WO2012112524A2 - Media, kits, systems and methods for the micropropagation of monocotyledonous plants - Google Patents

Media, kits, systems and methods for the micropropagation of monocotyledonous plants Download PDF

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Publication number
WO2012112524A2
WO2012112524A2 PCT/US2012/025018 US2012025018W WO2012112524A2 WO 2012112524 A2 WO2012112524 A2 WO 2012112524A2 US 2012025018 W US2012025018 W US 2012025018W WO 2012112524 A2 WO2012112524 A2 WO 2012112524A2
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WIPO (PCT)
Prior art keywords
media
iii
shoots
explants
stage
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PCT/US2012/025018
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French (fr)
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WO2012112524A3 (en
Inventor
Randall W. Burr
Jackie R. HEINRICHER
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The Red Triangle, LLC
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Priority claimed from PCT/US2011/024936 external-priority patent/WO2011100762A1/en
Priority claimed from US13/303,433 external-priority patent/US20120159847A1/en
Application filed by The Red Triangle, LLC filed Critical The Red Triangle, LLC
Priority to AU2012217902A priority Critical patent/AU2012217902B2/en
Publication of WO2012112524A2 publication Critical patent/WO2012112524A2/en
Publication of WO2012112524A3 publication Critical patent/WO2012112524A3/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/002—Culture media for tissue culture
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants

Definitions

  • Micropropagation also known as tissue culturing with the terms used interchangeably herein
  • tissue culturing is an excellent method to achieve large scale production of many plants.
  • Micropropagation is not unlike growing plants from cuttings.
  • the media comprises agar, with the addition of various compounds such as nutrients, inorganic salts, growth regulators, sugars, vitamins and other compounds.
  • a benefit to tissue culturing plants is that the plants can be grown in a sterile environment so that they 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.
  • micropropagation is very flexible and rapid upscaling is possible (within 1 year nearly one million plants can be produced from any 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.
  • the present disclosure provides effective media, kits, systems and methods for tissue culturing plants on a commercial scale.
  • One embodiment disclosed herein is a media for micropropagating plants wherein said media comprises meta-topolin and/or thidiazuron. In another embodiment, the media comprises meta-topolin.
  • the media is b-9-i media, b-9-ii media, b-9-iii media, b-9-iv media, b-9-v media, CW2-i media, CW2-N media, CW2-iii media, CW2-iv media, CW2-v media, b-10-i media, b-10-ii media, b-10-iii media, b- 10-iv media, b-10-v media, b-1 1 -i media, b-1 1 -ii media, b-1 1 -iii media, b-1 1 -iv media, b-1 1 -v media, b-12c-i media, b-12c-ii media, b-12c-iii media, b-12c-iv media, b-12c-v media, b-1 -i media, b-1 -ii media, b-1 -ii media, b-1 -iv media, b- 1
  • kits for micropropagating plants wherein said kit includes a media comprising meta-topolin and/or thidiazuron.
  • the media comprises meta-topolin.
  • the media is b-9-i media, b-9-ii media, b-9-iii media, b-9-iv media, b-9-v media, CW2-i media, CW2-N media, CW2-iii media, CW2-iv media, CW2-v media, b-10-i media, b-10-ii media, b-10-iii media, b-10-iv media, b-10-v media, b-1 1 -i media, b-1 1 -ii media, b-1 1 -iii media, b-1 1 -iv media, b-1 1 -v media, b-12c-i media, b-12c-ii media, b-12c-ii media, b-12c-ii media, b-12c-ii media, b-12c
  • Embodiments disclosed herein also include systems for the micropropagation of plants.
  • the system comprises a kit comprising a media comprising meta-topolin and/or thidiazuron.
  • the media is b-9-i media, b-9-ii media, b-9-iii media, b-9-iv media, b-9-v media, CW2-i media, CW2-N media, CW2-iii media, CW2-iv media, CW2-v media, b-10-i media, b-10-ii media, b-10-iii media, b-10-iv media, b-10-v media, b-1 1 -i media, b-1 1 -ii media, b-1 1 -iii media, b-1 1 -iv media, b-1 1 -v media, b-12c-i media, b-12c-ii media, b-12c-ii media, b-12c-ii media, b-12c-i
  • Embodiments disclosed herein also include methods of micropropagating plants.
  • the method comprises culturing explants, cultures and/or shoots in a media comprising meta-topolin and/or thidiazuron.
  • the growth media is b-9-i media, b-9-ii media, b-9-iii media, b-9-iv media, b-9-v media, CW2-i media, CW2-N media, CW2-iii media, CW2-iv media, CW2-v media, b-10-i media, b-10-ii media, b- 10-iii media, b-10-iv media, b-10-v media, b-10-v media, b-1 1 -i media, b-1 1 -ii media, b-1 1 -iii media, b-1 1 -iv media, b-1 1 -v media, b-12c-i media, b-12c-ii media, b-12c-ii media
  • a media for micropropagating plants wherein said media supports 10- 120 day multiplication cycles at least 1 month, for at least 3 months, for at least 6 months, for at least 9 months, for at least 12 months, for at least 15 months, for at least 18 months, for at least 21 months, for at least 24 months or for at least 36 months.
  • a media of embodiment 1 said media comprises meta-topolin or an analogue thereof.
  • a media according to embodiment 1 wherein said media comprises, consists essentially of or consists of b-9-i media, b-9-ii media, b-9-iii media, b- 9-iv media, b-9-v media, CW2-i media, CW2-ii media, CW2-iiv media, CW2-v media, b-10-i media, b-10-ii media, b-10-iii media, b-10-iv media, b-10-v media, b-1 1 -i media, b-1 1 -ii media, b-1 1 -iii media, b-1 1 -iv media, b-1 1 -v media, b-12c-i media, b-12c-ii media, b-12c-iii media, b-12c-iv media, b-12c-v media, b-1 -i media, b-1 -i media, b-1 -ii media, b-1 -iii media
  • kit for the micropropagation of plants wherein said kit comprises a media comprising meta-topolin or an analogue thereof wherein said media supports 10-120 day multiplication cycles at least 1 month, for at least 3 months, for at least 6 months, for at least 9 months, for at least 12 months, for at least 15 months, for at least 18 months, for at least 21 months, for at least 24 months or for at least 36 months.
  • a kit according to embodiment 4 where said media comprises, consists of or consists essentially of b-9-i media, b-9-ii media, b-9-iii media, b-9-iv media, b-9-v media, CW2-i media, CW2-N media, CW2-iii media, CW2-iv media, CW2-v media, b-10-i media, b-10-ii media, b-10-iii media, b-10-iv media, b-10-v media, b-1 1 -i media, b-1 1 -ii media, b-1 1 -iii media, b-1 1 -iv media, b-1 1 -v media, b-12c-i media, b-12c-ii media, b-12c-iii media, b-12c-iv media, b-12c-v media, b-1 -i media, b-1 -i media, b-1 -ii media, b-1 -
  • kit according to any of embodiments 4 or 5 wherein said system further comprises a media for transition to ex vivo conditions
  • a kit according to embodiment 6 wherein said media for transition to ex vivo conditions comprises, consists essentially of or consists of Br-2-i media, Br-2-ii media, Br-2-iii media, Br-2-iv media, Br-2-v media, Ech-i media, Ech-ii media, Ech-iii media, Ech-iv media, Ech-v media, Amel-i media, Amel-ii media, Amel-ii media, Amel-iv media, Amel-v media; FS1 -i media, FS1 -N media, FS1 -iii media, FS1 -iv media, FS1 -v media; FS2-i media, FS2-N media, FS2-iii media, FS2-iv media, FS2-v media; FS3-i media, FS3-N media, FS3-iii media, FS3-iv media, FS3-v media, FS4-i media, FS
  • kits described above further comprising an explant, wherein the explant can be any part of the plant including but not limited to internode, node, stolon, shoot, dormant lateral bud, immature flower bud, crown, rhizome or parts thereof.
  • a method of micropropagating plants comprising utilizing a media or kit of embodiments 1 , 2, 3, 4 or 5.
  • a method of transitioning micropropagated plants to ex vitro conditions comprising utilizing a kit of embodiment 6 or 7.
  • a method according to embodiment 1 1 wherein said method produces 100,000 plantlets tracing back to an individual an explant.
  • a method of micropropagting monocots comprising culturing monocot explants and/or shoots in a media of claim 14, 15, 16, 17 or 18.
  • a method of micropropagating monocots according to embodiment 19 wherein said monocot is Aegilops (goatgrass), Agave, Agropyron (crested wheatgrass), Agrostis (bentgrass), Allium (onion), Alopecurus (meadow foxtail), Amaranthus (amaranth), Ammophila (beach grass), Ananas (pineapple), Andropogon (beardgrass), Arrhenatherum (oat grass), Avena (oat), Axonopus (carpet grass), Beckmannia (slough grass), Bouteloua (grama grass), Bromus (brome grass), Calamagrostis (reed grass), Calamus (palm), Cortaderia (pampas grass), Dactylis (orchard grass), Elymus (wheat grass), Festuca (fescue), Geranium, Gladiolus, Hakonechloa (hakone grass), Hordeum (barley), Iridaceae
  • a method of micropropagating monocots according to embodiment 19 wherein said monocot is Aegilops (goatgrass), Agave, Agropyron (crested wheatgrass), Agrostis (bentgrass), Allium (onion), Alopecurus (meadow foxtail), Amaranthus (amaranth), Ammophila (beach grass), Ananas (pineapple), Andropogon (beardgrass), Arrhenatherum (oat grass), Avena (oat), Axonopus (carpet grass), Beckmannia (slough grass), Bouteloua (grama grass), Bromus (brome grass), Calamagrostis (reed grass), Calamus (palm), Cortaderia (pampas grass), Dactylis (orchard grass), Elymus (wheat grass), Festuca (fescue), Geranium, Gladiolus, Hakonechloa (hakone grass), Hordeum (barley), !ridacea
  • media for transitioning shoots to ex vitro conditions wherein media comprises, consists essentially of or consists of Br-2-i media, Br-2-ii media, Br-2-iii media, Br-2-iv media, Ech-i media, Ech-ii media, Ech-iii media, Ech-iv, Amel-i media, Amel-ii media, Amel-iii media, Amel-iv media, Amel-v media, FS1 -i media, FS1 -N media, FS1 -iii media, FS1 -iv media, FS1 -v media; FS2-i media, FS2-N media, FS2-iii media, FS2-iv media, FS2-v media; FS3-i media, FS3-M media, FS3-iii media, FS3-iv media, FS3-v media, FS4-i media, FS4-N media, FS4-ii media, FS4-
  • a media for micropropagating monocots wherein said media comprises thidiazuron or an analogue thereof and supports 10-120 day multiplication cycles for at least six months.
  • a method of micropropagting monocot comprising culturing monocot explants and/or shoots in a media of embodiment 22, 23, 24, 25, 26, 27 or 28.
  • a method of micropropagating monocot wherein said monocot is Aegilops (goatgrass), Agave, Agropyron (crested wheatgrass), Agrostis (bentgrass), Allium (onion), Alopecurus (meadow foxtail), Amaranthus (amaranth), Ammophila (beach grass), Ananas (pineapple), Andropogon (beardgrass), Arrhenatherum (oat grass), Avena (oat), Axonopus (carpet grass), Beckmannia (slough grass), Bouteloua (grama grass), Bromus (brome grass), Calamagrostis (reed grass), Calamus (palm), Cortaderia (pampas grass), Dactylis (orchard grass), Elymus (wheat grass), Festuca (fescue), Geranium, Gladiolus, Hakonechloa (hakone grass), Hordeum (barley), !ridaceae
  • a method of micropropagating monocot wherein said monocot is Aegilops (goatgrass), Agave, Agropyron (crested wheatgrass), Agrostis (bentgrass), Allium (onion), Alopecurus (meadow foxtail), Amaranthus (amaranth), Ammophila (beach grass), Ananas (pineapple), Andropogon (beardgrass), Arrhenatherum (oat grass), Avena (oat), Axonopus (carpet grass), Beckmannia (slough grass), Bouteloua (grama grass), Bromus (brome grass), Calamagrostis (reed grass), Calamus (palm), Cortaderia (pampas grass), Dactylis (orchard grass), Elymus (wheat grass), Festuca (fescue), Geranium, Gladiolus, Hakonechloa (hakone grass), Hordeum (barley), Iridaceae (
  • kit comprising a media according to any one of embodiments 14, 15, 16, 17, 18, 37, 22, 23, 24, 25, 26, 27 or 28.
  • 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.
  • 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.
  • the terms "dicotyledon” and “dicot” refer to a flowering plant having an embryo containing two seed halves or cotyledons. Examples include citrus; geranium; tobacco; tomato; the legumes, including peas, alfalfa, clover and soybeans; oaks; maples; roses; mints; squashes; daisies; walnuts; cacti; violets and buttercups.
  • the term "genotype” refers to the genetic makeup of an individual cell, cell culture, tissue, organism (e.g., a plant), or group of organisms.
  • line is used broadly to include, but is not limited to, a group of plants vegetatively propagated from a single parent plant, via tissue culture techniques or a group of inbred plants which are genetically very similar due to descent from a common parent(s).
  • a plant is said to "belong” to a particular line if it (a) is a primary transformant (TO) plant regenerated from material of that line; (b) has a pedigree comprised of a TO plant of that line; or (c) is genetically very similar due to common ancestry ⁇ e.g., via inbreeding or selfing).
  • TO primary transformant
  • the term "pedigree” denotes the lineage of a plant, e.g. in terms of the sexual crosses effected such that a gene or a combination of genes, in heterozygous (hemizygous) or homozygous condition, imparts a desired trait to the plant.
  • 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.
  • phenotype refers to the observable characters of an individual cell, cell culture, organism (e.g., a plant), or group of organisms which results from the interaction between that individual's genetic makeup (i.e., genotype) and the environment.
  • plant refers to a plant of any plant species of the Plant Kingdom (Plantae), including but not limited to the level of divisions of species into subspecies, hybrid, varieties, variants, mutations, chimera(s), or other subspecies designation.
  • Particular non-limiting examples include: amaranth, palm, palmetto, coconut, pineapple, grapefruit, lemon, lime, chestnut, mistletoe, banana, fig, holly, cherry, mountain ash, apple, orange, elm, willow tree, pine tree, eucalyptus, hemlock, cypress, Douglas fir, spruce, cedar, yew, magnolia, ginkgo, oak, maple, strawberry, blueberry, blackberry, raspberry, asparagus, rhubarb, bamboo, cabbage, pea (dry and fresh), chickpea, bean (garden, common, castor, dry, faba, garbanzo, lima, mung, navy and winged), lettuce, broccoli, pepper, tomato, radish, rape, camelina, wheat, millet, sedge, buckwheat, barley, rye, oat, corn (sweet and dent), rice, soybean, sorghum, alfalfa,
  • plant line is used broadly to include, but is not limited to, a group of plants vegetatively propagated from a single parent plant, via tissue culture techniques or a group of inbred plants which are genetically very similar due to descent from a common parent(s).
  • a plant is said to "belong” to a particular line if it (a) is a primary transformant (TO) plant regenerated from material of that line; (b) has a pedigree comprised of a TO plant of that line; or (c) is genetically very similar due to common ancestry ⁇ e.g., via inbreeding or selfing).
  • TO primary transformant
  • the term "pedigree” denotes the lineage of a plant, e.g. in terms of the sexual crosses effected such that a gene or a combination of genes, in heterozygous (hemizygous) or homozygous condition, imparts a desired trait to the plant.
  • plant tissue refers to any part of a plant.
  • 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.
  • variable refers to a subdivision of a species, consisting of a group of individuals within the species that are distinct in form or function from other similar arrays of individuals.
  • Micropropagation also known as tissue culturing with the terms used interchangeably herein, is an excellent method to achieve this aim.
  • Micropropagated plants are grown in vitro in sterile media.
  • the sterile media can be liquid, semi-solid, or solid, and the physical state of the media can be varied 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 BactoTM Agar.
  • Other exemplary gelling agents include carrageenan, gellan gum (commercially available as PhytaGelTM, GelriteTM and GelzanTM), 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.
  • the media comprises agar, with the addition of various compounds such as nutrients, inorganic salts, growth regulators, sugars, vitamins and other compounds.
  • Other media additives can include, but are not limited to, amino acids, macroelements, iron, microelements, inositol and undefined media components such as casein hydrolysates or yeast extracts.
  • the media can include any combination of NH 4 NO3; KNO3; Ca(NOs) 2 ; K 2 SO 4 ; MgSO 4 ; MnSO 4 ; ZnSO 4 ; CuSO 4 ; CaCI 2 ; Kl; CoCI 2 ; H 3 BO 3 ; Na 2 MoO 4 ; KH 2 PO 4 ; FeSO ; Na 2 EDTA; Na 2 H 2 PO ; myo-inositol; thiamine; pyridoxine; nicotinic acid; glycine; riboflavin; ascorbic acid; silicon standard solution; ⁇ - naphthoxyacetic acid (NAA); indole butyric acid (IBA); 3-indoleacetic acid (IAA); benzylaminopurine (BAP); 6-Y-Y-(dimethylallylamino)-purine (2-ip); sugar; agar; carrageenan and charcoal.
  • NAA ⁇ - naphthoxyacetic acid
  • IBA
  • plant growth regulators include auxins and compounds with auxin-like activity, cytokinins and compounds with cytokinin-like activity.
  • auxins include 2,4- dichlorophenoxyacetic acid, IBA, picloram and combinations thereof.
  • cytokinins in addition to meta-topolin and thidiazuron, include adenine hemisulfate, benzyladenine, dimethylallyladenine, kinetin, zeatin and combinations thereof.
  • Gibberellic acid also can be included in the media.
  • a sugar 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 sucrose, glucose, maltose, galactose and sorbitol or combinations thereof.
  • Certain media described herein include the cytokinins meta-topolin and/or thidiazuron. While certain embodiments utilize meta-topolin and/or thidiazuron defined as the particular compounds below, other related compounds can also be successful.
  • W is an aryl or heteroaryl
  • R 1 is substituted or unsubstituted alkyl wherein any C in the alkyl can be substituted with O, N or S;
  • each R 2 is independently H, OH, C1-C6 alkyl, C1-C6 alkylene, C1-C6 alkylyl, halogen, cyano, Ci-C 6 alkyloxy, aryl or heteroaryl each optionally substituted with a Ci-C 6 alkyl, SH, NHR 3 , CO 2 R 3 or halogen;
  • R 3 is H, OH, C1-C6 alkyl, C1-C6 alkylene, C1-C6 alkylyl, halogen, carboxylic group, ester group, aldehyde or cyano;
  • r 0 to 8.
  • W is
  • X1 -X 7 is each independently selected from C, N, O, S with the proviso that the
  • X linking the ring to N is C.
  • the compounds have a structure
  • the compounds have a structure
  • X 8 -X 12 is each independently selected from C, N, O, S;
  • each R 4 is independently H, OH, Ci-C 6 alkyl, Ci-C 6 alkylene, Ci-C 6 alkylyl, halogen, cyano, C1-C6 alkyloxy, aryl or heteroaryl each optionally substituted with a Ci-C 6 alkyl, SH, NHR 3 , CO 2 R 3 or halogen;
  • R 3 is H, OH, C1-C6 alkyl, C1-C6 alkylene, C1-C6 alkylyl, halogen, carboxylic group, ester group, aldehyde or cyano;
  • p 0 to 5;
  • q 0 to 6.
  • the compounds have a structure
  • the compounds have a structure
  • compounds can have structures selected from
  • R 4 is OH
  • compounds have a structure selected from
  • the compounds have a structure
  • the compounds have a structure
  • X 8 -X 12 is each independently selected from C, N, O, S;
  • each R 4 is independently H, OH, C1-C6 alkyl, C1-C6 alkylene, C1-C6 alkylyl, halogen, cyano, C1-C6 alkyloxy, aryl or heteroaryl each optionally substituted with a Ci-C 6 alkyl, SH, NHR 3 , CO 2 R 3 or halogen;
  • R 3 is H, OH, C1-C6 alkyl, C1-C6 alkylene, C1-C6 alkylyl, halogen, carboxylic group, ester group, aldehyde or cyano;
  • p 0 to 5;
  • q 0 to 6.
  • the compounds have a structure
  • the compounds have a structure
  • the compound is meta-topolin, also known as 6- (3-hydroxybenzylamino)-purine, and by the abbreviation mT, having a molecular formula of C12H10N 5 OH, a molecular weight of 241 .25, and the following structural formula:
  • meta-topolin is a derivative of a willow tree or a poplar tree.
  • V is an aryl or heteroaryl
  • each R 5 and R 6 is each independently H, OH, Ci-C6 alkyl, Ci-C6 alkylene, Ci- C6 alkylyl, halogen, cyano, C1-C6 alkyloxy, aryl or heteroaryl each optionally substituted with a Ci-C6 alkyl or halogen;
  • n 0 to 4.
  • o 0 to 5
  • X 13 -X 16 is each independently selected from C, N, O, S;
  • Z 1 and Z 2 are each independently NH, O, SH or CH or Z 1 and Z 2 can be combined to form a substituted or unsubstituted aryl or heteroaryl;
  • Y 1 is O or S.
  • X 17 -X 21 is each independently selected from C, N, O, S.
  • compounds include
  • the compound is Thidiazuron, also known as 1 - phenyl-3-(1 ,2,3-thiadiazol-5-yl)urea and 5-phenylcarbamoylamino-1 ,2,3- thiadiazole has the molecular formula of CgHsNUOS, a molecular weight of 220.25 and the following structural formula
  • each cytokinin can be present in an amount from 0.001 mg/L-100 mg/L and all amounts in between.
  • meta-topolin 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 , 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,
  • 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..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,
  • meta-topolin and/or its analogues and thidiazuron and/or its analogues can also be included in ratios.
  • the amount of meta-topolin and/or its analogues to thidiazuron and/or its analogues can be 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
  • NAA, BAP, 2ip and/or IBA can similarly can be present at 0.001 mg/L, 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,
  • a pharmaceutically acceptable salt is any salt of the parent compound that is suitable for use in the methods disclosed herein.
  • a pharmaceutically acceptable salt also refers to any salt which may form as a result of administration of an acid, another salt, or a prodrug which is converted into an acid or salt.
  • a salt comprises one or more ionic forms of the compound, such as a conjugate acid or base, associated with one or more corresponding counter-ions.
  • Salts can form from or incorporate one or more deprotonated acidic groups (e.g. carboxylic acids), one or more protonated basic groups (e.g. amines), or both (e.g. zwitterions).
  • tautomer refers to the migration of protons between adjacent single and double bonds. The tautomerization process is reversible. Other tautomers are possible when the compound includes, for example but not limited to, enol, keto, lactamin, amide, imidic acid, amine, and imine groups. Tautomers will generally reach an equilibrium state wherein the double bond is resonantly shared between the two bond lengths.
  • Alternate solid forms are different solid forms than those that may result from practicing the procedures described herein.
  • alternate solid forms may be polymorphs, different kinds of amorphous solid forms, glasses, and the like.
  • Non-covalent complexes are complexes that may form between the compound and one or more additional chemical species that do not involve a covalent bonding interaction between the compound and the additional chemical species. They may or may not have a specific ratio between the compound and the additional chemical species. Examples might include solvates, hydrates, charge transfer complexes, and the like.
  • plants are placed in various media that stimulate physiological processes such as growth and multiplication by and/or within the plant.
  • the process includes 3 steps (following explant preparation and disinfection, discussed below): (1 ) initiation of in vitro growth and/or multiplication of the explant in a media ; (2) further in vitro multiplication in a second media; and (3) transition to ex vitro conditions.
  • steps can be combined or skipped. For example, while there is commonly a change in media types between steps 1 and 2, in certain embodiments, a media change is not included.
  • media are defined as Stage 1 media (1 st media of a process); Stage 2 media (2 nd media of a process); Stage 3 media (3 rd media of a process); etc.
  • Particular media can change stage based on the number of steps within a particular process and where the particular media resides within their order.
  • 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).
  • the Stage 1 media is then placed into test tubes or other appropriate containers (including jars, boxes, jugs, cups, 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.
  • sterilization is achieved by autoclaving at 5-25 pounds pressure psi at a temperature of 200° F - for 200° F 10-25 minutes.
  • sterilization is achieved by autoclaving at 15 pounds pressure psi at a temperature of 250° F for 15-18 minutes.
  • Sterility can also be assessed by an accepted number of contaminated tubes per hundred tubes, for example and without limitation, 0 contaminated tubes per hundred tubes, no more than 1 contaminated tube per hundred tubes, no more than 2 contaminated tubes per hundred tubes, no more than 3 contaminated tubes per hundred tubes, no more than 4 contaminated tubes per hundred tubes, no more than 5 contaminated tubes per hundred tubes, no more than 6 contaminated tubes per hundred tubes, no more than 7 contaminated tubes per hundred tubes, no more than 8 contaminated tubes per hundred tubes, no more than 9 contaminated tubes per hundred tubes, no more than 10 contaminated tubes per hundred tubes, etc.
  • media containing a gelling agent such as agar, agarose, gellan gum, carrageenan or combinations thereof
  • the media solidifies upon cooling and serves to provide the micropropagated plant tissues with support, nutrients, growth regulators, water and other compounds as described below.
  • a gelling agent such as agar, agarose, gellan gum, carrageenan or combinations thereof
  • tubes and jars contain 15-25 ml_ media while boxes contain 40-50 ml_ media.
  • Cups can include 30-40 ml_ while jugs generally contain more than 50 ml_.
  • media containing a gelling agent such as agar, agarose, gellan gum, carrageenan or combinations thereof
  • the media solidifies upon cooling and serves to provide the micropropagated plant tissues with support, nutrients, growth regulators, water and other compounds as described below.
  • a gelling agent such as agar, agarose, gellan gum, carrageenan or combinations thereof
  • tubes and jars contain 20-25 ml_ media while boxes contain 40-50 ml_ media. Cups can inlcude 30-40 ml_ while jugs generall contain more than 50 ml_.
  • Micropropagated plants 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.
  • the explant can be any segment or collection of cells including but not limited to those from apical meristems, axillary buds, cambium, dormant lateral buds, immature flower buds, crowns, lateral meristems, shoot apices, stem segments, immature nodal sections from stems, internodes, lateral shoots, rhizomes, roots, seedlings, stolons (aka runners), or leaf segments.
  • the explant is taken from a 1 year old plant.
  • the explant is taken from a 2 year old plant. In another embodiment, the explant is taken from a plant that is 5 years old or less. In another embodiment, the explant is taken from a plant that is 4 years old or less. In another embodiment, the explant is taken from a plant that is 3 years old or less. In another embodiment, the explant is taken from a plant that is 2 years old or less. In another embodiment, the explant is taken from a plant that is 1 years old or less. In another embodiment, the explant is taken from a plant that is 6 months old or less. In another embodiment, the explant is taken from a plant that is 3 months old or less.
  • the plant from which the explant is obtained can be grown in any suitable husbandry situation, including but not limited to growing in a growth chamber, growing in a greenhouse or growing in a field.
  • explants can be used in accordance with the present disclosure.
  • immature nodal sections from stems can be used as the explant material .
  • 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.
  • explant material includes or is limited to the third node from the base of a cane. Additional sources of explants include those described in Examples 19-23.
  • Nodal section(s) or other explant types can be cut into 3-5, 1 -10, 2-9, 3-8, 4-6, 3-6 or 2-7 millimeter sections. Explants can be left intact and disinfected to remove pathogens on the exterior of the explant. Any disinfection method known in the art can be used.
  • Exemplary disinfection methods include application of a disinfectant, such as a disinfectant selected from among bleach (sodium and/or potassium and/or calcium hypochlorite), alcohol (e.g., ethanol, isopropyl), ozone, chlorine gas, iodine solution or antibiotic or anti-fungal solution or combinations thereof, or subjecting the exposed surface of the explant to ultraviolet light or to a temperature of -20° C or lower or to a temperature higher than 40° C or 50° C for a short period of time.
  • a disinfectant such as a disinfectant selected from among bleach (sodium and/or potassium and/or calcium hypochlorite), alcohol (e.g., ethanol, isopropyl), ozone, chlorine gas, iodine solution or antibiotic or anti-fungal solution or combinations thereof, or subjecting the exposed surface of the explant to ultraviolet light or to a temperature of -20° C or lower or to a temperature higher than 40° C or 50° C for a short period of time.
  • outer sheaths or other portions of the explant can be peeled off and discarded and the remaining piece put into an approximately 1 %, 5%, 10%, 15%, 20%, 25% or 30% solution of a commercial bleach or a similar disinfecting solution.
  • the peeled explant or other explant part in disinfecting solution can be put onto a shaker table, such as for example, a Lab Rotators, Adjustable speed, Barnstead/Lab line orbital Shaker (model number KS 260) for 10 minutes, 20 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes or 210 minutes at 6-9 or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12, 13, 14, or 15 revolutions per minute.
  • a shaker table such as for example, a Lab Rotators, Adjustable speed, Barnstead/Lab line orbital Shaker (model number KS 260) for 10 minutes, 20 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes or 210 minutes at 6-9 or 1 , 2, 3, 4, 5, 6,
  • the explant portions can then be put into an approximately 1 % solution of bleach or similar disinfecting solution, and placed back onto the shaker table for 30 minutes. In another embodiment, this 1 % bleach or similar disinfecting solution step can be repeated. In another embodiment, these described steps are progressive and include the entire disinfection process. As will be understood by one of ordinary skill in the art, a variety of appropriate disinfecting procedures can be used in accordance with the present disclosure.
  • the explants can be placed onto a Stage 1 media within the tube and the tubes can be placed in a regulated growth chamber.
  • growth chambers can include a number of configurations and sizes including table-top boxes, stand-alone chambers, closets, small rooms, large rooms, etc.
  • variables such as light or temperature can be appropriately controlled in such a growth chamber.
  • Appropriate ranges for tissue culturing bamboo include from 65°F-70°F, 60°F-75°F or 55°F-80°F at 200-500, 150-550 or 100-600 foot candles. Lighting can be full spectrum, although alternative lighting systems can also be utilized according to the present disclosure.
  • the explants are allowed to establish themselves within the tubes while in the growth chamber on Stage 1 media.
  • tissue culturing once established (i.e. growing without visible contamination), the cell cultures grown from the explants are transferred into a second, Stage 2 media.
  • Stage 2 media once established, the cell cultures can remain in Stage 1 media.
  • a large number of plants can be created within a relatively short period of time because each cell culture can develop multiple shoots and each shoot can be separated and placed into an individual tube where it will develop additional shoots to separate and multiply.
  • Non-limiting examples of media that commonly serve as Stage 1 and/or Stage 2 media include:
  • KH 2 PO 4 85-255 120-210 150-190 170 170 ⁇ 2
  • KH 2 PO 4 85-255 120-210 150-190 170 170 ⁇ 2
  • KH 2 PO 4 85-255 120-210 150-190 170 170 ⁇ 2
  • KH 2 PO 4 85-255 120-210 150-190 170 170 ⁇ 2
  • KH 2 PO 4 85-255 120-210 150-190 170 170 ⁇ 2
  • KH 2 PO 4 85-255 120-210 150-190 170 170 ⁇ 2
  • KH 2 PO 4 85-255 120-210 150-190 170 170 ⁇ 2
  • media can be transformed into a liquid media by removing agar or
  • the expected number of shoots or plantlets may be different at
  • multiplication can depend on the chosen container. For example,
  • multiplication can range from, without limitation, 1 -10 or 2-6 plants per tube, 1 - 15 or 4-9 plants per jar, 1 -20 or 9-17 plants per box or 1 -50 or 20-35 plants
  • the number 1 is included because certain species or particular cell
  • number of shoots or plantlets from a single explant 100, 500, 1 ,000; 5,000; 10,000, 20,000, 50,000, 100,000, 250,000, 500,000, 750,000 1 ,000,000 or more.
  • media that support transition to ex vitro conditions represent a Stage 2, Stage 3, Stage 4 or Stage 5 media.
  • Non-limiting examples of such media include:
  • plantlets and media can be placed in air permeable or air impermeable containers.
  • kits can include 2 of the different media provided herein, or include 3 of the different media provided herein, or include more than 3 of the different media described herein). While not explicitly describing each possible combination herein, one of ordinary skill in the art should understand that this disclosure supports all possible combinations.
  • the plants can undergo a series of treatments designed to acclimate them to an unregulated growing environment. This is because some plants, when microcultured, do not develop adequate defensive structures, such as waxy cuticles to protect them from ordinary environmental conditions.
  • the treatments that plants can undergo prior to being placed in an unregulated environment can include, without limitation, acclimatization to humidity, acclimatization to variations in temperature, and acclimatization to wind pressure. These acclimatization factors can be introduced gradually and/or in a staggered fashion.
  • Representative genus of bamboo appropriate for use with the disclosures herein include: Acidosasa; Ampelocalamus; Arundinaria; Bambusa; Bashania; Borinda; Brachystachyum; Cephalostachyum; Chimonobambusa; Chimonocalamus; Chusquea; Dendrocalamus; Dinochloa; Drepanostachyum; Eremitis; Fargesia; Gaoligongshania; Gigantochloa; Guadua; Hibanobambusa; Himalayacalamus; Indocalamus; Indosasa; Lithachne; Melocalamus; Melocanna; Menstruocalamus; Nastus; Neohouzeaua; Neololeba; Ochlandra; Oligostachyum; Olmeca; Oaji; Oxytenanthera; Phyllostachys; Pleioblastus; Pseudosasa; Raddia;
  • Non-limiting examples of species within these genus include:
  • Arundinaria Arundinaria appalachiana; Arundinaria funghomii; Arundinaria gigantea; Arundinaria gigantea 'Macon'; and Arundinaria Tecta
  • Bambusa arnhemica; balcooa; bambos; basihirsuta; beecheyana; beecheyana var pubescens; blumeana; boniopsis; burmanica; chungii; chungii var.
  • Albostriata textilis var. Glabra; textilis var. Gracilis; tulda; tulda 'Striata';
  • Tuldoides variostriata; ventricosa; ventricosa 'Kimmei'; vulgaris; vulgaris
  • Bashania Fargesii; and Qingchengshanensis
  • Borinda KR 5288; Albocerea; Angustissima; Contracta; Frigidorum;
  • Brachystachyum densiflorum; and densiflorum var. villosum
  • Cephalostachyum Pergracile; and Virgatum
  • Dendrocalamus Asper; asper 'Betung Hitam'; Brandisii; brandisii 'Black'; brandisii (variegated); Calostachyus; Giganteus; giganteus (Quail Clone); giganteus (variegated); Hamiltonii; Jianshuiensis; jianshuiensis (variegated); Latiflorus; latiflorus 'Mei-nung'; Membranaceus; Minor; minor 'Amoenus'; Sikkimensis; Sinicus; sp. 'Maroochy'; sp. 'Parker's Giant'; Strictus; Validus; and Yunnanicus
  • Drepanostachyum falcatum var. sengteeanum; and Khasianum
  • Gaoligongshania Gaoligongshania and Megalothyrsa
  • Guadua Amplexifolia; Angustifolia; angustifolia 'Bicolor'; angustifolia 'Less Thorny'; Chacoensis; Longifolia; Paniculata; sp. 'Aureocaulis'; and Velutina
  • Hibanobambusa Tranquillans; and tranquillans 'Shiroshima'
  • Himalayacalamus Falconeri; falconeri 'Damarapa'; Hookerianus; Planatus; and Porcatus
  • Indocalamus Cordatus; Latifolius; latifolius 'Hopei'; Longiauritus; sp. 'Hamadae'; sp. 'Solidus'; Tessellatus; and Victorialis
  • Melocalamus Arrectus
  • Neohouzeaua Mekongensis
  • Neololeba Atra
  • Oxytenanthera Abyssinica; and Braunii
  • Pleioblastus Akebono; Amarus; Argenteostriatus; Chino; chino 'Angustifolia'; chino 'Elegantissimus'; chino 'Kimmei'; chino 'Murakamiansus'; chino 'Vaginatus Variegatus'; Distichus; distichus 'Mini'; Fortunei; Gauntlettii; Gramineus; gramineus 'Monstrispiralis'; Hindsii; Humilis; humilis 'Albovariegatus'; humilis 'Variegatus'; Juxianensis; Kodzumae; Kongosanensis; kongosanensis 'Akibensis'; kongosanensis 'Aureostriatus'; Linearis; linearis 'Nana'; Nagashima; Oleosus; Py
  • Pseudosasa Amabilis; Cantori; Guanxianensis; Japonica; japonica 'Akebono'; japonica 'Akebono-suji'; japonica 'Pleioblastoides'; japonica sutsumiana'; japonica 'Variegata'; Longiligula; Owatarii; Usawai; and Viridula
  • Radd ia Brasiliensis; and Distichophylla
  • Sasa Cernua; Gracillima; Hayatae; Kagamiana; kagamiana ssp. Yoshinoi; Kurilensis; kurilensis 'Shimofuri'; Megalophylla; Nagimontana; nipponica (hort.); Oshidensis; Palmata; Senanensis; Shimidzuana; sp. Tsuboiana; and Veitchii
  • Sasaella Bitchuensis; hidaensis 'muraii'; Masamuneana; masamuneana 'Albostriata'; masamuneana 'Aureostriata'; Ramosa; Sasakiana; and Shiobarensis
  • Shibataea Chinensis; Kumasaca; kumasaca 'Albostriata'; kumasaca 'Aureostriata'; Lancifolia; and Nanpingensis
  • Thamnocalamus aristatus 'Aristatus hort. US'; Crassinodus; crassinodus 'Kew Beauty'; crassinodus 'Mendocino'; crassinodus 'Merlyn'; nepalensis 'Nyalam'; Spathiflorus; and Tessellatus
  • Yushania Alpina; Anceps; anceps 'Pitt White'; Boliana; Brevipaniculata; Exilis; Maculata; and maling
  • Particularly useful species include: edulis; scandens; ArundinariaGigantea; ArundinariaTecta; BambusaBalcooa; BambusaBambos; Bambusa Oldhamii; BambusaTextilis; BambusaTulda; BashaniaFargesii; BrachystachyumDensiflorum; Chusquea Gigantea; DendrocalamusAsper; DendrocalamusBrandisii; DendrocalamusGiganteus; DendrocalamusHamiltonii; DendrocalamusStrictus; FargesiaDenudata; Fargesiadracocephala'Ruia'; FargesiaMurieliae; FargesiaNitida;
  • FargesiaRobusta FargesiaRobusta
  • Fargesiarobusta 'Wolong'
  • Fargesiasp. 'Scabrida' GuaduaAmplexifolia
  • Guadua Paniculata Himalayacalamus Falconeri
  • Indocalamus Tessellatus Ochlandra Stridula
  • Additional representative monocots for which the media, systems and methods described and disclosed herein are applicable include but are not limited to monocotyledonous plants of the following genera: Aegilops (goatgrass), Agave, Agropyron (crested wheatgrass), Agrostis (bentgrass), Allium (onion), Alopecurus (meadow foxtail), Amaranthus (amaranth), Ammophila (beach grass), Ananas (pineapple), Andropogon (beardgrass), Arrhenatherum (oat grass), Avena (oat), Axonopus (carpet grass), Beckmannia (slough grass), Bouteloua (grama grass), Bromus (brome grass), Calamagrostis (reed grass), Calamus (palm), Cortaderia (pampas grass), Dactylis (orchard grass), Elymus (wheat grass), Festuca (fescue), Geranium, Gladiolus, Hakon
  • rolling tissue culture means that the multiplication process can continue substantially indefinitely by continuing to separate and multiply plantlets.
  • one shoot or plantlet is placed in a tube and the shoot or plantlet multiplies into a number of additional shoots or plantlet. After multiplication, each shoot or plantlet or a subset of the shoots or plantlets are separated and each placed in a subsequent tube for further multiplication.
  • This process can continue while at various times, some or all shoots or plantlet can be removed from the multiplication process and transitioned to ex vitro conditions.
  • indefinitely it is meant that 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, etc. day multiplication cycles can be repeated without initiating new explants for at least 1 month, for at least 3 months, for at least 6 months, for at least 9 months, for at least 12 months, for at least 15 months, for at least 18 months, for at least 21 months, for at least 24 months or for at least 36 months.
  • day multiplication cycles can be repeated without initiating new explants for at least 1 month, for at least 3 months, for at least 6 months, for at least 9 months, for at least 12 months, for at least 15 months, for at least 18 months, for at least 21 months, for at least 24 months or for at least 36 months.
  • Particular ranges of days in multiplication cycles include 10-120 days; 10-100 days; 10-80 days; 10-60 days; 10-120 days; 10-40 days; 10-20 days; 14-120 days; 14-90 days; 14-70 days; 14-50; 14-42 days; 14-30 days; 14-21 days; 12-42 days; 20-60 days; 10-15 days; 14-20 days; 14-18 days etc.
  • Kits can include, without limitation, one or more of the following in a package or container: (1 ) one or more media; and (2) one or more explants from one or more species of plants.
  • the media can be b-9-i media, b-9-ii media, b-9-iii media, b-9-iv media, b-9-v media, CW2-i media, CW2-ii media, CW2-iii media, CW2-iv media, CW2-v media, b-10-i media, b-10-ii media, b-10-iii media, b-10-iv media, b-10-v media, b-1 1 -i media, b-1 1 -ii media, b-1 1 -iv media, b-1 1 -v media, b-12c-i media, b-12c-ii media, b-12c-iii media, b-12c-iv media, b-12c-v media, b-1 -i media, b-1 -ii media, b-1 -ii media, b-1 -iv media, b-12c-v media, b-1 -i media, b-1 -
  • kits can comprise one or more containers for the tissue culturing process including without limitation, tubes, jars, boxes or jugs.
  • the kits can comprise instructions for the tissue culturing of bamboo.
  • 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.
  • kits can be supplied in containers of any sort such that the life of the different ingredients are preserved and are not adsorbed or altered by the materials of the container.
  • sealed glass ampules may contain ingredients that have been packaged under a neutral, non-reacting gas, such as nitrogen.
  • Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, etc., ceramic, metal or any other material typically employed to hold similar ingredients.
  • suitable containers include simple bottles that may be fabricated from similar substances as ampules, and envelopes, that may comprise foil-lined interiors, such as aluminum or an alloy.
  • Containers include test tubes, vials, flasks, bottles, syringes, or the like.
  • Containers may have a sterile access port, such as a bottle having a stopper that can be pierced.
  • Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the ingredients to be mixed.
  • Removable membranes may be glass, plastic, rubber, etc.
  • kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable media, such as a floppy disc, CD-ROM, DVD-ROM, Zip disc, videotape, audiotape, etc. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an internet web site specified by the manufacturer or distributor of the kit, or supplied as electronic mail.
  • One advantage of the disclosed embodiments is that the methods are more robust than those previously used producing plants that do not require special treatments required by those produced using methods of the prior art. For example, methods disclosed herein do not require the use of seeds or inflorescence to start plants; do not require selection of diseased starting plants (such as those exhibiting symptoms of witches broom or little leaf disease); do not require use of somatic embryogenesis and do not utilize pseudospiklets. 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. These improvements over prior methods provide even additional advantages related to the health of produced plants and efficiency of growth and processing.
  • One advantage of the disclosed embodiments is that the methods are more robust than those previously used producing plants that do not require special treatments required by those produced using methods of the prior art. For example, methods disclosed herein do not require the use of seeds or inflorescence to start plants; do not require selection of diseased starting plants (such as those exhibiting symptoms of witches broom or little leaf disease); do not require use of somatic embryogenesis and do not utilize pseudospiklets. 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. These improvements over prior methods provide even additional advantages related to the health of produced plants and efficiency of growth and processing.
  • Non-limiting embodiments encompassed by the present disclosure include:
  • a node from the cane with the lateral shoot just breaking the sheath can be used as the explant.
  • Each nodal section can be cut into 3-5 millimeter sections with the shoot intact.
  • 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.
  • 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.
  • Stage 1 media 15-25 ml_
  • 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 200-500 foot candles.
  • the initial Stage 1 media can be b-12c-iv at a pH of 5.7.
  • the explants can then be transferred to fresh b-12c-iv media every 10-120 days (usually every 21 days), with contaminated tubes being discarded. Contaminated tubes can be identified by bacterial discoloration of the agar or by visible surface contamination.
  • These explants can stay on b-12c-iv media for 3-4 10-120 day cycles (usually 21 day cycles).
  • Explants can then 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.
  • Live shoots can next be transferred to a Stage 2 media, such as b- 9, CW1 , CW2, CW3, CW4, CW5, CW6 or b-6 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.
  • 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 in Stage 2 media or transitioned to a Stage 3 media, for example, b-10-iv or b-1 1 -iv at a pH of 5.7 for further multiplication.
  • One-ten shoots per tube can be obtained per multiplication cycle.
  • the shoots can transferred to small tissue culturing boxes (known as "magenta boxes") for 10-120 days (usually 14-21 days) containing a Stage 3 or Stage 4 media, in this Example, BR-2 at a pH of 5.7 for 10-120 days (usually 14-21 days) or Amel at a pH of 5.7 for 10-120 days (usually 14-21 days).
  • magenta boxes small tissue culturing boxes
  • a node from the cane with the lateral shoot just breaking the sheath can be used as the explant.
  • Each nodal section can be cut into 3-5 millimeter sections with the shoot intact.
  • Some explants, including explants taken from canes 1 year or older can be pre-rinsed by shaking them in a jar of 70% isopropyl alcohol for 3 seconds followed by rinsing them under running tap water for 1 minute. Other explants are not pre-rinsed.
  • the outer sheaths can be peeled off and discarded and the remaining nodal section piece put into a 10% bleach solution.
  • 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. For some implants, including those taken from canes 1 year or older, this step can be modified by adding a few drops of Tween 20 to the 10% bleach solution and soaking the explants for 45 minutes rather than 1 hour. The explants can then be put into a 1 % bleach solution, and placed back onto the shaker table for 30 minutes. This 1 % bleach solution step can then be repeated.
  • Stage 1 media 15-25 ml_
  • the Stage 1 media can be b-12c-iv at a pH of 5.7.
  • the explants can be transferred to fresh b-12c-iv media every 10-120 days (usually every 21 days), with contaminated tubes being discarded.
  • These explants can stay on b-12c-iv media for 2 10-120 day cycles (usually 21 day cycles). Between cycles, excess sheaths can be removed.
  • explants can be transitioned to a Stage 2 media, in this Example, b-12- c supplemented with 7 g/L carageenan rather than the 5.5 g/L provided above.
  • explants can be cleaned.
  • the explants can be kept on b-12-c 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.
  • Non- limiting Stage 3 media include, a b-9 media, a CW1 media, a CW2 media a CW3 media, a CW4 media, a CW5 media, a CW6 media or a b-6 media at a pH of 5.7.
  • the cultures can stay on Stage 2 or Stage 3 media until the desired number of shoots is obtained by separation into new tubes and further expansion.
  • the range of time includes10-120 day cycles (usually 21 day cycles) between which the cultures can be assigned to go through another multiplication round or transitioned to a Stage 3 or Stage 4 media, such as a BR-2 media at a pH of 5.7 for 10-120 days (usually 21 days) in "magenta boxes" or a Amel media at a pH of 5.7 for 10-120 days (usually 14- 21 days).
  • a Stage 3 or Stage 4 media such as a BR-2 media at a pH of 5.7 for 10-120 days (usually 21 days) in "magenta boxes" or a Amel media at a pH of 5.7 for 10-120 days (usually 14- 21 days).
  • the following species can be multiplied in the following media according to procedures described in the proceeding paragraphs [000137]-[000145] at a pH of 5.5-5.7:
  • Arundinaria gigantea b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
  • Bambusa balcoa b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
  • Bambusa vulgaris b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
  • Bambusa vulgaris 'Vitatta' b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
  • Bambusa Oldhamii b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6- v;
  • Bambusa tulda b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
  • Dendrocalamus giganteus b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
  • Gigantochloa aspera b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
  • Gigantochloa scortechini b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
  • Guadua culeata b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
  • Guadua aculeata 'Nicaragua' b-9-v, CW1 -v, CW3-v, CW4-v, CW5- v or CW6-v;
  • Guadua amplexifolia b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
  • Guadua angustifolia b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
  • Guadua angustofolia bi-color b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
  • Guadua paniculata b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6- v;
  • Melocanna bambusoides b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
  • Neohouzeaua dullooa (Teinostachyum): b-9-v, CW1 -v, CW3-v, CW4-V, CW5-V or CW6-v;
  • Ochlandra travancorica b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
  • Phyllostachys edulis 'Moso' b-9-v, CW1 -v, CW3-V, CW4-V, CW5-V or CW6-v;
  • Schizostachyum lumampao b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-V.
  • Exemplary Paper Types Freesheet; Stock; Acid-free; A4; Board; Bond; Book; Bristol; Carbonless; Catalog; Coated; Cover; Dual-Purpose Bond; Duplex; English Finish; Equivalent; Fine; Free Sheet; Grain Long; Grain Short; Groundwood; Kraft; Lightweight; News Print; Publishing; Rag; Recycled; Tag; Uncoated; Virgin; Absorbent; Acid; Album; Albumin; Akaline; Bank Note; Tissue; Toilet; Towels; Fluff; Card Stock; OTC; OCR; Tissue Overlay; Napkins
  • Exemplary Pulp Types Air-dried; Alpha; bamboo; Bisulfate; Sulphite; Bleached; Chemical Cellulose (Dissolving ); Fluff; Fodder; Free; Fully Bleached; Hard; High Alpha Cellulose; Groundwood; Hot Groundwood; Jute; Knotter; Kraft; Long Fiber; Baled; Rolled; Market; Non-Wood; Board; Pressurized Groundwood;; Rag; Recycled; Reinforcement; Secondary; Semi- alkaline; Semi-bleached; Semi-chemical; Short Fiber; Soda; Specialty; Sulfate; Themochemical; Unbleached; Viscose; Wood
  • Board/Containers Linerboard; Containerboard; Cardboard; OCC; Paperboard
  • Wood Structural Wood Panels (including Structural Plywood; Oriented Strand Board; Structural Composite Panels); Glued Laminated Timber; Structural Composite Lumber (including Laminated Veneer Lumber; Parallel Strand Lumber; Oriented Strand Lumber); Prefabricated Wood I- Joists; Floor Joints; Railroad Ties; Flooring; Composites (including Auto; Aero; Music).
  • Textiles Feedstock; Filament Yarn; Knitted Fabric; Knitting; Narrow Width Fabric; Non Woven Fabric; Spun Yarn; Woven Fabric; Viscose Rayon; Batting; Ginned Fiber; Cloth
  • time on rooting media can be in permeable or impermeable containers.
  • time on rooting media can be in permeable or impermeable containers.
  • Live shoots were next transferred to a Stage 2 media, in this Example, b-9-iv at a pH of 5.7.
  • b-9-iv a pH of 5.7
  • one of the CW1 media at a pH of 5.7 can also be used.
  • the cultures stayed on b-9-iv media until the desired number of shoots was obtained by separation into new tubes and further expansion.
  • the range of time included 10-120 day cycles (usually 14-21 day cycles) between which the cultures were assigned to go through another multiplication round in Stage 2 media or transitioned to a Stage 3 media, in this Example, b-10-iv at a pH of 5.7 for further multiplication.
  • One-ten shoots per tube were obtained per multiplication cycle.
  • Example 1 In the example of Pleioblastus viridistriatus and Thamnocalamus crassinodus, the explants were chosen and disinfected as in Example 1 . The explants were then transferred into tubes containing a Stage 1 media, in this Example, b-12c-iv also as described in Example 1 . Plants were then transferred into a Stage 2 media, in this Example, b-4-iv at a pH of 5.5 for 10- 120 day cycles (usually 21 day cycles) until the desired number of shoots was obtained by separation into new tubes and further expansion. One-ten shoots per tube were obtained per multiplication cycle. The shoots were then placed in a Stage 3 media, in this Example, Br-2-iv at a pH of 5.7 for 10-120 days (usually 14-21 days).
  • Example 1 In the example of Chusquea Culeo "Cana Prieta", the explants were chosen and disinfected as in Example 1 . The explants were then transferred into tubes containing a Stage 1 media, in this Example, b-12c-iv media also as described in Example 1 . Plants were then transferred into a Stage 2 media, in this Example, b-9-iv at a pH of 5.5 for 10-120 day cycles (usually 21 day cycles) until the desired number of shoots was obtained by separation into new tubes and further expansion. B-6 media at a pH of 5.5 can also be used. One-ten shoots per tube were obtained per multiplication cycle. The shoots were then placed in a Stage 3 media, in this Example, Amel-iv at a pH of 5.7 for 10-120 days (usually 14-21 days).
  • Example 1 Bambusa Old amii
  • the explants were chosen and disinfected as in Example 1 .
  • the explants were then transferred into boxes containing a Stage 1 media, in this Example, b-10-iv (40-50 ml_) also as described in Example 1 but for the change to boxes.
  • Shoots were maintained on b-10-iv media for 10-120 day cycles (usually 21 day cycles) until the desired number of shoots was obtained by separation into new boxes and further expansion.
  • One-twenty shoots per box were obtained per multiplication cycle.
  • the shoots were then placed in a Stage 2 media, in this Example, Amel-iv at a pH of 5.7 for 10-120 days (usually 14-21 days).
  • Example 1 In the example of Phyllostachys Moso, Phyllostachys Atrovaginata & Dendrocalamus Asper, the explants were chosen and disinfected as in Example 1 . The explants were then transferred into tubes containing a Stage 1 media, in this Example, b-12c-iv also as described in Example 1 . Plants were then transferred into a Stage 2 media, in this Example, b-9-iv at a pH of 5.5 for 10-120 day cycles (usually 21 day cycles) until the desired number of shoots was obtained by separation into new tubes and further expansion. A B- 6 media at a pH of 5.5 can also be used. One-ten shoots per tube were obtained per multiplication cycle. The shoots were then placed in a Stage 3 media, in this Example, Amel-iv at a pH of 5.7 for 10-120 days (usually 14-21 days).
  • Example 1 the explants were chosen and disinfected as in Example 1 .
  • the explants were then transferred into tubes containing a Stage 1 media, in this Example, b-12c-iv also as described in Example 1 .
  • Shoots were then transferred into a Stage 2 media, in this Example, b-10-iv at a pH of 5.5 for 10-120 day cycles (usually 21 day cycles) until the desired number of shoots was obtained by separation into new tubes and further expansion.
  • One-ten shoots per tube were obtained per multiplication cycle.
  • the shoots were then placed in a Stage 3 media, in this Example, Amel-iv at a pH of 5.7 for 10-120 days (usually 14-21 days).
  • Stage 1 media 15-25 ml_
  • the tubes were placed into a regulated clean growth chamber at a temperature of from 65°F-70°F and a full spectrum light level of 200-500 foot candles.
  • the Stage 1 media was b-12c-iv at a pH of 5.7.
  • the explants were transferred to fresh b-12c-iv media every 10-120 days (usually every 21 days), with contaminated tubes being discarded. These explants stayed on b-12c-iv media for 2 10-120 day cycles (usually 21 day cycles). Between cycles, excess sheaths were removed.
  • explants were transitioned to a Stage 2 media, in this Example, b-12-c supplemented with 7 g/L carageenan rather than the 5.5 g/L provided above.
  • explants were cleaned. The explants were kept on b-12-c supplemented with 7 g/L carageenan for 10-120 day cycles (usually 21 day cycles) until multiple shoots were observed. Observation of multiple shoots occurred within 3-15 months.
  • the explant exhibited multiple shoots, it was either maintained on its Stage 2 media or transferred to a Stage 3 media, in this Example, when used b-9-iv at a pH of 5.7.
  • a CW1 media at a pH of 5.7 can also be used.
  • the cultures stayed on Stage 2 or Stage 3 media until the desired number of shoots was obtained by separation into new tubes and further expansion.
  • the range of time included 10-120 day cycles (usually 21 day cycles) between which the cultures were assigned to go through another multiplication round or were transitioned to a Stage 3 or Stage 4 media, in this Example, BR-2-iv at a pH of 5.7 for 10-120 days (usually 21 days) in "magenta boxes".
  • Example 13 Sasa Veitchii - alternate procedure [000213] In the example of Sasa Veitchii, the explants were chosen and disinfected as in Example 10.
  • the explants were kept on b-12-c supplemented with 7 g/L carageenan for 10-120 day cycles (usually 21 day cycles) until multiple shoots were observed. Observation of multiple shoots occurred within 3-15 months. [000217] Once the explant exhibited multiple shoots, it was either maintained on its Stage 2 media or transferred to a Stage 3 media, in this Example b-4-iv at a pH of 5.5 for 10-120 day cycles (usually 21 day cycles) until the desired number of shoots was obtained by separation into new tubes and further expansion. One-ten shoots per tube were obtained per multiplication cycle. The shoots were then placed in a Stage 3 or Stage 4 media, in this Example, Br-2-iv at a pH of 5.7 for 10-120 days (usually 14-21 days).
  • the explants were kept on b-10-c supplemented with 7 g/L carageenan for 10-120 day cycles (usually 21 day cycles) until multiple shoots were observed. Observation of multiple shoots occurred within 3-15 months. Cultures were maintained on Stage 2 media until the desired number of shoots was obtained. One-twenty shoots per box were obtained per multiplication cycle. The shoots were then placed in a Stage 3 media, in this Example, Amel-iv at a pH of 5.7 for 10-120 days (usually 14-21 days).
  • explants were cleaned.
  • the explants were kept on b-12-c supplemented with 7 g/L carageenan for 10-120 day cycles (usually 21 day cycles) until multiple shoots were observed. Observation of multiple shoots occurred within 3-15 months.
  • the explant exhibited multiple shoots, it was either maintained on its Stage 2 media or transferred to a Stage 3 media, in this Example b-9-iv at a pH of 5.5 for 10-120 day cycles (usually 21 day cycles) until the desired number of shoots was obtained by separation into new tubes and further expansion.
  • a B-6 media at a pH of 5.5 can also be used.
  • One-ten shoots per tube were obtained per multiplication cycle.
  • the shoots were then placed in a Stage 3 or Stage 4 media, in this Example, Amel-iv at a pH of 5.7 for 10-120 days (usually 14-21 days).
  • Example 18 Guadua Angustifolia - alternate procedure [000223]
  • the explants were chosen and disinfected as in Example 10.
  • the explants were then transferred into tubes containing a Stage 1 media, in this Example, b-12c-iv also as described in Example 10. These explants stayed on b-12c-iv media for 2 10-120 day cycles (usually 21 day cycles). Between cycles, excess sheaths were removed.
  • explants were transitioned to a Stage 2 media, in this Example, b-12-c supplemented with 7 g/L carageenan rather than the 5.5 g/L provided above.
  • explants were cleaned.
  • the explants were kept on b-12-c supplemented with 7 g/L carageenan for 10-120 day cycles (usually 21 day cycles) until multiple shoots were observed. Observation of multiple shoots occurred within 3-15 months.
  • Explants were from new shoots starting to rise from the ground in the spring. The new shoots were 1 -2 cm long when harvested. They were cleaned and cut down to 5-10 mm. They were then placed in a 10% bleach solution for 35 minutes. Following this first disinfection, the explants were further cut to 3-5 mm and then placed in a 10% bleach solution for another 35 minutes. Explants were then rinsed in a 1 % bleach solution for 30 minutes 2 times.
  • Plantlets were cycled every 10-60 days (usually every 42 days) into fresh media. Once the desired number of plantlets has been formed through separation and expansion in new tubes the plantlets were placed on a Stage 2 media, in this Example, FS2 at a pH of 5.3-6.0 for 10-120 days (usually 28 days) under normal culture room conditions including 65°F - 70°F at full spectrum 200 - 500 foot candles.
  • Example 20 Food Crop - Cabbage
  • the explants were from the dormant lateral buds. The buds were removed from the stems by cutting a wedge out of the stem with the bud attached. The explant was placed in a 10% bleach solution for 30 minutes. The wedge was removed from the bleach and the bud was cleaned of any stem pieces. The outer layer of sheath was removed resulting in an explant of 2-4 mm. This explant was then placed into a 10% bleach solution for another 30 minutes. Explants were then rinsed in a 1 % bleach solution for 30 minutes 2 times.
  • Plantlets where then transferred to a Stage 2 media, in this Example, FS3 for 10-120 days (usually 21 days).
  • Explants vary according to the variety of geranium. One explant type was immature flower bud taken as it appeared on the plant. These were cleaned of the outer sheath with a little of the stem attached. The explants were placed in a 10% bleach solution for 30 minutes. They were then rinsed in a 1 % bleach solution for 30 minutes 2 times. Following these rinses the explants were cut down further.
  • a second explant type was the crown of the geranium obtained by cutting the crown out and cleaning it down to 1 -2 cm. This explant was placed in a 10% bleach solution for 30 minutes. It was then removed, cleaned further and cut down to 5-10 mm. The explants were then placed back in a 10% bleach solution for another 30 minutes and then rinsed in a 1 % bleach solution for 30 minutes 2 times.
  • Example 22 Perennial - Trillium [000232] Explants were from the tips of the rhizomes in late winter just before emergence from the ground. The growing part of the rhizome was cleaned and cut down to 1 cm. This material was placed in a 10% bleach solution for 30 minutes. The tip was then removed and cleaned and cut down further to 5- 6 mm. It was then placed in a 10% bleach solution for another 30 minutes, after which it was rinsed in a 1 % bleach solution for 30 minutes 2 times.
  • tissue culturing method for individual species includes slight variations in media, timing and growth conditions. These variations for individual species require optimization based on factors including location, desired outcome, starting material, etc.
  • each can be initiated and/or multiplied in b-9-i media, b-9-ii media, b-9-iii media, b-9-iv media, b-9-v media, CW2-i media, CW2-N media, CW2-iii media, CW2-iv media, CW2-v media, b-10-i media, b-10-ii media, b-10-iii media, b-10-iv media, b-10-v media, b-1 1 -i media, b-1 1 -ii media, b-1 1 -iii media, b-1 1 -iv media, b-1 1 -v media, b-12c-i media, b-12c-ii media, b-12c-iii media, b-12c-iv media, b-12c-v media, b-1 -i media, b-1 -i media, b-1 -ii media, b-1 -i media, b-12c-i
  • tissue culturing method for individual species includes slight variations in media, timing and growth conditions. These variations for individual species require optimization based on factors including location, desired outcome, starting material, etc. As long as meta-topolin is included in the media used, however, successful commercial scale production following optimization will be achieved.

Abstract

Disclosed herein are media, systems and methods for achieving micropropagation of monocotyledonous plants.

Description

MEDIA, KITS, SYSTEMS AND METHODS FOR THE MICROPROPAGATION OF MONOCOTYLEDONOUS PLANTS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001]This application claims priority to and the benefit of U.S. Patent Application No. 13/303,433, filed November 23, 201 1 , which is a continuation- in-part of U.S. Patent Application No. 13/258,653, filed September 22, 201 1 , which is a National Stage Entry of International Patent Application No. PCT/US201 1/024936, filed February 15, 201 1 . This application also claims priority to and the benefit of U.S. Provisional Patent Application No. 61/442,744, filed February 14, 201 1 . The entire disclosures of U.S. Patent Application Nos. 13/303,433 and 13/258,653, International Patent Application No. PCT/US201 1/024936, and U.S. Provisional Patent Application No. 61/442,744 are incorporated by reference herein in their entireties.
FIELD OF THE DISCLOSURE
[0002] Disclosed herein are media, kits, systems and methods for achieving micropropagation of plants, particularly monocotyledonous plants ("monocots").
BACKGROUND OF THE DISCLOSURE
[0003] Micropropagation (also known as tissue culturing with the terms used interchangeably herein) is an excellent method to achieve large scale production of many plants. 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 media comprises agar, with the addition of various compounds such as nutrients, inorganic salts, growth regulators, sugars, vitamins and other compounds.
[0004] A benefit to tissue culturing plants is that the plants can be grown in a sterile environment so that they 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 any 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.
[0005] A number of plants have never been successfully micropropagated on a large scale. For example, while some papers have been published which address tissue culturing of monocot, in practice, the described methods have not translated into commercially viable propagation systems.
SUMMARY OF THE DISCLOSURE
[0006] The present disclosure provides effective media, kits, systems and methods for tissue culturing plants on a commercial scale.
[0007] One embodiment disclosed herein is a media for micropropagating plants wherein said media comprises meta-topolin and/or thidiazuron. In another embodiment, the media comprises meta-topolin.
[0008] In another embodiment, the media is b-9-i media, b-9-ii media, b-9-iii media, b-9-iv media, b-9-v media, CW2-i media, CW2-N media, CW2-iii media, CW2-iv media, CW2-v media, b-10-i media, b-10-ii media, b-10-iii media, b- 10-iv media, b-10-v media, b-1 1 -i media, b-1 1 -ii media, b-1 1 -iii media, b-1 1 -iv media, b-1 1 -v media, b-12c-i media, b-12c-ii media, b-12c-iii media, b-12c-iv media, b-12c-v media, b-1 -i media, b-1 -ii media, b-1 -iii media, b-1 -iv media, b- 1 -v media, b-4-i media, b-4-ii media, b-4-iii media, b-4-iv media, b-4-v media, b-6-i media, b-6-ii media, b-6-iii media, b-6-iv media, b-6-v media, CW1 -i media, CW1 -ii media, CW1 -iii media, CW1 -iv media, CW1 -v media, CW3-i media, CW3-ii media, CW3-iii media, CW3-iv media, CW3-v media, CW4-i media, CW4-ii media, CW4-iii media, CW4-iv media, CW4-v media, CW5-i media, CW5-ii media, CW5-iii media, CW5-iv media, CW5-v media, CW6-i media, CW6-N media, CW6-iii media, CW6-iv media, CW6-v media, R1 -i media, R1 -N media, R1 -iii media, R1 -iv media, R1 -v media, R2-i media, R2-N media, R2-iii media, R2-iv media, R2-v media, R3-i media, R3-N media, R3-iii media, R3-iv media, R3-v media, R4-i media, R4-N media, R4-iii media, R4-iv media or R4-v media.
[0009] One embodiment disclosed herein is a kit for micropropagating plants wherein said kit includes a media comprising meta-topolin and/or thidiazuron. In another embodiment, the media comprises meta-topolin. In another embodiment, the media is b-9-i media, b-9-ii media, b-9-iii media, b-9-iv media, b-9-v media, CW2-i media, CW2-N media, CW2-iii media, CW2-iv media, CW2-v media, b-10-i media, b-10-ii media, b-10-iii media, b-10-iv media, b-10-v media, b-1 1 -i media, b-1 1 -ii media, b-1 1 -iii media, b-1 1 -iv media, b-1 1 -v media, b-12c-i media, b-12c-ii media, b-12c-iii media, b-12c-iv media, b-12c-v media, b-1 -i media, b-1 -ii media, b-1 -iii media, b-1 -iv media, b- 1 -v media, b-4-i media, b-4-ii media, b-4-iii media, b-4-iv media, b-4-v media, b-6-i media, b-6-ii media, b-6-iii media, b-6-iv media, b-6-v media, CW1 -i media, CW1 -ii media, CW1 -iii media, CW1 -iv media, CW1 -v media, CW3-i media, CW3-ii media, CW3-iii media, CW3-iv media, CW3-v media, CW4-i media, CW4-ii media, CW4-iii media, CW4-iv media, CW4-v media, CW5-i media, CW5-ii media, CW5-iii media, CW5-iv media, CW5-v media, CW6-i media, CW6-ii media, CW6-iii media, CW6-iv media, CW6-v media, R1 -i media, R1 -ii media, R1 -iii media, R1 -iv media, R1 -v media, R2-i media, R2-ii media, R2-iii media, R2-iv media, R2-v media, R3-i media, R3-ii media, R3-iii media, R3-iv media, R3-v media, R4-i media, R4-ii media, R4-iii media, R4-iv media or R4-v media.
[00010] Embodiments disclosed herein also include systems for the micropropagation of plants. In one embodiment, the system comprises a kit comprising a media comprising meta-topolin and/or thidiazuron. In another embodiment, the media is b-9-i media, b-9-ii media, b-9-iii media, b-9-iv media, b-9-v media, CW2-i media, CW2-N media, CW2-iii media, CW2-iv media, CW2-v media, b-10-i media, b-10-ii media, b-10-iii media, b-10-iv media, b-10-v media, b-1 1 -i media, b-1 1 -ii media, b-1 1 -iii media, b-1 1 -iv media, b-1 1 -v media, b-12c-i media, b-12c-ii media, b-12c-iii media, b-12c-iv media, b-12c-v media, b-1 -i media, b-1 -ii media, b-1 -iii media, b-1 -iv media, b- 1 -v media, b-4-i media, b-4-ii media, b-4-iii media, b-4-iv media, b-4-v media, b-6-i media, b-6-ii media, b-6-iii media, b-6-iv media, b-6-v media, CW1 -i media, CW1 -ii media, CW1 -iii media, CW1 -iv media, CW1 -v media, CW3-i media, CW3-ii media, CW3-iii media, CW3-iv media, CW3-v media, CW4-i media, CW4-ii media, CW4-iii media, CW4-iv media, CW4-v media, CW5-i media, CW5-ii media, CW5-iii media, CW5-iv media, CW5-v media, CW6-i media, CW6-ii media, CW6-iii media, CW6-iv media, CW6-v media, R1 -i media, R1 -N media, R1 -iii media, R1 -iv media, R1 -v media, R2-i media, R2-ii media, R2-iii media, R2-iv media, R2-v media, R3-i media, R3-ii media, R3-iii media, R3-iv media, R3-v media, R4-i media, R4-ii media, R4-iii media, R4-iv media or R4-v media.
[00011] Embodiments disclosed herein also include methods of micropropagating plants. In one embodiment, the method comprises culturing explants, cultures and/or shoots in a media comprising meta-topolin and/or thidiazuron. In another embodiment, the growth media is b-9-i media, b-9-ii media, b-9-iii media, b-9-iv media, b-9-v media, CW2-i media, CW2-N media, CW2-iii media, CW2-iv media, CW2-v media, b-10-i media, b-10-ii media, b- 10-iii media, b-10-iv media, b-10-v media, b-1 1 -i media, b-1 1 -ii media, b-1 1 -iii media, b-1 1 -iv media, b-1 1 -v media, b-12c-i media, b-12c-ii media, b-12c-iii media, b-12c-iv media, b-12c-v media, b-1 -i media, b-1 -ii media, b-1 -iii media, b-1 -iv media, b-1 -v media, b-4-i media, b-4-ii media, b-4-iii media, b-4-iv media, b-4-v media, b-6-i media, b-6-ii media, b-6-iii media, b-6-iv media, b-6- v media, CW1 -i media, CW1 -N media, CW1 -iii media, CW1 -iv media, CW1 -v media, CW3-i media, CW3-N media, CW3-iii media, CW3-iv media, CW3-v media, CW4-i media, CW4-N media, CW4-iii media, CW4-iv media, CW4-v media, CW5-i media, CW5-ii media, CW5-iii media, CW5-iv media, CW5-v media, CW6-i media, CW6-ii media, CW6-iii media, CW6-iv media, CW6-v media, R1 -i media, R1 -ii media, R1 -iii media, R1 -iv media, R1 -v media, R2-i media, R2-ii media, R2-iii media, R2-iv media, R2-v media, R3-i media, R3-ii media, R3-iii media, R3-iv media, R3-v media, R4-i media, R4-ii media, R4-iii media, R4-iv media or R4-v media.
[00012] Further Embodiments
1. A media for micropropagating plants wherein said media supports 10- 120 day multiplication cycles at least 1 month, for at least 3 months, for at least 6 months, for at least 9 months, for at least 12 months, for at least 15 months, for at least 18 months, for at least 21 months, for at least 24 months or for at least 36 months.
2. A media of embodiment 1 said media comprises meta-topolin or an analogue thereof.
3. A media according to embodiment 1 wherein said media comprises, consists essentially of or consists of b-9-i media, b-9-ii media, b-9-iii media, b- 9-iv media, b-9-v media, CW2-i media, CW2-ii media, CW2-iii media, CW2-iv media, CW2-v media, b-10-i media, b-10-ii media, b-10-iii media, b-10-iv media, b-10-v media, b-1 1 -i media, b-1 1 -ii media, b-1 1 -iii media, b-1 1 -iv media, b-1 1 -v media, b-12c-i media, b-12c-ii media, b-12c-iii media, b-12c-iv media, b-12c-v media, b-1 -i media, b-1 -ii media, b-1 -iii media, b-1 -iv media, b- 1 -v media, b-4-i media, b-4-ii media, b-4-iii media, b-4-iv media, b-4-v media, b-6-i media, b-6-ii media, b-6-iii media, b-6-iv media, b-6-v media, CW1 -i media, CW1 -ii media, CW1 -iii media, CW1 -iv media, CW1 -v media, CW3-i media, CW3-ii media, CW3-iii media, CW3-iv media, CW3-v media, CW4-i media, CW4-ii media, CW4-iii media, CW4-iv media, CW4-v media, CW5-i media, CW5-ii media, CW5-iii media, CW5-iv media, CW5-v media, CW6-i media, CW6-N media, CW6-iii media, CW6-iv media, CW6-v media, R1 -i media, R1 -N media, R1 -iii media, R1 -iv media, R1 -v media, R2-i media, R2-N media, R2-iii media, R2-iv media, R2-v media, R3-i media, R3-N media, R3-iii media, R3-iv media, R3-v media, R4-i media, R4-N media, R4-iii media, R4-iv media or R4-v media.
4. A kit for the micropropagation of plants wherein said kit comprises a media comprising meta-topolin or an analogue thereof wherein said media supports 10-120 day multiplication cycles at least 1 month, for at least 3 months, for at least 6 months, for at least 9 months, for at least 12 months, for at least 15 months, for at least 18 months, for at least 21 months, for at least 24 months or for at least 36 months.
5. A kit according to embodiment 4 where said media comprises, consists of or consists essentially of b-9-i media, b-9-ii media, b-9-iii media, b-9-iv media, b-9-v media, CW2-i media, CW2-N media, CW2-iii media, CW2-iv media, CW2-v media, b-10-i media, b-10-ii media, b-10-iii media, b-10-iv media, b-10-v media, b-1 1 -i media, b-1 1 -ii media, b-1 1 -iii media, b-1 1 -iv media, b-1 1 -v media, b-12c-i media, b-12c-ii media, b-12c-iii media, b-12c-iv media, b-12c-v media, b-1 -i media, b-1 -ii media, b-1 -iii media, b-1 -iv media, b- 1 -v media, b-4-i media, b-4-ii media, b-4-iii media, b-4-iv media, b-4-v media, b-6-i media, b-6-ii media, b-6-iii media, b-6-iv media, b-6-v media, CW1 -i media, CW1 -ii media, CW1 -iii media, CW1 -iv media, CW1 -v media, CW3-i media, CW3-ii media, CW3-iii media, CW3-iv media, CW3-v media, CW4-i media, CW4-ii media, CW4-iii media, CW4-iv media, CW4-v media, CW5-i media, CW5-ii media, CW5-iii media, CW5-iv media, CW5-v media, CW6-i media, CW6-ii media, CW6-iii media, CW6-iv media, CW6-v media, R1 -i media, R1 -ii media, R1 -iii media, R1 -iv media, R1 -v media, R2-i media, R2-ii media, R2-iii media, R2-iv media, R2-v media, R3-i media, R3-ii media, R3-iii media, R3-iv media, R3-v media, R4-i media, R4-ii media, R4-iii media, R4-iv media or R4-v media.
6. A kit according to any of embodiments 4 or 5 wherein said system further comprises a media for transition to ex vivo conditions
7. A kit according to embodiment 6 wherein said media for transition to ex vivo conditions comprises, consists essentially of or consists of Br-2-i media, Br-2-ii media, Br-2-iii media, Br-2-iv media, Br-2-v media, Ech-i media, Ech-ii media, Ech-iii media, Ech-iv media, Ech-v media, Amel-i media, Amel-ii media, Amel-iii media, Amel-iv media, Amel-v media; FS1 -i media, FS1 -N media, FS1 -iii media, FS1 -iv media, FS1 -v media; FS2-i media, FS2-N media, FS2-iii media, FS2-iv media, FS2-v media; FS3-i media, FS3-N media, FS3-iii media, FS3-iv media, FS3-v media, FS4-i media, FS4-N media, FS4-iii media, FS4-iv media or FS4-v media.
8. Any of the kits described above further comprising an explant, wherein the explant can be any part of the plant including but not limited to internode, node, stolon, shoot, dormant lateral bud, immature flower bud, crown, rhizome or parts thereof.
9. A method of micropropagating plants comprising utilizing a media or kit of embodiments 1 , 2, 3, 4 or 5.
10. A method of transitioning micropropagated plants to ex vitro conditions comprising utilizing a kit of embodiment 6 or 7.
11. A method of micropropagating a plant utilizing a media or kit of embodiments 1 , 2, 3, 4 or 5 wherein an explant is obtained from a bamboo, a grass, a food crop plant or a perennial.
12. A method according to embodiment 1 1 wherein said method produces 100,000 plantlets tracing back to an individual an explant.
13. Any of the embodiments above wherein the meta-topolin or thidiazuron within a media is replaced with a meta-topolin or thidiazuron analogue described herein. 14. A media for micropropagating plants wherein said media comprises meta-topolin or an analogue thereof and supports 10-120 day multiplication cycles for at least six months.
15. A media according to claim 14 wherein said media supports 10-120 day multiplication cycles for at least one year.
16. A media according to claim 14 or 15 wherein said meta-topolin or analogue thereof is present in an amount from 0.0125 mg/mL - 10 mg/mL.
17. A media according to claim 14, 15 or 16 wherein said media further comprises thidiazuron or an analogue thereof.
18. A media according to claim 14, 15, 16, or 17 wherein said media further comprises NAA, BAP, 2ip and/or IBA.
19. A method of micropropagting monocots comprising culturing monocot explants and/or shoots in a media of claim 14, 15, 16, 17 or 18.
20. A method of micropropagating monocots according to embodiment 19 wherein said monocot is Aegilops (goatgrass), Agave, Agropyron (crested wheatgrass), Agrostis (bentgrass), Allium (onion), Alopecurus (meadow foxtail), Amaranthus (amaranth), Ammophila (beach grass), Ananas (pineapple), Andropogon (beardgrass), Arrhenatherum (oat grass), Avena (oat), Axonopus (carpet grass), Beckmannia (slough grass), Bouteloua (grama grass), Bromus (brome grass), Calamagrostis (reed grass), Calamus (palm), Cortaderia (pampas grass), Dactylis (orchard grass), Elymus (wheat grass), Festuca (fescue), Geranium, Gladiolus, Hakonechloa (hakone grass), Hordeum (barley), Iridaceae (iris), Lilium (lilies), Linum (flax), Lolium (rye grass), Miscanthus, Musa (bananas, plantains), Orchidaceae (orchids), Oryza (rice), Pennisetum (millet), Phalaris (canary grass), Phleum (timothy), Poa (blue grass), Phoenix (dates), Saccharum (sugarcane), Secale (rye), Sorghum, Trillium, Tripsacum (gama grass), Triticosecale (triticale), Triticum (wheat), Zea (corn) or Zoysia (zoysia grass).
21. A method of micropropagating monocots according to embodiment 19 wherein said monocot is Aegilops (goatgrass), Agave, Agropyron (crested wheatgrass), Agrostis (bentgrass), Allium (onion), Alopecurus (meadow foxtail), Amaranthus (amaranth), Ammophila (beach grass), Ananas (pineapple), Andropogon (beardgrass), Arrhenatherum (oat grass), Avena (oat), Axonopus (carpet grass), Beckmannia (slough grass), Bouteloua (grama grass), Bromus (brome grass), Calamagrostis (reed grass), Calamus (palm), Cortaderia (pampas grass), Dactylis (orchard grass), Elymus (wheat grass), Festuca (fescue), Geranium, Gladiolus, Hakonechloa (hakone grass), Hordeum (barley), !ridaceae (iris), Lilium (lilies), Linum (flax), Lolium (rye grass), Miscanthus, Musa (bananas, plantains), Orchidaceae (orchids), Oryza (rice), Pennisetum (millet), Phalaris (canary grass), Phleum (timothy), Poa (blue grass), Phoenix (dates), Saccharum (sugarcane), Secale (rye), Sorghum, Trillium, Tripsacum (gama grass), Triticosecale (triticale), Triticum (wheat), Zea (corn) or Zoysia (zoysia grass).
22. A media for transitioning shoots to ex vitro conditions wherein media comprises, consists essentially of or consists of Br-2-i media, Br-2-ii media, Br-2-iii media, Br-2-iv media, Ech-i media, Ech-ii media, Ech-iii media, Ech-iv, Amel-i media, Amel-ii media, Amel-iii media, Amel-iv media, Amel-v media, FS1 -i media, FS1 -N media, FS1 -iii media, FS1 -iv media, FS1 -v media; FS2-i media, FS2-N media, FS2-iii media, FS2-iv media, FS2-v media; FS3-i media, FS3-M media, FS3-iii media, FS3-iv media, FS3-v media, FS4-i media, FS4-N media, FS4-iii media, FS4-iv media or FS4-v media.
23. A media for micropropagating monocots wherein said media comprises thidiazuron or an analogue thereof and supports 10-120 day multiplication cycles for at least six months.
24. A media according to embodiment 23 wherein said media supports 10- 120 day multiplication cycles for at least one year.
25. A media according to embodiment 23 or 24 wherein said thidiazuron or analogue thereof is present in an amount from 0.0001 mg/mL - 5 mg/mL.
26. A media according to embodiment 23, 24 or 25 wherein said media further comprises meta-topolin or an analogue thereof, NAA, BAP, 2ip and/or IBA.
27. A media according to embodiment 26 wherein said meta-topolin or analogue thereof is present in an amount from 0.0125 mg/mL - 10 mg/mL.
28. A media according to embodiment 22, 23, 24, 25, 26 or 27 wherein said media comprises, consists essentially of or consists of b-9-i media, b-9-ii media, b-9-iii media, b-9-iv media, b-9-v media, CW2-i media, CW2-N media, CW2-iii media, CW2-iv media, CW2-v media, b-10-i media, b-10-ii media, b- 10-iii media, b-10-iv media, b-10-v media, b-1 1 -i media, b-1 1 -ii media, b-1 1 -iii media, b-1 1 -iv media, b-1 1 -v media, b-12c-i media, b-12c-ii media, b-12c-iii media, b-12c-iv media, b-12c-v media, b-1 -i media, b-1 -ii media, b-1 -iii media, b-1 -iv media, b-1 -v media, b-4-i media, b-4-ii media, b-4-iii media, b-4-iv media, b-4-v media, b-6-i media, b-6-ii media, b-6-iii media, b-6-iv media, b-6- v media, CW1 -i media, CW1 -N media, CW1 -iii media, CW1 -iv media, CW1 -v media, CW3-i media, CW3-N media, CW3-iii media, CW3-iv media, CW3-v media, CW4-i media, CW4-N media, CW4-iii media, CW4-iv media, CW4-v media, CW5-i media, CW5-N media, CW5-iii media, CW5-iv media, CW5-v media, CW6-i media, CW6-N media, CW6-iii media, CW6-iv media and/or CW6-V media.
29. A method of micropropagting monocot comprising culturing monocot explants and/or shoots in a media of embodiment 22, 23, 24, 25, 26, 27 or 28.
30. A method of micropropagating monocot according to embodiment 29 wherein said monocot is Aegilops (goatgrass), Agave, Agropyron (crested wheatgrass), Agrostis (bentgrass), Allium (onion), Alopecurus (meadow foxtail), Amaranthus (amaranth), Ammophila (beach grass), Ananas (pineapple), Andropogon (beardgrass), Arrhenatherum (oat grass), Avena (oat), Axonopus (carpet grass), Beckmannia (slough grass), Bouteloua (grama grass), Bromus (brome grass), Calamagrostis (reed grass), Calamus (palm), Cortaderia (pampas grass), Dactylis (orchard grass), Elymus (wheat grass), Festuca (fescue), Geranium, Gladiolus, Hakonechloa (hakone grass), Hordeum (barley), !ridaceae (iris), Lilium (lilies), Linum (flax), Lolium (rye grass), Miscanthus, Musa (bananas, plantains), Orchidaceae (orchids), Oryza (rice), Pennisetum (millet), Phalaris (canary grass), Phleum (timothy), Poa (blue grass), Phoenix (dates), Saccharum (sugarcane), Secale (rye), Sorghum, Trillium, Tripsacum (gama grass), Triticosecale (triticale), Triticum (wheat), Zea (corn) or Zoysia (zoysia grass).
31. A method of micropropagating monocot according to embodiment 29 wherein said monocot is Aegilops (goatgrass), Agave, Agropyron (crested wheatgrass), Agrostis (bentgrass), Allium (onion), Alopecurus (meadow foxtail), Amaranthus (amaranth), Ammophila (beach grass), Ananas (pineapple), Andropogon (beardgrass), Arrhenatherum (oat grass), Avena (oat), Axonopus (carpet grass), Beckmannia (slough grass), Bouteloua (grama grass), Bromus (brome grass), Calamagrostis (reed grass), Calamus (palm), Cortaderia (pampas grass), Dactylis (orchard grass), Elymus (wheat grass), Festuca (fescue), Geranium, Gladiolus, Hakonechloa (hakone grass), Hordeum (barley), Iridaceae (iris), Lilium (lilies), Linum (flax), Lolium (rye grass), Miscanthus, Musa (bananas, plantains), Orchidaceae (orchids), Oryza (rice), Pennisetum (millet), Phalaris (canary grass), Phleum (timothy), Poa (blue grass), Phoenix (dates), Saccharum (sugarcane), Secale (rye), Sorghum, Trillium, Tripsacum (gama grass), Triticosecale (triticale), Triticum (wheat), Zea (corn) or Zoysia (zoysia grass).
32. A kit comprising a media according to any one of embodiments 14, 15, 16, 17, 18, 37, 22, 23, 24, 25, 26, 27 or 28.
DEFINITIONS
[00013] 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.
[00014] 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.
[00015] As used herein, the terms "dicotyledon" and "dicot" refer to a flowering plant having an embryo containing two seed halves or cotyledons. Examples include citrus; geranium; tobacco; tomato; the legumes, including peas, alfalfa, clover and soybeans; oaks; maples; roses; mints; squashes; daisies; walnuts; cacti; violets and buttercups.
[00016] 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.
[00017] As used herein, the term "line" is used broadly to include, but is not limited to, a group of plants vegetatively propagated from a single parent plant, via tissue culture techniques or a group of inbred plants which are genetically very similar due to descent from a common parent(s). A plant is said to "belong" to a particular line if it (a) is a primary transformant (TO) plant regenerated from material of that line; (b) has a pedigree comprised of a TO plant of that line; or (c) is genetically very similar due to common ancestry {e.g., via inbreeding or selfing). In this context, the term "pedigree" denotes the lineage of a plant, e.g. in terms of the sexual crosses effected such that a gene or a combination of genes, in heterozygous (hemizygous) or homozygous condition, imparts a desired trait to the plant.
[00018] 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.
[00019] As used herein, the term "phenotype" refers to the observable characters of an individual cell, cell culture, organism (e.g., a plant), or group of organisms which results from the interaction between that individual's genetic makeup (i.e., genotype) and the environment.
[00020] As used herein, "plant" or "plants" refers to a plant of any plant species of the Plant Kingdom (Plantae), including but not limited to the level of divisions of species into subspecies, hybrid, varieties, variants, mutations, chimera(s), or other subspecies designation. Particular non-limiting examples include: amaranth, palm, palmetto, coconut, pineapple, grapefruit, lemon, lime, chestnut, mistletoe, banana, fig, holly, cherry, mountain ash, apple, orange, elm, willow tree, pine tree, eucalyptus, hemlock, cypress, Douglas fir, spruce, cedar, yew, magnolia, ginkgo, oak, maple, strawberry, blueberry, blackberry, raspberry, asparagus, rhubarb, bamboo, cabbage, pea (dry and fresh), chickpea, bean (garden, common, castor, dry, faba, garbanzo, lima, mung, navy and winged), lettuce, broccoli, pepper, tomato, radish, rape, camelina, wheat, millet, sedge, buckwheat, barley, rye, oat, corn (sweet and dent), rice, soybean, sorghum, alfalfa, trefoil, vetch, flax, lespedeza, lucerne, lupine, clover, trefoil, geranium, cotton, hydrangea, clematis, trillium, hyacinth, daffodil, tulip, bent grass, fescue, Bermuda grass, Miscanthus, orchard grass, timothy, oat grass, wheat grass, pampas grass, cassava, potato, taro, onion, carrot, eggplant, cucumber, watermelon, cantaloupe, peanut, artichoke, foxtail, guayule, hemp, kudzu, peppermint, safflower, sunflower, tobacco, fern, begonia, iris, impatiens, camellia, lily, coleus, orchid, kohlrabi, gladiolus, grape, cactus, poinsettia, Venus's fly-trap, Kalanchoe, aloe, basil, caraway, catnip, chicory, chives, cinnamon, cloves, coffee, dill, dock, fennel, foxglove, ginger, hawthorn, hop, lavender, licorice, may apple, mustard, oregano, parsley, plantain, rose, rosemary, sage, thyme, violet, witch hazel, ginseng, Echinacea, dandelion, periwinkle, azalea and poppy .
[00021] As used herein, the term "plant line" is used broadly to include, but is not limited to, a group of plants vegetatively propagated from a single parent plant, via tissue culture techniques or a group of inbred plants which are genetically very similar due to descent from a common parent(s). A plant is said to "belong" to a particular line if it (a) is a primary transformant (TO) plant regenerated from material of that line; (b) has a pedigree comprised of a TO plant of that line; or (c) is genetically very similar due to common ancestry {e.g., via inbreeding or selfing). In this context, the term "pedigree" denotes the lineage of a plant, e.g. in terms of the sexual crosses effected such that a gene or a combination of genes, in heterozygous (hemizygous) or homozygous condition, imparts a desired trait to the plant.
[00022] 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.
[00023] As used herein, the term "variety" refers to a subdivision of a species, consisting of a group of individuals within the species that are distinct in form or function from other similar arrays of individuals.
DETAILED DESCRIPTION OF THE DISCLOSURE
[00024] A method that allows the large scale commercial production of plants is highly desirable. Micropropagation (also known as tissue culturing with the terms used interchangeably herein), is an excellent method to achieve this aim.
[00025] Micropropagated plants are grown in vitro in sterile media. The sterile media can be liquid, semi-solid, or solid, and the physical state of the media can be varied 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. Typically, the media comprises agar, with the addition of various compounds such as nutrients, inorganic salts, growth regulators, sugars, vitamins and other compounds. Other media additives can include, but are not limited to, amino acids, macroelements, iron, microelements, inositol and undefined media components such as casein hydrolysates or yeast extracts. For example, the media can include any combination of NH4NO3; KNO3; Ca(NOs)2; K2SO4; MgSO4; MnSO4; ZnSO4; CuSO4; CaCI2; Kl; CoCI2; H3BO3; Na2MoO4; KH2PO4; FeSO ; Na2EDTA; Na2H2PO ; myo-inositol; thiamine; pyridoxine; nicotinic acid; glycine; riboflavin; ascorbic acid; silicon standard solution; β- naphthoxyacetic acid (NAA); indole butyric acid (IBA); 3-indoleacetic acid (IAA); benzylaminopurine (BAP); 6-Y-Y-(dimethylallylamino)-purine (2-ip); sugar; agar; carrageenan and charcoal. Examples of plant growth regulators include auxins and compounds with auxin-like activity, cytokinins and compounds with cytokinin-like activity. Exemplary auxins include 2,4- dichlorophenoxyacetic acid, IBA, picloram and combinations thereof. Exemplary cytokinins, in addition to meta-topolin and thidiazuron, include adenine hemisulfate, benzyladenine, dimethylallyladenine, kinetin, zeatin and combinations thereof. Gibberellic acid also can be included in the media. A sugar 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 sucrose, glucose, maltose, galactose and sorbitol or combinations thereof. [00026] Disclosed herein are specialized media, systems and methods that allow the successful tissue culturing of plants on a commercial scale. Certain media described herein include the cytokinins meta-topolin and/or thidiazuron. While certain embodiments utilize meta-topolin and/or thidiazuron defined as the particular compounds below, other related compounds can also be successful.
[00027] Compounds useful according to the present disclosure include meta- topolin analogues having a general formula
Figure imgf000015_0001
wherein W is an aryl or heteroaryl;
R1 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, C1-C6 alkylene, C1-C6 alkylyl, halogen, cyano, Ci-C6 alkyloxy, aryl or heteroaryl each optionally substituted with a Ci-C6 alkyl, SH, NHR3, CO2R3 or halogen;
R3 is H, OH, C1-C6 alkyl, C1-C6 alkylene, C1-C6 alkylyl, halogen, carboxylic group, ester group, aldehyde or cyano;
r is 0 to 8.
[00028] In one embodiment, W is
Figure imgf000015_0002
wherein a dashed line represents the presence or absence of a bond;
X1 -X7 is each independently selected from C, N, O, S with the proviso that the
X linking the ring to N is C.
[00029] In another embodiment, the compounds have a structure
Figure imgf000016_0001
wherein a dashed line represents the presence or absence of a bond.
[00030] In another embodiment, the compounds have a structure
Figure imgf000016_0002
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, Ci-C6 alkyl, Ci-C6 alkylene, Ci-C6 alkylyl, halogen, cyano, C1-C6 alkyloxy, aryl or heteroaryl each optionally substituted with a Ci-C6 alkyl, SH, NHR3, CO2R3 or halogen;
R3 is H, OH, C1-C6 alkyl, C1-C6 alkylene, C1-C6 alkylyl, halogen, carboxylic group, ester group, aldehyde or cyano;
p is 0 to 5; and
q is 0 to 6.
ents, the compounds have a structure
Figure imgf000016_0003
[00032] In still another embodiment, the compounds have a structure
Figure imgf000016_0004
[00033] Further still, compounds can have structures selected from
Figure imgf000017_0001
[00034] In one embodiment, R4 is OH.
[00035] In another embodiment, compounds have a structure selected from
Figure imgf000017_0002
[00036] In another embodiment, the compounds have a structure
Figure imgf000017_0003
wherein a dashed line represents the presence or absence of a bond.
[00037] In another embodiment, the compounds have a structure
Figure imgf000018_0001
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 alkylyl, halogen, cyano, C1-C6 alkyloxy, aryl or heteroaryl each optionally substituted with a Ci-C6 alkyl, SH, NHR3, CO2R3 or halogen;
R3 is H, OH, C1-C6 alkyl, C1-C6 alkylene, C1-C6 alkylyl, halogen, carboxylic group, ester group, aldehyde or cyano;
p is 0 to 5; and
q is 0 to 6.
[00038] In other embodiments, the compounds have a structure
Figure imgf000018_0002
[00039] In still another embodiment, the compounds have a structure
Figure imgf000018_0003
[00040] In one embodiment, the compound is meta-topolin, also known as 6- (3-hydroxybenzylamino)-purine, and by the abbreviation mT, having a molecular formula of C12H10N5OH, a molecular weight of 241 .25, and the following structural formula:
Figure imgf000019_0001
[00041] wherein said meta-topolin is a derivative of a willow tree or a poplar tree.
[00042] Compounds useful according to the present disclosure include thiadiazuron analogues having a general formula
Figure imgf000019_0002
wherein V is an aryl or heteroaryl;
each R5 and R6 is each independently H, OH, Ci-C6 alkyl, Ci-C6 alkylene, Ci- C6 alkylyl, halogen, cyano, C1-C6 alkyloxy, aryl or heteroaryl each optionally substituted with a Ci-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;
Z1 and Z2 are each independently NH, O, SH or CH or Z1 and Z2 can be combined to form a substituted or unsubstituted aryl or heteroaryl; and
Y1 is O or S.
[00043] In another embodiment, compounds have a structure
)o
Figure imgf000019_0003
wherein X17-X21 is each independently selected from C, N, O, S.
[00044] In other embodiments, compounds include
Figure imgf000020_0001
[00045] In one embodiment, the compound is Thidiazuron, also known as 1 - phenyl-3-(1 ,2,3-thiadiazol-5-yl)urea and 5-phenylcarbamoylamino-1 ,2,3- thiadiazole has the molecular formula of CgHsNUOS, a molecular weight of 220.25 and the following structural formula
Figure imgf000020_0002
[00046] If present in a media, each cytokinin can be present in an amount from 0.001 mg/L-100 mg/L and all amounts in between. In certain embodiments, meta-topolin 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 , 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, 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 particular 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..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. [00047] When both are utilized, meta-topolin and/or its analogues and thidiazuron and/or its analogues can also be included in ratios. For example, the amount of meta-topolin and/or its analogues to thidiazuron and/or its analogues can be 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. When both meta- topolin and thidiazuron are used, they can be present in the same or different media.
[00048] NAA, BAP, 2ip and/or IBA can similarly can be present at 0.001 mg/L, 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 100 mg/L or 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..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.
[00049] The structures or formula for a number of chemical compounds, including meta-topolin, have been provided above. One of ordinary skill in the art will recognize reference to a compound should be construed broadly to include pharmaceutically acceptable salts, prodrugs, tautomers, alternate solid forms, non-covalent complexes, analogs, derivatives and combinations thereof, of a chemical entity of the depicted structure or chemical name. [00050] A pharmaceutically acceptable salt is any salt of the parent compound that is suitable for use in the methods disclosed herein. A pharmaceutically acceptable salt also refers to any salt which may form as a result of administration of an acid, another salt, or a prodrug which is converted into an acid or salt. A salt comprises one or more ionic forms of the compound, such as a conjugate acid or base, associated with one or more corresponding counter-ions. Salts can form from or incorporate one or more deprotonated acidic groups (e.g. carboxylic acids), one or more protonated basic groups (e.g. amines), or both (e.g. zwitterions).
[00051] Not intended to be limited by the above described compounds, various tautomers of the above compounds may be possible. As used herein, "tautomer" refers to the migration of protons between adjacent single and double bonds. The tautomerization process is reversible. Other tautomers are possible when the compound includes, for example but not limited to, enol, keto, lactamin, amide, imidic acid, amine, and imine groups. Tautomers will generally reach an equilibrium state wherein the double bond is resonantly shared between the two bond lengths.
[00052] Unless stereochemistry is explicitly depicted, a structure is intended to include every possible stereoisomer, both pure or in any possible mixture.
[00053] Alternate solid forms are different solid forms than those that may result from practicing the procedures described herein. For example, alternate solid forms may be polymorphs, different kinds of amorphous solid forms, glasses, and the like.
[00054] Non-covalent complexes are complexes that may form between the compound and one or more additional chemical species that do not involve a covalent bonding interaction between the compound and the additional chemical species. They may or may not have a specific ratio between the compound and the additional chemical species. Examples might include solvates, hydrates, charge transfer complexes, and the like.
[00055] As an overview, in typical micropropagation, plants are placed in various media that stimulate physiological processes such as growth and multiplication by and/or within the plant. Generally the process includes 3 steps (following explant preparation and disinfection, discussed below): (1 ) initiation of in vitro growth and/or multiplication of the explant in a media ; (2) further in vitro multiplication in a second media; and (3) transition to ex vitro conditions. Not every tissue culture process requires each step, however, and in certain processes, steps can be combined or skipped. For example, while there is commonly a change in media types between steps 1 and 2, in certain embodiments, a media change is not included. In other processes, plants may not require a particular step promoting transition to ex vitro conditions but instead complete the process in a same media that supports multiplication. Accordingly, as described herein, media are defined as Stage 1 media (1 st media of a process); Stage 2 media (2nd media of a process); Stage 3 media (3rd media of a process); etc. Particular media can change stage based on the number of steps within a particular process and where the particular media resides within their order.
[00056] 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). The Stage 1 media is then placed into test tubes or other appropriate containers (including jars, boxes, jugs, cups, 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. Sterility can also be assessed by an accepted number of contaminated tubes per hundred tubes, for example and without limitation, 0 contaminated tubes per hundred tubes, no more than 1 contaminated tube per hundred tubes, no more than 2 contaminated tubes per hundred tubes, no more than 3 contaminated tubes per hundred tubes, no more than 4 contaminated tubes per hundred tubes, no more than 5 contaminated tubes per hundred tubes, no more than 6 contaminated tubes per hundred tubes, no more than 7 contaminated tubes per hundred tubes, no more than 8 contaminated tubes per hundred tubes, no more than 9 contaminated tubes per hundred tubes, no more than 10 contaminated tubes per hundred tubes, etc.
[00057] In media containing a gelling agent, such as agar, agarose, gellan gum, carrageenan or combinations thereof, the media solidifies upon cooling and serves to provide the micropropagated plant tissues with support, nutrients, growth regulators, water and other compounds as described below. Generally, tubes and jars contain 15-25 ml_ media while boxes contain 40-50 ml_ media. Cups can include 30-40 ml_ while jugs generally contain more than 50 ml_.
[00058] In media containing a gelling agent, such as agar, agarose, gellan gum, carrageenan or combinations thereof, the media solidifies upon cooling and serves to provide the micropropagated plant tissues with support, nutrients, growth regulators, water and other compounds as described below. Generally, tubes and jars contain 20-25 ml_ media while boxes contain 40-50 ml_ media. Cups can inlcude 30-40 ml_ while jugs generall contain more than 50 ml_.
[00059] Micropropagated plants 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. The explant can be any segment or collection of cells including but not limited to those from apical meristems, axillary buds, cambium, dormant lateral buds, immature flower buds, crowns, lateral meristems, shoot apices, stem segments, immature nodal sections from stems, internodes, lateral shoots, rhizomes, roots, seedlings, stolons (aka runners), or leaf segments. In one embodiment, the explant is taken from a 1 year old plant. In another embodiment, the explant is taken from a 2 year old plant. In another embodiment, the explant is taken from a plant that is 5 years old or less. In another embodiment, the explant is taken from a plant that is 4 years old or less. In another embodiment, the explant is taken from a plant that is 3 years old or less. In another embodiment, the explant is taken from a plant that is 2 years old or less. In another embodiment, the explant is taken from a plant that is 1 years old or less. In another embodiment, the explant is taken from a plant that is 6 months old or less. In another embodiment, the explant is taken from a plant that is 3 months old or less. The plant from which the explant is obtained can be grown in any suitable husbandry situation, including but not limited to growing in a growth chamber, growing in a greenhouse or growing in a field.
[00060] As will be understood by one of ordinary skill in the art, 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. Additional sources of explants include those described in Examples 19-23.
[00061] Nodal section(s) or other explant types can be cut into 3-5, 1 -10, 2-9, 3-8, 4-6, 3-6 or 2-7 millimeter sections. Explants can be left intact and disinfected to remove pathogens on the exterior of the explant. Any disinfection method known in the art can be used. Exemplary disinfection methods include application of a disinfectant, such as a disinfectant selected from among bleach (sodium and/or potassium and/or calcium hypochlorite), alcohol (e.g., ethanol, isopropyl), ozone, chlorine gas, iodine solution or antibiotic or anti-fungal solution or combinations thereof, or subjecting the exposed surface of the explant to ultraviolet light or to a temperature of -20° C or lower or to a temperature higher than 40° C or 50° C for a short period of time. In certain embodiments, small amounts (a few drops) of Tween 20 can be added to the disinfecting solutions.
[00062] Following initial disinfection, outer sheaths or other portions of the explant can be peeled off and discarded and the remaining piece put into an approximately 1 %, 5%, 10%, 15%, 20%, 25% or 30% solution of a commercial bleach or a similar disinfecting solution. The peeled explant or other explant part in disinfecting solution can be put onto a shaker table, such as for example, a Lab Rotators, Adjustable speed, Barnstead/Lab line orbital Shaker (model number KS 260) for 10 minutes, 20 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes or 210 minutes at 6-9 or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12, 13, 14, or 15 revolutions per minute. In another embodiment, the explant portions can then be put into an approximately 1 % solution of bleach or similar disinfecting solution, and placed back onto the shaker table for 30 minutes. In another embodiment, this 1 % bleach or similar disinfecting solution step can be repeated. In another embodiment, these described steps are progressive and include the entire disinfection process. As will be understood by one of ordinary skill in the art, a variety of appropriate disinfecting procedures can be used in accordance with the present disclosure.
[00063] Once disinfected, the explants can be placed onto a Stage 1 media within the tube and the tubes can be placed in a regulated growth chamber. As used herein, "growth chambers" can include a number of configurations and sizes including table-top boxes, stand-alone chambers, closets, small rooms, large rooms, etc. As is understood by one of ordinary skill in the art, variables such as light or temperature can be appropriately controlled in such a growth chamber. Appropriate ranges for tissue culturing bamboo include from 65°F-70°F, 60°F-75°F or 55°F-80°F at 200-500, 150-550 or 100-600 foot candles. Lighting can be full spectrum, although alternative lighting systems can also be utilized according to the present disclosure.
[00064] The explants are allowed to establish themselves within the tubes while in the growth chamber on Stage 1 media. In more common 3 stage tissue culturing, once established (i.e. growing without visible contamination), the cell cultures grown from the explants are transferred into a second, Stage 2 media. Alternatively, once established, the cell cultures can remain in Stage 1 media. At this stage in the tissue culturing process, a large number of plants can be created within a relatively short period of time because each cell culture can develop multiple shoots and each shoot can be separated and placed into an individual tube where it will develop additional shoots to separate and multiply.
[00065] Without limiting the media to a particular stage, non-limiting examples of media that commonly serve as Stage 1 and/or Stage 2 media include:
Media b-12c(i-v):
Media b-12c(i-v):
Figure imgf000026_0001
Component b-12c-i b-12c-ii b-12c-iii b- b-12c-v (mg/L in all 12c-iv
unless
otherwise
noted)
MnSO4 8.0-26.0 12.0-22.0 15.0-19.0 16.9 16.9±0.2
ZnSO4 4.0-12.0 6.0-10.0 8.0-9.0 8.6 8.6±2.0
CuSO4 0.012-0.378 0.020-0.030 0.022-0.028 0.025 0.025±.002
CaCI2 220-660 330-350 400-480 440 440±2
Kl 0.40-1 .25 0.60-1 .05 0.75-0.90 0.83 0.83±.02
CoCI2 0.012-0.378 0.020-0.030 0.022-0.028 0.025 0.025±.002
H3BO3 3.0-9.0 4.0-8.0 5.0-7.0 6.2 6.2±0.2
Na2MoO 0.12-0.36 0.18-0.31 .22-.28 0.25 0.25±.02
KH2PO4 85-255 120-210 150-190 170 170±2
FeSO4 27.0-84.0 40.0-70.0 50.0-60.0 55.7 55.7±0.2
Na2EDTA 37.0-1 12.0 55.0-94.0 67.0-82.0 74.6 74.6±0.2
Na2H2PO4 85-255 120-210 150-190 170 170±2 myo-lnositol 50-150 75-125 90-1 10 100 100±2
Thiamine 0.2-0.6 0.3-0.5 0.36-0.44 0.4 0.4±0.2
NAA 0.02 - 0.08 0.03-0.07 0.04-0.06 0.05 0.05±.02
BAP 0.5 - 1 .5 0.7 - 1 .3 0.9-1 .1 1 1 ±0.5
Thidiazuron 0.36 - 1 .12 0.56-0.94 0.67-.083 0.75 0.75±.02
Meta-topolin 2.5-7.5 3.7-6.2 4.5-5.5 5 5±2
Sugar g/L 15-45 22-37 27-33 30 30±2
Agar g/L 2.7-8.2 4.1 -6.8 4.9-6.1 5.5 5.5±0.2
Media CW2(i-v):
Component CW2-i CW2-N CW2-iii CW2- CW2-V iv
NH4NO3 825-2475 1237-2063 1485-1815 1650 1650±2
KNO3 950-2850 1425-2375 1710-2090 1900 1900±2
Ca(NO3)2 225-775 410-690 495-605 550 550±2
MgSO4 185-555 275-465 330-410 370 370±2
MnSO4 8.0-26.0 12.0-22.0 15.0-19.0 16.9 16.9±0.2
ZnSO4 4.0-12.0 6.0-10.0 8.0-9.0 8.6 8.6±0.2
CuSO4 0.012-0.378 0.020-0.030 0.022-0.028 0.025 0.025±.002
CaCI2 220-660 330-350 400-480 440 440±2
Kl 0.40-1 .25 0.60-1 .05 0.75-0.90 0.83 0.83±.02
CoCI2 0.012-0.378 0.020-0.030 0.022-0.028 0.025 0.025±.002
H3BO3 3.0-9.0 4.0-8.0 5.0-7.0 6.2 6.2±0.2
Na2MoO 0.12-0.36 0.18-0.31 .22-.28 0.25 0.25±.02
KH2PO4 85-255 120-210 150-190 170 170±2
FeSO4 27.0-84.0 40.0-70.0 50.0-60.0 55.7 55.7±0.2
Na2EDTA 37.0-1 12.0 55.0-94.0 67.0-82.0 74.6 74.6±0.2
Na2H2PO4 85-255 120-210 150-190 170 170±2 myo-lnositol 50-150 75-125 90-1 10 100 100±2
Thiamine 0.2-0.6 0.3-0.5 0.36-0.44 0.4 0.4±.02
NAA 0.02 - 0.08 0.03-0.07 0.04-0.06 0.05 0.05±.02 Component CW2-i CW2-N CW2-iii CW2- CW2-V iv
BAP 0.5-1.5 0.7-1.3 0.9-1.1 1 1±0.5
Meta-topolin 2.5-7.5 3.7-6.2 4.5-5.5 5 5±2
Sugar g/L 15-45 22-37 27-33 30 30±2
Agar g/L 2.7-8.2 4.1-6.8 4.9-6.1 5.5 5.5±0.2
Media CW3(i-iv)
Figure imgf000028_0001
Media b-9(i-v):
Figure imgf000028_0002
Figure imgf000029_0001
Media CW4(i-v):
Figure imgf000029_0002
Media CW6(i-v): Component CW6-1 CW6-M CW6-iii CW6- CW6-v iv
NH4NO3 825-2475 1237-2063 1485-1815 1650 1650±2
KNOs 950-2850 1425-2375 1710-2090 1900 1900±2
MgSO4 185-555 275-465 330-410 370 370±2
MnSO4 8.0-26.0 12.0-22.0 15.0-19.0 16.9 16.9±0.2
ZnSO4 4.0-12.0 6.0-10.0 8.0-9.0 8.6 8.6±0.2
CuSO4 0.012-0.378 0.020-0.030 0.022-0.028 0.025 0.025±.002
CaCI2 220-660 330-350 400-480 440 440±2
Kl 0.40-1 .25 0.60-1 .05 0.75-0.90 0.83 0.83±.02
C0CI2 0.012-0.378 0.020-0.030 0.022-0.028 0.025 0.025±.002
H3BO3 3.0-9.0 4.0-8.0 5.0-7.0 6.2 6.2±0.2
Na2MoO 0.12-0.36 0.18-0.31 .22-.28 0.25 0.25±.02
KH2PO4 85-255 120-210 150-190 170 170±2
K2SO5 181 .85- 272.80- 327.45- 363.75 363.75±.02
545.63 454.69 400.05
FeSO4 27.0-84.0 40.0-70.0 50.0-60.0 55.7 55.7±0.2
Na2EDTA 37.0-1 12.0 55.0-94.0 67.0-82.0 74.6 74.6±0.2
Na2H2PO4 85-255 120-210 150-190 170 170±2 myo-lnositol 50-150 75-125 90-1 10 100 100±2
Thiamine 0.2-0.6 0.3-0.5 0.36-0.44 0.4 0.4±0.2
NAA 0.05-0.15 0.07-0.12 0.09-0.1 1 0.1 0.1 ±0.02
BAP 0.5 - 1 .5 0.7 - 1 .3 0.9-1 .1 1 1 ±0.2
IBA 0.1 -0.3 0.15-0.25 0.17-0.22 0.2 0.2±0.1
Meta-topolin 2.5-7.5 3.7-6.2 4.5-5.5 5 5±2
Thidiazuron 0.12-0.36 0.18-0.31 .22-.28 0.25 0.25±.02
Sugar g/L 15-45 22-37 27-33 30 30±2
Agar g/L 2.7-8.2 4.1 -6.8 4.9-6.1 5.5 5.5±0.2
Media CW1 (i-v):
Figure imgf000030_0001
Component CW1-i CW1-N CW1-iii CW1- CW1-V iv
myo-lnositol 50-150 75-125 90-110 100 100±2
Thiamine 0.2-0.6 0.3-0.5 0.36-0.44 0.4 0.4±.02
NAA 0.02-0.08 0.03-0.07 0.04-0.06 0.05 0.05±.02
BAP 0.5-1.5 0.7-1.3 0.9-1.1 1 1±0.5
Meta-topolin 2.5-7.5 3.7-6.2 4.5-5.5 5 5±2
Sugar g/L 15-45 22-37 27-33 30 30±2
Agar g/L 2.7-8.2 4.1-6.8 4.9-6.1 5.5 5.5±0.2
Silicon 0.5-1.5 0.7-1.3 0.9-1.1 1 1±0.5 Solution ml_
Media CW5(i-v)
Figure imgf000031_0001
Media b-10(i-v):
Figure imgf000031_0002
Component b-10-i b-10-ii b-10-iii b-10- b-10-v iv
MnSO4 8.0-26.0 12.0-22.0 15.0-19.0 16.9 16.9±2.0
ZnSO4 4.0-12.0 6.0-10.0 8.0-9.0 8.6 8.6±2.0
CuSO4 0.012-0.378 0.020-0.030 0.022-0.028 0.025 0.025±.002
CaCI2 220-660 330-350 400-480 440 440±2
Kl 0.40-1 .25 0.60-1 .05 0.75-0.90 0.83 0.83±.02
CoCI2 0.012-0.378 0.020-0.030 0.022-0.028 0.025 0.025±.002
H3BO3 3.0-9.0 4.0-8.0 5.0-7.0 6.2 6.2±.2
Na2MoO 0.12-0.36 0.18-0.31 .22-.28 0.25 0.25±.02
KH2PO4 85-255 120-210 150-190 170 170±2
FeSO4 27.0-84.0 40.0-70.0 50.0-60.0 55.7 55.7±.2
Na2EDTA 37.0-1 12.0 55.0-94.0 67.0-82.0 74.6 74.6±0.2
Na2H2PO4 85-255 120-210 150-190 170 170±2 myo-lnositol 50-150 75-125 90-1 10 100 100±2
Thiamine 0.2-0.6 0.3-0.5 0.36-0.44 0.4 0.4±.02
NAA 0.02 - 0.08 0.03-0.07 0.04-0.06 0.05 0.05±.02
BAP 0.5 - 1 .5 0.7 - 1 .3 0.9-1 .1 1 1 ±0.5
Meta-topolin 2.5-7.5 3.7-6.2 4.5-5.5 5 5±2
Sugar g/L 15-45 22-37 27-33 30 30±2
Agar g/L 2.5-7.5 3.7-6.2 4.5-5.5 5 5±2
Media b-1 1 (i-v):
Component b-11-i b-11 -ii b-11-iii b-11 - b-11 -v iv
NH4NO3 825-2475 1237-2063 1485-1815 1650 1650±2
KNO3 950-2850 1425-2375 1710-2090 1900 1900±2
MgSO4 185-555 275-465 330-410 370 370±2
MnSO4 8.0-26.0 12.0-22.0 15.0-19.0 16.9 16.9±2.0
ZnSO4 4.0-12.0 6.0-10.0 8.0-9.0 8.6 8.6±2.0
CuSO4 0.012-0.378 0.020-0.030 0.022-0.028 0.025 0.025±.002
CaCI2 220-660 330-350 400-480 440 440±2
Kl 0.40-1 .25 0.60-1 .05 0.75-0.90 0.83 0.83±.02
CoCI2 0.012-0.378 0.020-0.030 0.022-0.028 0.025 0.025±.002
H3BO3 3.0-9.0 4.0-8.0 5.0-7.0 6.2 6.2±.2
Na2MoO4 0.12-0.36 0.18-0.31 .22-.28 0.25 0.25±.02
KH2PO4 85-255 120-210 150-190 170 170±2
FeSO4 27.0-84.0 40.0-70.0 50.0-60.0 55.7 55.7±.2
Na2EDTA 37.0-1 12.0 55.0-94.0 67.0-82.0 74.6 74.6±0.2
Na2H2PO4 85-255 120-210 150-190 170 170±2 myo-lnositol 50-150 75-125 90-1 10 100 100±2
Thiamine 0.2-0.6 0.3-0.5 0.36-0.44 0.4 0.4±.02
NAA 0.02 - 0.08 0.03-0.07 0.04-0.06 0.05 0.05±.02
BAP 0.5 - 1 .5 0.7 - 1 .3 0.9-1 .1 1 1 ±0.5
Thidiazuron 0.2-0.8 0.3-0.7 0.4-0.6 0.5 0.5±0.2
Meta-topolin 2.5-7.5 3.7-6.2 4.5-5.5 5 5±2
Sugar g/L 15-45 22-37 27-33 30 30±2 Media b-1 (i-v):
Figure imgf000033_0001
Figure imgf000034_0001
Media b-6(i-v):
Component b-6-i b-6-ii b-6-iii b-6-iv b-6-v
NH4NO3 600-1800 900-1500 1080-1320 1200 1200±2
Ca(NO3)2 838-2515 1257-2096 1510-1844 1677 1677±2
K2SO4 121 -363 181 -302 218-266 242 242±2
MgSO4 270-830 410-690 500-610 555 555±2
MnSO4 12.60-38.00 19.00-31 .70 22.80-27.80 25.35 25.35±.02
ZnSO4 6.4-19.5 9.6-16.2 1 1 .5-14.0 12.9 12.9±0.2
CuSO4 0.018-0.055 0.027-0.046 0.033-0.041 0.037 0.037±0.002
CaCI2 48-144 72-120 85-105 96 96±2
H3BO3 3.0-9.0 4.0-8.0 5.0-7.0 6.2 6.2±.2
Na2MoO4 0.12-0.36 0.18-0.31 .22-.28 0.25 0.25±.02
KH2PO4 85-255 120-210 150-190 170 170±2
FeSO4 27.0-84.0 40.0-70.0 50.0-60.0 55.7 55.7±.2
Na2EDTA 37.0-1 12.0 55.0-94.0 67.0-82.0 74.6 74.6±0.2
Na2H2PO4 42-128 63-106 75-95 85 85±2 myo-lnositol 100-300 150-250 180-220 200 200±2
Thiamine 0.4-1 .4 0.6-1 .1 0.8-1 .0 0.9 0.9±0.2
Pyridoxine 0.2-0.8 0.3-0.7 0.4-0.6 0.5 0.5±0.2
Nicotinic 0.2-0.8 0.3-0.7 0.4-0.6 0.5 0.5±0.2 acid
Glycine 1 -3 1 .5-2.5 1 .75-2.25 2 2±1
Riboflavin 10-30 15-25 18-22 20 20±2
NAA 0.5 - 1 .5 0.7 - 1 .3 0.9-1 .1 1 1 ±0.5
Thidiazuron 0.12-0.36 0.18-0.31 .22-.28 0.25 0.25±.02
2ip 2.5-7.5 3.7-6.2 4.5-5.5 5 5±2
Sugar g/L 12-37 15-35 20-30 25 25±2
Agar g/L 2.7-8.2 4.1 -6.8 4.9-6.1 5.5 5.5±0.2
Carrageenan 1 -3 1 .5-2.5 1 .75-2.25 2 2±1 g/L Media R1(i-v):
Figure imgf000035_0001
Media R2(i-v):
Figure imgf000035_0002
Figure imgf000036_0001
Media R3(i-v):
Figure imgf000036_0002
Media R4(i-v):
Figure imgf000036_0003
Component 50% Range 25% 10% R4-iv +/- Colun
Range Range
Kl 0.40-1 .25 0.60-1 .05 0.75-0.90 0.83 0.83±.02
CoCI2 0.012-0.378 0.020-0.030 0.022-0.028 0.025 0.025±.0(
H3BO3 3.0-9.0 4.0-8.0 5.0-7.0 6.2 6.2±.2
Na2MoO 0.12-0.36 0.18-0.31 0.22-0.28 0.25 0.25±.02
KH2PO4 85-255 120-210 150-190 170 170±2
FeSO4 13.0-42.0 20.8-34.7 25.1 -30.5 27.8 27.8±0.2
Na2EDTA 18.6-56.0 28.0-46.6 33.6-41 .0 37.3 37.3±0.2
Na2H2PO4 85-255 120-210 150-190 170 170±2 myo-lnositol 50-150 75-125 90-1 10 100 100±2
Thiamine 0.2-0.6 0.3-0.5 0.35-0.45 0.4 0.4±0.2
NAA 0.2-0.8 0.3-0.7 0.4-0.6 0.5 0.5±0.2
Meta-topolin 2.5-7.5 3.7-6.2 4.5-5.5 5 5±2
IAA 0.5-1 .5 0.7-1 .3 0.9-1 .1 1 1 ±0.5
BAP 0.5-1 .5 0.7-1 .3 0.9-1 .1 1 1 ±0.5
Sugar g/L 15-45 22-37 27-33 30 30±2
Agar g/L 2.5-7.5 3.7-6.2 4.5-5.5 5 5±2
[00066] Note that for each of these media, its solid form is provided. Each
media can be transformed into a liquid media by removing agar or
carageenan and liquid forms of these media and their uses are expressly
included within the scope of the present disclosure.
[00067] The expected number of shoots or plantlets may be different at
different stages of the tissue culturing process and can also depend on the
species of plant. In general, however, at the beginning of the process,
multiplication is from 1 .0-2.0, 1 .0-3.0 or 2.0-3.0 times. Once established,
multiplication can depend on the chosen container. For example,
multiplication can range from, without limitation, 1 -10 or 2-6 plants per tube, 1 - 15 or 4-9 plants per jar, 1 -20 or 9-17 plants per box or 1 -50 or 20-35 plants
per jug. The number 1 is included because certain species or particular cell
cultures require more time in Stage 1 or Stage 2 media before multiplication
begins. By carrying them through the process, however, most if not all begin
multiplication within a number of cycles. For example, some cell cultures may
begin to multiply only after 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16,
17 or 18 months in culture.
[00068] Methods disclosed herein can produce the following non-limiting
number of shoots or plantlets from a single explant: 100, 500, 1 ,000; 5,000; 10,000, 20,000, 50,000, 100,000, 250,000, 500,000, 750,000 1 ,000,000 or more.
[00069] Following multiplication through culturing and subculturing, particular plant shoots can be selected for transition to ex vitro conditions. Generally, media that support transition to ex vitro conditions represent a Stage 2, Stage 3, Stage 4 or Stage 5 media. Non-limiting examples of such media include:
Media Ech(i-v):
Figure imgf000038_0001
Media BR-2(i-v):
Figure imgf000038_0002
Figure imgf000039_0001
Charcoal g/L 150-450 220-370 270-330 300 300±2
Media Amel(i-v):
Figure imgf000039_0002
Figure imgf000040_0001
Charcoal g/L 2.5-7.5 3.7-6.2 4.5-5.5 5 5±2
Media FS1 (i-v)
Figure imgf000040_0002
Figure imgf000041_0001
Charcoal g/L 0.075-.225 0.1 125-0.1875 0.135-0.165 0.15 0.15±.02
Media FS3(i-v)
Component FS3-i FS3-N FS3-iii FS3-iv FS3-v
NH4NO3 825-2475 1237-2063 1485-1815 1650 1650±2
KNO3 950-2850 1425-2375 1710-2090 1900 1900±2
MgSO4 185-555 275-465 330-410 370 370±2
MnSO4 8.0-26.0 12.0-22.0 15.0-19.0 16.9 16.9±0.2
ZnSO4 4.0-12.0 6.0-10.0 8.0-9.0 8.6 8.6±0.2 cuSO4 0.012-0.378 0.020-0.030 0.022-0.028 0.025 0.025±.002
CaCI2 220-660 330-350 400-480 440 440±2
Kl 0.40-1 .25 0.60-1 .05 0.75-0.90 0.83 0.83±.02
CoCI2 0.012-0.378 0.020-0.030 0.022-0.028 0.025 0.025±.002
H3BO3 3.0-9.0 4.0-8.0 5.0-7.0 6.2 6.2±0.2
Na2MoO4 0.12-0.36 0.18-0.31 .22-.28 0.25 0.25±.02
KH2PO4 85-255 120-210 150-190 170 170±2
FESO4 13.0-42.0 20.8-34.7 25.1 -30.5 27.8 27.8±0.2
Na2EDTA 18.6-56.0 28.0-46.6 33.6-41 .0 37.3 37.3±0.2
Na2H2PO4 85-255 120-210 150-190 170 170±2 myo-lnositol 50-150 75-125 90-1 10 100 100±2
Thiamine 0.2-0.6 0.3-0.5 0.35-0.45 0.4 0.4±0.2
Pyridoxine 0.2-0.8 0.3-0.7 0.4-0.6 0.5 0.5±0.2
Nicotinic 0.2-0.8 0.3-0.7 0.4-0.6 0.5 0.5±0.2 acid
Riboflavin 5-15 7.5-12.5 9-1 1 10 10±2
IAA 5-15 7.5-12.5 9-1 1 10 10±2
Sugar g/L 15-45 22-37 27-33 30 30±2
Agar g/L 2.5-7.5 3.7-6.2 4.5-5.5 5 5±2
Carrageenan 2-6 3-5 3.6-4.4 4 4±2 g/L Media FS4
Figure imgf000042_0001
[00070] During transition to ex vitro conditions, plantlets and media can be placed in air permeable or air impermeable containers.
[00071] Each of the media described herein can be used in combination with each other media in a method, system or kit described herein. Moreover, the media can be combined in combinations greater than two (e.g., a kit may include 2 of the different media provided herein, or include 3 of the different media provided herein, or include more than 3 of the different media described herein). While not explicitly describing each possible combination herein, one of ordinary skill in the art should understand that this disclosure supports all possible combinations.
[00072] Following transition to ex vitro conditions, but before plants are placed in soil, or exposed to less regulated growing conditions, the plants can undergo a series of treatments designed to acclimate them to an unregulated growing environment. This is because some plants, when microcultured, do not develop adequate defensive structures, such as waxy cuticles to protect them from ordinary environmental conditions. The treatments that plants can undergo prior to being placed in an unregulated environment can include, without limitation, acclimatization to humidity, acclimatization to variations in temperature, and acclimatization to wind pressure. These acclimatization factors can be introduced gradually and/or in a staggered fashion.
[00073] Representative genus of bamboo appropriate for use with the disclosures herein include: Acidosasa; Ampelocalamus; Arundinaria; Bambusa; Bashania; Borinda; Brachystachyum; Cephalostachyum; Chimonobambusa; Chimonocalamus; Chusquea; Dendrocalamus; Dinochloa; Drepanostachyum; Eremitis; Fargesia; Gaoligongshania; Gigantochloa; Guadua; Hibanobambusa; Himalayacalamus; Indocalamus; Indosasa; Lithachne; Melocalamus; Melocanna; Menstruocalamus; Nastus; Neohouzeaua; Neololeba; Ochlandra; Oligostachyum; Olmeca; Otatea; Oxytenanthera; Phyllostachys; Pleioblastus; Pseudosasa; Raddia; Rhipidocladum; Sarocalamus; Sasa; Sasaella; Sasamorpha; Schizostachyum; Semiarundinaria; Shibataea; Sinobambusa; Thamnocalamus; Thyrsostachys; and Yushania.
[00074] Non-limiting examples of species within these genus include:
[00075] Acidosasa: Edulis
[00076] Ampelocalamus: Scandens
[00077] Arundinaria: Arundinaria appalachiana; Arundinaria funghomii; Arundinaria gigantea; Arundinaria gigantea 'Macon'; and Arundinaria Tecta
[00078] Bambusa: arnhemica; balcooa; bambos; basihirsuta; beecheyana; beecheyana var pubescens; blumeana; boniopsis; burmanica; chungii; chungii var. Barbelatta; cornigera; dissimulator; dissimulator 'Albinodia'; distegia; dolichoclada; dolichoclada 'Stripe'; dolichomerithalla 'Green stripe'; dolichomerithalla 'Silverstripe'; emeiensis 'Chrysotrichus'; emeiensis 'Flavidovirens'; emeiensis 'Viridiflavus'; eutuldoides; eutuldoides 'Viridivittata'; gibba; glaucophylla; intermedia; lako; lapidea; longispiculata; maculata; malingensis; multiplex; multiplex 'Alphonse Karr'; multiplex 'Fernleaf Stripestem'; multiplex 'Fernleaf; multiplex 'Golden Goddess'; multiplex 'Goldstripe'; multiplex 'Midori Green'; multiplex 'Riviereorum'; multiplex 'Silverstripe'; multiplex 'Tiny Fern Striped'; multiplex 'Tiny Fern'; multiplex 'Willowy'; nutans; odashimae; odashimae X B. Tuldoides; oldhamii; oliveriana; pachinensis; pervariabilis; pervariabilis 'Viridistriatus'; rigida; rutila; sinospinosa; sp 'Hirose'; sp. 'Clone X'; sp. 'Nana'; sp. 'Polymorpha'; sp.
'Richard Waldron'; stenostachya; suberecta; textilis; textilis 'Dwarf; textilis
'Kanapaha'; textilis 'Maculata'; textilis 'Mutabilis'; textilis 'Scranton'; textilis var.
Albostriata; textilis var. Glabra; textilis var. Gracilis; tulda; tulda 'Striata';
Tuldoides; variostriata; ventricosa; ventricosa 'Kimmei'; vulgaris; vulgaris
'Vittata'; vulgaris 'Wamin Striata'; and vulgaris 'Wamin'
[00079] Bashania: Fargesii; and Qingchengshanensis
[00080] Borinda: KR 5288; Albocerea; Angustissima; Contracta; Frigidorum;
Fungosa; fungosa 'White Cloud'; Lushuiensis; Macclureana; Nujiangensis;
Papyrifera; Perlonga; sp. 'Muliensis'; and Yulongshanensis
[00081] Brachystachyum: densiflorum; and densiflorum var. villosum
[00082] Cephalostachyum: Pergracile; and Virgatum
[00083] Chimonobambusa: macrophylla 'Intermedia'; Marmorea; marmorea
'Variegata'; Quadrangularis; quadrangularis 'Joseph de Jussieu'; quadrangularis 'Suow'; quadrangularis 'Yellow Groove'; Szechuanensis; and
Tumidissinoda
[00084] Chimonocalamus: Pallens
[00085] Chusquea: Andina; Circinata; circinata 'Chiapas' Coronalis; Culeou; culeou 'Argentina'; culeou 'Caha Prieta'; culeou 'Hillier's Form'; Cumingii; Delicatula; Foliosa; Galeottiana; Gigantea; Glauca; Liebmannii; Macrostachya; mimosa ssp. Austral is; Montana; Muelleri; Pittieri; Simpliciflora; sp. 'Chiconquiaco'; sp. 'Las Vigas'; Subtilis; Sulcata; Tomentosa; Uliginosa; Valdiviensis; and Virgata
[00086] Dendrocalamus: Asper; asper 'Betung Hitam'; Brandisii; brandisii 'Black'; brandisii (variegated); Calostachyus; Giganteus; giganteus (Quail Clone); giganteus (variegated); Hamiltonii; Jianshuiensis; jianshuiensis (variegated); Latiflorus; latiflorus 'Mei-nung'; Membranaceus; Minor; minor 'Amoenus'; Sikkimensis; Sinicus; sp. 'Maroochy'; sp. 'Parker's Giant'; Strictus; Validus; and Yunnanicus
[00087] Dinochloa: Malayana; and Scandens
[00088] Drepanostachyum: falcatum var. sengteeanum; and Khasianum
[00089] Eremitis: Eremitis
[00090] Fargesia: Adpressa; Apircirubens; apircirubens 'White Dragon'; Denudata; dracocephala 'Rufa'; Murieliae; murieliae 'SABE 939'; murieliae 'Vampire'; murieliae (next generation seedlings); Nitida; nitida 'Jiuzhaigou'; Robusta; robusta 'Campbell'; robusta 'Wolong'; sp. 'Scabrida'; and Utilis
[00091] Gaoligongshania: Gaoligongshania and Megalothyrsa
[00092] Gigantochloa: Hitam Hijau; Albociliata; Apus; Atroviolacea; Atter; Hasskarliana; Levis; Luteostriata; Maxima; Pseudoarundinacea; Ridleyi; Robusta; sp 'Rachel Carson'; sp. 'Bali White Stripe'; sp. 'Sumatra 3751 '; sp. 'Widjaja 3827'; and Wrayii
[00093] Guadua: Amplexifolia; Angustifolia; angustifolia 'Bicolor'; angustifolia 'Less Thorny'; Chacoensis; Longifolia; Paniculata; sp. 'Aureocaulis'; and Velutina
[00094] Hibanobambusa: Tranquillans; and tranquillans 'Shiroshima'
[00095] Himalayacalamus: Falconeri; falconeri 'Damarapa'; Hookerianus; Planatus; and Porcatus
[00096] Indocalamus: Cordatus; Latifolius; latifolius 'Hopei'; Longiauritus; sp. 'Hamadae'; sp. 'Solidus'; Tessellatus; and Victorialis
[00097] Indosasa: Crassiflora; and Gigantea
[00098] Lithachne: Humilis
[00099] Melocalamus: Arrectus
[000100] Melocanna: Baccifera
[000101] Menstruocalamus: Sichuanensis
[000102] Nastus: Elatus
[000103] Neohouzeaua: Mekongensis
[000104] Neololeba: Atra
[000105] Ochlandra: Stridula
[000106] Oligostachyum: Glabrescens
[000107] Olmeca: Recta
[000108] Otatea: acuminata 'Michoacan'; acuminata ssp. Acuminata acuminata ssp. Aztecorum; acuminata ssp. aztecorum 'Dwarf; Fimbriata; and glauca 'Mayan Silver'
[000109] Oxytenanthera: Abyssinica; and Braunii
[000110] Phyllostachys: Acuta; Angusta; Arcana; arcana 'Luteosulcata'; Atrovaginata; Aurea; aurea 'Albovariegata'; aurea Or Don'; aurea 'Flavescens-inversa'; aurea 'Holochrysa'; aurea 'Koi'; aurea 'Takemurai'; Aureosulcata; aureosulcata 'Alata'; aureosulcata 'Aureocaulis'; aureosulcata 'Harbin Inversa'; aureosulcata 'Harbin'; aureosulcata 'Pekinensis'; aureosulcata 'Spectabilis'; Aurita; Bambusoides; bambusoides 'Albovariegata'; bambusoides 'Castillon Inversa'; bambusoides 'Castillon'; bambusoides 'Golden Dwarf; bambusoides 'Job's Spot'; bambusoides 'Kawadana'; bambusoides 'Marliac'; bambusoides 'Rib Leaf; bambusoides 'Richard Haubrich'; bambusoides 'Slender Crookstem'; bambusoides 'Subvariegata'; bambusoides 'Tanakae'; bambusoides 'White Crookstem'; Bissetii; bissetii 'Dwarf; Dulcis; Edulis; edulis 'Anderson'; edulis 'Bicolor'; edulis 'Goldstripe'; edulis 'Heterocycla'; Elegans; Flexuosa; flexuosa 'Kimmei'; Glauca; glauca 'Notso'; glauca 'Yunzhu'; Heteroclada; heteroclada 'Purpurata'; heteroclada 'Solidstem'; Hispida; Humilis; Incarnata; Iridescens; Kwangsiensis; Lithophila; Lofushanensis; Makinoi; mannii 'Decora'; mannii 'Mannii'; Meyeri; Nidularia; nidularia 'Farcta'; nidularia 'Smoothsheath'; Nigra; nigra 'Bory'; nigra 'Daikokuchiku'; nigra 'Hale'; nigra 'Henon'; nigra 'Megurochiku'; nigra 'Mejiro'; nigra 'Muchisasa'; nigra 'Othello'; nigra 'Punctata'; nigra 'Shimadake'; nigra 'Tosaensis'; Nuda; nuda 'Localis'; Parvifolia; Platyglossa; Praecox; praecox 'Prevernalis'; praecox 'Viridisulcata'; Prominens; Propinqua; propinqua 'Beijing'; Robustiramea; Rubromarginata; Stimulosa; Varioauriculata; Violascens; Viridiglaucescens; Viridis; viridis 'Houzeau'; viridis 'Robert Young'; Vivax; vivax 'Aureocaulis'; vivax 'Black Spot'; vivax 'Huangwenzhu Inversa'; and vivax 'Huangwenzhu'
[000111] Pleioblastus: Akebono; Amarus; Argenteostriatus; Chino; chino 'Angustifolia'; chino 'Elegantissimus'; chino 'Kimmei'; chino 'Murakamiansus'; chino 'Vaginatus Variegatus'; Distichus; distichus 'Mini'; Fortunei; Gauntlettii; Gramineus; gramineus 'Monstrispiralis'; Hindsii; Humilis; humilis 'Albovariegatus'; humilis 'Variegatus'; Juxianensis; Kodzumae; Kongosanensis; kongosanensis 'Akibensis'; kongosanensis 'Aureostriatus'; Linearis; linearis 'Nana'; Nagashima; Oleosus; Pygmaeus; pygmaeus 'Greenstripe'; pygmaeus 'Ramosissimus'; shibuyanus 'Tsuboi'; Simonii; simonii 'Variegatus'; Viridistriatus; viridistriatus 'Chrysophyllus'; and Xestrophyllus
[000112] Pseudosasa: Amabilis; Cantori; Guanxianensis; Japonica; japonica 'Akebono'; japonica 'Akebono-suji'; japonica 'Pleioblastoides'; japonica sutsumiana'; japonica 'Variegata'; Longiligula; Owatarii; Usawai; and Viridula
[000113] Radd ia : Brasiliensis; and Distichophylla
[000114] Rhipidocladum: Pittieri; and Racemiflorum
[000115] Sarocalamus: Faberi; and Fangianus
[000116] Sasa: Cernua; Gracillima; Hayatae; Kagamiana; kagamiana ssp. Yoshinoi; Kurilensis; kurilensis 'Shimofuri'; Megalophylla; Nagimontana; nipponica (hort.); Oshidensis; Palmata; Senanensis; Shimidzuana; sp. Tsuboiana; and Veitchii
[000117] Sasaella: Bitchuensis; hidaensis 'muraii'; Masamuneana; masamuneana 'Albostriata'; masamuneana 'Aureostriata'; Ramosa; Sasakiana; and Shiobarensis
[000118] Sasamorpha : Borealis
[000119] Schizostachyum: Brachycladum; brachycladum 'Bali Kuning';
Caudatum; Glaucifolium; Jaculans; Lima; and sp. 'Murray Island'
[000120] Semiarundinaria: Fastuosa; fastuosa 'Viridis'; Fortis;
Kagamiana; Lubrica; Makinoi; Okuboi; sp. Maruyamana; sp. 'Korea';
Yashadake; yashadake 'Kimmei'; and yashadake 'kimmei inversa'
[000121] Shibataea: Chinensis; Kumasaca; kumasaca 'Albostriata'; kumasaca 'Aureostriata'; Lancifolia; and Nanpingensis
[000122] Sinobambusa: Gigantea; Intermedia; Tootsik; and tootsik
'Albostriata'
[000123] Thamnocalamus: aristatus 'Aristatus hort. US'; Crassinodus; crassinodus 'Kew Beauty'; crassinodus 'Mendocino'; crassinodus 'Merlyn'; nepalensis 'Nyalam'; Spathiflorus; and Tessellatus
[000124] Thyrsostachys: Oliveri; and Siamensis
[000125] Yushania: Alpina; Anceps; anceps 'Pitt White'; Boliana; Brevipaniculata; Exilis; Maculata; and maling
[000126] Particularly useful species include: edulis; scandens; ArundinariaGigantea; ArundinariaTecta; BambusaBalcooa; BambusaBambos; Bambusa Oldhamii; BambusaTextilis; BambusaTulda; BashaniaFargesii; BrachystachyumDensiflorum; Chusquea Gigantea; DendrocalamusAsper; DendrocalamusBrandisii; DendrocalamusGiganteus; DendrocalamusHamiltonii; DendrocalamusStrictus; FargesiaDenudata; Fargesiadracocephala'Ruia'; FargesiaMurieliae; FargesiaNitida;
FargesiaRobusta; Fargesiarobusta 'Wolong'; Fargesiasp. 'Scabrida'; GuaduaAmplexifolia; Guadua Paniculata; Himalayacalamus Falconeri; Indocalamus Tessellatus; Ochlandra Stridula; Otatea acuminata ssp. Aztecorum; Phyllostachys Atrovaginata; Phyllostachys Aurea; Phyllostachys Bambusoides; Phyllostachys Bissetii; Phyllostachys Edulis; Phyllostachys edulis 'Heterocycla'; Phyllostachys Glauca; Phyllostachys Iridescens; Phyllostachys Kwangsiensis; Phyllostachys Nidularia; Phyllostachys Nigra; Phyllostachys nigra 'Henon'; Phyllostachys Nuda; Phyllostachys Parvifolia; Phyllostachys Praecox; Phyllostachys Propinqua; Phyllostachys Viridis; Phyllostachys Vivax; Pleioblastus Distichus; Pleioblastus Fortunei; Pleioblastus Linearis; Pseudosasa Japonica; Sasa Kurilensis; Sasa Veitchii; Sasaella Masamuneana; Sasamorpha Borealis; Schizostachyum Brachycladum; Schizostachyum brachycladum 'Bali Kuning'; Schizostachyum Caudatum; Schizostachyum Glaucifolium; Schizostachyum Jaculans; Schizostachyum Lima; Schizostachyum sp. 'Murray Island'; Semiarundinaria Fastuosa; Semiarundinaria Yashadake; Shibataea Kumasaca; Sinobambusa Gigantea; Thamnocalamus Crassinodus; Thamnocalamus Tessellatus; Yushania Alpina; and Yushania maling.
[000127] As one of ordinary skill in the art appreciates, many species of bamboo have different common names. Accordingly, the following terminology and language comparisons are provided.
Figure imgf000048_0001
BAM BUS A dissemulator BAMBUSA dissimulator
BAMBUSA edulis BAMBUSA odashimae
BAMBUSA forbesii NEOLOLEBA atra
BAMBUSA glaucescens BAMBUSA multiplex
BAMBUSA multiplex 'Green
BAMBUSA multiplex 'Midori Green' Alphonse'
Bambusa mutabilis Bambusa textilis 'Mutabilis'
Bambusa tuldoides 'Clone X' Bambusa sp. 'Clone X'
Bambusa tuldoides 'ventricosa' Bambusa ventricosa
Bambusa variegata (hort.) Bambusa glaucophylla
BAMBUSA vulgaris 'Striata' BAMBUSA vulgaris 'Vittata'
BASH AN 1 A faberi SAROCALAMUS faberi
Borinda boliana Yushania boliana
CHIMONOBAMBUSA falcata HIMALYACALAMUS hookerianus
CHUSQUEA breviglumis CHUSQUEA gigantea
Chusquea breviglumis Chusquea culeou
CHUSQUEA nigricans CHUSQUEA culeou 'Ca?a Prieta'
CHUSQUEA quila CHUSQUEA valdiviensis
DENDROCALAMUS affinis BAMBUSA emeiensis
DENDROCALAMUS membranaceus BAMBUSA membranacea
DREPANOSTACHYUM falcatum HIMALAYACALAMUS hookerianus
DREPANOSTACHYUM falcatum 'var.
DREPANOSTACHYUM falconeri
sengteeanum'
HIMALAYACALAMUS falconeri
DREPANOSTACHYUM hookerianum
'Damarapa'
DREPANOSTA CHYUM
HIMALAYACALAMUS falconeri sengteeanum
FARGESIA angustissima BORINDA angustissima
FARGESIA crassinodus THAMNOCALAMUS crassinodus
Fargesia dracocephala Fargesia apircirubens
Fargesia dracocephala 'White
Fargesia apircirubens 'White Dragon' Dragon'
FARGESIA frigida BORINDA frigidorum
FARGESIA fungosa BORINDA fungosa
FARGESIA sp "A-4" FARGESIA adpressa
Fargesia sp. 'rufa' Fargesia dracocephala 'Rufa'
GELIDOCALAMUS fangianus SAROCALAMUS fangianus
GIGANTOCHLOA atroviolacea 'Timor
BAMBUSA lako
Black'
GIGANTOCHLOA luteostriata BAMBUSA luteostriata
GIGANTOCHLOA
GIGANTOCHLOA verticillata
pseudoarundinacea
Himalayacalamus asper Himalayacalamus planatus
HIMALAYACALAMUS falconeri DREPANOSTACHYUM falcatum 'var.
'glomeratum' sengteeanum'
HIMALAYACALAMUS intermedius Yushania boliana
HIMALAYACALAMUS planatus HIMALAYACALAMUS asper (hort.)
HIMALAYACALAMUS planatus Neomicrocalamus microphyllus (hort.)
NEOMICROCALAMUS microphyllus HIMALAYACALAMUS planatus NEOSINOCALAMUS affinis BAMBUSA emeiensis 'Chrysotrichus'
Otatea acuminata 'Mayan Silver' Otatea glauca 'Mayan Silver'
OTATEA aztecorum OTATEA acuminata ssp. aztecorum
PHYLLOSTACHYS cerata PHYLLOSTACHYS heteroclada
PHYLLOSTACHYS congesta PHYLLOSTACHYS atrovaginata
PHYLLOSTACHYS decora PHYLLOSTACHYS mannii 'Decora'
PHYLLOSTACHYS edulis
PHYLLOSTACHYS heterocycla
'Heterocycla'
PHYLLOSTACHYS heterocycla
PHYLLOSTACHYS edulis
pubescens
PHYLLOSTACHYS heterocycla
PHYLLOSTACHYS edulis 'Anderson' pubescens Anderson'
PHYLLOSTACHYS heteroclada
PHYLLOSTACHYS purpurata
'Purpurata'
PHYLLOSTACHYS purpurata PHYLLOSTACHYS heteroclada
'Solid stem' 'Solidstem'
PHYLLOSTACHYS purpurata
PHYLLOSTACHYS heteroclada 'Straightstem'
PLEIOBLASTUS kongosanensis
PLEIOBLASTUS akibensis
'Akibensis'
PLEIOBLASTUS gramineus PLEIOBLASTUS gramineus
'Raseetsu-chiku' 'Monstrispiralis'
PLEIOBLASTUS variegatus PLEIOBLASTUS fortunei
Oiongzhuea tumidissinoda Chimonobambusa tumidissinoda
SASA asahinae SASA shimidzuana
SASA humilis PLEIOBLASTUS humilis
SASA pygmaea PLEIOBLASTUS pygmaeus
SASA tessellata INDOCALAMUS tessellatus
SASA variegata PLEIOBLASTUS fortunei
Sasa veitchii 'Minor' Sasa hayatae
SASAELLA masamuneana
SASAELLA glabra lbostriata'
'Albostriata'
SASAELLA masamuneana
SASAELLA masamuneana
rhyncantha
SASAELLA rhyncantha SASAELLA masamuneana
SEMIARUNDINARIA villosa SEMIARUNDINARIA okuboi
SINARUNDINARIA FARGESIA
TETRAGONOCALAMUS angulatus CHIMONOBAMBUSA quadranqularis
THAMNOCALAMUS spathaceus FARGESIA murieliae
YUSHANIA aztecorum OTATEA acuminata ssp. aztecorum
Chinese & Japanese Names
Figure imgf000050_0001
Figure imgf000051_0001
English names
Figure imgf000051_0002
1 Technically not a bamboo but included within the meaning of bamboo herein.
Figure imgf000052_0001
Timber bambusoides ιιΐ
[000128] Additional representative monocots for which the media, systems and methods described and disclosed herein are applicable include but are not limited to monocotyledonous plants of the following genera: Aegilops (goatgrass), Agave, Agropyron (crested wheatgrass), Agrostis (bentgrass), Allium (onion), Alopecurus (meadow foxtail), Amaranthus (amaranth), Ammophila (beach grass), Ananas (pineapple), Andropogon (beardgrass), Arrhenatherum (oat grass), Avena (oat), Axonopus (carpet grass), Beckmannia (slough grass), Bouteloua (grama grass), Bromus (brome grass), Calamagrostis (reed grass), Calamus (palm), Cortaderia (pampas grass), Dactylis (orchard grass), Elymus (wheat grass), Festuca (fescue), Geranium, Gladiolus, Hakonechloa (hakone grass), Hordeum (barley), Iridaceae (iris), Lilium (lilies), Linum (flax), Lolium (rye grass), Miscanthus, Musa (bananas, plantains), Orchidaceae (orchids), Oryza (rice), Pennisetum (millet), Phalaris (canary grass), Phleum (timothy), Poa (blue grass), Phoenix (dates), Saccharum (sugarcane), Secale (rye), Sorghum, Trillium, Tripsacum (gama grass), Triticosecale (triticale), Triticum (wheat), Zea (corn) and Zoysia (zoysia grass).
[000129] By means of the media, systems and methods described and disclosed herein, it is possible for one of ordinary skill in the art to achieve rolling tissue cultures of plants including without limitation, bamboo, grasses, foodcrops, geraniums, trillium, etc.. As used herein, "rolling tissue culture" means that the multiplication process can continue substantially indefinitely by continuing to separate and multiply plantlets. In one embodiment, one shoot or plantlet is placed in a tube and the shoot or plantlet multiplies into a number of additional shoots or plantlet. After multiplication, each shoot or plantlet or a subset of the shoots or plantlets are separated and each placed in a subsequent tube for further multiplication. This process can continue while at various times, some or all shoots or plantlet can be removed from the multiplication process and transitioned to ex vitro conditions. By continuing indefinitely, it is meant that 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, etc. day multiplication cycles can be repeated without initiating new explants for at least 1 month, for at least 3 months, for at least 6 months, for at least 9 months, for at least 12 months, for at least 15 months, for at least 18 months, for at least 21 months, for at least 24 months or for at least 36 months. By continuing indefinitely, it is meant that 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, etc. day multiplication cycles can be repeated without initiating new explants for at least 1 month, for at least 3 months, for at least 6 months, for at least 9 months, for at least 12 months, for at least 15 months, for at least 18 months, for at least 21 months, for at least 24 months or for at least 36 months. Particular ranges of days in multiplication cycles include 10-120 days; 10-100 days; 10-80 days; 10-60 days; 10-120 days; 10-40 days; 10-20 days; 14-120 days; 14-90 days; 14-70 days; 14-50; 14-42 days; 14-30 days; 14-21 days; 12-42 days; 20-60 days; 10-15 days; 14-20 days; 14-18 days etc.
[000130] These media systems and methods can be packaged and/or described in various kits. Kits can include, without limitation, one or more of the following in a package or container: (1 ) one or more media; and (2) one or more explants from one or more species of plants. In certain non-limiting embodiments, the media can be b-9-i media, b-9-ii media, b-9-iii media, b-9-iv media, b-9-v media, CW2-i media, CW2-ii media, CW2-iii media, CW2-iv media, CW2-v media, b-10-i media, b-10-ii media, b-10-iii media, b-10-iv media, b-10-v media, b-1 1 -i media, b-1 1 -ii media, b-1 1 -iii media, b-1 1 -iv media, b-1 1 -v media, b-12c-i media, b-12c-ii media, b-12c-iii media, b-12c-iv media, b-12c-v media, b-1 -i media, b-1 -ii media, b-1 -iii media, b-1 -iv media, b- 1 -v media, b-4-i media, b-4-ii media, b-4-iii media, b-4-iv media, b-4-v media, b-6-i media, b-6-ii media, b-6-iii media, b-6-iv media, b-6-v media, CW1 -i media, CW1 -ii media, CW1 -iii media, CW1 -iv media, CW1 -v media, CW3-i media, CW3-ii media, CW3-iii media, CW3-iv media, CW3-v media, CW4-i media, CW4-ii media, CW4-iii media, CW4-iv media, CW4-v media, CW5-i media, CW5-ii media, CW5-iii media, CW5-iv media, CW5-v media, CW6-i media, CW6-ii media, CW6-iii media, CW6-iv media, CW6-v media, R1 -i media, R1 -N media, R1 -iii media, R1 -iv media, R1 -v media, R2-i media, R2-N media, R2-iii media, R2-iv media, R2-v media, R3-i media, R3-N media, R3-iii media, R3-iv media, R3-v media, R4-i media, R4-N media, R4-iii media, R4-iv media or R4-v media, Br-2-i media, Br-2-ii media, Br-2-iii media, Br-2-iv media, Ech-i media, Ech-ii media, Ech-iii media, Ech-iv, Amel-i media, Amel-ii media, Amel-iii media, Amel-iv media, Amel-v media; FS1 -i media, FS1 -N media, FS1 -iii media, FS1 -iv media, FS1 -v media; FS2-i media, FS2-N media, FS2-iii media, FS2-iv media, FS2-v media; FS3-i media, FS3-ii media, FS3-iii media, FS3-iv media, FS3-v media. In another embodiment, the kits can comprise one or more containers for the tissue culturing process including without limitation, tubes, jars, boxes or jugs. In another embodiment the kits can comprise instructions for the tissue culturing of bamboo. 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.
[000131] The ingredients included in the kits can be supplied in containers of any sort such that the life of the different ingredients are preserved and are not adsorbed or altered by the materials of the container. For example, sealed glass ampules may contain ingredients that have been packaged under a neutral, non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, etc., ceramic, metal or any other material typically employed to hold similar ingredients. Other examples of suitable containers include simple bottles that may be fabricated from similar substances as ampules, and envelopes, that may comprise foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, or the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the ingredients to be mixed. Removable membranes may be glass, plastic, rubber, etc.
[000132] As stated, kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable media, such as a floppy disc, CD-ROM, DVD-ROM, Zip disc, videotape, audiotape, etc. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an internet web site specified by the manufacturer or distributor of the kit, or supplied as electronic mail.
[000133] One advantage of the disclosed embodiments is that the methods are more robust than those previously used producing plants that do not require special treatments required by those produced using methods of the prior art. For example, methods disclosed herein do not require the use of seeds or inflorescence to start plants; do not require selection of diseased starting plants (such as those exhibiting symptoms of witches broom or little leaf disease); do not require use of somatic embryogenesis and do not utilize pseudospiklets. 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. These improvements over prior methods provide even additional advantages related to the health of produced plants and efficiency of growth and processing.
[000134] One advantage of the disclosed embodiments is that the methods are more robust than those previously used producing plants that do not require special treatments required by those produced using methods of the prior art. For example, methods disclosed herein do not require the use of seeds or inflorescence to start plants; do not require selection of diseased starting plants (such as those exhibiting symptoms of witches broom or little leaf disease); do not require use of somatic embryogenesis and do not utilize pseudospiklets. 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. These improvements over prior methods provide even additional advantages related to the health of produced plants and efficiency of growth and processing.
[000135] Non-limiting embodiments encompassed by the present disclosure include:
[000136] I. The following species: Arundinaria gigantea; Bambusa balcoa; Bambusa vulgaris; Bambusa vulgaris 'Vitatta'; Bambusa Oldhamii; Bambusa tulda; endrocalamus brandesii; Dendrocalamus asper; Dendrocalamus hamiltoni; Dendrocalamus giganteus; Dendrocalamus membranaceus; Dendrocalamus strictus; Gigantochloa aspera; Gigantochloa scortechini; Guadua culeata; uadua aculeata 'Nicaragua'; Guadua amplexifolia; Guadua angustifolia; Guadua angustofolia bi-color; Guadua paniculata; Melocanna bambusoides; eohouzeaua dullooa (Teinostachyum); Ochlandra travancorica; Phyllostachys edulis 'Moso'; Phyllostachys nigra; Phyllostachys nigra 'Henon'; Schizostachyum lumampao;
[000137] II. Initiated using Media b-12-c-v media or b-10-v media;
[000138] III. Multiplied on CW1 -v media; CW2-v media; CW3-v media; CW4- v media; CW5-v media; or CW6-v media for 10-120 day cycles; and
[000139] IV. Transitioned to ex vitro conditions on Br-2-v media; Ech-v media or Amel-v media.
[000140] More particularly, the following embodiments can be used:
[000141] Starting with a bamboo plant between the ages of 3 months and 3 years, a node from the cane with the lateral shoot just breaking the sheath can be used as the explant. Each nodal section can be cut into 3-5 millimeter sections with the shoot intact. 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. 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.
[000142] 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 200-500 foot candles. The initial Stage 1 media can be b-12c-iv at a pH of 5.7. The explants can then be transferred to fresh b-12c-iv media every 10-120 days (usually every 21 days), with contaminated tubes being discarded. Contaminated tubes can be identified by bacterial discoloration of the agar or by visible surface contamination. These explants can stay on b-12c-iv media for 3-4 10-120 day cycles (usually 21 day cycles). Explants can then 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.
[000143] Live shoots can next be transferred to a Stage 2 media, such as b- 9, CW1 , CW2, CW3, CW4, CW5, CW6 or b-6 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 in Stage 2 media or transitioned to a Stage 3 media, for example, b-10-iv or b-1 1 -iv at a pH of 5.7 for further multiplication. One-ten shoots per tube can be obtained per multiplication cycle.
[000144] Following removal from the multiplication process, the shoots can transferred to small tissue culturing boxes (known as "magenta boxes") for 10-120 days (usually 14-21 days) containing a Stage 3 or Stage 4 media, in this Example, BR-2 at a pH of 5.7 for 10-120 days (usually 14-21 days) or Amel at a pH of 5.7 for 10-120 days (usually 14-21 days).
[000145] The following procedures may also be used:
[000146] Starting with a bamboo plant between the ages of 3 months and 3 years, a node from the cane with the lateral shoot just breaking the sheath can be used as the explant. Each nodal section can be cut into 3-5 millimeter sections with the shoot intact. Some explants, including explants taken from canes 1 year or older can be pre-rinsed by shaking them in a jar of 70% isopropyl alcohol for 3 seconds followed by rinsing them under running tap water for 1 minute. Other explants are not pre-rinsed.
[000147] The outer sheaths can be peeled off and discarded and the remaining nodal section piece put into a 10% bleach solution. 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. For some implants, including those taken from canes 1 year or older, this step can be modified by adding a few drops of Tween 20 to the 10% bleach solution and soaking the explants for 45 minutes rather than 1 hour. The explants can then be put into a 1 % bleach solution, and placed back onto the shaker table for 30 minutes. This 1 % bleach solution step can then be repeated.
[000148] 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 200-500 foot candles. The Stage 1 media can be b-12c-iv at a pH of 5.7. The explants can be transferred to fresh b-12c-iv media every 10-120 days (usually every 21 days), with contaminated tubes being discarded. These explants can stay on b-12c-iv 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, in this Example, b-12- c supplemented with 7 g/L carageenan rather than the 5.5 g/L provided above. Following the third cycle, explants can be cleaned. The explants can be kept on b-12-c 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.
[000149] Once the explant exhibits multiple shoots, it can be either maintained on its Stage 2 media or transferred to a Stage 3 media. Non- limiting Stage 3 media include, a b-9 media, a CW1 media, a CW2 media a CW3 media, a CW4 media, a CW5 media, a CW6 media or a b-6 media at a pH of 5.7. The cultures can stay on Stage 2 or Stage 3 media until the desired number of shoots is obtained by separation into new tubes and further expansion. Generally, the range of time includes10-120 day cycles (usually 21 day cycles) between which the cultures can be assigned to go through another multiplication round or transitioned to a Stage 3 or Stage 4 media, such as a BR-2 media at a pH of 5.7 for 10-120 days (usually 21 days) in "magenta boxes" or a Amel media at a pH of 5.7 for 10-120 days (usually 14- 21 days).
[000150] In even more particular non-limiting embodiments, the following species can be multiplied in the following media according to procedures described in the proceeding paragraphs [000137]-[000145] at a pH of 5.5-5.7:
[000151] Arundinaria gigantea: b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
[000152] Bambusa balcoa: b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
[000153] Bambusa vulgaris: b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
[000154] Bambusa vulgaris 'Vitatta': b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
[000155] Bambusa Oldhamii: b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6- v; [000156] Bambusa tulda: b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
[000157] Dendrocalamus brandesii:b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
[000158] Dendrocalamus asper: b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
[000159] Dendrocalamus hamiltoni: b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
[000160] Dendrocalamus giganteus: b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
[000161] Dendrocalamus membranaceus: b-9-v, CW1 -v, CW3-v, CW4-v, CW5-V or CW6-v;
[000162] Dendrocalamus strictus: b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
[000163] Gigantochloa aspera: b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
[000164] Gigantochloa scortechini: b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
[000165] Guadua culeata: b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
[000166] Guadua aculeata 'Nicaragua': b-9-v, CW1 -v, CW3-v, CW4-v, CW5- v or CW6-v;
[000167] Guadua amplexifolia: b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
[000168] Guadua angustifolia: b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
[000169] Guadua angustofolia bi-color: b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
[000170] Guadua paniculata: b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6- v;
[000171] Melocanna bambusoides: b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
[000172] Neohouzeaua dullooa (Teinostachyum): b-9-v, CW1 -v, CW3-v, CW4-V, CW5-V or CW6-v;
[000173] Ochlandra travancorica: b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v; [000174] Phyllostachys edulis 'Moso': b-9-v, CW1 -v, CW3-V, CW4-V, CW5-V or CW6-v;
[000175] Phyllostachys nigra: b-9-v, CW1 -v, CW3-V, CW4-V, CW5-V or CW6- v;
[000176] Phyllostachys nigra 'Henon': b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-v;
[000177] Schizostachyum lumampao:b-9-v, CW1 -v, CW3-v, CW4-v, CW5-v or CW6-V.
[000178] As stated previously, there are many uses for plants produced according to the embodiments disclosed herein. Bamboos alone are used in, without limitation,
[000179] Exemplary Paper Types: Freesheet; Stock; Acid-free; A4; Board; Bond; Book; Bristol; Carbonless; Catalog; Coated; Cover; Dual-Purpose Bond; Duplex; English Finish; Equivalent; Fine; Free Sheet; Grain Long; Grain Short; Groundwood; Kraft; Lightweight; News Print; Publishing; Rag; Recycled; Tag; Uncoated; Virgin; Absorbent; Acid; Album; Albumin; Akaline; Bank Note; Tissue; Toilet; Towels; Fluff; Card Stock; OTC; OCR; Tissue Overlay; Napkins
[000180] Exemplary Pulp Types: Air-dried; Alpha; Bamboo; Bisulfate; Sulphite; Bleached; Chemical Cellulose (Dissolving ); Fluff; Fodder; Free; Fully Bleached; Hard; High Alpha Cellulose; Groundwood; Hot Groundwood; Jute; Knotter; Kraft; Long Fiber; Baled; Rolled; Market; Non-Wood; Board; Pressurized Groundwood;; Rag; Recycled; Reinforcement; Secondary; Semi- alkaline; Semi-bleached; Semi-chemical; Short Fiber; Soda; Specialty; Sulfate; Themochemical; Unbleached; Viscose; Wood
[000181] Board/Containers: Linerboard; Containerboard; Cardboard; OCC; Paperboard
[000182] Wood: Structural Wood Panels (including Structural Plywood; Oriented Strand Board; Structural Composite Panels); Glued Laminated Timber; Structural Composite Lumber (including Laminated Veneer Lumber; Parallel Strand Lumber; Oriented Strand Lumber); Prefabricated Wood I- Joists; Floor Joints; Railroad Ties; Flooring; Composites (including Auto; Aero; Musical). [000183] Textiles: Feedstock; Filament Yarn; Knitted Fabric; Knitting; Narrow Width Fabric; Non Woven Fabric; Spun Yarn; Woven Fabric; Viscose Rayon; Batting; Ginned Fiber; Cloth
[000184] Textile Products: Clothing; Towels; Sheets/Bedding; Pillows; Curtains
[000185] Food sources: Shoots; Any bi-product for food consumption
[000186] Consumer Goods: Animal Feed; Carpeting; Light Bulbs; Household Cleaning Products; Chopsticks & Toothpicks; Cleaning Brooms; Bicycles; Wheel Chairs; Fishing rods; Beer; Liquor; Pharmaceuticals; Cosmetics; Soap/Shampoo; Kitchenware; Crafts; Furniture; Nutraceuticals; Paper cups; Paper plates
[000187] Energy & Bioenergy: Charcoal; Insulation; Feedstock; Biomass
[000188] The following non-limiting examples are provided. In all examples, time on rooting media can be in permeable or impermeable containers.
[000189] The following non-limiting examples are provided. In all examples, time on rooting media can be in permeable or impermeable containers.
EXAMPLES
Example 1. Phyllostachys bissetti
[000190] Starting with a bamboo plant between the ages of 3 months and 3 years, a node from the cane with the lateral shoot just breaking the sheath was used as the explant. Each nodal section was cut into 3-5 millimeter sections with the shoot intact. The outer sheaths were 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. The explant in bleach solution was 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 were then put into a 1 % bleach solution with a final concentration of 0.6% sodium hydrochloride, and placed back onto the shaker table for 30 minutes. This 1 % bleach solution step was then repeated.
[000191] Individual explants were then placed on a Stage 1 media (15-25 mL) within a tube and the tubes were placed into a regulated clean growth chamber at a temperature of from 65°F-70°F and a full spectrum light level of 200-500 foot candles. The initial Stage 1 media in this Example was b-12c-iv at a pH of 5.7. The explants were transferred to fresh b-12c-iv media every 10-120 days (usually every 21 days), with contaminated tubes being discarded. Contaminated tubes were identified by bacterial discoloration of the agar or by visible surface contamination. These explants stayed on b-12c- iv media for 3-4 10-120 day cycles (usually 21 day cycles). Explants were taken off the media after the third cycle if multiplication was occurring. If multiplication was not occurring or not occurring to a significant degree, explants were left on the media for a fourth cycle.
[000192] Live shoots were next transferred to a Stage 2 media, in this Example, b-9-iv at a pH of 5.7. Alternatively to using one of the B-9 media, one of the CW1 media at a pH of 5.7 can also be used. The cultures stayed on b-9-iv media until the desired number of shoots was obtained by separation into new tubes and further expansion. Generally, the range of time included 10-120 day cycles (usually 14-21 day cycles) between which the cultures were assigned to go through another multiplication round in Stage 2 media or transitioned to a Stage 3 media, in this Example, b-10-iv at a pH of 5.7 for further multiplication. One-ten shoots per tube were obtained per multiplication cycle.
[000193] Following removal from the multiplication process, the shoots were transferred to small tissue culturing boxes (known as "magenta boxes") for 10-120 days (usually 14-21 days) containing a Stage 3 or Stage 4 media, in this Example. BR-2-iv at a pH of 5.7 for 10-120 days (usually 14-21 days). Example 2. Fargesia denudata
[000194] In the example of Fargeria denudata, the explants were chosen and disinfected as in Example 1 .
[000195] The explants were then transferred into jars containing a Stage 1 media, in this Example, b-12c-iv (liquid; 30-40 ml_) as described in Example 1 but for the use of jars. Explants were taken off the media after the third cycle if multiplication was occurring. If multiplication was not occurring or not occurring to a significant degree, explants were left on the media for a fourth cycle. Contaminated tubes were discarded.
[000196] The cultures were then transferred onto a Stage 2 media, in this Example, b-1 1 -iv (liquid) in jars on a rotating shelf that provides 6-9 revolutions per minute. The cultures remained on b-1 1 -iv media at a pH of 5.7 for 10-120 day cycles (usually 14 day cycles) until the desired number of shoots was obtained by separation into new jars and further expansion. One- fifteen shoots per jar were obtained per multiplication cycle. The shoots were then placed in a Stage 3 media, in this Example, Ech-iv at a pH of 6 for 10- 120 days (usually 14-21 days).
Example 3. Pleioblastus fortunei
[000197] In the example of Pleioblastus fortunei, the explants were chosen and disinfected as in Example 1 . The explants were then transferred into tubes containing a Stage 1 media, in this Example, b-12c-iv also as described in Example 1 . Shoots were then transferred to a Stage 2 media, in this Example, b-9-iv in magenta boxes (40-50 ml_). Alternatively, a CW1 media can also be used. They remained on b-9-iv media for 10-120 day cycles (usually 14 day cycles) until the desired number of shoots was obtained by separation into new boxes and further expansion. One-twenty shoots per box were obtained per multiplication cycle. The shoots were then placed on a Stage 3 media, in this Example, BR-2-iv for 10-120 days (usually 14-21 days). Example 4. Sasa Veitchii
[000198] In the example of Sasa Veitchii, the explants were chosen and disinfected as in Example 1 .
[000199] The explants were then transferred into tubes containing a Stage 1 media, in this Example, b-12c-iv also as described in Example 1 . Shoots were then transferred into a Stage 2 media, in this Example, b-1 -iv at a pH of 5.5 for 10-120 day cycles (usually 21 day cycles) until the desired number of shoots was obtained by separation into new tubes and further expansion One- ten shoots per tube were obtained per multiplication cycle. The shoots were then placed in a Stage 3 media, in this Example, Br-2-iv at a pH of 5.7 for 14- 21 days.
Example 5. Pleioblastus viridistriatus and Thamnocalamus crassinodus
[000200] In the example of Pleioblastus viridistriatus and Thamnocalamus crassinodus, the explants were chosen and disinfected as in Example 1 . The explants were then transferred into tubes containing a Stage 1 media, in this Example, b-12c-iv also as described in Example 1 . Shoots were then transferred into a Stage 2 media, in this Example, b-4-iv at a pH of 5.5 for 10- 120 day cycles (usually 21 day cycles) until the desired number of shoots was obtained by separation into new tubes and further expansion. One-ten shoots per tube were obtained per multiplication cycle. The shoots were then placed in a Stage 3 media, in this Example, Br-2-iv at a pH of 5.7 for 10-120 days (usually 14-21 days).
Example 6. Chusquea Culeo "Cana Prieta"
[000201] In the example of Chusquea Culeo "Cana Prieta", the explants were chosen and disinfected as in Example 1 . The explants were then transferred into tubes containing a Stage 1 media, in this Example, b-12c-iv media also as described in Example 1 . Shoots were then transferred into a Stage 2 media, in this Example, b-9-iv at a pH of 5.5 for 10-120 day cycles (usually 21 day cycles) until the desired number of shoots was obtained by separation into new tubes and further expansion. B-6 media at a pH of 5.5 can also be used. One-ten shoots per tube were obtained per multiplication cycle. The shoots were then placed in a Stage 3 media, in this Example, Amel-iv at a pH of 5.7 for 10-120 days (usually 14-21 days).
Example 7. Bambusa Old Hamii
[000202] In the example of Bambusa Old amii, the explants were chosen and disinfected as in Example 1 . The explants were then transferred into boxes containing a Stage 1 media, in this Example, b-10-iv (40-50 ml_) also as described in Example 1 but for the change to boxes. Shoots were maintained on b-10-iv media for 10-120 day cycles (usually 21 day cycles) until the desired number of shoots was obtained by separation into new boxes and further expansion. One-twenty shoots per box were obtained per multiplication cycle. The shoots were then placed in a Stage 2 media, in this Example, Amel-iv at a pH of 5.7 for 10-120 days (usually 14-21 days).
Example 8. Phyllostachys Moso, Phyllostachys Atrovaginata & Dendrocalamus Asper
[000203] In the example of Phyllostachys Moso, Phyllostachys Atrovaginata & Dendrocalamus Asper, the explants were chosen and disinfected as in Example 1 . The explants were then transferred into tubes containing a Stage 1 media, in this Example, b-12c-iv also as described in Example 1 . Shoots were then transferred into a Stage 2 media, in this Example, b-9-iv at a pH of 5.5 for 10-120 day cycles (usually 21 day cycles) until the desired number of shoots was obtained by separation into new tubes and further expansion. A B- 6 media at a pH of 5.5 can also be used. One-ten shoots per tube were obtained per multiplication cycle. The shoots were then placed in a Stage 3 media, in this Example, Amel-iv at a pH of 5.7 for 10-120 days (usually 14-21 days).
Example 9. Guadua Angustifolia
[000204] In the example of Guadua Angustofolia, the explants were chosen and disinfected as in Example 1 . The explants were then transferred into tubes containing a Stage 1 media, in this Example, b-12c-iv also as described in Example 1 . Shoots were then transferred into a Stage 2 media, in this Example, b-10-iv at a pH of 5.5 for 10-120 day cycles (usually 21 day cycles) until the desired number of shoots was obtained by separation into new tubes and further expansion. One-ten shoots per tube were obtained per multiplication cycle. The shoots were then placed in a Stage 3 media, in this Example, Amel-iv at a pH of 5.7 for 10-120 days (usually 14-21 days).
Example 10. Phyllostachys bissetti - alternate procedure
[000205] Starting with a bamboo plant between the ages of 3 months and 3 years, a node from the cane with the lateral shoot just breaking the sheath was used as the explant. Each nodal section was cut into 3-5 millimeter sections with the shoot intact. Some explants, including explants taken from canes 1 year or older were pre-rinsed by shaking them in a jar of 70% isopropyl alcohol for 3 seconds followed by rinsing them under running tap water for 1 minute. Other explants were not pre-rinsed.
[000206] The outer sheaths were peeled off and discarded and the remaining nodal section piece put into a 10% bleach solution. The explant in bleach solution was 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. For some implants, including those taken from canes 1 year or older, this step was modified by adding a few drops of Tween 20 to the 10% bleach solution and soaking the explants for 45 minutes rather than 1 hour. The explants were then put into a 1 % bleach solution, and placed back onto the shaker table for 30 minutes. This 1 % bleach solution step was then repeated. [000207] Individual explants were then placed on a Stage 1 media (15-25 ml_) within a tube and the tubes were placed into a regulated clean growth chamber at a temperature of from 65°F-70°F and a full spectrum light level of 200-500 foot candles. In this Example, the Stage 1 media was b-12c-iv at a pH of 5.7. The explants were transferred to fresh b-12c-iv media every 10-120 days (usually every 21 days), with contaminated tubes being discarded. These explants stayed on b-12c-iv media for 2 10-120 day cycles (usually 21 day cycles). Between cycles, excess sheaths were removed. At the time of transfer to the third cycle, explants were transitioned to a Stage 2 media, in this Example, b-12-c supplemented with 7 g/L carageenan rather than the 5.5 g/L provided above. Following the third cycle, explants were cleaned. The explants were kept on b-12-c supplemented with 7 g/L carageenan for 10-120 day cycles (usually 21 day cycles) until multiple shoots were observed. Observation of multiple shoots occurred within 3-15 months.
[000208] Once the explant exhibited multiple shoots, it was either maintained on its Stage 2 media or transferred to a Stage 3 media, in this Example, when used b-9-iv at a pH of 5.7. Alternatively to using one of the B-9 media, a CW1 media at a pH of 5.7 can also be used. The cultures stayed on Stage 2 or Stage 3 media until the desired number of shoots was obtained by separation into new tubes and further expansion. Generally, the range of time included 10-120 day cycles (usually 21 day cycles) between which the cultures were assigned to go through another multiplication round or were transitioned to a Stage 3 or Stage 4 media, in this Example, BR-2-iv at a pH of 5.7 for 10-120 days (usually 21 days) in "magenta boxes".
Example 11. Fargesia denudata - alternate procedure
[000209] In the example of Fargeria denudata, the explants were chosen and disinfected as in Example 10. The explants were then transferred into jars containing a Stage 1 media, in this Example, b-12c-iv (liquid; 30-40 ml_) as described in Example 10 but for the use of jars. These explants stayed on b- 12c-iv media for 2 10-120 day cycles (usually 21 day cycles). Between cycles, excess sheaths were removed. At the time of transfer to the third cycle, explants were transitioned to a Stage 2 media, in this Example, b-12-c supplemented with 7 g/L carageenan rather than the 5.5 g/L provided above. Following the third cycle, explants were cleaned. The explants were kept on b-12-c supplemented with 7 g/L carageenan for 10-120 day cycles (usually 21 day cycles) until multiple shoots were observed. Observation of multiple shoots occurred within 3-15 months.
[000210] Once the explant exhibited multiple shoots, it was either maintained on its Stage 2 media or transferred to a Stage 3 media, in this Example, b-1 1 - iv (liquid) at a pH of 5.7 in jars on a rotating shelf that provides 6-9 revolutions per minute. The cultures remained on Stage 2 or Stage 3 media for 10-120 day cycles (usually 14 day cycles) until the desired number of shoots was obtained by separation into new jars and further expansion. One-fifteen shoots per jar were obtained per multiplication cycle. The shoots were then placed in a Stage 3 or Stage 4 media, in this Example, Ech-iv at a pH of 6 for 10-120 days (usually 21 days).
Example 12 Pleioblastus fortune/ - alternate procedure
[000211] In the example of Pleioblastus fortunei, the explants were chosen and disinfected as in Example 10. The explants were then transferred into tubes containing a Stage 1 media in this Example, b-12c-iv also as described in Example 10. These explants stayed on b-12c-iv media for 2 10-120 day cycles (usually 21 day cycles). Between cycles, excess sheaths were removed. At the time of transfer to the third cycle, explants were transitioned to a Stage 2 media, in this Example, b-12-c supplemented with 7 g/L carageenan rather than the 5.5 g/L provided above. Following the third cycle, explants were cleaned. The explants were kept on b-12-c supplemented with 7 g/L carageenan for 10-120 day cycles (usually 21 day cycles) until multiple shoots were observed. Observation of multiple shoots occurred within 3-15 months.
[000212] Once the explant exhibited multiple shoots, it was either maintained on its Stage 2 media or transferred to a Stage 3 media, in this Example b-9-iv in magenta boxes (40-50 mL). (CW1 media can also be used). They remained on b-9-iv media for 10-120 day cycles (usually 14 day cycles) until the desired number of shoots was obtained by separation into new boxes and further expansion. One-twenty shoots per box were obtained per multiplication cycle. The shoots were then placed in a Stage 3 or Stage 4 media, in this Example, BR-2-iv for 10-120 days (usually 14-21 days).
Example 13. Sasa Veitchii - alternate procedure [000213] In the example of Sasa Veitchii, the explants were chosen and disinfected as in Example 10.
[000214] The explants were then transferred into tubes containing a Stage 1 media, in this Example, b-12c-iv also as described in Example 10. These explants stayed on b-12c-iv media for 2 10-120 day cycles (usually 21 day cycles). Between cycles, excess sheaths were removed. At the time of transfer to the third cycle, explants were transitioned to a Stage 2 media, in this Example, b-12-c supplemented with 7 g/L carageenan rather than the 5.5 g/L provided above. Following the third cycle, explants were cleaned. The explants were kept on b-12-c supplemented with 7 g/L carageenan for 10-120 day cycles (usually 21 day cycles) until multiple shoots were observed. Observation of multiple shoots occurred within 3-15 months.
[000215] Once the explant exhibited multiple shoots, it was either maintained on its Stage 2 media or transferred to a Stage 3 media, in this Example b-1 -iv at a pH of 5.5 for 10-120 day cycles (usually 21 day cycles) until the desired number of shoots was obtained by separation into new tubes and further expansion One-ten shoots per tube were obtained per multiplication cycle. The shoots were then placed in a Stage 3 or Stage 4 media, in this Example, Br-2-iv at a pH of 5.7 for 10-120 days (usually 14-21 days).
Example 14. Pleioblastus viridistriatus and Thamnocalamus crassinodus - alternate procedure
[000216] In the example of Pleioblastus viridistriatus and Thamnocalamus crassinodus, the explants were chosen and disinfected as in Example 10. The explants were then transferred into tubes containing a Stage 1 media, in this Example, b-12c-iv also as described in Example 10. These explants stayed on b-12c-iv media for 2 10-120 day cycles (usually 21 day cycles). Between cycles, excess sheaths were removed. At the time of transfer to the third cycle, explants were transitioned to a Stage 2 media, in this Example, b-12-c supplemented with 7 g/L carageenan rather than the 5.5 g/L provided above. Following the third cycle, explants were cleaned. The explants were kept on b-12-c supplemented with 7 g/L carageenan for 10-120 day cycles (usually 21 day cycles) until multiple shoots were observed. Observation of multiple shoots occurred within 3-15 months. [000217] Once the explant exhibited multiple shoots, it was either maintained on its Stage 2 media or transferred to a Stage 3 media, in this Example b-4-iv at a pH of 5.5 for 10-120 day cycles (usually 21 day cycles) until the desired number of shoots was obtained by separation into new tubes and further expansion. One-ten shoots per tube were obtained per multiplication cycle. The shoots were then placed in a Stage 3 or Stage 4 media, in this Example, Br-2-iv at a pH of 5.7 for 10-120 days (usually 14-21 days).
Example 15. Chusquea Culeo "Cana Prieta" - alternate procedure
[000218] In the example of Chusquea Culeo "Cana Prieta", the explants were chosen and disinfected as in Example 10. The explants were then transferred into tubes containing a Stage 1 media, in this Example, b-12c-iv also as described in Example 10. These explants stayed on b-12c-iv media for 2 10- 120 day cycles (usually 21 day cycles). Between cycles, excess sheaths were removed. At the time of transfer to the third cycle, explants were transitioned to a Stage 2 media, in this Example, b-12-c supplemented with 7 g/L carageenan rather than the 5.5 g/L provided above. Following the third cycle, explants were cleaned. The explants were kept on b-12-c supplemented with 7 g/L carageenan for 10-120 day cycles (usually 21 day cycles) until multiple shoots were observed. Observation of multiple shoots occurred within 3-15 months.
[000219] Once the explant exhibited multiple shoots, it was either maintained on its Stage 2 media or transferred to a Stage 3 media, in this Example b-9-iv at a pH of 5.5 for 10-120 day cycles (usually 21 days) until the desired number of shoots was obtained by separation into new tubes and further expansion. A B-6 media at a pH of 5.5 can also be used. One-ten shoots per tube were obtained per multiplication cycle. The shoots were then placed in a Stage 3 or Stage 4 media, in this Example, Amel-iv media at a pH of 5.7 for 10-120 days (usually 14-21 days).
Example 16. Bambusa Old Hamii - alternate procedure
[000220] In the example of Bambusa Old amii, the explants were chosen and disinfected as in Example 10. The explants were then transferred into boxes containing a Stage 1 media, in this Example, b-10-iv (40-50 mL) also as described in Example 10 but for the change to boxes. These explants stayed on b-10-iv media for 2 10-120 day cycles (usually 21 day cycles). Between cycles, excess sheaths were removed. At the time of transfer to the third cycle, explants were transitioned to a Stage 2 media, in this Example, b-10-c supplemented with 7 g/L carageenan rather than the 5.5 g/L provided above. Following the third cycle, explants were cleaned. The explants were kept on b-10-c supplemented with 7 g/L carageenan for 10-120 day cycles (usually 21 day cycles) until multiple shoots were observed. Observation of multiple shoots occurred within 3-15 months. Cultures were maintained on Stage 2 media until the desired number of shoots was obtained. One-twenty shoots per box were obtained per multiplication cycle. The shoots were then placed in a Stage 3 media, in this Example, Amel-iv at a pH of 5.7 for 10-120 days (usually 14-21 days).
Example 17. Phyllostachys Moso, Phyllostachys Atrovaginata & Dendrocalamus Asper - alternate procedure
[000221] In the example of Phyllostachys Moso, Phyllostachys Atrovaginata and Dendrocalamus Asper, the explants were chosen and disinfected as in Example 10. The explants were then transferred into tubes containing a Stage 1 media, in this Example, b-12c-iv also as described in Example 10. These explants stayed on b-12c-iv media for 2 10-120 day cycles (usually 21 day cycles). Between cycles, excess sheaths were removed. At the time of transfer to the third cycle, explants were transitioned to a Stage 2 media, in this Example, b-12-c supplemented with 7 g/L carageenan rather than the 5.5 g/L provided above. Following the third cycle, explants were cleaned. The explants were kept on b-12-c supplemented with 7 g/L carageenan for 10-120 day cycles (usually 21 day cycles) until multiple shoots were observed. Observation of multiple shoots occurred within 3-15 months.
[000222] Once the explant exhibited multiple shoots, it was either maintained on its Stage 2 media or transferred to a Stage 3 media, in this Example b-9-iv at a pH of 5.5 for 10-120 day cycles (usually 21 day cycles) until the desired number of shoots was obtained by separation into new tubes and further expansion. A B-6 media at a pH of 5.5 can also be used. One-ten shoots per tube were obtained per multiplication cycle. The shoots were then placed in a Stage 3 or Stage 4 media, in this Example, Amel-iv at a pH of 5.7 for 10-120 days (usually 14-21 days).
Example 18 Guadua Angustifolia - alternate procedure [000223] In the example of Guadua Angustofolia, the explants were chosen and disinfected as in Example 10. The explants were then transferred into tubes containing a Stage 1 media, in this Example, b-12c-iv also as described in Example 10. These explants stayed on b-12c-iv media for 2 10-120 day cycles (usually 21 day cycles). Between cycles, excess sheaths were removed. At the time of transfer to the third cycle, explants were transitioned to a Stage 2 media, in this Example, b-12-c supplemented with 7 g/L carageenan rather than the 5.5 g/L provided above. Following the third cycle, explants were cleaned. The explants were kept on b-12-c supplemented with 7 g/L carageenan for 10-120 day cycles (usually 21 day cycles) until multiple shoots were observed. Observation of multiple shoots occurred within 3-15 months.
[000224] Once the explant exhibited multiple shoots, it was either maintained on its Stage 2 media or transferred to a Stage 3 media, in this Example b-10- iv at a pH of 5.5 for 10-120 day cycles (usually 21 day cycles) until the desired number of shoots was obtained by separation into new tubes and further expansion. One-ten shoots per tube were obtained per multiplication cycle. The shoots were then placed in a Stage 3 or Stage 4 media, in this Example, Amel-iv rooting media at a pH of 5.7 for 10-120 days (usually 14-21 days). Example 19. Grass - Hakonechloa
[000225] Explants were from new shoots starting to rise from the ground in the spring. The new shoots were 1 -2 cm long when harvested. They were cleaned and cut down to 5-10 mm. They were then placed in a 10% bleach solution for 35 minutes. Following this first disinfection, the explants were further cut to 3-5 mm and then placed in a 10% bleach solution for another 35 minutes. Explants were then rinsed in a 1 % bleach solution for 30 minutes 2 times.
[000226] Plantlets were cycled every 10-60 days (usually every 42 days) into fresh media. Once the desired number of plantlets has been formed through separation and expansion in new tubes the plantlets were placed on a Stage 2 media, in this Example, FS2 at a pH of 5.3-6.0 for 10-120 days (usually 28 days) under normal culture room conditions including 65°F - 70°F at full spectrum 200 - 500 foot candles.
Example 20. Food Crop - Cabbage [000227] The explants were from the dormant lateral buds. The buds were removed from the stems by cutting a wedge out of the stem with the bud attached. The explant was placed in a 10% bleach solution for 30 minutes. The wedge was removed from the bleach and the bud was cleaned of any stem pieces. The outer layer of sheath was removed resulting in an explant of 2-4 mm. This explant was then placed into a 10% bleach solution for another 30 minutes. Explants were then rinsed in a 1 % bleach solution for 30 minutes 2 times.
[000228] Explants were then placed in tubes containing a Stage 1 media, in this Example, R2 at a pH of 5.7 for 10-120 day cycles (usually 21 day cycles) until the
desired number of plantlets was reached through separation and expansion in new tubes. Plantlets where then transferred to a Stage 2 media, in this Example, FS3 for 10-120 days (usually 21 days).
Example 21. Perennial - Geranium
[000229] Explants vary according to the variety of geranium. One explant type was immature flower bud taken as it appeared on the plant. These were cleaned of the outer sheath with a little of the stem attached. The explants were placed in a 10% bleach solution for 30 minutes. They were then rinsed in a 1 % bleach solution for 30 minutes 2 times. Following these rinses the explants were cut down further.
[000230] A second explant type was the crown of the geranium obtained by cutting the crown out and cleaning it down to 1 -2 cm. This explant was placed in a 10% bleach solution for 30 minutes. It was then removed, cleaned further and cut down to 5-10 mm. The explants were then placed back in a 10% bleach solution for another 30 minutes and then rinsed in a 1 % bleach solution for 30 minutes 2 times.
[000231] Explants were then placed in tubes containing a Stage 1 media, in this Example R3 at a pH of 5.7 for 10-120 day cycles (usually 28 day cycles) until the desired number of plantlets is reached through separation and expansion into new tubes. The plantlets were then placed on a Stage 2 media, in this Example, FS4 for 10-120 days (usually 28 days).
Example 22. Perennial - Trillium [000232] Explants were from the tips of the rhizomes in late winter just before emergence from the ground. The growing part of the rhizome was cleaned and cut down to 1 cm. This material was placed in a 10% bleach solution for 30 minutes. The tip was then removed and cleaned and cut down further to 5- 6 mm. It was then placed in a 10% bleach solution for another 30 minutes, after which it was rinsed in a 1 % bleach solution for 30 minutes 2 times.
[000233] Explants were then placed in tubes containing a Stage 1 media, in this Example, R4 at a pH of 5.7 for 10-60 day cycles (usually 42 day cycles) until the desired number of plantlets was reached through separation and expansion into new tubes. Bulbs were transferred to a Stage 2 media, in this Example, FS1 at a pH of 5.0-6.0 in the dark for 10-60 days (usually 42 days) at 70°F.
[000234] As will be understood by one of ordinary skill from the provided examples, the tissue culturing method for individual species includes slight variations in media, timing and growth conditions. These variations for individual species require optimization based on factors including location, desired outcome, starting material, etc.
[000235] For each of the species provided in the examples listed above, it particular embodiments, each can be initiated and/or multiplied in b-9-i media, b-9-ii media, b-9-iii media, b-9-iv media, b-9-v media, CW2-i media, CW2-N media, CW2-iii media, CW2-iv media, CW2-v media, b-10-i media, b-10-ii media, b-10-iii media, b-10-iv media, b-10-v media, b-1 1 -i media, b-1 1 -ii media, b-1 1 -iii media, b-1 1 -iv media, b-1 1 -v media, b-12c-i media, b-12c-ii media, b-12c-iii media, b-12c-iv media, b-12c-v media, b-1 -i media, b-1 -ii media, b-1 -iii media, b-1 -iv media, b-1 -v media, b-4-i media, b-4-ii media, b-4- iii media, b-4-iv media, b-4-v media, b-6-i media, b-6-ii media, b-6-iii media, b- 6-iv media, b-6-v media, CW1 -i media, CW1 -N media, CW1 -iii media, CW1 -iv media, CW1 -v media, CW3-i media, CW3-N media, CW3-iii media, CW3-iv media, CW3-v media, CW4-i media, CW4-N media, CW4-iii media, CW4-iv media, CW4-v media, CW5-i media, CW5-N media, CW5-iii media, CW5-iv media, CW5-v media, CW6-i media, CW6-N media, CW6-iii media, CW6-iv media, CW6-v media, R1 -i media, R1 -N media, R1 -iii media, R1 -iv media, R1 - v media, R2-i media, R2-N media, R2-iii media, R2-iv media, R2-v media, R3-i media, R3-N media, R3-iii media, R3-iv media, R3-v media, R4-i media, R4-ii media, R4-iii media, R4-iv media or R4-v media.
[000236] As used herein "in" and "on" are interchangeable in the context of placing explants, shoots or plantlets within a tube, jar, box or jug containing a media.
[000237] As will be understood by one of ordinary skill from the provided examples, the tissue culturing method for individual species includes slight variations in media, timing and growth conditions. These variations for individual species require optimization based on factors including location, desired outcome, starting material, etc. As long as meta-topolin is included in the media used, however, successful commercial scale production following optimization will be achieved.
[000238] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification, including the embodiments, and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
[000239] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
[000240] As used herein "in" and "on" are interchangeable in the context of placing explants, shoots or plantlets within a tube, jar, box or jug containing a media.
[000241] The terms "a," "an," "the" and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[000242] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
[000243] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. [000244] Specific embodiments disclosed herein may be further limited in the claims using consisting of or and consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term "consisting of excludes any element, step, or ingredient not specified in the claims. The transition term "consisting essentially of limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.
[000245] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

Claims

CLAIMS We claim:
1 . A method of micropropagating a monocot plant comprising initiating a shoot in vitro from a monocot plant explant on a first media, and multiplying the shoot initiated from the explant in vitro on a second media, wherein the first media and/or the second media comprises (1 ) meta-topolin (mT) or analog thereof, (2) thidiazuron (TDZ) or analog thereof, (3) benzylaminopurine (BAP), and (4) β-naphthoxyacetic acid (NAA).
2. The method of claim 1 , wherein the monocot plant is a plant of a genus selected from the group consisting of Agave, Amaranthus, Ananas, Calamus, Geranium, Gladiolus, Hakonechloa, Iridaceae, Lilium, Miscanthus, Musa, Orchidaceae, Phoenix (dates), Saccharum, Trillium and Zea.
3. The method of claim 1 , wherein the explant is selected from the group consisting of internode, node, stolon, shoot, dormant lateral bud, immature flower bud, crown, rhizome and parts thereof.
4. The method of claim 1 , wherein at least three in vitro shoots are obtained from the explant following multiplication.
5. The method of claim 4, wherein the method further comprises separating and individually transferring the at least three in vitro shoots to fresh first media or fresh second media.
6. The method of claim 1 , wherein the method further comprises two or more cycles of growing the in vitro shoots on fresh first media and then on fresh second media.
7. The method of claim 1 , wherein the method further comprises transferring the multiplied in vitro shoots to a third media that supports transition to ex vitro conditions.
8. A method of micropropagating a monocot plant comprising initiating a shoot in vitro from a monocot plant explant on a first media comprising (1 ) meta-topolin (mT) or analog thereof, (2) thidiazuron (TDZ) or analog thereof, (3) benzylaminopurine (BAP), and (4) β-naphthoxyacetic acid (NAA); and multiplying the in vitro shoot on a second media comprising (1 ) meta-topolin (mT) or analog thereof, (2) benzylaminopurine (BAP), and (3) β- naphthoxyacetic acid (NAA).
9. The method of claim 8, wherein the monocot plant is a plant of a genus selected from the group consisting of Agave, Amaranthus, Ananas, Calamus, Geranium, Gladiolus, Hakonechloa, Iridaceae, Lilium, Miscanthus, Musa, Orchidaceae, Phoenix (dates), Saccharum, Trillium and Zea.
10. The method of claim 8, wherein the explant is selected from the group consisting of internode, node, stolon, shoot, dormant lateral bud, immature flower bud, crown, rhizome and parts thereof.
1 1 . The method of claim 8, wherein at least three in vitro shoots are obtained from the explant following multiplication.
12. The method of claim 1 1 , wherein the method further comprises separating and individually transferring the at least three in vitro shoots to fresh first media or fresh second media.
13. The method of claim 8, wherein the method further comprises two or more cycles of growing the in vitro shoots on fresh first media and then on fresh second media.
14. The method of claim 8, wherein the method further comprises transferring the multiplied in vitro shoots to a third media that supports transition to ex vitro conditions.
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