetic modification comprises genome editing that increases the expression or activity of one or more of CYP82E4, CYP82E5, and CYP85E10 polypeptides. In another aspect, the CYP82E4 nicotine demethylase is CYP82E4v 2.

In another aspect, a tobacco plant of the present disclosure, or a portion thereof, comprises a genetic modification that increases the content of one or more antioxidants as compared to a control plant lacking the genetic modification. In another aspect, the genetic modification comprises a transgene encoding or targeting an antioxidant biosynthetic enzyme, a regulatory transcription factor for an antioxidant, an antioxidant transporter, an antioxidant metabolic enzyme, or a combination thereof. In another aspect, the genetic modification comprises a modification of one or more endogenous genes encoding an antioxidant biosynthetic enzyme, a regulatory transcription factor for an antioxidant, an antioxidant transporter, an antioxidant metabolic enzyme, or a combination thereof. In another aspect, the tobacco plant comprises a transgene encoding one or more polypeptides selected from the group consisting of: AtPAP1, Ntmyb3A, Ntmyb3B, Ntmyb3C, NtJAF13, sta 1, NtAN1, and NtAN 2. See U.S. patent application publication US2018/0119163, which is incorporated herein by reference in its entirety.

In one aspect, a tobacco plant of the present disclosure, or a portion thereof, comprises an increased amount of one or more antioxidants. In one aspect, the antioxidant with increased amount is selected from the group consisting of: anthocyanins, flavanones, flavanols, flavones, flavonols, isoflavones, hydroxybenzoic acids, hydroxycinnamic acids, ellagitannins, stilbenes, lignans, carotenoids and glycyrrhizins. In another aspect, the antioxidant with an increased amount is selected from the group consisting of: delphinidin, anthocyanidin, procyanidin, delphinidin, hesperetin, naringenin, catechin, epicatechin, apigenin, luteolin (Luteonin), quercetin, myricetin, rutarin, genistein, daidzein, gallic acid, vanillic acid, protocatechuic acid, ferulic acid, cinnamic acid, coumaric acid, chlorogenic acid, caffeic acid, ferulic acid, sanguinarine, resveratrol, sesamin, carotenoids (carotonoids) and vitamin C.

In one aspect, the tobacco plants of the present disclosure are capable of producing, upon curing, leaves having a USDA grade index value selected from the group consisting of: 55 or higher, 60 or higher, 65 or higher, 70 or higher, 75 or higher, 80 or higher, 85 or higher, 90 or higher and 95 or higher. In another aspect, the tobacco plant, when cured, is capable of producing leaves having a USDA grade index value comparable to the USDA grade index value of a control plant grown and cured under similar conditions, wherein the control plant has substantially the same genetic background as the tobacco plant except for the mutation or modification of interest.

In another aspect, a tobacco plant of the present disclosure is capable of producing, upon ripening, leaves having a USDA rating 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 rating index value of a control plant grown under similar conditions, wherein the control plant has substantially the same genetic background as the tobacco plant except for the mutation or modification of interest.

In another aspect, the tobacco plants of the present disclosure are capable of producing, upon curing, leaves having a USDA rating index value that is 65% -130%, 70% -130%, 75% -130%, 80% -130%, 85% -130%, 90% -130%, 95% -130%, 100% -130%, 105% -130%, 110% -130%, 115% -130%, or 120% -130% of the USDA rating index value of a control plant. In another aspect, the tobacco plant, when cured, is capable of producing leaves having a USDA rating index value that is 70% -125%, 75% -120%, 80% -115%, 85% -110%, or 90% -100% of the USDA rating index value of the control plant. In one aspect, the tobacco plant comprises a nicotine content that is less than 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of the nicotine content of a control plant grown under similar growth conditions, wherein the control plant has substantially the same genetic background as the tobacco plant except for the mutation or modification of interest.

In one aspect, cured leaves from the modified tobacco plant produce or comprise less than 2, less than 1.8, less than 1.5, less than 1.2, less than 1.0, less than 0.8, less than 0.6, less than 0.4, less than 0.3, less than 0.2, less than 0.15, less than 0.1, or less than 0.05ppm total TSNAs. In one aspect, cured leaf from a modified tobacco plant comprises a total TSNA of 2ppm to 0.05ppm, 1.8ppm to 0.05ppm, 1.5ppm to 0.05ppm, 1.2ppm to 0.05ppm, 1.0ppm to 0.05ppm, 0.8ppm to 0.05ppm, 0.6ppm to 0.05ppm, 0.4ppm to 0.05ppm, 0.3ppm to 0.05ppm, 0.2ppm to 0.05ppm, 0.15ppm to 0.05ppm, or 0.1ppm to 0.05 ppm. In one aspect, cured leaf from the modified tobacco plant comprises 2ppm to 0.05ppm, 1.8ppm to 0.1ppm, 1.5ppm to 0.15ppm, 1.2ppm to 0.2ppm, 1.0ppm to 0.3ppm, 0.8ppm to 0.4ppm, or 0.6ppm to 0.5ppm total TSNA.

In one aspect, the modified tobacco plant produces or comprises less than 2, less than 1.8, less than 1.5, less than 1.2, less than 1.0, less than 0.8, less than 0.6, less than 0.4, less than 0.3, less than 0.2, less than 0.15, less than 0.1, or less than 0.05ppm total NNN. In one aspect, cured leaf from a modified tobacco plant comprises 2ppm to 0.05ppm, 1.8ppm to 0.05ppm, 1.5ppm to 0.05ppm, 1.2ppm to 0.05ppm, 1.0ppm to 0.05ppm, 0.8ppm to 0.05ppm, 0.6ppm to 0.05ppm, 0.4ppm to 0.05ppm, 0.3ppm to 0.05ppm, 0.2ppm to 0.05ppm, 0.15ppm to 0.05ppm, or 0.1ppm to 0.05ppm total NNN. In one aspect, cured leaf from a modified tobacco plant comprises 2ppm to 0.05ppm, 1.8ppm to 0.1ppm, 1.5ppm to 0.15ppm, 1.2ppm to 0.2ppm, 1.0ppm to 0.3ppm, 0.8ppm to 0.4ppm, or 0.6ppm to 0.5ppm total NNN.

In one aspect, the modified tobacco plant produces or comprises less than 2, less than 1.8, less than 1.5, less than 1.2, less than 1.0, less than 0.8, less than 0.6, less than 0.4, less than 0.3, less than 0.2, less than 0.15, less than 0.1, or less than 0.05ppm total NNK. In one aspect, cured leaf from a modified tobacco plant comprises 2ppm to 0.05ppm, 1.8ppm to 0.05ppm, 1.5ppm to 0.05ppm, 1.2ppm to 0.05ppm, 1.0ppm to 0.05ppm, 0.8ppm to 0.05ppm, 0.6ppm to 0.05ppm, 0.4ppm to 0.05ppm, 0.3ppm to 0.05ppm, 0.2ppm to 0.05ppm, 0.15ppm to 0.05ppm, or 0.1ppm to 0.05ppm total NNK. In one aspect, cured leaf from a modified tobacco plant comprises 2ppm to 0.05ppm, 1.8ppm to 0.1ppm, 1.5ppm to 0.15ppm, 1.2ppm to 0.2ppm, 1.0ppm to 0.3ppm, 0.8ppm to 0.4ppm, or 0.6ppm to 0.5ppm total NNK.

In another aspect, the tobacco plant of the present disclosure comprises a total alkaloid content 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 aspect, the tobacco plant comprises a nicotine or total alkaloid content 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 aspect, the tobacco plant further comprises a transgene or mutation that directly inhibits the expression or activity of one or more genes encoding products 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.

In another aspect, the tobacco plant, when cured, is capable of producing leaves having a USDA grade index value selected from the group consisting of: 55 or higher, 60 or higher, 65 or higher, 70 or higher, 75 or higher, 80 or higher, 85 or higher, 90 or higher and 95 or higher. In another aspect, the tobacco plant, when cured, is capable of producing leaves having 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 another aspect, the tobacco plant, when cured, is capable of producing leaves having a USDA rating 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 USDA rating index value of the control plant. In another aspect, the tobacco plant, when cured, is capable of producing leaves having a USDA rating index value that is 65% -130%, 70% -130%, 75% -130%, 80% -130%, 85% -130%, 90% -130%, 95% -130%, 100% -130%, 105% -130%, 110% -130%, 115% -130%, or 120% -130% of the USDA rating index value of the control plant. In a further aspect, the tobacco plant, when cured, is capable of producing leaves having a USDA rating index value that is 70% -125%, 75% -120%, 80% -115%, 85% -110%, or 90% -100% of the USDA rating index value of the control plant. In another aspect, the tobacco plant comprises a nicotine or total alkaloid content 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 or total alkaloid content of a control plant grown under similar growth conditions. In another aspect, the tobacco plant further comprises a transgene or mutation that directly inhibits 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.

In one aspect, the present disclosure also provides a tobacco variety, cultivar, or line comprising a nic1b or nic2 mutation comprising a genetic modification that increases the conversion of nicotine to nornicotine and optionally further comprising another genetic modification that increases the amount of one or more antioxidants, wherein the tobacco variety, cultivar, or line has a leaf grade comparable to the leaf grade of a control tobacco variety, cultivar, or line when grown under similar growth conditions, wherein the control tobacco variety has substantially the same genetic background as the tobacco variety, cultivar, or line except for the mutation or modification of interest.

In one aspect, the present disclosure provides a population of any of the tobacco plants disclosed herein.

In one aspect, the present disclosure provides cured tobacco material of any of the tobacco plants disclosed herein. In a further aspect, the present disclosure provides a tobacco blend comprising cured tobacco material of any of the tobacco plants disclosed herein. In another aspect, the present disclosure provides a tobacco product comprising a cured tobacco material provided herein.

In another aspect, the tobacco blend of cured tobacco material of the present disclosure constitutes about at least 10 weight percent, at least 15 weight percent, at least 20 weight percent, at least 25 weight percent, at least 30 weight percent, at least 35 weight percent, at least 40 weight percent, at least 45 weight percent, at least 50 weight percent, at least 55 weight percent, at least 60 weight percent, at least 65 weight percent, at least 70 weight percent, at least 75 weight percent, at least 80 weight percent, at least 85 weight percent, at least 90 weight percent, or at least 95 weight percent of cured tobacco in the tobacco blend. In another aspect, a tobacco blend of cured tobacco material of the present disclosure constitutes about at least 10 volume percent, at least 15 volume percent, at least 20 volume percent, at least 25 volume percent, at least 30 volume percent, at least 35 volume percent, at least 40 volume percent, at least 45 volume percent, at least 50 volume percent, at least 55 volume percent, at least 60 volume percent, at least 65 volume percent, at least 70 volume percent, at least 75 volume percent, at least 80 volume percent, at least 85 volume percent, at least 90 volume percent, or at least 95 volume percent of cured tobacco in the tobacco blend.

In one aspect, tobacco plants are provided that further comprise one or more transgenes (e.g., CYP82E4, CYP82E5, CYP82E10) expressing one or more nicotine demethylase proteins that confer an increased amount of nornicotine as compared to a control plant lacking one or more transgenes expressing nicotine demethylase (see U.S. Pat. Nos. 8,319,011; 8,124,851; 9,187,759; 9,228,194; 9,228,195; 9,247,706). In one aspect, provided tobacco plants further comprise one or more transgenes encoding one or more nicotine demethylase proteins (e.g., CYP82E4, CYP82E5, CYP82E10) that confer an increased amount of nornicotine compared to a control plant lacking the one or more transgenes encoding nicotine demethylase. In one aspect, the modified tobacco plants described further comprise increased nicotine demethylase activity when grown and cured under comparable conditions, as compared to control plants.

In one aspect, the present disclosure provides a modified tobacco plant capable of producing cured tobacco leaf, said modified tobacco plant comprising a reduced content of one or more Tobacco Specific Nitrosamines (TSNAs) and further comprising an increased content of one or more antioxidants, wherein the reduced and increased content is comparable to cured leaf of a control tobacco plant or a control tobacco plant from the same variety when grown and cured under comparable conditions. In one aspect, the reduced content of the one or more TSNAs is less than 50% of the content of the one or more TSNAs in cured leaves from control plants. In another aspect, the modified tobacco plant further comprises an increased level of Oxygen Radical Absorbance Capacity (ORAC) as compared to a control tobacco plant when grown and cured under comparable conditions.

In another aspect, cured leaves from the modified tobacco plant comprise reduced levels of total TSNAs as compared to cured leaves from a control tobacco plant when grown and cured under comparable conditions. In one aspect, the one or more TSNAs reduced are selected from N ' -nitrosonornicotine (NNN), 4- (methylnitrosamino) -l- (3-pyridyl) -l-butanone (NNK), N ' -Nitrosoanatabine (NAT), N ' -Nitrosoanabasine (NAB), and any combination thereof. In one aspect, the amount of total TSNA or individual TSNA is measured on a freeze-dried cured leaf sample using liquid chromatography with tandem mass spectrometry (LC/MS).

In one aspect, the present disclosure provides a tobacco plant comprising at least one mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes) and a transgene comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% homology to SEQ ID No. 1. In one aspect, the present disclosure provides a tobacco plant comprising at least one mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes) and a transgene comprising a sequence having at least 70% homology to SEQ ID NO: 1. In one aspect, the transgene comprises a sequence having at least 75% homology to SEQ ID No. 1. In one aspect, the transgene comprises a sequence having at least 80% homology to SEQ ID No. 1. In one aspect, the transgene comprises a sequence having at least 85% homology to SEQ ID No. 1. In one aspect, the transgene comprises a sequence having at least 90% homology to SEQ ID No. 1. In one aspect, the transgene comprises a sequence having at least 91% homology to SEQ ID No. 1. In one aspect, the transgene comprises a sequence having at least 92% homology to SEQ ID No. 1. In one aspect, the transgene comprises a sequence having at least 93% homology to SEQ ID No. 1. In one aspect, the transgene comprises a sequence having at least 94% homology to SEQ ID No. 1. In one aspect, the transgene comprises a sequence having at least 95% homology to SEQ ID No. 1. In one aspect, the transgene comprises a sequence having at least 96% homology to SEQ ID No. 1. In one aspect, the transgene comprises a sequence having at least 97% homology to SEQ ID No. 1. In one aspect, the transgene comprises a sequence having at least 98% homology to SEQ ID No. 1. In one aspect, the transgene comprises a sequence having at least 99% homology to SEQ ID No. 1. In one aspect, the transgene comprises a sequence having at least 100% homology to SEQ ID No. 1.

In one aspect, the present disclosure provides a tobacco plant comprising at least one mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes) and comprising an amino acid sequence identical to SEQ ID NO:1, and further comprising a second transgene, and a transgene having a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% homologous thereto, the second transgene comprises a sequence identical to SEQ ID NO:2, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% homologous.

In one aspect, the present disclosure provides a tobacco plant comprising at least one mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes) and comprising an amino acid sequence identical to SEQ ID NO:1, and further comprising a second transgene, and a transgene having a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% homologous thereto, the second transgene comprises a sequence identical to SEQ ID NO:3, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% homologous.

In one aspect, the present disclosure provides a tobacco plant comprising at least one mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes) and comprising an amino acid sequence identical to SEQ ID NO:1, and further comprising a second transgene, and a transgene having a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% homologous thereto, the second transgene comprises a sequence identical to SEQ ID NO:4, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% homologous.

In one aspect, the disclosure provides a tobacco plant comprising at least one mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes) and a transgene comprising SEQ ID No. 1and further comprising a second transgene comprising a sequence having 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% homology to SEQ ID No. 2.

In one aspect, the disclosure provides a tobacco plant comprising at least one mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes) and a transgene comprising SEQ ID No. 1and further comprising a second transgene comprising a sequence having 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% homology to SEQ ID No. 3.

In one aspect, the disclosure provides a tobacco plant comprising at least one mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes) and a transgene comprising SEQ ID No. 1and further comprising a second transgene comprising a sequence having 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% homology to SEQ ID No. 4.

In one aspect, the present disclosure provides a tobacco plant comprising at least one mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes) and a transgene comprising SEQ ID NO: 1and further comprising a second transgene comprising a sequence having at least 70% homology to SEQ ID NO: 2. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 75% homology to SEQ ID No. 2. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 80% homology to SEQ ID No. 2. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 85% homology to SEQ ID No. 2. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 90% homology to SEQ ID No. 2. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 91% homology to SEQ ID No. 2. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 92% homology to SEQ ID No. 2. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 93% homology to SEQ ID No. 2. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 94% homology to SEQ ID No. 2. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 95% homology to SEQ ID No. 2. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 96% homology to SEQ ID No. 2. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 97% homology to SEQ ID No. 2. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 98% homology to SEQ ID No. 2. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 99% homology to SEQ ID No. 2. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 100% homology to SEQ ID No. 2.

In one aspect, the present disclosure provides a tobacco plant comprising at least one mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes) and a transgene comprising SEQ ID NO: 1and further comprising a second transgene comprising a sequence having at least 70% homology to SEQ ID NO: 3. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 75% homology to SEQ ID No. 3. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 80% homology to SEQ ID No. 3. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 85% homology to SEQ ID No. 3. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 90% homology to SEQ ID No. 3. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 91% homology to SEQ ID No. 3. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 92% homology to SEQ ID No. 3. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 93% homology to SEQ ID No. 3. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 94% homology to SEQ ID No. 3. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 95% homology to SEQ ID No. 3. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 96% homology to SEQ ID No. 3. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 97% homology to SEQ ID No. 3. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 98% homology to SEQ ID No. 3. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 99% homology to SEQ ID No. 3. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 100% homology to SEQ ID No. 3.

In one aspect, the present disclosure provides a tobacco plant comprising at least one mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes) and a transgene comprising SEQ ID NO: 1and further comprising a second transgene comprising a sequence having at least 70% homology to SEQ ID NO: 4. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 75% homology to SEQ ID No. 4. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 80% homology to SEQ ID No. 4. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 85% homology to SEQ ID No. 4. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 90% homology to SEQ ID No. 4. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 91% homology to SEQ ID No. 4. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 92% homology to SEQ ID No. 4. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 93% homology to SEQ ID No. 4. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 94% homology to SEQ ID No. 4. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 95% homology to SEQ ID No. 4. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 96% homology to SEQ ID No. 4. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 97% homology to SEQ ID No. 4. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 98% homology to SEQ ID No. 4. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 99% homology to SEQ ID No. 4. In another aspect, the tobacco plant comprises a transgene comprising SEQ ID No. 1, and further comprises a second transgene comprising a sequence having at least 100% homology to SEQ ID No. 4.

In one aspect, the present disclosure provides a tobacco plant comprising at least one mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes) and a transgene comprising a nucleotide sequence encoding a protein sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to SEQ ID No. 5. In one aspect, the present disclosure provides a tobacco plant comprising at least one mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes) and a transgene comprising a nucleotide sequence encoding a protein sequence having at least 70% identity to SEQ ID NO: 5. In one aspect, the transgene comprises a nucleotide sequence encoding a protein sequence having at least 75% identity to SEQ ID No. 5. In one aspect, the transgene comprises a nucleotide sequence encoding a protein sequence having at least 80% identity to SEQ ID No. 5. In one aspect, the transgene comprises a nucleotide sequence encoding a protein sequence having at least 85% identity to SEQ ID No. 5. In one aspect, the transgene comprises a nucleotide sequence encoding a protein sequence having at least 90% identity to SEQ ID No. 5. In one aspect, the transgene comprises a nucleotide sequence encoding a protein sequence having at least 91% identity to SEQ ID No. 5. In one aspect, the transgene comprises a nucleotide sequence encoding a protein sequence having at least 92% identity to SEQ ID No. 5. In one aspect, the transgene comprises a nucleotide sequence encoding a protein sequence having at least 93% identity to SEQ ID No. 5. In one aspect, the transgene comprises a nucleotide sequence encoding a protein sequence having at least 94% identity to SEQ ID No. 5. In one aspect, the transgene comprises a nucleotide sequence encoding a protein sequence having at least 95% identity to SEQ ID No. 5. In one aspect, the transgene comprises a nucleotide sequence encoding a protein sequence having at least 96% identity to SEQ ID No. 5. In one aspect, the transgene comprises a nucleotide sequence encoding a protein sequence having at least 97% identity to SEQ ID No. 5. In one aspect, the transgene comprises a nucleotide sequence encoding a protein sequence having at least 98% identity to SEQ ID No. 5. In one aspect, the transgene comprises a nucleotide sequence encoding a protein sequence having at least 99% identity to SEQ ID No. 5. In one aspect, the transgene comprises a nucleotide sequence encoding a protein sequence having at least 100% identity to SEQ ID No. 5.

In one aspect, the present disclosure provides a tobacco plant comprising at least one mutation in the Nic1b or Nic2 locus (e.g., one or more Nic1b _ ERF or Nic2_ ERF genes) and a transgene comprising a nucleotide sequence encoding a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to SEQ ID No.5, and further comprising a second transgene comprising a nucleotide sequence encoding a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% >, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% >, or at least 100% identity to SEQ ID No.6, A nucleotide sequence of a protein that is at least 99% or at least 100% identical.

In one aspect, the present disclosure provides a tobacco plant comprising at least one mutation in the Nic1b or Nic2 locus (e.g., one or more Nic1b _ ERF or Nic2_ ERF genes) and a transgene comprising a nucleotide sequence encoding a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to SEQ ID No.5, and further comprising a second transgene comprising a nucleotide sequence encoding a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% >, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% >, or at least 100% identity to SEQ ID No.7, A nucleotide sequence of a protein that is at least 99% or at least 100% identical.

In one aspect, the present disclosure provides a tobacco plant comprising at least one mutation in the Nic1b or Nic2 locus (e.g., one or more Nic1b _ ERF or Nic2_ ERF genes) and a transgene comprising a nucleotide sequence encoding a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to SEQ ID No.5, and further comprising a second transgene comprising a nucleotide sequence encoding a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% >, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% >, or at least 100% identity to SEQ ID No. 8, A nucleotide sequence of a protein that is at least 99% or at least 100% identical.

In one aspect, the present disclosure provides a tobacco plant comprising at least one mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes) and a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:5, and further comprising a second transgene comprising a nucleotide sequence encoding a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to SEQ ID NO: 6.

In one aspect, the present disclosure provides a tobacco plant comprising at least one mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes) and a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:5, and further comprising a second transgene comprising a nucleotide sequence encoding a protein sequence having 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to SEQ ID NO: 7.

In one aspect, the present disclosure provides a tobacco plant comprising at least one mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes) and a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:5, and further comprising a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to SEQ ID NO: 8.

In one aspect, the present disclosure provides a tobacco plant comprising at least one mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes) and a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:5, and further comprising a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 70% identity to SEQ ID NO: 6. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 75% identity to SEQ ID No. 6. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 80% identity to SEQ ID No. 6. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 85% identity to SEQ ID No. 6. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 90% identity to SEQ ID No. 6. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 91% identity to SEQ ID No. 6. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 92% identity to SEQ ID No. 6. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 93% identity to SEQ ID No. 6. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 94% identity to SEQ ID No. 6. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 95% identity to SEQ ID No. 6. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 96% identity to SEQ ID No. 6. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 97% identity to SEQ ID No. 6. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 98% identity to SEQ ID No. 6. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 99% identity to SEQ ID No. 6. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 100% identity to SEQ ID No. 6.

In one aspect, the present disclosure provides a tobacco plant comprising at least one mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes) and a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:5, and further comprising a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 70% identity to SEQ ID NO: 7. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 75% identity to SEQ ID No. 7. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 80% identity to SEQ ID No. 7. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 85% identity to SEQ ID No. 7. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 90% identity to SEQ ID No. 7. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 91% identity to SEQ ID No. 7. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 92% identity to SEQ ID No. 7. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 93% identity to SEQ ID No. 7. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 94% identity to SEQ ID No. 7. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 95% identity to SEQ ID No. 7. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 96% identity to SEQ ID No. 7. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 97% identity to SEQ ID No. 7. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 98% identity to SEQ ID No. 7. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 99% identity to SEQ ID No. 7. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 100% identity to SEQ ID No. 7.

In one aspect, the present disclosure provides a tobacco plant comprising at least one mutation in the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes) and a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:5, and further comprising a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 70% identity to SEQ ID NO: 8. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 75% identity to SEQ ID No. 8. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 80% identity to SEQ ID No. 8. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 85% identity to SEQ ID No. 8. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 90% identity to SEQ ID No. 8. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 91% identity to SEQ ID No. 8. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 92% identity to SEQ ID No. 8. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 93% identity to SEQ ID No. 8. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 94% identity to SEQ ID No. 8. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 95% identity to SEQ ID No. 8. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 96% identity to SEQ ID No. 8. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 97% identity to SEQ ID No. 8. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 98% identity to SEQ ID No. 8. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 99% identity to SEQ ID No. 8. In another aspect, the tobacco plant comprises a transgene comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID No.5, and further comprises a second transgene comprising a nucleotide sequence encoding a protein sequence having at least 100% identity to SEQ ID No. 8.

In one aspect, the low nicotine and/or low TSNA tobacco plants (e.g., overexpression or elevated levels of CYP82E4v2 and Myb3 (or AtPAP 1)) further comprise one or more genetic modifications that provide reduced levels of anatabine. Exemplary genetic modifications that provide for reduction of anatabine can be found in US 20160010103a 1and US 10375910B 2. In one aspect, the genetic modification to reduce anatabine comprises a mutation in the Quinolinate Synthase (QS) gene. In another aspect, the QS gene mutation comprises a mutation resulting in an amino acid substitution at a position corresponding to cysteine residue 487 and/or valine residue 516 of SEQ ID No. 8 of US 20160010103a 1. In another aspect, the genetic modification to reduce anatabine is present in, introgressed from, or derived from tobacco strain dm 932, wherein a representative sample of seed of said tobacco plant is deposited under ATCC accession No. PTA-124990. In another aspect, the genetic modification to reduce anatabine is present in, introgressed from, or derived from a tobacco strain of US 10375910B2 selected from the group consisting of: MS108, MS445, MS170, and MS 3908.

In one aspect, the present disclosure also provides a tobacco plant or part thereof comprising a nicotine or total alkaloid content 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 rating 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 aspect, the non-transgenic tobacco plant comprises a nicotine content of less than 2.0% and is capable of producing leaves having a USDA grade index value of 70 or higher when cured. In another aspect, the non-transgenic tobacco plant comprises less than 1.0% nicotine content and is capable of producing leaves having a USDA grade index value of 70 or higher when cured.

In one aspect, the present disclosure also provides a tobacco plant or part thereof comprising a non-transgenic mutation, wherein said non-transgenic mutation reduces the nicotine or total alkaloid content of said 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 content of a control plant when grown under similar growth conditions, wherein said tobacco plant, when cured, is capable of producing leaves having a USDA grade index value comparable to the USDA grade index value of said control plant, and wherein the control plant has substantially the same genetic background as the tobacco plant except for the mutation or modification of interest.

In one aspect, the reduced or increased content is within about 10%, within about 20%, within about 30%, within about 40%, within about 50%, within about 60%, within about 70%, within about 80%, within about 90%, within about 92%, within about 94%, within about 95%, within about 96%, within about 97%, within about 98%, or within about 99% less than or greater than the content of a control tobacco plant or cured leaf from a control tobacco plant when grown and cured under comparable conditions.

In another aspect, the reduced or increased content is less than or within about 1-fold, within about 2-fold, within about 3-fold, within about 4-fold, within about 5-fold, within about 6-fold, within about 7-fold, within about 8-fold, within about 9-fold, within about 10-fold, within about 15-fold, within about 20-fold, within about 25-fold, or within about 30-fold greater than the content of a control tobacco plant or cured leaf from a control tobacco plant when grown and cured under comparable conditions.

In one aspect, the reduced or increased content is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 92%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% less than or greater than the content in a control tobacco plant or cured leaf from a control tobacco plant when grown and cured under comparable conditions.

In another aspect, the reduced or increased content is at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, or at least about 30-fold less than or greater than the content in a control tobacco plant or cured leaf from a control tobacco plant when grown and cured under comparable conditions.

In one aspect, the reduced or increased content is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% less or greater than the content in a control tobacco plant or cured leaf from a control tobacco plant when grown and cured under comparable conditions.

In another aspect, the reduced or increased content is about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, or about 30-fold less than or greater than the content in a control tobacco plant or cured leaf from a control tobacco plant when grown and cured under comparable conditions.

In one aspect, the reduced or increased content is about 1-2 times less than or about 2-3 times greater than, about 3-4 times greater than, about 4-5 times greater than, about 5-6 times greater than, about 6-7 times greater than, about 7-8 times greater than, about 8-9 times greater than, about 9-10 times greater than, about 10-15 times greater than, about 15-20 times greater than, about 20-25 times greater than, about 25-30 times greater than, or about 30-50 times greater than the content in a control tobacco plant or cured leaf from a control tobacco plant when grown and cured under comparable conditions.

In another aspect, the reduced or increased content is about 1-10 times less than or about 2-10 times greater than, about 3-10 times greater than, about 4-10 times greater than, about 5-10 times greater than, about 6-10 times greater than, about 7-10 times greater than, about 8-10 times greater than, about 9-10 times greater than, about 10-50 times greater than, about 15-50 times greater than, about 20-50 times greater than, about 25-50 times greater than, or about 30-50 times greater than the content in a control tobacco plant or cured leaf from a control tobacco plant when grown and cured under comparable conditions.

In one aspect, cured leaves from the modified tobacco plant produce or comprise less than 2, less than 1.8, less than 1.5, less than 1.2, less than 1.0, less than 0.8, less than 0.6, less than 0.4, less than 0.3, less than 0.2, less than 0.15, less than 0.1, or less than 0.05ppm total TSNAs. In one aspect, cured leaf from a modified tobacco plant comprises a total TSNA of 2ppm to 0.05ppm, 1.8ppm to 0.05ppm, 1.5ppm to 0.05ppm, 1.2ppm to 0.05ppm, 1.0ppm to 0.05ppm, 0.8ppm to 0.05ppm, 0.6ppm to 0.05ppm, 0.4ppm to 0.05ppm, 0.3ppm to 0.05ppm, 0.2ppm to 0.05ppm, 0.15ppm to 0.05ppm, or 0.1ppm to 0.05 ppm. In one aspect, cured leaf from the modified tobacco plant comprises 2ppm to 0.05ppm, 1.8ppm to 0.1ppm, 1.5ppm to 0.15ppm, 1.2ppm to 0.2ppm, 1.0ppm to 0.3ppm, 0.8ppm to 0.4ppm, or 0.6ppm to 0.5ppm total TSNA.

In one aspect, lower nicotine content refers to an average nicotine content that is less than 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of the average nicotine content of a control tobacco plant. In another aspect, lower nicotine content refers to an average nicotine content that is about 0.5% -1%, 1% -2%, 2% -3%, 3% -4%, 4% -5%, 5% -6%, 7% -8%, 8% -9%, 9% -10%, 11% -12%, 12% -13%, 14% -15%, 15% -16%, 17% -18%, 18% -19%, 19% -20%, 21% -22%, 22% -23%, 23% -24%, 25% -26%, 26% -27%, 27% -28%, 28% -29%, 29% -30% of the average nicotine content of a control tobacco plant. In another aspect, lower nicotine content refers to an average nicotine content that is about 0.5% -5%, 5% -10%, 10% -20%, 20% -30% of the average nicotine content of a control tobacco plant.

In one aspect, the tobacco plants provided herein comprise an average nicotine or total alkaloid content selected from the group consisting of: 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%, 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% by dry weight. In another aspect, provided herein are tobacco plants comprising an average nicotine or total alkaloid content selected from the group consisting of: about 0.01% -0.02%, 0.02% -0.05%, 0.05% -0.75%, 0.75% -0.1%, 0.1% -0.15%, 0.15% -0.2%, 0.2% -0.3%, 0.3% -0.35%, 0.35% -0.4%, 0.4% -0.5%, 0.5% -0.6%, 0.6% -0.7%, 0.7% -0.8%, 0.8% -0.9%, 0.9% -1%, 1% -1.1%, 1.1% -1.2%, 1.2% -1.3%, 1.3% -1.4%, 1.4% -1.5%, 1.5% -1.6%, 1.6% -1.7%, 1.7% -1.8%, 1.8% -1.9%, 1.9% -2%, 2% -2.1%, 2.1% -2.2%, 2.2.3% -2.3%, 2.3% -2.3%, 2.5% -2.3%, 2.6% -2.7%, 2.5% -2.6%, 2.5% -2.5%, 2.6%, 2.5% -2.7%, 2.5% -2.6%, 2.5% -2.5%, 2.5% -2.6%, 2.5% -2.5%, 2.5% -2.6%, 2.5% -2.4%, 2.5%, 2.6%, 2.5% -2.5%, 2.4% and 0.4% of a, 2.7% -2.8%, 2.8% -2.9%, 2.9% -3%, 3% -3.1%, 3.1% -3.2%, 3.2% -3.3%, 3.3% -3.4%, 3.4% -3.5% and 3.5% -3.6%. In a further aspect, provided herein are tobacco plants comprising an average nicotine or total alkaloid content selected from the group consisting of: about 0.01% -0.1%, 0.02% -0.2%, 0.03% -0.3%, 0.04% -0.4%, 0.05% -0.5%, 0.75% -1%, 0.1% -1.5%, 0.15% -2%, 0.2% -3%, and 0.3% -3.5% by dry weight.

LA burley tobacco 21 (also known as LABU21) is a low total alkaloid tobacco line produced by incorporating low alkaloid genes from a gumba variety into burley tobacco 21 by several backcrosses (Legg et al 1970). It has about 0.2% total alkaloids (dry weight), while its parent (burley tobacco 21) has about 3.5% total alkaloids (dry weight). The leaf grade of LA BU21 is well below commercially acceptable standards. LA BU21 also exhibits other unfavorable leaf phenotypes characterized by lower yield, delayed maturation and senescence, higher sensitivity to insect herbivores and poorer end product quality after maturation (Chaplin and Weeks, Crop Sci.16: 416-. LABU21 leaf also showed traits such as higher polyamine content, higher chlorophyll content and more mesophyll cells per unit leaf area.

The LA burley tobacco 21 line comprises nic 1and nic2 lesions and comprises a mean nicotine amount of about 0.3% on a dry weight basis (ranging from about 0.2-0.6%). Low Intermediate (LI) burley tobacco 21 comprises a Nic1 lesion and the wild type Nic2 locus and comprises an average amount of nicotine of about 2.3% (range of about 1.5-3.0%). High Intermediate (HI) burley tobacco 21 comprises the wild type Nic1 locus and the Nic2 lesion and the average nicotine amount is about 3.7% (range of about 2.5-5.0%). Wild type burley tobacco 21 (also known as "BU 21") (Nic1 Nic2) contains an average amount of nicotine of about 4.7% (ranging from about 4.0-6.0%). See U.S. patent application publication US2016/0374387, the entire contents of which are incorporated herein by reference. Previous Shoji et al (2010) reported the nic2 locus to contain a deletion of 7 Ethylene Response Factor (ERF) genes. These deleted ERF genes were mapped to a single contiguous region of the TN90 genome.

In one aspect, the modified tobacco plant comprises one or more mutations or modifications that can provide reduced levels of one or more TSNAs. In another aspect, the one or more mutations can also provide one or more traits selected from the group consisting of: i. an increased level of Oxygen Radical Absorbance Capacity (ORAC), and ii. Wherein said reduced or increased content is comparable to a control tobacco plant or cured leaf from a control tobacco plant when grown and cured under comparable conditions. See U.S. patent application publication US2018/0119163, which is incorporated herein by reference in its entirety.

Unless otherwise indicated, reference herein to a measurement of alkaloid, polyamine, or nicotine content (or another foliar chemical or characteristic characterization) or a foliar ranking index value of a tobacco plant, variety, cultivar, or line refers to an average measurement, including, for example, an average of a plurality of leaves of an individual plant or an average measurement of a population of tobacco plants from an individual variety, cultivar, or line. Unless otherwise indicated, the nicotine, alkaloid or polyamine content (or another leaf chemical or characteristic characterization) of the tobacco plants described herein was measured 2 weeks after topping in pooled leaf samples collected from leaves No. 3, 4 and 5 after topping. In another aspect, the nicotine, alkaloid, or polyamine content (or another foliar chemical or characteristic) of the tobacco plant is measured after topping in the leaf having the highest nicotine, alkaloid, or polyamine content (or another foliar chemical or characteristic). In one aspect, the nicotine, alkaloid, or polyamine content of the tobacco plant is measured in leaf No. 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 aspect, the nicotine, alkaloid or polyamine content (or another leaf chemical or characteristic characterization) of the tobacco plant is measured after topping in a collection of two or more leaves having consecutive leaf numbers selected from the group consisting of: 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 aspect, the nicotine, alkaloid or polyamine content (or another leaf chemical or characteristic characterization) of the tobacco plant is measured after topping in leaves with a leaf number selected from the group consisting of: 1-5, 6-10, 11-15, 16-20, 21-25 and 26-30. In another aspect, the nicotine, alkaloid or polyamine content (or another leaf chemical or characteristic characterization) of the tobacco plant is measured after topping in a collection of two or more leaves having a leaf number selected from the group consisting of: 1-5, 6-10, 11-15, 16-20, 21-25 and 26-30. In another aspect, the nicotine, alkaloid or polyamine content (or another leaf chemical or characteristic characterization) of the tobacco plant is measured after topping in a collection of three or more leaves having a leaf number selected from the group consisting of: 1-5, 6-10, 11-15, 16-20, 21-25 and 26-30.

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

The population of tobacco plants or collection of tobacco leaves used to determine the average measurement (e.g., alkaloid or nicotine content or leaf grade) can be any size, e.g., 5,10, 15, 20, 25, 30, 35, 40, or 50. The average measurement or grade index value is determined following industry-accepted standards.

As used herein, "topping" refers to removing the stem tip, including SAM, flowers and up to several adjacent leaves, when the tobacco plant is near the vegetative maturity and at about the beginning of reproductive growth. Typically, tobacco plants are topped at the button stage (shortly after flower onset occurs). For example, tobacco plants grown in greenhouses or in the field may be topped when 50% of the plants have at least one open flower. Topping tobacco plants result in a loss of apical dominance and also induce an increase in alkaloid production.

Typically, the nicotine, alkaloid or polyamine content (or another foliar chemical or characteristic characterization) of tobacco plants is measured about 2 weeks after topping. Other time points may also be used. In one aspect, the nicotine, alkaloid, or polyamine content (or another leaf chemical or characteristic characterization) of the tobacco plant is measured about 1, 2,3, 4, or 5 weeks after topping. In another aspect, the nicotine, alkaloid, or polyamine content (or another foliar chemical or characteristic characterization) of the tobacco plant is measured about 3, 5,7, 10, 12, 14, 17, 19, or 21 days after topping.

Unless otherwise indicated, nicotine or alkaloid content (or another leaf chemical or characteristic characterization) of tobacco plants was measured in pooled leaf samples collected from post-topping leaves No. 3, 4 and 5 after topping. As used herein, whenever reference is made to a comparison between leaves from two plants (e.g., a mutant plant VS control plant), it is expected that leaves from the same or comparable stem position and developmental stage make the comparison demonstrable an effect due to genotype differences, but not from other factors.

As used herein, "similar growth conditions" or "equivalent conditions" refer to similar environmental conditions and/or agronomic practices for growing and making meaningful comparisons between two or more plant genotypes, such that neither the environmental conditions nor the agronomic practices contribute to or explain any differences observed between the two or more plant genotypes. Environmental conditions include, for example, light, temperature, water (humidity), and nutrients (e.g., nitrogen and phosphorous). Agronomic practices include, for example, seeding, trimming, undercutting, transplanting, topping, and sucking. See Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford (1999), pp 70-103, chapters 4B and 4C.

"alkaloids" are complex nitrogen-containing compounds that occur naturally in plants and have pharmacological effects in humans and animals. "Nicotine" is the major natural alkaloid in commercial cigarette tobacco and accounts for about 90% of the alkaloid content in R.rubrum tobacco. Other major alkaloids in tobacco include cotinine, nornicotine, mesmine, diennicotinyl, anabasine and anatabine. The minor tobacco alkaloids include nicotine-N-oxide, N-methylanatabine, N-methylanabasine, pseudonicotine oxide, 2, 3-bipyridine and others.

The alkaloid content can be determined by methods known in the art, for example by quantification based on gas liquid chromatography, high performance liquid chromatography, radioimmunoassay and enzyme-linked immunosorbent assay. For example, nicotine alkaloid content can be measured by gas liquid chromatography using a capillary column and FID detector equipped based on CORESTA recommended method No.7,1987 and ISO standards (ISO TC 126N 394E.See also Hibi et al, Plant Physiology 100:826-35 (1992)). Unless otherwise indicated, all alkaloid levels described herein are measured using those methods according to CORESTA method No 62, Determination of the levels in Tobacco and Tobacco Products by Gas Chromatographic Analysis, February 2005, and the preservation's Protocol for Analysis of Nicotine, Total Moisture and pH in Smokel Tobacco Products, defined at the center for disease control and Federal Register volume 64, published on 23.3.1999, No. 55 (and 74. 2009, revised day 1.7, No. 4).

Alternatively, Tobacco total alkaloids can be measured using a segmented flow colorimetry developed for analyzing Tobacco samples, modified by Skalar Instrument Co (West Chester, Pa.) and described in Collins et al, Tobacco Science 13:79-81 (1969). Briefly, tobacco samples were dried, ground and extracted prior to analysis for total alkaloids and reducing sugars. The process then uses acetic acid/methanol/water extraction and charcoal for decolorization. The determination of total alkaloids is based on the reaction of cyanogen chloride with nicotine alkaloids in the presence of aromatic amines to form a colored complex, which is measured at 460 nm. Unless otherwise indicated, the total alkaloid content or nicotine content indicated herein is on a dry weight basis (e.g., percent total alkaloid or percent nicotine).

As used herein, "reduced" or "increased" levels refer to statistically significant changes (decreases or increases) compared to a reference point. As used herein, "statistically significant" refers to a p-value of less than 0.05, a p-value of less than 0.025, a p-value of less than 0.01, or a p-value of less than 0.001, when an appropriate measure of statistical significance (e.g., a one-sided two-sample t-test) is used.

As used herein, "control plant" refers to a comparative plant, which is an unmodified tobacco plant of the same variety or a tobacco plant without a transgene of interest, depending on the context or purpose of the control plant. Control tobacco plants and target plants were grown and cured under comparable conditions.

The present disclosure also provides tobacco plants having altered nicotine content without negatively affecting other tobacco traits (e.g., leaf grade index values). In one aspect, the low nicotine or smokeless tobacco variety provides a commercially acceptable grade of cured tobacco. Tobacco grade is evaluated based on factors including, but not limited to, leaf stem position, leaf size, leaf color, leaf uniformity and integrity, maturity, texture, elasticity, luster (related to leaf color strength and depth, and luster), hygroscopicity (the ability of tobacco leaves to absorb and retain ambient moisture), and green nuances or abscission. Leaf grades may be determined, for example, using official standard grades (7u.s.c. § 511) published by Agricultural Marketing Service of the united states department of agriculture. See, for example, the official standard ratings of burley tobacco (us type 31 and foreign type 93) in effect at 11/5/1990 (55 f.r.40645); official standard grades of flue-cured tobacco (us types 11, 12, 13, 14 and foreign type 92) in effect at 3/27/1989 (54 f.r.7925); official standard grade (u.s.type41) of wide leaf tobacco, pennsylvania, effective 1/8/1965 (29 f.r.16854); official standard grades of cigar tobacco, ohio (U.S. types 42, 43 and 44) (28f.r.11719 and 28f.r.11926), effective 12, 8, 1963; official standard grades of Wisconsin cigar inner leafing tobacco (U.S. model 54 and 55), effective 11/20/1969 (34 F.R.17061); official standard grades of Wisconsin cigar inner leafing tobacco (U.S. model 54 and 55), effective 11/20/1969 (34 F.R.17061); official standard grades of cigar overwrap tobacco were planted in shadow in georgia and florida, effective in 1971 at 4 months (U.S. model 62). The USDA grade index value may be determined based on an industrially acceptable grade index. See, e.g., Bowman et al, Tobacco Science,32:39-40 (1988); the conventional Tobacco documentation library (Bates documentation # 523267826) 523267833, 1.7.1988, Melorandum on the deployed Burley Tobacco Grade Index); and Miller et al, 1990, tobaco lnn., 192:55-57 (all of the above references are incorporated by reference in their entirety). Unless otherwise indicated, the USDA grade index is a 0-100 numerical representation of the received federal grade and is a weighted average of all stem positions. Higher grade index indicates higher quality. Alternatively, the leaf rank may be determined via hyperspectral imaging. See, e.g., WO2011/027315 (published on 3/10/2011 and incorporated by reference in its entirety).

In one aspect, modified tobacco plants provided herein comprise tobacco leaves having a reduced total TSNA and further comprise one or more desirable or enhanced properties, such as inhibition or reduced tobacco branch growth (packer growth) before or after topping. In one aspect, the modified plants provided herein comprise fewer total branches, fewer branches, or both, when grown and cured under comparable conditions, as compared to a control plant lacking such modification. In one aspect, the smaller smoke branch of a modified plant provided herein comprises a reduced mass, a reduced length, a reduced diameter, or a combination thereof, as compared to the smoke branch of a control plant grown and cured under comparable conditions.

In one aspect, the tobacco plants provided herein comprise similar amounts of one or more tobacco aroma compounds selected from the group consisting of: 3-methylvaleric acid, valeric acid, isovaleric acid, labdenoid, cembrenoid, sugar esters, and reducing sugars. In another aspect, the tobacco plants provided herein comprise a nic1 mutation, a nic2 mutation, or a combination thereof, which has no effect on the content of one or more tobacco aroma compounds selected from the group consisting of: 3-methylvaleric acid, valeric acid, isovaleric acid, labdenoid, cembrenoid, sugar esters, and reducing sugars.

As used herein, tobacco aroma compounds are compounds that are related to the flavor and aroma of tobacco smoke. These compounds include, but are not limited to, 3-methylvaleric acid, valeric acid, isovaleric acid, cembrane and labdenoid diterpenes and sugar esters. The concentration of tobacco aroma compounds can be determined by any metabolite profiling method known in the art, including but not limited to gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance spectroscopy, liquid chromatography-link mass spectrometry. See The Handbook of Plant metabolism, edited by Weckwerth and Kahl, (Wiley-Blackwell) (2013, 28.5 months).

As used herein, a "reducing sugar" is any sugar (mono-or polysaccharide) having free or potentially free aldehyde or ketone groups. Glucose and fructose act as nicotine buffers in cigarette smoke by lowering the smoke pH and effectively reducing the amount of "free" unprotonated nicotine. For example, reducing sugars balance smoke taste by altering the sensory impact of nicotine and other tobacco alkaloids. An inverse relationship between sugar content and alkaloid content has been reported between tobacco varieties, within the same variety, and within the same line as a result of planting conditions. The reducing sugar content can be measured using a step flow colorimetry developed for the analysis of Tobacco samples, modified by Skalar Instrument Co (West Chester, Pa.) and described in Davis, Tobacco Science 20: 139-. For example, the sample is dialyzed against a sodium carbonate solution. Copper neocopper reagent was added to the sample and the solution was heated. The copper neocopper reagent chelate is reduced in the presence of a sugar to give a colored complex measured at 460 nm.

In one aspect, the tobacco plants provided herein comprise one or more non-naturally occurring mutant alleles at the Nic1b or Nic2 locus (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes) that reduce or eliminate the genetic activity of one or more genes (e.g., one or more of the Nic1b _ ERF or the Nic2_ ERF genes) of the Nic1b or Nic2 locus. In one aspect, these mutant alleles result in lower nicotine levels. The mutant Nic2 allele can be introduced by any method known in the art, including random or targeted mutagenesis pathways.

As used herein, a mutation refers to a heritable genetic modification introduced into a gene to reduce, inhibit, or eliminate the expression or activity of a product encoded by the gene. The modification may be in any sequence region of the gene, for example in the promoter, 5'UTR, exon, intron, 3' UTR or terminator regions. In one aspect, the mutation is not a natural polymorphism present in a particular tobacco variety or cultivar. As used herein, "mutant allele" refers to an allele from a locus in which the allele comprises a mutation.

Mutagenesis methods provided herein include, but are not limited to, treatment of seeds with Ethyl Methyl Sulfate (EMS) (Hildering and Verkerk, In, The use of induced mutations In plant Breeding, Pergamon press, pp 317-. EMS-induced mutagenesis consists of chemically inducing random point mutations over the length of the genome. Fast neutron mutagenesis involves exposing seeds to neutron bombardment, which results in a large number of deletions by double-stranded DNA breaks. Transposon tagging involves inserting a transposon within an endogenous gene to reduce or eliminate expression of the gene. The types of mutations that may be present in the tobacco gene include, for example, point mutations, deletions, insertions, duplications and inversions. These mutations are desirably present in the coding region of the tobacco gene; however, mutations in promoter and intron or untranslated regions of the tobacco gene may also be desirable.

In addition, rapid and automatable methods of screening for chemically induced mutant TILLING (target-induced local lesions in the genome) using denaturing HPLC or selective endonuclease digestion of selected PCR products are also suitable for use in the present disclosure. See, McCallum et al (2000) nat. Biotechnol.18: 455-. Mutations that affect gene expression or interfere with gene function can be determined using methods well known in the art. Insertional mutations in exons of genes often result in null mutants. Mutations in conserved residues are particularly effective in inhibiting protein function. In one aspect, a tobacco plant comprising a nonsense (e.g., stop codon) mutation is one or more NCG genes described herein.

In one aspect, the present disclosure also provides tobacco lines having altered nicotine content while maintaining commercially acceptable leaf quality. These lines can be generated by introducing mutations into one or more genes of the Nic1b or Nic2 loci (e.g., one or more of the Nic1b _ ERF or Nic2_ ERF genes) via precise genomic engineering techniques, e.g., transcription activator-like effector nucleases (TALENs), meganucleases, zinc finger nucleases, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 systems, CRISPR/CasX systems, CRISPR/CasY systems, CRISPR/Cpf1 systems, CRISPR/Csm1 systems, and combinations thereof (see, e.g., U.S. patent application publication 2017/0233756). See, e.g., Gaj et al, Trends in Biotechnology,31(7):397-405 (2013).

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

In one aspect, the modified tobacco plant comprises one or more mutations or modifications capable of activating one or more genes encoding biosynthetic enzymes, regulatory transcription factors, transporters, catabolic enzymes, or a combination thereof, for one or more antioxidants. In another aspect, one or more mutations or modifications in one or more genes encoding a biosynthetic enzyme, a regulatory transcription factor, a transporter, a catabolic enzyme, or a combination thereof for one or more antioxidants selected from the group consisting of: anthocyanins, flavanones, flavanols, flavones, flavonols, isoflavones, hydroxybenzoic acids, hydroxycinnamic acids, ellagitannins, stilbenes, lignans, carotenoids and glycyrrhizin. In another aspect, the one or more mutations or modifications are in one or more genes encoding biosynthetic enzymes, regulatory transcription factors, transport proteins, catabolic enzymes, or a combination thereof for one or more antioxidants selected from the group consisting of: delphinidin, anthocyanidin, procyanidin, delphinidin, hesperetin, naringenin, catechin, epicatechin, apigenin, luteolin, quercetin, myricetin, rutin, genistein, daidzein, gallic acid, vanillic acid, protocatechuic acid, ferulic acid, cinnamic acid, coumaric acid, chlorogenic acid, caffeic acid, ferulic acid, sanguinarine, resveratrol, sesamin, carotenoid and vitamin C.

In one aspect, the modified tobacco plants of the present description comprise tobacco leaves having increased anthocyanin content. In another aspect, the modified tobacco plant with increased anthocyanin content further comprises leaves having a purple or deep red visual appearance. In one aspect, the modified tobacco plant of the present specification comprises tobacco leaf having increased antioxidant content without increased anthocyanin content. In another aspect, a modified tobacco plant comprising increased levels of antioxidants without increased levels of anthocyanins further comprises leaves having a similar visual appearance as an unmodified tobacco plant.

As used herein, "antioxidant modulator protein" refers to a biosynthetic enzyme, regulatory transcription factor, or transporter protein that, when mutated or expressed in a tobacco plant, can alter the amount of one or more antioxidants as compared to a control plant that does not comprise the mutated or expressed biosynthetic enzyme, regulatory transcription factor, or transporter protein.

As used herein, "biosynthetic enzyme" refers to a protein that plays a role in the synthesis of antioxidants, alkaloids, TSNAs, chlorogenic acids, or other proteins that affect the activity or stability of antioxidants, alkaloids, TSNAs, or chlorogenic acids. These proteins catalyze reactions that result in the transformation of one molecular structure into another structure that is part of the biosynthetic pathway. Exemplary biosynthetic enzymes include, but are not limited to, anthocyanin synthase 2(NtANS2), dihydroflavonol-4-reductase (NtDFR2), shikimate O-hydroxycinnamoyl transferase (HCT), and hydroxycinnamoyl coenzyme A quinuclidine transferase (HQT). The activity of biosynthetic enzymes affects the overall concentration of the different molecular species that make up the biosynthetic pathway.

As used herein, a "regulatory transcription factor" is a protein that binds to a promoter element of a target gene to regulate transcription of one or more genes involved in antioxidant biosynthesis, transport, catabolism or other processes affecting the level of one or more antioxidants. Exemplary regulatory transcription factors include AtPAP1, ntpp 1, NtMYB 3-like, NtJAF13, and AtTTG1, see U.S. patent application publication US2018/0119163, the entire contents of which are incorporated herein by reference. Regulatory transcription factors may bind to DNA as part of a protein complex or alone. Regulatory transcription factors can have a single target or multiple targets and can bind different targets with different affinities. Modulating the activity of a transcription factor may be activating, inhibiting or attenuating transcription at a target locus.

As used herein, a "transporter" may be a transmembrane protein that actively or passively moves a molecule through a biological membrane. Transport proteins may facilitate the movement of ions, small molecules or large molecules. The transporter may be referred to as a transmembrane transporter, transmembrane pump, anion transporter, cation transporter, or convulsant protein. Transport proteins may also facilitate the movement of molecules or proteins in vesicles composed of biological membranes. The transporter may be integrated into a biological membrane. The transporter may be anchored to the biofilm via various modifications, such as, but not limited to, myristylation, prenylation, or palmitoylation.

In one aspect, the tobacco plants or plant genomes provided herein are mutated or edited by a nuclease selected from the group consisting of: meganucleases, Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 nuclease, CRISPR/CasX nuclease, CRISPR/CasY nuclease, CRISPR/Cpf1 nuclease, or CRISPR/Csm1 nuclease.

As used herein, "editing" or "genome editing" refers to 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 removal or replacement of an endogenous plant genomic nucleic acid sequence. In one aspect, 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, an edited nucleic acid sequence is provided that 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 a polynucleotide selected from the group consisting of: 1-4 and fragments thereof. In another aspect, an edited nucleic acid sequence is provided that 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 a polynucleotide encoding a polypeptide selected from the group consisting of: 5-8 of SEQ ID NO.

In one aspect, sequence identity may be altered by any means known in the art capable of substituting, adding, or deleting nucleotides or amino acids of a sequence relative to a reference sequence.

Meganucleases, ZFNs, TALENs, CRISPR/Cas9, CRISPR/CasX, CRISPR/CasY, CRISPR/Csm1, and CRISPR/Cpf1 induce double-stranded DNA breaks at target sites of the genomic sequence, which are then repaired by the natural process of Homologous Recombination (HR) or non-homologous end joining (NHEJ). Sequence modifications then occur at the cleavage site, which may include deletions or insertions leading to gene disruption in the case of NHEJ, or integration of the donor nucleic acid sequence by HR. In one aspect, provided methods include editing a plant genome with 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 than 10 nucleotides in the plant genome with a donor polynucleotide via HR. In one aspect, the mutations provided result from genome editing using a nuclease. In another aspect, the mutations provided result from non-homologous end joining or homologous recombination.

In one aspect, the mutations provided herein provide a dominant mutant that activates expression or activity of a target gene, such as a gene selected from the group consisting of: biosynthetic enzymes, regulatory transcription factors, transport proteins, catabolic enzymes, or combinations thereof, for one or more antioxidants.

Meganucleases commonly identified in microorganisms are unique enzymes with high activity and long recognition sequences (>14bp), leading to site-specific digestion of the target DNA. Engineered versions of naturally occurring meganucleases typically have extended DNA recognition sequences (e.g., 14-40 bp). Engineering of meganucleases can be more challenging than engineering of ZFNs and TALENs because the DNA recognition and cleavage functions of meganucleases are interwoven in 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.

ZFNs are synthetic proteins consisting of an engineered zinc finger DNA binding domain fused to the cleavage domain of a FokI restriction endonuclease. ZFNs can be designed to cut double stranded DNA of almost any length to modify the zinc finger DNA binding domain. ZFNs form dimers from monomers consisting of a non-specific DNA cleavage domain fused to a FokI endonuclease engineered to bind to a zinc finger array of a target DNA sequence.

The DNA binding domain of ZFNs typically consists of 3-4 zinc finger arrays. The amino acids at positions-1, +2, +3, and +6 relative to the start of the zinc finger ∞ -helix (which facilitate site-specific binding to the target DNA) can be altered and tailored to suit a specific target sequence. Other amino acids form a common backbone to generate ZFNs with different sequence specificities. Rules for selecting target sequences for ZFNs are known in the art.

FokI nuclease domains require dimerization to cleave DNA, so two ZFNs with their C-terminal regions are required to bind to opposite DNA strands (5-7 bp apart) of the cleavage site. If both-ZF-binding sites are palindromic, the ZFN monomer may cleave the target site. The term ZFN as used herein is broad-based and includes monomeric ZFNs that can cleave double-stranded DNA without the aid of a signal from another ZFN. The term ZFN is also used to refer to one or both members of a pair of ZFNs that are engineered to work together to cut DNA at the same site.

Without being bound by any scientific theory, because the DNA binding specificity of the zinc finger domain can in principle be re-engineered using one of a variety of methods, custom ZFNs can theoretically be constructed to target almost any gene sequence. Publicly available methods for engineering zinc finger domains include background dependent assembly (CoDA), Oligomerization Pool Engineering (OPEN), and modular assembly.

TALENs are artificial restriction enzymes produced by fusing a transcription activator-like effector (TALE) DNA binding domain to a fokl nuclease domain. When each member of the TALEN pair binds to a DNA site flanking the target site, the fokl monomers dimerize and cause a double-stranded DNA break at the target site. The term TALEN as used herein is broad and includes monomeric TALENs that can cleave double-stranded DNA without the aid of a signal from another TALEN. The term TALEN is also used to refer to one or both members of a pair of TALENs that act together to cleave DNA at the same site.

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 of the genus Xanthomonas (Xanthomonas). The xanthomonas pathogen secretes TALE into the host plant cell during infection. TALEs move to the nucleus where they recognize and bind to specific DNA sequences in the promoter region of specific genes in the host genome. TALEs have a central DNA binding domain consisting of 13-28 repeating monomers of 33-34 amino acids. The amino acids of each monomer are highly conserved, except for the hypervariable amino acid residues at positions 12 and 13. These two variable amino acids are called Repeat Variable Diresidue (RVD). 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 can recognize contiguous DNA bases. This simple relationship between amino acid sequence and DNA recognition allows the engineering of specific DNA binding domains by selecting combinations of repeats that contain appropriate RVDs.

In addition to the wild-type fokl cleavage domain, variants with mutated fokl cleavage domains have been designed to improve cleavage specificity and cleavage activity. The FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites with the correct orientation and spacing in the target genome. The number of amino acid residues between the TALEN DNA binding domain and the fokl cleavage domain and the number of bases between two individual TALEN binding sites are both parameters for obtaining a high level of activity.

The relationship between the amino acid sequence of the TALE binding domain and DNA recognition allows for programmable proteins. Software programs (such as DNA Works) can be used to design TALE constructs. Other methods of designing TALE constructs are known to those skilled in the art. See Doyle et al,. Nucleic Acids Research (2012)40: W117-122; cerak et al, Nucleic Acids Research (2011).39: e 82; and tall-nt. cac. corn. edu/about.

CRISPR/Cas9, CRISPR/CasX, CRISPR/CasY, CRISPR/Csm1, or CRISPR/Cpf1 systems are alternatives to fokl-based methods, ZFNs, and TALENs. CRISPR systems are based on RNA-guided engineered nucleases that use complementary base pairing to recognize DNA sequences at target sites.

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 obtained by integrating a short segment of invasive DNA, called a spacer, between two adjacent repeats proximal to the CRISPR locus. The CRISPR array (including the spacer) is transcribed when it subsequently encounters invasive DNA and is processed into small interference CRISPR RNA (crRNA) of about 40NT in length, which is combined with trans-activation CRISPRRNA(tracrRNA) to activate and guide the Cas9 nuclease. This cleaves the homologous double-stranded DNA sequence called the protospacer in the invaded DNA. A prerequisite for cleavage is the presence of a conserved protospacer-adjacent motif (PAM) downstream of the target DNA, which usually has the sequence 5-NGG-3, but less often the sequence NAG. Specificity is provided by the so-called "seed sequence" of about 12 bases upstream of the PAM, which must be matched between the RNA and the target DNA. Cpf 1and Csm1 function in a similar manner to Cas9, but Cpf 1and Csm1 do not require tracrRNA.

In yet another aspect, tobacco plants are provided that further comprise one or more transgenes encoding a nicotine demethylase (e.g., CYP82E4, CYP82E5, CYP82E10) that confers an increased amount of nornicotine as compared to a control plant lacking the one or more transgenes (for exemplary nicotine demethylase sequences, see U.S. patent nos. 8,319,011; 8,124,851; 9,187,759; 9,228,194; 9,228,195; 9,247,706). In one aspect, the modified tobacco plants described further comprise increased nicotine demethylase activity when grown and cured under comparable conditions, as compared to control plants.

The term "inhibitory sequence" encompasses any polynucleotide or polypeptide sequence, 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 sense orientation, a nucleotide sequence in antisense orientation, a complement of a nucleotide sequence in 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, combinations thereof, or the like, that is capable of inhibiting expression or function of a gene involved in the regulation of nicotine biosynthesis from the Nic1b or Nic2 locus in a plant (e.g., one or more Nic1b _ ERF or Nic2_ ERF genes). The term "polynucleotide sequence" includes sequences of RNA, DNA, chemically modified nucleic acids, nucleic acid analogs, combinations thereof, and the like.

The inhibitory sequence is designated by the name of the target gene product. Thus, a "Nic 2 inhibitory sequence" refers to an inhibitory sequence capable of inhibiting, e.g., at the transcriptional and/or translational level, the expression of a gene from the Nic2 locus in a plant involved in the regulation of nicotine biosynthesis, or capable of inhibiting the function of the gene product. When the phrase "capable of inhibiting" is used in the context of a polynucleotide inhibitory sequence, it is intended that the inhibitory sequence itself exerts an inhibitory effect; alternatively, when 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), the product of transcription or translation exerts an inhibitory effect on the target gene product (e.g., inhibits the expression or function of the target gene product), respectively, 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 (in the case of an inhibitory sequence encoding an inhibitory polypeptide).

In one aspect, the genetic modification that inhibits one or more of the Nic1b or Nic2 genes comprises one or more of the Nic1b or Nic2 inhibitory sequences. The disclosed Nic1b or Nic2 inhibitory sequences may be sequences that elicit 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 result, the Nic1b or Nic2 inhibitory sequence may be at least about 20 nucleotides, about 50 nucleotides, about 70 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 350 nucleotides, about 400 nucleotides, and up to the full-length polynucleotide encoding the disclosed protein. In one aspect, the Nic1b or Nic2 inhibitory sequence may be a fragment 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 length. In some embodiments, fragments of the cytochrome P450 polynucleotide are about 50, about 60, about 70, about 80, about 90, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 400 nucleotides in length, and other such values between about 70 and about 400 nucleotides in length. In one aspect, the Nic1b or Nic2 inhibitory sequence may comprise about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

The use of the term "polynucleotide" is not intended to limit the present disclosure to polynucleotides comprising DNA. One of ordinary skill in the art will recognize that polynucleotides can include ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. These deoxyribonucleotides and ribonucleotides include naturally occurring molecules and synthetic analogs. Polynucleotides of the present disclosure also encompass all forms of sequences, including but not limited to single stranded forms, double stranded forms, hairpins, stem-loop structures, and the like.

In one aspect, the present disclosure provides a recombinant DNA construct comprising a promoter functional in a tobacco cell operably linked to a polynucleotide encoding an RNA molecule capable of binding to an 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%, at least 99% identical to an amino acid sequence selected from the group consisting of seq id nos: 5-8 and fragments thereof, and wherein said RNA molecule inhibits expression of said 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 material or tobacco products comprising these recombinant DNA constructs. In one aspect, the transgenic plants, cured tobacco material, or tobacco products comprise lower levels of nicotine as compared to control tobacco plants that do not contain the recombinant DNA construct. Further provided are methods of reducing the nicotine content of a tobacco plant comprising transforming a tobacco plant with any of these recombinant DNA constructs.

As used herein, "operably linked" refers to a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional linkage that allows expression of the polynucleotide of interest. The operably linked elements may be continuous or discontinuous.

As used herein and when applied to sequences, "heterologous" refers to sequences derived from a foreign species, or if from the same species, substantially modified from their native form in composition and/or genomic locus by deliberate human intervention. The term also applies to nucleic acid constructs, also referred to herein as "polynucleotide constructs" or "nucleotide constructs". In this manner, a "heterologous" nucleic acid construct means a construct that is derived from a foreign species, or, if derived from the same species, is substantially modified from its native form at the composition and/or genomic locus by deliberate human intervention. Heterologous nucleic acid constructs include, but are not limited to, recombinant nucleotide constructs that have been introduced into a plant or plant part thereof, e.g., via a transformation method or subsequent breeding of a transgenic plant with another plant of interest. In one aspect, the promoter used is heterologous to the sequence driven by the promoter. In another aspect, the promoter used is heterologous to tobacco. In a further aspect, the promoter used is native to tobacco.

In one aspect, the modified tobacco plant described is a homologously transgenic (cisgenic) plant. As used herein, "homologous transgene" or "homotransgenic" refers to a genetic modification of a plant, plant cell, or plant genome in which all components (e.g., promoters, donor nucleic acids, selection genes) are of plant origin only (i.e., components not of plant origin are not used). In one aspect, modified plants, plant cells, or plant genomes provided are homotransgenic. The provided homologous transgenic plants, plant cells, and plant genomes can result in ready-to-use tobacco lines. In another aspect, modified tobacco plants are provided that do not comprise non-tobacco genetic material or sequences.

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

Also provided herein are compositions and methods for overexpressing one or more nicotine demethylase polypeptides in plants having transgene or mutagenic suppression of one or more genes from the Nic1b or Nic2 locus (e.g., one or more Nic1b _ ERF or Nic2_ ERF genes) in plants, particularly plants of the nicotiana species, including tobacco plants of various commercial varieties, and further optionally comprising another genetic modification that increases the content of one or more antioxidants.

In one aspect, the recombinant DNA construct or expression cassette may further comprise a selectable marker gene for selection of transgenic cells. Selectable marker genes include, but are not limited to, genes encoding antibiotic resistance, such as the genes encoding neomycin phosphotransferase ii (neo) and Hygromycin Phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate, bromoxynil, imidazolinone, and 2, 4-dichlorophenoxyacetate (2, 4-D). Other selectable markers include phenotypic markers such as beta-galactosidase and fluorescent proteins such as Green Fluorescent Protein (GFP).

In one aspect, the recombinant DNA construct or expression cassette comprises a promoter selected from the group consisting of: constitutive promoters, inducible promoters, and tissue-preferred promoters (e.g., leaf-specific or root-specific promoters). Exemplary constitutive promoters include the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in U.S. patent 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) the or. appl. Genet.81: 581-588); MAS (Velten et al (1984) EMBO J3: 2723-2730); ALS promoter (U.S. Pat. No.5,659,026), and the like. Exemplary chemically inducible promoters include the tobacco PR-1a promoter activated by salicylic acid. Other chemically inducible promoters of interest include the steroid responsive promoters (Schena et al (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al (1998) Plant J.14(2): 247-257) and the tetracycline inducible promoters (see, e.g., Gatz et al (1991) mol. Gen. Genet.227:229-237 and U.S. Pat. Nos.5,814, and 618 5,789,156). Other exemplary promoters which may be used are the promoters responsible for the expression of thermally regulated genes, the expression of photoregulated genes (e.g., pea rbcS-3A; maize rbcS promoter; chlorophyll alb-binding protein gene found in pea; or the Arassu promoter), hormone-regulated gene expression (e.g., abscisic acid (ABA) response sequence of the Em gene from wheat; ABA-inducible HVA 1and HVA22 and the rd29A promoter of barley and Arabidopsis; and wound-induced gene expression (e.g., of wunl), organ-specific gene expression (e.g., of tuber-specific storage protein genes); the 23-kDa zein gene from maize; or lentils (. beta. -phaseolin gene), or pathogen-inducible promoters (e.g., PR-1, prp-1, or. beta. -1,3 dextranase promoter, a wheat fungal inducible wirla promoter and a nematode inducible promoter, TobRB7-5A and Hmg-1 of tobacco dried celery).

Various types of promoters may be used in the transgenes or recombinant nucleic acids described herein (e.g., for expression of nicotine demethylase, NtMyb3, or AtPAP1), classified according to various criteria, such as constitutive, developmental, tissue-specific, tissue-preferred, inducible, etc., in relation to the coding sequence or expression pattern of the gene (including the transgene) operably linked to the promoter. Promoters that initiate transcription in all or most tissues of a plant are referred to as "constitutive" promoters. Promoters that initiate transcription at certain stages or stages of development are referred to as "developmental" promoters. Promoters whose expression is enhanced in certain tissues of a plant relative to other plant tissues are referred to as "tissue-enhanced" or "tissue-preferred" promoters. Thus, a "tissue-preferred" promoter causes relatively higher or preferred expression in a particular tissue of a plant, but has lower expression levels in other tissues of the plant. Promoters that express in a particular tissue of a plant and little or no expression in other plant tissues are referred to as "tissue-specific" promoters. Promoters that are expressed in a certain cell type of a plant are referred to as "cell type specific" promoters. An "inducible" promoter is a promoter that initiates transcription in response to environmental stimuli (such as cold, drought, heat, or light) or other stimuli (such as injury) or chemical application. Promoters classified according to their origin, such as heterologous, homologous, chimeric, synthetic, and the like, are also used herein. A "heterologous" promoter is a promoter sequence of a different origin relative to its associated transcribable sequence, coding sequence or gene (or transgene), and/or a promoter sequence not naturally present in the plant species to be transformed. The term "heterologous" more broadly includes a combination of two or more DNA molecules or sequences, when such a combination is not normally found in nature. For example, two or more DNA molecules or sequences are heterologous to each other if they are typically present at different loci in different genomes or in the same genome, or if they are combined differently in nature.

In one aspect, provided tobacco plants further comprise increased or decreased expression of an activity of a gene involved in nicotine biosynthesis or transport. Genes involved in nicotine biosynthesis include, but are not limited to, Arginine Decarboxylase (ADC), Methyl Putrescine Oxidase (MPO), NADH dehydrogenase, Ornithine Decarboxylase (ODC), phosphoribosyl anthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), Quinolinate Phosphoribosyltransferase (QPT), and S-adenosyl-methionine synthetase (SAMS). Although two candidate genes have been proposed (a622 and NBB1), nicotine synthases that catalyze the condensation step between nicotinic acid derivatives and the methylpyrrolidine cation have not been elucidated. See US2007/0240728A 1and US2008/0120737A 1. A622 encodes an isoflavone reductase-like protein. In addition, several transport proteins may be involved in the transport of nicotine. The transporter gene designated MATE has been cloned and characterized (Morita et al, PNAS 106:2447-52 (2009)).

In one aspect, tobacco plants are provided that further comprise one or more increased or decreased levels of mRNA, protein, or both of a gene encoding a product selected from the group consisting of: PMT, MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622 and NBB 1. In another aspect, provided are tobacco plants further comprising a transgene that directly inhibits the 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 NBB 1. In another aspect, provided are tobacco plants further comprising a transgene or mutation that inhibits the 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 NBB 1. In another aspect, provided are tobacco plants further comprising a transgene expressing one or more genes encoding a product selected from the group consisting of: PMT, MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622 and NBB 1.

Enhancer elements are regions of DNA that can be bound by proteins to activate transcription of RNA. In one aspect, a promoter sequence as used herein is operably linked to an enhancer element. In one aspect, the enhancer element provided herein is the CsVMV promoter.

Transformation of tobacco plants with the recombinant constructs or expression cassettes using any suitable transformation method known in the art is also disclosed. Methods for introducing polynucleotide sequences into tobacco plants are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods. "Stable transformation" refers to a transformation in which a nucleotide construct of interest introduced into a plant is integrated into the genome of the plant and is capable of being inherited by its progeny. By "transient transformation" is meant that the sequence is introduced into a plant and is expressed only transiently or is present only transiently in the plant.

In one aspect, the methods and compositions provided herein comprise a carrier. As used herein, the terms "vector" or "plasmid" are used interchangeably to refer to a circular double stranded DNA molecule that is physically separated from chromosomal DNA. In one aspect, the plasmid or vector used herein is capable of replication in vivo. As used herein, a "transformation vector" is a plasmid capable of transforming a plant cell. In one aspect, the plasmids provided herein are bacterial plasmids. In another aspect, the plasmids provided herein are or are derived from an agrobacterium Ti plasmid.

In one aspect, the plasmid or vector provided herein is a recombinant vector. As used herein, the term "recombinant vector" refers to a vector formed by a laboratory method of genetic recombination (such as molecular cloning). In another aspect, the plasmids provided herein are synthetic plasmids. As used herein, a "synthetic plasmid" is an artificially generated plasmid that is capable of the same function (e.g., replication) as a native plasmid (e.g., a Ti plasmid). Without limitation, one skilled in the art can recreate a synthetic plasmid via synthesis of the plasmid from a single nucleotide, or by splicing together nucleic acid molecules from different pre-existing plasmids.

Vectors are commercially available or can be produced by recombinant DNA techniques conventional in the art. A vector containing a nucleic acid can have expression elements operably linked to the nucleic acid, and can also include, for example, sequences encoding a selectable marker (e.g., an antibiotic resistance gene). The vector containing the nucleic acid may encode a chimeric or fusion polypeptide (i.e., a polypeptide operably linked to a heterologous polypeptide, which may be at the N-terminus or C-terminus of the polypeptide). Representative heterologous polypeptides are those that can be used to purify the encoded polypeptide (e.g., 6XHis tag (SEQ ID NO:45), glutathione S-transferase (GST)).

Suitable methods for introducing polynucleotides into plant cells of the present disclosure include microinjection (Crossway et al (1986) Biotechniques 4: 320-; riggs et al (1986) Proc. Natl. Acad. Sci. USA 83: 5602-; tomes et al (1995) in Plant Cell, Tissue, and organic Culture fundamentals Methods, ed. gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al (1988) Biotechnology 6: 923-. See also Weising 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-; finer and McMullen (1991) In Vitro Cell Dev.biol.27P: 175-; singh et al (1998) the or. appl. Genet.96:319-324 (soybean); de Wet et al (1985) in The Experimental management of Ovule Tissues, ed. Chapman et al (Longman, N.Y.), pp.197-209 (pollen); kaeppler et al (1990) Plant Cell Reports 9: 415-; d' Hall et al (1992) Plant Cell 4:1495-1505 (electroporation).

It is understood that any modified tobacco plant of the present disclosure may further comprise other agronomically desirable traits, such as by transformation with a genetic construct or transgene using techniques known in the art. Examples of desirable traits are, without limitation, herbicide resistance, pest resistance, disease resistance, high yield, high grade index values, ripeness, ripening quality, mechanical harvestability, retention capacity, leaf quality, height, plant maturity (e.g., early maturity, early middle maturity, middle late maturity, or late maturity), stem size (e.g., small, medium, or large stem), or leaf count per plant (e.g., small (e.g., 5-10 leaves), medium (e.g., 11-15 leaves), or large (e.g., 16-21) leaves), or any combination. In one aspect, tobacco plants provided herein that are capable of producing cured leaves or seeds with reduced TSNA comprise one or more transgenes expressing one or more insecticidal proteins, such as a crystal protein of Bacillus thuringiensis (Bacillus thuringiensis) or a nutritive insecticidal protein from Bacillus cereus, such as VIP3 (see, e.g., escherichia et al (1997) nat. biotechnol.15: 137). In another aspect, the tobacco plants provided herein further comprise an introgression trait conferring resistance to brown stalk rot (U.S. patent No.5,689,035) or resistance to cyst nematode (U.S. patent No.5,491,081).

The content and/or activity of the polypeptides provided herein can be modulated by employing polynucleotides that are incapable of directing the expression of a protein or RNA in a transformed plant. For example, the polynucleotides of the invention may be used to design polynucleotide constructs that may be used in methods of altering or mutating genomic nucleotide sequences in organisms. Such polynucleotide constructs include, but are not limited to, RNA-DNA vectors, RNA-DNA mutation vectors, RNA-DNA repair vectors, mixed double-stranded oligonucleotides, self-complementary RNA-DNA oligonucleotides, and recombinant oligonucleotide bases. Such nucleotide constructs and methods of use are known in the art. See, U.S. Pat. nos.5,565,350; 5,731,181, respectively; 5,756,325, respectively; 5,760,012, respectively; 5,795,972 and 5,871,984; each of which is incorporated herein by reference as if set forth in its entirety. See also International patent application publication Nos. WO98/149350, WO99/107865, and WO 99/125921; and Beetham et al (1999) Proc. Natl. Acad. Sci. USA 96: 8774-8778; each of which is incorporated herein by reference as if set forth in its entirety.

The present disclosure also provides compositions and methods for inhibiting the expression or function of one or more polypeptides that directly or indirectly inhibit the production or accumulation of one or more antioxidants in plants, particularly plants of the nicotiana species, including tobacco plants of various commercial varieties.

In one aspect, inhibition of expression of one or more polypeptides provided herein (e.g., the Nic1b _ ERF or the Nic2_ ERF gene) can be obtained by RNA interference (RNAi) expressing a transgene capable of producing the inhibitory sequences provided herein. In one aspect, RNAi includes expression of a non-coding RNA. As used herein, "non-coding RNA" is selected from the group consisting of: micro rna (mirna), small interfering rna (siRNA), trans-acting siRNA (ta-siRNA), transfer rna (trna), ribosomal rna (rrna), introns, hairpin rna (hprna), and intron-containing hairpin rna (ihprnna). In one aspect, a single non-coding RNA provided herein inhibits the expression 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, at least 10, or more than 10 polypeptides. In one aspect, the non-coding RNA provided herein is stably transformed into a plant genome. In another aspect, the non-coding RNA provided herein is transiently transformed into a plant genome.

As used herein, the terms "inhibit", "inhibition" and "down regulation" are defined as any method known in the art or described herein that reduces the expression or function of a gene product (e.g., mRNA, protein, non-coding RNA). "inhibition" can be in the context of a comparison between two cells (e.g., a modified cell VS control cell). Inhibition of expression or function of a gene product may also be in the context of, and include comparison between developmental stages or time stages within the same plant or plant component or between plants or plant components, comparison between plant cells, organelles, organs, tissues, or plant components within the same plant or between different plants. "inhibit" includes any relative reduction 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 "inhibit" encompasses any method or composition that down-regulates translation and/or transcription of a target gene product or functional activity of a target gene product. "inhibiting" does not necessarily include completely eliminating expression of the gene product. In one aspect, the expression of the gene product in the modified cells provided herein is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% lower than the expression of the gene product in control cells. In another aspect, a gene product in a modified cell provided herein comprises, or is less than, 1% -100%, 1% -95%, 1% -90%, 1% -80%, 1% -70%, 1% -50%, 1% -40%, 1% -30%, 1% -25%, 1% -20%, 1% -15%, 1% -10%, 1% -5%, 5% -25%, 5% -75%, 5% -100%, 10% -25%, 10% -75%, 10% -100%, 25% -50%, 25% -75%, 25% -100%, 50% -100% expression of the gene product in a control cell.

As used herein, "target site" refers to the location of a polynucleotide sequence that binds to and is cleaved by a site-specific nuclease that introduces a double-stranded break into the nucleic acid backbone. In another aspect, the target site comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 29, or at least 30 consecutive nucleotides. In another aspect, a target site provided herein is at least 10, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 125, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 nucleotides. In one aspect, the site-specific nuclease binds to a target site. In another aspect, the site-specific nuclease binds to the target site via a guide-directed non-coding RNA, i.e., such as, but not limited to CRISPR RNA or a single guide RNA (both described in detail below). In one aspect, the non-coding RNA provided herein is complementary to a target site. It is understood that the non-coding RNA-binding target sites need not be fully complementary; at least 1, at least 2, at least 3, at least 4, or at least 5, at least 6, at least 7, or at least 8 mismatches between the target site and the non-coding RNA can be tolerated. As used herein, "target region" or "targeting region" refers to a polynucleotide sequence that requires modification. In one aspect, the "target region," "targeting region," or "target gene" flanks two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more target sites. "target gene" refers to a polynucleotide sequence encoding a gene from which modification or regulation of transcriptional expression is desired. In one aspect, the polynucleotide sequence comprising the target gene further comprises one or more target sites. In another aspect, the transgene is said to target a target site or target gene. In another aspect, the target region comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more target genes. Without limitation, in one aspect, the target region may be deleted or reversed. As used herein, "flanking" when used to describe a target area refers to two or more target sites that physically surround the target area, with one target site on each side of the target area.

As used herein, "upstream" refers to a nucleic acid sequence located before the 5' end of a linked nucleic acid sequence. As used herein, "downstream" refers to a nucleic acid sequence located after the 3' end of a linked nucleic acid sequence. As used herein, "5'" refers to the beginning of the coding DNA sequence or the beginning of the RNA molecule. As used herein, "3'" refers to the end of the coding DNA sequence or the end of the RNA molecule. It is understood that "reversing" refers to reversing the orientation of a given polynucleotide sequence. For example, if the sample sequence 5 '-ATGATC-3' is inverted, it will read in the opposite direction as 5 '-CTAGTA-3'. In addition, the sample sequence 5 '-ATGATC-3' is considered to be in the "opposite direction" to the sample sequence 5 '-CTAGTA-3'.

In another aspect, the recombinant construct or expression cassette can be introduced into a plant by contacting the plant with a virus or viral nucleic acid. Typically, such methods involve incorporating the expression cassettes of the present disclosure into viral DNA or RNA molecules. It is recognized that promoters for use in expression cassettes also include promoters for transcription by viral RNA polymerases. Methods for introducing polynucleotides into plants and expressing the proteins encoded therein, including viral DNA or RNA molecules, are known in the art. See, for example, U.S. Pat. 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-.

As used herein, a "locus" is a chromosomal region in which a polymorphic nucleic acid, trait determinant, gene, or marker is located. The loci of the present disclosure comprise one or more polymorphisms in a population; for example, alternative alleles are present in some individuals. As used herein, "allele" refers to an alternative nucleic acid sequence at a particular locus. Alleles can be as small as 1 nucleotide base in length, but are typically larger. For example, a first allele may occur on one chromosome while a second allele occurs on a second homologous chromosome, e.g., for a different chromosome of a heterozygous individual, or between different homozygous or heterozygous individuals in a population. As used herein, a chromosome in a diploid plant is "hemizygous" when there is only one copy of the locus. For example, when an inserted transgene is inserted into only one sister chromosome (i.e., the second sister chromosome does not contain the inserted transgene), it is hemizygous.

In one aspect, the modified plant, seed, plant component, plant cell, or plant genome is homozygous for a transgene provided herein. In another aspect, the modified plant, seed, plant component, plant cell, or plant genome is heterozygous for a transgene provided herein. In one aspect, the modified plant, seed, plant component, plant cell, or plant genome is hemizygous for a transgene provided herein. In one aspect, the modified plant, seed, plant component, plant cell, or plant genome is homozygous for the mutation provided herein. In another aspect, the modified plant, seed, plant component, plant cell, or plant genome is heterozygous for a mutation provided herein. In one aspect, the modified plant, seed, plant component, plant cell, or plant genome is hemizygous for a mutation provided herein.

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

As used herein, "cross" or "crosses" means the production of progeny by fertilization (e.g., a cell, seed, or plant) and includes crossing (sexual) and self-pollination (selfing) between different plants.

As used herein, "backcross (backsrossing)" and "backcrossing" refer to the process by which a progeny plant repeatedly backcrosses with one of its parents. In a backcrossing scheme, the "donor" parent refers to the parent plant having the desired gene or locus to be introgressed. The "recipient" parent (one or more uses) or "recurrent" parent (two or more uses) refers to the parent plant into which the gene or locus has been introgressed. Initial hybridization yields F₁And (4) generation. The term "BC₁"isSecond use of the finger recurrent parent, "BC₂"refers to the third use of the recurrent parent, and so on. In one aspect, backcrossing is repeated, with the progeny individuals of each successive backcross generation backcrossing themselves to the same parental genotype.

As used herein, "elite variety" means any variety produced by breeding and selection that has superior agronomic performance.

As used herein, "selecting" or "selection" in the breeding context refers to the act of generally picking or selecting a desired individual from a population based on certain predetermined criteria.

In one aspect, the tobacco plants provided herein are hybrid plants. A hybrid can be produced by preventing self-pollination of a female parent plant (e.g., the seed parent) of a first variety, allowing pollen from a male parent plant of a second variety to fertilize the female parent plant, and allowing F to fertilize the female parent plant₁Hybrid seed is produced on female plants. Self-pollination of female plants can be prevented by emasculating the flowers during early stages of flower development. Alternatively, a male sterile form may be used to prevent pollen formation on the female parent plant. For example, male sterility can result from Male Sterility (MS) or transgenic male sterility, wherein the transgene inhibits microsporogenesis and/or pollen formation, or is self-incompatible. Female parent plants containing MS are particularly useful. In the aspect where the female parent plant is MS, pollen may be harvested from the male fertile plant and applied manually to the style of the MS female parent plant, and the resulting F harvested₁And (4) seeds. In addition, female sterile plants can also be used to prevent self-fertilization.

Plants can be used to form single cross tobacco F₁And (4) hybridizing. Manually transferring pollen from a male parent plant to a detasseled female parent plant or a male sterile female parent plant to form F₁And (4) seeds. Alternatively, a three-way cross may be performed, in which case a single cross F is made₁Hybrids are used as female parents and are crossed with different male parents. Alternatively, two-hybrid hybrids can be generated in which two different single crosses of F₁The progeny cross themselves. When a double hybrid is formedSelf-incompatibility can be used particularly advantageously to prevent self-pollination of female parents when cross-breeding.

In one aspect, the tobacco variety provided herein is male sterile. In another aspect, the tobacco variety provided herein is Cytoplasmic Male Sterility (CMS). Male sterile tobacco plants can be produced by any method known in the art. Methods for producing male sterile tobacco are described In Wernsman, E.A., and Rufty, R.C.1987.Chapter Seven. Tobacco. pages 669. 698In: clinical development. crop specifications. W.H.Fehr (ed.), MacMillan Publishing Go., Inc., New York, N.Y.761pp. In another aspect, the tobacco variety provided herein is female sterile. As a non-limiting example, female sterile plants can be prepared by mutating the STIG1 gene. See, e.g., Goldman et al 1994, EMBO Journal 13: 2976-.

As used herein, the term "sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refers to the residues that are the same in the two sequences when aligned for maximum correspondence over a specified comparison window. When percentage sequence identity is used with reference to proteins, it is recognized that residue positions that are not identical typically differ by conservative amino acid substitutions, wherein an amino acid residue is substituted for another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity), and thus do not alter the functional properties of the molecule. When conservative substitutions of sequences are different, the percent sequence identity may be adjusted upward to correct for the conservative nature of the substitutions. Sequences that differ due to such conservative substitutions are said to have "sequence similarity" or "similarity". For any protein sequence provided herein, functionally homologous proteins that differ in one or more amino acids by one or more well-known conservative amino acid substitutions are also contemplated, e.g., a conservative substitution where valine is alanine and threonine is serine. Conservative substitutions of amino acids in a native sequence may be selected from other members of the class to which the naturally occurring amino acid belongs. Representative amino acids in these different classes include, but are not limited to: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Conservative substitutions of amino acids within a natural amino acid sequence may be selected from other members of the group to which the naturally occurring amino acid belongs. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine and tryptophan; a group of amino acids having basic side chains is lysine, arginine and histidine; one group of amino acids having sulfur-containing side chains is cysteine and methionine. The natural conservative amino acid substitution group is as follows: valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. Another aspect of the present disclosure includes proteins that differ in one or more amino acids from the protein sequence by virtue of deletion or insertion of one or more amino acids in the native sequence.

The nucleic acid molecules, polypeptides or proteins provided may be isolated or substantially purified. An "isolated" or "purified" nucleic acid molecule, polypeptide, protein, or biologically active portion thereof, is substantially or essentially free of components normally associated with or interacting with a 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.

In one aspect, the present disclosure provides methods of detecting one or more of the recombinant nucleic acids and polypeptides described herein in a plant cell. Without limitation, nucleic acids may also be detected using hybridization. Hybridization between nucleic acids is discussed in detail in Sambrook et al (1989, Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

The polypeptide may be detected using an antibody. Techniques for detecting polypeptides using antibodies include enzyme-linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. The antibodies provided herein can be polyclonal or monoclonal antibodies. Antibodies having specific binding affinity for the polypeptides provided herein can be generated using methods well known in the art. The antibodies provided herein can be attached to a solid support (e.g., a microtiter plate) using methods known in the art.

Detection of (e.g., amplification product, hybridization complex, polypeptide) can be achieved using a detectable label. The term "tag" is intended to include the use of direct tags as well as indirect tags. Detectable labels include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.

Any plant tissue that can subsequently be propagated using cloning methods, whether by organogenesis or embryogenesis, can be transformed with the recombinant construct or expression cassette. By "organogenesis" is meant the process of developing shoots and roots sequentially from the center of a meristem. By "embryogenesis" is meant the process by which shoots and roots develop together in a consistent manner (not sequentially), whether from somatic cells or gametes. Exemplary tissues suitable for use in the various transformation protocols include, but are not limited to, callus, existing meristems (e.g., apical meristem, axillary buds, and root meristems) and induced meristems (e.g., cotyledon meristem and hypocotyl meristem), hypocotyls, cotyledons, leaf discs, pollen, embryos, and the like.

In one aspect, a tobacco plant is provided from a tobacco type selected from the group consisting of: cured tobacco, cured tobacco cured by air curing in dark color, cured tobacco cured by open fire in dark color, Galpao tobacco and oriental tobacco. In another aspect, tobacco plants are provided from a tobacco type selected from the group consisting of: burley tobacco, maryland tobacco, and dark tobacco.

In one aspect, tobacco plants are provided that are in a cured tobacco background or exhibit one or more of the flue-cured tobacco characteristics described herein. Cured tobacco (also known as virginia or golden yellow) accounts for about 40% of the tobacco yield in the world. Because it reaches a golden to dark orange color during curing, cured tobacco is also often referred to as "golden smoke". The cured tobacco has a light, bright aroma and taste. Cured tobacco typically has a high sugar content and a low oil content. The major countries where cured tobacco is grown are argentina, brazil, china, india, tanzania and the united states. In one aspect, a low alkaloid or low nicotine tobacco plant or seed is provided in a cured tobacco background selected from the group consisting of: CC 13, CC 27, CC 33, CC 37, CC 65, CC 67, CC 700, GF 318, GL 338, GL 368, GL 939, K346, K399, K326, NC 102, NC196, NC 291, NC 297, NC 299, NC 471, NC 55, NC 606, NC 71, NC 72, NC 92, PVH 1118, PVH 1452, PVH 2110, speech 168, speech 220, speech 225, speech 227, speech 236, and any species substantially derived from any of the foregoing species. In another aspect, a low alkaloid or low nicotine tobacco plant or seed is provided in a cured tobacco background selected from the group consisting of: coker 48, Coker 176, Coker 371-Gold, Coker 319, Coker 347, GL 939, K149, K326, K340, K346, K358, K394, K399, K730, NC 27NF, NC 37NF, NC 55, NC 60, NC 71, NC 72, NC 82, NC 95, NC 297, NC 606, NC 729, NC 2326, McNairr 373, McNair 944, Ox 207, Ox 414NF, Reams 126, Reams 713, Reams 744, RG 8, RG 11, RG 13, RG 17, RG 22, RG 81, RG H4, RG H51, Speight H-20, Speight G-28, Speight G-58, Speight G-70, Speight G-108, Speight G-l11, Speight G-l17, Speight 168, Speight 179, Speight NF-3, Va 116, Va 182, and any species derived substantially from any of the foregoing species. See WO2004/041006A 1. In another aspect, the low alkaloid or low nicotine tobacco plant, seed, hybrid, variety, or line is in any curing setting selected from the group consisting of: k326, K346, and NC 196. In one aspect, the tobacco plant or seed or modified tobacco plant or seed provided herein is a cured tobacco variety selected from the group consisting of the varieties listed in table 1and any variety derived substantially from any of the foregoing varieties. See WO2004/041006A 1. In another aspect, the modified tobacco plant or seed provided herein is a cured variety selected from the group consisting of: k326, K346, and NC 196.

TABLE 1 cured tobacco variety.

In one aspect, tobacco plants are provided that exhibit one or more of the characteristics of air cured tobacco described herein in an air cured tobacco background. Cured tobacco includes burley tobacco, maryland tobacco and dark tobacco. The common factor is that maturation is mainly without artificial heat and humidity sources. Burley tobacco is light to dark brown in color, high in oil and low in sugar. The burley tobacco is aired and cured in a storehouse. The major countries in which burley tobacco grows are argentina, brazil, italy, maraviroc, and american maryland tobacco is very fluffy, with good burning properties, low nicotine and neutral aroma. The major countries in which maryland grows include the united states and italy. In one aspect, a low alkaloid or low nicotine tobacco plant or seed is provided in a burley tobacco background selected from the group consisting of: clay 402, Clay 403, Clay 502, Ky 14, Ky907, Ky 910, Ky 8959, NC 2, NC 3, NC 4, NC 5, NC 2000, TN86, TN90, TN97, R610, R630, R711, R712, NCBH 129, Bu21 xKy 10, HB04P, Ky 14xL8, Kt 200, Newton 98, Pedigo 561, Pf561 and Va 509. In another aspect, the low alkaloid or low nicotine tobacco plant, seed, hybrid, variety or line is in any burley tobacco background selected from the group consisting of: TN90, KT209, KT206, KT212 and HB 4488. In one aspect, the tobacco plant or seed or modified tobacco plant or seed provided herein is a burley tobacco variety selected from the group consisting of the tobacco varieties listed in table 2 and any variety derived substantially from any of the foregoing varieties. In another aspect, the modified tobacco plant or seed provided herein is a burley tobacco variety selected from the group consisting of: TN90, KT209, KT206, KT212 and HB 4488.

TABLE 2 Burley tobacco variety.

In another aspect, a low alkaloid or low nicotine tobacco plant or seed is provided in a maryland tobacco background selected from the group consisting of: md 10, Md 40, Md 201, Md 609, Md 872 and Md 341. In another aspect, the tobacco plant or seed or modified tobacco plant or seed provided herein is a maryland tobacco variety selected from the group consisting of the tobacco varieties listed in table 3 and any variety derived substantially from any of the foregoing varieties.

TABLE 3 Maryland tobacco variety.

Maryland 10(TC 498)
	Maryland 14D2(TC 499)
Maryland 201(TC 503)
	Maryland 21(TC 500)
Maryland 341(TC 504)
	Maryland 40
Maryland 402
	Maryland 59(TC 501)
Maryland 601
	Maryland 609(TC 505)
Maryland 64(TC 502)
	Maryland 872(TC 506)
Maryland Mammoth(TC 507)

In one aspect, tobacco plants are provided that are in a dark air cured tobacco background or exhibit one or more dark air cured tobacco characteristics described herein. Dark air cured tobacco differs from other types primarily in that the curing process imparts a medium to dark brown color and a unique aroma to dark air cured tobacco. The dark color air cured tobacco is mainly used for producing chewing tobacco and snuff. In one aspect, provided low alkaloid or low nicotine tobacco plants or seeds are in a dark air cured tobacco background selected from the group consisting of: sumatra, Jatim, Dominican Cubano, Besuki, One packer, Green River, Virginia sun-cured, and Paraguan Passado, as well as any species derived substantially from any of the foregoing species.

In one aspect, tobacco plants are provided that are in a dark bright fire cured tobacco background or exhibit one or more of the dark bright fire cured tobacco characteristics described herein. Dark bright fire cured tobacco is typically cured with a low fire wood fire on the floor of an enclosed curing barn. Their leaves have a low sugar content but a high nicotine content. Dark-color, open-fire cured tobacco is used to make tube mixes, cigarettes, chewing tobacco, snuff, and strong-smoke cigars. The main growth areas for dark and open fire cured tobacco are Tennessee, Kentucky and Virginia, USA. In one aspect, a low alkaloid or low nicotine tobacco plant or seed is provided in a dark open fire 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 Stack Black Mammoth, DT 508, DT 518, DT 592, KY171, DF911, DF 485, TN D94, TN D950, VA 309 and VA 359. In one aspect, the tobacco plant or seed or modified tobacco plant or seed provided herein is selected from the group consisting of dark-fired flue-cured tobacco varieties of the group consisting of the tobacco varieties listed in table 4 and any variety derived substantially from any of the foregoing varieties.

TABLE 4 dark flame curing tobacco varieties.

In one aspect, a tobacco plant is provided that is in an oriental tobacco background or exhibits one or more of the oriental tobacco characteristics described herein. Oriental tobaccos are also known as greek aroma and turkey tobaccos because they are typically planted in eastern mediterranean areas such as turkey, greek, bulgaria, maachton, syria, libamon, italy and romania. The oriental varieties today are characterized by small plant and leaf size, and by their unique aroma characteristics, which are the result of plants adapting to growing barren soil and harsh climatic conditions over the past centuries. In one aspect, a low alkaloid or low nicotine tobacco plant or seed is provided in an oriental tobacco background selected from the group consisting of: izmir, Katerini, Samsun, Basma and Krumovgrad, Trambzon, Thesalian, Tasova, Sinop, Izmit, Hendek, Edire, Semdrili, Adiyanman, Yayladag, Iskenderun, Duzce, Macedonian, Mavra, Prilep, Bafra, Bursa, Bucak, Billis, Balikesi and any species substantially derived from any of the foregoing species. In one aspect, the tobacco plant or seed or modified tobacco plant or seed provided herein is an oriental tobacco variety selected from the group consisting of the tobacco varieties listed in table 5 and any variety derived substantially from any of the foregoing varieties.

TABLE 5 Oriental tobacco varieties.

In one aspect, the tobacco plant or seed or modified tobacco plant or seed provided herein is a cigar tobacco variety selected from the group consisting of the tobacco varieties listed in table 6and any variety derived substantially from any of the foregoing varieties.

TABLE 6 cigar tobacco variety

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

TABLE 7 other tobacco varieties

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

In one aspect, the modified tobacco plant, seed or cell described herein is from a variety selected from the group consisting of the tobacco varieties listed in table 1, table 2, table 3, table 4, table 5, table 6and table 7.

In one aspect, the low alkaloid or low nicotine tobacco plant, seed, hybrid, variety or line is derived substantially from or in the following genetic background: 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 371Gold, Coker 48, CU 263, DF911, Galpao tobacao, GL 26H, GL, GL 600, GL 737, GL 939, GL 973, HB04P, K149, K326, K346, K358, K394, K399, K730, KDH 959, KT 200, KT204LC, KY 10, KY 14, KY 160, KY17, KY171, KY907, KY LC, KTY14 x L8 LC, Little Critten, McNairr 373, McNairr 944, msKY 14xL8, Naow Leaf horse, NC 100, NC 102, NC 2000, NC 291, NC 297, NC 3, NC 4, NC 5, NC 3, NC 5, NC 3, NC 5, NC 5964 NC 5, NC 3, NC 5, NC 5964 NC 3, NC 5, NC 3, NC 5, NC 3, NC 10, NC 17, K346, K3, K346, K3, K346 of K3, K346 of K3, K346 of K3, K346 of K3, K3, K, NC 2002, Neal Smith Madole, OXFORD 207, 'Perique' tobaca, PVH03, PVH09, PVH19, PVH50, PVH51, R610, R630, R7-11, R7-12, RG 17, RG 81, RGH 51, 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, TN86LC, TN90, TN97LC, TN D94, TN D950, tr (tom rosson) Madole, VA 309, or VA359, Maryland 609, HB3307PLC, HB4488PLC, KT206LC, KT209LC, KT210LC, KT212LC, R610LC, PVH2310, NC196, KTD14LC, KTD6LC, KTD8LC, PD7302LC, PD7305LC, PD7309LC, PD7318LC, PD7319LC, PD7312LC, ShireyLC, or any commercial tobacco variety according to standard tobacco breeding techniques known in the art.

All particular varieties of dark air cured, burley, maryland, dark open fire cured or oriental types mentioned above are listed for exemplary purposes only. Any other dark air curing, burley, maryland, dark open fire curing or oriental tobacco varieties are also contemplated in this application.

Also provided are populations of said tobacco plants. In one aspect, the population of tobacco plants has a planting density of about 5,000 to about 8000, 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 aspect, the tobacco plant population is a low to moderate fertility soil type.

Also provided are containers of seeds from the tobacco plants. 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 or greater than about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000 or more seeds. Alternatively, the container can hold at least or greater than about 1 ounce, 5 ounces, 10 ounces, 1 pound, 2 pounds, 3 pounds, 4 pounds, 5 pounds or more of seeds, or at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000 grams. The container of tobacco seeds may be any container available in the art. As non-limiting examples, the container may be a box, bag, pouch, roll of tape, tube, or bottle.

Cured tobacco material made from the low alkaloid or low nicotine tobacco plant is also provided. Cured tobacco materials having a higher content of total alkaloids or nicotine made from the tobacco plant are also provided.

"curing" is an aging process that reduces moisture and causes destruction of chlorophyll, giving the tobacco a golden yellow color, and starch is also converted to sugar. Thus, cured tobacco has a higher reducing sugar content and a lower starch content than harvested green leaves. In one aspect, green leaf tobacco is provided that can be cured using conventional methods, such as, for example, flue-cured, barn-cured, open-fire cured, air-cured, or sun-cured. For a description of various types of maturation methods see, for example, (1999, Chapter 1in tobaco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). Cured tobacco is typically aged in wooden barrels (e.g., vats) or cartons for years (e.g., 2 to 5 years) under compression conditions having a moisture content of 10% to about 25%. See U.S. patent nos. 4,516,590 and 5,372,149. The cured and aged tobacco may 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. Typically, fermentation is characterized by high initial moisture content, heat generation and a loss of 10 to 20% of dry weight. See, e.g., U.S. patent nos. 4,528,993, 4,660,577, 4,848,373, 5,372,149; U.S. publication No. 2005/0178398; and Tso (1999, Chapter 1inTobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell publishing, Oxford). Cured, aged, and fermented tobacco can be further processed (e.g., cut, shredded, puffed, or mixed). See, e.g., U.S. patent nos. 4,528,993; 4,660,577, and 4,987,907. In one aspect, the cured tobacco material of the present disclosure is sun cured. In one aspect, cured tobacco materials of the present disclosure are oven cured, sun cured, or open fire cured.

Tobacco material obtained from the tobacco lines, varieties, or hybrids of the present disclosure can be used to prepare tobacco products. As used herein, "tobacco product" is defined as any product made from or derived from tobacco that is intended for human use or consumption.

Smoking articles provided include, but are not limited to, smoking articles (e.g., cigarettes and bidis), cigar articles (e.g., cigar wrappers and cigarillos), pipe smoking articles, tobacco-derived nicotine articles, smokeless tobacco articles (e.g., moist snuff, dry snuff, and chewing tobacco), films, chewables, labels, tablets, shaped portions, gels, consumable units, insoluble matrices, hollow shapes, reconstituted tobacco, expanded tobacco, and the like. See, for example, U.S. patent publication No. US 2006/0191548.

As used herein, "cigarette" refers to a tobacco product having a "rod" and a "filler". Cigarette "sticks" include cigarette paper, filters, filter rods (for containing filter material), tipping paper to secure cigarette paper (including filler) to the filter, and all glues that secure these components together. "filler" includes (1) all tobaccos, including but not limited to reconstituted and expanded tobaccos, (2) non-tobacco substitutes (including but not limited to herbs, non-tobacco plant materials, and other flavors that may accompany tobacco in a cigarette paper), (3) casings, (4) flavors, and (5) all other additives (incorporated into tobacco and substitutes and rolled into cigarettes).

As used herein, "reconstituted tobacco" refers to a portion of tobacco filler made from tobacco powder and other tobacco waste that is processed into a sheet and cut into strips to simulate tobacco. In addition to cost savings, reconstituted tobacco is very important because flavor development contributes to the flavor contribution of the cigarette through the use of the reaction between ammonia and sugar.

As used herein, "expanded tobacco" refers to a portion of a tobacco filler that is treated by expansion of a suitable gas such that the tobacco is "expanded," resulting in a decrease in density and an increase in filling capacity. It reduces the weight of tobacco used in cigarettes.

Tobacco products derived from plants of the present disclosure also include cigarettes and other smoking articles, particularly those that include a filter element, wherein the rod of smokeable material comprises cured tobacco in a tobacco blend. In one aspect, the smoking article of the present disclosure is selected from the group consisting of: cigarillos, non-ventilated filter cigarettes, cigars (cigar), snuff, pipe tobacco, cigar tobacco (cigar tobaco), cigarettes, chewing tobacco, leaf tobacco, hookah, shredded tobacco (shredded tobaco) and cut tobacco (cut tobaco). In another aspect, the smoking article of the present disclosure is a smokeless smoking article. Smokeless tobacco products do not burn, including but not limited to chewing tobacco, moist smokeless tobacco, snuff, and dry snuff. Chewing tobacco is coarsely divided tobacco leaves typically packaged in large bag-like packages and used in stoppers or twists. Moist smokeless tobacco is a moist, more finely divided tobacco, provided in loose form or in the form of a sachet, and typically packaged in a round can and used as a clip (ping) in or placed in the sachet between the cheek and gums of an adult tobacco consumer. Snuff is heat-treated smokeless tobacco. Dry snuff is finely ground tobacco that is placed in the mouth or used nasally. In another aspect, the smoking article of the present disclosure is selected from the group consisting of: loose leaf chewing tobacco, plug chewing tobacco (pluggage tobaco), snuff and snuff (nasal snuff). In yet another aspect, the smoking article of the present disclosure is selected from the group consisting of: an electronic heating cigarette, an e-cigarette and an electronic evaporation device.

In one aspect, the smoking article of the present disclosure can be a blended smoking article. In one aspect, the blended tobacco product comprises cured tobacco material. In one aspect, the cured tobacco material comprises about 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 cured tobacco of the tobacco blend. In one aspect, the cured tobacco material comprises about 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 cured tobacco of the tobacco blend.

In one aspect, a smoking article of the present disclosure can be a reduced nicotine smoking article. In a further aspect, a smoking article of the present disclosure can comprise nornicotine at a level of less than about 3 mg/g. For example, the nornicotine content in such a preparation can be about 3.0mg/g, 2.5mg/g, 2.0mg/g, 1.5mg/g, 1.0mg/g, 750 μ g/g, 500pg/g, 250pg/g, 100pg/g, 75pg/g, 50pg/g, 25pg/g, 10pg/g, 7.0pg/g, 5.0pg/g, 4.0pg/g, 2.0pg/g, 1.0pg/g, 0.5pg/g, 0.4pg/g, 0.2pg/g, 0.1pg/g, 0.05pg/g, 0.01pg/g, or undetectable.

In one aspect, a cured tobacco material or tobacco product is provided comprising an average nicotine or total alkaloid content selected from the group consisting of: 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%, 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, a cured tobacco material or tobacco product is provided comprising an average nicotine or total alkaloid content selected from the group consisting of: about 0.01% -0.02%, 0.02% -0.05%, 0.05% -0.75%, 0.75% -0.1%, 0.1% -0.15%, 0.15% -0.2%, 0.2% -0.3%, 0.3% -0.35%, 0.35% -0.4%, 0.4% -0.5%, 0.5% -0.6%, 0.6% -0.7%, 0.7% -0.8%, 0.8% -0.9%, 0.9% -1%, 1% -1.1%, 1.1% -1.2%, 1.2% -1.3%, 1.3% -1.4%, 1.4% -1.5%, 1.5% -1.6%, 1.6% -1.7%, 1.7% -1.8%, 1.8% -1.9%, 1.9% -2%, 2% -2.1%, 2.1% -2.2%, 2.2% -2.2%, 2.3% -2.3%, 2.3% -2.4% -2.5%, 2.3.4% -2.5%, 2.5% -2.5%, 2.3.5% -2.5%, 2.5% -2.5%, 2.5% -2.5%, 2.5% -2.5%, 2.5.5.5%, 2.5% and 2.5% -2.5.5.5.5.5.5.5.5.5%, 2.5.5.5.5.2.2.5%, 2.5.5%, 2.5%, 0.5%, 2.2.5% of a 2.5%, 0.5% -2.5.5% of a 2.5% of a 2.2.5% of a 2.2.2.2.2.3% of a 2.2.3% of a 2.2.2.2.3% of a 2.2.2.2.2.2.3% of a 2.3% of a 2.2.2.2.2.2.2.5% of a 2.2.2.2.5% of a 2.2.5% of a 2.2.2.2.2.3% of a 2.3% of a 3% of a mixture, 2.7% -2.8%, 2.8% -2.9%, 2.9% -3%, 3% -3.1%, 3.1% -3.2%, 3.2% -3.3%, 3.3% -3.4%, 3.4% -3.5% and 3.5% -3.6%. In a further aspect, a cured tobacco material or tobacco product is provided comprising, on a dry weight basis, an average nicotine or total alkaloid content selected from the group consisting of: about 0.01% -0.1%, 0.02% -0.2%, 0.03% -0.3%, 0.04% -0.4%, 0.05% -0.5%, 0.75% -1%, 0.1% -1.5%, 0.15% -2%, 0.2% -3%, and 0.3% -3.5%.

The present disclosure also provides methods of making a tobacco product comprising tobacco material from the tobacco plant. In one aspect, a method includes conditioning an aged tobacco material made from a tobacco plant provided herein to increase its moisture content from about 12.5% to about 13.5% to about 21%, mixing the conditioned tobacco material to produce a desired mixture. In one aspect, the method of making a tobacco product further comprises casing or flavoring the mixture. Typically, in the addition process, materials for addition or flavoring are added to the mixture to improve their quality and develop certain desirable flavor profiles by balancing the chemical composition. Further details of the feeding process can be found in Tobacco Production, Chemistry and Technology, Edited by L.Davis and M.Nielsen, Blackwell Science, 1999.

The provided tobacco material may also be processed using methods including, but not limited to, heat treatment (e.g., cooking, baking), flavoring, enzymatic treatment, puffing, and/or curing. Fermented and non-fermented tobacco can be processed using these techniques. Examples of suitable cured tobacco include dark air cured, dark open fire cured, burley, cured and cigar filler or wrapper, and products from whole leaf stuffing operations. In one aspect, the tobacco fiber comprises up to 70% dark tobacco based on fresh weight. For example, tobacco may be prepared by heating, sweating, and/or pasteurization steps, as described in U.S. publication Nos. 2004/0118422 or 2005/0178398.

The tobacco material provided may be fermented. Typically, fermentation is characterized by high initial moisture content, heat generation and a loss of 10 to 20% of dry weight. See, e.g., U.S. patent nos. 4,528,993; 4,660,577, respectively; 4,848,373, and 5,372,149. In addition to changing the aroma of the leaves, fermentation can also change the color and texture or both of the leaves. Also during fermentation, evolved gases may be produced, oxygen may be absorbed, pH may be changed, and the amount of water retained may be changed. See, for example, U.S. publication Nos. 2005/0178398 and Tso (1999, Chapter 1in Tobacco, Production, chemistry and technology, Davis & Nielsen, eds., Blac kwell Publishing, Oxford). The cured or cured and fermented tobacco may be further processed (e.g., cut, puffed, mixed, ground, or comminuted) prior to being added to the oral article. In some instances, the tobacco is a long cut fermented cured moist tobacco having an oven volatiles content of 48 to 50 weight percent prior to blending with the copolymer and optional flavorants and other additives.

In one aspect, a tobacco material is provided that can be processed to a desired size. In certain aspects, the tobacco fibers can be processed to have an average fiber size of less than 200 microns. In one aspect, the tobacco fiber is 75 to 125 microns. In another aspect, the tobacco fibers are processed to have a size of 75 microns or less. In one aspect, the tobacco fibers comprise long cut tobacco that can be cut or shredded to a width of about 10 cuts/inch (cut/inch) up to about 110 cuts/inch and a length of about 0.1 inch to about 1 inch. The double cut tobacco fibers may have a range of particle sizes such that about 70% of the double cut tobacco fibers fall between a-20 mesh to 80 mesh size.

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

The present disclosure also provides methods for breeding tobacco lines, cultivars or varieties comprising desired levels of total alkaloids or nicotine (e.g., low nicotine or no nicotine). Breeding can be performed by any known procedure. DNA fingerprinting, SNP mapping, haplotype mapping, or similar techniques can be used in marker-assisted selection (MAS) breeding programs to transfer or breed desired traits or alleles into tobacco plants. For example, a breeder may use the F1 hybrid plant disclosed herein at F₂Segregating populations are generated in generations or backcross generations, or the F1 hybrid plant is further crossed with other donor plants having an agronomically desirable genotype. F can be screened using techniques known in the art or one of the techniques listed herein₂Or a desired agronomic trait or a desired chemical characteristic of a plant of a backcross generation. Depending on the expected genetic pattern or MAS technique used, selected plants can be self-pollinated prior to each backcross cycle to help identify the individual plants of interest. Backcrossing or other breeding procedures can be repeated until the desired phenotype of the recurrent parent is restored. The recurrent parent in this disclosure may be a flue-cured variety, a burley variety, a dark air-cured variety, a dark open-fired flue-cured variety, or an oriental variety. For example, other breeding techniques can be found In Wernsman, E.A., and Rufty, R.C.1987.Chapter Seven. Tobacco. pages 669. 698In: Cultivar development. crop specifications. W.H.Fehr.,. MacMillan Publishing Go., Inc., New York, N.Y., the entire contents of which are incorporated herein by reference.

The results of plant breeding programs using the described tobacco plants include lines, cultivars, varieties, progeny, inbreds, and hybrids useful in the present disclosure. As used herein, the term "variety" refers to a population of plants having constant characteristics that segregate them from other plants of the same species. Varieties are usually, although not always, commercially sold. A further feature of a variety, despite having one or more distinguishing traits, is that the overall variation between individuals within the variety is very small. "pure line" varieties can be generated from individual parents by selfing and selection for generations, or by vegetative propagation using tissue or cell culture techniques. The breed may be substantially derived from another line or breed. As defined for the protection of new plant varieties by international convention (day 2 of 12 in 1961, day 10 of 11 in 1972, day 23 of 10 in 1978 and day 19 of 3 in 1991, revised geneva), the varieties "derive substantially" from the original varieties if: a) it is derived predominantly from an initial variety, or from a variety derived predominantly from an initial variety, while retaining the expression of the essential features resulting from the genotype or genotype combination of said initial variety; b) it is clearly different from the original variety; c) in addition to the differences resulting from the derivation behavior, it is consistent with the original breed in the expression of the essential characteristics resulting from the genotype or genotype combination of the original breed. For example, substantially derived varieties may be obtained by selection of natural or induced mutants, somaclonal variation, variant individuals from the original variety of plants, backcrossing or transformation. The first tobacco variety and the second tobacco variety (the first tobacco variety being substantially derived from the second tobacco variety) are considered to have substantially the same genetic background. In contrast to varieties, "lines" most commonly refer to a group of plants that are not commercially used, for example, for plant research. Lines typically show very little overall variation in one or more traits of interest among individuals, although there may be some variation in other traits among individuals.

In another aspect, plant parts provided include, but are not limited to, leaves, stems, roots, seeds, flowers, pollen, anthers, ovules, pedicles, fruits, meristems, cotyledons, hypocotyls, pods, embryos, endosperm, explants, callus, tissue cultures, shoots, cells, and protoplasts. In one aspect, provided tobacco plants do not include seeds. In one aspect, the present disclosure provides tobacco plant cells, tissues and organs that are not propagation material and that do not mediate the natural propagation of the plant. In another aspect, the present disclosure also provides tobacco plant cells, tissues and organs that are propagation material and mediate the natural propagation of plants. In another aspect, the invention provides tobacco plant cells, tissues and organs that are unable to sustain themselves through photosynthesis. In another aspect, the invention provides a somatic tobacco plant cell. In contrast to germ cells, somatic cells do not mediate plant propagation.

The cells, tissues and organs provided may be from seeds, fruits, leaves, cotyledons, hypocotyls, meristems, embryos, endosperm, roots, shoots, stems, pods, flowers, inflorescences, petioles, pedicels, style, stigma, receptacle, petals, sepals, pollen, anthers, filaments, ovaries, ovules, pericarp, phloem, vascular tissue. In another aspect, the disclosure provides a tobacco plant chloroplast. In another aspect, the present disclosure provides epidermal cells, stomatal cells, leaves or root hairs, storage roots or tubers. In another aspect, the present disclosure provides tobacco protoplasts.

The skilled artisan understands that tobacco plants propagate naturally via seeds, not by asexual or vegetative propagation. In one aspect, the present disclosure provides tobacco endosperm. In another aspect, the present disclosure provides a tobacco endosperm cell. In another aspect, the present disclosure provides a male-or female-sterile tobacco plant that is incapable of reproduction without human intervention. The skilled artisan further understands that cured tobacco does not constitute a living organism and is unable to grow or reproduce.

The following are exemplary embodiments of the present disclosure.

Embodiment 1. a tobacco plant, or a part thereof, comprising a first genetic modification that inhibits one or more genes from the NIC1b locus, the NIC2 locus, or both, and further comprising a second genetic modification that increases the conversion of nicotine to nornicotine.

Embodiment 2. the tobacco plant of embodiment 1 or a part thereof, wherein said first genetic modification comprises the nic1b mutant allele.

Embodiment 3 the tobacco plant of embodiment 2, or a portion thereof, wherein said nic1b mutant allele is derived from the cuba cigar tobacco variety, low alkaloid burley 21(LABU21), low intermediate burley 21(LIBU21), low alkaloid flue-cured tobacco 53(LAFC53), low nicotine KY171(LNKY171), or a variety derived therefrom.

Embodiment 4. the tobacco plant of embodiment 1, or a part thereof, wherein said first genetic modification comprises a transgene or mutation that reduces the expression or activity of one or more Ethylene Response Factor (ERF) genes from the NIC1b locus.

Embodiment 5 the tobacco plant of embodiment 4 or part thereof, wherein the first genetic modification comprises a transgene targeting one or more genes selected from the group consisting of: ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210, and ERF91L 2.

Embodiment 6 the tobacco plant of embodiment 1, or a portion thereof, wherein said first genetic modification comprises a transgene or mutation that reduces the expression or activity of one or more non-ERF genes from the NIC1b locus.

Embodiment 7. the tobacco plant of embodiment 1, or a part thereof, wherein said first genetic modification comprises a transgene or mutation that increases the expression or activity of one or more Ethylene Response Factor (ERF) genes from the NIC1b locus.

Embodiment 8 the tobacco plant of embodiment 1, or a portion thereof, wherein said first genetic modification comprises a transgene or mutation that reduces the expression or activity of one or more non-ERF genes from the NIC1b locus.

Embodiment 9 the tobacco plant of embodiment 1 or a part thereof, wherein said first genetic modification comprises the nic2 mutant allele.

Embodiment 10 the tobacco plant of embodiment 9, or a portion thereof, wherein said nic2 mutant allele is derived from a cuba cigar tobacco variety, low alkaloid burley tobacco 21(LABU21), low intermediate burley tobacco 21(LIBU21), low alkaloid flue-cured tobacco 53(LAFC53), low nicotine KY171(LNKY171), or a variety derived therefrom.

Embodiment 11 the tobacco plant of embodiment 1, or a portion thereof, wherein said first genetic modification comprises a transgene or mutation that reduces the expression or activity of one or more Ethylene Response Factor (ERF) genes from the NIC2 locus.

Embodiment 12 the tobacco plant of embodiment 11 or part thereof, wherein said first genetic modification comprises a transgene targeting one or more genes selected from the group consisting of: ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF 168.

Embodiment 13. the tobacco plant, or part thereof, of any one of embodiments 1-12, wherein the second genetic modification overexpresses nicotine demethylase.

Embodiment 14 the tobacco plant, or part thereof, of any one of embodiments 13, wherein said second genetic modification comprises a transgene encoding said nicotine demethylase.

Embodiment 15 the tobacco plant, or part thereof, of any one of embodiments 13, wherein said nicotine demethylase is from a plant of the genus nicotiana.

Embodiment 16 the tobacco plant, or part thereof, of any one of embodiments 13, wherein said nicotine demethylase comprises a non-native, mutated or engineered amino acid sequence.

Embodiment 17 the tobacco plant, or part thereof, of any one of embodiments 13, wherein said nicotine demethylase comprises a native or tobacco native amino acid sequence.

Embodiment 18. the tobacco plant, or part thereof, of any one of embodiments 1-12, wherein the second genetic modification comprises a transgene encoding one or more of CYP82E4, CYP82E5, and CYP85E10 polypeptides.

Embodiment 19. the tobacco plant, or part thereof, of any one of embodiments 1-12, wherein the second genetic modification comprises genome editing that increases the expression or activity of one or more of CYP82E4, CYP82E5, and CYP85E10 polypeptides.

Embodiment 20 the tobacco plant, or part thereof, of embodiment 18 or 19, wherein said second genetic modification increases the expression or activity of CYP82E4v2, or comprises a transgene encoding CYP82E4v 2.

Embodiment 21. the tobacco plant, or part thereof, of any one of embodiments 1-20, wherein the tobacco plant further comprises a third genetic modification that increases the level of one or more antioxidants as compared to a control plant lacking the third genetic modification.

Embodiment 22 the tobacco plant of embodiment 21, or a portion thereof, wherein said third genetic modification comprises a transgene encoding or targeting an antioxidant biosynthetic enzyme, a regulatory transcription factor for an antioxidant, an antioxidant transporter, an antioxidant metabolic enzyme, or a combination thereof.

Embodiment 23 the tobacco plant of embodiment 21, or part thereof, wherein the third genetic modification comprises a modification of one or more endogenous genes encoding an antioxidant biosynthetic enzyme, a regulatory transcription factor for an antioxidant, an antioxidant transporter, an antioxidant metabolic enzyme, or a combination thereof.

Embodiment 24 the tobacco plant, or part thereof, of embodiment 21, wherein said one or more antioxidants are selected from the group consisting of: anthocyanins, flavanones, flavanols, flavones, flavonols, isoflavones, hydroxybenzoic acids, hydroxycinnamic acids, ellagitannins, stilbenes, lignans, carotenoids and glycyrrhizin.

Embodiment 25 the tobacco plant of embodiment 21, or part thereof, wherein the one or more antioxidants are selected from the group consisting of: delphinidin, anthocyanidin, procyanidin, delphinidin, hesperetin, naringenin, catechin, epicatechin, apigenin, luteolin, quercetin, myricetin, rutin, genistein, daidzein, gallic acid, vanillic acid, protocatechuic acid, ferulic acid, cinnamic acid, coumaric acid, chlorogenic acid, caffeic acid, ferulic acid, sanguinarine, resveratrol, sesamin, carotenoid and vitamin C.

Embodiment 26 the tobacco plant of embodiment 21, or a part thereof, wherein said third genetic modification comprises a transgene encoding one or more polypeptides selected from the group consisting of: AtPAP1, Ntmyb3A, Ntmyb3B, Ntmyb3C, NtJAF13, sta 1, NtAN1, and NtAN 2.

Embodiment 27. a tobacco plant, or part thereof, comprising a first genetic modification that increases the conversion of nicotine to nornicotine and further comprising a second genetic modification that increases the content of one or more antioxidants.

Embodiment 28 the tobacco plant of embodiment 27, or a part thereof, further comprising a third genetic modification that reduces nicotine in said tobacco plant.

Embodiment 29 the tobacco plant of embodiment 28 or a part thereof, wherein said third genetic modification comprises a nic1b mutant allele, a nic2 mutant allele, or both.

Embodiment 30 the tobacco plant of embodiment 29, or a portion thereof, wherein said nic1b mutant allele is derived from the cuba cigar tobacco variety, low alkaloid burley 21(LABU21), low intermediate burley 21(LIBU21), low alkaloid flue-cured tobacco 53(LAFC53), low nicotine KY171(LNKY171), or a variety derived therefrom.

Embodiment 31 the tobacco plant of embodiment 28, or a part thereof, wherein said third genetic modification comprises a transgene or mutation that reduces the expression or activity of one or more Ethylene Response Factor (ERF) genes from the NIC1b locus.

Embodiment 32 the tobacco plant, or part thereof, of embodiment 28, wherein said third genetic modification comprises a transgene targeting one or more genes selected from the group consisting of: ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210, and ERF91L 2.

Embodiment 33 the tobacco plant of embodiment 28 or a part thereof, wherein said third genetic modification comprises a transgene or a mutation that reduces the expression or activity of one or more non-ERF genes from the NIC1b locus.

Embodiment 34 the tobacco plant of embodiment 28, or a part thereof, wherein said third genetic modification comprises a transgene or mutation that increases the expression or activity of one or more Ethylene Response Factor (ERF) genes from the NIC1b locus.

Embodiment 35 the tobacco plant of embodiment 28 or a part thereof, wherein said third genetic modification comprises a transgene or mutation that increases the expression or activity of one or more non-ERF genes from the NIC1b locus.

Embodiment 36 the tobacco plant of embodiment 29, or a portion thereof, wherein said nic2 mutant allele is derived from the cuba cigar tobacco variety, low alkaloid burley tobacco 21(LABU21), low intermediate burley tobacco 21(LIBU21), low alkaloid flue-cured tobacco 53(LAFC53), low nicotine KY171(LNKY171), or a variety derived therefrom.

Embodiment 37 the tobacco plant of embodiment 28, or a part thereof, wherein said third genetic modification comprises a transgene or mutation that reduces the expression or activity of one or more Ethylene Response Factor (ERF) genes from the NIC2 locus.

Embodiment 38 the tobacco plant of embodiment 37 or a part thereof, wherein said third genetic modification comprises a transgene from one or more genes selected from the group consisting of: ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF 168.

Embodiment 39. the tobacco plant, or part thereof, of any one of embodiments 28-38, wherein said second genetic modification comprises a transgene encoding a nicotine demethylase.

Embodiment 40 the tobacco plant, or part thereof, of any one of embodiments 39, wherein said nicotine demethylase is from a plant of the genus Nicotiana.

Embodiment 41 the tobacco plant, or part thereof, of any one of embodiments 39, wherein said nicotine demethylase comprises a non-native, mutated or engineered amino acid sequence.

Embodiment 42 the tobacco plant, or part thereof, of any one of embodiments 39, wherein said nicotine demethylase comprises a native or tobacco native amino acid sequence.

Embodiment 43 the tobacco plant, or part thereof, of any one of embodiments 28-38, wherein the second genetic modification comprises a transgene encoding one or more of CYP82E4, CYP82E5 and CYP85E10 polypeptides.

Embodiment 44 the tobacco plant of embodiment 43 or a part thereof, wherein said second genetic modification comprises a transgene encoding CYP82E4v 2.

Embodiment 45 the tobacco plant, or part thereof, of any one of embodiments 28-38, wherein the second genetic modification comprises genome editing that increases the expression or activity of one or more of CYP82E4, CYP82E5 and CYP85E10 polypeptides.

Embodiment 46 the tobacco plant of embodiment 45 or part thereof, wherein the second genetic modification comprises genome editing that increases the expression or activity of CYP82E4v 2.

Embodiment 47 the tobacco plant, or part thereof, of any one of embodiments 28-46, wherein said second genetic modification increases the level of one or more antioxidants and decreases the level of one or more TSNAs as compared to a control plant lacking said second genetic modification.

Embodiment 48 the tobacco plant of embodiment 47, or a portion thereof, wherein said second genetic modification comprises a transgene encoding or targeting an antioxidant biosynthetic enzyme, a regulatory transcription factor for an antioxidant, an antioxidant transporter, an antioxidant metabolic enzyme, or a combination thereof.

Embodiment 49 the tobacco plant of embodiment 47, or a part thereof, wherein said second genetic modification comprises a modification of one or more endogenous genes encoding an antioxidant biosynthetic enzyme, a regulatory transcription factor for an antioxidant, an antioxidant transporter, an antioxidant metabolic enzyme, or a combination thereof.

Embodiment 50 the tobacco plant of embodiment 47, or a part thereof, wherein said one or more antioxidants are selected from the group consisting of: anthocyanins, flavanones, flavanols, flavones, flavonols, isoflavones, hydroxybenzoic acids, hydroxycinnamic acids, ellagitannins, stilbenes, lignans, carotenoids and glycyrrhizin.

Embodiment 51 the tobacco plant of embodiment 47, or part thereof, wherein said one or more antioxidants are selected from the group consisting of: delphinidin, anthocyanidin, procyanidin, delphinidin, hesperetin, naringenin, catechin, epicatechin, apigenin, luteolin, quercetin, myricetin, rutin, genistein, daidzein, gallic acid, vanillic acid, protocatechuic acid, ferulic acid, cinnamic acid, coumaric acid, chlorogenic acid, caffeic acid, ferulic acid, sanguinarine, resveratrol, sesamin, carotenoid and vitamin C.

Embodiment 52 the tobacco plant of embodiment 47, or a part thereof, wherein said second genetic modification comprises a transgene encoding one or more polypeptides selected from the group consisting of: AtPAP1, Ntmyb3A, Ntmyb3B, Ntmyb3C, NtJAF13, sta 1, NtAN1, and NtAN 2.

Embodiment 53 the tobacco plant, or part thereof, of any of the preceding embodiments, wherein said plant is capable of producing leaves having a USDA rating 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 rating index value of the leaves of a control plant grown and processed under similar conditions, and wherein said control plant has substantially the same genetic background as said tobacco plant except for one or both of said genetic modifications.

Embodiment 54. the tobacco plant, or part thereof, of any of the preceding embodiments, wherein said plant is capable of producing leaves exhibiting a USDA grade index value selected from the group consisting of: 55 or higher, 60 or higher, 65 or higher, 70 or higher, 75 or higher, 80 or higher, 85 or higher, 90 or higher and 95 or higher.

Embodiment 55. the tobacco plant, or part thereof, of any of the preceding embodiments, wherein said plant is capable of producing leaves having a nicotine content that is less than 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70% or 80% of the nicotine content of a control plant, wherein said control plant has substantially the same genetic background as said tobacco plant except for one or both of said genetic modifications.

Embodiment 56. the tobacco plant, or part thereof, of any of the preceding embodiments, wherein said plant is capable of producing leaves comprising a nicotine content 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% by dry weight.

Embodiment 57. the tobacco plant, or part thereof, of any of the preceding embodiments, wherein said plant is capable of producing cured leaves comprising less than 2, less than 1.9, less than 1.8, less than 1.7, less than 1.6, less than 1.5, less than 1.4, less than 1.3, less than 1.2, less than 1.1, less than 1.0, 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 0.15, less than 0.1, or less than 0.05ppm total TSNA content.

Embodiment 58. the tobacco plant, or part thereof, of any of the preceding embodiments, wherein said plant is capable of producing a cured leaf comprising na from 2ppm to 0.05ppm, 1.9ppm to 0.05ppm, 1.8ppm to 0.05ppm, 1.7ppm to 0.05ppm, 1.6ppm to 0.05ppm, 1.5ppm to 0.05ppm, 1.4ppm to 0.05ppm, 1.3ppm to 0.05ppm, 1.2ppm to 0.05ppm, 1.1ppm to 0.05ppm, 1.0ppm to 0.05ppm, 0.9ppm to 0.05ppm, 0.8ppm to 0.05ppm, 0.7ppm to 0.05ppm, 0.6ppm to 0.05ppm, 0.5ppm to 0.05ppm, 0.4ppm to 0.05ppm, 0.3ppm to 0.05ppm, 0.2ppm to 0.05ppm, 0.15ppm to 0.05ppm, or 1.0.05 ppm.

Embodiment 59. the tobacco plant, or part thereof, of any of the preceding embodiments, wherein said plant is capable of producing cured leaves comprising less than 2, less than 1.9, less than 1.8, less than 1.7, less than 1.6, less than 1.5, less than 1.4, less than 1.3, less than 1.2, less than 1.1, less than 1.0, 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 0.15, less than 0.1, or less than 0.05ppm total NNN content.

Embodiment 60. the tobacco plant, or part thereof, of any of the preceding embodiments, wherein said plant is capable of producing a cured leaf content comprising 2ppm to 0.05ppm, 1.9ppm to 0.05ppm, 1.8ppm to 0.05ppm, 1.7ppm to 0.05ppm, 1.6ppm to 0.05ppm, 1.5ppm to 0.05ppm, 1.4ppm to 0.05ppm, 1.3ppm to 0.05ppm, 1.2ppm to 0.05ppm, 1.1ppm to 0.05ppm, 1.0ppm to 0.05ppm, 0.9ppm to 0.05ppm, 0.8ppm to 0.05ppm, 0.7ppm to 0.05ppm, 0.6ppm to 0.05ppm, 0.5ppm to 0.05ppm, 0.4ppm to 0.05ppm, 0.3ppm to 0.05ppm, 0.2ppm to 0.05ppm, 0.15ppm to 0.05ppm, or 1.05 ppm of n.

Embodiment 61. the tobacco plant, or part thereof, of any of the preceding embodiments, wherein said plant is capable of producing cured leaves comprising less than 2, less than 1.9, less than 1.8, less than 1.7, less than 1.6, less than 1.5, less than 1.4, less than 1.3, less than 1.2, less than 1.1, less than 1.0, 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 0.15, less than 0.1, or less than 0.05ppm total NNK content.

Embodiment 62. the tobacco plant, or part thereof, of any of the preceding embodiments, wherein said plant is capable of producing a cured leaf content comprising 2ppm to 0.05ppm, 1.9ppm to 0.05ppm, 1.8ppm to 0.05ppm, 1.7ppm to 0.05ppm, 1.6ppm to 0.05ppm, 1.5ppm to 0.05ppm, 1.4ppm to 0.05ppm, 1.3ppm to 0.05ppm, 1.2ppm to 0.05ppm, 1.1ppm to 0.05ppm, 1.0ppm to 0.05ppm, 0.9ppm to 0.05ppm, 0.8ppm to 0.05ppm, 0.7ppm to 0.05ppm, 0.6ppm to 0.05ppm, 0.5ppm to 0.05ppm, 0.4ppm to 0.05ppm, 0.3ppm to 0.05ppm, 0.2ppm to 0.05ppm, 0.15ppm to 0.05ppm, or 1.05 ppm k.

Embodiment 63. the tobacco plant population of any one of embodiments 28-62.

Example 64. cured tobacco material from the tobacco plant of any one of embodiments 1-62.

Embodiment 65 the cured tobacco material of embodiment 64, wherein said cured tobacco material is prepared by a curing process selected from the group consisting of: baking and curing, airing and curing, baking and curing with open fire and airing and curing.

Embodiment 66. tobacco blend comprising cured tobacco material of embodiment 64.

Embodiment 67 the tobacco blend of embodiment 66, wherein the cured tobacco material comprises about 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 percent by weight of the cured tobacco in the tobacco blend.

Embodiment 68 the tobacco blend of embodiment 66, wherein said cured tobacco material comprises about 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 cured tobacco in said tobacco blend.

Embodiment 69. a tobacco product comprising the cured tobacco material of embodiment 64.

Embodiment 70 the tobacco product of embodiment 69, wherein said tobacco product is selected from the group consisting of: cigarettes, cigarillos, non-ventilated filter cigarettes, cigars, snuff, pipe tobacco, cigars, cigarettes, chewing tobacco, leaf tobacco, cut tobacco and cut tobacco.

Embodiment 71 the tobacco product of embodiment 69, wherein said tobacco product is a smokeless tobacco product.

Embodiment 72 the tobacco product of embodiment 71, wherein said smokeless tobacco product is selected from the group consisting of: loose leaf chewing tobacco, plug chewing tobacco, moist snuff and snuff.

Embodiment 73. reconstituted tobacco comprising cured tobacco material of embodiment 64.

Having now generally described the present disclosure, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present disclosure unless otherwise specified.

Examples

Example 1 plant transformation

Tobacco plants expressing the gene of interest are produced via agrobacterium-mediated transformation. The expression vector p45-2-7 was used as a backbone for the generation of multiple transformation vectors. p45-2-7 contains the CsVMV promoter, the NOS terminator and a cassette containing the kanamycin selection marker (NPT II) operably linked to the actin 2 promoter and the NOS terminator. The nucleic acid vector containing the transgene of interest is introduced into the tobacco leaf disc via agrobacterium transformation. See, for example, Mayo et al, 2006, Nat Protoc.1:1105-11and Horsch et al, 1985, Science 227: 1229-.

Tobacco plants of high and medium burley tobacco 21(HI BU21) or low and medium burley tobacco 21(LI BU21) were grown in Magenta^TMGrowth in GA-7 cassettes, leaf disks were excised and placed in Petri dishes. Agrobacterium tumefaciens cells containing the transformation vector were collected by centrifugation of 20mL of the cell suspension in a 50mL centrifuge tube at 3500RPM for 10 minutes. The supernatant was removed and the Agrobacterium tumefaciens cell pellet resuspended in 40mL of liquid resuspension medium. Tobacco leaves (avoiding the middle rib) were cut into 8 0.6cm disks with a #15 razor blade and placed upside down in a Petri dishIn a petri dish. Murashige with B5 vitamin liquid resuspension medium&Skoog lamellae were added to the petri dish and leaf discs were uniformly punctured with a fine needle. Approximately 25mL of agrobacterium tumefaciens suspension was added to the petri dish and the leaf discs were incubated in suspension for 10 minutes.

The leaf discs were transferred to co-culture Petri dishes (1/2MS medium) and the discs were placed upside down to cover on co-culture TOM medium (MS medium with 20g/L sucrose; 1mg/L indole-3-acetic acid; and 2.5 mg/L6-Benzylaminopurine (BAP)). The Petri dishes were sealed with parafilm and then incubated at 24 degrees Celsius in dim light (60-80mE/ms) for three days with a photoperiod of 18 hours on and 6 hours off. After incubation, the leaf discs were transferred to regeneration/selection TOM K medium petri dishes (TOM medium plus 300mg/L kanamycin). Leaf disks were sub-cultured every two weeks at 24 ℃ in a 18 hour on, 6 hour off photoperiod in dim light into fresh TOMK medium until shoots became excisable. Shoots regenerated from leaf disks were removed with forceps and inserted into MS basal medium containing 100mg/L kanamycin. Explants were placed on MS basal medium containing 100mg/L kanamycin, incubated at 24 ℃ for 18 hours, and subjected to 6 hour light-off cycle under high intensity illumination (6080mE/MS) to induce rooting.

When plantlets containing shoots and roots grow large enough (e.g., reach Magenta)^TMAbout half the height of the GA-7 cassette) and they were transferred into the soil. Established seedlings were transferred to the greenhouse for further analysis and seed set. Control plants are untransformed HI BU21 or LI BU21 plants, or HI BU21 or LI BU21 plants that have been transformed with the empty p45-2-7 vector.

Example 2: overexpression of nicotine demethylase (cytochrome P450 monooxygenase CYP82E4v2) to reduce nicotine levels in tobacco

According to example 1, the coding sequence for nicotine demethylase CYP82E4v2(SEQ ID NO:1) was integrated into the p45-2-7 transformation vector and a modified tobacco plant was produced. Modified HI BU21 tobacco plant (T)₀And T₁Generations) and control tobacco plants were grown for 4-6 weeks after transplantation to soil. Beating the plants in the flowering periodAnd (6) carrying out top lifting. Two weeks after topping, the 3 rd to 5 th leaves from the top were harvested. Freeze drying or oven drying the leaves. Alkaloids were measured from dried leaves using standard protocols. CYP82E4v 2-expressing T, in comparison to control HI BU21 plants₀The transgenic plants had reduced nicotine content (figure 1). When CYP82E4v2 is overexpressed in LI BU21 and TN90 LC plants, in T₀Reduced nicotine was also observed in the transgenic plants (figure 1). The nornicotine, anabasine and anatabine content of these transgenic plants was also measured. Nornicotine levels increased when CYP82E4v2 was overexpressed in all three backgrounds (figure 2). At various T₀In transgenic plants, both anabasine and anatabine showed similar trends in their content, as their contents correlated with each other without decisive changes due to CYP82E4v2 overexpression (fig. 3 and 4). Further testing of T in TN90 LC₁The alkaloid content of generations, e.g. nicotine, decreased but no nicotine was increased, and anabasine and anatabine slightly increased (fig. 5-8). Notably, CYP82E4v2 overexpresses TN90 LC T₁The average nicotine content in the plants was about 1% of that of the control plants (fig. 5).

Example 3: overexpression of antioxidant regulatory genes reduces TSNA

According to example 1, the coding sequences for the antioxidant regulator genes Arabidopsis PAP1(SEQ ID NO:2), Nicotiana tabacum Myb3(SEQ ID NO:3) and Solanum tuberosum AN1(SEQ ID NO:4) were incorporated into the p45-2-7 transformation vector and modified tobacco plants were produced. Expresses CYP82E4v2 and at least one antioxidant regulator gene (T)₀And T₁Generations) of modified HI BU21 tobacco plants and control tobacco plants were grown for 4-6 weeks after being transplanted into soil. Plants were topped at flowering time. Two weeks after topping, the 3 rd to 5 th leaves from the top were harvested. Freeze drying or oven drying the leaves. Alkaloids were measured from dried leaves using standard protocols. Plants with low alkaloid content and high antioxidant content were selected and grown in the field. Selected plants as well as control plants were topped, leaves were air cured and tested for TSNA. Plants expressing Arabidopsis PAP1, R.tabacum Myb3, or Potato AN1 showed increased antioxidant capacity compared to unmodified control plantsAnd reduced amounts of TSNAs. Overexpression of any gene that increases antioxidant capacity in tobacco plants can be used to reduce TSNA in modified HIBU 21. As shown in FIG. 9, T overexpressing Myb3 in LATN90₀The transgenic plants exhibited increased antioxidant content as measured by the iron reduction antioxidant capacity (FRAP) assay (fig. 9). This increase in antioxidants was observed in 3 generations of plants (figure 10). Similarly, T overexpressing Myb3 in LATN90₂The transgenic plants showed increased antioxidant capacity (fig. 11). The LATN90 used herein is a TN90 variety with a nic1 nic2 double mutation introgressed from LA BU 21.

The iron reduction antioxidant capacity (FRAP) method is based on the reduction of a complex of 2,4, 6-tripyridyl-s-triazine (TPTZ) with ferric chloride hexahydrate (FeCl 3.6H 2O) which, after reduction, forms a blue ferrous complex (Benzie)&Strain,1996, Analytical Biochemistry 239, 70-76). Three solutions were used for the experiments: solution 1) TPTZ (0.07802g/25mL) in 10mmol.L of 40mM hydrochloric acid^-1A solution; solution 2) a 20mM solution of ferric chloride hexahydrate (0.13513g/25mL) in ACS water; solution 3)20mM acetate buffer, pH 3.6 (weight of sodium acetate trihydrate 0.27216g in 100mL of CS water, adjusted to the desired pH using HCl). The three solutions (TPTZ, FeCl3, acetate buffer) were mixed in a ratio of 1:1: 10. A volume of 245 μ L of the mixture was pipetted into a plastic cuvette, followed by the addition of 5 μ L of the sample (gallic acid,). The absorbance was measured at a wavelength of 593nm, the dominant lambda. Using different concentrationsA standard curve is prepared and the sample is compared to the standard curve. Total antioxidant was calculated using the following equation

Example 4: overexpression of CYP82E4v2 and tobacco Myb3 was used to reduce nicotine and TSNA and improve leaf quality

Tobacco carthamus Myb3(SEQ ID NO:3) and CYP82E4v2(SEQ ID NO:1) were introduced into the p45-2-7 transformation vector and used to generate modified tobacco plants as described in example 1. Modified tobacco plants (T)₀And T₁Generations) and control tobacco plants were grown for 4-6 weeks after being transplanted into soil. Plants were topped at flowering time. Two weeks after topping, the 3 rd to 5 th leaves from the top were harvested. Freeze drying or oven drying the leaves. Alkaloids were measured from dried leaves using standard protocols. Plants overexpressing CYP82E4v2 had reduced nicotine content compared to control plants. Plants with low alkaloid content and high antioxidant content were selected and grown in the field. Overexpression of Myb3 was confirmed using standard molecular techniques. Modified and control plants were topped, leaves were air-cured, and tested for alkaloids and TSNAs using standard protocols. Plants expressing arabidopsis PAP1, nicotiana tabacum Myb3, or potato AN1, and also expressing CYP82E4v2, were tested for nicotine and TSNA content compared to unmodified control plants. As shown in FIG. 12, T overexpressing CYP82E4v2 and Myb3, as compared to vector control plants, was observed in all three tobacco backgrounds TN90, HI BU21, and LI BU21₀The transgenic plants contain reduced nicotine. In contrast, in all three tobacco backgrounds TN90, HI BU21 and LI BU21, the T overexpressing CYP82E4v2 and Myb3 were compared to vector control plants₀Transgenic plants contained increased levels of nornicotine (figure 13). Likewise, anabasine and anatabine are in various T₀The contents in the transgenic plants showed similar trends, as their contents correlated with each other without decisive changes due to CYP82E4v2 and Myb3 overexpression (fig. 14 and 15). TN90 may show a slightly higher average content of anabasine and anatabine, if any, relative to the average control content. In addition, FRAP assays demonstrated that CYP82E4v2 and Myb3 co-overexpression also increased antioxidant content in all three tobacco backgrounds TN90, HI BU21, and LI BU21 (fig. 16).

Alternatively, HI BU21 (or LI BU21) plants were co-transformed with nicotine demethylase and Myb3 overexpression constructs. Callus color (e.g., purple or deep red) can be used to select Myb3 transformants. In addition, tobacco plants overexpressing AtPAP1 were transformed with nicotine demethylase overexpression constructs. In addition, nicotine demethylase overexpressing plants were transformed with the AtPAP1 (or Myb3) overexpressing construct. In addition, a single construct with two or more stacked expression cassettes (e.g., CsVMV-NtMYB3-Nos-T-CsVMV-CYP82E4-Nos-T as shown in SEQ ID No. 44) was used to transform HI BU21 (or LI BU21) plants. Transformants were selected and tested for nicotine and TSNA content.

Example 5: crossing of HI BU21 (or LI BU21) plants expressing antioxidant regulatory genes into burley tobacco transformed lines

Burley tobacco lines showing high conversion of nicotine to nornicotine (stably transformed lines) were crossed with HI BU21 (or LI BU21) plants expressing antioxidant regulatory genes using standard techniques. Testing for Stable F₂Nicotine content of plants. Antioxidant assays were performed to test the antioxidant capacity of these plants compared to the parent burley tobacco transformed line. TSNA analysis was used to assess the TSNA content compared to the parent burley tobacco transformed line. Alkaloids, antioxidant capacity and TSNA were measured using standard techniques.