WO2014137802A1 - Tobacco inbred and hybrid plants and uses thereof - Google Patents

Tobacco inbred and hybrid plants and uses thereof Download PDF

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Publication number
WO2014137802A1
WO2014137802A1 PCT/US2014/019381 US2014019381W WO2014137802A1 WO 2014137802 A1 WO2014137802 A1 WO 2014137802A1 US 2014019381 W US2014019381 W US 2014019381W WO 2014137802 A1 WO2014137802 A1 WO 2014137802A1
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src
tobacco
cultivar
seed
plant
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PCT/US2014/019381
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French (fr)
Inventor
Ramsey S. Lewis
Ralph E. Dewey
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North Carolina State University
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Priority to EP14714825.8A priority Critical patent/EP2964016A1/en
Publication of WO2014137802A1 publication Critical patent/WO2014137802A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/12—Leaves
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/82—Solanaceae, e.g. pepper, tobacco, potato, tomato or eggplant
    • A01H6/823—Nicotiana, e.g. tobacco
    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B13/00—Tobacco for pipes, for cigars, e.g. cigar inserts, or for cigarettes; Chewing tobacco; Snuff
    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B13/00—Tobacco for pipes, for cigars, e.g. cigar inserts, or for cigarettes; Chewing tobacco; Snuff
    • A24B13/02—Flakes or shreds of tobacco
    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
    • A24B15/10—Chemical features of tobacco products or tobacco substitutes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)

Definitions

  • NNN N- Nitrosonornicotine
  • the predominant alkaloid found in commercial tobacco varieties is nicotine, typically accounting for 90 - 95% of the total alkaloid pool.
  • the remaining alkaloid fraction is comprised primarily of three additional pyridine alkaloids: nornicotine, anabasine, and anatabine.
  • Nornicotine is generated directly from nicotine through the activity of the enzyme nicotine N-demethylase.
  • the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, produced by growing the seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573, where the leaf has a reduced amount of nomicotine and/or N'-nitrosonornicotine (NNN).
  • NNN N'-nitrosonornicotine
  • NN N'-nitrosonomicotine
  • the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573, where the plant of tobacco cultivar CMS TN90 SRC is the female parent.
  • the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a plant of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13573.
  • CMS cytoplasmic male sterile
  • the present disclosure also includes a harvested leaf of an ⁇ ⁇ progeny plant produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573.
  • CMS cytoplasmic male sterile
  • PTA-13573 with a second tobacco plant that comprises a desired trait to produce an ⁇ * ⁇ progeny seed; (b) growing the Fj progeny seed and selecting an Fi progeny plant that comprises the desired trait; (c) crossing the selected Fi progeny plant with a plant of said first tobacco cultivar to produce a backcross BQFi progeny seed;
  • PTA-13567 or CMS TN90 SRC a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573, to produce a backcross progeny plant seed; (d) growing the backcross progeny plant seed into a backcross progeny plant and selecting the backcross progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of the first tobacco cultivar, TN90 SRC or CMS TN90 SRC, respectively; and (e) repeating steps (c) and (d) one or more times in succession to produce a selected second, third, fourth or higher backcross progeny plant that comprises the desired trait and essentially all of the physiological and morphological characteristics of the first tobacco cultivar, TN90 SRC or CMS TN90 SRC.
  • the present disclosure includes a method for producing a tobacco plant having decreased nicotine conversion comprising: identifying a first tobacco plant comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ I D NO: 1 , SEQ ID NO: 2, SEQ ID NO: 1 1 , and any combination thereof: crossing the first tobacco plant with a second tobacco plant and collecting an Fi seed;
  • the present disclosure includes an herbicide resistant tobacco plant produced by a method comprising transforming a tobacco plant, or part thereof, produced by growing a seed of a tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13567 or a seed of a tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573 with at least one transgene (nucleic acid construct), where the at least one transgene (nucleic acid construct) confers resistance to an herbicide selected from the group consisting of
  • the present disclosure includes a method of producing a pest and/or insect resistant tobacco plant where the method comprises transforming a tobacco plant produced by growing a seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13567 or a seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573, with at least one transgene (nucleic acid construct) that confers pest and/or insect resistance.
  • the present disclosure further includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is produced by growing a seed of tobacco cultivar KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No.
  • the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572, where at least one tobacco plant is cytoplasmic male sterile (CMS).
  • CMS cytoplasmic male sterile
  • the present disclosure also includes a container of Fj progeny seeds produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572, where at least one tobacco plant is cytoplasmic male sterile (CMS).
  • CMS cytoplasmic male sterile
  • the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of hybrid tobacco cultivar KYI 4 X L8 SRC, where the product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
  • the present disclosure includes a method for producing a tobacco seed of KYI 4 X L8 SRC comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13571 and a second tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13572.
  • PTA-13572 with a second tobacco plant that comprises a desired trait to produce an Fi progeny seed; (b) growing the Fi progeny seed and selecting an Fi progeny plant that comprises the desired trait; (c) crossing the selected F
  • CMS cytoplasmic male sterility
  • progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of the first tobacco cultivar KYI 4 SRC or L8 SRC; and (e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny that comprise the desired trait, where the trait is cytoplasmic male sterility (CMS) and the CMS trait is obtained from the cytoplasm of Nicotiana suaveolens, and where the second tobacco plant is selected from the group consisting of CMS KYI 4 SRC or CMS L8 SRC.
  • CMS cytoplasmic male sterility
  • steps (c) and (d) can be repeated one or more times in succession to produce second or higher backcross progeny comprising the desired trait.
  • the present disclosure includes a method for producing a tobacco plant having decreased nicotine conversion comprising: identifying a first tobacco plant comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 2. SEQ ID NO: 1 1.
  • PTA-120312 where at least one tobacco plant is cytoplasmic male sterile, and where the tobacco product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
  • the present disclosure includes an Fi progeny plant produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563.
  • CMS cytoplasmic male sterile
  • the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, where the product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco where the product has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
  • a tobacco product prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, where the product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar
  • the present disclosure includes a part of a tobacco plant, produced by growing the seed of tobacco cultivar NC645 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. ⁇ - 13566, where the part is selected from the group consisting of leaf, pollen, ovule, embryo, cotyledon, hypocotyl, meristematic cell, protoplast, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
  • PTA- 13566 where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole, where the regenerated plant has all or essentially all of the morphological and physiological characteristics of cultivar NC645 SRC.
  • the present disclosure includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is produced by growing a seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, where at least one tobacco plant is cytoplasmic male sterile, and where the tobacco product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.
  • the present disclosure includes a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of hybrid tobacco cultivar NC7 SRC, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
  • PTA- 1 3566 with a plant of a second tobacco cultivar that comprises a desired trait to produce a progeny plant where the desired trait is selected from the group consisting of herbicide resistance, pest resistance, disease resistance, high yield, high grade index, curability, curing quality, mechanical harvestability, holding ability, leaf quality, height, plant maturation, early maturing, early to medium maturing, medium maturing, medium to late maturing, late maturing, small stalk, medium stalk, large stalk, leaf number per plant, 5-10 leaves per plant, 1 1-15 leaves per plant, 16-21 leaves per plant, and any combination thereof, to produce an Fi progeny seed; (b) growing the Fi progeny seed into an Fi progeny plant and selecting the Fi progeny plant having the desired trait; (c) crossing the selected Fi progeny plant with a plant of said first tobacco cultivar to produce a backcross progeny plant seed; (d) growing the backcross progeny plant seed into a backcross progeny plant and selecting the backcross progeny plant comprising the desired trait and
  • the present disclosure includes a method of producing an herbicide resistant tobacco plant comprising transforming a tobacco plant, or part thereof, produced by growing a seed of a tobacco cultivar selected from the group consisting of NC775 S R C. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No.
  • the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, where the leaf has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
  • NNN N'-nitrosonornicotine
  • the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, where the product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
  • the present disclosure includes an Fi progeny seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, where at least one tobacco plant is cytoplasmic male sterile (CMS).
  • CMS cytoplasmic male sterile
  • the present disclosure also includes a container of Fj progeny seeds produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, where at least one tobacco plant is cytoplasmic male sterile (CMS).
  • CMS cytoplasmic male sterile
  • the present disclosure also includes a harvested leaf of an Fj progeny plant having a reduced amount of nornicotine and/or N ' -nitrosonornicotine (NNN) in a smoke stream produced from the leaf, where the plant is produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC638 S RC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, where at least one tobacco plant is cytoplasmic male sterile (CMS).
  • CCS cytoplasmic male sterile
  • the present disclosure includes a tobacco plant regenerated from a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No.
  • the present disclosure includes a part of a tobacco plant, produced by growing the seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, where the part is selected from the group consisting of leaf, pollen, ovule, embryo, cotyledon, hypocotyl, meristematic cell, protoplast, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
  • the present disclosure includes a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13570.
  • the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS TN86 SRC , a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570, where the product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
  • the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13570.
  • the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570, where the plant of tobacco cultivar CMS TN86 SRC is the female parent.
  • the present disclosure includes a method of vegetatively propagating a plant of a tobacco cultivar comprising the steps of (a) collecting tissue capable of being propagated from a plant of a tobacco cultivar selected from the group consisting of NC638 SRC, a representative sample seed of the cultivar having been deposited with the
  • the present disclosure includes a tobacco plant produced by a method comprising introducing a desired trait into a tobacco cultivar comprising:
  • steps (c) and (d) can be repeated one or more times in succession to produce second or higher backcross progeny that comprise the desired trait.
  • PTA-13570 with a plant of a second tobacco cultivar that comprises a desired trait to produce a progeny plant where the desired trait is selected from the group consisting of herbicide resistance, pest resistance, disease resistance, high yield, high grade index, curability, curing quality, mechanical harvestability, holding ability, leaf quality, height, plant maturation, early maturing, early to medium maturing, medium maturing, medium to late maturing, late maturing, small stalk, medium stalk, large stalk, leaf number per plant, 5-10 leaves per plant, 11-15 leaves per plant, 16-21 leaves per plant, and any combination thereof, to produce an Fi progeny seed; (b) growing the F 1 progeny seed into an Fi progeny plant and selecting the progeny plant having the desired trait; (c) crossing the selected F) progeny plant with a plant of said first tobacco cultivar to produce a backcross progeny plant seed; (d) growing the backcross progeny plant seed into a backcross progeny plant and selecting the backcross progeny plant comprising the desired trait and essentially all
  • the third tobacco plant is a plant of a tobacco cultivar selected from the group consisting of the tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No.
  • the present disclosure includes a pest and/or insect resistant tobacco plant produced by a method comprising transforming a tobacco plant produced by growing a seed of a tobacco cultivar selected from the group consisting of NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13568, TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565.
  • the present disclosure includes a method of producing a disease resistant tobacco plant where the method comprises transforming a tobacco plant produced by growing a seed of a tobacco cultivar selected from the group consisting of NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13568, TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570, and hybrid cultivar NCBH129 SRC with at least one transgene (nucleic acid construct) that confers disease resistance.
  • a tobacco cultivar selected from the group consisting of NC638 SRC, a representative sample seed of the cultivar having been deposited with the AT
  • SEQ ID NO 1 sets forth a cyp82e4 W329Stop nucleotide sequence.
  • SEQ ID NO 3 sets forth a cyp82e4 W329Stop amino acid sequence.
  • SEQ ID NO 5 sets forth a CYP82E4 wild-type nucleotide sequence.
  • SEQ ID NO 7 sets forth a CYP82E4 wild-type amino acid sequence.
  • Figure 1 A comparison between populations of TN90 LC plants and TN90 SRC plants showing a more stable and lower average percent nicotine conversion in TN90 SRC. Leaves from 26 plants of each variety are sampled at the layby stage and ethephon- treated prior to analysis for nicotine and nornicotine levels using gas chromatography equipment.
  • Figure 2 Tobacco plants grown in the field treated with different amounts of nitrogen fertilizers.
  • TN90 SRC contains a significantly reduced level of nornicotine in tobacco filler compared to TN90 LC when grown under a same nitrogen fertilizer level.
  • Asterisks (*) indicate significant differences for filler nornicotine based on Tukey-Kramer USD (P ⁇ .05).
  • 0% E, 33% E, 67% E, and 100% E shown on the x-axis represents control, 33% Experimental, 67% Experimental, and 100% Experimental blends respectively.
  • KYI 4 SRC is a result of the introduction of three mutated CYP82E genes in a burley tobacco cultivar KYI 4.
  • the three genes are a mutated CYP82E4 gene recited as 325-6 #775 in Lewis et al.
  • CMS KYI 4 SRC is a male-sterile (CMS) version of K YI 4 SRC (CMS KYI 4 SRC) produced by crossing a plant of CMS KYI 4 as a female with pollen of KY14 SRC BC 6 Fi to produce male-sterile plants heterozygous for all three mutations.
  • CMS progeny plants of the CMS KY14 x KY14 SRC BC 6 F] cross are male sterile.
  • BC7F 1 CMS progeny homozygous for the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82el0 P381 S mutations are identified by genotyping and designated as CMS KY14 SRC. Because the CMS KY14 SRC line is male sterile, it is maintained via pollination with KY14 SRC. KY14 SRC is crossed as the male parent to CMS KY14 SRC to prepare CMS KY14 SRC Fi progeny plants.
  • a single BC F 2 plant is self- pollinated to produce BC 7 F 3 (L8 SRC) in which the wild-type CYP82E4, CYP82E5v2 and CYP82E10 alleles of L8 are replaced by the mutant (e.g. , cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381S).
  • the mutant e.g. , cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381S.
  • KYI 4 X L8 SRC has a genetic background that is at least 95%, at least 97%>, at least 98%, or at least 99% similar to burley tobacco cultivar KYI 4 x L8, a hybrid generated by pollinating plants of a male-sterile breeding line CMS KYI 4 with pollen produced by fertile breeding line L8.
  • KY14 X L8 SRC exhibits low nornicotine and is not subject to conversion to high nornicotine.
  • NC775 SRC is a result of the
  • NC775 SRC progeny plants have genetic backgrounds that are at least 95%, at least 97%, at least 98%, or at least 99% similar to NC775.
  • NC775 SRC plants exhibit low nornicotine levels and produce leaves with reduced potential for accumulating derived NNN during curing, storage, and smoking.
  • CMS NC775 SRC is a male-sterile (CMS) version of NC775 SRC (CMS NC775 SRC) produced by crossing a plant of CMS NC775 as a female with pollen of NC775 SRC BC 6 Fi to produce male-sterile plants heterozygous for all three mutations.
  • CMS progeny plants of the CMS NC775 x NC775 SRC BC ⁇ F, cross are male sterile.
  • BC 7 F 1 CMS progeny homozygous for the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82el0 P381S mutations are identified by genotyping and designated as CMS NC775 SRC. Because the CMS NC775 SRC line is male sterile, it is maintained via pollination with NC775 SRC. NC775 SRC is crossed as the male parent to CMS NC775 SRC to prepare CMS NC775 SRC F, progeny plants.
  • the present disclosure provides tobacco cultivars, and parts thereof, from NC638 SRC, representative sample seeds of this cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313.
  • the present disclosure provides a tobacco plant, or part thereof, produced by growing a seed of NC638 SRC.
  • a plant of the present disclosure can further include a plant with all, or essentially all of the morphological and physiological characteristics of cultivar NC638 SRC.
  • NC638 SRC is a result of the
  • CYP82E genes introduction of three mutated CYP82E genes in a hurley tobacco cultivar NC638.
  • the three genes are a mutated CYP82E4 gene recited as 325-6 #775 in Lewis et al. ("Three nicotine demethylase genes mediate nornicotine biosynthesis in Nicotiana tabacum L.: Functional characterization of the CYP82E10 gene.”
  • a mutated CYP82E5v2 recited in Lewis et al.
  • CMS NC638 SRC is a male-sterile (CMS) version of NC638 SRC (CMS NC638 SRC) produced by crossing a plant of CMS NC638 as a female with pollen of NC638 SRC BC 6 Fi to produce male-sterile plants heterozygous for all three mutations.
  • CMS progeny plants of the CMS NC638 x NC638 SRC BC 6 F, cross are male sterile.
  • a plurality of CMS NC638 x NC638 SRC BC 6 Fi x CMS plants e.g.
  • a plurality of CMS TN86 x TN86 SRC BC complicatF, x CMS plants are screened for the cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381 S mutations to identify plants heterozygous for all three mutations.
  • CMS plants e.g., CMS F, progeny plants
  • cyp82e4 W329Stop e.g., CMS F, progeny plants
  • cyp82e5v2 W422Stop cyp82el0 P381 S mutations
  • CMS TN86 SRC and CMS T 86 SRC I ⁇ progeny plants have genetic backgrounds that are at least 95%>, at least 97%, at least 98%o, or at least 99%> similar to TN86.
  • CMS TN86 SRC and CMS TN86 SRC F progeny plants exhibit low nornicotine levels and produce leaves with reduced potential for accumulating derived NNN during curing, storage, and smoking.
  • a plant of the present disclosure that is homozygous at the cyp82e4, cyp82e5v2, and cyp82E10 loci for SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ I D NO: 1 1 , respectively.
  • a plant of the present disclosure has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to NC638 or CMS NC638.
  • a plant of the present disclosure exhibits low nornicotine and is not subject to conversion to high NNN's.
  • a plant of the present disclosure that is homozygous at the cyp82e4, cyp82e5v2, and cyp82E10 loci for SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 1 1 , respectively.
  • a plant of the present disclosure has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to NC638 SRC and CMS NC638 SRC.
  • a plant of the present disclosure exhibits low nornicotine and is not subject to conversion to high nornicotine.
  • the nornicotine content in such plants can be in a range from 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 % dry weight.
  • the tobacco product typically has a reduced amount of NNN of less than about 10 pg/g.
  • the NNN content in such a product can be about 10 pg/g, 7.0 pg/g, 5.0 pg/g, 4.0 pg/g, 2.0 pg/g, 1.0 pg/g, 0.5 pg/g, 0.4 pg/g, 0.2 pg/g, 0.1 pg/g, 0.05 pg/g, 0.01 pg/g, and the like, or undetectable, or any range therein.
  • Nicotine and nornicotine can be measured in ethylene-treated leaves using methods known in the art (e.g. , gas chromatography). Percent nicotine demethylation in a sample is calculated by dividing the level of nornicotine by the combined level of nicotine and nornicotine as measured in the sample, and multiplying by 100. Percent nicotine demethylation in a sample from a plant of the present disclosure is about 50, 40, 30, 20, or 10 percent of a sample from an individual plant grown from a commercial seedlot of a cultivar selected from the group consisting of TO90, KY14, L8, NC775, NC645, NC638, TN86, KY I 4 X L8. C7. and NCBH 129.
  • a plant of the present disclosure including TN90 SRC, CMS TN90 SRC, KY14 SRC, CMS KY14 SRC, L8 SRC, NC775 SRC, CMS NC775 SRC, NC645 SRC, NC638 SRC. CMS NC638 SRC.
  • TN86 SRC, CMS TN86 SRC, KY 14 X L8 SRC, NC7 SRC. or NCBH129 SRC can have any yield potential, including high (e.g., over 3000 lbs/ A), moderately high (e.g., 2200-3000 lbs/ A), and moderate (e.g., less than 2000 lbs/ A) yield potential.
  • Seeds of hybrid cultivar KY14 X L8 SRC are obtainable by crossing plants of cultivars L8 SRC and CMS KYI 4 SRC and collecting seeds.
  • the present disclosure provides a seed of a KY I 4 SRC. CMS KYI 4 SRC, T8 SRC, or hybrid KY14XT8 SRC plant or other plant of the present disclosure in which a plant grown from a seed is male sterile.
  • Representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-120311 for KY 14 SRC, ATCC Accession No. PTA- 13571 for CMS KYI 4 SRC, ATCC Accession No. PTA- 13572 for L8 SRC. Seeds of hybrid cultivar KYI 4 X L8 SRC are obtainable by crossing plants of cultivars L8 SRC and CMS KY14 SRC and collecting seeds.
  • Seeds of hybrid cultivar NC7 SRC are obtainable by crossing plants of cultivars NC645 SRC and CMS NC775 SRC and collecting seeds.
  • the container of NC775 SRC, CMS NC775 SRC. NC645 SRC. or hybrid NC7 SRC seeds or other seeds of the present disclosure may contain any number, weight, or volume of seeds.
  • a 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.
  • the container can 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 seeds.
  • SRC seeds or other seeds of the present disclosure may be any container available in the art.
  • a container may be a box, a bag, a packet, a pouch, a tape roll, a pail, a foil, or a tube.
  • Representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-120312 for NC775 SRC.
  • ATCC Accession No. PTA-13563 for CMS NC775 SRC ATCC Accession No.
  • Seeds of hybrid cultivar NC7 SRC are obtainable by crossing plants of cultivars NC645 SRC and CMS NC775 SRC and collecting seeds.
  • the present disclosure provides a cured tobacco comprising the leaves and stems of the tobacco plants designated NC638 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No.
  • the present disclosure provides a cured tobacco comprising the leaves and stems of the hybrid tobacco plants designated NCBH129 SRC. Seeds of hybrid cultivar NCBH129 SRC are obtainable by crossing plants of cultivars TN86 SRC and CMS NC638 SRC and collecting seeds. [0586]
  • the present disclosure also provides a container of NC638 SRC, CMS NC638 SRC. T 86 SRC, CMS TN86 SRC. or hybrid NCBH 129 SRC seeds or other seeds of the present disclosure in which alkaloids obtained from tobacco plants grown from greater than 50% of the seeds have decreased nornicotine.
  • alkaloids obtained from NC638 SRC, CMS NC638 SRC, TN86 SRC, CMS TN86 SRC are examples of the present disclosure.
  • Seeds of hybrid cultivar NCBH 129 SRC are obtainable by crossing plants of cultivars TN86 SRC and CMS NC638 SRC and collecting seeds.
  • Containers of NC638 SRC. CMS NC638 SRC. TN86 SRC, CMS TN86 SRC, or hybrid NCBH129 SRC seeds or other seeds of the present disclosure may be any container available in the art.
  • a container may be a box, a bag, a packet, a pouch, a tape roll, a pail, a foil, or a tube.
  • Representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-120313 for NC638 SRC. ATCC Accession No. PTA- 13568 for CMS NC638 SRC. ATCC Accession No.
  • a tobacco product of the instant disclosure is a smokeless tobacco product.
  • a tobacco product of the instant disclosure is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff.
  • a tobacco product of the instant disclosure is selected from the group consisting of an electronically heated cigarette, an e-cigarette, an electronic vaporing device.
  • the nornicotine content in such a product can be 3.0 mg/g, 2.5 mg/g, 2.0 mg/g, 1.5 mg/g, 1.0 mg/g, 750 ⁇ g/g, 500 ⁇ g/g, 250 ⁇ /g, 100 ⁇ g/g, 75 ⁇ g/g, 50 ⁇ , 25 ⁇ , 10 ⁇ , 5 ⁇ , 1 ⁇ , 750 ng/g, 500 ng/g, 250 ng/g, 100 ng/g, 75 ng/g, 50 ng/g, 25 ng/g, 10 ng/g, 5 ng/g, 1 ng/g, 750 pg/g, 500 pg/g, 250 pg/g, 100 pg/g, 75 pg/g, 50 pg/g, 25 pg/g, 10 pg/g, 7.0 pg/g, 5.0 pg/g, 4.0 pg/g, 2.0 pg/g, 1.0 pg/g, 1.0
  • the tobacco product typically has a reduced amount of NNN of less than about 10 pg/g.
  • the NNN content in such a product can be about 10 pg/g, 7.0 pg/g, 5.0 pg/g, 4.0 pg/g, 2.0 pg/g, 1.0 pg/g, 0.5 pg/g, 0.4 pg/g, 0.2 pg/g, 0.1 pg/g, 0.05 pg/g, 0.01 pg/g, or undetectable, or any range therein.
  • the percentage of secondary alkaloids relative to total alkaloid content contained in a plant of the present disclosure may not be statistically different than from a commercial seedlot of a cultivar selected from the group consisting of TN90, KYI 4, 1.8, NC775, NC645, NC638, TN86, KY I 4 X 1.8. NC7, and NCBH129.
  • methods of the present disclosure further include self- pollinating or pollinating a male sterile pollen acceptor with a pollen donor capable of being used in production of a progeny plant of the present disclosure, such as a male sterile hybrid of the present disclosure.
  • a male sterile pollen acceptor plant or the pollen donor plant has at least one mutant allele, two, or even three mutant alleles at a nicotine demethylase locus, such as the cyp82e4 W329Stop allele, the cyp82e5v2 W422Stop allele, and/or the cyp82e!0 P381S allele.
  • CYP82E10 cyp82el0 P381 S (SEQ ID NO: 11)
  • CYP82E10 cyp82el0 P381 S (SEQ ID NO: 11)
  • N N N- nitrosonornicotine
  • BC4F 1 , BC5F1 , BC(,F] , and BC 7 F1 stages individuals heterozygous for all three mutations from each pedigree are self-pollinated to produce BC7F2 seed.
  • BC7F2 progeny from each pedigree are genotyped to identify individuals homozygous for all three mutations.
  • a single BC7F2 plant homozygous for all three mutations is self-pollinated to produce a BC-F?
  • the original tobacco cultivar KY I 4 X L8 LC is a hybrid generated by pollinating plants of a male-sterile breeding line CMS KYI 4 with pollen produced by fertile breeding line L8.
  • Hybrid cultivar KY I 4 X L8 SRC is a hybrid generated by pollinating plants of a male-sterile breeding line CMS KYI 4 SRC with pollen produced by fertile breeding line L8 SRC with each breeding line carrying introduced deleterious mutations in three genes (CYP82E4 (SEQ ID NO: 5), CYP82E5 (SEQ ID NO: 8), and CYP82E10 (SEQ ID NO: 9)) previously documented to encode for nicotine demethylase enzymes (Lewis et al. ,
  • CYP82E4 cyp82e4 W329Stop (SEQ ID NO: 1)
  • CYP82E5 cyp82e5v2 W422Stop (SEQ ID NO: 2)
  • CYP82E10 cyp82el0 P381 S (SEQ ID NO: 1 1)
  • CYP82el0 P381 S SEQ ID NO: 1 1
  • BC 7 F 2 seed A large number of BC 7 F 2 progeny from each pedigree are genotyped to identify individuals homozygous for all three mutations.
  • BC 7 F 2 plants homozygous for all three mutations are self-pollinated to produce BC 7 F 3 families (KY I 4 SRC) in which the wild-type CYP82E4, CYP82E5v2, and CYP82E10 alleles of KY 14 are replaced by the mutant alleles (cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381S).
  • the female parental line of KYI 4, CMS KYI 4, is cytoplasmic male sterile which causes pollen to not be produced.
  • CMS KY14 SRC To develop CMS KY14 SRC, a plant of CMS KY 14 is crossed with KYI 4 SRC to produce male-sterile plants heterozygous for all three mutations. A single male-sterile plant resulting from this cross is backcrossed as a female to KYI 4 SRC to produce progeny that are segregating for individuals homozygous for all three mutations. Triple homozygous individuals are identified by DNA genotyping to produce the CMS KYI 4 SRC line. Because the line is male-sterile, it is maintained via pollination with KYI 4 SRC (see above).
  • Hybrid cultivar KYI 4 X L8 SRC is produced by pollinating plants of CMS
  • Hybrid cultivar KYI 4 X L8 SRC is evaluated for cured leaf chemistry, yield, and physical quality at three North Carolina field research locations during 2012 (Laurel Springs, Reidsville. Waynesville). Hybrid cultivar KYI 4 X 1.8 LC is included for comparison.
  • the experimental design at each location is a randomized complete block design with four replications. Experimental units are single 20-plant plots. Plots are harvested and air-cured. Plot weights are used to determine per acre yields. Cured leaf is evaluated by a former US DA tobacco grader. Fifty gram composite leaf samples are collected from each plot and analyzed for percent nicotine, nornicotine, anatabine, anabasine, and percent nicotine conversion using gas chromatography equipment.
  • NC775 SRC is a backcross-derived version of burley tobacco cultivar NC775 carrying introduced mutations in three genes (CYP82E4 (SEQ ID NO: 5), CYP82E5 (SEQ ID NO: 8), and CYP82E10 (SEQ ID NO: 9)) previously documented to encode for nicotine demethylase enzymes (Lewis et al, Phy to chemistry, 71 (2010), 1988-1998).
  • CYP82E10 cyp82el0 P381 S (SEQ ID NO: 11)
  • CYP82E10 cyp82el0 P381 S (SEQ ID NO: 11)
  • NNN V- nitrosonornicotine
  • BC 7 F 2 seed A large number of BC 7 F 2 progeny from each pedigree are genotyped to identify individuals homozygous for all three mutations.
  • a single BC 7 F 2 plant homozygous for all three mutations is self-pollinated to produce a BC 7 F 3 family (NC775 SRC) in which the wild- type CYP82E4, CYP82E5v2, and CYP82E10 alleles of NC775 were replaced by the mutant alleles (cyp82e4 W329Stop, cy P 82e5v2 W422Stop, and cyp82el0 P381 S).
  • NC775 SRC line Because the line is male-sterile, it is maintained via pollination with NC775 SRC.
  • NC775 SRC is produced by pollinating plants of CMS NC775 SRC with pollen of NC775 SRC (see below).
  • NC775 SRC is evaluated for cured leaf chemistry, yield, and physical quality at three North Carolina field research locations during 2012 (Laurel Springs, Reidsville, Waynesville). NC775 is included for comparison.
  • the experimental design at each location is a randomized complete block design with four replications. Experimental units are single 20-plant plots. Plots are harvested and air-cured. Plot weights are used to determine per acre yields. Cured leaf is evaluated by a former USD A tobacco grader. Fifty gram composite leaf samples are collected from each plot and analyzed for percent nicotine, nomicotine, anatabine, anabasine, and percent nicotine conversion using gas chromatography equipment.
  • NC645 SRC is a backcross-derived version of burley tobacco cultivar NC645 carrying introduced null mutations in three genes (CYP82E4 (SEQ ID NO: 5), CYP82E5 (SEQ ID NO: 8), and CYP82E10 (SEQ ID NO: 9)) previously documented to encode for nicotine demethylase enzymes (Lewis et al. , Phytochemistry, 71 (2010), 1988- 1998).
  • the introduced mutations in CYP82E4 cyp82e4 W329Stop (SEQ ID NO: 1)
  • CYP82E5 cyp82e5v2 W422Stop (SEQ ID NO: 2)
  • CYP82E10 cyp82el0 P381 S (SEQ ID NO: 1 1)
  • docs not encode for a premature stop codon, but docs render the gene product non-functional for converting nicotine to nornicotine (Id. ).
  • the three mutations result in tobacco plants with (1) reduced genetic capacity to demethylate nicotine to form nornicotine, and (2) a corresponding diminished potential to accumulate JV-nitrosonornicotine (NNN), a potent carcinogen found in many tobacco products.
  • NNN JV-nitrosonornicotine
  • CYP82E4 cyp82e4 W329Stop (SEQ ID NO: 1)
  • CYP82E4 W329Stop SEQ ID NO: 1
  • LC cumbersome
  • the original tobacco cultivar NC645 is a fertile inbred line.
  • To develop NC645 SRC an individual plant of NC645 is initially pollinated with a plant of the genetic background DH98-325-6 carrying mutations in each of the three nicotine demethylase genes. Fi individuals originating from this cross and heterozygous for each mutation are backcrossed to the recurrent parent, NC645, to produce BCiFi progeny. BQFi progeny are screened using genotyping methodologies to identify individuals heterozygous for all three mutations. A single triple heterozygous BCiFi plant is backcrossed to NC645to produce BC 2 Fi progeny.
  • BC 7 F 1 stage individuals heterozygous for all three mutations from each pedigree are self- pollinated to produce BC 7 F 2 seed.
  • BC7F 2 progeny from each pedigree are genotyped to identify individuals homozygous for all three mutations.
  • BC7 F 2 plant homozygous for all three mutations is self-pollinated to produce a BC7F3 family (NC645 SRC) in which the wild-type CYP82E4, CYP82E5v2, and CYP82E10 alleles of NC645 were replaced by the mutant alleles (cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381 S).
  • NC645 SRC is evaluated for cured leaf chemistry, yield, and physical quality at three North Carolina field research locations during 2012 (Laurel Springs, Rcidsville, Waynesville). NC645 is included for comparison.
  • the experimental design at each location is a randomized complete block design with four replications. Experimental units are single 20-plant plots. Plots are harvested and air-cured. Plot weights are used to determine per acre yields. Cured leal " is evaluated by a former USDA tobacco grader. Fifty gram composite leaf samples are collected from each plot and analyzed for percent nicotine, nornicotine, anatabine, anabasine, and percent nicotine conversion using gas chromatography equipment.
  • NC645 SRC has significantly (P ⁇ 0.05) lower levels of nornicotine and percent nicotine conversion relative to NC645 (Table 2). NC645 and NC645 SRC are not significantly different from each other for percent nicotine, yield, or cured leaf quality indices.
  • CYP82E4 cyp82e4 W329Stop (SEQ ID NO: 1)
  • CYP82E4 cyp82e4 W329Stop (SEQ ID NO: 1)
  • LC cumbersome
  • the original tobacco cultivar NC7 is a hybrid generated by pollinating plants of a male-sterile breeding line CMS NC775 with pollen produced by fertile breeding line NC645.
  • hybrid cultivar NC7 SRC individual plants of fertile NC775 are first pollinated with a plant of the genetic background Dl 198-325-6 carrying mutations in each of the three nicotine demethylase genes. Fi individuals originating from each cross and heterozygous for each mutation are backcrossed to the recurrent parent (NC775 ) to produce BCiFi progenies.
  • Hybrid cultivar NC7 SRC is evaluated for cured leaf chemistry, y ield, and physical quality at three North Carolina field research locations during 2012 (Laurel Springs, Reidsville, Waynesville). Hybrid cultivar NC7 LC is included for comparison.
  • the experimental design at each location is a randomized complete block design with four replications. Experimental units are single 20-plant plots. Plots are harvested and air-cured. Plot weights are used to determine per acre yields. Cured leaf is evaluated by a former USDA tobacco grader. Fifty gram composite leaf samples are collected from each plot and analyzed for percent nicotine, nornicotine, anatabine, anabasine, and percent nicotine conversion using gas chromatography equipment.

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Abstract

The present disclosure provides tobacco inbred plants TN90 SRC, CMS TN90 SRC, KY14 SRC, CMS KY14 SRC, L8 SRC, NC775 SRC, CMS NC775 SRC, NC645 SRC, NC638 SRC, CMS NC638 SRC, TN86 SRC, and CMS TN86 SRC, and hybrids KY14 X L8 SRC, NC7 SRC, and NCBH129 SRC. The present disclosure also provides parts of such plants and products made from those parts. The present disclosure also includes progeny of the provided plants including hybrids.

Description

TOBACCO INBRED AND HYBRID PLANTS AND USES THEREOF
STATEMENT OF PRIORITY
[0001] This application claims the benefit, under 35 U.S.C. § 1 19 (e), of U.S.
Provisional Patent Application No. 61/772, 786, filed on March 5, 2013; U.S. Provisional Patent Application No. 61/772, 788, filed on March 5, 2013; U.S. Provisional Patent
Application No. 61/772, 792, filed on March 5, 2013; U.S. Provisional Patent Application No. 61/772,797, filed on March 5, 2013; U.S. Provisional Patent Application No.
61/915,951 , filed on December 13, 2013; U.S. Provisional Patent Application No.
61/915,964, filed on December 13, 2013; U.S. Provisional Patent Application No.
61/915,970, filed on December 13, 2013; and U.S. Provisional Patent Application No.
61/915,976, filed on December 13, 2013 in the United States Patent and Trademark Office. Each of the foregoing applications is incorporated by reference in their entireties.
INCORPORATION OF SEQUENCE LISTING
[0002] The Sequence Listing is hereby incorporated by reference in its entirety, including the file named P34050US02.txt, which is 38,238 bytes in size and was created on February 20, 2014, which is likewise herein incorporated by reference in its entirety.
FIELD
[0003] The present disclosure provides tobacco inbred plants TN90 SRC and CMS TN90 SRC. The present disclosure also provides tobacco inbred plants KYI 4 SRC, CMS KY 14 SRC, L8 SRC, and hybrid cultivar KYI 4 X L8 SRC. The present disclosure further provides tobacco inbred plants NC775 SRC, CMS NC775 SRC, NC645 SRC, and hybrid cultivar NC7 SRC. The present disclosure further provides tobacco inbred plants NC638 SRC. CMS NC638 SRC, TN86 SRC, CMS TN86 SRC, and hybrid cultivar NCBI I 129 SRC. The present disclosure also provides parts of such plants and products made from those parts. The present disclosure also includes progeny of the provided plants including hybrids.
BACKGROUND
100041 Tobacco (Nicotiana tabacum L.) is an important commercial crop in the United States as well as in other countries. In tobacco plants, N-demethylation of nicotine results in nornicotine, a secondary alkaloid known to be a precursor for formation of N- Nitrosonornicotine ("NNN") in cured leaves. NNN is an undesired component of cured leaves. 10005] The predominant alkaloid found in commercial tobacco varieties is nicotine, typically accounting for 90 - 95% of the total alkaloid pool. The remaining alkaloid fraction is comprised primarily of three additional pyridine alkaloids: nornicotine, anabasine, and anatabine. Nornicotine is generated directly from nicotine through the activity of the enzyme nicotine N-demethylase. Nornicotine usually represents less than 5% of the total pyridine alkaloid pool, but through a process termed "conversion," tobacco plants that initially produce very low amounts of nornicotine give rise to progeny that metabolically "convert" a large percentage of leaf nicotine to nornicotine. In tobacco plants that have genetically converted (termed "converters"), the great majority of nornicotine production occurs during the senescence and curing of the mature leaf (Wernsman and Matzinger (1968), Tob. Sci. , 72:226-228). Burley tobaccos are particularly prone to genetic conversion, with rates as high as 20% per generation observed in some cultivars.
[0006] During the curing and processing of the tobacco leaf, a portion of the nornicotine is metabolized to the compound NNN, a tobacco-specific nitrosamine (TSNA) that has been asserted to be carcinogenic in laboratory animals (Hecht and Hoffmann (1990), Cancer Surveys, 5:273-294; Hoffmann et al. (1994), J. Toxicol. Environ. Health, ¥7: 1-52; Hecht (1998), Chem. Res. Toxicol, 77:559-603). In flue-cured tobaccos, TSNAs are found to be predominantly formed through the reaction of alkaloids with the minute amounts of nitrogen oxides present in combustion gases formed by the direct-fired heating systems found in traditional curing barns (Peele and Gentry (1999), "Formation of Tobacco-specific
Nitrosamines in Flue-cured Tobacco," CORES 1 A Meeting, Agro-Phyto Groups, Suzhou, China). Retrofitting these curing barns with heat-exchangers virtually eliminated the mixing of combustion gases with the curing air and dramatically reduced the formation of TSNAs in tobaccos cured in this manner (Boyette and Hamm (2001), Rec. Adv. Tob. Sci., 27:17-22.). In contrast, in the air-cured Burley tobaccos, TSNA formation proceeds primarily through reaction of tobacco alkaloids with nitrite, a process catalyzed by leaf-borne microbes (Bush et al. (2001), Rec. Adv. Tob. Sci., 27:23-46). Thus far, attempts to reduce TSNAs through modification of curing conditions while maintaining acceptable quality standards have not proven to be successful for the air-cured tobaccos.
SUMMARY
[0007] In an aspect, the present disclosure includes a seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the American Type Culture Collection (ATCC) under ATCC Accession No. PTA- 13567. [00081 In another aspect, the present disclosure includes a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar TN90 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567.
[0009] In a further aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. ΡΊ A- 13567.
[0010] In an aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, produced by growing the seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567, where the leaf has a reduced amount of nomicotine and/or N'-nitrosonornicotine (NNN).
[0011] In an aspect, the present disclosure includes a harvested leaf, or part thereof, produced by growing the seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567, where the leaf has a reduced amount of nomicotine and/or N'-nitrosonomicotine (NNN), where the reduced amount of nomicotine and/or N'-nitrosonomicotine (NNN) is reduced in a smoke stream produced from the leaf.
[0012] In a further aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar TN90 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567, where the product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco and any combination thereof.
[0013] In an aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567, where the product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
[0014] In another aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13567, where the product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco, and where the product has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0015] In a further aspect, the present disclosure includes a second tobacco product prepared or produced from a first tobacco product prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567, where first product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco and any combination thereof, and the second tobacco product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco. In some aspects, the first and second product comprises a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0016] In an aspect, the present disclosure includes a part of a tobacco plant, produced by growing the seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567, where the part is selected from the group consisting of leaf, pollen, ovule, embryo, cotyledon, hypocotyl, meristematic cell, protoplast, root, root tip, pistil, anther, flower, shoot, stem, pod and petiole.
[0017] In another aspect, the present disclosure includes a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
[0018] In an aspect, the present disclosure includes a tobacco plant regenerated from a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole where the regenerated plant has all, or essentially all of the morphological and physiological characteristics of cultivar TN90 SRC.
10019] In an aspect, the present disclosure includes an Fi progeny plant of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the A l'CC under ATCC Accession No. PTA-13567.
[0020] In another aspect, the present disclosure includes an F| progeny plant of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13567, where the Fi plant is cytoplasmic male sterile (CMS).
[0021 ] In another aspect, the present disclosure includes an F| progeny seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar TN90 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13567, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0022] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567.
[0023] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13567, where the plant of tobacco cultivar TN90 SRC is the male parent.
[0024] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567. where the plant of tobacco cultivar TN90 SRC is the female parent.
[0025] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[Θ026] In an aspect, the present disclosure also includes a container of F] progeny seeds produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0027] In an aspect, the present disclosure includes an F | progeny plant produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567, and where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0028] In an aspect, the present disclosure also includes a harvested leaf of an F i progeny plant produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0029] In another aspect, the present disclosure further includes a harvested leaf of an F] progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) produced by growing a seed produced by a method of comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0030] In an aspect, the present disclosure also includes a harvested leaf of an Ft progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) in a smoke stream produced from the leaf, where the plant is produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[00311 In an aspect, the present disclosure includes a tobacco product prepared from an F] progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is produced by growing a seed of tobacco cultivar TN90 SRC , a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567, where at least one tobacco plant is cytoplasmic male sterile, and where the tobacco product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.
1 0321 In an aspect, the present disclosure further includes a tobacco product prepared from an F] progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is produced by growing a seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567, where at least one tobacco plant is cytoplasmic male sterile, and where the tobacco product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
[0033] In another aspect, the present disclosure includes a seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573.
[0034] In an aspect, the present disclosure includes a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13573.
[0035] In another aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573.
[0036] In an aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, produced by growing the seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573, where the leaf has a reduced amount of nomicotine and/or N'-nitrosonornicotine (NNN).
[0037] In another aspect, the present disclosure includes a harvested leaf, or part thereof, produced by growing the seed of tobacco cultivar CMS IN 90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573, where the leaf has a reduced amount of nomicotine and/or N'- nitrosonornicotine (NNN), where the reduced amount of nomicotine and/or
N'-nitrosonomicotine (NNN) is reduced in a smoke stream produced from the leaf.
[0038] In a further aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573, where the product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco and any combination thereof.
[0039] In an aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573, where the product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
[0040] In another aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573, where the product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco where the product has a reduced amount of nomicotine and/or N'-nitrosonomicotine (NNN).
[0041] In a further aspect, the present disclosure includes a second tobacco product prepared or produced from a first tobacco product prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573, where first product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco and any combination thereof, and the second tobacco product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco. In some aspects, the first and/or the second product comprises a reduced amount of nornicotine and/or N'-nitrosonornicotine ( NN).
[0042] In an aspect, the present disclosure includes a part of a tobacco plant, produced by growing the seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13573, where the part is selected from the group consisting of leaf, pollen, ovule, embryo, cotyledon, hypocotyl, meristematic cell, protoplast, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
1 04 1 In another aspect, the present disclosure includes a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13573, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
[0044] In an aspect, the present disclosure includes a tobacco plant regenerated from a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13573, where the cell or protoplast of the tissue culture can be produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole where the regenerated plant has all, or essentially all of the morphological and physiological characteristics of cultivar CMS TN90 SRC.
[0045] In an aspect, the present disclosure includes an Fj progeny plant of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573.
10046] In another aspect, the present disclosure includes an Fj progeny plant of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13573, where the Ft plant is cytoplasmic male sterile (CMS). [0047] In another aspect, the present disclosure includes an F1 progeny seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a plant of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No . PTA- 13573.
[0048] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13573.
[0049] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573, where the plant of tobacco cultivar CMS TN90 SRC is the female parent.
[0050] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a plant of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13573.
100511 In an aspect, the present disclosure also includes a container of Fj progeny seeds produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS 'I N 90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573.
[0052] In an aspect, the present disclosure includes an F] progeny plant produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573. [0053] In an aspect, the present disclosure also includes a harvested leaf of an ¥\ progeny plant produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573.
[0054] In another aspect, the present disclosure further includes a harvested leaf of an ~F\ progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile
(CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573.
[0055] In an aspect, the present disclosure also includes a harvested leaf of an Fj progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) in a smoke stream produced from the leaf, where the plant is produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573.
[0056] In an aspect, the present disclosure includes a tobacco product prepared from an Fj progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573, and where the tobacco product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco and any combination thereof.
[0057] In an aspect, the present disclosure further includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a F] progeny seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573, and where the tobacco product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
[0058] In an aspect, the present disclosure further includes a tobacco product prepared from an F progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573, and where the tobacco product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco, and further where the product has a reduced amount of nornicotine and/or N'- nitrosonornicotine (NNN).
[0059] In an aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile, where the cytoplasmic male sterile plant is a plant of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13573.
[0060] In an aspect, the present disclosure includes a method of vegetatively propagating a plant of a tobacco cultivar comprising the steps of (a) collecting tissue capable of being propagated from a plant of a tobacco cultivar TN90 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13567 or a plant of a tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573;
(b) cultivating the tissue to obtain a proliferated shoot; and (c) rooting the proliferated shoots to obtain a rooted plantlet.
[0061] In an aspect, the present disclosure includes a method of vegetatively propagating a plant of a tobacco cultivar comprising the steps of (a) collecting tissue capable of being propagated from a plant of a tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13567 or a plant of a tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573; (b) cultivating the tissue to obtain a proliferated shoot; (c) rooting the proliferated shoots to obtain a rooted plantlet; and (d) growing a plant from the rooted plantlet.
[0062] In an aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567 or a plant of a tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573, with a second tobacco plant that comprises a desired trait to produce an Ι*Ί progeny seed; (b) growing the Fj progeny seed and selecting an Fi progeny plant that comprises the desired trait; (c) crossing the selected Fi progeny plant with a plant of said first tobacco cultivar to produce a backcross BQFi progeny seed;
(d) growing the BQFi progeny seed and selecting a backcross BCiFj progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of said first tobacco cultivar, TN90 SRC or CMS TN90 SRC; and
(e) repeating steps (c) and (d) three or more times (e.g., 3, 4, 5, 6, 7, 8, 9, 10, and the like) in succession to produce selected fourth or higher backcross progeny that comprise the desired trait. In additional aspects, steps (c) and (d) can be repeated one or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and the like) in succession to produce second or higher backcross progeny comprising the desired trait.
[0063] In an aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567, with a second tobacco plant that comprises a desired trait to produce an Fi progeny seed; (b) growing the Fi progeny seed and selecting an Fi progeny plant that comprises the desired trait; (c) crossing the selected Fi progeny plant with a plant of the first tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567 to produce a backcross BC |Fi progeny seed; (d) growing the BCiFi progeny seed and selecting a backcross BCiFi progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of the first tobacco cultivar, TN90 SRC; and (e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny that comprise the desired trait, where the trait is cytoplasmic male sterility (CMS). In additional aspects, steps (c) and (d) can be repeated one or more times in succession to produce second or higher backcross progeny comprising the desired trait, where the trait is CMS.
[0064] In an aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567, with a second tobacco plant that comprises a desired trait to produce an Fi progeny seed; (b) growing the Fi progeny seed and selecting an Fi progeny plant that comprises the desired trait; (c) crossing the selected Fi progeny plant with a plant of the first tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567 to produce a backcross BCj Fi progeny seed; (d) growing the BC|Fi progeny seed and selecting a backcross BCiFi progeny plant comprising the desired trait and the physiological and essentially all of morphological characteristics of the first tobacco cultivar TN90 SRC; and (e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny that comprise the desired trait, where the trait is cytoplasmic male sterility (CMS) and the CMS trait is obtained from the cytoplasm of Nicotiana suaveolens or Nicotiana glauca. In additional aspects, steps (c) and (d) can be repeated one or more times in succession to produce second or higher backcross progeny comprising the desired trait, where the trait is CMS and the CMS trait is obtained from the cytoplasm of Nicotiana suaveolens or Nicotiana glauca.
[0065] In an aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567 with a second tobacco plant that comprises a desired trait to produce an Fi progeny seed; (b) growing the Fj progeny seed and selecting an Fi progeny plant that comprises the desired trait; (c) crossing the selected Fj progeny plant with a plant of the first tobacco cultivar, TN90 SRC to produce a backcross BCiFi progeny seed; (d) growing the BQFi progeny seed and selecting a backcross BCiFi progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of tobacco cultivar TN90 SRC; and (e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny that comprise the desired trait, where the trait is cytoplasmic male sterility (CMS) and the CMS trait is obtained from the cytoplasm of Nicotiana suaveolens, and where the second tobacco plant is CMS TN90 SRC. In additional aspects, steps (c) and (d) can be repeated one or more times in succession to produce second or higher backcross progeny comprising the desired trait, where the trait is CMS and the CMS trait is obtained from the cytoplasm of Nicotiana suaveolens.
[0066] In an aspect, the present disclosure includes a tobacco plant produced by a method comprising introducing a desired trait into a tobacco cultivar comprising:
(a) crossing a plant of a first tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13567 or CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13573, with a second tobacco plant that comprises a desired trait to produce an Fi progeny seed; (b) growing the Y\ progeny seed and selecting an F] progeny plant that comprises the desired trait; (c) crossing the selected Fi progeny plant with a plant of the first tobacco cultivar TN90 SRC or CMS TN90 SRC to produce a backcross BCiFj progeny seed; (d) growing the BCiFj progeny seed and selecting a backcross BCjFi progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of the first tobacco cultivar TN90 SRC or CMS TN90 SRC; and (e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny that comprise the desired trait. In additional aspects, steps (c) and (d) can be repeated one or more times in succession to produce second or higher backcross progeny that comprise the desired trait.
[0067] In another aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar, TN90 SRC, a representative sample seed of the cultivar having been deposited with the
ATCC under ATCC Accession No. PTA- 13567 or CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. ΡΤΛ- 13573, with a plant of a second tobacco cultivar that comprises a desired trait to produce a progeny plant where the desired trait is selected from the group consisting of herbicide resistance, pest resistance, disease resistance, high yield, high grade index, curability, curing quality, mechanical harvestability, holding ability, leaf quality, height, plant maturation, early maturing, early to medium maturing, medium maturing, medium to late maturing, late maturing, small stalk, medium stalk, large stalk, leaf number per plant, 5-10 leaves per plant, 11-15 leaves per plant, 16-21 leaves per plant, and any combination thereof, to produce an Fi progeny seed; (b) growing the F i progeny seed into an F\ progeny plant and selecting the Fi progeny plant having the desired trait; (c) crossing the selected
Figure imgf000018_0001
progeny plant with a plant of the first tobacco cultivar, TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567 or CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573, to produce a backcross progeny plant seed; (d) growing the backcross progeny plant seed into a backcross progeny plant and selecting the backcross progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of the first tobacco cultivar, TN90 SRC or CMS TN90 SRC, respectively; and (e) repeating steps (c) and (d) one or more times in succession to produce a selected second, third, fourth or higher backcross progeny plant that comprises the desired trait and essentially all of the physiological and morphological characteristics of the first tobacco cultivar, TN90 SRC or CMS TN90 SRC.
[0068] In another aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar, TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567 or CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13573, with a plant of a second tobacco cultivar that comprises a desired trait to produce a progeny plant where the desired trait is selected from the group consisting of herbicide resistance, pest resistance, disease resistance, high yield, high grade index, curability, curing quality, mechanical harvestability, holding ability, leaf quality, height, plant maturation, early maturing, early to medium maturing, medium maturing, medium to late maturing, late maturing, small stalk, medium stalk, large stalk, leaf number per plant, 5-10 leaves per plant, 1 1 -15 leaves per plant, 16-21 leaves per plant, and any combination thereof, to produce an Fi progeny seed; (b) growing the Fj progeny seed into an Fi progeny plant and selecting the Fi progeny plant having the desired trait; (c) crossing the selected Fj progeny plant with a plant of the first tobacco cultivar to produce a backcross progeny plant seed; (d) growing the backcross progeny plant seed into a backcross progeny plant and selecting the backcross progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of the first tobacco cultivar, TN90 SRC or CMS TN90 SRC, respectively; and (e) repeating steps (c) and (d) one or more times in succession to produce a selected second, third, fourth or higher backcross progeny plant that comprises the desired trait and essentially all of the physiological and morphological characteristics of the first tobacco cultivar. TN90 SRC or CMS TN90 SRC.
[0069] In another aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar, TN90 SRC, a representative sample seed of the cultivar having been deposited with the
ATCC under ATCC Accession No. PTA- 13567 or CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13573, with a plant of a second tobacco cultivar that comprises a desired trait to produce a progeny plant where the desired trait is selected from the group consisting of herbicide resistance, pest resistance, disease resistance, high yield, high grade index, curability, curing quality, mechanical harvestability, holding ability, leaf quality, height, plant maturation, early maturing, early to medium maturing, medium maturing, medium to late maturing, late maturing, small stalk, medium stalk, large stalk, leaf number per plant, 5-10 leaves per plant, 11-15 leaves per plant, 16-21 leaves per plant, and any combination thereof, to produce an F] progeny seed; (b) growing the Fi progeny seed into an Fi progeny plant and selecting the Fi progeny plant having the desired trait; (c) crossing the selected Fi progeny plant with a plant of the first tobacco cultivar to produce a backcross progeny plant seed; (d) growing the backcross progeny plant seed into a backcross progeny plant and selecting the backcross progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of the first tobacco cultivar, TN90 SRC or CMS TN90 SRC, respectively; and (e) repeating steps (c) and (d) one or more times in succession to produce a selected second, third, fourth or higher backcross progeny plant that comprises the desired trait and essentially all of the physiological and morphological characteristics of the first tobacco cultivar, TN90 SRC or CMS TN90 SRC, respectively, where the plant has a desired trait of disease resistance.
[0070] In another aspect, the present disclosure includes a method for producing a tobacco plant having decreased nicotine conversion comprising: identifying a first tobacco plant comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 1 1 , and any combination thereof; crossing the first tobacco plant with a second tobacco plant and collecting an Fi seed;
crossing a plant grown from the Fi seed to a third tobacco plant and collecting a second tobacco seed; and identifying a second tobacco seed or a plant grown from the second seed that is homozygous for the nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 . SEQ ID NO: 2, SEQ ID NO: 1 1 . and an combination thereof.
[0071] In an aspect, the present disclosure includes a method for producing a tobacco plant having decreased nicotine conversion comprising: identifying a first tobacco plant comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2. SEQ ID NO: 1 1. and any combination thereof; crossing the first tobacco plant with a second tobacco plant and collecting an Έ\ seed;
crossing a plant grown from the Fi seed to a third tobacco plant and collecting a second tobacco seed; and identifying a second tobacco seed or a plant grown from the second seed that is homozygous for the nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 1 1 , and any combination thereof, where the second tobacco plant comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 1 1, and any combination thereof.
10072] In an aspect, the present disclosure includes a method for producing a tobacco plant having decreased nicotine conversion comprising: identifying a first tobacco plant comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 1 1. and any combination thereof- crossing the first tobacco plant with a second tobacco plant and collecting an Fi seed;
crossing a plant grown from the Fi seed to a third tobacco plant and collecting a second tobacco seed; and identifying a second tobacco seed or a plant grown from the second seed that is homozygous for the nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 . SEQ ID NO: 2. SEQ ID NO: 1 1, and any combination thereof, where the second tobacco plant does not have the nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 1 1 , and any combination thereof, and the third tobacco plant is a tobacco plant comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 2. SEQ ID NO: 1 1, and any combination thereof.
[0073] In an aspect, the present disclosure includes a method for producing a tobacco plant having decreased nicotine conversion comprising: identifying a first tobacco plant comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ I D NO: 1 . SEQ ID NO: 2. SEQ ID NO: 1 1 . and any combination thereof; crossing the first tobacco plant with a second tobacco plant and collecting an Fi seed;
crossing a plant grown from the Fi seed to a third tobacco plant and collecting a second tobacco seed; and identifying a second tobacco seed or a plant grown from the second seed that is homozygous for the nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1. SEQ ID NO: 2, SEQ ID NO: 1 1. and any combination thereof, where the first tobacco plant comprises the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.
[0074] In an aspect, the present disclosure includes a method for producing a tobacco plant having decreased nicotine conversion comprising: identifying a first tobacco plant comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 . SEQ ID NO: 2, SEQ ID NO: 1 1, and any combination thereof; crossing the first tobacco plant with a second tobacco plant and collecting an F1 seed;
crossing a plant grown from the Fi seed to a third tobacco plant and collecting a second tobacco seed; and identifying a second tobacco seed or a plant grown from the second seed that is homozygous for the nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 1 1 , and any combination thereof, where the third tobacco plant is a tobacco plant comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 11 , and any combination thereof.
[0075] In an aspect, the present disclosure includes a method for producing a tobacco plant having decreased nicotine conversion comprising: identifying a first tobacco plant comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ I D NO: 1 , SEQ ID NO: 2, SEQ ID NO: 1 1 , and any combination thereof: crossing the first tobacco plant with a second tobacco plant and collecting an Fi seed;
crossing a plant grown from the Fi seed to a third tobacco plant and collecting a second tobacco seed; and identifying a second tobacco seed or a plant grown from the second seed that is homozygous for the nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 1 1 , and any combination thereof, where the first tobacco plant is a plant of tobacco cultivar TN90 SRC, a
representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1 3567. or a plant of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573.
[0076] In an aspect, the present disclosure includes a method for producing a tobacco plant having decreased nicotine conversion comprising: identifying a first tobacco plant comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ I D NO: 1, SEQ ID NO: 2, SEQ I D NO: 1 1 , and any combination thereof; crossing the first tobacco plant with a second tobacco plant and collecting an Fi seed;
crossing a plant grown from the Fi seed to a third tobacco plant and collecting a second tobacco seed; and identifying a second tobacco seed or a plant grown from the second seed that is homozygous for the nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, and any combination thereof, where the third tobacco plant is a plant of tobacco cultivar TN90 SRC, a
representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13567, or a plant of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13573.
[0077] In another aspect, the present disclosure includes a method of producing a plant of a tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13567 or tobacco cultivar CMS TN90 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13573 comprising at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, and the like) additional desired trait comprising the steps of: (a) collecting tissue capable of being propagated from a plant of a tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13567 or a plant of a tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13573; and (b) introducing a at least one transgene (nucleic acid construct) conferring at least one desired trait into the tissue.
[0078J In another aspect, the present disclosure includes a method of producing an herbicide resistant tobacco plant comprising transforming a tobacco plant, or part thereof, produced by growing a seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13567 or a seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13573 with at least one transgene (nucleic acid construct), where the at least one transgene (nucleic acid construct) confers resistance to an herbicide selected from the group consisting of imidazolinone, cyclohexanedione, sulfonylurea, glyphosate, glufosinate, phenoxy proprionic acid, L-phosphinothricin, triazine, benzonitrile, and any combination thereof. [0079] In another aspect, the present disclosure includes an herbicide resistant tobacco plant produced by a method comprising transforming a tobacco plant, or part thereof, produced by growing a seed of a tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13567 or a seed of a tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573 with at least one transgene (nucleic acid construct), where the at least one transgene (nucleic acid construct) confers resistance to an herbicide selected from the group consisting of
imidazolinone, cyclohexanedione, sulfonylurea, glyphosate, glufosinate, phenoxy proprionic acid, L-phosphinothricin, triazine, benzonitrile, and any combination thereof.
[0080] In another aspect, the present disclosure includes a method of producing a pest and/or insect resistant tobacco plant where the method comprises transforming a tobacco plant produced by growing a seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13567 or a seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573, with at least one transgene (nucleic acid construct) that confers pest and/or insect resistance.
[0081] In a further aspect, the present disclosure includes a pest and/or insect resistant tobacco plant produced by a method comprising transforming a tobacco plant produced by growing a seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13567 or a seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573, with at least one transgene (nucleic acid construct) that confers pest and/or insect resistance.
10082] In a further aspect, the present disclosure includes a pest and/or insect resistant tobacco plant produced by a method comprising transforming a tobacco plant produced by growing a seed of tobacco cultivar TN90 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13567 or a seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573, with at least one transgene (nucleic acid construct) that confers pest and/or insect resistance, where the transgene (nucleic acid construct) encodes a Bacillus thuringiensis (BT) endotoxin.
[0083] In another aspect, the present disclosure includes a method of producing a disease resistant tobacco plant, the method comprising transforming a tobacco plant produced by growing a seed of tobacco cultivar TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567 or a seed of tobacco cultivar CMS TN90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573 with at least one transgene (nucleic acid construct) that confers disease resistance.
[0084] In a further aspect, the present disclosure includes a disease resistant tobacco plant produced by transforming a tobacco plant produced by growing a seed of tobacco cultivar TN90 S RC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567 or a seed of tobacco cultivar CMS IN 90 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573 with at least one transgene (nucleic acid construct) that confers disease resistance.
[0085] In an aspect, the present disclosure includes a seed of tobacco cultivar KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the American Type Culture Collection (ATCC) under ATCC Accession No. PTA- 120311.
[0086] In another aspect, the present disclosure includes a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120311.
[0087] In a further aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar KYI 4 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 120311 .
[0088] In an aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, produced by growing the seed of tobacco cultivar KY14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 12031 1. where the leaf has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0089] In an aspect, the present disclosure includes a harvested leaf, or part thereof, produced by growing the seed of tobacco cultivar KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 12031 1, where the leaf has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN), where the reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) is reduced in a smoke stream produced from the leaf. [0090] In a further aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 12031 1. where the product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.
100 11 In an aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 120311, where the product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
[0092] In another aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 12031 1 , where the product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco, and where the product has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0093] In a further aspect, the present disclosure includes a second tobacco product prepared or produced from a first tobacco product prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar KY14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 12031 1 , where first product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco and any combination thereof, and the second tobacco product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco. In some aspects, the first and/or second product comprises a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0094] In an aspect, the present disclosure includes a part of a tobacco plant, produced by growing the seed of tobacco cultivar KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. ΡΤΛ- 120311, where the part is selected from the group consisting of leaf, pollen, ovule, embryo, cotyledon, hypocotyl, meristematic cell, protoplast, root, root tip, pistil, anther, flower, shoot, stem, pod and petiole.
[0095] In another aspect, the present disclosure includes a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120311, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
[0096] In an aspect, the present disclosure includes a tobacco plant regenerated from a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 120311, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole where the regenerated plant has all, or essentially all of the morphological and physiological characteristics of cultivar KYI 4 SRC.
[0097] In an aspect, the present disclosure includes an F i progeny plant of tobacco cultivar KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-12031 1.
[0098] In another aspect, the present disclosure includes an Fj progeny plant of tobacco cultivar KY14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120311 , where the Fi plant is cytoplasmic male sterile (CMS).
[0099] In another aspect, the present disclosure includes an Fi progeny seed produced crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar KY14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-12031 1, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0100] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120311.
[0101] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120311 , where the plant of tobacco cultivar KYI 4 SRC is the male parent.
[0102] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar KYI 4 S RC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120311, where the plant of tobacco cultivar KYI 4 SRC is the female parent.
[0103] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120311, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0104] In an aspect, the present disclosure also includes a container of Fj progeny seeds produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-12031 1, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0105] In an aspect, the present disclosure includes an Fi progeny plant produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-12031 1, and where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0106] In an aspect, the present disclosure also includes a harvested leaf of an F1 progeny plant produced by growing a seed produced by a method of comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-12031 1, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0107] In another aspect, the present disclosure further includes a harvested leaf of an Fi progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) produced by growing a seed produced by a method of comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-12031 1 , where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0108] In an aspect, the present disclosure also includes a harvested leaf of an Fi progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) in a smoke stream produced from the leaf, where the plant is produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-12031 1 , where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0109] In an aspect, the present disclosure includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is produced by growing a seed of tobacco cultivar KY14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-12031 1 , where at least one tobacco plant is cytoplasmic male sterile, and where the tobacco product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco and any combination thereof.
[0110] In an aspect, the present disclosure further includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is produced by growing a seed of tobacco cultivar KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-12031 1, where at least one tobacco plant is cytoplasmic male sterile, and where the tobacco product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
[01 1 1J In an aspect, the present disclosure further includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is produced by growing a seed of tobacco cultivar KY I 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 12031 1 , where at least one tobacco plant is cytoplasmic male sterile, and where the tobacco product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco, and further where the product has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0112] In another aspect, the present disclosure includes a seed of tobacco cultivar CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1 3571 .
[0113] In an aspect, the present disclosure includes a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PT A- 13571 .
[0114] In another aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PT A- 1 3571 .
[0115] In an aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, produced by growing the seed of tobacco cultivar CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13571 , where the leaf has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0116] In another aspect, the present disclosure includes a harvested leaf, or part thereof, produced by growing the seed of tobacco cultivar CMS KY14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1 3571. where the leaf has a reduced amount of nornicotine and/or N'- nitrosonornicotine (NNN), where the reduced amount of nornicotine and/or
N'-nitrosonornicotine (NNN) is reduced in a smoke stream produced from the leaf.
[0117] In a further aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13571, where the product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.
[0118] In an aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13571, where the product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
[0119] In another aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13571, where the product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco where the product has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0120] In a further aspect, the present disclosure includes a second tobacco product prepared or produced from a first tobacco product prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13571, where first product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco and any combination thereof, and the second tobacco product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco. In some aspects, the first and/or second product comprises a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0121] In an aspect, the present disclosure includes a part of a tobacco plant, produced by growing the seed of tobacco cultivar CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13571 , where the part is selected from the group consisting of leaf, pollen, ovule, embryo, cotyledon, hypocotyl, meristematic cell, protoplast, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
[0122] In another aspect, the present disclosure includes a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS KY14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13571 , where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
[0123] In an aspect, the present disclosure includes a tobacco plant regenerated from a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13571 , where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole where the regenerated plant has all, or essentially all of the morphological and physiological characteristics of cultivar CMS KY I 4 SRC.
[0124] In an aspect, the present disclosure includes an Fj progeny plant of tobacco cultivar CMS KY14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13571 .
[0125] In another aspect, the present disclosure includes an Fi progeny plant of tobacco cultivar CMS KY14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1 3571 . where the F] plant is cytoplasmic male sterile (CMS).
[0126] In another aspect, the present disclosure includes an Fj progeny seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a plant of tobacco cultivar CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1 3571 .
[0127] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS KY14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13571.
[0128] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13571. where the plant of tobacco cultivar CMS KYI 4 SRC is the female parent.
[0129] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a plant of tobacco cultivar CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13571.
[0130] In an aspect, the present disclosure also includes a container of Fi progeny seeds produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS KY 14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13571.
[0131] In an aspect, the present disclosure includes an Fi progeny plant produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS KY14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13571 .
[0132] In an aspect, the present disclosure also includes a harvested leaf of an Fi progeny plant produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13571.
[0133] In another aspect, the present disclosure further includes a harvested leaf of an Fi progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No . PTA- 13571.
[0134] In an aspect, the present disclosure also includes a harvested leaf of an Fi progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) in a smoke stream produced from the leaf, where the plant is produced by growing a seed produced by a method of comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13571.
[0135] In an aspect, the present disclosure includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13571 , and where the tobacco product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.
[0136] In an aspect, the present disclosure further includes a tobacco product prepared from an F\ progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fj progeny seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS KY14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13571, and where the tobacco product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco. [0137] In an aspect, the present disclosure further includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13571 , and where the tobacco product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco, and further where the product has a reduced amount of nomicotine and/or N' - nitrosonornicotine (NNN).
[0138] In an aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile, where the cytoplasmic male sterile plant is a plant of tobacco cultivar CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13571.
[0139] In another aspect, the present disclosure includes a seed of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572.
[0140] In an aspect, the present disclosure includes a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572.
[0141] In another aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar L8 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572.
[0142] In an aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, produced by growing the seed of tobacco cultivar L8 SRC, a
representative sample seed of the cultivar having been deposited with the ATCC under
ATCC Accession No. PTA-13572, where the leaf has a reduced amount of nomicotine and/or N'-nitrosonomicotine (NNN).
[0143] In another aspect, the present disclosure includes a harvested leaf, or part thereof, produced by growing the seed of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13572, where the leaf has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN), where the reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) is reduced in a smoke stream produced from the leaf.
10144] In a further aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572, where the product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.
[0145] In an aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572. where the product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
[0146] In another aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13572. where the product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco where the product has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0147] In a further aspect, the present disclosure includes a second tobacco product prepared or produced from a first tobacco product prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar T8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13572, where first product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco and any combination thereof, and the second tobacco product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco. In some aspects, the first and/or second product comprises a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN). [0148] In an aspect, the present disclosure includes a part of a tobacco plant, produced by growing the seed of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13572. where the part is selected from the group consisting of leaf, pollen, ovule, embryo, cotyledon, hypocotyl, meristematic cell, protoplast, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
[0149] In another aspect, the present disclosure includes a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13572, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
[0150] In an aspect, the present disclosure includes a tobacco plant regenerated from a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13572, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole, where the regenerated plant has all, or essentially all of the morphological and physiological characteristics of cultivar L8 SRC.
[0151] In an aspect, the present disclosure includes an Fi progeny plant of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13572.
[0152] In another aspect, the present disclosure includes an F| progeny plant of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13572, where the Fi plant is cytoplasmic male sterile (CMS).
[0153] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13572. [0154] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar L8 SRC , a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572, where the plant of tobacco cultivar L8 SRC is the male parent.
[0155] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar L8 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572, where the plant of tobacco cultivar L8 SRC is the female parent.
[0156] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0157] In another aspect, the present disclosure includes an Fi progeny seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0158] In an aspect, the present disclosure also includes a container of Fj progeny seeds produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0159] In an aspect, the present disclosure includes an Fi progeny plant produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572, and where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0160] In an aspect, the present disclosure also includes a harvested leaf of an F i progeny plant produced by growing a seed produced by a method of comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572. where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0161] In another aspect, the present disclosure further includes a harvested leaf of an Fi progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) produced by growing a seed produced by a method of comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0162] In an aspect, the present disclosure also includes a harvested leaf of an Fi progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) in a smoke stream produced from the leaf, where the plant is produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0163] In an aspect, the present disclosure includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is produced by growing a seed of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572, where at least one tobacco plant is cytoplasmic male sterile, and where the tobacco product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.
[0164] In an aspect, the present disclosure further includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is produced by growing a seed of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. P I A- 13572. where at least one tobacco plant is cytoplasmic male sterile, and where the tobacco product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
[0165] In an aspect, the present disclosure further includes a tobacco product prepared from an F] progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fj progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is produced by growing a seed of tobacco cultivar L8 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572, where at least one tobacco plant is cytoplasmic male sterile, and where the tobacco product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco, and further where the product has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0166] In another aspect, the present disclosure includes a seed of hybrid tobacco cultivar KY14 X L8 SRC (ATCC Accession No. PTA- 13569). Seeds of hybrid cultivar KY14 X L8 SRC are obtainable by crossing plants of cultivars L8 SRC and CMS KY14 SRC and collecting the seeds.
[0167] In an aspect, the present disclosure includes a tobacco plant, or part thereof, produced by growing a seed of hybrid tobacco cultivar KYI 4 X L8 SRC.
[0168] In a further aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant produced by growing the seed of hybrid tobacco cultivar KYI 4 X L8 SRC.
[0169] In another aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, produced by growing the seed of hybrid tobacco cultivar KYI 4 X L8 SRC, where the leaf has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0170] In an aspect, the present disclosure includes a harvested leaf, or part thereof, produced by growing the seed of hybrid tobacco cultivar KY14 X L8 SRC, where the leaf has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN), where the reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) is reduced in a smoke stream produced from the leaf.
[01711 In a further aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of hybrid tobacco cultivar KY14 X L8 SRC, where the product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.
[0172] In an aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of hybrid tobacco cultivar KYI 4 X L8 SRC, where the product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
[0173] In another aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of hybrid tobacco cultivar K YI 4 X L8 SRC, where the product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco where the product has a reduced amount of nornicotine and/or N'- nitrosonornicotine (NNN).
[0174] In a further aspect, the present disclosure includes a second tobacco product prepared or produced from a first tobacco product prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar KYI 4 X L8 SRC, where the first tobacco product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco and any combination thereof, and the second tobacco product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco. In some aspects, the first and/or second product comprises a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0175] In an aspect, the present disclosure includes a part of a tobacco plant, produced by growing a seed of hybrid tobacco cultivar KY14 X L8 SRC, where the part is selected from the group consisting of leaf, pollen, ovule, embryo, cotyledon, hypocotyl, meristematic cell, protoplast, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
[0176] In a further aspect, the present disclosure includes a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of hybrid tobacco cultivar KYI 4 X L8 SRC, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
[0177] In an aspect, the present disclosure includes a tobacco plant regenerated from a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of hybrid tobacco cultivar KYI 4 X L8 SRC, where the cell or protoplast of the tissue culture can be produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole, where the regenerated plant has all, or essentially all, of the morphological and physiological characteristics of hybrid cultivar KYI 4 X L8 SRC.
[0178] In an aspect, the present disclosure includes an Fi progeny plant of hybrid tobacco cultivar KYI 4 X L8 SRC.
[0179] In an aspect, the present disclosure includes an Fj progeny plant of hybrid tobacco cultivar KYI 4 X L8 SRC, where the plant of tobacco cultivar KYI 4 X L8 SRC is the female parent.
[0180] In another aspect, the present disclosure includes an Fi progeny plant of hybrid tobacco cultivar KYI 4 X L8 SRC where the Fi plant is cytoplasmic male sterile (CMS).
[0181] In another aspect, the present disclosure includes a method for producing a tobacco seed of KY14 X L8 SRC comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS KYI 4 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13571 .
[0182] In another aspect, the present disclosure includes a method for producing a tobacco seed of KYI 4 X L8 SRC comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13572.
[0183] In another aspect, the present disclosure includes a method for producing a tobacco seed of KYI 4 X L8 SRC comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13571 and a second tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13572.
[0184] In an aspect, the present disclosure includes a method of vegetatively propagating a plant of a tobacco cultivar comprising the steps of (a) collecting tissue capable of being propagated from a plant of a tobacco cultivar selected from the group consisting of KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 12031 1 , CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PT A- 13571 , L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13572, and hybrid cultivar KY14 X L8 SRC;
(b) cultivating the tissue to obtain a proliferated shoot; and (c) rooting the proliferated shoots to obtain a rooted plantlet.
[0185] In an aspect, the present disclosure includes a method of vegetatively propagating a plant of a tobacco cultivar comprising the steps of (a) collecting tissue capable of being propagated from a plant of a tobacco cultivar selected from the group consisting of KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 12031 1 , CMS KY14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1 3571 , L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572, and hybrid cultivar KY14 X L8 SRC;
(b) cultivating the tissue to obtain a proliferated shoot; (c) rooting the proliferated shoots to obtain a rooted plantlet; and (d) growing a plant from the rooted plantlet.
[0186] In an aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar selected from the group consisting of K Y 14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 12031 1 , CMS KY14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13571 , and L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572, with a second tobacco plant that comprises a desired trait to produce an ¥i progeny seed; (b) growing the Fi progeny seed and selecting an Fi progeny plant that comprises the desired trait; (c) crossing the selected Fi progeny plant with a plant of said first tobacco cultivar to produce a backcross BCjFi progeny seed; (d) growing the BCiFi progeny seed and selecting a backcross BC)F] progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of said first tobacco cultivar, KYI 4 SRC, CMS KYI 4 SRC, or L8 SRC, respectively; and e) repeating steps (c) and (d) three or more times (e.g., 3, 4, 5, 6, 7, 8, 9, 10, and the like) in succession to produce selected fourth or higher backcross progeny that comprise the desired trait. In additional aspects, steps (c) and (d) can be repeated one or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and the like) in succession to produce second or higher backcross progeny comprising the desired trait.
[0187] In an aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar selected from the group consisting of KY14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 12031 1. CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13571, and L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572, with a second tobacco plant that comprises a desired trait to produce an Fi progeny seed; (b) growing the Fi progeny seed and selecting an Fj progeny plant that comprises the desired trait; (c) crossing the selected Fi progeny plant with a plant of said first tobacco cultivar to produce a backcross BCjFi progeny seed; (d) growing the BCiFi progeny seed and selecting a backcross BCiFi progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of the first tobacco cultivar, KYI 4 SRC, CMS KY14 SRC, or L8 SRC, respectively; and (e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny that comprise the desired trait, where the trait is cytoplasmic male sterility (CMS). In additional aspects, steps (c) and (d) can be repeated one or more times in succession to produce second or higher backcross progeny comprising the desired trait, where the trait is CMS.
[0188] In an aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar selected from the group consisting of KY14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 12031 1 and L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572, with a second tobacco plant that comprises a desired trait to produce an Fi progeny seed; (b) growing the Fi progeny seed and selecting an Fi progeny plant that comprises the desired trait; (c) crossing the selected F| progeny plant with a plant of said first tobacco cultivar; (d) growing the BC1F1 progeny seed and selecting a backcross BC' iFi progeny plant comprising the desired trait and the physiological and essentially all of morphological characteristics of the first tobacco cultivar KYI 4 SRC or L8 SRC, respectively; and (e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny that comprise the desired trait, where the trait is cytoplasmic male sterility (CMS) and the CMS trait is obtained from the cytoplasm of Nicotiana suaveolens or Nicotiana glauca. In additional aspects, steps (c) and (d) can be repeated one or more times in succession to produce second or higher backcross progeny comprising the desired trait, where the trait is CMS and the CMS trait is obtained from the cytoplasm of Nicotiana suaveolens or Nicotiana glauca.
[0189] In an aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar selected from the group consisting of KYI 4 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. P TA-12031 1 and L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572 with a second tobacco plant that comprises a desired trait to produce an Fi progeny seed; (b) growing the Fi progeny seed and selecting an Fi progeny plant that comprises the desired trait; (c) crossing the selected Fi progeny plant with a plant of said first tobacco cultivar to produce a backcross BQFi progeny seed; (d) growing the BC iFi progeny seed and selecting a backcross BCiFi. progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of the first tobacco cultivar KYI 4 SRC or L8 SRC; and (e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny that comprise the desired trait, where the trait is cytoplasmic male sterility (CMS) and the CMS trait is obtained from the cytoplasm of Nicotiana suaveolens, and where the second tobacco plant is selected from the group consisting of CMS KYI 4 SRC or CMS L8 SRC. In additional aspects, steps (c) and (d) can be repeated one or more times in succession to produce second or higher backcross progeny comprising the CMS trait obtained from the cytoplasm of Nicotiana suaveolens, and where the second tobacco plant is selected from the group consisting of CMS KY I 4 SRC or CMS L8 SRC.
[0190] In an aspect, the present disclosure includes a tobacco plant produced by a method comprising introducing a desired trait into a tobacco cultivar comprising:
(a) crossing a plant of a first tobacco cultivar selected from the group consisting of KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-12031 1, CMS KY14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13571, and L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572, with a second tobacco plant that comprises a desired trait to produce an Fj progeny seed; (b) growing the Fj progeny seed and selecting an Fi progeny plant that comprises the desired trait; (c) crossing the selected Fi progeny plant with a plant of said first tobacco cultivar to produce a backcross BCiFi progeny seed;
(d) growing the BQFi progeny seed and selecting a backcross BQFi progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of the first tobacco cultivar KY I 4 SRC. CMS KY14 SRC. or L8 SRC, respectively; and (e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny that comprise the desired trait. In additional aspects, steps (c) and (d) can be repeated one or more times in succession to produce second or higher backcross progeny comprising the desired trait.
[0191] In another aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar selected from the group consisting of KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120311, CMS KY14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13571, and L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572, with a plant of a second tobacco cultivar that comprises a desired trait to produce a progeny plant where the desired trait is selected from the group consisting of herbicide resistance, pest resistance, disease resistance, high yield, high grade index, curability, curing quality, mechanical harvestability, holding ability, leaf quality, height, plant maturation, early maturing, early to medium maturing, medium maturing, medium to late maturing, late maturing, small stalk, medium stalk, large stalk, leaf number per plant, 5-10 leaves per plant, 11-15 leaves per plant, 16-21 leaves per plant, and any combination thereof, to produce an Fi progeny seed; (b) growing the Fi progeny seed into an Fi progeny plant and selecting the Fi progeny plant having the desired trait; (c) crossing the selected Fi progeny plant with a plant of said first tobacco cultivar, to produce a backcross progeny plant seed; (d) growing the backcross progeny plant seed into a backcross progeny plant and selecting the backcross progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of the first tobacco cultivar, KY I 4 SRC, CMS KY14 SRC, or L8 SRC, respectively; and (e) repeating steps (c) and (d) one or more times in succession to produce a selected second, third, fourth or higher backcross progeny plant that comprises the desired trait and essentially all of the physiological and morphological characteristics of the first tobacco cultivar. KY I 4 SRC, CMS KY I 4 SRC. or L8 SRC, respectively.
[0192] In another aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar selected from the group consisting of KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1203 1 1 , CMS KYI 4 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1 3571. and L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13572, with a plant of a second tobacco cultivar that comprises a desired trait to produce a progeny plant where the desired trait is selected from the group consisting of herbicide resistance, pest resistance, disease resistance, high yield, high grade index, curability, curing quality, mechanical harvestability, holding ability, leaf quality, height, plant maturation, early maturing, early to medium maturing, medium maturing, medium to late maturing, late maturing, small stalk, medium stalk, large stalk, leaf number per plant, 5-10 leaves per plant, 1 1 -15 leaves per plant, 16-21 leaves per plant, and any combination thereof, to produce an
Figure imgf000046_0001
progeny seed; (b) growing the Fi progeny seed into an Fi progeny plant and selecting the ¥i progeny plant having the desired trait; (c) crossing the selected Fi progeny plant with a plant of said first tobacco cultivar to produce a backcross progeny plant seed; (d) growing the backcross progeny plant seed into a backcross progeny plant and selecting the backcross progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of the first tobacco cultivar, KY14 SRC, CMS KYI 4 SRC, or L8 SRC, respectively; and (e) repeating steps (c) and (d) one or more times in succession to produce a selected second, third, fourth or higher backcross progeny plant that comprises the desired trait and essentially all of the physiological and morphological characteristics of said first tobacco cultivar, KY I 4 SRC, CMS KY 14 SRC, or L8 SRC, respectively, where the desired trait is disease resistance.
[0193] In an aspect, the present disclosure includes a method for producing a tobacco plant having decreased nicotine conversion comprising: identifying a first tobacco plant comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 2. SEQ ID NO: 1 1. and any combination thereof- crossing the first tobacco plant with a second tobacco plant and collecting an f seed; crossing a plant grown from the Fi seed to a third tobacco plant and collecting a second tobacco seed; and identifying a second tobacco seed or a plant grown from the second seed that is homozygous for the nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 11 , and any combination thereof, where the first tobacco plant is a plant of a tobacco cultivar KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-12031 1, a plant of tobacco cultivar CMS KY14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13571 , or a plant of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13572. and hybrid cultivar KY14 X L8 SRC.
[0194] In an aspect, the present disclosure includes a method for producing a tobacco plant having decreased nicotine conversion comprising: identifying a first tobacco plant comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 1 1. and any combination thereof- crossing the first tobacco plant with a second tobacco plant and collecting an Fi seed;
crossing a plant grown from the Fi seed to a third tobacco plant and collecting a second tobacco seed; and identifying a second tobacco seed or a plant grown from the second seed that is homozygous for the nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 1 1 , and any combination thereof, where the third tobacco plant is a plant of tobacco cultivar KYI 4 SRC, a
representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1203 1 1. a plant of tobacco cultivar CMS KY I 4 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1 3571 , a plant of tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1 3572. or hybrid cultivar KYI 4 X L8 SRC.
[0195] In another aspect, the present disclosure includes a method of producing a plant of a tobacco cultivar selected from the group consisting of tobacco cultivar KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-12031 1 , tobacco cultivar CMS KY14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1 3571 , tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1 3572, and hybrid cultivar KY14 X L8 SRC comprising at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, and the like) additional desired trait comprising the steps of: (a) collecting tissue capable of being propagated from a plant of a tobacco cultivar selected from the group consisting of KYI 4 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120311, CMS KY14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13571. L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13572, and hybrid cultivar KY14 X L8 SRC; and (b) introducing a transgene (nucleic acid construct) conferring at least one desired trait into the tissue.
[0196] In another aspect, the present disclosure includes a method of producing an herbicide resistant tobacco plant comprising transforming a tobacco plant, or part thereof, produced by growing a seed of a tobacco cultivar selected from the group consisting of tobacco cultivar KYI 4 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 120311 , tobacco cultivar CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the
ATCC under ATCC Accession No. PTA- 13571. tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13572. and hybrid cultivar KY14 X L8 SRC with at least one transgene (nucleic acid construct), where the at least one transgene (nucleic acid construct) confers resistance to an herbicide selected from the group consisting of imidazolinone, cyclohexanedione, sulfonylurea, glyphosate, glufosinate, phenoxy proprionic acid, T-phosphinothricin, triazine, benzonitrile and any combination thereof.
[0197] In another aspect, the present disclosure includes an herbicide resistant tobacco plant produced by a method comprising transforming a tobacco plant, or part thereof, produced by growing a seed of a tobacco cultivar selected from the group consisting of tobacco cultivar KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 120311, tobacco cultivar CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13571, tobacco cultivar L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572, and hybrid cultivar KY14 X L8 SRC, with at least one transgene (nucleic acid construct), where the at least one transgene (nucleic acid construct) confers resistance to an herbicide selected from the group consisting of imidazolinone, cyclohexanedione, sulfonylurea, glyphosate, glufosinate, phenoxy proprionic acid, L-phosphinothricin, triazine, benzonitrile, and any combination thereof.
[0198] In another aspect, the present disclosure includes a method of producing a pest and/or insect resistant tobacco plant where the method comprises transforming a tobacco plant produced by growing a seed of a tobacco cultivar selected from the group consisting of KY14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120 1 1. CMS KY 14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13571, L8 SRC . a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572, and hybrid cultivar KYI 4 X L8 SRC, with at least one transgene (nucleic acid construct) that confers pest and/or insect resistance.
[ 1 1 In a further aspect, the present disclosure includes a pest and/or insect resistant tobacco plant produced by a method comprising transforming a tobacco plant produced by growing a seed of a tobacco cultivar selected from the group consisting of KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120311, CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13571 , 1.8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572, and hybrid cultivar KY14 X L8 SRC with at least one transgene (nucleic acid construct) that confers pest and/or insect resistance.
102001 In a further aspect, the present disclosure includes a pest and/or insect resistant tobacco plant produced by a method comprising transforming a tobacco plant produced by growing a seed of a tobacco cultivar selected from the group consisting of KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-12031 1, CMS KY14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13571 , L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13572, and hybrid cultivar KY14 X T8 SRC, with at least one transgene (nucleic acid construct) that confers pest and/or insect resistance, where the transgene (nucleic acid construct) encodes a Bacillus thuringiensis (BT) endotoxin.
[0201] In another aspect, the present disclosure includes a method of producing a disease resistant tobacco plant, the method comprising transforming a tobacco plant produced by growing a seed of a tobacco cultivar selected from the group consisting of KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 12031 1. CMS KY14 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13571, L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13572. and hybrid cultivar KY14 X L8 SRC with at least one transgene (nucleic acid construct) that confers disease resistance.
[0202] In a further aspect, the present disclosure includes a disease resistant tobacco plant produced by transforming a tobacco plant produced by growing a seed of a tobacco cultivar selected from the group consisting of KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 120311, CMS KYI 4 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13571, L8 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. P A- 13572. and hybrid cultivar KYI 4 X L8 SRC with at least one transgene (nucleic acid construct) that confers disease resistance.
[0203] In an aspect, the present disclosure includes a seed of tobacco cultivar
NC775 SRC , a representative sample seed of the cultivar having been deposited with the American Type Culture Collection (ATCC) under ATCC Accession No. PTA- 120312.
[0204] In another aspect, the present disclosure includes a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312.
[0205] In a further aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312.
[0206] In an aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, produced by growing the seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, where the leaf has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0207] In an aspect, the present disclosure includes a harvested leaf, or part thereof, produced by growing the seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PT A- 120312. where the leaf has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN), where the reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) is reduced in a smoke stream produced from the leaf.
10208] In a further aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, where the product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.
[0209] In an aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, where the product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
[0210] In another aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, where the product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco, and where the product has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0211] In a further aspect, the present disclosure includes a second tobacco product prepared or produced from a first tobacco product prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, where first product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco and any combination thereof, and the second tobacco product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco. In some aspects, the first and/or second product comprises a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0212] In an aspect, the present disclosure includes a part of a tobacco plant, produced by growing the seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 120312, where the part is selected from the group consisting of leaf, pollen, ovule, embryo, cotyledon, hypocotyl, meristematic cell, protoplast, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
[0213] In another aspect, the present disclosure includes a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
[0214] In an aspect, the present disclosure includes a tobacco plant regenerated from a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 120312, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole, where the regenerated plant has all or essentially all of the morphological and physiological
characteristics of cultivar NC775 SRC.
[0215] In an aspect, the present disclosure includes an Ft progeny plant of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312.
[0216] In another aspect, the present disclosure includes an Fi progeny plant of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, where the ¥ plant is cytoplasmic male sterile (CMS).
[0217] In another aspect, the present disclosure includes an F| progeny seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0218] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312.
[0219] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, where the plant of tobacco cultivar NC775 SRC is the male parent.
[U220] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, where the plant of tobacco cultivar NC775 SRC is the female parent.
[0221] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0222] In an aspect, the present disclosure also includes a container of Fi progeny seeds produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0223] In an aspect, the present disclosure includes an F i progeny plant produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, and where at least one tobacco plant is cytoplasmic male sterile (CMS). [0224] In an aspect, the present disclosure also includes a harvested leaf of an Fi progeny plant produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 120312, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0225] In another aspect, the present disclosure further includes a harvested leaf of an Fi progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0226] In an aspect, the present disclosure also includes a harvested leaf of an Fi progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) in a smoke stream produced from the leaf, where the plant is produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0227] In an aspect, the present disclosure includes a tobacco product prepared from an F1 progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is produced by growing a seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, where at least one tobacco plant is cytoplasmic male sterile, and where the tobacco product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.
[0228] In an aspect, the present disclosure further includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is produced by growing a seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, where at least one tobacco plant is cytoplasmic male sterile, and where the tobacco product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
[0229] In an aspect, the present disclosure further includes a tobacco product prepared from an Fj progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a F progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is produced by growing a seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, where at least one tobacco plant is cytoplasmic male sterile, and where the tobacco product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco, and further where the product has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0230] In another aspect, the present disclosure includes a seed of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563.
[0231] In an aspect, the present disclosure includes a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13563.
[0232] In another aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant produced by growing the seed of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563.
[0233] In an aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, produced by growing the seed of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563, where the leaf has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0234] In another aspect, the present disclosure includes a harvested leaf, or part thereof, produced by growing the seed of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563, where the leaf has a reduced amount of nornicotine and/or N'- nitrosonornicotine (NNN), where the reduced amount of nornicotine and/or
N'-nitrosonornicotine (NNN) is reduced in a smoke stream produced from the leaf.
[0235] In a further aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563, where the product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.
[0236] In an aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563, where the product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
[0237] In another aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563, where the product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco where the product has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0238] In a further aspect, the present disclosure includes a second tobacco product prepared or produced from a first tobacco product prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS NC775 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563, where first product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco and any combination thereof, and the second tobacco product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco. In some aspects, the first and/or second product comprises a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0239] In an aspect, the present disclosure includes a part of a tobacco plant, produced by growing the seed of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13563, where the part is selected from the group consisting of leaf, pollen, ovule, embryo, cotyledon, hypocotyl, meristematic cell, protoplast, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
[0240] In another aspect, the present disclosure includes a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
[0241] In an aspect, the present disclosure includes a tobacco plant regenerated from a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13563, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole, where the regenerated plant has all or essentially all of the morphological and physiological characteristics of cultivar CMS NC775 SRC.
[0242] In an aspect, the present disclosure includes an Ft progeny plant of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563.
[0243] In another aspect, the present disclosure includes an Fi progeny plant of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563, where the F| plant is cytoplasmic male sterile (CMS).
[0244] In another aspect, the present disclosure includes an Fi progeny seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a plant of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563.
[0245] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563.
[0246] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13563, where the plant of tobacco cultivar CMS NC775 SRC is the female parent.
[0247] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a plant of tobacco cultivar CMS NC775 SRC, a
representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1 3563.
[0248] In an aspect, the present disclosure also includes a container of Fi progeny seeds produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS NC775 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1 3563.
[0249] In an aspect, the present disclosure includes an Fi progeny plant produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563.
[0250] In an aspect, the present disclosure also includes a harvested leaf of an Fi progeny plant produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563. [0251] In another aspect, the present disclosure further includes a harvested leaf of an Fi progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13563.
[0252] In an aspect, the present disclosure also includes a harvested leaf of an Fi progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) in a smoke stream produced from the leaf, where the plant is produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13563.
[0253] In an aspect, the present disclosure includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13563, and where the tobacco product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.
[0254] In an aspect, the present disclosure further includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13563, and where the tobacco product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
102551 In an aspect, the present disclosure further includes a tobacco product prepared from an F ] progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a F progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. ΡΤΛ- 13563, and where the tobacco product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco, and further where the product has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0256] In another aspect, the present disclosure includes a seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13566.
[0257] In an aspect, the present disclosure includes a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. ΡΤΛ- 13566.
[0258] In another aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13566.
[0259] In an aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, produced by growing the seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13566, where the leaf has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0260] In another aspect, the present disclosure includes a harvested leaf, or part thereof, produced by growing the seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13566, where the leaf has a reduced amount of nornicotine and/or N'- nitrosonornicotine (NNN), where the reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) is reduced in a smoke stream produced from the leaf.
[0261] In a further aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, where the product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.
[0262] In an aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC645 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, where the product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
[0263] In another aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, where the product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco where the product has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0264] In a further aspect, the present disclosure includes a second tobacco product prepared or produced from a first tobacco product prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, where first product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco and any combination thereof, and the second tobacco product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco. In some aspects, the first and/or second product comprises a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0265] In an aspect, the present disclosure includes a part of a tobacco plant, produced by growing the seed of tobacco cultivar NC645 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. ΡΤΛ- 13566, where the part is selected from the group consisting of leaf, pollen, ovule, embryo, cotyledon, hypocotyl, meristematic cell, protoplast, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
[0266] In another aspect, the present disclosure includes a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. P i A- 13566. where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
[0267] In an aspect, the present disclosure includes a tobacco plant regenerated from a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13566, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole, where the regenerated plant has all or essentially all of the morphological and physiological characteristics of cultivar NC645 SRC.
[0268] In an aspect, the present disclosure includes an Fi progeny plant of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566.
[0269] In another aspect, the present disclosure includes an Fi progeny plant of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, where the Fj plant is cytoplasmic male sterile (CMS).
[0270] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566.
[0271] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, where the plant of tobacco cultivar NC645 SRC is the male parent.
[0272] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, where the plant of tobacco cultivar NC645 SRC is the female parent.
[0273] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0274] In another aspect, the present disclosure includes an F1 progeny seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0275] In an aspect, the present disclosure also includes a container of Fi progeny seeds produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0276] In an aspect, the present disclosure includes an Fj progeny plant produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, and where at least one tobacco plant is cytoplasmic male sterile (CMS). [0277] In an aspect, the present disclosure also includes a harvested leaf of an Fi progeny plant produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13566, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0278] In another aspect, the present disclosure further includes a harvested leaf of an Fi progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0279] In an aspect, the present disclosure also includes a harvested leaf of an F) progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) in a smoke stream produced from the leaf, where the plant is produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0280] In an aspect, the present disclosure includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is produced by growing a seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, where at least one tobacco plant is cytoplasmic male sterile, and where the tobacco product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.
[0281] In an aspect, the present disclosure further includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is produced by growing a seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, where at least one tobacco plant is cytoplasmic male sterile, and where the tobacco product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
[0282] In an aspect, the present disclosure further includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is produced by growing a seed of tobacco cultivar NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, where at least one tobacco plant is cytoplasmic male sterile, and where the tobacco product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco, and further where the product has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0283] In another aspect, the present disclosure includes a seed of hybrid tobacco cultivar NC7 SRC (ATCC Accession No. PTA- 13564). Seeds of hybrid cultivar NC7 SRC are obtainable by crossing plants of cultivars NC775 SRC and CMS NC775 SRC and collecting the seeds.
[0284] In an aspect, the present disclosure includes a tobacco plant, or part thereof, produced by growing a seed of hybrid tobacco cultivar NC7 SRC.
[0285] In a further aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, or part thereof, produced by growing the seed of hybrid tobacco cultivar NC7 SRC.
[0286] In another aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, produced by growing the seed of hybrid tobacco cultivar NC7 SRC, where the leaf has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0287] In an aspect, the present disclosure includes a harvested leaf, or part thereof, produced by growing the seed of hybrid tobacco cultivar NC7 SRC, where the leaf has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN), where the reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) is reduced in a smoke stream produced from the leaf. [0288] In a further aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of hybrid tobacco cultivar NC7 SRC, where the product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.
[0289] In an aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of hybrid tobacco cultivar NC7 SRC, where the product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
[0290] In another aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of hybrid tobacco cultivar NC7 SRC, where the product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco where the product has a reduced amount of nornicotine and/or N'- nitrosonornicotine (NN ).
[0291] In a further aspect, the present disclosure includes a second tobacco product prepared or produced from a first tobacco product prepared from a tobacco plant, or part thereof, produced by growing the seed of hybrid tobacco cultivar NC7 SRC, where the first tobacco product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco and any combination thereof, and the second tobacco product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco. In some aspects, the first and/or second product comprises a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0292] In an aspect, the present disclosure includes a part of a tobacco plant, produced by growing a seed of hybrid tobacco cultivar NC7 SRC, where the part is selected from the group consisting of leaf, pollen, ovule, embryo, cotyledon, hypocotyl, meristematic cell, protoplast, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
[029 1 In a further aspect, the present disclosure includes a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of hybrid tobacco cultivar NC7 SRC, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
[0294] In an aspect, the present disclosure includes a tobacco plant regenerated from a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of hybrid tobacco cultivar NC7 SRC, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole, where the regenerated plant has all or essentially all of the morphological and physiological characteristics of hybrid cultivar NC7 SRC.
[0295] In an aspect, the present disclosure includes an Fi progeny plant of hybrid tobacco cultivar NC7 SRC.
[0296] In an aspect, the present disclosure includes an Fj progeny plant of hybrid tobacco cultivar NC7 SRC, where the plant of tobacco cultivar NC7 SRC is the female parent.
[0297] In another aspect, the present disclosure includes an F t progeny plant of hybrid tobacco cultivar NC7 SRC where the Fi plant is cytoplasmic male sterile (CMS).
[0298] In another aspect, the present disclosure includes a method for producing a tobacco seed of NC7 SRC comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312.
[0299] In another aspect, the present disclosure includes a method for producing a tobacco seed of NC7 SRC comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563.
[0300] In another aspect, the present disclosure includes a method for producing a tobacco seed of NC7 SRC comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563 and a second tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312.
[0301] In an aspect, the present disclosure includes a method of vegetatively propagating a plant of a tobacco cultivar comprising the steps of (a) collecting tissue capable of being propagated from a plant of a tobacco cultivar selected from the group consisting of NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. ΡΊ Α- 13563, NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1 566. and hybrid cultivar NC7 SRC:
(b) cultivating the tissue to obtain a proliferated shoot; and (c) rooting the proliferated shoots to obtain a rooted plantlet.
[0302] In an aspect, the present disclosure includes a method of vegetatively propagating a plant of a tobacco cultivar comprising the steps of (a) collecting tissue capable of being propagated from a plant of a tobacco cultivar selected from the group consisting of NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1 203 12, CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13563, NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, and hybrid cultivar NC7 SRC;
(b) cultivating the tissue to obtain a proliferated shoot; (c) rooting the proliferated shoots to obtain a rooted plantlet; and (d) growing a plant from the rooted plantlet.
[0303] In an aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar selected from the group consisting of NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1203 12, CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13563, and NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, with a second tobacco plant that comprises a desired trait to produce an Fj progeny seed; (b) growing the Fi progeny seed and selecting an Fi progeny plant that comprises the desired trait; (c) crossing the selected Fi progeny plant with a plant of said first tobacco cultivar to produce a backcross BC) Fi progeny seed; (d) growing the BCJ F J progeny seed and selecting a backcross BCjFi progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of said first tobacco cultivar, NC775 SRC, CMS NC775 SRC. or NC645 SRC, respectively, and (e) repeating steps (c) and (d) three or more times (e.g., 3, 4, 5, 6, 7, 8, 9, 10, and the like) in succession to produce selected fourth or higher backcross progeny that comprise the desired trait. In additional aspects, steps (c) and (d) can be repeated one or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and the like) in succession to produce second or higher backcross progeny comprising the desired trait.
[0304] In an aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar selected from the group consisting of NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312 and NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, with a second tobacco plant that comprises a desired trait to produce an Fi progeny seed; (b) growing the Fi progeny seed and selecting an ¥\ progeny plant that comprises the desired trait; (c) crossing the selected Fi progeny plant with a plant of said first tobacco cultivar to produce a backcross BQFi progeny seed;
(d) growing the BQFi progeny seed and selecting a backcross BCiFi progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of the first tobacco cultivar, NC775 SRC or NC645 SRC, respectively; and (e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny that comprise the desired trait, where the trait is cytoplasmic male sterility (CMS). In additional aspects, steps (c) and (d) can be repeated one or more times in succession to produce second or higher backcross progeny comprising the desired trait, where the trait is CMS.
[0305] In an aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar selected from the group consisting of NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312 and NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, with a second tobacco plant that comprises a desired trait to produce an F] progeny seed; (b) growing the Fi progeny seed and selecting an Fi progeny plant that comprises the desired trait; (c) crossing the selected Fi progeny plant with a plant of said first tobacco cultivar to produce a backcross BQF) progeny seed;
(d) growing the BCiFi progeny seed and selecting a backcross BCj Fi progeny plant comprising the desired trait and the physiological and essentially all of morphological characteristics of the first tobacco cultivar NC775 SRC or NC645 SRC, respectively; and (e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny that comprise the desired trait, where the trait is cytoplasmic male sterility (CMS) and the CMS trait is obtained from the cytoplasm of Nicotiana suaveolens or Nicotiana glauca. In additional aspects, steps (c) and (d) can be repeated one or more times in succession to produce second or higher backcross progeny comprising the desired trait, where the trait is CMS and the CMS trait is obtained from the cytoplasm of Nicotiana suaveolens or Nicotiana glauca.
10306] In an aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar selected from the group consisting of NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312 and NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13566 with a second tobacco plant that comprises a desired trait to produce an Ft progeny seed; (b) growing the Fi progeny seed and selecting an Fi progeny plant that comprises the desired trait; (c) crossing the selected F] progeny plant with a plant of said first tobacco cultivar to produce a backcross BCiFi progeny seed;
(d) growing the BC'i Fi progeny seed and selecting a backcross BCi f'i progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of tobacco cultivar NC775 SRC or NC645 SRC, respectively; and
(e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny that comprise the desired trait, where the trait is cytoplasmic male sterility (CMS) and the CMS trait is obtained from the cytoplasm of Nicotiana suaveolens, and where the second tobacco plant is CMS NC775 SRC. In additional aspects, steps (c) and (d) can be repeated one or more times in succession to produce second or higher backcross progeny comprising the CMS trait obtained from the cytoplasm of Nicotiana suaveolens, and where the second tobacco plant is CMS NC775 SRC.
[0307] In an aspect, the present disclosure includes a tobacco plant produced by a method comprising introducing a desired trait into a tobacco cultivar comprising:
(a) crossing a plant of a first tobacco cultivar selected from the group consisting of NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13563, and NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13566, with a second tobacco plant that comprises a desired trait to produce an ¥\ progeny seed; (b) growing the Fi progeny seed and selecting an Fj progeny plant that comprises the desired trait; (c) crossing the selected Fj progeny plant with a plant of said first tobacco cultivar to produce a backcross BC iF] progeny seed; (d) growing the BCjFi progeny seed and selecting a backcross BQFi progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of the first tobacco cultivar NC775 SRC, CMS NC775 SRC, or NC645 SRC, respectively; and (e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny that comprise the desired trait. In additional aspects, steps (c) and (d) can be repeated one or more times in succession to produce second or higher backcross progeny comprising the desired trait.
[0308] In another aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar selected from the group consisting of NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13563. and NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13566, with a plant of a second tobacco cultivar that comprises a desired trait to produce a progeny plant where the desired trait is selected from the group consisting of herbicide resistance, pest resistance, disease resistance, high yield, high grade index, curability, curing quality, mechanical harvestability, holding ability, leaf quality, height, plant maturation, early maturing, early to medium maturing, medium maturing, medium to late maturing, late maturing, small stalk, medium stalk, large stalk, leaf number per plant, 5-10 leaves per plant, 11-15 leaves per plant, 16-21 leaves per plant, and any combination thereof, to produce an Fi progeny seed; (b) growing the Fi progeny seed into an Fi progeny plant and selecting the F1 progeny plant having the desired trait; (c) crossing the selected Fi progeny plant with a plant of said first tobacco cultivar to produce a backcross progeny plant seed; (d) growing the backcross progeny plant seed into a backcross progeny plant and selecting the backcross progeny plant comprising the desired trait and essentially all of the
physiological and morphological characteristics of the first tobacco cultivar, NC775 SRC, CMS NC775 SRC, or NC645 SRC, respectively; and (e) repeating steps (c) and (d) one or more times in succession to produce a selected second, third, fourth or higher backcross progeny plant that comprises the desired trait and essentially all of the physiological and morphological characteristics of the first tobacco cultivar, NC775 SRC, CMS NC775 SRC, or NC645 SRC, respectively.
[0309] In another aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar selected from the group consisting of NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563, and NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, with a plant of a second tobacco cultivar that comprises a desired trait to produce a progeny plant where the desired trait is selected from the group consisting of herbicide resistance, pest resistance, disease resistance, high yield, high grade index, curability, curing quality, mechanical harvestability, holding ability, leaf quality, height, plant maturation, early maturing, early to medium maturing, medium maturing, medium to late maturing, late maturing, small stalk, medium stalk, large stalk, leaf number per plant, 5-10 leaves per plant, 11-15 leaves per plant, 16-21 leaves per plant, and any combination thereof, to produce an Fi progeny seed; (b) growing the Fj progeny seed into an Fi progeny plant and selecting the Fi progeny plant having the desired trait; (c) crossing the selected Ft progeny plant with a plant of said first tobacco cultivar to produce a backcross progeny plant seed; (d) growing the backcross progeny plant seed into a backcross progeny plant and selecting the backcross progeny plant comprising the desired trait and essentially all of the
physiological and morphological characteristics of a tobacco cultivar, NC775 SRC, CMS NC775 SRC, or NC645 SRC, respectively; and (e) repeating steps (c) and (d) one or more times in succession to produce a selected second, third, fourth or higher backcross progeny plant that comprises the desired trait and essentially all of the physiological and
morphological characteristics of the first tobacco cultivar, NC775 SRC, CMS NC775 SRC, or NC645 SRC, respectively, where the plant has a desired trait.
[0310] In another aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar selected from the group consisting of NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563, and NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1 3566, with a plant of a second tobacco cultivar that comprises a desired trait to produce a progeny plant where the desired trait is selected from the group consisting of herbicide resistance, pest resistance, disease resistance, high yield, high grade index, curability, curing quality, mechanical harvestability, holding ability, leaf quality, height, plant maturation, early maturing, early to medium maturing, medium maturing, medium to late maturing, late maturing, small stalk, medium stalk, large stalk, leaf number per plant, 5-10 leaves per plant, 1 1-15 leaves per plant, 16-21 leaves per plant, and any combination thereof, to produce an Fi progeny seed; (b) growing the Fi progeny seed into an Fi progeny plant and selecting the Fi progeny plant having the desired trait; (c) crossing the selected Fi progeny plant with a plant of said first tobacco cultivar to produce a backcross progeny plant seed; (d) growing the backcross progeny plant seed into a backcross progeny plant and selecting the backcross progeny plant comprising the desired trait and essentially all of the
physiological and morphological characteristics of the first tobacco cultivar, NC775 SRC, CMS NC775 SRC, or NC645 SRC, respectively; and (e) repeating steps (c) and (d) one or more times in succession to produce a selected second, third, fourth or higher backcross progeny plant that comprises the desired trait and essentially all of the physiological and morphological characteristics of a tobacco cultivar, NC775 SRC, CMS NC775 SRC, or NC645 SRC, respectively, where the plant has a desired trait of disease resistance.
[0311] In an aspect, the present disclosure includes a method for producing a tobacco plant having decreased nicotine conversion comprising: identifying a first tobacco plant comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 1 1, and any combination thereof; crossing the first tobacco plant with a second tobacco plant and collecting an Fi seed;
crossing a plant grown from the Fi seed to a third tobacco plant and collecting a second tobacco seed; and identifying a second tobacco seed or a plant grown from the second seed that is homozygous for the nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 1 1 . and any combination thereof, where the first tobacco plant is a plant of a tobacco cultivar selected from the group consisting of tobacco cultivar selected from the group consisting of NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 120312. CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13563, NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, and hybrid cultivar NC7 SRC.
[0312] In an aspect, the present disclosure includes a method for producing a tobacco plant having decreased nicotine conversion comprising: identifying a first tobacco plant comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 1 1 , and any combination thereof; crossing the first tobacco plant with a second tobacco plant and collecting an seed;
crossing a plant grown from the F seed to a third tobacco plant and collecting a second tobacco seed; and identifying a second tobacco seed or a plant grown from the second seed that is homozygous for the nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, and any combination thereof, where the third tobacco plant is a plant of a tobacco cultivar selected from the group consisting of NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, CMS NC775 SRC, a
representative sample seed of the cultivar having been deposited with the ATCC under
ATCC Accession No. PTA-13563, NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, and hybrid cultivar NC7 SRC.
[0313] In another aspect, the present disclosure includes a method of producing a plant of a tobacco cultivar selected from the group consisting of NC775 SRC, a
representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563, and NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, comprising at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, and the like) additional desired trait comprising the steps of: (a) collecting tissue capable of being propagated from a plant of a tobacco cultivar selected from the group consisting of NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under A CC Accession No. PTA-120312. CMS NC775 SRC, a
representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563, and NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566; and (b) introducing at least one transgene (nucleic acid construct) conferring at least one desired trait into the tissue. [0314] In another aspect, the present disclosure includes a method of producing an herbicide resistant tobacco plant comprising transforming a tobacco plant, or part thereof, produced by growing a seed of a tobacco cultivar selected from the group consisting of NC775 S R C. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13563, NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13566, and hybrid cultivar NC7 SRC, with at least one transgene (nucleic acid construct), where the at least one transgene (nucleic acid construct) confers resistance to an herbicide selected from the group consisting of
imidazolinone, cyclohexanedione, sulfonylurea, glyphosate, glufosinate, phenoxy proprionic acid, L-phosphinothricin, triazine, benzonitrile, and any combination thereof.
[0315] In another aspect, the present disclosure includes an herbicide resistant tobacco plant produced by a method comprising transforming a tobacco plant, or part thereof, produced by growing a seed of a tobacco cultivar selected from the group consisting of NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13563, NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13566. and hybrid cultivar NC7 SRC, with at least one transgene (nucleic acid construct), where the at least one transgene (nucleic acid construct) confers resistance to an herbicide selected from the group consisting of
imidazolinone, cyclohexanedione, sulfonylurea, glyphosate, glufosinate, phenoxy proprionic acid, L-phosphinothricin, triazine, benzonitrile, and any combination thereof.
[0316] In another aspect, the present disclosure includes a method of producing a pest and/or insect resistant tobacco plant where the method comprises transforming a tobacco plant produced by growing a seed of a tobacco cultivar selected from the group consisting of NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13563, NC645 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13566, and hybrid cultivar NC7 SRC, with at least one transgene (nucleic acid construct) that confers pest and/or insect resistance. [0317] In a further aspect, the present disclosure includes a pest and/or insect resistant tobacco plant produced by a method comprising transforming a tobacco plant produced by growing a seed of a tobacco cultivar selected from the group consisting of NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, CMS NC775 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13563, NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, and hybrid cultivar NC7 SRC with at least one transgene (nucleic acid construct) that confers pest and/or insect resistance.
[0318] In a further aspect, the present disclosure includes a pest and/or insect resistant tobacco plant produced by a method comprising transforming a tobacco plant produced by growing a seed of a tobacco cultivar selected from the group consisting of NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13563, NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, and hybrid cultivar NC7 SRC, with at least one transgene (nucleic acid construct) that confers pest and/or insect resistance, where the transgene encodes a Bacillus thuringiensis (BT) endotoxin.
[0319] In another aspect, the present disclosure includes a method of producing a disease resistant tobacco plant where the method comprises transforming a tobacco plant produced by growing a seed of a tobacco cultivar selected from the group consisting of NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13563, NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566, and hybrid cultivar NC7 SRC with at least one transgene (nucleic acid construct) that confers disease resistance.
[0320] In a further aspect, the present disclosure includes a disease resistant tobacco plant produced by a method comprising transforming a tobacco plant produced by growing a seed of a tobacco cultivar selected from the group consisting of NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312, CMS NC775 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13563, NC645 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13566, and hybrid cultivar NC7 SRC, with at least one transgene (nucleic acid construct) that confers disease resistance.
[0321] In an aspect, the present disclosure includes a seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the American Type Culture Collection (ATCC) under ATCC Accession No. PTA-120313.
[0322] In another aspect, the present disclosure includes a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313.
[0323] In a further aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313.
[0324] In an aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, where the leaf has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0325] In an aspect, the present disclosure includes a harvested leaf, or part thereof, produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 120313, where the leaf has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN), where the reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) is reduced in a smoke stream produced from the leaf.
[0326] In a further aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 120313, where the product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.
[0327] In an aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, where the product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
[0328] In another aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, where the product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco, and where the product has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0329] In a further aspect, the present disclosure includes a second tobacco product prepared or produced from a first tobacco product prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC638 S RC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, where first product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco and any combination thereof, and the second tobacco product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco. In some aspects, the first and second product comprises a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[ 33 1 In an aspect, the present disclosure includes a part of a tobacco plant, produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 120313, where the part is selected from the group consisting of leaf, pollen, ovule, embryo, cotyledon, hypocotyl, meristematic cell, protoplast, root, root tip, pistil, anther, flower, shoot, stem, pod and petiole.
[0331] In another aspect, the present disclosure includes a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole. [0332] In an aspect, the present disclosure includes a tobacco plant regenerated from a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 120313, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole, where the regenerated plant has all, or essentially all of the morphological and physiological characteristics of cultivar NC638 SRC.
[0333] In an aspect, the present disclosure includes an Fi progeny plant of tobacco cultivar NC638 S RC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313.
[0334] In another aspect, the present disclosure includes an Fj progeny plant of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, where the Fi plant is cytoplasmic male sterile (CMS).
[0335] In another aspect, the present disclosure includes an Fi progeny seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0336] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313.
[0337] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, where the plant of tobacco cultivar NC638 SRC is the male parent. [0338] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, where the plant of tobacco cultivar NC638 SRC is the female parent.
[0339] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0340] In an aspect, the present disclosure also includes a container of Fj progeny seeds produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0341] In an aspect, the present disclosure includes an F i progeny plant produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, and where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0342] In an aspect, the present disclosure also includes a harvested leaf of an Fi progeny plant produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. Ρ Λ- 120313, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0343] In another aspect, the present disclosure further includes a harvested leaf of an Fi progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0344] In an aspect, the present disclosure also includes a harvested leaf of an Fj progeny plant having a reduced amount of nornicotine and/or N ' -nitrosonornicotine (NNN) in a smoke stream produced from the leaf, where the plant is produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC638 S RC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0345] In an aspect, the present disclosure includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a F progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is produced by growing a seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, where at least one tobacco plant is cytoplasmic male sterile, and where the tobacco product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.
[0346] In an aspect, the present disclosure further includes a tobacco product prepared from an Fj progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is produced by growing a seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, where at least one tobacco plant is cytoplasmic male sterile, and where the tobacco product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
[0347] In an aspect, the present disclosure further includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Ft progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is produced by growing a seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, where at least one tobacco plant is cytoplasmic male sterile, and where the tobacco product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco, and further where the product has a reduced amount of nomicotine and/or N'-nitrosonornicotine (NNN).
[0348] In another aspect, the present disclosure includes a seed of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568.
[0349] In an aspect, the present disclosure includes a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13568.
[0350] In another aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568.
[0351] In an aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, produced by growing the seed of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568, where the leaf has a reduced amount of nomicotine and/or N'-nitrosonomicotine (NNN).
[0352] In another aspect, the present disclosure includes a harvested leaf, or part thereof, produced by growing the seed of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568, where the leaf has a reduced amount of nomicotine and/or N'- nitrosonomicotine (NNN), where the reduced amount of nomicotine and/or
N'-nitrosonomicotine (NNN) is reduced in a smoke stream produced from the leaf.
[0353] In a further aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568, where the product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof. 103541 In an aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568, where the product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
[0355] In another aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568, where the product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco where the product has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0356] In a further aspect, the present disclosure includes a second tobacco product prepared or produced from a first tobacco product prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568, where first product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco and any combination thereof, and the second tobacco product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco. In some aspects, the first and second product comprises a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0357] In an aspect, the present disclosure includes a part of a tobacco plant, produced by growing the seed of tobacco cultivar CMS NC638 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13568, where the part is selected from the group consisting of leaf, pollen, ovule, embryo, cotyledon, hypocotyl, meristematic cell, protoplast, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
[0358] In another aspect, the present disclosure includes a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
[0359] In an aspect, the present disclosure includes a tobacco plant regenerated from a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13568, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole, where the regenerated plant has all, or essentially all of the morphological and physiological characteristics of cultivar CMS NC638 SRC.
[0360] In an aspect, the present disclosure includes an F | progeny plant of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568.
[0361] In another aspect, the present disclosure includes an F] progeny plant of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568, where the F | plant is cytoplasmic male sterile (CMS).
[0362] In another aspect, the present disclosure includes an Fi progeny seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a plant of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568.
[0363] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568.
[0364] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568, where the plant of tobacco cultivar CMS NC638 SRC is the female parent. [0365] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS ), where the cytoplasmic male sterile plant is a plant of tobacco cultivar CMS NC638 SRC, a
representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13568.
[Θ366] In an aspect, the present disclosure also includes a container of Fi progeny seeds produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13568.
[0367] In an aspect, the present disclosure includes an Fi progeny plant produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. TA- 13568.
[0368] In an aspect, the present disclosure also includes a harvested leaf of an Fi progeny plant produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13568.
[0369] In another aspect, the present disclosure further includes a harvested leaf of an Fj progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568.
[0370] In an aspect, the present disclosure also includes a harvested leaf of an Ft progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) in a smoke stream produced from the leaf, where the plant is produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568.
[0371] In an aspect, the present disclosure includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568, and where the tobacco product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.
[0372] In an aspect, the present disclosure further includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13568, and where the tobacco product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
[0373] In an aspect, the present disclosure further includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13568, and where the tobacco product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco, and further where the product has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0374] In an aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile, where the cytoplasmic male sterile plant is a plant of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13568.
[0375] In another aspect, the present disclosure includes a seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565.
[0376] In an aspect, the present disclosure includes a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565.
[0377] In another aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565.
[0378] In an aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, produced by growing the seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, where the leaf has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0379] In another aspect, the present disclosure includes a harvested leaf, or part thereof, produced by growing the seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, where the leaf has a reduced amount of nornicotine and/or N'- nitrosonornicotine (NNN), where the reduced amount of nornicotine and/or
N'-nitrosonornicotine (NNN) is reduced in a smoke stream produced from the leaf.
[0380] In a further aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar TN86 SRC , a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, where the product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.
[0381] In an aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, where the product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
[0382] In another aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, where the product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco where the product has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0383] In a further aspect, the present disclosure includes a second tobacco product prepared or produced from a first tobacco product prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, where first product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco and any combination thereof, and the second tobacco product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco. In some aspects, the first and second product comprises a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0384] In an aspect, the present disclosure includes a part of a tobacco plant, produced by growing the seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, where the part is selected from the group consisting of leaf, pollen, ovule, embryo, cotyledon, hypocotyl, meristematic cell, protoplast, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
[0385] In another aspect, the present disclosure includes a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
[0386] In an aspect, the present disclosure includes a tobacco plant regenerated from a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole, where the regenerated plant has all, or essentially all of the morphological and physiological characteristics of cultivar TN86 SRC.
[0387] In an aspect, the present disclosure includes an Fi progeny plant of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565.
[0388] In another aspect, the present disclosure includes an Ft progeny plant of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, where the Fi plant is cytoplasmic male sterile (CMS).
[0389] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565.
[0390] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, where the plant of tobacco cultivar TN86 SRC is the male parent.
[0391] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, where the plant of tobacco cultivar TN86 SRC is the female parent.
[0392] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0393] In another aspect, the present disclosure includes an F| progeny seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0394] In an aspect, the present disclosure also includes a container of F1 progeny seeds produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0395] In an aspect, the present disclosure includes an F] progeny plant produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, and where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0396] In an aspect, the present disclosure also includes a harvested leaf of an Fi progeny plant produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, where at least one tobacco plant is cytoplasmic male sterile (CMS). [0397] In another aspect, the present disclosure further includes a harvested leaf of an Fi progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (N ) produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. P I A- 1 3565. where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0398] In an aspect, the present disclosure also includes a harvested leaf of an F1 progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) in a smoke stream produced from the leaf, where the plant is produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13565, where at least one tobacco plant is cytoplasmic male sterile (CMS).
[0399] In an aspect, the present disclosure includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fj progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is produced by growing a seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13565. where at least one tobacco plant is cytoplasmic male sterile, and where the tobacco product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.
[0400] In an aspect, the present disclosure further includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is produced by growing a seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13565, where at least one tobacco plant is cytoplasmic male sterile, and where the tobacco product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco. 10401 ] In an aspect, the present disclosure further includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is produced by growing a seed of tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13565, where at least one tobacco plant is cytoplasmic male sterile, and where the tobacco product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco, and further where the product has a reduced amount of nomicotine and/or N'-nitrosonornicotine (NNN).
[0402] In another aspect, the present disclosure includes a seed of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13570.
[0403] In an aspect, the present disclosure includes a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13570.
[0404] In another aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS TN86SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570.
[0405] In an aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, produced by growing the seed of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570, where the leaf has a reduced amount of nomicotine and/or N'-nitrosonomicotine (NNN).
1040 1 In another aspect, the present disclosure includes a harvested leaf, or part thereof, produced by growing the seed of tobacco cultivar CMS T 86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570, where the leaf has a reduced amount of nomicotine and/or N'- nitrosonomicotine (NNN), where the reduced amount of nomicotine and/or
N'-nitrosonomicotine (NNN) is reduced in a smoke stream produced from the leaf.
[0407] In a further aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570, where the product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.
[0408] In an aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS TN86 SRC , a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570, where the product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
[0409] In another aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570, where the product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco where the product has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0410] In a further aspect, the present disclosure includes a second tobacco product prepared or produced from a first tobacco product prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570, where first product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco and any combination thereof, and the second tobacco product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco. In some aspects, the first and second product comprises a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0411] In an aspect, the present disclosure includes a part of a tobacco plant, produced by growing the seed of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13570, where the part is selected from the group consisting of leaf, pollen, ovule, embryo, cotyledon, hypocotyl, meristematic cell, protoplast, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole. [0412] In another aspect, the present disclosure includes a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS T 86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13570, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
[0413] In an aspect, the present disclosure includes a tobacco plant regenerated from a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13570, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole, where the regenerated plant has all, or essentially all of the morphological and physiological characteristics of cultivar CMS TN86SRC.
[0414] In an aspect, the present disclosure includes an Fi progeny plant of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570.
[0415] In another aspect, the present disclosure includes an Fi progeny plant of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570, where the Fi plant is cytoplasmic male sterile (CMS).
[0416] In another aspect, the present disclosure includes an ¥i progeny seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a plant of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1 570.
[0417] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13570. [0418] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570, where the plant of tobacco cultivar CMS TN86 SRC is the female parent.
[0419] In another aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a plant of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570.
[0420] In an aspect, the present disclosure also includes a container of F] progeny seeds produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570.
[0421] In an aspect, the present disclosure includes an F} progeny plant produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570.
[0422] In an aspect, the present disclosure also includes a harvested leaf of an Fi progeny plant produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570.
[0423] In another aspect, the present disclosure further includes a harvested leaf of an F'i progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13570.
[0424] In an aspect, the present disclosure also includes a harvested leaf of an F1 progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) in a smoke stream produced from the leaf, where the plant is produced by growing a seed produced by crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570.
[0425] In an aspect, the present disclosure includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a F1 progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570, and where the tobacco product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.
[0426] In an aspect, the present disclosure further includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fj progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PT A- 1 570, and where the tobacco product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
[0427] In an aspect, the present disclosure further includes a tobacco product prepared from an Fi progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a Fi progeny seed produced by a method comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile (CMS), where the cytoplasmic male sterile plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PT A- 13570, and where the tobacco product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco, and further where the product has a reduced amount of nornicotine and/or N ' -nitro sonornicotine (NNN) .
[0428] In an aspect, the present disclosure includes a method for producing a tobacco seed comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where at least one tobacco plant is cytoplasmic male sterile, where the cytoplasmic male sterile plant is a plant of tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13570.
[0429] In another aspect, the present disclosure includes a seed of hybrid tobacco cultivar NCBH129 SRC (ATCC Accession No. PTA-13562). Seeds of hybrid cultivar NCBH129 SRC are obtainable by crossing plants of cultivars TN86 SRC and CMS NC638 SRC and collecting the seeds.
[0430] In an aspect, the present disclosure includes a tobacco plant, or part thereof, produced by growing a seed of hybrid tobacco cultivar NCBH129 SRC.
[0431] In a further aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, or part thereof, produced by growing the seed of hybrid tobacco cultivar NCBI 1129 SRC.
[0432] In another aspect, the present disclosure includes a harvested leaf, or part thereof, of a tobacco plant, produced by growing the seed of hybrid tobacco cultivar
NCBH129 SRC, where the leaf has a reduced amount of nornicotine and/or N- nitrosonomicotine (NNN).
[0433] In an aspect, the present disclosure includes a harvested leaf, or part thereof, produced by growing the seed of hybrid tobacco cultivar NCBH129 SRC, where the leaf has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN), where the reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) is reduced in a smoke stream produced from the leaf.
[0434] In a further aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of hybrid tobacco cultivar NCBH129 SRC, where the product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.
[0435] In an aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of hybrid tobacco cultivar NCBH129 SRC, where the product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco.
[0436] In another aspect, the present disclosure includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of hybrid tobacco cultivar NCBH129 SRC, where the product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco where the product has a reduced amount of nornicotine and/or N'- nitrosonornicotine (NNN).
[0437] In a further aspect, the present disclosure includes a second tobacco product prepared or produced from a first tobacco product prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NCBH 129 SRC, where first product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco and any combination thereof, and the second tobacco product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and chewing tobacco. In some aspects, the first and second product comprises a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
[0438] In an aspect, the present disclosure includes a part of a tobacco plant, produced by growing a seed of hybrid tobacco cultivar NCBH129 SRC, where the part is selected from the group consisting of leaf, pollen, ovule, embryo, cotyledon, hypocotyl, meristematic cell, protoplast, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole.
1 43 1 In a further aspect, the present disclosure includes a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of hybrid tobacco cultivar NCBH129 SRC, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole. [0440] In an aspect, the present disclosure includes a tobacco plant regenerated from a tissue culture produced from a protoplast or cell from a tobacco plant, or part thereof, produced by growing the seed of hybrid tobacco cultivar NCBH129 SRC, where the cell or protoplast of the tissue culture is produced from a plant part selected from the group consisting of a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, pod, and petiole, where the regenerated plant has all, or essentially all of the morphological and physiological characteristics of hybrid cultivar NCBH129 SRC.
[0441 ] In an aspect, the present disclosure includes an Fi progeny plant of hybrid tobacco cultivar NCBH129 SRC.
[0442] In an aspect, the present disclosure includes an Fi progeny plant of hybrid tobacco cultivar NCBH129 SRC, where the plant of tobacco cultivar NCBH129 SRC is the female parent.
[0443] In another aspect, the present disclosure includes an Fj progeny plant of hybrid tobacco cultivar NCBH129 SRC where the Fi plant is cytoplasmic male sterile (CMS).
[0444] In another aspect, the present disclosure includes a method for producing a tobacco seed of NCBH129 SRC comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313.
[0445] In another aspect, the present disclosure includes a method for producing a tobacco seed of NCBH 129 SRC comprising crossing two tobacco plants and harvesting the resultant tobacco seed, where one tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568 and a second tobacco plant is a tobacco plant produced by growing the seed of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313.
[0446] In an aspect, the present disclosure includes a method of vegetatively propagating a plant of a tobacco cultivar comprising the steps of (a) collecting tissue capable of being propagated from a plant of a tobacco cultivar selected from the group consisting of NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13568, TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570, and hybrid cultivar NCBH129 SRC; (b) cultivating the tissue to obtain a proliferated shoot; and (c) rooting the proliferated shoots to obtain a rooted plantlet.
[0447] In an aspect, the present disclosure includes a method of vegetatively propagating a plant of a tobacco cultivar comprising the steps of (a) collecting tissue capable of being propagated from a plant of a tobacco cultivar selected from the group consisting of NC638 SRC, a representative sample seed of the cultivar having been deposited with the
ATCC under ATCC Accession No. PTA-120313, CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13568, TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570, and hybrid cultivar NCBH129 SRC; (b) cultivating the tissue to obtain a proliferated shoot; (c) rooting the proliferated shoots to obtain a rooted plantlet; and (d) growing a plant from the rooted plantlet.
[0448] In an aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar selected from the group consisting of NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568, TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PT A- 13565. and CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570, with a second tobacco plant that comprises a desired trait to produce an Fi progeny seed; (b) growing the Fi progeny seed and selecting an Fj progeny plant that comprises the desired trait; (c) crossing the selected Fi progeny plant with a plant of said first tobacco cultivar to produce a backcross BC' j Fi progeny seed; (d) growing the B ' i F) progeny seed and selecting a backcross BCiF( progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of said first tobacco cultivar, NC638 SRC, CMS NC638 SRC, TN86 SRC, or CMS TN86 SRC, respectively; and (e) repeating steps (c) and (d) three or more times (e.g., 3, 4, 5, 6, 7, 8, 9, 10, and the like) in succession to produce selected fourth or higher backcross progeny that comprise the desired trait. In additional aspects, steps (c) and (d) can be repeated one or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and the like) in succession to produce second or higher backcross progeny comprising the desired trait.
10449] In an aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar selected from the group consisting of NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, and TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13565. with a second tobacco plant that comprises a desired trait to produce an Fi progeny seed; (b) growing the Fi progeny seed and selecting an Fi progeny plant that comprises the desired trait; (c) crossing the selected F1 progeny plant with a plant of said first tobacco cultivar to produce a backcross BCiFi progeny seed;
(d) growing the BCiFj progeny seed and selecting a backcross BC1F1 progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of the first tobacco cultivar, NC638 SRC, or TN86 SRC, respectively; and
(e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny that comprise the desired trait, where the trait is cytoplasmic male sterility (CMS). In additional aspects, steps (c) and (d) can be repeated one or more times in succession to produce second or higher backcross progeny comprising the desired trait, where the trait is CMS.
[0450] In an aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar selected from the group consisting of NC638 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, and TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13565. with a second tobacco plant that comprises a desired trait to produce an Fi progeny seed; (b) growing the Fi progeny seed and selecting an Fx progeny plant that comprises the desired trait; (c) crossing the selected Fj progeny plant with a plant of said first tobacco cultivar to produce a backcross BCiFi progeny seed;
(d) growing the BCiFi progeny seed and selecting a backcross BQFi progeny plant comprising the desired trait and the physiological and essentially all of morphological characteristics of the first tobacco cultivar NC638 SRC or TN86 SRC, respectively; and
(e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny that comprise the desired trait, where the trait is cytoplasmic male sterility (CMS) and the CMS trait is obtained from the cytoplasm of Nicotiana suaveolens or Nicotiana glauca. In additional aspects, steps (c) and (d) can be repeated one or more times in succession to produce second or higher backcross progeny comprising the desired trait, where the trait is CMS and the CMS trait is obtained from the cytoplasm of Nicotiana suaveolens or Nicotiana glauca.
10451 ] In an aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar selected from the group consisting of NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, and TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13565 with a second tobacco plant that comprises a desired trait to produce an Fi progeny seed; (b) growing the Fi progeny seed and selecting an F progeny plant that comprises the desired trait; (c) crossing the selected Fi progeny plant with a plant of said first tobacco cultivar to produce a backcross BCiFi progeny seed;
(d) growing the BC i F i progeny seed and selecting a backcross BC |Fi progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of tobacco cultivar NC638 SRC or TN86 SRC, respectively; and (e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny that comprise the desired trait, where the trait is cytoplasmic male sterility (CMS) and the CMS trait is obtained from the cytoplasm of Nicotiana suaveolens, and where the second tobacco plant is selected from the group consisting of CMS NC638 SRC and CMS TN86 SRC. In additional aspects, steps (c) and (d) can be repeated one or more times in succession to produce second or higher backcross progeny comprising the desired trait, where the trait is CMS and the CMS trait is obtained from the cytoplasm of Nicotiana suaveolens.
[0452] In an aspect, the present disclosure includes a tobacco plant produced by a method comprising introducing a desired trait into a tobacco cultivar comprising:
(a) crossing a plant of a first tobacco cultivar selected from the group consisting of NC638 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13568, TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13565. and CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570, with a second tobacco plant that comprises a desired trait to produce an Fj progeny seed; (b) growing the Fj progeny seed and selecting an Fi progeny plant that comprises the desired trait; (c) crossing the selected Fi progeny plant with a plant of said first tobacco cultivar to produce a backcross BQFi progeny seed; (d) growing the BCiFj. progeny seed and selecting a backcross BCiFi progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of the first tobacco cultivar NC638 SRC, CMS NC638 SRC, TN86 SRC. or CMS ΊΝ86 SRC. respectively; and (e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny that comprise the desired trait. In additional aspects, steps (c) and (d) can be repeated one or more times in succession to produce second or higher backcross progeny that comprise the desired trait.
[0453] In another aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar selected from the group consisting of NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568, TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13565, and CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570, with a plant of a second tobacco cultivar that comprises a desired trait to produce a progeny plant where the desired trait is selected from the group consisting of herbicide resistance, pest resistance, disease resistance, high yield, high grade index, curability, curing quality, mechanical harvestability, holding ability, leaf quality, height, plant maturation, early maturing, early to medium maturing, medium maturing, medium to late maturing, late maturing, small stalk, medium stalk, large stalk, leaf number per plant, 5-10 leaves per plant, 11-15 leaves per plant, 16-21 leaves per plant, and any combination thereof, to produce an Fi progeny seed; (b) growing the F1 progeny seed into an Fi progeny plant and selecting the
Figure imgf000103_0001
progeny plant having the desired trait; (c) crossing the selected F) progeny plant with a plant of said first tobacco cultivar to produce a backcross progeny plant seed; (d) growing the backcross progeny plant seed into a backcross progeny plant and selecting the backcross progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of the first tobacco cultivar. NC638 SRC. CMS NC638 SRC. Ν86 SRC, or CMS TN86 SRC, respectively; and (e) repeating steps (c) and (d) one or more times in succession to produce a selected second, third, fourth or higher backcross progeny plant that comprises the desired trait and essentially all of the physiological and morphological characteristics of the first tobacco cultivar, NC638 SRC. CMS NC638 SRC, TN86 SRC or CMS TN86 SRC, respectively.
[0454] In another aspect, the present disclosure includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a first tobacco cultivar selected from the group consisting of NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568, TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13565, and CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13570. with a plant of a second tobacco cultivar that comprises a desired trait to produce a progeny plant where the desired trait is selected from the group consisting of herbicide resistance, pest resistance, disease resistance, high yield, high grade index, curability, curing quality, mechanical harvestability, holding ability, leaf quality, height, plant maturation, early maturing, early to medium maturing, medium maturing, medium to late maturing, late maturing, small stalk, medium stalk, large stalk, leaf number per plant, 5-10 leaves per plant, 1 1-15 leaves per plant, 16-21 leaves per plant, and any combination thereof, to produce an Fi progeny seed; (b) growing the ΐι progeny seed into an Fi progeny plant and selecting the Fi progeny plant having the desired trait; (c) crossing the selected Fi progeny plant with a plant of said first tobacco cultivar to produce a backcross progeny plant seed; (d) growing the backcross progeny plant seed into a backcross progeny plant and selecting the backcross progeny plant comprising the desired trait and essentially all of the physiological and morphological characteristics of said first tobacco cultivar. NC638 SRC, CMS NC638 SRC, TN86 SRC, or CMS TN86 SRC. respectively; and (e) repeating steps (c) and (d) one or more times in succession to produce a selected second, third, fourth or higher backcross progeny plant that comprises the desired trait and essentially all of the physiological and morphological characteristics of a tobacco cultivar. NC638 SRC, CMS NC638 SRC, TN86 SRC, or CMS TN86 SRC, respectively, where the desired trait is disease resistance.
[0455] In an aspect, the present disclosure includes a method for producing a tobacco plant having decreased nicotine conversion comprising: identifying a first tobacco plant comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 1 1, and any combination thereof; crossing the first tobacco plant with a second tobacco plant and collecting an ¥\ seed;
crossing a plant grown from the Fi seed to a third tobacco plant and collecting a second tobacco seed; and identifying a second tobacco seed or a plant grown from the second seed that is homozygous for the nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 1 1 , and any combination thereof, where the first tobacco plant is a plant of a tobacco cultivar selected from the group consisting of tobacco cultivar NC638 SRC. a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. P I A- 13568, tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, tobacco cultivar CMS T 86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13570, and hybrid cultivar NCBH129 SRC.
[0456] In an aspect, the present disclosure includes a method for producing a tobacco plant having decreased nicotine conversion comprising: identifying a first tobacco plant comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 1 1 , and any combination thereof; crossing the first tobacco plant with a second tobacco plant and collecting an ¥i seed;
crossing a plant grown from the Fi seed to a third tobacco plant and collecting a second tobacco seed; and identifying a second tobacco seed or a plant grown from the second seed that is homozygous for the nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 11, and any combination thereof, where the third tobacco plant is a plant of a tobacco cultivar selected from the group consisting of the tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, tobacco cultivar CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. ΡΤΛ- 13568. tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13570, and hybrid cultivar NCBH129 SRC. [0457] In another aspect, the present disclosure includes a method of producing a plant of a tobacco cultivar selected from the group consisting of tobacco cultivar NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, tobacco cultivar CMS NC638 SRC. a
representative sample seed of the cultivar having been deposited with the ATCC under
ATCC Accession No. PTA-13568, tobacco cultivar TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13565, tobacco cultivar CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570, and hybrid cultivar NCBH129 SRC comprising at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, and the like) additional desired trait comprising the steps of: (a) collecting tissue capable of being propagated from a plant of a tobacco cultivar selected from the group consisting of NC638 SRC, a
representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568, TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13570, and hybrid cultivar NCBH129 SRC; and (b) introducing at least one transgene (nucleic acid construct) conferring at least one desired trait into the tissue.
[0458] In another aspect, the present disclosure includes a method of producing an herbicide resistant tobacco plant comprising transforming a tobacco plant, or part thereof, produced by growing a seed of a tobacco cultivar selected from the group consisting of NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120 13, CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13568, TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570, and hybrid cultivar NCBH129 SRC with at least one transgene (nucleic acid construct), where the at least one transgene (nucleic acid construct) confers resistance to an herbicide selected from the group consisting of imidazolinone, cyclohexanedione, sulfonylurea, glyphosate, glufosinate, phenoxy proprionic acid, L-phosphinothricin, triazine, benzonitrilc, and any combination thereof. [0459] In another aspect, the present disclosure includes an herbicide resistant tobacco plant produced by a method comprising transforming a tobacco plant, or part thereof, produced by growing a seed of a tobacco cultivar selected from the group consisting of NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13568, TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565. CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13570. and hybrid cultivar NCBH129 SRC, with at least one transgene (nucleic acid construct), where the at least one transgene (nucleic acid construct) confers resistance to an herbicide selected from the group consisting of imidazolinone, cyclohexanedione, sulfonylurea, glyphosate, glufosinate, phenoxy proprionic acid, L-phosphinothricin, triazine, benzonitrile, and any combination thereof.
[0460] In another aspect, the present disclosure includes a method of producing a pest and/or insect resistant tobacco plant where the method comprises transforming a tobacco plant produced by growing a seed of a tobacco cultivar selected from the group consisting of NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13568, TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13570, and hybrid cultivar NCBH129 SRC, with at least one transgene (nucleic acid construct) that confers pest and/or insect resistance.
[0461] In a further aspect, the present disclosure includes a pest and/or insect resistant tobacco plant produced by a method comprising transforming a tobacco plant produced by growing a seed of a tobacco cultivar selected from the group consisting of NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. Ρ ΓΑ- 13568, TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570, and hybrid cultivar NCBH129 SRC with at least one transgene (nucleic acid construct) that confers pest and/or insect resistance.
[0462] In a further aspect, the present disclosure includes a pest and/or insect resistant tobacco plant produced by a method comprising transforming a tobacco plant produced by growing a seed of a tobacco cultivar selected from the group consisting of NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13568, TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565. CMS TNS6 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570, and hybrid cultivar NCBH129 SRC, with at least one transgene (nucleic acid construct) that confers pest and/or insect resistance, where the transgene encodes a Bacillus thuringiensis (BT) endotoxin.
[0463] In another aspect, the present disclosure includes a method of producing a disease resistant tobacco plant where the method comprises transforming a tobacco plant produced by growing a seed of a tobacco cultivar selected from the group consisting of NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313, CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13568, TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570, and hybrid cultivar NCBH129 SRC with at least one transgene (nucleic acid construct) that confers disease resistance.
[0464] In a further aspect, the present disclosure includes a disease resistant tobacco plant produced by a method comprising transforming a tobacco plant produced by growing a seed of a tobacco cultivar selected from the group consisting of NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120 13, CMS NC638 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13568, I 86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565, CMS TN86 SRC, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570, and hybrid cultivar NCBH129 SRC with at least one transgene (nucleic acid construct) that confers disease resistance.
BRIEF DESCRIPTION OF THE SEQUENCES
[0465] SEQ ID NO 1 sets forth a cyp82e4 W329Stop nucleotide sequence.
[04661 SEQ ID NO 2 sets forth a cyp82e5v2 W422Stop nucleotide sequence.
[04671 SEQ ID NO 3 sets forth a cyp82e4 W329Stop amino acid sequence.
[0468] SEQ ID NO 4 sets forth a cyp82e5v2 W422Stop amino acid sequence.
[0469] SEQ ID NO 5 sets forth a CYP82E4 wild-type nucleotide sequence.
10470] SEQ ID NO 6 sets forth a CYP82E5v2 wild-type nucleotide sequence.
[0471] SEQ ID NO 7 sets forth a CYP82E4 wild-type amino acid sequence.
[0472] SEQ ID NO 8 sets forth a CYP82E5v2 wild-type amino acid sequence.
[0473] SEQ ID NO 9 sets forth a CYP82E10 wild-type nucleotide sequence.
[0474] SEQ ID NO 10 sets forth a CYP82E10 wild-type amino acid sequence.
[0475] SEQ ID NO 1 1 sets forth a CYP82E10 P381 S nucleotide sequence.
[0476] SEQ ID NO 12 sets forth a CYP82E10 P381 S amino acid sequence.
[0477J All three tobacco Nicotine Demethylase genes (CYP82E4, CYP82E5v2, CYP82E10) share a common structure: a 939 bp exon 1 and a 612 bp exon 2 separated by a large intron, whose length varies among the three genes. See Lewis et al, "Three nicotine demethylase genes mediate nornicotine biosynthesis in Nicotiana tabacum L.: Functional characterization of the CYP82E10 gene," Phytochemistry, 71 (2010), 1988-1998. SEQ ID NOs: 1, 2, 5, 6, 9, and 1 1 set forth wild-type or mutant versions of coding sequences of CYP82E4, CYP82E5v2, and CYP82E10. It is understood that, used herein, a plant
comprising, having, or homozygous for a sequence selected from SEQ ID NOs: 1, 2, 5, 6, 9, and 11 refers to a plant comprising at the CYP82E4, CYP82E5v2, or CYP82E10 endogenous locus a genomic sequence comprising the coding sequence of SEQ ID NO: 1, 2, 5, 6, 9, or 11.
BRIEF DESCRIPTION OF THE DRAWINGS
[0478] Figure 1 : A comparison between populations of TN90 LC plants and TN90 SRC plants showing a more stable and lower average percent nicotine conversion in TN90 SRC. Leaves from 26 plants of each variety are sampled at the layby stage and ethephon- treated prior to analysis for nicotine and nornicotine levels using gas chromatography equipment. [0479] Figure 2: Tobacco plants grown in the field treated with different amounts of nitrogen fertilizers.
[0480] Figure 3 : Comparable nitrate levels in tobacco filler from TN90 LC and TN90 SRC grown under a same nitrogen fertilizer level. No statistical difference is observed for nitrate concentrations between TN90 SRC and TN90 LC tobacco filler grown under a same nitrogen fertilizer level.
[0481 ] Figure 4: Comparable tobacco yields from TN90 LC and TN90 SRC grown under a same nitrogen fertilizer level. No statistical difference is observed for tobacco yields between ΊΓΝ90 SRC and TN90 LC tobacco filler grown under a same nitrogen fertilizer level.
[0482] Figure 5: Comparable levels of total alkaloids from TN90 LC and TN90
SRC grown under a same nitrogen fertilizer level. No statistical difference is observed for total alkaloids levels between TN90 SRC and TN90 LC tobacco filler grown under a same nitrogen fertilizer level.
[0483] Figure 6: TN90 SRC contains a significantly reduced level of nornicotine in tobacco filler compared to TN90 LC when grown under a same nitrogen fertilizer level. Asterisks (*) indicate significant differences for filler nornicotine based on Tukey-Kramer USD (P<.05).
[0484] Figure 7: TN90 SRC contains a significantly reduced level of NNN in tobacco filler compared to TN90 LC when grown under a high nitrogen fertilizer level (448 kg/ha). The asterisk (*) indicates a significant difference for NNN levels in tobacco filler based on Tukey-Kramer HSD (P<.05).
[0485] Figure 8: Comparisons of NNN levels in cigarette smoke from cigarettes comprising 100% TN90 SRC or 100% TN90 LC. TN90 SRC cigarette smoke contains a significantly reduced level of NNN compared to TN90 LC cigarette smoke when both varieties are grown under a same nitrogen fertilizer level. Asterisks (*) indicate significant differences for NNN levels in tobacco filler based on Tukey-Kramer HSD (P<.05).
[0486] Figures 9A-9B: Evaluations of smoke NNN levels in blended cigarettes made with an Extramural Blend comprising varying amounts of TN90 SRC as the Burley component. Experimental blends with increasing percentages of TN90 SRC (33%
Experimental comprising 8% TN90 SRC and 15% TN90 LC, 67% Experimental comprising 15% TN90 SRC and 8% TN90 LC, and 100% Experimental comprising 23% TN90 SRC) are compared to a control blend comprising 23% TN90 LC and no TN90 SRC (0%
Experimental). Figure 9 A shows formulations of control and experimental blends. Each component is shown by their percent dry weight. Both the International Organization of Standardization (ISO) smoking method (shown as Smoke ISO on the x-axis) and the Health Canada Intense (HCI) smoking method (shown as Smoke HC Intense on the x-axis) are used. Figure 9B shows that the inclusion of TN90 SRC filler in an Extramural Blend (even when TN90 SRC only constitutes 33% or 67% of the total Burly component of the blend, e.g., 33% Experimental or 67% Experimental) reduces the level of smoke NNN measured by either the ISO smoking method or the HCI smoking method. Further, blended cigarettes with an Extramural Blend comprising I 90 SRC filler as 100% of the Burley component (100% Experimental; TN90 SRC = 23% of overall blend) show a reduction of 38% (ISO method) and 35% (HCI method) in smoke NNN compared to a control blend comprising TN90 LC filler as 100% of the Burley component (0% Experimental; TN90 LC - 23% of overall blend). 0% E, 33% E, 67% E, and 100% E shown on the x-axis represents control, 33% Experimental, 67% Experimental, and 100% Experimental blends respectively.
DETAILED DESCRIPTION
[0487] This description is not intended to be a detailed catalog of all the different ways in which the disclosure may be implemented, or all the features that may be added to the instant disclosure. 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, the disclosure contemplates that in some embodiments of the disclosure, any feature or combination of features set forth herein can be excluded or omitted. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant disclosure. Hence, the following descriptions are intended to illustrate some particular embodiments of the disclosure, and not to exhaustively specify all permutations, combinations and variations thereof.
[0488] Unless otherwise defined, 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 disclosure belongs. The terminology used in the description of the disclosure herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.
[0489] All publications, patent applications, patents and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art.
[0490] Unless the context indicates otherwise, it is specifically intended that the various features of the disclosure described herein can be used in any combination.
Moreover, the present disclosure also contemplates that in some embodiments of the disclosure, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a composition comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.
[0491] As used in the description of the disclosure and the appended claims, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
[0492] As used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of
combinations when interpreted in the alternative ("or").
[0493] The term "about," as used herein when referring to a measurable value such as a dosage or time period and the like, refers to variations of ± 20%, ± 10%, + 5%, + 1%, ± 0.5%, or even ± 0.1% of the specified amount.
[0494] 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" mean "between about X and about Y" and phrases such as "from about X to Y" mean "from about X to about Y."
[0495] The terms "comprise," "comprises" and "comprising" as used herein, specify the presence of the stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
[0496] As used herein, the transitional phrase "consisting essentially of" means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed disclosure. Thus, the term "consisting essentially of when used in a claim of this disclosure is not intended to be interpreted to be equivalent to "comprising."
[0497] "Introducing," in the context of a polynucleotide sequence (e.g., a recombinant polynucleotide and/or expression cassette of the disclosure), means presenting a polynucleotide sequence to the plant, plant part, and/or plant cell in such a manner that the polynucleotide sequence gains access to the interior of a cell. Where more than one polynucleotide sequence is to be introduced these polynucleotide sequences can be assembled as part of a single polynucleotide or nucleic acid construct, or as separate polynucleotide or nucleic acid constructs, and can be located on the same or different transformation vectors. Accordingly, these polynucleotides can be introduced into plant cells in a single
transformation event, in separate transformation events, or, e.g., as part of a breeding protocol. Thus, the term "transformation" as used herein refers to the introduction of a heterologous nucleic acid into a cell. Transformation of a cell may be stable or transient. Thus, in some embodiments, a plant cell, plant part or plant of this disclosure can be stably transformed with a recombinant polynucleotide of the disclosure. In other embodiments, a plant cell, plant part or plant of this disclosure can be transiently transformed with a recombinant polynucleotide of the disclosure.
104981 "Tobacco product" is defined as "any product made or derived from tobacco that is intended for human use or consumption, including any component, part, or accessory of a tobacco product (except for raw materials other than tobacco used in manufacturing a component, part, or accessory of a tobacco product)" (section 201 of the FD&C Act; 21 U.S.C. 321). The label and packaging is part of a tobacco product.
[0499] Terms "nicotine conversion rate," "percent nicotine conversion," and "percentage nicotine conversion" are used interchangeably. Percent nicotine demethylation in a sample is calculated by dividing the level of nomicotine by the combined level of nicotine and nomicotine as measured in the sample, and multiplying by 100.
TN90 SRC
[05001 In some aspects, the present disclosure provides tobacco cultivars, and parts thereof, from TN90 SRC, representative sample seeds of this cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13567. In other aspects, the present disclosure provides a tobacco plant, or part thereof, produced by growing a seed of TN90 SRC. In further aspects, a plant of the present disclosure can include a plant with all, or essentially all, of the morphological and physiological characteristics of cultivar TN90 SRC.
[0501] While not being limited by process, TN90 SRC is a result o the introduction of three mutated CYP82E genes in a burley tobacco cultivar TN90. The three genes are a mutated CYP82E4 gene recited as 325-6 #775 in Lewis et al. ("Three nicotine demethylase genes mediate nomicotine biosynthesis in Nicotiana tabacum L.: Functional characterization of the CYP82E10 gene," Phytochemistry, 71 (2010), 1988-1998 (SEQ ID NO: 1, which sets forth a cyp82e4 W329Stop, hereby incorporated by reference in its entirety)), a mutated CYP82E5v2 recited in Lewis et al. (supra) as 325-6 #1-13 (SEQ ID NO: 2, which sets forth a cyp82e5v2 W422Stop, hereby incorporated by reference in its entirety), and a mutated CYP82E10 recited in Lewis et al. (supra) as 325-6 #1041 (SEQ ID NO: 1 1 , which sets forth a cyp82el0 P381S, hereby incorporated by reference in its entirety). Mutations cyp82e4 W329Stop and cyp82e5v2 W422Stop result in truncated proteins while cyp82E10 P381S results in a nonfunctional protein. A cyp82e4 W329Stop ("e "), a cyp82e5v2 W422Stop ("e5"), and a cyp82el0 P381S ("ei0") mutation are introduced from a e4e4\e5e5\e!0el0 triple mutant from a strong converter burley background, line DH98-325-6, as listed in Table 4 of Lewis et al. (supra) into a burley tobacco cultivar TN90 background.
[0502] TN90 SRC is the result of seven backcrosses with burley cultivar TN90 as the recurrent parent, followed by two rounds of selfing with selection for homozygosity for the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82el0 P381 S mutations to yield BC-F3 plants (TN90 SRC) in which the wild-type CYP82E4, CYP82E5v2 and CYP82E10 alleles of TN90 are replaced by the mutant alleles (e.g., cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyP82elO P381 S).
[0503] TN90 SRC progeny plants have genetic backgrounds that are at least 95%, at least 97%, at least 98%, or at least 99% similar to TN90. TN90 SRC plants exhibit low nornicotine levels and produce leaves with reduced potential for accumulating derived NNN during curing, storage, and smoking.
CMS TN90 SRC
[0504] In some aspects, the present disclosure provides tobacco cultivars, and parts thereof, from CMS FN 90 SRC, representative sample seeds of this cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13573. In further aspects, the present disclosure also includes a tobacco plant, or part thereof, produced by growing a seed of CMS TN90 SRC. In still further aspects, a plant of the present disclosure can include a plant with all, or essentially all, of the morphological and physiological characteristics of cultivar CMS TN90 SRC. CMS TN90 SRC is a male-sterile (CMS) version of TN90 SRC (CMS TN90 SRC) produced by crossing a plant of CMS TN90 as a female with pollen of TN90 SRC BC6Fi to produce male-sterile plants heterozygous for all three mutations. The CMS progeny plants of the CMS TN90 x TN90 SRC BC6Fi cross are male sterile. A pluralit of CMS TN90 x TN90 SRC BC6F, x CMS plants (e.g., CMS F, progeny plants) are screened for the cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381S mutations to identify plants heterozygous for all three mutations. A single male-sterile plant resulting from this cross is backcrossed as a female to TN90 SRC to prepare BC7F1 CMS progeny. BC7F 1 CMS progeny homozygous for the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82el0 P381 S mutations are identified by genotyping and designated as CMS TN90 SRC. Because the CMS TN90 SRC line is male sterile, it is maintained via pollination with TN90 SRC. TN90 SRC is crossed as the male parent to CMS ΤΓ 90 SRC to prepare CMS TN90 SRC Fi progeny plants.
[0505] CMS TN90 SRC and CMS TN90 SRC F, progeny plants have genetic backgrounds that are at least 95%, at least 97%, at least 98%, or at least 99% similar to TN90. CMS TN90 SRC and CMS TN90 SRC Fi progeny plants exhibit low nornicotine levels and produce leaves with reduced potential for accumulating derived NNN during curing, storage, and smoking.
Y14 SRC
[0506] In one aspect, the present disclosure provides tobacco cultivars, and parts thereof, from KYI 4 SRC, representative sample seeds of this cultivar having been deposited with the ATCC under ATCC Accession No. P I A- 120311. In another aspect, the present disclosure provides a tobacco plant, or part thereof, produced by growing a seed of KYI 4 SRC. In a further aspect, a plant of the present disclosure can include a plant with all, or essentially all, of the morphological and physiological characteristics of cultivar KYI 4 SRC.
[0507] While not being limited by process. KYI 4 SRC is a result of the introduction of three mutated CYP82E genes in a burley tobacco cultivar KYI 4. The three genes are a mutated CYP82E4 gene recited as 325-6 #775 in Lewis et al. ("Three nicotine demethylase genes mediate nornicotine biosynthesis m Nicotiana tabacum L.: Functional characterization of the CYP82E10 gene," Phytochemistry, 71 (2010), 1988-1998 (SEQ ID NO: 1, which sets forth a cyp82e4 W329Stop, hereby incorporated by reference in its entirety)), a mutated CYP82E5v2 recited in Lewis et al. {supra) as 325-6 #1-13 (SEQ ID NO: 2, which sets forth a cyp82e5v2 W422Stop, hereby incorporated by reference in its entirety), and a mutated CYP82E10 recited in Lewis et al. (supra) as 325-6 #1041 (SEQ ID NO:l 1 , which sets forth a cyp82el0 P381 S, hereby incorporated by reference in its entirety). Mutations cyp82e4 W329Stop and cyp82e5v2 W422Stop result in truncated proteins while cyp82E10 P381 S results in a nonfunctional protein. A cyp82e4 W329Stop ("e4"), a cyp82e5v2 W422Stop ("e5"), and a cyp82el0 P381S ("eiO") mutation are introduced from a e4e4\e5e5\el0el0 triple mutant from a strong converter burley background, line DH98-325-6, as listed in Table 4 of Lewis et al. {supra) into a burley tobacco cultivar KYI 4 background.
[0508] KYI 4 SRC is the result of seven backcrosses with burley culti var KYI 4 as the recurrent parent, followed by two rounds of selfing with selection for homozygosity for the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82el 0 P381 S mutations to yield BC7F3 plants (KYI 4 SRC) in which the wild-type CYP82E4, CYP82E5v2 and CYP82E10 alleles of KY14 are replaced by the mutant (e.g., cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381 S) alleles.
[0509] KYI 4 SRC progeny plants have genetic backgrounds that are at least 95%, at least 97%, at least 98%>, or at least 99%> similar to KY14. KY14 SRC plants exhibit low nornicotine levels and produce leaves with reduced potential for accumulating derived NNN during curing, storage, and smoking.
CMS KY14 SRC
[0510] In other aspects, the present disclosure also provides tobacco cultivars, and parts thereof, from CMS KYI 4 SRC, representative sample seeds of this cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13571. In further aspects, the present disclosure provides a tobacco plant, or part thereof, produced by growing a seed of CMS KYI 4 SRC. In still further aspects, the plant of the present disclosure can include a plant with all, or essentially all, of the morphological and physiological characteristics of cultivar CMS KY14 SRC. CMS KYI 4 SRC is a male-sterile (CMS) version of K YI 4 SRC (CMS KYI 4 SRC) produced by crossing a plant of CMS KYI 4 as a female with pollen of KY14 SRC BC6Fi to produce male-sterile plants heterozygous for all three mutations. The CMS progeny plants of the CMS KY14 x KY14 SRC BC6F] cross are male sterile. A plurality of CMS KY14 x KY14 SRC BC F, x CMS plants (e.g., CMS F, progeny plants) are screened for the cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el 0 P381 S mutations to identify plants heterozygous for all three mutations. A single male-sterile plant resulting from this cross is backcrossed as a female to KYI 4 SRC to prepare BC7F] CMS progeny. BC7F 1 CMS progeny homozygous for the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82el0 P381 S mutations are identified by genotyping and designated as CMS KY14 SRC. Because the CMS KY14 SRC line is male sterile, it is maintained via pollination with KY14 SRC. KY14 SRC is crossed as the male parent to CMS KY14 SRC to prepare CMS KY14 SRC Fi progeny plants.
[0511] CMS KYI 4 SRC and CMS KYI 4 SRC F, progeny plants have genetic backgrounds that are at least 95%, at least 97%, at least 98%, or at least 99% similar to KY I 4. CMS KY14 SRC and CMS KY I 4 SRC F, progeny plants exhibit low nomicotine levels and produce leaves with reduced potential for accumulating derived NNN during curing, storage, and smoking.
L8 SRC
[0512] In some aspects, the present disclosure provides tobacco cultivars, and parts thereof, from L8 SRC, where representative sample seeds of this cultivar have been deposited with the ATCC under ATCC Accession No. PTA-13572. In other aspects, the present disclosure provides a tobacco plant, or part thereof, produced by growing a seed of L8 SRC. In still further aspects, a plant of the present disclosure can include a plant with all, or essentially all, of the morphological and physiological characteristics of cultivar L8 SRC. While not being limited by process, L8 SRC is a result of introducing the cyp82e4
W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381S mutations from DH98-325-6. Fi individuals originating from a cross between L8 and DH98-325-6 and heterozygous for each mutation are then backcrossed seven times to L8 to produce BC7F 1 progeny. BC7Fi individuals heterozygous for all three mutations are self-pollinated to produce BC7F2 seed and individuals homozygous for all three mutations identified. A single BC F2 plant is self- pollinated to produce BC7F3 (L8 SRC) in which the wild-type CYP82E4, CYP82E5v2 and CYP82E10 alleles of L8 are replaced by the mutant (e.g. , cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381S).
[0513] L8 SRC has a genetic background that is at least 95%, at least 97%, at least
98%, or at least 99% similar to L8. L8 SRC exhibits low nomicotine levels and produce leaves with reduced potential for accumulating derived NNN during curing, storage, and smoking.
KYI 4 X L8 SRC
[0514] In other aspects, the present disclosure provides tobacco cultivars, and parts thereof, from KY14 X L8 SRC. In another aspect, the present disclosure provides a tobacco plant, or part thereof, produced by growing the seed of KY14 X L8 SRC. In a further aspect, a plant of the present disclosure can include a plant with all, or essentially all, of the morphological and physiological characteristics of cultivar KY14 X L8 SRC.
[0515] While not being limited by process, K Y 14 X 1.8 SRC is produced by pollinating plants of CMS KY14 SRC with pollen of L8 SRC. Again, not limited by any particular scientific theory, cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381S mutations all encode for proteins with reduced or eliminated ability to convert nicotine to nomicotine. KYI 4 X L8 SRC has a genetic background that is at least 95%, at least 97%>, at least 98%, or at least 99% similar to burley tobacco cultivar KYI 4 x L8, a hybrid generated by pollinating plants of a male-sterile breeding line CMS KYI 4 with pollen produced by fertile breeding line L8. KY14 X L8 SRC exhibits low nornicotine and is not subject to conversion to high nornicotine.
NC775 SRC
[0516] In one aspect, the present disclosure provides tobacco cultivars, and parts thereof, from NC775 SRC, representative sample seeds of this cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120312. In another aspect, the present disclosure additionally provides a tobacco plant, or part thereof, produced by growing a seed of NC775 SRC. In a further aspect, a plant of the present disclosure can include a plant with all or essentially all of the morphological and physiological characteristics of cultivar NC775 SRC.
[0517] While not being limited by process, NC775 SRC is a result of the
introduction of three mutated CYP82E genes in a burley tobacco cultivar NC775. The three genes are a mutated CYP82E4 gene recited as 325-6 #775 in Lewis et al. ("Three nicotine demethylase genes mediate nornicotine biosynthesis in Nicotiana tabacum L.: Functional characterization of the CYP82E10 gene," P yto chemistry, 71 (2010), 1988-1998 (SEQ ID NO: 1, which sets forth a cyp82e4 W329Stop, hereby incorporated by reference in its entirety)), a mutated CYP82E5v2 recited in Lewis et al. {supra) as 325-6 #1-13 (SEQ ID NO: 2, which sets forth a cyp82e5v2 W422Stop, hereby incorporated by reference in its entirety), and a mutated CYP82E10 recited in Lewis et al. {supra) as 325-6 #1041 (SEQ ID NO: ] 1 , which sets forth a cyp82el0 P381 S, hereby incorporated by reference in its entirety).
Mutations cyp82e4 W329Stop and cyp82e5v2 W422Stop result in truncated proteins while cyp82E10 P381 S results in a nonfunctional protein. A cyp82e4 W329Stop (W), a cyp82e5v2 W422Stop ("e5"), and a cyp82el0 P381 S ("el 0") mutation are introduced from a e4e4\e5e5\el0el0 triple mutant from a strong converter burley background, line DH98-325-6, as listed in Table 4 of Lewis et al. {supra) into a burley tobacco cultivar NC775 background.
[0518] NC775 SRC is the result of seven backcrosses with burley culti var NC775 as the recurrent parent, followed by two rounds of selling with selection for homozygosity for the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82el 0 P381 S mutations to yield BC7F3 plants (NC775 SRC) in which the wild-type CYP82E4, CYP82E5v2, and CYP82E10 alleles of NC775 are replaced by the mutant (e.g., cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e!0 P381 S) alleles. [0519] NC775 SRC progeny plants have genetic backgrounds that are at least 95%, at least 97%, at least 98%, or at least 99% similar to NC775. NC775 SRC plants exhibit low nornicotine levels and produce leaves with reduced potential for accumulating derived NNN during curing, storage, and smoking.
CMS NC775 SRC
[0520] In some aspects, the present disclosure provides tobacco cultivars, and parts thereof, from CMS NC775 SRC, representative sample seeds of this cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13563. In further aspects, the present disclosure additionally provides a tobacco plant, or part thereof, produced by growing a seed of CMS NC775 SRC. In still further aspects, a plant of the present disclosure can include a plant with all or essentially all, of the morphological and physiological
characteristics of cultivar CMS NC775 SRC. CMS NC775 SRC is a male-sterile (CMS) version of NC775 SRC (CMS NC775 SRC) produced by crossing a plant of CMS NC775 as a female with pollen of NC775 SRC BC6Fi to produce male-sterile plants heterozygous for all three mutations. The CMS progeny plants of the CMS NC775 x NC775 SRC BC^F, cross are male sterile. A plurality of CMS NC775 x NC775 SRC BC6Fi x CMS plants (e.g., CMS Fi progeny plants) are screened for the cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381 S mutations to identify plants heterozygous for all three mutations. A single male-sterile plant resulting from this cross is backcrossed as a female to NC775 SRC to prepare BC7F1 CMS progeny. BC7F1 CMS progeny homozygous for the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82el0 P381S mutations are identified by genotyping and designated as CMS NC775 SRC. Because the CMS NC775 SRC line is male sterile, it is maintained via pollination with NC775 SRC. NC775 SRC is crossed as the male parent to CMS NC775 SRC to prepare CMS NC775 SRC F, progeny plants.
[0521] CMS NC775 SRC and CMS NC775 SRC F, progeny plants have genetic backgrounds that are at least 95%, at least 97%, at least 98%, or at least 99% similar to NC775. CMS NC775 SRC and CMS NC775 SRC F, progeny plants exhibit low nornicotine levels and produce leaves with reduced potential for accumulating derived NNN during curing, storage, and smoking.
NC645 SRC
[0522] In some aspects, the present disclosure provides tobacco cultivars, and parts thereof, from NC645 SRC, where representative sample seeds of this cultivar have been deposited with the ATCC under ATCC Accession No. PTA- 13566. In further aspects, the present disclosure additionally provides a tobacco plant, or part thereof, produced by growing a seed of NC645 SRC. In still further aspects, a plant of the present disclosure can include a plant with all or essentially all of the morphological and physiological characteristics of cultivar NC645 SRC. While not being limited by process, NC645 SRC is a result of introducing the cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381 S mutations from Dl 198-325-6. F, individuals originating from a cross between NC645 and DH98-325-6 and heterozygous for each mutation are then backcrossed seven times to NC645 to produce BC7F1 progeny. BC7F 1 individuals heterozygous for all three mutations are self-pollinated to produce BC7F2 seed and individuals homozygous for all three mutations identified. A single BC7F2 plant is self-pollinated to produce BC7F3 (NC645 SRC) in which the wild-type CYP82E4, CYP82E5v2 and CYP82E10 alleles of NC645 are replaced by the mutant (e.g., cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381S).
[0523] NC645 SRC has a genetic background that is at least 95%, at least 97%, at least 98%o, or at least 99% similar to NC645. NC645 SRC exhibits low nomicotine levels and produce leaves with reduced potential for accumulating derived N N during curing, storage, and smoking.
NC7 SRC
[0524] In some aspects, the present disclosure provides tobacco cultivars, and parts thereof, from NC7 SRC. In another aspect, the present disclosure provides a tobacco plant, or part thereof, produced by growing the seed of NC7 SRC. In still further aspects, a plant of the present disclosure can include a plant with all or essentially all of the morphological and physiological characteristics of cultivar NC7 SRC.
[0525] While not being limited by process, NC7 SRC is produced by pollinating plants of CMS NC775 SRC with pollen of NC645 SRC. Again, not limited by any particular scientific theory, cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381S mutations all encode for proteins with reduced or eliminated ability to convert nicotine or nomicotine. NC7 SRC has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to burley tobacco cultivar NC7, a hybrid generated by pollinating plants of a male-sterile breeding line NC775 with pollen produced by fertile breeding line NC645. NC7 SRC exhibits low nomicotine and is not subject to conversion to high nomicotine.
NC638 SRC
[0526] In one aspect, the present disclosure provides tobacco cultivars, and parts thereof, from NC638 SRC, representative sample seeds of this cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120313. In another aspect, the present disclosure provides a tobacco plant, or part thereof, produced by growing a seed of NC638 SRC. In a further aspect, a plant of the present disclosure can further include a plant with all, or essentially all of the morphological and physiological characteristics of cultivar NC638 SRC.
[0527] While not being limited by process, NC638 SRC is a result of the
introduction of three mutated CYP82E genes in a hurley tobacco cultivar NC638. The three genes are a mutated CYP82E4 gene recited as 325-6 #775 in Lewis et al. ("Three nicotine demethylase genes mediate nornicotine biosynthesis in Nicotiana tabacum L.: Functional characterization of the CYP82E10 gene." Phytoc e istry. 71 (2010), 1988-1998 (SEQ ID NO: 1, which sets forth a cyp82e4 W329Stop, hereby incorporated by reference in its entirety)), a mutated CYP82E5v2 recited in Lewis et al. {supra) as 325-6 #1-13 (SEQ ID NO: 2, which sets forth a cyp82e5v2 W422Stop, hereby incorporated by reference in its entirety), and a mutated CYP82E10 recited in Lewis et al. (supra) as 325-6 #1041 (SEQ ID NO: l 1, which sets forth a cyp82el0 P381S, hereby incorporated by reference in its entirety).
Mutations cyp82e4 W329Stop and cyp82e5v2 W422Stop result in truncated proteins while cyp82El 0 P381 S results in a nonfunctional protein. A cyp82e4 W329Stop ( W), cyp82e5v2 W422Stop ("e5"), and a cyp82el0 P381 S ("e/0") mutation are introduced from a e4e4\e5e5\elOelO triple mutant from a strong converter burley background, line DH98-325-6, as listed in Table 4 of Lewis et al. (supra) into a burley tobacco cultivar NC638 background.
[0528] NC638 SRC is the result of seven backcrosses with burley cultivar NC638 as the recurrent parent, followed by two rounds of selfmg with selection for homozygosity for the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82el0 P381S mutations to yield BC7F3 plants (NC638 SRC) in which the wild-type CYP82E4, CYP82E5v2 and CYP82E10 alleles of NC638 are replaced by the mutant (e.g. , cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381 S) alleles.
[0529] NC638 SRC progeny plants have genetic backgrounds that are at least 95%, at least 97%, at least 98%, or at least 99% similar to NC638. NC638 SRC plants exhibit low nornicotine levels and produce leaves with reduced potential for accumulating derived NNN during curing, storage, and smoking.
CMS NC638 SRC
[0530] In some aspects, the present disclosure also provides tobacco cultivars, and parts thereof, from CMS NC638 SRC, representative sample seeds of this cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13568. In further aspects, the present disclosure also includes a tobacco plant, or part thereof, produced by growing a seed of CMS NC638 SRC. In still further aspects, a plant of the present disclosure can further include a plant with all, or essentially all, of the morphological and physiological characteristics of cuhivar CMS NC638 SRC. CMS NC638 SRC is a male-sterile (CMS) version of NC638 SRC (CMS NC638 SRC) produced by crossing a plant of CMS NC638 as a female with pollen of NC638 SRC BC6Fi to produce male-sterile plants heterozygous for all three mutations. The CMS progeny plants of the CMS NC638 x NC638 SRC BC6F, cross are male sterile. A plurality of CMS NC638 x NC638 SRC BC6Fi x CMS plants (e.g. , CMS F] progeny plants) are screened for the cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381 S mutations to identify plants heterozygous for all three mutations. A single male-sterile plant resulting from this cross is backcrossed as a female to NC638 SRC to prepare BC7F1 CMS progeny. BC7F] CMS progeny homozygous for the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82el0 P381 S mutations are identified by genotyping and designated as CMS NC638 SRC. Because the CMS NC638 SRC line is male sterile, it is maintained via pollination with NC638 SRC. NC638 SRC is crossed as the male parent to CMS NC638 SRC to prepare CMS NC638 SRC F, progeny plants.
[0531] CMS NC638 SRC and CMS NC638 SRC F , progeny plants have genetic backgrounds that are at least 95%, at least 97%, at least 98%>, or at least 99% similar to NC638. CMS NC638 SRC and CMS NC638 SRC Fi progeny plants exhibit low nomicotine levels and produce leaves with reduced potential for accumulating derived N during curing, storage, and smoking.
TN86 SRC
105321 In some aspects, the present disclosure also provides tobacco cultivars, and parts thereof, from TN86 SRC, where representative sample seeds of this cultivar have been deposited with the ATCC under ATCC Accession No. PTA-13565. In further aspects, the present disclosure also includes a tobacco plant, or part thereof, produced by growing a seed of TN86 SRC. . In still further aspects, plant of the present disclosure can include a plant with all, or essentially all, of the morphological and physiological characteristics of cultivar FN 86 SRC. While not being limited by process, I N 86 SRC is a result of introducing the cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381 S mutations from DH98-325- 6. F] individuals originating from a cross between TN86 and DH98-325-6 and heterozygous for each mutation are then backcrossed seven times to TN86 to produce BC7F] progeny.
BC7F1 individuals heterozygous for all three mutations are self-pollinated to produce BC7F2 seed and individuals homozygous for all three mutations identified. A single BC7F2 plant is self-pollinated to produce BC7F3 (TN86 SRC) in which the wild-type CYP82E4, CYP82E5v2 and CYP82E10 alleles of TN86 are replaced by the mutant (e.g. , cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381S).
[0533] TN86 SRC has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to I N 86. T 86 SRC exhibits low nornicotine levels and produce leaves with reduced potential for accumulating derived NNN during curing, storage, and smoking.
CMS TN86 SRC
[0534] In some aspects, the present disclosure also provides tobacco cultivars, and parts thereof, from CMS TN86 SRC, representative sample seeds of this cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13570. In other aspects, the present disclosure also includes a tobacco plant, or part thereof, produced by growing a seed of CMS TN86 SRC. In still other aspects, the plant of the present disclosure can further include a plant with all, or essentially all, of the morphological and physiological
characteristics of cultivar CMS ΪΝ86 SRC. CMS TN86 SRC is a male-sterile (CMS) version of TN86 SRC (CMS TN86 SRC) produced by crossing a plant of CMS TN86 as a female with pollen of TN86 SRC BC6Fi to produce male-sterile plants heterozygous for all three mutations. The CMS progeny plants of the CMS TN86 x FN 86 SRC BC(,Fi cross are male sterile. A plurality of CMS TN86 x TN86 SRC BC„F, x CMS plants (e.g., CMS F, progeny plants) are screened for the cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381 S mutations to identify plants heterozygous for all three mutations. A single male-sterile plant resulting from this cross is backcrossed as a female to TN86 SRC to prepare BCyFi CMS progeny. BCyFi CMS progeny homozygous for the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82el0 P381S mutations are identified by genotyping and designated as CMS TN86 SRC. Because the CMS TN86 SRC line is male sterile, it is maintained via pollination with TN86 SRC. FN 86 SRC is crossed as the male parent to CMS TO 86 SRC to prepare CMS TN86 SRC F) progeny plants.
[0535] CMS TN86 SRC and CMS T 86 SRC I· , progeny plants have genetic backgrounds that are at least 95%>, at least 97%, at least 98%o, or at least 99%> similar to TN86. CMS TN86 SRC and CMS TN86 SRC F, progeny plants exhibit low nornicotine levels and produce leaves with reduced potential for accumulating derived NNN during curing, storage, and smoking.
NCBH129 SRC
[0536] In some aspects, the present disclosure includes tobacco cultivars, and parts thereof, from NCBH129 SRC. In another aspect, the present disclosure includes a tobacco plant, or part thereof, produced by growing the seed of NCBH129 SRC. In other aspects, the plant of the present disclosure can include a plant with all, or essentially all, of the morphological and physiological characteristics of cultivar NCBH129 SRC.
10537] While not being limited by process, NCBH129 SRC is produced by pollinating plants of CMS NC638 SRC with pollen of TN86 SRC. Again, not limited by any particular scientific theory, cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381 S mutations all encode for proteins with reduced or eliminated ability to convert nicotine or nornicotine. NCBI 11 29 SRC has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to burley tobacco cultivar NCBH129, a hybrid generated by pollinating plants of a male-sterile breeding line NC638 with pollen produced by fertile breeding line TN86. NCBH129 SRC exhibits low nornicotine and is not subject to conversion to high nornicotine.
Other plants
{0538] The present disclosure includes a tobacco seed produced by crossing two parent tobacco plants and harvesting the resultant tobacco seed, where at least one parent tobacco plant is TN90 SRC. In one aspect, the TN90 SRC is the male parent plant. In another aspect, the CMS TN90 SRC is the female parent plant. One aspect of the present disclosure provides tobacco plants that are homozygous at the cyp82e4, cyp82e5v2, and cyp82E10 loci for SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 1 1 , respectively, and which share a genetic background that is greater than about 75%, 80%, 85%, 90%, 95%, 98%), or 99% similar to TN90 or CMS TN90. In one aspect, approximately or greater than about 50%, 75%, or 100% of a progeny's genetics is provided by a plant of the present disclosure that is homozygous at the cyp82e4, cyp82e5v2, and cyp82E10 loci for SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 1 1 , respectively. In one aspect, a plant of the present disclosure has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to TN90 or CMS TN90. In another aspect, a plant of the present disclosure exhibits low nornicotine and is not subject to conversion to high nornicotine. In one aspect, a plant of the present disclosure is the progeny plant of a female or male parent plant that is Fusarium wilt resistant. In another aspect, a plant of TN90 SRC has low resistance to black shank and moderate resistance to bacterial wilt.
[0539] In some aspects, the present disclosure provides a tobacco seed produced by crossing two parent tobacco plants and harvesting the resultant tobacco seed, where at least one parent tobacco plant is KY14 SRC. In one aspect, the KY14 SRC is the male parent plant. In another aspect, the CMS KYI 4 SRC is the female parent plant. One aspect of the present disclosure provides tobacco plants that are homozygous at the cyp82e4, cyp82e5v2, and cyp82E10 loci for SEQ ID NO: 1 , SEQ ID NO: 2. and SEQ ID NO: 1 1, respectively, and which share a genetic background that is greater than about 75%, 80%, 85%, 90%, 95%, 98%, or 99% similar to KY14 or CMS KY14. In one aspect, approximately or greater than about 50%o, 15%, or 100%o of a progeny's genetics is provided by a plant of the present disclosure that is homozygous at the cyp82e4, cyp82e5v2, and cyp82E10 loci for SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 1 1 , respectively. In one aspect, a plant of the present disclosure has a genetic background that is at least 95%o, at least 97%, at least 98%>, or at least 99% similar to KY I 4 or CMS KY14. In another aspect, a plant of the present disclosure exhibits low nornicotine and is not subject to conversion to high nornicotine. In one aspect, a plant of the present disclosure is the progeny plant of a female or male parent plant that is Fusarium wilt resistant. In another aspect, a plant of KYI 4 SRC has low resistance to black shank and moderate resistance to bacterial wilt.
[0540] The present disclosure includes a tobacco seed produced by crossing two parent tobacco plants and harvesting the resultant tobacco seed, where at least one parent tobacco plant is KYI 4 SRC. In one aspect, the KYI 4 SRC is the male parent plant. In another aspect, the CMS KYI 4 SRC is the female parent plant. One aspect of the present disclosure provides tobacco plants that are homozygous at the cyp82e4, cyp82e5v2, and cyp82E10 loci for SEQ I D NO: 1 , SEQ ID NO: 2. and SEQ ID NO: 1 1 , respectively, and which share a genetic background that is greater than about 75%o, 80%>, 85%>, 90%>, 95%o, 98%o, or 99%o similar to KY14 or CMS KY14. In one aspect, approximately or greater than about 50%), 75%o, or 100%) of a progeny's genetics is provided by a plant of the present disclosure that is homozygous at the cyp82e4, cyp82e5v2, and cyp82E10 loci for SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 1 1 , respectively. In one aspect, a plant of the present disclosure has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to KY14 SRC and CMS KY14 SRC. In another aspect, a plant of the present disclosure exhibits low nornicotine and is not subject to conversion to high nornicotine. In one aspect, a plant of the present disclosure is the progeny plant of a female or male parent plant that is Fusarium wilt resistant. In another aspect, a plant of KYI 4 X L8 SRC has moderate resistance to black shank and moderate resistance to bacterial wilt.
[0541] In some aspects, the present disclosure includes a tobacco seed produced by crossing two parent tobacco plants and harvesting the resultant tobacco seed, where at least one parent tobacco plant is NC775 SRC. In one aspect, the NC775 SRC is the male parent plant. In another aspect, the CMS NC775 SRC is the female parent plant. One aspect of the present disclosure provides tobacco plants that are homozygous at the cyp82e4, cyp82e5v2, and cyp82E10 loci for SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 1 1. respectively, and which share a genetic background that is greater than 75%, 80%, 85%, 90%, 95%, 98%, or 99%) similar to NC775 or CMS NC775. In one aspect, approximately or greater than 50%, 75%, or 100% of a progeny's genetics is provided by a plant of the present disclosure that is homozygous at the cyp82e4, cyp82e5v2, and cyp82E10 loci for SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 11, respectively. In one aspect, a plant of the present disclosure has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to NC775 or CMS NC775. In another aspect, a plant of the present disclosure exhibits low nornicotine and is not subject to conversion to high nornicotine. In one aspect, a plant of the present disclosure is the progeny plant of a female or male parent plant that is Fusarium wilt resistant. In another aspect, a plant of NC775 SRC has low resistance to black shank and moderate resistance to bacterial wilt.
[0542] The present disclosure includes a tobacco seed produced by crossing two parent tobacco plants and harvesting the resultant tobacco seed, where at least one parent tobacco plant is NC775 SRC. In one aspect, the NC775 SRC is the male parent plant. In another aspect, the CMS NC775 SRC is the female parent plant. One aspect of the present disclosure provides tobacco plants that are homozygous at the cyp82e4, cyp82e5v2, and cyp82E10 loci for SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 1 1. respectively, and which share a genetic background that is greater than 75%, 80%, 85%, 90%, 95%>, 98%, or 99%) similar to NC775 or CMS NC775. In one aspect, approximately or greater than 50%, 75%o, or 100% of a progeny's genetics is provided by a plant of the present disclosure that is homozygous at the cyp82e4, cyp82e5v2, and cyp82E10 loci for SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 11, respectively. In one aspect, a plant of the present disclosure has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to NC775 SRC and CMS NC775 SRC. In another aspect, a plant of the present disclosure exhibits low nornicotine and is not subject to conversion to high nornicotine. In one aspect, a plant of the present disclosure is the progeny plant of a female or male parent plant that is Fusarium wilt resistant. In another aspect, a plant of NC7 SRC has moderate resistance to black shank and moderate resistance to bacterial wilt.
[0543] The present disclosure includes a tobacco seed produced by crossing two parent tobacco plants and harvesting the resultant tobacco seed, where at least one parent tobacco plant is NC638 SRC. In one aspect, the NC638 SRC is the male parent plant. In another aspect, the CMS NC638 SRC is the female parent plant. One aspect of the present disclosure provides tobacco plants that are homozygous at the cyp82e4, cyp82e5v2, and cyp82E10 loci for SEQ I D NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 1 1 , respectively, and which share a genetic background that is greater than about 75%, 80%, 85%, 90%, 95%, 98%o, or 99% similar to NC638 or CMS NC638. In one aspect, approximately or greater than about 50%), 75%, or 100% of a progeny's genetics is provided by a plant of the present disclosure that is homozygous at the cyp82e4, cyp82e5v2, and cyp82E10 loci for SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ I D NO: 1 1 , respectively. In one aspect, a plant of the present disclosure has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to NC638 or CMS NC638. In another aspect, a plant of the present disclosure exhibits low nornicotine and is not subject to conversion to high NNN's. In one aspect, a plant of the present disclosure is the progeny plant of a female or male parent plant that is Fusarium wilt resistant. In another aspect, a plant of NC638 SRC has low resistance to black shank and moderate resistance to bacterial wilt.
[0544] The present disclosure includes a tobacco seed produced by crossing two parent tobacco plants and harvesting the resultant tobacco seed, where at least one parent tobacco plant is NC638 SRC. In one aspect, the NC638 SRC is the male parent plant. In another aspect, the CMS NC638 SRC is the female parent plant. One aspect of the present disclosure provides tobacco plants that are homozygous at the cyp82e4, cyp82e5v2, and cyp82E10 loci for SEQ ID NO: 1 , SEQ I D NO: 2, and SEQ ID NO: 1 1. respectively, and which share a genetic background that is greater than about 75%, 80%, 85%, 90%, 95%, 98%), or 99%) similar to NC638 or CMS NC638. In one aspect, approximately or greater than about 50%, 75%, or 100% of a progeny's genetics is provided by a plant of the present disclosure that is homozygous at the cyp82e4, cyp82e5v2, and cyp82E10 loci for SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 1 1 , respectively. In one aspect, a plant of the present disclosure has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to NC638 SRC and CMS NC638 SRC. In another aspect, a plant of the present disclosure exhibits low nornicotine and is not subject to conversion to high nornicotine. In one aspect, a plant of the present disclosure is the progeny plant of a female or male parent plant that is Fusarium wilt resistant. In another aspect, a plant of NC638 SRC has moderate resistance to black shank and moderate resistance to bacterial wilt.
[0545] In one aspect, a plant of the present disclosure is a medium-late maturing variety with moderately high yield potential. In another aspect, a plant of the present disclosure offers a broad range of important agronomic characteristics. In a further aspect, a plant of the present disclosure has one, two, three, four or more of the traits including moderate resistance to black shank, some tolerance to blue mold, black root rot resistance, and resistance to common virus diseases. In another aspect, a plant of the present disclosure has blue mold tolerance and level 4 resistance to both races of black shank and high root rot resistance. In one aspect, a plant of the present disclosure, such as TN90 SRC, CMS TN90 SRC. KY14 SRC, CMS KY14 SRC. L8 SRC, NC775 SRC. CMS NC775 SRC. NC645 SRC. NC638 SRC. CMS C638 SRC, TN86 SRC. CMS TN86 SRC, KYI 4 X L8 SRC. NC7 SRC, and/or NCBH129 SRC, lacks Fusarium wilt resistance. In another aspect, a plant of the present disclosure is Fusarium wilt resistant. In another aspect, a plant of the present disclosure has low resistance to black shank and moderate resistance to bacterial wilt.
[0546] In an aspect, the plants of the present disclosure have reduced or eliminated ability to convert nicotine to nornicotine. In an aspect, the percentage nicotine conversion can be less than about 75%, 70%, 60%, 50%, or 25% of that found in a cultivar selected from the group consisting of TN90, KY14, L8, NC775, NC645, NC638, I 86. KY14 X L8, NC7, and NCBH129. In other aspects, the nicotine conversion in plants of the present disclosure, including but not limited to TN90 SRC. CMS TN90 SRC. KYI 4 SRC. CMS KY I 4 SRC. L8 SRC, NC775 SRC, CMS NC775 SRC, NC645 SRC, NC638 SRC, CMS NC638 SRC, TN86 SRC. CMS TN86 SRC. KY I 4 X L8 SRC. NC7 SRC, and NCBH 129 SRC, can 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 still other aspects, the nicotine conversion in plants of the present disclosure, including but not limited to TN90 SRC, CMS TN90 SRC, KYI 4 SRC, CMS KY14 SRC. L8 SRC, NC775 SRC, CMS NC775 SRC, NC645 SRC, NC638 SRC, CMS NC638 SRC, F 86 SRC. CMS ΓΝ86 SRC. KY I 4 X L8 SRC. NC7 SRC. and/or NCBH129 SRC. can be in a range from about 3% to about 1%, about 3% to about 0.5%, or about 2% to about 0.5%. In a preferred aspect, the percentage nicotine conversion is less than about 25%, 10%, 5%, or 2% of that found in a cultivar selected from the group consisting of TN90. KYI 4, L8, NC775, NC645, NC638, TN86, KY14 X L8, NC7, and NCBH129 without the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82el0 P381S mutations: In an aspect, the tobacco plants of the present disclosure 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 tobacco plants of the present disclosure 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 tobacco plants of the present disclosure 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 rates can be in a range from 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 rates can be in a range from 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 disclosure 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.
[0547] In another aspect, the tobacco plants of the present disclosure typically have a reduced amount of nornicotine of less than about 0.10 % dry weight. For example, the nornicotine content in such 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 content 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 such plants can be in a range from 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 % dry weight. In some aspects, in a plant of the present disclosure, the nornicotine is a relatively small percentage of total alkaloids in the plant compared to a commercial seedlot of a cultivar selected from the group consisting of TN90, KY14, L8, NC775, NC645, NC638, TN86, KY14 X L8, NC7, and NCBH129. In some aspects, the nornicotine in a plant of the present disclosure 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 a reduced amount of nitrosamine content can be manufactured using tobacco plant material from plants and plant parts of the present disclosure. Thus, in some embodiments, a tobacco product manufactured using tobacco plant material from plants and plant parts of the present disclosure can comprise a reduced amount of nornicotine of less than about 3 mg/g. For example, the nornicotine content in such a product can be 3.0 mg/g, 2.5 mg/g, 2.0 mg/g, 1.5 mg/g, 1.0 mg/g, 750 μ§/§, 500 μ§/§, 250 μξ/g, 100 μ§/& 75 μ§/Ε, 50 μ^, 25 μ^, 10 μ^, 5 μ^, 1 μ^, 750 ng/g, 500 ng/g, 250 ng/g, 100 ng/g, 75 ng/g, 50 ng/g, 25 ng/g, 10 ng/g, 5 ng/g, 1 ng/g, 750 pg/g, 500 pg/g, 250 pg/g, 100 pg/g, 75 pg/g, 50 pg/g, 25 pg/g, 10 pg/g, 7.0 pg/g, 5.0 pg/g, 4.0 pg/g, 2.0 pg/g, 1.0 pg/g, 0.5 pg/g, 0.4 pg/g, 0.2 pg/g, 0.1 pg/g, 0.05 pg/g, 0.01 pg/g, 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 such a product can be about 10 pg/g, 7.0 pg/g, 5.0 pg/g, 4.0 pg/g, 2.0 pg/g, 1.0 pg/g, 0.5 pg/g, 0.4 pg/g, 0.2 pg/g, 0.1 pg/g, 0.05 pg/g, 0.01 pg/g, and the like, or undetectable, or any range therein. The percentage of secondary alkaloids relative to total alkaloid content contained in a plant of the present disclosure may not be statistically different than from a commercial seedlot of a cultivar selected from the group consisting of TN90, KY I 4. L8, NC775, NC645, NC638, TN86, KYI 4 X L8, NC7, and NCBH129.
[0548] Differences between two inbred tobacco varieties or two hybrid tobacco varieties can be evaluated using statistical approaches. Statistical analysis includes the calculation of mean values, determination of the statistical significance of the sources of variation, and the calculation of the appropriate variance components. Methods for determining statistical significance are known in the art. Statistical software is available, for example, the PROC GLM function of SAS. Significance is generally presented as a "p- value." A statistically significant p-value is 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, a statistically significant value is less than 0.01. In yet another aspect, it can 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.
[0549] Tobacco plants of the present disclosure that are homozygous for the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82el0 P381S alleles have a reversion rate that is statistically significantly lower than corresponding control low-converter plants having wild type nicotine demethylase CYP82E4, E5, and E10 genes. In addition, homozygous CYP82E4, CYP82E5, and CYP82E10 triple mutant tobacco plants have a percent conversion to nornicotine of less than about 2.0%, e.g., undetectable to about 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%, or any range therein. In some aspects, the triple mutant tobacco plants have a percent conversion to nornicotine in a range from, for example, about 1.0% to 2.0%, 0.8% to 1.8%, 0.8% to 2.0%, or 1.0% to 2.0%.
[0550] Nicotine and nornicotine can be measured in ethylene-treated leaves using methods known in the art (e.g. , gas chromatography). Percent nicotine demethylation in a sample is calculated by dividing the level of nornicotine by the combined level of nicotine and nornicotine as measured in the sample, and multiplying by 100. Percent nicotine demethylation in a sample from a plant of the present disclosure is about 50, 40, 30, 20, or 10 percent of a sample from an individual plant grown from a commercial seedlot of a cultivar selected from the group consisting of TO90, KY14, L8, NC775, NC645, NC638, TN86, KY I 4 X L8. C7. and NCBH 129.
[0551] In an aspect, the tobacco plants of the present disclosure have a USDA quality index of about 73, about 72, about 71, about 70, about 69, about 68, about 67 or about 66 or any range therein. In an aspect, the tobacco plants of the present disclosure have a USDA quality index of about 65. In another aspect, the quality index may be at least about 55, 60, 62.5 or greater, or any range therein. In another aspect, tobacco plants of the present disclosure can have a quality 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.
[0552] A plant of the present disclosure, including TN90 SRC, CMS TN90 SRC, KY14 SRC, CMS KY14 SRC, L8 SRC, NC775 SRC, CMS NC775 SRC, NC645 SRC, NC638 SRC. CMS NC638 SRC. TN86 SRC, CMS TN86 SRC, KY 14 X L8 SRC, NC7 SRC. or NCBH129 SRC, can have any yield potential, including high (e.g., over 3000 lbs/ A), moderately high (e.g., 2200-3000 lbs/ A), and moderate (e.g., less than 2000 lbs/ A) yield potential.
[0553] In another aspect, the present disclosure also provides for a plant grown from the seed of a TN90 SRC or CMS TN90 SRC plant in which alkaloids obtained from tobacco plants grown for the seed have decreased nornicotine, as well as plant parts and tissue cultures from such plants, representative sample seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA- 13567 for TN90 SRC and ATCC Accession No. PTA- 13573 for CMS TN90 SRC.
[0554] In another aspect, the present disclosure also provides for a plant grown from the seed of a KY14 SRC, CMS KY14 SRC, L8 SRC, or a hybrid KY14 X L8 SRC plant in which alkaloids obtained from tobacco plants grown for the seed have decreased nornicotine, as well as plant parts and tissue cultures from such plants, representative sample seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA- 12031 1 for KY I 4 SRC. A CC Accession No. P I A- 13571 for CMS KY I 4 SRC.
ATCC Accession No. PTA-13572 for L8 SRC. Seeds of hybrid cultivar KY14 X T8 SRC are obtainable by crossing plants of cultivars T8 SRC and CMS KYI 4 SRC and collecting seeds.
[0555] In another aspect, the present disclosure also provides for a plant grown from the seed of a NC775 SRC. CMS NC775 SRC, NC645 SRC, or a hybrid NC7 SRC plant in which alkaloids obtained from tobacco plants grown for the seed have decreased nomicotine, as well as plant parts and tissue cultures from such plants, representative sample seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-120312 for NC775 SRC, ATCC Accession No. PTA-13563 for CMS NC775 SRC. ATCC Accession No. PTA-13566 for NC645 SRC. Seeds of hybrid cultivar NC7 SRC are obtainable by crossing plants of cultivars NC645 SRC and CMS NC775 SRC and collecting seeds.
[0556] In another aspect, the present disclosure also provides for a plant grown from the seed of a NC638 SRC, CMS NC638 SRC, TN86 SRC, CMS TN86 SRC, or a hybrid NCBH129 SRC plant in which alkaloids obtained from tobacco plants grown for the seed have decreased nomicotine, as well as plant parts and tissue cultures from such plants, representative sample seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-120313 for NC638 SRC, ATCC Accession No. PTA-13568 for CMS NC638 SRC, ATCC Accession No. PTA-13565 for TN86 SRC, and ATCC Accession No. PTA-13570 for CMS TN86 SRC. Seeds of hybrid cultivar NCBH129 SRC are obtainable by crossing plants of cultivars TN86 SRC and CMS NC638 SRC and collecting seeds.
[0557] An aspect of the present disclosure provides for parts of a cultivar selected from the group consisting of TN90 SRC. CMS TN90 SRC. KYI 4 SRC, CMS KY I 4 SRC, 1.8 SRC, NC775 SRC, CMS NC775 SRC, NC645 SRC, NC638 SRC, CMS NC638 SRC, TN86 SRC. CMS TN86 SRC. KY I 4 X 1.8 SRC. NC7 SRC, and NCBH 129 SRC. A part of a cultivar can comprise any plant part and includes, but is not limited to, leaves, pollen, embryos, cotyledons, hypocotyls, roots, root tips, anthers, flowers, ovules, shoots, stems, stalks, pith and capsules, tissue culture comprising tissue, callus, cells or protoplasts. In another aspect, the present disclosure provides for parts from hybrids derived from a cultivar selected from the group consisting of TN90 SRC, CMS TN90 SRC, KY14 SRC, CMS KY14 SRC, L8 SRC, NC775 SRC, CMS NC775 SRC, NC645 SRC, NC638 SRC, CMS NC638 SRC, TN86 SRC, CMS TN86 SRC. KY I 4 X L8 SRC, NC7 SRC. and NCBH129 SRC. In yet another aspect, the present disclosure provides for parts from genetically modified (e.g., by conventional breeding or genetic engineering techniques) forms of the foregoing plants and tissue culture.
[0558] Additional aspects of the present disclosure provide products comprising tobacco from the plants of the present disclosure, and parts thereof. Other aspects of the disclosure provide cured plant parts, which include, but are not limited to, a leaf, pollen, ovule, embryo, cotyledon, hypocotyl, meristematic cell, protoplast, root, root tip, pistil, anther, flower, shoot, stem, pod, petiole, and the like, and combinations thereof.
[0559] Thus, in some aspects, the present disclosure provides a cured tobacco comprising the leaves of the tobacco plant designated TN90 SRC. a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567. In another aspect, the present disclosure provides a cured tobacco comprising the leaves of the tobacco plant designated CMS ΊΓΝ90 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13573.
[0560] In an aspect, the present disclosure provides a cured tobacco comprising the stems of the tobacco plant designated TN90 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567. In another aspect, the present disclosure provides a cured tobacco comprising the stems of the tobacco plant designated CMS TN90 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13573.
[0561] In an aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the tobacco plants designated TN90 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13567. In another aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the tobacco plants designated CMS TN90 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13573.
[0562] The present disclosure also provides a container of TN90 SRC or CMS TN90 SRC seeds or other seeds of the present disclosure in which alkaloids obtained from tobacco plants grown from greater than about 50% of the seeds have decreased nornicotine. In another aspect, alkaloids obtained from TN90 SRC or CMS TN90 SRC plants or other plants of the present disclosure grown from greater than about 55%, 60%>, 65%, 70%, 75%, 80%), 85%o, 90%), or 95% of the seeds in the container have decreased nornicotine, representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-13567 for TN90 SRC and/or ATCC Accession No. PTA- 13573 for CMS TN90 SRC.
10563] The container of TN90 SRC or CMS TN90 SRC seeds or other seeds of the present disclosure may contain any number, weight or volume of seeds. For example, a container can contain at least, or greater than, about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000 or more seeds. Alternatively, the container can 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 seeds. Representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-13567 for TN90 SRC and/or ATCC Accession No. PTA-13573 for CMS TN90 SRC.
[0564] Containers of TN90 SRC or CMS TN90 SRC seeds or other seeds of the present disclosure may be any container available in the art. By way of a non-limiting example, a container may be a box, a bag, a packet, a pouch, a tape roll, a pail, a foil, or a tube. Representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-13567 for TN90 SRC and/or ATCC Accession No. PTA-13573 for CMS TN90 SRC.
[0565] In another aspect, the present disclosure also provides a container of TN90
SRC or CMS TN90 SRC seeds in which greater than about 50% of T 90 SRC or CMS TN90 SRC seeds or other seeds of the present disclosure have decreased nornicotine.
Representative samples of seeds of these cultivars having been deposited with the ATCC, for example under ATCC Accession No. PTA-13567 for TN90 SRC and/or ATCC Accession No. PTA-13573 for CMS TN90 SRC.
[0566] In one aspect, the present disclosure provides a seed of a TN90 SRC or CMS TN90 SRC plant or other plant of the present disclosure in which a plant grown from said seed is male sterile. Representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-13567 for TN90 SRC and/or ATCC Accession No. PTA-13573 for CMS TN90 SRC.
[0567] In some aspects, the present disclosure provides a cured tobacco comprising the leaves of the tobacco plant designated Y14 SRC , a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120311. In another aspect, the present disclosure provides a cured tobacco comprising the leaves of the tobacco plant designated CMS KYI 4 SRC. a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13571. In another aspect, the present disclosure provides a cured tobacco comprising the leaves of the tobacco plant designated L8 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13572. In yet another aspect, the present disclosure provides a cured tobacco comprising the leaves of the hybrid tobacco plant designated KY14 X L8 SRC. Seeds of hybrid cultivar KY14 X L8 SRC are obtainable by crossing plants of cultivars L8 SRC and CMS KY14 SRC and collecting seeds.
[0568] In an aspect, the present disclosure provides a cured tobacco comprising the stems of the tobacco plant designated KY14 S RC . a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-120311. In another aspect, the present disclosure provides a cured tobacco comprising the stems of the tobacco plant designated CMS KYI 4 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13571. In an aspect, the present disclosure provides a cured tobacco comprising the stems of the tobacco plant designated L8 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13572. In yet another aspect, the present disclosure provides a cured tobacco comprising the stems of the hybrid tobacco plant designated KYI 4 X L8 SRC. Seeds of hybrid cultivar KYI 4 X L8 SRC are obtainable by crossing plants of cultivars L8 SRC and CMS KYI 4 SRC and collecting seeds.
[0569] In an aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the tobacco plants designated KY ! 4 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No.
PTA-120311. In another aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the tobacco plants designated CMS KYI 4 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13571. In an aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the tobacco plants designated L8 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13572. In yet another aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the hybrid tobacco plants designated KYI 4 X L8 SRC. Seeds of hybrid cultivar KY14 X L8 SRC are obtainable by crossing plants of cultivars L8 SRC and CMS KY14 SRC and collecting seeds.
[0570] The present disclosure also provides a container of KYI 4 SRC, CMS KY I 4 SRC, L8 SRC, or hybrid KY14 X L8 SRC seeds or other seeds of the present disclosure in which alkaloids obtained from tobacco plants grown from greater than about 50% of the seeds have decreased nomicotine. In another aspect, alkaloids obtained from KYI 4 SRC. CMS KY14 SRC, L8 SRC, or hybrid KY14 X L8 SRC plants or other plants of the present disclosure grown from greater than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the seeds in the container have decreased nomicotine, representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC
Accession No. P i A- 12031 1 for KY14 SRC. ATCC Accession No. PTA-13571 for CMS KYI 4 SRC, and/or ATCC Accession No. PTA- 13572 for L8 SRC. Seeds of hybrid cultivar KY14 X L8 SRC are obtainable by crossing plants of cultivars L8 SRC and CMS KYI 4 SRC and collecting seeds.
[0571] The container of KY14 SRC. CMS KY I 4 SRC, L8 SRC. or hybrid KYI 4 X L8 SRC seeds or other seeds of the present disclosure may contain any number, weight or volume of seeds. For example, a container can contain at least, or greater than, about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000 or more seeds. Alternatively, the container can 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 seeds.
Representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-12031 1 for KY14 SRC, ATCC Accession No. PTA- 13571 for CMS KY I 4 SRC, or ATCC Accession No. PTA- 13572 for 1.8 SRC. Seeds of hybrid cultivar KY14 X L8 SRC are obtainable by crossing plants of cultivars L8 SRC and CMS KYI 4 SRC and collecting seeds.
[0572] Containers of KY I 4 SRC, CMS KY I 4 SRC, L8 SRC or hybrid KY 14 X L8 SRC seeds or other seeds of the present disclosure may be any container available in the art. By way of a non-limiting example, a container may be a box, a bag, a packet, a pouch, a tape roll, a pail, a foil, or a tube. Representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-120311 for KY14 SRC, ATCC Accession No. PTA- 13571 for CMS KY14 SRC, and/or ATCC Accession No. PTA- 13572 for L8 SRC. Seeds of hybrid cultivar KY14 X T8 SRC are obtainable by crossing plants of cultivars L8 SRC and CMS KYI 4 SRC and collecting seeds.
[0573] In another aspect, the present disclosure also provides a container of KYI 4 SRC, CMS KY14 SRC, L8 SRC, or hybrid cultivar KY14 X L8 SRC seeds in which greater than about 50% of KY14 SRC, CMS KY14 SRC, T8 SRC, or hybrid KY14 X L8 SRC seeds or other seeds of the present disclosure have decreased nornicotine. Representative samples of seeds of these cultivars having been deposited with the ATCC, for example under ATCC Accession No. PTA- 12031 1 for KY I 4 SRC, ATCC Accession No. ΡΤΛ- 13571 for CMS KY14 SRC, and/or ATCC Accession No. PTA- 13572 for L8 SRC. Seeds of hybrid cultivar KY14 X L8 SRC are obtainable by crossing plants of cultivars L8 SRC and CMS KY14 SRC and collecting seeds.
[0574] In one aspect, the present disclosure provides a seed of a KY I 4 SRC. CMS KYI 4 SRC, T8 SRC, or hybrid KY14XT8 SRC plant or other plant of the present disclosure in which a plant grown from a seed is male sterile. Representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-120311 for KY 14 SRC, ATCC Accession No. PTA- 13571 for CMS KYI 4 SRC, ATCC Accession No. PTA- 13572 for L8 SRC. Seeds of hybrid cultivar KYI 4 X L8 SRC are obtainable by crossing plants of cultivars L8 SRC and CMS KY14 SRC and collecting seeds.
[0575] In some aspects, the present disclosure provides a cured tobacco comprising the leaves of the tobacco plant designated NC775 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 120312. In another aspect, the present disclosure provides a cured tobacco comprising the leaves of the tobacco plant designated CMS NC775 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563. In another aspect, the present disclosure provides a cured tobacco comprising the leaves of the tobacco plant designated NC645 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566. In yet another aspect, the present disclosure provides a cured tobacco comprising the leaves of the hybrid tobacco plant designated NC7 SRC. Seeds of hybrid cultivar NC7 SRC are obtainable by crossing plants of cultivars NC645 SRC and CMS NC775 SRC and collecting seeds.
[0576] In an aspect, the present disclosure provides a cured tobacco comprising the stems of the tobacco plant designated NC775 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 120312. In another aspect, the present disclosure provides a cured tobacco comprising the stems of the tobacco plant designated CMS NC775 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563. In an aspect, the present disclosure provides a cured tobacco comprising the stems of the tobacco plant designated NC645 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13566. In yet another aspect, the present disclosure provides a cured tobacco comprising the stems of the hybrid tobacco plant designated NC7 SRC. Seeds of hybrid cultivar NC7 SRC are obtainable by crossing plants of cultivars NC645 SRC and CMS NC775 SRC and collecting seeds.
[0577] In an aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the tobacco plants designated NC775 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No.
PTA- 120312. In another aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the tobacco plants designated CMS NC775 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13563. In an aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the tobacco plants designated NC645 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No.
PTA-13566. In yet another aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the hybrid tobacco plants designated NC7 SRC. Seeds of hybrid cultivar NC7 SRC are obtainable by crossing plants of cultivars NC645 SRC and CMS NC775 SRC and collecting seeds.
[0578] The present disclosure also provides a container of NC775 SRC. CMS NC775 SRC, NC645 SRC, or hybrid NC7 SRC seeds or other seeds of the present disclosure in which alkaloids obtained from tobacco plants grown from greater than about 50% of the seeds have decreased nornicotine. In another aspect, alkaloids obtained from NC775 SRC. CMS NC775 SRC, NC645 SRC, or hybrid NC7 SRC plants or other plants of the present disclosure grown from greater than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the seeds in the container have decreased nornicotine, representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC
Accession No. PTA-120312 for NC775 SRC, ATCC Accession No. PTA- 13563 for CMS NC775 SRC, ATCC Accession No. PTA-13566 for NC645 SRC. Seeds of hybrid cultivar NC7 SRC are obtainable by crossing plants of cultivars NC645 SRC and CMS NC775 SRC and collecting seeds.
[0579] The container of NC775 SRC, CMS NC775 SRC. NC645 SRC. or hybrid NC7 SRC seeds or other seeds of the present disclosure may contain any number, weight, or volume of seeds. For example, a container can contain at least, or greater than, about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, or more seeds. Alternatively, the container can 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 seeds.
Representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-120312 for NC775 SRC, ATCC Accession No. PTA- 13563 for CMS NC775 SRC. ATCC Accession No. PTA-1 566 for NC645 SRC. Seeds of hybrid cultivar NC7 SRC are obtainable by crossing plants of cultivars NC645 SRC and CMS NC775 SRC and collecting seeds.
[05801 Containers of NC775 SRC. CMS NC775 SRC, NC645 SRC or hybrid NC7
SRC seeds or other seeds of the present disclosure may be any container available in the art. By way of a non-limiting example, a container may be a box, a bag, a packet, a pouch, a tape roll, a pail, a foil, or a tube. Representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-120312 for NC775 SRC. ATCC Accession No. PTA-13563 for CMS NC775 SRC, ATCC Accession No.
PTA- 13566 for NC645 SRC. Seeds of hybrid cultivar NC7 SRC are obtainable by crossing plants of cultivars NC645 SRC and CMS NC775 SRC and collecting seeds.
[0581] In another aspect, the present disclosure also provides a container of NC775 SRC. CMS NC775 SRC. NC645 SRC, or hybrid cultivar NC7 SRC in which greater than 50% of NC775 SRC. CMS NC775 SRC. NC645 SRC, or hybrid NC7 SRC seeds or other seeds of the present disclosure have decreased nornicotine. Representative samples of seeds of these cultivars having been deposited with the ATCC, for example under ATCC Accession No. PTA- 120312 for NC775 SRC. ATCC Accession No. PTA-13563 for CMS NC775 SRC. ATCC Accession No. PTA- 13566 for NC645 SRC. Seeds of hybrid cultivar NC7 SRC are obtainable by crossing plants of cultivars NC645 SRC and CMS NC775 SRC and collecting seeds.
10582] In one aspect, the present disclosure provides a seed of a NC775 SRC, CMS NC775 SRC, NC645 SRC, or hybrid NC7 SRC plant or other plant of the present disclosure in which a plant grown from a seed is male sterile. Representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA- 120312 for NC775 SRC. ATCC Accession No. PTA-13563 for CMS NC775 SRC. ATCC Accession No. PTA- 13566 for NC645 SRC. Seeds of hybrid cultivar NC7 SRC are obtainable by crossing plants of cultivars NC645 SRC and CMS NC775 SRC and collecting seeds.
[0583] In some aspects, the present disclosure provides a cured tobacco comprising the leaves of the tobacco plant designated NC638 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 120313. In another aspect, the present disclosure provides a cured tobacco comprising the leaves of the tobacco plant designated CMS NC638 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568. In another aspect, the present disclosure provides a cured tobacco comprising the leaves of the tobacco plant designated TN86 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13565. In further aspect, the present disclosure provides a cured tobacco comprising the leaves of the tobacco plant designated CMS TN86 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13570. In yet another aspect, the present disclosure provides a cured tobacco comprising the leaves of the hybrid tobacco plant designated NCBH129 SRC. Seeds of hybrid cultivar NCBH129 SRC are obtainable by crossing plants of cultivars TN86 SRC and CMS NC638 SRC and collecting seeds.
[0584] In an aspect, the present disclosure provides a cured tobacco comprising the stems of the tobacco plant designated NC638 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 120313. In another aspect, the present disclosure provides a cured tobacco comprising the stems of the tobacco plant designated CMS NC638 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568. In an aspect, the present disclosure provides a cured tobacco comprising the stems of the tobacco plant designated TN86 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565. In further aspect, the present disclosure provides a cured tobacco comprising the stems of the tobacco plant designated CMS TN86 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13570. In yet another aspect, the present disclosure provides a cured tobacco comprising the stems of the hybrid tobacco plant designated NCBH129 SRC. Seeds of hybrid cultivar NCBH129 SRC are obtainable by crossing plants of cultivars TN86 SRC and CMS NC638 SRC and collecting seeds.
[0585] In an aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the tobacco plants designated NC638 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No.
PTA- 120313. In another aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the tobacco plants designated CMS NC638 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13568. In an aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the tobacco plants designated TN86 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-13565. In further aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the tobacco plant designated CMS TN86 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 13570. In yet another aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the hybrid tobacco plants designated NCBH129 SRC. Seeds of hybrid cultivar NCBH129 SRC are obtainable by crossing plants of cultivars TN86 SRC and CMS NC638 SRC and collecting seeds. [0586] The present disclosure also provides a container of NC638 SRC, CMS NC638 SRC. T 86 SRC, CMS TN86 SRC. or hybrid NCBH 129 SRC seeds or other seeds of the present disclosure in which alkaloids obtained from tobacco plants grown from greater than 50% of the seeds have decreased nornicotine. In another aspect, alkaloids obtained from NC638 SRC, CMS NC638 SRC, TN86 SRC, CMS TN86 SRC. or hybrid NCBH129 SRC plants or other plants of the present disclosure grown from greater than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the seeds in the container have decreased nornicotine, representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-120313 for NC638 SRC, ATCC Accession No. PTA- 13568 for CMS NC638 SRC, ATCC Accession No. PTA- 13565 for
TN86 SRC, and/or ATCC Accession No. PTA- 13570 for CMS TN86 SRC. Seeds of hybrid cultivar NCBH129 SRC are obtainable by crossing plants of cultivars TN86 SRC and CMS NC638 SRC and collecting seeds.
[0587] The container of NC638 SRC, CMS NC638 SRC, TN86 SRC, CMS TN86 SRC, or hybrid NCBH129 SRC seeds or other seeds of the present disclosure may contain any number, weight or volume of seeds. For example, a container can contain at least, or greater than, about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000 or more seeds. Alternatively, the container can 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 seeds. Representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA- 120313 for NC638 SRC, ATCC Accession No. PTA- 13568 for CMS NC638 SRC, A CC Accession No. PTA- 13565 for TN86 SRC, and/or ATCC Accession No. PTA-13570 for CMS TN86 SRC. Seeds of hybrid cultivar NCBH 129 SRC are obtainable by crossing plants of cultivars TN86 SRC and CMS NC638 SRC and collecting seeds.
[05881 Containers of NC638 SRC. CMS NC638 SRC. TN86 SRC, CMS TN86 SRC, or hybrid NCBH129 SRC seeds or other seeds of the present disclosure may be any container available in the art. By way of a non-limiting example, a container may be a box, a bag, a packet, a pouch, a tape roll, a pail, a foil, or a tube. Representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-120313 for NC638 SRC. ATCC Accession No. PTA- 13568 for CMS NC638 SRC. ATCC Accession No. PTA-13565 for TN86 SRC, and/or ATCC Accession No. PTA-13570 for CMS TN86 SRC. Seeds of hybrid cultivar NCBH129 SRC are obtainable by crossing plants of cultivars TN86 SRC and CMS NC638 SRC and collecting seeds. [0589 j In another aspect, the present disclosure also provides a container of NC638 SRC, CMS NC638 SRC. T 86 SRC, CMS TN86 SRC. or hybrid cultivar NCBH129 SRC seeds in which greater than about 50% of NC638 SRC. CMS NC638 SRC. TN86 SRC. CMS TN86 SRC, or hybrid NCBH129 SRC seeds or other seeds of the present disclosure have decreased nornicotine. Representative samples of seeds of these cultivars having been deposited with the ATCC, for example under ATCC Accession No. PTA-120313 for NC638 SRC, ATCC Accession No. PTA- 13568 for CMS NC638 SRC, ATCC Accession No.
PTA- 13565 for TN86 SRC and/or A CC Accession No. PTA- 13570 for CMS TN86 SRC. Seeds of hybrid cultivar NCBH129 SRC are obtainable by crossing plants of cultivars TN86 SRC and CMS NC638 SRC and collecting seeds.
[0590] In one aspect, the present disclosure provides a seed of a NC638 SRC. CMS NC638 SRC, TN86 SRC, CMS TN86 SRC, or hybrid NCBH129 SRC plant or other plant of the present disclosure in which a plant grown from a seed is male sterile. Representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-120313 for NC6 SRC, ATCC Accession No. PTA- 13568 for CMS NC638 SRC, ATCC Accession No. PTA- 1 3565 for TN86 SRC and/or ATCC
Accession No. PTA- 13570 for CMS TN86 SRC. Seeds of hybrid cultivar NCBH129 SRC are obtainable by crossing plants of cultivars TN86 SRC and CMS NC638 SRC and collecting seeds.
[0591] Tobacco material obtained from the tobacco lines, varieties or hybrids of the present disclosure can be used to make tobacco products including, without limitation, cigarette products (e.g. , cigarettes and bidi cigarettes), cigar products (e.g., cigar wrapping tobacco and cigarillos), pipe tobacco products, smokeless cigarette products, smokeless tobacco products (e.g. , moist snuff, dry snuff, and chewing tobacco), films, chewables, tabs, shaped parts, gels, consumable units, insoluble matrices, hollow shapes and the like. See, e.g., U.S. Patent Publication No. US 2006/0191548, which is herein incorporated by reference in its entirety.
[0592] Tobacco products derived from plants of the present disclosure also include cigarettes and other smoking articles, particularly those smoking articles including filter elements, wherein the rod of smokeable material includes cured tobacco within a tobacco blend. In an aspect, a tobacco product can include but is not limited to pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and/or cut tobacco or any combination thereof. [0593] In an aspect, a tobacco product of the instant disclosure is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, hookah tobacco, shredded tobacco, and cut tobacco. In another aspect, a tobacco product of the instant disclosure is a smokeless tobacco product. In a further aspect, a tobacco product of the instant disclosure is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff. In yet another aspect, a tobacco product of the instant disclosure is selected from the group consisting of an electronically heated cigarette, an e-cigarette, an electronic vaporing device.
[0594] In an aspect, the tobacco product of the present disclosure can be a blended tobacco product. In other aspects of the disclosure, the tobacco product of the present disclosure can be a reduced nicotine tobacco product. In still other aspects, the tobacco product of the present disclosure can be a blended tobacco product with reduced nicotine content. Thus, the tobacco product of the present disclosure can be a blended reduced nicotine tobacco product. Tobacco product material comprises a blend of tobacco materials from the present disclosure, wherein the blend comprises at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95 percent by weight of a cured tobacco, or any range therein, based on the dry weight of the tobacco material. US 2008/0245377 is herein incorporated by reference for blend mixtures in its entirety.
[0595] In an aspect, tobacco products having a reduced amount of nitrosamine content can be manufactured using tobacco plant material from plants and plant parts of the present disclosure. Thus, in some aspects, a tobacco product manufactured using tobacco plant material from plants and plant parts of the present disclosure can comprise a reduced amount of nornicotine of less than about 3 mg/g. For example, the nornicotine content in such a product can be 3.0 mg/g, 2.5 mg/g, 2.0 mg/g, 1.5 mg/g, 1.0 mg/g, 750 μg/g, 500 μg/g, 250 μζ/g, 100 μg/g, 75 μg/g, 50 μ^, 25 μ^, 10 μ^, 5 μ^, 1 μ^, 750 ng/g, 500 ng/g, 250 ng/g, 100 ng/g, 75 ng/g, 50 ng/g, 25 ng/g, 10 ng/g, 5 ng/g, 1 ng/g, 750 pg/g, 500 pg/g, 250 pg/g, 100 pg/g, 75 pg/g, 50 pg/g, 25 pg/g, 10 pg/g, 7.0 pg/g, 5.0 pg/g, 4.0 pg/g, 2.0 pg/g, 1.0 pg/g, 0.5 pg/g, 0.4 pg/g, 0.2 pg/g, 0.1 pg/g, 0.05 pg/g, 0.01 pg/g, or undetectable, 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 such a product can be about 10 pg/g, 7.0 pg/g, 5.0 pg/g, 4.0 pg/g, 2.0 pg/g, 1.0 pg/g, 0.5 pg/g, 0.4 pg/g, 0.2 pg/g, 0.1 pg/g, 0.05 pg/g, 0.01 pg/g, or undetectable, or any range therein. The percentage of secondary alkaloids relative to total alkaloid content contained in a plant of the present disclosure may not be statistically different than from a commercial seedlot of a cultivar selected from the group consisting of TN90, KYI 4, 1.8, NC775, NC645, NC638, TN86, KY I 4 X 1.8. NC7, and NCBH129.
[0596] The instant disclosure further provides a method of manufacturing a tobacco product, where the method comprising conditioning aged tobacco material made from an tobacco plant disclosed herein to increase its moisture content from between about 12.5% and about 13.5% to about 21%, blending the conditioned tobacco material to produce a desirable blend. In one aspect, the method of manufacturing a tobacco product disclosed herein uses tobacco material from a cultivar selected from the group consisting of TN90 SRC, CMS TN90 SRC. KY I 4 SRC. CMS KY I 4 SRC, 1.8 SRC. NC775 SRC. CMS NC775 SRC, NC645 SRC. NC638 SRC. CMS NC638 SRC, TN86 SRC. CMS TN86 SRC. KY 14 X L8 SRC. NC7 SRC, and NCBH129 SRC. In another aspect, the method of manufacturing a tobacco product disclosed herein further comprises casing or flavoring the blend. Generally, during the casing process, casing or sauce materials are added to blends to enhance their quality by balancing the chemical composition and to develop certain desired flavor characteristics. Further details for the casing process can be found in Tobacco Production, Chemistry and
Technology, Edited by L. Davis and M. Nielsen, Blackwell Science, 1999.
[0597] A tobacco plant of the present disclosure designated TN90 SRC. CMS I N 0 SRC, KYI 4 SRC. CMS KY I 4 SRC. L8 SRC, NC775 SRC, CMS NC775 SRC. NC645 SRC. NC638 SRC. CMS NC638 SRC, TN86 SRC. CMS TN86 SRC, KY I 4 X 1.8 SRC. NC7 SRC. and NCBH129 SRC carrying the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82el0 P381 S alleles can be used in a plant breeding program to create useful lines, cultivars, varieties, progeny, inbreds, and hybrids. Thus, in some aspects, an F1; F2, F3, or later generation tobacco plant containing the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82el0 P381S alleles is crossed with a second Nicotiana plant, and progeny of the cross are identified in which the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82el 0 P381 S alleles are present. It will be appreciated that the second Nicotiana plant can be TN90, KY14, 1 ,8. NC775, NC645, NC638, TN86, or any other Nicotiana species or line, optionally with an additional desirable trait, such as herbicide resistance.
[0598] In still other aspects, methods of the present disclosure further include self- pollinating or pollinating a male sterile pollen acceptor with a pollen donor capable of being used in production of a progeny plant of the present disclosure, such as a male sterile hybrid of the present disclosure. Either the male sterile pollen acceptor plant or the pollen donor plant has at least one mutant allele, two, or even three mutant alleles at a nicotine demethylase locus, such as the cyp82e4 W329Stop allele, the cyp82e5v2 W422Stop allele, and/or the cyp82e!0 P381S allele. In an aspect, all three alleles at each nicotine demethylase locus are mutant alleles, making the plant homozygous for cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381S.
[0599] Breeding can be carried out via any known procedures. DNA fingerprinting,
SNP or similar technologies may be used in a marker-assisted selection (MAS) breeding program to transfer or breed mutant alleles of a nicotine demethylase gene into other tobaccos. For example, a breeder can create segregating populations from hybridizations of a genotype containing cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381S alleles with an agronomically desirable genotype. Plants in the F2 or backcross generations can be screened using a marker developed from cyp82e4 W329Stop, cyp82e5v2 W422Stop, or cyp82e!0 P381S alleles or a fragment thereof, using one of the techniques known in the art or disclosed herein. Plants identified as possessing one or more cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381S alleles can be backcrossed or self-pollinated to create a second population to be screened. Depending on the expected inheritance pattern or the MAS technology used, it may be necessary to self-pollinate the selected plants before each cycle of backcrossing to aid identification of the desired individual plants. Backcrossing or other breeding procedure can be repeated until the desired phenotype of the recurrent parent is recovered. A recurrent parent in the present disclosure can be a cultivar selected from the group consisting of TN90 SRC. CMS TN90 SRC. KY14 SRC, CMS KY I 4 SRC. 1.8 SRC, NC775 SRC, CMS NC775 SRC, NC645 SRC, NC638 SRC, CMS NC638 SRC, TN86 SRC, CMS TN86 SRC. KY14 X L8 SRC. NC7 SRC, and NCBH129 SRC. Other breeding techniques can be found, for example, in Wernsman, E.A., and Riifty. R.C., 1987. Chapter Seventeen. Tobacco, pages 669-698 In: Cultivar Development. Crop Species. W.H. Fehr (ed.), MacMillan Publishing Go., Inc., New York, N.Y., incorporated herein by reference in their entireties.
[0600] Nicotiana species which exhibit breeding compatibility with Nicotiana tabacum include Nicotiana amplexicaulis, PI 271989; Nicotiana benthamiana PI 555478; Nicotiana bigelovii PI 555485; Nicotiana debneyi; Nicotiana excelsior PI 224063; Nicotiana glutinosa PI 555507; Nicotiana goodspeedii PI 241012; Nicotiana gossei PI 230953;
Nicotiana hesperis PI 271991 ; Nicotiana knightiana PI 555527; Nicotiana maritima PI 555535; Nicotiana megalosiphon PI 555536; Nicotiana nudicaulis PI 555540; Nicotiana paniculata PI 555545; Nicotiana plumbaginifolia PI 555548; Nicotiana repanda PI 555552; Nicotiana rustica; Nicotiana suaveolens PI 230960; Nicotiana sylvestris PI 555569; Nicotiana tomentosa PI 266379; Nicotiana tomentosiformis; and Nicotiana trigonophylla PI 555572. See also, Compendium of Tobacco Diseases published by American
Phytopathology Society, or The Genus Nicotiana Illustrated, published by Japan Tobacco Inc, hereby incorporated by reference in their entireties.
[0601] The result of a plant breeding program using the mutant tobacco plants described herein includes useful lines, cultivars, varieties, progeny, inbreds, and hybrids. As used herein, the term "cultivar" or "variety" refers to a population of plants that share constant characteristics which separate them from other plants of the same species. A cultivar or variety is often, although not always, sold commercially. While possessing one or more distinctive traits, a cultivar or variety is further characterized by a very small overall variation between individuals within that cultivar or variety. A "pure line" variety may be created by several generations of self-pollination and selection, or vegetative propagation from a single parent using tissue or cell culture techniques. A cultivar or variety can be essentially derived from another cultivar, line, or variety. As defined by the International Convention for the Protection of New Varieties of Plants (Dec. 2, 1961, as revised at Geneva on Nov. 10, 1972, on Oct. 23, 1978, and on Mar. 19, 1991), a cultivar or variety is
"essentially derived" from an initial cultivar or variety if: a) it is predominantly derived from the initial cultivar or variety, or from a cultivar or variety that is predominantly derived from the initial cultivar or variety, while retaining the expression of the essential characteristics that result from the genotype or combination of genotypes of the initial cultivar or variety; b) it is clearly distinguishable from the initial cultivar or variety; and c) except for the differences which result from the act of derivation, it conforms to the initial cultivar or variety in the expression of the essential characteristics that result from the genotype or combination of genotypes of the initial cultivar or variety. Essentially derived varieties can be obtained, for example, by the selection of a natural or induced mutant, a somaclonal variant, a variant individual from plants of the initial cultivar or variety, backcrossing, or transformation. A "line" as distinguished from a cultivar or variety most often denotes a group of plants used non-commercially, for example in plant research. A line typically displays little overall variation between individuals for one or more traits of interest, although there may be some variation between individuals for other traits.
[0602] Hybrid tobacco varieties can be produced by preventing self-pollination of female parent plants ( . e. , seed parents) of a first variety, permitting pollen from male parent . plants of a second variety to fertilize the female parent plants, and allowing F] hybrid seeds to form on the female plants. Self-pollination of female plants can be prevented by emasculating the flowers at an early stage of flower development. Alternatively, pollen formation can be prevented on the female parent plants using a form of male sterility. For example, male sterility can be produced by cytoplasmic male sterility (CMS), or transgenic male sterility wherein a transgene inhibits microsporogenesis and/or pollen formation, or self- incompatibility. Female parent plants containing CMS are particularly useful. In aspects in which the female parent plants are CMS, pollen may be harvested from male fertile plants and applied manually to the stigmas of CMS female parent plants, and the resulting F\ seed is harvested.
1 60 J Plants can be used to form single-cross tobacco Fi hybrids. Alternatively. three-way crosses can be carried out wherein a single-cross Fi hybrid is used as a female parent and is crossed with a different male parent. As another alternative, double-cross hybrids can be created wherein the Fi progeny of two different single-crosses are themselves crossed. Self-incompatibility can be used to particular advantage to prevent self-pollination of female parents when forming a double-cross hybrid.
[0604] Successful crosses yield Fi plants that are fertile, have cyp82e W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381 S alleles, and can be backcrossed with one of the parents, such as a cultivar selected from the group consisting of TN90 SRC, CMS TN90 SRC, KYI 4 SRC, CMS KYI 4 SRC, L8 SRC, NC775 SRC, CMS NC775 SRC, NC645 SRC, NC638 SRC. CMS NC638 SRC, TN86 SRC, CMS TN86 SRC. KY14 X L8 SRC, NC7 SRC, and NCBH129 SRC, if desired. In some aspects, a plant population in the F2 generation is screened for cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82el0 P381S alleles. Selected plants can be crossed with one of the parents and the first backcross (BC1) generation plants are self-pollinated to produce a BC1 F2 population that is again screened for variant nicotine demethylase gene expression (e.g., the null version of the nicotine demethylase gene). The process of backcrossing, self-pollination, and screening is repeated, for example, at least four times, until the final screening produces a plant that is fertile and reasonably similar to the recurrent parent. This plant, if desired, is self-pollinated and the progeny are subsequently screened again to confirm that the plant exhibits the same low nicotine conversion phenotype as a cultivar selected from the group consisting of TN90 SRC, CMS TN90 SRC. KY14 SRC. CMS KY I 4 SRC. L8 SRC. NC775 SRC, CMS NC775 SRC, NC645 SRC. NC638 SRC, CMS NC638 SRC. TN86 SRC, CMS TN86 SRC. KYI 4 X 1.8 SRC, NC7 SRC, and NCBH129 SRC. Breeder's seed of the selected plant is produced using standard methods including, for example, field testing, confirmation of the null condition for nicotine demethylase, chemical analyses of cured leaf to determine the level of alkaloids and/or chemical analyses of cured leaf to determine the ratio of nornicotine to nicotine+nornicotine.
[0605] In one aspect, a ΓΊ progeny is the result of a cross between TN90 SRC and CMS TN90 SRC to generate Fi progeny that are male sterile. In one aspect, a Fi progeny is the result of a cross between KY14 SRC and CMS KY14 SRC to generate Fi progeny that are male sterile. In another aspect, a Ft progeny is the result of a cross between L8 SRC and CMS KYI 4 SRC to generate Fi progeny that are male sterile. In one aspect, a
Figure imgf000148_0001
progeny is the result of a cross between NC775 SRC and CMS NC775 SRC to generate Fi progeny that are male sterile. In another aspect, a Fi progeny is the result of a cross between NC645 SRC and CMS NC775 SRC to generate F] progeny that are male sterile. In one aspect, a F | progeny is the result of a cross between NC638 SRC and CMS NC638 SRC to generate F, progeny that are male sterile. In another aspect, a Fi progeny is the result of a cross between TN86 SRC and CMS TN86 SRC to generate F, progeny that are male sterile. Male sterile tobacco plants may be produced by any method known in the art. Methods of producing male sterile tobacco are described in Wernsman, E.A., and Rufty, R.C., 1987. Chapter Seventeen. Tobacco, pages 669-698 In: Cultivar Development. Crop Species. W.H. Fehr (ed.), MacMillan Publishing Go., Inc., New York, N.Y. 761 pp.
[0606] The present disclosure further provides methods of producing a tobacco plant by crossing a cultivar selected from the group consisting of TN90 SRC, CMS TN90 SRC. KY I 4 SRC, CMS KY I 4 SRC. L8 SRC. NC775 SRC. CMS NC775 SRC. NC645 SRC, NC638 SRC, CMS NC638 SRC. TN86 SRC, CMS TN86 SRC, KY I 4 X L8 SRC, NC7 SRC, and NCBH129 SRC with itself or a different tobacco line. The disclosure further relates to methods for producing other tobacco cultivars or breeding lines derived from a cultivar selected from the group consisting of ΓΝ90 SRC, CMS TN90 SRC. KY I 4 SRC, CMS KY I 4 SRC, L8 SRC, NC775 SRC, CMS NC775 SRC, NC645 SRC, NC638 SRC, CMS NC638 SRC. T 86 SRC. CMS T 86 SRC. KYI 4 X L8 SRC, NC7 SRC, and NCBH129 SRC by crossing a plant of a cultivar selected from the group consisting of TN90 SRC, CMS TN90 SRC, KY14 SRC, CMS KY14 SRC, L8 SRC, NC775 SRC, CMS NC775 SRC, NC645 SRC, NC638 SRC. CMS NC638 SRC, TN86 SRC. CMS TN86 SRC, KY14 X 1.8 SRC, NC7 SRC, and NCBH129 SRC with a second tobacco plant and growing the progeny seed to yield a tobacco plant derived from TN90 SRC. CMS TN90 SRC. KY I 4 SRC, CMS KY 14 SRC. L8 SRC, NC775 SRC, CMS NC775 SRC, NC645 SRC, NC638 SRC, CMS NC638 SRC, TN86 SRC. CMS TN86 SRC. KY14 X 1.8 SRC, NC7 SRC. or NCBH129 SRC. An additional aspect of the present disclosure provides a method for producing a tobacco plant that contains in its genetic material one or more transgenes, comprising crossing cultivars of the present disclosure with a second cultivar containing one or more transgenes wherein progeny are produced, so that the genetic material of the progeny that result from the cross comprise the transgene(s) optionally operably linked to one or more regulatory elements. In one aspect, the second cultivar may be a plant derived from a cultivar selected from the group consisting of TN90 SRC, CMS TN90 SRC. KYI 4 SRC, CMS KYI 4 SRC. L8 SRC, NC775 SRC. CMS NC775 SRC, NC645 SRC. NC638 SRC. CMS NC638 SRC. TN86 SRC. CMS TN86 SRC, KY14 X L8 SRC, NC7 SRC, and NCBH129 SRC, transformed with one or more transgenes.
[0607] The disclosure further provides for the vegetative propagation of a plant, hybrids and progeny thereof, of a cultivar selected from the group consisting of TN90 SRC, CMS TN90 SRC, KY I 4 SRC. CMS KY I 4 SRC. L8 SRC. NC775 SRC. CMS NC775 SRC. NC645 SRC, NC638 SRC. CMS NC638 SRC, TN86 SRC, CMS TN86 SRC, KY14 X 1,8 SRC, NC7 SRC, and NCBH129 SRC. In one aspect, the disclosure provides for a method of vegetatively propagating a plant of a tobacco cultivar comprising collecting tissue capable of being propagated from a plant of a cultivar selected from the group consisting of TN90 SRC, CMS TN90 SRC. KY I 4 SRC. CMS KYI 4 SRC. L8 SRC. NC775 SRC, C S NC775 SRC, NC645 SRC, NC638 SRC. CMS NC638 SRC, TN86 SRC, CMS I N 86 SRC. KY14 X L8 SRC, NC7 SRC, and NCBH129 SRC, cultivating the tissue to obtain a proliferated shoot and rooting the proliferated shoots to obtain a rooted plantlet. In another aspect, the plant tissue may be collected from an Fi hybrid of a plant of a cultivar selected from the group consisting of TN90 SRC. CMS TN90 SRC, KYI 4 SRC. CMS KYI 4 SRC. 1.8 SRC, NC775 SRC, CMS NC775 SRC, NC645 SRC, NC638 SRC, CMS NC638 SRC, TN86 SRC, CMS TN86 SRC, KY I 4 X 1.8 SRC, NC7 SRC, and NCBH129 SRC. In an aspect, the plant tissue may be collected from an F2, F3, F4 or later progeny plant obtained by breeding a plant of a cultivar selected from the group consisting of TN90 SRC. CMS TN90 SRC. KYI 4 SRC, CMS KY 14 SRC, L8 SRC, NC775 SRC, CMS NC775 SRC, NC645 SRC, NC638 SRC, CMS NC638 SRC. TN86 SRC. CMS TN86 SRC. KYI 4 X 1.8 SRC. NC7 SRC, and NCBH 129 SRC.
[0608] A plant comprising a mutation in a nicotine demethylase gene can be identified by selecting or screening the mutagenized plant material, or progeny thereof. Such screening and selection methodologies are known to those having ordinary skill in the art. Examples of screening and selection methodologies include, but are not limited to, Southern analysis, PGR amplification for detection of a polynucleotide, Northern blots, RNase protection, primer-extension, RT-PCR amplification for detecting RNA transcripts, enzymatic assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides, and protein gel electrophoresis, Western blots, immunoprecipitation, and enzyme-linked immunoassays to detect polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining also can be used to detect the presence or expression of polypeptides and/or polynucleotides. Methods for performing all of the referenced techniques are known.
[0609] It is understood that a tobacco plant of the present disclosure, including, but not limited to, TN90 SRC. CMS TN90 SRC. KY I 4 SRC. CMS KY I 4 SRC, L8 SRC. NC775 SRC. CMS C775 SRC, NC645 SRC, NC638 SRC, CMS NC638 SRC, TN86 SRC, CMS TN86 SRC, KYI 4 X L8 SRC, NC7 SRC, and NCBH129 SRC, can be transformed by a genetic construct (nucleic acid construct) or transgene using any technique known in the art. Without limitation, an example of a desired trait can include herbicide resistance, pest resistance, disease resistance, high yield, high grade index, curability, curing quality, mechanical harvestability, holding ability, leaf quality, height, plant maturation (e.g. , early maturing, early to medium maturing, medium maturing, medium to late maturing, or late maturing), stalk size (e.g. , small, medium, or large stalk), 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) number of leaves), or any combination. Any plant of the present disclosure can be used as a basis for tissue culture, regeneration, transformed, or a combination of any of these. In an aspect, a plant of the present disclosure derived by tissue culture, transformation, or both has all, or essentially all, of the morphological and physiological characteristics of a cultivar selected from the group consisting of TN90 SRC. CMS T 90 SRC. KY14 SRC, CMS KY I 4 SRC. L8 SRC, NC775 SRC, CMS NC775 SRC, NC645 SRC, NC638 SRC, CMS NC638 SRC, ΪΝ86 SRC, CMS TN86 SRC, KY I 4 X L8 SRC. NC7 SRC, and NCBH129 SRC.
[0610] Having now generally described the disclosure, the same will be more readily understood through reference to the following examples that are provided by way of illustration, and are not intended to be limiting of the present disclosure, unless specified.
EXAMPLES
Example 1
Breeding of homozygous cyp82e4 W329Stop, the cyp82e5v2 \V422Stop, and cyp82el0 P381S mutant plants into the TN90 Burley Tobacco Cultivar
[061 1 ] TN90 SRC is a backcross-deri vcd version of burley tobacco cultivar TN90 carrying introduced mutations in three genes (CYP82E4 (SEQ ID NO: 5), CYP82E5 (SEQ ID NO: 8), and CYP82E10 (SEQ ID NO: 9)) previously documented to encode for nicotine demethylase enzymes (Lewis et al , Phytochemistry, 71 (2010), 1988-1998). The introduced mutations in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) and CYP82E5 (cyp82e5v2 W422Stop (SEQ ID NO: 2)) encode for premature stop codons which render the genes non- functional. The introduced mutation in CYP82E10 (cyp82el0 P381 S (SEQ ID NO: 11)) does not encode for a premature stop codon, but does render the gene product non-functional for converting nicotine to nornicotine (Id). When in homozygous condition, the three mutations result in tobacco plants with (1) reduced genetic capacity to demethylate nicotine to form nornicotine, and (2) a corresponding diminished potential to accumulate N-nitrosonornicotine (NNN), a potent carcinogen found in many tobacco products. The mutation in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) also provides phenotypic stability for the "nicotine conversion" trait and eliminates the requirement to utilize the cumbersome "LC" method for reducing levels of nornicotine in tobacco cultivars (see e.g., Jack et al. 2007. Implications of reducing nornicotine accumulation in burley tobacco: appendix A - the LC protocol. Rec. Adv. Tob. Sci. 33: 58-79).
[0612J The original tobacco cultivar TN90 is a fertile inbred line. CMS TN90 is a cytoplasmic male-sterile version of TN90. To develop TN90 SRC, an individual plant of TN90 is pollinated with a plant of the genetic background DH98-325-6 carrying mutations in each of the three nicotine demethylase genes. Fi individuals originating from this cross and heterozygous for each mutation are backcrossed to the recurrent parent, TN90, to produce BC i F i progeny. BCYFi progeny are screened using genotyping methodologies to identify individuals heterozygous for all three mutations. A single triple heterozygous BCiFi plant is backcrossed to TN90 to produce BCVFi progeny. The process of backcrossing and identification of individuals heterozygous for all three mutations is repeated through the BC3F 1. BC4F1, BC F, . BC6Fi; and BC7Fi stages. At the BC7F1 stage, individuals
heterozygous for all three mutations from each pedigree are self-pollinated to produce BC7F2 seed. A large number of BC7F2 progeny from each pedigree are genotyped to identify individuals homozygous for all three mutations. A single BC7F2 plant homozygous for all three mutations is self-pollinated to produce a BC7F3 family (TN90 SRC) in which the wild- type CYP82E4, CYP82E5v2, and CYP82E10 alleles of TN90 were replaced by the mutant alleles (cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381 S).
[0613] The male-sterile (CMS) version of TN90 SRC (CMS TN90 SRC) is produced by crossing a plant of CMS TN90 as a female with pollen of TN90 SRC to produce male-sterile plants heterozygous for all three mutations. A single male-sterile plant resulting from this cross is then subsequently backcrossed as a female to TN90 SRC to produce progeny that are segregating for individuals homozygous for all three mutations. Triple homozygous individuals are identified by DNA genotyping to produce the CMS TN90 SRC line. Because the line is male-sterile, it is maintained via pollination with TN90 SRC.
[0614] Commercial TN90 SRC is produced by pollinating plants of CMS TN90
SRC with pollen of TN90 SRC.
Testing of TN90 SRC
[0615] TN90 SRC is evaluated for cured leaf chemistry, yield, and physical quality at three North Carolina field research locations during 2012 (Laurel Springs, Reidsville, Waynesville). TN90 is included for comparison. The experimental design at each location is a randomized complete block design with four replications. Experimental units are single 20- plant plots. Plots are harvested and air-cured. Plot weights are used to determine per acre yields. Cured leaf is evaluated by a former USDA tobacco grader. Fifty gram composite leaf samples are collected from each plot and analyzed for percent nicotine, nornicotine, anatabine, anabasine, and percent nicotine conversion using gas chromatography equipment.
[0616] Comparisons using the least significant difference (LSD) test indicates that TN90 SRC has significantly (P < 0.05) lower levels of nornicotine and percent nicotine conversion relative to TN90 (Table 1). TN90 and TN90 SRC are not significantly different from each other for percent nicotine, yield, or cured leaf quality indices.
[0617] TN90 SRC is further evaluated for percent nicotine conversion in a field at
Blackstone, Virginia during 2013. A commercial TN90 LC variety is included for comparison. Leaves are sampled at the layby stage and ethephon-treated prior to analysis for nicotine and nornicotine levels using gas chromatography equipment. Random sampling of 26 plants indicates that TN90 LC has an average percent nicotine conversion of 2.61%, whereas TN90 SRC has an average percent nicotine conversion of 0.47% (Figure 1). The low percent nicotine conversion in TN90 SRC is also more stable compared to TN90 LC.
Analysis of NNN levels in TN90 SRC tobacco filler and cigarette smoke
[0618] TN90 SRC is also evaluated for NNN levels in both tobacco filler and cigarette smoke at two Virginia field research locations during 2013 (Glade Spring and Blackstone). A commercial TN90 LC variety is included for comparison. Various levels of nitrogen fertilizer (0, 224, and 448 kg/ha) are tested to evaluate the impact of nitrate levels on NNN levels in tobacco filler and smoke (Figure 2). [0619] Both FN 90 SRC and TN90 LC contains increasing levels of nitrate in tobacco filler as the level of nitrogen fertilizer increases from 0, to 224, and to 448 kg ha. No statistical difference is observed for nitrate concentrations between TN90 SRC and TN90 LC tobacco filler grown under a same nitrogen fertilizer level (Figure 3). Further, no statistical difference is observed for tobacco yields between TN90 SRC and TN90 LC grown under a same nitrogen fertilizer level (Figure 4). Neither is a statistical difference observed for the total level of alkaloids between TN90 SRC and TN90 LC tobacco filler grown under a same nitrogen fertilizer level (Figure 5).
106201 TN90 SRC contains a significantly reduced level of nornicotine in tobacco filler compared to TN90 LC when grown under a same nitrogen fertilizer level (Figure 6). Specifically, a 75% reduction of nornicotine is observed in TN90 S RC filler compared to TN90 LC filler when both are grown with 0 kg/ha nitrogen fertilizer. Reductions of 79% and 81 % are observed for nornicotine levels in TN90 SRC filler compared to TN90 LC filler when both are grown with 224 and 448 kg/ha nitrogen fertilizers, respectively. Notably, the level of nornicotine increases in TN90 LC filler as the level of nitrogen fertilizer increases. However, TN90 SRC filler contains a relatively constant level of nornicotine across different levels of nitrogen fertilizer treatments (Figure 6).
[0621 ] TN90 SRC also contains a significantly reduced level of NNN in tobacco filler compared to TN90 LC when grown under a same nitrogen fertilizer level (Figure 7). Specifically, a 66% reduction of NNN is observed in TN90 SRC filler compared to TN90 LC filler when both are grown with 0 kg/ha nitrogen fertilizer. Reductions of 82% and 89% are observed for NNN levels in TN90 SRC filler compared to TN90 LC filler when both are grown with 224 and 448 kg/ha nitrogen fertilizers, respectively.
[0622] The level of NNN is further tested in cigarette smoke from cigarettes comprising 100% TN90 SRC or 100% TN90 LC. TN90 SRC cigarette smoke contains a significantly reduced level of NNN compared to TN90 LC cigarette smoke when both varieties are grown under a same nitrogen fertilizer level (Figure 8). Specifically, a 65% reduction of NNN is observed in TN90 SRC cigarette smoke compared to TN90 LC cigarette smoke when both are grown with 0 kg/ha nitrogen fertilizer. Reductions of 73% and 79% are observed for NNN levels in TN90 SRC cigarette smoke compared to TN90 LC cigarette smoke when both are grown with 224 and 448 kg/ha nitrogen fertilizers, respectively. Analysis of smoke NN levels in blended cigarettes comprising TN90 SRC
[0623] NNN levels are further evaluated in cigarette smoke from blended cigarettes made with an Extramural Blend comprising varying amounts of TN90 SRC as the Burley component. An Extramural Blend comprise a blend formulation of about 23% Burley tobacco, 35% Bright tobacco (also known as flue-cured tobacco), 15% Oriental tobacco, and 27% reconstitute leaves (RL). Experimental blends with increasing percentages of TN90 SRC (33% Experimental comprising 8% TN90 SRC and 15% TN90 LC, 67% Experimental comprising 15% TN90 SRC and 8%> TN90 LC, and 100% Experimental comprising 23%> TN90 SRC) are compared to a control blend comprising 23% TN90 LC and no TN90 SRC (Figure 9A). Both the International Organization of Standardization (ISO) smoking method (35 cm3 puff volume, 2 second duration, 60 second frequency, 0% vent blocking; see Routine analytical cigarette-smoking machine - Definitions and standard conditions (ISO 3308:2012) and the Health Canada Intense (HCI) smoking method (55cm puff volume, 2 second duration, 30 second frequency, 100%) vent blocking; see Health Canada Test Method T-l 15. Determination of Tar, Water, Nicotine and Carbon Monoxide in Mainstream Tobacco Smoke, 1999-12-31) are used to assess smoke NNN levels.
[0624] The inclusion of TN90 SRC tiller in an Extramural Blend (even when TN90 SRC only constitutes 33% or 67% of the total Burly component of the blend, e.g. , 33% Experimental or 67% Experimental) reduces the level of smoke NNN measured by either the ISO smoking method or the HCI smoking method. Specifically, blended cigarettes with an Extramural Blend comprising TN90 SRC filler as 100% of the Burley component (100% Experimental; TN90 SRC = 23% of overall blend) show a reduction of 38%> (ISO method) and 35% (HCI method) in smoke NNN compared to a control blend comprising TN90 LC filler as 100% of the Burley component (0% Experimental; TN90 LC = 23%) of overall blend) (Figure 9B).
Table 1: Means for experiment EX13-132 entries evaluated in three 2012 NC environments.
Figure imgf000155_0001
Nic = nicotine; nor = nomicotine; anab = anabasine; anat = anatabine
Table 2. Means for experiment EX13-131 entries evaluated in three 2012 NC environments.
Figure imgf000156_0001
Example 2
Breeding of homozygous cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82el0 P381S mutant plants into the KYI 4 Burley Tobacco Cultivar
[0625] KY I 4 SRC is a backcross-derived version of burley tobacco cultivar K Y 14 carrying introduced mutations in three genes (CYP82E4 (SEQ ID NO: 5), CYP82E5 (SEQ ID NO: 8), and CYP82E10 (SEQ ID NO: 9)) previously documented to encode for nicotine demethylase enzymes (Lewis et ai , 2010). The introduced mutations in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) and CYP82E5 (cyp82e5v2 W422Stop (SEQ ID NO: 2)) encode for premature stop codons which render the genes non-functional. The introduced mutation in CYP82E10 (cyp82el0 P381 S (SEQ ID NO: 1 1 )) does not encode for a premature stop codon. but does render the gene product non- functional for converting nicotine to nornicotine (Lewis et al. 2010). When in homozygous condition, the three mutations result in tobacco plants with (1) reduced genetic capacity to demethylate nicotine to form nornicotine, and (2) a corresponding diminished potential to accumulate N-nitrosonornicotine (NNN), a potent carcinogen found in many tobacco products. The mutation in CYP82E4 (cyp82e4 W329Stop (SEQ I D NO: 1)) also provides phenotypic stability for the "nicotine conversion" trait and eliminates the requirement to utilize the cumbersome "LC" method for reducing levels of nornicotine in tobacco cultivars.
[0626] The original tobacco cultivar KYI 4 is a fertile inbred line. CMS K Y 14 is a cytoplasmic male-sterile version of KYI 4. To develop K Y 14 SRC, an individual plant of
KYI 4 is pollinated with a plant of the genetic background DH98-325-6 carrying mutations in each of the three nicotine demethylase genes. Fi individuals originating from this cross and heterozygous for each mutation are backcrossed to the recurrent parent, KYI 4, to produce BCiFi progeny. BCiFi progeny are screened using genotyping methodologies to identify individuals heterozygous for all three mutations. A single triple heterozygous BCiFi plant is backcrossed to KYI 4 to produce BC2Fi progeny. The process of backcrossing and identification of individuals heterozygous for all three mutations is repeated through the BC3F1, BC4F1, BC5F1, BC6F,, and BC7F, stages. At the BC7F 1 stage, individuals
heterozygous for all three mutations from each pedigree are sell-pollinated to produce BC7F2 seed. A large number of BC7F2 progeny from each pedigree are genotyped to identify individuals homozygous for all three mutations. A single BC7F2 plant homozygous for all three mutations is self-pollinated to produce a BC7F3 family (K Y 14 SRC) in which the wild- type CYP82E4, CYP82E5v2, and CYP82E10 alleles of KYI 4 were replaced by the mutant alleles (cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381 S). [0627] The male-sterile (CMS) version of KYI 4 SRC (CMS KY 14 SRC) is produced by crossing a plant of CMS KYI 4 as a female with pollen of K Y 14 SRC to produce male-sterile plants heterozygous for all three mutations. A single male-sterile plant resulting from this cross is then subsequently backcrossed as a female to KYI 4 SRC to produce progeny that are segregating for individuals homozygous for all three mutations. Triple homozygous individuals are identified by DNA genotyping to produce the CMS KYI 4 SRC line. Because the line is male-sterile, it is maintained via pollination with KYI 4 SRC.
[06281 Commercial KY I 4 SRC is produced by pollinating plants of CMS KY I 4 SRC with pollen of KY I 4 SRC.
Testing of KYI 4 SRC
[0629] KYI 4 SRC is evaluated for cured leaf chemistry, yield, and physical quality at three North Carolina field research locations during 2012 (Laurel Springs, Reidsville, Waynesville). K Y 14 is included for comparison. The experimental design at each location is a randomized complete block design with four replications. Experimental units are single 20- plant plots. Plots are harvested and air-cured. Plot weights are used to determine per acre yields. Cured leaf is evaluated by a former USD A tobacco grader. Fi ty gram composite leaf samples are collected from each plot and analyzed for percent nicotine, nornicotine, anatabine. anabasine, and percent nicotine conversion using gas chromatography equipment.
[0630] Comparisons using the least significant difference (LSD) test indicates that K Y 14 SRC has significantly (P < 0.05) lower levels of nornicotine and percent nicotine conversion relative to KY I 4 (Table 2). K Y 14 and KYI 4 SRC are not significantly different from each other for percent nicotine, yield, or cured leaf quality indices.
Example 3
Breeding of homozygous cyp82e4 W329Stop, the cyp82eSv2 W422Stop, and cyp82el0 P381S mutant plants into the L8 Burley Tobacco Cultivar
[0631 ] L8 SRC is a backcross-derived version of burley tobacco cultivar L8 carrying introduced null mutations in three genes (CYP82E4 (SEQ ID NO: 5), CYP82E5 (SEQ ID NO: 8), and CYP82E10 (SEQ ID NO: 9)) previously documented to encode for nicotine demethylase enzymes (Lewis et al, Phytochemistry, 71 (2010), 1988-1998). The introduced mutations in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1 )) and CYP82E5
(cyp82e5v2 W422Stop (SEQ ID NO: 2)) encode for premature stop codons which render the genes non-functional. The introduced mutation in CYP82E10 (cyp82el0 P381 S (SEQ ID NO: 11)) does not encode for a premature stop codon, but does render the gene product nonfunctional for converting nicotine to nornicotine (Id.). When in homozygous condition, the three mutations result in tobacco plants with (1) reduced genetic capacity to demethylate nicotine to form nornicotine, and (2) a corresponding diminished potential to accumulate N- nitrosonornicotine (N N), a potent carcinogen found in many tobacco products. The mutation in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1 )) also provides phenotypic stability for the "nicotine conversion'' trait and eliminates the requirement to utilize the cumbersome "LC" method for reducing levels of nornicotinc in tobacco cultivars (see e.g., Jack et al. 2007. Implications of reducing nornicotinc accumulation in burley tobacco:
appendix A - the LC protocol. Rec. Adv. Tab. Sci. 33: 58-79).
10632 J The original tobacco cultivar L8 is a fertile inbred line. To develop L8 SRC, an individual plant of L8 is initially pollinated with a plant of the genetic background DH98- 325-6 carrying mutations in each of the three nicotine dcmethylase genes. F] individuals originating from this cross and heterozygous for each mutation are backcrossed to the recurrent parent, L8, to produce BQFj progeny. BCjFi progeny are screened using genotyping methodologies to identify individuals heterozygous for all three mutations. A single triple heterozygous BCj Fi plant is backcrossed to L8 to produce BC2F) progeny. The process of backcrossing and identification of individuals heterozygous for all three mutations is repeated through the BC3F 1. BC4F1, BC5F1 , BC(,F], and BC7F1 stages. At the BC7F1 stage, individuals heterozygous for all three mutations from each pedigree are self-pollinated to produce BC7F2 seed. A large number of BC7F2 progeny from each pedigree are genotyped to identify individuals homozygous for all three mutations. A single BC7F2 plant homozygous for all three mutations is self-pollinated to produce a BC-F? family (L8 SRC) in which the wild-type CYP82E4, CYP82E5v2, and CYP82E10 alleles of L8 were replaced by the mutant alleles (cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381 S).
Testing of L8 SRC
10633] 1,8 SRC is evaluated for cured leaf chemistry, yield, and physical quality at three North Carolina field research locations during 2012 (Laurel Springs, Reidsville, Waynesville). L8 is included for comparison. The experimental design at each location is a randomized complete block design with four replications. Experimental units are single 20- plant plots. Plots are harvested and air-cured. Plot weights are used to determine per acre yields. Cured leaf is evaluated by a former USDA tobacco grader. Fifty gram composite leaf samples are collected from each plot and analyzed for percent nicotine, nornicotine, anatabine, anabasine, and percent nicotine conversion using gas chromatography equipment.
[0634] Comparisons using the least significant difference (LSD) test indicated that L8 SRC has significantly (P < 0.05) lower levels of nornicotine and percent nicotine conversion relative to L8 (Table 2). L8 and L8 SRC are not significantly different from each other for percent nicotine, yield, or cured leaf quality indices.
Example 4
Breeding of homozygous cyp82e4 \V329Stop, the cyp82e5v2 W422Stop, and cyp82el0 P381S mutant plants into the KYI 4 X L8 Burley Tobacco Cultivar
[0635] The original tobacco cultivar KY I 4 X L8 LC is a hybrid generated by pollinating plants of a male-sterile breeding line CMS KYI 4 with pollen produced by fertile breeding line L8. Hybrid cultivar KY I 4 X L8 SRC is a hybrid generated by pollinating plants of a male-sterile breeding line CMS KYI 4 SRC with pollen produced by fertile breeding line L8 SRC with each breeding line carrying introduced deleterious mutations in three genes (CYP82E4 (SEQ ID NO: 5), CYP82E5 (SEQ ID NO: 8), and CYP82E10 (SEQ ID NO: 9)) previously documented to encode for nicotine demethylase enzymes (Lewis et al. ,
Phytochemistry, 71 (2010), 1988- 1998). The introduced mutations in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) and CYP82E5 (cyp82e5v2 W422Stop (SEQ ID NO: 2)) encode for premature stop codons which render the genes non-functional. The introduced mutation in CYP82E10 (cyp82el0 P381 S (SEQ ID NO: 1 1)) does not encode for a premature stop codon, but does render the gene product non-functional for converting nicotine to nornicotine (Id.). When in homozygous condition, the three mutations result in tobacco plants with (1) reduced genetic capacity to demethylate nicotine to form nornicotine, and (2) a corresponding diminished potential to accumulate N-nitrosonornicotine (NNN), a potent carcinogen found in many tobacco products. The mutation in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) also provides phenotypic stability for the "nicotine conversion" trait and eliminates the requirement to utilize the cumbersome "LC" method for reducing levels of nornicotine in tobacco cultivars (see e.g., Jack et al. 2007. Implications of reducing nornicotine accumulation in burley tobacco: appendix A - the LC protocol. Rec. Adv. Tob. Sci. 33 : 58-79).
[0636] The original tobacco cultivar KY I 4 X L8 LC is a hybrid generated by pollinating plants of a male-sterile breeding line CMS KYI 4 with pollen produced by fertile breeding line L8. To develop hybrid cultivar KYI 4 X L8 SRC, individual plants of fertile KYI 4 are first pollinated with a plant of the genetic background DH98-325-6 carrying mutations in each of the three nicotine demethylase genes. Fi individuals originating from each cross and heterozygous for each mutation are backcrossed to the recurrent parent (KYI 4) to produce BCjFi progenies. [0637] BCiFi progenies are screened using genotyping methodologies to identify individuals heterozygous for all three mutations. A single BQFi plant from each pedigree is backcrossed to KYI 4, to produce BC2Fi progenies. The process of backcrossing and identification of individuals heterozygous for all three mutations is repeated through the BC3Fi, BC4F1, BC5F1, BC6Fi, and BC7F1 stages. At the BC7Fi stage, individuals
heterozygous for all three mutations from each pedigree are self-pollinated to produce BC7F2 seed. A large number of BC7F2 progeny from each pedigree are genotyped to identify individuals homozygous for all three mutations. BC7F2 plants homozygous for all three mutations are self-pollinated to produce BC7F3 families (KY I 4 SRC) in which the wild-type CYP82E4, CYP82E5v2, and CYP82E10 alleles of KY 14 are replaced by the mutant alleles (cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381S).
[0638] The female parental line of KYI 4, CMS KYI 4, is cytoplasmic male sterile which causes pollen to not be produced. To develop CMS KY14 SRC, a plant of CMS KY 14 is crossed with KYI 4 SRC to produce male-sterile plants heterozygous for all three mutations. A single male-sterile plant resulting from this cross is backcrossed as a female to KYI 4 SRC to produce progeny that are segregating for individuals homozygous for all three mutations. Triple homozygous individuals are identified by DNA genotyping to produce the CMS KYI 4 SRC line. Because the line is male-sterile, it is maintained via pollination with KYI 4 SRC (see above).
[0639] Hybrid cultivar KYI 4 X L8 SRC is produced by pollinating plants of CMS
KYI 4 SRC with pollen of L8 SRC.
Testing of Hybrid Cultivar KY14 X L8 SRC
[0640] Hybrid cultivar KYI 4 X L8 SRC is evaluated for cured leaf chemistry, yield, and physical quality at three North Carolina field research locations during 2012 (Laurel Springs, Reidsville. Waynesville). Hybrid cultivar KYI 4 X 1.8 LC is included for comparison. The experimental design at each location is a randomized complete block design with four replications. Experimental units are single 20-plant plots. Plots are harvested and air-cured. Plot weights are used to determine per acre yields. Cured leaf is evaluated by a former US DA tobacco grader. Fifty gram composite leaf samples are collected from each plot and analyzed for percent nicotine, nornicotine, anatabine, anabasine, and percent nicotine conversion using gas chromatography equipment.
[06411 Comparisons using the least significant difference (LSD) test indicates that hybrid cultivar KY14 X L8 SRC has significantly (P < 0.05) lower levels of nornicotine and percent nicotine conversion relative to KYI 4 X L8 LC (Table 2). KYI 4 X L8 LC and hybrid cultivar KYI 4 X L8 SRC are not significantly different from each other for percent nicotine, yield, or cured leaf quality indices.
Example 5
Breeding of homozygous cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82el0 P381S mutant plants into the NC775 Burley Tobacco Cultivar
[0642] NC775 SRC is a backcross-derived version of burley tobacco cultivar NC775 carrying introduced mutations in three genes (CYP82E4 (SEQ ID NO: 5), CYP82E5 (SEQ ID NO: 8), and CYP82E10 (SEQ ID NO: 9)) previously documented to encode for nicotine demethylase enzymes (Lewis et al, Phy to chemistry, 71 (2010), 1988-1998). The introduced mutations in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) and CYP82E5
(cyp82e5v2 W422Slop (SEQ ID NO: 2)) encode for premature stop codons which render the genes non-functional. The introduced mutation in CYP82E10 (cyp82el0 P381 S (SEQ ID NO: 11)) does not encode for a premature stop codon, but does render the gene product nonfunctional for converting nicotine to nornicotine (Id.). When in homozygous condition, the three mutations result in tobacco plants with (1) reduced genetic capacity to demethylate nicotine to form nornicotine, and (2) a corresponding diminished potential to accumulate V- nitrosonornicotine (NNN), a potent carcinogen found in many tobacco products. The mutation in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) also provides phenotypic stability for the "nicotine conversion" trait and eliminates the requirement to utilize the cumbersome "LC" method for reducing levels of nornicotine in tobacco cultivars (see e.g., Jack et al. 2007. Implications of reducing nornicotine accumulation in burley tobacco:
appendix A - the LC protocol. Rec. Adv. Tob. Sci. 33 : 58-79).
[0643] The original tobacco cultivar NC775 is a fertile inbred line. CMS NC775 is a cytoplasmic male-sterile version of NC775. To develop NC775 SRC, an individual plant of NC775 is pollinated with a plant of the genetic background Dl 198-325-6 carrying mutations in each of the three nicotine demethylase genes. Fi individuals originating from this cross and heterozygous for each mutation are backcrossed to the recurrent parent. NC775, to produce BC'i F] progeny. BCiFj progeny are screened using genotyping methodologies to identify individuals heterozygous for all three mutations. A single triple heterozygous BC| F] plant is backcrossed to NC775 to produce BC2Fi progeny. The process of backcrossing and identification of individuals heterozygous for all three mutations is repeated through the BC3F1, BC4F1 , BC F,, BC6F,, and BC7F, stages. At the BC7F1 stage, individuals
heterozygous for all three mutations from each pedigree are sel -pollinated to produce BC7F2 seed. A large number of BC7F2 progeny from each pedigree are genotyped to identify individuals homozygous for all three mutations. A single BC7F2 plant homozygous for all three mutations is self-pollinated to produce a BC7F3 family (NC775 SRC) in which the wild- type CYP82E4, CYP82E5v2, and CYP82E10 alleles of NC775 were replaced by the mutant alleles (cyp82e4 W329Stop, cyP82e5v2 W422Stop, and cyp82el0 P381 S).
[0644] The male-sterile (CMS) version of NC775 SRC (CMS NC775 SRC) is produced by crossing a plant of CMS NC775 as a female with pollen of NC775 SRC to produce male-sterile plants heterozygous for all three mutations. A single male-sterile plant resulting from this cross is then subsequently backcrossed as a female to NC775 SRC to produce progeny that are segregating for individuals homozygous for all three mutations. Triple homozygous individuals are identified by DNA genotyping to produce the CMS
NC775 SRC line. Because the line is male-sterile, it is maintained via pollination with NC775 SRC.
[0645] Commercial NC775 SRC is produced by pollinating plants of CMS NC775 SRC with pollen of NC775 SRC (see below).
Testing of NC775 SRC
[0646] NC775 SRC is evaluated for cured leaf chemistry, yield, and physical quality at three North Carolina field research locations during 2012 (Laurel Springs, Reidsville, Waynesville). NC775 is included for comparison. The experimental design at each location is a randomized complete block design with four replications. Experimental units are single 20-plant plots. Plots are harvested and air-cured. Plot weights are used to determine per acre yields. Cured leaf is evaluated by a former USD A tobacco grader. Fifty gram composite leaf samples are collected from each plot and analyzed for percent nicotine, nomicotine, anatabine, anabasine, and percent nicotine conversion using gas chromatography equipment.
[0647] Comparisons using the least significant difference (LSD) test indicates that NC775 SRC has significantly (P < 0.05) lower levels of nomicotine and percent nicotine conversion relative to NC775 (Table 2). NC775 and NC775 SRC are not significantly different from each other for percent nicotine, yield, or cured leaf quality indices.
Example 6
Breeding of homozygous cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82el0 P381S mutant plants into the NC645 Burley Tobacco Cultivar
[0648] NC645 SRC is a backcross-derived version of burley tobacco cultivar NC645 carrying introduced null mutations in three genes (CYP82E4 (SEQ ID NO: 5), CYP82E5 (SEQ ID NO: 8), and CYP82E10 (SEQ ID NO: 9)) previously documented to encode for nicotine demethylase enzymes (Lewis et al. , Phytochemistry, 71 (2010), 1988- 1998). The introduced mutations in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) and CYP82E5 (cyp82e5v2 W422Stop (SEQ ID NO: 2)) encode for premature stop codons which render the genes non-functional. The introduced mutation in CYP82E10 (cyp82el0 P381 S (SEQ ID NO: 1 1)) docs not encode for a premature stop codon, but docs render the gene product non-functional for converting nicotine to nornicotine (Id. ). When in homozygous condition, the three mutations result in tobacco plants with (1) reduced genetic capacity to demethylate nicotine to form nornicotine, and (2) a corresponding diminished potential to accumulate JV-nitrosonornicotine (NNN), a potent carcinogen found in many tobacco products. The mutation in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) also provides phenotypic stability for the "nicotine conversion" trait and eliminates the requirement to utilize the cumbersome "LC" method for reducing levels of nornicotine in tobacco cultivars (see e.g., Jack et al. 2007. Implications of reducing nornicotine accumulation in burley tobacco: appendix A - the LC protocol. Rec. Adv. Tob. Sci. 33: 58-79).
[0649] The original tobacco cultivar NC645 is a fertile inbred line. To develop NC645 SRC, an individual plant of NC645 is initially pollinated with a plant of the genetic background DH98-325-6 carrying mutations in each of the three nicotine demethylase genes. Fi individuals originating from this cross and heterozygous for each mutation are backcrossed to the recurrent parent, NC645, to produce BCiFi progeny. BQFi progeny are screened using genotyping methodologies to identify individuals heterozygous for all three mutations. A single triple heterozygous BCiFi plant is backcrossed to NC645to produce BC2Fi progeny. The process of backcrossing and identification of individuals heterozygous for all three mutations is repeated through the BC3Fl5 BC4Fl5 BC5Fi, BC(,l7i, and BC7F1 stages. At the BC7F1 stage, individuals heterozygous for all three mutations from each pedigree are self- pollinated to produce BC7F2 seed. A large number of BC7F2 progeny from each pedigree are genotyped to identify individuals homozygous for all three mutations. A single BC7F2 plant homozygous for all three mutations is self-pollinated to produce a BC7F3 family (NC645 SRC) in which the wild-type CYP82E4, CYP82E5v2, and CYP82E10 alleles of NC645 were replaced by the mutant alleles (cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381 S).
Testing of C645 SRC
[0650] NC645 SRC is evaluated for cured leaf chemistry, yield, and physical quality at three North Carolina field research locations during 2012 (Laurel Springs, Rcidsville, Waynesville). NC645 is included for comparison. The experimental design at each location is a randomized complete block design with four replications. Experimental units are single 20-plant plots. Plots are harvested and air-cured. Plot weights are used to determine per acre yields. Cured leal" is evaluated by a former USDA tobacco grader. Fifty gram composite leaf samples are collected from each plot and analyzed for percent nicotine, nornicotine, anatabine, anabasine, and percent nicotine conversion using gas chromatography equipment.
[0651] Comparisons using the least significant difference (LSD) test indicated that
NC645 SRC has significantly (P < 0.05) lower levels of nornicotine and percent nicotine conversion relative to NC645 (Table 2). NC645 and NC645 SRC are not significantly different from each other for percent nicotine, yield, or cured leaf quality indices.
Example 7
Breeding of homozygous cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82el0 P381S mutant plants into the NC7 Hybrid Burley Tobacco Cultivar
[0652] The original tobacco cultivar NC7 is a hybrid generated by pollinating plants of a male-sterile breeding line CMS NC775 with pollen produced by fertile breeding line NC645. I lybrid culti var NC7 SRC is a a hybrid generated by pollinating plants of a male- sterile breeding line CMS NC775 SRC with pollen produced by fertile breeding line NC645 SRC with each breeding line carrying introduced deleterious mutations in three genes (CYP82E4 (SEQ ID NO: 5), CYP82E5 (SEQ ID NO: 8), and CYP82E10 (SEQ ID NO: 9)) previously documented to encode for nicotine demethylase enzymes (Lewis et al..
Phytochemistry, 71 (2010), 1988-1998). The introduced mutations in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) and CYP82E5 (cyp82e5v2 W422Stop (SEQ ID NO: 2)) encode for premature stop codons which render the genes non-functional. The introduced mutation in CYP82E10 (cyp82el0 P381 S (SEQ ID NO: 1 1)) does not encode for a premature stop codon, but does render the gene product non-functional for converting nicotine to nornicotine (Id.). When in homozygous condition, the three mutations result in tobacco plants with (1) reduced genetic capacity to demethylate nicotine to form nornicotine, and (2) a corresponding diminished potential to accumulate N-nitrosonornicotine (NNN), a potent carcinogen found in many tobacco products. The mutation in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) also provides phenotypic stability for the "nicotine conversion" trait and eliminates the requirement to utilize the cumbersome "LC" method for reducing levels of nornicotine in tobacco cultivars (see e.g., Jack et al. 2007. Implications of reducing nornicotine accumulation in burley tobacco: appendix A. - the LC protocol. Rec. Adv. Tab. Sci. 33: 58-79).
[0653] The original tobacco cultivar NC7 is a hybrid generated by pollinating plants of a male-sterile breeding line CMS NC775 with pollen produced by fertile breeding line NC645. To develop hybrid cultivar NC7 SRC, individual plants of fertile NC775 are first pollinated with a plant of the genetic background Dl 198-325-6 carrying mutations in each of the three nicotine demethylase genes. Fi individuals originating from each cross and heterozygous for each mutation are backcrossed to the recurrent parent (NC775 ) to produce BCiFi progenies.
[0654] BCjFi progenies are screened using genotyping methodologies to identify individuals heterozygous for all three mutations. A single BCiFj plant from each pedigree is backcrossed to NC775, to produce BC2F[ progenies. The process of backcrossing and identification of individuals heterozygous for all three mutations is repeated through the BC3F1 , BC4F1 , BC5F1, BC6Fi, and BC7F, stages. At the BC7F, stage, individuals
heterozygous for all three mutations from each pedigree are self-pollinated to produce BC7F2 seed. A large number of BC7F2 progeny from each pedigree are genotyped to identify individuals homozygous for all three mutations. BC7F2 plants homozygous for all three mutations are self-pollinated to produce BC7F3 families (NC775 SRC) in which the wild-type CYP82E4, CYP82E5v2, and CYP82E10 alleles of NC775 are replaced by the mutant alleles (cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e!0 P381 S).
[0655] The female parental line of NC7, CMS NC775, is cytoplasmic male sterile which causes pollen to not be produced. To develop CMS NC775 SRC, a plant of CMS NC775 is crossed with NC775 SRC to produce male-sterile plants heterozygous for all three mutations. A single male-sterile plant resulting from this cross is backcrossed as a female to NC775 SRC to produce progeny that are segregating for individuals homozygous for all three mutations. Triple homozygous individuals are identified by DNA genotyping to produce the CMS NC775 SRC line. Because the line is male-sterile, it is maintained via pollination with NC775 SRC (see above).
[0656] Hybrid cultivar NC7 SRC is produced by pollinating plants of CMS NC775
SRC with pollen of NC645 SRC.
Testing of Hybrid Cultivar NC7 SRC
[0657] Hybrid cultivar NC7 SRC is evaluated for cured leaf chemistry, y ield, and physical quality at three North Carolina field research locations during 2012 (Laurel Springs, Reidsville, Waynesville). Hybrid cultivar NC7 LC is included for comparison. The experimental design at each location is a randomized complete block design with four replications. Experimental units are single 20-plant plots. Plots are harvested and air-cured. Plot weights are used to determine per acre yields. Cured leaf is evaluated by a former USDA tobacco grader. Fifty gram composite leaf samples are collected from each plot and analyzed for percent nicotine, nornicotine, anatabine, anabasine, and percent nicotine conversion using gas chromatography equipment.
[0658] Comparisons using the least significant di ference (LSD) test indicates that hybrid cultivar NC7 SRC has significantly (P < 0.05) lower levels of nornicotine and percent nicotine conversion relative to NC7 LC (Table 2). NC7 LC and hybrid cultivar NC'7 SRC are not significantly different from each other for percent nicotine, yield, or cured leaf quality indices.
Example 8
Breeding of homozygous cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82el0 P381S mutant plants into the NC638 Burley Tobacco Cultivar
[0659] NC638 SRC is a backcross-derived version of burley tobacco cultivar NC638 carrying introduced mutations in three genes (CYP82E4 (SEQ ID NO: 5). CYP82E5 (SEQ ID NO: 8), and CYP82E10 (SEQ ID NO: 9)) previously documented to encode for nicotine demethylase enzymes (Lewis et al , Phytochemistry, 71 (2010), 1988-1998). The introduced mutations in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1 )) and CYP82E5
(cyp82e5v2 W422Stop (SEQ ID NO: 2)) encode for premature stop codons which render the genes non-functional. The introduced mutation in CYP82E10 (cyp82el0 P381 S (SEQ ID NO: 1 1)) does not encode for a premature stop codon, but does render the gene product nonfunctional for converting nicotine to nornicotine (Id). When in homozygous condition, the three mutations result in tobacco plants with (1) reduced genetic capacity to demethylate nicotine to form nornicotine, and (2) a corresponding diminished potential to accumulate N- nitrosonornicotine (NNN), a potent carcinogen found in many tobacco products. The mutation in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) also provides phenotypie stability for the "nicotine conversion" trait and eliminates the requirement to utilize the cumbersome "LC" method for reducing levels of nornicotine in tobacco cultivars (see e.g., Jack et al. 2007. Implications of reducing nornicotine accumulation in burley tobacco:
appendix A - the LC protocol. Rec. Adv. Tob. Sci. 33 : 58-79).
[0660] The original tobacco cultivar NC638 is a fertile inbred line. CMS NC638 is a cytoplasmic male-sterile version of NC638. To develop NC638 SRC, an individual plant of NC638 is pollinated with a plant of the genetic background DI 198-325-6 carrying mutations in each of the three nicotine demethylase genes. Fi individuals originating from this cross and heterozygous for each mutation are backcrossed to the recurrent parent, NC638, to produce BC'i Fi progeny. BQFi progeny are screened using genotyping methodologies to identify individuals heterozygous for all three mutations. A single triple heterozygous BC 1F 1 plant is backcrossed to NC638 to produce BC2F] progeny. The process of backcrossing and identification of individuals heterozygous for all three mutations is repeated through the BC3F1, BC4F1, BC5F1 , BC6Fi. and BC7F, stages. At the BC7F, stage, individuals
heterozygous for all three mutations from each pedigree are self-pollinated to produce BC7F2 seed. A large number of BC7F2 progeny from each pedigree are gcnotyped to identify individuals homozygous for all three mutations. A single BC7F2 plant homozygous for all three mutations is self-pollinated to produce a BC7F3 family (NC638 SRC) in which the wild- type CYP82E4, CYP82E5v2, and CYP82E10 alleles of NC638 were replaced by the mutant alleles (cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el 0 P381 S).
[0661] The male-sterile (CMS) version of NC638 SRC (CMS NC638 SRC) is produced by crossing a plant of CMS NC638 as a female with pollen of NC638 SRC to produce male-sterile plants heterozygous for all three mutations. A single male-sterile plant resulting from this cross is then subsequently backcrossed as a female to NC638 SRC to produce progeny that are segregating for individuals homozygous for all three mutations. Triple homozygous individuals are identified by DNA genotyping to produce the CMS NC638 SRC line. Because the line is male-sterile, it is maintained via pollination with NC638 SRC.
[06621 Commercial NC638 SRC is produced by pollinating plants of CMS NC638 SRC with pollen of TN86 SRC (see below).
Testing of NC638 SRC
[0663] NC638 SRC is evaluated for cured leaf chemistry, yield, and physical quality at three North Carolina field research locations during 2012 (Laurel Springs, Rcidsville, Waynesville). NC638 is included for comparison. The experimental design at each location is a randomized complete block design with four replications. Experimental units are single 20-plant plots. Plots are harvested and air-cured. Plot weights are used to determine per acre yields. Cured leaf is evaluated by a former USDA tobacco grader. Fifty gram composite leaf samples are collected from each plot and analyzed for percent nicotine, nornicotine, anatabine. anabasine. and percent nicotine conversion using gas chromatography equipment.
[0664] Comparisons using the least significant difference (LSD) test indicates that
NC638 SRC has significantly (P < 0.05) lower levels of nornicotine and percent nicotine conversion relative to NC638 (Table 2). NC638 and NC638 SRC are not significantly different from each other for percent nicotine, yield, or cured leaf quality indices. Example 9
Breeding of homozygous cyp82e4 W329Stop, the cyp82eSv2 W422Stop, and cyp82el0 P381S mutant plants into the TN86 Burley Tobacco Cultivar
[0665] TN86 SRC is a backcross-derived version of burley tobacco culti var TN86 carrying introduced null mutations in three genes (CYP82E4 (SEQ ID NO: 5), CYP82E5 (SEQ ID NO: 8), and CYP82E10 (SEQ ID NO: 9)) previously documented to encode for nicotine demethylase enzymes (Lewis et al , Phyto chemistry, 71 (2010), 1 988-1 998). The introduced mutations in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) and CYP82E5 (cyp82e5v2 W422Stop (SEQ ID NO: 2)) encode for premature stop codons which render the genes non-functional. The introduced mutation in CYP82E10 {cyp82el0 P381 S (SEQ ID NO: 1 1)) does not encode for a premature stop codon, but does render the gene product nonfunctional for converting nicotine to nornicotine (Id). When in homozygous condition, the three mutations result in tobacco plants with (1) reduced genetic capacity to demethylate nicotine to form nornicotine, and (2) a corresponding diminished potential to accumulate N- nitrosonomicotine (NNN), a potent carcinogen found in many tobacco products. The mutation in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) also provides phenotypic stability for the "nicotine conversion" trait and eliminates the requirement to utilize the cumbersome "LC" method for reducing levels of nornicotine in tobacco cultivars (see e.g., Jack et al. 2007. Implications of reducing nornicotine accumulation in burley tobacco:
appendix A - the LC protocol. Rec. Adv. Tob. Sci. 33 : 58-79).
[0666] The original tobacco cultivar TN86 is a fertile inbred line. To develop TN86 SRC, an individual plant of TN86 is initially pollinated with a plant of the genetic
background DH98-325-6 carrying mutations in each of the three nicotine demethylase genes. Fi individuals originating from this cross and heterozygous for each mutation are backcrossed to the recurrent parent, TN86, to produce BCi Fj progeny. BCjFi progeny are screened using genotyping methodologies to identify individuals heterozygous for all three mutations. A single triple heterozygous BCjFi plant is backcrossed to TN86 to produce BC Fi progeny. The process of backcrossing and identification of indi viduals heterozygous for all three mutations is repeated through the BC3F1 , BC4F 1 , BC5F1 , BC6Fi. and BC7F1 stages. At the BC7Fi stage, individuals heterozygous for all three mutations from each pedigree are sel - pollinated to produce BC7F2 seed. A large number of BC7F2 progeny from each pedigree are genotyped to identify individuals homozygous for all three mutations. A single BC7F2 plant homozygous for all three mutations is self-pollinated to produce a BC7F3 family (TN86 SRC) in which the wild-type CYP82E4, CYP82E5v2, and CYP82E10 alleles of TN86 were replaced by the mutant alleles (cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381S).
[0667] The male-sterile (CMS) version of TN86 SRC (CMS TN86 SRC) is produced by crossing a plant of CMS TN86 as a female with pollen of TN86 SRC to produce male-sterile plants heterozygous for all three mutations. A single male-sterile plant resulting from this cross is then subsequently backcrossed as a female to TN86 SRC to produce progeny that are segregating for individuals homozygous for all three mutations. Triple homozygous individuals are identified by DNA genotyping to produce the CMS TN86 SRC line. Because the line is male-sterile, it is maintained via pollination with TN86 SRC.
10668] Commercial TN86 SRC is produced by pollinating plants of CMS TN86
SRC with pollen of TN86 SRC.
Testing of TN86 SRC
[0669] TN86 SRC is evaluated for cured leaf chemistry, yield, and physical quality at three North Carolina field research locations during 2012 (Laurel Springs, Reidsville, WaynesviUe). TN86 is included for comparison. The experimental design at each location is a randomized complete block design with four replications. Experimental units are single 20- plant plots. Plots are harvested and air-cured. Plot weights are used to determine per acre yields. Cured leaf is evaluated by a former USDA tobacco grader. Fifty gram composite leaf samples are collected from each plot and analyzed for percent nicotine, nornicotine, anatabine, anabasine, and percent nicotine conversion using gas chromatography equipment.
[0670] Comparisons using the least significant difference (LSD) test indicated that TN86 SRC has significantly (P < 0.05) lower levels of nornicotine and percent nicotine conversion relative to TN86 (Table 1). TN86 and TN86 SRC arc not significantly different from each other for percent nicotine, yield, or cured leaf quality indices. Example 10
Breeding of homozygous cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82el0 P381S mutant plants into the NCBH129 Burley Tobacco Hybrid Cultivar
[0671] The original tobacco cultivar NCBH129 is a hybrid generated by pollinating plants of a male-sterile breeding line CMS NC638 with pollen produced by fertile breeding line TN86. Hybrid cultivar NCBH 129 SRC is a hybrid generated by pollinating plants of a male-sterile breeding line CMS NC638 SRC with pollen produced by fertile breeding line TN86 SRC with each breeding line carrying introduced deleterious mutations in three genes (CYP82E4 (SEQ ID NO: 5), CYP82E5 (SEQ ID NO: 8), and CYP82E10 (SEQ ID NO: 9)) previously documented to encode for nicotine demethylase enzymes (Lewis et al. ,
Phytochemistry, 71 (2010), 1988-1998). The introduced mutations in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) and CYP82E5 (cyp82e5v2 W422Stop (SEQ ID NO: 2)) encode for premature stop codons which render the genes non-functional. The introduced mutation in CYP82E10 (cyp82e!0 P381S (SEQ ID NO: 1 1)) does not encode for a premature stop codon, but docs render the gene product non-functional for converting nicotine to nornicotine (Id.). When in homozygous condition, the three mutations result in tobacco plants with (1) reduced genetic capacity to demethylate nicotine to form nornicotine, and (2) a corresponding diminished potential to accumulate N-nitrosonornicotine (NNN), a potent carcinogen found in many tobacco products. The mutation in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) also provides phenotypic stability for the "nicotine conversion" trait and eliminates the requirement to utilize the cumbersome "LC" method for reducing levels of nornicotine in tobacco cultivars (see e.g., Jack et al. 2007. Implications of reducing nornicotine accumulation in burley tobacco: appendix A - the LC protocol. Rec. Adv. Tob. Scz\ 33 : 58-79).
[0672] The original tobacco cultivar NCBl l 129is a hybrid generated by pollinating plants of a male-sterile breeding line CMS NC638 with pollen produced by fertile breeding line TN86. To develop hybrid cultivar NCBI 1129 SRC, individual plants of fertile NC638 and TN86 are first pollinated with a plant of the genetic background DH98-325-6 carrying mutations in each of the three nicotine demethylase genes. F] individuals originating from each cross and heterozygous for each mutation are backcrossed to the recurrent parent (NC638 or TN86, respectively) to produce BCjFi progenies.
10673] BCiFi progenies are screened using genotyping methodologies to identify individuals heterozygous for all three mutations. A single BCj Fi plant from each pedigree is backcrossed to either NC638 or TN86, respectively, to produce BCVl' i progenies. The process of backcrossing and identification of individuals heterozygous for all three mutations is repeated through the BC3Fl 5 BC4F,, BC5F1, BC6F,. and BC7Fj stages. At the BC7Fi stage, individuals heterozygous for all three mutations from each pedigree are self-pollinated to produce BC7F2 seed. A large number of BC7F2 progeny from each pedigree are genotyped to identify individuals homozygous for all three mutations. BC7F2 plants homozygous for all three mutations are self-pollinated to produce BC7F3 families (NC638 SRC and TN86 SRC) in which the wild-type CYP82E4, CYP82E5v2, and CYP82E10 alleles of NC638 and TN86 are replaced by the mutant alleles (cyp82 4 W329Stop, cyp82e5v2 W422Stop, and cyp82el0 P381 S).
[0674] The female parental line of NCBH129, CMS NC638, is cytoplasmic male sterile which causes pollen to not be produced. To develop CMS NC638 SRC, a plant of CMS NC638 is crossed with NC638 SRC to produce male-sterile plants heterozygous for all three mutations. A single male-sterile plant resulting from this cross is backcrossed as a female to NC638 SRC to produce progeny that are segregating for individuals homozygous for all three mutations. Triple homozygous individuals are identified by DNA genotyping to produce the CMS NC638 SRC line. Because the line is male-sterile, it is maintained via pollination with NC638 SRC (see above).
[0675] Hybrid cultivar NCBH129 SRC is produced by pollinating plants of CMS NC638 SRC with pollen of TN86 SRC.
Testing of Hybrid Cultivar NCBH129 SRC
[0676] Hybrid cultivar NCBH129 SRC is evaluated for cured leaf chemistry, yield, and physical quality at three North Carolina field research locations during 2012 (Laurel
Springs, Reidsvillc, WaynesviUe). 1 lybrid cultivar NCBI 1129 LC is included for comparison.
The experimental design at each location is a randomized complete block design with four replications. Experimental units are single 20-plant plots. Plots are harvested and air-cured.
Plot weights are used to determine per acre yields. Cured leaf is evaluated by a former USD A tobacco grader. Fifty gram composite leaf samples are collected from each plot and analyzed for percent nicotine, nornicotine. anatabine. anabasine, and percent nicotine conversion using gas chromatography equipment.
[0677] Comparisons using the least significant difference (LSD) test indicates that hybrid cultivar NCBH129 SRC has significantly (P < 0.05) lower levels of nornicotine and percent nicotine conversion relative to NCBH129 (Table 2). NCBH129 LC and hybrid cultivar NCBH 129 SRC are not significantly different from each other for percent nicotine, yield, or cured leaf quality indices. DEPOSIT INFORMATION
[0678] A deposit of the proprietary inbred plant lines disclosed above and recited in the appended claims have been made with American Type Culture Collection (ATCC), 10801 University Boulevard. Manassas, VA 201 10. The date of deposit for TN90 SRC, CMS TN90 SRC, KY14 SRC, CMS KY14 SRC, L8 SRC, NC775 SRC, CMS NC775 SRC, NC645 SRC, NC638 SRC, CMS NC638 SRC, TN86 SRC, CMS TN86 SRC, KY14 X L8 SRC, NC7 SRC, and NCB1 I 129 SRC was February 26, 2013. The deposits of 2500 seeds for each variety was taken from the same deposits maintained since prior to the filing date of this application. Upon issuance of a patent, all restrictions upon the deposits will be removed, and the deposits are intended to meet all of the requirements of 37 C.F.R. § 1.801 -1.809. The ATCC has issued the following accession numbers: ATCC Accession No. PTA-13567 for TN90 SRC. ATCC Accession No. PTA- 13573 for CMS TN90 SRC. ATCC Accession No. PTA- 12031 1 for KY14 SRC, ATCC Accession No. PTA- 13571 for CMS KY I 4 SRC, ATCC Accession No. ΡΤΛ-13572 for L8 SRC, ATCC Accession No. PTA- 13569 for hybrid cultivar KYI 4 X L8 SRC. ATCC Accession No. PTA-120312 for NC775 SRC, ATCC Accession No. Ρ ΓΑ- 13563 for CMS NC775 SRC. ATCC Accession No. PTA- 13566 for NC645 SRC, ATCC Accession No. PTA- 13564 for hybrid cultivar NC7 SRC, ATCC Accession No. PTA- 120313 for NC638 SRC, ATCC Accession No. PTA- 13568 for CMS NC638 SRC, ATCC Accession No. PTA- 13565 for TN86 SRC, ATCC Accession No. PTA- 13570 for CMS TN86 SRC, and ATCC Accession No. PTA- 13562 for hybrid cultivar NCBH129 SRC. These deposits will be maintained in the depository for a period of 30 years, or 5 years after the last request, or for the effective li e of the patent, whichever is longer, and will be replaced as necessary during that period. Applicants do not waive any infringement of their rights granted under this patent or under the Plant Variety Protection Act (7 U.S.C. 2321 et seq.).

Claims

What is claimed is:
1. Cured tobacco or a tobacco product prepared therefrom,
wherein said cured tobacco is from a tobacco plant, or part thereof, of a hybrid, a cultivar, or a progeny hybrid produced from said cultivar,
wherein said hybrid is selected from the group consisting of KY 14 X L8 SRC. NC7
SRC, and NCBH129 SRC,
wherein said cultivar is selected from the group consisting of TN90 SRC, CMS TN9Q SRC, KY14 SRC, CMS KY14 SRC, L8 SRC, NC775 SRC, CMS NC775 SRC, NC645 SRC, NC638 SRC, CMS NC638 SRC, TN86 SRC, and CMS TN86 SRC;
and wherein a representative sample seed of said TN90 SRC, CMS TN90 SRC, KYI 4
SRC, CMS KY14 SRC, L8 SRC, NC775 SRC, CMS NC775 SRC, NC645 SRC, NC638 SRC, CMS NC638 SRC, TN86 SRC, CMS TN86 SRC, KYI 4 X L8 SRC, NC7 SRC, and NCBH129 SRC has been deposited at the ATCC with the following ATCC Accession Nos.: ATCC Accession No. PTA- 13567 for TN90 SRC. ATCC Accession No. ΡΤΛ- 13573 for CMS TN90 SRC, ATCC Accession No. PTA- 12031 1 for KY I 4 SRC, ATCC
Accession No. PTA-13571 for CMS KY I 4 SRC. ATCC Accession No. PTA- 13572 for L8 SRC, ATCC Accession No. PTA- 13569 for hybrid cultivar KYI 4 X L8 SRC, ATCC Accession No. PTA-120312 for NC775 SRC, ATCC Accession No. PTA- 13563 for CMS NC775 SRC, ATCC Accession No. PTA- 13566 for NC645 SRC, ATCC Accession No. PTA- 13564 for hybrid cultivar NC7 SRC, ATCC Accession No. PTA- 120313 for NC638
SRC, ATCC Accession No. PTA- 13568 for CMS NC638 SRC, ATCC Accession No. PTA- 13565 for TN86 SRC. ATCC Accession No. PTA- 13570 for CMS TN86 SRC, and ATCC Accession No. PTA- 13562 for hybrid cultivar NCBH 129 SRC.
2. The tobacco product of claim 1 , wherein said tobacco product is selected from the group consisting of a tobacco blend, a pipe tobacco, a cigar tobacco, a cigarette tobacco, a chewing tobacco, a leaf tobacco, a shredded tobacco, and a cut tobacco.
3. The tobacco product of claim 1, wherein said tobacco product is selected from the group consisting of a cigarillo, a non- ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and a chewing tobacco.
4. The tobacco product of claim 3, wherein said tobacco product has an amount of
nornicotine of less than about 3 mg/g.
5. The tobacco product of claim 4, wherein said amount of nicotine is selected from the group consisting of 3.0 mg/g, 2.5 mg/g, 2.0 mg/g, 1.5 mg/g, 1.0 mg/g. 750 μg/g, 500 pg/g, 250 pg/g, 100 pg/g, 75 pg/g, 50 pg/g, 25 pg/g, 10 pg/g, 7.0 pg/g, 5.0 pg/g, 4.0 pg/g, 2.0 pg/g, 1.0 pg/g, 0.5 pg/g, 0.4 pg/g, 0.2 pg/g, 0.1 pg/g, 0.05 pg/g, 0.01 pg/g, and undetectable.
6. The tobacco product of claim 3, wherein said tobacco product has an amount of
N * -nitrosonornicotine (NNN) of less than about 10 pg/g.
7. The tobacco product of claim 6, wherein said amount of NNN is selected from the group consisting of 10 pg/g, 7.0 pg/g, 5.0 pg/g, 4.0 pg/g, 2.0 pg/g, 1.0 pg/g, 0.5 pg/g, 0.4 pg/g, 0.2 pg/g, 0.1 pg/g, 0.05 pg/g, 0.01 pg/g, and undetectable.
8. The tobacco product of claim 1 , wherein said cured tobacco is at least about 5% by dry weight of the total tobacco in said tobacco product.
9. The tobacco product of claim 8, wherein said cured tobacco has a dry weight percentage of said total cured tobacco selected from the group consisting of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%), at least about
60%, at least about 70%, at least about 80%, at least about 90%, and at least about 95%.
10. A method of producing a tobacco product, comprising:
a. providing cured tobacco from a tobacco plant, or part thereof, of a hybrid, a
cultivar, or a hybrid produced from said cultivar, wherein said hybrid is selected from the group consisting of K YI 4 X L8 SRC, NC7 SRC, and NCBH 129 SRC, and wherein said cultivar is selected from the group consisting of TN90 SRC, CMS TN90 SRC, KY14 SRC, CMS KY14 SRC, L8 SRC, NC775 SRC, CMS NC775 SRC, NC645 SRC, NC638 SRC, CMS NC638 SRC, TN86 SRC, and CMS TN86 SRC;
b. preparing a tobacco product from said cured tobacco, wherein a representative sample seed of said TN90 SRC, CMS TN90 SRC, KY14 SRC, CMS KYI 4 SRC. L8 SRC, NC775 SRC, CMS NC775 SRC, NC645 SRC, NC638 SRC, CMS NC638 SRC, TN86 SRC, CMS TN86 SRC, KY14 X L8 SRC, NC7 SRC, and NCBH129 SRC has been deposited at the ATCC with the following ATCC Accession Nos.: ATCC Accession No. PTA- 13567 for TN90 SRC, ATCC
Accession No. PTA- 13573 for CMS TN90 SRC, ATCC Accession No. PTA- 12031 1 for KYI 4 SRC, ATCC Accession No. PTA- 13571 for CMS KYI 4 SRC. ATCC Accession No. PTA- 13572 for L8 SRC. ATCC Accession No. P TA- 13569 for hybrid cultivar KYI 4 X L8 SRC, ATCC Accession No. PTA-120312 for NC775 SRC, ATCC Accession No. PTA- 13563 for CMS NC775 SRC, ATCC Accession No. PTA- 13566 for NC645 SRC. ATCC Accession No. PTA- 13564 for hybrid cultivar NC7 SRC, ATCC Accession No. PTA- 120313 for NC638 SRC, ATCC Accession No. PTA- 13568 for CMS NC638 SRC, ATCC Accession No. PTA- 13565 for TN86 SRC, ATCC Accession No. PTA- 13570 for CMS TN86 SRC, and ATCC Accession No. PTA- 13562 for hybrid cultivar NCBH 129 SRC.
1 1. A seed of tobacco cultivar selected from the group consisting of TN90 SRC, CMS TN90 SRC, KYI 4 SRC, CMS KY I 4 SRC, 1.8 SRC, NC775 SRC, CMS NC775 SRC, NC645 SRC, NC638 SRC, CMS NC638 SRC, TN86 SRC, CMS TN86 SRC. KYI 4 X L8 SRC, NC7 SRC, and NCB1 1129 SRC; wherein a representative sample seed of said TN90 SRC. CMS TN90 SRC, KY I 4 SRC, CMS KYI 4 SRC. L8 SRC, NC775 SRC, CMS NC775
SRC, NC645 SRC, NC638 SRC, CMS NC638 SRC, TN86 SRC, CMS TN86 SRC, KY I 4 X L8 SRC, NC7 SRC, and NCBH129 SRC has been deposited at the ATCC with the following ATCC Accession Nos.: ATCC Accession No. PTA- 13567 for TN90 SRC, ATCC Accession No. PTA- 13573 for CMS TN90 SRC, ATCC Accession No. ΡΤΛ- 12031 1 for KY14 SRC, ATCC Accession No. PTA- 13571 for CMS KYI 4 SRC, ATCC
Accession No. PTA- 13572 for 1,8 SRC, ATCC Accession No. PTA- 13569 for hybrid cultivar KYI 4 X L8 SRC, ATCC Accession No. PTA- 120312 for NC775 SRC, ATCC Accession No. PTA- 13563 for CMS NC775 SRC, ATCC Accession No. PTA- 13566 for NC645 SRC, ATCC Accession No. PTA- 13564 for hybrid cultivar NC7 SRC, ATCC Accession No. PTA-120313 for NC638 SRC, ATCC Accession No. P TA- 13568 for CMS
NC638 SRC, ATCC Accession No. PTA- 13565 for TN86 SRC, ATCC Accession No. PTA- 13570 for CMS TN86 SRC, and ATCC Accession No. PTA- 13562 for hybrid cultivar NCBH 129 SRC.
12. A tobacco plant, or part thereof, produced from said seed of claim 1 1.
13. The part of the plant of claim 12, wherein said part is selected from the group consisting of a harvested leaf, a pollen, an ovule, an embryo, a cotyledon, a hypocotyl, a meristematic cell, a protoplast, a root, a root tip, a pistil, an anther, a flower, a shoot, a stem, a pod, a petiole, and combinations thereof.
14. A harvested leaf of said tobacco plant of claim 12.
15. The harvested leaf of claim 14, wherein said leaf has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN).
16. The harvested leaf of claim 15, wherein said reduced amount of nornicotine and/or N- nitrosonornicotine (NNN) is reduced in a smoke stream produced from said leaf.
17. An F] progeny plant of a tobacco cultivar selected from the group consisting of TN90 SRC, CMS TN90 SRC, KY I 4 SRC, CMS KY I 4 SRC. 18 SRC, NC775 SRC, CMS NC775 SRC, NC645 SRC, NC638 SRC, CMS NC638 SRC, TN86 SRC, CMS TN86
SRC, KYI 4 X 18 SRC, NC7 SRC, and NCBH129 SRC; wherein a representative sample seed of said TN90 SRC, CMS TN90 SRC, KY I 4 SRC, CMS KYI 4 SRC. L8 SRC, NC775 SRC, CMS NC775 SRC. NC645 SRC, NC638 SRC, CMS NC638 SRC, TN86 SRC. CMS TN86 SRC, KYI 4 X 18 SRC, NC7 SRC. and NCBH 129 SRC has been deposited at the ATCC with the following ATCC Accession Nos.: ATCC Accession No.
PTA- 13567 for TN90 SRC. ATCC Accession No. PTA- 13573 for CMS TN90 SRC. ATCC Accession No. PTA-12031 1 for KY I 4 SRC, ATCC Accession No. PTA- 13571 for CMS KYI 4 SRC, ATCC Accession No. PTA- 13572 for 18 SRC, ATCC Accession No. PTA- 13569 for hybrid cultivar KY I 4 X 18 SRC, ATCC Accession No. PTA- 1203 12 for NC775 SRC. ATCC Accession No. PTA- 13563 for CMS NC775 SRC. ATCC Accession
No. PTA- 13566 for NC645 SRC. ATCC Accession No. PTA- 13564 for hybrid cultivar NC7 SRC, ATCC Accession No. PTA- 120313 for NC638 SRC, ATCC Accession No. PTA- 13568 for CMS NC63 SRC, ATCC Accession No. PTA- 13565 for TN86 SRC. ATCC Accession No. PTA- 13570 for CMS TN86 SRC. and ATCC Accession No. PTA- 13562 for hybrid cultivar NCBI 1129 SRC. 8. The Fl progeny plant of claim 17, wherein said Fl plant is male sterile.
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