Genetic approaches for achieving ultra-low nicotine content in tobacco

Abstract

The present invention relates to tobacco products derived from plants containing mutated berberine bridge enzyme-like nucleic acids and recessive nic1 and / or nic2 alleles, and methods for making same.

Description

Technical Field

[0001] [Cross - Reference to Related Applications] This application claims the benefit of priority to U.S. Application No. 62 / 958,505, filed on January 8, 2020, the content of which is hereby incorporated by reference in its entirety as part of this specification.

[0002] The present invention relates to mutant berberine bridge enzyme - like nucleic acids and tobacco products derived from plants containing recessive nic1 and / or nic2 alleles, and methods for producing the same.

Background Art

[0003] The pyridine alkaloids of tobacco (Nicotiana tabacum L.) are one of the most investigated groups of plant secondary compounds. Nicotine constitutes more than 90% of the total alkaloid pool in most tobacco genotypes and is mainly involved in the pharmacological responses experienced by users of tobacco products. In order of relative abundance, the remaining major alkaloids in tobacco include anatabine, nornicotine, and anabasine. The alkaloid levels in tobacco are affected by environmental conditions, interactions with plant pests, and plant genetics.

[0004] Although nicotine is the main compound that gives tobacco product users the pharmacological effects they seek, there are several situations where it would be desirable to develop products using tobacco plants that produce and accumulate very low levels of nicotine. For example, some studies have shown that the use of low-nicotine cigarettes as a component of a smoking cessation strategy can help smokers who are trying to quit (Hatsukami et al., 2010a; Donny et al., 2014). Other reports have demonstrated that by reducing nicotine levels in tobacco products below a critical threshold, they can no longer cause or sustain a toxic response (Benowitz and Henningfield, 1994; Benowitz et al., 2007). Such studies can ultimately influence regulatory authorities such as the US Food and Drug Administration, which have the authority to determine what levels of various tobacco components (including nicotine) would be acceptable in cigarettes and other tobacco products.

Summary of the Invention

[0005] In one aspect, the disclosure of the present technology is a method of curing one or more leaves of a Nicotiana plant comprising (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1 or a recessive allele of nic1 and a recessive allele of nic2, the method comprising: (a) heating the leaves starting at a temperature of 92°F to 96°F and increasing the temperature to 104°F to 108°F at a rate of about 1°F per hour until the maximum upper temperature is reached, and holding for a period of about 52 to 58 hours at the upper temperature, a yellowing step; (b) drying for about 22 hours at a temperature of about 120°F, a leaf drying step; and (c) drying for about 50 to about 65 hours at a temperature of about 132°F to 138°F, a stem drying step, thereby curing one or more leaves of the Nicotiana plant and producing one or more cured leaves of the Nicotiana plant.

[0006] In some embodiments, the Nicotiana plant comprises (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1 and a recessive allele of nic2. In some embodiments, the recessive allele of nic1 is a homozygous recessive allele. In some embodiments, the recessive allele of nic2 is a homozygous recessive allele. In some embodiments, the Nicotiana plant has a reduced nicotine alkaloid content compared to a plant modified by (A) only (e.g., not modified by (A) and (B)).

[0007] In some embodiments, the present technology provides a dried leaf of a Nicotiana plant produced by the method, wherein the dried leaf comprises: (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc; and (B) a recessive allele of nic1 or a recessive allele of nic1 and a recessive allele of nic2. In some embodiments, the dried leaf comprises: (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc; and (B) a recessive allele of nic1 and a recessive allele of nic2. In some embodiments, the recessive allele of nic1 is a homozygous recessive allele. In some embodiments, the recessive allele of nic2 is a homozygous recessive allele. In some embodiments, the leaf has a reduced nicotine alkaloid content compared to the leaf of a wild-type control Nicotiana plant or compared to a Nic1 / Nic2 control Nicotiana plant. In some embodiments, the nicotine alkaloid is nicotine. In some embodiments, the dried leaf has a nicotine content of 0.5 mg / g or less. In some embodiments, the dried leaf has a nicotine content of 0.4 mg / g or less. In some embodiments, the leaf comprises an increased level of saccharides and / or a reduced level of ammonia compared to a leaf dried according to a standard drying method. In some embodiments, the Nicotiana plant further comprises a reduction in the expression of a polynucleotide encoding an additional nicotine alkaloid biosynthetic enzyme selected from the group consisting of aspartate oxidase, quinolinate synthase, quinolinate phosphoribosyl transferase, ornithine decarboxylase, putrescine N-methyl transferase, methyl putrescine oxidase, and A622.

[0008] In some embodiments, the present technology provides a tobacco product comprising dried leaves. In some embodiments, the tobacco is selected from the group consisting of leaf tobacco, shredded tobacco, cut tobacco, ground tobacco, powdered tobacco, tobacco extract, smokeless tobacco, moist or dry snuff, pipe tobacco, cigars, cigarillos, cigarettes, and chewing tobacco. In some embodiments, the product is selected from the group consisting of cigarillos, kretek cigarettes, non-ventilated recess filter cigarettes, ventilated recess filter cigarettes, cigars, snuff, tobacco-containing gums, tobacco-containing troches, and chewing tobacco.

[0009] In some embodiments, the present disclosure provides a method for improving the yield of Nicotiana plants, the method comprising: (a) fertilizing the seedlings at the germination stage of about 90%, wherein the fertilizing step is performed through fertigation, and optionally the concentration of N in the fertilizer is about 200 ppm; (b) applying a plastic mulch treatment; and (c) applying nitrogen to the seedlings at a rate of about 90-120 pounds (lbs) / acre of total N at about 4-5 weeks after transplantation, at about 6-7 weeks after transplantation, and at about 8-9 weeks after transplantation to provide an increase in the yield and quality of the Nicotiana plants. In some embodiments, the nitrogen is applied using fertigation. In some embodiments, the substrate is covered with a surface covering sheet, thereby providing a substrate covered with the surface covering sheet. In some embodiments, the substrate and / or the substrate covered with the surface covering sheet is covered with a mulch.

[0010] In one aspect, the disclosure of the present technology provides a dried tobacco leaf derived from a Nicotiana plant, wherein the Nicotiana plant: (1) contains one or both of the recessive nic1 and nic2 alleles; and (2) contains BBLa, BBLb, and BBLc genes modified compared to the wild type such that the activity of BBLa, BBLb, and BBLc is reduced, or the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc is reduced, whereby the nicotine alkaloid content of the Nicotiana plant is decreased compared to a control Nicotiana plant. In some embodiments, the Nicotiana plant contains both of the recessive nic1 and nic2 alleles. In some embodiments, the recessive allele of nic1 is a homozygous recessive allele. In some embodiments, the recessive allele of nic2 is a homozygous recessive allele. In some embodiments, the dried leaf has a USDA grade index comparable to or better than that of the dried leaf derived from a control Nicotiana plant. In some embodiments, the leaf has a USDA grade index of about 60 or higher. In some embodiments, the yield of the dried leaf is comparable to or increased compared to a control Nicotiana plant. In some embodiments, the nicotine alkaloid is nicotine.

[0011] In some embodiments, the present technology provides a tobacco product comprising the dried leaf. In some embodiments, the tobacco is selected from the group consisting of leaf tobacco, shredded tobacco, cut tobacco, ground tobacco, powdered tobacco, tobacco extract, smokeless tobacco, moist or dry snuff, pipe tobacco, cigars, cigarillos, cigarettes, and chewing tobacco. In some embodiments, the tobacco product is selected from the group consisting of cigarillos, kretek cigarettes, non-ventilated recessed filter cigarettes, ventilated recessed filter cigarettes, cigars, snuff, tobacco-containing gum, tobacco-containing troches, and chewing tobacco.

[0012] In one aspect, the disclosure of the present technology provides a method for producing a Nicotiana plant having a reduced nicotine alkaloid content, the method comprising the step of combining, in a Nicotiana plant, (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1, or a recessive allele of nic1 and a recessive allele of nic2, wherein the Nicotiana plant has a reduced nicotine alkaloid content compared to a plant modified by (A) only (e.g., not modified by (A) and (B)). In some embodiments, the Nicotiana plant comprises a recessive allele of nic1. In some embodiments, the Nicotiana plant comprises a recessive allele of nic1 and a recessive allele of nic2. In some embodiments, the recessive allele of nic1 is a homozygous recessive allele. In some embodiments, the recessive allele of nic2 is a homozygous recessive allele. In some embodiments, the method further comprises the step of reducing the expression of a polynucleotide encoding an additional nicotine alkaloid biosynthetic enzyme selected from the group consisting of aspartate oxidase, quinolinic acid synthetase, quinolinate phosphoribosyl transferase, ornithine decarboxylase, putrescine N-methyl transferase, methyl putrescine oxidase, and A622.

[0013] In some embodiments, the Nicotiana plant has a nicotine alkaloid content that is at least 40% reduced compared to a plant modified only by (A) (e.g., not modified by (A) and (B)). In some embodiments, the nicotine alkaloid is nicotine, and the nicotine content is reduced by about 40% to about 90% compared to a plant modified only by (A) (e.g., not modified by (A) and (B)). In some embodiments, the nicotine alkaloid is nicotine, and the nicotine content is from about 0.014% to about 0.098%. In some embodiments, the plant has a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1 or a recessive allele of nic1 and a recessive allele of nic2. In some embodiments, the recessive allele of nic1 is a homozygous recessive allele. In some embodiments, the recessive allele of nic2 is a homozygous recessive allele.

[0014] In some embodiments, the technology provides a progeny plant or seed produced from the plant, wherein the progeny plant or seed has a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1 or a recessive allele of nic1 and a recessive allele of nic2. In some embodiments, the recessive allele of nic1 is a homozygous recessive allele. In some embodiments, the recessive allele of nic2 is a homozygous recessive allele.

[0015] In one aspect, the disclosure of the present technology provides a tobacco product comprising tobacco derived from Nicotiana plants, wherein the Nicotiana plants have (A) a modification that reduces the activities of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acids encoding BBLa, BBLb, and BBLc, and (B) a recessive allele of nic1 or recessive alleles of nic1 and nic2, and the Nicotiana plants have a reduced nicotine alkaloid content as compared to plants modified only by (A) (e.g., not modified by (A) and (B)). In some embodiments, the recessive allele of nic1 is a homozygous recessive allele. In some embodiments, the recessive allele of nic2 is a homozygous recessive allele. In some embodiments, the Nicotiana plants are modified to reduce the activities of BBLa, BBLb, and BBLc and / or the expression of the nucleic acids encoding BBLa, BBLb, and BBLc and comprise a recessive allele of nic1. In some embodiments, the Nicotiana plants are modified to reduce the activities of BBLa, BBLb, and BBLc and / or the expression of the nucleic acids encoding BBLa, BBLb, and BBLc and comprise recessive alleles of nic1 and nic2. In some embodiments, the Nicotiana plants have a nicotine alkaloid content that is at least 40% reduced as compared to plants modified only by (A). In some embodiments, the nicotine alkaloid is nicotine, and the nicotine content is reduced by about 40% to about 90% as compared to plants modified only by (A). In some embodiments, the nicotine alkaloid is nicotine, and the nicotine content is from about 0.014% to about 0.098%. In some embodiments, the nicotine alkaloid is nicotine, and the nicotine content is about 0.014%. In some embodiments, the tobacco is selected from the group consisting of leaf tobacco, shredded tobacco, cut tobacco, ground tobacco, powdered tobacco, tobacco extract, smokeless tobacco, moist or dry snuff, pipe tobacco, cigar tobacco, cigarillo tobacco, cigarette tobacco, and chewing tobacco.In some embodiments, the product is selected from the group consisting of cigarettes, kretek cigarettes, non-ventilated concave filter cigarettes, ventilated concave filter cigarettes, cigars, snuffs, tobacco-containing gums, tobacco-containing troches, and chewing tobacco. In some embodiments, the Nicotiana plant further comprises a decrease in the expression of a polynucleotide encoding an additional nicotine alkaloid biosynthetic enzyme selected from the group consisting of aspartate oxidase, quinolate synthase, quinolinate phosphoribosyltransferase, ornithine decarboxylase, putrescine N-methyltransferase, methylputrescine oxidase, and A622.

[0016] In one aspect, the present invention provides a dried tobacco leaf derived from a Nicotiana plant, wherein: (A) the Nicotiana plant: (1) comprises one or both of the recessive nic1 and nic2 alleles; (2) comprises modified BBLa, BBLb, and BBLc genes compared to the wild type, whereby the nicotine alkaloid content of the Nicotiana plant is decreased compared to the wild type (e.g., without BBLa, nic1, and nic2); and (B) the dried tobacco leaf has the characteristics of a good-quality to high-quality leaf with good color intensity, normal width, and uniform texture, and optionally, the dried tobacco leaf has a USDA grade index greater than 60, or is of good quality to high quality to low quality or excellent quality to high quality to good quality according to the USDA standard grades (“Official Standard Grades for Flue-Cured Tobacco U.S. Types 11, 12, 13, 14, and Foreign Type 92”).

[0017] In one aspect, a method for producing a Nicotiana plant having a reduced nicotine alkaloid content, the method comprising the step of combining in a Nicotiana plant: (A) a modification that reduces the activity of BBLa, BBLb and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb and the nucleic acid encoding BBLc; and (B) the recessive allele of nic1 or the recessive allele of nic1 and the recessive allele of nic2, wherein the Nicotiana plant has a reduced nicotine alkaloid content compared to a plant modified by (A) alone.

[0018] In one aspect, a method of drying one or more leaves of a Nicotiana plant comprising (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1 or recessive alleles of nic1 and nic2, wherein the Nicotiana plant has a reduced nicotine alkaloid content compared to a plant modified by (A) only, the method comprising: (a) a yellowing step that begins heating the leaves at a temperature of 92°F to 96°F and increases the temperature to 104°F to 108°F at a rate of about 1°F per hour until the maximum upper temperature is reached, and holds at the upper temperature for a period of about 52 to 58 hours; (b) a leaf drying step at a temperature of about 120°F for about 22 hours; and (c) a stem drying step at a temperature of about 132°F to 138°F for about 50 to about 65 hours with continuous monitoring, thereby drying one or more leaves of the Nicotiana plant and producing one or more dried leaves of the Nicotiana plant. In some embodiments, the leaf drying step occurs at a temperature of about 116°F to 120°F for about 28 to 34 hours. In some embodiments, the leaf drying step occurs at a temperature of about 116°F to 118°F for about 28 to 34 hours. In some embodiments, the present disclosure relates to dried leaves of a Nicotiana plant produced by the method of drying. In some embodiments, the leaves are ultra-low nicotine leaves containing 0.04% nicotine or less. In some embodiments, the leaves contain increased levels of sugars and / or ammonia compared to leaves dried according to standard drying methods. In some embodiments, the Nicotiana plant further comprises a reduction in the expression of a polynucleotide encoding an additional nicotine alkaloid biosynthetic enzyme selected from the group consisting of aspartate oxidase, quinolinate synthase, quinolinate phosphoribosyltransferase, ornithine decarboxylase, putrescine N-methyltransferase, methylputrescine oxidase, and A622. In some embodiments, the present disclosure relates to a tobacco product comprising the dried leaves produced by the drying method described herein.In some embodiments of the tobacco product, the tobacco is selected from the group consisting of leaf tobacco, shredded tobacco, cut tobacco, ground tobacco, powdered tobacco, tobacco extract, smokeless tobacco, wet or dry snuff, pipe tobacco, cigars, cigarillos, cigarettes, and chewing tobacco. In some embodiments, the tobacco product is selected from the group consisting of cigarillos, kretek cigarettes, non-ventilated concave filter cigarettes, ventilated concave filter cigarettes, cigars, snuff, tobacco-containing gums, tobacco-containing lozenges, and chewing tobacco.

[0019] In one aspect, a method for improving the yield and quality of Nicotiana plants of the present invention, comprising: (a) fertilizing the seedlings at the germination stage of about 90%, wherein the fertilizing step is carried out through fertilization irrigation (e.g., drip irrigation), and optionally the concentration of N is about 200 ppm; (b) applying a plastic mulch treatment, for example, the plastic mulch is added to cover the fertilization irrigation equipment (e.g., drip tape / tube); and (c) applying nitrogen to the seedlings at about 4-5 weeks, about 6-7 weeks, and about 8-9 weeks after transplantation at a rate of about 90-120 pounds per acre of total N to provide an increase in the yield and quality of the Nicotiana plants.

[0020] In one aspect, a tobacco product, wherein the tobacco product comprises tobacco derived from Nicotiana plants, and the Nicotiana plants comprise (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1 or a recessive allele of nic1 and a recessive allele of nic2, and the Nicotiana plants have a reduced nicotine alkaloid content compared to plants modified only by (A).

[0021] The present invention further provides plants and plant parts produced by the method of the present invention, as well as agricultural crops, progeny, and products produced from said plants and parts thereof, and seeds derived from said plants. The present invention further provides vectors and expression cassettes for carrying out the method of the present invention.

[0022] These and other aspects of the present invention are described in more detail in the description of the invention below.

Brief Description of the Drawings

[0023]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0024] [Detailed Description of the Invention] Here, the present invention will be described below with reference to the accompanying drawings and examples in which embodiments of the present invention are shown. This description is not intended to be an exhaustive catalog of all the different ways in which the present invention can be implemented or of all the features that can be added to the present invention. For example, features illustrated with reference to one embodiment can be incorporated into other embodiments, and features illustrated with reference to a particular embodiment can be omitted from that embodiment. Thus, the present invention contemplates that in some embodiments of the present invention, any feature or combination of features described herein can be excluded or omitted. In addition, numerous modifications and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of this disclosure, and such are not departures from the present invention. Therefore, the following description is intended to illustrate some particular embodiments of the present invention and is not intended to specify exhaustively all permutations, combinations, and variations thereof.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used in the description of the invention herein are for the purpose of describing particular embodiments only and are not intended to limit the present invention.

[0026] All publications, patent applications, patents, and other references cited herein are hereby incorporated by reference in their entirety into this specification for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

[0027] Unless otherwise indicated in context, it is specifically intended that the various features of the invention described herein can be used in any combination. Further, the invention contemplates that in some embodiments of the invention, any feature or combination of features described herein may be excluded or omitted. By way of illustration, if a composition is specified herein as including components A, B, and C, it is specifically intended that any one of A, B, or C, or combinations thereof, can be omitted and disclaimed, either singly or in any combination.

[0028] As used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0029] Also, as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted alternatively (“or”).

[0030] As used herein, the term “about,” when referring to a measurable value such as an amount or concentration, is intended to encompass the specified value and variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value. For example, “about X” is intended to include X and variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of X, when X is a measurable value. The ranges provided herein for measurable values may include any other range and / or individual value therein.

[0031] As used herein, phrases such as “between X and Y” and “about between X and Y” should be interpreted as including X and Y. As used herein, phrases such as “about between X and Y” mean “about between about X and about Y,” and phrases such as “about X and Y” mean “about X and about Y.”

[0032] The description of ranges of values herein is merely intended to serve as a concise way of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually recited herein. For example, when a range of 10-15 is disclosed, 11, 12, 13, and 14 are also disclosed.

[0033] As used herein, the terms "comprise", "comprises", and "comprising" 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.

[0034] As used herein, the transitional phrase "consisting essentially of" is to be interpreted to mean that a claim's scope is to include the specified materials or steps recited in the claim, and those that do not materially affect the basic and novel characteristics of the claimed invention. Thus, the term "consisting essentially of" is not intended to be interpreted as equivalent to "comprising" when used in the claims of the present invention.

[0035] As used herein, the terms "increase", "increasing", "increased", "enhance", "enhanced", "enhancing", and "enhancement" (and grammatical variations thereof) describe an increase of at least about 15%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500%, or more as compared to a control.

[0036] As used herein, the terms "reduce", "reduced", "reducing", "reduction", "lower", "suppress", and "decrease" (and their grammatical variants) describe, for example, a reduction of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% as compared to a control. In some embodiments, the reduction can result in an activity or amount that is undetectable or essentially undetectable (i.e., a small amount, such as less than about 10% or even 5%). Thus, for example, a reduction in the transcription of one or more target DNAs means a reduction in the transcription of the target gene of at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% as compared to a control (e.g., a plant that does not contain mutations in the BBLa, BBLb, and BBLc nucleic acids).

[0037] As used herein, the term "very low nicotine" or "VLN" tobacco refers to tobacco containing 0.5 mg nicotine / gram tobacco or less.

[0038] As used herein, terms such as "standard drying treatment method", "standard method of drying treatment", "standard drying treatment protocol", or "standard tobacco drying treatment method" refer to known tobacco drying treatment methods (e.g., the methods described in Powell 1987 (Powell Manufacturing, Co.’s Bulk Curing / Drying Owner’s Operator’s Manual for Flue Cured Tobaccos, a manual entitled Powell Manufacturing, Co. (Jan. 20, 1987))).

[0039] As used herein, "chimeric" refers to a nucleic acid molecule or polypeptide in which at least two components are derived from different sources (e.g., different organisms, different coding regions).

[0040] As used herein, the term "complement" can mean 100% complementarity or identity with a reference nucleotide sequence, or it can mean less than 100% complementarity (e.g., complementarity of about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.).

[0041] As used herein, the terms "complementary" or "complementarity" refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base pairing. For example, the sequence "A-G-T" binds to the complementary sequence "T-C-A". Complementarity between two single-stranded molecules can be "partial" where only a portion of the nucleotides bind, or it can be complete where there is complete complementarity between the single-stranded molecules. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between the nucleic acid strands.

[0042] As used herein, terms such as "express", "expresses", "expressed", or "expression" with respect to a nucleic acid molecule and / or nucleotide sequence (e.g., RNA or DNA) indicate that the nucleic acid molecule and / or nucleotide sequence is transcribed and optionally translated. Thus, a nucleic acid molecule and / or nucleotide sequence can express, for example, a polypeptide or functional non-translated RNA of interest.

[0043] The "fragment" or "portion" of a nucleotide sequence refers to a nucleotide sequence of a length that is reduced (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides) compared to a reference nucleic acid or nucleotide sequence, and that contains, consists essentially of, and / or consists of contiguous nucleotides that are identical or substantially identical (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence. Such a nucleic acid fragment or portion according to the present invention may, where appropriate, be included in a larger polynucleotide of which it is a constituent.

[0044] As used herein, the term "gene" refers to a nucleic acid molecule that can be used to produce mRNA, antisense RNA, RNAi (miRNA, siRNA, shRNA), anti-microRNA antisense oligodeoxynucleotides (AMO), etc. A gene may or may not have the ability to be used to produce a functional protein or gene product. A gene may contain both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences, and / or 5' and 3' untranslated regions). A gene may be "isolated" in the sense that it means a nucleic acid that does not substantially or essentially contain the components that are normally found associated with nucleic acids in their natural state. Such components include other cellular materials, culture media from recombinant production, and / or various chemicals used in the chemical synthesis of nucleic acids.

[0045] A "heterologous" or "recombinant" nucleic acid is a nucleotide sequence that is not naturally associated with the host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleotide sequence. Alternatively, a heterologous nucleotide sequence can be one that does not naturally occur together with another nucleotide sequence to which it is related. For example, a nucleic acid construct containing a "heterologous promoter" operably associated with a nucleic acid molecule is a promoter that does not naturally occur together with the nucleic acid molecule to which it is related.

[0046] Different nucleic acids or proteins having homology are referred to herein as "homologs". The term homolog includes homologous sequences from the same and other species, as well as orthologous sequences from the same and other species. "Homology" refers to the level of similarity between two or more nucleic acid and / or amino acid sequences from the perspective of the percentage of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties between different nucleic acids or proteins. Thus, the compositions and methods of the present invention further include homologs to the nucleotide sequences and polypeptide sequences of the present invention. As used herein, "orthologous" refers to homologous nucleotide sequences and / or amino acid sequences in different species that arose from a common ancestral gene during speciation. Homologs of the nucleotide sequences of the present invention have substantial sequence identity with the said nucleotide sequences of the present invention (e.g., at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and / or 100%).

[0047] As used herein, hybridization, hybridize, hybridizing, and grammatical variations thereof refer to the binding of two perfectly complementary nucleotide sequences, or substantially complementary sequences in which some mismatched base pairs may be present. Conditions for hybridization are well known in the art and vary based on the length of the nucleotide sequence and the degree of complementarity between nucleotide sequences. In some embodiments, the conditions for hybridization can be of high stringency, depending on the amount and length of complementarity of the sequences to be hybridized, or they can be of medium or low stringency. Conditions constituting low, medium, and high stringency for the purpose of hybridization between nucleotide sequences are well known in the art (see, for example, Gasiunas et al. (2012) Proc. Natl. Acad. Sci. 109:E2579-E2586; M.R. Green and J. Sambrook (2012) Molecular Cloning: A Laboratory Manual. 4th Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0048] As used herein, "modify", "modifying", or "modification" (and grammatical variations thereof) means any change to a BBL polynucleotide (e.g., BBLa, BBLb, BBLc) and / or a BBL polypeptide, or another polypeptide or polynucleotide that results in a decrease or elimination of the expression of the nucleic acid and / or the production and / or activity of the polypeptide. Such modifications can include, but are not limited to, deleting or inserting one or more nucleotides or an entire nucleic acid region (transcribed and non-transcribed regions) that decreases or eliminates the expression of the nucleic acid and / or the production and / or activity of the polypeptide, and / or introducing one or more point mutations.

[0049] As used herein, the terms "modulate", "modulates", "modulated", or "modulation" refer to the enhancement (e.g., increase) or inhibition (e.g., decrease) of a specified activity (e.g., modulated nicotine production / content). Thus, in some embodiments, an increase or enhancement in activity (e.g., nuclease activity) of about 15%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500%, or more can be observed as compared to a control. In other embodiments, a decrease in expression level or activity (e.g., BBLa, BBLb, and BBLc expression levels, or BBLa, BBLb, and BBLc polypeptide activities) of about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% can be observed as compared to a control.

[0050] A "natural" or "wild-type" nucleic acid, nucleotide sequence, polypeptide, or amino acid sequence refers to a nucleic acid, nucleotide sequence, polypeptide, or amino acid sequence that occurs in nature or is endogenous. Thus, for example, a "wild-type nucleic acid" is a nucleic acid that occurs in nature or is endogenous to an organism. A "homologous" nucleic acid sequence is a nucleotide sequence that is naturally associated with the host cell into which it is introduced. As used herein, a "wild-type" strain or variety of tobacco refers to a tobacco strain or variety that does not contain the BBLabc mutation, or the recessive nic1 and / or nic2 alleles. As used herein, a "Nic1 / Nic2 control" strain or variety of tobacco refers to a tobacco strain or variety that does not contain the homozygous recessive nic1 and / or nic2 alleles.

[0051] In some embodiments, as described herein, the plants of the present technology are homozygous for three major bbl mutations (e.g., bbl-a / bbl-a, bbl-b / bbl-b, bbl-c / bbl-c) and also homozygous for the recessive nic1 and / or nic2 alleles (e.g., nic1 / nic1, nic2 / nic2), and include the BBLabc mutation.

[0052] Also, as used herein, the terms "nucleic acid", "nucleic acid molecule", "nucleic acid construct", "nucleotide sequence", and "polynucleotide" refer to RNA or DNA that is linear or branched, single-stranded or double-stranded, or a hybrid thereof. The term also encompasses RNA / DNA hybrids. When dsRNA is produced synthetically, less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, and others can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides containing C-5 propyne analogs of uridine and cytidine have been shown to bind RNA with high affinity and be potent antisense inhibitors of gene expression. Other modifications can also be made, such as modifications to the phosphodiester backbone or to the 2'-hydroxy of the ribose sugar of RNA. The nucleic acid constructs of the present disclosure can be DNA or RNA, but are preferably DNA. Thus, although the nucleic acid constructs of the present invention can be described and used in the form of DNA depending on the intended use, they can also be described and used in the form of RNA.

[0053] As used herein, the term "nucleotide sequence" refers to a heteropolymer of nucleotides, or the sequence of these nucleotides from the 5' to the 3' end of a nucleic acid molecule, and includes DNA or RNA molecules, including cDNA, DNA fragments or portions, genomic DNA, synthetic (e.g., chemically synthesized) DNA, plasmid DNA, mRNA, and antisense RNA, any of which may be single-stranded or double-stranded. The terms "nucleotide sequence", "nucleic acid", "nucleic acid molecule", "oligonucleotide", and "polynucleotide" are also used interchangeably herein to refer to a heteropolymer of nucleotides. All nucleic acids provided herein have 5' and 3' ends. Further, unless otherwise indicated, the nucleic acid molecules and / or nucleotide sequences provided herein are presented in the 5' to 3' direction from left to right herein, and are represented using the standard codes representing nucleotide letters as described in the U.S. sequence rules, U.S. Patent Rules 1.821-1.825, and the World Intellectual Property Organization (WIPO) Standard ST.25.

[0054] As used herein, the term "percent sequence identity" or "identity percent" refers to the percentage of identical nucleotides in the linear polynucleotide sequence of a reference ("query") polynucleotide molecule (or its complementary strand) compared to a test ("subject") polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned. In some embodiments, "identity percent" may refer to the percentage of identical amino acids in an amino acid sequence.

[0055] As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide or peptide sequences are invariant over the entire window of alignment of components, e.g., nucleotides or amino acids. "Identity" can be readily calculated by known methods including, but not limited to, those described in Computational Molecular Biology (Lesk, A.M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D.W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A.M., and Griffin, H.G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).

[0056] As used herein, "target DNA", "target region", or "target region in the genome" refers to a region of a gene that has been engineered to be bound and cleaved by any class of custom-designed nuclease (e.g., ZFN, TALEN, meganuclease, CRISPR-Cas, etc.), and a region of a biological genome that is fully or substantially complementary thereto (e.g., at least 70% complementary (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater)).

[0057] As used herein, the terms "substantially identical" or "substantial identity" in the context of at least two nucleic acid molecules, nucleotide sequences, or protein sequences refer to two or more sequences or subsequences that, when compared and aligned for maximum correspondence using one of the following sequence comparison algorithms or by visual inspection, have at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and / or 100% nucleotide or amino acid residue sequence identity.

[0058] For sequence comparison, typically one sequence acts as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are determined if necessary, and sequence algorithm program parameters are determined. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence relative to the reference sequence based on the determined program parameters.

[0059] Optimal alignment of arrays for aligning comparison windows is well known to those skilled in the art and can be performed by tools such as the local homology algorithms of Smith and Waterman, the homology alignment algorithms of Needleman and Wunsch, the similarity search methods of Pearson and Lipman, and optionally, by computer execution of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA, which are available as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA). The "identity fraction" for an aligned segment of a test array and a reference array is the number of identical components shared by the two aligned arrays, divided by the total number of components in the reference array segment, i.e., the entire reference array or a smaller defined portion of the reference array. The percent sequence identity is expressed as the identity fraction multiplied by 100. Comparison of one or more polynucleotide sequences can be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence. For the purposes of the present invention, the "percent identity" can also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.

[0060] Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy a threshold score T that is somewhat optimistically estimated when aligned with words of the same length in the database sequences. T is referred to as the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits serve as seeds to initiate a search for longer HSPs that contain them. The word hits are then extended in both directions along each sequence as long as the cumulative alignment score can increase. The cumulative score is calculated using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for a mismatched residue; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction stops when the cumulative score becomes zero or less, due to the accumulation of one or more negatively scored residue alignments that cause the cumulative alignment score to fall by an amount X from its maximum achieved value, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses, as initial settings, a word length (W) of 11, an expectation value (E) of 10, a cutoff of 100, M = 5, N = -4, and comparison of both strands. For amino acid sequences, the BLASTP program uses, as initial settings, a word length (W) of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0061] In addition to calculating the percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat’l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, if the smallest sum probability in a comparison of a test nucleotide sequence to a reference nucleotide sequence is less than about 0.1 to less than about 0.001, the test nucleic acid sequence is considered to be similar to the reference sequence. Thus, in some embodiments of the invention, the smallest sum probability in a comparison of a test nucleotide sequence to a reference nucleotide sequence is less than about 0.001.

[0062] Two nucleotide sequences can be considered to be substantially complementary even if the two sequences hybridize to each other under stringent conditions. In some representative embodiments, two nucleotide sequences that are considered to be substantially complementary hybridize to each other under highly stringent conditions.

[0063] "Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern hybridization are sequence-dependent and vary under different environmental parameters. General guidelines for nucleic acid hybridization can be found in Tijssen Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes part I chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays” Elsevier, New York (1993). Generally, highly stringent hybridization and wash conditions are selected to be approximately 5°C below the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.

[0064] T m is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe (under defined ionic strength and pH). Extremely stringent conditions are T mis selected to be equal. Examples of stringent hybridization conditions for hybridization of a complementary nucleotide sequence having more than 100 complementary residues on a filter in a Southern or Northern blot are 50% formamide with 1 mg of heparin at 42° C., and the hybridization is carried out overnight. An example of highly stringent washing conditions is 0.15 M NaCl at 72° C. for about 15 minutes. An example of stringent washing conditions is washing with 0.2× SSC at 65° C. for 15 minutes (see Sambrook below for an explanation of the SSC buffer). Often, low stringency washing precedes high stringency washing to remove background probe signal. An example of medium stringency washing for a double-stranded molecule of more than 100 nucleotides is 1× SSC at 45° C. for 15 minutes. An example of low stringency washing for a double-stranded molecule of more than 100 nucleotides is 4-6× SSC at 40° C. for 15 minutes. For short probes (e.g., about 10-50 nucleotides), stringent conditions typically involve a salt concentration of less than about 1.0 M Na ions, typically about 0.01-1.0 M Na ion concentration (or other salts), at a pH of 7.0-8.3, and a temperature of typically at least about 30° C. Stringent conditions can also be achieved using the addition of destabilizing agents such as formamide. In general, a signal-to-noise ratio of 2× (or higher) than that observed for non-related probes in a particular hybridization assay indicates detection of specific hybridization. Nucleotide sequences that do not hybridize to each other under stringent conditions are still substantially identical if the proteins they encode are substantially identical. This can occur, for example, when copies of a nucleotide sequence are created using the maximum codon degeneracy tolerated by the genetic code.

[0065] The following are examples of sets of hybridization / washing conditions that can be used to clone homologous nucleotide sequences that are substantially identical to the reference nucleotide sequences of the present invention. In one embodiment, the reference nucleotide sequence hybridizes to the "test" nucleotide sequence at 50°C in 7% SDS, 0.5 M NaPO4, 1 mM EDTA, with washing at 50°C in 2× SSC, 0.1% sodium dodecyl sulfate (SDS). In another embodiment, the reference nucleotide sequence hybridizes to the "test" nucleotide sequence at 50°C in 7% SDS, 0.5 M NaPO4, 1 mM EDTA, with washing at 50°C in 1× SSC, 0.1% sodium dodecyl sulfate (SDS), or at 50°C in 7% SDS, 0.5 M NaPO4, 1 mM EDTA, with washing at 50°C in 0.5× SSC, 0.1% sodium dodecyl sulfate (SDS). In yet a further embodiment, the reference nucleotide sequence hybridizes to the "test" nucleotide sequence at 50°C in 7% SDS, 0.5 M NaPO4, 1 mM EDTA, with washing at 50°C in 0.1× SSC, 0.1% sodium dodecyl sulfate (SDS), or at 50°C in 7% SDS, 0.5 M NaPO4, 1 mM EDTA, with washing at 65°C in 0.1× SSC, 0.1% sodium dodecyl sulfate (SDS).

[0066] Nucleotide sequences of interest (e.g., nucleic acids encoding nucleases useful for mutating BBL nucleic acids) can be operably linked to a variety of promoters, terminators, and / or other regulatory elements for expression in plant cells. Any promoter, terminator, or other regulatory element that is functional in plant cells can be used with the nucleic acids of the present invention. In some embodiments, the promoter can be operably linked to polynucleotides and / or nucleic acids useful in practicing the present invention. In some embodiments, the terminator can be operably linked to the polynucleotides and / or nucleic acids of the present invention.

[0067] A "promoter" is a nucleotide sequence that controls or regulates the transcription of a nucleotide sequence (i.e., a coding sequence) that is operably associated with the promoter. The coding sequence can encode a polypeptide and / or a functional RNA. Typically, a "promoter" refers to a nucleotide sequence that contains a binding site for RNA polymerase II or RNA polymerase III and directs the initiation of transcription. Generally, a promoter is found 5', i.e., upstream, of the start of the coding region of the corresponding coding sequence. The promoter region may contain other elements that function as regulators of gene expression. These include the TATA box consensus sequence and often the CAAT box consensus sequence (Breathnach and Chambon, (1981) Annu. Rev. Biochem. 50:349). In plants, the CAAT box can be replaced by the AGGA box (Messing et al., (1983) in Genetic Engineering of Plants, T. Kosuge, C. Meredith and A. Hollaender (eds.), Plenum Press, pp. 211-227).

[0068] Useful promoters for the present invention can include, for example, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred, and / or tissue-specific promoters for use in the preparation of recombinant nucleic acid molecules, i.e., "chimeric genes" or "chimeric polynucleotides". These various types of promoters are known in the art. The selection of a promoter will vary depending on the temporal and spatial requirements of expression and on the host cell to be transformed. Promoters for many different organisms are well known in the art. Based on the extensive knowledge that exists in the art, an appropriate promoter can be selected for a particular host organism of interest. Thus, for example, much is known about promoters that are upstream of genes that are highly constitutively expressed in model organisms, and such knowledge can be readily accessed and implemented in other systems as needed. In some embodiments, expression of a nucleotide sequence of interest can be in any plant and / or plant part (e.g., in leaves, petioles or stems, ears, inflorescences, roots, seeds, and / or seedlings, etc.), and the promoter is selected accordingly.

[0069] In some embodiments, one or more of the polynucleotides and nucleic acids of the invention can be operably associated with a promoter, as well as a terminator, and / or other regulatory elements for expression in a plant cell. Any promoter, terminator, or other regulatory element that is functional in a plant cell can be used with the nucleic acids of the invention. Non-limiting examples of useful promoters for the present invention include, but are not limited to, the Arabidopsis thaliana U6 RNA polymerase III promoter, the 35S promoter, the actin promoter, the ubiquitin promoter, the Rubisco small subunit promoter, the AlcR / AlcA (ethanol-inducible) promoter, the glucocorticoid receptor (GR) fusion, GVG, the pOp / LhGR (dexamethasone-inducible) promoter, the XVE / OlexA (β-estradiol-inducible) promoter, inducible promoters including, but not limited to, heat shock promoters, and / or bidirectional promoters (see, e.g., Gatz, Christine. Current Opinion in Biotechnology 7(2):168-172(1996); Borghi L. Methods Mol Biol. 655:65-75(2010); Baron et al. Nucleic acids research 23(17)(1995), 3605; Kumar et al. Plant molecular biology 87(4-5):341-353(2015)).

[0070] As used herein, "operatively linked" or "operatively associated" means that the recited elements are functionally related to each other and generally also physically related. Thus, as used herein, the terms "operatively linked" or "operatively associated" refer to nucleotide sequences on a single nucleic acid molecule that are functionally associated. Thus, a first nucleotide sequence that is operatively linked to a second nucleotide sequence means that the first nucleotide sequence is placed in a functional relationship to the second nucleotide sequence. For example, if a promoter causes transcription or expression of the nucleotide sequence, the promoter is operatively associated with the nucleotide sequence. One of ordinary skill in the art will understand that a control sequence (e.g., a promoter) need not be contiguous with the nucleotide sequence to which it is operatively associated, so long as the control sequence functions to direct its expression. Thus, for example, intervening untranslated but transcribed sequences may be present between the promoter and the nucleotide sequence, and the promoter may still be considered to be "operatively linked" to the nucleotide sequence.

[0071] In some embodiments, components for modifying or mutating BBL nucleic acids and any other polynucleotide of interest (e.g., other polynucleotides encoding nicotinic alkaloid biosynthetic enzyme transcription factors that positively regulate nicotinic alkaloid biosynthesis) can be included in an "expression cassette". As used herein, an "expression cassette" means a nucleic acid construct that includes a nucleotide sequence of interest (e.g., a nuclease useful for mutating a BBL nucleic acid), wherein the nucleotide sequence is operably associated with at least a control sequence (e.g., a promoter). The expression cassette can be chimeric, meaning that at least one of its components is heterologous to at least one of the other components. Thus, for example, the nucleic acid to be expressed can be operably linked to a promoter or other regulatory element that is heterologous to the nucleic acid to be expressed (e.g., heterologous to the CRISPR guide DNA). The expression cassette can be naturally occurring but obtained in a recombinant form useful for heterologous expression.

[0072] In addition to the promoter, the expression cassette can optionally also include additional regulatory elements functional in plant cells, including but not limited to transcription and / or translation termination regions (i.e., termination regions). A variety of transcription terminators are available for use in expression cassettes and are involved in the termination of transcription beyond the heterologous nucleotide sequence of interest and proper mRNA polyadenylation. The termination region can be native to the transcription initiation region, native to the operably linked nucleotide sequence of interest, native to the host cell, or can be derived from another source (i.e., foreign or heterologous to the promoter, to the nucleotide sequence of interest, to the host, or to any combination thereof). Non-limiting examples of terminators that are functional in plants and useful in the present invention include the actin terminator; the rubisco small subunit terminator, the rubisco large subunit terminator, the nopaline synthase terminator, and / or the ubiquitin terminator.

[0073] Several untranslated leader sequences derived from viruses are known to enhance gene expression. Specifically, leader sequences from Tobacco Mosaic Virus (TMV, "ω sequence"), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (Gallie et al. (1987) Nucleic Acids Res. 15:8693-8711; and Skuzeski et al. (1990) Plant Mol. Biol. 15:65-79). Other leader sequences known in the art include picornavirus leaders such as the Encephalomyocarditis virus (EMCV) 5' untranslated region leader (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders such as the Tobacco Etch Virus (TEV) leader (Allison et al. (1986) Virology 154:9-20); the Maize Dwarf Mosaic Virus (MDMV) leader (Allison et al. (1986) supra); the Human Immunoglobulin Heavy Chain Binding Protein (BiP) leader (Macejak & Samow (1991) Nature 353:90-94); the untranslated leader from the coat protein mRNA of AMV (AMV RNA 4; Jobling & Gehrke (1987) Nature 325:622-625); the Tobacco Mosaic TMV leader (Gallie et al. (1989) Molecular Biology of RNA 237-256); and the MCMV leader (Lommel et al. (1991) Virology 81:382-385), but are not limited thereto. See also Della-Cioppa et al. (1987) Plant Physiol. 84:965-968.

[0074] The expression cassette may also optionally include transcriptional and / or translational termination regions (i.e., termination regions) that are functional in plants. A variety of transcriptional terminators are available for use in expression cassettes and are involved in the termination of transcription and correct mRNA polyadenylation beyond the heterologous nucleotide sequence of interest. The termination region can be native to the transcription start region, native to the operably linked nucleotide sequence of interest, native to the plant host, or can be derived from another source (i.e., foreign or heterologous to the promoter, the nucleotide sequence of interest, the plant host, or any combination thereof). Suitable transcriptional terminators include, but are not limited to, the CAMV 35S terminator, the tml terminator, the nopaline synthase terminator, and / or the pea rbcs E9 terminator. These can be used in both monocotyledonous and dicotyledonous plants. Additionally, the native transcriptional terminator of the coding sequence can be used.

[0075] The expression cassette may also include a nucleotide sequence for a selectable marker that can be used to select transformed host cells. As used herein, "selectable marker" means a nucleotide sequence that, when expressed, confers a distinct phenotype on a host cell expressing the marker and thus enables such transformed cells to be distinguished from those that do not have the marker. Such a nucleotide sequence can encode either a selectable or screenable marker depending on whether the marker confers a trait that can be selected by chemical means such as by using a selection agent (e.g., an antibiotic, etc.) or whether the marker is a trait (e.g., fluorescence) that can simply be identified through observation or testing such as by screening. Many examples of suitable selectable markers are known in the art and can be used in the expression cassettes described herein.

[0076] In addition to the expression cassette, the nucleic acids described herein can be used in connection with a vector. The term "vector" refers to a composition for moving, delivering, or introducing one or more nucleic acids into a cell. A vector includes a nucleic acid molecule that contains the nucleotide sequence to be moved, delivered, or introduced. Vectors for use in the transformation of host organisms are well known in the art. Non-limiting examples of common classes of vectors include viral vectors, plasmid vectors, phage vectors, phagemid vectors, cosmid vectors, fosmid vectors, bacteriophages, artificial chromosomes, or Agrobacterium binary vectors, which may or may not be self-infectious or mobile and which may be in double-stranded or single-stranded linear or circular form. The vectors defined herein can transform eukaryotic hosts by integration into the cell genome or by existing extrachromosomally (e.g., an autonomously replicating plasmid having an origin of replication). Additionally, shuttle vectors are included, which by nature or design are DNA vehicles capable of replication in two different host organisms. In some representative embodiments, the nucleic acid in the vector is under the control of and operably linked to an appropriate promoter or other regulatory element for transcription in the host cell. The vector can be a bifunctional expression vector that functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory element, and in the case of cDNA, this may be under the control of an appropriate promoter or other regulatory element for expression in the host cell. Thus, the polynucleotide and / or expression cassette can be included in vectors described herein and known in the art.

[0077] As used herein, "nicotinic alkaloid" refers to an alkaloid derived from nicotinic acid. These alkaloids generally contain a 3-pyridyl ring structure, and nicotine, nornicotine, anatabine, and anabasine are the main nicotinic alkaloids within the genus Nicotiana. In some embodiments, the nicotinic alkaloid can comprise, consist essentially of, or consist of nicotine, nornicotine, anatabine, and / or anabasine. In some embodiments, the nicotinic alkaloid is nicotine.

[0078] As used herein, "alkaloid content" means the total amount of alkaloids found in a plant, for example, from the perspective of dry weight percent (dry weight %) or fresh weight percent (fresh weight %).

[0079] Useful plants with the present invention can be any Nicotiana plant that produces nicotine and / or other related alkaloids. Thus, in some embodiments, the plant can be Nicotiana tabacum, Nicotiana rustica, or Nicotiana benthamiana. Any variety of tobacco, including but not limited to, aromatic fire-cured brightleaf tobacco, Burley; Cavendish; Corojo; Criollo; Oriental tobacco; Perique; Shade tobacco; Thuoc lao; Type 22; NC95, K326, K346, White Burley, wild tobacco, Y1, etc., is useful with the present invention.

[0080] As used herein, the term "plant part" includes, but is not limited to, reproductive tissues (e.g., petals, sepals, stamens, pistils, receptacles, anthers, pollen, flowers, fruits, flower buds, ovules, seeds, embryos); vegetative tissues (e.g., petioles, stems, roots, root hairs, root tips, pith, coleoptiles, stalks, shoots, branches, apical meristems, axillary buds, cotyledons, hypocotyls, and leaves); vascular tissues (e.g., phloem and xylem); specialized cells such as epidermal cells, parenchyma cells, collenchyma cells, sclerenchyma cells, stomata, guard cells, cuticles, mesophyll cells, etc.; callus tissue; and cuttings. The term "plant part" also includes plant cells, plant protoplasts, plant tissues, plant organs, plant cell tissue cultures, plant calli, plant clumps, etc., including plant cells that are intact in a plant and / or part of a plant. As used herein, "shoot" refers to the part above the ground surface, including leaves and stems. As used herein, the term "tissue culture" encompasses cultures of tissues, cells, protoplasts, and calli.

[0081] As used herein, "plant cell" refers to the structural and physiological unit of a plant, typically including a cell wall, but also including protoplasts. The plant cells of the present invention can be in the form of isolated single cells, or can be cultured cells, or can be part of a more highly organized unit such as a plant tissue (including callus) or a plant organ. In some embodiments, the plant cells can be algal cells.

[0082] "Plant cell culture" means a culture of plant units such as protoplasts, cultured cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos at various stages of development. In some embodiments of the present invention, there are provided transgenic tissue cultures or transgenic plant cell cultures, wherein the transgenic tissue or cell cultures contain the nucleic acid molecule / nucleotide sequence of the present invention.

[0083] As used herein, "plant organ" means a distinct, visible, structured, and differentiated part of a plant, such as a root, stem, leaf, flower bud, or embryo.

[0084] As used herein, "plant tissue" means a group of plant cells organized into structural and functional units. Any tissue of a plant in an implant or in culture is included. This term includes, but is not limited to, an entire plant, a plant organ, a plant seed, a tissue culture, and any group of plant cells organized into structural and / or functional units. The use of this term in conjunction with or in the absence of any specific type of plant tissue listed above or otherwise subsumed by this definition is not intended to exclude any other type of plant tissue.

[0085] "Introducing," "introduce," "introduced" (and their grammatical variants) in the context of a polynucleotide of interest (e.g., a nuclease useful for mutating a BBL nucleic acid) means presenting the polynucleotide of interest to a host organism or a cell of said organism (e.g., a host cell) in such a manner that the polynucleotide gains access to the interior of the cell. Where more than one polynucleotide is to be introduced, these polynucleotides can be assembled as part of a single polynucleotide or nucleic acid construct, or as separate polynucleotides or nucleic acid constructs, and can be arranged in the same or different expression constructs or transformation vectors. Thus, these polynucleotides can be introduced into cells in a single transformation event, in separate transformation / transfection events, or, for example, they can be incorporated into an organism by conventional breeding protocols. Thus, in some embodiments, one or more polynucleotides encoding a nuclease useful for modifying or mutating a BBL nucleic acid (e.g., a Crispr-Cas nuclease, a meganuclease, a zinc finger nuclease (ZFN), and / or a transcription activator-like effector nuclease (TALEN)) can be introduced into a host organism or a cell of said host organism alone or in combination in a single expression cassette and / or vector. In some embodiments, introducing a recessive allele of nic1, or recessive alleles of recessive alleles of nic1 and nic2, can include incorporating one or more of the recessive alleles into a plant, including modifications in, e.g., BBLa, BBLb, and BBLc, by conventional breeding, whereby the BBLa, BBLb, and BBLc genes have reduced or no expression, or the polypeptides encoded by the modified BBLa, BBLb, and BBLc genes have reduced or no activity.

[0086] As used herein, the terms "transformation" or "transfection" refer to the introduction of a heterologous nucleic acid, such as a nucleic acid encoding a nuclease, into a cell. Transformation of a cell can be stable or transient, or can be partially stably transformed and partially transiently transformed. Thus, in some embodiments, modification of the plant genome can be stable, and in some embodiments, the modification can be transient. In some embodiments, after stable transformation, the nucleic acid construct introduced into the plant genome can be removed, for example, by crossing with an unmodified plant or segregation of a non-homozygous plant.

[0087] "Transient transformation" in the context of a polynucleotide means that the polynucleotide is introduced into a cell and not integrated into the genome of the cell.

[0088] "Stably introduced" or "stably introduced into" in the context of a polynucleotide means that the introduced polynucleotide is stably integrated into the genome of the cell, and thus the cell is stably transformed with the polynucleotide.

[0089] As used herein, "stable transformation" or "stably transformed" means that a nucleic acid construct is introduced into a cell and integrated into the genome of the cell. Thus, the integrated nucleic acid construct can be inherited by its progeny, more particularly by the progeny of multiple generations. As used herein, "genome" can include the nuclear, plastid, and / or mitochondrial genome, and thus can include the integration of a nucleic acid construct into the nuclear, plastid, and / or mitochondrial genome. Stable transformation as used herein can also refer to a transgene maintained extrachromosomally, for example, as a minichromosome or plasmid.

[0090] Transient transformation can be detected by, for example, an enzyme-linked immunosorbent assay (ELISA) or Western blot, which can detect the presence of a peptide or polypeptide encoded by one or more transgenes introduced into a plant or plant cell. Stable transformation of a cell can be detected, for example, by a Southern blot hybridization assay of the genomic DNA of the cell with a nucleic acid sequence that specifically hybridizes to the nucleotide sequence of a transgene introduced into an organism (e.g., bacteria, archaea, yeast, algae, etc.). Stable transformation of a cell can be detected, for example, by a Southern blot hybridization assay of the DNA of the cell with a nucleic acid sequence that specifically hybridizes to the nucleotide sequence of a transgene introduced into a plant or other organism. Stable transformation of a cell can also be detected by, for example, a polymerase chain reaction (PCR) or other amplification reaction well known in the art that employs a specific primer sequence that hybridizes to the target sequence of the transgene and results in amplification of the transgene sequence that can be detected according to standard methods. Transformation can also be detected by direct sequencing and / or hybridization protocols well known in the art.

[0091] Procedures for transforming plants are well known and routine in the art and are described throughout the literature. Non-limiting examples of methods for plant transformation include transformation by bacterial-mediated nucleic acid delivery (e.g., by Agrobacterium), virus-mediated nucleic acid delivery, silicon carbide or nucleic acid whisker-mediated nucleic acid delivery, liposome-mediated nucleic acid delivery, microinjection, particle bombardment, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated nucleic acid uptake, and any other electrical, chemical, physical (mechanical), and / or biological mechanism that results in the introduction of nucleic acid into plant cells, including any combination thereof. General guidelines for various plant transformation methods known in the art include Miki et al. (“Procedures for Introducing Foreign DNA into Plants” in Methods in Plant Molecular Biology and Biotechnology, Glick, B.R. and Thompson, J.E., Eds. (CRC Press, Inc., Boca Raton, 1993), pages 67-88) and Rakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)).

[0092] Agrobacterium-mediated transformation is a method commonly used to transform plants, particularly dicotyledonous plants, because of its high efficiency of transformation and its broad utility with many different species. Agrobacterium-mediated transformation typically involves the transfer of a binary vector carrying foreign DNA of interest into a suitable Agrobacterium strain, which may rely on a complement of vir genes carried by the host Agrobacterium strain on a co-resident Ti plasmid or on the chromosome (Uknes et al. (1993) Plant Cell 5:159-169). The transfer of the recombinant binary vector into Agrobacterium can be accomplished by a triparental mating procedure using Escherichia coli carrying the recombinant binary vector and a helper E. coli strain carrying a plasmid that can mobilize the recombinant binary vector into the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred into Agrobacterium by nucleic acid transformation (Hofgen & Willmitzer (1988) Nucleic Acids Res. 16:9877).

[0093] Transformation of plants by recombinant Agrobacterium usually involves co-cultivation of Agrobacterium with plant-derived explants and follows methods well known in the art. Transformed tissues are regenerated on selective media containing an antibiotic or herbicide resistance marker between the binary plasmid T-DNA borders.

[0094] Another method for transforming plants, plant parts, and / or plant cells involves propelling inert or biologically active particles into plant tissues and cells. See, for example, U.S. Patent Nos. 4,945,050; 5,036,006; and 5,100,792. Generally, this method involves propelling inert or biologically active particles into plant cells under conditions effective to penetrate the outer surface of the cells and effect incorporation into the interior thereof. When inert particles are utilized, the vector can be introduced into the cells by coating the particles with a vector containing the nucleic acid of interest. Alternatively, one or more cells can be surrounded by the vector, whereby the vector is carried to the cells by the wake of the particles. Biologically active particles (e.g., dried yeast cells, dried bacteria, or bacteriophages, each containing one or more nucleic acids to be introduced) can also be propelled into plant tissue.

[0095] Therefore, nucleotide sequences can be introduced into plants, plant parts, and / or plant cells in a number of ways well known in the art. The methods of the present invention do not depend on a particular method for introducing one or more nucleotide sequences into a plant, only that they gain access to the interior of at least one cell of the plant. Thus, in certain embodiments of the present invention, intact plants can be regenerated from these transformed cells using any of a variety of known techniques. Plant regeneration from plant cells, plant tissue cultures, and / or cultured protoplasts is described, for example, in Evans et al. (Handbook of Plant Cell Cultures, Vol. 1, MacMilan Publishing Co. New York (1983)); as well as in Vasil I.R. (ed.) (Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I (1984) and Vol. II (1986)). Methods for selecting transformed transgenic plants, plant cells, and / or plant tissue cultures are routine in the art and can be employed in the methods of the present invention provided herein.

[0096] As used herein, the term "tobacco product" refers to a product containing material produced by Nicotiana plants, including, for example, nicotine gum and patches for smoking cessation, cigarette tobacco including expanded (puffed) and reconstituted tobacco, cigar tobacco, pipe tobacco, cigarettes, cigars, and all forms of smokeless tobacco such as chewing tobacco, snuff, snus, and troches. "Cigarette" includes electronic cigarettes and "heat not burn" products, which are cigarette-like devices that heat tobacco rather than burn it. In some embodiments, tobacco products can include, but are not limited to, cigarillos, kretek cigarettes, non-ventilated concave filter cigarettes, ventilated concave filter cigarettes, cigars, snuff, tobacco-containing gums, tobacco-containing troches, and / or chewing tobacco.

[0097] The present invention relates in part to the discovery that Nicotiana plants comprising (a) polynucleotides encoding BBLa, BBLb, and BBLc, which are berberine bridge-like (BBL) polypeptides, which are modified to reduce or eliminate the expression of the polynucleotide and / or the activity of the polypeptide produced therefrom, and (b) a recessive allele of nic1, or recessive alleles of nic1 and nic2, provide a reduced nicotine alkaloid content compared to Nicotiana plants that do not contain the modified BBLa, BBLb, and BBLc polynucleotides, and the recessive allele of nic1, or recessive alleles of nic1 and nic2, or compared to Nicotiana plants containing only the modified BBLa, BBLb, and BBLc polynucleotides.

[0098] Nicotine is a well-studied plant natural product that is produced in significant amounts by the Nicotiana tabacum L. species, commonly known as tobacco, and numerous other members of the Nicotiana genus. This pyridine alkaloid is synthesized in the tobacco roots and then translocates to the above-ground plant parts in a process stimulated by plant wounding or loss of the apical inflorescence. Nicotine accumulation likely plays a role in natural plant defense against herbivores. Nicotine also plays an important role in human society because it is the main toxicant in manufactured tobacco products such as combustible cigarettes, which have a well-studied toxicant profile. The U.S. Food and Drug Administration (FDA) has listed 93 chemical constituents of tobacco and tobacco smoke that are designated as "harmful and potentially harmful" due to their association with cancer, toxicity, or respiratory, circulatory, reproductive, or developmental toxicity (U.S. Food and Drug Administration 2012).

[0099] Although nicotine itself is not recognized as a carcinogen, the World Health Organization (2015) and the United States Food and Drug Administration (2018) recommend obliging to lower the nicotine level in combustible cigarettes to a non-toxic level in order to reduce overall poisoning to such products and corresponding toxic exposures. The nicotine percentage on a dry weight basis in conventional tobacco cultivars typically ranges between 1.0 - 5.0% and variability is observed due to the sales type (burley, flue-cured, dark, cigar wrapper, or oriental), plant genetics, growing environment, and stalk position. Manufacturers blend the supplied dried leaves to produce cigarette tobacco filters having nicotine between 1.0 - 2.0% on a dry weight basis. The specific concentration at which nicotine becomes non-toxic in combustible cigarettes can be difficult to determine and can vary among individuals, but Benowitz and Henningfield (New Engl. J. Med. 331, 123 - 125 (1994)) predicted that a tobacco filter nicotine content between 0.02 - 0.03% is below the "threshold level of intoxication". The World Health Organization (2015) recommends lowering the nicotine content of tobacco filters to below 0.04%.

[0100] Various methods of chemical extraction are reportedly being used to achieve an 80 - 98% reduction in the nicotine content of tobacco filters. However, the increased cost associated with chemical extraction and the possibility of co-extracting compounds that positively affect sensory properties make these techniques unattractive. The use of modified plant genetics is a preferred route to achieve a reduction in cigarette nicotine levels.

[0101] Genetic methods for developing new tobacco cultivars with reduced nicotine accumulation potential involve the use of (1) genetic variability naturally present within N. tabacum or related species, (2) genetic variability induced by gene editing or mutagenesis, or (3) novel variability created by genetic manipulation. Among the diverse tobacco materials in the United States Nicotiana Germplasm Collection, there is a wide range of variability in alkaloid accumulation, ranging from 0.02 to 6.55% on a dry weight basis. However, the ultra-low nicotine level of 0.04% recommended by the World Health Organization is not readily observed in the lowest alkaloid materials. Recessive alleles at the Nic1 and Nic2 (also known as A and B) loci have been found to contribute to a large reduction in nicotine and related alkaloids (nornicotine, anabasine, and anatabine), from between 1.5 and 4.5% to approximately 0.20 to 0.45%. However, this allelic variability is well-known to be associated with a decrease in the yield and quality of dried leaves, rendering the dried leaves commercially undesirable.

[0102] Knowledge of specific genes identified as being involved in nicotine biosynthesis enables the use of techniques such as RNA interference to downregulate their expression and achieve a corresponding reduction in nicotine accumulation. However, the commercialization of "GMO" tobacco cultivars is subject to variable levels of complex regulatory oversight worldwide. As an alternative to genetic manipulation, gene silencing achieved by induced mutagenesis or gene editing can be used to achieve nicotine reduction. Due to the polyploid nature of N. tabacum, mutations in multiple gene copies are often required to achieve the desired phenotype. The increased complexity of mutation breeding in polyploid species can be offset by the fact that the products of such breeding methods are not regarded as regulated products worldwide.

[0103] The berberine bridge-like (BBL) gene family has previously been identified as encoding enzymes involved in one of the final steps of the N. tabacum nicotine biosynthetic pathway, although their exact roles are not currently understood. This family consists of six closely related members, three of which are expressed to a significant extent (BBL-a, BBL-b, and BBL-c), and three of which are expressed at low levels (BBL-d1, BBL-d2, and BBL-e). The inventors have previously demonstrated substantially reduced nicotine content in conventionally outdoor-grown pipe-dried tobacco plants with RNA interference transgenes that reduce the expression of this gene family (Lewis et al., PLOS ONE 10, e0117273 (2015)). The inventors have also reported preliminary results regarding the effects of inducible mutations in three of the most highly expressed BBL gene family members (Lewis et al., PLOS ONE 10, e0117273 (2015)). The present invention is directed to the development and testing of tobacco strains combining the BBLabc mutation with naturally occurring recessive alleles at the Nic1 and Nic2 loci.

[0104] Accordingly, the present invention provides a method for producing a Nicotiana plant having a reduced nicotine alkaloid content, the method comprising the steps of: (A) modifying to reduce the activity of BBLa, BBLb and BBLc, or reducing the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb and the nucleic acid encoding BBLc; and (B) combining in a Nicotiana plant a recessive allele of nic1, or a recessive allele of nic1 and a recessive allele of nic2, wherein the Nicotiana plant has a reduced nicotine alkaloid content compared to a plant modified by (A) only (e.g., not modified by (A) and (B)). In some embodiments, the method comprises the steps of: (A) modifying to reduce the activity of BBLa, BBLb and BBLc, or reducing the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb and the nucleic acid encoding BBLc; and (B) combining in a Nicotiana plant a recessive allele of nic1. In some embodiments, the method comprises the steps of: (A) modifying to reduce the activity of BBLa, BBLb and BBLc, or reducing the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb and the nucleic acid encoding BBLc; and (B) combining in a Nicotiana plant a recessive allele of nic1 and a recessive allele of nic2. In some embodiments, the method for producing a Nicotiana plant having a reduced nicotine alkaloid content may further comprise a modification to reduce the expression of a polynucleotide encoding an additional nicotine alkaloid biosynthetic enzyme. In some embodiments, the additional nicotine alkaloid biosynthetic enzyme may include, but is not limited to, aspartate oxidase, quinolinic acid synthetase, quinolinate phosphoribosyl transferase, ornithine decarboxylase, putrescine N-methyl transferase, methylputrescine oxidase, and / or A622.

[0105] In some embodiments, a Nicotiana plant produced by the method of the invention can have a nicotine alkaloid content that is reduced by at least 40% (e.g., at least about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96%, or more; or any range or value therein) as compared to a plant modified by (A) only (e.g., not modified by (A) and (B)). In some embodiments, the nicotine alkaloid that is reduced in a Nicotiana plant produced by the method of the invention can be nicotine, and the nicotine content can be reduced by about 40% to about 90% (e.g., about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or 91%, or more; or any range or value therein) as compared to a plant modified by (A) only (e.g., not modified by (A) and (B)). In some embodiments, a Nicotiana plant produced by the method of the invention that reduces the activity of (A) BBLa, BBLb, and BBLc, or modifies the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and has a recessive allele of nic1 or recessive alleles of nic1 and nic2 can have a nicotine content of about 0.014% to about 0.098%.

[0106] In some embodiments, a Nicotiana plant produced by the method of the invention having (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1 may contain a nicotine alkaloid content that is reduced by at least about 30% (e.g., about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56%, or a rate greater than that, or any range or value therein) compared to a plant modified by (A) only (e.g., not modified by (A) and (B)). In some embodiments, the nicotine alkaloid that is reduced in the Nicotiana plant of the invention may be nicotine, and the nicotine content may be reduced by about 40% compared to a plant modified by (A) only (e.g., not modified by (A) and (B)). In some embodiments, a Nicotiana plant of the invention having (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1 may contain a nicotine content of about 0.098%.

[0107] In some embodiments, a Nicotiana plant produced by the method of the invention having (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) the recessive alleles of nic1 and nic2 may contain a nicotine alkaloid content that is reduced by at least about 40% (e.g., at least about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96%, or a rate above that, or any range or value therein) compared to a plant modified by (A) only (e.g., not modified by (A) and (B)). In some embodiments, the nicotine alkaloid that is reduced in the Nicotiana plant of the invention may be nicotine, and the nicotine content may be reduced by about 90% (e.g., about 90, 91, 92, 93, 94, 95, 96, 97%, or a rate above that) compared to a plant modified by (A) only (e.g., not modified by (A) and (B)). In some embodiments, a Nicotiana plant of the invention having (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) the recessive alleles of nic1 and nic2 may contain a nicotine content of about 0.014%.

[0108] In some embodiments, a Nicotiana plant produced by the method of the invention may contain increased chlorophyll (e.g., increased green) compared to a plant modified by (A) only (e.g., not modified by (A) and (B)), optionally, the increase in chlorophyll is in the leaves of the plant or in the leaves and stems of the plant. In some embodiments, a Nicotiana plant produced by the method of the invention may contain a lower sugar content compared to a plant modified by (A) only (e.g., not modified by (A) and (B)), optionally, the sugar content may be reduced by about 7% to 28% (e.g., about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28%, or a proportion above that, or any range or value therein) compared to a plant modified by (A) only (e.g., not modified by (A) and (B)). In some embodiments, a Nicotiana plant produced by the method of the invention may contain a lower nitrogen content compared to a plant modified by (A) only (e.g., not modified by (A) and (B)), optionally, the nitrogen content may be reduced by about 10% to 25% (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25%, or a proportion above that, or any range or value therein) compared to a plant modified by (A) only (e.g., not modified by (A) and (B)).

[0109] The present invention further provides a method for drying the leaves and stems of the Nicotiana plants of the present invention. The Nicotiana plants of the present invention containing the genetic modifications described herein produce leaves and stems that do not provide tobacco of sufficient quality for use in tobacco products when dried using standard tobacco drying methods (e.g., the methods described in Powell 1987 (Powell Manufacturing, Co.’s Bulk Curing / Drying Owner’s Operator’s Manual for Flue Cured Tobaccos, manual titled Powell Manufacturing, Co. (Jan. 20, 1987))).In contrast to standard methods of drying, the drying methods described herein, when applied to the leaves and stems of the Nicotiana plants of the present invention (e.g., (A) modifications that reduce the activity of BBLa, BBLb, and BBLc, or reduce the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) having a recessive allele of nic1 or a recessive allele of nic1 and a recessive allele of nic2), provide tobacco having improved quality compared to the leaves and stems of the Nicotiana plants of the present invention dried using a standard drying protocol [e.g., heating the leaves at a start temperature of about 92°F to about 96°F (e.g., about 92°F, 93°F, 94°F, 95°F, or 96°F, or any range or value therein), increasing to a maximum upper temperature of about 104°F to about 108°F (e.g., about 104°F, 105°F, 106°F, 107°F, 108°F, or any range or value therein) at a rate of about 1°F per hour until the maximum upper temperature is reached, and holding the upper temperature for an approximate period of about 52 - 58 hours (e.g., about 52, 53, 54, 55, 56 hours, or any range or value therein), a yellowing step; a leaf drying step at a temperature of about 120°F for about 22 hours; and (c) a stem drying step at a temperature of about 132°F to 138°F (e.g., about 132°F, 133°F, 134°F, 135°F, 136°F, 137°F, or 138°F, or any range or value therein) for about 50 hours to about 65 hours (e.g., about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 hours, or any range or value therein) with continuous monitoring, thereby drying one or more leaves of the Nicotiana plant and producing one or more dried leaves of the Nicotiana plant]. In some embodiments, the leaf drying step is carried out at a temperature of about 116°F to 120°F or about 116°F to 118°F (e.g., about 116°F, 117°F, 118°F, 119°F, 120°F, or any range or value therein) for about 28 - 34 hours (e.g., about 28, 29, 30, 31, 32, 33, or 34 hours, or any range or value therein).The improvement in the quality of the Nicotiana plants of the present technology dried according to the method of the present technology includes, but is not limited to, better index grades, improved texture, improved color, high high-quality, increased sugar levels, and reduced ammonia levels as compared to the Nicotiana plants of the present technology dried using a standard drying treatment protocol. The sugar levels in the types dried by iron pipe drying are indicators of bright leaves with good aroma, and the ammonia levels are typically used as indicators of off-flavors in tobacco.

[0110] Thus, in some embodiments, the present invention is a method for drying one or more leaves and / or stems of a Nicotiana plant, wherein the Nicotiana plant has (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1, or a recessive allele of nic1 and a recessive allele of nic2, and the Nicotiana plant has a reduced nicotine alkaloid content compared to a plant modified by (A) only. This method includes a yellowing step that (a) starts heating the leaves at a temperature of 92°F to 96°F and increases the temperature to 104°F to about 108°F (e.g., 104°F, 105°F, 106°F, 107°F, or 108°F, or any range or value therein) at a rate of about 1°F per hour until the maximum upper temperature is reached, and holds for a period of about 52 - 58 hours (e.g., about 52, 53, 54, 55, 56, 57, or 58 hours, and any range or value therein) at the upper temperature. Optionally, this step is a yellowing step with a length of about 54 hours; (b) optionally, a leaf drying step at a temperature of about 120°F for about 22 hours; and (c) a stem drying step at about 132°F to 138°F (132°F, 133°F, 134°F, 135°F, 136°F, 137°F, or 118°F, or any range or value therein), optionally at a temperature of 134°F, for about 50 hours to about 65 hours (e.g., about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 6, 61, 62, 63, 64, or 65 hours, and any range or value therein), with continuous monitoring. Thereby, one or more leaves of the Nicotiana plant are dried to produce one or more dried leaves of the Nicotiana plant. In some embodiments, the leaf drying step is performed at a temperature of about 116°F to 120°F or about 116°F to 118°F (116°F, 117°F, 118°F, 119°F, 120°F, or any range or value therein) for about 28 - 34 hours.

[0111] In some embodiments, the present invention provides dried tobacco produced by the method of the present invention. In some embodiments, the dried tobacco of the present invention (e.g., leaves, smoking leaves, lugs, cutters, primings) has high-quality to premium-quality characteristics with good color intensity, normal width, and uniform texture. Optionally, the dried tobacco has a USDA grade index greater than 60. In some embodiments, the dried tobacco is of high-quality to good-quality to low-quality or excellent-quality to high-quality to good-quality according to the USDA standard grades (“Official Standard Grades for Flue-Cured Tobacco U.S. Types 11, 12, 13, 14, and Foreign Type 92”) (published on the USDA Agriculture Marketing Service website (ams.usda.gov / grades-standards / tobacco)) and Title 7, Chapter 1, Part 29 issued under the authority of The Tobacco Inspection Act (49 Stat. 731; 7 U.S.C. 511) (effective date of March 27, 1989 (54 F.R. 7925)). Example USDA standard grades are listed in Table 1.

[0112] [Table 1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

[0113] In some embodiments, a dried tobacco leaf derived from a Nicotiana plant, wherein (A) the Nicotiana plant comprises (1) one or both of the recessive nic1 and nic2 alleles, and (2) modified BBLa, BBLb, and BBLc genes compared to the wild type, whereby the nicotine alkaloid content of the Nicotiana plant is reduced compared to the wild type; and (B) the dried tobacco leaf (including leaves, smoking leaves, tips, cutters, and rags) has not been cultivated and dried using the method of the present invention, and as a result, has the characteristics of high-quality to good-quality leaves with good color intensity, normal width, and uniform texture compared to leaves derived from the same Nicotiana plant that produce poor-quality to low-quality leaves. In some embodiments, the dried tobacco of the present invention may have a USDA grade index greater than 60.

[0114] In some embodiments, the dried tobacco produced by the method of the present invention from Nicotiana plants having one or both of the recessive nic1 and nic2 alleles (compared to wild type) and the modified BBLa, BBLb, and BBLc genes, which have a reduced nicotine alkaloid content compared to wild type, may include, but is not limited to, leaves, smoking leaves, tips, cutters, primings, and / or lugs. In some embodiments, the smoking leaves provided by the present invention have a USDA quality grade of H3F (high-quality orange smoking leaves) - 4F (good-quality orange smoking leaves) - 5F (low-quality orange smoking leaves) compared to H6F (low-quality orange smoking leaves) - H6FR (low-quality orange-red smoking leaves) - H6K (low-quality mottled smoking leaves) for smoking leaves of Nicotiana plants having one or both of the recessive nic1 and nic2 alleles and the modified BBLa, BBLb, and BBLc genes that have not been dried or cultivated using the method of the present invention. In some embodiments, the leaves (Group B) provided by the present invention have a USDA quality grade of B2L (excellent-quality lemon-colored leaves) - B3L (high-quality lemon-colored leaves) - B4L (good-quality lemon-colored leaves) compared to B5L (low-quality lemon-colored leaves) - B6L (low-quality lemon-colored leaves) for leaves (Group B) of Nicotiana plants having one or both of the recessive nic1 and nic2 alleles and the modified BBLa, BBLb, and BBLc genes that have not been dried or cultivated using the method of the present invention.

[0115] In some embodiments, the cutters (Group C) provided by the present invention may have USDA quality grades of C2L (high-quality lemon-colored cutter) to C3L (superior-quality lemon-colored cutter) to C4L (good-quality lemon-colored cutter) as compared to C5L (low-quality lemon-colored cutter) for cutters (Group C) of Nicotiana plants having one or both of the recessive nic1 and nic2 alleles and the altered BBLa, BBLb, and BBLc genes that have not been dried or cultivated using the method of the present invention. In some embodiments, the cutters (Group C) provided by the present invention may have USDA quality grades of C2F (high-quality orange-colored cutter) to C3F (superior-quality orange-colored cutter) to C4F (good-quality orange-colored cutter) as compared to C5F (low-quality orange-colored cutter) for cutters (Group C) of Nicotiana plants having one or both of the recessive nic1 and nic2 alleles and the altered BBLa, BBLb, and BBLc genes that have not been dried or cultivated using the method of the present invention.

[0116] In some embodiments, the rags (Group X) provided by the present invention may have USDA quality grades of X2L (high-quality lemon-colored rag) to X3L (superior-quality lemon-colored rag) to X4L (good-quality lemon-colored rag) as compared to X5L (low-quality lemon-colored rag) for rags (Group X) of Nicotiana plants having one or both of the recessive nic1 and nic2 alleles and the altered BBLa, BBLb, and BBLc genes that have not been dried or cultivated using the method of the present invention. In some embodiments, the rags (Group X) provided by the present invention may have USDA quality grades of X2F (high-quality orange-colored rag) to X3F (superior-quality orange-colored rag) to X4F (good-quality orange-colored rag) as compared to X5F (low-quality orange-colored rag) for rags (Group X) of Nicotiana plants having one or both of the recessive nic1 and nic2 alleles and the altered BBLa, BBLb, and BBLc genes that have not been dried or cultivated using the method of the present invention.

[0117] In some embodiments, the primings (P group) provided by the present invention may have USDA quality grades of P2L (excellent quality lemon-colored priming) to P3L (high-quality lemon-colored priming) to P4L (good quality lemon-colored priming) as compared to P5L (low-quality lemon-colored priming) for primings (P group) of Nicotiana plants having one or both of the recessive nic1 and nic2 alleles and the altered BBLa, BBLb, and BBLc genes that have not been dried or cultivated using the method of the present invention. In some embodiments, the primings (P group) provided by the present invention may have USDA quality grades of P2F (excellent quality orange-colored priming) to P3F (high-quality orange-colored priming) to P4F (good quality orange-colored priming) as compared to P5F (low-quality orange-colored priming) for primings (P group) of Nicotiana plants having one or both of the recessive nic1 and nic2 alleles and the altered BBLa, BBLb, and BBLc genes that have not been dried or cultivated using the method of the present invention.

[0118] As used herein, "leaf" refers to the main component of a tobacco plant, and the size, shape, and position on the stalk can be indicators of leaf quality. "Leaf" on a tobacco plant dried by iron pipe drying refers to the second group of leaves from the top, while in flue-cured and dark air-dried tobacco, "leaf" is a general term for all of the leaves located third from the top of the tobacco plant.

[0119] "Smoking leaves" grow just above the center of the stalk.

[0120] As used herein, "rag" refers to the second group of leaves from the ground on a tobacco plant dried by iron pipe drying; the largest leaves on a burley tobacco plant located near the group at the center of the stalk and / or the center of the leaf on a flue-cured and dark air-dried tobacco plant.

[0121] As used herein, "cutter" refers to the largest leaf on a tobacco plant that has been dried in an iron pipe and is located near the central part of the stalk.

[0122] As used herein, "tip" refers to the uppermost leaf on a tobacco plant or burley tobacco plant that has been dried in an iron pipe and is often removed during processing, and / or the pointed end of a tobacco leaf (the one located farthest from the stalk).

[0123] In some embodiments, there is provided a tobacco product comprising tobacco derived from a Nicotiana plant, wherein the Nicotiana plant comprises (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1, or a recessive allele of nic1 and a recessive allele of nic2, and the Nicotiana plant has a reduced nicotine alkaloid content compared to a plant modified only by (A) (e.g., not modified by (A) and (B)). In some embodiments, there is provided a tobacco product comprising tobacco derived from a Nicotiana plant, wherein the Nicotiana plant comprises (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1. In some embodiments, there is provided a tobacco product comprising tobacco derived from a Nicotiana plant, wherein the Nicotiana plant comprises (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1 and a recessive allele of nic2.

[0124] In some embodiments, a Nicotiana plant of the invention having (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, for use in producing a tobacco product, and (B) a recessive allele of nic1, or a recessive allele of nic1 and a recessive allele of nic2, may contain a nicotine alkaloid content that is reduced by at least about 40% (e.g., at least about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96%, or a rate above that, or any range or value therein) as compared to a plant modified by (A) only (e.g., not modified by (A) and (B)). In some embodiments, the nicotine alkaloid that is reduced in the Nicotiana plant of the invention for use in producing a tobacco product may be nicotine, and the nicotine content may be reduced by about 40% to about 90% (e.g., about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or 91%, or a rate above that; or any range or value therein) as compared to a plant modified by (A) only (e.g., not modified by (A) and (B)).In some embodiments, a Nicotiana plant of the invention having (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1 or recessive alleles of nic1 and nic2 for use in producing a tobacco product can contain a nicotine content of from about 0.014% to about 0.098%.

[0125] In some embodiments, a Nicotiana plant of the invention having (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1 for use in producing a tobacco product can contain a nicotine alkaloid content that is at least about 30% (e.g., about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56% or more, or any range or value therein) reduced compared to a plant modified by (A) only (e.g., not modified by (A) and (B)). In some embodiments, the nicotine alkaloid that is reduced in the Nicotiana plant of the invention for use in producing a tobacco product can be nicotine, and the nicotine content can be reduced by about 40% compared to a plant modified by (A) only. In some embodiments, a Nicotiana plant of the invention having (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1 for use in producing a tobacco product can contain a nicotine content of about 0.098%.

[0126] In some embodiments, a Nicotiana plant of the invention having (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) the recessive alleles of nic1 and nic2, for use in producing a tobacco product, may contain a nicotine alkaloid content that is at least 40% (e.g., at least about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96%, or a rate greater than that, or any range or value therein) decreased compared to a plant modified only by (A) (e.g., not modified by (A) and (B)). In some embodiments, the nicotine alkaloid that is decreased in the Nicotiana plant of the invention for use in producing a tobacco product may be nicotine, and the nicotine content may be decreased by about 90% (e.g., about 90, 91, 92, 93, 94, 95, 96, 97%, or a rate greater than that, or any range or value therein) compared to a plant modified only by (A). In some embodiments, a Nicotiana plant of the invention having (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) the recessive alleles of nic1 and nic2, for use in producing a tobacco product, may contain a nicotine content of about 0.014%.

[0127] Procedures for determining nicotine alkaloid content are well known and routine in the art and are described in the literature. Non-limiting examples of such methods include gas chromatography, mass spectrometry (Domino et al. 1992 Med Sci Res. 20:859-860; Sheen et al. 2006 J Food Sci 53(5):1572-1573), HPLC (Keinanen et al. 2001 J Agric Food Chem 49:3553-3558; Halitschke and Baldwin 2003 Plant J 36:794-807), UV absorption (Willits et al. 2005 Analytical Chemistry 22:430-433), and the like.

[0128] In some embodiments, Nicotiana plants useful for producing the tobacco and tobacco products described herein may include further genetic modifications. For example, a Nicotiana plant may further include a decrease in the expression of a polynucleotide encoding an additional nicotine alkaloid biosynthetic enzyme. In some embodiments, additional nicotine alkaloid biosynthetic enzymes may include, but are not limited to, aspartate oxidase, quinolinic acid synthase, quinolinate phosphoribosyl transferase, ornithine decarboxylase, putrescine N-methyl transferase, methyl putrescine oxidase, and / or A622.

[0129] The Nicotiana plants of the present invention can be further modified to reduce the activity of additional nicotine alkaloid biosynthetic enzymes or to reduce the expression of nucleic acids encoding additional nicotine alkaloid biosynthetic enzymes. Such additional nicotine alkaloid biosynthetic enzymes include, but are not limited to, additional berberine bridge enzyme-like polypeptides, aspartate oxidase, quinolinic acid synthetase, quinolinate phosphoribosyl transferase, ornithine decarboxylase, putrescine N-methyl transferase, methylputrescine oxidase, and A622. Thus, for example, the Nicotiana plants can be further modified to reduce the expression of BBLe, BBLd-1 and / or BBLd-2 and / or to reduce the activity of additional berberine bridge enzyme-like polypeptides such as BBLe, BBLd-1 and / or BBLd-2.

[0130] In some embodiments, the invention further comprises reducing the expression of a polynucleotide encoding a transcription factor that positively regulates nicotine alkaloid biosynthesis in a Nicotiana plant or plant part. Thus, in some embodiments, the Nicotiana plant or plant part can be further modified to reduce the expression of at least one polynucleotide encoding a transcription factor that positively regulates nicotine alkaloid biosynthesis. Non-limiting examples of transcription factors that positively regulate nicotine alkaloid biosynthesis include ERF family transcription factors such as ERF189, ERF221 and ERF32, and / or bHLH family transcription factors such as NtMYC1 and NtMYC2, and COI1.

[0131] In some embodiments, the Nicotiana plants of the present invention can be further modified to overexpress at least one polynucleotide encoding a transcription factor that negatively regulates nicotine alkaloid biosynthesis. Non-limiting examples of transcription factors that negatively regulate nicotine alkaloid biosynthesis include JAZ.

[0132] As used herein, "overexpressed," "overexpression," "overexpressing" (and grammatical variations thereof) refer to the production of a gene product in a transgenic Nicotiana plant or plant part that exceeds the level of production of the same gene product in a control Nicotiana plant or plant part, where the transgenic Nicotiana plant or plant part is transformed with a recombinant nucleic acid construct that confers an increase in the production of the gene product, while the control Nicotiana plant or plant part is not transformed with said recombinant nucleic acid construct.

[0133] Expression of any of the additional polynucleotides to be modified in the Nicotiana plants of the present invention can be decreased by any known means for introducing mutations, including gene editing, and / or via introduction into the Nicotiana plants of interfering RNAs developed to target nucleic acids encoding any one or more of the additional nicotine alkaloid biosynthetic enzymes. As is well known in the art, "interfering RNA" is RNA that can cause gene silencing. Interfering RNAs used herein include, but are not limited to, sense RNA, antisense RNA, short interfering RNA (siRNA), microRNA (miRNA), double-stranded RNA (dsRNA), hairpin RNA (RNA), and any type of RNA molecule that can downregulate or silence the expression of a target nicotine alkaloid biosynthetic nucleic acid.

[0134] In some embodiments, provided are seeds of Nicotiana plants of the present invention and Nicotiana plants produced from the seeds, wherein the seeds comprise: (A) a modification that reduces the activities of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1, or a recessive allele of nic1 and a recessive allele of nic2, and the Nicotiana plants have a reduced nicotine alkaloid content as compared to plants modified by (A) only (e.g., not modified by (A) and (B)). In some embodiments, provided are seeds of Nicotiana plants of the present invention and Nicotiana plants produced from the seeds, wherein the seeds comprise: (A) a modification that reduces the activities of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1, and the Nicotiana plants have a reduced nicotine alkaloid content as compared to plants modified by (A) only (e.g., not modified by (A) and (B)). In some embodiments, provided are seeds of Nicotiana plants of the present invention and Nicotiana plants produced from the seeds, wherein the seeds comprise: (A) a modification that reduces the activities of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1 and a recessive allele of nic2, and the Nicotiana plants have a reduced nicotine alkaloid content as compared to plants modified by (A) only (e.g., not modified by (A) and (B)).

[0135] In some embodiments, the present invention provides progeny Nicotiana plants produced from the Nicotiana plants of the present invention. In some embodiments, there is further provided a crop comprising a plurality of Nicotiana plants of the present invention planted together in an agricultural land.

[0136] Additional aspects of the present invention include harvest products produced from the Nicotiana plants or plant parts of the present invention, as well as processed products produced from said harvest products. The harvest product can be the whole plant or any plant part, and said harvest product comprises the recombinant nucleic acid molecule / construct of the present invention. Thus, in some embodiments, non-limiting examples of the harvest product include seeds, fruits, flowers, or parts thereof (e.g., stamens, stigmas, etc.), leaves, stems, etc.

[0137] Any tobacco or tobacco product can be produced using the Nicotiana plants of the present invention. In some embodiments, the tobacco produced can include, but is not limited to, leaf tobacco, shredded tobacco, cut tobacco, ground tobacco, powdered tobacco, tobacco extract, smokeless tobacco, moist or dry snuff, pipe tobacco, cigar tobacco, cigarillo tobacco, cigarette tobacco, and / or chewing tobacco. In some embodiments, the tobacco products made using the tobacco of the present invention can include, but are not limited to, cigarillos, kretek cigarettes, non-ventilated concave filter cigarettes, ventilated concave filter cigarettes, cigars, snuff, tobacco-containing gums, tobacco-containing troches, and / or chewing tobacco.

[0138] In some embodiments, the present invention provides a tobacco product, which can be a blended tobacco product. In some embodiments of the present invention, the tobacco product of the present invention can be a reduced nicotine tobacco product. In still other embodiments, the tobacco product of the present invention can be a blended tobacco product having a reduced nicotine content. Thus, the tobacco product of the present invention can be a blended reduced nicotine tobacco product.

[0139] In some embodiments, the present invention provides a reduced-nicotine alkaloid tobacco product having a reduced nicotine alkaloid content and having a modification that (A) reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) includes a recessive allele of nic1, or recessive alleles of nic1 and nic2, wherein the Nicotiana plant or plant part from which the product is produced has a reduced nicotine alkaloid content as compared to a plant modified by (A) only (e.g., not modified by (A) and (B)).

[0140] The present invention further provides a method for producing a blended tobacco, comprising: a) providing a first tobacco; b) providing a second tobacco, wherein the second tobacco has a reduced nicotine alkaloid content and has a modification that (A) reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) includes a recessive allele of nic1, or recessive alleles of nic1 and nic2, and is produced from a Nicotiana plant of the present invention; and c) blending the first tobacco and the second tobacco to produce a blended tobacco. In some embodiments, the first and second tobaccos can be produced from a Nicotiana plant of the present invention. In some embodiments, both the first tobacco and the second tobacco have a reduced nicotine alkaloid content and have a modification that (A) reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) includes a mutation in a recessive allele of nic1, or recessive alleles of nic1 and nic2, and are from different Nicotiana plant types.

[0141] In another aspect of the invention, a method for producing a blended reduced nicotine tobacco, comprising: a) providing a first tobacco; b) providing a second tobacco, wherein the second tobacco has a reduced nicotine alkaloid content and is produced from a Nicotiana plant of the invention that has (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1, or a recessive allele of nic1 and a recessive allele of nic2; and c) blending the first tobacco and the second tobacco to produce the blended reduced nicotine tobacco. In some embodiments, the first and second tobaccos can be produced from Nicotiana plants of the invention. In some embodiments, both the first tobacco and the second tobacco have a reduced nicotine alkaloid content and are from different Nicotiana plant types that have (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1, or a recessive allele of nic1 and a recessive allele of nic2. As an example, flue-cured and air-cured tobaccos are components of a typical American blend cigarette. Thus, in some embodiments of the invention, a low nicotine alkaloid tobacco product can be produced by blending low nicotine Burley types and high nicotine flue-cured types, each having a reduced nicotine alkaloid content and having (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1, or a recessive allele of nic1 and a recessive allele of nic2, thereby reducing the overall nicotine alkaloid (e.g., nicotine, anatabine, nornicotine, anabasine, etc.) content of the low alkaloid tobacco product.

[0142] As is well known in the art, tobacco formulations for tobacco products can, in addition to tobacco, alter the bitterness, sweetness, sourness, or saltiness of the formulation; the perceived dryness or wetness of the formulation; or other components that can enhance the degree of tobacco flavor presented by the formulation. Such other components can include salts (e.g., sodium chloride, potassium chloride, sodium citrate, potassium citrate, sodium acetate, potassium acetate, etc.); natural sweeteners (e.g., fructose, sucrose, glucose, maltose, mannose, galactose, lactose, etc.); artificial sweeteners (e.g., sucralose, saccharin, aspartame, acesulfame K, etc.), organic and inorganic fillers (e.g., grains, processed grains, puffed grains, maltodextrin, dextrose, calcium carbonate, calcium phosphate, corn starch, lactose, mannitol, xylitol, sorbitol, microcrystalline cellulose, etc.); binders (e.g., povidone, sodium carboxymethylcellulose, and other modified cellulose type binders, sodium alginate, xanthan gum, starch-based binders, gum arabic, lecithin, etc.); pH adjusters or buffering agents (e.g., metal hydroxides, preferably alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, and other alkali metal buffers such as potassium carbonate, sodium carbonate, sodium bicarbonate, etc.); colorants (e.g., dyes and pigments including caramel colorants and titanium dioxide); humectants (e.g., glycerin, propylene glycol, etc.); preservatives (e.g., potassium sorbate, etc.); syrups (e.g., honey, high fructose corn syrup, etc.); disintegration aids (e.g., crystalline cellulose, croscarmellose sodium, crospovidone, sodium starch glycolate, pregelatinized corn starch, etc.); antioxidants (e.g., ascorbic acid, polyphenol-containing materials such as grape seed extract and oil, green tea extract and black tea extract, peanut endocarp, potato peel, etc. (see Santhosh et al., Phytomedicine, 122:16-220 (2005); incorporated herein by reference)); and flavoring agents.The flavorant can be natural or synthetic and includes, but is not limited to, fresh, sweet, herbal, confectionery, floral, fruity, or spice flavors. Specific types of flavors include, but are not limited to, vanilla, coffee, chocolate, cream, mint, spearmint, menthol, peppermint, wintergreen, lavender, cardamom, nutmeg, cinnamon, clove, cascarilla, patchouli, honey, jasmine, ginger, anise, sage, licorice, grape, lemon, orange, apple, peach, lime, raspberry, and strawberry. (See Leffingwill et al., Tobacco Flavoring for Smoking Products, R.J. Reynolds Tobacco Company (1972)). The flavorant can also include components considered to be humectants, coolants, or smoothing agents, including, but not limited to, eucalyptus. These flavors can be provided alone or as blends (e.g., spearmint and menthol, or orange and cinnamon). Representative types of components are also described in U.S. Patent No. 5,387,416 to White et al. and PCT Publication No. WO2005 / 041699 to Quinter et al., the relevant portions of each of which are incorporated herein by reference. Thus, in some embodiments, the tobacco product of the present invention can include flavor components or aromas.

[0143] The amount of tobacco in the tobacco formulation can vary. In certain embodiments, the amount of tobacco in the tobacco formulation is at least about 25 percent to at least about 40 percent on a dry weight basis (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 dry weight %, and any value or range therein). The amount of other components in the tobacco formulation preferably exceeds about 25 percent to about 40 percent on a dry weight basis.

[0144] In some embodiments of the present invention, there is provided a method for reducing the amount of nicotine in a human who uses tobacco, the method comprising providing to the human any of the tobacco products of the present invention.

[0145] In yet another aspect of the present invention, there is provided a method for reducing the nicotine consumption of a smoker, the method comprising: (a) providing to the smoker a first tobacco product comprising tobacco produced from a Nicotiana plant of the present invention, the first tobacco product having a reduced nicotine alkaloid content and comprising a modification that (A) reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1, or a recessive allele of nic1 and a recessive allele of nic2; (b) providing to the smoker a second tobacco product comprising tobacco produced from a Nicotiana plant of the present invention, the second tobacco product having a reduced nicotine alkaloid content and comprising a modification that (A) reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1, or a recessive allele of nic1 and a recessive allele of nic2, wherein the second tobacco product contains less nicotine than the first tobacco product.

[0146] In some aspects of the present invention, a smoker may be provided with additional tobacco products comprising tobacco produced from a Nicotiana plant of the present invention, the additional tobacco products having a reduced nicotine alkaloid content and comprising a modification that (A) reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1, or a recessive allele of nic1 and a recessive allele of nic2, wherein the additional tobacco products contain sequentially decreasing amounts of nicotine starting from a third product that contains less nicotine than the first or second tobacco product.

[0147] In some embodiments of the present invention, there is provided a smoking cessation kit, which comprises a tobacco product selected from any of the products of the present invention produced from Nicotiana plants of the present invention, having a reduced nicotine alkaloid content and having (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1, or a recessive allele of nic1 and a recessive allele of nic2.

[0148] In some embodiments, the present invention provides a kit comprising a first tobacco product containing nicotine and a second tobacco product containing an amount of nicotine less than that in the first tobacco product, wherein the first or second tobacco product has a reduced nicotine alkaloid content and has (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1, or a recessive allele of nic1 and a recessive allele of nic2, and the kit comprises a tobacco product produced from a Nicotiana plant of the present invention.

[0149] In some aspects, the present invention provides a product produced from a Nicotiana plant of the present invention, having a reduced nicotine alkaloid content and having (A) a modification that reduces the activity of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) a recessive allele of nic1, or a recessive allele of nic1 and a recessive allele of nic2, and the product produced is selected from the group consisting of industrial enzymes, pharmaceuticals, cosmetic components, human and livestock food, food additives, and fermentation products.

[0150] The present invention further provides a method for improving / increasing the yield and quality of a Nicotiana plant of the present invention, which (A) reduces the activities of BBLa, BBLb, and BBLc, or modifies to reduce the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) has a recessive allele of nic1, or recessive alleles of nic1 and nic2. In some embodiments, the method for improving the yield of a Nicotiana plant of the present invention comprises: (a) fertilizing the seedlings at the stage of about 90% germination, wherein the fertilizing step is carried out through fertilizing irrigation (e.g., drip irrigation), and optionally the concentration of N in the fertilizer is about 200 ppm; (b) applying a plastic mulch treatment, wherein, for example, the plastic mulch is added to cover the fertilizing irrigation facility (e.g., drip tape / tube); and (c) applying nitrogen to the seedlings at about 4-5 weeks after transplantation, about 6-7 weeks after transplantation, and about 8-9 weeks after transplantation at a rate of about 90-120 pounds / acre of total N to provide an increase in the yield and quality of the Nicotiana plant.

[0151] As used herein, the "substrate" on which a Nicotiana plant and / or its plant part (e.g., seed) is grown / planted refers to any medium for germinating the seed and / or for planting / growing the Nicotiana plant, and may include, but is not limited to, natural soil, synthetic soil, planting medium, soil-free medium (e.g., sphagnum moss, perlite, vermiculite, etc.), and / or any combination thereof.

[0152] As used herein, "increase in yield" means, compared to the same plants grown under standard tobacco growing conditions (e.g., fertilizers applied to the ground surface and later added to the sides (e.g., laid-by), and separately supplied water (rain or external irrigation); plastic mulch is not part of the standard growing procedure, nor is the use of fertigation (water + fertilizer) by drip tape), when grown under the modified conditions described herein, (A) a modification that reduces the activities of BBLa, BBLb, and BBLc, or reduces the expression of the nucleic acid encoding BBLa, the nucleic acid encoding BBLb, and the nucleic acid encoding BBLc, and (B) an increase in biomass, an increase in the number of leaves, an increase in leaf area, and / or an increase in petiole circumference observed for tobacco plants containing the recessive allele of nic1, or the recessive allele of nic1 and the recessive allele of nic2.

[0153] In some embodiments, nitrogen can be applied to the substrate using fertigation (before and / or after planting). As used herein, "fertigation" refers to a fertilizer application where the fertilizer is incorporated into the irrigation water by a drip system. Fertigation provides for an even distribution of the fertilizer solution by irrigation.

[0154] In some embodiments, the substrate on which Nicotiana plants are planted can be coated to reduce weed growth and water loss, and to better control the temperature of the substrate (e.g., keep it more constant and / or increase the soil temperature). In some embodiments, the coating can be a plastic mulch (e.g., a ground cover sheet), including but not limited to, a plastic ground cover sheet (e.g., a white plastic sheet, a black plastic sheet).

[0155] Here, the present invention will be described with reference to the following examples. These examples are not intended to limit the invention to the claims, but rather should be understood to be illustrative of certain specific embodiments. Any variations in the exemplified methods that occur to those skilled in the art are intended to fall within the scope of the present invention.

Example

[0156] [Example 1: Evaluation of K326 iron pipe-dried tobacco isolines containing mutations in BBL-a, BBL-b, and BBL-c (BBL mutations (a, b, and c))] Evaluation of eight K326 homozygous BBL mutant strains and their corresponding controls in six field environments showed that adding no BBL gene mutations had a significant effect of reducing nicotine accumulation (Figure 1). Only the genotypes with mutations in both BBL-a and BBL-b showed a significant (P<0.05) decrease in nicotine compared to K326. The K326(220) and K326(222) mutant strains accumulated 0.45 and 0.38% nicotine, respectively, compared to 2.69% for K326. A progressive decrease in nicotine was observed as the number of recessive alleles at the nic1 and nic2 loci increased in the genetic background of NC95. The nicotine level in the triple homozygous mutant strain K326(222) was not lower than that of LAFC53, which is a nic1 / nic1 nic2 / nic2 isoline of the iron pipe-dried tobacco cultivar NC95.

[0157] Some slightly significant (P<0.05) decreases were observed for anatabine percentage in the single mutant strains, but not for anabasine (Figure 1). An extreme decrease in anatabine percentage was measured for K326(220) and K326(222). A more modest but still significant decrease was found for anabasine percentage for these two genotypes. Similar to nicotine, a progressive decrease in anatabine and anabasine accumulation was found with the addition of the recessive alleles at the nic1 and nic2 loci in the NC95 genetic background. A significant (P<0.05) increase in nornicotine percentage was observed for the double mutant genotypes K326(220) and K326(222) (Figure 1). No significant changes in reducing sugar percentage were found among the K326 isogenic strains or among the NC95 isogenic strains. Table 2 provides a summary of the genotypes evaluated for alkaloid accumulation and agronomic traits in this example.

[0158] [Table 2]

[0159] [Example 2: Gene transfer of recessive alleles at the Nic1 and Nic2 loci to BBL mutations (a, b, and c), and field evaluation] The present invention relates in part to determining whether combining an inducible BBL mutation with naturally occurring recessive alleles at the Nic1 and Nic2 loci can further reduce nicotine accumulation in tobacco. For this purpose, the recessive alleles at the Nic1 and Nic2 loci were introgressed into K326(222) which has a genetic background containing inducible mutations in the three most highly expressed BBL gene family members (BBL-a, BBL-b, and BBL-c). First, K326(222) was hybridized with LAFC53(nic1 / nic1 nic2 / nic2). Then, after backcrossing to K326(222), with selection for homozygous mutant conditions at the BBL-a, BBL-b, and BBL-c loci using previously described KASP markers, and selection for the recessive nic1 and nic2 alleles using SNP markers described by Adams et al. (2016), BC2F1 progeny were generated. The bbl-a / bbl-a bbl-b / bbl-b bbl-c / bbl-c Nic1 / nic1 Nic2 / nic2 BC2F1 individuals were self-pollinated, and the triple homozygous bbl mutant BC2F2 were identified by genotyping as being either Nic1 / Nic1 nic2 / nic2, nic1 / nic1 Nic2 / Nic2, or nic1 / nic1 nic2 / nic2. Such BC2F2 plants were self-pollinated to produce BC2F3 families, which were evaluated for nicotine accumulation in comparison to K326, K326(222), and LAFC53 in a single 2019 field environment near Clayton, NC. The plants were managed according to standard North Carolina flue-cured production practices. The experimental design was a completely randomized design using each genotype represented by between 12 and 37 plants. As previously described, the top two leaves of each plant were harvested 21 days after topping, air-dried, and analyzed for alkaloid profile.

[0160] [Example 3: Alkaloid analysis for BBL mutations (a, b, and c) with various recessive alleles at the Nic1 and Nic2 loci] As shown in Figure 2, the genotype of the bbl-a / bbl-a bbl-b / bbl-b bbl-c / bbl-c genetic combination generally presented lower, but not significantly lower, alkaloid levels (except for nornicotine) when combined with the recessive allele (「222 Nic1 / Nic1 nic2 / nic2」) only at the Nic2 locus in the K326 genetic background. A significant decrease (P<0.05) in all alkaloids was observed for bbl-a / bbl-a bbl-b / bbl-b bbl-c / bbl-c individuals that also had the recessive allele (「222 nic1 / nic1 Nic2 / Nic2」) only at the Nic1 locus (Figure 2). The lowest alkaloid levels were measured for bbl-a / bbl-a bbl-b / bbl-b bbl-c / bbl-c plants that were also homozygous for the recessive alleles at both the Nic1 and Nic2 loci (「222 nic1 / nic1 nic2 / nic2」) (Figure 2). The average nicotine content of plants with this genotype was very low (0.014%) (Figure 2).

[0161] [Example 4: Evaluation of iron pipe-dried tobacco containing BBL mutations (a, b, and c) with various recessive alleles at the Nic1 and Nic2 loci] The Nicotiana plants of the present technology (K326(222)nic1 / nic1 nic2 / nic2) were evaluated for yield, quality, and chemical properties of dried leaves in comparison with K326(WT) and K326(222) plants. Along with K326(WT), four K326(222)nic1 / nic1 nic2 / nic2 strains and plants derived from two K326(222) strains were grown in two rows with approximately 60 - 90 plants per strain with three replicates and managed according to standard flue - cured production practices in North Carolina. Based on publicly available flue - cured tobacco price indices, the price per hundredweight ($ / Cwt) and price per acre ($ / A) were calculated. After harvesting and curing, the alkaloid levels and grade index values of the cured leaves were measured. As previously outlined by Lewis et al., PLOS ONE 10, e0117273 (2015), oven - dried samples were ground and passed through a 1 - mm sieve and analyzed for alkaloid profiles (expressed as percentage of dry weight).

[0162] As shown in Table 3, the Nicotiana plants of the present technology (K326(222)nic1 / nic1 nic2 / nic2) with reduced nicotine levels maintained comparable or improved yields of cured leaves and USDA grade indices compared to wild - type control (K326) and Nic1 / Nic2 control (K326(222)).

[0163]

Table 3

[0164] [Example 5: Optimization of field - grown tobacco containing various recessive alleles (a, b, and c) of the BBL mutation at the Nic1 and Nic2 loci] <Tobacco seedling production> The production of tobacco seedlings against the BBLabc / nic1-2 strain is carried out in the same manner as the wild type, except for some modifications. The greenhouse needs to be clean and free of weeds, decaying plant matter, debris, algae, and any other items that can provide food or habitats for insects and other pests. Different float beds were used for seedlings of different ages and genetic characteristics. At least 1 foot of buffer material was maintained between the float beds and ensured to be frequently renovated. Filling the floating trays with a standard seedling substrate / growing mix (e.g., Seedling Pro-Mix). When germination reaches 90%, wash away the float bed water and replace it with fresh water mixed with a fertilizer (40-10-20) (N-P-K) having a nitrogen concentration of about 200 ppm. The first trimming should occur 2 or 3 weeks after the fertilizer is added to the water, and subsequent trimmings should be carried out regularly to ensure the uniformity of the seedlings. When the stems become thick and they grow at least about 3 inches above the soil line, the seedlings should be ready for transplantation.

[0165] <Cultivation and Harvest> The soil composition for seedling cultivation should be approximately 20% clay, 25% silt, and 55% sand. The drainage class should have a pH of about 6.5 and be moderately to well-drained. Using fertigation, apply half of the N as pre-planting, with the other half over three separate application periods at about 4, 6, and 8 weeks after transplantation. Add plastic mulch to cover the fertigation drip tape or tube. The timing of fertigation is intended to capture the N demand of the plants in three periods as follows. Four weeks after transplantation, the plants enter the "rapid growth" stage where they will "settle" and reach about 80% of the total biomass and nitrogen is in high demand by the plants. Two weeks later, the plants are expected to be in the "rapid growth" stage. Eight weeks after transplantation, the plants enter "flowering" where water and N are needed to assist in fully elongated leaf maturity and reach a high GRI quality.

[0166] The experiment was assembled in a randomized complete block design with three blocks containing different strains to be tested each. The ground surface was rotated, pre-plant nitrogen was applied at a predetermined rate, and then subsequently followed by spraying of herbicides and fungicides. Plastic mulch was applied between this spraying and the transplanting of seedlings to the bed. Four to five weeks after transplanting, the plasticulture was fertigated. Another application was made six to seven weeks later, and finally, the last application was eight to nine weeks after transplanting. At the flowering stage, the plants were pinched and suckering control was sprayed. The BBLabc / nic1-2 combination was not harvested at the same time or in the conventional manner due to the fact that it matures at a different rate from the wild-type control. Typically, wild-type tobacco leaves are harvested when they are fully mature and ripened. Mature leaves exhibit slight yellowing and shrinkage between the veins and are more easily snapped off at the stalk than immature leaves. In the BBLabc / nic1-2 combination strain, this process is accelerated in the bottom leaves and different from the upper leaves compared to the wild-type strain. The yellowing and texture of the leaves are different from the normal wild-type. The yield data were measurements of biomass achieved in two internal sample rows estimated in terms of acres.

[0167] [Example 6: Optimization of the galvanized pipe drying treatment process for BBL mutations (a, b, and c) with various recessive alleles at the Nic1 and Nic2 loci] The kiln was filled (packed full and evenly) with newly harvested leaves from one genetic background at a time. The leaf material placed in the kiln was of as similar quality as possible. An analog thermometer and a digital sensor were used to maintain the dry bulb temperature (DBT) and wet bulb temperature (WBT) trajectories. The leaves were monitored during the drying process. The initial parameters were as follows, with some modifications, based on Powell 1987 (the manual entitled Powell Manufacturing, Co.’s Bulk Curing / Drying Owner’s Operator’s Manual for Flue Cured Tobaccos, Powell Manufacturing, Co. (Jan. 20, 1987)). For example, the upper limit temperature was increased from 104 - 108°F at a rate of 1°F increase per hour until the maximum upper limit temperature was reached, the lower limit temperature was decreased from 92 - 96°F, and the upper limit temperature was held for a period of about 52 - 58 hours. The yellowing step in the drying process is typically 48 hours, but in this specification, this step was extended to 52 - 58 hours with continuous monitoring of temperature and leaf color. The length of time and temperature modifications were due to the higher chlorophyll (leaf green) content resulting from the introduction of the Nic1 and / or Nic2 recessive alleles. The yellowing phase ends when the majority of each leaf has turned yellow and most of the green color is limited to the leaf veins.

[0168] The next step is the “leaf drying” phase. This phase is typically about 24 hours, but in this specification, it was reduced to 22 hours. For leaf drying, the upper limit temperature (ULT) was raised to 120°F. After this step, the leaves were orange / red. The final phase of the drying process is stem drying. Stem drying was initiated by raising the ULT to 135°F, resulting in complete drying (18% moisture) of the leaves.

[0169] When cultivated and dried by the method of the present technology, the dried tobacco leaves of the present technology (including leaves, smoking leaves, tips, cutters, lugs) have the characteristics of high-quality to good-quality leaves with good color intensity, normal width, and uniform texture when compared to the standard procedures of tobacco growth (including rain-fed) and drying that produce low-quality to poor-quality leaves using this Nicotiana plant. For example, the BBLabc / nic1-2 line cultivated and dried by the method of the present technology produced good-quality orange (H4F) for smoking leaves, good-quality lemon-colored cutters (C4L), and good-quality orange (P4F) for priming leaves. In some embodiments, generally, the dried tobacco leaves of the present invention can have a USDA grade index greater than 60. Thus, the cultivation and drying method of the present technology is useful for producing commercially quality dried tobacco leaves with ultra-low nicotine levels.

[0170] [Example 7: Optimization of field-grown tobacco plants containing BBL mutations (a, b, and c) with various recessive alleles at the Nic1 and Nic2 loci] The ultra-low nicotine level of 0.04% recommended by the World Health Organization is not readily observable in the lowest alkaloid tobacco available today. To date, the only known tobacco strain that has achieved an ultra-low nicotine level below 0.04% is of the Vector 21-41 GMO type. This type was developed using antisense RNA that specifically suppresses the expression of the gene encoding quinolinate phosphoribosyltransferase, an enzyme involved in nicotine biosynthesis in tobacco roots, under the genetic background of recessive alleles at the Nic1 and Nic2 loci (also known as A and B) (Xie et al., 2004). These loci have been found to contribute to a large reduction in nicotine and related alkaloids (e.g., nornicotine, anabasine, and anatabine) from between 1.5 and 4.5% to approximately 0.20 to 0.45% (Legg et al. Can. J. Genet Cytol. 13:287-291 (1971); Lewis, Nicotine & Tobacco Res. Pages 1-5 (2018) doi:10109 / ntr / nty022).

[0171] The influential work by Chaplin and Weeks (Crop Sci. 16:416-418 (1976)), in which they introgressed the recessive alleles of Nic1 and Nic2 into 10 different tobacco genetic backgrounds, showed that the yields of 7 out of 10 lines decreased. Furthermore, 3 lines showed no decrease in nicotine compared to the recurrent parent. The USDA grade index was lower for 3 out of 7 lines due to the darker color of the dried leaves for the 7 low-alkaloid lines compared to the recurrent parent. Three of the nic1 and nic2 introgression lines exhibited grade indices equal to or better than the parent. There may be genetic background components that can help mitigate the effects of the nic1 and nic2 recessive alleles in those lines. In addition, most of the nic1 and nic2 introgression lines exhibited decreases in sugar content (7-28%) and nitrogen content (10-25%) compared to the recurrent parent. The general characteristics were that the leaves were greener in appearance compared to the parent and probably contained a higher content of chlorophyll. In addition, abnormal leaf ripening was observed (i.e., not the same as the recurrent parent plant).

[0172] These same characteristics were observed in combinations of the nic1 and nic2 recessive alleles that were introduced into tobacco plants with a more moderate expression that included the BBL mutations (a, b, and c). Strains containing the BBL mutations (a, b, and c) provide lower yields (10 - 30%) and lower grade indices (9 - 16%) depending on the growing conditions (Lewis et al., PLOS ONE 10:e0117273 (2015)); however, the chlorophyll content and sugar content are not affected. Tobacco plants containing the BBL mutations (a, b, and c) in combination with the recessive Nic1 and Nic2 alleles (either individually or together) provide lower nicotine levels, but this combination can have a negative impact on the yield and leaf grade index of the tobacco plants. The methods and modifications of the present technology for growing and curing tobacco strains containing the BBLabc mutation in combination with the recessive Nic1 and Nic2 alleles (either individually or together) aim to alleviate problems such as a decrease in nitrogen content, a decrease in sugar content, and a high chlorophyll content that can have a negative impact on the yield and quality of the leaves produced by these strains. Therefore, useful tobacco cultivation and curing protocols of the present technology with the tobacco strains of the present invention (e.g., BBLabc + nic1; BBLabc + nic1 and nic2) are as follows: (a) At the stage of approximately 90% seedling germination, flush the floating bed water and replace it with fresh water mixed with fertilizer (40 - 10 - 20) (N - P - K). The amount of nitrogen is approximately 200 ppm, which is higher than under standard growing conditions. The seedlings should be transplanted when their height reaches at least about 3 inches above the soil line. (b) Using fertigation, apply approximately half of the N, as before planting (e.g., by drip tape), over three separate applications during specific periods of plant growth at approximately 4 - 5, 6 - 7, and 8 - 9 weeks after transplanting, with the other half having the other split (see page 43, last paragraph). The fertilization step is carried out mainly through drip irrigation at a rate of approximately 90 - 120 pounds / acre of total N. (c) To help raise the temperature and improve seedling growth, use plastic mulch to cover the raised beds. (d) The leaf ripening process in the BBLabc / nic1-2 strain is not typical for tobacco. In this case, the process is accelerated in the bottom leaves and different from the upper leaves compared to the wild type strain. Apply additional nitrogen before topping (at a rate of about 90 - 120 pounds per acre). The leaf yellowing and texture are different from typical wild type tobacco leaves. The leaves should be harvested when they are green to yellow in color. (e) Regarding the drying process, it was determined that a wider temperature range and a longer yellowing step were required. Therefore, the upper limit temperature was increased to 108°F at a rate of 1°F increase per hour from the lower limit temperature, and the lower limit temperature was decreased to 92°F. The yellowing step in the drying process is usually 48 hours; however, in this specification, this step was extended to 52 - 58 hours (or more depending on yellowing) with continuous monitoring of temperature and leaf color.

[0173] [Example 8: Tobacco leaves from plants containing BBL (a, b, and c) mutations with various recessive alleles at the Nic1 and Nic2 loci, grown and dried according to the method of the present technology, are characterized by an improvement in quality compared to tobacco leaves from plants grown and dried according to standard methods.] Control tobacco plants (K326(WT)) and K326(222) tobacco plants containing BBLa, b, and c mutations (bbl-a / bbl-a, bbl-b / bbl-b, bbl-c / bbl-c) prepared according to Example 2, and K326(222) nic1 / nic1 nic2 / nic2 tobacco plants containing BBLa, b, and c mutations (bbl-a / bbl-a, bbl-b / bbl-b, bbl-c / bbl-c) that are also homozygous for the recessive alleles at both the Nic1 and Nic2 loci (nic1 / nic1, nic2 / nic2) were cultivated and dried according to either standard procedures known in the art such as Powell 1987 (Powell Manufacturing, Co.’s Bulk Curing / Drying Owner’s Operator’s Manual for Flue Cured Tobaccos, a manual entitled Powell Manufacturing, Co. (Jan. 20, 1987)) (“standard NC”), or the method of the present technology described in Examples 5 - 7 (“new H”).

[0174] Two field trials were conducted. Tobacco plants from both the “standard NC” and “new H” groups were grown in the same field. Leaves were harvested from several (>10) plants for each individual genetic background. Various tobacco quality parameters were measured by sending 100 - gram samples of the dried leaves to laboratories (Global Lab Services and Enthalpy Analytical) for chemical property evaluation.

[0175] <Results> As shown in Table 4, several parameters indicating tobacco leaf quality were measured in leaves harvested from control and K326(222) and K326(222) nic1 / nic1 tobacco plants cultivated and dried according to either the standard method or the method of the present technology.

[0176]

Table 4

[0177] Overall, leaves from K326(222) and K326(222) nic1 / nic1 nic2 / nic2 tobacco plants grown and dried according to the method of the present technology had a better grade, texture, color, and higher quality compared to leaves from plants grown and dried according to standard methods. As demonstrated by the results shown in Table 4, leaves from K326(222) and K326(222) nic1 / nic1 nic2 / nic2 tobacco plants grown and dried according to the method of the present technology (i.e., K326(222) and K326(222) nic1 / nic1 nic2 / nic2 cultivated and dried according to the "new H" method) contained more sugars compared to leaves from plants cultivated and dried according to standard methods (i.e., K326(222) and K326(222) nic1 / nic1 nic2 / nic2 tobacco plants cultivated and dried according to the "standard NC" method). Sugar levels in the type dried by iron pipe are a good indicator of bright leaves with good flavor. In addition, leaves from K326(222) and K326(222) nic1 / nic1 nic2 / nic2 strains grown and dried according to the method of the present technology were characterized by a reduced percentage of ammonia compared to leaves from K326(222) and K326(222) nic1 / nic1 nic2 / nic2 strains grown and dried according to standard methods. The ammonia percentage is typically used as an indicator of off-flavors in tobacco. Similar results were observed for total nitrogen (data not shown). Evaluations for acetaldehyde and formaldehyde showed no significant differences (data not shown).

[0178] Therefore, these results demonstrate that the cultivation and drying methods of the present technology are useful for producing dried tobacco leaves with improved quality and very low nicotine levels.

[0179] The foregoing are examples of the present invention and should not be received as limitations thereof. The present invention is defined by the following claims, and equivalents of the claims should be included therein.

Claims

Claim 1 (A) a modification that reduces the activities of BBL a, BBL b, and BBL c, or reduces the expression of the nucleic acid encoding BBL a, the nucleic acid encoding BBL b, and the nucleic acid encoding BBL c, and (B) a homozygous recessive allele of nic1, or a homozygous recessive allele of nic1 and a homozygous recessive allele of nic2 A method for drying one or more leaves of a Nicotiana plant comprising: (a) a yellowing step that begins heating the leaves starting at a temperature of 92°F to 96°F and increases the temperature to 104°F to 108°F at a rate of about 1°F per hour until a maximum upper temperature is reached, and holding at the maximum upper temperature for a period of about 52 to 58 hours; (b) a leaf drying step that includes drying at a temperature of about 120°F for about 22 hours; and (c) a stem drying step that includes drying at a temperature of about 132°F to 138°F for about 50 to about 65 hours thereby drying the one or more leaves of the Nicotiana plant and producing one or more dried leaves of the Nicotiana plant. Claim 2 The method of claim 1, wherein the Nicotiana plant comprises (A) a modification that reduces the activities of BBL a, BBL b, and BBL c, or reduces the expression of the nucleic acid encoding BBL a, the nucleic acid encoding BBL b, and the nucleic acid encoding BBL c, and (B) a homozygous recessive allele of nic1 and a homozygous recessive allele of nic2. Claim 3 The method of claim 1 or 2, wherein the Nicotiana plant has a reduced nicotine alkaloid content as compared to a plant modified by (A) only (e.g., not modified by (A) and (B)). Claim 4 Dried leaves of a Nicotiana plant comprising (A) a modification that reduces the activities of BBL a, BBL b, and BBL c, or reduces the expression of the nucleic acid encoding BBL a, the nucleic acid encoding BBL b, and the nucleic acid encoding BBL c, and (B) a homozygous recessive allele of nic1, or a homozygous recessive allele of nic1 and a homozygous recessive allele of nic2. Claim 5 (A) Modifications that reduce the activities of BBL a, BBL b, and BBL c, or reduce the expression of the nucleic acid encoding BBL a, the nucleic acid encoding BBL b, and the nucleic acid encoding BBL c, and (B) the homozygous recessive alleles of nic1 and the homozygous recessive alleles of nic2, the dried leaf according to claim 4.

6. The dried leaf according to claim 5, having a reduced nicotine alkaloid content as compared to the leaf of a wild-type control Nicotiana plant or as compared to a Nic1 / Nic2 control Nicotiana plant.

7. The dried leaf according to claim 6, wherein the nicotine alkaloid is nicotine.

8. The dried leaf according to claim 7, having a nicotine content of 0.5 mg / g or less.

9. The dried leaf according to claim 8, having a nicotine content of 0.4 mg / g or less.

10. The dried leaf according to any one of claims 4 to 9, comprising increased levels of saccharides and / or decreased levels of ammonia as compared to leaves dried according to standard drying methods.

11. The dried leaf according to any one of claims 4 to 10, wherein the Nicotiana plant further comprises a decrease in the expression of a polynucleotide encoding an additional nicotine alkaloid biosynthetic enzyme selected from the group consisting of aspartate oxidase, quinolinic acid synthetase, quinolinate phosphoribosyltransferase, ornithine decarboxylase, putrescine N-methyltransferase, methylputrescine oxidase, and A622.

12. A tobacco product comprising the dried leaf according to any one of claims 4 to 11.

13. The tobacco product according to claim 12, wherein the tobacco product is selected from the group consisting of leaf tobacco, cut tobacco, shredded tobacco, ground tobacco, powdered tobacco, tobacco extract, smokeless tobacco, moist or dry snuff, pipe tobacco, cigar tobacco, cigarillo tobacco, cigarette tobacco, and chewing tobacco.

14. The tobacco product according to claim 13, wherein the tobacco product is selected from the group consisting of cigarillos, kretek cigarettes, non-ventilated concave filter cigarettes, ventilated concave filter cigarettes, cigars, snuff, tobacco-containing gums, tobacco-containing troches, and chewing tobacco.

15. The Nicotiana plant is (1) comprising one or both of the homozygous recessive nic1 and nic2 alleles, and (2) comprising the BBL a, BBL b, and BBL c genes, including modifications that reduce the activity of BBL a, BBL b, and BBL c, or reduce the expression of the nucleic acid encoding BBL a, the nucleic acid encoding BBL b, and the nucleic acid encoding BBL c, whereby the nicotine alkaloid content of said Nicotiana plant is reduced compared to a control Nicotiana plant, Dried tobacco leaves derived from a Nicotiana plant. **Claim 16** The dried tobacco leaf according to claim 15, wherein the Nicotiana plant comprises both of the homozygous recessive nic1 and nic2 alleles. **Claim 17** The dried tobacco leaf according to claim 15 or 16, comprising a USDA grade index comparable to or better than that of dried leaves derived from a control Nicotiana plant. **Claim 18** The dried tobacco leaf according to claim 16, comprising a USDA grade index of about 60 or higher. **Claim 19** The dried tobacco leaf according to any one of claims 15 to 18, wherein the yield of the dried leaf is comparable to or increased compared to a control Nicotiana plant. **Claim 20** The dried tobacco leaf according to any one of claims 15 to 19, wherein the nicotine alkaloid is nicotine. **Claim 21** A tobacco product comprising the dried leaf according to any one of claims 15 to 20. **Claim 22** The tobacco product according to claim 21, wherein the tobacco product is selected from the group consisting of leaf tobacco, shredded tobacco, cut tobacco, ground tobacco, powdered tobacco, tobacco extract, smokeless tobacco, moist or dry snuff, pipe tobacco, cigar tobacco, cigarillo tobacco, cigarette tobacco, and chewing tobacco. **Claim 23** The tobacco product according to claim 22, wherein the tobacco product is selected from the group consisting of cigarillos, kretek cigarettes, non-ventilated concave filter cigarettes, ventilated concave filter cigarettes, cigars, snuff, tobacco-containing gums, tobacco-containing troches, and chewing tobacco. **Claim 24** (A) modifications that reduce the activity of BBL a, BBL b, and BBL c, or reduce the expression of the nucleic acid encoding BBL a, the nucleic acid encoding BBL b, and the nucleic acid encoding BBL c, and (B) Homozygous recessive alleles of nic1 or homozygous recessive alleles of nic1 and homozygous recessive alleles of nic2 A method for producing a Nicotiana plant having a reduced nicotine alkaloid content, comprising the step of combining in a Nicotiana plant, wherein said Nicotiana plant has a reduced nicotine alkaloid content compared to a plant modified only by (A) (e.g., not modified by (A) and (B)). **Claim 25** The method according to claim 24, wherein the Nicotiana plant comprises the homozygous recessive allele of nic1. **Claim 26** The method according to claim 24, wherein the Nicotiana plant comprises the homozygous recessive allele of nic1 and the homozygous recessive allele of nic2. **Claim 27** The method according to any one of claims 24 to 26, further comprising the step of reducing the expression of a polynucleotide encoding an additional nicotine alkaloid biosynthetic enzyme selected from the group consisting of aspartate oxidase, quinolinic acid synthetase, quinolinate phosphoribosyl transferase, ornithine decarboxylase, putrescine N-methyl transferase, methyl putrescine oxidase, and A622. **Claim 28** The method according to any one of claims 24 to 27, wherein the Nicotiana plant has a nicotine alkaloid content reduced by at least 40% compared to a plant modified only by (A) (e.g., not modified by (A) and (B)). **Claim 29** The method according to any one of claims 24 to 28, wherein the nicotine alkaloid is nicotine and the nicotine content is reduced by about 40% to about 90% compared to a plant modified only by (A) (e.g., not modified by (A) and (B)). **Claim 30** The method according to any one of claims 24 to 29, wherein the nicotine alkaloid is nicotine and the nicotine content is about 0.014% to about 0.098%. **Claim 31** A Nicotiana plant comprising (A) a modification that reduces the activity of BBL a, BBL b, and BBL c, or reduces the expression of the nucleic acid encoding BBL a, the nucleic acid encoding BBL b, and the nucleic acid encoding BBL c, and (B) a homozygous recessive allele of nic1, or a homozygous recessive allele of nic1 and a homozygous recessive allele of nic2.

32. A progeny plant or seed produced from the plant according to claim 31, comprising (A) a modification that reduces the activity of BBL a, BBL b, and BBL c, or reduces the expression of the nucleic acid encoding BBL a, the nucleic acid encoding BBL b, and the nucleic acid encoding BBL c, and (B) a homozygous recessive allele of nic1, or a homozygous recessive allele of nic1 and a homozygous recessive allele of nic2.

33. A tobacco product comprising tobacco derived from a Nicotiana plant, wherein the Nicotiana plant comprises (A) a modification that reduces the activity of BBL a, BBL b, and BBL c, or reduces the expression of the nucleic acid encoding BBL a, the nucleic acid encoding BBL b, and the nucleic acid encoding BBL c, and (B) a homozygous recessive allele of nic1 or a homozygous recessive allele of nic1 and a homozygous recessive allele of nic2 and has a reduced nicotine alkaloid content compared to a plant modified only by (A) (for example, not modified by (A) and (B)).

34. The tobacco product according to claim 33, wherein the Nicotiana plant is modified to reduce the activity of BBL a, BBL b, and BBL c, and / or the expression of the nucleic acid encoding BBL a, BBL b, and BBL c, and comprises the homozygous recessive allele of nic1.

35. The tobacco product according to claim 33, wherein the Nicotiana plant is modified to reduce the activity of BBL a, BBL b, and BBL c, and / or the expression of the nucleic acid encoding BBL a, BBL b, and BBL c, and comprises the homozygous recessive allele of nic1 and the homozygous recessive allele of nic2.

36. The tobacco product according to any one of claims 33 to 35, wherein the Nicotiana plant has a nicotine alkaloid content reduced by at least 40% compared to a plant modified only by (A).

37. The tobacco product according to any one of claims 33 to 35, wherein the nicotine alkaloid is nicotine, and the nicotine content is reduced by about 40% to about 90% as compared with a plant modified only by (A).

38. The tobacco product according to any one of claims 33 to 37, wherein the nicotine alkaloid is nicotine, and the nicotine content is about 0.014% to about 0.098%.

39. The tobacco product according to claim 35, wherein the nicotine alkaloid is nicotine, and the nicotine content is about 0.014%.

40. The tobacco product according to any one of claims 33 to 39, wherein the tobacco product is selected from the group consisting of leaf tobacco, shredded tobacco, cut tobacco, ground tobacco, powdered tobacco, tobacco extract, smokeless tobacco, wet or dry snuff, pipe tobacco, cigars, cigarillos, cigarettes, and chewing tobacco.

41. The tobacco product according to any one of claims 33 to 39, wherein the tobacco product is selected from the group consisting of cigarillos, kretek cigarettes, non-ventilated concave filter cigarettes, ventilated concave filter cigarettes, cigars, snuff, tobacco-containing gums, tobacco-containing lozenges, and chewing tobacco.

42. The tobacco product according to any one of claims 33 to 41, further comprising a decrease in the expression of a polynucleotide encoding an additional nicotine alkaloid biosynthetic enzyme selected from the group consisting of aspartate oxidase, quinolate synthase, quinolinate phosphoribosyl transferase, ornithine decarboxylase, putrescine N-methyl transferase, methyl putrescine oxidase, and A622.

43. A method for improving the yield of the Nicotiana plant according to claim 1, comprising: (a) fertilizing the seedlings at the stage of about 90% germination, wherein the fertilizing step is performed through fertigation, and optionally the concentration of N in the fertilizer is about 200 ppm; (b) applying a plastic mulch treatment; and (c) applying nitrogen to the seedlings at about 4 to 5 weeks after transplantation, at about 6 to 7 weeks after transplantation, and at about 8 to 9 weeks after transplantation, at a rate of about 90 to 120 pounds / acre of total N, to provide an increase in the yield and quality of the Nicotiana plant The method comprising.

44. The method according to claim 43, wherein the nitrogen is applied using fertigation. **Claim 45** The method according to claim 43 or 44, comprising coating a substrate with a ground cover sheet, thereby providing a substrate coated with the ground cover sheet. **Claim 46** The method according to claim 45, comprising multi-coating the substrate and / or the substrate coated with the ground cover sheet.

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