WO2021183636A1 - Transformation et régénération de cannabaceae - Google Patents

Transformation et régénération de cannabaceae Download PDF

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Publication number
WO2021183636A1
WO2021183636A1 PCT/US2021/021693 US2021021693W WO2021183636A1 WO 2021183636 A1 WO2021183636 A1 WO 2021183636A1 US 2021021693 W US2021021693 W US 2021021693W WO 2021183636 A1 WO2021183636 A1 WO 2021183636A1
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Prior art keywords
plant
cannabaceae
explant
plants
donor
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PCT/US2021/021693
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English (en)
Inventor
Anders UPPGAARD
Alexandre DA SILVA CONCEIÇÃO
Alisha HERSHMAN
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Calyxt, Inc.
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Publication of WO2021183636A1 publication Critical patent/WO2021183636A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/28—Cannabaceae, e.g. cannabis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation

Definitions

  • Cannabis sativa is cultivated for a wide variety of products including fiber, oilseed, food, and medicinal/psychoactive compounds for industrial, agricultural, healthcare and recreational use.
  • Humulus lupulus (hop) is a valuable source of several secondary metabolites, such as flavonoids, bitter acids, and essential oils.
  • cannabis and hop have only recently started to gain genomic resources, but the information about functionally associated gene expression is limited. Further resources are needed for functional genomics and breeding to provide new Cannabaceae cultivars with desired traits.
  • Stable transgenic lines are essential for functional genomics; however, members of the Cannabaceae family are notoriously recalcitrant to transformation, in part, because the regeneration efficiencies are quite low.
  • Cannabaceae plant material such as clonal plant material, and regenerating the transformed tissue into various differentiated plant tissues and whole Cannabaceae plants, and the transformed plant cells, tissues, plant parts and whole plants.
  • Methods described herein include systematic design of genotype-specific transformation and regeneration systems for members of the Cannabaceae family including Cannabis and Humulus. Further embodiments implement the genotype- specific transformation and regeneration systems to provide transformed plants from clonal material derived from a mother plant that has one or more desirable traits.
  • Methods described herein include transforming clonal tissue with a transgene capable of editing the genome of members of the Cannabaceae family, regenerating the transformed tissue into a transgenic plant and, after the desired gene editing has been achieved, segregating the transgene region out in progeny via self-fertilization ⁇
  • materials and methods described herein can be used for efficient and reliable generation of new transgenic Cannabaceae cultivars and non-transgenic Cannabaceae cultivars with improved agronomic and/or non- agronomic traits.
  • a first aspect of the present disclosure includes a method for transforming a Cannabaceae plant comprising: excising an explant from a donor Cannabaceae plant having at least one set of true leaves; contacting the explant with a polynucleotide encoding a polypeptide to transform a plant cell; selecting, from the explant, a plant cell expressing the polynucleotide; and growing the selected plant cell into a Cannabaceae plant, wherein the Cannabaceae plant is genetically distinguishable from the donor Cannabaceae plant.
  • the explant can comprise apical or axillary meristem.
  • the donor Cannabaceae plant can be a plant that has been maintained in a vegetative state using a photoperiod of 16 hours light/8 hours dark.
  • the donor Cannabaceae plant can be a clone produced from a mother plant with a tissue capable of regenerating a shoot.
  • the step of growing can include culturing the selected plant cell to produce one or more shoots, culturing the shoots to produce roots, and acclimatizing the rooted shoots.
  • the polypeptide can be selected from the group of selectable markers, screenable markers and combinations thereof, and optionally wherein the screenable marker is a fluorescent protein.
  • the polypeptide can be a rare cutting endonuclease.
  • the rare cutting endonuclease is a TALE nuclease, Cas9/gRNA, zinc-finger nuclease, or meganuclease.
  • the method can further include selecting a Cannabaceae plant with a mutation induced by the rare-cutting endonuclease; self-pollinating the selected Cannabaceae plant to provide seed; growing the seed to provide progeny Cannabaceae plants; and selecting progeny plants having the induced mutation but lacking the polynucleotide encoding the rare cutting endonuclease.
  • the method can further include Agrobacterium- mediated transformation. The explant can be infected with an A.
  • the explant can include a node.
  • the node can be wounded to permit the A. tumefaciens to penetrate meristem tissue of the node.
  • the wounded node can be obtained by slicing an excised node at least partially.
  • the explant can be infected by immersing the explant in the A. tumefaciens solution. In some cases, infecting includes applying a vacuum to the immersed explant or sonicating the immersed explant.
  • the method can further include rinsing the immersed explant with water after soaking for about 1 minute to about 20 minutes and allowing the rinsed explant to dry.
  • the polynucleotide can further encode a gene endogenous to the Cannabaceae plant operably linked to a regulatory element to modulate expression of the endogenous gene in the Cannabaceae plant.
  • the polynucleotide can further encode a gene conferring an agronomic trait to the Cannabaceae plant.
  • the apical meristem of the donor Cannabaceae plant can have been removed.
  • the method can further include immersing the contacted explant in a solution comprising at least one preservative and at least one antibiotic for about 1 to about 60 minutes.
  • the Cannabaceae plant is a cannabis plant or a hop plant, in any of the embodiments above.
  • the present disclosure describes a transformed Cannabaceae plant, plant part, or plant cell made by any method of the first aspect.
  • the present disclosure further describes a transformed Cannabaceae plant, plant part, or plant cell made by a method comprising: excising an explant from a donor Cannabaceae plant having at least one set of true leaves; contacting the explant with a polynucleotide encoding a rare cutting endonuclease to induce a mutation in a gene of interest in a plant cell; selecting, from the explant, a plant cell with the induced mutation in the gene of interest, and growing the selected plant cell into a Cannabaceae plant, wherein expression of the gene of interest is modified in the Cannabaceae plant as compared to the donor Cannabaceae plant.
  • the method can further include self-pollinating the Cannabaceae plant to provide seed; growing the seed to provide progeny Cannabaceae plants; and selecting progeny plants having the induced mutation but lacking the polynucleotide encoding the rare cutting endonuclease.
  • the method can further include immersing the contacted explant in a solution comprising at least one preservative and at least one antibiotic for about 1 to about 60 minutes.
  • the Cannabaceae plant can be a cannabis plant or a hop plant.
  • the present disclosure describes a method of improving Cannabaceae regeneration comprising immersing an explant obtained from a Cannabaceae plant in a solution comprising at least one preservative and at least one antibiotic for a duration sufficient to remove endophytes in the explant, wherein when plated on regeneration medium the immersed explant exhibits improved regeneration efficiency compared with an untreated explant obtained from the same Cannabaceae plant and has not been immersed in the solution.
  • the improved regeneration efficiency comprises greater survival rate, greater shoot induction, greater shoot multiplication, or shoot elongation compared with the untreated explant.
  • the at least one antibiotic includes one or more antibiotics selected from the group consisting of Nystatin, streptomycin, cephalexin, cefotaxime, carbenicillin, ticarcillin, clavulanate, and penicillin-G, or a salt thereof.
  • the at least one preservative can include 5-chloro-2-methyl-3(2H)-isothiazolone, 2-methyl-3(2H)- isothiazolone, magnesium chloride, magnesium nitrate, sodium benzoate, or potassium sorbate.
  • the method can include agitating the explant in the solution.
  • the explant can be immersed in the solution for about 1 minute to about 60 minutes.
  • the solution can include about 1-500 mg/L of the at least one antibiotic.
  • the solution can include about 0.01-5% (v/v) of the at least one preservative.
  • the Cannabaceae plant can be a cannabis plant or a hop plant.
  • FIG. 1 is a flowchart describing method 100 for identifying a mother plant having a desired phenotype and which can provide clonal material capable of being transformed and regenerated, according to one or more embodiments of the present disclosure.
  • FIG. 2 shows the expression pattern of fluorescent protein in a YFP-transformed cannabis explant with an emerging shoot (left) and a non-transformed control explant (right), according to one or more embodiments of the present disclosure.
  • FIG. 3 is a flowchart describing method 200 for propagating, transforming, and regenerating transformed clonal material into whole cannabis plants, according to one or more embodiments of the present disclosure.
  • FIG. 4 is a flowchart describing method 600 for preparing, transforming, sterilizing, and regenerating transformed explants according to one or more embodiments of the present disclosure.
  • FIGs. 5A-5D show selection and harvesting cannabis material from a mother plant for micropropagation or a donor plant for transformation and regeneration according to one or more embodiments of the present disclosure.
  • A shows a whole cannabis plant (var. Cherry Diesel) with branches with several nodes (inset);
  • B shows a suitable excision site for harvesting nodal tissue (highlighted);
  • C shows the harvested branch from 5B; and
  • D shows the trimmed branch of 5C, ready for sterilization.
  • FIGs. 6A-6C include tables of exemplary media formulations according to one or more embodiments of the present disclosure.
  • A describes a co-cultivation medium (“Co- Cult-G DKW”);
  • B) describes a regeneration medium (“Page DKW-T05 (-PPM)”); and
  • C) describes a rooting medium (“Hemp RM DKW + 0.5IBA”).
  • FIGs. 7A-7H show stages of tissue culture (i.e., micropropagation) according to one or more embodiments of the present disclosure.
  • A shows a top view of a petri dish with nodal explants plated on a regeneration medium
  • B shows a perspective view of 7A
  • C shows a top view of regenerated shoots obtained from the explants shown in 7A
  • D shows a perspective view of 7C
  • E shows a top view of rooted shoots obtained from the regenerated shoots of 7C after plating on rooting medium
  • F shows a perspective view of 7E
  • G shows a top view of a regenerated Cannabis plantlet generated from a rooted shoot of 7E
  • H shows a perspective view of 7G.
  • FIG. 8 shows the expression pattern of fluorescent protein in a YFP-transformed cannabis explant with an emerging shoot.
  • FIGs. 9A, 9B and 9C show expression patterns of fluorescent protein in YFP- Talen pair-transformed Cannabis explants (i.e., transgenic calli).
  • FIG. 10 show an exemplary result of Illumina sequencing of DNA extracted from transgenic calli indicating the transformation was effective for Talen-induced gene editing at the targeted locus of the Cannabis genome.
  • the present disclosure features methods and materials for transforming Cannabaceae plants, plant parts, and plant cells using Agrobacterium- mediated delivery of plasmid DNA.
  • Methods of the present disclosure feature transforming clonal tissue and thereby ensuring a more reliable phenotype than obtainable using Cannabaceae seed or seedlings.
  • the methods and materials described herein are optimized for enhancing the transformation efficiency of Cannabaceae explants derived from clonal tissue maintained in a vegetative state and for promoting regeneration of the transformed explants.
  • the plants, plant parts, and plant cells of the present disclosure can be used to produce new Cannabaceae varieties. In some embodiments, the plants, plant parts and plant cells can be used to develop new, unique and superior Cannabaceae varieties or hybrids with desired phenotypes.
  • an “endogenous gene” refers to a nucleic acid molecule having the sequence of a wild-type sequence occurring in the wild-type plant, or a sequence having a percent identity that allows it to retain the function of the encoded product, such as a sequence with at least 90% identity, and may be obtained from the plant or plant part of cell, or may be synthetically produced. Further embodiments provide the sequence has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. In embodiments described herein, an endogenous gene nucleotide 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.
  • Cannabaceae plant is used to include any member of the Cannabaceae family at any stage of development.
  • a “Cannabaceae plant part” refers to any part of a Cannabaceae plant, including a plant cutting, a plant cell, a plant cell culture, a plant organ, a plant seed (achenes), and a plantlet.
  • the family includes about 170 species grouped in about 11 genera, including economically important genera of Cannabis , Humulus and Celtis.
  • the term “cannabis plant” is used broadly to include a Cannabis sativa L. plant from any landrace, cultivar, or variety, at any stage of development.
  • a cannabis plant part refers to any part of a cannabis plant, including a plant cutting, a plant cell, a plant cell culture, a plant organ, a plant seed (achenes), and a plantlet.
  • C. sativa L. may be divided into three sub-species: C. sativa ssp. sativa, C. sativa ssp. indica, and C. sativa ssp. ruderalis.
  • the different varieties of cannabis can be grouped into four basic categories, including: (1) “wild” plants that have escaped from cultivation and grow independently in nature; (2) plants grown for fiber from the main stalk; (3) plants grown for oilseed; and (4) plants grown for psychoactive compounds.
  • the cannabis plant can be any variety, including a commercial variety, or a variety contributing to one or more commercial varieties, such as “Afghani #1”, “Acapulco Gold” or “Mexican sativa”, “Blueberry”, “Grand daddy purple”, “Haze” or “Original Haze”, “Hindu Kush”, “Northern lights #5”, “OG Kush”, “Skunk #1”, and “Thai” or “Thai stick”.
  • the cannabis plant can be identified by chemical phenotype or “chemotype”, which indicates the chemical composition of cannabinoids.
  • the cannabis plant can be categorized as Type I, Type II, Type III, Type IV, or Type V, based on the phytocannabinoid content (e.g., tetrahydrocannabinolic acid dominant, equal parts cannabidiolic acid and tetrahydrocannabinolic acid, cannabidiolic acid dominant, cannabigerolic acid dominant, or lacking cannabinoids, respectively).
  • a “cannabis plant” includes high- and low-THC subspecies with both domesticated and ruderal varieties, including marijuana and hemp types of cannabis (e.g., cannabis known as C. indica using folk classification).
  • hop plant is used broadly to include plants of any species of Humulus from any landrace, cultivar, or variety, thereof, at any stage of development.
  • a hop plant part refers to any part of a hop plant, including a plant cutting (e.g., rhizome), a plant cell, a plant cell culture, a plant organ (e.g., cones or hops), and a plantlet.
  • Varieties of hop plants include hop plants cultivated for use in brewing (“Brewer’s varieties) and ornamental varieties of Humulus lupulus.
  • a “landrace” refers to a local variety of a domesticated plant species which has developed largely by natural processes, by adaptation to the natural and cultural environment in which it lives. The development of a landrace may also involve some selection by humans, but it differs from a formal breed which has been selectively bred deliberately to conform to a particular formal, purebred standard of traits.
  • cultivar means a group of similar plants that by structural features and performance (i.e., morphological and physiological characteristics) can be identified from other varieties within the same species. Furthermore, the term “cultivar” variously refers to a variety, strain or race of plant that has been produced by horticultural or agronomic techniques and is not normally found in wild populations. The terms cultivar, variety, strain and race are often used interchangeably by plant breeders, agronomists and farmers.
  • “Variety” means a plant grouping within a single botanical taxon of the lowest known rank, which grouping, irrespective of whether the conditions for the grant of a breeder’s right are fully met, can be: i) defined by the expression of the characteristics resulting from a given genotype or combination of genotypes, ii) distinguished from any other plant grouping by the expression of at least one of the said characteristics, and iii) considered as a unit with regard to its suitability for being propagated unchanged.
  • a “plant cell” is the structural and physiological unit of the plant, comprising a protoplast and a cell wall.
  • a plant cell can be in the form of an isolated single cell or aggregate of cells such as a friable callus, or a cultured cell, or can be part of a higher organized unit, for example, a plant tissue, plant organ, or plant.
  • a plant cell can be a protoplast, a gamete producing cell, or a cell or collection of cells that can regenerate into a whole plant.
  • a seed which comprises multiple plant cells and is capable of regenerating into a whole plant, is considered a plant cell for purposes of this disclosure.
  • a “plant tissue” or “plant organ” can be a seed, protoplast, callus, or any other groups of plant cells that is organized into a structural or functional unit.
  • Plant parts include harvestable parts and parts useful for propagation of progeny plants.
  • a harvestable part of a plant can be any useful part of a plant, for example, flowers, strobiles, pollen, seedlings, leaves, bracts, buds (meristems), stems, seed pods, seeds, roots, nodules, trichome (including lupulin glands of hop cones), branch, petiole, intemode, fibers, and the like, and includes extracts such as kief or hash which includes cannabis trichomes or glands.
  • a part of a plant useful for propagation includes, for example, seeds, seed pods, cuttings, seedlings, rootstocks, and the like.
  • “Meristem” refers to a plant tissue containing undifferentiated cells (meristematic cells), found in zones of the plant where growth can take place. Meristematic cells give rise to various organs of the plant and keep the plant growing. There are three types of meristematic tissues: apical (at the tips), intercalary (in the middle) and lateral (axial, at the sides).
  • “Seed” refers to any plant structure that is formed by continued differentiation of the ovule of the plant, following its normal maturation point at flower opening, irrespective of whether it is formed in the presence or absence of fertilization and irrespective of whether the seed structure is fertile or infertile.
  • “Germplasm” refers to the overall genetic potential of the Cannabaceae plant, including the seeds and cuttings.
  • Skunk #1 is recognized as good cannabis germplasm with a well-known, recorded breeding history.
  • Plant breeding preferably begins with the analysis and definition of problems and weaknesses of the current germplasm, the establishment of program goals, and the definition of specific breeding objectives.
  • the next step is preferable selection of germplasm that possess the traits to meet the program goals.
  • the goal is to combine in a single variety or hybrid an improved combination of desirable traits from the parental germplasm.
  • Explant refers to a plant part having regeneration potential via micropropagation.
  • the explant can be capable of regenerating a shoot, root, or whole plant.
  • Explants can be nodal segments, roots, rhizome, leaf disc, leaf blade, petiole, or other vegetative tissue.
  • Donor plant refers to a source of explants.
  • “Mother plant” refers to a source of cuttings capable of propagating donor plants using conventional cloning techniques.
  • the term “mother plant” can also refer to a source of explants.
  • transformation refers to the transfer of nucleic acid (i.e., a nucleotide polymer) into a cell
  • gene transformation refers to the transfer and incorporation of DNA, especially recombinant DNA, into a cell
  • transformant refers to a cell, tissue or organism that has undergone transformation.
  • Agrobacterium tumefaciens and “ Rhizobium radiobacter” are used interchangeably in the present disclosure.
  • Agrobacterium rhizogenes and “ Rhizobium rhizogenes” are used interchangeably in the present disclosure.
  • “Expression cassette” means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to a nucleotide sequence of interest, which is optionally operably linked to termination signals and/or other regulatory elements.
  • An expression cassette may also include sequences required for proper translation of the nucleotide sequence.
  • the coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a non-translated RNA, in the sense or antisense direction.
  • the expression cassette with the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • the expression cassette may also be one, which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
  • An expression cassette may be assembled entirely extracellularly (e.g., by recombinant cloning techniques).
  • an expression cassette may also be assembled using in part endogenous components.
  • an expression cassette may be obtained by placing (or inserting) a promoter sequence upstream of an endogenous sequence, which thereby becomes functionally linked and controlled by said promoter sequences.
  • a mother Cannabaceae plant can be any type of Cannabaceae plant, as described above.
  • the mother plant is a female plant (i.e., not a hermaphrodite).
  • the mother plant is a Cannabaceae genotype that is susceptible to Agrobacterium infection, and which exhibits a desired regeneration response.
  • the mother Cannabaceae plant can be from an elite line having one or more certain desired traits.
  • FIG. 1 describes method 100 for identifying a mother Cannabaceae plant, or mother plant.
  • a mother plant has a desired phenotype and can provide explant material for transformation and regeneration into a new transgenic Cannabaceae cultivar.
  • Method 100 can include systematic design of a genotype- specific transformation and regeneration system optimized for each identified mother plant.
  • the Cannabaceae plant is a cannabis plant or a hop plant.
  • a potential mother plant is selected based on its phenotype.
  • desired traits include but are not limited to traits resulting in increased biomass production, production of specific chemicals (e.g., relative levels of one or more cannabinoids, terpenes, and flavonoids), increased seed production, improved plant material quality, increased seed oil content, etc.
  • Additional examples of desired traits include pest resistance, vigor, development time (time to harvest), enhanced nutrient content, novel growth patterns, flavor or color profiles observed under defined conditions (e.g., temperature- , growing medium-, nutrient-, or photoperiod-specific profiles), salt, heat, drought and cold tolerance, and the like.
  • a potential mother plant can be selected based on a photoperiod sensitivity that allows clonal tissue to be maintained in a vegetative state (i.e., a photoperiod that suppresses transition to a reproductive state).
  • the mother plant is a transgenic plant.
  • desired traits also include selectable marker genes (e.g., genes encoding herbicide or antibiotic resistance used only to facilitate detection or selection of transformed cells), hormone biosynthesis genes leading to the production of a plant hormone (e.g., auxins, gibberellins, cytokinins, abscisic acid and ethylene that are used only for selection), or reporter genes (e.g., luciferase, b-glucuronidase, chloramphenicol acetyl transferase (CAT), etc.).
  • the mother plant can also be a plant with preferred chemical compositions for a specific application, e.g., compositions preferred for medical use or industrial applications.
  • the mother plant’s phenotype can be assessed in a growth chamber or under field conditions.
  • method 100 includes step 104 for propagating donor plants from the potential mother plant.
  • New Cannabaceae plants can be propagated from the selected potential mother by any means known in the art.
  • potential mother plants can be grown from seed until the main stem bears a suitable number of lateral branches with a meristem of a pre-determined length (e.g., about 20 cm).
  • These branches can be used to grow potential donor plants by cutting the branch from the potential mother and placing it in a dilute disinfectant solution.
  • the cut branches can be stripped of lower fan leaves, and these can be re-cut at a 45° angle to a uniform length (e.g., about 15 cm).
  • each cutting can be dipped in a rooting hormone and placed in a rooting medium in a seedling tray.
  • the cuttings generally produce roots between 5 to 35 days of placement, depending on the genotype and environmental conditions. Genotype can have a significant impact on both the number of cuttings available and their rate of rooting.
  • Step 106 of method 100 includes testing explants obtained directly from the potential mother plant or from the donor plants to determine the potential mother plant’s capacity for regeneration via micropropagation. Not all Cannabaceae cultivars or explants excised therefrom exhibit the same capacity for regeneration.
  • Establishing a regeneration system can be the first step for producing transformed plants.
  • the system can include one or more types of regeneration media based on the response of the tissue.
  • a desired response can be regeneration into a viable shoot.
  • the shoot can be of any length, such as 2 cm or 5 cm or regeneration into a cluster of viable shoots.
  • step 106 can include optimizing the regeneration system to improve the efficiency of regeneration.
  • the regeneration system can be optimized so that the explant regenerates a viable shoot with greater than 50% efficiency or greater than 75% efficiency.
  • the efficiency can be calculated as the number of regenerated explants of at least 5 cm in size/the total number of cultured explants x 100. Capacity for regeneration can be assessed by testing various explants for callus induction or node regeneration under several conditions.
  • the regeneration system can be optimized by testing regenerable explants for response to various media, such as shoot formation, shoot multiplication, and rooting. In some cases, the regeneration system can be optimized by testing rooted explants for their ability to be acclimatized to the environmental conditions associated with expression of the desired traits of the potential mother plant.
  • Step 106 can include testing explants from several different plant parts (e.g., leaf, petiole, nodal segments, or roots) at different locations on the plant.
  • the characteristics of the donor plant (and therefore mother plant) cannot be perfectly inherited during the process of genetic transformation using explants from embryos or seedling plants.
  • the explants of the present disclosure are vegetative explants (i.e., not generative explants).
  • explants include meristem tissue, leaf tissue, petiole tissue, or node tissue.
  • Different explants can include the same tissue derived from different locations on the plant. For example, the extent of lignification of nodal segments or cuticle thickness of leaf tissue can vary with its location on the plant.
  • the donor plant is a Cannabaceae cultivar that produces, or can be induced to produce, axillary meristems.
  • Axillary meristems, the origin of lateral branches, are formed in the center of the boundary zone separating the shoot apical meristem (SAM) from the developing leaf primordium.
  • SAM shoot apical meristem
  • axillary meristem initiation is induced by removing the apical meristem of the plant (e.g., by topping or pinching).
  • the removal of the apical meristem stimulates many axillary meristems to grow from the sides of the plants.
  • the removal of the apical meristem allows for ten times, or 30 times more meristems to be harvested from the donor plant through its life. These additional meristems increase the chance of obtaining positive response to regeneration and transformation.
  • Nodal segments harboring an axillary meristem can be cultured to regenerate multiple shoots with or without intervention of callus phase.
  • the explant can be a nodal segment comprising axillary meristem induced by apical meristem removal.
  • the relative suitability of a potential mother plant can be determined by calculating the regeneration rate (%), counting the number of shoots, and measuring shoot length (cm) after 2, 3, 4, 5, or 6 weeks using a variety of media (e.g., MS or WPM), modified with one or more auxins, cytokinins and/or growth regulators at different concentrations.
  • an explant can be cultured in solid basal medium with one or more growth regulators to induce callus or shoot formation.
  • the basal medium and/or growth regulators for inducing roots can be the same as or different than the basal medium and/or growth regulators required to induce callus formation, to induce shoot formation, or to multiply shoots.
  • An exemplary regeneration medium is described in FIG. 6B.
  • Root induction can be assayed in a similar manner to shoot formation.
  • the method can include transferring shoots that have achieved a desired length (e.g., 2.5 cm to 5 cm) into a rooting medium supplemented with activated charcoal and/or one or more auxins at different concentrations and determining average root number and root length.
  • a desired length e.g., 2.5 cm to 5 cm
  • An exemplary rooting medium is described in FIG. 6C.
  • the explants can be tested in a variety of regeneration medium formulations comprising different plant growth regulators (e.g., Basal medium (e.g., Driver and Kuniyaki Walnut (DKW), Murashige and Skoog medium (MS) and Woody Plant Medium (WPM)), which includes macro and microelements and vitamins, further including auxins (e.g., naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), Indole-3 -butyric acid (IBA), and 2,4-dichlorophenoxyacetic acid (2,4-D)); cytokinins (e.g., kinetin, benzylaminopurine (BAP), zeatin,6-Y-Y-(dimethylallylamino)-purine (2-iP) and thidiazuron (TDZ)); gibberellins (gibberellic acid (GA3)); abscisic acid (ABA); organic components -
  • Methods of the present disclosure also include propagating regenerable explants via tissue culture methods (micropropagation) to produce a plurality of new plants having the same genetics as the mother (i.e., untransformed plants), but with greater space efficiency than can be achieved by cuttings (see, e.g., FIGs. 7A-7H).
  • Micropropagation provides better control over plant growth than using larger cuttings and preserves strain genetics while avoiding some of the risk of contamination as conventional propagation.
  • the regenerated plantlets can be transferred at the rooting stage to be acclimated the final growing environment.
  • step 108 method 100 includes assessing the transformation capacity of the regenerable explants identified in step 106.
  • Step 108 can include optimizing the transformation system to improve the efficiency of transformation, for example when a specific cultivar is identified as susceptible to transformation.
  • Methods described herein can include Agrobacterium- mediated transformation.
  • A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria which genetically transform plant cells. The Ti and Ri plasmids of Agrobacterium carry genes responsible for genetic transformation of the plant.
  • an Agrobacterium- mediated plant transformation system of the present disclosure uses a disarmed strain that retains full vir gene functions to mediate T-DNA processing, transfer and integration but removes the oncogenes in the helper Ti plasmid.
  • This system includes an alternative T-DNA region containing the gene of interest in a binary vector.
  • the virulence proteins encoded by the helper Ti plasmid act in trans to help T-DNA on the binary vector transfer from bacteria into plants.
  • Agrobacterium-mediated plant transformation includes the activation of the
  • the T-DNA includes the DNA sequence of a screenable marker that permits rapid, visual confirmation of transformation and/or the location of transformants in the explant within days of infection.
  • a screenable marker such as a fluorescent protein (a green fluorescent protein (GFP or eGFP), yellow fluorescent protein (YFP), or red fluorescent protein (RFP)) can be detected using fluorescence microscopy. The expression pattern of the fluorescent protein can be used to further enhance the transformation efficiency.
  • the explant can be examined using fluorescence microscopy to determine the extent to which the bacteria were able to penetrate all the tissues of the explant (see, e.g., FIG. 2).
  • the expression pattern can be used to direct future explant preparation to improve transformation efficiency of desired tissues or cells.
  • Exemplary Agrobacterium strains include C58, A136, A208, A348, Ach5,
  • Th Q Agrobacterium can be cultured using traditional methods. For example, an aliquot of bacteria can be placed into a sterile microfuge tube and precipitated (e.g., at 12,000 rpm for 3 minutes). The supernatant can be removed, and the bacteria can be resuspended with an inoculation medium to a desired concentration.
  • Step 108 can include determining optimal concentration of bacteria in the infection medium and the optimal time the explant is contacted with the infection medium.
  • concentration of Agrobacterium used for infection can be varied to prevent overgrowth.
  • a range of Agrobacterium concentrations from 102 to 1010 cfu/mL can be used.
  • the bacterial density can be varied within the range of about 0.01 to about 2.00, such as about 0.05, 0.10, 0.15, 0.20, 0.40, 0.60, 0.80, 1.00, 1.30, 1.60, and 2.00 at OD 6 oo( nm) .
  • the culture can be grown or diluted to a bacterial density of about 0.4-0.6 ODeoo. In some cases, the culture is grown or diluted to a bacterial density of no greater than 0.5 ODeoo, such as 0.3- 0.5 OD600.
  • Step 108 can include performing one or more steps for preparing the explant tissue for infection.
  • Preparing the explant tissue can include wounding a portion of the explant.
  • Many methods of wounding can be assessed, including, for example, cutting, abrading, piercing, poking, penetration with fine particles or pressurized fluids, plasma wounding, application of hyperbaric pressure, or sonication.
  • the tissue can be chopped into 1 cm pieces (e.g., petiole tissue) or sliced partially or fully (e.g., node tissue).
  • Wounding can be performed using, scalpels, scissors, needles, abrasive objects, airbrush, particles, electric gene guns, or sound waves.
  • the tissue of the explant is subjected to a chemical treatment that renders the cell walls more permeable (e.g., treatment with macerating enzymes such as cellulase, pectinase or macerozyme).
  • a chemical treatment that renders the cell walls more permeable (e.g., treatment with macerating enzymes such as cellulase, pectinase or macerozyme).
  • the explant can be treated during inoculation to enhance transformation efficiency. Treatments include subjecting the explant to vacuum infiltration, heat shock and/or centrifugation, and sonication. For example, a vacuum can be applied to the explant in Agrobacterium solution (e.g., for about 10 minutes) or the explant is sonicated in Agrobacterium solution followed by soaking the explant in th Agrobacterium solution (e.g., for 10 minutes). In some cases, the explant is sonicated in Agrobacterium solution (e.g., for about 80 seconds) and then soaked in the Agrobacterium solution (e.g., for about 19 minutes). Combinations of more than one type of wounding and/or treatment during inoculation can be utilized, and can be based on the plant variety and/or other characteristics, such as lignin content.
  • One or more phenolic compounds can be included in the inoculation medium prior to or during the Agrobacterium co-cultivation.
  • the phenolic compound can be acetosyringone, acetophenone, chalcone, or cinnamic acid.
  • the plant phenolic compound can be added to the medium prior to contacting the starting material with Agrobacterium (for e.g., several hours to one day). Possible concentrations of plant phenolic compounds in the inoculation medium range from about 25 mM to 700 pM, such as 100-200 pM.
  • Acetosyringone 50, 100, 150, 200, or 250 pM
  • co-cultivation media can be added to co-cultivation media.
  • step 108 also includes testing explants from several different plant parts (e.g., leaf, petiole, nodal segments, or roots).
  • Donor plants for transformation and regeneration of identified mother plants can include propagating donor plants to provide clonal material for the generation of new transgenic Cannabaceae plants.
  • Donor material can include, without limitation, a clone or a seedling.
  • step 202 of method 200 includes propagating a population of donor plants from the mother plant (e.g., a mother plant identified using method 100).
  • the donor plant is grown under optimized conditions to provide suitable explants for transformation and regeneration.
  • Optimal conditions can include maintaining a donor plant in a vegetative state throughout its life. For example, a cannabis plant enters the vegetative stage after the plant develops seven sets of true leaves, and the 8 th is barely visible in the center of the growth tip.
  • the plant directs its energy resources primarily to the growth of leaves, stems, and roots.
  • Harvesting clonal material from the donor plant, transforming it, and growing the transformed material into mature plants capable of producing viable seed can require that the donor plants have been maintained in a vegetative state.
  • the donor plant can be an in vitro rooted plant in soil, or an ex vitro plant rooted in soil in a greenhouse or other growth chamber.
  • the conditions of step 204 can be optimized to ensure that the donor plant is not a plant transitioning from the vegetative state to the reproductive stage, or a plant in its reproductive stage.
  • the conditions of step 204 can be optimized based on the physiologic age of the plants.
  • Donor plants are clonally propagated from a mother plant, and therefore, plant age can be based on the day the donor plant was transferred to soil.
  • the donor plant can have an age of from 1 to 120 months, from 1.5 to 100 months, or from 2 to 80 months, e.g., from 2 to 24 months, from 2.5 to 12 months, from 3 to 6 months, or about 3.5 months.
  • Serial cuttings can be removed from a donor plant, rooted, grown under long day length, and used to replace older donor plants using conventional cloning techniques, such as stem propagation.
  • the propagation process creates a genetic replica of the mother through relatively large cuttings (conventional cloning) or micropropagation.
  • the process can be repeated, indefinitely, without loss of vigor, if the cutting material is kept free of pathogens such as viruses and insect vectors.
  • step 204 can include maintaining the vegetative state of donor plant using day lengths of 14 hours or longer in a controlled growth chamber, depending on the cultivar.
  • the vegetative state can be maintained under 16/8 hr- light/dark conditions.
  • a wide variety of lighting types can be used to maintain the vegetative state; these include metal halide bulbs, HPS lamps, LEDs, or a combination of different lighting types. LEDs can be optimized to specific production conditions by controlling periodicity, quantity, and spectrum of the light provided.
  • Donor Cannabaceae plants can be cultivated under the control of a vegetative state environmental management system configured to ensure that the donor plants are maintained in the vegetative state, in optimal health, and free from pathogens or insect vectors.
  • the environment management system can include computer control of grow room environment, sensors, and fertigation devices. The system facilitates monitoring the parameters that must be optimized to grow the highest quality and healthiest plants, such as real time measurement of temperature, relative humidity, and carbon dioxide content. Water and/or fertigation parameters can be measured, including pH, flow rate, Nitrogen-Phosphorous-Potassium, ppm of certain compounds (e.g., micronutrients).
  • Donor plants can be maintained at a temperature between 25 and 30 °C, with relative humidity (RH) levels of 75% or higher during the propagation and development stages of the donor plants and 55-60% during their vegetative stage.
  • RH relative humidity
  • vegetative stage donor plants can be maintained in a growth chamber at 25 °C, 70% RH.
  • Optimal values for fertilizer e.g., nitrogen/liter and pH
  • step 204 includes inducing axillary meristem growth in donor plants that have been maintained in a vegetative state.
  • the apical meristems can be removed. This creates a steady donor tissue pipeline comprising axillary meristem that can be used for creating explant tissues for transformation in tissue culture.
  • Step 206 can include the excision and preparation of explants.
  • Methods of the present disclosure e.g., methods 100 and 200
  • the explant can be sourced from pathogen- indexed stock.
  • explants can be selected as single-node segments.
  • the meristem can be exposed or isolated from the segments under the stereomicroscope in the laminar hood using dissection instruments.
  • Methods of the present disclosure can include disinfecting the explants before culture to remove surface contaminants such as bacteria and bacterial and fungal spores, and endophytic microbes (Endophytes) with minimal damage to plant cells.
  • Endophytes include viruses, bacteria and fungi that may colonize plant tissue and organs without eliciting symptoms of disease. Endophytes can be symbiotic, but they can also be pathogenic to host tissue. Endophytic bacteria and fungi are frequently observed in plant tissue cultures, and often affect in vitro propagation. Endophytic colonization of plant tissues can persist and progress from surface- sterilized tissues including stem, leaf, petiole, and axillary buds.
  • the disinfecting solution can include one or more disinfecting agents selected from the group consisting of ethanol, hypochlorite (NaCIO or Ca(C10) 2 ), benzalkonium chloride, silver nitrate, mercuric chloride and hydrogen peroxide.
  • the disinfecting solution can include preservatives and inorganic salts that target microbial enzymes in the Krebs cycle and electron transport chain (e.g., as sold by the trade name Plant Preservative Mixture (PPMTM)).
  • PPMTM Plant Preservative Mixture
  • the disinfecting solution includes one or more antibiotics.
  • Non limiting examples of suitable antibiotics include nystatin, streptomycin, cephalexin, cefotaxime, carbenicillin, ticarcillin, clavulanic acid, penicillin-G, and salts, and combinations thereof (e.g., TIMENTIN®).
  • the disinfecting solution can contain the disinfecting agent within a range of 0.01% to about 95% by volume (e.g., 0.5-0.9% by volume) or e.g., up to about 500 pg/ml in the case of antibiotics.
  • the explant can be exposed to the disinfecting solution for a period of 0.1 to about 60 minutes (e.g., about 30 minutes).
  • the disinfecting solution can further include a mild detergent such as a polysorbate (e.g., TWEEN 20 or TWEEN 80) or other non-ionic surfactant.
  • a mild detergent such as a polysorbate (e.g., TWEEN 20 or TWEEN 80) or other non-ionic surfactant.
  • the explants can be washed with a disinfecting solution including about 0.5-2% NaCIO (e.g., 0.5% NaCIO) and up to 0.1% Tween (e.g., Tween 20). Tween can improve contact of the NaCIO with the explant tissue.
  • the epidermis from stem segments is removed and the explants washed under running tap water (e.g., for 5 minutes) before disinfecting the explants in a laminar flow cabinet.
  • the explants can be placed in a sterilized beaker (e.g., autoclaved) or 50 mL centrifuge tube, and the disinfecting solution added (e.g., by stirring on magnetic mini stirrer). In some cases, the explant is agitated in the disinfecting solution for 1-60 minutes by stirring or shaking (e.g., at 60 RPM). After immersion in the solution for the desired duration, the explants can be rinsed several times. For example, the disinfected explants can be rinsed with sterile distilled water 3 times, for 3, 5 and 10 minutes respectively. After surface disinfection, the plant material can be kept in distilled water in Petri dishes in the laminar flow cabinet to prevent drying.
  • a sterilized beaker e.g., autoclaved
  • 50 mL centrifuge tube e.g., 50 mL centrifuge tube
  • the disinfecting solution e.g., by stirring on magnetic mini stirrer
  • the explant is agitated in the disinfecting
  • the cut ends of the explants can be trimmed with a sterile scalpel before placing the explants on the culture medium.
  • the apical or axillary buds can be placed into a gauze bag to facilitate manipulation during disinfection.
  • the disinfecting treatment can increase regeneration rates.
  • the treated explants can demonstrate greater shoot induction, shoot multiplication, and shoot elongation compared with explants plated without treatment with the disinfecting solution.
  • the regeneration rate of explants can be determined using the methods described above.
  • step 208 can include preparing the explant for infection by pre culturing the disinfected explant.
  • the explant can be pre-cultured using the co cultivation medium before inoculation.
  • the donor Cannabaceae plants, plant parts or plant cells are modified by introducing one or more nucleic acid sequences (e.g., a transgenes) which when expressed lead to a desired phenotype.
  • the nucleic acid sequences can be introduced by direct or indirect plant transformation methods.
  • Embodiments of the present disclosure include the use the bacterial pathogen Agrobacterium tumefaciens to inject a piece of DNA.
  • the piece of DNA can be part of an expression cassette introduced into a binary plasmid for Agrobacterium-mediated transformation.
  • the methods of the present disclosure can include constructing an expression cassette that will function in Cannabaceae plant cells.
  • a vector may comprise DNA comprising a gene under control of or operatively linked to a regulatory element (for example, a promoter).
  • the expression cassette encodes a transgene.
  • the transgene can be isolated from any source and encode specific protein products.
  • the plasmid can include DNA of foreign genes, or additional, or modified versions of native, or endogenous genes.
  • the plasmid can include regulatory elements that drive expression of additional native or endogenous genes differently than the native regulatory elements.
  • the plasmid can be used alone or in combination with other plasmids to provide transformed plants using transformation methods as described below.
  • the expression cassette can include at least one genetic marker that allows transformed cells containing the marker to be either recovered by negative or positive selection.
  • a key factor in production of transformed plants involves the selection and regeneration of transformed explants containing a stably integrated gene of interest. Only a small percentage of cells receive the foreign gene during plant genetic transformation.
  • a marker gene for a selective agent can be introduced simultaneously with the novel foreign DNA. The marker can be harbored on the same vector as the sequence-specific nuclease(s) or can be delivered as a separate vector.
  • markers for plant transformation include selectable markers conferring resistance to a toxic substance such as an antibiotic or herbicide, and screenable markers (e.g., b-glucuronidase (GUS), b- galactosidase, luciferase and chloramphenicol acetyltransferase).
  • GUS b-glucuronidase
  • b- galactosidase b- galactosidase
  • luciferase luciferase
  • chloramphenicol acetyltransferase chloramphenicol acetyltransferase
  • Genes encoding screenable markers are particularly useful to quantify or visualize the spatial pattern of expression of a gene in specific tissues and are frequently referred to as reporter genes because they can be fused to a gene or gene regulatory sequence for the investigation of gene expression.
  • the GFP mutant Yellow Fluorescent Protein (YFP) can be used to indicate which plant tissues of the explant the Agrobacterium was able to integrate the foreign DNA vector.
  • Genes included in expression cassettes can be driven by a nucleotide sequence comprising a regulatory element, such as a promoter for expression in cannabis or hop.
  • the promoter is typically upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
  • the promoter can be a promoter under developmental control, such as a promoter that preferentially initiate transcription in certain tissues (e.g., “tissue-preferred” and “tissue-specific” promoters).
  • the promoter is a “cell type” specific promoter which drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves.
  • the promoter is an inducible under environmental control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions or the presence of light.
  • the promoter is a under most environmental conditions and most tissues/cells (i.e., a constitutive promoter).
  • a regulatory element of the expression cassette can be a signal sequence for targeting proteins to subcellular compartments.
  • transport of protein produced by transgenes to a subcellular compartment such as the nucleus, chloroplast, vacuole, peroxisome, glyoxysome, cell wall or mitochondrion or for secretion into the apoplast, is accomplished by means of operably linking the nucleotide sequence encoding a signal sequence to the 5' and/or 3' region of a gene encoding the protein of interest.
  • Targeting sequences at the 5' and/or 3' end of the structural gene may determine, during protein synthesis and processing, where the encoded protein is ultimately compartmentalized.
  • the expression cassette can include a gene encoding a foreign protein or a protein conferring an agronomic trait.
  • Agronomic genes include genes that confer resistance to pests or disease, genes that confer resistance to an herbicide, genes that confer or contribute to a value-added trait, which for cannabis can be increased iron content, decreased nitrate content of leaves, improved flavor (e.g., sweetness), improved fragrance, modified fatty acid metabolism, or modified carbohydrate composition.
  • Value-added traits for hop plants can include improved brewing profiles, such as bitterness or aroma qualities, and/or flavonoid profile.
  • the expression cassette can include a nucleotide sequence encoding a rare-cutting endonuclease, or a portion (e.g., a subunit) of a rare-cutting endonuclease.
  • Rare-cutting endonucleases are natural or engineered proteins that have endonuclease activity directed to nucleic acid sequences containing a recognition sequence (target sequence). Rare-cutting endonucleases generally cause cleavage inside their recognition site, leaving 2 to 4 nucleotides (nt) staggered cut with 3' OH or 5' OH overhangs. Further, active rare-cutting endonucleases can be multimeric or associated with accessory molecules.
  • a rare-cutting endonuclease can be a meganuclease, such as a wild type or variant homing endonuclease (e.g., a homing endonuclease belonging to the dodecapeptide family.
  • a wild type or variant homing endonuclease e.g., a homing endonuclease belonging to the dodecapeptide family.
  • Another type of rare-cutting endonuclease is referred to herein as “Cas9/CRISPR system”. This system is characterized by the combined use of an endonuclease from the bacterial Cas9 family and of a single stranded guide RNA that guides said endonuclease to a DNA target sequence generally of 20 base pairs.
  • This DNA target is generally chosen to be located in the genome upstream so-called PAM (protospacer adjacent motif) sequence motives (NGG or NAG) recognized by Cas9.
  • PAM protospacer adjacent motif sequence motives
  • the guide RNA molecule which is generally a single stranded RNA is introduced into the living cell to confer cleavage and specificity to Cas9. It is a synthetic RNA designed to match the desired 20 bp sequence in the genome upstream the PAM.
  • a rare-cutting endonuclease can be a fusion protein that contains a DNA binding domain and a catalytic domain with cleavage activity.
  • TALE- nucleases and ZFNs are examples of fusions of DNA binding domains with the catalytic domain of the endonuclease Fokl.
  • Customized TALE-nucleases are commercially available under the trade name TALENTM (Cellectis, Paris, France).
  • TALENTM Cellellectis, Paris, France
  • the specificity of transcription activator-like (TAL) effectors depends on an effector-variable repeat. Polymorphisms are present primarily at repeat positions 12 and 13, which are referred to herein as the repeat variable-diresidue (RVD).
  • the RVDs of TAL effectors correspond to the nucleotides in their target sites in a direct, linear fashion, one RVD to one nucleotide, with some degeneracy and no apparent context dependence.
  • This mechanism for protein-DNA recognition enables target site prediction for new target specific TAL effectors, as well as target site selection and engineering of new TAL effectors with binding specificity for the selected sites.
  • TAL effector DNA binding domains can be fused to other sequences, such as endonuclease sequences, resulting in chimeric endonucleases targeted to specific, selected DNA sequences, and leading to subsequent cutting of the DNA at or near the targeted sequences.
  • Such cuts double-stranded breaks
  • TALE-nucleases can be used to facilitate site directed mutagenesis in complex genomes, knocking out or otherwise altering gene function with great precision and high efficiency.
  • TALE-nucleases targeted to an endogenous Cannabaceae gene can be used to mutagenize the endogenous gene, resulting in Cannabaceae plants or plant tissue with modified expression of the endogenous gene.
  • Some endonucleases e.g., Fokl
  • a pair of TALE-nuclease monomers targeted to different DNA sequences can be used.
  • the relevant sequences useful in the processes include “functional variants” of the sequences disclosed. Functional variants include, for example, sequences having one or more nucleotide substitutions, deletions or insertions and wherein the variant retains desired activity. Functional variants can be created by methods available to one skilled in the art, such as site-directed mutagenesis, induced mutation, identified as allelic variants, cleaving through use of restriction enzymes, or the like.
  • the inactive monomers can come together to create a functional enzyme that cleaves the DNA.
  • a highly site-specific restriction enzyme can be created.
  • Methods for selecting endogenous target sequences and generating TALE- nucleases targeted to such sequences can be performed. See, for example, U.S. Pat. App. Pub. No. US 2011/0145940 A1 (Jun. 2011), which is incorporated by reference.
  • software that specifically identifies TALE-nuclease recognition sites can be used.
  • RNA-guided endonucleases are a new genome engineering tool that has been developed based on the RNA-guided CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)- associated nuclease (Cas9) from the type II prokaryotic CRISPR adaptive immune system. This system can cleave DNA sequences that are flanked by a short sequence motif known as a proto-spacer adjacent motif (PAM).
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas9 CRISPR adaptive immune system. This system can cleave DNA sequences that are flanked by a short sequence motif known as a proto-spacer adjacent motif (PAM).
  • PAM proto-spacer adjacent motif
  • crRNA CRISPR RNA
  • tracrRNA trans-activating crRNA
  • sgRNA single guide RNA
  • step 208 includes preparing the disinfected explants generated in step 206 for transformation.
  • step 208 includes preparing the explant for infection by activating Agrobacterium.
  • Activating Agrobacterium includes selection or construction of binary plasmids for infection and integration of the DNA, as described above.
  • the DNA is introduced into the Cannabaceae plant, plant part or plant cell using the bacterial strain, bacterial density, inoculation steps, and co-cultivation steps identified as being optimal for the mother plant.
  • the Agrobacterium strain can be grown and used under conditions that enhance transformation efficiency as determined using the steps of method 100.
  • step 208 can include wounding the explant to prepare it for inoculation, as described above.
  • the explants are wounded in specific areas of the explant based on the expression pattern of the screenable marker observed in step 108.
  • the tissue can be chopped into 1 cm pieces (e.g., petiole tissue) or sliced at least partially or to a depth required to expose meristem tissue (e.g., within node tissue).
  • the explant can also be mechanically wounded (e.g., using a scalpel) to permit infiltration into the interstitial spaces of the meristem; this can be advantageous, for example, when lignin content of the tissue is high.
  • Method 200 further includes step 210 for transformation of the explant, including inoculation and co-cultivation of the explant and the Agrobacterium.
  • Inoculating the explant with the activated Agrobacterium culture can require a few minutes to a few hours, such as about 10 minutes to 3 hours (e.g., about 20 minutes).
  • a vacuum can be applied to the explant in Agrobacterium solution (e.g., for about 10 minutes), followed by soaking the explant in the Agrobacterium solution (e.g., for 10 minutes).
  • the explant is sonicated in Agrobacterium solution (e.g., for about 80 seconds) and then soaked in the Agrobacterium solution (e.g., for about 19 minutes).
  • the excess inoculation medium can be drained, and the Agrobacterium permitted to co-cultivate with the explant for several days, generally carried out for 1 to 14, preferably 2 to 4 days. Normally no selection agent is present during this step.
  • the method of inoculation can be based on the optimized transformation conditions identified in step 108. For example, the explant can be soaked in Agrobacterium solution for 20 minutes and can be rinsed of the liquid inoculation medium and allowed to dry on sterile blotting paper before co-cultivating on solid medium.
  • An exemplary co-cultivation medium is described in FIG. 6A.
  • a phenolic compound can be included in the inoculation medium prior to or during the Agrobacterium co-cultivation, as described above.
  • the concentration of phenolic compound during inoculation or co-cultivation can be different than the concentration determined during step 108.
  • expression of the above-described selectable marker genes allows for preferential selection of transformed cells, tissues and/or plants.
  • a selection method must be employed such that whole plants are regenerated from a single transformed cell and every cell of the transformed plant carries the DNA of interest.
  • step 210 can include removing, suppressing the growth of, or killing the Agrobacterium. These steps may include one or more washes.
  • the medium employed after the co-cultivation step contains an antibiotic to kill the remaining Agrobacterium cells as well as any untransformed tissue of the explant.
  • Step 210 can include a second disinfecting step. For example, disinfecting the explants after co culture advantageously removes the Agrobacterium as well as endophytic bacteria, fungi, and viruses that may contaminate the tissue culture.
  • the disinfecting solution can include one or more preservatives such as 5-chloro-2-methyl-3(2H)-isothiazolone and 2-methyl-3(2H)- isothiazolone, one or more inorganic salts such as magnesium chloride, magnesium nitrate, sodium benzoate, and potassium sorbate (e.g., Plant Preservative Mixture, PPMTM) and one or more antibiotics (e.g., Nystatin, streptomycin, cephalexin, cefotaxime, carbenicillin, ticarcillin, clavulanic acid, and penicillin- G).
  • preservatives such as 5-chloro-2-methyl-3(2H)-isothiazolone and 2-methyl-3(2H)- isothiazolone
  • inorganic salts such as magnesium chloride, magnesium nitrate, sodium benzoate, and potassium sorbate
  • PPMTM Plant Preservative Mixture
  • antibiotics e.g., Nystatin, streptomycin, ce
  • the disinfecting solution can contain the disinfecting agent within a range of 0.01% to about 95% by volume or e.g., up to about 500 mg/ml in the case of antibiotics.
  • the explant can be exposed to the disinfecting solution for a period of 0.1 to about 60 minutes.
  • the explant can be placed in a sterilized beaker, 50 mL centrifuge tube, or other suitable vessel, and sufficient the disinfecting solution added to cover the explant. In some cases, the explant is agitated in the solution for 1-60 minutes (e.g., about 30 minutes). After immersion in the solution for the desired duration, the explants can be rinsed several times. After disinfection, the explant can be transferred to regeneration media.
  • Immersing the transformed explant in a disinfecting solution that includes at least one antibiotic and at least one preservative for 1 to 60 minutes can improve the regeneration efficiency of the explant by removing endophytes.
  • the improvement can be demonstrated by one or more of greater shoot induction, shoot multiplication, and shoot elongation compared with explants plated without removing endophytes using the methods described above.
  • Methods of the present disclosure include regeneration of the explant.
  • a fraction of the explant is genetically transformed, and a plant regenerated therefrom is a chimeric plant.
  • the gene of interest can be transformed into cells that give rise to pollen or ovule, and thus into seeds (i.e., germline transformation).
  • Step 212 of method 200 the transformed tissue is regenerated.
  • Step 212 can include introducing the transformants into culture, followed by initiation of shoot growth (Stage I).
  • Stage I is stabilized when the explants produce a constant number of normal shoots after subculture (about 3 weeks).
  • the explant has expanded into a cluster of small shoots.
  • the shoots can be separated and transplanted to new culture medium (Stage II). Material may be sub-cultured several times to new medium to maximize the quantity of shoots produced.
  • the shoots can be sub-cultured every 2 to 8 weeks.
  • the next stage (Stage III) includes transferring the multiplied shoots to rooting medium for root formation and to prepare the regenerated plants to be transplanted into soil.
  • the plants can be conditioned to increase their potential to acclimatize for survival during transplanting.
  • Stage IV includes transfer of the regenerated plants to soil for adaptation to the environmental conditions of the donor plant. Stages I- IV can be completed within 2 months (e.g., in about 55 days).
  • step 212 refers to a cannabis plant, this is merely exemplary.
  • Method 200 is applicable to any Cannabaceae plant.
  • the regenerated plant can be a hop plant cultured from an explant obtained from a hop plant.
  • the co-cultivated explant can be incubated on a regeneration medium comprising at least one plant growth factor (e.g., the plant growth factors and concentrations identified in step 106).
  • the employed medium may further contain at least one compound, which in combination with the selectable marker gene allows for identification and/or selection of plant cells (e.g., a selective agent).
  • the transformants may be incubated for a certain time (e.g., 5 to 14 days) after the co-cultivation step on a medium lacking a selection compound. Establishment of a reliable resistance level against the selection compound may need some time to prevent unintended damage by the selection compound.
  • Transformed cells i.e., those in which the DNA has integrated into the genome of a host cell of the target material, can be selected from untransformed cells.
  • the tissue can be grown upon this medium for a period of 1 to 8 weeks, preferably about 2-4 weeks, until shoots have developed.
  • Embodiments of the present disclosure include seeds, plants, plant cells and parts of plants of the transformed or transformed and regenerated donor Cannabaceae according to one or more of the methods described herein.
  • Cannabaceae plants produced by growing seeds of transformed plants, as well as a cell or tissue culture of regenerable cells from such plants are and Cannabaceae plants regenerated from such cell or tissue culture having the morphological and physiological characteristics of transformed Cannabaceae plants are within scope of the present disclosure.
  • the tissue culture will preferably be capable of regenerating plants having the physiological and morphological characteristics of transformed Cannabaceae plants, and of regenerating plants having substantially the same genotype as the Cannabaceae plant of the present disclosure.
  • the regenerable cells in such tissue cultures will be from embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, pistils, roots, root tips, flowers, seeds, pods, bolls, buds, stems, or the like.
  • the plants of the present disclosure can be used to produce new plant varieties.
  • the plants are used to develop new, unique and superior varieties or hybrids with desired phenotypes.
  • Transgenic plants of the present invention enable commercial- scale production of desired compounds extracted from the plant.
  • the foregoing methods can be used to produce a transgenic line.
  • the transgenic line could then be crossed, with another (non-transformed or transformed) line to produce a new transgenic Cannabaceae line.
  • a genetic trait that has been engineered into a particular Cannabaceae cultivar using the foregoing transformation techniques could be moved into another line using traditional backcrossing techniques that are well known in the plant breeding arts.
  • a backcrossing approach could be used to move an engineered trait from a public, non-elite inbred line into an elite inbred line, or from an inbred line containing a foreign gene in its genome into an inbred line or lines which do not contain that gene.
  • crossing can refer to a simple X by Y cross, or the process of backcrossing, depending on the context.
  • Embodiments of the methods provided herein can include removing the transgene to provide a new non-transgenic Cannabaceae plant.
  • genetic techniques can be used to provide progeny of a transformed plant with a transgene-induced deletion (e.g., targeted deletion induced by expression of a transgene expressing a rare cutting endonuclease) that lack the transgene.
  • progeny plants can be obtained by self-pollinating (selfing) a transformed Cannabaceae plant that is heterozygous for the transgene by segregation. Selfing of such heterozygous plants provides for the transgene to segregate out of a subset of the progeny plant population.
  • Artisans skilled in the art of plant breeding, cannabis breeding, or hop breeding will be familiar with techniques for selfing Cannabaceae plants and producing feminized Cannabaceae seeds.
  • a transformed Cannabaceae plant of the present disclosure includes at least a first transgene, and which is otherwise capable of expressing all the physiological and morphological characteristics of the donor plant.
  • a transformed Cannabaceae plant of the present disclosure includes a cis-genic modification that alters expression of one or more genes compared with the mother Cannabaceae plant, while expressing the desired trait of the mother plant to the same extent as the mother plant, when grown under the same conditions.
  • a transformed Cannabaceae plant of the present disclosure includes one or more targeted mutation (e.g., deletion) that alters the expression of one or more genes of the donor Cannabaceae plant.
  • a transformed cannabis plant can exhibit lower levels of D 9 - T c t a h y d o c a n n a b i n o 1 (THC) than the donor cannabis plant as a result of the altered gene expression.
  • a plant is provided that comprises a single locus conversion.
  • the single locus conversion can include a dominant or recessive allele.
  • the locus conversion may confer a desired trait upon the transformed Cannabaceae plant.
  • a Cannabaceae plant of generated by methods of the present disclosure can include stacked traits that provide a combined effect, resulting from the use of multiple separate nucleic acid constructs or transformation events.
  • multiple constructs as described above may be introduced into cannabis plant cells by the same or different methods, including the introduction of such a trait by the inclusion of two transcription cassettes in a single transformation vector, the simultaneous transformation of two expression constructs, retransformation using plant tissue expressing one construct with an expression construct for the second gene, or by crossing transgenic plants via traditional plant breeding methods, so long as the resulting product is a plant having both characteristics (i.e., integrated into its genome.
  • Plant parts of the present disclosure can include products and compositions produced or purified from plants produced by the methods described herein, including the stalks, cones (hops), fibers, pulp, flowers, seeds, hemp and the like.
  • Products produced from cannabis plants include industrial textiles, building materials, foods and nutritional supplements, personal care products such as soap, lotions, balms and the like, animal bedding, industrial products such as paints, inks, solvents and lubricants, consumer textiles, animal feed, etc.
  • the Cannabaceae plants and plant parts are used to provide extracts, which can be used as a flavoring or aromatic component, or for obtaining Cannabaceae -derived medicinal compounds.
  • the transformed Cannabaceae plants or plant parts can be used for medical purpose.
  • the plants can be used by patients having a disease or condition known to be alleviated or treated by one or more compounds derived from a Cannabaceae plant.
  • the cannabis plants of the present disclosure can be used by a patient experiencing chronic pain, chemotherapy-induced nausea and vomiting, or multiple sclerosis spasticity symptoms.
  • cannabis plants were evaluated for their ability to perform in the field amongst photoperiod sensitive varieties.
  • Germplasm designated as “Cultivar 1” and “Cultivar 2” were identified as potential mother plants for a photoperiod sensitivity that allowed the plants to be kept vegetative under long day length conditions.
  • the potential mother plants were cloned by conventional cloning and the donor plants grown in 25° C, 70% RH, 16/8hr-light/dark conditions in growth chambers to maintain the vegetative state of the clonal material.
  • the donor plants were scouted weekly to ensure insect pests were not inhabiting the plants.
  • Donor plants had their apical meristems removed once they reached 10” in height. Removal of the apical meristem stimulated axillary meristems to grow from the sides of the plants can allow >30-50 meristems to be harvested from the donor plant through its life.
  • Optimal formulations for callus formulation included 5mM TDZ and ImM NAA or Zeatin 0.5mM, ImM, 2mM with IAA O.ImM, 0.5mM, ImM.
  • node segments were regenerated using a variety of media. Node tissue demonstrated an ability to regenerate into 5 cm shoots at a rate greater than 75% efficiency.
  • the Agrobacterium tumefaciens was transformed using a binary vector containing a YFP selectable marker, and grown using methods known in the art.
  • the final bacterial cell concentration (ODeoo) was adjusted to 0.3-1.3 for the transformation of the explants.
  • a bacterial density greater than 0.5 caused major Agrobacterium overgrowth, tissue necrosis and ultimately explant death. All plants putatively transformed with YFP were tested for expression. An OD of 0.4 gave good transformation. Cultivar 2 outperformed Cultivar 1 in transformation efficiency.
  • Strain LBA4404 delivered and stably integrated YFP vectors into the cannabis node tissue using acetosyringone to stimulate Agrobacterium to infect plant tissues.
  • the explants were contacted with th Agrobacterium solution using various methods: (1) soaking explants in Agrobacterium solution for 20 minutes, (2) applying a vacuum to the explants for 10 minutes in Agrobacterium solution, then allowing the explants to soak for another 10 minutes, and (3) subjecting the explants to sonication in the Agrobacterium solution for 80 seconds, then soaking for 19 minutes. Overgrowth was avoided by rinsing the explants in sterile water after soaking in Agrobacterium.
  • YFP expression showed exactly where the Agrobacterium was able to integrate the DNA vector into the plant tissue and allowed fast screening of explants to determine where the Agrobacterium was able to penetrate, and where penetration was prevented.
  • the lignified tissue appears to prevent the Agrobacterium from penetrating. More extreme wounding by piercing the compact and lignified tissue of the base of the nodal segments to access axillary meristem with a syringe needle was also insufficient for the Agrobacterium to reach the meristem area.
  • Meristem penetration was accomplished in node tissue using a sharp #10 scalpel to slice the node in half, leaving half of a meristem on each explant. Combining the half node slicing and scalpel disruption at the base of the meristem to cause maximal wounding is expected to allow the Agrobacterium to penetrate the lignified and compact tissues. 5. Transformation and regeneration
  • the prepared explants are transformed and regenerated in optimized media according to the following schedule:
  • transgenic plants After being transferred to soil, transgenic plants are grown to be self-pollinated to produce seed for further breeding experiments. Keeping the plants vegetative can be critical for being able to harvest clonal material, transform it, and grow the transformed plants into mature plants that can produce viable seed.
  • Hemp donor plants of varieties “Cherry Blossom” and “Cherry Diesel” were grown vegetatively at 25 °C +/- 2 °C at 16/8-hr light/dark conditions. Lateral branches containing one or more nodes (see FIG. 5A, inset) were harvested from donor hemp plants using sharp scissors (FIGs 5B and 5C). The leaves were removed (FIG. 5D). The harvested segments ranged in length from about l-20cm (FIG. 4, Step 602). The segments were sterilized with 70% EtOH.
  • step 604 the harvested lateral branches were placed into 50ml centrifuge tubes containing 10- 15ml sterile ddHiO. Next, the ddHiO was removed, and 30-50ml 10%
  • H2O2 was added to each 50ml centrifuge tube containing the lateral branches.
  • the 50ml centrifuge tubes were sealed and placed on rotary shaker at 20rpm for 10 minutes. After 10 minutes, the 50ml centrifuge tubes are removed from the rotary shaker. Next, the H2O2 solution is removed, and 30-50ml of fresh 10% H2O2 was added to each 50ml centrifuge tube, which were then sealed and placed on rotary shaker at 20rpm for 15 minutes.
  • the 50ml centrifuge tubes were removed from the rotary shaker and placed into a sterile hood (biosafety, laminar, etc.).
  • a sterile hood biosafety, laminar, etc.
  • the harvested tissue was rinsed as follows: the H2O2 solution was removed, and 30-50ml of sterile ddthO was added to each 50ml centrifuge tube, the 50ml centrifuge tubes were sealed and shaken by hand for 1 minute and the ddthO was removed. The rinse procedure was repeated for a total of 3 times.
  • Step 606 Lateral branch segments were cut into individual node explants (about 1 cm) under a microscope using forceps and a sharp scalpel (Step 606). Each node explant was wounded with the scalpel at the meristem area (i.e., by slicing, piercing, or otherwise incising the tissue to expose meristem) to enhance penetration of the Agrobacterium deep into the tissue. The node explants were placed into sterile ddtbO until the Agrobacterium transformation treatment.
  • Step 608 node explants were placed in a petri dish containing
  • Agrobacterium inoculum with acetosyringone at an OD600 of 0.4-0.6 Hemp node explants from the hemp variety “Cherry Blossom” were transformed using the Agrobacterium strain AGL1 with a binary vector that contained a screenable marker (YFP) and a TALEN pair targeting a locus of the Cannabis genome.
  • YFP screenable marker
  • Agrobacterium solution was added to the petri dish after sonication to ensure high viability Agrobacterium.
  • the node explants were allowed to soak in th Agrobacterium inoculum for 10-30 minutes. Then, the node explants were removed from the Agrobacterium inoculum, rinsed in sterile ddH 2 0, and blotted dry on sterile filter paper. The dry node explants were then plated on co-cultivation medium (FIG. 8A) for 1-2 days to stimulates th Agrobacterium to transform the explant cells using t-DNA delivery of the binary vector.
  • co-cultivation medium FIG. 8A
  • step 610 was performed by immersing the node explants in a sterilization solution of cefotaxime (l-500mg/L) and Plant Preservative Mixture (PPMTM) (0.01-5%) and agitated (rotary shaker at about 60RPM) for 1-60 minutes (e.g., about 30 minutes).
  • the cefotaxime/PPMTM sterilization solution was 95-100% effective in eliminating endophytic bacteria, fungi, and viruses, as well as the Agrobacterium used to transform the explants.
  • the sterilization step reduced labor costs associated with salvaging contaminated explants and the risk of further contamination, and reduced the likelihood of genetic changes in response to extreme treatments.
  • solutions of harsher sterilants e.g., bleach, EtOH, H2O2, and HgCh
  • harsher sterilants e.g., bleach, EtOH, H2O2, and HgCh
  • solutions of harsher sterilants were only 25-75% effective in eliminating endophytic bacteria, fungi, and viruses, and more likely to cause tissue death.
  • Explants that were not immersed in a solution including at least one preservative and at least one antibiotic for at least 1 minute and up to about 60 minutes showed signs of endophytic contamination.
  • the endophytic contamination typically appeared one or more days after the explant was plated on growth medium.
  • the endophytic contamination quickly engulfed the untreated explant(s) with rapidly growing fungi or bacteria and usually killed the explant(s) within a few days.
  • the node explants were removed from the sterilization solution, rinsed with sterile ddtbO, blotted dry on filter paper, and plated onto regeneration medium (FIG. 6B).
  • the regeneration medium further contained 50mg/L Kanamycin to select against wild type tissue and favor the growth of the explant cells that have been transformed by the Agrobacterium.
  • the explants were grown on the kanamycin selection medium for six weeks and were sub-cultured onto fresh medium every two weeks during this period.

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Abstract

L'invention concerne des matériels et des méthodes de transformation et de régénération de plantes, de parties de plantes et de cellules végétales de cannabaceae. L'invention concerne donc par exemple une partie de plante ou une cellule végétale dérivée de matériel clonal qui a été transformée avec un polynucléotide d'intérêt et régénérée en une plante de cannabaceae, telle qu'une plante de cannabis ou une plante de humulus.
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Citations (4)

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US20120311744A1 (en) * 2011-06-06 2012-12-06 Erich E. Sirkowski Marked Cannabis For Indicating Medical Marijuana
WO2019234750A1 (fr) * 2018-06-07 2019-12-12 The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization (Aro) (Volcani Center) Procédés de régénération et de transformation de cannabis
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US20110145940A1 (en) 2009-12-10 2011-06-16 Voytas Daniel F Tal effector-mediated dna modification
US20120311744A1 (en) * 2011-06-06 2012-12-06 Erich E. Sirkowski Marked Cannabis For Indicating Medical Marijuana
WO2019234750A1 (fr) * 2018-06-07 2019-12-12 The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization (Aro) (Volcani Center) Procédés de régénération et de transformation de cannabis
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