Methods and compositions for producing tobacco plants with reduced levels of nicotinic alkaloids

Abstract

The present disclosure provides compositions and methods related to tobacco plants. In particular, the present disclosure provides novel methods for producing tobacco plants and any related tobacco product having low nicotine alkaloid content. Tobacco plants produced according to the methods of the present disclosure exhibit reduced levels of nicotine alkaloids (e.g., nicotine) as compared to naturally occurring and transgenic tobacco plants, and thus represent commercial value substitutes for currently available tobacco varieties.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 317,272, filed on March 7, 2022, which is incorporated herein by reference in its entirety for all purposes.

[0003] Electronically Submitted Materials Incorporated by Reference

[0004] Incorporated herein by reference in its entirety is a computer-readable nucleotide / amino acid sequence listing filed concurrently with this document, and the sequence listing is identified as follows: a 9,320-byte ASCII (text) file named "NCSU-40676.601" created on February 14, 2023. Technical Field

[0005] The disclosure provides compositions and methods relevant to tobacco plants. Specifically, the disclosure provides novel methods for producing tobacco plants and any related tobacco products with low nicotinic alkaloid content. Compared with naturally occurring and genetically modified tobacco plants, the tobacco plants produced according to the disclosed method show the level of nicotinic alkaloids (e.g., nicotine) that reduces, and therefore represent substitutes with commercial value of currently available tobacco varieties. Background Art

[0006] Nicotine is generally the most abundant pyridine alkaloid produced by tobacco (Nicotiana tabacum L), although nornicotine can predominate in some plants due to increased activity of genes encoding nicotine demethylases (Sisson and Saunders, 1982; Lewis et al., 2010). Because nicotine contributes to the addictiveness of cigarettes, some public health agencies have recommended that research that could mandate reductions in nicotine levels in these products be conducted to reduce human exposure to tobacco smoke-related toxicants (U.S. Food and Drug Administration, 2018). The World Health Organization (WHO) recommends reducing nicotine levels in cigarette tobacco to 0.4 mg g / d. -1 At or below the level reported as non-addictive (WHO, 201 5). This represents a reduction of approximately 95% compared to the levels currently present in cigarette tobacco. There are no commercially available tobacco cultivars that routinely exhibit such ultra-low leaf nicotine accumulation levels (Lewis, 2018). Therefore, some people in the tobacco community are interested in developing new cultivars with nicotine levels below the proposed tolerance threshold level. Summary of the invention

[0007] Embodiments of the present disclosure include tobacco cultivars, or any part thereof, comprising reduced levels of at least one nicotinic alkaloid as compared to a corresponding naturally occurring tobacco plant or part thereof.

[0008] In some embodiments, the cultivar is non-transgenic.

[0009] In some embodiments, at least one nicotinic alkaloid is selected from the group consisting of nicotine, nornicotine, anatabine and anabasine. In some embodiments, at least one nicotinic alkaloid is nicotine, and wherein the cultivar contains no more than 0.35% nicotine. In some embodiments, at least one nicotinic alkaloid is nornicotine, and wherein the cultivar contains no more than 0.04% nornicotine. In some embodiments, at least one nicotinic alkaloid is anatabine, and wherein the cultivar contains no more than 0.06% anatabine. In some embodiments, at least one nicotinic alkaloid is anabasine, and wherein the cultivar contains no more than 0.008% anabasine. In some embodiments, at least one nicotinic alkaloid is nicotine and nornicotine, and wherein the cultivar contains no more than 0.35% nicotine and no more than 0.04% nornicotine.

[0010] In some embodiments, the cultivar comprises a nic1 allele having reduced expression and / or function compared to wild-type nic1. In some embodiments, the cultivar comprises a nic2 allele having reduced expression and / or function compared to wild-type nic2. In some embodiments, the nic1 allele and / or the nic2 allele are derived from at least one of the following lines: LAFC53, MAFC5, LMAFC34, LA Burley 21, LI Burley21, and HI Burley 21.

[0011] In some embodiments, cultivars include functional CYP82E2 alleles. In some embodiments, CYP82E2 alleles are derived from forest tobacco (N.sylvestris). In some embodiments, cultivars include functional CYP82E3 alleles. In some embodiments, CYP82E3 alleles are derived from fluffy tobacco (N.tomentosiformis) (or related species, such as from Nicotiana Section Tomentosae). In some embodiments, compared with naturally occurring tobacco plants, the nicotine N-demethylation activity of proteins encoded by CYP82E2 alleles and / or CYP82E3 alleles increases.

[0012] Embodiments of the present disclosure also include progeny plants, seeds, or cells produced from any of the tobacco cultivars described herein.

[0013] Embodiments of the present disclosure also include tobacco products derived from any of the tobacco cultivars described herein, or portions thereof.

[0014] In some embodiments, the tobacco product is selected from the group consisting of leaf tobacco, shredded tobacco, cut tobacco, tobacco dust, tobacco powder, tobacco extract, smokeless tobacco, moist or dry snuff, pipe tobacco, cigar tobacco, cigarillo tobacco, cigarette tobacco, and chewing tobacco.

[0015] In some embodiments, the tobacco product is selected from the group consisting of cigarillos, clove cigarettes, unventilated recessed filter cigarettes, ventilated recessed filter cigarettes, cigars, snuff, tobacco-containing gum, tobacco-containing lozenges, and chewing tobacco.

[0016] Embodiments of the present disclosure also include methods for producing tobacco cultivars, the tobacco cultivars comprising at least one nicotinic alkaloid at a reduced level compared to a corresponding naturally occurring tobacco plant or part thereof. According to these embodiments, the method comprises hybridizing a first tobacco variety comprising a first low nicotine trait with a second tobacco variety comprising a second low nicotine trait to produce a progeny plant. In some embodiments, the progeny plant comprises at least one nicotinic alkaloid at a reduced concentration.

[0017] In some embodiments, the method comprises backcrossing.

[0018] In some embodiments, at least one nicotinic alkaloid is selected from the group consisting of nicotine, nornicotine, anatabine and anabasine. In some embodiments, at least one nicotinic alkaloid is nicotine, and wherein the cultivar contains no more than 0.35% nicotine. In some embodiments, at least one nicotinic alkaloid is nornicotine, and wherein the cultivar contains no more than 0.04% nornicotine. In some embodiments, at least one nicotinic alkaloid is anatabine, and wherein the cultivar contains no more than 0.06% anatabine. In some embodiments, at least one nicotinic alkaloid is anabasine, and wherein the cultivar contains no more than 0.008% anabasine. In some embodiments, at least one nicotinic alkaloid is nicotine and nornicotine, and wherein the cultivar contains no more than 0.35% nicotine and no more than 0.04% nornicotine.

[0019] In some embodiments, the first low nicotine trait and / or the second low nicotine trait include nic1 alleles with reduced expression and / or function compared to wild type nic1. In some embodiments, the first low nicotine trait and / or the second low nicotine trait include nic2 alleles with reduced expression and / or function compared to wild type nic2. In some embodiments, the first tobacco variety and / or the second tobacco variety include nic1 alleles and / or nic2 alleles derived from at least one of the following strains: MAFC5, LMAFC34, LA Burley 21, LI Burley 21 and HI Burley21. In some embodiments, the first tobacco variety and / or the second tobacco variety include nic1 and / or nic2 invalid alleles (deletions). In some embodiments, nic1 and / or nic2 invalid alleles are derived from naturally occurring tobacco (Ntabacum) germplasm.

[0020] In some embodiments, the first tobacco variety and / or the second tobacco variety comprise functional CYP82E2 alleles.In some embodiments, CYP82E2 alleles are derived from forest tobacco.In some embodiments, the first tobacco variety and / or the second tobacco variety comprise functional CYP82E3 alleles.In some embodiments, CYP82E3 alleles are derived from fluffy tobacco (or related species, for example Nicotiana fluffy tobacco group).In some embodiments, compared with naturally occurring tobacco plants, the nicotine N-demethylation activity of the protein encoded by CYP82E2 alleles and / or CYP82E3 alleles increases.

[0021] In some embodiments, the first low nicotine trait comprises a nic1 allele and / or a nic2 allele having reduced expression and / or function compared to wild type nic1 and / or nic2, and wherein the second low nicotine trait comprises a functional CYP82E2 allele and / or a functional CYP82E3 allele.

[0022] In some embodiments, the first tobacco variety, the second tobacco variety, and the progeny plants are non-transgenic.

[0023] Embodiments of the present disclosure also include seeds or cells obtained from a progeny plant produced according to any of the methods described herein.

[0024] Embodiments of the present disclosure also include tobacco products derived from progeny plants produced according to any of the methods described herein.

[0025] In some embodiments, the product is selected from the group consisting of leaf tobacco, shredded tobacco, raw cut tobacco, tobacco dust, tobacco powder, tobacco extract, smokeless tobacco, moist or dry snuff, pipe tobacco, cigar tobacco, cigarillo tobacco, cigarette tobacco, and chewing tobacco.

[0026] In some embodiments, the product is selected from the group consisting of cigarillos, clove cigarettes, unventilated recessed filter cigarettes, ventilated recessed filter cigarettes, cigars, snuff, tobacco-containing gum, tobacco-containing lozenges, and chewing tobacco.

[0027] In some embodiments, the cultivar comprises a CYP82E2 allele comprising a nucleotide sequence encoding a K375E point mutation and / or a L422W point mutation relative 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 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.

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

[0029] In some embodiments, the cultivar comprises a CYP82E2 allele comprising a nucleotide sequence encoding a K375E point mutation and / or a L422W point mutation relative 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 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.

[0030] In some embodiments, the CYP82E3 allele comprises a nucleotide sequence encoding a C330W point mutation relative to the wild-type CYP82E3 amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the CYP82E3 allele comprises a nucleotide sequence encoding a CYP82E3 protein comprising the amino acid sequence set forth in SEQ ID NO: 6.

[0031] In some embodiments, the cultivated variety further includes inhibition of expression of at least one of the following within the cultivated variety: NBB1, A622, quinolinate 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.

[0032] Embodiments of the present disclosure also include a method for generating point mutations in a target gene in a tobacco plant cell. According to these embodiments, the method includes introducing into the cell a clustered regularly spaced short palindromic repeat (CRISPR)-related (CRISPR-Cas) system, the system including: (a) at least one guide RNA (gRNA), wherein the at least one gRNA comprises a nucleotide sequence capable of hybridizing with a target sequence or a portion thereof in a target gene, wherein the target gene comprises a nucleotide sequence encoding a CYP82E2 protein; (b) a nucleic acid comprising a donor DNA sequence, the donor DNA sequence comprising: (i) a DNA fragment, wherein the DNA fragment replaces the target sequence in the target gene or inserts into a target site in the target gene; and (ii) two homologous arms, each arm being flanked on opposite sides of the DNA fragment; and (c) an effector protein, or one or more polynucleotides encoding an effector protein. In some embodiments, at least one gRNA forms a complex with the effector protein. In some embodiments, the effector protein is a Cas protein comprising a nuclease.

[0033] In some embodiments, (a) the point mutation is a K375E point mutation relative to the wild-type CYP82E2 amino acid sequence as shown in SEQ ID NO: 1; or (b) the point mutation is a L422W point mutation relative to the wild-type CYP82E2 amino acid sequence as shown in SEQ ID NO: 1.

[0034] In some embodiments, at least two point mutations are generated. In some embodiments, the at least two point mutations include a K375E point mutation relative to the wild-type CYP82E2 amino acid sequence shown in SEQ ID NO: 1 and a L422W point mutation relative to the wild-type CYP82E2 amino acid sequence shown in SEQ ID NO: 1.

[0035] In some embodiments, the tobacco plant further comprises a recessive allele of nic1 and / or a recessive allele of nic2.

[0036] In some embodiments, the method further comprises inhibiting expression of at least one of the following in the cultivar: NBB1, A622, quinolinate 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.

[0037] Embodiments of the present disclosure also include a method for generating point mutations in a target gene in a tobacco plant cell. According to these embodiments, the method includes introducing into the cell a clustered regularly spaced short palindromic repeat (CRISPR)-related (CRISPR-Cas) system, the system including: (a) at least one guide RNA (gRNA), wherein the at least one gRNA comprises a nucleotide sequence capable of hybridizing with a target sequence or a portion thereof in a target gene, wherein the target gene comprises a nucleotide sequence encoding a CYP82E3 protein; (b) a nucleic acid comprising a donor DNA sequence, the donor DNA sequence comprising: (i) a DNA fragment, wherein the DNA fragment replaces the target sequence in the target gene or inserts into a target site in the target gene; and (ii) two homologous arms, each arm being flanked on opposite sides of the DNA fragment; and (c) an effector protein, or one or more polynucleotides encoding an effector protein. In some embodiments, at least one gRNA forms a complex with the effector protein. In some embodiments, the effector protein is a Cas protein comprising a nuclease.

[0038] In some embodiments, the point mutation is a C330W point mutation relative to the wild-type CYP82E3 amino acid sequence as shown in SEQ ID NO:5.

[0039] In some embodiments, the tobacco plant further comprises a recessive allele of nic1 and / or a recessive allele of nic2.

[0040] In some embodiments, the method further comprises inhibiting the expression of at least one of the following in the tobacco plant: NBB1, A622, quinolinate 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.

[0041] Embodiments of the present disclosure also include methods for producing tobacco plants with reduced nicotinic alkaloid content. According to these embodiments, the method includes combining in tobacco plants: (a) one or more genetic modifications that increase CYP82E2 and / or CYP82E3 activity; and (b) recessive alleles of nic1 and / or recessive alleles of nic2. In some embodiments, compared with corresponding naturally occurring or non-converted control tobacco plants, the tobacco plants have reduced nicotinic alkaloid content.

[0042] In some embodiments, the tobacco plant comprises a homozygous recessive allele for nic1 and / or a homozygous recessive allele for nic2.

[0043] In some embodiments, one or more genetic modifications that increase CYP82E2 and / or CYP82E3 activity 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 techniques or technologies, and any combination thereof.

[0044] In some embodiments, the one or more genetic modifications that increase CYP82E2 activity include a point mutation of a target gene in a tobacco plant cell, wherein the point mutation is selected from one or more of a K375E point mutation relative to the wild-type CYP82E2 amino acid sequence as shown in SEQ ID NO: 1 and a L422W point mutation relative to the wild-type CYP82E2 amino acid sequence as shown in SEQ ID NO: 1.

[0045] In some embodiments, the one or more genetic modifications that increase CYP82E3 activity include a point mutation in a target gene in a tobacco plant cell, wherein the point mutation is a C330W point mutation relative to the wild-type CYP82E3 amino acid sequence as shown in SEQ ID NO:5.

[0046] In some embodiments, point mutations are introduced into plants by a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (CRISPR-Cas) system, the system comprising: (a) at least one guide RNA (gRNA), wherein the at least one gRNA comprises a nucleotide sequence capable of hybridizing with a target sequence in a target gene or a portion thereof, wherein the target gene comprises a nucleotide sequence encoding a CYP82E2 protein; (b) a nucleic acid comprising a donor DNA sequence, the donor DNA sequence comprising: (i) a DNA fragment, wherein the DNA fragment replaces the target sequence in the target gene or is inserted into a target site in the target gene; and (ii) two homology arms, each arm flanking the opposite side of the DNA fragment; and (c) an effector protein, or one or more polynucleotides encoding an effector protein. In some embodiments, at least one gRNA forms a complex with the effector protein. In some embodiments, the effector protein is a Cas protein comprising a nuclease.

[0047] Embodiments of the present disclosure also include tobacco plants produced by any of the methods described herein, wherein the plant comprises: (a) one or more genetic modifications that increase CYP82E2 and / or CYP82E3 activity; and (b) recessive alleles of nic1 and / or recessive alleles of nic2. Embodiments of the present disclosure also include offspring plants or seeds produced by the plant, wherein the offspring plants or seeds include: (A) one or more genetic modifications that increase CYP82E2 and / or CYP82E3 activity; and (B) recessive alleles of nic1 and / or recessive alleles of nic2. Embodiments of the present disclosure also include tobacco products comprising tobacco from tobacco plants, wherein the plant comprises: (A) one or more genetic modifications that increase CYP82E2 and / or CYP82E3 activity; and (B) recessive alleles of nic1 and / or recessive alleles of nic2.

[0048] According to these embodiments, (a) the tobacco is selected from the group consisting of leaf tobacco, shredded tobacco, raw cut tobacco, tobacco dust, tobacco powder, tobacco extracts, smokeless tobacco, moist or dry snuff, pipe tobacco, cigar tobacco, cigarillo tobacco, cigarette tobacco, and chewing tobacco; or (b) the product is a reduced-nicotine tobacco product selected from the group consisting of cigarillos, clove cigarettes, unventilated recessed-filter cigarettes, ventilated recessed-filter cigarettes, cigars, snus, snus, tobacco-containing gum, tobacco-containing lozenges, and chewing tobacco. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figures 1A to 1D: Representative graphical data of alkaloid composition of low alkaloid hybrid lines tested (Nic1 and Nic2 loci derived from LAFC53 in combination with CYP82E3 alleles derived from Nicotiana villosa) and related controls according to one embodiment of the present disclosure. Figure 1A includes nicotine percentage; Figure 1B includes nornicotine percentage; Figure 1C includes anatabine percentage; Figure 1D includes anabasine percentage. Averages are averaged for three field environments.

[0050] Figures 2A to 2D: Representative graphical data of yield and quality determination of low alkaloid hybrid lines tested (Nic1 locus and Nic2 locus derived from LAFC53 in combination with CYP82E3 alleles derived from Nicotiana villosa) and related controls according to one embodiment of the present disclosure. Figure 2A includes yield (lbs / A); Figure 2B includes hundredweight (Cwt) value ($); Figure 2C includes acre value ($); Figure 2D includes grade index. DETAILED DESCRIPTION

[0051] Embodiments of the present disclosure provide novel methods for producing tobacco plants and any related tobacco products with low nicotinic alkaloid content. Compared with naturally occurring and genetically modified tobacco plants, tobacco plants produced according to the methods of the present disclosure show reduced levels of nicotinic alkaloids (e.g., nicotine), and therefore represent commercially valuable substitutes for currently available tobacco varieties.

[0052] The section headings as used in this section and the entire disclosure herein are for organizational purposes only and are not intended to be limiting.

[0053] 1. Definition

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. In the event of a conflict, this document (including definitions) shall prevail. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are described below. 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.

[0055] The term "about" will be understood by those of ordinary skill in the art and will vary to some extent based on the context in which it is used. If there is a use of the term that is unclear to those of ordinary skill in the art given its context of use, "about" will mean plus or minus 10% of a particular term at most. For example, in some embodiments, it will mean plus or minus 5% of a particular term. Numerical values ​​preceded by the term "about" are used herein to present certain ranges. The term "about" is used herein to provide textual support for the exact number after it and the number that is close to or approximates the number after the term. When determining whether a number is close to or approximates a specifically listed number, a number that is close to or approximates an unlisted number can be a number that provides a substantially equivalent value of a specifically listed number in the context in which it is proposed.

[0056] As used herein, the terms "comprise," "include," "having," "has," "can," "contain," and variations thereof are intended to be open transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates additional embodiments "comprising," "consisting of," and "consisting essentially of," whether or not explicitly stated.

[0057] For the recitation of numerical ranges herein, each intermediate value with the same degree of precision is expressly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range of 6.0-7.0, 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.

[0058] As used herein, "correlating to" means compared to.

[0059] As used herein, the term "nucleic acid molecule" refers to any molecule containing nucleic acid, including but not limited to DNA or RNA. The term encompasses sequences containing base analogs of any known DNA and RNA, including but not limited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 1-methylcytosine, 2-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 1-methylcytosine, 2-methylcytosine, 2-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 1-methylcytosine, 2-methylcytosine, 5-methylcytosine, N6-methyladenine, 7 ... Purine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5′-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, oxybutoxythymidine (oxybutoxosine), pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine and 2,6-diaminopurine.

[0060] The term "gene" refers to a nucleic acid (e.g., DNA) sequence comprising a coding sequence for producing 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, as long as the desired activity or functional properties (e.g., enzyme activity, ligand binding, signal transduction, immunogenicity, etc.) of the full length or fragment are retained. The term also encompasses the coding region of a structural gene and sequences located near the coding region at the 5' and 3' ends, with a distance of about 1 kb or longer at either end, such that the gene corresponds to the length of the full-length mRNA. The sequence located 5' of the coding region and present on the mRNA is referred to as a 5' non-translated sequence. The sequence located 3' or downstream of the coding region and present on the mRNA is referred to as a 3' non-translated sequence. The term "gene" encompasses cDNA and genomic forms of a gene. The genomic form or clone of a gene contains a coding region interrupted by a non-coding sequence referred to as an "intron" or "insertion region" or "insertion sequence." Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements, such as enhancers. Introns are removed or "spliced ​​out" from the nuclear or primary transcript; therefore, introns are not present in the messenger RNA (mRNA) transcript. mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0061] As used herein, the term "heterologous gene" refers to a gene that is not present in its natural environment. For example, a heterologous gene includes a gene introduced from one species into another species. A heterologous gene also includes a native gene of an organism that has been altered in some way (e.g., mutated, added in multiple copies, connected to a non-native regulatory sequence, etc.). The difference between a heterologous gene and an endogenous gene is that a heterologous gene sequence is usually connected to a DNA sequence that is not found to be naturally associated with a gene sequence in a chromosome and is associated with a part of a chromosome not found in nature (e.g., a gene expressed at a locus where the gene is not normally expressed).

[0062] As used herein, "operably linked" refers to a functional connection between two or more elements. For example, an operable connection between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional connection that allows the expression of the polynucleotide of interest. Operably linked elements can be continuous or non-continuous.

[0063] As used herein, "gene expression" refers to the biosynthesis or production of a gene product, including the transcription and / or translation of the gene product.

[0064] As used herein, the term "oligonucleotide" refers to a single-stranded polynucleotide chain of short length. The length of an oligonucleotide is generally less than about 300 residues (e.g., between 15 and 100 residues), however, as used herein, the term is also intended to encompass longer polynucleotide chains. Oligonucleotides are generally referred to by their length. For example, an oligonucleotide of 24 residues is referred to as a "24-mer". Oligonucleotides can form secondary structures and tertiary structures by self-hybridization or by hybridization with other polynucleotides. Such structures may include but are not limited to duplexes, hairpins, crosses, bends and triplexes.

[0065] The terms "homology" and "homologous" refer to a degree of identity. There can be partial homology or complete homology. A partially homologous sequence is a sequence that is less than 100% identical to another sequence.

[0066] As used herein, the terms "complementary" and "complementarity" are used to refer to polynucleotides (e.g., sequences of nucleotides, such as oligonucleotides or target nucleic acids) that are related by base pairing rules. For example, the sequence "5'-AGT-3'" is complementary to the sequence "3'-TCA-5'". Complementarity can be "partial", in which only the bases of some nucleic acids match according to the base pairing rules. Alternatively, there can be "complete" or "full" complementarity between nucleic acids. The degree of complementarity between nucleic acid chains has a significant effect on the efficiency and intensity of hybridization between nucleic acid chains. This is particularly important in amplification reactions and detection methods that rely on binding between nucleic acids. Either term can also be used to refer to individual nucleotides, particularly in the context of polynucleotides. For example, a particular nucleotide within an oligonucleotide can be noted for its complementarity or lack of complementarity with a nucleotide within another nucleic acid chain, in contrast or comparison to the complementarity between the rest of the oligonucleotide and the nucleic acid chain.

[0067] In some contexts, the term "complementarity" 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, such as nucleotides that can base pair, such as by Watson-Crick base pairing or other base pairing. Nucleotides that can form base pairs (e.g., complementary to each other) are the following pairs: cytosine and guanine, thymine and adenine, adenine and uracil, and guanine and uracil. Percent complementarity does not need to be calculated based on the entire length of the nucleic acid sequence. The percentage of complementarity can be limited to a specific region of the nucleic acid sequence of base pairing, such as starting from the first base-paired nucleotide to the end of the last base-paired nucleotide. As used herein, the complement of a nucleic acid sequence refers to an oligonucleotide that is in "antiparallel association" when aligned with a nucleic acid sequence so that the 5' end of one sequence is paired with the 3' end of another sequence. Certain bases that are not common in natural nucleic acids may be included in the nucleic acids of the present invention, and include, for example, inosine and 7-deazaguanine. Complementarity need not be perfect; a stable duplex may contain mismatched base pairs or unmatched bases. One skilled in the art of nucleic acid technology can determine duplex stability empirically taking into account many variables, including, for example, the length of the oligonucleotide, the base composition and sequence of the oligonucleotide, ionic strength, and the incidence of mismatched base pairs.

[0068] Thus, in some embodiments, "complementary" means that a first nucleic acid sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of a second nucleic acid 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 nucleic acid bases, or that the two sequences hybridize under stringent hybridization conditions. "Fully complementary" means that each nucleic acid base of the first nucleic acid is capable of pairing with each nucleic acid base at the corresponding position in 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 a 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.

[0069] As used herein, a "double-stranded nucleic acid" can be a portion of a nucleic acid, a region of a longer nucleic acid, or an entire nucleic acid. A "double-stranded nucleic acid" can be, for example, but not limited to, a double-stranded DNA, a double-stranded RNA, a double-stranded DNA / RNA hybrid, etc. Single-stranded nucleic acids having secondary structures (e.g., base-paired secondary structures) and / or higher-order structures include "double-stranded nucleic acids." For example, a triplex structure is considered to be "double-stranded." In some embodiments, any base-paired nucleic acid is a "double-stranded nucleic acid."

[0070] When used in connection with nucleic acids, such as in "isolated oligonucleotides" or "isolated polynucleotides", the term "isolated" refers to a nucleic acid sequence identified and separated from at least one component or contaminant that is usually associated with it in its natural source. An isolated nucleic acid exists in a form or setting different from that 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 exist in nature. For example, a given DNA sequence (e.g., a gene) is found near a neighboring gene on a host cell chromosome; RNA sequences, such as specific mRNA sequences encoding a specific protein, are found in cells as a mixture with many other mRNAs encoding a variety of proteins. However, isolated nucleic acids encoding a given protein include, for example, such nucleic acids in cells that normally express a given protein, wherein the nucleic acid is in a chromosomal position different from that of a natural cell, or is otherwise flanked by a nucleic acid sequence different from that found in nature. An isolated nucleic acid, oligonucleotide, or polynucleotide may exist in single-stranded or double-stranded form. When an isolated nucleic acid, oligonucleotide or polynucleotide is used to express a protein, the oligonucleotide or polynucleotide will contain at least a sense or coding strand (i.e., the oligonucleotide or polynucleotide may be single-stranded), but may contain both a sense strand and an antisense strand (i.e., the oligonucleotide or polynucleotide may be double-stranded).

[0071] As used herein, a "locus" is a chromosomal region where a polymorphic nucleic acid, a trait determinant, a gene or a marker is located. The loci of the present disclosure include one or more polymorphisms in a population; for example, alternative alleles are present in some individuals. As used herein, an "allele" refers to an alternative nucleic acid sequence at a specific locus. The length of an allele can be as small as 1 nucleotide base, but is typically larger. For example, a first allele may 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 located on a single chromosome.

[0072] As used herein, "introgression" or "introgress" refers to the transmission of a desired allele of a genetic locus from one genetic background to another.

[0073] As used herein, "hybridized" or "crossing" means the production of offspring (eg, cells, seeds, or plants) via fertilization, and includes crossing between plants (sexual crossing) and self-fertilization (selfing).

[0074] As used herein, "backcross" and "backcrossing" refer to the process of repeatedly crossing offspring plants back to one of their parents. In a backcrossing scheme, a "donor" parent refers to a parent plant with a desired gene or locus to be introgressed. A "recipient" parent (used once or multiple times) or a "recurrent" parent (used twice or more times) refers to a parent plant into which a gene or locus is introgressed. The initial hybridization produces an F1 generation. The term "BC 1" refers to the second use of the recurrent parent, and "BC2" refers to the third use of the recurrent parent, and the rest may be deduced by analogy. In some aspects, backcrossing is repeated, and the offspring individuals of each continuous backcross generation are themselves backcrossed with the same parental genotype.

[0075] As used herein, "single gene converted" or "single gene convertion" refers to a plant developed using a plant breeding technique called backcrossing or through genetic engineering in which essentially all of the desired morphological and physiological characteristics of the variety are restored in addition to a single gene transferred into the variety through the backcrossing technique or through genetic engineering.

[0076] As used herein, the term "variety" refers to a population of plants that share constant characteristics that distinguish them from other plants of the same species. Varieties are often sold commercially, although not always. Although varieties have one or more unique traits, varieties are further characterized by very little overall variation between individuals within the variety. "Pure line" varieties can be created by several generations of self-pollination and selection, or by vegetative propagation from a single parent using tissue or cell culture techniques. A variety may be substantially derived from another line or variety. As defined in " International Convention for the Protection of New Varieties of Plants " (December 2, 1961, November 10, 1972; October 23, 1978; and revised in Geneva on March 19, 1991), if meet the following, then variety "basically derived from " initial variety: a) described variety is mainly derived from initial variety, or is derived from the variety mainly derived from initial variety, retains the expression of the basic characteristics produced by the genotype of initial variety or genotype combination simultaneously; b) described variety is significantly different from initial variety; and c) except the difference produced by derivative behavior, described variety meets initial variety in the expression of the basic characteristics produced by the genotype of initial variety or genotype combination. The variety basically derived can be obtained, for example, by selecting natural or induced mutant, somatic clone variant, variant individual from the plant of initial variety, backcrossing or conversion. The second tobacco variety that the first tobacco variety and the first variety are basically derived from is considered to have basically the same genetic background. Different from variety, " strain " usually represents a group of plants used for non-commercial purposes, for example, in plant research. Lines typically show little overall variation between individuals for one or more traits of interest, although there may be some variation between individuals for other traits.

[0077] As used herein, "elite variety" means any variety produced by breeding and selection with superior agronomic performance.

[0078] As used herein, "selecting" or "selection" in the context of marker assisted selection or breeding refers to the act of picking or choosing desired individuals, usually from a population, based on certain predetermined criteria.

[0079] As used herein, the term "trait" refers to one or more detectable characteristics of a cell or organism that can be affected by genotype. The phenotype can be observed with the naked eye, or by any other evaluation means known in the art, such as microscopy, biochemical analysis, genomic analysis, determination of specific disease tolerance, etc. In some cases, the phenotype is directly controlled by a single gene or locus, such as a "monogenic trait". In other cases, the phenotype is the result of multiple genes.

[0080] As used herein, the term "gene mutation" or "genetic alteration" refers to the heritable genetic modification of the expression or activity of the product of the gene encoding by introducing a gene. Such modification can be in any sequence region of the gene, such as in a promoter, 5'UTR, exon, intron, 3'UTR or terminator region. In one aspect, the mutation reduces, suppresses or eliminates the expression or activity of the gene product. In another aspect, the mutation increases, promotes, strengthens or enhances the expression or activity of the gene product. In one aspect, the mutation is not a natural polymorphism present in a specific tobacco variety or cultivar. As used herein, "mutant allele" refers to an allele from a locus in which the allele comprises a mutation. As used herein, "mutation" refers to the generation of mutations without involving a transgenic or without remaining a transgenic associated with the mutation in the final mutant. In one aspect, mutagenesis is cisgenic. In another aspect, mutagenesis is via gene or genome editing. In yet another aspect, mutagenesis is via random mutagenesis, such as chemical mutagenesis (e.g., EMS) or physical mutagenesis (r- radiation).

[0081] As used herein, "polymorphism" means that there are one or more changes in a population. Polymorphism can be manifested as a change in the nucleotide sequence of a nucleic acid, or a variation in the amino acid sequence of a protein. Polymorphism includes one or more changes in the presence of a nucleic acid sequence or a nucleic acid signature at one or more loci in a population of one or more individuals. Changes may include, but are not limited to, one or more nucleotide base changes, the insertion of one or more nucleotides, or the deletion of one or more nucleotides. Polymorphism can be produced by random processes in nucleic acid replication, by mutagenesis, due to mobile genomic elements, by copy number changes, and in meiotic processes such as unequal exchange, genome replication, and chromosome breakage and fusion. Changes can be common or exist at low frequencies in a population, the former having greater practicality in general plant breeding, and the latter can be associated with rare but important phenotypic changes. Useful polymorphisms may include single nucleotide polymorphisms (SNPs), insertions or deletions (Indels) in DNA sequences, simple sequence repeats (SSRs) of DNA sequences, restriction fragment length polymorphisms (RFLPs), and tag SNPs. Genetic markers, genes, DNA-derived sequences, RNA-derived sequences, promoters, 5′ untranslated regions of genes, 3′ untranslated regions of genes, microRNAs, siRNAs, tolerance loci, satellite markers, transgenics, mRNAs, ds mRNAs, transcription profiles, and methylation patterns may also include polymorphisms. In addition, the presence, absence, or copy number variation of the aforementioned may include polymorphisms.

[0082] As used herein, the term "plant" encompasses whole plants, grafted plants, ancestors and offspring of plants, and plant parts, including seeds, buds, stems, roots (including tubers), stock, scion, and plant cells, tissues, and organs. Plants can be in any form, including suspension cultures, embryos, meristems, calli, leaves, gametophytes, sporophytes, pollen, and microspores. Plants particularly useful in the disclosed method include all plants belonging to the Nicotiana family.

[0083] As used herein, the term "tobacco" refers to any plant in the genus Nicotiana that produces nicotinic alkaloids. Tobacco also refers to products containing materials produced by plants of the genus Nicotiana, thus including, for example, expanded tobacco, reconstituted tobacco, cigarettes, cigars, chewing tobacco or smokeless tobacco forms, snuff, and buccal tobacco made from tobacco with increased GE nicotine. Examples of Nicotiana species include, but are not limited to, the following: Nicotiana acaulis, Nicotiana acunminata, Nicotiana acuminata var.multiflora, Nicotiana africana, Nicotiana alata, Nicotiana amplexicaulis, Nicotiana arentsii, Nicotiiana attenuata, Nicotiana benavidesii, Nicotiana benthaniana, Nicotiana bigelovii, Nicotiana bonariensis, Nicotianacavicola, 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, Nicotianakawakamii, Nicotiana knightiana, Nicotiana langsdorffii, Nicotiana linearis, Nicotiana longiflora, Nicotiana maritima, Nicotiana megalosiphon, Nicotiana miersii, Nicotiana noctifiora, Nicotiana anudicaulis, Nicotiana obtusifolia, Nicotiana occidentakis, Nicotiana subsp. Hesperis occidentalissubsp.hesperis), Nicotiana otophora, Nicotiana paniculata, Nicotiana auciflora, Nicotiana petunioides, Nicotiana lumbaginifolia, Nicotiana quadrivalvis, Nicotiana araimondii, Nicotiana repanda, Nicotiana rosulata, Nicotiana rosulata subsp. ingulba, Nicotiana arotundifolia, Nicotiana rustica, Nicotiana setchellii, Nicotiana simulans, Nicotiana solanifolia, Nicotiana spegazzinii, Nicotiana stocktonii), sweet tobacco (Nicotiana suaveolens), forest tobacco, common tobacco, blue tobacco (Nicotiana thyrsiflora), downy tobacco (Nicotiana tomentosa), downy tobacco, triangular leaf tobacco (Nicotiana trigonophylla), shade tobacco (Nicotiana umbratica), wavy leaf tobacco (Nicotiana undulata), trembling tobacco (Nicotianavelutina), celery tobacco (Nicotiana wigandioides) and flowering tobacco (Nicotiana x sanderae).

[0084] As used herein, the term "transgenic plant" refers to a plant that contains a nucleic acid sequence that is also present in another organism or species or is optimized from another organism or species with respect to host codon usage. Monocotyledonous and dicotyledonous angiosperm or gymnosperm cells can be transformed in various ways known in the art. For example, see Klein et al., Biotechnology 4: 583-590 (1993); Bechtold et al., CR Acad. 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).

[0085] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. For example, any nomenclature used in conjunction with cell and tissue culture, molecular biology, plant biology, genetics, and protein and nucleic acid chemistry and hybridization as described herein and the techniques of the field are those nomenclatures and techniques well-known and commonly used in the art. The meaning and scope of the terms should be clear; however, if there is any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definition. In addition, unless the context requires otherwise, singular terms shall include plural terms and plural terms shall include singular terms.

[0086] 2. Low-nicotine tobacco plants

[0087] Tobacco alkaloid accumulation is considered to be subject to complex genetic control. Researchers have always studied the use of Nic1 and Nic2 loci (also referred to as A loci and B loci in some documents) at naturally occurring allele variability 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 do not usually produce average nicotine levels below 0.4 mg g -1 of flue-cured tobacco leaves (Lewis, 2018).

[0088] The Nic2 locus has been found to contain a series of genes on tobacco linkage group 19 that encode ethylene response factor (ERF) transcription factors that comprehensively affect the expression of structural genes in the tobacco alkaloid biosynthetic pathway. This set of genes was found to be deleted in 'LA Burley21' (a backcross-derived nic1 / nic1 nic2 / nic2 version of Burley 21). A similar array of genes is located at or near the Nic1 locus on tobacco linkage group 7 (Sui et al., 2020; Qin et al., 2021), where the epigenetically silent allele of ERF199 may explain the effect on alkaloid accumulation at this locus in LA Burley 21 (Qin et al., 2021).

[0089] Naturally occurring variability at the locus encoding nicotine demethylase (NDM) may also significantly affect nicotine levels by increasing / decreasing its demethylation to form nornicotine. However, the biochemical conversion of nicotine to nornicotine has historically been considered an unattractive method for reducing nicotine levels in tobacco plants, because during the baking and storage of flue-cured tobacco, nornicotine has an increased tendency to form its corresponding carcinogenic tobacco-specific nitrosamines (TSNAs), i.e., N′-nitrosonornicotine (NNN). The large accumulation of this alkaloid can also undesirably change the physical and sensory properties of flue-cured tobacco leaves. The degree of nicotine demethylation in tobacco is considered to be a genetically unstable trait, while the ancestral species of tobacco, Nicotiana tabacum and Nicotiana villosa, exhibit stable and high levels of nicotine demethylation.

[0090] As further described herein, embodiments of the present disclosure include plant breeding methods for developing new tobacco genotypes in which genetic variability at the Nic1 locus and the Nic2 locus derived from LAFC53 is combined with genetic variability encoding stable and high levels of NDM activity derived from forest tobacco or tomentosa. Using these genetic combinations, variability at the Nic1 locus and the Nic2 locus reduces the expression of structural genes involved in nicotine biosynthesis, but does not reduce nicotine to zero, and generally does not even reduce nicotine to less than 0.4 mg g -1 Most of the remaining nicotine is removed via the activity of a forest tobacco or tobacco-derived gene encoding an NDM enzyme. The result of this strategy is a genotype that produces the lowest publicly reported and commercially available nicotine levels in field-grown tobacco, and a corresponding reduction in anabasine, anatabine, and nornicotine (relative to standard flue-cured tobacco cultivars). Importantly, there is no increase in nornicotine levels relative to standard tobacco cultivars.

[0091] The source of genetic variability at the Nic1 locus and Nic2 locus that affect low nicotine content is derived from the flue-cured tobacco breeding line LAFC53. The source of genetic changes encoding stable and high levels of nicotine demethylase activity is ultimately derived from forest tobacco or velvety tobacco. Breeding line SC58 CsCs was previously developed by using conventional interspecific hybridization followed by backcross breeding to transfer the main NDM gene from forest tobacco to the genetic background of old flue-cured tobacco cultivar 'SC 58'. Similarly, breeding line SC58 CtCt was also developed using a similar breeding method that transferred the main NDM gene from velvety tobacco to the genetic background of 'SC 58'. It was later discovered that SC58 CsCs possessed an NDM gene designated as CYP82E2, which is different from the naturally occurring gene in cultivated tobacco, in which the tobacco version was inactivated by two degenerate mutations (Chakrabarti et al., 2007). Leaf senescence can stimulate the expression of CYP82E2 derived from SC58 CsCs. It was later discovered that SC58 CtCt possesses a nicotine demethylase gene designated CYP82E3 that is distinct from the gene naturally present in cultivated tobacco, with the tobacco version being inactivated by a single base pair mutation (Gavilano et al., 2007). CYP82E3 derived from SC58 CtCt is active in green leaves, making this gene extremely efficient (more efficient than CYP82E2) in demethylating nicotine to form nornicotine.

[0092] In the excellent flue-cured tobacco genetic background, backcross breeding method is used to combine the nic1 allele and nic2 allele derived from LAFC53 with CYP82E2 or CYP82E3 derived from forest tobacco or fluffy tobacco respectively. Six generations of backcrossing are used to transfer the allele of interest to the genetic background of cultivar 'K326' (open pollinated flue-cured tobacco cultivar). After the sixth generation backcrossing, the BC6F1 plant carrying the allele variability of interest is self-pollinated, and the BC6F2 individual of the homozygous allele of interest is identified. The BC6F2 plant selected is self-pollinated to produce stable BC6F3 provenance. Cytoplasmic male sterility (Cms) F1 hybrid is produced by hybridizing the aforementioned inbred line as a pollen parent with the Cms version of K326, and the nic1 allele and nic2 allele derived from LAFC53 are transferred thereto via the backcross breeding program. NDM genes derived from either Nicotiana tabacum or Nicotiana velutipes were deployed in the heterozygous state in the F1 hybrids in order to reduce the effects of any deleterious linkage drag that may be present.

[0093] In accordance with the foregoing, embodiments of the present disclosure include tobacco cultivars, or any part thereof, comprising a reduced level of at least one nicotinic alkaloid as compared to a corresponding naturally occurring tobacco plant or part thereof. In some embodiments, the tobacco cultivars produced according to the methods of the present disclosure include at least one nicotinic alkaloid selected from the group consisting of nicotine, nornicotine, anatabine, and anabasine.

[0094] In some embodiments, the tobacco cultivars of the present disclosure contain reduced levels of nicotine. In some embodiments, the tobacco cultivars contain no more than 0.35% nicotine. In some embodiments, the tobacco cultivars contain no more than 0.30% nicotine. In some embodiments, the tobacco cultivars contain no more than 0.25% nicotine. In some embodiments, the tobacco cultivars contain no more than 0.20% nicotine. In some embodiments, the tobacco cultivars contain no more than 0.15% nicotine. In some embodiments, the tobacco cultivars contain no more than 0.10% nicotine. In some embodiments, the tobacco cultivars contain no more than 0.09% nicotine. In some embodiments, the tobacco cultivars contain no more than 0.08% nicotine. In some embodiments, the tobacco cultivars contain no more than 0.07% nicotine. In some embodiments, the tobacco cultivars contain no more than 0.06% nicotine. In some embodiments, the tobacco cultivars contain no more than 0.05% nicotine. In some embodiments, the tobacco cultivars contain no more than 0.04% nicotine. In some embodiments, tobacco cultivars contain no more than 0.03% nicotine. In some embodiments, tobacco cultivars contain no more than 0.02% nicotine. In some embodiments, tobacco cultivars contain no more than 0.01% nicotine. In some embodiments, tobacco cultivars contain about 0.1% to about 0.35% nicotine. In some embodiments, tobacco cultivars contain about 0.1% to about 0.25% nicotine. In some embodiments, tobacco cultivars contain about 0.1% to about 0.15% nicotine. In some embodiments, tobacco cultivars contain about 0.1% to about 0.10% nicotine. In some embodiments, tobacco cultivars contain about 0.15% to about 0.35% nicotine. In some embodiments, tobacco cultivars contain about 0.25% to about 0.35% nicotine. In some embodiments, tobacco cultivars contain about 0.10% to about 0.20% nicotine. In some embodiments, the tobacco cultivar comprises about 0.01% to about 0.10% nicotine. In some embodiments, the tobacco cultivar comprises about 0.01% to about 0.5% nicotine. In some embodiments, the tobacco cultivar comprises about 0.01% to about 0.03% nicotine.

[0095] In some embodiments, the tobacco cultivars of the present disclosure include reduced levels of nornicotine. In some embodiments, the cultivars include no more than 0.04% nornicotine. In some embodiments, the cultivars include no more than 0.03% nornicotine. In some embodiments, the cultivars include no more than 0.02% nornicotine. In some embodiments, the cultivars include no more than 0.01% nornicotine. In some embodiments, the cultivars include no more than 0.005% nornicotine. In some embodiments, the cultivars include about 0.005% to about 0.04% nornicotine. In some embodiments, the cultivars include about 0.01% to about 0.04% nornicotine. In some embodiments, the cultivars include about 0.02% to about 0.04% nornicotine. In some embodiments, the cultivars include about 0.03% to about 0.04% nornicotine. In some embodiments, the cultivars include about 0.005% to about 0.03% nornicotine. In some embodiments, the cultivar comprises from about 0.005% to about 0.02% nornicotine. In some embodiments, the cultivar comprises from about 0.005% to about 0.01% nornicotine.

[0096] In some embodiments, the tobacco cultivars of the present disclosure include reduced levels of anatabine. In some embodiments, the cultivars include no more than 0.06% anatabine. In some embodiments, the cultivars include no more than 0.05% anatabine. In some embodiments, the cultivars include no more than 0.04% anatabine. In some embodiments, the cultivars include no more than 0.03% anatabine. In some embodiments, the cultivars include no more than 0.02% anatabine. In some embodiments, the cultivars include no more than 0.01% anatabine. In some embodiments, the cultivars include no more than 0.005% anatabine. In some embodiments, the cultivars include no more than 0.001% anatabine. In some embodiments, the cultivars include about 0.001% to about 0.06% anatabine. In some embodiments, the cultivars include about 0.005% to about 0.06% anatabine. In some embodiments, the cultivars include about 0.01% to about 0.06% anatabine. In some embodiments, the cultivar contains about 0.02% to about 0.06% anatabine. In some embodiments, the cultivar contains about 0.03% to about 0.06% anatabine. In some embodiments, the cultivar contains about 0.04% to about 0.06% anatabine. In some embodiments, the cultivar contains about 0.05% to about 0.06% anatabine. In some embodiments, the cultivar contains about 0.005% to about 0.05% anatabine. In some embodiments, the cultivar contains about 0.005% to about 0.04% anatabine. In some embodiments, the cultivar contains about 0.005% to about 0.03% anatabine. In some embodiments, the cultivar contains about 0.005% to about 0.02% anatabine. In some embodiments, the cultivar contains about 0.005% to about 0.01% anatabine. In some embodiments, the cultivar contains about 0.01% to about 0.05% anatabine. In some embodiments, the cultivar comprises about 0.02% to about 0.04% anatabine.

[0097] In some embodiments, the tobacco cultivars of the present disclosure contain reduced levels of anabasine. In some embodiments, the cultivars contain no more than 0.008% anabasine. In some embodiments, the cultivars contain no more than 0.007% anabasine. In some embodiments, the cultivars contain no more than 0.006% anabasine. In some embodiments, the cultivars contain no more than 0.005% anabasine. In some embodiments, the cultivars contain no more than 0.004% anabasine. In some embodiments, the cultivars contain no more than 0.003% anabasine. In some embodiments, the cultivars contain no more than 0.002% anabasine. In some embodiments, the cultivars contain no more than 0.001% anabasine. In some embodiments, the cultivars contain no more than 0.0005% anabasine. In some embodiments, the cultivars contain about 0.0005% to about 0.008% anabasine. In some embodiments, the cultivar contains about 0.001% to about 0.008% anabasine. In some embodiments, the cultivar contains about 0.002% to about 0.008% anabasine. In some embodiments, the cultivar contains about 0.003% to about 0.008% anabasine. In some embodiments, the cultivar contains about 0.004% to about 0.008% anabasine. In some embodiments, the cultivar contains about 0.005% to about 0.008% anabasine. In some embodiments, the cultivar contains about 0.006% to about 0.008% anabasine. In some embodiments, the cultivar contains about 0.007% to about 0.008% anabasine. In some embodiments, the cultivar contains about 0.0005% to about 0.007% anabasine. In some embodiments, the cultivar contains about 0.0005% to about 0.006% anabasine. In some embodiments, the cultivar contains about 0.0005% to about 0.005% anabasine. In some embodiments, the cultivar contains about 0.0005% to about 0.005% anabasine. In some embodiments, the cultivar contains about 0.0005% to about 0.004% anabasine. In some embodiments, the cultivar contains about 0.0005% to about 0.003% anabasine. In some embodiments, the cultivar contains about 0.0005% to about 0.002% anabasine. In some embodiments, the cultivar contains about 0.0005% to about 0.001% anabasine. In some embodiments, the cultivar contains about 0.001% to about 0.006% anabasine. In some embodiments, the cultivar comprises from about 0.002% to about 0.005% anabasine. In some embodiments, the cultivar comprises from about 0.003% to about 0.006% anabasine.

[0098] In some embodiments, cultivar comprises nic1 alleles with expression and / or function reduced compared with wild type nic1. In some embodiments, cultivar comprises nic2 alleles with expression and / or function reduced compared with wild type nic2. In some embodiments, cultivar comprises nic1 alleles and nic2 alleles with expression and / or function reduced compared with wild type nic1 alleles and wild type nic2 alleles. In some embodiments, nic1 alleles 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 tobacco variety and / or the second tobacco variety comprise nic1 and / or nic2 invalid alleles (deletions). In some embodiments, nic1 and / or nic2 invalid alleles are derived from naturally occurring tobacco germplasm.

[0099] In some embodiments, cultivars include functional CYP82E2 alleles. In some embodiments, CYP82E2 alleles are derived from forest tobacco. In some embodiments, cultivars include functional CYP82E3 alleles. In some embodiments, the CYP82E3 alleles are derived from fluffy tobacco, or related species (e.g., Nicotiana fluffy tobacco group). In some embodiments, cultivars include functional CYP82E2 alleles derived from forest tobacco and functional CYP82E3 alleles derived from fluffy tobacco or related species (e.g., Nicotiana fluffy tobacco group). In some embodiments, compared with naturally occurring tobacco plants, the nicotine N-demethylation activity of proteins encoded by CYP82E2 alleles and / or CYP82E3 alleles increases.

[0100] In some embodiments, the tobacco cultivars of the present disclosure are non-transgenic (e.g., do not have a heterologous transgene). According to these embodiments, the tobacco cultivars of the present disclosure can be produced by breeding (e.g., backcrossing) various tobacco lines with a desired trait corresponding to a reduced level of at least one nicotinic alkaloid. Therefore, the tobacco cultivars of the present disclosure are not naturally occurring.

[0101] As those of ordinary skill in the art will understand based on the disclosure, transgenic methods can also be used to produce the tobacco cultivar of at least one nicotinic alkaloid of the level of reduction as described herein.In some embodiments, tobacco cultivar of the present disclosure can be engineered to comprise one or more traits of at least one nicotinic alkaloid corresponding to the level of reduction.For example, tobacco plant can be engineered to comprise nic1 allele and / or nic2 allele substantially similar to those alleles found in LAFC53, MAFC5, LMAFC34, LA Burley 21, LI Burley 21 and HI Burley 21.These nic1 allele and / or nic2 allele can have one or more genetic changes, and the genetic change causes the level of at least one nicotinic alkaloid to reduce (for example, loss of function or hylomorphic mutation (hylomorphic mutation)). Such genetic alterations 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., Trendis in Biotechnology, 31(7): 397-405 (2013).

[0102] The tobacco cultivar of at least one nicotinic alkaloid of the level with reduction as described herein can also be engineered to comprise one or more traits of at least one nicotinic alkaloid of the level corresponding to reduction except the change of nic1 and / or nic2.For example, compared with the corresponding naturally occurring tobacco plant, tobacco plant can be engineered to comprise functional CYP82E2 allele and / or functional CYP82E3 allele.In some embodiments, CYP82E2 allele and / or CYP82E3 allele are engineered to recover the wild-type function that becomes non-functional corresponding CYP82E2 albumen and / or CYP82E3 albumen by genetic mutation.In some embodiments, CYP82E2 allele and / or CYP82E3 allele can be engineered to have one or more genetic changes, and the genetic change causes the level of at least one nicotinic alkaloid to reduce (for example, gain of function or supermorphological mutation). Such genetic alterations 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).

[0103] In some embodiments, the tobacco plant is engineered to include a functional CYP82E2 allele and / or CYP82E3 allele compared to a corresponding naturally occurring or non-transformed control tobacco plant. For example, in some embodiments, the CYP82E2 allele is engineered to restore the function of a corresponding CYP82E2 protein that has become non-functional through a genetic mutation, or to increase the activity of a corresponding CYP82E2 protein compared to a corresponding naturally occurring or non-transformed control tobacco plant. In some embodiments, the CYP82E2 allele of a tobacco plant is engineered to include a nucleotide sequence encoding a K375E point mutation and / or a L422W point mutation relative to a wild-type CYP82E2 amino acid sequence (e.g., SEQ ID NO: 1). An exemplary wild-type CYP82E2 amino acid sequence is shown in SEQ ID NO: 1 (GenBank Accession No. ABA07806):

[0104] An exemplary CYP82E2 amino acid sequence comprising a K375E point mutation relative to the wild-type CYP82E2 amino acid sequence is shown in SEQ ID NO: 2 (the point mutation is shown below in bold and underlined text):

[0105]

[0106] An exemplary CYP82E2 amino acid sequence comprising the L422W point mutation relative to the wild-type CYP82E2 amino acid sequence is shown in SEQ ID NO: 3 (the point mutation is shown below in bold and underlined text):

[0107] An exemplary CYP82E2 amino acid sequence comprising a K375E point mutation and a L422W point mutation relative to the wild-type CYP82E2 amino acid sequence is shown in SEQ ID NO: 4 (the point mutations are shown below in bold and underlined text):

[0108]

[0109] In some embodiments, the CYP82E3 allele is engineered to restore the function of a corresponding CYP82E3 protein that has become non-functional through a genetic mutation, or to increase the activity of a corresponding CYP82E3 protein compared to a corresponding naturally occurring or non-transformed control tobacco plant. In some embodiments, the CYP82E3 allele of a tobacco plant is engineered to include a nucleotide sequence encoding a C330W point mutation relative to a wild-type CYP82E3 amino acid sequence (e.g., SEQ ID NO: 5). An exemplary wild-type CYP82E3 amino acid sequence is shown in SEQ ID NO: 5 (NCBI Reference Sequence: NM_001326063.1):

[0110]

[0111] An exemplary CYP82E3 amino acid sequence comprising a C330W point mutation relative to the wild-type CYP82E3 amino acid sequence is shown in SEQ ID NO: 6 (the point mutation is shown below in bold and underlined text):

[0112] In some embodiments, the present technology provides methods for producing tobacco plants with reduced nicotinic alkaloid content, the methods comprising combining in a tobacco plant (e.g., Nicotiana tabacum): (A) a genetic modification that increases the activity of CYP82E2 and / or CYP82E3 compared to a corresponding naturally occurring or non-transformed control tobacco plant; and (B) a recessive allele of nic1 and / or a recessive allele of nic2.

[0113] In some embodiments, introducing a recessive allele of nic1 and / or a recessive allele of nic2 can include incorporating one or more recessive alleles into, for example, a tobacco plant (e.g., Nicotiana tabacum) via conventional breeding, wherein the tobacco plant comprises one or more genetic modifications that increase the activity of CYP82E2 and / or CYP82E3 in the tobacco plant compared to a wild-type tobacco plant.

[0114] In some embodiments, tobacco plants produced by the methods of the present technology having (A) a genetic modification to increase CYP82E2 and / or CYP82E3 activity; and (B) a recessive allele of nic1 and / or a recessive allele of nic2 may contain at least about 40% (e.g., at least about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125 94%, 95%, 96%, 97%, 98%, 99%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, In some embodiments, the nicotinic alkaloid reduced in a tobacco plant can be nicotine, wherein 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.

[0115] In some embodiments, tobacco plants produced by the methods of the present technology with (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 comprise reduced levels of nicotine. In some embodiments, the tobacco plant comprises no more than about 0.35% nicotine. In other embodiments, the tobacco plant comprises no more than about 0.30% nicotine, no more than about 0.25% nicotine, no more than about 0.20% nicotine, no more than about 0.15% nicotine, no more than about 0.10% nicotine, no more than about 0.09% nicotine, no more than about 0.08% nicotine, no more than about 0.07% nicotine, no more than about 0.06% nicotine, no more than about 0.05% nicotine, no more than about 0.04% nicotine, no more than about 0.03% nicotine, no more than about 0.02% nicotine, or no more than about 0.01% nicotine.

[0116] 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.

[0117] In some embodiments, the tobacco plants produced by the methods of the present technology with (A) genetic modifications that increase CYP82E2 and / or CYP82E3 activity; and (B) recessive alleles of nic1 and / or recessive alleles of nic2 comprise reduced levels of nornicotine. In some embodiments, the plant comprises no more than about 0.04% nornicotine, no more than about 0.03% nornicotine, no more than about 0.02% nornicotine, no more than about 0.01% nornicotine, or no more than about 0.005% nornicotine. In some embodiments, the plant comprises 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.

[0118] In some embodiments, the tobacco plants produced by the methods of the present technology with (A) increased CYP82E2 and / or CYP82E3 activity genetic modification; and (B) recessive alleles of nic1 and / or recessive alleles of nic2 include reduced levels of anatabine. In some embodiments, the plant includes no more than about 0.06% anatabine, no more than about 0.05% anatabine, no more than about 0.04% anatabine, no more than about 0.03% anatabine, no more than about 0.02% anatabine, no more than about 0.01% anatabine, no more than about 0.005% anatabine, or no more than about 0.001% anatabine. In other embodiments, the plant comprises 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, about 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.

[0119] In some embodiments, the tobacco plants produced by the methods of the present technology with (A) increased CYP82E2 and / or CYP82E3 activity genetic modification; and (B) recessive alleles of nic1 and / or recessive alleles of nic2 include reduced levels of anabasine. In some embodiments, the plant includes no more than about 0.008% anabasine, no more than about 0.007% anabasine, no more than about 0.006% anabasine, no more than about 0.005% anabasine, no more than about 0.004% anabasine, no more than about 0.003% anabasine, no more than about 0.002% anabasine, no more than about 0.001% anabasine, or no more than about 0.0005% anabasine. In other embodiments, the plant comprises from about 0.0005% to about 0.008% anabasine, from about 0.001% to about 0.008% anabasine, from about 0.002% to about 0.008% anabasine, from about 0.003% to about 0.008% anabasine, from about 0.004% to about 0.008% anabasine, from about 0.005% to about 0.008% anabasine, from about 0.006% to about 0.008% anabasine, from about 0.007% to about 0.008% anabasine, from about 0.0005% to about 0.007% anabasine, from about 0.0005% to about 0.007% anabasine, from about 0.0005% to about 0.007% anabasine. % to about 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.

[0120] As described herein, genetic modifications 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, CRISPR / Cas9 systems, CRISPR / Cpf1 systems, CRISPR / Csm1 systems, gene knock-in techniques or technologies, or any combination thereof.

[0121] For example, in some embodiments, the methods of the present technology involve the use of a CRISPR / Cas system that binds to a target site in a region of interest in a genome, wherein the CRISPR / Cas system comprises a CRISPR / Cas nuclease and an engineered crRNA / tracrRNA (or a single guide RNA (sgRNA) or a guide RNA (gRNA)). In some embodiments, the CRISPR system generally comprises (i) a polynucleotide encoding a Cas protein, and (ii) at least one sgRNA engineered for RNA-guided genome 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, Csyl, 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 known. For example, the amino acid sequence of the Streptococcus pyogenes Cas9 protein can be found in the SwissProt database with accession number Q99ZW2. In some embodiments, the methods of the present technology involve the use of a CRISPR / Cpf1 system that binds to a target site in a region of interest in the genome. In some embodiments, the methods of the present technology involve the use of a CRISPR / Csm1 system that binds to a target site in a region of interest in the genome.

[0122] In some embodiments, CRISPR / Cas, CRISPR / Cpf1 or CRISPR / Csm1 systems identify target sites in one or more of CYP82E2 or CYP82E3. In some embodiments, CRISPR / Cas, CRISPR / Cpf1 or CRISPR / Csm1 systems produce specific sequence changes in CYP82E2 or CYP82E3 genes, such as mutations that cause K375E point mutations and / or L422W point mutations in the CYP82E2 amino acid sequence or mutations that cause C330W point mutations in the CYP82E3 amino acid sequence. In some embodiments, CRISPR / Cas, CRISPR / Cpf1 or CRISPR / Csm1 systems produce specific sequence changes via gene knock-in or gene replacement. The methods of gene knock-in or gene replacement are well known in the art. Gene knock-in or gene replacement methods based on CRISPR can utilize homology-directed repair (HDR) mechanisms, non-homologous end joining (NHEJ) mechanisms, or HDR and NHEJ mechanisms. A non-limiting example of a CRISPR-based approach that utilizes HDR and NHEJ mechanisms is called tandem repeat-HDR (TR-HDR), which 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), which is incorporated herein by reference in its entirety.

[0123] In some embodiments, the method described herein uses a meganuclease DNA binding domain to bind to a region of interest in a plant cell genome. A meganuclease is an engineered version of a naturally occurring restriction enzyme, which typically has an extended DNA recognition sequence (e.g., a length of about 14 to about 40 base pairs). Meganucleases (also referred to as homing endonucleases) are typically divided into five families based on sequence and structural motifs: LAGLIDADG family, GIY-YIG family, His-Cyst box family, PD-(D / E)XK family, and HNH family. In some embodiments, meganucleases include engineered homing endonucleases. Homing endonucleases and meganucleases 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 recognition sequences are known.

[0124] In some embodiments, the meganuclease is tailored to recognize a target in one or more of CYP82E2 or CYP82E3. In some embodiments, the meganuclease produces a specific sequence change in the CYP82E2 or CYP82E3 gene, such as a mutation resulting in a K375E point mutation and / or a L422W point mutation in the CYP82E2 amino acid sequence or a mutation resulting in a C330W point mutation in the CYP82E3 amino acid sequence.

[0125] In some embodiments, the methods described herein use transcription activator-like effector nucleases (TALENs) to edit plant genomes by inducing double-strand breaks (DSBs). TALENs are restriction enzymes that can be engineered to cleave specific DNA sequences. TALENs are constructed by fusing TAL effector DNA binding domains with DNA cleavage domains (e.g., nuclease domains, such as nuclease domains derived from FokI endonucleases). Transcription activator-like effectors (TALEs) can be engineered to bind to desired DNA sequences according to methods known in the art, and when combined with nucleases, provide techniques for cutting DNA at specific locations. For example, in some embodiments, the use of TALEN technology produces specific sequence changes in CYP82E2 or CYP82E3 genes, such as mutations that cause K375E point mutations and / or L422W point mutations in the CYP82E2 amino acid sequence or mutations that cause C330W point mutations in the CYP82E3 amino acid sequence.

[0126] In some embodiments, the compositions and methods described herein use zinc finger nucleases (ZFNs) to edit plant genomes by inducing double-strand breaks (DSBs). ZFNs are artificial restriction enzymes produced by fusing zinc finger DNA binding domains with DNA cleavage domains (e.g., nuclease domains, such as nuclease domains derived from FokI endonucleases). 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 eukaryotic transcription factors and zinc fingers. The second domain is a nuclease domain, which contains the FokI restriction enzyme responsible for cleaving DNA. ZFNs can be engineered to bind to desired DNA sequences and cleave DNA at specific locations according to methods known in the art. For example, after identifying the target sequence in the nicotine biosynthesis gene, the corresponding ZFN sequence is engineered and inserted into a plasmid. The plasmid is inserted into a target cell, translated in the target cell to produce a functional ZFN, and then the ZFN enters the nucleus, where it binds and cleaves its target sequence, thereby introducing a double-strand break (DSB). When DSBs are repaired by homology-directed repair or non-homologous end joining, this method can be used to introduce exogenous DNA sequences into the target gene. For example, in some embodiments, the use of ZFN technology produces specific sequence changes in the CYP82E2 or CYP82E3 gene, such as a mutation that results in a K375E point mutation and / or a L422W point mutation in the CYP82E2 amino acid sequence or a mutation that results in a C330W point mutation in the CYP82E3 amino acid sequence.

[0127] In some embodiments, the present technology provides a method for producing a tobacco plant with reduced nicotinic alkaloid content (e.g., a reduction in total nicotinic alkaloid content, nicotine, nornicotine, anatabine and / or anabasine in a plant produced by the present technology as described above with (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 method comprising combining in a tobacco plant (a) a genetic modification that increases CYP82E2 and / or CYP82E3 activity as described herein; and (b) a modification that inhibits the following: (i) the activity of ERF199 and / or ERF189, or (ii) the expression of a nucleic acid encoding ERF199 and / or a nucleic acid encoding ERF189, as compared to a corresponding naturally occurring or non-transformed control tobacco plant.

[0128] In some embodiments, the present technology further comprises inhibiting the expression of endogenous genes encoding transcription factors that positively regulate alkaloid production (such as NtERF221, NtMYC1a, NtMYC1b, NtMYC2a and / or NtMYC2b genes) to reduce the level of nicotinic alkaloids in the plant.

[0129] In some embodiments, the present technology further comprises inhibiting the expression of one or more nicotinic alkaloid biosynthesis genes such as A622, NBB1, QPT (quinolinate phosphoribosyltransferase), PMT (putrescine methyltransferase), ODC (ornithine decarboxylase), AO (aspartate oxidase), QS (quinolinate synthase) and MPO (N-methylputrescine oxidase) genes to reduce the level of nicotinic alkaloids in the plant.

[0130] Examples of methods that can be used to inhibit 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 targeted genome engineering 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.

[0131] According to the foregoing, embodiments of the present disclosure also include offspring plants, seeds or cells produced from any tobacco cultivar as described herein using transgenic and / or non-transgenic methods.Embodiments of the present disclosure also include tobacco products derived from any tobacco cultivar as described herein or its part.In some embodiments, tobacco products are selected from the group consisting of tobacco leaf, shredded tobacco, raw cut tobacco, tobacco dust, tobacco powder, tobacco extract, smokeless tobacco, moist snuff or dry snuff, pipe tobacco, cigar tobacco, cigarillo tobacco, cigarette tobacco and chewing tobacco.In some embodiments, tobacco products are selected from the group consisting of cigarillo, clove cigarette, non-ventilated recessed filter cigarette, ventilated recessed filter cigarette, cigar, snuff, containing tobacco chewing gum, containing tobacco lozenge and chewing tobacco.

[0132] Embodiments of the present disclosure also include methods for producing tobacco cultivars, and the tobacco cultivars include at least one nicotinic alkaloid of the level reduced compared with corresponding naturally occurring tobacco plants or their parts. According to these embodiments, the method includes hybridizing the first tobacco cultivar comprising the first low nicotinic trait with the second tobacco cultivar comprising the second low nicotinic trait to produce offspring plants. In some embodiments, offspring plants include at least one nicotinic alkaloid of the concentration reduced. In some embodiments, the method includes backcrossing. As those of ordinary skill in the art can recognize, transgenic and / or non-transgenic methods can be used to produce this type of tobacco cultivar of at least one nicotinic alkaloid of the level with reduced.

[0133] In some embodiments, and as further described above, at least one nicotinic alkaloid is selected from the group consisting of nicotine, nornicotine, anatabine and anabasine. In some embodiments, at least one nicotinic alkaloid is nicotine, and wherein the cultivar contains no more than 0.35% nicotine. In some embodiments, at least one nicotinic alkaloid is nornicotine, and wherein the cultivar contains no more than 0.04% nornicotine. In some embodiments, at least one nicotinic alkaloid is anatabine, and wherein the cultivar contains no more than 0.06% anatabine. In some embodiments, at least one nicotinic alkaloid is anabasine, and wherein the cultivar contains no more than 0.008% anabasine. In some embodiments, at least one nicotinic alkaloid is nicotine and nornicotine, and wherein the cultivar contains no more than 0.35% nicotine and no more than 0.04% nornicotine.

[0134] In some embodiments, the first low nicotine trait and / or the second low nicotine trait include a nic1 allele with reduced expression and / or function compared to wild type nic1. In some embodiments, the first low nicotine trait and / or the second low nicotine trait include a nic2 allele with reduced expression and / or function compared to wild type nic2. In some embodiments, the first tobacco variety and / or the second tobacco variety include a nic1 allele and / or a nic2 allele derived from at least one of the following strains: LAFC53, MAFC5, LMAFC34, LA Burley 21, LI Burley21 and Sichuan Burley 21. In some embodiments, the first tobacco variety and / or the second tobacco variety include nic1 and / or nic2 invalid alleles (deletions). In some embodiments, nic1 and / or nic2 invalid alleles are derived from naturally occurring tobacco germplasm.

[0135] In some embodiments, the first tobacco variety and / or the second tobacco variety comprise functional CYP82E2 alleles. In some embodiments, CYP82E2 alleles are derived from forest tobacco. In some embodiments, the first tobacco variety and / or the second tobacco variety comprise functional CYP82E3 alleles. In some embodiments, the CYP82E3 alleles are derived from fluffy tobacco, or related species (e.g., Nicotiana fluffy tobacco group). In some embodiments, compared with naturally occurring tobacco plants, the nicotine N-demethylation activity of the protein encoded by CYP82E2 alleles and / or CYP82E3 alleles increases.

[0136] In some embodiments, the first low nicotine trait comprises a nic1 allele and / or a nic2 allele having reduced expression and / or function compared to wild type nic1 and / or nic2, and wherein the second low nicotine trait comprises a functional CYP82E2 allele and / or a functional CYP82E3 allele.

[0137] 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 transgenic.

[0138] Embodiments of the present disclosure also include seeds or cells obtained from offspring plants produced according to any method described herein. Embodiments of the present disclosure also include tobacco products derived from offspring plants produced according to any method described herein. In some embodiments, the product is selected from the group consisting of tobacco leaves, shredded tobacco, raw cut tobacco, tobacco dust, tobacco powder, tobacco extract, smokeless tobacco, moist snuff or dry snuff, pipe tobacco, cigar tobacco, cigarillo tobacco, cigarette tobacco and chewing tobacco. In some embodiments, the product is selected from the group consisting of cigarillos, clove cigarettes, unventilated recessed filter cigarettes, ventilated recessed filter cigarettes, cigars, snuff, tobacco-containing chewing gum, tobacco-containing lozenges and chewing tobacco.

[0139] In some embodiments, the cultivar comprises a CYP82E2 allele comprising a nucleotide sequence encoding a K375E point mutation and / or a L422W point mutation relative 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 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.

[0140] In some embodiments, the CYP82E3 allele comprises a nucleotide sequence encoding a C330W point mutation relative to the wild-type CYP82E3 amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the CYP82E3 allele comprises a nucleotide sequence encoding a CYP82E3 protein comprising the amino acid sequence set forth in SEQ ID NO: 6.

[0141] In some embodiments, the cultivar comprises a CYP82E2 allele comprising a nucleotide sequence encoding a K375E point mutation and / or a L422W point mutation relative 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 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.

[0142] In some embodiments, the CYP82E3 allele comprises a nucleotide sequence encoding a C330W point mutation relative to the wild-type CYP82E3 amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the CYP82E3 allele comprises a nucleotide sequence encoding a CYP82E3 protein comprising the amino acid sequence set forth in SEQ ID NO: 6.

[0143] In some embodiments, the cultivated variety further includes inhibition of expression of at least one of the following within the cultivated variety: NBB1, A622, quinolinate 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 a method for generating point mutations in a target gene in a tobacco plant cell. According to these embodiments, the method includes introducing into the cell a clustered regularly spaced short palindromic repeat (CRISPR)-related (CRISPR-Cas) system, the system including: (a) at least one guide RNA (gRNA), wherein the at least one gRNA comprises a nucleotide sequence capable of hybridizing with a target sequence or a portion thereof in a target gene, wherein the target gene comprises a nucleotide sequence encoding a CYP82E2 protein; (b) a nucleic acid comprising a donor DNA sequence, the donor DNA sequence comprising: (i) a DNA fragment, wherein the DNA fragment replaces the target sequence in the target gene or inserts into a target site in the target gene; and (ii) two homologous arms, each arm being flanked on opposite sides of the DNA fragment; and (c) an effector protein, or one or more polynucleotides encoding an effector protein. In some embodiments, at least one gRNA forms a complex with the effector protein. In some embodiments, the effector protein is a Cas protein comprising a nuclease.

[0145] In some embodiments, (a) the point mutation is a K375E point mutation relative to the wild-type CYP82E2 amino acid sequence as shown in SEQ ID NO: 1; or (b) the point mutation is a L422W point mutation relative to the wild-type CYP82E2 amino acid sequence as shown in SEQ ID NO: 1.

[0146] In some embodiments, at least two point mutations are generated. In some embodiments, the at least two point mutations include a K375E point mutation relative to the wild-type CYP82E2 amino acid sequence shown in SEQ ID NO: 1 and a L422W point mutation relative to the wild-type CYP82E2 amino acid sequence shown in SEQ ID NO: 1.

[0147] In some embodiments, the tobacco plant further comprises a recessive allele of nic1 and / or a recessive allele of nic2.

[0148] In some embodiments, the method further comprises inhibiting expression of at least one of the following in the cultivar: NBB1, A622, quinolinate 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.

[0149] Embodiments of the present disclosure also include a method for generating point mutations in a target gene in a tobacco plant cell. According to these embodiments, the method includes introducing into the cell a clustered regularly spaced short palindromic repeat (CRISPR)-related (CRISPR-Cas) system, the system including: (a) at least one guide RNA (gRNA), wherein the at least one gRNA comprises a nucleotide sequence capable of hybridizing with a target sequence or a portion thereof in a target gene, wherein the target gene comprises a nucleotide sequence encoding a CYP82E3 protein; (b) a nucleic acid comprising a donor DNA sequence, the donor DNA sequence comprising: (i) a DNA fragment, wherein the DNA fragment replaces the target sequence in the target gene or inserts into a target site in the target gene; and (ii) two homologous arms, each arm being flanked on opposite sides of the DNA fragment; and (c) an effector protein, or one or more polynucleotides encoding an effector protein. In some embodiments, 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] In some embodiments, the point mutation is a C330W point mutation relative to the wild-type CYP82E3 amino acid sequence as shown in SEQ ID NO: 5. In some embodiments, the tobacco plant further comprises a recessive allele of nic1 and / or a recessive allele of nic2.

[0151] In some embodiments, the method further comprises inhibiting the expression of at least one of the following in the tobacco plant: NBB1, A622, quinolinate 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.

[0152] Embodiments of the present disclosure also include methods for producing tobacco plants with reduced nicotinic alkaloid content. According to these embodiments, the method includes combining in tobacco plants: (a) one or more genetic modifications that increase CYP82E2 and / or CYP82E3 activity; and (b) recessive alleles of nic1 and / or recessive alleles of nic2. In some embodiments, compared with corresponding naturally occurring or non-converted control tobacco plants, the tobacco plants have reduced nicotinic alkaloid content.

[0153] In some embodiments, the tobacco plant comprises a homozygous recessive allele for nic1 and / or a homozygous recessive allele for nic2.

[0154] In some embodiments, one or more genetic modifications that increase CYP82E2 and / or CYP82E3 activity 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 techniques or technologies, and any combination thereof.

[0155] In some embodiments, the one or more genetic modifications that increase CYP82E2 activity include a point mutation of a target gene in a tobacco plant cell, wherein the point mutation is selected from one or more of a K375E point mutation relative to the wild-type CYP82E2 amino acid sequence as shown in SEQ ID NO: 1 and a L422W point mutation relative to the wild-type CYP82E2 amino acid sequence as shown in SEQ ID NO: 1.

[0156] In some embodiments, the one or more genetic modifications that increase CYP82E3 activity include a point mutation in a target gene in a tobacco plant cell, wherein the point mutation is a C330W point mutation relative to the wild-type CYP82E3 amino acid sequence as shown in SEQ ID NO:5.

[0157] In some embodiments, point mutations are introduced into plants by a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (CRISPR-Cas) system, the system comprising: (a) at least one guide RNA (gRNA), wherein the at least one gRNA comprises a nucleotide sequence capable of hybridizing with a target sequence in a target gene or a portion thereof, wherein the target gene comprises a nucleotide sequence encoding a CYP82E2 protein; (b) a nucleic acid comprising a donor DNA sequence, the donor DNA sequence comprising: (i) a DNA fragment, wherein the DNA fragment replaces the target sequence in the target gene or is inserted into a target site in the target gene; and (ii) two homology arms, each arm flanking the opposite side of the DNA fragment; and (c) an effector protein, or one or more polynucleotides encoding an effector protein. In some embodiments, at least one gRNA forms a complex with the effector protein. In some embodiments, the effector protein is a Cas protein comprising a nuclease.

[0158] Embodiments of the present disclosure also include tobacco plants produced by any of the methods described herein, wherein the plant comprises: (a) one or more genetic modifications that increase CYP82E2 and / or CYP82E3 activity; and (b) recessive alleles of nic1 and / or recessive alleles of nic2. Embodiments of the present disclosure also include offspring plants or seeds produced by the plant, wherein the offspring plants or seeds include: (A) one or more genetic modifications that increase CYP82E2 and / or CYP82E3 activity; and (B) recessive alleles of nic1 and / or recessive alleles of nic2. Embodiments of the present disclosure also include tobacco products comprising tobacco from tobacco plants, wherein the plant comprises: (A) one or more genetic modifications that increase CYP82E2 and / or CYP82E3 activity; and (B) recessive alleles of nic1 and / or recessive alleles of nic2.

[0159] According to these embodiments, (a) the tobacco is selected from the group consisting of leaf tobacco, shredded tobacco, raw cut tobacco, tobacco dust, tobacco powder, tobacco extracts, smokeless tobacco, moist or dry snuff, pipe tobacco, cigar tobacco, cigarillo tobacco, cigarette tobacco, and chewing tobacco; or (b) the product is a reduced-nicotine tobacco product selected from the group consisting of cigarillos, clove cigarettes, unventilated recessed-filter cigarettes, ventilated recessed-filter cigarettes, cigars, snus, snus, tobacco-containing gum, tobacco-containing lozenges, and chewing tobacco.

[0160] 3. Example

[0161] It will be apparent to those skilled in the art that other suitable modifications and adjustments to the disclosed methods described herein are readily applicable and understandable, and may be performed using suitable equivalents without departing from the scope of the disclosure or the aspects and embodiments disclosed herein. Having now described the disclosure in detail, it will be more clearly understood by reference to the following examples, which are intended only to illustrate some aspects and embodiments of the disclosure and should not be construed as limiting the scope of the disclosure. All journal references, U.S. patents, and published disclosures cited herein are hereby incorporated by reference in their entirety.

[0162] The present disclosure has several aspects, illustrated by the following non-limiting examples.

[0163] Example 1

[0164] Cms NCLA hybrid. In view of the fact that countries around the world (including the United States) may force the nicotine level in conventional cigarettes to be reduced to below the so-called "sub-addiction threshold level", the following tobacco cultivars have commercial interests: the tobacco cultivars can produce flue-cured tobacco leaves with nicotine levels that enable manufacturers to reach target nicotine levels. Currently, the nicotine level of cigarette tobacco fillers is being discussed and / or suggested to be 0.04%. There is currently no flue-cured tobacco cultivars (grown under conventional agronomic management systems and averaged for all stem positions) that usually accumulate such low levels of nicotine.

[0165] Thus, embodiments of the present disclosure include newly established cytoplasmic male sterile (Cms) flue-cured tobacco hybrids NCLA Hybrid 1, NCLA Hybrid 2, and NCLA Hybrid 3 (derived from the Nic1 locus and Nic2 locus of LAFC53 in combination with the CYP82E3 allele derived from tobacco velvet) with ultra-low nicotine content. These hybrid cultivars accumulate the lowest levels of nicotine of any genetic material that have been publicly reported. As described herein, these new F1 hybrids were developed using naturally occurring genetic variation and conventional breeding methods (i.e., gene editing, genetic engineering, or new breeding methods were not used to develop these materials). These hybrids can be grown anywhere in the world without worrying about patent infringement, or regulations regarding gene editing or genetic engineering breeding results.

[0166] Data supporting the low-nicotine phenotype of these new hybrids were generated from field evaluations at three North Carolina research stations. The experimental design at each location was a randomized complete block design with four replications. Plots consisted of 20 plant rows and were managed according to North Carolina flue-cured tobacco production recommendations. Leaves were harvested, cured, and graded by former USDA tobacco graders over four passes. Alkaloid composition of composite flue-cured tobacco leaf samples was analyzed using gas chromatography.

[0167] The materials evaluated in the first experiment were as follows: K326-standard flue-cured tobacco cultivar; LAFC53-near isogenic version of flue-cured tobacco cultivar NC95 containing recessive alleles Nic1 locus and Nic2 locus; K326 nic1 / nic2-near isogenic version of flue-cured tobacco cultivar K326 containing recessive alleles Nic1 locus and Nic2 locus. Figures 1A to 1D provide an alkaloid profile of the aforementioned materials evaluated at three NC positions. Figures 2A to 2D present the yield and quality determination of these materials. Overall, in the experiment, the Cms NCLA hybrid produced the lowest nicotine level of all materials. The nornicotine level of the CmsNCLA hybrid was similar to that of K326, but slightly lower than that of K326. The levels of anatabine and anabasine of the new hybrid were lower than those of K326.

[0168] Table 1 below provides data from additional experiments. The alkaloid data highlighted in bold are approximately half of the lowest detection level of the analytical equipment because these numbers are below the detection limit. (This was done to allow analysis of variance to be performed.)

[0169]

[0170]

[0171]

[0172]

[0173] Example 2

[0174] Targeted mutagenesis using CRISPR / Cas9 to regulate nicotine biosynthesis in tobacco. This example demonstrates the use of CRISPR / Cas9 to reduce nicotine biosynthesis in tobacco plants by combining in the plant: (A) one or more genetic modifications that increase CYP82E2 and / or CYP82E3 activity; and (B) a recessive allele of nic1 and / or a recessive allele of nic2.

[0175] Methods. The nic1 recessive allele and / or the nic2 recessive allele may be derived from one of the following tobacco plant lines: LAFC53 (nic1 / nic1), MAFC5 (Nic1 / Nic1nic2 / nic2), LMAFC34 (nic1 / nic1 Nic2 / Nic2), LA Burley 21 (nic1 / nic1nic2 / nic2), LI Burley 21 (nic1 / nic1 Nic2 / Nic2), or HI Burley21 (Nic1 / Nic1 nic2 / nic2), or any tobacco plant line having a genome into which a recessive allele has been incorporated at the Nic1Nic2 locus.

[0176] Cas9 and sgRNA vectors are prepared using standard methods known in the art to introduce a K375E point mutation and / or a L422W point mutation in the CYP82E2 protein of tobacco plants and / or a C330W point mutation in the CYP82E3 protein of tobacco plants. See, for example, Schiml et al., Methods in Molecular Biology, 1469: 11 1-122 (2016). Using sequence analysis software, the expected sgRNA targeting sequence 5′ of the protospacer adjacent motif (PAM) sequence that matches the canonical form 5′-NGG can be determined.

[0177] A repair template comprising a donor DNA comprising a desired mutation (e.g., a K375E point mutation and / or a L422W point mutation relative to the wild-type CYP82E2 amino acid sequence (e.g., SEQ ID NO: 1) and / or a C330W point mutation relative to the wild-type CYP82E3 amino acid sequence (e.g., SEQ ID NO: 5)) is prepared in the form of a single-stranded DNA donor oligonucleotide or a DNA donor plasmid according to methods 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 comprising Cas9 and sgRNA and donor oligonucleotide or plasmid are transformed into Agrobacterium tumefacien. Stable Agrobacterium tumefacien-mediated transformation is carried out and transformed into one of the following strains: MAFC5, LMAFC34, LA Burley 21, LI Burley 21 or HI Burley 21 (for example, by flower dip transformation or agrobacterium infiltration method). 1 0-14 days after transformation, DNA samples are extracted from plants and mutagenic events are determined. These CRISPR / Cas induced mutations can be identified by, for example, PCR / restriction enzyme assays, Surveyor nuclease assays and / or sequencing.

[0178] Results: It is predicted that a genetically engineered tobacco plant comprising a nic1 recessive allele and / or a nic2 recessive allele and a K375E point mutation and / or a L422W point mutation and / or a C330W point mutation in the CYP82E2 protein and / or a C330W point mutation in the CYP82E3 protein of the tobacco plant will have reduced accumulation of nicotinic alkaloids such as nicotine, nornicotine, anatabine and / or anabasine as compared to a corresponding naturally occurring or non-transformed control tobacco plant or plants.

[0179] Therefore, these results will show that the methods of the present technology can be used to produce tobacco plants with reduced nicotinic alkaloid content, wherein one or more genetic modifications that increase the activity of CYP82E2 and / or CYP82E3 are combined with a recessive allele of nic1 and / or a recessive allele of nic2.

[0180] Biological deposits

[0181] Regeneration material of breeding lines SC58 CsCs and SC58 CtCt are deposited separately in the Department of Crop and Soil Sciences, North Carolina State University, Raleigh, NC 27695. SC58 CsCs, identified as LA K326 CsCs, is deposited under accession number GH21-190-17. SC58 CtCt, identified as LA K326 CtCt, is deposited under accession number GH21-191-13. Third-party experts with prior approval from NCSU may obtain samples of biological material from either line by making a written request to the Director, Office of Research Commercialization, North Carolina State University, located in the 2nd Floor of the Bolton Innovation Center, 1021 Main Campus Boulevard, Raleigh, NC 27606.

[0182] Except as permitted by 37 CFR §1.808(b), upon issuance of a patent derived from this application or any application citing this application for priority purposes, all restrictions imposed on publicly available deposited biological material will be irrevocably removed.

Claims

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

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

3. The tobacco cultivar 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. The tobacco cultivar of any one of claims 1 to 3, wherein the at least one nicotinic alkaloid is nicotine, and wherein the cultivar contains no more than 0.35% nicotine.

5. The tobacco cultivar of any one of claims 1 to 4, wherein the at least one nicotinic alkaloid is nornicotine, and wherein the cultivar contains no more than 0.04% nornicotine.

6. The tobacco cultivar of any one of claims 1 to 5, wherein the at least one nicotinic alkaloid is anatabine, and wherein the cultivar contains no more than 0.06% anatabine.

7. The tobacco cultivar of any one of claims 1 to 6, wherein the at least one nicotinic alkaloid is anabasine, and wherein the cultivar contains no more than 0.008% anabasine.

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

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

10. The tobacco cultivar of any one of claims 1 to 9, wherein the cultivar comprises a nic2 allele having reduced expression and / or function compared to wild-type nic2.

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

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

13. The tobacco cultivar of claim 12, wherein the CYP82E2 allele is derived from Nicotiana tabacum.

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

15. The tobacco cultivar of claim 14, wherein the CYP82E3 allele is derived from Nicotiana villosa.

16. The tobacco cultivar of any one of claims 12 to 15, wherein the nicotine N-demethylating activity of the protein encoded by the CYP82E2 allele and / or the CYP82E3 allele is increased compared to a naturally occurring tobacco plant.

17. A progeny plant, seed or cell produced from any one of the tobacco cultivars of claims 1 to 16.

18. A tobacco product derived from any of the tobacco cultivars or parts thereof according to claims 1 to 16.

19. The tobacco product of claim 18, wherein the product is selected from the group consisting of leaf tobacco, shredded tobacco, raw cut tobacco, tobacco dust, tobacco powder, tobacco extract, smokeless tobacco, moist or dry snuff, pipe tobacco, cigar tobacco, cigarillo tobacco, cigarette tobacco, and chewing tobacco.

20. The tobacco product of claim 18, wherein the product is selected from the group consisting of cigarillos, clove cigarettes, unventilated recessed filter cigarettes, ventilated recessed filter cigarettes, cigars, snuff, tobacco-containing gum, tobacco-containing lozenges, and chewing tobacco.

21. A method of producing a tobacco cultivar comprising a reduced level of at least one nicotinic alkaloid compared to a corresponding naturally occurring tobacco plant or part thereof, the method comprising: crossing a first tobacco variety comprising a first low-nicotine trait with a second tobacco variety comprising a second low-nicotine trait to produce progeny plants; Wherein the progeny plants comprise a reduced concentration of at least one nicotinic alkaloid.

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

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

24. The method of any one of claims 21 to 23, wherein the at least one nicotinic alkaloid is nicotine, and wherein the cultivar contains no more than 0.35% nicotine.

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

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

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

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

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

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

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

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

33. The method of claim 32, wherein the CYP82E2 allele is derived from Nicotiana tabacum.

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

35. The method of claim 34, wherein the CYP82E3 allele is derived from Nicotiana vulgaris.

36. The method of any one of claims 32 to 35, wherein the nicotine N-demethylating activity of the protein encoded by the CYP82E2 allele and / or the CYP82E3 allele is increased compared to a naturally occurring tobacco plant.

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

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

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

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

41. The tobacco product of claim 40, wherein the product is selected from the group consisting of leaf tobacco, shredded tobacco, raw cut tobacco, tobacco dust, tobacco powder, tobacco extracts, smokeless tobacco, moist or dry snuff, pipe tobacco, cigar tobacco, cigarillo tobacco, cigarette tobacco, and chewing tobacco.

42. The tobacco product of claim 40, wherein the product is selected from the group consisting of cigarillos, clove cigarettes, unventilated recessed filter cigarettes, ventilated recessed filter cigarettes, cigars, snuff, tobacco-containing gum, tobacco-containing lozenges, and chewing tobacco.

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

1.

44. The tobacco cultivar of claim 43, wherein: (a) the CYP82E2 allele comprises a nucleotide sequence encoding a CYP82E2 protein, and the CYP82E2 protein comprises an amino acid sequence as shown in SEQ ID NO: 2; (b) the CYP82E2 allele comprises a nucleotide sequence encoding a CYP82E2 protein, and the CYP82E2 protein comprises an amino acid sequence as shown in SEQ ID NO: 3; or (c) The CYP82E2 allele comprises a nucleotide sequence encoding a CYP82E2 protein, and the CYP82E2 protein comprises an amino acid sequence as shown in SEQ ID NO:

4.

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

5.

46. ​​The tobacco cultivar 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. The tobacco cultivar of any one of claims 43-46, wherein the cultivar further comprises inhibition of expression of at least one of the following within the cultivar: NBB1, A622, quinolinate phosphoribosyltransferase (QPT), putrescine N-methyltransferase (PMT), ornithine decarboxylase (ODC), aspartate oxidase (AO), quinolinate synthase (QS), N-methylputrescine oxidase (MPO), NtERF221, NtMYC1a, NtMYC1b, NtMYC-2a, or NtMYC2b.

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

1.

49. The method of claim 48, wherein: (a) the CYP82E2 allele comprises a nucleotide sequence encoding a CYP82E2 protein, and the CYP82E2 protein comprises an amino acid sequence as shown in SEQ ID NO: 2; (b) the CYP82E2 allele comprises a nucleotide sequence encoding a CYP82E2 protein, and the CYP82E2 protein comprises an amino acid sequence as shown in SEQ ID NO: 3; or (c) The CYP82E2 allele comprises a nucleotide sequence encoding a CYP82E2 protein, and the CYP82E2 protein comprises an amino acid sequence as shown in SEQ ID NO:

4.

50. The method of claim 34 or 35, wherein the CYP82E3 allele comprises a nucleotide sequence encoding a C330W point mutation relative to the wild-type CYP82E3 amino acid sequence shown in SEQ ID NO:

5.

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. A method for generating a point mutation in a target gene in a tobacco plant cell, the method comprising introducing into the cell a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (CRISPR-Cas) system, the system comprising: (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, wherein the target gene comprises a nucleotide sequence encoding a CYP82E2 protein, (b) a nucleic acid comprising a donor DNA sequence, the donor DNA sequence comprising: (i) a DNA fragment, wherein the DNA fragment replaces the target sequence in the target gene or is inserted into the target site in the target gene; and (ii) two homology arms, each arm flanking opposite sides of the DNA fragment, and (c) an effector protein, or one or more polynucleotides encoding the effector protein; wherein the at least one gRNA forms a complex with the effector protein; and The effector protein is a Cas protein comprising a nuclease.

53. The method of claim 52, wherein: (a) the point mutation is a K375E point mutation 2 or (b) The point mutation is L422W point mutation relative to the wild-type CYP82E2 amino acid sequence as shown in SEQ ID NO:

1.

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

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

1.

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

57. The method of any one of claims 52-56, further comprising inhibiting expression of at least one of the following in the tobacco plant: NBB1, A622, quinolinate 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. A method for generating a point mutation in a target gene in a tobacco plant cell, the method comprising introducing into the cell a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (CRISPR-Cas) system, the system comprising: (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, wherein the target gene comprises a nucleotide sequence encoding a CYP82E3 protein, (b) a nucleic acid comprising a donor DNA sequence, the donor DNA sequence comprising: (i) a DNA fragment, wherein the DNA fragment replaces the target sequence in the target gene or is inserted into the target site in the target gene; and (ii) two homology arms, each arm flanking opposite sides of the DNA fragment, and (c) an effector protein, or one or more polynucleotides encoding the effector protein; wherein the at least one gRNA forms a complex with the effector protein; and The effector protein is a Cas protein comprising a nuclease.

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

5.

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

61. The method of any one of claims 58-60, further comprising inhibiting expression of at least one of the following in the tobacco plant: NBB1, A622, quinolinate 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. A method of producing a tobacco plant having a reduced nicotinic alkaloid content, the method comprising combining in a tobacco 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, Wherein the tobacco plant has a reduced nicotinic alkaloid content compared to a corresponding naturally occurring or non-transformed control tobacco plant.

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

64. The method of claim 62 or claim 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 techniques or technologies, and any combination thereof.

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

1.

66. The method of any one of claims 62-64, wherein the one or more genetic modifications that increase CYP82E3 activity comprise a point mutation in a target gene in a tobacco plant cell, wherein the point mutation is a C330W point mutation relative to the wild-type CYP82E3 amino acid sequence as shown in SEQ ID NO:

5.

67. The method of claim 65 or claim 66, wherein 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), 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, wherein the target gene comprises a nucleotide sequence encoding a CYP82E2 protein, (b) a nucleic acid comprising a donor DNA sequence, the donor DNA sequence comprising: (i) a DNA fragment, wherein the DNA fragment replaces the target sequence in the target gene or is inserted into the target site in the target gene; and (ii) two homology arms, each arm flanking opposite sides of the DNA fragment, and (c) an effector protein, or one or more polynucleotides encoding the effector protein; wherein the at least one gRNA forms a complex with the effector protein; and The effector protein is a Cas protein comprising a nuclease.

68. The method of any one of claims 62-67, further comprising inhibiting expression of at least one of the following in the tobacco plant: NBB1, A622, quinolinate phosphoribosyltransferase (QPT), putrescine N-methyltransferase (PMT), ornithine decarboxylase (ODC), aspartate oxidase (AO), quinolinate synthase (QS), N-methylputrescine oxidase (MPO), NtERF221, NtMYC1a, NtMYC1b, NtMY2a or NtMYC2b.

69. A tobacco plant produced by the method of any one of claims 62-68, wherein the plant comprises: (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.

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 the activity of CYP82E2 and / or CYP82E3; and (B) a recessive allele of nic1 and / or a recessive allele of nic2.

71. A tobacco product comprising tobacco from a tobacco plant as described in claim 69, wherein the plant comprises: (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.

72. The tobacco product of claim 71, wherein: (a) the tobacco is selected from the group consisting of leaf tobacco, shredded tobacco, raw cut tobacco, tobacco dust, tobacco powder, tobacco extract, smokeless tobacco, moist or dry snuff, pipe tobacco, cigar tobacco, cigarillo tobacco, cigarette tobacco, and chewing tobacco; or (b) the product is a reduced-nicotine tobacco product selected from the group consisting of cigarillos, clove cigarettes, unventilated recessed-filter cigarettes, ventilated recessed-filter cigarettes, cigars, snus, snus, tobacco-containing chewing gum, tobacco-containing lozenges, and chewing tobacco.

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