Transformation of Cannabaceae Cells

Abstract

An exemplary method of the present disclosure includes soaking Cannabaceae seeds in a hydration solution, excising a subset of embryonic tissue from the soaked Cannabaceae seeds to extract a Cannabaceae meristem region or hypocotyl (EA), and exposing the Cannabaceae meristem region or EA to a heterologous nucleotide sequence to transform Cannabaceae cells of the Cannabaceae meristem region or EA.

Description

[Background technology]

[0001] Incorporation by reference of material Electronic submission Incorporated by reference in its entirety is a computer readable nucleotide / amino acid sequence listing, an ASCII text file 433 kb in size, which was submitted concurrently herewith and identified as follows: "C1633116111_SequenceListing_ST25" and created on June 13, 2022.

[0002] Cannabaceae is a family of dicotyledonous flowering plants that includes a variety of trees, upright herbs, and bicotyledonous herbs. Examples of Cannabaceae include cannabis plants, such as industrial hemp. Industrial hemp is a cannabis plant variety that has less than 0.3% tetrahydrocannabinol (THC). Industrial hemp can be cultivated to produce fiber, grain, or non-intoxicating pharmaceutical compounds such as cannabidiol (CBD) and terpenes. Functional genomics and breeding can be used to provide new Cannabaceae cultivars with desired traits. Stable transgenic lines are useful for functional genomics. Cannabaceae plants can be resistant to transformation. There is a need to improve the transformation and regeneration efficiency of Cannabaceae for genetic breeding and other uses. Summary of the Invention [Problem to be solved by the invention]

[0003] The present disclosure features methods and materials for transforming Cannabaceae plant cells, such as Cannabaceae meristem regions or hypocotyls (EA), and regenerating the transformed cells into various differentiated plant tissues, plant parts, and whole plants. The methods described herein include the systematic design of genotype-specific transformation and regeneration systems. Further embodiments implement the genotype-specific transformation and regeneration system to provide transformed Cannabaceae plants from clonal material derived from the Cannabaceae meristem regions or EAs having one or more traits of interest. [Means for solving the problem]

[0004] Some aspects of the present disclosure relate to a method comprising soaking Cannabaceae seeds in a hydration solution, excising a subset of embryonic tissue from the soaked Cannabaceae seeds to extract a Cannabaceae meristem region or EA, and exposing the Cannabaceae meristem region or EA to a heterologous nucleotide sequence to transform Cannabaceae cells of the Cannabaceae meristem region or EA.

[0005] Some embodiments relate to a method comprising soaking Cannabaceae seeds in a hydration solution, excising a subset of embryonic tissue from the soaked Cannabaceae seeds to extract a Cannabaceae meristem region or EA, and exposing the Cannabaceae meristem region or EA to a heterologous nucleotide sequence to transform Cannabaceae cells of the Cannabaceae meristem region or EA.

[0006] In some embodiments, excising the subset of embryonic tissue comprises removing the seed coat without removing any of the cotyledons of the soaked Cannabaceae seeds.

[0007] In some embodiments, excising the subset of embryonic tissue comprises removing the seed coat and one of the cotyledons of the soaked Cannabaceae seed.

[0008] In some embodiments, excising the subset of embryonic tissue comprises removing the seed coat and cutting the rootlet of the soaked Cannabaceae seed.

[0009] In some aspects, excising the subset of embryonic tissue comprises removing both the seed coat, the cotyledons, and the leaf primordia of a soaked Cannabaceae seed.

[0010] In some embodiments, excising the subset of embryonic tissues comprises removing the seed coat, one of the cotyledons, and a leaf primordium of a soaked Cannabaceae seed.

[0011] In some embodiments, the method further comprises regenerating tissue from the transformed Cannabaceae cells using a culture medium comprising thidiazuron (TDZ), the tissue comprising one or more of a shoot, a root, a root hair structure, and a whole plant.

[0012] In some aspects, regenerating tissue comprises inducing shoot formation from the transformed Cannabaceae cells using a culture medium.

[0013] In some embodiments, the culture medium comprises about 1 milligram (mg) / liter (L) to about 20 mg / L of TDZ.

[0014] In some embodiments, the method further comprises screening the transformed Cannabaceae cell or tissue regenerated from the transformed Cannabaceae cell for expression of the heterologous nucleotide sequence using a selection agent to screen the transformed Cannabaceae cell or tissue regenerated from the transformed Cannabaceae cell, the selection agent being selected from kanamycin A (kan), g418, spectinomycin and glyphosate.

[0015] In some embodiments, exposing the Cannabaceae meristem region or EA to a heterologous nucleotide sequence comprises contacting the Cannabaceae meristem region or EA with a bacterial strain carrying the heterologous nucleotide sequence.

[0016] In some embodiments, exposing the Cannabaceae meristem region or EA to a heterologous nucleotide sequence comprises exposing the Cannabaceae meristem region or EA to an infection medium comprising a bacterial strain transformed to carry the heterologous nucleotide sequence.

[0017] In some embodiments, the method further comprises removing the infection medium, co-cultivating the Cannabaceae meristem regions or EAs for a threshold period of time, and culturing the Cannabaceae meristem regions or EAs in a selection medium to select for transformed Cannabaceae meristem regions or EAs.

[0018] In some embodiments, the infection medium comprises thidiazuron (TDZ), metolachlor, magnesium sulfate, Tween, acetosyringone (MTA), thiols, GA3, Gamborg's B5 vitamins, DKW salts, AB salts, glucose, Silwet L-77, and combinations thereof.

[0019] In some aspects, the heterologous nucleotide sequence encodes a rare-cutting endonuclease operably linked to a promoter and optionally a screening marker.

[0020] Some embodiments relate to a method comprising soaking Cannabaceae seeds in a hydration solution, excising a subset of embryonic tissue from the soaked Cannabaceae seeds to extract a Cannabaceae meristem region or EA, exposing the Cannabaceae meristem region or EA to a heterologous nucleotide sequence to transform Cannabaceae cells of the Cannabaceae meristem region or EA, and regenerating tissue from the transformed Cannabaceae cells using a culture medium containing thidiazuron (TDZ).

[0021] In some embodiments, the culture medium comprises a shoot induction medium comprising about 1 mg / L to about 20 mg / L of TDZ.

[0022] In some embodiments, transforming a Cannabaceae cell comprises exposing a Cannabaceae meristem region or EA to an infection medium comprising a bacterial strain carrying a heterologous nucleotide sequence and comprising about 0.1 mg / L to about 2 mg / L of TDZ.

[0023] In some embodiments, regenerating tissue comprises transferring and culturing the Cannabaceae meristem region or EA in a shoot induction medium (SIM) containing the TDZ, and after culturing in the SIM, transferring and culturing the Cannabaceae meristem region or EA in a shoot elongation medium (SEM) to induce shoot formation.

[0024] In some embodiments, regenerating the tissue further comprises recovering by transferring and culturing the Cannabaceae meristem region or EA after exposure to the heterologous nucleotide sequence and prior to transfer to the SIM in a regeneration medium comprising from about 0.1 mg / L to about 10 mg / L of TDZ, wherein the SIM comprises from about 1 mg / L to about 20 mg / L of TDZ.

[0025] In some embodiments, regenerating tissue comprises inducing shoot formation from the transformed Cannabaceae cells using a culture medium containing TDZ, and inducing roots from the formed shoots.

[0026] In some aspects, inducing roots from the formed shoots includes screening the formed shoots for shoots of a minimum height, rooting the selected shoots of a minimum height to induce primary roots, and transferring and rooting the shoots with the induced primary roots to induce the formation of new primary root and root hair structures.

[0027] In some aspects, the method further comprises transferring the selected shoots having the primary root and root hair structures to soil.

[0028] Some embodiments relate to transformed Cannabaceae explants produced using any of the methods described herein.

[0029] Various exemplary embodiments can be more fully understood when considered in conjunction with the following detailed description of the accompanying drawings. [Brief description of the drawings]

[0030] [Figure 1] FIG. 1 is a flow diagram showing an exemplary method for transforming Cannabaceae cells from a Cannabaceae meristem region or EA consistent with the present disclosure. [Diagram 2] FIG. 1 is another flow diagram showing an exemplary method for transforming Cannabaceae cells from a Cannabaceae meristem region or EA consistent with the present disclosure. [Diagram 3]FIG. 1 shows an exemplary scheme for regenerating transformed Cannabaceae tissue from transformed Cannabaceae cells of the Cannabaceae meristem region or EA, consistent with the present disclosure. [Figure 4] FIG. 1 shows an exemplary expression construct for delivery of heterologous sequences to Cannabaceae cells of the Cannabaceae meristem region or EA, consistent with the present disclosure. [Figure 5A] FIG. 1 shows an exemplary expression construct for transforming Cannabaceae cells from a Cannabaceae meristem region or EA consistent with the present disclosure. [Figure 5B] FIG. 1 shows an exemplary expression construct for transforming Cannabaceae cells from a Cannabaceae meristem region or EA consistent with the present disclosure. [Figure 5C] FIG. 1 shows an exemplary expression construct for transforming Cannabaceae cells from a Cannabaceae meristem region or EA consistent with the present disclosure. [Figure 5D] FIG. 1 shows an exemplary expression construct for transforming Cannabaceae cells from a Cannabaceae meristem region or EA consistent with the present disclosure. [Figure 5E] FIG. 1 shows an exemplary expression construct for transforming Cannabaceae cells from a Cannabaceae meristem region or EA consistent with the present disclosure. [Figure 5F] FIG. 1 shows an exemplary expression construct for transforming Cannabaceae cells from a Cannabaceae meristem region or EA consistent with the present disclosure. [Figure 5G] FIG. 1 shows an exemplary expression construct for transforming Cannabaceae cells from a Cannabaceae meristem region or EA consistent with the present disclosure. [Figure 5H] FIG. 1 shows an exemplary expression construct for transforming Cannabaceae cells from a Cannabaceae meristem region or EA consistent with the present disclosure. [Figure 5I] FIG. 1 shows an exemplary expression construct for transforming Cannabaceae cells from a Cannabaceae meristem region or EA consistent with the present disclosure. [Figure 6A] 1 includes exemplary images from samples in Table 1 consistent with the present disclosure. [Figure 6B]1 includes exemplary images from samples in Table 1 consistent with the present disclosure. [Figure 7A] FIG. 1 is an image of a cannabis seedling explant transformed with a bacterial strain consistent with the present disclosure. [Figure 7B] FIG. 1 is an image of a cannabis seedling explant transformed with a bacterial strain consistent with the present disclosure. [Figure 8] 7A-7B are images of plants regenerated from cannabis seedling explants transformed with the bacterial strains shown in FIGS. [Figure 9] 9 is an image of PCR data from regenerated plants shown in FIG. 8 consistent with the present disclosure. [Figure 10A] FIG. 1 is an image of a meristematic region of cannabis transformed with a bacterial strain consistent with the present disclosure. [Figure 10B] FIG. 1 is an image of a meristematic region of cannabis transformed with a bacterial strain consistent with the present disclosure. [Figure 11] FIG. 1 is an image of a cannabis seedling explant transiently transformed with a bacterial strain consistent with the present disclosure. [Figure 12A] FIG. 1 is an image of a cannabis seedling explant stably transformed with a bacterial strain consistent with the present disclosure. [Figure 12B] FIG. 1 is an image of a cannabis seedling explant stably transformed with a bacterial strain consistent with the present disclosure. [Figure 12C] FIG. 1 is an image of a cannabis seedling explant stably transformed with a bacterial strain consistent with the present disclosure. [Figure 13A] FIG. 1 is an image of a cannabis EA stably transformed in a bacterial strain consistent with the present disclosure. [Figure 13B] FIG. 1 is an image of a cannabis EA stably transformed in a bacterial strain consistent with the present disclosure. [Figure 14A] FIG. 1 is an image of a cannabis seedling explant stably transformed with a bacterial strain consistent with the present disclosure. [Figure 14B] FIG. 1 is an image of a cannabis seedling explant stably transformed with a bacterial strain consistent with the present disclosure. [Figure 15A] FIG. 1 is an image of a cannabis seedling explant stably transformed with a bacterial strain consistent with the present disclosure. [Figure 15B]FIG. 1 is an image of a cannabis seedling explant stably transformed with a bacterial strain consistent with the present disclosure. [Figure 16A] FIG. 13 shows data results from an experiment evaluating different culture media and light conditions consistent with the present disclosure. [Figure 16B] FIG. 13 shows data results from an experiment evaluating different culture media and light conditions consistent with the present disclosure. [Figure 16C] FIG. 13 shows data results from an experiment evaluating different culture media and light conditions consistent with the present disclosure. [Figure 16D] FIG. 13 shows data results from an experiment evaluating different culture media and light conditions consistent with the present disclosure. [Figure 16E] FIG. 13 shows data results from an experiment evaluating different culture media and light conditions consistent with the present disclosure. [Figure 16F] FIG. 13 shows data results from an experiment evaluating different culture media and light conditions consistent with the present disclosure. [Figure 16G] FIG. 13 shows data results from an experiment evaluating different culture media and light conditions consistent with the present disclosure. [Figure 17A] 13A-13C are images of explants from an experiment evaluating transformation of explants in different media consistent with the present disclosure. [Fig. 17B-17C] 13A-13C are images of explants from an experiment evaluating transformation of explants in different media consistent with the present disclosure. [Figure 17D] 13A-13C are images of explants from an experiment evaluating transformation of explants in different media consistent with the present disclosure. [Figure 17E] 13A-13C are images of explants from an experiment evaluating transformation of explants in different media consistent with the present disclosure. [Figure 17F] 13A-13C are images of explants from an experiment evaluating transformation of explants in different media consistent with the present disclosure. [Figure 18A] FIG. 1 shows the results of transforming explants with different plasmid vectors consistent with the present disclosure. [Figure 18B] FIG. 1 shows the results of transforming explants with different plasmid vectors consistent with the present disclosure. [Figure 18C] FIG. 1 shows the results of transforming explants with different plasmid vectors consistent with the present disclosure. [Figure 18D] FIG. 1 shows the results of transforming explants with different plasmid vectors consistent with the present disclosure. [Figure 18E] FIG. 1 shows the results of transforming explants with different plasmid vectors consistent with the present disclosure. [Figure 18F] FIG. 1 shows the results of transforming explants with different plasmid vectors consistent with the present disclosure. [Figure 18G] FIG. 1 shows the results of transforming explants with different plasmid vectors consistent with the present disclosure. [Figure 18H] FIG. 1 shows the results of transforming explants with different plasmid vectors consistent with the present disclosure. [Figure 18I] FIG. 1 shows the results of transforming explants with different plasmid vectors consistent with the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Aspects of the present disclosure relate to various methods for producing transformed Cannabaceae cell lines, plants and plant parts, such as meristematic regions of seed parts or Cannabaceae EAs, from Cannabaceae seeds. These methods may include extracting Cannabaceae meristem regions or EAs from Cannabaceae seeds and transforming cells of the Cannabaceae meristem regions or EAs by exposure to a heterologous nucleotide sequence. The exposure may cause transformation of the cells, and the transformed cells may be used to regenerate transformed Cannabaceae tissues, such as plant parts or whole plants. The plants, plant parts and plant cells of the present disclosure may be used to produce specific Cannabaceae varieties. In some embodiments, the plants, plant parts and plant cells may be used to develop new varieties or hybrids with specific traits and / or phenotypes. The present invention is not necessarily limited to such applications, but various aspects of the present invention may be understood through the description of various embodiments using this context.

[0032] Cannabis plants are used for a variety of purposes. For example, cannabis, a type of Cannabis plant, is a fast-growing plant that can be used as a low-cost source of food, building or clothing materials, biomass, paint, paper, and other materials, as well as for medicinal or recreational purposes. To provide specific traits and / or varieties, plants can be transformed using gene editing techniques in tissue culture. However, Cannabis plant cells in tissue culture can be non-responsive or resistant to produce embryogenic cells that continue to form clonal plants and intact plants. An embodiment of the present disclosure relates to transforming Cannabis plant cells from Cannabis meristem regions or EAs, which can be regenerated to form transformed plant tissues, such as plant parts or whole plants.

[0033] In various embodiments, the Cannabaceae meristem region or EA is prepared from Cannabaceae seeds. For example, whole Cannabaceae seeds can be sterilized and soaked in a hydration solution, and a subset of embryonic tissue can be excised from the soaked Cannabaceae seeds to extract the Cannabaceae meristem region or EA. The Cannabaceae meristem region or EA is then exposed to a heterologous nucleotide sequence to transform Cannabaceae cells of the Cannabaceae meristem region or EA. The heterologous nucleotide sequence can encode a polypeptide and can cause mutations in the Cannabaceae cells. Tissues can be regenerated from transformed Cannabaceae cells expressing the heterologous nucleotide sequence. In some embodiments, the regenerated tissue can include plant parts or entire Cannabaceae plants that exhibit specific traits caused by transformation.

[0034]

[0023] Referring now to the drawings, Figure 1 is a flow diagram illustrating an exemplary method for transforming Cannabaceae cells from Cannabaceae meristem regions or EAs consistent with the present disclosure. Method 100 can be used to regenerate transformed Cannabaceae tissue from Cannabaceae cells transformed to express a heterologous nucleotide sequence.

[0035] At 101, the method includes soaking Cannabaceae seeds in a hydration solution. In some embodiments, the seeds are fully mature, have intact seed coats, and / or are free of bacteria, fungi, or other pest vectors. For example, the hydration solution may be sterile distilled water or ddH2O. 2The hydration solution may include water such as 0.25 mL of water. In some embodiments, the Cannabaceae seeds can be soaked by exposing the Cannabaceae seeds to a hydration solution and / or using a rotary shaker for a period of time. For example, the Cannabaceae seeds can be placed in a 50 mL tube containing sterile water and placed on a rotary shaker for 10-24 hours. In some embodiments, the Cannabaceae seeds can be placed on a rotary shaker for 16-20 hours, 10-14 hours, 12-14 hours, 14-16 hours, 16-18 hours, or 18-20 hours, among other ranges. In some embodiments, the Cannabaceae seeds can be placed in a 100×25 mm Petri dish containing sterile filter paper and 2-3 mL of sterile water, sealed, and soaked for 16-20 hours, 10-14 hours, 12-14 hours, 14-16 hours, 16-18 hours, or 18-20 hours, among other ranges.

[0036] In some embodiments, prior to soaking the Cannabaceae seeds, the method 100 can include sterilizing the Cannabaceae seeds. In some embodiments, the Cannabaceae seeds can be sterilized using scarification and hydrogen peroxide. Scarification can be effected by exposing the Cannabaceae seeds to an acid, such as sulfuric acid. In some embodiments, the Cannabaceae seeds can be exposed to the acid for 1 to 30 seconds, then the acid is removed, the seeds are rinsed with sterile water, and the Cannabaceae seeds are exposed to hydrogen peroxide or another sterilizing agent for 1 to 30 minutes. However, the sterilizing agent can be, for example, but not limited to, ethanol, hypochlorite (NaClO or Ca(ClO) 2), benzalkonium chloride, silver nitrate, mercury chloride, and hydrogen peroxide. The sterilizing solution may contain 0.01% to about 95% by volume of the sterilizing agent. The Cannabaceae seeds may be exposed to the sterilizing solution for 0.1 to about 30 minutes. The sterilizing solution may further include a mild detergent such as polysorbate (e.g., TWEEN 20 or TWEEN 80) or other non-ionic surfactant. In some embodiments, the Cannabaceae seeds may be washed with a sterilizing solution containing about 10% hydrogen peroxide. The Cannabaceae seeds may be placed in a sterile 50 mL conical tube and the sterilizing solution may be added (e.g., by placing the 50 mL tube on a rotating shaker). After immersion in the solution, the Cannabaceae seeds may be rinsed several times. For example, the sterilized Cannabaceae seeds may be rinsed with sterile distilled water three to five times, for one to ten minutes each rinse. After sterilization, the Cannabaceae seeds can be kept in distilled water in sealed Petri dishes in a laminar flow cabinet to prevent drying.

[0037] At 103, the method 100 includes excising a subset of embryonic tissue from the soaked Cannabaceae seed to extract the Cannabaceae meristem region or EA. As used herein, the "Cannabaceae EA" includes the part of the seed between the shoot and the rootlet, not including the cotyledons. The "meristem region" includes plant tissue that includes undifferentiated cells (meristem cells) found in the area of ​​the plant where growth can occur. The Cannabaceae EA may include the shoot, the rootlet, and the hypocotyl. The part of the embryo between the attachment point of the cotyledons and the rootlet is called the hypocotyl. The EA ends in the rootlet, which is the area where the root develops. After germination, the embryo can give rise to a seedling. In some embodiments, the undifferentiated cells of the meristematic region can have the ability to grow and regenerate into a plant (meristem cells) found in the area of ​​the plant where growth can occur.

[0038] In some embodiments, removing the subset of embryonic tissues may include removing the seed coat without removing any of the cotyledons of the soaked Cannabaceae seeds. For example, sterile forceps or other cutting instruments (e.g., scalpels, scissors) can be used to remove the seed coat and the embryonic tissue. In some embodiments, removing the subset of embryonic tissues includes removing the seed coat and one of the cotyledons of the soaked Cannabaceae seeds. Removing a subset of the embryonic tissues, such as one of the cotyledons, can reduce the time and cost of extraction compared to removing all of the embryonic tissues, and can also reduce the risk of destroying tissue. In some embodiments, removing the subset of embryonic tissues includes removing the seed coat and cutting the rootlet of the soaked Cannabaceae seeds. In some embodiments, removing the subset of embryonic tissues includes removing both the seed coat, the cotyledons, and the leaf primordia of the soaked Cannabaceae seeds. In further embodiments, removing the subset of embryonic tissues includes removing the seed coat, one of the cotyledons, and the leaf primordia of the soaked Cannabaceae seeds. As described above, removing one of the cotyledons can reduce tissue damage compared to excising all of the embryonic tissue, thereby improving transformation efficiency and reducing the time and costs associated with transformation. In various embodiments, sterile forceps can be used to remove the seed coat, and the embryo is then ready for meristematic region or EA extraction. For example, forceps can be used to hold the seed, and a scalpel blade can be used to cut off one or both of the cotyledons. Care can be taken to prevent or reduce damage to the meristematic tissue. In some embodiments, after removing one or more cotyledons, a scalpel blade can be used to remove the leaf primordia, taking care to prevent or reduce damage to the meristematic tissue.

[0039] At 105, the method 100 includes exposing the Cannabaceae meristem region or EA to a heterologous nucleotide sequence to transform Cannabaceae cells of the Cannabaceae meristem region or EA. The heterologous nucleotide sequence can encode a polypeptide that is expressed by the Cannabaceae cells and / or causes transformation of the Cannabaceae cells. For example, the heterologous sequence can encode a gene of interest. In response to expression of the heterologous nucleotide sequence, the transformed Cannabaceae cells can be used to regenerate tissues, plant parts, or whole plants that exhibit one or more traits associated with the gene of interest.

[0040] In some embodiments, the Cannabaceae cell is transformed using bacteria-mediated transformation. For example, exposing the Cannabaceae meristem region or EA to a heterologous nucleotide sequence comprises contacting the Cannabaceae meristem region or EA with a bacterial strain having a heterologous nucleotide sequence. The bacterial strain can be transformed to have a heterologous nucleotide sequence. In some embodiments, the method comprises transforming the Cannabaceae cell by exposing the Cannabaceae meristem region or EA to an infection medium containing a transformed bacterial strain, as further described herein.

[0041] Bacterial strains can include any strain transformed to induce expression of a heterologous nucleotide sequence, such as to express a gene of interest. Bacterial strains can include Rhizobium strains, such as Rhizobium strains or Agrobacterium strains. In some embodiments, bacterial strains include Agrobacterium tumefaciens or Rhizobium rhizogenes strains (R. rhizogenes), formerly known as Agrobacterium rhizogenes (A. rhizogenes). Rhizobium strains include T-DNA that causes disease symptoms in infected plants and is contained in a root-inducing (Ri) plasmid or a tumor-inducing (Ti) plasmid. In some embodiments, wild-type or disarmed strains carrying the Ti or Ri plasmid can be used. For example, Rhizobia strains can be cured in that they retain complete vir gene function that mediates T-DNA processing, transfer and integration, but remove the oncogene in the helper Ti or Ri plasmid. The bacterial strain contains a second T-DNA that contains the gene of interest in a binary vector. During transformation, virulence proteins encoded by the helper Ti plasmid act in trans to aid in the transfer of the T-DNA on the binary vector from the bacteria to the plant. The T-DNA from the bacterial strain can be stably integrated into plant parts. For example, tissues resulting from Rhizobia infection of plant tissues carry the T-DNA from the Ri or Ti plasmid and form vascular connections with their plant host.

[0042] Generally, the bacterial strain is prepared for infection by introducing a heterologous nucleotide sequence into the bacterial strain (e.g., by electroporation) and culturing the transformed bacterial strain under conditions that select for positively transformed cells. In some embodiments, the method 100 includes selecting a particular bacterial strain. The selection of a bacterial strain effective for producing transformed cells may depend on the plant species to be infected and can be determined empirically.

[0043] In some embodiments, wild-type and disarmed strains of A. tumefaciens or A. rhizobium carrying Ti or Ri plasmids can be used for gene transfer into plants. Exemplary strains include, but are not limited to, nopaline-type strains, octopine-type strains such as LBA4404, succinamopine-type strains such as EHA101 or EHA105, and A. tumefaciens derived from A. rhizogenes agropine, mannopine, and cucumopine-type strains (e.g., MSU440, A13, 1855, 1193, A4, Qual, K599 (AKANCPPB2659), and C58C1). Optionally, the A. tumefaciens strain is selected from the group consisting of AGL1, EHA105, GV3101, ICF320, CryX, LBA4404, C58, A136, A208, A348, Ach5, EHA101, NT1RE, NT1RE(pJK270), 1D1108, 1D1460, 1D1609, 1D132, 1D1478, and 1D1487, and the A. rhizobium strain is K599 or a strain derived from K599.

[0044] The nucleic acid sequence can be introduced by direct or indirect plant transformation methods. An embodiment of the present disclosure includes the use of an activated Rhizobium bacterium to inject a piece of DNA into the Cannabaceae meristem region or EA. The piece of DNA can be part of an expression cassette introduced into a binary plasmid for Rhizobium-mediated transformation. The expression cassette can be introduced into the bacterium using conventional methods.

[0045] Bacteria-mediated plant transformation involves the activation of a Rhizobium strain with a DNA fragment cloned into a plasmid. The activated Rhizobium strain is then used to transform the meristem region or EA into individual plant cells. In some embodiments, the T-DNA includes a DNA sequence of a screening marker that confirms the transformation and / or the location of the transformant or EA within the cell in the Cannabaceae meristem region. For example, the screening marker may include a gene that confers a selection marker or a labeling marker. The screening marker can be embedded in the same vector as the gene of interest or can be delivered as a separate vector. The labeling marker can confer a detectable label that can be detected visually, electrically, or otherwise, such as a fluorescent protein (green fluorescent protein (GFP or eGFP), yellow fluorescent protein (YFP), or red fluorescent protein (RFP)) that can be detected using fluorescence microscopy. The selection marker can confer resistance to a toxic substance (e.g., a selection agent). Examples of selection markers for plant transformation include selection markers that confer resistance to a toxic substance such as an antibiotic or herbicide. For example, the selection marker may include kanamycin resistance gene, g4198 resistance gene, spectinomycin resistance gene, or glyphosate resistance gene. Examples of labeling markers include β-glucuronidase (GUS), β-galactosidase, luciferase, Ruby, and chloramphenicol acetyltransferase. Screening markers can be useful for quantifying or visualizing the spatial pattern of expression of genes in specific tissues, and are often called reporter genes because they can be fused to genes or gene regulatory sequences for gene expression investigations. For example, a GFP variant, yellow fluorescent protein (YFP), can be used to indicate plant cells in which Rhizobium can take up foreign DNA.

[0046] In some embodiments, the heterologous nucleotide sequence comprises a gene editing reagent. The gene editing reagent can comprise a rare-cutting endonuclease or a part (e.g., a subunit) thereof. The rare-cutting endonuclease can be a transcription activator-like effector nuclease (TALEN), a meganuclease, a zinc finger nuclease (ZFN), or a clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) nuclease reagent. For example, rare-cutting endonucleases have been described in Baker, Nature Methods 9:23-26, 2012; Belahj et al., Plant Methods, 9:39, 2013; Gu et al., Nature, 435:1122-1125, 2005; Yang et al., Proc Natl Acad Sci USA, 103:10503-10508, 2006; Kay et al. Science, 318:648-651, 2007; Sugio et al., Proc Natl Acad Sci USA, 104:10720-10725, 2007; Romer et al. Science, 318:645-648, 2007; Schornack et al., J Plant Physiol, 163:256-272, 2006; and WO 2011 / 072246, each of which is incorporated herein in its entirety for its teachings.

[0047] In some embodiments, the vector can include a TALEN sequence that encodes a first and a second TALEN and a binding domain that binds to a target site and causes a mutation at the target site. The first TALEN can generate a double-stranded break at or near a first target site associated with the first binding domain, and the second TALEN can generate a double-stranded break at or near a second target site associated with the second binding domain. In some embodiments, the first and the second binding domain can be associated with a target gene. In some embodiments, the TALEN sequence can be co-delivered with a secondary transgene into plant tissue to cause expression of the secondary transgene together with a viral (e.g., hairy root) transgene.

[0048] As mentioned above, examples are not limited to TALENs and may include, among others, CRISPR / Cas systems (see, e.g., Belahj et al., Plant Methods, 9:39, 2013), or may not include gene editing reagents. In some embodiments, Cas9 endonuclease and guide RNA can be used (either a complex of CRISPRRNA (crRNA) and transactivating crRNA (tracrRNA), or a synthetic fusion (sgRNA) of the 3' end of crRNA and the 5' end of tracrRNA). The guide RNA directs Cas9 binding and DNA cleavage to a homologous sequence adjacent to a protospacer adjacent motif (PAM). Upon reaching the target DNA sequence, Cas9 generates a DNA double-strand break 3 nucleotides from the 3' end of the crRNA targeting sequence. In some embodiments, this approach or other approaches, such as ZFNs and / or meganucleases, can be used in addition to TALE nucleases to obtain modified plant parts.

[0049] In various embodiments, the bacterial strain can form part of the infection medium used to infect and transform the Cannabaceae meristem region or EA and associated cells. The infection medium can include the transformed bacterial strain, basal salts, sugars, and growth hormones and / or plant growth regulators. Plant growth regulators refer to or include compounds that modify plant growth, e.g., induce regeneration. In some embodiments, the plant growth regulator includes thidiazuron (TDZ). For example, the infection medium can include about 1 milligram (mg) / liter (L) of TDZ. However, embodiments are not so limited and can include other growth hormones or regulators and / or other concentrations of TDZ, such as metatopolin, metolachlor, or magnesium sulfate, among others. In some embodiments, the infection medium includes magnesium sulfate, Tween, and acetosyringone (MTA). In some embodiments, the infection medium comprises about 0.1 mg / L to about 2 mg / L of TDZ. In some embodiments, the infection medium comprises about 0.1 mg / L to about 1.5 mg / L, about 0.1 mg / L to about 1.0 mg / L, about 0.1 mg / L to about 0.5 mg / L, about 0.5 mg / L to about 2 mg / L, about 1.0 mg / L to about 2 mg / L, about 1.5 mg / L to about 2 mg / L, about 0.5 mg / L to about 1.5 mg / L, about 0.5 mg / L to about 1.0 mg / L, or about 1.0 mg / L to about 1.5 mg / L TDZ, among other ranges.

[0050] Exposing the meristematic region or EA to a bacterial strain may include introducing the bacterial strain into the intracellular space of cells of the meristematic region or EA. For example, the bacterial strain carrying the nucleic acid sequence can be introduced into the intracellular space by immersing the Cannabaceae meristematic region or EA in a bacterial suspension in an infection medium. In some embodiments, the meristematic region or EA can be subjected to a chemical treatment that makes the cell wall more permeable (e.g., treatment with a macerating enzyme such as cellulase, pectinase or macerozyme). Transformation efficiency can be increased by subjecting the donor material to vacuum infiltration, heat shock and / or centrifugation, and sonication.

[0051] In various embodiments, transformation may include preparing, inoculating, and co-cultivating bacterial cultures. For example, they can be prepared by culturing and suspending in infection medium at a target concentration for inoculating Cannabaceae meristem regions or EAs. Bacterial cultures can be inoculated from streaked plates, and bacterial cells are washed and resuspended in a culture medium suitable for inoculation of Cannabaceae meristem regions or EAs. In some embodiments, bacteria can be grown in nutrient-rich liquid medium as an initial starter culture for about 8 hours, and the starter culture can be used to inoculate shake flasks containing Agrobacterium minimal growth (AB) medium and grown for about 20-24 hours.

[0052] The density of the bacterial culture used for inoculation and the ratio of bacterial cells to Cannabaceae meristem regions or EA can be varied. For example, the concentration of bacteria in the infection medium can be altered to prevent overgrowth. Optimizing the bacterial density can facilitate transformation. Bacterial density is adjusted to an OD of 0.4 to 0.9, which corresponds to 10,000,000 to 1,000,000,000 cfu / mL. 600 (nm). In some embodiments, the culture is grown or diluted to a bacterial density of at least 0.7 to 1.5.

[0053] The infection medium can be supplemented with one or more compounds to improve infection and transformation rates by mitigating oxidative stress and / or bacterial overgrowth. For example, the infection medium can include an effective amount of an antioxidant, such as a thiol or related sulfur-containing compound. Suitable thiol compounds include L-cysteine, dithiothreitol (DTT) and sodium thiosulfate. The infection medium can include other chemicals (e.g., TDZ, metatopolin, glyphosate), as described above. The infection medium can also include compounds that enhance Agrobacterium transformation efficiency, such as acetosyringone.

[0054] In some embodiments, the method 100 may further include screening the transformed Cannabaceae cells, for example, using the screening markers described above. Such embodiments include the use of a selection protocol to select cells expressing one or more proteins encoded by the Cannabaceae meristem region, EA, and / or heterologous nucleotide sequence. As described above, the heterologous nucleotide sequence may optionally encode a screening marker. The screening marker may be used to identify transformed Cannabaceae meristem regions, EA, and / or cells, and / or to distinguish transformed Cannabaceae meristem regions, EA, and / or cells from untransformed Cannabaceae meristem regions. For example, selection for phosphomannose isomerase (PMI) expression includes the addition of mannose to the medium with limitation of other carbon sources. Transformed cells and / or tissues may be identified by a variety of methods, some of which are described below.

[0055] As described above, the screening marker can include a label marker and / or a selection marker. The label marker allows visual, electrical, or other identification of the label. Examples of label markers include YFP, RFP, betaline, and PDS editing that causes bleaching. Usually, expression occurs spontaneously. In some embodiments, expression of the nucleotide sequence is induced, for example, by a change in a biotic or abiotic factor. Expression of the gene associated with the label marker 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, Western blotting, etc. Tissues can be assayed for expression of the label marker. Expression of the fluorescent protein marker can be visualized by UV excitation, fluorescence microscopy, or flow cytometry. Transformation efficiency can be calculated and compared to conventional methods.

[0056] In some embodiments, a selection agent can be used to neutralize (e.g., kill) Cannabaceae cells and / or tissues that are not transformed to express the selection marker. For example, the selection marker can be associated with a gene that confers resistance to the selection agent, which can be a toxic substance such as an antibiotic or herbicide. In some embodiments, the method 100 includes using a selection agent to screen the transformed Cannabaceae cells or tissues regenerated from the transformed Cannabaceae cells. For example, the selection marker can include or be associated with an antibiotic, such as kanamycin, to provide resistance to kanamycin. When exposed to a sufficient amount of kanamycin, the untransformed Cannabaceae meristem regions and / or EAs will die and the transformed Cannabaceae meristem regions and / or EAs can survive. Examples of selection agents include kanamycin A (kan), g418, spectinomycin, and glyphosate.

[0057] The method 100 may include Cannabaceae meristem region and / or EA preparation at 101 and 103, transformation of Cannabaceae cells at 105, and regeneration of tissue from the transformed Cannabaceae cells, as further illustrated and described by FIG. 2. The preparation may include soaking Cannabaceae seeds and removing a subset of embryonic material to produce Cannabaceae meristem regions or EAs. The transformation of Cannabaceae cells may include preparation of bacterial cultures, inoculation, and co-cultivation. After co-cultivation, tissue may be regenerated. Regenerating tissue may include inducing shoot formation from the infected Cannabaceae meristem region or EA, elongating the shoots, and inducing root formation from the shoots by rooting the shoots. The method 100 may provide transformed Cannabaceae plant parts within 2-15 weeks after the Cannabaceae meristem region or EA is exposed to a heterologous nucleotide sequence. The materials and methods of the present disclosure allow for the development of an efficient transformation system for Cannabaceae.

[0058] Inoculation can be performed at a temperature of about 20-28°C (°C), about 23-28°C, about 24-26°C, or about 25°C. Contact time with the suspension can range from about less than 1 minute (short immersion) to about 3 hours. After inoculation, excess bacterial suspension can be removed (e.g., by blotting or rinsing in sterile ddH2O) and the EA is plated onto the co-culture medium.

[0059] Different plant tissue culture media can be used for the co-cultivation step. Co-cultivation of Cannabaceae meristem regions or EA with bacteria under in vitro conditions can be optimized with respect to duration, temperature, irradiance, and / or medium composition and pH. In other embodiments, sterile filter paper moistened with sterile water can be used for the co-cultivation step. In some embodiments, Cannabaceae meristem regions or EA are co-cultivated for a threshold period of time. For example, 1-4 days of co-cultivation may be sufficient for successful transformation, while longer periods (e.g., 5-7 days) can be utilized for recalcitrant genotypes that require enhanced transformation efficiency. Temperatures for incubation can range from 18-25°C, or 20-23°C, for example, at about 23°C. Co-cultivation can be performed in light or light-limited conditions. Lighting conditions can be optimized for the plant genotype. In some embodiments, co-cultivation is performed in ambient light at 23±1°C for 2-4 days of co-cultivation.

[0060] After co-cultivation, in some embodiments, the transformed meristem regions and / or EAs can be rinsed with an antibiotic solution to remove excess bacteria. The antibiotic rinse solution can consist of one or more antibiotics, such as cefotaxime, timentin, or carbenicillin. After the antibiotic rinse, the transformed meristem regions and / or EAs can be plated on a selective medium (e.g., SIM). The selective medium can include basal salts and an agent that inhibits bacterial growth, such as an antibiotic (e.g., carbenicillin, ticarcillin, clavulanic acid, ampicillin, spectinomycin, and / or cefotaxime). Exemplary selective media may include basal salts (e.g., DKW) and vitamins (e.g., B5 vitamins), as well as effective amounts of the following: sugars (e.g., sucrose, glucose, maltose), a buffer to maintain a pH within the range of about 5.4 to 6 (e.g., 2-(N-morpholino)ethanesulfonic acid (MES)), and antibiotics (e.g., cefotaxime and a combination of ticarcillin and clavulanic acid (timentin) and / or spectinomycin). In some embodiments, the selective media may further include a selective agent. For example, if the selective marker is spCN, a spectinomycin resistance gene, the selective media may include an effective amount of spectinomycin (e.g., up to 150 mg / L).

[0061] In some embodiments, the recovery step is carried out by transferring the converted meristem regions and / or EA to a regeneration medium, such as a CL medium, to help reduce or eliminate contamination. The CL medium can include a biocide, such as a plant preservative mixture (PPM), to reduce or eliminate contaminants. The regeneration medium can further include a plant growth regulator, such as vitamins, sugars, basal salts, and / or TDZ. In some embodiments, the CL medium includes about 0.1 mg / L to about 10 mg / L of TDZ. In some embodiments, the CL medium includes about 1 mg / L of TDZ or about 2 mg / L of TDZ. In some embodiments, the CL medium contains from about 0.1 to about 9 mg / L, from about 0.1 to about 8 mg / L, from about 0.1 to about 7 mg / L, from about 0.1 to about 6 mg / L, from about 0.1 to about 5 mg / L, from about 0.1 to about 4 mg / L, from about 0.1 to about 3 mg / L, from about 0.1 to about 2.5 mg / L, from about 0.1 to about 2 ... Approximately 1.5 mg / L, approximately 0.1 to approximately 1.0 mg / L, approximately 0.1 to approximately 0.5 mg / L, 0.5 to approximately 10 mg / L, approximately 1 to approximately 10 mg / L, approximately 1.5 to approximately 10 mg / L, About 2.0 to about 10 mg / L, about 2.5 to about 10 mg / L, about 2 to about 8 mg / L, about 2 to about 6 mg / L, about 2 to about 4 mg / L, about 2 to about 3 mg / L, including TZ / L.

[0062] 2 is a flow diagram showing another exemplary method for transforming Cannabaceae cells from a Cannabaceae meristem region or EA consistent with the present disclosure. Method 200 includes steps 101, 103, and 105 described by FIG. 1, the details of which will not be repeated.

[0063] The method 200 further includes, at 207, regenerating tissue from the transformed Cannabaceae cells, the tissue including one or more of a shoot, a root, a root hair structure, and a complete plant. As further illustrated by FIG. 3, regenerating tissue may include inducing shoot formation from the transformed Cannabaceae cells. For example, inducing shoot formation may include inducing shoot formation and inducing shoot elongation. In some embodiments, regenerating tissue may include inducing shoot formation from the transformed Cannabaceae cells and inducing roots from the formed shoots.

[0064] In some embodiments, inducing shoot formation may include transferring and culturing the Cannabaceae meristem region or EA on shoot induction medium (SIM), such as SIM+S100 (TDZ2) or (TDZ10), as further described below. In some embodiments, the Cannabaceae meristem region or EA may be cultured on SIM containing 10 mg / L TDZ for about 2 to about 6 weeks, in some embodiments, about 2 to 3 weeks. The Cannabaceae meristem region or EA may be oriented in SIM with the rootlet placed in the SIM medium and the apical meristem placed on top, similar to how a plant with roots is placed in the ground and the rootlet is allowed to bathe the SIM medium. The SIM medium may include a plant growth regulator of TDZ. Somewhat surprisingly, a concentration of about 0.1 mg / L to about 20 mg / L of TDZ may be used in the SIM medium. In some embodiments, the SIM medium includes about 2 mg / L or about 10 mg / L, although embodiments are not so limited. TDZ can be present in SIM at a concentration sufficient to break the apical dominance of Cannabaceae meristem cells, thereby increasing transformation efficiency and resulting in more transgenic events. In some embodiments, the SIM medium contains at least about 0.1 mg / L to about 15 mg / L, 0.1 mg / L to about 10 mg / L, 0.1 mg / L to about 8 mg / L, 0.1 mg / L to about 6 mg / L, 0.1 mg / L to about 5 mg / L, 0.1 mg / L to about 4 mg / L, 0.1 mg / L to about 3 mg / L, 0.1 mg / L to about 2 mg / L, 0.1 mg / L to about 1 mg / L, 0.1 mg / L to about 0.5 mg / L, about 0.5 mg / L to about 1 mg / L, among other ranges. 20mg / L, approximately 1mg / L to approximately 20mg / L, approximately 2mg / L to approximately 20mg / L, approximately 3mg / L to approximately 20mg / L, approximately 4mg / L to approximately 20mg / L, approximately 5mg / L to approximately 20mg / L, approximately 6mg / L to approximately 20mg / L, approximately It may contain 8 mg / L to about 20 mg / L, about 10 mg / L to about 20 mg / L, about 15 mg / L to about 20 mg / L, about 2 mg / L to about 15 mg / L, about 2 mg / L to about 10 mg / L, about 2 mg / L to about 5 mg / L.

[0065] After culturing in the SIM medium, the method 200 may include transferring and culturing the Cannabaceae meristem region or EA in a first shoot elongation medium (SEM). In some embodiments, a scalpel blade or other tool may be used to cut the shoot rootlet prior to transfer to the first SEM. After culturing in the first SEM, in some embodiments (although the embodiments are not so limited), the method 200 may further include transferring and culturing the Cannabaceae meristem region or EA in a second SEM. For both the first SEM and the second SEM, the rootlet of the Cannabaceae meristem region or EA is oriented downwards in the SEM and the apical meristem is positioned upwards. The first SEM and the second SEM may include different amounts of a selection agent, such as different amounts of spectinomycin.

[0066] In some embodiments, roots can be induced from the shoots. Inducing roots from formed shoots can include screening formed shoots for shoots of a minimum height, such as shoots 1-2 inches tall. Shoots of minimum height can be selected, and method 100 further includes rooting the selected shoots to induce primary root formation, and transferring the shoots having the induced primary shoots to be rooted to induce new primary shoot and root hair structure formation. In various embodiments, the selected shoots are rooted in a rooting medium (RM) and subcultured in fresh RM to induce new primary root and root hair structures. Method 200 can further include transferring the selected shoots having primary root and root hair structures to soil to regenerate partial or whole Cannabaceae plants expressing a polypeptide, such as one or more proteins of interest.

[0067] The above-mentioned infection medium, co-cultivation medium, selection medium, regeneration medium (e.g., CL medium), SIM, first SEM, second SEM, and / or RM generally include water, a basal salt mixture, sugars, and one or more other components, such as vitamins, selection agents, amino acids, and plant hormones. Each of the SIM, first SEM, and second SEM can include sugars, basal salts, growth hormones, and antibiotics, among other reagents, such as water and vitamins. For example, the SIM and SEM can include nutrient sources of nitrogen, phosphorus, potassium, sulfur, calcium, magnesium, iron, boron, molybdenum, manganese, cobalt, zinc, copper, chlorine, and iodine. Macro elements include NH 4 NO 3 , (NH 4 ) 2 SO 4 , KNO 3 , CaCl 2 2H 2 O, MgSO 4 7H 2 O, and KH 2 PO 4 The microelements are KI, H 3 BO 3 , MnSO 4 4H 2 O, ZnSO 4 , Na 2 MoO 4 2H 2 O, CuSO 4 5H 2 O, CoCl 2 6H 2 O, CoSO 4 7H 2 O, FeSO 4 7H 2 O and Na 2 EDTA 2H 2The solid plant culture medium may be provided as 0.0. Organic supplements such as nicotinic acid, pyridoxine-HCl, thiamine-HCl, and glycine may be included. Typically, the pH of the medium is adjusted to 5.7±0.5 with dilute KoH and / or HCl. The solid plant culture medium may further include a gelling agent, such as Gelrite, agar, or agarose. In various embodiments, the infection medium, regeneration medium, and / or SIM may include a plant growth regulator of TDZ, as described in the exemplary embodiments above. In some embodiments, the SIM may include a higher concentration of TDZ than the infection medium and / or regeneration medium.

[0068] Any suitable plant culture medium can be used. Examples of medium formulations include, but are not limited to, Murashige and Skoog (1962), N6, Linsmaier and Skoog (1965), L3 (Lin and Zhang (2005)), Uchimiya and Murashige (1962), Gamborg's media (1968), D medium, Nitsch and Nitsch (1969), DKW, and Schenk and Hildebrandt (1972).

[0069] The SIM and SEM may include a selection agent, a plant hormone and / or a plant growth regulator, such as an auxin, a cytocanin or a gibberellin. The plant hormone may be selected from the free and conjugated forms of naturally occurring plant hormones or plant growth regulators, or their synthetic analogs and precursors. Naturally occurring and synthetic analogs of auxins include, but are not limited to, indoleacetic acid (IAA), 3-indolebutyric acid (IBA), α-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), 4-(2,4-dichlorophenoxy)butyric acid, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 3-amino-2,5-dichlorobenzoic acid (chloramben), (4-chloro-2-methylphenoxy)acetic acid (MCPA), 4-(4-chloro-2-methylphenoxy)butanoic acid (MCPB), mecoprop, dicloprop, quinclorac, picloram, triclopyr, clopyralid, fluoroxypyr, dicamba, and combinations thereof. Any combination of two or more auxins may be present in the nutrient medium. Natural and synthetic analogs of cytokinins include, but are not limited to, kinetin, zeatin, zeatin riboside, zeatin riboside phosphate, dihydrozeatin, isopentyl adenine 6-benzyladenine, and combinations thereof. Any combination of two or more cytokinins may be present in the medium.

[0070] The presence of an effective amount of auxin, and optionally an effective amount of cytokinin, can promote cell division, improve regenerative capacity, and / or induce the growth of more regenerative tissue. The effect of exogenous auxin in producing a morphological response can be enhanced by the addition of one or more of antioxidants, amino acids, cobalt or AgNO. 3 The Cas amino acids provide an organic nitrogen source in the form of amino acids hydrolyzed from casein that can tolerate high salt conditions without degradation. Glutamine, asparagine, and methionine play complex roles in regulating biosynthetic pathways that result in morphogenetic responses.

[0071] 3 shows an exemplary scheme for regenerating transformed Cannabaceae tissue from transformed Cannabaceae cells of the Cannabaceae meristem region or EA consistent with the present disclosure. Scheme 310 can include an embodiment of method 100 of FIG. 1 and / or method 200 of FIG. 2.

[0072] The Cannabaceae meristem region or EA 320 can be prepared (311) by sterilizing the Cannabaceae seeds 312 at 315 to produce sterilized Cannabaceae seeds 314. Sterilization can include the use of a sterilizing agent, as described above. The sterilized Cannabaceae seeds 314 can be soaked at 317 by soaking the Cannabaceae seeds 312 in a hydrating solution. A subset of embryonic tissue can be excised at 319 from the soaked Cannabaceae seeds 318 to extract the Cannabaceae meristem region or EA 320.

[0073] The Cannabaceae meristem region or EA320 may be exposed to a heterologous nucleotide sequence at 321. In some embodiments, the exposing comprises inoculating and co-cultivating the Cannabaceae meristem region or EA320 with a bacterial strain by contacting the Cannabaceae meristem region or EA320 with an infection medium containing a bacterial strain having a heterologous nucleotide sequence, as described above, and co-cultivating under in vitro conditions sufficient for transformation.

[0074] Exposure to the heterologous nucleotide sequence may cause transformation of Cannabaceae cells 322 within the Cannabaceae meristem region or EA 320. In some embodiments, the Cannabaceae cells may be screened to identify transformed Cannabaceae cells 322. Transformed cells or tissues may be screened by a variety of methods. As described above, cells or tissues may be screened using a selection agent in a selection medium and / or a screening marker expressed by the transformed cells. In some instances, the transfer of the non-nucleotidic sequence is induced, for example, by a change in a biological or non-biological factor. Expression of the gene of interest may be measured by RT-PCR, 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. Tissues may be assayed for expression of the marker. Expression of the fluorescent protein marker may be visualized by UV excitation, fluorescence microscopy, or flow cytometry. Transformation efficiency may be calculated and compared to conventional methods. However, the embodiments are not so limited, and in some embodiments, screening may not be performed.

[0075] In some embodiments, in 323, the Cannabaceae meristem region or EA having the transformed Cannabaceae cells 322 can be transferred (e.g., to a regeneration medium (e.g., CL medium) and cultured for a period of time, e.g., 1-5 days, to perform the recovery step. The regeneration medium (which may be interchangeably referred to as recovery medium) can include the CL medium described above containing a biocide such as PPM. In some embodiments, the regeneration medium includes one or more selection agents and antibiotics. For example, the regeneration medium can include reagents that neutralize non-transformed cells and / or tissues and neutralize bacteria, while transformed cells and / or tissues can survive due to expression of a selection marker encoded by a heterologous nucleotide sequence.

[0076] After co-cultivation and recovery (with optional selection and / or screening), the Cannabaceae meristem region or EA with transformed Cannabaceae cells 322 can be used to regenerate tissue at 329. For example, the Cannabaceae meristem region or EA with transformed Cannabaceae cells 322 can be transferred to and cultured in the SIM at 323 under conditions that induce shoot formation for a period of time, such as 15-20 days. Conditions can include sterilization, white light, and 23° C. The SIM can include a reagent that neutralizes the bacterial strain among other reagents, such as TDZ, as described above. In response, shoot formation can be induced. The formed shoot 324 can be removed from the SIM and the rootlet of the shoot 324 can be cut. The formed shoot 324 with the cut rootlet can be transferred to an SEM at 325 and cultured under conditions that induce elongated shoots 326 for a period of time, such as 30-50 days. In some embodiments, the formed shoots 324 can be subcultured to a fresh SEM every 10-15 days, e.g., every 14 days, for that period. Conditions can include sterility, white light, and room temperature. In some embodiments, the formed shoots 324 with the cut rootlets can be transferred to a first SEM for cultivation for a first period, e.g., 15-25 days, and then transferred to a second SEM for a second period, e.g., 15-25 days. In the second SEM, in some embodiments, the formed shoots 324 can be subcultured. At 327, the elongated shoots 326 can be screened for selected shoots of minimum height, and the selected shoots can be rooted on an RM to induce formed roots 328. In some embodiments, rooting the elongated shoots 326 can include cutting the shoots and rooting the cut shoots on an RM until primary roots develop that are a threshold length, such as developing at least two primary roots that are at least 1 cm in length (and for about 14 days). Rooted shoots can be subcultured onto fresh RM approximately every 7-14 days until new primary root and root hair structures are formed. Rooted shoots 327 can be sent for acclimatization in soil to form partial or complete cannabis plants.

[0077] In various embodiments, the above methods and schemes can be used to obtain Cannabaceae plants or plant parts transformed to express a heterologous nucleotide sequence and / or a gene encoded thereby, and produced using a method according to any one of the above claims. Some embodiments relate to transformed Cannabaceae explants produced using a method according to any one of the above claims.

[0078] In some embodiments, the bacterial-mediated method can be used to obtain Cannabaceae plant tissue transformed from Cannabaceae meristem regions or EA obtained from Cannabaceae seeds. Cannabaceae meristem regions or EA can be obtained from any Cannabaceae strain, species or variety of interest, including but not limited to. The method of the present invention can be used with Cannabaceae meristem regions or EA of any Cannabaceae genotype, including hybrids and inbreds.

[0079] In some embodiments, biological transformation methods can be used to obtain transformed Cannabaceae plant tissue from a Cannabaceae meristem region or EA. For example, the Cannabaceae meristem region or EA can be modified by bombardment techniques, such as by particle bombardment with an expression construct. In some embodiments, the Cannabaceae meristem region or EA can be exposed to the expression construct by bombardment with a bombardment gun and / or particles coated with the expression construct. Specific examples of bombardment-driven transformation are known to those of skill in the art.

[0080] 4 shows an exemplary expression construct for delivering heterologous sequences to Cannabaceae cells of the Cannabaceae meristem region or EA consistent with the present disclosure. The exemplary expression construct 440 is or includes a binary vector including an expression cassette 441 and a vector backbone 446. The expression cassette 441 includes a transgene causing expression of a gene of interest 445. The transgene of the expression cassette 441 includes the gene of interest 445, a promoter 447, a left border 449, and a right border 448. In some embodiments, the left border 449 and the right border 448 can be separate from the expression cassette 441. The expression construct 440 and / or the expression cassette 441 can include various additional components such as TALE sequences, screening markers, terminators, and additional expression cassettes, among other components such as signaling peptides.

[0081] The expression cassette 441 can include genes encoding foreign proteins or proteins that confer agronomic traits. Agronomic genes include genes that confer resistance to pests or diseases, genes that confer resistance to herbicides, and / or genes that confer or contribute to value-added traits.

[0082] In some embodiments, the expression construct 440 can be used to transform a bacterial strain. As used herein, an expression construct refers to or includes a nucleic acid sequence (e.g., a DNA sequence) that includes one or more vectors or binary vectors carrying genes. A vector or binary vector includes or refers to a DNA sequence that includes one or more genes or transgenes, sometimes referred to as an "insert," and a backbone. A vector or binary vector can include an expression cassette 441 that includes a gene or transgene and a regulatory sequence that is expressed by the transformed plant cell. Upon successful transformation, the expression cassette 441 causes the plant cell to express one or more proteins of interest and / or otherwise express the target mutation.

[0083] The expression cassette 441 may include a heterologous sequence encoding a gene of interest 445, T-DNA border sequences 448, 449, and a promoter 447. An expression cassette typically includes a promoter operably linked to a nucleotide sequence encoding a gene of interest 445, optionally operably linked to a termination signal and / or other regulatory elements. For example, the expression cassette 441 may include TALENT-DNA. The expression cassette 441 may also include sequences required for proper translation of the nucleotide sequence, post-translational processing, localization and accumulation in a cellular compartment or tissue, or secretion into tissue culture medium. As an example, the gene of interest 445 may be associated with a protein that includes a signal peptide of plant origin (e.g., an N-terminal signal peptide from tobacco PR1a protein or calreticulin) or a signal peptide from a eukaryotic secreted polypeptide, such as a mammalian signal peptide, and may be efficiently secreted through the plasma membrane and cell wall into the extracellular medium. In some embodiments, the heterologous nucleotide sequence encoding the gene of interest 445 includes an N-terminal tag. For example, in the case of a transmembrane or membrane-anchored protein, an expression construct 440 can be prepared which modifies the N-terminus by replacing the transmembrane or membrane-anchored domain with an N-terminal secretion signal sequence.

[0084] The expression cassette 441 comprising the nucleotide sequence may be chimeric, meaning that at least one of its components is heterologous to at least one of the other components. In some embodiments, the expression cassette 441 may be naturally occurring or completely assembled extracellularly (e.g., by recombinant cloning techniques). The expression cassette 441 may be obtained by placing (or inserting) a promoter sequence upstream of an endogenous sequence such that it is operably linked to and controlled by the inserted promoter sequence.

[0085] In some embodiments, the promoter 447 can include an inducible promoter, a strong promoter, or a tissue-specific promoter. For example, the heterologous nucleotide sequence encoding the gene of interest 445 can be operably linked to an inducible promoter, a strong promoter, or a tissue-specific promoter. In some embodiments, the promoter 447 can include a constitutive promoter. An inducible promoter can be switched on and off, whereas a constitutive promoter can be always active. For example, the heterologous nucleotide sequence encoding the gene of interest 445 can be operably linked to a ubiquitin promoter (Ubi) or a 35S cauliflower mosaic virus (CMV) promoter.

[0086] A promoter typically includes at least a core (basal) promoter, but may also include at least one control element. Such elements include upstream activation regions (UARs) and other DNA sequences that affect transcription of the nucleic acid, which may optionally include synthetic upstream elements. Factors for selecting a promoter to drive expression of a copy include efficiency, selectivity, inducibility, desired expression level, and cell or tissue type specificity. The promoter 447 may be one that is preferentially expressed in Cannabaceae EA or under certain conditions, such as a tissue-specific promoter. The promoter 447 may be regulated by factors such as temperature, light, or stress. For example, an inducible promoter may be used to drive expression in response to an external stimulus (e.g., exposure to an inducer). Suitable promoters include, but are not limited to, the light-inducible promoter from ssRUBISCO, the MAS promoter, the rice actin promoter, the maize ubiquitin promoter, the PR-I promoter, the CZ19B1 promoter, the mirp promoter, the CesA promoter, the Gama-zein promoter, the Glob-1 promoter, the maize 15 kDa zein promoter, the 22 kDa zein promoter, the 27 kDa zein promoter, the delta-zein promoter, the waxy promoter, the contracted 1 promoter, the contracted 2 promoter, the globulin 1 promoter, the pEMU promoter, the maize H3 histone promoter, the beta estradiol promoter, and the dexamethasone-inducible promoter. Non-limiting examples of constitutive promoters include 35S promoters such as the 35SCMV promoter, the 2x35S promoter, the nopaline synthase (NOS) promoter, and the ubi3 promoter, among others.

[0087] The promoter for driving expression may have strong transcriptional activity. A strong promoter drives expression at high levels, or at about 1 / 10 to about 1 / 100 to about 1 / 1,000 transcripts. Enhancers may be used in combination with the promoter region to increase transcription levels. If the gene of interest 445 is endogenous to the plant species, the expression cassette may be effective to achieve at least a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold increase in expression level compared to the expression level of the endogenous gene of interest in wild-type plant tissue.

[0088] Heterologous nucleotide sequences encoding a gene of interest 445 may include DNA sequences derived from various organisms, including, but not limited to, humans and other mammals and / or vertebrates, invertebrates, plants, sponges, bacteria, fungi, algae, and archaea. Heterologous nucleotide sequences may encode proteins having at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence of the corresponding wild-type gene. In some cases, heterologous nucleotide sequences have significant similarity with sequences encoding proteins and share functional domains. Heterologous nucleotide sequences may be obtained from related organisms that have homologous, orthologous, or paralogous genes with the gene encoding the protein. Methods for identifying conserved or similar heterologous nucleotide sequences and constructing recombinant genes encoding proteins, optionally with various modifications (e.g., codon-optimized sequences) to improve expression, include conventional techniques in molecular biology. For example, PCR amplification or design and synthesis of overlapping complementary synthetic oligonucleotides can be annealed and ligated together to obtain a gene with restriction sites for cloning, or subcloning from another already cloned source, or cloning from a library.

[0089] In some embodiments, the heterologous nucleotide sequence may comprise a sequence of a gene present in a wild-type Cannabaceae plant, or a sequence with a percent identity that allows the function of the gene-encoded product to be preserved, for example, a sequence with at least 90% identity. The sequence may be obtained from an organism or organism part, or may be synthetically produced. The sequence may have at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the gene occurring in the wild-type organism. The sequence may be inserted into a different locus than the wild-type gene and operably linked to a different promoter than the wild-type gene. The bacterial-mediated method of the present disclosure does not depend on a particular expression construct. Any expression construct that can be introduced into a plant cell can be used in the method. The nucleic acid sequence may be part of an expression cassette introduced into the T-DNA region of a plasmid for bacterial-mediated transformation. The T-DNA may be present in a binary vector. The nucleotide sequence may include the DNA sequence of interest and other sequences, such as regulatory sequences for the expression of the DNA sequence of interest. Binary vectors that can be used in the present invention are known to those skilled in the art. Binary vectors typically carry an antibiotic resistance gene to allow selection in bacteria. To increase transfection efficiency, the bacteria can carry the virG gene. The virG can be present on the same plasmid as the heterologous nucleic acid sequence or in a helper plasmid.

[0090] The disclosed method may include constructing an expression cassette and / or expression construct that functions in a Cannabaceae plant cell. The construction may include the selection of various components necessary for introduction and expression by the Cannabaceae plant cell. For example, the expression cassette may be incorporated into a recombinant double-stranded plasmid or vector molecule that includes a promoter that functions in a Cannabaceae plant cell to cause the production of an RNA sequence, (b) a structural DNA sequence that causes the production of an RNA sequence that encodes a desired polypeptide, and (c) a 3' non-translated DNA sequence that functions in a plant cell to cause the addition of polyadenylated nucleotides to the 3' end of the RNA sequence. Methods for preparing plasmids or vectors containing the desired components are well known in the art.

[0091] In some embodiments, the expression cassette also contains sequences encoding screening markers and associated regulatory elements as described, as well as nucleic acid sequences that, when expressed, confer a particular trait. These transduction sequences include genes of agronomic interest (e.g., genes for insect or pest resistance, environmental or stress resistance, herbicide resistance), genes for quality improvement such as nutritional enhancement, or any desired change in plant physiology, growth, development, morphology, or plant products.

[0092] In some embodiments, the expression cassette may include a nucleotide sequence for generating a targeted mutation in the Cannabaceae genome. For example, a sequence encoding a rare-cutting endonuclease or a portion thereof (e.g., a subunit) may be introduced by an expression cassette. The methods provided herein may include transient expression of a programmable RNA-guided endonuclease or a portion thereof (e.g., a subunit). The rare-cutting endonuclease may be a fusion protein comprising a DNA-binding domain and a catalytic domain having cleavage activity. TALE nucleases and ZFNs are examples of fusions of a DNA-binding domain with the catalytic domain of the endonuclease FokI. In other examples, the rare-cutting endonuclease is a meganuclease, such as a wild-type or variant homing endonuclease.

[0093] In some embodiments, the Cannabaceae plants of the present disclosure can be used to produce new plant varieties. In some embodiments, the plants are used to develop new, unique and superior varieties or hybrids with specific traits and / or phenotypes.

[0094] The described method can be used to create transgenic lines. A transgenic line can be crossed with another (non-transformed or transformed) line to create a new transgenic Cannabaceae line. Alternatively, the genetic trait engineered into a particular Cannabaceae cultivar using the above techniques can be transferred to another line using backcrossing techniques. For example, a backcrossing approach can be used to transfer an engineered trait from a public non-elite population inbred line to an elite population inbred line, or from an inbred line that contains a foreign gene in its genome to an inbred line that does not contain the gene. "Crossing" can refer to a simple X and Y crossing, or a process of backcrossing.

[0095] An embodiment of the method provided herein may include removing the transgene to provide a new non-transgenic Cannabaceae plant. For example, genetic techniques can be used to provide progeny of transformed plants with a transgene-induced deletion (e.g., a targeted deletion induced by expression of a transgene expressing a rare-cutting endonuclease) that lacks the transgene. In an exemplary embodiment, progeny plants can be obtained by self-pollinating (selfing) transformed Cannabaceae plants that are heterozygous for the transgene by segregation. Selfing such heterozygous plants provides that the transgene segregates from a subset of the progeny plant population.

[0096] In some embodiments, the transformed Cannabaceae plant contains at least a first transgene and is otherwise capable of expressing all physiological and morphological characteristics of the donor Cannabaceae plant. In other embodiments, the transformed Cannabaceae plant contains cis-gene modifications that alter the expression of one or more genes compared to the Cannabaceae plant when grown under the same conditions, while expressing the traits of the parental strain to the same extent as the parental strain. In other embodiments, the transformed Cannabaceae plant contains one or more targeted mutations (e.g., deletions) that alter the expression of one or more genes of the donor Cannabaceae plant. For example, the transformed plant can exhibit lower levels of THC than the donor Cannabaceae plant as a result of the altered gene expression. In some embodiments, the Cannabaceae plant contains a single locus conversion. The single locus conversion can include a dominant or recessive allele. The locus conversion can confer a trait to the transformed Cannabaceae plant.

[0097] Cannabis plants may contain stacked traits that provide a combined effect resulting from the use of multiple nucleic acid constructs or transformation events. For example, multiple constructs such as those described above can be introduced into Cannabis plant cells by the same or different methods, including the introduction of such traits by including two transcription cassettes in a single transformation vector, co-transformation of two expression constructs, re-transformation of plant tissue expressing one construct with an expression construct for a second gene, or crossing transgenic plants via traditional plant breeding methods, with the resulting product being a plant with both characteristics.

[0098] Plant parts can include products and compositions produced or purified from plants produced by the methods described herein, including stems, fiber, pulp, flowers, seeds, etc. Products produced from Cannabaceae plants include industrial textiles, building materials, food and dietary supplements, personal care products such as soaps, lotions, balms, etc., industrial products such as animal bedding, paints, inks, solvents and lubricants, consumer textiles, animal feed, etc. In some cases, Cannabaceae plants and plant parts are used to provide extracts that can be used as flavoring or fragrance ingredients, or to obtain pharmaceutical compounds derived from Cannabaceae.

[0099] Various terms used herein (including the claims) include their obvious meanings in the art unless otherwise indicated. As used herein, the terms "comprise(s)", "include(s)", "having", "has", "can", "contain(s)", and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The present disclosure also contemplates other embodiments that "comprising", "consisting of", and "consisting essentially of" the embodiments or elements presented herein, whether or not expressly stated. The singular forms "a", "and", and "the" include plural references unless the context clearly dictates otherwise.

[0100] The various ranges provided herein include the stated range and any value or subrange within the stated range. Additionally, when "about" is used to describe a value or percentage, this includes, refers to, and / or encompasses a variation (up to + / - 10%) from the stated value or percentage.

[0101] "Explant" refers to a plant part capable of regenerating by micropropagation. An explant can regenerate a shoot, a root, or the entire plant.

[0102] "Donor plant" refers to the source of the explant. The donor Cannabaceae plant can be any type of Cannabaceae plant. In some cases, the donor plant is a female plant (e.g., not hermaphrodite). In some embodiments, the donor plant is a Cannabaceae genotype that is susceptible to Rhizobium infection and exhibits a desired regeneration response. The donor Cannabaceae plant can be derived from an elite line that has one or more desired traits.

[0103] "Transformation" refers to the transfer of a nucleotide sequence 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.

[0104] An "expression cassette" can refer to a DNA sequence capable of directing the expression of a particular nucleotide sequence in a suitable host cell, comprising a promoter operably linked to a nucleotide sequence of interest, optionally operably linked to termination signals and / or other regulatory elements. An expression cassette can also include sequences necessary for proper translation of the nucleotide sequence. The coding region can code for a protein of interest, but can also code for a functional RNA of interest, such as an antisense RNA or a non-translated RNA, in the sense or antisense orientation. An expression cassette can be chimeric, meaning that at least one of its components is heterologous to at least one of the other components. An expression cassette can be partially assembled using endogenous components. For example, an expression cassette can be obtained by placing (or inserting) a promoter sequence upstream of an endogenous sequence, such that it is operably linked to and controlled by the inserted promoter sequence.

[0105] As used herein, Cannabaceae refers to plants of the Cannabaceae family. For example, Cannabaceae plants or plant parts can include plants or plant parts belonging to the Cannabaceae genus, sometimes referred to as cannabis plants or plant parts, including Cannabis sativa, Cannabis indica, and Cannabis ruderalis. However, embodiments are not so limited, and Cannabaceae plants or plant parts can include plants or plant parts of Humulus (e.g., Hops), Celtis, Alfonante, Chaetachrome, Dironniella, Rosanella, Parasponia, Pteroceltis, and / or Trema, among other plants or plant parts. The term "plant" generally refers to the entire plant, but when used as an adjective, "plant" refers to any material present in, obtained from, derived from, or associated with a plant, such as plant organs (e.g., leaves, stems, roots, flowers), single cells (e.g., pollen), seeds, plant cells, including tissue culture cells, products produced from plants, etc. The term "Cannabaceae plant parts" refers to one or more plant tissues or organs obtained from an entire plant of the Cannabaceae family. Cannabaceae plant parts include vegetative structures (e.g., leaves, stems), roots (e.g., hairy or non-hairy roots), floral organs / structures, seeds (including embryos, endosperm, and seed coats), plant tissues (e.g., vascular tissue, ground tissue, etc.), cells, and their progeny.

[0106] A "cannabaceae plant cell" is a structural and physiological unit of a plant, including a protoplast and a cell wall. A plant cell may be a cell in culture. A cannabaceae plant cell may be in the form of an isolated single cell or an aggregate of cells, such as a friable callus, or a cultured cell, or may be a higher order tissue unit, such as a cannabaceae plant tissue, a plant organ, or a part of a plant. A cannabaceae plant cell may be a protoplast, a gamete-producing cell, or a cell or a collection of cells that can regenerate into a whole plant. By "cannabaceae plant tissue" is meant differentiated tissue in a plant, or differentiated tissue obtained from a plant ("explant"), or undifferentiated tissue derived from various forms of aggregates of plant cells in culture, such as immature or mature embryos, seeds, roots, shoots, fruits, pollen, and callus. Plant tissue in or from seeds, such as cannabaceae seeds, includes the seed coat or testa, storage cotyledons, and embryos.

[0107] "Meristem" or "meristem region" refers to plant tissue that contains undifferentiated cells (meristem cells) found in the area of ​​a plant where growth can occur. Meristem cells give rise to plant organs and maintain plant growth.

[0108] The term "variety" or "cultivar" refers to a population of plants that share characteristics that separate them from other plants of the same species. Although having one or more distinctive traits, varieties can be further characterized by minor overall variations among individuals within the variety. "Pure line" varieties can be produced by several generations of self-pollination and selection, or vegetative propagation from a single parent, using tissue or cell culture techniques. A variety may be essentially derived from another line or variety. A variety is "essentially derived" from an earlier variety if (a) it is derived primarily from an earlier variety, or is derived primarily from an earlier variety, but retains the expression of characteristics resulting from the genotype or combination of genotypes of the earlier variety, (b) it is distinguishable from the earlier variety, and (c) it matches the earlier variety in the expression of characteristics resulting from the genotype or combination of genotypes of the earlier variety, except for differences resulting from the act of derivation. Essentially derived varieties can be obtained by selection of natural or induced mutants, somatic variants, variant individuals from plants of the earlier variety, backcrossing, or transformation. A "line", as distinguished from a variety, may denote a group of plants used non-commercially, often for botanical research, etc. Lines typically show little variation among individuals for one or more traits of interest, but may have some variation among individuals for other traits.

[0109] "Transformation frequency" refers to the percentage of plant cells that are successfully transformed with a heterologous nucleotide sequence after performing a transformation protocol on the cells to introduce the nucleic acid. Increasing "conversion efficiency" refers to improvements such as increased conversion frequency and quality events that affect the overall efficiency of the conversion process by reducing resource usage.

[0110] "Regeneration" refers to a morphogenetic response that results in the production of new tissues, organs, embryos, whole plants or parts of whole plants from a single cell or group of cells. In the disclosed method, regeneration proceeds from meristematic regions or EAs. "Regeneration potential" refers to the ability of a plant cell to undergo regeneration. "Regeneration efficiency" can be calculated from the number of plantlets regenerated from embryogenic callus or individual explants.

[0111] The term "gene of interest" or "trait of interest" corresponds to a gene or trait that is expressed by a Cannabaceae cell that has been transformed using the techniques described herein.

[0112] Various embodiments are made in accordance with the underlying provisional application, U.S. Provisional Application No. 63 / 245,301, filed September 17, 2021, and entitled "Transformed Cannabaceae Cells and Methods Thereof," to which the benefit is claimed and which is hereby fully incorporated by reference in its entirety for the teachings thereof.

[0113] Experimental Embodiments Various experimental embodiments are directed to transforming Cannabaceae cells from Cannabaceae meristem regions or EAs. Such embodiments include preparing Cannabaceae meristem regions and / or EAs, transforming Cannabaceae cells of the Cannabaceae family, and regenerating tissues for the transformed Cannabaceae cells. Some embodiments are directed to providing a reproducible, reliable, and simple transformation and regeneration system from Cannabaceae meristem regions and / or EAs.

[0114] 5A-5I show exemplary expression constructs for transforming Cannabaceae cells from the Cannabaceae meristem region or EA consistent with the present disclosure. In some experimental embodiments, expression constructs were used in bacterial strain transformation (e.g., A. rhizogenes transformation) to include right and left T-DNA border sequences, allowing the bacterial strain to deliver DNA to Cannabaceae plant cells. The expression constructs are plasmids and can be referred to as plasmid vectors. The expression constructs further include a DNA sequence encoding a transgene that is codon-optimized according to the codon bias used by the target, cloned into a vector, and under the control of a promoter and a terminator. Constitutive and root-specific promoters are selected for tissue-specific approaches.

[0115] FIG. 5A shows an exemplary plasmid vector 550, sometimes referred to herein as a "betalain vector," which includes DNA sequences encoding at least one enzyme associated with the production of betalains. Plasmid vector 550 includes genes encoding the enzymes CYP76AD1, DODA, and glucosyltransferase driven by an FMV promoter, where CYP76AD1 is linked to DODA, and DODA is linked to glucosyltransferase by a 2A self-cleaving peptide P2A, sometimes referred to herein as a "betalain cassette." Plasmid vector 550 further includes a plant selectable marker cassette and a LacZ cassette, which are in inverted orientation on the plasmid, as described further below. The plant selectable marker cassette encodes a selectable marker that, when expressed, confers resistance to a selection agent (e.g., bacteria or other toxic substances), a promoter, and a terminator for selection of transformed plant cells. The LacZ cassette encodes the LacZ gene and the LacZ promoter used as a selectable marker. The gene cassette is flanked by left border (LB) and right border (RB) T-DNA sequences, allowing the transfer of the entire sequence or transgenes into cannabis plant cells by bacterial strains of A. rhizogenes, such as 18R12. The plasmid backbone also contains a bacterial selection marker cassette that is in the opposite orientation on the plasmid, encoding a kanamycin resistance (KanR) gene for selection and maintenance of the plasmid in A. rhizogenes strains. The 18R12 strain was used to transform the cannabis plant parts. The sequence of the plasmid vector 550 is shown in SEQ ID NO:1. Further identified are the sequences of the betalain cassette (SEQ ID NO:2), the plant selection marker cassette (SEQ ID NO:10), the T-DNA borders of RB (SEQ ID NO:15) and LB (SEQ ID NO:16), the LacZ cassette (SEQ ID NO:17) and the bacterial selection marker cassette (SEQ ID NO:20). The betalain cassette (SEQ ID NO:2) encodes the FMV promoter (SEQ ID NO:3), CYP76AD1 (SEQ ID NO:4), P2A1 (SEQ ID NO:5), DODA (SEQ ID NO:6), P2A2 (SEQ ID NO:7), glucosyltransferase (SEQ ID NO:8) and the rbcS terminator (SEQ ID NO:9).The plant selection marker cassette (SEQ ID NO:10) encodes the VaUbi3 promoter (SEQ ID NO:11), chloroplast transit peptide (SEQ ID NO:12), SpcN (SEQ ID NO:13) and Nos terminator (SEQ ID NO:14). The LacZ cassette (SEQ ID NO:17) encodes the LacZ promoter (SEQ ID NO:18) and LacZ gene (SEQ ID NO:19). The bacterial selection marker cassette (SEQ ID NO:20) encodes the KanR promoter (SEQ ID NO:21) and KanR gene (SEQ ID NO:22).

[0116] FIG. 5B shows an exemplary plasmid vector 560 containing a DNA sequence encoding a TALEN associated with a low-THC transgene, sometimes referred to herein as a "low-THC TALEN vector." The plasmid vector 560 contains genes encoding a left half TALEN and a right half TALEN associated with a transgene that reduces the THC content in a transformed Cannabaceae plant or plant part, hereafter referred to as the left TALEN cassette and the right TALEN cassette. The plasmid vector 560 contains a YFP reporter cassette encoding a YFP used to select transformed plant parts. The plasmid vector 560 further contains a plant selectable marker cassette in the reverse orientation on the plasmid as described above, and the features are not repeated. The plasmid backbone also contains a bacterial selectable marker cassette in the reverse orientation on the plasmid, encoding a KanR gene for selection and maintenance of the plasmid in the A. rhizogenes strain. The Cannabaceae plant part was transformed using the 18R12 strain. The sequence of the plasmid vector 560 is shown in SEQ ID NO: 23. Further identified are the sequences of the left TALEN cassette (SEQ ID NO:24), the right TALEN cassette (SEQ ID NO:32), the YFP reporter cassette (SEQ ID NO:36), the plant selection marker cassette (SEQ ID NO:38) and the bacterial selection marker cassette (SEQ ID NO:20). The left TALEN cassette (SEQ ID NO:24) encodes the Nos promoter (SEQ ID NO:25), the left TAL effector N-terminus (SEQ ID NO:26), the CsTHCAS_T22-L1 binding domain (SEQ ID NO:27), the left TAL effector C-terminus (SEQ ID NO:28), the linker (SEQ ID NO:29), Fok1 (SEQ ID NO:30) and the Nos terminator (SEQ ID NO:31). The right TALEN cassette (sequence number 32) encodes the Nos promoter (sequence number 25), the right TAL effector N-terminus (sequence number 33), the CsTHCAS_T22-R1 binding domain (sequence number 34), the right TAL effector C-terminus (sequence number 35), a linker (sequence number 29), Fok1 (sequence number 30) and a Nos terminator (sequence number 31).The YFP reporter cassette (SEQ ID NO:36) encodes the FMV promoter (SEQ ID NO:3), the YFP CDS (SEQ ID NO:37) and the Rbcs-E9 terminator (SEQ ID NO:9). The plant selection marker cassette (SEQ ID NO:38) encodes the 35S promoter (SEQ ID NO:39), the STLS1nptII intron (SEQ ID NO:40), the NptII exon (SEQ ID NO:41) and the 35S terminator (SEQ ID NO:42). The bacterial selection marker cassette (SEQ ID NO:20) encodes the KanR promoter (SEQ ID NO:21) and the KanR gene (SEQ ID NO:22).

[0117] FIG. 5C shows an exemplary plasmid vector 570 comprising a DNA sequence encoding a TALEN associated with a low-THC transgene, sometimes referred to herein as a "low-THC-specific YFP vector." The plasmid vector 570 contains genes encoding a left half TALEN and a right half TALEN associated with a transgene that reduces the THC content in a transformed Cannabaceae plant or plant part, hereafter referred to as the left TALEN cassette and the right TALEN cassette. The plasmid vector 570 contains a YFP reporter cassette encoding a YFP used to select transformed plant parts. The plasmid vector 570 further comprises a plant selectable marker cassette in the reverse orientation on the plasmid as described above, and the features are not repeated. The plasmid backbone also contains a bacterial selectable marker cassette in the reverse orientation on the plasmid, encoding a KanR gene for selection and maintenance of the plasmid in the A. rhizogenes strain. The Cannabaceae plant part was transformed using the 18R12 strain. The sequence of the plasmid vector 570 is shown in SEQ ID NO: 43. Further identified are the sequences of the left TALEN cassette (SEQ ID NO: 44), the right TALEN cassette (SEQ ID NO: 48), the YFP reporter cassette (SEQ ID NO: 36), the plant selection marker cassette (SEQ ID NO: 10) and the bacterial selection marker cassette (SEQ ID NO: 20). The left TALEN cassette (SEQ ID NO: 44) encodes the VaUbi3 promoter (SEQ ID NO: 11), the left TAL effector N-terminus (SEQ ID NO: 45), the CsTHCAS_T22-L1 binding domain (SEQ ID NO: 46), the left TAL effector C-terminus (SEQ ID NO: 47), the linker (SEQ ID NO: 29), Fok1 (SEQ ID NO: 30) and the Nos terminator (SEQ ID NO: 31). The right TALEN cassette (sequence number 48) encodes the VaUbi3 promoter (sequence number 11), the right TAL effector N-terminus (sequence number 49), the CsTHCAS_T22-R1 binding domain (sequence number 50), the right TAL effector C-terminus (sequence number 51), a linker (sequence number 29), Fok1 (sequence number 30) and a Nos terminator (sequence number 31).The YFP reporter cassette (SEQ ID NO:36) encodes the FMV promoter (SEQ ID NO:3), the YFP CDS (SEQ ID NO:37) and the Rbcs-E9 terminator (SEQ ID NO:9). The plant selection marker cassette (SEQ ID NO:10) encodes the VaUbi3 promoter (SEQ ID NO:11), the chloroplast transit peptide (SEQ ID NO:12), SpcN (SEQ ID NO:13) and the Nos terminator (SEQ ID NO:14). The bacterial selection marker cassette (SEQ ID NO:20) encodes the KanR promoter (SEQ ID NO:21) and the KanR gene (SEQ ID NO:22).

[0118] FIG. 5D shows an exemplary plasmid vector 572 comprising a DNA sequence encoding a TALEN associated with a phytoene desaturase (PDS) transgene. Plasmid vector 572 encodes a left half TAL effector with a PDS binding domain fused to SSP DnaE intein-N and a right half TAL effector with a PDS binding domain fused to SSP intein-N, hereinafter referred to as the left TALE intein cassette and the right TALE intein cassette. The plasmid vector further encodes a Fok1 endonuclease fused to SSP DnaE intein-C, hereinafter referred to as the endonuclease cassette. Plasmid vector 572 contains a YFP reporter cassette, as described above, which is in a reverse orientation on the plasmid. The plasmid backbone also encodes a KanR gene and contains a bacterial selection marker cassette, which is in a reverse orientation on the plasmid. The sequence of plasmid vector 572 is shown in SEQ ID NO:52. Further identified are the sequences of the Left TALE Intein Cassette (SEQ ID NO:57), the Right TALE Intein Cassette (SEQ ID NO:64), the YFP Reporter Cassette (SEQ ID NO:53), the Endonuclease Cassette (SEQ ID NO:66), the Bacterial Selection Marker Cassette (SEQ ID NO:74), the Right T-DNA Border (SEQ ID NO:72), and the Left T-DNA Border (SEQ ID NO:73). The Left TALE Intein Cassette (SEQ ID NO:57) encodes the VaUbi3 Promoter (SEQ ID NO:58), the N-Terminus (SEQ ID NO:59), the PDS Left Binding Domain (SEQ ID NO:60), Feature 10 (SEQ ID NO:61), Intein-N (SEQ ID NO:62) and the Nos Terminator (SEQ ID NO:63). Feature 10 is part of the C40 domain of the Yeast_1NLS_HAtag_N152_C40 sequence, which does not encode the Fok1 endonuclease. For example, in the Yeast_1NLS_HAtag_N152_C40 sequence containing a repeating variable di-residue (RVD), Fok1 is removed and replaced with an intein, such as Gp41-1int-N or SSP DnaE intein-N. The N-terminus contains the N-terminal domain of Yeast_1NLS_HAtag_N152_C40.The right TALE intein cassette (SEQ ID NO:64) encodes the VaUbi3 promoter (SEQ ID NO:58), N-terminus (SEQ ID NO:59), PDS right binding domain (SEQ ID NO:65), feature 10 (SEQ ID NO:61), intein-N (SEQ ID NO:62) and Nos terminator (SEQ ID NO:63). The endonuclease cassette (SEQ ID NO:66) encodes the pMtEF1A promoter (SEQ ID NO:67), feature 10 (SEQ ID NO:68), intein-C CDS (SEQ ID NO:69), Fok1 (SEQ ID NO:70), yeast_1NLS_HAtag_N152_C40 (SEQ ID NO:71) and Nos terminator (SEQ ID NO:63). Yeast_1NLS_HAtag_N152_C40 is a small protein containing a nuclear localization signal (NLS) into which a half TALEN has been inserted between the N-terminal and C-terminal domains (e.g., at the N-terminus) of the protein. The YFP reporter cassette (SEQ ID NO:53) encodes the FMV promoter (SEQ ID NO:54), the YFP CDS (SEQ ID NO:55) and the Rbcs-E9 terminator (SEQ ID NO:56). The bacterial selection marker cassette (SEQ ID NO:74) encodes the KanR promoter (SEQ ID NO:75) and the KanR gene (SEQ ID NO:76).

[0119] FIG. 5E shows an exemplary plasmid vector 574 containing a DNA sequence encoding a TALEN associated with a PDS transgene. Plasmid vector 574 encodes a left half TAL effector with a PDS binding domain fused to Gp41-1 intein-N and a right half TAL effector with a PDS binding domain fused to Gp41-1 intein-N, hereafter referred to as the left TALE intein cassette and the right TALE intein cassette. The plasmid vector further encodes a Fok1 endonuclease fused to Gp41-1 intein-C, hereafter referred to as the endonuclease cassette. Plasmid vector 574 contains a YFP reporter cassette that is in the reverse orientation on the plasmid as described above, and the features are not repeated. The plasmid backbone also contains a bacterial selection marker cassette that is in the reverse orientation on the plasmid. The sequence of plasmid vector 574 is shown in SEQ ID NO: 77. Further identified are the sequences of the Left TALE Intein Cassette (SEQ ID NO: 78), the Right TALE Intein Cassette (SEQ ID NO: 81), the YFP Reporter Cassette (SEQ ID NO: 53), the Endonuclease Cassette (SEQ ID NO: 82), the Bacterial Selection Marker Cassette (SEQ ID NO: 74), the Right T-DNA Border (SEQ ID NO: 72), and the Left T-DNA Border (SEQ ID NO: 73). The Left TALE Intein Cassette (SEQ ID NO: 78) encodes the VaUbi3 promoter (SEQ ID NO: 58), the N-terminus (SEQ ID NO: 59), the PDS left binding domain (SEQ ID NO: 60), feature 10 (SEQ ID NO: 79), the Intein-N (SEQ ID NO: 80), and the Nos terminator (SEQ ID NO: 63). The right TALE intein cassette (SEQ ID NO: 81) encodes the VaUbi3 promoter (SEQ ID NO: 58), N-terminus (SEQ ID NO: 59), PDS right binding domain (SEQ ID NO: 65), feature 10 (SEQ ID NO: 79), intein-N (SEQ ID NO: 80) and Nos terminator (SEQ ID NO: 63). The endonuclease cassette (SEQ ID NO: 82) encodes the pMtEF1A promoter (SEQ ID NO: 67), feature 10 (SEQ ID NO: 83), intein-C CDS (SEQ ID NO: 84), Fok1 (SEQ ID NO: 70), yeast_1NLS_HAtag_N152_C40 (SEQ ID NO: 71) and Nos terminator (SEQ ID NO: 63).The YFP reporter cassette (SEQ ID NO:53) encodes the FMV promoter (SEQ ID NO:54), the YFP CDS (SEQ ID NO:55) and the Rbcs-E9 terminator (SEQ ID NO:56). The bacterial selection marker cassette (SEQ ID NO:74) encodes the KanR promoter (SEQ ID NO:75) and the KanR gene (SEQ ID NO:76).

[0120] FIG. 5F shows an exemplary plasmid vector 576 containing a DNA sequence encoding a TALEN associated with the THCAS transgene. Plasmid vector 576 encodes a left half TAL effector with a THCAS binding domain fused to SSP DnaE intein-N and a right half TAL effector with a THCAS binding domain fused to SSP intein-N, hereafter referred to as the left TALE intein cassette and the right TALE intein cassette. The plasmid vector further encodes a Fok1 endonuclease fused to SSP DnaE intein-C, hereafter referred to as the endonuclease cassette. Plasmid vector 576 contains a YFP reporter cassette that is in the reverse orientation on the plasmid as described above, and the features are not repeated. The plasmid backbone also contains a bacterial selection marker cassette that is in the reverse orientation on the plasmid. The sequence of plasmid vector 576 is shown in SEQ ID NO:85. Further identified are the sequences of the Left TALE Intein Cassette (SEQ ID NO: 86), the Right TALE Intein Cassette (SEQ ID NO: 90), the YFP Reporter Cassette (SEQ ID NO: 53), the Endonuclease Cassette (SEQ ID NO: 66), the Bacterial Selection Marker Cassette (SEQ ID NO: 74), the Right T-DNA Border (SEQ ID NO: 72), and the Left T-DNA Border (SEQ ID NO: 73). The Left TALE Intein Cassette (SEQ ID NO: 86) encodes the VaUbi3 promoter (SEQ ID NO: 58), the N-terminus (SEQ ID NO: 59), the THCAS left binding domain (SEQ ID NO: 87), feature 10 (SEQ ID NO: 88), Intein-N (SEQ ID NO: 89), and the Nos terminator (SEQ ID NO: 63). The right TALE intein cassette (SEQ ID NO:90) encodes the VaUbi3 promoter (SEQ ID NO:58), N-terminus (SEQ ID NO:59), THCAS right binding domain (SEQ ID NO:91), feature 10 (SEQ ID NO:88), intein-N (SEQ ID NO:89) and Nos terminator (SEQ ID NO:63). The endonuclease cassette (SEQ ID NO:66) encodes the pMtEF1A promoter (SEQ ID NO:67), feature 10 (SEQ ID NO:68), intein-C CDS (SEQ ID NO:69), Fok1 (SEQ ID NO:70), yeast_1NLS_HAtag_N152_C40 (SEQ ID NO:71) and Nos terminator (SEQ ID NO:63).The YFP reporter cassette (SEQ ID NO:53) encodes the FMV promoter (SEQ ID NO:54), the YFP CDS (SEQ ID NO:55) and the Rbcs-E9 terminator (SEQ ID NO:56). The bacterial selection marker cassette (SEQ ID NO:74) encodes the KanR promoter (SEQ ID NO:75) and the KanR gene (SEQ ID NO:76).

[0121] FIG. 5G shows an exemplary plasmid vector 578 containing a DNA sequence encoding a TALEN associated with the THCAS transgene. Plasmid vector 578 encodes a left half TAL effector with a THCAS binding domain fused to Gp41-1 intein-N and a right half TAL effector with a THCAS binding domain fused to Gp41-1 intein-N, hereafter referred to as the left TALE intein cassette and the right TALE intein cassette. The plasmid vector further encodes a Fok1 endonuclease fused to Gp41-1 intein-C, hereafter referred to as the endonuclease cassette. Plasmid vector 576 contains a YFP reporter cassette in the reverse orientation on the plasmid as described above, and the features are not repeated. The plasmid backbone also contains a bacterial selection marker cassette in the reverse orientation on the plasmid. The sequence of plasmid vector 576 is shown in SEQ ID NO:92. Further identified are the sequences of the Left TALE Intein Cassette (SEQ ID NO: 93), the Right TALE Intein Cassette (SEQ ID NO: 97), the YFP Reporter Cassette (SEQ ID NO: 53), the Endonuclease Cassette (SEQ ID NO: 82), the Bacterial Selection Marker Cassette (SEQ ID NO: 74), the Right T-DNA Border (SEQ ID NO: 72), and the Left T-DNA Border (SEQ ID NO: 73). The Left TALE Intein Cassette (SEQ ID NO: 93) encodes the VaUbi3 promoter (SEQ ID NO: 58), the N-terminus (SEQ ID NO: 59), the THCAS left binding domain (SEQ ID NO: 94), feature 10 (SEQ ID NO: 95), Intein-N (SEQ ID NO: 96), and the Nos terminator (SEQ ID NO: 63). The right TALE intein cassette (SEQ ID NO:97) encodes the VaUbi3 promoter (SEQ ID NO:58), N-terminus (SEQ ID NO:59), THCAS right binding domain (SEQ ID NO:98), feature 10 (SEQ ID NO:95), intein-N (SEQ ID NO:96) and Nos terminator (SEQ ID NO:63). The endonuclease cassette (SEQ ID NO:82) encodes the pMtEF1A promoter (SEQ ID NO:67), feature 10 (SEQ ID NO:83), intein-C CDS (SEQ ID NO:84), Fok1 (SEQ ID NO:70), yeast_1NLS_HAtag_N152_C40 (SEQ ID NO:71) and Nos terminator (SEQ ID NO:63).The YFP reporter cassette (SEQ ID NO:53) encodes the FMV promoter (SEQ ID NO:54), the YFP CDS (SEQ ID NO:55) and the Rbcs-E9 terminator (SEQ ID NO:56). The bacterial selection marker cassette (SEQ ID NO:74) encodes the KanR promoter (SEQ ID NO:75) and the KanR gene (SEQ ID NO:76).

[0122] FIG. 5H shows an exemplary plasmid vector 580 containing a DNA sequence encoding a TALEN associated with a PDS transgene. Plasmid vector 580 encodes a left half TAL effector with a PDS binding domain fused to Gp41 intein-N and a right half TAL effector with a PDS binding domain fused to Gp41-1 intein-N, hereafter referred to as the left TALE intein cassette and the right TALE intein cassette. Plasmid vector further encodes a Fok1 endonuclease fused to Gp41-1 intein-C, hereafter referred to as the endonuclease cassette. Plasmid vector 580 contains a YFP reporter cassette that is in the reverse orientation on the plasmid as described above, and the features are not repeated. The plasmid backbone also contains a bacterial selection marker cassette that is in the reverse orientation on the plasmid. The sequence of plasmid vector 580 is shown in SEQ ID NO:99. Further identified are the sequences of the Left TALE Intein Cassette (SEQ ID NO: 100), the Right TALE Intein Cassette (SEQ ID NO: 104), the YFP Reporter Cassette (SEQ ID NO: 53), the Endonuclease Cassette (SEQ ID NO: 106), the Bacterial Selection Marker Cassette (SEQ ID NO: 74), the Right T-DNA Border (SEQ ID NO: 72), and the Left T-DNA Border (SEQ ID NO: 73). The Left TALE Intein Cassette (SEQ ID NO: 100) encodes the VaUbi3 promoter (SEQ ID NO: 58), the N-terminus (SEQ ID NO: 59), the PDS left binding domain (SEQ ID NO: 101), feature 10 (SEQ ID NO: 102), Intein-N (SEQ ID NO: 103), and the Nos terminator (SEQ ID NO: 63). The right TALE intein cassette (SEQ ID NO:104) encodes the VaUbi3 promoter (SEQ ID NO:58), N-terminus (SEQ ID NO:59), PDS right binding domain (SEQ ID NO:105), feature 10 (SEQ ID NO:102), intein-N (SEQ ID NO:103) and Nos terminator (SEQ ID NO:63). The endonuclease cassette (SEQ ID NO:106) encodes the pMtEF1A promoter (SEQ ID NO:67), feature 10 (SEQ ID NO:107), intein-C CDS (SEQ ID NO:84), Fok1 (SEQ ID NO:70), yeast_1NLS_HAtag_N152_C40 (SEQ ID NO:71) and Nos terminator (SEQ ID NO:63).The YFP reporter cassette (SEQ ID NO:53) encodes the FMV promoter (SEQ ID NO:54), the YFP CDS (SEQ ID NO:55) and the Rbcs-E9 terminator (SEQ ID NO:56). The bacterial selection marker cassette (SEQ ID NO:74) encodes the KanR promoter (SEQ ID NO:75) and the KanR gene (SEQ ID NO:76).

[0123] FIG. 5I shows an exemplary plasmid vector 582 containing a DNA sequence encoding a TALEN associated with a THCAS transgene. Plasmid vector 582 contains genes encoding a left half TAL effector with a THCAS binding domain fused to Gp41-1 intein-N and a right half TAL effector with a THCAS binding domain fused to Gp41-1 intein-N, hereinafter referred to as the left TALE intein cassette and the right TALE intein cassette. The plasmid vector further encodes a Fok1 endonuclease fused to Gp41-1 intein-C, hereinafter referred to as the endonuclease cassette. Plasmid vector 582 contains a YFP reporter cassette in the reverse orientation on the plasmid as described above, and the features are not repeated. The plasmid backbone also contains a bacterial selection marker cassette in the reverse orientation on the plasmid. The sequence of plasmid vector 582 is shown in SEQ ID NO: 108. Further identified are the sequences of the Left TALE Intein Cassette (SEQ ID NO: 109), the Right TALE Intein Cassette (SEQ ID NO: 112), the YFP Reporter Cassette (SEQ ID NO: 53), the Endonuclease Cassette (SEQ ID NO: 114), the Bacterial Selection Marker Cassette (SEQ ID NO: 74), the Right T-DNA Border (SEQ ID NO: 72), and the Left T-DNA Border (SEQ ID NO: 73). The Left TALE Intein Cassette (SEQ ID NO: 109) encodes the VaUbi3 promoter (SEQ ID NO: 58), the N-terminus (SEQ ID NO: 59), the THCAS left binding domain (SEQ ID NO: 110), feature 10 (SEQ ID NO: 111), Intein-N (SEQ ID NO: 103), and the Nos terminator (SEQ ID NO: 63). The right TALE intein cassette (sequence number 112) encodes the VaUbi3 promoter (sequence number 58), N-terminus (sequence number 59), THCAS right binding domain (sequence number 113), feature 10 (sequence number 111), intein-N (sequence number 103) and Nos terminator (sequence number 63).The endonuclease cassette (SEQ ID NO:114) encodes the pMtEF1A promoter (SEQ ID NO:67), feature 10 (SEQ ID NO:115), intein-C CDS (SEQ ID NO:84), Fok1 (SEQ ID NO:70), yeast_1NLS_HAtag_N152_C40 (SEQ ID NO:71), and Nos terminator (SEQ ID NO:63). The YFP reporter cassette (SEQ ID NO:53) encodes the FMV promoter (SEQ ID NO:54), YFP CDS (SEQ ID NO:55), and Rbcs-E9 terminator (SEQ ID NO:56). The bacterial selection marker cassette (SEQ ID NO:74) encodes the KanR promoter (SEQ ID NO:75) and KanR gene (SEQ ID NO:76).

[0124] Some embodiments relate to preparing starter cultures of bacterial strains and preparing bacterial culture media containing the bacterial strains. The bacterial strains included A. rhizogenes bacterial strain (18r12). The A. rhizogenes strain was streaked onto a plate on AB+Kan50 medium. A single colony was inoculated into 15 mL of YEP culture + 7.5 uL of Kan50, all in a 50 mL vented conical tube. In some experiments, a second culture is inoculated with another single colony as a backup. The cultures were placed at an angle on a shaker (220 rpm) at 28° C. for approximately 8 hours. OD STARTER Ideally, the optical density (OD) should be between 0.2 and 0.4. 600 A 250 mL flask was prepared with 49 mL of liquid AB minimal culture medium, 1 mL of YEP starter culture, and 25 uL of Kan50 and grown for 20 hours at 28° C. on a shaker (220 rpm). As previously described, bacterial strains can be transformed to carry heterologous nucleotide sequences.

[0125] Cannabaceae seeds were prepared by sterilizing and soaking the seeds. The following steps were performed in a fume hood: 1) A 50 mL tube containing approximately 100 seeds was taken and 10 mL of 12 M sulfuric acid was added using a serological pipette. 2) The 50 mL tube was closed and gently shaken for 10 seconds. 3) The 50 mL tube was opened and the sulfuric acid was removed using a serological pipette. 4) The sulfuric acid waste was removed by dissolving in 400 mL of ddH2O. 25) To rinse, add 45 ml of sterile ddHO. 2 6) The 50 ml tube was closed and gently shaken for 10 seconds. 7) Using a serological pipette, ddHO was added from the 50 ml tube. 2 8) To rinse, add 45 mL of fresh ddHO. 2 ddHO was added to the 50 mL tube, the tube was closed, and the tube was brought to a laminar flow hood. 2 Remove the 50 mL tube and add 45 mL of 30% HO. 2 O 2 The 50 mL tube was closed and placed on a rotating shaker at 20 rpm for 10-20 min. After 10-20 min, the 50 mL tube was removed from the rotating shaker and placed back in the laminar flow hood. Using a serological pipette, remove H from the 50 mL tube. 2 O 2 The seeds were then added to the 50 mL tube containing ddHO. 2 The O rinse was performed five times (5 times). The fifth ddH 2 After O-rinsing, the sterilized seeds were poured out onto a plate containing solid MS medium and 8 mL of 2% v / v PPM was added directly to the plate and placed on a shaker set at 70 rpm overnight. This was used to avoid over-soaking and PPM to eliminate endophytic contamination.

[0126] In some experimental embodiments, starter cultures of bacterial strains, seed soaks were performed on day 1.

[0127] Starter cultures were prepared for infection. The OD, bottles, and three 50 mL flasks of starter culture were centrifuged at 5400 rpm for 10 minutes. The supernatant was removed and 150 mL of TDZ infection medium was added to resuspend the pellet. Infection medium was prepared by adding 75 uL of 40 mg / mL acetosyringone for a final [acetosyringone] = 100 uM. 0.02% v / v of Silwet L-77 was added to the resuspended bacteria in infection medium.

[0128] The soaked seeds were used to extract Cannabaceae meristem regions and / or EAs. For example, 20 mL of TDZ infection medium was added to a 100×25 mm Petri dish to form an infection plate, and the soaked seeds were left on the plate with solid MS medium containing 2% v / v PPM. A sufficient number of infection plates were used to provide approximately 75 Cannabaceae meristem regions and / or EAs per plate, and one infection plate per soaked seed plate. Sterile forceps were used to gently remove the cotyledons, primary leaves, and seed coats from each EA. Once the seed coats, cotyledons, and primary leaves were removed, the EAs were placed into a Petri dish containing 20 mL of TDZ infection medium before being inoculated with bacteria (such as 18r12).

[0129] Bacterial strains cultured and suspended in TDZ infection medium or similar are sonicated and inoculated into submerged EA and / or meristem regions. For example, TDZ infection medium was pipetted off of the infection plates and 10 mL of resuspended bacteria was added. The infection plates were parafilmed and sonicated one by one for 80 seconds. After sonication, 20 mL of fresh bacteria was added. The plates were then incubated at room temperature for 30 minutes in a laminar flow hood.

[0130] After sonication and inoculation, the meristematic regions and / or EA were co-cultured with the bacterial strains. For example, the remaining bacteria were pipetted from the infection plate. The meristematic regions and / or EA were then transferred to 750 uL of sterile ddHO, sometimes referred to herein as the "co-culture plate." 2 Transfer the plates to a new 100 × 15 mm Petri dish containing a piece of sterile filter paper moistened with 5% H2O. This facilitated drying of excess bacteria. Collect the EA from the infected plate (e.g., around 75% total) into a pile using curved jaw forceps and transfer the pile of EA, with the EA in the pile still attached together, into sterile ddHO. 2 The cells were then transferred to the prepared co-culture plate with sterile filter paper moistened with O. The co-culture plate was wrapped in a layer of parafilm and incubated at 23 °C, 40% humidity and ambient light (0 umol / m -2 / s -2 ) for 2 to 4 days.

[0131] In other experiments, co-culture medium was used. For example, single meristem regions and / or EAs were transferred to co-culture plates with co-culture medium on top of them by gently picking up one meristem region and / or EA at a time and plating approximately 10 EAs by spreading them one by one on each co-culture plate. The co-culture plates were wrapped in a layer of parafilm and incubated at 23 °C, 40% humidity for 16 / 8 h in ambient light (30 μmol / m -2 / s -1 ) for 2 to 4 days.

[0132] In some experimental embodiments, preparation of infection medium, extraction of Cannabaceae meristem regions and / or EA, sonication and inoculation, and co-cultivation were performed or at least started on day 2.

[0133] After co-cultivation, the meristematic regions and / or EAs were transferred to Cannabaceae Liquid (CL) medium as a recovery step to help reduce or eliminate contamination, such as CL medium disclosed below. For example, the meristematic regions and / or EAs were transferred to CL medium in 100 x 25 mm Petri dishes and sealed with parafilm. In some experiments, approximately 50-100 EAs per plate were cultured in a Conviron incubator at 23°C and 40% humidity, with 50-100 umol / m -2 / s -1 The plates were plated at 16 / 8 h light for 3 days.

[0134] After the recovery step, the meristem regions and / or EA are transferred to SIM containing TDZ in the range of 2-10 mg / L. The EA are oriented with the rootlet facing down and / or the meristem regions are oriented with the SIM and apical meristem facing up. In some embodiments, the EA are immersed in solid SIM. The bottom of a second sterile 100 x 25 mm plate is used as a lid and sealed with micropore tape. In some embodiments, the EA are transferred to SIM containing TDZ in the range of 2-10 mg / L. 2 The plates were incubated at room temperature (23±1°C) under white fluorescent light at 1000 Hz for 18 days.

[0135] After shoot induction, the first shoot elongation was performed. For example, the meristem region and / or EA were transferred to a first SEM, such as SEMI+S100 medium. The rootlet was cut and the EA was oriented with the rootlet lowered into the first SEM medium, apical meristem up. The bottom of a second sterile 100×25 mm plate was used as a lid and sealed with micropore tape. In some embodiments, EA was incubated at 100 μmol / m 2 Five EAs per plate were cultured for 21 days under white fluorescent light per second.

[0136] After the first shoot elongation, a second shoot elongation was performed. For example, the meristem region and / or EA were then transferred to a second SEM, such as SEMI+S150 medium. The EA was oriented with the rootlet lowered and the apical meristem up in the second SEM medium. The bottom of a second sterile 100×25 mm plate was used as a lid and sealed with micropore tape. In some embodiments, the EA was incubated at 100 μmol / m 2 Five EAs per plate were cultured for 21 days under white fluorescent light per second.

[0137] After the second shoot elongation, the elongated shoots were rooted. Positive looking shoots (preferably 2 inch tall, minimum 1 inch tall shoots with unbleached leaves) were cut and rooted on Cannabaceae RM DKW+0.5IBA medium in a phytotray under LEDs for approximately 14 days until the shoots developed at least two primary roots that were at least 1 cm long. Shoots were subcultured on fresh RM approximately every 7-14 days until new primary root and root hair structures developed and before being sent for acclimation in soil.

[0138] To make AB salt (20X), the following protocol and volumes were used: 700 mL ddH 2 O; 20 g NH 4 Cl; 6 g MgSO 4 *7H 2 HO; 3 g KCl; 0.2 g CaCl 2 ; 50 mg FeSO 4 *7H 2Adjust the pH to 7.0 with KOH and dilute the solution in 1000 mL of ddHO. 2 The volume was adjusted to O.

[0139] To make AB Buffer (20X), the following protocol and volumes were used to make 1 L of media: 700 mL ddH 2 O;60g K 2 HPO 4 ;20g NaH 2 PO 4 Dissolve the solution in 1000 mL of ddHO. 2 The volume was adjusted to O.

[0140] To make AB Minimal Agar Medium (Solid), the following protocol and volumes were used to make 1 L of medium: 700 mL ddH 2 HO; 5 g sucrose; 15 g molecular grade agar; dissolve the solution in 900 mL of ddHO. 2 Volume to 50 mL of 20x AB salts; and 50 mL of 20x AB buffer. The medium was autoclaved for 25 min on a liquid cycle. To make AB+Kan50 medium, 50 mg / L kanamycin was added to AB medium.

[0141] To make AB minimal medium (liquid), the following protocol and volumes were used to make 1 L of medium: 700 mL ddH 2 HO; 5 g sucrose; solution in 900 mL ddHO. 2 Volume to 50 mL of 20x AB salts; and 50 mL of 20x AB buffer. The medium was autoclaved for 25 minutes on a liquid cycle, cooled to 55°C, and poured into 100 x 15 mm plates.

[0142] To make YEP medium (liquid), the following protocol and volumes were used to make 1 L of medium: 800mL ddH- 2 HO; 10 g Bacto-peptone; 5 g yeast extract; 5 g NaCl; Dissolve the solution in 1000 mL ddHO. 2The volume was brought to 0. The medium was filter sterilized.

[0143] To make the TDZ infection medium, the following protocol and volumes were used to make 1 L of medium: 800mL ddH- 2 25 mL of 20×AB salts; 25 mL of 20×AB buffer; 1 g of potassium nitrate; 20 g of glucose; 5 g of MES (M825); 0.1 mL of Gamborg's B5 vitamins (G219) [1000X]; and dissolve the solution in ddHO. 2 Volume was adjusted to 1000 mL with 500 mL of HO. The pH was adjusted to 5.4 by titration with KOH / HCl. 1.0 mL [1 mg / mL] of TDZ (T8118). The medium was filter sterilized and thiols were added on the day of use, which included 2.0 mL dithiothreitol [77 mg / ml], 4.96 mL sodium thiosulfate·5H2O [50 mg / ml], and 8 mL L-cysteine ​​[50 mg / ml]. In some embodiments, 0.02% v / v Silwet L-77 was added on the day of use.

[0144] To make CL medium, the following protocol and volumes were used to form 1 L of medium: 800mL ddH- 2 HO; 30 g sucrose (S391); 5.22 g DKW basal salts (D190); 1 g MES (M825); 1 mL PPM; 1 mL Gamborg's B5 vitamins (G219) [1000x]; and dissolve the solution in 168.9 mL ddHO. 2 The volume was adjusted to 0.5 mL / min. The pH was adjusted to 5.7 by titration with KOH. The medium was filter sterilized and the following was added after autoclaving: 2 mL of TDZ (T8119) [1 mg / mL]; 2.0 mL of asparagine (A107) [25 mg / mL], 2.0 mL of glutamine (G229) [25 mg / mL], 1.0 mL of timentin (T104) [300 mg / mL], 1.2 mL of cefotaxime (C380) [250 mg / mL] and 2 mL of carbencillin (C346) [250 mg / mL].

[0145] To make co-culture medium (e.g., co-cult G DKW), the following protocol and volumes were used to form 1 L of medium: 800mL ddH- 2 HO; 20 g glucose; 5.22 g G DKW basal salts (D190); 1 g MES (M825); solution in 183.75 mL ddHO. 2 Volume of 100. pH adjusted to 5.8 by titration with KOH. 6 g agar, plant TC (A296). The medium was autoclaved using a MediaClave on an AGAR cycle, and the following was added after autoclaving: 0.125 mL of IAA (I364) [1 mg / ml], 2 mL of trans-Zeatin Riboside (Z899) [1 mg / ml], and 2 mL of acetosyringone (A1104) [20 mg / ml = 100 mM]. As used herein, the G in DKW refers to gelzan (e.g., a solidifying agent), and DKW is a type of salt. However, in various experiments, the co-culture was incubated in sterile ddHO. 2 This was performed using a piece of sterile filter paper moistened with O and without the use of co-culture medium.

[0146] To make SIM+S10(TDZ2) medium, the following protocol and volumes were used to form 1 L of medium: 800mL ddH- 2 HO; 30 g sucrose (S391); 5.22 g DKW basal salts (D190); 1 g MES (M825); 1 mL Gamborg's B5 vitamin (G219) [1000x]; 8 mL iron chelate (F318); Dissolve the solution in 163.532 mL ddHO. 2 Volume of 100. pH adjusted to 5.7 by titration with KOH. 7 g agar, plant TC (A296). The medium was autoclaved using a MediaClave on an AGAR cycle and the following was added after autoclaving: 2 mL TDZ (T8118) [1 mg / mL], 2 mL asparagine (A107) [25 mg / mL], 2 mL glutamine (G229) [25 mg / mL], 1.2 mL carbenicillin (C346) [250 mg / mL], 1.2 mL cefotaxime (C380) and 0.2 mL spectinomycin (S4014) [50 mg / mL].

[0147] To make SIM+S10 (TDZ10) medium, the following protocol and volumes were used to form 1 L of medium: 800mL ddH- 2 HO; 30 g sucrose (S391); 5.22 g DKW basal salts (D190); 1 g MES (M825); 1 mL Gamborg's B5 vitamins (G219); 8 mL iron chelate (F318); Dissolve the solution in 155.532 mL ddHO. 2 Volume of 100. pH adjusted to 5.7 by titration with KOH. 7 g agar, plant TC (A296). The medium was autoclaved using a MediaClave on an AGAR cycle and the following was added after autoclaving: 10 mL TDZ (T8118) [1 mg / mL], 2 mL asparagine (A107) [25 mg / mL], 2 mL glutamine (G229) [25 mg / mL], 1.2 mL carbenicillin (C346) [250 mg / mL], 1.2 mL cefotaxime (C380) and 0.2 mL spectinomycin (S4014) [50 mg / mL].

[0148] To make SIM+S50(TDZ2) medium, the following protocol and volumes were used to form 1 L of medium: 800mL ddH- 2 HO; 30 g sucrose (S391); 5.22 g DKW basal salts (D190); 1 g MES (M825); 1 mL Gamborg's B5 vitamins (G219); 8 mL iron chelate (F318); Dissolve the solution in 163.532 mL ddHO. 2Volume of 100. pH adjusted to 5.7 by titration with KOH. 7 g agar, plant TC (A296). The medium was autoclaved using a MediaClave on an AGAR cycle and the following was added after autoclaving: 2 mL TDZ (T8118) [1 mg / mL], 2 mL asparagine (A107) [25 mg / ml], 2 mL glutamine (G229) [25 mg / ml], 1.2 mL carbenicillin (C346) [250 mg / ml], 1.2 mL cefotaxime (C380) and 1 mL spectinomycin (S4014) [50 mg / ml].

[0149] To make SIM+S50(TDZ10) medium, the following protocol and volumes were used to form 1 L of medium: 800mL ddH- 2 HO; 30 g sucrose (S391); 5.22 g DKW basal salts (D190); 1 g MES (M825); 1 mL Gamborg's B5 vitamins (G219); 8 mL iron chelate (F318); Dissolve the solution in 155.532 mL ddHO. 2 Volume of 100. pH adjusted to 5.7 by titration with KOH. 7 g agar, plant TC (A296). The medium was autoclaved using a MediaClave on an AGAR cycle and the following was added after autoclaving: 10 mL TDZ (T8118) [1 mg / mL], 2 mL asparagine (A107) [25 mg / mL], 2 mL glutamine (G229) [25 mg / mL], 1.2 mL carbenicillin (C346) [250 mg / mL], 1.2 mL cefotaxime (C380) and 1 mL spectinomycin (S4014) [50 mg / mL].

[0150] To make SIM+S100(TDZ2) medium, the following protocol and volumes were used to form 1 L of medium: 800mL ddH- 2HO; 30 g sucrose (S391); 5.22 g DKW basal salts (D190); 1 g MES (M825); 1 mL Gamborg's B5 vitamins (G219); 8 mL iron chelate (F318); Dissolve the solution in 163.532 mL ddHO. 2 Volume of 100. pH adjusted to 5.7 by titration with KOH. 7 g agar, plant TC (A296). The medium was autoclaved using a MediaClave on an AGAR cycle and the following was added after autoclaving: 2 mL TDZ (T8118) [1 mg / mL], 2 mL asparagine (A107) [25 mg / ml], 2 mL glutamine (G229) [25 mg / ml], 1.2 mL carbenicillin (C346) [250 mg / ml], 1.2 mL cefotaxime (C380) and 2 mL spectinomycin (S4014) [50 mg / ml].

[0151] To make SIM+S100 (TDZ10) medium, the following protocol and volumes were used to form 1 L of medium: 800mL ddH- 2 HO; 30 g sucrose (S391); 5.22 g DKW basal salts (D190); 1 g MES (M825); 1 mL Gamborg's B5 vitamins (G219); 8 mL iron chelate (F318); Dissolve the solution in 155.532 mL ddHO. 2 Volume of 100. pH adjusted to 5.7 by titration with KOH. 7 g agar, plant TC (A296). The medium was autoclaved using a MediaClave on an AGAR cycle and the following was added after autoclaving: 10 mL TDZ (T8118) [1 mg / mL], 2 mL asparagine (A107) [25 mg / ml], 2 mL glutamine (G229) [25 mg / ml], 1.2 mL carbenicillin (C346) [250 mg / ml], 1.2 mL cefotaxime (C380) and 2 mL spectinomycin (S4014) [50 mg / ml].

[0152] To make SEM I+S50 medium, the following protocol and volumes were used to make 1 L of medium: 800mL ddH- 2 HO; 30 g sucrose (S391); 5.22 g DKW basal salts (D190); 1 g MES (M825); 1 mL Gamborg's B5 vitamins (G219); 8 mL iron chelate (F318); Dissolve the solution in 154.594 mL ddHO. 2 Volume of 100. pH adjusted to 5.7 by titration with KOH. 7 g agar, plant TC (A296). The medium was autoclaved using a MediaClave on an AGAR cycle and the following was added after autoclaving: 0.0385 mL gibberellic acid (G362) [13 mg / ml], 0.1 mL IAA (I364) [1 mg / ml], 10 mL L-ascorbic acid [10 mg / ml], 2 mL asparagine (A107) [25 mg / ml], 2 mL glutamine (G229) [25 mg / ml], 1.2 mL carbenicillin (C346) [250 mg / ml], 1.2 mL cefotaxime (C380) and 1 mL spectinomycin (S4014) [50 mg / ml].

[0153] To make SEM I+S100 medium, the following protocol and volumes were used to make 1 L of medium: 800mL ddH- 2 HO; 30 g sucrose (S391); 5.22 g DKW basal salts (D190); 1 g MES (M825); 1 mL Gamborg's B5 vitamins (G219); 8 mL iron chelate (F318); Dissolve the solution in 154.594 mL ddHO. 2 Volume of 100. pH adjusted to 5.7 by titration with KOH. 7 g agar, plant TC (A296). The medium was autoclaved using a MediaClave on the AGAR cycle and the following was added after autoclaving: 0.0385 mL gibberellic acid (G362) [13 mg / ml], 0.1 mL IAA (I364) [1 mg / ml], 10 mL L-ascorbic acid [10 mg / ml], 2 mL asparagine (A107) [25 mg / ml], 2 mL glutamine (G229) [25 mg / ml], 1.2 mL carbenicillin (C346) [250 mg / ml], 1.2 mL cefotaxime (C380) and 2 mL spectinomycin (S4014) [50 mg / ml].

[0154] To make SEM I+S150 medium, the following protocol and volumes were used to make 1 L of medium: 800mL ddH- 2 HO; 30 g sucrose (S391); 5.22 g DKW basal salts (D190); 1 g MES (M825); 1 mL Gamborg's B5 vitamins (G219); 8 mL iron chelate (F318); Dissolve the solution in 154.594 mL ddHO. 2 Volume of 100. pH adjusted to 5.7 by titration with KOH. 7 g agar, plant TC (A296). The medium was autoclaved using a MediaClave on an AGAR cycle and the following was added after autoclaving: 0.0385 mL gibberellic acid (G362) [13 mg / ml], 0.1 mL IAA (I364) [1 mg / ml], 10 mL L-ascorbic acid [10 mg / ml], 2 mL asparagine (A107) [25 mg / ml], 2 mL glutamine (G229) [25 mg / ml], 1.2 mL carbenicillin (C346) [250 mg / ml], 1.2 mL cefotaxime (C380) and 3 mL spectinomycin (S4014) [50 mg / ml].

[0155] To make Cannabaceae RM DKW+0.5IBA medium, the following protocol and volumes were used to make 1 L of medium: 800mL ddH- 2HO; 30 g sucrose (S391); 5.22 g DKW basal salts (D190); 1 mL Gamborg's B5 vitamins (G219); 5 mL plant preservative mixture; and dissolve the solution in 174.63 mL of ddHO to a volume of 1. 2 The pH was adjusted to 5.8 by titration with KOH. 7 g agar, plant TC (A296) The medium was autoclaved in an AGAR cycle with MediaClave and 0.5 mL of IBA [1 mg / 1 mL] was added.

[0156] Infection media (e.g., EA TDZ infection medium), co-culture medium, regeneration medium (e.g., CL medium), SIM (e.g., SIM+S10(TDZ2) medium, SIM+S10(TDZ10) medium, SIM+S50(TDZ2) medium, SIM+S50(TDZ10) medium, SIM+S100(TDZ2) medium, and SIM+S100(TDZ10) medium), SEM (e.g., SEMI+S50 medium, SEMI+S100 medium, and SEMI+S150 medium), and RM DKW generally include water, a basal salt mixture, sugars, and one or more other components, such as vitamins, selection agents, amino acids, and plant hormones. SIM and SEM can include nutritional sources of nitrogen, phosphorus, potassium, sulfur, calcium, magnesium, iron, boron, molybdenum, manganese, cobalt, zinc, copper, chlorine, and iodine. In some experimental embodiments, the following was performed according to Table 1: [Table 1] In such an experimental embodiment, cannabis EA was transformed with plasmid vector 570 as shown in FIG. 5C. Cannabis EA was transformed using bacteria containing a binary vector using the Cannabaceae transformation protocol. The binary vector contains different elements including a spectinomycin selection marker, a YFP visual selection marker, and a TALEN pair designed to knock out part of the THCAS gene. Cannabis EA with editing frequencies of 21% and 31% were plated on SIM+S10 (TDZ10) for 3 weeks.

[0157] Figures 6A-6B include exemplary images from samples in Table 1 consistent with the present disclosure. The images from Figures 6A-6B are Illumina aligned images from Sample 2. The white area within the vertical yellow dotted line indicates successful editing of the targeted THCAS region. Figure 6B is an expanded version of Figure 6A showing a lead exhibiting a knockout edit of the THCAS region.

[0158] Figures 7A-7B are images of cannabis seedling explants transformed with bacterial strains consistent with the present disclosure. In some experiments, cannabis seedling explants with cotyledons intact during transformation were transformed with plasmid vector 560, as shown in Figure 5B. In such experiments, cannabis seedlings with cotyledons intact were transformed using the Cannabaceae transformation protocol, with bacteria containing a binary vector. The binary vector contains different elements, including a kanamycin selection marker, a YFP visual selection marker, and a TALEN pair designed to knock out a portion of the THCAS gene. As shown by the image in Figure 7A, YFP is found in the transformed explants in the stem of the explant, indicating that the transgene is stably integrated into the cannabis genome. As shown by the image in Figure 7B, YFP fluorescence can also be seen in the root region of the explant.

[0159] Figure 8 is an image of a plant regenerated from a cannabis seedling explant transformed with the bacteria shown in Figures 7A-7B, consistent with the present disclosure. The plant was tissue sampled and analyzed using PCR. The PCR data shows that the plant contains the transgene delivered by the transformation method.

[0160] Figure 9 is an image of PCR data from the regenerated plants shown in Figure 8, consistent with the present disclosure. As shown, the plants have a positive band for the transgene, which aligns well with the positive control DNA.

[0161] Figures 10A-10B are images of cannabis meristematic regions transformed with bacterial strains consistent with the present disclosure. In some embodiments, cannabis explants were transformed with plasmid vector 550 as shown in Figure 5A. In experiments, cannabis explants containing or implemented as meristematic regions were transformed using the above transformation protocol and Agrobacterium strains containing binary vectors. The binary vectors contained different elements including a spectinomycin selection marker and a betalain visual selection marker. The cannabis explants showed highly efficient transformation, with the entire explant expressing betalain color. Furthermore, explants derived from the cannabis explants demonstrate that the transgene was successfully integrated into the regenerating tissue. Betalains are shown to be expressed in the meristematic, petiole and leaf tissues in addition to the high levels of betalain expression in the original explant. Figure 10A is an image of a transformed cannabis explant showing expression of betalains, indicating integration of the T-DNA vector. More specifically, the image in Figure 10A shows the conversion of the meristem region. Figure 10B shows a plant regenerated from the explant, which shows betalain expression in the meristem, petiole and leaf tissue. The original explant shows highly efficient integration of the T-DNA vector. Furthermore, the regenerated plant derived from this explant shows betalain expression in the meristem, petiole and leaf tissue.

[0162] Figure 11 is an image of cannabis seedlings transiently transformed with bacterial strains consistent with the present disclosure. In some experiments, transient expression of seedlings transformed with plasmid vector 550 was performed. In such an embodiment, cannabis seedlings were transformed using the Agrobacterium strain containing a binary vector using the transformation protocol described above. The binary vector contained different elements including a spectinomycin selection marker and a betalain visual selection marker. The seedlings showed efficient transformation, with some explants transiently expressing betalain color 45 minutes after transformation. The image in Figure 11 shows cannabis seedlings showing transient expression of betalain color 45 minutes after transformation. The circled explants are transiently expressing betalains, while the rest are not.

[0163] Figures 12A-C are images of cannabis seedling explants stably transformed with bacterial strains consistent with the present disclosure. In some embodiments, cannabis seedling explants were stably transformed with plasmid vector 550. Seedlings showed efficient transformation, with exemplary explants showing stable transformation of the RUBY vector compared to controls. Figure 12A shows an image of a control explant not transformed with the RUBY vector. 12B and 12C show stably transformed cannabis seedling explants. As shown in Figures 12B-C, betalain expression can be seen in the explants.

[0164] Figures 13A-B are images of cannabis EA stably transformed with bacterial strains consistent with the present disclosure. In some embodiments, the cannabis EA was transformed with plasmid vector 550. The EA showed stable expression of the transgene compared to no betalain expression in the wild type EA control. Figure 13A shows an image of a control explant not transformed with the RUBY vector. Figure 13B shows a stably transformed cannabis seedling explant. As shown by Figure 13B, betalain expression can be seen in the explant. The transformed cannabis EA shows stable expression of betalains throughout the explant.

[0165] 14A-B are images of cannabis seedling explants stably transformed with bacterial strains consistent with the present disclosure. In some embodiments, the cannabis seedling explants were transformed with plasmid vector 550. The seedling explants were thinly sliced ​​and imaged using a microscope. The sections show cells expressing betalains. FIG. 14A-B show cross-sections of cannabis seedlings stably transformed with transgenes.

[0166] Figures 15A-B are images of cannabis seedling explants stably transformed with bacterial strains consistent with the present disclosure. In some embodiments, cannabis seedling explants were transformed with a plasmid vector 560 as shown in Figure 5B. The seedling explants were stably transformed with a transformation protocol using Agrobacterium containing a binary vector as described above. The binary vector contained various elements including a kanamycin selection marker and a YFP reporter (e.g., a visual selection marker). High levels of YFP fluorescence were identified in the meristematic region 7 days after transformation. Figure 15A is an image of a cannabis explant transformed with the plasmid vector 560 under white light. Figure 15B is an image of the cannabis explant of Figure 15A placed under YFP fluorescent light. The explants show stable YFP fluorescence in the meristematic region due to integration of the vector into the cannabis genome.

[0167] Various experiments were aimed at testing different culture media and different concentrations of components of the culture media, such as SIM with different TDZ concentrations. Cannabis is recalcitrant and in some experiments, the resulting explants did not show transformed shoots. Based on various experiments, it is believed that cannabis plants have strong apical dominance and breaking apical dominance increases transformation efficiency and results in more transgenic events. Somewhat surprisingly, concentrations of about 2 mg / L to about 10 mg / L of TDZ were used in the SIM to break apical dominance and increase shoots of meristematic cannabis cells. Some experiments were aimed at testing different SIMs with different light conditions on cannabis EA. For example, seeds were sterilized, soaked, and EA was extracted. In some experiments, EA was not transformed and 40 to 50 EA were regenerated in each different SIM. In further experiments, a subset of EAs was plated on SIM without a plant growth regulator such as TDZ, and the remaining EAs were plated on SIM with 10 mg / L TDZ, and different groups of each subset were exposed to different light conditions. Tables 2 and 3 summarize exemplary media and light conditions. [Table 2] [Table 3] The B, G, R values ​​in Table 3 include values ​​for blue, green, and red light along with the light intensity.

[0168] 16A-16G are diagrams of data results from experiments evaluating different culture media and light conditions consistent with the present disclosure.

[0169] Figure 16A shows images 1680, 1681, 1683, 1684 of cannabis explants regenerated from cannabis EA cultivated on different SIM. Although not shown, IAA, adenine hemisulfate and glucose had no significant positive effect. Images 1680, 1681, 1683, 1684 show the resulting explants regenerated on SIM containing 1 mg / L TDZ, 2.5 mg / L TDZ, 5 mg / L TDZ and 10 mg / L TDZ, respectively. After about 3 weeks, explants on SIM containing 10 mg / L TDZ showed the greatest shoot growth. After about 6 weeks, explants on SIM containing 10 mg / L TDZ showed signs of stress, such as chlorosis, callus formation and growth inhibition. Thus, cannabis EA can be cultured on SIM containing 10 mg / L TDZ for about 2 to about 6 weeks, and in some embodiments, for about 2 to 3 weeks.

[0170] Figure 16B is a graph showing the number of explants and resulting meristems regenerated in different SIMs as previously described by Figure 16A. As shown, SIM with 10 mg / L TDZ had most explants with multiple meristems. SIM with 5 mg / L TDZ had most explants with numerous meristems.

[0171] FIG. 16C is an image of the resulting explants regenerated on SIM containing 10 mg / L TDZ.

[0172] Examples are not limited to media containing TDZ and / or TDZ concentrations shown in Figures 16B-16C. In some experiments, additional TDZ concentrations were tested and different combinations of ingredients in the SIM were tested. Tables 4 and 5 show the different SIMs tested and the resulting meristems at 2 and 6 weeks. [Table 4] [Table 5]

[0173] Figure 16D is an image of the resulting growth of cannabis EA on SIM containing 10 mg / L TDZ and no plant growth regulator. In particular, the image shows growth under light I conditions as described in Table 3 above. The medium without plant growth regulator had good root growth, especially under light I and light II conditions, and under light III conditions with significantly longer shoots.

[0174] FIG. 16E shows images of the resulting growth of cannabis EA in SIM under different light conditions (e.g., light I, light II, and light III conditions) without plant growth regulators and with 10 mg / L TDZ.

[0175] Figures 16F and 16G are graphs showing the results of different light conditions on the number of meristems per explant in cultures without plant growth regulators (Figure 16F) and with 10 mg / L TDZ (Figure 16G). As shown, light conditions alone did not significantly affect shoot number, but the use of TDZ did. For example, about half of the 10 mg / L TDZ had multiple meristems, the light II condition resulted in 40% of explants with 3+ meristems, the light II condition resulted in 24% of explants with 3+ meristems, and the light I condition resulted in 12% of explants with 3+ meristems.

[0176] Various experiments were directed to transforming cannabis EA using the protocol described above, including the use of SIM medium containing infection, CL, and TDZ. For example, 50-75 EA were isolated in 20 mL of TDZ infection medium + thiol / infection plate. After isolation was complete, the TDZ infection medium + thiol was removed. 18r12 with an OD600 of approximately 0.8 were grown in YEP / AB culture medium. 18r12 was resuspended in TDZ infection medium + thiol + 100 uM acetosyringone (AS) + 0.02% v / v Silwet L-77. 10 mL of resuspended 18r12 was sonicated for 80 seconds, where sonication resulted in little or no YFP or other transformant expression. Other examples included sonication for 20-60 seconds. 20 mL of freshly resuspended 18r12 was then added to the 10 mL already in the infection plate and inoculated for 30 minutes to an hour. Remove 30 mL of resuspended 18r12 from the infected plate. Take 50-75 EA from the infected plate and resuspend them in 750 µL of ddHO in a 100 x 15 mm Petri dish. 2 Co-culture for 5 days was performed by attaching the EA to sterile paper moistened with 200 mL of HO to form a co-culture plate. The co-culture plate was parafilmed and placed in an incubator with conditions including 24 h ambient light, 0 μmol / m-2 / s-1, 40% RH, and 23 °C. EA were then collected in 100 × 24 mm Petri dishes containing 20 ml of CL medium (0.1% PPM, TDZ), parafilmed, and placed in a Comviron for 3 days, during which time they were filled with 1 x sterilized ddHO. 2 The CL medium was removed and EA was replaced with 1x sterile ddHO. 2 The plants were rinsed with 0 and placed in SIM+S10 (TDZ2) solid medium for approximately 3 weeks. Tables 6 and 7 below describe the different culture media and conditions used to transform cannabis EA. In various experiments, the resulting transformed explants were tested using PCR to identify the transformation frequency. Transformed cultures with copy numbers between 0.5 and 2.5 were identified, and from such cultures, 7 transgenic T0s were observed out of a total of 47 T0s, resulting in a transformation efficiency of 14.89%. [Table 6] [Table 7]

[0177] 17A-17F are images of explants from an experiment evaluating the transformation of explants in different media consistent with the present disclosure. FIG. 17A shows images of explants obtained from different infection media including CsEA TDZ+0.02% v / v Silwet L-77, GmEA TDZ+0.02% v / v Silwet L-77 (which has the same salt composition as CsEA TDZ+0.02% v / v Silwet L-77, which further contains gibberellic acid), MTA+TDZ, and MTA. As shown by FIG. 17A, different infection media treatments resulted in different YFP expression. More specifically, CsEA TDZ and GmEA TDZ were performed between MTA+TDZ and MTA.

[0178] Figures 17B-C show the explants obtained when transformed using GmEA TDZ + 0.02% v / v Silwet L-77 (Figure 17B) and MTA infection medium (Figure 17C). As shown, leaf primordia are not transformed and give rise to non-transgenic primary shoots.

[0179] Figures 17D-E are images of the resulting stably transformed explants imaged 4 weeks after infection with 18r12 plasmid vector 570, harvested and regenerated using CL (e.g., 2 mg / L TDZ) and SIM (2 mg / L TDZ).

[0180] FIG. 17F is an image of the resulting stably transformed explants that were imaged 5 weeks after infection with 18r12-plasmid vector 570, harvested, and regenerated using CL (e.g., 2 mg / L TDZ) and SIM (2 mg / L TDZ).

[0181] In further experimental embodiments, cannabis EA were transformed with plasmid vectors 572, 576, 578, 580, 582 plasmid vectors as shown by Figures 5D-5I. Cannabis EA were transformed using bacteria containing each binary vector using the Cannabaceae transformation protocol described above and regenerated using the steps of the experimental embodiments described above, including the use of infection medium, CL (containing PPM and TDZ2) and SIM (TDZ10). In various embodiments, editing efficiency was compared between different plasmid vectors. The different plasmid vectors included a first set targeting the PDS gene (e.g., plasmid vector 10, 572 in Figure 5D, 574 in Figure 5E, 580 in Figure 5H) and a second set targeting the THCAS gene (e.g., plasmid vector 11, 576 in Figure 5F, 578 in Figure 5G, 582 in Figure 5I). Within each of the first and second sets, the respective vectors did not include an intein (e.g., plasmid vector 10 and plasmid vector 11, not shown herein), an intein between SSP DnaE and a native ectein (e.g., 572 in FIG. 5D, 576 in FIG. 5F), an intein between GP41-1 and a non-native ectein (e.g., 574 in FIG. 5E, 578 in FIG. 5G), and an intein between GP41-1 and a native ectein (e.g., 580 in FIG. 5H, 582 in FIG. 5I).

[0182] 18A-18I are diagrams showing the results of transforming explants with different plasmid vectors consistent with the present disclosure. FIG. 18A is a graph comparing the editing efficiency of the first and second set of different plasmid vectors, showing the editing efficiency of no intein (e.g., plasmid vector 10 and plasmid vector 11, SSP DnaE and native ectein intein (e.g., 572 in FIG. 5D, 574 in FIG. 5F), GP41-1 and non-native ectein intein (e.g., 574 in FIG. 5E, 578 in FIG. 5G, and GP41-1 and native ectein intein (e.g., 580 in FIG. 5H, 582 in FIG. 5I). FIG. 18B-18C are graphs comparing the percent editing events of the first and second set of different plasmid vectors as shown by FIG. 18A.

[0183] 18D-18I are images of explants from experiments evaluating transformation of explants with the different plasmid vectors of FIGS. 5D-5I, consistent with the present disclosure.

[0184] Various experimental embodiments were directed to transforming Cannabaceae EA using various media. In some experiments, the infection medium, CL medium and SIM medium contained TDZ. Somewhat surprisingly, SIM medium sometimes contained 2 or 10 mg / L TDZ, resulting in regenerated Cannabaceae explants with transformed cells.

Claims

【Claim 1】 soaking flax family seeds in an aqueous solution, excising a subset of embryonic tissue from the soaked flax family seeds to extract a flax family meristem region or embryonic axis (EA), exposing the flax family meristem region or EA to a heterologous nucleotide sequence to transform flax family cells of the flax family meristem region or EA, A method comprising the above steps.

Images