Methods and compositions for producing tobacco plants with reduced nicotinic alkaloid levels

Abstract

The present disclosure provides compositions and methods related to tobacco plants. In particular, the present disclosure provides novel methods for producing tobacco plants with reduced nicotinic alkaloid content, and any related tobacco products. Tobacco plants produced according to the methods of the present disclosure have reduced levels of nicotinic alkaloids (e.g., nicotine) compared to both naturally occurring and transgenic tobacco plants, and therefore represent a commercially valuable alternative to currently available tobacco varieties.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 317,272, filed March 7, 2022, which is incorporated by reference in its entirety for all purposes.

[0002] Incorporation by Reference of Electronically Submitted Materials The computer-readable nucleotide / amino acid sequence listing, submitted contemporaneously herewith and identified as follows, is incorporated herein by reference in its entirety: One 9,320 byte ASCII (text) file entitled "NCSU-40676.601", created on February 14, 2023.

[0003] The present disclosure provides compositions and methods related to tobacco plants. In particular, the present disclosure provides novel methods for producing tobacco plants with reduced nicotinic alkaloid content, and any related tobacco products. Tobacco plants produced according to the methods of the present disclosure have reduced levels of nicotinic alkaloids (e.g., nicotine) compared to both naturally occurring and transgenic tobacco plants, and therefore represent a commercially valuable alternative to currently available tobacco varieties. [Background technology]

[0004] Nicotine is usually the most abundant pyridine alkaloid produced by tobacco (Nicotiana tabacum L.), although in some plants, nornicotine may predominate due to increased activity of the gene encoding the nicotine demethylase enzyme (Sisson and Saunders, 1982;Lewis et al., 2010). Because nicotine contributes to the addictive properties of cigarettes, some public health agencies have recommended future research into mandating lowering the nicotine levels in these products to lower human exposure to toxicants associated with tobacco smoke (U.S. Food and Drug Administration, 2018). The reported nicotine levels in cigarettes are 0.4 mg g -1 The World Health Organization (WHO) has recommended that nicotine levels in tobacco be reduced to non-addictive levels below 500 mg / kg / day (WHO, 2015). This represents an approximately 95% reduction in the levels present in current cigarettes. There are no commercially available tobacco varieties that routinely exhibit such extremely low levels of nicotine accumulation in the leaves (Lewis, 2018). Several players in the tobacco industry are consequently interested in developing new varieties with nicotine levels below the proposed tolerance thresholds. Summary of the Invention

[0005] Embodiments of the present disclosure include tobacco varieties, or any portion thereof, that contain reduced levels of at least one nicotinic alkaloid as compared to a naturally occurring corresponding tobacco plant, or portion thereof.

[0006] In some embodiments, the variety is non-transgenic.

[0007] In some embodiments, the at least one nicotinic alkaloid is selected from the group consisting of nicotine, nornicotine, anatabine, and anabasine. In some embodiments, the at least one nicotinic alkaloid is nicotine, and the variety contains 0.35% or less nicotine. In some embodiments, the at least one nicotinic alkaloid is nornicotine, and the variety contains 0.04% or less nornicotine. In some embodiments, the at least one nicotinic alkaloid is anatabine, and the variety contains 0.06% or less anatabine. In some embodiments, the at least one nicotinic alkaloid is anabasine, and the variety contains 0.008% or less anabasine. In some embodiments, the at least one nicotinic alkaloid is nicotine and nornicotine, and the variety contains 0.35% or less nicotine and 0.04% or less nornicotine.

[0008] In some embodiments, the cultivar comprises a nic1 allele that has reduced expression and / or function compared to wild-type nic1. In some embodiments, the cultivar comprises a nic2 allele that has reduced expression and / or function compared to wild-type nic2. In some embodiments, the nic1 and / or nic2 alleles are derived from at least one of the following strains: LAFC53, MAFC5, LMAFC34, LA Burley 21, LI Burley 21, and HI Burley 21.

[0009] In some embodiments, the cultivar comprises a functional CYP82E2 allele. In some embodiments, the CYP82E2 allele is derived from N. sylvestris. In some embodiments, the cultivar comprises a functional CYP82E3 allele. In some embodiments, the CYP82E3 allele is derived from N. tomentosiformis (or a related species, e.g., Nicotiana Section Tomentosae). In some embodiments, the nicotine N-demethylation activity of the proteins encoded by the CYP82E2 and / or CYP82E3 alleles is increased compared to naturally occurring tobacco plants.

[0010] Embodiments of the present disclosure also include progeny plants, seeds, or cells produced from any of the tobacco varieties described herein.

[0011] Embodiments of the present disclosure also include tobacco products derived from any of the tobacco varieties, or portions thereof, described herein.

[0012] In some embodiments, 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, cigarettes, cigarillos, cigars, and chewing tobacco.

[0013] In some embodiments, the tobacco product is selected from the group consisting of cigarillos, krettic cigarettes, non-breathable recessed filter cigarettes, breathable recessed filter cigarettes, cigars, snuff, tobacco-containing gums, tobacco-containing lozenges, and chewing tobacco.

[0014] The present disclosure also includes a method for producing a tobacco variety that includes a reduced level of at least one nicotinic alkaloid compared to a naturally occurring corresponding tobacco plant, or a portion thereof. According to these embodiments, the method includes crossing a first tobacco variety that includes a first low-nicotine trait with a second tobacco variety that includes a second low-nicotine trait to produce a progeny plant. In some embodiments, the progeny plant includes a reduced concentration of at least one nicotinic alkaloid.

[0015] In some embodiments, the method comprises backcrossing.

[0016] In some embodiments, the at least one nicotinic alkaloid is selected from the group consisting of nicotine, nornicotine, anatabine, and anabasine. In some embodiments, the at least one nicotinic alkaloid is nicotine, and the variety contains 0.35% or less nicotine. In some embodiments, the at least one nicotinic alkaloid is nornicotine, and the variety contains 0.04% or less nornicotine. In some embodiments, the at least one nicotinic alkaloid is anatabine, and the variety contains 0.06% or less anatabine. In some embodiments, the at least one nicotinic alkaloid is anabasine, and the variety contains 0.008% or less anabasine. In some embodiments, the at least one nicotinic alkaloid is nicotine and nornicotine, and the variety contains 0.35% or less nicotine and 0.04% or less nornicotine.

[0017] In some embodiments, the first and / or second low nicotine trait comprises a nic1 allele that is reduced in expression and / or function compared to wild type nic1. In some embodiments, the first and / or second low nicotine trait comprises a nic2 allele that is reduced in expression and / or function compared to wild type nic2. In some embodiments, the first and / or second tobacco variety comprises a nic1 and / or nic2 allele derived from at least one of the following strains: MAFC5, LMAFC34, LA Burley 21, LI Burley 21, and HI Burley 21. In some embodiments, the first and / or second tobacco variety comprises a nic1 and / or nic2 null allele (deletion). In some embodiments, the nic1 and / or nic2 null allele is derived from naturally occurring N. tabacum germplasm.

[0018] In some embodiments, the first and / or second tobacco varieties comprise a functional CYP82E2 allele. In some embodiments, the CYP82E2 allele is derived from N. sylvestris. In some embodiments, the first and / or second tobacco varieties comprise a functional CYP82E3 allele. In some embodiments, the CYP82E3 allele is derived from N. tomentosiformis (or a related species, e.g., Nicotiana Section Tomentosae). In some embodiments, the nicotine N-demethylation activity of the proteins encoded by the CYP82E2 and / or CYP82E3 alleles is increased compared to a naturally occurring tobacco plant.

[0019] In some embodiments, the first low nicotine trait comprises nic1 and / or nic2 alleles with reduced expression and / or function compared to wild-type nic1 and / or nic2, and the second low nicotine trait comprises a functional CYP82E2 allele and / or a functional CYP82E3 allele.

[0020] In some embodiments, the first tobacco variety, the second tobacco variety, and the progeny plants are non-transgenic.

[0021] Embodiments of the present disclosure also include seeds or cells obtained from a progeny plant produced according to any of the methods described herein.

[0022] Embodiments of the present disclosure also include tobacco products derived from progeny plants produced according to any of the methods described herein.

[0023] In some embodiments, the 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, cigarettes, cigarillos, cigars, and chewing tobacco.

[0024] In some embodiments, the product is selected from the group consisting of cigarillos, krettic cigarettes, non-breathable recessed filter cigarettes, breathable recessed filter cigarettes, cigars, snuff, tobacco-containing gums, tobacco-containing lozenges, and chewing tobacco.

[0025] In some embodiments, the cultivar comprises a CYP82E2 allele that comprises a nucleotide sequence encoding a K375E and / or a L422W point mutation compared to the wild-type CYP82E2 amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, (a) the CYP82E2 allele comprises a nucleotide sequence encoding a CYP82E2 protein comprising an amino acid sequence set forth in SEQ ID NO: 2, (b) the CYP82E2 allele comprises a nucleotide sequence encoding a CYP82E2 protein comprising an amino acid sequence set forth in SEQ ID NO: 3, or (c) the CYP82E2 allele comprises a nucleotide sequence encoding a CYP82E2 protein comprising an amino acid sequence set forth in SEQ ID NO: 4.

[0026] In some embodiments, a CYP82E3 allele comprises a nucleotide sequence that encodes a C330W point mutation compared to the wild-type CYP82E3 amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, a CYP82E3 allele comprises a nucleotide sequence that encodes a CYP82E3 protein comprising the amino acid sequence set forth in SEQ ID NO: 6.

[0027] In some embodiments, the cultivar comprises a CYP82E2 allele that comprises a nucleotide sequence encoding a K375E and / or a L422W point mutation compared to the wild-type CYP82E2 amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, (a) the CYP82E2 allele comprises a nucleotide sequence encoding a CYP82E2 protein comprising an amino acid sequence set forth in SEQ ID NO: 2, (b) the CYP82E2 allele comprises a nucleotide sequence encoding a CYP82E2 protein comprising an amino acid sequence set forth in SEQ ID NO: 3, or (c) the CYP82E2 allele comprises a nucleotide sequence encoding a CYP82E2 protein comprising an amino acid sequence set forth in SEQ ID NO: 4.

[0028] In some embodiments, a CYP82E3 allele comprises a nucleotide sequence that encodes a C330W point mutation compared to the wild-type CYP82E3 amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, a CYP82E3 allele comprises a nucleotide sequence that encodes a CYP82E3 protein comprising the amino acid sequence set forth in SEQ ID NO: 6.

[0029] In some embodiments, the cultivar further comprises suppression of expression in the cultivar of at least one of NBBl, A622, quinolate phosphoribosyltransferase (QPT), putrescine N-methyltransferase (PMT), ornithine decarboxylase (ODC), aspartate oxidase (AO), quinolinate synthase (QS), N-methylputrescine oxidase (MPO), NtERF221, NtMYC1a, NtMYC1b, NtMYC2a, or NtMYC2b.

[0030] Embodiments of the present disclosure also include methods of making a point mutation in a target gene in a Nicotiana tabacum plant cell. According to these embodiments, the method includes introducing into the cell a clustered regularly interspaced short palindromic repeats (CRISPR) associated (CRISPR-Cas) system comprising: (a) at least one guide RNA (gRNA), the at least one gRNA comprising a nucleotide sequence capable of hybridizing with a target sequence or a portion thereof in a target gene, the target gene comprising a nucleotide sequence encoding a CYP82E2 protein; (b) (i) a DNA fragment, the DNA fragment replacing a target sequence in the target gene or being inserted into a target site in the target gene, and (ii) two homologous arms, each arm flanking opposite sides of the DNA fragment; and (c) a nucleic acid comprising an effector protein or a donor DNA sequence comprising one or more polynucleotides encoding the effector protein. In some embodiments, at least one gRNA forms a complex with an effector protein, in some embodiments, the effector protein is a Cas protein that comprises a nuclease.

[0031] In some embodiments, (a) the point mutation is a K375E point mutation compared to the wild-type CYP82E2 amino acid sequence set forth in SEQ ID NO:1, or (b) the point mutation is a L422WE point mutation compared to the wild-type CYP82E2 amino acid sequence set forth in SEQ ID NO:1.

[0032] In some embodiments, at least two point mutations are made, hi some embodiments, the at least two point mutations include a K375E point mutation compared to the wild-type CYP82E2 amino acid sequence set forth in SEQ ID NO:1, and a L422W point mutation compared to the wild-type CYP82E2 amino acid sequence set forth in SEQ ID NO:1.

[0033] In some embodiments, the Nicotiana tabacum plant further comprises a recessive allele of nic1 and / or a recessive allele of nic2.

[0034] In some embodiments, the method further comprises suppressing expression in at least one variety of NBBl, A622, quinolate phosphoribosyltransferase (QPT), putrescine N-methyltransferase (PMT), ornithine decarboxylase (ODC), aspartate oxidase (AO), quinolinate synthase (QS), N-methylputrescine oxidase (MPO), NtERF221, NtMYC1a, NtMYC1b, NtMYC2a, or NtMYC2b.

[0035] Embodiments of the present disclosure also include methods of making a point mutation in a target gene in a Nicotiana tabacum plant cell. According to these embodiments, the method includes introducing into the cell a clustered regularly interspaced short palindromic repeats (CRISPR) associated (CRISPR-Cas) system comprising: (a) at least one guide RNA (gRNA), the at least one gRNA comprising a nucleotide sequence capable of hybridizing with a target sequence or a portion thereof in a target gene, the target gene comprising a nucleotide sequence encoding a CYP82E3 protein; (b) (i) a DNA fragment, the DNA fragment replacing a target sequence in the target gene or being inserted into a target site in the target gene, and (ii) two homologous arms, each arm flanking opposite sides of the DNA fragment; and (c) a nucleic acid comprising an effector protein or a donor DNA sequence comprising one or more polynucleotides encoding the effector protein. In some embodiments, at least one gRNA forms a complex with an effector protein, in some embodiments, the effector protein is a Cas protein that comprises a nuclease.

[0036] In some embodiments, the point mutation is a C330W point mutation compared to the wild-type CYP82E3 amino acid sequence set forth in SEQ ID NO:5.

[0037] In some embodiments, the Nicotiana tabacum plant further comprises a recessive allele of nic1 and / or a recessive allele of nic2.

[0038] In some embodiments, the method further comprises suppressing expression in the Nicotiana tabacum plant of at least one of NBBl, A622, quinolate phosphoribosyltransferase (QPT), putrescine N-methyltransferase (PMT), ornithine decarboxylase (ODC), aspartate oxidase (AO), quinolinate synthase (QS), N-methylputrescine oxidase (MPO), NtERF221, NtMYC1a, NtMYC1b, NtMYC2a, or NtMYC2b.

[0039] Embodiments of the present disclosure also include methods for producing Nicotiana tabacum plants with reduced nicotinic alkaloid content. According to these embodiments, the methods include combining in a Nicotiana tabacum plant (a) one or more genetic modifications that increase the activity of CYP82E2 and / or CYP82E3, and (b) a recessive allele of nic1 and / or a recessive allele of nic2. In some embodiments, the Nicotiana tabacum plant has reduced nicotinic alkaloid content compared to a corresponding naturally occurring or non-transformed control tobacco plant.

[0040] In some embodiments, the Nicotiana tabacum plant comprises a homozygous recessive allele of nic1 and / or a homozygous recessive allele of nic2.

[0041] In some embodiments, one or more genetic modifications that increase the activity of CYP82E2 and / or CYP82E3 are introduced by transcription activator-like effector nucleases (TALENs), meganucleases, zinc finger nucleases, CRISPR / Cas9 systems, CRISPR / Cpf1 systems, CRISPR / Csm1 systems, gene knock-in technology or scientific techniques, and any combination thereof.

[0042] In some embodiments, the one or more genetic modifications that increase the activity of CYP82E2 comprise a point mutation in the target gene of the Nicotiana tabacum plant cell, the point mutation being selected from one or more of a K375E point mutation compared to the wild-type CYP82E2 amino acid sequence set forth in SEQ ID NO:1, and a L422W point mutation compared to the wild-type CYP82E2 amino acid sequence set forth in SEQ ID NO:1.

[0043] In some embodiments, the one or more genetic modifications that increase the activity of CYP82E3 include a point mutation in the target gene of the Nicotiana tabacum plant cell, wherein the point mutation is a C330W point mutation compared to the wild-type CYP82E3 amino acid sequence set forth in SEQ ID NO:5.

[0044] In some embodiments, the point mutation is introduced into the plant by a clustered regularly interspaced short palindromic repeats (CRISPR) associated (CRISPR-Cas) system comprising: (a) at least one guide RNA (gRNA), the at least one gRNA comprising a nucleotide sequence capable of hybridizing to a target sequence or a portion thereof in a target gene, the target gene comprising a nucleotide sequence encoding a CYP82E2 protein; (b) (i) a DNA fragment, the DNA fragment replacing a target sequence in the target gene or being inserted into a target site in the target gene, and (ii) two homologous arms, each flanking opposite sides of the DNA fragment; and (c) a nucleic acid comprising an effector protein or a donor DNA sequence comprising one or more polynucleotides encoding the effector protein. In some embodiments, the at least one gRNA forms a complex with the effector protein. In some embodiments, the effector protein is a Cas protein comprising a nuclease.

[0045] Embodiments of the disclosure also include Nicotiana tabacum plants produced according to any of the methods described herein, the plants comprising (a) one or more genetic modifications that increase CYP82E2 and / or CYP82E3 activity, and (b) a recessive allele at nic1 and / or a recessive allele at nic2. Embodiments of the disclosure also include progeny plants or seeds produced from the plants, the progeny plants or seeds comprising (A) one or more genetic modifications that increase CYP82E2 and / or CYP82E3 activity, and (B) a recessive allele at nic1 and / or a recessive allele at nic2. Embodiments of the disclosure also include tobacco products comprising tobacco from a Nicotiana tabacum plant, the plants comprising (A) one or more genetic modifications that increase CYP82E2 and / or CYP82E3 activity, and (B) a recessive allele at nic1 and / or a recessive allele at nic2.

[0046] According to these embodiments, (a) 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, cigarettes, cigarillos, cigarillos, leaf tobacco, and chewing tobacco, or (b) the product is a reduced-nicotine tobacco product selected from the group consisting of cigarillos, krettic cigarettes, non-ventilated recessed filter cigarettes, ventilated recessed filter cigarettes, cigars, snuff, snus, tobacco-containing gum, tobacco-containing lozenges, and chewing tobacco. [Brief description of the drawings]

[0047] [Figure 1A] FIG. 1 is an exemplary graphical data of alkaloid composition for tested low alkaloid hybrid strains (Nic1 and Nic2 loci from LAFC53 in combination with CYP82E3 alleles from N. tomentosiformis) and related controls, including nicotine percentage, according to one embodiment of the present disclosure. [Figure 1B] 1 is an exemplary graphical data of alkaloid composition for tested low alkaloid hybrid strains (Nic1 and Nic2 loci from LAFC53 in combination with CYP82E3 alleles from N. tomentosiformis) and related controls, including nornicotine percentage, according to one embodiment of the present disclosure. [Figure 1C] FIG. 1 is an exemplary graphical data of alkaloid composition for tested low alkaloid hybrid strains (Nic1 and Nic2 loci from LAFC53 in combination with CYP82E3 alleles from N. tomentosiformis) and related controls according to one embodiment of the present disclosure, including anatabine percentage. [Figure 1D]FIG. 1 is an exemplary graphical data of alkaloid composition for tested low alkaloid hybrid strains (Nic1 and Nic2 loci from LAFC53 in combination with CYP82E3 alleles from N. tomentosiformis) and related controls according to one embodiment of the present disclosure. Includes % anabasine. Mean values ​​are averaged for three field environments. [Figure 2A] 1 is an exemplary graphical data of yield and quality measurements for a tested low alkaloid hybrid strain (Nic1 and Nic2 loci from LAFC53 in combination with a CYP82E3 allele from N. tomentosiformis) and related controls according to one embodiment of the present disclosure, including yield (lbs / A). [Figure 2B] 1 is an exemplary graphical data of yield and quality measurements for tested low alkaloid hybrid strains (Nic1 and Nic2 loci from LAFC53 in combination with CYP82E3 alleles from N. tomentosiformis) and related controls according to one embodiment of the present disclosure, including hundredweight (Cwt) values ​​($). [Figure 2C] FIG. 1 is an exemplary graphical data of yield and quality measurements for a tested low alkaloid hybrid line (Nic1 and Nic2 loci from LAFC53 in combination with CYP82E3 allele from N. tomentosiformis) and related controls according to one embodiment of the present disclosure. Acre values ​​($) included. [Figure 2D] 1 is an exemplary graphical data of yield and quality measurements for a tested low alkaloid hybrid strain (Nic1 and Nic2 loci from LAFC53 in combination with CYP82E3 allele from N. tomentosiformis) and related controls according to one embodiment of the present disclosure, including a grade index. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] Embodiments of the present disclosure relate to novel methods for producing tobacco plants with reduced nicotinic alkaloid content, and any related tobacco products. Tobacco plants produced according to the methods of the present disclosure have reduced levels of nicotinic alkaloids (e.g., nicotine) compared to both naturally occurring and transgenic tobacco plants, and therefore represent a commercially valuable alternative to currently available tobacco varieties.

[0049] The section headings used in this section and throughout this disclosure are for organizational purposes only and are not intended to be limiting.

[0050] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of conflict, the present specification, including definitions, will control. Although preferred methods and materials are described below, methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0051] The term "about" is understood by those skilled in the art and will vary to some extent depending on the context in which the word is used. If there is a use of an item that is not clear to those skilled in the art, "about" will mean up to plus or minus 10% of the particular item, taking into account the context in which the item is used. For example, in some embodiments, "about" means plus or minus 5% of the particular term. Certain ranges are indicated herein by numbers preceded by the term "about". The term "about" is used herein to provide a literal indication of the exact number that it precedes, as well as a number that is close to or approximately the number that it precedes. In determining whether a number is close to or approximately a specifically recited number, the close or approximate unrecited number may be a number that provides a substantial equivalent to the specifically recited number in the context in which the number is indicated.

[0052] As used herein, the terms "comprise," "include," "having," "has," "can," "containing," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether or not expressly stated.

[0053] In the description of numerical ranges herein, each intervening number is expressly contemplated with the same precision. For example, for the range 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0054] As used herein, "correlates to" refers to compared to.

[0055] As used herein, the term "nucleic acid molecule" refers to any nucleic acid-containing molecule, including, but not limited to, DNA or RNA. This term includes, but is not limited to, 4 acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudoracil, 1-methylguanine, 1-methylirinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguadenine, 3-methylcytosine, 5-methylisocytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxy ... The bases encompass sequences containing any of the known DNA and RNA base analogs, including alpha-2-thiouracil, beta-D mannoylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, oxybutoxosine, pseudosil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, pseudosil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

[0056] The term "gene" refers to a nucleic acid (e.g., DNA) sequence that comprises a coding sequence for the production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA, sRNA, microRNA, lincRNA). A polypeptide may be encoded by a full-length coding sequence or by any portion of a coding sequence, so long as the desired activity or functional properties of the full length or fragment (e.g., enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) are retained. The term also encompasses the coding region of a structural gene and sequences located adjacent to the coding region at both the 5' and 3' ends for a distance of about 1 kb or more on either end, such that the gene corresponds to the length of the full-length mRNA. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed either "introns," "intervening regions," or "intervening sequences." Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA) and may contain regulatory elements such as enhancers. Introns are removed or "spliced ​​out" from the nuclear or primary transcript and therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0057] As used herein, the term "heterologous gene" refers to a gene that is not present in its natural environment. For example, heterologous genes include genes from one species that are introduced into another species. Heterologous genes also include genes that are native to an organism that have been altered in some way (e.g., mutated, added to multiple copies, linked to non-native regulatory sequences, etc.). Heterologous genes are distinct from native genes in that they are not normally found naturally associated with gene sequences in chromosomes or are linked to DNA sequences that are associated with parts of chromosomes not found in nature (e.g., genes that are expressed at loci where the gene is not normally expressed).

[0058] As used herein, "operably linked" refers to a functional link between two elements. For example, an operably linked link between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional link that allows expression of the polynucleotide of interest. The operably linked elements may be contiguous or non-contiguous.

[0059] As used herein, "gene expression" refers to the biosynthesis or production of a gene product, including transcription and / or translation of the gene product.

[0060] As used herein, the term "oligonucleotide" refers to a single-stranded polynucleotide chain that is short in length. Oligonucleotides are usually less than about 300 residues long (e.g., 15-100), but as used herein, the term is also intended to encompass longer polynucleotide chains. Oligonucleotides are often referred to by their length. For example, an oligonucleotide of 24 residues is called a "24mer." Oligonucleotides can form secondary and tertiary structures by self-hybridization or by hybridization to other polynucleotides. Such structures can include, but are not limited to, duplexes, hairpins, cruciforms, bends, and triplexes.

[0061] The terms "homology" and "homologous" refer to the degree of identity. There may be partial or complete homology. A partially homologous sequence is one that is less than 100% identical to another sequence.

[0062] As used herein, the term "complementary" or "complementarity" is used to refer to a polynucleotide (e.g., a sequence of nucleotides, such as an oligonucleotide or a target nucleic acid) that is related by the rules of base pairing. For example, the sequence "5'-AGT-3'" is complementary to the sequence "3'-TCA-5'". Complementarity can be "partial", where only a portion of the nucleic acid bases match according to the rules of base pairing. Alternatively, there can be "complete" or "total" complementarity between nucleic acids. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands. Complementarity is particularly important in amplification reactions and detection methods that depend on binding between nucleic acids. Either term can also be used to refer to individual nucleotides, especially in the context of polynucleotides. For example, a particular nucleotide in an oligonucleotide can be described as being complementary or lacking complementarity to a nucleotide in another nucleic acid strand, as compared to the complementarity between the oligonucleotide and the remainder of the nucleic acid strand.

[0063] In some contexts, the term "complementary" and related terms (e.g., "complementary," "complement") refer to nucleotides of a nucleic acid sequence that can bind to another nucleic acid sequence by hydrogen bonds, e.g., nucleotides capable of base pairing by Watson-Crick base pairing or other base pairing. Nucleotides capable of base pairing, e.g., complementary to each other, are cytosine and guanine, thymine and adenine, adenine and uracil, and guanine and uracil pairs. The percentage of complementarity need not be calculated over the entire length of the nucleic acid sequence. The percentage of complementarity can be limited to a particular region of the base-paired nucleic acid sequence, e.g., beginning at the first base-paired nucleotide and ending at the last base-paired nucleotide. As used herein, the complement of a nucleic acid sequence refers to an oligonucleotide that "antiparallel associates" with the nucleic acid sequence when aligned such that the 5' end of one is paired with the 3' end of the other. Certain bases that are not commonly found in natural nucleic acids may be included in the nucleic acids of the present invention, such as inosine and 7-deazaguanine. Complementarity does not need to be perfect. Stable duplexes may contain mismatched base pairs or unmatched bases. Those skilled in the art of nucleic acid technology can experimentally measure duplex stability, taking into account a number of variables, including, for example, the length of the oligonucleotide, the base composition and sequence of the oligonucleotide, ionic strength, and the occurrence rate of mismatched base pairs.

[0064] Thus, in some embodiments, "complementary" refers to a first nucleobase sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of a second nucleobase sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleobases, or the two sequences hybridize under stringent hybridization conditions. "Fully complementary" refers to each nucleobase of the first nucleic acid being capable of pairing with each nucleobase at the corresponding position of the second nucleic acid. For example, in certain embodiments, an oligonucleotide in which each nucleobase has complementarity to a nucleic acid has a nucleobase sequence that is identical to the complement of the nucleic acid over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleobases.

[0065] As used herein, a "double-stranded nucleic acid" can be a portion of a nucleic acid, a region of a longer nucleic acid, or the entire nucleic acid. A "double-stranded nucleic acid" can be, for example, but not limited to, double-stranded DNA, double-stranded RNA, double-stranded DNA / RNA hybrids, etc. Single-stranded nucleic acids that have secondary structures (e.g., base-paired secondary structures) and / or higher order structures include "double-stranded nucleic acids." For example, triplex structures are considered "double stranded." In some embodiments, any base-paired nucleic acid is a "double-stranded nucleic acid."

[0066] The term "isolated", when used in reference to a nucleic acid, as in "isolated oligonucleotide" or "isolated polynucleotide", refers to a nucleic acid sequence that is identified and separated from at least one component or contaminant that is normally associated with its natural source. An isolated nucleic acid is so present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids, such as nucleic acids such as DNA and RNA, are found in the state in which they naturally occur. For example, a given DNA sequence (e.g., a gene) is found on a host cell chromosome adjacent to adjacent genes, and an RNA sequence, such as a particular mRNA sequence that encodes a particular protein, is found in a cell as a mixture with many other mRNAs that encode many proteins. However, an isolated nucleic acid that encodes a given protein includes, by way of example, a nucleic acid in a cell that normally expresses the given protein, where the nucleic acid is in a different chromosomal location than that of the natural cell, or is otherwise adjacent to a different nucleic acid sequence than that found in nature. An isolated nucleic acid, oligonucleotide, or polynucleotide can be present in single-stranded or double-stranded form. When the isolated nucleic acid, oligonucleotide, or polynucleotide is used to express a protein, the oligonucleotide or polynucleotide will, at a minimum, contain a sense or coding strand (i.e., the oligonucleotide or polynucleotide may be single-stranded), but may contain both the sense and antisense strands (i.e., the oligonucleotide or polynucleotide may be double-stranded).

[0067] As used herein, a "locus" is a chromosomal region where a polymorphic nucleic acid, trait determinant, gene, or marker is located. The locus of the present disclosure includes one or more polymorphisms in a population. For example, alternative alleles exist in some individuals. As used herein, "allele" refers to alternative nucleic acid sequences at a particular locus. The length of an allele can be as small as one nucleotide base, but is usually larger. For example, a first allele can occur on one chromosome, while a second allele occurs on a second homologous chromosome, for example, on a different chromosome in a heterozygous individual, or between different homozygous or heterozygous individuals in a population. As used herein, the term "chromosomal interval" refers to a continuous linear span of genomic DNA present on a single chromosome.

[0068] As used herein, "introgression" or "introject" refers to the transfer of a desired allele at a locus from one genetic background to another.

[0069] As used herein, "mated" or "mating" means to produce offspring by fertilization (e.g., a cell, seed, or plant), and includes the mating of a plant (sexual) with a self-fertilizing (self).

[0070] As used herein, "backcross" and "backcrossing" refer to the process in which a progeny plant is repeatedly backcrossed to one of its parents. In a backcrossing scheme, the "donor" parent refers to the parent plant that has the desired gene or locus to be introduced. The "recipient" parent (used one or more times), or the "recurrent" parent (used two or more times), refers to the parent plant to which the gene or locus is transferred. This initial cross produces the F1 generation. The term "BC1" refers to the second use of the recurrent parent, "BC2" refers to the third use of the recurrent parent, and so on. In some embodiments, backcrossing is performed repeatedly, with the progeny individuals of each successive backcross generation being themselves backcrossed to the same parental genotype.

[0071] As used herein, "single-gene converted" or "single-gene conversion" means a plant developed using the plant breeding technique known as backcrossing or by genetic modification, where essentially all of the desired morphological and physiological characteristics of the variety are restored in addition to a single gene transferred to the variety by the backcrossing technique or by genetic modification.

[0072] As used herein, the term "variety" refers to a group of plants that share certain characteristics that distinguish them from other plants of the same variety. Varieties are often, but not always, commercially available. While having one or more different traits, varieties are also characterized by very little overall variation between individuals within the variety. "Pure strain" varieties can be produced by several generations of self-pollination and selection, or by vegetative propagation from a single parent using tissue or cell culture techniques. Varieties can be essentially derived from another strain or variety. As defined by the International Convention for the Protection of New Varieties of Plants (December 2, 1961; revised at Geneva on November 10, 1972, October 23, 1978, and March 19, 1991), a variety is "essentially derived" from an original variety if: a) it is derived primarily from the original variety, or from a variety derived primarily from the original variety, while retaining the expression of essential characteristics resulting from a single genotype or combination of genotypes of the original variety; b) it is clearly distinguishable from the original variety; and c) it corresponds to the original variation in the expression of essential characteristics resulting from a single genotype or combination of genotypes of the original variety, except for differences caused by induction effects. Essentially derived varieties can be obtained, for example, by selection of variant individuals from natural or induced mutations, somaclonal variants, original varieties, backcrossing, or transgenic plants. A first tobacco variety and a second tobacco variety from which the first variety is essentially derived are considered to have essentially the same genetic background. A "strain," as distinguished from a variety, most often refers to a group of plants used non-commercially, for example, in botanical research. A strain typically exhibits little overall variation among individuals for one or more traits of interest, although some variation may exist among individuals for other traits.

[0073] As used herein, "superior variety" means any variety that has resulted from breeding and selection for superior agronomic performance.

[0074] As used herein, "selecting" or "selection" in the context of marker-assisted selection or breeding refers to the act of picking out or selecting desired individuals, usually from a population, based on some particular predetermined criteria.

[0075] As used herein, the term "trait" refers to one or more detectable characteristics of a cell or organism that can be influenced by a genotype. A phenotype can be observable with the naked eye or by any other means of assessment known in the art, such as microscopy, biochemical analysis, genomic analysis, assays for specific disease resistance, etc. In some cases, a phenotype is directly controlled by a single gene or locus, e.g., a "single gene trait." In other cases, a phenotype is the result of several genes.

[0076] As used herein, the term "genetic mutation" or "genetic change" refers to a heritable genetic modification that is introduced into a gene and alters the expression or activity of the product encoded by the gene. Such modifications can be present in any sequence region of the gene, for example, in the promoter, 5'UTR, exons, introns, 3'UTR, or terminator regions. In one aspect, the mutation reduces, inhibits, or eliminates the expression or activity of the gene product. In another aspect, the mutation increases, elevates, enhances, or enhances the expression or activity of the gene product. In one aspect, the mutation is not a natural polymorphism present in a particular tobacco variety or cultivar. As used herein, "mutation allele" refers to an allele that originates from a locus where the allele contains a mutation. As used herein, "mutagenesis" refers to the generation of a mutation without a transgene or where the mutation-associated transgene does not remain in the final mutant. In one aspect, the mutagenesis is cisgenic. In another aspect, the mutagenesis is by gene or genome editing. In a further embodiment, mutagenesis is by random mutagenesis, for example, by chemical (eg, EMS) or physical (r-irradiation) mutagenesis.

[0077] As used herein, "polymorphism" means the presence of one or more variations in a population. Polymorphism may be manifested as a variation in the nucleotide sequence of a nucleic acid or as a variation in the amino acid sequence of a protein. Polymorphism includes the presence of variation in one or more nucleic acid sequences or nucleic acid features at one or more loci in a population of one or more individuals. Variations may include, but are not limited to, the change of one or more nucleotide bases, the insertion of one or more nucleotides, or the deletion of one or more nucleotides. Polymorphisms may arise from random processes in nucleic acid replication, such as unequal crossing over, genome duplication, and chromosome breakage and fusion, by mutation introduction, as a result of mobile genomic elements, from copy number variation, and during the process of meiosis. Variations can be commonly found or can exist at low frequencies in a population, the former of great utility in general plant breeding, and the latter of rare but important phenotypic variation. Useful polymorphisms can include single nucleotide polymorphisms (SNPs), insertions or deletions (indels) in DNA sequences, simple sequence repeats (SSRs) in DNA sequences, restriction fragment length polymorphisms (RELPs), and tag SNPs. Genetic markers, genes, sequences derived from DNA, sequences derived from RNA, promoters, 5' untranslated regions of genes, 3' untranslated regions of genes, microRNAs, siRNAs, tolerance loci, satellite markers, transgenes, mRNAs, ds mRNAs, transcriptional profiles, and methylation patterns can also comprise polymorphisms. In addition, the presence, absence, or copy number variation of the above can comprise polymorphisms.

[0078] As used herein, the term "plant" includes whole plants, transplants, plant ancestor(s) and progeny, as well as plant parts including seeds, shoots, stems, roots (including tubers), rhizomes, scions, and plant cells, tissues, and organs. Plants can be in any form, including suspension cultures, embryos, meristematic regions, callus cells, leaves, gametophytes, sporophytes, pollen, and microspores. Plants that are particularly useful in the methods of the present disclosure include all plants belonging to the Nicotiana family.

[0079] As used herein, the term "tobacco" refers to any plant of the genus Nicotiana that produces nicotinic alkaloids. Tobacco also refers to products that contain substances produced by Nicotiana plants, and thus includes, for example, expanded tobacco, reconstituted tobacco, cigarettes, cigars, chewing tobacco, or smokeless tobacco, snuff, and snus forms made from GE-enhanced nicotine tobacco. Examples of Nicotiana species include, but are not limited to: Nicotiana acaulis, Nicotiana acuminata, Nicotiana acuminata var.multiflora, Nicotiana africana, Nicotiana alata, Nicotiana amplexicaulis, Nicotiana arentsii, Nicotiana attenuata, Nicotiana benavidesii, Nicotiana benthamiana, Nicotiana bigelovii, Nicotiana bonariensis, Nicotiana cavicola, Nicotiana clevelandii, Nicotiana cordifolia, Nicotiana corymbosa, Nicotiana debneyi, Nicotiana excelsior, Nicotiana forgetiana, Nicotiana fragrans, Nicotiana glauca, Nicotiana glutinosa, Nicotiana goodspeedii, Nicotiana gossei, Nicotiana hybrid, Nicotiana ingulba, Nicotiana kawakamii, Nicotiana knightiana, Nicotiana langsdorffii, Nicotiana linearis, Nicotiana longiflora, Nicotiana maritima, Nicotiana megalosiphon, Nicotiana miersii, Nicotiana noctiflora, Nicotiana nudicaulis, Nicotiana obtusifolia, Nicotiana occidentalis, Nicotiana occidentalis subsp. hesperis、Nicotiana otophora、Nicotiana paniculata、Nicotiana pauciflora、Nicotiana petunioides、Nicotiana plumbaginifolia、Nicotiana quadrivalvis、Nicotiana raimondii、Nicotiana repanda、Nicotiana rosulata、Nicotiana rosulata subsp.ingulba, Nicotiana rotundifolia, Nicotiana rustica, Nicotiana setchellii, Nicotiana simulans, Nicotiana solanifolia, Nicotiana spegazzinii, Nicotiana stocktonii, Nicotiana suaveolens, Nicotiana sylvestris, N. tabacum, Nicotiana thyrsiflora, Nicotiana tomentosa, Nicotiana tomentosiformis, Nicotiana trigonophylla, Nicotiana umbratica, Nicotiana undulata, Nicotiana velutina, Nicotiana wigandioides, and Nicotiana x sanderae. .

[0080] As used herein, the term "transgenic plant" refers to a plant that contains a nucleic acid sequence that is itself also present in another organism or species, or that is optimized from another organism or species, as compared to the host codon usage. Both monocotyledonous and dicotyledonous angiosperms, or gymnosperms, plant cells can be transformed by a variety of methods known to those skilled in the art. See, for example, Klein et al., Biotechnology 4: 583-590 (1993); Bechtold et al., CRAcad. Sci. Paris 316: 1194-1199 (1993); Bent et al., Mol. Gen. Genet. 204: 383-396 (1986); Paszowski et al., EMBO J. 3: 2717-2722 (1984); Sagi et al., Plant Cell Rep. 13: 262-266 (1994).

[0081] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. For example, any technical terms used in connection with cell and tissue culture, molecular biology, plant biology, genetics, and protein and nucleic acid chemistry and hybridization described herein, and these techniques are well known and commonly used in the art. The meaning and scope of the terms must be clear, but in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms include the plural and plural terms include the singular.

[0082] 2. Low-nicotine tobacco plants Tobacco alkaloid accumulation is thought to be under complex genetic control. Researchers have historically explored using naturally occurring allelic variability at the Nic1 and Nic2 loci (also referred to as the A and B loci in some literature) to achieve lower nicotine levels in tobacco. Such variability has been used to develop low-alkaloid breeding lines, such as LA Burley 21 (Legg et al., 1970) and LAFC53 (Chaplin, 1975). However, these genotypes routinely produce nicotine levels below 0.4 mg g -1 (Lewis, 2018).

[0083] The Nic2 locus has been found to contain a set of genes in N. tabacum linkage 19 that encode ethylene response factor (ERF) transcription factors that globally affect the expression of structural genes in the tobacco alkaloid biosynthetic pathway. This gene cluster was found to be deleted in "LA Burley 21" (a backcross-derived nic1 / nic1 nic2 / nic2 version of Burley 21). A similar gene array is located at or near the Nic1 locus in N. tabacum linkage 7 (Sui et al., 2020; Qin et al., 2021), where an epigenetically licensed allele of ERF199 may explain the effect of alkaloid accumulation at this locus in LA Burley 21 (Qin et al., 2021).

[0084] Naturally occurring variability in loci encoding nicotine demethylase (NDM) enzymes can also significantly affect nicotine levels by increasing / decreasing nornicotine formation from demethylation. Biochemical conversion of nicotine to nornicotine has historically been considered an unattractive method to reduce nicotine levels in tobacco plants due to the increased tendency of nornicotine to form its corresponding carcinogenic tobacco-specific nitrosamine (TSMA), N'-nitrosonornicotine (NNN), during curing and storage of dry tobacco. This high accumulation of alkaloids also undesirably alters the physical and organoleptic properties of the dry leaves. The degree of nicotine demethylation in N. tabacum is considered a genetically unstable trait, but the progenitor species of N. tabacum, N. sylvestris, and tomentosiformis exhibit a stable and high degree of nicotine demethylation.

[0085] As further described herein, embodiments of the present disclosure include plant breeding methodologies for developing novel tobacco genotypes in which genetic variability at the Nic1 and Nic2 loci from LAFC53 is combined with genetic variability from either N. sylvestris or N. tomentosiformis that encodes stable, high levels of NDM activity. Using these genetic combinations, the variability at the Nic1 and Nic2 loci acts to reduce expression of structural genes involved in nicotine biosynthesis, but does not reduce nicotine to zero, and even typically reduces nicotine to less than 0.4 mg g -1 The majority of the remaining nicotine is removed by the activity of genes from N. sylvestris or N. tomentosiformis that code for the NDM enzyme. The result of this strategy is a genotype that produces the lowest publicly reported and commercially available nicotine for field-grown tobacco, with corresponding reductions in anabasine, anatabine, and nornicotine (compared to standard flue-cured tobacco varieties). Importantly, nornicotine levels are not increased compared to standard tobacco varieties.

[0086] The source of genetic variability at the Nic1 and Nic2 loci, which affect low nicotine content, was derived from the bark-cured tobacco breeding line LAFC53. The source of genetic variability encoding stable and high levels of nicotine demethylase activity was ultimately derived from either N. sylvestris or N. tomentosiformis. The breeding line SC58 CsCs was previously developed by transferring the major NDM genes from N. sylvestris into the genetic background of "SC 58", an old bark-cured tobacco variety, using conventional interspecific hybridization followed by backcross breeding. Similarly, the breeding line SC58 CtCt was also developed using a similar breeding methodology to transfer the major NDM genes from N. tomentosiformis into the genetic background of "SC 58". SC58 csCs was later found to have a NDM gene, designated CYP82E2, which is different from the gene naturally occurring in cultivated tobacco, and which the N. tabacum version is inactivated by two degenerative mutations (Chakrabarti et al., 2007). Expression of CYP82E2 from SC58 CsCs is stimulated by leaf senescence. SC58 CtCt was later found to have a nicotine demethylase gene, designated mCYP82E3, which is different from the gene naturally occurring in cultivated tobacco, and which the N. tabacum version is inactivated by a single base pair mutation (Gavilano et al., 2007). CYP82E3 from SC58 CtCt is active in green leaves, making this gene extremely efficient at demethylating nicotine to form nornicotine (more so than in CYP82E2).

[0087] Backcross breeding was used to combine the nic1 and nic2 alleles from LAFC53 with either CYP82E2 or CYP82E3 from either N. sylvestris or N. tomentosiformis, respectively, in a select tubular-cured genetic background. Six generations of backcrossing were used to transfer the alleles of interest into the genetic background of cultivar "K326," an open-pollinated tubular-cured tobacco cultivar. After the sixth backcross generation, BC6F1 plants with the allele variability of interest were self-pollinated and BC6F2 individuals homozygous for the allele of interest were identified. Selected BC6F2 plants were self-pollinated to generate stable BC6F3 seed lots. Cytoplasmic male sterile (Cms) F1 hybrids were generated by hybridizing the inbred lines described above as pollen parents with the Cms version of K326, in which the nic1 and nic2 alleles from LAFC53 were transferred by a backcross breeding procedure. The NDM genes from either N. sylvestris or N. tomentosiformis are deployed in a heterozygous condition in the F1 hybrids to reduce the effects of any deleterious linkage drag that may be present.

[0088] In accordance with the above, embodiments of the present disclosure include tobacco varieties, or any portion thereof, that contain reduced levels of at least one nicotinic alkaloid compared to a naturally occurring corresponding tobacco plant, or portion thereof. In some embodiments, the tobacco varieties produced according to the methods of the present disclosure contain at least one nicotinic alkaloid selected from the group consisting of nicotine, nornicotine, anatabine, and anabasine.

[0089] In some embodiments, the tobacco varieties of the present disclosure comprise reduced levels of nicotine. In some embodiments, the tobacco varieties comprise 0.35% or less nicotine. In some embodiments, the tobacco varieties comprise 0.30% or less nicotine. In some embodiments, the tobacco varieties comprise 0.25% or less nicotine. In some embodiments, the tobacco varieties comprise 0.20% or less nicotine. In some embodiments, the tobacco varieties comprise 0.15% or less nicotine. In some embodiments, the tobacco varieties comprise 0.10% or less nicotine. In some embodiments, the tobacco varieties comprise 0.09% or less nicotine. In some embodiments, the tobacco varieties comprise 0.08% or less nicotine. In some embodiments, the tobacco varieties comprise 0.07% or less nicotine. In some embodiments, the tobacco varieties comprise 0.06% or less nicotine. In some embodiments, the tobacco varieties comprise 0.05% or less nicotine. In some embodiments, the tobacco varieties comprise 0.04% or less nicotine. In some embodiments, the tobacco varieties comprise 0.03% or less nicotine. In some embodiments, the tobacco variety contains 0.02% or less nicotine. In some embodiments, the tobacco variety contains 0.01% or less nicotine. In some embodiments, the tobacco variety contains about 0.1% to about 0.35% nicotine. In some embodiments, the tobacco variety contains about 0.1% to about 0.25% nicotine. In some embodiments, the tobacco variety contains about 0.1% to about 0.15% nicotine. In some embodiments, the tobacco variety contains about 0.1% to about 0.10% nicotine. In some embodiments, the tobacco variety contains about 0.15% to about 0.35% nicotine. In some embodiments, the tobacco variety contains about 0.25% to about 0.35% nicotine. In some embodiments, the tobacco variety contains about 0.10% to about 0.20% nicotine. In some embodiments, the tobacco variety contains about 0.01% to about 0.10% nicotine. In some embodiments, the tobacco variety contains about 0.01% to about 0.5% nicotine. In some embodiments, the tobacco variety contains about 0.01% to about 0.03% nicotine.

[0090] In some embodiments, tobacco varieties of the present disclosure include reduced levels of nornicotine. In some embodiments, the variety includes 0.04% or less nornicotine. In some embodiments, the variety includes 0.03% or less nornicotine. In some embodiments, the variety includes 0.02% or less nornicotine. In some embodiments, the variety includes 0.01% or less nornicotine. In some embodiments, the variety includes 0.005% or less nornicotine. In some embodiments, the variety includes about 0.005% to about 0.04% nornicotine. In some embodiments, the variety includes about 0.01% to about 0.04% nornicotine. In some embodiments, the variety includes about 0.02% to about 0.04% nornicotine. In some embodiments, the variety includes about 0.03% to about 0.04% nornicotine. In some embodiments, the variety includes about 0.005% to about 0.03% nornicotine. In some embodiments, the variety contains about 0.005% to about 0.02% nornicotine. In some embodiments, the variety contains about 0.005% to about 0.01% nornicotine.

[0091] In some embodiments, tobacco varieties of the present disclosure contain reduced levels of anatabine. In some embodiments, the variety contains 0.06% or less anatabine. In some embodiments, the variety contains 0.05% or less anatabine. In some embodiments, the variety contains 0.04% or less anatabine. In some embodiments, the variety contains 0.03% or less anatabine. In some embodiments, the variety contains 0.02% or less anatabine. In some embodiments, the variety contains 0.01% or less anatabine. In some embodiments, the variety contains 0.005% or less anatabine. In some embodiments, the variety contains 0.001% or less anatabine. In some embodiments, the variety contains about 0.001% to about 0.06% anatabine. In some embodiments, the variety contains about 0.005% to about 0.06% anatabine. In some embodiments, the variety contains about 0.01% to about 0.06% anatabine. In some embodiments, the variety contains about 0.02% to about 0.06% anatabine. In some embodiments, the variety contains about 0.03% to about 0.06% anatabine. In some embodiments, the variety contains about 0.04% to about 0.06% anatabine. In some embodiments, the variety contains about 0.05% to about 0.06% anatabine. In some embodiments, the variety contains about 0.005% to about 0.05% anatabine. In some embodiments, the variety contains about 0.005% to about 0.04% anatabine. In some embodiments, the variety contains about 0.005% to about 0.03% anatabine. In some embodiments, the variety contains about 0.005% to about 0.02% anatabine. In some embodiments, the variety contains about 0.005% to about 0.01% anatabine. In some embodiments, the variety contains about 0.01% to about 0.05% anatabine. In some embodiments, the variety contains about 0.02% to about 0.04% anatabine.

[0092] In some embodiments, tobacco varieties of the present disclosure contain reduced levels of anabasine. In some embodiments, the variety contains 0.008% or less anabasine. In some embodiments, the variety contains 0.007% or less anabasine. In some embodiments, the variety contains 0.006% or less anabasine. In some embodiments, the variety contains 0.005% or less anabasine. In some embodiments, the variety contains 0.004% or less anabasine. In some embodiments, the variety contains 0.003% or less anabasine. In some embodiments, the variety contains 0.002% or less anabasine. In some embodiments, the variety contains 0.001% or less anabasine. In some embodiments, the variety contains 0.0005% or less anabasine. In some embodiments, the variety contains about 0.0005% to about 0.008% anabasine. In some embodiments, the variety contains about 0.001% to about 0.008% anabasine. In some embodiments, the variety contains about 0.002% to about 0.008% anabasine. In some embodiments, the variety contains about 0.003% to about 0.008% anabasine. In some embodiments, the variety contains about 0.004% to about 0.008% anabasine. In some embodiments, the variety contains about 0.005% to about 0.008% anabasine. In some embodiments, the variety contains about 0.006% to about 0.008% anabasine. In some embodiments, the variety contains about 0.007% to about 0.008% anabasine. In some embodiments, the variety contains about 0.005% to about 0.007% anabasine. In some embodiments, the variety contains about 0.005% to about 0.007% anabasine. In some embodiments, the variety contains about 0.005% to about 0.006% anabasine. In some embodiments, the variety contains about 0.0005% to about 0.005% anabasine. In some embodiments, the variety contains about 0.0005% to about 0.005% anabasine. In some embodiments, the variety contains about 0.0005% to about 0.004% anabasine. In some embodiments, the variety contains about 0.0005% to about 0.003% anabasine. In some embodiments, the variety contains about 0.0005% to about 0.002% anabasine.In some embodiments, the variety contains about 0.0005% to about 0.001% anabasine. In some embodiments, the variety contains about 0.001% to about 0.006% anabasine. In some embodiments, the variety contains about 0.002% to about 0.005% anabasine. In some embodiments, the variety contains about 0.003% to about 0.006% anabasine.

[0093] In some embodiments, the cultivar comprises a nic1 allele that is reduced in expression and / or function compared to wild-type nic1. In some embodiments, the cultivar comprises a nic2 allele that is reduced in expression and / or function compared to wild-type nic2. In some embodiments, the cultivar comprises a nic1 allele and a nic2 allele that are reduced in expression and / or function compared to wild-type nic1 and wild-type nic2. In some embodiments, the nic1 and / or nic2 alleles are derived from at least one of the following strains: LAFC53, MAFC5, LMAFC34, LA Burley 21, LI Burley 21, and HI Burley 21. In some embodiments, the first and / or second tobacco varieties comprise a nic1 and / or nic2 null allele (deletion). In some embodiments, the nic1 and / or nic2 null alleles are derived from naturally occurring N. tabacum germplasm.

[0094] In some embodiments, the cultivar comprises a functional CYP82E2 allele. In some embodiments, the CYP82E2 allele is derived from N. sylvestris. In some embodiments, the cultivar comprises a functional CYP82E3 allele. In some embodiments, the CYP82E3 allele is derived from N. tomentosiformis, or a related species (e.g., Nicotiana Section Tomentosae). In some embodiments, the cultivar comprises a functional CYP82E2 allele from N. sylvestris and a functional CYP82E3 allele from N. tomentosiformis, or a related species (e.g., Nicotiana Section Tomentosae). In some embodiments, the nicotine N-demethylation activity of the proteins encoded by the CYP82E2 and / or CYP82E3 alleles is increased compared to naturally occurring tobacco plants.

[0095] In some embodiments, the tobacco varieties of the present disclosure are non-transgenic (e.g., have no heterologous transgenes). According to these embodiments, the tobacco varieties of the present disclosure can be produced by breeding (e.g., backcrossing) various tobacco strains having a desired trait(s) corresponding to reduced levels of at least one nicotinic alkaloid. As such, the tobacco varieties of the present disclosure do not exist in nature.

[0096] Based on the present disclosure, as will be understood by those skilled in the art, tobacco varieties having reduced levels of at least one nicotinic alkaloid described herein can also be produced using transgenic approaches. In some embodiments, the tobacco varieties of the present disclosure can be engineered to include one or more traits corresponding to reduced levels of at least one nicotinic alkaloid. For example, tobacco plants can be engineered to include nic1 and / or nic2 alleles substantially similar to those found in LAFC53, MAFC5, LMAFC34, LA Burley 21, LI Burley 21, and HI Burley 21. These nic1 and / or nic2 alleles can have one or more genetic changes (e.g., loss of function or mass morphological mutations) that result in reduced levels of at least one nicotinic alkaloid. Such genetic changes can be engineered using any means known in the art, including, but not limited to, transcription activator-like effector nucleases (TALENs), meganucleases, zinc finger nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 systems, CRISPR / Cpf1 systems, CRISPR / Csm1 systems, and combinations thereof (see, e.g., Gaj et al., Trends in Biotechnology, 31(7):397-405 (2013)).

[0097] Tobacco varieties having reduced levels of at least one nicotinic alkaloid described herein can also be engineered to include one or more traits corresponding to the reduced levels of at least one nicotinic alkaloid in addition to the alterations to nic1 and / or nic2. For example, tobacco plants can be engineered to include functional CYP82E2 and / or CYP82E3 alleles compared to the corresponding naturally occurring tobacco plants. In some embodiments, the CYP82E2 and / or CYP82E3 alleles are engineered to restore wild-type function of the corresponding CYP82E2 and / or CYP82E3 proteins that have become non-functional due to genetic mutations. In some embodiments, the CYP82E2 and / or CYP82E3 alleles can be engineered to have one or more genetic changes that result in reduced levels of at least one nicotinic alkaloid (e.g., gain-of-function or upregulation mutations). Such genetic changes can be engineered using any means known in the art, including, but not limited to, transcription activator-like effector nucleases (TALENs), meganucleases, zinc finger nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 systems, CRISPR / Cpf1 systems, CRISPR / Csm1 systems, and combinations thereof (see, e.g., Gaj et al., Trends in Biotechnology, 31(7):397-405 (2013)).

[0098] In some embodiments, tobacco plants are engineered to contain functional CYP82E2 and / or CYP82E3 alleles as compared to a corresponding naturally occurring or non-transformed control tobacco plant. For example, in some embodiments, CYP82E2 alleles are engineered to restore function of a corresponding CYP82E2 protein that has been rendered non-functional by genetic mutation or to increase activity of the corresponding CYP82E2 protein as compared to a corresponding naturally occurring or non-transformed control tobacco plant. In some embodiments, CYP82E2 alleles of tobacco plants are engineered to contain nucleotide sequences encoding K375E and / or L422W point mutations as compared to a wild-type CYP82E2 amino acid sequence (e.g., SEQ ID NO: 1). An exemplary wild-type CYP82E2 amino acid sequence is set forth in SEQ ID NO: 1 (GenBank Accession No. ABA07806): MVFPIEAFVGLVTFTFLLYFLWTKKSQKLPKPLPPKIPGGWPVIGHLFHFNNDGDDRPLARKLGDLADKYGPVFTFRLGLPLVLVVSSYEAIKDCFSTNDAIFSNRPAFLYGEYLGYNNTMLFLANYGPYW RKNRKLVIQEVLSASRLEKFKQVRFTRIQTSIKNLYTRINGNSSTINLTDWLEELNFGLIVKMIAGKNYESGKGDEQVERFKNAFKDFMVLSMEFVLWDAFPIPLFKWVDFQGHIKAMKRTFKDIDSVFQN WLEEHINKREKMEVGAEGNEQDFIDVVLSKLSKEYLDEGYSRDTVIKATVFSLVLDAADTVALHINWGMTLLINNQNALMKAQEEIDTKVGKDRWVEESDIKDLVYLQAIVKKVLRLYPPGPLLVPHENVKDCVVSGYHIPKGTRLFANVMKLQRDPKLLSNPDKFDPERFIAGDIDFRGHHYEFIPFGSGRRSCPGMTYALQVEHLTMAHLIQGFNYKTPNDEALDMKEGAGITIRKVNPVELIITPRLAPELY (SEQ ID NO: 1).

[0099] An exemplary CYP82E2 amino acid sequence containing a K375E point mutation compared to the wild-type CYP82E2 amino acid sequence is set forth in SEQ ID NO:2 (the point mutation is represented below by bold and underlined letters): [ka]

[0100] An exemplary CYP82E2 amino acid sequence that includes a L422W point mutation, as compared to the wild-type CYP82E2 amino acid sequence, is set forth in SEQ ID NO:3 (the point mutation is represented below by bold and underlined letters): [ka]

[0101] An exemplary CYP82E2 amino acid sequence containing a K375E point mutation and a L422W point mutation, as compared to the wild-type CYP82E2 amino acid sequence, is set forth in SEQ ID NO:4 (point mutations are represented below by bold and underlined letters): [ka]

[0102] In some embodiments, a CYP82E3 allele is engineered to restore function of a corresponding CYP82E3 protein that has been rendered non-functional by genetic mutation, or to increase the activity of the corresponding CYP82E3 protein compared to a corresponding naturally occurring or non-transformed control tobacco plant. In some embodiments, a CYP82E3 allele of a tobacco plant is engineered to include a nucleotide sequence encoding a C330W point mutation compared to a wild-type CYP82E3 amino acid sequence (e.g., SEQ ID NO: 5). An exemplary wild-type CYP82E3 amino acid sequence is set forth in SEQ ID NO: 5 (NCBI Reference Sequence: NM_001326063.1): MVFPVEAIVGLVTFTFLFYFLWTKKSQKPSKPLPPKIPGGWPVIGHLFYFDDDGDDRPLARKLGDLADKYGPVFTFRLGLPLVLVVSSYEAIKDCFSTNDAIFSNRPAFLYGEYLGYKNAMLFLANYGSYW RKNRKLIIQEVLSASRLEKFKHVRFARIQTSIKNLYTRIDGNSSTINLTDWLEELNFGLIVKMIAGKNYESGKGDEQVERFKKAFKDFMILSMEFVLWDAFPIPLFKWVDFQGHVKAMKRTFKDIDSVFQNW LEEHIKKREKIMEVGTEGNEQDFIDVVLSKMSNEYLGEGYSRDTVIKATVFSLVLDAADTVALHINCGMALLINNQNALKKAQEEIDTKVGKDRWVEESDIKDLVYLQAIVKEVLRLYPPGPLLVPHENVEDCVVSGYHIPKGTRLFANVMKLQRDPKLWSNPDKFNPERFIARDIDFHGQHYEYIPFGSGRRSCPGMTYALQVEHLTMAHLIQGFNYRTPTDEPLDMKEGAGITIRKVNPVKVIITPRLAPELY (sequence number 5).

[0103] An exemplary CYP82E3 amino acid sequence containing a C330W point mutation, as compared to the wild-type CYP82E3 amino acid sequence, is set forth in SEQ ID NO:6 (the point mutation is represented below by bold and underlined letters): [ka]

[0104] In some embodiments, the technology provides a method for producing a tobacco plant with reduced nicotinic alkaloid content, the method comprising combining in a tobacco plant (e.g., Nicotiana tabacum) (A) a genetic modification that increases CYP82E2 and / or CYP82E3 activity relative to a corresponding, naturally occurring, or untransformed control tobacco plant, and (B) a recessive allele of nic1 and / or a recessive allele of nic2.

[0105] In some embodiments, introducing a recessive allele of nic1 and / or a recessive allele of nic2 can include incorporating one or more of the recessive alleles by conventional breeding into a tobacco plant (e.g., Nicotiana tabacum) that contains one or more genetic modifications that increase the activity of CYP82E2 and / or CYP82E3 in the tobacco plant, e.g., compared to a wild-type tobacco plant.

[0106] In some embodiments, tobacco plants produced by the methods of the present technology having (A) a genetic modification that increases activity of CYP82E2 and / or CYP82E3 and (B) a recessive allele of nic1 and / or a recessive allele of nic2 have an increased CYP82E2 activity and / or CYP82E3 activity and / or a ... %, 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. In some embodiments, the nicotinic alkaloid reduced in the tobacco plant can be nicotine, and the nicotine content can be reduced by about 90% or more (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) compared to a corresponding naturally occurring or non-transformed control tobacco plant.

[0107] In some embodiments, tobacco plants produced by the methods of the present technology having (A) a genetic modification that increases the activity of CYP82E2 and / or CYP82E3 and (B) a recessive allele of nic1 and / or a recessive allele of nic2 contain reduced levels of nicotine. In some embodiments, the tobacco plants contain about 0.35% or less nicotine. In other embodiments, the tobacco plants contain about 0.30% or less nicotine, about 0.25% or less nicotine, about 0.20% or less nicotine, about 0.15% or less nicotine, about 0.10% or less nicotine, about 0.09% or less nicotine, about 0.08% or less nicotine, about 0.07% or less nicotine, about 0.06% or less nicotine, about 0.05% or less nicotine, about 0.04% or less nicotine, about 0.03% or less nicotine, about 0.02% or less nicotine, or about 0.01% or less nicotine.

[0108] In some embodiments, the tobacco plant comprises about 0.1% to about 0.35% nicotine, about 0.1% to about 0.25% nicotine, about 0.1% to about 0.15% nicotine, about 0.1% to about 0.10% nicotine, about 0.15% to about 0.35% nicotine, about 0.25% to about 0.35% nicotine, about 0.10% to about 0.20% nicotine, about 0.01% to about 0.10% nicotine, about 0.01% to about 0.5% nicotine, or about 0.01% to about 0.03% nicotine.

[0109] In some embodiments, tobacco plants produced by the methods of the present technology having (A) a genetic modification that increases the activity of CYP82E2 and / or CYP82E3 and (B) a recessive allele of nic1 and / or a recessive allele of nic2 contain reduced levels of nornicotine. In some embodiments, the plants contain about 0.04% nornicotine or less, about 0.03% nornicotine or less, about 0.02% nornicotine or less, about 0.01% nornicotine or less, or about 0.005% nornicotine. In some embodiments, the plant contains about 0.005% to about 0.04% nornicotine, about 0.01% to about 0.04% nornicotine, about 0.02% to about 0.04% nornicotine, about 0.03% to about 0.04% nornicotine, about 0.005% to about 0.03% nornicotine, about 0.005% to about 0.02% nornicotine, or about 0.005% to about 0.01% nornicotine.

[0110] In some embodiments, tobacco plants produced by the methods of the present technology having (A) a genetic modification that increases CYP82E2 and / or CYP82E3 activity and (B) a recessive allele of nic1 and / or a recessive allele of nic2 contain reduced levels of anatabine, hi some embodiments, the plants contain less than about 0.06% anatabine, less than about 0.05% anatabine, less than about 0.04% anatabine, less than about 0.03% anatabine, less than about 0.02% anatabine, less than about 0.01% anatabine, less than about 0.005% anatabine, or less than about 0.001% anatabine. In other embodiments, the plant contains about 0.001% to about 0.06% anatabine, about 0.005% to about 0.06% anatabine, about 0.01% to about 0.06% anatabine, about 0.02% to about 0.06% anatabine, about 0.03% to about 0.06% anatabine, about 0.04% to about 0.06% anatabine, about 0.05% to about 0.06% anatabine. 0.005% to about 0.05% anatabine, about 0.005% to about 0.04% anatabine, about 0.005% to about 0.03% anatabine, about 0.005% to about 0.02% anatabine, about 0.005% to about 0.01% anatabine, about 0.01% to about 0.05% anatabine, or about 0.02% to about 0.04% anatabine.

[0111] In some embodiments, tobacco plants produced by the methods of the present technology having (A) a genetic modification that increases CYP82E2 and / or CYP82E3 activity and (B) a recessive allele of nic1 and / or a recessive allele of nic2 contain reduced levels of anabasine, hi some embodiments, the plants contain less than about 0.008% anabasine, less than about 0.007% anabasine, less than about 0.006% anabasine, less than about 0.005% anabasine, less than about 0.004% anabasine, less than about 0.003% anabasine, less than about 0.002% anabasine, less than about 0.001% anabasine, or less than about 0.0005% anabasine. In other embodiments, the plant is provided with about 0.0005% to about 0.008% anabasine, about 0.001% to about 0.008% anabasine, about 0.002% to about 0.008% anabasine, about 0.003% to about 0.008% anabasine, about 0.004% to about 0.008% anabasine, about 0.005% to about 0.008% anabasine, about 0.000% to about 0.008% anabasine, about 0.007% to about 0.008% anabasine, about 0.0005% to about 0.007% anabasine, about 0.0005% to about 0.007% anabasine, about 0.0005% to about Contains 0.006% anabasine, about 0.0005% to about 0.005% anabasine, about 0.0005% to about 0.005% anabasine, about 0.0005% to about 0.004% anabasine, about 0.0005% to about 0.003% anabasine, about 0.0005% to about 0.002% anabasine, about 0.0005% to about 0.001% anabasine, about 0.001% to about 0.006% anabasine, about 0.002% to about 0.005% anabasine, or about 0.003% to about 0.006% anabasine.

[0112] As described herein, genetic modification can be achieved using any means known in the art, including, but not limited to, transcription activator-like effector nucleases (TALENs), meganucleases, zinc finger nucleases, CRISPR / Cas9 systems, CRISPR / Cpf1 systems, CRISPR / Csm1 systems, gene knock-in technology or scientific techniques, or any combination thereof.

[0113] For example, in some embodiments, methods of the technology involve the use of a CRISPR / Cas system that binds to a target site in a region of interest in a genome, the CRISPR / Cas system comprising a CRISPR / Cas nuclease and a recombinant crRNA / tracrRNA (or a single guide RNA (sgRNA) or guide RNA (gRNA)). In some embodiments, the CRISPR system generally comprises (i) a polynucleotide encoding a Cas protein and (ii) at least one sgRNA for RNA-guided genome recombination in a plant cell. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Cys3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Smr1, Cmr3, Cmr4, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. In some embodiments, the Cas protein is a Streptococcus pyogenes Cas9 protein. These enzymes are well known. For example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under the accession number Q99ZW2. In some embodiments, the methods of the technology relate to the use of the CRISPR / Cpf1 system to bind to a target site in a region of interest in a genome. In some embodiments, the methods of the technology relate to the use of the CRISPR / Csm1 system to bind to a target site in a region of interest in a genome.

[0114] In some embodiments, the CRISPR / Cas, CRISPR / Cpf1, or CRISPR / Csm1 system recognizes a target site in one or more of CYP82E2 or CYP82E3. In some embodiments, the CRISPR / Cas, CRISPR / Cpf1, or CRISPR / Csm1 system generates a specific sequence change in the CYP82E2 or CYP82E3 gene, for example, a mutation resulting in a K375E and / or L422W point mutation in the CYP82E2 amino acid sequence, or a mutation resulting in a C330W point mutation in the CYP82E3 amino acid sequence. In some embodiments, the CRISPR / Cas, CRISPR / Cpf1, or CRISPR / Csm1 system generates a specific sequence change by gene knock-in or gene replacement. Methods for gene knock-in or gene replacement are well known in the art. CRISPR-based methods of gene knock-in or gene replacement can utilize the homology-directed repair (HDR) mechanism, the non-homologous end joining (NHEJ) mechanism, or both HDR and NHEJ mechanisms. A non-limiting example of a CRISPR-based method that utilizes both HDR and NHEJ mechanisms is called tandem repeat HDR (TR-HDR) and is described in Lu et al., “Targeted, efficient sequence insertion and replacement in rice,” Nature Biotechnology, 38(12):1402-1407.doi: 10.1038 / s41587-020-0581-5, (2020), the entire contents of which are incorporated herein by reference.

[0115] In some embodiments, the methods described herein use a meganuclease DNA binding domain to bind to a region of interest in the genome of a plant cell. Meganucleases are typically engineered versions of naturally occurring restriction enzymes with extended DNA recognition sequences (e.g., about 14 to about 40 base pairs in length). Meganucleases (also known as homing endonucleases) are generally classified into five families based on sequence and structural motifs: the LAGLIDADG family, the GIY-YIG family, the His-Cyst box family, the PD-(D / E)XK family, and the HNH family. In some embodiments, the meganuclease comprises an engineered homing endonuclease. Recognition sequences for homing endonucleases and meganucleases are well known, such as I-Sce, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII, and I-TevIII.

[0116] In some embodiments, the meganuclease is adapted to recognize a target in one or more of CYP82E2 or CYP82E3. In some embodiments, the meganuclease generates a specific sequence change in the CYP82E2 or CYP82E3 gene, for example, a mutation resulting in a K375E and / or L422W point mutation in the CYP82E2 amino acid sequence, or a mutation resulting in a C330W point mutation in the CYP82E3 amino acid sequence.

[0117] In some embodiments, the methods described herein use transcription activation-like effector nucleases (TALENs) to edit plant genomes by introducing double-stranded breaks (DSBs). TALENs are restriction enzymes that can be engineered to cleave specific sequences in DNA. TALENs are constructed by fusing a TAL effector DNA binding domain to a DNA cleavage domain (e.g., a nuclease domain, such as that derived from FokI endonuclease). Transcription activation-like effectors (TALEs) can be engineered and bound to a desired DNA sequence according to methods well known in the art, resulting in a technique for cleaving DNA at a specific location when combined with a nuclease. For example, in some embodiments, the use of TALEN technology generates specific sequence changes in the CYP82E2 or CYP82E3 genes, such as mutations resulting in K375E and / or L422W point mutations in the CYP82E2 amino acid sequence, or C330W point mutations in the CYP82E3 amino acid sequence.

[0118] In some embodiments, the compositions and methods described herein use zinc finger nucleases (ZFNs) to edit plant genomes by introducing double strand breaks (DSBs). ZFNs are artificial restriction enzymes generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain (e.g., a nuclease domain such as that derived from FokI endonuclease). ZFNs can be engineered to bind and cleave DNA at specific locations. ZFNs contain two protein domains. The first domain is a DNA binding domain, which contains a eukaryotic transcription factor and a zinc finger. The second domain is a nuclease domain containing the FokI restriction enzyme, which is responsible for DNA cleavage. ZFNs can be engineered to bind to a desired DNA sequence and cleave DNA at specific locations according to methods well known in the art. For example, after a target sequence is identified in a nicotine biosynthesis gene, the corresponding (ZFN) sequence is engineered and inserted into a plasmid. The plasmid is inserted into a target cell, where it is translated to produce a functional ZFN, which then enters the nucleus, where it binds to and cuts its target sequence, introducing a double-strand break (DSB). Since the DSB is repaired either by homology-directed repair or by non-homologous end joining, such an approach can be used to introduce exogenous DNA sequences into target genes. For example, in some embodiments, the use of ZFN technology generates specific sequence changes in the CYP82E2 or CYP82E3 gene, such as mutations that result in K375E and / or L422W point mutations in the CYP82E2 amino acid sequence, or mutations that result in C330W point mutations in the CYP82E3 amino acid sequence.

[0119] In some embodiments, the subject technology provides methods for producing tobacco plants with reduced nicotinic alkaloid content (e.g., reduced total nicotinic alkaloid content, nicotine, nornicotine, anatabine, and / or anabasine, as described above for plants produced by the subject technology having (A) a genetic modification that increases CYP82E2 and / or CYP82E3 activity and (B) a recessive allele of nic1 and / or a recessive allele of nic2), the methods comprising combining within the tobacco plant (a) a genetic modification that increases CYP82E2 and / or CYP82E3 activity as described herein, with (b) a modification that inhibits (i) the activity of ERF199 and / or ERF189, or (ii) expression of a nucleic acid encoding ERF199 and / or a nucleic acid encoding ERF189, relative to a corresponding, naturally occurring, or non-transformed control tobacco plant.

[0120] In some embodiments, the technology further includes suppressing expression of endogenous genes encoding transcription factors that positively regulate alkaloid production, such as NtERF221, NtMYC1a, NtMYC1b, NtMYC2a, and / or NtMYC2b genes, thereby reducing nicotinic alkaloid levels in the plant.

[0121] In some embodiments, the technology further includes suppressing expression of one or more nicotinic alkaloid biosynthetic genes, such as A622, NBBl, QPT (quinolate phosphoribosyltransferase), PMT (putrescine methyltransferase), ODC (ornithine decarboxylase), AO (aspartate oxidase), QS (quinolinic acid synthase), and MPO (N-methylputrescine oxidase) genes, to reduce nicotinic alkaloid levels in the plant.

[0122] Examples of methods that can be used to suppress the ERF199, ERF189, NtERF221, NtMYC1a, NtMYC1b, NtMYC2a, NtMYC2b, A622, NBB1, QPT, PMT, ODC, AO, QS, and / or MPO genes include, but are not limited to, antisense-sense co-suppression, RNAi, artificial microRNA, virus-induced gene silencing (VIGS), antisense-sense co-suppression, targeted mutagenesis, and related genome modification methods including, but not limited to, transcription activator-like effector nucleases (TALENs), meganucleases, zinc finger nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 systems, CRISPR / Cpf1 systems, CRISPR / Csm1 systems, and any combination thereof.

[0123] In accordance with the above, embodiments of the present disclosure also include progeny plants, seeds, or cells produced from any of the tobacco varieties described herein, produced using transgenic and / or non-transgenic methods.Embodiments of the present disclosure also include tobacco products derived from any of the tobacco varieties described herein, or portions thereof.In some embodiments, 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, cigarettes, cigarillos, cigars, and chewing tobacco.In some embodiments, the tobacco product is selected from the group consisting of cigarillos, krettic cigarettes, non-breathable recessed filter cigarettes, breathable recessed filter cigarettes, cigars, snuff, tobacco-containing gum, tobacco-containing lozenges, and chewing tobacco.

[0124] The embodiments of the present disclosure also include methods for producing tobacco varieties that include reduced levels of at least one nicotinic alkaloid compared to a naturally occurring corresponding tobacco plant, or portion thereof. According to these embodiments, the method includes crossing a first tobacco variety that includes a first low-nicotine trait with a second tobacco variety that includes a second low-nicotine trait to produce a progeny plant. In some embodiments, the progeny plant includes a reduced concentration of at least one nicotinic alkaloid. In some embodiments, the method includes backcrossing. Such tobacco varieties with reduced levels of at least one nicotinic alkaloid can be produced using transgenic and / or non-transgenic methods, as will be recognized by those skilled in the art.

[0125] In some embodiments, and as further described above, the at least one nicotinic alkaloid is selected from the group consisting of nicotine, nornicotine, anatabine, and anabasine. In some embodiments, the at least one nicotinic alkaloid is nicotine, and the variety contains 0.35% or less nicotine. In some embodiments, the at least one nicotinic alkaloid is nornicotine, and the variety contains 0.04% or less nornicotine. In some embodiments, the at least one nicotinic alkaloid is anatabine, and the variety contains 0.06% or less anatabine. In some embodiments, the at least one nicotinic alkaloid is anabasine, and the variety contains 0.08% or less anabasine. In some embodiments, the at least one nicotinic alkaloid is nicotine and nornicotine, and the variety contains 0.35% or less nicotine and 0.04% or less nornicotine.

[0126] In some embodiments, the first and / or second low nicotine trait comprises a nic1 allele that is reduced in expression and / or function compared to wild type nic1. In some embodiments, the first and / or second low nicotine trait comprises a nic2 allele that is reduced in expression and / or function compared to wild type nic2. In some embodiments, the first and / or second tobacco variety comprises a nic1 and / or nic2 allele from at least one of the following strains: LAFC53, MAFC5, LMAFC34, LA Burley 21, LI Burley 21, and HI Burley 21. In some embodiments, the first and / or second tobacco variety comprises a nic1 and / or nic2 null allele (deletion). In some embodiments, the nic1 and / or nic2 null allele is derived from naturally occurring N. tabacum germplasm.

[0127] In some embodiments, the first and / or second tobacco varieties comprise a functional CYP82E2 allele. In some embodiments, the CYP82E2 allele is derived from N. sylvestris. In some embodiments, the first and / or second tobacco varieties comprise a functional CYP82E3 allele. In some embodiments, the CYP82E3 allele is derived from N. tomentosiformis or a related species (e.g., Nicotiana Section Tomentosae). In some embodiments, the nicotine N-demethylation activity of the proteins encoded by the CYP82E2 and / or CYP82E3 alleles is increased compared to a naturally occurring tobacco plant.

[0128] In some embodiments, the first low nicotine trait comprises nic1 and / or nic2 alleles with reduced expression and / or function compared to wild-type nic1 and / or nic2, and the second low nicotine trait comprises a functional CYP82E2 allele and / or a functional CYP82E3 allele.

[0129] In some embodiments, the first tobacco variety, the second tobacco variety, and the progeny plants are non-transgenic. In some embodiments, one or more of the first tobacco variety, the second tobacco variety, and / or the progeny plants are non-transgenic.

[0130] Embodiments of the present disclosure also include seeds or cells obtained from a progeny plant produced according to any of the methods described herein.Embodiments of the present disclosure also include tobacco products derived from a progeny plant produced according to any of the methods described herein.In some embodiments, the 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, cigarettes, cigarillos, cigars, and chewing tobacco.In some embodiments, the product is selected from the group consisting of cigarillos, krettic cigarettes, non-breathable recessed filter cigarettes, breathable recessed filter cigarettes, cigars, snuff, tobacco-containing gum, tobacco-containing lozenges, and chewing tobacco.

[0131] In some embodiments, the cultivar comprises a CYP82E2 allele that comprises a nucleotide sequence encoding a K375E and / or a L422W point mutation compared to the wild-type CYP82E2 amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, (a) the CYP82E2 allele comprises a nucleotide sequence encoding a CYP82E2 protein comprising an amino acid sequence set forth in SEQ ID NO: 2, (b) the CYP82E2 allele comprises a nucleotide sequence encoding a CYP82E2 protein comprising an amino acid sequence set forth in SEQ ID NO: 3, or (c) the CYP82E2 allele comprises a nucleotide sequence encoding a CYP82E2 protein comprising an amino acid sequence set forth in SEQ ID NO: 4.

[0132] In some embodiments, a CYP82E3 allele comprises a nucleotide sequence that encodes a C330W point mutation compared to the wild-type CYP82E3 amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, a CYP82E3 allele comprises a nucleotide sequence that encodes a CYP82E3 protein comprising the amino acid sequence set forth in SEQ ID NO: 6.

[0133] In some embodiments, the cultivar comprises a CYP82E2 allele that comprises a nucleotide sequence encoding a K375E and / or a L422W point mutation compared to the wild-type CYP82E2 amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, (a) the CYP82E2 allele comprises a nucleotide sequence encoding a CYP82E2 protein comprising an amino acid sequence set forth in SEQ ID NO: 2, (b) the CYP82E2 allele comprises a nucleotide sequence encoding a CYP82E2 protein comprising an amino acid sequence set forth in SEQ ID NO: 3, or (c) the CYP82E2 allele comprises a nucleotide sequence encoding a CYP82E2 protein comprising an amino acid sequence set forth in SEQ ID NO: 4.

[0134] In some embodiments, a CYP82E3 allele comprises a nucleotide sequence that encodes a C330W point mutation compared to the wild-type CYP82E3 amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, a CYP82E3 allele comprises a nucleotide sequence that encodes a CYP82E3 protein comprising the amino acid sequence set forth in SEQ ID NO: 6.

[0135] In some embodiments, the cultivar further comprises suppression of expression in the cultivar of at least one of NBBl, A622, quinolate phosphoribosyltransferase (QPT), putrescine N-methyltransferase (PMT), ornithine decarboxylase (ODC), aspartate oxidase (AO), quinolinate synthase (QS), N-methylputrescine oxidase (MPO), NtERF221, NtMYC1a, NtMYC1b, NtMYC2a, or NtMYC2b.

[0136] Embodiments of the present disclosure also include methods of making a point mutation in a target gene in a Nicotiana tabacum plant cell. According to these embodiments, the method includes introducing into the cell a clustered regularly interspaced short palindromic repeats (CRISPR) associated (CRISPR-Cas) system comprising: (a) at least one guide RNA (gRNA), the at least one gRNA comprising a nucleotide sequence capable of hybridizing with a target sequence or a portion thereof in a target gene, the target gene comprising a nucleotide sequence encoding a CYP82E2 protein; (b) (i) a DNA fragment, the DNA fragment replacing a target sequence in the target gene or being inserted into a target site in the target gene, and (ii) two homologous arms, each arm flanking opposite sides of the DNA fragment; and (c) a nucleic acid comprising an effector protein or a donor DNA sequence comprising one or more polynucleotides encoding the effector protein. In some embodiments, at least one gRNA forms a complex with an effector protein, in some embodiments, the effector protein is a Cas protein that comprises a nuclease.

[0137] In some embodiments, (a) the point mutation is a K375E point mutation compared to the wild-type CYP82E2 amino acid sequence set forth in SEQ ID NO:1, or (b) the point mutation is a L422WE point mutation compared to the wild-type CYP82E2 amino acid sequence set forth in SEQ ID NO:1.

[0138] In some embodiments, at least two point mutations are made, hi some embodiments, the at least two point mutations include a K375E point mutation compared to the wild-type CYP82E2 amino acid sequence set forth in SEQ ID NO:1, and a L422W point mutation compared to the wild-type CYP82E2 amino acid sequence set forth in SEQ ID NO:1.

[0139] In some embodiments, the Nicotiana tabacum plant further comprises a recessive allele of nic1 and / or a recessive allele of nic2.

[0140] In some embodiments, the method further comprises suppressing expression in at least one variety of NBBl, A622, quinolate phosphoribosyltransferase (QPT), putrescine N-methyltransferase (PMT), ornithine decarboxylase (ODC), aspartate oxidase (AO), quinolinate synthase (QS), N-methylputrescine oxidase (MPO), NtERF221, NtMYC1a, NtMYC1b, NtMYC2a, or NtMYC2b.

[0141] Embodiments of the present disclosure also include methods of making a point mutation in a target gene in a Nicotiana tabacum plant cell. According to these embodiments, the method includes introducing into the cell a clustered regularly interspaced short palindromic repeats (CRISPR) associated (CRISPR-Cas) system comprising: (a) at least one guide RNA (gRNA), the at least one gRNA comprising a nucleotide sequence capable of hybridizing with a target sequence or a portion thereof in a target gene, the target gene comprising a nucleotide sequence encoding a CYP82E3 protein; (b) (i) a DNA fragment, the DNA fragment replacing a target sequence in the target gene or being inserted into a target site in the target gene, and (ii) two homologous arms, each arm flanking opposite sides of the DNA fragment; and (c) a nucleic acid comprising an effector protein or a donor DNA sequence comprising one or more polynucleotides encoding the effector protein. In some embodiments, at least one gRNA forms a complex with an effector protein, in some embodiments, the effector protein is a Cas protein that comprises a nuclease.

[0142] In some embodiments, the point mutation is a C330W point mutation compared to the wild-type CYP82E3 amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the Nicotiana tabacum plant further comprises a recessive allele of nic1 and / or a recessive allele of nic2.

[0143] In some embodiments, the method further comprises suppressing expression in the Nicotiana tabacum plant of at least one of NBBl, A622, quinolate phosphoribosyltransferase (QPT), putrescine N-methyltransferase (PMT), ornithine decarboxylase (ODC), aspartate oxidase (AO), quinolinate synthase (QS), N-methylputrescine oxidase (MPO), NtERF221, NtMYC1a, NtMYC1b, NtMYC2a, or NtMYC2b.

[0144] Embodiments of the present disclosure also include methods for producing Nicotiana tabacum plants with reduced nicotinic alkaloid content. According to these embodiments, the methods include combining in a Nicotiana tabacum plant (a) one or more genetic modifications that increase the activity of CYP82E2 and / or CYP82E3, and (b) a recessive allele of nic1 and / or a recessive allele of nic2. In some embodiments, the Nicotiana tabacum plant has reduced nicotinic alkaloid content compared to a corresponding naturally occurring or non-transformed control tobacco plant.

[0145] In some embodiments, the Nicotiana tabacum plant comprises a homozygous recessive allele of nic1 and / or a homozygous recessive allele of nic2.

[0146] In some embodiments, one or more genetic modifications that increase the activity of CYP82E2 and / or CYP82E3 are introduced by transcription activator-like effector nucleases (TALENs), meganucleases, zinc finger nucleases, CRISPR / Cas9 systems, CRISPR / Cpf1 systems, CRISPR / Csm1 systems, gene knock-in technology or scientific techniques, and any combination thereof.

[0147] In some embodiments, the one or more genetic modifications that increase the activity of CYP82E2 comprise a point mutation in the target gene of the Nicotiana tabacum plant cell, the point mutation being selected from one or more of a K375E point mutation compared to the wild-type CYP82E2 amino acid sequence set forth in SEQ ID NO:1, and a L422W point mutation compared to the wild-type CYP82E2 amino acid sequence set forth in SEQ ID NO:1.

[0148] In some embodiments, the one or more genetic modifications that increase the activity of CYP82E3 include a point mutation in the target gene of the Nicotiana tabacum plant cell, wherein the point mutation is a C330W point mutation compared to the wild-type CYP82E3 amino acid sequence set forth in SEQ ID NO:5.

[0149] In some embodiments, the point mutation is introduced into the plant by a clustered regularly interspaced short palindromic repeats (CRISPR) associated (CRISPR-Cas) system comprising: (a) at least one guide RNA (gRNA), the at least one gRNA comprising a nucleotide sequence capable of hybridizing to a target sequence or a portion thereof in a target gene, the target gene comprising a nucleotide sequence encoding a CYP82E2 protein; (b) (i) a DNA fragment, the DNA fragment replacing a target sequence in the target gene or being inserted into a target site in the target gene, and (ii) two homologous arms, each flanking opposite sides of the DNA fragment; and (c) a nucleic acid comprising an effector protein or a donor DNA sequence comprising one or more polynucleotides encoding the effector protein. In some embodiments, the at least one gRNA forms a complex with the effector protein. In some embodiments, the effector protein is a Cas protein comprising a nuclease.

[0150] Embodiments of the disclosure also include Nicotiana tabacum plants produced according to any of the methods described herein, the plants comprising (a) one or more genetic modifications that increase CYP82E2 and / or CYP82E3 activity, and (b) a recessive allele at nic1 and / or a recessive allele at nic2. Embodiments of the disclosure also include progeny plants or seeds produced from the plants, the progeny plants or seeds comprising (A) one or more genetic modifications that increase CYP82E2 and / or CYP82E3 activity, and (B) a recessive allele at nic1 and / or a recessive allele at nic2. Embodiments of the disclosure also include tobacco products comprising tobacco from a Nicotiana tabacum plant, the plants comprising (A) one or more genetic modifications that increase CYP82E2 and / or CYP82E3 activity, and (B) a recessive allele at nic1 and / or a recessive allele at nic2.

[0151] According to these embodiments, (a) 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, cigarettes, cigarillos, cigarillos, and chewing tobacco, or (b) the product is a reduced-nicotine tobacco product selected from the group consisting of cigarillos, krettic cigarettes, non-ventilated recessed filter cigarettes, ventilated recessed filter cigarettes, cigars, snuff, snus, tobacco-containing gum, tobacco-containing lozenges, and chewing tobacco.

[0152] 3. Working Example It will be appreciated by those skilled in the art that other suitable modifications and adaptations of the disclosed methods described herein are readily applicable and recognizable, and may be made using suitable equivalents without departing from the scope of the disclosure or the aspects and embodiments disclosed herein. Having described the disclosure in detail, the disclosure will be more clearly understood by reference to the following examples, which are intended merely to illustrate some aspects and embodiments of the disclosure, and should not be considered as limiting the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referenced herein are incorporated herein by reference in their entirety.

[0153] The present disclosure has multiple aspects, which are illustrated by the following non-limiting examples.

[0154] Example 1 Cms NCLA Hybrid. Given the possibility that nicotine levels in conventional cigarettes will be forcibly lowered below the so-called "threshold of addiction" in various countries around the world (including the United States), there is commercial interest in tobacco varieties that produce cured tobacco with nicotine levels that allow manufacturers to achieve their target nicotine levels. Currently, nicotine levels in cigarette tobacco filler of 0.04% are being discussed and / or recommended. No iron-cured tobacco varieties are currently available that routinely accumulate nicotine at such low levels (when grown under conventional agricultural management systems and averaged over all stem positions).

[0155] Thus, embodiments of the present disclosure include NCLA Hybrid 1, NCLA Hybrid 2, and NCLA Hybrid 3 (Nic1 and Nic2 loci from LAFC53 combined with CYP82E3 alleles from N. tomentosiformis), which are novel established cytoplasmic male sterile (Cms) tube-cured tobacco hybrids with extremely low nicotine content. These hybrid varieties accumulate nicotine at the lowest levels of any publicly reported genetic material. As described herein, naturally occurring genetic variation and traditional breeding approaches were used to develop these new F1 hybrids (i.e., no gene editing, genetic modification, or novel breeding approaches were used to develop these materials). These hybrids can be grown anywhere in the world without concerns about patent infringement or regulations regarding the results of gene editing or genetic modification breeding.

[0156] Data supporting the low-nicotine phenotype of these new hybrids was generated from field evaluations at three North Carolina research stations. The experimental design at each location was a four-way randomized block design. Plots consisted of 20 plant rows that were managed according to North Carolina recommendations for flue-cured tobacco products. Leaves were collected at four primings, dried, and graded by a former USDA tobacco grader. Composite leaf samples were analyzed for alkaloid composition using gas chromatographic methods.

[0157] The materials evaluated in the first experiment were: K326 - a standard iron-cured tobacco variety, LAFC53 - a near-isogenic version of iron-cured tobacco variety NC95 containing recessive alleles at the Nic1 and Nic2 loci, and K326 nic1 / nic2 - a near-isogenic version of iron-cured tobacco variety K326 containing recessive alleles at the Nic1 and Nic2 loci. The alkaloid profiles for the above materials evaluated at the three NC positions are shown in Figures 1A-1D. Yield and quality measurements of these materials are shown in Figures 2A-2D. The Cms NCLA hybrid produced the lowest nicotine levels of all materials in the experiment. Nornicotine levels for the Cms NCLA hybrid were similar to, but slightly lower than, those for K326. Anatabine and anabasine levels of the new hybrids were below those for K326.

[0158] Data from the additional experiments are shown below in Table 1. Alkaloid data highlighted in bold type are approximately half of the lowest level of detection for the analytical run, as numbers were below the lower limit of detection. (This was done to allow for analysis of variance.) [Table 1-1] [Table 1-2] [Table 1-3]

[0159] Example 2. Targeted mutagenesis using CRISPR / Cas9 to control nicotine biosynthesis in Nicotiana. This example demonstrates the use of CRISPR / Cas9 to reduce nicotine biosynthesis in Nicotiana plants by combining (A) one or more genetic modifications that increase the activity of CYP82E2 and / or CYP82E3 in the plant with (B) a recessive allele of nic1 and / or a recessive allele of nic2.

[0160] Methods. The nic1 and / or nic2 recessive alleles can be derived from one of the following tobacco plant lines: LAFC53 (nic1 / nic1), MAFC5 (Nic1 / Nic1 nic2 / nic2), LMAFC34 (nic1 / nic1 Nic2 / Nic2), LABurley21 (nic1 / nic1 nic2 / nic2), LI Burley 21 (nic1 / nic1 Nic2 / Nic2), or HI Burley 21 (Nic1 / Nic1 nic2 / nic2), or any tobacco plant line having a genome with integrated recessive alleles at the Nic1 and / or Nic2 loci.

[0161] To introduce the K375E and / or L422W point mutations into the CYP82E2 protein of tobacco plants and / or the C330W point mutation into the CYP82E3 protein, Cas9 and sgRNA vectors are prepared using standard methods well known in the art.See, for example, Schiml et al., Methods in Molecular Biology, 1469:111-122 (2016).Using sequence analysis software, the sgRNA targeting sequence of interest can be determined to be immediately 5' to the protospacer adjacent motif (PAM) sequence, which matches the standard form 5'-NGG.

[0162] A repair template containing donor DNA containing the desired mutation (e.g., K375E and / or L422W point mutations compared to the wild-type CYP82E2 amino acid sequence (e.g., SEQ ID NO: 1), and / or C330W point mutations compared to the wild-type CYP82E3 amino acid sequence (e.g., SEQ ID NO: 5)) in the form of either a single-stranded DNA donor oligo or a DNA donor plasmid is prepared according to methods well known in the art. See, e.g., Ran et al., Nature Protocols, 8(11):2281-2308(2013) and Lu et al., “Targeted, efficient sequence insertion and replacement in rice,” Nature Biotechnology, 38 (12):1402-1407.doi: 10.1038 / s41587-020-0581-5, (2020). The vector containing Cas9 and sgRNA and donor oligo or plasmid is transformed into Agrobacterium tumefaciens. Stable Agrobacterium-mediated transformation into one of the following strains is performed: MAFC5, LMAFC34, LA Burley 21, LI Burley 21, or HI Burley 21 (e.g., by floral dip transformation or agroinfiltration). 10-14 days after transformation, DNA samples are extracted from the plants and assayed for mutagenesis events. These CRISPR / Cas-induced mutations can be identified, for example, by PCR / restriction enzyme assays, Surveyor nuclease assays, and / or sequencing.

[0163] Results: Transgenic Nicotiana plants containing nic1 and / or nic2 recessive alleles and the K375E and / or L422W point mutations in the CYP82E2 protein and / or the C330W point mutation in the CYP82E3 protein of tobacco plants are expected to have reduced accumulation of nicotinic alkaloids, such as nicotine, nornicotine, anatabine, and / or anabasine, compared to corresponding naturally occurring or non-transformed control tobacco plants.

[0164] Thus, these results indicate that the methods of the present technology are useful for producing tobacco plants with reduced nicotinic alkaloid content that combine one or more genetic modifications that increase the activity of CYP82E2 and / or CYP82E3 with a recessive allele of nic1 and / or a recessive allele of nic2.

[0165] biological deposit Reproduction material of breeding strains SC58 CsCs and SC58 CtCt, respectively, will be deposited at the Crop and Soil Science Department, North Carolina State University, Raleigh, NC 27695. SC58 CsCs, identified as LA K326 CsCs, will be deposited under accession number GH21-190-17. SC58 CtCt, identified as LA K326 CtCt, will be deposited under accession number GH21-191-13. Third party experts previously certified as such by NCSU may obtain samples of biological material for either strain by written request to the Director of Office of Research Commercialization, North Carolina State University, Poulton Innovation Center, 2nd Floor, 1021 Main Campus Drive, Raleigh, NC 27606.

[0166] Except as permitted by 37 C.F.R. §1.808(b), all restrictions imposed on the availability to the public of the deposited biological material will be irreversibly removed upon the grant of a patent from this application or any application incorporating this for priority.

Claims

1. A tobacco variety, or any part thereof, that contains a reduced level of at least one nicotinic alkaloid compared to a naturally occurring corresponding tobacco plant, or part thereof.

2. The tobacco variety of claim 1 , wherein the variety is non-transgenic.

3. 3. The tobacco variety of claim 1 or claim 2, wherein the at least one nicotinic alkaloid is selected from the group consisting of nicotine, nornicotine, anatabine, and anabasine.

4. 4. The tobacco variety according to claim 1, wherein the at least one nicotinic alkaloid is nicotine, and the variety contains no more than 0.35% nicotine.

5. 5. The tobacco variety of claim 1, wherein the at least one nicotinic alkaloid is nornicotine, and the variety contains no more than 0.04% nornicotine.

6. 6. The tobacco variety of claim 1, wherein the at least one nicotinic alkaloid is anatabine, and the variety contains no more than 0.06% anatabine.

7. 7. The tobacco variety of claim 1, wherein the at least one nicotinic alkaloid is anabasine, and the variety contains no more than 0.008% anabasine.

8. 3. The tobacco variety of claim 1 or claim 2, wherein the at least one nicotinic alkaloid is nicotine and nornicotine, and the variety contains no more than 0.35% nicotine and no more than 0.04% nornicotine.

9. The tobacco variety according to any one of claims 1 to 8, wherein the variety comprises a nic1 allele that has reduced expression and / or function compared to wild-type nic1.

10. 10. The tobacco variety of claim 1 , wherein the variety comprises a nic2 allele that has reduced expression and / or function compared to wild-type nic2.

11. 11. The tobacco variety of claim 9 or claim 10, wherein the nic1 and / or the nic2 allele is derived from at least one of the following strains: LAFC53, MAFC5, LMAFC34, LA Burley 21, LI Burley 21, and HI Burley 21

12. The tobacco variety of any one of claims 1 to 11, wherein the variety comprises a functional CYP82E2 allele.

13. 13. The tobacco variety of claim 12, wherein the CYP82E2 allele is derived from N. sylvestris.

14. The tobacco variety of any one of claims 1 to 13, wherein the variety comprises a functional CYP82E3 allele.

15. 15. The tobacco variety of claim 14, wherein the CYP82E3 allele is derived from N. tomentosiformis.

16. 16. The tobacco variety of claim 12, wherein the nicotine N-demethylation activity of the proteins encoded by the CYP82E2 and / or CYP82E3 alleles is increased compared to a naturally occurring tobacco plant.

17. A progeny plant, seed, or cell produced from any of the tobacco varieties according to claims 1-16.

18. A tobacco product derived from a tobacco variety according to claims 1 to 16, or any of their parts.

19. 19. The tobacco product of claim 18, wherein the 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, cigars, cigarillos, cigarettes, and chewing tobacco.

20. 20. The tobacco product of claim 18, wherein the product is selected from the group consisting of cigarillos, krettic cigarettes, non-breathable recessed filter cigarettes, breathable recessed filter cigarettes, cigars, snuff, tobacco-containing gums, tobacco-containing lozenges, and chewing tobacco.

21. 1. A method for producing a tobacco variety that contains a reduced level of at least one nicotinic alkaloid compared to a naturally occurring corresponding tobacco plant, or part thereof, said method comprising: crossing a first tobacco variety including a first low-nicotine trait with a second tobacco variety including a second low-nicotine trait to produce a progeny plant; The progeny plant comprises a reduced level of at least one nicotinic alkaloid.

22. 22. The method of claim 21 , wherein the method comprises backcrossing.

23. 23. The method of claim 21 or 22, wherein the at least one nicotinic alkaloid is selected from the group consisting of nicotine, nornicotine, anatabine, and anabasine.

24. 24. The method of any one of claims 21 to 23, wherein the at least one nicotinic alkaloid is nicotine and the variety contains 0.35% or less nicotine.

25. 25. The method of any one of claims 21 to 24, wherein the at least one nicotinic alkaloid is nornicotine and the variety contains no more than 0.04% nornicotine.

26. 26. The method of any one of claims 21 to 25, wherein the at least one nicotinic alkaloid is anatabine and the variety contains no more than 0.06% anatabine.

27. 27. The method of any one of claims 21 to 26, wherein the at least one nicotinic alkaloid is anabasine and the variety contains no more than 0.008% anabasine.

28. 28. The method of any one of claims 21 to 27, wherein the at least one nicotinic alkaloid is nicotine and nornicotine, and the variety contains no more than 0.35% nicotine and no more than 0.04% nornicotine.

29. 29. The method of any one of claims 21 to 28, wherein the first and / or second low nicotine trait comprises a nic1 allele that has reduced expression and / or function compared to wild-type nic1.

30. 30. The method of any one of claims 21 to 29, wherein the first and / or second low nicotine trait comprises a nic2 allele that has reduced expression and / or function compared to wild-type nic2.

31. 31. The method of any one of claims 21 to 30, wherein the first and / or second tobacco variety comprises a nic1 and / or nic2 allele derived from at least one of the following strains: LAFC53, MAFC5, LMAFC34, LA Burley 21, LI Burley 21, and HI Burley 21

32. 32. The method of any one of claims 21 to 31, wherein the first and / or second tobacco variety comprises a functional CYP82E2 allele.

33. 33. The method of claim 32, wherein the CYP82E2 allele is derived from N. sylvestris.

34. 34. The method of any one of claims 21 to 33, wherein the first and / or second tobacco variety comprises a functional CYP82E3 allele.

35. 35. The method of claim 34, wherein the CYP82E3 allele is derived from N. tomentosiformis.

36. 36. The method of any one of claims 32 to 35, wherein the nicotine N-demethylation activity of the proteins encoded by the CYP82E2 and / or CYP82E3 alleles is increased compared to naturally occurring tobacco plants.

37. 37. The method of any one of claims 21 to 36, wherein the first low nicotine trait comprises a nic1 and / or nic2 allele that has reduced expression and / or function compared to wild-type nic1 and / or nic2, and the second low nicotine trait comprises a functional CYP82E2 allele and / or a functional CYP82E3 allele.

38. 38. The method of any one of claims 21 to 37, wherein the first tobacco variety, the second tobacco variety, and the progeny plant are non-transgenic.

39. 22. A seed or cell obtained from a progeny plant produced according to the method of claim 21.

40. 22. A tobacco product derived from a progeny plant produced according to the method of claim 21.

41. 41. The tobacco product of claim 40, wherein the 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, cigars, cigarillos, cigarettes, and chewing tobacco.

42. 41. The tobacco product of claim 40, wherein the product is selected from the group consisting of cigarillos, krettic cigarettes, non-breathable recessed filter cigarettes, breathable recessed filter cigarettes, cigars, snuff, tobacco-containing gums, tobacco-containing lozenges, and chewing tobacco.

43. 14. The tobacco variety of claim 12 or 13, wherein the CYP82E2 allele comprises a nucleotide sequence encoding a K375E and / or a L422W point mutation compared to the wild-type CYP82E2 amino acid sequence set forth in SEQ ID NO:

1.

44. (a) the CYP82E2 allele comprises a nucleotide sequence encoding a CYP82E2 protein comprising the amino acid sequence set forth in SEQ ID NO:2; (b) the CYP82E2 allele comprises a nucleotide sequence encoding a CYP82E2 protein comprising the amino acid sequence set forth in SEQ ID NO:3; or (c) the CYP82E2 allele comprises a nucleotide sequence encoding a CYP82E2 protein comprising the amino acid sequence set forth in SEQ ID NO:4; 44. The tobacco variety of claim 43.

45. 16. The tobacco variety of claim 14 or 15, wherein the CYP82E3 allele comprises a nucleotide sequence encoding a C330W point mutation compared to the wild-type CYP82E3 amino acid sequence set forth in SEQ ID NO:

5.

46. 46. ​​The tobacco variety of claim 45, wherein the CYP82E3 allele comprises a nucleotide sequence encoding a CYP82E3 protein comprising the amino acid sequence set forth in SEQ ID NO:

6.

47. 47. The tobacco variety of any one of claims 43-46, wherein the variety further comprises suppression of expression in at least one of the following: NBB1, A622, quinolate phosphoribosyltransferase (QPT), putrescine N-methyltransferase (PMT), ornithine decarboxylase (ODC), aspartate oxidase (AO), quinolinate synthase (QS), N-methylputrescine oxidase (MPO), NtERF221, NtMYC1a, NtMYC1b, NtMYC2a, or NtMYC2b.

48. 34. The method of claim 32 or 33, wherein the CYP82E2 allele comprises a nucleotide sequence encoding a K375E and / or a L422W point mutation compared to the wild-type CYP82E2 amino acid sequence set forth in SEQ ID NO:

1.

49. (a) the CYP82E2 allele comprises a nucleotide sequence encoding a CYP82E2 protein comprising the amino acid sequence set forth in SEQ ID NO:2; (b) the CYP82E2 allele comprises a nucleotide sequence encoding a CYP82E2 protein comprising the amino acid sequence set forth in SEQ ID NO:3; or (c) the CYP82E2 allele comprises a nucleotide sequence encoding a CYP82E2 protein comprising the amino acid sequence set forth in SEQ ID NO:4; 49. The method of claim 48.

50. 36. The method of claim 34 or 35, wherein the CYP82E3 allele comprises a nucleotide sequence that encodes a C330W point mutation compared to the wild-type CYP82E3 amino acid sequence set forth in SEQ ID NO:

5.

51. 51. The method of claim 50, wherein the CYP82E3 allele comprises a nucleotide sequence encoding a CYP82E3 protein comprising the amino acid sequence set forth in SEQ ID NO:

6.

52. 1. A method for creating a point mutation in a target gene in a Nicotiana tabacum plant cell, the method comprising introducing into the cell a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (CRISPR-Cas) system, (a) at least one guide RNA (gRNA), wherein the at least one gRNA comprises a nucleotide sequence capable of hybridizing to a target sequence or a portion thereof in the target gene, the target gene comprising a nucleotide sequence encoding a CYP82E2 protein; (b) (i) a DNA fragment, the DNA fragment replacing a target sequence in a target gene or being inserted at a target site in the target gene; and (ii) two homologous arms, each flanking opposite sides of the DNA fragment; A nucleic acid comprising a donor DNA sequence comprising: (c) an effector protein or one or more polynucleotides encoding said effector protein, said at least one gRNA forms a complex with said effector protein; The method, wherein the effector protein is a Cas protein that comprises a nuclease.

53. (a) the point mutation is a K375E point mutation compared to the wild-type CYP82E2 amino acid sequence set forth in SEQ ID NO:1; or (b) the point mutation is a L422WE point mutation compared to the wild-type CYP82E2 amino acid sequence set forth in SEQ ID NO:1; 53. The method of claim 52.

54. 53. The method of claim 52, wherein at least two point mutations are made.

55. 55. The method of claim 54, wherein the at least two point mutations include a K375E point mutation compared to the wild-type CYP82E2 amino acid sequence set forth in SEQ ID NO:1, and a L422W point mutation compared to the wild-type CYP82E2 amino acid sequence set forth in SEQ ID NO:

1.

56. 56. The method of any one of claims 52 to 55, wherein the Nicotiana tabacum plant further comprises a recessive allele of nic1, and / or a recessive allele of nic2.

57. 57. The method of any one of claims 52-56, further comprising suppressing expression in the Nicotiana tabacum plant of at least one of NBB1, A622, quinolate phosphoribosyltransferase (QPT), putrescine N-methyltransferase (PMT), ornithine decarboxylase (ODC), aspartate oxidase (AO), quinolinate synthase (QS), N-methylputrescine oxidase (MPO), NtERF221, NtMYC1a, NtMYC1b, NtMYC2a, or NtMYC2b.

58. 1. A method for creating a point mutation in a target gene in a Nicotiana tabacum plant cell, the method comprising introducing into the cell a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (CRISPR-Cas) system, (a) at least one guide RNA (gRNA), wherein the at least one gRNA comprises a nucleotide sequence capable of hybridizing to a target sequence or a portion thereof in the target gene, the target gene comprising a nucleotide sequence encoding a CYP82E3 protein; (b) (i) a DNA fragment, the DNA fragment replacing a target sequence in a target gene or being inserted at a target site in the target gene; and (ii) two homologous arms, each flanking opposite sides of the DNA fragment; A nucleic acid comprising a donor DNA sequence comprising: (c) an effector protein or one or more polynucleotides encoding said effector protein, said at least one gRNA forms a complex with said effector protein; The method, wherein the effector protein is a Cas protein that comprises a nuclease.

59. 59. The method of claim 58, wherein the point mutation is a C330W point mutation compared to the wild-type CYP82E3 amino acid sequence set forth in SEQ ID NO:

5.

60. 60. The method of claim 58 or claim 59, wherein the Nicotiana tabacum plant further comprises a recessive allele of nic1 and / or a recessive allele of nic2.

61. 61. The method of any one of claims 58-60, further comprising suppressing expression in the Nicotiana tabacum plant of at least one of NBB1, A622, quinolate phosphoribosyltransferase (QPT), putrescine N-methyltransferase (PMT), ornithine decarboxylase (ODC), aspartate oxidase (AO), quinolinate synthase (QS), N-methylputrescine oxidase (MPO), NtERF221, NtMYC1a, NtMYC1b, NtMYC2a, or NtMYC2b.

62. 1. A method for producing a Nicotiana tabacum plant having reduced nicotinic alkaloid content, comprising: (a) one or more genetic modifications that increase the activity of CYP82E2 and / or CYP82E3; and (b) a recessive allele of nic1 and / or a recessive allele of nic2; [0033] The method, wherein the Nicotiana tabacum plant has reduced nicotinic alkaloid content as compared to a corresponding naturally occurring or non-transformed control tobacco plant.

63. 63. The method of claim 62, wherein the Nicotiana tabacum plant comprises a homozygous recessive allele of nic1 and / or a homozygous recessive allele of nic2.

64. The method of claim 62 or 63, wherein the one or more genetic modifications that increase the activity of CYP82E2 and / or CYP82E3 are introduced by transcription activator-like effector nucleases (TALENs), meganucleases, zinc finger nucleases, CRISPR / Cas9 systems, CRISPR / Cpf1 systems, CRISPR / Csm1 systems, gene knock-in technology or scientific techniques, and any combination thereof.

65. 65. The method of any one of claims 62-64, wherein the one or more genetic modifications that increase the activity of CYP82E2 comprise a point mutation in a target gene of the Nicotiana tabacum plant cell, the point mutation being selected from one or more of a K375E point mutation compared to the wild-type CYP82E2 amino acid sequence set forth in SEQ ID NO:1, and a L422W point mutation compared to the wild-type CYP82E2 amino acid sequence set forth in SEQ ID NO:

1.

66. 65. The method of any one of claims 62-64, wherein the one or more genetic modifications that increase the activity of CYP82E3 comprise a point mutation in a target gene of the Nicotiana tabacum plant cell, the point mutation being a C330W point mutation compared to the wild-type CYP82E3 amino acid sequence set forth in SEQ ID NO:

5.

67. The point mutation is (a) at least one guide RNA (gRNA), wherein the at least one gRNA comprises a nucleotide sequence capable of hybridizing to a target sequence or a portion thereof in the target gene, the target gene comprising a nucleotide sequence encoding a CYP82E2 protein; (b) (i) a DNA fragment, the DNA fragment replacing a target sequence in a target gene or being inserted at a target site in the target gene; and (ii) two homologous arms, each flanking opposite sides of the DNA fragment; A nucleic acid comprising a donor DNA sequence comprising: (c) an effector protein or one or more polynucleotides encoding said effector protein; The plant is introduced into the plant by a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (CRISPR-Cas) system comprising: said at least one gRNA forms a complex with said effector protein; The effector protein is a Cas protein that includes a nuclease.

67. The method of claim 65 or 66.

68. 68. The method of any one of claims 62-67, further comprising suppressing expression in the Nicotiana tabacum plant of at least one of NBB1, A622, quinolate phosphoribosyltransferase (QPT), putrescine N-methyltransferase (PMT), ornithine decarboxylase (ODC), aspartate oxidase (AO), quinolinate synthase (QS), N-methylputrescine oxidase (MPO), NtERF221, NtMYC1a, NtMYC1b, NtMYC2a, or NtMYC2b.

69. 69. A Nicotiana tabacum plant produced by the method of any one of claims 62-68, the plant comprising: (A) one or more genetic modifications that increase activity of CYP82E2 and / or CYP82E3; and (B) a recessive allele of nic1, and / or a recessive allele of nic2.

70. 70. A progeny plant or seed produced from the plant of claim 69, wherein the progeny plant or seed comprises: (A) one or more genetic modifications that increase activity of CYP82E2 and / or CYP82E3; and (B) a recessive allele of nic1, and / or a recessive allele of nic2.

71. 70. A tobacco product comprising tobacco derived from the Nicotiana tabacum plant of claim 69, the plant comprising: (A) one or more genetic modifications that increase activity of CYP82E2 and / or CYP82E3; and (B) a recessive allele of nic1, and / or a recessive allele of nic2.

72. (a) 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; or (b) the product is a reduced-nicotine tobacco product selected from the group consisting of cigarillos, kretique cigarettes, non-breathable recessed filter cigarettes, breathable recessed filter cigarettes, cigars, snuff, snus, tobacco-containing gums, tobacco-containing lozenges, and chewing tobacco; 72. The tobacco product of claim 71.

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