WO2022198106A1

WO2022198106A1 - Producing betalains using plant cell matrices

Info

Publication number: WO2022198106A1
Application number: PCT/US2022/021042
Authority: WO
Inventors: Benjamin Campbell; Lauren Harrison; Rachelle LAPHAM; Jonathan Mayers
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 contacting a plant part with a nucleotide sequence encoding a gene that induces plant cell matrix (PCM) formation and a nucleotide sequence encoding an enzyme associated with production of a betalain, and culturing the plant part to enhance the production of the betalain.

Description

PRODUCING BETALAINS USING PLANT CELL MATRICES

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 178 kb in size, submitted concurrently herewith, and identified as follows: “06331201 ll_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] A variety of aspects of the present disclosure are directed to a method of transforming a plant part to induce formation of a collection of plant cells, referred to herein as “a plant cell matrix (PCM)”, and production of a betalain.

[0005] Various aspects are directed to a method comprising contacting a plant part with a nucleotide sequence encoding a gene that induces PCM formation and a nucleotide sequence encoding an enzyme associated with production of a betalain, and culturing the plant part to enhance the production of the betalain.

[0006] In some aspects, contacting the plant part with the nucleotide sequences comprises contacting the plant part with a bacterium strain comprising the nucleotide sequence encoding a gene that induces PCM formation and the nucleotide sequence encoding the enzyme.

[0007] In some aspects, in the PCM comprises plant cells transformed by the contact with the nucleotide sequence and includes a plurality of different plant cells types, the plurality of different plant cells types comprises cells selected from (such as being selected from the group consisting of) plant stem cells, maturing cells, mature cells, and a combination thereof.

[0008] In some aspects, wherein the enzyme is selected from (such as being selected from the group consisting of): dihydroxyphenylalanine (DOPA) 4,5-dioxygenase (DODA), Cytochrome P450 (CYP76AD1), CYP76AD6, glucosyltransferase, and a combination thereof.

[0009] 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.

[0010] In some aspects, the betalain comprises a betacyanin or a betaxanthin.

[0011] In some aspects, the betalain is selected from (such as being selected from the group consisting of): betanin, isobetanin, probetanin, neobetanin, vulgaxanthin, miraxanthin, portulaxanthin, and indicaxanthin, a combination thereof.

[0012] In some aspects, the plant part is from a monocotyledon plant or a dicotyledon plant.

[0013] 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.

[0014] 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.

[0015] In some aspects, the plant part is a seedling, a petiole, an intemode, a node, a meristem, or a leaf.

[0016] In some aspects, the plant part is from a Cannabaceae plant, a Brassicaceae plant, a Solanaceae plant, a Fabaceae plant, or an Apiacea plant.

[0017] In some aspects, the method further comprises culturing the plant part under growth conditions to enhance transformation, induce the PCM formation, and induce production of the betalain.

[0018] In some aspects, the growth conditions are selected from (such as being selected from the 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 plant part under the growth conditions comprises intermittently contacting the plant part with a culture medium containing sugar and basal salt.

[0020] In some aspects, contacting the plant part with nucleotide sequences and culturing the plant part comprises infecting the plant part with a Rhizobium or Agrobacterium strain comprising a root-inducing (Ri) plasmid or a tumor-inducing (Ti) plasmid, a nucleotide sequence encoding a gene that induces PCM formation, and a nucleotide sequence encoding the enzyme associated with production of the betalain, and culturing the infected plant part to enhance PCM formation, and induce expression of the enzyme and production of the betalain.

[0021] In some aspects, the Rhizobium or Agrobacterium strain comprises the Ri plasmid comprising the nucleotide sequence encoding the gene that induces PCM formation, and the nucleotide sequence encoding the enzyme.

[0022] In some aspects, the Rhizobium or Agrobacterium strain comprises the Ri plasmid, the nucleotide sequence encoding the gene that induces PCM formation, and the nucleotide sequence encoding the enzyme.

[0023] In some aspects, the Rhizobium or Agrobacterium strain comprises a disarmed Ti plasmid or disarmed Ri plasmid, a nucleotide sequence encoding a gene that induces PCM formation, and a nucleotide sequence encoding the enzyme.

[0024] In some aspects, contacting the plant part with the nucleotide sequences and culturing the plant part comprises contacting the plant part with the nucleotide sequence encoding the gene that induces PCM formation, culturing the plant part to enhance PCM formation, contacting formed PCM tissue from the PCM with the nucleotide sequence encoding the enzyme associated with production of the betalain, and culturing the PCM tissue to enhance production of the betalain by the PCM.

[0025] In some aspects, contacting the plant part with the nucleotide sequences and culturing the plant part comprises simultaneously introducing to the 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, a hypocotyl segment, a petiole, an intemode, a node, a meristem, or a leaf. [0026] In some aspects, the method further comprises identifying the bacterium strain from a plurality of bacterium strains.

[0027] In some aspects, the nucleotide sequence encoding the enzyme is operably connected to a promoter.

[0028] In some aspects, the nucleotide sequence encoding the enzyme is operably connected to an ubiquitin promoter, a figwort mosaic promoter, or a 35 S Cauliflower Mosaic Virus promoter.

[0029] In some aspects, the method further compnses screening new growth from the cultured plant part for PCM formation.

[0030] In some aspects, the method further comprises screening and selecting cultured plant part for production of the betalain.

[0031] A method of generating a bacterium strain comprising transforming a bacterium strain with a nucleotide sequence encoding an enzyme associated with production of a betalain, 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.

[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] Various aspects are directed to a method comprising contacting a plant part with a bacterium strain containing a Ri plasmid or a Ti plasmid, a nucleotide sequence encoding an enzyme associated with production of a betalain, and a nucleotide sequence encoding a gene that induces PCM formation, inducing formation of PCM tissue from the plant part under infection conditions, and culturing the PCM tissue in a culture medium under growth conditions to induce expression of the nucleotide sequences and production of the betalain.

[0036] In some aspects, the bacterium strain comprises a Rhizobium or Agrobacterium strain and the method further comprises transforming the Rhizobium or Agrobacterium 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.

[0037] In some aspects, the culture medium is selected from a liquid culture medium and a solid growth medium, and optionally comprises a selection agent.

[0038] In some aspects, the method further composes selecting PCM tissue from the plant part as transformed by the contact with the bacterium strain for culturing in the culture medium, and screening the cultured PCM tissue for production of the betalain. [0039] In some aspects, contacting the plant part with the bacterium strain comprises simultaneously introducing to the 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, a hypocotyl segment, a petiole, an intemode, or a leaf.

[0040] In some aspects, contacting the plant part with the bacterium strain and culturing the plant part comprises contacting the plant part with a first bacterium strain comprising the nucleotide sequence encoding the gene that induces PCM formation, culturing the 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.

[0041] In some aspects, the method further comprises capturing the betalain by isolating and purifying the betalain from the culture medium, PCM tissue of the PCM, or a combination thereof.

[0042] Various aspects are directed to plant cell matrix (PCM) culture for producing a betalain, the PCM culture being induced from a plant part, wherein a cell of the PCM culture comprises a nucleotide sequence encoding an enzyme associated with production of a betalain. [0043] In some aspects, the nucleotide sequence encoding the enzyme encodes a plurality of enzymes and is operably linked to a promoter and comprises 2A self-cleaving peptides located between respective enzymes of the plurality of enzymes.

[0044] In some aspects, the nucleotide sequence comprises a sequence selected from (such as being selected from the group consisting of): SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 36, and a combination thereof.

[0045] Various aspects are directed to a PCM culture that produces betalain in plant cells of the PCM culture.

[0046] In some aspects, the plant cells express a sequence selected from (such as being selected from the group consisting ol): SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 36, and a combination thereof.

[0047] In some aspects, the plant cells are transformed by 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.

[0048] In some aspects, the PCM culture is generated from a Cannabaceae plant, a Brassicaceae plant, a Solanaceae plant, a Fabaceae plant, or an Apiacea plant.

[0049] Various aspects are directed to an expression construct comprising SEQ ID NO:

2, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 35, or SEQ ID NO: 39.

[0050] In some aspects, the expression construct comprises SEQ ID NO: 1, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 38, or SEQ ID NO: 40.

[0051] Various aspects are directed to a system for producing a betalain from PCM tissue comprising a plurality of bioreactors in serial connection, wherein each bioreactor is inoculated with a PCM culture according to the method of claim 1 or 2, and configured for growth and maintenance of the PCM culture in a culture medium.

[0052] In some aspects, the culture medium comprises a liquid culture medium and the system is configured to recover the betalain from the liquid culture medium.

[0053] In some aspects, at least one bioreactor is a flask, 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.

[0054] In some aspects, each bioreactor of the plurality is structurally and operationally similar. [0055] Various aspects are directed to a betalain produced by using the PCM according to the method of claim 1 or 2.

[0056] Various aspects are directed to a method comprising transforming a plurality of plant parts with a plurality of bacterium strains to induce PCM formation, and optionally to induce expression of an enzyme associated with production of a betalain, therefrom, assessing transformation frequencies of the plurality of bacterium strains, and selecting respective ones of the plurality of bacterium strains based on the transformation frequencies.

[0057] In some aspects, the selected respective ones of the plurality of bacterium strains comprise ATCC 43057, ATCC 43056, ATCC 13333, ATCC 15834, K599, or a combination thereof.

DESCRIPTION OF THE DRAWINGS

[0058] 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:

[0059] FIGs. 1 A-1B illustrate example methods for producing a betalain using a PCM, consistent with the present disclosure.

[0060] FIG. 2 illustrates an example method for transforming a bacterium strain to comprise a sequence encoding an enzyme associated with production of betalain, consistent with the present disclosure.

[0061] FIG. 3 illustrates an example method for transforming a plant part to induce PCM formation and production of a betalain, consistent with the present disclosure.

[0062] FIGs. 4A-4C illustrate an example expression construct for delivery of a sequence encoding an enzyme, consistent with the present disclosure.

[0063] FIGs. 5A-5F illustrate example expression constructs for delivery of a sequence encoding an enzyme, consistent with the present disclosure.

[0064] FIGs. 6A-6D illustrate example images of PCM cultures producing betacyanin, consistent with the present disclosure.

[0065] FIGs. 7A-7B illustrate example images of PCM cultures producing betacyanin at different levels, consistent with the present disclosure. [0066] FIGs. 8A-8F illustrate example images of PCM cultures producing betacyanin at different levels, consistent with the present disclosure.

[0067] FIGs. 9A-9C illustrate example images of PCM cultures producing betanidin and betaxanthin, consistent with the present disclosure.

[0068] FIGs. 10A-10B illustrate example images of PCM cultures producing betaxanthin, consistent with the present disclosure.

[0069] FIGs. 11 A-l IB illustrate example images of betalains in liquid from PCM cultures, consistent with the present disclosure.

[0070] FIGs. 12A-12B illustrate example experimental results from PCM cultures producing betalains, consistent with the present disclosure.

[0071] FIGs. 13A-13B illustrate example images of PCM cultures generated from solanum tuberosum plants, consistent with the present disclosure.

[0072] FIGs. 14A-14B illustrate example images showing betalain production in solanum tuberosum PCM cultures, consistent with the present disclosure.

[0073] FIGs. 15A-15B illustrate example images of betalain production in solanum tuberosum PCM cultures using different bacterium strains, consistent with the present disclosure.

DETAILED DESCRIPTION

[0074] The present disclosure is directed to methods, materials, and systems for transforming plant parts to induce a PCM phenotype and produce a betalain. Various aspects are directed to PCM cultures transformed to express an enzyme associated with production of a betalain and systems for production and recovery of the betalain using the transformed PCM tissue.

[0075] 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. Betalams 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. For example, plant- based biomass production of betalains 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.

[0076] Embodiments in accordance with the present disclosure are directed to transforming a plant part to induce PCM formation and induce production of a betalain.

In some embodiments, a bacterium strain can be used to transform the plant part. For example, Rhizobium strains, Agrobacterium strains, and other Rhizobia strains capable of inducing PCM phenotype in plants can be used to non-transiently transform the plant part and/or to produce the betalain. The bacterial strain can be any strain harboring a Ri plasmid or otherwise being transformed to induce PCM formation, as further described herein. The transformed plant part can be transiently or stably modified by the bacterium strain and can be cultured to maximum production of the betalain. In some embodiments, the plant part can be infected with a first bacterium strain to produce PCM tissue and then the formed PCM tissue can be transformed with a second bacterium strain to produce the betalain, sometimes herein referred to as “re-transformation” or “re-transformed”. Embodiments are not limited to use of a bacterium strain to induce PCM formation, and can include contacting a plant part with a nucleotide sequence to transform the plant part and without a bacterium strain. For example, the plant part can otherwise be contacted with a (heterologous) nucleotide sequence encoding the Ri plasmid and/or the gene that induces PCM formation, which transforms plant cells to express the nucleotide sequence. The resulting PCM 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] As used herein, a PCM includes and/or refers to plant cells transformed by a nucleotide sequence encoding a gene that induces PCM formation, which can include a plurality of different plant cell types and can be used to produce a betalain. The PCM can include plant cell types including, but not limited to, plant stem cells, maturing cells, and mature cells. In some embodiments, the PCM is a tissue culture including the transformed plant cells, e.g., the plurality of different plant cell types. 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.

[0078] Turning now to the figures, FIGs. 1A-1B illustrate example methods for producing a betalain using a PCM, consistent with the present disclosure.

[0079] As shown by FIG. 1 A, at 1 101, the method 100 includes contacting a plant part with a (first) nucleotide sequence encoding a gene that induces PCM formation (e.g.,

PCM gene), and a (second) nucleotide sequence encoding an enzyme associated with production of a betalain. The plant part can be a seedling or a hypocotyl segment, although examples are not so limited and can include plant cells or other plant parts, such as a petiole, intemode, or leaf. The plant part can be from a monocotyledon plant or a dicotyledon plant.

[0080] The nucleotide sequences can be heterologous to the plant. As described below, the contact with the nucleotide 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. In some embodiments, as further described below, the (first) nucleotide sequence encoding the gene that induces PCM formation can include or encode a Ri gene or plasmid that is expressed by plant cells of the plant part in response to the contact. As further described below, in some embodiments, at 101, the plant can be co-transformed with both 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. In various embodiments, the gene that induces PCM formation 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 sequence can encode the plurality of PCM genes, such as a plurality of different PCM genes.

[0081] Such techniques and/or methods for contacting the plant part with the (first and/or second) nucleotide sequences to transform the plant part and induce PCM formation 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), 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.

[0082] In some embodiments, the contact of the plant part with the nucleotide sequences to transform the plant part, induce PCM formation, and/or produce the betalain 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 gene(s) in a plant part to produce the PCM and/or betalain production by injecting a suspension including the bacterium strain containing the gene or genes of interest into the 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 segment(s) which integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the 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).

[0083] 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 “agroinfiltration protocol”.

[0084] 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 minipores 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. In some embodiments, the transformation or the re-transformation, as further described herein, 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.

[0085] As described above, in various embodiments, contacting the plant part with the nucleotide sequences, at 102 of method 100, includes contacting the plant part with a bacterium strain comprising the nucleotide sequence encoding the gene that induces PCM formation to transform the plant part and induce PCM formation. For example, the plant part can be contacted with the bacterium 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 induce production of the betalain.

[0086] In some embodiments, the bacterium strain can earn- 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 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: 39.1n some embodiments, the bacterium strain can be transformed with 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, although embodiments are not so limited.

[0087] In some embodiments, the contact with the bacterium strain can be under infection conditions that induce and/or enhance transformation of the 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 bacterium. 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.

[0088] The enzyme can be associated with the pathway for converting tyrosine to the betalain. Tyrosine can be naturally synthesized by the 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. In some embodiments, the enzyme can include a plurality of enzymes, such as L-DOPA, dihydroxyphenylalanine (DOPA) 4,5-dioxygenase (DODA) , Cytochrome P450 (CYP76AD1), CYP76AD6, glucosyltransferase, among other enzymes and combinations thereof. As such, a combination of multiple enzymes are expressed in various embodiments, such as DODA, CYP76AD1, and glucosyltransferase. Example glucosyltransferase include, without limitation, betanidin-5-O-glucosyltransferase and cyclo-DOPA-5-O-glucosyltransferase. In some embodiments, the three enzy mes 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.

[0089] In some embodiments, the enzyme and/or the betalain can be exogenous or heterologous to the 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 plant species (e.g., a wild-type plant expresses the betalain), and contacting the plant part with the (second) 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 and/or a betaxanthin. For example, the betalain can be selected from betanin, isobetanin, probetanin, neobetanin, vulgaxanthin, miraxanthin, portulaxanthin, and indicaxanthin, among other types ofbetalains and combinations thereof.

[0090] The bacterium strain can include any strain capable of inducing PCM formation and/or transformed to induce expression of the enzyme and production of the betalain. As previously described, 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. In some embodiments, the plant part can be transiently or stably transformed or modified in response to the contact with the bacterium strain, and which causes formation of the PCM.

[0091] In some embodiments, the PCM transgene is transferred in response to the contact with the bacterium strain, which can cause an infection, and along with (e.g., simultaneously with) the secondary transgene that encodes the enzyme introduced into the bacterium strain using electroporation and other cloning techniques. For example, in addition to the T-DNA from the Ri plasmid, additional T-DNAs can be co-delivered to the plant part and expressed in PCMs as transgenic PCM tissue, such as those from vectors carrying a gene encoding the enzyme. The T-DNA(s) from the bacterium strain can be stably integrated in the plant part. In other embodiments, the plant part can be transformed with a first bacterium strain to induce the PCM formation and then the formed PCM can be transformed with a second bacterium strain to produce the enzyme, sometimes herein referred to as “re-transformation” or “re-transformed” and as further described herein. In other embodiments, the transformation and/or re-transformation can be performed.

[0092] Contacting the plant part with the nucleotide sequences encoding the gene that induces PCM formation and the enzyme associated with betalain production, such as contact with the bacterium strain, can transform the plant part to express the PCM phenotype, and optionally, to produce the betalain, as further described below. The transformation of the plant part can be transient or non-transient, e.g., stable. A stable transformation includes or refers to the nucleotide sequence being integrated into the plant genome and as such represents a stable and inherited trait. A transient transformation includes or refers to a nucleotide sequence being expressed by the 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. [0093] Generally, the bacterium strain is prepared for infecting the plant part by introducing a nucleotide sequence encoding the enzy me into the bacterium strain (e.g., by electroporation) and culturing the transformed bacterium strain under conditions to select positively transformed cells.

[0094] In some embodiments, the method 100 includes selecting the particular bacterium strain. Selection of an effective strain for the production of transformed PCM cultures can depend on the plant species to be infected and can be determined empirically. Parameters such as the PCM tissue induction percentage per total explants, the PCM tissue initiation days per total explants, and the PCM tissue induction frequency per single explant can be measured to select the bacterium strain.

[0095] Many strains of R. rhizogenes exist and can be used for 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 plant part can include a 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. Other example bacterium strains, which can be used for re-transforming PCMs, include 18rl2, GV3101, AGL1, and EHA105.

[0096] However, embodiments are not so limited. In some embodiments, the bacterium strain can be transformed to can ' 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. [0097] In some embodiments, the bacterium strain can be transformed using a vector or vectors carrying the genes. In other embodiment, vector(s) carrying the genes can be used to transform the plant cells of the plant part without the use of a bacterium strain.

[0098] In some embodiments, a vector or binary vector carrying the gene associated with the enzyme can include a nucleic acid sequence 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 embodiments, 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 ak, Nature, 435:1122-1125, 2005; Yang et ak, ProcNatl Acad Sci USA, 103:10503-10508, 2006; Kay et ak Science, 318:648-651, 2007; Sugio et ak, ProcNatl 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.

[0099] In some embodiments, the 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 plant tissue with the secondary transgene to cause expression of the secondary transgene along with the PCM transgene. In some embodiments, the TALEN sequence can encode tyrosine and/or otherwise cause upregulation or overexpression of tyrosine in the formed PCM tissue. [0100] 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 embodiments, 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 plant parts. [0101] 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. Cocultivation of the plant part on media can facilitate delivery of both a Ri plasmid (or a disarmed Ti plasmid) and 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 plant cells. In various embodiments, as further illustrated by FIG. 2, the method 100 can include transforming a wild-type bacterium strain with the nucleotide sequence encoding the enzyme, and, in some embodiments, with the gene that induces PCM formation. In some embodiments, two bacterium strains can be prepared: the first bacterium strain to induce PCM and the second bacterium strain transformed to include the nucleotide sequence encoding the enzyme.

[0102] The method 100 can also include preparing a plant part, such as an explant, to be inoculated with the prepared bacterium strain or otherwise contacted with the nucleotide sequences encoding the gene that induces PCM formation and encoding the enzyme (both being heterologous to the plant). Cells of the plant part can be transformed with an expression construct suitable for expression of the enzyme and production of the betalain. Different plant parts, such as hypocotyl, leaf, stem, stalk, petiole, menstem, a node, an intemode. shoot tip, cotyledon, protoplast, storage root, or tuber, can be used to induce PCM formation. 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 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 contacted with the nucleotide sequence. For example, the plant tissue can be immersion into and/or submerged in the bacterium strain culture. Appropriate media and incubation conditions for contact or infection, co-cultivation, and PCM induction can depend on the explant to be transformed. The transformed explant can be cultured to enhance or optimize transformation and PCM induction and development, as further described herein.

[0103] At 103, the method 100 further includes culturing the plant part to enhance transformation or production of the betalain. In various embodiments, the 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 sequences encoding the PCM gene and the enzyme associated with betalain production, and then cultured in another culture medium or a plurality of culture mediums to enhance further PCM tissue growth and production of the betalain. More particularly, and in some embodiments, the plant part is contacted and co-cultured with the bacterium strain under the infection conditions to transform the 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 plant part is cultured using a culture medium.

[0104] In some embodiments, the plant part can be cultured under growth condition 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.

[0105] The plant type can include a dicotyledonous plant. In some embodiments, the plant type can include a Cannabaceae plant, a Brassicaceae plant, a Solanaceae plant, a Fabaceae plant, or an Apiacea plant. In some embodiments, the plant type can include a Cannabaceae plant, a Brassicaceae plant, or a Solanaceae plant. In some embodiments, the plant type include a plant part selected from a seedling (e.g., hypocotyl), a petiole, meristem, a node, an intemode, or a leaf

[0106] In some embodiments, the plant type can include a specific plant line and/or clone of the plant that exhibits greater PCM formation and/or betalam production than other plant lines and/or clones. For example, within a plant species, there can be genetic variability which causes different optimized tissue formation from the PCM compared to other plant lines and/or clones. A plurality of 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 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 plant can be screened for and/or selected by culturing the plurality of plant clones of different plant lines (and/or plurality of plant clones of a plant line), as transformed by the nucleotide sequence, 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.

[0107] In some embodiments, as noted above, the type of contact can comprise intermittently contacting the 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 plant part with the culture medium and no contact of the plant part with the culture medium. By providing an intermittent contact, the transformed plant part is provided with nutrients (e.g., sugars and basal salts) for growth during times of contact with the culture medium, and is provided with air or other gases for growth during times of no contact with the 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 medium 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 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.

[0108] In some embodiments, the intermittent contact comprises cycling between contacting (e.g., submerging, dripping, or other types of contact) the plant part with the culture medium and not contacting (e.g., not submerging, dripping, 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 plant part is in contact with the culture medium as compared to the time the plant part is not in contact. For example, the 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.

[0109] In various embodiments, contacting the plant part with the nucleotide sequences encoding the gene that induces PCM formation and encoding the enzyme, such as contact with the bacterium strain, comprises simultaneously introducing a first transgene and a second transgene to the plant part, and cultivating the transformed plant part to generate PCM tissue, such as cultivating using or under the above-described growth conditions. The first transgene can be associated with PCM formation, and the second transgene can be associated with the enzyme which can cause production of the betalain by converting tyrosine to the 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 plant of the plant part.

[0110] As noted above, embodiments are not limited to simultaneously introducing a first transgene and a second transgene to the plant part. For example, the method 100 can include two infections. In some embodiments, the plant part is first transformed using the first transgene that induces the PCM phenoty pe to produce PCM tissue and the PCM tissue is isolated from wild-type tissue and retransformed using the second transgene associated with the betalain. 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.

[0111] As an example, contacting the plant part with the bacterium strain and culturing the plant part, at 101 and 103 of the method 100, can include contacting the plant part with a first bacterium strain comprising the nucleotide encoding the gene that induces PCM formation and culturing the plant part to enhance PMC formation, such as under the above-described growth conditions. For example, the first bacterium strain can comprise an Ri plasmid or Ti plasmid and including the nucleotide sequence encoding PCM gene. The method 100 can further include contacting the formed PCM tissue from the PCM with a second bacterium strain comprising the nucleotide sequence encoding the enzy me, and culturing the PCM tissue to enhance production of the betalain by the PCM, as under the above-described growth conditions. In some embodiments, the second bacterium strain may comprise an Ri plasmid or a Ti plasmid. In some embodiments, the second bacterium strain may induce further PCM formation, and in other embodiments, may not (e.g., may include or 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 tissue in a solution containing the bacterium strain or pipetting bacterium strain onto the PCM tissue.

[0112] In some embodiments, the first and/or second transformation can include other transformation techniques which may or may not include use of bacterium strain(s). For example, the first transformation can include 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 of the betalain. [0113] As described above, culturing the plant part can include inducing formation of PCM tissue from the i plant part as transformed or infected and/or culturing the PCM tissue in a culture medium or culture mediums under conditions for expression of the nucleotide sequence(s), such as those encoding the PCM gene or the enzyme. For example, the method 100 can include screening new growth from the cultured plant part for PCM formation. For PCM induction, the plant part can be transferred into liquid or solid media with antibiotics two or three days after infection or co-cultivation, and for up to two or three months. 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 PCMs can be induced within a short period of time, which can vary from one week to over three months depending on the plant species. In some embodiments, the PCM can be induced for two weeks to eight weeks via the contact with the culture medium (e.g., intermittent contact with a liquid culture medium). After the PCM is established (e.g., after culturing under the growth conditions), the PCM can be maintained in culture, and in some embodiments, so long as the PCM is transferred to fresh media every one to three weeks. In some embodiments, the decontaminated PCM tissue can be sub-cultured on hormone- free medium regularly (e.g., every one to two weeks).

[0114] The various described culture mediums (e.g., used to infect or an liquid infection medium, co-cultivate medium, selection medium, solid media) can each generally comprise water, a basal salt mixture, a sugar, and optionally other components such as vitamins, selection agents, amino acids, and phytohormones. At least some of the medias (e.g., for enhancing growth) can include sugars, basal salts, growth hormones, selection agents, and/or antibiotic agents, among other reagents, such as water and vitamins. For example, the various medias can include nutritional 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, KN03, CaCk-2H20, MgS04-7H₂0, and KH2PO4. Micro elements can be provided as KI, H3BO3, MnSOr 4FhO, ZnS0₄, Na₂Mo0₄2H₂0, CuS0₄ 5H₂0,

C0CI2 6H2O, C0SO4 7H2O. FeS04-7H20, and Na₂EDTA 2H2O. 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.

[0115] In some embodiments, a respective culture medium, such as the above-noted solid culture medium, 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 occurnng and synthetic analogues of auxins include, but are not limited to, indoleacetic acid (IAA), 3-indolebutyric acid (IB A), a-napthaleneacetic acid (NAA), 2, 4-dichlorophenoxy acetic acid (2,4-D), 4-(2,4-dichlorophenoxy)butyric acid, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 3-ammo-2,5-dichlorobenzoic acid (chloramben), (4-chloro-2- methylphenoxyjacetic 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.

[0116] 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 antioxidants, amino acids, cobalt, or AgNCb. 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.

[0117] In some embodiments, the new growth is screened to identify the PCM tissue and the identified PCM tissue is separated and sub-cultured in the culture medium under conditions for expression of the nucleotide sequence(s) and production of the betalain. Selection can be accomplished in multiple ways. 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 is one option. 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 PCM tissue over wild type as the wild-type tissue can die in the absence of the growth hormone when grown in the dark. However, embodiments are not so limited and the culture medium can include hormones in some embodiments.

In various embodiments, the PCM 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. A further selection technique can be used, such as a 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 PCM tissue that produce the betalain. For example, the cultured PCM tissue can be screened for the production of 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 plant part and to allow for selection of the PCM tissue that contains the second transgene.

[0118] In some embodiments, the transgenic PCM strains can be isolated and characterized. For example, the method 100 can include screening and selecting cultured plant parts for expression of the enzyme using end point RT-PCR, fluorescent protein reporter expression (e.g., RFP or YFP), or visually identifying betalain production from the PCM 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.

[0119] Due to the site uncertainty of integration of the nucleotide sequence(s) into the plant cell genome, PCM strains can show different expression patterns for the enzyme(s) and/or production of the betalam. Expression and/or production levels can be measured using biochemical analysis to quantity betalain concentration in the medium (e.g., Lowry, Bradford, BCA, Spectroscopy, HPLC, LC-MS, and UV spectroscopic protein assays). PCM 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 betalain-producing PCM lines that secrete the betalain at high levels on a biomass basis (e g., per gram dry weight).

[0120] To initiate a PCM culture in liquid medium, a piece of a transformed PCM tissue (e.g., 1 gm piece) can be transferred to a culture vessel. Any conventional plant or PCM 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.

[0121] In some embodiments, the method 100 (and/or method 102) can include capturing the betalain. The betalain can be captured by isolating and purifying the betalain from the culture medium and/or from PCM tissue of the PCM. 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 reducing the media volume (e.g., by cross-flow filtration).

[0122] The betalain can be isolated and purified from other components of the spent media. For example, a betalain or betalains can be isolated and purified from the spent media using recovery steps. 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.

[0123] To enhance recovery of the betalain secreted by the PCM cultures, an effective amount of a 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 enzyme and/or betalain concentration and activity by preventing degradation and denaturation, or any substance that blocks nonspecific interactions between the secreted betalain and walls of the culture vessel. A compound stabilizing agent for use in PCM 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 PCM culture cell viability and integrity, enzyme expression, and growth and cell division. In addition, the compound stabilizing agent may not interfere with purification of the secreted betalain. An “effective amount” of a compound stabilizing agent is an amount, when added to a given volume of a PCM culture medium, that significantly improves recovery of a secreted 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. 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 amino acids and betalain), and polymers (e.g., polyethylene glycol, polyvinylpyrrolidone).

[0124] In some embodiments, the betalain is not secreted, or not fully secreted, by the PCM culture. The betalain can accumulate in root tissue or cells of the PCM culture. When the PCM culture has 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 betalain product can then be purified in accordance with the nature of the product. [0125] Purifying typically starts with extraction of the betalain and removal of any plant insoluables. 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 variety 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/ani on-exchange, reversed phase chromatography or anion, hydrophobic), various 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 purify 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. [0126] FIG IB illustrates another example method 102 for producing a betalain using a PCM. In some examples, the method 102 of FIG. IB may include an implementation of the method 100 of FIG. 1A.

[0127] At 104, the method 102 includes contacting a plant part with a bacterium strain containing a Ri plasmid or a Ti plasmid, a nucleotide sequence encoding an enzyme associated with production of the betalain, and a nucleotide sequence encoding a gene that induces PCM formation. At 106, the method 102 includes inducing the formation of PCM tissue from the plant part under infection conditions. For example, the contact with the bacterium strain can simultaneously introduced the first transgene associated with PCM formation and the second transgene associated with the enzy me. In other embodiments, the contact with the bacterium strain can include multiple contacts with multiple bacterium strains, such as the previously described “re-transformation”.

[0128] At 108, the method 102 includes culturing the PCM tissue in a culture medium under growth conditions to induce expression of the nucleotide sequence and production of the betalain, such as the previously described growth condition.

[0129] In various embodiments, the growth conditions can enhance PCM formation and/or production of betalain, resulting in production of PCM tissue at a greater level than production than production of tissue (e.g., root tissue) by a wild-type plant or plant grown in the field. For example, the production of the PCM tissue by the PCM 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 grown 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 PCM tissue produced by the PCM in 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 PCM tissue) by the PCM, such as grams of PCM tissue or root tissue. For example, the PCM can produce PCM tissue at a greater mass level than root tissue produced by a wild-type-plant or as grown in the field.

[0130] In some embodiments, the PCM can produce PCM 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 increase, at least a 100-fold, at least 200-fold, or at least 500 fold as compared to the root tissue produced by a wild-type plant and/or as grown in the field. In some embodiments, PCM can include PCM tissue 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 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, among other range increases in the PCM tissue mass as compared tissue mass (e.g., root tissue) produced by a wild-type plant and/or as grown in the field.

[0131] In some embodiments, the method 100 includes culturing the plant part under the growth conditions to induce and enhance PCM formation and to induce production of betalain by the PCM formed. In various embodiments, the plant part can be cultured with the bacterium strain to induce PCM formation or otherwise contacted with the nucleotide sequence(s) encoding the PCM gene and/or the enzyme, 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.

[0132] FIG. 2 illustrates an example method for transforming a bacterium strain to comprise a sequence encoding an enzyme, consistent with the present disclosure. The method 200 can be combined with the method 100 and/or method 102 of FIGs. 1A-1B, in some embodiments.

[0133] At 205, the method 202 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, although embodiments are not so limited. In some embodiments, 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: 3.

[0134] In some embodiments, the bacterium strain is a w ild-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, among other types of bacterium. In some such embodiments, transforming the bacterium strain can include disarming the Ti plasmid and transforming with both the nucleotide sequence encoding the enzy me and the gene that induces PCM formation. 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 202 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.

[0135] The bacterium strain can be transformed using an expression construct, such as a vector that includes an expression cassette. As used herein, an expression construct refers to or includes a nucleic acid sequence (e.g., DNA sequence) including a single or a plurality' of vectors or binary vectors carrying genes. A vector or binary vector includes 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 plant cell. Successful transformation can result in the expression cassette directing plant cells to make the enzyme and the betalain as a secondary' metabolite. In some examples, the bacterium strain can be 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, 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.

[0136] In some embodiments, the expression cassette includes the sequence encoding the enzyme, T-DNA border sequences, and a promoter. 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 regulator elements. For example, the expression cassette can include TALEN T-DNA. The expression cassette 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 PCM culture media Enzymes comprising signal peptides of plant origin (e.g., the N-terminal signal peptide from the tobacco PR1 a protein or calreticulin) or signal peptides from eukaryotic secreted polypeptides, e.g., mammalian signal peptides, can be efficiently secreted through the plasma membrane and cell wall into the extracellular medium.

[0137] The expression cassette comprising the nucleotide sequence encoding the gene 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 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.

[0138] In some embodiments, the bacterium strain is transformed using an expression construct that includes the vector(s) or binary vector(s) carrying genes. The vector or binary vector(s) 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 as previously described. 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. 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). [0139] In some embodiment, multiple bacterium strains can be transformed. For example, a first bacterium strain can 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 200 can include transforming a first bacterium strain to carry the PCM gene and a second bacterium strain to carrying the nucleotide sequence encoding enzyme.

[0140] 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. 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. [0141] 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 kDazein promoter, 22 kDazein promoter, 27 kDa zein promoter, d-zein promoter, 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.

[0142] 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 transcnpts 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 or more 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.

[0143] 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, bacteria, 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 enzyme. 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. [0144] 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 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.

[0145] An example expression construct including a vector is illustrated by FIG. 4A and discussed further herein.

[0146] In some embodiments, the DNA sequence can include the sequence of a gene occurring in the wild-type plant or other organism, 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% identity. 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.

[0147] At 207, the method 202 includes culturing the transformed bacterium strain. For example, the bacterium strain can be cultured under the infection conditions, such as 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.

[0148] Although FIGs. 2-3 illustrate contacting a plant part with the bacterium strain to induce PCM formation and/or transforming a bacterium strain, embodiments are not so limited. In some embodiments, a wild-type bacterium strain can be used to transform the plant part to form a PCM, which can be enhanced by culturing the transformed plant part under growth conditions. In some embodiments, the plant part can be transformed using other transformation techniques which may not include use of a bacterium strain, as described above. [0149] FIG. 3 illustrates an example method for transforming a plant part to induce PCM formation and expression of a betalain, consistent with the present disclosure. The method 310 can include an implementation of method 100 of FIG. 1A or method 102 of FIG. IB. At 315, the method 310 includes preparing a wild-type plant part, such as a cutting (e.g., hypocotyl segment), seedling, an intemode, or a leaf excised from a host plant. In some embodiments, the host plant is Cannabaceae plant, however embodiments are not so limited and can be different to other plants such as other monocotyledon plants or dicotyledon plants.

[0150] At 317, the method 310 includes inoculating the wild-type plant part with a bacterium strain solution. The bactenum strain can be transformed to carry the nucleotide sequence encoding the enzyme associated with production of a betalain, such as using an expression construct. Once such a construct is available, bacterium strains can be transformed to carry the expression construct and used to infect wild-type plant part. Selection of transgenic PCM tissue (e.g., hairy roots) using a plant selective agent (e.g., spectinomycin) can enrich the formation of high expressing transgenic PCM tissue (versus non-transgenic roots carrying only the Ri plasmid T-DNA or wild-type roots) and, optionally, increase the expression of genome editing reagents in root tissues.

[0151] At 319, the method 310 includes culturing and screening the infected plant part. For example, the method 310 can include transferring cuttings and/or whole seedlings infected with the bacterium strain to a medium for selection of transgenic tissue, e.g., PCM tissue. Other types of plant tissue can be used, such as a petiole, an intemode, or a leaf. The degree of editing in the plant part can be directly related to the abundance of betalain in tissue and can be tracked using various methods of betalain detection. For instance, plant parts can be assayed for accumulation of the betalain in new PCM tissue. New root growth can be sampled for detection of the betalain using visual screening, RT- PCR, Spectroscopy, microscopy, LC-MS, HPLC, and/or western blot. PCM 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 plants, depending on the species. [0152] At 321, the method 310 can include isolating the betalain, such as previously described and/or using a system as described below.

[0153] As described above, embodiments are not limited to co-transformation and may include infecting plant parts to induce PCM formation and re-transforming the PCM to produce the betalain. The transformation and re-transformation can be accomplished by a wide variety of techniques, as described above by the examples provided for contacting the plant part with the nucleotide sequence.

[0154] Various embodiments of the present disclosure are directed to a non-naturally occurring plant part, such as a PCM culture and/or PCM tissue generated by the methods of FIGs. 1A-1B, FIG. 2 and/or FIG. 3.

[0155] Various embodiments of the present disclosure are directed to a PCM culture generated by the methods of FIGs. 1A-1B, FIG. 2, and/or FIG. 3. For example, the PCM culture can be used for producing a betalain, the PCM culture being induced from a plant part and a bacterium strain, wherein a cell of the PCM culture comprises a nucleotide sequence encoding an enzyme associated with 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 encodes a plurality of enzymes and is operably linked to a promoter and includes 2A self-cleaving peptides located between respective enzymes of the plurality of enzymes.

[0156] In some embodiments, the nucleotide sequence encoding the enzyme is selected from SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 36, and a combination thereof.

[0157] Some embodiments are directed to a PCM culture that produces betalain from plant cells of the PCM culture. For example, the plant cells of the PCM culture may- express a sequence selected from: EQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 36, and a combination thereof. In some embodiments, plant cells are transformed by 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. In some embodiments, PCM culture is generated from a Cannabaceae plant, a Brassicaceae plant, a Solanaceae plant, a Fabaceae plant, or an Apiacea plant. [0158] Other embodiments directed to expression constructs. The expression construct 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, the expression construct can comprise SEQ ID NO: 1, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 38, or SEQ ID NO: 40. [0159] In some embodiments, a method can include and/or the methods of FIGs. 1A-1B, FIG. 2, and/or FIG. 3 can further include identifying the bacterium strain from a plurality of bacterium strains. For example, a method can include transforming a plurality of plant parts with a plurality of bacterium strains to induce PCM formation, and optionally to induce production of a betalain, and assessing transformation frequencies of the plurality of bacterium strains therefrom. In some embodiments, the plurality of plant parts are transformed with modified bacterium strains, such as bactenum strains carrying a nucleotide sequence encoding a gene that induces PCM formation and/or a nucleotide sequence encoding enzyme, as described above. In some embodiments, the plurality of plant parts are transformed with wild-type 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.

[0160] In some embodiments, the PCM culture can be used to produce the betalain. Various embodiments are directed to a system for producing the betalain from the PCM tissue. For example, the system can include a plurality of bioreactors in serial connection, wherein each bioreactor is inoculated with the PCM culture according to and/or obtained using any of above-described methods and configured for growth and maintenance of the PCM culture in a culture medium.

[0161] In embodiments of the present disclosure, the transgenic PCM cultures are maintained in a bioreactor system. The PCM culture can be grown in 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).

[0162] 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 PCM culture, an inlet and an outlet. In some cases, one inlet of each bioreactor is connected to an air compressor configured to provide sterilized air to the PCM 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 PCM 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.

[0163] 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 intended relative proportions. The media can be delivered from the mixer by a delivery pipe having an aseptic connector. [0164] 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 PCM culture achieves a steady state of betalain 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.

[0165] 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. [0166] Some embodiments are directed to a betalain produced by a plant part infected with a bacterium strain using a method and/or PCM culture of any of the methods, PCM culture, system, and/or PCM tissue provided herein.

[0167] FIGs. 4A-4C illustrates example expression construct for delivery of a gene encoding the enzyme, consistent with the present disclosure. The example expression construct 420 of FIG. 4A is or includes a vector containing an expression cassette 421 and a vector backbone 426. The expression cassette 421 includes a transgene that causes expression of the betalain. The transgene of the expression cassette 421 includes a gene of interest 425 associated with the betalain, a promoter 427, a left border 429, and a right border 428. In some embodiments, the left border 429 and the right border 428 can be separate from the expression cassette 421. The expression construct 420 and/or expression cassette 421 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.

[0168] FIG. 4B illustrates different example expression cassettes, which can form part of the expression construct 420 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.

[0169] 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.

[0170] As used herein, a “plant” refers to any organism of the kingdom Plantae. In some embodiments, the plant includes a plant species selected from the families of Cannabaceae, Brassicaceae, Solanaceae, Fabaceae, and Apiaceae. In some embodiments, the plant includes a plant selected from the families of Cannabaceae, Brassicaceae, and Solanaceae. In some embodiments, the plant is selected from the families of Cannabaceae and Solanaceae. Non-limiting example plants can be from the families of Cannabaceae, Brassicecea, Fabaceae, Poaceae, Solanaceae, Apiaceae, Malvaceae, and Asteraceae, among other plant families. Non-limiting example plants include but are not limited to Cannabis sativa, Cannabis indica, Cannabis ruderalis, Humulus, Celtis, Alphananthe, Chaetachme, Gironniera, Lozanella, Parasponia, Pteroceltis, Trema, Glycine max, Phaseolus, Pisum sativum, Civer aretinum, Medicago sativa, Arachis hypogaea,

Ceratonia siliqua, Glycyrrhiza glabra, Avena sativa, Zea mays, Triticum aestivum, Oryza sativa, Oryza glaberrima, Hordeum vulgare, Eleusme coracana, Panicum miliaceum, Daucus carota, Solanum lycopersicon, Solanum aviculare, Solanum nigrum, Catharanthus roseus, Panax quinquefolius, Nicotiana tabacum, Atropa belladoma, Thlaspi caerulescens, Brassica napus, Brassica juncea, Ipomoea batatas, Helianthus annuus, and Gossypium plants or plant parts, among other plants or plant parts.

[0171] In some embodiments, the plant includes a Cannabaceae plant or plant part. 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 plant parts.

[0172] In some embodiments, the plant includes a Brassicaceae plant or plant part. As used herein, Brassicaceae refers to a plant of the family Brassicaceae. For example, the Brassicacee plant or plant part can belong to the genus of Draba, Erysium, Lepidium, Cardamine, or Alyssum, among others. In some embodiments, the Brassicacee plant or plant part includes Brassica oleracea (e.g., broccoli, cabbage, cauliflower, kale, collards), Brassica rapa (e.g., turnip, Chinese cabbage, etc.), Brassica napus, Raphamis sativus (e.g., common radish), Armoracia rusticana (e.g., horseradish), or Arabidopsis thaliana (e.g., thale cress), among other plants.

[0173] In some embodiments, the plant includes a Solanaceae plant or plant part. As used herein, Solanaceae refers to a plant of the family Solanaceae. For example, the Solanaceae plant or plant can belong to the genus of Solanum, such as Solanum tuberosum, Solanum dulcamara, Solanum lycopersicum, Solanum melongena, Solanum aethiopicum, Solanum quitoense, Solanum torvum, Solanum muricatum, Solanum betaceum, Solanum lycocarpum, and Solanum scabrum, among others. However, embodiments are not so limited, and the Solanaceae plant or plant part can include Lycianthes, Cestmm, Nolana, Physalis, Lycium, Nicotiana, Brunfelsia, Sessea, Vestia, Reyesia, Salpiglossis, Coeloneurum, Goetzea, Anthocercis, Cypanthera, Benthamiella, Brunfelsia, Calibrachoa, Leptoglossis, Nierembergia, Petunia, Schizanthus, Schwenckia, Iochroma, Chamaersaracha, or Jaltomata, among others. [0174] In some embodiments, the plant includes a Fabaceae plant or plant part. As used herein, Fabaceae refers to a plant of the family Fabaceae. For example, the Fabaceae plant or plant can belong to the genus of Glycine, such as Glycine max (e.g., soybean), Glycine soja, Glycine albicans, Glycine curvata, or Glycine pemota, among others. However, embodiments are not so limited, and the Fabaceae plant or plant part can include Phaselous, Pisum (e.g., Pisum sativum), Cicer (e.g., Cicer arietinum), Medicago (e g., Medicago sativa), Arachis (e.g., Arachis hypogaea), Ceratonia (e.g., Ceratonia siliqua), Glycyrrhiza (e.g., Glycyrnhiza glabra), Cytisus (e.g., Cytisus scoparus), Robinia (e.g., Robinia pseudoacacia), Ulex (e.g., Ulex eropaeus), Puerana (e.g., Pueraria montant), or Lupinus, among others.

[0175] In some embodiments, the plant includes an Apiaceae plant or plant part. As used herein, Apiaceae refers to a plant of the family Apiaceae. In some embodiments,

Apiaceae plant or plant part can belong to the genus of Daucus, Pastmaca, Petroselinum, Coriandnirn, Anethum, Foeniculum, Cumin um, Carum, Anthnscus, Apiurn, Arracacia, Ferula, Pimpinella, or Myrrhis, among others. In some embodiments, the Apiaceae plant or plant part includes Daucus carota, Pastinaca sativa, Petroselinum crispum, Coriandrum sativum, Anethum graveolens, Foeniculum vulgare, Curnmurn cyminum, Carum carvi, Anthriscus cereoliuni, Apium graveolens, Arracacia xanthorrhiza, Ferula asafetida, Ferula gummosa, Pimpinella ansium, Myrrhis odorata, or Levisticum officinale, among others. [0176] 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 “plant part” refers to plant tissues, organs, or cells which are obtained from a whole plant. Plant parts include vegetative structures (for example, leaves, stems), roots, 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 “plant cell” refers to a cell obtained from a plant or in a plant, 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. “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 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 “bacterial line” (or strain) refers to a particular strain of the plant or bacteria.

[0177] The expression “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 plant, or exogenous. In a case where the betalain is endogenous to the plant, e.g., produced naturally by the plant, the betalain of interest is overproduced with respect to an untransformed plant.

[0178] 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.

[0179] 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.

[0180] 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 Matnces”; and (iii) U.S. Provisional Application No. 63/240,660, filed on September 3, 2021 and entitled “Producing Betalains Using Hairy Roots” 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 varying 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.

[0181] 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

[0182] As further illustrated below in connection with the experimental embodiments, plant parts were transformed to induce PCM formation and produce a betalain in different plant species. Different experiments were conducted to illustrate successfully transforming bacterial strains and transforming a Cannabaceae plant part with the bacterial strains and solanum tuberosum plant parts. Further experiments were conducted to transform the plant part to induce PCM formation and to re-transform the PCM to produce betalain. The transformed and re-transformed Cannabaceae plant parts exhibited PCM tissue and 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.

[0183] FIGs. 5A-5F 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.

[0184] FIG. 5A illustrates an example plasmid vector 540 that includes a DNA sequence coding for a plurality of enzymes to produce betalain (e.g., betanidin). The plasmid vector 540 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 540 further includes a plant selectable marker cassette and a LacZ cassette, which are in reverse orientation on the plasmid vector 540, 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 540. The Cannabaceae plant part (e.g., cannabis) was transformed using the R. rhizogenes strain A4 (ATCC43057). The plasmid vector 540 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 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).

[0185] FIG. 5B illustrates an example plasmid vector 550 that includes a DNA sequence coding for a plurality of enzymes to produce betalain (e.g., betanidin). The plasmid vector 550 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 550 further includes a plant selectable marker cassette and a LacZ cassette, which are in reverse orientation on the plasmid vector 550 and as described by plasmid vector 540, 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 550. The Cannabaceae plant part (e.g., cannabis) was transformed using the R. rhizogenes strain A4 (ATCC43057). The plasmid vector 550 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 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).

[0186] FIG. 5C illustrates an example plasmid vector 560 that includes a DNA sequence coding for a plurality of enzymes to produce betalain (e.g., betanidin). The plasmid vector 560 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 560 further includes a plant selectable marker cassette and a LacZ cassette, which are in reverse orientation on the plasmid vector 560. In the particular plasmid vector 560, the plant selectable marker cassette includes the CP4 gene. 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 560. The Cannabaceae plant part (e.g., cannabis) was transformed using the R. rhizogenes strain A4 (ATCC43057). The plasmid vector 560 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 cassete (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 cassete (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 cassete (SEQ ID NO: 17) encodes the LacZ promoter (SEQ ID NO: 18) and LacZ gene (SEQ ID NO: 19). The bacterial selection marker cassete (SEQ ID NO: 20) encodes the KanR promoter (SEQ ID NO: 21) and KanR gene (SEQ ID NO: 22).

[0187] FIG. 5D illustrates an example plasmid vector 565 that includes a DNA sequence coding for a plurality of enzymes to produce betalains (e.g., betaxanthin). The plasmid vector 565 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 565 further includes a plant selectable marker cassete and a LacZ cassete, which are in reverse orientation on the plasmid vector 565 and as described by plasmid vector 540, the features of which are not repeated. The gene cassetes are flanked by the LB and RB T-DNA sequences, as described above. The plasmid backbone also contains a bacterial selection marker cassete that encodes the KanR gene, and which is in reverse orientation on the plasmid vector 565. The Cannabaceae plant part (e.g., cannabis) was transformed using the R. rhizogenes strain A4 (ATCC43057). The plasmid vector 565 sequence is illustrated by SEQ ID NO: 34 below. Further identified is the sequence of the betalain cassete (SEQ ID NO: 35), the plant selectable marker cassete (SEQ ID NO: 10), the T-DNA borders of the RB (SEQ ID NO: 15) and LB (SEQ ID NO: 16), the LacZ cassete (SEQ ID NO: 17) and the bacterial selection marker cassete (SEQ ID NO: 20). The betalain cassete (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 cassete (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 cassete (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). [0188] FIG. 5E illustrates an example plasmid vector 567 that includes a DNA sequence coding for a plurality of enzymes to produce betalain (e.g., betanidin and betaxanthin). The plasmid vector 567 contains the gene encoding the enzy mes CYP76AD1 and DODA driven by a FMV promoter, with CYP76AD1 linked to DODA by a 2A self-cleaving peptide P2A. The plasmid vector 567 further includes a plant selectable marker cassette and a LacZ cassette, which are in reverse orientation on the plasmid vector 567 and as described by plasmid vector 540, 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 567. The Cannabaceae plant part (e.g., cannabis) was transformed using the R. rhizogenes strain A4 (ATCC43057). The plasmid vector 567 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 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).

[0189] FIG. 5F illustrates an example plasmid vector 570 that includes a DNA sequence coding for a plurality of enzymes to produce betalain (e.g., betanidin). The plasmid vector 570 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 570 further includes a plant selectable marker cassette and a LacZ cassette, which are in reverse orientation on the plasmid vector 570 and as described by plasmid vector 540, 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 570. The Cannabaceae plant part (e.g., cannabis) was transformed using the R. rhizogenes strain A4 (ATCC43057). The plasmid vector 570 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).

[0190] Consistent with the above description, bacterium strains and 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. Six (6) days prior to the experiment, 50-100 cannabis seeds 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 embry os onto 8P-MS-G media plates (see media recipes) with a maximum of five embry os 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.

[0191] 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.

[0192] To infect whole cannabis seedlings, the point of a scalpel was used to make a small wound in 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°C to 23 degrees C). The plates w'ere 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.

[0193] 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 Phytatrays™, were closed and placed in a light chamber for two weeks.

[0194] 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 w'as 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 parafdm and placed in the dark overnight (22°C to 23°C). The plates were removed from the dark chamber and transferred to a 16/8-hour light/dark incubator (75 lumens, 23°C) for one additional day for a total of two days of co-cultivation.

[0195] 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.

[0196] 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 some particular experiments, spectinomycin was added. This will allow for the growth of only PCM clones containing and expressing both the binary vector and PCM T-DNAs.

[0197] 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.

[0198] Plates of sub-cultured roots w'ere transferred to fresh PCM media every two to three weeks with the concentration of cefotaxime in the medium being gradually reduced from 500 mg/nlL (for two rounds of transfers) to 300 mg/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. [0199] 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.

[0200] 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.

[0201] 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 in 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. [0202] 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.

[0203] 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°C and poured 100 mL per Phytatray™ or 25 mL or 50 mL per 100 x 25 mm plates.

[0204] 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.

[0205] 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°C and poured into 25mL into 100 x 15 mm plates.

[0206] 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 FeSOAVfhO: the pH was adjusted to 7.0 with KOH; and the solution was brought to volume with 1000 ml of ddftO.

[0207] 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 ofNaH2P04; and the solution was brought to volume with 1000 ml of dcTHO.

[0208] To create AB minimal agar media (liquid), the following protocol and volumes were used to form 1L of media:

700 ml of ddH20; 5 g of Sucrose; the solution was brought to volume with 1000 ml of dcUrhO; 50 ml of 20x AB Salts; and 50 ml of 20X AB Buffer.

The media was autoclaved on liquid cycle for 25 minutes.

[0209] To create AB minimal media (plates), 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; 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°C and poured into 100 x 15 mm plates.

[0210] To create YEP media (liquid), the following protocol and volumes were used to form 1L of media:

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

The media was filter sterilized.

[0211] 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).

[0212] 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°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 in place of MS basal salts.

[0213] 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 ddfkO; 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 ddfhO; 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°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.

[0214] 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.

[0215] 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 ddH20; 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 ddH20; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0216] 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 ddH20; 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 diRLO; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0217] 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 ddfhO; 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.

[0218] 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 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.

[0219] 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 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 300 mg of Cefotaxime [250 mg/L] was added.

[0220] 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 dd¾0; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave. [0221] 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 ddfhO; 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 ddPLO; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0222] 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 dcbLO; 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 ddH20; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0223] 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 dcbLO; 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.

[0224] 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. 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.

[0225] 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 dcbnO; 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.

[0226] 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 dcbLO; 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 ddH20; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0227] 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.

[0228] 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 dcbLO; 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.

[0229] 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.

[0230] 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 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.

[0231] 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 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 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] was added. [0232] 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 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 178.932 mL of ddthO; 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. [0233] 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. [0234] 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:

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. [0235] 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: 800 mL of dcll hO; 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 ddbbO; 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 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.

[0236] 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 dchBO; 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 5 mg G419 Sulfate [50 mg/mL] was added.

[0237] To create another culture media containing DKW, B5, Cefotaxime, and Spectinomycin, referred to as DKW-B5 + 0300 + Sped 00, the following protocol and volumes were used to create 1L of media:

800 mL of dcbBO; 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 300 mg of Cefotaxime [250 mg/L] and 100 mg Spectinomycin [50 mg/mL] (S4014) was added. [0238] 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 dcbBO; 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 ddfkO; 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. [0239] 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:

800 mL of dchLO; 30 g of Sucrose (S9378); 5.22 g of DKW basal salt mixture (D190); the solution was brought to volume with 179.932 mL of ddftO; the pH was adjusted to 5.8 with KOH; 6 g Agarose (A6013) was added.

[0240] 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 ddH₂0; 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 ddH20; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0241] 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 dcbLO; 30 g of Sucrose (S9378); the solution was brought to volume with 181.132 mL of ddftO; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0242] 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 dcbLO; 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.

[0243] 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 dcll hO; 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 ddtbO; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0244] 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 dchGO; 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 ddH20; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0245] 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.

[0246] 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 ddH20; 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.

[0247] 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 dcU hO; 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.

[0248] 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 ddThO; 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 ddtbO; the pH was adjusted to 5.8 with KOH.

The media was autoclaved on AGAR cycle with MediaClave.

[0249] 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 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 180.131 mL of ddH20; the pH was adjusted to 5.8 with KOH.

The media was filter sterilized.

[0250] In various experimental embodiments, the expression constructs illustrated by FIGs. 5A-5F were used to transform plant parts to induce PCM formation and production of betalains, such as the production of betanidin, betaxanthin, and/or betaxanthin. In some experiments, Cannabaceae plant parts were transformed, such as cannabis plant parts, however embodiments are not so limited.

[0251] FIGs. 6A-6D illustrate example images of PCM cultures producing betacyanin, consistent with the present disclosure. In some experiments, cannabis hypocotyls were co-transformed to generate PCMs and produce betalains using an R. rhizogenes strain A4 transformed with the plasmid vector 550 illustrated by FIG. 5B. The resulting PCMs produced a betacyanin, specifically, betanidin. FIG. 6A is an image showing the cannabis hypocotyl segment forming a PCM that is expressing the betacyanin. FIGs. 6B-6D are images showing a cannabis PCM explant producing the betacyanin. The betacyanin is seen in the root tip, primary root, and root hairs. In FIGs. 6C-6D, multiple transgenic events are shown with different levels of betacyanin expression. [0252] FIGs. 7A-7B 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 550 illustrated by FIG. 5B. FIGs. 7A-7B are images showing cannabis PCM explants expressing different levels of betacyanin after transforming whole seedlings.

[0253] FIGs. 8A-8F illustrate example images of PCM cultures producing betacyanin at different levels, consistent with the present disclosure. . In some experiments, the cannabis PCMs, such as those illustrated by FIGs. 6A-7B, were transformed with the plasmid vector 550 illustrated by FIG. 5B. FIGs. 8A-8F are images of the PCM tissue that were re-transformed and that express different levels of betacyanin. The betacyanin, e.g., betanidin, is expressed in roots, root tips, root hairs, and wounding sites.

[0254] FIGs. 9A-9C 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 retransformed to produce betalains using a disarmed R. rhizogenes strain A4 transformed with the plasmid vector 567 illustrated by FIG. 5E. The resulting PCM cultures produced multiple betalains including betanidin and betaxanthin. FIG. 9A is an image of the resulting PCM transformed with the plasmid vector 567 imaged in white light, and FIG. 9B is an image of the PCM of FIG. 9B 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. 9C is an image verifying the presence of betanidin and betaxanthin in the transformed PCM tissue.

[0255] FIGs. 10A-10B illustrate example images of PCM cultures producing betaxanthin, consistent with the present disclosure. In some experiments, cannabis plant parts were transformed to express the PCM phenotype using a first R. rhizogenes strain A4 and then the PCM tissues were re-transformed to produce a betalain using a disarmed R. rhizogenes strain A4 strain, e.g., 18rl2, transformed with the plasmid vector 565 illustrated by FIG. 5D. FIGs. 10A-10B are images of plant PCM tissue was retransformed and producing betaxanthin.

[0256] FIGs. 11 A-l IB 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 540 and a second bacterium strain as described above, and the resulting PCM culture secreted or otherwise presented the betalain into the liquid media. FIGs. 11 A-l IB are images showing the betalain present in the liquid media after the cannabis PCMs were re-transformed with the plasmid vector 540.

[0257] FIGs. 12A-12B illustrate example experimental results from PCM cultures producing betalains, consistent with the present disclosure. FIG. 12A 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. 12A, the wild-type beet standard has a peak for betanin at around 10.446. The cannabis PCM was generated using the plasmid vector 570 illustrated by FIG. 5F and has a similar peak around 10.446, as shown by the bottom graph of FIG. 12A. FIG. 12B illustrates a chromatogram of chemistnes 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 567 of FIG. 5E, and a second cannabis PCM in the bottom graph that was generated using the plasmid vector 565 of FIG. 5D. As shown by the top graph of FIG. 12B, the wild-type beet standard has peaks for betaxanthin at around 6.687, betanin at around 10.446, and isobetanm at around 11.387. As shown by the middle graph of FIG. 12B, the first cannabis PCM has a peak around 10.446 that corresponds with betacyanin. As shown by the bottom graph of FIG. 12B, the second cannabis PCM has a peak around 6.687 that corresponds with betaxanthin.

[0258] Embodiments in accordance with the present disclosure are not limited to transforming Cannabaceae plant parts. Betalain can be produced in plant parts of other plant species by identifying a bacterium strain to transform the plant part, designing and generating a plasmid vector that includes heterozygous sequence encoding the betalain, transforming the bacterium strain with the plasmid vector, and infecting the plant part of the plant with the transformed bacterium strain. Betalains can be produced in various different plant species and include various types of betalains by designing an expression construct to infect the plant species, developing a tissue culture and transformation methodology, and transforming the plant part to produce the betalain.

[0259] An example process for designing the expression construct includes the following: 1) cloning and sequencing the gene of interest (e.g., the target enzyme(s) for a betalain) into an entry vector, 2) optionally adding a purification tag for isolation, 3) cloning into a binary vector containing a selectable marker and/or reporter gene (e.g., YFP), 4) testing a variety of promoters for protein expression using protoplasts, tissue infiltration, and/or transient transformation assays, 5) selecting a promoter which shows the highest expression among the tested variety of promoters in the target species to drive the expression of the gene of interest, and 6) introducing the binary vector into a Rhizogenes strain which gives the highest frequency of PCM formation (see below) among a set of strains or into another bacterium strain that does not induce PCM formation (e.g., 18rl2) . [0260] An example process for developing a tissue culture and transformation methodology includes the following: 1) infecting the target plant species with a variety of wild-type Rhizogenes strains, 2) determining the transformation frequencies of the variety of wild-type Rhizogenes strains based on the number of explants which form PCMs out the total explants, 3) testing a variety of tissue from the target species (cotyledon, hypocotyl, stem, leaf, root, immature embryo, etc.) to determine which tissue is the most amenable to PCM formation, 4) subculturing formed PCMs and optimize media formulations to maximize growth and biomass accumulation via repeat adjustment and testing, 5) optimizing the subculturing technique, medias, and timing via repeat adjustment and testing, 6) determining which Rhizogenes strain produces the highest transformation frequency and produces PCM clones which grow well and for long periods of time in tissue culture, 7) determining the optimal selectable marker to be used for selection of the gene of interest by performing kill curve assays using a number of different selection agents on isolated PCM clones, 8) introducing the optimized binary vector (see above) into the selected Rhizogenes strain and infect tissue most amenable to transformation in target plant species, and 9) subculturing and selecting for PCM clones which express the gene of interest.

[0261] 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 570 illustrated by FIG. 5F.

[0262] Some experiments were conducted that transformed the solanum tuberosum plant part to generate a PCM. FIGs. 13A-13B illustrate example images of PCM cultures generated from solanum tuberosum plants, consistent with the present disclosure. FIG.

13 A illustrates a PCM culture generated from a first solanum tuberosum strain and FIG. 13B 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.

[0263] FIGs. 14A-14B 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 570, as noted above, and bacterium strain A. rhizogenes ATCC15834 transformed with the plasmid vector 570.

[0264] FIGs. 15A-15B illustrate example images of betalain production in solanum tuberosum PCM cultures using different bacterium strains, consistent with the present disclosure. For example, FIG. 15A illustrates betalain production in a PCM culture generated using A4 bacterium strain transformed with the plasmid vector 570 and FIG. 15B illustrates betalain production in a PCM culture generated using bacterium strain A. rhizogenes ATCC15834 transformed with the plasmid vector 570.

[0265] 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. [0266] 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.

[0267] 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.

[0268] 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.

[0269] 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.

[0270] 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).

[0271] 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 ofMgS04*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.

[0272] 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.

[0273] To create the MG media, the following protocol and volumes were used to make a 1L solution of media:

700 mL of ddH₂0; 5 g of glucose; and the solution was brought to volume with 1000 mL of ddH₂0; 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).

[0274] 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.

[0275] 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

[0276] 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 aeroponics. 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

[0277] 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 enzyme in PCM tissue.

Claims

1. A method comprising: contacting a plant part with a nucleotide sequence encoding a gene that induces plant cell matrix (PCM) formation and a nucleotide sequence encoding an enzyme associated with production of a betalain; and culturing the plant part to enhance the production of the betalain.

2. The method of claim 1, wherein contacting the plant part with the nucleotide sequences comprises contacting the plant part with a bacterium strain comprising the nucleotide sequence encoding a gene that induces PCM formation and the nucleotide sequence encoding the enzyme.

3. The method of claim 1 or 2, wherein the PCM comprises plant cells transformed by the contact with the nucleotide sequence and includes a plurality of different plant cells types, the plurality of different plant cells types comprises cells selected from: plant stem cells, maturing cells, mature cells, and a combination thereof.

4. The method of claim 1 or 2, wherein the enzyme is selected from: dihydroxyphenylalanine (OOP A) 4,5-dioxygenase (DODA), Cytochrome P450

(CYP76AD1), CYP76AD6, glucosyltransferase, and a combination thereof.

5. The method of claim 1 or 2, wherein 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.

6. The method of claim 1 or 2, wherein the betalain comprises a betacyanin or a betaxanthin.

7. The method of claim 1 or 2, wherein the betalain is selected from: betanin, isobetanm, probetanin, neobetanin, vulgaxanthin, miraxanthin, portulaxanthin, and indicaxanthin, a combination thereof.

8. The method of claim 1 or 2, wherein the plant part is from a monocotyledon plant or a dicotyledon plant.

9. The method of claim 1 or 2, 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.

10. The method of claim 1 or 2, 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.

11. The method of claim 1 or 2, wherein the plant part is a seedling, a petiole, an intemode, a node, a meristem, or a leaf.

12. The method of claim 1 or 2, wherein the plant part is from a Cannabaceae plant, a Brassicaceae plant, a Solanaceae plant, a Fabaceae plant, or an Apiacea plant.

13. The method of claim 1 or 2, further comprising culturing the plant part under growth conditions to enhance transformation, induce the PCM formation, and induce production of the betalain.

14. The method of claim 13, 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.

15. The method of claim 13, wherein culturing the plant part under the growth conditions comprises intermittently contacting the plant part with a culture medium containing sugar and basal salt.

16. The method of claim 1 or 2, wherein contacting the plant part with nucleotide sequences and culturing the plant part comprises: infecting the plant part with a Rhizobium or Agrobacterium strain comprising a root-inducing (Ri) plasmid or a tumor-inducing (Ti) plasmid, a nucleotide sequence encoding a gene that induces PCM formation, and a nucleotide sequence encoding the enzyme associated with production of the betalain; and culturing the infected plant part to enhance PCM formation, and induce expression of the enzyme and production of the betalain.

17. The method of claim 16, wherein the Rhizobium or Agrobacterium strain comprises: the Ri plasmid comprising the nucleotide sequence encoding the gene that induces PCM formation; and the nucleotide sequence encoding the enzyme.

18. The method of claim 16, wherein the Rhizobium or Agrobacterium strain comprises: the Ri plasmid; the nucleotide sequence encoding the gene that induces PCM formation; and the nucleotide sequence encoding the enzyme.

19. The method off claim 16, wherein the Rhizobium or Agrobacterium strain comprises: a disarmed Ti plasmid or disarmed Ri plasmid; a nucleotide sequence encoding a gene that induces PCM formation; and a nucleotide sequence encoding the enzyme.

20. The method of claim 1 or 2, wherein contacting the plant part with the nucleotide sequences and culturing the plant part comprises: contacting the plant part with the nucleotide sequence encoding the gene that induces PCM formation; culturing the plant part to enhance PCM formation; contacting formed PCM tissue from the PCM with the nucleotide sequence encoding the enzyme associated with production of the betalain; and culturing the PCM tissue to enhance production of the betalain by the PCM.

21. The method of claim 1 or 2, wherein contacting the plant part with the nucleotide sequences and culturing the plant part comprises simultaneously introducing to the 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.

22. The method of claim 1 or 2, further comprising identifying the bacterium strain from a plurality of bacterium strains.

23. The method of claim 1 or 2, wherein the nucleotide sequence encoding the enzyme is operably connected to a promoter.

24. The method of claim 1 or 2, wherein the nucleotide sequence encoding the enzyme is operably connected to an ubiquitin promoter, a figwort mosaic promoter, or a 35S Cauliflower Mosaic Virus promoter.

25. The method of claim 1 or 2, further comprising screening new growth from the cultured plant part for PCM formation.

26. The method of claim 1 or 2, further comprising screening and selecting cultured plant part for production of the betalain.

27. A method of generating a bacterium strain comprising: transforming a bacterium strain with a nucleotide sequence encoding an enzyme associated with production of a betalain, 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.

28. The method of claim 27, 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.

29. The method of claim 27, wherein the bacterium strain is transformed using an expression cassette comprises SEQ ID NO: 2, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 35, or SEQ ID NO: 39.

30. The method of claim 27, 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.

31. A method comprising: contacting a plant part with a bacterium strain containing a root-inducing (Ri) plasmid or a tumor-inducing (Ti) plasmid, a nucleotide sequence encoding an enzyme associated with production of a betalain, and a nucleotide sequence encoding a gene that induces plant cell matrix (PCM) formation; inducing formation of PCM tissue from the plant part under infection conditions; and culturing the PCM tissue in a culture medium under growth conditions to induce expression of the nucleotide sequences and production of the betalain.

32. The method of claim 31 , wherein the bacterium strain comprises a Rhizobium or Agrobacterium strain and the method further comprises transforming the Rhizobium or Agrobacterium 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.

33. The method of claim 31, wherein the culture medium is selected from a liquid culture medium and a solid growth medium, and optionally comprises a selection agent.

34. The method of claim 31 , further comprising: selecting PCM tissue from the plant part as transformed by the contact with the bacterium strain for culturing in the culture medium; and screening the cultured PCM tissue for production of the betalain.

35. The method of claim 31 , wherein contacting the plant part with the bactenum strain comprises simultaneously introducing to the 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, or a leaf.

36. The method of claim 31 , wherein contacting the plant part with the bacterium strain and culturing the plant part comprises: contacting the plant part with a first bacterium strain comprising the nucleotide sequence encoding the gene that induces PCM formation; culturing the 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.

37. The method of claim 31 , further comprising capturing the betalain by isolating and purifying the betalain from the culture medium, PCM tissue of the PCM, or a combination thereof.

38. A plant cell matrix (PCM) culture for producing a betalain, the PCM culture being induced from a plant part, wherein a cell of the PCM culture comprises a nucleotide sequence encoding an enzyme associated with production of a betalain.

39. The PCM culture of claim 38, wherein the nucleotide sequence encoding the enzyme encodes a plurality of enzymes and is operably linked to a promoter and comprises 2A self-cleaving peptides located between respective enzymes of the plurality of enzymes.

40. The PCM culture of claim 38, wherein the nucleotide sequence comprises a sequence selected from:

SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 36, and a combination thereof.

41. A plant cell matrix (PCM) culture that produces betalain in plant cells of the PCM culture.

42. The PCM culture of claim 41, wherein the plant cells express a sequence selected from:

43. The PCM culture of claim 41, wherein the plant cells are transformed by 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.

44. The PCM culture of claim 41, wherein the PCM culture is generated from a Cannabaceae plant, a Brassicaceae plant, a Solanaceae plant, a Fabaceae plant, or an Apiacea plant.

45. An expression construct comprising SEQ ID NO: 2, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 35, or SEQ ID NO: 39.

46. The expression construct of claim 45, wherein the expression construct comprises SEQ ID NO: 1, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 38, or SEQ ID NO: 40.

47. A system for producing a betalain from plant cell matrix (PCM) tissue comprising: a plurality of bioreactors in serial connection, wherein each bioreactor is inoculated with a PCM culture according to the method of claim 1 or 2, and configured for growth and maintenance of the PCM culture in a culture medium.

48. The system of claim 47, wherein the culture medium comprises a liquid culture medium and the system is configured to recover the betalain from the liquid culture medium.

49. The system of claim 47, wherein at least one bioreactor is a flask, 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.

50. The system of claim 47, wherein each bioreactor of the plurality is structurally and operationally similar.

51. A betalain produced by using the PCM according to the method of claim 1 or 2.

52. A method comprising: transforming a plurality of plant parts with a plurality of bacterium strains to induce plant cell matrix (PCM) formation, and optionally to induce expression of an enzyme associated with production of a betalain; therefrom, assessing transformation frequencies of the plurality of bacterium strains; and selecting respective ones of the plurality of bacterium strains based on the transformation frequencies.

53. The method of claim 52, wherein the selected respective ones of the plurality of bacterium strains compnse ATCC 43057, ATCC 43056, ATCC 13333, ATCC 15834, K599, or a combination thereof.