Tobacco inbred and hybrid plants and tobacco products made thereof

Abstract

The present disclosure provides tobacco inbred plants NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, CMS NC1426-11 SRC, and NC1426-17 SRC, and hybrids NC 3 SRC, NC 6 SRC, and NC 4 SRC. The present disclosure also provides parts of such plants, cured tobacco, and tobacco products made from those parts. The present disclosure also includes progenies of the provided plants including hybrids.

Claims

1. Cured tobacco or a tobacco product prepared therefrom, wherein said cured tobacco is from a tobacco plant, or part thereof, of a hybrid, a cultivar, or a progeny hybrid produced from said cultivar, wherein said hybrid is selected from the group consisting of NC 3 SRC, NC 6 SRC, and NC 4 SRC, wherein said cultivar is selected from the group consisting of NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, CMS NC1426-11 SRC, and NC1426-17 SRC, and wherein representative sample seeds of said NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, NC 3 SRC, NC 6 SRC, and NC 4 SRC have been deposited with the ATCC with the following ATCC Accession Nos.: PTA-121049 for NC1209-23 SRC, PTA-121052 for DH19 SRC, PTA-121051 for DH98-325-5 SRC, PTA-121031 for NC1426-11 SRC, PTA-121070 for CMS NC1426-11 SRC, PTA-121032 for NC1426-17 SRC, PTA-121055 for NC 3 SRC, PTA-121042 for NC 6 SRC, and PTA-121040 for NC 4 SRC, wherein said progeny hybrid comprises essentially all of the morphological and physiological characteristics of the corresponding deposited cultivar used to produce said progeny hybrid.

2. The tobacco product of claim 1, wherein said tobacco product is selected from the group consisting of a pipe tobacco, a cigar tobacco, a cigarette tobacco, a chewing tobacco, a leaf tobacco, a shredded tobacco, and a cut tobacco.

3. The tobacco product of claim 1, wherein said tobacco product is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, and a chewing tobacco.

4. The tobacco product of claim 3, wherein said tobacco product has an amount of nornicotine of less than about 3 mg/g.

5. The tobacco product of claim 4, wherein said amount of nornicotine is selected from the group consisting of 3.0 mg/g, 2.5 mg/g, 2.0 mg/g, 1.5 mg/g, 1.0 mg/g, 750 g/g, 500 pg/g, 250 pg/g, 100 pg/g, 75 pg/g, 50 pg/g, 25 pg/g, 10 pg/g, 7.0 pg/g, 5.0 pg/g, 4.0 pg/g, 2.0 pg/g, 1.0 pg/g, 0.5 pg/g, 0.4 pg/g, 0.2 pg/g, 0.1 pg/g, 0.05 pg/g, 0.01 pg/g, and undetectable.

6. The tobacco product of claim 3, wherein said tobacco product has an amount of N-nitrosonornicotine (NNN) of less than about 10 pg/g.

7. The tobacco product of claim 6, wherein said amount of NNN is selected from the group consisting of 10 pg/g, 7.0 pg/g, 5.0 pg/g, 4.0 pg/g, 2.0 pg/g, 1.0 pg/g, 0.5 pg/g, 0.4 pg/g, 0.2 pg/g, 0.1 pg/g, 0.05 pg/g, 0.01 pg/g, and undetectable.

8. The tobacco product of claim 1, wherein said cured tobacco is at least about 5% by dry weight of the total tobacco material in said tobacco product.

9. The tobacco product of claim 8, wherein said cured tobacco has a dry weight percentage of said total tobacco material selected from the group consisting of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, and at least about 95%.

10. A method of producing a tobacco product, comprising: a. providing cured tobacco from a tobacco plant, or part thereof, of a hybrid, a cultivar, or a progeny hybrid produced from said cultivar, wherein said hybrid is selected from the group consisting of NC 3 SRC, NC 6 SRC, and NC 4 SRC, wherein said cultivar is selected from the group consisting of NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, CMS NC1426-11 SRC, and NC1426-17 SRC, and wherein representative sample seeds of said NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, NC 3 SRC, NC 6 SRC, and NC 4 SRC have been deposited with the ATCC with the following ATCC Accession Nos.: PTA-121049 for NC1209-23 SRC, PTA-121052 for DH19 SRC, PTA-121051 for DH98-325-5 SRC, PTA-121031 for NC1426-11 SRC, PTA-121070 for CMS NC1426-11 SRC, PTA-121032 for NC1426-17 SRC, PTA-121055 for NC 3 SRC, PTA-121042 for NC 6 SRC, and PTA-121040 for NC 4 SRC, wherein said progeny hybrid comprises essentially all of the morphological and physiological characteristics of the corresponding deposited cultivar used to produce said progeny hybrid; b. preparing a tobacco product from said cured tobacco.

11. A seed of tobacco hybrid or cultivar, wherein said hybrid is selected from the group consisting of NC 3 SRC, NC 6 SRC, and NC 4 SRC, wherein said cultivar is selected from the group consisting of NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, CMS NC1426-11 SRC, and NC1426-17 SRC, and wherein representative sample seeds of said NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, NC 3 SRC, NC 6 SRC, and NC 4 SRC have been deposited with the ATCC with the following ATCC Accession Nos.: PTA-121049 for NC1209-23 SRC, PTA-121052 for DH19 SRC, PTA-121051 for DH98-325-5 SRC, PTA-121031 for NC1426-11 SRC, PTA-121070 for CMS NC1426-11 SRC, PTA-121032 for NC1426-17 SRC, PTA-121055 for NC 3 SRC, PTA-121042 for NC 6 SRC, and PTA-121040 for NC 4 SRC.

12. A tobacco plant, or part thereof, produced from said seed of claim 11.

13. The part of the plant of claim 12, wherein said part is selected from the group consisting of a harvested leaf, a pollen, an ovule, an embryo, a cotyledon, a hypocotyl, a meristematic cell, a protoplast, a root, a root tip, a pistil, an anther, a flower, a shoot, a stem, a pod, a petiole, and combinations thereof.

14. A harvested leaf of said tobacco plant of claim 12.

15. The harvested leaf of claim 14, wherein said leaf has a reduced amount of nornicotine and/or N-nitrosonornicotine (NNN).

16. The harvested leaf of claim 15, wherein said reduced amount of nornicotine and/or N-nitrosonornicotine (NNN) is reduced in a smoke stream produced from said leaf.

17. The cured tobacco material or a tobacco product of claim 1, wherein said cured tobacco material is from a tobacco plant, or part thereof, of said hybrid or said cultivar.

Description

DETAILED DESCRIPTION OF THE DISCLOSURE

(1) This description is not intended to be a detailed catalog of all the different ways in which the disclosure may be implemented, or all the features that may be added to the instant disclosure. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the disclosure contemplates that in some embodiments of the disclosure, any feature or combination of features set forth herein can be excluded or omitted. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant disclosure. Hence, the following descriptions are intended to illustrate some particular embodiments of the disclosure, and not to exhaustively specify all permutations, combinations and variations thereof.

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

(3) All publications, patent applications, patents and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art.

(4) Unless the context indicates otherwise, it is specifically intended that the various features of the disclosure described herein can be used in any combination. Moreover, the present disclosure also contemplates that in some embodiments of the disclosure, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a composition comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

(5) As used in the description of the disclosure and the appended claims, the singular forms a, an and the are intended to include the plural forms as well, unless the context clearly indicates otherwise.

(6) 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).

(7) The term about, as used herein when referring to a measurable value such as a dosage or time period and the like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

(8) As used herein, phrases such as between X and Y and between about X and Y should be interpreted to include X and Y. As used herein, phrases such as between about X and Y mean between about X and about Y and phrases such as from about X to Y mean from about X to about Y.

(9) The terms comprise, comprises and comprising as used herein, specify the presence of the stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

(10) As used herein, the transitional phrase consisting essentially of means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed disclosure. Thus, the term consisting essentially of when used in a claim of this disclosure is not intended to be interpreted to be equivalent to comprising.

(11) Introducing, in the context of a polynucleotide sequence (e.g., a recombinant polynucleotide and/or expression cassette of the disclosure), means presenting a polynucleotide sequence to the plant, plant part, and/or plant cell in such a manner that the polynucleotide sequence gains access to the interior of a cell. Where more than one polynucleotide sequence is to be introduced these polynucleotide sequences can be assembled as part of a single polynucleotide or nucleic acid construct, or as separate polynucleotide or nucleic acid constructs, and can be located on the same or different transformation vectors. Accordingly, these polynucleotides can be introduced into plant cells in a single transformation event, in separate transformation events, or, e.g., as part of a breeding protocol. Thus, the term transformation as used herein refers to the introduction of a heterologous nucleic acid into a cell. Transformation of a cell may be stable or transient. Thus, in some embodiments, a plant cell, plant part or plant of this disclosure can be stably transformed with a recombinant polynucleotide of the disclosure. In other embodiments, a plant cell, plant part or plant of this disclosure can be transiently transformed with a recombinant polynucleotide of the disclosure.

(12) Tobacco product is defined as any product made or derived from tobacco that is intended for human use or consumption, including any component, part, or accessory of a tobacco product (except for raw materials other than tobacco used in manufacturing a component, part, or accessory of a tobacco product) (section 201 of the FD&C Act; 21 U.S.C. 321). The label and packaging is part of a tobacco product.

(13) Terms nicotine conversion rate, percent nicotine conversion, and percentage nicotine conversion are used interchangeably. Percent nicotine demethylation in a sample is calculated by dividing the level of nornicotine by the combined level of nicotine and nornicotine as measured in the sample, and multiplying by 100.

(14) NC1209-23 SRC

(15) In one aspect, the present disclosure provides tobacco cultivars, and parts thereof, from NC1209-23 SRC, representative sample seeds of this cultivar having been deposited with the ATCC under ATCC Accession No. PTA-121049. In another aspect, the present disclosure provides a tobacco plant, or part thereof, produced by growing a seed of NC1209-23 SRC. In a further aspect, a plant of the present disclosure can include a plant with all, or essentially all, of the morphological and physiological characteristics of cultivar NC1209-23 SRC.

(16) While not being limited by process, NC1209-23 SRC is a result of the introduction of three mutated CYP82E genes in a burley tobacco cultivar NC1209-23. The three genes are a mutated CYP82E4 gene recited as 325-6 #775 in Lewis et al. (Three nicotine demethylase genes mediate nornicotine biosynthesis in Nicotiana tabacum L.: Functional characterization of the CYP82E10 gene, Phytochemistry, 71 (2010), 1988-1998 (SEQ ID NO: 1, which sets forth a cyp82e4 W329Stop, hereby incorporated by reference in its entirety)), a mutated CYP82E5v2 recited in Lewis et al. (supra) as 325-6 #1-13 (SEQ ID NO: 2, which sets forth a cyp82e5v2 W422Stop, hereby incorporated by reference in its entirety), and a mutated CYP82E10 recited in Lewis et al. (supra) as 325-6 #1041 (SEQ ID NO:11, which sets forth a cyp82e10 P381S, hereby incorporated by reference in its entirety). Mutations cyp82e4 W329Stop and cyp82e5v2 W422Stop result in truncated proteins while cyp82E10 P381S results in a nonfunctional protein. A cyp82e4 W329Stop (e4), a cyp82e5v2 W422Stop (e5), and a cyp82e10 P381S (e10) mutation are introduced from a e4e4|e5e5|e10e10 triple mutant from a strong converter burley background, line DH98-325-6, as listed in Table 4 of Lewis et al. (supra) into a burley tobacco cultivar NC1209-23 background.

(17) NC1209-23 SRC is the result of seven backcrosses with burley cultivar NC1209-23 as the recurrent parent, followed by two rounds of selfing with selection for homozygosity for the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82e10 P381S mutations to yield BC.sub.7F.sub.3 plants (NC1209-23 SRC) in which the wild-type CYP82E4, CYP82E5v2 and CYP82E10 alleles of NC1209-23 are replaced by the mutant (e.g., cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S) alleles.

(18) NC1209-23 SRC progeny plants have genetic backgrounds that are at least 95%, at least 97%, at least 98%, or at least 99% similar to NC1209-23. NC1209-23 SRC plants exhibit low nornicotine levels and produce leaves with reduced potential for accumulating derived NNN during curing, storage, and smoking.

(19) CMS NC1209-23 SRC

(20) In other aspects, the present disclosure also provides tobacco cultivars, and parts thereof, from CMS NC1209-23 SRC. In further aspects, the present disclosure provides a tobacco plant, or part thereof, produced by growing a seed of CMS NC1209-23 SRC. In still further aspects, the plant of the present disclosure can include a plant with all, or essentially all, of the morphological and physiological characteristics of cultivar CMS NC1209-23 SRC. CMS NC1209-23 SRC is a male-sterile (CMS) version of NC1209-23 SRC (CMS NC1209-23 SRC) produced by crossing a plant of CMS NC1209-23 as a female with pollen of NC1209-23 SRC BC.sub.6F.sub.1 to produce male-sterile plants heterozygous for all three mutations. The CMS progeny plants of the CMS NC1209-23NC1209-23 SRC BC.sub.6F.sub.1 cross are male sterile. A plurality of CMS NC1209-23NC1209-23 SRC BC.sub.6F.sub.1CMS plants (e.g., CMS F.sub.1 progeny plants) are screened for the cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S mutations to identify plants heterozygous for all three mutations. A single male-sterile plant resulting from this cross is backcrossed as a female to NC1209-23 SRC to prepare BC.sub.7F.sub.1 CMS progeny. BC.sub.7F.sub.1 CMS progeny homozygous for the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82e10 P381S mutations are identified by genotyping and designated as CMS NC1209-23 SRC. Because the CMS NC1209-23 SRC line is male sterile, it is maintained via pollination with NC1209-23 SRC. NC1209-23 SRC is crossed as the male parent to CMS NC1209-23 SRC to prepare CMS NC1209-23 SRC F.sub.1 progeny plants.

(21) CMS NC1209-23 SRC and CMS NC1209-23 SRC F.sub.1 progeny plants have genetic backgrounds that are at least 95%, at least 97%, at least 98%, or at least 99% similar to NC1209-23. CMS NC1209-23 SRC and CMS NC1209-23 SRC F.sub.1 progeny plants exhibit low nornicotine levels and produce leaves with reduced potential for accumulating derived NNN during curing, storage, and smoking.

(22) DH19 SRC

(23) In some aspects, the present disclosure provides tobacco cultivars, and parts thereof, from DH19 SRC, where representative sample seeds of this cultivar have been deposited with the ATCC under ATCC Accession No. PTA-121052. In other aspects, the present disclosure provides a tobacco plant, or part thereof, produced by growing a seed of DH19 SRC. In still further aspects, a plant of the present disclosure can include a plant with all, or essentially all, of the morphological and physiological characteristics of cultivar DH19 SRC. While not being limited by process, DH19 SRC is a result of introducing the cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S mutations from DH98-325-6. F.sub.1 individuals originating from a cross between DH19 and DH98-325-6 and heterozygous for each mutation are then backcrossed seven times to DH19 to produce BC.sub.7F.sub.1 progeny. BC.sub.7F.sub.1 individuals heterozygous for all three mutations are self-pollinated to produce BC.sub.7F.sub.2 seed and individuals homozygous for all three mutations identified. A single BC.sub.7F.sub.2 plant is self-pollinated to produce BC.sub.7F.sub.3 (DH19 SRC) in which the wild-type CYP82E4, CYP82E5v2 and CYP82E10 alleles of DH19 are replaced by the mutant (e.g., cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S).

(24) DH19 SRC has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to DH19. DH19 SRC exhibits low nornicotine levels and produce leaves with reduced potential for accumulating derived NNN during curing, storage, and smoking.

(25) NC 3 SRC

(26) In other aspects, the present disclosure provides tobacco cultivars, and parts thereof, from NC 3 SRC. In another aspect, the present disclosure provides a tobacco plant, or part thereof, produced by growing the seed of NC 3 SRC. In a further aspect, a plant of the present disclosure can include a plant with all, or essentially all, of the morphological and physiological characteristics of cultivar NC 3 SRC.

(27) While not being limited by process, NC 3 SRC is produced by pollinating plants of CMS NC1209-23 SRC with pollen of DH19 SRC. Again, not limited by any particular scientific theory, cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S mutations all encode for proteins with reduced or eliminated ability to convert nicotine to nornicotine. NC 3 SRC has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to burley tobacco cultivar NC1209-23DH19, a hybrid generated by pollinating plants of a male-sterile breeding line CMS NC1209-23 with pollen produced by fertile breeding line DH19. NC 3 SRC exhibits low nornicotine and is not subject to conversion to high nornicotine.

(28) DH98-325-5 SRC

(29) In some aspects, the present disclosure provides tobacco cultivars, and parts thereof, from DH98-325-5 SRC, where representative sample seeds of this cultivar have been deposited with the ATCC under ATCC Accession No. PTA-121051. In other aspects, the present disclosure provides a tobacco plant, or part thereof, produced by growing a seed of DH98-325-5 SRC. In still further aspects, a plant of the present disclosure can include a plant with all, or essentially all, of the morphological and physiological characteristics of cultivar DH98-325-5 SRC. While not being limited by process, DH98-325-5 SRC is a result of introducing the cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S mutations from DH98-325-6. F.sub.1 individuals originating from a cross between DH98-325-5 and DH98-325-6 and heterozygous for each mutation are then backcrossed seven times to DH98-325-5 to produce BC.sub.7F.sub.1 progeny. BC.sub.7F.sub.1 individuals heterozygous for all three mutations are self-pollinated to produce BC.sub.7F.sub.2 seed and individuals homozygous for all three mutations identified. A single BC.sub.7F.sub.2 plant is self-pollinated to produce BC.sub.7F.sub.3 (DH98-325-5 SRC) in which the wild-type CYP82E4, CYP82E5v2 and CYP82E10 alleles of DH98-325-5 are replaced by the mutant (e.g., cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S).

(30) DH98-325-5 SRC has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to DH98-325-5. DH98-325-5 SRC exhibits low nornicotine levels and produce leaves with reduced potential for accumulating derived NNN during curing, storage, and smoking.

(31) NC 6 SRC

(32) In other aspects, the present disclosure provides tobacco cultivars, and parts thereof, from NC 6 SRC. In another aspect, the present disclosure provides a tobacco plant, or part thereof, produced by growing the seed of NC 6 SRC. In a further aspect, a plant of the present disclosure can include a plant with all, or essentially all, of the morphological and physiological characteristics of cultivar NC 6 SRC.

(33) While not being limited by process, NC 6 SRC is produced by pollinating plants of CMS NC1209-23 SRC with pollen of DH98-325-5 SRC. Again, not limited by any particular scientific theory, cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S mutations all encode for proteins with reduced or eliminated ability to convert nicotine to nornicotine. NC 6 SRC has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to burley tobacco cultivar NC1209-23DH98-325-5, a hybrid generated by pollinating plants of a male-sterile breeding line CMS NC1209-23 with pollen produced by fertile breeding line DH98-325-5. NC 6 SRC exhibits low nornicotine and is not subject to conversion to high nornicotine.

(34) NC1426-11 SRC

(35) In one aspect, the present disclosure provides tobacco cultivars, and parts thereof, from NC1426-11 SRC, representative sample seeds of this cultivar having been deposited with the ATCC under ATCC Accession No. PTA-121031. In another aspect, the present disclosure provides a tobacco plant, or part thereof, produced by growing a seed of NC1426-11 SRC. In a further aspect, a plant of the present disclosure can include a plant with all, or essentially all, of the morphological and physiological characteristics of cultivar NC1426-11 SRC.

(36) While not being limited by process, NC1426-11 SRC is a result of the introduction of three mutated CYP82E genes in a burley tobacco cultivar NC1426-11. The three genes are a mutated CYP82E4 gene recited as 325-6 #775 in Lewis et al. (Three nicotine demethylase genes mediate nornicotine biosynthesis in Nicotiana tabacum L.: Functional characterization of the CYP82E10 gene, Phytochemistry, 71 (2010), 1988-1998 (SEQ ID NO: 1, which sets forth a cyp82e4 W329Stop, hereby incorporated by reference in its entirety)), a mutated CYP82E5v2 recited in Lewis et al. (supra) as 325-6 #1-13 (SEQ ID NO: 2, which sets forth a cyp82e5v2 W422Stop, hereby incorporated by reference in its entirety), and a mutated CYP82E10 recited in Lewis et al. (supra) as 325-6 #1041 (SEQ ID NO:11, which sets forth a cyp82e10 P381S, hereby incorporated by reference in its entirety). Mutations cyp82e4 W329Stop and cyp82e5v2 W422Stop result in truncated proteins while cyp82E10 P381S results in a nonfunctional protein. A cyp82e4 W329Stop (e4), a cyp82e5v2 W422Stop (e5), and a cyp82e10 P381S (e10) mutation are introduced from a e4e4|e5e5|e10e10 triple mutant from a strong converter burley background, line DH98-325-6, as listed in Table 4 of Lewis et al. (supra) into a burley tobacco cultivar NC1426-11 background.

(37) NC1426-11 SRC is the result of seven backcrosses with burley cultivar NC1426-11 as the recurrent parent, followed by two rounds of selfing with selection for homozygosity for the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82e10 P381S mutations to yield BC.sub.7F.sub.3 plants (NC1426-11 SRC) in which the wild-type CYP82E4, CYP82E5v2 and CYP82E10 alleles of NC1426-11 are replaced by the mutant (e.g., cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S) alleles.

(38) NC1426-11 SRC progeny plants have genetic backgrounds that are at least 95%, at least 97%, at least 98%, or at least 99% similar to NC1426-11. NC1426-11 SRC plants exhibit low nornicotine levels and produce leaves with reduced potential for accumulating derived NNN during curing, storage, and smoking.

(39) CMS NC1426-11 SRC

(40) In other aspects, the present disclosure also provides tobacco cultivars, and parts thereof, from CMS NC1426-11 SRC, representative sample seeds of this cultivar having been deposited with the ATCC under ATCC Accession No. PTA-121070. In further aspects, the present disclosure provides a tobacco plant, or part thereof, produced by growing a seed of CMS NC1426-11 SRC. In still further aspects, the plant of the present disclosure can include a plant with all, or essentially all, of the morphological and physiological characteristics of cultivar CMS NC1426-11 SRC. CMS NC1426-11 SRC is a male-sterile (CMS) version of NC1426-11 (CMS NC1426-11 SRC) produced by crossing a plant of CMS NC1426-11 as a female with pollen of NC1426-11 SRC BC.sub.6F.sub.1 to produce male-sterile plants heterozygous for all three mutations. The CMS progeny plants of the CMS NC1426-11NC1426-11 SRC BC.sub.6F.sub.1 cross are male sterile. A plurality of CMS NC1426-11NC1426-11 SRC BC.sub.6F.sub.1CMS plants (e.g., CMS F.sub.1 progeny plants) are screened for the cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S mutations to identify plants heterozygous for all three mutations. A single male-sterile plant resulting from this cross is backcrossed as a female to NC1426-11 SRC to prepare BC.sub.7F.sub.1 CMS progeny. BC.sub.7F.sub.1 CMS progeny homozygous for the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82e10 P381S mutations are identified by genotyping and designated as CMS NC1426-11 SRC. Because the CMS NC1426-11 SRC line is male sterile, it is maintained via pollination with NC1426-11 SRC. NC1426-11 SRC is crossed as the male parent to CMS NC1426-11 SRC to prepare CMS NC1426-11 SRC F.sub.1 progeny plants.

(41) CMS NC1426-11 SRC and CMS NC1426-11 SRC F.sub.1 progeny plants have genetic backgrounds that are at least 95%, at least 97%, at least 98%, or at least 99% similar to NC1426-11. CMS NC1426-11 SRC and CMS NC1426-11 SRC F.sub.1 progeny plants exhibit low nornicotine levels and produce leaves with reduced potential for accumulating derived NNN during curing, storage, and smoking.

(42) NC1426-17 SRC

(43) In some aspects, the present disclosure provides tobacco cultivars, and parts thereof, from NC1426-17 SRC, where representative sample seeds of this cultivar have been deposited with the ATCC under ATCC Accession No. PTA-121032. In other aspects, the present disclosure provides a tobacco plant, or part thereof, produced by growing a seed of NC1426-17 SRC. In still further aspects, a plant of the present disclosure can include a plant with all, or essentially all, of the morphological and physiological characteristics of cultivar NC1426-17 SRC. While not being limited by process, NC1426-17 SRC is a result of introducing the cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S mutations from DH98-325-6. F.sub.1 individuals originating from a cross between NC1426-17 and DH98-325-6 and heterozygous for each mutation are then backcrossed seven times to NC1426-17 to produce BC.sub.7F.sub.1 progeny. BC.sub.7F.sub.1 individuals heterozygous for all three mutations are self-pollinated to produce BC.sub.7F.sub.2 seed and individuals homozygous for all three mutations identified. A single BC.sub.7F.sub.2 plant is self-pollinated to produce BC.sub.7F.sub.3 (NC1426-17 SRC) in which the wild-type CYP82E4, CYP82E5v2 and CYP82E10 alleles of NC1426-17 are replaced by the mutant (e.g., cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S).

(44) NC1426-17 SRC has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to NC1426-17. NC1426-17 SRC exhibits low nornicotine levels and produce leaves with reduced potential for accumulating derived NNN during curing, storage, and smoking.

(45) NC 4 SRC

(46) In other aspects, the present disclosure provides tobacco cultivars, and parts thereof, from NC 4 SRC. In another aspect, the present disclosure provides a tobacco plant, or part thereof, produced by growing the seed of NC 4 SRC. In a further aspect, a plant of the present disclosure can include a plant with all, or essentially all, of the morphological and physiological characteristics of cultivar NC 4 SRC.

(47) While not being limited by process, NC 4 SRC is produced by pollinating plants of CMS NC1426-11 SRC with pollen of NC1426-17 SRC. Again, not limited by any particular scientific theory, cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S mutations all encode for proteins with reduced or eliminated ability to convert nicotine to nornicotine. NC 4 SRC has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to burley tobacco cultivar NC 4LC, a hybrid generated by pollinating plants of a male-sterile breeding line CMS NC1426-11 with pollen produced by fertile breeding line NC1426-17. NC 4 SRC exhibits low nornicotine and is not subject to conversion to high nornicotine.

(48) Other Plants

(49) In some aspects, the present disclosure provides a tobacco seed produced by crossing two parent tobacco plants and harvesting the resultant tobacco seed, where at least one parent tobacco plant is NC1209-23 SRC. In one aspect, the NC1209-23 SRC is the male parent plant. In another aspect, the CMS NC1209-23 SRC is the female parent plant. One aspect of the present disclosure provides tobacco plants that are homozygous at the cyp82e4, cyp82e5v2, and cyp82E10 loci for SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 11, respectively, and which share a genetic background that is greater than about 75%, 80%, 85%, 90%, 95%, 98%, or 99% similar to NC1209-23 or CMS NC1209-23. In one aspect, approximately or greater than about 50%, 75%, or 100% of a progeny's genetics is provided by a plant of the present disclosure that is homozygous at the cyp82e4, cyp82e5v2, and cyp82E10 loci for SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 11, respectively. In one aspect, a plant of the present disclosure has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to NC1209-23 or CMS NC1209-23. In another aspect, a plant of the present disclosure exhibits low nornicotine and is not subject to conversion to high nornicotine. In one aspect, a plant of the present disclosure is the progeny plant of a female or male parent plant that is Fusarium wilt resistant. In another aspect, a plant of NC1209-23 SRC has low resistance to black shank and moderate resistance to bacterial wilt.

(50) The present disclosure includes a tobacco seed produced by crossing two parent tobacco plants and harvesting the resultant tobacco seed, where at least one parent tobacco plant is NC1209-23 SRC. In one aspect, the NC1209-23 SRC is the male parent plant. In another aspect, the CMS NC1209-23 SRC is the female parent plant. One aspect of the present disclosure provides tobacco plants that are homozygous at the cyp82e4, cyp82e5v2, and cyp82E10 loci for SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 11, respectively, and which share a genetic background that is greater than about 75%, 80%, 85%, 90%, 95%, 98%, or 99% similar to NC1209-23 or CMS NC1209-23. In one aspect, approximately or greater than about 50%, 75%, or 100% of a progeny's genetics is provided by a plant of the present disclosure that is homozygous at the cyp82e4, cyp82e5v2, and cyp82E10 loci for SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 11, respectively. In one aspect, a plant of the present disclosure has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to NC1209-23 SRC and CMS NC1209-23 SRC. In another aspect, a plant of the present disclosure exhibits low nornicotine and is not subject to conversion to high nornicotine. In one aspect, a plant of the present disclosure is the progeny plant of a female or male parent plant that is Fusarium wilt resistant. In another aspect, a plant of NC 3 SRC has moderate resistance to black shank and moderate resistance to bacterial wilt. In another aspect, a plant of NC 6 SRC has moderate resistance to black shank and moderate resistance to bacterial wilt.

(51) In some aspects, the present disclosure provides a tobacco seed produced by crossing two parent tobacco plants and harvesting the resultant tobacco seed, where at least one parent tobacco plant is NC1426-11 SRC. In one aspect, the NC1426-11 SRC is the male parent plant. In another aspect, the CMS NC1426-11 SRC is the female parent plant. One aspect of the present disclosure provides tobacco plants that are homozygous at the cyp82e4, cyp82e5v2, and cyp82E10 loci for SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 11, respectively, and which share a genetic background that is greater than about 75%, 80%, 85%, 90%, 95%, 98%, or 99% similar to NC1426-11 or CMS NC1426-11. In one aspect, approximately or greater than about 50%, 75%, or 100% of a progeny's genetics is provided by a plant of the present disclosure that is homozygous at the cyp82e4, cyp82e5v2, and cyp82E10 loci for SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 11, respectively. In one aspect, a plant of the present disclosure has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to NC1426-11 or CMS NC1426-11. In another aspect, a plant of the present disclosure exhibits low nornicotine and is not subject to conversion to high nornicotine. In one aspect, a plant of the present disclosure is the progeny plant of a female or male parent plant that is Fusarium wilt resistant. In another aspect, a plant of NC1426-11 SRC has low resistance to black shank and moderate resistance to bacterial wilt.

(52) The present disclosure includes a tobacco seed produced by crossing two parent tobacco plants and harvesting the resultant tobacco seed, where at least one parent tobacco plant is NC1426-11 SRC. In one aspect, the NC1426-11 SRC is the male parent plant. In another aspect, the CMS NC1426-11 SRC is the female parent plant. One aspect of the present disclosure provides tobacco plants that are homozygous at the cyp82e4, cyp82e5v2, and cyp82E10 loci for SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 11, respectively, and which share a genetic background that is greater than about 75%, 80%, 85%, 90%, 95%, 98%, or 99% similar to NC1426-11 or CMS NC1426-11. In one aspect, approximately or greater than about 50%, 75%, or 100% of a progeny's genetics is provided by a plant of the present disclosure that is homozygous at the cyp82e4, cyp82e5v2, and cyp82E10 loci for SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 11, respectively. In one aspect, a plant of the present disclosure has a genetic background that is at least 95%, at least 97%, at least 98%, or at least 99% similar to NC1426-11 SRC and CMS NC1426-11 SRC. In another aspect, a plant of the present disclosure exhibits low nornicotine and is not subject to conversion to high nornicotine. In one aspect, a plant of the present disclosure is the progeny plant of a female or male parent plant that is Fusarium wilt resistant. In another aspect, a plant of NC 4 SRC has moderate resistance to black shank and moderate resistance to bacterial wilt.

(53) In one aspect, a plant of the present disclosure is a medium-late maturing variety with moderately high yield potential. In another aspect, a plant of the present disclosure offers a broad range of important agronomic characteristics. In a further aspect, a plant of the present disclosure has one, two, three, four or more of the traits including moderate resistance to black shank, some tolerance to blue mold, black root rot resistance, and resistance to common virus diseases. In another aspect, a plant of the present disclosure has blue mold tolerance and level 4 resistance to both races of black shank and high root rot resistance. In one aspect, a plant of the present disclosure, such as NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, and hybrid cultivars NC 3 SRC, NC 6 SRC, and NC 4 SRC, lacks Fusarium wilt resistance. In another aspect, a plant of the present disclosure is Fusarium wilt resistant. In another aspect, a plant of the present disclosure has low resistance to black shank and moderate resistance to bacterial wilt.

(54) In an aspect, the plants of the present disclosure have reduced or eliminated ability to convert nicotine to nornicotine. In an aspect, the percentage nicotine conversion is less than about 75%, 70%, 60%, 50%, or 25% of that found in NC1209-23, DH19, DH98-325-5, or hybrid cultivar NC 3 LC or NC 6 LC. The nicotine conversion in plants of the present disclosure, including NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, and hybrid cultivar NC 3 SRC and NC 6 SRC, can be less than about 4%, about 3.5%, about 3%, about 2.5%, about 2%, about 1.5%, or about 1%, or any range therein. In still other aspects, the nicotine conversion in plants of the present disclosure, including NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, and hybrid cultivars NC 3 SRC and NC 6 SRC, can be in a range from about 3% to about 1%, about 3% to about 0.5%, or about 2% to about 0.5%. In a preferred aspect, the percentage nicotine conversion is less than about 25%, 10%, 5%, or 2% of that found in NC1209-23, DH19, DH98-325-5, or hybrid cultivar NC 3 LC or NC 6 LC without the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82e10 P381S mutations.

(55) In an aspect, the plants of the present disclosure have reduced or eliminated ability to convert nicotine to nornicotine. In an aspect, the percentage nicotine conversion is less than about 75%, 70%, 60%, 50%, or 25% of that found in CMS NC1426-11, NC1426-17 or hybrid cultivar NC 4LC. The nicotine conversion in plants of the present disclosure, including NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, and hybrid cultivar NC 4 SRC, can be less than about 4%, about 3.5%, about 3%, about 2.5%, about 2%, about 1.5%, or about 1%, or any range therein. In still other aspects, the nicotine conversion in plants of the present disclosure, including NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, and hybrid cultivar NC 4 SRC, can be in a range from about 3% to about 1%, about 3% to about 0.5%, or about 2% to about 0.5%. In a preferred aspect, the percentage nicotine conversion is less than about 25%, 10%, 5%, or 2% of that found in NC1426-11, NC1426-17, or hybrid cultivar NC 4 LC without the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82e10 P381S mutations.

(56) In an aspect, the tobacco plants of the present disclosure can have a nicotine conversion rate of about 3.5, 3.25, 3.0 or 2.75% or less. In another aspect, the nicotine conversion rate of tobacco plants of the present disclosure can be about 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5% or less or any range therein. In another aspect, the nicotine conversion rate of tobacco plants of the present disclosure can be about 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6% or less or any range therein. In another aspect, the nicotine conversion rates can be in a range from about 0.5% to about 0.9%, about 0.5% to about 1.5%, about 0.5% to about 2.0%, about 0.5% to about 2.5%, about 0.5% to about 2.75%, and about 0.5% to about 3.0%. In another aspect, the nicotine conversion rates can be in a range from about 1.0% to about 1.5%, about 1.0% to about 1.75%, about 1.0% to about 2.0%, about 1.0% to about 2.5%, about 1.0% to about 2.75%, or about 1.0% to about 3.0%. In another aspect, the nicotine conversion rate in a plant of the present disclosure can be less than about 2.9, 2.75, 2.5, 2.25, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1 or 1.0% or any range therein.

(57) In another aspect, the tobacco plants of the present disclosure typically have a reduced amount of nornicotine of less than about 0.10% dry weight. For example, the nornicotine content in such plants can be about 1.2, 1.0, 0.7, 0.5, 0.4, 0.2, 0.1, 0.09, 0.085, 0.08, 0.075, 0.07, 0.065, 0.06, 0.055, 0.05, 0.045, 0.04, 0.035, 0.025, 0.01, 0.009, 0.0075, 0.005, 0.0025, 0.001, 0.0009, 0.00075, 0.0005, 0.00025, or 0.0001% dry weight, or undetectable, or any range therein. In another aspect, the nornicotine content can be less than about 1.2, 1.0, 0.9, 0.8, 0.7, 0.5, 0.4, 0.2, 0.1, 0.075, 0.05, 0.025, 0.01, 0.009, 0.0075, 0.005, 0.0025, 0.001, 0.0009, 0.00075, 0.0005, 0.00025, or 0.0001% dry weight, or any range therein. In another aspect, the nornicotine content in such plants can be in a range from about 1.2% to about 1.0%, about 0.7% to about 0.5%, about 0.4% to about 0.2%, about 0.1% to about 0.075%, about 0.05% to about 0.025%, about 0.01% to about 0.0075%, about 0.005% to about 0.0025%, about 0.001% to about 0.00075%, about 0.0005% to about 0.00025%, or about 0.0005% to about 0.0001% dry weight. In some aspects, in a plant of the present disclosure, the nornicotine is a relatively small percentage of total alkaloids in the plant compared to a commercial seedlot of NC1209-23, DH19, DH98-325-5, NC1426-11, NC1426-17, or hybrid cultivar NC 3 LC, NC 6 LC, or NC 4 LC. In some aspects, the nornicotine in a plant of the present disclosure can be about 2% to about 1%, less than 3%, about 2%, about 1.5%, about 1%, or 0.75% of total alkaloids. Tobacco products having a reduced amount of nitrosamine content can be manufactured using tobacco plant material from plants and plant parts of the present disclosure. Thus, in some embodiments, a tobacco product manufactured using tobacco plant material from plants and plant parts of the present disclosure can comprise a reduced amount of nornicotine of less than about 3 mg/g. For example, the nornicotine content in such a product can be 3.0 mg/g, 2.5 mg/g, 2.0 mg/g, 1.5 mg/g, 1.0 mg/g, 750 g/g, 500 g/g, 250 g/g, 100 g/g, 75 g/g, 50 g/g, 25 g/g, 10 g/g, 5 g/g, 1 g/g, 750 ng/g, 500 ng/g, 250 ng/g, 100 ng/g, 75 ng/g, 50 ng/g, 25 ng/g, 10 ng/g, 5 ng/g, 1 ng/g, 750 pg/g, 500 pg/g, 250 pg/g, 100 pg/g, 75 pg/g, 50 pg/g, 25 pg/g, 10 pg/g, 7.0 pg/g, 5.0 pg/g, 4.0 pg/g, 2.0 pg/g, 1.0 pg/g, 0.5 pg/g, 0.4 pg/g, 0.2 pg/g, 0.1 pg/g, 0.05 pg/g, 0.01 pg/g, and the like, or undetectable, or any range therein. The tobacco product typically has a reduced amount of NNN of less than about 10 pg/g. For example, the NNN content in such a product can be about 10 pg/g, 7.0 pg/g, 5.0 pg/g, 4.0 pg/g, 2.0 pg/g, 1.0 pg/g, 0.5 pg/g, 0.4 pg/g, 0.2 pg/g, 0.1 pg/g, 0.05 pg/g, 0.01 pg/g, and the like, or undetectable, or any range therein. The percentage of secondary alkaloids relative to total alkaloid content contained in a plant of the present disclosure may not be statistically different than from a commercial seedlot of NC1209-23, DH19, DH98-325-5, NC1426-11, NC1426-17, or hybrid cultivar NC 3 LC, NC 6 LC, or NC 4LC.

(58) Differences between two inbred tobacco varieties or two hybrid tobacco varieties can be evaluated using statistical approaches. Statistical analysis includes the calculation of mean values, determination of the statistical significance of the sources of variation, and the calculation of the appropriate variance components. Methods for determining statistical significance are known in the art. Statistical software is available, for example, the PROC GLM function of SAS. Significance is generally presented as a p-value. A statistically significant p-value is less than 0.10. In a preferred aspect, the p-value is less than, or equal to, 0.05. In another aspect, the p-value is 0.04 or less, 0.03 or less, or 0.02 or less. In yet another aspect, a statistically significant value is less than 0.01. In yet another aspect, it can be less than 0.009, less than 0.008, less than 0.007, less than 0.006, less than 0.005, less than 0.004, less than 0.003, less than 0.002, or less than 0.001.

(59) Tobacco plants of the present disclosure that are homozygous for the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82e10 P381S alleles have a reversion rate that is statistically significantly lower than corresponding control low-converter plants having wild type nicotine demethylase CYP82E4, E5, and E10 genes. In addition, homozygous CYP82E4, CYP82E5, and CYP82E10 triple mutant tobacco plants have a percent conversion to nornicotine of less than about 2.0%, e.g., undetectable to about 2.0%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, or any range therein. In some aspects, the triple mutant tobacco plants have a percent conversion to nornicotine in a range from, for example, about 1.0% to 2.0%, 0.8% to 1.8%, 0.8% to 2.0%, or 1.0% to 2.0%.

(60) Nicotine and nornicotine can be measured in ethylene-treated leaves using methods known in the art (e.g., gas chromatography). Percent nicotine demethylation in a sample is calculated by dividing the level of nornicotine by the combined level of nicotine and nornicotine as measured in the sample, and multiplying by 100. Percent nicotine demethylation in a sample from a plant of the present disclosure is about 50, 40, 30, 20, or 10 percent of a sample from an individual plant grown from a commercial seedlot of NC1209-23, DH19, DH98-325-5, NC1426-11, NC1426-17, or hybrid cultivar NC 3 LC, NC 6 LC, or NC 4LC.

(61) In an aspect, the tobacco plants of the present disclosure have a USDA quality index of about 73, about 72, about 71, about 70, about 69, about 68, about 67 or about 66 or any range therein. In an aspect, the tobacco plants of the present disclosure have a USDA quality index of about 65. In another aspect, the quality index may be at least about 55, 60, 62.5 or greater, or any range therein. In another aspect, tobacco plants of the present disclosure can have a quality index in the range of about 60 to about 65, about 60 to about 70, about 62.5 to about 65, about 62.5 to about 70, or about 65 to about 70.

(62) A plant of the present disclosure, including NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, and hybrid cultivar NC 3 SRC, NC 6 SRC, or NC 4 SRC, can have any yield potential, including high (e.g., over 3000 lbs/A), moderately high (e.g., 2200-3000 lbs/A), and moderate (e.g., less than 2000 lbs/A) yield potential.

(63) In another aspect, the present disclosure also provides for a plant grown from the seed of a NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, or a hybrid NC 3 SRC, NC 6 SRC, plant in which alkaloids obtained from tobacco plants grown for the seed have decreased nornicotine, as well as plant parts and tissue cultures from such plants, representative sample seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-121049 for NC1209-23 SRC, ATCC Accession No. PTA-121052 for DH19 SRC, and ATCC Accession No. PTA-121051 for DH98-325-5 SRC. Seeds of hybrid cultivar NC 3 SRC are obtainable by crossing plants of cultivars DH19 SRC and CMS NC1209-23 SRC and collecting seeds. Seeds of hybrid cultivar NC 6 SRC are obtainable by crossing plants of cultivars DH98-325-5 SRC and CMS NC1209-23 SRC and collecting seeds.

(64) An aspect of the present disclosure provides for parts of the cultivars NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, and hybrid cultivars NC 3 SRC and NC 6 SRC. A part of a cultivar can comprise any plant part and includes, but is not limited to, leaves, pollen, embryos, cotyledons, hypocotyls, roots, root tips, anthers, flowers, ovules, shoots, stems, stalks, pith and capsules, tissue culture comprising tissue, callus, cells or protoplasts of the cultivars NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, and hybrid cultivars NC 3 SRC and NC 6 SRC. In another aspect, the present disclosure provides for parts from hybrids of cultivars NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, and hybrid cultivars NC 3 SRC and NC 6 SRC derived tobacco plants. In yet another aspect, the present disclosure provides for parts from genetically modified (e.g., by conventional breeding or genetic engineering techniques) forms of the foregoing plants and tissue culture.

(65) In another aspect, the present disclosure also provides for a plant grown from the seed of a NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, or a hybrid NC 4 SRC plant in which alkaloids obtained from tobacco plants grown for the seed have decreased nornicotine, as well as plant parts and tissue cultures from such plants, representative sample seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-121031 for NC1426-11 SRC, ATCC Accession No. PTA-121070 for CMS NC1426-11 SRC, ATCC Accession No. PTA-121032 for NC1426-17 SRC. Seeds of hybrid cultivar NC 4 SRC are obtainable by crossing plants of cultivars NC1426-17 SRC and CMS NC1426-11 SRC and collecting seeds.

(66) An aspect of the present disclosure provides for parts of the cultivars NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, and hybrid cultivar NC 4 SRC. A part of a cultivar can comprise any plant part and includes, but is not limited to, leaves, pollen, embryos, cotyledons, hypocotyls, roots, root tips, anthers, flowers, ovules, shoots, stems, stalks, pith and capsules, tissue culture comprising tissue, callus, cells or protoplasts of the cultivars NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, and hybrid cultivar NC 4 SRC. In another aspect, the present disclosure provides for parts from hybrids of cultivars NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, and hybrid cultivar NC 4 SRC derived tobacco plants. In yet another aspect, the present disclosure provides for parts from genetically modified (e.g., by conventional breeding or genetic engineering techniques) forms of the foregoing plants and tissue culture.

(67) Additional aspects of the present disclosure provide products comprising tobacco from the plants of the present disclosure, and parts thereof. Other aspects of the disclosure provide cured plant parts, which include, but are not limited to, a leaf, pollen, ovule, embryo, cotyledon, hypocotyl, meristematic cell, protoplast, root, root tip, pistil, anther, flower, shoot, stem, pod, petiole, and the like, and combinations thereof.

(68) Thus, in some aspects, the present disclosure provides a cured tobacco comprising the leaves of the tobacco plant designated NC1209-23 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-121049. In another aspect, the present disclosure provides a cured tobacco comprising the leaves of the tobacco plant designated CMS NC1209-23 SRC. In another aspect, the present disclosure provides a cured tobacco comprising the leaves of the tobacco plant designated DH19 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-121052. In yet another aspect, the present disclosure provides a cured tobacco comprising the leaves of the hybrid tobacco plant designated NC 3 SRC. Seeds of hybrid cultivar NC 3 SRC are obtainable by crossing plants of cultivars DH19 SRC and CMS NC1209-23 SRC and collecting seeds.

(69) In another aspect, the present disclosure provides a cured tobacco comprising the leaves of the tobacco plant designated DH98-325-5 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-121051. In yet another aspect, the present disclosure provides a cured tobacco comprising the leaves of the hybrid tobacco plant designated NC 6 SRC. Seeds of hybrid cultivar NC 6 SRC are obtainable by crossing plants of cultivars DH98-325-5 SRC and CMS NC1209-23 SRC and collecting seeds.

(70) In an aspect, the present disclosure provides a cured tobacco comprising the stems of the tobacco plant designated NC1209-23 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-121049. In another aspect, the present disclosure provides a cured tobacco comprising the stems of the tobacco plant designated CMS NC1209-23 SRC. In an aspect, the present disclosure provides a cured tobacco comprising the stems of the tobacco plant designated DH19 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-121052. In yet another aspect, the present disclosure provides a cured tobacco comprising the stems of the hybrid tobacco plant designated NC 3 SRC.

(71) In an aspect, the present disclosure provides a cured tobacco comprising the stems of the tobacco plant designated DH98-325-5 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-121051. In yet another aspect, the present disclosure provides a cured tobacco comprising the stems of the hybrid tobacco plant designated NC 6 SRC.

(72) In an aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the tobacco plants designated NC1209-23 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-121049. In another aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the tobacco plants designated CMS NC1209-23 SRC. In an aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the tobacco plants designated DH19 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-121052. In yet another aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the hybrid tobacco plants designated NC 3 SRC.

(73) In an aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the tobacco plants designated DH98-325-5 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-121051. In yet another aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the hybrid tobacco plants designated NC 6 SRC.

(74) In some aspects, the present disclosure provides a cured tobacco comprising the leaves of the tobacco plant designated NC1426-11 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-121031. In another aspect, the present disclosure provides a cured tobacco comprising the leaves of the tobacco plant designated CMS NC1426-11 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-121070. In another aspect, the present disclosure provides a cured tobacco comprising the leaves of the tobacco plant designated NC1426-17 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-121032. In yet another aspect, the present disclosure provides a cured tobacco comprising the leaves of the hybrid tobacco plant designated NC 4 SRC. Seeds of hybrid cultivar SRC NC 4 SRC are obtainable by crossing plants of cultivars NC1426-17 SRC and CMS NC1426-11 SRC and collecting seeds.

(75) In an aspect, the present disclosure provides a cured tobacco comprising the stems of the tobacco plant designated NC1426-11 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-121031. In another aspect, the present disclosure provides a cured tobacco comprising the stems of the tobacco plant designated CMS NC1426-11 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-121070. In an aspect, the present disclosure provides a cured tobacco comprising the stems of the tobacco plant designated NC1426-17 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-121032. In yet another aspect, the present disclosure provides a cured tobacco comprising the stems of the hybrid tobacco plant designated NC 4 SRC.

(76) In an aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the tobacco plants designated NC1426-11 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-121031. In another aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the tobacco plants designated CMS NC1426-11 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-121070. In an aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the tobacco plants designated NC1426-17 SRC, a representative sample seed of said cultivar having been deposited with the ATCC under ATCC Accession No. PTA-121032. In yet another aspect, the present disclosure provides a cured tobacco comprising the leaves and stems of the hybrid tobacco plants designated NC 4 SRC.

(77) The present disclosure also provides a container of NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, or hybrid NC 3 SRC or NC 6 SRC seeds or other seeds of the present disclosure in which alkaloids obtained from tobacco plants grown from greater than about 50% of the seeds have decreased nornicotine. In another aspect, alkaloids obtained from NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, or hybrid NC 3 SRC or NC 6 SRC plants or other plants of the present disclosure grown from greater than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the seeds in the container have decreased nornicotine, representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-121049 for NC1209-23 SRC, ATCC Accession No. PTA-121051 for DH98-325-5 SRC and/or ATCC Accession No. PTA-121052 for DH19 SRC.

(78) The container of NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, or hybrid NC 3 SRC or NC 6 SRC seeds or other seeds of the present disclosure may contain any number, weight or volume of seeds. For example, a container can contain at least, or greater than, about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000 or more seeds. Alternatively, the container can contain at least, or greater than, about 1 ounce, 5 ounces, 10 ounces, 1 pound, 2 pounds, 3 pounds, 4 pounds, 5 pounds or more seeds. Representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-121049 for NC1209-23 SRC ATCC Accession No. PTA-121051 for DH98-325-5 SRC, or ATCC Accession No. PTA-121052 for DH19 SRC.

(79) Containers of NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, or hybrid NC 3 SRC or NC 6 SRC seeds or other seeds of the present disclosure may be any container available in the art. By way of a non-limiting example, a container may be a box, a bag, a packet, a pouch, a tape roll, a pail, a foil, or a tube. Representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-121049 for NC1209-23 SRC ATCC Accession No. PTA-121051 for DH98-325-5 SRC, and/or ATCC Accession No. PTA-121052 for DH19 SRC.

(80) In another aspect, the present disclosure also provides a container of NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, or hybrid cultivar NC 3 SRC or NC 6 SRC in which greater than about 50% of NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, or hybrid NC 3 SRC seeds or other seeds of the present disclosure have decreased nornicotine. Representative samples of seeds of these cultivars having been deposited with the ATCC, for example under ATCC Accession No. PTA-121049 for NC1209-23 SRCATCC Accession No. PTA-121051 for DH98-325-5 SRC, and/or ATCC Accession No. PTA-121052 for DH19 SRC.

(81) In one aspect, the present disclosure provides a seed of a NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, or hybrid NC 3 SRC or NC 6 SRC plant or other plant of the present disclosure in which a plant grown from a seed is male sterile. Representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-121049 for NC1209-23 SRCATCC Accession No. PTA-121051 for DH98-325-5 SRC, ATCC Accession No. PTA-121052 for DH19 SRC.

(82) The present disclosure also provides a container of NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, or hybrid NC 4 SRC seeds or other seeds of the present disclosure in which alkaloids obtained from tobacco plants grown from greater than about 50% of the seeds have decreased nornicotine. In another aspect, alkaloids obtained from NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, or hybrid NC 4 SRC plants or other plants of the present disclosure grown from greater than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the seeds in the container have decreased nornicotine, representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-121031 for NC1426-11 SRC, ATCC Accession No. PTA-121070 for CMS NC1426-11 SRC, and/or ATCC Accession No. PTA-121032 for NC1426-17 SRC. Seeds of hybrid cultivar NC 4 SRC are obtainable by crossing plants of cultivars NC1426-17 SRC and CMS NC1426-11 SRC and collecting seeds.

(83) The container of NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, or hybrid NC 4 SRC seeds or other seeds of the present disclosure may contain any number, weight or volume of seeds. For example, a container can contain at least, or greater than, about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000 or more seeds. Alternatively, the container can contain at least, or greater than, about 1 ounce, 5 ounces, 10 ounces, 1 pound, 2 pounds, 3 pounds, 4 pounds, 5 pounds or more seeds.

(84) Containers of NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC or hybrid NC 4 SRC seeds or other seeds of the present disclosure may be any container available in the art. By way of a non-limiting example, a container may be a box, a bag, a packet, a pouch, a tape roll, a pail, a foil, or a tube. Representative samples of seeds of these cultivars having been deposited with the ATCC, for example, under ATCC Accession No. PTA-121031 for NC1426-11 SRC, ATCC Accession No. PTA-121070 for CMS NC1426-11 SRC, and/or ATCC Accession No. PTA-121032 for NC1426-17 SRC. Seeds of hybrid cultivar NC 4 SRC are obtainable by crossing plants of cultivars NC1426-17 SRC and CMS NC1426-11 SRC and collecting seeds.

(85) In another aspect, the present disclosure also provides a container of NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, or hybrid cultivar NC 4 SRC in which greater than about 50% of NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, or hybrid NC 4 SRC seeds or other seeds of the present disclosure have decreased nornicotine.

(86) In one aspect, the present disclosure provides a seed of a NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, or hybrid NC 4 SRC plant or other plant of the present disclosure in which a plant grown from a seed is male sterile.

(87) Tobacco material obtained from the tobacco lines, varieties or hybrids of the present disclosure can be used to make tobacco products including, without limitation, cigarette products (e.g., cigarettes and bidi cigarettes), cigar products (e.g., cigar wrapping tobacco and cigarillos), pipe tobacco products, smokeless cigarette products, smokeless tobacco products (e.g., moist snuff, dry snuff, and chewing tobacco), films, chewables, tabs, shaped parts, gels, consumable units, insoluble matrices, hollow shapes and the like. See, e.g., U.S. Patent Publication No. US 2006/0191548, which is herein incorporated by reference in its entirety.

(88) Tobacco products derived from plants of the present disclosure also include cigarettes and other smoking articles, particularly those smoking articles including filter elements, wherein the rod of smokeable material includes cured tobacco within a tobacco blend. In an aspect, a tobacco product can include but is not limited to pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and/or cut tobacco or any combination thereof.

(89) In an aspect, a tobacco product of the instant disclosure is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, hookah tobacco, shredded tobacco, and cut tobacco. In another aspect, a tobacco product of the instant disclosure is a smokeless tobacco product. In a further aspect, a tobacco product of the instant disclosure is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff. In yet another aspect, a tobacco product of the instant disclosure is selected from the group consisting of an electronically heated cigarette, an e-cigarette, an electronic vaporing device.

(90) In an aspect, the tobacco product of the present disclosure can be a blended tobacco product. In other aspects of the disclosure, the tobacco product of the present disclosure can be a reduced nicotine tobacco product. In still other aspects, the tobacco product of the present disclosure can be a blended tobacco product with reduced nicotine content. Thus, the tobacco product of the present disclosure can be a blended reduced nicotine tobacco product. Tobacco product material comprises a blend of tobacco materials from the present disclosure, wherein the blend comprises at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95 percent by weight of a cured tobacco, or any range therein, based on the dry weight of the tobacco material. US 2008/0245377 is herein incorporated by reference for blend mixtures in its entirety.

(91) In an aspect, tobacco products having a reduced amount of nitrosamine content can be manufactured using tobacco plant material from plants and plant parts of the present disclosure. Thus, in some aspects, a tobacco product manufactured using tobacco plant material from plants and plant parts of the present disclosure can comprise a reduced amount of nornicotine of less than about 3 mg/g. For example, the nornicotine content in such a product can be 3.0 mg/g, 2.5 mg/g, 2.0 mg/g, 1.5 mg/g, 1.0 mg/g, 750 g/g, 500 g/g, 250 g/g, 100 g/g, 75 g/g, 50 g/g, 25 g/g, 10 g/g, 5 g/g, 1 g/g, 750 ng/g, 500 ng/g, 250 ng/g, 100 ng/g, 75 ng/g, 50 ng/g, 25 ng/g, 10 ng/g, 5 ng/g, 1 ng/g, 750 pg/g, 500 pg/g, 250 pg/g, 100 pg/g, 75 pg/g, 50 pg/g, 25 pg/g, 10 pg/g, 7.0 pg/g, 5.0 pg/g, 4.0 pg/g, 2.0 pg/g, 1.0 pg/g, 0.5 pg/g, 0.4 pg/g, 0.2 pg/g, 0.1 pg/g, 0.05 pg/g, 0.01 pg/g, or undetectable, or any range therein. The tobacco product typically has a reduced amount of NNN of less than about 10 pg/g. For example, the NNN content in such a product can be about 10 pg/g, 7.0 pg/g, 5.0 pg/g, 4.0 pg/g, 2.0 pg/g, 1.0 pg/g, 0.5 pg/g, 0.4 pg/g, 0.2 pg/g, 0.1 pg/g, 0.05 pg/g, 0.01 pg/g, or undetectable, or any range therein. The percentage of secondary alkaloids relative to total alkaloid content contained in a plant of the present disclosure may not be statistically different than from a commercial seedlot of NC1209-23, DH19, DH98-325-5, NC1426-11, NC1426-17, or hybrid cultivar NC 3 LC, NC 6 LC, NC 4 LC.

(92) A tobacco plant of the present disclosure designated NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, or hybrid cultivar NC 3 SRC, NC 6 SRC, or NC 4 SRC, carrying the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82e10 P381 S alleles can be used in a plant breeding program to create useful lines, cultivars, varieties, progeny, inbreds, and hybrids. Thus, in some aspects, an F.sub.1, F.sub.2, F.sub.3, or later generation tobacco plant containing the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82e10 P381S alleles is crossed with a second Nicotiana plant, and progeny of the cross are identified in which the cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82e10 P381S alleles are present. It will be appreciated that the second Nicotiana plant can be NC1209-23, CMS NC1209-23, DH19, DH98-325-5, NC1426-11, CMS NC1426-11, NC1426-17, or any other Nicotiana species or line, optionally with an additional desirable trait, such as herbicide resistance.

(93) In still other aspects, methods of the present disclosure further include self-pollinating or pollinating a male sterile pollen acceptor with a pollen donor capable of being used in production of a progeny plant of the present disclosure, such as a male sterile hybrid of the present disclosure. Either the male sterile pollen acceptor plant or the pollen donor plant has at least one mutant allele, two, or even three mutant alleles at a nicotine demethylase locus, such as the cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S alleles. In an aspect, all three alleles at each nicotine demethylase locus are mutant alleles, making the plant homozygous for cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82e10 P381S.

(94) Breeding can be carried out via any known procedures. DNA fingerprinting, SNP or similar technologies may be used in a marker-assisted selection (MAS) breeding program to transfer or breed mutant alleles of a nicotine demethylase gene into other tobaccos. For example, a breeder can create segregating populations from hybridizations of a genotype containing cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S alleles with an agronomically desirable genotype. Plants in the F.sub.2 or backcross generations can be screened using a marker developed from cyp82e4 W329Stop, the cyp82e5v2 W422Stop, or cyp82e10 P381S alleles or a fragment thereof, using one of the techniques known in the art or disclosed herein. Plants identified as possessing one or more cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82e10 P381S alleles can be backcrossed or self-pollinated to create a second population to be screened. Depending on the expected inheritance pattern or the MAS technology used, it may be necessary to self-pollinate the selected plants before each cycle of backcrossing to aid identification of the desired individual plants. Backcrossing or other breeding procedure can be repeated until the desired phenotype of the recurrent parent is recovered. A recurrent parent in the present disclosure can be NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, NC 3 SRC, NC 6 SRC, or NC 4 SRC. 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 entireties.

(95) Nicotiana species which exhibit breeding compatibility with Nicotiana tabacum include Nicotiana amplexicaulis, PI 271989; Nicotiana benthamiana PI 555478; Nicotiana bigelovii PI 555485; Nicotiana debneyi; Nicotiana excelsior PI 224063; Nicotiana glutinosa PI 555507; Nicotiana goodspeedii PI 241012; Nicotiana gossei PI 230953; Nicotiana hesperis PI 271991; Nicotiana knightiana PI 555527; Nicotiana maritima PI 555535; Nicotiana megalosiphon PI 555536; Nicotiana nudicaulis PI 555540; Nicotiana paniculata PI 555545; Nicotiana plumbaginifolia PI 555548; Nicotiana repanda PI 555552; Nicotiana rustica; Nicotiana suaveolens PI 230960; Nicotiana sylvestris PI 555569; Nicotiana tomentosa PI 266379; Nicotiana tomentosiformis; and Nicotiana trigonophylla PI 555572. See also, Compendium of Tobacco Diseases published by American Phytopathology Society, or The Genus Nicotiana Illustrated, published by Japan Tobacco Inc, hereby incorporated by reference in their entireties.

(96) The result of a plant breeding program using the mutant tobacco plants described herein includes useful lines, cultivars, varieties, progeny, inbreds, and hybrids. As used herein, the term cultivar or variety refers to a population of plants that share constant characteristics which separate them from other plants of the same species. A cultivar or variety is often, although not always, sold commercially. While possessing one or more distinctive traits, a cultivar or variety is further characterized by a very small overall variation between individuals within that cultivar or variety. A pure line variety may be created by several generations of self-pollination and selection, or vegetative propagation from a single parent using tissue or cell culture techniques. A cultivar or variety can be essentially derived from another cultivar, line, or variety. As defined by the International Convention for the Protection of New Varieties of Plants (Dec. 2, 1961, as revised at Geneva on Nov. 10, 1972, on Oct. 23, 1978, and on Mar. 19, 1991), a cultivar or variety is essentially derived from an initial cultivar or variety if: a) it is predominantly derived from the initial cultivar or variety, or from a cultivar or variety that is predominantly derived from the initial cultivar or variety, while retaining the expression of the essential characteristics that result from the genotype or combination of genotypes of the initial cultivar or variety; b) it is clearly distinguishable from the initial cultivar or variety; and c) except for the differences which result from the act of derivation, it conforms to the initial cultivar or variety in the expression of the essential characteristics that result from the genotype or combination of genotypes of the initial cultivar or variety. Essentially derived varieties can be obtained, for example, by the selection of a natural or induced mutant, a somaclonal variant, a variant individual from plants of the initial cultivar or variety, backcrossing, or transformation. A line as distinguished from a cultivar or variety most often denotes a group of plants used non-commercially, for example in plant research. A line typically displays little overall variation between individuals for one or more traits of interest, although there may be some variation between individuals for other traits.

(97) 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.sub.1 hybrid seeds to form on the female plants. Self-pollination of female plants can be prevented by emasculating the flowers at an early stage of flower development. Alternatively, pollen formation can be prevented on the female parent plants using a form of male sterility. For example, male sterility can be produced by cytoplasmic male sterility (CMS), or transgenic male sterility wherein a transgene inhibits microsporogenesis and/or pollen formation, or self-incompatibility. Female parent plants containing CMS are particularly useful. In aspects in which the female parent plants are CMS, pollen may be harvested from male fertile plants and applied manually to the stigmas of CMS female parent plants, and the resulting F.sub.1 seed is harvested.

(98) Plants can be used to form single-cross tobacco F.sub.1 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 F.sub.1 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.sub.1 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.sub.1 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 F.sub.1 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.

(99) Successful crosses yield F.sub.1 plants that are fertile, have cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S alleles, and can be backcrossed with one of the parents, such as NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, NC 3 SRC, NC 6 SRC, or NC 4 SRC, if desired. In some aspects, a plant population in the F.sub.2 generation is screened for cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82e10 P381S alleles. Selected plants can be crossed with one of the parents and the first backcross (BC1) generation plants are self-pollinated to produce a BC1 F.sub.2 population that is again screened for variant nicotine demethylase gene expression (e.g., the null version of the nicotine demethylase gene). The process of backcrossing, self-pollination, and screening is repeated, for example, at least four times, until the final screening produces a plant that is fertile and reasonably similar to the recurrent parent. This plant, if desired, is self-pollinated and the progeny are subsequently screened again to confirm that the plant exhibits the same low nicotine conversion phenotype as NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, NC 3 SRC, NC 6 SRC, or NC 4 SRC. Breeder's seed of the selected plant is produced using standard methods including, for example, field testing, confirmation of the null condition for nicotine demethylase, chemical analyses of cured leaf to determine the level of alkaloids and/or chemical analyses of cured leaf to determine the ratio of nornicotine to nicotine+nornicotine.

(100) In one aspect, an F.sub.1 progeny is the result of a cross between NC1209-23 SRC and CMS NC1209-23 SRC to generate F.sub.1 progeny that are male sterile. In another aspect, an F.sub.1 progeny is the result of a cross between DH19 SRC and CMS NC1209-23 SRC to generate F.sub.1 progeny that are male sterile. In another aspect, an F.sub.1 progeny is the result of a cross between DH98-325-5 SRC and CMS NC1209-23 SRC to generate F.sub.1 progeny that are male sterile. In one aspect, an F.sub.1 progeny is the result of a cross between NC1426-11 SRC and CMS NC1426-11 SRC to generate F.sub.1 progeny that are male sterile. In another aspect, an F.sub.1 progeny is the result of a cross between NC1426-17 SRC and CMS NC1426-11 SRC to generate F.sub.1 progeny that are male sterile. Male sterile tobacco plants may be produced by any method known in the art. Methods of producing male sterile tobacco are described in Wernsman, E. A., and Rufty, R. C. 1987. Chapter Seventeen. Tobacco. Pages 669-698 In: Cultivar Development. Crop Species. W. H. Fehr (ed.), MacMillan Publishing Go., Inc., New York, N.Y. 761 pp.

(101) The present disclosure further provides methods of producing a tobacco plant by crossing one of cultivars NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, and hybrids NC 3 SRC, NC 6 SRC, and NC 4 SRC with itself or a different tobacco line. The disclosure further relates to methods for producing other tobacco cultivars or breeding lines derived from cultivars NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, and hybrids NC 3 SRC, NC 6 SRC, and NC 4 SRC by crossing a plant of cultivars NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, and hybrids NC 3 SRC, NC 6 SRC, and NC 4 SRC with a second tobacco plant and growing the progeny seed to yield a NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, or hybrid NC 3 SRC, NC 6 SRC, or NC 4 SRC-derived tobacco plant. An additional aspect of the present disclosure provides a method for producing a tobacco plant that contains in its genetic material one or more transgenes, comprising crossing cultivars of the present disclosure with a second cultivar containing one or more transgenes wherein progeny are produced, so that the genetic material of the progeny that result from the cross comprise the transgene(s) optionally operably linked to one or more regulatory elements. In one aspect, the second cultivar may be a plant derived from cultivars NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, or hybrid NC 3 SRC, NC 6 SRC, or NC 6 SRC transformed with one or more transgenes.

(102) The disclosure further provides for the vegetative propagation of a plant of cultivars NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, and hybrids NC 3 SRC, NC 6 SRC, and NC 4 SRC, hybrids and progeny thereof. In one aspect, the disclosure provides for a method of vegetatively propagating a plant of a tobacco cultivar comprising collecting tissue capable of being propagated from a plant of cultivars NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, and hybrids NC 3 SRC, NC 6 SRC, and NC 4 SRC, cultivating the tissue to obtain a proliferated shoot and rooting the proliferated shoots to obtain a rooted plantlet. In another aspect, the plant tissue may be collected from an F.sub.1 hybrid of a plant of cultivars NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, and hybrids NC 3 SRC, NC 6 SRC, and NC 4 SRC. In an aspect, the plant tissue may be collected from an F.sub.2, F.sub.3, F.sub.4 or later progeny plant obtained by breeding a plant of cultivars NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, and hybrids NC 3 SRC, NC 6 SRC, and NC 4 SRC.

(103) 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.

(104) It is understood that a tobacco plant of the present disclosure, including, but not limited to, NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, and hybrid cultivars NC 3 SRC, NC 6 SRC, and NC 4 SRC can be transformed by a genetic construct (nucleic acid construct) or transgene using any technique known in the art. Without limitation, an example of a desired trait can include herbicide resistance, pest resistance, disease resistance, high yield, high grade index, curability, curing quality, mechanical harvestability, holding ability, leaf quality, height, plant maturation (e.g., early maturing, early to medium maturing, medium maturing, medium to late maturing, or late maturing), stalk size (e.g., small, medium, or large stalk), or leaf number per plant (e.g., small (e.g., 5-10 leaves), medium (e.g., 11-15 leaves), or large (e.g., 16-21) number of leaves), or any combination. Any plant of the present disclosure can be used as a basis for tissue culture, regeneration, transformed, or a combination of any of these. In an aspect, a plant of the present disclosure derived by tissue culture, transformation, or both has all, or essentially all, of the morphological and physiological characteristics of cultivar NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, CMS NC1426-11 SRC, NC1426-17 SRC, or hybrid NC 3 SRC, NC 6 SRC or NC 4 SRC.

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

EXAMPLES

Example 1

Breeding of Homozygous cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82e10 P381S Mutant Plants into the NC1209-23 Burley Tobacco Cultivar

(106) NC1209-23 SRC is a backcross-derived version of burley tobacco cultivar NC1209-23 carrying introduced mutations in three genes (CYP82E4 (SEQ ID NO: 5), CYP82E5 (SEQ ID NO: 8), and CYP82E10 (SEQ ID NO: 9)) previously documented to encode for nicotine demethylase enzymes (Lewis et al., 2010). The introduced mutations in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) and CYP82E5 (cyp82e5v2 W422Stop (SEQ ID NO: 2)) encode for premature stop codons which render the genes non-functional. The introduced mutation in CYP82E10 (cyp82e10 P381S (SEQ ID NO: 11)) does not encode for a premature stop codon, but does render the gene product non-functional for converting nicotine to nornicotine (Lewis et al. 2010). When in homozygous condition, the three mutations result in tobacco plants with (1) reduced genetic capacity to demethylate nicotine to form nornicotine, and (2) a corresponding diminished potential to accumulate N-nitrosonornicotine (NNN), a potent carcinogen found in many tobacco products. The mutation in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) also provides phenotypic stability for the nicotine conversion trait and eliminates the requirement to utilize the cumbersome LC method for reducing levels of nornicotine in tobacco cultivars.

(107) The original tobacco cultivar NC1209-23 is a fertile inbred line. CMS NC1209-23 is a cytoplasmic male-sterile version of NC1209-23. To develop NC1209-23 SRC, an individual plant of NC1209-23 is pollinated with a plant of the genetic background DH98-325-6 carrying mutations in each of the three nicotine demethylase genes. F.sub.1 individuals originating from this cross and heterozygous for each mutation are backcrossed to the recurrent parent, NC1209-23, to produce BC.sub.1F.sub.1 progeny. BC.sub.1F.sub.1 progeny are screened using genotyping methodologies to identify individuals heterozygous for all three mutations. A single triple heterozygous BC.sub.1F.sub.1 plant is backcrossed to NC1209-23 to produce BC.sub.2F.sub.1 progeny. The process of backcrossing and identification of individuals heterozygous for all three mutations is repeated through the BC.sub.3F.sub.1, BC.sub.4F.sub.1, BC.sub.5F.sub.1, BC.sub.6F.sub.1, and BC.sub.7F.sub.1 stages. At the BC.sub.7F.sub.1 stage, individuals heterozygous for all three mutations from each pedigree are self-pollinated to produce BC.sub.7F.sub.2 seed. A large number of BC.sub.7F.sub.2 progeny from each pedigree are genotyped to identify individuals homozygous for all three mutations. A single BC.sub.7F.sub.2 plant homozygous for all three mutations is self-pollinated to produce a BC.sub.7F.sub.3 family (NC1209-23 SRC) in which the wild-type CYP82E4, CYP82E5v2, and CYP82E10 alleles of NC1209-23 were replaced by the mutant alleles (cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S).

(108) The male-sterile (CMS) version of NC1209-23 SRC (CMS NC1209-23 SRC) is produced by crossing a plant of CMS NC1209-23 as a female with pollen of NC1209-23 SRC to produce male-sterile plants heterozygous for all three mutations. A single male-sterile plant resulting from this cross is then subsequently backcrossed as a female to NC1209-23 SRC to produce progeny that are segregating for individuals homozygous for all three mutations. Triple homozygous individuals are identified by DNA genotyping to produce the CMS NC1209-23 SRC line. Because the line is male-sterile, it is maintained via pollination with NC1209-23 SRC.

(109) Commercial NC1209-23 SRC is produced by pollinating plants of CMS NC1209-23 SRC with pollen of NC1209-23 SRC.

Example 2

Breeding of Homozygous cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82e10 P381S Mutant Plants into the DH19 Burley Tobacco Cultivar

(110) DH19 SRC is a backcross-derived version of burley tobacco cultivar DH19 carrying introduced null mutations in three genes (CYP82E4 (SEQ ID NO: 5), CYP82E5 (SEQ ID NO: 8), and CYP82E10 (SEQ ID NO: 9)) previously documented to encode for nicotine demethylase enzymes (Lewis et al., Phytochemistry, 71 (2010), 1988-1998). The introduced mutations in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) and CYP82E5 (cyp82e5v2 W422Stop (SEQ ID NO: 2)) encode for premature stop codons which render the genes non-functional. The introduced mutation in CYP82E10 (cyp82e10 P381S (SEQ ID NO: 11)) does not encode for a premature stop codon, but does render the gene product non-functional for converting nicotine to nornicotine (Id). When in homozygous condition, the three mutations result in tobacco plants with (1) reduced genetic capacity to demethylate nicotine to form nornicotine, and (2) a corresponding diminished potential to accumulate N-nitrosonornicotine (NNN), a potent carcinogen found in many tobacco products. The mutation in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) also provides phenotypic stability for the nicotine conversion trait and eliminates the requirement to utilize the cumbersome LC method for reducing levels of nornicotine in tobacco cultivars (see e.g., Jack et al. 2007. Implications of reducing nornicotine accumulation in burley tobacco: appendix Athe LC protocol. Rec. Adv. Tob. Sci. 33: 58-79).

(111) The original tobacco cultivar DH19 is a fertile inbred line. To develop DH19 SRC, an individual plant of DH19 is initially pollinated with a plant of the genetic background DH98-325-6 carrying mutations in each of the three nicotine demethylase genes. F.sub.1 individuals originating from this cross and heterozygous for each mutation are backcrossed to the recurrent parent, DH19, to produce BC.sub.1F.sub.1 progeny. BC.sub.1F.sub.1 progeny are screened using genotyping methodologies to identify individuals heterozygous for all three mutations. A single triple heterozygous BC.sub.1F.sub.1 plant is backcrossed to DH19 to produce BC.sub.2F.sub.1 progeny. The process of backcrossing and identification of individuals heterozygous for all three mutations is repeated through the BC.sub.3F.sub.1, BC.sub.4F.sub.1, BC.sub.5F.sub.1, BC.sub.6F.sub.1, and BC.sub.7F.sub.1 stages. At the BC.sub.7F.sub.1 stage, individuals heterozygous for all three mutations from each pedigree are self-pollinated to produce BC.sub.7F.sub.2 seed. A large number of BC.sub.7F.sub.2 progeny from each pedigree are genotyped to identify individuals homozygous for all three mutations. A single BC.sub.7F.sub.2 plant homozygous for all three mutations is self-pollinated to produce a BC.sub.7F.sub.3 family (DH19 SRC) in which the wild-type CYP82E4, CYP82E5v2, and CYP82E10 alleles of DH19 were replaced by the mutant alleles (cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S).

Example 3

Testing of DH19 SRC

(112) DH19 SRC is evaluated for cured leaf chemistry, yield, and physical quality at two North Carolina field research locations during 2013 (Reidsville and Waynesville). DH19 is included for comparison. The experimental design at each location is a randomized complete block design with four replications. Experimental units are single 20-plant plots. Plots are harvested and air-cured. Plot weights are used to determine per acre yields. Cured leaf is evaluated by a former USDA tobacco grader. Fifty gram composite leaf samples are collected from each plot and analyzed for percent nicotine, nornicotine, anatabine, anabasine, and percent nicotine conversion using gas chromatography equipment.

(113) Comparisons using the least significant difference (LSD) test indicated that DH19 SRC has significantly (P<0.05) lower levels of nornicotine and percent nicotine conversion relative to DH19 (Table 1). DH19 and DH19 SRC are not significantly different from each other for percent nicotine, yield, or cured leaf quality indices.

Example 4

Breeding of Homozygous cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82e10 P381S Mutant Plants into the NC 3 Burley Tobacco Cultivar

(114) The original tobacco cultivar NC 3 LC is a hybrid generated by pollinating plants of a male-sterile breeding line CMS NC1209-23 with pollen produced by fertile breeding line DH19. Hybrid cultivar NC 3 SRC is a hybrid generated by pollinating plants of a male-sterile breeding line CMS NC1209-23 SRC with pollen produced by fertile breeding line DH19 SRC with each breeding line carrying introduced deleterious mutations in three genes (CYP82E4 (SEQ ID NO: 5), CYP82E5 (SEQ ID NO: 8), and CYP82E10 (SEQ ID NO: 9)) previously documented to encode for nicotine demethylase enzymes (Lewis et al., Phytochemistry, 71 (2010), 1988-1998). The introduced mutations in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) and CYP82E5 (cyp82e5v2 W422Stop (SEQ ID NO: 2)) encode for premature stop codons which render the genes non-functional. The introduced mutation in CYP82E10 (cyp82e10 P381S (SEQ ID NO: 11)) does not encode for a premature stop codon, but does render the gene product non-functional for converting nicotine to nornicotine (Id.). When in homozygous condition, the three mutations result in tobacco plants with (1) reduced genetic capacity to demethylate nicotine to form nornicotine, and (2) a corresponding diminished potential to accumulate N-nitrosonornicotine (NNN), a potent carcinogen found in many tobacco products. The mutation in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) also provides phenotypic stability for the nicotine conversion trait and eliminates the requirement to utilize the cumbersome LC method for reducing levels of nornicotine in tobacco cultivars (see e.g., Jack et al. 2007. Implications of reducing nornicotine accumulation in burley tobacco: appendix Athe LC protocol. Rec. Adv. Tob. Sci. 33: 58-79).

(115) The original tobacco cultivar NC 3 LC is a hybrid generated by pollinating plants of a male-sterile breeding line CMS NC1209-23 with pollen produced by fertile breeding line DH19. To develop hybrid cultivar NC 3 SRC, individual plants of fertile NC1209-23 are first pollinated with a plant of the genetic background DH98-325-6 carrying mutations in each of the three nicotine demethylase genes. F.sub.1 individuals originating from each cross and heterozygous for each mutation are backcrossed to the recurrent parent (NC1209-23) to produce BC.sub.1F.sub.1 progenies.

(116) BC.sub.1F.sub.1 progenies are screened using genotyping methodologies to identify individuals heterozygous for all three mutations. A single BC.sub.1F.sub.1 plant from each pedigree is backcrossed to NC1209-23, to produce BC.sub.2F.sub.1 progenies. The process of backcrossing and identification of individuals heterozygous for all three mutations is repeated through the BC.sub.3F.sub.1, BC.sub.4F.sub.1, BC.sub.5F.sub.1, BC.sub.6F.sub.1, and BC.sub.7F.sub.1 stages. At the BC.sub.7F.sub.1 stage, individuals heterozygous for all three mutations from each pedigree are self-pollinated to produce BC.sub.7F.sub.2 seed. A large number of BC.sub.7F.sub.2 progeny from each pedigree are genotyped to identify individuals homozygous for all three mutations. BC.sub.7F.sub.2 plants homozygous for all three mutations are self-pollinated to produce BC.sub.7F.sub.3 families (NC1209-23 SRC) in which the wild-type CYP82E4, CYP82E5v2, and CYP82E10 alleles of NC1209-23 are replaced by the mutant alleles (cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S).

(117) The female parental line of NC1209-23, CMS NC1209-23, is cytoplasmic male sterile which causes pollen to not be produced. To develop CMS NC1209-23 SRC, a plant of CMS NC1209-23 is crossed with NC1209-23 SRC to produce male-sterile plants heterozygous for all three mutations. A single male-sterile plant resulting from this cross is backcrossed as a female to NC1209-23 SRC to produce progeny that are segregating for individuals homozygous for all three mutations. Triple homozygous individuals are identified by DNA genotyping to produce the CMS NC1209-23 SRC line. Because the line is male-sterile, it is maintained via pollination with NC1209-23 SRC (see above).

(118) Hybrid cultivar NC 3 SRC is produced by pollinating plants of CMS NC1209-23 SRC with pollen of DH19 SRC.

Example 5

Testing of Hybrid Cultivar NC 3 SRC

(119) Hybrid cultivar NC 3 SRC is evaluated for cured leaf chemistry, yield, and physical quality at two North Carolina field research locations during 2013 (Reidsville and Waynesville). Hybrid cultivar NC 3 LC is included for comparison. The experimental design at each location is a randomized complete block design with four replications. Experimental units are single 20-plant plots. Plots are harvested and air-cured. Plot weights are used to determine per acre yields. Cured leaf is evaluated by a former USDA tobacco grader. Fifty gram composite leaf samples are collected from each plot and analyzed for percent nicotine, nornicotine, anatabine, anabasine, and percent nicotine conversion using gas chromatography equipment.

(120) Comparisons using the least significant difference (LSD) test indicates that hybrid cultivar NC 3 SRC has significantly (P<0.05) lower levels of nornicotine and percent nicotine conversion relative to NC 3 LC (Table 1). NC 3 LC and hybrid cultivar NC 3 SRC are not significantly different from each other for percent nicotine, yield, or cured leaf quality indices.

Example 6

Breeding of Homozygous cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82e10 P381S Mutant Plants into the DH98-325-5 Burley Tobacco Cultivar

(121) DH98-325-5 SRC is a backcross-derived version of burley tobacco cultivar DH98-325-5 carrying introduced null mutations in three genes (CYP82E4 (SEQ ID NO; 5), CYP82E5 (SEQ ID NO: 8), and CYP82E10 (SEQ ID NO: 9)) previously documented to encode for nicotine demethylase enzymes (Lewis et al., Phytochemistry, 71 (2010), 1988-1998). The introduced mutations in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) and CYP82E5 (cyp82e5v2 W422Stop (SEQ ID NO: 2)) encode for premature stop codons which render the genes non-functional. The introduced mutation in CYP82E10 (cyp82e10 P381S (SEQ ID NO: 11)) does not encode for a premature stop codon, but does render the gene product non-functional for converting nicotine to nornicotine (Id.). When in homozygous condition, the three mutations result in tobacco plants with (1) reduced genetic capacity to demethylate nicotine to form nornicotine, and (2) a corresponding diminished potential to accumulate N-nitrosonornicotine (NNN), a potent carcinogen found in many tobacco products. The mutation in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) also provides phenotypic stability for the nicotine conversion trait and eliminates the requirement to utilize the cumbersome LC method for reducing levels of nornicotine in tobacco cultivars (see e.g., Jack et al. 2007. Implications of reducing nornicotine accumulation in burley tobacco: appendix Athe LC protocol. Rec. Adv. Tob. Sci. 33: 58-79).

(122) The original tobacco cultivar DH98-325-5 is a fertile inbred line. To develop DH98-325-5 SRC, an individual plant of DH98-325-5 is initially pollinated with a plant of the genetic background DH98-325-6 carrying mutations in each of the three nicotine demethylase genes. F.sub.1 individuals originating from this cross and heterozygous for each mutation are backcrossed to the recurrent parent, DH98-325-5, to produce BC.sub.1F.sub.1 progeny. BC.sub.1F.sub.1 progeny are screened using genotyping methodologies to identify individuals heterozygous for all three mutations. A single triple heterozygous BC.sub.1F.sub.1 plant is backcrossed to DH98-325-5 to produce BC.sub.2F.sub.1 progeny. The process of backcrossing and identification of individuals heterozygous for all three mutations is repeated through the BC.sub.3F.sub.1, BC.sub.4F.sub.1, BC.sub.5F.sub.1, BC.sub.6F.sub.1, and BC.sub.7F.sub.1 stages. At the BC.sub.7F.sub.1 stage, individuals heterozygous for all three mutations from each pedigree are self-pollinated to produce BC.sub.7F.sub.2 seed. A large number of BC.sub.7F.sub.2 progeny from each pedigree are genotyped to identify individuals homozygous for all three mutations. A single BC.sub.7F.sub.2 plant homozygous for all three mutations is self-pollinated to produce a BC.sub.7F.sub.3 family (DH98-325-5 SRC) in which the wild-type CYP82E4, CYP82E5v2, and CYP82E10 alleles of DH98-325-5 were replaced by the mutant alleles (cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S).

Example 7

Testing of DH98-325-5 SRC

(123) DH98-325-5 SRC is evaluated for cured leaf chemistry, yield, and physical quality at two North Carolina field research locations during 2013 (Reidsville and Waynesville). DH98-325-5 is included for comparison. The experimental design at each location is a randomized complete block design with four replications. Experimental units are single 20-plant plots. Plots are harvested and air-cured. Plot weights are used to determine per acre yields. Cured leaf is evaluated by a former USDA tobacco grader. Fifty gram composite leaf samples are collected from each plot and analyzed for percent nicotine, nornicotine, anatabine, anabasine, and percent nicotine conversion using gas chromatography equipment.

(124) Comparisons using the least significant difference (LSD) test indicated that DH98-325-5 SRC has significantly lower levels (P<0.05) of nornicotine and percent nicotine conversion relative to DH98-325-5 (Table 1). DH98-325-5 and DH98-325-5 SRC are not significantly different from each other for percent nicotine, yield, or cured leaf quality indices.

Example 8

Breeding of Homozygous cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82e10 P381S Mutant Plants into the NC 6LC Burley Tobacco Cultivar

(125) The original tobacco cultivar NC 6 LC is a hybrid generated by pollinating plants of a male-sterile breeding line CMS NC1209-23 with pollen produced by fertile breeding line DH98-325-5. Hybrid cultivar NC 6 SRC is a hybrid generated by pollinating plants of a male-sterile breeding line CMS NC1209-23 SRC with pollen produced by fertile breeding line DH98-325-5 SRC with each breeding line carrying introduced deleterious mutations in three genes (CYP82E4 (SEQ ID NO: 5), CYP82E5 (SEQ ID NO: 8), and CYP82E10 (SEQ ID NO: 9)) previously documented to encode for nicotine demethylase enzymes (Lewis et al., Phytochemistry, 71 (2010), 1988-1998). The introduced mutations in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) and CYP82E5 (cyp82e5v2 W422Stop (SEQ ID NO: 2)) encode for premature stop codons which render the genes non-functional. The introduced mutation in CYP82E10 (cyp82e10 P381S (SEQ ID NO: 11)) does not encode for a premature stop codon, but does render the gene product non-functional for converting nicotine to nornicotine (Id.). When in homozygous condition, the three mutations result in tobacco plants with (1) reduced genetic capacity to demethylate nicotine to form nornicotine, and (2) a corresponding diminished potential to accumulate N-nitrosonornicotine (NNN), a potent carcinogen found in many tobacco products. The mutation in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) also provides phenotypic stability for the nicotine conversion trait and eliminates the requirement to utilize the cumbersome LC method for reducing levels of nornicotine in tobacco cultivars (see e.g., Jack et al. 2007. Implications of reducing nornicotine accumulation in burley tobacco: appendix Athe LC protocol. Rec. Adv. Tob. Sci. 33: 58-79).

(126) The original tobacco cultivar NC 6 LC is a hybrid generated by pollinating plants of a male-sterile breeding line CMS NC1209-23 with pollen produced by fertile breeding line DH98-325-5. To develop hybrid cultivar NC 6 SRC, individual plants of fertile NC1209-23 are first pollinated with a plant of the genetic background DH98-325-6 carrying mutations in each of the three nicotine demethylase genes. F.sub.1 individuals originating from each cross and heterozygous for each mutation are backcrossed to the recurrent parent (NC1209-23) to produce BC.sub.1F.sub.1 progenies.

(127) BC.sub.1F.sub.1 progenies are screened using genotyping methodologies to identify individuals heterozygous for all three mutations. A single BC.sub.1F.sub.1 plant from each pedigree is backcrossed to NC1209-23, to produce BC.sub.2F.sub.1 progenies. The process of backcrossing and identification of individuals heterozygous for all three mutations is repeated through the BC.sub.3F.sub.1, BC.sub.4F.sub.1, BC.sub.5F.sub.1, BC.sub.6F.sub.1, and BC.sub.7F.sub.1 stages. At the BC.sub.7F.sub.1 stage, individuals heterozygous for all three mutations from each pedigree are self-pollinated to produce BC.sub.7F.sub.2 seed. A large number of BC.sub.7F.sub.2 progeny from each pedigree are genotyped to identify individuals homozygous for all three mutations. BC.sub.7F.sub.2 plants homozygous for all three mutations are self-pollinated to produce BC.sub.7F.sub.3 families (NC1209-23 SRC) in which the wild-type CYP82E4, CYP82E5v2, and CYP82E10 alleles of NC1209-23 are replaced by the mutant alleles (cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S).

(128) The female parental line of NC1209-23, CMS NC1209-23, is cytoplasmic male sterile which causes pollen to not be produced. To develop CMS NC1209-23 SRC, a plant of CMS NC1209-23 is crossed with NC1209-23 SRC to produce male-sterile plants heterozygous for all three mutations. A single male-sterile plant resulting from this cross is backcrossed as a female to NC1209-23 SRC to produce progeny that are segregating for individuals homozygous for all three mutations. Triple homozygous individuals are identified by DNA genotyping to produce the CMS NC1209-23 SRC line. Because the line is male-sterile, it is maintained via pollination with NC1209-23 SRC (see above).

(129) Hybrid cultivar NC 6 SRC is produced by pollinating plants of CMS NC1209-23 SRC with pollen of DH98-325-5 SRC.

Example 9

Testing of Hybrid Cultivar NC 6 SRC

(130) Hybrid cultivar NC 6 SRC is evaluated for cured leaf chemistry, yield, and physical quality at two North Carolina field research locations during 2013 (Reidsville and Waynesville). Hybrid cultivar NC 6 LC is included for comparison. The experimental design at each location is a randomized complete block design with four replications. Experimental units are single 20-plant plots. Plots are harvested and air-cured. Plot weights are used to determine per acre yields. Cured leaf is evaluated by a former USDA tobacco grader. Fifty gram composite leaf samples are collected from each plot and analyzed for percent nicotine, nornicotine, anatabine, anabasine, and percent nicotine conversion using gas chromatography equipment.

(131) Comparisons using the least significant difference (LSD) test indicates that hybrid cultivar NC 6 SRC has significantly (P<0.05) lower levels of nornicotine and percent nicotine conversion relative to NC 6 LC (Table 1). NC 6 LC and hybrid cultivar NC 6 SRC are not significantly different from each other for percent nicotine, yield, or cured leaf quality indices.

Example 10

Breeding of Homozygous cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82e10 P381S Mutant Plants into the NC1426-11 Burley Tobacco Cultivar

(132) NC1426-11 SRC is a backcross-derived version of burley tobacco cultivar NC1426-11 carrying introduced mutations in three genes (CYP82E4 (SEQ ID NO: 5), CYP82E5 (SEQ ID NO: 8), and CYP82E10 (SEQ ID NO: 9)) previously documented to encode for nicotine demethylase enzymes (Lewis et al., 2010). The introduced mutations in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) and CYP82E5 (cyp82e5v2 W422Stop (SEQ ID NO: 2)) encode for premature stop codons which render the genes non-functional. The introduced mutation in CYP82E10 (cyp82e10 P381S (SEQ ID NO: 11)) does not encode for a premature stop codon, but does render the gene product non-functional for converting nicotine to nornicotine (Lewis et al. 2010). When in homozygous condition, the three mutations result in tobacco plants with (1) reduced genetic capacity to demethylate nicotine to form nornicotine, and (2) a corresponding diminished potential to accumulate N-nitrosonornicotine (NNN), a potent carcinogen found in many tobacco products. The mutation in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) also provides phenotypic stability for the nicotine conversion trait and eliminates the requirement to utilize the cumbersome LC method for reducing levels of nornicotine in tobacco cultivars.

(133) The original tobacco cultivar NC1426-11 is a fertile inbred line. CMS NC1426-11 is a cytoplasmic male-sterile version of NC1426-11. To develop NC1426-11 SRC, an individual plant of NC1426-11 is pollinated with a plant of the genetic background DH98-325-6 carrying mutations in each of the three nicotine demethylase genes. F.sub.1 individuals originating from this cross and heterozygous for each mutation are backcrossed to the recurrent parent, NC1426-11, to produce BC.sub.1F.sub.1 progeny. BC.sub.1F.sub.1 progeny are screened using genotyping methodologies to identify individuals heterozygous for all three mutations. A single triple heterozygous BC.sub.1F.sub.1 plant is backcrossed to NC1426-11 to produce BC.sub.2F.sub.1 progeny. The process of backcrossing and identification of individuals heterozygous for all three mutations is repeated through the BC.sub.3F.sub.1, BC.sub.4F.sub.1, BC.sub.5F.sub.1, BC.sub.6F.sub.1, and BC.sub.7F.sub.1 stages. At the BC.sub.7F.sub.1 stage, individuals heterozygous for all three mutations from each pedigree are self-pollinated to produce BC.sub.7F.sub.2 seed. A large number of BC.sub.7F.sub.2 progeny from each pedigree are genotyped to identify individuals homozygous for all three mutations. A single BC.sub.7F.sub.2 plant homozygous for all three mutations is self-pollinated to produce a BC.sub.7F.sub.3 family (NC1426-11 SRC) in which the wild-type CYP82E4, CYP82E5v2, and CYP82E10 alleles of NC1426-11 were replaced by the mutant alleles (cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S).

(134) The male-sterile (CMS) version of NC1426-11 SRC (CMS NC1426-11 SRC) is produced by crossing a plant of CMS NC1426-11 as a female with pollen of NC1426-11 SRC to produce male-sterile plants heterozygous for all three mutations. A single male-sterile plant resulting from this cross is then subsequently backcrossed as a female to NC1426-11 SRC to produce progeny that are segregating for individuals homozygous for all three mutations. Triple homozygous individuals are identified by DNA genotyping to produce the CMS NC1426-11 SRC line. Because the line is male-sterile, it is maintained via pollination with NC1426-11 SRC.

(135) Commercial NC1426-11 SRC is produced by pollinating plants of CMS NC1426-11 SRC with pollen of NC1426-11 SRC.

Example 11

Testing of NC1426-11 SRC

(136) NC1426-11 SRC is evaluated for cured leaf chemistry, yield, and physical quality at two North Carolina field research locations during 2013 (Reidsville and Waynesville). NC1426-11 is included for comparison. The experimental design at each location is a randomized complete block design with four replications. Experimental units are single 20-plant plots. Plots are harvested and air-cured. Plot weights are used to determine per acre yields. Cured leaf is evaluated by a former USDA tobacco grader. Fifty gram composite leaf samples are collected from each plot and analyzed for percent nicotine, nornicotine, anatabine, anabasine, and percent nicotine conversion using gas chromatography equipment.

(137) Comparisons using the least significant difference (LSD) test indicates that NC1426-11 SRC has significantly lower levels (P<0.05) of nornicotine and percent nicotine conversion relative to NC1426-11 (Table 1). NC1426-11 and NC1426-11 SRC are not significantly different from each other for percent nicotine but are significantly different for yield, cured leaf quality indices, and percent anatabine.

Example 12

Breeding of Homozygous cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82e10 P381S Mutant Plants into the NC1426-17 Burley Tobacco Cultivar

(138) NC1426-17 SRC is a backcross-derived version of burley tobacco cultivar NC1426-17 carrying introduced null mutations in three genes (CYP82E4 (SEQ ID NO: 5), CYP82E5 (SEQ ID NO: 8), and CYP82E10 (SEQ ID NO: 9)) previously documented to encode for nicotine demethylase enzymes (Lewis et al., Phytochemistry, 71 (2010), 1988-1998). The introduced mutations in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) and CYP82E5 (cyp82e5v2 W422Stop (SEQ ID NO: 2)) encode for premature stop codons which render the genes non-functional. The introduced mutation in CYP82E10 (cyp82e10 P381S (SEQ ID NO: 11)) does not encode for a premature stop codon, but does render the gene product non-functional for converting nicotine to nornicotine (Id.). When in homozygous condition, the three mutations result in tobacco plants with (1) reduced genetic capacity to demethylate nicotine to form nornicotine, and (2) a corresponding diminished potential to accumulate N-nitrosonornicotine (NNN), a potent carcinogen found in many tobacco products. The mutation in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) also provides phenotypic stability for the nicotine conversion trait and eliminates the requirement to utilize the cumbersome LC method for reducing levels of nornicotine in tobacco cultivars (see e.g., Jack et al. 2007. Implications of reducing nornicotine accumulation in burley tobacco: appendix Athe LC protocol. Rec. Adv. Tob. Sci. 33: 58-79).

(139) The original tobacco cultivar NC1426-17 is a fertile inbred line. To develop NC1426-17 SRC, an individual plant of NC1426-17 is initially pollinated with a plant of the genetic background DH98-325-6 carrying mutations in each of the three nicotine demethylase genes. F.sub.1 individuals originating from this cross and heterozygous for each mutation are backcrossed to the recurrent parent, NC1426-17, to produce BC.sub.1F.sub.1 progeny. BC.sub.1F.sub.1 progeny are screened using genotyping methodologies to identify individuals heterozygous for all three mutations. A single triple heterozygous BC.sub.1F.sub.1 plant is backcrossed to NC1426-17 to produce BC.sub.2F.sub.1 progeny. The process of backcrossing and identification of individuals heterozygous for all three mutations is repeated through the BC.sub.3F.sub.1, BC.sub.4F.sub.1, BC.sub.5F.sub.1, BC.sub.6F.sub.1, and BC.sub.7F.sub.1 stages. At the BC.sub.7F.sub.1 stage, individuals heterozygous for all three mutations from each pedigree are self-pollinated to produce BC.sub.7F.sub.2 seed. A large number of BC.sub.7F.sub.2 progeny from each pedigree are genotyped to identify individuals homozygous for all three mutations. A single BC.sub.7F.sub.2 plant homozygous for all three mutations is self-pollinated to produce a BC.sub.7F.sub.3 family (NC1426-17 SRC) in which the wild-type CYP82E4, CYP82E5v2, and CYP82E10 alleles of NC1426-17 were replaced by the mutant alleles (cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S).

Example 13

Testing of NC1426-17 SRC

(140) NC1426-17 SRC is evaluated for cured leaf chemistry, yield, and physical quality at two North Carolina field research locations during 2013 (Reidsville and Waynesville). NC1426-17 is included for comparison. The experimental design at each location is a randomized complete block design with four replications. Experimental units are single 20-plant plots. Plots are harvested and air-cured. Plot weights are used to determine per acre yields. Cured leaf is evaluated by a former USDA tobacco grader. Fifty gram composite leaf samples are collected from each plot and analyzed for percent nicotine, nornicotine, anatabine, anabasine, and percent nicotine conversion using gas chromatography equipment.

(141) Comparisons using the least significant difference (LSD) test indicated that NC1426-17 SRC has significantly lower levels (P<0.05) of nornicotine and percent nicotine conversion relative to NC1426-17 (Table 1). NC1426-17 and NC1426-17 SRC are not significantly different from each other for percent nicotine, yield, or cured leaf quality indices.

Example 14

Breeding of Homozygous cyp82e4 W329Stop, the cyp82e5v2 W422Stop, and cyp82e10 P381S Mutant Plants into the NC4LC Burley Tobacco Cultivar

(142) The original tobacco cultivar NC4LC is a hybrid generated by pollinating plants of a male-sterile breeding line CMS NC1426-11 with pollen produced by fertile breeding line NC1426-17. Hybrid cultivar NC 4 SRC is a hybrid generated by pollinating plants of a male-sterile breeding line CMS NC1426-11 SRC with pollen produced by fertile breeding line NC1426-17 SRC with each breeding line carrying introduced deleterious mutations in three genes (CYP82E4 (SEQ ID NO: 5), CYP82E5 (SEQ ID NO: 8), and CYP82E10 (SEQ ID NO: 9)) previously documented to encode for nicotine demethylase enzymes (Lewis et al., Phytochemistry, 71 (2010), 1988-1998). The introduced mutations in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) and CYP82E5 (cyp82e5v2 W422Stop (SEQ ID NO: 2)) encode for premature stop codons which render the genes non-functional. The introduced mutation in CYP82E10 (cyp82e10 P381S (SEQ ID NO: 11)) does not encode for a premature stop codon, but does render the gene product non-functional for converting nicotine to nornicotine (Id.). When in homozygous condition, the three mutations result in tobacco plants with (1) reduced genetic capacity to demethylate nicotine to form nornicotine, and (2) a corresponding diminished potential to accumulate N-nitrosonornicotine (NNN), a potent carcinogen found in many tobacco products. The mutation in CYP82E4 (cyp82e4 W329Stop (SEQ ID NO: 1)) also provides phenotypic stability for the nicotine conversion trait and eliminates the requirement to utilize the cumbersome LC method for reducing levels of nornicotine in tobacco cultivars (see e.g., Jack et al. 2007. Implications of reducing nornicotine accumulation in burley tobacco: appendix Athe LC protocol. Rec. Adv. Tob. Sci. 33: 58-79).

(143) The original tobacco cultivar NC 4LC is a hybrid generated by pollinating plants of a male-sterile breeding line CMS NC1426-11 with pollen produced by fertile breeding line NC1426-17. To develop hybrid cultivar NC 4 SRC, individual plants of fertile NC1426-11 are first pollinated with a plant of the genetic background DH98-325-6 carrying mutations in each of the three nicotine demethylase genes. F.sub.1 individuals originating from each cross and heterozygous for each mutation are backcrossed to the recurrent parent (NC1426-11) to produce BC.sub.1F.sub.1 progenies.

(144) BC.sub.1F.sub.1 progenies are screened using genotyping methodologies to identify individuals heterozygous for all three mutations. A single BC.sub.1F.sub.1 plant from each pedigree is backcrossed to NC1426-11, to produce BC.sub.2F.sub.1 progenies. The process of backcrossing and identification of individuals heterozygous for all three mutations is repeated through the BC.sub.3F.sub.1, BC.sub.4F.sub.1, BC.sub.5F.sub.1, BC.sub.6F.sub.1, and BC.sub.7F.sub.1 stages. At the BC.sub.7F.sub.1 stage, individuals heterozygous for all three mutations from each pedigree are self-pollinated to produce BC.sub.7F.sub.2 seed. A large number of BC.sub.7F.sub.2 progeny from each pedigree are genotyped to identify individuals homozygous for all three mutations. BC.sub.7F.sub.2 plants homozygous for all three mutations are self-pollinated to produce BC.sub.7F.sub.3 families (NC1426-11 SRC) in which the wild-type CYP82E4, CYP82E5v2, and CYP82E10 alleles of NC1426-11 are replaced by the mutant alleles (cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S).

(145) The female parental line of NC1426-11, CMS NC1426-11, is cytoplasmic male sterile which causes pollen to not be produced. To develop CMS NC1426-11 SRC, a plant of CMS NC1426-11 is crossed with NC1426-11 SRC to produce male-sterile plants heterozygous for all three mutations. A single male-sterile plant resulting from this cross is backcrossed as a female to NC1426-11 SRC to produce progeny that are segregating for individuals homozygous for all three mutations. Triple homozygous individuals are identified by DNA genotyping to produce the CMS NC1426-11 SRC line. Because the line is male-sterile, it is maintained via pollination with NC1426-11 SRC (see above).

(146) Hybrid cultivar NC 4 SRC is produced by pollinating plants of CMS NC1426-11 SRC with pollen of NC1426-17 SRC.

Example 15

Testing of Hybrid Cultivar NC 4 SRC

(147) Hybrid cultivar NC 4 SRC is evaluated for cured leaf chemistry, yield, and physical quality at two North Carolina field research locations during 2013 (Reidsville and Waynesville). The experimental design at each location is a randomized complete block design with four replications. Experimental units are single 20-plant plots. Plots are harvested and air-cured. Plot weights are used to determine per acre yields. Cured leaf is evaluated by a former USDA tobacco grader. Fifty gram composite leaf samples are collected from each plot and analyzed for percent nicotine, nornicotine, anatabine, anabasine, and percent nicotine conversion using gas chromatography equipment. Results are presented in Table 1.

(148) TABLE-US-00001 TABLE 1 Entry means for experiment EX14-157 entries evaluated in two 2013 North Carolina environments (Reidsville and Waynesville). Total Nicotine Yield Grade Nicotine Nornicotine Anabasine Anatabine Alkaloids Conversion Variety (lbs/A) Index (%) (%) (%) (%) (%) (%) Banket A1 2071 72.4 2.757 0.049 0.017 0.258 3.081 1.796 Banket A1 SRC 1948 72.2 3.214 0.018* 0.019 0.284 3.535 0.594* Burley 21 1985 72.7 2.250 0.060 0.012 0.174 2.495 2.649 Burley 21 SRC 1897 74.7 2.881 0.018* 0.014 0.218 3.130 0.631* NC 3LC 2168 73.9 1.921 0.117 0.012 0.180 2.230 5.523 NC 3 SRC 2097 75.9 2.154 0.011* 0.011 0.175 2.351 0.504* NC 4 SRC 2143 74.6 2.395 0.013* 0.012 0.217 2.637 0.554* NC 6LC 2275 75.7 2.093 0.221 0.012 0.204 2.530 8.965 NC 6 SRC 2245 72.1 2.380 0.010* 0.012 0.199 2.601 0.431* DH98-325-5 2187 72.1 2.066 0.045 0.012 0.200 2.323 2.205 DH98-325-5 SRC 2086 72.5 2.332 0.013* 0.012 0.205 2.562 0.533* DH19 1898 70.7 1.854 0.044 0.011 0.158 2.066 2.257 DH19 SRC 2242 72.5 1.870 0.018* 0.012 0.159 2.058 0.998* NC1426-17 1924 71.8 1.802 0.046 0.012 0.211 2.072 2.565 NC1426-17 SRC 2056 75.2 2.030 0.012* 0.012 0.214 2.269 0.614* NC1426-11 2415 71.2 2.228 0.078 0.013 0.232 2.551 3.513 NC1426-11 SRC 2056* 75.4* 2.121 0.011* 0.011 0.181* 2.324 0.524* VA 509 LC 1975 72.9 2.197 0.169 0.013 0.187 2.567 7.107 Ky 14 LC 2237 74.2 2.151 0.072 0.012 0.209 2.444 3.407 Banket A1 is a fertile burley inbred variety. Banket A1 SRC is a backcross-derived version of Banket A1 carrying introduced mutations in three genes (cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S). Burley 21 is a fertile burley inbred variety. Burley 21 SRC is a backcross-derived version of Burley 21 carrying introduced mutations in three genes (cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S). Tobacco cultivar NC 3LC is a hybrid generated by pollinating plants of a male-sterile breeding line CMS NC1209-23 with pollen produced by fertile breeding line DH19. Tobacco cultivar NC 3 SRC is a hybrid generated by pollinating plants of a male-sterile breeding line CMS NC1209-23 SRC with pollen produced by fertile breeding line DH19 SRC. Tobacco cultivar NC 4 SRC is a hybrid generated by pollinating plants of a male-sterile breeding line CMS NC1426-11 SRC with pollen produced by fertile breeding line NC1426-17 SRC. Tobacco cultivar NC 6LC is a hybrid generated by pollinating plants of a male-sterile breeding line CMS NC1209-23 with pollen produced by fertile breeding line DH98-325-5. Tobacco cultivar NC 6 SRC is a hybrid generated by pollinating plants of a male-sterile breeding line CMS NC1209-23 SRC with pollen produced by fertile breeding line DH98-325-5 SRC. DH98-325-5 is a fertile burley inbred variety. DH98-325-5 SRC is a backcross-derived version of DH98-325-5 carrying introduced mutations in three genes (cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S). DH 19 is a fertile burley inbred variety. DH 19 SRC is a backcross-derived version of DH 19 carrying introduced mutations in three genes (cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S). NC1426-17 is a fertile burley inbred variety. NC1426-17 SRC is a backcross-derived version of NC1426-17 carrying introduced mutations in three genes (cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S). NC1426-11 is a fertile burley inbred variety. NC1426-11 SRC is a backcross-derived version of NC1426-11 carrying introduced mutations in three genes (cyp82e4 W329Stop, cyp82e5v2 W422Stop, and cyp82e10 P381S). KY 14 LC and VA 509 LC are LC-selected burley tobacco varieties KY 14 and VA 509. *Indicates significantly different (P < 0.05) from the nearly isogenic LC variety or regular breeding line. Significance tests were based upon log transformed valued for % nicotine, % nornicotine, % anabasine, % anatabine, and % nicotine conversion. Significance tests were based upon non-transformed values for the remainder of the measured characteristics.

DEPOSIT INFORMATION

(149) A deposit of the proprietary inbred plant lines disclosed above and recited in the appended claims have been made with American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110. The date of deposit for NC1209-23 SRC, CMS NC1209-23 SRC, DH19 SRC, DH98-325-5 SRC, NC1426-11 SRC, and NC1426-17 SRC was Feb. 26, 2014. The date of deposit for CMS NC1426-11 SRC was Mar. 7, 2014. The deposits of 2500 seeds for each variety was taken from the same deposits maintained since prior to the filing date of this application. Upon issuance of a patent, all restrictions upon the deposits will be irrevocably removed, and the deposits are intended by Applicant to meet all of the requirements of 37 C.F.R. 1.801-1.809. The ATCC has issued the following accession numbers: PTA-121049 for NC1209-23 SRC, PTA-121052 for DH19 SRC, PTA-121051 for DH98-325-5 SRC, PTA-121031 for NC1426-11 SRC, PTA-121070 for CMS NC1426-11 SRC, PTA-121032 for NC1426-17 SRC, PTA-121055 for NC 3 SRC, PTA-121042 for NC 6 SRC, and PTA-121040 for NC 4 SRC. These deposits will be maintained in the depository for a period of 30 years, or 5 years after the last request, or for the effective 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.).