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(12) STANDARD PATENT (11) Application No. AU 2016244230 B2 (19) AUSTRALIAN PATENT OFFICE

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(12) STANDARD PATENT (11) Application No. AU 2016244230 B2 (19) AUSTRALIAN PATENT OFFICE (12) STANDARD PATENT (11) Application No. AU 2016244230 B2 (19) AUSTRALIAN PATENT OFFICE (54) Title COMPOSITIONS, METHODS, AND SYSTEMS FOR MICROPROPAGATION OF PLANTS (51) International Patent Classification(s) C12N 5/04 (2006.01) A01C 1/00 (2006.01) (21) Application No: 2016244230 (22) Date of Filing: 2016.10.12 (43) Publication Date: 2016.10.27 (43) Publication Journal Date: 2016.10.27 (44) Accepted Journal Date: 2018.07.19 (62) Divisional of: 2012287184 (71) Applicant(s) Amplex Bioresources, LLC (72) Inventor(s) Burr, Randall W.;Heinricher, Jackie R.;Victor, Jerrin (74) Agent / Attorney Pizzeys Patent and Trade Mark Attorneys Pty Ltd, GPO Box 1374, BRISBANE, QLD, 4001, AU (56) Related Art US 2002/0086425 Al ABSTRACT The present invention provides compositions, methods, and systems for achieving very high multiplication rate of plants in vitro micropropagation. WO 2013/016198 PCT/US2012/047622 COMPOSITIONS, METHODS AND SYSTEMS FOR MICROPROPAGATION OF PLANTS CROSS-REFERENCE TO RELATED APPLICATIONS 5 This application claims priority to and the benefit of U.S. Provisional Patent Application Serial Nos. 61/510,955, filed July 22, 2011; 61/514,331, filed August 2, 2011; 61/515,735, filed August 05, 2011; 61/523,162, filed August 12, 2011; 61/523,205, filed August 12, 2011; 61/552,834, filed August 28, 2011; 61/607,838, filed March 7, 2012; and 61/618,344, filed March 30, 2012, each of which is hereby incorporated by reference in their 0 entireties for all purposes. TECHNICAL FIELD The present invention relates generally to compositions, methods, and systems for plant propagation. In some embodiments, the present invention provides compositions, 5 methods, and systems for the micropropagation of plants. BACKGROUND With increasing burdens on land to produce food and biomass for energy and materials additional attention is being placed on identifying and utilizing faster growing and '0 more productive plants. Although many plants are suitable for such purposes, there is still a great need to develop compositions, methods, and systems for fast, economical plant propagation. SUMMARY OF THE INVENTION 25 The present invention provides compositions, methods and systems for plant tissue culture, for example, compositions, methods and systems for plant micropropagation. In some embodiments, the compositions, methods and systems are used for plant in vitro micropropagation. Media are provided for plant in vitro micropropagation. In some embodiments, bud 30 induction media and shoot elongation/maintenance media are provided. In some embodiments, the bud induction media comprise one or more strong cytokinins, and the shoot elongation/maintenance media comprise one or more relatively weaker cytokinins. In some embodiments, the bud induction media comprise thidiazuron (TDZ) or analog thereof, and 1 WO 2013/016198 PCT/US2012/047622 the elongation and maintenance media comprise one or more cytokinins other than TDZ or an analog thereof. In some embodiments, the cytokinins other than TDZ are selected from the group consisting of N6-benzylaminopurine (BAP), meta-topolin (mT), zeatin, zeatin riboside, dihydrozeatin, kinetin, isopentenyladenine (ip, e.g., 2ip), adenine hemisulfate, 5 dimethylallyladenine, N-(2-chloro-4-pyridyl)-N'- phenylurea) (4-CPPU), and analogs thereof. In some embodiments, the media can be used for plants in vitro micropropagation of monocots or dicots. In some embodiments, the media can be used for bamboo plants in vitro micropropagation. In some embodiments, the bud induction medium comprises an effective amount of 0 thidiazuron (TDZ) or analog thereof, and wherein the shoot elongation/maintenance medium comprises an effective amount of one or more cytokinins other than TDZ or an analog thereof. In some embodiments, the one or more cytokinins other than TDZ or an analog thereof in the shoot elongation/maintenance medium is selected from the group consisting of N6 benzylaminopurine (BAP), meta-topolin (mT), zeatin, zeatin riboside, dihydrozeatin, kinetin, 5 2-isopentenyladenine (2ip), adenine hemisulfate, dimethylallyladenine, N-(2-chloro-4 pyridyl)-N'- phenylurea) (4-CPPU), and analogs thereof. In some embodiments, the concentration of TDZ or analog thereof in the bud induction medium is effective to induce shoot buds. In some embodiments, the concentration of TDZ or analog in the bud induction media thereof is about 0.25 mg/L to about 100 mg/L, '0 for example, about 0.5 mg/L to about 2 mg/L. Thus, the concentration of TDZ or analog thereof in the bud induction media can, for example, be about 0.25 mg/L, about 0.3 mg/L, about 0.4 mg/L, about 0.5 mg/L, about 0.6 mg/L, about 0.7 mg/L, about 0.8 mg/L, about 0.9 mg/L, about 1.0 mg/L, about out 1.5 mg/L, about 2.0 mg/L, about 2.5 mg/L, about 3 mg/L, about 4 mg/L, about 5 mg/L, about 6 mg/L, about 7 mg/L, about 8 mg/L, about 9 mg/L, about 25 10 mg/L, about 15 mg/L, about 20 mg/L, about 25 mg/L, about 30 mg/L, about 40 mg/L, about 50 mg/L, about 60 mg/L, about 70 mg/L, about 80 mg/L, about 90 mg/L or about 100 mg/L. In some embodiments, the concentration of the one or more cytokinins other than TDZ or an analog thereof in the shoot elongation/maintenance medium is effective to 30 elongate shoots. In some embodiments, the concentration of the one or more cytokinins other than TDZ or an analog thereof in the shoot elongation/maintenance medium from about 0.01 mg/L to about 100 mg/L, for example, from about 0.25 mg/L to about 5 mg/L. Thus, the concentration of one or more cytokinins other than TDZ or analog thereof in the shoot 2 WO 2013/016198 PCT/US2012/047622 elongation/maintenance media can, for example, be about 0.01 mg/L, about 0.02 mg/L, about 0.03 mg/L, about 0.04 mg/L, about 0.05 mg/L, about 0.06 mg/L, about 0.07 mg/L, about 0.08 mg/L, about 0.09 mg/L, about 0.10 mg/L, about 0.15 mg/L, about 0.20 mg/L, about 0.25 mg/L, about 0.3 mg/L, about 0.35 mg/L, about 0.4 mg/L, about 0.45 mg/L, about 0.5 mg/L, 5 about 0.6 mg/L, about 0.7 mg/L, about 0.8 mg/L, about 0.9 mg/L, about 1.0 mg/L, about 1.5 mg/L, about 2.0 mg/L, about 2.5 mg/L, about 3 mg/L, about 4 mg/L, about 5 mg/L, about 6 mg/L, about 7 mg/L, about 8 mg/L, about 9 mg/L, about 10 mg/L, about 15 mg/L, about 20 mg/L, about 25 mg/L, about 30 mg/L, about 40 mg/L, about 50 mg/L, about 60 mg/L, about 70 mg/L, about 80 mg/L, about 90 mg/L or about 100 mg/L. 0 In some embodiments, the bud induction medium and/or the shoot elongation/maintenance medium further comprise one or more auxins. In some embodiments, the auxins are selected from the group consisting of -naphthoxyacetic acid (NAA), 2,4 Dichlorophenoxy acetic acid (2,4-D), indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), picloram, and analogs of each thereof. For example, the auxin is NAA or analogs thereof. 5 In some embodiments, the bud induction medium is a liquid medium. In some embodiments, the bud induction medium is a solid medium. In some embodiments, the shoot elongation/maintenance medium is a liquid medium. In some embodiments, the shoot elongation/maintenance media is a solid media. Also provided are methods of in vitro micropropagation of plants. In some '0 embodiments, the methods are used for micropropagating plants in vitro. In some embodiments, the methods comprise (a) incubating a plant tissue culture, explant or seed in a first medium, and (b) then incubating the plant tissue obtained from step (a) in a second medium. In some embodiments, the first medium is a bud induction medium, and the second medium is a shoot elongation and maintenance medium. 25 In some embodiments, when a bud induction medium and a shoot elongation and maintenance medium are used, the methods comprise: (a) incubating a plant tissue culture, explant or seed in a bud induction medium to induce shoot bud formation; and (b) incubating the shoot buds obtained in step (a) in a shoot elongation/maintenance medium. In some embodiments, when a bud induction medium and a shoot elongation and 30 maintenance medium are used, the methods further comprise: (c) incubating the shoots from step (b) in a bud induction medium to induce shoot bud formation; and (d) incubating the shoot buds obtained in step (c) in a shoot elongation/maintenance medium. 3 WO 2013/016198 PCT/US2012/047622 In some embodiments, when a bud induction medium and a shoot elongation and maintenance medium are used, the methods further comprise: (e) repeating the incubating step (c) and step (d) for at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or more additional cycles. There is no limit to how 5 many times the cycling of step (c) and step (d) can be repeated. Buds and/or shoots obtained in step (a) or step (c) can be separated prior to the buds and/or shoots entering step (b) or step (d), respectively, wherein such separation can result in a single bud or shoot, 2 buds and/or shoots, 3 buds and/or shoots, or 4 or more buds and/or shoots per separation.
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