BACKGROUND

[0001] Members of the Cannabaceae family are erect or climbing plants with petalless flowers and dry, one-seeded fruits. Several genera are economically important. For example, Cannabis sativa is cultivated for a wide variety of products including fiber, oilseed, food, and medicinal/psychoactive compounds for industrial, agricultural, healthcare and recreational use. Humulus lupulus (hop) is a valuable source of several secondary metabolites, such as flavonoids, bitter acids, and essential oils. Unlike other economically important crops, cannabis and hop has only recently started to gain genomic resources, but the information about functionally associated gene expression is limited. Further resources are needed for functional genomics and breeding to provide new Cannabaceae cultivars with desired traits. For example, the interaction between transcription factors and cis- acting regulatory sequences present in plant promoters is a key step involved in the regulation of plant gene expression. Stable transgenic lines are essential for functional genomics; however, gene functional characterization in cannabis and hop lags behind other economically important crops due to the low efficiency, lengthiness and technical complexity of the available stable transformation methods.

[0002] Transient expression of nucleic acids provides a means to predict performance of a gene, promoter, expression cassette, or other element in a stable transgenic plant. Heterologous systems can be useful, but, as the activity of many promoters or proteins frequently depends on specific interactions that only occur in homologous backgrounds, a homologous expression system is desirable. Direct transfection and bacterium-mediated gene transfer of this family of plants is complicated by plant morphology, which can limit entry into the parenchyma. Biotechnology tools for improving various traits desired by growers and consumers of economically important members, such as cannabis and hops, providing genetic solutions to problems affecting cultivation, and providing the means for efficient improvement of Cannabaceae cultivars, requires Cannabaceae- specific methods of transient transformation. SUMMARY

[0003] The present disclosure features methods and materials related to a transient transformation system in Cannabaceae obviating the need to perform functional genetics studies in a heterologous system. The methods described herein detail a systematic approach for evaluating the effect of various parameters on transformation efficiency in cannabis, such as Agrobacterium strain, bacterial density, tissue selection, infiltration method, volume infiltrated, length of co-cultivation, surfactants, and other chemicals. These methods and materials can be used for characterization of gene functions and identification of regulatory elements such as promoter characterization and protein subcellular localization using gene- targeted and site-directed mutagenesis, gene silencing, overexpression of wild-type or mutant Cannabaceae genes, analysis of spatial and temporal expression of Cannabaceae genes. These methods and materials can be used to develop precision breeding techniques (e.g., cisgenic and intragenic approaches) that enable precise genetic modification of Cannabaceae plants, plant parts and plant cells.

[0004] In a first aspect, this disclosure features a method for transient transformation of Cannabaceae including: (a) infiltrating a target tissue of a Cannabaceae plant with an Agrobacterium suspension, wherein the Agrobacterium includes an expression cassette having a nucleic acid sequence operably linked to a regulatory element; and (b) performing an assay to detect expression of the nucleic acid sequence. The expression cassette can include a reporter gene. The reporter gene can encode a fluorescent protein. The nucleic acid sequence can encode a polypeptide or a RNA transcript. The RNA transcript can be capable of silencing a target gene via RNA interference. The polypeptide encoded by the nucleic acid sequence can be a rare cutting endonuclease. The rare cutting endonuclease can be a TALE nuclease, Cas9/CRISPR system, zinc-finger nuclease, or meganuclease. The regulatory element can include a promoter. The regulatory element can be a Cannabaceae- derived regulatory element. The method can further include further performing an assay to detect expression of the reporter gene. The Agrobacterium can further include a second expression cassette having a second reporter gene operably linked to a 35S promoter from cauliflower mosaic vims (CMV) and the method can further include comparing relative expression of the first and second reporter gene. Infiltrating can include injecting the target tissue with a syringe. The syringe can be a needleless syringe. The target tissue can be in an aerial part of the Cannabaceae plant. The target tissue can be leaf tissue or floral tissue. The aerial part can be leaf tissue bordered by veins or a leaf vein. The floral tissue can include a lateral female flower or an apical female flower. Infiltrating can include vacuum infiltration. The Agrobacterium suspension can have an optical density of about 0.01 to about 2.00 ODeoo _. The optical density can be no greater than 0.5 ODeoo. The Cannabaceae plant can have been grown under 16/8hr light/dark conditions. In some embodiments the Cannabaceae plant is a cannabis plant or a hop plant.

[0005] In a second aspect, this disclosure features a transformed Cannabaceae plant, plant part, or plant cell made by a method of one or more embodiments of the first aspect. [0006] In a third aspect, this disclosure features a method for identifying regulatory elements of endogenous Cannabaceae genes including: (a) infiltrating a target tissue of a Cannabaceae plant with an Agrobacterium suspension, wherein the Agrobacterium includes an expression cassette having a reporter gene operably linked to a putative Cannabaceae- derived regulatory element; and (b) performing an assay to detect expression of the reporter gene. The reporter gene can encode a fluorescent protein. The fluorescent protein can be a yellow fluorescent protein (YFP). The regulatory element can be a putative promoter. The Agrobacterium can further include a second expression cassette having a second reporter gene operably linked to a 35S promoter from cauliflower mosaic vims (CMV) and the method can further include comparing relative expression of the first and second reporter gene. Infiltrating can include syringe infiltration. The infiltration syringe can be a needleless syringe. The target tissue can be leaf tissue or floral tissue. The leaf tissue can be bordered by leaf veins or a leaf vein. Infiltrating can include vacuum infiltration. The floral tissue can be a lateral female flower or an apical female flower. The Agrobacterium suspension can have an optical density of about 0.01 to about 2.00 ODeoo. The optical density can be no greater than 0.5 ODeoo. The Cannabaceae plant can have been grown under 16/8hr light/dark conditions. The Cannabaceae plant can be a cannabis plant or a hop plant.

[0007] In a fourth aspect, this disclosure features an expression cassette including a Cannabaceae- derived regulatory sequence identified by a method according to one or more embodiments of the third aspect, which sequence is operably linked to a nucleic acid encoding a polypeptide or RNA transcript. The RNA transcript can be capable of silencing a target gene via RNA interference. The nucleic acid that is operably linked to the Cannabaceae- derived regulatory sequence can encode a rare-cutting endonuclease.

[0008] In a fifth aspect, this disclosure features a Cannabaceae plant, plant part or plant cell including the expression cassette of one or more embodiments of the fourth aspect, wherein the Cannabaceae plant, plant part or plant cell has a modified phenotype compared to a Cannabaceae plant that does not include the expression cassette.

[0009] The details of one or more examples are set forth in the description below. Other features, objects, and advantages will be apparent from the description and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

[0010] This written disclosure describes illustrative embodiments that are non-limiting and non-exhaustive. The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments discussed in the present document.

[0011] Reference is made to illustrative embodiments that are depicted in the figures, in which:

[0012] FIG. 1 shows yellow fluorescent protein (YFP) expression in Agrobacterium transformed leaf tissue, according to at least one embodiment of the present disclosure. [0013] FIG. 2 shows a cannabis flower that has been infiltrated with Agrobacterium carrying a GUS vector, according to one or more embodiments of the present disclosure. Black outlines surround regions of floral tissue with GUS-induced color indicating the presence of transformed cannabis cells that are transgenic for the GUS gene.

DETAILED DESCRIPTION

[0014] The present disclosure features methods and materials related to a transient transformation system in Cannabaceae. The methods described herein optimize various parameters for enhanced transformation efficiency in Cannabaceae ; and can be used to characterize Cannabaceae gene function, show protein subcellular localization, and identify trans- or cis-acting regulatory elements such as promoters and terminators, including tissue specific regulatory elements. Transient expression is achieved by transient transfection of Cannabaceae tissue without selection of transfected cells for stable incorporation of the nucleic acid sequence into a plant chromosome. These methods and materials can facilitate precise genetic modification of Cannabaceae by stable transformation methods.

Definitions

For the present disclosure, terms are defined as follows:

[0015] An “endogenous gene” refers to a nucleic acid molecule comprising the sequence occurring in the wild-type plant, or a sequence having a percent identity that allows it to retain the function of the encoded product, such as a sequence with at least 90% identity, and may be obtained from the plant or plant part cell, or may be synthetically produced. Further embodiments provide the sequence has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to the sequence occurring in the wild-type plant.

[0016] The term “ Cannabaceae plant” is used to include any member of the Cannabaceae family at any stage of development. A “ Cannabaceae plant part” refers to any part of a Cannabaceae plant, including a plant cutting, a plant cell, a plant cell culture, a plant organ, a plant seed (achenes), and a plantlet. The family includes about 170 species grouped in about 11 genera, including economically important genera of Cannabis , Humulus and Celtis.

[0017] The term “cannabis plant” is used broadly to include a Cannabis sativa L. plant from any landrace, cultivar, or variety, at any stage of development. A cannabis plant part refers to any part of a cannabis plant, including a plant cutting, a plant cell, a plant cell culture, a plant organ, a plant seed (achenes), and a plantlet. C. sativa L. may be divided into three sub-species: C. sativa ssp. sativa, C. sativa ssp. indica, and C. sativa ssp. ruderalis. The different varieties of cannabis can be grouped into four basic categories, including: (1) “wild” plants that have escaped from cultivation and grow independently in nature; (2) plants grown for fiber from the main stalk; (3) plants grown for oilseed; and (4) plants grown for psychoactive compounds. The cannabis plant can be any variety, including a commercial variety, or a variety contributing to one or more commercial varieties, such as “Afghani #1”, “Acapulco Gold” or “Mexican sativa”, “Blueberry”, “Grand daddy purple”, “Haze” or “Original Haze”, “Hindu Kush”, “Northern lights #5”, “OG Kush”, “Skunk #1”, and “Thai” or “Thai stick”. The cannabis plant can be identified by chemical phenotype or “chemotype”, which indicates the chemical composition of cannabinoids. For example, the cannabis plant can be categorized as Type I, Type II, Type III, Type IV, or Type V, based on the phytocannabinoid content (e.g., tetrahydrocannabinolic acid dominant, equal parts cannabidiolic acid and tetrahydrocannabinolic acid, cannabidiolic acid dominant, cannabigerolic acid dominant, or lacking cannabinoids, respectively). Thus, a “cannabis plant” includes high- and low-THC subspecies with both domesticated and ruderal varieties, including marijuana and hemp types of cannabis (e.g., cannabis known as C. indica using folk classification).

[0018] The term “hop plant” is used broadly to include plants of any species of

Humulus from any landrace, cultivar, or variety, thereof, at any stage of development. A hop plant part refers to any part of a hop plant, including a plant cutting (e.g., rhizome), a plant cell, a plant cell culture, a plant organ (e.g., cones or hops), and a plantlet. Varieties of hop plants include hop plants cultivated for use in brewing (“Brewer’s varieties) and ornamental varieties of Humulus lupulus.

[0019] A “landrace” refers to a local variety of a domesticated plant species which has developed largely by natural processes, by adaptation to the natural and cultural environment in which it lives. The development of a landrace may also involve some selection by humans, but it differs from a formal breed which has been selectively bred deliberately to conform to a particular formal, purebred standard of traits.

[0020] The term “cultivar” means a group of similar plants that can be identified from other varieties within the same species by structural features and performance (i.e., morphological and physiological characteristics). Furthermore, the term “cultivar” variously refers to a variety, strain or race of plant that has been produced by horticultural or agronomic techniques and is not normally found in wild populations. The terms cultivar, variety, strain and race are often used interchangeably by plant breeders, agronomists and farmers.

[0021] “Variety” means a plant grouping within a single botanical taxon of the lowest known rank, which grouping, irrespective of whether the conditions for the grant of a breeder’s right are fully met, can be: (i) defined by the expression of the characteristics resulting from a given genotype or combination of genotypes, (ii) distinguished from any other plant grouping by the expression of at least one of the said characteristics, and (iii) considered as a unit with regard to its suitability for being propagated unchanged.

[0022] A “plant cell” is the structural and physiological unit of the plant, including a protoplast and a cell wall. A plant cell can be in the form of an isolated single cell or aggregate of cells such as a friable callus, or a cultured cell, or can be part of a higher organized unit, for example, a plant tissue, plant organ, or plant. Thus, a plant cell can be a protoplast, a gamete producing cell, or a cell or collection of cells that can regenerate into a whole plant. As such, a seed, which includes multiple plant cells and is capable of regenerating into a whole plant, is considered a plant cell for purposes of this disclosure.

[0023] A “plant tissue” or “plant organ” can be a seed, protoplast, callus, or any other groups of plant cells that is organized into a structural or functional unit. Plant parts include harvestable parts and parts useful for propagation of progeny plants. A harvestable part of a plant can be any useful part of a plant, for example, flowers (male and female), strobiles, pollen, seedlings, leaves, bracts, buds (meristems, including vegetative (e.g., nodal) and floral meristems), stems, seed pods, seeds, roots, nodules, trichomes (including lupulin glands of hop cones), branch, petiole, internode, fibers, and the like, and includes extracts such as kief or hash which includes cannabis trichomes or glands. A part of a plant useful for propagation includes, for example, seeds, seed pods, cuttings, seedlings, rootstocks, and the like.

[0024] “Meristem” refers to a plant tissue containing undifferentiated cells

(meristematic cells), found in zones of the plant where growth can take place. Meristematic cells give rise to various organs of the plant and keep the plant growing. There are three types of meristematic tissues: apical (at the tips), intercalary (in the middle) and lateral (axial, at the sides).

[0025] “Seed” (or “achene”) refers to any plant structure that is formed by continued differentiation of the ovule of the plant, following its normal maturation point at flower opening, irrespective of whether it is formed in the presence or absence of fertilization and irrespective of whether or not the seed structure is fertile or infertile.

[0026] “Germplasm” refers to the overall genetic potential of the Cannabaceae plant, including the seeds and cuttings. For example, Skunk #1 is recognized as good cannabis germplasm with a well-known, recorded breeding history. There are numerous steps in the development of any novel, desirable plant germplasm. Plant breeding preferably begins with the analysis and definition of problems and weaknesses of the current germplasm, the establishment of program goals, and the definition of specific breeding objectives. The next step is preferable selection of germplasm that possess the traits to meet the program goals. The goal is to combine in a single variety or hybrid an improved combination of desirable traits from the parental germplasm.

[0027] “Transformation” refers to the transfer of nucleic acid (i.e., a nucleotide polymer) into a cell, and “genetic transformation” refers to the transfer and incorporation of DNA, especially recombinant DNA, into a cell. The term “transformant” refers to a cell, tissue or organism that has undergone transformation.

[0028] The terms “ Agrobacterium tumefaciens” and “ Rhizobium radiobacter” are used interchangeably in the present disclosure.

[0029] The terms “ Agrobacterium rhizogenes” and “ Rhizobium rhizogenes” are used interchangeably in the present disclosure.

[0030] “Expression cassette” means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, including a promoter operably linked to a nucleotide sequence of interest, which is optionally operably linked to termination signals and/or other regulatory elements. An expression cassette may also include sequences required for proper translation of the nucleotide sequence. The coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a non-translated RNA, in the sense or antisense direction. The expression cassette including the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be naturally occurring but has been obtained in a recombinant form useful for heterologous expression. An expression cassette may be assembled entirely extracellularly (e.g., by recombinant cloning techniques). However, an expression cassette may also be assembled using in part endogenous components. For example, an expression cassette may be obtained by placing (or inserting) a promoter sequence upstream of an endogenous sequence, which thereby becomes functionally linked and controlled by said promoter sequences.

[0031] “Regulatory element” refers to a nucleic acid molecule having gene regulatory activity, i.e., one that has the ability to affect the transcriptional and/or translational expression pattern of an operably linked transcribable polynucleotide. Regulatory elements include nucleic acid molecules such as promoters, enhancers, leaders, and intron regions that have gene regulatory activity. For example, a “promoter” is a nucleic acid molecule that is involved in recognition and binding of RNA polymerase to and other proteins (e.g., trans acting transcription factors) to initiate transcription. A promoter may be initially isolated from the 5' flanking region of a genomic copy of the gene or synthetically produced. Gene regulatory activity may be positive or negative and the effect may be characterized by its temporal, spatial, developmental, tissue, environmental, physiological, pathological, cell cycle, and/or chemically responsive qualities. The gene regulatory activity can be described by quantitative or qualitative measures.

[0032] “Donor plant” refers to a source of tissue for Agrobacterium mediated infiltration.

[0033] “Mother plant” refers to a source of cuttings capable of propagating donor plants using conventional cloning techniques.

[0034] Embodiments of the present disclosure feature methods and materials for transient transformation of Cannabaceae tissue. The tissue to be transformed can be any Cannabaceae tissue in planta or tissue excised from a Cannabaceae plant. The donor plant can be, without limitation, a clone or a seedling. Thus, in some cases, the methods described herein include propagating a donor plant from a mother plant, or growing seeds to obtain a donor plant. The donor plant is grown under optimized conditions to provide suitable tissue for transformation. Optimal conditions can include maintaining a donor plant in a vegetative state throughout its life. A Cannabaceae plant such as cannabis, for example, enters the vegetative stage after the plant develops seven sets of true leaves, and the 8 ^th is barely visible in the center of the growth tip. During the vegetative phase, the plant directs its energy resources primarily to the growth of leaves, stems, and roots. The donor plant can be an in vitro rooted plant in soil, or an ex vitro plant rooted in soil in a greenhouse or other growth chamber. The conditions can be optimized to ensure that the donor plant is not a plant transitioning from the vegetative state to the reproductive stage, or a plant in its reproductive stage.

[0035] The growth conditions can be optimized based on the physiologic age of the plants. For example, a donor plant grown for a limited period of time or to a specific physiological age. The age of donor plants clonally propagated from a mother plant, can be based on the day the donor plant was transferred to soil. The donor Cannabaceae plant can have an age of from 1 to 120 months, from 1.5 to 100 months, or from 2 to 80 months, e.g., from 2 to 24 months, from 2.5 to 12 months, from 3 to 6 months, or about 3.5 months. Serial cuttings can be removed from a donor plant, rooted, grown under long day length, and used to replace older donor plants using conventional cloning techniques, such as stem propagation. The propagation process creates a genetic replica of the mother through relatively large cuttings (conventional cloning) or micropropagation. The process can be repeated, indefinitely, without loss of vigor, if the cutting material is kept free of pathogens such as viruses and insect vectors.

[0036] The growing environment can influence efficiency of the agroinfiltration. For example, donor Cannabaceae plants can be grown under conditions that promote growth of the tissue targeted for infiltration. The target tissue can be an aerial plant part, such as an immature leaf, mature leaf, male flower or male flower parts, female flower or female flower parts (e.g., cones or strobili), stem, bine, or the target tissue can be root tissue (e.g., hop rhizomes). Depending on the cultivar, a donor plant can be grown using day lengths of 14 hours or longer in a controlled growth chamber. For example, a vegetative state can be maintained under 16/8 hr-light/dark conditions. A wide variety of lighting types can be used to maintain the vegetative state; these include metal halide bulbs, HPS lamps, LEDs, or a combination of different lighting types. LEDs can be optimized to specific production conditions by controlling periodicity, quantity, and spectrum of the light provided.

[0037] Donor Cannabaceae plants can be cultivated under the control of an environmental management system configured to ensure that the donor plants are maintained in the vegetative state, in optimal health, and free from pathogens or insect vectors. In some embodiments, the environment management system can include computer control of grow room environment, sensors, and fertigation devices. The system facilitates monitoring the parameters that must be optimized to grow the highest quality and healthiest plants, such as real time measurement of temperature, relative humidity, and carbon dioxide content. Water and/or fertigation parameters can be measured, including pH, flow rate, Nitrogen- Phosphorous-Potassium levels, ppm of certain compounds (e.g., micronutrients). In the growing chamber, constant airflow and drier air can be utilized to prevent plant diseases and mold formation. Donor plants can be maintained at a temperature between 25 and 30 °C, with relative humidity (RH) levels of 75% or higher during the propagation and development stages of the donor plants and 55-60% during their vegetative stage. For example, vegetative stage donor plants can be maintained in a growth chamber at 25 °C, 70% RH. Optimal values for fertilizer (e.g., nitrogen/liter and pH) can vary with the selected cultivar.

[0038] Using methods described herein, Cannabaceae donor material is transformed via Agrobacterium- mediated infiltration. The donor material can be the whole plant, plant part or portion thereof. For example, the plant part can be a whole leaf in planta or a leaf harvested from the plant. Sometimes, the donor material is excised from the plant (e.g., a leaf disk). Thus, the methods can include handling harvested plant parts or excised tissue in an axenic (aseptic; sterile) environment under defined conditions.

[0039] Methods of the present disclosure can include disinfecting the tissue before it is contacted with the Agrobacterium to remove surface contaminants such as bacteria and fungal spores with minimal damage to plant cells. The disinfecting solution can include one or more disinfecting agents selected from the group consisting of ethanol, hypochlorite (NaCIO or Ca(C10) ₂ ), benzalkonium chloride, silver nitrate, mercuric chloride and hydrogen peroxide. The disinfecting solution can contain the disinfecting agent within a range of 0.01% to about 95% by volume. The tissue can be exposed to the disinfecting solution for a period of 0.1 to about 30 minutes. The disinfecting solution can further include a mild detergent such as a polysorbate (e.g., TWEEN 20 or TWEEN 80) or other non-ionic surfactant. The tissue can be washed with a disinfecting solution including about 0.5-2% NaCIO (e.g., 0.5% NaCIO) and up to 0.1% Tween (e.g., Tween 20). Tween can improve contact of the NaCIO with the tissue. In an exemplary method, donor material is washed under running tap water (e.g., for 5 minutes) before disinfecting the material in a laminar flow cabinet. The donor material can be placed in a sterilized beaker (e.g., autoclaved), and the disinfecting solution added (e.g., by stirring on magnetic mini- stirrer). After immersion in the solution, the donor material can be rinsed several times, in distilled water in the laminar flow cabinet to prevent drying. The cut ends of the donor material can be trimmed with a sterile scalpel before donor material is contacted with the Agrobacterium.

[0040] The Cannabaceae plant, plant part or plant cell is contacted with an

Agrobacterium harboring a heterologous nucleic acid sequence. The nucleic acid sequence can be part of an expression cassette introduced into a T-DNA region of a plasmid for Agrobacterium- mediated transformation. The T-DNA can be present in a binary vector. The nucleic acid construct can include a DNA sequence of interest and other sequences such as regulatory sequences for expression of the DNA sequence of interest. Binary vectors usable in the invention are known to the skilled person. The binary vector typically has an antibiotic resistance gene for allowing selection in bacteria. For increasing transfection efficiency, the Agrobacterium can harbor a virG gene. The virG can be present on the same plasmid as the heterologous nucleic acid sequence, or in a helper plasmid.

[0041] The methods of the present disclosure can include constructing an expression cassette that will function in Cannabaceae plant cells. Such a vector may can include DNA including a gene under control of or operatively linked to a regulatory element (for example, a promoter), or a DNA for gene silencing (i.e., producing RNA interference). In one or more embodiments of the present disclosure, the expression cassette encodes a transgene. The transgene can be isolated from any source and encode specific protein products. The plasmid can include DNA of foreign genes, or additional, or modified versions of native, or endogenous genes. The plasmid can include regulatory elements that drive expression of additional native or endogenous genes differently than the native regulatory elements, and combinations of the native or endogenous regulatory elements with different regulatory elements. The plasmid can be a transcriptional fusion where the regulatory sequences of promoters or terminators are provided by the expression cassette of the vector and only the coding sequence from the gene has to be inserted. The plasmid can be used alone or in combination with other plasmids to provide transformed Cannabaceae plants using transformation methods as described below. [0042] The expression cassette can include at least one genetic marker that allows transformed cells containing the marker to be identified. A marker gene can be introduced alone or simultaneously with a gene of interest. Exemplary markers for transient transformation include screenable markers (e.g., b-glucuronidase (GUS), b-galactosidase, luciferase and chloramphenicol acetyltransferase). In some cases, GFP and mutants of GFP may be used as screenable markers. Genes encoding screenable markers are particularly useful to quantify or visualize the spatial pattern of expression of a gene in specific tissues and are frequently referred to as reporter genes because they can be fused to a gene or gene regulatory sequence for the investigation of gene expression. For example, the GFP mutant, Yellow Fluorescent Protein (YFP) can be used to observe regions of the donor material where the Agrobacterium was able to penetrate.

[0043] The expression of genes included in expression cassettes can be driven by a nucleotide sequence including a regulatory element, such as an endogenous promoter of a Cannabaceae gene. The promoter is typically upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. The promoter can be a promoter under developmental control, such as a promoter that preferentially initiates transcription in certain tissues (e.g., “tissue-preferred” and “tissue- specific” promoters). In some cases, the promoter is a “cell type” specific promoter which drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. In one or more embodiments, the promoter is an inducible promoter under environmental control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions or the presence of light. Alternatively, the promoter is a under most environmental conditions and most tissues/cells (i.e., a constitutive promoter).

[0044] A regulatory element of the expression cassette can be a signal sequence for targeting proteins to subcellular compartments. For example, transport of protein produced by transgenes to a subcellular compartment such as the nucleus, chloroplast, vacuole, peroxisome, glyoxysome, cell wall or mitochondrion or for secretion into the apoplast, is accomplished by means of operably linking the nucleotide sequence encoding a signal sequence to the 5' and/or 3' region of a gene encoding the protein of interest. Targeting sequences at the 5' and/or 3' end of the structural gene may determine, during protein synthesis and processing, where the encoded protein is ultimately compartmentalized.

[0045] The expression cassette can include a gene encoding a foreign protein or a protein conferring an agronomic trait. Agronomic genes include genes that confer resistance to pests or disease, genes that confer resistance to an herbicide, genes that confer or contribute to a value-added trait, which for Cannabaceae can be increased iron content, decreased nitrate content of leaves, improved flavor (e.g., sweetness), improved fragrance, modified fatty acid metabolism, or modified carbohydrate composition.

[0046] In some cases, the expression cassette can include a nucleotide sequence encoding a rare-cutting endonuclease, or a portion (e.g., a subunit) of a rare-cutting endonuclease. Rare-cutting endonucleases are natural or engineered proteins that have endonuclease activity directed to nucleic acid sequences containing a recognition sequence (target sequence). Rare-cutting endonucleases generally cause cleavage inside their recognition site, leaving 2 to 4 nucleotides (nt) staggered cut with 3' OH or 5' OH overhangs. Further, active rare-cutting endonucleases can be multimeric or associated with accessory molecules.

[0047] Sometimes, the rare-cutting endonuclease is a “Cas9/CRISPR system”. The methods provided herein can include the transient expression of programmable RNA-guided endonucleases, or portions (e.g., subunits) thereof. The RNA-guided CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-associated nuclease (Cas9) was developed from the type II prokaryotic CRISPR adaptive immune system. This system can cleave DNA sequences that are flanked by a short sequence motif known as a proto-spacer adjacent motif (PAM). Cleavage is achieved by engineering a specific CRISPR RNA (crRNA) that is complementary to the target sequence that associates with the Cas9 endonuclease. In this complex, the trans-activating crRNA (tracrRNA): crRNA complex acts as a guide RNA that directs the Cas9 endonuclease to the cognate target sequence. A synthetic single guide RNA (sgRNA) also has been developed that, on its own, is capable of targeting the Cas9 endonuclease. The DNA target sequence is generally 20 base pairs. This DNA target is generally chosen to be located in the genome upstream so-called PAM (protospacer adjacent motif) sequence motives (NGG or NAG) recognized by Cas9. The guide RNA molecule (gRNA), which is generally a single stranded RNA is introduced into the living cell to confer cleavage and specificity to Cas9. It is a synthetic RNA designed to match the desired 20 bp sequence in the genome upstream the PAM.

[0048] The rare-cutting endonuclease can be a fusion protein that contains a DNA binding domain and a catalytic domain with cleavage activity. TALE-nucleases and ZFNs are examples of fusions of DNA binding domains with the catalytic domain of the endonuclease Fokl. Customized TAFE-nucleases are commercially available under the trade name TAFEN™ (Cellectis, Paris, France). The specificity of transcription activator- like (TAF) effectors depends on an effector-variable repeat. Polymorphisms are present primarily at repeat positions 12 and 13, which are referred to herein as the repeat variable- diresidue (RVD).

[0049] The RVDs of TAF effectors correspond to the nucleotides in their target sites in a direct, linear fashion, one RVD to one nucleotide, with some degeneracy and no apparent context dependence. This mechanism for protein-DNA recognition enables target site prediction for new target specific TAF effectors, as well as target site selection and engineering of new TAF effectors with binding specificity for the selected sites.

[0050] TAF effector DNA binding domains can be fused to other sequences, such as endonuclease sequences, resulting in chimeric endonucleases targeted to specific, selected DNA sequences, and leading to subsequent cutting of the DNA at or near the targeted sequences. Such cuts (double-stranded breaks) in DNA can induce mutations into the wild type DNA sequence via NHEJ or homologous recombination, for example. In some cases, TAFE-nucleases can be used to facilitate site directed mutagenesis in complex genomes, knocking out or otherwise altering gene function with great precision and high efficiency. As described herein, TAFE-nucleases targeted to an endogenous Cannabaceae gene can be used to mutagenize the endogenous gene, resulting in Cannabaceae plants or plant tissue with modified expression of the endogenous gene (e.g., such as loss or gain of function). The fact that some endonucleases (e.g., Fokl) function as dimers can be used to enhance the target specificity of the TAFE-nuclease. For example, a pair of TAFE-nuclease monomers targeted to different DNA sequences can be used. The relevant sequences useful in the processes include “functional variants” of the sequences disclosed. Functional variants include, for example, sequences having one or more nucleotide substitutions, deletions or insertions and wherein the variant retains desired activity. Functional variants can be created by methods available to one skilled in the art, such as site-directed mutagenesis, induced mutation, identified as allelic variants, cleaving through use of restriction enzymes, or the like.

[0051] When the two TALE-nuclease recognition sites are in close proximity, the inactive monomers can come together to create a functional enzyme that cleaves the DNA. By requiring DNA binding to activate the nuclease, a highly site-specific restriction enzyme can be created. Methods for selecting endogenous target sequences and generating TALE- nucleases targeted to such sequences can be performed. See, for example, U.S. Pat. App. Pub. No. US 2011/0145940 A1 (Jun. 2011), which is incorporated by reference. In some embodiments, software that specifically identifies TALE-nuclease recognition sites can be used.

[0052] In other embodiments, the rare-cutting endonuclease is a meganuclease, such as a wild type or variant homing endonuclease (e.g., a homing endonuclease belonging to the dodecapeptide family.

[0053] The Agrobacterium harboring the heterologous nucleic acid sequence can be grown and used under conditions that enhance transformation efficiency. A. tumefaciens and A. rhizo genes are plant pathogenic soil bacteria which genetically transform plant cells. The Ti and Ri plasmids of Agrobacterium carry genes responsible for genetic transformation of the plant. Exemplary Agrobacterium strains include C58, AGL1, A136, A208, A348, Ach5, 18rl2v, G3101, LBA4404, EHA105, EHA101, NT IRE, NTlRE(pJK270), 1D1108, 1D1460, 1D1609, 1D132, 1D1478, and 1D1487. Th Q Agrobacterium can be cultured using traditional methods and suspended in infiltration medium at a desired concentration. Thus, a range of Agrobacterium concentrations from 102 to 1010 cfu/mL can be used. The bacterial density can be varied within the range of about 0.01 to about 2.00, such as about 0.05, 0.10, 0.15, 0.20, 0.40, 0.60, 0.80, 1.00, 1.30, 1.60, and 2.00 at OD ₆ oo (nm). The concentration of Agrobacterium in the infiltration medium can be varied to prevent overgrowth. In some cases, the culture is grown or diluted to a bacterial density of no greater than 0.5 ODeoo, such as 0.3- 0.5 OD600. [0054] One or more phenolic compounds can be included in the infiltration medium prior to or during the Agrobacterium co-cultivation. The phenolic compound can be acetosyringone, acetophenone, chalcone, or cinnamic acid. The plant phenolic compound can be added to the medium prior to contacting the starting material with Agrobacterium (for e.g., several hours to one day). Possible concentrations of plant phenolic compounds in the inoculation medium range from about 25 mM to 700 pM, such as 100-200 pM. Acetosyringone (50, 100, 150, 200, or 250 pM) can be added to co-cultivation media.

[0055] Agrobacterium harboring the nucleic acid sequence can be introduced into the intracellular space by injecting the Agrobacterium suspension into the target tissue parenchyma, soaking the target tissue in Agrobacterium suspension, or spraying the Agrobacterium on the target tissue. Injection can include contacting the target tissue with a syringe with or without a needle. The syringe has small aperture and permits variable pressure that causes the suspension of Agrobacterium to facially enter into the injection site and spread outwardly. The injection site can be defined by morphological features of the tissue. For example, the injection site can be an interveinal portion of a leaf. The infiltration of tissue around the injection site can be result in the infiltrated area being visibly darker green than un-infiltrated tissues. The entire plant part can be infiltrated by repeatedly injecting the suspension at different sites on the tissue until the whole part is darker green than other parts of the plant. Alternatively, discrete areas on the same plant part can be injected. Thus, syringe infiltration provides flexibility to either infiltrate the entire tissue with one target gene, or to introduce genes of multiple targets on one plant part.

[0056] The target tissue can be contacted with the Agrobacterium in planta, i.e., the target tissue is within, on, or attached to the donor Cannabaceae plant. The target tissue can be selected based on a tissue- specific expression of endogenous Cannabaceae genes. For example, genes encoding enzymes that synthesize specialized metabolites, such as medicinal compounds, can be expressed at high levels in leaf tissue and expressed at low levels or not at all in root tissue. Agroinfiltration of target tissue that expresses a target gene at high levels can be used to efficiently demonstrate the efficacy of the nucleic acid sequence to silence target gene expression. [0057] V arious infiltration methods can be combined to maximize the number of cells and tissues are transfected. Infiltration of the bacterium into the intracellular space of the donor material can be facilitated by wounding the tissue before it is contacted with the Agrobacterium suspension. The donor material can be wounded by cutting, abrading, nicking, piercing, poking, penetration with fine particles or pressurized fluids, plasma wounding, application of hyperbaric pressure, or sonication. Wounding can be performed using, scalpels, scissors, needles, abrasive objects, airbrush, particles, electric gene guns, or sound waves. In some cases, the donor material is subjected to a chemical treatment that renders the cell walls more permeable (e.g., treatment with macerating enzymes such as cellulase, pectinase or macerozyme). The transformation efficiency can be enhanced by subjecting the donor material to vacuum infiltration, heat shock and/or centrifugation, and sonication. For example, a vacuum can be applied to a sealable vessel containing the donor material and the Agrobacterium suspension, and the change in pressure may facilitate penetration of the bacteria into the intercellular or interstitial spaces of the donor material. The vacuum applied can be any suitable pressure for the tissue being infiltrated, typically within the range of about 25 mmHg to about 700 mmHg. Combinations of more than one type of wounding and/or treatment during infiltration can be utilized, and can be based on the plant variety and/or other characteristics, such as lignin content.

[0058] Infection of the donor material can require a few minutes to a few hours, such as about 10 minutes to 3 hours (e.g., about 20 minutes). For example, a vacuum can be applied to the donor material in agrobacterium solution (e.g., for about 10 minutes), followed by soaking the donor material in the Agrobacterium solution (e.g., for 10 minutes). In some cases, the donor material is sonicated in Agrobacterium solution (e.g., for about 80 seconds) and then soaked in the Agrobacterium solution (e.g., for about 19 minutes). The excess infiltration medium can be drained, and the Agrobacterium permitted to co-cultivate with the donor material for several days, generally carried out for 1 to 14, preferably 2 to 4 days. Normally no selection agent is present during this step. For example, the donor material can be soaked in Agrobacterium solution for 20 minutes and can be rinsed of the liquid inoculation medium and allowed to dry on sterile blotting paper before co-cultivating. [0059] After the co-cultivation period, the transformed tissue can be identified by various methods. Usually expression occurs spontaneously after agroinfiltration. In some cases, expression of the gene is induced by a change in biotic or abiotic factors, for example. Expression of the gene can be measured by reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time polymerase chain reaction (qPCR), Northern blotting, dot- blot hybridization, in situ hybridization, nuclear run-on and/or nuclear run-off, RNase protection, or immunological and enzymatic methods such as ELISA, radioimmunoassay, and western blotting. Expression of a fluorescent protein marker can be visualized by UV excitation, fluorescence microscopy or flow cytometry.

[0060] To determine the efficacy of transient expression of a rare cutting endonuclease, the transformed cells be analyzed to determine whether mutations have been introduced at the target site(s), through nucleic acid-based assays or protein-based assays to detect expression levels as described above, for example, or using nucleic acid-based assays (e.g., PCR and DNA sequencing, or PCR followed by a T7E1 assay) to detect mutations at the genomic loci. In a T7E1 assay, genomic DNA can be isolated, and sequences flanking TALE- nuclease recognition sites for the target genomic sequences can be PCR-amplified. Amplification products then can be denatured and re-annealed. If the re-annealed fragments form a heteroduplex, T7 endonuclease I cuts at the site of mismatch. The digested products can be visualized by gel electrophoresis to quantify mutagenesis activity of the endonuclease. [0061] Methods and materials described herein can be applied for rapid analysis of regulatory elements in vivo , including Cannabaceae derived regulatory elements, such as endogenous promoters, or for elucidating promoter function, gene expression, subcellular protein localization, protein-protein interactions, and metabolism. For example, agroinfiltration can be used to test various promoter/terminator combinations, and identify endogenous Cannabaceae promoter/terminators. The promoter also may include at least one control element such as an upstream element. Such elements include upstream activation regions (UARs) and, optionally, other DNA sequences that affect transcription of a polynucleotide such as a synthetic upstream element. The choice of promoters useful in the methods depends upon the type of desired expression to be achieved. Factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell or tissue specificity. For example, tissue-, organ- and cell-preferred promoters that confer transcription only or predominantly in a particular tissue, organ, and cell type, respectively, can be used. In some embodiments, promoters specific to vegetative tissues such as the stem, parenchyma, ground meristem, vascular bundle, cambium, phloem, cortex, shoot apical meristem, lateral shoot meristem, root apical meristem, lateral root meristem, leaf primordium, leaf mesophyll, or leaf epidermis can be suitable regulatory regions. Other classes of promoters include, but are not limited to, inducible promoters, such as promoters that confer transcription in response to inducers, such as external stimuli such as chemical agents, developmental stimuli, or environmental stimuli. The promoter may be one which preferential expresses to particular tissue, organ or other part of a plant, or may express during a certain stage of development or under certain conditions. When referring to preferential expression, what is meant is expression at a higher level in the particular plant tissue than in other plant tissue.

[0062] A promoter of interest may have strong or weak transcriptional activity. A skilled person appreciates a promoter sequence can be modified to provide for a range of expression levels of and operably linked heterologous nucleic acid molecule. Generally, by “weak promoter” is intended a promoter that drives expression of a coding sequence at a low level. By “low level” is intended levels of about 1/10,000 transcripts to about 1/100,000 transcripts to about 1/500,000 transcripts. Conversely, a strong promoter drives expression of a coding sequence at a high level, or at about 1/10 transcripts to about 1/100 transcripts to about 1/1,000 transcripts. It is recognized that to increase transcription levels, enhancers can be utilized in combination with the promoter regions.

[0063] Expression cassettes including a Cannabaceae- derived regulatory sequence identified by these methods are within the scope of the present disclosure. For example, an expression cassette can include a Cannabaceae- derived regulatory element operably linked to a nucleic acid encoding a polypeptide or RNA transcript for silencing a target gene via RNA interference. In some cases, the operably linked nucleic acid encodes a rare-cutting endonuclease.

[0064] Embodiments of the present disclosure include Cannabaceae plants, plant parts, and plant cells transiently transformed according to one or more of the methods described herein. In some cases, although the expression is transient, the effect of expression can persist in the plant, plant part or plant cell. For example, a plant part can contain exogenous compounds or higher levels of endogenous compounds than non-transformed plants, plant parts or plant cells, as a result of transient expression of the introduced genes.

EXAMPLES

[0065] The following Examples are intended to illustrate the above invention and should not be construed as to narrow its scope. One skilled in the art will readily recognize that the Examiners suggest many other ways in which the invention could be practiced. Numerous variations and modifications may be made while remaining within the scope of the invention.

Introduction

[0066] The construction of transgenic, cis-genic, or gene editing vectors to genetically engineer Cannabaceae plant cells requires identification of genetic elements useful for transformation. N. benthamiana has been widely used to study the function of genes in plant through transient expression. Unlike the leaf tissue of N. benthamiana , Cannabaceae leaf tissue, such as cannabis and hop, is coated in a waxy lipid layer comprising epicuticular waxes controlling water loss and functioning as a defensive barrier). In addition, cannabis leaves are significantly more compact than tobacco leaves. The wax and compact nature of the leaf tissue restricts infiltration of Agrobacterium into the intracellular spaces where the bacterium can transform individual plant cells.

A. In planta Infiltration

[0067] Agrobacterium containing a YFP plasmid was grown in 5ml cultures of Luria Broth (LB) medium with 50pg kanamycin per ml for 8 hours at 28 °C. 1ml of the Agrobacterium culture was then grown overnight for 16-18 hours in 100ml of LB medium containing 50pg kanamycin per ml at 28 °C. The culture was centrifuged and resuspended to an Oϋ _ό oo of 0.4 in MMA solution (lOmM methysulfonate (MES), lOmM MgCh, lOOuM acetosyringone). [0068] Cannabis plants were grown from seed or clone for 1-2 months in growth chambers with 16/8hr light/dark conditions at 26° C. These plants were used as donor plants to provide leaf tissue for infiltration. Leaves ranging in length from 0.5cm- 10cm were selected as donor material.

[0069] A blunt end 20ml syringe without a needle was filled with the agrobacterium solution. Various areas of the leaf tissues (lamina, midrib, vein) were perforated with a needle (separate from the syringe). The blunt-ended syringe was then pressed against the axial or abaxial side of the leaf at the areas where the needle had penetrated. Using a finger to apply pressure on the leaf opposite to the side of perforation, the Agrobacterium solution was delivered into the target leaf tissue. Varying degrees of pressure were tested; in this method, large amounts of pressure were most effective in delivering Agrobacterium solution into the leaf tissue. Small leaves were damaged by the pressure of the syringe. Optimal results were obtained with leaves of 5- 10cm length.

[0070] Following Agrobacterium infiltration, the plants were maintained in the growth chamber with 16/8hr light/dark conditions at 26 °C. 48 to 72 hours after infiltration the leaves were harvested and viewed under a fluorescent microscope. High levels of expression were detected at the leaf veins, where th Agrobacterium was able to penetrate. (FIG. 1). Cannabis leaf that has been infiltrated with Agrobacterium carrying an empty vector showed no fluorescent cells, indicating that the control does not cause autofluorescence in the leaf tissue (not shown).

[0071] Different promoter and terminator combinations were tested in the YFP vector to determine the strength of response.

B. Leaf Disc Infiltration

[0072] Agrobacterium containing the YFP plasmid is grown in 5ml cultures of LB medium with 50pg kanamycin per ml for 8 hours at 28 °C. 1ml of the agrobacterium culture is then grown overnight for 16-18 hours in 100ml of LB medium containing 50pg kanamycin per ml at 28 °C. The culture is centrifuged and the pellet resuspended to an ODeoo of 0.4 in an infiltration solution (lOmM MES, lOmM MgCh, and lOOuM acetosyringone (MMA solution)). [0073] Cannabis plants are grown from seed or clone for 1-2 months in growth chambers with 16/8hr light/dark conditions at 26 °C. These plants are used as donor plants to provide leaf tissue for infiltration.

[0074] Leaf tissue is harvested from the donor plants by cutting off entire leaves at the petiole. The leaf tissue is then sterilized. Leaf tissue is placed into centrifuge tubes containing 40ml of sterile ddfLO until all the tissue harvesting is complete. The ddfLO is removed, and 45ml of 70% EtOH is added to each centrifuge tube containing leaves. The tubes are sealed, and shaken slowly by hand for one minute. After one minute, the 70% EtOH is removed, and the leaf tissue is rinsed with sterile ddHiO three times. The centrifuge tubes are placed into a laminar flow hood. 1% NaCIO solution containing 0.1% tween is added to each centrifuge tube containing leaves. The tubes are sealed, and shaken slowly by hand for ten minutes. The 1% NaCIO sterilization is repeated twice. After the second NaCIO sterilization, the NaCIO solution is removed, and the leaf tissue is rinsed with sterile ddH20 three times.

[0075] Sterile leaf tissue is sliced into l-5mm pieces using a scalpel. Each piece is placed into a well in a 96 well plate. Each well contains 0.5ml of the agrobacterium solution. The 96 well plate is placed into a 4L vacuum chamber, and vacuumed for 15 minutes at . The 96-well plate was then placed in the dark at 25 °C for one hour, where the leaf pieces are allowed to soak. After one hour, the leaf pieces are moved to a new 96 well plate, where they are rinsed three times with 500mg/L cefotaxime solution. 0.5ml of W5 wash buffer (2 mM MES, 154 mM NaCl, 125 mM CaCh and 5 mM KC1 at pH 5.7) was added to each well containing leaf tissue. The 96 well plate is sealed with parafilm and placed in an incubator with 16/8hr light/dark at 25 °C. 48 to 72 hours after infiltration, leaves are harvested and viewed under a fluorescent microscope.

[0076] Different promoter and terminator combinations are tested in the YFP vector to determine the strength of response.

C. Agrobacterium vacuum infiltration of Cannabis tissue [0077] The infiltration of five Agrobacterium strains into different cannabis genotypes and tissues were investigated. For each of the strains GV3101, EHA105, LBA4404, 18rl2v, and AGL1, the Agrobacterium containing a b-glucuronidase (GUS) plasmid was grown in 5ml cultures of Luria Broth (LB) medium with 50pg kanamycin per ml for 8 hours at 28 °C. 1ml of the Agrobacterium culture was then grown overnight for 16-18 hours in 100ml of LB medium containing 50mg kanamycin per ml at 28 °C. The culture was centrifuged and resuspended to an ODeoo of 0.4 in MMA solution (lOmM methysulfonate (MES), lOmM MgCh, IOOmM acetosyringone).

[0078] Cannabis plants were grown from seed or clone for 2 months in growth chambers with 16/8hr light/dark conditions at 26° C. After 2 months of vegetative growth, the plants were moved to growth chambers with 12/12hr light/dark conditions for 1-2 months. These plants were used as donor plants to provide flower tissue for infiltration experiments. [0079] Cannabis plant parts were harvested from the donor plants and placed into 50ml centrifuge tubes. Seedlings from the genotype Fiber & Seed were harvested and placed into 50ml centrifuge tubes to be used as a control tissue for infiltration with GV3101, 18rl2v and AGL1.

[0080] Agrobacterium solution was added to each of the 50ml centrifuge tubes containing a cannabis plant part or seedling. After Agrobacterium was added, the tubes were placed into a vacuum chamber, and vacuum applied (~635mmHg) for 5, 10, or 15 minutes. After vacuum, the explants were removed from Agrobacterium solution, and placed into co cultivation.

[0081] Following Agrobacterium infiltration, the plant parts were maintained on co cultivation conditions for 48 to 72 hours. After infiltration, the plant parts were stained using conventional GUS staining protocols. Varying levels GUS expression were observed and graded (See FIG. 2 and TABLES 1-5). For samples of each tissue type, the degree of GUS staining is defined as follows: o indicates no staining, + indicates spotty staining; ++ indicates sectional staining, and +++ indicates strong staining.

[0082] The foregoing description of various preferred embodiments of the disclosure have been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosure to the precise embodiments; many modifications and variations are possible in light of the above teaching. The example embodiments, as described above, were chosen and described in order to best explain the principles of the disclosure and its practical application to thereby enable others skilled in the art to best utilize the disclosure in various embodiments and with various modifications as are suited to the particular use contemplated. Combinations or sub-combinations of the specific features and aspects of the embodiments disclosed herein may be made and fall within the scope of this disclosure. Various features and aspects of the disclosed embodiments can be combined with or substituted for one another in order to form various embodiments. It is intended that the scope of the disclosure be defined by the claims appended hereto.