WO2022198107A1

WO2022198107A1 - Producing betalain in cannabaceae plant parts

Info

Publication number: WO2022198107A1
Application number: PCT/US2022/021043
Authority: WO
Inventors: Benjamin Campbell; Lauren Harrison; Rachelle LAPHAM; Jonathan Mayers; Anders UPPGAARD
Original assignee: Calyxt, Inc.
Priority date: 2021-03-18
Filing date: 2022-03-18
Publication date: 2022-09-22

Abstract

Example embodiments in accordance with the present disclosure are directed to methods for transforming a Cannabaceae plant part by exposing the Cannabaceae plant part to an expression construct comprising a nucleotide sequence encoding an enzyme associated with production of a betalain to transform the Cannabaceae plant part with the expression construct, and inducing production of the betalain in the transformed Cannabaceae plant part.

Description

PRODUCING BETALAIN IN CANNABACEAE PLANT PARTS

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED

ELECTRONICALLY

[0001] Incorporated by reference in its entirety is a computer-readable nucleotide/amino acid sequence listing, an ASCII text file which is 177 kb in size, submitted concurrently herewith, and identified as follows: “C1633121111_SequenceListing_ST25” and created on March 17, 2022.

BACKGROUND

[0002] Betalains are a class of red, orange, yellow, and purple tyrosine-derived pigments that are found in Caryophyllales plants and in some fungi. Betalains can be used as a colorant, such as a food dye, as well as for therapeutic and nutritional purposes, such as an antioxidant and/or for anti-inflammatory properties, among other uses.

SUMMARY

[0003] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description.

[0004] Various aspects of the present disclosure are directed to a method comprising transforming a Cannabaceae plant part to induce production of a betalain.

[0005] Various aspects are directed to a method comprising exposing a Cannabaceae plant part to an expression construct comprising a nucleotide sequence encoding an enzyme associated with production of a betalain to transform the Cannabaceae plant part with the expression construct, and inducing production of the betalain in the transformed Cannabaceae plant part.

[0006] In some aspects, the enzyme comprises dihydroxyphenylalanine (DOF A) 4,5- di oxygenase (DODAj. Cytochrome P450 (CYP76AD1), CYP76AD6, glucosyltransferase, or a combination thereof. [0007] In some aspects, the enzyme comprises a plurality of enzymes and the nucleotide sequence encodes the plurality of enzymes linked by a plurality of 2A self-cleaving peptides.

[0008] In some aspects, the betalain comprises a betacyanin or a betaxanthin.

[0009] In some aspects, the betalain is selected from (such as being selected from a group consisting of): betanin, isobetanin, probetanin, neobetanin, vulgaxanthin, miraxanthin, portulaxanthin, and indicaxanthin.

[0010] In some aspects, the nucleotide sequence encoding the enzyme comprises SEQ ID NO: 2, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 35, or SEQ ID NO: 39.

[0011] In some aspects, the Cannabaceae plant part is a leaf, a root, a seed, a meristem, a node, an intemode, a meristem, a petiole, a hypocotyl segment, or a cell.

[0012] In some aspects, the Cannabaceae plant part is stably transformed by the exposure to expression construct.

[0013] In some aspects, the Cannabaceae plant part is transiently transformed by the exposure to expression construct.

[0014] In some aspects, the method further comprises generating the expression construct comprising the nucleotide sequence that encodes the enzyme.

[0015] In some aspects, exposing the Cannabaceae plant part to the expression construct and inducing production of the betalain comprises contacting the Cannabaceae plant part with a bacterium strain comprising the expression construct that comprises the nucleotide sequence encoding the enzyme, and culturing the Cannabaceae plant part to induce production of the betalain in the Cannabaceae plant part.

[0016] In some aspects, inducing the production comprises culturing the Cannabaceae plant part under growth conditions to enhance transformation, PCM formation, and induce production of the betalain.

[0017] In some aspects, the Cannabaceae plant part is contacted with the expression construct to transform the Cannabaceae plant part and induce formation of a collection of plant cells, referred to herein as “a plant cell matrix (PCM)”, and production of an betalain.

[0018] In some aspects, the growth conditions are selected from (such as being selected from a group consisting of): a liquid culture medium, a type of culture medium, an amount of contact with the culture medium, a type of contact with the culture medium, a plant type, and a combination thereof.

[0019] In some aspects, culturing the Cannabaceae plant part under the growth conditions comprises intermittently contacting the Cannabaceae plant part with a culture medium containing sugar and basal salt.

[0020] In some aspects, the method further comprises identifying a bacterium strain from a plurality of bacterium strains.

[0021] In some aspects, contacting the Cannabaceae plant part with the bacterium strain comprises simultaneously introducing to the Cannabaceae plant part a first transgene associated with PCM formation, and a second transgene associated with the enzyme.

And, the method further comprises cultivating the plant part as transformed to generate PCM tissue, wherein the plant part is a seedling, ahypocotyl segment, a petiole, an intemode, a node, a meristem or a leaf.

[0022] In some aspects, contacting the Cannabaceae plant part with the bacterium strain and culturing the Cannabaceae plant part comprises contacting the Cannabaceae plant part with a first bacterium strain comprising the nucleotide sequence encoding the gene that induces PCM formation, culturing the Cannabaceae plant part to enhance PCM formation, contacting formed PCM tissue from the PCM with a second bacterium strain comprising the nucleotide sequence encoding the enzyme, and culturing the PCM tissue to enhance production of the betalain by the PCM.

[0023] In some aspects, infecting the Cannabaceae plant part with the bacterium strain comprises injecting the bacterium strain into the Cannabaceae plant part, agroinfiltrating the bacterium strain into the Cannabaceae plant part, or culturing the infected Cannabaceae plant part to enhance transformation, induce PCM formation, and induce expression of the enzyme.

[0024] In some aspects, exposing the Cannabaceae plant part to the expression construct and inducing the production of the betalain comprises simultaneously introducing to the Cannabaceae plant part a first transgene associated with PCM formation, and a second transgene associated with the enzyme, the method further comprising cultivating the Cannabaceae plant part as transformed to generate PCM tissue, wherein the Cannabaceae plant part is a seedling, a hypocotyl segment, a petiole, an intemode, a node, a meristem, or a leaf.

[0025] In some aspects, exposing the Cannabaceae plant part to the expression construct and inducing the production of the betalain comprises contacting the Cannabaceae plant part a nucleotide sequence encoding a gene that induces PCM formation, culturing the Cannabaceae plant part to enhance PCM formation, contacting formed PCM tissue from the PCM with the nucleotide sequence encoding the enzyme, and culturing the PCM tissue to enhance production of the betalain by the PCM.

[0026] In some aspects, transforming the Cannabaceae plant part comprises exposing the Cannabaceae plant part to the expression construct to transform the Cannabaceae plant part with the expression construct via bombardment, and inducing expression of the enzyme in tissue of the transformed Cannabaceae plant part.

[0027] In some aspects, exposing the Cannabaceae plant part to the expression construct via bombardment comprises using a bombardment gun, particles coated with the expression construct, or a combination thereof.

[0028] In some aspects, the nucleotide sequence encoding the enzyme is operably connected to a promoter.

[0029] In some aspects, the method further compnses screening the transformed Cannabaceae plant part or new growth from the transformed Cannabaceae plant part for production of the betalain and tissue formation.

[0030] Various aspects are directed to method of generating a bacterium strain comprising transforming a bacterium strain with a nucleotide sequence encoding an enzyme, wherein the bacterium strain comprises a nucleotide sequence encoding a gene that induces PCM formation or is transformed to comprise the nucleotide sequence encoding the gene that induces PCM formation, and culturing the transformed bacterium strain.

[0031] In some aspects, generating an expression construct comprising the nucleotide sequence encoding the enzyme, and forming a solution or suspension comprising the expression construct or attaching the expression construct to particles for bombardment or injection. [0032] In some aspects, the nucleotide sequence encoding the enzyme comprises SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 36, or a combination thereof.

[0033] In some aspects, the bacterium strain is transformed using an expression cassette comprising SEQ ID NO: 2, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 35, or SEQ ID NO: 39.

[0034] In some aspects, the bacterium strain is transformed using an expression construct comprising SEQ ID NO: 1, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 38, or SEQ ID NO: 40.

[0035] In some aspects, the method further comprises transforming the bacterium strain to carry the nucleotide sequence encoding the enz me using a vector containing a right and left transferred DNA (T-DNA) border sequence, the nucleotide sequence encoding the enzy me, and a promoter.

[0036] Various aspects are directed to a method comprising contacting a Cannabaceae plant part with a bacterium strain containing a nucleotide sequence encoding an enzyme associated with production of a betalain, and inducing formation of tissue from the Cannabaceae plant part, wherein the tissue expresses the nucleotide sequence.

[0037] In some aspects, the method further compnses selecting tissue from the Cannabaceae plant part as transformed for culturing in a culture medium; and screening the cultured tissue for production of the betalain.

[0038] In some aspects, the method further comprises capturing the betalain by isolating and purifying the betalain from the Cannabaceae plant part as transformed, the culture medium, or a combination thereof.

[0039] Various aspects are directed to a tissue culture that produces a betalain, the tissue culture being induced from a Cannabaceae plant part and an expression construct comprising a nucleotide sequence encoding an enzyme associated with production of a betalain, wherein plant cells of the tissue culture comprises the nucleotide sequence encoding the enzyme.

[0040] In some aspects, the nucleotide sequence encoding the enzyme comprises SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 36, or a combination thereof. [0041] In some aspects, the expression construct comprises an expression cassette comprising SEQ ID NO: 2, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 35, or SEQ ID NO: 39.

[0042] Various aspects are directed to a system for producing a betalain from a Cannabaceae plant part, the system comprising a plurality of bioreactors in serial connection, wherein each bioreactor is inoculated with a Cannabaceae plant part according to method of claim 1, and configured for growth and maintenance of the Cannabaceae plant part or a tissue culture formed from the Cannabaceae plant part in a culture medium.

[0043] In some aspects, the culture medium comprises a liquid culture medium and the system is configured to recover the betalain from the culture medium.

[0044] In some aspects, at least one bioreactor is a flask, temporary immersion system, plastic sleeve reactor, a bubble reactor, a mist reactor, an airlift reactor, a liquid-dispersed reactor or a bioreactor configured to generate micro- or nano-bubbles.

[0045] In some aspects, each bioreactor of the plurality is structurally and operationally similar.

[0046] Various aspects are directed to a Cannabaceae tissue generated using a method of claim 1.

[0047] Various aspects are directed to a betalain produced by a Cannabaceae plant or plant part according to the method of claim 1.

[0048] Various aspects are directed to a method comprising transforming a plurality of Cannabaceae plant parts with an expression construct to induce production of a betalain, and screening the transformed plurality of Cannabaceae plant parts for the production of the betalain.

BRIEF DESCRIPTION OF THE DRAWINGS [0049] The foregoing aspects and many of the advantages of this invention will become more readily understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

[0050] FIG. 1 illustrates an example method for producing a betalain in a Cannabaceae plant part, consistent with the present disclosure. [0051] FIG. 2 illustrates an example method for transforming a Cannabaceae plant part to induce expression of an enzyme and production of a betalain, consistent with the present disclosure.

[0052] FIGs. 3A-3B illustrate different example methods for expressing an enzyme in a Cannabaceae plant part, consistent with the present disclosure.

[0053] FIGs. 4A-4C illustrate example expression construct for delivery of a sequence encoding an enzyme, consistent with the present disclosure.

[0054] FIGs. 5A-5B illustrate different example methods for generating an expression construct comprising a sequence encoding an enzyme, consistent with the present disclosure.

[0055] FIGs. 6A-6F illustrate example expression constructs for delivery of a sequence encoding an enzyme, consistent with the present disclosure.

[0056] FIGs. 7A-7D illustrate example images of PCM cultures producing betacyanin, consistent with the present disclosure.

[0057] FIGs. 8A-8B illustrate example images of PCM cultures producing betacyanin at different levels, consistent with the present disclosure.

[0058] FIGs. 9A-9F illustrate example images of PCM cultures producing betacyanin at different levels, consistent with the present disclosure.

[0059] FIGs. 10A-10C illustrate example images of PCM cultures producing betanidin and betaxanthin, consistent with the present disclosure.

[0060] FIGs. 11A-11B illustrate example images of PCM cultures producing betaxanthin, consistent with the present disclosure.

[0061] FIGs. 12A-12B illustrate example images of betalains in liquid from PCM cultures, consistent with the present disclosure.

[0062] FIGs. 13A-13C illustrate example images of a Cannabaceae EA transformed with an Agrobacterium strain, consistent with the present disclosure.

[0063] FIG. 14 illustrates example image of a Cannabaceae seedling transiently transformed with an Agrobacterium strain, consistent with the present disclosure.

[0064] FIGs. 15A-15C illustrate example images of a Cannabaceae seedling explant stably transformed with an Agrobacterium strain, consistent with the present disclosure. [0065] FIGs. 16A-16B illustrate example images of a Cannabaceae EA stably transformed with an Agrobacterium strain, consistent with the present disclosure.

[0066] FIGs. 17A-17B illustrate example images of a Cannabaceae seedling explant stably transformed with an Agrobacterium strain, consistent with the present disclosure. [0067] FIGs. 18A-18D illustrate example images of Cannabaceae node explant stably transformed with an expression construct associated with a betalain, consistent with the present disclosure.

[0068] FIGs. 19A-19B illustrate example experimental results from PCM cultures producing betalains, consistent with the present disclosure.

[0069] FIGs. 20A-20B illustrate example images of PCM cultures generated from solanum tuberosum plants, consistent with the present disclosure.

[0070] FIGs. 21A-21B illustrate example images showing betalain production in solanum tuberosum PCM cultures, consistent with the present disclosure.

[0071] FIGs. 22A-22B illustrate example images of betalain production in solanum tuberosum PCM cultures using different bacterium strains, consistent with the present disclosure.

DETAILED DESCRIPTION

[0072] The present disclosure is directed to methods, materials, and systems for transforming Cannabaceae plant parts to induce expression an enzyme and production of a betalain. Various aspects are directed to Cannabaceae tissue cultures transformed to express an enzyme associated with production of a betalain and systems for production and recovery of the betalain.

[0073] Betalains can be used for a variety of different purposes, including medical, food, and industrial applications. In some examples, the betalains can be used as natural colorants and flavorings, which can be challenging for food and beverage formulators to generate. Betalains, as used herein, include tyrosine-derived pigments, which can be red, red-violet, violet, yellow, orange, and yellow-orange. Betalains include betacyanins, which are red to violet betalain pigments, and betaxanthms, which are yellow to orange betalain pigments. Example betacyanins include betanin, isobetanin, probetanin, and neobetanin. Example betaxanthins include vulgaxanthm, miraxanthin, portulaxanthin, and indicaxanthin. Betalains can be found in plants of Caryophyllales and in some fungi, such as in Basidiomycota phylum. Betalains are produced by converting tyrosine in the plant or other organism to L-3,4-dihydroxyphenylalanine (L-DOPA) and then converting L- DOPA to the different betalains through different enzymatic pathways, as further illustrated herein by FIG. 4C. Betalains can be produced at low concentrations within plants or fungi, which can be improved through plant transformation. Further, plant-based biomass production of a betalain via outdoor agriculture, such as growing plants in the field and for the harvesting of compounds from the plant biomass, can be labor and time intensive, as well as requiring large areas of land to produce sufficient amounts of biomass. Plant transformation and tissue culture present significant limitations to genome editing efforts, requiring extensive time, labor and materials to develop and implement specialized protocols.

[0074] Embodiments in accordance with the present disclosure are directed to transforming a Cannabaceae plant part to induce production of a betalain. The Cannabaceae plant part can be transformed by exposing the plant part to an expression construct that includes a nucleotide sequence encoding an enzyme associated with the betalain to transform the plant part with the expression construct, and inducing production of the betalain in the transformed Cannabaceae plant part.

[0075] In some embodiments, the Cannabaceae plant part can be contacted (e.g., infected) with a bacterium strain and/or bacterium strains. For example, Rhizobium strains, Agrobacterium strains, Ochrobactrum strains, Ensifer strains (e.g., Ensifer adhaeren strains), and other bacterium strains can be capable of inducing PCM formation and/or transiently or non-transiently transforming the Cannabaceae plant part to produce the betalain. In some embodiments, the Cannabaceae plant part can be stably modified by the bacterium strain and/or can be cultured to maximize production of the betalain. The bacterium strain can be any strain that harbors a Ri plasmid or is otherwise transformed to induce PCM formation.

[0076] However, embodiments are not so limited and can include other ty pes of transformations, which may or may not induce PCM formation. In some embodiments, the plant part can be exposed to an expression construct including a nucleotide sequence for transformation and without using a bacterium strain. For example, the Cannabaceae plant part can be contacted with a (heterologous) nucleotide sequence encoding the Ri plasmid and/or the gene that induces PCM formation, which transforms Cannabaceae plant cells to express the nucleotide sequence. In some embodiments, the Cannabaceae plant part can be infected with a first bacterium strain to produce PCMs and then the formed PCM tissue of the PCM can be transformed with a second bacterium strain to produce the betalain, sometimes herein referred to as “re-transformation” or “re transformed”. In some embodiments, the Cannabaceae plant part can be transiently transformed using an agroinfiltration and/or injection process. In some embodiments, the Cannabaceae plant part is stably modified by bombardment technique, such as via particle bombardment with the expression construct. The methodologies can be used to produce betalains in a sustainable (environmentally and/or otherwise) and more-reliable manner, and can provide a secure and reliable supply source of betalains.

[0077] Turning now to the figures, FIG. 1 illustrates an example method for producing a betalain in a Cannabaceae plant part, consistent with the present disclosure.

[0078] At 101, the method 100 includes exposing a Cannabaceae plant part to an expression construct including a nucleotide sequence encoding an enzyme associated with production of a betalain to transform the Cannabaceae plant part with the expression construct. The Cannabaceae plant part can be a seed or seedling, a hypocotyl segment, a leaf, a root, a node, an intemode, a petiole, a meristem, a plant cell, although embodiments are not so limited and can include other Cannabaceae plant parts. As further described herein, the transformation can be performed by exposing the Cannabaceae plant part to the expression construct using a particular technique, such as infecting the Cannabaceae plant part with a bacterium strain, inducing PCM formation, agroinfiltration, viral injection, and/or bombardment, among other techniques.

[0079] The nucleotide sequence can be heterologous to the plant. As described below, the contact with the nucleotide sequence (or sequences) can be performed using a variety of different techniques and which may transform cells of the plant part to express the nucleotide sequences and form a PCM and produce the betalain. As further described below, in some embodiments, at 101, the plant can be co-transformed with nucleotide sequences encoding the PCM gene and the enzyme. In other embodiments, the plant part can be contacted with the nucleotide sequence encoding the PCM gene to form a PCM and then PCM tissue of the PCM can be contacted with nucleotide sequence encoding the enzyme.

[0080] The enzyme can be associated with the pathway for converting tyrosine to the betalain. Tyrosine can be naturally synthesized by the Cannabaceae plant. In some embodiments, the nucleotide sequence can additionally encode a reactant, such as tyrosine. For example, tyrosine can be upregulated or overexpressed due to the transformation. The betalain can be referred to as a secondary metabolite which is produced and/or increased in production due to transformation and expression of the enzyme. The enzyme can include a plurality of enzymes, such as different combinations of dihydroxyphenylalanine (DOPA) 4,5 -di oxygenase (DODA), Cytochrome P450 (CYP76AD1), CYP76AD6, glucosyltransferase, among other enzymes and combinations thereof. Example glucosyltransferase include, without limitation, betani din-5 -O- glucosyltransferase and cyclo-DOPA-5-O-glucosyltransferase. In some embodiments, a combination of multiple enzymes are expressed, such as DODA, CYP76AD1, and glucosyltransferase. For example, the three enzymes of DODA, CYP76AD1, and glucosyltransferase can create a heterologous pathway with a natively produced reactant of tyrosine to produce betanin. In other embodiments, two enzymes can be expressed. For example, the enzymes of DODA and CYP76AD1 can create a heterologous pathway with a natively produced reactant of tyrosine to produce betanidin. As another example, enzymes of DODA and CYP76AD6 can create a heterologous pathway with a natively produced reactant of tyrosine to produce betaxanthins. The different enzymes can be separated or linked by 2A self-cleaving peptides, such as P2A, F2A, T2A, and E2A. The 2A self-cleaving peptides induce ribosomal skipping during translation, thereby assisting in generating the separate enzymes during translation by causing the ribosome to fail at making a peptide bond.

[0081] In some embodiments, the enzyme and/or the betalain can be exogenous or heterologous to the Cannabaceae plant species (e.g., a wild-type plant does not express the betalain). In other embodiments, the enzyme and/or the betalain can be endogenous to the Cannabaceae plant species (e g., a wild-type plant expresses the betalain), and contacting the plant part with the nucleotide sequence encoding the enzyme and/or the bacterium strain can result in an increased level of expression of the enzyme(s), and/or betalain as compared to the wild-type plant and/or to expression of a derivative of the wild-type betalain. In some embodiments, the betalain includes a betacyanin or a betaxanthin. For example, the betalain can be selected frombetanin, isobetanin, probetanin, neobetanin, vulgaxanthin, miraxanthin, portulaxanthin, and indicaxanthin, among other types of betalain.

[0082] The transformation of the Cannabaceae plant part via the exposure to the expression construct can be transient or non-transient, e.g., stable. A stable transformation includes or refers to the nucleotide sequence encoding the enzyme being integrated into the Cannabaceae plant genome and as such represents a stable and inherited trait. A transient transformation includes or refers to the nucleotide sequence encoding the enzyme being expressed by the Cannabaceae plant cell transformed but may not integrated into the genome, and as such represents a transient trait. As used herein the term “transformation” or “transforming” can include or refer to a process by which foreign DNA, such as an expression construct including the DNA, enters and changes wild-type DNA.

[0083] The exposure to the expression construct and transformation can be accomplished by a wide variety of techniques. Such methods include, but are not limited to, particle bombardment mediated transformation (e.g., Finer et al, 1999, Curr. Top. Microbiol. Immunol., 240:59), protoplast electroporation (e.g., Bates, 1999, Methods Mol. Biol., 111:359), viral infection (e.g., Porta and Lomonossoff, 1996, Mol. Biotechnol. 5:209), microinjection, liposome injection, polyethylene glycol (PEG) delivery to protoplasts, and agroinfiltration. Other example techniques can be used to facilitate uptake by a cell of the nucleic acid include calcium phosphate and other chemical mediators of intracellular transport, microinjection compositions, and homologous recombination compositions (e.g., for integrating a gene into a preselected location within the chromosome of the cell). Other example techniques can involve the use of liposomes, electroporation, or chemicals that increase free DNA uptake, transformation using viruses or pollen and the use of microprojection. Various molecular biology techniques are common in the art (e.g., Sambrook et al, 1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York). Transformation methods can include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses and microprojection.

[0084] In some embodiments, the exposure to the expression construct and transformation can include contacting the Cannabaceae plant part with a bacterium strain that includes or carries the nucleotide sequence encoding the enzyme, as further described herein.

[0085] In some embodiments, the exposure to the expression construct and transformation of the Cannabaceae plant part can be provided via agroinfiltration. In some embodiments, the transformation is provided via Agrobacterium-mediated transformation (e.g., Komari et al., 1998, Curr. Opin. Plant Biol., 1:161), including floral dip transformation. Agroinfiltration can induce transient expression of genes in a plant to produce the betalain, by injecting a suspension including the bacterium strain containing the gene or genes of interest into the Cannabaceae plant part. In some embodiments, the transformation can be performed by an Agrobacterium-mediated gene transfer. The Agrobacterium-mediated gene transfer can include the use of plasmid vectors that contain DNA segments which integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the Cannabaceae plant species and the Agrobacterium delivery system. The transformation can be performed with any suitable tissue explant that provides a source for initiation of whole-plant differentiation (See Horsch et al., 1988. Plant Molecular Biology Manual A5, 1-9, Kluwer Academic Publishers, Dordrecht).

[0086] In some embodiments, the agroinfiltration technique can be implemented as described in PCT application PCT/US21/25067, entitled “Agrobacterium-mediated Infiltration of Cannabis”, filed on March 31, 2021, which is fully incorporated herein for its teaching and sometimes herein referred to as the “Cannabaceae agroinfiltration protocol”.

[0087] In some embodiments, the nucleotide sequence that encodes the enzyme is provided to a Cannabaceae cell via viral transformation (transduction) using a suitable plant virus, using gene gun techniques or electroporation.

[0088] In some embodiments, when the nucleotide sequence can be transformed into a plant cell, a Cannabaceae cell culture can be produced. [0089] In some embodiments, the transformation can be performed by a direct DNA uptake. There are various methods of direct DNA transfer into plant cells. In electroporation, the protoplasts are exposed to a strong electric field, opening up mini pores to allow DNA to enter. In microinjection, the DNA is mechanically injected directly into the cells using micropipettes. In microparticle bombardment, the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues.

[0090] In some embodiments, the transformation is performed in tissue culture on media and in a controlled environment. “Media” refers to the numerous nutrient mixtures that are used to grow cells in vitro, that is, outside of the intact living organism. Recipient cell targets include, but are not limited to, meristem cells, callus, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells. Callus can be initiated from tissue sources including, but not limited to, immature embryos, seedling apical meristems, microspores and the like. Cells capable of proliferating as callus are also recipient cells for genetic transformation.

[0091] In some embodiments, the transformation is performed on a Cannabaceae node explant. For example, a Cannabaceae node plant part can be transformed using a technique, which is performed using at least substantially the same features as disclosed in PCT application PCT/US21/21693, entitled “Transformation and Regeneration of Cannabaceae”, filed on March 10, 2021, which is fully incorporated herein for its teaching and sometimes herein referred to as the “Cannabaceae node transformation protocol.”

[0092] In some embodiments, the transformation is performed on a seedling explant or an embry onic axis (EA). For example a Cannabaceae seed can be imbibed in a hydration solution. The seed can be grown to full maturity, has an umblemished seed coat, and/or is free of bacteria, fungi, and other pest vectors. The hydration solution can include water, such as sterile distilled water or ddFhO. In some embodiments, the Cannabaceae seed can be imbibed by exposing the Cannabaceae seed to the hydration solution for a period of time and/or using a rotary shaker. In some embodiments, the Cannabaceae seed can be placed on a rotary shaker for between sixteen and twenty hours, between ten and fourteen hours, between twelve and fourteen hours, between fourteen and sixteen hours, between sixteen and eighteen hours, or between eighteen and twenty hours, among other ranges. [0093] Prior to imbibing the Cannabaceae seed, the Cannabaceae seed can be sterilized. The Cannabaceae seed can be sterilized using scarification and hydrogen peroxide, in some embodiments. The scarification can be provided by exposing the Cannabaceae seed to an acid, such as sulfuric acid. In some embodiments, the Cannabaceae seed can be exposed to the acid for a period of 1 second to 30 seconds, and then the acid is removed and the Cannabaceae seed is exposed to hydrogen peroxide or another sterilization agent, for a period of between 1 to 30 minutes. However, examples are not so limited, and can include sterilizing the Cannabaceae seed using a sterilization solution including sterilizing agents, such as a group consisting of ethanol, hypochlorite (NaCIO or Ca(C10)2), benzalkonium chloride, silver nitrate, mercuric chloride and hydrogen peroxide. The sterilization solution can contain the sterilizing agent within a range of 0.01% to about 95% by volume. The Cannabaceae seed can be exposed to the sterilization solution for a period of 0.1 to about 30 minutes. The sterilization solution can further include a mild detergent such as a polysorbate (e.g., TWEEN 20 or TWEEN 80) or other non-ionic surfactant. In some embodiments, Cannabaceae seed can be washed with a sterilization solution including about 10% hydrogen peroxide. The Cannabaceae seed can be placed in a sterile 50ml conical tube, and the sterilization solution added (e.g., by placing the 50ml tube on a rotary shaker). After immersion in the solution, the Cannabaceae seed can be rinsed several times. For example, the sterilized Cannabaceae seed can be rinsed with sterile distilled water 3-5 times, for 1-10 minutes each rinse. After sterilizing, the Cannabaceae seed can be kept in distilled water in closed Petri dishes in the laminar flow cabinet to prevent drying.

[0094] The seedling or EA explant may be prepared by excising a subset of embryonic tissue from the imbibed Cannabaceae seed to extract the seedling or EA explant. As used herein, a “Cannabaceae EA” includes and/or refers to a portion of the seed between the plumule and radicle, not including the cotyledons. The Cannabaceae EA can include the plumule, radicle, and hypocotyl. The portion of the embryo between the cotyledon attachment point and the radicle is referred to as the hypocotyl. The embryonic axis terminates in a radicle, which is the region from which the root develops. [0095] In some embodiments, excising the subset of the embryonic tissue can include removing a seed coat without removing either of the cotyledons of the imbibed Cannabaceae seed. For example, the seed coat and the embryonic tissue can be removed using sterile forceps or other excision tools (e.g., scalpel, scissors). In some embodiments, excising the subset of the embryonic tissue includes removing a seed coat and one of the cotyledons of the imbibed Cannabaceae seed. In some embodiments, excising the subset of the embryonic tissue includes removing a seed coat and cutting a radicle of the imbibed Cannabaceae seed. In some embodiments, excising the subset of the embryonic tissue includes removing a seed coat, both cotyledons, and leaf primordia of the imbibed Cannabaceae seed. In further embodiments, excising the subset of the embryonic tissue includes removing a seed coat, one of the cotyledons, and leaf primordia of the imbibed Cannabaceae seed. As noted above, removing the subset of the embryonic tissue can mitigate tissue damage, compared to excising all of the embryonic tissue, and thereby improving transformation efficiency and reducing time and costs associated with transformation. In various embodiments, the seed coat is removed using sterile forceps and then the embryos is ready for EA extraction. For example, the forceps can be used to hold the seedling, and a scalpel blade can be used to slice off one or both of the cotyledons. Care can be taken to prevent or mitigate damage to the meristem. In some embodiments, after removing a cotyledon(s), the scalpel blade can be used to remove the leaf primordia, taking care to prevent or mitigate damage to the meristem.

[0096] In some aspects, exposing the Cannabaceae plant part to the expression construct and inducing production of the betalain comprises contacting the Cannabaceae plant part with a bacterium strain transformed by the expression construct comprises the nucleotide sequence encoding the enzyme and culturing the Cannabaceae plant part to induce production of the betalain in the Cannabaceae plant part, as further described herein. [0097] In some embodiments, the exposure to the expression construct and transformation of the Cannabaceae plant part can include exposing the Cannabaceae plant part to a (first) nucleotide sequence encoding a gene that induces PCM and a (second) nucleotide sequence encoding the enzyme associated with production of the betalain. The contact with the (first and second) nucleotide sequences can be performed using any of above described techniques and which may transform cells of the plant part to express the nucleotide sequences and form a PCM and produce the betalain. In some embodiments, as further described below, the (first) nucleotide sequence encoding the gene that induces PCM formation can include or encode a root inducing (Ri) gene or plasmid that is expressed by plant cells of the plant part in response to the contact. In some embodiments, a bacterium strain that carries or is modified to carry the Ri gene, and optionally, the nucleotide that encodes the enzyme can be used to infect and transform the Cannabaceae plant part.

[0098] Using any of the above example transformation techniques and as previously described, the Cannabaceae plant part (e.g., node explant, EA explant, seedling explant, among others) is transformed using an expression construct. As used herein, an expression construct includes and/or refers to a nucleic acid sequence (e.g., DNA sequence) including a vector or vectors carrying genes. In various embodiments, the method 100 can include generating the expression construct that includes the nucleotide sequence that encodes the enzyme. A vector or binary vector includes and/or refers to a DNA sequence that includes a transgene, sometimes referred to as “inserts”, and a backbone. The vector or binary vector can include an expression cassette that includes the transgene and a regulatory sequence to be expressed by a transformed Cannabaceae plant cell. Successful transformation can result in the expression cassette directing Cannabaceae plant cells to make the enzyme and produce the betalain as a secondary metabolite.

[0099] As some non-limiting examples, the nucleotide sequence encoding the enzyme can comprise SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 36, or a combination thereof. In some embodiments, the expression cassette encoding a plurality of enzymes can comprise SEQ ID NO: 2, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 35, or SEQ ID NO: 39. In some embodiments, an expression construct can comprise SEQ ID NO: 1, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 38, or SEQ ID NO: 40, although embodiments are not so limited.

[0100] In some embodiments, a vector or binary vector carrying the gene associated with the enzyme and/or the betalain can include nucleic acid sequences encoding other gene editing reagents, such as rare-cutting endonucleases. The rare-cutting endonuclease(s) can be a transcription activator-like effector nuclease (TALE nuclease), a meganuclease, a zinc finger nuclease (ZFN), or a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) nuclease reagent. In some examples, a rare-cutting endonuclease can be implemented as 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; Schomack et al., J Plant Physiol, 163:256-272, 2006; and WO 2011/072246, each of which are incorporated herein in their entireties for their teachings.

[0101] In some embodiments, the vector or binary vector can include a transcription activator like effector nuclease (TALEN) sequence that encodes first and second TALE nucleases and binding domains to bind to target sites and cause a mutation at the target sites. The first TALE nuclease can generate a double stranded break at or near the first target site associated with a first binding domain and the second TALE nuclease can generate a double stranded break at or near the second target site associated with a second binding domain. In some embodiments, the first and second binding domains can be associated with a target gene. In some embodiments, the TALEN sequence can be co delivered to the Cannabaceae plant tissue with the secondary transgene to cause expression of the secondary transgene along with the PCM transgene. In some embodiments, the TALEN sequence and PCM transgene (as further described herein) are delivered separately. In some embodiments, the TALEN sequence can encode tyrosine and/or otherwise cause upregulation or overexpression of tyrosine in the formed PCM. [0102] As noted above, examples are not limited to TALENs and can include CRISPR/Cas systems (see, e.g., Belahj et al., Plant Methods, 9:39, 2013), among others or may not include the gene editing reagents. In some examples, a Cas9 endonuclease and a guide RNA can be used (either a complex between a CRISPR RNA (crRNA) and trans activating crRNA (tracrRNA), or a synthetic fusion between the 3' end of the crRNA and 5 'end of the tracrRNA (sgRNA)). The guide RNA directs Cas9 binding and DNA cleavage to homologous sequences that are adjacent to a proto-spacer adjacent motif (PAM). Once at the target DNA sequence, Cas9 generates a DNA double-strand break at a position three nucleotides from the 3' end of the crRNA targeting sequence. In some embodiments, this approach or other approaches, such as ZFN and/or meganucleases, can be used in addition to TALE nucleases to obtain modified Cannabaceae plant parts.

[0103] The method 100 can include preparing a Cannabaceae plant part, such as an explant, to be exposed to the prepared expression construct. Cells of the Cannabaceae plant part can be transformed with the expression construct suitable for expression of the enzyme and production of the betalain. Different Cannabaceae plant parts, such as hypocotyl, leaf, stem, stalk, petiole, meristem, a node, an intemode, shoot tip, seed, cotyledon, protoplast, storage root, or tuber, can be used. For different species, the most efficient explant material can vary in tissue/organ source and age. Juvenile material (e.g., from one to five days germinated seed, three to ten day seedling) can be optimal for at least some plants. The explant can include Cannabaceae plant tissue that has been wounded. The wounded tissue can be infected by contact with or immersion into a prepared bacterium strain culture or otherwise exposed to the expression construct via other techniques. For example, the plant tissue can be immersion into and/or submerged in the bacterium strain culture. Appropriate media and incubation conditions for transformation and/or infection, co-cultivation, and tissue induction can depend on the explant to be transformed. The transformed explant can be cultured to enhance or optimize transformation and development, as further described herein.

[0104] At 103, the method 100 further includes inducing production of the betalain in the transformed Cannabaceae plant part. Expression of the enzyme and resulting production of the betalain can be induced using various techniques, such as regeneration and/or culturing techniques and respective culture mediums, among other techniques, and can result in production of the betalain. Culturing the transformed Cannabaceae plant part can include inducing formation of tissue from the transformed Cannabaceae plant part and/or culturing the tissue in a culture medium(s) under growth conditions for expression of the nucleotide sequence. Example culture mediums are provided below. For example, the method 100 can include screening new growth from the cultured Cannabaceae plant part for tissue formation. For tissue induction, the Cannabaceae plant part can be transferred into liquid or solid media with antibiotics two or three days after exposure to the expression construct or co-cultivation. Suitable antibiotics include cefotaxime sodium, carbencilin disodium, vancomycin, ampicillin sodium, claforan, streptomycin sulphate, and tetracycline, and combinations thereof. The amount of antibiotic to kill or eliminate redundant bacteria can range in concentration from 100 to 500 pg/mL. The Cannabaceae tissue, such as PCM tissue, can be induced within a short period of time, which can vary from one week to over a month depending on the plant species.

[0105] In some embodiments, the explant is transformed and tissue is regenerated from the transformed Cannabaceae cells. The tissue may include shoots, roots, root hair structures, and full plants. In some embodiments, PCM tissue are regenerated. In other embodiments, regenerating tissue can include inducing formation of shoots from the transformed Cannabaceae cells. For example, the induction of shoot formation can include inducing shoot formation and inducing shoot elongation. In some embodiments, regenerating the tissue can include inducing formation of shoots from the transformed Cannabaceae cells and inducing roots from the formed shoots.

[0106] In some embodiments, inducing shoot formation can include transferring and culturing a Cannabaceae EA in a shoot inducing medium (SIM), such as SIM +S100 as further illustrated below. The Cannabaceae EA can be oriented in the SIM with the radicle placed down in the SIM medium and the apical meristem placed up, similar to placing a plant with roots in the ground and such that the radicle can soak the SIM medium up. After culturing in the SIM medium, the method 200 can include transferring and culturing the Cannabaceae EA in a first shoot elongation medium (SEM). In some embodiments, prior to transferring to the first SEM, the radicle of the shoots can be cut using a scalpel blade or other tool. After culturing in the first SEM, the method 200 can further include transferring and culturing the Cannabaceae EA in a second SEM. For both the first SEM and the second SEM, the radicle of the Cannabaceae EA is oriented down into the SEMs and the apical meristem placed up. The first SEM and second SEM can include different amounts of a selection agent, such as different amounts of spectinomycin.

[0107] In some embodiments, roots can be induced from the shoots. Inducing roots from the formed shoots can include screening the formed shoots for shoots of a minimum height, such as shoots that are one to two inches in height. Shoots of the minimum height can be selected and the method 100 further includes rooting the selected shoots to induce primary root formation, and transferring and rooting shoots with the induced primary shoots to induce new primary shoots and root hair structure formation. In various embodiments, the select shoots are rooted in a rooting medium (RM) and subcultured in fresh RM to induce the new primary roots and root hair structures. The method 200 can further include transferring selected shoots with primary roots and root hair structures to soil to regenerate partial or whole Cannabaceae plants that express the polypeptide, such as proteins of interest.

[0108] The infection medium, co-cultivation medium, selection medium, SIM, first SEM, second SEM, and RM, as described above, generally comprise water, a basal salt mixture, a sugar, and other components such as vitamins, selection agents, amino acids, and phytohormones. Each of the SIM, the first SEM, and the second SEM can include sugars, basal salts, growth hormones, and antibiotic agents, among other reagents, such as water and vitamins. For example, the SIM and SEMs can include nutntional sources of nitrogen, phosphorus, potassium, sulfur, calcium, magnesium, iron, boron, molybdenum, manganese, cobalt, zinc, copper, chlorine, and iodine. Macroelements can be provided as NH4NO3, (NH4)2S04, KNO3, CaCb 2H2O, MgS04-7H₂0, and KH2PO4. Micro elements can be provided as KI, H3BO3, MnSC>4-4H₂0, ZnSCb, Na₂MoC>42H₂0, CuSC>4-5H₂0, C0CI2 6H2O, CoS04-7H₂0, FeSCri 7H2O, and Na₂EDTA 2H₂0. Organic supplements such as nicotinic acid, Pyridoxine-HCl, Thiamine-HCl, and glycine can be included. Generally, the pH of the medium is adjusted to 5.7±0.5 using dilute KoH and/or HC1. Solid plant culture media can further include a gelling agent such as, for example, gelrite, agar or agarose.

[0109] Any suitable plant culture medium can be used. Examples of media formulations include but are not limited to Driver and Kuniyuki Walnut Basal Salt (DKW, 1984), Murashige and Skoog (1962), N6, Linsmaier and Skoog (1965), L3 (Lin and Zhang (2005)), Uchimiya and Murashige (1962), Gamborg's media (1968), Nitsch andNitsch (1969), and Schenk and Hildebrandt (1972).

[0110] The SIM and SEMs can include can include selection agents, phytohormones and/or plant growth regulators such as, for example, auxins, cytokinins, or gibberellins. The phytohormones can be selected from free and conjugated forms of naturally occurring phytohormones or plant growth regulators, or their synthetic analogues and precursors. Naturally occurring and synthetic analogues of auxins include, but are not limited to, indoleacetic acid (IAA), 3-indolebutyric acid (IBA), a-napthaleneacetic 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 can be present in the nutritive media. Natural cytokinins and synthetic analogues of cytokinins include, but are not limited to, kinetin, zeatin, zeatin riboside, zeatin riboside phosphate, dihydrozeatin, isopentyl adenine 6-benzyladenine and combinations thereof Any combinations of two or more cytokinins can be present in the mediums.

[0111] Presence of an effective amount of the auxin, and optionally an effective amount of the cytokinin, can promote cell division, improve regenerability, and/or induce the growth of more regenerative tissue. The effect of exogenous auxin to produce a morphological response can be enhanced by the addition of an antioxidant, amino acids, cobalt, or AgNC . Casamino acids provide a source of organic nitrogen in the form of amino acids hydrolyzed from Casein that can tolerate high salt conditions without degrading. Glutamine, asparagine, and methionine play complex roles in regulation of biosynthetic pathways that result in morphogenic response.

[0112] In some embodiments, the new growth is screened to identify the transformed tissue and the identified transformed tissue is separated and sub-cultured in the culture medium under conditions for expression of the nucleotide sequence. For example, the method 100 can include selecting tissue from the transformed Cannabaceae plant part for culturing in the culture medium or culture mediums and screening the cultured tissue for expression of the nucleotide sequence. In other embodiments, the identified PCM tissues are re-transformed with a second bacterium strain, as described above, and then sub cultured. As used herein, the PCM phenotype includes and/or refers to roots that tend to resemble thick, fluffy cords as compared to wild-type roots that are long, thin, and smooth. Thus, visual phenotype selection can be used. PCM tissue, as used herein, includes and/or refers to tissue (e.g., roots) exhibiting the PCM phenotype. PCM tissue, in accordance with various embodiments, is isolated from photosynthetic wild-type tissue and, therefore, may not contain any remaining photosynthetic wild-type tissue. Alternatively or additionally, the culture medium can include a liquid culture medium or a solid growth medium which is hormone-free, e.g., has an absence of added plant growth hormones. The absence of the added plant growth hormones can be used to select transformed Cannabaceae tissue over wild type as the wild-type tissue can die in the absence of the growth hormone when grown in the dark. A further selection technique can be used, such as selection agent or reporter gene. In some embodiments, the culture medium can further include a selection agent, such as an antibiotic or herbicide to select Cannabaceae tissue that produces the betalain. In some embodiments, a reporter gene, such as yellow fluorescent protein (YFP) or red fluorescent protein (RFP), can be used to further transform the Cannabaceae plant part and to allow for selection of the Cannabaceae tissue that contains the second transgene. In various embodiments, the PCM tissue or other types of transformed tissue is isolated from photosynthetic wild-type tissue and, therefore, may not contain any remaining photosynthetic wild-type tissue. The resulting PCM culture similarly may not contain any remaining photosynthetic wild-type tissue.

[0113] In some embodiments, the transgenic Cannabaceae tissue strains can be isolated and characterized. For example, the method 100 can include screening and selecting transformed tissue (such as cultured infected plant parts or other types of transformed Cannabaceae plant parts) for expression of the enzyme using end point RT-PCR or fluorescent protein reporter expression (e.g., RFP or YFP) in formed Cannabaceae tissue. However, embodiments are not so limited and other molecular biology methods can be used, such as DNA-sequencing, southern blot analysis, northern blot analysis, and/or western blot analysis.

[0114] Due to the site uncertainty of integration of the nucleotide sequence(s) into the plant cell genome, Cannabaceae tissue strains can show different expression patterns for the enzyme(s) and/or production of the betalain. Expression and/or production levels can be measured using biochemical analysis to quantify betalain concentration in the medium (e.g., Lowry. Bradford, BCA, Spectroscopy, HPLC, LC-MS, and UV spectroscopic protein assays). Cannabaceae tissue strains having the desired pattern and level of expression can be identified by the presence of the betalain in the media. Subculture and selection can be performed repeatedly to obtain betalam-producing Cannabaceae tissue lines that secrete the betalain at high levels on a biomass basis (e.g., per gram dry weight).

[0115] To initiate a Cannabaceae tissue culture in liquid medium, a piece of a transformed Cannabaceae plant part can be transferred to a culture vessel. Any conventional plant culture medium can be used in the practice of the present invention; multiple plant culture media are commercially available as dry (powdered) media and dry basal salts mixtures, for example.

[0116] In some embodiments, the method 100 can include capturing the betalain. The betalain can be captured by isolating and purifying the betalain from the culture medium and/or from the transformed Cannabaceae plant tissue (e.g., PCM tissue). Recovery of the produced betalain from the spent media can include primary recovery steps (e.g., conditioning and pretreatment) and purification steps (e.g., capture and polishing). The spent media is typically concentrated, clarified, and conditioned prior to a chromatography (capture) step. Conditioning and pretreatment of the betalain can include steps to maximize product binding by capture chromatography and the lifetime of capture chromatography media (e.g., affinity resins), reduce binding of plant components to the betalain, and stabilize the betalain for purification, such as conditioning by crossflow filtration, pH adjustment, and dead-end filtration, in any order. Typically, conditioning can include adjusting media pH, ionic strength, and buffer composition. Conditioning can further include removing plant impurities that can interfere with the method of purification, reducing overall plant protein burden; and reducing betalain exposure to phenolics and proteases, such as by two-phase partitioning, adsorption, precipitation, and membrane filtration. Conditioning can further include a reducing the media volume (e.g., by cross-flow filtration).

[0117] The betalain can be isolated and purified from other components of the spent media. For example, a betalain can be isolated and purified from the spent media using a recovery step. In some embodiments, the recovered betalain is at least 60% pure, e.g., greater than 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% pure.

[0118] To enhance recovery of the betalain produced and secreted by the transformed Cannabaceae plant part or resulting tissue culture, an effective amount of a compound stabilizing agent can be added to the growth medium. In general, compound stabilizing agents can include any substance conventionally employed during purification of a particular polypeptide to maintain compound concentration and activity by preventing protein degradation and denaturation, or any substance that blocks nonspecific interactions between the secreted betalain and walls of the culture vessel. In some embodiments, resins can be used for betalain recovery and/or purification. The resins can be used to remove phenolics that may impact plant health or the resins (or other absorbents) may be used for in situ product removal, such as for removing the betalain(s) out of the media. A compound stabilizing agent for use in tissue culture media should not support or encourage bacterial growth in the culture medium or be phytotoxic at the concentrations employed. Preferably, the compound stabilizing agent is used at levels that does not substantially reduce tissue culture cell viability and integrity, protein expression, and growth and cell division. In addition, the stabilizing agent may not interfere with purification of the produced betalain. An “effective amount” of a compound stabilizing agent is an amount, when added to a given volume of a tissue culture medium that significantly improves recovery of a betalain from the medium, e.g., increasing betalain recovery by a statistically significant amount. Preferably, recover is increased by at least 20%, as compared with control medium that is otherwise identical except that it lacks the compound stabilizing agent. Compound stabilizing agents include without limitation preservatives and antimicrobials (e.g., benzalkonium chloride, glycerol, sodium azide, thymol), carbohydrates (e.g., sucrose, lactose, sorbitol, trehalose), antioxidants and reducing agents (e.g., Dithiothreitol, EDTA, 2-Mercaptoethanol), amino acids, derivatives of ammo acids and betalain), and polymers (e.g., polyethylene glycol, polyvinylpyrrolidone).

[0119] In some embodiments, the betalain is not secreted, or not fully secreted, by the Cannabaceae tissue culture. The betalain can accumulate in tissue or cells of the Cannabaceae tissue culture. When the tissue culture has been grown to the desired stage, the culture or a portion thereof, can be harvested, and the betalain can be isolated from the harvested material using conventional methods. For example, harvested tissue can be ground and the betalain extracted with appropriate solvents. The crude product can then be purified in accordance with the nature of the product. [0120] Purifying typically starts with extraction of the betalain and removal of any plant insolubles. Betalain yields and purity in the crude extracts can be improved through screening of different solvent systems. This can be done by adjusting solvent, pH, and buffer condition. For betalains intended for use in high purity applications (e.g., food, cosmetics, and drug colorants), multiple purification steps can be implemented to remove impurities and to ensure high product quality. Purification of plant-derived betalain is dependent on betalain properties and impurities that coextract. The skilled artisan is capable of adapting strategies for extraction and purification employed with other systems that endogenously and/or heterologously produce betalain (e.g., plant and microbial systems). Thus, after extracting, purification procedures can use techniques developed for betalain products. Purification of the betalain can include adsorption chromatography, solid-phase extraction, or other forms of extraction to enrich betalain while removing impurities. A v ariety of resins and solvent conditions are available for these purification steps. The skilled artisan can select an appropriate resin based on the expression level of the betalain, spent media complexity and its effect on purification efficiency, product stability during processing, and removal methods for critical impurities. Resin selection is determined by betalain and impurity properties, such as charge, hydrophobicity, and biospecificity. Selecting a resin based on the property most unique to the betalain compared to the other products of the PCM system can improve purification efficiency by increasing binding capacity and/or product purity. Once suitable chromatography resin functionality is determined (cation/anion-exchange, reversed phase chromatography or anion, hydrophobic), vanous resins with different particle sizes, surface areas, and resin backbones can be screened for purification efficiency at different binding conditions, such as solvent, pH and ionic strength conditions. Further purification steps can be implemented to maximize separation of betalain from impurities, to achieve target purity based on the product application. These purifications can include a variety of orthogonal steps such gel permeation column chromatography, normal phase column chromatography, reverse-phase column chromatography, ion-exchange chromatography, aqueous two-phase extraction, reverse- phase high performance liquid chromatography. [0121] FIG. 2 illustrates an example method for transforming a Cannabaceae plant part to induce expression an enzyme, consistent with the present disclosure. At 215, the method 210 includes preparing a wild-type Cannabaceae plant part, such as a cutting (e.g., hypocotyl segment) or seedling excised from a host Cannabaceae plant.

[0122] At 217, the method 210 includes exposing the wild-type Cannabaceae plant part to an expression construct. In some embodiments, the exposure can include inoculating, injecting, and/or agroinfiltratmg the wild-type Cannabaceae plant part with a bacterium strain solution. For example, the bacterium strain can be transformed to carry the nucleotide sequence encoding the enzyme. Once such an expression construct is available, bacterium strains can be transformed to carry the vector or binary vector and used to infect wild-type Cannabaceae plant part. In other embodiments, the wild-type Cannabaceae plant part can be exposed to the expression construct via bombardment technique, as previously described. Selection of transformed Cannabaceae plant parts can be performed using a plant selective agent (e.g., spectinomycin), which can enrich the formation of high expressing transformed Cannabaceae plant parts (versus non- transformed Cannabaceae plant part, such as PCM tissue carrying only the Ri plasmid T- DNA or wild-type roots) and, optionally, increase the expression of genome editing reagents in Cannabaceae tissues.

[0123] At 219, the method 210 includes culturing and/or screening the transformed Cannabaceae plant part. For example, the method 210 can include transferring tissue, such as leafs, cuttings and/or whole seedlings, exposed to the expression construct to a medium for selection of transgenic tissue, such as transgenic PCM tissue or other types of transformed Cannabaceae plant tissue. Other types of plant tissue can be used, such as a petiole, an intemode, or a leaf. The degree of editing in the Cannabaceae plant part can be directly related to the abundance of the enzyme and/or betalain in tissue and can be tracked using various methods of enzyme and/or betalain detection. For instance, Cannabaceae plant parts can be assayed for accumulation of the betalain in new plant tissue. The tissue from the transformed new plant growth can be sampled for detection of the betalain using RT-PCR, Spectroscopy, microscopy, LC-MS, F1PLC, or western blot, respectively. Plant growth positive for the betalain can be screened for detection of edits using Illumina® amplicon sequencing of the target gene. Root growth positive for edits can be propagated either vegetatively or through other methods known to stabilize edits in individual Cannabaceae plants.

[01 4] However, embodiments are not limited to culturing and can include other techniques, such as de novo induction or in plant dipping and/or vacuum infiltration and which would be well known to one of ordinary skill. Further, embodiments are not limited to co-transformation and may include infecting plant parts to induce PCM formation and re-transforming the PCM tissue to produce the betalain.

[0125] At 221, the method 210 can include isolating the betalain, such as previously described and/or using a system as described below.

[0126] Various embodiments described above and including method 210 transforming and, optionally re-transforming a Cannabaceae plant part to produce the betalain. The transformation and re-transformation can be accomplished by a wide variety of techniques as previously described.

[0127] FIGs. 3A-3B illustrate different example methods for expressing an enzyme in a Cannabaceae plant part, consistent with the present disclosure.

[0128] FIG. 3A illustrates a method 330 for expressing the enzyme in a Cannabaceae plant part using a bacterium strain. At 332, the method 330 includes contacting a Cannabaceae plant part with a bacterium strain comprising the expression construct that includes the nucleotide sequence encoding the enzyme. For example, the plant part can be contacted with the bactenum strain via submersion, spraying, dripping, and/or other forms of contact. In some embodiments, as further described herein, the contact can include contact with a liquid culture containing the bacterium strain, sometimes herein referred to as a “liquid bacterium medium”. In some examples, as further described herein, a bacterium strain can include multiple strains, such as a first bacterium strain to induce PCM formation and a second bacterium strain to induced production of the betalain.

[0129] The bacterium strain can include a strain capable of infecting the Cannabaceae plant part and, in response, inducing expression of the enzyme. In various embodiments, contacting the Cannabaceae plant part with the bacterium can include injecting the bacterium strain into the Cannabaceae plant part, agromfiltrating the bacterium strain into the Cannabaceae plant part, and/or culturing the Cannabaceae plant part to enhance transformation, induce tissue formation, and induce expression of the enzyme and production of an associated betalain.

[0130] In some embodiments, the bacterium strain is capable of inducing PCM formation and/or transformed to induce expression of the enzyme. For example, the bacterium strain can comprise an Ri plasmid or a Ti plasmid, a nucleotide sequence encoding a gene that induces PCM formation (e.g., PCM gene), and/or a nucleotide sequence encoding the enzyme. For example, the gene can induce PCM phenotype in Cannabaceae plant parts and can non-transiently transform the Cannabaceae plant parts. The transformed Cannabaceae plant part is stably modified by the bacterium strain to form the PCM and the PCM can be cultured to maximum production of the betalain.

[0131] As used herein, a PCM includes and/or refers to plant cells transformed by a nucleotide sequence encoding a gene that includes PCM formation, which can include a plurality of different plant cell types and can be used to produce the betalain. In some embodiments, the PCM is a tissue culture including the transformed plant cells, e g., the plurality of different plant cell types. The PCM can include plant cell types including, but not limited to, plant stem cells, maturing cells, and mature cells. In some examples, the PCM is produced by infecting plant cells with the bacterium strain, or otherwise contacting with the nucleotide encoding the gene that induces PCM formation, to induce PCM phenotype and to form the PCM. For example, the PCM is formed by isolating the tissue associated with the PCM phenotype from the wild-type tissue. Although the above describes forming the PCM using a bacterium strain, embodiments are not so limited. In some embodiments, the Cannabaceae plant part can otherwise be contacted with a (heterologous) nucleotide sequence encoding the Ri plasmid and/or gene that induces PCM formation, which transforms plant cells to express the nucleotide sequence.

[0132] In some embodiments, the bacterium strain can carry the nucleotide sequence encoding the enzyme that includes SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 36, or a combination thereof. In some examples, the bacterium strain can be transformed with an expression cassette that includes SEQ ID NO: 2, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 35, or SEQ ID NO: 39 and/or an expression constmct SEQ ID NO: 1, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 38, or SEQ ID NO: 40, although embodiments are not so limited. [0133] In some embodiments, the contact with the bacterium strain can be under infection conditions that induce and/or enhance transformation of the Cannabaceae plant part to express the PCM phenotype and/or produce the betalain. The infection conditions can include use of the liquid bacterium medium, a type of bacterium, and/or a type or an amount of contact with the bacterium stain, among other conditions. The bacterium strain can include the specific species or line of bacteria. The type or amount of contact with the bacterium strain can include immersion, spraying, dripping, and/or other contact in a time range of one to five days for co-cultivation.

[0134] The bacterium strain can include a Rhizobia strain, such as a Rhizobium strain or Agrobacterium strain. In some embodiments, the bacterium strain includes a Rhizobium rhizogenes strain (R. rhizogenes), formerly known as Agrobacterium rhizogenes. R. rhizogenes is a Rhizobium species that can be used to transform plant cells and is sometimes preferred due to high virulence and rapid development of transgenic materials in the form of hairy roots and/or a PCM. These Rhizobium strains have not been disarmed, meaning that the Rhizobium strains contain original T-DNA which causes hairy root disease symptoms on infected plants contained on the Ri plasmid. PCMs resulting from R. rhizogenes infection of plant tissue carry the T-DNA from the Ri plasmid and form vascular connections with their plant hosts. These vascular connections allow the PCM tissue (e.g., roots) to function similarly to wild-type roots, and can grow aggressively and out-compete wild-type roots. The PCM transgene can be transferred during infection along with any secondary transgene introduced into the bacterium strain using electroporation and other cloning techniques. For example, in addition to the TDNA from the Ri plasmid, additional TDNAs can be co-delivered or delivered separately to the plant part and expressed in PCM cultures as transgenic PCM tissue, such as those from vectors carrying a gene associated with the betalain production. The T- DNA(s) from the bacterium strain can be stably integrated in the Cannabaceae plant part. [0135] Many strains of R. rhizogenes exist and can be used for Cannabaceae plant transformation. The strain can be an octopine, agropine, nopaline, mannopine, or cucumopine strain. Suitable strains of R. rhizogenes for use can include American Type Cell Culture (ATCC) 43057, ATCC 43056, ATCC 13333, ATCC 15834, and K599. In some embodiments, the bacterium strain is ATCC 43057, ATCC 43056, ATCC 13333, or ATCC 15834. In some embodiments, the bacterium strain used to infect the Cannabaceae plant part can include an Ri plasmid that includes the nucleotide sequence encoding the gene that induces PCM formation and can include the nucleotide sequence encoding the enzyme. For example, the Ri plasmid carries the gene that induces PCMs, sometimes herein referred to as “the PCM gene” for ease of reference, and a separate T-DNA carries the nucleotide sequence encoding the enzyme. Other example bacterium strains, which can be used for re-transforming PCM tissue, include 18rl2, GV3101, AGL1, and EHA105.

[0136] However, embodiments are not so limited. In some embodiments, the bacterium strain can be transformed to cany' the PCM gene. For example, the bacterium strain can include a Ti plasmid and may not carr the gene that induces PCM formation. A Ti plasmid can carry a gene capable of inducing tumors. The Ti plasmid can be disarmed by deleting the tumor inducing gene and introducing the gene that induces PCM formation using a T-DNA. In some embodiments, the bacterium strain can be transformed to include a disarmed Ti plasmid, the nucleotide sequence encoding the gene that induces PCM formation, and the nucleotide sequence encoding the enzyme. In some embodiments, the bacterium strain can be transformed to include a disarmed Ri plasmid, the nucleotide sequence encoding the gene that induces PCM formation, and the nucleotide sequence encoding the enzyme. For example, a first T-DNA can carry the PCM gene and a second T-DNA can carry the nucleotide sequence encoding the enzyme. In some embodiments, multiple bacterium strains may be used, as further described below. The bacterium strain can be transformed using a vector or vectors carrying the genes. In some embodiments, no bacterium strain is used to transform the plant cells and/or induce PCM formation. For example, the vector(s) carrying the genes can be used to transform the plant cells of the plant part without the use of a bacterium strain.

[0137] Embodiments are not limited to PCM formation and other types of bacterium strains can be used and/or different Cannabaceae plant parts can be transformed. As described above, the Cannabaceae plant parts can be transformed via injection, agroinfiltration, bombardment, among other techniques. Further, other types of bacteria stains can be used. In other embodiments, PCMs can be formed without the use of a bacterium strain by otherwise contacting Cannabaceae plant part with a nucleotide sequence encoding the PCM gene.

[0138] Generally, the bacterium strain is prepared for contacting (e.g., infecting) the Cannabaceae plant part by introducing a nucleotide sequence encoding the enzyme into the bacterium strain (e.g., by electroporation) and culturing the transformed bacterium strain under infection conditions to select positively transformed cells. The nucleotide sequence encoding the enzyme is heterologous to the bacterium strain.

[0139] In some embodiments, any of the above described methods can include selecting the particular bacterium strain. Parameters such as the transformed tissue induction percentage per total explants, the transformed tissue initiation days per total explants, and the transformed tissue induction frequency per single explant can be measured to select the bacterium strain.

[0140] The method used for bacterium strain infection can vary but can include the preparation of a fresh wild-type shoot (cut at the stem) or seedling (cut at the hypocotyl) cuttings, and inoculation of the cut end with the bacterium strain. In some embodiments, cocultivation of the Cannabaceae plant part on media can facilitate delivery of both a Ri plasmid (or a disarmed Ti plasmid) and vector or binary vector T-DNAs to the wild-type tissue. Binary, superbinary, pGreen or co-integrate vectors containing appropriate genes (e.g., encoding the enzyme) and selectable markers and/or reporter genes can be prepared and transferred into the bacterium strain. Suitable vectors contain right and left T-DNA border sequences to allow for delivery of the DNA into the Cannabaceae plant cells. In various embodiments, the method 330 can include transforming a wild-type bacterium strain with the nucleotide sequence encoding the enzyme, and, in some embodiments, with the gene that induces transformed Cannabaceae tissue formation. In some embodiments, two bacterium strains can be prepared: the first bacterium strain to induce PCMs and the second bacterium strain transformed to include the nucleotide sequence. [0141] The method 330 can also include preparing a Cannabaceae plant part, such as an explant, to be inoculated with the prepared bacterium strain, as previously described in connection with the method 100.

[0142] In various embodiments, transforming the Cannabaceae plant part includes simultaneously or sequentially introducing a first transgene and a second transgene to the plant part, and cultivating the transformed plant part to generate transformed tissue, such as cultivating the PCM tissue under the below-described growth condition (e.g., culturing performed under the growth conditions). The first transgene can be associated with PCM formation, and the second transgene can be associated with the enzyme and/or production of betalain. In some embodiments, the first transgene is naturally occurring in the bacterium strain and the second transgene is non-naturally occurring and/or transgenic. In some embodiments, both the first transgene and the second transgene are non-naturally occurring and/or transgenic. In some embodiments, no bacterium strain is used and the first and second transgene can both be heterologous to the Cannabaceae plant part, and are simultaneously introduced to the Cannabaceae plant part using any of the above- described techniques.

[0143] In some embodiments, the Cannabaceae plant part is first transformed using the first transgene that induces the PCM phenotype to produce PCM tissue and the PCM tissue is isolated from wild-ty pe Cannabaceae tissue and retransformed using the second transgene associated with the enzyme. For example, the first transformation can include a protocol involving a first bacterium strain as described above (e g., culturing to form PCM tissue), and the second retransformation can include exposing the formed PCM tissue to the second bacterium strain, such as 18rl2. Other types of bacterium strains can be used as the second bacterium strain, including GV3101, AGL1, and EHA105. Examples are not limited to use of bacterium strains for either transformation.

[0144] As an example, contacting the Cannabaceae plant part with the bacterium strain and culturing the plant part, at 332 of the method 330, can include contacting the Cannabaceae plant part with a first bacterium strain comprising the nucleotide sequence encoding the gene that induces PCM formation, culturing the Cannabaceae plant part to enhance PCM formation, such as under the growth conditions as further described below, and contacting the formed PCM tissue from the PCM with a second bacterium strain comprising the nucleotide sequence encoding the enzyme, and culturing the PCM tissue to enhance production of the betalain by the PCM, as under the below-described growth conditions.

[0145] Although FIG. 3 A illustrates contact with a bacterium strain to induce PCM formation and production of the betalain, PCM can be formed without use of bacterium strain in various embodiment. In some embodiments, the above-described simultaneous exposure or re-transformation can be performed using other transformation techniques which may or may not include use of bacterium strain(s).

[0146] In various embodiments, PCM formation can be induced by contacting the Cannabaceae plant part with a nucleotide sequence encoding the gene that induces PCM formation and a nucleotide sequence encoding the enzyme. The contact can be performed with and/or without bacterium strain by simultaneously contacting the Cannabaceae plant part with both nucleotide sequences or performing a re-transformation. For example, exposing the Cannabaceae plant part to the expression construct and culturing the Cannabaceae plant part can comprise simultaneously introducing a first transgene and a second transgene to the Cannabaceae plant part, and cultivating the Cannabaceae plant part as transformed to generate PCM tissue and produce betalain, such as under the above-described growth conditions.

[0147] In other embodiments, PCM formation can be induced using a re-transformation methodology. For example, exposing the Cannabaceae plant part to the expression construct and culturing the Cannabaceae plant part can comprise contacting the plant part with the (first) nucleotide sequence encoding the gene that induces PCM formation, culturing the formed PCM tissue under growth conditions to enhance PCM formation, contacting PCM tissue of the PCM with the (second) nucleotide sequence encoding the enzyme, and culturing the formed PCM tissue under growth conditions to enhance production or expression of the betalain

[0148] At 334, the method 330 includes culturing the Cannabaceae plant part to induce expression of enzyme and production of the betalain in the Cannabaceae plant part. Inducing expression of the enzyme can include culturing the Cannabaceae plant part as transformed to enhance transformation and induce expression of the enzyme. As previously described, culturing an infected Cannabaceae plant part can include inducing formation of tissue (e.g., PCM or other tissue) from the infected Cannabaceae plant part and culturing the tissue in a culture medium under conditions for expression of the nucleotide sequence encoding the enzyme. In response to the expression and production of the enzyme, the enzyme can convert tyrosine, produced by the plant part, to the betalain. [0149] In various embodiments, the Cannabaceae plant part can be cultured with the bacterium strain to induce PCM formation, and optionally production of betalain, or otherwise is contacted with the nucleotide sequence(s) encoding the PCM gene and/or the betalain, and then cultured in another culture medium or a plurality of culture mediums to enhance further PCM tissue growth and/or production of the betalain. In some embodiments, the Cannabaceae plant part is contacted and co-cultured with the bacterium strain under the infection conditions to transform the Cannabaceae plant part and for a period of time (e.g., one to five days). After the period of time, the bacterium strain is removed and/or killed, such as using antibiotics, and the transformed Cannabaceae plant part is cultured using a culture medium.

[0150] In some embodiments, the Cannabaceae plant part transformed using any of the above-described techniques and methods described herein (and not limited to infection with a bacterium strain) can be cultured under growth conditions to enhance PCM formation and/or production of the betalain. The growth conditions can include a liquid culture medium, a type of culture medium, a type or amount of contact with the culture medium, and a plant type. The liquid culture medium can include a culture medium in a liquid form. The type of culture medium can include a liquid-based medium containing sugar and Driver and Kuniyuki Walnut (DKW) basal salts, Murashige and Skoog (MS) basal salts, or Woody Plant basal salt mixtures (WPM), herein sometimes generally referred to as “DKW”, “MS”, and “WPM” for ease of reference. In some embodiments, the type of culture medium can include a culture medium containing a pH buffer, such as 2-(N morpholino) ethanesulfonic acid (MES) buffer (e.g., lg/L of MES buffer), among other types of buffers, such as bis-tris buffer. The pH buffer can prevent or mitigate pH shifts. In some embodiments, the culture medium can include a liquid-based medium containing sugar, DKW or MS, and a pH buffer, among other components. However, embodiments are not limited to liquid culture mediums and can include solid culture mediums with sugar, DKW, MS, and/or a pH buffer. The type or amount of contact with the culture medium can include an intermittent contact, spraying, dripping, and/or contact or contact cycle in a time range of one week to three months. As further described below, in some embodiments, the growth conditions can additionally include providing supplemental gas, such as oxygen, to the plant tissue. [0151] The plant type can include the specific Cannabaceae species. In some embodiments, the plant type include a Cannabacea plant part selected from a seedling (e g., hypocotyl), a petiole, meristem, a node, an intemode, or a leaf.

[0152] In some embodiments, the Cannabaceae plant type can include a specific plant line and/or clone of the Cannabaceae plant that exhibits greater PCM formation and/or betalain production than other plant lines and/or clones. For example, within a Cannabaceae plant species, there can be genetic variability which causes different optimized tissue formation from the PCM or other transformed plant tissue compared to other plant lines and/or clones. A plurality of Cannabaceae plant lines and/or clones of the plant line(s) can be transformed to form PCMs and screened to identify the particular plant line and/or clone with the optimized PCM formation and/or betalain production among the plurality of plant lines and/or clones after the exposure to the expression construct and/or contact with the nucleotide sequence that induced PCM formation followed by culturing with a culture medium, such as a liquid culture medium. In some embodiments, the specific plant line and/or clone of the Cannabaceae plant can be screened for and/or selected by culturing the plurality of Cannabaceae plant clones of different plant lines (and/or plurality of plant clones of a plant line), as transformed by expression construct and/or nucleotide sequences, using an intermittent contact with the liquid culture medium or other type of culture medium containing the sugar and basal salt, as described above and further described below, and which can result in enhanced growth rates among the plurality of PCMs formed and with a greater dynamic range of growth rates among the plurality of PCMs as compared to PCMs formed using a constant contact with the liquid culture medium and/or use of other types of culture mediums (e.g., solid mediums) for inducing tissue growth of the plant part transformed to express the PCM phenotype. A dynamic range of growth rates can include a difference between the fastest growing PCM and the slowest growing PCM among the plurality of PCMs formed. By having a greater dynamic range, selection of the optimal or subset of optimal PCMs among the plurality can occur faster and/or more easily as compared to a lower dynamic range. An optimized or optimal PCM includes and/or refers to a PCM or subset of PCMs exhibiting the greatest growth rate(s) among the plurality of PCMs. For example, a user can visually select the optimized or subset of optimized PCMs among the plurality of PCMs. In some embodiments, the growth rates of the plurality of PCMs can be measured and compared to select the optimized PCM or subset of optimized PCMs. [0153] In some embodiments, as noted above, the type of contact can comprise intermittently contacting the Cannabaceae plant part with the culture medium, such as with a liquid culture medium. Intermittent contact, as used herein, includes and/or refers to cycling between contact of the Cannabaceae plant part with the culture medium and no contact of the Cannabaceae plant part with the culture medium. By providing an intermittent contact, the transformed Cannabaceae plant part is provided with nutrients (e.g., sugars and basal salts) for growth during times of contact with the liquid culture medium, and is provided with air or other gases for growth during times of no contact with the liquid culture medium. In contrast, with constant contact, parts of the PCM tissue of the PCM formed may be in the liquid or other types of media at all times and may not have access to air or other gases as needed for survival and/or growth. In some embodiments, the growth conditions can further include exposure to a supplemental gas and a type of gas. The supplemental gas can be provided to the Cannabaceae plant part, such as during no contact times (e.g., no contact with the liquid culture medium). The liquid culture medium or other type of media can be drained or otherwise removed during the no contact times.

[0154] In some embodiments, the intermittent contact comprises cycling between contacting (e.g., submerging, dripping, or other types of contact) the Cannabaceae plant part with the culture medium and not contacting (e.g., submerging, dnpping, or other types of contact) the plant part with the culture medium at a duty cycle of between 1 percent and 25 percent, such as with a liquid culture medium. A duty cycle, as used herein, refers to the percentage of time that the Cannabaceae plant part is in contact with the culture medium as compared to the time the Cannabaceae plant part is not in contact. For example, the Cannabaceae plant part can be contacted for ten minutes and not contacted by the culture medium, such as the liquid culture medium, for fifty minutes, every hour over a total period of time of about one week (e.g., seven days) to about three months (e.g., ninety days) or more, resulting in a duty cycle of 16.67 percent over the total period of time. In some embodiments, the total period of time includes between about two weeks (e.g., fourteen days) and about three months, about two weeks and about two months (e.g., sixty days), about two weeks and about one month (e.g., thirty days), about twenty days and about three months, about twenty days and about two months, about twenty days and about one month, about one month and about three month, or about one month and about two months, among other ranges of periods of time.

[0155] According to various embodiments, the producing the betalain from the Cannabaceae plant part can include contacting a plant part with a bacterium strain containing a Ri plasmid or a Ti plasmid, a nucleotide sequence encoding an enzyme, and a nucleotide sequence encoding a gene that induces PCM formation. After contacting, the PCM tissue is cultured in a culture medium under the growth conditions to induce expression of the nucleotide sequence. However, other types of bacterium strains can be used to transform the tissue and the tissue is cultured under the growth conditions to enhance betalain production.

[0156] In various embodiments, the growth conditions can enhance tissue formation and/or production of betalain, resulting in production of transformed Cannabaceae tissue at a greater level than production than production of Cannabaceae tissue (e.g., root tissue) by a wild-type plant or plant grown in the field. For example, the production of the transformed Cannabaceae tissue can be at least about two-fold to about 500-fold compared to production of tissue (e.g., root tissue) by the wild-type plant or plant grown in the field and/or at a growth rate of at least about 2-fold to about 500-fold compared to the production of the tissue by wild-type plant or plant grown in the field. In some embodiments, the production of the PCM tissue by the PCM can be at a growth rate that is about 2-fold to about a 500-fold, about a 4-fold to about a 500-fold, about an 8-fold to about a 500-fold, about a 10-fold to about a 500-fold, about a 15-fold to about a 500-fold, about a 20-fold to about a 500-fold, about a 20-fold to about a 400-fold, about a 20-fold to about a 300-fold, about a 20-fold to about a 100-fold, about a 15 -fold to about a 400- fold, about a 15-fold to about a 300-fold, about a 15-fold to about a 200-fold, about a 15- fold to about a 100-fold, about a 15-fold to about a 50-fold, or about a 15-fold to about a 30-fold, compared to the production of the tissue by wild-type plant or plant grow n in the field. As used herein, growth rate includes and/or refers to an amount of root biomass produced in a period of time, which can include a mass level (e.g., grams (g)) of transformed Cannabaceae tissue produced by the Cannabaceae tissue culture m a period of time and can optionally be per unit of area. Mass level or mass includes and/or refers to the amount of biomass produced (e.g., grams per square meter per month of dry tissue) by the Cannabaceae tissue culture, such as grams of Cannabaceae tissue or root tissue.

For example, the Cannabaceae tissue culture, such as a PCM, can produce Cannabaceae tissue at a greater mass level than root tissue produced by a wild-type-plant or as grown in the field.

[0157] In some embodiments, the Cannabaceae tissue culture can produce transformed tissue at a mass level that is at least a 2-fold (or times), at least a 3-fold, at least a 4-fold, at least an 8-fold, at least a 10-fold, at least a 15-fold, at least an 18-fold, at least a 20- fold, at least a 25-fold at least a 30-fold, at least a 40-fold, at least a 50-fold, at least a 100-fold, at least 200-fold, or at least 500 fold increase as compared to the root tissue produced by a wild-type plant and/or as grown in the field. In some embodiments, the Cannabaceae tissue culture can include transformed tissue produced at a mass level that is at about a 2-fold to about a 500-fold, about a 4-fold to about a 500-fold, about an 8-fold to about a 500-fold, about a 10-fold to about a 500-fold, about a 15 -fold to about a 500- fold, about a 20-fold to about a 500-fold, about a 20-fold to about a 400-fold, about a 20- fold to about a 300-fold, about a 200-fold to about a 100-fold, about a 15-fold to about a 400-fold, about a 15-fold to about a 300-fold, about a 15-fold to about a 200-fold, about a 15-fold to about a 100-fold, about a 15-fold to about a 50-fold, or about a 15-fold to about a 30-fold, among other range increases in the tissue mass as compared tissue mass (e.g., root tissue) produced by a wild-type plant and/or as grown in the field.

[0158] In some embodiments, the methods described herein can include culturing the Cannabaceae plant part under the growth conditions to induce and enhance transformed Cannabaceae tissue formation and to induce production of betailain by the transformed Cannabaceae tissue. In various embodiments, the Cannabaceae plant part can be cultured with the bacterium strain to induce transformed tissue formation or otherwise contacted with the nucleotide sequence(s) encoding the gene that induces PCM formation (e.g., the PCM gene) and/or encoding the enzyme (both being heterologous to the plant), and then cultured in another culture medium or a plurality of culture mediums to induce production of the betalain, such as in liquid or solid culture mediums. [0159] As described above, in some embodiments, the method 330 can include two infections. For example, the method 330 can include infecting the plant part with a first bacterium strain comprising the Ri plasmid or the Ti plasmid and including the nucleotide sequence encoding PCM gene and culturing the infected plant part to induce PCM formation. The method 330 can further include infecting the formed PCM tissue with a second bacterium strain, which can comprise an Ri plasmid or a Ti plasmid, and includes the nucleotide sequence encoding the enzyme. In some embodiments, the second bacterium strain can induce further PCM formation, and in other embodiments, may not (e g., may include or may not include the PCM gene). The second transformation can be caused by exposing the PCM tissue to the second bacterium strain, such as by dipping the PCM in a solution containing the bacterium strain or pipetting bacterium strain onto the PCM tissue.

[0160] In various embodiments, the gene that induces PCM formation, which can be encoded by the nucleotide sequence, can include a plurality of genes that induce PCM formation (e.g., a plurality of PCM genes) and/or a plurality of nucleotide sequences can encode the plurality of PCM genes, such as a plurality of different PCM genes.

[0161] In some embodiments, a method can include and/or the methods of FIG. 1, FIG.

2, and/or FIG. 3A can further include identifying the bacterium strain from a plurality of bacterium strains. For example, a method can include transforming a plurality of Cannabaceae plant parts with a plurality of bacterium strains to induce transformed tissue formation and/or production of the betalain, and assessing transformation frequencies of the plurality of bacterium strains therefrom. In some embodiments, the plurality of Cannabaceae plant parts are transformed with modified bacterium strains, such as bacterium strains carrying a nucleotide sequence encoding a gene that induces PCM formation and/or a nucleotide sequence encoding the enzyme as described above. In some embodiments, the plurality of Cannabaceae plant parts are transformed with wild-ty pe bacterium strains, such as those that induce PCM formation. In some embodiments, the plurality' of bacterium strains can include a plurality of Rhizobium strains, a plurality of Agrobacterium strains, or combinations thereof. In some embodiments, the plurality of bacterium strains can include a plurality of R. Rhizogenes strains. In some embodiments, the plurality of bacterium strains can include a plurality of Agrobacterium tumefaciens strains. The method can further include selecting respective ones of the plurality of bacterium strains based on the transformation frequencies. For example, the respective ones of the plurality of bacterium strains with the highest transformation frequency or frequencies among the plurality of bacterium strains can be selected. In some examples, the selected bacterium strain is ATCC 43057, ATCC 43056, ATCC 13333, ATCC 15834, and/or K599. In some embodiments, the selected bacterium strain is ATCC 43057, ATCC 43056, ATCC 13333, or ATCC 15834. In some embodiments, the selected bacterium strain is ATCC 43057, ATCC 43056, or ATCC 13333.

[0162] FIG. 3B illustrates a method 335 for expressing the enzyme in a Cannabaceae plant part using an expression construct and a bombardment technique. At 337, the method 335 includes exposing Cannabaceae plant part to the expression construct that includes the nucleotide sequence encoding the enzyme via bombardment and to transform Cannabaceae plant part with the expression construct. For example, exposing the Cannabaceae plant part to the expression construct via bombardment can include using a bombardment gun and/or particles coated with the expression construct. Specific examples of bombardment driven transformation is provided above, and would be known to one of ordinary skill in the art.

[0163] At 339, the method 335 includes inducing expression of the enzyme in tissue of the transformed Cannabaceae plant part. Inducing expression of the enzyme can include culturing the transformed Cannabaceae plant part to enhance transformation and induce expression of the enzyme and/or production of the associated betalain, such as under the above-described growth conditions. In some embodiments, the method 335 can be used to simultaneously (or sequentially) induce PCM formation and production of betalain using a nucleotide sequence encoding the gene that induces PCM and the nucleotide sequence encoding the enzyme, as previously described.

[0164] FIGs. 4A-4C illustrate example expression constructs for delivery of a gene encoding an enzyme, consistent with the present disclosure. The example expression construct 440 is or includes a vector containing an expression cassette 441 and a vector backbone 446. The expression cassette 441 includes a transgene that causes production of a betalain. The transgene of the expression cassette 441 includes a gene of interest 445 associated with the betalain, a promoter 447, a left border 449, and a right border 448. The expression construct 440 and/or expression cassette 441 can include various additional components, such as TALE sequences, a selection agent, a terminator, and an additional expression cassette, among other components, such as signaling peptides, compound markers, and/or compound purification tags.

[0165] As described above, the expression cassette 441 includes the sequence encoding the enzyme (e.g., gene of interest 445), T-DNA border sequences 448, 449, and a promoter 445. Expression cassettes typically include a promoter operably linked to a nucleotide sequence of interest (e.g., that encodes the enzyme), which is optionally operably linked to termination signals and/or other regulatory elements. For example, the expression cassette 441 can include TALEN T-DNA. The expression cassette 441 can 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 the tissue culture media. Enzymes comprising signal peptides of plant origin (e.g., the N-terminal signal peptide from the tobacco PRla protein or calreticulin) or signal peptides from eukary otic secreted polypeptides, e.g., mammalian signal peptides, can be efficiently secreted through the plasma membrane and cell wall into the extracellular medium.

[0166] The expression cassette 441 compnsing the nucleotide sequence of interest can be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette 441 can also be one which is naturally occurring or assembled entirely extracellularly (e.g., by recombinant cloning techniques). An expression cassette can be obtained by placing (or inserting) a promoter sequence upstream of an endogenous sequence, which thereby becomes functionally linked and controlled by the inserted promoter sequence.

[0167] In some embodiments, the promoter can include an inducible promoter, a strong promoter, or a root-tissue specific promoter. For example, the nucleotide sequence encoding the enzyme can be operably connected to the inducible promoter, strong promoter, or root-tissue specific promoter. However, embodiments are not so limited and the promoter can include a weak or low-level promoter. In some embodiments, the promoter can include a constitutive promoter. An inducible promoter can be switched on and off, whereas a constitutive promoter can always be active. For example, the nucleotide sequence encoding the enzyme can be operably connected to an ubiquitin promoter, a figwort mosaic promoter (FMV), or a 35S Cauliflower Mosaic Virus (CMV) promoter.

[0168] A promoter typically includes at least a core (basal) promoter, but can also include a control element. Such elements include upstream activation regions (UARs) and, optionally, other DNA sequences that affect transcription of a nucleic acid, which can include synthetic upstream elements. Factors for selecting a promoter to drive expression of the copy include efficiency, selectability, inducibility, desired expression level, and cell- or tissue-type specificity. The promoter can be one which preferentially expresses in root tissue or under certain conditions, e.g., is a root-tissue specific promoter. The promoter can be modulated by factors such as temperature, light or stress. For example, inducible promoters can be used to drive expression in response to external stimuli (e.g., exposure to an inducer). Suitable promoters include, but are not limited to, a light- inducible promoter from ssRUBISCO, MAS promoter, rice actin promoter, maize ubiquitin promoter, PR-I promoter, CZ19B1 promoter, milps promoter, CesA promoter, Gama-zein promoter, Glob-1 promoter, maize 15 kDa zein promoter, 22 kDa zein promoter, 27 kDa zein promoter, d-zein, waxy promoter, shrunken 1 promoter, shrunken 2 promoter, globulin 1 promoter, pEMU promoter, maize H3 histone promoter, beta- estradiol promoter, and dexamethasone-inducible promoters. Non-limiting examples of constitutive promoters include 35S promoter, such as 35S CMV promoter, 2x 35S promoter, nopaline synthase (NOS) promoter, ubiquitin-3(ubi3), among others.

[0169] A promoter for driving expression in the PCM culture can have strong transcriptional activity. A strong promoter drives expression of the enzyme encoding nucleic acid at a high level, or at about 1/10 transcripts to about 1/100 transcripts to about 1/1,000 transcripts. Enhancers can be utilized in combination with the promoter regions to increase transcription levels. When the enzyme and/or betalain is endogenous to the plant species, the expression cassette can be effective for achieving at least a 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold increase in the level of expression compared to the expression level of the endogenous enzyme and/or betalain in the plant tissue in which it is normally found. [0170] The nucleotide sequence encoding the enzyme can include a DNA sequence derived from various organisms, including but not limited to, humans and other mammals and/or vertebrates, invertebrates, plants, sponges, bactena, fungi, algae, archaebacteria, etc. Additionally, synthetic betalains are expressly contemplated, as are derivatives and analogs of any betalain. The DNA sequence can encode an enzyme having at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of a corresponding wild-type enzyme. In some embodiments, the DNA sequence has significant similarity and shared functional domains with the sequence encoding the betalain. The DNA sequence can be obtained from a related organism having a homologous, orthologous, or paralogous gene to a gene encoding the enzyme. In general, the methods for identifying conserved or similar DNA sequences and constructing recombinant genes encoding betalains, optionally with various modifications for improved expression (e.g., codon optimized sequences), 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 yield a gene with convenient restriction sites for cloning, or subcloning from another already cloned source, or cloning from a library.

[0171] A number of nucleic acids can encode the enzyme having a particular amino acid sequence. Codons in the coding sequence for a given enzyme can be modified such that optimal expression in Cannabaceae plants is obtained using appropriate codon bias tables. For example, at least some of the codons present a heterologous gene sequence that can be modified from a triplet code that is infrequently used in plants to a triplet code that is more common in plants.

[0172] In some embodiments, the DNA sequence can include the sequence of a gene occurring in the wild-type Cannabaceae plant, or a sequence having a percent identity that allows it to retain the function of the gene encoded product, such as a sequence with at least 90% identify. This sequence can be obtained from the organism or organism part or can be synthetically produced. The sequence can have at least 91%, 92%, 93%, 94%,

95%, 96%, 97%, 98%, 99% identity to the gene occurring in the wild-type organism. The sequence can be inserted at a different locus than that of the wild-type gene and be operably linked to a different promoter than the wild-type gene. [0173] FIG. 4B illustrates different example expression cassettes, which can form part of the expression construct 440 of FIG. 4A, consistent with the present disclosure. More particularly, FIG. 4B illustrates expression cassettes 430, 431, 432 associated with production of different betalains. Expression cassette 430 is associated with production of betanin, and encodes a promoter, CYP76AD1, a first 2A self-cleaving peptide, DODA, a second 2A self-cleaving peptide, glycosyltransferase, and a terminator. Expression cassette 431 is associated with production of betanidin, and encodes a promoter, CYP76AD1, a 2A self-cleaving peptide, DODA, and a terminator. Expression cassette 432 is associated with production of betaxanthins, and encodes a promoter, CYP76AD6, a 2A self-cleaving peptide, DODA, and a terminator. In some embodiments, betaxanthins can include a mixture of molecules, such as amines or amino acids which can be spontaneously added to the betalamic acid.

[0174] FIG. 4C illustrates example pathways for converting tyrosine to a betalain using the enzymes expressed in response to transformation of plant cells with an expression construct illustrated by FIGs. 4A-4B, consistent with the present disclosure.

[0175] FIGs. 5A-5B illustrate different example methods for generating an expression construct comprising a sequence encoding an enzyme, consistent with the present disclosure. The methods 550 and/or 550 of FIGs. 5A-5B can be combined with any of the methods 100, 210, 330, 335 of FIGs. 1-3B, in some embodiments.

[0176] FIG. 5A illustrates an example method 550 for transforming a bacterium strain to comprise a sequence encoding an enzyme, consistent with the present disclosure.

[0177] At 552, the method 550 includes transforming a bacterium strain with the nucleotide sequence encoding the enzyme. For example, the nucleotide sequence encoding the enzyme can include SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 36, or a combination thereof. In some embodiments, the bacterium strain is a wild- type bacterium strain including a Ri plasmid that carries the nucleotide sequence encoding a gene that induces PCM formation (e.g., PCM gene). In some embodiments, the bacterium strain is a wild-type bacterium strain that does not carry the gene that induces PCM formation, such as bacterium strain including a Ti plasmid or a bacterium strain without a Ri plasmid or a Ti plasmid. In such embodiments, transforming the bacterium strain can include disarming the Ti plasmid and transforming with both the nucleotide sequences encoding the enzyme and the gene that induces PCM formation. [0178] However, embodiments are not so limited and other types of bacterium strains can be used that may not include a Ri plasmid and/or Ti plasmid. For example, the bacterium strain can include a Ochrobactrum strain and/or an Ensifer adhaeren strain, among other bacteria strains and combinations thereof.

[0179] In other embodiments, two bacterium strain may be used, the first including a wild-type bacterium strain that carries the PCM gene or that is transformed to carry the PCM gene, and a second that is transformed to carry the nucleotide sequence encoding the enzyme. For example, the method 550 may include transforming a first bacterium strain to carry the PCM gene and a second bacterium strain to carrying the nucleotide sequence encoding the enzyme.

[0180] The bacterium strain can be transformed using an expression construct, such as a vector that includes an expression cassette, as previously described. In some embodiments, the bacterium strain is transformed using an expression construct that includes the vector or plurality of vectors or binary vectors carrying genes. The expression construct can include the vector(s) carrying gene(s). The vector or binary vector can include a right T-DNA border sequence, a left T-DNA border sequence, the nucleotide sequence encoding the enzyme, and a promoter, such as including an expression cassette and vector backbone. An example expression construct can include a first vector that includes the nucleotide sequence encoding the enzyme and a second vector that includes the sequence encoding the PCM gene. Each of the first and second vectors can include right and left T-DNA border sequences and a promoter. However embodiments are not so limited, and in some embodiments the bacterium strain already carries the PCM gene or does not carry the PCM gene. In some embodiments, the bacterium strain can be transformed in two separate transformation processes or using a vector carrying two or more transgenes (e.g., including multiple expression cassettes). In some examples, the bacterium strain can be transformed using an expression constmct comprising SEQ ID NO: 1, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 38, or SEQ ID NO: 4 and/or a nucleotide sequence encoding the enzyme comprising SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 36, or a combination thereof, although embodiments are not so limited. As described above, embodiments are not limited use of a bacterium strain. In various embodiments, an expression construct, as described herein, can be used to transform plant cells of a plant part without use of a bacterium strain. [0181] An example expression construct including a vector is illustrated by FIG. 4A, as previously discussed. In various embodiments, the method 550 further includes generating the expression construct including the nucleotide sequence encoding the enzyme.

[0182] At 554, the method 550 includes culturing the transformed bacterium strain. For example, the bacterium strain can be cultured under the above-described infection conditions, such in a rich media, such as Luria-Bertani (LB), a yeast extract peptone (YEP) media, and other rich media known in the art, or a minimal media, such as AB media (see media recipes below) and other minimal media known in art with appropriate antibiotics, as further described below. In some embodiments, a first bacterium strain and a second bacterium strain can be cultured. In some embodiments, a first bacterium strain and a second bacterium strain can be cultured.

[0183] FIG 5B illustrates another example method 555 for transforming a bacterium strain to comprise a sequence encoding an enzyme, consistent with the present disclosure. At 557, the method 555 includes generating an expression construct including a nucleotide sequence encoding the enzyme, as described above. At 559, the method 555 includes generating a solution and/or a suspension including the expression construct for transforming a Cannabaceae plant part. The solution and/or suspension can including particles which the expression construct is attached to. For example, the method 555 can further include attaching the expression construct to the particles in suspension for performing bombardment.

[0184] In either methods 550 and 555, the transformed bacterium strain and/or the solution or suspension can be used to transform a Cannabaceae plant part to express the enzyme and to produce the betalain, as previously described.

[0185] Although FIGs. 5 A-5B illustrate transforming a bacterium strain for transforming a Cannabaceae plant part, embodiments are not so limited. In some embodiments, a wild- type bacterium strain can be used to transform the Cannabaceae plant part to form a PCM, which can be enhanced by culturing the transformed plant part under growth conditions. In some embodiments, the Cannabaceae plant part can be transformed using other transformation techniques which may not include use of a bacterium strain, as described above.

[0186] Various embodiments of the present disclosure are directed to a non-naturally occurring Cannabaceae plant part, such as transformed Cannabaceae tissue generated by the methods of FIG. 1, FIG. 2 and/or FIGs. 3A-3B.

[0187] Various embodiments of the present disclosure are directed to a Cannabaceae tissue culture generated by the methods of FIG. 1, FIG. 2, and/or FIGs. 3A-3B, such as a PCM culture. For example, the tissue culture can be used for producing a betalain, the tissue culture being induced from a Cannabaceae plant part and an expression construct, wherein a plant cell of the tissue culture comprises a s nucleotide sequence encoding the enzyme associated with the production of the betalain. In some embodiment, the PCM culture is induced using nucleotide sequence(s) encoding the gene that induces PCM formation and encoding the enzyme. In some embodiments, the nucleotide sequence encoding the enzyme comprises SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 36, or a combination thereof. In some embodiments, the expression construct includes an expression cassette comprising SEQ ID NO: 2, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 35, or SEQ ID NO: 39.

[0188] In some embodiments, the tissue culture can be used to produce the betalain. Various embodiments are directed to a system for producing the betalain from the Cannabaceae tissue. For example, the system can include a plurality of bioreactors in serial connection, wherein each bioreactor is inoculated with the tissue culture according to and/or obtained using any of above-described methods, and configured for growth and maintenance of the tissue culture in a culture medium.

[0189] In embodiments of the present disclosure, the transgenic tissue cultures are maintained in a bioreactor system. The tissue culture can be grown in a temporary immersion system, a plastic sleeve reactor, a bubble reactor, a mist reactor, an airlift reactor, a liquid-dispersed reactor, or a bioreactor configured to generate micro- or nano bubbles. A bioreactor can be any vessel adapted for receiving sterile growth media and enclosing the plant tissue therein. In some cases, a bioreactor is a flask (e.g., an Erlenmeyer flask). In some embodiments, the bioreactor can include or be implemented substantially similar to a commercially available bioreactor, such as commercially available temporary immersion systems including Plantform bioreactor™ and RITA®. [0190] A system comprising a plurality of bioreactors in serial connection for large scale production of the betalain of interest is described herein. Each of the connected bioreactors can be structurally and operationally similar. Each bioreactor is configured with a growth chamber for housing the tissue culture, an inlet. In some embodiments, each bioreactor includes an outlet. In some cases, one inlet of each bioreactor is connected to an air compressor configured to provide sterilized air to the tissue cultures. The air can be oxygen-enriched air. Substantially pure molecular oxygen can be provided. The bioreactors can include a separate inlet in fluid connection with a media supply system configured to provide growth media to the tissue cultures. The connections can be made at the beginning of a growth/harvesting cycle (e.g., when the bioreactor is inoculated with the PCM culture) under anoxic conditions. The sterilized air and/or media can be provided continuously, or in predetermined pulses, during each culturing cycle.

The system can be configured to remove excess air and/or waste gases by one of the outlets.

[0191] The bioreactor system can include holding tanks for media and additives. For example, micro elements, macro elements and vitamins, and additives such as antibiotics or fungicides can be held in different tanks. The system can include a mixer fed by a pump that delivers each component of the media at the desired relative proportions. The media can be delivered from the mixer by a delivery pipe having an aseptic connector. [0192] The bioreactor system is configured to permit collection of the media for betalain recovery. For example, the bioreactor can include a media outlet that can be closed by a valve. A portion of spent media can be removed from each bioreactor by opening the valve before or as fresh media is supplied to the bioreactor. The removal can be achieved under gravity, whereby the spent media flows into a conduit connected to each of the bioreactors to pool spent media. In addition, the system can permit media to be harvested from each bioreactor separately. The conduit can include a sample port that allows for collection of smaller samples of the spent media for detecting secretion of the betalain. The conduit can be configured for conditioning and pretreatment of the spent media. In some embodiments, the conduit is in fluid connection with components for capture of the secreted betalain (e g., ion-exchange columns). The system can be configured for continuous recovery of the secreted betalain once the tissue culture achieves a steady state of protein secretion. In some embodiments, the spent media can flow into a removable recovery tank for batch-wise purification of the secreted betalain. The recovery tank can be removed from the bioreactor system periodically and the contents decanted for isolation and purification of the secreted betalain.

[0193] The operation of the bioreactor system can be controlled by circuitry, such as a processor and/or computer that includes a processor and memory. The circuitry can be configured to control parameters such as temperature, amount and timing of air entering the bioreactors and/or exit of waste gases, amount and timing of the addition of culture medium, and/or amount of light. The circuitry can be connected to the conduit or a sample port. The circuitry can control an automated sampler and/or media harvester for removing portions of the spent media for testing and/or recovery. The circuitry can also optionally be connected to an analyzer to provide feedback for operation of the circuitry. [0194] Some embodiments are directed to a betalain produced by a Cannabaceae plant part transformed by an expression construct using a method and/or tissue culture of any of the methods, culture, system, and/or tissue provided herein.

[0195] As used herein, Cannabaceae refers to a plant of the family Cannabaceae. For example, the Cannabaceae plant or plant part can include a plant or plant part that belongs to the genus of Cannabis, sometimes referred to as a cannabis plant or plant part, and which includes Cannabis sativa, Cannabis indica, and Cannabis ruderalis). However, embodiments are not so limited, and the Cannabaceae plant or plant part can include Humulus (e.g., hops), Celtis, Alphananthe, Chaetachme, Gironniera, Lozanella, Parasponia, Pteroceltis, and/or Trema plants or plant parts, among other plants or p parts. The term ^‘‘plant” generally refers to whole plants, but when “plant” is used as an adjective, refers to any substance which is present in, obtained from, derived from, or related to a plant, such as plant organs (e.g., leaves, stems, roots, flowers), single cells (e.g., pollen), seeds, plant cells including tissue cultured cells, products produced from the plant. The term “Cannabaceae plant part” refers to plant tissues or organs which are obtained from a whole plant of the family Cannabaceae. Cannabaceae plant parts include vegetative structures (for example, leaves, stems), roots (for example, PCM tissues or non-PCM tissue), floral organs/structures, seed (including embryo, endosperm, and seed coat), plant tissue (for example, vascular tissue, ground tissue, and the like), cells and progeny of the same. The term “Cannabaceae plant cell” refers to a cell obtained from a plant or in a plant of the family Cannabaceae, and includes protoplasts or other cells derived from plants, gamete-producing cells, and cells which regenerate into whole plants. Plant cells can be cells in culture. “Cannabaceae plant tissue” means differentiated tissue in a plant or obtained from a plant (“explant”) or undifferentiated tissue derived from immature or mature embryos, seeds, roots, shoots, fruits, pollen, and various forms of aggregations of plant cells in culture, such as calli. Plant tissues in or from seeds, such as Cannabaceae seeds, include a seed coat or testa, storage cotyledon, and embryo. A “plant clone” is a plant or plant part produced via well-known plant cloning processes. A plurality of clones can be produced from a single individual plant through asexual reproduction. A “plant line” or “bacterium line” (or strain) refers to a particular strain of the plant or bacteria.

[0196] The production “betalain of interest” corresponds to any betalain that can be produced by the method according to the present disclosure. The betalain of interest can be endogenous to the Cannabaceae plant, or exogenous. In a case where the betalain is endogenous to the plant, e.g., produced naturally by the Cannabaceae plant, the betalain of interest is overproduced with respect to an untransformed Cannabaceae plant.

[0197] The skilled artisan would recognize that various terminology as used in the Specification (including claims) connote a plain meaning in the art unless otherwise indicated. The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. Furthermore, when “about” is utilized to describe a value or percentage this includes, refers to, and/or encompasses variations (up to +/- 10%) from the stated value or percentage.

[0198] The singular forms “a”, “and”, and “the” include plural references unless the context clearly dictates otherwise. For example, singular forms of “a PCM gene” or a “gene that induces PCM formation”, as used herein, includes a single PCM gene and a plurality of PCM genes in different embodiments (e.g., one or more PCM genes). As other non-limiting examples, “a nucleotide sequence”, “a vector”, “an expression construct”, “an expression cassette”, “a plant part”, “a culture medium”, “an enzyme”, “a transgene”, “a PCM culture”, “a bacterium strain”, among others singular forms of elements or components includes a singular form and a plurality form of the element or component, such as one or more nucleotide sequences, one or more vectors, one or more expression constructs, one or more expression cassettes, one or more a plant parts, one or more culture mediums, one or more enzy mes, one or more transgene, one or more PCM cultures, one or more bacterium strains, among others.

[0199] Various embodiments are implemented in accordance with the underlying provisional applications: (i) U.S. Provisional Application No. 63/162,702, filed on March 18, 2021 and entitled “Expressing Recombinant Compounds Using Cannabaceae Hairy Roots”; (ii) U.S. Provisional Application No. 63/304,850, filed on January 31, 2022 and entitled “Expressing Recombinant Compounds Using Plant Cell Matrices”; (iii) U.S. Provisional Application No. 63/240,660, filed on September 3, 2021 and entitled “Producing Betalains Using Hairy Roots”; and (iv) U.S. Provisional Application No. 63/241,857, filed on September 8, 2021 and entitled “Betalain in Cannabaceae” and to each of which benefit is claimed and each are fully incorporated herein by reference. For instance, embodiments herein and/or in the provisional applications can be combined in vary ing degrees (including wholly). Embodiments discussed in the provisional applications are not intended, in any way, to be limiting to the overall technical disclosure, or to any part of the claimed invention unless specifically noted.

[0200] Based upon the above discussion and illustrations, those skilled in the art will recognize that various modifications and changes can be made to the embodiments without strictly following the exemplary embodiments and applications illustrated and described herein. For example, methods as exemplified in the Figures can involve steps carried out in various orders, with aspects of the embodiments herein retained, or can involve fewer or more steps. Such modifications do not depart from the scope of various aspects of the disclosure, including aspects set forth in the claims. EXPERIMENTAL EMBODIMENTS

[0201] As further illustrated below in connection with the experimental embodiments, expression constructs were generated and used to transform a Cannabaceae plant part to produce a betalain. Different experiments were conducted to illustrate successfully transforming bacterium strains and transforming a Cannabaceae plant part and solanum tuberosum plant part. The transformed Cannabaceae plant parts exhibited production of a betalain. Example constructs and sequences used to experimental embodiments include the nucleotide sequences set forth in SEQ ID NOs: 1-45. SEQ ID NOs: 1-45 are each synthetic DNA.

[0202] FIGs. 6A-6F illustrate example expression constructs for delivery of a sequence encoding an enzyme, consistent with the present disclosure. Each expression construct for bacterium strain transformation (e.g., R. rhizogenes transformation) contains a right T- DNA border sequence and a left T-DNA border sequence, to allow the bacterium strain to deliver the DNA into the plant cells. The expression construct are plasmids and can be referred to as plasmid vectors. The expression constructs further include a betalain cassette, such as a DNA sequence coding the enzyme for producing betalain from tyrosine, which is codon optimized according to the codon bias used by the target, and cloned in binary' vectors, are under the regulation of a promoter, such as a FMV promoter, and a terminator. The enzymes includes CYP76AD1, CYP76AD6, DODA, and/or glucosyltransferase, and various combinations thereof. The enzymes are separated by 2A self-cleaving sequences, such a sequences encoding F2A or P2A. For example, a first 2A self-cleaving sequence links CYP76AD1 to DODA and a second 2A self-cleaving sequence links DODA to glucosyltransferase. Constitutive promoters and root specific promoters are selected for tissue-specific approaches. The expression constructs further include additional cassettes, such as a plant selectable marker cassette, a LacZ cassette, and a bacterial selection maker cassette. The additional cassettes are oriented in reverse on the plasmid as compared to the betalain cassette.

[0203] FIG. 6A illustrates an example plasmid vector 640 that includes a DNA sequence coding for a plurality of enzymes to produce betalain (e.g., betanidin). The plasmid vector 640 contains the gene encoding the enzymes CYP76AD1, DODA, and glucosyltransferase driven by a FMV promoter, with CYP76AD1 linked to DODA and DODA linked to glucosyltransferase by 2A self-cleaving peptides F2A. The plasmid vector 640 further includes a plant selectable marker cassette and a LacZ cassette, which are in reverse orientation on the plasmid vector 640, as further described below. The plant selectable marker cassette encodes a selection marker that when expressed, confers resistance to a selection agent (e.g., bacteria or other toxic substances) for selection of transformed plant cells, a promoter, and a terminator. The LacZ cassette encodes a LacZ gene and LacZ promoter used as a selectable marker. The gene cassettes are flanked by the left border (LB) and right border (RB) T-DNA sequences allowing for transfer of the entire sequence or transgene into the plant cells by the bacterium strain of R. rhizogenes. The plasmid backbone also contains a bacterial selection marker cassette that encodes the kanamycin resistance (KanR) gene for selection and maintenance of the plasmid within the R. rhizogenes strain, and which is in reverse orientation on the plasmid vector 640. The Cannabaceae plant part (e.g., cannabis) was transformed using the R. rhizogenes strain A4 (ATCC43057). The plasmid vector 640 sequence is illustrated by SEQ ID NO:

1 below. Further identified is the sequence of the betalain cassette (SEQ ID NO: 2), the plant selectable marker cassette (SEQ ID NO: 10), the T-DNA borders of the 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), F2A 1 (SEQ ID NO: 5), DODA (SEQ ID NO: 6), F2A 2 (SEQ ID NO: 7), glucosyltransferase (SEQ ID NO: 8), and aribulose bisphosphate carboxylase (rbcS) terminator (SEQ ID NO: 9). The plant selectable marker cassette (SEQ ID NO: 10) encodes a 35S promoter (SEQ ID NO: 11), an ST LSI nptll mtron (SEQ ID NO: 12), anptll exon (SEQ ID NO: 13), and a 35S 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).

[0204] FIG. 6B illustrates an example plasmid vector 650 that includes a DNA sequence coding for a plurality of enzymes to produce betalain (e.g., betanidin). The plasmid vector 650 contains the gene encoding the enzymes CYP76AD1, DODA, and glucosyltransferase driven by a FMV promoter, with CYP76AD1 linked to DODA and DODA linked to glucosyltransferase by 2A self-cleaving peptides P2A. The plasmid vector 650 further includes a plant selectable marker cassette and a LacZ cassette, which are in reverse orientation on the plasmid vector 650 and as described by plasmid vector 640, the features of which are not repeated. The gene cassettes are flanked by the LB and RB T-DNA sequences, as described above. The plasmid backbone also contains a bacterial selection marker cassette that encodes the KanR gene, and which is in reverse orientation on the plasmid vector 650. The Cannabaceae plant part (e.g., cannabis) was transformed using the R. rhizogenes strain A4 (ATCC43057). The plasmid vector 650 sequence is illustrated by SEQ ID NO: 23 below. Further identified is the sequence of the betalain cassette (SEQ ID NO: 24), the plant selectable marker cassette (SEQ ID NO: 10), the T-DNA borders of the 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: 24) encodes the FMV promoter (SEQ ID NO: 3), CYP76AD1 (SEQ ID NO: 4), P2A 1 (SEQ ID NO: 25), DODA (SEQ ID NO: 6), P2A 2 (SEQ ID NO: 26), glucosyltransferase (SEQ ID NO: 8), and a rbcS terminator (SEQ ID NO: 9). The plant selectable marker cassette (SEQ ID NO: 10) encodes a 35S promoter (SEQ ID NO: 11), an ST LSI nptll intron (SEQ ID NO: 12), anptll exon (SEQ ID NO: 13), and a 35S 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).

[0205] FIG. 6C illustrates an example plasmid vector 660 that includes a DNA sequence coding for a plurality of enzymes to produce betalain (e.g., betanidin). The plasmid vector 660 contains the gene encoding the enzymes CYP76AD1, DODA, and glucosyltransferase driven by a FMV promoter, with CYP76AD1 linked to DODA and DODA linked to glucosyltransferase by 2A self-cleaving peptides P2A. The plasmid vector 660 further includes a plant selectable marker cassette and a LacZ cassette, which are in reverse orientation on the plasmid vector 660 and as described by plasmid vector 640, the features of which are not repeated. The gene cassettes are flanked by the LB and RB T-DNA sequences, as described above. The plasmid backbone also contains a bacterial selection marker cassette that encodes the KanR gene, and which is in reverse orientation on the plasmid vector 660. The Cannabaceae plant part (e.g., cannabis) was transformed using the R. rhizogenes strain A4 (ATCC43057). The plasmid vector 660 sequence is illustrated by SEQ ID NO: 27 below. Further identified is the sequence of the betalain cassette (SEQ ID NO: 28), the plant selectable marker cassette (SEQ ID NO: 29), the T-DNA borders of the 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:28) encodes the FMV promoter (SEQ ID NO: 3), CYP76AD1 (SEQ ID NO: 4), P2A 1 (SEQ ID NO: 25), DODA (SEQ ID NO: 6), P2A 2 (SEQ ID NO: 26), glucosyltransferase (SEQ ID NO: 8), and a rbcS terminator (SEQ ID NO: 9). The plant selectable marker cassette (SEQ ID NO: 29) encodes a VuUbi promoter (SEQ ID NO: 30), a chloroplast transit peptide (SEQ ID NO: 31), a Cp4 gene (SEQ ID NO: 32), and a Nos terminator (SEQ ID NO: 33). 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).

[0206] FIG. 6D illustrates an example plasmid vector 665 that includes a DNA sequence coding for a plurality of enzymes to produce betalain (e.g., betaxanthin). The plasmid vector 665 contains the gene encoding the enzymes CYP76AD6 and DODA driven by a FMV promoter, with CYP76AD6 linked to DODA by a 2A self-cleaving peptide P2A. The plasmid vector 665 further includes a plant selectable marker cassette and a LacZ cassette, which are in reverse orientation on the plasmid vector 665 and as described by plasmid vector 640, the features of which are not repeated. The gene cassettes are flanked by the LB and RB T-DNA sequences, as described above. The plasmid backbone also contains a bacterial selection marker cassette that encodes the KanR gene, and which is in reverse orientation on the plasmid vector 665. The Cannabaceae plant part (e.g., cannabis) was transformed using the R. rhizogenes strain A4 (ATCC43057). The plasmid vector 665 sequence is illustrated by SEQ ID NO: 34 below. Further identified is the sequence of the betalain cassette (SEQ ID NO: 35), the plant selectable marker cassette (SEQ ID NO: 10), the T-DNA borders of the 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: 34) encodes the FMV promoter (SEQ ID NO: 3), CYP76AD6 (SEQ ID NO: 36), P2A 1 (SEQ ID NO: 25), DODA with stop (SEQ ID NO: 37), and a rbcS terminator (SEQ ID NO: 9). The plant selectable marker cassette (SEQ ID NO: 10) encodes a 35S promoter (SEQ ID NO: 11), an ST LSI nptll intron (SEQ ID NO: 12), a nptll exon (SEQ ID NO: 13), and a 35S 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). [0207] FIG. 6E illustrates an example plasmid vector 667 that includes a DNA sequence coding for a plurality of enzymes to produce betalain (e.g., betanidin and betaxanthin). The plasmid vector 667 contains the gene encoding the enzymes CYP76AD1 and DODA driven by a FMV promoter, with CYP76AD1 linked to DODA by a 2 A self-cleaving peptide P2A. The plasmid vector 667 further includes a plant selectable marker cassette and a LacZ cassette, which are in reverse orientation on the plasmid vector 667 and as described by plasmid vector 640, the features of which are not repeated. The gene cassettes are flanked by the LB and RB T-DNA sequences, as described above. The plasmid backbone also contains a bacterial selection marker cassette that encodes the KanR gene, and which is in reverse orientation on the plasmid vector 667. The Cannabaceae plant part (e.g., cannabis) was transformed using the R. rhizogenes strain A4 (ATCC43057). The plasmid vector 667 sequence is illustrated by SEQ ID NO: 38 below. Further identified is the sequence of the betalain cassette (SEQ ID NO: 39), the plant selectable marker cassette (SEQ ID NO: 10), the T-DNA borders of the 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: 39) encodes the FMV promoter (SEQ ID NO: 3), CYP76AD1 (SEQ ID NO: 4), P2A 1 (SEQ ID NO: 25), DODA with stop (SEQ ID NO: 37), and a rbcS terminator (SEQ ID NO: 9). The plant selectable marker cassette (SEQ ID NO: 10) encodes a 35S promoter (SEQ ID NO: 11), an ST LSI nptll intron (SEQ ID NO: 12), a nptll exon (SEQ ID NO: 13), and a 35S 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). [0208] FIG. 6F illustrates an example plasmid vector 670 that includes a DNA sequence coding for a plurality of enzymes to produce betalain (e.g., betanidin). The plasmid vector 670 contains the gene encoding the enzymes CYP76AD1, DODA, and glucosyltransferase driven by a FMV promoter, with CYP76AD1 linked to DODA and DODA linked to glucosyltransferase by 2A self-cleaving peptides P2A. The plasmid vector 670 further includes a plant selectable marker cassette and a LacZ cassette, which are in reverse orientation on the plasmid vector 670 and as described by plasmid vector 640, the features of which are not repeated. The gene cassettes are flanked by the LB and RB T-DNA sequences, as described above. The plasmid backbone also contains a bacterial selection marker cassette that encodes the KanR gene, and which is in reverse orientation on the plasmid vector 670. The Cannabaceae plant part (e.g., cannabis) was transformed using the R. rhizogenes strain A4 (ATCC43057). The plasmid vector 670 sequence is illustrated by SEQ ID NO: 40 below. Further identified is the sequence of the betalain cassette (SEQ ID NO: 24), the plant selectable marker cassette (SEQ ID NO: 10), the T-DNA borders of the 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: 24) encodes the FMV promoter (SEQ ID NO: 3), CYP76AD1 (SEQ ID NO: 4), P2A 1 (SEQ ID NO: 25), DODA (SEQ ID NO: 6), P2A 2 (SEQ ID NO: 26), glucosyltransferase (SEQ ID NO: 8), and a rbcS terminator (SEQ ID NO: 9). The plant selectable marker cassette (SEQ ID NO: 41) encodes a VuUbi promoter (SEQ ID NO: 42), chloroplast transit peptide (SEQ ID NO: 43), an SpcN (e.g., Spec) (SEQ ID NO: 44), and a Nos terminator (SEQ ID NO: 45). 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).

[0209] Consistent with the above description, bacterium strains and Cannabaceae plant source material were prepared as follows. Five to seven days prior to the experiment, the desired R. rhizogenes strain was streaked out onto an AB minimal media agar plate (see media recipes) with appropriate antibiotics. The plates were incubated at 28 degrees C until the day of the experiment. In some embodiments, the colony was inoculated in a 15 mL YFP culture plus 7.5 uL Kan, which were all in a 50 m L vented conical tube [0210] In some experiments, six (6) days prior to the experiment, 50-100 cannabis seeds or other plant parts were surface sterilized with 10 mL concentrated sulfuric acid and washed twice with sterile water. The seeds were soaked in 30% hydrogen peroxide (H2O2) for 20 minutes and washed twice with sterile water. The seeds were allowed to imbibe in sterile water overnight (e.g., for 16-24 hours) with some gentle agitation, either in a conical tube placed in a motorized invertor or a sealed petri dish on a rotary shaker. On the following day, the water was removed, and the imbibed seeds were washed one time for 5 minutes in 30% hydrogen peroxide before rinsing three times with sterile water. Using forceps and a stereomicroscope, the seed coats and endosperm were removed before plating the embryos onto 8P-MS-G media plates (see media recipes) with a maximum of five embryos per plate. The plates were sealed with parafilm and placed in the dark for three 3 days. The plates were transferred to a 16/8-hour light/dark incubator (75 lumens, 23 degrees C) for two additional days.

[0211] Infection of cannabis hypocotyl tissue for PCM production was performed as follows. Five hours prior to infection, a loopful of bacteria from the plates was suspended in 1 mL of sterile water containing 100 mM acetosyringone. The bacterial suspension was maintained in a dark lab drawer at room temperature. The cannabis seedlings were removed from the incubator and the following steps were performed.

[0212] To infect whole cannabis seedlings, the point of a scalpel was used to make a small wound m the hypocotyl of each seedling, approximately 5-10 mm above the top of the radicle. Immediately after wounding, the wound was inoculated with 20 pL of bacterial suspension. After a minimum of 10 minutes, the seedlings were transferred to PCM co-cultivation media (a Murashige and Skoog (MS) media plate, see media recipes). The number of seedlings per plate was limited to five inoculated seedlings. The plates were sealed with parafilm and placed in the dark overnight (22 degrees C to 23 degrees C). The plates were removed from the dark chamber and transferred to a 16/8-hour light/dark incubator (75 lumens, 23 degrees C) for one additional day for a total of two days of co-cultivation.

[0213] For whole cannabis seedlings, each seedling was transferred to an individual PCM media plate (e.g., an MS media plate, see media recipes) containing 500 pg/mL of cefotaxime (PCM + Cef500), and care was exercised to ensure that the previously wounded part of the hypocotyl was touching the medium. The plates, such Phytotrays™, were closed and placed in a light chamber for two weeks.

[0214] To infect cannabis hypocotyl tissue, the point of a scalpel was used to cut off the radicle and cotyledon of each seedling. The hypocotyl were then cut into 5-10 mm segments. The cut segments were gathered into piles and immediately inoculated with 50- 100 pL of bacterial suspension. Care was taken to ensure that all segments, particularly the cut ends, were covered with a layer of bacterium strain. After a minimum of 10 minutes, the segments were transferred to PCM co-cultivation media (MS media, see media recipes) keeping the segments in a pile. The number of piles of segment per plate was limited to four piles of segments. The plates were sealed with parafilm and placed in the dark overnight (22 degrees C to 23 degrees C). The plates were removed from the dark chamber and transferred to a 16/8-hour light/dark incubator (75 lumens, 23 degrees C) for one additional day for a total of two days of co-cultivation.

[0215] For cannabis hypocotyl tissue, each pile was transferred to an individual PCM media plate (an MS media plate, see media recipes) containing 500 pg/mL of cefotaxime (PCM+ Cef500), and care was exercised to ensure that the segments were spread out evenly over the surface of the plates. The plates, such Phytatrays™, were closed and placed in a light chamber for two weeks.

[0216] To isolate and maintain the PCM clones, after two weeks, the tissue was transferred to fresh PCM media plate (see media recipes) containing 500 pg/mL of cefotaxime (PCM +Cef500) before returning the plates to the light chamber for two more weeks. After another two weeks, the tissue was screened for root formation. Any developing roots were removed using a scalpel and transferred to another PCM media plate (PCM + Cef500) for sub-culturing. These plates were sealed with parafilm and placed in the dark (22 degrees C or 23 degrees C). When selecting for the presence of a secondary T-DNA (a fluorescent protein or TALEN expressing cassette) insertion originating from a binary vector, an appropriate selection compound is added to the above media at the root sub-culturing stage. In the particular experiment including YFP, spectinomycin was added. This will allow for the growth of only PCM clones containing and expressing both the binary vector and PCM T-DNAs. [0217] Any whole seedlings or hypocotyl segments which have not developed roots were returned to the light chamber after transferring to fresh media for another two weeks. If the tissue has not developed roots after three rounds of media transfers, for a total of 8 weeks, it was discarded. For a particularly successful transformation, root tissue was excised from the same original explant multiple times. The original explant will usually start to die after 8 weeks of culture.

[0218] Plates of sub-cultured roots were transferred to fresh PCM media every two to three weeks with the concentration of cefotaxime in the medium being gradually reduced from 500 pg/mL (for two rounds of transfers) to 300 pg/mL (for one to two rounds of transfers) to 100 pg/mL (for one to two rounds of transfers). Other selection agents should be maintained at the same concentration throughout. Healthy PCM clones grow to be quite large (will cover the surface of the plate) so a portion of each clone was transferred to fresh media (between 1-2 square cm) while the remaining tissue is discarded. In some experimental embodiments, only one clone is maintained per plate. [0219] After the final round of sub-culturing on PCM media containing 100 pg/mL cefotaxime (PCM + CeflOO), the clones were transferred to medium containing no antibiotics (same as PCM co-cultivation media media) because all R. rhizogenes should be eliminated from the tissue. The clones can be maintained indefinitely on this media type and will only need to be transferred to fresh media every two to three weeks.

[0220] To grow PCM tissue in liquid media, a piece of root tissue approximately one cm² is placed in a 250 mL flask containing 50 mL of liquid medium. This should be done with root tissue that has already had any R. rhizogenes eliminated resulting in a “clean” clone. Sterility should be maintained at all times. The flask was placed on a shaker in the dark with a speed of 100 rotations per minute (rpm) and allowed to grow for 7 days before removing the spent media with a sterile pipette and adding 50 mL of fresh media. After two to three weeks, the tissue should double in size. If no bacterial or fungal contamination is present, a larger flask was used, for example a 500 mL flask containing 250 mL of MS liquid media or a 6 L flask containing 3 L of media, to bulk up the root tissue. In other experiments, DKW liquid media was used. The tissue was cultured and maintained in the same manner as described above. [0221] For EA or seedling explant the following was performed. The Cannabaceae seeds were prepped by sterilizing and imbibing the seed. The following steps were performed in a fume hood: 1) a 50ml tube containing around 100 seeds was taken and serological pipette was used to add 10ml of 12M sulfuric acid; 2) the 50ml tube was closed, and gently shaken for 10 seconds; 3) the 50ml tube was opened and a serological pipette was used to remove the sulfuric acid; 4) the sulfuric acid waste was pipetted into a glass waste beaker containing 400ml of ddFLO; 5) to rinse, 45ml of stenle ddEhO was added to the 50ml tube containing the seeds; 6) the 50ml tube was closed and gently shaken for 10 seconds; 7) a serological pipette was used to remove the ddELO from the 50ml tube; and 8) to rinse, 45ml of fresh ddELO was added to the 50ml tube, the tube was closed, and the tube was brought to a laminar flow hood. The ddELO was removed from the 50ml tube and 45ml of 30% H2O2 was added. The 50ml tube was closed and placed on a rotary shaker at 20 rpm for 20 minutes. After 20 minutes, the 50ml tube was removed from the rotary shaker and brought back to the laminar flow hood. A serological pipette was used to remove the EhChfrom the 50ml tube. A ddELO rinse was performed five times (5x) on the 50ml tube containing the seeds. After the fifth ddELO rinse, 45ml of sterile ddEEO was added to the 50ml tube, and the tube was placed on the rotary shaker at 13rpm for 16 hours.

[0222] The starter culture of the bacterium strain, the seed imbibition occurred on day 1, in some experimental embodiments.

[0223] The starter culture was prepared for infection. OD, bottle, and centrifuge three 50 mL flask of starter culture at 5400 rpm for 10 minute. The supernatant was removed and a 150 mL of MTA infection medium was added to resuspend the pellet. 150 uL of 40 mg/mL Acetosyringone was added to give final [Acetosyringone] = 200 uM to prepare the infection medium.

[0224] The imbibed seeds were used to extract Cannabaceae EA. For example, 15 mL of sterile ddELO was added to 100x25mm petri plates and the imbibed seeds were placed into a new 100x25mm petri dish. A sufficient number of infection plates were used to provide around 75 Cannabaceae EAs per plate and one infection plate per plate of imbibed seeds. Sterile forceps were used to gently remove the seed coat from each seed. Once the seed coats were removed from all seeds, the embryos were placed in the petri plates with the 15 mL sterile ddFbO and were ready for EA extraction. The forceps were used to gently hold a seedling and a #11 scalpel was used to gently break off the outer cotyledon. Once the outer cotyledon was broken off, the scalpel was used to slice off the inner cotyledon, making sure not to damage the meristem. Once the cotyledons was removed, the scalpel was used to gently brush off the leaf primordia, taking care the meristem is not touched or damaged. The Cannabaceae EAs were then placed in 15 mL of sterile ddFLO in a fresh 100x25mm petri plate.

[0225] The bacterium strain that is cultured and suspended, such as in an EA TDZ infection medium or MTA infection medium, is sonicated and inoculated with the imbibed EAs. For example, the MTA infection medium was pipetted off from the infection plates and 12mL of the resuspended agrobacterium was added. The infection plates were parafilmed and sonicated one plate at a time for 80 seconds. After the sonication, 12 mL of fresh agrobacterium was added. The plates were then incubated for 30 minutes at room temperature in a laminar flow hood.

[0226] After sonicating and inoculating, the EAs were co-cultivated with the bacterium strain. For example, the remaining agrobacterium was pipetted off the infection plates. 20mL of sterile ddFEO was added to each plate to rinse off excess agrobacterium. The EAs were then transferred to a new 100x25mm petri dish containing a piece of sterile filter pater. This assisted in drying off excess bacterium. The single EAs were transferred onto the co-cultivation plates by gently picking up one EA at a time and plating around 10 EA in a spread out fashion one each co-cultivation plate. The co-cultivation plates were wrapped in layer of parafilm and incubated for two to four days in 16/8 hour ambient light (SOumol/nr s ¹) at 23°C, 40% humidity in a Conviron incubator.

[0227] The preparation of the infection medium, the extraction of the Cannabaceae EA, the sonication and inoculation, and the co-cultivation occurred or at least started on day 2, in some experimental embodiments.

[0228] After the co-cultivation, the EAs were transferred to a shoot induction medium, such as EAL +S50 media or EA SIM +S10. For example, the EA were transferred to EA SIM +S10 media in a 100x25mm petri dish and sealed with parafilm. The EAs were oriented with the radicle down into SIM media and apical meristem up. In some experiments, around 10 EAs were plated per plate for 7 days inl6/8 hour ambient light (SOumol/nrVs ¹) at 23°C, 40% humidity in a Conviron incubator.

[0229] After the first shoot induction medium, the EAs are transferred to a second shoot induction medium containing higher levels of selection. The regenerating EAs were transferred to a SIM, such as EA SIM + S100 or EA SIM+S50 media. The EAs were oriented with the radicle down into SIM media and apical meristem up. The bottom of a second sterile 100x25mm plate was used as a lid and sealed with micropore tape. In some embodiments, the EAs were cultured 10 EAs per plate for 18 days under 100 umol/m²/sec white fluorescent light at room 23 +/- 1 degrees C.

[0230] After shoot induction, a first shoot elongation was performed. For example, the EAs were transferred to a first SEM, such as to EA SEM I + SI 00 media. The radicles were cut and the EAs were oriented with the radicle down into the first SEM media and apical meristem up. The bottom of a second sterile 100x25mm plate was used as a lid and sealed with micropore tape. In some embodiments, the EAs were cultured 5 EAs per plate for 21 days under 100 umol/m²/sec white fluorescent light at 23 +/- 1 degrees C.

[0231] After the first shoot elongation, a second shoot elongation was performed. For example, the EAs were then transferred to a second SEM, such as to EA SEM I + SI 50 media. The EAs were oriented with the radicle down into the second SEM media and apical meristem up. The bottom of a second sterile 100x25mm plate was used as a lid and sealed with micropore tape. In some embodiments, the EAs were cultured 5 EAs per plate for 21 days under 100 umol/m²/sec white fluorescent light at 23 +/- 1 degrees C.

[0232] After the second shoot elongation, the elongated shoots were rooted. Positive looking shoots (preferably 2” in height, minimum of 1” tall shoots) were cut and rooted on a Cannabaceae RM DKW+ 0.5 IBA media in phytatrays under LEDs for around 14 days until the shoots have developed at least two primary roots that are at least one cm in length. The shoots were subcultured onto fresh RM around every 7-14 days until new primary roots and root hair structures developed and before sending to acclimation in soil. [0233] R. rhizogenes was prepared as follows for infecting and transforming Cannabaceae plant parts. 25 ml of cells of the desired R. rhizogenes strain were grown overnight in YEP media with appropriate antibiotics. The overnight culture was inoculated with a single colony from a fresh AB solid media plate (see media recipes) and with the appropriate antibiotics. With the protocol the cells were kept on ice. Cells were collected from the 25 ml culture by centrifugation of the cells into a sterile conical tube with shaking at 4 degrees C at 6000 rpm for ten minutes. The cells were washed three times with 5 ml of ice-cold sterile water, with the tube in an ice bucket with a mixture of ice to ensure a low temperature. Care was taken to ensure that the outside surface of the tube was clean to prevent contamination of the cells. The cells were then washed one time with 5 ml of ice-cold 10% glycerol. 800 mΐ of the 10% glycerol was used to suspend cells, resulting in approximately 1000 mΐ of cell suspension. The competent cells were aliquoted into two microfuge tubes with 60 mΐ m each tube. Electroporation was then performed or the tubes were stored in -80 degrees C freezer for later electroporation. In some embodiments, electroporation can be implemented using at least some of the features described in Chassy, et al., “Transformation of Bacteria by Electroporation”, Trends Biotechnol, Vol. 6, Issue 12, 303-309, 1988, which is herein incorporated in its entirety for its teaching. In some experiments, after electroporation, 1 ml of YEP medium was used to resuspend the cells. The cells were then transferred into a sterile test tube and incubated at 28-30 degrees C with shaking for two hours. The cells were transferred to a microfuge tube, and a series of 10-fold dilutions with 0.9 % sterile NaCl or YEP liquid media were made. 100 mΐ of the undiluted culture was plated and each dilution (e.g. 10¹, 10²) onto separate AB sucrose media plates with appropriate antibiotics. The original tube was kept at 4 degrees C. Pinprick colonies should appear within 48 hours. The number of colonies were counted three days after transformation and this number can be used to determine the competency of the cells. Care was taken to obtain single colonies (not confluent lawn) from the selection plates before proceeding with the experiments. [0234] In some experiments, multiple bacterium strains were assessed to identify transformation frequencies. R. rhizogenes strains of K599, A4 (ATCC43057), R1000 (ATCC43056), and TR104 (ATCC13333) were used to infect cannabis whole seedlings and/or hypocotyl segments. Transformation frequency was determined by the number of plants or segments which exhibited PCM formation out of the total assayed over multiple experiments. A4 gave a transformation frequency of 68% to 89%, TR104 a frequency of 28% to 67%, K599 a frequency of around 2%, and R1000 a frequency of less than 2%. PCM clones isolated from tissues infected with A4 also had the best growth in tissue culture and have been able to be maintained indefinitely. TR104 derived clones eventually lose vitality after two or three times of being sub-cultured. In various experiments, A4 was used for transformation experiments.

[0235] To create the 8P-MS-G media (Phytatrays™ or plates), the following protocol and volumes were used to make a 1L solution of media:

800 ml ddH20; 10 g Sucrose; 4.43 g MS Basal Salts + Vitamins (Phytotech, M519); the solution was brought to volume with 1000 ml ddH20; the pH was adjusted to 5.7 with titration of KOH; and 3.58 g Gelzan™ (Phytotech, G3251).

The media was autoclaved on the liquid cycle for 25 minutes and cooled to 55 degrees C and poured 100 mL per Phytatray™ or 25 mL or 50 mL per 100 x 25 mm plates.

[0236] To create the LB media (culture tubes), the following protocol and volumes were used to make a 1L solution of media:

800 ml of ddH20; 25 g of LB (Sigma: L3522); and the solution was brought to volume with 1000ml of ddftO.

The media was autoclaved on liquid cycle for 25 minutes.

[0237] To create the LB agar media (plates), the following protocol and volumes were used to make a 1L solution of media:

800 ml of ddH20; 25 g of LB (Sigma: L3522); 15 g of Agar (Sigma: A5306); and the solution was brought to volume with 1000 ml of ddftO.

The media was autoclaved on liquid cycle for 25 minutes and cooled to 55 degrees C and poured into 25mL into 100 x 15 mm plates.

[0238] To create AB Salts (20X), the following protocol and volumes were used:

700 ml of ddH₂0; 20 g of NH₄C1; 6 g of MgS04*7H₂0; 3 g of KC1;

0.2 g of CaCh; 50 mg of FeS04*7H20; the pH was adjusted to 7.0 with KOH; and the solution was brought to volume with 1000 ml of ddftO.

[0239] To create AB Buffer (20X), the following protocol and volumes were used to form 1L of the media:

700 ml of dd¾0; 60 g of K2HPO4; 20 g ofNaftPCri; and the solution was brought to volume with 1000 ml of dcTHO.

[0240] To create AB minimal agar media (liquid), the following protocol and volumes were used to form 1L of media: 700 ml of ddfhO; 5 g of Sucrose; the solution was brought to volume with 1000 ml of ddfkO; 50 ml of 20x AB Salts; and 50 ml of 20X AB Buffer.

The media was autoclaved on liquid cycle for 25 minutes. To create the AB +Kan50 medium, 50mg/L of Kanamycin was added to the AB medium.

[0241] To create AB minimal media (plates), the following protocol and volumes were used to form 1L of media:

700 ml of ddTBO; 5 g of Sucrose; the solution was brought to volume with 1000 ml of ddThO; 50 ml of 20x AB Salts; 50 ml of 20X AB Buffer; and 15 g of Agar (Sigma: A5306).

The media was autoclaved on liquid cycle for 25 minutes and cooled to 55 degrees C and poured into 100 x 15 mm plates.

[0242] To create YEP media (liquid), the following protocol and volumes were used to form 1L of media:

800 mL of ddfhO; 10 g of Bacto-peptone; 5 g of Yeast extract; 5 g ofNaCl; and the solution was brought to volume with 1000 mL of ddTLO.

The media was filter sterilized.

[0243] To create YEP media (plates), the following protocol and volumes were used to form 1L of media:

800 mL of ddLLO; 10 g of Bacto-peptone; 5 g of Yeast extract; 5 g of NaCl; the solution w as brought to volume with 1000 mL of ddLLO; and 15 g of Agar (Sigma: A5306).

[0244] To create PCM media (plates), which can be referred to as an MS media with antibiotics, the following protocol and volumes were used:

800 ml of ddLLO; 30 g of Sucrose (Phytotech: S9378); 4.43 g of MS Basal Salts + Vitamins (Phytotech: M519); the solution was brought to volume with 1000 ml of ddLLO; the pH was adjusted to 5.8 by titration of KOH; and 6 g of Agarose (Phytotech: A6013). The media was autoclaved on liquid cycle for 25 minutes and cooled to 55 degrees C before adding 500 mg/L, 300 mg/L, or 100 mg/L of cefotaxime and pouring 50 mL per 100 x 25 mm plates. In some experimental embodiments, DKW basal salts were used. [0245] To create PCM co-cultivation media (plates), which can be referred to as an MS media without antibiotics, the following protocol and volumes were used to create 1L of media:

800 ml of ddThO; 30 g of Sucrose (Phytotech: S9378); 4.43 g of MS Basal Salts + Vitamins (Phytotech: M519); the solution was brought to volume with 1000 ml of ddThO; the pH was adjusted to 5.8 by titration of KOH; and 6 g of Agarose (Phytotech: A6013).

The media was autoclaved on liquid cycle for 25 minutes and cooled to 55 degrees C and poured 50 mL per 100 x 25 mm plate. In some experimental embodiments, DKW basal salts were used in place of MS basal salts.

[0246] To create EA TDZ infection media, the following protocol and volumes were used to form 1L of media:

800 mL of ddH₂0; 1 mL B5-Macro; 1 mL B5-Micro; 25 mL 20X AB Salts; 25 mL 20X AB Buffer; 1 g Potassium Nitrate; 30 g Glucose; and 5 g MES (M825); the solution was brought to volume with 131.895 mL ddH20; the pH was adjusted to 5.4 with titration of KOH/HC1; 0.1 mL Gamborg's B5 Vitamins (G219) [1000X]; 38.4 uL Giberellic Acid (G362) [13mg/ml]; 1.0 mL TDZ (T8118) [lmg/ml].

The media was filter sterilized and thiols were added the day of use. The thiols added included 2.0 mL Dithiothreitol [77mg/ml], 4.96 mL Sodium Thiosulfate· 5H20 [50mg/ml], and 8 mL L-Cysteine [50mg/ml]

[0247] To create the MTA infection medium, the following protocol and volumes were used to form 1L of media:

800 mL of ddH20; 2.4647 g of MgS047H20 (lOmM); the solution was brought to volume with 1000 mL ddftO; 10 drops Tween 10; and was filter sterilized. [0248] To create EA liquid (EAL) +S10 media, the following protocol and volumes were used to form 1L of media: 800 mL of ddH₂0; 15 g Sucrose (S391); 5.22 g DKW (D190); 1 g MES (M825); 1 mL B5 Vitamins (G219); the solution was brought to volume with 183.7 mL ddLbO; and the pH was adjusted to 5.7 with titration of KOH.

The media was filter sterilized and the following were added post autoclave: 2.0 mL Asparagine (A107) [25mg/ml], 2.0 mL Glutamine (G229) [25mg/ml], 0.5 mL Timentin (T104) [300mg/ml], 1.2 mL Cefotaxime (C380) [250mg/ml], and 0.2 mL Spectinomycin (S4014) [50mg/ml]

[0249] To create EAL +S100 media, the following protocol and volumes were used to form 1L of media:

800 mL of ddH₂0; 15 g Sucrose (S391); 5.22 g DKW (D190); 1 g MES (M825); 1 mL B5 Vitamins (G219); the solution was brought to volume with 181.9 mL ddH20; and the pH was adjusted to 5.7 with titration of KOH.

The media was filter sterilized and the following were added post autoclave: 2.0 mL Asparagine (A107) [25mg/ml], 2.0 mL Glutamine (G229) [25mg/ml], 0.5 mL Timentin (T104) [300mg/ml], 1.2 mL Cefotaxime (C380) [250mg/ml], and 2 mL Spectinomycin (S4014) [50mg/ml].

[0250] To create the co-cultivation media, such as co-cult G DKW, the following protocol and volumes were used to form 2L of media:

1600 mL of ddH₂0; 40 g Glucose; 10.44 g DKW (D190); 2 g MES (M825); the solution was brought to volume with 367.5 mL ddLLO: the pH was adjusted to 5.8 with titration of KOH; and 12 g Agar, Plant TC (A296).

The media was autoclaved on AGAR cycle with the MediaClave and the following were added post autoclave: 0.25mL IAA (1364) [lmg/ml], 2 mL trans-Zeatin Riboside (Z899) [lmg/ml], and 4 mL Acetosyringone (A1104) 120 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.

[0251] To create EA SIM +S10 media, the following protocol and volumes were used to form 2L of media:

1600 mL of ddH₂0; 60 g Sucrose (S391); 10.44 g DKW (D190); 2 g MES (M825); 2 mL B5 Vitamins (G219); 16 mL Iron Chelate (F318); the solution was brought to volume with 328.787 mL ddLLO: the pH was adjusted to 5.7 with titration of KOH; and 14 g Agar, Plant TC (A296). The media was autoclaved on AGAR cycle with the MediaClave and the following were added post autoclave: 0.0769 mL Gibberellic Acid (G362) [13mg/ml], 0.2 mL IAA (1364) [lmg/ml], 2 mL trans-Zeatin Riboside (Z899) [lmg/ml], 4 mL Asparagine (A107) [25mg/ml], 4 mL Glutamine (G229) [25mg/ml], 2.4 mL Carbenicillin (C346)

[250mg/ml], 2.4 mL Cefotaxime (C380), and 0.4 mL Spectinomycin (S4014) [50mg/ml]. [0252] To create EA SIM +S10 media, the following protocol and volumes were used to form 4L of media:

3200 mL of ddH 0; 120 g Sucrose (S391); 20.88 g DKW (D190); 4 g MES (M825); 4 mL B5 Vitamins (G219); 32 mL Iron Chelate (F318); the solution was brought to volume with 654.374 mL ddLLO; the pH was adjusted to 5.7 with titration of KOH; and 28 g Agar, Plant TC (A296).

The media was autoclaved on AGAR cycle with the MediaClave and the following were added post autoclave: 0.1538 mL Gibberellic Acid (G362) [13mg/ml], 0.4 mL IAA (1364) [lmg/ml], 4 mL trans-Zeatin Riboside (Z899) [lmg/ml], 8 mL Asparagine (A107) [25mg/ml], 7 mL Glutamine (G229) [25mg/ml], 4.8 mL Carbenicillin (C346)

[250mg/ml], 4.8 mL Cefotaxime (C380), and 0.8 mL Spectinomycin (S4014) [50mg/ml]. [0253] To create EA SIM +S50 media, the following protocol and volumes were used to form 4L of media:

3200 mL of ddH₂0; 120 g Sucrose (S391); 20.88 g DKW (D190); 4 g MES (M825); 4 mL B5 Vitamins (G219); 32 mL Iron Chelate (F318); the solution was brought to volume with 654.374 mL ddftO; the pH was adjusted to 5.7 with titration of KOH; and 28 g Agar, Plant TC (A296).

[250mg/ml], 4.8 mL Cefotaxime (C380), and 4 mL Spectinomycin (S4014) [50mg/ml]. [0254] To create EA SIM +S100 media, the following protocol and volumes were used to form 1L of media:

800 mL of dd¾0; 30 g Sucrose (S391); 5.22 g DKW (D190); 1 g MES (M825); 1 mL B5 Vitamins (G219); 8 mL Iron Chelate (F318); the solution was brought to volume with 162.594 mL dcll kO; the pH was adjusted to 5.7 with titration of KOH; and 7 g Agar, Plant TC (A296).

The media was autoclaved on AGAR cycle with the MediaClave and the following were added post autoclave: 0.0385 mL Gibberellic Acid (G362) [13mg/ml], 0.1 mL IAA (1364) [1 mg/ml], 1 mL trans-Zeatin Riboside (Z899) [lmg/ml], 2 mL Asparagine (A107) [25 mg/ml], 2 mL Glutamine (G229) [25mg/ml], 1.2 mL Carbenicillin (C346)

[250mg/ml], 1.2 mL Cefotaxime (C380), and 2 mL Spectinomycin (S4014) [50mg/ml] [0255] To create EA SEM I +S50 media, the following protocol and volumes were used to form 4L of media:

3200 mL of ddH₂0; 120 g Sucrose (S391); 20.88 g DKW (D190); 4 g MES (M825); 4 mL B5 Vitamins (G219); 32 mL Iron Chelate (F318); the solution was brought to volume with 623.174 mL ddftO; the pH was adjusted to 5.7 with titration of KOH; and 28 g Agar, Plant TC (A296).

The media was autoclaved on AGAR cycle with the MediaClave and the following were added post autoclave: 0.1538 mL Gibberellic Acid (G362) [13mg/ml], 0.4 mL IAA (1364) [lmg/ml], 4 mL trans-Zeatin Riboside (Z899) [lmg/ml], 40 mL L-ascorbic acid [lOmg/ml], 8 mL Asparagine (A107) [25mg/ml], 8 mL Glutamine (G229) [25mg/ml], 2.4 mL Carbenicillin (C346) [250mg/ml], 2.4 mL Cefotaxime (C380), and 4 mL Spectinomycin (S4014) [50mg/ml]

[0256] To create EA SEM I +S100 media, the following protocol and volumes were used to form 9L of media:

7200 mL of ddH₂0; 270 g Sucrose (S391); 46.98 g DKW (D190); 9 g MES (M825); 9 mL B5 Vitamins (G219); 72 mL Iron Chelate (F318); the solution was brought to volume with 1402.14 mL ddftO; the pH was adjusted to 5.7 with titration of KOH; and 63 g Agar, Plant TC (A296).

The media was autoclaved on AGAR cycle with the MediaClave and the following were added post autoclave: 0.3462 mL Gibberellic Acid (G362) [13mg/ml], 0.9 mL IAA (1364) [lmg/ml], 9 mL trans-Zeatin Riboside (Z899) [lmg/ml], 90 mL L-ascorbic acid [lOmg/ml], 18 mL Asparagine (A107) [25mg/ml], 18 mL Glutamine (G229) [25mg/ml], 5.4 mL Carbenicillin (C346) [250mg/ml], 5.4 mL Cefotaxime (C380), and 18 mL Spectinomycin (S4014) [50mg/ml] [0257] To create EA SEM I +S150 media, the following protocol and volumes were used to form 1L of media:

800 mL of ddfEO; 30 g Sucrose (S391); 5.22 g DKW (D190); 1 g MES (M825); 1 mL B5 Vitamins (G219); 8 mL Iron Chelate (F318); the solution was brought to volume 153.794 mL ddEEO; the pH was adjusted to 5.7 with titration of KOH; and 7 g Agar, Plant TC (A296).

The media was autoclaved on AGAR cycle with the MediaClave and the following were added post autoclave: 0.0385 mL Gibberellic Acid (G362) [13mg/ml], 0.1 mL IAA (1364) [1 mg/ml], 1 mL trans-Zeatin Riboside (Z899) [lmg/ml], 10 mL L-ascorbic acid [lOmg/ml], 2 mL Asparagine (A107) [25mg/ml], 2 mL Glutamine (G229) [25mg/ml], 0.6 mL Carbenicillin (C346) [250mg/ml], 0.6 mL Cefotaxime (C380), and 3 mL Spectinomycin (S4014 [50mg/ml]).

[0258] To create the Cannabaceae RM DKW+ 0.5 IBA media, the following protocol and volumes were used to form 1L of media:

800 mL of ddH20; 30 g Sucrose (S391); 5.22 g DKW basal salts (D190); 1 mL B5 Vitamins (G219); 5 mL Plant Preservation Mixture; the solution was brought to volume 174.63 mL ddH20; the pH was adjusted to 5.8 with titration of KOH; and 7 g Agar, Plant TC (A296).

The media was autoclaved on AGAR cycle with the MediaClave and 0.5 mL of IBA [lmg/lmL) was added.

[0259] The infection medium (e.g., EA TDZ infection media), co-cultivation medium, regeneration medium (e.g., EAL +S10 media, EAL +S100 media, or EA SIM +S10), SIM (e.g., EA SIM +S10 media, EA SIM +S50 media, and EA SIM +S100 media), first SEM (e.g., EA SEM I +S50 media, EA SEM I +S100 media, and EA SEM I +S150 media), and RM described above generally comprise water, a basal salt mixture, a sugar, and other components such as vitamins, selection agents, amino acids, and phytohormones. The SIM and SEMs can include nutritional sources of nitrogen, phosphorus, potassium, sulfur, calcium, magnesium, iron, boron, molybdenum, manganese, cobalt, zinc, copper, chlorine, and iodine.

[0260] As noted above, other types of culture media was used. For example, some experimental embodiments were directed to assessing various different types of culture mediums were used to enhance growth of PCM tissue using transformed plant parts. Various experimental embodiments were directed to use and assessment of different culture mediums. The following provides different example culture media used and protocols creating the same.

[0261] To create a liquid culture media containing DKW and B5, referred to as PCM DKW-B5 liquid, the following protocol and volumes were used to create 1L of media:

800 mL of ddhkO; 30 g of Sucrose (S9378); 5.22 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was brought to volume with 180.132 mL of ddfhO; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0262] To create a culture media containing DKW and MES, referred to as PCM 5gL MES, the following protocol and volumes were used to create 1L of media:

800 mL of dcbLO; 30 g of Sucrose (S9378); 5.22 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); 5 g MES (M825); the solution was brought to volume with 180.132 mL of dcU hO; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0263] To create a liquid culture media containing DKW and MES, referred to as PCM DKW-MES liquid, the following protocol and volumes were used to create 1L of media: 800 mL of dcbLO; 30 g of Sucrose (S9378); 5.22 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); 1 g MES (M825); the solution was brought to volume with 180.132 mL of dcU hO; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave. In some experimental embodiments, it was identified that culture media with 1 g MES (e.g., the DKW-MES liquid) performed better than 5 g MES, such as PCM 5gL MES.

[0264] To create another culture media containing DKW and MES (solid media), referred to as PCM MES, the following protocol and volumes were used to create 1L of media:

800 mL of dcbLO; 30 g of Sucrose (S9378); 5.22 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); 1 g MES (M825); the solution was brought to volume with 180.132 mL of ddfhO; the pH was adjusted to 5.8 with KOH; 6 g Agarose (A6013) was added.

The media was autoclaved on AGAR cycle with MediaClave.

[0265] To create a culture media containing DKW, MES, and Cefotaxime, referred to as PCM + Cef300 +MES, the following protocol and volumes were used to create 1L of media:

800 mL of dcbLO; 30 g of Sucrose (S9378); 5.22 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); 1 g MES (M825); the solution was brought to volume with 180.132 mL of ddftO; the pH was adjusted to 5.8 with KOH; 6 g Agarose (A6013) was added.

The media was autoclaved on AGAR cycle with MediaClave, and post autoclave 300 mg of Cefotaxime [250 mg/L] was added.

[0266] To create a liquid culture media containing DKW and B5, referred to as PCM DKW-B5-15g/L liquid, the following protocol and volumes were used to create 1L of media:

800 mL of dcbLO; 15 g of Sucrose (S9378); 5.22 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was brought to volume with 189.566 mL of ddH20; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0267] To create a liquid culture media containing DKW and B5, referred to as PCM DKW-B5-45g/L liquid, the following protocol and volumes were used to create 1L of media:

800 mL of dcbLO; 45 g of Sucrose (S9378); 5.22 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was brought to volume with 170.698 mL of dd¾0; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0268] To create a liquid culture media containing DKW and B5, referred to as PCM DKW-B5-5g/L liquid, the following protocol and volumes were used to create 1L of media: 800 mL of dcll hO; 5 g of Sucrose (S9378); 5.22 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was brought to volume with 195.855 mL of ddhbO; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0269] To create a liquid culture media containing DKW and B5, referred to as PCM DKW-B5-60g/L liquid, the following protocol and volumes were used to create 1L of media:

800 mL of dchHO; 60 g of Sucrose (S9378); 5.22 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was brought to volume with 161.264 mL of ddH20; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0270] To create a liquid culture media containing DKW and B5, referred to as PCM DKW-B5-15g/L liquid, the following protocol and volumes were used to create 1L of media:

800 mL of dchHO; 15 g of Sucrose (S9378); 5.22 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was brought to volume with 189.566 mL of ddH20; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave. In some experiments, the liquid culture media with different sucrose concentrations, as listed above, were assessed and were not selected for optimized growth conditions. However, embodiments are not so limited.

[0271] To create a liquid culture media containing DKW and B5, referred to as 0.25x DKW-B5 liquid, the following protocol and volumes were used to create 1L of media:

800 mL of dcftHO; 30 g of Sucrose (S9378); 1.31 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was brought to volume with 180.132 mL of ddH20; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave. [0272] To create a liquid culture media containing DKW and B5, referred to as 0.5x DKW-B5 liquid, the following protocol and volumes were used to create 1L of media:

800 mL of ddfhO; 30 g of Sucrose (S9378); 2.61 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was brought to volume with 180.132 mL of ddthO; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0273] To create a liquid culture media containing DKW and B5, referred to as 0.75x DKW-B5 liquid, the following protocol and volumes were used to create 1L of media:

800 mL of dcbLO; 30 g of Sucrose (S9378); 3.92 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was brought to volume with 180.132 mL of ddH20; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0274] To create a liquid culture media containing DKW and B5, referred to as 1.5x DKW-B5 liquid, the following protocol and volumes were used to create 1L of media:

800 mL of ddH20; 30 g of Sucrose (S9378); 7.83 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was brought to volume with 180.132 mL of ddH20; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave. In some experiments, the liquid culture media with different DKW concentrations, as listed above, were assessed and were not selected for optimized growth conditions. However, embodiments are not so limited.

[0275] To create a culture media containing DKW, B5 and Cefotaxime, referred to as DKW-B5 + CflOO, the following protocol and volumes were used to create 1L of media: 800 mL of dcbLO; 30 g of Sucrose (S9378); 5.22 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was brought to volume with 179.732 mL of ddH20; the pH was adjusted to 5.8 with KOH; 6 g Agarose (A6013) was added. The media was autoclaved on AGAR cycle with MediaClave, and post autoclave 100 mg of Cefotaxime [250 mg/L] was added.

[0276] To create another culture media containing DKW, B5 and Cefotaxime, referred to as DKW-B5 + Cf300. the following protocol and volumes were used to create 1L of media:

800 mL of ddThO; 30 g of Sucrose (S9378); 5.22 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was brought to volume with 178.932 mL of ddTLO; the pH was adjusted to 5.8 with KOH; 6 g Agarose (A6013) was added.

[0277] To create another culture media containing DKW, B5 and Cefotaxime, referred to as DKW-B5 + Cf500, the following protocol and volumes were used to create 1L of media:

800 mL of dchLO; 30 g of Sucrose (S9378); 5.22 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was brought to volume with 178.132 mL of ddH20; the pH was adjusted to 5.8 with KOH; 6 g Agarose (A6013) was added.

The media was autoclaved on AGAR cycle with MediaClave, and post autoclave 500 mg of Cefotaxime [250 mg/L] was added.

[0278] To create a culture media containing DKW, B5, Cefotaxime, and Spectinomycin, referred to as DKW-B5 + Cf500 + Spec 10, the following protocol and volumes were used to create 1L of media:

800 mL of dchLO; 30 g of Sucrose (S9378); 5.22 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was brought to volume with 178.932 mL of dd¾0; the pH was adjusted to 5.8 with KOH; 6 g Agarose (A6013) was added.

The media was autoclaved on AGAR cycle with MediaClave, and post autoclave 500 mg of Cefotaxime [250 mg/L] and 10 mg Spectinomycin [50 mg/mL] (S4014) was added. [0279] To create another culture media containing DKW, B5, Cefotaxime, and Spectinomycin, referred to as DKW-B5 + Cf500 + Spec20, the following protocol and volumes were used to create 1L of media:

The media was autoclaved on AGAR cycle with MediaClave, and post autoclave 500 mg of Cefotaxime [250 mg/L] and 20 mg Spectinomycin [50 mg/mL] (S4014) was added. [0280] To create another culture media containing DKW, B5, Cefotaxime, and Spectinomycin, referred to as DKW-B5 + Cf500 + Spec30, the following protocol and volumes were used to create 1L of media:

800 mL of dcbLO; 30 g of Sucrose (S9378); 5.22 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was brought to volume with 178.932 mL of ddH20; the pH was adjusted to 5.8 with KOH; 6 g Agarose (A6013) was added.

The media was autoclaved on AGAR cycle with MediaClave, and post autoclave 500 mg of Cefotaxime [250 mg/L] and 30 mg Spectinomycin [50 mg/mL] (S4014) was added. [0281] To create another culture media containing DKW, B5, Cefotaxime, and Spectinomycin, referred to as DKW-B5 + Cf500 + Spec40, the following protocol and volumes were used to create 1L of media:

The media was autoclaved on AGAR cycle with MediaClave, and post autoclave 500 mg of Cefotaxime [250 mg/L] and 40 mg Spectinomycin [50 mg/mL] (S4014) was added. In some experiments, the above (and below) example media were used to assess different Spectinomycin concentrations. [0282] To create another culture media containing DKW, B5, Cefotaxime, and G419 Sulfate, referred to as DKW-B5 + Cf500 + G418, the following protocol and volumes were used to create 1L of media:

800 mL of ddThO; 30 g of Sucrose (S9378); 5.22 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was brought to volume with 178.132 mL of ddtLO; the pH was adjusted to 5.8 with KOH; 6 g Agarose (A6013) was added.

The media was autoclaved on AGAR cycle with MediaClave, and post autoclave 500 mg of Cefotaxime [250 mg/L] and 5 mg G419 Sulfate [50 mg/mL] was added.

[0283] To create another culture media containing DKW, B5, Cefotaxime, and Spectinomycin, referred to as DKW-B5 + Cf300 + Sped 00, the following protocol and volumes were used to create 1L of media:

The media was autoclaved on AGAR cycle with MediaClave, and post autoclave 300 mg of Cefotaxime [250 mg/L] and 100 mg Spectinomycin [50 mg/mL] (S4014) was added. [0284] To create another culture media containing DKW, B5, Cefotaxime, and Spectinomycin, referred to as DKW-B5 + Cf500 + Sped 00, the following protocol and volumes were used to create 1L of media:

800 mL of dcbLO; 30 g of Sucrose (S9378); 5.22 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was brought to volume with 178.132 mL of ddH20; the pH was adjusted to 5.8 with KOH; 6 g Agarose (A6013) was added.

The media was autoclaved on AGAR cycle with MediaClave, and post autoclave 500 mg of Cefotaxime [250 mg/L] and 100 mg Spectinomycin [50 mg/mL] (S4014) was added. [0285] To create a culture media containing DKW and Cefotaxime, referred to as DKW + Cef300, the following protocol and volumes were used to create 1L of media: (D190); the solution was brought to volume with 179.932 mL of ddLLO; the pH was adjusted to 5.8 with KOH; 6 g Agarose (A6013) was added.

The media was autoclaved on AGAR cycle with MediaClave. and post autoclave 300 mg of Cefotaxime [250 mg/L] was added.

[0286] To create a culture media containing WPM and B5, referred to as WPM-B5-30, the following protocol and volumes were used to create 1L of media:

800 mL of ddH 0; 30 g of Sucrose (S9378); 2.3 g of WPM (LI 54); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was brought to volume with 180.132 mL of dcbHO; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0287] To create a culture media containing Sucrose, referred to as WPM-B5-30, the following protocol and volumes were used to create 1L of media:

800 mL of dcbnO; 30 g of Sucrose (S9378); the solution was brought to volume with 181.132 mL of dcbHO; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0288] To create an infection culture media containing DKW, referred to as 0.5x DKW infection, the following protocol and volumes were used to create 1L of media:

800 mL of dcbnO; 2.61 of DKW basal salt mixture (D190); the solution was brought to volume with 200 mL of ddH₂0; the pH was adjusted to 5.8 with KOH. The media was autoclaved on AGAR cycle with MediaClave.

[0289] To create a liquid culture media containing DKW and B5, referred to as DKW- B5-0 liquid, the following protocol and volumes w ere used to create 1L of media:

800 mL of dcbnO; 5.22 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was brought to volume with 199 mL of ddH₂0; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0290] To create another liquid culture media containing DKW and B5, referred to as DKW-B5-5 liquid, the following protocol and volumes were used to create 1L of media: 800 mL of ddH20; 5 g of Sucrose (S9378); 5.22 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was

80 brought to volume with 195.855 mL of ddfkO; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0291] To create another liquid culture media containing DKW and B5, referred to as

DKW-B5-10 liquid, the following protocol and volumes were used to create 1L of media: 800 mL of ddH20; 10 g of Sucrose (S9378); 5.22 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was brought to volume with 192.711 mL of ddH20; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0292] To create another liquid culture media containing DKW and B5, referred to as

DKW-B5-20 liquid, the following protocol and volumes were used to create 1L of media: 800 mL of dchLO; 20 g of Sucrose (S9378); 5.22 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was brought to volume with 186.421 mL of ddH20; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0293] To create another liquid culture media containing DKW and B5, referred to as

DKW-B5-40 liquid, the following protocol and volumes were used to create 1L of media: 800 mL of dcbLO; 40 g of Sucrose (S9378); 5.22 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was brought to volume with 173.843 mL of ddH20; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0294] To create another liquid culture media containing DKW and B5, referred to as

DKW-B5-50 liquid, the following protocol and volumes were used to create 1L of media: 800 mL of ddH20; 50 g of Sucrose (S9378); 5.22 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was brought to volume with 167.554 mL of ddH20; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave. [0295] To create another liquid culture media containing DKW and B5, referred to as DKW-B5-filter sterilize (FS) liquid, the following protocol and volumes were used to create 1L of media:

800 mL of ddFhO; 30 g of Sucrose (S9378); 5.22 g of DKW basal salt mixture (D190); 1 mL of Gamborg Vitamin solution [lOOOx] (G219); the solution was brought to volume with 180.131 mL of ddFLO; the pH was adjusted to 5.8 with KOH.

The media was filter sterilized.

[0296] In various experimental embodiments, the expression constructs illustrated by FIGs. 6A-6F were used to transform Cannabaceae plant parts to induce production of betalains, such as the production of betanidin, betaxanthin, and/or betaxanthin. Examples are not limited to PCM formation and transformation, and include other plant parts.

[0297] FIGs. 7A-7D illustrate examples images of PCM cultures producing betacyanin, consistent with the present disclosure. In some experiments, cannabis hypocotyls were co-transformed to express PCMs and betalains using an R. rhizogenes strain A4 transformed with the plasmid vector 650 illustrated by FIG. 6B. The resulting PCMs produced a betacyanin, specifically, betanidin. FIG. 7A is an image showing the cannabis hypocotyl segment forming a PCM that is producing the betacyanin. FIGs. 7B-7D are images showing a cannabis PCM explant expressing the betacyanin. The betacyanin is seen in the root tip, primary root, and root hairs. In FIGs. 7C-7D, multiple transgenic events are show n with different levels of betacyanin expression.

[0298] FIGs. 8A-8B illustrate example images of PCM cultures producing betacyanin at different levels, consistent with the present disclosure. In some experiments, cannabis whole seedlings were co-transformed to express PCMs and betalains using an R. rhizogenes strain A4 transformed with the plasmid vector 650 illustrated by FIG. 6B. FIGs. 8A-8B are images showing cannabis PCM explants producing different levels of betacyanin after transforming whole seedlings.

[0299] FIGs. 9A-9F illustrate example images of PCM cultures producing betacyanin at different levels, consistent with the present disclosure. In some experiments, the cannabis PCMwere re-transformed with the plasmid vector 650 illustrated by FIG. 6B. FIGs. 9A- 9F are images of the Cannabaceae PCM tissue that were transformed and that produce different levels of betacyanin. The betacyanin, e.g., betanidin, is produced in roots, root tips, root hairs, and wounding sites.

[0300] FIGs. 10A-10C illustrate example images of PCM cultures producing betanidin and betaxanthin, consistent with the present disclosure. In some experiments, cannabis plant parts were transformed to generate PCMs using a R. rhizogenes strain A4 and then re-transformed to produce betalains using a disarmed R. rhizogenes strain A4 transformed with the plasmid vector 667 illustrated by FIG. 6E. The resulting cannabis PCMs produced multiple betalains including betanidin and betaxanthin. FIG. 10A is an image of the resulting cannabis PCM transformed with the plasmid vector 667 imaged in white light, and FIG. 10B is an image of the cannabis PCM of FIG. 10B imaged in fluorescent light under excitation at 488mm. The first transformation included a protocol involving a first bacterium strain as described above (e.g., culturing to form a PCM culture), and the second or retransformation included exposing the formed PCM tissue to the second bacterium strain, such as 18rl2. Other types of bacterium strains may be used as the second bacterium strain, including GV3101, AGL1, and EHA105. FIG. IOC is an image verifying the presence of betanidin and betaxanthin in the transformed cannabis PCM tissue.

[0301] FIGs. 11A-11B illustrate example images of PCM cultures producing betaxanthin, consistent with the present disclosure. In some experiments, cannabis plant parts were transformed to express PCM phenotype using a first R. rhizogenes strain A4 and then the PCM tissue was re-transformed to produce a betalain using a disarmed R. rhizogenes strain A4 strain, e.g., 18rl2, transformed with the plasmid vector 665 illustrated by FIG. 6D. FIGs. 11A-11B are images of Cannabaceae PCMs re-transformed and producing betaxanthin.

[0302] FIGs. 12A-12B illustrate example images of betalains in liquid from PCM cultures, consistent with the present disclosure. In some experiments, the cannabis plant parts were transformed to form PCMs using a first bacterium strain and then re transformed to produce betalains using plasmid vector 650 and a second bacterium strain as described above, and the resulting PCM culture secreted or otherwise produced the betalain into the liquid media. FIGs. 12A-12B are images showing the betalain present in the liquid media after the cannabis PCMswere re-transformed with the plasmid vector 650.

[0303] Embodiments are not limited to transforming Cannabaceae plant parts using a bacterium strain and/or inducing PCM formation. Betalain can be produced in Cannabaceae plant parts by identifying a bacterium strain to transform the plant part, designing and generating a plasmid vector that includes sequence encoding an enzyme, transforming the bacterium strain with the plasmid vector, and infecting the plant part of the plant with the transformed bacterium strain via an injection and/or agroinfiltration technique.

[0304] In some embodiments, a cannabis EA explant and/or seed explant was transformed using an EA protocol, as described above.

[0305] FIGs. 13A-13C illustrate example images of a Cannabaceae EA transformed with an Agrobacterium strain, consistent with the present disclosure. In some embodiments, cannabis EAs were transformed with the plasmid vector 670 as illustrated by FIG. 6F. In the experiments, the cannabis EAs were transformed using the transformation protocol, as described above, and using Agrobacterium containing a binary vector. The binary vector contained different elements including spectinomycin selection marker and a betalain visual selection marker. The cannabis EA shows a highly efficient transformation, with the entire explant expressing the betalain color. Additionally, the explant derived from the cannabis EA evidences that the transgene successfully integrated into the regenerating tissues. Betalain is shown as being expressed in the meristem, petiole, and leaf tissue, in addition to the high levels of betalain expression in the original explant. FIG. 13 A is an image of the transformed cannabis EA explant that shows expression of betalain and illustrates integration of the T-DNA vector. FIG. 13B illustrates a plant regenerated from the explant which shows betalain expression in the meristem, petiole, and leaf tissues.

The original explant shows highly efficient integration of the T-DNA vector. FIG. 13C is an image of a regenerated plant derived from this explant shows betalain expression in the meristem, petiole, and leaf tissues.

[0306] FIG. 14 illustrates an example image of a Cannabaceae seedling transiently transformed with an Agrobacterium strain, consistent with the present disclosure. In some experiments, transient expression of seedlings transformed with plasmid vector 670 was performed. In such embodiments, the cannabis seedlings were transformed using the transformation protocol as described above and using Agrobacterium containing a binary vector. The binary vector contained different elements including spectinomycin selection marker and a betalain visual selection marker. The seedlings showed efficient transformation, with some explants transiently expressing the betalain color 45 minutes after transformation. The image of FIG. 14 illustrates the cannabis seedling showing transient expression of the betalain color 45 minutes after transformation. The circled explants are transiently expressing betalain, while the remaining are not.

[0307] FIGs. 15A-15C illustrate example images of a Cannabaceae seedling explant stably transformed with an Agrobacterium strain, consistent with the present disclosure.

In some embodiments, cannabis seedling explants were stably transformed with a plasmid vector 670 of FIG. 6F. The seedlings showed efficient transformation, with example explants showing stable transformation of the plasmid vector 670 compared to the control. FIG. 15 A illustrates an image of a control explant that is not transformed with the plasmid vector 670. FIGs. 15B and 15C show stably transformed cannabis seedling explants. As shown by FIGs. 15B-15C, the betalain expression can be seen in the explant. [0308] FIG. 16A-16B illustrate example images of a Cannabaceae EA stably transformed with an Agrobacterium strain, consistent with the present disclosure. In some embodiments, cannabis EAs were transformed with a plasmid vector 670 of FIG. 6F. The EAs showed stable expression of the transgene, compared to no betalain expression in the wild-type EA control. FIG. 16A illustrates an image of a control explant that is not transformed with the plasmid vector. FIG. 16B shows a stably transformed cannabis seedling explant. As shown by FIG. 16B, the betalain expression can be seen in the explant. The transformed cannabis EA shows stable expression of betalain throughout the entire explant.

[0309] FIGs. 17A-17B illustrate example images of a Cannabaceae seedling explant stably transformed with an Agrobacterium strain, consistent with the present disclosure.

In some embodiments, cannabis seedling explants were transformed with the plasmid vector 670. The seedling explants were thinly sliced and imaged using a microscope. The sections show cells that are expressing betalain. FIGs. 17A-17B illustrate cross sections of a cannabis seedling that has been stably transformed with the transgene. It is clear that certain cells have been transformed and are expressing the transgene, while others have not.

[0310] In some embodiments, a Cannabaceae node was transformed using an Agrobacterium-mediated transformation. In other embodiments, the Cannabaceae plant part can be transformed using the expression construct and a bombardment technique. In some embodiments, the Cannabaceae plant part was transformed using at least substantially the same features as disclosed in PCT application PCT/US21/25067, entitled “Agrobacterium-mediated Infiltration of Cannabis”, filed on March 31, 2021, which is fully incorporated herein for its teaching and sometimes herein referred to as the “Cannabaceae agroinfiltration protocol”. In some embodiments, a Cannabaceae node plant part was transformed using a technique, which was performed using at least substantially the same features as disclosed in PCT application PCT/US21/21693, entitled “Transformation and Regeneration of Cannabaceae”, filed on March 10, 2021, which is fully incorporated herein for its teaching and sometimes herein referred to as the “Cannabaceae node transformation protocol.”

[0311] FIGs. 18A-18D illustrate example images of Cannabaceae node explant stably transformed with an expression construct associated with a betalain, consistent with the present disclosure. In some experiments, node explants were stably transformed with the plasmid vector 670. In such embodiments, cannabis node explants were transformed using the Cannabaceae node transformation protocol as described above and using agrobacterium containing a binary vector. The image of FIG. 18A is control node explant that has not been transformed with the plasmid vector 670. The image of FIG. 18B is a node explant that has been transformed with the plasmid vector 670. The transformed cannabis node explants show stable expression of the betalain throughout the explant.

FIG. 18C is an image of a cannabis node explant transformed to produce betalain throughout the stem region. Additionally, betalain expression is also be seen on the meristematic tissues that are regenerating from the nodal explants. FIG. 18D is an image of a cannabis node explant transformed with the plasmid vector 670 which exhibits betalain expression throughout the leaf and meristem regions. The betalain be seen on the meristematic tissues that are regenerating from the nodal explants. Certain leaves and meristems in this image are expressing betalain, while others are not, showing that this is a chimeric explant.

[0312] FIGs. 19A-19B illustrate example experimental results from PCM cultures producing betalains, consistent with the present disclosure. FIG. 19A illustrates a chromatogram of chemistries derived from a wild-type beet tissue extract on the top graph as compared to and lined up with a cannabis PCM line on the bottom graph. The box illustrates a betalain, such as a betanin and/or betanidin. As shown by the top graph of FIG. 19A, the wild-type beet standard has a peak for betanin at around 10.446. The cannabis PCM was generated using the plasmid vector 670 illustrated by FIG. 6F and has a similar peak around 10.446, as shown by the bottom graph of FIG. 19A. FIG. 19B illustrates a chromatogram of chemistries derived from a wild-type beet tissue extract on the top graph as compared to a first cannabis PCM in the middle graph that was generated using the plasmid vector 667 of FIG. 6E, and a second cannabis PCM in the bottom graph that was generated using the plasmid vector 665 of FIG. 6D. As shown by the top graph of FIG. 19B, the wild-type beet standard has peaks for betaxanthin at around 6.687, betanin at around 10.446, and isobetanin at around 11.387. As shown by the middle graph of FIG. 19B, the first cannabis PCM has a peak around 10.446 that corresponds with betacyanin. As shown by the bottom graph of FIG. 19B, the second cannabis PCM has a peak around 6.687 that corresponds with betaxanthin.

[0313] Some experiment embodiments were directed to transforming Solanaceae plant parts to produce a PCM. For example, a solanum tuberosum plant part was transformed using an A. rhizogenes ATCC15834 strain, and under conditions described herein, and using a A. rhizogenes ATCC15834 strain containing a plasmid vector encoding for a plurality of enzymes associated with a betalain, such as the plasmid vector 670 illustrated by FIG. 6F.

[0314] Some experiments were conducted that transformed the solanum tuberosum plant part to generate a PCM. FIGs. 20A-20B illustrate example images of PCM cultures generated from solanum tuberosum plants, consistent with the present disclosure. FIG. 20A illustrates a PCM culture generated from a first solanum tuberosum strain and FIG. 20B illustrates a PCM culture generated from a second solanum tuberosum strain. As previously described, genetic variability between strains and even clones of a strain results in different mass of PCM tissue produced in the PCM cultures. Some experimental embodiments were directed to assessing different biomass growth rates of PCM cultures generated from different clones. The resulting growth rates ranged from 1.5 to around 10 grams of biomass in two weeks of growth.

[0315] FIGs. 21A-21B illustrate example images showing betalain production in solanum tuberosum PCM cultures, consistent with the present disclosure. The solanum tuberosum plant parts were transformed using the plasmid vector 670, as noted above, and bacterium strain A. rhizogenes ATCC15834 transformed with the plasmid vector 670.

[0316] FIGs. 22A-22B illustrate example images of betalain production in solanum tuberosum PCM cultures using different bacterium strains, consistent with the present disclosure. For example, FIG. 22A illustrates betalain production in a PCM culture generated using A4 bacterium strain transformed with the plasmid vector 670 and FIG. 22B illustrates betalain production in a PCM culture generated using bacterium strain A. rhizogenes ATCC15834 transformed with the plasmid vector 670.

[0317] The Solanaceae plant parts were transformed using the below described protocols. In vitro solanum tuberosum plants were sub-cultured 3-5 weeks prior to use.

[0318] The bacterium stain was prepared by inoculating 25 mL of minimum growth (MG) media (in 50 mL sterile centrifuge tubes) supplemented with appropriate antibiotics (e.g. 50 mg/mL kanamyacin) loop/colony of A. rhizogenes carrying a binary plasmid (e.g. YFP reporter, incubated at 28 degrees C with shaking for 2 days, OD600 around 0.5, and spun at 6000 RPM for 10 minutes in the large centrifuge at 4 degrees C. Supernatant and resuspended pellet was discarded in 25 mL MG media supplemented with 200 mM acetosyringone.

[0319] Solanaceae stem explant were then prepared by harvesting stems from 3-5 week old tissue-culture plants with thick (2-3mm diameter) stems growing on a modified MS (MMS) media. The plant were cut at the intemode below the lowest leaf to be harvested, and the container was covered in between harvests to prevent wilting. Excised shoot were placed on a sterile petri dish lid and stem intemodes into 2-3 cm explants discarding any meristematic (nodal) tissue. The prepared stem explants were transferred to petri dish containing Agrobacterium solution and infected or co-cultivated. After 15-20 minutes in Agrobacterium solution, the infected stem explants were transferred to MS media with no antibiotics (100x15mm petri dish) with 15-18 stem explants per petri dish, and sealed with parafilm and place in the dark (28 degrees C) for 48 hours.

[0320] Regeneration was the performed following the co-cultivation. The explants were transferred to MS media petri dishes (100 x 15mm) supplemented with 250 mg/L Cefotaxime and 150 mg/L Timentin, sealed with micropore tape, and then transferred to 16/8-hour light/dark (75 lumens, approximately 28 degrees C) growth incubator, with the plates being transferred to fresh media every two weeks. PCMs growing were screened from the stem ends using fluorescent markers (e.g., YFP) and harvested as needed.

[0321] For transforming Solanaceae plant parts, to create the MS media, the following protocol and volumes were used to make a 1L solution of media:

600 mL ddH20; 10 g Sucrose; 4.43 g MS Basal Salts + Vitamins (Phytotech, M519); the solution was brought to volume with 1000 mL ddH20; the pH was adjusted to 5.7 with titration of KOH; and 3.58 g Gelzan™ (Phytotech, G3251) was added for a solid media.

[0322] To create the MMS media, the following protocol and volumes were used to make a 1L solution of media:

800 mL of ddH20; 25 g of sucrose; 4.45 g of MS Basal Salts + Vitamins (Phytotech, M519); the solution was brought to volume with 1000 mL ddH20; the pH was adjusted to 5.7 with titration of KOH; and 7.5 g of Agar (Phytotech, A296) was added.

The media was autoclaved on liquid cycle for 25 minutes, followed by adding 0.8 mL of Cefotaxime (250 mg/ml) and 01. mL of 6-BAP (1 mg/ml).

[0323] To create the MG salts (20X), the following protocol and volumes were used to make a 1L solution of media:

700 mL of ddH₂0; 20 g NH4Cl; 6 g of MgS04*7H20; 3 g ofKCl; 0.2 g of CaC12; 50 mg of FeS04*7H20; and the solution was brought to volume with 1000 mL of ddH₂0.

[0324] To create the MG buffer (20X), the following protocol and volumes were used to make a 1L solution of media:

700 mL of ddH₂0; 60 g of K2HP04; 20 g of NaH2P04; and the solution was brought to volume with 100 mL of ddH₂0. [0325] To create the MG media, the following protocol and volumes were used to make a 1L solution of media:

700 mL of ddTkO; 5 g of glucose; and the solution was brought to volume with

1000 mL of ddtbO; followed by adding 50 mL of 20X MG salts, 50 mL of 20X

MG buffer, and 15 g of Agar (Sigma: A5306) (for solid media).

[0326] Various experimental embodiments were directed to assessing different growth conditions and resulting growth rates of PCM tissue, as well as assessing growth rates over wild-types of tissues. The different growth conditions included assessing the above- listed culture mediums including liquid forms, solid forms, different basal salts, different sugar amounts, and pH buffers. In various embodiments, different light/dark conditions were assessed. In some experimental embodiments, culture mediums that were liquid- based and included DKW performed better than those containing WPM or MS.

[0327] Some experimental embodiments were directed to infecting different plant clones to generate PCMs and selecting the optimal PCM from the plurality of clones based on increases in biomass while culturing under growth conditions. The plant clones were transformed with an A4 bacterium strain containing the RI plasmids and placed in flasks. The weight gain was tracked over a period of around one month. Such experiments illustrated the genetic variability between clones. Table 1 illustrates different example clone results from the experiments. Additional clones were tested.

Table 1

[0328] Various experiments were directed to assessing growth rates and increases in growth rates of PCM tissue in PCM cultures as compared to wild-type roots grown in the field and via aeropomcs. For assessing growth rates of wild-type roots, the calculation was based on grams of dried wild-type root per meter squared per month (g/m²/month). For assessing growth rates of PCM tissue, the calculation was based on dried PCM tissue g/m²/month. In various experimental embodiments and using calculations described above, it was estimated that wild- type plants grown in a field produce about 6-7 root g/m²/month and grown using aeroponics produce about 13 root g/m²/month. In contrast, PCM cultures produced PCM tissue at about 120-190 PCM g/m²/month, which was about 9-14 fold increase in root tissue production over production of wild-type roots grown using aeroponics and about 18-28 fold increase in root tissue production over wild-type roots grown in the field. Further increases in growth can be shown via additional optimization. Tables 2-4 illustrate example mass and growth rate calculations.

Table 2: Root Tissue Biomass

Table 3: Aeroponics Growth Calculations

Table 4: PCM Growth Calculations

[0329] Embodiments are not limited to the transformations illustrated by the experimental embodiments and can be directed to variety of different transformations and PCM generations in a variety of different plant species to achieve different growth rates and/or production of betalains in PCM tissue.

Claims

1. A method comprising: exposing a Cannabaceae plant part to an expression construct comprising a nucleotide sequence encoding an enzyme associated with production of a betalain to transform the Cannabaceae plant part with the expression construct; and inducing production of the betalain in the transformed Cannabaceae plant part.

2. The method of claim 1, wherein fee enzyme comprises: dihydroxyphenylalanine (DOPA) 4J-dioxygenase (DODA). Cytochrome P450

(CYP76AD1), CYP76AD6, glucosyl transferase or a combination thereof.

3. The method of claim 1, wherein the enzyme comprises a plurality of enzymes and the nucleotide sequence encodes the plurality of enzymes linked by a plurality of 2A selfcleaving peptides.

4. The method of claim 1, wherein the betalain comprises a betacyanin or a betaxanthin.

5. The method of claim 1, wherein the betalain is selected from: betanin, isobetanm, probetanin, neobetanin, vulgaxanthin, miraxanthin, portulaxanthin, and indicaxanthin.

6. The method of claim 1, wherein the nucleotide sequence encoding the enzyme comprises SEQ ID NO: 2, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 35, or SEQ ID NO: 39.

7. The method of claim 1, wherein the Cannabaceae plant part is a leaf, a root, a seed, a meristem, a node, an intemode, a meristem, a petiole, a hypocotyl segment, or a cell.

8. The method of claim 1, wherein the Cannabaceae plant part is stably transformed by the exposure to expression construct.

9. The method of claim 1, wherein the Cannabaceae plant part is transiently transformed by the exposure to expression construct.

10. The method of claim 1, further comprising generating the expression construct comprising the nucleotide sequence that encodes the enzyme.

11. The method claim 1 , wherein exposing the Cannabaceae plant part to the expression construct and inducing production of the betalain comprises: contacting the Cannabaceae plant part with a bacterium strain comprising the expression construct that comprises the nucleotide sequence encoding the enzyme; and culturing the Cannabaceae plant part to induce production of the betalain in the Cannabaceae plant part.

12. The method of claim 11 , wherein inducing the production comprises culturing the Cannabaceae plant part under growlh conditions to enhance transformation, induce plant cell matrix (PCM) formation, and induce production of the betalain.

13. The method of claim 12, wherein the growth conditions are selected from: a liquid culture medium, a type of culture medium, an amount of contact with the culture medium, a type of contact with the culture medium, a plant type, and a combination thereof.

14. The method of claim 12, wherein culturing the Cannabaceae plant part under the growth conditions comprises intermittently contacting the Cannabaceae plant part with a culture medium containing sugar and basal salt.

15. The method of claim 1 , further comprising identifying a bacterium strain from a plurality of bacterium strains.

16. The method of claim 11, wherein contacting the Cannabaceae plant part with the bacterium strain comprises simultaneously introducing to the Cannabaceae plant part: a first transgene associated with plant cell matrix (PCM) formation, and a second transgene associated with the enzyme, the method further comprising cultivating the plant part as transformed to generate PCM tissue, wherein the plant part is a seedling, a hypocotyl segment, a petiole, an intemode, a node, a meristem or a leaf.

17. The method of claim 11, wherein contacting the Cannabaceae plant part with the bacterium strain and culturing the Cannabaceae plant part comprises: contacting the Cannabaceae plant part with a first bacterium strain comprising the nucleotide sequence encoding the gene that induces PCM formation; culturing the Cannabaceae plant part to enhance PCM formation; contacting formed PCM tissue from the PCM with a second bacterium strain comprising the nucleotide sequence encoding the enzyme; and culturing the PCM tissue to enhance production of the betalain by the PCM.

18. The method of claim 11 , wherein infecting the Cannabaceae plant part with the bacterium strain comprises: injecting the bacterium strain into the Cannabaceae plant part; agroinfiltrating the bacterium strain into the Cannabaceae plant part; or culturing the infected Cannabaceae plant part to enhance transformation, induce plant cell matrix (PCM) formation, and induce expression of the enzyme.

19. The method of claim 1, wherein exposing the Cannabaceae plant part to the expression construct and inducing the production of the betalain comprises simultaneously introducing to the Cannabaceae plant part: a first transgene associated with plant cell matrix (PCM) formation, and a second transgene associated with the enzyme, the method further comprising cultivating the Cannabaceae plant part as transformed to generate PCM tissue, wherein the Cannabaceae plant part is a seedling, ahypocotyl segment, a petiole, an intemode, a node, a meristem, or a leaf.

20. The method of claim 1, wherein exposing the Cannabaceae plant part to the expression construct and inducing the production of the betalain comprises: contacting the Cannabaceae plant part a nucleotide sequence encoding a gene that induces plant cell matrix (PCM) formation; culturing the Cannabaceae plant part to enhance PCM formation; contacting formed PCM tissue from the PCM with the nucleotide sequence encoding the enzyme; and culturing the PCM tissue to enhance production of the betalain by the PCM.

21. The method of claim 1 , wherein transforming the Cannabaceae plant part comprises: exposing the Cannabaceae plant part to the expression construct to transform the Cannabaceae plant part with the expression construct via bombardment; and inducing expression of the enzyme in tissue of the transformed Cannabaceae plant part.

22. The method of claim 21, wherein exposing the Cannabaceae plant part to the expression construct via bombardment comprises using a bombardment gun, particles coated with the expression construct, or a combination thereof.

23. The method of claim 1, wherein the nucleotide sequence encoding the enzyme is operably connected to a promoter.

24. The method of claim 1, further comprising screening the transformed Cannabaceae plant part or new growth from the transformed Cannabaceae plant part for production of the betalain and tissue formation.

25. A method of generating a bacterium strain comprising: transforming a bacterium strain with a nucleotide sequence encoding an enzyme, wherein the bacterium strain comprises a nucleotide sequence encoding a gene that induces plant cell matrix (PCM) formation or is transformed to comprise the nucleotide sequence encoding the gene that induces PCM formation; and culturing the transformed bacterium strain.

26. The method of claim 25, further comprising: generating an expression construct comprising the nucleotide sequence encoding the enzyme: and forming a solution or suspension comprising the expression construct or attaching the expression construct to particles for bombardment or injection.

27. The method of claim 25, wherein the nucleotide sequence encoding the enzyme comprises SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 36, or a combination thereof.

28. The method of claim 25, wherein the bacterium strain is transformed using an expression cassette comprising SEQ ID NO: 2, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 35, or SEQ ID NO: 39.

29. The method of claim 25, wherein the bacterium strain is transformed using an expression construct comprising SEQ ID NO: 1, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 38, or SEQ ID NO: 40.

30. The method of claim 25, wherein the method further comprises transforming the bacterium strain to carry the nucleotide sequence encoding the enzyme using a vector containing: a right and left transferred DNA (T-DNA) border sequence; the nucleotide sequence encoding the enzyme; and a promoter.

31. A method comprising: contacting a Cannabaceae plant part with a bacterium strain containing a nucleotide sequence encoding an enzyme associated with production of a betalain; and inducing formation of tissue from the Cannabaceae plant part, wherein the tissue expresses the nucleotide sequence.

32. The method of claim 31, further comprising: selecting tissue from the Cannabaceae plant part as transformed for culturing in a culture medium; and screening the cultured tissue for production of the betalain.

33. The method of claim 32, further comprising capturing the betalain by isolating and purifying the betalain from the Cannabaceae plant part as transformed, the culture medium, or a combination thereof.

34. A tissue culture that produces a betalain, the tissue culture being induced from a Cannabaceae plant part and an expression construct comprising a nucleotide sequence encoding an enzyme associated with production of a betalain, wherein plant cells of the tissue culture comprises the nucleotide sequence encoding the enzyme.

35. The tissue culture of claim 34, wherein the nucleotide sequence encoding the enzyme compnses SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 36, or a combination thereof.

36. The tissue culture of claim 34, wherein the expression construct comprises an expression cassette comprising SEQ ID NO: 2, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 35, or SEQ ID NO: 39.

37. A system for producing a betalain from a Cannabaceae plant part, the system comprising: a plurality of bioreactors in serial connection, wherein each bioreactor is inoculated with a Cannabaceae plant part according to method of claim 1, and configured for growth and maintenance of the Cannabaceae plant part or a tissue culture formed from the Cannabaceae plant part in a culture medium.

38. The system of claim 37, wherein the culture medium comprises a liquid culture medium and the system is configured to recover the betalain from the culture medium.

39. The system of claim 37, wherein at least one bioreactor is a flask, temporary immersion system, plastic sleeve reactor, a bubble reactor, a mist reactor, an airlift reactor, a liquid-dispersed reactor or a bioreactor configured to generate micro- or nano bubbles.

40. The system of claim 37, wherein each bioreactor of the plurality is structurally and operationally similar.

41. Cannabaceae tissue generated using a method of claim 1.

42. A betalain produced by a Cannabaceae plant or plant part according to the method of claim 1.

43. A method comprising: transforming a plurality of Cannabaceae plant parts with an expression construct to induce production of a betalain; and screening the transformed plurality of Cannabaceae plant parts for the production of the betalain.