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WO 2019/006466 Al 03 January 2019 (03.01.2019) W !P O PCT

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WO 2019/006466 Al 03 January 2019 (03.01.2019) W !P O PCT (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2019/006466 Al 03 January 2019 (03.01.2019) W !P O PCT (51) International Patent Classification: (71) Applicant: BOOSHOOT LLC [US/US]; P.O. Box 3609, A 01C 1/02 (2006 .0 1) A 01H 6/28 (20 18.0 1) Hailey, Idaho 83333 (US). A01G 31/02 (2006 .0 1) C12N 5/04 (2006 .01) (72) Inventor: HEINRICHER, Jackie; c/o BOOSHOOT LLC, A01G 31/06 (2006.01) P.O. Box 3609, Hailey, Idaho 83333 (US). (21) International Application Number: (74) Agent: VEITENHEIMER, Erich et al; COOLEY LLP, PCT/US2018/040637 1299 Pennsylvania Avenue, N.W., Suite 700, Washington, (22) International Filing Date: District of Columbia 20004-2400 (US). 02 July 2018 (02.07.2018) (81) Designated States (unless otherwise indicated, for every (25) Filing Language: English kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, (26) Publication Language: English CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, (30) Priority Data: DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, 62/527,946 30 June 2017 (30.06.2017) US HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, 62/61 1,858 29 December 2017 (29.12.2017) US KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (54) Title: COMPOSITIONS AND METHODS FOR LARGE-SCALE IN VITRO PLANT BIOCULTURE F G. 1 Shoot tip necrosis in pistachio plant cultured in vitro (B0012 iu (57) Abstract: The present invention provides media, kits, systems, and methods for achieving (1) large scale pistachio production within a short time via bioculture, (2) large scale yam production within a short time via bioculture, (3) high multiplication rate of plants including cannabis via in vitro micropropagation, (4) high induction rates of somatic embryos from later buds in bamboo, (5) reduced production of phenolic compounds in plants, (6) high production of virus-free plants, including potato, and (7) large scale hemp o production via culturing. The present invention for pistachio and yam production results in shorter tuber development phase and higher © yield. In some embodiments, the present invention provides compositions, methods, and systems for the micropropagation and mass production of perennials, grasses, bamboos, cannabis and phyto-pharmaceutical plants as well as hemp plants. In some embodiments, e present invention provides compositions, methods, and systems for reducing the production of a phenolic by a plant, such as bamboo. [Continued on nextpage] WO 2019/006466 Al llll II II 11III II I II II II i II III II I II SC, SD, SE, SG, SK, SL, SM, ST, SV, SY,TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). Declarations under Rule 4.17: — as to applicant's entitlement to apply for and be granted a patent (Rule 4.1 7(H)) — as to the applicant's entitlement to claim the priority of the earlier application (Rule 4.17(in)) Published: — with international search report (Art. 21(3)) — before the expiration of the time limit for amending the claims and to be republished in the event of receipt of amendments (Rule 48.2(h)) COMPOSITIONS AND METHODS FOR LARGE-SCALE IN VITRO PLANT BIOCULTURE CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of priority to U.S. Provisional Application No. 62/527,946, filed June 30, 2017; and U.S. Provisional Application No. 62/611,858, filed December 29, 2017; each of which is herein incorporated by reference in its entirety. TECHNICAL FIELD [0002] This invention provides compositions, systems, and methods for efficient, rapid and large scale production of plants using bioculture in vitro. In some embodiments, the present invention provides compositions, methods, and systems for the micropropagation and mass production of perennials, grasses, monocots, dicots, and phyto-pharmaceutical plants. In some embodiments, the present invention provides compositions, methods, and systems for the production of virus-free plants. BACKGROUND [0003] This invention generally relates to a method of rapid regeneration/proliferation of plant tissues. More particularly, it relates to an improved method of plant tissue proliferation which comprises cuituring a tissue or an organ of a plant, a part of the same or cultured cells to proliferate the tissue, organ or cultured cells, thereby regenerating a plant body or producing a useful substance formed by that plant, for the purposes of rapidly generating plants. [0004] The state of the art s such that the demand for plants and plant products far outweight the availability possible with the techniques known in the art. There exists a clear nead in the art for the rapid proliferation of viable plants, and the present application seeks to meet the demand. SUMMARY OF THE INVENTION [0005] The present invention provides compositions, methods, kits, bioreactors, and systems for efficient and rapid propagation of pistachio plants at a large scale via bioculture. [0006] In some embodiments, the present invention describes an automated, or semi-automated, low-cost system for the production of pistachio plants, which significantly increases the quantity and quality of pistachio plants, the number and size of the resulting plants, reduces the cost and shortens the cultivation time. [0007] This invention provides novel compositions and an efficient and rapid system for mass propagation of pistachio plants in vitro. [0008] In one embodiment, the present invention provides media for plant micropropagation. In some further embodiments, the media are used for micropropagation of pistachio plants. [0009] In some embodiments, the media are initiation media, multiplication media, and rooting media, such as the BOOS, B004, B005, B006, B007, BOOS, B009, BOOH, BOOK), B0013, B0014, B0015, B0016, combination thereof, or functional equivalents thereof (e.g., by reducing or increasing one or more component concentration, or by adding or removing one or more component, wherein the media maintain the same function). As used herein, the media named "BOO" is equivalent to "BOOS." For example, the media of the present invention are referred to herein as a "BOOS, BQ04, BOOS, B006, BOOT, BOOS, B009, BOOH, BOOIO, B0013, B0014, B0015, BOO 16, etc.", which are also known as (a.k.a) "BOOS3, BOOS4, BOOS5, BOOS6, BOOS7, BOOS8, BOOS9, BOOS . BOOS . BOOS13, BOQS14, BOOS15, BOOS16, etc.", respectively. Therefore, the media desginated as "BOO" herein is interchangeably used as "BOOS" in the present invention. [00 Θ] In some embodiments, the initiation media comprise Murashige & Skoog (MS) salts, Woody Plant (WPM) tissue culture salts, and/or Driver Kuniyukt Walnut (DKW ) tissue culture salts, and sucrose. In some embodiments, the concentration of one or more components in the MS, WPM, or DKW salts is modified. In some embodiments, the sucrose concentration is about 25 to 35 g/L, for example, about 30 g L. [0011] In some embodiments, the initiation media comprises at least one cytokinm. In some embodiments, the initiation media further comprises at least one auxin. [0012] In some embodiments, the cytokinm is meta-topolin (raT) or functional derivatives thereof. In some embodiments, the mT concentration in the initiation media is about 0.1 to 30 mg/L, for example, about 1-3 mg/L. [0013] In some embodiments, the auxin is Naphthaleneacetic acid or functional derivatives thereof. In some embodiments, the NAA concentration in the initiation media is about 0.01 to 1 mg/L, for example, about 0. mg/L. [0014] In some embodiments, the auxin is IBA or functional derivatives thereof. In some embodiments, the IBA concentration in the initiation media is about 0.01 to 1 mg/L, for example, about 0. mg/'L. [0015] In some embodiments, the media further comprises a gibberellin acid. In some embodiments, the gibberellin acid is GAS or functional derivatives thereof. In some embodiments, the gibberellin acid concentration in the initiation media is about 0.2 to 20 mg/'L, for example, about 2 mg/'L. [0016] In some embodiments, the initiation media are liquid, semi-liquid, solid, or semi-solid media. In some embodiments, the initiation media comprise about 4 to about 10 grams gelling agent, such as agar. [0017] In some embodiments, the initiation media has a p of about 5.0 to 6.0, for example, about 5.7. [0018] In some embodiments, the multiplication media are similar to, or as the same as the initiation media. [0019] In some embodiments, the rooting media comprise Murashige & Skoog (MS) salts. Woody Plant (WPM) tissue culture salts, and/or Driver Kuniyuki Walnut (DKW) tissue culture salts, and sucrose. In some embodiments, the sucrose concentration is about 25 to 35 g/L, for example, about 30 g/L. In some embodiments, the sucrose concentration is much higher, for example, at least 60 g/L, such as about 60 g/L to about 20 g/L, In some embodiments, the rooting media comprises at least one auxin in some embodiments, the rooting media do not comprise any cytokinin.
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