WO2019006470A1 - Media for rapid and reliable tissue culturing of plants - Google Patents

Abstract

The present invention provides media, kits, systems, and methods for achieving large scale plant production within a short time via bioculture. The present disclosure provides for the production of a host of plants from monocots to dicots, including herbs and phyto-pharmaceutical plants. In some aspects, the invention allows for the mass culture of plants that are otherwise difficult to grow at scale for commercial agricultural production.

Description

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 62/527,862, filed June 30, 2017; which is herein incorporated by reference in its entirety.

TECHNICAL FIELD

[0002] This invention provides compositions, systems, and methods for efficient, rapid and large scale in vitro plant bioculture. In some embodiments, the present invention provides compositions, methods, and systems for the micropropagation and mass production of perennials, grasses, monocots, dicots, and phyto-pharmaceutical plants. In some embodiments, the present invention provides compositions, methods, and systems for the production of virus-free plants.

BACKGROUND

[00Θ3] Medicinal plants play an important role in health care throughout the world— especially in non-industrialized continents such as Africa, South America and parts of Asia. Even in many industrialized countries, a number of traditional plants are widely used by a majority of people for minor to moderate everyday ailments through self-medication.

[0004] Although many traditional medicinal plant remedies do not undergo extensive scientific testing, they are very popular and their sale is not restricted by government regulatory agencies. Some medicinal plants do have substantial laboratoiy and clinical testing and those that fall into this category are referred to as phytopharmaceuticals.

[0005] One of the major problems associated with phytopharmaceutical plant preparations is the variability in the content of the medicinally active ingredients. This problem was highlighted in Belgium in 1997, when more than 100 people were diagnosed with total destruction of their kidneys through irreversible interstitial fibrosis caused by a mis-identified Chinese medicinal plant (Betz, 1998). This has led to strict government controls on purity and levels of active constituents in phytopharmaceutical products in Europe. Such strict regulation does not currently exist, however, in most countries, including Canada and the United States.

[0006] The variability in medicinal content of phytopharmaceutical plants is likely the result of a variety of factors including: year-to-year and plant-to-plant variability in medicinal content; adulteration of m edicinal preparations with misidentified plant species; a lack of adequate methods for production and standardization of the crop; a lack of understanding of the unique plant physiology or efficacy with human consumption; and consumer fraud. In addition, phytopharmaceutical plant preparations are typically produced from field-grown crops and therefore are susceptible to infestation by bacteria, fungi and insects that can alter the medicinal content of the preparations.

[0007] Echinacea products are currently among the best-se!Ung herbal remedies in North America and have been for several years (Schardt, 1998). Preparations of Echinacea sp. have historically been used for the treatment of common human ailments such as colds and flu (Kindscher, 1992). Commercially prepared extracts and whole dried tissue preparations are made from the root of Echinacea species, a crop which takes about 3 years to produce a saleable product.

[0008] With increasing burdens on land to produce food and biomass for energy and materials additional attention is being placed on identifying and utilizing faster growing and more productive plants. Although many plants are suitable for such purposes, there is still a great need to develop compositions, methods, and systems for fast, economical plant propagation

[0009] There is, therefore, a need within the phytopharmaceutical plant industry for the development of an in vitro system for the reliable and reproducible propagation of phytopharmaceutical plants, and when desired the phytofortification of phytopharmaceutical plants with desired compounds of interest.

[0010] It is an object of the invention to overcome disadvantages of the prior art. The above object is met by the combinations of features of the main claims, the sub-claims disclose further advantageous embodiments of the invention.

SUMMARY OF THE INVENTION

[0011] The present invention provides compositions, methods, kits, bioreactors, and systems for efficient and rapid propagation of plants at a large scale via bioculture through tissue culture and the use of bioreactors.

[0012] In some emobidments, the present disclosure is drawn to medium for producing cannabis micropropagations wherein said medium comprises sucrose and at least one cytokinin and at least one auxin; at least one growth retardant; or at least one cytokimn, at least one auxin, and at least one growth retardant; wherein the medium is selected from any one of tables 1 -9. [0013] In some embodiments, the medium is selected from any one or more of the following: IKK) i 01 . BOO102, BOO103, BOO104, BOO105, BOO105, BOO106, BOO107, BOO108, BOO109, BOO110, BOOl l l, B00112, B00113, B00114, B00115, B001 16, B00117, B00118, B00119, BOOI 2G, B00121 , B00122, BOOI 23, B00124, B00125, B00126, B00127, B00128, B00129, BOO130, B00131 , B00132, B00133, B00134, B00135, B00136, B00137, and combinations thereof.

[0014] in some embodiments, the disclosure is drawn to a method for producing cannabis micropropagations comprising utilizing a temporary immersion bioreactor, wherein the bioreactor comprises: a growth vessel for incubating plant tissue in a sterile or substantially sterile environment; a first media container having a first fluid port and a second fluid port, the first fluid port fluidically coupleable to the growth vessel; a second media container having a first fluid port and a second fluid port, the first fluid port fluidically coupleable to the growth vessel; a gas source fluidically coupleable to the second fluid port of the first media container and second fluid port of the second media container; and a controller operable in a first operating mode in which pressurized gas is delivered from the gas source to the first media container to displace a first volume of liquid contained therein to the growth vessel, and a second operating mode in which pressurized gas is delivered from the gas source to the second media container to displace a second volume of liquid contained therein to the growth vessel; wherein the medium is selected from tables 1-9.

[0015] In some embodiments, the disclosure is drawn to a termporary immersion bioreactor, comprising: a growth vessel for incubating cannabis plant tissue in a sterile or substantially sterile environment; a first media container having a first fluid port and a second fluid port, the first fluid port fluidically coupleable to the growth vessel; a second media container having a first fluid port and a second fluid port, the first fluid port fluidically coupleable to the growt vessel; a gas source fluidically coupleable to the second fluid port of the first media container and second fluid port of the second media container; and a controller operable in a first operating mode in which pressurized gas is delivered from the gas source to the first media container to displace a first volume of liquid contained therein to the growth vessel, and a second operating mode in which pressurized gas is delivered from the gas source to the second media container to displace a second volume of liquid contained therein to the growth vessel; wherein the medium is selected from tables 1-9, [0016] In some embodiments of the temporary immersion bioreactor, the controller is operable in a third operating mode in which liquid contained in the growth vessel is allowed to flow from the growt vessel into at least one of the first media container and the second media container, in some embodiments of the temporary immersion bioreactor, the controller is operable in a first incubation sequence in which the third operating mode is executed subsequent to the first operating mode. In some embodiments of the temporary immersion bioreactor, the controller is operable in a second incubation sequence in which the third operating mode is executed subsequent to the second operating mode. In some embodiments of the temporary immersion bioreactor, the controller is further operable in a plant propagation mode in which the first incubation sequence and the second incubation sequence are executed.

[0017] In some embodiments, the present invention describes an automated, or semi-automated, low-cost system for the production of plants, which significantly increases the quantity and quality of plants, the number and size of the resulting plants, reduces the cost and shortens the cultivation time.

[0018] This invention provides novel compositions and an efficient and rapid system for mass propagation of plants in vitro

[0019] In one embodiment, the present invention provides media for plant micropropagation. In some further embodiments, the media are used for micropropagation of cannabis plants.

[0020] In some embodiments, the media are initiation media, multiplication media, and rooting media, such as the BOO101, BOO102, BOO103, BOO104, BOO105, BOO106, BOO107, BQO108, BOO 109, BOO110, combination thereof, or functional equivalents thereof (e.g., by reducing or increasing one or more component concentration, or by adding or removing one or more component, wherein the media maintain the same function). As used herein, the media named "BOO" is equivalent to "BOOS." For example, the media of the present invention are referred to herein as BOO101, BOO102, BOO103, BOO104, BOO105, BOO106, BOO107, BOO108, BOO109, BOO110, etc., which are also known as (a.k.a) BOOS101, BOOS102, BOOS103, BOOS 104, BOOS 105, BOOS 106, BOOS 107, BOOS 108, BOOS 109, BOOS 110, etc., respectively. Therefore, the media desginated as "BOO" herein is interchangeably used as "BOOS" in the present invention.

[0021] In some embodiments, the present invention describes a medium for producing plant rnicropropagations wherein said media comprises sucrus and (1) at least one cytokinin and at least one auxin, (2) at least one growth retardant; or (3) at least one cytokinin, at least one auxin, and at least one growth retardant. In one embodiment, the sucrose has a concentration of about 25-35 g/L. In one embodiment, the at least one cytokinin is 2ip. In one embodiment, the2ip has a concentration of about 1 to 10 mg/L. In one embodiment, the at least one auxin is IAA. In one embodiment, the IAA has a concentration of about 0.1 to 10 mg/L. In one embodiment, the growth retardant is a gibberellms acid antagonist. In one embodiment, the media is selected from tables 1 -9. In one embodiment, the media is selected from tables 2-8. In one embodiment, the gibbereilins acid antagonist is ancymidol. In one embodiment, ancymidol has a concentration of about 0.1 to 10 mg/L. In one embodiment, the medium is a solid, semi-solid, liquid, or semi -liquid medium. In one embodiment, the medium has a pH of about 5.5 to 6.2.

[0022] In some embodiments the present invention describes a medium for producing phyto- pharmaceuticai plant micropropagations wherein said media comprises sucrose and (i) at least one auxm; (ii) at least one growth retardant; or (iii) at least one auxm and at least one growth retardant. In one embodiment, the sucrose has a concentration of about 50-100 g/L. In one embodiment, the medium does not comprise any cytokinin. In one embodiment, the at least one auxin is NAA. In one embodiment, the NAA has a concentration of about 0.01 to about 0.1 mg/L. In one embodiment, the growth retardant is a gibbereilins acid antagonist. In one embodiment, the gibbereilins acid antagonist is ancymidol. In one embodiment, the ancymidol has a concentration of about 0.1 to 10 mg/L. In one embodiment, the medium is a solid, semi-solid, liquid, or semi- liquid medium. In one embodiment, the medium has a pH of about 5.5 to 6.2.

[0023] In some embodiments, the present disclosure is drawn to a set of media for producing phyto-pharmaceutical plant micropropagations wherein the set of media comprises: one or more propagation and multiplication medium; one or more pre-tuberization medium; and one or more tuberization medium; wherein the propagation and multiplication medium does not contain any plant hormone or plant growth regulator; wherein the pre-tuberization medium comprises sucrose at concentration S 1 and at least one cytokinin and at least one auxin; at least one growth retardant; or at least one cytokinin, at least one auxm, and at least one growth retardant; wherein the tuberization medium comprises sucrose at concentration S2 and at least one auxin; at least one growth retardant; or at least one auxin and at least one growth retardant; wherein S I is smaller than S2; and, wherein the propagation and multiplication medium, the pre-tuberization medium, and the tuberization medium are used to produce microtubers. In one embodiment, SI is about 25-35 g/L and S2 is about 50-100 g/L. In one embodiment, wherein the at least one cytokinin in the pre- tuberization medium is 2ip. In one embodiment, the 2ip has a concentration of about 1 to 10 mg/L. In one embodiment, the at least in one auxin in the pre- tuberization medium is IAA. In one embodiment, the IAA has a concentration of about 0.1 to 10 mg/L, In one embodiment, the at least one auxin in the tuberization medium is NAA. In one embodiment, the NAA has a concentration of about 0.01 to about 0.05 mg/L. In one embodiment, the growth retardant in the pre-tuherization medium and/or the tuberization medium is a gibberellins acid antagonist. In one embodiment, the gibberellins acid antagonist is ancymidol. in one embodiment, the ancymidol has a concentration of about 0.1 to 10 mg/L. in one embodiment, the one or more medium is a solid, semi-solid, liquid, or semi-liquid medium. In one embodiment, the one or more medium has a pH of about 5.5 to 6.2. In one embodiment, the present disclosure is drawn to methods for producing plant micropropagations comprising utilizing any media/medium or any set of media/medium of the present disclosure. In some aspects, the present disclosure is drawn to a kit for producing microtubers, wherein the kit comprises any medium or set of media of the present disclosure.

[0024] In some aspects, the present disclosure is drawn to a method for producing phyto- pharmaceutical plant micropropagations comprising utilizing a temporary immersion bioreactor, wherein the bioreactor comprises: a growth vessel for incubating plant tissue in a sterile or substantially sterile environment; a first media container having a first fluid port and a second fluid port, the first fluid port fluidicallv coupleable to the growth vessel; a second media container having a first fluid port and a second fluid port, the first fluid port fluidicaliy coupleable to the growth vessel; a gas source fluidicallv coupieable to the second fluid port of the first media container and second fluid port of the second media container; and a controller operable in a first operating mode in which pressurized gas is delivered from the gas source to the first media container to displace a first volume of liquid contained therein to the growth vessel, and a second operating mode in which pressurized gas is delivered from the gas source to the second media container to displace a second volume of liquid contained therein to the growth vessel.

[0025] In some aspects, the present disclosure is drawn to a temporary immersion bioreactor, comprising: a growth vessel for incubating plant tissue in a sterile or substantially sterile environment; a first media container having a first fluid port and a second fluid port, the first fluid port fluidicallv coupleable to the growth vessel; a second media container having a first fluid port and a second fluid port, the first fluid port fluidicaliy coupleable to the growth vessel; a gas source fluidically coupleable to the second fluid port of the first media container and second fluid port of the second media container; and a controller operable in a first operating mode in which pressurized gas is delivered from the gas source to the first media container to displace a first volume of liquid contained therein to the growth vessel, and a second operating mode in which pressurized gas is delivered from the gas source to the second media container to displace a second volume of liquid contained therein to the growth vessel.

[0026] in one aspect, the controller is operable in a third operating mode in which liquid contained in the growth vessel is allowed to flow from the growth vessel into at least one of the first media container and the second media container.

[0027] In one aspect, the temporary immersion bioreactor of claim 39, wherein the controller is operable in a first incubation sequence in which the third operating mode is executed subsequent to the first operating mode.

[0028] In one aspect, the is operable in a second incubation sequence in which the third operating mode is executed subsequent to the second operating mode.

[0029] In one aspect, the controller is further operable in a plant propagation mode in which the first mcubation sequence and the second incubation sequence are executed.

[0030] In one aspect, the growth vessel is elevated above the first and second media containers to allow liquid to flow from the growth vessel into at least one of the first media container and the second media container in the third operating mode.

[0031] In one aspect the bioreactor further comprising a manifold fluidically coupleable to the growth vessel, the first media container, and the second media container, wherein the manifold is operable to control liquid flow between the growth vessel and the first media container and between the growth vessel and the second media container.

[0032] In one aspect, the growth vessel includes a fluid conduit configured to siphon liquid from the growth vessel to at least one of the first media container and the second media container.

[0033] In one aspect, the growth vessel is an ebb and flow bioreactor. In one aspect, the controller is operable to control fluid communication between the growth vessel and the first media container, between the growth vessel and the second media container, between the gas source and the first media container, and between the gas source and the second media container.

[0034] In some aspects, the present disclosure is drawn to a system for production of phyto- pharmaceutical plant micropropagations, comprising; a temporary immersion bioreactor of the disclosure; a ginseng explant; a pre-tuberization medium, wherein the media is any one from tables

1- 9; a tuberization medium, wherein the media is any one from tables 1-9, In one embodiment, the ginseng explant is a pathogen-free seedling. In one embodiment, the ginseng seedling comprises about 4 to 7 axillary buds,

[0035] In some aspects, the plant is selected from an herb. In one aspect, the plant is selected from a phytopharmaceutical-producing plant. In some aspects, the plant is selected from the group consisting of basil, cannabis, pistachio, ginseng, St. John's wort, willow, lotus, vvasabi, and echinacea. In some aspects, the medium is selected from any one of tables 1 -9 or any one of tables

2- 8. In some aspects, the plant tissue is selected from an herb. In some aspects, the plant tissue is from a phytopharmaceutical-producing plant. In some aspects, the plant tissue is selected from the group consisting of basil, cannabis, pistachio, ginseng, St. John's wort, willow, lotus, wasabi, and echinacea.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1 is a block diagram of an example of one system of the invention.

[0037] FIG. 2 is a schematic illustration of a non-limiting embodiment of the system of FIG. 1.

[0038] FIG. 3 is a schematic illustration of a media container of the system of FIG. 2.

[0039] FIG. 4 is a schematic illustration of a manifold of the system of FIG. 2.

[0040] FIG. 5A is a front view of a gro wth vessel of the system of FIG. 2.

[0041] FIG. SB is a side view of the growth vessel of FIG. 5A.

[0042] FIG. 5C is a top view of the growth vessel of FIG. 5A.

[0043] FIG. 6 is a flowchart of a plant propagation sequence of the invention.

[0044] FIG. 7 is a front perspective view of an oscillating rack, according to an embodiment.

[0045] FIG. 8 is a side perspective view of the oscillating rack of FIG. 7.

[0046] FIG. 9 is an enlarged exploded view of a portion of the oscillating rack labeled as Region Z in FIG. 8.

[0047] FIG. 10 is a cross-sectional view of an upright included in the oscillating rack of FIG. 8, taken along line 4-4 in FIG. 7.

[0048] FIG. 11 is a perspective view of a shelf assembly included in the oscillating rack of FIG. 7.

[0049] FIG. 12 is a perspective view of a portion of the shelf assembly of FIG. 11, [0050] FIG. 13 is a cross-sectional view of a platform included in the portion of the shelf assembly taken along line 7-7 in FIG. 12.

[0051] FIG. 14 is a perspective view of bushings included in the shelf assembly of FIG. 10.

[0052] FIG. 15 is an exploded view of a drive assembly included in the oscillating rack of FIG, 7.

[0053] FIG. 16 is a side view of a portion of the oscillating rack of FIG. 7, in a first configuration.

[0054] FIG. 17 is a side view of the portion of the oscillating rack of FIG. 7, in a second configuration.

[0055] FIG. 18 is a side view of the portion of the oscillating rack of FIG. 7, in a third configuration.

DETAILED DESCRIPTION

Definition

[0056] As used herein, the verb "comprise" as is used in this description and in the claims and its conjugations are used in its non- limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.

[0057] The term "a" or "an" refers to one or more of that entity; for example, "a gene" refers to one or more genes or at least one gene. As such, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein. In addition, reference to "an element" by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements are present, unless the context clearly requires that there is one and only one of the elements.

[0058] As used herein, the term "plant" refers to any living organism belonging to the kingdom Plantae (i.e., any genus/species in the Plant Kingdom) to trees, herbs, bushes, grasses, vines, ferns, mosses and green algae. The term refers to both monocotyledonous plants, also called monocots, and dicotyledonous plants, also called dicots. Examples of particular plants include but are not limited to bamboo, corn, potatoes, roses, apple trees, sunflowers, wheat, rice, bananas, tomatoes, opo, pumpkins, squash, lettuce, cabbage, oak trees, guzmania, geraniums, hibiscus, clematis, poinsettias, sugarcane, taro, duck weed, pine trees, Kentucky blue grass, zoysia, coconut trees, brassica leafy vegetables (e.g. broccoli, broccoli raab, Brussels sprouts, cabbage, Chinese cabbage (Bok Choy and Napa), cauliflower, cavalo, collards, kale, kohlrabi, mustard greens, rape greens, and other brassica leafy vegetable crops), bulb vegetables (e.g. garlic, leek, onion (dry bulb, green, and Welch), shallot, and other bulb vegetable crops), citrus fruits (e.g. grapefruit, lemon, lime, orange, tangerine, citrus hybrids, pummel o, and other citrus fruit crops), cucurbit vegetables (e.g. cucumber, citron melon, edible gourds, gherkin, muskmelons (including hybrids and/or cultivars of cucumis melons), water-melon, cantaloupe, and other cucurbit vegetable crops), fruiting vegetables (including eggplant, ground cherry, pepino, pepper, tomato, tomatillo, and other fruiting vegetable crops), grape, leafy vegetables (e.g. romaine), root/tuber and corm vegetables (e.g. potato), and tree nuts (almond, pecan, pistachio, and walnut), berries (e.g., tomatoes, barberries, currants, elderberries, gooseberries, honeysuckles, mayapples, nannyberries, Oregon- grapes, see-buckthorns, hackberries, bearbernes, lingonbernes, strawberries, sea grapes, lackberries, cloudberries, loganberries, raspberries, salmonberries, thimbleberries, and wineberries), cereal crops (e.g., com, rice, wheat, barley, sorghum, millets, oats, ryes, triticales, buckwheats, fomo, and qumoa), pome fruit (e.g., apples, pears), stone fruits (e.g., coffees, jujubes, mangos, olives, coconuts, oil palms, pistachios, almonds, apricots, cherries, damsons, nectarines, peaches and plums), vine (e.g., table grapes, wine grapes), fibber crops (e.g. hemp, cotton), ornamentals, and the like. For example, the plant is a species in the tribe of Camelmeae, such as C. alyssum, C. anomala, C. grandiflora, C. hispida, C. laxa, C. microcarpa, C. microphylla, C. persistens, C. rumelica, C. saliva, C, Stiefelhagenii, or any hybrid thereof. For example, in some embodiments, the plant is a species in the Pistachioa genus. In some embodiments, the plant is W. japonica. In some embodiments, the plant is a species in the Solarium genus, such as S. tuberosum S. stenotomum, S. phureja, S. goniocalyx, S. ajanhuiti. S. chaucha, S. juzepczukii, and Λ^'. curtilobum. In some embodiments, the plant is a yam variety of the S. tuberosum species.

[0059] In some embodiments, the compositions, methods, and systems are useful for crop plant in vitro propagation. In some embodiments, a crop plant is an agricultural plant. As used herein, the term "crop plant" refers to any plant grown for any commercial purpose, including, but not limited to the following purposes: seed production, hay production, ornamental use, fruit production, berry production, vegetable production, oil production, protein production, forage production, animal grazing, golf courses, lawns, flower production, landscaping, erosion control, green manure, improving soil tilth/health, producing pharmaceutical products/drugs, producing food or food additives, smoking products, pulp production and wood production. For example, an agricultural plant can be potato, tomato, yam, sugar beet, cassava, cucumber, or cauliflower. [0060] In some embodiments, the compositions, methods, and systems are useful for monocotyledon plants propagation. As used herein, the term "monocotyledon" or "monocot" refer to any of a subclass (Monocotyledoneae) of flowering plants having an embryo containing only one seed leaf and usually having parallel- veined leaves, flower parts in multiples of three, and no secondary growth in stems and roots. Examples include lilies; orchids; rice; corn, grasses, such as tall fescue, goat grass, and Kentucky bluegrass; grains, such as wheat, oats and barley; irises; onions and palms.

[0061] In some embodiments, the compositions, methods, and systems are useful for propagation of perennials. The perennial can be an evergreen, deciduous, monoearpic, woody, or herbaceous perennial. In some embodiments, the perennial is Begonia, banana, goldenrod, mint, agave, maple tree, pine tree, apple tree, alfalfa or red clover.

[0062] In some embodiments, the compositions, methods, and systems are useful for propagation of grasses. The grass can be of the Poaceae (or Gramineae), Cyperaceae or Juncaceae family. The grass can be a perennial grass or a cereal grass. The grass can be switchgrass, big biuestem, miscanthus, alfalfa, orchard grass, or reed canarygrass. The grass can be bamboo, tall fescue, goat grass, or Kentucky bluegrass. Other types of grasses include wheat, rye, oat, barley, soy, and hemp, as well as straws derived therefrom.

[0063] In some embodiments, the compositions, methods, and systems are useful for propagation of phyto-pharmaceutical plants. A phyto-pharmaceutical plant is a plant that can be used for a plant-based medicament. In one embodiment, one or more active ingredients in a phyto- pharmaceutical is derived from a plant disclosed herein. In some embodiments, the active ingredient is a plant disclosed herein.

[0064] In some embodiments, the compositions, methods, and systems are useful for propagation of Aloe vera. Ginger, Grape, Cannabis, Garlic, Onion, Echinacea, Geranium, Hakonechloa, Miscanthus, Arundo donax, Switch grass. Rice, or Sugar cane.

[0065] In some embodiments, the compositions, methods, and systems are useful for bamboo plant in vitro propagation. As used herein, the term "bamboo" refers to plants in the subfamily of Bambusoideae. Representative genera of bamboo are described in International Patent Application Publication No. WO2011100762, which is incorporated herein by reference in its entirety. [0066] As used herein, the terms "herb", "herbs" and "herbal" all refer to an annual, biennial, or perennial plant that does not develop persistent woody tissue but dies down at the end of a growing season. Herbal plants typically are capable of flowering and producing seeds. In some contexts the terms refer to a plant or plant part valued for its medicinal, savory, or aromatic qualities. Examples of herbs include, but are not limited to, sage, rosemary, parsley, basil, catnip and marijuana.

[0067] As used herein, "herbal medicine" or "herbal medicinal" refer to herbs, herbal materials, herbal preparations, and finished herbal products that conta in parts of plants, other plant materials, or combinations thereof as active ingredients. Herbs include crude plant material, for example, leaves, flowers, fruit, seed, and stems. Herbal materials include, in addition to herbs, fresh juices, gums, fixed oils, essential oils, resins, and dr powders of herbs. Herbal preparations are the basis for finished herbal products and may include comminuted or powdered herbal materials, or extracts, tinctures, and fatty oils of herbal materials. Finished herbal products consist of herbal preparations made from one or more herbs. See, e.g., Perspectives in Clinical Research, Apr-Jun 2016, 7(2): 59-61.

[0068] As used herein, the term "phytopharmaceutical" (aka "phyto-pharmaceutical") refers to a pharmaceutical of plant origin.

[0069] As used herein, the term "plant part" refers to any part of a plant including but not limited to the shoot, root, stem, axillary buds, seeds, stipules, leaves, petals, flowers, ovules, bracts, branches, petioles, node, internodes, bark, pubescence, tillers, rhizomes, fronds, blades, pollen, stamen, microtubers, and the like. The two mam parts of plants grown in some sort of media, such as soil, are often referred to as the "above-ground" part, also often referred to as the "shoots", and the "below-ground" part, also often referred to as the "roots".

[0070] As used herein, the term "germpiasm" refers to the genetic material with its specific molecular and chemical makeup that comprises the physical foundation of the hereditary qualities of an organism.

[0071] As used herein, the phrase "derived from" refers to the origin or source, and may include naturally occurring, recombinant, unpurified, or purified molecules. A nucleic acid or an amino acid derived from an origin or source may have all kinds of nucleotide changes or protein modification as defined elsewhere herein. [0072] As used herein, the term "offspring" refers to any plant resulting as progeny from a vegetative or sexual reproduction from one or more parent plants or descendants thereof. For instance an offspring plant may be obtained by cloning or selfing of a parent plant or by crossing two parent plants and include sellings as well as the Fl or F2 or still further generations. An Fl is a first-generation offspring produced from parents at least one of which is used for the first time as donor of a trait, while offspring of second generation (F2) or subsequent generations (F3, F4, etc.) are specimens produced from selfings of Fl's, F2's etc. An Fl may thus be (and usually is) a hybrid resulting from a cross between two true breeding parents (true-breeding is homozygous for a trait), while an F2 may be (and usually is) an offspring resulting from self-pollination of said Fl hybrids.

[0073] As used herein, the term "plant tissue" refers to any part of a plant. Examples of plant organs include, but are not limited to the leaf, stem, root, tuber, seed, branch, pubescence, nodule, leaf axil, flower, pollen, stamen, pistil, petal, peduncle, stalk, stigma, style, bract, fruit, trunk, carpel, sepal, anther, ovule, pedicel, needle, cone, rhizome, stolon, shoot, pericarp, endosperm, placenta, berry, stamen, and leaf sheath.

[0074] As used herein, the term "variety" or "cultivar" means a group of similar plants that by structural features and performance can be identified from other varieties within the same species. The term "variety" as used herein has identical meaning to the corresponding definition in the International Convention for the Protection of New Varieties of Plants (UPQV treaty), of Dec. 2, 1961, as Revised at Geneva on Nov. 10, 1972, on Oct. 23, 1978, and on Mar. 19, 1991. Thus, "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 expressi on of at least one of the said characteristics and iii) considered as a unit with regard to its suitability for being propagated unchanged.

[0075] As used herein, the term "cross", "crossing", "cross pollination" or "cross-breeding" refer to the process by which the pollen of one flower on one plant is applied (artificially or naturally) to the ovule (stigma) of a flower on another plant.

[0076] As used herein, the term "cultivar" 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. [0077] As used herein, the term "cross", "crossing", "cross pollination" or "cross-breeding" refer to the process by which the pollen of one flower on one plant is applied (artificially or naturally) to the ovule (stigma) of a flower on another plant.

[0078] As used herein, the term "cultivar" 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.

[0079] As used herein, the term "genotype" refers to the genetic makeup of an individual cell, cell culture, tissue, organism (e.g., a plant), or group of organisms.

[0080] As used herem, the term "clone" refers to a cell, group of cells, a part, tissue, organism (e.g., a plant), or group of organisms that is descended or derived from and genetically identical or substantially identical to a single precursor, in some embodiments, the clone is produced in a process comprising at least one asexual step.

[0081] As used herein, the term "hybrid" refers to any individual cell, tissue or plant resulting from a cross between parents that differ in one or more genes.

[0082] As used herein, the term "inbred" or "inbred line" refers to a relatively true-breeding strain.

[0083] As used herein, the term "population" means a genetically homogeneous or heterogeneous collection of plants sharing a common genetic derivation.

[0084] As used herein, the term "bioreactor" refers to any vessel, device or system capable of holding, supporting and/or growing viable tissue. In other words, the term "bioreactor" as used herein may refer to a growth vessel that holds viable plant tissue, various other components internal or external to the growth vessel that are required for or aid the holding, supporting and/or growing of viable plant tissue, and any subsystem thereof.

[0085] As used herein, the phrase "temporary immersion bioreactor" refers to any bioreactor designed to temporarily wet a part or entire culture or plant tissue with nutrient medium (e.g., liquid or semi-liquid) followed by draining a part or all of the excess nutrient medium.

[0086] As used herein, a "plant propagation system" is a bioreactor for growing viable plant tissue.

[0087] More rapidly growing plants are available for cultivation and industrial uses, including plants in the subfamily Bambusoidea. The subfamily Bambusoideae (of the family Poaceae), comprises both woody and herbaceous bamboos. At present roughly 120 genera of temperate and tropical woody bamboos are recognized. Bamboos are versatile plants with many different applications. It has been estimated that approximately 2.2 billion people worldwide use bamboo to some extent, and in 1985 the global revenue attributable to bamboo was estimated around U.S. $4.5 billion. The market for bamboo is also expanding. Bamboo shoots are a staple of Asian cuisine, and bamboo is found in a number of products including toothpicks, brooms, poles for viticulture and arboriculture, landscaping materials, parquet flooring, laminate materials, furniture, handicrafts and other household items. In addition, bamboo is becoming an important source of textile material as a component of paper production and as a source of structural timber.

[0088] Plant species such as bamboo are considered environmentally friendly "green" products, which have extremely rapid growth rates. Despite rapid growth rates, other characteristics of these plants make it difficult to rapidly propagate these plants at an industrial scale. For example, many commercially important bamboos only flower at intervals of as long as 60-130 years. Compounding the difficulties of this long flowering cycle is the fact that many bamboos exhibit mass (or gregarious) flowering, with all plants in the population flowering simultaneously. For example, Phyllostachys bambusoides flowers at an interval of 130 years, and in this species all plants of the same stock flower at the same time, regardless of differences in geographic locations or climatic conditions. After flowering, the bamboo dies. Bamboo's lengthy flowering interval and propensity for mass flowering makes it very difficult to obtain seeds for propagation. Compounding this problem is the fact that bamboo seeds, even when they are available, remain viable for no more than 3-6 months.

[0089] As a result of these difficulties with the propagation of bamboo and other fast growing plant species using traditional sexual reproduction, these plants are often propagated by asexual techniques such as clump division and cutting. These asexual propagation techniques, however, are insufficient to meet projected world demand because both their capacity to produce mass scale production, and their practical efficiency, are too low. In addition many asexual propagation methods have the downside of failing to eliminate pathogens present in the parent plants. Therefore, compositions, methods, and systems to achieve large scale production of plants are highly desirable. Micropropagation (also known as tissue culturing with the terms used interchangeably herein), is an excellent potential method that could be used to achieve this aim.

[0090] Micropropagation is not unlike growing plants from cuttings. However, unlike plants grown from cuttings, micropropagated plants are grown in vitro in sterile media. Typically, the growth media comprises a solid or semi-solid material such as agar, with the addition of various compounds such as nutrients, inorganic salts, growth regulators, sugars, vitamins and other compounds. [0091] A benefit of tissue culturing plants is that the plants can be grown in a sterile environment so that they may more likely remain disease free. Other benefits include the ability to grow very large numbers of plants in a small space, the reduced water and nutrient needs of micropropagated plants, and the rapid multiplication of tissues that can in turn be used to yield more tissue culture material. Moreover micropropagation is very flexible and rapid upscaling is possible (within 1 year nearly one million plants can be produced from a single genotype). Such short time frames and large numbers cannot be rivaled by any conventional method. Tissue culturing also provides for the production of high quality plants which are easy to transport and deliver.

[0092] Some papers have published which address tissue culturing of plants. In practice, however (i.e., for large or mass scale propagation of bamboos), the compositions, methods, and systems described in these papers do not translate into commercially viable propagation systems.

[0093] The difficulties encountered in tissue culturing of plant species include high incidences of endogenous or surface contaminations and browning, factors related to dormancy or topophysis and hyperhydncity. The present disclosure provides compositions, methods, and systems that overcome these difficulties allowing the commercial-scale asexual production of plants.

[0094] Micropropagation in liquid culture media increases nutrient uptake and promotes growth. However, the advantages of in vitro culture in a liquid media are often counterbalanced by technical problems such as asphyxia, hyperhydncity, shear forces and the need for complex equipment.

[0095] International Patent Application Publication No. WO/2011/100762, which is incorporated herein by reference in its entirety, describes compositions and methods that are useful for bamboo in vitro propagation.

[0096] The present application discloses novel compositions, methods, and systems for the rapid in vitro propagation of plants. The present application also discloses novel compositions, methods, and systems for the reduction of phenolic production by plants, such as bamboo.

[0097] The present invention provides compositions and methods that can significantly increase plant tissue culture multiplication rate within a shorter time. In some embodiments, a strong cytokinin such as thidiazuron is utilized for a very brief period of time in either a solid or liquid induction medium to induce multiple shoot bud formation in explants of plant species. This bud induction treatment utilizing a media containing a strong cytokinin such as thidiazuron is followed by a shoot elongation and maintenance treatment whereby a relatively weaker cytokinin such as BAP, meta-topolin, 2ip, zeatin and or zeatin riboside is used to accomplish the shoot elongation and maintenance of the culture. This process, when alternated methodically resulted in culture multiplication rates between 2X and 28X within a 3 -week culture cycle.

Plants

[0098] Pistachio. As used herein, the term pistachio refers to all species in the genus Pistacia, including Pistacia vera, Pistacia atlantica, and Pistacia intergerrina

[0099] Yam. Yam is the common name for some plant species in the genus Dioscorea (family dioscoreaceae) which produce tubers, bulbils, or rhizomes having medicinal and economic importance. These are perennial herbaceous vines cultivated for the consumption of their starchy tubers in Africa, Asia, Latin America, the Caribbean, and Oceania.

100100] True yams are botanically distinct from the sweet potato, but moist-fleshed varieties of sweet potato are often called yams in the United States. D. bulhifera, the air-potato yam, is one of the few true yams cultivated for food in the United States. Exemplary yam varieties for which the present invention applies include, but are not limited to, white yam, yellow yam, Kokoro yam, water yam, winged yam, purple yam, Chinese yam, air potato, lesser yam, bitter yam, and cush- cush yam.

[00101] Potato. There are about five thousand potato varieties worldwide. The major species grown worldwide is Solanum tuberosum (a tetraploid with 48 chromosomes), and modern varieties of this species are the most widely cultivated. There are also four diploid species (with 24 chromosomes): Λ^'. stenotomum, S. phureja, S. goniocafyx, and .V ajanhuiri. There are two triploid species (with 36 chromosomes): S. chaucha and S.juzepczukii. There is one pentaploid cultivated species (with 60 chromosomes): S. curtilobum. There are two major subspecies of Solanum tuberosum: andigena, or Andean; and tuberosum, or Chilean. The Andean potato is adapted to the short-day conditions prevalent in the mountainous equatorial and tropical regions where it originated. The Chilean potato, native to the Chiloe Archipelago, is adapted to the long-day conditions prevalent in the higher latitude region of southern Chile.

[00102] Most modern potatoes grown in North America arrived through European settlement and not independently from the South American sources. However, at least one wild potato species, Sokmum fendleri, is found as far north as Texas and used in breeding for resistance to a nematode species that attacks cultivated potatoes. A secondary center of genetic variability of the potato is Mexico, where important wild species that have been used extensively in modern breeding are found, such as the hexaploid Solanum demissiim, as a source of resistance to the devastating late blight disease. Another relative native to this region, Solanum bulbocastanum, has been used to genetically engineer the potato to resist potato blight.

[00103] Exemplary potato varieties for which the present invention applies include, but are not limited to, Adirondack Blue, Adirondack Red, Agata, Almond, Apline, Alturas, Amandine, Annabelle, Anya, Arran Victory, Atlantic, Avalanche, Bamberg, Bannock Russet, Belle de Fontenay, BF-15, Bildtstar, Bintje, Blazer, Busset, Blue Congo, Bonnotte, British Queens, Cabritas, Camota, Canela Russet, Cara, Carola, Chelina, Chiloe, Cielo, Clavela Blanca, Desiree, Estima, Fianna, Fingerling, Flava, German Butterball, Golden Wonder, Goldrush, Home Guard, Innovator, Irish Cobbler, Jersey Royal, Kennebec, Kerr's Pink, Kestrel, Keuka Gold, King Edward, Kipfler, Lady Balfour, Langlade, Linda, Marcy, Marfona, Maris Piper, Marquis, Megachip, Monalisa, Nicola, Pachacona, Pike, Pink Eye, Pink , Fir Apple, Primura, Ranger Russet, Ratte, Record, Red LaSoda, Red Norland, Red Pontiac, Rooster, Russet Burbank, Russet Norkotah, Selma, Shepody, Sieglmde, Silverton, Russet, Sirco, Snowden, Spunta, Stobrawa, Superior, Vivaldi, Vitelotte, Yellow Finn, Yukon Gold, blue potato varieties (e.g., Cream of the Crop), Igorota, Solibao, Ganza, Eliane, BelRus, Centennial Russet, Century Russet, Frontier Russet, Hilite Russet, Krantz, Lemhi Russet, Nooksack, Norgold Russet, Norking Russet, Ranger Russet, Russet Burbank, Russet Norkotah, Russet Nugget, Allegany, Atlantic, Beacon Chipper, CalWhite, Cascade, Castile, Chipeta, Gemchip, Irish Cobbler, Itasca, Ivory Crisp, Kanona, Katahdin, Kennebec, Kennebec Story, La Chipper, Lamoka, Monona, Monticello, Norchip, Norwis, Onaway, Chieftain, La Rouge, NorDonna, Norland, Red La Soda, Red Pontiac, Red Ruby, Sangre, Viking, Ontario, Pike, Sebago, Shepody, Snowden, Superior, Waneta, White Pearl, White Roseand, Mazama, and all genetically modified varieties. More potato varieties are described in Clough et al.. lort Technology, 2010, 20(1 ): 250-256; Potato Variety Handbook, National Institute of Agricultural Botany, 2000; Chase et al, North American Potato Variety Inventory, Potato Association of America, 1988, each of which is incorporated by reference in its entirety.

Cannabis [00104] Cannabis, more commonly known as marijuana, is a genus of flowering plants that includes at least three species, Cannabis sativa. Cannabis indica, and Cannabis ruderalis as determined by plant phenotypes and secondary metabolite profiles. In practice however, cannabis nomenclature is often used incorrectly or interchangeably. Cannabis literature can be found referring to all cannabis varieties as "sativas" or all cannabinoid producing plants as "indicas". indeed the promiscuous crosses of indoor cannabis breeding programs have made it difficult to distinguish varieties, with most cannabis being sold in the United States having features of both sativa and indica species.

[00105] Cannabis is one of the world's oldest and most useful cultivated genus of plants.

Humans have used hemp varieties of cannabis for the production of industrial materials, including food, paper, textiles, plastics, detergents, and biofuels. Humans also have a long history of using psychoactive varieties of cannabis for medical and recreational applications. Cannabis has long been used for drug and industrial purposes, fiber (hemp), for seed and seed oils, for medicinal purposes, and as a recreational drug. Industrial hemp products are made from Cannabis plants selected to produce an abundance of fiber. Some Cannabis strains have been bred to produce minimal levels of THC, the principal psychoactive constituent responsible for the psychoactivity associated with marijuana. Marijuana has historically consisted of the dried flowers of Cannabis plants selectively bred to produce high levels of THC and other psychoactive cannabinoids. Various extracts including hashish and hash oil are also produced from the plant.

[00106] Interest in psychoactive varieties of cannabis has recently exploded following the relaxation drug laws within the United States, and with the discovery of previously unrecognized applications for cannabis in the treatment of human diseases such as diabetes, epilepsy, schizophrenia, and cancer.

[00107] Cannabis is diploid, having a chromosome complement of 2n=20, although polyploid individuals have been artificially produced. The first genome sequence of Cannabis, which is estimated to be 820 Mb in size, was published in 2011 by a team of Canadian scientists (Bakel et al, "The draft genome and transcriptome of Cannabis sativa" Genome Biology 12:R102).

[00108] All known strains of Cannabis are wind-pollinated and the fruit is an achene. Most strains of Cannabis are short day plants, with the possible exception of C. sativa subsp. sativa var. spontanea (= C. ruderalis), which is commonly described as "auto-flowering" and may be day- neutral . [00109] The genus Cannabis was formerly placed in the Nettle (Urticaceae) or Mulberry

(Moraceae) family, and later, along with the Hamulus genus (hops), in a separate family, the Hemp family (Cannabaceae sensu stricto). Recent phylogenetic studies based on cpDNA restriction site analysis and gene sequencing strongly suggest that the Cannabaceae sensu stricto arose from within the former Celtidaceae family, and that the two families should be merged to form a single monophyletic family, the Cannabaceae sensu lato.

[00110] Cannabis plants produce a unique family of terpeno-phenolic compounds called cannabinoids. Cannabinoids, terpenoids, and other compounds are secreted by glandular trichomes that occur most abundantly on the floral calyxes and bracts of female plants. As a drug it usually comes in the form of dried flower buds (marijuana), resin (hashish), or various extracts collectively known as hashish oil. There are at least 483 identifiable chemical constituents known to exist in the cannabis plant (Rudolf Brenneisen, 2007, Chemistry and Analysis of Phytocannabinoids (cannabinoids produced by cannabis) and other Cannabis Constituents, In Marijuana and the Cannabinoids, ElSohly, ed.; incorporated herein by reference) and at least 85 different cannabinoids have been isolated from the plant (El-Alfy, Abir T, et a!., 2010, "Antidepressant-like effect of delta-9-tetrahydrocannabinol and other cannabinoids isolated from Cannabis sativa L", Pharmacology Biochemistry and Behavior 95 (4): 434-42; incorporated herein by reference). The two cannabinoids usually produced in greatest abundance are cannabidiol (CBD) and/or Δ⁹ -tetrahydrocannabinol (THC). THC is psychoactive while CBD is not. See, ElSohly, ed. (Marijuana and the Cannabinoids, Humana Press Inc., 321 papers, 2007), which is incorporated herein by reference in its entirety, for a detailed description and literature review on the cannabinoids found in marijuana.

[00111] Cannabinoids are the most studied group of secondary metabolites in cannabis.

Most exist in two forms, as acids and in neutral (decarboxylated) forms. The acid form is designated by an "A" at the end of its acronym (i.e. THC A). The phytocannabinoids are synthesized in the plant as acid forms, and while some decarboxylation does occur in the plant, it increases significantly post-harvest and the kinetics increase at high temperatures. (Sanchez and Verpoorte 2008). The biologically active forms for human consumption are the neutral forms. Decarboxylation is usually achieved by thorough drying of the plant material followed by heating it, often by either combustion, vaporization, or heating or baking in an oven. Unless otherwise noted, references to cannabinoids in a plant include both the acidic and decarboxylated versions (e.g., CBD and CBDA).

[00112] The cannabinoids in cannabis plants include, but are not limited to, Δ⁹-

Tetrahydrocannabinol (A⁹-THC), A⁸-Tetrahydrocannabinol (A⁸-THC), Cannabichromene (CBC), Cannabicyclol (CBL), Cannabidiol (CBD), Cannabielsoin (CBE), Cannabigerol (CBG), Cannabinidiol (CBND), Cannabinol (CBN), Cannabitriol (CBT), and their propyl homologs, including, but are not limited to cannabidivarin (CBDV), A⁹-Tetrahydrocannabivarin (THCV), cannab j chromevarin (CBCV), and cannabigerovarm (CBGV). See Holley et al. (Constituents of Cannabis saliva L. XT Cannabidiol and cannabichromene in samples of known geographical origin, J. Pharm. Sci. 64:892-894, 1975) and De Zeeuw et al. (Cannabinoids with a propyl side chain in Cannabis, Occurrence and chromatographic behavior, Science 175:778-779), each of which is herein incorporated by reference in its entirety for all purposes. Non-THC cannabinoids can be collectively referred to as "CBs", wherein CBs can be one of THCV, CBDV, CBGV, CBCV, CBD, CBC, CBE, CBG, CBN, CBND, and CBT cannabinoids.

[00113] In addition to cannabinoids, cannabis also produces over 120 different terpenes (Russo 201 1 , Taming THC: potential cannabis sy nergy and phytocannabmoid-terpenoid entourage effects, British Journal of Pharmacology, 163 : 1344-1364). Within the context and verbiage of this document the terms 'terpenoid' and 'terpene' are used interchangeably. Examples of representative terpines include, but are not limited to, terpinolene, alpha pheiladrene, beta ocimene, carene, limonene, gamma terpinene, alpha pinene, alpha terpinene, beta pinene, fenchol, camphene, alpha terpineol, alpha humulene, beta caryophyllene, iinalooi, caiy oxide, and myrcene.

[00114] Cannabinoids are odorless, so terpenoids are responsible for the unique odor of cannabis, and each variety has a slightly different profile that can potentially be used as a tool for identification of different varieties or geographical origins of samples (Hillig 2004. "A chemotaxonomic analysis of terpenoid variation in Cannabis" Biochem System and Ecology 875- 891). It also provides a unique and complex flavor smell, and effect profile for each variety that is appreciated by both novice users and connoisseurs. In addition to many circulatory and muscular effects, some terpenes interact with neurological receptors. A few terpenes produced by cannabis plants also bind weakly to Cannabinoid receptors. Some terpenes can alter the permeability of cell membranes and allow in either more or less THC, while other terpenes can affect serotonin and dopamine chemistry as neurotransmitters. Terpenoids are lipophilic, and can interact with lipid membranes, ion channels, a variety of different receptors (including both G-protein coupled odorant and neurotransmitter receptors), and enzymes. Some are capable of absorption through human skin and passing the blood brain barrier.

[00115] Cannabis is an annual, dioecious, flowering herb. The leaves are palmately compound or digitate, with serrate leaflets. Cannabis normally has imperfect flowers, with staminate "male" and pistillate "female" flowers occurring on separate plants. It is not unusual, however, for individual plants to separately bear both male and female flowers (i.e., have monoecious plants). Although monoecious plants are often referred to as "hermaphrodites," true hermaphrodites (which are less common in cannabis) bear staminate and pistillate structures on individual flowers, whereas monoecious plants bear male and female flowers at different locations on the same plant.

[00116] The life cy cle of cannabis varies with each variety but can be generally summarized into germination (or rooting/recovery after asexual propagation), vegetative growth, and reproductive stages. Because of heavy breeding and selection by humans, most cannabis seeds have lost dormancy mechanisms and do not require any pre-treatments or winterization to induce germination (See Clarke, RC et al. "Cannabis: Evolution and Ethnobotany" University of California Press 2013). Seeds placed in viable growth conditions are expected to germinate in about 3 to 7 days. The first true leaves of a cannabis plant contain a single leaflet, with subsequent leaves developing in opposite formation. In some embodiments, subsequent leaves develop with increasing number of leaflets. Leaflets can be narrow or broad depending on the morphology of the plant grown. Cannabis plants are normally allowed to grow vegetatively for the first 4 to 8 weeks. During this period, the plant responds to increasing light with faster and faster growth. Under ideal conditions, cannabis plants can grow up to 2.5 inches a day, and are capable of reaching heights of up to 20 feet. Indoor growth pruning techniques tend to limit cannabis size through careful pruning of apical or side shoots.

[00117] Although, some cannabis varieties will flower without the need for external stimuli, most varieties have an absolute requirement for inductive photoperiods in the form of short days or long nights to induce fertile flowering. The first sign of flowering in cannabis is the appearance of undifferentiated flower primordial along the main stem of the nodes. At this stage, the sex of the plants are still not distinguishable. As the flower primordia continue to develop, female (pistillate), and male (staminate) flowers can be distinguished. [00118] For most cannabinoid producing purposes, only female plants are desired. The presence of male flowers is considered undesirable as pollination is known to reduce the cannabinoid yield, and potentially ruin a crop. For this reason, most cannabis is grown "sinsemilla" through vegetative (i.e., asexual) propagation. In this way, only female plants are produced and no space is wasted on male plants.

[00119] Commercial production of these medicinal and recreational cannabis varieties however, has been slowed down by the lack of true-breeding psychoactive genetics. Indeed, most popular cannabis strains in the market do not have fixed genetics, and are unable to produce uniform progeny when propagated through seeds. Modern cannabis production techniques thus rely on asexual cuttings of single cannabis "mother" plants to produce uniform crops of genetically- identical plants. Current asexual reproduction techniques however, still represent a major bottleneck in cannabis production yields. Improper techniques and incorrect hormones and nutrient formulations result in low propagation yields, and slow rooting and recovery of successful clones. Although asexual reproduction of cannabis is somewhat easily performed, its inherent constraints of time, space and resources severely limits the total number of plants that can be produced in large scale commercial operations. Furthermore, asexual reproduction of cannabis is constantly plagued by a host of other problems, including, but not limited to, abiotic disorders (e.g., nutrition, light quality and quantity, water availability, etc.); pathogens (e.g., Powdeiy Mildew and Pythium root rots); mites (e.g., two spotted spider mites and hemp russet mite); apliids (e.g., rice root aphid and hop aphid); white flies; viruses (e.g., Tobacco Mosaic Virus) and fungus gnats.

[00120] The present disclosure generally relates to compositions, systems, and methods for cannabis tissue culture and the Cannabis cells, calli, tissues, plant parts and whole plants produced and/or regenerated from such tissue culture. The disclosures of the present invention circumvent many of the problems associated with the asexual reproduction of cannabis.

[00121] This disclosure describes, inter alia, compositions, systems and methods for producing and maintaining Cannabis cell explants, Cannabis tissue explants, Cannabis cell cultures, isolated Cannabis cell cultures, Cannabis tissue cultures, isolated Cannabis tissue cultures. Cannabis callus, and isolated Cannabis callus. In some embodiments, the compositions, systems and methods of the present invention are used to produce clones of Cannabis plants, genotypes, strains, and/or varieties. This can be accomplished, e.g., via regeneration of whole plants from the Cannabis tissue cultures produced according to the present invention. [00122] The compositions, systems and methods of the present invention can be used for the tissue culturing and plant regeneration of any Cannabis germplasm. With twenty-six states and the District of Columbia in the United States legalizing marijuana in some form (i.e., for medical and/or recreational use), Cannabis germplasms, strains, varieties and/or lines are publicly and commercially available. U.S. Patent No. 6,630,507 issued on October 7, 2003 and assigned on the patent face to The United States of America, is directed to methods of treating diseases caused by oxidative stress by administering therapeutically effective amounts of a cannabidiol (CBD) cannabinoid from cannabis plants that has substantially no binding to the N-methyl-D- aspartate (NMD A.) receptor, wherein the CBD acts as an antioxidant and neuroprotectant. A search of the U.S.P.T.O Patent Application Information Retrieval (PAIR) system also reveals the existence of thousands of cannabis-related applications and issued patents including US 8,034,843 (use of cannabinoids for treating nausea, vomiting, emesis, motion sickness), US 7,698,594 (cannabinoid compositions for treatment of pam), and US 8,632,825 (anti-tumoural effects of cannabinoid combinations) among many others. Some examples of publicly-disclosed Cannabis germplasms, strains, varieties and/or lines each of which produce different amounts and/or ratios of cannabis metabolites can be found, e.g., in U.S. Patent Nos. 9,095,554; 9,370,164; and 9,642,317; and U.S. Published Patent Application Nos. 20110098348; 20140287068; 20160324091; and 20160360721, each of which is specifically incorporated by reference herein in its entireties, including ail of the tables and figures. Specific strains of cannabis are disclosed in U.S. Published Patent Application Nos. 20140245494 and 20160073567 ('Cannabis Plant Named Erez'); 20140245495 20160073568 ('Cannabis Plant Named Midnight'); 20140259228 and 20160073566 ('Cannabis Plant Named Avidekel'); 20160000843 ('High Cannabmol Cannabis Strains'); 20160345477 ('Cannabis Plant Named Ecuadorian Sativa'); and 20170172040 ('Cannabis Plant Named Katelyn Faith').

[00123] In some embodiments, any medium, or combinations thereof, of the present disclosure may be utilized in cannabis cultivation.

Media

[00124] The present invention provides media comprising compounds with unique types, concentrations, and combinations. In some embodiments, the medium is a liquid, semi-liquid, solid or semi-solid medium. [00125] In some embodiments, liquid cultures offer several advantages. The liquid cultivation sa ves time, because it enables replacement of the full medium in the vessel containing multiple explants be made at once, instead of individual transfers of single plant. In addition, a liquid culture results in increased shoot length because a larger area of the explant can get in contact with the medium.

[00126] The present invention provides media used for in vitro micropropagation of plants, such as bamboo plants and agricultural plants. Media useful for the production of perennials, grasses and phyto-pharmaceutical plants, is also provided herein.

[00127] Medium and methods used for plant micropropagation have been described at least in M, R. Ahuja, Micropropagation of woody plants, Springer, 1993, ISBN 0792318072, 9780792318071 ; Narayanaswamy, Plant cell and tissue culture, Tata McGraw-Hill Education, 1994, ISBN 0074602772, 9780074602775; Singh and Kumar, Plant Tissue Culture, APH Publishing, 2009, ISBN 8131304396, 9788131304396; Y.P.S. Bajaj High-tech and micropropagation V, Springer, 1997, ISBN 3540616063, 9783540616061 ; Tngiano and Gray, Plant Tissue Culture, Development and Biotechnology, CRC Press, 2010, ISBN 1420083260, 9781420083262; Gupta and Ibaraki, Plant tissue culture engineering Volume 6 of Focus on biotechnology, Springer, 2006, ISBN 1402035942, 9781402035944; Jam and Ishii, Micropropagation of woody trees and fruits Volume 75 of Forestry sciences, Springer, 2003, ISBN 1402011350, 9781402011351 ; and Aitken-Christie et al., Automation and environmental control in plant tissue culture, Springer, 1995, ISBN 0792328418, 9780792328414, each of which is incorporated herein by reference in its entirety.

[00128] Medium and methods for bamboo micropropagation have been described in International Patent Application Publication No. WO2011100762, which is incorporated herein by reference in its entirety.

[00129] The physical state of the media can vary by the incorporation of one or more gelling agents. Any gelling agent known in the art that is suitable for use in plant tissue culture media can be used. Agar is most commonly used for this purpose. Examples of such agars include Agar Type A, E or M and Bacto™ Agar. Other exemplary gelling agents include carrageenan, gellan gum (commercially available as PhytaGel™, Gelrite® and Gelzan™), alginic acid and its salts, and agarose. Blends of these agents, such as two or more of agar, carrageenan, gellan gum, agarose and alginic acid or a salt thereof also can be used. In some embodiments, no gelling agent or very little gelling agent is used for a liquid medium.

[00130] In some embodiments, the media comprise one or more minimum nutrition necessaiy for plant growth, such as amino acids, macroelements, microelements, aluminum, boron, chlorine (chloride), chromium, cobalt, copper, iodine, iron, lead, magnesium, manganese, molybdenum, nitrogen (nitrates), potassium, phosphorous (phosphates), silicon, sodium, sulphur (sulphates), titanium, vanadium, zinc, inositol and undefined media components such as casein hydrolysat.es or yeast extracts. For example, the media can include any combination of NH4NO3; KNO3; Ca(N03)2; K2SO4; MgSO.i; MnSO.i; ZnSC ; 2SO5; CuSO,. CaC3₂; I; C0CI2; H3BO3; Na?.Mo04; KH2PO4; FeSCU; Na?.EDTA; Na2H_P04; inositol (e.g., myo-inositol); thiamine; pyridoxine; nicotinic acid; glycine; and riboflavin. It is known to those in the art that one or more components mentioned above can be omitted without affecting the function of the media.

[00131] The media can comprise one or more carbon source, such as a sugar. Non-limiting exemplary sugars include sucrose, glucose, maltose, galactose and sorbitol or combinations thereof.

[00132] In some embodiments, the media can comprise inorganic salts, growth regulators, carbon source, and/or vitamins. In some embodiments, at least one of the vitamins is provided by the Murashige and Skoog medium salts (Murashige and Skoog, 1962), Woody Plant (WPM) tissue culture salts, Driver Kuniyuki Walnut (DKW) tissue culture, and/or functional variations thereof.

[00133] The media further comprise one or more effective amount of plant growth regulators (PGRs). Examples of plant growth regulators include plant hormones, such as auxms and compounds with auxin-like activity, cytokinins and compounds with cytokimn-like activity. The term "cytokinin" refers to a class of plant growth regulators that are characterized by their ability to stimulate cell division and shoot organogenesis in tissue culture. Non-limiting examples of cytokinins include, N⁶ - benzyiaminopurine (BAP) (a.k.a. N ⁶ -benzyladenine (BA)), meta- topolin, zeatin, kinetin, thiadiazuron (TDZ), meta-topolin, 2-isopentenyladenine (a.k.a., 6-γ-γ- (dimethylallylamino)-purine or 2ip), adenine hemisulfate, dimethylallyladenine, 4-CPPU (N-(2- chioro-4-py ridy 1)-N' - phenylurea)), and analogs thereof. The term "auxin" refers to a class of plant growth regulators that are characterized principally by their capacity to stimulate cell division in excised plant tissues, in addition to their role in cell division and cell elongation, auxins influence other developmental processes, including root initiation. Non-limiting examples of β- naphthoxyacetic acid (NAA), 2,4- Dichlorophenoxy acetic acid (2,4-D), indole-3 -butyric acid (IBA), indole-3 -acetic acid (LAA), picloram, and analogs thereof. More cytokinins and auxins are described in WO2011100762, US5211738, US20100240537, US20060084577, US20030158043, and Aremu et al,, 201 1 , which are incorporated by reference in their entireties, in some embodiments, the cytokinin is BAP or any functional variant thereof. In some embodiments, the auxin is IAA or any functional variant thereof.

[00134] In some embodiments, other plant growth regulators can be added in the media to improve cell growth and development. In some embodiments, growth inhibitors and/or growth retardants are used.

[00135] Non-limiting examples of growth inhibitors include, abscisic acid, ancymidol, butralin, carbaryl, chlorphonium, chlorpropham, dikegulac, flumetrafm, fluoridamid, fosamine, glyphosme, isopyrimol, jasmonic acid, maleic hydrazide, mepiquat, piproctanyl, prohydrojasmon, propham, tiaojiean, and 2,3,5-tri-iodobenzoic acid, derivatives thereof, or combinations thereof.

[00136] Non-limiting examples of growth retardant include, ancymidol (e.g., A-Rest®, Abide®), chlormequat chloride (e.g., Chlormequat E-Pro®, Citadel®, Cycocel®), daminozide (e.g., B-Nine®, Dazide®), ethephon (e.g., Florel®), flurprimidol (e.g., Topflor®), paclobutrazol (e.g., Bonzi®, Downsize®, Paczoi®, Piccolo®), mefluidide, paclobutrazol, tetcyclacis and uniconazole (e.g., Concise®, Sumagic®). In some embodiments, the growth retardant is an gibbereliic acid (GA3) antagonist which can inhibit GA3 pathway, for example, ancymidol, tannins, paclobutrazol (PBZ), (2-Chloroethyl) trimethylammonium chloride, abscisin, exogenous ABA, derivatives thereof, or combinations thereof.

[00137] Exemplary concentrations of the components described above are shown in Table

1. The concentrations of these components can be adjusted based on plant species, tissue type, and purposes, etc, without substantially affecting the media function. The exemplary concentrations are by no means limiting, and merely encompass some of the embodiments. In some embodiments, the media formulations of tables 2-8 may be modified within the scope of the component concentrations disclosed in Table .

[00138] Table 1: Exemplary Concentrations Component Concentrations

(mg/L i all unless otherwise noted)

NH NO3 about 800-about 2500

KNO3 about 900-about 3000

Ca(N()₃)2 0-about 800

K2SO4 0-about 800

MgS()4 about 1 50-about 550

nS04 about 8.0-about 26.0

ZnS(>4 about 4.0-about 12.0

CtsS04 about 0.010-about 0.4

CaCh about 200-about 660

KI about 0.4-about 1.5

Coch about 0.010-about 0.4

H3B03 about 3.0-about 9.0

Na2Mo()₄ about 0.10-about 0.4

KH2PO4 about 80-about 250

FeS04 about 25-about 90

Na₂EDTA about 35-about 120

Na₂H₂P04 about 0-250 / about 80-250 myo-Inositol about 50-about 150

Thiamine about 0.2-about 0.6

Pyridoxine about 0.1 -about 10

Nicotinic acid about 0.1 -about 10

Sugar about 10 g/L -about 1 00 g/L

Glycine about 0 - about 5

Riboflavin about 0 - about 5

Ascorbic Acid about 0 - about 5

Gelling agent* about 2.5 g/L -about 8.0 g/L

*The amount of gelling agent may vary depending on the type of the ageni, and the type of the media (e.g., semi-solid or solid media) [00139] As used herein and in the claims, where the term "about" is used with a numerical value, the numerical value may vary from the explicit number; the variation will be ±10%.

[00140] Optionally, the media further comprise one or more buffering agent. The buffering agent can buffer the salt concentration and/or the pH in the medium. For example, the buffering agent can maintain the pH of the liquid mixture so the pH is kept around about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5 about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0. For example, the pH of the liquid mixture is in a range between about 5.0 to about 7.0. In some embodiments, the buffering agent is 2-(N-morpholino)ethanesulfonic acid (MES), Adenosine deaminase (ADA), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), N-(2- Acetamido)-2-aminoethanesulfonic acid (ACES), Cholamine chloride, etc. In some embodiments, the buffering agent is MES, and its concentration is about 500-1200 mg/L. In some embodiments, the pH of the medium is maintained at about 5.5 to 6.5, for example, about 5.8. In some embodiments, the pH of the medium is maintained at about 5.0 to 6.0, for example, about 5.7.

[00141] If present in a media, each cytokinin can be present in an amount from about 0.001 mg L-about 10 mg/L and all amounts in between. For example, the concentration of a cytokmin is about 0.001, 0.01, 0.1, 1, 2, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99. or about 100 mg/L. In some embodiment, at least one cytokinin is meta-topolin, and its concentration is about 0.2 to 20 mg/L, for example, about 2 mg/L. In some embodiment, at least one cytokinin is 2ip, and its concentration is about 1 to 10 mg/L, for example, about 4-5 mg/L.

[00142] In some embodiments, the media comprise meta-topolin and/or its analogues. In some embodiments, meta-topolin is present in an amount equal to or greater than 1.5 mg/L, equal to or greater than 2.0 mg/L, equal to or greater than 2.5 mg/L, equal to or greater than 3.0 mg/L, equal to or greater than 3.5 mg/L, equal to or greater than 4.5 mg/L or equal to or greater than 5.0 mg/L. In other embodiments, meta-topolin is present in an amount of 3.2 mg/L or 5.36 mg/L. In another embodiment, the amount of meta-topolin cannot be less than 1.5 mg/1,, cannot be less than 2.0 mg/L, cannot be less than 2.5 mg L, cannot be less than 3.0 mg L, cannot be less than 3.5 mg/L, cannot be less than 4.5 mg/L or cannot be less than 5.0 mg/L. In some embodiments, meta-topolin and/or its analogues can be included in any amount up to 200 mg/L. In some embodiments, the media is used for bamboo micropropagation. In some embodiments, the bamboo plant is selected from the species consisting of Arundinana, Bambusa, Borinda, chusquea, Dendrocalamus, fargesia, Guadua, Phyllostachys, Pleioblastus and Thamnocalamus.

[00143] In some embodiments, the media comprise thidiazuron and/or its analogues. In some embodiments, thidiazuron and/or its analogues can be present at 0.001 mg/L, 0.01, 0.025, 0.05, 0,075, 0, 1 , 0.15, 0.2, 0.25, 0.3, 0,35, 0.4, 0.45, 0.5, 0.55, 0.6, 0,65, 0.7, 0.75, 0.8, 0.85, 0,9, 0.95, 1.0, 1.25, 1.50, 1.75, 2.0, 2.25, 2.5, 2.75, 3.5, 4.5, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 mg/L. In some embodiments, thidiazuron and or its analogues can also be included in any amount up to 200 mg/L.

[00144] If present in a media, each auxm can be present in an amount from about 0.01 mg/L- about 100 mg L and all amounts in between. For example, the concentration of an auxm is about 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.5, 1, 2, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or about 100 mg/L, In some embodiments, at least one auxin is IAA, and the concentration is about 0.1 to 10 mg/L, for example, about 1 mg/L. In some embodiments, at least one auxin is NAA, and the concentration is about 0.001 to I mg/L, for example, about 0.02 mg/L.

[00145] In some embodiments, at least one auxin is NAA, IBA or combination thereof. In some embodiments, at least one auxin is IBA. In some embodiments, NAA or IBA is presented in an initiation medium or a multiplication medium, and the concentration is about 0.01 - 10 mg/L, for example, about 0.02-1 mg/L. In some embodiments, NAA or IBA is presented in a rooting medium. In some embodiments, the NAA/IBA concentration in a rooting medium is about 1 to 10 mg/L, for example, about 1-3 mg/L. In some embodiments, the NAA IBA concentration in a rooting medium is about 100-1500 mg/L, for example, about 250-1000 mg/L,

[00146] In some embodiments, the present invention provides several types of media that are used in in vitro micropropagation of plants of the present disclosure.

[00147] The first type of media, referred herein as the initiation media, is similar to or essentially the same as the Murashige and Skoog medium (Murashige and Skoog, 1962), Wood - Plant (WPM) tissue culture salts, Driver Kuniyuki Walnut (DKW), but comprise at least one auxin and/or at least one cytokrain. In some embodiments, the sucrose concentration in the propagation and multiplication media is about 20-40 g/L or higher. In some embodiments, at least one auxin is ml In some embodiments, at least one cytokinin is NAA. In some embodiments, the media comprise at least one gelling agent such as agar. In some embodiments, the concentration of the gelling agent is about 4 to about 10 grams, for example, about 7 grams. In some embodiments, the initiation media do not contain NaaLLPCk In some embodiments, the initiation media do not contain pyridoxine, nicotinic acid, and/or riboflavin.

[00148] The second type of media, referred herein as the micropropagation media or multiplication media, are as the same as, or similar to the initiation media. In some embodiments, the sucrose concentration in the propagation and multiplication media is about 20-40 g/L or higher. In some embodiments, at least one auxin is m'T. In some embodiments, at least one cvtokinm is IBA. In some embodiments, the media comprise at least one gelling agent such as agar. In some embodiments, the media comprise at least one gelling agent such as agar. In some embodiments, the concentration of the gelling agent is about 4 to about 10 grams, for example, about 5 grams.

[00149] In some embodiments, the initiation medium and/or the multiplication medium comprise MS medium containing double concentration of meso elements (e.g., one or more of CaC12.2H20, MgS04.7H20, and KH2P04), double iron, and one or more Gamborg's vitamins (e.g., one or more of

Nictotinic acid, pyridoxine salts, and thiamine salts).

[00150] The third type of media, referred herein as the rooting media, are similar to or essentially the same as the Murashige and Skoog medium, Woody Plant (WPM) tissue culture salts, Driver Kuniyuki Walnut (DKW), but comprise at least one auxin. In some embodiments, the auxin is IBA. In some embodiments, the media do not comprise any cytokinin. In some embodiments, the sucrose concentration in the propagation and multiplication media is about 20- 40 g/L or at least 60 g/L, In some embodiments, the concentration of IB A is about 0, 1 to 10 mg L. In some embodiments, the concentration of IB A is about 100 to 1500 mg L. In some embodiments, the concentration of the gelling agent is about 4 to about 10 grams, for example, about 6 grams.

[00151] In some embodiments, the micropropagation and multiplication media (or elongation and multiplication media), is similar to or essentially the same as the Murashige and Skoog medium (Murashige and Skoog, 1962), but without any hormone or growth regulator. In some embodiments, the sucrose concentration in the propagation and multiplication media is about

[00152] In some embodiments, the pre-tuberization media, comprises one or more cytokinins and one or more auxin. In some embodiments, the cytokinin is 2ip and the auxin is LAA. Alternatively, instead of cytokinin and auxin, the pre-tuberization media comprises one or more plant retardant in low amount, such as ancymidol or analog thereof. In some embodiments, the pre-tuberization media comprise one or more cytokinin, one or more auxin, and one or more growth retardant. In some embodiments, the concentration of 2ip is about 1 to 10 mg/L, for example, about 4-5 mg/L. In some embodiments, the concentration of IAA is about 0.1 to 10 mg/L, for example, about 1 mg L. In some embodiments, the concentration of ancymidol is about 0.1 to 10 mg/L. In any case, the sucrose concentration is about 20 g/L to about 40 g/L, for example, about 30 g/L.

[00153] In some embodiments, the tuberization media, comprises one or more auxin, but does not comprise any cytokinin. Alternatively, instead of auxin, the tuberization media comprises one or more plant retardant, such as ancymidol or analog thereof. In some embodiments, the tuberization media comprise one or more auxin and one or more growth retardant. In some embodiments, the auxin is NAA. In some embodiments, the NAA concentration is about 0.01 to about 0.05 mg/L. In some embodiments, the concentration of ancymidol is about 0.1 to 10 mg/L. In any case, the sucrose concentration in the media is higher compared to the sucrose concentration in the pre-tuberization media. For examples, the sucrose concentration in the tuberization media is about 50 g/L to about 1 OOg/L or more.

[00154] In some embodiments, the media comprise NAA and/or its analogues. In some embodiments, NAA and/or its analogues can be present at 0.001 mg/L, 0.01 , 0.0125, 0.015, 0.0175, 0.02, 0,0225, 0.025, 0.0275, 0,03, 0.0325, 0.035, 0,0375, 0.04, 0.0425, 0.045, 0.0475, 0,05, 0.0525, 0.055, 0.0575, 0.06, 0.0625, 0,065, 0.0675, 0,07, 0.0725, 0,075, 0.0775, 0,08, 0.0825, 0.085, 0,0875, 0.09, 0.0925, 0,095, 0.0957, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0,45, 0.5, 0.55, 0,6, 0.65, 0.7, 0,75, 0.8, 0.85, 0,9, 0.95, 1.0, 1 ,25, 1.50, 1.75, 2,0, 2.25, 2.5, 2.75, 3.5, 4.5, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100 mg/L, in some embodiments, NAA and/or its analogues can be included any amount up to 200 mg/L.

[00155] In some embodiments, the media comprise IBA and/or its analogues. In some embodiments, IBA and/or its analogues can be present at 0.001 mg/L, 0.01 , 0.025, 0.05, 0.075, 0.08, 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1.0, 1.25, 1.50, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.5, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 1 5, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 mg/L. In some embodiments, IBA and/or its analogues can be included any amount up to 200 mg/L.

[00156] In some embodiments, the media comprise BAP and/or its analogues. In some embodiments, BAP and/or its analogues can be present at 0.001 mg/L, 0.01, 0.025, 0.05, 0.06, 0.07, 0.0725, 0.075, 0.0775, 0.08, 0.0825, 0.085, 0.0875, 0.09, 0.0925, 0.095, 0.0975, 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0,575, 0.6, 0.625, 0.65, 0,675, 0.7, 0.75, 0.8, 0,85, 0.9, 0.95, 1.0, 1 ,25, 1.50, 1.75, 2.0, 2,25, 2.5, 2.75, 3.5, 4.5, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 mg L. In some embodiments, BAP and/or its analogues can be included any amount up to 200 mg/L.

[00157] In some embodiments, the media comprise 2ip and/or its analogues. In some embodiments, 2ip and/or its analogues can be present at 0.001 mg/L, 0.01 , 0.025, 0.05, 0.075, 0.08, 0.1, 0.15, 0,2, 0.25, 0.3, 0.35, 0.4, 0,45, 0.5, 0.55, 0.6, 0.65, 0,7, 0.75, 0,8, 0.85, 0.9, 0.95, 1.0, 1,25, 1.50, 1.75, 2,0, 2.25, 2,5, 2.75, 3.0, 3,5, 4,0, 4,5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 mg/L. In some embodiments, 2ip and/or its analogues can be included any amount up to 200 mg/L.

[00158] In some embodiments, the media comprise DPU and/or its analogues. In some embodiments, DPU and/or its analogues can be present at 0,001 mg/L, 0.01, 0,025, 0,05, 0,075, 0.1, 0,15, 0.2, 0,25, 0.3, 0.35, 0.4, 0.45, 0,5, 0.55, 0.6, 0.65, 0.7, 0,75, 0.8, 0.85, 0.9, 0.95, 1,0, 1.25, 1.50, 1,75, 2.0, 2.25, 2,5, 2.75, 3.5, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 mg/L. In some embodiments, DPU and/or its analogues can be included any amount up to

|00159] In some embodiments, the media comprise CPPU and/or its analogues. In some embodiments, CPPU and/or its analogues can be present at 0,001 mg/L, 0.01, 0.025, 0.05, 0,075, 0.1, 0.15, 0.2, 0,25, 0.3, 0.35, 0.4, 0.45, 0,5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.25, 1.50, 1,75, 2.0, 2.25, 2.5, 2.75, 3.5, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 mg/L. In some embodiments, CPPU and/or its analogues can be included any amount up to 200 mg/L,

[ΟΟίόθ] In some embodiments, one or more cytokmins in combination with one or more other cytokmins or auxins, and auxins in combination with other auxins or cytokinins can also be utilized in ratios. For example, in some embodiments, any two cytokinins and/or auxins in pairs disclosed herein can be included in the following exemplary ratios: 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1,21:1,20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1; 9:1, 8:1, 7:1, 6.9:1, 6.8:1, 6.7:1,6.6:1,6.5:1,6.4: 1, 6.3:1, 6.2:1, 6,1:1, 6:1, 5.9:1, 5.8:1, 5.7:1, 5.6:1, 5,5:1, 5,4:1, 5.3:1, 5.2:1, 5.1 : 1, 5: 1; 4: 1 , 3: 1, 2: 1, 1 : 1 , 0.75: 1, 0.5: 1, 0.25: 1 , 0, 1 : 1, 0.075: 1, 0.05: 1, 0.025: 1 or 0,001 : 1. These ratios can also be utilized between meta-topolin (and analogues) with thidiazuron (and analogues), with NAA (and analogues), with BAP (and analogues), with IBA (and analogues), with 2ip (and analogues), with DPU (and analogues) and/or with CPPU (and analogues). Similarly, the ratios can be utilized between thidiazuron (and analogues) with NAA (and analogues), with BAP (and analogues), with IBA (and analogues), with 2ip (and analogues), with DPU (and analogues) and/or with CPPU (and analogues). The ratios can also be utilized between NAA (and analogues) with BAP (and analogues), with IBA (and analogues), with 2ip (and analogues), with DPU (and analogues) and/or with CPPU (and analogues). The ratios can also be utilized between BAP (and analogues) with IBA (and analogues), with 2ip (and analogues), with DPU (and analogues) and/or with CPPU (and analogues). In short, each of the cytokinins and/or auxins or its analogues can be included with a second cytokinm and/or auxin disclosed herein according to any of the disclosed ratios.

[00161] In some embodiments, the present invention utilizes a sterility test medium

(BOO101). Preparing BOO101 in liquid tubes requires 45.22 grams of L & W medium in lL for 160 tubes. In some embodiments, the solid sterility test media is prepared with 45.22 grams in 1L with 6g carrageenan in 40 sterile disposable Petri dishes.

Table 2

Table 3

Table 4

Table 5

Table 6

Table 7

Table 8

Component B00132 BOO 133 B00134 BOO 135 B00136 B00137

(IL) (IL) (IL) (IL) (IL) (IL)

Charcoal 0.15g 0.03g

Rifampicin 0.05g 0.05g 0.05g 0.05g pH 5.75 5,75 5.7 6.5 6,0 5.7 00162J In one embodiment, the numbers listed without units in the media formulations of tables 2-8 represent a concentration of milligrams/liter. In another embodiment, the numbers listed without units in the media formulations of tables 2-8 represent a concentration of micrograms/liter.

[00163] In some embodiments, the present invention provides different types of media that are useful in the production of perennials, grasses and phyto-pharmaceutical plants. In some embodiments, the medium useful for producing perennials, grasses and phyto-pharmaceutical plants is a liquid medium. In some embodiments, the medium is a solid medium.

[00164] The media useful for the production of perennials, grasses, and phtyo- pharmaceutieai plants can be any one of the media described in the above tables. In one embodiment, the media is selected from any of the above tables. In one embodiment, the media is Pulsing media I or Pulsing media 2. In another embodiment, the media is Pulsing media 1 , wherein TDZ is substituted with a different cytokinin, such as BAP, Zeatin, CPPU, or DPU.

[00165] In some embodiments, a combination of the media described in the aforementioned tables are useful for the production of any variety of plants, including perennials, grasses, and phyto-pharmaceutical plants. The combination of media can be used sequentially. In one embodiment, the combination comprises Pulsing media 1, Pulsmg media 2 or both; and BOOl 01, BOO102, BOO103, BOOl 04, BOO105, BOO106, BOOl 07, BOO108, BOOl 09, BOOl 10, BOOl l l, B00112, BOOl 13, B00114, BOOl 15, BOOl 16, B00117, B00118, BOOl 19, BOO 120, BOOl 21 , BOO 122, BOOl 23, BOO 124, BOO 125, BOOl 26, BOO 127, BOO 128, BOOl 29, BOO130, B00131, B00132, B00133, B00134, B00135, B00136, and B00137. In some embodiments, the Pulsing media 1 in the combination is Pulsing media 1 , wherein TDZ is substituted with a different cytokinin, such as BAP, Zeatin, CPPU, or DPU.

[00166] In some embodiments, amelanchier is grown in BOO102, broccoli is grown in

BOO103, cabbage is pre-rooted in BOO 104, cabbage is rooted in BOO105, cabbage is grown or suspended in BOO106, cauliflower is grown in BOO107, Dicentra is grown in BOO108, Dicentra is grown in BOO 109, Dicentra is grown in BOOl 10, Dicentra is grown in BOOl 11, Dicentra is grown in BOO! 12, Dicentra is grown in BOOl 13, Dicentra is grown in B00114, Dicentra is grown in B00115, Dicentra is grown in BOO 116, Dicentra is grown in BOOl 17, echinacea is grown in BOOl 18, echinacia is grown/rooted in BOO 119, echinacia is grown/rooted in BOO 120, eehmacia is grown/rooted in B00121, gypsophilia is grown/rooted in B00122, gypsophilia is grown/rooted in BOOl 23, Gaillaudin is grown in BOOl 24, lily is grown in BOOl 25, lily is grown in BOOl 26, lily is grown in BOOl 27, Miscanthus is grown in BOOl 28, Miscanthus is grown in BOOl 29, phlox is grown in BOO130, peonie is grown in B00131, peonie is grown in BOO 132, peonie is grown in B00133, peonie is is rooted/grown in B00134, gypsum is grown/rooted in BOOl 35, rose is grown/rooted in B00136, and wasahi is grown in B00137

[00167] In some embodiments, the present invention provides different types of media that are useful in the production of virus-free plants, such as agricultural plants. The media useful for the production of virus-free plants can comprise an antiviral. The antiviral can be acemannan, acyclovir, adefovir, alovudine, alvircept, amantadine, aranotin, arildone, atevirdine, pyridine, cidofovir, cipamfylline, cytarabme, desciclovir, disoxaril, edoxudme, enviradene, enviroxime, famdclovir, famotine, fiacitabine, fialuridine, floxuridine, fosarilate, fosfonet, ganciclovir, idoxuridme, kethoxal, lobucavir, memotine, methisazone, penciclovir, pirodavir, somantadine, sorivudine, tilorone, trifiuridme, valaciclovir, vidarabine, viroxime, zinviroxime, moroxydme, podophyliotoxm, ribavirine, rimantadine, staliimycine, statoion, tromantadine and xenazoic acid, and their pharmaceutically acceptable salts. In one embodiment, the antiviral is ribavirine (also known as Virazole) or derivatives thereof, such as viramidine (also known as Taribavirin). In some embodiments, a media useful for the production of virus-free plants comprises one or more antivirals.

[00168] In other embodiments, the present invention provides at least two types of media that are used in in vitro micropropagation. The first media, referred herein as the "bud induction media", comprises at least one strong cytokinin, such as a thidiazuron, or analogs thereof. The second media, referenced herein as the "shoot elongation/maintenance media", comprises one or more cytokinins other than the cytokinin in the bud induction media. For example, the cytokinins are selected from meta-topolin, kinetin, isopentenyl adenine (iP), zeatin, trans-zeatin, zeatin riboside, dihydrozeatin, benzyleadenin (BAP), benzyladenosine ([9RJBAP), analogs thereof, or combination thereof. Other cytokinins available for use in tissue culture can also be substituted for the above cytokinins to achieve similar effects. [00169] In some embodiments, the bud induction medium is a liquid medium. In some embodiments, the bud induction medium is a solid medium. In some embodiments, the shoot elongation/maintenance medium is a liquid medium. In some embodiments, the shoot elongation/maintenance media is a solid media.

[00170] The bud induction media, the shoot elongation/maintenance media, and media useful for producing perennials, grasses and phy to-pharmaceutical plants comprise components of a minimum media for plant tissue culture, such as carbon source and salts. In some embodiments, the media can comprise one or more components selected from NH4NO3, KNO3, Ca(N0₃)2, K2SO4, MgS04, MnS()4, ZnS()₄, CuS()₄, K2SO5, ⁽ aCi :. Kl, C0CI2, H3BO3, Na₂Mo04, KH2PO4, FeS( Na2.EDTA, Na₂H₂P04, Glycine, myo-Inositol, Thiamine, Pyridoxine, Nicotinic acid, and Riboflavin.

[00171] In some embodiments, the media useful for producing perennials, grasses and phyto-pharmaceutical plants comprises only one strong cytokinin, for example, thidiazuron (TDZ), or an analog thereof. In some embodiments, the media comprises one additional cytokinin. In some embodiments, the media further comprises one or more auxin, such as NAA, 2,4-D, IBA, IAA, picloram, or analogs thereof. In other embodiments, the media useful for producing perennials, grasses and phyto-pharmaceutical plants does not comprise a cytokinin or auxin. In yet other embodiments, the media useful for producing perennials, grasses and phyto- pharmaceutical plants does not comprise a plant hormone.

[00172] In some embodiments, the concentration of the strong cytokinin (e.g., TDZ) in the media useful for producing perennials, grasses and phyto-pharmaceutical plants, is about 0.25 mg/L (± 10%) to about 100 mg/L (± 10%), for example, is about 0.2 mg/L (± 10%), about 0.5 mg/L (± 10%), about 1.0 mg L (± 10%), about 5 mg/L (± 10%), about 10 mg/L (± 10%), about 20 mg/L (± 10%), about 30 mg/L (± 0%), about 40 mg/L (± 10%), about 50 mg/L (± 10%), about 60 mg/L (± 10%), about 70 mg/L (± 10%), about 80 mg/L (± 10%), about 90 mg/L (± 10%), or about 100 mg/L (± 10%). For example, the concentration of TDZ is about 0.2 (± 10%) to about 20 (± 10%) mg/L, about 0.4 (± 10%) to about 10 (± 10%) mg L, or about 0.5 (± 10%) to about 2 (± 10%) mg/L.

[00173] In some embodiments, the concentration of TDZ or analog thereof in the media useful for producing perennials, grasses and phyto-pharmaceutical plants, is about 0.25 mg/L to about 100 mg/L, for example, about 0.5 mg/L to about 2 mg/L. Thus, the concentration of TDZ or analog thereof can, for example, he about 0.25 mg/L, about 0.3 mg L, about 0.4 mg L, about 0.5 mg/L, about 0.6 mg/L, about 0.7 mg/L, about 0.8 mg/L, about 0.9 mg/L, about 1.0 mg/L, about out 1.5 mg/L, about 2.0 mg/L, about 2.5 mg/L, about 3 mg/L, about 4 mg/L, about 5 mg/L, about 6 mg/L, about 7 mg/L, about 8 mg/L, about 9 mg/L, about 10 mg/L, about 15 mg/L, about 20 mg/L, about 25 mg/L, about 30 mg/L, about 40 mg/L, about 50 mg/L, about 60 mg/L, about 70 mg/L, about 80 mg/L, about 90 mg/L or about 100 mg/L.

[00174] In some embodiments, the bud induction media (BOOl) comprises only one strong cytokinin, for example, thidiazuron (TDZ), or an analog thereof. In some embodiments, the embryo induction media or bud induction media (BOOl ) comprises one additional cytokinin. In some embodiments, the embryo induction media or bud induction media (BOOl) further comprises one or more auxin, such as NAA, 2,4-D, IB A, IAA, picloram, or analogs thereof.

[00175] In some embodiments, the concentration of the strong cytokinin (e.g., TDZ) in the bud induction media (BOOl ) is about 0.25 mg/L (± 10%) to about 100 mg/L (± 10%), for example, is about 0.2 mg/L (± 10%), about 0.5 mg/L (± 10%), about 1.0 mg/L (± 10%), about 5 mg/L (± 10%), about 10 mg/L (± 10%), about 20 mg/L (± 10%), about 30 mg/L (± 10%), about 40 mg/L (± 0%), about 50 mg/L (± 10%), about 60 mg/L (± 10%), about 70 mg/L (± 10%), about 80 mg/L (± 10%), about 90 mg/L (± 10%), or about 100 mg/L (± 10%). For example, the concentration of TDZ is about 0.2 (± 10%) to about 20 (± 10%) mg/L, about 0.4 (± 10%) to about 10 (± 10%) mg/L, or about 0.5 (± 10%) to about 2 (± 10%) mg/L.

[00176] In some embodiments, the concentration of the strong cytokinin (e.g., TDZ) in the media useful for producing virus-free plants, such as in agricultural plants, is about 0.25 mg/L (± 10%) to about 100 mg/L (± 10%), for example, is about 0.2 mg/L (± 10%), about 0.5 mg/L (± 10%), about 1.0 mg/L (± 10%), about 5 mg/L (± 10%), about 10 mg/L (± 10%), about 20 mg/L (± 10%), about 30 mg/L (± 10%), about 40 mg/L (± 10%), about 50 mg/L (± 10%), about 60 mg/L (± 10%), about 70 mg L (± 10%), about 80 mg/L (± 10%), about 90 mg/L (± 10%), or about 100 mg/L (± 10%). For example, the concentration of TDZ is about 0.2 (± 10%) to about 20 (± 10%) mg/L, about 0.4 (± 10%) to about 10 (± 10%) mg L, or about 0,5 (± 10%) to about 2 (± 10%) mg/L.

[00177] In some embodiments, the concentration of TDZ or analog thereof in the media useful for producing virus-free plants, such as agricultural plants, is about 0.25 mg/L to about 100 mg/L, for example, about 0.5 mg/L to about 2 mg/L. Thus, the concentration of TDZ or analog thereof can, for example, be about 0,25 mg L, about 0.3 mg/L, about 0.4 mg/L, about 0.5 mg/L, about 0.6 mg/L, about 0.7 mg/L, about 0.8 mg/L, about 0.9 mg/L, about 1.0 mg/L, about out 1.5 mg/L, about 2.0 mg/L, about 2.5 mg/L, about 3 mg/L, about 4 mg/L, about 5 mg/L, about 6 mg/L, about 7 mg/L, about 8 mg/L, about 9 mg/L, about 10 mg/L, about 15 mg/L, about 20 mg/L, about 25 mg/L, about 30 mg/L, about 40 mg/L, about 50 mg/L, about 60 mg/L, about 70 mg/L, about 80 mg/L, about 90 mg/L or about 100 mg/L.

[00178] In some embodiments, the concentration of TDZ or analog thereof in the bud induction medium is effective to induce shoot buds. In some embodiments, the concentration of TDZ or analog in the bud induction media thereof is about 0.25 mg L to about 100 mg/L, for example, about 0.5 mg/L to about 2 mg/L. Thus, the concentration of TDZ or analog thereof in the bud induction media can, for example, be about 0.25 mg/L, about 0.3 mg/L, about 0.4 mg L, about 0.5 mg/L, about 0.6 mg/L, about 0.7 mg/L, about 0.8 mg L, about 0.9 mg/L, about 1.0 mg/L, about out 1.5 mg/L, about 2.0 mg/L, about 2.5 mg/L, about 3 mg/L, about 4 mg/L, about 5 mg L, about 6 mg/L, about 7 mg/L, about 8 mg/L, about 9 mg/L, about 10 mg/L, about 15 mg/L, about 20 mg/L, about 25 mg/L, about 30 mg/L, about 40 mg/L, about 50 mg/L, about 60 mg/L, about 70 mg/L, about 80 mg L, about 90 mg/L or about 100 mg/L.

[00179] In some embodiments, the bud induction medium and/or the shoot elongation/maintenance medium further comprise one or more auxins. In some embodiments, the auxins are selected from the group consisting of β-naphthoxyacetic acid (NAA), 2,4- Dichlorophenoxy acetic acid (2,4-D), indole- 3 -butyric acid (IBA), indole-3-acetic acid (LAA), picloram, and analogs of each thereof. For example, the auxin is NAA or analogs thereof.

[00180] In some embodiments, the concentration of the auxin in the bud induction media

(BOOl) is about 0.01 mg/L (± 10%) to about 10 mg/L (± 10%), for example, is about 0.01 mg/L (± 10%), about 0,05 mg/L (± 10%), about 0.1 mg L (± 10%), about 0.5 mg/L (± 10%), about 1 mg L (± 10%), about 5 mg/L (± 10%), or about 10 mg/L (± 10%).

[00181] In some embodiments, the shoot elongation/maintenance media comprises one or more cytokinins other than TDZ, such as meta-topolin, kinetin, isopentenyladenine (ip, e.g., 2ip), zeatin, trans-zeatin, zeatin riboside, dihydrozeatin, benzyleadenin (BAP), benzyladenosine

([9R]BAP), analogs thereof. In some embodiments, the shoot elongation/maintenance media further comprise one or more auxm, such as NAA, 2,4-D, IBA, IAA, picloram, or analogs thereof. [00182] In some embodiments, the concentration of cytokinin in the shoot elongation/maintenance media is about 0.01 mg/L (± 10%) to about 100 mg/L (± 10%), for example, is about 0.01 mg/L (± 10%), about 0.05 mg/L (± 10%), about 0.1 mg/L (± 10%), about 0.5 mg L (± 10%), about 1 mg L (± 10%), about 5 mg/L (± 10%), about 10 mg/L(± 10%), about 20 mg/L(± 10%), about 30 mg L(± 10%), about 40 mg/L(± 10%), about 50 mg/L(± 10%), about 60 mg/L(± 10%), about 70 mg/L(± 10%), about 80 mg/L(± 10%), about 90 mg/L(± 10%), or about 100 mg/L(± 10%). In some embodiments, the concentration of the cytokinin in the shoot elongation/maintenance media is about 0.01 (± 10%) to about 20 (± 10%) mg/L, about 0.1 (± 10%) to about 10 (± 10%) mg/L, or about 0,25 (± 10%) to about 5 (± 10%) mg/L.

[00183] In some embodiments, the concentration of the one or more cytokimns other than

TDZ or an analog thereof in the shoot elongation/maintenance medium is effective to elongate shoots. In some embodiments, the concentration of the one or more cytokinins other than TDZ or an analog thereof in the shoot elongation/maintenance medium from about 0.01 mg/L to about 100 mg/L, for example, from about 0.25 mg/L to about 5 mg/L. Thus, the concentration of one or more cytokinins other than TDZ or analog thereof in the shoot elongation/maintenance media can, for example, be about 0.01 mg/L, about 0.02 mg/L, about 0.03 mg/L, about 0.04 mg/L, about 0.05 mg/L, about 0.06 mg L, about 0.07 mg/L, about 0.08 mg/L, about 0.09 mg L, about 0.10 mg/L, about 0.15 mg/L, about 0.20 mg/L, about 0.25 mg/L, about 0.3 mg/L, about 0.35 mg/L, about 0.4 mg/L, about 0.45 mg/L, about 0.5 mg/L, about 0.6 mg/L, about 0.7 mg/L, about 0.8 mg/L, about 0.9 mg/L, about 1.0 mg/L, about 1.5 mg/L, about 2.0 mg/L, about 2.5 mg/L, about 3 mg/L, about 4 mg/L, about 5 mg/L, about 6 mg/L, about 7 mg/L, about 8 mg/L, about 9 mg L, about 10 mg/L, about 15 mg/L, about 20 mg/L, about 25 mg L, about 30 mg/L, about 40 mg/L, about 50 mg/L, about 60 mg/L, about 70 mg/L, about 80 mg/L, about 90 mg/L or about 100 mg/L.

[00184] In some embodiments, the concentration of the auxin in the bud induction media

(BOOl) is about 0.01 mg/L (± 10%) to about 50 mg/L (± 10%), for example, is about 0.01 mg/L (± 10%), about 0,05 mg/L (± 10%), about 0.1 mg/L (± 10%), about 0.5 mg/L (± 10%), about 1 mg/L (± 10%), about 5 mg/L (± 10%), about 10 mg/L(± 10%), about 20 mg/L(± 10%), about 30 mg/L(± 10%), about 40 mg/L(± 10%), or about 50 mg/L(± 10%). In some embodiments, the concentration of the auxin in the shoot elongation/maintenance media is about 0.01 (± 10%) to about 20 (± 10%) mg/L, about 0.02 (± 10%) to about 10 (± 10%) mg/L, or about 0,05 (± 10%) to about 5 (± 10%) mg/L, [00185] Non-limiting concentrations of the components in the bud induction media and shoot elongation/maintenance media are shown in Table 9. One or more components in table 9 can be omitted or replaced without affecting the function of the media. The concentration of each component can be adjusted without affecting the function of the media.

[00186] Table 9: Exemplary concentrations of hud induction media and shoot elongation and maintenance media.

[00187] In some embodiments, the bud induction media comprise thidiazuron (TDZ) or analog thereof, and the elongation and maintenance media comprise one or more cytokinins other than TDZ or an analog thereof. In some embodiments, the cytokinins other than TDZ are selected from the group consisting of N6-benzylaminopurine (BAP), meta-topolin (mT), zeatin, zeatin riboside, dihydrozeatin, kinetin, isopentenyladenine (ip, e.g., 2ip), adenine hemisulfate, dimethylallyladenine, N-(2-chloro-4-pyridyl)-N'- phenylurea) (4-CPPU), and analogs thereof. In some embodiments, the media can be used for plants in vitro micropropagation of monocots or dicots. In some embodiments, the media can be used for bamboo plants in vitro micropropagation.

[00188] In some embodiments, the bud induction medium comprises an effective amount of thidiazuron (TDZ) or analog thereof!, and wherein the shoot elongation/maintenance medium comprises an effective amount of one or more cytokinins other than TDZ or an analog thereof. In some embodiments, the one or more cytokinins other than TDZ or an analog thereof in the shoot elongation/maintenance medium is selected from the group consisting of N6-benzylaminopurine (BAP), meta-topolin (mT), zeatin, zeatin riboside, dihydrozeatin, kinetin, 2-isopentenyladenine (2ip), adenine hemisulfate, dimethylallyladenine, N-(2-chloro-4-pyridyl)-N'- phenylurea) (4- CPPU), and analogs thereof.

[00189] In addition to the "bud induction media" and "shoot elongation/maintenance media" combination described above, the present invention provides other alternative media combinations for plant propagation. For example, provided are media combinations comprising at least one Stage 1 medium and at least one Stage 2 medium. In some embodiments, the number assigned to a media within a given process is maintained when a certain media is used more than one time. For example, certain embodiments disclosed herein include cycling explants or shoots in a rotation of media. For example, an explant may be placed in a Stage I media followed by a Stage 2 media and then returned back to its Stage 1 media. In this context, when exposure to a media is repeated, it retains its lowest Stage number within the particular process. In some embodiments, the alternative media are selected from Stage 1 media, Stage 2 media, Stage 3 media, Stage 4 media, Stage 5 media. Stage 6 media, Stage 7 media, etc. as described herein. The media of the present disclosure can be utilized in any order, thus media of the present disclosure are contemplated as any number of stages.

[00190] In some embodiments, the alternative media comprise meta-topolin or an analogue thereof. In some embodiments, the alternative media comprise at least two other cytokinins. In some embodiments, the alternative media comprise at least three cytokinins. In some embodiments, said alternative media comprise at least one auxm and at least two cytokinms. In some embodiments, said alternative media comprise at least two auxins and at least two cytokinins, in some embodiments, said alternative media comprise at least two auxins and at least three cytokinins. In some embodiments, the media supports multiplication cycles for a predetermined period of time. In some embodiments, the media support multiplication cycles for at least six months.

[00191] In some embodiments, to begin the process, a Stage 1 media can be obtained or prepared. Stage 1 media include a pH that is generally hospitable to plants (typically from 4.0-7.0 or 4.5-6.5). Stage 1 media disclosed herein can include (i) meta-topolin; (2) at least three cytokinins; (3) the cytokinin meta-topolin or an analogue thereof in combination with at least two other cytokinins; (4) at least one auxin and at least two cytokinins; (5) at least two auxins and at least two cytokinins or (6) at least two auxins and at least three cytokinms. In certain embodiments, Stage 1 media must include more than 1 auxin. In other embodiments, Stage 1 media must include more than 1 cvtokinm. In further embodiments, Stage 1 media must include more than 1 auxin and more than 1 cytokinin.

[00192] In some embodiments, the cytokinins and auxins are chosen from one or more of meta-topolin, thidiazuron, NAA, IBA, BAP, 2ip, CCPU and DPU. In additional embodiments, the cytokinms and auxins are chosen from one or more of meta-topolin or analogues thereof, thidiazuron or analogues thereof, NAA or analogues thereof, IBA or analogues thereof, BAP or analogues thereof, 2ip or analogues thereof, CCPU or analogues thereof and DPU or analogues thereof.

[00193] In some embodiments, the media and at least two other cytokinins. In some embodiments, the media supports multiplication cycles for at least six months.

[00194] In some embodiments, the media comprise at least three cytokinins. In some embodiments, the media can support multiplication cycles for at least six months. In some embodiments, provided are media comprising the cytokinin meta-topolin or an analogue thereof in combination with at least two other cytokinins.

[00195] In some embodiments, the media comprise at least one auxin and at least two cytokinins. In some embodiments, the media can support multiplication cycles for at least six months. In some embodiments, at least one cytokinin is meta-topolin or an analogue thereof. In some embodiments, the media comprise at least two auxins and at least two cytokinins. In some embodiments, the media can support multiplication cycles for at least six months.

[00197] In some embodiments, the media comprise at least two auxins and at least three cytokinins. In some embodiments, the media can support multiplication cycles for at least six months.

[00198] In some embodiments, the micropropagated plants are grown in vitro in sterile media.

[00199] In some embodiments, the media can be liquid, semi-solid, or solid, and the physical state of the media can be varied by the incorporation or removal of one or more gelling agents. Any gelling agent known in the art that is suitable for use in plant tissue culture media can be used. Agar is most commonly used for this purpose. Examples of such agars include Agar Type A, E or M and Bacto® Agar (Becton Dickinson & Co.). Other exemplary gelling agents include carrageenan, gellan gum (commercially available as PhytaGel™ (Sigma-Aldnch), Gelnte® (Sigma- Aldrich) and Gelzan™ (Caisson Labs)), alginic acid and its salts, and agarose. Blends of these agents, such as two or more of agar, carrageenan, gellan gum, agarose and alginic acid or a salt thereof also can be used.

[00200] Examples of plant growth regulators include abscisic acid (ABA), triacontanol, phlorogiuciiiol, auxms and compounds with auxm-like activity, cytokinins and compounds with eytokmin-like activity. Exemplary auxms include 4-fluorophenoxyacetic acid (FA), 2,4,5- trichlorophenoxyacetic acid (2,4,5-T), 3-bromooxindole-3-aceitc acid, 4-bromophenoxyacetic acid, dicamba, p-chlorophenoxyacetic acid (CPA) indole-3-propinoic acid (IPA), 2,4- dichlorophenoxyacetic acid (2,4-D), indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), picloram and combinations thereof. Exemplary cytokinins include meta-topolin, thidiazuron, N~ (2-chloro-4-pyridyl)-N-phenylurea (CCPU), 1,3-diphenylurea (DPU), adenine hemisulfate, benzyladenine, dimethylallyladenine, kinetin, zeatin, riboside, adenosine, meta-topolin riboside, meta-topolin-9-glucoside, ortho-topoiin, ortho-topolin riboside, ortho-topolin-9-glucoside, para- topolin, para-topolin riboside, para-topolin-9~glucoside, ortho-methoxytopolin, ortho- methoxytopolin riboside, meta-methoxytopolin, meta-methoxytopolin riboside, meta- methoxytopolin-9-glucoside and combinations thereof as well as plant extracts having cytokinin- like activity, such as coconut water, banana powder, malt extract, pineapple powder or tomato powder.

[00201] Gibberellic acid also can be included i n the media. A sugar or combination of sugars can be included in the media and can serve as a carbon source. Such sugars are known to those of ordinary skill in the art. Exemplary sugars include fructose, sucrose, glucose, maltose, galactose manmtol and sorbitol or combinations thereof. Other exemplary additives (with suggested but non- limiting functions) include polyamines (regeneration enhancer); citric acid, polyvinylpyrodine (PVP) and sodium thiosulfate (anti-browning agents); CaNC or calcium gluconate (hy erhydncity reducer); paclobutrazol or ancymidol (multiplication enhancer); acetyl salicylic acid (ethylene inhibitor) and p-chlorophenoxyisobutyric acid (PCIB) and truodobenzoic acid (TIBA) (anti-auxins).

[00202] In some embodiments, basal media can be Murashige and Skoog (MS). Suitable nutrient salts also include, without limitation, Anderson's Rhododendron, Chu's N-6, DKW, Gamborg's B-5, Hoagiands No. 2, Kao & Michayluk, Nitsch & Nitsch, Schenk and Hildebrant, Vacin and Went, Whites and WPM, available from commercial sources such as Caisson Laboratories, Inc or Phytotechnology Laboratories.

[00203] One example of a Stage 1 media includes meta-topolin. Another non-limiting example includes meta-topolin, thidiazuron, NAA and BAP. Another non- limiting example includes meta-topolin, NAA and BAP. Another non-limiting example includes meta-topolin, NAA, BAP and IBA. Another non- limiting example includes meta-topolin, thidiazuron, NAA, BAP and IBA. Another non-limiting example includes thidiazuron, NAA, BAP and 2ip. Another non-limiting example includes thidiazuron, NAA and 2ip. Another non-limiting example includes meta-topolin, thidiazuron, NAA, BAP, IBA and 2ip. Another non-limiting example includes meta- topolin, IBA, 2ip and BAP. Another non-limiting example includes meta-topolin, thidiazuron, CPPU, NAA and B AP. Another non-limiting example includes meta-topolin, thidiazuron, DPU, NAA and BAP. Another non-limiting example includes thidiazuron, CPPU, BAP, IBA and 2ip. Another non-limiting example includes CPPU, DPU, NAA and BAP. Another non-limiting example includes meta-topolin, thidiazuron, CPPU, DPU, NAA, BAP, IBA and 2ip. Each of these non-limiting examples can also include analogues of meta-topolin, thidiazuron, NAA, BAP, DPU, CPPU, IBA and/or 2ip. [00204] The Stage 1 media is then placed into test tubes or other appropriate containers

(including jars, boxes, jugs, cups, sterile bag technology, bioreactors, temporary immersion vessels, etc. wherein when not specified are collectively referred to as "tubes"). These tubes can be capped or covered and autoclaved to sterilize the tubes and media. In another embodiment, sterilization is achieved by autoclaving at 5-25 pounds pressure psi at a temperature of 200° F - for 200° F 10-25 minutes. In another embodiment, sterilization is achieved by autoclaving at 15 pounds pressure psi at a temperature of 250° F for 15-18 minutes. Liquid media can be subjected to filter sterilization.

[00205] After the explants are allowed to establish themselves on Stage 1 media, the cell cultures grown from the explants are transferred into a Stage 2 media. Stage 2 media disclosed herein can include (i) meta-topolin; (2) at least three cytokinms; (3) the cytokinin meta-topolin or an analogue thereof in combination with at least two other cytokinms; (4) at least one auxm and at least two cytokinms; (5) at least two auxins and at least two cytokinms or (6) at least two auxins and at least three cytokinms. In certain embodiments, Stage 2 media must include more than 1 auxin. In other embodiments, Stage 2 media must include more than 1 cytokinin. In further embodiments, Stage 2 media must include more than 1 auxin and more than 1 cytokinin.

[00206] In some embodiments, the cytokinins and auxins are chosen from one or more of meta-topolin, thidiazuron, NAA, IBA, BAP, 2ip, CCPU and DPU. In additional embodiments, the cytokinms and auxins are chosen from one or more of meta-topolin or analogues thereof, thidiazuron or analogues thereof, NAA or analogues thereof, IBA or analogues thereof, BAP or analogues thereof, 2ip or analogues thereof, CCPU or analogues thereof and DPU or analogues thereof.

[00207] In some embodiments, one example of a Stage 2 media includes meta-topolin. Another non-limiting example includes meta-topolin, thidiazuron, NA A and BAP. Another non- limiting example includes meta-topolin, NAA and BAP. Another non-limiting example includes meta-topolin, NAA, BAP and IBA. Another non-limiting example includes meta-topolin, thidiazuron, NAA, BAP and IBA. Another non-limiting example includes thidiazuron, NAA, BAP and 2ip. Another non-limiting example includes thidiazuron, NAA and 2ip. Another non-limiting example includes meta-topolin, thidiazuron, NAA, BAP, IBA and 2ip. Another non-limiting example includes meta-topolin, IBA, 2ip and BAP. Another non-limiting example includes meta- topolin, thidiazuron, CPPIJ, NAA and BAP. Another non-limiting example includes meta-topolin, thidiazuron, DPU, NAA and BAP. Another non-lirniting example includes thidiazuron, CPPU, BAP, IBA and 2ip. Another non-limiting example includes CPPU, DPU, NAA and BAP. Another non-limiting example includes meta-topolin, thidiazuron, CPPU, DPU, NAA, BAP, IBA and 2ip. Each of these non-limiting examples can also include analogues of meta-topolin, thidiazuron, NAA, BAP, DPU, CPPU, IBA and/or 2ip,

[00208] In particular embodiments disclosed herein, both Stage 1 and Stage 2 media include meta-topolin. In another non-limiting embodiment, both Stage 1 and Stage 2 media include meta- topolin, thidiazuron, NAA and BAP. In another non-limiting embodiment, both Stage 1 and Stage 2 media include meta-topolin, NAA and BAP. In another non-limiting embodiment, both Stage 1 and Stage 2 media include meta-topolin, NAA, BAP and IBA. In another non-limiting embodiment, both Stage 1 and Stage 2 media include meta-topolin, thidiazuron, NAA, BAP and IBA. In another non-limiting embodiment, both Stage 1 and Stage 2 media include thidiazuron, N AA, BAP and 2ip. In another non- limiting embodiment, both Stage 1 and Stage 2 media include thidiazuron, NAA and 2ip. In another non- limiting embodiment, both Stage 1 and Stage 2 media include meta-topolin, thidiazuron, NAA, BAP, IBA and 2ip. In another non-limiting embodiment, both Stage 1 and Stage 2 media include meta-topolin, IBA, 2ip and BAP. In another non-limiting embodiment, both Stage 1 and Stage 2 media include meta-topolin, thidiazuron, CPPU, NAA and BAP. In another non-limiting embodiment, both Stage 1 and Stage 2 media include meta-topolin, thidiazuron, DPU, NAA and BAP. In another non-limiting embodiment, both Stage 1 and Stage 2 media include thidiazuron, CPPU, BAP, IBA and 2ip. In another non-limiting embodiment, both Stage 1 and Stage 2 media include CPPU, DPU, NAA and BAP. In another non-limiting embodiment, both Stage 1 and Stage 2 media include meta-topolin, thidiazuron, CPPU, DPU, NAA, BAP, IBA and 2ip. Each of these non-limiting examples can also include analogues of meta-topolin, thidiazuron, NAA, BAP, DPU, CPPU, IB A and/or 2ip.

[00209] In some embodiments, the non-cytokinin components are those found in

Anderson's Rhododendron, Chu's N-6, DKW, Gamborg's B-5, Hoaglands No. 2, Kao & Michayluk, Nitsch & Nitsch, Schenk and Hildebrant, Vacin and Went, Whites and WPM, available from commerci al sources. Particular media can have higher or lower levels of macron utrients than those provided in the preceding tables and others will lack nitrates. In some embodiments, the media have higher or lower levels of macronutrients and lack nitrates. In some embodiments, the media have higher levels of macronutrients and lack nitrates. [00210] Media disclosed herein also include spiked media. Spiked media are those in which the concentration of at least one cytokinin and/or auxin in the media described above is increased by, for example and without limitation, 1%, 3%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% 95%, 100%, 105%, 110% or 200%. In other embodiments, the concentration of at least one cytokinin and/or auxin in the media described above is increased by, for example and without limitation, 1-10%, 5-15%, 10-20%, 15-25%, 20- 30%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60-70%, 65-75%, 70-80%, 75-85%, 80-90%, 85-95%, 90-100%, 95-105%, 100-1 10%, 105-115%, 110-120%, 1 15-125%, 120-130%, 125-135%, 130-140%, 135-145%, 140-150%, 145-155%, 150-160%, 155-165%, 160- 170%, 165-175%, 170-180%, 175-185%, 180-190%, 185-195%, 190-200%, 195-205%, 3-6%, 7- 17%, 12-22%, 17-27%, 22-32%, 27-37%, 32-42%, 37-47%, 42-52%, 47-57%, 52-62%, 57-67%, 62-72%, 67-77%, 72-82%, 77-87%, 82-92%, 87-97%, 92-102%, 97-107%, 102-1 12%, 107-1 17%, 112-122%, 117-127%, 122-132%, 127-137%, 132-142%, 137-147%, 142-152%, 147-157%, 152- 162%, 157-167%, 162-172%, 167-177%, 172-182%, 177-187%, 162-172%, 167-177%, 182- 192%, 187-197%, 192-202%, 197-207%, or 200-210%. When more than one cytokinin and/or auxin is spiked, the concentrations of each raised cytokinin and/or auxin can be raised by the same amount or a different amount than other cytokinins and/or auxins in the media.

[00211] The following tables provide non-limiting examples of spiked media disclosed herein. Each media includes the components described in its respective table above or adjusted as described in the next paragraph with the following adjustments to cytokinin levels.

Media B0038(i-v):

[00212] Additional spiked media can include any standard media described above with the addition or adjustment to the following cytokinin and/or auxin concentrations:

Media AA

Media AC Component AC-i AC-ii AC-iii AC-iv AC-v

BAP 1 1 1 1 1

CPPU 2,5 7.5 10 25 50

[00213] When a spiked media is utilized, explants or shoots generally (but not necessarily) remain on the spiked media for a shorter period of time than those kept on non-spiked media and following culture on a spiked media, the explants or shoots are transferred to a media containing standard, reduced or no levels of cytokmins and/or auxins (those containing reduced or no cytokinms and/or auxins are both referred to as "reduced" media herein).

[00214] One or more compositions of the media disclosed herein can also be adjusted based on the plant species.

[00215] In some embodiments, non-limiting examples of Stage 2, Stage 3, Stage 4, Stage 5, Stage 6, Stage 7 media include:

Cytokinins and Analogs

[00216] Compounds useful according to the present disclosure include meta-topolin analogues having a general formula

wherein W is an aryl or heteroaryl;

Rl is substituted or unsubstituted alkyl wherein any C in the alkyl can be substituted with O, N or S;

each R2 is independently H, OH, C1-C6 alkyl, C -C6 alkvlene, C1-C6 alkynvl, halogen, cyano, C1-C6 alkyloxy, aryl or heteroaryl each optionally substituted with a C1-C6 alkyl, SH, NHR3, C02R3 or halogen;

R3 is H, OH, C1-C6 alkyl, C1-C6 alkvlene, CI -C6 aikyny], halogen, carboxylic group, ester group, aldehyde or cyano;

r is 0 to 8.

In some embodiments, W is

wherem a dashed line represents the presence or absence of a bond:

XI -X⁷ is each independently selected from C, N, O, S with the proviso that the X linking the ring to N is C.

[00217] In some embodiments, the compounds have a structure,

_wherein a dashed line represents the presence or absence of a bond.

[00218] In some embodiments, the compounds have a structure,

wherein a dashed line represents the presence or absence of a bond;

X8-X12 is each independently selected from C, N, O, S;

each R4 is independently H, OH, C1-C6 alkyl, C1-C6 alkylene, C1-C6 alkynyl, halogen, cyano, C1-C6 alkyloxy, aryl or heteroaryl each optionally substituted with a C1-C6 alkyl, SH, NHR3, C02R3 or halogen;

R3 is H, OH, C1-C6 alkyl, C1-C6 alkylene, C1-C6 alkynyl, halogen, carboxvlic group, ester group, aldehyde or cyano; p is 0 to 5; and q is 0 to 6.

iments, the compounds have a structure,

iments, the compounds have a structure,

[00221] Further still, compounds can have structures selected from

ome embodiments, the compounds have a structure.

wherein a dashed line represents the presence or absence of a bond.

e compounds have a structure

wherein a dashed line represents the presence or absence of a bond;

X8-X12 is each independently selected from C, N, O, S;

each R4 is independently H, OH, C1-C6 alkyl, C1 -C6 alkylene, C1-C6 alkynyi, halogen, cyano, C1-C6 alkyloxy, aryl or heteroaryl each optionally substituted with a C1-C6 alkyl, SH, NHR3, C02R3 or halogen;

R3 is H, OH, C1 -C6 alkyl, CI -C6 alkylene, CI -C6 alkynyi, halogen, carboxylic group, ester group, aldehyde or cyano;

p is 0 to 5; and q is 0 to 6.

[00225] In some embodiments, the compounds have a structure

[00226] In still some embodiments, the compounds have a structure

17] In some embodiments, the compound is meta-topolin, also known as 6~(3~ hydroxybenzylamino)-purine, and by the abbreviation mT, having a empirical formula of C12H10N5OH, a molecular weight of 241.25, and the following structural formula:

wherem said meta-topolin is a derivative of a willow tree or a poplar tree.

[00228] Meta-topolin analogues particularly include, without limitation, meta-topolin riboside, meta-topolin-9-glucoside, ortho-topolin, ortho-topolin riboside, ortho-topolin-9- glucoside, para-topolin, para-topolin riboside, para-topolin-9-glucoside, ortho-methoxytopolin, ortho-methoxytopolin riboside, meta-methoxytopolin, meta-methoxytopolin riboside and meta- methoxytopolin-9-glucoside. In particular embodiments, referred to herein as "mT limited embodiments", 6-(3-fluorobenzylamino)purine (FmT), 6-(3-flurobenzyiamino)purine-9-riboside (FmTR) and/or 6-(3-methoxybenzylamino)purine-9-riboside (memTR) can be excluded from the class of meta-topolin analogs.

[00229] Compounds useful according to the present disclosure include thidiazuron analogues having a general formula

wherein V is an aryl or heteroaryl;

each R5 and R6 is each independently H, OH, C1-C6 alkyl, C1-C6 alkylene, C1-C6 alkynyl, halogen, cyano, C -C6 alkyloxy, aryl or heteroaryl each optionally substituted with a C1-C6 alkyl or halogen;

n is 0 to 4;

o is 0 to 5

X13-X16 is each independently selected from C, N, O, S;

Zl and Z2 are each independently NH, O, SH or CH or Zl and Z2 can be combined to form a substituted or unsubstituted aryl or heteroaryl; and

Yl is O or S.

[ compounds have a structure

wherein X17-X21 is each independently selected from C, N, (), S.

[ compounds include

[00232] In one embodiment, the compound is thidiazuron, also known as l~phenyl-3-(l ,2,3- thiadiazol-5-y])urea and 5-phenylcarbamoylamino~l,2,3-thiadiazole, has the empirical formula of of 220.25 and the following structural formula

[00233] Compounds useful according to the present disclosure include B-naphthoxyacetic analogues havin a general formula:

or a salt thereof;

wherem Ra is COR3, C02R3, CONR3R4, or CN;

each Rb is independently R3; OR3; F; CI; Br; I; CN; N02; OCF3; CF3; NR2R3; SR3, SOR3,

S02R3, C02R3, COR3, CONR3R4, CSNR4R5; or optionally substituted aryl or optionally substituted heteroaryl, wherem each substituent of aryl or heteroaryl is independently C1-C6 alkyl,

F, CI, Br, or I;

a is 1 , 2, 3, 4, 5, 6, or 7;

Xa is Ni l. S or O;

each R3 is independently II, C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl; and

each R4 is independently R3 or optionally substituted phenyl, wherein each substituent of phenyl is independently C1-C6 alkyl, F, Ci, Br, or I

[00234] In another embodiment, compounds have a structure:

[00235] In one embodiment, the compound is B-naphthoxyacetic acid (NAA), also known as acetic acid, (2-naphthalenoxy)-(9CI) and has a CAS Number of 120-23-0, has the empirical formula and the following structural formula:

Other examples of NAA analogues may include, but are not limited to:

or a salt thereof;

wherein Rl is COR3, C02R3, CONR3R4, each R2 is independently R3; OR3; F; CI; Br; I; CN; N02; OCF3; CF3; NR2R3; SR3, SOR3, S02R3, C02R3, COR3, CONR3R4, CSNR4R5; or optionally substituted aryl or optional Iv substituted heteroaryl, wherein each substituent of aryl or heteroaryl is independently C1-C6 alkyl, F, CI, Br, or I;

n is 1, 2, 3, or 4;

X is NH, S or O;

each R3 is independently H, C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl; and

each R4 is independently R3 or optionally substituted phenyl, wherein each substituent of phenyl is independently C1-C6 alkyl, F, CL Br, or I.

[00238] In another embodiment, compounds have a structure:

[00239] In one embodiment, the compound is indole butyric acid (IB A), also known as 1-

Indole-3-butanoic acid, and has a CAS Number of 133-32-4, has the empirical formula of C12H13N02, a molecular weight of 203.24, and the following structural formula:

[00240] Other examples of IB A. analogues may include, but are not limited to:

(BAP) analogues having a general formula:

or a salt thereof;

wherein a dashed line represents the presence or absence of a bond;

each R5 and each R6 is independently R3; OR3; F; CI; Br; I; CN; N02; OCF3; CF3; NR2R3; SR3,

SOR3, S02R3, C02R3, COR3, CONR3R4, CSNR4R5; or optionally substituted aryl or optionally substituted heteroaryl, wherein each substituent of aryl or heteroaryl is independently C1-C6 alkyl,

F, CI, Br, or I;

o is 0, 1 , 2, 3, 4, or 5;

p is 0, 1 , or 2;

XI is Nil, S or O;

X4 is -N= and X5 is M l -. -S-, or -^■()-; or X5 is -N= and X4 is -NH-, -S-, or -0-;

X2 and X3 and are independently N or CR6;

each R3 is independently H, C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl; and

each R4 is independently R3 or optionally substituted phenyl, wherem each substituent of phenyl is independently C1-C6 alkyl, F, CI, Br, or I.

|00242J In some embodiments, X4 is -N= and X5 is -NH-, -S-, or -0-, and the dashed line represents the presence or absence of a bond. Thus, compounds of according to the formula below are contemplated.

[00243] In other embodiments, X5 is and X4 is -NH-, -S-, or -0-. Thus, compounds of the formula below are contemplated.

the compounds have a structure:

he compounds have a structure:

[00246] In one embodiment, the compound is benzylaminopurine (BAP), also known as

9H-Purm-6-amine, N-(phenylmethyl)-, which has a CAS Number of 1214-39-7, an empirical ecular weight of 225.25, and the following structural formula:

[00247 Other examples of BAP analogues may include, but are not limited to:

Compounds useful according to the present disclosure include 6-y (dimethylallylamino)-purine 2ip analogues having a general formula:

or a salt thereof;

wherein a dashed line represents the presence or absence of a bond;

wherein R7, R8, and each R9 are independently R3; OR3; F; CI; Br; I; CN; N02; OCF3; CF3; NR2R3; SR3, SGR3, S02R3, C02R3, COR3, CONR3R4, CSNR4R5; or optionally substituted aryi or optionally substituted heteroaryl, wherein each substituent of aryl or heteroaryl is independently C1-C6 alkyl, F, CI, Br, or I;

q is 0, , or 2;

X6 is NH, S or O;

X9 is -N= and XI 0 is -NH-, -S-, or -0-; or XI 0 is -N= and X9 is - H-, -S-, or -0-; X7 and X8 and are independently N or CR9; and

each R3 is independently H, C1 -C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl; and

each R4 is independently R3 or optionally substituted phenyl, wherein each substituent of phenyl is independently C1-C6 alkyl, F, CI, Br, or I.

[00249] In some embodiments, the dashed line represents the presence or absence of a bond. Thus, compounds of according to the formulas below are contemplated.

00251] In other embodiments, XI 0 is -N= and X9 is -NH-, -S-, or -()-. Thus, compounds having the structure shown below are contemplated.

[00252] In another embodiment, compounds have a structure:

[00253] In one embodiment, the compound is 6-y,y,-(dimethylallylamino)-purine (2ip) or

DAP, also known as 9H-purin-6-amme, N-(3-methyl-2-butene~l~yl)-, having a CAS No. 2365-40- 4, an empirical formula of C10H13N5, a molecular weight of 203.24, and the following structural formula:

[00255] Compounds useful according to the present disclosure include N,N-diphenylurea

(DPU) analogues having a general formula:

or a salt thereof;

wherein each R10 and each Rl 1 is independently R3; OR3; F; CI; Br; I; CN; N02; OCF3; CF3; NR2R3; SR3, SOR3, S02R3, C02R3, COR3, CONR3R4, CSNR4R5; or optionally substituted aryl or optionally substituted heteroaryl, wherein each substituent of aryl or heteroaryl is independently C1-C6 alkyl, F, CI, Br, or I;

r and s are independently 0, 1, 2, 3, 4, or 5;

XI 1 and X12 are independently NR10, S, or O;

each R3 is independently H, C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl; and

each R4 is independently R3 or optionally substituted phenyl, wherein each substituent of phenyl is independently C1-C6 alkyl, F, CI, Br, or I.

] In another embodiment, compounds have a structure:

[00257] In one embodiment, the compound is Ν,Ν-diphenylurea (DPU), which is represented by a formula:

or a salt thereof;

wherein each R 2 and each R13 is independently R3; OR3; F; CI; Br; I; CN; N02; OCF3; CF3; NR2R3; SR3, SOR.3, S02R3, C02R3, ( OR . CONR3R4, CSN 4R5; or optionally substituted aryl or optionally substituted heteroaryl, wherein each substituent of aryl or heteroaryl is independently C1.-C6 alkyl, F, CI, Br, or I;

t and u are independently 0, 1 , 2, 3, 4, or 5;

XI 3 and XI 4 are independently NR12, S, or O;

each R3 is independently H, C1 -C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl; and

each R4 is independently R3 or optionally substituted phenyl, wherein each substituent of phenyl is independently C1.-C6 alkyl, F, CI, Br, or I.

[00260] In one embodiment, compounds have a structure:

[00263] Other examples of PPU analogues may include, but are not limited to:

Propagation / Micropropagation

[00264] Micropropagation is the practice of rapidly multiplying stock plant material to produce a large number of progeny plants, using modern plant tissue culture methods, Micropropagation is used to multiply novel plants, such as those that have been genetically modified or bred through conventional plant breeding methods. It is also used to provide a sufficient number of plantlets for planting from a stock plant w nch does not produce seeds, or does not respond well to vegetative reproduction,

[00265] Micropropagation can first begin with the selection of plant material to be propagated. Clean stock materials that are free of viruses and fungi are important in the production of the healthiest plants.

[00266] Once the plant material is chosen for culture, the collection of explant(s) begins and is dependent on the type of tissue to be used, including stem tips, anthers, petals, pollen and others plant tissues. The explant material is then surface sterilized, usually in multiple courses of bleach and alcohol washes and finally rinsed in sterilized water. This small portion of plant tissue, sometimes only a single cell, is placed on a growth medium, typically containing sucrose as an energy source and one or more plant growth regulators (plant hormones). Usually the medium is thickened with agar to create a gel which supports the explant during growth. Some plants are easily grown on simple media but others require more complicated media for successful growth; the plant tissue grows and differentiates into new tissues depending on the medium. For example, media containing cytokmins are used to create branched shoots from plant buds.

[00267] Multiplication is the taking of tissue samples produced during the first stage and increasing their number. Following the successful introduction and growth of plant tissue, the establishment stage is followed by multiplication. Through repeated cycles of this process, a single explant sample may be increased from one to hundreds or thousands of plants. Depending on the type of tissue grown, multiplication can involve different methods and media. If the plant material grown is callus tissue, it can be placed in a blender and cut into smaller pieces and recultured on the same type of culture medium to grow more callus tissue. If the tissue is grown as small plants called plantlets, hormones are often added that cause the plantlets to produce many small offshoots that can be removed and recultured.

[00268] The next stage ("pretransplant" stage) involves treating the plantlets/shoots produced to encourage root growth and "hardening." It is performed in vitro, or in a sterile or substantially sterile environment. "Hardening" refers to the preparation of the plants for a natural growth environment. Until this stage, the plantlets have been grown in "ideal" conditions, designed to encourage rapid growth. Due to lack of necessity, the plants are likely to be highly susceptible to disease and often do not have fully functional dermal coverings and will be inefficient in their use of water and energy. In vitro conditions are high in humidity and plants grown under these conditions do not form a working cuticle and stomata that keep the plant from drying out, when taken out of culture the plantlets need time to adjust to more natural environmental conditions. Hardening typically involves slowly weaning the plantlets from a high-humidity, low light, warm environment to what would be considered a normal growth environment for the species in question.

[00269] In the final stage of plant micropropagation, the plantlets are removed from the plant media and transferred to soil or (more commonly) potting compost for continued growth by conventional methods. This stage is often combined with the "pretransplant" stage.

[00270] Modern plant tissue culture is performed under aseptic conditions under filtered air.

Living plant materials from the environment are naturally contaminated on their surfaces (and sometimes interiors) with microorganisms, so surface sterilization of starting materials (explants) in chemical solutions (usually alcohol or bleach) is required. Explants are then usually placed on the surface of a solid culture medium, but are sometimes placed directly into a liquid medium, particularly when cell suspension cultures are desired. Solid and liquid media are generally composed of inorganic salts plus a few organic nutrients, vitamins and plant hormones. Solid media are prepared from liquid media with the addition of a gelling agent, usually purified agar.

[00271] The composition of the medium, particularly the plant hormones and the nitrogen source (nitrate versus ammonium salts or amino acids) have profound effects on the morphology of the tissues that grow from the initial explant. For example, an excess of auxin will often result in a proliferation of roots, while an excess of cytokinin may yield shoots. A balance of both auxin and cytokinin will often produce an unorganized growth of cells, or callus, but the morphology of the outgrowth will depend on the plant species as well as the medium composition. As cultures grow, pieces are typically sliced off and transferred to new media (subcultured) to allow for growth or to alter the morphology of the culture. The skill and experience of the tissue culturist are important in judging which pieces to culture and which to discard. As shoots emerge from a culture, they may be sliced off and rooted with auxin to produce plantlets which, when mature, can be transferred to potting soil for further growth in the greenhouse as normal plants.

[00272] The tissue obtained from the plant to culture is called an explant. Based on work with certain model systems, particularly tobacco, it has often been claimed that a totipotent explant can be grown from any part of the plant. However, this concept has been vitiated in practice. In many species expiants of various organs vary in their rates of growth and regeneration, while some do not grow at all. The choice of explant material also determines if the plantlets developed via tissue culture are haploid or diploid. Also the risk of microbial contamination is increased with inappropriate expiants. Thus it is very important that an appropriate choice of explant be made prior to tissue culture.

[00273] The specific differences in the regeneration potential of different organs and expiants have various explanations. The significant factors include differences in the stage of the cells in the cell cycle, the availability of or ability to transport endogenous growth regulators, and the metabolic capabilities of the cells. The most commonly used tissue expiants are the meristematic ends of the plants like the stem tip, auxiliary bud tip and root tip. These tissues have high rates of cell division and either concentrate or produce required growth regulating substances including auxins and cytokinins. Some expiants, like the root tip, are hard to isolate and are contaminated with soil microflora that become problematic during the tissue culture process. Certain soil microflora can form tight associations with the root systems, or even grow within the root. Soil particles bound to roots are difficult to remove without injury to the roots that then allows microbial attack. These associated microflora will generally overgrow the tissue culture medium before there is significant growth of plant tissue. Aerial (above soil) explants are also rich in undesirable microflora. However, they are more easily removed from the explant by gentle rinsing, and the remainder usually can be killed by surface sterilization. Most of the surface microflora do not form tight associations with the plant tissue. Such associations can usually be found by visual inspection as a mosaic, de-colorization or localized necrosis on the surface of the explant.

[00274] An alternative for obtaining un contaminated explants is to take explants from seedlings which are aseptically grown from surface-sterilized seeds. The hard surface of the seed is less permeable to penetration of harsh surface sterilizing agents, such as hypochlorite, so the acceptable conditions of sterilization used for seeds can be much more stringent than for vegetative tissues.

[00275] Tissue cultured plants are clones, if the original mother plant used to produce the first explants is susceptible to a pathogen or environmental condition, the entire crop would be susceptible to the same problem, and conversely any positive traits would remain within the line also. Plant tissue culture is used widely in plant science; it also has a number of commercial applications. Applications include:

[00276] 1. Micropropagation is widely used in forestry and in floriculture.

Micropropagation can also be used to conserve rare or endangered plant species.

[00277] 2. A plant breeder may use tissue culture to screen cells rather than plants for advantageous characters, e.g. pathogen resistance/tolerance.

[00278] 3. Large-scale growth of plant cells in liquid culture inside bioreactors as a source of secondary products, like recombinant proteins used as biopharmaceuticals.

[00279] 4. To cross distantly related species by protoplast fusion and regeneration of the novel hybrid.

[00280] 5. To cross-pollinate distantly related species and then tissue culture the resulting embryo, which would otherwise normally die (Embryo Rescue). [00281] 6. For production of doubled monoploid (dihaploid) plants from haploid cultures to achieve homozygous lines more rapidly in breeding programs, usually by treatment with colchicine which causes doubling of the chromosome number.

[00282] 7. As a tissue for transformation, followed by either short-term testing of genetic constructs or regeneration of transgenic plants.

[00283] 8. Certain techniques such as meristem tip culture can be used to produce clean plant material from infected stock, such as potatoes and many species of soft fruit.

[00284] 9. Micropropagation using meristem and shoot culture to produce large numbers of identical individuals.

[00285] Micropropagated plants can begin from a selected piece of plant tissue, called an "explant" or "mother plant." This explant is the source of cells to be developed during the tissue culturing process. For example, the explant can be any segment or collection of cells from apical meristems, terminal buds, axillary buds, adventitious buds, accessory buds, pseudo-terminal buds, cambium, lateral meristem, lateral bud, vegetative buds, reproductive buds, mixed buds, shoot segments, shoot apices, stem segments, immature nodal sections from stems, lateral shoots, seedlings, seeds, shoots starting to rise from the ground, immature flower buds, inflorescences, crown segments, leaf segments, or any part thereof. In one embodiment, the explant is taken from a plant of about 1 week old, about 2 weeks old, about 3 weeks old, about 1 month old, about 2 month old, about 3 months old, about half year old, about 1 year old, about 2 years old plant, about 3 years old, about 5 years old, or more. The plant from which the explant is obtained can be grown in any suitable conditions, including but not limited to growing in a growth chamber, growing in a greenhouse, growing in a field, or growing in a tissue culture container (petri dish, margenta box, etc.). In some embodiments, the explant is tissue culture obtained from shoot clumps maintained as stock on growth media. In some embodiments, the explant is a nodal section having one or more axillary bud, which can be dormant or active. In some other embodiments, the explant is a seed or a part thereof.

[00286] In some embodiments, the tissue culture is obtained from shoot clumps maintained on growth media as stock. In some embodiments, the explant is a segment of bamboo cane. In some embodiments, the segment of bamboo cane comprises an internode. In some embodiments, the segment of bamboo cane comprises a nodal section. In some embodiments, the nodal section comprises a single bud. In some embodiments, the bud is dormant or active. In some embodiments, the explant is taken from a plant of about 1 week old, about 2 weeks old, about 3 weeks old, about

1 month old, about 2 month old, about 3 months old, about half year old, about I year old, about

2 years old plant, about 3 years old, about 5 years old, or more. In some embodiments, a bamboo seed or a part thereof is used.

[00287] Availability of virus free starting material is desirable for an agricultural seed production program. Thus, in some embodiments, the virus-free micropropagated plants begin from an explant that is subjected to one or more antiviral treatments, such as a chemical antiviral, thermotherapy, and/or meri stem- tip culture. Meristem culture is one procedure used to produce a virus-free plant. In this method, apical or axillary growing tips (0.1-0.3 mm) are dissected and allowed to grow into plantlets on special culture medium under controlled conditions. The meristem culture for virus elimination is based on the principle that many viruses are unable to infect the apical/axillary meristem of a growing plant and that a virus-free plant can be produced if a small (e.g. 0.1-0.3 mm) piece of meristemic tissue is propagated. Excision of very small meristems typically requires a high degree of expertise and the development of plants from these small meristems (mericlones) can be lengthy (i.e. 4 to 8 months). To increase the percentage of virus freedom in regenerated mericlones, meristem culture can be combined with other antiviral treatments, such as thermotherapy (high temperature treatment) or chemotherapy (treatment with antiviral compounds) to increase the production of virus-free plants.

[00288] Thus, in some embodiments, the method comprises using meristem culture, thermotherapy, chemotherapy, or any combination thereof to produce a virus-free plant. In one embodiment, meristem culture, thermotherapy, and chemotherapy, are used to produce a virus- free plant. In some embodiments, the use of an antiviral can increase the success in producing a virus-free plant by at least two or three times. In one embodiment, chemotherapy comprises using an antiviral in a medium to culture the explant. In another embodiment, thermotherapy comprises incubating an explant under a 16h light photoperiod at 30-40

light intensity at 37 °C. In some embodiments, the thermotherapy is for one week.

[00289] Accordingly, in one aspect of the present invention, a method for producing a virus- free plant comprises incubating an explant with medium, optionally comprising an antiviral; optionally, subjecting an explant of the plant culture to thermotherapy, wherein the explant grows into a plantlet; excising an apical meristem from the plantlet; and placing the apical meristem into a regeneration media; wherein a virus-free plantlet is produced. [00290] In some other embodiments, the regeneration media comprises an antiviral, such as

Ribavirain (also known as Virazole). The method for producing a virus-free plant can also comprise culturing or subculturing, using conditions such as disclosed in PCX Publication No. WO2013016198, which is incorporated by reference in its entirety. For example, culturing or subculturing can be of the explant, apical meristem, the plantlet, or any combination thereof. The culturing or subculturing can be performed every one, two to three weeks. In one embodiment, culturing comprises incubating the explant under a 16h light photoperiod at 80-100 pmol/rn²/x light intensity at 24 °C, In some embodiments, the method for producing a virus-free plant uses one or more different regeneration media.

[00291] The plantlet produced by a method disclosed herein can be subcultured or tested for viruses. Any method known for testing for the presence of a virus can be used, such as by enzyme-linked immunosorbent assay (ELISA). The plantlet can be multiplied and subcultured, and used for further propagation. The pssent invention is applicable to a whole range of agricultural crops where a protocol for isolation and culture of meristematic cells or meristematic zones in vitro are available. Plantlets could be further induced and regenerated from the above cultures using either organogenesis or somatic embryogenesis.

[00292] In some embodiments, the tissue culture is obtained from shoot clumps maintained on growth media as stock. In some embodiments, the explant is a segment of bamboo cane. In some embodiments, the segment of bamboo cane comprises an internode. In some embodiments, the segment of bamboo cane comprises a nodal section. In some embodiments, the nodal section comprises a single bud. In some embodiments, the bud is dormant or active. In some embodiments, the explant is taken from a plant of about 1 week old, about 2 weeks old, about 3 weeks old, about

1 month old, about 2 month old, about 3 months old, about half year old, about 1 year old, about

2 years old plant, about 3 years old, about 5 years old, or more. In some embodiments, a bamboo seed or a part thereof is used.

[00293] The present inventi on provides methods for in vitro micropropagation of plant, for example, gymnosperm plants, angiosperm plants, monocot plants, dicot plants, crops, agriculturally/economically/environmentally important plants, etc.

[00294] The present invention provides methods for in vitro micropropagation of plant, for example, gymnosperm plants, angiosperm plants, monocot plants, dicot plants, crops, agriculturally/economically /'e vironmentally important plants, etc. [00295] In some embodiments, the methods disclosed herein can be used for in vitro micropropagation of a gymnosperm plant. For example, the methods can be used for in vitro micropropagation of the plants in the family/order of Cycadaceae, Zamiaceae, Ginkgoaceae, Wehvitschiaceae, Gnetaceae, Ephedraceae, Pinaceae, Araucariaceae, Podocarpaceae, Sciadopityaceae, Ciipressaceae, or Taxaceae.

[00296] In some embodiments, the methods disclosed herein can be used for in vitro micropropagation of an angiosperm plant. For example, the methods can be used for in vitro micropropagation of the plants in the family /order of Ceratophyllum, Chloranthaceae, eudicots, magnoliids, or monocots.

[00297] In some embodiments, the methods disclosed herein can be used for in vitro micropropagation of a dicot plant. For example, the methods can be used for in vitro micropropagation of the plants in the family/order of Biixaceae, Cannabaceae, Didymelaceae, Sabiaceae, Trochodendraceae, Tetracentraceae, Ranuneidales, Proteales, Aexioxicaceae, Berberidopsidaceae, Ditteniaceae, Gunnerales, Caryophyllales, Saxifragales, Santalales, rosids, Aphloiaceae, Geissoiomataceae, Ixerbaceae, Picramniaceae, Strassburgeriaceae, Vitaceae, Crossosomaiales, Geraniales, Myrtales, Zygophyllaceae, Krameriaceae, Hitaceae, Celastrales, Malpighiales, Oxalidales, Fabales, Rosales, Cucurbitales, Fagales, Tapisciaceae, Brassicales, Malvales, Sapindales, asterids, Cornales, Ericales, Boraginaceae, Icacinaceae, Oncothecaceae, Vahliaceae, Garryales, Solanales, Gentianales, Lamiales, Brimiaceae, Columelliaceae, Desfontainiaceae, Eremosynaceae, Escalloniaceae, Paracryphiaceae, Polyosmaceae, Sphenostemonacae, Tribelaceae, Aquifoiiales, Apiales, Dipsacales, or Asteraies.

[00298] In some embodiments, the methods disclosed herein can be used for in vitro micropropagation of a monocot plant. For example, the methods can be used for in vitro micropropagation of the plants in the family/order of Acorales, Alismatales, Asparagales, Dioscoreales, Li Hales, Pandanales, Petrosaviales, Dasypogonaceae, Arecales, Commelinales, Poales, or ZingiberaJes.

[00299] In some embodiments, the methods disclosed herein can be used for in vitro micropropagation of a bamboo species, such as Phyllostachys edulis (e.g., Phyllostackys edulisi 'MosoV, Phyllostachys bissetti, Fargesia denudata, Pleioblastus fortunei, Sasa Veitchii, Pleioblastus viridisiriatus, Thamnocalamus crassinodus, Chusquea Culeo "Carta Prieta ", Bambusa OMHamii, Phyllostachys Moso, Phyllostachys Atrovaginata, Dendrocalamus Asper, or Guadua Angustifolia. In some embodiments, the bamboo species is Phyllostachys edulis, Moso.

[00300] In some embodiments, the plant is a non-bamboo species. In some embodiments, the non-bamboo plant species is Geranium spp. (e.g., Geranium rozanne), Hakonechloa macra (e.g., Hakonechloa macra 'Aureola', Hakonechloa macra. 'All gold'), Hellehorus (e,g., Hellehorus 'Ivory Prince'), Phormium, Wasabi (e.g., Wasabi C2), Ariindinaria (e.g. , Arundinaria gigantean), or Solarium (e.g., Solarium tuberosum and Solarium tuberosum),

[00301] In some embodiments, the methods are used for rapid bamboo in vitro micropropagation. High shoot multiplication rate can be achieved in the methods disclosed herein. As used herein, the phrase "multiplication rate" refers to the multiplication fold of plant shoots obtained in a micropropagation process by starting from a single explant. For example, in the situation where the explant is a nodal section comprising a single bud, and 3 shoots are obtained after a micropropagation cycle, the multiplication rate is 3X. In some embodiments, by using the bud induction media and the shoot elongation/maintenance media, a multiplication rate of at least about 2X to about 30X can be achieved after micropropagation. For example, about 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 11X, 12X, 13X, 14X, 15X, 6X, 17X, 18X, 19X, 20X, 2 IX, 22X, 23X, 24X, 25X, 26X, 27X, 28X, 29X, about 3 OX, or more can be achieved within about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or about 28 days, or more.

[00302] In some embodiments, the present invention is based on the unexpected discovery that a pulsed treatment of an explant on a first medium comprising a strong cytokinin, such as TDZ, followed by a treatment of the explant on a second medium comprising one or more cytokinins other than TDZ, e.g., cytokinins that are relatively weaker than TDZ, such as meta- topolin, kinetin, isopentenyl adenine (iP, e.g., 2ip), zeatin, trans- zeatin, zeatin riboside, dihydrozeatin, benzyleadenin (BAP), or benzyladenosine ([9RJBAP), can provide rapid in vitro micropropagation with unexpected high multiplication rate.

[00303] In some embodiments, the methods comprise using a bud induction medium and a shoot elongation/maintenance media, wherein the bud induction medium comprises a strong cytokinin, such as TDZ, and the shoot elongation/maintenance medium (B002) comprises a relatively weaker cytokinin, such as meta-topolin, kinetin, isopentenyl adenine (iP, e.g., 2ip), zeatin, trans-zeatin, zeatin riboside, dihydrozeatin, benzyleadenin (BAP), or benzyladenosine ([9R]BAP).

[00304] Examples of a bud induction medium are described herein. In some embodiments, a bud induction medium comprises one or more strong cytokinin or analog thereof. In some embodiments, the bud induction medium comprises only one strong cytokmin, wherein the cytokinin is TDZ or analog thereof. In some embodiments, the concentration of the strong cytokinin (e.g., TDZ) in the bud induction media is about 0.25 mg L to about 100 mg L, for example, about 0.5 mg L to about 2 mg/L.

[00305] Examples of a shoot elongation/maintenance media are described herein, in some embodiments, a shoot elongation/maintenance medium comprises one or more cytokmin that is relatively weaker cytokinin, such as a cytokinin other than TDZ. In some embodiments, the shoot elongation/maintenance medium comprises only one relatively weaker cytokmin, such as BAP, meta-topolin, ip (e.g., 2ip), zeatin, zeatin riboside, or combination thereof. In some embodiments, the shoot elongation/maintenance medium comprises more than one cytokinins. In some embodiments, the concentration of a cytokinin in a shoot elongation/maintenance medium is about 0.01 mg/L to about 100 mg/L, for example, 0.25 mg/L to about 5 mg/L.

[00306] In some embodiments, the bud induction medium and/or the shoot elongation/maintenance medium comprises one or more auxin, such as β-naphthoxyacetic acid (NAA), 2,4- Dichlorophenoxy acetic acid (2,4-D), indole-3 -butyric acid (IBA), indoie-3 -acetic acid (IAA), piclorani, or analogs thereof. In some embodiments, the bud induction medium and/or the shoot elongation/maintenance medium comprises NAA. In some embodiments, the concentration of an auxin in the media is 0.01 mg/L to about 50 mg/L, for example, about 0.25 mg/L, to about 0.5 mg/L.

[00307] In some embodiments, the methods are used for micropropagating plants in vitro.

In some embodiments, the methods comprise (a) incubating a plant tissue culture, explant or seed in a first medium, and (b) then incubating the plant tissue obtained from step (a) in a second medium. In some embodiments, the first medium is a bud induction medium, and the second medium is a shoot elongation and maintenance medium.

[003Θ8] In some embodiments, the methods comprise (a) incubating a tissue culture, explant or seed/seed part in a bud induction medium to induce shoot bud formation; (b) incubating the shoot buds obtained in step (a) in a shoot elongatkm-maintenanee medium. [00309] The methods can further comprise (c) incubating the shoots from step (b) in a bud induction medium to induce shoot bud formation; and (d) incubating the shoot buds obtained in step (c) in a shoot elongation/maintenance medium.

[00310] In some embodiments, the shoot buds obtained in step (a) and/or step (c) are separated prior to incubating the shoot buds in step (b) and/or step (d). In some embodiments, the separation produces groups of 1 to 3 shoot buds per separation prior to incubating the shoot buds in step (b) and/or step (d).

[00311] In some embodiments, the methods further comprises (e) repeating the incubating steps (c) and step (d) for at least once.

[00312] In some embodiments, when a bud induction medium and a shoot elongation and maintenance medium are used, the methods further comprise: (e) repeating the incubating step (c) and step (d) for at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or more additional cycles. There is no limit to how many times the cycling of step (c) and step (d) can be repeated. Buds and/or shoots obtained in step (a) or step (c) can be separated prior to the buds and/or shoots entering step (b) or step (d), respectively, wherein such separation can result in a single bud or shoot, 2 buds and/or shoots, 3 buds and/or shoots, or 4 or more buds and/or shoots per separation. Optimum separation for maximum, rapid production of bamboo copies of a single species, genotype or clone usually involves separating the buds and/or shoots obtained in step (a) or step (c) into 1-3 buds and/or shoots prior to entering into step (b) or step (d), respectively. Where there are 2 or more buds and/or shoots per separation this is known in the art as a clumping or "clump" of buds and/or shoots. Some variation in the methodologies of the present invention may be necessary so as to fine-tune the process for specific species, genotypes or clones of bamboo and such process variations are within the disclosure of this invention.

[00313] In some embodiments, both the bud induction medium and the shoot elongation/maintenance medium are liquid media. The advantage of liquid media is that the one can replace old media with fresh media, or replace one type of media with another type of media quickly and easily, without transferring the seedlings of plant from one container to another. Therefore, in some embodiments, the whole micropropagation process is achieved in a single container, for example, in a bioreactor. [00314] In some embodiments, the bud induction media and/or the shoot elongation/maintenance media are semi-solid or solid media. In some embodiments, liquid media and semi-solid or solid media can be used subsequently with any desired order. For example, the bud induction medium in step (a) and/or step (c) is liquid, semi-solid, or solid; the shoot elongation/maintenance medium in step (b) and/or step (d) is liquid, semi-solid, or solid. Thus, in some embodiments, the bud induction medium of step (a) and/or step (c) is a liquid medium. In some embodiments, the bud mduction medium of step (a) and/or step (c) is a solid medium. In some embodiments, the shoot elongation/maintenance medium of step (b) and/or step (d) is a liquid medium. In some embodiments, the shoot elongation/maintenance media of step (b) and/or step (d) is a solid media.

[00315] In some embodiments the methods of the present invention may involve using a liquid media for one step and a solid media for the next step of a particular cycle. For example, the present invention encompasses methods whereby step (a) is accomplished using liquid media and step (b) is accomplished using solid media. Alternatively, if both steps (a) and (b) and/or steps (c) and (d) are both done using liquid media, then the present invention contemplates that the liquid media may be changed without moving the buds and/or shoots to another container (e.g., test tube, bioreactor, jar, etc.). For example, if both steps (a) and (b) are accomplished using liquid media in a hydroponic setup, then the buds and/or shoots may remain in their fixed or unfixed position while the liquid media is replaced.

[00316] In some embodiments, the incubation of step (a) and/or step (c) lasts for a period that is sufficient to produce more than one shoot bud. For example, the period is set so as to produce at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 0, at least 15, at least 20, at least 25, at least 26, at least 27, at least 28, at least 30, or more shoot buds for each plant tissue culture, explant or seed placed in the bud induction medium of step (a) or (c).

[00317] In some embodiments, the incubation period of step (a) or step (c) lasts for about one hour to about three weeks, or more. For example, the incubation period of step (a) or step (c) lasts for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 1 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, about 1 week, about 1.5 weeks, about 2 weeks, about 2.5 weeks, about 3 weeks, about 3.5 weeks, about 4 weeks, or more. In some embodiments, the incubation of step (a) and/or step (c) lasts from about 24 hours to about 60 hours. Thus, the incubation of step (a) and/or step (c) can last for about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 35 hours, about 40 hours, about 45 hours, about 50 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59, hours, or about 60 hours. In some embodiments the incubation stage of step (a) or step (c) can last longer than 60 hours, e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, or longer.

[00318] In some embodiments, the mcubation of step (b) and/or step (d) lasts for any desired period. In some embodiments, the mcubation of step (b) and/or step (d) lasts from about 24 hours to about four weeks, or more. For example, the mcubation of step (b) and/or step (d) lasts from about three days to about five days, or more. Thus, the incubation of step (b) and/or step (d) can last for about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 35 hours, about 40 hours, about 45 hours, about 48 hours, about 50 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59, hours, about 60 hours, about 72 hours, about 96 hours or about 120 hours. In some embodiments the mcubation of step (b) and/or (d) can last longer than 120 hours, e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, or longer. In some embodiments, the incubation period of step (b) or step (d) lasts for about 0.5 week, about 1 week, about 1.5 weeks, about 2 weeks, about 2.5 weeks, about 3 weeks, about 3.5 weeks, about 4 weeks, about 4.5 weeks, about 5 weeks, about 5.5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, or more.

[00319] The incubation periods in the steps can be adjusted depending on the species of the plant, type of the explant, a desired multiplication rate. Without wishing to be bound by any theory, in some embodiments, for a bamboo species, the incubation period in step (a) or step (c) can be about 1 hour to about 3 weeks, for example, about 24 hours to about 60 hours; and the mcubation period in step (b) or step (d) can be about 24 hours to about 4 weeks, for example, about 3 days to about 5 days.

[00320] The shoot multiplication rate can be further improved by repeating step (c) and step

(d). For example, the multiple shoots developed after treatment of step (a) and treatment of step (b) can be subjected to one or more round of treatment of step (c) and treatment of step (d). In some embodiments, treatment in step (c) and treatment of step (d) are conducted at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight time, or more. Since the treatments in all steps are in short periods, a very short total time is needed to reach a very high shoot multiplication rate.

[00321] In some embodiments, each step of (a) to (e) can also be repeated before conducting the next step, by replacing old media with fresh media, for once, twice, three times, or more. In some embodiments, step (e) is repeated at least once, twice, three times, or more. In some embodiments, steps (a) to (e) take approximately one week, two weeks, three weeks, four weeks, five weeks, six weeks, or more.

[00322] In some embodiments, starting from a single explant, the present methods can provide about 10X to about 3 OX shoot multiplication rate in approximately three weeks. In addition, at least about 500, at least about 1,000, at least about 2,000, at least about 3,000, at least about 4,000, at least about 5,000, at least about 6,000, at least about 7,000, at least about 8,000, at least about 9,000, at least about 0,000, at least about 20,000, at least about 30,000, at least about 40,000, at least about 50,000, at least about 60,000, at least about 70,000, at least about 80,000, at least about 90,000, at least about 100,000, or more plant shoots can be obtained within about 6 weeks, about 10 weeks, about 2 months, about 2.5 months, about 3 months, about 4 months, about 5 months, about 6 months.

[00323] To further improve the shoot multiplication rate, a separation step can be added during or immediately after one or more steps selected from steps (a), (b), (c), and (d). For example, multiple shoot buds produced in step (a) and/or step (c), or multiple shoots produced in step (c) and/or step (c) can be separated into individual pieces, and each of the separated pieces can be placed in an individual container comprising fresh media. For example, multiple shoot buds developed in a bud induction medium can be divided into individual pieces, and placed either on a fresh bud induction medium, or on a fresh shoot elongation/maintenance medium; multiple shoots developed in a shoot elongation/maintenance medium can be separated into individual pieces, and placed either on a fresh shoot elongation/maintenance medium, or a fresh bud induction medium. Each separated piece may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more shoot buds or shoots.

[00324] The present invention also provides methods for plant micropropagation using a plant growth system described herein. [00325] In some embodiments, a plant growth system of the present invention is used for plant micropropagation. In some embodiments, it is used for bamboo micropropagation. In some embodiments, it is used for micropropagation of Phyttostachys edulisi 'Moso', Phyllostachys bissetti, Fargesia denudata, Pieioblastns fortunei, Sasa Veitchii, P!eiobiastns viridistriatus, Thamnocalamus crassinodiis, Chusqiiea Cuieo "Cana Prieta ", Bambusa Old Hamii, Phyllostachys Moso, Phyllostachys Atrovaginata, Dendrocalamus Asper, or Guadna Angus ti folia, Nigra Henon, Rufa, or Nigra.

[00326] In some embodiments, a plant growth system of the present invention is used for plant micropropagation, wherein the plant is a perennial, grass, or phtyo-pharmaceutical plant. In some embodiments, it is used for micropropagation of a perennial. The perennial can be an evergreen, deciduous, monocarpic, woody, or herbaceous perennial. In some embodiments, the perennial is Begonia, banana, goldenrod, mint, agave, maple tree, pine tree, apple tree, alfalfa or red clover. In some embodiments, it is used for micropropagation of a grass. The grass can be of the Poaceae (or Gramineae), Cyperaceae or Juncaceae family. The grass can be a perennial grass or a cereal grass. The grass can be switchgrass, big biuestem, miseanthus, alfalfa, orchard grass, or reed canarygrass. The grass can be bamboo, tall fescue, goat grass, or Kentucky bluegrass. Other types of grasses include wheat, rye, oat, barley, soy, and hemp, as well as straws derived therefrom. In some embodiments, it is used for micropropagation of a phyto-pharmaceutical plant. In some embodiments, it is used for micropropagation of Aloe vera, Ginger, Grape, Cannabis, Garlic, Onion, Echinacea, Geranium, Hakonechloa, Miseanthus, Arundo donax, Switch grass, Rice, or Sugar cane.

[00327] Referring again to the system 100 in FIG. 1, in use for plant micropropagation, the plant propagation sequence starts with placing an explant into the growth vessel 1 10. In some embodiments, the first media container 130 comprises a bud induction medium as described herein, and the second media container 150 comprises a shoot elongation/maintenance medium.

[00328] In some embodiments, the bud induction medium comprises an effective amount of thidiazuron (TDZ) or analog thereof, and wherein the shoot elongation/maintenance medium comprises an effective amount of one or more cytokinins other than TDZ or an analog thereof. In some embodiments, the concentration of TDZ or analog thereof in the bud induction medium is about 0.25 mg/L to about 100 rng/L, e.g., from 0.5 mg L to about 2 mg/L. In some embodiments, one or more cytokinins other than TDZ or an analog thereof in the shoot elongation/maintenance medium is selected from the group consisting of N⁶-benzylaminopurine (BAP), meta-topolin (mT), zeatin, kinetin, 2-isopentenyladenine (2ip), adenine hemisulfate, dimethylallyladenine, N-(2- chloro-4-pyridyl)-N'- phenylurea) (4-CPPU), and analogs of each thereof. In some embodiments, the concentration of the one or more cytokinins other than TDZ or an analog thereof is from about 0.01 mg/L to about 100 mg/L, e.g., from about 0.25 mg/L to about 5 mg L. In some embodiments, the bud induction medium and/or the shoot elongation-'maintenance medium further comprises one or more auxins, such as β-naphthoxyacetic acid (NAA), 2,4- Dichlorophenoxy acetic acid (2,4-D), indole-3-butyric acid (IB A), indole-3-acetic acid (IAA), picloram, and analogs of each thereof.

[00329] In some embodiments, the first incubation sequence of 304 lasts for about half hour to about three weeks, e.g., for about 24 hours to about 60 hours, and the second incubation sequence of 314 lasts for about 24 hours to about four weeks, e.g., for about three days to about five days.

[00330] In some embodiments, the length the plant propagation sequence is determined by the multiplication rate reached. In some embodiments, the multiplication rate is from at least about 1,000 to at least about 100,000 within about 3 weeks to about 6 months.

[00331] Bioreactor methods

[00332] 1. The methods are suitable for both small scale (e.g., laboratory) and large scale

(e.g., industrial) plant micropropagation.

[00333] 2. The methods allow the pulsing micropropagation technology described herein to be used in a more efficient way (e.g., recycled medium; less labor: more accurate control: less contamination; etc.).

[00334] 3. The methods enable greatly improved shoot/plant multiplication over prior methods (e.g., micropropagation using solid medium, and micropropagation using liquid medium without a bioreactor).

[00335] 4. The methods enable greater plant survival rate over prior methods, particularly for certain plant species, such as Moso bamboo.

[00336] 5. Bamboo releases phenolics which are harmful to the shoots/plants when they buildup in the media/environment, which has been a problem with using solid medium.

[00337] Moving from solid growth environment (e.g., plant micropropagation in tissue culture tubes/boxes) to the liquid environment and combining pulsing methods and a bioreactor system, the present invention achieves a major improvement in number of shoots/plants that are obtained, as well as improving the resultant plants' healt and ability to produce full size plants. Without wishing to be bound by any theory, the inventors believe these achievements are the result of controlling/reducing the exposure of the shoots/plantlets to toxic components in the growth compositions (e.g., certain plant hormones, such as TDZ) and/or plant produced by-products (e.g., phenolics), by utilizing the bioreactor systems of the present invention.

[00338] In addition to the methods described above which are based on using "bud induction media" and "shoot elongation/maintenance media" combination, the present invention also provides alternative plant micropropagation methods based on using "Stage 1 media", "Stage 2 media", "Stage 3 media", and/or more media.

[00339] In embodiments, the methods comprising using at least one "Stage 1 media" and at least one "Stage 2 media", and an explant. In some embodiments, the Stage 1 and Stage 2 media are used sequentially during plant propagation. In some embodiments, the explants remain on the Stage 1 medium for about 1 to about 36 hours (e.g., when spiked media are used). In some embodiments, the explants remain on the Stage 1 medium for 10-120 days (e.g., when standard or reduced media are used). In some embodiments, the explants stay on Stage 1 medium for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more rounds (e.g., in each round, explants are transferred from an old Stage 1 medium to a fresh Stage 1 medium) before being transferred to the Stage 2 medium. In some embodiments, the explants remain on the Stage 2 medium for about 1 to about 36 hours (e.g., when spiked media are used). In some embodiments, the explants remain the Stage 2 medium for about 10-120 days (e.g., when standard or reduced media are used). In some embodiments, the explants stay on Stage 2 medium for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more rounds (e.g., in each round, explants are transferred from an old Stage 2 medium to a fresh Stage 2 medium).

[0034Θ] In some embodiments, the Stage 1 and Stage 2 media are used in rotation during plant propagation. In some embodiments, the rotation is continuous for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cycles. In some embodiments, the explants stay on Stage 1 medium for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more rounds first before being transferred to the Stage 2 medium. In some embodiments, the explants stay on Stage 2 medium for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more rounds first before being transferred back to the Stage 1 medium. In some embodiments, the explants are on the Stage 1 medium for about 1 -36 hours (e.g., when spiked media are used) or more (e.g., when standard or reduced media are used) followed by being transferred to the Stage 2 medium. In some embodiments, the rotation is continuous until multiple shoots are observed. In some embodiments, the rotation takes about 1 week to about 24 months, depending on plant species and media.

[00341] Optionally, the multiplied shoots are then placed on a Stage 3 medium for further multiplication until desired number of shoots is obtained, depending on previous treatments. The explants on the Stage 3 medium can be further transferred to a Stage 4 medium, in some embodiments, the explants are on the Stage 3 medium for about 1-36 hours (e.g., when spiked media are used) followed by being transferred to the Stage 4 medium. In some embodiments, the explants are on the Stage 3 medium for about 10- 120 days or more (e.g., when standard or reduced media are used) followed by being transferred to the Stage 4 medium. In some embodiments, the explants remain the Stage 4 medium for about 10-120 days.

[00342] Alternatively, the multiplied shoots obtained from a Stage 2 medium can be rotated between at least one Stage 3 medium and at least one Stage 4 medium. In some embodiments, the rotation is continuous for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cycles. In some embodiments, the rotation is continuous until desired number of shoots. In some embodiments, the desired number of shoots is obtained by separation into new tubes and further expansion. In some embodiments, about one to ten shoots per tube are obtained per multiplication cycle.

[00343] In some embodiments, the explants are placed on a Stage 1 medium, a Stage 2 medium, and a Stage 3 medium in rotation, until desired number of shoots is obtained. In some embodiments, the multiplied shoots are then placed on a Stage 4 medium.

[00344] In some embodiments, the explants are placed on a Stage 1 medium and a Stage 2 medium in rotation, until desired number of shoots is obtained. In some embodiments, the multiplied shoots are then placed on a rotation of a Stage 3 medium and a Stage 4 medium.

[00345] In some embodiments, the explants are placed on a Stage 1 medium for about 1, 2,

3, 4, 5, 6, 7, 8, 9, 10, or more rounds, and then transferred to a Stage 2 medium for about 1, 2, 3,

4, 5, 6, 7, 8, 9, 10, or more rounds, until desired number of shoots is obtained. In some embodiments, the multiplied shoots are then placed on a rotation of a Stage 3 medium and a Stage 4 medium.

[00346] In some embodiments, the explants are placed on a Stage I medium first. In some embodiments, the explants are on the Stage 1 medium for about 1-36 hours (e.g., when spiked media are used). In some embodiments, the explants are on the Stage 1 medium for about 10-120 days or more (e.g., when standard or reduced media are used). In some embodiments, this step comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more rounds of fresh Stage 1 medium. Then, the explants are kept on a rotation of a Stage 2 medium and a Stage 3 medium, until desired number of shoots is obtained. In some embodiments, the multiplied shoots are then placed on a Stage 4 medium. In some embodiments, the multiplied shoots remain on the Stage 4 medium for about 10-120 days.

[00347] Still optionally, the multiplied shoots obtained from a Stage 4 medium can be transferred onto a Stage 5 medium as described herein. In some embodiments, the shoots are placed on a Stage 5 medium for about 1-24 hours or more (e.g., when spiked media are used). In some embodiments, the shoots are placed on a Stage 5 medium for about 10 to 120 days (e.g., when standard or reduced media are used). In some embodiments, the shoots are transferred to small tissue culturing boxes, such as the magenta boxes.

[00348] Still optionally, the explants are kept on the Stage 5 medium first until the desired number of shoots is obtained, then transferred to a Stage 6 medium as described herein. In some embodiments, the shoots are placed on a Stage 6 medium for about 1-24 hours or more (e.g., when spiked media are used). In some embodiments, the shoots are placed on a Stage 6 medium for about 10 to 120 days (e.g., when standard or reduced media are used).

[00349] In some embodiments, the explants obtained from the Stage 4 medium are placed on a rotation of a Stage 5 medium and a Stage 6 media, until the desired number of shoots is obtained.

[00350] Still optionally, the explants kept on the Stage 6 medium are transferred to a Stage

7 medium as described herein. In some embodiments, the shoots are placed on a Stage 7 medium for about 1-24 hours or more (e.g., when spiked media are used). In some embodiments, the shoots are placed on a Stage 7 medium for about 10 to 120 days (e.g., when standard or reduced media are used).

[00351] Still optionally, the multiplied shoots obtained from a Stage 4 medium can be transferred onto a rotation of a Stage 4 medium, a Stage 5 medium, and a Stage 6 medium as described herein.

[00352] Still optionally, the multiplied shoots obtained from a Stage 7 medium can be transferred one or more other additional media (e.g., a Stage 8, a Stage 9, etc) for further propagation if needed,

[00353] A variety of appropriate explants can be used in accordance with the present disclosure, in certain embodiments according to the present disclosure, immature nodal sections from stems can be used as the explant material. In one embodiment, the explants can be new growth canes with the lateral shoots just breaking the sheath at nodal section(s). New growth canes include those obtained from the plant within a current season or year, wherein such new growth canes can be obtained from any node on the plant. In one particular embodiment, explant material includes or is limited to the third node from the base of a cane.

[00354] In some embodiments, the plant is a bamboo. Detailed methods for collecting and initially disinfecting bamboo explants are described in WO/2011/100762, which is incorporated herein by reference in its entirety. In some embodiments, the disinfectant such as dichloroisocyanuric acid, dichloroisanuric acid, triehlorotriazinetriona, mercuric chloride, hydrogen peroxide, FungiGoneTM (bioWorkl, Inc., Dublin, OH), plant preservatives can be used. In some embodiments, following the initial disinfection, the outer sheaths of a bamboo can be peeled off and discarded and the remaining piece can be put into an approximately 1% to about 50% solution of a commercial bleach or a similar disinfecting solution. In some embodiments, the bleach can be heated to about 20-60°C, such as 23-50°C. In some embodiments, sonication and vacuum infiltration of the tissue can also be used with the described disinfection procedures. 00355] In some embodiments, the multiplication process can continue substantially indefinitely by continuing to separate and multiply shoots. In some embodiments, the multiplication cycles can be repeated without initiating new explants for at least 1 month, at least 3 months, at least 6 months, at least 12 months, at least 24 months, at least 36 months, or more. In some embodiments, the multiplication cycles include 1 -10 days per cycle, 2-9 days per cycle, 3-6 days per cycle, 0.5-3 days per cycle, 4-5 days per cycle, 0.5-1 day per cycle, 10-120 days per cycle, etc.

[00356] The present invention has many advantages. Without wishing to be bound by any theory, the methods disclosed herein do not require the use of seeds or inflorescence to start plants, or selection of diseased starting plants, or the use of antibiotics, somatic embryogenesis, pseudospiklets, or induction and/or reversion of flowering. For successful growth following tissue culture, the produced plants do not require watering directly on the pot but remain robust with overhead watering and do not require multiple adjustments to light intensity or humidity conditions prior to transfer to a greenhouse or other growing conditions. Moreover, media can be free from polyaspartic acid(s), seaweed concentrates and/or surfactants. These improvements over prior methods provide even additional advantages related to the health of produced plants and efficiency of growth and processing.

[00357] In some embodiments, the present invention can be used for grass propagation. In some embodiments, the micropropagated plants have not been genetically modified. Other particular embodiments exclude the use of timentin and/or kanamycin in the micropropagation procedure.

[00358] In some embodiments, when the methods are used for bamboo propagation, explants from a bamboo plant between the age of 3 months and 3 years are used. In some embodiments, a node from the cane with the lateral shoot just breaking the sheath can be used as the explants. In some embodiments, each nodal section can be cut into 3-5 millimeter sections with the shoot intact. In some embodiments, the outer sheaths can be peeled off and discarded and the remaining nodal section piece put into a 10% bleach solution with a final concentration of 0.6% sodium hydrochloride. In some embodiments, the explant in bleach solution can be placed onto a Lab Rotators, Adjustable speed, Barnstead/Lab line orbital Shaker (model number KS 260) shaker table for 1 hour at 6-9 revolutions per minute. The explants can then be put into a 1% bleach solution with a final concentration of 0.06% sodium hydrochloride, and be placed back onto the shaker table for 30 minutes. This 1% bleach solution step can then be repeated.

[00359] In some embodiments, individual explants can then be placed on a Stage 1 media (15-25 mL) within a tube and the tubes can be placed into a regulated clean growth chamber at a temperature of from 65°F-70°F and a full spectrum light level of 36-54 nmole/m²/s². The initial Stage 1 media can be B0038-iv at a pH of 5.7. The explants can then be transferred to fresh B0038-iv media every 10-120 days (usually every 21 days), with contaminated tubes being discarded.

[00360] In some embodiments, if a spiked version of the B0038-iv media is utilized, the explants can be placed in the spiked media for 0.25, 0.5, 0.75, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47 or 48 hours before transition to a "standard" media disclosed herein or to a media containing substantially reduced or no cytokinins ("reduced" media as used herein) for the remainder of the 10-120 day cycle. Additional time periods for placement in a spiked media include anywhere between 0.1 and 240 hours and can include, without limitation, 0.1-0.5 hours, 0.3-2,5 hours, 2.5-6 hours, 1-10 hours, 5-15 hours, 10-20 hours, 15-25 hours, 20-30 hours, 25-35 hours, 30-40 hours, 35-45 hours, 40-50 hours, 45-55 hours, 50-60 hours, 55-65 hours, 60-70 hours, 65-75 hours, 70-80 hours, 75-85 hours, 80-90 hours, 85-95 hours, 90-100 hours, 95- 105 hours, 100-l lO hours, 105-115 hours, 1 10-120 hours, 15-125 hours, 120-130 hours, 125-135 hours, 130-140 hours, 135-145 hours, 140-150 hours, 145-155 hours, 150-160 hours, 155-165 hours, 160-170 hours, 165-175 hours, 170-180 hours, 175-185 hours, 180-190 hours, 185-195 hours, 190-200 hours, 195-205 hours, 200-210 hours, 205-215 hours, 210-220 hours, 215-225 hours, 220-230 hours, 225-235 hours, 230-240 hours, 235-245 hours, 240-250 hours, 3-6 hours, 7-17 hours, 12-22 hours, 17-27 hours, 22-32 hours, 27-37 hours, 32-42 hours, 37-47 hours, 42-52 hours, 47-57 hours, 52-62 hours, 57-67 hours, 62-72 hours, 67-77 hours, 72-82 hours, 77-87 hours, 82-92 hours, 87-97 hours, 92-102 hours, 97-107 hours, 102-112 hours, 107-117 hours, 112-122 hours, 117-127 hours, 122-132 hours, 127-137 hours, 132-142 hours, 137-147 hours, 142-152 hours, 147-157 hours, 152-162 hours, 157-167 hours, 162-172 hours, 167-177 hours, 172-182 hours, 177-187 hours, 62-172 hours, 167- 77 hours, 182-192 hours, 187-197 hours, 192-202 hours, 197-207 hours, 202-212 hours, 207-217 hours, 212-222 hours, 217-227 hours, 222-232 hours, 227-237 hours, 232-242 hours, 237-247 hours or 242-252 hours. Placement in spiked media can also be 0.5 hours less than a cycle in standard or reduced media, 1 hour less than a cycle in standard or reduced media and all time periods in between 1 and 240 hours less than a cycle in standard or reduced media. Alternatively, in place of spending the remainder of the cycle in the standard or reduced media, explants can be placed on a spiked media for a period of time followed by culture on a standard or reduced media for the full cycle time (i.e. 10-120 days not reduced by time spent in the spiked media).

[00361] Media containing no cytokinins or substantially reduced cytokinins can be a reduced media with all cytokinins and/or auxins removed or can have at least one eytokmin and/or auxin's amount reduced by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, 1 -10%, 5-15%, 10-20%, 15-25%, 20-30%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60-70%, 65-75%, 70- 80%, 75-85%, 80-90%, 85-95%, 90-100%, 3-6%, 7-17%, 12-22%, 17-27%, 22-32%, 27-37%, 32- 42%, 37-47%, 42-52%, 47-57%, 52-62%, 57-67%, 62-72%, 67-77%, 72-82%, 77-87%, 82-92% or 87-97%,

[00362] Alternatively, the cytokinins noted above can be replaced with weaker cytokinms at similar or higher levels. Exemplary weaker cytokinins include zeatin and kinetin. [00363] Contaminated tubes can be identified by bacterial discoloration of the agar or by visible surface contamination. These explants can stay on the chosen media for 3-4 10-120 day cycles (usually 21 day cycles) or as modified in the spiked procedure (spiked media for a period of 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47 or 48 hours before transition to a standard or reduced media for the remainder of the 10-120 day cycle or for a full 10-120 day cycle). Additional time periods for placement in a spiked media include anywhere between 0.1 and 240 hours and can include, without limitation, 0.1-0.5 hours, 0.3-2.5 hours, 2,5-6 hours, 1 -10 hours, 5-15 hours, 10-20 hours, 15-25 hours, 20-30 hours, 25-35 hours, 30-40 hours, 35-45 hours, 40-50 hours, 45-55 hours, 50-60 hours, 55-65 hours, 60-70 hours, 65-75 hours, 70- 80 hours, 75-85 hours, 80-90 hours, 85-95 hours, 90-100 hours, 95-105 hours, 100-110 hours, 105- 115 hours, 110-120 hours, 115-125 hours, 120-130 hours, 125-135 hours, 130-140 hours, 135-145 hours, 140-150 hours, 145-155 hours, 150-160 hours, 155-165 hours, 160-170 hours, 165-175 hours, 170-180 hours, 175-185 hours, 180-190 hours, 185-195 hours, 190-200 hours, 195-205 hours, 200-210 hours, 205-215 hours, 210-220 hours, 215-225 hours, 220-230 hours, 225-235 hours, 230-240 hours, 235-245 hours, 240-250 hours, 3-6 hours, 7-17 hours, 12-22 hours, 17-27 hours, 22-32 hours, 27-37 hours, 32-42 hours, 37-47 hours, 42-52 hours, 47-57 hours, 52-62 hours, 57-67 hours, 62-72 hours, 67-77 hours, 72-82 hours, 77-87 hours, 82-92 hours, 87-97 hours, 92- 102 hours, 97-107 hours, 102-112 hours, 107-117 hours, 112-122 hours, 117-127 hours, 122-132 hours, 127-137 hours, 132-142 hours, 137-147 hours, 142-152 hours, 147-157 hours, 152-162 hours, 157-167 hours, 162-172 hours, 167-177 hours, 172-182 hours, 177-187 hours, 162-172 hours, 167-177 hours, 182-192 hours, 187-197 hours, 192-202 hours, 197-207 hours, 202-212 hours, 207-217 hours, 212-222 hours, 217-227 hours, 222-232 hours, 227-237 hours, 232-242 hours, 237-247 hours or 242-252 hours. Placement in spiked media can also be 0.5 hours less than a cycle in standard or reduced media, 1 hour less than a cycle in standard or reduced media and all time periods in between 1 and 240 hours less than a cycle in standard or reduced media. If an explant is cultured on a particular spiked media type, when transferred to a standard or reduced media, the standard or reduced media can be of the same type (e.g. standard or reduced) or of a different type.

[00364] In particular embodiments disclosed herein, culture periods are less than 12 weeks, less than 9 weeks, or less than 6 weeks. [00365] Explants can be taken off the media after the third cycle if multiplication is occurring. If multiplication is not occurring or not occurring to a significant degree, explants can be left on the media for a fourth cycle.

[00366] Live shoots can next be transferred to a Stage 2 media at a pH of 5.7. The cultures can stay on this Stage 2 media until the desired number of shoots is obtained by separation into new tubes and further expansion. Generally, the range of time includes 10-120 day cycles (usually 14-21 day cycles) between which the cultures are assigned to go through another multiplication round or transitioned to a Stage 3 or Stage 4 media at a pH of 5.7 for further multiplication.

[00367] In particular embodiments disclosed herein, culture periods are less than 12 weeks, less than 9 weeks, or less than 6 weeks.

[00368] Alternatively, live shoots can also be placed on a spiked media for a period of 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 1 5, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 hours before transition to a same or different type of standard or reduced media for the remainder of the 10-120 day cycle or for a full 10-120 day cycle. Additional time periods for placement in a spiked media include anywhere between 0.1 and 240 hours and can include, without limitation, 0.1-0.5 hours, 0.3-2.5 hours, 2.5-6 hours, 1-10 hours, 5-15 hours, 10-20 hours, 15-25 hours, 20-30 hours, 25-35 hours, 30-40 hours, 35-45 hours, 40-50 hours, 45-55 hours, 50-60 hours, 55-65 hours, 60-70 hours, 65-75 hours, 70-80 hours, 75-85 hours, 80-90 hours, 85-95 hours, 90-100 hours, 95-105 hours, 100-l lO hours, 105-115 hours, 110-120 hours, 115-125 hours, 120-130 hours, 125-135 hours, 130- 140 hours, 135-145 hours, 140-150 hours, 145-155 hours, 150-160 hours, 155-165 hours, 160-170 hours, 165-175 hours, 170-180 hours, 175-185 hours, 180-190 hours, 185-195 hours, 190-200 hours, 195-205 hours, 200-210 hours, 205-215 hours, 210-220 hours, 215-225 hours, 220-230 hours, 225-235 hours, 230-240 hours, 235-245 hours, 240-250 hours, 3-6 hours, 7-17 hours, 12- 22 hours, 17-27 hours, 22-32 hours, 27-37 hours, 32-42 hours, 37-47 hours, 42-52 hours, 47-57 hours, 52-62 hours, 57-67 hours, 62-72 hours, 67-77 hours, 72-82 hours, 77-87 hours, 82-92 hours, 87-97 hours, 92-102 hours, 97-107 hours, 102-112 hours, 107-117 hours, 112-122 hours, 1 17-127 hours, 122-132 hours, 127-137 hours, 132-142 hours, 137-147 hours, 142-152 hours, 147-157 hours, 152-162 hours, 157-167 hours, 162-172 hours, 167-177 hours, 172-182 hours, 177-187 hours, 162-172 hours, 167-177 hours, 182-192 hours, 1 87-197 hours, 192-202 hours, 197-207 hours, 202-212 hours, 207-217 hours, 212-222 hours, 217-227 hours, 222-232 hours, 227-237 hours, 232-242 hours, 237-247 hours or 242-252 hours. Placement in spiked media can also be 0,5 hours less than a cycle in standard or reduced media, 1 hour less than a cycle in standard or reduced media and all time periods in between 1 and 240 hours less than a cycle in standard or reduced media,

[00369] Generally, one-ten shoots per tube can be obtained per multiplication cycle.

[00370] Following removal from the multiplication process, the shoots can be transferred to small tissue culturing boxes (known as "magenta boxes") for 10-120 days (usually 14-21 days) containing a Stage 3, Stage 4 or Stage 5 media. As above, shoots can be placed in spiked media for shorter time periods followed by placement into a standard or reduced media for the remainder of or for a full 10-120 day cycle,

[00371] In particular embodiments disclosed herein, culture periods are less than 12 weeks, less than 9 weeks, or less than 6 weeks.

[00372] As will be understood by one of ordinary skill in the art, when spiked media are used, the use of the spiked media increases the number of media stages within a particular process due the following use of a standard or reduced media. If spiked media are used at only one stage, the process generally expands by 1 media stage. If spiked media are used at two stages, the process generally expands by 2 media stages. If spiked media are used at three stages, the process generally expands by 3 media stages, etc.

[00373] Alternatively, the following procedures may also be used (Stage 1, Stage 2, Stage 3, etc, media are defined elsewhere herein):

[00374] Individual explants can then be placed on a Stage 1 media (15-25 mL) within a tube and the tubes placed into a regulated clean growth chamber at a temperature of from 65°F-70°F and a full spectrum light level of 36-90 μηιοΙε/ηΐ'Α:². The Stage I media can be standard or spiked media. If placed on standard media, the explants can be transferred to fresh media every 0-120 days (usually every 2 days), with contaminated tubes being discarded. If on spiked media, the explants can remain on the spiked media for 0.25, 0.5, 0.75, I, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 hours and then be transferred to a media without spiked components (standard or reduced) for the remainder of the 10-120 day cycle or for a full 10-120 day cycle. Additional time periods for placement in a spiked media include anywhere between 0.1 and 240 hours and can include, without limitation, 0.1-0.5 hours, 0.3-2.5 hours, 2.5-6 hours, 1-10 hours, 5-15 hours, 10-20 hours, 5-25 hours, 20-30 hours, 25-35 hours, 30-40 hours, 35-45 hours, 40-50 hours, 45-55 hours, 50-60 hours, 55-65 hours, 60-70 hours, 65-75 hours, 70-80 hours, 75- 85 hours, 80-90 hours, 85-95 hours, 90-100 hours, 95-105 hours, 100-110 hours, 105-115 hours, 1 10-120 hours, 115-125 hours, 120-130 hours, 125-135 hours, 130-140 hours, 135-145 hours, 140- 150 hours, 145-1 55 hours, 150-160 hours, 155-165 hours, 160-170 hours, 165-175 hours, 170-1 80 hours, 175-1 85 hours, 180-190 hours, 185-195 hours, 190-200 hours, 195-205 hours, 200-210 hours, 205-215 hours, 210-220 hours, 215-225 hours, 220-230 hours, 225-235 hours, 230-240 hours, 235-245 hours, 240-250 hours, 3-6 hours, 7-17 hours, 12-22 hours, 17-27 hours, 22-32 hours, 27-37 hours, 32-42 hours, 37-47 hours, 42-52 hours, 47-57 hours, 52-62 hours, 57-67 hours, 62-72 hours, 67-77 hours, 72-82 hours, 77-87 hours, 82-92 hours, 87-97 hours, 92-102 hours, 97- 107 hours, 102-112 hours, 107-117 hours, 1 12-122 hours, 1 17-127 hours, 122-132 hours, 127-137 hours, 132-142 hours, 137-147 hours, 142-152 hours, 147-157 hours, 152-162 hours, 157-167 hours, 162-172 hours, 167-177 hours, 172-182 hours, 177- 87 hours, 162-172 hours, 167-177 hours, 182-192 hours, 187-197 hours, 192-202 hours, 197-207 hours, 202-212 hours, 207-217 hours, 212-222 hours, 217-227 hours, 222-232 hours, 227-237 hours, 232-242 hours, 237-247 hours or 242-252 hours. Placement in spiked media can also be 0.5 hours less than a cycle in standard or reduced media, 1 hour less than a cycle in standard or reduced media and all time periods in between 1 and 240 hours less than a cycle in standard or reduced media. These explants can stay on standard media or spiked media for 2 10-120 day cycles (usually 21 day cycles). Between cycles, excess sheaths can be removed. At the time of transfer to the third cycle, explants can be transitioned to a Stage 2 media or Stage 3 media (depending on whether spiked procedures are used), in this Example, standard media supplemented with 7 g/L carageenan rather than the 5.5 g/L provided above or a spiked B0038-iv supplemented with 7 g/L carageenan rather than the 5.5 g L provided above for 0,25, 0.5, 0,75, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 hours followed by transition to standard or reduced media. Additional time periods for placement in a spiked media include anywhere between 0, 1 and 240 hours and can include, without limitation, 0.1-0.5 hours, 0.3-2.5 hours, 2,5-6 hours, 1-10 hours, 5-15 hours, 10-20 hours, 15-25 hours, 20-30 hours, 25-35 hours, 30-40 hours, 35-45 hours, 40-50 hours, 45-55 hours, 50- 60 hours, 55-65 hours, 60-70 hours, 65-75 hours, 70-80 hours, 75-85 hours, 80-90 hours, 85-95 hours, 90-100 hours, 95-105 hours, 100-1 10 hours, 105-1 15 hours, 1 10-120 hours, 1 15-125 hours, 120-130 hours, 125-135 hours, 130-140 hours, 135-145 hours, 140-150 hours, 145-155 hours, 150- 160 hours, 155-165 hours, 160-170 hours, 165-175 hours, 170-180 hours, 175-185 hours, 180-190 hours, 185-195 hours, 190-200 hours, 195-205 hours, 200-210 hours, 205-215 hours, 210-220 hours, 215-225 hours, 220-230 hours, 225-235 hours, 230-240 hours, 235-245 hours, 240-250 hours, 3-6 hours, 7-17 hours, 12-22 hours, 17-27 hours, 22-32 hours, 27-37 hours, 32-42 hours, 37-47 hours, 42-52 hours, 47-57 hours, 52-62 hours, 57-67 hours, 62-72 hours, 67-77 hours, 72- 82 hours, 77-87 hours, 82-92 hours, 87-97 hours, 92-102 hours, 97-107 hours, 102-112 hours, 107- 1 17 hours, 112-122 hours, 117-127 hours, 122-132 hours, 127-137 hours, 132-142 hours, 137-147 hours, 142-152 hours, 147-157 hours, 152-162 hours, 1 57-167 hours, 162-172 hours, 167-177 hours, 172-1 82 hours, 177-1 87 hours, 162-172 hours, 167-177 hours, 182-192 hours, 187-197 hours, 192-202 hours, 97-207 hours, 202-212 hours, 207-217 hours, 212-222 hours, 217-227 hours, 222-232 hours, 227-237 hours, 232-242 hours, 237-247 hours or 242-252 hours. Placement in spiked media can also he 0.5 hours less than a cycle in standard or reduced media, 1 hour less than a cycle in standard or reduced media and all time periods in between 1 and 240 hours less than a cycle in standard or reduced media. Following the third cycle, explants can be cleaned. The explants can be kept on media supplemented with 7 g/'L carageenan rather than the 5.5 g/L provided above for 10-120 day cycles (usually 21 day cycles) until multiple shoots are observed. Observation of multiple shoots can occur within 3-15 months. When multiple cycles are used, explants can be cultured in standard media for all cycles, on spiked media followed by standard media for all cycles or on spiked media followed by reduced media for all cycles. Alternatively, explants can be exposed to one or more of these treatments across cycles in any combination and order.

[00375] In particular embodiments disclosed herein, culture periods are less than 12 weeks, less than 9 weeks, or less than 6 weeks.

[00376] Once an explant exhibits multiple shoots, it can be either maintained on its current media when shooting occurred (with transfer to fresh media every 10-120 days) or transferred to a subsequent media. The cultures can stay on the current or subsequent media until the desired number of shoots is obtained by separation into new tubes and further expansion. Generally, the range of time includes 10-120 day cycles (usually 21 day cycles) between which the cultures can be assigned to go through another multiplication round or transitioned to a next stage media. [00377] In particular embodiments disclosed herein, culture periods are less than 12 weeks, less than 9 weeks, or less than 6 weeks.

[00378] In some embodiments, the methods comprise using the bioreactors of the present invention. In some embodiments, the methods comprising using the racks of the present invention. In some embodiments, the methods comprise usmg the bioreactors and the racks of the present invention. In some embodiments, when two or more media described above are used in rotation during micropropagation, a bioreactor described herein can be used, for example, when a Stage 1 and a Stage 2 media are used in rotation, when a Stage 3 and a Stage 4 media are used in rotation, and/or when a Stage 1 , a Stage 2, and a Stage 3 media are used in rotation, etc.

Plant Propagation Bioreactor

[00379] Bioreactors can be used for plant micropropagation to more efficiently increase shoot mass than in stationar cultures. In some embodiments, bioreactors can be used for micropropagation to induce the formation of microtubers. In some embodiments, the induction of microtuber formation is done so more synchronously and in greater numbers in a bioreactor than in stationar cultures. Bioreactors offer a promising way of scaling-up micropropagation processes, making it possible to work in large containers with a high degree of control over culture parameters (e.g., pH, aeration, oxygen, carbon dioxide, hormones, nutrients, etc.). Bioreactors are also compatible with the automation of micropropagation procedures, utilizing artificial intelligence, which reduces production costs.

[00380] Previously bioreactors have been mostly applied to microbial technology, cell culture, and somatic embryogenesis and prior to the present invention, bioreactors for plant micropropagation were rare, complicated, and expensive.

[00381] To solve these problems, the present invention provides novel compositions, methods, and systems for the micropropagation of plants using a bioreactor. Without wishing to be bound by any theory, the present invention achieves greatly improved plant micropropagation through controlling/reducing exposure of the shoots/plantlets to toxic components that build-up in the growth compositions/environment (e.g., certain plant hormones, such as TDZ) and/or are produced as by-products of plant growt (e.g., phenolics).

[00382] The present application also provides a system for plant micropropagation. The system is also useful for the production of perennials, grasses and phy to-pharmaceutical plants. In some embodiments, the system comprises a growth vessel, two or more media containers, and a power source for driving fluid into and/or out of the growth vessel. In some embodiments, the system further comprises a controller. In some embodiments, the system further comprises a light source and/or a gas source providing C02, 02, N2, or mixture thereof to the growth vessel

[00383] In some embodiments, a temporary immersion bioreactor (TIB, a.k.a. temporary immersion system (TIS) or "Ebb and Flow" bioreactor) is used. Non-limiting examples of temporary immersion bioreactors include nutrient mist bioreactors, tilting and rocking vessels, twin flask system, or single containers with at least two compartments, such as Recipient for Automated Temporary Immersion (RITA®).

[00384] In some embodiments, the temporary immersion bioreactor is disposable. In some embodiments, the temporary immersion bioreactor is reusable.

[00385] In some embodiments, the temporary immersion bioreactor involves a wetting and drying cycle which occurs periodically in a predetermined period of time and hence it can also be termed as periodic, temporary immersion. In some embodiments, the temporary immersion bioreactor has a mere up-and-down motion of the nutrient medium without renewal. In some embodiments, renewal of nutrient medium in the bioreactor is involved.

[00386] Temporary immersion bioreactors provide an excellent way of using liquid medium at the same time controlling the gaseous environment. Moreover, it can provide the possible automation of the production system which facilitates low production costs. In other words, increasing the rate of growth and multiplication by using bioreactors more plants per unit area of the growth room are produced, which reduces the cost per plant per unit space of growth room. Liquid culture bioreactors are mainly suitable for the large-scale production of small size somatic embryos, growth of bulb, corms, micro tubers, compact shoot cultures etc. Major features of a temporary immersion bioreactor are:

• Reduction of hyperhydricity, compared with that of permanent immersion, is the major achievement of a temporary immersion system. As plants are immersed in the medium for short time, the physiological disorders are reduced and the plants become healthier. ® Plant growth and development can be controlled by manipulating the frequency and

duration of immersion in liquid medium.

® Plant growth is improved because during every immersion the plant is in direct contact with the medium and a thin film of liquid covers the plant throughout the interval period. ® Air vents attached to the vessel prevent the cultures from contamination.

• Due to the lack of agitation or aeration, the mechanical stress on plant tissues are

generally low compared with the other bioreactor systems.

[00387] Temporary immersion bioreactors, which represent simple plastic vessels with medium (e.g., liquid, semi-liquid, etc) moving from one side to another every several minutes, can be used to generate microtubers. This temporary immersion system has been shown to stimulate shoot multiplication in many plant species. For example, the multiplication rates for sugarcane and pineapple were 6 and 3-4 times, respectively, higher compared with the rates obtained in liquid or solid media (Lorenzo et al., 1998; Escalona et al., 1999). In some embodiments, the bioreactor used in the present invention is a bioreactor described in International Patent Application No. PCT/US2012/047622, which is incorporated by reference in its entirety.

[00388] Other non-limiting examples of plant micropropagation systems include those described in U.S. Patent. Nos. 3,578,431; 4,320,594; 4,669,217; 4,908,315; 4,934,096; 5,049,505; 5,088,231 ; 5,104,527; 5,119,588; 5,139,956; 5,171,683; 5,184,420; 5,186,895; 5,212,906; 5,225,342; 5,558,984; 5,597,731; and US 6,753,178. More examples of plant micropropagation systems can be found in Etienne et al. (Bioreactors in coffee micropropagation, Braz. J. Plant Physiol., 18(l):45-54, 2006); Ziv (Bioreactor Technology for Plant Micropropagation Horticultural Reviews, Volume 24, Edited by Jules Janick ISBN 0-471-33374-3); and Paek et al. (Application of bioreactors for large-scale micropropagation systems of plants. In Vitro Cell. Dev. Biol. -Plant 37: 149-157, March-April 2001). It is understood that plant propagation systems that can be used in the present invention includes those derived from the ones described above by adding or reducing one or more parts/features of the systems known to one skilled in the art.

[00389] In some embodiments, the present invention provides novel compositions, methods, and systems for the induction, establishment, and maturation of embryos from plants using a bioreactor.

[00390] In some embodiments, the media can be used for in vitro propagation through somatic embryogenesis.

[00391] In some embodiments, provided are the first type of media that can initiate an embryogenic response in a plant or a plant part.

[00392] In some embodiments, the media can comprise nutrients selected from the group consisting of amino acids, macroelements, microelements, aluminum, boron, chlorine (chloride), chromium, cobalt, copper, iodine, iron, lead, magnesium, manganese, molybdenum, nitrogen (nitrates), potassium, phosphorous (phosphates), silicon, sodium, sulphur (sulphates), titanium, vanadium, zinc, inositol and undefined media components such as casein hydrolysat.es or yeast extracts. For example, the media can include any combination of NH4NO3; KNO3; Ca(N0₃)₂; K2SO4; MgS0₄; MnS()₄; ZnSC ; K2SO5; CuSO.i; CaCb; KI; C0CI2; H3BO3; NSP.MOOU; H2PO4; FeS()₄; Na₂EDTA; Na₂H₂P04; inositol (e.g., myo-inositol); thiamine; pyridoxine; nicotinic acid; glycine; riboflavin; ascorbic acid; and silicon standard solution. It is known to those in the art that one or more components mentioned above can be omitted without affecting the function of the media.

[00393] In some embodiments, the media comprise macronutrients (e.g., ammonium nitrate, ammonium sulfate, calcium chloride anhydrous, magnesium sulfate anhydrous, potassium nitrate, potassium phosphate monobasic), micronutrients (e.g., boric acid, cobalt chloride anhydrous, cupric sulfate anhydrous, ferrous sulfate, manganese sulfate, molybdic acid sodium salt, Na₂- EDTA, potassium iodide, zmc sulfate), and vitamins (e.g., glycerine, myo-Inositol, nicotinic acid Pyridoxine-HCl, thiamine-HCl), or those found in the MS media (Murashige and Skoog, 1962). In some embodiments, the amount of one or more components of the MS media is doubled. In some embodiments, the amount of NH4N03, KN03, Ca(N03)2, K2S04, MgS04, MnS04, ZnS04, CuS04, and/or CaC12 is doubled. In some embodiments, the liquid media are supplemented with one or more amino acids. In some embodiments, the ammo acids can be any one of or combinations of the 20 fundamental amino acids known to one skilled in the art. In some em bodiments, the amino acids can be any derivatives or analogs of the 20 fundamental amino acids. In some embodiments, the ammo acids can be any synthetic amino acids, non-natural amino acids, or combinations thereof. In some embodiments, the amino acids comprise prolin and/or serine.

[00394] The present invention also provides a second type of media used for in vitro propagation of plants through somatic embryogenesis. In some embodiments, the second type of media are liquid or solid media. In some embodiments, the second media comprises one or more salts as described herein, e.g., the salts that can be found in MS media. In some embodiments, the amount of one or more components of the MS media is doubled. In som e embodiments, the amount of NH4N03, KN03, Ca(N03)2, K2S04, MgS04, MnS04, ZnS04, CuSCM, and/or CaC12 is doubled. In some embodiments, the liquid media are supplemented with one or more amino acids. In some embodiments, the amino acids can be any one of or combinations of the 20 fundamental amino acids known to one skilled in the art. In some embodiments, the amino acids can be any derivatives or analogs of the 20 fundamental amino acids. In some embodiments, the amino acids can be any synthetic amino acids, non-natural amino acids, or combinations thereof. In some embodiments, the ammo acids comprise prolin and/or serine.

[00395] The present invention also provides a third type of media used for in vitro propagation of bamboo through somatic embryogenesis. In some embodiments, a third type of medium is a solid medium. In some embodiments, the second media comprises one or more salts as described herein, e.g., the salts that can be found in MS media. In some embodiments, the amount of one or more components of the MS media is doubled. In some embodiments, the amount of NH4N03, KN03, Ca(N03)2, K2S04, MgS04, MnS04, ZnS04, CuS04, and/or CaC12 is doubled.

[00396] In some embodiments, the third media are supplemented with abscisic acid (ABA), derivatives thereof, analogs thereof, or any combinations thereof. In some embodiments, the concentration of ABA is about 1.0 to about 100 μΜ. For example, the concentration of ABA is about 1, 2, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 μΜ or more. In some embodiments, the ABA concentration is about 40-60 mg/L, e.g., about 52.8 mg L.

[00397] In some embodiments, the third media comprise charcoal, such as active charcoal.

Surprisingly, addition of charcoal to this type of media can greatly enhance embryo production and maturation. In some embodiments, the concentration of charcoal is about 0.01% to about 10%, such as about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, or more, by weight. In some embodiments, the liquid media are supplemented with one or more amino acids. In some embodiments, the amino acids can be any one of or combinations of the 20 fundamental amino acids known to one skilled in the art. In some embodiments, the amino acids can be any derivatives or analogs of the 20 fundamental amino acids. In some embodiments, the amino acids can be any synthetic amino acids, non-natural amino acids, or combinations thereof. In some embodiments, the amino acids comprise proline and/or serine.

[00398] The present invention also provides a fourth type of media used for in vitro propagation of bamboo through somatic embryogenesis. in some embodiments, a fourth type of medium is a solid medium, in some embodiments, the fourth type of media is used for embryo germination. In some embodiments, the media of this type are solid media.

[00399] In some embodiments, the fourth type of media comprises one or more salts described herein, e.g., the salts that can be found in MS media. In some embodiments, the liquid media are supplemented with one or more ammo acids. In some embodiments, the amino acids can be any one of or combinations of the 20 fundamental amino acids known to one skilled in the art. In some embodiments, the ammo acids can be any derivatives or analogs of the 20 fundamental ammo acids. In some embodiments, the ammo acids can be any synthetic amino acids, non-natural amino acids, or combinations thereof. In some embodiments, the amino acids comprise prolin and/or serine.

[00400] The present application also provides a system for plant micropropagation. The system is also useful for induction, establishment, and maturation of embryos. The present application also provides a system for plant micropropagation. The system is also useful for the reduction of phenolics in plants. In some embodiments, the system comprises a growth vessel, two or more media containers, and a power source for driving fluid into and/or out of the growth vessel. In some embodiments, the system further comprises a controller. In some embodiments, the system further comprises a light source and/or a gas source providing Co?., 02, N2, or mixture thereof to the growth vessel.

[00401] In some embodiments, the system comprises:

[00402] a growth vessel for incubating plant tissue in a sterile or substantially sterile environment;

[00403] a first media container having a first fluid port and a second fluid port, the first fluid port fluidically coupleable to the growth vessel;

[00404] a second media container having a first fluid port and a second fluid port, the first fluid port fluidically coupleable to the growth vessel; and [00405] a gas source fluidically coupleable to the second fluid port of the first media container and second fluid port of the second media container.

[00406] In some embodiments, the system further comprises a controller operable in a first operating mode in which pressurized gas is delivered from the gas source to the first media container to displace a first volume of liquid contained therein to the growth vessel, and a second operating mode in which pressurized gas is delivered from the gas source to the second media container to displace a second volume of liquid contained therein to the growth vessel.

[00407] In some embodiments, the controller is operable in a third operating mode in which liquid contained in the growth vessel is allowed to flow from the growth vessel into at least one of the first media container and the second media container.

[00408] In some embodiments, the controller is operable in a first incubation sequence in which the third operating mode is executed subsequent to the first operating mode.

[00409] In some embodiments, the controller is operable in a second incubation sequence in which the third operating mode is executed subsequent to the second operating mode.

[00410] In some embodiments, the controller is further operable in a plant propagation mode, in which the first incubation sequence and the second incubation sequence are executed.

[00411] In some embodiments, the growth vessel is elevated above the first and second media containers to allow liquid to flow from the growth vessel into at least one of the first media container and the second media container in the third operating mode.

[00412] In some embodiments, the system further comprises a manifold fluidically coupleable to the growth vessel, the first media container, and the second media container,

[00413] In some embodiments, the manifold is operable to control liquid flow between the growth vessel and the first media container and between the growth vessel and the second media container.

[00414] In some embodiments, the growth vessel includes a fluid conduit configured to siphon liquid from the growth vessel to at least one of the first media container and the second media container.

[00415] In some embodiments, the growth vessel is an ebb and flow bioreactor.

[00416] In some embodiments, the controller is operable to control fluid communication between the growth vessel and the first media container, between the growth vessel and the second media container, between the gas source and the first media container, and between the gas source and the second media container.

[00417] In some embodiments, the first media container comprises a bud induction medium as described herein, and the second media container comprises a shoot elongation/maintenance medium. In some embodiments, the bud induction medium comprises an effective amount of thidiazuron (TDZ) or analog thereof!, and the shoot elongation/maintenance medium comprises an effective amount of one or more cytokinins other than TDZ or an analog thereof.

[00418] The present application also provides methods for exchanging liquid media in a bioreactor/plant growth system for the micropropagation of plant or plant tissue.

[00419] In some embodiments, the bioreactor comprising a growth vessel for incubating the plant tissue, a first media container fluidically coupleable to the growth vessel, a second media container fluidically coupleable to the growth vessel, and a gas source fluid fluidically coupleable to the first media container and the second media container

In some embodiments, the methods comprise:

establishing fluid communication between the first media container and the growth vessel;

fluidically isolating the second media container from the growth vessel;

establishing fluid communication between the gas source and the first media container:

delivering compressed gas to the first media container to displace a first volume of liquid from the first media container to the growth vessel;

[00425] allowing at least a portion of the first volume of liquid to flow from the growth vessel back into the first media container;

[00426] establishing fluid communication between the second media container and the growth vessel;

17] fluidically isolating the first media container from the growth vessel;

establishing fluid communication between the gas source and the second media container;

[00429] delivering compressed gas to the second media container to displace a second volume of liquid from the first media container to the growth vessel; and [00430] allowing at least a portion of the second volume of liquid to flow from the growth vessel back into the second media container.

[00431] In some embodiments, the compressed gas is delivered to the first media container for approximately one minute.

[00432] In some embodiments, the compressed gas is delivered to the second media container for approximately one minute.

[00433] In some embodiments, the liquid is allowed to flow from the growth vessel back into the first media container for approximately 8 minutes.

[00434] In some embodiments, the liquid is allowed to flow from the growth vessel back into the second media container for approximately 8 minutes.

[00435] In some embodiments, said systems are used for plant propagation comprising a bud induction medium and a shoot elongation and maintenance medium. For example, the systems are used for plant propagations wherein a rotation of a bud induction medium and a shoot elongation and maintenance medium is involved.

[00436] In some embodiments, said systems are used for plant propagation comprising the alternative media as described herein. For example, the systems are used for plant propagations wherein (1) a rotation of a Stage 1 medium and a Stage 2 medium is involved; (2) a rotation of a Stage 2 medium and a Stage 3 medium is involved; (3) a rotation of a Stage 1 medium, a Stage 2 medium, and a Stage 3 medium is involved; (4) a rotation of a Stage 3 medium and a Stage 4 medium is involved; (5) a rotation of a Stage 4 medium and a Stage 5 medium is involved; (6) a rotation of a Stage 5 medium and a Stage 6 medium is involved; (7) a rotation of a Stage 4 medium, a Stage 5 medium, and a Stage 6 medium is involved; and/or (7) a rotation of a Stage 6 medium and a Stage 7 medium is involved.

[00437] In some embodiments, a temporary immersion bioreactor 100 is schematically illustrated in FIG. 1. The system 100 is configured for large scale multiplication of plants. In some embodiments, the system 100 is used for large scale multiplication of dicot plants. In some embodiments, the system 100 is used for large scale multiplication of yam. The system 100 includes a growth vessel 110, a first media container 130, a second media container 150, a gas source 170, and a controller 190.

[00438] The growth vessel 110 is configured to incubate plant tissue in a sterile or substantially sterile environment. The growth vessel 1 10 may be any suitable container capable of providing a sterile or substantially sterile environment for the plant tissue and nutrient media. The growth vessel 110 may further be of any suitable material and any desirable shape. For example, the growth vessel 1 10 may be transparent to permit visual observation and light stimulation of the plant tissue, and may be constructed to reduce shear forces on the incubated tissue.

[00439] In some embodiments, the growth vessel 1 10 comprises one or more type of light source suitable for plant growth. Alternatively, the growth vessel is transparent to permit light stimulation provided outside of the growth vessel 110.

[00440] In some embodiments, the growth vessel 1 10 is connected to a gas source. In some embodiments, the gas source provides carbon dioxide, oxygen, nitrogen, or combinations thereof. In some embodiments, the provided gas or mixture of gas is sterile or substantially sterile. The ratio of the gas mixture provided to the growth vessel 1 10 can be predetermined readily controlled depending on any of a variety of factors including, for example, the type of plants being grown in the plant propagation system 100.

[00441] The first media container 130 and second media container 150 are configured to contain a liquid and a gas and are each fluidically coupleable to both the growth vessel 1 10 and the gas source 170. Additional media containers can be includes depending on any of a variety of factors including, for example, the type of plants being grown in the plant propagation system 100. The media containers 130, 150 may contain identical liquid or semi-liquid media, or media that differs in content and/or composition. The media containers 130, 150 can be fluidically coupled to the growth vessel 110 in any suitable manner. For example, in some embodiments, the growth vessel 1 10 can have multiple fluid exchange ports (not shown), and each media container 130, 150 can be coupled to a separate media exchange port. Each connection may be direct and continuous, or include a controllable valve (e.g., manual or under electronic control of the controller 190). In some embodiments, the growth vessel 1 10 can have a single fluid exchange port (not shown) for connecting all media containers 130, 150, and a manifold (not shown) to control exchange of liquid media between the media containers 130, 150 and the growth vessel 1 10. The manifold can include any number of fluid communicators and valves (manual, electronically actuated, hydrauiicaliy actuated, etc.) to control liquid exchange between the media containers 130, 150 and the growth vessel 1 10. In some embodiments, the growth vessel 1 10 can have multiple fluid exchange ports (not shown) for connecting each media container.

I l l [00442] The gas source 170 can be any device or system suitable for delivering pressurized gas to the media containers 130, 150. The gas source 170 can include one or more of, but is not limited to, compressed tanks of gas and gas pumps. Any number of gas sources 170 may be employed. For example, in some embodiments, each media container 130, 150 may be connected to a different gas source 170. In some embodiments, a manifold can be used to connect the gas source 170 to the media containers 130, 150. The manifold can include any number of fluid communicators and valves (manual, electronically actuated, hydraulically actuated, etc.) to control the pressurized gas supply to the media containers 130, 150. The manifold and valves can be coupled to the controller 190 to allow automated and/or electronic control of the gas supply to the medial containers 130, 150. The gas employed by the gas source 170 may be any gas that does not compromise the liquid media in the media containers 130, 150. Examples of such gases include any inert gas, oxygen, nitrogen, carbon dioxide, gas of an atmospheric composition, and combinations thereof.

[00443] The gas source 170 is operable to change the gas pressure in the media containers

130, 150 by delivering a mass of pressurized gas to one or both of the medial containers 130, 150. In some embodiments, the gas source 170 is configured to deliver pressurized gas to the first media container 130 or the second media container 150 to raise the gas pressure in the first media container 130 or the second media container 150 up to about 1 pound per square inch (psi), e.g., about 0.7 psi, about 0.8 psi, about 0.9 psi, about 1 psi, about 1.1 psi, or about 1.2 psi. The increased gas pressure in the first media container 130 or the second media 150 causes at least a portion of the liquid media contained in the first media container 130 to be displaced from the first media container 130 to the growth vessel 110, or at least a portion of the liquid media contained in the second media container 150 to be displaced from the second media container 150 to the growth vessel 110, The gas source 170 can be deactivated or isolated to allow the gas pressure in the first media container 130 or the second media container 50 to return to its original (e.g., atmospheric) value. As a result, the displaced porti on of the liquid media in the growth vessel 1 10 returns back to the first or the second media container. The combination of pressurization and deactivation of the gas source 170 results in "pulsing" of the first media and/or the second media contacting the plant tissue in the growth vessel 1 10.

[00444] In some embodiments, during the deactivation stage, media container equalizes pressure, and automatic siphoning drainage begins, emptying media back to original media container. In some embodiments, the siphoning drainage rate is about 500 to 1000 mis/minute, such as 600-720 mis/minute.

[00445] In some embodiments, during the drainage, the plants are partially submerged in media for about 2 to about 4 minutes, such as about 2,5 to about 3 minutes.

[00446] In some embodiments, after the media is drained, and before the next media comes into the vessel, the plants inside the vessels are dried for a predetermined time. In some embodiments, the plants are dried for about 1 -10 minutes, for example, about 5 minutes.

[00447] The pulsing process can be repeated for the first media container 130 or the second media container 150 for any number of cycles depending on, for example, the type of plants being grown in the plant propagation system 100. For example, the repeated cycles in total take about half hour to about six weeks, e.g., about 24 hours to about 60 hours, about 60 hours to about one week, or about one week to about six weeks., e.g., about 2 weeks to about 3 weeks. In some embodiments, the first media container and the second media container hold the same medium. 100448] In some embodiments, the pulsing process can be alternated between the first media container 130 and the second media container 150. In some embodiments, the pulsing process can be repeated for the first media container 130 for a predetermined number of cycles and then switched to the second media container for a predetermined number of cycles. In some embodiments, the pulsing process can be repeated and alternated between the first media container 130 and the second media container 150 according to any of a variety of predetermined patterns depending on, for example, the type of plants being grown in the plant propagation system 100. For example, the pulsing process can repeated for the first media for about half hour to about six weeks, e.g., about 24 hours to about 60 hours, about 60 hours to about one week, or about one week to about six weeks, e.g., about 2 weeks to about 3 weeks.

[00449] Operation of the plant propagation system 00 is controlled either manually, or by the controller 190 which may be a processor, a computing device, or any programmable/configurable device or system as is known in the art. The controller 190 is configured for electronically control of the gas source 170, and controls at least activation and deactivation of the gas source 170. In some embodiments, the controller 190 is configured for electronic control of a manifold that connects the gas source 170 to each of the media containers 130, 150 to enable selection of one of the media con ta iners. In some embodiments, the controller 190 is configured for electronic control of a manifold that connects the media containers 130, 150 with the growth vessel 1 10 to enable control of liquid flow between the media containers 130, 150 with the growth vessel 1 10. Furthermore, and not inconsistent with various embodiments and combinations thereof, the controller 190 may be connected to, and configured for control of, multiple gas sources, multiple manifolds, and/or multiple valves. Control of other aspects of the system 1 00 not illustrated herein (e.g., control of a gas exchange system, a temperature control system, etc.) are within the scope of this invention.

[00450] The controller 190 is operable in a first operating mode in which it causes the gas source 170 to deliver pressurized gas to the first media container 130 to displace a first volume of liquid contained therein to the growth vessel 1 1 0. Additionally, the controller 190 is operable in a second operating mode in which it causes the gas source 170 to deliver pressurized gas to the second media container 150 to displace a second volume of liquid contained therein to the growth vessel 1 10. In some embodiments, the first and second operating modes are run for a predetermined time. In some embodiments, the first and second operating modes are run for about one minute ± half minute, e.g., about 30 seconds, about 40 seconds, about 50 seconds, about 60 seconds, about 70 seconds, about 80 seconds, or about 90 seconds. As described above, the first and second operating modes can be repeated and/or alternated according to a predetermined pattern.

[00451] Optionally, after the first operating mode or second operating mode has been executed (i.e., the growth vessel 110 contains liquid from one of the media containers 130, 150), the controller 190 is operable to be run in a third operating mode. In the third operating mode, the liquid in the growth vessel 1 10 is allowed to return to its respective media container, for example, by deactivating the gas source 170. In some embodiments, the third operating mode is run for a predetermined time. In some embodiments, the third operating mode is run for about eight minutes, e.g., about 7 minutes, 7.5 minutes, 8 minutes, 8.5 minutes, etc.

[00452] In some embodiments, the controller 190 is operable to run a first incubation sequence comprising one or more cycles of the first operating mode-third operating mode sequence. In some embodiments, the first incubation sequence is run from about 1 hour to about 3 weeks, e.g. , from about 24 hours to about 60 hours. The controller 190 is also operable to run a second incubation sequence comprising one or more cycles of the second operating mode-third operating mode sequence. In some embodiments, the second incubation sequence is run from about 24 hours to about 4 weeks, and e.g., 3 days to about 5 days. [00453] In some embodiments, the controller 90 is operable to run a plant propagation sequence comprising one or more cycles of the first incubation sequence each followed by the second incubation sequence. The number of cycles of the plant propagation sequence may range from one to eight, or more.

[00454] In some embodiments, one, or more, or all of the parts of the growth system can be sterilized by any known methods, such as autoclaving.

[00455] In some embodiments, the media is driven into or out of the growth vessel by gas pressure. In some embodiments, the media is driven into or out of the growth vessel by other forces, such as gravity, electricity, etc.

[00456] Referring now to FIG. 2-5C, an exemplary embodiment of a plant propagation system 200 is shown. The system 200 is similar in operation to the system 100 described above, thus unless stated otherwise, various components of the system 200 may be of similar design and function to that of other embodiments. For example, the growth vessel 210 may be similar to the growth vessel 1 10.

[00457] The illustrated embodiment of FIG. 2 includes a growth vessel 210, a manifold

240, a first media container 230, a second media container 250, a gas pump 270 as the gas source, and a timer-controlled circuit 290 as the controller. The single gas source 270 is attached to the first media container 230, and may be removed and reattached to the second media container 250. Advantageously, the use of a filter 252 (see FIG. 3 also) for each media container 230, 250 prevents any potential contamination of the liquid media in the media containers by the air pump 270, even upon switching. Alternatively, two gas sources can be used, with each source attached to media container 230 or 250, under the control of the timer-controlled circuit 290, so no reattachment is needed. As illustrated in FIG. 3, each media container 230, 250 has a first fluid port 232 in fluid communication or otherwise coupleable with port 222 of the growth vessel 210 to enable exchange of fluid between the media containers and the growth vessel. Each media container 230, 250 also has a second fluid port 236 in fluid communication or otherwise coupleable with the gas source 270 to change the gas pressure inside the media container. The ports 232 and 236 are formed on an adapter 238 that seals the media container to prevent contamination, a bulkhead adapter for example. As illustrated, the second fluid port 236 is additionally fitted with a filter 252 (e.g. a vent filter with stepped hose barbs) to prevent contamination of the fluid in the media container during gas exchange with the gas pump 270. [00458] FIG. 2 and 4 illustrate a non-limiting design of a manifold 240 that connects to tubing 242 and 244 from the media containers 230, 250, respectively. FIG. 4 illustrates valves 246 and 248 formed on manifold 240 for controlling flow from each tubing 242, 244, respectively. Any suitable 2-way valve may be employed such as, for example, ball valves, gate valves, butterfly valves, etc. Valves 246, 248 may be under electronic control of the timer 290, and/or under manual control. In the setup illustrated in FIG, 2, valve 246 is open to fluidly couple the growth vessel 210 and the first media container 230. Valve 248 is closed to fluidly isolate the second media container 250 from the growth vessel 210 as well as the first media container 230. In this manner, intermixing of fluids between the first media container 230 and the second media container 250 is prevented.

[00459] Control of the gas pump 270 may be achieved by switching on/off the power supply of the gas pump by the timer-controlled circuit 290. In an embodiment, the gas pump is a 1 psi pump which when powered by the circuit 290 (during the first or second operating mode, for example), pumps gas into the connected first media container 230 to increase the pressure to 1 psi. The gas pump 270 can be deactivated or other wise turned off when circuit 290 shuts off the power to the gas pump 270 (during the third operating mode, for example), thereby allowing the pressure in the first media container to equalize by allowing the pumped gas to flow back into the gas pump.

[00460] As illustrated in FIG. 5A-5C, the growth vessel 210 includes a closure 212 for accessing the interior of the growth vessel, and a handle 216 for ease of transportation. Though illustrated as formed on a front portion, the closure 212 and the handle 216 may be formed on any other part of the growth vessel 210. In an embodiment, the growth vessel 210 is an ebb and flow bioreactor.

[00461] The growth vessel 210 can also have a gas exchange port 220 and a fluid exchange port 222 formed on the growth vessel, although any number of gas exchange ports and fluid exchange ports are within the scope of the invention. The ports 220 and 222 are fitted with adapters for enabling fluid communication with the interior of the growth vessel 210, while maintaining sterility. In an embodiment, the ports 220 and 222 are fitted with bulkhead adapters. The growth vessel 210 also includes a fluid conduit 226 attached to the port 222 for exchanging fluid with the interior of the growth vessel. The conduit 226 is of sufficient length and has a lumen of appropriate cross-section to enable siphoning of fluid from the floor of the growth vessel 210 and into the selected media container 230 as is described in more detail below for the system 100 of FIG. 1. [00462] In an embodiment, growth vessel 210 is used for large scale multiplication of plants.

In some embodiments, growth vessel 210 is used for large scale multiplication of yam. In some embodiments, growth vessel further is used for pre-rooting and rooting the cultures. Referring again to the system 100 in FIG. 1, in use, the plant tissue to be incubated (e.g. yam tissue) is placed in the growth vessel 1 10, which is then placed at a height relative to the media containers 130, 150 to achieve the siphoning effect. First and second liquid media are placed in media containers 130, 150, respectively. In an embodiment, the first and second liquid media are different. The gas source 170 is connected to media containers 130, 1 0, and the controller 190 is connected to the gas source, and any other components requiring electronic control, as discussed.

[00463] Operation of an exemplary plant propagation sequence 300 of the controller 190 is described herein as illustrated in FIG. 6. While illustrated in a stepwise manner, it is noted that the order of execution of these steps need not necessarily follow so. At 302, controller 190 starts the plant propagation sequence. At 304, controller 190 starts the first incubation sequence. At 306, the controller 190 establishes fluid communication between the first media container 130 and the growth vessel 110. The controller 190 also establishes fluid communication between the gas source 170 and the first media container 130. The controller 190 also fluidically isolates the second media container 150 from the growth vessel 110. At 308, the controller enters the first operating mode and drives the gas source 170 to deliver pressurized gas to the first media container 130 to displace a first volume of liquid contained therein to the growth vessel 110. At 310, the controller then enters the third communication mode and allows at least a portion of the first volume of liquid in the growth vessel 110 to return to the first media container 130 by deactivating the gas source 170.

[00464] At 312, if the first incubation sequence is not complete, the controller 190 returns to 308, and enters the first operating mode again. If the first incubation sequence is complete, the controller, at 3 4, starts the second incubation sequence. At 316, the controller establishes fluid communication between the second media container 150 and the growth vessel 1 10. The controller also establishes fluid communication between the gas source 170 and the second media container 150. The controller also fluidically isolates the first media container 130 from the growth vessel 110. At 318, the controller enters the second operating mode and drives the gas source 170 to deliver pressurized gas to the second media container 150 to displace a second volume of liquid contained therein to the growth vessel 110. At 320, the controller then enters the third communication mode and allows at least a portion of the second volume of liquid in the growth vessel 110 to return to the second media container 150 by deactivating the gas source 170.

[00465] At 322, if the second incubation sequence is not complete, the controller 190 returns to 318 and enters the second operating mode again. If the second incubation sequence is complete but (as determined at 324) the plant propagation sequence is not complete, the controller 190 returns to step 304 and starts the first incubation sequence again, if the plant propagation sequence is complete, the controller 190 exits the plant propagation process at 326. In an embodiment, the controller 190 includes a visual and/or audio indicator for signaling the end of the plant propagation sequence.

[00466] Aspects of the invention are hence beneficial for providing a semi-fully or fully automated, enclosed plant propagation system that is fully programmable for independently controlling, for multiple media, the pulsing time (i.e. the activation/deactivation time) and the incubation time, as well as for controlling the total number of incubation cycles for the entire set of available liquid media. Desirably, all components of the system are autoclavable, and hence reusable. Significant cost savings are realized by reduction in labor, oversight, and contamination loss.

[00467] Non-limiting examples of plant micropropagation systems include those described in U.S. Patent. Nos. 3,578,431; 4,320,594; 4,669,217; 4,908,315; 4,934,096; 5,049,505; 5,088,231; 5,104,527; 5,119,588; 5,139,956; 5,171,683; 5,184,420; 5,186,895; 5,212,906; 5,225,342; 5,558,984; 5,597,731 ; and US 6,753,178. More examples of plant micropropagation systems can be found in Etienne et al. (Bioreactors in coffee micropropagation, Braz. J. Plant Physiol, 18(l):45-54, 2006); Ziv (Bioreactor Technology for Plant, Micropropagation Horticultural Reviews, Volume 24, Edited by Jules Janick ISBN 0-471-33374-3); and Paek et al. (Application of bioreactors for large-scale micropropagation systems of plants, In Vitro Cell. Dev. Biol. -Plant 37: 149-157, March- April 2001).

[00468] It is understood that plant propagation systems of the invention also include the systems derived from the exemplary systems described herein, by adding one or more parts/features of the systems known to one skilled in the art. Racks for Plsmt Micropropagation

[00469] For many plant species there are well known processes for micropropagation. In some instances, for example, it is desirable to intermittently expose cultivated plant tissue within a growth medium to a liquid nutrient solution. Some known systems designed to perform this function are inadequate and prone to breakage and/or mechanical failure. Furthermore, known systems are not sufficiently robust for a large-scale application. Thus, a need exists for improved apparatus for intermittently exposing microenvironments of tissue culture plantlets to a liquid nutrient solution. The present invention meets this need by providing devices and methods for intermittently exposing microenvironments of tissue culture plantlets to a liquid nutrient solution are described herein.

[00470] In some embodiments, an apparatus includes a frame, a shelf assembly supported on the frame, and a drive assembly coupled to the shelf assembly. In such embodiments, the drive assembly can be configured to impart an oscillating motion to the shelf assembly relative to the frame such that tissue culture plantlets in propagation vessels and supported on the shelf assembly are intermittently exposed to a liquid nutrient solution. In some embodiments, described herein relate generally to a rack and more particularly, to an oscillating rack for plant propagation vessels.

[00471] FIG. 7 asid 8 are perspective views of an oscillating rack 100, according to an embodiment. An oscillating rack 100 includes a frame 1 0, a shelf assembly 140, and a drive assembly 170. The frame 100 includes upnghts 111, an upper cross member 120, and a base 125. The components of the frame 100 can be formed from any suitable material. For example, in some embodiments, the frame 100 can be formed from aluminum. In other embodiments, the frame 100 can be formed from an aluminum alloy, steel, and/or steel alloy and can be of any suitable gauge or thickness.

[00472] The uprights 111 can be any suitable configuration and extend upwardly from the base 125, as described in further detail herein. The upper cross member 120 can be any suitable size, shape, or configuration. For example, in some embodiments, the upper cross member 120 can be a formed (e.g., mechanically bent) or extruded C-channei. In other embodiments, the upper cross member 120 can be a substantially closed or solid structure, such as, for example, box tubing or bar stock. The upper cross member 120 is configured to be coupled to an upper portion of the uprights 1 1 1. In this manner, the upper cross member 120 can increase the rigidity and/or strength of an upper portion of the oscillating rack 100. [00473] The base 25 can be any suitable platform or structure. For example, while shown in FIG, 7-9 as being substantially I-shaped (e.g., including a cross member coupled to two support members perpendicularly aligned to the cross member), in other embodiments, the base 125 can be any shape or configuration. In some embodiments, for example, the base 125 can be a substantially rectangular structure. In other embodiments, the base 125 can include stiffening members and/or the like. For example, in some embodiments, the base 125 can include a sheet metal portion coupled (e.g., screwed, welded, riveted, or otherwise fastened) to a top surface of the base 125 configured to increase the rigidity and/or strength of the base 125. Furthermore, in the embodiments shown in FIG. 7-9, the base 125 includes a set of caster wheels such that the oscillating rack 100 can be moved or repositioned.

[00474] As shown in FIG. 9, the base 125 includes a support member 126 that extends from a top surface of the base 125. More specifically, the support members 126 are fixedly coupled (e.g., welded) to the top surface of the base 125 and receive at least a portion of the one of the uprights 111. Furthermore, one of the support members 126 includes a drive shaft opening 127 and a rocker shaft opening 128 configured to receive a drive shaft 172 and a rocker shaft 182 of the drive assembly 170, respectively.

[00475] The uprights 111 include a set of walls that define a substantially C- Shaped cross- section (FIG. 10) and a cover 112 (FIG. 9). As shown in FIG. 7-9, the uprights 111 are configured to extend from the top surface of the base 125. More specifically, the uprights 11 1 are disposed around the support members 126 such that the uprights 11 1 extend away from the top surface of the base 125. Similarly stated, the support members 126 are disposed within a volume 115 defined by the set of wails that define the C-shaped cross-section. In this manner, the uprights 1 11 can be coupled (e.g., welded and/or fastened) to the base 125 and/or support members 126. The uprights 111 can further define any number of holes configured to receive portions of the oscillating rack 100. For example, at least one upright 1 1 1 can include a drive shaft opening 113 and a rocker shaft opening 1 14 configured to receive a drive shaft 172 and a rocker shaft 182, respectively, included in the drive assembly 170, respectively, and positioned to align with the drive shaft opening 127 and the rocker shaft opening 128, respectively, in the support member 126.

[00476] In some embodiments, the uprights 111 can define any number of holes and/or protrusions configured to engage a portion of a lighting system (not shown) and/or control system (not shown). In some embodiments, each cover 1 12 can be coupled to a respective upright 111 such that the cover 112 and the upright 1 1 1 house a set of electrical components (not shown) within the volume 1 15. For example, in some embodiments, the volume 115 can contain wires, switches, relays, electronic devices (e.g., a programmable logic controller (PLC) including, for example, at least a processor, a memory, and a network interface), and/or the like. In some embodiments, at least one upright 1 1 1 can include a sensor bracket 121. In such embodiments, a sensor can be disposed on the sensor bracket 121 and can indicate and/or monitor the position of the shelf assembly 140 relative to the frame 110, as further described herein.

[00477] The shelf assembly 140 is rotatably coupled to the uprights 111 (see e.g., FIG. 7) and includes a set of shelves 141, a set of outer bushings 150, a set of inner bushings 155, and a set of linkages 145. For example, as shown in FIG. 11, the shelf assembly 140 can include an - suitable number of shelves 141 configured to be vertically stacked. Furthermore, the shelves 141 are operatively coupled together via the linkages 145 (e.g., the linkages 145 transfer at least a portion of a force to cause each shelf 141 of the shelf assembly 140 to pivot simultaneously, as further described herein). While shown in FIG. 11 as including two linkages 145, in some embodiments, a shelf assembly can include any suitable number of linkages 141. For example, in some embodiments, a shelf assembly can include a set of four linkages 141 such that a first set of two linkages 145 are disposed on a first side of the shelves 141 and a second set of two linkages 145 are disposed on a second side of the shelves 141.

[00478] As shown in FIG. 12, each shelf 141 includes a set of platforms 142 coupled together by support tubes 144 (e.g., a first support tube 144 is disposed on a first side of a shelf 141 and a second support tube 144 is disposed on a second side of a shelf 141). As shown in FIG. 13, the platforms 142 define a cross-sectional shape defining a double return, thereby increasing the strength and rigidity of the platform 142. Furthermore, at least one of the support tubes 144 of a shelf 141 is configured to be coupled to the linkages 145.

[00479] The outer bushing 150 and the inner bushing 155 (FIG. 14) are configured to rotatably couple the shelf assembly 40 to the uprights 1 1 1. More specifically, the inner bushings 155 are rigidly coupled to the support tubes 144 of the shelves 141 and the outer bushings 150 are rigidly coupled to the uprights 1 1 1. In this manner, the inner bushings 155 can be rotatably disposed within an opening 151 defined by the outer bushings 150. Thus, the shelves 141 can pivot about the inner bushings 155, disposed within the openings 151 of the outer bushings 150, in response to at least a portion of a force exerted by the drive assembly 170. [00480] Referring now to FIG. 15, the drive assembly includes a motor 171, a drive gear

173, and a rocker assembly 180. The motor 171 can be any suitable motor defining any suitable torque and/or output speed. For example, in some embodiments, the motor 171 is a Bison 650AC. As shown in FIG, 9, the motor 171 is configured to be coupled to at least one of the uprights 111 such that a drive shaft 172 extends from the motor 171 through the drive shaft opening 113 of the upright 1 11 and the drive shaft opening 127 of the support member 126. The drive gear 173 is configured to be disposed about the drive shaft 172 and is housed within the volume 115 defined by the upright 111.

[00481] The rocker assembly 180 includes a rocker gear 181, a rocker shaft 182, a bearing

183, a mounting bracket 184, and a rocker bushing 186. The rocker gear 181 can be any suitable size and/or define any suitable number of teeth. Furthermore, the rocker gear 181 is disposed within the volume 5 and is operably coupled to the drive gear 73, for example via a chain (not shown). The arrangement of the drive gear 73 and the rocker gear 181 can be such that a desired gear ratio is defined between the drive gear 73 and the rocker gear 181.

[00482] The rocker shaft 182 is configured to be inserted into the rocker gear 81 and the bearing 183 and extends through the rocker shaft opening 128 of the support member 126 and the rocker shaft opening 114 of the upright 111. The bearing 183 can be used to facilitate the rotation of the rocker shaft 182 and/or to reduce wear on the rocker assembly 180. The rocker shaft 182 is further configured to be inserted through the rocker bushing 186 and is fixedly coupled (e.g., welded) to the mounting bracket 184. With the rocker shaft 182 coupled to the mounting bracket

184, the mounting bracket 184 can be coupled to the support tube 144 of a first shelf 141. Thus, with the mounting bracket 184 coupled to the first shelf 141 and the rocker shaft 182, the shelf assembly 140 is operably coupled to the motor 171.

[00483] For example, FIG. 16-18 illustrate a portion of the oscillating rack 100 in a first configuration, a second configuration, and a third configuration, respectively. As seen in FIG. 16, the oscillating rack 100 can be in the first configuration such that platforms 142 of the shelves 141 are substantially parallel to a horizontal axis (e.g., the shelves 141 are parallel to the ground). In some embodiments, the shelves 141 can be substantially perpendicular to the linkages 145, while the oscillating rack 100 is in the first configuration.

[00484] [1031 ] As shown in FIG. 17, the oscillating rack 100 can be moved towards the second configuration by rotating the rocker gear 181 in the direction of the arrow AA. More specifically, the motor 171 (not shown in FIG. 17) can be electrically engaged (e.g., placed in the "on" position via, for example, a control panel) such that the motor 171 rotates the drive shaft 172 and the drive gear 73. The motor 171 can be configured to rotate the drive shaft 172 at any given output speed. For example, in some embodiments, the motor 171 can be configured to rotate the drive shaft 172 at a rate between 0.5 RPM and 1 RPM.

[00485] As described above, the rocker gear 181 is operably coupled to the drive gear 173 via a chain. Thus, the chain transfers a portion of the rotational force produced by the motor 171 to the rocker gear 181 such that the rocker gear 181 rotates in the direction of the arrow AA. With the mounting bracket 184 coupled to the first shelf 141 (as described above), a portion of the rotational force, produced by the motor 171 , is applied to the first shelf 141. In this manner, a first end of the first shelf 141 is urged to move in the direction of the arrow BB and a second end of the first shelf 141 is urged to move in the direction of the arrow CC. Moreover, with the linkages 145 coupled to the each of the shelves 141, the linkages 145 transfer a portion of the rotational force produced by the motor 171 to each of the shelves 141. Therefore, each shelf 141 is configured to move concurrently with the first shelf 141 in response to at least a portion of the rotational force produced by the motor 171. In addition, when in use with, for example, cultivated plant tissue, the pivoting motion of the shelves 141 can be such that a set of portions of the plant tissue, such as the roots, disposed on a surface of the platforms 142 are intermittently tilted so that the portions (e.g., roots) are alternately immersed in, and free of, a liquid nutrient contained in the vessels. Expanding further, the pivoting motion of the shelves 141 is such that the shelves 141 are placed at an angle relative to the horizontal axis, thus, the liquid nutrients flow in the direction of the arrow DD.

[00486] As shown in FIG, 18, the oscillating rack 100 can be moved from the second configuration towards the third configuration by rotating the rocker gear 181 in the direction of the arrow EE (substantially opposite the direction AA). With the rocker gear 181 being moved in the direction of the arrow EE, the first end of the first shelf 141 is urged to move in the direction of the arrow FF (substantially opposite the direction BB) and the second end portion of the first shelf 141 is urged to move in the direction of the arrow GG (substantially opposite the direction CC). Furthermore, the linkages 145 urge each of the shelves 141 of the shelf assembly 140 to move concurrently with the first shelf 141. Thus, when in use with, for example, cultivated plant tissue, the pivoting motion of the shelves 141 in the direction EE can be such that the liquid nutrient can be urged to flow in the direction of the arrow HH such that the cultured plant tissues (e.g., the roots) are alternately immersed in, and free of, the liquid nutrient contained in the vessels.

[00487] When in use, the oscillating rack 100 can be configured to oscillate between the second configuration and the third configuration, in some embodiments, the oscillating rack 100 can oscillate between the second configuration and the third configuration with a given cycle time. For example, in some embodiments, the cycle time can be 25 seconds (e.g., an oscillating time of 15 seconds and a hold time in the second configuration or the third configuration for 10 seconds before moving in the opposite direction). In other embodiments, the cycle time can be any other suitable length of time. In some embodiments, the oscillating rack 100 can include a sensor (described above). In such embodiments, the sensor, such as a magnetic sensor, can be configured to sense the position of the shelf assembly 140 relative to the frame 100. The sensor can be configured to be in electrical communication with, for example, a programmable logic controller. The programmable logic controller and the sensor can detect a system malfunction. For example, in some embodiments, the programmable logic controller can be configured to send an electrical signal to an output device to generate a suitable output if the sensor does not sense the position of the shelf assembly 140 for predetermined time period (e.g., 35 seconds). The output can be an audible alarm, a flashing light, a telephone call, an email, and/or any other suitable notification.

[00488] The components described herein can be made using any suitable manufacturing technique. For example, in some embodiments, some components can be extruded. In some embodiments, the components can be formed (e.g., bent). In such embodiments, the components can include any suitable feature such that the component defines a specific material characteristic. For example, the platforms 142 are described above as including a double return configured to increase the strength and/or rigidity of the platforms 142. In some embodiments, other components can include similar features. For example, in some embodiments, the uprights 1 11 can include a double return. In other embodiments, the linkages 145 can include a double return.

[00489] The components described herein can be assembled in any suitable manner. For example, in some embodiments, components can be welded. In other embodiments, at least a portion of the components can be mechanically fastened. For example, in some embodiments, portions of the components described herein can be assembled (e.g., coupled) via bolts and nuts, screws, pins, and/or the like. In some embodiments, a portion of the components can be assembled using self-clinching nuts (e.g., PEM nuts) in conjunction with bolts or screws. [00490] While various embodiments have been described above, it should be understood that they have been presented by way of example only, and not limitation. Where schematics and/or embodiments described above indicate certain components arranged in certain orientations /or positions, the arrangement of components may be modified. Similarly, where methods and/or events described above indicate certain events and/or procedures occurring in certain order, the ordering of certain events and/or procedures may be modified. While the embodiments have been particularly shown and described, it will be understood that various changes in form and details may be made.

[00491] Although various embodiments have been described as having particular features and/or combinations of components, other embodiments are possible having a combination of any features and/or components from any of embodiments as discussed above.

Methods for Plant Bioculture 1

[00492] The methods can be conducted with or without a bioreactor.

[00493] In some embodiments, the methods comprise (a) obtaining a plant explant. Any suitable plant parts may be used. In some embodiments, single-node explants, shoot tips, basal (bottom) parts of plants with multiple buds, are used as starting material as explants. The explants can be treated to substantially reduce the chance of contamination. Any suitable methods can be used. In some embodiments, commercial bleach can be used. For example, explant can be sterilized in about 1%, 5%, 10%, 15% or more commercial bleach for about 10 minutes, 20 minutes, 30 minutes, or more depending on the condition of the explant. In some embodiments, to further reduce the chance of contamination, explant can be cut into small piece, such as about 3mm, about 5 mm, or more in length. The small pieces can be rinsed again once, twice, or more in about 1%, 5%, 10%, or 15% commercial bleach solution and then placed on an initiation medium. The initiation medium can be any suitable medium as described herein.

[00494] This step is completed when shoot tips start breaking and forming multiple shoots from the explant. During the process, explant can be sub cultured on a fresh initiation medium every 3 to 4 weeks or any suitable period of time.

[00495] The multiple shoots initiated from the explant can be dived into small clumps, for example, clumps of 2 to 3 shoots each and transferred to a multiplication medium as described herein. This step can be conducted in a bioreactor. [00496] Any suitable growth condition can be used. In some embodiments, the plants are grown under about 20-28 °C (e.g., about 22-24°C), day-night cycle (e.g., 16/8 hour day/night), with a photon flux density of about 10-150 ,umol/m²/s (e.g, 80-100 ,umol/m²/s).

[00497] In some embodiments, a temporary immersion bioreactor is used. In some embodiments, in a single cultivation cycle, it takes about 0.1, 0.2, 0.3, 0.4, 0.5, 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 15, 20, 25, 30 minutes or more to fill the cultivation chamber of the bioreactor in which the plants are grown with a predetermined amount of liquid or semi-liquid medium. The medium is kept in the chamber for about I, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes or more, and then drained from the chamber. In some embodiments, it takes about 0, 1 , 0.2, 0.3, 0,4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5,5, 6, 7, 8, 9, 10, 15, 20, 25, 30 minutes or more minutes to drain the medium out of the chamber. Then optionally the chamber is dried for about 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 15, 20, 25, 30 minutes or more minutes. In some embodiments, the cultivation cycle described above takes about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes or more.

[00498] In some embodiments, a relatively small amount of liquid medium is used, such as about 25-100 ml of liquid medium per bioreactor. Advantages of using a relatively small amount of liquid medium include, but are not limited to, better control of temporal immersion of the explants, preventing drowning of the explains, and reducing chances of contamination.

[00499] In some embodiments, the liquid medium in the bioreactors is changed with fresh one every 1 week, 2 weeks, 3 weeks, 4 weeks or more, each of which is called a growth cycle (or cycle). Advantages of using refreshed liquid medium include, but are not limited to, providing more nutrients to plants, removing accumulated detrimental chemicals in the medium due to plant metabolism, and reducing chances of contamination.

[00500] In some embodiments, an oscillating rack system is used to move liquid from one side to another. In some embodiments, the oscillation cycle is about once per two minutes, once per minute, one and a half per minute, twice per minute or more. In some embodiments, the oscillating rack system is used in the initiation and/or multiplication step. A non-limiting example of oscillating rack system is described in International Patent Application No. PCT/US2012/047622, which is incorporated herein in its entirety including any figures therein.

[00501] In some embodiments, the pistachio plant tissue biomass are multiplied for about

1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6.0 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, or more during each growth cycle.

[00502] Any suitable plant, plant part, plant tissue culture, or plant cell can be used as the explant. In some embodiments, the explant is pathogen-free, e.g., bacteria-free, fungi-free and/or virus-free. In some embodiments, the explant is a pistachio rhizome shoot tip. In some embodiments, the explant is taken from a plant of about 1 week old, about 2 weeks old, about 3 weeks old, about 1 month old, about 2 month old, about 3 months old, about half year old, about 1 year old, or more.

[00503] The explant can be any segment or collection of cells from apical meristems, terminal buds, axillary buds, adventitious buds, accessory buds, pseudo-terminal buds, cambium, lateral meristem, lateral bud, vegetative buds, reproductive buds, mixed buds, shoot segments, shoot apices, stem segments, immature nodal sections from stems, lateral shoots, seedlings, seeds, shoots starting to rise from the ground, immature flower buds, inflorescences, crown segments, leaf segments, roots, rhizome, or any part thereof.

[00504] The multiplied shoots are then transferred to a rooting medium as described herein.

Optionally, the multiplied shoots are divided into clumps of about 3 to 6 shoots before the transfer. It usually takes about 2-4 weeks for the shoots to develop roots. Once the roots are formed, the plants can be transfer to either in vitro or in vivo conditions for further growth.

Methods for Plant Biocultisre 2 (Tuber Producing Plants, among others)

[00505] In some embodiments, this step comprises breaking field tuber dormancy to induce buds, sprouting of buds, sterilization of sprout, and subsequent cycles of cultivation in vivo or in vitro.

[00506] The tuber dormancy can be broken naturally, or by treatment with GA3, ethanol, temperature treatment, thiourea, ethylene chlorohydrins, rindite, carbon disulphide, and/or bromoethane, etc., or by methods described in Bryan, 1989 and Claassens et al, 2005, each of which is incorporated by reference in its entirety.

[00507] Any sterilization method suitable for plant can be used. In some embodiments, the sprouts are sterilized in 0.5% solution of NaDCC. [00508] The sterilized sprouts are then cultivated in vitro (e.g. , in a tube) on a solid or semisolid medium. In some embodiments, the sprouts are first cultivated in solid medium, wherein the medium comprises MS salts, IAA, 2ip, and sucrose. In some embodiments, the concentration of IAA. is about 0.1 to 1 mg/L, e.g., about 1 mg/L; the concentration of 2ip is about 1 to 10 mg/L, e.g., about 4-5 mg/L; and the concentration of sucrose is about 10 to 40 g/L, e.g., about 30 g/L. Then the sprouts are grown on a medium comprising MS salts and sucrose without any hormones. In some embodiments, the concentration of sucrose is about 10 to 40 g/L, e.g., about 20 g/L, e.g., the media as described herein. Any suitable growth condition can be used. In some embodiments, the sprouts are grown under about 20-28°C (e.g., about 24°C), day-night cycle (e.g., 16/8 hour day/night), with a photon flux density of about 50-150 p.mol/m²/s (e.g., about 85-100 μηιο1/ηι²/3). In some embodiments, the cultivation takes about one to three months, e.g., about two months, or takes as long as needed until the plants are pathogen-free. As used herein, one skilled in the art would understand that the standard of "pathogen-free" vanes from one pathogen species to another, and the plant can be regarded as pathogen-free as long as the population of a specific pathogen contained in the plants does not substantially affect future microtuber production.

[00509] Optionally, the step of obtaining pathogen-free sprouts comprises testing plants for the presence of pathogens, such as one or more bacteria species, fungal species, and/or virus species. In some embodiments, the virus species is selected from Dioscorea bacilliform virus (DBV, genus Badnavirus), Yam mosaic virus (YMV, genus Potyvirus), Yam mild mosaic virus (YMMV, genus Potyvirus), Potato leaf roll virus (PLRV), Potato virus A (PVA), Potato virus M (PVM), Potato virus S (PVS), Potato virus X (PVX), Potato virus Potato virus S (PVS), Potato virus X (PVX), Potato virus Y (PVY), and Potato spindle tuber viroid (PSTVd). In some embodiments, the testing methods comprise detecting one or more nucleotides (e.g., DNA or RNA) and/or one or more polypeptide that is specific to the pathogen, by using any suitable technologies known to one skilled in the art.

[00510] In some embodiments, the methods comprise (b) propagating the pathogen-free sprouts obtained in step (a) or any other sources to produce plants. The step is also called elongation stage in which stems of plants are elongated. In some embodiments, the propagation is in vitro or in vivo. In some embodiments, the propagation is done in a bioreactor of the present application or any other suitable bioreactors known to one skilled in the art, or simply in any suitable culture tubes. In some embodiments, solid, semi-solid, liquid or semi-liquid medium is used. In some embodiments, one 4-5-week-old well-developed plant contained multiple axillary buds is used as the starting materials. In some embodiments, such well-developed plant has about 3-10 axillary buds, e.g., about 4 to 7 axillary buds.

[00511] In some embodiments, the well -developed plant is grown either on solid medium or semi-solid medium in a culture tube, or in liquid or semi-liquid medium in a bioreactor. In some embodiments, the medium comprises MS salts and sucrose without any hormones, e.g., the propagation and multiplication media as described above. In some embodiments, the concentration of sucrose is about 10 to 40 g/L, e.g., about 20 g/L, e.g., the media as described herein.

[00512] Any suitable growth condition can be used. In some embodiments, the plants are grown under about 20-28°C (e.g., about 24°C), day-night cycle (e.g., 16/8 hour day/night), with a photon flux density of about 50-150 μιηοΙ/ηΓ/β (e.g., about 85-100 μηιο1/ηι²/8) when a culture tuber is used, or about 10-100 μηιο1/ηι²/8 (e.g., about 30-80

when a bioreactor is used.

[00513] In some embodiments, the cultivation takes about 3-8 weeks in a solid or semi-solid medium in culture tubes, e.g., about 4-6 weeks, or about 1 -4 weeks in a liquid medium in bioreactors, e.g., about 2.5-3 weeks, depending on plant type and variety.

[00514] In some embodiments, a temporary immersion bioreactor is used. In some embodiments, in a single cultivation cycle, it takes about 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 15, 20, 25, 30 minutes or more to fill the cultivation chamber of the bioreactor in which the plants are grown with a predetermined amount of liquid or semi-liquid medium. The medium is kept in the chamber for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes or more, and then drained from the chamber. In some embodiments, it takes about 0.1, 0.2, 0.3, 0,4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5,5, 6, 7, 8, 9, 10, 15, 20, 25, 30 minutes or more minutes to drain the medium out of the chamber. Then optionally the chamber is dried for about 0.1, 0,2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 15, 20, 25, 30 minutes or more minutes. In some embodiments, the cultivation cycle described above takes about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes or more.

[00515] In some embodiments, the same medium is used in each cultivation cycle. In some embodiments, two or more different media are used sequentially, of which each is used in a cycle.

[00516] In some embodiments, a relatively small amount of liquid medium is used, such as about 25-100 ml of liquid medium per bioreactor. Advantages of using a relatively small amount of liquid medium include, but are not limited to, better control of temporal immersion of the explants, preventing drowning of the explains, and reducing chances of contamination.

[00517] In some embodiments, the liquid medium is the bioreaetors is changed with fresh one every 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, every week, every 10 days, or every two weeks. Advantages of using refreshed liquid medium include, but are not limited to, providing more nutrients to plants, removing accumulated detrimental chemicals in the medium due to plant metabolism, and reducing chances of contamination.

[00518] In some embodiments, an oscillating rack system is used to move liquid from one side to another. In some embodiments, the oscillation cycle is about once per two minutes, once per minute, one and a half per minute, twice per minute or more. In some embodiments, the oscillating rack system is used in the propagation stage (elongation stage) and/or microtuberization stages (e.g., the pre-tuberization stage and tubenzation stage). A non-limiting example of oscillating rack system is described in U.S. Provisional Patent Application US 61/618,344, filed on March 30, 2012, which is incorporated herein in its entirety including any figures therein.

[00519] In some embodiments, the plants are multiplied for about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6.0 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, or more. This step results in increased shoot length and more internodes per plant.

[00520] Any suitable plant, plant part, plant tissue culture, or plant cell can be used as the explant for micropropagation. In some embodiments, the explant is pathogen-free, e.g., bacteria- free, fugi-free and/or virus-free. In some embodiments, the explant is a stock plant maintained by serial in vitro subculture. In some embodiments, the explant is a segment of seedlings. In some embodiments, the segment of plant material comprises one or more axillary bud. In some embodiments, the bud is dormant or active. In some embodiments, the explant is taken from a plant of about 1 week old, about 2 weeks old, about 3 weeks old, about 1 month old, about 2 month old, about 3 months old, about half year old, about I year old, or more.

[00521] The explant can be any segment or collection of cells from apical meristems, terminal buds, axillary buds, adventitious buds, accessory buds, pseudo-terminal buds, cambium, lateral meristem, lateral bud, vegetative buds, reproductive buds, mixed buds, shoot segments, shoot apices, stem segments, immature nodal sections from stems, lateral shoots, seedlings, seeds, shoots starting to rise from the ground, immature flower buds, inflorescences, crown segments, leaf segments, roots, recycled microtubers, sprouts from cold-stored seed tubers, or any part thereof.

[00522] In some embodiments, the methods further comprise (c) pretreating the plants obtained from step (b) or any other sources to produce pretreated plants. This step is also called pre-tuberization stage. In some embodiments, this step was performed in a bioreactor. In some embodiments, the bioreactor is a temporary immersion bioreactor. In some embodiments, liquid or semi-liquid medium is used, e.g. the pre-tuberization media as described above.

[00523] In some embodiments, the plants obtained from step (b) or any other resources are cultured in a liquid medium, wherein the each plant has about 3-10 axillary buds, e.g., about 4 to 7 axillary buds. In some embodiments, the medium comprises MS salts, sucrose, at least one cytokinin, and at least one auxin, e.g., B0018 as described herein. In some embodiments, the cytokinin is 2ip or analog thereof. In some embodiments, the auxin is IAA or analog thereof. Alternatively, the medium comprises MS salts, sucrose, and at least one growth retardant, e.g., the B0023, BOO 19, BOO20, B0024 media described herein, or combination thereof. In some embodiment, at least one retardant is ancymidol or analog thereof. Still in some embodiments, the pre-tuberization media comprise one or more cytokinin, one or more auxin, and one or more growth retardant. In some embodiments, the concentration of 2ip is about 1 to 10 mg/L, for example, about 4-5 mg/L. In some embodiments, the concentration of IAA is about 0.1 to 10 mg/L, for example, about 1 mg L. In some embodiments, the concentration of ancymidol is about 0.1 to 10 mg/L, for example, about 0.5 mg/L, about 1 mg/L, about 2 mg/L, or about 5 mg/L. In any case, the sucrose concentration is about 20 g/L to about 40 g/L, for example, about 30 g/L. In some embodiments, a medium comprising MS salts, sucrose, at least one cytokinin, and at least one auxin, and a medium comprising MS salts, sucrose, and at least one growth retardant are used in combination, or sequentially during the pretreatment stage in any order, in one or more culture cycles.

[00524] Any suitable growth condition can be used. In some embodiments, the plants are grown under about 20~28°C (e.g., about 24°C), day-night cycle (e.g., 16/8 hour day/night), with a photon flux density of about 10-100 ,umol/nr/s (e.g., about 30-80

In some embodiments, the duration of the pretreatment step is about 1-4 weeks, e.g., about 1 to 2 weeks or about 2-3 weeks. [00525] In some embodiments, the methods further comprise (d) initiating microtubers from the pretreated plants obtained from step (c) or any other sources. This step is also called tuberization stage.

[00526] One or more ways to initiate tuberization of plants in vitro can be utilized in step

(d). in some embodiments, the methods of present application comprise initiating tuberization in vitro by supplying relatively high concentration of sucrose. For example, the sucrose concentration in the tuberization induction media is about 5% w/v, about 6% w/'v, about 7% w/v, about 8% w/v, about 9% w/v, about 10% w/'v, or more.

[00527] In some embodiments, the methods of present application comprise triggering tuberization in vitro by shifting the tissue culture from long-day light conditions to short-day light conditions. For example, the photoperiod condition is changed from long-day conditions, such as about 23/1 hours (light/dark), about 22/2 hours (light/dark), about 21/3 hours (light/dark), about 20/4 hours (light/dark), about 19/5 hours (light/dark), about 18/6 hours (light/dark), about 17/7 hours (light/dark), about 16/8 hours (light/dark), about 15/9 hours (light/dark), about 14/8 hours (light/dark), or about 13/11 hours (light/dark) to short-day conditions, such as about 11/13 hours (light'dark), about 10/14 hours (light/dark), about 9/15 hours (light/dark), about 8/16 hours (light/dark), about 7/17 hours (light/dark), about 6/18 hours (light/dark), about 5/19 hours (light'dark), about 4/20 hours (light/dark), about 3/21 hours (light/ dark), about 2/22 hours (light/dark), or about 1/23 hours (light/ dark).

[00528] In some embodiments, the methods of present application comprise triggering tuberization in vitro by using a total darkness condition.

[00529] In some embodiments, the methods of present application comprise triggering tuberization in vitro by using cool temperature conditions. For example, the temperature during the day time and/or the night time is about 25°±1 °C, 2 °±1 °C, 23°±1⁰C, 22⁰±1 °C, 21°±1 °C, 20°±1 °C, 19°±1 °C, 18°±1 °C, 17⁰±1°C, 16^C'±1°C, 15°±1°C, 14°±1 °C, or lower. In some embodiments, the day time temperature is about 20°±2°C and night time temperature is about 18°±2°C. In some embodiments, the temperature during the night time is lower than the temperature during the day time, for example, about 0.5°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, or more.

[00530] In some embodiments, the methods of present application comprise triggering tuberization in vitro by using one or more phytohorrnones or growth regulators, such as cytokins or growth retardants. In some embodiments, the cytokm is selected from the group consisting of thidiazuron (TDZ), N⁶-benzylaminopurine (BAP, a.k.a. BA), meta-topolin (ml), zeatin, zeatin riboside, dihydrozeatin, kinetin, isopentenyladenine (ip, e.g., 2ip), adenine hemisulfate, dimethylallyladenine, N-(2-chloro-4-pyridyl)-N'- phenyl urea) (4-CPPU), analog thereof, and combination thereof. In some embodiments, the growth retardants is selected from the group consisting of alar, ancymidol, chJorocholme chloride (CCC), coumarin, fl undone, tetcyclacis (TET), ancymidol, analog thereof, and combination thereof. In some embodiments, the growth retardant is a gibberellic acid (GA3) antagonist, such as ancymidol and its functional derivatives.

[00531] In some embodiments, the methods of present application comprise triggering tuberization in vitro by increased nitrate:ammonium ratio and/or increased nitrogen: carbon ratio.

[00532] In some embodiments, more than one way of triggering tuberization described above are simultaneously and/or sequentially used. More methods for triggering tuberization can be found in Donnelly et al. 2003, Seabrook et al. 1993, Gopal et al. 998, and Gopal et ai. 1997, Garner and Biake et al. 1989, Bizan et al. 1995, Nasiruddin and Blake 1994, each of which is incorporated by reference in its entirety for all purposes.

[00533] In some embodiments, step fd) was performed in a bioreactor. In some embodiments, the bioreactor is a temporary immersion bioreactor. In some embodiments, liquid or semi-liquid medium is used, e.g. the pre- tuberization media as described above. In some embodiments, the plants obtained from step (c) or any other resources are cultured in a liquid or semi-liquid medium. In some embodiments, the liquid medium or semi-liquid medium comprises one or more auxin, but does not comprise any cytokinin media described herein. Alternatively, instead of auxm, the tuberization medium comprises one or more plant retardant, such as ancymidol or analog thereof. In some embodiments, the tuberization medium comprises one or more auxin and one or more growth retardant. In some embodiments, the auxin is NAA. In some embodiments, the NAA concentration is about 0.01 to about 0.05 mg/L, for example, about 0.01 mg/L, or about 0.02 mg/L. In some embodiments, the concentration of ancymidol is about 0.1 to 10 mg/L, for example, about 0.5 mg/L, about 1 mg/L, or about 5 mg/L. In any case, the sucrose concentration in the tuberization medium is higher compared to the sucrose concentration in the pre-tuberization medium used in step (c). For examples, the sucrose concentration in the tuberization media is about 50 g/L to about 100 g/L or more, for example, about 60 g/L, about 70 g/L, or about 80 g/L. In some embodiments, the sucrose concentration is about 80 g/L. Any suitable growth condition can be used,

[00534] In some embodiments, the plants are grown under a temperature that is lower than the temperature used in step (c). For example, when the temperature used in step (c) is about 24°C, the temperature used in step (d) can be about 15-24°C. In some embodiments, the plants are cultured with continuous darkness. In some embodiments, this step lasts for about 5-6 weeks, or any period of time that is suitable for a specific species or a specific goal (e.g., with predetermined microtubers production number and/or size),

[00535] The methods can further comprise (e) harvesting the microtubers produced in step

(d). The microtubers propagated by methods described herein can be either stored under suitable conditions for future use, or could be directly transplanted to soil without any acclimation. Optionally, this step includes washing, dr ing, weighing, counting and/or storing the microtubers. In some embodiments, the microtubers are stored at a temperature above 0 °C but below about 10 °C, e.g., at about 4°C.

[00536] The methods described herein can be further modified and optimized, depending on the purposes and goals. For example, factors affecting yam tissue culture disclosed in Nistor et al, 2010, Rosu et al, 2004, Badoni et al., 2009, Wang et al., 1982, Abbott et al, 1986, Ewing et a!.. 1992, Khun et al, 1995, Perl et al, 1991, Leclerc et al, 1994, I. ex yd et al, 1993, Ahmad et al., 1993, and others can be considered. More information can be found in Bajaj, 2009 (Yam Volume 3 of Biotechnology in agriculture and forestry. Springer- Verlag, 1987, ISBN 3540179666, 9783540179665); Haverkort and Anisimov, 2007 (Yam production and innovative technologies, Wagemngen Academic Pub, 2007, ISBN 9086860427, 9789086860425); Rajan and Markose 2007 (Propagation of Horticultural Crops: Vol.06. Horticulture Science Series, New India Publishing, 2007, ISBN 8189422480, 9788189422486); Leclerc 1993 (The production and utili zation of yam microtubers, McGiil University, 1993), Krasteva and Panayotov, 2009 (Proceedings of the fourth Balkan Symposium on Vegetables and Yams, ISHS, 2009, ISBN 9066055529, 9789066055520), each of which is incorporated herein by reference in its entirety for all purposes.

Propagation / Culture of Grasses

[00537] The present invention also provides methods for in vitro propagation of grassy plants. In some embodiments, the methods comprise propagating grass through somatic embryogenesis. [00538] In some embodiments, the methods start with (a) initiating embryos from an explant. In some embodiments, this step comprises culturing vegetative explants obtained from a plant on the first type of media described herein,

[00539] One or more types of explants obtained from a plant can be used. As used herein, an "explant" (a.k.a. a "mother plant") is the source of cells to be developed during the tissue culturing process. For example, the explant can be any segment or collection of cells from apical meristems, terminal buds, axillary buds, adventitious buds, accessory buds, pseudo-terminal buds, cambium, lateral meristem, lateral bud, vegetative buds, reproductive buds, mixed buds, shoot segments, shoot apices, stem segments, immature nodal sections from stems, iateral shoots, seedlings, seeds, shoots starting to rise from the ground, immature flower buds, inflorescences, crown segments, leaf segments, or any part thereof. In some embodiments, the explants can be node segments, immature leaves, immature embryos, or mature seeds. In some embodiments, the explants can be tissue comprising meristematic cells, such as the cells located in axillary or lateral buds of a bamboo plant.

[00540] In some embodiments, the bamboo species is selected from Phyllostachys bissetti, Fargesia denudata, Pieioblastus fortunei, Sasa Veitchii, Pleioblastus viridistriatus, Thamnocalamus erassinodus, Chusquea Culeo "Cana Prieta", Bambusa Old Hamii, Phyllostachys Moso, Phyllostachys Atrovaginata, Dendrocalamus Asper, or Guadua Angustifolia, or the ones described in WO/2011/100762, which is incorporated herein by reference in its entirety. Grasses may be further selected from maize, wheat, fescues, barley, oat, fountaingrass, ryegrass, lye, sorghum, needlegrass, vetiver, panicgrass, scotch grass, rice, millet, and generally any member of the Poaceae family.

[00541] In one embodiment, the explants are taken from a juvenile or a mature plant. In some embodiments, the explants are taken from a plant of about 1 week old, about 2 weeks old, about 3 weeks old, about 1 month old, about 2 month old, about 3 months old, about half year old, about 1 year old, about 2 years old plant, about 3 years old, about 5 years old, or more. The plant from which the explant is obtained can be grown in any suitable conditions, including but not limited to growing in a growth chamber, growing in a greenhouse, growing in a field, or growing in a tissue culture container (petri dish, margenta box, etc.). In some embodiments, the explant is tissue culture obtained from shoot clumps maintained as stock on growth media. In some embodiments, the explant is a nodal section having one or more axillary bud, which can be dormant or active. In some other embodiments, the explant is a seed or a part thereof. In some embodiments, the explants are free or substantially free of pathogens. In some embodiments, before culturing the explants on the media, the explants are sterilized. Non-limiting examples of sterilizing explants are described in WO/201 1 /100762, which is incorporated herein by reference in its entirety.

[00542] In some embodiments, the explants are cultured on the first type of media until one or more embryos initiate, in some embodiments, the explants are cultured on the first type of media for about 1 -24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 2 months, about 2.5 months, about 3 months, about 3.5 months, about 4 months, about 4.5 months, about 5 months, about 5.5 months, about 6 months, or more. 00543] In some embodiments, the explants are transferred from an old medium to a fresh medium of the same type after a pre- determined period of time, or to separate from contaminated tissue culture when needed.

[00544] In some embodiments, the explants are placed under a suitable temperature and a suitable light level. In some embodiments, the explants are placed under a temperature of from 65°F-70°F or more and a full spectrum light level of 36-54

or more.

[00545] The embryos generated by step described above can be used for multiple purposes, or subjected to any suitable methods to produce mature embryos. In some embodiments, once an explant exhibits initiated embryos, the embryos obtained from the initiation stage can be collected and cultured in the second media as described herein to produce an embryogenic suspension. In some embodiments, the second media are liquid media.

[00546] In some embodiments, the embryos are cultured on the second media for about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 0 hours, 1 1 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 7 hours, 18 hours, 19 hours, 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 2 months, about 2.5 months, about 3 months, about 3.5 months, about 4 months, about 4.5 months, about 5 months, about 5.5 months, about 6 months, or more. In some embodiments, the step is continued until a sufficient amount of embryogenic-like structures are obtained. In some embodiments, pulsing methods described herein are used.

[00547] In some embodiments, the embryogenic-like structures are transferred from an old medium to a fresh medium of the same type after a pre-determined period of time or to separate them from contaminated tissue culture when needed.

[00548] In some embodiments, the embryogenic-like structures are cultured in a bioreactor, such as a temporary immersion bioreactor (e.g. an ebb and flow bioreactor). Bioreactors offer a promising way of scalmg-up micropropagation processes, making it possible to work in large containers with a high degree of control over the culture parameters (e.g., pH, aeration, oxygen, carbon dioxide, hormones, nutrients, etc.). Bioreactors are also compatible with the automation of micropropagation procedures, utilizing artificial intelligence, which reduces production costs. Non-limiting examples of bioreactors include those described in U.S. Patent. Nos. 3,578,431 ; 4,320,594; 4,669,217; 4,908,315; 4,934,096; 5,049,505; 5,088,231 ; 5,104,527; 5,119,588; 5,139,956; 5,171,683; 5,184,420; 5,186,895; 5,212,906; 5,225,342; 5,558,984; 5,597,731; and US 6,753, 78. More examples of plant micropropagation systems can be found in Etienne et al. (Bioreactors in coffee micropropagation, Braz. J. Plant Physiol., 18(l):45-54, 2006); Ziv (Bioreactor Technology for Plant Micropropagation Horticultural Reviews, Volume 24, Edited by Jules Jamck ISBN 0-471-33374-3); and Paek et al. (Application of bioreactors for large-scale micropropagation systems of plants, In vitro Cell. Dev. Biol. -Plant 37: 149-157, March- April 2001).

[00549] In some embodiments, the bioreactor is placed under a suitable temperature and a suitable light level. In some embodiments, the explants are placed under a temperature of from

65°F-70°F or more and a full spectrum light level of 36-54 pmole/nr/s² or more.

[00550] In some embodiments, during this step, the density of the embryogenic-like structures can be estimated or measured in order to determine if more cycles of eu!turing are needed.

[00551] The embryos suspension generated by the step described above can be used for multiple purposes, or subjected to any suitable methods to produce mature embryos. In some embodiments, once a desired embryogenic suspension is obtained, the embryos in the suspension can be transferred onto the third media as described herein. The media can be either liquid or solid. In some embodiments, the media are solid. [00552] The embryos are further multiplied and/or induced into a maturation stage during this step. Abscisic acid in the media is helpful to induce embryo maturation. In some embodiments, charcoal (e.g., active charcoal) can be added, which surprisingly can greatly enhance embryo production and maturation. In some embodiments, the charcoal is about 0.01% to 10% of the media by weight.

[00553] In some embodiments, the embryos are cultured on the third media for about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 2 months, about 2.5 months, about 3 months, about 3.5 months, about 4 months, about 4.5 months, about 5 months, about 5.5 months, about 6 months, or more. In some embodiments, the step is continued until enough mature embryos are obtained. In some embodiments, mature embryos can be isolated from the media while the culturing is continued to obtain more mature embryos. Embroys will establish on the third media.

[00554] In some embodiments, the embryos are transferred from an old medium to a fresh medium of the same type after a pre-determined period of time or to separate them from contaminated tissue culture when needed.

[00555] The mature embryos generated by the methods described above can be used for multiple purposes, or subjected to any suitable methods to germinate. In some embodiments, the mature embryos are germinated. In some embodiments, the mature somatic embryos are germinated on the fourth type of media of the present invention. In some embodiments, the media are solid media.

[00556] In some embodiments, the mature somatic embryos are germinated on the fourth media for about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 2 months, about 2.5 months, about 3 months, about 3.5 months, about 4 months, about 4.5 months, about 5 months. about 5.5 months, about 6 months, or more. In some embodiments, the step is continued until germination is accomplished.

[00557] The mature embryos or the germinated plants can be used for multiple purposes, in some embodiments, the mature somatic embryos can be treated and stored under suitable conditions before germination. In some embodiments, the mature embryos or the germinated plants can be used as a stock to produce more plants through tissue culture, by using methods known to one skilled in the art, such as those described in WO/2011/100762, which is incorporated herein by reference in its entirety. In some embodiments, the bamboo plants can be transferred to an /// vitro or an /// vivo condition to produce mature pla ts.

[00558] The present invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures, are incorporated herein by reference in their entirety for all purposes.

Kits

[00559] The present invention also provides kits for plant propagation. In some embodiments, the kits include one or more media of the present invention. In some embodiments, the kits include one or more explants of a plant species.

[00560] In another embodiment, the kits can comprise one or more containers for the tissue culturing process including without limitation, tubes, jars, boxes, jugs, cups, sterile bag technology, bioreactors, temporary immersion vessels, etc. In another embodiment the kits can comprise instructions for the tissue culturing of specific plants/strains. In another embodiment, the kits comprise combinations of the foregoing. Components of various kits can be found in the same or different containers. Additionally, when a kit is supplied, the different components of the media can be packaged in separate containers and admixed immediately before use. Such packaging of the components separately may permit long-term storage without losing the active components' functions. Alternatively, media can be provided pre-mixed.

[00561] The present invention also provides kits for bamboo plant propagation. In some embodiments, the kits include one or more media of the present invention. In some embodiments, the kits include one or more explants of a bamboo species. [00562] In addition, also provided are kits for growing bamboo plant from somatic embryos.

In some embodiments, the kits comprise one or more somatic embryos of the same bamboo species or of different bamboo species. Optionally, the kits comprise one or more media for germinating bamboo embryos. In some embodiments, the media for germinating bamboo embryos are selected from the fourth type of media as described herein.

[00563] The present invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures, are incorporated herein b - reference in their entirety for all purposes.

Propagation / Culture of Herbs and Phyto-Pharmacuetical Plants

[00564] The present invention also provides methods for in vitro propagation of herbs and phyto-pharmaceutical plants. The methods and/or media described herein may be used to culture and micropropagate any one of the following plants/plant part (Romanized Chinese nomenclature): Ai Ye, Ai Ye Tan, Ba Ji Tian, Ba Ji Tian, Bai Bian Dou (Bian Dou), Bai Bu, Bai Fan Shu Gen, Bat Fu Zi, Bai Guo (Yin Guo), Bai He, Bat Hua She She Cao, Bai Jiang Cao, Bai Mao Gen, Bai Qian, Bai Shao, Bai Tou Weng, Bat Wei, Bai Xian Pi, Bai Zhi, Bai Zhu, Bai Zi Ren, Ban Lan Gen, Ban Xia (Jiang), Ban Zhi Lian, Bei Sha Shen, Bian Xu, Bo He, Bu Gu Zhi, Gang Er Zi (Chao), Gang Zhu, Ce Bai Ye, Ce Bai Ye Tan, Cha Chi Huang, Cha Ye (Lu Cha), Chai Hu, Chang Chun Hua (Ri Ri Chun), Che Qian Cao, Che Qian Zi, Chen Pi, Chi Fu Ling, Chi Shao, Chong Wei Zi, Chuan Lian Zi, Chuan Niu Xi, Chuan Po Shi, Chuan Xin Lian, Chuan Xiong, Chun Pi (Chun Gen Pi), Ci, Shi, Ci Wu Jia (Wu Jia Shen), Da Ding, Huang, Da Fu Pi, Da Huang, Da Huang (Zhi), Da Qing Ye, Da Xiao Ji, Da Zao (Hong), Dan Dou Chi, Dan Nan Xing, Dan Shen, Dan Zhu Ye, Dang Gui, Dang Gui Wei, Dang Shen, Dao Di Wu Gong, Deng Xin Cao, Di Fu Zi, Di Gu Pi, Di Huang (Sheng), Di Yu, Di Yu Tan, Ding Shu Xiu, Ding Xiang, Dong Chong Xia Cao, Dong Gua Zi, Dou Kou (Bai Dou Kou), Du Huo, Du Zhong, E Bu Shi Cao, E Jiao, E Zhu, Er Cha, Fan Xie Ye, Fang Feng, Fang Ji (Han Fang Ji), Fen Bi Xie (Bi Xie), Feng Wei Cao, Fo Shou, Fu Hai Shi, Fu Ling, Fu Lmg Pi, Fu Pen Zi, Fu Rong, Fe Shen Fu Xiao Mai, Fu Zi (Zhi), Gan Cao, Gan Jiang, Gao Ben, Gao Liang Jiang, Ge Gen, Ge Hua, Ge Qiao (Hai Ge Fen), Geng Mi, Gou Qi Zi, Gou Teng, Gu Gui Bu, Gua Lou (Gua Lou Shi), Gua Lou Pi, Gua Lou Zi, Guan Ye Jin Si Tao, Gui Zhi, Hai Jin Sha, Hai Piao Xiao, Hai Tong Pi, Hai Zao, Han Xiu Cao Gen, He Huan Hua, He Huan Pi, He Shou Wu, He Ye, He Zi, Hei Da Zao, Hei Zhi Ma, Hong Hua, Hong Jing Tian, Hou Po, Hu Huang Lian, Hu Po, Ho yao huang, Hu zhang, Hua jiao, Hua shi, Hua shi cao, Huai hua, Huang ho, huang jin gui, Huang jmg, Huang lian, Huang Qi, Huang Qin, Huang shut qte, Hou man ren, huo xiang, Ji 3i, Ji net jin, Ji xiang teng, Ji xue cao, Ji xue teng, Jiang huang, Jiang xiang, Jiao gu lan, Jiao zhi zi, Jie geng, Jie zi, Jin qian cao, Jin yin hua, Jin ying zi, Jmg jie, Jing jie tan, Jiu cai zi, Jiu ceng ta, Ju he, Ju hong, Ju hua, Jue ming zi, Ku shen, Ku xmg ren, Kun bu, Lai fu zi, Li zhi he, Lian Qiao, Lian xu, Lian zi, Lian zi xin, Liang mi an zhen, Ling zhi, Liu zhi huang, Long dan, Long gu, Long van rou, Lu gen, Lu jiao shuang, Lu lu tong, Lu rong, Lu xian cao, Luo bu ma, Luo shi teng, Ma bian cao, Ma bo, Ma chi xian, Mai dong, Mai ya, Man jing zi, Mang Xiao, Mao dong qmg, Mian ma guan zhong, Mo gu xiao, Mo han lian, Mo yao, Mu dan pi, Mu gua, Mu ii, Mu tong, Mu xiang, Mu zei, Nan sha shen, Niu bank Zi, Niu xi, Nu zhen, O jie, Pang da hai, Pao jiang, Pao zai cao, Pi pa ye, Po bu zi ye, Pu gong ying, Pu huang, Pu huang tan, Pu tao zi, Pu yin, Qi ye lian, Qian cao, Qian ceng ta, Qian hu, Qian nian jian, Qian shi, Qiang huo, Qin jiao, Qin pi, Qing hao, Qing pi, Qu mai, Ren dong teng, Ren shen, Rou cong rong, Rou dou kou, Rou gui, Ru xiang, San leng, San Qi, Sang bat pi, San ji sheng, Sang shen Sang ye, Sang zhi, Sha ren, Sha yuan zi, Shan dou gen, Shan yao, Shan zha, Shan zha tan, Shan zhu yu, She chuang zi, She gan, Shen jin cao, Sheng jiang, Sheng ma, Shi chiang pu, Sha di, Shi gao, Shi jian chuan, Shi jue ming, Shi iiu pi, Shi wei, Shou wu teng, Shu di huang, Shu wei huang, S ui ding xiang, Shu fei ji. Si gua lou Su mu, Suan zao ren Suo yang, Tai zi shen, Tao ren, Tian dong, Tian hua fen, Tian ma, tian nan xing, Tian zhu huang, Ting li zi, Tong cao, Tou gu cao, Tu fu ling, Tu si zi, Wan dian jin, Wang bu liu xing, Wei ling xian, Wu bei zhi, Wu jia pi, Wu mei, Wu tian, Wu wei zi, Wu yao, Wu zhu yu Wu tian, Xi xian cao, Xi yang shen, Xia ku cao, Xia tian wu, Xian feng cao, Xian he cao, Xian mao, Xiang fu, Xiao hui xiang, Xiao mai, Xie bai, Xie cao, Xin yi, Xu chang qing, Xu duan, Xuan fu hua, Xuan shen, Ya she huang, Yan hu suo, Ye ju hua, Yi mu cao, Yi yi ren Yi zhi, Yi zhi xiang, Yin chen, Yin Xing ye, Yin yang huo, Yu jin, Yu li ren, Yu mi xu, Yu xing cao, Yu zhu, Yuan zhi, Zao jiao, Zao jian ci, Ze lan, Ze xie, Zhe bei mu, zhen zhu mu, Zhi cao wu, Zhi Chuan wu, Zhi gan cao, Zhi huang qi, Zhi mu, Zhi qiao, Zhi Shi, Zhi zi, Zhu ling, Zhu ru, Zi hua di ding, Zi ran tong, Zi shi ting, Zi u ye, Zi su zi, and Zi wan.

[00565] The methods and/or media described herein may be used to culture and micropropagate any one of the following plants: Artemisia argyi, brown artimisia, morinda, dolichos nut, stemona, stinking flueggea root, typhonium, ginkgo, lily, oldenlandia, thlaspi, imperata, cynanchum stauntoni, peony, Pulsatilla, Cynanchum atratum, dictamnus, Angelica, Atractylodes (alba), biota seed, isatis root, pinellia, scute barbata, glehnia, polygonum aviculare, mint, psorales, xanthmm fruit, atractylodes, biota., brown biota, stellaria, tea leaf, bupleurum, Madagascar periwinkle, plantago leaf, plantago seed, citrus peel, red pona, red peony, leonurus fruit, melia, cyathula, cudrama root, andrographis, ligusticum ailanthus bark, loadstone, eleuthero, euonymus, areca husk, rhubarb, isatis leaf, cirsium, red jujube, soia, Arisaema pulvis, salvia root lophatherium, tangkuei root, tangkuei tails, codonopsis, lysimachia, juncus, kochia, lyceum root bark, rehmannia, sanguisorba, brown sanguisorba, elephantopi, Flox caryophylli, Cordyceps sinensis, benincasa, cardamom, tuhuo angelica, eucommia bark, centipede herb, zedoaria, catechu, senna leaf, siler, stephania, tokoro, pteris, citrus sarcodactylus, pona poria cortex, rubus, hibiscus root, fushen poria, levis wheat, aconite, licorice, ginger, ligusticia (kaopen), galangal, pueraria root, pueraria flower, cyclina, rice, gambir, drynaria, trichosanthes fruit, St. John's wort, cinnamon, wasabi (Wasabia japonica), lyceum fruit, lygodium spores, erythrina, ascophyllum, mimosa pudicae, albizzia flower, polygonum, lotus, chebule, black jujube, sesame seed, carthamus, rhodiola, magnolia bark, picrorrhiza, succinium, leucas, polygonum cuspidatum, zanthoxylum, orthosiphon, sophora, phellodendron bark, vameria, polygonatum root, coptis, astragalus, scute, solanum, hemp, agastache, tribuius, paederia, gotu kola, willow, spatholobi, turmeric, dalbergia, gynostemma, brown gardenia, pratycodon, mustard, lysimachia, ionicera flower, rosa laevigata, schizonepeta, allium seed, basil, Ocimum basihciim, citrus plants, red tangerine, chrysanthemum (Chrysanthemum sp.), cassia seed, sophora flavescens, apricot, kelp, raphanus, litchi seed, forsythia, lotus stamen, lotus embryo, shiny leaf prickly ash root, Zanthoxyli nitidi, Ganoderma, solidago, gentian, longan fruit, phragmites, cornus cervi fragments, liquidambar, pyrolae, apocynum venetum, star jasmine vine, verbena, lasiosphaera, portulacae, ophiopogon, barley, vitex, mirabilitum Ilex pubescentis, aspidum, hyptix, eclipta, moutan, chaenomeles, caulis akebiae, vladimiria, equisetum, adenophora, arctium, achyranthes, ligustrum, lotus node, sterculia, physalis angulate, eriobotrya, sebastan plum cordia, dandelion, bulrush, grape seed, indian stringbush, schefflerae, rhizome rubiae, lycopodium, peucedanum, homalomena, Euryale, notopterygium, gentian macrophylla root, fraxinus Artemisia, citrus viride, dianthus, Ionicera vine, ginseng, cistanche, myristica, mastic, scirpus, notoginseng, mulberry, loranthus, amomum, astragalus seed, sophora subprostrata dioscorea, Crataegus, cormus, cnidium fruit, belamcanda, lycopodium, cimicifuga, acorus, kaki calyx, Chinese sage, haliotis, granatum rind, folium pyrrosiae, polygonum multifloru vine, justicia, ludwigia, milk thistle, luffa fiber, sappan wood, zizyphus, cynomorium, rehmannia, pseudostellaria, persica, asparagus, trichosanthes root, gastrodia, arisaema, bamboo, lepidium, tetrapanax, centella herba, smilax, cuscuta, ilex, vaccana seed, clematis, gallnut, acanthopanax, mume, wutien, schizandra, lindera eyodia, siegesbeckia, American ginseng, prunella, decumbent corydalis rhizome, bidens, agrimony, curculigo, cyperus fennel, wheat, bakeri, valerian root, magnolia flower, paniculate swallow wort root, dtpsacus, inula flowers, scrophularia, lippie, corydalis, chrysanthemum, leonurus, coix, alpima fruit, veronica cinerea, capillans, epimedium, curcuma, prunus seed, corn silk, houttuynia, polygonatum odorati, polygala, gleditsia fruit, gleditsia spine, lycopus, alisma, aconite tsao wu, astragalus, anemarrhena, aurantium fruit, aurantium immaturus, gardenia, polyporus viola, perilla, perilla seed, aster, and rhizome asteris.

[00566] In some embodiments, the list in the preceding paragraph includes common names, scientific names, plants, and plant parts. If reciting plant parts or plant products, the disclosure contemplates culturing and micropropagatmg the plants from which the products or parts are from.

EXAMPLES

Example 1

In vitro Initiation

[00567] Fully developed pistachio plants were cut into single-node explants, shoot tips, basal (bottom) parts of plants with multiple buds, and cultured on different agar-solidified culture media under standard tissue culture conditions (16 h light photoperiod, cool white fluorescent lights at 80-100 ,umol/m²/s light intensity, 24°C).

[00568] Culture vessels (baby food jars) with ventilated lids are used in all experiments: 1-

3 explants per 1 vessel, 12 vessels per each experimental medium, 40 ml of medium in each vessel. The explants are subcultured every 30 days, photographed every week, and observed daily.

[00569] The culture media differed in composition of major macronutrients (e.g., MS vs.

WPM vs. DKW), individual elements (calcium, magnesium, phosphorus, zinc copper, boron, etc.), as well as in type and concentration of plant growth regulators (PGRs).

Example 2

In vitro multiplication on solid medium [00570] From a series of experiments examining the reactions of plants in vitro, new culture media - BOO 101 . BOO102, BOO103, BOO104, BOO105, BOO106, BOO107, BOO108, BOO109, BOOl lO, BOOl l l, B00112, B00113, B00114, B00115, B001 16, B00117, B00118, B00119, BOO120, B00121 , B00122, B00123, B00124, B00125, B00126, B00127, B00128, B00129, BOO130, B00131 , B00132, B00133, B00134, B00135, B00136, and B00137, - were developed for increasing multiplication rate and overall quality of plants. Using these media plant culture consistently resulted in well-developed healthy plants with minimal tissue necrosis. The compositions of these media are presented in Table 1, and their ke - advantages are based on data presented below.

Because the type and dosage of PGRs are usually the key factors controlling the morphogenetic response of plant cells in vitro, the mam work is focused on testing various concentrations of cytokinins or cytokinin-like compounds (TDZ, 2iP, BA, meta-Topolin, ZR), alone or in combination with auxins (NAA, IAA, IBA), to induce organogenesis and high frequency multiplication. In the absence of PGRs (i.e., on hormone-free media), the plant growth is expected to be poor. In contrast, the culture media with 1-3 mg/l meta-Topolin, alone or in combination with 0.02-1 mg/l NAA or IBA, is expected to provide the highest rate of shoot organogenesis with the fastest response: each explant forming multiple meristematic buds, and the first axillary shoots appearing as early as 3 days after the explants are placed on the media. Without wishing to be bound by any particular theory, meta-topolin can reduce or eliminate phenolics in the tissue culture, therefore leads to increased survival rate.

Example 3

In vitro multiplication in liquid medium

[00571] Pistachio cultures are also multiplied in liquid medium using temporary immersion bioreactor vessels. The size of bioreactor vanes from 0.1 to 20 L depending on production requirements. Bioreactors are inoculated with pistachio material produced in tubes, jars, or boxes. Bioreactors is kept under standard conditions (22- 24 °C and 16/8 hours day /night photoperiod). Media are refreshed every day, every two days, every three days, every four days, every five days, every six days, or every one to four weeks. After each cycle the amount of biomass increased between about 1, 2, 3, 4, 5 times or more. After several multiplication cycles the shoots were be further subjected to in vitro rooting under solid or liquid conditions. [00572] Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials, similar or equivalent to those described herein, can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.

Example 4

Propagation of Stock Plants

[00573] The stock plants are propagated through single-node cuttings (containing one axillary bud) or shoot-tip explants, and cultured aseptically in tubes (Sigma) on BOOl 17 medium without growth regulators at 24° C under a 16-h light period. Light is provided by cool white fluorescent tubes (Sylvania) at a photon flux density of 85-100 mol/m²/s.

[00574] Alternatively, an indirect shoot organogenesis method is used to produce a plant shoot explant, which is then used to produce the stock plants. Meristemic clones are grown on the solid BOO103 medium under standard tissue culture conditions, and plant shoots were regenerated from the meristemic clones after incubation.

[00575] The phrase "meristemic clones" as used herein is equivalent to the phrase

"meristem derived clones". "Meristemic clones" means that these clones were derived from a meristamatic tissue such as the apical meristem.

Example 5

Micropropagation of Herbs and Phyto-Pharmaceutical Plants

[00576] Initial explants taken from greenhouse or field grown plants are surface sterilized, using a procedure such as described in PCT Publication No. WO2013016198. In vitro cultures are initiated in culture vessels containing BOQ1Q1, BOO102, BQO103, BOO104, BOO105, BOOl 06, BOO 107, BOO 108, BOO 109, BOOl 10, BOOl 11, BOOl 12, BOOl 13, BOOl 14, BOOl 15, BOOl 16, BOOl 17, BOOl 18, BOOl 19, BOO120, BOQ121, B00122, B00123, B00124, BOO i 25. B00126, B00127, B00128, B00129, BOO130, B00131 , B00132, B00133, B00134, B00135, and/or B00136 solid media. After several 4-week subculture cycles, microshoots are moved into bioreactors. The initial liquid media is similar to the solid media but without agar. Every 7 to 10 days the initial media is replaced with Pulsing media 1. The material is maintained in the Pulsing media 1 for 3 days. All media is refreshed every 4 weeks. The cultures in bioreactors are maintained under standard environmental conditions (16/8 hour photoperiod and 25°C ± 2°C). Microshoots are harvested at maturity (6 weeks). Singulated shoots are moved to a rooting medium. Established plants are planted in soil or hydroponics.

Example 6

Micropropagation of Cannabis species

[00577] Initial explants of Cannabis saliva, or hybrids thereof, are taken from greenhouse or field grown plants are surface sterilized, using a procedure such as described in PCT Publication No. WO2013016198. In vitro cultures are initiated in culture vessels containing BOOl 16 solid media. After several 4-week subculture cycles, microshoots are moved into bioreactors. The initial liquid media is similar liquid media but without agar. The material is maintained in B00128 media for up to 6 weeks. The cultures are exposed to pulsing medium containing TDZ (Pulsing medium 1) followed by medium containing meta-topoline, once every 3 to 7 days. After the 6 weeks period the culture in bioreactor is pulsed weekly with Pulse 2 media (Pulsing media 2). All media is refreshed every 4 weeks. The cultures in bioreactors are maintained under standard environmental conditions (16/8 hour photoperiod and 25°C ± 2°C). Microshoots are harvested at maturity (8 to 10 weeks). Singulated shoots are moved to rooting media for rooting. Established plants are planted in soil or hydroponic applications.

Example 7

Culture initiation of Hemp

[00578] Explants for hemp culture initiation are collected from healthy hemp stocks grown in greenhouse and/or field. Apical and lateral buds of plants are isolated and washed thoroughly with a mild detergent and surface sterilized under aseptic conditions. Surface sterilization is achieved by immersing the shoot buds in solutions of bleach for a period of time ranging from about 15 minutes to about 2 hours. After surface sterilization, the shoots are placed in a sterile surface in the laminar flow hood, dead tissues are removed using a sharp scalpel, and the buds are inoculated in a suite of test tubes, each containing a different medium, from the media disclosed herein. [00579] Initiated hemp cultures are multipled in liquid media in different culture vessels varying in size and volume from about 00 mis to about 10 gallons. Each culture cycle rannges from between about 4 to about 8 weeks based on the vessel size. The size vessels used in the trial result in production of a total of about 5 plants to about 15,000 plants per vessel for each culture cycle. Upon conclusion of the multiplication cycle, a rooting cycle is initiated using any one or more the media disclosed hereing. Cultures are incubated with temperature ranges from 25°C + 2° and a photoperiod set at 16 hrs/8 hrs of light/dark

[00580] In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only- examples and should not be taken as limiting the scope of the invention.

[00581] Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials, similar or equivalent to those described herein, can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.

NUMBERED EMBODIMENTS OF THE DISCLOSURE

[00582] Notwithstanding the appended claims, the disclosure sets forth the following numbered embodiments:

1. A medium for producing cannabis micropropagations wherein said medium comprises sucrose and

(1) at least one eyiokinin and at least one auxin;

(2) at least one growth retardant; or

(3) at least one cytokinin, at least one auxin, and at least one growth retardant;

wherein the medium is selected from any one of tables 1 -9.

2. The medium of embodiment 1, wherein the medium is selected from any one or more of the following: BOO101 , BOO102, BOO103, BOO104, BOO105, BOO105, BOO106, BQO107, BOO 108, BOO 109, BOO 110, BQOl l l , BOO 112, BOO 1 13, BOO 1 14, B00115, BOO 1 16, B001 17, B00118, B00119, BOO120, B00121 , B00122, B00123, B00124, B00125, B00126, B00127, B00128, B00129, BOO130, B00131 , B00132, B00133, B00134, B00135, B00136, B00137, and combinations thereof,

3. A method for producing cannabis micropropagations comprising utilizing a temporary immersion bioreactor, wherein the bioreactor comprises:

a growth vessel for incubating plant tissue in a sterile or substantially sterile environment; a first media container having a first fluid port and a second fluid port, the first fluid port fluidically coupleable to the growth vessel;

a second media container having a first fluid port and a second fluid port, the first fluid port fluidically coupleable to the growth vessel;

a gas source fluidically coupleable to the second fluid port of the first media container and second fluid port of the second media container; and

a controller operable in a first operating mode in which pressurized gas is delivered from the gas source to the first media container to displace a first volume of liquid contained therein to the growth vessel, and a second operating mode in which pressurized gas is delivered from the gas source to the second media container to displace a second volume of liquid contained therein to the growth vessel; wherein the medium is selected from tables 1-9.

4. A temporary immersion bioreactor, comprising:

a growth vessel for incubating cannabis plant tissue in a sterile or substantially sterile environment;

a first media container having a first fluid port and a second fluid port, the first fluid port fluidically coupleable to the growth vessel;

a second media container having a first fluid port and a second fluid port, the first fluid port fluidically coupleable to the growth vessel;

a gas source fluidically coupleable to the second fluid port of the first media container and second fluid port of the second media container; and

a controller operable in a first operating mode in which pressurized gas is delivered from the gas source to the first media container to displace a first volume of liquid contained therein to the growth vessel, and a second operating mode in which pressurized gas is delivered from the gas source to the second media container to displace a second volume of liquid contained therein to the growth vessel; wherein the medium is selected from tables 1 -9. 5. The temporary immersion bioreactor of embodiment 4, wherein the controller is operable in a third operating mode in which liquid contained in the growth vessel is allowed to flow from the growth vessel into at least one of the first media container and the second media container.

6. The temporary immersion bioreactor of any one of embodiments 4-5, wherein the controller is operable in a first incubation sequence in which the third operating mode is executed subsequent to the first operating mode.

7. The temporary immersion bioreactor of any one of embodiments 4-6, wherein the controller is operable in a second incubation sequence in which the third operating mode is executed subsequent to the second operating mode.

8. The temporary immersion bioreactor of any one of embodiments 4-7, wherein the controller is further operable in a plant propagation mode in which the first incubation sequence and the second incubation sequence are executed.

9. A medium for producing plant micropropagations wherem said medium comprises sucrose and

(1) at least one cytokinin and at least one auxin;

(2) at least one growth retardant; or

(3) at least one cytokinin, at least one auxin, and at least one growth retardant;

wherein the medium is selected from any one of tables 1-9.

10. The medium of embodiment 9, wherem the sucrose has a concentration of about 25-35 g/L.

11. The medium of any one of embodiments 9-10, wherein the at least one cytokinin is 2ip.

12. The medium of embodiment 11, wherein the 2ip has a concentration of about 1 to 10 rng L,

13. The medium of any one of embodiments 9-12, wherein the at least one auxin is IAA.

14. The medium of embodiment 3, wherein the IAA has a concentration of about 0.1 to 10 mg/L.

15. The medium of any one of embodiments 9-14, wherein the growth retardant is a gibberellins acid antagonist.

16. The medium of embodiment 5, wherein the gibberellins acid antagonist is ancymidol.

17. The medium of embodiment 16, wherem the ancymidol has a concentration of about 0.1 to 10 mg/L. 18. The medium of any one of embodiments 9-17, wherein the medium is a solid, semi -solid, liquid, or semi-liquid medium.

19. The medium of any one of embodiments 9-18, wherem the medium has a pH of about 5.5 to 6.2.

20. A medium for producing phyto-pharmaceutical plant micropropagations wherein said media comprises sucrose and

(i) at least one auxin;

(ii) at least one growth retardant; or

(lii) at least one auxin and at least one growth retardant; wherein the medium is selected from tables 1-9.

21. The medium of embodiment 20, wherein the sucrose has a concentration of about 50-100 g L.

22. The medium of any one of embodiments 20-21, wherein the medium does not comprise any cytokinin.

23. The medium of any one of embodiments 20-22, wherein the at least one auxin is NAA.

24. The medium of any one of embodiments 20-23, wherein the NAA has a concentration of about 0.01 to about 0.1 mg L.

25. The medium of any one of embodiments 20-24, wherein the growth retardant is a gibberelims acid antagonist.

26. The medium of embodiment 25, wherem the gibberelims acid antagonist is ancvmidol.

27. The medium of embodiment 26, wherein the ancymidol has a concentration of about 0.1 to 10 mg/L.

28. The medium of any one of embodiments 20-27, wherein the medium is a solid, semisolid, liquid, or semi-liquid medium.

29. The medium of any one of embodiments 20-29, wherein the medium has a pH of about 5.5 to 6.2,

30. A set of media for producing phyto-pharmaceutical plant micropropagations wherein the set of media comprises:

(1) one or more propagation and multiplication medium;

(2) one or more pre-tuberization medium; and (3) one or more tuberization medium;

wherein the propagation and multiplication medium does not contain any plant hormone or plant growt regulator;

wherein the pre- tuberization medium comprises sucrose at concentration SI and

(i) at least one cytokinin and at least one auxin;

(ii) at least one growth retardant; or

(in) at least one cytokinin, at least one auxin, and at least one growth retardant; wherein the tubenzation medium comprises sucrose at concentration S2 and

(i) at least one auxin;

(ii) at least one growth retardant; or

(in) at least one auxin and at least one growth retardant;

wherein SI is smaller than S2; and,

wherein the propagation and multiplication medium, the pre-tuberization medium, and the tubenzation medium are used to produce microtubers; wherein the medium is selected from tables 1 -9.

31. The set of media of embodiment 30, wherein SI is about 25-35 g/'L and S2 is about 50- 100 g/L.

32. The set of media of any one of embodiments 30-31, wherein the at least one cytokinin in the pre-tuberization medium is 2ip.

33. The set of media of embodiment 32, wherein the 2ip has a concentration of about 1 to 10 mg L.

34. The set of media of any one of embodiments 30-33, wherein the at least in one auxin in the pre-tuberization medium is IAA.

35. The set of media of embodiment 34, wherein the IAA has a concentration of about 0.1 to lO mg L.

36. The set of media of any one of embodiments 30-35, wherein the at least one auxin in the tuberization medium is NAA.

37. The set of media of embodiment 36, wherein the NAA has a concentration of about 0.01 to about 0.05 mg/L.

38. The set of media of any one of embodiments 30-37, wherein the growth retardant in the pre-tuberization medium and/or the tuberization medium is a gibberellms acid antagonist. 39. The set of media of embodiment 38, wherein the gibberellins acid antagonist is ancymidol.

40. The set of media of embodiment 39, wherein the ancymidol has a concentration of about 0.1 to l O mg/L.

41. The set of media of any one of embodiments 30-40, wherein one or more medium is a solid, semi-solid, liquid, or semi-liquid medium.

42. The set of media of any one of embodiments 30-41 , wherein one or more medium has a pH of about 5.5 to 6.2.

43. A kit for producing microtubers, wherein the kit comprises a medium of any one of embodiments 9 to 29 or a set of media of any one of embodiments 30 to 42; wherein the medium is selected from tables 1-9.

44. A method for producing plant micropropagations comprising utilizing a set of medium of any one of embodiments 30-42 or a kit of embodiment 43.

45. A method for producing plant micropropagations comprising utilizing a temporary immersion bioreactor, wherein the bioreactor comprises:

a growth vessel for incubating plant tissue in a sterile or substantially sterile environment; a first media container having a first fluid port and a second fluid port, the first fluid port fluidicallv coupleable to the growth vessel;

a second media container having a first fluid port and a second fluid port, the first fluid port fluidically coupleable to the growth vessel;

a gas source fluidicallv coupleable to the second fluid port of the first media container and second fluid port of the second media container; and

a controller operable in a first operating mode in which pressurized gas is delivered from the gas source to the first media container to displace a first volume of liquid contained therein to the growth vessel, and a second operating mode in which pressurized gas is delivered from the gas source to the second media container to displace a second volume of liquid contained therein to the growth vessel; wherein the medium is selected from tables 1-9.

46. A temporary immersion bioreactor, comprising:

a growth vessel for incubating plant tissue in a sterile or substantially sterile environment; a first media container having a first fluid port and a second fluid port, the first fluid port fluidicallv coupleable to the growth vessel; a second media container having a first fluid port and a second fluid port, the first fluid port fluidically coupleable to the growth vessel;

a gas source fluidically coupleable to the second fluid port of the first media container and second fluid port of the second media container; and

a controller operable in a first operating mode in which pressurized gas is delivered from the gas source to the first media container to displace a first volume of liquid contained therein to the growth vessel, and a second operating mode in which pressurized gas is delivered from the gas source to the second media container to displace a second volume of liquid contained therein to the growth vessel; wherein the medium is selected from tables 1 -9.

47. The temporary immersion bioreactor of embodiment 46, wherein the controller is operable in a third operating mode in which liquid contained in the growth vessel is allowed to flow from the growth vessel into at least one of the first media container and the second media contamer.

48. The temporary immersion bioreactor of embodiment 47, wherein the controller is operable in a first incubation sequence in which the third operating mode is executed subsequent to the first operating mode.

49. The temporary immersion bioreactor of embodiment 48, wherein the controller is operable in a second incubation sequence in which the third operating mode is executed subsequent to the second operating mode.

50. The temporary immersion bioreactor of embodiment 49, wherein the controller is further operable in a plant propagation mode in which the first incubation sequence and the second incubation sequence are executed.

51. The temporary immersion bioreactor of embodiment 47, wherein the growth vessel is elevated above the first and second media containers to allow liquid to flow from the growth vessel into at least one of the first media container and the second media container in the third operating mode.

52. The temporary immersion bioreactor of any one of embodiments 46-51 , further comprising a manifold fluidically coupleable to the growth vessel, the first media container, and the second media container,

wherein the manifold is operable to control liquid flow between the growth vessel and the first media container and between the growth vessel and the second media contamer. 53. The temporary immersion bioreactor of any one of embodiments 46-52, wherein the growth vessel includes a fluid conduit configured to siphon liquid from the growth vessel to at least one of the first media container and the second media container.

54. The temporary immersion bioreactor of any one of embodiments 46-53, wherein the growth vessel is an ebb and flow bioreactor.

55. The temporary immersion bioreactor of any one of embodiments 46-54, wherein the controller is operable to control fluid communication between the growth vessel and the first media container, between the growth vessel and the second media container, between the gas source and the first media container, and between the gas source and the second media container.

56. A system for production of phyto-pharmaceutical plant micropropagations, comprising: a temporary immersion bioreactor of any one of embodiments 46 to 55;

a ginseng explant;

a pre-tuberization medium, wherein the media is any one of embodiments 9 to 19;

a tuberization medium, wherein the media is any one of embodiments 20 to 29.

57. The system of embodiment 56, wherein the ginseng explant is a pathogen-free seedling.

58. The system of embodiment 57, wherein the ginseng seeding comprises about 4 to 7 axillary buds.

59. The medium of any one of embodiments 9-29, wherein the plant is an herb.

60. The medium of any one of embodiments 9-29, wherein the plant is a

phytopharmaceutical-producing plant.

61. The medium of any one of embodiments 9-29, wherein the plant is selected from the group consisting of basil, cannabis, pistachio, ginseng, St. John's wort, willow, lotus, wasabi, and echinacea.

62. The medium of any one of embodiments 9-29, wherein the medium is selected from any one of tables 1 -9.

63. The medium of any one of embodiments 9-29, wherein the medium is selected from any one of tables 2-8.

[00583] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. [00584] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following Claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby

INCORPORATION BY REFERENCE

[00585] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

REFERENCES

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[00605] Nasiruddin and Blake 1994, Production of potato microtubers with and without growth regulators. In: PJ Lmnsden, JR Nicholas, WJ Davies (eds), Physiology, Growth and Development of Plants. Kluwer, Dordrecht, pp. 254-260.

[00606] Jimenez et al., 1999, Improved production of potato microtubers using a temporary immersion system, Plant Cell, Tissue and Organ Culture 59: 19-23, 1999. [00607] Akita and Takayama, 1994, Stimulation of potato (Solatium tuberosum L.) tuberization by semicontmuous liquid medium surface level control, Plant Cell Reports (1994) 13: 184 187

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Claims

1. A medium for producing cannabis micropropagations wherein said medium comprises sucrose and

(1 ) at least one cytokinin and at least one auxin;

(2) at least one growth retardant; or

(3) at least one cytokinin, at least one auxin, and at least one growth retardant; wherein the medium is selected from any one of tables 1-9.

2. The medium of claim 1, wherein the medium is selected from any one or more of the following: BOO101, BOO102, BOO103, BOO104, BOO105, BOO105, BOO106, BOO107, BOO! 08, BOO 109, BOOl 10, BOOl 1 1 , BOO 1 12, BOOl 13, BOO 1 14, BOOl 15, BOOl 16, B00117, BOOl l S, B00119, BOO120, B00121, B00122, B00123, B00124, B00125, B00126, B00127, B00128, B00129, BOO130, B00131 , BOOl 32, B00133, B00134, BOOl 35, BOOl 36, BOOl 37, and combinations thereof.

3. A method for producing cannabis micropropagations comprising utilizing a temporar immersion bioreactor, wherein the bioreactor comprises:

a growth vessel for incubating plant tissue in a sterile or substantially sterile environment; a first media container having a first fluid port and a second fluid port, the first fluid port fluidically coupleable to the growth vessel;

a second media container having a first fluid port and a second fluid port, the first fluid port fluidically coupleable to the growth vessel;

a gas source fluidically coupleable to the second fluid port of the first media container and second fluid port of the second media container; and

a controller operable in a first operating mode in which pressurized gas is delivered from the gas source to the first media container to displace a first volume of liquid contained therein to the growth vessel, and a second operating mode in which pressurized gas is delivered from the gas source to the second media container to displace a second volume of liquid contained therein to the growth vessel; wherein the medium is selected from tables 1-9.

4. A temporary immersion bioreactor, comprising: a growth vessel for incubating cannabis plant tissue in a sterile or substantially sterile environment;

a first media container having a first fluid port and a second fluid port, the first fluid port f!uidically coupleable to the growth vessel;

a second media container having a first fluid port and a second fluid port, the first fluid port fluidical!y coupleable to the growth vessel;

a gas source fluidically coupleable to the second fluid port of the first media container and second fluid port of the second media container; and

a controller operable in a first operating mode in which pressurized gas is delivered from the gas source to the first media container to displace a first volume of liquid contained therein to the growth vessel, and a second operating mode in which pressurized gas is delivered from the gas source to the second media container to displace a second volume of liquid contained therein to the growth vessel; wherein the medium is selected from tables 1-9.

5. The temporary immersion bioreactor of claim 4, wherein the controller is operable in a third operating mode in which liquid contained in the growth vessel is allowed to flow from the growth vessel into at least one of the first media container and the second media container.

6. The temporary immersion bioreactor of claim 5, wherein the controller is operable in a first incubation sequence in which the third operating mode is executed subsequent to the first operating mode.

7. The temporary immersion bioreactor of claim 6, wherein the controller is operable in a second incubation sequence in which the third operating mode is executed subsequent to the second operating mode.

8. The temporary immersion bioreactor of claim 7, wherein the controller is further operable in a plant propagation mode in which the first incubation sequence and the second incubation sequence are executed.

9. A medium for producing plant micropropagations wherein said medium comprises sucrose and

(1) at least one cytokinm and at least one auxin;

(2) at least one growth retardant; or

(3) at least one cytokinm, at least one auxin, and at least one growth retardant; wherein the medium is selected from any one of tables 1-9.

10. The medium of claim 9, wherein the sucrose has a concentration of about 25-35 g/L.

11. The medium of claim 9, wherein the at least one cytokinin is 2ip.

12. The medium of claim 1 1 , wherein the 2ip has a concentration of about 1 to 10 mg/L.

13. The medium of claim 9, wherein the at least one auxin is IAA.

14. The medium of claim 13, wherein the IA A has a concentration of about 0, 1 to 10 mg/L.

15. The medium of claim 9, wherem the growth retardant is a gibberellins acid antagonist.

16. The medium of claim 15, wherein the gibberellins acid antagonist is ancymidol .

17. The medium of claim 16, wherein the ancymidol has a concentration of about 0.1 to 10 mg/L.

18. The medium of claim 9, wherein the medium is a solid, semi-solid, liquid, or semi-liquid medium.

19. The medium of claim 9, wherein the medium has a pH of about 5.5 to 6.2.

20. A medium for producing phyto-pharmaceutical plant micropropagations wherein said media comprises sucrose and

(i) at least one auxin;

(i) at least one growth retardant; or (iii) at least one auxin and at least one growth retardant; wherein the medium is selected from tables 1 -9.

21. The medium of claim 20, wherein the sucrose has a concentration of about 50-100 g L.

22. The medium of claim 20, wherein the medium does not comprise any cytokimn.

23. The medium of claim 20, wherein the at least one auxin is NAA.

24. The medium of claim 20, wherein the NA A has a concentration of about 0.0 to about 0.1 mg/L,

25. The medium of claim 20, wherein the growth retardant is a gibberellins acid antagonist.

26. The medium of claim 25, wherein the gibberellins acid antagonist is ancymidol.

27. The medium of claim 26, wherein the ancymidol has a concentration of about 0. to 10 mg/L.

28. The medium of claim 20, wherein the medium is a solid, semi-solid, liquid, or semi- liquid medium.

29. The medium of claim 20, wherein the medium has a pH of about 5.5 to 6.2.

30. A set of media for producing phyto-pharmaceutical plant micropropagations wherein the set of media comprises:

(1) one or more propagation and multiplication medium;

(2) one or more pre-tubenzation medium; and

(3) one or more tuberization medium;

wherein the propagation and multiplication medium does not contain any plant hormone or plant growth regulator;

wherein the pre- tuberization medium comprises sucrose at concentration SI and

(i) at least one cytokimn and at least one auxin;

(ii) at least one growth retardant; or (iii) at least one cytokinin, at least one auxin, and at least one growth retardant;

wherein the tuberization medium comprises sucrose at concentration S2 and

(iv) at least one auxin;

(v) at least one growth retardant; or

(vi) at least one auxm and at least one growth retardant;

wherein S I is smaller than S2; and,

wherein the propagation and multiplication medium, the pre-tuberization medium, and the tuberization medium are used to produce microtubers; wherein the medium is selected from tables 1 -9.

31. The set of media of claim 30, wherein S I is about 25-35 g/L and S2 is about 50-100 g/L.

32. The set of media of claim 30, wherein the at least one cytokinin in the pre-tuberization medium is 2ip.

33. The set of media of claim 32, wherein the 2ip has a concentration of about 1 to 10 mg/L.

34. The set of media of claim 30, wherein the at least in one auxin in the pre-tuberization medium is IAA.

35. The set of media of claim 34, wherein the IAA has a concentration of about 0.1 to 1 0 mg/L.

36. The set of media of claim 30, wherein the at least one auxin in the tuberization medium is NAA.

37. The set of media of claim 36, wherein the NAA has a concentration of about 0.01 to about 0.05 mg/L.

38. The set of media of claim 30, wherein the growth retardant in the pre-tuberization medium and/or the tuberization medium is a gibberellins acid antagonist.

39. The set of media of claim 38, wherein the gibberellins acid antagonist is ancymido

40. The set of media of claim 39, wherein the ancymidol has a concentration of about 0.1 to

41. The set of media of claim 30, wherein one or more medium is a solid, semi-solid, li quid, or semi-liquid medium.

42. The set of media of claim 30, wherein one or more medium has a pH of about 5.5 to 6.2.

43. A kit for producing microtubers, wherein the kit comprises a medium of any one of claims 9 to 29 or a set of media of any one of claims 30 to 42; wherein the medium is selected from tables 1-9.

44. A method for producing plant micropropagations comprising utilizing a set of medium of any one of claims 30-42 or a kit of claim 43.

45. A method for producing plant micropropagations comprising utilizing a temporary immersion bioreactor, wherein the bioreactor comprises:

a growth vessel for incubating plant tissue in a sterile or substantially sterile environment; a first media container having a first fluid port and a second fluid port, the first fluid port fluidically coupleable to the growth vessel;

a second media container having a first fluid port and a second fluid port, the first fluid port fluidically coupleable to the growth vessel;

a gas source fluidically coupleable to the second fluid port of the first media container and second fluid port of the second media container; and

a controller operable in a first operating mode in which pressurized gas is delivered from the gas source to the first media container to displace a first volume of liquid contained therein to the growth vessel, and a second operating mode in which pressurized gas is delivered from the gas source to the second media container to displace a second volume of liquid contained therein to the growth vessel; wherein the medium is selected from tables 1-9.

46. A temporary immersion bioreactor, comprising:

a growth vessel for incubating plant tissue in a sterile or substantially sterile environment; a first media container having a first fluid port and a second fluid port, the first fluid port fluidically coupleable to the growth vessel; a second media container having a first fluid port and a second fluid port, the first fluid port fluidically coupleable to the growth vessel;

a gas source fluidically coupleable to the second fluid port of the first media container and second fluid port of the second media container; and

a controller operable in a first operating mode in which pressurized gas is delivered from the gas source to the first media container to displace a first volume of liquid contained therein to the growth vessel, and a second operating mode in which pressurized gas is delivered from the gas source to the second media container to displace a second volume of liquid contained therein to the growth vessel; wherein the medium is selected from tables 1 -9..

47. The temporary immersion bioreactor of claim 46, wherein the controller is operable in a third operating mode in which liquid contained in the growth vessel is allowed to flow from the growth vessel into at least one of the first media container and the second media container.

48. The temporary immersion bioreactor of claim 47, wherein the controller is operable in a first incubation sequence in which the third operating mode is executed subsequent to the first operating mode.

49. The temporary immersion bioreactor of claim 48, wherein the controller is operable in a second incubation sequence in which the third operating mode is executed subsequent to the second operating mode.

50. The temporary immersion bioreactor of claim 49, wherein the controller is further operable in a plant propagation mode in which the first incubation sequence and the second incubation sequence are executed.

51. The temporary immersion bioreactor of claim 47, wherein the growth vessel is elevated above the first and second media containers to allow liquid to flow from the growth vessel into at least one of the first media container and the second media container in the third operating mode.

52. The temporary immersion bioreactor of claim 46, further comprising a manifold fluidically coupleable to the growth vessel, the first media container, and the second media container, wherein the manifold is operable to control liquid flow between the growth vessel and the first media container and between the growth vessel and the second media container.

53. The temporary immersion bioreactor of claim 46, wherein the growth vessel includes a fluid conduit configured to siphon liquid from the growth vessel to at least one of the first media container and the second media contamer.

54. The temporary immersion bioreactor of claim 46, wherein the growth vessel is an ebb and flow bioreactor.

55. The temporary immersion bioreactor of claim 46, wherein the controller is operable to control fluid communication between the growth vessel and the first media container, between the growth vessel and the second media container, between the gas source and the first media container, and between the gas source and the second media container.

56. A system for production of phyto-pharmaceutical plant micropropagations, comprising: a temporary immersion bioreactor of any one of claims 46 to 55:

a ginseng explant;

a pre-tuberization medium, wherein the media is any one of claims 9 to 19;

a tuberization medium, wherein the media is any one of claims 20 to 29.

57. The system of claim 56, wherein the ginseng explant is a pathogen-free seedling.

58. The system of claim 57, wherein the ginseng seeding comprises about 4 to 7 axillary buds.

59. The medium of any one of claims 9-29, wherein the plant is an herb.

60. The medium of any one of claims 9-29, wherein the plant is a phytopharmaceutical- producing plant.

61. The medium of any one of claims 9-29, wherein the plant is selected from the group consisting of basil, cannabis, pistachio, ginseng, St. John's wort, willow, lotus, wasabi, and echinacea.

62. The medium of any one of claims 9-29, wherein the medium is selected from any one of tables 1-9.

63. The medium of any one of claims 9-29, wherein the medium is selected from any one of tables 2-8.