阅读说明：本技术 低烟碱烟草植物及由其制备的烟草制品 (Low nicotine tobacco plants and tobacco products made therefrom ) 是由 A·亚当斯 M·德戈多伊·卢索 S·普拉莫德 许冬梅 于 2019-12-03 设计创作，主要内容包括：本申请提供了烟草近交植物dS1746、dS1746MS、dS1564和dS1564MS。本申请还提供了这类植物的部分和由这些部分制成的制品。本申请还包括所提供的植物的后代,包括杂交系。(The present application provides tobacco inbred plants dS1746, dS1746MS, dS1564 and dS1564 MS. Also provided are parts of such plants and articles made from these parts. Progeny of the provided plants, including hybrid lines, are also included.)

1. Seed of tobacco cultivar dS1746 or dS1746MS, a representative sample seed of said cultivar dS1746 was deposited with the ATCC under ATCC accession No. PTA-125409, and a representative sample seed of said cultivar dS1746MS was deposited with the ATCC under ATCC accession No. PTA-126414.

2. A tobacco plant, or part thereof, grown from the seed of claim 1.

3. Harvested leaf of the tobacco plant of claim 2.

4. The harvested leaf of claim 3, wherein the leaf comprises a nicotine level of less than 0.2% dry weight.

5. Cured tobacco comprising tobacco material from the tobacco plant or portion thereof of claim 2.

6. A smoking article comprising cured tobacco according to claim 5, wherein said article is selected from the group consisting of: pipe tobacco, cigars, cigarettes, chewing tobacco, tobacco leaves, tobacco shreds and tobacco shreds.

7. A smoking article comprising cured tobacco according to claim 5, wherein the smoking article is selected from the group consisting of: cigarillos, non-ventilated filter cigarettes, cigars, snuff and chewing tobacco.

8. A smoking article as defined in claim 6, wherein the article has a nicotine level of less than 0.2% dry weight.

9. A smoking article as claimed in claim 7, wherein the nicotine level of said article is less than 0.2% by dry weight.

10. Tobacco cultivar dS1746 or F of dS1746MS₁A progeny plant, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC accession No. PTA-125409, and a representative sample seed of said cultivar dS1746MS having been deposited with the ATCC under ATCC accession No. PTA-126414.

11. Seed of tobacco cultivar dS1564 or dS1564MS, a representative sample seed of said cultivar dS1564 having been deposited with the ATCC under ATCC accession No. PTA-125408, and a representative sample seed of said cultivar dS1564MS having been deposited with the ATCC under ATCC accession No. PTA-126413.

12. A tobacco plant, or part thereof, grown from the seed of claim 11.

13. Harvested leaf of the tobacco plant of claim 12.

14. The harvested leaf of claim 13, wherein said leaf comprises a nicotine level of less than 0.2% dry weight.

15. Cured tobacco comprising tobacco material from the tobacco plant or portion thereof of claim 12.

16. A smoking article comprising cured tobacco according to claim 15, wherein said article is selected from the group consisting of: pipe tobacco, cigars, cigarettes, chewing tobacco, tobacco leaves, tobacco shreds and tobacco shreds.

17. A smoking article comprising cured tobacco according to claim 5, wherein the smoking article is selected from the group consisting of: cigarillos, non-ventilated filter cigarettes, cigars, snuff and chewing tobacco.

18. The smoking article of claim 16, wherein said article has a nicotine level of less than 0.2% dry weight.

19. The smoking article of claim 17, wherein said article has a nicotine level of less than 0.2% dry weight.

20. Tobacco cultivar dS1564 or F of dS1564MS₁A progeny plant, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC accession No. PTA-125408, and a representative sample seed of said cultivar dS1564MSThe sample seeds are deposited with the ATCC under ATCC accession number PTA-126413.

Technical Field

The present application provides tobacco inbred plants dS1746 (and male sterile, dS1746MS), dS1564 (and male sterile, dS1564MS), and progeny thereof. Also provided are parts of such plants and articles made from these parts. Progeny of the provided plants, including hybrid lines, are also included.

Background

Tobacco (Nicotiana tabacum L.) is an important commercial crop in both the united states and other countries. Four major alkaloids are found in tobacco: nicotine, nornicotine, anabasine and anatabine. Nicotine is the major alkaloid, and usually accounts for more than 90% of total alkaloids in commercial tobacco cultivars. Nicotine biosynthesis occurs primarily in the tobacco root. The tobacco plant then transports the nicotine through the vascular bundle to the leaves where it is stored in the vacuole.

A variety of factors affect tobacco alkaloid levels, including genotype, environment, fertilization, and agronomic use (e.g., stimulation of nicotine production by topping, wounding, and herbivore damage). The low alkaloid trait originally found in lines of the cuba cigar variety was introduced into cigarette varieties through a series of backcrosses. The low alkaloid tobacco germplasm was subsequently enrolled in the genetic background of cultivar Burley 21 (Legg et al, Crop Science,10:212 (1970)). Genetic studies using the low alkaloid burley 21(LA BU21) line showed that two unlinked loci contribute to nicotine levels in tobacco leaves. These two loci are designated as Nic1 and Nic 2. The nic1 and nic2 mutations in LA BU21 are semi-dominant. They exhibit a dose-dependent effect at nicotine levels with a nic1 effect about 2.4 times stronger than the nic2 effect. Molecular characterization of the Nic2 locus has been reported. The nic2 mutation was shown to contain a deletion in the transcription factor gene cluster from the Ethylene Response Factor (ERF) family (Shoji et al, Plant Cell, (10):3390-409 (2010)).

Reducing the total alkaloid content in tobacco can have a number of benefits. Nicotinic alkaloid levels in tobacco plants are reported to play an important role in protecting plants from insects and herbivores.

Consistent with the role of alkaloids in insect defense, LA BU21 has been reported to be extremely sensitive to insect damage (Legg et al, Crop Science,10:212 (1970)). Further studies comparing isogenic lines of flue-cured tobacco with low percent total alkaloids (about 0.20%) to their "normal" repeat parent (total alkaloids 1.85-2.70%) reported that yield, grade index, total N and reducing sugar content in low alkaloid lines were less than normal flue-cured tobacco cultivars (Chaplin and Weeks, Crop Science,16(3):416-18 (1976)).

There is a need to develop tobacco plants and products that contain altered levels of nicotine (e.g., reduced levels of nicotine) while maintaining, if not making more optimal, the quality of the tobacco leaf.

Brief description of the invention

In one aspect, the present disclosure provides seeds of tobacco cultivar dS1746 or dS1746MS, a representative sample seed of said cultivar dS1746 was deposited with the ATCC under ATCC accession No. PTA-125409, and a representative sample seed of said cultivar dS1746MS was deposited with the ATCC under ATCC accession No. PTA-126414.

In another aspect, the present disclosure provides a tobacco plant, or a portion thereof, grown from a seed of tobacco cultivar dS1746 or dS1746 MS.

In one aspect, the present disclosure provides harvested leaves of a tobacco plant of tobacco cultivar dS1746 or dS1746 MS.

In one aspect, the present disclosure provides harvested leaves of a tobacco plant of tobacco cultivar dS1746 or dS1746MS, wherein the leaves have a USDA grade index value of 50 or greater.

In one aspect, the present disclosure provides harvested leaves of a tobacco plant of tobacco cultivar dS1746 or dS1746MS, wherein the leaves comprise a nicotine level of less than 0.2% dry weight.

In one aspect, the present disclosure provides cured tobacco comprising tobacco material from a tobacco cultivar dS1746 or dS1746MS tobacco plant.

In one aspect, the present disclosure provides a tobacco product comprising cured tobacco from a tobacco plant of tobacco cultivar dS1746 or dS1746MS, wherein the product is selected from the group consisting of: pipe tobacco, cigars, cigarettes, chewing tobacco, tobacco leaves, shredded tobacco (shredded tobaco) and cut tobacco (cut tobaco).

In one aspect, the present disclosure provides a tobacco product comprising cured tobacco from a tobacco plant of tobacco cultivar dS1746 or dS1746MS, wherein the tobacco product is selected from the group consisting of: cigarillos, non-ventilated filter cigarettes, cigars, snuff and chewing tobacco.

In one aspect, the present disclosure provides a tobacco product comprising cured tobacco from a tobacco plant of tobacco cultivar dS1746 or dS1746MS, wherein the product has a nicotine level of less than 0.2% dry weight.

In one aspect, the disclosure provides seeds of tobacco cultivar dS1564 or dS1564MS, a representative sample seed of said cultivar dS1564 was deposited with the ATCC under ATCC accession No. PTA-125408, and a representative sample seed of said cultivar dS1564MS was deposited with the ATCC under ATCC accession No. PTA-126413.

In another aspect, the present disclosure provides a tobacco plant, or a portion thereof, grown from a seed of tobacco cultivar dS1564 or dS1564 MS.

In one aspect, the disclosure provides harvested leaves of a tobacco plant of tobacco cultivar dS1564 or dS1564 MS.

In one aspect, the present disclosure provides harvested leaves of a tobacco plant of tobacco cultivar dS1564 or dS1564MS, wherein the leaves have a USDA grade index value of 50 or greater.

In one aspect, the present disclosure provides harvested leaves of a tobacco plant of tobacco cultivar dS1564 or dS1564MS, wherein the leaves comprise a nicotine level of less than 0.2% dry weight.

In one aspect, the present disclosure provides cured tobacco comprising tobacco material from a tobacco plant of tobacco cultivar dS1564 or dS1564 MS.

In one aspect, the present disclosure provides a tobacco product comprising cured tobacco from a tobacco plant of tobacco cultivar dS1564 or dS1564MS, wherein the product is selected from the group consisting of: pipe tobacco, cigars, cigarettes, chewing tobacco, tobacco leaves, tobacco shreds and tobacco shreds.

In one aspect, the present disclosure provides a tobacco product comprising cured tobacco from a tobacco plant of tobacco cultivar dS1564 or dS1564MS, wherein the tobacco product is selected from the group consisting of: cigarillos, non-ventilated filter cigarettes, cigars, snuff and chewing tobacco.

In one aspect, the present disclosure provides a tobacco product comprising cured tobacco from a tobacco plant of tobacco cultivar dS1564 or dS1564MS, wherein the product has a nicotine level of less than 0.2% dry weight.

Brief description of the sequences

SEQ ID NOS 1-5 show the nucleotide sequences of five SNP markers for the genotype nic1 or nic2 loci.

SEQ ID NOS.6-10 show the nucleotide sequences of the low alkaloid alleles of the corresponding SNP markers.

SEQ ID NOS 11-15 show the nucleotide sequences of the high alkaloid alleles of the corresponding SNP markers.

The various sequences include the "N" in the nucleotide sequence. "N" may be any nucleotide, such as A, T, G, C, or a deletion or insertion of one or more nucleotides. In some examples, a string "N" is displayed. The number of "N" does not necessarily correlate with the actual number of undetermined nucleotides at that position. The actual nucleotide sequence may be longer or shorter than the "N" segment shown.

Detailed Description

This description is not an exhaustive list of all the different ways in which the application can be implemented or all the features that can be added to the application. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, in some embodiments of the present application, it is contemplated that any feature or combination of features set forth herein may be excluded or omitted. In addition, many variations and additions to the various embodiments set forth herein will be apparent to those skilled in the art in light of the present disclosure without departing from the present application. The following description is, therefore, intended to be illustrative of certain specific embodiments of the application and is not exhaustive of all permutations, combinations and variations thereof.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety for the purpose of presenting the teachings associated with the sentences and/or paragraphs of the reference therein. References to techniques used herein are intended to refer to techniques commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to those skilled in the art.

The various features described herein can specifically be used in any combination, unless the context indicates otherwise. Furthermore, the present application also contemplates that in some embodiments of the present application, any feature or combination of features set forth herein may be excluded or omitted. For purposes of illustration, if the specification states that the composition comprises components A, B and C, in particular, either one of A, B or C, or a combination thereof, may be omitted and discarded individually or in any combination.

As used in the description of the present application and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, and not combinations when interpreted in an alternative manner ("or").

As used herein, the term "about" when referring to a measurable value (such as nicotine amount or level, etc.) is meant to encompass variations of ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5% or even ± 0.1% of the defined amount.

As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" refer to "between about X and about Y" and phrases such as "from about X to Y" refer to "from about X to about Y".

In the context of a polynucleotide sequence (e.g., a recombinant polynucleotide and/or expression cassette of the present application), "introduced" refers to the presentation of the polynucleotide sequence into a plant, plant part, and/or plant cell in such a way that the polynucleotide sequence enters the interior of the cell. When more than one polynucleotide sequence is introduced, these polynucleotide sequences may be assembled as part of a single polynucleotide or nucleic acid construct, or as separate polynucleotides or nucleic acid constructs, and may be positioned on the same or different transformation vectors. Thus, these polynucleotides can be introduced into a plant cell in a single transformation event, in separate transformation events, or, for example, as part of a breeding program. Thus, the term "transformation" as used herein refers to the introduction of a heterologous nucleic acid into a cell. Transformation of the cells may be stable or transient. Thus, in some embodiments, a plant cell, plant part, or plant of the present application can be stably transformed with a recombinant polynucleotide of the present application. In other embodiments, a plant cell, plant part, or plant of the present application can be transiently transformed with a recombinant polynucleotide of the present application.

In one aspect, the present application includes seed of tobacco cultivar dS1746, a representative sample seed of said cultivar having been deposited with the American Type Culture Collection (ATCC) under ATCC accession No. PTA-125409.

In another aspect, the present application includes a tobacco plant, or a portion thereof, produced by growing seeds of tobacco cultivar dS 1746.

In another aspect, the present application includes a harvested leaf or portion thereof of a tobacco plant or portion thereof produced by growing a seed of tobacco cultivar dS 1746.

In one aspect, the present application includes harvested leaf of a tobacco plant, or a portion thereof, produced by growing seed of tobacco cultivar dS1746, wherein the leaf has a reduced amount of nicotine relative to a control tobacco variety having substantially the same genetic background as dS1746 (except that the nic1 and the nic2 loci introgress dS1746) when grown under similar growth conditions.

In another aspect, the present application includes a tobacco product prepared from a tobacco plant, or a portion thereof, produced by growing seeds of tobacco cultivar dS1746, wherein the product is selected from the group consisting of: pipe tobacco, cigars, cigarettes, chewing tobacco, tobacco leaves, tobacco shreds and tobacco shreds, and any combination thereof.

In one aspect, the present application includes a tobacco product prepared from a tobacco plant, or a portion thereof, produced by growing seeds of tobacco cultivar dS1746, wherein the product is selected from the group consisting of: cigarillos, non-ventilated filter cigarettes, cigars, snuff and chewing tobacco.

In another aspect, the tobacco product has a reduced amount of nicotine relative to a tobacco product that is not prepared from dS 1746.

In another aspect, the present application includes a second tobacco product prepared or produced from a first tobacco product prepared from a tobacco plant, or a portion thereof, produced by growing seeds of tobacco cultivar dS1746, wherein the first product is selected from the group consisting of: pipe tobacco, cigars, cigarettes, chewing tobacco, tobacco leaves, tobacco shreds, and any combination thereof, and the second tobacco product is selected from the group consisting of: cigarillos, non-ventilated filter cigarettes, cigars, snuff and chewing tobacco. In one aspect, the first and second articles comprise reduced amounts of nicotine.

In one aspect, the present application includes a portion of a tobacco plant produced by growing a seed of tobacco cultivar dS1746, wherein the portion is selected from the group consisting of: leaves, pollen, ovules, embryos, cotyledons, hypocotyls, meristematic cells, protoplasts, roots, root tips, pistils, anthers, flowers, buds, stems, pods and petioles.

In another aspect, the present application includes a tissue culture produced from protoplasts or cells from a tobacco plant or part thereof produced by growing seeds of tobacco cultivar dS1746, wherein the cells or protoplasts of the tissue culture are produced from a plant part selected from the group consisting of: leaves, pollen, embryos, cotyledons, hypocotyls, meristematic cells, roots, root tips, pistils, anthers, flowers, buds, stems, pods and petioles.

In one aspect, the present application includes a tobacco plant regenerated from a tissue culture produced from protoplasts or cells from a tobacco plant or part thereof produced by growing seeds of tobacco cultivar dS1746, wherein the cells or protoplasts of the tissue culture are produced from a plant part selected from the group consisting of: leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root tip, pistil, anther, flower, bud, stem, pod and petiole, wherein the regenerated plant has all or substantially all of the morphological and physiological characteristics of cultivar dS 1746.

In one aspect, the present application includes F of tobacco cultivar dS1746₁A progeny plant.

In another aspect, the present application includes F of tobacco cultivar dS1746₁A progeny plant, a representative sample seed of said cultivar being deposited with the ATCC under ATCC accession No. PTA-125409, whereinF₁Plants are Male Sterile (MS). In another aspect, F₁Plants are Cytoplasmic Male Sterile (CMS).

In another aspect, the present application includes F produced by crossing two tobacco plants and harvesting the resulting tobacco seeds₁Progeny seed, wherein at least one tobacco plant is a tobacco plant produced by growing seed of tobacco cultivar dS1746, wherein at least one tobacco plant is MS or CMS.

In another aspect, the present application includes a method for producing tobacco seed comprising crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one tobacco plant is a tobacco plant produced by growing seed of tobacco cultivar dS 1746.

In another aspect, the present application includes a method for producing tobacco seed comprising crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one tobacco plant is a tobacco plant produced by growing seed of tobacco cultivar dS1746, wherein the plant of tobacco cultivar dS1746 is the male parent.

In another aspect, the present application includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one tobacco plant is a tobacco plant produced by growing seed of tobacco cultivar dS1746, wherein the plant of tobacco cultivar dS1746 is the female parent.

In another aspect, the present application includes a method for producing tobacco seed comprising crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one tobacco plant is a tobacco plant produced by growing seed of tobacco cultivar dS1746, wherein at least one tobacco plant is MS or CMS.

In one aspect, the present application also includes F produced by crossing two tobacco plants and harvesting the resulting tobacco seeds₁A container of progeny seed, wherein at least one tobacco plant is a tobacco plant produced by growing seeds of tobacco cultivar dS1746, wherein at least one isThe nicotiana species plant is MS or CMS.

In one aspect, the present application includes F produced by growing seed produced by crossing two tobacco plants and harvesting the resulting tobacco seed₁A progeny plant, wherein at least one tobacco plant is a tobacco plant produced by growing seeds of tobacco cultivar dS1746, and wherein at least one tobacco plant is MS or CMS.

In one aspect, the present application also includes F produced by growing seed produced by crossing two tobacco plants and harvesting the resulting tobacco seed₁Harvested leaf of a progeny plant, wherein at least one tobacco plant is a tobacco plant produced by growing seed of tobacco cultivar dS1746, wherein at least one tobacco plant is MS or CMS. On the other hand, when grown under similar growth conditions, relative to a control tobacco variety having substantially the same genetic background as dS1746 (except that the nic1 and nic2 loci introgress dS1746), from F₁The harvested leaves of the progeny plants have a reduced amount of nicotine.

In one aspect, the present application includes a composition consisting of F₁A tobacco product made from a progeny tobacco plant or part thereof, wherein said plant or part thereof is produced by growing F₁Progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one tobacco plant is produced by growing seed of tobacco cultivar dS1746, wherein at least one tobacco plant is cytoplasmic male sterile, and wherein the tobacco product is selected from the group consisting of: pipe tobacco, cigars, cigarettes, chewing tobacco, tobacco leaves, tobacco shreds and any combination thereof.

In one aspect, the present application also includes a composition consisting of F₁A tobacco product made from a progeny tobacco plant or part thereof, wherein said plant or part thereof is produced by growing F₁Progeny seed production, said progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one tobacco plant is produced by growing seed of tobacco cultivar dS1746, wherein at least one tobacco plant is cytoplasmic male sterile, and wherein said progeny seed is produced by a method comprising crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one tobacco plant is cytoplasmic male sterile, and wherein said progeny seed is produced by a method comprising growing seed of tobacco cultivar dS1746, and wherein said progeny seed is produced by a method comprising crossing two tobacco plants and harvesting the resulting tobacco seed, and wherein said progeny seed is produced by a method comprising growing seed of tobacco cultivar dS1746, and wherein said at least one tobacco plant is cytoplasmic male sterile, and wherein said progeny seed is produced by a method comprising growing seed of tobacco cultivar dS1746, and wherein said progeny seed is produced by a method comprising crossing a method comprising growing seed and harvesting the sameThe tobacco product is selected from the group consisting of: cigarillos, non-ventilated filter cigarettes, cigars, snuff and chewing tobacco.

In another aspect, the present application includes seed of tobacco cultivar dS1746MS, a representative sample seed of the cultivar deposited with the ATCC under ATCC accession No. PTA-126414.

In one aspect, the present application includes a tobacco plant, or a portion thereof, produced by growing seeds of tobacco cultivar dS1746 MS.

In another aspect, the present application includes a harvested leaf or portion thereof of a tobacco plant, or portion thereof, produced by growing seeds of tobacco cultivar dS1746 MS.

In one aspect, the present application includes harvested leaves of a tobacco plant, or a portion thereof, produced by growing seeds of tobacco cultivar dS1746MS, wherein the leaves have a reduced amount of nicotine relative to a control tobacco variety having substantially the same genetic background as dS1746MS (except that the nic1 and nic2 loci introgress dS1746MS) when grown under similar growth conditions.

In another aspect, the present application includes a tobacco product prepared from a tobacco plant, or a portion thereof, produced by growing seeds of tobacco cultivar dS1746MS, wherein the product is selected from the group consisting of: pipe tobacco, cigars, cigarettes, chewing tobacco, tobacco leaves, tobacco shreds and any combination thereof.

In one aspect, the present application includes a tobacco product prepared from a tobacco plant, or a portion thereof, produced by growing seeds of tobacco cultivar dS1746MS, wherein the product is selected from the group consisting of: cigarillos, non-ventilated filter cigarettes, cigars, snuff and chewing tobacco.

In another aspect, the present application includes a tobacco product prepared from a tobacco plant, or a portion thereof, produced by growing seeds of tobacco cultivar dS1746MS, wherein the product is selected from the group consisting of: a cigarillo, a non-ventilated filter cigarette, a cigar, snuff and a chewing tobacco, wherein the article has a reduced amount of nicotine relative to a control article prepared from a control tobacco plant having substantially the same genetic background as dS1746MS (except that the nic1 and nic2 loci have been introgressed into dS1746MS) when grown under similar growth conditions.

In another aspect, the present application includes a second smoking article prepared or produced from a first smoking article prepared from a tobacco plant, or part thereof, produced by growing seeds of tobacco variety dS1746MS, wherein the first article is selected from the group consisting of: pipe tobacco, cigar, cigarette, chewing tobacco, tobacco leaf, tobacco shred, and any combination thereof, and the second tobacco product is selected from the group consisting of: cigarillos, non-ventilated filter cigarettes, cigars, snuff and chewing tobacco. In one aspect, the first and/or second article comprises a reduced amount of nicotine.

In one aspect, the present application includes a portion of a tobacco plant produced by growing seeds of tobacco cultivar dS1746MS, wherein the portion is selected from the group consisting of: leaves, pollen, ovules, embryos, cotyledons, hypocotyls, meristematic cells, protoplasts, roots, root tips, pistils, anthers, flowers, buds, stems, pods and petioles.

In another aspect, the present application includes a tissue culture produced from protoplasts or cells from a tobacco plant or part thereof produced by growing seeds of tobacco cultivar dS1746MS, wherein the cells or protoplasts of the tissue culture are produced from a plant selected from the group consisting of: leaves, pollen, embryos, cotyledons, hypocotyls, meristematic cells, roots, root tips, pistils, anthers, flowers, buds, stems, pods and petioles.

In one aspect, the present application includes a tobacco plant regenerated from a tissue culture produced from protoplasts or cells from a tobacco plant or part thereof produced by growing seeds of tobacco cultivar dS1746MS, wherein the cells or protoplasts of the tissue culture can be produced from a plant part selected from the group consisting of: leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root tip, pistil, anther, flower, bud, stem, pod and petiole, wherein the regenerated plant has all or substantially all of the morphological and physiological characteristics of cultivar dS1746 MS.

In one aspect, the present application includes F of tobacco cultivar dS1746MS₁A progeny plant.

In another aspect, the present application includes F produced by crossing two tobacco plants and harvesting the resulting tobacco seeds₁Progeny seed, wherein at least one tobacco plant is a plant of tobacco variety dS1746 MS.

In another aspect, the present application includes a method for producing tobacco seed comprising crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one tobacco plant is a tobacco plant produced by growing seed of tobacco cultivar dS1746 MS.

In another aspect, the present application includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one tobacco plant is a tobacco plant produced by growing seed of tobacco cultivar dS1746MS, wherein the plant of tobacco cultivar dS1746MS is the female parent.

In another aspect, the present application includes a method for producing tobacco seed comprising crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one of the tobacco plants is a plant of tobacco cultivar dS1746 MS.

In one aspect, the present application also includes F₁A container of progeny seed produced by crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one tobacco plant is a tobacco plant produced by growing seed of tobacco cultivar dS1746 MS.

In one aspect, the application includes F produced by growing seeds₁A progeny plant produced by crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one tobacco plant is a tobacco plant produced by growing seed of tobacco cultivar dS1746 MS.

In one aspect, the application also includes F produced by growing seeds₁Harvesting of progeny plantsObtaining leaves, the seeds produced by crossing two tobacco plants and harvesting the resulting tobacco seeds, wherein at least one tobacco plant is a tobacco plant produced by growing seeds of tobacco cultivar dS1746 MS.

In another aspect, the present application also includes F produced by growing seeds having a reduced amount of nicotine₁Harvested leaf of a progeny plant, said seed produced by crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one tobacco plant is a tobacco plant produced by growing seed of tobacco cultivar dS1746 MS.

In one aspect, the present application includes a composition consisting of F₁A tobacco product made from a progeny tobacco plant or part thereof, wherein said plant or part thereof is produced by growing F₁Progeny seed, said F₁Progeny seed is produced by crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one tobacco plant is a tobacco plant produced by growing seed of tobacco cultivar dS1746MS, and wherein the tobacco product is selected from the group consisting of: pipe tobacco, cigars, cigarettes, chewing tobacco, tobacco leaves, tobacco shreds and any combination thereof.

In one aspect, the present application also includes a composition consisting of F₁A tobacco product made from a progeny tobacco plant or part thereof, wherein the plant or part thereof is produced by growing F resulting from crossing two tobacco plants and harvesting the resulting tobacco seeds₁Progeny seed, wherein at least one tobacco plant is a tobacco plant produced by growing seeds of tobacco cultivar dS1746MS, and wherein the tobacco product is selected from the group consisting of: cigarillos, non-ventilated filter cigarettes, cigars, snuff and chewing tobacco.

In one aspect, the present application also includes a composition consisting of F₁A tobacco product made from a progeny tobacco plant or part thereof, wherein the plant or part thereof is produced by growing F resulting from crossing two tobacco plants and harvesting the resulting tobacco seeds₁Progeny seed, wherein at least one tobacco plant is a tobacco plant produced by growing seeds of tobacco cultivar dS1746MS, andwherein the tobacco product is selected from the group consisting of: cigarillos, non-ventilated filter cigarettes, cigars, snuff and chewing tobacco and further wherein the article has a reduced amount of nicotine.

In one aspect, the present application includes a method for producing tobacco seed comprising crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one tobacco plant is a plant of tobacco cultivar dS1746 MS.

In one aspect, the present application includes a method of asexually propagating a plant of the tobacco cultivar comprising the steps of: (a) collecting tissue capable of propagating from a plant of tobacco cultivar dS1746 or dS1746 MS; (b) culturing the tissue to obtain multiplied shoots; and (c) rooting the proliferated shoots to obtain rooted shoots.

In one aspect, the present application includes a method of asexually propagating a plant of the tobacco cultivar comprising the steps of: (a) collecting tissue capable of propagating from a plant of tobacco cultivar dS1746 or dS1746 MS; (b) culturing the tissue to obtain multiplied shoots; (c) rooting the proliferated buds to obtain rooted seedlings; and (d) growing plants from the rooted shoots.

In one aspect, the application includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar dS1746 or dS1746MS with a second tobacco plant comprising a desired trait to produce F₁Progeny seed; (b) growth F₁Progeny seed and selection for F comprising a desired trait₁A progeny plant; (c) will select F₁Crossing the progeny plant with the first tobacco cultivar plant to produce backcross BC₁F₁Progeny seed; (d) growing BC₁F₁Progeny seeds and selecting backcross BC comprising the desired trait and substantially all physiological and morphological characteristics of the first tobacco cultivar₁F₁A progeny plant; and (e) repeating steps (c) and (d) three or more times (e.g., 3, 4,5, 6, 7, 8,9, 10, etc.) in succession to produce backcross progeny of the selected fourth or higher generation comprising the desired trait. In one aspect, the weight may be continuously heavyRepeating steps (c) and (d) one or more times (e.g., 1, 2,3, 4,5, 6, 7, 8,9, 10, etc.) to produce second or higher generation backcross progeny comprising the desired trait.

In one aspect, the application includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar dS1746 with a second tobacco plant comprising a desired trait to produce F₁Progeny seed; (b) growth F₁Progeny seed and selection for F comprising a desired trait₁A progeny plant; (c) will select F₁Crossing the progeny plant with a plant of the first tobacco cultivar dS1746 to produce backcross BC₁F₁Progeny seed; (d) growing BC₁F₁Progeny seeds and selection of backcross BC comprising the desired trait and substantially all of the physiological and morphological characteristics of first tobacco cultivar dS1746₁F₁A progeny plant; and (e) repeating steps (c) and (d) three or more times in succession to produce backcross progeny of the selected fourth or higher generation that comprise the desired trait, wherein the trait is Cytoplasmic Male Sterility (CMS). In one aspect, steps (c) and (d) may be repeated one or more times in succession to produce backcross progeny of the second or higher generation that comprise the desired trait, wherein the trait is CMS.

In one aspect, the application includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a first tobacco cultivar dS1746 plant with a second tobacco plant comprising a desired trait to produce F₁Progeny seed; (b) growth F₁Progeny seed and selection for F comprising a desired trait₁A progeny plant; (c) will select F₁Crossing the progeny plant with a plant of the first tobacco cultivar to produce backcross BC₁F₁Progeny seed; (d) growing BC₁F₁Progeny seeds and selecting backcross BC comprising the desired trait and substantially all physiological and morphological characteristics of the first tobacco cultivar₁F₁A progeny plant; and (e) repeating steps (c) and (d) three or more times in succession to produce backcross progeny of the selected fourth or higher generation that comprise the desired traitWherein the trait is Cytoplasmic Male Sterility (CMS) and the CMS trait is obtained from the cytoplasm of sweet tobacco (Nicotiana suaveolens) or Nicotiana glauca. In one aspect, steps (c) and (d) may be repeated one or more times in succession to produce backcross progeny of the second or higher generation that comprise the desired trait, wherein the trait is CMS, and the CMS trait is obtained from the cytoplasm of sweet tobacco or nigella sativa.

In one aspect, the application includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar dS1746 with a second tobacco plant comprising a desired trait to produce F₁Progeny seed; (b) growth F₁Progeny seed and selection for F comprising a desired trait₁A progeny plant; (c) will select F₁Progeny plants are crossed with plants of the first tobacco cultivar dS1746 to produce backcross BC₁F₁Progeny seed; (d) growing BC₁F₁Progeny seeds and selection of backcross BC comprising the desired trait and substantially all of the physiological and morphological characteristics of tobacco cultivar dS1746₁F₁A progeny plant; and (e) repeating steps (c) and (d) three or more times in succession to produce backcross progeny of the selected fourth or higher generation that comprise the desired trait, wherein the trait is Cytoplasmic Male Sterility (CMS) and the CMS trait is obtained from the cytoplasm of sweet tobacco, and wherein the second tobacco plant is dS1746 MS. In one aspect, steps (c) and (d) may be repeated one or more times in succession to produce backcross progeny of a second or higher generation that comprise the desired trait, wherein the trait is CMS and the CMS trait is obtained from the cytoplasm of sweet tobacco.

In one aspect, the present application includes a tobacco plant produced by a method comprising introducing a desired trait into a tobacco cultivar, the method comprising: (a) crossing a plant of a first tobacco cultivar dS1746 or dS1746MS with a second tobacco plant comprising a desired trait to produce F₁Progeny seed; (b) growth F₁Progeny seed and selection for F comprising a desired trait₁A progeny plant; (c) will select F₁Progeny plant and said first cigarettePlant crossing of grass cultivars dS1746 or dS1746MS to produce backcross BC₁F₁Progeny seed; (d) growing BC₁F₁Progeny seeds and selection of backcross BC comprising the desired trait and substantially all of the physiological and morphological characteristics of the first tobacco cultivar dS1746 or dS1746MS₁F₁A progeny plant; and (e) repeating steps (c) and (d) three or more times in succession to produce backcross progeny of the selected fourth or higher generation that comprise the desired trait. In one aspect, steps (c) and (d) can be repeated one or more times in succession to produce backcross progeny of the second or higher generation that comprise the desired trait.

In another aspect, the present application includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar dS1746 or dS1746MS with a plant of a second tobacco cultivar comprising a desired trait to produce a progeny plant, wherein the desired trait is selected from the group consisting of: herbicide resistance, pest resistance, disease resistance, high yield, high grade index, curativeness, ripening quality, mechanical harvestability, holding power, leaf quality, height, plant maturity, precocity, early middle maturity, middle late maturity, small stem, medium stem, large stem, number of leaves per plant, 5-10 leaves per plant, 11-15 leaves per plant, 16-21 leaves per plant, and any combination thereof, to produce F₁Progeny seed; (b) growth F₁The progeny seed is F₁Progeny plants and selection of F having desired traits₁A progeny plant; (c) will select F₁Crossing a progeny plant with a plant of the first tobacco cultivar dS1746 or dS1746MS to produce backcross progeny plant seed; (d) growing the backcross progeny plant seed into a backcross progeny plant and selecting a backcross progeny plant comprising the desired trait and substantially all of the physiological and morphological characteristics of the first tobacco cultivar dS1746 or dS1746 MS; and (e) repeating steps (c) and (d) one or more times in succession to produce selected second, third, fourth or higher generation backcross progeny plants comprising the desired trait and substantially all of the physiological and morphological characteristics of first tobacco cultivar dS1746 or dS1746MS。

In another aspect, the present application includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar dS1746 or dS1746MS with a plant of a second tobacco cultivar comprising a desired trait to produce a progeny plant, wherein the desired trait is selected from the group consisting of: herbicide resistance, pest resistance, disease resistance, high yield, high grade index, curativeness, ripening quality, mechanical harvestability, holding power, leaf quality, height, plant maturity, precocity, early middle maturity, middle late maturity, small stem, medium stem, large stem, number of leaves per plant, 5-10 leaves per plant, 11-15 leaves per plant, 16-21 leaves per plant, and any combination thereof to produce F₁Progeny seed; (b) growth F₁The progeny seed is F₁Progeny plants and selection of F having desired traits₁A progeny plant; (c) let selected F₁Crossing a progeny plant with a plant of the first tobacco cultivar to produce backcross progeny plant seed; (d) growing the backcross progeny plant seed into a backcross progeny plant and selecting a backcross progeny plant comprising the desired trait and substantially all of the physiological and morphological characteristics of the first tobacco cultivar dS1746 or dS1746 MS; and (e) repeating steps (c) and (d) one or more times in succession to produce selected second, third, fourth or higher generation backcross progeny plants comprising the desired trait and substantially all of the physiological and morphological characteristics of said first tobacco cultivar dS1746 or dS1746 MS.

In another aspect, the present application includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar dS1746 or dS1746MS with a plant of a second tobacco cultivar comprising a desired trait to produce a progeny plant, wherein the desired trait is selected from the group consisting of: herbicide resistance, pest resistance, disease resistance, high yield, high grade index, cure quality, mechanical harvest, retention capacity, leaf quality, height, plant maturity, precocity, early middle maturity, middle late maturity, small stem, medium stem, large stem, number of leaves per plant, plant resistance, high yield, high disease resistance, cure quality, mechanical harvest, retention capacity, leaf quality, plant maturity, and/or plant maturity5-10 leaves per plant, 11-15 leaves per plant, 16-21 leaves per plant and any combination thereof to produce F₁Progeny seed; (b) growth F₁The progeny seed is F₁Progeny plants and selection of F having desired traits₁A progeny plant; (c) will select F₁Crossing a progeny plant with a plant of the first tobacco cultivar to produce backcross progeny plant seed; (d) growing the backcross progeny plant seed into a backcross progeny plant and selecting a backcross progeny plant comprising the desired trait and substantially all of the physiological and morphological characteristics of the first tobacco cultivar dS1746 or dS1746 MS; and (e) repeating steps (c) and (d) one or more times in succession to produce selected second, third, fourth or higher generation backcross progeny plants comprising the desired trait and substantially all of the physiological and morphological characteristics of the first tobacco cultivar dS1746 or dS1746MS, wherein the plants have the desired disease resistance trait.

In another aspect, the present application includes a method for producing a tobacco plant comprising: identifying a first tobacco plant comprising a nucleotide sequence selected from the group consisting of: 6-10 and any combination thereof; crossing the first tobacco plant with the second tobacco plant, and collecting F₁Seeds; will be composed of F₁Crossing the plant with the grown seed with a third tobacco plant and collecting the second tobacco seed; and identifying a second tobacco seed homozygous for the nucleotide sequence or a plant grown from the second seed. In one aspect, the second tobacco plant comprises a nucleotide sequence selected from the group consisting of seq id no:6-10 and any combination thereof. In another aspect, the third tobacco plant comprises a nucleotide sequence selected from the group consisting of seq id no:6-10 and any combination thereof. In one aspect, the first tobacco plant is a plant of tobacco cultivar dS1746 or dS1746 MS. In another aspect, the second tobacco plant is a plant of tobacco cultivar dS1746 or dS1746 MS. In another aspect, the third tobacco plant is a plant of tobacco cultivar dS1746 or dS1746 MS.

In another aspect, the present application includes a method of producing a plant of tobacco cultivar dS1746 or dS1746MS comprising at least one (e.g., 1, 2,3, 4,5, 6, 7, 8, etc.) additional desired trait comprising the steps of: (a) collecting tissue capable of propagating from a plant of tobacco cultivar dS1746 or dS1746 MS; and (b) introducing into said tissue at least one transgene (nucleic acid construct) conferring at least one desired trait.

In another aspect, the present application includes a method of producing a herbicide resistant tobacco plant comprising transforming a tobacco plant, or part thereof, produced by growing seeds of tobacco cultivar dS1746 or dS1746MS with at least one transgene (nucleic acid construct), wherein the at least one transgene (nucleic acid construct) confers resistance to an herbicide. In another aspect, the herbicide is selected from the group consisting of: imidazolinones, cyclohexanediones, sulfonylureas, glyphosate, glufosinate, phenoxypropionic acid, L-phosphinothricin, triazines, benzonitrile, and any combination thereof.

In another aspect, the present application includes herbicide-resistant tobacco plants produced by a method comprising transforming a tobacco plant, or a portion thereof, produced by growing seeds of tobacco variety dS1746 or dS1746MS with at least one transgene (nucleic acid construct), wherein the at least one transgene (nucleic acid construct) confers resistance to an herbicide. In another aspect, the herbicide is selected from the group consisting of: imidazolinones, cyclohexanediones, sulfonylureas, glyphosate, glufosinate, phenoxypropionic acid, L-phosphinothricin, triazines, benzonitrile, and any combination thereof.

In another aspect, the present application includes a method of producing a pest and/or insect resistant tobacco plant, wherein the method comprises transforming a tobacco plant produced by growing seeds of tobacco cultivar dS1746 or dS1746MS with at least one transgene (nucleic acid construct) that confers pest and/or insect resistance.

In another aspect, the present application includes pest and/or insect resistant tobacco plants produced by a method comprising transforming a tobacco plant produced by growing seeds of tobacco cultivar dS1746 or dS1746MS with at least one transgene (nucleic acid construct) that confers pest and/or insect resistance.

In another aspect, the present application includes a pest and/or insect resistant tobacco plant produced by a method comprising transforming a tobacco plant produced by growing seed of tobacco variety dS1746 or dS1746MS with at least one transgene (nucleic acid construct) conferring pest and/or insect resistance, wherein the transgene (nucleic acid construct) encodes a Bacillus Thuringiensis (BT) endotoxin.

In another aspect, the present application includes a method of producing a disease-resistant tobacco plant, comprising transforming a tobacco plant produced by growing seeds of tobacco cultivar dS1746 or dS1746MS with at least one transgene (nucleic acid construct) conferring disease resistance.

In another aspect, the present application includes disease resistant tobacco plants produced by transforming a tobacco plant produced by growing seeds of tobacco cultivar dS1746 or dS1746MS with at least one transgene (nucleic acid construct) conferring disease resistance.

As used herein, "similar growth conditions" refers to similar environmental conditions and/or agronomic practices for growing and making meaningful comparisons between two or more plant genotypes, such that neither the environmental conditions nor the agronomic practices contribute to or 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, pruning, undercutting, transplanting, topping and sprouting. See Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford (1999), pp 70-103, chapters 4B and 4C.

"alkaloids" are complex nitrogen-containing compounds that occur naturally in plants and have pharmacological effects in humans and animals. "Nicotine" is the major natural alkaloid in commercial cigarettes, and accounts for about 90% of the alkaloid content in tobacco. Other major alkaloids in tobacco include cotinine, nornicotine, mesmine, diennicotinyl, anabasine and anatabine. The small amount of tobacco alkaloid includes nicotine-N-oxide, N-methylanatabine, N-methylanabasine, pseudonicotine, 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, nicotinic alkaloid levels can be measured by the GC-FID method, which is based on CORESTA's recommended method No.7,1987 and ISO standards (ISO TC 126N 394E, also see Hibi et al, Plant Physiology 100:826-35(1992)), for a method using gas-liquid chromatography equipped with a capillary column and a FID detector.

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

In one aspect, the tobacco plants provided herein comprise an average nicotine or total alkaloid level 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, provided herein are tobacco plants comprising an average nicotine or total alkaloid level selected from the group consisting of: 0.01% -0.02%, 0.02% -0.05%, 0.05% -0.75%, 0.75% -0.1%, 0.1% -0.15%, 0.15% -0.2%, 0.2% -0.3%, 0.3% -0.35%, 0.35% -0.4%, 0.4% -0.5%, 0.5% -0.6%, 0.6% -0.7%, 0.7% -0.8%, 0.8% -0.9%, 0.9% -1%, 1% -1.1%, 1.1% -1.2%, 1.2% -1.3%, 1.3% -1.4%, 1.4% -1.5%, 1.5% -1.6%, 1.6% -1.7%, 1.7% -1.8%, 1.8% -1.9%, 1.9% -2%, 2% -2.1%, 2.1% -2.2%, 2.2.2% -2.2%, 2.3% -2.3%, 2.4% -2.5%, 2.3% -2.5%, 2.5% -2.5%, 2.5% -2.5%, 0.5% -2.4.5%, 0.5%, 0.4.4% and 2.4.4.4.4.4.5% -2.5% -2.4.5% -2.5% 2.8-2.9%, 2.9-3%, 3-3.1%, 3.1-3.2%, 3.2-3.3%, 3.3-3.4%, 3.4-3.5%, 3.5-3.6%. In another aspect, provided herein are tobacco plants comprising an average nicotine or total alkaloid level selected from the group consisting of: about 0.01% -0.1%, 0.02% -0.2%, 0.03% -0.3%, 0.04% -0.4%, 0.05% -0.5%, 0.75% -1%, 0.1% -1.5%, 0.15% -2%, 0.2% -3%, and 0.3% -3.5% on a dry weight basis.

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, the low nicotine or smokeless tobacco variety provides a commercially acceptable grade of cured tobacco. Tobacco grade is evaluated based on factors including, but not limited to, petiole position, leaf size, leaf color, leaf uniformity and integrity, maturity, texture, elasticity, luster (related to leaf color strength and depth and luster), hygroscopicity (the ability of the tobacco leaf to absorb and retain ambient moisture), and green notes or shades. Leaf ratings may be determined, for example, using official standard ratings published by agricultural sales services of the U.S. department of agriculture (7 u.s.c. § 511). See, for example, official standard grade burley tobacco (us type 31 and foreign type 93) effective at 5/11/1990 (55 f.r.40645); official standard grades (us types 11, 12, 13, 14 and foreign type 92) of flue-cured tobacco validated at 27.3.15.1989 (54 f.r.7925); official standard grades of Pennsylvania cotyledon tobacco (type 41 USA) that were validated 1, 8/1965 (29 F.R.16854); official standard grades (U.S. 42, 43 and 44 types) of solanum temp. tobacco, ohio, validated at 12, 8, 1963 (28 f.r.11719 and 28 f.r.11926); official standard grades of mixed cigar tobacco (U.S. types 54 and 55) in wisconsin, effective at 11/20/1969 (34 f.r.17061); official standard grades of mixed cigar tobacco (U.S. types 54 and 55) in wisconsin, effective at 11/20/1969 (34 f.r.17061); official standard grades of cigar wrap tobacco (us type 62) planted in shade in georgia and florida, effective in 4 months 1971. The USDA grade index may be determined from industry accepted grade indices. See, for example, Bowman et al, Tobacco Science,32:39-40 (1988); legacy Tobacco Document Library (Bates Document # 523267826) 523267833, July 1,1988, proposed Burley Scale index Memo); and Miller et al, 1990, Tobacco Intern, 192:55-57 (the foregoing references are incorporated herein by reference in their entirety). In one aspect, the USDA grade index represents the received federal grade by a number from 0 to 100 and is a weighted average of all stem positions. Higher grade index indicates higher quality. Alternatively, blade grade may be determined by hyperspectral imaging. See, for example, WO 2011/027315 (published 3/10/2011, and incorporated herein by reference in its entirety).

dS1746

In one aspect, the present application provides tobacco cultivars and portions thereof grown or developed from dS 1746. In another aspect, the present application provides a tobacco plant, or part thereof, produced by growing the seed of dS 1746. In another aspect, the plants of the present application can include plants having all or substantially all of the morphological and physiological characteristics of cultivar dS 1746.

Although not limited by the method, dS1746 is a backcross-derived form of burley tobacco cultivar TN90 carrying a homozygous introgression at two loci (nic1 and nic2 loci) that results in an overall reduction in alkaloid content in the lines. dS1746 is the result of three backcrosses with burley cultivar TN90 as the recurrent parent, followed by four rounds of selfing and selection for homozygosity at the nic1 and nic2 loci. The dS1746 progeny plants have a genetic background at least 95%, at least 97%, at least 98% or at least 99% similar to TN 90. dS1746 plants exhibit low nicotine levels.

dS1746MS

In one aspect, the present application provides tobacco cultivars and portions thereof grown or developed by dS1746 MS. In another aspect, the present application also includes seeds produced by growing dS1746MSA tobacco plant or part thereof. In yet another aspect, the plants of the present application can include plants having all or substantially all of the morphological and physiological characteristics of cultivar dS1746 MS. dS1746MS is a Male Sterile (MS) version of dS 1746. Because the dS1746MS line is male sterile, it is maintained by pollination with dS 1746. Preparation of F of dS1746MS by crossing dS1746 as male parent with dS1746MS₁A progeny plant. The dS1746MS progeny plants may have a genetic background at least 95%, at least 97%, at least 98% or at least 99% similar to TN 90. dS1746MS and dS1746MS F₁Progeny plants exhibit low nicotine levels.

Other plants

The present application includes tobacco seeds produced by crossing two parent tobacco plants and harvesting the resulting tobacco seeds, wherein at least one parent tobacco plant is dS 1746. In one aspect, dS1746 is a paternal plant. In another aspect, dS1746MS is a female parent plant. One aspect of the present application provides a tobacco plant homozygous for the nic1 and nic2 loci that has a similar genetic background to TN90 or MS TN90 (male sterile TN 90) of greater than about 75%, 80%, 85%, 90%, 95%, 98% or 99%. In one aspect, about or greater than about 50%, 75% or 100% of the progeny genes are provided by plants of the present application that are homozygous for the nic1 and nic2 loci. In one aspect, the plants of the present application have a genetic background that is at least 95%, at least 97%, at least 98% or at least 99% similar to TN90 or MS TN 90. In another aspect, the plants of the present application exhibit low nicotine. In one aspect, the plant of the present application is a progeny plant of a female or male parent plant resistant to Fusarium wilt. On the other hand, the plants of dS1746 have low resistance to blackleg and moderate resistance to bacterial wilt.

In one aspect, the plants of the present application are mid-late maturing varieties with moderately high yield potential. In another aspect, the plants of the present application provide a wide range of important agronomic characteristics. In another aspect, the plants of the present application have one, two, three, four or more traits, including moderate resistance to blackleg, tolerance to penicillium, resistance to black root rot, and resistance to common viral diseases. In another aspect, the plants of the present application have penicillium resistance and grade 4 resistance to black shank and high root rot. In one aspect, a plant of the present application, such as dS1746, dS1746MS, dS1564, or dS1564MS, lacks wilt resistance. In another aspect, the plants of the present application are resistant to fusarium wilt. In another aspect, the plants of the present application have low resistance to blackleg and moderate resistance to bacterial wilt.

In one aspect, the tobacco plants of the present application comprise one or more mutations (e.g., nonsense or loss-of-function mutations) in one or more nicotine demethylase genes (CYP82E4, CYP82E5v2, CYP85E 10). In another aspect, the tobacco plant comprises one or more of a cyp82e 4W 329Stop, cyp82e5v 2W 422Stop, and cyp82e 10P 381S mutation.

In one aspect, the plants of the present application have a reduced or eliminated ability to convert nicotine to nornicotine. In one aspect, the percent nicotine conversion may be less than about 75%, 70%, 60%, 50%, or 25% of those found in TN90 or K326. In another aspect, nicotine conversion in a plant of the present application (including dS1746, dS1746MS, dS1564, or dS1564MS) may be less than about 4%, about 3.5%, about 3%, about 2.5%, about 2%, about 1.5%, about 1%, or any range therein. In yet other aspects, nicotine conversion in plants of the present application (including dS1746, dS1746MS, dS1564, or dS1564MS) can range from about 3% to about 1%, from about 3% to about 0.5%, or from about 2% to about 0.5%. In one aspect, the tobacco plants of the present application can have a nicotine conversion rate of about 3.5, 3.25, 3.0, or 2.75% or less. In another aspect, the nicotine conversion rate of a tobacco plant of the present application can be about 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5% or less or any range therein. In another aspect, the nicotine conversion rate of a tobacco plant of the present application can be about 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6% or less or any range therein. In another aspect, the nicotine conversion may be in the range of about 0.5% to about 0.9%, about 0.5% to about 1.5%, about 0.5% to about 2.0%, about 0.5% to about 2.5%, about 0.5% to about 2.75%, and about 0.5% to about 3.0%. In another aspect, the nicotine conversion may be in the range of about 1.0% to about 1.5%, about 1.0% to about 1.75%, about 1.0% to about 2.0%, about 1.0% to about 2.5%, about 1.0% to about 2.75%, or about 1.0% to about 3.0%. In another aspect, the nicotine conversion rate in a plant of the present application can be less than about 2.9, 2.75, 2.5, 2.25, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, or 1.0% or any range therein.

In another aspect, the tobacco plants of the present application typically have a reduced nicotine reduction amount of less than about 0.10% dry weight. For example, the nornicotine content in these plants can be about 1.2, 1.0, 0.7, 0.5, 0.4, 0.2, 0.1, 0.09, 0.085, 0.08, 0.075, 0.07, 0.065, 0.06, 0.055, 0.05, 0.045, 0.04, 0.035, 0.025, 0.01, 0.009, 0.0075, 0.005, 0.0025, 0.001, 0.0009, 0.00075, 0.0005, 0.00025, or 0.0001% dry weight, or undetectable, or any range therein. In another aspect, the nornicotine level can be less than about 1.2, 1.0, 0.9, 0.8, 0.7, 0.5, 0.4, 0.2, 0.1, 0.075, 0.05, 0.025, 0.01, 0.009, 0.0075, 0.005, 0.0025, 0.001, 0.0009, 0.00075, 0.0005, 0.00025, or 0.0001% dry weight, or any range therein. In another aspect, the nornicotine content in these plants can be in the dry weight range of about 1.2% to about 1.0%, about 0.7% to about 0.5%, about 0.4% to about 0.2%, about 0.1% to about 0.075%, about 0.05% to about 0.025%, about 0.01% to about 0.0075%, about 0.005% to about 0.0025%, about 0.001% to about 0.00075%, about 0.0005% to about 0.00025%, or about 0.0005% to about 0.0001%. In one aspect, nornicotine comprises a relatively small percentage of total alkaloids in the plant as compared to a commercial seed lot of TN90 or K326 in the plants of the present application. In one aspect, the nornicotine in a plant of the present application can be about 2% to about 1%, less than 3%, about 2%, about 1.5%, about 1%, or 0.75% of total alkaloids. Tobacco products having reduced nitrosamine content can be made using tobacco plant material from plants and plant parts of the present application. Thus, in some embodiments, tobacco products manufactured using tobacco plant material from plants and plant parts of the present application can comprise a reduced nicotine reduction amount of less than about 3 mg/g. For example, the nornicotine content in these articles of manufacture may be 3.0mg/g, 2.5mg/g, 2.0mg/g, 1.5mg/g, 1.0mg/g, 750 μ g/g, 500 μ g/g, 250 μ g/g, 100 μ g/g, 75 μ g/g, 50 μ g/g, 25 μ g/g, 10 μ g/g, 5 μ g/g, 1 μ g/g, 750ng/g, 500ng/g, 250ng/g, 100ng/g, 75ng/g, 50ng/g, 25ng/g, 10ng/g, 5ng/g, 1ng/g, 750pg/g, 500pg/g, 250pg/g, 100ng/g, 75 g, 50 g/g, 25, 10 g/g, 7.0pg/g, 5.0pg, 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, and the like, or undetectable, or any range therein. The tobacco product typically has a reduced amount of NNN of less than about 10 pg/g. For example, the NNN content in these articles can be about 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 the like, or undetectable, or any range therein.

The difference between two inbred tobacco varieties or two hybrid tobacco varieties can be assessed using statistical methods. The statistical analysis includes calculation of a mean, determination of statistical significance of the source of variation, and calculation of an appropriate variance component. Methods for determining statistical significance are known in the art. Statistical software is available, for example, PROC GLM functionality of SAS. Significance is often expressed as "p-value". Statistically significant p-values were less than 0.10. In a preferred aspect, the p-value is less than or equal to 0.05. In another aspect, the p value is 0.04 or less, 0.03 or less, or 0.02 or less. In yet another aspect, the statistically significant value is less than 0.01. In yet another aspect, it may be less than 0.009, less than 0.008, less than 0.007, less than 0.006, less than 0.005, less than 0.004, less than 0.003, less than 0.002, or less than 0.001.

In one aspect, the tobacco plant of the present application has a USDA leaf mass index of at least about 85, 80, 75, 73, 72, 71, 70, 69, 68, 67, or 66, or any range therein. In one aspect, the tobacco plants of the present application have a USDA leaf mass index of at least about 65, in another aspect, the mass index can be at least about 55, 60, 62.5, or any range therein. In another aspect, the tobacco plant of the present application may have a lamina mass index in the range of about 60 to about 65, about 60 to about 70, about 62.5 to about 65, about 62.5 to about 70, or about 65 to about 70.

Plants of the present application, including dS1746, dS1746MS, dS1564, 1564MS, or progeny thereof, may have any yield potential, including high (e.g., greater than 3000lbs/a), medium (e.g., 2200-.

One aspect of the present application provides a portion of cultivars dS1746, dS1746MS, dS1564, or dS1564 MS. Parts of a cultivar can comprise any plant part including, but not limited to, leaves, pollen, embryos, cotyledons, hypocotyls, roots, root tips, anthers, flowers, ovules, shoots, stems, stalks, pith and pods, tissue cultures comprising tissue, callus, cells or protoplasts of cultivars dS1746, dS1746MS, dS1564 or dS1564 MS. In another aspect, the present application provides a portion from a hybrid line derived from cultivar dS1746 or dS1746 MS. In yet another aspect, the present application provides portions from genetically modified (e.g., by conventional breeding or genetic engineering techniques) forms of the plants and tissue cultures described above.

Another aspect of the present application provides an article of manufacture comprising tobacco and portions thereof from a plant of the present application. Another aspect of the present application provides a cured plant part including, but not limited to, leaves, pollen, ovules, embryos, cotyledons, hypocotyls, meristematic cells, protoplasts, roots, root tips, pistils, anthers, flowers, buds, stems, pods, petioles, and the like, and combinations thereof.

Thus, in one aspect, the present application provides cured tobacco comprising the leaves of a tobacco plant designated dS 1746. In another aspect, the present application provides cured tobacco comprising the leaves of a tobacco plant designated dS1746 MS.

In one aspect, the present application provides cured tobacco comprising the stem of a tobacco plant designated dS 1746. In another aspect, the present application provides cured tobacco comprising the stem of a tobacco plant designated dS1746 MS.

In one aspect, the present application provides cured tobacco comprising the leaves and stems of a tobacco plant designated dS 1746. In another aspect, the present application provides cured tobacco comprising the leaves and stems of a tobacco plant designated dS1746 MS.

Also provided herein are containers of dS1746 or dS1746MS seeds or other seeds of the present application, wherein the alkaloids obtained from tobacco plants grown from greater than about 50% of the seeds have reduced nicotine. In another aspect, in the container, the alkaloid obtained from a dS1746 or dS1746MS plant or other plant grown from greater than about 5%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the seeds in the container of the present application has reduced nicotine.

The containers of dS1746 or dS1746MS seeds, or other seeds of the present application, 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 contain 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 dS1746 or dS1746MS seeds or other seeds of the present application may be any container available in the art. As non-limiting examples, the container may be a box, bag, pouch, roll of film (tape roll), tub, foil, or tube.

In another aspect, the present application also provides a container of dS1746 or dS1746MS, wherein greater than about 50% of the dS1746 or dS1746MS seeds, or other seeds of the present application, have reduced nicotine.

In one aspect, the tobacco plant population is also provided. In one aspect, the tobacco plant population has a planting density of about 5,000 to about 8000, about 5,000 to about 7,600, about 5,000 to about 7,200, about 5,000 to about 6,800, about 5,000 to about 6,400, about 5,000 to about 6,000, about 5,000 to about 5,600, about 5,000 to about 5,200, about 5,200 to about 8,000, about 5,600 to about 8,000, about 6,000 to about 8,000, about 6,400 to about 8,000, about 6,800 to about 8,000, about 7,200 to about 8,000, or about 7,600 to about 8,000 plants per acre. In another aspect, the tobacco plant population is in a soil type having low to moderate fertility.

In one aspect, the present application provides seeds of the dS1746 or dS1746MS plants, or other plants of the present application, wherein the plants grown from the seeds are male sterile.

In one aspect, the present application includes seeds of tobacco cultivar dS1564, a representative sample seed of which was deposited with the American Type Culture Collection (ATCC) under ATCC accession No. PTA-125408.

In another aspect, the present application includes a tobacco plant, or portion thereof, produced by growing seeds of tobacco cultivar dS 1564.

In another aspect, the present application includes a harvested leaf or portion thereof of a tobacco plant, or portion thereof, produced by growing a seed of tobacco cultivar dS 1564.

In one aspect, the present application includes harvested leaf of a tobacco plant produced by growing seed of tobacco cultivar dS1564, or a portion thereof, wherein the leaf has a reduced amount of nicotine relative to a control tobacco variety having substantially the same genetic background as dS1564 (except for the ni 1 and ni 2 loci introgressed dS1746) when grown under similar growth conditions.

In another aspect, the present application includes a tobacco product prepared from a tobacco plant, or a portion thereof, produced by growing seeds of tobacco variety dS1564, wherein the product is selected from the group consisting of: pipe tobacco, cigars, cigarettes, chewing tobacco, tobacco leaves, tobacco shreds and any combination thereof.

In one aspect, the present application includes a tobacco product prepared from a tobacco plant, or a portion thereof, produced by growing seeds of tobacco variety dS1564, wherein the product is selected from the group consisting of: cigarillos, non-ventilated filter cigarettes, cigars, snuff and chewing tobacco.

In another aspect, the tobacco product has a reduced amount of nicotine relative to a tobacco product not prepared from dS 1564.

In another aspect, the present application includes a second tobacco product prepared or produced from a first tobacco product prepared from a tobacco plant, or part thereof, produced by growing seeds of tobacco variety dS1564, wherein the first product is selected from the group consisting of: pipe tobacco, cigar, cigarette, chewing tobacco, tobacco leaf, tobacco shred, and any combination thereof, and the second tobacco product is selected from the group consisting of: cigarillos, non-ventilated filter cigarettes, cigars, snuff and chewing tobacco. In one aspect, the first and second articles comprise reduced amounts of nicotine.

In one aspect, the present application includes a portion of a tobacco plant produced by growing seeds of tobacco cultivar dS1564, wherein the portion is selected from the group consisting of: leaves, pollen, ovules, embryos, cotyledons, hypocotyls, meristematic cells, protoplasts, roots, root tips, pistils, anthers, flowers, buds, stems, pods and petioles.

In another aspect, the present application includes a tissue culture produced from protoplasts or cells from a tobacco plant or part thereof produced by growing seeds of tobacco cultivar dS1564, wherein the cells or protoplasts of the tissue culture are produced from a plant part selected from the group consisting of: leaves, pollen, embryos, cotyledons, hypocotyls, meristematic cells, roots, root tips, pistils, anthers, flowers, buds, stems, pods and petioles.

In one aspect, the present application includes a tobacco plant regenerated from a tissue culture produced from protoplasts or cells from a tobacco plant or part thereof produced by growing seeds of tobacco cultivar dS1564, wherein the cells or protoplasts of the tissue culture are produced from a plant part selected from the group consisting of: leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root tip, pistil, anther, flower, bud, stem, pod and petiole, wherein the regenerated plant has all or substantially all of the morphological and physiological characteristics of cultivar dS 1564.

In one aspect, the application includes F of tobacco cultivar dS1564₁A progeny plant.

At another placeIn one aspect, the present application includes F of tobacco cultivar dS1564₁A progeny plant, a representative sample seed of the cultivar having been deposited with the ATCC as ATCC accession number PTA-125408, wherein F₁Plants are Male Sterile (MS). In another aspect, F₁Plants are Cytoplasmic Male Sterile (CMS).

In another aspect, the present application includes F produced by crossing two tobacco plants and harvesting the resulting tobacco seeds₁Progeny seed, wherein at least one tobacco plant is a tobacco plant produced by growing seed of tobacco cultivar dS1564, wherein at least one tobacco plant is MS or CMS.

In another aspect, the present application includes a method for producing tobacco seed comprising crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one tobacco plant is a tobacco plant produced by growing seed of tobacco cultivar dS 1564.

In another aspect, the present application includes a method for producing tobacco seed comprising crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one tobacco plant is a tobacco plant produced by growing seed of tobacco cultivar dS1564, wherein the plant of tobacco cultivar dS1564 is the male parent.

In another aspect, the present application includes a method for producing tobacco seed comprising crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one tobacco plant is a tobacco plant produced by growing seed of tobacco cultivar dS1564, wherein the plant of tobacco cultivar dS1564 is the female parent.

In another aspect, the present application includes a method for producing tobacco seed comprising crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one tobacco plant is a tobacco plant produced by growing seed of tobacco cultivar dS1564, wherein at least one tobacco plant is MS or CMS.

In one aspect, the application also includes a plant produced by crossing two tobacco plants and harvesting the resulting tobacco seedsF₁A container of progeny seed, wherein at least one tobacco plant is a tobacco plant produced by growing seeds of tobacco cultivar dS1564, wherein at least one tobacco plant is MS or CMS.

In one aspect, the application includes F produced by growing seeds₁A progeny plant, said seed produced by crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one tobacco plant is a tobacco plant produced by growing seed of tobacco cultivar dS1564, and wherein at least one tobacco plant is MS or CMS.

In one aspect, the application also includes F produced by growing a hybrid of two tobacco plants and harvesting the resulting tobacco seeds₁Harvested leaf of a progeny plant, wherein at least one tobacco plant is a tobacco plant produced by growing seeds of tobacco cultivar dS1564, wherein at least one tobacco plant is MS or CMS. On the other hand, when grown under similar growth conditions, from F compared to a control tobacco variety with essentially the same genetic background as dS1564 (except for the nic1 and nic2 locus introgression into dS1746)₁The harvested leaves of the progeny plants have a reduced amount of nicotine.

In one aspect, the present application includes a composition consisting of F₁A tobacco product made from a progeny tobacco plant or part thereof, wherein said plant or part thereof is produced by growing F₁Progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one tobacco plant is produced by growing seed of tobacco cultivar dS1564, wherein at least one tobacco plant is cytoplasmic male sterile, and wherein the tobacco product is selected from the group consisting of: pipe tobacco, cigars, cigarettes, chewing tobacco, tobacco leaves, tobacco shreds and any combination thereof.

In one aspect, the present application also includes a composition consisting of F₁A tobacco product made from a progeny tobacco plant or part thereof, wherein said plant or part thereof is produced by growing F₁Progeny seed production by a method comprising crossing two tobacco plants and harvesting the resulting tobacco seed, whichWherein at least one tobacco plant is produced by growing seeds of tobacco cultivar dS1564, wherein at least one tobacco plant is cytoplasmic male sterile, and wherein the tobacco product is selected from the group consisting of: cigarillos, non-ventilated filter cigarettes, cigars, snuff and chewing tobacco.

In another aspect, the present application includes seeds of tobacco cultivar dS1564MS, a representative sample seed of which was deposited with the ATCC under ATCC accession No. PTA-126413.

In one aspect, the present application includes a tobacco plant, or portion thereof, produced by growing seeds of tobacco cultivar dS1564 MS.

In another aspect, the present application includes harvested leaves or portions thereof of a tobacco plant, or portion thereof, produced by growing seeds of tobacco cultivar dS1564 MS.

In one aspect, the present application includes harvested leaves of a tobacco plant produced by growing seeds of tobacco variety dS1564MS, or a portion thereof, wherein the leaves have a reduced amount of nicotine relative to a control tobacco variety having substantially the same genetic background as dS1564MS (except for the nic1 and nic2 loci introgressed into dS1746MS) when grown under similar growth conditions.

In another aspect, the present application includes a tobacco product prepared from a tobacco plant, or a portion thereof, produced by growing seeds of tobacco cultivar dS1564MS, wherein the product is selected from the group consisting of: pipe tobacco, cigars, cigarettes, chewing tobacco, tobacco leaves, tobacco shreds and any combination thereof.

In one aspect, the present application includes a tobacco product prepared from a tobacco plant, or a portion thereof, produced by growing seeds of tobacco cultivar dS1564MS, wherein the product is selected from the group consisting of: cigarillos, non-ventilated filter cigarettes, cigars, snuff and chewing tobacco.

In another aspect, the present application includes a tobacco product made from a tobacco plant, or a portion thereof, produced by growing seeds of tobacco cultivar dS1564MS, wherein the product is selected from the group consisting of: a cigarillo, a non-ventilated filter cigarette, a cigar, snuff and a chewing tobacco, wherein the article has a reduced amount of nicotine when grown under similar growth conditions relative to a control article prepared from a control tobacco plant having substantially the same genetic background as dS1564MS (except for the ni 1 and ni 2 loci introgressed into dS 1746).

In another aspect, the present application includes a second tobacco product prepared or produced from a first tobacco product prepared from a tobacco plant, or part thereof, produced by growing seeds of tobacco cultivar dS1564MS, wherein the first product is selected from the group consisting of: pipe tobacco, cigar, cigarette, chewing tobacco, tobacco leaf, tobacco shred, and any combination thereof, and the second tobacco product is selected from the group consisting of: cigarillos, non-ventilated filter cigarettes, cigars, snuff and chewing tobacco. In one aspect, the first and/or second article comprises a reduced amount of nicotine.

In one aspect, the present application includes a portion of a tobacco plant produced by growing seeds of tobacco cultivar dS1564MS, wherein the portion is selected from the group consisting of: leaves, pollen, ovules, embryos, cotyledons, hypocotyls, meristematic cells, protoplasts, roots, root tips, pistils, anthers, flowers, buds, stems, pods and petioles.

In another aspect, the present application includes a tissue culture produced from protoplasts or cells from a tobacco plant or part thereof produced by growing seeds of tobacco cultivar dS1564MS, wherein the cells or protoplasts of the tissue culture are produced from a plant part selected from the group consisting of: leaves, pollen, embryos, cotyledons, hypocotyls, meristematic cells, roots, root tips, pistils, anthers, flowers, buds, stems, pods and petioles.

In one aspect, the present application includes a tobacco plant regenerated from a tissue culture produced from protoplasts or cells from a tobacco plant or portion thereof produced by growing seeds of tobacco cultivar dS1564MS, wherein the cells or protoplasts of the tissue culture can be produced from a plant portion selected from the group consisting of: leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root tip, pistil, anther, flower, bud, stem, pod and petiole, wherein the regenerated plant has all or substantially all of the morphological and physiological characteristics of cultivar dS1564 MS.

In one aspect, the present application includes F of tobacco cultivar dS1564MS₁A progeny plant.

In another aspect, the present application includes F produced by crossing two tobacco plants and harvesting the resulting tobacco seeds₁Progeny seed, wherein at least one tobacco plant is a plant of tobacco cultivar dS1564 MS.

In another aspect, the present application includes a method for producing tobacco seed comprising crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one tobacco plant is a tobacco plant produced by growing seed of tobacco cultivar dS1564 MS.

In another aspect, the present application includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one tobacco plant is a tobacco plant produced by growing seed of tobacco cultivar dS1564MS, wherein the plant of tobacco cultivar dS1564MS is the female parent.

In another aspect, the present application includes a method for producing tobacco seed comprising crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one of the tobacco plants is a plant of tobacco cultivar dS1564 MS.

In one aspect, the present application also includes F produced by crossing two tobacco plants and harvesting the resulting tobacco seeds₁A container of progeny seed, wherein at least one tobacco plant is a tobacco plant produced by growing seeds of tobacco cultivar dS1564 MS.

In one aspect, the application includes F produced by growing seeds₁Progeny plants produced by crossing two tobacco plants and harvesting the resulting tobacco seeds, wherein at least one tobacco plant is a tobacco plant produced by growing seed of tobacco cultivar dS1564MS。

In one aspect, the present application also includes harvested leaves of a F1 progeny plant produced by growing seeds produced by crossing two tobacco plants and harvesting the resulting tobacco seeds, wherein at least one tobacco plant is a tobacco plant produced by growing seeds of tobacco cultivar dS1564 MS.

In another aspect, the present application also includes F produced by growing seeds having a reduced amount of nicotine₁Harvested leaf of a progeny plant, said seed produced by crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one tobacco plant is a tobacco plant produced by growing seed of tobacco cultivar dS1564 MS.

In one aspect, the present application includes a composition consisting of F₁A tobacco product made from a progeny tobacco plant or part thereof, wherein the plant or part thereof is produced by growing F resulting from crossing two tobacco plants and harvesting the resulting tobacco seeds₁Progeny seed, wherein at least one tobacco plant is a tobacco plant produced by growing seeds of tobacco variety dS1564MS, and wherein the tobacco product is selected from the group consisting of: pipe tobacco, cigars, cigarettes, chewing tobacco, tobacco leaves, tobacco shreds and any combination thereof.

In one aspect, the present application also includes a composition consisting of F₁A tobacco product made from a progeny tobacco plant or part thereof, wherein the plant or part thereof is produced by growing F resulting from crossing two tobacco plants and harvesting the resulting tobacco seeds₁Progeny seed, wherein at least one tobacco plant is a tobacco plant produced by growing seeds of tobacco cultivar dS1564MS, and wherein the tobacco product is selected from the group consisting of: cigarillos, non-ventilated filter cigarettes, cigars, snuff and chewing tobacco.

In one aspect, the present application also includes a composition consisting of F₁A tobacco product made from a progeny tobacco plant or part thereof, wherein the plant or part thereof is produced by growing F resulting from crossing two tobacco plants and harvesting the resulting tobacco seeds₁Progeny seed, wherein at least one tobacco is plantedThe plant is a tobacco plant produced by growing seeds of tobacco cultivar dS1564MS, and wherein the tobacco product is selected from the group consisting of: cigarillos, non-ventilated filter cigarettes, cigars, snuff and chewing tobacco and further wherein the article has a reduced amount of nicotine.

In one aspect, the present application includes a method for producing tobacco seed comprising crossing two tobacco plants and harvesting the resulting tobacco seed, wherein at least one tobacco plant is a plant of tobacco cultivar dS1564 MS.

In one aspect, the present application includes a method of asexually propagating a plant of the tobacco cultivar comprising the steps of: (a) collecting tissue capable of propagating from a plant of tobacco cultivar dS1564 or dS1564 MS; (b) culturing the tissue to obtain multiplied shoots; and (c) rooting the proliferated shoots to obtain rooted shoots.

In one aspect, the present application includes a method of asexually propagating a plant of the tobacco cultivar comprising the steps of: (a) collecting tissue capable of propagating from a plant of tobacco cultivar dS1564 or dS1564 MS; (b) culturing the tissue to obtain multiplied shoots; (c) rooting the proliferated buds to obtain rooted seedlings; and (d) growing plants from the rooted shoots.

In one aspect, the application includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar dS1564 or dS1564MS with a second tobacco plant comprising a desired trait to produce F₁Progeny seed; (b) growth F₁Progeny seed and selection for F comprising a desired trait₁A progeny plant; (c) will select F₁Crossing the progeny plant with a plant of the first tobacco cultivar to produce backcross BC₁F₁Progeny seed; (d) growing BC₁F₁Progeny seeds and selecting backcross BC comprising the desired trait and substantially all physiological and morphological characteristics of the first tobacco cultivar₁F₁A progeny plant; and (e) repeating steps (c) and (d) three or more times (e.g., 3, 4,5, 6, 7, 8,9, 10, etc.) in succession to produce a plant comprising the desired traitBackcross progeny of the selected fourth or higher generation. In one aspect, steps (c) and (d) can be repeated one or more times (e.g., 1, 2,3, 4,5, 6, 7, 8,9, 10, etc.) in succession to produce backcross progeny of a second or higher generation that comprise the desired trait.

In one aspect, the application includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar dS1564 with a second tobacco plant comprising a desired trait to produce F₁Progeny seed; (b) growth F₁Progeny seed and selection for F comprising a desired trait₁A progeny plant; (c) will select F₁Progeny plants are crossed with a first tobacco cultivar dS1564 plant to produce backcross BC₁F₁Progeny seed; (d) growing BC₁F₁Progeny seeds and selecting backcross BC comprising the desired trait and substantially all of the physiological and morphological characteristics of the first tobacco cultivar dS1564₁F₁A progeny plant; and (e) repeating steps (c) and (d) three or more times in succession to produce backcross progeny of the selected fourth or higher generation that comprise the desired trait, wherein the trait is Cytoplasmic Male Sterility (CMS). In one aspect, steps (c) and (d) may be repeated one or more times in succession to produce backcross progeny of the second or higher generation that comprise the desired trait, wherein the trait is CMS.

In one aspect, the application includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a first tobacco cultivar dS1564 plant with a second tobacco plant comprising a desired trait to produce F₁Progeny seed; (b) growth F₁Progeny seed and selection for F comprising a desired trait₁A progeny plant; (c) will select F₁Crossing the progeny plant with a plant of the first tobacco variety to produce backcross BC₁F₁Progeny seed; (d) growing BC₁F₁Progeny seeds and selecting backcross BC comprising the desired trait and substantially all physiological and morphological characteristics of the first tobacco cultivar₁F₁A progeny plant; and (e) repeating steps (c) and (d) three or more times in succession to produce a packet(ii) backcross progeny of the selected fourth or higher generation that comprise the desired trait, wherein the trait is Cytoplasmic Male Sterility (CMS) and the CMS trait is obtained from the cytoplasm of sweet tobacco or nicotiana tabacum. In one aspect, steps (c) and (d) may be repeated one or more times in succession to produce backcross progeny of the second or higher generation that comprise the desired trait, wherein the trait is CMS, and the CMS trait is obtained from the cytoplasm of sweet tobacco or nigella sativa.

In one aspect, the application includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar dS1564 with a second tobacco plant comprising a desired trait to produce F₁Progeny seed; (b) growth F₁Progeny seed and selection for F comprising a desired trait₁A progeny plant; (c) will select F₁Progeny plants are crossed with plants of first tobacco cultivar dS1564 to produce backcross BC₁F₁Progeny seed; (d) growing BC₁F₁Progeny seeds and selection of backcross BC comprising the desired trait and substantially all of the physiological and morphological characteristics of tobacco cultivar dS1564₁F₁A progeny plant; and (e) repeating steps (c) and (d) three or more times in succession to produce backcross progeny of the selected fourth or higher generations that comprise the desired trait, wherein the trait is Cytoplasmic Male Sterility (CMS) and the CMS trait is obtained from the cytoplasm of sweet tobacco, and wherein the second tobacco plant is dS1564 MS. In one aspect, steps (c) and (d) may be repeated one or more times in succession to produce backcross progeny of the second or higher generation that comprise the desired trait, wherein the trait is CMS, and the CMS trait is obtained from the cytoplasm of sweet tobacco.

In one aspect, the present application includes a tobacco plant produced by a method comprising introducing a desired trait into a tobacco cultivar, the method comprising: (a) crossing a plant of a first tobacco cultivar dS1564 or dS1564MS with a second tobacco plant comprising a desired trait to produce F₁Progeny seed; (b) growth F₁Progeny seed and selection for F comprising a desired trait₁A progeny plant; (c) will select F₁Progeny plants are crossed with a first tobacco cultivar dS1564 or dS1564MS plant to produce backcross BC₁F₁Progeny seed; (d) growing BC₁F₁Progeny seeds and selecting backcross BC comprising the desired trait and substantially all of the physiological and morphological characteristics of the first tobacco cultivar dS1564 or dS1564MS₁F₁A progeny plant; and (e) repeating steps (c) and (d) three or more times in succession to produce backcross progeny of the selected fourth or higher generation that comprise the desired trait. In one aspect, steps (c) and (d) can be repeated one or more times in succession to produce backcross progeny of the second or higher generation that comprise the desired trait.

In another aspect, the present application includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar dS1564 or dS1564MS with a plant of a second tobacco cultivar comprising a desired trait to produce a progeny plant, wherein the desired trait is selected from the group consisting of: herbicide resistance, pest resistance, disease resistance, high yield, high grade index, curativeness, ripening quality, mechanical harvestability, holding power, leaf quality, height, plant maturity, precocity, early middle maturity, middle late maturity, small stem, medium stem, large stem, number of leaves per plant, 5-10 leaves per plant, 11-15 leaves per plant, 16-21 leaves per plant, and any combination thereof, to produce F₁Progeny seed; (b) growth F₁The progeny seed is F₁Progeny plants and selection of F having desired traits₁A progeny plant; (c) will select F₁Crossing the progeny plant with a first tobacco cultivar dS1564 or dS1564MS plant to produce backcross progeny plant seed; (d) growing the backcross progeny plant seeds into backcross progeny plants and selecting a backcross progeny plant comprising the desired trait and substantially all physiological and morphological characteristics of first tobacco cultivar dS1564 or dS1564 MS; and (e) repeating steps (c) and (d) one or more times in succession to produce selected backcross progeny plants of the second, third, fourth or higher generation that comprise the desired trait and substantially all of the progeny of said first tobacco cultivar dS1564 or dS1564MSPhysical and morphological characteristics.

In another aspect, the present application includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar dS1564 or dS1564MS with a plant of a second tobacco cultivar comprising a desired trait to produce a progeny plant, wherein the desired trait is selected from the group consisting of: herbicide resistance, pest resistance, disease resistance, high yield, high grade index, curativeness, ripening quality, mechanical harvestability, holding power, leaf quality, height, plant maturity, precocity, early middle maturity, middle late maturity, small stem, medium stem, large stem, number of leaves per plant, 5-10 leaves per plant, 11-15 leaves per plant, 16-21 leaves per plant, and any combination thereof, to produce F₁Progeny seed; (b) growth F₁The progeny seed is F₁Progeny plants and selection of F having desired traits₁A progeny plant; (c) will select F₁Crossing the progeny plant with a plant of the first tobacco cultivar to produce backcross progeny plant seed; (d) growing the backcross progeny plant seeds into backcross progeny plants and selecting a backcross progeny plant comprising the desired trait and substantially all physiological and morphological characteristics of first tobacco cultivar dS1564 or dS1564 MS; and (e) repeating steps (c) and (d) one or more times in succession to produce selected second, third, fourth or higher generation backcross progeny plants comprising the desired trait and substantially all of the physiological and morphological characteristics of first tobacco cultivar dS1564 or dS1564 MS.

In another aspect, the present application includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar dS1564 or dS1564MS with a plant of a second tobacco cultivar comprising a desired trait to produce a progeny plant, wherein the desired trait is selected from the group consisting of: herbicide resistance, pest resistance, disease resistance, high yield, high grade index, cure quality, mechanical harvestability, retention capacity, leaf quality, height, plant maturity, precocity, early middle maturity, middle late maturity, small stem, medium stem, large stem, per plantThe number of leaves of the plant, 5-10 leaves per plant, 11-15 leaves per plant, 16-21 leaves per plant, and any combination thereof, to produce F₁Progeny seed; (b) growth F₁Progeny seed to F₁Progeny plants and selection of F having desired traits₁A progeny plant; (c) let selected F₁Crossing the progeny plant with a plant of the first tobacco cultivar to produce backcross progeny plant seed; (d) growing the backcross progeny plant seeds into backcross progeny plants and selecting a backcross progeny plant comprising the desired trait and substantially all of the physiological and morphological characteristics of the first tobacco cultivar dS1564 or dS1564 MS; and (e) repeating steps (c) and (d) one or more times in succession to produce selected second, third, fourth or higher generation backcross progeny plants comprising the desired trait and substantially all of the physiological and morphological characteristics of first tobacco cultivar dS1564 or dS1564MS, wherein the plants have the desired trait of disease resistance.

In another aspect, the present application includes a method for producing a tobacco plant comprising: identifying a first tobacco plant comprising a nucleotide sequence selected from the group consisting of: 6-10 and any combination thereof; crossing the first tobacco plant with the second tobacco plant, and collecting F₁Seeds; will be composed of F₁Crossing the plant with the grown seed with a third tobacco plant and collecting the second tobacco seed; and identifying a second tobacco seed homozygous for the nucleotide sequence or a plant grown from the second seed. In one aspect, the second tobacco plant comprises a nucleotide sequence selected from the group consisting of seq id no: SEQ ID NO:6-10, and any combination thereof. In another aspect, the third tobacco plant comprises a nucleotide sequence selected from the group consisting of seq id no: SEQ ID NO:6-10, and any combination thereof. In one aspect, the first tobacco plant is a plant of tobacco cultivar dS1564 or dS1564 MS. In another aspect, the second tobacco plant is a plant of tobacco cultivar dS1564 or dS1564 MS. In another aspect, the third tobacco plant is a plant of tobacco cultivar dS1564 or dS1564 MS.

In another aspect, the present application includes a method of producing a plant of tobacco cultivar dS1564 or dS1564MS comprising at least one (e.g., 1, 2,3, 4,5, 6, 7, 8, etc.) additional desired trait comprising the steps of: (a) collecting tissue capable of propagating from a plant of tobacco cultivar dS1564 or dS1564 MS; and (b) introducing into the tissue at least one transgene (nucleic acid construct) conferring at least one desired trait.

In another aspect, the present application includes a method of producing a herbicide resistant tobacco plant comprising transforming a tobacco plant, or a portion thereof, produced by growing seeds of tobacco cultivar dS1564 or dS1564MS with at least one transgene (nucleic acid construct), wherein the at least one transgene (nucleic acid construct) confers resistance to a herbicide. In another aspect, the herbicide is selected from the group consisting of: imidazolinones, cyclohexanediones, sulfonylureas, glyphosate, glufosinate, phenoxypropionic acid, L-phosphinothricin, triazines, benzonitrile, and any combination thereof.

In another aspect, the present application includes herbicide resistant tobacco plants produced by a method comprising transforming a tobacco plant, or part thereof, produced by growing seeds of the plant tobacco cultivar dS1564 or dS1564MS with at least one transgene (nucleic acid construct), wherein the at least one transgene (nucleic acid construct) confers resistance to herbicides. In another aspect, the herbicide is selected from the group consisting of: imidazolinones, cyclohexanediones, sulfonylureas, glyphosate, glufosinate, phenoxypropionic acid, L-phosphinothricin, triazines, benzonitrile, and any combination thereof.

In another aspect, the present application includes a method of producing a pest and/or insect resistant tobacco plant, wherein the method comprises transforming a tobacco plant produced by growing seeds of tobacco cultivar dS1564 or dS1564MS with at least one transgene (nucleic acid construct) that confers pest and/or insect resistance.

In another aspect, the present application includes pest and/or insect resistant tobacco plants produced by a method comprising transforming a tobacco plant produced by growing seeds of tobacco cultivar dS1564 or dS1564MS with at least one transgene (nucleic acid construct) that confers pest and/or insect resistance.

In another aspect, the present application includes a pest and/or insect resistant tobacco plant produced by a method comprising transforming a tobacco plant produced by growing seeds of tobacco cultivar dS1564 or dS1564MS with at least one transgene (nucleic acid construct) conferring pest and/or insect resistance, wherein the transgene (nucleic acid construct) encodes a Bacillus Thuringiensis (BT) endotoxin.

In another aspect, the present application includes a method of producing a disease-resistant tobacco plant, comprising transforming a tobacco plant produced by growing seeds of tobacco cultivar dS1564 or dS1564MS with at least one transgene (nucleic acid construct) that confers disease resistance.

In another aspect, the present application includes disease resistant tobacco plants produced by transforming tobacco plants produced by growing seeds of tobacco cultivar dS1564 or dS1564MS with at least one transgene (nucleic acid construct) that confers disease resistance.

dS1564

In one aspect, the present application provides tobacco cultivars and portions thereof grown or developed from dS 1564. In another aspect, the present application provides a tobacco plant, or part thereof, produced by growing seeds of dS 1564. In another aspect, the plants of the present application can include plants having all or substantially all of the morphological and physiological characteristics of cultivar dS 1564.

Although not limited by the method, dS1564 is an inbred-derived form of flue-cured tobacco cultivar K326 carrying a homozygous introgression at two loci (the nic1 and the nic2 loci) that results in an overall reduction in alkaloid content in the lines. The dS1564 progeny plants have a genetic background at least 55%, at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% similar to K326. dS1564 plants exhibit low nicotine levels.

dS1564MS

In one aspect, the present application provides tobacco cultivars and portions thereof grown or developed from dS1564 MS. In another aspect, the present application further includes production by growing seeds of dS1564MSOr a part thereof. In yet another aspect, the plants of the present application can include plants having all or substantially all of the morphological and physiological characteristics of cultivar dS1564 MS. dS1564MS is a Male Sterile (MS) form of dS 1564. Because the dS1564MS line is male sterile, it is maintained by pollination with dS 1564. By crossing dS1564 as a male parent with dS1564MS to produce dS1564MS F₁A progeny plant. The dS1564MS progeny plants may have a genetic background at least 55%, at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% similar to K326. dS1564MS and dS1564MS F₁Progeny plants exhibit low nicotine levels.

Other plants

The present application includes tobacco seeds produced by crossing two parent tobacco plants and harvesting the resulting tobacco seeds, wherein at least one parent tobacco plant is dS 1564. In one aspect, dS1564 is a paternal plant. In another aspect, dS1564MS is a maternal plant. One aspect of the present application provides a tobacco plant homozygous at the nic1 and nic2 loci that has a genetic background greater than about 75%, 80%, 85%, 90%, 95%, 98% or 99% similarity to K326 or MS K326 (male sterile K326). In one aspect, about or greater than about 50%, 75% or 100% of the progeny genes are provided by plants homozygous at the nic1 and nic2 loci of the present application. In one aspect, the plants of the present application have a genetic background that is at least 95%, at least 97%, at least 98% or at least 99% similar to K326 or MS K326. In another aspect, the plants of the present application exhibit low nicotine. In one aspect, the plant of the present application is a progeny plant of a female or male parent plant that is resistant to blight. On the other hand, the dS1564 plants have low resistance to blackleg and moderate resistance to bacterial wilt.

Thus, in one aspect, the present application provides cured tobacco comprising the leaves of the tobacco plant designated dS 1564. In another aspect, the present application provides cured tobacco comprising the leaves of the tobacco plant designated dS1564 MS.

In one aspect, the present application provides cured tobacco comprising the stem of a tobacco plant designated dS 1564. In another aspect, the present application provides cured tobacco comprising the stem of the tobacco plant designated dS1564 MS.

In one aspect, the present application provides cured tobacco comprising the leaves and stems of a tobacco plant designated dS 1564. In another aspect, the present application provides cured tobacco comprising the leaves and stems of the tobacco plant designated dS1564 MS.

The present application also provides containers of dS1564 or dS1564MS seeds or other seeds of the present application, wherein the alkaloids obtained from tobacco plants grown from greater than about 50% of the seeds have reduced nicotine. In another aspect, the alkaloid obtained from a dS1564 or dS1564MS plant grown from greater than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the seeds or other plant of the present application in the container has reduced nicotine.

The dS1564 or dS1564MS seed or other seed containers of the present application 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 contain 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 containers of dS1564 or dS1564MS seeds or other seeds of the present application may be any available in the art. As non-limiting examples, the container may be a box, bag, pouch, film roll, tub, foil, or tube.

In another aspect, the present application also provides a container of dS1564 or dS1564MS, wherein greater than about 50% of the dS1564 or dS1564MS seeds or other seeds of the present application have reduced nicotine.

Tobacco materials obtained from the tobacco lines, varieties, or hybrid lines of the present application can be used to make tobacco products, including, but not limited to, smoking products (e.g., cigarettes and bidis), cigar products (e.g., cigar wrap tobacco and cigarillos), pipe tobacco products, smokeless smoking products, smokeless tobacco products (e.g., moist snuff, dry snuff, and chewing tobacco), films (films), chewables, sheets, shaped parts, gels, consumable units, insoluble matrices, hollow shaped parts, and the like. See, for example, U.S. patent publication No. US 2006/0191548, which is incorporated by reference herein in its entirety.

Tobacco products derived from the plants of the present application also include cigarettes and other smoking products, particularly those that include a filter element, wherein the rod of smokable material comprises cured tobacco within a tobacco blend. In one aspect, the tobacco product may include, but is not limited to, pipe, cigar, cigarette, chewing tobacco, tobacco shreds, and/or tobacco shreds, or any combination thereof.

In one aspect, the smoking article of the present application can be a blended smoking article. In another aspect of the present application, the smoking article of the present application can be a reduced nicotine smoking article. In yet another aspect, the smoking article of the present application can be a blended smoking article having a reduced nicotine content. Thus, the smoking article of the present application can be a blended nicotine-reduced smoking article. The smoking article material comprises a mixture of tobacco materials from the present application, wherein the mixture comprises at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95 wt% cured tobacco, or any range therein, based on the dry weight of the tobacco material. The entire contents of US 2008/0245377 for the blend mixture are incorporated herein by reference.

In one aspect, tobacco plant material from plants and plant parts of the present application can be used to make tobacco products having reduced amounts of nicotine. Thus, in one aspect, tobacco products manufactured using tobacco plant material from plants and plant parts of the present application may comprise a reduced amount of nicotine or total alkaloids 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 0.04%, less than 0.03%, less than 0.02%, less than 0.01%, less than 0.005%, less than 0.025%, less than 0.001%, and less than 0.0005%. In another aspect, tobacco products made using tobacco plant material from plants and plant parts of the present application can comprise nicotine or total alkaloid levels 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 0.04%, less than 0.03%, less than 0.02%, less than 0.01%, less than 0.005%, less than 0.025%, less than 0.001%, and less than 0.0005%.

Reference herein to a measurement of an alkaloid or nicotine level (or another leaf chemical or characteristic characterization) or leaf rank index value of a tobacco plant, variety, cultivar, or line, unless otherwise indicated, refers to an average measurement, including, for example, an average of a plurality of leaves of an individual plant or an average measurement of a population of tobacco plants from an individual variety, cultivar, or line. In one aspect, after topping, nicotine or alkaloid levels (or another leaf chemical or characteristic characterization) of tobacco plants after topping are measured in pooled leaf samples collected from leaves No. 3, 4, and 5. In another aspect, the nicotine or alkaloid level (or another leaf chemical or characteristic characterization) of the tobacco plant is measured after topping the leaf with the highest level of nicotine or alkaloid (or another leaf chemical or characteristic characterization). In one aspect, the nicotine or alkaloid level (or another leaf chemical or characteristic characterization) of the tobacco plant is measured after topping leaf numbers 1, 2,3, 4,5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In another aspect, the nicotine or alkaloid level (or another leaf chemical or characteristic characterization) of the tobacco plant is measured after topping a collection of two or more pieces of consecutive leaf numbers selected from the group consisting of: 1.2, 3, 4,5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30. In another aspect, the nicotine or alkaloid level (or another leaf chemical or characteristic characterization) of the tobacco plant is measured after topping leaves of a leaf number selected from the group consisting of: 1-5, 6-10, 11-15, 16-20, 21-25 and 26-30. In another aspect, the nicotine or alkaloid level (or another leaf chemical or characteristic characterization) of the tobacco plant is measured after topping a collection of two or more leaves of a leaf number selected from the group consisting of: 1-5, 6-10, 11-15, 16-20, 21-25 and 26-30. In another aspect, the nicotine or alkaloid level (or another leaf chemical or characteristic characterization) of the tobacco plant is measured after topping a collection of three or more leaves of a leaf number selected from the group consisting of: 1-5, 6-10, 11-15, 16-20, 21-25 and 26-30.

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

The tobacco plant population or tobacco collection used to determine the average measurement (e.g., alkaloid or nicotine level or lamina 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 according to an industry-accepted standard protocol.

As used herein, "topping" refers to the removal of the stem tip, including SAM, flowers and up to several adjacent leaves, as the tobacco plant approaches 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, greenhouse or field grown tobacco plants 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.

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

Designated dS174 of the present application6. The tobacco plants of dS1746MS, dS1564, or dS1564MS can be used in plant breeding programs to produce useful lines, cultivars, varieties, progeny, inbreds, and hybrid lines. Thus, in one aspect, F will contain the nic1 and nic2 loci₁、F₂、F₃Or a progeny tobacco plant is crossed with a second tobacco plant and progeny of the cross in which the nic1 and nic2 loci are present are identified. It is to be understood that the second tobacco plant may be TN90, K326 or any other tobacco species or line, optionally with other desirable traits, such as herbicide resistance.

In yet another aspect, the methods of the present application further comprise self-pollination or pollination of a male sterile pollen recipient with a pollen donor that can be used to produce a progeny plant of the present application (such as a male sterile hybrid line of the present application).

Breeding can be carried out by any known method. DNA fingerprinting, SNPs, or similar techniques can be used in Marker Assisted Selection (MAS) breeding programs to transfer or breed mutant alleles of nicotine demethylase genes into other tobacco. For example, a breeder may generate an isolated population from a cross of a genotype containing nic1 and nic2 with an agronomically desirable genotype. F₂Or plants in backcrossing generations can be screened using markers (e.g., SNP markers having the sequences shown in SEQ ID NOS: 1-5) using one of the techniques known in the art or disclosed herein. Plants identified as having one or both of nic1 and nic2 can be backcrossed or self-pollinated to produce a second population to be screened. Depending on the expected genetic pattern or MAS technique used, self-pollination of the selected plants may be required 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 application may be dS1746, dS1746MS, dS1564 or dS1564 MS. Other breeding techniques can be found, for example, In Wernsman, E.A., and Rufty, R.C.,1987, Chapter Seven. Tobacco. pages 669-.

Species of Nicotiana species that are reproduction compatible with Nicotiana tabacum (Nicotiana tabacum) include: nicotiana amplexicaulis, PI 271989; burley tobacco (Nicotiana benthamiana) PI 555478; nicotiana bigelovii PI 555485; tobacco di bonnie (Nicotiana debneyi); high tobacco (Nicotiana excelsior) PI 224063; nicotiana glutinosa (Nicotiana glutinosa) PI 555507; gutesbi tobacco (Nicotiana goodspedii) PI 241012; -Nicotiana tabacum (Nicotiana gossei) PI 230953; western tobacco (Nicotiana hepteris) PI 271991; nicotiana tabacum (Nicotiana knightiana) PI 555527; seashore tobacco (Nicotiana maritima) PI 555535; super large tube tobacco (Nicotiana megasiphon) PI 555536; naked stem tobacco (Nicotiana nudifloris) PI 555540; cone tobacco (Nicotiana paniculata) PI 555545; blue jasmine leaf tobacco (Nicotiana plumbaginifolia) PI 555548; tabacco remana (Nicotiana repanda) PI 555552; yellow flower tobacco (Nicotiana rustica); sweet tobacco (Nicotiana suaveolens) PI 230960; forest tobacco (Nicotiana sylvestris) PI 555569; nicotiana tomentosa PI 266379; tobacco fluff (Nicotiana tomentosa); and Nicotiana triangularis (Nicotiana trigenophylla) PI 555572. See also The Complex of Tobacco Diseases published by The American microbiological Society, or The Genus Nicotiana illuminated published by Japan Tobacco Inc, The entire contents of which are incorporated herein by reference.

The results of plant breeding programs using the mutant tobacco plants described herein include useful lines, cultivars, varieties, progeny, inbreds, and hybrid lines. As used herein, the term "variety" refers to a group of plants having constant characteristics that separate it from other plants of the same species. Although not always, varieties are often sold commercially. Despite having one or more unique traits, a variety is also characterized by very small overall variation between individuals within the variety. "inbred" varieties can be produced by several generations of self-pollination and selection, or by vegetative propagation from a single parent using tissue or cell culture techniques. A breed may be substantially derived from another line or breed. The variety "is" essentially derived "from the starting variety, as defined by the international plant new species protection convention (Dec.2,1961, as revised at Geneva on Nov.10,1972, on Oct.23,1978, and on Mar.19,1991), if: a) derived predominantly from the original variety, or derived from a variety derived predominantly from the original variety while retaining expression of essential characteristics resulting from the genotype or genotype combination of the original variety; b) it is significantly different from the original variety; and c) which, apart from the differences resulting from the derivation, corresponds to the original variety in the expression of the essential characteristics resulting from the genotype or genotype combination of the original variety. For example, substantially derived varieties may be obtained by selection of natural or induced mutants, somaclonal variants, variant individuals from plants of the original variety, backcrossing or transformation. In contrast to varieties, "lines" most commonly refer to a group of plants that are not commercially used, for example, for plant research. Although there may be some variation in other traits between individuals, lines often show very little overall variation in one or more traits of interest between individuals.

By preventing self-pollination of the female parent plant of the first variety (i.e., the seed parent), pollen from the male parent plant of the second variety is allowed to fertilize the female parent plant and allow F₁Hybrid seeds are formed on the maternal plant, and hybrid tobacco varieties can be produced. Self-pollination of female plants can be prevented by detasseling the stamens during 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 be produced by Cytoplasmic Male Sterility (CMS) or transgenic male sterility, wherein the transgene inhibits microsporogenesis and/or pollen formation, or self-incompatibility. The female parent plant containing CMS is particularly useful. In one aspect where the maternal plant is CMS, pollen may be harvested from a male fertile plant and applied artificially to the stigma of the CMS maternal plant, and the resulting F harvested₁And (4) seeds.

Plants can be used to form single cross tobacco F₁And (3) hybridizing. In this regard, the parent variety plants can be grown as a substantially homogeneous contiguous population to facilitate natural cross-pollination from the parent plant to the female plant. Selective harvesting of F formed on maternal plants by conventional methods₁And (4) seeds. It is also possible to grow two parent plant species in bulk and harvest them on the female parent due to self-pollinationFormed F₁A mixture of hybrid seed and seed formed on the male parent. Alternatively, a three-way cross may be performed in which a single cross F is made₁The hybrid line is used as female parent and hybridized with different male parents. Alternatively, double cross hybrid lines can be generated in which two different single cross F's are present₁The progeny cross themselves. Self-incompatibility can be particularly advantageous for preventing female parents from self-pollinating when forming a double-crossed hybrid.

Successful hybridization yielded fertile F with nic1 and nic2₁Plants, and if desired, can be backcrossed to one of the parents, such as dS1746, dS1746MS, dS1564, or dS1564 MS. In one aspect, for F₂The plants in the generations were screened for nic1 and nic 2. The selected plant can be crossed with one of the parents and a first Backcross (BC) can be performed₁) Self-pollination of progeny plants to produce BC₁F₂The population was screened again for nic1 and nic 2. The backcrossing, self-pollination, and selection processes are repeated, for example, at least four times, until the final selection yields plants that are fertile and reasonably similar to the recurrent parent. If desired, the plant is self-pollinated and the progeny are then screened again to confirm that the plant exhibits the same low nicotine phenotype as dS1746, dS1746MS, dS1564 or dS1564 MS. Propagules for selected plants are produced using standard methods including, for example, field trials, confirmation of the nic1 and nic2 genotypes, chemical analysis of cured leaves, to determine alkaloid or nicotine levels.

In one aspect, F₁The progeny is F which crosses between dS1746 and dS1746MS (or between dS1564 and dS1564MS) to produce male sterility₁The results of the offspring. Male sterile tobacco plants can be produced by any method known in the art. Methods for producing male sterile tobacco are described In Wernsman, E.A., and Rufty, R.C.,1987, Chapter Seven. Tobacco. pages 669. 698In: Cultivar development. crop specifications. W.H.Fehr.,. MacMillan Publishing Go., Inc., New York, N.Y.761pp.

Also provided are methods of producing a tobacco plant by crossing one of cultivars dS1746 or dS1746MS with itself or a different tobacco line. The present application also relates to methods of producing other tobacco cultivars or breeding lines derived from cultivars dS1746 or dS1746MS by crossing a cultivar dS1746 or dS1746MS plant with a second tobacco plant and growing progeny seed growth to produce a dS1746 or dS1746 MS-derived tobacco plant. Another aspect of the present application provides a method for producing a tobacco plant containing one or more transgenes in its genetic material, comprising crossing a cultivar of the present application with a second cultivar containing one or more transgenes, wherein progeny are produced such that the genetic material of the progeny produced by the cross comprises a transgene optionally operably linked to one or more regulatory elements. In one aspect, the second cultivar can be a dS1746 or dS1746MS plant derived from a cultivar transformed with one or more transgenes.

The present application further provides for the asexual propagation of hybrid plants of cultivar dS1746 or dS1746MS, and progeny thereof. In one aspect, the present application provides a method of clonally propagating plants of tobacco cultivars comprising collecting tissue capable of propagating from a plant of cultivars dS1746 or dS1746MS, culturing the tissue to obtain a propagated shoot, and rooting the propagated shoot to obtain a rooted shoot. In another aspect, the plant tissue may be from cultivar dS1746 or F of dS1746MS₁And (5) collecting the hybrid lines. In one aspect, the plant tissue can be F obtained from a plant obtained by breeding cultivars dS1746 or dS1746MS₂、F₃、F₄Or later progeny plants.

Also provided are methods of producing a tobacco plant by crossing one of cultivars dS1564 or dS1564MS with itself or a different tobacco line. The present application also relates to methods of producing other tobacco cultivars or breeding lines derived from cultivars dS1564 or dS1564MS by crossing a plant of cultivar dS1564 or dS1564MS with a second tobacco plant and growing progeny seed to produce a dS1564 or dS1564MS derived tobacco plant. Another aspect of the present application provides a method for producing a tobacco plant containing one or more transgenes in its genetic material, comprising crossing a cultivar of the present application with a second cultivar containing one or more transgenes, wherein progeny are produced such that the genetic material of the progeny produced by the cross comprises a transgene optionally operably linked to one or more regulatory elements. In one aspect, the second cultivar can be a plant derived from cultivar dS1564 or dS1564MS transformed with one or more transgenes.

The present application further provides for the asexual propagation of hybrid plants of cultivar dS1564 or dS1564MS, and progeny thereof. In one aspect, the present application provides a method of clonally propagating plants of tobacco cultivars comprising collecting tissue capable of propagation from plants of cultivar dS1564 or dS1564MS, culturing the tissue to obtain propagated shoots, and rooting the propagated shoots to obtain rooted shoots. In another aspect, the plant tissue can be from cultivar dS1564 or F of dS1564MS₁And (5) collecting the hybrid lines. In one aspect, plant tissue can be obtained from F obtained by growing cultivar dS1564 or dS1564MS plants₂、F₃、F₄Or later progeny plants.

In one aspect, the plant of cultivars dS1746, dS1746MS, dS1564, or dS1564MS is further modified to comprise a mutation in the gene of interest. By selecting or screening mutagenized plant material or progeny thereof, plants comprising mutations in the gene of interest can be identified. Such screening and selection methods are known to those of ordinary skill in the art. Examples of screening and selection methods include, but are not limited to, Southern analysis, PCR amplification to detect polynucleotides, Northern blot, RNase protection, primer extension, RT-PCR amplification to detect RNA transcription, enzymatic assays to detect enzymatic or ribozyme activity of polypeptides and polynucleotides, and protein gel electrophoresis, Western blot, immunoprecipitation, and enzyme-linked immunoassay to detect 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 performing all of the cited techniques are known.

In another aspect, the tobacco plants provided herein (e.g., dS1746MS, dS1564, or dS1564MS) are further subjected to genome editing by one or more precise genome engineering techniques, e.g., transcription activator-like effector nucleases (TALENs), meganucleases, Zinc Finger Nucleases (ZFNs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 systems, CRISPR/Cpf1 systems, CRISPR/Csm1 systems, and combinations thereof (e.g., see U.S. patent application publication 2017/0233756). See, for example, Gaj et al, Trends in Biotechnology,31(7):397-405 (2013). In one aspect, the tobacco plants or plant genomes provided herein are mutated or edited by a nuclease selected from the group consisting of: meganucleases, ZFNs, TALENs, CRISPR/Cas9 nuclease, CRISPR/Cpf1 nuclease or CRISPR/Csm1 nuclease.

It is understood that the tobacco plants of the present application, including dS1746, dS1746MS, dS1564, or dS1564MS, can be transformed by genetic constructs (nucleic acid constructs) or transgenes using any technique known in the art. Without limitation, examples of desirable traits may include herbicide resistance, pest resistance, disease resistance, high yield, high grade index, curable, ripening quality, mechanical harvestability, retention capacity, leaf quality, height, plant maturity (e.g., early, middle, late or late), stem size (e.g., small, middle or large stem), or leaf number 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. Any plant of the present application may be used as a basis for tissue culture, regeneration, transformation, or any combination of these. In one aspect, a plant of the present application derived by tissue culture, transformation, or both has all or substantially all of the morphological and physiological characteristics of cultivar dS1746 or dS1746 MS.

"ripening" is an aging process that reduces moisture and causes chlorophyll breakdown, giving the tobacco leaves a golden yellow color, and by which starch is converted to sugar. Thus, cured tobacco has a higher reducing sugar content and a lower starch content than harvested green leaves. In one aspect, the green leaf tobacco provided can be cured using conventional methods, for example, flue cure, barn cure, open fire cure, air cure, or sun cure. See, for example, Tso (1999, Chapter 1 in tobaco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford) for a description of different types of maturation methods. Cured tobacco is typically aged for years (e.g., two to five years) under pressure in wooden casks (e.g., Hogshead) or cardboard boxes at moisture contents ranging from 10% to about 25%. See U.S. patent nos. 4,516,590 and 5,372,149. The cured and aged tobacco may then be further processed. Further processing includes conditioning the tobacco under vacuum with or without the introduction of steam at various temperatures, under pasteurization and fermentation. In general, fermentations are characterized by high initial moisture content, exotherms, and a dry weight loss of 10-20%. See, for example, U.S. patent nos. 4,528,993, 4,660,577, 4,848,373, 5,372,149; U.S. publication No. 2005/0178398; and Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). Cured, aged, and fermented tobacco can be further processed (e.g., cut, shredded, puffed, or mixed). See, for example, U.S. patent nos. 4,528,993; 4,660,577, respectively; 4,987,907. In one aspect, the cured tobacco material of the present disclosure is sun cured. In another aspect, cured tobacco materials of the present disclosure are flue cured, air cured, or open fire cured.

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

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

As used herein, "cigarette" refers to a tobacco product having a "rod" and a "filler". Cigarette "sticks" include cigarette paper, filters, filter rods (for containing filter material), tipping paper that holds the cigarette paper (including filler) to the filter, and all glue that holds 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 flavorants that may be enwrapped in cigarette paper with the tobacco), (3) outer shells, (4) flavorings, and (5) all other additives (incorporated into the tobacco and substitutes and enwrapped in cigarettes).

As used herein, "reconstituted tobacco" refers to a portion of tobacco filler made from tobacco dust and other tobacco waste, which is processed into sheet form and cut into strips to simulate tobacco. In addition to cost savings, reconstituted tobacco is very important for its contribution to the taste of cigarettes by using the reaction between ammonia and sugar to process flavour.

As used herein, "expanded tobacco" refers to a portion of a tobacco filler that is processed by expansion of a suitable gas such that the tobacco is "expanded," resulting in a reduction in density and greater packing capacity. Which reduces the weight of tobacco used in the cigarette.

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

In one aspect, the smoking article of the present disclosure can be a blended smoking article. In another aspect, a smoking article of the present disclosure can be a reduced nicotine smoking article. In another 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 these preparations 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, a cured tobacco material or tobacco product is provided comprising an average nicotine or total alkaloid level 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 one aspect, a cured tobacco material or tobacco product is provided comprising less than the average nicotine or total alkaloid level selected from the group consisting of: 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, a cured tobacco material or tobacco product is provided comprising an average nicotine or total alkaloid level selected from the group consisting of: about 0.01% -0.02%, 0.02% -0.05%, 0.05% -0.75%, 0.75% -0.1%, 0.1% -0.15%, 0.15% -0.2%, 0.2% -0.3%, 0.3% -0.35%, 0.35% -0.4%, 0.4% -0.5%, 0.5% -0.6%, 0.6% -0.7%, 0.7% -0.8%, 0.8% -0.9%, 0.9% -1%, 1% -1.1%, 1.1% -1.2%, 1.2% -1.3%, 1.3% -1.4%, 1.4% -1.5%, 1.5% -1.6%, 1.6% -1.7%, 1.7% -1.8%, 1.8% -1.9%, 1.9% -2%, 2% -2.1%, 2.1% -2.2%, 2.1% -2.2.2%, 2.3% -2.3%, 2.3% -2.4% -2.5%, 2.5% -2.3% -2.5%, 2.3% -2.5%, 2.6%, 2.7% -2.5%, 2.5% -2.6%, 2.5% -2.5%, 2.3%, 2.5% -2.4% and 2.4.4.4.4.5% of a, 2.7% -2.8%, 2.8% -2.9%, 2.9% -3%, 3% -3.1%, 3.1% -3.2%, 3.2% -3.3%, 3.3% -3.4%, 3.4% -3.5% and 3.5% -3.6%. In another aspect, a cured tobacco material or tobacco product is provided comprising an average nicotine or total alkaloid level selected from the group consisting of: about 0.01% -0.1%, 0.02% -0.2%, 0.03% -0.3%, 0.04% -0.4%, 0.05% -0.5%, 0.75% -1%, 0.1% -1.5%, 0.15% -2%, 0.2% -3%, and 0.3% -3.5% on a dry weight basis.

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

Detailed Description

Example 1: breeding homozygous nic1/nic2 Low alkaloid loci into TN90 background

Line dS1746 is a backcross-derived form of burley tobacco cultivar TN90 carrying a homozygous introgression at two loci (the nic1 and the nic2 loci) that results in an overall reduction in alkaloid content in the line. To produce dS1746, individual plants grown from a TN90 commercial seed lot were selected and originally crossed with plants grown from a LA Burley 21 USDA germplasm seed lot, a tobacco cultivar homozygous at the nic1 and nic2 loci. Using the KASP test shown in Table 1, multiple F were screened₁The presence of the nic1 and nic2 loci in plants. Selecting a single F₁Plants and backcrossing with TN90 in the greenhouse to produce BC₁F₁The progeny. Screening multiple BC₁F₁Progeny, and individual plants heterozygous for the nic1 and nic2 loci were identified. The whole genome SNP profile of the 175K SNP Axiome array was used to select plants with heterozygous loci and highest recovery of genomic material from the TN90 parent. Hybrid BC to be selected₁F₁Plants were backcrossed with TN90 in the greenhouse to produce BC₂F₁And (4) seeds. Using this backcrossing method, at BC₂F₁And BC₃F₁Individual heterozygous plants with the nic1 and nic2 loci were identified in the generation.

To generate plants homozygous for the nic1 and nic2 loci, BC were screened₃F₁The locus of the progeny plant to identify heterozygous plants. Self-pollination of individual plants heterozygous for the nic1 and nic2 loci to produce BC₃F₂And (4) seeds. For multiple BC₃F₂Progeny are genotyped to identify individuals homozygous for the nic1 and nic2 loci. Self-pollination of individual plants homozygous for the nic1 and nic2 loci to produce BC₃F₃And (4) seeds. Using this self-pollination method (inbreeding), at BC₃F₃And BC₃F₄Plants were selected in generations to produce variety dS1746, which is homozygous for the nic1 and nic2 loci and has greater than 94% of the genome derived from elite cultivar TN 90.

Table 1: KASP test for genotyping and selection of nic1 and nic2 loci

Example 2: preparation of male sterile line

To prepare Male Sterile (MS) lines, the BC prepared in example 1 (described above) was selected₃F₄The progeny plant of (a), which is homozygous for the nic1 and nic2 loci, and crosses as male parent with MS TN 90. BC₃F₄×MS TN 90 hybrid MS F₁The progeny plant is male sterile. Screening for the nic1 and nic2 loci for these MS F₁Progeny plants (e.g., BC)₄F₁MS). Similarly, BC₃F₁Hybridization with TN90 to obtain fertile BC screening for said loci₄F₁A plant. MS F identified as having said locus₁The progeny plant is then combined with a fertile male parent BC also having said locus₃F₄The female parent of the cross. Progeny of this cross (e.g., MS F)₂Progeny) were male sterile, and those homozygous for the nic1 and nic2 loci were identified by genotyping and designated dS1746 MS. To maintain the male sterile line, dS1746MS plants were pollinated with a fertile dS1746 plant.

Example 3: field testing of dS1746

Plants of the dS1746 line were grown in random complete block plan, three to four replicates, during the 2017 and 2018 seasons, in the Blackstone field research site and in the Chilean nursery of Rangcagua, to evaluate the chemical nature, yield and physical quality of the cured leaves. Each replicate block of Blackstone is a row (One-row) of plots, each plot having 25 plants. Each replicate block in Rancagua is a 2-row (2-row) plot with 100 plants per plot. Plants were cut by stem at maturity, air cured and rated from the previous USDA tobacco. The weight of the plot was used to determine the yield per acre. After curing, the fourth leaf counted from the top of twelve different test plants of the air-cured variety was collected to prepare fifty-gram samples of composite leaves from each plot. The composite samples were analyzed by gas chromatography for the percentage of nicotine, nornicotine, anatabine and anabasine. Alkaloid measurement data are shown as the average of four replicates (table 2A).

Agronomic measurements were performed on 5 consecutive plants per replicate. Terminal plants are not included. The measurements were performed immediately prior to harvest and included: plant height, leaf number (after topping), leaf width and leaf length (top down on the 5 th leaf, just before first harvest) and 50% days to flowering (tables 2B-2D). The measurements of five consecutive plants per replicate were then averaged.

Table 2A: dS1746 and alkaloid levels in control plants.

Table 2B: dS1746 and leaf measurements of control plants.

Table 2C: number of leaves in dS1746 and control plants at flowering.

	Days of flowering:	61 days after planting
				Average number of leaves	Variety of (IV) C
Repetition of 4	20	dS1746
				20.8	LA BU21
	18.6	TN90 LC
			Repetition of 3	21	LA BU21
	21	TN90 LC
				19.25	dS1746
Repetition 2	19.8	TN90 LC
				20.4	dS1746
	21.2	LA BU21
			Repetition of 1	19.2	dS1746
	20	LA BU21
				18.8	TN90 LC

Table 2D: dS1746 and plant height of control plants.

	Average plant height (cm)	Variety of (IV) C
			Repetition of 4	153.2	dS1746
	150.8	LA BU21
				150	TN90 LC
Repetition of 3	153.4	LA BU21
				154.8	TN90
	151.2	dS1746
			Repetition 2	149.4	TN90 LC
	151.8	dS1746
				153.8	LA BU21
Repetition of 1	150.6	dS1746
				152.4	LA BU21
	149.4	TN90 LC

Example 4: breeding homozygous nic1/nic2 Low alkaloid loci into K326 background

Line dS1564 is inbred-derived form of flue-cured tobacco cultivar K326, which carries two introgression loci (the nic1 and the nic2 loci), resulting in an overall reduction in alkaloid content in plants. To produce dS1564, individual plants grown from commercial seed lots of K326 were selected and initially crossed with plants grown from USDA germplasm seed lots of LAFC53, LAFC53 being a tobacco cultivar homozygous for the nic1 and nic2 loci. Screening for multiple F's with the nic1 and nic2 loci₁A plant. Selecting a single F₁Plants and self-pollinated to produce F₂A progeny plant. Screening of multiple F₂Progeny plants and individual plants homozygous for the nic1 and nic2 loci were identified. The genome-wide SNP profile using the 175K SNP Axiome array was used to select plants with homozygous nic1 and nic2 loci and highest recovery of genetic material from the K326 parent. Self-pollinating the selected individuals to produce F₃The progeny. Using the inbreeding method, at F₄Individual plants homozygous for the nic1 and nic2 loci were identified in generations to produce F homozygous for the nic1 and nic2 loci₅Seed (designated line dS1564) and which has greater than 42% genetic material derived from elite cultivar K326. A male sterile form of line dS1564 (designated "dS 1564 MS") was also developed.

Example 5: field testing of dS1564

Plants from the dS1564 line were grown in three to four replicates in a random complete block plan during the 2017 and 2018 seasons, in the Blackstone field research site and in the Chilean nursery of Rangcagua to evaluate the chemical, yield and physical quality of the cured leaves. Each replicate block in Blackstone is a row of plots, each plot having 25 plants. Each replicate block in Rancagua is a 2-row plot with 100 plants per plot. Plants were cut by the stem at maturity, cured and rated from the previous USDA tobacco. Cured tobacco varieties are pretreated and cured in a tobacco curing house. The shelves are completely and uniformly filled (approximately 45-50KG of green tobacco per shelf). The weight of the plot was used to determine the yield per acre. After curing, twelve random leaves from the third pretreatment of the flue-cured tobacco variety of each replicate plot were collected to prepare fifty-gram samples of composite leaves from each plot. The composite samples were analyzed by gas chromatography for the percentage of nicotine, nornicotine, anatabine and anabasine. Alkaloid measurement data are shown as the average of four replicates (table 3A).

Agronomic measurements were performed on 5 consecutive plants per replicate. Terminal plants are not included. Measurements were taken prior to harvest, including: plant height, leaf number (after topping), leaf width and leaf length (top down, 5 th leaf, just before first harvest) and days to flowering 50% (tables 3B-3D). The measurements of five consecutive plants per replicate were then averaged.

Table 3A: levels of alkaloids in dS1564 and control plants.

Table 3B: dS1564 and control plants.

Table 3B: number of leaves in dS1564 and control plants at flowering.

Table 3D: dS1564 and plant height of control plants.

	Average plant height (cm)	Variety of (IV) C
			Repetition of 4	150.4	K326
	151.6	dS1564
				148.2	LA FC53
Repetition of 3	148.2	LA FC53
				148.2	K326
	146.8	dS1564
			Repetition 2	147.8	LA FC53
	148	dS1564
				148	K326
Repetition of 1	141.4	dS1564
				147.6	K326
	156.4	LA FC53

Preservation information

The proprietary inbred plant lines disclosed above and recited in the appended claims have been deposited with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, VA 20110. The dS1746 and dS1564 deposit dates were 2018, 10 and 9. The deposit dates of dS1746MS and dS1564MS were 11 months and 22 days 2019. Prior to the filing date of this application, a deposit of 2500 seeds per variety was made from the same deposit that was preserved. All restrictions on the deposit will be irreversibly removed after the patent is issued, and the deposit is intended to satisfy all requirements of 37c.f.r. § 1.801-1.809. The ATCC has issued the following accession numbers: ATCC accession number PTA-125409 to dS1746, ATCC accession number PTA-126414 to dS1746MS, ATCC accession number PTA-125408 to dS1564, and ATCC accession number PTA-126413 to dS1564 MS. These deposits will be held at the depository facility for 30 years, or 5 years after the last request, or within the period of validity of the patent, whichever is longer, and will be replaced as needed during that period. The applicant does not give up any act of infringing the rights granted under the patent or plant variety protection act (7 u.s.c.2321, etc.).

PCT/RO/134 Table