Methods for producing terpenes in plants

Abstract

Compositions and methods are provided for modifying the secondary metabolic function of glandular trichomes in plants such as tobacco or cannabis, which controls the formation of terpenes that give plant leaves specific aroma and fragrance characteristics. Enhanced terpene production and composition is achieved through targeted modification in the biochemical synthesis pathway of menthol and cis-abienol. The present application provides novel nucleotide sequences that code the enzymology for the production of these terpenes in tobacco and cannabis, and their application in plants. TIFF2024505755000019.tif168167

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION OF SEQUENCE LISTING This application claims the benefit of U.S. Provisional Patent Application No. 63 / 145,259, filed February 3, 2021; U.S. Provisional Patent Application No. 63 / 145,262, filed February 3, 2021; and U.S. Provisional Patent Application No. 63 / 145,263, filed February 3, 2021, all of which are incorporated by reference in their entireties. This application also includes a sequence listing, which has been submitted electronically in ASCII format and is incorporated by reference in its entirety. The ASCII copy, created on January 25, 2022, is named P34829WO00_SL.txt and is 744,449 bytes in size as measured on Microsoft Windows®.

[0002] Field The present disclosure relates to terpenoid biosynthetic genes and their genetic manipulation in plants, including tobacco and cannabis.

[0003] array Table 1 provides the nucleic acid and amino acid sequences used in this disclosure.

[0004] Table 1: Sequences used in this disclosure TIFF2024505755000002.tif91162TIFF2024505755000003.tif227162TIFF2024505755000004.tif231162TIFF20245057550 00005.tif229162TIFF2024505755000006.tif228162TIFF2024505755000007.tif224162TIFF2024505755000008.tif241162 [Background technology]

[0005] background Plants can produce a wide variety of secondary metabolites, many of which are produced in aboveground structures called glandular trichomes. Terpenes, produced by a wide variety of plants and some insects, represent a large and diverse class of organic compounds composed of the five-carbon building block isoprene. Terpenoids are modified terpenes that contain additional functional groups.

[0006] Terpenoids comprise the largest and most diverse family of secondary metabolites present in plants. Terpenoids, which can be cyclic or acyclic, vary in size from 5-carbon hemiterpenes to long complex molecules containing thousands of isoprene units. The olefinic backbone of terpenoids is made up of multiples of 5-carbon (C) isoprene units, with the major groups being monoterpenes (10C), sesquiterpenes (15C), and diterpenes (20C). These terpenoids are produced through the condensation of 5-C isoprene units (dimethylallyl diphosphate [DMAPP] and isopentenyl diphosphate [IPP]) with each other, mostly by sequential head-to-tail addition of DMAPP to IPP.

[0007] To improve the aroma and flavor profiles of downstream plant-based products, terpenoid levels in plants such as tobacco and cannabis can be enhanced and modified by targeted manipulation of gene expression of genes in the terpene biosynthetic pathway. Summary of the Invention

[0008] overview In one aspect, the present disclosure provides a modified tobacco plant, tobacco seed or part thereof, or a modified cannabis plant, cannabis seed or part thereof, comprising a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter.

[0009] In one aspect, the disclosure provides a modified tobacco plant, tobacco seed or portion thereof, or a modified cannabis plant, cannabis seed or portion thereof, comprising at least one non-naturally occurring mutation in an endogenous gene encoding a polypeptide involved in the biosynthesis of at least one terpene, wherein the modified tobacco plant or modified cannabis plant exhibits increased mRNA expression of the endogenous gene or increased accumulation of the polypeptide compared to a control tobacco plant or control cannabis plant lacking the at least one non-naturally occurring mutation when grown under equivalent conditions.

[0010] In one aspect, the disclosure provides a modified tobacco plant, tobacco seed or part thereof, or a modified cannabis plant, cannabis seed or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene.

[0011] In one aspect, the disclosure provides a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter.

[0012] In one aspect, the disclosure provides a method of producing a modified tobacco plant or a modified cannabis plant, comprising: (a) introducing a recombinant nucleic acid molecule into at least one tobacco or cannabis cell, wherein the recombinant nucleic acid molecule comprises a nucleic acid sequence encoding a polypeptide involved in terpene biosynthesis operably linked to a heterologous promoter; (b) selecting at least one tobacco or cannabis cell comprising the recombinant nucleic acid molecule; and (c) regenerating a modified tobacco or cannabis plant from the at least one tobacco or cannabis cell selected in step (b), wherein the modified tobacco or cannabis plant comprises an increased amount of at least one terpene in at least one tissue compared to a control tobacco plant lacking the recombinant nucleic acid molecule when grown under equivalent conditions.

[0013] In one aspect, the disclosure provides a method of producing a modified tobacco plant or a modified cannabis plant comprising the steps of: (a) inducing in at least one tobacco or cannabis cell at least one non-naturally occurring mutation in an endogenous gene encoding a polypeptide involved in terpene biosynthesis; (b) selecting at least one tobacco or cannabis cell comprising the at least one non-naturally occurring mutation from step (a); and (c) regenerating a modified tobacco or cannabis plant from the at least one tobacco or cannabis cell selected in step (b), wherein the modified tobacco or cannabis plant comprises an increased amount of at least one terpene in at least one tissue compared to a control tobacco or cannabis plant lacking the at least one non-naturally occurring mutation when grown under equivalent conditions.

[0014] In one aspect, the disclosure provides a method of producing a modified tobacco plant or a modified cannabis plant, comprising: (a) introducing a recombinant DNA construct into at least one tobacco or cannabis cell, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene; (b) selecting at least one tobacco or cannabis cell comprising the recombinant DNA construct; and (c) regenerating at least one modified tobacco or cannabis plant from the at least one tobacco or cannabis cell selected in step (b), wherein the modified tobacco or cannabis plant comprises a reduced amount of at least one terpene in at least one tissue compared to a control tobacco or cannabis plant lacking the recombinant DNA construct when grown under equivalent conditions.

[0015] In one aspect, the disclosure provides a method comprising preparing a tobacco product or cannabis product using cured tobacco or cannabis material from a modified tobacco or cannabis plant, wherein the modified tobacco or cannabis plant comprises a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter.

[0016] In one aspect, the disclosure provides a method comprising preparing a tobacco product or cannabis product using cured tobacco or cannabis material from a modified tobacco or cannabis plant, wherein the modified tobacco or cannabis plant comprises at least one non-naturally occurring mutation in an endogenous gene encoding a polypeptide involved in the biosynthesis of at least one terpene, and wherein the modified tobacco or cannabis plant exhibits increased mRNA expression of the endogenous gene or increased accumulation of the polypeptide compared to a control tobacco or cannabis plant lacking the at least one non-naturally occurring mutation when grown under equivalent conditions.

[0017] In one aspect, the disclosure provides a method comprising preparing a tobacco or cannabis product using cured tobacco or cannabis material from a modified tobacco or cannabis plant, wherein the modified tobacco or cannabis plant comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid molecule encoding at least one small RNA molecule capable of binding to and reducing expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene.

[0018] In one aspect, the disclosure provides a method comprising transforming a tobacco or cannabis cell with a recombinant nucleic acid molecule, the recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter.

[0019] In one aspect, the disclosure provides a method comprising transforming a tobacco or cannabis cell with a recombinant nucleic acid molecule, the recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene.

[0020] In one aspect, the disclosure provides a method for producing a tobacco plant or a cannabis plant, comprising: (a) crossing at least one tobacco or cannabis plant of a first tobacco or cannabis variety with at least one tobacco or cannabis plant of a second tobacco or cannabis variety to produce at least one progeny tobacco or cannabis seed, wherein the at least one tobacco or cannabis plant of the first tobacco or cannabis variety comprises a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter; and (b) selecting at least one progeny tobacco or cannabis seed, or a plant germinated therefrom, wherein the at least one progeny tobacco or cannabis seed, or a plant germinated therefrom, comprises the recombinant nucleic acid molecule.

[0021] In one aspect, the disclosure provides a method for producing a tobacco plant or a cannabis plant, comprising the steps of: (a) crossing at least one tobacco or cannabis plant of a first tobacco or cannabis variety with at least one tobacco or cannabis plant of a second tobacco or cannabis variety to produce at least one progeny tobacco or cannabis seed, wherein the at least one tobacco or cannabis plant of the first tobacco or cannabis variety comprises at least one non-naturally occurring mutation in an endogenous gene encoding a polypeptide involved in the biosynthesis of at least one terpene, and wherein the at least one tobacco or cannabis plant of the first tobacco or cannabis variety exhibits increased mRNA expression of the endogenous gene or increased accumulation of the polypeptide when grown under equivalent conditions, compared to a control tobacco or cannabis plant of the first tobacco or cannabis variety lacking the at least one non-naturally occurring mutation; and (b) selecting at least one progeny tobacco or cannabis seed, or a plant germinated therefrom, wherein the at least one progeny tobacco or cannabis seed, or a plant germinated therefrom, comprises at least one non-naturally occurring mutation.

[0022] In one aspect, the disclosure provides a method for producing a tobacco plant or a cannabis plant, comprising: (a) crossing at least one tobacco or cannabis plant of a first tobacco or cannabis variety with at least one tobacco or cannabis plant of a second tobacco or cannabis variety to produce at least one progeny tobacco or cannabis seed, wherein the at least one tobacco or cannabis plant of the first tobacco or cannabis variety comprises a recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a nucleic acid molecule encoding at least one small RNA molecule capable of binding to and reducing expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene; and (b) selecting at least one progeny tobacco or cannabis seed, or a plant germinated therefrom, wherein the at least one progeny tobacco or cannabis seed, or a plant germinated therefrom, comprises the recombinant nucleic acid molecule. [Brief description of the drawings]

[0023] [Figure 1] Depicts the phenylpropanoid, cytoplasmic mevalonate (MVA), plastidic methylerythritol phosphate (MEP), and polyketide biosynthetic pathways in plants. Precursor transport is represented by dashed arrows, while direct catalysis is depicted by bold arrows. Abbreviations used: IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; MVA, mevalonate; MEP, methylerythritol phosphate. [Diagram 2] 1 depicts the menthol biosynthetic pathway in plants. [Diagram 3] Depicting the cis-abienol biosynthetic pathway in plants. [Figure 4] 1 depicts the cembratrienediol biosynthetic pathway in plants. [Diagram 5] 1 depicts the levopimaric acid biosynthetic pathway in plants. [Figure 6] 1 depicts the L-leucine biosynthetic pathway in plants. [Figure 7] 1 depicts gene expression in trichomes of terpene biosynthetic genes. [Figure 8] Includes panels A and B. Panel A depicts the abundance of NtNMD transcripts generated by RNA sequencing in flowers and leaves of tobacco cultivars TN90 and K326 at different plant development stages. Panel B depicts the abundance of NtABS transcripts generated by RNA sequencing in flowers and leaves of tobacco cultivars TN90 and K326 at different plant development stages (d: day; w: week). [Figure 9] FIG. 1 depicts a representative vector construct for overexpression of candidate genes in tobacco, where each promoter is operably linked to a sequence encoding the candidate gene and green fluorescent protein (G3GFP). [Figure 10]1 depicts the map of pGWB551, a representative vector that can be used for overexpression and knockdown of candidate terpene biosynthetic genes in tobacco. [Figure 11] The figures, including panels A, B, C, D, E, F, and G, depict green fluorescent protein (GFP) expression (driven by the CaMV 35S promoter) in the calli of transgenic tobacco plant lines. FIG. 11A shows geranylgeranyl diphosphate synthase-TN90; FIG. 11B shows geranylgeranyl diphosphate synthase-Nb; FIG. 11C shows cembratrienol synthase 2a-Nb; FIG. 11D shows neomenthol dehydrogenase-Nb; FIG. 11E shows cis-abienol synthase-TN90; FIG. 11F shows menthofuran synthase-Nb; and FIG. 11G shows GFP-TN90. Nb=N. benthamiana; TN90=N. tabacum cultivar "TN90". [Figure 12] Includes panels A and B. Panel A depicts the relative gene expression of NtaABS and panel B depicts the relative gene expression of NtABS in TO transgenic tobacco plants transformed with a cis-abienol synthase overexpression construct. NtaABS and NtABS are each operably linked to the CaMV 35S promoter. [Figure 13] FIG. 1 depicts the relative gene expression of NtNMD operably linked to the CaMV 35S promoter in T0 transgenic tobacco plants transformed with a menthone reductase overexpression construct. [Figure 14] Includes panels A and B. Panel A depicts the relative gene expression of neophytadiene in NtNMD-overexpressing T0 transgenic tobacco plants compared to T90 control plants. Panel B depicts the relative gene expression in NtABS / NtaABS-overexpressing T0 transgenic tobacco plants compared to TN90 control plants. [Figure 15]The relative gene expression levels of NtNMD (SEQ ID NO:15) in T1 transgenic plants of the tobacco line TN90 overexpressing NtNMD are depicted compared to wild-type TN90 plants (not shown). [Figure 16] The relative gene expression levels of NtaABS (SEQ ID NO:4) in T1 transgenic plants of tobacco line TN90 overexpressing NtaABS are depicted compared to wild-type TN90 plants (not shown). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Detailed Description Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Those skilled in the art will recognize that many methods can be used in the practice of this disclosure. Indeed, this disclosure is in no way limited to the methods and materials described. When a term is provided in the singular form, the inventors also contemplate aspects of this disclosure described by the plural form of that term, and vice versa. In the event of differences in terms and definitions used in references incorporated by reference, the terms used in this application shall have the definitions provided herein. Other technical terms used have their common meaning in the technical field in which they are used, as exemplified by various technical dictionaries, such as "The American Heritage® Science Dictionary" (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), "McGraw-Hill Dictionary of Scientific and Technical Terms" (6th edition, 2002, McGraw-Hill, New York), or "Oxford Dictionary of Biology" (6th edition, 2008, Oxford University Press, Oxford and New York).

[0025] All references cited herein, including, for example, all patents, published patent applications and non-patent publications, are hereby incorporated by reference in their entirety.

[0026] When a grouping of options is presented, any and all combinations of the members of that grouping of options are specifically contemplated. For example, if an item is selected from the group consisting of A, B, C, and D, the inventors specifically contemplate each option individually (e.g., A alone, B alone, etc.), as well as combinations such as A and B and D; A and C; B and C, etc. The term "and / or," when used in a list of two or more items, means any one of the listed items alone or in combination with any one or more of the other listed items. For example, the phrase "A and / or B" is intended to mean either or both A and B, i.e., A alone, B alone, or a combination of A and B. The phrase "A, B, and / or C" is intended to mean A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0027] When a range of numerical values ​​is provided herein, the range is understood to include the endpoints of the range as well as any number between the endpoints of the stated range. For example, "1-10" includes any number between 1 and 10, as well as the number 1 and the number 10.

[0028] The term "about" is used herein to mean approximately, roughly, around, or within the region thereof. When the term "about" is used in conjunction with a numerical range, it is understood to mean +10% or -10% modifying the range by extending the boundaries above and below the numerical values ​​set forth. For example, "about 100" would include 90 to 110.

[0029] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include a plurality of compounds, including mixtures thereof.

[0030] As used herein, when a term or phrase such as "about," "at least," "at least about," "at most," "less than," "more than," "within," or "as" is followed by a series or range of percentage numerical values, such term or phrase is deemed to modify each and every percentage numerical value in that series or range, regardless of whether an adverb, preposition or other modifying phrase is repeated before each and every member.

[0031] Any tobacco plant provided herein, or a part thereof, is specifically contemplated for use with any method provided herein.Similarly, any modified tobacco plant, or a part thereof, is specifically contemplated for use with any method provided herein.Any cannabis plant provided herein, or a part thereof, is specifically contemplated for use with any method provided herein.Similarly, any modified cannabis plant, or a part thereof, is specifically contemplated for use with any method provided herein.

[0032] Any nucleic acid sequence, amino acid sequence, or other composition provided herein is specifically contemplated for use with any of the methods provided herein.

[0033] Terpenes represent a large and diverse class of organic compounds consisting of the five-carbon building block isoprene. Terpenes are produced by a wide variety of plants and some insects. Terpenes often have a strong odor and may protect the plants that produce them by deterring herbivores. Terpenoids are modified terpenes that contain additional functional groups.

[0034] The largest and most diverse family of secondary metabolites in plants are the terpenoids. Terpenoids are synthesized through the condensation of 5-C isoprene units (dimethylallyl diphosphate [DMAPP] and isopentenyl diphosphate [IPP]) with each other, mostly by sequential head-to-tail addition of DMAPP to IPP. The plastid methylerythritol phosphate (MEP) pathway consists of seven steps that convert pyruvate and glyceraldehyde-3-phosphate to IPP and DMAPP. See Figure 1. The initial cyclization process is catalyzed by different terpene synthases (TPS), and mutations in the enzymes lead to variations in monoterpene structures. Terpenes can range in size from five-carbon hemiterpenes to long complex molecules containing thousands of isoprene units. Some monoterpene synthases can produce multiple products.

[0035] In addition to terpene synthases, trans-isoprenyl diphosphate synthase (IDS) is also one of the core enzymes involved in the creation of diverse terpenoids. TPS and IDS share a conserved alpha-terpenoid synthase fold and a trinuclear metal cluster for catalysis.

[0036] Monoterpenes are cyclic molecules composed of two isoprene units. They are the C 10 Monoterpenes are colorless, lipid-soluble, structurally diverse (cyclic and acyclic) volatile molecules. These molecules also play important roles as chemoattractants between plants and their pollinators, and as inhibitors in plant-plant and plant-herbivore communication, and are important components of plant defense. Monoterpene production and accumulation is generally restricted to glandular trichomes, and plants lacking such specialized structures typically accumulate only trace amounts of monoterpenes.

[0037] Terpene synthase (TPS) genes can be grouped into seven clades (TPS-a, TPS-b, TPS-c, TPS-d, TPS-e / f, TPS-g, and TPS-h), where TPS-d and TPS-h are specific to gymnosperms and the club moss Selaginalla moellendorffii, and TPS-a, TPS-b, and TPS-g are specific to angiosperms. TPS-a often contains sesquiterpene synthases and diterpene synthases. Neophytadiene is an example of an abundant sesquiterpenoid in tobacco. The TPS-b and TPS-g clades often contain monoterpene synthases. The genome of the cultivated tomato (S. lycopersicum), a close relative of tobacco, contains 44 TPS genes, 29 of which appear to be functional or potentially functional. Expression patterns of tomato TPS genes in leaf and stem trichomes differ quantitatively and qualitatively, consistent with the distinct volatile profiles of these two organs. Tissue-specific differences in gene expression in Solanaceae species are due, in part, to the distinct intra- and inter-species chemical variability observed. Many terpene synthases form multiple products from the same substrate.

[0038] The aromatic (odor) and organoleptic (taste / aroma) properties of tobacco leaf tissue can be enhanced by targeted genetic manipulation of the biosynthetic pathways that control terpenoid formation. Similarly, cannabis resin also contains a variety of mono- and sesquiterpenes. Two major biosynthetic pathways control terpenoid formation: one pathway controls the formation of menthol and related compounds (see Figure 2), and the other controls the formation of labdanoids such as cis-abienol (see Figure 3).

[0039] Targeted overexpression of genes in the menthol biosynthetic pathway can enhance menthol accumulation in tobacco plants. (-)-Menthol, a C10 monoterpene, is a characteristic constituent of Mentha piperita (peppermint), an economically important plant for the production of essential oils used for various industrial and medicinal purposes. In Mentha species, menthol biosynthesis and storage are restricted to the shield-like glandular trichomes (glandular scales) on the aboveground surface of the plant.

[0040] The biosynthesis of (-)-menthol involves eight enzymatic steps. The formation of the ubiquitous monoterpene precursor geranyl diphosphate and its subsequent cyclization to the parent olefin (-)-(4S)-limonene is the first committed enzymatic reaction in the menthol biosynthetic pathway.

[0041] Genes encoding enzymes of the menthol biosynthetic pathway include geranyl diphosphate synthase (GDP) (SEQ ID NOs: 7, 28, and 49), limonene synthase (LS) (SEQ ID NOs: 8, 29, and 50), limonene 3-hydroxylase (L3OH) (SEQ ID NOs: 10, 31, and 52), limonene synthase chloroplast isoform X1 (SEQ ID NOs: 165, 181, and 197), isopiperitenol dehydrogenase (IPD) (SEQ ID NOs: 13, 34, 55, 169, 185, and 201), pulegone reductase (SEQ ID NOs: 14, 35, and 56), menthofuran synthase (MFS) (SEQ ID NOs: 16, 37, and 58), and neomenthol dehydrogenase (NtNMD) (SEQ ID NOs: 17, 18, and 201). NO:15, 36, and 57) (see FIG. 2).

[0042] The second major terpenoid biosynthetic pathway involves the formation of labdanoids such as cis-abienol (see FIG. 3). The major labdanoids found in some aromatic plants, including some forms of tobacco, are cis-abienol and labdenediol. Cis-abienol is a diterpenoid that is abundant in some Oriental tobacco varieties, but is uncommon in commercial flue and Burley-type tobaccos such as TN90.

[0043] The biosynthesis of cis-abienol proceeds in two steps (see FIG. 3): a class II terpene synthase (8-hydroxy-copalyl diphosphate synthase) (SEQ ID NOs: 3, 24, 45, 115, 129, and 143) synthesizes 8-hydroxy-copalyl diphosphate from geranylgeranyl diphosphate, and a kaurene synthase-like (KSL) enzyme (cis-abienol synthase) (SEQ ID NOs: 4, 5, 6, 25, 26, 27, 46, 47, 48, 119, 133, and 147) converts 8-hydroxy-copalyl diphosphate to cis-abienol.

[0044] Further biosynthetic pathways used for targeted aroma enhancement include the cembratrienediol biosynthetic pathway (see FIG. 4), the levopimaric acid biosynthetic pathway (see FIG. 5), and the L-leucine biosynthetic pathway (see FIG. 6). Cembratrienol synthase 2a (SEQ ID NOs: 17, 38, 59, 114, 128, and 142) is a key enzyme in the cembratrienediol biosynthetic pathway (see FIG. 4). The diterpenes cembratrienol (CBTol) and cembratrienediol (CBTdiol) are considered important defense compounds and are produced in trichomes. Levopimaradiene synthase (SEQ ID NOs: 18, 39, and 60) is a key enzyme in the levopimaric acid biosynthetic pathway (see FIG. 5). Levopimaric acid has been reported to be involved in diterpene resin acid production in conifers. 2-Isopropylmalate synthetase (IPS) (SEQ ID NOs: 19, 40, 61, 116, 124, 130, 138, 144, 152, 175, 191, and 207) and 2-oxoisovalerate dehydrogenase (SEQ ID NOs: 20, 21, 41, 42, 62, and 63) are key enzymes in the L-leucine biosynthetic pathway (see FIG. 6). Leucine is one of the branched-chain amino acids that contribute to the aroma properties in Oriental tobacco.

[0045] In one aspect, the present disclosure provides a modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof, comprising a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter.

[0046] In a further aspect, the disclosure provides a modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof, comprising at least one non-naturally occurring mutation in an endogenous gene encoding a polypeptide involved in the biosynthesis of at least one terpene, wherein the modified tobacco or cannabis plant exhibits increased mRNA expression of the endogenous gene or increased accumulation of the polypeptide compared to a control tobacco or cannabis plant lacking the at least one non-naturally occurring mutation when grown under equivalent conditions.

[0047] Terpenes Terpenes are a class of aromatic organic compounds produced by plants and some insects. Terpenes are members of a group of volatile unsaturated hydrocarbons found in the essential oils of plants and have the formula C 10 H 16 and related structures, such as, but not limited to, those having the formula C 15 H 24 The terpene compounds are based on compounds such as sesquiterpenes having the formula: ##STR00011## or simple derivatives of such compounds. Terpenes are hydrocarbon molecules that are often used by plants to directly prevent feeding or to attract predators or parasites of plant herbivores. Non-limiting examples of terpenes include citral, menthol, camphor, salvinorin A, cannabinoids, and curcuminoids.

[0048] In cannabis, several classes of terpenes have been identified, including but not limited to myrcene, pinene, limonene, caryophyllene, linalool, terpinolene, camphene, terpineol, phellandrene, carene, humulene, pulegone, and geraniol. Myrcene, specifically beta-myrcene, is a monoterpene and is the most common terpene produced by cannabis. Pinene is a bicyclic monoterpenoid. Two naturally occurring structural isomers of pinene are known: alpha-pinene and beta-pinene. Limonene is a monocyclic monoterpenoid and is one of the two main compounds formed from pinene. Caryophyllene, specifically beta-caryophyllene, is a sesquiterpene common to many plants except cannabis, including cloves, cinnamon leaves, and pepper. Beta-caryophyllene is the only terpene known to interact with the endocannabinoid system, as it is a functional cannabinoid-2 receptor (CB2) agonist. Linalool is an acyclic monoterpenoid that is a crucial precursor in the formation of vitamin E and has been isolated from many different plants. Delta-3-carene is a bicyclic monoterpene. Alpha-terpineol, terpinen-4-ol, and 4-terpineol are three closely related monoterpenoids. Humulene is a sesquiterpene, also known as alpha-humulene. Pulegone, a monocyclic monoterpenoid, is a minor component of cannabis.

[0049] In some aspects, terpenes are terpenoids.Terpenoids (also called isoprenoids) are modified terpenes that often contain additional functional groups, including oxygen.Terpenoids, which can be cyclic or acyclic, vary in size from 5-carbon hemiterpenes to long complex molecules that contain thousands of isoprene units.Terpenoids are produced through the condensation of 5-carbon isoprene units (e.g., dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP)) with each other, mostly by sequential head-to-tail addition of DMAPP to IPP.The initial cyclization process is catalyzed by different terpene synthases, and mutations in the enzymes lead to mutations in monoterpene structures.

[0050] Terpenoids are classified according to the number of isoprene units that comprise the parent terpene: Hemiterpenoids contain one isoprene unit; Monoterpenoids contain two isoprene units; Sesquiterpenoids contain three isoprene units; Diterpenoids contain four isoprene units; Sesterterpenoids contain five isoprene units; Triterpenoids contain six isoprene units; Tetraterpenoids contain eight isoprene units; Polyterpenoids contain more than eight isoprene units.

[0051] In some aspects, the polypeptide is involved in the biosynthesis of at least one terpene. In some aspects, the polypeptide is involved in the biosynthesis of at least one terpenoid. In some aspects, the polypeptide is involved in the biosynthesis of at least one terpenoid selected from the group consisting of hemiterpenoids, monoterpenoids, sesquiterpenoids, diterpenoids, sesterterpenoids, triterpenoids, tetraterpenoids, and polyterpenoids. In some aspects, the polypeptide is involved in the biosynthesis of at least one terpene selected from the group consisting of hemiterpenes, monoterpenes, sesquiterpenes, diterpenes, sesterterpenes, triterpenes, tetraterpenes, and polyterpenes.

[0052] As used herein, the term "biosynthesis" refers to the production of complex molecules (such as, but not limited to, terpenes or terpenoids) in plants or plant cells. To "participate" in the biosynthesis of a compound, a polypeptide may directly interact with a substrate during the biosynthesis of the compound, or a polypeptide may affect (positively or negatively) the expression of a polypeptide that directly interacts with the substrate (e.g., a transcription factor that promotes the expression of an enzyme that converts a substrate to a new form, or a repressor that inhibits the expression of an enzyme that converts a substrate to a new form). Examples of biosynthetic pathways can be found in Figures 1-6. In one aspect, the transcription factor is MYB61 isoform X1. As a non-limiting example, SEQ ID NOs:208 and 176 are the amino acid and nucleic acid sequences of transcription factor MYB61 isoform X1, respectively.

[0053] In some aspects, the polypeptide is involved in the biosynthesis of hemiterpenes. In some aspects, the polypeptide is involved in the biosynthesis of hemiterpenoids. In some aspects, the polypeptide is involved in the biosynthesis of monoterpenes. In some aspects, the polypeptide is involved in the biosynthesis of monoterpenoids. In some aspects, the polypeptide is involved in the biosynthesis of sesquiterpenes. In some aspects, the polypeptide is involved in the biosynthesis of sesquiterpenoids. In some aspects, the polypeptide is involved in the biosynthesis of diterpenes. In some aspects, the polypeptide is involved in the biosynthesis of diterpenoids. In some aspects, the polypeptide is involved in the biosynthesis of sesterterpenes. In some aspects, the polypeptide is involved in the biosynthesis of sesterterpenoids. In some aspects, the polypeptide is involved in the biosynthesis of triterpenes. In some aspects, the polypeptide is involved in the biosynthesis of triterpenoids. In some aspects, the polypeptide is involved in the biosynthesis of tetraterpenes. In some aspects, the polypeptide is involved in the biosynthesis of polyterpenoids. In some aspects, the polypeptide is involved in the biosynthesis of a monoterpene. In some aspects, the polypeptide is involved in the biosynthesis of a polyterpenoid.

[0054] Terpene synthase (TPS) genes can be grouped into seven clades: TPS-a, TPS-b, TPS-c, TPS-d, TPS-e / f, TPS-g, and TPS-h. TPS-a, TPS-b, and TPS-g are restricted to angiosperms, while TPS-d and TPS-h are specific to gymnosperms and the club moss Selaginella moellendorffii. The TPS-a clade often contains sesquiterpene synthases and diterpene synthases, whereas the TPS-b and TPS-g clades often contain monoterpene synthases.

[0055] Terpene synthase-like genes In some aspects, the polypeptide involved in the biosynthesis of at least one terpenoid is a TPS-a clade member. In some aspects, the polypeptide involved in the biosynthesis of at least one terpenoid is a TPS-b clade member. In some aspects, the polypeptide involved in the biosynthesis of at least one terpenoid is a TPS-c clade member. In some aspects, the polypeptide involved in the biosynthesis of at least one terpenoid is a TPS-e / f clade member. In some aspects, the polypeptide involved in the biosynthesis of at least one terpenoid is a TPS-g clade member. In some aspects, the polypeptide involved in the biosynthesis of at least one terpenoid is a member of a clade selected from the group consisting of TPS-a, TPS-b, TPS-c, TPS-e / f and TPS-g.

[0056] In some aspects, the terpene is menthol. In some aspects, the terpene is menthol or a related compound. In some aspects, the terpene is a labdanoid. In some aspects, the terpene is a cembratrienediol. In some aspects, the terpene is levopimaric acid. In some aspects, the terpene is L-leucine. In some aspects, the terpene is neophytadiene. In some aspects, the labdanoid is cis-abienol. In some aspects, the labdanoid is labdane-diol. In some aspects, the terpene is selected from the group consisting of menthol or a related compound, labdanoid, cembratrienediol, levopimaric acid, and L-leucine. In some aspects, the terpene is selected from the group consisting of menthol or a related compound, labdanoid, cembratrienediol, levopimaric acid, L-leucine, and neophytadiene. In some aspects, the terpene is selected from the group consisting of menthol, labdanoids, cembratrienediol, levopimaric acid, and L-leucine.In some aspects, the terpene is selected from the group consisting of menthol, labdanoids, cembratrienediol, levopimaric acid, L-leucine, and neophytadiene.

[0057] In some aspects the terpene is myrcene. In some aspects the terpene is pinene. In some aspects the terpene is limonene. In some aspects the terpene is caryophyllene. In some aspects the terpene is linalool. In some aspects the terpene is terpinolene. In some aspects the terpene is camphene. In some aspects the terpene is terpineol. In some aspects the terpene is phellandrene. In some aspects the terpene is carene. In some aspects the terpene is humulene. In some aspects the terpene is pulegone. In some aspects the terpene is geraniol.

[0058] As used herein, "menthol" has the chemical formula: 10 H 20Menthol refers to an organic compound having the formula: 5-methyl-2-(propan-2-yl)cyclohexan-1-ol and the International Union of Pure and Applied Chemistry (IUPAC) name: 5-methyl-2-(propan-2-yl)cyclohexan-1-ol. Menthol is also referred to as "(-)-menthol". Menthol-related compounds include, but are not limited to, (+)-menthol, (+)-isomenthol, (+)-neomenthol, (+)-neoisomenthol, (-)-isomenthol, (-)-neomenthol, and (-)-neoisomenthol. In one aspect, the menthol-related compound is selected from the group consisting of (+)-menthol, (+)-isomenthol, (+)-neomenthol, (+)-neoisomenthol, (-)-isomenthol, (-)-neomenthol, and (-)-neoisomenthol.

[0059] As used herein, "neophytadiene" has the chemical formula: 20 H 38 and the IUPAC name: 7,11,15-trimethyl-3-methylidenehexadec-1-ene.

[0060] As used herein, "cembratrienediol" refers to a compound having the chemical formula: 20 H 34 O 2 and the IUPAC name: (1R,3R,4Z,8Z,12S,13Z)-1,5,9-trimethyl-12-propan-2-ylcyclotetradeca-4,8,13-triene-1,3-diol. Cembratriene diol is also known as "beta-cembrenediol".

[0061] As used herein, "levopimaric acid" refers to a compound having the chemical formula: 20 H 30 O 2and IUPAC name: (1R,4aR,4bS,10aR)-1,4a-dimethyl-7-propan-2-yl-2,3,4,4b,5,9,10,10a-octahydrophenanthrene-1-carboxylic acid. Levopimaric acid is also called "L-pimaric acid." In some aspects, levopimaric acid is involved in diterpene resin acid production.

[0062] As used herein, "L-leucine" has the chemical formula: 6 H 12 NO 2 and the amino acid with the IUPAC name: (2S)-2-amino-4-methylpentanoic acid.

[0063] As used herein, "labdanoid" refers to a diterpene that is a terpenoid derivative of the basic parent labdane. Labdane has the chemical formula: 20 H 38 and has the IUPAC name: (1S,2S,4aS,8aR)-2,5,5,8a-tetramethyl-1-[(3R)-3-methylpentyl]-1,2,3,4,4a,6,7,8-octahydronaphthalene.

[0064] A non-limiting example of a labdanoid is cis-abienol. As used herein, "cis-abienol" has the chemical formula: 20 H 34 It refers to the organic compound having the following IUPAC name: (1R,2R,4aS,8aS)-2,5,5,8a-tetramethyl-1-[(2Z)-3-methylpenta-2,4-dienyl]-3,4,4a,6,7,8-hexahydro-1H-naphthalen-2-ol.

[0065] As used herein, “myrcene” has the chemical formula: 10 H 16and IUPAC name: 7-methyl-3-methylene-octa-1,6-diene. Myrcene, also known as beta-myrcene or alpha-myrcene, belongs to a class of organic compounds known as acyclic monoterpenoids. Thus, myrcene is considered to be an isoprenoid lipid molecule.

[0066] As used herein, “pinene” has the chemical formula: 10 H 16 and IUPAC name: (1S,5S)-2,6,6-trimethylbicyclo[3.1.1]hept-2-ene ((-)-α-pinene). (+)-α-pinene, also known as 2-pinene or acintene a, belongs to a class of organic compounds known as bicyclic monoterpenoids. These are monoterpenoids that contain exactly two rings fused together. Alpha-pinene is pinene, a bicyclo[3.1.1]hept-2-ene, substituted at the 2, 6 and 6 positions by methyl groups, respectively. It has a role as a plant metabolite.

[0067] As used herein, “limonene” has the chemical formula: 10 H 16 and the compound with IUPAC name: 1-methyl-4-(1-methylethenyl)-cyclohexene. Limonene (+ / -)- is a racemic mixture of the naturally occurring cyclic monoterpene limonene.

[0068] As used herein, “caryophyllene” or “beta-caryophyllene” has the chemical formula: 15 H 24and IUPAC name: (1R,4E,9S)-4,11,11-trimethyl-8-methylidenebicyclo[7.2.0]undec-4-ene. Beta-caryophyllene is classified as a sesquiterpenoid. (-)-beta-caryophyllene is beta-caryophyllene in which the stereocenter adjacent to the exocyclic double bond has the S configuration and the remaining stereocenter has the R configuration. It is the most commonly occurring form of beta-caryophyllene and is present in many essential oils.

[0069] As used herein, "linalool" refers to the compound having the chemical formula: CHO and the IUPAC name: 3,7-dimethylocta-1,6-dien-3-ol. Beta-linalool, also known as 3,7-dimethyl-1,6-octadien-3-ol or 2,6-dimethylocta-2,7-dien-6-ol, is classified as an acyclic monoterpenoid.

[0070] As used herein, “terpinolene” has the chemical formula: 10 H 16 and IUPAC name: 1-methyl-4-propan-2-ylidenecyclohexene. Terpinolene, also known as alpha-terpinolene or iso-terpinene, is classified as a menthane monoterpenoid. These are monoterpenoids with structures based on the o-, m- or p-menthane skeleton.

[0071] As used herein, "camphene" has the chemical formula: 10 H 16 and IUPAC name: 2,2-dimethyl-3-methylidenebicyclo[2.2.1]heptane. Camphene is classified as a bicyclic monoterpenoid containing two fused rings. Camphene is a monoterpene with a bicyclic skeleton that is a bicyclo[2.2.1]heptane substituted at the 2-position by a geminal methyl group and at the 3-position by a methylidene group.

[0072] As used herein, "terpineol" refers to a monoterpene alcohol of which there are four isomers: alpha-terpineol, beta-terpineol, gamma-terpineol, and terpinen-4-ol. Beta-terpineol and gamma-terpineol differ only in the position of the double bond. Terpineol is usually a mixture of these isomers with alpha-terpineol as the major component. As used herein, "alpha- ... is a chemical formula: 10 H 18 It refers to the organic compound having the following IUPAC name: 2-(4-methylcyclohex-3-en-1-yl)propan-2-ol.

[0073] As used herein, "phellandrene" refers to the cyclic monoterpene "alpha-phellandrene" or "beta-phellandrene." As used herein, "alpha-phellandrene" has the chemical formula: 10 H 16 and IUPAC name: 2-methyl-5-propan-2-ylcyclohexa-1,3-diene. Alpha-phellandrene is also known as alpha-phellandren or dihydro-p-cymene. As used herein, "beta-phellandrene" refers to the organic compound having the chemical formula: 10 H 16 and IUPAC name: 3-methylidene-6-propan-2-ylcyclohexene. Beta-phellandrene is also known as beta-phellandren or 2-p-menthadiene.

[0074] As used herein, "carene" or "alphacarene" has the chemical formula: 10 H 16and IUPAC name: 3,7,7-trimethylbicyclo[4.1.0]hept-3-ene. Alpha-carene, also known as 3-carene or delta-carene, is classified as a bicyclic monoterpenoid.

[0075] As used herein, "humulene" has the chemical formula: 15 H 24 and the organic compound with the IUPAC name: (1E,4E,8E)-2,6,6,9-tetramethylcycloundeca-1,4,8-triene.

[0076] As used herein, "pulegone" has the chemical formula: 10 H 16 It refers to the organic compound with the IUPAC name: (5R)-5-methyl-2-propan-2-ylidenecyclohexan-1-one. (+)-Pulegone, also known as D-pulegone or cis-isopulegone, is classified as a menthane monoterpenoid.

[0077] As used herein, "geraniol" has the chemical formula: 10 H 18 Geraniol is an acyclic monoterpenoid and is also called 2E-geraniol, (e)-geraniol, or geranyl alcohol.

[0078] In some aspects, the polypeptide is a geranylgeranyl diphosphate synthase. In some aspects, the polypeptide is an 8-hydroxy-copalyl diphosphate synthase. In some aspects, the polypeptide is a cis-abienol synthase. In some aspects, the polypeptide is a cembratrienol synthase 2a. In some aspects, the polypeptide is a levopimaradiene synthase. In some aspects, the polypeptide is a 2-isopropylmalate synthetase. In some aspects, the polypeptide is a 2-oxoisovalerate dehydrogenase. In some aspects, the polypeptide is a geranyl diphosphate synthase. In some aspects, the polypeptide is a limonene synthase. In some aspects, the polypeptide is a limonene 3-hydroxylase. In some aspects, the polypeptide is an isopiperitenol dehydrogenase. In some aspects, the polypeptide is a pulegone reductase. In some aspects, the polypeptide is a menthofuran synthase. In some aspects, the polypeptide is a neomenthol dehydrogenase. In some aspects, the polypeptide is a geranylgeranyl pyrophosphate synthase. In some aspects, the polypeptide is a kolavenyl diphosphate synthase. In some aspects, the polypeptide is a solanesyl diphosphate synthase. In some aspects, the polypeptide is a terpene synthase. In some aspects, the polypeptide is a 2-alkenal reductase. In some aspects, the polypeptide is a germacrene synthase. In some aspects, the polypeptide is a cytochrome P450. In some aspects, the polypeptide is a carveol dehydrogenase. In some aspects, the polypeptide is a MYB61 transcription factor.

[0079] In one aspect, the polypeptide is selected from the group consisting of geranyl diphosphate synthase (GDP), limonene synthase (LS), limonene 3-hydroxylase (L3OH), isopiperitenol dehydrogenase (IPD), pulegone reductase, menthofuran synthase, and GGPPS neomenthol dehydrogenase (NtNMD).

[0080] In some aspects, the polypeptide is selected from the group consisting of geranylgeranyl diphosphate synthase (GGPPS2), 8-hydroxy-copalyl diphosphate synthase, and cis-abienol synthase.

[0081] In certain aspects, the polypeptide is selected from the group consisting of geranylgeranyl diphosphate synthase, 8-hydroxy-copalyl diphosphate synthase, cis-abienol synthase, cembratrienol synthase 2a, levopimaradiene synthase, 2-isopropylmalate synthetase, 2-oxoisovalerate dehydrogenase, geranyl diphosphate synthase, limonene synthase, limonene 3-hydroxylase, isopiperitenol dehydrogenase, pulegone reductase, menthofuran synthase, neomenthol dehydrogenase, phylloplanin, and premnaspirodiene oxygenase.

[0082] In some aspects, the polypeptide is selected from the group consisting of geranylgeranyl pyrophosphate synthase, kolavenyl diphosphate synthase, solanesyl diphosphate synthase, terpene synthase, limonene synthase, isopipeperitenol dehydrogenase, 2-alkenal reductase, germacrene synthase, 2-isopropylmalate synthase, and MYB61.

[0083] In certain aspects, the polypeptide is selected from the group consisting of geranylgeranyl diphosphate synthase, 8-hydroxy-copalyl diphosphate synthase, cis-abienol synthase, cembratrienol synthase 2a, levopimaradiene synthetase, 2-isopropylmalate synthetase, 2-oxoisovalerate dehydrogenase, geranyl diphosphate synthase, limonene synthase, limonene 3-hydroxylase, isopiperitenol dehydrogenase, pulegone reductase, menthofuran synthase, neomenthol dehydrogenase, phylloplanin, premnaspirodiene oxygenase, geranylgeranyl pyrophosphate synthase, kolavenyl diphosphate synthase, solanesyl diphosphate synthase, terpene synthase, 2-alkenal reductase, germacrene synthase, and MYB61.

[0084] As a non-limiting example, SEQ ID NOs:45 and 3 are representative of the amino acid and nucleic acid sequences of 8-hydroxy-copalyl diphosphate synthase, respectively. As a non-limiting example, SEQ ID NOs:59 and 17 are representative of the amino acid and nucleic acid sequences of cembratrienol synthase 2a, respectively. As a non-limiting example, SEQ ID NOs:60 and 18 are representative of the amino acid and nucleic acid sequences of levopimaradiene synthase, respectively. As a non-limiting example, SEQ ID NOs:61 and 207 are representative of the amino acid sequence of 2-isopropylmalate synthase. As a non-limiting example, SEQ ID NOs:61 and 174 are representative of the nucleic acid sequence of 2-isopropylmalate synthase.

[0085] As a non-limiting example, SEQ ID NOs:160 and 158 are representative of the amino acid and nucleic acid sequences of Premna spirodiene oxygenase (Gene id g89817), respectively. As a non-limiting example, SEQ ID NOs:157 and 155 are representative of the amino acid and nucleic acid sequences of phylloplanin (Gene id g62191), respectively. As a non-limiting example, SEQ ID NOs:194 and 164 are representative of the amino acid and nucleic acid sequences of terpene synthase, respectively.

[0086] In some aspects, geranylgeranyl diphosphate synthase is a component of the cis-abienol biosynthetic pathway. As a non-limiting example, SEQ ID NOs: 43 and 1 are representative of the amino acid and nucleic acid sequences of geranylgeranyl diphosphate synthase, respectively. In some aspects, geranylgeranyl pyrophosphate synthase is a component of the cis-abienol biosynthetic pathway. As a non-limiting example, SEQ ID NOs: 44 and 191 are representative of the amino acid sequence of geranylgeranyl pyrophosphate synthase. As a non-limiting example, SEQ ID NOs: 2 and 161 are representative of the nucleic acid sequence of geranylgeranyl pyrophosphate synthase.

[0087] In some aspects, 2-oxoisovalerate dehydrogenase is a component of the L-leucine biosynthesis pathway in plants.As a non-limiting example, SEQ ID NOs:62 and 20 are representative of the amino acid and nucleic acid sequences of dehydrogenase subunit alpha (Gene id g50844), respectively.As a non-limiting example, SEQ ID NOs:63 and 21 are representative of the amino acid and nucleic acid sequences of 2-oxoisovalerate dehydrogenase subunit alpha (Gene id g63865), respectively.

[0088] In some aspects, the cis-abienol synthase is a component of the cis-abienol biosynthetic pathway. As a non-limiting example, SEQ ID NO:147 and 119 are representative of the amino acid and nucleic acid sequences of cis-abienol synthase, respectively. In some aspects, the cis-abienol synthase is selected from the group consisting of cis-abienol synthase isoform 1 and cis-abienol synthase isoform 2. In some aspects, the cis-abienol synthase is selected from the group consisting of cis-abienol synthase isoform 1, cis-abienol synthase isoform 2, and Ent-kaurene synthase. In some aspects, the cis-abienol synthase is cis-abienol synthase isoform 1. In some aspects, the cis-abienol synthase is cis-abienol synthase isoform 2. In one aspect, the cis-abienol synthase is an ent-kaurene synthase. As a non-limiting example, SEQ ID NOs:46 and 4 are representative of the amino acid and nucleic acid sequences of cis-abienol synthase isoform 1, respectively. As a non-limiting example, SEQ ID NOs:47 and 5 are representative of the amino acid and nucleic acid sequences of cis-abienol synthase isoform 2, respectively. As a non-limiting example, SEQ ID NOs:48 and 6 are representative of the amino acid and nucleic acid sequences of ent-kaurene synthase, respectively.

[0089] In one aspect, kolavenyl diphosphate synthase catalyzes the first reaction of Salvinorin A biosynthesis, which is the formation of (-)-kolavenyl diphosphate ((-)-KPP). (-)-KPP is then dephosphorylated to produce (-)-kolavenol. As a non-limiting example, SEQ ID NO:192 and 162 are representative of the amino acid and nucleic acid sequences of kolavenyl diphosphate synthase, respectively.

[0090] In some aspects, NAD(P)-dependent oxidoreductases catalyze the NADPH-dependent reduction of a wide range of substrates. In some aspects, the oxidoreductase protein contains an oxidoreductase domain. In some aspects, SDR family oxidoreductases share a conserved oxidoreductase domain with identified [mammalian] carbonyl reductases. In some aspects, an example of a protein containing an SDR family oxidoreductase conserved domain is the uncharacterized protein LOC115704491. As a non-limiting example, SEQ ID NOs:201 and 171 are representative of the amino acid and nucleic acid sequences of the uncharacterized protein LOC115704491, respectively.

[0091] In one aspect, another example of the enzyme in the oxidoreductase family is 2-alkenal reductase. The substrates of 2-alkenal reductase are n-alkanal, NAD+ and NADP+. 2-alkenal reductase is also called NADPH-dependent alkenal / one oxidoreductase. As a non-limiting example, SEQ ID NO:200 and 170 are representative of the amino acid and nucleic acid sequences of 2-alkenal reductase, respectively.

[0092] In one aspect, cytochrome P450 (CYP) is a large enzyme family that catalyzes the hydroxylation of a variety of terpene substrates. In one aspect, Premna spirodiene oxygenase (PSO) (SEQ ID NOs: 158 and 160) catalyzes the hydroxylation of a variety of sesquiterpene substrates. The hydroxylation reactions catalyzed by various cytochrome P450s are regiospecific and stereospecific. As non-limiting examples, SEQ ID NOs: 31-33, 37, 159, 166-168, and 172 are representative of nucleic acid sequences of cytochrome P450. As non-limiting examples, SEQ ID NOs: 52-54, 58, 160, 196-198, and 202 are representative of amino acid sequences of cytochrome P450.

[0093] In some aspects, the germacrene D synthase is a sesquiterpene synthase that catalyzes the formation of germacrene D. In some aspects, the germacrene D synthase is a (-)-germacrene D synthase. As non-limiting examples, SEQ ID NOs:203 and 173 are representative of the amino acid and nucleic acid sequences, respectively, of a (-)-germacrene D synthase.

[0094] In some aspects, modified plants, seeds or plant parts comprising the recombinant nucleic acids provided herein comprise an increased amount of at least one terpene when grown under equivalent conditions, compared to a control plant, seed or plant part lacking the recombinant nucleic acid molecule. In some aspects, modified tobacco or cannabis plants, tobacco or cannabis seeds, or tobacco plant parts comprising the recombinant nucleic acids provided herein comprise an increased amount of at least one terpene when grown under equivalent conditions, compared to a control tobacco or cannabis plant, tobacco or cannabis seeds, or tobacco or cannabis plant part lacking the recombinant nucleic acid molecule.

[0095] In some aspects, the increased amount of the at least one terpene comprises an increase of at least 0.5%. In some aspects, the increased amount of the at least one terpene comprises an increase of at least 1%. In some aspects, the increased amount of the at least one terpene comprises an increase of at least 2%. In some aspects, the increased amount of the at least one terpene comprises an increase of at least 3%. In some aspects, the increased amount of the at least one terpene comprises an increase of at least 4%. In some aspects, the increased amount of the at least one terpene comprises an increase of at least 5%. In some aspects, the increased amount of the at least one terpene comprises an increase of at least 10%. In some aspects, the increased amount of the at least one terpene comprises an increase of at least 12.5%. In some aspects, the increased amount of the at least one terpene comprises an increase of at least 15%. In some aspects, the increased amount of the at least one terpene comprises an increase of at least 17.5%. In some aspects, the increased amount of the at least one terpene comprises an increase of at least 20%. In some aspects, the increased amount of at least one terpene includes at least a 25% increase. In some aspects, the increased amount of at least one terpene includes at least a 30% increase. In some aspects, the increased amount of at least one terpene includes at least a 40% increase. In some aspects, the increased amount of at least one terpene includes at least a 50% increase. In some aspects, the increased amount of at least one terpene includes at least a 60% increase. In some aspects, the increased amount of at least one terpene includes at least a 70% increase. In some aspects, the increased amount of at least one terpene includes at least an 80% increase. In some aspects, the increased amount of at least one terpene includes at least a 90% increase. In some aspects, the increased amount of at least one terpene includes at least a 100% increase. In some aspects, the increased amount of at least one terpene includes at least a 150% increase. In some aspects, the increased amount of at least one terpene includes at least a 200% increase. In one aspect, the increased amount of at least one terpene comprises an increase of at least 250%.In one aspect, the increased amount of at least one terpene comprises an increase of at least 500%.

[0096] In some aspects, the increased amount of the at least one terpene includes an increase of 0.5% to 500%. In some aspects, the increased amount of the at least one terpene includes an increase of 0.5% to 250%. In some aspects, the increased amount of the at least one terpene includes an increase of 0.5% to 100%. In some aspects, the increased amount of the at least one terpene includes an increase of 0.5% to 75%. In some aspects, the increased amount of the at least one terpene includes an increase of 0.5% to 50%. In some aspects, the increased amount of the at least one terpene includes an increase of 0.5% to 25%. In some aspects, the increased amount of the at least one terpene includes an increase of 0.5% to 10%. In some aspects, the increased amount of the at least one terpene includes an increase of 0.5% to 5%. In some aspects, the increased amount of the at least one terpene includes an increase of 0.5% to 500%. In some aspects, the increased amount of the at least one terpene includes an increase of 5% to 250%. In some aspects, the increased amount of the at least one terpene includes an increase of 5% to 100%. In some aspects, the increased amount of the at least one terpene includes an increase of 5% to 50%. In some aspects, the increased amount of the at least one terpene includes an increase of 25% to 500%. In some aspects, the increased amount of the at least one terpene includes an increase of 25% to 250%. In some aspects, the increased amount of the at least one terpene includes an increase of 50% to 100%. In some aspects, the increased amount of the at least one terpene includes an increase of 100% to 500%.

[0097] Terpenes can be extracted using any method known in the art, such as solvent extraction.See, for example, Jiang et al., Curr Protoc Plant Biol., 1:345-358 (2016). Solvent-free extraction methods are also known in the art.See, for example, Yang and Xie, Science Direct, 162: 135-139 (2006).

[0098] The amount of terpene in plant can be measured by any method known in the art, including but not limited to gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry.See The Handbook of Plant Metabolomics, edited by Weckwerth and Kahl, (Wiley-Blackwell) (May 28, 2013).In some aspects, the amount of at least one terpene refers to the concentration of that terpene in the tissue that is collected.

[0099] In one aspect, the modified tobacco or cannabis plant provided herein further comprises a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide operably linked to a heterologous promoter, wherein the polypeptide causes an increase in average trichome density compared to a control tobacco or cannabis plant lacking the transgene or modification when grown under equivalent conditions.Non-limiting examples of genes that can be overexpressed to increase average trichome density include NtGIS, NtMYB86, NbGIS, cannabis MYB61, cannabis GIS3, and cannabis non-specific lipid transfer protein 1-like.

[0100] Cannabinoids Cannabinoids are naturally occurring compounds found in the Cannabis plant. Many cannabinoids are concentrated in the resin produced in the glandular trichomes, and at least 113 cannabinoids are known. The two major cannabinoids are tetrahydrocannabinol (THC) and cannabidiol (CBD), but over 100 additional cannabinoids have been identified. CBD constitutes approximately 40% of the plant resin extract.

[0101] Cannabinoids exert their effects by interacting with specific cannabinoid receptors. The two major types of cannabinoid receptors in humans are cannabinoid receptor type 1 (CB1) and cannabinoid receptor type 2 (CB2).

[0102] In some aspects, the heterologous polynucleotide is involved in the biosynthesis of cannabinoids. There are several main classes of natural cannabinoids. Cannabinoids derived from the cannabis plant are called phytocannabinoids. All classes derived from the cannabigerol type (CBG) compound differ primarily in the manner in which this precursor is cyclized.

[0103] In one aspect the cannabinoid is selected from the group consisting of cannabigerol-type (CBG) cannabinoids, cannabichromene-type (CBC) cannabinoids, cannabidiol-type (CBD) cannabinoids, tetrahydrocannabinol-type (THC) cannabinoids, cannabinol-type (CBN) cannabinoids, cannabinodiol-type (CBDL) cannabinoids, cannabielsoin-type (CBE) cannabinoids, iso-tetrahydrocannabinol-type (iso-THC) cannabinoids, cannabicyclol-type (CBL) cannabinoids, and cannabidiol-type (CBT) cannabinoids.

[0104] Cannabigerol (CBG) is one of the major phytocannabinoids present in Cannabis sativa. CBG is the non-acidic form of cannabigerolic acid, the parent molecule from which other cannabinoids are synthesized. Cannabichromene (CBC) is a non-psychoactive cannabinoid. CBC is also known as cannabichromene, cannabichromene, pentylcannabichromene, or cannabinochromene.

[0105] Cannabidiol (CBD) is a naturally occurring compound found in the resinous flowers of cannabis. CBD is the primary phytocannabinoid that comprises up to 40% of the extract of the cannabis plant and binds to a wide variety of physiological targets in the endocannabinoid system. Tetrahydrocannabinol (THC) is also known as delta-9-tetrahydrocannabinol. Some cannabis plants contain very small amounts of THC and are considered "industrial hemp."

[0106] Cannabinol (CBN) is a weakly psychoactive cannabinoid found in trace amounts in cannabis, mostly in aged cannabis. THC breaks down into CBN after a long period of time. The breakdown of THC to CBN can be accelerated by exposure of dry plant material to oxygen and heat. Cannabinodiol (CBDL) is a fully aromatized extract of CBD. CBDL is a psychoactive cannabinoid present in low concentrations in Cannabis sativa.

[0107] Cannabielsoin (CBE) is a non-psychoactive cannabinoid metabolite derived from CBD. Cannabicyclol (CBL) is a non-psychoactive cannabinoid found in cannabis. Cannabichromene decomposes to CBL by natural radiation or under acidic conditions. Cannabidiol (CBT) is a cannabinoid that is not always detected in cannabis, but when detectable, CBT is often present in low concentrations. CBT is structurally similar to THC. There are currently nine known types of CBT.

[0108] In one aspect, the cannabis-derived cannabinoid, or phytocannabinoid, is selected from the group consisting of tetrahydrocannabinol, tetrahydrocannabinolic acid, cannabidiol, cannabidiol acid, cannabinol, cannabigerol, cannabichromene, cannabicyclol, cannabivarin, tetrahydrocannabivarin, cannabidivarin, cannabichromevarin, cannabigerovarin, cannabigerol monomethyl ether, cannabiersoin, and cannabicitran.

[0109] As used herein, “tetrahydrocannabinol” (“THC”) has the chemical formula: 21 H 30 O 2 and the organic compound with the IUPAC name: (-)-(6aR,10aR)-6,6,9-trimethyl-3-pentyl-6a,7,8,10a-tetrahydro-6H-benzo[c]chromen-1-ol.

[0110] As used herein, “tetrahydrocannabinolic acid” (“THCA” or “2-COOH-THC”) has the chemical formula: 22 H 30 O 4 This refers to an organic compound having the following structure:

[0111] As used herein, “cannabidiol” (“CBD”) has the chemical formula: 21 H 30 O 2 It refers to an organic compound with a molecular weight of 314.469 g / mol, and IUPAC name: 2-[(1R,6R)-3-methyl-6-(prop-1-en-2-yl)cyclohex-2-en-1-yl]-5-pentylbenzene-1,3-diol. It is a 21-carbon terpenophenolic compound formed following decarboxylation from the cannabidiolic acid precursor, but can also be produced synthetically. CBD is usually taken to refer to the naturally occurring (-)-enantiomer. (+)CBD has been synthetically produced.

[0112] As used herein, “cannabidiol acid” (“CBDA”) has the chemical formula: 22 H 30 O 4 and the IUPAC name: 2,4-dihydroxy-3-[(1R,6R)-3-methyl-6-prop-1-en-2-ylcyclohex-2-en-1-yl]-6-pentylbenzoic acid.

[0113] In plants, THC and CBD arise from their acidic precursors, THCA and CBDA, respectively, which both arise from cannabigerolic acid (CBGA). The final biosynthetic steps differ, with THCA synthase producing THCA and CBDA synthase producing CBDA, respectively, from CBGA. Subsequent decarboxylation of THCA and CBDA by light exposure, heating or aging results in the biosynthesis of THC or CBD. THCA synthase catalyzes the oxidative cyclization of the monoterpene moiety of CBGA to form THCA. CBDA synthase catalyzes the oxidative cyclization of the monoterpene moiety of CBGA to form CBDA. THC is made from THCA by nonenzymatic decarboxylation. CBD is made from CBDA by nonenzymatic decarboxylation.

[0114] As used herein, “cannabinol” refers to a compound having the chemical formula: 21 H 26 O 2 and the organic compound with the IUPAC name: 6,6,9-trimethyl-3-pentylbenzo[c]chromen-1-ol.

[0115] As used herein, “cannabigerol” has the chemical formula: 21 H 32 O 2 and the IUPAC name: 2-[(2E)-3,7-dimethylocta-2,6-dienyl]-5-pentylbenzene-1,3-diol.

[0116] As used herein, “cannabichromene” refers to a compound having the chemical formula:21 H 30 O 2 and the organic compound with the IUPAC name: 2-methyl-2-(4-methylpent-3-enyl)-7-pentylchromen-5-ol.

[0117] As used herein, “cannabicyclol” refers to a compound having the chemical formula: 21 H 30 O 2 and the organic compound with the IUPAC name: 9,13,13-trimethyl-5-pentyl-8-oxatetracyclo[7.4.1.0.0]tetradeca-2,4,6-trien-3-ol.

[0118] As used herein, "cannabivarin" refers to a compound having the chemical formula: 19 H 22 O 2 and the organic compound with the IUPAC name: 6,6,9-trimethyl-3-propylbenzo[c]chromen-1-ol.

[0119] As used herein, "tetrahydrocannabivarin" refers to a compound having the chemical formula: 19 H 26 O 2 and the organic compound with IUPAC name: (6aR,10aR)-6,6,9-trimethyl-3-propyl-6a,7,8,10a-tetrahydrobenzo[c]chromen-1-ol.

[0120] As used herein, “cannabidivarin” refers to a compound having the chemical formula: 19 H 26 O 2 and the organic compound with the IUPAC name: 2-[(1R,6R)-3-methyl-6-prop-1-en-2-ylcyclohex-2-en-1-yl]-5-propylbenzene-1,3-diol.

[0121] As used herein, "cannabichromevaline" refers to a compound having the chemical formula: 19 H 26 O2 and the organic compound with the IUPAC name: 2-methyl-2-(4-methylpent-3-enyl)-7-propylchromen-5-ol.

[0122] As used herein, “cannabigerovarin” has the chemical formula: 19 H 28 O 2 and the organic compound with the IUPAC name: 2-[(2E)-3,7-dimethylocta-2,6-dienyl]-5-propylbenzene-1,3-diol.

[0123] As used herein, “cannabigerol monomethyl ether” refers to a compound having the chemical formula: 22 H 28 O 2 and the organic compound with the IUPAC name: 1-methoxy-6,6,9-trimethyl-3-pentylbenzo[c]chromene.

[0124] As used herein, “cannabielsoin” refers to a compound having the chemical formula: 21 H 30 O 3 and the organic compound having the IUPAC name: (5aS,6S,9R,9aR)-6-methyl-3-pentyl-9-prop-1-en-2-yl-7,8,9,9a-tetrahydro-5aH-dibenzofuran-1,6-diol.

[0125] As used herein, “cannabinoid” refers to a compound having the chemical formula: 21 H 30 O 2 and the IUPAC name: 1,5,5-trimethyl-9-pentyl-6,15-dioxatetracyclo[9.3.1.0.0]pentadeca-7(12),8,10-triene.

[0126] promoter As generally understood in the art, the term "promoter" refers to a DNA sequence that contains an RNA polymerase binding site, a transcription initiation site, and / or a TATA box, and supports or promotes the transcription and expression of an associated transcribable polynucleotide sequence and / or gene (or transgene). Promoters can be synthetically produced, modified, or derived from known or naturally occurring promoter sequences or other promoter sequences. Promoters can also include chimeric promoters that include a combination of two or more heterologous sequences. Thus, promoters of the present application can include variants of promoter sequences that are similar in composition to, but not identical to, other promoter sequences known or provided herein.

[0127] In one aspect, the present disclosure provides a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene, operably linked to a heterologous promoter.In one aspect, the present disclosure provides a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene, operably linked to a heterologous promoter.In one aspect, the present disclosure provides a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene, operably linked to a heterologous promoter.

[0128] It is recognized in the art that the fragment of promoter sequence can function to drive the transcription of functionally linked nucleic acid molecule.For example, but not limited to, if a 1000bp promoter is shortened to 500bp, and this 500bp fragment can drive transcription, this 500bp fragment is called "functional fragment".

[0129] In some aspects, the heterologous promoter comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 104-111, 206-208, or a functional fragment thereof. In some aspects, the heterologous promoter comprises a nucleic acid sequence that is at least 92.5% identical to SEQ ID NO: 104-111, 206-208, or a functional fragment thereof. In some aspects, the heterologous promoter comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 104-111, 206-208, or a functional fragment thereof. In some aspects, the heterologous promoter comprises a nucleic acid sequence that is at least 96% identical to SEQ ID NO: 104-111, 206-208, or a functional fragment thereof. In some aspects, the heterologous promoter comprises a nucleic acid sequence that is at least 97% identical to SEQ ID NO: 104-111, 206-208, or a functional fragment thereof. In some aspects, the heterologous promoter comprises a nucleic acid sequence that is at least 98% identical to SEQ ID NO: 104-111, 206-208, or a functional fragment thereof. In some aspects, the heterologous promoter comprises a nucleic acid sequence that is at least 99% identical to SEQ ID NO: 104-111, 206-208, or a functional fragment thereof. In some aspects, the heterologous promoter comprises a nucleic acid sequence that is at least 99.9% identical to SEQ ID NO: 104-111, 206-208, or a functional fragment thereof. In some aspects, the heterologous promoter comprises a nucleic acid sequence that is 100% identical to SEQ ID NO: 104-111, 206-208, or a functional fragment thereof.

[0130] A promoter that drives expression in all or most tissues of a plant is referred to as a "constitutive" promoter. In one aspect, the constitutive promoter is selected from the group consisting of the cauliflower mosaic virus 35S promoter, the ubiquitin promoter, the actin promoter, the opine promoter, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the alcohol dehydrogenase promoter.

[0131] Promoters that drive expression during specific periods or stages of development are referred to as "developmental" promoters.

[0132] A promoter that drives enhanced expression in a certain tissue of an organism compared to other tissues of the organism is called a "tissue-preferred" promoter. Thus, a "tissue-preferred" promoter causes relatively high or preferential expression in certain tissues of a plant, but lower expression levels in other tissues of the plant. As a non-limiting example, a trichome tissue-preferred promoter may show higher activity in trichomes, but also show activity in additional tissues, such as stem, leaf and flower tissue, albeit at lower levels. A "tissue-specific" promoter causes expression only in a certain tissue. As a non-limiting example, a trichome tissue-specific promoter drives expression only in trichomes. In one aspect, the tissue-specific promoter is a trichome tissue-specific promoter. In another aspect, the tissue-preferred promoter is a trichome tissue-preferred promoter.

[0133] In one aspect, the promoter is selected from the group consisting of a cyclase promoter, a Premna spirodiene oxygenase (PSO) promoter, and a ribulose biphosphate carboxylase promoter.

[0134] In some aspects, the disclosure provides a modified tobacco plant, tobacco seed, or part thereof, wherein the tissue-preferred promoter comprises a trichome-preferred promoter. In some aspects, the disclosure provides a modified tobacco plant, tobacco seed, or part thereof, wherein the tissue-specific promoter comprises a trichome-specific promoter. In some aspects, the disclosure provides a modified tobacco plant, tobacco seed, or part thereof, wherein the tissue-preferred or tissue-specific promoter comprises a trichome-preferred or trichome-specific promoter. In some aspects, the trichome-specific promoter sequence is selected from the group consisting of SEQ ID NOs: 104-111 and 206-208. In some aspects, the trichome-preferred promoter sequence is selected from the group consisting of SEQ ID NOs: 104-111 and 206-208.

[0135] An "inducible" promoter is a promoter that initiates transcription in response to an environmental stimulus, such as heat, cold, sun, light, or other stimulus, such as wounding or the application of a chemical.

[0136] In one aspect, the promoter provided herein is a constitutive promoter. In another aspect, the promoter provided herein is an inducible promoter. In a further aspect, the promoter provided herein is a developmental promoter. In another aspect, the promoter is a tissue-preferred or tissue-specific promoter. In a further aspect, the promoter is selected from the group consisting of a constitutive promoter, a tissue-preferred promoter, a tissue-specific promoter, and an inducible promoter.

[0137] In one aspect, the disclosure provides a heterologous promoter. In another aspect, the disclosure provides a promoter operably linked to a heterologous polynucleotide. In another aspect, the disclosure provides a polynucleotide sequence operably linked to a heterologous promoter.

[0138] As used herein, "operably linked" refers to the functional linkage between two or more elements.For example, the functional linkage between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional relationship that allows the expression of the polynucleotide of interest.The elements that are functionally linked may be contiguous or non-contiguous.In one aspect, the promoter provided herein is functionally linked to a heterologous nucleic acid molecule.

[0139] plant In one aspect, the present disclosure provides a modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof, comprising a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter.

[0140] In one aspect, the disclosure provides a modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof, comprising at least one non-naturally occurring mutation in an endogenous gene encoding a polypeptide involved in the biosynthesis of at least one terpene, wherein the modified tobacco or cannabis plant exhibits increased mRNA expression of the endogenous gene or increased accumulation of the polypeptide compared to a control tobacco or cannabis plant lacking the at least one non-naturally occurring mutation when grown under equivalent conditions.

[0141] In some aspects, the plants provided herein are modified plants. In some aspects, the seeds provided herein are modified seeds. In some aspects, the plant parts provided herein are modified plant parts. As used herein, "modified" in the context of a plant, seed or plant part refers to a plant, seed or plant part that is introduced for a specific purpose and contains genetic variations beyond natural polymorphisms. Without being limited thereto, modified plants, seeds or plant parts include recombinant nucleic acid molecules. In another aspect, modified plants, seeds or plant parts include genetic modifications. In some aspects, modified plants, seeds or plant parts are transgenic.

[0142] Cannabis cultivars range from plants grown to produce recreational cannabis to plants grown for use in hemp fiber derived from the plant's stem (e.g., industrial hemp). In recreational cultivars, the amount of THC exceeds the amount of CBD in the dried female inflorescences used for smoking and oral administration. Industrial hemp cultivars produce substantially lower levels of THC and higher levels of CBD.

[0143] The plant genus Cannabis encompasses the species C. sativa, C. indica, and C. ruderalis. Another classification scheme applied to Cannabis is the chemotaxonomic classification system. There are five chemotaxonomic types of cannabis: those with high levels of THC, those with high levels of CBD, those with equal levels of THC and CBD, those with high levels of CBG, and those with low to undetectable cannabinoids.

[0144] In addition to genetic characteristics, cultivated cannabis plants are influenced by environmental conditions and production techniques during their life cycle. Studies evaluating the effects of ambient temperature and humidity, soil temperature and precipitation on the content of THC and CBD in industrial hemp state that these agro-climatic conditions affect THC and CBD differently. For example, CBD content is positively affected by soil temperature and ambient temperature, but negatively affected by precipitation. See, for example, Sikora, V., et al., 2011, Genetika, 43(3):449-456.

[0145] In some aspects, the plant is a tobacco plant. In some aspects, the plant is a Nicotiana plant. In some aspects, the tobacco plant is a Nicotiana tabacum plant.

[0146] In one aspect, the Nicotiana plant, seed, or plant part is selected from the group consisting of Nicotiana tabacum, Nicotiana amplexicaulis PI 271989; Nicotiana benthamiana PI 555478; Nicotiana bigelovii PI 555485; Nicotiana debneyi; Nicotiana excelsior PI 224063; Nicotiana glutinosa PI 555507; Nicotiana goodspeedii PI 241012; Nicotiana gossei PI 241012; 230953;Nicotiana hesperis PI 271991;Nicotiana knightiana PI 555527;Nicotiana maritima PI 555535;Nicotiana megalosiphon PI 555536;Nicotiana nudicaulis PI 555540;Nicotiana paniculata PI 555545;Nicotiana plumbaginifolia PI 555548;Nicotiana repanda PI 555552;Nicotiana rustica rustica);Nicotiana suaveolens PI 230960;Nicotiana sylvestris PI 555569;Nicotiana tomentosa PI 266379;Nicotiana tomentosiformis;and Nicotiana trigonophylla PI 555572;

[0147] In some aspects, the plant is a cannabis plant. In some aspects, the plant is a Cannabis plant. In some aspects, the cannabis plant is a Cannabis sativa plant. In some aspects, the cannabis plant is a Cannabis indica plant. In some aspects, the cannabis plant is a Cannabis ruderalis plant. In some aspects, the cannabis plant is selected from the group consisting of Cannabis sativa, Cannabis indica, and Cannabis ruderalis.

[0148] In some aspects, cannabis plants can be divided into chemical species or chemotaxonomic groups based on the ratio of THC / CBD. In some aspects, the ratio of THC / CBD is based on the relative levels of THC and CBD within a given cannabis plant. Cannabis plants with a THC / CBD ratio greater than 1.0 have high levels of THC. Cannabis plants with a THC / CBD ratio of approximately 1.0 have comparable levels of THC and CBD. Cannabis plants with a THC / CBD ratio less than 1.0 have high levels of CBD.

[0149] In some aspects, the cannabis plant is placed in a chemotaxonomic type that contains high levels of THC compared to other evaluated cannabinoids. In some aspects, the cannabis plant is placed in a chemotaxonomic type that contains high levels of CBD compared to other evaluated cannabinoids. In some aspects, the cannabis plant is placed in a chemotaxonomic type that contains comparable levels of THC and CBD. In some aspects, the cannabis plant is placed in a chemotaxonomic type that contains high levels of CBG compared to other evaluated cannabinoids. In some aspects, the cannabis plant is placed in a chemotaxonomic type that contains low to non-detectable cannabinoids.

[0150] In some aspects, the cannabis plant, plant part, or seed contains low to undetectable levels of THC. In some aspects, the cannabis plant, plant part, or seed contains low to undetectable levels of CBD. In some aspects, the cannabis plant, plant part, or seed contains low to undetectable levels of CBG.

[0151] In some aspects, the seed is a tobacco seed. In some aspects, the seed is a Nicotiana seed. In some aspects, the tobacco seed is a Nicotiana tabacum seed.

[0152] In some aspects, the seed is a cannabis seed. In some aspects, the seed is a Cannabis seed. In some aspects, the cannabis seed is a Cannabis sativa seed. In some aspects, the cannabis seed is a Cannabis indica seed. In some aspects, the cannabis seed is a Cannabis ruderalis seed. In some aspects, the cannabis seed is selected from the group consisting of Cannabis sativa, Cannabis indica, and Cannabis ruderalis.

[0153] In some aspects, the cannabis seeds are placed in a chemotaxonomic type that contains high levels of THC compared to other evaluated cannabinoids. In some aspects, the cannabis seeds are placed in a chemotaxonomic type that contains high levels of CBD compared to other evaluated cannabinoids. In some aspects, the cannabis seeds are placed in a chemotaxonomic type that contains comparable levels of THC and CBD. In some aspects, the cannabis seeds are placed in a chemotaxonomic type that contains high levels of CBG compared to other evaluated cannabinoids. In some aspects, the cannabis seeds are placed in a chemotaxonomic type that contains low to non-detectable cannabinoids.

[0154] In some aspects, the plant part is a tobacco plant part. In some aspects, the plant part is a Nicotiana plant part. In some aspects, the tobacco plant part is a Nicotiana tabacum plant part.

[0155] In some aspects, the plant part is a cannabis plant part. In some aspects, the plant part is a Cannabis plant part. In some aspects, the cannabis plant part is a Cannabis sativa plant part. In some aspects, the cannabis plant part is a Cannabis indica plant part. In some aspects, the cannabis plant part is a Cannabis ruderalis plant part. In some aspects, the cannabis plant part is selected from the group consisting of Cannabis sativa, Cannabis indica, and Cannabis ruderalis.

[0156] In some aspects, the cannabis plant parts are placed in a chemotaxonomic type that contains high levels of THC compared to other evaluated cannabinoids. In some aspects, the cannabis plant parts are placed in a chemotaxonomic type that contains high levels of CBD compared to other evaluated cannabinoids. In some aspects, the cannabis plant parts are placed in a chemotaxonomic type that contains comparable levels of THC and CBD. In some aspects, the cannabis plant parts are placed in a chemotaxonomic type that contains high levels of CBG compared to other evaluated cannabinoids. In some aspects, the cannabis plant parts are placed in a chemotaxonomic type that contains low to non-detectable cannabinoids.

[0157] In some aspects, the plant parts provided include, but are not limited to, leaves, stems, roots, trichomes, seeds, flowers, pollen, anthers, ovules, pedicels, fruits, meristems, cotyledons, hypocotyls, pods, embryos, endosperms, explants, calli, tissue cultures, shoots, cells, and protoplasts. In some aspects, the plant parts do not include seeds. In some aspects, the present disclosure provides plant cells, tissues, and organs that are not reproductive material and do not mediate natural reproduction of plants. In another aspect, the present disclosure also provides plant cells, tissues, and organs that are reproductive material and mediate natural reproduction of plants. In another aspect, the present disclosure provides plant cells, tissues, and organs that cannot sustain themselves through photosynthesis. In another aspect, the present disclosure provides somatic plant cells. Somatic cells, in contrast to germ line cells, do not mediate plant reproduction.

[0158] The cells, tissues and organs can be from seeds, fruits, leaves, cotyledons, hypocotyls, meristems, embryos, endosperms, roots, shoots, stems, trichomes, pods, flowers, inflorescences, stalks, pedicels, styles, stigmas, receptacles, petals, calyxes, pollen, anthers, filaments, ovaries, ovules, pericarp, phloem, vascular tissues. In another aspect, the present disclosure provides plant chloroplasts. In a further aspect, the present disclosure provides epidermal cells, stoma cells, leaves or root hairs, storage roots, or tubers. In another aspect, the present disclosure provides tobacco protoplasts. In another aspect, the present disclosure provides cannabis protoplasts.

[0159] Those skilled in the art will appreciate that tobacco and cannabis plants reproduce naturally through seeds, rather than through asexual or vegetative reproduction. In one aspect, the present disclosure provides a plant endosperm.

[0160] The present disclosure provides cells derived from the plants provided herein.

[0161] As used herein, a "progeny plant" or "progeny seed" refers to any hybrid generation, e.g., F 1 , F 2 , F 3 , F 4 , F 5 , F 6 , F 7 etc.

[0162] In one aspect, the tobacco plant, seed or plant part is of a tobacco variety selected from the group consisting of a flue cured variety, a bright variety, a Burley variety, a Virginia variety, a Maryland variety, a dark variety, a Galpao variety, an Oriental variety, and a Turkish variety.

[0163] In one aspect, the tobacco cells are of a tobacco variety selected from the group consisting of flue-cured, bright, burley, Virginia, Maryland, dark, Galpao, Oriental, and Turkish.

[0164] In one aspect, the tobacco leaf is of a tobacco variety selected from the group consisting of flue-cured, bright, burley, Virginia, Maryland, dark, Galpao, Oriental, and Turkish.

[0165] In one aspect, the cured tobacco leaves or plant parts are of a tobacco variety selected from the group consisting of flue-cured, bright, burley, Virginia, Maryland, dark, Galpao, Oriental, and Turkish. One of skill in the art will further appreciate that cured tobacco is not a component of a living organism and is incapable of growth or reproduction.

[0166] Flue-cured tobacco (also called "Virginia" or "Bright" tobacco) accounts for approximately 40% of the world's tobacco production. Flue-cured tobacco is often also referred to as "Bright tobacco" due to the golden yellow to deep orange color it attains during curing. Flue-cured tobacco has a light and elegant aroma and flavor. Flue-cured tobacco is generally high in sugar and low in oil. The major flue-cured tobacco growing countries are Argentina, Brazil, China, India, Tanzania and the United States. In one aspect, the tobacco plants, seeds or plant parts provided herein are of a flue-cured tobacco variety selected from the group consisting of the varieties listed in Table 2, and any variety essentially derived from any one of the aforementioned varieties. See WO 2004 / 041006 A1. In a further aspect, the tobacco plants, seeds or plant parts provided herein are of a flue-cured variety selected from the group consisting of K326, K346, and NC196.

[0167] Table 2. Flue-cured tobacco varieties TIFF2024505755000009.tif154145TIFF2024505755000010.tif158145

[0168] Air-cured tobaccos include "Burley," "Maryland," and "Dark" tobaccos. The common factor that unites air-cured tobaccos is that curing occurs primarily without artificial sources of heat and moisture. Burley tobacco is light to dark brown in color and is high in oils and low in sugars. Burley tobacco is typically air-cured in warehouses. Major burley-growing countries include Argentina, Brazil, Italy, Malawi, and the United States.

[0169] Maryland tobacco is very fluffy, has good burning characteristics, low nicotine and a neutral flavor. Major Maryland-growing countries include the United States and Italy.

[0170] In one aspect, the tobacco plants, seeds or plant parts provided herein are of a Burley tobacco variety selected from the group consisting of the tobacco varieties listed in Table 3, and any variety essentially derived from any one of the foregoing varieties. In a further aspect, the tobacco plants, seeds or plant parts provided herein are of a Burley variety selected from the group consisting of TN 90, KT 209, KT 206, KT212, and HB 4488.

[0171] (Table 3) Burley tobacco varieties TIFF2024505755000011.tif200162

[0172] In another aspect, the tobacco plant, seed or plant part provided herein is of a Maryland tobacco variety selected from the group consisting of the tobacco varieties listed in Table 4, and any variety essentially derived from any one of the foregoing varieties.

[0173] Table 4. Maryland Tobacco Varieties TIFF2024505755000012.tif72128

[0174] Dark air-cured tobacco is distinguished from other tobacco types primarily by its medium to dark brown color and its curing process, which gives it a distinct aroma. Dark air-cured tobacco is primarily used in the production of chewing tobacco and snuff. In one aspect, the tobacco plants, seeds, or plant parts provided herein are of a dark air-cured tobacco variety selected from the group consisting of Sumatra, Jatim, Dominican Cubano, Besuki, One sucker, Green River, Virginia sun-cured, and Paraguan Passado, as well as any variety essentially derived from any one of the aforementioned varieties.

[0175] Dark flue-cured tobacco is generally dried in a low-burning wood fire on the floor of a closed drying warehouse. Dark flue-cured tobacco is typically used to produce pipe blends, cigarettes, chewing tobacco, snuff, and strong-tasting cigars. The main growing areas of dark flue-cured tobacco are Tennessee, Kentucky, and Virginia in the United States. In one aspect, the tobacco plant, seed, or plant part provided herein is of a dark flue-cured tobacco variety selected from the group consisting of the tobacco varieties listed in Table 5, and any variety essentially derived from any one of the aforementioned varieties.

[0176] Table 5. Dark, flue-cured tobacco varieties TIFF2024505755000013.tif158147

[0177] Oriental tobacco is also referred to as Greek, aroma and Turkish tobacco, as it is typically grown in the eastern Mediterranean region, such as Turkey, Greece, Bulgaria, Macedonia, Syria, Lebanon, Italy and Romania. The small plant size, small leaf size and unique aroma characteristics of Oriental tobacco varieties are the result of their adaptation to have evolved in poor soil and stressful climatic conditions. In one aspect, the tobacco plant, seed or plant part provided herein is of an Oriental tobacco variety selected from the group consisting of the tobacco varieties listed in Table 6, and any variety essentially derived from any one of the aforementioned varieties.

[0178] Table 6. Oriental tobacco varieties TIFF2024505755000014.tif107140

[0179] In one aspect, the tobacco plants, seeds or plant parts provided herein are of a cigar tobacco variety selected from the group consisting of the tobacco varieties listed in Table 7, and any variety essentially derived from any one of the foregoing varieties.

[0180] (Table 7) Cigar Tobacco Varieties TIFF2024505755000015.tif51157

[0181] In one aspect, the tobacco plants, seeds or plant parts provided herein are of a tobacco variety selected from the group consisting of the tobacco varieties listed in Table 8, and any variety essentially derived from any one of the foregoing varieties.

[0182] (Table 8) Other tobacco varieties TIFF2024505755000016.tif38128

[0183] In one aspect, the tobacco plant or plant part is from a variety selected from the group consisting of tobacco varieties listed in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 and Table 8. In another aspect, the tobacco plant or plant part is from a variety listed in Table 2. In another aspect, the tobacco plant or plant part is from a variety listed in Table 3. In another aspect, the tobacco plant or plant part is from a variety listed in Table 4. In another aspect, the tobacco plant or plant part is from a variety listed in Table 5. In another aspect, the tobacco plant or plant part is from a variety listed in Table 6. In another aspect, the tobacco plant or plant part is from a variety listed in Table 7. In another aspect, the tobacco plant or plant part is from a variety listed in Table 8.

[0184] In one aspect, the tobacco seed is from a variety selected from the group consisting of tobacco varieties listed in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 and Table 8. In another aspect, the tobacco seed is from a variety listed in Table 2. In another aspect, the tobacco seed is from a variety listed in Table 3. In another aspect, the tobacco seed is from a variety listed in Table 4. In another aspect, the tobacco seed is from a variety listed in Table 5. In another aspect, the tobacco seed is from a variety listed in Table 6. In another aspect, the tobacco seed is from a variety listed in Table 7. In another aspect, the tobacco seed is from a variety listed in Table 8.

[0185] In one aspect, the tobacco cell is from a variety selected from the group consisting of tobacco varieties listed in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 and Table 8. In another aspect, the tobacco cell is from a variety listed in Table 2. In another aspect, the tobacco cell is from a variety listed in Table 3. In another aspect, the tobacco cell is from a variety listed in Table 4. In another aspect, the tobacco cell is from a variety listed in Table 5. In another aspect, the tobacco cell is from a variety listed in Table 6. In another aspect, the tobacco cell is from a variety listed in Table 7. In another aspect, the tobacco cell is from a variety listed in Table 8.

[0186] All of the above specific varieties of air-cured, dark air-cured, burley, Maryland, dark flame-cured, cigar, or Oriental type are listed for illustrative purposes only. Any additional air-cured, dark air-cured, burley, Maryland, dark flame-cured, cigar, or Oriental varieties are also contemplated in the present application.

[0187] In some aspects, the plants or varieties provided herein are inbred plants or varieties. As used herein, an "inbred" variety is a variety that has been bred for genetic homogeneity.

[0188] As used herein, a "hybrid" is produced by crossing two plants from different varieties or species, so that the offspring contains genetic material from each parent. Those skilled in the art will recognize that higher order hybrids can be produced as well. For example, a first hybrid can be produced by crossing variety C with variety D to produce a C×D hybrid, and a second hybrid can be produced by crossing variety E with variety F to produce an E×F hybrid. The first and second hybrids can be further crossed to produce a higher order hybrid (C×D)×(E×F) that contains all the genetic information of the four parent varieties. In some aspects, the plants or seeds provided herein are hybrid plants or seeds.

[0189] In some aspects, the tobacco plants provided herein are inbred tobacco plants. In some aspects, the tobacco seeds provided herein are inbred tobacco seeds. In some aspects, the tobacco plants provided herein are hybrid tobacco plants. In another aspect, the tobacco seeds provided herein are hybrid tobacco seeds.

[0190] In some aspects, the cannabis plant provided herein is an inbred cannabis plant. In some aspects, the cannabis seed provided herein is an inbred cannabis seed. In some aspects, the cannabis plant provided herein is a hybrid cannabis plant. In another aspect, the cannabis seed provided herein is a hybrid cannabis seed.

[0191] Unless otherwise specified, all comparisons with control plants require similar or equivalent growing conditions for the two plants to be compared. As used herein, "grown under equivalent conditions", "similar growing conditions" or "equivalent growing conditions" refers to similar environmental conditions and / or agricultural practices for growth and meaningful comparison between two or more plant genotypes, which do not contribute to or explain the differences observed between two or more plant genotypes. Environmental conditions include, for example, light, temperature, moisture (humidity), and nutrients (e.g., nitrogen and phosphorus). Agricultural practices include, for example, sowing, pruning, stubbling, planting, topping, and division. See Chapters 4B and 4C of Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford (1999), pp 70-103. See also Amaducci et al. Field Crops Research, 107:161-169 (2008). As used herein, a "control plant" refers to a plant of identical or nearly identical genetic make-up to the modified plant being compared, except for the non-naturally occurring mutation or recombinant DNA provided herein that has been introduced into the modified plant.

[0192] In some aspects, the first plant variety and the second plant variety are the same variety. In some aspects, the first plant variety and the second plant variety are two different varieties. In some aspects, the second plant variety comprises the recombinant nucleic acid molecule.

[0193] In some aspects, the first plant variety is heterozygous for the recombinant nucleic acid molecule. In some aspects, the first plant variety is hemizygous for the recombinant nucleic acid molecule. In some aspects, the first plant variety is homozygous for the recombinant nucleic acid molecule. In some aspects, the second plant variety is heterozygous for the recombinant nucleic acid molecule. In some aspects, the second plant variety is hemizygous for the recombinant nucleic acid molecule. In some aspects, the second plant variety is homozygous for the recombinant nucleic acid molecule. In some aspects, the progeny seeds, or plants sprouted therefrom, are heterozygous for the recombinant nucleic acid molecule. In some aspects, the progeny seeds, or plants sprouted therefrom, are hemizygous for the recombinant nucleic acid molecule. In some aspects, the progeny seeds, or plants sprouted therefrom, are homozygous for the recombinant nucleic acid molecule.

[0194] In some aspects, the first plant variety is a tobacco plant variety. In some aspects, the second plant variety is a tobacco plant variety.

[0195] In some aspects, the first plant variety is a cannabis plant variety. In some aspects, the second plant variety is a cannabis plant variety.

[0196] As used herein, the term "crossing" refers to the intentional crossing of two plants. In some aspects, crossing includes pollination and / or fertilization of a first plant by a second plant. The two plants being crossed can be distantly related, closely related, or identical. In some aspects, both of the two plants being crossed are modified plants. In some aspects, the two plants being crossed are of the same variety. In some aspects, the two plants being crossed are of two different varieties. In some aspects, one of the two plants being crossed is male sterile. In some aspects, one of the two plants being crossed is female sterile. In some aspects, at least one of the two plants being crossed is a hybrid tobacco plant. In some aspects, at least one of the two plants being crossed is a hybrid cannabis plant. In some aspects, at least one of the two plants being crossed is a modified plant.

[0197] In some aspects, a plant of a first variety is the male parent in a crossbreeding process. In some aspects, a plant of a first variety is the female parent in a crossbreeding process. In some aspects, a plant of a second variety is the male parent in a crossbreeding process. In some aspects, a plant of a second variety is the female parent in a crossbreeding process.

[0198] In one aspect, the plant or variety provided herein is male sterile. In another aspect, the plant or variety provided herein is cytoplasmic male sterile (CMS). Male sterile plants can be produced by any method known in the art. Methods for producing male sterile tobacco are described in Wernsman, EA, and Rufty, RC 1987. Chapter Seventeen. Tobacco. Pages 669-698 in: Cultivar Development. Crop Species. WH Fehr (ed.), MacMillan Publishing Go., Inc., New York, NY 761 pp. Also see kush[dot]com / blog / feminized-cannabis-seeds-all-you-need-to-know, which describes processes such as spraying female cannabis plants with colloidal silver daily after flowering to reduce rodelization and ethylene production.

[0199] In another aspect, the plant or variety provided herein is female-sterile.As a non-limiting example, female-sterile plants can be produced by mutating STIG1 gene.See, for example, Goldman et al. 1994, EMBO Journal 13:2976-2984.

[0200] In one aspect, the disclosure provides a method for producing a modified tobacco plant, comprising the steps of: (a) introducing a recombinant nucleic acid molecule into at least one tobacco cell, wherein the recombinant nucleic acid molecule comprises a nucleic acid sequence encoding a polypeptide involved in terpene biosynthesis operably linked to a heterologous promoter; (b) selecting at least one tobacco cell comprising the recombinant nucleic acid molecule; and (c) regenerating a modified tobacco plant from the at least one tobacco cell selected in step (b), wherein the modified tobacco plant comprises an increased amount of at least one terpene in at least one tissue compared to a control tobacco plant lacking the recombinant nucleic acid molecule when grown under equivalent conditions.

[0201] In one aspect, the disclosure provides a method of producing a modified cannabis plant comprising: (a) introducing a recombinant nucleic acid molecule into at least one cannabis cell, wherein the recombinant nucleic acid molecule comprises a nucleic acid sequence encoding a polypeptide involved in terpene biosynthesis operably linked to a heterologous promoter; (b) selecting at least one cannabis cell comprising the recombinant nucleic acid molecule; and (c) regenerating a modified cannabis plant from the at least one cannabis cell selected in step (b), wherein the modified cannabis plant comprises an increased amount of at least one terpene in at least one tissue compared to a control cannabis plant lacking the recombinant nucleic acid molecule when grown under equivalent conditions.

[0202] Transformation Numerous methods for introducing recombinant DNA constructs into plant cells are known in the art and can be used according to the methods of the present application to produce transgenic plant cells and plants. Any suitable method or technique for transformation of plant cells known in the art can be used according to the present methods. Effective methods for transformation of plants include bacterially mediated transformation, such as Agrobacterium-mediated or Rhizobium-mediated transformation, and biolistic-mediated transformation. See, for example, Feeney and Punja, In Vitro Cell. and Dev. Biol. - Plant, 39:578-585 (2003). A variety of methods are known in the art for transforming explants with transformation vectors via bacterially mediated transformation or biolistic bombardment, and then subsequently culturing or otherwise regenerating transgenic plants from the explants. Other methods for plant transformation, such as microinjection, electroporation, vacuum infiltration, pressurization, sonication, silicon carbide fiber agitation, polyethylene glycol (PEG)-mediated transformation, and the like, are also known in the art. Transgenic plants produced by these transformation methods can be chimeric or non-chimeric for the transformation event depending on the method and explant used.

[0203] The method of transforming plant cells is well known to those skilled in the art.For example, the specific teaching of transforming plant cells by particle bombardment with recombinant DNA-coated particles (for example, biolistics transformation) can be found in U.S. Patent No. 5,550,318; No. 5,538,880, No. 6,160,208; No. 6,399,861; and No. 6,153,812, and Agrobacterium-mediated transformation is described in U.S. Patent No. 5,159,135; No. 5,824,877; No. 5,591,616; No. 6,384,301; No. 5,750,871; No. 5,463,174; and No. 5,188,958, all of which are incorporated herein by reference. Further methods for transforming plants can be found, for example, in Compendium of Transgenic Crop Plants (2009) Blackwell Publishing. Any suitable method known to those skilled in the art can be used to transform tobacco or cannabis cells with any of the nucleic acid molecules provided herein.

[0204] In one aspect, the disclosure provides a method comprising transforming a tobacco or cannabis cell with a recombinant nucleic acid molecule, the recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter.

[0205] In one aspect, the disclosure provides a method for producing a tobacco or cannabis plant, comprising: (a) crossing at least one tobacco or cannabis plant of a first tobacco or cannabis variety with at least one tobacco or cannabis plant of a second tobacco or cannabis variety to produce at least one progeny tobacco or cannabis seed, wherein the at least one tobacco or cannabis plant of the first tobacco or cannabis variety comprises a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter; and (b) selecting at least one progeny tobacco or cannabis seed, or a plant germinated therefrom, wherein the at least one progeny tobacco or cannabis seed, or a plant germinated therefrom, comprises the recombinant nucleic acid molecule.

[0206] In one aspect, the disclosure provides a method for producing a tobacco or cannabis plant, comprising: (a) crossing at least one tobacco or cannabis plant of a first tobacco or cannabis variety with at least one tobacco or cannabis plant of a second tobacco or cannabis variety to produce at least one progeny tobacco or cannabis seed, wherein the at least one tobacco or cannabis plant of the first tobacco or cannabis variety comprises at least one non-naturally occurring mutation in an endogenous gene encoding a polypeptide involved in the biosynthesis of at least one terpene, and wherein the at least one tobacco or cannabis plant of the first tobacco or cannabis variety exhibits increased mRNA expression of the endogenous gene or increased accumulation of the polypeptide when grown under equivalent conditions, compared to a control tobacco or cannabis plant of the first tobacco or cannabis variety lacking the at least one non-naturally occurring mutation; and (b) selecting at least one progeny tobacco or cannabis seed, or a plant germinated therefrom, wherein the at least one progeny tobacco or cannabis seed, or a plant germinated therefrom, comprises at least one non-naturally occurring mutation.

[0207] In one aspect, the method of introducing a nucleic acid molecule into a tobacco or cannabis cell comprises Agrobacterium-mediated transformation. In another aspect, the method of introducing a nucleic acid molecule into a tobacco or cannabis cell comprises PEG-mediated transformation. In another aspect, the method of introducing a nucleic acid molecule into a tobacco or cannabis cell comprises biolistec transformation. In another aspect, the method of introducing a nucleic acid molecule into a tobacco or cannabis cell comprises liposome-mediated transfection (lipofection). In another aspect, the method of introducing a nucleic acid molecule into a tobacco or cannabis cell comprises lentiviral transfection.

[0208] Lipofection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787; and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those in International Publication No. WO 1991 / 017424 and International Publication No. WO 1991 / 016024. Delivery can be to cells (e.g., in vitro or ex vivo administration) or to target tissues (e.g., in vivo administration).

[0209] Any tobacco cell capable of regenerating a fertile tobacco plant is contemplated as a useful recipient cell for the practice of the present disclosure.Similarly, any cannabis cell capable of regenerating a fertile cannabis plant is contemplated as a useful recipient cell for the practice of the present disclosure.

[0210] In some aspects, the recombinant DNA construct is introduced into a tobacco cell. In some aspects, the recombinant DNA construct is introduced into a tobacco protoplast cell. In other aspects, the recombinant DNA construct is introduced into a tobacco callus cell. In some aspects, the recombinant DNA construct is introduced into a tobacco cell selected from the group consisting of a seed cell, a fruit cell, a leaf cell, a cotyledon cell, a hypocotyl cell, a meristem cell, an embryo cell, an endosperm cell, a root cell, a shoot cell, a stem cell, a flower cell, an inflorescence cell, a stalk cell, a pedicel cell, a style cell, a stigma cell, a receptacle cell, a petal cell, a calyx cell, a pollen cell, anther cell, a filament cell, an ovary cell, an ovule cell, a pericarp cell, and a phloem cell.

[0211] In some aspects, the recombinant DNA construct is introduced into a cannabis cell. In some aspects, the recombinant DNA construct is introduced into a cannabis protoplast cell. In another aspect, the recombinant DNA construct is introduced into a cannabis callus cell. In some aspects, the recombinant DNA construct is introduced into a cannabis cell selected from the group consisting of a seed cell, a fruit cell, a leaf cell, a cotyledon cell, a hypocotyl cell, a meristem cell, an embryo cell, an endosperm cell, a root cell, a shoot cell, a stem cell, a flower cell, an inflorescence cell, a stalk cell, a pedicel cell, a style cell, a stigma cell, a receptacle cell, a petal cell, a calyx cell, a pollen cell, anther cell, a filament cell, an ovary cell, an ovule cell, a pericarp cell, and a phloem cell.

[0212] Callus can be initiated from various tissue sources, including but not limited to immature embryos or parts of embryos, seedling apical meristems, microspores, etc. Those cells that can grow as callus can serve as recipient cells for transformation. Practical transformation methods and materials for making the transgenic plants of the present disclosure (e.g., various media and recipient target cells, transformation of immature embryos, and subsequent regeneration of fertile transgenic plants) are disclosed, for example, in U.S. Patents 6,194,636 and 6,232,526 and U.S. Patent Application Publication 2004 / 0216189, all of which are incorporated herein by reference.

[0213] In one aspect, the disclosure provides a method of producing a modified tobacco or cannabis plant, comprising: (a) introducing a recombinant DNA construct into at least one tobacco or cannabis cell, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene; (b) selecting at least one tobacco or cannabis cell comprising the recombinant DNA construct; and (c) regenerating at least one modified tobacco or cannabis plant from the at least one tobacco or cannabis cell selected in step (b), wherein the modified tobacco or cannabis plant comprises a reduced amount of at least one terpene in at least one tissue compared to a control tobacco or cannabis plant lacking the recombinant DNA construct when grown under equivalent conditions.

[0214] In one aspect, the disclosure provides a method comprising transforming a tobacco or cannabis cell with a recombinant nucleic acid molecule, the recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene.

[0215] In one aspect, the disclosure provides a method for producing a tobacco or cannabis plant, comprising: (a) crossing at least one tobacco or cannabis plant of a first tobacco or cannabis variety with at least one tobacco or cannabis plant of a second tobacco or cannabis variety to produce at least one progeny tobacco or cannabis seed, wherein the at least one tobacco or cannabis plant of the first tobacco or cannabis variety comprises a recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene; and selecting at least one progeny tobacco or cannabis seed, or a plant germinated therefrom, wherein the at least one progeny tobacco or cannabis seed, or a plant germinated therefrom, comprises the recombinant nucleic acid molecule.

[0216] mutation Without limitation, modified plants can include non-natural mutations or recombinant DNA constructs. In one aspect, modified tobacco plants include non-natural mutations. In another aspect, modified tobacco plants include recombinant DNA constructs. In another aspect, modified tobacco plants include genetic modifications. In one aspect, modified cannabis plants include non-natural mutations. In another aspect, modified cannabis plants include recombinant DNA constructs. In another aspect, modified cannabis plants include genetic modifications.

[0217] In one aspect, the disclosure provides a method of producing a modified tobacco or cannabis plant comprising the steps of: (a) inducing in at least one tobacco or cannabis cell at least one non-naturally occurring mutation in an endogenous gene encoding a polypeptide involved in terpene biosynthesis; (b) selecting at least one tobacco or cannabis plant comprising the at least one non-naturally occurring mutation from step (a); and (c) regenerating a modified tobacco or cannabis plant from the at least one tobacco or cannabis cell selected in step (b), wherein the modified tobacco or cannabis plant comprises an increased amount of at least one terpene in at least one tissue compared to a control tobacco or cannabis plant lacking the at least one non-naturally occurring mutation when grown under equivalent conditions.

[0218] As used herein, "mutation" refers to a heritable genetic modification that is introduced into a gene to change the expression or activity of the product encoded by the reference sequence of the gene. For example, a mutation in a certain gene, such as limonene synthase (LS), is referred to as a LS mutant. Such a modification can be in any sequence region of the gene, such as the promoter, 5' untranslated region (UTR), exon, intron, 3'-UTR, or terminator region. In one aspect, the mutation reduces, inhibits, or eliminates the expression or activity of the gene product. In another aspect, the mutation increases, elevates, enhances, or increases the expression or activity of the gene product.

[0219] In some aspects, the mutation is not a natural polymorphism present in a particular tobacco variety or cultivar. In some aspects, the mutation is a "non-natural" or "non-naturally occurring" mutation. As used herein, "non-natural" or "non-naturally occurring" mutation refers to the non-naturally occurring mutation that is created through human intervention, and does not correspond to the naturally occurring mutation that is created without human intervention. Non-limiting examples of human intervention include mutagenesis (e.g., chemical mutagenesis, ionizing radiation mutagenesis) and targeted genetic modification (e.g., CRISPR-based, TALEN-based, zinc finger-based). Non-natural and non-natural mutations do not include naturally occurring mutations that occur naturally (e.g., through abnormal DNA replication in the germ line of plants).

[0220] In some aspects, the mutation is not a natural polymorphism present in a particular tobacco variety or cultivar. It will be appreciated that when identifying a mutation, the reference DNA sequence should be from the same tobacco variety. For example, if the modified tobacco plant containing the mutation is from variety TN90, the endogenous reference sequence must be the endogenous TN90 sequence, not a homologous sequence from a different tobacco variety (e.g., K326). Similarly, if the modified tobacco cell containing the mutation is a TN90 cell, the endogenous reference sequence must be the endogenous TN90 sequence, not a homologous sequence from a tobacco cell from a different tobacco variety (e.g., K326).

[0221] In some aspects, the mutation is not a natural polymorphism present in a particular cannabis species, variety or cultivar. When identifying a mutation, it will be recognized that the reference DNA sequence should be from the same cannabis species, variety or cultivar. For example, if the modified cannabis plant containing the mutation is a strain of C. indica, the endogenous reference sequence must be the corresponding endogenous C. indica sequence, not a homologous sequence from a different cannabis species or strain (e.g., a sequence from a different strain of C. sativa or C. indica). Similarly, if the modified cannabis cell containing the mutation is a cell from C. indica, the endogenous reference sequence must be the corresponding endogenous C. indica sequence, not a homologous sequence from a cannabis cell from a different cannabis variety or species (e.g., a sativa strain).

[0222] In one aspect, the tobacco plant, tobacco seed, or part thereof is homozygous for at least one non-natural mutation. In another aspect, the tobacco plant, tobacco seed, or part thereof is heterozygous for at least one non-natural mutation. In one aspect, the tobacco plant, tobacco seed, or part thereof is hemizygous for at least one non-natural mutation. In another aspect, the tobacco plant, tobacco seed, or part thereof is homozygous for the introduced recombinant DNA construct. In another aspect, the tobacco plant, tobacco seed, or part thereof is hemizygous for the introduced recombinant DNA construct. In a further aspect, the tobacco plant, tobacco seed, or part thereof is heterozygous for the introduced recombinant DNA construct.

[0223] In one aspect, the cannabis plant, cannabis seed, or part thereof is homozygous for at least one non-natural mutation. In another aspect, the cannabis plant, cannabis seed, or part thereof is heterozygous for at least one non-natural mutation. In one aspect, the cannabis plant, cannabis seed, or part thereof is hemizygous for at least one non-natural mutation. In another aspect, the cannabis plant, cannabis seed, or part thereof is homozygous for the introduced recombinant DNA construct. In another aspect, the cannabis plant, cannabis seed, or part thereof is hemizygous for the introduced recombinant DNA construct. In a further aspect, the cannabis plant, cannabis seed, or part thereof is heterozygous for the introduced recombinant DNA construct.

[0224] In some aspects, the tobacco seed, or a plant germinated therefrom, is heterozygous for the recombinant nucleic acid molecule. In some aspects, the tobacco seed, or a plant germinated therefrom, is hemizygous for the recombinant nucleic acid molecule. In some aspects, the tobacco seed, or a plant germinated therefrom, is homozygous for the recombinant nucleic acid molecule.

[0225] In some aspects, the cannabis seed, or the plant germinated therefrom, is heterozygous for the recombinant nucleic acid molecule. In some aspects, the cannabis seed, or the plant germinated therefrom, is hemizygous for the recombinant nucleic acid molecule. In some aspects, the cannabis seed, or the plant germinated therefrom, is homozygous for the recombinant nucleic acid molecule.

[0226] In some aspects, the mutations provided herein create a dominant allele at the mutated locus. A dominant allele is an allele that eliminates the contribution of a second allele at the same locus. A dominant allele can be a "dominant negative allele" or a "dominant positive allele". A dominant negative allele, or antimorph, is an allele that acts opposite to the normal allele function. A dominant negative allele typically does not function normally, either directly inhibiting the activity of the wild-type protein (e.g., through dimerization) or inhibiting the activity of a second protein (e.g., a pathway activator or downstream component) that is required for the normal function of the wild-type protein. For example, a dominant negative allele abolishes or reduces the normal function of the allele in the heterozygous or homozygous state. A dominant positive allele can increase normal gene function (e.g., hypermorph) or confer new function to the gene (e.g., neomorph). A semidominant allele occurs when the penetrance of the associated phenotype in an individual heterozygous for the allele is less than that observed in an individual homozygous for the allele.

[0227] In one aspect, the mutations provided herein create a dominant negative allele at the mutant locus. In another aspect, the mutations provided herein create a dominant positive allele at the mutant locus.

[0228] As used herein, "inducing" mutation refers to causing mutation in polynucleotide sequence through human intervention.Many suitable methods for inducing mutation in tobacco and cannabis are known in the art.Non-limiting examples of such methods include using chemical mutagens, using radiation, using nucleases, using transposons, and using Agrobacterium.In some aspects, inducing mutation includes using agents selected from the group consisting of chemical mutagens, radiation, transposons, Agrobacterium, and nucleases.

[0229] In some aspects, inducing mutations comprises the use of a chemical mutagen. In some aspects, the chemical mutagen comprises ethyl methanesulfonate (EMS).

[0230] In another aspect, inducing mutations comprises the use of radiation, hi some aspects, the radiation comprises gamma rays, X-rays, ionizing radiation, or fast neutrons.

[0231] In one aspect, inducing the mutation comprises the use of a transposon, hi another aspect, inducing the mutation comprises the use of Agrobacterium.

[0232] In a further aspect, inducing the mutation comprises the use of a nuclease. In some aspects, the nuclease is selected from the group consisting of meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, CRISPR / Cas9 nucleases, CRISPR / Cpf1 nucleases, CRISPR / CasX nucleases, CRISPR / CasY nucleases, and Csm1 nucleases. In some aspects, inducing the mutation comprises the use of a CRISPR / Cas9 nuclease. In some aspects, inducing the mutation comprises the use of a CRISPR / Cpf1 nuclease. In some aspects, inducing the mutation comprises the use of a CRISPR / CasX nuclease. In some aspects, inducing the mutation comprises the use of a CRISPR / CasY nuclease. In some aspects, inducing the mutation comprises the use of a Csm1 nuclease.

[0233] Several types of mutations are known in the art. In one aspect, the mutation includes an insertion. An "insertion" refers to the addition of one or more nucleotides or amino acids to a given polynucleotide or amino acid sequence, respectively, compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, the mutation includes a deletion. A "deletion" refers to the removal of one or more nucleotides or amino acids to a given polynucleotide or amino acid sequence, respectively, compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, the mutation includes a substitution. A "substitution" refers to the replacement of one or more nucleotides or amino acids to a given polynucleotide or amino acid sequence, respectively, compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, the mutation includes an inversion. An "inversion" refers to when a segment of a polynucleotide or amino acid sequence is inverted end-to-end. A "duplication" refers to when a segment of a polynucleotide or amino acid sequence is repeated. The repeated segment may immediately follow the original segment, or may be separated from the original segment by one or more nucleotides or amino acids. In some aspects, the mutations provided herein include mutations selected from the group consisting of insertions, deletions, substitutions, duplications, and inversions.

[0234] In one aspect, the non-naturally occurring mutation comprises a mutation selected from the group consisting of one or more nucleotide substitutions, deletions, insertions, duplications, and inversions compared to an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190.

[0235] In one aspect, the non-naturally occurring mutation comprises a mutation selected from the group consisting of one or more nucleotide substitutions, deletions, insertions, duplications, and inversions compared to an endogenous nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205.

[0236] In some aspects, the non-natural mutation comprises at least one mutation selected from the group consisting of nonsense mutation, missense mutation, frameshift mutation, splice site mutation and any combination thereof.As used herein, "nonsense mutation" refers to a mutation to a nucleic acid sequence that introduces a premature stop codon into the amino acid sequence by the nucleic acid sequence.As used herein, "missense mutation" refers to a mutation to a nucleic acid sequence that causes a substitution within the amino acid sequence encoded by the nucleic acid sequence.As used herein, "frameshift mutation" refers to an insertion or deletion to a nucleic acid sequence that shifts the frame for translating the nucleic acid sequence into an amino acid sequence."Splice site mutation" refers to a mutation in a nucleic acid sequence that causes an intron to be retained for protein translation or, alternatively, causes an exon to be excluded from protein translation.Splice site mutation can cause nonsense, missense, or frameshift mutation.

[0237] Mutations in the coding region of a gene (e.g., exon mutations) can result in a truncated protein or polypeptide when the mutated messenger RNA (mRNA) is translated into a protein or polypeptide. In some aspects, the present disclosure provides mutations that result in truncation of a protein or polypeptide. As used herein, a "truncated" protein or polypeptide contains at least one less amino acid compared to an endogenous control protein or polypeptide. For example, if endogenous protein A contains 100 amino acids, a truncated form of protein A can contain 1-99 amino acids. In some aspects, non-naturally occurring mutations result in truncation of a polypeptide.

[0238] Without being limited to any scientific theory, one way to cause the truncation of a protein or polypeptide is by the introduction of a premature stop codon in the mRNA transcript of an endogenous gene. In one aspect, the present disclosure provides a mutation that causes a premature stop codon in the mRNA transcript of an endogenous gene. As used herein, a "stop codon" refers to a nucleotide triplet in an mRNA transcript that signals the end of protein translation. A "premature stop codon" refers to a stop codon that is located before (e.g., 5' on) the normal stop codon position in an endogenous mRNA transcript. Several stop codons are known in the art, including, but not limited to, "UAG", "UAA", "UGA", "TAG", "TAA" and "TGA".

[0239] In some aspects, the mutation provided herein comprises null mutation. In some aspects, at least one non-natural mutation provided herein comprises null mutation. As used herein, "null mutation" refers to a mutation that confers complete loss of function to the protein encoded by the gene that comprises the mutation, or alternatively, a mutation that confers complete loss of function to the small RNA encoded by the genomic locus. Null mutation can cause the lack of mRNA transcript production, the lack of small RNA transcript production, the lack of protein function, or a combination thereof.

[0240] The mutations provided herein can be located at any site of the endogenous gene. In one aspect, the mutations provided herein are located in the exons of the endogenous gene. In another aspect, the mutations provided herein are located in the introns of the endogenous gene. In a further aspect, the mutations provided herein are located in the 5'-UTR of the endogenous gene. In yet another aspect, the mutations provided herein are located in the 3'-UTR of the endogenous gene. In yet another aspect, the mutations provided herein are located in the promoter of the endogenous gene. In yet another aspect, the mutations provided herein are located in the terminator of the endogenous gene. In one aspect, the non-natural mutations provided herein include mutations in sequence regions selected from the group consisting of promoter, 5'-UTR, 3'-UTR, exon, intron, and terminator.

[0241] Screening and selection of mutagenized tobacco plants can be performed through any methodology known to those skilled in the art. Examples of screening and selection methodologies include, but are not limited to, Southern analysis, PCR amplification for detection of polynucleotides, Northern blot, RNase protection, primer extension, RT-PCR amplification for detection of RNA transcripts, Sanger sequencing, next generation sequencing technology (e.g., Illumina, PacBio, Ion Torrent, 454, and Oxford nanopore), enzyme assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides, and protein gel electrophoresis, Western blot, immunoprecipitation, and enzyme-linked immunoassays for detecting polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining can also be used to detect the presence or expression of polypeptides and / or polynucleotides. Methods for carrying out all of the reference and similar techniques are known.

[0242] Reduced Expression / Activity In one aspect, the small RNA molecule reduces expression of any nucleic acid sequence to which it is capable of binding. In another aspect, the non-naturally occurring mutations provided herein reduce expression of the mutated nucleic acid sequence compared to the non-mutated nucleic acid sequence in a control plant grown under comparable conditions.

[0243] The reduced expression of endogenous nucleic acid sequence can be measured using any suitable method known in the art. Non-limiting examples of measuring expression include quantitative reverse transcription polymerase chain reaction (qRT-PCR), digital PCR, RNA blot (e.g., Northern blot), RNA sequencing. The difference in expression can be described as absolute or relative quantification. For example, see Livak and Schmittgen, Methods, 25:402-408 (2001). If the endogenous nucleic acid sequence codes for a protein, the change in expression can be inferred by examining the accumulation of the encoded protein. Non-limiting examples of measuring protein accumulation include Western blot and enzyme-linked immunosorbent assay (ELISA).

[0244] In some aspects, the reduction in expression is measured using qRT-PCR. In other aspects, the reduction in expression is measured using RNA blot. In other aspects, the reduction in expression is measured using RNA sequencing. In some aspects, the reduction in expression is measured using digital PCR. In further aspects, the reduction in expression is measured using Western blot. In yet further aspects, the reduction in expression is measured using ELISA.

[0245] In some aspects, non-naturally occurring variations in a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190 result in a reduced level of expression of the nucleic acid sequence compared to a nucleic acid sequence lacking the non-naturally occurring variations in a control plant grown under comparable conditions. In some aspects, non-naturally occurring variations in a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205 result in a reduced level of expression of the nucleic acid sequence compared to a nucleic acid sequence lacking the non-naturally occurring variations in a control plant grown under comparable conditions.

[0246] In one aspect, the reduction in expression comprises at least a 1% reduction compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, the reduction in expression comprises at least a 5% reduction compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, the reduction in expression comprises at least a 10% reduction compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, the reduction in expression comprises at least a 25% reduction compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, the reduction in expression comprises at least a 50% reduction compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, the reduction in expression comprises at least a 75% reduction compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, the reduction in expression comprises at least a 90% reduction compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, reduced expression comprises at least a 95% reduction compared to expression in the same tissue of a control plant grown under equivalent conditions.

[0247] In one aspect, the reduction in expression comprises a 1% to 99% reduction compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, the reduction in expression comprises a 1% to 90% reduction compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, the reduction in expression comprises a 1% to 75% reduction compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, the reduction in expression comprises a 1% to 50% reduction compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, the reduction in expression comprises a 1% to 25% reduction compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, the reduction in expression comprises a 25% to 90% reduction compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, the reduction in expression comprises a 50% to 90% reduction compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, reduced expression includes a 25% to 75% reduction compared to expression in the same tissue of a control plant grown under equivalent conditions.

[0248] In some aspects, the reduction in expression comprises a statistically significant reduction compared to the expression in the same tissue of a control plant grown under the same conditions.Those skilled in the art will recognize that any level of reduction is envisioned, as long as the level of reduction is determined to be statistically significant using a recognized statistical hypothesis test.As a non-limiting example, the Student's t-test is one statistical hypothesis test that can be used to determine whether the reduction in expression between modified plants and control plants is statistically significant.As used herein, "statistically significant" refers to a p-value that is less than or equal to 0.05.

[0249] In one aspect, the non-naturally occurring mutation results in a reduced level of activity by the protein or polypeptide encoded by the nucleic acid sequence provided herein, compared to the activity of a control plant grown under equivalent conditions. In another aspect, the non-naturally occurring mutation in an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190 reduces the level of activity by the protein or polypeptide encoded by the nucleic acid sequence, compared to the activity of the protein or polypeptide encoded by the endogenous nucleic acid sequence in a control tobacco or cannabis plant, when grown under equivalent conditions, wherein the nucleic acid sequence lacks the non-naturally occurring mutation in the control tobacco or cannabis plant. In another aspect, the non-naturally occurring mutation in an endogenous nucleic acid sequence (wherein the endogenous nucleic acid sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205) reduces the level of activity by a protein or polypeptide encoded by the nucleic acid sequence compared to the activity of the protein or polypeptide encoded by the endogenous nucleic acid sequence in a control tobacco or cannabis plant (wherein the nucleic acid sequence lacks the non-naturally occurring mutation in the control tobacco or cannabis plant) when grown under equivalent conditions.

[0250] As used herein, "activity" when referring to a protein or polypeptide refers to the ability to perform enzyme function.As a non-limiting example, the activity of a dehydrogenase protein or polypeptide refers to the ability of dehydrogenase to remove hydrogen group from organic molecule substrate.Without being limited by any scientific theory, when a mutated dehydrogenase protein has reduced activity compared to a control dehydrogenase protein that is not mutated, the mutated dehydrogenase can (a) not remove hydrogen group from any organic molecule substrate; (b) only remove hydrogen group from organic molecule substrate at a reduced rate compared to the non-mutated dehydrogenase protein; (c) only remove hydrogen group from substrate that is not suitable for the non-mutated dehydrogenase protein; or (d) be a combination of (b) and (c). Conversely, and without being limited to any scientific theory, if a mutated dehydrogenase protein has increased activity compared to a non-mutated control dehydrogenase protein, the mutated dehydrogenase can (a) remove hydrogen groups from organic molecule substrates at an increased rate compared to the non-mutated dehydrogenase protein; (b) remove hydrogen groups from substrates that are not suitable substrates for non-mutated dehydrogenase; or (c) both (a) and (b). The activity of dehydrogenase can be measured using standard techniques in the art. See, for example, Yu et al., Enzyme Microb. Technol., 49:272-276 (2011); Seely and Pegg, J. Biol. Chem., 258:2496-2500 (1983); and Mizusaki et al., Plant and Cell Physiology, 14:103-110 (1973).

[0251] Increased expression / activity In some aspects, the non-naturally occurring mutation results in increased expression of the nucleic acid sequence. In some aspects, the non-naturally occurring mutation results in an increased level of expression of the nucleic acid sequence compared to the expression of the nucleic acid sequence in the same tissue of a control tobacco or cannabis plant when grown under equivalent conditions, wherein the nucleic acid sequence lacks at least one non-naturally occurring mutation in the control tobacco or cannabis plant.

[0252] In some aspects, the disclosure provides a tobacco plant comprising increased expression of a sequence that is at least 70% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In some aspects, the disclosure provides a tobacco plant comprising increased expression of a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In some aspects, the disclosure provides a tobacco plant comprising increased expression of a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In some aspects, the disclosure provides a tobacco plant comprising increased expression of a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In some aspects, the disclosure provides a tobacco plant comprising increased expression of a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In some aspects, the disclosure provides a tobacco plant comprising increased expression of a sequence that is at least 92.5% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In some aspects, the disclosure provides a tobacco plant comprising increased expression of a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In some aspects, the disclosure provides a tobacco plant comprising increased expression of a sequence that is at least 96% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In some aspects, the disclosure provides a tobacco plant comprising increased expression of a sequence that is at least 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190.In some aspects, the disclosure provides a tobacco plant comprising increased expression of a sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In some aspects, the disclosure provides a tobacco plant comprising increased expression of a sequence that is at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In some aspects, the disclosure provides a tobacco plant comprising increased expression of a sequence that is at least 99.5% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In one aspect, the disclosure provides a tobacco plant comprising increased expression of a sequence that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190.

[0253] In some aspects, the disclosure provides a cannabis plant comprising increased expression of a sequence that is at least 70% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In some aspects, the disclosure provides a cannabis plant comprising increased expression of a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In some aspects, the disclosure provides a cannabis plant comprising increased expression of a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In some aspects, the disclosure provides a cannabis plant comprising increased expression of a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In some aspects, the disclosure provides a cannabis plant comprising increased expression of a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In some aspects, the disclosure provides a cannabis plant comprising increased expression of a sequence that is at least 92.5% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In some aspects, the disclosure provides a cannabis plant comprising increased expression of a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In some aspects, the disclosure provides a cannabis plant comprising increased expression of a sequence that is at least 96% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In some aspects, the disclosure provides a cannabis plant comprising increased expression of a sequence that is at least 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175.In one aspect, the disclosure provides a cannabis plant comprising increased expression of a sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In one aspect, the disclosure provides a cannabis plant comprising increased expression of a sequence that is at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In one aspect, the disclosure provides a cannabis plant comprising increased expression of a sequence that is at least 99.5% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In one aspect, the present disclosure provides a cannabis plant comprising increased expression of a sequence that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175.

[0254] In some aspects, the disclosure provides a tobacco plant comprising increased expression of a sequence that is at least 70% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In some aspects, the disclosure provides a tobacco plant comprising increased expression of a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In some aspects, the disclosure provides a tobacco plant comprising increased expression of a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In some aspects, the disclosure provides a tobacco plant comprising increased expression of a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In some aspects, the disclosure provides a tobacco plant comprising increased expression of a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In some aspects, the disclosure provides a tobacco plant comprising increased expression of a sequence that is at least 92.5% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In some aspects, the disclosure provides a tobacco plant comprising increased expression of a sequence at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In some aspects, the disclosure provides a tobacco plant comprising increased expression of a sequence at least 96% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In some aspects, the disclosure provides a tobacco plant comprising increased expression of a sequence at least 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175.In some aspects, the disclosure provides a tobacco plant comprising increased expression of a sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In some aspects, the disclosure provides a tobacco plant comprising increased expression of a sequence that is at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In some aspects, the disclosure provides a tobacco plant comprising increased expression of a sequence that is at least 99.5% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In one aspect, the disclosure provides a tobacco plant comprising increased expression of a sequence that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175.

[0255] In some aspects, the disclosure provides a cannabis plant comprising increased expression of a sequence that is at least 70% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In some aspects, the disclosure provides a cannabis plant comprising increased expression of a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In some aspects, the disclosure provides a cannabis plant comprising increased expression of a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In some aspects, the disclosure provides a cannabis plant comprising increased expression of a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In some aspects, the disclosure provides a cannabis plant comprising increased expression of a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In some aspects, the disclosure provides a cannabis plant comprising increased expression of a sequence that is at least 92.5% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In some aspects, the disclosure provides a cannabis plant comprising increased expression of a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In some aspects, the disclosure provides a cannabis plant comprising increased expression of a sequence that is at least 96% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In some aspects, the disclosure provides a cannabis plant comprising increased expression of a sequence that is at least 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190.In some aspects, the disclosure provides a cannabis plant comprising increased expression of a sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In some aspects, the disclosure provides a cannabis plant comprising increased expression of a sequence that is at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In some aspects, the disclosure provides a cannabis plant comprising increased expression of a sequence that is at least 99.5% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In one aspect, the present disclosure provides a cannabis plant comprising increased expression of a sequence that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190.

[0256] In another aspect, the disclosure provides increased expression of sequences encoding proteins at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, the disclosure provides increased expression of sequences encoding proteins at least 75% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, the disclosure provides increased expression of sequences encoding proteins at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, the disclosure provides increased expression of sequences encoding proteins at least 85% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, the disclosure provides increased expression of sequences encoding proteins at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, the disclosure provides increased expression of sequences encoding proteins at least 92.5% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, the disclosure provides increased expression of sequences encoding proteins at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, the disclosure provides increased expression of sequences encoding proteins at least 96% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205.In another aspect, the disclosure provides increased expression of sequences encoding proteins at least 97% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, the disclosure provides increased expression of sequences encoding proteins at least 98% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, the disclosure provides increased expression of sequences encoding proteins at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, the disclosure provides increased expression of sequences encoding proteins at least 99.5% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, the disclosure provides increased expression of sequences encoding proteins 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205.

[0257] In one aspect, the increased level of expression comprises at least a 1% increase compared to expression in the same tissue of a control plant grown under comparable conditions. In one aspect, the increased level of expression comprises at least a 5% increase compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, the increased level of expression comprises at least a 10% increase compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, the increased level of expression comprises at least a 25% increase compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, the increased level of expression comprises at least a 50% increase compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, the increased level of expression comprises at least a 75% increase compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, the increased level of expression comprises at least a 100% increase compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, the increased level of expression comprises at least a 200% increase compared to expression in the same tissue of a control plant grown under comparable conditions, hi another aspect, the increased level of expression comprises at least a 500% increase compared to expression in the same tissue of a control plant grown under comparable conditions.

[0258] In one aspect, the non-naturally occurring mutation results in an increased level of activity by a protein or polypeptide encoded by the nucleic acid sequence compared to the activity of the protein or polypeptide encoded by the nucleic acid sequence in a control tobacco or cannabis plant when grown under equivalent conditions, wherein the nucleic acid sequence lacks at least one non-naturally occurring mutation in the control tobacco or cannabis plant.

[0259] Nucleic acids and amino acids As used herein, "heterologous" refers to a sequence (nucleic acid or amino acid) that originates from a foreign species or, if derived from the same species, has been substantially modified in composition and / or genomic loci from its native form by deliberate human intervention. This term is also applicable to nucleic acid constructs, also referred to herein as "polynucleotide constructs". Thus, a "heterologous" nucleic acid construct is intended to mean a construct that originates from a foreign species or, if derived from the same species, has been substantially modified in composition and / or genomic loci from its native form by deliberate human intervention. Heterologous nucleic acid constructs include, but are not limited to, recombinant nucleotide constructs that are introduced into a plant or plant part thereof, for example, via transformation methods or subsequent breeding of a transgenic plant with another plant of interest. It will be understood that an endogenous promoter can be considered heterologous to the endogenous gene to which it is operably linked if the two are not naturally operably linked (e.g., human intervention is required to place them in operably linked state). As used herein, an "endogenous" nucleic acid sequence refers to a nucleic acid sequence that is naturally present in the genome of an organism.

[0260] In some aspects, the heterologous polynucleotide comprises a gene. In some aspects, the heterologous polynucleotide encodes a small RNA molecule or a precursor thereof. In some aspects, the heterologous polynucleotide encodes a polypeptide.

[0261] As used herein, "gene" refers to a polynucleotide capable of producing a functional unit (such as, but not limited to, a polypeptide or a small RNA molecule). A gene can include a promoter, an enhancer sequence, a leader sequence, a transcription start site, a transcription stop site, a polyadenylation site, one or more exons, one or more introns, a 5'-UTR, a 3'-UTR, or any combination thereof. A "gene sequence" can include a polynucleotide sequence that encodes a promoter, an enhancer sequence, a leader sequence, a transcription start site, a transcription stop site, a polyadenylation site, one or more exons, one or more introns, a 5'-UTR, a 3'-UTR, or any combination thereof. In one aspect, a gene encodes a small RNA molecule or a precursor thereof. In another aspect, a gene encodes a polypeptide.

[0262] In one aspect, the gene comprises a nucleic acid sequence that is at least 70% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In one aspect, the gene comprises a nucleic acid sequence that is at least 75% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In one aspect, the gene comprises a nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In one aspect, the gene comprises a nucleic acid sequence that is at least 85% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In one aspect, the gene comprises a nucleic acid sequence that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In one aspect, the gene comprises a nucleic acid sequence that is at least 92.5% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In one aspect, the gene comprises a nucleic acid sequence that is at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In one aspect, the gene comprises a nucleic acid sequence that is at least 96% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In one aspect, the gene comprises a nucleic acid sequence that is at least 97% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In one aspect, the gene comprises a nucleic acid sequence that is at least 98% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190.In one aspect, the gene comprises a nucleic acid sequence that is at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In one aspect, the gene comprises a nucleic acid sequence that is at least 99.5% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In one aspect, the gene comprises a nucleic acid sequence that is 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190.

[0263] In one aspect, the gene comprises a nucleic acid sequence that is at least 70% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In one aspect, the gene comprises a nucleic acid sequence that is at least 75% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In one aspect, the gene comprises a nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In one aspect, the gene comprises a nucleic acid sequence that is at least 85% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In one aspect, the gene comprises a nucleic acid sequence that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In one aspect, the gene comprises a nucleic acid sequence that is at least 92.5% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In one aspect, the gene comprises a nucleic acid sequence that is at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In one aspect, the gene comprises a nucleic acid sequence that is at least 96% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In one aspect, the gene comprises a nucleic acid sequence that is at least 97% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In one aspect, the gene comprises a nucleic acid sequence that is at least 98% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175.In one aspect, the gene comprises a nucleic acid sequence that is at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In one aspect, the gene comprises a nucleic acid sequence that is at least 99.5% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In one aspect, the gene comprises a nucleic acid sequence that is 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175.

[0264] In one aspect, the gene comprises a nucleic acid sequence encoding an amino acid sequence at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In one aspect, the gene comprises a nucleic acid sequence encoding an amino acid sequence at least 75% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In one aspect, the gene comprises a nucleic acid sequence encoding an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In one aspect, the gene comprises a nucleic acid sequence encoding an amino acid sequence at least 85% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In one aspect, the gene comprises a nucleic acid sequence encoding an amino acid sequence at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In one aspect, the gene comprises a nucleic acid sequence encoding an amino acid sequence at least 92.5% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In one aspect, the gene comprises a nucleic acid sequence encoding an amino acid sequence at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In one aspect, the gene comprises a nucleic acid sequence encoding an amino acid sequence at least 96% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205.In one aspect, the gene comprises a nucleic acid sequence encoding an amino acid sequence at least 97% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In one aspect, the gene comprises a nucleic acid sequence encoding an amino acid sequence at least 98% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In one aspect, the gene comprises a nucleic acid sequence encoding an amino acid sequence at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In one aspect, the gene comprises a nucleic acid sequence encoding an amino acid sequence at least 99.9% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In one aspect, the gene comprises a nucleic acid sequence encoding an amino acid sequence 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205.

[0265] In one aspect, the polypeptide comprises an amino acid sequence at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In one aspect, the polypeptide comprises an amino acid sequence at least 75% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In one aspect, the polypeptide comprises an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In some aspects, the polypeptide comprises an amino acid sequence at least 85% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In some aspects, the polypeptide comprises an amino acid sequence at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In some aspects, the polypeptide comprises an amino acid sequence at least 92.5% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In some aspects, the polypeptide comprises an amino acid sequence at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In some aspects, the polypeptide comprises an amino acid sequence at least 96% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In some aspects, the polypeptide comprises an amino acid sequence at least 97% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205.In one aspect, the polypeptide comprises an amino acid sequence at least 98% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In one aspect, the polypeptide comprises an amino acid sequence at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In one aspect, the polypeptide comprises an amino acid sequence at least 99.9% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In one aspect, the polypeptide comprises an amino acid sequence 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205.

[0266] The term "percent identity" or "percent identical" when used herein with respect to two or more nucleotide or amino acid sequences is calculated by (i) comparing two optimally aligned sequences (nucleotide or amino acid) over a comparison window (one or more regions that can be "aligned"); (ii) determining the number of positions in both sequences where the same nucleic acid base (for nucleotide sequences) or amino acid residue (for proteins and polypeptides) occurs to obtain the number of matched positions; (iii) dividing the number of matched positions by the total number of positions in the comparison window; and then (iv) multiplying this quotient by 100% to obtain the percent identity. When "percent identity" is calculated with respect to a reference sequence without specifying a specific comparison window, the percent identity is determined by dividing the number of matched positions over the region of alignment by the total length of the reference sequence. Thus, for the purposes of this application, when two sequences (query and subject) are optimally aligned (gaps allowed in their alignment), "percent identity" to the query sequence equals the number of identical positions between the two sequences divided by the total number of positions in the query sequence over its length (or comparison window) multiplied by 100%.

[0267] When percentages of sequence identity are used with respect to amino acids, it is recognized that non-identical residue positions often differ by conservative amino acid substitutions, in which an amino acid residue is replaced with another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity), and therefore does not alter the functional properties of the molecule. When sequences differ by conservative substitutions, the percent sequence identity can be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity."

[0268] Various pairwise or multiple sequence alignment algorithms and programs are known in the art, such as ClustalW or Basic Local Alignment Search Tool® (BLAST™), which can be used to compare sequence identity or similarity between two or more nucleotide or amino acid sequences for optimal alignment of sequences to calculate percent identity. Although other alignment and comparison methods are known in the art, alignment and percent identity between two sequences (including the percent identity ranges described above) can be determined by the Clustal W algorithm, see, e.g., Chenna et al., "Multiple sequence alignment with the Clustal series of programs," Nucleic Acids Research 31: 3497-3500 (2003); Thompson et al., "Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice," Nucleic Acids Research 22: 4673-4680 (1994); Larkin MA et al., "Clustal W and Clustal X version 2.0," Bioinformatics 23: 2947-48 (2007); and Altschul et al. "Basic local alignment search tool." J. Mol. Biol. 215:403-410. (1990), the entire contents and disclosures of which are incorporated herein by reference.

[0269] The term "percent complementarity" or "percent complementary" as used herein with respect to two nucleotide sequences is similar to the concept of percent identity, but refers to the percentage of nucleotides in the query sequence that are optimally base-paired or hybridized to the nucleotides of the subject sequence when the query and subject sequences are aligned linearly and optimally base-paired, without secondary folding structures such as loops, stems, or hairpins. Such percent complementarity can be between two DNA strands, two RNA strands, or a DNA strand and an RNA strand. "Percent complementarity" can be calculated by (i) optimally base-pairing or hybridizing two nucleotide sequences in a linear and fully extended alignment (i.e., without folding or secondary structures) over a comparison window, (ii) determining the number of base-paired positions between the two sequences over the comparison window to obtain the number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions in the comparison window, and (iv) multiplying this quotient by 100% to obtain the percent complementarity of the two sequences. The optimal base pairing of two sequences can be determined based on known pairing of nucleotide bases through hydrogen bonds (such as GC, AT and AU). When "percent complementarity" is calculated with respect to a reference sequence without a specific comparison window, the percent identity is determined by dividing the number of complementary positions between two linear sequences by the total length of the reference sequence. Therefore, for the purposes of this application, when two sequences (query and subject) are optimally base-paired (mismatched or non-base-paired nucleotides are allowed), the "percent complementarity" to a query sequence is equal to the number of base-paired positions between two sequences divided by the total number of positions over the length of the query sequence, multiplied by 100%.

[0270] The use of the term "polynucleotide" or "nucleic acid molecule" is not intended to limit the present disclosure to polynucleotides that include deoxyribonucleic acid (DNA). For example, ribonucleic acid (RNA) molecules are also contemplated. Those skilled in the art will recognize that polynucleotides and nucleic acid molecules can include ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. Polynucleotides of the present disclosure also encompass sequences of any form, including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-loop structures, and the like. In one aspect, the nucleic acid molecules provided herein are DNA molecules. In another aspect, the nucleic acid molecules provided herein are RNA molecules. In one aspect, the nucleic acid molecules provided herein are single-stranded. In another aspect, the nucleic acid molecules provided herein are double-stranded. The nucleic acid molecules can encode polypeptides or small RNAs.

[0271] As used herein, a "recombinant nucleic acid molecule" refers to a nucleic acid molecule formed by experimental methods of genetic recombination, including but not limited to molecular cloning. Similarly, a "recombinant DNA construct" refers to a DNA molecule formed by experimental methods of genetic recombination.

[0272] In one aspect, the present disclosure provides a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene, operably linked to a heterologous promoter.In one aspect, the present disclosure provides a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene, operably linked to a heterologous promoter.In one aspect, the present disclosure provides a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene, operably linked to a heterologous promoter.

[0273] Nucleic acids can be isolated using techniques conventional in the art. For example, nucleic acids can be isolated using any method, including but not limited to recombinant nucleic acid techniques and / or polymerase chain reaction (PCR). General PCR techniques are described, for example, in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation, which can be used to isolate nucleic acids. Isolated nucleic acids can also be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides. Non-limiting examples of primers that can be used to amplify specific candidate genes provided in this disclosure are provided in Table 1 (SEQ ID NO: 64-103).

[0274] In one aspect, the present disclosure provides a method for detecting the mRNA expression of endogenous genes encoding polypeptides involved in the biosynthesis of at least one terpene.Messenger RNA (mRNA) levels can be measured by standard techniques in the art, such as reverse transcription polymerase chain reaction (RT-PCR) and microarray analysis.RNA can be reverse transcribed into cDNA (complementary DNA) by methods known in the art.Synthesized cDNA can be amplified by methods known in the art, such as real-time PCR and digital PCR for relative or absolute quantification of mRNA transcript targets.

[0275] As used herein, the term "polypeptide" refers to a chain of at least two covalently linked amino acids. A polypeptide can be encoded by a polynucleotide provided herein. A protein provided herein can be encoded by a nucleic acid molecule provided herein. A protein can include a polypeptide provided herein. As used herein, "protein" refers to a chain of amino acid residues that can provide structure or enzymatic activity to a cell.

[0276] Polypeptides can be purified from natural sources (e.g., biological samples) by known methods such as DEAE ion exchange, gel filtration and hydroxyapatite chromatography.Polypeptides can also be purified, for example, by expressing nucleic acid in an expression vector.In addition, purified polypeptides can be obtained by chemical synthesis.The degree of purity of polypeptides can be measured using any suitable method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0277] In one aspect, the present disclosure provides a method for detecting recombinant nucleic acid and polypeptide in plant cells.Nucleic acid can also be detected using hybridization, but is not limited to this.Nucleic acid-nucleic acid hybridization is discussed in detail in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).mRNA expression can be localized in tissue samples using in situ hybridization.

[0278] In one aspect, the disclosure provides a method for detecting the accumulation of a polypeptide in a modified tobacco plant relative to a control tobacco plant lacking at least one non-naturally occurring mutation when grown under equivalent conditions, where increased accumulation of the polypeptide in the modified plant is determined relative to detection of the same polypeptide in a control plant lacking the non-naturally occurring mutation and grown under equivalent conditions.

[0279] Polypeptides can be detected using antibodies. Techniques for detecting polypeptides using antibodies include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation and immunofluorescence. The antibodies provided herein can be polyclonal or monoclonal. Antibodies with specific binding affinity for the polypeptides provided herein can be produced using methods well known in the art. The antibodies provided herein can be attached to a solid support, such as a microtiter plate, using methods well known in the art.

[0280] Detection (e.g., of an amplification product, of a hybridization complex, of a polypeptide) can be accomplished using a detectable label. The term "label" is intended to encompass the use of direct and indirect labels. Detectable labels include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials and radioactive materials.

[0281] In one aspect, the disclosure provides a modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene.

[0282] In one aspect, the disclosure provides a small RNA molecule, or a precursor thereof. As used herein, "small RNA molecule" refers to a non-coding RNA molecule between 16 nucleotides and 50 nucleotides in length. In one aspect, the small RNA molecule comprises between 16 nucleotides and 40 nucleotides. In another aspect, the small RNA molecule comprises between 16 nucleotides and 30 nucleotides. In another aspect, the small RNA molecule comprises between 18 nucleotides and 50 nucleotides. In another aspect, the small RNA molecule comprises between 18 nucleotides and 40 nucleotides. In another aspect, the small RNA molecule comprises between 18 nucleotides and 30 nucleotides. In another aspect, the small RNA molecule comprises between 18 nucleotides and 25 nucleotides. In another aspect, the small RNA molecule comprises between 20 nucleotides and 28 nucleotides. In another aspect, the small RNA molecule comprises between 20 nucleotides and 24 nucleotides. In another aspect, the small RNA molecule comprises between 21 nucleotides and 23 nucleotides. In another aspect, the small RNA molecule comprises 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.

[0283] In some aspects, the small RNA molecule is at least 75% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In some aspects, the small RNA molecule is at least 80% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In some aspects, the small RNA molecule is at least 85% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In some aspects, the small RNA molecule is at least 90% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In some aspects, the small RNA molecule is at least 92.5% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In some aspects, the small RNA molecule is at least 95% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In some aspects, the small RNA molecule is at least 97.5% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In some aspects, the small RNA molecule is at least 99% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In some aspects, the small RNA molecule is 100% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190.

[0284] In some aspects, the small RNA molecule is at least 75% identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In some aspects, the small RNA molecule is at least 80% identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In some aspects, the small RNA molecule is at least 85% identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In some aspects, the small RNA molecule is at least 90% identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In some aspects, the small RNA molecule is at least 92.5% identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In some aspects, the small RNA molecule is at least 95% identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In one aspect, the small RNA molecule is at least 97.5% identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In one aspect, the small RNA molecule is at least 99% identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205.In one aspect, the small RNA molecule is 100% identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205.

[0285] In one aspect, the small RNAs provided herein comprise a nucleic acid sequence that is at least 88.7% identical or complementary to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In another aspect, the small RNAs provided herein comprise a nucleic acid sequence that is at least 94.3% identical or complementary to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In another aspect, the small RNAs provided herein comprise a nucleic acid sequence that is 100% identical or complementary to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In another aspect, the small RNAs provided herein comprise a nucleic acid sequence that is at least 85% identical or complementary to at least 20 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In another aspect, the small RNAs provided herein comprise a nucleic acid sequence that is at least 90% identical or complementary to at least 20 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In another aspect, the small RNAs provided herein comprise a nucleic acid sequence that is at least 95% identical or complementary to at least 20 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In another aspect, the small RNAs provided herein comprise a nucleic acid sequence that is 100% identical or complementary to at least 20 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190.

[0286] In certain aspects, the small RNA molecule is selected from the group consisting of double-stranded RNA, small interfering RNA (siRNA), trans-acting siRNA, and microRNA (miRNA).

[0287] miRNAs are generally about 19 to about 25 nucleotides (usually about 20 to 24 nucleotides in plants) that guide the trans cleavage of target transcripts and negatively regulate the expression of genes involved in various regulatory and developmental pathways. In some cases, miRNAs serve to guide in-phase processing of siRNA primary transcripts.

[0288] Many microRNA genes (MIR genes) have been identified and published in databases ("miRBase," available online at microrna[dot]sanger[dot]ac[dot]uk / sequences; see also Griffiths-Jones et al. (2003) Nucleic Acids Res., 31:439-441). MIR genes have been reported to occur in intergenic regions (both singly and in clusters in the genome), but may also be located entirely or partially within introns of other genes (both protein-coding and non-protein-coding). For a review of miRNA biogenesis, see Kim (2005) Nature Rev. Mol. Cell. Biol., 6:376-385. Transcription of MIR genes can, at least in some cases, be under the facilitatory control of the MIR gene's own promoter. The primary transcript, termed the "pri-miRNA", can be quite large (several kilobases) and polycistronic, containing one or more pre-miRNAs (folded structures containing stem-loop arrangements that are processed into mature miRNAs) as well as the usual 5' "cap" and polyadenylated tail of an mRNA.

[0289] The maturation of mature miRNA from its corresponding precursor (pri-miRNA and pre-miRNA) is significantly different between animals and plants.For example, in plant cells, microRNA precursor molecules are mostly processed into mature miRNA completely in the nucleus, whereas in animal cells, pri-miRNA transcripts are processed by animal-specific enzyme Drosha in the nucleus, and then pre-miRNA is transported to the cytoplasm, where it is further processed into mature miRNA.The mature miRNA in plants is typically 21 nucleotides long.

[0290] Transgenic expression of miRNAs (whether naturally occurring or artificial sequences) can be employed to regulate the expression of the miRNA's target gene(s). Inclusion of miRNA recognition sites in transgenically expressed transcripts is also useful for regulating the expression of the transcripts. Recognition sites for miRNAs have been demonstrated in all regions of the mRNA, including the 5' untranslated, coding and 3' untranslated regions, showing that the location of the miRNA target site relative to the coding sequence does not necessarily affect repression. Since miRNAs are important regulatory elements in eukaryotes, transgenic repression of miRNAs is useful for manipulating biological pathways and responses. Finally, promoters of MIR genes can have highly specific expression patterns (e.g., cell-specific, tissue-specific, time-specific or inducible), and are therefore useful for inducing such specific transcription of DNA sequences to which they are operably linked in recombinant constructs. Various uses of miRNAs, their precursors, their recognition sites, and their promoters are detailed in US Patent Application Publication No. 2006 / 0200878 A1, which is incorporated herein by reference. Non-limiting examples of these uses include: (1) expression of native miRNAs or miRNA precursor sequences to repress target genes; (2) expression of artificial miRNAs or miRNA precursor sequences to repress target genes; (3) expression of transgenes with miRNA recognition sites, where the transgene is repressed when the mature miRNA is expressed; (4) expression of transgenes driven by miRNA promoters.

[0291] Designing an artificial miRNA sequence can be as simple as replacing nucleotides in the miRNA stem region of a miRNA precursor with a sequence that is complementary to the intended target, as demonstrated by Zeng et al. (2002) Mol. Cell, 9:1327-1333. One non-limiting example of a general method for determining nucleotide changes in a native miRNA sequence to produce an engineered miRNA precursor includes the steps of: (a) selecting a unique target sequence of at least 18 nucleotides specific to the target gene, for example, by using sequence alignment tools such as BLAST (see, e.g., Altschul et al. (1990) J. Mol. Biol., 215:403-410; Altschul et al. (1997) Nucleic Acids Res., 25:3389-3402) against both tobacco cDNA and genomic DNA databases to identify any potential matches with the target transcript orthologs and unrelated genes, thereby avoiding unintended silencing of non-target sequences; (b) analyzing the target gene for undesirable sequences (e.g., those that match sequences from non-target species) and analysing each potential 19-mer segment based on GC content, Reynolds score (Reynolds et al. (2004) Nature Biotechnol., 22:326-330), and for functional asymmetry, characterized by a negative difference in free energy (".DELTA..DELTA.G" or "ΔΔG") (see Khvorova et al. (2003) Cell, 115:209-216). Preferably, 19-mers are selected that have all or most of the following characteristics: (1) Reynolds score >4, (2) GC content of about 40% to about 60%, (3) negative ΔΔG, (4) terminal adenosine, (5) absence of four or more consecutive identical nucleotides; (6) location near the 3' end of the target gene; and (7) minimal divergence from the miRNA precursor transcript.It has been reported that the position of every third nucleotide in siRNA is particularly important in affecting RNAi efficacy, and an algorithm "siExplorer" has been published at rna[dot]chem[dot]t[dot]u-tokyo[dot]ac[dot]jp / siexplorer.htm (see Katoh and Suzuki (2007) Nucleic Acids Res., 10.1093 / nar / gkl1120); (c) determining the reverse complement sequence of the selected 19-mer for use in generating modified mature miRNA. The additional nucleotide at position 20 preferably matches the selected target sequence, and the nucleotide at position 21 is preferably selected to be unpaired to prevent the spread of silencing to the target transcript or to be paired with the target sequence to promote the spread of silencing to the target transcript; and (d) transforming a plant with the artificial miRNA.

[0292] In some aspects, the vector can be used, for example, for overexpression and knockdown of candidate terpene biosynthetic genes. A non-limiting example of a vector is pGWB551. In some aspects, the vector comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO:154. In some aspects, the vector comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO:154. In some aspects, the vector comprises a nucleic acid sequence that is 100% identical to SEQ ID NO:154.

[0293] product In some aspects, the present disclosure provides plant material from any plant or plant part provided herein. In some aspects, the present disclosure provides cannabis material from any cannabis plant or cannabis plant part provided herein. In some aspects, the present disclosure provides dried plant material from any plant or plant part provided herein. In some aspects, the present disclosure provides dried tobacco material from any tobacco plant or tobacco plant part provided herein. In some aspects, the present disclosure provides dried cannabis material from any cannabis plant or cannabis plant part provided herein.

[0294] In one aspect, the disclosure provides a method comprising preparing a tobacco product or cannabis product using a cured tobacco material or cannabis material derived from a modified tobacco or cannabis plant, the modified tobacco or cannabis plant comprising a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter. In one aspect, the disclosure provides a method comprising preparing a tobacco product or cannabis product using a cured tobacco material or cannabis material derived from a modified tobacco or cannabis plant, the modified tobacco or cannabis plant comprising at least one non-naturally occurring mutation in an endogenous gene encoding a polypeptide involved in the biosynthesis of at least one terpene, the modified tobacco or cannabis plant exhibiting increased mRNA expression of the endogenous gene or increased accumulation of the polypeptide when grown under comparable conditions, compared to a control tobacco or cannabis plant lacking the at least one non-naturally occurring mutation.

[0295] In some aspects, the cured tobacco plant material is produced by a drying process selected from the group consisting of hot air curing, air drying, flame curing, and sun drying. In some aspects, the cured tobacco material is produced by a drying process selected from the group consisting of hot air curing, air drying, flame curing, and sun drying.

[0296] "Curing" is a aging process that reduces the moisture content and causes the destruction of chlorophyll, thereby giving tobacco leaves a golden color and converting starch to sugars. Cured tobacco therefore has a higher reducing sugar content and a lower starch content than harvested green leaves. In one aspect, the tobacco plants or plant components provided herein can be dried using conventional means, such as hot air drying, barn drying, fire drying, air drying, or sun drying. For a description of different types of curing methods, see, for example, Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). Cured tobacco is typically aged in wooden drums (e.g., vats) or cardboard cartons under compressed conditions for several years (e.g., 2-5 years) at a moisture content ranging from 10% to about 25%. See U.S. Patent Nos. 4,516,590 and 5,372,149. The cured and aged tobacco can then be further processed, which includes conditioning the tobacco under vacuum with or without steam injection at various temperatures, pasteurization, and fermentation.

[0297] Following harvest of the cannabis plant, the cannabis plant material is typically dried prior to curing. Cannabis can be dried in a dark room with a fan for gentle air circulation, maintaining a temperature of about 15°C to about 22°C and a humidity of 45% to 55%. The first drying cycle lasts from 5 to 15 days. The dried cannabis can then be dried to enhance aroma, flavor and potency. The length of the cannabis drying process depends on the strain. For example, some cannabis strains benefit from a drying process of 2 to 3 weeks, others from 4 to 8 weeks, while others benefit from a drying process of 6 months or more.

[0298] Information regarding harvesting burley and dark tobacco varieties can be found in the 2019-2020 Burley and Dark Tobacco Production Guide (December 2018) published by the University of Kentucky, the University of Tennessee, Virginia Polytechnic Institute and University, and North Carolina State University, which is incorporated by reference in its entirety.

[0299] In some aspects, the dried tobacco material comprises a tobacco material selected from the group selected from dried leaf material, dried stem material, dried bud material, dried flower material, and dried root material. In some aspects, the dried tobacco material comprises dried leaf material, dried stem material, or both. In some aspects, the dried tobacco material comprises dried leaf material. In some aspects, the dried tobacco material comprises dried stem material.

[0300] In some aspects, the cannabis material comprises cannabis material selected from the group selected from leaf material, stem material, bud material, flower material, root material, hairy roots, cell cultures, and callus. In some aspects, the cannabis material comprises leaf material, stem material, or both. In some aspects, the cannabis material comprises leaf material. In some aspects, the cannabis material comprises stem material.

[0301] In some aspects, the cured tobacco material comprises flue-cured tobacco material. In some aspects, the cured tobacco material comprises air-cured tobacco material. In some aspects, the cured tobacco material comprises flue-cured tobacco material. In some aspects, the cured tobacco material comprises sun-cured tobacco material. In some aspects, the cured tobacco material provided herein is selected from the group consisting of air-cured tobacco material, flue-cured tobacco material, sun-cured tobacco material, and flue-cured tobacco material. In some aspects, the cured tobacco material is derived from a tobacco variety selected from the group consisting of flue-cured, bright, burley, Virginia, Maryland, dark, Oriental, and Turkish.

[0302] In some aspects, the cured tobacco provided herein is selected from the group consisting of air-cured, flue-cured, sun-cured, and flue-cured tobacco. In some aspects, the cured tobacco is from a tobacco variety selected from the group consisting of flue-cured, bright, burley, Virginia, Maryland, dark, Oriental, and Turkish.

[0303] Fermentation is typically characterized by high initial moisture content, heat generation, and a loss of 10-20% of dry weight. See, e.g., U.S. Patent Nos. 4,528,993, 4,660,577, 4,848,373, 5,372,149; U.S. Patent Publication No. 2005 / 0178398; and Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). The dried, aged and fermented tobacco can be further processed (e.g., cut, shredded, expanded, or blended). See, e.g., U.S. Patent Nos. 4,528,993; 4,660,577; and 4,987,907. The dried, aged and fermented cannabis can be further processed (e.g., cut, shredded, expanded, or blended). In one aspect, the present disclosure provides a fermented tobacco material derived from any tobacco plant, or part thereof, provided herein. In another aspect, the present disclosure provides a fermented tobacco material derived from any modified tobacco plant, or part thereof, provided herein. In one aspect, the present disclosure provides a fermented cannabis material derived from any cannabis plant, or part thereof, provided herein. In another aspect, the present disclosure provides a fermented cannabis material derived from any modified cannabis plant, or part thereof, provided herein.

[0304] Tobacco materials obtained from the tobacco lines, varieties or hybrids of the present disclosure can be used to manufacture tobacco products. As used herein, "tobacco products" are defined as any product made from or derived from tobacco intended for human use or consumption. In one aspect, the present disclosure provides tobacco products comprising plant material from the tobacco plants provided herein. In another aspect, the present disclosure provides tobacco products comprising plant material from the modified tobacco plants provided herein. In another aspect, the present disclosure provides tobacco products comprising dried tobacco materials. In another aspect, the present disclosure provides tobacco products comprising fermented tobacco materials. In another aspect, the present disclosure provides tobacco products comprising tobacco blends.

[0305] Cannabis material obtained from the cannabis line, strain, variety or hybrid of the present disclosure can be used to produce cannabis products. As used herein, "cannabis product" is defined as any product produced or derived from cannabis intended for human use or consumption. In one aspect, the present disclosure provides a cannabis product comprising plant material from a cannabis plant provided herein. In another aspect, the present disclosure provides a cannabis product comprising plant material from a modified cannabis plant provided herein. In another aspect, the present disclosure provides a cannabis product comprising cannabis material. In another aspect, the present disclosure provides a cannabis product comprising dried cannabis material. In another aspect, the present disclosure provides a cannabis product comprising fermented cannabis material. In another aspect, the present disclosure provides a cannabis product comprising a cannabis blend.

[0306] Tobacco products include, but are not limited to, cigarette products (e.g., cigarettes and bidi cigarettes), cigar products (e.g., cigar wrappers and cigarillos), pipe tobacco products, tobacco-derived products, tobacco-derived nicotine products, smokeless tobacco products (e.g., moist snuff, dry snuff, and chewing tobacco), films, chewables, tabs, shaped parts, gels, consumable units, insoluble matrices, hollow shapes, reconstituted tobacco, expanded tobacco, etc. See, e.g., U.S. Patent Publication No. US 2006 / 0191548.

[0307] As used herein, a "cigarette" refers to a tobacco product having a "rod" and a "filler." The "rod" of a cigarette includes the cigarette paper, the filter, the plug wrap (used to contain the filtration material), the tipping paper that secures the cigarette paper (including the filler) to the filter, and any adhesives that secure these components together. The "filler" includes (1) all tobacco, including but not limited to reconstituted tobacco and expanded tobacco, (2) non-tobacco substitutes (including but not limited to herbs, non-tobacco plant materials, and other spices that may accompany tobacco that is wrapped in cigarette paper), (3) casing, (4) flavorings, and (5) all other additives that are mixed with the tobacco and substitutes and wrapped into the cigarette.

[0308] In one aspect, the tobacco product comprises reconstituted tobacco. In another aspect, the present disclosure provides a reconstituted tobacco comprising dried tobacco material. As used herein, "reconstituted tobacco" refers to a member of tobacco filler made from tobacco dust and other tobacco scrap materials that are processed into sheets and cut into strips to resemble tobacco. In addition to cost savings, reconstituted tobacco is also highly valued for its contribution to cigarette flavor, since it uses a reaction between ammonia and sugars to drive flavor development.

[0309] In one aspect, the cannabis product comprises reconstituted cannabis. In another aspect, the present disclosure provides reconstituted cannabis comprising cannabis material. As used herein, "reconstituted cannabis" refers to a member of cannabis filling made from cannabis dust and other cannabis scrap materials that are processed into sheets and cut into strips to resemble cannabis.

[0310] In some aspects, the tobacco product includes expanded tobacco. As used herein, "expanded tobacco" refers to a member of tobacco filler that has been processed through a suitable gas expansion to "expand" the tobacco resulting in reduced density and greater filling capacity. Expanded tobacco reduces the weight of the tobacco used in the cigarette.

[0311] In some aspects, the cannabis product includes expanded cannabis. As used herein, "expanded cannabis" refers to a member of cannabis filling that has been processed through the expansion of a suitable gas to "expand" the cannabis resulting in reduced density and greater filling capacity.

[0312] Tobacco products derived from the plants of the present disclosure also include cigarettes and other smoking articles, particularly smoking articles that include a filter element, where the rod of smokable material includes cured tobacco within a tobacco blend. In some aspects, the tobacco products of the present disclosure are selected from the group consisting of kretek, bidi cigarettes, cigarillos, non-ventilated recess filter cigarettes, vented recess filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, hookah, shredded tobacco, and cut tobacco.

[0313] The cannabis products derived from the plants of the present disclosure also include cigarettes and other smoking articles, particularly smoking articles that include a filter element, where the rod of smokable material includes dried cannabis in a cannabis blend. In one aspect, the cannabis products of the present disclosure are selected from the group consisting of kreteks, cigarillos, cigars, snuff, pipe cannabis, cigar cannabis, cigarette cannabis, chewing cannabis, water cannabis, shredded cannabis, and cut cannabis.

[0314] In certain aspects, the tobacco product of the present disclosure is selected from the group consisting of cigarettes, heated tobacco products, kreteks, bidi cigarettes, cigars, cigarillos, non-ventilated cigarettes, ventilated recessed filter cigarettes, pipe tobacco, snuff, snus, chewing tobacco, moist smokeless tobacco, fine cut chewing tobacco, long cut chewing tobacco, pouched chewing tobacco products, gum, tablets, lozenges, and dissolving strips.

[0315] In some aspects, the cannabis products of the present disclosure are selected from the group consisting of cigarettes, heated cannabis products, kreteks, cigars, cigarillos, pipe cannabis, snuff, snus, chewing cannabis, moist smokeless cannabis, fine cut chewing cannabis, long cut chewing cannabis, pouched chewing cannabis products, gum, tablets, lozenges, and dissolving strips.

[0316] In some aspects, the cannabis products of the present disclosure are consumable products. In further aspects, the consumable products are edible products. In some aspects, the consumable products are oils.

[0317] In some aspects, the tobacco product of the present disclosure is a smokeless tobacco product. In some aspects, the smokeless tobacco product is selected from the group consisting of loose leaf chewing tobacco, solid chewing tobacco, moist snuff, nasal snuff, dry snuff, and snus.

[0318] In some aspects, the cannabis product of the present disclosure is a smokeless cannabis product. In some aspects, the smokeless cannabis product is selected from the group consisting of chewing cannabis, moist snuff, dry snuff, and snus.

[0319] Smokeless tobacco or smokeless cannabis products are not combusted and include, but are not limited to, chewing tobacco, moist smokeless tobacco, moist smokeless cannabis, snus, and dry snuff. Chewing tobacco or cannabis is coarsely divided tobacco or cannabis leaves, typically packaged in large pouch-like packages and used in a solid or twisted form. Moist smokeless tobacco or cannabis is more finely divided tobacco or cannabis with a moist feel, provided in loose form or in the form of a bag, typically packaged in a round tin and used as a pinch or in a bag between the cheek and gums of an adult tobacco or cannabis consumer. Snus is smokeless tobacco or cannabis that has been heat-treated. Dry snuff is finely powdered tobacco or cannabis that is placed in the mouth or used intranasally.

[0320] In yet another aspect, the tobacco or cannabis product of the present disclosure is selected from the group consisting of an electronically heated cigarette, an e-cigarette, an electronic vaporization device.

[0321] In some aspects, the tobacco or cannabis products of the present disclosure can be blended tobacco or cannabis products.

[0322] In another aspect, the present disclosure provides a tobacco blend comprising a dried tobacco material. The tobacco blend may include any combination of dried tobacco, never-cured tobacco, fermented tobacco, unfermented tobacco, expanded tobacco, and reconstituted tobacco.

[0323] In another aspect, the present disclosure provides a cannabis blend comprising cannabis material. The cannabis blend can include any combination of fermented cannabis, unfermented cannabis, expanded cannabis, and reconstituted cannabis.

[0324] In some aspects, the tobacco blend comprises at least 5% by weight of cured tobacco. In some aspects, the tobacco blend comprises at least 10% by weight of cured tobacco. In some aspects, the tobacco blend comprises at least 15% by weight of cured tobacco. In some aspects, the tobacco blend comprises at least 20% by weight of cured tobacco. In some aspects, the tobacco blend comprises at least 25% by weight of cured tobacco. In some aspects, the tobacco blend comprises at least 30% by weight of cured tobacco. In some aspects, the tobacco blend comprises at least 35% by weight of cured tobacco. In some aspects, the tobacco blend comprises at least 40% by weight of cured tobacco. In some aspects, the tobacco blend comprises at least 45% by weight of cured tobacco. In some aspects, the tobacco blend comprises at least 50% by weight of cured tobacco. In some aspects, the tobacco blend comprises at least 55% by weight of cured tobacco. In some aspects, the tobacco blend comprises at least 60% by weight of cured tobacco. In some aspects, the tobacco blend comprises at least 65% by weight of cured tobacco. In some aspects, the tobacco blend comprises at least 70% by weight of cured tobacco. In some aspects, the tobacco blend comprises at least 75% by weight of cured tobacco. In some aspects, the tobacco blend comprises at least 80% by weight of cured tobacco. In some aspects, the tobacco blend comprises at least 85% by weight of cured tobacco. In some aspects, the tobacco blend comprises at least 90% by weight of cured tobacco. In some aspects, the tobacco blend comprises at least 95% by weight of cured tobacco.

[0325] In some aspects the cannabis blend comprises at least 5% cannabis by weight. In some aspects the cannabis blend comprises at least 10% cannabis by weight. In some aspects the cannabis blend comprises at least 15% cannabis by weight. In some aspects the cannabis blend comprises at least 20% cannabis by weight. In some aspects the cannabis blend comprises at least 25% cannabis by weight. In some aspects the cannabis blend comprises at least 30% cannabis by weight. In some aspects the cannabis blend comprises at least 35% cannabis by weight. In some aspects the cannabis blend comprises at least 40% cannabis by weight. In some aspects the cannabis blend comprises at least 45% cannabis by weight. In some aspects the cannabis blend comprises at least 50% cannabis by weight. In some aspects the cannabis blend comprises at least 55% cannabis by weight. In some aspects the cannabis blend comprises at least 60% cannabis by weight. In some aspects the cannabis blend comprises at least 65% cannabis by weight. In some aspects, the cannabis blend comprises at least 70% cannabis by weight. In some aspects, the cannabis blend comprises at least 75% cannabis by weight. In some aspects, the cannabis blend comprises at least 80% cannabis by weight. In some aspects, the cannabis blend comprises at least 85% cannabis by weight. In some aspects, the cannabis blend comprises at least 90% cannabis by weight. In some aspects, the cannabis blend comprises at least 95% cannabis by weight.

[0326] In some aspects, the tobacco blend comprises at least 5% by volume of cured tobacco. In some aspects, the tobacco blend comprises at least 10% by volume of cured tobacco. In some aspects, the tobacco blend comprises at least 15% by volume of cured tobacco. In some aspects, the tobacco blend comprises at least 20% by volume of cured tobacco. In some aspects, the tobacco blend comprises at least 25% by volume of cured tobacco. In some aspects, the tobacco blend comprises at least 30% by volume of cured tobacco. In some aspects, the tobacco blend comprises at least 35% by volume of cured tobacco. In some aspects, the tobacco blend comprises at least 40% by volume of cured tobacco. In some aspects, the tobacco blend comprises at least 45% by volume of cured tobacco. In some aspects, the tobacco blend comprises at least 50% by volume of cured tobacco. In some aspects, the tobacco blend comprises at least 55% by volume of cured tobacco. In some aspects, the tobacco blend comprises at least 60% by volume of cured tobacco. In some aspects, the tobacco blend comprises at least 65% by volume of cured tobacco. In some aspects, the tobacco blend comprises at least 70% by volume of cured tobacco. In some aspects, the tobacco blend comprises at least 75% by volume of cured tobacco. In some aspects, the tobacco blend comprises at least 80% by volume of cured tobacco. In some aspects, the tobacco blend comprises at least 85% by volume of cured tobacco. In some aspects, the tobacco blend comprises at least 90% by volume of cured tobacco. In some aspects, the tobacco blend comprises at least 95% by volume of cured tobacco.

[0327] In some aspects, the cannabis blend comprises at least 5% cannabis by volume. In some aspects, the cannabis blend comprises at least 10% cannabis by volume. In some aspects, the cannabis blend comprises at least 15% cannabis by volume. In some aspects, the cannabis blend comprises at least 20% cannabis by volume. In some aspects, the cannabis blend comprises at least 25% cannabis by volume. In some aspects, the cannabis blend comprises at least 30% cannabis by volume. In some aspects, the cannabis blend comprises at least 35% cannabis by volume. In some aspects, the cannabis blend comprises at least 40% cannabis by volume. In some aspects, the cannabis blend comprises at least 45% cannabis by volume. In some aspects, the cannabis blend comprises at least 50% cannabis by volume. In some aspects, the cannabis blend comprises at least 55% cannabis by volume. In some aspects, the cannabis blend comprises at least 60% cannabis by volume. In some aspects, the cannabis blend comprises at least 65% cannabis by volume. In some aspects, the cannabis blend comprises at least 70% cannabis by volume. In some aspects, the cannabis blend comprises at least 75% cannabis by volume. In some aspects, the cannabis blend comprises at least 80% cannabis by volume. In some aspects, the cannabis blend comprises at least 85% cannabis by volume. In some aspects, the cannabis blend comprises at least 90% cannabis by volume. In some aspects, the cannabis blend comprises at least 95% cannabis by volume.

[0328] In one aspect, the disclosure provides a method comprising preparing a tobacco or cannabis product using cured tobacco or cannabis material from a modified tobacco or cannabis plant, wherein the modified tobacco or cannabis plant comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene.

[0329] Aspects The following non-limiting example embodiments are contemplated. 1. A modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof, comprising a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter. 2. A modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof, comprising at least one non-naturally occurring mutation in an endogenous gene encoding a polypeptide involved in the biosynthesis of at least one terpene, wherein the modified tobacco or cannabis plant exhibits increased mRNA expression of the endogenous gene or increased accumulation of the polypeptide compared to a control tobacco or cannabis plant lacking the at least one non-naturally occurring mutation when grown under equivalent conditions. 3. A modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene. 4. The modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of any of aspects

[0059] to 3, wherein the polypeptide is selected from the group consisting of geranyl diphosphate synthase (GDP), limonene synthase (LS), limonene 3-hydroxylase (L3OH), isopiperitenol dehydrogenase (IPD), pulegone reductase, menthofuran synthase (MFS), geranylgeranyl diphosphate synthase (GGPPS), neomenthol dehydrogenase (NtNMD), phylloplanin, premnaspirodiene oxygenase (PSO), kolavenyl diphosphate synthase (KPS), solanesyl phosphate synthase 3, terpene synthase 10-like, and germacrene D synthase. 5. A modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of any of embodiments

[0059] to 3, wherein at least one terpene is menthol or a related compound. 6. A modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of any of embodiments

[0059] to 3, wherein the polypeptide is selected from the group consisting of geranylgeranyl diphosphate synthase (GGPPS2), 8-hydroxy-copalyl diphosphate synthase, and cis-abienol synthase. 7. The modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of embodiment 6, wherein the cis-abienol synthase is selected from the group consisting of cis-abienol synthase isoform 1 and cis-abienol synthase isoform 2. 8. A modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of any of embodiments

[0059] to 3, wherein at least one terpene is a labdanoid. 9. The modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of any of embodiments

[0059] to 3, wherein at least one terpene is neophytadiene. 10. The modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of embodiment 8, wherein the labdanoid is cis-abienol. 11. A modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof according to any one of embodiments

[0059] to 3, wherein the polypeptide is cembratrienol synthase 2a. 12. A modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of any of embodiments

[0059] to 3, wherein at least one terpene is cembratrienediol. 13. A modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof according to any one of embodiments

[0059] to 3, wherein the polypeptide is levopimaradiene synthase. 14. The modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of any of embodiments

[0059] to 3, wherein at least one terpene is levopimaric acid. 15. The modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of any of embodiments

[0059] to 3, wherein the polypeptide is selected from the group consisting of 2-isopropylmalate synthase (IPS), 2-oxoisovalerate dehydrogenase, and 2-alkenal reductase. 16. A modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of any of embodiments

[0059] to 3, wherein at least one terpene is L-leucine. 17. The modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of embodiment

[0059] , wherein the nucleic acid sequence comprises a sequence that is at least 70% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. 18. The modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of embodiment 2, wherein the endogenous gene comprises a sequence that is at least 70% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. 19. The modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of any of embodiments

[0059] to 3, wherein the polypeptide comprises an amino acid sequence that is at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. 20. A modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of any of embodiments

[0059] to 3, wherein the heterologous promoter comprises a constitutive promoter. 21. A modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of any of embodiments

[0059] to 3, wherein the heterologous promoter comprises a tissue-preferred promoter or a tissue-specific promoter. 22. The modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of embodiment 21, wherein the tissue-preferred or tissue-specific promoter comprises a trichome-preferred or trichome-specific promoter. 23. A modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of any of embodiments

[0059] to 3, wherein the heterologous promoter comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 104-111, 206-208, or a functional fragment thereof. 24. The modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of embodiment

[0059] , wherein the modified tobacco or cannabis plant comprises an increased amount of at least one terpene compared to a control tobacco or cannabis plant lacking the recombinant nucleic acid molecule when grown under equivalent conditions. 25. The modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of embodiment 2, wherein said modified tobacco or cannabis plant comprises an increased amount of at least one terpene compared to said control tobacco or cannabis plant. 26. The modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of embodiment 24 or 25, wherein the increased amount of the at least one terpene comprises an increase of at least 5% compared to the control tobacco or cannabis plant. 27. The modified tobacco plant, tobacco seed, or part thereof of any of embodiments

[0059] to 3, wherein the tobacco plant, tobacco seed, or part thereof is of a tobacco variety selected from the group consisting of a flue-cured variety, a bright variety, a Burley variety, a Virginia variety, a Maryland variety, a dark variety, a Galpao variety, an Oriental variety, and a Turkish variety. 28. A modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof according to any one of aspects

[0059] to 3, wherein the tobacco or cannabis plant is male sterile or cytoplasmically male sterile. 29. The modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of embodiment 2, which is homozygous for at least one non-naturally occurring mutation. 30. The modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of embodiment 2, which is heterozygous for at least one non-naturally occurring mutation. 31. The modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of embodiment 2, wherein at least one non-naturally occurring mutation comprises a mutation selected from the group consisting of an insertion, a deletion, a substitution, a duplication, and an inversion. 32. The modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of embodiment 2, wherein the at least one non-naturally occurring mutation comprises at least one mutation selected from the group consisting of a nonsense mutation, a missense mutation, a frameshift mutation, a splice site mutation, and any combination thereof. 33. The modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of embodiment 2, wherein at least one non-naturally occurring mutation comprises a null mutation. 34. The modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of embodiment 2, wherein at least one non-naturally occurring mutation results in a truncation of said polypeptide. 35. The modified tobacco or cannabis plant, tobacco or cannabis seed, or part thereof of embodiment 2, wherein at least one non-naturally occurring mutation comprises a mutation in a sequence region selected from the group consisting of a promoter, a 5'-untranslated region (UTR), an exon, an intron, a 3'-UTR, and a terminator. 36. A dried tobacco material or cannabis material derived from any of the modified tobacco or cannabis plants, tobacco or cannabis seeds, or parts thereof of any of embodiments

[0059] to 35. 37. The cured tobacco material of embodiment 36, produced by a drying process selected from the group consisting of hot air drying, air drying, flame drying, and sun drying. 38. A tobacco product comprising the dried tobacco material or cannabis material of embodiment 36 or 37, wherein the tobacco or cannabis product is selected from the group consisting of kretek, bidi cigarettes, cigarillos, non-ventilated recess filter cigarettes, vented recess filter cigarettes, cigars, snuff, pipe tobacco, pipe cannabis, cigar tobacco, cannabis cigars, cigarette tobacco, cannabis cigarettes, chewing tobacco, leaf tobacco, water pipe, water cannabis, shredded tobacco, shredded cannabis, cut tobacco, and cut cannabis. 39. The tobacco or cannabis product of embodiment 38, which is a smokeless tobacco or smokeless cannabis product. 40. The tobacco or cannabis product of embodiment 39, wherein the smokeless tobacco or smokeless cannabis product is selected from the group consisting of loose-leaf chewing tobacco, loose-leaf chewing cannabis, solid chewing tobacco, solid chewing cannabis, moist snuff, nasal snuff, dry snuff, and snus. 41. Reconstituted tobacco or cannabis comprising the dried tobacco material or cannabis material of embodiment 36 or 37. 42. Fermented tobacco or fermented cannabis, comprising the dried tobacco material or cannabis material of embodiment 36 or 37. 43. The tobacco or cannabis product of embodiment 38, comprising reconstituted tobacco or reconstituted cannabis. 44. The tobacco or cannabis product of embodiment 38, comprising fermented tobacco or fermented cannabis. 45. A recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter. 46. ​​A method for producing a modified tobacco or cannabis plant, comprising the steps of: (a) introducing a recombinant nucleic acid molecule into at least one tobacco or cannabis cell, the recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in terpene biosynthesis operably linked to a heterologous promoter; (b) selecting at least one tobacco or cannabis cell containing the recombinant nucleic acid molecule; and (c) regenerating a modified tobacco or cannabis plant from at least one tobacco or cannabis cell selected in step (b), wherein the modified tobacco or cannabis plant comprises an increased amount of at least one terpene in at least one tissue compared to a control tobacco or cannabis plant lacking the recombinant nucleic acid molecule when grown under equivalent conditions. 47. A method for producing a modified tobacco or cannabis plant, comprising the steps of: (a) inducing in at least one tobacco or cannabis cell at least one non-naturally occurring mutation in an endogenous gene encoding a polypeptide involved in terpene biosynthesis; (b) selecting at least one tobacco or cannabis plant containing the at least one non-naturally occurring mutation from step (a); and (c) regenerating a modified tobacco or cannabis plant from the at least one tobacco or cannabis cell selected in step (b), wherein the modified tobacco or cannabis plant comprises an increased amount of at least one terpene in at least one tissue compared to a control tobacco or cannabis plant lacking the at least one non-naturally occurring mutation when grown under equivalent conditions. 48. A method for producing a modified tobacco or cannabis plant, comprising the steps of: (a) introducing a recombinant DNA construct into at least one tobacco or cannabis cell, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene; (b) selecting at least one tobacco or cannabis cell containing the recombinant DNA construct; and (c) regenerating at least one modified tobacco or cannabis plant from the at least one tobacco or cannabis cell selected in step (b), wherein the modified tobacco or cannabis plant comprises a reduced amount of at least one terpene in at least one tissue compared to a control tobacco or cannabis plant lacking the recombinant DNA construct when grown under equivalent conditions. 49. A method comprising preparing a tobacco or cannabis product using cured tobacco material from a modified tobacco or cannabis plant, wherein the modified tobacco or cannabis plant comprises a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter. 50. A method comprising preparing a tobacco or cannabis product using cured tobacco or cannabis material from a modified tobacco or cannabis plant, wherein the modified tobacco or cannabis plant comprises at least one non-naturally occurring mutation in an endogenous gene encoding a polypeptide involved in the biosynthesis of at least one terpene, and wherein the modified tobacco or cannabis plant exhibits increased mRNA expression of the endogenous gene or increased accumulation of the polypeptide compared to a control tobacco or cannabis plant lacking the at least one non-naturally occurring mutation when grown under equivalent conditions. 51. A method comprising preparing a tobacco or cannabis product using cured tobacco or cannabis material from a modified tobacco or cannabis plant, wherein the modified tobacco or cannabis plant comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene. 52. A method comprising transforming a tobacco or cannabis cell with a recombinant nucleic acid molecule, the recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter. 53. A method comprising transforming a tobacco or cannabis cell with a recombinant nucleic acid molecule, the recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene. 54. A method for producing a tobacco or cannabis plant, comprising the steps of: (a) crossing at least one tobacco or cannabis plant of a first tobacco variety with at least one tobacco or cannabis plant of a second tobacco or cannabis variety to produce at least one progeny tobacco or cannabis seed, wherein the at least one tobacco or cannabis plant of the first tobacco or cannabis variety contains a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter; and (b) selecting at least one progeny tobacco or cannabis seed, or a plant germinated therefrom, wherein the at least one progeny tobacco or cannabis seed, or a plant germinated therefrom, comprises the recombinant nucleic acid molecule. 55. A method for producing a tobacco or cannabis plant, comprising the steps of: (a) crossing at least one tobacco or cannabis plant of a first tobacco or cannabis variety with at least one tobacco plant of a second tobacco or cannabis variety to produce at least one progeny tobacco or cannabis seed, wherein the at least one tobacco or cannabis plant of the first tobacco or cannabis variety comprises a recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene; and (b) selecting at least one progeny tobacco seed, or a plant germinated therefrom, wherein the at least one progeny tobacco or cannabis seed, or a plant germinated therefrom, comprises the recombinant nucleic acid molecule. 56. A method for producing a tobacco or cannabis plant, comprising the steps of: (a) crossing at least one tobacco or cannabis plant of a first tobacco variety with at least one tobacco or cannabis plant of a second tobacco or cannabis variety to produce at least one progeny tobacco or cannabis seed, wherein the at least one tobacco or cannabis plant of the first tobacco variety comprises at least one non-naturally occurring mutation in an endogenous gene encoding a polypeptide involved in the biosynthesis of at least one terpene, and the at least one tobacco or cannabis plant of the first tobacco or cannabis variety exhibits increased mRNA expression of the endogenous gene or increased accumulation of the polypeptide when grown under equivalent conditions compared to a control tobacco or cannabis plant of the first tobacco or cannabis variety lacking the at least one non-naturally occurring mutation; and (b) selecting at least one progeny tobacco or cannabis seed, or a plant germinated therefrom, wherein the at least one progeny tobacco or cannabis seed, or a plant germinated therefrom, comprises the at least one non-naturally occurring mutation. 57. The method of any of aspects 54-56, wherein the first tobacco variety and the second tobacco variety are the same tobacco variety, or the first cannabis variety and the second cannabis variety are the same cannabis variety. 58. The method of any of aspects 54-55, wherein at least one tobacco or cannabis plant of a second tobacco or cannabis variety comprises the recombinant nucleic acid molecule. 59. The method of any of aspects 54-55, wherein the at least one progeny tobacco or cannabis seed, or a plant germinated therefrom, is heterozygous for the recombinant nucleic acid molecule. 60. The method of any of aspects 54-55, wherein the at least one progeny tobacco or cannabis seed, or a plant germinated therefrom, is homozygous for the recombinant nucleic acid molecule. 61. The method of embodiment 56, wherein at least one tobacco or cannabis plant of a second tobacco or cannabis variety comprises the at least one non-naturally occurring mutation. 62. The method of embodiment 56, wherein the at least one progeny tobacco or cannabis seed, or a plant germinated therefrom, is heterozygous for the at least one non-naturally occurring mutation. 63. The method of embodiment 56, wherein the at least one progeny tobacco or cannabis seed, or a plant germinated therefrom, is homozygous for the at least one non-naturally occurring mutation.

[0330] Having now generally described the present disclosure, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the disclosure, unless specifically stated. EXAMPLES

[0331] Example 1. Identification of candidate trichome / terpenoid genes Little is known about the proteins that carry out the biosynthetic steps to produce some terpenes (see Figures 1-6). Published genes involved in terpene biosynthesis were used as queries in the National Center for Biotechnology Information (NCBI) database. The resulting sequences were then extracted from a second internal database. This methodology generated gene candidates and homologs putatively involved in terpene biosynthesis (SEQ ID NOs: 1-63, 112-153, and 155-205). See Table 1.

[0332] Example 2. Survey of native expression of candidate genes The expression profile of the candidate genes identified in Example 1 is investigated in tobacco trichomes. See Figure 7. Trichomes are isolated from mature leaves of tobacco cultivar "TN90" by brushing the leaves with a brush that has been submerged in liquid nitrogen. Trichomes are collected in a container containing liquid nitrogen for RNA extraction. Total RNA is isolated using TRIzol™, and cDNA is synthesized using methods known in the art. PCR is performed using gene-specific primers. Candidate genes for evaluation include cyclase (SEQ ID NO:17); phylloplanin (SEQ ID NO:155); premnaspirodiene oxygenase (PSO) (SEQ ID NO:158); neomenthol dehydrogenase (SEQ ID NO:15); and cis-abienol synthase (SEQ ID NO:4). CYP71D16 is a tobacco trichome-specific P450 hydroxylase gene used as a positive control for trichome gene expression. CYP71D16 shares approximately 98.5% homology with PSO.

[0333] Example 3. Investigation of NMD and ABS expression profiles The expression profiles of the candidate genes identified in Example 1 are investigated in flowers and leaves from tobacco cultivars TN90 and K326 and N. benthamiana at various stages of tobacco plant development.

[0334] Transcript abundance of neomenthol dehydrogenase (NMD; SEQ ID NO:15) and cis-abienol (ABS) isoforms 1 (SEQ ID NO:4) and 2 (SEQ ID NO:5) is examined at different plant developmental stages in TN90 and K326 tobacco cultivars using RNA sequencing.

[0335] Approximately 22-25 plants per variety are grown in two rows. Experiments are performed in triplicate using a randomized method. Because the flowering stage varies among plants, all plants are pinched when at least 50% of the plants begin to flower. Plants approaching uniform size are selected for sampling at different time points. Plant samples evaluated include flowers (before pinching) and leaves at various post-pinching times (e.g., 1 day after pinching; 3 days after pinching; 1 week after pinching; 2 weeks after pinching; 3 weeks after pinching; and at harvest). See Figures 8A-8B.

[0336] Example 4. Vector constructs for generating modified tobacco or cannabis plants Candidate genes identified in Example 1 encoding enzymes of the cis-abienol biosynthetic pathway (e.g., geranylgeranyl diphosphate synthase (GGPPS2) (SEQ ID NOs:1 and 113), 8-hydroxy-copalyl diphosphate synthase (SEQ ID NOs:3 and 115), cis-abienol synthase isoform 1 (SEQ ID NO:4), and cis-abienol synthase isoform 2 (SEQ ID NO:5)) are cloned into the construct.

[0337] Separate transformation vectors containing each one of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190 under the control of the CaMV 35S promoter are constructed using methods known in the art and vector pGWB551 (SEQ ID NO: 154). Representative vector constructs used to transform tobacco or cannabis plants are depicted in Figure 9. Maps of the vector backbones used to generate the vector constructs are depicted in Figure 10.

[0338] Using standard methods in the art, each vector construct produced is transformed into tobacco or cannabis cells separately in separate experiments.Briefly, vector is introduced into tobacco or cannabis leaf disk via Agrobacterium-mediated transformation.See, for example, Mayo et al., Nat. Protoc., 1:1105-1111 (2006); and Horsch et al., Science, 227:1229-1231 (1985).

[0339] Representative images of calli from control and transgenic tobacco lines expressing GFP in different backgrounds (Nb = N. benthamiana; TN90 = Nicotiana tabacum cultivar "TN90") are provided in Figures 11A-11F. FIG. 11A depicts geranylgeranyl diphosphate synthase-TN90 (SEQ ID NOs: 1 and 43); FIG. 11B depicts geranylgeranyl diphosphate synthase-Nb (SEQ ID NOs: 1 and 43); FIG. 11C depicts cembratrienol synthase 2a-Nb (SEQ ID NOs: 17 and 59); FIG. 11D depicts neomenthol dehydrogenase-Nb (SEQ ID NOs: 15 and 57); FIG. 11E depicts cis-abienol synthase-TN90 (SEQ ID NOs: 4 and 46); FIG. 11F depicts menthofuran synthase-Nb (SEQ ID NOs: 16 and 58); and FIG. 11G depicts the positive control GFP-TN90.

[0340] Example 5. Regeneration of modified tobacco plants Tobacco genotypes (TN90 and K326) and N. benthamiana plants are transformed with vector constructs as described in Example 4 and grown in Magenta™ GA-7 boxes, and leaf disks are cut and placed in Petri dishes. Agrobacterium tumefaciens cells containing the transformation vector are harvested by centrifuging 20 mL of cell suspension in a 50 mL centrifuge tube at 3500 RPM for 10 minutes. The supernatant is removed and the Agrobacterium tumefaciens cell pellet is resuspended in 40 mL of liquid resuspension medium. Tobacco leaves, avoiding the midrib, are cut into eight 0.6 cm disks with a #15 razor blade and placed upside down in a Petri dish. A thin layer of Murashige & Skoog (MS) containing B5 Vitamin Liquid Resuspension Medium is added to the Petri dish and the leaf disks are poked evenly with a fine-tipped needle. Add approximately 25 mL of Agrobacterium tumefaciens suspension to the Petri dish and incubate the leaf discs in the suspension for 10 min.

[0341] Transfer the leaf discs to a co-cultivation Petri dish (1 / 2 MS medium) and place the discs upside down in contact with a filter paper laid on co-cultivation TOM medium (MS medium containing 30 g / L sucrose; 0.1 mg / L indole-3-acetic acid; and 1 mg / L 6-benzylaminopurine (BAP)). After sealing the Petri dish with parafilm, incubate at 24 °C in dim light (60-80 mE / ms) with a photoperiod of 18 h on and 6 h off for 2 days.

[0342] After incubation, the leaf discs are transferred to regeneration / selection TOM Hyg medium Petri dishes (TOM medium + 200 mg / L cefotaxime and 50 mg / L hygromycin). Leaf discs are subcultured to fresh TOM K medium in dim light (60-80 mE / ms) at 24 °C with a photoperiod of 18 hours on and 6 hours off once every 2 weeks until shoots can be excised. Shoots from the leaves are removed with forceps and placed in MS rooting medium (MS Gamborg B5, 3 g / L sucrose and 7 g / L dextrose) with 50 mg / L hygromycin and 200 mg / L cefotaxime. Shoots on MS rooting medium with 50 mg / L hygromycin and 200 mg / L cefotaxime are incubated in the light (approximately 60-80 mE / ms) at 24 °C with a photoperiod of 18 hours on and 6 hours off to induce rooting.

[0343] Once the plantlets, including both shoots and roots, are large enough (e.g., greater than half the height of a Magenta™ GA-7 box), they are transferred to Jiffy peat pellets for acclimation in a growth chamber. Once rooted, the seedlings are transferred to the greenhouse for further growth, propagation and analysis.

[0344] Example 6. Confirmation of expression of terpene biosynthetic genes in modified tobacco plants The modified tobacco plants (T 0 RNA is isolated from young leaves of the first generation (100-1500 seq.). RNA is also isolated from control tobacco plants lacking the recombinant nucleic acid construct grown under equivalent conditions. Total RNA is isolated using TRIzol™ and cDNA is synthesized using methods known in the art. Terpenoid gene expression in the different transgenic lines of each gene construct is quantified via quantitative real-time PCR (qRT-PCR). Primers specific for the candidate genes are provided in Table 1 (SEQ ID NO: 64-103).

[0345] Gene expression of NtNMD (SEQ ID NO:15), NtaABS (SEQ ID NO:4), and NtABS (SEQ ID NO:5) in the modified tobacco plants is quantified using qRT-PCR and compared to NtNMD, NtaABS, and NtABS expression in control tobacco plants. Relative gene expression of cis-abienol synthase isoform 1 (aABS) (SEQ ID NO:4) and cis-abienol synthase isoform 2 (ABS) (SEQ ID NO:5) is compared to the T of transgenic tobacco plants. 0 The relative gene expression of NtNMD (SEQ ID NO: 15) was calculated for each line. See Figures 12A-12B. 0 The relative expression of NtNMD (see FIG. 15) and NtaABS (see FIG. 16) is calculated for each line. 1 The relative expression is measured in transgenic tobacco plants using the standard method in the art. (-ΔΔC(t)) The overexpression rate is determined using a methodology in which the native expression of the overexpressed gene in a wild-type plant is used as a baseline (e.g., set equal to 1). See, e.g., Livak and Schmittgen, Methods, 25:402-408 (2001).

[0346] Example 7. Measurement of terpenoids in engineered tobacco plants overexpressing candidate genes Quantitative metabolic profile analysis was performed on the T 0It is carried out using transgenic plants. Leaf samples from young or old leaves are ground separately in liquid nitrogen for solvent extraction of terpenes. Ground leaves are mixed with a 60:40 hexane:ethyl acetate solvent mixture (v / v) supplemented with heptadecanol as an internal standard and then incubated overnight in a shaker. The solvent extract is concentrated in a chilled SpeedVac and loaded onto a silica column. The column is washed with hexane and drained into a collection tube. Samples are aliquoted from the flow-through fraction and used for metabolite analysis by gas chromatography-mass spectrometry (GC-MS).

[0347] Increased levels of neophytadiene are observed in transgenic tobacco plants overexpressing NtNMD (SEQ ID NO:15). See Figure 14A. Increased levels of neophytadiene are also observed in transgenic tobacco plants overexpressing NtABS (SEQ ID NO:5) and NtaABS (SEQ ID NO:4). See Figure 15B.

[0348] As detected by GC-MS, neomenthol over-accumulates in transgenic tobacco plants overexpressing NtNMD (SEQ ID NO:15) compared to TN90 control plants. See Table 9.

[0349] Table 9. Accumulation of aroma volatiles in NtNMD transgenic tobacco plants TIFF2024505755000017.tif150165

[0350] Enhanced volatile aroma compounds accumulate in transgenic tobacco plants overexpressing NtABS (SEQ ID NO:5) and NtaABS (SEQ ID NO:4), as detected by GC-MS. See Table 10.

[0351] Table 10. Accumulation of aroma volatiles in NtABS and NtaABS transgenic tobacco plants TIFF2024505755000018.tif215165

[0352] Example 8. Creating mutations in candidate genes SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190 are separately specifically edited in the tobacco or cannabis genome to create mutations in each of the genes identified in Example 1. Tobacco or cannabis protoplasts are transfected using polyethylene glycol (PEG) with plasmids encoding CRISPR proteins or CRISPR proteins and specific guide RNAs (gRNAs) that target individual genes at the desired location.

[0353] The transfected protoplasts are then fixed in 1% agarose beads and subjected to tissue culture. When the calli grow to approximately 1 millimeter in diameter, they are spread on TOM2 plates. Using fragment analysis, the calli are screened for mutations (e.g., insertions or deletions (indels)) at the target position. Candidates that show a size shift compared to the wild-type control are selected for further culture, and the resulting shoots are tested by fragment analysis to again confirm the presence of the mutation.

[0354] Modified tobacco or cannabis plants (T 0 Generations) are grown as described in Example 5. Terpenoid levels are then measured as described in Example 7.

[0355] Example 9. Knockdown of candidate genes using small RNA molecules in tobacco and cannabis Reducing the expression of the genes identified in Example 1 is tested for its effect on the levels of specific terpenoids.

[0356] Separate transformation vectors are constructed containing artificial miRNAs designed to reduce the transcription or translation of each one of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190 driven by CaMV 35S.

[0357] Modified tobacco plants (T 0 Generations) are grown as described in Example 5. Terpenoid levels are then measured as described in Example 7.

[0358] Example 10. Expressing terpenoid biosynthetic genes with trichome-specific promoters Trichome-specific promoters are used to drive the expression of genes involved in terpenoid biosynthesis. Constructs are made as described in Example 4, with each trichome-specific promoter (e.g., SEQ ID NO:104-111 and 206-208, see Table 1) driving the expression of neomenthol dehydrogenase (NtNMD; SEQ ID NO:15), or one of the two isoforms of cis-abienol synthase (NtaABS (isoform 1; SEQ ID NO:4) and NtABS (isoform 2; SEQ ID NO:5)) or its homologues, such as cis-abienol synthase (e.g., SEQ ID NO:119) in separate constructs.

[0359] Each of the constructs is transformed separately into tobacco or cannabis cells and modified tobacco or cannabis plants are regenerated as described in Example 5. The modified tobacco or cannabis plants are tested for terpenes as provided in Example 7.

Claims

1. A modified tobacco plant, tobacco seed, or part thereof, comprising a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter, wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 57, 43, 46, 47, 58, and 59.

2. 2. The modified tobacco plant, tobacco seed, or part thereof of claim 1, wherein the nucleic acid sequence comprises a sequence that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15, 1, 4, 5, 16, and 17.

3. 3. The modified tobacco plant, tobacco seed, or part thereof of claim 1 or 2, wherein the heterologous promoter comprises a constitutive promoter.

4. 3. The modified tobacco plant, tobacco seed, or part thereof of claim 1 or 2, wherein the heterologous promoter comprises a tissue-preferred or tissue-specific promoter.

5. 5. The modified tobacco plant, tobacco seed, or part thereof of claim 4, wherein the tissue-preferred or tissue-specific promoter comprises a trichome-preferred or trichome-specific promoter.

6. 3. The modified tobacco plant, tobacco seed, or part thereof of claim 1 or 2, wherein the heterologous promoter comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 104-111 or 206-208.

7. 2. The modified tobacco plant, tobacco seed, or part thereof of claim 1, wherein the modified tobacco plant comprises an increased amount of at least one terpene compared to a control tobacco plant lacking the recombinant nucleic acid molecule when grown under equivalent conditions.

8. 8. The modified tobacco plant, tobacco seed, or part thereof of claim 7, wherein the increased amount of the at least one terpene comprises an increase of at least 5% compared to the control tobacco plant.

9. 9. The modified tobacco plant, seed, or part thereof of any one of claims 1 to 8, wherein the tobacco plant, seed, or part thereof is of a tobacco variety selected from the group consisting of a flue-cured variety, a bright variety, a Burley variety, a Virginia variety, a Maryland variety, a dark variety, a Galpao variety, an Oriental variety, and a Turkish variety.

10. 10. The modified tobacco plant, tobacco seed, or part thereof of any one of claims 1 to 9, wherein the tobacco plant is male sterile or cytoplasmically male sterile.

11. 11. A cured tobacco material derived from the modified tobacco plant or part thereof according to any one of claims 1 to 10.

12. 12. The cured tobacco material of claim 11, wherein the cured tobacco material is selected from the group consisting of flue-cured tobacco material, air-cured tobacco material, flue-cured tobacco material, and sun-cured tobacco material.

13. A tobacco product comprising the dried tobacco material of claim 11 or 12, selected from the group consisting of kreteks, bidi cigarettes, cigarillos, non-ventilated recess filter cigarettes, vented recess filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, hookah, shredded tobacco, and cut tobacco.

14. 13. A tobacco product comprising the dried tobacco material of claim 11 or 12, which is a smokeless tobacco product.

15. 15. The tobacco product of claim 14, wherein the smokeless tobacco product is selected from the group consisting of loose-leaf chewing tobacco, solid chewing tobacco, moist snuff, nasal snuff, dry snuff, and snus.

16. 13. A reconstituted tobacco comprising the dried tobacco material of claim 11 or 12.

17. 13. A fermented tobacco comprising the dried tobacco material of claim 11 or 12.

18. 15. The tobacco product of claim 13 or 14, comprising reconstituted tobacco.

19. 15. The tobacco product of claim 13 or 14, comprising fermented tobacco.

20. 1. A method for producing a modified tobacco plant, comprising the steps of: (a) introducing into at least one tobacco cell a recombinant nucleic acid molecule, the recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in terpene biosynthesis operably linked to a heterologous promoter, the polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 57, 43, 46, 47, 58, and 59; (b) selecting at least one tobacco cell containing the recombinant nucleic acid molecule; and (c) regenerating a modified tobacco plant from at least one tobacco cell selected in step (b), wherein the modified tobacco plant comprises an increased amount of at least one terpene in at least one tissue compared to a control tobacco plant lacking the recombinant nucleic acid molecule when grown under equivalent conditions.

21. A method comprising the step of preparing a tobacco product using the dried tobacco material according to claim 11 or 12.

22. A method comprising a step of transforming a tobacco cell with a recombinant nucleic acid molecule, the recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter, the polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 57, 43, 46, 47, 58, and 59.

23. 1. A method for producing a tobacco plant, comprising the steps of: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter, wherein the polypeptide comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 57, 43, 46, 47, 58, and 59; and (b) selecting at least one progeny tobacco seed, or a plant germinated therefrom, wherein said at least one progeny tobacco seed, or a plant germinated therefrom, comprises said recombinant nucleic acid molecule.

24. 24. The method of claim 23, wherein the first tobacco variety and the second tobacco variety are the same tobacco variety.

25. 24. The method of claim 23, wherein at least one tobacco plant of a second tobacco variety comprises the recombinant nucleic acid molecule.

26. 26. The method of any one of claims 23 to 25, wherein said at least one progeny tobacco seed, or a plant germinated therefrom, is heterozygous for said recombinant nucleic acid molecule.

27. 26. The method of any one of claims 23 to 25, wherein said at least one progeny tobacco seed, or a plant germinated therefrom, is homozygous for said recombinant nucleic acid molecule.

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