阅读说明：本技术 用于生产具有改变的生物碱水平的烟草植物和制品的基于pmt工程改造的组合物和方法 (PMT-based engineered compositions and methods for producing tobacco plants and products with altered levels of alkaloids ) 是由 C·库迪西普迪 亓栋 沈燕新 U·瓦雷克 J·斯特里克兰德 许冬梅 于 2019-07-26 设计创作，主要内容包括：本公开提供了与具有改变的总生物碱和尼古丁水平和商业上可接受的叶等级的烟草植物、它们通过育种或转基因方法的开发以及从这些烟草植物生产烟草制品有关的组合物和方法。(The present disclosure provides compositions and methods related to tobacco plants having altered levels of total alkaloids and nicotine and commercially acceptable leaf grades, their development by breeding or transgenic methods, and the production of tobacco products from these tobacco plants.)

1. A tobacco plant, or a portion thereof, comprising one or more mutant alleles in at least one PMT gene selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4, wherein the tobacco plant is capable of producing leaves having a nicotine level that is lower than the nicotine level of leaves from a control tobacco plant not having the one or more mutant alleles when grown and processed under comparable conditions.

2. The tobacco plant, or portion thereof, of claim 1, wherein said tobacco plant comprises one or more mutant alleles in at least two PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT 4.

3. The tobacco plant, or portion thereof, of claim 1, wherein said tobacco plant comprises one or more mutant alleles in at least three PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT 4.

4. The tobacco plant, or portion thereof, of claim 1, wherein said tobacco plant comprises one or more mutant alleles in at least four PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT 4.

5. The tobacco plant, or portion thereof, of claim 1, wherein said tobacco plant comprises one or more mutant alleles in five PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT 4.

6. The tobacco plant, or part thereof, according to any one of claims 1 to 5, wherein the tobacco plant is capable of producing leaves having a nicotine level that is less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.25% of the nicotine level of leaves from a control tobacco plant not having the one or more mutant alleles when grown and processed under comparable conditions.

7. A tobacco plant, or part thereof, according to any one of claims 1 to 6, wherein said tobacco plant is capable of producing leaves having a total alkaloid level that is less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.25% of the total alkaloid level of leaves from said control tobacco plant when grown and processed under comparable conditions.

8. The tobacco plant, or part thereof, according to claim 7, wherein said tobacco plant is capable of producing leaves having a total alkaloid level that is less than 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of the total alkaloid level of leaves from said control tobacco plant when grown and processed under comparable conditions.

9. The tobacco plant, or part thereof, according to any one of claims 1 to 8, wherein said one or more mutant alleles comprise a mutation in a sequence region selected from the group consisting of a promoter, a 5 'UTR, a first exon, a first intron, a second exon, a second intron, a third exon, a 3' UTR, a terminator, and any combination thereof.

10. The tobacco plant, or part thereof, according to any one of claims 1 to 9, wherein said one or more mutant alleles comprise one or more mutation types selected from the group consisting of nonsense mutations, missense mutations, frameshift mutations, splice site mutations, and any combination thereof.

11. The tobacco plant, or part thereof, according to any one of claims 1 to 10, wherein the one or more mutant alleles result in one or more of: PMT protein truncations, nontranslatable PMT gene transcripts, non-functional PMT proteins, premature stop codons in PMT genes, and any combination thereof.

12. The tobacco plant, or part thereof, according to any one of claims 1 to 11, wherein the one or more mutant alleles comprise a mutation selected from the group consisting of a substitution, deletion, insertion, duplication, and inversion of one or more nucleotides relative to a wild-type PMT gene.

13. The tobacco plant, or part thereof, according to any one of claims 1 to 12, wherein the one or more mutant alleles comprise a zygote status selected from the group consisting of homozygous, heterozygous, and heteroallelic genes.

14. The tobacco plant, or part thereof, according to any one of claims 1 to 12, wherein the one or more mutant alleles are homozygous or heteroallelic for at least 1-5 PMT genes.

15. The tobacco plant, or part thereof, according to any one of claims 1 to 12, wherein the one or more mutant alleles are homozygous or heteroallelic in at least 4 PMT genes.

16. The tobacco plant, or part thereof, according to any one of claims 1 to 12, wherein the one or more mutant alleles are homozygous or heteroallelic in all five PMT genes.

17. The tobacco plant, or part thereof, according to any one of claims 1 to 16, wherein said at least two PMT genes are PMT1a and PMT 3.

18. The tobacco plant, or part thereof, of any one of the preceding claims, wherein the tobacco plant is capable of producing leaves with a nicotine level selected from the group consisting of: less than 0.15%, less than 0.125%, less than 0.1%, less than 0.08%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, and less than 0.01% dry weight.

19. The tobacco plant, or part thereof, according to any of the preceding claims, wherein said tobacco plant is capable of producing leaves with a total alkaloid level selected from the group consisting of: less than 1%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, and less than 0.2% dry weight.

20. The tobacco plant, or part thereof, according to any of the preceding claims, wherein the tobacco plant is capable of producing cured leaves having a total TSNA level of: 2 to 0.05, 1.9 to 0.05, 1.8 to 0.05, 1.7 to 0.05, 1.6 to 0.05, 1.5 to 0.05, 1.4 to 0.05, 1.3 to 0.05, 1.2 to 0.05, 1.1 to 0.05, 1.0 to 0.05, 0.9 to 0.05, 0.8 to 0.05, 0.7 to 0.05, 0.6 to 0.05, 0.5 to 0.05, 0.4 to 0.05, 0.3 to 0.05, 0.2 to 0.05, 0.15 to 0.05, or 0.1 to 0.05 ppm.

21. A population of tobacco plants according to any one of claims 1 to 20.

22. Cured tobacco material from a tobacco plant according to any one of claims 1 to 20.

23. The cured tobacco material of claim 22, wherein the cured tobacco material is prepared by a curing process selected from the group consisting of: baking, air-curing, smoking, and sun-curing.

24. The cured tobacco material of claim 22, wherein the cured tobacco material comprises tobacco leaf, and wherein the tobacco leaf exhibits reduced mold infection as compared to a control cured tobacco material from variety LA Burley 21.

25. A phyllotactic formulation comprising a cured tobacco material according to claim 22.

26. The leaf set formulation of claim 25, 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% by weight of cured tobacco in the leaf set formulation.

27. The leaf set formulation of claim 25, 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% by volume of cured tobacco in the leaf set formulation.

28. A smoking article comprising the cured tobacco material of claim 22.

29. A smoking article according to claim 28, wherein the smoking article is selected from the group consisting of: cigarettes, cigarillos, non-ventilated recess-filter cigarettes, cigars, tobacco-containing, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, tobacco leaves, shredded tobacco and cut tobacco.

30. A smoking article according to claim 28, wherein the smoking article is a smokeless tobacco article.

31. The tobacco product of claim 30, wherein the smokeless tobacco product is selected from the group consisting of loose-leaf chewing tobacco, cut tobacco rod chewing tobacco, wet tobacco powder, and snuff.

32. A reconstituted tobacco comprising the cured tobacco material of claim 22.

Technical Field

The present disclosure provides tobacco genetic engineering for modulating alkaloid and nicotine levels.

Background

Nicotine is the major alkaloid and typically accounts for over 90-95% of total alkaloids in commercial tobacco cultivars. The remaining alkaloid fraction mainly contains three additional alkaloids: nornicotine, anabasine and anatabine. Tobacco plants with reduced nicotine levels have been achieved with different and inconsistent results by regulating different nicotine biosynthesis genes and transcriptional regulators. New techniques for reducing the level of nicotine in tobacco leaves are needed.

Disclosure of Invention

The present disclosure provides tobacco plants having altered levels of total alkaloids and nicotine and commercially acceptable leaf grades, their development by breeding or transgenic methods, and the production of tobacco products from these tobacco plants.

In one aspect, the present disclosure provides a tobacco plant, or a portion thereof, comprising one or more mutant alleles in at least one PMT gene selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4, wherein the tobacco plant is capable of producing leaves having nicotine levels that are lower than the nicotine levels of leaves from a control tobacco plant not having the one or more mutant alleles when grown and processed under comparable conditions.

In another aspect, the tobacco plant comprises one or more mutant alleles in at least two PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT 4.

In a further aspect, the tobacco plant comprises one or more mutant alleles in at least three PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT 4.

In another aspect, the tobacco plant comprises one or more mutant alleles in at least four PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT 4.

In a further aspect, the tobacco plant comprises one or more mutant alleles in five PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT 4.

In one aspect, the disclosure provides a tobacco plant selected from the group consisting of a single pmt mutant, a double pmt mutant, a triple mutant, a quadruple mutant, and a quintet mutant, as set forth in tables 8A through 8E.

In one aspect, the present disclosure provides a tobacco plant as set forth in tables 4A to 4E or table 10. In another aspect, the present disclosure provides a progeny plant of a tobacco plant of table 4A to 4E or table 10 from selfing or crossing with another plant of table 4A to 4E or table 10.

In another aspect, the disclosure provides a tobacco plant comprising various combinations of the pmt mutant alleles set forth in tables 5A through 5E or tables 12A through 12E to produce a single pmt mutant, a double pmt mutant, a triple mutant, a quadruple mutant, or a penta mutant. In one aspect, the present disclosure provides a tobacco plant comprising a pmt mutant allele sequence selected from the group consisting of SEQ ID nos.21 to 200, 410 to 441, 474 to 505, 538 to 569, 602 to 633, and 666 to 697.

The present disclosure further provides cured tobacco, leaf set formulations, tobacco products comprising plant material from the disclosed tobacco plants, lines, varieties, or hybrids.

Brief description of the sequences

SEQ ID Nos: exemplary genomic sequences of PMT1b, PMT1a, PMT2, PMT3, and PMT4 from the TN90 reference genome are set forth, 1 through 5, respectively.

SEQ ID Nos: exemplary cDNA sequences for PMT1b, PMT1a, PMT2, PMT3, and PMT4 from TN90 are set forth, respectively, 6 through 10.

SEQ ID Nos: exemplary polypeptide sequences of PMT1b, PMT1a, PMT2, PMT3, and PMT4 from TN90 are set forth, respectively, at 11 through 15.

SEQ ID Nos: 16 to 22 illustrate exemplary guide RNA sequences.

SEQ ID Nos: exemplary edited pmt mutant sequences are set forth in FIGS. 23-200, 410-441, 474-505, 538-569, 602-633, and 666-697.

Drawings

FIG. 1: FIG. TN90 RNA expression of five PMT genes in roots

FIG. 2: various low nicotine strains: nicotine levels in CS15 (five PMT knockout mutant line CS15 in nlm (ph) background, PMT RNAi transgenic line in VA359 background) and low nicotine KY171 ("LN KY 171") cultivars (KY 171 background contained the nic1 and nic2 double mutations), in comparison to their respective normal alkaloid control lines: nlm (ph), VA359 and KY171 background.

FIG. 3: various low alkaloid strains: total alkaloid levels in CS15, PMT RNAi and LN KY171, and their respective normal alkaloid control lines: nlm (ph), VA359, and KY171 background.

FIG. 4: various low alkaloid strains: leaf yield in CS15, PMT RNAi and LN KY171, compared to their respective normal alkaloid control lines: nlm (ph), VA359, and KY171 background.

FIG. 5: various low alkaloid strains: leaf quality in CS15, PMT RNAi and LN KY171, compared to their respective normal alkaloid control lines: nlm (ph), VA359, and KY171 background.

FIG. 6: photographs depicting the modulation of mold growth on tobacco, including TN90 LC (fig. 6A), LA BU 21 (fig. 6B), TN90 comprising RNAi constructs that down-regulate PR50 (fig. 6C), TN90 comprising RNAi constructs that down-regulate PMT genes (fig. 6D), and TN90 comprising edits to all five PMT genes (fig. 6E).

FIG. 7: a depiction of the fungal infection observed in the lines examined in figures 6A-6E.

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 as follows.

Any references cited herein (including, for example, all patents and publications) are incorporated by reference in their entirety.

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

When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the upper and lower bounds of the numerical values by 10%.

As used herein, phrases such as "less than," "greater than," "at least," "at most," "about," "less than," "higher than," and "about" when used in connection with a range of values, modify each value in the range. For example, a statement of "less than 1%, 2%, or 3%" is equivalent to "less than 1%, less than 2%, or less than 3%".

As used herein, a tobacco plant refers to a plant from the nicotiana species.

The biosynthesis of nicotine in tobacco begins with: polyamines (putrescine) are methylated to N-methylputrescine by an enzyme (putrescine N-methyltransferase (PMT)) using S-adenosyl-methionine as a cofactor. This is the step of participating the precursor metabolite in nicotine biosynthesis. PMT enzymes are classified as EC 2.1.1.53 under the enzyme classification system. In tobacco, five genes encode putrescine N-methyltransferases, designated PMT1a, PMT1b, PMT2, PMT3, and PMT 4. The genomic DNA sequences, cDNA sequences and protein sequences of these five PMT genes in TN90 plants are listed in table 1A. The present disclosure describes compositions and methods for editing PMT genes to produce PMT mutant plants with reduced nicotine levels while maintaining leaf mass.

As used herein, "PMT 1 b" or "PMT 1b gene" refers to a gene encoded in tobacco having the sequence as set forth in SEQ ID No: 11, or a sequence of an exemplary amino acid sequence described in seq id no.

As used herein, "PMT 1 a" or "PMT 1a gene" refers to the locus in tobacco that encodes a polypeptide having the exemplary amino acid sequence set forth in SEQ ID No.12 in TN 90.

As used herein, "PMT 2" or "PMT 2 gene" refers to a locus that encodes a polypeptide in tobacco having an exemplary amino acid sequence as set forth in SEQ ID No.13 in TN 90.

As used herein, "PMT 3" or "PMT 3 gene" refers to a locus that encodes a polypeptide in tobacco having an exemplary amino acid sequence as set forth in SEQ ID No.14 in TN 90.

As used herein, "PMT 4" or "PMT 4 gene" refers to a locus that encodes a polypeptide in tobacco having an exemplary amino acid sequence as set forth in SEQ ID No.15 in TN 90.

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. 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 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, "PMT mutant" refers to a tobacco plant that comprises one or more mutations in one or more PMT genes. The pmt mutants may be single mutants, double mutants, triple mutants, quadruple mutants or penta mutants. As used herein, a single, double, triple, quadruple or quintuple PMT mutant refers to a mutant having a modification in one, two, three, four or five PMT genes, respectively. The PMT mutants may also be homozygous, heterozygous or a combination of heteroallelic mutants in one or more PMT genes.

As used herein, gene names or locus names are capitalized and displayed in italics, such as PMT1a, PMT1b, PMT2, PMT3, and PMT 4. Italics, such as PMT1a, PMT1b, PMT2, PMT3, and PMT4 are not used for protein or polypeptide name capitalization. Mutant names (used to refer to a gene or a general mutation in a group of genes, or to a particular mutant allele) are shown in lower case and italics, for example pmt, pmt1a, pmt1b, pmt2, pmt3, and pmt 4.

In one aspect, the present disclosure provides a tobacco plant, or a portion thereof, comprising one or more mutant alleles in at least one PMT gene selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4, wherein the tobacco plant is capable of producing leaves having a nicotine level that is lower than the nicotine level of leaves from a control tobacco plant not having the one or more mutant alleles when grown and processed under comparable conditions. In one aspect, a single pmt mutant tobacco plant is provided. In another aspect, a single pmt mutant tobacco plant comprises a nicotine level that is less than 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the nicotine level of a control plant that does not have a single pmt mutation, when grown under similar growth conditions. In a further aspect, a single pmt mutant tobacco plant comprises a nicotine level that is 1% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 95% of the nicotine level of a control plant that does not have a single pmt mutation, when grown under similar growth conditions.

In another aspect, the tobacco plant comprises one or more mutant alleles in at least two PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT 4. In one aspect, a double pmt mutant tobacco plant is provided. In another aspect, the dual pmt mutant tobacco plant comprises a nicotine level that is less than 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the nicotine level of a control plant that does not have the dual pmt mutation, when grown under similar growth conditions. In a further aspect, the dual pmt mutant tobacco plant comprises a nicotine level that is 1% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 95% of the nicotine level of a control plant that does not have the dual pmt mutation, when grown under similar growth conditions.

In a further aspect, the tobacco plant comprises one or more mutant alleles in at least three PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT 4. In one aspect, a three pmt mutant tobacco plant is provided. In another aspect, the triple pmt mutant tobacco plant comprises a nicotine level that is less than 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the nicotine level of a control plant that does not have the triple pmt mutation, when grown under similar growth conditions. In a further aspect, the triple pmt mutant tobacco plant comprises a nicotine level that is 1% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 95% of the nicotine level of a control plant that does not have the triple pmt mutation, when grown under similar growth conditions.

In another aspect, the tobacco plant comprises one or more mutant alleles in at least four PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT 4. In one aspect, a tetrapmt mutant tobacco plant is provided. In another aspect, the tetrapmt mutant tobacco plant comprises a nicotine level that is less than 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the nicotine level of a control plant that does not have the tetrapmt mutation when grown under similar growth conditions. In a further aspect, the tetrapmt mutant tobacco plant comprises a nicotine level that is 1% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 95% of the nicotine level of a control plant that does not have the tetrapmt mutation, when grown under similar growth conditions.

In a further aspect, the tobacco plant comprises one or more mutant alleles in five PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT 4. In one aspect, a five pmt mutant tobacco plant is provided. In another aspect, the five pmt mutant tobacco plant comprises a nicotine level that is less than 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the nicotine level of a control plant that does not have the five pmt mutation, when grown under similar growth conditions. In a further aspect, the five pmt mutant tobacco plant comprises a nicotine level that is 1% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 95% of the nicotine level of a control plant that does not have the five pmt mutation, when grown under similar growth conditions.

In one aspect, the tobacco plant is a single pmt mutant, a double pmt mutant, a triple mutant, a quadruple mutant, or a penta mutant as set forth in tables 8A through 8E. In another aspect, a tobacco plant comprises one or more pmt mutant alleles set forth in tables 5A through 5E and tables 12A through 12E. Also provided are each combination of the pmt mutant alleles listed in tables 5A through 5E and tables 12A through 12E to generate a single pmt mutant, a double pmt mutant, a triple mutant, a quadruple mutant, or a penta mutant. The locus of each mutation may be homozygous or heterozygous, or comprise a heteroallelic combination. In another aspect, the tobacco plant comprises a pmt mutant genotype combination, as shown for each individual line listed in tables 4A through 4E and table 10. In one aspect, a tobacco plant comprises a pmt mutant allele sequence selected from the group consisting of SEQ ID nos.21 to 200, 410 to 441, 474 to 505, 538 to 569, 602 to 633, and 666 to 697. In another aspect, the disclosure provides a double, triple, quadruple or penta mutant comprising a pmt mutant allele sequence selected from the group consisting of SEQ ID nos.21 to 200, 410 to 441, 474 to 505, 538 to 569, 602 to 633, and 666 to 697.

In one aspect, the tobacco plant is capable of producing leaves having a nicotine level that is less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.25% of the nicotine level of leaves from a control tobacco plant when grown and processed under comparable conditions. In another aspect, the tobacco plant is capable of producing leaves having a nicotine level of 1% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 95% of the nicotine level of a control tobacco plant when grown and processed under comparable conditions.

In another aspect, the tobacco plant is capable of producing leaves having a total alkaloid level that is less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.25% of the total alkaloid level of a control tobacco plant when grown and processed under comparable conditions. In another aspect, the tobacco plant is capable of producing leaves having a total alkaloid level that is 1% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 95% of the total alkaloid level of a control tobacco plant when grown and processed under comparable conditions.

In a further aspect, the tobacco plant is capable of producing leaves having a total alkaloid level that is less than 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of the total alkaloid level of leaves from a control tobacco plant when grown and processed under comparable conditions.

In one aspect, the mutant PMT allele comprises a mutation in a PMT sequence region selected from the group consisting of: a promoter, a 5 'UTR, a first exon, a first intron, a second exon, a second intron, a third exon, a third intron, a fourth exon, a fourth intron, a fifth exon, a fifth intron, a sixth exon, a sixth intron, a seventh exon, a seventh intron, an eighth exon, a 3' UTR, a terminator, and any combination thereof. In another aspect, the mutant PMT allele comprises a mutation in a region of the PMT genomic sequence listed in tables 1D through 1H.

In another aspect, the mutant pmt allele comprises one or more mutation types selected from the group consisting of a nonsense mutation, a missense mutation, a frameshift mutation, a splice site mutation, and any combination thereof. In one aspect, the mutant pmt allele is a null allele or a knockout allele.

In one aspect, mutating the pmt allele results in one or more of: PMT protein truncations, nontranslatable PMT gene transcripts, non-functional PMT proteins, premature stop codons in PMT genes, and any combination thereof.

In another aspect, the mutant pmt allele comprises a mutation selected from the group consisting of: substitution, deletion, insertion, duplication, and inversion of one or more nucleotides relative to the wild-type PMT gene.

In one aspect, the pmt mutant comprises a zygotic state selected from the group consisting of homozygous, heterozygous, and heteroallelic. In another aspect, the PMT mutant is homozygous or heteroallelic for at least 1, 2,3, 4, or 5 PMT genes. In one aspect, the PMT mutant is homozygous or heteroallelic for at least 4 PMT genes. In another aspect, the PMT mutant is homozygous or heteroallelic in all five PMT genes. In another aspect, the PMT mutant comprises a mutation in PMT1a and PMT 3.

In one aspect, the tobacco plant is capable of producing leaves with nicotine levels selected from the group consisting of: less than 0.15%, less than 0.125%, less than 0.1%, less than 0.08%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, and less than 0.01% dry weight.

In another aspect, the tobacco plant is capable of producing leaves having a total alkaloid level selected from the group consisting of: less than 1%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, and less than 0.2% dry weight.

In a further aspect, the tobacco plant is capable of producing cured leaves having a total TSNA level of: 2 to 0.05ppm, 1.9 to 0.05ppm, 1.8 to 0.05ppm, 1.7 to 0.05ppm, 1.6 to 0.05ppm, 1.5 to 0.05ppm, 1.4 to 0.05ppm, 1.3 to 0.05ppm, 1.2 to 0.05ppm, 1.1 to 0.05ppm, 1.0 to 0.05ppm, 0.9 to 0.05ppm, 0.8 to 0.05ppm, 0.7 to 0.05ppm, 0.6 to 0.05ppm, 0.5 to 0.05ppm, 0.4 to 0.05ppm, 0.3 to 0.05ppm, 0.2 to 0.05ppm, 0.15 to 0.05ppm or 0.1 to 0.05 ppm.

In one aspect, the tobacco plant is capable of producing leaves upon modulation having a USDA grade index value selected from the group consisting of 50 or higher, 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 is capable of producing leaves upon conditioning having a USDA grade index value comparable to that of a control plant when grown and conditioned under similar conditions, wherein the control plant shares substantially the same genetic background as the tobacco plant except for the modification. In a further aspect, the tobacco plant, when conditioned, 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 a control plant when grown under similar conditions, wherein the control plant shares substantially the same genetic background as the tobacco plant except for the modification. In a further aspect, the tobacco plant, when conditioned, is capable of producing leaves having a USDA grade index value of 65% to 130%, 70% to 130%, 75% to 130%, 80% to 130%, 85% to 130%, 90% to 130%, 95% to 130%, 100% to 130%, 105% to 130%, 110% to 130%, 115% to 130%, or 120% to 130% of the USDA grade index value of the control plant. In a further aspect, the tobacco plant, when conditioned, is capable of producing leaves having a USDA grade index value of 70% to 125%, 75% to 120%, 80% to 115%, 85% to 110%, or 90% to 100% of the USDA grade index value of the control plant.

In one aspect, the tobacco plant comprises a nicotine level that is less than 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70% or 80% of the nicotine level of a control plant when grown under similar growth conditions, wherein the control plant shares substantially the same genetic background as the tobacco plant except for the modification.

In a further aspect, the tobacco plant comprises one or more pmt mutant alleles, and 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: MPO, QPT, BBL, A622, aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, ornithine decarboxylase, arginine decarboxylase, nicotine absorption permease (NUP), and MATE transporter.

In one aspect, the tobacco plant comprises one or more pmt mutant alleles, and further comprises a mutation in the ERF gene of the Nic2 locus. In one aspect, the tobacco plant further comprises one or more mutations in two or more, three or more, four or more, five or more, six or more, or all seven genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF 168. See Shoji et al, Plant Cell, (10):3390-409 (2010); and Kajikawa et al, Plant physiol.2017,174: 999-. In one aspect, the tobacco plant further comprises one or more mutations in ERF189, ERF115, or both.

In one aspect, the tobacco plant comprises one or more qpt mutant alleles, and further comprises a mutation in the ERF gene of the Nic1 locus (or the Nic1b gene in PCT/US2019/013345 disclosed in WO/2019/140297 as filed on 11/1/2019). See also WO/2018/237107. In one aspect, the tobacco plant further comprises one or more mutations in two or more, three or more, four or more, five or more, six or more, or seven or more genes selected from the group consisting of ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210, and ERF91L 2. See Kajikawa et al, Plant physiol.2017,174: 999-. In one aspect, the tobacco plant further comprises one or more mutations in one or more, two or more, three or more, four or more, five or more, or all six genes selected from the group consisting of ERFnew, ERF199, ERF19, ERF29, ERF210, and ERF91L 2.

In one aspect, the disclosure further provides a pmt mutant tobacco plant, or portion thereof, comprising a nicotine or total alkaloid level selected from the group consisting of: less than 3%, less than 2.75%, less than 2.5%, less than 2.25%, less than 2.0%, less than 1.75%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.025%, less than 0.01%, and less than 0.005%, wherein said 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, such pmt mutant tobacco plants comprise nicotine levels of less than 0.02% and are capable of producing leaves having a USDA grade index value of 70 or greater when formulated. In a further aspect, such tobacco plants comprise nicotine levels below 0.01% and are capable of producing leaves having a USDA grade index value of 70 or higher when formulated.

In one aspect, the invention also provides a tobacco plant or part thereof comprising a non-transgenic mutation, wherein said non-transgenic mutation reduces nicotine or total alkaloid levels of a tobacco plant to less than 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70% or 80% of the nicotine level of a control plant when grown under similar 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 the control plant, and wherein said control plant shares substantially the same genetic background with said tobacco plant except for the non-transgenic mutation.

In one aspect, the tobacco plant comprises a pmt mutation introduced by a method selected from the group consisting of random mutagenesis and targeted mutagenesis. In another aspect, the pmt mutation is introduced by a targeted mutagenesis method selected from the group consisting of a meganuclease, a zinc finger nuclease, a TALEN, and a CRISPR.

Unless otherwise indicated, reference herein to a measurement of an alkaloid or nicotine level (or another leaf chemical or property characterization) or leaf rank 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 a single plant or an average measurement of a population of tobacco plants from a single variety, cultivar, or line.

Unless otherwise indicated, nicotine or alkaloid levels (or another leaf chemical or characteristic characterization) of tobacco plants were measured post-topping in mixed leaf samples taken from the post-topping leaves nos. 3, 4 and 5. As used herein, whenever reference is made to a comparison between leaves from two plants (e.g., a mutant plant and a control plant), leaves from the same or comparable tobacco leaf site (talk position) and developmental stage are intentional, such that the comparison may demonstrate an effect due to genotype differences and not from other factors. Illustratively, leaf 3 of the wild-type control plant is intended as a reference point to compare with leaf 3 of the pmt mutant plant. In one aspect, a tobacco plant comprising at least one pmt mutation is compared to a control tobacco plant of the same tobacco variety.

The nicotine or alkaloid level (or another leaf chemical or characteristic feature) of the tobacco plant can also be measured using alternative methods. In one aspect, the nicotine or alkaloid level (or another leaf chemical or property characteristic) of the tobacco plant is measured in the leaf with the highest nicotine or alkaloid level (or another leaf chemical or property characteristic) after topping. In one aspect, the nicotine or alkaloid level of the tobacco plant is measured in the leaf of number 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 or alkaloid level (or another leaf chemical or characteristic feature) of the tobacco plant is measured after topping in two or more leaves having a mixture of consecutive leaf numbers selected from the group consisting of leaf numbers 1, 2,3, 4,5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. In another aspect, the nicotine or alkaloid level (or another leaf chemical or property characteristic) of the tobacco plant is measured after topping in leaves having a leaf number selected from the group consisting of: 1 to 5,6 to 10, 11 to 15, 16 to 20, 21 to 25 and 26 to 30. In another aspect, the nicotine or alkaloid level (or another leaf chemical or property characteristic) of the tobacco plant is measured after topping in two or more leaves having a mixture of leaf numbers selected from the group consisting of: 1 to 5,6 to 10, 11 to 15, 16 to 20, 21 to 25 and 26 to 30. In another aspect, the nicotine or alkaloid level (or another leaf chemical or property characteristic) of the tobacco plant is measured after topping in three or more leaves having a mix of leaf numbers selected from the group consisting of: 1 to 5,6 to 10, 11 to 15, 16 to 20, 21 to 25 and 26 to 30.

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

The population of tobacco plants or collection of tobacco leaves used to determine the average measure (e.g., alkaloid or nicotine level 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 an industry recognized standard protocol.

As used herein, "topping" refers to the removal of the shoot tips, including SAM, flowers and up to several adjacent leaves, when the tobacco plant is near vegetative maturity and near the beginning of reproductive growth. Typically, tobacco plants are topped at the bud stage (shortly after flower initiation occurs). For example, tobacco plants grown in a greenhouse or field may be topped when 50% of the plants have at least one open flower. Topping tobacco plants results in loss of apical dominance and also induces an increase in alkaloid production.

Unless otherwise indicated, nicotine or alkaloid levels (or another leaf chemical or characteristic characterization) of tobacco plants were measured 2 weeks after topping. Alternatively, other points in time may be used. In one aspect, the nicotine or alkaloid level (or another leaf chemical or property 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 levels (or another leaf chemical or property characterization) of the tobacco plant are measured about 3, 5, 7, 10, 12, 14, 17, 19 or 21 days after topping.

As used herein, "similar growth conditions" or "equivalent growth 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 environmental conditions nor agronomic practices contribute to or account for 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, leaf cutting, undercutting, transplanting, topping and sprouting. See Chapters 4B and 4C of Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford (1999), pp 70-103.

"alkaloids" are complex nitrogen-containing compounds that occur naturally in plants and have pharmacological effects on humans and animals. "nicotine" is the major natural alkaloid in commercial cigarettes and comprises about 90% of the alkaloid content in tobacco. Other major alkaloids in tobacco include cotinine, nornicotine, macystine, diennicotinine, anabasine and anatabine. The minor alkaloid in tobacco comprises nicotine-N-oxide, N-methylanatabine, N-methylanabasine, pseudooxystrobin, 2, 3-bipyridine, etc.

Alkaloid levels can be determined by methods known in the art, for example by quantitation based on gas-liquid chromatography, high performance liquid chromatography, radioimmunoassay and enzyme-linked immunosorbent assay. For example, nicotine alkaloid levels can be measured by GC-FID methods based on CORESTA recommendation method No.7(1987) and ISO standards (ISO TC 126N 394E, see also Hibi et al, Plant Physiology 100:826-35(1992)) using gas-liquid chromatography equipped with a capillary column and FID detector.

Unless specifically stated otherwise, alkaloid and Nicotine levels are measured using a method defined in compliance with CORESTA method 62(Determination of Nicotine in Tobacco and Tobacco Products by Gas Chromatographic Analysis, month 2 2005) and in Protocol for Analysis of Nicotine, Total Moisture and pH in Smokeless Tobacco Products published by the American centers for disease control and prevention, 3.23.1999, Federal Register, volume 64, phase 55 (and revised 2009, 7.1.7.74, phase 4). Alternatively, Tobacco total alkaloids can be measured using a segmented flow colorimetry developed for analyzing Tobacco samples, adapted from Skalar Instrument Co (West Chester, Pa.) and described by Collins et al, Tobacco Science 13:79-81 (1969). In short, a sample of tobacco may be dried, ground and extracted prior to analysis for total and reducing sugars. The process then used acetic acid/methanol/water extraction and charcoal for decolorization. The total alkaloid content is determined based on: the reaction of cyanogen chloride with nicotine alkaloid in the presence of aromatic amine forms a colored complex measured at 460 nm. Unless otherwise indicated, levels of total alkaloids or nicotine indicated herein are on a dry weight basis (e.g., percent total alkaloids or percent nicotine).

In one aspect, the average nicotine or total alkaloid level of the tobacco plant is 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, the average nicotine or total alkaloid level of the tobacco plant is selected from the group consisting of: about 0.01% to 0.02%, 0.02% to 0.05%, 0.05% to 0.75%, 0.75% to 0.1%, 0.1% to 0.15%, 0.15% to 0.2%, 0.2% to 0.3%, 0.3% to 0.35%, 0.35% to 0.4%, 0.4% to 0.5%, 0.5% to 0.6%, 0.6% to 0.7%, 0.7% to 0.8%, 0.8% to 0.9%, 0.9% to 1%, 1% to 1.1%, 1.1% to 1.2%, 1.2% to 1.3%, 1.3% to 1.4%, 1.4% to 1.5%, 1.5% to 1.6%, 1.6% to 1.7%, 1.7% to 1.8%, 1.8% to 1.9%, 1.9% to 2%, 1.2% to 2.5%, 3.3%, 3% to 1.6%, 3.6% to 1.7%, 3.2% to 2%, 3.3.3% to 2%, 3.3% to 2%, 3.2% to 2%, 3.3% to 2.3%, 3.3% to 2% to 2.5%, 3.3%, 3%, 3.6% to 2% to 2.3.3.2% to 2%, 3% to 2.3.3.3%, 3.2% to 2% to 2.3%, 3%, 3.3.3.3.3.3%, 3% to 2% to 2.3%, 3%, 3.2% to 2% to 2.3.3%, 3.3%, 3%, 3.3. In a further aspect, the average nicotine or total alkaloid level of the tobacco plant is selected from the group consisting of: about 0.01% to 0.1%, 0.02% to 0.2%, 0.03% to 0.3%, 0.04% to 0.4%, 0.05% to 0.5%, 0.75% to 1%, 0.1% to 1.5%, 0.15% to 2%, 0.2% to 3%, and 0.3% to 3.5% dry weight.

The present disclosure also provides tobacco plants having altered nicotine levels without negatively affecting other tobacco traits (e.g., leaf grade index values). In one aspect, a low nicotine or nicotine-free tobacco variety provides a commercially acceptable grade of reconstituted tobacco. Tobacco grade is evaluated based on factors including, but not limited to: petiole location, leaf size, leaf color, uniformity and integrity of the leaf, maturity, texture, elasticity, luster (related to leaf color strength and depth, and luster), hygroscopicity (the ability of the tobacco leaf to absorb and retain ambient moisture), and nuances or off-colors of green. For example, official standards grades (7u.s.c. § 511) published by the agricultural market service of the united states department of agriculture may be used to determine leaf grades. See, e.g., Burley tobacco official standards ratings (US type 31 and foreign 93), effective at 11/5/1990 (55 F.R.40645); flue-cured tobacco official standard grades (U.S. types 11, 12, 13, 14 and foreign type 92), effective 3 months and 27 days in 1989 (54 f.r.7925); official standard grade of pennsylvania seed-leaf tobacco (us type 41), effective 1/8 days 1965 (29 f.r.16854); official standard grades of solanum torvum leaf tobacco, ohio (U.S. types 42, 43 and 44), effective 12 months and 8 days in 1963 (28f.r.11719 and 28 f.r.11926); official standard grades of cigar hybrid tobacco, wisconsin (us types 54 and 55), effective 11 months and 20 days 1969 (34 f.r.17061); official standard grades of cigar hybrid tobacco, wisconsin (us types 54 and 55), effective 11 months and 20 days 1969 (34 f.r.17061); official standard grades of cigar wrap tobacco for shade in georgia and florida (us type 62), effective in 1971 in 4 months. The USDA grade index value may be determined based on industry accepted grade indices. See, e.g., Bowman et al, Tobacco Science,32:39-40 (1988); traditional library of Tobacco literature (Bates document # 523267826) 523267833, 1.7.1988, Melorandum on the deployed Burley Tobacco Grade Index); and Miller et al, 1990, Tobacco Intern, 192:55-57 (all of the aforementioned references are incorporated by reference in their entirety). In one aspect, the USDA grade index is a 0-100 numerical representation of the received federal grade and is a weighted average of all tobacco leaf parts. A higher ranking index indicates a higher quality. Alternatively, the leaf rank may be determined by hyperspectral imaging. See, for example, WO 2011/027315 (published on 3/10/2011 and incorporated by reference in its entirety).

In one aspect, the tobacco plants provided herein comprise similar levels of one or more tobacco aroma compounds as compared to a control tobacco plant when grown under similar growth conditions. In another aspect, the tobacco plants provided herein comprise similar levels of one or more tobacco aroma compounds selected from the group consisting of: 3-methylvaleric acid, valeric acid, isovaleric acid, 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 measured 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-mass spectrometry. See "The Handbook of Plant Metabolomics" (Wiley-Blackwell) edited by Weckwerth and Kahl (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 a nicotine buffer in cigarette smoke by lowering smoke pH and effectively reducing the amount of "free" unprotonated nicotine. Reducing sugars balance smoke taste, for example, by altering the sensory impact of nicotine and other tobacco alkaloids. The inverse relationship between sugar content and alkaloid content due to planting conditions has been reported between various tobacco varieties, within the same variety, and within the same plant line. The level of reducing sugar can be measured using a step flow colorimetry developed by Skalar Instrument Co (West Chester, Pa.) and used to analyze Tobacco samples as described by Davis, Tobacco Science 20:139-144 (1976). For example, the sample is dialyzed against a sodium carbonate solution. Copper neocuprin reagent (Copper neocuprin) was added to the sample and the solution was heated. In the presence of sugar, the copper neocupron reagent chelate is reduced, forming a colored complex measured at 460 nm.

In one aspect, the tobacco plant comprises one or more non-naturally occurring mutant alleles at one or more PMT loci that reduce or eliminate PMT enzymatic activity at the one or more PMT loci. In one aspect, these mutant alleles result in lower nicotine levels. The mutant pmt alleles can be introduced by any method known in the art, including random or targeted mutagenesis methods.

Such mutagenesis methods 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 causes a 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. For example, the types of mutations that may be present in a tobacco gene include point mutations, deletions, insertions, duplications, and inversions. Such mutations are expected to be present in the coding region of the tobacco gene. However, mutations in the promoter region, and intron or untranslated regions of the tobacco gene may also be desirable.

Furthermore, a fast and automatable method for screening for chemically induced mutations, TILLING (directed induction of local mutations in the genome) is also suitable for the present disclosure, which uses denaturing HPLC or selective endonuclease digestion of selected PCR products. See McCallum et al (2000) nat. Biotechnol.18: 455-. Mutations that affect gene expression or interfere with the function of a gene 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 may be particularly effective in inhibiting protein function. In one aspect, the tobacco plant comprises a nonsense (e.g., stop codon) mutation of one or more PMT genes described herein.

It will be appreciated that when identifying mutations, the endogenous reference DNA sequence should be from the same variety of tobacco. For example, if the modified tobacco plant comprising the mutation is from variety TN90, the endogenous reference sequence must be an endogenous TN90 sequence, rather than a homologous sequence from a different tobacco variety (e.g., K326). Similarly, if the modified tobacco cell comprising the mutation is a TN90 cell, the endogenous reference sequence must be an endogenous TN90 sequence, rather than a homologous sequence of tobacco cells from a different tobacco variety (e.g., K326).

In one aspect, the present disclosure also provides a tobacco line having an altered nicotine level while maintaining a commercially acceptable leaf quality. This line can be generated by introducing mutations into one or more PMT genes via precise genome engineering techniques, such as transcription activator-like effector nucleases (TALENs), meganucleases, zinc finger nucleases, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 systems, CRISPR/Cpf1 systems, CRISPR/Csm1 systems, and combinations thereof (see, e.g., U.S. patent application publication 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 performed 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, as well as 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 the cited techniques are known.

In one aspect, the tobacco plant or plant genome provided herein is mutated or edited by an enzyme selected from the group consisting of: meganuclease, Zinc Finger Nuclease (ZFN), transcription activator-like effector nuclease (TALEN), CRISPR/Cas9 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, the edited nucleic acid sequences provided have at least 99.9%, at least 99.5%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80%, or at least 75% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs:1 to 10, and fragments thereof. In another aspect, the edited nucleic acid sequences provided have at least 99.9%, at least 99.5%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80%, or at least 75% sequence identity to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs:11 to 15.

Meganucleases, ZFNs, TALENs, CRISPR/Cas9, CRISPR/Csm1, and CRISPR/Cpf1 induce double-stranded DNA breaks at target sites of genomic sequences, which are then repaired by the natural process of Homologous Recombination (HR) or non-homologous end joining (NHEJ). Sequence modifications then occur at the 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 nuclease that functions 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 provided mutations result from genome editing using a nuclease. In another aspect, the provided mutations result from non-homologous end joining or homologous recombination.

Meganucleases, which are commonly identified in microorganisms, are unique enzymes with high activity and long recognition sequences (> 14bp) that result in site-specific digestion of target DNA. Engineered versions of naturally occurring meganucleases typically have extended DNA recognition sequences (e.g., 14 to 40 bp). Since the DNA recognition and cleavage functions of meganucleases are interwoven in a single domain, engineering of meganucleases can be more challenging than ZFNs and TALENs. 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 enzyme. ZFNs can be designed to cut double stranded DNA of almost any length for use in modifying zinc finger DNA binding domains. ZFNs form dimers from monomers consisting of the nonspecific DNA cleavage domain of FokI endonucleases fused to a zinc finger array engineered to bind a target DNA sequence.

The DNA binding domain of ZFNs is typically composed of 3-4 zinc finger arrays. The amino acids at the-1, +2, +3, and +6 positions relative to the start of the zinc finger ∞ -helix (which facilitate site-specific binding to the target DNA) can be varied and tailored to suit a particular target sequence. The other amino acids form a consensus backbone (consensus 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 C-terminal regions are required to bind opposite DNA strands of the cleavage site (5-7 bp apart). If both ZF binding sites are palindromic, the ZFN monomer can modify the target site. As used herein, the term "ZFN" is broad and includes monomeric ZFNs that can cleave double-stranded DNA without the aid of 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, it is theoretically possible to construct custom ZFNs to target almost any gene sequence. Publicly available methods for engineering zinc finger domains include context dependent assembly (CoDA), oligomer library 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. As used herein, the term TALEN is broad and includes monomeric TALENs that can cleave double-stranded DNA without the aid of 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 to almost any DNA sequence. TALE proteins are DNA binding domains derived from various plant bacterial pathogens of the genus xanthomonas. During infection, the xanthomonas pathogen secretes TALE into the host plant cell. 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 repeat 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 repetitive segments containing 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 distinct DNA binding domains for properly oriented and spaced sites 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 separate TALEN binding sites are both parameters for achieving 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.

The CRISPR/Cas9 system, CRISPR/Csm1, or CRISPR/Cpf1 system are alternatives to fokl-based methods, ZFNs, and TALENs. CRISPR systems are based on RNA-guided engineered nucleases that utilize 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 invasion by nucleic acids (such as viruses) by cleaving exogenous DNA in a sequence-dependent manner. Immunity is obtained by the integration of short fragments of the invading DNA, referred to as the spacer between two adjacent repeats proximal to the CRISPR locus. The CRISPR array (including the spacer sequence) is transcribed when it subsequently encounters invasive DNA and is processed into a small interference CRISPR RNA (cr RNA) of about 40nt in length, which binds to trans-activation CRISPR RNA (tracrRNA) to activate and guide the Cas9 nuclease. This will cleave the homologous double stranded DNA sequence in the invaded DNA, called the protospacer. A prerequisite for cleavage is the presence of a conserved prepro-spacer sequence adjacent motif (PAM) downstream of the target DNA, which usually has the sequence 5-NGG-3, but usually NAG is low. 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. Cpf1 and Csm1 function in a similar manner as Cas9, but Cpf1 and Csm1 do not require tracrRNA.

In yet another aspect, a tobacco plant provided herein comprises one or more pmt mutations, and further comprises one or more mutations in one or more loci encoding a nicotine demethylase (e.g., CYP82E4, CYP82E5, CYP82E10) that result in a reduced amount of nornicotine as compared to a control plant lacking the one or more mutations in the one or more loci encoding the 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, the described tobacco plants further comprise reduced nicotine demethylase activity when grown and conditioned under comparable conditions, as compared to control plants.

In one aspect, the pmt mutant tobacco plant further comprises a mutation that is capable of producing leaves with a lower level of anabasine than the levels of anabasine from leaves of a wild-type control tobacco plant when grown and processed under comparable conditions. In another aspect, the pmt mutant tobacco plant further comprises a mutation that is capable of producing leaves with a scouring alkali level that is 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70% or 80% lower than the scouring alkali level of leaves from a wild-type control tobacco plant, when grown and processed under comparable conditions.

In one aspect, the pmt mutant tobacco plant comprises a further mutation that is capable of producing a leaf having a greater than 2-fold reduction in anatabine levels when grown and processed under comparable conditions as compared to a leaf from a control tobacco plant. In another aspect, the pmt mutant tobacco plant comprises a further mutation that is capable of producing a leaf having a greater than 3, 4,5, 6, 7, 8,9, 10, 11, 12, or 13-fold reduction in anatabine levels as compared to a leaf from a wild-type control tobacco plant when grown and processed under comparable conditions. In one aspect, the mutations that provide lower levels of anatabine are the mutations described in U.S. application publication No. 2014/0283165 and U.S. application publication No. 2016/0010103. In another aspect, the pmt mutant further comprises a mutation in a Quinolinate Phosphoribosyltransferase (QPT) or Quinolinate Synthetase (QS) gene. In a further aspect, the pmt mutant plant further comprises a transgene or mutation that inhibits expression or activity of a QPT or QS gene.

In one aspect, the pmt mutant tobacco plant further comprises a mutation capable of providing a level of nornicotine that is less than 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, or 35% of the level of nornicotine from the leaves of a wild-type control tobacco plant, when grown and processed under comparable conditions.

In one aspect, the pmt mutant tobacco plant is capable of producing modulated leaves having a total N-nitrosodemethylated nicotine (NNN) level of: 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 0.05 ppm.

In another aspect, the pmt mutant tobacco plant is capable of producing modulated leaves having a total NNN level of: 2 to 0.05, 1.9 to 0.05, 1.8 to 0.05, 1.7 to 0.05, 1.6 to 0.05, 1.5 to 0.05, 1.4 to 0.05, 1.3 to 0.05, 1.2 to 0.05, 1.1 to 0.05, 1.0 to 0.05, 0.9 to 0.05, 0.8 to 0.05, 0.7 to 0.05, 0.6 to 0.05, 0.5 to 0.05, 0.4 to 0.05, 0.3 to 0.05, 0.2 to 0.05, 0.15 to 0.05, or 0.1 to 0.05 parts per million (ppm).

In one aspect, the pmt mutant tobacco plant is capable of producing cured leaves having a total nicotine-derived nitrosamine ketone (NNK) level of: 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 0.05 ppm.

In another aspect, the pmt mutant tobacco plant is capable of producing modulated leaves having a total NNK level of: 2 to 0.05, 1.9 to 0.05, 1.8 to 0.05, 1.7 to 0.05, 1.6 to 0.05, 1.5 to 0.05, 1.4 to 0.05, 1.3 to 0.05, 1.2 to 0.05, 1.1 to 0.05, 1.0 to 0.05, 0.9 to 0.05, 0.8 to 0.05, 0.7 to 0.05, 0.6 to 0.05, 0.5 to 0.05, 0.4 to 0.05, 0.3 to 0.05, 0.2 to 0.05, 0.15 to 0.05 or 0.1 to 0.05 ppm.

In one aspect, the pmt mutant tobacco plant further comprises a mutation or transgene that provides an increased level of one or more antioxidants. In another aspect, the pmt mutant tobacco plant further comprises a genetic modification in an endogenous gene encoding an antioxidant biosynthetic enzyme, a regulatory transcription factor for an antioxidant, an antioxidant transporter protein, an antioxidant metabolic enzyme, or a combination thereof, and further comprises an increased level of one or more antioxidants in the cured leaf as compared to a control cured leaf lacking the genetic modification. In a further aspect, the pmt mutant tobacco plant further comprises a transgene and further comprises an increased level of one or more antioxidants in the cured leaf as compared to a control cured leaf lacking the transgene, wherein the transgene encodes or directly modulates an antioxidant biosynthetic enzyme, a regulatory transcription factor for an antioxidant, an antioxidant transporter protein, an antioxidant metabolic enzyme, or a combination thereof. In one aspect, the pmt mutant tobacco plant further comprises a transgenic or homologous transgenic construct that expresses one or more genes selected from the group consisting of AtPAP1, NtAN2, NtAN1, NtJAF13, NtMyb3, chorismate mutase, and Aroate Dehydratase (ADT). In another aspect, the pmt mutant tobacco plant further comprises one or more transgenes or genetic modifications to increase antioxidants or decrease one or more TSNAs, as described in WIPO publication No. 2018/067985 or U.S. publication No. 2018/0119163.

In one aspect, the tobacco plants described are modified tobacco plants. As used herein, in the context of plants, "modified" refers to plants that comprise genetic alterations introduced for some purpose and beyond natural polymorphisms.

In one aspect, the tobacco plants described are homologous transgenic plants. 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, the provided plants, plant cells, or plant genomes are homologously transgenic. The provided homologous transgenic plants, plant cells, and plant genomes can result in a ready-to-use tobacco line. In another aspect, provided tobacco plants do not comprise non-tobacco genetic material or sequences.

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

In one aspect, provided tobacco plants comprise one or more pmt mutations, and further comprise reduced expression or activity of one or more genes involved in nicotine biosynthesis or transport. Genes involved in nicotine biosynthesis include, but are not limited to, Arginine Decarboxylase (ADC), methylcytosine oxidase (MPO), NADH dehydrogenase, Ornithine Decarboxylase (ODC), phosphoribosyl anthranilate isomerase (PRAI), Quinolinate Phosphoribosyltransferase (QPT), and S-adenosylmethionine synthetase (SAMS). Although two candidate genes have been proposed (a622 and NBB1), nicotine synthases catalyzing the condensation step between nicotinic acid derivatives and the methylpyrrolidium cation have not been elucidated. See US 2007/0240728 a1 and US 2008/0120737 a 1. A622 encodes an isoflavone reductase-like protein. In addition, some transport proteins may be involved in the transport of nicotine. A transporter gene named MATE has been cloned and characterized (Morita et al, PNAS 106:2447-52 (2009)).

In one aspect, provided tobacco plants comprise one or more pmt mutations, and further comprise a reduced level of mRNA, protein, or both of one or more genes encoding a product selected from the group consisting of: MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622 and NBB 1. In another aspect, provided are tobacco plants comprising one or more pmt mutations, and further comprising a transgene that directly inhibits the expression of one or more genes encoding a product selected from the group consisting of: MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622 and NBB 1. In another aspect, provided are tobacco plants comprising one or more pmt mutations, and 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: MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622 and NBB 1.

In one aspect, provided tobacco plants are from a tobacco type selected from the group consisting of: flue-cured, air-cured, dark-cured, Galpao and oriental. In another aspect, provided is a tobacco plant from the group consisting of tobacco types selected from the group consisting of: burley, maryland and dark tobacco.

In one aspect, provided are tobacco plants in a flue-cured tobacco background or exhibiting one or more of the flue-cured tobacco characteristics described herein. Flue-cured tobacco (also known as virginia or light-colored tobacco) accounts for approximately 40% of the world's tobacco production. Flue-cured tobacco is also commonly referred to as "light-coloured smoke" because it appears golden to dark orange during conditioning. The flue-cured tobacco has light fragrance and taste. Flue-cured tobacco generally has a high sugar content and a low oil content. The major tobacco-growing countries are argentina, brazil, china, india, tanzania and the united states. In one aspect, in a flue-cured tobacco context, a low alkaloid or low nicotine tobacco plant or seed provided is 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 variety derived primarily from any of the foregoing. In another aspect, in a flue-cured tobacco context, a low alkaloid or low nicotine tobacco plant or seed is provided that is 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 variety derived primarily from any of the foregoing. See WO 2004/041006 a 1. In a further aspect, the low alkaloid or low nicotine tobacco plant, seed, hybrid, variety, or line is selected from the group consisting of K326, K346, and NC196 in any flue-cured tobacco context.

In one aspect, provided tobacco plants are in an air-cured tobacco background or exhibit one or more air-cured tobacco characteristics described herein. Cured tobacco includes burley tobacco, maryland tobacco and dark tobacco. A common factor is that the conditioning is mainly performed without artificial heat source and humidity. Burley tobacco is from light to dark brown in color, high in oil content, and low in sugar content. The burley tobacco is aired in a curing barn. The major burley tobacco planting countries are argentina, brazil, italy, maraca and the united states. Maryland tobacco is very fluffy, and has good combustion characteristics, low nicotine and neutral fragrance. The major maryland countries include the united states and italy. In one aspect, a low alkaloid or low nicotine tobacco plant or seed provided is 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, Bu 21 xKy 10, HB04P, Ky 14xL8, Kt 200, Newton 98, Pedigo 561, Pf561 and Va 509. In a further aspect, the low alkaloid or low nicotine tobacco plant, seed, hybrid, variety, or line is selected from the group consisting of TN90, KT209, KT206, KT212, and HB4488 in any flue-cured tobacco background. In another aspect, a low alkaloid or low nicotine tobacco plant or seed is provided that is selected from the group consisting of Md 10, Md 40, Md 201, Md 609, Md 872 and Md 341 in a maryland tobacco background.

In one aspect, provided tobacco plants are on a dark tobacco-curing background or exhibit one or more dark tobacco-curing characteristics described herein. Dark air-cured tobacco differs from other types primarily in its formulation process, which imparts a medium to dark brown color and a unique aroma to dark air-cured tobacco. Dark air-cured tobacco is mainly used for producing chewing tobacco and tobacco containing tobacco. In one aspect, in a dark tobacco-curing background, provided low alkaloid or low nicotine tobacco plants or seeds are selected from the group consisting of: sumatra, Jatim, Dominican Cubano, Besuki, One packer, Green River, Virginia sun-cured tobacco, and Paraguan Passuado.

In one aspect, tobacco plants are provided that are under a dark smoking background or exhibit one or more of the dark smoking characteristics described herein. Dark smoke is typically smoked with low-fire wood on the floor of a closed brew house. Their leaves have low sugar content but high nicotine content. Dark flue-cured tobacco is used to make mixed pipe tobacco, cigarettes, chewing tobacco, cigars containing tobacco and having a strong taste. The major areas of planting for dark smoke are Tennessee, Kentucky and Virginia, USA. In one aspect, in a dark flue-cured tobacco background, a low alkaloid or low nicotine tobacco plant or seed is provided selected from the group consisting of: narrow leaf Madole, modified Madole, Tom Rosson Madole, Newton's VH Madole, Little Crittenden, Green Wood, Little Wood, Small Stack Black Mammooth, DT 508, DT 518, DT 592, KY171, DF911, DF 485, TN D94, TN D950, VA 309 and VA 359.

In one aspect, provided tobacco plants are in the context of oriental tobacco or exhibit one or more of the oriental tobacco characteristics described herein. Oriental tobaccos are also known as greek, aromatic and turkish tobaccos because they are commonly grown in the eastern midsea area, such as turkey, greek, bulgaria, mauton, syria, libamon, italy and romania. The plantlet and leaf size, as well as its unique aroma characteristics, characteristic of today's oriental varieties are the result of plants being adapted to poor soils and harsh climatic conditions in which they have been grown for many centuries. In one aspect, in the context of oriental tobacco, a low alkaloid or low nicotine tobacco plant or seed is provided 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, Balikesir, and any variety derived substantially from any of the foregoing.

In one aspect, the low alkaloid or low nicotine tobacco plant, seed, hybrid, variety, or line is derived from or within 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, K, 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, Madole, NC 100, NC 102, NC 2000, NC 291, NC 297, NC 3, NC 299, NC 5, NC 4, NC 5 NC 4, NC 3, NC 5, NC 3, NC 4, NC 3, NC 5, NC 5964, NC 3, NC 5, NC 5964, NC 3, NC 606, NC 5, NC 3, NC, Neal Smith Madole, OxFORD 207, 'Perique' tobaco, PVH03, PVH09, PVH19, PVH50, PVH51, R610, R630, R7-11, R7-12, RG 17, RG 81, RG H51, RGH 4, RGH 51, RS 1410, Speight 168, Speight 172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI 1269, KT 86, TN86LC, TN97, PD LC, PD D5, Speight 950, Speight H20, NF3, TI 1406, KT 86, TN 609 TN 80, TN97, PD 7348, PD 7327, TN 7327, TF LC, PLC 7327, TF 8626, PLC 7327, PLC 739, KT 27, KT 46K 46, PD 7353, PD 739, PLC 7327, PLC 739, PLC 7323, PD 27, PLC 7323, PLC 739, PLC 7348, PLC 739, PLC 7353, PLC 4153, PLC 739, or PLC 4153, PLC 4135, PLC 4153, PLC 4127, PLC 4135, PLC 4126, PLC 4153, PLC 4135, PLC 4127, PLC 4153, PLC 4135, PD 27.

All of the aforementioned dark air-curing, burley, maryland, dark smoking or oriental types of specific varieties are listed for exemplary purposes only. Any additional dark air-cured, white rib, maryland, dark smoked, oriental varieties are also contemplated in this application.

Populations of the described tobacco plants are also provided. In one aspect, the population of tobacco plants has the following planting density: about 5,000 to about 8,000, about 5,000 to about 7,600, about 5,000 to about 7,200, about 5,000 to about 6,800, about 5,000 to about 6,400, about 5,000 to about 6,000, about 5,000 to about 5,600, about 5,000 to about 5,200, about 5,200 to about 8,000, about 5,600 to about 8,000, about 6,000 to about 8,000, about 6,400 to about 8,000, about 6,800 to about 8,000, about 7,200 to about 8,000, or about 7,600 to about 8,000 plants per acre. In another aspect, the population of tobacco plants is in a low to medium fertility soil type.

Also provided are containers of the seeds from the tobacco plants. The container of tobacco seeds of the present process may 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 may 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. The container of tobacco seeds may be any container available in the art. As non-limiting examples, the container may be a box, a bag, a pouch, a coil, a tube, or a bottle.

Also provided are reconstituted tobacco materials made from the low alkaloid or low nicotine tobacco plants. Further provided is a reconstituted tobacco material made from the tobacco plant having higher levels of total alkaloids or nicotine.

"conditioning" is an alcoholization process that reduces moisture and destroys chlorophyll, giving the leaves a golden yellow colour, and by this process converts starch to sugar. Thus, the cured tobacco has a higher reducing sugar content and a lower starch content than harvested flue-cured tobacco. In one aspect, the provided flue-cured tobacco may be cured using conventional means, e.g., baking, barn curing, smoking, air curing, or sun curing. For a description of different types of modulation methods see, for example, Tso (1999, Chapter 1in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). Cured tobacco is typically alcoholized under compression conditions in a wooden barrel (e.g., pig-head barrel) or cardboard box for years (e.g., two to five years) with moisture content ranging from 10% to about 25%. See, U.S. patent nos. 4,516,590 and 5,372,149. The conditioned and alcoholized tobacco may then be further processed. Further processing includes warming and moistening the tobacco under vacuum with or without the introduction of steam at different temperatures, pasteurization and fermentation. Fermentation is generally characterized by high initial moisture content, heat generation and a loss of dry weight of 10% to 20%. 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 1in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). The conditioned, alcoholized, and fermented tobacco may be further processed (e.g., cut, shredded, expanded, or mixed). See, for example, U.S. patent nos. 4,528,993, 4,660,577, and 4,987,907. In one aspect, the reconstituted tobacco material of the present disclosure is sun-cured. In another aspect, the reconstituted tobacco material of the present disclosure is cured, air-cured, or smoked.

The presence of mold on cured tobacco can significantly reduce the quality and marketability (e.g., leaf grade) of cured leaves. Mold growth is a common problem that can occur during extended periods of high humidity (e.g., relative humidity greater than 70%) at temperatures of about 10 ℃ (50 ° F) to 32.2 ℃ (90 ° F). At higher temperatures, mold is more likely to be present.

Tobacco plants, varieties, and lines provided herein that comprise mutant alleles in one or more PMT genes, two or more PMT genes, three or more PMT genes, four or more PMT genes, or five PMT genes exhibit reduced fungal infection as compared to a low alkaloid tobacco variety, LA Burley 21(LA BU 21). Similarly, tobacco plants, varieties, and lines comprising RNAi constructs that down-regulate expression or translation of one or more PMT genes, two or more PMT genes, three or more PMT genes, four or more PMT genes, or five PMT genes provided herein exhibit reduced fungal infection as compared to low alkaloid tobacco variety LA Burley 21(LA BU 21).

LA BU 21 is a low total alkaloid tobacco line that is produced by the incorporation of low alkaloid genes from a variety of Cuba cigars into burley tobacco 21 by several backcrosses (Legg et al, Crop Science,10:212 (1970)). It has about 0.2% total alkaloids (dry weight), while the parent body burley 21 has about 3.5% total alkaloids (dry weight). LA BU 21 has a leaf grade well below the commercially acceptable standard.

In one aspect, cured tobacco comprising the mutant allele of pmt1a did not comprise observable mold infection. In another aspect, cured tobacco comprising the mutant allele of pmt1b did not comprise an observable mold infection. In another aspect, cured tobacco comprising the mutant allele of pmt2 did not comprise an observable mold infection. In another aspect, cured tobacco comprising the mutant allele of pmt3 did not comprise an observable mold infection. In another aspect, pmt4 is includedIs/are as followsCured tobacco leaves with mutant alleles do not contain observable fungal infections. In another aspect, cured tobacco comprising the mutant allele of pmt1a, the mutant allele of pmt1b, the mutant allele of pmt2, the mutant allele of pmt3, and the mutant allele of pmt4 does not comprise an observable mold infection.

In one aspect, cured tobacco comprising the mutant allele of pmt1a comprises reduced mold infection as compared to a control cured tobacco from variety LA BU 21. In another aspect, cured tobacco comprising the mutant allele of pmt1b comprises reduced mold infection as compared to a control cured tobacco from variety LA BU 21. In another aspect, cured tobacco comprising the pmt2 mutant allele comprises reduced mold infection as compared to a control cured tobacco from variety LA BU 21. In another aspect, cured tobacco comprising the pmt3 mutant allele comprises reduced mold infection as compared to a control cured tobacco from variety LA BU 21. In another aspect, cured tobacco comprising the pmt4 mutant allele comprises reduced mold infection as compared to a control cured tobacco from variety LA BU 21. In another aspect, a cured tobacco comprising the mutant allele of pmt1a, the mutant allele of pmt1b, the mutant allele of pmt2, the mutant allele of pmt3, and the mutant allele of pmt4 comprises reduced mold infection as compared to a control cured tobacco from variety LA BU 21.

In one aspect, a cured leaf from a tobacco plant, variety, or line provided in any of tables 4A-4E, table 10, or table 14 does not comprise an observable mold infection. In another aspect, a cured leaf from a tobacco plant, variety, or line provided in any of tables 4A-4E, 10, or 14 comprises reduced mold infection as compared to a control cured leaf from variety LA BU 21.

In one aspect, cured leaves from a tobacco plant, variety, or line comprising one or more pmt mutations provided in any one of tables 5A-5E and tables 12A-12E do not comprise an observable fungal infection. In another aspect, a cured leaf from a tobacco plant, variety, or line comprising one or more pmt mutations provided in any one of tables 5A-5E and tables 12A-12E comprises reduced fungal infection as compared to a control cured leaf from variety LA BU 21.

In one aspect, cured leaves from a tobacco plant, variety, or line comprising the mutant allele of pmt1a comprise a higher leaf grade than control cured leaves from variety LA BU 21. In one aspect, cured leaves from a tobacco plant, variety, or line comprising the mutant allele of pmt1b comprise a higher leaf grade than control cured leaves from variety LA BU 21. In one aspect, cured leaves from a tobacco plant, variety, or line comprising the mutant allele of pmt2 comprise a higher leaf grade than control cured leaves from variety LA BU 21. In one aspect, cured leaves from a tobacco plant, variety, or line comprising the mutant allele of pmt3 comprise a higher leaf grade than control cured leaves from variety LA BU 21. In one aspect, cured leaves from a tobacco plant, variety, or line comprising the mutant allele of pmt4 comprise a higher leaf grade than control cured leaves from variety LA BU 21. In another aspect, modulation leaves from a plant, variety, or line comprising a pmt1a mutant allele, a pmt1b mutant allele, a pmt2 mutant allele, a pmt3 mutant allele, and a pmt4 mutant allele comprise a higher leaf grade than control modulation leaves from variety LA BU 21.

In one aspect, cured leaf from a tobacco plant, variety, or line provided in any one of tables 4A-4E, table 10, or table 14 comprises a higher leaf grade than a control cured leaf from variety LA BU 21.

In one aspect, cured leaves from a tobacco plant, variety, or line comprising one or more pmt mutations provided in any one of tables 5A-5E and tables 12A-12E comprise a higher leaf grade than control cured leaves from variety LA BU 21.

In one aspect, "reduced mold infection" refers to a reduction in the area of infected leaves. In another aspect, "reduced mold infection" refers to a reduction in the number of mold spores that survive on the infected leaf. Standard methods for detecting and enumerating viable mold spores are known and available in the art.

In one aspect, the reduced mold infection comprises at least a 1% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced mold infection comprises at least a 2% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced mold infection comprises at least a 3% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced mold infection comprises at least a 4% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced mold infection comprises at least a 5% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced mold infection comprises at least a 10% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced mold infection comprises at least a 15% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced mold infection comprises at least a 20% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced mold infection comprises at least a 25% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced mold infection comprises at least a 30% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced mold infection comprises at least a 35% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced mold infection comprises at least a 40% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced mold infection comprises at least a 50% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced mold infection comprises at least a 60% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced mold infection comprises at least a 70% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced mold infection comprises at least a 75% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced mold infection comprises at least an 80% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced mold infection comprises at least a 90% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced mold infection comprises at least a 95% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced fungal infection comprises a 100% reduction in infected leaf area as compared to control leaf.

In one aspect, the reduced fungal infection comprises a 1% to 100% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced fungal infection comprises a 1% to 90% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced fungal infection comprises a 1% to 80% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced fungal infection comprises a 1% to 70% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced fungal infection comprises a 1% to 60% reduction in infected leaf area as compared to control leaves. In one aspect, the reduced fungal infection comprises a 1% to 50% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced fungal infection comprises a 1% to 40% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced fungal infection comprises a 1% to 30% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced fungal infection comprises a 1% to 20% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced fungal infection comprises a 1% to 10% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced fungal infection comprises a 10% to 100% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced fungal infection comprises a 20% to 100% reduction in infected leaf area as compared to control leaves. In one aspect, the reduced fungal infection comprises a 30% to 100% reduction in infected leaf area as compared to control leaves. In one aspect, the reduced fungal infection comprises a 40% to 100% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced fungal infection comprises a 50% to 100% reduction in infected leaf area as compared to control leaves. In one aspect, the reduced fungal infection comprises a 60% to 100% reduction in infected leaf area as compared to control leaves. In one aspect, the reduced fungal infection comprises a 70% to 100% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced fungal infection comprises an 80% to 100% reduction in infected leaf area as compared to control leaves. In one aspect, the reduced fungal infection comprises a 90% to 100% reduction in infected leaf area as compared to control leaves. In one aspect, the reduced fungal infection comprises a 10% to 75% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced fungal infection comprises a 25% to 75% reduction in infected leaf area as compared to control leaf. In one aspect, the reduced fungal infection comprises a 25% to 50% reduction in infected leaf area as compared to control leaves.

In one aspect, the genus of mold infecting cured tobacco is selected from the group consisting of cladosporium, penicillium, alternaria, aspergillus, and trichoderma.

Tobacco material obtained from a tobacco line, variety, or hybrid of the present disclosure can be used to make a tobacco product. As used herein, a "tobacco product" is defined as any product made from or derived from tobacco that is intended for human use or consumption.

Tobacco products provided include, but are not limited to, smoking products (e.g., cigarettes and bidi cigarettes), cigar products (e.g., cigar wrappers and cigarillos), pipe tobacco products, tobacco-derived nicotine products, smokeless tobacco products (e.g., wet tobacco powders, dry tobacco powders, and chewing tobacco), tobacco films, chewables, tabs, shaped parts, gels, consumable units, insoluble matrices, hollow shapes, reconstituted tobacco leaves, expanded tobacco leaves, 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 "rods" include cigarette paper, filters, plugwrap (for containing the filter material), tipping paper to secure the 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 rolled in cigarette paper), (3) liquor, (4) flavors, and (5) all other additives (which are blended into the tobacco and substitutes and rolled into the cigarette).

As used herein, "reconstituted tobacco" refers to a portion of tobacco filler, made from tobacco dust and other tobacco waste, processed into sheets and cut into strips to resemble tobacco. In addition to cost savings, reconstituted tobacco is also very important because it contributes to cigarette taste by processing flavor development using the reaction between ammonia and sugar.

As used herein, "expanded tobacco" refers to a portion of tobacco filler that is processed by expansion with a suitable gas such that the tobacco is "puffed", resulting in a reduction in density and greater 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 comprising a filter element, wherein the rod of smokable material comprises cured tobacco in a leaf formulation. In one aspect, a smoking article of the present disclosure is selected from the group consisting of: cigarillo, non-ventilated recess filter cigarette, cigar, tobacco containing, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, tobacco leaf, hookah tobacco, shredded tobacco and cut tobacco. In another aspect, the smoking article of the present disclosure is a smokeless tobacco product. Smokeless tobacco products do not burn and include, but are not limited to, chewing tobacco, moist smokeless tobacco, tobacco-containing, and dry tobacco powders. Chewing tobacco is a coarsely divided tobacco leaf, which is typically packaged in large bag-like packages and used in either cut tobacco strips (plugs) or rotary cut (twist). Moist smokeless tobacco is a moist, more finely divided tobacco that is provided in loose form or in a pouched form and is typically packaged in a round can, serving as a pouch or placed in a pocket between the cheek and gums of an adult tobacco consumer. The tobacco is a heat-treated smokeless tobacco. Dry tobacco powder is finely ground tobacco for oral or nasal use. In a further aspect, the tobacco product of the present disclosure is selected from the group consisting of loose chewing tobacco, tobacco rod chewing tobacco, wet tobacco powder, and snuff. In yet another aspect, the smoking article of the present disclosure is selected from the group consisting of an electronic heated cigarette, an electronic vaporization device.

In one aspect, the smoking article of the present disclosure can be a blended smoking article. In another aspect, the smoking article of the present disclosure can be a low 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 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, the average nicotine or total alkaloid level of a cured tobacco material or tobacco product provided is 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% on a dry weight basis. In another aspect, the average nicotine or total alkaloid level of a cured tobacco material or tobacco product provided is selected from the group consisting of: about 0.01% to 0.02%, 0.02% to 0.05%, 0.05% to 0.75%, 0.75% to 0.1%, 0.1% to 0.15%, 0.15% to 0.2%, 0.2% to 0.3%, 0.3% to 0.35%, 0.35% to 0.4%, 0.4% to 0.5%, 0.5% to 0.6%, 0.6% to 0.7%, 0.7% to 0.8%, 0.8% to 0.9%, 0.9% to 1%, 1% to 1.1%, 1.1% to 1.2%, 1.2% to 1.3%, 1.3% to 1.4%, 1.4% to 1.5%, 1.5% to 1.6%, 1.6% to 1.7%, 1.7% to 1.8%, 1.8% to 1.9%, 1.9% to 1.4%, 1.4% to 2%, 3.5% to 1.6%, 1.6% to 1.7%, 1.7% to 2%, 3.2% to 2%, 3.3.3% to 2%, 3.3% to 2%, 3.2% to 2.3%, 3.3.3%, 3% to 2%, 3.2.3.3%, 3% to 2% to 2.3.3.3%, 3%, 3.2% to 2% to 2.3%, 3.3.3%, 3.3.3.3.3%, 3% to 2% to 2.3%, 3%, 3.2% to 2% to 2.3%, 3.3%, 3%, 3.3.3%, 3. In a further aspect, the average nicotine or total alkaloid level of a cured tobacco material or tobacco product provided is selected from the group consisting of: about 0.01% to 0.1%, 0.02% to 0.2%, 0.03% to 0.3%, 0.04% to 0.4%, 0.05% to 0.5%, 0.75% to 1%, 0.1% to 1.5%, 0.15% to 2%, 0.2% to 3%, and 0.3% to 3.5% on a dry weight basis.

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. Incubation may 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 a F1 hybrid plant or further cross a F1 hybrid plant with other donor plants having an agronomically desirable genotype at F₂Or generation of backcross. F can be screened using one of the techniques known in the art or listed herein₂Or a desired agronomic trait or a desired chemical profile of a plant in a backcross generation. Depending on the expected genetic pattern or MAS technique used, the 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 can be a baked variety, burley tobacco variety, dark air-cured variety, dark smoked variety, or oriental variety. Other breeding techniques may be found In, for example, Wernsman, E.A., and Rufty, R.C.1987 Chapter Seven. Tobacco. pages 669. 698 In: Current development. crop specifications. W.H.Fehr (ed.), MacMillan Publishing Go., Inc., New York, N.Y., which are incorporated herein by reference In their entirety.

The results of plant breeding programs using the 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 that share constant characteristics that separate them from other plants of the same species. Although not always the case, varieties are often sold commercially. A further feature of a variety, albeit with one or more distinguishing traits, is that the overall variation between individuals within the variety is very small. "pure line" varieties can be produced by self-pollination and selection for several generations, or vegetative propagation from a single parent using tissue or cell culture techniques. A breed may be derived essentially from another line or breed. As defined by the international convention for the protection of new varieties of plants (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, the varieties "substantially derive" from the original varieties if: a) it is derived primarily from the original variety, or from a variety derived primarily from the original variety, while retaining expression of the essential features resulting from the genotype or combination of genotypes of the original variety; b) it is clearly different from the original variety; and c) in addition to the differences resulting from the derivation behavior, it corresponds to the original breed in the expression of the essential characteristic caused by the genotype or combination of genotypes of the original breed. For example, substantially derived varieties may be obtained by selecting natural or induced mutants, somaclonal variants, variant individuals from the original variety of plants, backcrosses or transformations. 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 one aspect, the disclosure provides a tobacco plant, variety, line, or cell comprising one or more pmt mutations provided in any one of tables 5A-5E and tables 12A-12E.

In another aspect, the present disclosure provides a tobacco plant, variety, line, or cell derived from any of the tobacco plants, varieties, or lines provided in any of tables 4A-4E, table 10, or table 14.

In one aspect, the disclosure provides tobacco line 18GH203, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH341, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1678,and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line 17GH1680, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1804, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1898, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH207, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH342, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH343, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH348, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH349, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH355, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH359, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH64, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH682, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH692, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. At one isIn one aspect, the disclosure provides tobacco line 18GH697, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH922, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH957, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1808, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1810, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1886, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1888 and, F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1889, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH189, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1893, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1901, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1902, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH3, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH125, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH208, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH403, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH414, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH434, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line 18GH436, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH437, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH449, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH706, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH709, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH710, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH716, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH729, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH731, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco lines18GH752, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH756, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH768, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH771, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH776, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH800, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH818, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH10, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH1004, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH1033, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH132, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH134, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH217, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH456, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant.In one aspect, the disclosure provides tobacco line 18GH457, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH460, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH465, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH71, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH830, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH831, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH836, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line 18GH841, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH974, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH981, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH994, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1905, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH128, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH130, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH131, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH133, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH136, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH216, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH227, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH5, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH6, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH65, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH66, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH69, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH72, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH73, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH74, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH78, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH79, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH8, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH9, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line 17GH1696, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1717, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1719, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1729, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1736, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1737, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1739, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1740, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1835, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1848, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant.In one aspect, the disclosure provides tobacco line 17GH1849, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1912, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1937, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1940, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1943, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1944, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH1051, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH22, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH34, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH473, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH49, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH50, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH848, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH850, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH851, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line 17GH1699, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1708, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1722, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1724, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1725, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1845, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1846, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1847, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1911, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1912, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1915, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1918, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosureTobacco line 17GH1928, and F derived therefrom, are provided₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1932, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1933, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1936, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH20, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH28, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH31, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH47, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH51, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH52, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line CS107, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line CS106, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line CS115, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1809-13, and F derived therefrom₁Or F₂Tobacco plant or male sexAnd (4) breeding tobacco plants. In one aspect, the disclosure provides tobacco line CS111, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line CS112, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1678-60, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line CS131, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH709-01, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH709-08, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH414-11, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH414-19, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH437-04, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH437-08, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH437-32, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH437-39, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line 18GH449-26, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH449-33, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH125-48, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line CS102, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line CS103, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1719-30, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1740-36, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line 17GH1698-22, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1700-13, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1702-17, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1849-01, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1849-48, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 17GH1737-24, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line CS118, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line CS133, and F derived therefrom₁Or F₂Tobacco plants orA male sterile tobacco plant. In one aspect, the disclosure provides tobacco line CS120, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH1108-07, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line 18GH2162, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line CS164, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line CS163, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line CS146, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line CS147, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line CS150, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line CS151, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line CS148, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line CS149, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line CS152, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line CS153, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line CS143, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line 18GH2169, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco strain 18GH2171, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line CS165, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the present disclosure provides tobacco line CS118, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant. In one aspect, the disclosure provides tobacco line 18GH2254-7, and F derived therefrom₁Or F₂A tobacco plant or a male sterile tobacco plant.

In one aspect, the present disclosure discloses a method of introducing a low nicotine trait into a tobacco variety, the method comprising: (a) crossing a first tobacco variety comprising a low-nicotine trait with a second tobacco variety not comprising the low-nicotine trait to produce one or more progeny tobacco plants; (b) genotyping one or more progeny tobacco plants for a pmt mutant allele selected from those listed in tables 4A to 4E, tables 5A to 5E, table 10, and tables 12A to 12E; and (c) selecting a progeny tobacco plant comprising the pmt mutant allele. In another aspect, the methods further comprise backcrossing the selected progeny tobacco plant with a second tobacco variety. In a further aspect, the methods further comprise: (d) crossing the selected progeny plant with itself or with a second tobacco variety to produce one or more further progeny tobacco plants; and (e) selecting a further progeny tobacco plant comprising the low nicotine trait. In one aspect, the step (e) of selecting comprises marker assisted selection. In one aspect, the methods result in a single gene transformation comprising a low nicotine trait. In one aspect, these methods result in a single gene transformation comprising a pmt mutant allele. In one aspect, the second tobacco variety is a elite variety. In another aspect, the genotyping step of these methods involves the determination of one or more molecular markers. In another aspect, genotyping may involve a polymorphic marker comprising a polymorphism selected from the group consisting of: single Nucleotide Polymorphisms (SNPs), insertions or deletions in DNA sequences (indels), simple sequence repeats of DNA sequences (SSRs), Restriction Fragment Length Polymorphisms (RFLPs), and tag SNPs.

As used herein, a "locus" is a chromosomal locus or region in which a polymorphic nucleic acid, trait determinant, gene, or marker is located. A "locus" may be shared by two homologous chromosomes to refer to their respective loci or regions. As used herein, "allele" refers to an alternative nucleic acid sequence at a gene or a particular locus (e.g., a nucleic acid sequence of a gene or locus that is different from other alleles of the same gene or locus). An allele can be considered to be (i) wild-type or (ii) mutant if there is one or more mutations or edits in the nucleic acid sequence of the mutant allele relative to the wild-type allele. A mutant allele of a gene may have reduced or eliminated gene activity or expression levels relative to a wild-type allele. For diploid organisms, such as tobacco, the first allele may occur on one chromosome and the second allele may occur at the same locus on a second homologous chromosome. A plant is described as being heterozygous for a mutant allele if one allele at a locus on one chromosome of the plant is the mutant allele and the other corresponding allele on a homologous chromosome of the plant is wild-type. However, if both alleles at a locus are mutant alleles, then the plant is described as being homozygous for the mutant allele. A plant homozygote for a mutant allele at a locus may contain the same mutant allele or different mutant alleles (if heteroallelic or biallelic).

As used herein, "introduced" or "introducing … …" refers to the transmission of a desired allele of a genetic locus from one genetic background to another.

As used herein, "crossed" or "cross" refers to the production of progeny by fertilization (e.g., a cell, seed, or plant) and includes crosses between a plant (sexual) and self-fertilization (selfing).

As used herein, "backcross (backcross)" and "backcrossing" refer to the process by which a progeny plant is repeatedly crossed back to one of its parents. In a backcrossing scheme, the "donor" parent refers to the parent plant having the desired gene or locus to be introduced. The "recipient" parent (used one or more times) or "recurrent" parent (used two or more times) refers to the parent plant into which the gene or locus is introduced. Initial hybridization yielded generation F1. The term "BC 1" refers to the second use of the recurrent parent, "BC 2" refers to the third use of the recurrent parent, and so on. In some aspects, backcrossing is performed iteratively, wherein the progeny individuals of each successive backcross generation are themselves backcrossed to the same parental genotype.

As used herein, "single-gene transformed" or "single-gene transformation" refers to a plant developed using a plant breeding technique known as backcrossing or by genetic engineering, wherein substantially all of the desired morphological and physiological characteristics of the variety are restored, except for a single gene transferred to the variety by the backcrossing technique or by genetic engineering.

As used herein, "elite variety" means any variety resulting from breeding and selection that is superior in agronomic performance.

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

As used herein, the term "trait" refers to one or more detectable characteristics of a cell or organism that can be affected by a genotype. The phenotype may be observed by the naked eye, or by any other means of assessment known in the art, such as microscopy, biochemical analysis, genetic analysis, determination of specific disease tolerance, and the like. In some cases, the phenotype is directly controlled by a single gene or genetic locus, e.g., a "monogenic trait". In other cases, the phenotype is the result of several genes.

As used herein, "marker assay" means a method for detecting a polymorphism at a particular locus using a particular method, e.g., measuring at least one phenotype such as seed color, flower color, or other visually detectable trait, Restriction Fragment Length Polymorphism (RFLP), single base extension, electrophoresis, sequence alignment, allele-specific oligonucleotide hybridization (ASO), random amplified polymorphic dna (rapd), microarray-based techniques, and nucleic acid sequencing techniques, among others.

As used herein, "marker assisted selection" (MAS) is a process of selecting a phenotype based on the genotype of a marker. "marker assisted selective breeding" refers to the process of selecting for a desired trait or traits in a plant or plants by detecting one or more nucleic acids from the plant, wherein the nucleic acids are associated with the desired trait, and then selecting for plants or germplasm having those nucleic acid or nucleic acids.

As used herein, "polymorphism" refers to the presence of one or more variation in a population. Polymorphisms can manifest as variations in the nucleotide sequence of a nucleic acid or variations in the amino acid sequence of a protein. Polymorphisms include the presence of one or more variations in a nucleic acid sequence or nucleic acid characteristic at one or more loci in a population of one or more individuals. Variations may include, but are not limited to, alterations of one or more nucleotide bases, insertions of one or more nucleotides, or deletions of one or more nucleotides. Polymorphisms can arise from random processes in nucleic acid replication as a result of moving genomic elements, from copy number variations, and during meiosis processes (such as unequal crossover, genome replication, and chromosome fragmentation and fusion) through mutations. Variations can be commonly found within a population or may exist at a low frequency, the former having greater utility in general plant breeding, while the latter may be associated with rare but important phenotypic variations. Useful polymorphisms may include Single Nucleotide Polymorphisms (SNPs), insertions or deletions in DNA sequences (indels), simple sequence repeats of DNA sequences (SSRs), Restriction Fragment Length Polymorphisms (RFLPs), and tag SNPs. Genetic markers, genes, DNA-derived sequences, RNA-derived sequences, promoters, 5 'untranslated regions of genes, 3' untranslated regions of genes, micrornas, sirnas, tolerance loci, satellite markers, transgenes, mrnas, ds mrnas, transcription profiles, and methylation patterns can also comprise polymorphisms. In addition, the presence, absence or variation of the aforementioned copy number may comprise a polymorphism.

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

As used herein, a "marker" or "molecular marker" or "marker locus" is a term used to refer to a nucleic acid or amino acid sequence sufficient to uniquely characterize a particular locus on a genome. Any detectable polymorphic trait may be used as a marker so long as it is differentially inherited and exhibits linkage disequilibrium with the phenotypic trait of interest. Thus, each marker is indicative of a specific segment of DNA, having a unique nucleotide sequence. The mapped positions provide a measure of the relative position of the particular markers with respect to each other. When a trait is described as being associated with a given marker, it will be understood that the actual DNA fragment whose sequence affects the trait is typically co-segregating with the marker. If markers are identified on both sides of the trait, a more accurate and more precise localization of the trait can be obtained. By measuring the presence of the marker in progeny of a cross, the presence of the trait can be detected by a relatively simple molecular test, without the need to actually assess the presence of the trait itself (which can be difficult and time consuming), as the actual assessment of the trait requires that the plant be grown to a stage and/or environmental conditions in which the trait can be expressed.

It is to be understood that any of the tobacco plants of the present disclosure may further comprise additional agronomically desirable traits, for example, by transformation with a genetic construct or a 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; modulation property; modulating quality; mechanical harvestability; holding ability; leaf mass; height, plant maturity (e.g., early to medium, medium to late, or late); handle size (e.g., small, medium, or large handle); or the number of leaves 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, the disclosed low-or nicotine-free tobacco plants or seeds comprise one or more transgenes expressing one or more insecticidal proteins, such as, for example, a crystallin protein of Bacillus thuringiensis (Bacillus cereus) or a plant insecticidal protein of Bacillus cereus, such as VIP3 (see, e.g., estuch et al, (1997) nat biotechnol.15:137) in another aspect, the tobacco plant further comprises a trait that confers resistance to brown rot (U.S. patent No.5,689,035) or resistance to nematodes (U.S. patent No.5,491,081) introduced.

The disclosure also provides pmt mutant tobacco plants comprising altered nicotine or total alkaloid levels, but yields comparable to those of a corresponding initial tobacco plant without such altered nicotine levels. In one aspect, the pmt mutant variety provides a yield selected from the group consisting of: about 1200 to 3500, 1300 to 3400, 1400 to 3300, 1500 to 3200, 1600 to 3100, 1700 to 3000, 1800 to 2900, 1900 to 2800, 2000 to 2700, 2100 to 2600, 2200 to 2500, and 2300 to 2400 lbs/acre. In another aspect, the pmt mutant tobacco variety provides a yield selected from the group consisting of: about 1200 to 3500, 1300 to 3500, 1400 to 3500, 1500 to 3500, 1600 to 3500, 1700 to 3500, 1800 to 3500, 1900 to 3500, 2000 to 3500, 2100 to 3500, 2200 to 3500, 2300 to 3500, 2400 to 3500, 2500 to 3500, 2600 to 3500, 2700 to 3500, 2800 to 3500, 2900 to 3500, 3000 to 3500 and 3100 to 3500 lbs/acre. In a further aspect, the pmt mutant tobacco plant provides a yield of 65% to 130%, 70% to 130%, 75% to 130%, 80% to 130%, 85% to 130%, 90% to 130%, 95% to 130%, 100% to 130%, 105% to 130%, 110% to 130%, 115% to 130%, or 120% to 130% of the yield of a control plant having substantially the same genetic background except for the pmt mutation. In a further aspect, the pmt mutant tobacco plant provides a yield that is 70% to 125%, 75% to 120%, 80% to 115%, 85% to 110%, or 90% to 100% of the yield of a control plant that has substantially the same genetic background except for the pmt mutation.

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

In one aspect, the disclosed tobacco plants comprise a modification that confers a desired trait (e.g., low nicotine, no nicotine, or low alkaloid), and further comprise a trait that is substantially equivalent to an unmodified control plant, wherein the trait is selected from the group consisting of: yield, maturity and senescence, susceptibility to insect feeding, polyamine content after topping, chlorophyll levels, mesophyll cell number per unit leaf area and final product quality after conditioning.

In one aspect, the disclosed tobacco plants comprise modifications that confer desirable traits (e.g., low nicotine, no nicotine, or low alkaloid), and further comprise the following yields: yield relative to unmodified control plants is greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 100%, greater than 105%, greater than 110%, greater than 115%, greater than 120%, greater than 125%, greater than 130%, greater than 135% or greater than 140%. In one aspect, the disclosed tobacco plants comprise modifications that confer desirable traits (e.g., low nicotine, no nicotine, or low alkaloid), and further comprise the following yields: yield relative to unmodified control plants of 70% to 140%, 75% to 135%, 80% to 130%, 85% to 125%, 90% to 120%, 95% to 115% or 100% to 110%. In one aspect, the disclosed tobacco plants comprise modifications that confer desirable traits (e.g., low nicotine, no nicotine, or low alkaloid), and further comprise the following yields: yield relative to unmodified control plants of 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, 90% to 100%, 95% to 105%, 105% to 115%, 110% to 120%, 115% to 125%, 120% to 130%, 125% to 135%, or 130% to 140%.

In one aspect, the disclosed low nicotine or nicotine-free tobacco varieties are suitable for machine harvesting. In another aspect, disclosed low nicotine or nicotine-free tobacco varieties are mechanically harvested.

In one aspect, the tobacco plant provided is a hybrid plant. Hybrids can be generated by: preventing self-pollination of a female parent plant of a first variety (e.g., the seed parent), allowing pollen from a male parent plant of a second variety to fertilize the female parent plant, and allowing the formation of F1 hybrid seed on the female plant. Self-pollination of female plants can be prevented by emasculating the flowers during the early stages of flower development. Alternatively, a form of male sterility can 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 (where the transgene inhibits microsporogenesis and/or pollen formation) or self-incompatibility. Female parent plants containing MS are particularly useful. In aspects in which the maternal plant is MS, pollen may be harvested from a male fertile plant and applied manually to the stigma of the MS maternal plant, and the resulting F1 seed harvested.

Plants can be used to form single cross tobacco F1 hybrids. Pollen from a paternal plant is manually transferred to a castrated maternal plant or a male sterile maternal plant to form F1 seeds. Alternatively, triple crossing may be performed, in which a single cross F1 hybrid is used as the female parent and crossed with a different male parent. Alternatively, a double cross hybrid can be generated in which two different single cross F1 progeny cross themselves. Particularly advantageously, self-incompatibility can be used to prevent self-pollination of the female parent when forming a double cross hybrid.

In one aspect, the low nicotine or nicotine-free tobacco variety is male sterile. In another aspect, the low nicotine or nicotine-free tobacco variety is cytoplasmic male sterile. Male sterile tobacco plants can be produced by any method known in the art. Methods for producing male sterile tobacco are described In Wernsman, E.A., and Rufty, R.C.1987.Chapter Seven. Tobacco. pages 669. 698 In: Cultivar development. crop specifications. W.H.Fehr.,. MacMillan Publishing Go., Inc., New York, N.Y.761pp.

In one aspect, the present disclosure provides a male sterile tobacco plant, variety, or line comprising one or more pmt mutations provided in any one of tables 5A to 5E and tables 12A to 12E.

In another aspect, the present disclosure provides a male sterile tobacco plant, variety or line derived from any of the tobacco plants, varieties or lines provided in any one of tables 4A to 4E, table 10 or table 14.

In one aspect, the present disclosure provides male sterile line dCS 11. In another aspect, the present disclosure provides male sterile line dCS 12. In another aspect, the present disclosure provides male sterile line dCS 13. In another aspect, the present disclosure provides male sterile line dCS 14. In another aspect, the present disclosure provides male sterile line dCS 15. In another aspect, the present disclosure provides male sterile line dCS 16. In another aspect, the present disclosure provides male sterile line dCS 17. In another aspect, the present disclosure provides male sterile line dCS 18. In another aspect, the present disclosure provides a male sterile line dS 697.

In a further aspect, the tobacco 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, the provided tobacco portion does 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 present disclosure provides tobacco plant cells, tissues and organs that are unable to sustain themselves through photosynthesis. In another aspect, the present disclosure provides a somatic tobacco plant cell. In contrast to propagating cells, somatic cells do not mediate plant propagation.

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

It is understood by those skilled in the art that tobacco plants are naturally propagated by seeds, not by vegetative or vegetative propagation. In one aspect, the present disclosure provides tobacco endosperm. In another aspect, the present disclosure provides a tobacco endosperm cell. In a further aspect, the present disclosure provides a male or female sterile tobacco plant that is incapable of reproduction without human intervention.

In one aspect, the disclosure provides a nucleic acid molecule comprising at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs:1 to 10, and fragments thereof. In one aspect, the disclosure provides a polypeptide or protein comprising at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:11 to 15.

As used herein, the term "sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refers to the residues in the two sequences that are the same when aligned for maximum correspondence over a particular comparison window. When percentage of sequence identity is used in reference to proteins, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, wherein other amino acid residues having similar chemical properties (e.g., charge or hydrophobicity) are substituted for the amino acid residue, and thus do not alter the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted up to correct for the conservative nature of the substitution.

The present disclosure further provides a method of making a tobacco product comprising a tobacco material from the disclosed tobacco plant. In one aspect, a method includes warming and moisturizing an alcoholized tobacco material prepared from tobacco plants to increase its moisture content from about 12.5% to about 13.5% to about 21%, mixing the warmed and moisturized tobacco material to produce a desired mixture. In one aspect, the method of making a smoking article further comprises flavoring or perfuming the mixture. Typically, a feed solution or flavoring material is added to the mixture during the addition process to improve its quality by balancing the chemical composition and to develop certain desirable flavor characteristics. Further details regarding 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 materials can also be processed using methods including, but not limited to, heat treatment (e.g., curing, brewing), flavoring, enzymatic treatment, expansion, and/or brewing. Fermented and unfermented tobaccos can be processed using these techniques. Examples of suitable processed tobacco include dark air-cured, dark smoked, burley, baked and cigar core or coat, and products from whole leaf destemming operations. In one aspect, the tobacco fiber comprises up to 70% dark smoke by fresh weight. For example, as described in U.S. publication Nos. 2004/0118422 or 2005/0178398, tobacco may be conditioned by heating, fermenting (sweasting), and/or pasteurizing steps.

The provided tobacco material can undergo fermentation. In general, fermentation is characterized by high initial moisture content, heat production, and a loss of dry weight of 10-20%. See, for example, U.S. patent nos. 4,528,993, 4,660,577, 4,848,373 and 5,372,149. In addition to altering the aroma of the leaves, fermentation can also alter the color, texture, or both of the leaves. Also, during the fermentation process, 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., Blackwell Publishing, Oxford). The cured, or cured and fermented, tobacco can be further processed (e.g., shredded, puffed, mixed, ground, or comminuted) prior to introduction into the oral article. In some cases, the tobacco is long cut fermented reconstituted wet tobacco having an oven volatiles content of 48 to 50 weight percent prior to mixing with the copolymer and optional flavoring and other additives.

In one aspect, the provided tobacco material can be processed to a desired size. In one aspect, the tobacco fibers can be processed to 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 shredded or chopped into a width of about 10 to about 110 filaments/inch and a length of about 0.1 inch up to about 1 inch. The bi-cut tobacco fibers can have a range of particle sizes such that about 70% of the bi-cut tobacco fibers fall within a mesh size of-20 mesh to 80 mesh.

The provided tobacco material can be processed to have about 10% by weight or greater; about 20 wt% or greater; about 40 wt% or greater; about 15 wt% to about 25 wt%; about 20 wt% to about 30 wt%; about 30 wt% to about 50 wt%; a total oven volatiles content of 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 generally 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 the sample after drying at 110 ℃ for 3.25 hours in a preheated, forced air oven. The oral article can have a different overall oven volatiles content as compared to the oven volatiles content of the tobacco fiber used to make the oral article. The described processing steps can reduce or increase oven volatiles content.

Having now generally described the 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 limit the disclosure unless otherwise specified.

Examples of the invention

Example 1: expression profiles of five PMT genes.

Biosynthesis of nicotine begins with conversion of polyamine putrescine to N-methyl putrescine by putrescine N-methyltransferase (PMT). This is the step of participating the precursor metabolite in nicotine biosynthesis. Genes encoding PMT (PMT1a, PMT1b, PMT2, PMT3, and PMT4) are present in the tobacco (Nicotiana tabacum) genome. The genomic DNA sequence, cDNA sequence and protein sequence of the five PMT genes are listed in table 1A. Table 1B and 1C provide sequence identity between the five PMT genes. The mixed expression levels from pre-topping to harvest provided support that, without being limited to any particular theory, PMT1a and PMT3 represent the two major PMT genes (fig. 1).

Table 1A: the sequence of five tobacco PMT genes.

Table 1B: cDNA sequence identity between five tobacco PMT genes as determined by Clustal 2.1

Table 1C: protein sequence identity between five tobacco PMT genes as determined by Clustal 2.1

Table 1D: PMT1b genomic sequence (SEQ ID No.1) annotation.

Table 1E: PMT1b genomic sequence (SEQ ID No.2) annotation.

Table 1F: PMT2 genome sequence (SEQ ID No.3) annotation.

Table 1G: PMT3 genome sequence (SEQ ID No.4) annotation.

Table 1H: PMT4 genome sequence (SEQ ID No.5) annotation.

Example 2: PMT genome editing and tobacco strain development

PMT knock-out mutants were generated by editing various PMT genes. Tobacco protoplasts are transfected with polyethylene glycol (PEG) using plasmids encoding the genome editing technology 1(GET 1) protein or the Genome Editing Technology (GET)2 protein and specific targeting rnas (grnas) targeting PMT genes at desired locations. Table 2 lists gRNA sequences used for PMT editing. Some grnas (e.g., nos.6 and 7) were mixed together for targeting multiple PMT genes in a single transfection.

Then, the transfected protoplasts were immobilized in 1% agarose beads and subjected to tissue culture. When calli were grown to about 1mm in diameter, they were plated on TOM2 plates. The calli were screened for insertions or deletions (indels) at the target location using fragment analysis. Candidates showing size shift compared to the wild-type control were selected for further culture and subsequent shoots were tested again by fragment analysis to confirm the presence of indels. Rooted shoots are potted and the target sites sequenced to determine the exact sequence of the deletion. Young leaves from each plant were harvested and PCR amplification of PMT fragments was performed using phirekit. PMT libraries for each line were indexed and 384 lines were pooled and sequenced using Miseq.

SNP analysis was performed to determine the exact edited PMT mutant allele sequence and the zygotic status at each PMT locus. Table 3 provides zygotic information for representative edited plants. Tables 4A through 4E provide indel sequence information for each edited line of various tobacco varieties (e.g., K326, TN90, NLM, oriental). Tables 5A through 5E provide genomic sequences of about 40 nucleotides from each pmt mutant allele, with the site of editing located in the middle of the genomic sequence (e.g., 20 nucleotides on each side of the deleted or inserted sequence site).

Table 2: gRNA sequences and their target genes used in 2 genome editing techniques. "Y" indicates that the gRNA targets the PMT gene, and "-" indicates that the gRNA does not target the PMT gene.

Table 3: zygosity of individual PMT loci in selected PMT mutants in various contexts generated by genome editing using GET 2. The number one (1) indicates a homozygote for a single mutant allele. Numbers 2 to 5 indicate heteroallelic combinations with 2 to 5 indels. Hyphens indicate no data. Detailed genotype information is shown in tables 4A to 4D.

Example 3: alkaloid analysis of PMT-edited lines

Genome edited tobacco plants were planted in 10 "pots with 75PPM fertilizer in the greenhouse along with controls. During the flowering phase, plants were topped and leaf samples were taken from 3, 4,5 leaves from the top two weeks after topping and alkaloid content was determined using a method as specified in CORESTA method 62 (2005-2, Determination of nicontine in tobacao and tobacao Products by Gas chromatography Analysis) and the american centers for disease control and prevention 1999 3-23, federal bulletin 64, vol 55, Protocol for Analysis of nicontine, Total sport and pH in Smokeless tobacao Products (and 2009-1-7, vol 4, revised version) were determined (tables 6A to 6C).

Briefly, about 0.5g of tobacco was extracted using liquid/liquid extraction into an organic solvent containing an internal standard and analyzed by Gas Chromatography (GC) using Flame Ionization Detection (FID). Results may be reported as weight percent (Wt%) of the as-received or dry weight. Reporting data on a dry weight basis requires determination of Oven Volatiles (OV). Unless otherwise indicated, levels of total alkaloids or nicotine or levels of alkaloids or nicotine alone as indicated herein are based on dry weight (e.g., percent total alkaloids or percent nicotine).

Plants were also grown, harvested in the field, and tested for alkaloid and TSNA levels in cured tobacco. The leaf yield and leaf grade of PMT edited plants were also evaluated. Further, different mutant combinations (e.g., single, double, triple, or quadruple mutants) of individual PMT genes were generated and tested.

Example 4: the five pmt knockout mutant was compared to other low alkaloid tobacco plants.

Five PMT knockout mutant line CS15 (for genotype, see table 4E, in nlm (ph) background) was grown side-by-side with PMT RNAi transgene line (in VA359 background, as described in US 2015/0322451) and low nicotine KY171 ("LN KY 171") variety (KY 171 background with the double mutations of nic1 and nic 2). The leaves were harvested and prepared by dark smoking. Each line was analyzed for nicotine and total alkaloid levels, leaf yield and leaf quality (fig. 2 to 5). The data indicate that inhibition of PMT gene activity by editing all five PMT genes can reduce nicotine levels without compromising leaf yield or quality.

Example 5: a tobacco line having an edited mutant allele in one or more PMT genes was obtained.

Tobacco lines having mutations in either the PMT genes alone or selected combinations of the PMT genes were obtained from the tobacco lines listed in table 3. Five, four, three or double mutants (having mutations in five, four, three or two PMT genes, respectively) were crossed with an unmutated control line and isolated progeny plants were selected for a particular PMT mutation combination. Tables 7A to 7E show the possible mutant combinations obtained. For mutations, each mutated gene may be homozygote or heterozygote. Each of the mutant alleles listed in tables 4A to 4E and table 10 can be used to generate single, double, triple, quinary or quadruple mutants. Exemplary individual pmt mutant alleles are listed in tables 9A through 9E.

Example 6: total alkaloid reduction was further achieved by combining pmt mutations with mutations in other genes.

To further reduce total alkaloids and/or selected individual alkaloids, the pmt mutants were combined with mutations in additional genes associated with alkaloid biosynthesis in tobacco, such as Quinolinate Phosphoribosyltransferase (QPT) or Quinolinate Synthase (QS). Briefly, gene editing is used to mutate selected QPT and/or QS genes in a desired pmt mutant background (e.g., four or five pmt mutants). Alkaloid and TSNA levels were tested in cured tobacco in the resulting combined qpt/pmt or qs/pmt mutants. Leaf yield and leaf grade were also evaluated.

Table 6A: alkaloid levels in PMT-edited lines in K326 (weight percent per gram leaf (dry weight) shown here and in tables 6B, 6C and 7)

Table 6B: alkaloid levels in PMT-edited lines in TN90

Table 6C: alkaloid levels in PMT-edited lines of narrow-leaf Madole (NLM)

Table 7: relative changes in nicotine and total alkaloid levels in the five pmt knockout mutants of different species. The average percent levels of nicotine and total alkaloids were calculated from the percent level data from the individual lines as shown in tables 6A to 6C. The relative change reflects the nicotine or total alkaloid levels of the five pmt mutants relative to their control.

Table 8A: list of mutants obtained with various genotype combinations of five PMT genes (single gene mutations)

	PMT1a	PMT1b	PMT2	PMT3	PMT4
						1	Mutants	WT	WT	WT	WT
2	WT	Mutants	WT	WT	WT
						3	WT	WT	Mutants	WT	WT
4	WT	WT	WT	MutationsBody	WT
						5	WT	WT	WT	WT	Mutants

Table 8B: list of mutants obtained with various genotype combinations of five PMT genes (double Gene mutations)

	PMT1a	PMT1b	PMT2	PMT3	PMT4
						1	Mutants	Mutants	WT	WT	WT
2	Mutants	WT	Mutants	WT	WT
						3	Mutants	WT	WT	Mutants	WT
4	Mutants	WT	WT	WT	Mutants
						5	WT	Mutants	Mutants	WT	WT
6	WT	Mutants	WT	Mutants	WT
						7	WT	Mutants	WT	WT	Mutants
8	WT	WT	Mutants	Mutants	WT
						9	WT	WT	Mutants	WT	Mutants
10	WT	WT	WT	Mutants	Mutants

Table 8C: list of mutants obtained with various genotype combinations of five PMT genes (three-gene combinations)

Table 8D: list of mutants obtained with various genotype combinations of five PMT genes (four-gene combination)

	PMT1a	PMT1b	PMT2	PMT3	PMT4
						1	Mutants	Mutants	Mutants	Mutants	WT
2	WT	Mutants	Mutants	Mutants	Mutants
						3	Mutants	WT	Mutants	Mutants	Mutants
4	Mutants	Mutants	WT	Mutants	Mutants
						5	Mutants	Mutants	Mutants	WT	Mutants

Table 8E: list of mutants obtained with various genotype combinations of five PMT genes (five-gene combination)

	PMT1a	PMT1b	PMT2	PMT3	PMT4
						1	Mutants	Mutants	Mutants	Mutants	Mutants

Example 7: PMT genome editing and tobacco strain development

Additional PMT knock-out mutants were generated by editing all five PMT genes (PMT1a, PMT1b, PMT2, PMT3, and PMT4) in different tobacco lines. Tobacco protoplasts were transfected with polyethylene glycol (PEG) using plasmids encoding genome editing technology (GET2) proteins and specific targeting rnas (grnas) targeting PMT genes at the desired locations. Table 9 lists gRNA sequences used for PMT editing. Some grnas (e.g., nos.6 and 7) were mixed together for targeting multiple PMT genes in a single transfection.

Table 9: targeting RNA for GET2 used in example 7. "Y" indicates that the gRNA is capable of targeting the PMT gene, and "-" indicates that the gRNA does not target the PMT gene.

Then, the transfected protoplasts were immobilized in 1% agarose beads and subjected to tissue culture. When calli were grown to about 1mm in diameter, they were plated on TOM2 plates. The calli were screened for insertions or deletions (indels) at the target location using fragment analysis. Candidates showing size shift compared to the wild-type control were selected for further culture and subsequent shoots were tested again by fragment analysis to confirm the presence of indels. Rooted shoots are potted and the target sites sequenced to determine the exact sequence of the deletion. Young leaves from each plant were harvested and PCR amplification of PMT fragments was performed using phirekit. PMT libraries for each line were indexed and 384 lines were pooled and sequenced using Miseq.

SNP analysis was performed to determine the exact edited PMT mutant allele sequence and the zygotic status at each PMT locus. Table 10 provides indel sequence information in each edited line of various tobacco varieties (e.g., Basma, K326, katrini, TN90, Izmir).

Table 11 provides the length (in nucleotides) of each PMT indel for each gene in each line as provided in table 10.

Table 11 length (in nucleotides) of each indel of the selected lines provided in table 10.

Tables 12A through 12E provide genomic sequences of about 90 nucleotides from each pmt mutant allele, with the sites of editing located in the middle of the genomic sequence (e.g., 45 nucleotides on each side of the deleted or inserted sequence site).

Example 8 alkaloid analysis of PMT edited lines.

Homozygous genomically edited tobacco lines from example 7 were grown in the field with control lines. During the flowering phase, plants were topped and leaf samples were taken from the third, fourth, and fifth leaves from the top of the plants two weeks after topping and measured using the method defined in accordance with CORESTA method 62(Determination of nico in tomato and tobaca Products by Gas chromatography Analysis, 2 months 2005) and Protocol for Analysis of nico, published by the american centers for disease control and prevention on feed Register, volume 64, phase 55, 3/23 of 1999 (and revised on volume 74, 7 of 2009, 4) (see tables 13A-13C).

About 0.5g of tobacco was extracted using liquid/liquid extraction into an organic solvent containing an internal standard and analyzed by Gas Chromatography (GC) using Flame Ionization Detection (FID). Results may be reported as weight percent (Wt%) of the as-received or dry weight. Reporting data on a dry weight basis requires determination of Oven Volatiles (OV). Unless otherwise indicated, levels of total alkaloids or nicotine or levels of alkaloids or nicotine alone as indicated herein are based on dry weight (e.g., percent total alkaloids or percent nicotine).

Plants were also grown, harvested in the field, and tested for alkaloid and TSNA levels in cured tobacco. The leaf yield and leaf grade of PMT edited plants were also evaluated.

Table 13A: nicotine analysis of K326 and TN90 PMT edited lines two weeks after flowering.

Table 13B: nicotine analysis of K326 and TN90 PMT edited lines two weeks after topping.

Table 13C: nicotine analysis of lines edited by katrini and Basma PMT two weeks after flowering.

Example 9 development of male sterile PMT edited lines.

Lines from example 7 were used to develop PMT edited hybrid lines. The hybrid lines are grown in the field and used as progenitor cells for male sterile lines. See table 14.

Table 14: PMT edited extremely low nicotine male sterile line

Example 10 PMT-edited lines resist mold during modulation

Tobacco leaves harvested from several low alkaloid tobacco lines were subjected to standard air-curing practices. After the modulation was completed, the tobacco leaves were checked for mold.

Tobacco from LA BU 21 exhibited more mold infestation than TN90 LC, TN90 variety containing RNAi constructs that down-regulate all five PMT genes, TN90 variety containing RNAi constructs that down-regulate alkaloid biosynthesis genes PR50, and four PMT edited lines (CS47, CS59, CS63, and CS64) in the TN90 genetic background. See table 15 and fig. 6A to 6E and 7.

TABLE 15 mould damage exhibited by tobacco lines. "G" means little/no mold; "s" refers to some molds; and "B" refers to a prominent mold. The percent mold refers to the percentage of air cured tobacco rods exhibiting damage from each type of mold.