WO2012118779A1

WO2012118779A1 - Tobacco inbred plants ncbex1f, ncbex1ms, and nc ex90

Info

Publication number: WO2012118779A1
Application number: PCT/US2012/026864
Authority: WO
Inventors: Ramsey S. Lewis; Ralph E. Dewey
Original assignee: North Carolina State University
Priority date: 2011-02-28
Filing date: 2012-02-28
Publication date: 2012-09-07
Also published as: CN103687480A; KR20140004760A; RU2013139871A; EP2680689A1; JP2014515596A; BR112013021515A2; CA2828488A1; US9096864B2; US20120216822A1

Abstract

The present invention provides tobacco inbred plants NCBEX1F and NCBEX1MS, and NC EX90. The present invention also provides parts of such plants and products made from those parts. The present invention also includes progeny of the provided plants including hybrids.

Description

TOBACCO INBRED PLANTS NCBEX1F, NCBEX1MS, AND NC EX90

Cross-Reference to Related Applications

This application claims the benefit under 35 U.S.C. § 1 19 of U.S. Provisional Application No. 61/447,443, filed February 28, 2011 (pending) and U.S. Provisional Application No. 61/546,573, filed October 13, 201 1 (pending), each of which is herein incorporated by reference in its entirety, including its respective sequence listing.

Incorporation of Sequence Listing

The Sequence Listing is hereby incorporated by reference in its entirety, including the file named P33831US02.txt, which is 28,672 bytes in size and was created on February 17, 2012, which is likewise herein incorporated by reference in its entirety.

Field of the Invention

The present invention provides tobacco inbred plants NCBEX1F, NCBEX1MS, and NC EX90. The present invention also provides parts of such plants and products made from those parts. The present invention also includes progeny of the provided plants including hybrids.

Background of the Invention

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.

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 metabohcally "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. 12:226-228). Burley tobaccos are particularly prone to genetic conversion, with rates as high as 20% per generation observed in some cultivars.

During the curing and processing of the tobacco leaf, a portion of the nornicotine is metabolized to the compound NN , a tobacco-specific nitrosamine (TSNA) that has been asserted to be carcinogenic in laboratory animals (Hecht and Hoffmann (1990) Cancer Surveys 8:273-294; Hoffmann et al. (1994) J. Toxicol. Environ. Health 41 : 1-52; Hecht (1998) Chem. Res. Toxicol. 1 1 :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," CORESTA 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 OF THE INVENTION

In an aspect, the present invention includes a seed of tobacco cultivar NCBEXIF, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718.

In another aspect, the present invention includes a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NCBEXIF, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718.

In a further aspect, the present invention includes a harvested leaf, or part thereof, of a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NCBEXIF, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 11718.

In an aspect, the present invention includes a harvested leaf, or part thereof, of a tobacco plant, produced by growing the seed of tobacco cultivar NCBEXIF, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, where the leaf has a reduced amount of nornicotine and/or N'- nitrosonornicotine (NNN) and/or more stably low nicotine conversion as compared to a leaf from TN 90 LC or most other commercial burley tobacco cultivars grown under similar conditions.

In an aspect, the present invention includes a harvested leaf, or part thereof, produced by growing the seed of tobacco cultivar NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718, where the leaf has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) and/or more stably low nicotine conversion as compared to-a leaf from TN 90 LC or most other commercial burley tobacco cultivars grown under similar conditions, and the reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) is reduced in a smoke stream produced from burning the leaf as compared to burning a leaf from TN 90 LC or most other commercial burley tobacco cultivars grown under similar conditions.

In a further aspect, the present invention includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, where the product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and cut tobacco.

In an aspect, the present invention includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718, 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.

In another aspect, the present invention includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, 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) and/or more stably low nicotine conversion as compared to a product prepared from TN 90 LC or most other commercial burley tobacco cultivars grown and processed under similar conditions.

In an aspect, the present invention includes a part of a tobacco plant, produced by growing the seed of tobacco cultivar NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1 1718, 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.

In another aspect, the present invention 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 NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, 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.

In an aspect, the present invention 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 NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, 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 and the regenerated plant has essentially all of the morphological and physiological characteristics of cultivar NCBEX IF when grown under the same environmental conditions.

In another aspect, the present invention includes a seed of tobacco cultivar NC EX90.

In an aspect, the present invention includes a tobacco plant, or part thereof, produced by growing a seed of tobacco cultivar NC EX90,

In a further aspect, the present invention includes a harvested leaf, or part thereof, of a tobacco plant, produced by growing the seed of tobacco cultivar NC EX90.

In another aspect, the present invention includes a harvested leaf, or part thereof, of a tobacco plant, produced by growing the seed of tobacco cultivar NC EX 90, where the leaf has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) and/or more stably low nicotine conversion as compared to a leaf from TN 90 LC or most other commercial burley tobacco cultivars grown under similar conditions.

In an aspect, the present invention includes a harvested leaf, or part thereof, of a tobacco plant, produced by growing the seed of tobacco cultivar NC EX90, where the leaf has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) and/or more stably low nicotine conversion as compared to a leaf from TN 90 LC or most other commercial burley tobacco cultivars grown under similar conditions, and the reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) is reduced in a smoke stream produced from burning the leaf as compared to burning a leaf from TN 90 LC or most other commercial burley tobacco cultivars when grown under similar conditions.

In a further aspect, the present invention includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC EX90, where the product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and cut tobacco.

In an aspect, the present invention includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC EX90, 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.

In another aspect, the present invention includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NC EX90, 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) and/or more stably low nicotine conversion as compared to a product prepared from TN 90 LC or most other commercial burley tobacco cultivars grown and processed under similar conditions.

In an aspect, the present invention includes a part of a tobacco plant, produced by growing a seed of tobacco cultivar NC EX90, 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.

In a further aspect, the present invention 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 NC EX90, 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,

In an aspect, the present invention 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 NC EX90, 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, and the regenerated plant has essentially all of the morphological and physiological characteristics of cultivar NC EX90 when grown under the same environmental conditions.

In another aspect, the present invention includes a seed of tobacco cultivar

NCBEX1MS, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1719.

In an aspect, the present invention includes a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1719.

In another aspect, the present invention includes a harvested leaf, or part thereof, of a tobacco plant produced by growing the seed of tobacco cultivar NCBEX1MS, a

representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1719.

In an aspect, the present invention includes a harvested leaf, or part thereof, of a tobacco plant, produced by growing the seed of tobacco cultivar NCBEX1MS, a

representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1719, where the leaf has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) and/or more stably low nicotine conversion as compared to a leaf from TN 90 LC or most other commercial burley tobacco cultivars grown under similar conditions.

In another aspect, the present invention includes a harvested leaf, or part thereof, of a tobacco plant, produced by growing the seed of tobacco cultivar NCBEX1MS, a

representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1719, where the leaf has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) and/or more stably low nicotine conversion as compared to a leaf from TN 90 LC or most other commercial burley tobacco cultivars when grown under similar conditions, and the reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) is reduced in a smoke stream produced from burning the leaf as compared to burning a leaf from TN 90 LC or most other commercial burley tobacco cultivars grown under similar conditions.

In a further aspect, the present invention includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar

NCBEX1MS, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11719, where the product is selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and cut tobacco.

In an aspect, the present invention includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar

NCBEX1MS, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1719, 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.

In another aspect, the present invention includes a tobacco product, prepared from a tobacco plant, or part thereof, produced by growing the seed of tobacco cultivar

NCBEX1MS, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1719, 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) and/or more stably low nicotine conversion as compared to a product prepared from TN 90 LC or most other commercial burley tobacco cultivars grown and processed under similar conditions.

In an aspect, the present invention includes a part of a tobacco plant, produced by growing the seed of tobacco cultivar NCBEX1MS, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1719, 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.

In another aspect, the present invention 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 NCBEX1MS, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1719, 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.

In an aspect, the present invention 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 NCBEX1MS, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1719, 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, and the plant has essentially all of the morphological and physiological characteristics of cultivar NCBEX1MS when grown under the same environmental conditions.

In an aspect, the present invention includes an Fi progeny plant of tobacco cultivar NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718.

In an aspect, the present invention includes an Fi progeny plant of tobacco cultivar NCBEX1MS, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11719.

In an aspect, the present invention includes an Fi progeny plant of tobacco cultivar NC EX90.

In another aspect, the present invention includes an Fi progeny plant of tobacco cultivar NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718, where the Fi plant is cytoplasmic male sterile (CMS).

In another aspect, the present invention 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 NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718.

In another aspect, the present invention 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 NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, where the plant of tobacco cultivar

NCBEX1F is the female parent.

In another aspect, the present invention 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 NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, where at least one tobacco plant is cytoplasmic male sterile (CMS).

In another aspect, the present invention includes an 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 a tobacco plant produced by growing the seed of tobacco cultivar NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, where at least one tobacco plant is cytoplasmic male sterile (CMS).

In art aspect, the present invention also includes a container of Fj progeny seeds produced by a method 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 NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718, where at least one tobacco plant is cytoplasmic male sterile (CMS).

In an aspect, the present invention includes an Fi progeny plant produced by growing a seed produced by a method 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 NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, and where at least one tobacco plant is cytoplasmic male sterile (CMS).

In an aspect, the present invention also includes a harvested leaf of an Fi progeny plant produced by growing a seed produced by a method 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 NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1 1718, and where at least one tobacco plant is cytoplasmic male sterile (CMS).

In another aspect, the present invention further includes a harvested leaf of an Fi progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) and/or more stably low nicotine conversion as compared to a leaf from TN 90 LC or most other commercial burley tobacco cultivars grown under similar conditions, produced by growing a seed produced by a method 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 NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, and where at least one tobacco plant is cytoplasmic male sterile (CMS).

In an aspect, the present invention also includes a harvested leaf of an F_t progeny plant, wherein the harvested leaf has a reduced amount of nornicotine and/or N'- nitrosonornicotine (NNN) in a smoke stream produced from burning the leaf as compared to burning a leaf from TN 90 LC or most other commercial burley tobacco cultivars grown under similar conditions, where the plant is produced by growing a seed produced by a method 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 NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, and where at least one tobacco plant is cytoplasmic male sterile (CMS).

In an aspect, the present invention 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 NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718, 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, and cut tobacco.

In an aspect, the present invention 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 NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718, 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.

In an aspect, the present invention 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 NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718, 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) and/or more stably low nicotine conversion as compared to a product prepared from TN 90 LC or most other commercial burley tobacco cultivars grown and processed under similar conditions.

In an aspect, the present invention includes a progeny plant of tobacco cultivar NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718.

In an aspect, the present invention includes a progeny plant of tobacco cultivar NCBEX1MS, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1719.

In an aspect, the present invention includes a progeny plant of tobacco cultivar NC

EX90.

In another aspect, the present invention includes a progeny plant of tobacco cultivar NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718, where the progeny plant is cytoplasmic male sterile (CMS).

In another aspect, the present invention 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 NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718.

In another aspect, the present invention 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 NCBEXIF, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718, where the plant of tobacco cultivar

NCBEXIF is the female parent.

In another aspect, the present invention 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 NCBEXIF, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, where at least one tobacco plant is cytoplasmic male sterile (CMS).

In another aspect, the present invention includes a progeny 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 NCBEXIF, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718, where at least one tobacco plant is cytoplasmic male sterile (CMS).

In an aspect, the present invention also includes a container of progeny seeds produced by a method 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 NCBEXIF, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, where at least one tobacco plant is cytoplasmic male sterile (CMS).

In an aspect, the present invention includes a progeny plant produced by growing a seed produced by a method 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 NCBEXIF, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, and where at least one tobacco plant is cytoplasmic male sterile (CMS). In an aspect, the present invention also includes a harvested leaf of a progeny plant produced by growing a seed produced by a method 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 NCBEXIF, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, and where at least one tobacco plant is cytoplasmic male sterile (CMS).

In another aspect, the present invention further includes a harvested leaf of a progeny plant having a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) and/or more stably low nicotine conversion as compared to a leaf from TN 90 LC or most other commercial burley tobacco cultivars grown under similar conditions, produced by growing a seed produced by a method 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 NCBEXIF, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, and where at least one tobacco plant is cytoplasmic male sterile (CMS).

In an aspect, the present invention also includes a harvested leaf of a progeny plant, wherein the harvested leaf has a reduced amount of nornicotine and/or N'-nitrosonornicotine (NNN) in a smoke stream produced from burning the leaf as compared to burning a leaf from TN 90 LC or most other commercial burley tobacco cultivars grown under similar conditions, where the plant is produced by growing a seed produced by a method 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 NCBEXIF, a

representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, and where at least one tobacco plant is cytoplasmic male sterile (CMS).

In an aspect, the present invention includes a tobacco product prepared from a progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a 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 NCBEXIF, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718, 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, and cut tobacco.

In an aspect, the present invention further includes a tobacco product prepared from a progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a 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 NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 11718, 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.

In an aspect, the present invention further includes a tobacco product prepared from a progeny tobacco plant, or part thereof, where the plant or part thereof is produced by growing a 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 NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718, 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) and/or more stably low nicotine conversion as compared to a product prepared from TN 90 LC or most other commercial burley tobacco cultivars grown and processed under similar conditions.

In an aspect, the present invention 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 NCBEX1MS, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1719.

In an aspect, the present invention includes a method of vegetatively propagating a plant of a tobacco cultivar comprising the steps of (a) culturing tissue capable of being propagated from a plant of a tobacco cultivar selected from the group consisting of

NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718, NCBEX1MS, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1 1719, and NC EX90 to obtain a proliferated shoot; and (b) rooting the proliferated shoots to obtain a rooted plantlet.

NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718, NCBEX1MS, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1 1719, and NC EX90 to obtain a proliferated shoot; (b) rooting the proliferated shoots to obtain a rooted plantlet; and (c) growing a plant from the rooted plantlet.

In an aspect, the present invention includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a tobacco cultivar selected from the group consisting of NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, NCBEX1MS, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1719, and NC EX90, 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 a tobacco cultivar selected from the group consisting of NCBEX1F,

NCBEX1MS, and NC EX90 to produce a backcross F₂ progeny seed; (d) growing the F₂ progeny seed and selecting a backcross F₂ progeny plant comprising the desired trait; 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 and essentially all of the

physiological and morphological characteristics of a tobacco cultivar selected from the group consisting of NCBEX1F, NCBEX1MS, and NC EX90, when grown under the same environmental conditions.

In an aspect, the present invention includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a tobacco cultivar selected from the group consisting of NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, NCBEX1MS, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1719, and NC EX90, 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 a tobacco cultivar selected from the group consisting of NCBEX1F,

NCBEX 1 MS, and NC EX90 to produce a backcross F₂ progeny seed; (d) growing the F₂ progeny seed and selecting a backcross F₂ progeny plant comprising the desired trait; 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 and essentially all of the

physiological and morphological characteristics of a tobacco cultivar selected from the group consisting of NCBEX1F, NCBEX1MS, and NC EX90 when grown under the same environmental conditions, where the trait is cytoplasmic male sterility (CMS).

In an aspect, the present invention includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a tobacco cultivar selected from the group consisting of NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, NCBEX1MS, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1 1719, and NC EX90, 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 Fj progeny plant with a plant of a tobacco cultivar selected from the group consisting of NCBEX1F,

NCBEX1 MS, and NC EX90 to produce a backcross F₂ progeny seed; (d) growing the F₂ progeny seed and selecting a backcross F₂ progeny plant comprising the desired trait; and (e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny that comprise essentially all of the physiological and

morphological characteristics of a tobacco cultivar selected from the group consisting of NCBEX1 F, NCBEX1MS, and NC EX90 when grown under the same environmental conditions, and the desired trait, where the trait is cytoplasmic male sterility (CMS) and the CMS trait is obtained from the cytoplasm of Nicotiana suaveolens.

In an aspect, the present invention includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a tobacco cultivar selected from the group consisting of NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, NCBEX1MS, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1 1719, and NC EX90, 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 Fj progeny plant with a plant of a tobacco cultivar selected from the group consisting of NCBEXIF,

NCBEXIMS, and NC EX90 to produce a backcross F₂ progeny seed; (d) growing the F₂ progeny seed and selecting a backcross F₂ progeny plant comprising the desired trait; and (e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny that comprise essentially all of the physiological and

morphological characteristics of a tobacco cultivar selected from the group consisting of NCBEXIF, NCBEXIMS, and NC EX90 when grown under the same environmental conditions, and the desired trait, where the trait is cytoplasmic male sterility (CMS) and the CMS trait is obtained from the cytoplasm of Nicotiana snaveolens, and where the second tobacco plant is TN 90 LC CMS.

In an aspect, the present invention includes a tobacco plant produced by a method comprising introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a tobacco cultivar selected from the group consisting of NCBEXIF, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, NCBEXIMS, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1719, and NC EX90, 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 ¥_\ progeny plant with a plant of a tobacco cultivar selected from the group consisting of NCBEXIF, NCBEXIMS, and NC EX90 to produce a backcross F₂ progeny seed; (d) growing the F₂ progeny seed and selecting a backcross F₂ progeny plant comprising the desired trait; 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 and essentially all of the physiological and morphological characteristics of a tobacco cultivar selected from the group consisting of NCBEXIF, NCBEXIMS, and NC EX90, when grown under the same environmental conditions.

In another aspect, the present invention includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a tobacco cultivar selected from the group consisting of NCBEXIF, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, NCBEXIMS, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11719, and NC EX90, with a plant of another 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 a tobacco cultivar selected from the group consisting of NCBEX1F, NCBEX1MS, and NC EX90 to produce a backcross F2 progeny plant seed; (d) growing the backcross F2 progeny plant seed into a backcross F2 progeny plant and selecting a F2 backcross progeny plant comprising the desired trait; and (e) repeating steps (c) and (d) one or more times in succession to produce a selected fourth or higher backcross progeny plant that comprises the desired trait and essentially all of the physiological and morphological characteristics of a tobacco cultivar selected from the group consisting of NCBEX1 F, NCBEX1MS, and NC EX90, when grown under the same environmental conditions.

In another aspect, the present invention includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a tobacco cultivar selected from the group consisting of NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, NCBEX1MS, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11719, and NC EX90, with a plant of another 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 a tobacco cultivar selected from the group consisting of NCBEXIF, NCBEXIMS, and NC EX90 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 (e) repeating steps (c) and (d) one or more times in succession to produce a selected fourth or higher backcross progeny plant that comprises the desired trait and essentially all of the physiological and morphological characteristics of a tobacco cultivar selected from the group consisting of NCBEXIF, NCBEXIMS, and NC EX90 when grown under the same environmental conditions.

In another aspect, the present invention includes a method of introducing a desired trait into a tobacco cultivar comprising: (a) crossing a plant of a tobacco cultivar selected from the group consisting of NCBEXIF, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718, NCBEXIMS, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1719, and NC EX90, with a plant of another 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 Fj progeny seed; (b) growing the F| progeny seed into an F[ progeny plant and selecting the Fi progeny plant having the desired trait;

(c) crossing the selected Fj progeny plant with a plant of a tobacco cultivar selected from the group consisting of NCBEXIF, NCBEXIMS, and NC EX90 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 selected from the group consisting of NCBEXI F, NCBEXIMS, and NC EX90, when grown under the same environmental conditions; and (e) repeating steps (c) and (d) one or more times in succession to produce a selected fourth or higher backcross progeny plant that comprises the desired trait and essentially all of the physiological and morphological characteristics of a tobacco cultivar selected from the group consisting of NCBEXIF, NCBEXIMS, and NC EX90, when grown under the same environmental conditions, where the plant has the desired trait of disease resistance.

In another aspect, the present invention includes a method for producing a tobacco plant having decreased and more stable nicotine conversion as compared to TN 90 LC or most other commercial burley tobacco cultivars grown and processed under similar conditions comprising: identifying a first tobacco plant having the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2; crossing the first tobacco plant with a second tobacco plant and collecting an Fj seed; selling a plant grown from the F] seed or crossing a plant grown from the Fi seed to a third tobacco plant to obtain a F₂ seed; and identifying a tobacco plant grown from the F₂ seed that is homozygous for the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

In an aspect, the present invention includes a method for producing a tobacco plant having decreased and more stable nicotine conversion as compared to TN 90 LC or most other commercial burley tobacco cultivars grown and processed under similar conditions comprising: identifying a first tobacco plant having the nucleotide sequence set forth in SEQ ID NO: T or SEQ ID NO: 2; crossing the first tobacco plant with a second tobacco plant and collecting an Fi seed; selfing a plant grown from the Fi seed or crossing a plant grown from the Fi seed to a third tobacco plant to obtain a F₂ seed; and identifying a tobacco plant grown from the F₂ seed that is homozygous for the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, where the second tobacco plant has the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

In an aspect, the present invention includes a method for producing a tobacco plant having decreased and more stable nicotine conversion as compared to TN 90 LC or most other commercial burley tobacco cultivars grown and processed under similar conditions comprising: identifying a first tobacco plant having the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2; crossing the first tobacco plant with a second tobacco plant and collecting an Fi seed; selfing a plant grown from the Fi seed or crossing a plant grown from the Fi seed to a third tobacco plant to obtain a F₂ seed; and identifying a tobacco plant grown from the F₂ seed that is homozygous for the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, where the second tobacco plant does not have the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 and the third tobacco plant is a tobacco plant having the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In an aspect, the present invention includes for producing a tobacco plant having decreased and more stable nicotine conversion as compared to TN 90 LC or most other commercial burley tobacco cultivars grown and processed under similar conditions comprising: identifying a first tobacco plant having the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2; crossing the first tobacco plant with a second tobacco plant and collecting an F] seed; selfmg a plant grown from the Fi seed or crossing a plant grown from the F| seed to a third tobacco plant to obtain a F₂ seed; and identifying a tobacco plant grown from the F₂ seed that is homozygous for the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, where the third tobacco plant is a tobacco plant having the nucleotide sequence set forth in SEQ ID NO: 1 and SEQ ID NO: 2.

In an aspect, the present invention includes a method for producing a tobacco plant having decreased and more stable nicotine conversion as compared to TN 90 LC or most other commercial burley tobacco cultivars grown and processed under similar conditions comprising: identifying a first tobacco plant having the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2; crossing the first tobacco plant with a second tobacco plant and collecting an Fi seed; selfmg a plant grown from the Fj seed or crossing a plant grown from the F] seed to a third tobacco plant to obtain a F₂ seed; and identifying a tobacco plant grown from the F₂ seed that is homozygous for the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, where the first tobacco plant is a plant of a tobacco cultivar selected from the group consisting of NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718, NCBEX1MS, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 11719, and NC EX90.

In an aspect, the present invention includes a method for producing a tobacco plant having decreased and more stable nicotine conversion as compared to TN 90 LC or most other commercial burley tobacco cultivars grown and processed under similar conditions comprising: identifying a first tobacco plant having the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2; crossing the first tobacco plant with a second tobacco plant and collecting an Fi seed; selfmg a plant grown from the Fi seed or crossing a plant grown from the Fi seed to a third tobacco plant to obtain a F₂ seed; and identifying a tobacco plant grown from the F₂ seed that is homozygous for the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, where the third tobacco plant is a plant of a tobacco cultivar selected from the group consisting of NCBEX1F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718, NCBEX1MS, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1719, and NC EX90.

In another aspect, the present invention includes a method of producing a plant of a tobacco cultivar selected from the group consisting of NCBEXIF, NCBEX1MS, and NC EX90 having an additional desired trait comprising the steps of: (a) introducing a trans gene conferring a desired trait into tissue capable of being propagated from a plant of a tobacco cultivar selected from the group consisting of NCBEXIF, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, NCBEX1MS, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11719, and NC EX90.

In another aspect, the present invention 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 NCBEXI F, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718, with a transgene where the transgene confers resistance to an herbicide. In some non-limiting examples, the herbicide is selected from the group consisting of imidazolinone,

cyclohexanedione, sulfonylurea, glyphosate, glufosinate, phenoxy proprionic acid, L- phosphinothricin, triazine and benzonitrile.

In another aspect, the present invention includes an herbicide resistant tobacco plant produced by a method comprising transforming a tobacco plant, or part thereof, produced by growing a seed of tobacco cultivar NCBEXIF, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, with a transgene where the transgene confers resistance to an herbicide selected from the group consisting of imidazolinone, cyclohexanedione, sulfonylurea, glyphosate, glufosinate, phenoxy proprionic acid, L-phosphinothricin, triazine and benzonitrile.

In another aspect, the present invention includes a method of producing a pest or insect resistant tobacco plant where the method comprises transforming a tobacco plant produced by growing a seed of tobacco cultivar NCBEXIF, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, with a transgene that confers pest or insect resistance.

In a further aspect, the present invention includes a pest or insect resistant tobacco plant produced by a method comprising transforming a tobacco plant produced by growing a seed of tobacco cultivar NCBEXIF, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718, with a transgene that confers pest or insect resistance.

In a further aspect, the present invention includes a pest or insect resistant tobacco plant produced by a method comprising transforming a tobacco plant produced by growing a seed of tobacco cultivar NCBEXIF, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718, with a transgene that confers pest or insect resistance. In some aspects, the transgene encodes a Bacillus

Ihuringiensis (BT) endotoxin.

In another aspect, the present invention includes a method of producing a disease resistant tobacco plant where the method comprises transforming a tobacco plant produced by growing a seed of tobacco cultivar NCBEXIF, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718, with a transgene that confers disease resistance.

In a further aspect, the present invention includes a disease resistant tobacco plant produced by a method comprising transforming a tobacco plant produced by growing a seed of tobacco cultivar NCBEXIF, a representative sample seed of the cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718, with a transgene that confers disease resistance.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 sets forth a cyp82e4 W329Stop nucleotide sequence.

SEQ ID NO: 2 sets forth a cyp82e5v2 W422Stop nucleotide sequence.

SEQ ID NO: 3 sets forth a cyp82e4 W329Stop amino acid sequence.

SEQ ID NO: 4 sets forth a cyp82e5v2 W422Stop amino acid sequence.

SEQ ID NO: 5 sets forth a CYP82E4 wild-type nucleotide sequence.

SEQ ID NO: 6 sets forth a CYP82E5v2 wild-type nucleotide sequence.

SEQ ID NO: 7 sets forth a CYP82E4 wild-type amino acid sequence.

SEQ ID NO: 8 sets forth a CYP82E5v2 wild-type amino acid sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts TN 90 LC certified plants and NC EX90 ("TN 90 RC") plants. Figure 2 sets forth nucleotide and amino acid sequences of the present invention. Figures 2A and 2B set forth the percentage of nicotine conversion for 50 plants of TN 90 LC and NCBEX1MS, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully hereinafter with reference to the accompanying drawings and specification, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein.

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 invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

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.

As used in the description of the invention 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.

Also 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").

The term "about," as used herein when referring to a measurable value such as an amount of a compound (e.g., an amount of nornicotine) and the like, is meant to encompass variations of ± 20%, ± 10%, + 5%, ± 1 %, ± 0.5%, or even ± 0.1% of the specified amount.

NC EX90

The present invention includes tobacco cultivars, and parts thereof, from NC EX90. In another aspect, the present invention includes a tobacco plant, or part thereof, produced by growing the seed of NC EX90. A plant of the present invention can include a plant with essentially all of the morphological and physiological characteristics of cultivar NC EX90, when grown under the same environmental conditions. While not being limited by process, NC EX90 is a result of the introduction of two mutated CYP82 genes in a Tennessee 90 Low Converter ("TN 90 LC") cultivar. The two genes are a mutated CYP82E4 gene recited as 325-6 #775 in Lewis et al. (Three nicotine demethylase genes mediate nornicotine biosynthesis 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)), and a mutated CYP82E5v2 recited in Lewis et al. (supra) as 325-6 #1-13 (SEQ ID NO: 2, which sets forth a cyp82e5v2 W422Stop, all references hereby incorporated by reference in their entirety). Both of these mutations result in truncated proteins. A cyp82e4 W329Stop and a cyp82e5\>2 W422Stop are introduced from a e4e5\e4e5 double mutant in a strong converter burley background, line DH98-325-6, as listed in Table 2 of Lewis et al. (supra) into a TN 90 LC background.

NC EX90 is generated by backcrossing with TN 90 LC five times as the recurrent parent and selling twice. NC EX90 is homozygous for both a cyp82e4 W329Stop and a cyp82e5v2 W422Stop. Again, not limited by any particular scientific theory _> a cyp82e4 W329Stop and a cyp82e5v2 W422Stop are recessive. A cyp82e4 W329Stop and a cyp82e5v2 W422Stop encode proteins with reduced or eliminated ability to convert nicotine or nornicotine. NC EX90 has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to TN 90 LC. NC EX90 exhibits low NNN and is not subject to conversion to high NNN's. (See, Figure 1)

NCBEXIMS

The present invention also provides tobacco cultivars, and parts thereof, from

NCBEXIMS, where representative sample seeds of this cultivar have been deposited with the ATCC under ATCC Accession No. PTA-1 1719. The present invention also includes a tobacco plant, or part thereof, produced by growing a seed of NCBEXIMS. A plant of the present invention can include a plant with essentially all of the morphological and

physiological characteristics of cultivar NCBEXIMS, when grown under the same environmental conditions. While not being limited by process, NCBEXIMS is a result of introducing the cyp82e4 W329Stop and cyp82e5v2 W422Stop mutations from NC EX90 BC₅F| into a TN 90 LC cytoplasmic male sterile ("CMS") plant by crossing NC EX90 BC5F 1 as the male parent with TN 90 LC CMS to prepare CMS Fi progeny plants.

The CMS F, progeny plants of the BC5F1 x TN 90 LC CMS cross are male sterile. A plurality of BC₅F] x TN 90 LC CMS Fj plants (e.g. , CMS F] progeny plants) are screened for the cyp82e4 W329Stop and cyp82e5v2 W422Stop mutations and crossed as the female parent to NC EX90 to prepare BC₇F] CMS progeny. BC7F1 CMS progeny homozygous for the cyp82e4 W329Stop and the cyp82e5v2 W422Stop mutations are identified by genotyping and designated as NCBEX1MS. NCBEX1MS has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to TN 90 LC. NCBEX1MS exhibits low NN and is not subject to conversion to high NNN's.

NCBEXIF

The present invention also provides tobacco cultivars, and parts thereof, from

NCBEXIF, representative sample seeds of this cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718. The present invention also includes a tobacco plant, or part thereof, produced by growing a seed of NCBEXIF. A plant of the present invention can further include a plant with essentially all of the morphological and physiological characteristics of cultivar NCBEXIF, when grown under the same

environmental conditions. NCBEXIF is the result of seven backcrosses with TN 90 LC as the recurrent parent, followed by two rounds of selfmg with selection for homozygosity for the cyp82e4 W329Stop and the cyp82e5v2 W422Stop mutations to yield BC7F3 plants.

NCBEXIF has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to TN 90 LC. NCBEXIF exhibits low NNN and is not subject to conversion to high NNN's,

Other plants

A progeny plant of the present application can be a plant of ¥_\, F₂, F₃, F₄ or later generation obtained by either crossing two parental plants or selfmg one plant.

Under similar conditions as defined in the present application can be under similar environmental conditions or under similar laboratory conditions.

The present invention 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 NCBEXIF. In one aspect, the NCBEXIF is the male parent plant. In another aspect, the NCBEX1MS is the female parent plant. One aspect of the present invention provides tobacco plants that are homozygous at both a cyp82e4 and a cyp82e5v2 loci for SEQ ID NO: 1 and SEQ ID NO: 2, respectively, which share a genetic background that is greater than 75%, 80%, 85%, 90%, 95%, 98%, or 99% TN90 or TN90 LC. In one aspect, approximate or greater than 50%, 75%, or 100% of a progeny's genetics is provided by a plant of the present invention that is homozygous at both a cyp82e4 and a cyp82e5v2 loci for SEQ ID NO: 1 and SEQ ID NO: 2. In one aspect, a plant of the present invention has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to TN 90 LC. In another ■ aspect, a plant of the present invention exhibits low NNN and is not subject to conversion to high NNN's. In one aspect, a plant of the present invention is the progeny plant of a female or male parent plant that is Fusarhim wilt resistant.

In one aspect, a plant of the present invention is a medium-late maturing variety with moderately high yield potential. In another aspect, a plant of the present invention offers a broad range of important agronomic characteristics. In a further aspect, a plant of the present invention 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 invention 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 invention, such as NCBEX1F, NCBEX1MS, and NC EX90, lacks Fusarhim wilt resistance. In another aspect, a plant of the present invention is Fusarhim wilt resistant.

In an aspect, the plants of the present invention have reduced or eliminated ability to convert nicotine to nornicotine. In an aspect, the percentage nicotine conversion is less than about 75%, about 70%, about 60%, about 50%, or about 25% of that found in TN 90 LC or most other commercial burley tobacco cultivars grown and processed under similar conditions. The nicotine conversion in plants of the present invention, including NCBEX1F, NCBEX1MS, and NC EX90, can be less than about 4%, about 3.5%, about 3%, about 2.5%, about 2%, about 1.5%, about 1%, about 3-1%, about 3-0.5%, or about 2-0.5%. In a preferred aspect, the percentage nicotine conversion is less than about 25%o, about 10%, about 5%, or about 2% of that found in TN90 without a cyp82e4 W329Stop and a cyp82e5v2 W422Stop. In an aspect, the tobacco plants of the present invention have a nicotine conversion rate of about 3.5, about 3.25, about 3.0 or about 2.75% or less. In another aspect, the nicotine conversion rate of tobacco plants of the present invention 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. In another aspect, the nicotine conversion rate of tobacco plants of the present invention 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. In another aspect, the nicotine conversion rates can be from about 0.5 to 0.9%, 0.5 to 1.5%, 0.5 to 2.0%, 0.5 to 2.5%, 0.5 to 2.75%, and 0.5 to 3.0%. In another aspect, the nicotine conversion rates can be from about 1.0 to 1.5%, 1.0 to 1.75%,1.0 to 2.0%, 1.0 to 2.5%, 1.0 to 2.75%, and 1.0 to 3.0%. In another aspect, the nicotine conversion rate in a plant of the present invention may 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%. In an aspect, the tobacco plants of the present invention have a nicotine conversion rate of 3.5, 3.25, 3.0 or 2.75% or less.

In another aspect, the tobacco plants of the present invention typically have a reduced amount of nornicotine of less than about 0.10 % dry weight as compared to TN 90 LC or most other commercial burley tobacco cultivars grown and processed under similar conditions. 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, 0.0001 % dry weight, or undetectable. 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, 0.0001 % dry weight. In another aspect, the nornicotine content in such plants can be from about 1.2-1.0, 0.7-0.5, 0.4-0.2, 0.1-0.075, 0.05-0.025, 0.01-0.0075, 0.005-0.0025, 0.001-0.00075, 0.0005-0.00025, or 0.0005-0.0001 % dry weight. In a plant of the present invention, the nornicotine is a relatively small percentage of total alkaloids in the plant compared to a commercial seedlot of TN90 LC. The nornicotine in a plant of the present invention may be 2-1 %, less than 3%, about 2%, about 1.5%, about 1%, or 0.75%> percentage of total alkaloids.

Tobacco products having a reduced amount of nitrosamine content and/or more stably low nicotine conversion as compared to a product prepared from TN 90 LC or most other commercial burley tobacco cultivars grown and processed under similar conditions can be manufactured using tobacco plant material from plants and plant parts of the present invention. The tobacco product produced from a tobacco plant of this invention can have an amount of nornicotine of less than about 3 mg/g, For example, the nornicotine content in such a product can be about 3.0 mg/g, 2.5 mg/g, 2.0 mg/g, 1.5 mg/g, 1.0 mg/g, 750 μg/g, 500 pg/g, 250 pg/g, 100 pg/g, 75 pg/g, 50 pg/g, 25 pg/g, 10 pg/g, 7.0 pg/g, 5.0 pg/g, 4.0 pg/g, 2.0 pg/g, 1.0 pg/g, 0.5 pg/g, 0.4 pg/g, 0.2 pg/g, 0.1 pg/g, 0.05 pg/g, 0.01 pg/g, or undetectable. In some aspects, a tobacco product produced from a tobacco plant of this invention can have a 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. The percentage of secondary alkaloids relative to total alkaloid content contained in a plant of the present invention may not be statistically different than from a commercial seedlot of TN90 LC.

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 P OC 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, 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.

Tobacco plants of the present invention that are homozygous for cyp82e4 W329Stop and cyp82e5v2 W422Stop alleles have a reversion rate that is statistically significantly lower than corresponding control low-converter plants having wild type nicotine demethylase CYP82E4 and E5 genes. In addition, homozygous CYP82E4 and CYP82E5 double mutant tobacco plants have a percent conversion of nicotine to nornicotine of less than about 2.0%, e.g. , undetectable to about 2.0%, about 1.0 to 2.0%, about 0.8 to 1.8%, about 0.8 to 2.0%, or about 1.0 to 2.0%.

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 invention is 50, 40, 30, 20, 10 percent of a sample from an individual plant grown from a commercial seedlot of TN 90.

In an aspect, the tobacco plants of the present invention have a USDA quality index of about 73, about 72, about 71, about 70, about 69, about 68, about 67 or about 66. In an aspect, the tobacco plants of the present invention 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, In another aspect, tobacco plants of the present invention can have a quality index in the range of, about 60-65, about 60-70, about 62.5-65, about 62.5-70, or about 65-70, or any range therein. A plant of the present invention, including NCBEXIMS, NCBEXIF, and NC EX90, can have any yield, 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.

In another aspect, the present invention also provides for a plant grown from the seed of a NCBEXIF, NCBEXIMS, or NC EX90 plant in which alkaloids obtained from tobacco plants grown for the seed have decreased nornicotine and/or more stably low nicotine conversion as compared to TN 90 LC or most other commercial burley tobacco cultivars grown and processed under similar conditions, 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-1 1718 for NCBEXIF and ATCC Accession No. PTA-1 1719 for NCBEXIMS.

An aspect of the present invention provides for parts of the cultivars NCBEXIF, NCBEX IMS, and NC EX90. For example, as used herein, a plant part includes but is not limited to leaves, pollen, embryos, cotyledons, hypocotyls, roots, root tips, anthers, flowers, ovules, shoots, stems, stalks, pith and capsules, petioles, pods, tissue culture comprising tissue, callus, cells or protoplasts of the cultivars NCBEXIF, NCBEXIMS, and/or NC EX90. In another aspect, the present invention provides for parts from hybrids having cultivars NCBEXI F, NCBEXIMS, and/or NC EX90 as parents or ancestors, and NCBEXIF,

NCBEXIMS, and/or NC EX90 derived tobacco plants. In yet another aspect, the present invention provides for parts from genetically modified (e.g., by conventional breeding or genetic engineering techniques) forms of the foregoing plants and tissue culture.

Additional aspects of the present invention provide products comprising tobacco wherein the tobacco further comprises tobacco from the plants of the present invention, and parts thereof. Other aspects of the invention provide cured plant leaves and other plant parts from the plants of the present invention. Accordingly, in some aspects the cured plant parts 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, capsules and the like, and combinations thereof.

Thus, in some aspects, the present invention provides a cured tobacco comprising the leaves of the tobacco plant designated NCBEXI F, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718. In another aspect the present invention provides a cured tobacco comprising the leaves of the tobacco plant designated NCBEXIMS, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1719. In yet another aspect, the present invention provides a cured tobacco comprising the leaves of the tobacco plant designated NC EX90.

In an aspect, the present invention provides a cured tobacco comprising the stems of the tobacco plant designated NCBEXIF, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718. In another aspect the present invention provides a cured tobacco comprising the stems of the tobacco plant designated NCBEX1MS, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1719. In yet another aspect, the present invention provides a cured tobacco comprising the stems of the tobacco plant designated NC EX90.

In still other aspects, the present invention provides a cured tobacco comprising the leaves and stems of the tobacco plants designated NCBEXIF, NCBEX1 MS, and/or NC EX90, representative sample seeds of these cultivars having been deposited with the ATCC under ATCC Accession No. PTA-1 1718 for NCBEXIF and ATCC Accession

No .PTA- 1 1719 for NCBEX IMS.

The present invention also provides a container of NCBEXI F, NCBEX1 MS, or NC EX90 seeds or other seeds of the present invention (e.g., hybrids, inbreds, and the like) in which alkaloids obtained from tobacco plants grown from greater than 50% of the seeds have decreased nornicotine and/or more stably low nicotine conversion as compared to TN 90 LC or most other commercial burley tobacco cultivars grown under similar conditions. In another aspect, alkaloids obtained from NCBEXIF, NCBEX1MS, or NC EX90 plants or other plants of the present invention grown from greater than 55%, 60%, 65%, 70%, 75%, 80%, 85%o, 90%, 95% of the seeds in the container have decreased nornicotine and/or more stably low nicotine conversion as compared to TN 90 LC or most other commercial burley tobacco cultivars grown under similar conditions, representative sample seeds of cultivars NCBEXI F and NCBEX IMS having been deposited with the ATCC under ATCC Accession No. PTA-1 1718 for NCBEXIF and ATCC Accession No. PTA-1 1719 for NCBEX IMS.

A container of NCBEXIF, NCBEX1MS, or NC EX90 seeds or other seeds of the present invention 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 sample seeds of NCBEXIF and NCBEXIMS cultivars having been deposited with the ATCC under ATCC Accession Nos. PTA- 1 1718 and PTA- 1 1719, respectively.

Containers of NCBEXI F, NCBEXIMS, or NC EX90 seeds or other seeds of the present invention 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 sample seeds of these cultivars having been deposited with the ATCC. Representative sample seeds of NCBEXIF and NCBEXIMS cultivars have been deposited with the ATCC under ATCC Accession Nos. PTA-1 1718 and PTA-1 1719, respectively.

In another aspect, the present invention also provides a container of NCBEXIF, NCBEXIMS, or NC EX90 seeds in which greater than 50% of NCBEXIF, NCBEXIMS, or NC EX90 seeds or other seeds of the present invention have decreased nornicotine and/or more stably low nicotine conversion as compared to TN 90 LC or most other commercial burley tobacco cultivars grown and processed under similar conditions. Representative sample seeds of NCBEXIF and NCBEXIMS cultivars have been deposited with the ATCC under ATCC Accession Nos. PTA-1 1718 and PTA- 1 1719, respectively.

In one aspect, the present invention provides a seed of a NCBEX I F, NCBEXIMS, or NC EX90 plant or other plant of the present invention in which a plant grown from the seed is male sterile. Representative sample seeds of NCBEXIF and NCBEXI MS cultivars have been deposited with the ATCC under ATCC Accession Nos. PTA-1 1718 and PTA-1 1719, respectively.

Tobacco material, including without limitation any plant parts, for example leaves, midveins, and stalks, obtained from the tobacco cultivars, lines, varieties or hybrids of the present invention 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.

Tobacco products derived from plants of the present invention 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 may be pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and cut tobacco.

In an aspect, the tobacco product of the present invention can be a blended tobacco product. In other aspects of the invention, the tobacco product of the present invention can be a reduced nicotine tobacco product. In still other aspects, the tobacco product of the present invention can be a blended tobacco product with reduced nicotine content. Thus, the tobacco product of the present invention can be a blended reduced nicotine tobacco product. Tobacco product material comprises a blend of tobacco materials from the present invention, 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, based on the dry weight of the tobacco material. US 2008/0245377 is herein incorporated by reference for blend mixtures in its entirety.

In an aspect, tobacco products having a reduced amount of nitrosamine content can be manufactured using tobacco plant material from plants and/or plant parts of the present invention. The tobacco product typically has a reduced amount of nornicotine of less than about 3 mg/g as compared to a product prepared from TN 90 LC or most other commercial burley tobacco cultivars grown and processed under similar conditions. 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 ^ig/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. The tobacco product comprising tobacco material from a plant or plant part thereof of this invention typically has a reduced amount of N N of less than about 10 pg/g as compared to a product prepared from TN 90 LC or most other commercial burley tobacco cultivars grown and processed under similar conditions. For example, the nornicotine 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. The percentage of secondary alkaloids relative to total alkaloid content contained in a plant of the present invention may not be statistically different than from a commercial seedlot of TN90 LC.

A tobacco plant of the present invention designated NCBEX1F, NCBEX1MS, or NC EX90, carrying cyp82e4 W329Stop and cyp82e5v2 W422Stop alleles can be used in a plant breeding program to create useful lines, cultivars, varieties, progeny, inbreds, and/or hybrids. Thus, in some aspects, an F_{l s} F₂, F₃, or later generation tobacco plant containing cyp82e4 W329Stop and cyp82e5v2 W422Stop alleles is crossed with a second Nicotiana plant, and progeny of the cross are identified in which the cyp82e4 W329Stop and cyp82e5v2

W422Stop alleles are present. It will be appreciated that the second Nicotiana plant will be TN90 or TN90 LC, optionally with an additional desirable trait. In some aspects the additional desirable trait can be herbicide resistance mentioned below. It will also be appreciated that the second TN90 or TN90 LC Nicotiana plant can contain cyp82e4

W329Stop and cyp82e5v2 W422Stop alleles.

In still other aspects, methods of the present invention 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 invention, such as a male sterile hybrid of the present invention. Either the male sterile pollen acceptor plant or the pollen donor plant has at least one mutant allele, preferably two, at a nicotine demethylase locus, such as cyp82e4 W329Stop and cyp82e5v2 W422Stop. In an aspect, both alleles at each nicotine demethylase locus are mutant alleles, making the plant homozygous for cyp82e4 W329Stop and cyp82e5v2 W422Stop.

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 and cyp82e5v2 W422Stop alleles with an agronomically desirable genotype. Plants in the F₂ or backcross generations can be screened using a marker developed from cyp82e4 W329Stop or cyp82e5v2 W422Stop alleles or a fragment thereof, using one of the techniques known in the art or listed herein. Plants identified as possessing cyp82e4 W329Stop and cyp82e5v2 W422Stop 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 invention can be NCBEX1F or NC EX90. Other breeding techniques can be found, for example, in Wernsman, E. A., and Rufty, R. C. 1987. Chapter Seventeen. Tobacco. Pages 669-698 In: Cultivar Development. Crop Species. W. H. Fehr (ed.), MacMillan Publishing Go., Inc., New York, N.Y., incorporated herein by reference in their entirety. Nicotiana species which exhibit breeding compatibility with Nicotiana tabac m include Nicotiana amplexicaulis, PI 271989; Nicotiana benthamiana PI 555478; Nicoticma bigelovii PI 555485; Nicotiana debneyi; Nicotiana excelsior PI 224063; Nicotiana ghttinosa PI 555507; Nicotiana goodspeedii PI 241012; Nicotiana gossei PI 230953; Nicotiana hesperis PI 271991 ; Nicotiana knightiana PI 555527; Nicotiana maritime! PI 555535;

Nicotiana megalosiphon PI 555536; Nicotiana nudicaiilis 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 tomeniosa 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 entirety.

The result of a plant breeding program using the tobacco plants described herein includes useful lines, cultivars, varieties, progeny, inbreds, and hybrids. As used herein, the term 'variety' refers to a population of plants that share constant characteristics which separate them from other plants of the same species. A variety is often, although not always, sold commercially. While possessing one or more distinctive traits, a variety is further characterized by a very small overall variation between individuals within that 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 variety can be essentially derived from another 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 variety is 'essentially derived' from an initial variety if: a) it is predominantly derived from the initial variety, or from a variety that is predominantly derived from the initial variety, while retaining the expression of the essential characteristics that result from the genotype or combination of genotypes of the initial variety; b) it is clearly distinguishable from the initial variety; and c) except for the differences which result from the act of derivation, it conforms to the initial variety in the expression of the essential characteristics that result from the genotype or combination of genotypes of the initial 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 variety, backcrossing, or transformation. A 'line' as distinguished from a 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.

Hybrid tobacco varieties can be produced by preventing self-pollination of female parent plants (i.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 Fi seed harvested.

Plants can be used to form single-cross tobacco F) hybrids. In such an aspect, the plants of the parent varieties can be grown as substantially homogeneous adjoining populations to facilitate natural cross-pollination from the male parent plants to the female parent plants. The Fi seed formed on the female parent plants is selectively harvested by conventional means. One also can grow the two parent plant varieties in bulk and harvest a blend of F| hybrid seed formed on the female parent and seed formed upon the male parent as the result of self-pollination. Alternatively, three-way crosses can be carried out wherein a single-cross F] 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.

In some aspects, successful crosses yield F| plants that are fertile, have cyp82e4 W329Stop and cyp82e5v2 W422Stop alleles, and can be backcrossed with one of the parents, such as NCBEX1F or NC EX90 if desired. In some aspects, a plant population in the F₂ generation is screened for cyp82e4 W329Stop and cyp82e5v2 W422Stop 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 4 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 and stable nicotine conversion phenotype as NCBEX1F. 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.

In one aspect, a Fi progeny is the result of a cross between NCBEX1F and

NCBEXIMS to generate Fi 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 ufty, 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.

The present invention further provides methods of producing a tobacco plant by crossing one of cultivars NCBEX1F, NCBEXIMS, and/or NC EX90 with itself or a different tobacco line. The invention further relates to methods for producing other tobacco cultivars or breeding lines derived from cultivars NCBEX1F, NCBEXIMS, and/or NC EX90 by crossing a plant of a cultivar of NCBEX1F, NCBEXIMS, and/or NC EX90 with a second tobacco plant and growing the progeny seed to yield a NCBEX1F-, NCBEXIMS-, or NC EX90-derived tobacco plant. An additional aspect of the present invention provides a method for producing a tobacco plant that contains in its genetic material one or more transgenes, comprising crossing cultivars of the present invention with a second tobacco plant containing one or more transgenes to produce progeny that 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 cultivars NCBEXIF, NCBEXIMS, and/or NC EX90 transformed with one or more transgenes.

The invention further provides for the vegetative propagation of a plant of cultivars NCBEXIF, NCBEXIMS, and/or NC EX90, hybrids and progeny thereof. In one aspect, the invention provides for a method of vegetatively propagating a plant of a tobacco cultivar comprising cultivating tissue capable of being propagated from a plant of a plant of cultivars NCBEXIF, NCBEXIMS, and/or NC EX90, to obtain a proliferated shoot and rooting the proliferated shoots to obtain a rooted plantlet. In another aspect, the plant tissue may be from an F, hybrid of a plant of cultivars NCBEXIF, NCBEXIMS, and/or NC EX90. In an aspect, the plant tissue may be from an F₂, F₃, F₄ or later progeny plant obtained by breeding a plant of cultivars NCBEX1F, NCBEX1MS, and/or NC EX90.

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, PCR 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.

It is understood that a tobacco plant of the present invention, including NCBEX1F, NCBEX1MS and/or NC EX90, can be transformed by a genetic construct or transgene using a technique known in the art. Without limitation, an example of a desired trait is 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. , a small, medium, or a large stalk); or leaf number per plant (e.g. , a small (e.g. , 5-10 leaves), medium (e.g. , 1 1 - 15 leaves), or large (e.g. , 16-21) number of leaves), or any combination thereof. Any plant of the present invention can be used as a basis for tissue culture, regenerated, transformed, or a combination of any of these. In an aspect, a plant of the present invention derived by tissue culture, transformation, or both has essentially all of the morphological and physiological characteristics of cultivar NCBEX1F,

NCBEX1MS or NC EX90 under similar conditions.

Having now generally described the invention, 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 invention, unless specified. EXAMPLES

Example 1

Breeding of homozygous c p82e4 W329Stop and cyp82e5v2 W422Stop mutant plants into the TN 90 LC background

NC EX90 is a backcross-derived version of burley tobacco cultivar TN 90 LC carrying introduced deleterious mutations in cyp82e4 and cyp82e5v2, two genes previously documented to encode for nicotine demethylase enzymes (Lewis et al., supra). To prepare NC EX90 an individual plant grown from a commercial seedlot of TN 90 LC is selected and initially crossed with plant GH08B-14 in a greenhouse. GH08B-14 is a burley tobacco cultivar heterozygous for the cyp82e4 W329Stop and the cyp82e5v2 W422Stop mutations described by Lewis et al. (2010). A plurality of Fi plants are screened for the presence of the cyp82e4 W329Stop and the cyp82e5v2 W422Stop mutations. Individual Fi plants are selected and backcrossed to TN 90 LC in a greenhouse to produce BCiFj progeny. A plurality of BCiFi progeny are screened and an individual plant heterozygous for the cyp82e4 W329Stop and cyp82e5v2 W422Stop mutations is identified. The heterozygous selected BCiFj plant is backcrossed to TN 90 LC in a greenhouse to produce BC₂Fi seed. A plurality of BC₂Fi plants are screened for the presence of the cyp82e4 W329Stop and cyp82e5v2 W422Stop mutations to identify a heterozygous progeny plant for a subsequent round of backcross breeding. Using this backcross procedure, individual heterozygous plants having the cyp82e4 W329Stop and cyp82e5v2 W422Stop mutations are identified in the BC3F1, BC4F1, and BC5F1 progeny.

To produce plants homozygous for the cyp82e4 W329Stop and cyp82e5v2 W422Stop mutations, BC5F1 progeny plants are screened for the cyp82e4 W329Stop and cyp82e5v2 W422Stop mutations to identify heterozygous plants. Individual plants heterozygous for the cyp82e4 W329Stop and cyp82e5v2 W422Stop mutations are self-pollinated to produce BC₅F₂ seed. A plurality of BC₅F₂ progeny are genotyped to identify individuals homozygous for both of the cyp82e4 \V329Stop and the cyp82e5v2 W422Stop mutations. Four individual BC₅F₂ progeny plants are self-pollinated to produce four BC5F3 progeny lines. BC5F3 are homozygous for the CYP82E4 W329Stop and CYP82E5v2 W422Stop mutations.

Identification of BC5F3 progeny lines with desirable traits

Plants from the four BC5F3 progeny lines are grown in a randomized complete block design with three replications for evaluation of cured leaf chemistry, yield, and physical quality at three North Carolina field research locations during 2010 (Waynesville, Laurel Springs, and Reidsville). Each replicated block is a 20-plant plot. Plants are stalk cut at maturity, air cured and evaluated by a former USDA tobacco grader. Plot weights are used to determine per acre yields. Fifty gram composite leaf samples are collected from each plot and analyzed for percent nicotine, nornicotine, anatabine, and anabasine by gas

chromatography. BC5F3 progeny line #3 is identified with superior yield, high grade and desirable alkaloid content and designated 'NC EX90.'

Analysis of NC EX90

Results of gas chromatography alkaloid analysis are presented in Table 1.

Analysis with single degree of freedom CONTRAST statements are performed using the PROC GLM function of SAS. This line exhibits significantly lower levels of nornicotine {P < 0.0001) and nicotine conversion {P = 0.0002) as compared to a commercial seedlot of TN 90 LC (Table 1). The ratio of secondary alkaloids to total alkaloids are significantly lower for NC EX90 relative to TN 90 LC (P = 0.0071). The two lines are not significantly different for nicotine, anatabine, anabasine, yield, or cured leaf quality.

Table 1. Comparisons between TN 90 LC and NC EX90 (TN 90 LC + e4e4 + e5e5) for alkaloid determinations, yield, and cured leaf quality. Means are from three 2010 North Carolina environments. The experimental design at each location was a randomized complete block design with three replications.

Ratio

Total Secondary

Nicotine Nornicotine Anatabine Anabasine Alkaloids Nicotine Alkaloids :

(% dry (% dry (% dry (% dry (% dry Conversion Total Yield Quality

Entry weight) weight) weight) weight) weight) (%) Alkaloids (lbs/A) Index

TN 90 LC 3.579 0.158 0.216 0.022 3.980 3.918 0.096 2403.3 62.59

NC EX90 3.571 0.076 0.221 0.022 3.890 2.043 0.081 2454.5 65.70

P-value^a 0.9622 <0.0001 0.7560 0.7059 0.6620 0.0002 0.0071 0.8856 0.5514 ^a P-values were obtained from single degree of freedom CONTRAST statements executed by PROC GLM of SAS.

Example 2

Preparation of inbred line NCBEXIF

To prepare inbred line NCBEXIF, two additional rounds of backcrossing of individual BC5F1 progeny plants prepared in Example 1 above are performed. A plurality of BC5F1 plants are screened for the presence of the cyp82e4 W329Stop and the cyp82e5v2 W422Stop mutations to identify a heterozygous progeny plant for a subsequent round of backcross breeding. Using this backcross procedure, individual heterozygous plants having the cyp82e4 W329Stop and the cyp82e5v2 W422Stop mutations are identified in the BC6F] and BC7F₁ progeny.

To produce plants homozygous for the cyp82e4 W329Stop and the cyp82e5v2 W422Stop mutations, BC7F1 progeny plants are screened for the cyp82e4 W329Stop and the cyp82e5v2 W422Stop mutations to identify heterozygous plants. Individual plants heterozygous for the cyp82e4 W329Stop and the cyp82e5v2 W422Stop mutations are self- pollinated to produce BC₇F₂ seed. A plurality of BC₇F₂ progeny are genotyped to identify individuals homozygous for both of the cyp82e4 W329Stop and the cyp82e5v2 W422Stop mutations. Individual BC₇F₂ progeny plants are self-pollinated to produce individual BC₇F₃ progeny lines. The NCBEXIF progeny lines are homozygous for the cyp82e4 W329Stop and the cyp82e5v2 W422Stop mutations.

Example 3

Preparation of cytoplasmic male sterile lines

To prepare a cytoplasmic male sterile (CMS) line, a BC5F1 progeny plant prepared as described in Example 1 above that is heterozygous for the cyp82e4 W329Stop and the cyp82e5v2 W422Stop mutations is selected and crossed as the pollen parent to a proprietary CMS sterile line of the North Carolina State University designated TN 90 LC CMS. TN 90 LC CMS is prepared by crossing a TN 90 LC plant with a plant of tobacco species Nicoiicma suaveolens. The Fj progeny plant is backcrossed nine times to TN 90 LC to produce BC9F1 plants and designated TN 90 LC CMS. The TN 90 LC CMS line is maintained by backcrossing to TN 90 LC.

The F, progeny plants of the BC₅F, x TN 90 LC CMS are male sterile. A plurality of BC5F 1 x TN 90 CMS Fi plants (e.g., the Fi progeny plants) are screened for the cyp82e4 W329Stop and the cyp82e5v2 W422Stop mutations and crossed as the female parent to TN 90 LC to prepare BC7F1 CMS progeny. BC7F 1 CMS progeny heterozygous for the cyp82e4 W329Stop and the cyp82e5v2 W422Stop mutations are identified by genotyping. Two rounds of crossing to NC EX90 are performed to prepare BC7F3 CMS plants and designated as line NCBEX1MS. To maintain the line, plants of line NCBEX1MS plants are pollinated with a NCBEXIF plant.

Example 4

Demonstration of trait stability in nicotine conversion for NCBEX1MS relative to TN 90LC

Leaves from 50 plants for NCBEX1MS and TN90LC are collected using the "LC" method as disclosed in Jack et al. Implications of reducing nornicotine accumulation in burley tobacco: Appendix A - The LC Protocol. Rec. Adv. Tob. Sci. 2007, 33, 58-79. Leaves are air cured and analyzed for alkaloid profiles using gas chromatography. See Figures 2A and 2B and Table 2.

Analysis with single degree of freedom CONTRAST statements are performed using the PROC GLM function of SAS. This line exhibits significantly lower levels of nornicotine (P < 0.05) and nicotine conversion (P < 0.05) as compared to a commercial seedlot of TN 90 LC. Rates of nicotine conversion and nornicotine levels are significantly reduced while yield and quality of NCBEX1MS lines is not significantly different from TN 90 LC (differences do not exceed the LSD at the 0.05 level of significance).

Table 2. Comparison of NCBEX1 MS relative to TN 90 LC and standard checks for yield, quality, and cured leaf chemistry during the 2011 growing season. Means are averages over three 2011

field environments. The experimental design was a randomized complete block design with four replications at each environment.

Nicotine

Yield Grade Nicotine Nornicotine Anabasine Anatabine Conversion

Genotype (lbs/A) Index (%) (%) (%) (%) (%)

VA509 2959.3 69.3 3.405 0.155 0.014 0.15 1 4.297

Ky 14 LC 3126.2 71.9 3.644 0.086 0.016 0.187 2.256

TN 90 LC

(certified) 3 139.7 70.7 3.845 0.159 0.016 0.220 3.932

NCBEX 1 MS 3 142.9 72.2 3.955 0.080 0.016 0.200 1.971

LSD (0.05) 473.5 8.4 0.570 0.053 0.004 0.054 1.085

DEPOSIT INFORMATION

A deposit of at least 2500 seeds 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 20110. The date of deposit for NCBEX1F and NCBEXIMS was September 6, 2011 and September 6, 201 1 , respectively. The deposit of 2500 seeds for each variety was taken from the same deposit maintained since prior to the filing date of this application. Upon issuance of a patent, all restrictions upon the deposit will be removed, and the deposit is intended to meet all of the requirements of 37 C.F.R. § 1.801-1.809. The ATCC accession numbers for inbred lines NCBEX1F and NCBEXIMS are PTA-1 1718, PTA-1 1719, respectively. 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 life 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 . A tobacco product, prepared from a tobacco plant, or part thereof, grown from a seed of tobacco cultivar NCBEX1F, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11718.

2. The tobacco product of Claim 1 , wherein said product 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, shredded tobacco, and cut tobacco.

3. The tobacco product of Claim 2, wherein said product has a reduced amount of

nornicotine.

4. A tobacco product, prepared from a tobacco plant, or part thereof, grown from a seed of tobacco cultivar NCBEXIMS, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-11719.

5. The tobacco product of Claim 4, wherein said product is selected from the group

6. The tobacco product of Claim 5, wherein said product has a reduced amount of

nornicotine.

7. A seed of tobacco cultivar NCBEX1F, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-1 1718.

8. A tobacco plant, or a part thereof, produced by growing the seed of Claim 7.

9. A harvested leaf of the tobacco plant, or part thereof, of Claim 8.

10. The harvested leaf of Claim 9, wherein said leaf has a redviced amount of nornicotine.

1 1. The harvested leaf of Claim 10, wherein said reduced amount of nornicotine is reduced in a smoke stream produced from burning said leaf.

12. A seed of tobacco cultivar NCBEX1MS, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA- 1 1719.

13. A tobacco plant, or a part thereof, produced by growing the seed of Claim 12.

14. A harvested leaf of the tobacco plant, or part thereof, of Claim 13.

15. The harvested leaf of Claim 14, wherein said leaf has a reduced amount of nornicotine.

16. The harvested leaf of Claim 15, wherein said reduced amount of nornicotine is reduced in a smoke stream produced from burning said leaf.

17. A F₂ progeny plant of tobacco cultivar NCBEXIF, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No.

PTA-1 1718.

18. A F₂ progeny plant of tobacco cultivar NCBEX1MS, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No.

PTA-1 1719.

19. The F₂ progeny plant of Claim 17, wherein said F₂ progeny plant is cytoplasmic male sterile (CMS).

20. The F₂ progeny plant of Claim 18, wherein said F₂ progeny plant is cytoplasmic male sterile (CMS).