WO2022115850A1 - Tissue culture of an autoflower cannabis plant - Google Patents

Tissue culture of an autoflower cannabis plant Download PDF

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WO2022115850A1
WO2022115850A1 PCT/US2021/072570 US2021072570W WO2022115850A1 WO 2022115850 A1 WO2022115850 A1 WO 2022115850A1 US 2021072570 W US2021072570 W US 2021072570W WO 2022115850 A1 WO2022115850 A1 WO 2022115850A1
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autoflower
plant
medium
cannabis
cannabis plant
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PCT/US2021/072570
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French (fr)
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Ian Davidson
Sma ZOBAYED
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Segra International Corporation
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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
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/12—Leaves
    • 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

Definitions

  • the present invention relates to compositions and methods for large-scale micropropagation of auto-flowered Cannabis plants.
  • Auto-flowering Cannabis refer to varieties of the Cannabis genus of flowering plants that switch from a vegetative growth stage to a flowering stage based on age, rather than length of day. Most Cannabis cultivars switch to the flowering stage by measuring the ratio of light to dark hours, and thereby flower according to the seasons.
  • Photoperiodism is the physiological reaction of plants to the length of night or a dark period.
  • the current invention is designed to keep Cannabis shoots in the vegetative stage (non-flowering) rather than the flowering stage of their life cycle through the manipulation of plant growth regulators in aseptic tissue culture media.
  • Most plants are photoperiod- sensitive, meaning their physiological queues to grow or flower are determined by the amount and duration of available light where they grow.
  • An autoflower plant also called an autoflowering plant, is a type of plant having a life cycle that does not depend on the shortening of the light period to induce flowering. Depending on the variety, an autoflower plant will automatically start to flower at a set number of days or within a narrow range of a set number of days, regardless of the amount of light it receives each day.
  • Some embodiments of the invention relate to a method of producing a true-to- type clonal autoflower Cannabis plant by micropropagation.
  • the method can include (a) initiating a tissue culture process from an explant of a seed or a flowering mother autoflower Cannabis plant; (b) incubating the explant in aseptic growth medium; (c) optionally detecting the absence of bacterial or fungal contaminants on the explant; (d) multiplying shoots in a multiplying medium, (e) rooting in a medium comprising Murashige and Skoog containing indole-3-acetic acid sucrose 30 g L-l and agar 8 g L- 1; pH 5.7; (f) rooting and acclimation of the explant ex vivo; and/or (g) obtaining a true- to-type clonal autoflower Cannabis plant.
  • the clonal autoflower Cannabis plant is not synchronous in flowering time with the mother autoflower Cannabis plant.
  • the initiating step can include sterilization of nodal and/or axillary microcuttings in a bleach solution (1% NaOCl w/v).
  • the growth medium of step (b) can be Murashige and Skoog salts and vitamins, supplemented with growth regulators including Flurprimidol (0.1-1 mg/L), Succinic Acid 2,2-Dimethylhydrazidec (0.1-1 mg/L), Meropenem Trihydrate (0.1-1 mg/L), 6-Benzylaminopurine (0.1-1 mg/L), Indole-3-Acetic Acid (IAA) (0.1-1 mg/L); sucrose 30 g L-l and agar 8 g L-l; pH 5.7.
  • growth regulators including Flurprimidol (0.1-1 mg/L), Succinic Acid 2,2-Dimethylhydrazidec (0.1-1 mg/L), Meropenem Trihydrate (0.1-1 mg/L), 6-Benzylaminopurine (0.1-1 mg/L), Indole-3-Acetic Acid (IAA) (0.1-1 mg/L); sucrose 30 g L-l and agar 8 g L-l; pH 5.7.
  • the multiplying medium of step (d) can be Murashige and Skoog salts and vitamins, supplemented with Succinic Acid 2,2-Dimethylhydrazidec (0.1-10 mg/L), Meropenem Trihydrate (0.1-10 mg/L), 6-Benzylaminopurine (0.1-10 mg/L), Indole-3 -Acetic Acid (IAA) (0.1-10 mg/L); sucrose 30 g L-l and agar 8 g L-l; pH 5.7.
  • the clonal Cannabis plant is disease-free.
  • Some embodiments of the invention relate to a system using the method disclosed herein for the in vitro clonal micropropagation of a Cannabis plant autoflower.
  • the system can include (a) an explant in the form of embryo from a seed, apical tissue, nodal tissue, meristematic tissue, young shoot tips, stems or leaves from a mothering plant in the flowering stage and/or (b) an aseptic tissue culture medium for micropropagation.
  • the mothering plant can be a photo-neutral or autoflower Cannabis variety.
  • the system can be capable of producing a clonal autoflower Cannabis plant.
  • FIG. 1 depicts photographs of a plant at different stages in the method described herein. Left: 21 days after initiation; middle: elongation/multiplication; right: in vitro.
  • the present invention relates to compositions, kits, methods, and systems designed to keep Cannabis shoots in the vegetative stage (non-flowering) rather than the flowering stage of their life cycle through the manipulation of plant growth regulators in aseptic tissue culture media.
  • the disclosure provides a system for the in vitro clonal micropropagation of an autoflower Cannabis plant.
  • the system can include an explant of Cannabis.
  • the explant can, for example, be in the form of embryo from a seed, apical tissue, nodal tissue, meristematic tissue, young shoot tip, stem, leaf, and/or the like from a mother plant in the flowering stage.
  • the system can also include an aseptic tissue culture medium for micropropagation.
  • the mother plant is a photo-neutral or autoflower Cannabis variety.
  • the system can produce a clonal autoflower Cannabis plant.
  • kits for the in vitro clonal micropropagation of a Cannabis autoflower plant can include an aseptic/sterile tissue culture medium in a sterile container suitable for the micropropagation of a Cannabis explant.
  • “photo-neutral plant” or “autoflower plant” can be defined as a plant in which flowering can occur irrespective of the day length.
  • the term “disease-free” is used with reference to plants that have been screened for certain viruses, bacteria, and fungi, determined to be “clean”, and maintained under controlled conditions without use of pesticides. Thus, in some examples disease-free is used to refer to plants that are determined to be “virus-free” and “free of bacteria and fungi”. For example, a culture can be determined to be “free of bacteria and fungi” using a culture indexing test or by visual examination.
  • such a method includes culturing for about 7 days in Leifert and Waites Solution, then visually inspecting the culture for turbidity, an indication of microbial contamination (See, e.g., Leifert, Ritchie and Waites, World Journal of Microbiology and Biotechnology 7:452- 469, 1991). Cultures having any observable turbidity are removed and those plants are discarded. Likewise, for example, a culture can be determined to be free of viruses using PCR.
  • virus-free is indicated when the plants do not product a positive test result for any of the following viruses, for example using PCR or ELISA methods: Arabis Mosaic Virus, Tobacco Mosaic Virus, Cucumber Mosaic Virus, Alfalfa Mosaic Virus, Tomato Ringspot Virus, Tobacco streak Virus, Tobacco Ringspot Virus, Potyvirus Group.
  • viruses for example using PCR or ELISA methods: Arabis Mosaic Virus, Tobacco Mosaic Virus, Cucumber Mosaic Virus, Alfalfa Mosaic Virus, Tomato Ringspot Virus, Tobacco streak Virus, Tobacco Ringspot Virus, Potyvirus Group.
  • plants can be screened for presence or absence of viroids such as, for example, hop latent viroid.
  • explant means living plant tissue that is removed from the natural site of growth and placed in sterile medium (e.g., DKW or MS) for culture. This can be of any tissue type such as meristems, leaves, roots, stems, or any portion taken from a plant and used to initiate tissue culture.
  • sterile medium e.g., DKW or MS
  • initiation is the tissue culture phase where explants are taken and placed into in vitro conditions in preparation for culture indexing and multiplication.
  • micropropagation is the practice of rapidly multiplying plant material to produce a large number of progeny plants (clones) using plant tissue culture methods. Micropropagation is used to multiply commercially useful quantities of plants, such as those that have bred through conventional plant methods or been genetically modified. It is also used to provide plantlets from a stock plant which does not produce seeds or for which conventional vegetative reproduction is not commercially viable.
  • micropropagation stage can mean a stage of plant tissue culture that has a duration of about 3-4 weeks, where the plant growth medium is DKW or Murashige and Skoog salts standard media (MS), and may include various vitamins known in the art of plant tissue culture, as well as plant hormones such as benzylaminopurine (BAP), thidiazuron (TDZ), meta - topolin (mT), gibberellic acid (GA3), and indole butyric acid (IB A).
  • BAP benzylaminopurine
  • TDZ thidiazuron
  • mT meta - topolin
  • G3 gibberellic acid
  • IB A indole butyric acid
  • mother plant can be used with reference to a selected young heathy plant exhibiting a desired phenotype and chemotype. These plants provide clean, true- to-type explants to begin the production of clean commercial plants.
  • MS Middle ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
  • growth media or “tissue culture media” or “basal media” or “incubation media”, means sterile liquid, semi- solid, or solid media containing nutrients and other ingredients.
  • medium is a singular form of the term “media.”
  • initiation can refer to a stage of plant tissue culture that typically has a duration of about 3-4 weeks, wherein the plant growth medium can be MS plus supplements.
  • the disclosure also provides a method of producing a true-to-type clonal Cannabis plant by micropropagation.
  • true-to-type plant refers to a genetically identical plant.
  • the method can include a step of initiating a tissue culture process from an explant of a seed or a flowering mother Cannabis plant.
  • the method can include a step of incubating the explant in aseptic tissue culture growth media.
  • the method can include a step of detecting the absence of bacterial or fungal contaminants on the explant by visual confirmation or testing.
  • the method can include a step of multiplying shoots in vitro in a multiplying medium.
  • the method can include a step of rooting in a rooting medium.
  • the method can include a step of rooting and acclimation of the explant ex vivo. Initiation step
  • the initiating step can include collection of seed or an explant from a mother plant of autoflowering Cannabis.
  • the initiating step can include sterilization of nodal and axillary microcuttings.
  • the sterilization can include incubation in a bleach solution such as, for example, 1% NaOCl w/v bleach.
  • the initiating step can include collecting seed.
  • the seed can be washed with an aseptic solution.
  • the aseptic solution can be about 0.1% antiseptic liquid detergent (e.g ., 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%).
  • the seed can be washed for about 5, 10, 15, 20, 25 minutes or more. This can be followed by dipping the seed in another sterilization solution such as, for example, 75% v/v ethanol for about 5, 10, 15, 20, 30 seconds or more. This can be followed by washing the seed in, for example, sterile distilled water.
  • sterilized seeds can be germinated in a container.
  • the container can be, for example, a plastic pot or any suitable container.
  • the pot can contain a growing medium such as perlite, peat soil, or the like, or a combination thereof. Germination can occur within about 48 hours (e.g., 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54 hours).
  • the seedling can be incubated at about 22-26 °C (e.g., 18, 29, 20, 21, 22, 23, 24, 25, 26, 27, or 28 °C) under a 16/8-hour (light/dark cycle) under about 100-200 pmol m 2 s 1 light (e.g., 50, 75, 100, 125, 150, 175, 200, 225, or 250 pmol m-2 s-1 light).
  • 22-26 °C e.g., 18, 29, 20, 21, 22, 23, 24, 25, 26, 27, or 28 °C
  • a 16/8-hour (light/dark cycle) under about 100-200 pmol m 2 s 1 light (e.g., 50, 75, 100, 125, 150, 175, 200, 225, or 250 pmol m-2 s-1 light).
  • nodal segments can be collected from mother plants.
  • the plants can be about 20-40 days old (e.g., 10, 15, 20, 25, 30, 25, 40, 45, or 50 days).
  • the segments can be cleaned in a sterilizing solution and cut into further segments, for example, segments of about 1-5 cm (e.g., 1 cm, 1.5 cm, 2 cm, 3 cm, 4 cm, 5 cm) with about one or two buds.
  • nodal cuttings or apical tips from 2 cm to 3 cm, 2 cm to 4 cm, or 1 cm to 5 cm in length with approximately 0.5 cm to 2.0 cm, 0.75 cm to 1.5 cm, or 1 cm to 1.5 cm of stem below the lowest node are used to initiate tissue culture of Cannabis.
  • a cutting that is shorter or longer than 1 cm to 5 cm in length is used to initiate tissue culture of Cannabis.
  • the further segments can be subjected to further sterilizing steps in a sterilizing solution.
  • the sterilizing solution can be, for example 5% (v/v) liquid detergent, 75% (v/v) ethanol, 1% (v/v) sodium hypochloride, and/or the like.
  • the segments can be surface- sterilized with 75% (v/v) ethanol for about 30 seconds (e.g., about 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 seconds), followed with 1% (v/v) sodium hypochloride solution for about 8-20 minutes (e.g., 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 minutes) and then rinsed with sterile distilled water about five times (e.g., 2, 3, 4, 5, 6, or 7 times).
  • 75% (v/v) ethanol e.g., about 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 seconds
  • 1% (v/v) sodium hypochloride solution for about 8-20 minutes (e.g., 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 minutes) and then rinsed with sterile distilled water about five times (e.g., 2, 3, 4, 5, 6, or 7 times).
  • the method includes screening the explants for pathogens. Screening can be done visually or by a test as known in the art. If a pathogen is detected, the explant can be treated according to methodology known in the art such as methodology described in, for example, U.S. Patent Application Publication No. US 2020-0352124 Al, which is fully incorporated by reference herein.
  • the sterilized explants can be inoculated vertically in growth medium supplemented with different concentrations and combinations of plant growth regulators.
  • the initiation step or stage takes about one week, with establishment of in vitro plants in about 3 to 7 weeks, 4 to 6 weeks, or 4 to 5 weeks.
  • Cannabis explants are transferred to multiplying media, such that they can be divided and multiplied. This stage serves to generate large numbers of shoots per explant.
  • the micropropagation step lasts three or more weeks and relies on use of a multiplying medium.
  • the micropropagation step comprises transfer of a nodal or apical cutting of about 1.5 cm in length to fresh micropropagation media.
  • individual shoots are cut off and transferred into a rooting medium.
  • the method includes acclimatization, wherein plantlets are transferred to soil for growth in a greenhouse. This may be followed by large scale propagation the plants.
  • Plant growth media for use in tissue culture micropropagation of Cannabis are (sterile) liquid, semi- solid, or solid media and contain nutrients and other reagents.
  • the initial and maintenance tissue culture growth medium for Cannabis is Murashige and Skoog salts plus vitamins (MS), a standard mixture of specific nutrients developed for plant tissue culture (Murashige T & Skoog F). MS salts and vitamins are available premixed from many sources. In some embodiments, MS medium is modified and/or supplemented.
  • MS Murashige and Skoog salts plus vitamins
  • compositions for an aseptic tissue culture medium can be used for the growth medium, the multiplying medium, and the rooting medium.
  • the medium can include a plant growth medium such as MS salts and vitamins, and/or the like.
  • MS can be used at about 25%, 50%, 75%, or 100% strength (e.g., 20%, 25%, 30%, 45%, 50%, 55%, 70%, 75%, 80%, 95%, 100%).
  • Tissue culture medium can be supplemented with one or more plant growth regulators.
  • Plant growth regulators are any substances, or mixtures of substances, intended to alter the germination, growth, maturation, or development of plants and/or their products. Plant growth regulators can be classified into subcategories including, but not limited to, antiauxins (e.g., clofibric acid; 2,3,5-tri- iodobenzoic acid; and/or the like), auxins (e.g., 4-CPA; 2,4-D; 2,4-DB; 2,4-DEP; dichlorprop; fenoprop; IAA; IBA; naphthaleneacetamide; a-naphthaleneacetic acid; 1- naphthol, naphthoxyacetic acid; potassium naphthenate; sodium naphthenate; 2,4,5-T; and/or the like), cytokinins (e.g., 2iP, benzyladenine, kinetin, zeatin, and/or the like), defoliants (e
  • the term can additionally include but is not limited to other active ingredients such as, for example, benzofluor, buminafos, carvone, ciobutide, clofencet, cloxyfonac, cyclanilide, cycloheximide, epocholeone, ethychlozate, ethylene, fenridazon, heptopargil, holosulf, inabenfide, karetazan, lead arsenate, methasulfocarb, prohexadione, pydanon, sintofen, triapenthenol, and/or trinexapac.
  • active ingredients such as, for example, benzofluor, buminafos, carvone, ciobutide, clofencet, cloxyfonac, cyclanilide, cycloheximide, epocholeone, ethychlozate, ethylene, fenrid
  • the plant growth regulator can be a hormone selected from cytokinins and auxins.
  • cytokinins can be, for example, kinetin, zeatin, 6- benzylaminopurine, diphenyl urea, thidiazuron, and/or the like.
  • Preferred auxins can be, for example, indole-3-actetic acid (IAA), 4-chloroindole- 3-acetic acid (4-CTIAA), 2- phenylacetic acid (PAA), indole- 3 -butyric acid (IBA), and/or the like.
  • the plant growth regulator can be in a concentration of about 0.01-lmg/L.
  • the plant grown regulator can be in a concentration of about O.Olmg/L, 0.05mg/L, O.lOmg/L, 0.25mg/L, 0.5mg/L, 0.75mg/L, l.Omg/L, or more.
  • the MS medium used for initiation/growth can, for example, be adjusted to pH 5.8, solidified with about 7 g/L agar (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 g/L agar), and autoclaved at about 121 °C (e.g., 114, 116, 118, 120, 121, 122, 124, or 126 °C) for about 15-18 minutes (e.g., 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes).
  • agar e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 g/L agar
  • 121 °C e.g., 114, 116, 118, 120, 121, 122, 124, or 126 °C
  • Basal culture medium is an enriched medium .
  • the basal culture medium can include, for example, about 50%, 75%, or 100% strength MS medium.
  • the 50% strength MS medium chosen as the basal culture medium for shoot induction can be supplemented with different compositions and concentrations of plant growth regulators, such as, for example about 0.5-2.0 mg/L 6-benzyladenine (BA) (e.g., 0.01, 0.05, 0.2, 0.5, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, or 2.4 mg/L); about 0.05-0.5 mg/L a-naphthaleneacetic acid (NAA) (e.g.
  • BA 6-benzyladenine
  • NAA a-naphthaleneacetic acid
  • gibberellic acid e.g., 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, or 2.4 mg/L.
  • Shoot multiplication medium can be defined as a medium that uses plant growth regulators, including hormones, to induce shoot multiplication.
  • the medium for shoot induction can contain, for example, about 30 g/L sucrose (e.g., 24, 26, 28, 30, 32, 34, or 36 g/L sucrose) and can be supplemented with different compositions and concentrations plant growth regulators.
  • the shoot multiplication medium can use, for example, about 50%, 75%, or 100% strength MS medium.
  • the MS medium can be supplemented with different compositions and concentrations of plant growth regulators, such as, for example, about 0.1-0.6 mg/L BA (e.g., 0.01, 0.05, 0.2, 0.3, 0.4, 0.5, 0.6, or 0.7 mg/L) and about 0.01-0.06 mg/L NAA (e.g., 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, or 0.07 mg/L).
  • plant growth regulators such as, for example, about 0.1-0.6 mg/L BA (e.g., 0.01, 0.05, 0.2, 0.3, 0.4, 0.5, 0.6, or 0.7 mg/L) and about 0.01-0.06 mg/L NAA (e.g., 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, or 0.07 mg/L).
  • Root induction medium can be defined as a medium that uses plant growth regulators, including hormones, to induce root formation.
  • root induction medium can use, for example, about 50%, 75%, or 100% strength MS medium.
  • the MS medium can be supplemented with different compositions and concentrations of plant growth regulators such as, for example, about 0.1-1.0 mg/L NAA (e.g., 0.1, 0.05, 0.2, 0.4, 0.6, 0.8, 1, or 1.2 mg/L); about 0.1- 1.0 mg/L IBA (e.g., 0.0.1, 0.05, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, or 1.2 mg/L); and about 0.1-0.5 mg/L cycocel (CCC) (e.g., 0.0.1, 0.05, 0.2, 0.2, 0.3, 0.4, 0.5, 0.6, or 0.7 mg/L).
  • CCC 0.1-0.5 mg/L cycocel
  • Seeds of Cannabis sativa autoflower varieties were first washed for 20 minutes with 0.1% antiseptic liquid detergent. Afterward, the seeds were thoroughly washed under running tap water and then surface sterilized by briefly being dipped in 75% (v/v) ethanol for 30 seconds followed by four washes with sterile distilled water. The sterilized seeds were germinated on a plastic pot (1 gallon) containing mixture of perlite and peat soil. One seed was sowed per pot. Typically, the seeds germinated within 48 hours. The seedlings were incubated in the growth room at 24 ⁇ 2 °C under a 16/8-hour (light/dark cycle) photoperiod provided with cool white, fluorescent light (100-200 pmol m 2 s 1 ).
  • Nodal segments were collected from 30-day-old plants. After cleaning in a solution of 5% (v/v) liquid detergent, the nodal segments were washed under running tap water. They were further cut into segments (2-5 cm) with one or two buds, then surface-sterilized with 75% (v/v) ethanol for 30 seconds, followed with 1% (v/v) sodium hypochloride solution for 8-20 minutes. Afterward, they were rinsed with sterile distilled water five times. [0049] After cutting off two ends, the sterilized explants were inoculated vertically on half- strength MS medium supplemented with different concentrations and combinations of plant growth regulators for shoot induction.
  • MS medium was used for initiation.
  • the medium was adjusted to pH 5.8, solidified with 7 g/L agar, and autoclaved at 121 °C for 15-18 minutes.
  • the medium used for the shoot bud induction/initiation contained 30 g/L sucrose and other growth regulators.
  • the cultures were incubated in the growth room at 24 ⁇ 2 °C under a 16/8-hour (light/dark cycle) photoperiod provided with cool, white-fluorescent light (80 pmol m-2 s-1).
  • the half-strength MS medium chosen as the basal culture medium for shoot induction was supplemented with the following plant growth regulators: 6- benzyladenine (BA): 0.5-2.0 mg/L; a-naphthaleneacetic acid (NAA): 0.05- 0.5 mg/L; gibberellic acid (GA3): 1.0-2.0 mg/L.
  • BA 6- benzyladenine
  • NAA a-naphthaleneacetic acid
  • GA3 gibberellic acid
  • Plantlets that were observed to have well-developed roots after four weeks were transferred to an indoor grow room and kept for approximately 5-7 days. Afterwards, the plantlets were gently removed from the culture vessels and the adhering medium was washed off. The plantlets were subsequently transplanted to plastic trays containing mixture of perlite and peat soil (quick plug), which had been disinfected. The survival rate was calculated after one month.
  • This Example describes an efficient protocol for large-scale micropropagation of auto-flowered Cannabis plants. Direct multiple shoot induction suppresses the risk of genetic instability.
  • the maximum shoot bud induction (79.0%) occurred on 1/2 MS medium supplemented with 2.0 mg/L BA, 0.1 mg/L NAA, and 2.0 mg/L GA3. It turned out that MS 1/2 medium was the best basic medium for in vitro propagation and the highest proliferation rate (310%) was obtained on this medium.
  • Half-strength MS medium supplemented with 0.5 mg/L NAA and 0.5 mg/L IBA was proven to be the best for rooting and the highest rooting percent (nearly 96%).
  • the regenerated plantlets were well-acclimatized. DNA fingerprinting confirmed the genetic uniformity of regenerated plants. Hence, this protocol can be successfully used for the commercial multiplication.
  • This Example describes an efficient and reproducible method for successful in vitro regeneration of autoflowered Cannabis plants by optimizing various growth regulators and basal media.
  • the Example demonstrates the in vitro regeneration of autoflowered Cannabis plants, and it was observed that these shootlets were able to produce roots and be transplanted under ex vitro conditions.
  • any numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the disclosure are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and any included claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the application are approximations, the numerical values set forth in the specific examples are usually reported as precisely as practicable.

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Abstract

This invention relates to compositions and methods for large-scale micropropagation of auto-flowered Cannabis plants.

Description

TISSUE CULTURE OF AN AUTOFLOWER CANNABIS PLANT
Claim of Priority under 35 U.S.C. §119
[0001] The present Application for Patent claims priority to Provisional Application No. 63/117,827 entitled “TISSUE CULTURE OF AN AUTOFLOWER CANNABIS PLANT” filed November 24, 2020, which is assigned to the assignee hereof, and which is hereby expressly incorporated by reference herein.
BACKGROUND
Field
[0002] The present invention relates to compositions and methods for large-scale micropropagation of auto-flowered Cannabis plants.
Background
[0003] Auto-flowering Cannabis refer to varieties of the Cannabis genus of flowering plants that switch from a vegetative growth stage to a flowering stage based on age, rather than length of day. Most Cannabis cultivars switch to the flowering stage by measuring the ratio of light to dark hours, and thereby flower according to the seasons.
[0004] Photoperiodism is the physiological reaction of plants to the length of night or a dark period. The current invention is designed to keep Cannabis shoots in the vegetative stage (non-flowering) rather than the flowering stage of their life cycle through the manipulation of plant growth regulators in aseptic tissue culture media. Most plants are photoperiod- sensitive, meaning their physiological queues to grow or flower are determined by the amount and duration of available light where they grow. An autoflower plant, also called an autoflowering plant, is a type of plant having a life cycle that does not depend on the shortening of the light period to induce flowering. Depending on the variety, an autoflower plant will automatically start to flower at a set number of days or within a narrow range of a set number of days, regardless of the amount of light it receives each day.
[0005] Recently, there has been an increased interest in growing autoflower Cannabis plants because they allow for a more consistent crop in terms of growth, yield, and harvest times. Autoflower Cannabis plants cannot be cloned through the traditional approach of vegetative propagation, as the harvested shoots will begin flowering at about the same time as the parent plant from which they were taken, and before vegetative growth can occur. Consequentially, the new plant will not grow to a commercially relevant size. Therefore, there is a need to develop specific methods, compositions, and systems for fast economical autoflower plant propagation, which also overcomes issues around variability, pathogens, and consistency of performance and other traits.
SUMMARY
[0006] Some embodiments of the invention relate to a method of producing a true-to- type clonal autoflower Cannabis plant by micropropagation. The method can include (a) initiating a tissue culture process from an explant of a seed or a flowering mother autoflower Cannabis plant; (b) incubating the explant in aseptic growth medium; (c) optionally detecting the absence of bacterial or fungal contaminants on the explant; (d) multiplying shoots in a multiplying medium, (e) rooting in a medium comprising Murashige and Skoog containing indole-3-acetic acid sucrose 30 g L-l and agar 8 g L- 1; pH 5.7; (f) rooting and acclimation of the explant ex vivo; and/or (g) obtaining a true- to-type clonal autoflower Cannabis plant. In some embodiments, the clonal autoflower Cannabis plant is not synchronous in flowering time with the mother autoflower Cannabis plant.
[0007] In some embodiment, the initiating step can include sterilization of nodal and/or axillary microcuttings in a bleach solution (1% NaOCl w/v).
[0008] In some embodiments, the growth medium of step (b) can be Murashige and Skoog salts and vitamins, supplemented with growth regulators including Flurprimidol (0.1-1 mg/L), Succinic Acid 2,2-Dimethylhydrazidec (0.1-1 mg/L), Meropenem Trihydrate (0.1-1 mg/L), 6-Benzylaminopurine (0.1-1 mg/L), Indole-3-Acetic Acid (IAA) (0.1-1 mg/L); sucrose 30 g L-l and agar 8 g L-l; pH 5.7.
[0009] In some embodiments, the multiplying medium of step (d) can be Murashige and Skoog salts and vitamins, supplemented with Succinic Acid 2,2-Dimethylhydrazidec (0.1-10 mg/L), Meropenem Trihydrate (0.1-10 mg/L), 6-Benzylaminopurine (0.1-10 mg/L), Indole-3 -Acetic Acid (IAA) (0.1-10 mg/L); sucrose 30 g L-l and agar 8 g L-l; pH 5.7. [0010] In some embodiments, the clonal Cannabis plant is disease-free.
[0011] Some embodiments of the invention relate to a system using the method disclosed herein for the in vitro clonal micropropagation of a Cannabis plant autoflower. The system can include (a) an explant in the form of embryo from a seed, apical tissue, nodal tissue, meristematic tissue, young shoot tips, stems or leaves from a mothering plant in the flowering stage and/or (b) an aseptic tissue culture medium for micropropagation. In some embodiments, the mothering plant can be a photo-neutral or autoflower Cannabis variety. In some embodiments, the system can be capable of producing a clonal autoflower Cannabis plant.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 depicts photographs of a plant at different stages in the method described herein. Left: 21 days after initiation; middle: elongation/multiplication; right: in vitro.
DETAILED DESCRIPTION
[0013] The present invention relates to compositions, kits, methods, and systems designed to keep Cannabis shoots in the vegetative stage (non-flowering) rather than the flowering stage of their life cycle through the manipulation of plant growth regulators in aseptic tissue culture media.
[0014] The disclosure provides a system for the in vitro clonal micropropagation of an autoflower Cannabis plant. In some embodiments, the system can include an explant of Cannabis. The explant can, for example, be in the form of embryo from a seed, apical tissue, nodal tissue, meristematic tissue, young shoot tip, stem, leaf, and/or the like from a mother plant in the flowering stage. The system can also include an aseptic tissue culture medium for micropropagation. Generally, the mother plant is a photo-neutral or autoflower Cannabis variety. In some embodiments, the system can produce a clonal autoflower Cannabis plant.
[0015] Some embodiments of the invention relate to a kit for the in vitro clonal micropropagation of a Cannabis autoflower plant. The kit can include an aseptic/sterile tissue culture medium in a sterile container suitable for the micropropagation of a Cannabis explant.
[0016] As used herein, “photo-neutral plant” or “autoflower plant” can be defined as a plant in which flowering can occur irrespective of the day length. [0017] The term “disease-free” is used with reference to plants that have been screened for certain viruses, bacteria, and fungi, determined to be “clean”, and maintained under controlled conditions without use of pesticides. Thus, in some examples disease-free is used to refer to plants that are determined to be “virus-free” and “free of bacteria and fungi”. For example, a culture can be determined to be “free of bacteria and fungi” using a culture indexing test or by visual examination. In some examples, such a method includes culturing for about 7 days in Leifert and Waites Solution, then visually inspecting the culture for turbidity, an indication of microbial contamination (See, e.g., Leifert, Ritchie and Waites, World Journal of Microbiology and Biotechnology 7:452- 469, 1991). Cultures having any observable turbidity are removed and those plants are discarded. Likewise, for example, a culture can be determined to be free of viruses using PCR. In some examples, “virus-free” is indicated when the plants do not product a positive test result for any of the following viruses, for example using PCR or ELISA methods: Arabis Mosaic Virus, Tobacco Mosaic Virus, Cucumber Mosaic Virus, Alfalfa Mosaic Virus, Tomato Ringspot Virus, Tobacco streak Virus, Tobacco Ringspot Virus, Potyvirus Group. In addition, plants can be screened for presence or absence of viroids such as, for example, hop latent viroid.
[0018] The term “explant”, used herein with reference to plant tissue culture, means living plant tissue that is removed from the natural site of growth and placed in sterile medium (e.g., DKW or MS) for culture. This can be of any tissue type such as meristems, leaves, roots, stems, or any portion taken from a plant and used to initiate tissue culture.
[0019] As used herein, the term “initiation” is the tissue culture phase where explants are taken and placed into in vitro conditions in preparation for culture indexing and multiplication.
[0020] As used herein, the term “micropropagation” is the practice of rapidly multiplying plant material to produce a large number of progeny plants (clones) using plant tissue culture methods. Micropropagation is used to multiply commercially useful quantities of plants, such as those that have bred through conventional plant methods or been genetically modified. It is also used to provide plantlets from a stock plant which does not produce seeds or for which conventional vegetative reproduction is not commercially viable. [0021] As used herein, the term “micropropagation stage” can mean a stage of plant tissue culture that has a duration of about 3-4 weeks, where the plant growth medium is DKW or Murashige and Skoog salts standard media (MS), and may include various vitamins known in the art of plant tissue culture, as well as plant hormones such as benzylaminopurine (BAP), thidiazuron (TDZ), meta - topolin (mT), gibberellic acid (GA3), and indole butyric acid (IB A).
[0022] The term “mother plant”, can be used with reference to a selected young heathy plant exhibiting a desired phenotype and chemotype. These plants provide clean, true- to-type explants to begin the production of clean commercial plants.
[0023] As used herein, the term “Murashige and Skoog salts standard medium” or “MS”, means Murashige and Skoog salts standard medium plus vitamins, as described in Murashige T & Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-97, 1962.
[0024] As used herein, the term “growth media” or “tissue culture media” or “basal media” or “incubation media”, means sterile liquid, semi- solid, or solid media containing nutrients and other ingredients. As used herein, the term “medium” is a singular form of the term “media.”
[0025] As used herein, the term “initiation” can refer to a stage of plant tissue culture that typically has a duration of about 3-4 weeks, wherein the plant growth medium can be MS plus supplements.
[0026] The disclosure also provides a method of producing a true-to-type clonal Cannabis plant by micropropagation. As used herein “true-to-type plant” refers to a genetically identical plant. In some embodiments, the method can include a step of initiating a tissue culture process from an explant of a seed or a flowering mother Cannabis plant. In some embodiments, the method can include a step of incubating the explant in aseptic tissue culture growth media. In some embodiments, the method can include a step of detecting the absence of bacterial or fungal contaminants on the explant by visual confirmation or testing. In some embodiments, the method can include a step of multiplying shoots in vitro in a multiplying medium. In some embodiments, the method can include a step of rooting in a rooting medium. In some embodiments, the method can include a step of rooting and acclimation of the explant ex vivo. Initiation step
[0027] In some embodiments, the initiating step can include collection of seed or an explant from a mother plant of autoflowering Cannabis. In some embodiments, the initiating step can include sterilization of nodal and axillary microcuttings. The sterilization can include incubation in a bleach solution such as, for example, 1% NaOCl w/v bleach.
[0028] In some embodiment, the initiating step can include collecting seed. The seed can be washed with an aseptic solution. For example, the aseptic solution can be about 0.1% antiseptic liquid detergent ( e.g ., 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%). The seed can be washed for about 5, 10, 15, 20, 25 minutes or more. This can be followed by dipping the seed in another sterilization solution such as, for example, 75% v/v ethanol for about 5, 10, 15, 20, 30 seconds or more. This can be followed by washing the seed in, for example, sterile distilled water.
[0029] In some embodiments, sterilized seeds can be germinated in a container. The container can be, for example, a plastic pot or any suitable container. The pot can contain a growing medium such as perlite, peat soil, or the like, or a combination thereof. Germination can occur within about 48 hours (e.g., 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54 hours). The seedling can be incubated at about 22-26 °C (e.g., 18, 29, 20, 21, 22, 23, 24, 25, 26, 27, or 28 °C) under a 16/8-hour (light/dark cycle) under about 100-200 pmol m2 s 1 light (e.g., 50, 75, 100, 125, 150, 175, 200, 225, or 250 pmol m-2 s-1 light).
[0030] In some embodiments, nodal segments can be collected from mother plants. The plants can be about 20-40 days old (e.g., 10, 15, 20, 25, 30, 25, 40, 45, or 50 days). The segments can be cleaned in a sterilizing solution and cut into further segments, for example, segments of about 1-5 cm (e.g., 1 cm, 1.5 cm, 2 cm, 3 cm, 4 cm, 5 cm) with about one or two buds. In some embodiments, nodal cuttings or apical tips from 2 cm to 3 cm, 2 cm to 4 cm, or 1 cm to 5 cm in length with approximately 0.5 cm to 2.0 cm, 0.75 cm to 1.5 cm, or 1 cm to 1.5 cm of stem below the lowest node are used to initiate tissue culture of Cannabis. In some embodiments, a cutting that is shorter or longer than 1 cm to 5 cm in length is used to initiate tissue culture of Cannabis. The further segments can be subjected to further sterilizing steps in a sterilizing solution. The sterilizing solution can be, for example 5% (v/v) liquid detergent, 75% (v/v) ethanol, 1% (v/v) sodium hypochloride, and/or the like. There can be multiple cleaning steps. Each cleaning step can be about 0.5, 1, 2, 3, 4, 5, 6, 7, 10, 15, 20 minutes or more. For example, the segments can be surface- sterilized with 75% (v/v) ethanol for about 30 seconds (e.g., about 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 seconds), followed with 1% (v/v) sodium hypochloride solution for about 8-20 minutes (e.g., 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 minutes) and then rinsed with sterile distilled water about five times (e.g., 2, 3, 4, 5, 6, or 7 times).
[0031] In some embodiments, the method includes screening the explants for pathogens. Screening can be done visually or by a test as known in the art. If a pathogen is detected, the explant can be treated according to methodology known in the art such as methodology described in, for example, U.S. Patent Application Publication No. US 2020-0352124 Al, which is fully incorporated by reference herein.
[0032] After cutting off two ends, the sterilized explants can be inoculated vertically in growth medium supplemented with different concentrations and combinations of plant growth regulators.
[0033] In some embodiments, the initiation step or stage takes about one week, with establishment of in vitro plants in about 3 to 7 weeks, 4 to 6 weeks, or 4 to 5 weeks.
Multiplying step
[0034] In some embodiments, Cannabis explants are transferred to multiplying media, such that they can be divided and multiplied. This stage serves to generate large numbers of shoots per explant. In some embodiments, the micropropagation step lasts three or more weeks and relies on use of a multiplying medium. In one exemplary embodiment, the micropropagation step comprises transfer of a nodal or apical cutting of about 1.5 cm in length to fresh micropropagation media.
Rooting step
[0035] In some embodiments, individual shoots are cut off and transferred into a rooting medium.
Acclimation step
[0036] In some embodiments, the method includes acclimatization, wherein plantlets are transferred to soil for growth in a greenhouse. This may be followed by large scale propagation the plants. Tissue Culture Media
[0037] Plant growth media for use in tissue culture micropropagation of Cannabis are (sterile) liquid, semi- solid, or solid media and contain nutrients and other reagents.
[0038] In some embodiments, the initial and maintenance tissue culture growth medium for Cannabis is Murashige and Skoog salts plus vitamins (MS), a standard mixture of specific nutrients developed for plant tissue culture (Murashige T & Skoog F). MS salts and vitamins are available premixed from many sources. In some embodiments, MS medium is modified and/or supplemented.
[0039] Some embodiments of the invention relate to compositions for an aseptic tissue culture medium. Different media can be used for the growth medium, the multiplying medium, and the rooting medium. The medium can include a plant growth medium such as MS salts and vitamins, and/or the like. MS can be used at about 25%, 50%, 75%, or 100% strength (e.g., 20%, 25%, 30%, 45%, 50%, 55%, 70%, 75%, 80%, 95%, 100%). Tissue culture medium can be supplemented with one or more plant growth regulators.
[0040] “Plant growth regulators” as used herein are any substances, or mixtures of substances, intended to alter the germination, growth, maturation, or development of plants and/or their products. Plant growth regulators can be classified into subcategories including, but not limited to, antiauxins (e.g., clofibric acid; 2,3,5-tri- iodobenzoic acid; and/or the like), auxins (e.g., 4-CPA; 2,4-D; 2,4-DB; 2,4-DEP; dichlorprop; fenoprop; IAA; IBA; naphthaleneacetamide; a-naphthaleneacetic acid; 1- naphthol, naphthoxyacetic acid; potassium naphthenate; sodium naphthenate; 2,4,5-T; and/or the like), cytokinins (e.g., 2iP, benzyladenine, kinetin, zeatin, and/or the like), defoliants (e.g., calcium cyanamide, dimethipin, endothal, ethephon, merphos, metoxuron, pentachlorophenol, thidiazuron, tribufos, and/or the like), ethylene inhibitors (e.g., aviglycine, 1- methylcyclopropene, and/or the like), ethylene releasers (e.g., ACC, etacelasil, ethephon, glyoxime, and/or the like), gibberellins (e.g., gibberellic acid; gibberellins, including but not limited to non-cyclopropene compounds that show gibberellin-like activity, such as, for example: helminthosporic acid, phaseolic acid, kaurenoic acid, and steviol; and/or the like), growth inhibitors (e.g., abscisic acid; ancymidol; butralin; carbaryl; chlorphonium; chlorpropham; dikegulac; flumetralin; fluoridamid; fosamine; glyphosine; isopyrimol; jasmonic acid; maleic hydrazide; mepiquat; piproctanyl; prohydrojasmon; propham 2,3,5-tri-iodobenzoic acid; and/or the like), morphactins (e.g., chlorfluren, chlorflurenol, dichlorflurenol, flurenol, and/or the like), growth retardants/modifiers (e.g., chlormequat, daminozide, flurprimidol, mefluidide, paclobutrazol, cyproconazole, tetcyclacis, uniconazole, ancymidol, trinexapac-ethyl, progexadione-CA, and/or the like), growth stimulators (e.g., brassinolide; forchlorfenuron; hymexazol; 2-amino-6-oxypurine derivatives as described below; indolinone derivates as described below; 3,4-disubstituted maleimide derivatives as described below; fused azepinone derivatives as described below; and/or the like). The term can additionally include but is not limited to other active ingredients such as, for example, benzofluor, buminafos, carvone, ciobutide, clofencet, cloxyfonac, cyclanilide, cycloheximide, epocholeone, ethychlozate, ethylene, fenridazon, heptopargil, holosulf, inabenfide, karetazan, lead arsenate, methasulfocarb, prohexadione, pydanon, sintofen, triapenthenol, and/or trinexapac.
[0041] In some embodiments, the plant growth regulator can be a hormone selected from cytokinins and auxins. Preferred cytokinins can be, for example, kinetin, zeatin, 6- benzylaminopurine, diphenyl urea, thidiazuron, and/or the like. Preferred auxins can be, for example, indole-3-actetic acid (IAA), 4-chloroindole- 3-acetic acid (4-CTIAA), 2- phenylacetic acid (PAA), indole- 3 -butyric acid (IBA), and/or the like.
[0042] The plant growth regulator can be in a concentration of about 0.01-lmg/L. For example, the plant grown regulator can be in a concentration of about O.Olmg/L, 0.05mg/L, O.lOmg/L, 0.25mg/L, 0.5mg/L, 0.75mg/L, l.Omg/L, or more.
[0043] In some embodiments, the MS medium used for initiation/growth can, for example, be adjusted to pH 5.8, solidified with about 7 g/L agar (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 g/L agar), and autoclaved at about 121 °C (e.g., 114, 116, 118, 120, 121, 122, 124, or 126 °C) for about 15-18 minutes (e.g., 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes).
[0044] Basal culture medium is an enriched medium . In some embodiments, the basal culture medium can include, for example, about 50%, 75%, or 100% strength MS medium. In some embodiments, the 50% strength MS medium chosen as the basal culture medium for shoot induction can be supplemented with different compositions and concentrations of plant growth regulators, such as, for example about 0.5-2.0 mg/L 6-benzyladenine (BA) (e.g., 0.01, 0.05, 0.2, 0.5, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, or 2.4 mg/L); about 0.05-0.5 mg/L a-naphthaleneacetic acid (NAA) (e.g. 0.01, 0.05, 0.2, 0.3, 0.4, 0.5, 0.6, or 0.7 mg/L); about 1.0-2.0 mg/L gibberellic acid (GA3), (e.g., 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, or 2.4 mg/L).
[0045] Shoot multiplication medium can be defined as a medium that uses plant growth regulators, including hormones, to induce shoot multiplication. In some embodiments, the medium for shoot induction can contain, for example, about 30 g/L sucrose (e.g., 24, 26, 28, 30, 32, 34, or 36 g/L sucrose) and can be supplemented with different compositions and concentrations plant growth regulators. In some embodiments, the shoot multiplication medium can use, for example, about 50%, 75%, or 100% strength MS medium. In some embodiments, the MS medium can be supplemented with different compositions and concentrations of plant growth regulators, such as, for example, about 0.1-0.6 mg/L BA (e.g., 0.01, 0.05, 0.2, 0.3, 0.4, 0.5, 0.6, or 0.7 mg/L) and about 0.01-0.06 mg/L NAA (e.g., 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, or 0.07 mg/L).
[0046] Root induction medium can be defined as a medium that uses plant growth regulators, including hormones, to induce root formation. In some embodiments, root induction medium can use, for example, about 50%, 75%, or 100% strength MS medium. In some embodiments, the MS medium can be supplemented with different compositions and concentrations of plant growth regulators such as, for example, about 0.1-1.0 mg/L NAA (e.g., 0.1, 0.05, 0.2, 0.4, 0.6, 0.8, 1, or 1.2 mg/L); about 0.1- 1.0 mg/L IBA (e.g., 0.0.1, 0.05, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, or 1.2 mg/L); and about 0.1-0.5 mg/L cycocel (CCC) (e.g., 0.0.1, 0.05, 0.2, 0.2, 0.3, 0.4, 0.5, 0.6, or 0.7 mg/L).
Table 1. Exemplary media for micropropagation
Figure imgf000012_0001
EXAMPLE
Example 1
[0047] Seeds of Cannabis sativa autoflower varieties were first washed for 20 minutes with 0.1% antiseptic liquid detergent. Afterward, the seeds were thoroughly washed under running tap water and then surface sterilized by briefly being dipped in 75% (v/v) ethanol for 30 seconds followed by four washes with sterile distilled water. The sterilized seeds were germinated on a plastic pot (1 gallon) containing mixture of perlite and peat soil. One seed was sowed per pot. Typically, the seeds germinated within 48 hours. The seedlings were incubated in the growth room at 24 ±2 °C under a 16/8-hour (light/dark cycle) photoperiod provided with cool white, fluorescent light (100-200 pmol m 2 s 1).
[0048] Nodal segments were collected from 30-day-old plants. After cleaning in a solution of 5% (v/v) liquid detergent, the nodal segments were washed under running tap water. They were further cut into segments (2-5 cm) with one or two buds, then surface-sterilized with 75% (v/v) ethanol for 30 seconds, followed with 1% (v/v) sodium hypochloride solution for 8-20 minutes. Afterward, they were rinsed with sterile distilled water five times. [0049] After cutting off two ends, the sterilized explants were inoculated vertically on half- strength MS medium supplemented with different concentrations and combinations of plant growth regulators for shoot induction.
[0050] In this example, MS medium was used for initiation. The medium was adjusted to pH 5.8, solidified with 7 g/L agar, and autoclaved at 121 °C for 15-18 minutes. The medium used for the shoot bud induction/initiation contained 30 g/L sucrose and other growth regulators. The cultures were incubated in the growth room at 24 ±2 °C under a 16/8-hour (light/dark cycle) photoperiod provided with cool, white-fluorescent light (80 pmol m-2 s-1).
[0051] The half-strength MS medium chosen as the basal culture medium for shoot induction was supplemented with the following plant growth regulators: 6- benzyladenine (BA): 0.5-2.0 mg/L; a-naphthaleneacetic acid (NAA): 0.05- 0.5 mg/L; gibberellic acid (GA3): 1.0-2.0 mg/L. After four weeks of incubation, the percentage of shoot induction, time taken for bud initiation, and the growth state of the buds were recorded.
[0052] For shoot multiplication, in vitro nodal segments (1-2 cm) were cut off and transferred into fresh half- strength MS medium supplemented with different concentrations of BA (0.10.6 mg/L) in combination with NAA (0.01-0.06 mg/L) and the maximum rate of shoot multiplication was standardized. In addition, three different basal culture media (MS, 1/2 MS, 3/4 MS) with the same plant growth regulators were compared during phase subculture phase, and the optimal medium was selected (1/2 MS). After four weeks of incubation, the multiplication rate and number of new shoots per explant (A 0.5 cm) were recorded.
[0053] Individual shoots (1-2 cm height) were cut off and transferred into rooting media. To optimize the best root induction medium, half- strength MS medium was chosen as the rooting medium and was supplemented with different compositions and concentrations of the plant growth regulator: NAA (0.1-1.0 mg/L), IB A (0.1-1.0 mg/L), and CCC (0.1 - 0.5 mg/L). The percentage of root induction, root numbers, and the growth state of roots were observed and recorded after four weeks.
[0054] Plantlets that were observed to have well-developed roots after four weeks were transferred to an indoor grow room and kept for approximately 5-7 days. Afterwards, the plantlets were gently removed from the culture vessels and the adhering medium was washed off. The plantlets were subsequently transplanted to plastic trays containing mixture of perlite and peat soil (quick plug), which had been disinfected. The survival rate was calculated after one month.
[0055] For genetic fidelity studies, total genomic DNA was extracted from fresh leaves of 18 randomly selected acclimatized plants and their mother plant.
[0056] This Example describes an efficient protocol for large-scale micropropagation of auto-flowered Cannabis plants. Direct multiple shoot induction suppresses the risk of genetic instability. The maximum shoot bud induction (79.0%) occurred on 1/2 MS medium supplemented with 2.0 mg/L BA, 0.1 mg/L NAA, and 2.0 mg/L GA3. It turned out that MS 1/2 medium was the best basic medium for in vitro propagation and the highest proliferation rate (310%) was obtained on this medium. Half-strength MS medium supplemented with 0.5 mg/L NAA and 0.5 mg/L IBA was proven to be the best for rooting and the highest rooting percent (nearly 96%). The regenerated plantlets were well-acclimatized. DNA fingerprinting confirmed the genetic uniformity of regenerated plants. Hence, this protocol can be successfully used for the commercial multiplication.
[0057] This Example describes an efficient and reproducible method for successful in vitro regeneration of autoflowered Cannabis plants by optimizing various growth regulators and basal media. The Example demonstrates the in vitro regeneration of autoflowered Cannabis plants, and it was observed that these shootlets were able to produce roots and be transplanted under ex vitro conditions.
[0058] The various methods and techniques described above provide a number of ways to carry out the application. Of course, it is to be understood that not necessarily all objectives or advantages described are achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as taught or suggested herein. A variety of alternatives are mentioned herein. It is to be understood that some embodiments specifically include one, another, or several features, while others specifically exclude one, another, or several features, while still others mitigate a particular feature by including one, another, or several other features.
[0059] Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be employed in various combinations by one of ordinary skill in this art to perform methods in accordance with the principles described herein. Among the various elements, features, and steps some will be specifically included, and others specifically excluded in diverse embodiments.
[0060] Although the application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the application extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.
[0061] In some embodiments, any numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the disclosure are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and any included claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the application are approximations, the numerical values set forth in the specific examples are usually reported as precisely as practicable.
[0062] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment of the application (especially in the context of certain claims) are construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application. [0063] Variations on preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the application can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the application unless otherwise indicated herein or otherwise clearly contradicted by context.
[0064] All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and/or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of same that may have a limiting effect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and/or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and/or the use of the term in the present document shall prevail.
[0065] In closing, it is to be understood that the embodiments of the application disclosed herein are illustrative of the principles of the embodiments of the application. Other modifications that can be employed can be within the scope of the application. Thus, by way of example, but not of limitation, alternative configurations of the embodiments of the application can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present application are not limited to that precisely as shown and described.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. A method of producing a true-to-type clonal autoflower Cannabis plant by micropropagation comprising: a. initiating a tissue culture process from an explant of a seed or a flowering mother autoflower Cannabis plant; b. incubating the explant in aseptic growth medium; c. optionally detecting the absence of bacterial or fungal contaminants on the explant; d. multiplying shoots in a multiplying medium, e. rooting in a medium comprising Murashige and Skoog containing indole-3- acetic acid sucrose 30 g L-l and agar 8 g L-l; pH 5.7; f. rooting and acclimation of the explant ex vivo; and g. obtaining a true-to-type clonal autoflower Cannabis plant, wherein the clonal autoflower Cannabis plant is not synchronous in flowering time with the mother autoflower Cannabis plant.
2. The method of claim 1 wherein the initiating step comprises sterilization of nodal and axillary microcuttings in a bleach solution (1% NaOCl w/v).
3. The method of claim 1 wherein the growth medium of step b. is Murashige and Skoog salts and vitamins, supplemented with growth regulators comprising Flurprimidol (0.1-1 mg/L), Succinic Acid 2,2-Dimethylhydrazidec (0.1-1 mg/L), Meropenem Trihydrate (0.1-1 mg/L), 6-Benzylaminopurine (0.1-1 mg/L), Indole- 3 -Acetic Acid (IAA) (0.1-1 mg/L); sucrose 30 g L-l and agar 8 g L-l; pH 5.7.
4. The method of claim 1, wherein the multiplying medium of step d is Murashige and Skoog salts and vitamins, supplemented with Succinic Acid 2,2- Dimethylhydrazidec (0.1-10 mg/L), Meropenem Trihydrate (0.1-10 mg/L), 6- Benzylaminopurine (0.1-10 mg/L), Indole-3 -Acetic Acid (IAA) (0.1-10 mg/L); sucrose 30 g L-l and agar 8 g L-l; pH 5.7.
5. The method of claim 1 wherein the clonal Cannabis plant is disease-free.
6. A system using the method of claim 1 for the in vitro clonal micropropagation of a Cannabis plant autoflower comprising: a. an explant in the form of embryo from a seed, apical tissue, nodal tissue, meristematic tissue, young shoot tips, stems or leaves from a mothering plant in the flowering stage; b. an aseptic tissue culture medium for micropropagation. wherein the mothering plant is a photo-neutral or autoflower Cannabis variety; and wherein the system is capable of producing a clonal autoflower Cannabis plant.
PCT/US2021/072570 2020-11-24 2021-11-23 Tissue culture of an autoflower cannabis plant WO2022115850A1 (en)

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Citations (4)

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WO2019006470A1 (en) * 2017-06-30 2019-01-03 Booshoot Llc Media for rapid and reliable tissue culturing of plants
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JP2012110280A (en) * 2010-11-25 2012-06-14 Osaka Univ Method for forming gene transferred plant body
US20150093776A1 (en) * 2012-04-19 2015-04-02 Dianaplantsciences, S.A.S. Polyphenol, terpenoid, glycoside, and alkaloid production by crocus sativus cell cultures
WO2019006470A1 (en) * 2017-06-30 2019-01-03 Booshoot Llc Media for rapid and reliable tissue culturing of plants
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