TW201321503A - Compositions, methods and systems for micropropagation of plants - Google Patents

Abstract

The present invention provides compositions, methods, and systems for achieving very high multiplication rate of plants in vitro micropropagation.

Description

植物微體繁殖之組成物、方法及系統 Composition, method and system for plant micropropagation

本發明整體而言係關於植物繁殖之組成物、方法及系統。在一些具體例中，本發明係提供用於植物微體繁殖(micropropagation)之組成物、方法和系統。 The present invention is generally directed to compositions, methods, and systems for plant propagation. In some embodiments, the invention provides compositions, methods, and systems for plant micropropagation.

(相關申請案之相互參照) (Reciprocal reference of related applications)

本案主張下列優先權及利益：2011年7月22日申請之美國臨時專利申請第61/510,955號；2011年8月2日申請之第61/514,331號；2011年8月5日申請第61/515,735號；2011年8月12日申請之第61/523,162號；2011年8月12日申請之第61/523,205號；2011年8月28日申請之第61/552,834號；2012年3月7日申請之第61/607,838號；及2012年3月30日申請之第61/618,344號，其各以參照方式全部併入本文用於所有目的。 The case claims the following priorities and benefits: US Provisional Patent Application No. 61/510,955, filed on July 22, 2011; No. 61/514,331, filed on August 2, 2011; Application No. 61/ on August 5, 2011 No. 515, 523; No. 61/523, 162, filed on August 12, 2011; No. 61/523, 205, filed on August 12, 2011; No. 61/552,834, filed on August 28, 2011; March 7, 2012 Japanese Application No. 61/607,838; and No. 61/618,344, filed on Mar.

隨著土地生產用於能量和原料的食物和生質的負擔增加，更多注意力被放在識別和利用生長更快和更多產的植物上。雖然有很多植物適用於這類目的，但對於開發快速、經濟的植物繁殖組成物、方法及系統仍存在極大的需求。 As the burden of land production for food and biomass for energy and raw materials increases, more attention is placed on identifying and utilizing plants that grow faster and produce more. Although many plants are suitable for this purpose, there is still a great need to develop rapid, economical plant propagation compositions, methods and systems.

本發明提供用於植物組織培養的組成物、方法及系統，例如植物微體繁殖的組成物、方法及系統。在一些具體例中， 所述組成物、方法及系統係用於植物體外微體繁殖。 The present invention provides compositions, methods and systems for plant tissue culture, such as compositions, methods and systems for plant micropropagation. In some specific examples, The compositions, methods, and systems are useful for in vitro micropropagation of plants.

提供用於植物體外微體繁殖的培養基，在一些具體例中，提供芽誘導培養基(bud induction media)及苗伸長/維持培養基(shoot elongation/maintenance media)。在一些具體例中，芽誘導培養基包含一種或多種強細胞***素，且苗伸長/維持培養基包含一種或多種相對弱的細胞***素。在一些具體例中，芽誘導培養基包含噻苯隆(thidiazuron，TDZ)或其類似物，且苗伸長及維持培養基包含除TDZ或其類似物之外的一種或多種細胞***素。在一些具體例中，所述除TDZ之外的細胞***素係選自由N6-苄胺基嘌呤(BAP)、間拓樸林(meta-topolin，mT)、玉米素(zeatin)、玉米素核苷(zeatin riboside)、二氫玉米素(dihydrozeatin)、激動素、異戊烯腺嘌呤(ip，如2ip)、腺嘌呤半硫酸鹽(adenine hemisulfate)、二甲基烯丙基腺嘌呤(dimethylallyladenine)、N-(2-氯-4-吡啶基)-N'-苯基脲(4-CPPU)，及其類似物所組成之群。在一些具體例中，所述培養基可用於單子葉植物或雙子葉植物的植物體外微體繁殖。在一些具體例中，所述培養基可用於竹植物的體外微體繁殖。 A medium for in vitro micropropagation of plants is provided, and in some embodiments, a bud induction medium and a shoot elongation/maintenance media are provided. In some embodiments, the shoot induction medium comprises one or more strong cytokinins and the shoot elongation/maintenance medium comprises one or more relatively weak cytokinins. In some embodiments, the shoot induction medium comprises thidiazuron (TDZ) or an analog thereof, and the shoot elongation and maintenance medium comprises one or more cytokinins in addition to TDZ or an analog thereof. In some embodiments, the cytokinin other than TDZ is selected from the group consisting of N6-benzylaminopurine (BAP), meta-topolin (mT), zeatin, and zeatin nucleus. Zeatin riboside, dihydrozeatin, kinetin, isopentenyl adenine (ip, such as 2ip), adenine hemisulfate, dimethylallyladenine a group consisting of N-(2-chloro-4-pyridyl)-N'-phenylurea (4-CPPU), and analogs thereof. In some embodiments, the medium can be used for in vitro micropropagation of monocotyledonous or dicotyledonous plants. In some embodiments, the medium can be used for in vitro micropropagation of bamboo plants.

在一些具體例中，所述芽誘導培養基包含有效量的噻苯隆(TDZ)或其類似物，且其中所述苗伸長/維持培養基包含有效量的除TDZ或其類似物之外的一種或多種細胞***素。在一些具體例中，所述苗伸長/維持培養基中一種或多種除 TDZ或其類似物之外的細胞***素選自由N6-苄胺基嘌呤(BAP)、間拓樸林(mT)、玉米素、玉米素核苷、二氫玉米素、激動素、2-異戊烯腺嘌呤(2ip)、腺嘌呤半硫酸鹽、二甲基烯丙基腺嘌呤、N-(2-氯-4-吡啶基)-N'-苯基脲(4-CPPU)，及其類似物所組成之群。 In some embodiments, the shoot induction medium comprises an effective amount of thiazolone (TDZ) or an analog thereof, and wherein the shoot elongation/maintenance medium comprises an effective amount of one other than TDZ or an analog thereof or A variety of cytokinins. In some embodiments, one or more of the seedling elongation/maintenance medium The cytokinin other than TDZ or its analog is selected from the group consisting of N6-benzylaminopurine (BAP), metatopia (mT), zeatin, zeatin nucleoside, dihydro zeatin, kinetin, 2-iso Pentylene adenine (2ip), adenine hemisulfate, dimethylallyl adenine, N-(2-chloro-4-pyridyl)-N'-phenylurea (4-CPPU), and a group of analogs.

在一些具體例中，所述芽誘導培養基中TDZ或其類似物的濃度能有效誘導苗芽。在一些具體例中，所述芽誘導培養基中TDZ或其類似物的濃度約0.25 mg/L至約100 mg/L，例如，約0.5 mg/L至約2 mg/L。因此，所述芽誘導培養基中TDZ或其類似物的濃度可為，例如，約0.25 mg/L、約0.3 mg/L、約0.4 mg/L、約0.5 mg/L、約0.6 mg/L、約0.7 mg/L、約0.8 mg/L、約0.9 mg/L、約1.0 mg/L、約1.5 mg/L、約2.0 mg/L、約2.5 mg/L、約3 mg/L、約4 mg/L、約5 mg/L、約6 mg/L、約7 mg/L、約8 mg/L、約9 mg/L、約10 mg/L、約15 mg/L、約20 mg/L、約25 mg/L、約30 mg/L、約40 mg/L、約50 mg/L、約60 mg/L、約70 mg/L、約80 mg/L、約90 mg/L或約100 mg/L。 In some embodiments, the concentration of TDZ or an analog thereof in the shoot induction medium is effective to induce shoot growth. In some embodiments, the concentration of TDZ or an analog thereof in the shoot induction medium is from about 0.25 mg/L to about 100 mg/L, for example, from about 0.5 mg/L to about 2 mg/L. Thus, the concentration of TDZ or an analog thereof in the shoot induction medium can be, for example, 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 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.

在一些具體例中，所述苗伸長/維持培養基中除TDZ或其類似物之外的一種或多種細胞***素的濃度能有效使苗伸長。在一些具體例中，所述苗伸長/維持培養基中除TDZ或其類似物之外的一種或多種細胞***素的濃度為自約0.01 mg/L至約100 mg/L，例如，自約0.25 mg/L至約5 mg/L。 因此，所述苗伸長/維持培養基中除TDZ或其類似物之外的一種或多種細胞***素的濃度可為，例如，約0.01 mg/L、約0.02 mg/L、約0.03 mg/L、約0.04 mg/L、約0.05 mg/L、約0.06 mg/L、約0.07 mg/L、約0.08 mg/L、約0.09 mg/L、約0.10 mg/L、約0.15 mg/L、約0.20 mg/L、約0.25 mg/L、約0.3 mg/L、約0.35 mg/L、約0.4 mg/L、約0.45 mg/L、約0.5 mg/L、約0.6 mg/L、約0.7 mg/L、約0.8 mg/L、約0.9 mg/L、約1.0 mg/L、約1.5 mg/L、約2.0 mg/L、約2.5 mg/L、約3 mg/L、約4 mg/L、約5 mg/L、約6 mg/L、約7 mg/L、約8 mg/L、約9 mg/L、約10 mg/L、約15 mg/L、約20 mg/L、約25 mg/L、約30 mg/L、約40 mg/L、約50 mg/L、約60 mg/L、約70 mg/L、約80 mg/L、約90 mg/L或約100 mg/L。 In some embodiments, the concentration of one or more cytokinins in the shoot elongation/maintenance medium other than TDZ or an analog thereof is effective to elongate the shoot. In some embodiments, the concentration of one or more cytokinins in the shoot elongation/maintenance medium other than TDZ or an analog thereof is from about 0.01 mg/L to about 100 mg/L, for example, from about 0.25. From mg/L to about 5 mg/L. Thus, the concentration of one or more cytokinins in the shoot elongation/sustainment medium other than TDZ or an analog thereof may be, for example, about 0.01 mg/L, about 0.02 mg/L, about 0.03 mg/L, About 0.04 mg/L, about 0.05 mg/L, about 0.06 mg/L, about 0.07 mg/L, about 0.08 mg/L, about 0.09 mg/L, about 0.10 mg/L, about 0.15 mg/L, about 0.20 Mg/L, about 0.25 mg/L, about 0.3 mg/L, about 0.35 mg/L, about 0.4 mg/L, about 0.45 mg/L, about 0.5 mg/L, about 0.6 mg/L, about 0.7 mg/ L, about 0.8 mg/L, about 0.9 mg/L, about 1.0 mg/L, about 1.5 mg/L, about 2.0 mg/L, about 2.5 mg/L, about 3 mg/L, about 4 mg/L, About 5 mg/L, about 6 mg/L, about 7 mg/L, about 8 mg/L, about 9 mg/L, about 10 mg/L, about 15 mg/L, about 20 mg/L, about 25 Mg/L, about 30 mg/L, about 40 mg/L, about 50 mg/L, about 60 mg/L, about 70 mg/L, about 80 mg/L, about 90 mg/L or about 100 mg/ L.

在一些具體例中，所述芽誘導培養基及/或所述苗伸長/維持培養基進一步包含一種或多種生長素。在一些具體例中，所述生長素選自由β-萘氧乙酸(NAA)、2,4-二氯苯氧乙酸(2,4-D)、吲哚-3-丁酸(IBA)、吲哚-3-乙酸(IAA)、毒莠定(picloram)，及其各別類似物所組成之群。例如，所述生長素是NAA或其類似物。 In some embodiments, the shoot induction medium and/or the shoot elongation/maintenance medium further comprises one or more auxins. In some embodiments, the auxin is selected from the group consisting of β-naphthyloxyacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-butyric acid (IBA), and hydrazine. A group consisting of indole-3-acetic acid (IAA), picloram, and its various analogs. For example, the auxin is NAA or an analog thereof.

在一些具體例中，所述芽誘導培養基是液體培養基。在一些具體例中，所述芽誘導培養基是固體培養基。在一些具體例中，所述苗伸長/維持培養基是液體培養基。在一些具體例中，所述苗伸長/維持培養基是固體培養基。 In some embodiments, the shoot induction medium is a liquid medium. In some embodiments, the shoot 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 medium is a solid medium.

本發明又提供植物體外微體繁殖的方法。在一些具體例中，所述方法用於體外微體繁殖植物。在一些具體例中，所述方法包括(a)在第一培養基中培養植物組織培養物、培植體(explant)或種子，及(b)然後在第二培養基中培養從步驟(a)獲得的植物組織。在一些具體例中，第一培養基是芽誘導培養基，且第二培養基是苗伸長及維持培養基。 The invention further provides a method of in vitro micropropagation of plants. In some embodiments, the method is for in vitro micropropagation of plants. In some embodiments, the method comprises (a) cultivating a plant tissue culture, an explant or a seed in a first medium, and (b) then cultivating the obtained from the step (a) in the second medium. Plant tissue. In some embodiments, the first medium is a shoot induction medium and the second medium is a shoot elongation and maintenance medium.

在一些具體例中，當使用芽誘導培養基以及苗伸長及維持培養基時，所述方法包括：(a)在芽誘導培養基中培養植物組織培養物、培植體或種子以誘導苗芽形成；及(b)在苗伸長/維持培養基中培養在步驟(a)獲得的苗芽。 In some embodiments, when a shoot induction medium and a shoot elongation and maintenance medium are used, the method comprises: (a) cultivating a plant tissue culture, a culture or a seed in a shoot induction medium to induce shoot formation; and b) cultivating the shoots obtained in step (a) in shoot elongation/maintenance medium.

在一些具體例中，當使用芽誘導培養基以及苗伸長及維持培養基時，所述方法進一步包括：(c)在芽誘導培養基中培養從步驟(b)獲得的苗以誘導苗芽形成；及(d)在苗伸長/維持培養基中培養在步驟(c)獲得的苗芽。 In some embodiments, when the shoot induction medium and the shoot elongation and maintenance medium are used, the method further comprises: (c) cultivating the seed obtained from the step (b) in the shoot induction medium to induce shoot formation; and d) cultivating the shoots obtained in step (c) in shoot elongation/maintenance medium.

在一些具體例中，當使用芽誘導培養基以及苗伸長及維持培養基時，所述方法進一步包括：(e)將培養步驟(c)和步驟(d)重複至少一次、至少兩次、至少三次、至少四次、至少五次、至少六次、至少七次、至少八次，或更多次額外的迴圈。對於步驟(c)和步驟(d)可重複的迴圈次數沒有限制。在步驟(a)或步驟(c)中獲得的芽及/或苗可以在該芽及/或苗分別進入步驟(b)或步驟(d)之前分離，其中這樣的分離每次分離可產生單個芽或苗、2個芽及/或苗、3個芽及/或苗、或4 個或更多芽及/或苗。最佳分離大量、快速生產單一物種、基因型或選殖株的複製竹通常係牽涉將步驟(a)或步驟(c)獲得的芽及/或苗在分別進入步驟(b)或步驟(d)之前分別分成1-3個芽及/或苗。當每次分離有2個或更多個芽及/或苗時，其在此領域係稱為聚叢(clumping)或芽及/或苗「團(clump)」。本發明的方法學中的一些變化可能是必需的，以針對竹的特定物種、基因型或選殖株精細調整方法，且這類方法的變化在本發明的公開之內。 In some embodiments, when the shoot induction medium and the shoot elongation and maintenance medium are used, the method further comprises: (e) repeating the culture step (c) and the step (d) at least once, at least twice, at least three times, At least four times, at least five times, at least six times, at least seven times, at least eight times, or more than one additional loop. There is no limit to the number of repeatable loops for step (c) and step (d). The shoots and/or shoots obtained in step (a) or step (c) may be separated before the shoots and/or shoots respectively enter step (b) or step (d), wherein such separation may result in a single separation Bud or seedling, 2 buds and / or seedlings, 3 buds and / or seedlings, or 4 One or more buds and / or seedlings. Optimal separation of large quantities, rapid production of single species, genotypes or selected strains of replicated bamboo usually involves the buds and/or shoots obtained in step (a) or step (c), respectively, into step (b) or step (d) ) previously divided into 1-3 buds and / or seedlings. When there are 2 or more shoots and/or shoots per separation, it is referred to in this field as clumping or buds and/or shoot clumps. Some variations in the methodology of the invention may be necessary to fine tune the method for a particular species, genotype or selection of bamboo, and variations of such methods are within the disclosure of the present invention.

在一些具體例中，所述芽誘導培養基包含有效量的噻苯隆(TDZ)或其類似物，且其中所述苗伸長/維持培養基包含有效量的一種或多種除TDZ或其類似物之外的細胞***素。在一些具體例中，所述苗伸長/維持培養基中一種或多種除TDZ或其類似物的細胞***素選自由N6-苄胺基嘌呤(BAP)、間拓樸林(mT)、玉米素、玉米素核苷、二氫玉米素、激動素、2-異戊烯腺嘌呤(2ip)、腺嘌呤半硫酸鹽、二甲基烯丙基腺嘌呤、N-(2-氯-4-吡啶基)-N'-苯基脲(4-CPPU)，及其類似物所組成之群。 In some embodiments, the shoot induction medium comprises an effective amount of thiazolone (TDZ) or an analog thereof, and wherein the shoot elongation/sustainment medium comprises an effective amount of one or more other than TDZ or an analog thereof Cytokinin. In some embodiments, one or more cytokinins other than TDZ or an analog thereof in the shoot elongation/maintenance medium are selected from the group consisting of N6-benzylaminopurine (BAP), metatopia (mT), zeatin, Zeatin nucleoside, dihydro zeatin, kinetin, 2-isopentene adenine (2ip), adenine hemisulfate, dimethylallyl adenine, N-(2-chloro-4-pyridyl a group of -N'-phenylurea (4-CPPU), and analogs thereof.

在一些具體例中，所述芽誘導培養基中TDZ或其類似物的濃度能有效誘導苗芽。在一些具體例中，所述芽誘導培養基中TDZ或其類似物的濃度是約0.25 mg/L至約100 mg/L，例如約0.5 mg/L至約2 mg/L。因此，所述芽誘導培養基中TDZ或其類似物的濃度可以是，例如，約0.25 mg/L、 約0.3 mg/L、約0.4 mg/L、約0.5 mg/L、約0.6 mg/L、約0.7 mg/L、約0.8 mg/L、約0.9 mg/L、約1.0 mg/L、約1.5 mg/L、約2.0 mg/L、約2.5 mg/L、約3 mg/L、約4 mg/L、約5 mg/L、約6 mg/L、約7 mg/L、約8 mg/L、約9 mg/L、約10 mg/L、約15 mg/L、約20 mg/L、約25 mg/L、約30 mg/L、約40 mg/L、約50 mg/L、約60 mg/L、約70 mg/L、約80 mg/L、約90 mg/L或約100 mg/L。 In some embodiments, the concentration of TDZ or an analog thereof in the shoot induction medium is effective to induce shoot growth. In some embodiments, the concentration of TDZ or an analog thereof in the shoot induction medium is from about 0.25 mg/L to about 100 mg/L, such as from about 0.5 mg/L to about 2 mg/L. Therefore, the concentration of TDZ or an analog thereof in the bud induction medium may be, for example, 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 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.

在一些具體例中，所述苗伸長/維持培養基中除TDZ或其類似物之外的一種或多種細胞***素的濃度能有效使苗伸長。在一些具體例中，所述苗伸長/維持培養基中除TDZ或其類似物之外的一種或多種細胞***素的濃度是從約0.01 mg/L至約100 mg/L，例如，從約0.25 mg/L至約5 mg/L。因此，所述苗伸長/維持培養基中除TDZ或其類似物之外的一種或多種細胞***素的濃度可以是，例如，約0.01 mg/L、約0.02 mg/L、約0.03 mg/L、約0.04 mg/L、約0.05 mg/L、約0.06 mg/L、約0.07 mg/L、約0.08 mg/L、約0.09 mg/L、約0.10 mg/L、約0.15 mg/L、約0.20 mg/L、約0.25 mg/L、約0.3 mg/L、約0.35 mg/L、約0.4 mg/L、約0.45 mg/L、約0.5 mg/L、約0.6 mg/L、約0.7 mg/L、約0.8 mg/L、約0.9 mg/L、約1.0 mg/L、約1.5 mg/L、約2.0 mg/L、約2.5 mg/L、約3 mg/L、約4 mg/L、約5 mg/L、約6 mg/L、約7 mg/L、約8 mg/L、約9 mg/L、約10 mg/L、約15 mg/L、約20 mg/L、 約25 mg/L、約30 mg/L、約40 mg/L、約50 mg/L、約60 mg/L、約70 mg/L、約80 mg/L、約90 mg/L或約100 mg/L。 In some embodiments, the concentration of one or more cytokinins in the shoot elongation/maintenance medium other than TDZ or an analog thereof is effective to elongate the shoot. In some embodiments, the concentration of one or more cytokinins in the shoot elongation/sustainment medium other than TDZ or an analog thereof is from about 0.01 mg/L to about 100 mg/L, for example, from about 0.25. From mg/L to about 5 mg/L. Therefore, the concentration of one or more cytokinins in the shoot elongation/maintenance medium other than TDZ or an analog thereof may be, for example, about 0.01 mg/L, about 0.02 mg/L, about 0.03 mg/L, About 0.04 mg/L, about 0.05 mg/L, about 0.06 mg/L, about 0.07 mg/L, about 0.08 mg/L, about 0.09 mg/L, about 0.10 mg/L, about 0.15 mg/L, about 0.20 Mg/L, about 0.25 mg/L, about 0.3 mg/L, about 0.35 mg/L, about 0.4 mg/L, about 0.45 mg/L, about 0.5 mg/L, about 0.6 mg/L, about 0.7 mg/ L, about 0.8 mg/L, about 0.9 mg/L, about 1.0 mg/L, about 1.5 mg/L, about 2.0 mg/L, about 2.5 mg/L, about 3 mg/L, about 4 mg/L, About 5 mg/L, about 6 mg/L, about 7 mg/L, about 8 mg/L, about 9 mg/L, about 10 mg/L, about 15 mg/L, about 20 mg/L, About 25 mg/L, about 30 mg/L, about 40 mg/L, about 50 mg/L, about 60 mg/L, about 70 mg/L, about 80 mg/L, about 90 mg/L or about 100 Mg/L.

在一些具體例中，所述芽誘導培養基及/或所述苗伸長/維持培養基進一步包含一種或多種生長素。在一些具體例中，所述生長素選自由β-萘氧乙酸(NAA)、2,4-二氯苯氧乙酸(2,4-D)、吲哚-3-丁酸(IBA)、吲哚-3-乙酸(IAA)、毒莠定及其各自的類似物。例如，所述生長素是NAA或其類似物。 In some embodiments, the shoot induction medium and/or the shoot elongation/maintenance medium further comprises one or more auxins. In some embodiments, the auxin is selected from the group consisting of β-naphthyloxyacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-butyric acid (IBA), and hydrazine. Indole-3-acetic acid (IAA), chlorpyrifos and their respective analogs. For example, the auxin is NAA or an analog thereof.

在一些具體例中，步驟(a)及/或步驟(c)所述芽誘導培養基是液體培養基。在一些具體例中，步驟(a)及/或步驟(c)所述芽誘導培養基是固體培養基。在一些具體例中，步驟(b)及/或步驟(d)所述苗伸長/維持培養基是液體培養基。在一些具體例中，步驟(b)及/或步驟(d)所述苗伸長/維持培養基是固體培養基。 In some embodiments, the shoot induction medium of step (a) and/or step (c) is a liquid medium. In some embodiments, the shoot induction medium of step (a) and/or step (c) is a solid medium. In some embodiments, the shoot elongation/sustainment medium of step (b) and/or step (d) is a liquid medium. In some embodiments, the shoot elongation/sustainment medium of step (b) and/or step (d) is a solid medium.

在一些具體例中，本發明的方法可牽涉特定迴圈中一個步驟使用液體培養基而下一個步驟使用固體培養基。例如，本發明涵蓋之方法，其中步驟(a)使用液體培養基完成而步驟(b)使用固體培養基完成。或者，如果步驟(a)及(b)及/或步驟(c)及(d)兩者都使用液體培養基完成，那麼本發明考慮的是可以改變該液體培養基而不將芽及/或苗移到另外的容器中(如試管、生物反應器、罐等)。例如，如果步驟(a)和(b)兩者都使用水耕(hydroponic)裝置的液體培養基完成，那麼在替換液體培養基時，該芽及/或苗可維持在其固定或非固定的 位置上。 In some embodiments, the method of the invention may involve the use of a liquid medium in one step and a solid medium in the next step in a particular loop. For example, the invention encompasses a method wherein step (a) is accomplished using a liquid medium and step (b) is accomplished using a solid medium. Alternatively, if steps (a) and (b) and/or steps (c) and (d) are both accomplished using a liquid medium, the present invention contemplates that the liquid medium can be altered without buds and/or shoots. Go to another container (such as test tubes, bioreactors, cans, etc.). For example, if both steps (a) and (b) are performed using a liquid medium of a hydroponic device, the shoot and/or shoot can be maintained at its fixed or non-fixed state when the liquid medium is replaced. Location.

在一些具體例中，步驟(a)及/或步驟(c)的培養持續足以產生多於一個苗芽的期間。例如，該期間係經設置以使置於步驟(a)或(c)的芽誘導培養基之各竹組織培養物、培植體或種子產生至少1個、至少2個、至少3個、至少4個、至少5個、至少6個、至少7個、至少8個、至少9個、至少10個、至少15個、至少20個、至少25個、至少26個、至少27個、至少28個、至少30個，或更多個苗芽。在一些具體例中，步驟(a)及/或步驟(c)的培養持續從約1小時至約3週或更長。例如，步驟(a)及/或步驟(c)的培養持續從約24小時至約60小時。因此，步驟(a)及/或步驟(c)的培養可持續約24小時、約25小時、約26小時、約27小時、約28小時、約29小時、約30小時、約35小時、約40小時、約45小時、約50小時、約55小時、約56小時、約57小時、約58小時、約59小時或約60小時。在一些具體例中，步驟(a)或步驟(c)的培養階段可持續長於60小時，如約1週、約2週、約3週、約4週或更長。 In some embodiments, the culture of step (a) and/or step (c) continues for a period of time sufficient to produce more than one shoot. For example, the period is such that at least one, at least two, at least three, at least four of the bamboo tissue cultures, cultures or seeds placed in the bud induction medium of step (a) or (c) are produced. At least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 26, at least 27, at least 28, at least 30 or more sprouts. In some embodiments, the culture of step (a) and/or step (c) lasts from about 1 hour to about 3 weeks or longer. For example, the cultivation of step (a) and/or step (c) lasts from about 24 hours to about 60 hours. Thus, the cultivation of step (a) and/or step (c) can last for about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 35 hours, about 40 hours, about 45 hours, about 50 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, or about 60 hours. In some embodiments, the culture phase of step (a) or step (c) can last longer than 60 hours, such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, or longer.

在一些具體例中，步驟(b)及/或步驟(d)的培養持續任何期望的期間。在一些具體例中，步驟(b)及/或步驟(d)的培養持續從約24小時到約4週或更長。例如，步驟(b)及/或步驟(d)的培養持續從約3天到約5天或更長。因此，步驟(b)及/或步驟(d)的培養可持續約24小時、約25小時、約26小時、 約27小時、約28小時、約29小時、約30小時、約35小時、約40小時、約45小時、約48小時、約50小時、約55小時、約56小時、約57小時、約58小時、約59小時、約60小時、約72小時、約96小時或約120小時。在一些具體例中，步驟(b)及/或(d)的培養可持續長於120小時，如約1週、約2週、約3週、約4週或更長。 In some embodiments, the incubation of step (b) and/or step (d) continues for any desired period of time. In some embodiments, the incubation of step (b) and/or step (d) continues from about 24 hours to about 4 weeks or longer. For example, the cultivation of step (b) and/or step (d) lasts from about 3 days to about 5 days or longer. Therefore, the culture of step (b) and/or step (d) can last for about 24 hours, about 25 hours, about 26 hours, About 27 hours, about 28 hours, about 29 hours, about 30 hours, about 35 hours, about 40 hours, about 45 hours, about 48 hours, about 50 hours, about 55 hours, about 56 hours, about 57 hours, about 58 Hours, about 59 hours, about 60 hours, about 72 hours, about 96 hours, or about 120 hours. In some embodiments, the culture of steps (b) and/or (d) can last for more than 120 hours, such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, or longer.

在一些具體例中，步驟(e)被重複至少一次、兩次、三次或更多次。 In some embodiments, step (e) is repeated at least once, twice, three times or more.

在一些具體例中，步驟(a)到(e)需要約1週、2週、3週、4週、5週、6週或更長。 In some embodiments, steps (a) through (e) require about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or longer.

在一些具體例中，所述植物是雙子葉植物的物種。在一些具體例中，所述植物是單子葉植物的物種。在一些具體例中，所述植物是竹物種。在一些具體例中，所述竹是毛竹(Phyllostachys edulisi‘Moso’)、蓉城竹(Phyllostachys bissetti)、缺苞箭竹(Fargesia denudata)、菲白竹(Pleioblastus fortunei)、維氏熊竹(Sasa Veitchii)、菲黃竹(Pleioblastus viridistriatus)、筱竹(Thamnocalamus crassinodus)、丘斯誇竹(Chusquea Culeo“Cana Prieta”)、白綠竹(Bambusa Old Hamii)、楠竹(Phyllostachys Moso)、烏芽竹(Phyllostachys Atrovaginata)、馬來甜龍竹(Dendrocalamus Asper)、瓜多竹(Guadua Angustifolia)、紫竹(Phylostachys Nigra)、青川箭竹(Fargesia rufa)、華西箭竹(Fargesia nitida)、伯齡達竹 (Borinda Boliana)、神農箭竹(Fargesia murielae)、菲白竹(Pleioblastus fortune)、拐棍竹(Fargesia robusta)和白綠竹(Bambusa OldHamii)。 In some embodiments, the plant is a species of a dicot. In some embodiments, the plant is a species of monocot. In some embodiments, the plant is a bamboo species. In some embodiments, the bamboo is Phyllostachys edulisi 'Moso', Phyllostachys bissetti , Fargesia denudata , Pleioblastus fortunei , Sasa Veitchii , Pleioblastus viridistriatus , Thamnocalamus crassinodus , Chusquea Culeo “Cana Prieta” , Bambusa Old Hamii , Phyllostachys Moso , Phyllostachys Atrovaginata ), Dendrocalamus Asper , Guadua Angustifolia , Phylostachys Nigra , Fargesia rufa , Fargesia nitida , Borinda Boliana , Shennong F argesia murielae , Pleioblastus fortune , Fargesia robusta and Bambusa Old Hamii .

在一些具體例中，所述植物為非竹之物種。在一些具體例中，所述非竹植物之物種是老鸛草屬(Geranium spp.，如天竺葵(Geranium rozanne))、知風草(Hakonechloa macra，如金知風草(Hakonechloa macra Aureola)、金葉知風草(Hakonechloa macra All gold)、聖誕玫瑰屬(Helleborus，如聖誕玫瑰(Helleborus Ivory Prince))、紐西蘭麻(Phormium)、山葵(Wasabi，如Wasabi C2)、青籬竹屬(Arundinaria，如大青籬竹(Arundinaria gigantean))、或茄屬(Solanum，如馬鈴薯及Solanum tuberosum)。 In some embodiments, the plant is a non-bamboo species. In some embodiments, the species of the non-bamboo plant of a Geranium (Geranium spp., Such as geranium (Geranium rozanne)), known wind grass (Hakonechloa macra, such as gold, known wind grass (Hakonechloa macra Aureola), gold leaf known Hakonechloa macra All gold, Helleborus (Helleborus Ivory Prince ), Phormium , Wasabi (as Wasabi C2), Arundinaria (such as large Arundinaria (Arundinaria gigantean)), or Solanum (Solanum, such as potato and Solanum tuberosum).

在一些具體例中，從維持在生長培養基上作為原種(stock)的苗團獲得所述組織培養物。在一些具體例中，所述培植體是竹莖(cane)的節(segment)。在一些具體例中，所述竹莖的節包含節間(internode)。在一些具體例中，所述竹莖的節包含節段(nodal section)。在一些具體例中，所述節段包含單一個芽。在一些具體例中，所述芽是休眠或有活性的。在一些具體例中，所述培植體係取自約1週齡、約2週齡、約3週齡、約1月齡、約2月齡、約3月齡、約半年齡、約1年齡、約2年齡植物、約3年齡、約5年齡或更老的植物。在一些具體例中，使用竹的種子或其一部分。 In some embodiments, the tissue culture is obtained from a shoot that is maintained as a stock on a growth medium. In some embodiments, the implant is a segment of a cane. In some embodiments, the knot of the bamboo stem comprises an internode. In some embodiments, the section of the bamboo stem comprises a nodal section. In some embodiments, the segment comprises a single bud. In some embodiments, the bud is dormant or active. In some embodiments, the culture system is taken from about 1 week old, about 2 weeks old, about 3 weeks old, about 1 month old, about 2 months old, about 3 months old, about half age, about 1 age, A plant of about 2 years old, about 3 years old, about 5 years old or older. In some embodiments, bamboo seeds or a portion thereof are used.

在一些具體例中，在步驟(b)及/或步驟(d)培養該苗芽之前，將步驟(a)及/或步驟(c)獲得的苗芽分離。在一些具體例中，在步驟(b)及/或步驟(d)培養該苗芽之前，所述分離每次分離產生1到3個苗芽之群。 In some embodiments, the shoots obtained in step (a) and/or step (c) are separated prior to culturing the shoots in step (b) and/or step (d). In some embodiments, the separation produces 1 to 3 shoots per separation prior to culturing the shoot in step (b) and/or step (d).

本發明還提供了用於植物繁殖之選擇性培養基及選擇性方法。在一些具體例中，所述選擇性培養基選自如本文描述的階段1培養基、階段2培養基、階段3培養基、階段4培養基、階段5培養基、階段6培養基、階段7培養基等。 The invention also provides selective media and alternative methods for plant propagation. In some embodiments, the selective medium is selected from the group consisting of Stage 1 medium, Stage 2 medium, Stage 3 medium, Stage 4 medium, Stage 5 medium, Stage 6 medium, Stage 7 medium, and the like as described herein.

在一些具體例中，使用至少一階段1培養基和至少一階段2培養基。 In some embodiments, at least one stage 1 medium and at least one stage 2 medium are used.

在一些具體例中，在植物繁殖中依序使用階段1及階段2培養基。在一些具體例中，所述培植體在階段1培養基上保留約1到約36小時(如使用強化培養基(spiked media)時)。在一些具體例中，所述培植體在階段1培養基上保留10-120天(如使用標準或減料培養基時)。在一些具體例中，所述培植體在被轉移到階段2培養基之前，在階段1培養基上保留至少1、2、3、4、5、6、7、8、9、10或更多輪(如在每一輪中，將培植體從舊的階段1培養基轉移到新鮮的階段1培養基)。在一些具體例中，所述培植體在階段2培養基上保留約1到約36小時(如使用強化培養基時)。在一些具體例中，所述培植體在階段2培養基上保留約10-120天(如使用標準或減料培養基時)。在一些具體例中，所述培植體在 階段2培養基上保留至少1、2、3、4、5、6、7、8、9、10或更多輪(如在每一輪中，將培植體從舊的階段2培養基轉移到新鮮的階段2培養基)。 In some embodiments, Stage 1 and Stage 2 media are used sequentially in plant propagation. In some embodiments, the culture is maintained on Stage 1 medium for about 1 to about 36 hours (eg, when using spiked media). In some embodiments, the culture is maintained on Stage 1 medium for 10-120 days (eg, when standard or reduced medium is used). In some embodiments, the culture body retains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more rounds on the Stage 1 medium prior to being transferred to the Stage 2 medium ( As in each round, the transplants were transferred from the old Stage 1 medium to the fresh Stage 1 medium). In some embodiments, the culture is maintained on Stage 2 medium for from about 1 to about 36 hours (eg, when a fortified medium is used). In some embodiments, the culture is retained on Stage 2 medium for about 10-120 days (eg, when standard or reduced medium is used). In some embodiments, the implant is Maintain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more rounds on Stage 2 medium (eg, in each round, transfer the culture from the old Stage 2 medium to the fresh stage) 2 medium).

在一些具體例中，在植物繁殖時交替使用所述階段1和階段2培養基。在一些具體例中，所述交替連續進行至少1、2、3、4、5、6、7、8、9、10或更多個迴圈。在一些具體例中，所述培植體在被轉移到階段2培養基之前，先在階段1培養基上保留至少1、2、3、4、5、6、7、8、9、10或更多輪。在一些具體例中，所述培植體在被轉移回階段1培養基之前，先在階段2培養基上保留至少1、2、3、4、5、6、7、8、9、10或更多輪。在一些具體例中，所述培植體在階段1培養基上約1-36小時(如使用強化培養基時)或更長(如使用標準或減料培養基時)，隨後被轉移到階段2培養基。在一些具體例中，所述交替係持續至觀察到多個苗。在一些具體例中，所述交替係根據植物的物種和培養基持續約1週至約24個月。 In some embodiments, the Stage 1 and Stage 2 media are used alternately during plant propagation. In some embodiments, the alternating is performed continuously for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more loops. In some embodiments, the culture body retains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more rounds on the Stage 1 medium prior to being transferred to the Stage 2 medium. . In some embodiments, the culture body retains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more rounds on the Stage 2 medium prior to being transferred back to the Stage 1 medium. . In some embodiments, the culture is transferred to Stage 2 medium on Stage 1 medium for about 1-36 hours (eg, when using fortified medium) or longer (eg, when standard or reduced medium is used). In some embodiments, the alternation continues until multiple shoots are observed. In some embodiments, the alternation is from about 1 week to about 24 months depending on the species and culture medium of the plant.

可選擇地，根據先前的處理，然後將增殖的苗置於階段3培養基上以進一步增殖直到獲得期望數量的苗。所述階段3培養基上的培植體可進一步被轉移到階段4培養基。在一些具體例中，所述培植體在階段3培養基上約1-36小時(如使用強化培養基時)，隨後被轉移到階段4培養基。在一些具體例中，所述培植體在階段3培養基上約10-120天或更長 (如使用標準或減料培養基時)，隨後被轉移到階段4培養基。在一些具體例中，所述培植體在階段4培養基上保留約10-120天。 Alternatively, according to the previous treatment, the propagated shoots are then placed on Stage 3 medium for further propagation until the desired number of shoots are obtained. The culture on the Stage 3 medium can be further transferred to Stage 4 medium. In some embodiments, the culture is on stage 3 medium for about 1-36 hours (as with the use of fortified medium) and subsequently transferred to stage 4 medium. In some embodiments, the culture is about 10-120 days or longer on Stage 3 medium. (If standard or reduced medium is used), then transferred to Stage 4 medium. In some embodiments, the culture is retained on Stage 4 medium for about 10-120 days.

或者，從階段2培養基所獲得的增殖苗可以在至少一種階段3培養基和至少一種階段4培養基之間交替。在一些具體例中，所述交替連續進行至少1、2、3、4、5、6、7、8、9、10或更多個迴圈。在一些具體例中，所述交替係持續至期望數量的苗。在一些具體例中，藉由分離到新管且進一步擴張(expansion)以獲得期望數量的苗。在一些具體例中，每個增殖週期可獲得每管約1到10個苗。 Alternatively, the proliferative shoots obtained from the Stage 2 medium can be alternated between at least one Stage 3 medium and at least one Stage 4 medium. In some embodiments, the alternating is performed continuously for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more loops. In some embodiments, the alternation continues to a desired number of shoots. In some embodiments, a desired number of shoots are obtained by separating into new tubes and further expanding. In some embodiments, about 1 to 10 shoots per tube can be obtained per proliferation cycle.

在一些具體例中，所述培植體被置於交替的階段1培養基、階段2培養基和階段3培養基上，直到獲得期望數量的苗。在一些具體例中，增殖的苗隨後被置於階段4培養基上。 In some embodiments, the implants are placed on alternating Phase 1 medium, Stage 2 medium, and Stage 3 medium until a desired number of shoots are obtained. In some embodiments, the propagated shoots are then placed on stage 4 medium.

在一些具體例中，所述培植體被置於交替的階段1培養基和階段2培養基上，直到獲得期望數量的苗。在一些具體例中，增殖的苗隨後被置於交替的階段3培養基和階段4培養基上。 In some embodiments, the implants are placed on alternating Phase 1 medium and Stage 2 medium until a desired number of shoots are obtained. In some embodiments, the propagated shoots are then placed on alternating Phase 3 medium and Stage 4 medium.

在一些具體例中，所述培植體被置於階段1培養基上約1、2、3、4、5、6、7、8、9、10，或更多輪，且隨後被轉移到階段2培養基上約1、2、3、4、5、6、7、8、9、10，或更多輪，直到獲得期望數量的苗。在一些具體例中，增殖的苗隨後被置於交替的階段3培養基和階段4培養基上。 In some embodiments, the implant is placed on stage 1, medium about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, and then transferred to stage 2 About 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more rounds on the medium until the desired number of shoots are obtained. In some embodiments, the propagated shoots are then placed on alternating Phase 3 medium and Stage 4 medium.

在一些具體例中，所述培植體首先被置於階段1培養基上。在一些具體例中，所述培植體在階段1培養基上約1-36小時(如使用強化培養基時)。在一些具體例中，所述培植體在階段1培養基上約10-120天或更長(如使用標準或減料培養基時)。在一些具體例中，該步驟包含1、2、3、4、5、6、7、8、9、10或更多輪新鮮的階段1培養基。然後將培植體保留在交替的階段2培養基和階段3培養基上，直到獲得期望數量的苗。在一些具體例中，增殖的苗被置於階段4培養基上。在一些具體例中，增殖的苗保留在階段4培養基上約10-120天。 In some embodiments, the culture is first placed on Stage 1 medium. In some embodiments, the culture is on stage 1 medium for about 1-36 hours (eg, when a fortified medium is used). In some embodiments, the culture is on stage 1 medium for about 10-120 days or longer (eg, when standard or reduced medium is used). In some embodiments, the step comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more rounds of fresh Stage 1 medium. The cultures are then retained on alternating Phase 2 medium and Stage 3 medium until the desired number of shoots are obtained. In some embodiments, the propagated shoots are placed on stage 4 medium. In some embodiments, the proliferated shoots remain on Stage 4 medium for about 10-120 days.

仍然可選擇地，從階段4培養基獲得的增殖苗可以被轉移到如在本文中描述的階段5培養基上。在一些具體例中，所述苗被置於階段5培養基上約1-24小時或更長(如使用強化培養基時)。在一些具體例中，所述苗被置於階段5培養基上約10到120天(如使用標準或減料培養基時)。在一些具體例中，所述苗被轉移到小組織培養箱中，如Magenta培養箱。 Still alternatively, the proliferative shoots obtained from Stage 4 medium can be transferred to Stage 5 medium as described herein. In some embodiments, the shoot is placed on Stage 5 medium for about 1-24 hours or longer (eg, when a fortified medium is used). In some embodiments, the shoot is placed on Stage 5 medium for about 10 to 120 days (eg, when standard or reduced medium is used). In some embodiments, the shoots are transferred to a small tissue culture incubator, such as a Magenta incubator.

仍然可選擇地，所述培植體首先被置於階段5培養基上直到獲得期望數量的苗，然後被轉移到如在本文中描述的階段6培養基上。在一些具體例中，所述苗被置於階段6培養基上約1-24小時或更長(如使用強化培養基時)。在一些具體例中，所述苗被置於階段6培養基上約10到120天(如使用標 準或減料培養基時)。 Still alternatively, the culture is first placed on stage 5 medium until the desired number of shoots are obtained and then transferred to stage 6 medium as described herein. In some embodiments, the shoot is placed on Stage 6 medium for about 1-24 hours or longer (as when using fortified medium). In some embodiments, the shoot is placed on stage 6 medium for about 10 to 120 days (eg, When quenching or reducing the medium).

在一些具體例中，從階段4培養基獲得的培植體被置於交替的階段5培養基和階段6培養基上，直到獲得期望數量的苗。 In some embodiments, the cultures obtained from the Stage 4 medium are placed on alternating Phase 5 medium and Stage 6 medium until the desired number of shoots are obtained.

仍然可選擇地，保留在階段6培養基上的培植體被轉移到如本文描述的階段7培養基上。在一些具體例中，所述苗被置於階段7培養基上約1-24小時或更長(如使用強化培養基時)。在一些具體例中，所述苗被置於階段7培養基上約10到120天(如使用標準或減料培養基時)。 Still alternatively, the cultures retained on stage 6 medium are transferred to stage 7 medium as described herein. In some embodiments, the shoot is placed on stage 7 medium for about 1-24 hours or longer (eg, when a fortified medium is used). In some embodiments, the shoot is placed on Stage 7 medium for about 10 to 120 days (eg, when standard or reduced medium is used).

仍然可選擇地，從階段4培養基獲得的增殖苗可以被轉移到所述交替的階段4培養基、階段5培養基和階段6培養基上。 Still alternatively, the proliferative shoots obtained from the Stage 4 medium can be transferred to the alternating Stage 4 medium, Stage 5 medium and Stage 6 medium.

仍然可選擇地，如需要的話，從階段7培養基獲得的增殖苗可以被轉移到一個或多個其他額外的培養基上(如階段8、階段9等)以用於進一步的繁殖。 Still alternatively, if desired, the proliferative shoots obtained from Stage 7 medium can be transferred to one or more other additional media (eg, Stage 8, Stage 9, etc.) for further propagation.

在一些具體例中，可選擇的培養基包含間拓樸林或其類似物。在一些具體例中，可選擇的培養基包含至少兩種其他細胞***素。在一些具體例中，所述培養基支持預定期間的增殖週期。在一些具體例中，所述培養基支持至少6個月的增殖週期。 In some embodiments, the selectable medium comprises metatopia or an analog thereof. In some embodiments, the selectable medium comprises at least two other cytokinins. In some embodiments, the medium supports a proliferative cycle for a predetermined period of time. In some embodiments, the medium supports a proliferation cycle of at least 6 months.

在一些具體例中，可選擇的培養基包含至少3種細胞***素。在一些具體例中，所述培養基支持預定的期間的增殖週 期。在一些具體例中，所述培養基支持至少6個月的增殖週期。 In some embodiments, the selectable medium comprises at least three cytokinins. In some embodiments, the medium supports a proliferating week for a predetermined period period. In some embodiments, the medium supports a proliferation cycle of at least 6 months.

在一些具體例中，可選擇的培養基包含至少一種生長素和至少兩種細胞***素。在一些具體例中，所述培養基支持預定的期間的增殖週期。在一些具體例中，所述培養基支持至少6個月的增殖週期。 In some embodiments, the selectable medium comprises at least one auxin and at least two cytokinins. In some embodiments, the medium supports a proliferative cycle for a predetermined period of time. In some embodiments, the medium supports a proliferation cycle of at least 6 months.

在一些具體例中，可選擇的培養基包含至少兩種生長素和至少兩種細胞***素。在一些具體例中，所述培養基支持預定的期間的增殖週期。在一些具體例中，所述培養基支持至少6個月的增殖週期。 In some embodiments, the selectable medium comprises at least two auxins and at least two cytokinins. In some embodiments, the medium supports a proliferative cycle for a predetermined period of time. In some embodiments, the medium supports a proliferation cycle of at least 6 months.

在一些具體例中，可選擇的培養基包含至少兩種生長素和至少三種細胞***素。在一些具體例中，所述培養基支持預定的期間的增殖週期。在一些具體例中，所述培養基支持至少6個月的增殖週期。 In some embodiments, the selectable medium comprises at least two auxins and at least three cytokinins. In some embodiments, the medium supports a proliferative cycle for a predetermined period of time. In some embodiments, the medium supports a proliferation cycle of at least 6 months.

本案還提供了用於植物微體繁殖的系統。在一些具體例中，所述系統包含生長器皿(growth vessel)、兩個或更多個培養基容器(container)以及用於驅動流體進及/或出生長器皿的能量來源。在一些具體例中，所述系統進一步包含控制器。在一些具體例中，系統進一步包含光源及/或提供CO₂、O₂、N₂或其混合物至生長器皿的氣體源。 The system also provides a system for plant micropropagation. In some embodiments, the system includes a growth vessel, two or more media containers, and a source of energy for driving fluid into and/or out of the growing vessel. In some embodiments, the system further includes a controller. In some embodiments, the system further comprising a light source and / or gas source provides CO _2, O _2, N ₂ or a mixture thereof to a growth vessel.

在一些具體例中，所述系統包含：用於在無菌或基本上無菌的環境中培養植物組織的生長 器皿；具有第一流體口(fluid port)和第二流體口的第一培養基容器，所述第一流體口與生長器皿是流體可連接的；具有第一流體口和第二流體口的第二培養基容器，所述第一流體口與生長器皿是流體可連接的；和氣體源，其與第一培養基容器的第二流體口和第二培養基容器的第二流體口是流體可連接的。 In some embodiments, the system comprises: for growing a plant tissue in a sterile or substantially sterile environment a first medium container having a first fluid port and a second fluid port, the first fluid port being fluidly connectable to the growth vessel; and a second fluid port having a first fluid port and a second fluid port a medium container, the first fluid port being fluidly connectable to the growth vessel; and a gas source fluidly connectable to the second fluid port of the first medium container and the second fluid port of the second medium container.

在一些具體例中，所述系統進一步包含控制器，其係可以第一操作模式和第二操作模式操作，該第一操作模式係將加壓氣體從氣體源輸送到第一培養基容器以將其中含有的第一體積的液體置換到生長器皿，以及該第二操作模式係將加壓氣體從氣體源輸送到第二培養基容器以將其中所含第二體積的液體置換到生長器皿。 In some embodiments, the system further includes a controller operable in a first mode of operation and a second mode of operation, the first mode of operation transporting pressurized gas from the gas source to the first medium container to The first volume of liquid contained is displaced to the growth vessel, and the second mode of operation delivers pressurized gas from the gas source to the second medium reservoir to displace the second volume of liquid contained therein into the growth vessel.

在一些具體例中，所述控制器在第三操作模式下是可操作的，該模式中允許生長器皿中所含液體從生長器皿流至第一培養基容器和第二培養基容器中的至少一個。 In some embodiments, the controller is operable in a third mode of operation that allows liquid contained in the growing vessel to flow from the growth vessel to at least one of the first medium container and the second medium container.

在一些具體例中，所述控制器在第一培養次序下是可操作的，其中在第一操作模式之後接著執行第三操作模式。 In some embodiments, the controller is operable in a first culture sequence, wherein the third mode of operation is then performed after the first mode of operation.

在一些具體例中，所述控制器在第二培養次序下是可操作的，其中在第二操作模式之後接著執行第三操作模式。 In some embodiments, the controller is operable in a second culture sequence, wherein the third mode of operation is then performed after the second mode of operation.

在一些具體例中，所述控制器在植物繁殖模式下是進一步可操作的，其中執行第一培養次序和第二培養次序。 In some embodiments, the controller is further operable in a plant propagation mode in which a first culture sequence and a second culture sequence are performed.

在一些具體例中，將所述生長器皿升高到第一和第二培養基容器之上，以允許在第三操作模式中液體從生長器皿流入第一培養基容器和第二培養基容器中的至少一個。 In some embodiments, the growth vessel is raised above the first and second medium containers to allow liquid to flow from the growth vessel into at least one of the first medium container and the second medium container in the third mode of operation. .

在一些具體例中，所述系統進一步包含與生長器皿、第一培養基容器和第二培養基容器流體可連接的支管， 在一些具體例中，所述支管是可操作的，以控制生長器皿和第一培養基容器之間以及生長器皿和第二培養基液體之間的液體流動。 In some embodiments, the system further comprises a branch tube fluidly connectable to the growth vessel, the first medium container, and the second medium container, In some embodiments, the manifold is operable to control fluid flow between the growth vessel and the first medium container and between the growth vessel and the second medium liquid.

在一些具體例中，所述生長器皿包括液體導管(conduit)，該液體導管係經配置以將液體從生長器皿虹吸(siphon)到第一培養基容器和第二培養基容器的至少一個。 In some embodiments, the growth vessel includes a liquid conduit configured to siphon liquid from the growth vessel to at least one of the first medium container and the second medium container.

在一些具體例中，所述生長器皿是間歇浸沒式生物反應器(ebb and flow bioreactor)。 In some embodiments, the growth vessel is an ebb and flow bioreactor.

在一些具體例中，所述控制器是可操作的，以控制生長器皿和第一培養基容器之間、生長器皿和第二培養基容器、氣體源和第一培養基容器和氣體源和第二培養基容器之間的流體連通。 In some embodiments, the controller is operable to control between the growth vessel and the first medium container, the growth vessel and the second medium container, the gas source and the first medium container, and the gas source and the second medium container Fluid communication between.

在一些具體例中，所述第一培養基容器包含如在本文中描述的芽誘導培養基，且所述第二培養基容器包含苗伸長/維持培養基。在一些具體例中，所述芽誘導培養基包含有效量的噻苯隆(TDZ)或其類似物，且所述苗伸長/維持培養基包含有效量的一種或多種除TDZ或其類似物之外的細胞*** 素。 In some embodiments, the first medium container comprises a shoot induction medium as described herein, and the second medium container comprises a shoot elongation/maintenance medium. In some embodiments, the shoot induction medium comprises an effective amount of thiazolone (TDZ) or an analog thereof, and the shoot elongation/sustainment medium comprises an effective amount of one or more other than TDZ or an analog thereof Cell division Prime.

本應用還提供了用於交換生物反應器/植物生長系統中的液體培養基的方法，以用於植物或植物組織的微體繁殖。 The present application also provides a method for exchanging a liquid medium in a bioreactor/plant growth system for micropropagation of plants or plant tissues.

在一些具體例中，所述生物反應器包含用於培養植物組織的生長器皿、與生長器皿流體可連接的第一培養基容器、與生長器皿流體可連接的第二培養基容器、和與第一培養基容器和第二培養基容器流體可連接的氣體源。 In some embodiments, the bioreactor comprises a growth vessel for culturing plant tissue, a first medium container fluidly connectable to the growth vessel, a second medium container fluidly connectable to the growth vessel, and the first medium A gas source to which the container and the second medium container are fluidly connectable.

在一些具體例中，所述方法包括：建立第一培養基容器和生長器皿之間的流體連通；生長器皿流體分離第二培養基容器；立氣體源和第一培養基容器之間的流體連通；壓縮氣體輸送到第一培養基容器以將第一體積的液體從第一培養基容器置換到生長器皿；許至少一部分第一體積的液體從生長器皿流回到第一培養基容器中；立第二培養基容器和生長器皿之間的流體連通；生長器皿流體分離第一培養基容器；立氣體源和第二培養基容器之間的流體連通；壓縮氣體輸送到第二培養基容器以將第二體積的液體從第一培養基容器置換到生長器皿；並許至少一部分第二體積的液體從生長器皿流回到第二培養基容器中。 In some embodiments, the method includes: establishing fluid communication between the first medium container and the growth vessel; growing the vessel fluid to separate the second medium container; fluid communication between the standing gas source and the first medium container; and compressing the gas Delivered to the first medium container to displace the first volume of liquid from the first medium container to the growth vessel; at least a portion of the first volume of liquid flows from the growth vessel back into the first medium container; the second medium container and growth Fluid communication between the vessels; the growth vessel fluid separates the first medium container; the fluid communication between the standing gas source and the second medium container; and the compressed gas is delivered to the second medium container to transfer the second volume of liquid from the first medium container Displacement into the growth vessel; and allowing at least a portion of the second volume of liquid to flow from the growth vessel back into the second medium reservoir.

在一些具體例中，將所述壓縮氣體以約1分鐘輸送到第一培養基容器。 In some embodiments, the compressed gas is delivered to the first medium container in about 1 minute.

在一些具體例中，將所述壓縮氣體以約1分鐘輸送到第二培養基容器。 In some embodiments, the compressed gas is delivered to the second medium container in about 1 minute.

在一些具體例中，允許液體以約8分鐘從生長器皿流回到第一培養基容器。 In some embodiments, the liquid is allowed to flow from the growth vessel back to the first medium container in about 8 minutes.

在一些具體例中，允許液體以約8分鐘從生長器皿流回到第二培養基容器。 In some embodiments, the liquid is allowed to flow from the growth vessel back to the second medium container in about 8 minutes.

在一些具體例中，將所述系統用於包含芽誘導培養基與苗伸長及維持培養基的植物繁殖。例如，將所述系統用於其中涉及芽誘導培養基與苗伸長及維持培養基交替的植物繁殖。 In some embodiments, the system is used for plant propagation comprising a shoot induction medium and shoot elongation and maintenance medium. For example, the system is used for plant propagation in which a shoot-inducing medium is involved with seedling elongation and maintenance medium is alternated.

在一些具體例中，將所述系統用於包含如在本文中描述可選擇培養基的植物繁殖。例如，將所述系統用於植物繁殖，其中(1)涉及階段1培養基和階段2培養基的交替；(2)涉及階段2培養基和階段3培養基的交替；(3)涉及階段1培養基、階段2培養基和階段3培養基的交替；(4)涉及階段3培養基和階段4培養基的交替；(5)涉及階段4培養基和階段5培養基的交替；(6)涉及階段5培養基和階段6培養基的交替；(7)涉及階段4培養基、階段5培養基和階段6培養基的交替；及/或(7)涉及階段6培養基和階段7培養基的交替。 In some embodiments, the system is used for plant propagation comprising a selectable medium as described herein. For example, the system is used for plant propagation, wherein (1) involves the alternation of Stage 1 medium and Stage 2 medium; (2) involves the alternating of Stage 2 medium and Stage 3 medium; (3) involves Stage 1 medium, Stage 2 Alternation of medium and stage 3 medium; (4) involving alternating of stage 3 medium and stage 4 medium; (5) involving alternating of stage 4 medium and stage 5 medium; (6) involving alternating of stage 5 medium and stage 6 medium; (7) Alternating with Stage 4 medium, Stage 5 medium, and Stage 6 medium; and/or (7) involving the alternating of Stage 6 medium and Stage 7 medium.

還提供用於將組織培養小植株(plantlet)的微環境間歇地 暴露於如在本文中描述的液體營養溶液的裝置和方法。在一些具體例中，設備包括框架(frame)、架組件(shelf assembly)和驅動組件(drive assembly)。在此具體例中，驅動組件可經配置以接合架組件，以促進架組件相對於框架的擺動運動，從而使組織培養小植株間歇地暴露於所述液體營養溶液。在一些具體例中，本文描述一般係涉及架(rack)，且更具體地係涉及用於植物繁殖器皿的擺動架。 Also provided is a microenvironment for tissue culture of plantlets intermittently Apparatus and methods for exposure to a liquid nutrient solution as described herein. In some embodiments, the device includes a frame, a shelf assembly, and a drive assembly. In this particular example, the drive assembly can be configured to engage the frame assembly to facilitate oscillating motion of the frame assembly relative to the frame to thereby intermittently expose tissue culture plantlets to the liquid nutritional solution. In some embodiments, the description herein generally relates to racks, and more particularly to swinging frames for plant breeding vessels.

已可獲得栽培和工業用途的更快生長的植物，包括竹亞科植物。所述竹亞科(屬於禾本科)包含木本和草本竹兩者，目前大致辨識出120種溫帶和熱帶的木本竹屬。竹是具有許多不同應用的多用途植物，據估計世界範圍內約22億人在一定程度上使用竹，且於1985年全球可歸因於竹的收入估計約為45億美元，竹的市場也在擴大。竹筍是亞洲烹飪的原料，且竹存在於許多產品中，包括牙籤、掃帚、用於葡萄栽培(viticulture)和樹木栽培(arboriculture)的杆(pole)、景觀材料、鑲花地板、層壓材料、傢俱、手工藝品和其他家庭用品。此外，竹正成為紡織材料的重要來源和紙張生產的成分和結構木料(structural timber)的來源。 Faster growing plants for cultivation and industrial use, including bamboo subfamilies, have been obtained. The bamboo subfamily (which belongs to the family Poaceae) contains both woody and herbaceous bamboos, and currently 120 species of temperate and tropical woody genus are generally identified. Bamboo is a versatile plant with many different applications. It is estimated that about 2.2 billion people worldwide use bamboo to some extent, and in 1985 the global income attributable to bamboo was estimated to be about $4.5 billion. The bamboo market is also Expanding. Bamboo shoots are a raw material for Asian cooking, and bamboo is found in many products, including toothpicks, brooms, poles for viticulture and arboriculture, landscape materials, parquet flooring, laminates, Furniture, handicrafts and other household items. In addition, bamboo is becoming an important source of textile materials and a source of paper production and a source of structural timber.

植物物種，諸如竹，係被認為是環境友好的「綠色」產品，其具有非常快的生長速度。儘管生長速度快，這些植物的其他特性使它們很難在工業規模上快速繁殖。例如，許多商業 上重要的竹僅以間隔長達60-130年的時間開花。使該長開花週期的困難性增加的是許多竹顯示大規模(或集體性)開花，即種群中的所有植物同時開花。例如，桂竹(Phyllostachys bambusoides)以間隔130年的時間開花，且在該物種中，同系(same stock)的所有植物在同一時間開花，而與地理位置或天氣條件的差異無關，在開花之後竹死亡。竹的漫長開花間隔和大規模開花的傾向使要獲得用於繁殖的種子非常困難。使該問題更複雜的是，即使能夠獲得竹的種子，卻無法保持存活超過3-6個月。 Plant species, such as bamboo, are considered to be environmentally friendly "green" products with very fast growth rates. Despite their fast growth, the other characteristics of these plants make them difficult to multiply on an industrial scale. For example, many commercially important bamboos only bloom at intervals of up to 60-130 years. The difficulty in making this long flowering cycle is that many bamboos show large-scale (or collective) flowering, ie all plants in the population flower at the same time. For example, Phyllostachys bambusoides blooms at intervals of 130 years, and in this species, all plants of the same stock bloom at the same time, regardless of geographical or weather conditions, and bamboo dies after flowering. . The long flowering interval of bamboo and the tendency to bloom on a large scale make it very difficult to obtain seeds for reproduction. To complicate the problem, even if the seeds of bamboo can be obtained, they cannot survive for more than 3-6 months.

由於這些使用傳統有性生殖繁殖竹及其他快速生長植物物種的困難，經常使用無性技術，如分株(clump division)和扦插(cutting)繁殖這些植物。然而，這些無性繁殖技術，因為它們產生大規模生產的能力和它們的實踐效率都很低，不足以達到預計的世界性需求。此外，許多無性繁殖方法的缺點是不能消除親本植物中存在的病原體。因此，實現植物大規模生產的組成物、方法和系統是高度期望的。微體繁殖(也稱為組織培養，此等術語在本文中可交互使用)是一種可被用於實現該目的優秀的潛在方法。 Due to these difficulties in using traditional sexual reproduction of bamboo and other rapidly growing plant species, these plants are often propagated using asexual techniques such as clump division and cutting. However, these asexual reproduction techniques are not sufficiently efficient to achieve the expected worldwide demand because of their ability to produce large-scale production and their practical efficiency. In addition, many asexual propagation methods have the disadvantage of not eliminating pathogens present in the parental plants. Therefore, compositions, methods and systems for achieving large-scale production of plants are highly desirable. Micropropagation (also known as tissue culture, which terms are used interchangeably herein) is a potential method that can be used to achieve this goal.

微體繁殖與從扦插生長植物並非沒有相似之處。然而，與從扦插生長的植物不同的是，微體繁殖植物在體外無菌培養基中生長。典型地，該生長培養基包含固體或半固體材料如瓊脂，還添加了各種化合物如營養物、無機鹽、生長調節物、 糖、維生素和其他化合物。 Micropropagation does not have similarities with growing plants from cuttings. However, unlike plants grown from cuttings, micropropagated plants are grown in sterile medium in vitro. Typically, the growth medium comprises a solid or semi-solid material such as agar, and various compounds such as nutrients, inorganic salts, growth regulators, Sugar, vitamins and other compounds.

組織培養植物的優勢在於植物可以在無菌環境中生長以使他們更可能保持無疾病。其他優勢包括在小空間內生長非常大量植物的能力、微體繁殖植物的水和營養需求降低、和可繼而用於產生更多組織培養材料的組織快速增殖。此外，微體繁殖非常靈活且快速擴大規模是可能的(1年中可從單一基因型生產接近1百萬個植物)。如此短時間範圍和如此大量係非任何習知方法所能匹敵的。組織培養還提供了易於運輸和輸送的高品質植物的生產。 The advantage of tissue culture plants is that plants can grow in a sterile environment to make them more likely to remain disease free. Other advantages include the ability to grow very large numbers of plants in small spaces, the reduced water and nutrient requirements of micropropagated plants, and the rapid proliferation of tissues that can be used to produce more tissue culture materials. In addition, micropropagation is very flexible and rapid expansion is possible (approximately 1 million plants can be produced from a single genotype in a year). Such a short time frame and such a large number of systems are not comparable to any conventional methods. Tissue culture also provides the production of high quality plants that are easy to transport and transport.

已經發表了一些關於植物的組織培養的論文。然而，在實施上(即，竹的大規模繁殖)，這些論文中描述的組成物、方法和系統並不能轉化為商業可行的繁殖系統。 A number of papers on tissue culture of plants have been published. However, in practice (ie, large-scale reproduction of bamboo), the compositions, methods, and systems described in these papers cannot be translated into commercially viable reproductive systems.

植物物種的組織培養中遇到的困難包括內源或表面污染和褐變(browning)的高發生率、與休眠或生長惰性(topophysis)和玻璃質化(hyperhydricity)相關的因素。本案揭示內容係提供了克服這些困難、允許植物以商業規模無性生產的組成物、方法和系統。 Difficulties encountered in tissue culture of plant species include high incidence of endogenous or surface contamination and browning, factors associated with dormancy or topophysis and hyperhydricity. The disclosure of the present invention provides compositions, methods and systems that overcome these difficulties and allow plants to be produced asexually on a commercial scale.

液體培養基的微體繁殖提高了營養的攝取且促進生長。然而，液體培養基中體外培養的優勢經常被技術問題抵消，諸如窒息(asphyxia)、玻璃質化、剪切力(shear forces)和複雜裝備的需求。 Micropropagation of liquid media increases nutrient uptake and promotes growth. However, the advantages of in vitro culture in liquid media are often offset by technical issues such as asphyxia, vitrification, shear forces, and the need for complex equipment.

國際專利申請公開號WO/2011/100762以引用方式全部併 入本文，其描述對竹體外繁殖有用的組成物和方法。 International Patent Application Publication No. WO/2011/100762 is incorporated by reference in its entirety. In this context, it describes compositions and methods useful for in vitro propagation of bamboo.

本案係揭示用於植物快速體外繁殖的新穎組成物、方法和系統。本發明提供可在更短時間內顯著提高植物組織培養增殖率的組成物和方法。在一些具體例中，在固體或液體誘導培養基中以很短時間段利用強細胞***素如噻苯隆，以在植物物種的培植體中誘導多個苗芽的形成。利用含有強細胞***素如噻苯隆的培養基的芽誘導處理後，接著苗伸長和維持處理係藉由使用相對弱的細胞***素如BAP、間拓樸林、2ip、玉米素和或玉米素核苷，以完成該培養物的苗伸長和維持。當依序地交替這個過程，在3週的培養週期內達成2X和28X間的培養增殖率。 This case discloses novel compositions, methods and systems for rapid in vitro propagation of plants. The present invention provides a composition and method which can significantly increase the proliferation rate of plant tissue culture in a shorter period of time. In some embodiments, strong cytokinins, such as thibenone, are utilized in solid or liquid-inducing media for a short period of time to induce the formation of multiple shoots in the culture of plant species. After bud induction treatment using a medium containing a strong cytokinin such as thifenone, subsequent elongation and maintenance treatment by using relatively weak cytokinins such as BAP, metatopia, 2ip, zeatin and or zeatin Nucleosides to complete shoot elongation and maintenance of the culture. When this process was alternated sequentially, a culture proliferation rate between 2X and 28X was achieved in a 3-week culture period.

(定義) (definition)

本發明提供了用於植物繁殖的組成物、方法和系統。在一些具體例中，所述組成物包含至少一種植物激素。如本文說明及申請專利範圍中所使用的動詞「包含」，連同其詞形變化，是以其非限制性含義使用，意指包括在該詞之後的物件，但未排除沒有特別提出的物件。 The present invention provides compositions, methods and systems for plant propagation. In some embodiments, the composition comprises at least one phytohormone. The verb "comprise" as used in the description and the scope of the claims, as well as the singular variations thereof, are used in their non-limiting sense, and are meant to include the item after the word, but the item not specifically recited is not excluded.

在一些具體例中，所述組成物、方法和系統對植物體外繁殖是有用的。如本文使用的術語「植物」指任何屬於植物界的活生物(即，任何植物界中的屬/種)。這包括熟悉的生物，例如但不限於樹木、草本植物、灌木、草、藤、蕨類、苔蘚和綠藻。該等術語係指單子葉植物(也稱為monocots)和雙 子葉植物(也稱為dicots)。特定的植物實例包括但不限於竹、玉米、馬鈴薯、薔薇、蘋果樹、向日葵、小麥、稻、香蕉、番茄、匏瓜(opo)、南瓜、櫛瓜(squash)、萵苣、高麗菜(cabbage)、橡樹、觀賞鳳梨(guzmania)、天竺葵、木槿(hibiscus)、鐵線蓮(clematis)、聖誕紅(poinsettias)、甘蔗、芋頭、浮萍(duck weed)、松樹、草地早熟禾(Kentucky blue grass)、結縷草(zoysia)、椰樹、十字花科葉菜(brassica leafy vegetables，如青花菜(broccoli)、蕪菁(broccoli raab)、球芽甘藍(Brussels sprouts)、高麗菜、大白菜(Chinese cabbage，Bok Choy和Napa)、花椰菜(cauliflower)、義大利甘藍(cavalo)、散葉甘藍(collards)、羽衣甘藍(kale)、球莖甘藍(kohlrabi)、芥菜(mustard greens)、油菜(rape greens)、和其他十字花科葉菜作物)、球莖蔬菜(如大蒜、韭、洋蔥(乾球洋蔥(dry bulb)、綠洋蔥(green)和青蔥(Welch))、珠蔥和其他球莖蔬菜作物)、柑橘類水果(如葡萄柚、檸檬、萊姆、橙、柑橘、雜交柑橘、柚和其他柑橘類水果作物)、葫蘆科蔬菜(如黃瓜、醃菜西瓜(citron melon)、可食葫蘆、醃漬用小黃瓜(gherkin)、甜瓜(muskmelons，包括甜瓜屬的雜交種及/或栽培種)、西瓜、洋香瓜(cantaloupe)和其他葫蘆科蔬菜作物)、果實蔬菜(包括茄子、酸漿(ground cherry)、茄瓜(pepino)、胡椒、番茄、黏果酸漿(tomatillo)和其他果實蔬菜作物)、葡萄、葉菜(如長葉萵苣(romaine))、根/塊莖和球莖蔬菜(如馬 鈴薯)和樹生堅果(杏仁、山胡桃、開心果和胡桃)、漿果(如番茄、小檗、醋栗、接骨木果、鵝莓、忍冬、盾葉鬼臼(mayapples)、莢迷果(nannyberries)、俄勒岡葡萄、沙棘、樸樹果、熊果、越橘、草莓、海葡萄、黑莓、雲莓、羅甘莓、懸鉤子、美莓(salmonberries)、果實樹莓(thimbleberries)和裹白樹莓(wineberries))、穀類作物(如玉米、稻、小麥、大麥、高粱、黍(millets)、燕麥(oats)、黑麥(ryes)、黑小麥(triticales)、蕎麥(buckwheats)、福尼奧米(fonio)和藜麥(quinoa))、梨果(pome fruit)(如蘋果、梨)、核果(stone fruits)(如咖啡、棗、芒果、橄欖、椰子、油椰子、開心果、杏仁、杏、櫻桃、西洋李、油桃、桃和李)、藤(如鮮食葡萄、釀酒葡萄)、纖維作物(如***、棉花)、觀賞植物(ornamentals)等。例如，所述植物為Camelineae族的一個物種，例如C.alyssum、C.anomala、C.grandiflora、C.hispida、C.laxa、C.microcarpa、C.microphylla、C.persistens、C.rumelica、C.sativa、C.Stiefelhagenii或其任何雜交種。 In some embodiments, the compositions, methods, and systems are useful for in vitro propagation of plants. The term "plant" as used herein refers to any living organism that belongs to the plant kingdom (ie, a genus/species in any plant kingdom). This includes familiar organisms such as, but not limited to, trees, herbs, shrubs, grasses, vines, ferns, mosses, and green algae. These terms refer to monocots (also known as monocots) and dicots (also known as dicots). Specific plant examples include, but are not limited to, bamboo, corn, potato, rose, apple, sunflower, wheat, rice, banana, tomato, opo, pumpkin, squash, lettuce, cabbage. , oak, ornamental guzmania, geranium, hibiscus, clematis, poinsettias, sugar cane, taro, duck weed, pine, Kentucky blue grass , zoysia, coconut, Brassica leafy vegetables, such as broccoli, broccoli raab, Brussels sprouts, cabbage, Chinese cabbage , Bok Choy and Napa), cauliflower, cavalo, collard, kale, kohlrabi, mustard greens, rape greens, And other cruciferous leafy crops), bulbous vegetables (such as garlic, alfalfa, onions (dry bulb, green onion (green) and shallot (Welch)), onion and other bulbous vegetable crops), citrus Fruits (such as grapefruit, lemon, Mulberry, orange, citrus, hybrid citrus, pomelo and other citrus fruit crops), cucurbit vegetables (eg cucumber, pickle watermelon (citron melon), edible gourd, pickled gherkin, melon melon (including melon) Hybrids and/or cultivars), watermelon, cantaloupe and other cucurbit vegetable crops, fruits and vegetables (including eggplant, ground cherry, pepino, pepper, tomato, sticky fruit) Physalis (tomatillo and other fruit and vegetable crops), grapes, leafy vegetables (such as romaine), roots/tubers and bulbous vegetables (such as potatoes) and tree nuts (almonds, hickory, pistachios and walnuts), Berries (eg tomatoes, sorghum, gooseberry, elderberry, gooseberry, honeysuckle, mayapples, nannyberries, Oregon grapes, sea buckthorn, arborvitae, bearberry, bilberry, strawberry, Sea grapes, blackberries, cloudberries, raspberries, raspberries, salmonberries, thimbleberries and wineberries, cereal crops (such as corn, rice, wheat, barley, sorghum, alfalfa) (millets), oats (oat s), ryes, triticales, buckwheats, fonio and quinoa, pome fruit (eg apples, pears), stone fruit ( Stone fruits) (eg coffee, dates, mangoes, olives, coconuts, coconut palms, pistachios, almonds, apricots, cherries, western plums, nectarines, peaches and plums), vines (eg table grapes, wine grapes), fiber crops (such as hemp, cotton), ornamentals, etc. For example, the plant is a species Camelineae group, e.g. C.alyssum, C.anomala, C.grandiflora, C.hispida, C.laxa, C.microcarpa, C.microphylla, C.persistens, C.rumelica, C .sativa , C.Stiefelhagenii or any of its hybrids.

在一些具體例中，所述組成物、方法和系統對於作物植物的體外繁殖是有用的。本文使用的術語「作物植物」指任何生長用於任何商業目的的植物，其包括但不限於下列目的：種子生產、乾草生產、裝飾用途、果實生產、漿果生產、蔬菜生產、油生產、蛋白質生產、草料生產、動物牧草、高爾夫球場、草坪、花卉生產、景觀、侵蝕防治(erosion control)、 綠色肥料、提高土壤耕作性(tilth)/健康、生產醫藥產品/藥品、生產食品或食品添加劑、吸煙產品、紙漿生產和木材生產。 In some embodiments, the compositions, methods, and systems are useful for in vitro propagation of crop plants. The term "crop plant" as used herein refers to any plant grown for any commercial purpose including, but not limited to, seed production, hay production, decorative use, fruit production, berry production, vegetable production, oil production, protein production. , forage production, animal pasture, golf course, lawn, flower production, landscape, erosion control, Green fertilizer, improved soil tillage/health, production of pharmaceutical products/pharmaceuticals, production of food or food additives, smoking products, pulp production and wood production.

在一些具體例中，本發明提供了從本文描述的組成物、方法和系統生產的植物所衍生的植物部分。本文使用的術語「植物部分」指植物的任何部分，其包括但不限於苗、根、莖、種子、托葉(stipules)、葉、花瓣、花、胚珠、苞片(bracts)、枝、葉柄、節間、樹皮、被短柔毛(pubescence)、分蘖(tillers)、根莖(rhizomes)、葉狀體(fronds)、葉片、花粉、雄蕊等等。在某種培養基如土壤中生長的植物的兩個主要部分，經常被稱為「地上」部分，也經常被稱為「苗」，以及「地下」部分，也經常被稱為「根」。本文使用的用語「衍生於」係指起源或來源，且可包括天然產生的、重組的、未純化的或純化的分子。衍生於某一起源或來源的核酸或胺基酸可具有如本文其他處定義的所有種類的核苷酸改變或蛋白質修飾。本文使用的用語「一種」或「一個」係指該實體的一或多者，例如「一種基因」係指一或多種基因，或至少一種基因。同樣地，用語「一種」(或「一個」)、「一或多種」和「至少一種」在本文中可互換使用。此外，由不定冠詞「一種」或「一個」所提及之「元件」未排除存在有多於一種該元件的可能性，除非上下文清楚地要求有且僅有一種該元件。 In some embodiments, the invention provides plant parts derived from plants produced by the compositions, methods, and systems described herein. The term "plant part" as used herein refers to any part of a plant including, but not limited to, seedlings, roots, stems, seeds, stipules, leaves, petals, flowers, ovules, bracts, branches, petioles , internodes, bark, pubescence, tillers, rhizomes, fronds, leaves, pollen, stamens, etc. The two main parts of plants that grow in a medium such as soil, often referred to as the "ground" part, are often referred to as "miao" and "underground" parts, often referred to as "roots." The term "derived from" as used herein refers to origin or source and may include naturally occurring, recombinant, unpurified or purified molecules. Nucleic acids or amino acids derived from a source or source may have all kinds of nucleotide alterations or protein modifications as defined elsewhere herein. The term "a" or "an" as used herein refers to one or more of the entities, such as "a gene" refers to one or more genes, or at least one gene. Similarly, the terms "a" (or "an", "one or more" and "said" are used interchangeably herein. In addition, the "element" referred to by the indefinite article "a" or "an" does not exclude the <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt;

在一些具體例中，本發明提供了從本文描述的組成物、方 法和系統產生的植物所衍生的植物組織。本文使用的術語「植物組織」指植物的任何部分。植物器官的實例包括但不限於葉、莖、根、塊莖、種子、枝、被短柔毛、根瘤(nodule)、葉腋、花、花粉、雄蕊、雌蕊、花瓣、花序梗(peduncle)、柄(stalk)、柱頭、花柱、苞片、果實、幹(trunk)、心皮(carpel)、萼片、花藥、胚珠、花梗(pedicel)、針葉(needle)、毬果(cone)、根莖、匍匐枝(stolon)、苗、果皮(pericarp)、胚乳(endosperm)、胎座(placenta)、漿果(berry)、雄蕊和葉鞘。 In some embodiments, the invention provides compositions, squares from the description herein Plant tissue derived from plants produced by the method and system. The term "plant tissue" as used herein refers to any part of a plant. Examples of plant organs include, but are not limited to, leaves, stems, roots, tubers, seeds, branches, pubescence, nodule, leaf mites, flowers, pollen, stamens, pistils, petals, peduncles, stalks ( Stalk), stigma, style, sepals, fruit, trunk, carpel, sepals, anthers, ovules, pedicels, needles, cones, rhizomes, lychees (stolon), seedlings, pericarp, endosperm, placenta, berries, stamens and sheaths.

在一些具體例中，所述組成物、方法和系統對於單子葉植物的繁殖是有用的。本文使用的術語「單子葉植物的」或「單子葉植物」係指有花植物的任何亞綱(單子葉植物綱(Monocotyledoneae))，其具有僅含有單種葉的胚且通常具有平行脈葉，花的部分為3的倍數，且在莖和根處沒有次生生長。實例包括百合；蘭；稻；玉米，草，諸如高羊茅(tall fescue)、山羊麥(goat grass)和草地早熟禾(Kentucky bluegrass)；穀物，諸如小麥、燕麥和大麥；鳶尾；洋蔥和棕櫚。 In some embodiments, the compositions, methods, and systems are useful for the propagation of monocots. The term "monocotyledonous" or "monocotyledonous plant" as used herein refers to any subclass of the flowering plant (Monocotyledoneae) having embryos containing only a single leaf and usually having parallel veins The part of the flower is a multiple of 3 and there is no secondary growth at the stem and root. Examples include lily; orchid; rice; corn, grass, such as tall fescue, goat grass, and Kentucky bluegrass; cereals such as wheat, oats and barley; iris; onion and palm .

在一些具體例中，所述組成物、方法和系統對於竹植物的體外繁殖是有用的。本文使用的術語「竹」係指竹亞科的植物。在國際專利申請公開號WO2011100762(以引用方式全部併入本文)中描述了代表性竹之屬。 In some embodiments, the compositions, methods, and systems are useful for in vitro propagation of bamboo plants. The term "bamboo" as used herein refers to a plant of the family Bamboo. Representative bamboo genus is described in International Patent Application Publication No. WO 2011100762, which is incorporated herein in its entirety by reference.

在一些具體例中，本發明提供了包含本文所描述的組成 物、方法和系統生產的植物種源(germplasm)的植物。本文使用的術語「種源」指具有其特定的分子和化學構成的遺傳物質，該遺傳物質包含了生物的遺傳素質的物質基礎。 In some embodiments, the invention provides compositions comprising the compositions described herein Plant, germplasm, and plant produced by the method, system, and system. As used herein, the term "species source" refers to a genetic material having its specific molecular and chemical composition that contains the material basis of the genetic qualities of the organism.

在一些具體例中，本發明提供了從本文所描述的組成物、方法和系統產生的植物衍生的後代。本文使用的術語「後代」指從一種或多種親本植物或其後代的無性或有性生殖作為後代產生的任何植物。例如，可藉由親本植物的選殖或自交(selfing)或兩種親本植物的雜交產生後代植物，且包括自交後代(selfings)以及F1或F2或之後的世代。F1是從親本產生的第一代後代，親本中至少一個是第一次作為某一性狀的供體使用，而第二代(F2)或後續世代(F3、F4等)的後代是從F1、F2等的自交產生的樣品。因此F1可以是(且通常是)從兩個繁殖純種(true breeding)的親本之間雜交產生的雜種(繁殖純種對於某一性狀為同型接合的)，而F2可以是(且通常是)從所述F1雜交體的自花授粉產生的後代。 In some embodiments, the invention provides plant derived progeny produced from the compositions, methods, and systems described herein. The term "progeny" as used herein, refers to any plant produced as a progeny from the asexual or sexual reproduction of one or more parent plants or their progeny. For example, progeny plants can be produced by colonization or selfing of the parental plants or by crossing of the two parental plants, and include selfings and F1 or F2 or subsequent generations. F1 is the first generation of offspring produced from the parent. At least one of the parents is used for the first time as a donor of a certain trait, while the descendants of the second generation (F2) or subsequent generations (F3, F4, etc.) are from A sample produced by selfing of F1, F2, and the like. Thus F1 can be (and usually is) a hybrid produced by crossing between two naturally-bred parents (the breeding pure is homozygous for a trait), and F2 can be (and usually a progeny produced from self-pollination of the F1 hybrid.

在一些具體例中，本發明提供了從本文描述的組成物、方法和系統產生的植物衍生的雜交，和製造和使用這類雜交的方法。本文使用的術語「雜交(cross)」、「雜交(crossing)」、「異花授粉」或「雜交育種」指一種藉由植物上一朵花的花粉被施加(人工地或自然地)至另一植物上的花的胚珠(柱頭)上之過程。 In some embodiments, the invention provides plant-derived hybridizations, and methods of making and using such hybrids, produced from the compositions, methods, and systems described herein. The term "cross", "crossing", "cross-pollination" or "hybrid breeding" as used herein refers to a method in which a pollen of a flower on a plant is applied (manually or naturally) to another The process of ovules (columns) on the flowers of a plant.

在一些具體例中，本發明提供了從本文描述的組成物、方 法和系統產生的植物衍生的栽培種(cultivars)。本文使用的術語「栽培種」係指已由園藝學(horticultural)或農藝學(agronomic)技術產生的植物的變種(variety)、品系(strain)或品種(race)，並且正常情況下不存在於野生種群中。 In some embodiments, the invention provides compositions, squares from the description herein Plant-derived cultivars produced by methods and systems. The term "cultivar" as used herein refers to a variety, strain, or race of a plant that has been produced by horticultural or agronomic techniques and does not normally exist in In the wild population.

在一些具體例中，本發明提供了從本文描述的組成物、方法和系統產生的植物衍生的變種。本文使用的術語「變種」係指物種的細分，由一組該物種中的個體組成，這組個體在形態或功能上與其他類似的一群個體是截然不同的。 In some embodiments, the invention provides plant-derived variants produced from the compositions, methods, and systems described herein. As used herein, the term "variant" refers to a subdivision of a species consisting of a group of individuals in the species that are distinctly morphologically or functionally distinct from other similar groups of individuals.

本文使用的術語「變種」或「栽培種」係指一組類似的植物，其藉由結構特徵和性能可以將該組植物與同一物種中的其他變種識別出來。本文使用的術語「變種」與於1961年12月2日生效、於1972年11月10日、1978年10月23日和1991年3月19日在日內瓦修訂的保護植物新品種國際公約(International Convention for the Protection of New Varieties of Plants)(UPOV條約)中相應的定義具有等同的含義。因此，「變種」指最低的已知層級的單個植物學分類(botanical taxon)中的植物族群(plant grouping)，該族群，不考慮育種者權利的授予條件是否完全達到，可以i)由特定的基因型或基因型組合產生特徵的表現來定義，ii)由至少一種所述特徵的表現與任何其他植物族群區別，和iii)就其進行不變繁殖的適應性而被認為是一個單元。 The term "variant" or "cultivated species" as used herein refers to a group of similar plants that are capable of recognizing the group of plants and other varieties in the same species by structural features and properties. The term "variant" as used herein and the International Convention for the Protection of New Varieties of Plants, which came into effect on December 2, 1961, and was amended in Geneva on November 10, 1972, October 23, 1978, and March 19, 1991 (International The corresponding definitions in the Convention for the Protection of New Varieties of Plants) have equivalent meanings. Thus, "variant" refers to the plant grouping in a single botanical taxon of the lowest known level, regardless of whether the conditions for granting the breeder's rights are fully met, i) by specific A genotype or genotype combination produces a characteristic representation to define, ii) is distinguished from any other plant population by at least one of said characteristics, and iii) is considered a unit for its adaptability to invariant reproduction.

在一些具體例中，本發明提供了從本文描述的組成物、方 法和系統產生的植物衍生的基因型。本文使用的術語「基因型」指個體細胞、細胞培養物、組織、生物(如植物)或生物群體的遺傳構成。 In some embodiments, the invention provides compositions, squares from the description herein Plant-derived genotypes produced by methods and systems. The term "genotype" as used herein refers to the genetic makeup of an individual cell, cell culture, tissue, organism (eg, a plant), or a biological population.

在一些具體例中，本發明提供了從本文描述的組成物、方法和系統產生的植物衍生的選殖株。本文使用的術語「選殖株」指從單個前體遺傳或衍生的，且遺傳上與該前體等同或基本上等同的細胞、細胞群體、部分、組織、生物(如植物)或生物群體。在一些具體例中，所述選殖株產生於包括至少一個無性步驟的過程。 In some embodiments, the invention provides plant-derived selection strains produced from the compositions, methods, and systems described herein. The term "selected strain" as used herein, refers to a cell, cell population, part, tissue, organism (eg, plant) or biological population that is inherited or derived from a single precursor and that is genetically equivalent or substantially equivalent to the precursor. In some embodiments, the selected strain is produced in a process comprising at least one asexual step.

在一些具體例中，本發明提供了從本文描述的組成物、方法和系統產生的植物衍生的雜種。本文使用的術語「雜種」指在一個或多個基因上不同的親體之間的雜交產生的任何個體細胞、組織或植物。 In some embodiments, the invention provides plant-derived hybrids produced from the compositions, methods, and systems described herein. The term "hybrid" as used herein, refers to any individual cell, tissue or plant produced by the crossing between one or more genetically distinct broods.

在一些具體例中，本發明提供了從本文描述的組成物、方法和系統產生的植物衍生的近交系(inbreds)。本文使用的術語「近交系」或「近交品系」指相對的純種品系。 In some embodiments, the invention provides plant-derived inbreds produced from the compositions, methods, and systems described herein. The term "inbred line" or "inbred line" as used herein refers to a relative pure line.

在一些具體例中，本發明提供了從本文描述的組成物、方法和系統產生的植物衍生的種群。本文使用的術語「種群」係指具有共同的遺傳衍生過程(genetic derivation)的遺傳上同質或異質的植物集合。 In some embodiments, the invention provides plant-derived populations produced from the compositions, methods, and systems described herein. The term "population" as used herein refers to a collection of genetically homogenous or heterogeneous plants having a common genetic derivation.

在一些具體例中，本發明提供了用於植物物種生物培養(bioculture)的生物反應器。本文使用的術語「生物反應器」 指任何能保存、支持和/或生長可存活組織的器皿、裝置或系統。換言之，本文使用的術語「生物反應器」可指保存可存活的植物組織的生長器皿，保存、支持和/或生長可存活組織需要的或說明保存、支持和/或生長可存活組織的生長器皿內部或外部的各種其他元件，和其任何子系統。 In some embodiments, the invention provides a bioreactor for use in plant species bioculture. The term "bioreactor" as used herein Refers to any vessel, device, or system that can hold, support, and/or grow viable tissue. In other words, the term "bioreactor" as used herein may refer to a growth vessel that preserves viable plant tissue, a growth vessel that preserves, supports, and/or grows viable tissue or that describes preservation, support, and/or growth of viable tissue. Various other components, internal or external, and any of its subsystems.

在一些具體例中，本發明提供了用於植物物種生物培養的間歇浸沒式生物反應器。本文使用的術語「間歇浸沒式生物反應器」指設計為分別由營養培養基迴圈的流入和流出到生物反應器滋養可存活組織的任何生物反應器。經常在水培中使用間歇浸沒式生物反應器。本文使用的「植物繁殖系統」是用於生長可存活的植物組織的生物反應器。 In some embodiments, the invention provides an intermittent immersion bioreactor for the biological cultivation of plant species. As used herein, the term "intermittent immersion bioreactor" refers to any bioreactor designed to nourish viable tissue by inflow and outflow into the bioreactor, respectively, from the nutrient medium loop. Intermittent immersion bioreactors are often used in hydroponics. As used herein, "plant propagation system" is a bioreactor for growing viable plant tissue.

(組成物) (composition)

微體繁殖是使用現代的植物組織培養方法，以實施快速增殖原種植物材料而產生大量後代植物。微體繁殖係用於增殖新的植物，如那些藉由習知植物育種方法已經被遺傳修飾的或育種的。其還被用於為從不產生種子或對無性生殖反應不佳的原種植物進行的種植提供足量的小植株。 Micropropagation is the use of modern plant tissue culture methods to produce rapid proliferative plant material to produce a large number of progeny plants. Micropropagation is used to propagate new plants, such as those that have been genetically modified or bred by conventional plant breeding methods. It is also used to provide sufficient plantlets for planting from native plants that do not produce seeds or that have poor asexual reproduction.

微體繁殖可首先從要繁殖的植物材料的選擇開始。清潔的沒有病毒和真菌的原種材料在最健康的植物的生產中是重要的。一旦選擇了用於培養的植物材料，即開始培植體的收集且根據將要使用的組織類型，所述組織類型包括莖尖(stem tips)、花藥、花瓣、花粉和其他植物組織。然後將該 培植體材料表面滅菌，通常以多個過程的漂白和酒精洗滌且最後在無菌水中漂洗。將這小部分植物組織，有時僅為單個細胞，置於通常含有蔗糖作為能量來源和一種或多種植物生長調節劑(植物激素)的生長培養基上。通常將該培養基用瓊脂稠化(thicken)以製造在生長中支撐該培植體的凝膠。一些植物在簡單的培養基上容易地生長，而其他植物需要更複雜的培養基以成功生長；植物組織依賴培養基生長並分化為新的組織。例如，含有細胞***素的培養基被用於從植物芽製造分枝的苗。 Micropropagation can begin with the selection of plant material to be propagated. Clean, original materials without viruses and fungi are important in the production of the healthiest plants. Once the plant material for cultivation is selected, the collection of the implant is initiated and depending on the type of tissue to be used, the tissue types include stem tips, anthers, petals, pollen, and other plant tissues. Then The body material is surface sterilized, usually bleached and washed with multiple processes and finally rinsed in sterile water. This small portion of plant tissue, sometimes only a single cell, is placed on a growth medium that typically contains sucrose as an energy source and one or more plant growth regulators (plant hormones). The medium is typically thickened with agar to make a gel that supports the culture during growth. Some plants grow easily on simple media, while others require more complex media for successful growth; plant tissues rely on media to grow and differentiate into new tissues. For example, a medium containing cytokinin is used to produce branched shoots from plant shoots.

增殖係為取出第一階段中產生的組織樣本並增加它們的數量。在成功的引入和生長植物組織之後，於建立階段(establishment stage)後接著增殖。藉由該過程的重複迴圈，可以將單個培植體樣本從一個增加到數百或上千的植物。根據生長的組織的類型，增殖可牽涉不同的方法和培養基。如果生長的植物材料是癒合組織(callus tissue)，可將其置於攪拌器中且切成更小的塊並在同一類型的培養基上重培養以生長更多的癒合組織。如果所述組織作為稱作小植株的小植物生長，則經常添加激素以使小植株產生許多可以被移除和重培養的分株(offshoots)。 The proliferation is to take out the tissue samples produced in the first stage and increase their number. After successful introduction and growth of plant tissue, proliferation is followed by an establishment stage. With a repeating loop of the process, a single implant sample can be increased from one to hundreds or thousands of plants. Proliferation can involve different methods and media depending on the type of tissue being grown. If the growing plant material is a callus tissue, it can be placed in a blender and cut into smaller pieces and re-cultured on the same type of medium to grow more healing tissue. If the tissue is grown as a small plant called a plantlet, hormones are often added to cause the plantlets to produce many offshoots that can be removed and re-cultured.

下一階段(「移植前(pretransplant)階段」)係牽涉處理產生的小植株/苗以促進根生長和「硬化(hardening)」，其係在體外或在無菌或基本無菌的環境中施行。「硬化」指用於自然 生長環境的植物的準備。在本階段之前，小植株係設計為促進快速生長的「理想」條件下生長。由於缺乏必要性，植物很可能對疾病是高度易感的且經常不具有完全功能的表皮覆蓋，而且在它們對水和能源的使用中效率會比較低。體外條件下的濕度高且在這些條件下生長的植物並不形成阻止植物脫水的有效角質層(cuticle)和氣孔(stomata)，在從培養中取出時，小植株需要時間來調節適應更自然的環境條件。硬化通常牽涉將小植株從高濕度、低光照、溫暖的環境緩慢脫離到可以看做是所述物種的自然生長環境的環境。 The next stage ("pretransplant stage") involves handling the resulting plantlets/emergence to promote root growth and "hardening", either in vitro or in a sterile or substantially sterile environment. "hardening" means used for nature Preparation of plants for growing environment. Prior to this stage, plantlets were designed to promote growth under "ideal" conditions for rapid growth. Due to the lack of necessity, plants are likely to be highly susceptible to disease and often do not have fully functional epidermal coverage, and are less efficient in their use of water and energy. Plants with high humidity under in vitro conditions and grown under these conditions do not form effective cuticles and stomatas that prevent plant dehydration. When removed from culture, plantlets need time to adjust to more natural adaptation. Environmental conditions. Hardening typically involves slowly detaching plantlets from high humidity, low light, warm environments to an environment that can be considered a natural growth environment for the species.

在植物微體繁殖的最終階段，將小植株從植物培養基中移出並轉移到土壤或(更普遍地)盆栽肥料中以用習知方法繼續生長。該階段經常和「移植前」階段組合。 In the final stage of plant micropropagation, the plantlets are removed from the plant medium and transferred to soil or (more generally) potted fertilizer for continued growth by conventional methods. This phase is often combined with the "pre-transplantation" phase.

現代植物組織培養在過濾空氣的無菌條件下施行。來自該環境的活的植物材料在其表面(且有時在內部)被微生物自然污染，因此起始材料(培植體)在化學溶液(通常是酒精或漂白劑)中的表面滅菌是必需的。然後通常將培植體置於固體培養基的表面，但有時直接置於液體培養基中，特別是期望細胞懸浮培養的時候。固體和液體培養基一般由無機鹽和一些有機營養物、維生素和植物激素組成。固體培養基由液體培養基添加通常為純化瓊脂的膠凝劑(gelling agent)製備而來。 Modern plant tissue culture is performed under sterile conditions that filter air. Living plant material from this environment is naturally contaminated by microorganisms on its surface (and sometimes inside), so surface sterilization of the starting material (cultivation) in a chemical solution (usually alcohol or bleach) is necessary. The culture is then typically placed on the surface of the solid medium, but sometimes placed directly in the liquid medium, especially when the cells are in suspension culture. Solid and liquid media generally consist of inorganic salts and some organic nutrients, vitamins and phytohormones. The solid medium is prepared by adding a gelling agent, usually a purified agar, to the liquid medium.

培養基的組成，特別是植物激素和氮源(硝酸鹽對比銨鹽 或胺基酸)對於從起始培植體生長的組織的形態具有深遠的影響。例如，植物生長素過量經常將導致根的增生(proliferation)，而細胞***素的過量可產生苗。植物生長素和細胞***素的平衡經常將產生細胞或癒合組織的無序生長，但是長出物(outgrowth)的形態將依賴於植物物種和培養基組成。隨著培養物的生長，通常將塊切下並轉移到新的培養基(繼代培養(subculture))以允許生長或改變培養物的形態。組織培養者的技能和經驗在判斷應培養哪塊和丟棄哪些塊時是重要的。當培養物中顯現出苗時，可以將其切下並用生長素生根以產生小植株，可以在小植株成熟時將其轉移到盆栽土以進一步在溫室中作為正常的植物生長。 The composition of the medium, especially the phytohormone and nitrogen source (nitrate versus ammonium salt) Or amino acid) has a profound effect on the morphology of the tissue grown from the starting culture. For example, an excess of auxin will often result in the proliferation of roots, while an excess of cytokinin can produce shoots. The balance of auxin and cytokinin will often produce disordered growth of cells or healing tissues, but the morphology of the outgrowth will depend on the plant species and medium composition. As the culture grows, the pieces are typically cut and transferred to a new medium (subculture) to allow growth or to alter the morphology of the culture. The skills and experience of the tissue cultivator is important in determining which pieces should be cultivated and which pieces are discarded. When the shoot appears in the culture, it can be cut and rooted with auxin to produce plantlets, which can be transferred to potting soil as the plant grows to further grow as a normal plant in the greenhouse.

從植物獲得的要培養的組織被稱為培植體。基於某些模式系統特別是煙草的工作，經常聲稱可以從植物的任何部分生長出全能的培植體。然而，在實施時已證明該觀念是無效的。在許多物種中，不同器官的培植體在它們的生長和再生的速度上不同，而一些完全不生長。培植體材料的選擇還決定了由組織培養產生的小植株是單倍體或雙倍體的。用不適當的培植體還會增加微生物污染的風險。因此在組織培養之前做出培植體的適當選擇是非常重要的。 The tissue to be cultured obtained from the plant is called a culture body. Based on the work of certain model systems, particularly tobacco, it is often claimed that pluripotent implants can be grown from any part of the plant. However, this concept has proven to be ineffective at the time of implementation. In many species, implants of different organs differ in their rate of growth and regeneration, while some do not grow at all. The choice of culture material also determines whether the plantlets produced by tissue culture are haploid or diploid. Improper implants also increase the risk of microbial contamination. Therefore, it is very important to make appropriate selection of the culture body before tissue culture.

不同的器官和培植體在再生潛力上的特定差異具有不同的解釋。顯著的因素包括細胞迴圈中的細胞階段、內源生長調節劑的可獲性或運輸能力和細胞的代謝能力的差異。最普 遍使用的組織培植體是植物的分生組織末端，如莖尖、腋芽尖(auxiliary bud tip)和根尖。這些組織具有高的細胞***速率且或集中或產生需要的生長調節物質，包括植物生長素和細胞***素。一些培植體，如根尖，係難以分離且被土壤微生物相(microflora)污染而在組織培養過程中成問題。某些土壤微生物相可與根系統形成緊密的結合，或甚至在根內生長。與根結合的土壤顆粒難以在對根沒有損傷的情況下移除，而損傷隨後會允許微生物的侵襲。這些結合的微生物相一般會在植物組織顯著生長之前在組織培養基上過度生長。氣生(土壤之上)的培植體也富含不想要的微生物相。然而，它們更容易藉由溫和的漂洗從培植體上移除，且通常可藉由表面滅菌將剩餘物殺死。大多數表面微生物相不與植物組織形成緊密的結合。這樣的結合通常可藉由培植體表面上的花斑(mosaic)、脫色或局部壞死之外觀檢查而發現。 Different organs and implants have different interpretations of specific differences in reproductive potential. Significant factors include differences in the cell stage in the cell cycle, the availability or transport capacity of the endogenous growth regulator, and the metabolic capacity of the cell. Most popular The tissue cultures used throughout are the meristematic ends of the plant, such as the tip of the stem, the auxiliary bud tip, and the apex. These tissues have a high rate of cell division and either concentrate or produce the desired growth regulating substances, including auxins and cytokinins. Some implants, such as root tips, are difficult to separate and are contaminated by soil microflora and are problematic during tissue culture. Certain soil microbial phases can form tight bonds with the root system or even grow within the roots. The soil particles combined with the roots are difficult to remove without damage to the roots, and the damage subsequently allows the invasion of the microorganisms. These combined microbial phases generally overgrow on tissue culture media prior to significant growth of plant tissue. The aerial (upper soil) implants are also rich in unwanted microbial phases. However, they are easier to remove from the implant by gentle rinsing and the residue can usually be killed by surface sterilization. Most surface microbial phases do not form a tight bond with plant tissue. Such combinations are typically found by visual inspection of mosaic, discoloration or local necrosis on the surface of the implant.

獲得未污染的培植體的一個選擇性方式是從幼苗(seedlings)取得培植體，所述幼苗是從表面滅菌的種子無菌生長的。對於強表面滅菌劑如次氯酸鹽的滲透，種子的硬表面較不可滲透，因此用於種子可接受的滅菌條件較用於無性組織者嚴格得多。 An alternative way to obtain uncontaminated implants is to obtain cultures from seedlings that are aseptically grown from surface sterilized seeds. For the penetration of strong surface sterilants such as hypochlorite, the hard surface of the seed is less impermeable, so the sterilization conditions acceptable for the seed are much more stringent than those used for the asexual organizer.

組織培養的植物為選殖株時，如果用以產生第一培植體的原始母系植物對於病原體或環境條件是易感的，整個作物對於同一問題也會是易感的，且相反地任何陽性性狀也會保留 在該系內。在植物科學中廣泛使用植物組織培養，其還具有許多商業應用。所述應用包括： When the tissue cultured plant is a selection strain, if the original mother plant used to produce the first culture is susceptible to pathogens or environmental conditions, the entire crop will be susceptible to the same problem, and conversely any positive trait Will also keep Within the department. Plant tissue culture is widely used in plant science and it also has many commercial applications. The applications include:

1.在林業和花卉栽培中廣泛使用微體繁殖。微體繁殖還可用於保存稀有的或瀕危的植物物種。 1. Micropropagation is widely used in forestry and flower cultivation. Micropropagation can also be used to preserve rare or endangered plant species.

2.植物育種者可使用組織培養來篩選細胞而非植物的有利特徵，如病原體抗性/耐受性。 2. Plant Breeders can use tissue culture to screen for favorable characteristics of cells other than plants, such as pathogen resistance/tolerance.

3.將生物反應器內液體培養中的植物細胞的大規模生長作為次產物的來源，如作為生物藥使用的重組蛋白。 3. Large-scale growth of plant cells in liquid culture in a bioreactor as a source of secondary products, such as recombinant proteins for use as biopharmaceuticals.

4.藉由原生質體(protoplast)融合和新的雜交體的再生將遠源物種雜交。 4. Hybridization of distant species by protoplast fusion and regeneration of new hybrids.

5.對遠源物種雜交授粉並隨後將產生的胚組織培養，否則所述胚在正常情況下會死亡(胚胎拯救(Embryo Rescue))。 5. Hybrid pollination of the distant species and subsequent propagation of the resulting embryo tissue, otherwise the embryo will die under normal conditions (Embryo Rescue).

6.用於從單倍體培養產生加倍的單倍體(雙倍體)植物以在育種程序中更快地獲得同型接合系，其通常係藉由秋水仙素(colchicine)處理導致染色體數目的加倍。 6. For the production of doubled haploid (diploid) plants from haploid culture to obtain homozygous junctions more quickly in the breeding program, which is usually caused by colchicine resulting in the number of chromosomes. double.

7.作為用於轉化的組織，隨後是遺傳構建體的短期測試或轉基因植物的再生。 7. As a tissue for transformation, followed by a short-term test of the genetic construct or regeneration of the transgenic plant.

8.可使用某些技術如分生組織尖培養以從如馬鈴薯和許多軟果實物種的被感染的原種產生乾淨的植物材料。 8. Certain techniques, such as meristematic tip culture, can be used to produce clean plant material from infected stocks such as potatoes and many soft fruit species.

9.使用分生組織和苗培養以產生大量等同個體的微體繁殖。 9. Use meristematic tissue and shoot culture to produce micropropagation of a large number of equivalent individuals.

用於植物微體繁殖的培養基和方法至少已描述於M.R. Ahuja，Micropropagation of woody plants，Springer，1993，ISBN 0792318072，9780792318071；Narayanaswamy，Plant cell and tissue culture，Tata McGraw-Hill Education，1994，ISBN 0074602772，9780074602775；Singh和Kumar，Plant Tissue Culture，APH Publishing，2009，ISBN 8131304396，9788131304396；Y.P.S.Bajaj High-tech and micropropagation V，Springer，1997，ISBN 3540616063，9783540616061；Trigiano和Gray，Plant Tissue Culture，Development and Biotechnology，CRC Press，2010，ISBN 1420083260，9781420083262；Gupta和Ibaraki，Plant tissue culture engineering Volume 6 of Focus on biotechnology，Springer，2006，ISBN 1402035942，9781402035944；Jain和Ishii，Micropropagation of woody trees and fruits Volume 75 of Forestry sciences，Springer，2003，ISBN 1402011350，9781402011351；和Aitken-Christie等，Automation and environmental control in plant tissue culture，Springer，1995，ISBN 0792328418，9780792328414，其各以引用方式全部併入本文中。 Media and methods for plant micropropagation have been described, at least, in MR Ahuja, Micropropagation of woody plants , Springer, 1993, ISBN 0792318072, 9780792318071; Narayanaswamy, Plant cell and tissue culture , Tata McGraw-Hill Education, 1994, ISBN 0074602772, 9780074602775; Singh and Kumar, Plant Tissue Culture , APH Publishing, 2009, ISBN 8131304396, 9788131304396; YPSBajaj High-tech and micropropagation V , Springer, 1997, ISBN 3540616063, 9783540616061; Trigiano and Gray, Plant Tissue Culture, Development and Biotechnology , CRC Press, 2010, ISBN 1420083260, 9781420083262; Gupta and Ibaraki, Plant tissue culture engineering Volume 6 of Focus on biotechnology , Springer, 2006, ISBN 1402035942, 9781402035944; Jain and Ishii, Micropropagation of woody trees and fruits Volume 75 of Forestry sciences, Springer , 2003, ISBN 1402011350,9781402011351; and Aitken-Christie et, Automation and environmental control in plant tissue culture, Springer, 1995 ISBN 0792328418,9780792328414, each of which is incorporated herein by reference in its entirety.

國際專利申請公告之WO2011100762(其以引用方式全部併入本文)中已描述了用於竹微體繁殖的培養基和方法。 Media and methods for bamboo micropropagation have been described in WO2011100762, the entire disclosure of which is hereby incorporated by reference.

本發明提供了可用於植物如竹植物的體外微體繁殖的培養基。所述培養基可以是液體的、半固體的或固體的，且所 述培養基的物理狀態可以藉由摻入一種或多種膠凝劑而變化。可以使用本領域已知的任何適用於植物組織培養基的膠凝劑。瓊脂是最普遍用於該目的，這類瓊脂的實例包括Agar Type A、E或M和Bacto^TMAgar。其他示例性的膠凝劑包括角叉膠(carrageenan)、吉蘭糖膠(gellan gum)(由市售PhytaGel^TM、Gelrite®和Gelzan^TM獲得)、褐藻酸(alginic acid)和其鹽，和瓊脂糖。也可使用這些試劑的混合物，如兩種或更多種瓊脂、角叉膠、吉蘭糖膠、瓊脂糖和褐藻酸或其鹽。典型地，所述培養基包含瓊脂，且添加了各種化合物如營養物、無機鹽、生長調節劑、碳源、維生素和其他化合物。 The present invention provides a medium that can be used for in vitro micropropagation of plants such as bamboo plants. The medium may be liquid, semi-solid or solid, and the physical state of the medium may be varied by incorporation of one or more gelling agents. Any gelling agent suitable for use in plant tissue culture media known in the art can be used. Agar is most commonly used for this purpose, examples of such include agar Agar Type A, E or M and Bacto ^TM Agar. Other exemplary gelling agents include carrageenan (carrageenan), gellan gum (gellan gum) (from commercially available PhytaGel ^TM, Gelrite® and obtained Gelzan ^TM), alginic acids (alginic acid) and its salts, agar and sugar. Mixtures of these agents may also be used, such as two or more of agar, carrageenan, gellan gum, agarose, and alginic acid or a salt thereof. Typically, the medium contains agar and various compounds such as nutrients, inorganic salts, growth regulators, carbon sources, vitamins and other compounds are added.

在一些具體例中，所述培養基包含一種或多種植物生長需要的最低限度的營養物，如胺基酸、大量元素(macroelements)、微量元素、鋁、硼、氯(氯化物)、鉻、鈷、銅、碘、鐵、鉛、鎂、錳、鉬、氮(硝酸鹽)、鉀、磷(磷酸鹽)、矽、鈉、硫(硫酸鹽)、鈦、釩、鋅、纖維醇(inositol)和未限定的培養基組分如酪蛋白水解物(casein hydrolysates)或酵母萃取物。例如，所述培養基可包括以下的任意組合：NH₄NO₃、KNO₃、Ca(NO₃)₂、K₂SO₄、MgSO₄、MnSO₄、ZnSO₄、K₂SO₅、CuSO₄、CaCl₂、KI、CoCl₂、H₃BO₃、Na₂MoO₄、KH₂PO₄、FeSO₄、Na₂EDTA、Na₂H₂PO4、纖維醇(如肌醇)、硫胺(thiamine)、吡哆醇(pyridoxine)、煙酸(nicotinic acid)、甘胺酸、核黃素(riboflavin)、抗壞血酸(ascorbic acid)及矽標 準溶液。所述培養基可進一步包含其他的已在例如國際專利申請公告之WO2011100762中描述的組分。本領域的技術人員已知可省去一種或多種上述組分而不影響培養基的功能。 In some embodiments, the medium comprises one or more minimal nutrients required for plant growth, such as amino acids, macroelements, trace elements, aluminum, boron, chloride (chloride), chromium, cobalt. , copper, iodine, iron, lead, magnesium, manganese, molybdenum, nitrogen (nitrate), potassium, phosphorus (phosphate), barium, sodium, sulfur (sulfate), titanium, vanadium, zinc, inositol And undefined medium components such as casein hydrolysates or yeast extracts. For example, the medium may include any combination of the following: NH ₄ NO ₃ , KNO ₃ , Ca(NO ₃ ) ₂ , K ₂ SO ₄ , MgSO ₄ , MnSO ₄ , ZnSO ₄ , K ₂ SO ₅ , CuSO ₄ , CaCl ₂ , KI, CoCl ₂ , H ₃ BO ₃ , Na ₂ MoO ₄ , KH ₂ PO ₄ , FeSO ₄ , Na ₂ EDTA, Na ₂ H ₂ PO4, cellulose alcohol (such as inositol), thiamine (thiamine), pyridine Pyridoxine, nicotinic acid, glycine, riboflavin, ascorbic acid and bismuth standard solutions. The medium may further comprise other components which have been described, for example, in WO2011100762 to the International Patent Application Publication. It is known to those skilled in the art that one or more of the above components can be omitted without affecting the function of the culture medium.

所述培養基可包含一種或多種碳源如糖。非限制的示例性的糖包括蔗糖、葡萄糖、麥芽糖、半乳糖和山梨糖醇(sorbitol)或其之組合。 The medium may comprise one or more carbon sources such as sugars. Non-limiting exemplary sugars include sucrose, glucose, maltose, galactose, and sorbitol, or combinations thereof.

表1顯示了上述組分的示例性濃度。可基於植物的物種、組織的類型和目的等調整這些組分的濃度，而基本上不影響培養基的功能。 Table 1 shows exemplary concentrations of the above components. The concentration of these components can be adjusted based on the species of the plant, the type and purpose of the tissue, etc., without substantially affecting the function of the medium.

所述培養基進一步包含一種或多種有效量的植物生長調節劑。植物生長調節劑的實例包括植物激素，如生長素和具有類生長素活性的化合物、細胞***素和具有類細胞***素活性的化合物。術語「細胞***素」指一類植物生長調節劑， 其特徵在於它們刺激組織培養中細胞***和苗器官發生(organogenesis)的能力。細胞***素的非限制性實例包括：N⁶-苄胺基嘌呤(BAP)(亦稱N⁶-苄基腺嘌呤(BA))、間拓樸林、玉米素、激動素、噻苯隆(TDZ)、間拓樸林、2-異戊烯腺嘌呤(亦稱6-γ-γ-(二甲基烯丙基胺基)-嘌呤或2ip)、腺嘌呤半硫酸鹽、二甲基烯丙基腺嘌呤、4-CPPU(N-(2-氯-4-吡啶基)-N'-苯基脲)，及其類似物。術語「生長素」指一類植物生長調節劑，其主要特徵為其在切離的植物組織中刺激細胞***的能力。除了在細胞***和細胞伸長上的作用之外，生長素還影響其他發育過程，包括根發生(root initiation)。非限制性的實例是β-萘氧乙酸(NAA)、2,4-二氯苯氧乙酸(2,4-D)、吲哚-3-丁酸(IBA)、吲哚-3-乙酸(IAA)、毒莠定及其類似物。更多細胞***素和植物生長素係描述於WO2011100762、US5211738、US20100240537、US20060084577、US20030158043、及Aremu等人之(Plant Cell Tiss.Organ.Cult.,DOI 10.1007/s11240-011-0007-7,2011)，其藉由引用方式全部併入本文。 The medium further comprises one or more effective amounts of a plant growth regulator. Examples of the plant growth regulator include plant hormones such as auxin and compounds having auxin-like activity, cytokinins, and compounds having cytokinin-like activity. The term "cytokinin" refers to a class of plant growth regulators characterized by their ability to stimulate cell division and organogenesis in tissue culture. Non-limiting examples of cytokinins include: N ⁶ -benzylaminopurine (BAP) (also known as N ⁶ -benzyl adenine (BA)), metatopolin, zeatin, kinetin, thiabendazole ( TDZ), metatopolin, 2-isopentene adenine (also known as 6-γ-γ-(dimethylallylamino)-hydrazine or 2ip), adenine hemisulfate, dimethylene Propyl adenine, 4-CPPU (N-(2-chloro-4-pyridyl)-N'-phenylurea), and analogs thereof. The term "auxin" refers to a class of plant growth regulators whose primary feature is its ability to stimulate cell division in excised plant tissue. In addition to its role in cell division and cell elongation, auxin also affects other developmental processes, including root initiation. Non-limiting examples are beta-naphthyloxyacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-butyric acid (IBA), indole-3-acetic acid ( IAA), chlorpyrifos and its analogues. Further cytokinins and auxin lines are described in WO2011100762, US5211738, US20100240537, US20060084577, US20030158043, and Aremu et al. ( Plant Cell Tiss. Organ. Cult. , DOI 10.1007/s 11240-011-0007-7, 2011). , which is incorporated herein by reference in its entirety.

在一些具體例中，在所述培養基中可包括其他植物激素，如脫落酸(abscisic acid)、激勃素(gibberellic acid)、其類似物或其之組合。在一些具體例中，可在培養基中添加活性炭以改善細胞生長和發育。 In some embodiments, other plant hormones may be included in the culture medium, such as abscisic acid, gibberellic acid, analogs thereof, or combinations thereof. In some embodiments, activated carbon can be added to the culture medium to improve cell growth and development.

如存在於培養基中，每種細胞***素可以以從約0.001 mg/L-約100 mg/L和所有兩者之間的量存在。例如，細胞***素的濃度是約0.001、0.01、0.1、1、2、3、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100 mg/L或更多。 If present in the medium, each cytokinin can be from about 0.001 Mg/L - about 100 mg / L and the amount between all of them is present. For example, the concentration of cytokinin is about 0.001, 0.01, 0.1, 1, 2, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5. 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 , 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 , 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 , 99, 100 mg / L or more.

在一些具體例中，所述培養基包含間拓樸林及/或其類似物。在一些具體例中，間拓樸林以如下量存在：等於或大於1.5 mg/L、等於或大於2.0 mg/L、等於或大於2.5 mg/L、等於或大於3.0 mg/L、等於或大於3.5 mg/L、等於或大於4.5 mg/L或等於或大於5.0 mg/L。在其他具體例中，間拓樸林以3.2 mg/L或5.36 mg/L的量存在。在另外的具體例中，間拓樸林的量不能低於1.5 mg/L、不能低於2.0 mg/L、不能低於2.5 mg/L、不能低於3.0 mg/L、不能低於3.5 mg/L、不能 低於4.5 mg/L或不能低於5.0 mg/L。在一些具體例中，可含括高至200 mg/L的任何量之間拓樸林及/或其類似物。在一些具體例中，將所述培養基用於竹的微體繁殖。在一些具體例中，所述竹植物選自下列組成之物種：青籬竹屬(Arundinaria)、莿竹屬(Bambusa)、北風竹屬(Borinda)、秋竹屬(chusquea)、麻竹屬(Dendrocalamus)、箭竹屬(fargesia)、瓜多竹屬(Guadua)、孟宗竹屬(Phyllostachys)、苦竹屬(Pleioblastus)和筱竹屬(Thamnocalamus)。 In some embodiments, the medium comprises an intermediate topology and/or an analog thereof. In some embodiments, the metatopia is present in an amount equal to or greater than 1.5 mg/L, equal to or greater than 2.0 mg/L, equal to or greater than 2.5 mg/L, equal to or greater than 3.0 mg/L, equal to or greater than or equal to 3.5 mg/L, equal to or greater than 4.5 mg/L or equal to or greater than 5.0 mg/L. In other specific examples, metatopia is present in an amount of 3.2 mg/L or 5.36 mg/L. In another specific example, the amount of metatopia should not be less than 1.5 mg/L, not less than 2.0 mg/L, not less than 2.5 mg/L, not less than 3.0 mg/L, and not less than 3.5 mg. /L, can't Less than 4.5 mg/L or not less than 5.0 mg/L. In some embodiments, any amount between up to 200 mg/L of topology and/or an analog thereof can be included. In some embodiments, the medium is used for micropropagation of bamboo. In some embodiments, the bamboo plant is selected from the group consisting of Arundinaria, Bambusa, Borinda, Chusquea, Dendrocalamus. ), Fargesia, Guadua, Phyllosachys, Pleioblastus, and Thamnocalamus.

在一些具體例中，所述培養基包含噻苯隆及/或其類似物。在一些具體例中，噻苯隆及/或其類似物可如下存在：0.001 mg/L、0.01、0.025、0.05、0.075、0.1、0.15、0.2、0.25、0.3、0.35、0.4、0.45、0.5、0.55、0.6、0.65、0.7、0.75、0.8、0.85、0.9、0.95、1.0、1.25、1.50、1.75、2.0、2.25、2.5、2.75、3.5、4.5、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99或100 mg/L。在一些具體例中，還可含括高至200 mg/L的任何量之噻苯隆及/ 或其類似物。 In some embodiments, the medium comprises thidiazuron and/or an analog thereof. In some embodiments, the thiabendazole and/or its analog may be present as follows: 0.001 mg/L, 0.01, 0.025, 0.05, 0.075, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.25, 1.50, 1.75, 2.0, 2.25, 2.5, 2.75, 3.5, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 mg/L. In some specific examples, any amount of thiabendone up to 200 mg/L and/or Or an analogue thereof.

如存在於培養基中的話，每種生長素可從約0.001 mg/L-約100 mg/L及所有兩者之間的量存在。例如，生長素的濃度是約0.001、0.01、0.1、1、2、3、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99或約100 mg/L。 Each auxin may be present in an amount from about 0.001 mg/L to about 100 mg/L, and all in between, if present in the culture medium. For example, the concentration of auxin is about 0.001, 0.01, 0.1, 1, 2, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or about 100 mg/L.

在一些具體例中，所述培養基包含NAA及/或其類似物。在一些具體例中，NAA及/或其類似物可如下存在：0.001 mg/L、0.01、0.0125、0.015、0.0175、0.02、0.0225、0.025、0.0275、0.03、0.0325、0.035、0.0375、0.04、0.0425、0.045、0.0475、0.05、0.0525、0.055、0.0575、0.06、0.0625、0.065、0.0675、0.07、0.0725、0.075、0.0775、0.08、0.0825、0.085、0.0875、0.09、0.0925、0.095、0.0957、0.1、0.15、0.2、0.25、 0.3、0.35、0.4、0.45、0.5、0.55、0.6、0.65、0.7、0.75、0.8、0.85、0.9、0.95、1.0、1.25、1.50、1.75、2.0、2.25、2.5、2.75、3.5、4.5、5、6、7、8、9、10、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或100 mg/L。在一些具體例中，可含括高至200 mg/L的任何量之NAA及/或其類似物。 In some embodiments, the medium comprises NAA and/or an analog thereof. In some embodiments, NAA and/or its analogs may be present as follows: 0.001 mg/L, 0.01, 0.0125, 0.015, 0.0175, 0.02, 0.0225, 0.025, 0.0275, 0.03, 0.0325, 0.035, 0.0375, 0.04, 0.0425, 0.045, 0.0475, 0.05, 0.0525, 0.055, 0.0575, 0.06, 0.0625, 0.065, 0.0675, 0.07, 0.0725, 0.075, 0.0775, 0.08, 0.0825, 0.085, 0.0875, 0.09, 0.0925, 0.095, 0.0957, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.25, 1.50, 1.75, 2.0, 2.25, 2.5, 2.75, 3.5, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 mg/L. In some embodiments, any amount of NAA and/or an analog thereof up to 200 mg/L can be included.

在一些具體例中，所述培養基包含IBA及/或其類似物。在一些具體例中，IBA及/或其類似物可如下存在：0.001 mg/L、0.01、0.025、0.05、0.075、0.08、0.1、0.125、0.15、0.175、0.2、0.225、0.25、0.275、0.3、0.325、0.35、0.375、0.4、0.425、0.45、0.475、0.5、0.525、0.55、0.575、0.6、0.625、0.65、0.675、0.7、0.725、0.75、0.775、0.8、0.825、0.85、0.875、0.9、0.925、0.95、0.975、1.0、1.25、1.50、1.75、2.0、2.25、2.5、2.75、3.0、3.25、3.5、3.75、4.0、4.5、5、6、7、8、9、10、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或100 mg/L。在一些具體例中，可含括高至200 mg/L的任何量之IBA及/或其類似物。 In some embodiments, the medium comprises IBA and/or an analog thereof. In some embodiments, IBA and/or its analogs may be present as follows: 0.001 mg/L, 0.01, 0.025, 0.05, 0.075, 0.08, 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1.0, 1.25, 1.50, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 mg/L. In some embodiments, any amount of IBA and/or an analog thereof up to 200 mg/L can be included.

在一些具體例中，所述培養基包含BAP及/或其類似物。在一些具體例中，BAP及/或其類似物可如下存在：0.001 mg/L、0.01、0.025、0.05、0.06、0.07、0.0725、0.075、0.0775、0.08、0.0825、0.085、0.0875、0.09、0.0925、0.095、0.0975、0.1、0.125、0.15、0.175、0.2、0.225、0.25、0.275、0.3、0.325、0.35、0.375、0.4、0.425、0.45、0.475、0.5、0.525、0.55、0.575、0.6、0.625、0.65、0.675、0.7、0.75、0.8、0.85、0.9、0.95、1.0、1.25、1.50、1.75、2.0、2.25、2.5、2.75、3.5、4.5、5、6、7、8、9、10、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或100 mg/L。在一些具體例中，可含括高至200 mg/L的任何量之BAP及/或其類似物。 In some embodiments, the medium comprises BAP and/or an analog thereof. In some embodiments, BAP and/or its analogs may be present as follows: 0.001 mg/L, 0.01, 0.025, 0.05, 0.06, 0.07, 0.0725, 0.075, 0.0775, 0.08, 0.0825, 0.085, 0.0875, 0.09, 0.0925, 0.095, 0.0975, 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.25, 1.50, 1.75, 2.0, 2.25, 2.5, 2.75, 3.5, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 mg/L. In some embodiments, any amount of BAP and/or an analog thereof up to 200 mg/L can be included.

在一些具體例中，所述培養基包含2ip及/或其類似物。在一些具體例中，2ip及/或其類似物可如下存在：0.001 mg/L、0.01、0.025、0.05、0.075、0.08、0.1、0.15、0.2、0.25、0.3、0.35、0.4、0.45、0.5、0.55、0.6、0.65、0.7、0.75、0.8、0.85、0.9、0.95、1.0、1.25、1.50、1.75、2.0、2.25、2.5、2.75、3.0、3.5、4.0、4.5、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99或100 mg/L。在一些具體例中，可含括高至200 mg/L的任何量之2ip及/或其類似物。 In some embodiments, the medium comprises 2 ip and/or an analog thereof. In some embodiments, 2ip and/or its analogs may be present as follows: 0.001 mg/L, 0.01, 0.025, 0.05, 0.075, 0.08, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.25, 1.50, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.5, 4.0, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 mg/L. In some embodiments, any amount of 2ip and/or an analog thereof up to 200 mg/L can be included.

在一些具體例中，所述培養基包含DPU及/或其類似物。在一些具體例中，DPU及/或其類似物可如下存在：0.001 mg/L、0.01、0.025、0.05、0.075、0.1、0.15、0.2、0.25、0.3、0.35、0.4、0.45、0.5、0.55、0.6、0.65、0.7、0.75、0.8、0.85、0.9、0.95、1.0、1.25、1.50、1.75、2.0、2.25、 2.5、2.75、3.5、4.5、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99或100 mg/L。在一些具體例中，可含括高至200 mg/L的任何量之DPU及/或其類似物。 In some embodiments, the medium comprises DPU and/or an analog thereof. In some embodiments, DPU and/or its analogs may be present as follows: 0.001 mg/L, 0.01, 0.025, 0.05, 0.075, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.25, 1.50, 1.75, 2.0, 2.25, 2.5, 2.75, 3.5, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 mg /L. In some embodiments, any amount of DPU and/or an analog thereof up to 200 mg/L can be included.

在一些具體例中，所述培養基包含CPPU及/或其類似物。在一些具體例中，CPPU及/或其類似物可如下存在：0.001 mg/L、0.01、0.025、0.05、0.075、0.1、0.15、0.2、0.25、0.3、0.35、0.4、0.45、0.5、0.55、0.6、0.65、0.7、0.75、0.8、0.85、0.9、0.95、1.0、1.25、1.50、1.75、2.0、2.25、2.5、2.75、3.5、4.5、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、 92、93、94、95、96、97、98、99或100 mg/L。在一些具體例中，可含括高至200 mg/L的任何量之CPPU及/或其類似物。 In some embodiments, the medium comprises CPPU and/or an analog thereof. In some embodiments, CPPU and/or its analogs may be present as follows: 0.001 mg/L, 0.01, 0.025, 0.05, 0.075, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.25, 1.50, 1.75, 2.0, 2.25, 2.5, 2.75, 3.5, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 mg/L. In some embodiments, any amount of CPPU and/or the like can be included up to 200 mg/L.

在一些具體例中，還可以按比率利用一種或多種細胞***素和一種或多種其他細胞***素或生長素的組合，和生長素和其他生長素或細胞***素的組合。例如，在一些具體例中，可含括下列示例性比率之本文公開的任何兩種成對的細胞***素及/或生長素：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或0.001：1。這些比率可以用於間拓樸林(及類似物)和噻苯隆(及類似物)之間、和NAA(及類似物)之間、和BAP(及類似物)之間、和IBA(及類似物)之間、和2ip(及類似物)之間、和DPU(及類似物)之間及/或和CPPU(及類似物)之間。類似地，該比率可用於噻苯隆(及類似物)和NAA(及類似物)之間、和BAP(及類似物)之間、和IBA(及類似物)之間、和2ip(及類似物)之間、和DPU(及類似物)之間及/或和CPPU(及類似 物)之間。該比率還可用於NAA(及類似物)和BAP(及類似物)之間、和IBA(及類似物)之間、和2ip(及類似物)之間、和DPU(及類似物)之間及/或和CPPU(及類似物)之間。該比率還可用於BAP(及類似物)和IBA(及類似物)之間、和2ip(及類似物)之間、和DPU(及類似物)之間及/或和CPPU(及類似物)之間。簡而言之，依據任何所述公開的比率，可含括每種細胞***素和/或生長素或其類似物與本文公開的另一種細胞***素和/或生長素。 In some embodiments, a combination of one or more cytokinins and one or more other cytokinins or auxins, and a combination of auxin and other auxins or cytokinins may also be utilized in a ratio. For example, in some embodiments, any two of the paired cytokinins and/or auxins disclosed herein may be included in the following exemplary ratios: 100:1, 95:1, 90:1, 85:1 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 29:1, 28: 1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1 15:1, 14:1, 13:1, 12:1, 11:1, 10:1; 9:1, 8:1, 7:1, 6.9:1, 6.8:1, 6.7:1, 6.6: 1, 6.5:1, 6.4:1, 6.3:1, 6.2:1, 6.1:1,6:1, 5.9:1, 5.8:1, 5.7:1, 5.6:1, 5.5:1, 5.4:1 5.3:1, 5.2:1, 5.1:1, 5:1; 4:1, 3:1, 2:1, 1:1, 0.75:1, 0.5:1, 0.25:1, 0.1:1, 0.075: 1, 0.05: 1, 0.025: 1 or 0.001:1. These ratios can be used between metatopia (and analogs) and thidiazuron (and analogs), and between NAA (and analogs), and between BAP (and analogs), and IBA (and Between the analogs, and between 2ip (and the like), and between the DPU (and the like) and/or between the CPPU (and the like). Similarly, this ratio can be used between thiexone (and analogs) and NAA (and analogs), and between BAP (and analogs), and between IBA (and analogs), and 2ip (and similar) Between), and between DPU (and similar) and / or with CPPU (and similar Between things). This ratio can also be used between NAA (and analogs) and BAP (and analogs), and between IBA (and analogs), and between 2ip (and analogs), and between DPUs (and analogs). And / or with CPPU (and similar). This ratio can also be used between BAP (and analogs) and IBA (and analogs), and between 2ip (and analogs), and between DPU (and analogs) and/or with CPPU (and analogs). between. Briefly, depending on any of the disclosed ratios, each cytokinin and/or auxin or analog thereof can be included with another cytokinin and/or auxin disclosed herein.

在一些具體例中，本發明提供了至少兩種類型的在體外微體繁殖中使用的培養基。第一培養基，在本文中稱為「芽誘導培養基」，包含至少一種強細胞***素，如噻苯隆或其類似物。第二培養基，在本文中稱為「苗伸長/維持培養基」，包含一種或多種除芽誘導培養基中細胞***素以外的細胞***素。例如，所述細胞***素選自：間拓樸林、激動素、異戊烯腺嘌呤(iP)、玉米素、反玉米素、玉米素核苷、二氫玉米素、苄胺基嘌呤(BAP)、苄基腺苷([9R]BAP)，其類似物，或其之組合。還可用其他可獲的用於組織培養基的細胞***素取代上述細胞***素以實現類似的效果。 In some embodiments, the invention provides at least two types of media for use in in vitro micropropagation. The first medium, referred to herein as the "bud induction medium," comprises at least one strong cytokinin, such as thidiazuron or an analog thereof. The second medium, referred to herein as "emergence elongation/maintenance medium", comprises one or more cytokinins other than cytokinin in the budding induction medium. For example, the cytokinin is selected from the group consisting of: intermittent forest, kinetin, isoprenoid adenine (iP), zeatin, anti-zein, zeatin nucleoside, dihydro zeatin, benzylaminopurine (BAP) ) benzyl adenosine ([9R]BAP), an analog thereof, or a combination thereof. It is also possible to replace the above cytokinins with other available cytokinins for tissue culture to achieve a similar effect.

芽誘導培養基和苗伸長/維持培養基兩者皆包含用於植物組織培養的基本培養基的組分，如碳源和鹽。在一些具體例中，兩種培養基都可包含一種或多種選自下列的組分：NH₄NO₃、KNO₃、Ca(NO₃)₂、K₂SO₄、MgSO₄、MnSO₄、ZnSO₄、 CuSO₄、K₂SO₅、CaCl₂、Kl、CoCl₂、H₃BO₃、Na₂MoO₄、KH₂PO₄、FeSO₄、Na₂EDTA、Na₂H₂PO₄、甘胺酸、肌醇、硫胺、吡哆醇、煙酸和核黃素。 Both the bud induction medium and the shoot elongation/maintenance medium contain components of a minimal medium for plant tissue culture, such as a carbon source and a salt. In some embodiments, both media may comprise one or more components selected from the group consisting of NH ₄ NO ₃ , KNO ₃ , Ca(NO ₃ ) ₂ , K ₂ SO ₄ , MgSO ₄ , MnSO ₄ , ZnSO ₄ , CuSO ₄ , K ₂ SO ₅ , CaCl ₂ , Kl, CoCl ₂ , H ₃ BO ₃ , Na ₂ MoO ₄ , KH ₂ PO ₄ , FeSO ₄ , Na ₂ EDTA, Na ₂ H ₂ PO ₄ , glycine, Inositol, thiamine, pyridoxine, niacin and riboflavin.

在一些具體例中，所述芽誘導培養基僅包含一種強細胞***素，例如噻苯隆(TDZ)或其類似物。在一些具體例中，所述芽誘導培養基包含一種另外的細胞***素。在一些具體例中，芽誘導培養基進一步包含一種或多種生長素，如NAA、2,4-D、IBA、IAA、毒莠定或其類似物。 In some embodiments, the shoot induction medium comprises only one strong cytokinin, such as thidiazuron (TDZ) or an analog thereof. In some embodiments, the shoot induction medium comprises an additional cytokinin. In some embodiments, the shoot induction medium further comprises one or more auxins, such as NAA, 2,4-D, IBA, IAA, chlorpyrifos or analogs thereof.

在一些具體例中，芽誘導培養基中所述強細胞***素(如TDZ)的濃度是約0.25 mg/L(±10%)至約100 mg/L(±10%)，例如，約0.2 mg/L(±10%)、約0.5 mg/L(±10%)、約1.0 mg/L(±10%)、約5 mg/L(±10%)、約10 mg/L(±10%)、約20 mg/L(±10%)、約30 mg/L(±10%)、約40 mg/L(±10%)、約50 mg/L(±10%)、約60 mg/L(±10%)、約70 mg/L(±10%)、約80 mg/L(±10%)、約90 mg/L(±10%)或約100 mg/L(±10%)。例如，TDZ的濃度是約0.2(±10%)至約20(±10%)mg/L、約0.4(±10%)至約10(±10%)mg/L，或約0.5(±10%)至約2(±10%)mg/L。 In some embodiments, the concentration of the strong cytokinin (eg, TDZ) in the shoot induction medium is from about 0.25 mg/L (±10%) to about 100 mg/L (±10%), for example, about 0.2 mg. /L (±10%), about 0.5 mg/L (±10%), about 1.0 mg/L (±10%), about 5 mg/L (±10%), about 10 mg/L (±10%) ), about 20 mg/L (±10%), about 30 mg/L (±10%), about 40 mg/L (±10%), about 50 mg/L (±10%), about 60 mg/ L (±10%), approximately 70 mg/L (±10%), approximately 80 mg/L (±10%), approximately 90 mg/L (±10%) or approximately 100 mg/L (±10%) . For example, the concentration of TDZ is from about 0.2 (±10%) to about 20 (±10%) mg/L, from about 0.4 (±10%) to about 10 (±10%) mg/L, or about 0.5 (±10) %) to about 2 (±10%) mg/L.

在一些具體例中，芽誘導培養基中生長素的濃度是約0.01 mg/L(±10%)至約10 mg/L(±10%)，例如，約0.01 mg/L(±10%)、約0.05 mg/L(±10%)、約0.1 mg/L(±10%)、約0.5 mg/L(±10%)、約1 mg/L(±10%)、約5 mg/L(±10%)或約10 mg/L (±10%)。 In some embodiments, the concentration of auxin in the shoot induction medium is from about 0.01 mg/L (±10%) to about 10 mg/L (±10%), for example, about 0.01 mg/L (±10%), About 0.05 mg/L (±10%), about 0.1 mg/L (±10%), about 0.5 mg/L (±10%), about 1 mg/L (±10%), about 5 mg/L ( ±10%) or about 10 mg/L (±10%).

在一些具體例中，所述苗伸長/維持培養基包含一種或多種除TDZ以外的細胞***素，如間拓樸林、激動素、異戊烯腺嘌呤(iP如2ip)、玉米素、反玉米素、玉米素核苷、二氫玉米素、苄胺基嘌呤(BAP)、苄基腺苷([9R]BAP)，其類似物。在一些具體例中，所述苗伸長/維持培養基進一步包含一種或多種生長素，如NAA、2,4-D、IBA、IAA、毒莠定或其類似物。 In some embodiments, the shoot elongation/maintenance medium comprises one or more cytokinins other than TDZ, such as mesomorphine, kinetin, isoprenoid adenine (iP such as 2ip), zeatin, anti-corn , zeatin nucleoside, dihydro zeatin, benzylaminopurine (BAP), benzyl adenosine ([9R] BAP), analogs thereof. In some embodiments, the shoot elongation/maintenance medium further comprises one or more auxins, such as NAA, 2,4-D, IBA, IAA, chlorpyrifos or the like.

在一些具體例中，所述苗伸長/維持培養基中細胞***素的濃度是約0.01 mg/L(±10%)至約100 mg/L(±10%)，例如，約0.01 mg/L(±10%)、約0.05 mg/L(±10%)、約0.1 mg/L(±10%)、約0.5 mg/L(±10%)、約1 mg/L(±10%)、約5 mg/L(±10%)、約10 mg/L(±10%)、約20 mg/L(±10%)、約30 mg/L(±10%)、約40 mg/L(±10%)、約50 mg/L(±10%)、約60 mg/L(±10%)、約70 mg/L(±10%)、約80 mg/L(±10%)、約90 mg/L(±10%)，或約100 mg/L(±10%)。在一些具體例中，所述苗伸長/維持培養基中細胞***素的濃度是約0.01(±10%)至約20(±10%)mg/L、約0.1(±10%)至約10(±10%)mg/L，或約0.25(±10%)至約5(±10%)mg/L。 In some embodiments, the concentration of cytokinin in the shoot elongation/sustainment medium is from about 0.01 mg/L (±10%) to about 100 mg/L (±10%), for example, about 0.01 mg/L ( ±10%), about 0.05 mg/L (±10%), about 0.1 mg/L (±10%), about 0.5 mg/L (±10%), about 1 mg/L (±10%), about 5 mg/L (±10%), approximately 10 mg/L (±10%), approximately 20 mg/L (±10%), approximately 30 mg/L (±10%), approximately 40 mg/L (± 10%), about 50 mg/L (±10%), about 60 mg/L (±10%), about 70 mg/L (±10%), about 80 mg/L (±10%), about 90 Mg/L (±10%), or about 100 mg/L (±10%). In some embodiments, the concentration of cytokinin in the shoot elongation/maintenance medium is from about 0.01 (±10%) to about 20 (±10%) mg/L, from about 0.1 (±10%) to about 10 ( ±10%) mg/L, or about 0.25 (±10%) to about 5 (±10%) mg/L.

在一些具體例中，所述芽誘導培養基中生長素的濃度是約0.01 mg/L(±10%)至約50 mg/L(±10%)，例如，約0.01 mg/L(±10%)、約0.05 mg/L(±10%)、約0.1 mg/L(±10%)、約 0.5 mg/L(±10%)、約1 mg/L(±10%)、約5 mg/L(±10%)、約10 mg/L(±10%)、約20 mg/L(±10%)、約30 mg/L(±10%)、約40 mg/L(±10%)，或約50 mg/L(±10%)。在一些具體例中，所述苗伸長/維持培養基中生長素的濃度是約0.01(±10%)至約20(±10%)mg/L、約0.02(±10%)至約10(±10%)mg/L，或約0.05(±10%)至約5(±10%)mg/L。 In some embodiments, the concentration of auxin in the shoot induction medium is from about 0.01 mg/L (±10%) to about 50 mg/L (±10%), for example, about 0.01 mg/L (±10%) ), about 0.05 mg/L (±10%), about 0.1 mg/L (±10%), about 0.5 mg/L (±10%), approximately 1 mg/L (±10%), approximately 5 mg/L (±10%), approximately 10 mg/L (±10%), approximately 20 mg/L (± 10%), about 30 mg/L (±10%), about 40 mg/L (±10%), or about 50 mg/L (±10%). In some embodiments, the concentration of auxin in the shoot elongation/sustainment medium is from about 0.01 (±10%) to about 20 (±10%) mg/L, from about 0.02 (±10%) to about 10 (± 10%) mg/L, or about 0.05 (±10%) to about 5 (±10%) mg/L.

表2顯示了所述芽誘導培養基和苗伸長/維持培養基中組分的非限制性的濃度。可以省去或替換表2中的一種或多種組分而不影響培養基的功能。可以調整每種組分的濃度而不影響培養基的功能。 Table 2 shows the non-limiting concentrations of the components in the shoot induction medium and shoot elongation/maintenance medium. One or more of the components of Table 2 can be omitted or replaced without affecting the function of the medium. The concentration of each component can be adjusted without affecting the function of the medium.

在一些具體例中，所述培養基可選自圖7顯示的表中所列者，或其任何同等的培養基。 In some embodiments, the medium can be selected from the ones listed in the table shown in Figure 7, or any equivalent medium thereof.

除了上述「芽誘導培養基」和「苗伸長/維持培養基」的組合之外，本發明還提供其他用於植物繁殖的選擇性培養基組合。例如，提供了包含至少一種階段1培養基和至少一種 階段2培養基的培養基組合。在一些具體例中，當使用某一培養基多於一次時，保留特定過程中指定培養基的編號。例如，本文揭露的某些具體例包括於交替的培養基中進行培植體或苗之迴圈。例如，可將培植體置於階段1培養基接著於階段2培養基中，然後返回到其階段1培養基。在此上下文中，當重複暴露於一種培養基時，該培養基保留其在該特定過程中的最低的階段編號。 In addition to the above combinations of "bud induction medium" and "emergence elongation/maintenance medium", the present invention also provides other selective medium combinations for plant propagation. For example, providing at least one stage 1 medium and at least one Medium combination of medium of phase 2. In some embodiments, when a medium is used more than once, the number of the designated medium in a particular process is retained. For example, some specific examples disclosed herein include the implantation of a culture or seedling loop in alternating media. For example, the culture body can be placed in Stage 1 medium followed by Stage 2 medium and then returned to its Stage 1 medium. In this context, when repeatedly exposed to a medium, the medium retains its lowest stage number in that particular process.

在一些具體例中，所述過程開始時，可獲得或製備階段1培養基。階段1培養基包括植物一般易於接受的pH(通常從4.0-7.0或4.5-6.5)。本文所揭示的階段1培養基可包括(i)間拓樸林；(2)至少三種細胞***素；(3)細胞***素間拓樸林或其類似物與至少兩種其他的細胞***素的組合；(4)至少一種生長素和至少兩種細胞***素；(5)至少兩種生長素和至少兩種細胞***素或(6)至少兩種生長素和至少三種細胞***素。在某些具體例中，階段1培養基必須包括多於1種的生長素。在其他的具體例中，階段1培養基必須包括多於1種的細胞***素。在進一步的具體例中，階段1培養基必須包括多於1種的生長素和多於1種的細胞***素。 In some embodiments, Stage 1 medium can be obtained or prepared at the beginning of the process. Stage 1 media includes pH that is generally acceptable to plants (typically from 4.0-7.0 or 4.5-6.5). The Stage 1 medium disclosed herein may comprise (i) an intermediate forest; (2) at least three cytokinins; (3) a cytokinin intermediate topology or an analogue thereof and at least two other cytokinins Combining; (4) at least one auxin and at least two cytokinins; (5) at least two auxins and at least two cytokinins or (6) at least two auxins and at least three cytokinins. In some embodiments, the Stage 1 medium must include more than one auxin. In other embodiments, the Stage 1 medium must include more than one cytokinin. In a further embodiment, the Stage 1 medium must include more than one auxin and more than one cytokinin.

在一些具體例中，所述細胞***素和生長素選自間拓樸林、噻苯隆、NAA、IBA、BAP、2ip、CCPU和DPU的一種或多種。在另外的具體例中，所述細胞***素和生長素選自間拓樸林或其類似物、噻苯隆或其類似物、NAA或其類 似物、IBA或其類似物、BAP或其類似物、2ip或其類似物、CCPU或其類似物和DPU或其類似物的一種或多種。 In some embodiments, the cytokinin and auxin are selected from one or more of the group consisting of Topoline, Thiabendron, NAA, IBA, BAP, 2ip, CCPU, and DPU. In another embodiment, the cytokinin and auxin are selected from the group consisting of metatopia or an analogue thereof, thidiazuron or an analogue thereof, NAA or the like One or more of analogs, IBA or an analogue thereof, BAP or an analog thereof, 2ip or an analog thereof, CCPU or the like and DPU or an analogue thereof.

在一些具體例中，所述培養基具有至少兩種其他的細胞***素。在一些具體例中，所述培養基維持至少6個月的繁殖週期。 In some embodiments, the medium has at least two other cytokinins. In some embodiments, the medium maintains a reproductive cycle of at least 6 months.

在一些具體例中，所述培養基至少包含三種細胞***素。在一些具體例中，所述培養基能維持至少6個月的繁殖週期。在一些具體例中，提供了包含細胞***素間拓樸林或其類似物與至少兩種其他的細胞***素的組合的培養基。 In some embodiments, the medium comprises at least three cytokinins. In some embodiments, the medium is capable of maintaining a reproductive cycle of at least 6 months. In some embodiments, a medium comprising a combination of a cytokinin topology forest or an analog thereof and at least two other cytokinins is provided.

在一些具體例中，所述培養基包含至少一種生長素和至少兩種細胞***素。在一些具體例中，所述培養基能維持至少6個月的繁殖週期。在一些具體例中，至少一種細胞***素是間拓樸林或其類似物。 In some embodiments, the medium comprises at least one auxin and at least two cytokinins. In some embodiments, the medium is capable of maintaining a reproductive cycle of at least 6 months. In some embodiments, the at least one cytokinin is metatuberculosis or an analog thereof.

在一些具體例中，所述培養基包含至少兩種生長素和至少兩種細胞***素。在一些具體例中，所述培養基能維持至少6個月的繁殖週期。 In some embodiments, the medium comprises at least two auxins and at least two cytokinins. In some embodiments, the medium is capable of maintaining a reproductive cycle of at least 6 months.

在一些具體例中，所述培養基包含至少兩種生長素和至少三種細胞***素。在一些具體例中，所述培養基能維持至少6個月的繁殖週期。 In some embodiments, the medium comprises at least two auxins and at least three cytokinins. In some embodiments, the medium is capable of maintaining a reproductive cycle of at least 6 months.

在一些具體例中，所述微體繁殖的植物在無菌的培養基中體外生長。 In some embodiments, the micropropagated plants are grown in vitro in a sterile medium.

在一些具體例中，所述培養基可以是液體的、半固體的或 固體的，且可以藉由摻入或移除一種或多種膠凝劑改變所述培養基的物理狀態。可以使用本領域已知的任何適用於植物組織培養基的膠凝劑。瓊脂是最通常用於該目的的。這類瓊脂的實例包括Agar Type A、E或M和Bacto® Agar(Becton Dickinson & Co.)。其他示例性的膠凝劑包括角叉膠、吉蘭糖膠(由市售PhytaGel^TM(Sigma-Aldrich)、Gelrite®(Sigma-Aldrich)和Gelzan^TM(Caisson Labs)獲得)、褐藻酸和其鹽、和瓊脂糖。還可使用這些試劑的混合物，如兩種或更多種的瓊脂、角叉膠、吉蘭糖膠、瓊脂糖和褐藻酸或其鹽。 In some embodiments, the medium can be liquid, semi-solid or solid, and the physical state of the medium can be altered by incorporation or removal of one or more gelling agents. Any gelling agent suitable for use in plant tissue culture media known in the art can be used. Agar is most commonly used for this purpose. Examples of such agars include Agar Type A, E or M and Bacto® Agar (Becton Dickinson & Co.). Other exemplary gelling agents include carrageenan, gellan gum (, Gelrite® (Sigma-Aldrich) and Gelzan ^TM (Caisson Labs) available commercially ^{PhytaGel TM (Sigma-Aldrich))} , alginic acid and its salts , and agarose. Mixtures of these agents may also be used, such as two or more of agar, carrageenan, gellan gum, agarose, and alginic acid or a salt thereof.

植物生長調節劑的實例包括脫落酸(ABA)、三十烷醇(triacontanol)、間苯三酚(phloroglucinol)、生長素和具有類生長素活性的化合物、細胞***素和具有類細胞***素活性的化合物。示例性的生長素包括4-氟苯氧基乙酸(FA)、2,4,5-三氯苯氧基乙酸(2,4,5-T)、3-溴氧化吲哚-3-乙酸、4-溴苯氧基乙酸、麥草畏(dicamba)、p-氯苯氧基乙酸(CPA)、吲哚-3-丙酸(IPA)、2,4-二氯苯氧基乙酸(2,4-D)、吲哚-3-丁酸(IBA)、吲哚-3-乙酸(IAA)、毒莠定和其組合。示例性的細胞***素包括間拓樸林、噻苯隆、N-(2-氯-4-吡啶)-N-苯脲(CCPU)、1,3-二苯脲(DPU)、腺嘌呤半硫酸鹽、苄基腺嘌呤、二甲基烯丙基腺嘌呤、激動素、玉米素、核苷、腺苷、間拓樸林核苷、間拓樸林-9-葡糖苷、正拓樸林(ortho-topolin)、正拓樸林核苷、正拓樸林-9-葡糖苷、對拓樸林(para-topolin)、對拓 樸林核苷、對拓樸林-9-糖苷、正甲氧拓樸林、正甲氧拓樸林核苷、間甲氧拓樸林、間甲氧拓樸林核苷、間甲氧拓樸林-9-葡糖苷和其組合，和具有類細胞***素活性的植物萃取物，如椰汁、香蕉粉、麥芽萃取物、鳳梨粉或番茄粉。 Examples of plant growth regulators include abscisic acid (ABA), triacontanol, phloroglucinol, auxin and compounds having auxin-like activity, cytokinins, and cytokinin-like activity. compound of. Exemplary auxins include 4-fluorophenoxyacetic acid (FA), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 3-bromo-phosphonium-3-acetate, 4-bromophenoxyacetic acid, dicamba, p-chlorophenoxyacetic acid (CPA), indole-3-propionic acid (IPA), 2,4-dichlorophenoxyacetic acid (2,4 -D), indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), chlorpyrifos and combinations thereof. Exemplary cytokinins include metatubinar, thiabendazole, N-(2-chloro-4-pyridyl)-N-phenylurea (CCPU), 1,3-diphenylurea (DPU), adenine and a half Sulfate, benzyl adenine, dimethylallyl adenine, kinetin, zeatin, nucleoside, adenosine, metatubinar nucleoside, metatopolin-9-glucoside, positive topping (ortho-topolin), Zhengtuopu nucleoside, Zhengtuopulin-9-glucoside, para-topolin, and extension Parkin nucleoside, totop forest 9-glycoside, orthomethoxytopocetoline, n-methoxy topology, methionine, m-methoxy topology, m-methoxy topology, m-methoxy Parkin-9-glucoside and combinations thereof, and plant extracts having cytokinin-like activity, such as coconut milk, banana powder, malt extract, pineapple powder or tomato powder.

所述培養基中還可包括激勃素。所述培養基中可包括一種糖或糖的組合且其可充當碳源。這類糖是本領域的通常技術人員已知的。示例性的糖包括果糖、蔗糖、葡萄糖、麥芽糖、半乳糖、甘露糖醇和山梨糖醇或其組合。其他示例性的添加劑(建議但非限制性的功能)包括聚胺(再生增強劑)；檸檬酸、聚乙烯基吡啶(PVP)和硫代硫酸鈉(抗褐化劑)；CaNO₃或葡萄糖酸鈣(玻璃質化還原劑)；多效唑(paclobutrazol)或嘧啶醇(ancymidol)(增殖增強劑)；乙醯基水楊酸(乙烯抑制劑)和p-氯苯氧基異丁酸(PCIB)和三碘苯甲酸(TIBA)(抗-生長素)。 The medium may also include activating hormone. A combination of sugars or sugars can be included in the medium and it can serve as a carbon source. Such sugars are known to those of ordinary skill in the art. Exemplary sugars include fructose, sucrose, glucose, maltose, galactose, mannitol, and sorbitol, or a combination thereof. Other exemplary additives (recommended but non-limiting functions) include polyamines (regeneration enhancers); citric acid, polyvinylpyridine (PVP), and sodium thiosulfate (anti-browning agent); CaNO ₃ or gluconic acid Calcium (glassy reducing agent); paclobutrazol or ancymidol (proliferation enhancer); acetylsalicylic acid (ethylene inhibitor) and p-chlorophenoxyisobutyric acid (PCIB) and Triiodobenzoic acid (TIBA) (anti-auxin).

在一些具體例中，基底培養基可以是Murashige及Skoog(MS)。合適的營養鹽還包括但不限於，Anderson’s Rhododendron、Chu’s N-6、DKW、Gamborg’s B-5、Hoaglands No.2、Kao & Michayluk、Nitsch & Nitsch、Schenk及Hildebrant、Vacin及Went、Whites及WPM，由市售來源如Caisson Laboratories,Inc或Phytotechnology Laboratories可獲得者。 In some embodiments, the basal medium can be Murashige and Skoog (MS). Suitable nutrient salts include, but are not limited to, Anderson's Rhododendron, Chu's N-6, DKW, Gamborg's B-5, Hoaglands No. 2, Kao & Michayluk, Nitsch & Nitsch, Schenk and Hildebrant, Vacin and Went, Whites and WPM. Available from commercial sources such as Caisson Laboratories, Inc or Phytotechnology Laboratories.

階段1培養基的一個實例包括間拓樸林。另一種非限制性 的實例包括間拓樸林、噻苯隆、NAA和BAP。另一種非限制性的實例包括間拓樸林、NAA和BAP。另一種非限制性的實例包括間拓樸林、NAA、BAP和IBA。另一種非限制性的實例包括間拓樸林、噻苯隆、NAA、BAP和IBA。另一種非限制性的實例包括噻苯隆、NAA、BAP和2ip。另一種非限制性的實例包括噻苯隆、NAA和2ip。另一種非限制性的實例包括間拓樸林、噻苯隆、NAA、BAP、IBA和2ip。另一種非限制性的實例包括間拓樸林、IBA、2ip和BAP。另一種非限制性的實例包括間拓樸林、噻苯隆、CPPU、NAA和BAP。另一種非限制性的實例包括間拓樸林、噻苯隆、DPU、NAA和BAP。另一種非限制性的實例包括噻苯隆、CPPU、BAP、IBA和2ip。另一種非限制性的實例包括CPPU、DPU、NAA和BAP。另一種非限制性的實例包括間拓樸林、噻苯隆、CPPU、DPU、NAA、BAP、IBA和2ip。這些非限制性實例中的每一個都可包括間拓樸林、噻苯隆、NAA、BAP、DPU、CPPU、IBA和/或2ip的類似物。 An example of a Stage 1 medium includes a meta-forest. Another non-restrictive Examples include Topoline, Thiabendron, NAA, and BAP. Another non-limiting example includes metatopolin, NAA, and BAP. Another non-limiting example includes metatopolin, NAA, BAP, and IBA. Another non-limiting example includes metatopolin, thidiazuron, NAA, BAP, and IBA. Another non-limiting example includes thidiazuron, NAA, BAP, and 2ip. Another non-limiting example includes thidiazuron, NAA, and 2ip. Another non-limiting example includes metatopolin, thidiazuron, NAA, BAP, IBA, and 2ip. Another non-limiting example includes metatopolin, IBA, 2ip, and BAP. Another non-limiting example includes metatopolin, thidiazuron, CPPU, NAA, and BAP. Another non-limiting example includes metatopolin, thidiazuron, DPU, NAA, and BAP. Another non-limiting example includes thidiazuron, CPPU, BAP, IBA, and 2ip. Another non-limiting example includes CPPU, DPU, NAA, and BAP. Another non-limiting example includes metatopolin, thidiazuron, CPPU, DPU, NAA, BAP, IBA, and 2ip. Each of these non-limiting examples can include analogs of metatopolin, thidiazuron, NAA, BAP, DPU, CPPU, IBA, and/or 2ip.

然後將階段1培養基置於試管或其他適當的容器中(包括罐、箱、壺、杯、無菌袋技術、生物反應器、暫時的浸漬器皿等，其中未指明的統稱為「管」)。可以將這些管加蓋或覆蓋並以高壓滅菌(autoclave)為滅菌管和培養基。在另一個具體例中，經由5-25磅壓力psi，在200℉的溫度高壓滅菌10-25分鐘完成滅菌。在另一個具體例中，經由15磅壓力 psi，在250℉的溫度高壓滅菌15-18分鐘完成滅菌。液體培養基可接受過濾滅菌。 Stage 1 medium is then placed in a test tube or other suitable container (including cans, boxes, pots, cups, sterile bag techniques, bioreactors, temporary impregnation vessels, etc., not specifically designated as "tubes"). These tubes can be capped or covered and autoclaveed as a sterile tube and medium. In another embodiment, sterilization is accomplished by autoclaving at a temperature of 200 °F for 10-25 minutes via 5-25 pounds pressure psi. In another specific example, via 15 pounds of pressure The psi is autoclaved at a temperature of 250 °F for 15-18 minutes to complete sterilization. The liquid medium can be sterilized by filtration.

在所述培植體可以在階段1培養基上自己安置之後，從這些培植體生長出的細胞培養物被轉移到階段2培養基中。本文揭示的階段2培養基可包括(i)間拓樸林；(2)至少三種細胞***素；(3)細胞***素間拓樸林或其類似物與至少兩種其他的細胞***素的組合；(4)至少一種生長素和至少兩種細胞***素；(5)至少兩種生長素和至少兩種細胞***素或(6)至少兩種生長素和至少三種細胞***素。在某些具體例中，階段2培養基必須包括多於1種生長素。在其他的具體例中，階段2培養基必須包括多於1種細胞***素。在進一步的具體例中，階段2培養基必須包括多於1種生長素和多於1種細胞***素。 After the culture bodies can be self-placed on the stage 1 medium, the cell cultures grown from these culture bodies are transferred to the stage 2 medium. The Stage 2 medium disclosed herein may comprise (i) an intermediate forest; (2) at least three cytokinins; (3) a combination of a cytokinin topology or an analog thereof and at least two other cytokinins (4) at least one auxin and at least two cytokinins; (5) at least two auxins and at least two cytokinins or (6) at least two auxins and at least three cytokinins. In some embodiments, the Stage 2 medium must include more than one auxin. In other embodiments, the Stage 2 medium must include more than one cytokinin. In a further embodiment, the Stage 2 medium must include more than one auxin and more than one cytokinin.

在一些具體例中，所述細胞***素和生長素選自間拓樸林、噻苯隆、NAA、IBA、BAP、2ip、CCPU和DPU的一種或多種。在另外的具體例中，所述細胞***素和生長素選自間拓樸林或其類似物、噻苯隆或其類似物、NAA或其類似物、IBA或其類似物、BAP或其類似物、2ip或其類似物、CCPU或其類似物和DPU或其類似物的一種或多種。 In some embodiments, the cytokinin and auxin are selected from one or more of the group consisting of Topoline, Thiabendron, NAA, IBA, BAP, 2ip, CCPU, and DPU. In another embodiment, the cytokinin and auxin are selected from the group consisting of metatopia or an analogue thereof, thidiazuron or an analogue thereof, NAA or an analogue thereof, IBA or an analog thereof, BAP or the like Or one or more of 2ip or an analog thereof, CCPU or the like and DPU or an analogue thereof.

在一些具體例中，所述階段2培養基的一個實例包括間拓樸林。另一個非限制性的實例包括間拓樸林、噻苯隆、NAA和BAP。另一個非限制性的實例包括間拓樸林、NAA和 BAP。另一個非限制性的實例包括間拓樸林、NAA、BAP和IBA。另一個非限制性的實例包括間拓樸林、噻苯隆、NAA、BAP和IBA。另一個非限制性的實例包括噻苯隆、NAA、BAP和2ip。另一個非限制性的實例包括噻苯隆、NAA和2ip。另一個非限制性的實例包括間拓樸林、噻苯隆、NAA、BAP、IBA和2ip。另一個非限制性的實例包括間拓樸林、IBA、2ip和BAP。另一個非限制性的實例包括間拓樸林、噻苯隆、CPPU、NAA和BAP。另一個非限制性的實例包括間拓樸林、噻苯隆、DPU、NAA和BAP。另一個非限制性的實例包括噻苯隆、CPPU、BAP、IBA和2ip。另一個非限制性的實例包括CPPU、DPU、NAA和BAP。另一個非限制性的實例包括間拓樸林、噻苯隆、CPPU、DPU、NAA、BAP、IBA和2ip。這些非限制性實例中的每一個都可包括下列的類似物：間拓樸林、噻苯隆、NAA、BAP、DPU、CPPU、IBA和/或2ip。 In some embodiments, one example of the Stage 2 medium comprises a meta-forest. Another non-limiting example includes metatopolin, thidia, NAA, and BAP. Another non-limiting example includes Topology, NAA, and BAP. Another non-limiting example includes metatopolin, NAA, BAP, and IBA. Another non-limiting example includes metatopolin, thidiazuron, NAA, BAP, and IBA. Another non-limiting example includes thidiazuron, NAA, BAP, and 2ip. Another non-limiting example includes thidiazuron, NAA, and 2ip. Another non-limiting example includes metatopolin, thidiazuron, NAA, BAP, IBA, and 2ip. Another non-limiting example includes Metaplin, IBA, 2ip, and BAP. Another non-limiting example includes metatopolin, thidiazuron, CPPU, NAA, and BAP. Another non-limiting example includes metatopolin, thidiazuron, DPU, NAA, and BAP. Another non-limiting example includes thidiazuron, CPPU, BAP, IBA, and 2ip. Another non-limiting example includes CPPU, DPU, NAA, and BAP. Another non-limiting example includes metatopolin, thidiazuron, CPPU, DPU, NAA, BAP, IBA, and 2ip. Each of these non-limiting examples may include the following analogs: metatopolin, thidiazuron, NAA, BAP, DPU, CPPU, IBA, and/or 2ip.

在本文揭示的特定具體例中，階段1和階段2培養基兩者都包括間拓樸林。在另一個非限制性的具體例中，階段1和階段2培養基兩者都包括間拓樸林、噻苯隆、NAA和BAP。在另一個非限制性的具體例中，階段1和階段2培養基兩者都包括間拓樸林、NAA和BAP。在另一個非限制性的具體例中，階段1和階段2培養基兩者都包括間拓樸林、NAA、BAP和IBA。在另一個非限制性的具體例中，階段1 和階段2培養基兩者都包括間拓樸林、噻苯隆、NAA、BAP和IBA。在另一個非限制性的具體例中，階段1和階段2培養基兩者都包括噻苯隆、NAA、BAP和2ip。在另一個非限制性的具體例中，階段1和階段2培養基兩者都包括噻苯隆、NAA和2ip。在另一個非限制性的具體例中，階段1和階段2培養基兩者都包括間拓樸林、噻苯隆、NAA、BAP、IBA和2ip。在另一個非限制性的具體例中，階段1和階段2培養基兩者都包括間拓樸林、IBA、2ip和BAP。在另一個非限制性的具體例中，階段1和階段2培養基兩者都包括間拓樸林、噻苯隆、CPPU、NAA和BAP。在另一個非限制性的具體例中，階段1和階段2培養基兩者都包括間拓樸林、噻苯隆、DPU、NAA和BAP。在另一個非限制性的具體例中，階段1和階段2培養基兩者都包括噻苯隆、CPPU、BAP、IBA和2ip。在另一個非限制性的具體例中，階段1和階段2培養基都包括CPPU、DPU、NAA和BAP。在另一個非限制性的具體例中，階段1和階段2培養兩者基都包括間拓樸林、噻苯隆、CPPU、DPU、NAA、BAP、IBA和2ip。這些非限制性實例中的每一個還可包括下列的類似物：間拓樸林、噻苯隆、NAA、BAP、DPU、CPPU、IBA和/或2ip。 In the particular embodiment disclosed herein, both Stage 1 and Stage 2 media include a meta-forest. In another non-limiting embodiment, both Stage 1 and Stage 2 media include metatopolin, thidia, NAA, and BAP. In another non-limiting embodiment, both stage 1 and stage 2 media include interspinning, NAA, and BAP. In another non-limiting embodiment, both Stage 1 and Stage 2 media include metatopolin, NAA, BAP, and IBA. In another non-limiting specific example, stage 1 Both the phase 2 and medium 2 include metatopolin, thidia, NAA, BAP, and IBA. In another non-limiting embodiment, both Stage 1 and Stage 2 media include thidia, NAA, BAP, and 2ip. In another non-limiting embodiment, both Stage 1 and Stage 2 media include thidiazuron, NAA, and 2ip. In another non-limiting embodiment, both Stage 1 and Stage 2 media include metatopolin, thidiazuron, NAA, BAP, IBA, and 2ip. In another non-limiting embodiment, both Stage 1 and Stage 2 media include metatopolin, IBA, 2ip, and BAP. In another non-limiting embodiment, both Stage 1 and Stage 2 media include metatublin, thidia, CPPU, NAA, and BAP. In another non-limiting embodiment, both Stage 1 and Stage 2 media include metatopolin, thidia, DPU, NAA, and BAP. In another non-limiting embodiment, both Stage 1 and Stage 2 media include thidia, CPPU, BAP, IBA, and 2ip. In another non-limiting embodiment, both Stage 1 and Stage 2 media include CPPU, DPU, NAA, and BAP. In another non-limiting embodiment, both phase 1 and phase 2 cultures include metatopolin, thidiazuron, CPPU, DPU, NAA, BAP, IBA, and 2ip. Each of these non-limiting examples may also include the following analogs: metatopolin, thidiazuron, NAA, BAP, DPU, CPPU, IBA, and/or 2ip.

在一些具體例中，階段1和階段2培養基的實例係描述於WO/2011/100762，其以引用方式全部併入本文。例如，階 段1和階段2培養基可選自由下列族群所組成的培養基：培養基b-12c(i-v)；培養基CW2(i-v)；培養基CW3(i-v)；培養基b-9(i-v)；培養基CW4(i-v)；培養基CW1(i-v)；培養基CW5(i-v)；培養基CW6(i-v)；培養基b-10(i-v)；培養基b-11(i-v)；培養基b-1(i-v)；培養基b-4(i-v)；培養基b-6(i-v)。 In some specific examples, examples of Stage 1 and Stage 2 media are described in WO/2011/100762, which is incorporated herein in its entirety by reference. For example, order The medium of Stage 1 and Stage 2 may be selected from the following medium: medium b-12c (iv); medium CW2 (iv); medium CW3 (iv); medium b-9 (iv); medium CW4 (iv); Medium CW1 (iv); medium CW5 (iv); medium CW6 (iv); medium b-10 (iv); medium b-11 (iv); medium b-1 (iv); medium b-4 (iv); Medium b-6 (iv).

在一些具體例中，階段1和階段2培養基的實例選自下列：培養基b-12c(i-v)： In some embodiments, examples of Stage 1 and Stage 2 media are selected from the group consisting of: medium b-12c (iv):

培養基CW2(i-v)： Medium CW2 (iv):

培養基CW3(i-v)： Medium CW3 (iv):

培養基b-9(i-v)： Medium b-9(iv):

培養基CW4(i-v)： Medium CW4 (iv):

培養基CW1(i-v)： Medium CW1 (iv):

培養基CW5(i-v)： Medium CW5(iv):

培養基CW6(i-v)： Medium CW6 (iv):

培養基b-10(i-v)： Medium b-10(iv):

培養基b-11(i-v)： Medium b-11 (iv):

培養基b-1(i-v)： Medium b-1 (iv):

培養基b-4(i-v)： Medium b-4 (iv):

培養基b-6(i-v)： Medium b-6(iv):

培養基B-9N2 Medium B-9N2

培養基B-12C CPPU Medium B-12C CPPU

培養基B-12C DPU Medium B-12C DPU

在一些具體例中，所述非細胞***素的組分是由市售來源可獲得之Anderson’s Rhododendron、Chu’s N-6、DKW、Gamborg’sB-5、Hoaglands No.2、Kao & Michayluk、Nitsch & Nitsch、Schenk及Hildebrant、Vacin及Went、Whites及WPM中所發現者。特定的培養基可具有比前述表中提供者 更高或更低程度的大量營養物(macronutrients)，且其他的將缺少硝酸鹽。在一些具體例中，所述培養基具有更高或更低程度的大量營養物且缺少硝酸鹽。在一些具體例中，所述培養基具有更高程度的大量營養物且缺少硝酸鹽。 In some embodiments, the non-cytokinin component is commercially available from Anderson's Rhododendron, Chu's N-6, DKW, Gamborg's B-5, Hoaglands No. 2, Kao & Michayluk, Nitsch & Found in Nitsch, Schenk and Hildebrant, Vacin and Went, Whites and WPM. Specific media may have a provider than in the previous table Higher or lower levels of macronutrients, and others will lack nitrate. In some embodiments, the medium has a higher or lower level of nutrients and lacks nitrate. In some embodiments, the medium has a higher degree of nutrients and lacks nitrate.

本文所揭示的培養基還包括強化培養基。所述強化培養基為將上述培養基中的至少一種細胞***素和/或生長素的濃度增加，例如且不限於，1%、3%、5%、7%、10%、15%、20%、25%、30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、100%、105%、110%或200%。在其他的具體例中，上述培養基中的至少一種細胞***素和/或生長素的濃度增加，例如且不限於，1-10%、5-15%、10-20%、15-25%、20-30%、25-35%、30-40%、35-45%、40-50%、45-55%、50-60%、55-65%、60-70%、65-75%、70-80%、75-85%、80-90%、85-95%、90-100%、95-105%、100-110%、105-115%、110-120%、115-125%、120-130%、125-135%、130-140%、135-145%、140-150%、145-155%、150-160%、155-165%、160-170%、165-175%、170-180%、175-185%、180-190%、185-195%、190-200%、195-205%、3-6%、7-17%、12-22%、17-27%、22-32%、27-37%、32-42%、37-47%、42-52%、47-57%、52-62%、57-67%、62-72%、67-77%、72-82%、77-87%、82-92%、87-97%、92-102%、97-107%、102-112%、107-117%、112-122%、 117-127%、122-132%、127-137%、132-142%、137-147%、142-152%、147-157%、152-162%、157-167%、162-172%、167-177%、172-182%、177-187%、162-172%、167-177%、182-192%、187-197%、192-202%、197-207%，或200-210%。當多於一種細胞***素和/或生長素經強化，每種增加的細胞***素和/或生長素的濃度可以與培養基中其他細胞***素和/或生長素相同或不同的量增加。 The medium disclosed herein also includes a fortified medium. The strengthening medium is an increase in concentration of at least one cytokinin and/or auxin in the above medium, such as, but not limited to, 1%, 3%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105% , 110% or 200%. In other specific examples, the concentration of at least one cytokinin and/or auxin in the above medium is increased, for example, and not limited to, 1-10%, 5-15%, 10-20%, 15-25%, 20-30%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60-70%, 65-75%, 70-80%, 75-85%, 80-90%, 85-95%, 90-100%, 95-105%, 100-110%, 105-115%, 110-120%, 115-125%, 120-130%, 125-135%, 130-140%, 135-145%, 140-150%, 145-155%, 150-160%, 155-165%, 160-170%, 165-175%, 170-180%, 175-185%, 180-190%, 185-195%, 190-200%, 195-205%, 3-6%, 7-17%, 12-22%, 17-27%, 22-32%, 27-37%, 32-42%, 37-47%, 42-52%, 47-57%, 52-62%, 57-67%, 62-72%, 67-77%, 72-82%, 77-87%, 82-92%, 87-97%, 92-102%, 97-107%, 102-112%, 107-117%, 112-122%, 117-127%, 122-132%, 127-137%, 132-142%, 137-147%, 142-152%, 147-157%, 152-162%, 157-167%, 162-172%, 167-177%, 172-182%, 177-187%, 162-172%, 167-177%, 182-192%, 187-197%, 192-202%, 197-207%, or 200-210% . When more than one cytokinin and/or auxin is fortified, the concentration of each increased cytokinin and/or auxin may be increased by the same or a different amount than other cytokinins and/or auxins in the medium.

下表提供了本文所揭示的強化培養基的非限制性的實例。每種培養基包括了在上述各自的表或如下述段落中所描述以調整為具有下列細胞***素程度的經調整組分：培養基b-12c(i-v)： The following table provides non-limiting examples of the fortified media disclosed herein. Each medium comprises an adjusted component adjusted to have the following degree of cytokinin as described in the respective tables above or as described in the following paragraph: medium b-12c(iv):

培養基CW2(i-v)： Medium CW2 (iv):

培養基CW3(i-v)： Medium CW3 (iv):

培養基b-9(i-v)： Medium b-9(iv):

培養基CW4(i-v)： Medium CW4 (iv):

培養基CW1(i-v)： Medium CW1 (iv):

培養基CW5(i-v)： Medium CW5(iv):

培養基CW6(i-v)： Medium CW6 (iv):

培養基b-10(i-v)： Medium b-10(iv):

培養基b-11(i-v)： Medium b-11 (iv):

培養基b-1(i-v)： Medium b-1 (iv):

培養基b-4(i-v)： Medium b-4 (iv):

培養基b-6(i-v)： Medium b-6(iv):

培養基B-9N2： Medium B-9N2:

培養基B-12C CPPU Medium B-12C CPPU

培養基B-12C DPU Medium B-12C DPU

另外的強化培養基可包括添加或調整成下列細胞***素和/或生長素濃度的任何上述標準的培養基：培養基AA Additional fortification medium may include any of the above standard media added or adjusted to the following cytokinin and/or auxin concentrations: medium AA

培養基AB Medium AB

培養基AC Medium AC

如在下述更完整解釋，當利用強化培養基時，培植體或苗一般(但並非必然)在強化培養基比置於非強化培養基者的時間更短，且在培養於強化培養基之後，將該培植體或苗轉移到含有標準程度的、降低程度的或沒有細胞***素和/或生長素的培養基上(含有降低或沒有細胞***素和/或生長素者在本文中都稱為「減料」培養基)。 As explained more fully below, when using a fortified medium, the culture or seedling is generally (but not necessarily) shorter in the time the fortified medium is placed than in the non-fortified medium, and after culturing the medium, the culture is grown. Or seedlings are transferred to a medium containing a standard degree of reduced or no cytokinin and/or auxin (containing reduced or no cytokinin and/or auxin, referred to herein as "reduced" medium ).

本文所揭示培養基的一種或多種成分可基於植物種類進行調整。 One or more components of the media disclosed herein can be adjusted based on the plant species.

在一些具體例中，本文所揭示培養基可用於竹的組織培養。WO/2011/100762係描述代表性竹之屬，其以引用方式全部併入本文中。 In some embodiments, the media disclosed herein can be used for tissue culture of bamboo. WO/2011/100762 describes a representative genus of bamboo, which is hereby incorporated by reference in its entirety.

在一些具體例中，階段1培養基係選自由下列族群之組成：b-12c-v培養基、B-12C-CPPU-v培養基、B-12C DPU-v、B-9N2-v培養基或b-10-v培養基或其強化版本。 In some embodiments, the Stage 1 medium is selected from the group consisting of b-12c-v medium, B-12C-CPPU-v medium, B-12C DPU-v, B-9N2-v medium or b-10 -v medium or an enhanced version thereof.

在一些具體例中，階段2培養基係選自由下列族群之組成：CW1-v培養基；CW2-v培養基；CW3-v培養基；CW4-v培養基；CW5-v培養基；CW6-v培養基、B-12C-CPPU-v培 養基、B-12C DPU-v、B-9N2-v培養基或其強化和減料/標準版本，進行10-120天的週期(如下述更完整描述的修飾強化培養基)。 In some embodiments, the Stage 2 medium is selected from the group consisting of CW1-v medium; CW2-v medium; CW3-v medium; CW4-v medium; CW5-v medium; CW6-v medium, B-12C. -CPPU-v training Nutrient, B-12C DPU-v, B-9N2-v medium or its fortified and reduced/standard versions are subjected to a 10-120 day cycle (modification-enhancing medium as described more fully below).

在一些具體例中，可以包括一種或多種階段3培養基。在一些具體例中，階段3培養基係選自由下列族群之組成：Br-2-v培養基；Ech-v培養基或Amel-v培養基，或其強化和減料/標準版本(如下述更完整描述的修飾強化培養基)。 In some embodiments, one or more Stage 3 media can be included. In some embodiments, the Stage 3 medium is selected from the group consisting of Br-2-v medium; Ech-v medium or Amel-v medium, or a reinforced and reduced/standard version thereof (as described more fully below) Modified medium ().

在一些具體例中，階段2、階段3、階段4、階段5、階段6、階段7培養基的非限制性實例包括：培養基Ech(i-v)： In some embodiments, non-limiting examples of Stage 2, Stage 3, Stage 4, Stage 5, Stage 6, Stage 7 media include: medium Ech (iv):

培養基BR-2(i-v)： Medium BR-2 (iv):

培養基Amel(i-v) Medium Amel (iv)

在一些具體例中，這些培養基的強化版本的非限制性實例包括：培養基Ech(i-v)： In some embodiments, non-limiting examples of enhanced versions of these media include: medium Ech (iv):

培養基BR-2(i-v)： Medium BR-2 (iv):

培養基Amel(i-v)： Medium Amel (iv):

(細胞***素和類似物) (cytokinins and analogs)

依據所揭示的有用化合物係包括間拓樸林類似物，其具有通式： Useful compounds according to the disclosure include interstitial analogs having the general formula:

其中W是芳基或雜芳基；R1是經取代的或未經取代的烷基，其中烷基中的任何C可以用O、N或S取代；各R2獨立地是H、OH、C1-C6烷基、C1-C6伸烷基、C1-C6炔基、鹵素、氰基、C1-C6烷氧基、芳基或雜芳基，各自可選擇性地經C1-C6烷基、SH、NHR3、CO2R3或鹵素取代；R3是H、OH、C1-C6烷基、C1-C6伸烷基、C1-C6炔基、鹵素、羧基基團、酯基基團、醛或氰基；R是0至8。 Wherein W is aryl or heteroaryl; R1 is substituted or unsubstituted alkyl, wherein any C in the alkyl group may be substituted with O, N or S; each R2 is independently H, OH, C1- C6 alkyl, C1-C6 alkylene, C1-C6 alkynyl, halogen, cyano, C1-C6 alkoxy, aryl or heteroaryl, each optionally C1-C6 alkyl, SH, NHR3, CO2R3 or halogen substituted; R3 is H, OH, C1-C6 alkyl, C1-C6 alkylene, C1-C6 alkynyl, halogen, carboxyl group, ester group, aldehyde or cyano; R is 0 to 8.

在一些具體例中，W是 In some specific examples, W is

其中虛線代表存在或不存在之鍵；X¹-X⁷各自獨立地選自C、N、O、S，條件是將環與N連接的X是C。 Wherein the dotted line represents the bond present or absent; X ¹ -X ⁷ are each independently selected from C, N, O, S, provided that the X connecting the ring to N is C.

在一些具體例中，所述化合物具有結構， In some embodiments, the compound has a structure,

其中虛線代表存在或不存在之鍵。 The dotted line represents the presence or absence of a bond.

在一些具體例中，所述化合物具有結構， In some embodiments, the compound has a structure,

其中虛線代表存在或不存在之鍵；X8-X12各自獨立地選自C、N、O、S；各R4獨立地是H、OH、C1-C6烷基、C1-C6伸烷基、C1-C6炔基、鹵素、氰基、C1-C6烷氧基、芳基或雜芳基，其各自可選擇性地經C1-C6烷基、SH、NHR3、CO2R3或鹵素取代；R3是H、OH、C1-C6烷基、C1-C6伸烷基、C1-C6炔基、鹵素、羧基基團羧基基團、酯基基團、醛或氰基；p是0到5；且q是0到6。 Wherein the dotted line represents the presence or absence of a bond; X8-X12 are each independently selected from C, N, O, S; each R4 is independently H, OH, C1-C6 alkyl, C1-C6 alkyl, C1- C6 alkynyl, halogen, cyano, C1-C6 alkoxy, aryl or heteroaryl, each of which may be optionally substituted by C1-C6 alkyl, SH, NHR3, CO2R3 or halogen; R3 is H, OH , C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkynyl, halogen, carboxyl group carboxyl group, ester group, aldehyde or cyano; p is 0 to 5; and q is 0 to 6.

在一些具體例中，所述化合物具有結構， In some embodiments, the compound has a structure,

在一些具體例中，所述化合物具有結構， In some embodiments, the compound has a structure,

又更進一步地，所述化合物可具有選自如下的結構： Still further, the compound may have a structure selected from the group consisting of:

在一些具體例中，R4是OH。 In some embodiments, R4 is OH.

在一些具體例中，所述化合物具有選自如下的結構： In some embodiments, the compound has a structure selected from the group consisting of:

在一些具體例中，所述化合物具有結構， In some embodiments, the compound has a structure,

其中虛線代表存在或不存在之鍵。 The dotted line represents the presence or absence of a bond.

在另一個具體例中，所述化合物具有結構 In another embodiment, the compound has a structure

其中虛線代表存在或不存在之鍵；X8-X12是各自獨立地選自C、N、O、S；各R4獨立地是H、OH、C1-C6烷基、C1-C6伸烷基、C1-C6炔基、鹵素、氰基、C1-C6烷氧基、芳基或雜芳基，其各自可選擇性地經C1-C6烷基、SH、NHR3、CO2R3或鹵素取代；R3是H、OH、C1-C6烷基、C1-C6伸烷基、C1-C6炔基、鹵素、羧基基團、酯基基團、醛或氰基；P是0到5；且q是0到6。 Wherein the dotted line represents the presence or absence of a bond; X8-X12 are each independently selected from C, N, O, S; each R4 is independently H, OH, C1-C6 alkyl, C1-C6 alkyl, C1 -C6 alkynyl, halogen, cyano, C1-C6 alkoxy, aryl or heteroaryl, each of which may be optionally substituted by C1-C6 alkyl, SH, NHR3, CO2R3 or halogen; R3 is H, OH, C1-C6 alkyl, C1-C6 alkylene, C1-C6 alkynyl, halogen, carboxyl group, ester group, aldehyde or cyano; P is 0 to 5; and q is 0 to 6.

在一些具體例中，所述化合物具有結構 In some embodiments, the compound has a structure

更有一些具體例中，所述化合物具有結構 In some specific examples, the compound has a structure

在一些具體例中，所述化合物是間拓樸林，也稱為6-(3-羥基苄基胺基)-嘌呤，且縮寫為mT，具有經驗分子式C12H10N5OH、分子量241.25和下列結構式： In some embodiments, the compound is metatuberculosis, also known as 6-(3-hydroxybenzylamino)-indole, and is abbreviated as mT, having the empirical formula C12H10N5OH, molecular weight 241.25, and the following structural formula:

其中所述間拓樸林是柳樹或楊樹的衍生物。 Wherein the meta-forest is a derivative of willow or poplar.

間拓樸林類似物特定地包括但不限於：間拓樸林核苷、間拓樸林-9-葡糖苷、正拓樸林、正拓樸林核苷、正拓樸林-9-葡糖苷、對拓樸林、對拓樸林核苷、對拓樸林-9-葡糖苷、正甲氧拓樸林、正甲氧拓樸林核苷、間甲氧拓樸林、間甲氧拓樸林核苷和間甲氧拓樸林-9-葡糖苷。在特定的具體例即本文中稱為「mT限制性具體例」中，可以將6-(3-氟代苄基胺基)嘌呤(FmT)、6-(3-氟代苄基胺基)嘌呤-9-核苷(FmTR) 和/或6-(3-甲氧苄基胺基)嘌呤-9-核苷(memTR)從間拓樸林類似物的類中排除。 The metatopical analogs specifically include, but are not limited to, inter-topical forest nucleosides, metatopical forests-9-glucosides, positive topological forests, positive topographical nucleosides, positive tops of forests-9-Portuguese Glycoside, on top of the forest, on the top of the forest nucleoside, on the top of the forest - 9 - glucoside, n-methoxy topology, n-methoxy topolin nucleoside, m-methoxy topolin, m-methoxy Topolin nucleoside and m-methoxytopolin-9-glucoside. In a specific specific example, which is referred to herein as "mT-restricted specific example", 6-(3-fluorobenzylamino)phosphonium (FmT), 6-(3-fluorobenzylamino) can be used.嘌呤-9-nucleoside (FmTR) And/or 6-(3-methoxybenzylamino)purine-9-nucleoside (memTR) is excluded from the class of metatopolin analogs.

依據所揭示有用的化合物包括噻苯隆類似物，其具有通式 Compounds useful according to the disclosure include thiabendazole analogs having the general formula

其中V是芳基或雜芳基；各R5和R6是各自獨立的H、OH、C1-C6烷基、C1-C6伸烷基、C1-C6炔基、鹵素、氰基、C1-C6烷氧基、芳基或雜芳基，其各自選擇性地經C1-C6烷基或鹵素取代；n是0到4；o是0到5 X13-X16各自獨立地選自C、N、O、S；Z1和Z2是各自獨立的NH、O、SH或CH，或可組合Z1和Z2以形成經取代或未經取代的芳基或雜芳基；且Y1是O或S。 Wherein V is aryl or heteroaryl; each R5 and R6 are independently H, OH, C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkynyl, halogen, cyano, C1-C6 alkane An oxy, aryl or heteroaryl group, each of which is optionally substituted by a C1-C6 alkyl group or a halogen; n is 0 to 4; o is 0 to 5 X13-X16 are each independently selected from C, N, O, S; Z1 and Z2 are each independently NH, O, SH or CH, or Z1 and Z2 may be combined to form a substituted or unsubstituted aryl or heteroaryl group; and Y1 is O or S.

在另一個具體例中，化合物具有結構 In another embodiment, the compound has a structure

其中X17-X21各自獨立地選自C、N、O、S。 Wherein X17-X21 are each independently selected from the group consisting of C, N, O, and S.

在其他的具體例中，化合物包括 In other specific examples, the compound includes

在一個具體例中，所述化合物是噻苯隆，亦稱為1-苯基-3-(1,2,3-噻二唑-5-基)脲和5-苯羰基胺基-1,2,3-噻二唑，具有經驗分子式C9H8N4OS、分子量220.25和下列結構式 In one embodiment, the compound is thidiazuron, also known as 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea and 5-phenylcarbonylamino-1, 2,3-thiadiazole, having the empirical formula C9H8N4OS, molecular weight 220.25 and the following structural formula

依據所揭示有用的化合物包括B-萘氧乙酸類似物，其具有通式： Compounds useful according to the disclosure include B-naphthyloxyacetic acid analogs having the general formula:

或其鹽；其中Ra是COR3、CO2R3、CONR3R4、或CN；各Rb獨立地是R3；OR3；F；Cl；Br；I；CN；NO2；OCF3；CF3；NR2R3；SR3、SOR3、SO2R3、CO2R3、COR3、CONR3R4、CSNR4R5；或選擇性地經取代的芳基或選擇性地經取代的雜芳基，其中芳基或雜芳基的各取代基獨立地是C1-C6烷基、F、Cl、Br、或I；A是1、2、3、4、5、6、或7；Xa是NH、S或O； 各R3獨立地是H、C1-C6烷基、C2-C6烯基、或C2-C6炔基；及各R4獨立地是R3或選擇性地經取代的苯基，其中苯基的各取代基獨立地是C1-C6烷基、F、Cl、Br、或I。 Or a salt thereof; wherein Ra is COR3, CO2R3, CONR3R4, or CN; each Rb is independently R3; OR3; F; Cl; Br; I; CN; NO2; OCF3; CF3; NR2R3; SR3, SOR3, SO2R3, CO2R3 , COR3, CONR3R4, CSNR4R5; or a selectively substituted aryl or a selectively substituted heteroaryl group, wherein each substituent of the aryl or heteroaryl group is independently a C1-C6 alkyl group, F, Cl , Br, or I; A is 1, 2, 3, 4, 5, 6, or 7; Xa is NH, S or O; Each R3 is independently H, C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl; and each R4 is independently R3 or a selectively substituted phenyl group, wherein each substituent of the phenyl group Independently C1-C6 alkyl, F, Cl, Br, or I.

在另一個具體例中，所述化合物具有結構： In another embodiment, the compound has a structure:

在一個具體例中，所述化合物是B-萘氧乙酸(NAA)，亦稱為(2-萘氧)-(9CI)乙酸且具有CAS號120-23-0，具有經驗分子式C12H10O3、分子量202.21和下列結構式： In one embodiment, the compound is B-naphthyloxyacetic acid (NAA), also known as (2-naphthyloxy)-(9CI) acetic acid and has a CAS number of 120-23-0, having the empirical formula C12H10O3, molecular weight 202.21 And the following structural formula:

NAA類似物的其他實例可包括但不限於： Other examples of NAA analogs can include, but are not limited to:

依據所揭示有用的化合物包括吲哚丁酸(IBA)的類似物，其具有通式： Compounds useful according to the disclosure include analogs of indolebutyric acid (IBA) having the general formula:

或其鹽；其中R1是COR3、CO2R3、CONR3R4或CN；各R2獨立地是R3；OR3；F；Cl；Br；I；CN；NO2；OCF3；CF3；NR2R3；SR3、SOR3、SO2R3、CO2R3、COR3、CONR3R4、CSNR4R5；或選擇性地經取代的芳基或選擇性地經取代的雜芳基，其中芳基或雜芳基的各取代基獨立地是C1-C6烷基、F、Cl、Br或I；n是1、2、3或4； X是NH、S或O；各R3獨立地是H、C1-C6烷基、C2-C6烯基或C2-C6炔基；及各R4獨立地是R3或選擇性地經取代的苯基，其中苯基的各取代基獨立地是C1-C6烷基、F、Cl、Br或I。 Or a salt thereof; wherein R1 is COR3, CO2R3, CONR3R4 or CN; each R2 is independently R3; OR3; F; Cl; Br; I; CN; NO2; OCF3; CF3; NR2R3; SR3, SOR3, SO2R3, CO2R3, COR3, CONR3R4, CSNR4R5; or a selectively substituted aryl or a selectively substituted heteroaryl group, wherein each substituent of the aryl or heteroaryl group is independently a C1-C6 alkyl group, F, Cl, Br or I; n is 1, 2, 3 or 4; X is NH, S or O; each R3 is independently H, C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl; and each R4 is independently R3 or a selectively substituted phenyl group, Wherein each substituent of the phenyl group is independently a C1-C6 alkyl group, F, Cl, Br or I.

在另一個具體例中，所述化合物具有結構： In another embodiment, the compound has a structure:

在一個具體例中，所述化合物是吲哚丁酸(IBA)，也稱為1-吲哚-3-丁酸，且具有CAS號133-32-4，具有經驗分子式C12H13NO2、分子量203.24和下列結構式： In one embodiment, the compound is indolebutyric acid (IBA), also known as 1-indole-3-butyric acid, and has CAS number 133-32-4, having the empirical formula C12H13NO2, molecular weight 203.24, and the following Structure:

IBA類似物的其他實例可包括但不限於： Other examples of IBA analogs can include, but are not limited to:

依據所揭示有用的化合物包括苄胺基嘌呤(BAP)類似物，其具有通式： Compounds useful according to the disclosure include benzylaminopurine (BAP) analogs having the general formula:

或其鹽；其中虛線代表存在或不存在之鍵；各R5和各R6獨立地是R3；OR3；F；Cl；Br；I；CN；NO2；OCF3；CF3；NR2R3；SR3、SOR3、SO2R3、CO2R3、COR3、CONR3R4、CSNR4R5；或選擇性地經取代的芳基或選擇性地經取代的雜芳基，其中芳基或雜芳基的各取代基獨立地是C1-C6烷基、F、Cl、Br或I；o是0、1、2、3、4或5；p是0、1或2； X1是NH、S或O；X4是-N=且X5是-NH-、-S-或-O-；或X5是-N=且X4是-NH-、-S-或-O-；X2和X3獨立地是N或CR6；各R3獨立地是H、C1-C6烷基、C2-C6烯基或C2-C6炔基；及各R4獨立地是R3或選擇性地經取代的苯基，其中苯基的各取代基獨立地是C1-C6烷基、F、Cl、Br或I。 Or a salt thereof; wherein the dotted line represents a bond present or absent; each R5 and each R6 is independently R3; OR3; F; Cl; Br; I; CN; NO2; OCF3; CF3; NR2R3; SR3, SOR3, SO2R3, CO2R3, COR3, CONR3R4, CSNR4R5; or a selectively substituted aryl or a selectively substituted heteroaryl group, wherein each substituent of the aryl or heteroaryl group is independently a C1-C6 alkyl group, F, Cl, Br or I; o is 0, 1, 2, 3, 4 or 5; p is 0, 1 or 2; X1 is NH, S or O; X4 is -N= and X5 is -NH-, -S- or -O-; or X5 is -N= and X4 is -NH-, -S- or -O-; X2 And X3 are independently N or CR6; each R3 is independently H, C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl; and each R4 is independently R3 or a selectively substituted phenyl Wherein each substituent of the phenyl group is independently C1-C6 alkyl, F, Cl, Br or I.

在一些具體例中，X4是-N=且X5是-NH-、-S-或-O-，且虛線代表存在或不存在之鍵。因此，考慮依據以下化學式的化合物。 In some embodiments, X4 is -N= and X5 is -NH-, -S-, or -O-, and the dashed line represents the presence or absence of a bond. Therefore, a compound according to the following chemical formula is considered.

在其他的具體例中，X5是-N=且X4是-NH-、-S-或-O-。因此，考慮依據以下化學式的化合物。 In other specific examples, X5 is -N= and X4 is -NH-, -S- or -O-. Therefore, a compound according to the following chemical formula is considered.

在另一個具體例中，所述化合物具有結構： In another embodiment, the compound has a structure:

在另一個具體例中，所述化合物具有結構： In another embodiment, the compound has a structure:

在一個具體例中，所述化合物是苄基胺基嘌呤(BAP)，亦稱為9H-嘌呤-6-胺，N-(苯甲基)-，其具有CAS編號1214-39-7、經驗分子式C12H11N5、分子量225.25和下列結構式： In one embodiment, the compound is benzylaminopurine (BAP), also known as 9H-indol-6-amine, N-(benzyl)-, having CAS number 1214-39-7, experience Molecular formula C12H11N5, molecular weight 225.25 and the following structural formula:

BAP類似物的其他實例可包括但不限於： Other examples of BAP analogs can include, but are not limited to:

依據所揭示有用的化合物包括6-y-y-(二甲基烯丙基胺基)-嘌呤(2ip)類似物，其具有通式： Compounds useful according to the disclosure include 6-yy-(dimethylallylamino)-indole (2ip) analogs having the general formula:

或其鹽；其中虛線代表存在或不存在之鍵；其中R7、R8和各R9獨立地是R3；OR3；F；Cl；Br；I；CN；NO2；OCF3；CF3；NR2R3；SR3、SOR3、SO2R3、CO2R3、COR3、CONR3R4、CSNR4R5；或選擇性地經取代的芳基或選擇性地經取代的雜芳基，其中芳基或雜芳基的各取代基獨立地是C1-C6烷基、F、Cl、Br或I；q是0、1或2；X6是NH、S或O；X9是-N=且X10是-NH-、-S-或-O-；或X10是-N=且X9 是-NH-、-S-或-O-；X7和X8獨立地是N或CR9；及各R3獨立地是H、C1-C6烷基、C2-C6烯基或C2-C6炔基；及各R4獨立地是R3或選擇性地經取代的苯基，其中苯基的各取代基獨立地是C1-C6烷基、F、Cl、Br或I。 Or a salt thereof; wherein the dotted line represents a bond present or absent; wherein R7, R8 and each R9 are independently R3; OR3; F; Cl; Br; I; CN; NO2; OCF3; CF3; NR2R3; SR3, SOR3, SO2R3, CO2R3, COR3, CONR3R4, CSNR4R5; or a selectively substituted aryl or a selectively substituted heteroaryl group, wherein each substituent of the aryl or heteroaryl group is independently a C1-C6 alkyl group, F, Cl, Br or I; q is 0, 1 or 2; X6 is NH, S or O; X9 is -N= and X10 is -NH-, -S- or -O-; or X10 is -N= And X9 Is -NH-, -S- or -O-; X7 and X8 are independently N or CR9; and each R3 is independently H, C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl; Each R4 is independently R3 or a selectively substituted phenyl group, wherein each substituent of the phenyl group is independently C1-C6 alkyl, F, Cl, Br or I.

在一些具體例中，虛線代表存在或不存在之鍵。因此，考慮依據以下結構式的化合物。 In some embodiments, the dashed line represents the presence or absence of a bond. Therefore, compounds according to the following structural formula are considered.

在一些具體例中，X9是-N=且X10是-NH-、-S-或-O-。因此，考慮依據以下結構式的化合物。 In some embodiments, X9 is -N= and X10 is -NH-, -S- or -O-. Therefore, compounds according to the following structural formula are considered.

在其他的具體例中，X10是-N=且X9是-NH-、-S-或-O-。因此，考慮具有以下結構的化合物。 In other specific examples, X10 is -N= and X9 is -NH-, -S- or -O-. Therefore, a compound having the following structure is considered.

在另一個具體例中，所述化合物具有結構： In another embodiment, the compound has a structure:

在一個具體例中，所述化合物是6-y,y,-(二甲基烯丙基胺基)-嘌呤(2ip)或DAP，亦稱為N-(3-甲基-2-丁烯-1-基)-9H-嘌呤-6-胺，具有CAS編號2365-40-4、經驗分子式C10H13N5、分子量203.24和下列結構式： In one embodiment, the compound is 6-y, y,-(dimethylallylamino)-indole (2ip) or DAP, also known as N-(3-methyl-2-butene) -1-yl)-9H-indole-6-amine, having CAS number 2365-40-4, empirical formula C10H13N5, molecular weight 203.24 and the following structural formula:

2ip類似物的其他實例包括但不限於： Other examples of 2ip analogs include, but are not limited to:

依據所揭示有用化合物包括N,N-二苯脲(DPU)類似物，其具有通式： Useful compounds according to the disclosure include N,N-diphenylurea (DPU) analogs having the general formula:

或其鹽；其中各R10和各R11獨立地是R3；OR3；F；Cl；Br；I；CN；NO2；OCF3；CF3；NR2R3；SR3、SOR3、SO2R3、 CO2R3、COR3、CONR3R4、CSNR4R5；或選擇性地經取代的芳基或選擇性地經取代的雜芳基，其中芳基或雜芳基的各取代基獨立地是C1-C6烷基、F、Cl、Br或I；r和s獨立地是0、1、2、3、4或5；X11和X12獨立地是NR10、S或O；各R3獨立地是H、C1-C6烷基、C2-C6烯基或C2-C6炔基；及各R4獨立地是R3或選擇性地經取代的苯基，其中苯基的各取代基獨立地是C1-C6烷基、F、Cl、Br或I。 Or a salt thereof; wherein each R10 and each R11 are independently R3; OR3; F; Cl; Br; I; CN; NO2; OCF3; CF3; NR2R3; SR3, SOR3, SO2R3, CO2R3, COR3, CONR3R4, CSNR4R5; or a selectively substituted aryl or a selectively substituted heteroaryl group, wherein each substituent of the aryl or heteroaryl group is independently a C1-C6 alkyl group, F, Cl, Br or I; r and s are independently 0, 1, 2, 3, 4 or 5; X11 and X12 are independently NR10, S or O; each R3 is independently H, C1-C6 alkyl, C2 a -C6 alkenyl or C2-C6 alkynyl; and each R4 is independently R3 or a selectively substituted phenyl group, wherein each substituent of the phenyl group is independently C1-C6 alkyl, F, Cl, Br or I.

在另一個具體例中，所述化合物具有結構： In another embodiment, the compound has a structure:

在一個具體例中，所述化合物是N,N-二苯脲(DPU)，其由如下化學式代表： In one embodiment, the compound is N,N-diphenylurea (DPU), which is represented by the following chemical formula:

DPU類似物的其他實例包括但不限於： Other examples of DPU analogs include, but are not limited to:

依據所揭示有用的化合物包括N-吡啶基-N’-苯脲(在本文中可交互使用PPU和CPPU)類似物，其具有通式： Compounds useful according to the disclosure include N-pyridyl-N'-phenylurea (PPU and CPPU can be used interchangeably herein) analogs having the general formula:

或其鹽；其中各R12和各R13獨立地是R3；OR3；F；Cl；Br；I；CN；NO2；OCF3；CF3；NR2R3；SR3、SOR3、SO2R3、CO2R3、COR3、CONR3R4、CSNR4R5；或選擇性地經取代的芳基或選擇性地經取代的雜芳基，其中芳基或雜芳基的各取代基獨立地是C1-C6烷基、F、Cl、Br或I；t和u獨立地是0、1、2、3、4或5；X13和X14獨立地是NR12、S或O；各R3獨立地是H、C1-C6烷基、C2-C6烯基或C2-C6炔 基；及各R4獨立地是R3或選擇性地經取代的苯基，其中苯基的各取代物獨立地是C1-C6烷基、F、Cl、Br或I。 Or a salt thereof; wherein each R12 and each R13 are independently R3; OR3; F; Cl; Br; I; CN; NO2; OCF3; CF3; NR2R3; SR3, SOR3, SO2R3, CO2R3, COR3, CONR3R4, CSNR4R5; a selectively substituted aryl or a selectively substituted heteroaryl wherein each substituent of the aryl or heteroaryl is independently C1-C6 alkyl, F, Cl, Br or I; t and u Independently 0, 1, 2, 3, 4 or 5; X13 and X14 are independently NR12, S or O; each R3 is independently H, C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkyne And each R4 is independently R3 or a selectively substituted phenyl group, wherein each substituent of the phenyl group is independently C1-C6 alkyl, F, Cl, Br or I.

在一個具體例中，化合物具有結構： In one embodiment, the compound has a structure:

在另一個具體例中，化合物具有結構： In another embodiment, the compound has a structure:

在一個具體例中，所述化合物是N-(2-氯吡啶-4-基)-N’-苯脲，其由如下化學式代表： In one embodiment, the compound is N-(2-chloropyridin-4-yl)-N'-phenylurea, which is represented by the following chemical formula:

PPU類似物的其他實例可包括但不限於： Other examples of PPU analogs can include, but are not limited to:

(植物繁殖系統) (Plant Reproduction System)

生物反應器提供一種有前途的大規模微體繁殖方法，使得在對培養參數(如pH、通氣、氧氣、二氧化碳、激素、營養物等)具有高度控制的大容器中工作成為可能。利用人工智慧，生物反應器和微體繁殖步驟的自動化也可相容，藉此降低生產成本。 Bioreactors provide a promising large-scale micropropagation method that makes it possible to work in large containers with high levels of control over culture parameters such as pH, aeration, oxygen, carbon dioxide, hormones, nutrients, and the like. With artificial intelligence, the automation of the bioreactor and micropropagation steps can also be compatible, thereby reducing production costs.

以前主要將生物反應器應用於微生物技術、細胞培養和體細胞胚胎發生(somatic embryogenesis)，而且在本發明之前，用於植物微體繁殖的生物反應器是罕見、複雜和昂貴的。 Bioreactors have previously been primarily applied to microbial technology, cell culture, and somatic embryogenesis, and prior to the present invention, bioreactors for plant micropropagation were rare, complex, and expensive.

為了解決這些問題，本發明提供了使用生物反應器於植物微體繁殖的新穎組成物、方法和系統。不希望受任何理論束縛，本發明藉由控制/降低苗/小植株對生長組成物/環境中累積的(例如某些植物激素，如TDZ)和/或作為植物生長的副產物產生的(如酚類化合物(phenolics))有毒組分的暴露，實 現了極大改進的植物微體繁殖。 In order to solve these problems, the present invention provides novel compositions, methods and systems for the propagation of plant microsomes using bioreactors. Without wishing to be bound by any theory, the present invention is produced by controlling/reducing seedlings/plantlets accumulated in growth compositions/environments (eg, certain plant hormones, such as TDZ) and/or as by-products of plant growth (eg, Exposure of toxic components of phenolics There has been a greatly improved plant micropropagation.

圖1係示意地說明依據一個具體例的植物繁殖系統100，所述系統100是為植物的大規模增殖而配置。在一些具體例中，將所述系統100用於單子葉植物的大規模增殖。在一些具體例中，將所述系統100用於竹的大規模增殖。所述系統100包括生長器皿110、第一培養基容器130、第二培養基容器150、氣體源170和控制器190。 Figure 1 is a schematic illustration of a plant propagation system 100 according to one particular example, the system 100 being configured for large-scale proliferation of plants. In some embodiments, the system 100 is used for large-scale proliferation of monocots. In some embodiments, the system 100 is used for large scale proliferation of bamboo. The system 100 includes a growth vessel 110, a first medium reservoir 130, a second medium reservoir 150, a gas source 170, and a controller 190.

生長器皿110是為了將植物組織在無菌或基本上無菌的環境中培養而配置。生長器皿110可以是任何能為植物組織和營養培養基提供無菌或基本上無菌的環境的適合容器。生長器皿110可進一步具有任何適合的材料和任何期望的形狀。例如，生長器皿110可以是透明的，以允許對植物組織的視覺觀察和光刺激，而且可構建為減少培養組織上的剪切力。 Growth vessel 110 is configured to culture plant tissue in a sterile or substantially sterile environment. Growth vessel 110 can be any suitable container that provides a sterile or substantially sterile environment for plant tissue and nutrient media. Growth vessel 110 can further have any suitable material and any desired shape. For example, the growth vessel 110 can be transparent to allow for visual observation and light stimulation of plant tissue, and can be constructed to reduce shear forces on the cultured tissue.

在一些具體例中，所述生長器皿110包含一種或多種適用於植物生長的光源。或者，所述生長器皿是透明的，以允許在生長器皿110外提供的光刺激。 In some embodiments, the growth vessel 110 comprises one or more light sources suitable for plant growth. Alternatively, the growth vessel is transparent to allow for light stimulation provided outside the growth vessel 110.

在一些具體例中，所述生長器皿110與氣體源連接。在一些具體例中，所述氣體源提供二氧化碳、氧氣、氮氣或其組合。在一些具體例中，提供的氣體或氣體混合物是無菌的或基本上無菌的。根據包括例如在植物繁殖系統100中生長的植物類型的多種因素中的任何一個，可以預先容易地控制向 生長器皿110提供的氣體混合物的比率。 In some embodiments, the growth vessel 110 is coupled to a source of gas. In some embodiments, the gas source provides carbon dioxide, oxygen, nitrogen, or a combination thereof. In some embodiments, the gas or gas mixture provided is sterile or substantially sterile. According to any of a variety of factors including, for example, the type of plant grown in the plant propagation system 100, it is possible to easily control the direction in advance. The ratio of the gas mixture provided by the growth vessel 110.

配置第一培養基容器130和第二培養基容器150以含有液體和氣體，且每個都可與生長器皿110和氣體源170流體連接。可以根據包括如在植物繁殖系統100中生長的植物類型在內的多種因素中的任何一個納入另外的培養基容器。培養基容器130、150可含有完全相同的液體培養基，或者含量及/或組成不同的培養基。培養基容器130、150可以任何合適的方式液體地連接到生長器皿110。例如，在一些具體例中，生長器皿110可具有多個流體交換口(未顯示)，且可將各培養基容器130、150連接到分別的培養基交換口。各個連接可以是直接且連續的，或包括可控閥(如手動的或在控制器190的電子控制下)。在一些具體例中，生長器皿110可具有單個流體交換口(未顯示)以連接所有的培養基容器130、150，和支管(未顯示)以控制培養基容器130、150和生長器皿110之間的液體培養基的交換。所述支管可包括任意數量的流體連通器(communicators)和閥(手動、電子致動、液壓致動等)以控制培養基容器130、150和生長器皿110之間的流體連通。在一些具體例中，生長器皿110可具有多個流體交換口(未顯示)以連接每個培養基容器。 The first medium container 130 and the second medium container 150 are configured to contain liquid and gas, and each can be fluidly coupled to the growth vessel 110 and the gas source 170. Additional media containers can be included according to any of a variety of factors including the type of plant grown as in plant propagation system 100. The culture medium containers 130, 150 may contain the same liquid medium, or a medium having a different content and/or composition. The media containers 130, 150 can be fluidly coupled to the growth vessel 110 in any suitable manner. For example, in some embodiments, the growth vessel 110 can have a plurality of fluid exchange ports (not shown) and the respective media containers 130, 150 can be coupled to separate media exchange ports. Each connection may be direct and continuous, or include a controllable valve (eg, manually or under electronic control of controller 190). In some embodiments, the growth vessel 110 can have a single fluid exchange port (not shown) to connect all of the media containers 130, 150, and a manifold (not shown) to control the liquid between the culture media containers 130, 150 and the growth vessel 110. Exchange of medium. The manifold can include any number of fluidic communicators and valves (manual, electronically actuated, hydraulically actuated, etc.) to control fluid communication between the culture medium containers 130, 150 and the growth vessel 110. In some embodiments, the growth vessel 110 can have a plurality of fluid exchange ports (not shown) to connect each of the media containers.

氣體源170可以是任何適用於將加壓氣體輸送到培養基容器130、150的裝置或系統。氣體源170可包括一種或多種，但不限於氣體壓縮罐和氣體泵。可以使用任意數量的氣 體源170。例如，在一些具體例中，可將每個培養基容器130、150連接到不同的氣體源170。在一些具體例中，可以使用支管將氣體源170連接到培養基容器130、150。所述支管可包括任意數量的流體連通器和閥(手動、電子致動、液壓致動等)以控制加壓氣體到培養基容器130、150的供應。可以將支管和閥連接到控制器190以允許自動及/或電子控制對培養基容器130、150的氣體供應。氣體源170使用的氣體可以是任何不損害培養基容器130、150中液體培養基的氣體。這類氣體的實例包括任何惰性氣體、氧氣、氮氣、二氧化碳、具有大氣組成的氣體及其組合。 Gas source 170 can be any device or system suitable for delivering pressurized gas to culture medium containers 130, 150. Gas source 170 may include one or more, but is not limited to, a gas compression canister and a gas pump. Can use any amount of gas Body source 170. For example, in some embodiments, each of the culture media containers 130, 150 can be coupled to a different gas source 170. In some embodiments, the gas source 170 can be connected to the culture medium containers 130, 150 using a manifold. The manifold can include any number of fluid communication and valves (manual, electronically actuated, hydraulically actuated, etc.) to control the supply of pressurized gas to the media containers 130, 150. The manifolds and valves can be connected to the controller 190 to allow automatic and/or electronic control of the gas supply to the culture media containers 130, 150. The gas used by the gas source 170 can be any gas that does not damage the liquid medium in the medium containers 130, 150. Examples of such gases include any inert gas, oxygen, nitrogen, carbon dioxide, gases having an atmospheric composition, and combinations thereof.

氣體源170可藉由向培養基容器130、150中的一個或兩個輸送大量的加壓氣體來改變培養基容器130、150中的氣壓而操作。在一些具體例中，配置氣體源170以向第一培養基容器130或第二培養基容器150輸送加壓氣體，將第一培養基容器130或第二培養基容器150中的氣壓提高到約1磅每平方英寸(psi)，如約0.7 psi、約0.8 psi、約0.9 psi、約1 psi、約1.1 psi或約1.2 psi。第一培養基容器130或第二培養基容器150中增加的氣壓使第一培養基容器130中含有的液體培養基的至少一部分從第一培養基容器130置換到生長器皿110，或第二培養基容器150中含有的液體培養基的至少一部分從第二培養基容器150置換到生長器皿110。經由解除(deactivate)或隔離氣體源170以允許第一培養基 容器130或第二培養基容器150中的氣壓回到其最初的(如大氣壓)值。如此，生長器皿110中的液體培養基的置換部分返回到第一或第二培養基容器中。加壓和解除氣體源170的組合係導致第一培養基及/或第二培養基與生長器皿110中植物組織接觸的「脈動」。 The gas source 170 can be operated by varying the gas pressure in the culture medium containers 130, 150 by delivering a large amount of pressurized gas to one or both of the culture medium containers 130, 150. In some embodiments, the gas source 170 is configured to deliver pressurized gas to the first medium container 130 or the second medium container 150 to increase the gas pressure in the first medium container 130 or the second medium container 150 to about 1 pound per square. Inches (psi), such as about 0.7 psi, about 0.8 psi, about 0.9 psi, about 1 psi, about 1.1 psi, or about 1.2 psi. The increased air pressure in the first medium container 130 or the second medium container 150 causes at least a portion of the liquid medium contained in the first medium container 130 to be displaced from the first medium container 130 to the growth vessel 110, or contained in the second medium container 150. At least a portion of the liquid medium is displaced from the second medium container 150 to the growth vessel 110. Deactivating or isolating gas source 170 to allow first medium The gas pressure in vessel 130 or second medium container 150 returns to its original (e.g., atmospheric pressure) value. As such, the displaced portion of the liquid medium in the growth vessel 110 is returned to the first or second medium container. The combination of the pressurized and degassed gas source 170 results in "pulsation" of the first medium and/or the second medium in contact with the plant tissue in the growing vessel 110.

在一些具體例中，在解除階段過程中，培養基容器使壓力均衡，且開始自動的虹吸排水，使培養基排空回到原始的培養基容器中。在一些具體例中，虹吸排水速率是約500到1000 mls/分，如600-720 mls/分。 In some embodiments, during the release phase, the media container equalizes the pressure and begins automatic siphon drainage to empty the medium back to the original media container. In some embodiments, the siphon drainage rate is about 500 to 1000 mls/min, such as 600-720 mls/min.

在一些具體例中，在排水時，植物部分地浸沒於培養基約2到約4分鐘，如約2.5到約3分鐘。 In some embodiments, the plant is partially submerged in the medium for about 2 to about 4 minutes, such as from about 2.5 to about 3 minutes, upon drainage.

在一些具體例中，在排空培養基之後且在下一種培養基進入容器之前，將容器中的植物以預定的時間乾燥。在一些具體例中，將所述植物乾燥約3-6分鐘，如約5分鐘。 In some embodiments, the plants in the container are dried for a predetermined period of time after emptying the medium and before the next medium enters the container. In some embodiments, the plant is dried for about 3-6 minutes, such as about 5 minutes.

對於第一培養基容器130或第二培養基容器150，可以根據如植物繁殖系統100中生長的植物類型將脈動過程重複任意迴圈數。例如，重複迴圈總共花費約半小時至約3週，如約24小時至約60小時，或約24小時至約4週，如約3天至約5天。在一些具體例中，第一培養基容器容納包括植物激素TDZ的芽誘導培養基，而第二培養基容器容納不包括TDZ的苗伸長/維持培養基。 For the first medium container 130 or the second medium container 150, the pulsation process can be repeated for any number of loops according to the type of plant grown in the plant propagation system 100, for example. For example, repeating the loop takes a total of about half an hour to about 3 weeks, such as from about 24 hours to about 60 hours, or from about 24 hours to about 4 weeks, such as from about 3 days to about 5 days. In some embodiments, the first medium container contains a shoot induction medium comprising the plant hormone TDZ, and the second medium container contains a shoot elongation/maintenance medium that does not include TDZ.

在一些具體例中，可以在第一培養基容器130和第二培養 基容器150之間交替所述脈動過程。在一些具體例中，可對第一培養基容器130的脈動過程重複預定的迴圈數，然後轉換到第二培養基容器進行預定的迴圈數。在一些具體例中，可依據多種預定模式中的任意一種以重複和交替第一培養基容器130和第二培養基容器150之間的脈動過程，例如，依據植物繁殖系統100中生長的植物類型。例如，可以約半小時至約3週的時間重複對第一培養基的脈動過程，如約24小時至約60小時，且隨後對第二培養基以約24小時至約4週重複，如約3天至約5天。 In some embodiments, the first medium container 130 and the second culture may be The pulsation process is alternated between the base containers 150. In some embodiments, the predetermined number of turns may be repeated for the pulsing process of the first medium container 130, and then converted to the second medium container for a predetermined number of turns. In some embodiments, the pulsation process between the first medium container 130 and the second medium container 150 can be repeated and alternated according to any of a plurality of predetermined modes, for example, depending on the type of plant grown in the plant propagation system 100. For example, the pulsation of the first medium can be repeated for about half an hour to about 3 weeks, such as from about 24 hours to about 60 hours, and then the second medium is repeated for about 24 hours to about 4 weeks, such as about 3 days. It will take about 5 days.

植物繁殖系統100的操作係為手動控制，或是由控制器190控制，所述控制器190可以是處理器、計算裝置或任何如本領域已知的可程式設計的/可配置的裝置或系統。配置所述控制器190以電子控制氣體源170，且至少控制氣體源170的啟動和解除。在一些具體例中，配置所述控制器190以電子控制將氣體源170與各個培養基容器130、150連接的支管，以使得選擇其中一個培養基容器成為可能。在一些具體例中，配置所述控制器190以電子控制將培養基容器130、150和生長器皿110連接的支管，以能夠控制培養基容器130、150和生長器皿110之間的液體流動。此外，且與各種具體例及其組合並非不一致的是，可以將控制器190連接到多個氣體源、多個支管及/或多個閥，並配置成用於其之控制。本文未舉例說明的系統100於其他方面的控制 (如氣體交換系統、溫度控制系統等的控制)係在本發明範圍內。 The operation of plant propagation system 100 is manually controlled or controlled by controller 190, which may be a processor, computing device, or any programmable/configurable device or system as is known in the art. . The controller 190 is configured to electronically control the gas source 170 and at least control the activation and deactivation of the gas source 170. In some embodiments, the controller 190 is configured to electronically control a manifold connecting the gas source 170 to each of the culture media containers 130, 150 to enable selection of one of the media containers. In some embodiments, the controller 190 is configured to electronically control a manifold connecting the culture medium containers 130, 150 and the growth vessel 110 to enable control of fluid flow between the culture medium containers 130, 150 and the growth vessel 110. Moreover, and not inconsistent with the various specific examples and combinations thereof, controller 190 can be coupled to a plurality of gas sources, a plurality of manifolds, and/or a plurality of valves, and configured for control thereof. Control of system 100 in other aspects not illustrated herein (Control of gas exchange systems, temperature control systems, etc.) is within the scope of the invention.

控制器190在第一操作模式下是可操作的，其中控制器引起氣體源170將加壓氣體輸送到第一培養基容器130以將其中含有的第一體積的液體置換到生長器皿110。此外，控制器190在第二操作模式下是可操作的，其中控制器引起氣體源170將加壓氣體輸送到第二培養基容器150以將其中含有的第二體積的液體置換到生長器皿110。在一些具體例中，以預定的時間運行第一和第二操作模式。在一些具體例中，將第一和第二操作模式運行約1分鐘±半分鐘，如約30秒、約40秒、約50秒、約60秒、約70秒、約80秒或約90秒。如上描述的，可以依據預定的模式重複及/或交替第一和第二操作模式。 The controller 190 is operable in a first mode of operation wherein the controller causes the gas source 170 to deliver pressurized gas to the first medium container 130 to displace the first volume of liquid contained therein to the growth vessel 110. Further, the controller 190 is operable in the second mode of operation wherein the controller causes the gas source 170 to deliver pressurized gas to the second medium container 150 to displace the second volume of liquid contained therein to the growth vessel 110. In some embodiments, the first and second modes of operation are run at predetermined times. In some embodiments, the first and second modes of operation are operated for about 1 minute ± half a minute, such as about 30 seconds, about 40 seconds, about 50 seconds, about 60 seconds, about 70 seconds, about 80 seconds, or about 90 seconds. . As described above, the first and second modes of operation may be repeated and/or alternated according to a predetermined pattern.

此外，在已執行第一操作模式或第二操作模式之後(即，生長器皿110含有來自培養基容器130、150其中之一的液體)，控制器190可在第三操作模式下運行而操作。在第三操作模式中，允許生長器皿110中的液體返回到其各自的培養基容器，例如，通過解除氣體源170。在一些具體例中，以預定的時間運行第三操作模式。在一些具體例中，第三操作模式係運行約8分鐘，如約7分鐘、7.5分鐘、8分鐘、8.5分鐘等。 Further, after the first mode of operation or the second mode of operation has been performed (ie, the growth vessel 110 contains liquid from one of the culture media containers 130, 150), the controller 190 can operate in a third mode of operation. In the third mode of operation, the liquid in the growing vessel 110 is allowed to return to its respective media container, for example, by releasing the gas source 170. In some embodiments, the third mode of operation is run at a predetermined time. In some embodiments, the third mode of operation is about 8 minutes, such as about 7 minutes, 7.5 minutes, 8 minutes, 8.5 minutes, and the like.

在一些具體例中，控制器190可藉由運行包含第一操作模 式-第三操作模式次序的一個或多個迴圈的第一培養次序而操作。在一些具體例中，第一培養次序係運行從約1小時至約3週，如從約24小時至約60小時。控制器190也可藉由運行包含第二操作模式-第三操作模式次序的一個或多個迴圈的第二培養次序而操作。在一些具體例中，第二培養次序係運行從約24小時至約4週，且例如，3天到約5天。 In some specific examples, the controller 190 can include the first operational mode by running The first culture sequence of one or more loops of the third operational mode sequence operates. In some embodiments, the first culture sequence is run from about 1 hour to about 3 weeks, such as from about 24 hours to about 60 hours. Controller 190 can also operate by running a second culture sequence that includes one or more loops of the second mode of operation - the third mode of operation mode. In some embodiments, the second culture sequence is run from about 24 hours to about 4 weeks, and for example, from 3 days to about 5 days.

在一些具體例中，控制器190可藉由運行包含每個第一培養次序後是第二培養次序的一個或多個迴圈的植物繁殖次序而操作。植物繁殖次序的迴圈數範圍可從1至8或更多。 In some embodiments, controller 190 can operate by running a plant breeding sequence that includes one or more loops of each of the first culture sequences followed by a second culture sequence. The number of loops in the order of plant propagation can range from 1 to 8 or more.

在一些具體例中，可藉由任何已知的方法，如高壓滅菌，對生長系統的一個或多個或所有部分滅菌。 In some embodiments, one or more or all portions of the growth system can be sterilized by any known method, such as autoclaving.

在一些具體例中，藉由氣壓將培養基驅入或驅出生長器皿。在一些具體例中，藉由其他力，如重力、電力等，將培養基驅入或驅出生長器皿。 In some embodiments, the culture medium is driven into or out of the growth vessel by gas pressure. In some embodiments, the culture medium is driven into or out of the growth vessel by other forces, such as gravity, electricity, and the like.

現參見圖2-5C，其顯示了植物繁殖系統200的示例性具體例。系統200的操作與上述系統100類似，因此除非另外說明的，系統200的各種元件可以與其他具體例中的具有類似的設計和功能。例如，生長器皿210可與生長器皿110類似。 Referring now to Figures 2-5C, an exemplary embodiment of a plant propagation system 200 is shown. The operation of system 200 is similar to system 100 described above, and thus the various elements of system 200 may have similar designs and functions as in other specific examples unless otherwise stated. For example, the growth vessel 210 can be similar to the growth vessel 110.

圖2舉例說明的具體例包括生長器皿210、支管240、第一培養基容器230、第二培養基容器250、作為氣體源的氣體泵270、和作為控制器的計時器控制電路290。將單個氣 體源270附於第一培養基容器230，且其可移除及重附著於第二培養基容器250。有利地，各個培養基容器230、250所使用的濾器252(另參見圖3)係防止培養基容器中任何可能的氣體泵270(甚至在轉換的時候)對液體培養基的污染。或者，可以使用兩個氣體源，各個源係在計時器控制電路290的控制下附著於培養基容器230或250，因此不需要重附著。如圖3之說明，各個培養基容器230、250具有第一流體口232，其與生長器皿210的口222為流體連通或者以其他方式可連接，以使培養基容器和生長器皿之間的流體可以交換。各個培養基容器230、250還具有第二流體口236，其與氣體源270為流體連通或者以其他方式可連接的以改變培養基容器中的氣壓。口232和236係形成於密封培養基容器的接合器238上以防止污染，例如一種艙壁接合器(bulkhead adapter)。如圖所示，第二液體口236另外與濾器252接合(如具有階形倒刺軟管(stepped hose barbs)的通氣口濾器)以防止在與氣體泵270交換氣體時培養基容器中流體的污染。 The specific example illustrated in Fig. 2 includes a growth vessel 210, a branch pipe 240, a first medium container 230, a second medium container 250, a gas pump 270 as a gas source, and a timer control circuit 290 as a controller. Single gas The body source 270 is attached to the first medium container 230 and is removable and reattached to the second medium container 250. Advantageously, the filter 252 (see also Figure 3) used by each of the media containers 230, 250 prevents contamination of the liquid medium by any possible gas pump 270 in the media container (even at the time of conversion). Alternatively, two gas sources can be used, each source being attached to the culture medium container 230 or 250 under the control of the timer control circuit 290, so that no reattachment is required. As illustrated in Figure 3, each of the media containers 230, 250 has a first fluid port 232 that is in fluid communication with or otherwise connectable to the port 222 of the growing vessel 210 to allow fluid exchange between the media container and the growing vessel. . Each of the media containers 230, 250 also has a second fluid port 236 that is in fluid communication with the gas source 270 or otherwise connectable to change the gas pressure in the media container. Ports 232 and 236 are formed on the adapter 238 that seals the media container to prevent contamination, such as a bulkhead adapter. As shown, the second fluid port 236 is additionally coupled to a filter 252 (such as a vent filter with stepped hose barbs) to prevent contamination of the fluid in the media container when the gas is exchanged with the gas pump 270. .

圖2和4說明了與分別來自培養基容器230、250的管242和244連接的支管240的非限制性設計。圖4說明了形成於支管240上以分別控制來自每個管242、244流動的閥246和248。可以使用任何合適的兩通閥(2-way valve)，例如球閥、門閥、蝶形閥等。閥246、248可以受計時器290的電 子控制，並且/或者是手動控制。在圖2描述的裝置中，閥246開啟，以將生長器皿210和第一培養基容器230流體連接。閥248關閉，以將第二培養基容器250從生長器皿210和第一培養基容器230流體隔離。以此種方式，阻止了第一培養基容器230和第二培養基容器250之間的液體混合。 2 and 4 illustrate a non-limiting design of branch 240 joined to tubes 242 and 244 from medium reservoirs 230, 250, respectively. FIG. 4 illustrates valves 246 and 248 formed on manifold 240 to control flow from each of tubes 242, 244, respectively. Any suitable 2-way valve can be used, such as ball valves, gate valves, butterfly valves, and the like. Valves 246, 248 can be energized by timer 290 Sub-control, and / or manual control. In the apparatus depicted in FIG. 2, valve 246 is opened to fluidly connect growth vessel 210 to first medium reservoir 230. Valve 248 is closed to fluidly isolate second medium container 250 from growth vessel 210 and first medium container 230. In this way, liquid mixing between the first medium container 230 and the second medium container 250 is prevented.

藉由計時器控制電路290開/關氣體泵的電源供應，實現對氣體泵270的控制。在一個具體例中，所述氣體泵是1 psi泵，當其由電路290供能時(例如在第一或第二操作模式下)，將氣體泵入連接的第一培養基容器230以將壓力提高到1 psi。當電路290切斷對氣體泵270的供能時，可以解除或者以其他方式關閉氣體泵270(例如在第三操作模式下)，由此通過使泵入的氣體流回到氣體泵中而使第一培養基容器中的壓力均衡。 The control of the gas pump 270 is achieved by the timer control circuit 290 turning on/off the power supply of the gas pump. In one embodiment, the gas pump is a 1 psi pump that, when energized by circuit 290 (eg, in the first or second mode of operation), pumps gas into the connected first medium container 230 to apply pressure Increase to 1 psi. When circuit 290 shuts off power to gas pump 270, gas pump 270 can be deactivated or otherwise shut down (e.g., in a third mode of operation), thereby causing the pumped gas to flow back into the gas pump. The pressure in the first medium container is equalized.

如圖5A-C之說明，生長器皿210包括用於進入生長器皿內部的閉合物212、便於運輸的柄216。儘管圖示為形成於正面部分，閉合物212和柄216可以形成於生長器皿210的任何其他部分。在一個具體例中，生長器皿210是間歇浸沒式生物反應器。 As illustrated in Figures 5A-C, the growth vessel 210 includes a closure 212 for accessing the interior of the growth vessel, and a handle 216 for transport. Although illustrated as being formed in the front portion, the closure 212 and the handle 216 can be formed in any other portion of the growth vessel 210. In one embodiment, the growth vessel 210 is an intermittent immersion bioreactor.

生長器皿210也可具有形成於生長器皿上的氣體交換口220和流體交換口222，儘管任何數量的氣體交換口和流體交換口都在本發明的範圍之內。口220和222與接合器接合以使生長器皿210內部的流體能夠連通，同時維持無菌。在 一個具體例中，所述口220和222與艙壁接合器接合。生長器皿210還包括附著於口222以與生長器皿內部流體連通的流體導管(conduit)226。所述導管226具有充足的長度且具有有適當的橫截面的內腔以使流體能從生長器皿210的底部虹吸到選擇的培養基的容器230中，如在下面以更多細節描述的圖1的系統100。 The growth vessel 210 can also have a gas exchange port 220 and a fluid exchange port 222 formed on the growth vessel, although any number of gas exchange ports and fluid exchange ports are within the scope of the present invention. Ports 220 and 222 engage the adapter to enable fluid communication within the growth vessel 210 while maintaining sterility. in In one embodiment, the ports 220 and 222 are engaged with the bulkhead adapter. Growth vessel 210 also includes a fluid conduit 226 attached to port 222 for fluid communication with the interior of the growth vessel. The conduit 226 is of sufficient length and has a lumen of suitable cross-section to enable fluid to be siphoned from the bottom of the growth vessel 210 into the container 230 of the selected medium, as described in more detail below in FIG. System 100.

在一個具體例中，生長器皿210用於植物的大規模增殖。在一些具體例中，生長器皿210係用於竹的大規模增殖。在一些具體例中，生長管進一步用於培養物的預生根和生根。再次參見圖1的系統100，在使用中，將待培養的植物組織(如竹組織)置於生長器皿110中，隨後將其置於相對於培養基容器130、150能達到虹吸作用的高度。將第一和第二液體培養基分別置於培養基容器130、150中。在一個具體例中，第一和第二液體培養基是不同的。如所討論，氣體源170與培養基容器130、150連接，且控制器190與氣體源和任何需要電子控制的其他元件連接。 In one embodiment, the growth vessel 210 is used for large-scale proliferation of plants. In some embodiments, the growth vessel 210 is used for large-scale proliferation of bamboo. In some embodiments, the growth tube is further used for pre-rooting and rooting of the culture. Referring again to system 100 of Figure 1, in use, plant tissue (e.g., bamboo tissue) to be cultured is placed in growth vessel 110, which is then placed at a height that is capable of siphoning relative to medium containers 130,150. The first and second liquid media are placed in the culture medium containers 130, 150, respectively. In one embodiment, the first and second liquid media are different. As discussed, gas source 170 is coupled to medium reservoirs 130, 150, and controller 190 is coupled to a gas source and any other components that require electronic control.

如圖6說明所描述控制器190的示例性植物繁殖次序300的操作。儘管以逐步的方式說明，但需要注意的是這些步驟的執行順序並不必要按此進行。在302，控制器190開始植物繁殖次序。在304，控制器190開始第一培養次序。在306，控制器190建立第一培養基容器130和生長器皿110之間的流體連通。控制器190還建立了氣體源170和第一培養基容 器130之間的流體連通。控制器190還將第二培養基容器150與生長器皿110流體隔離。在308，控制器進入第一操作模式並驅動氣體源170將加壓氣體輸送到第一培養基容器130，以將其中含有的第一體積的液體置換到生長器皿110中。在310，該控制器隨後進入第三連通模式且藉由解除氣體源170允許生長器皿110中至少一部分第一體積的液體返回到第一培養基容器130。 The operation of the exemplary plant breeding sequence 300 of the controller 190 is illustrated as illustrated in FIG. Although explained in a step-by-step manner, it should be noted that the order in which these steps are performed is not necessarily performed as such. At 302, controller 190 begins the plant breeding sequence. At 304, controller 190 begins a first culture sequence. At 306, controller 190 establishes fluid communication between first medium container 130 and growth vessel 110. Controller 190 also establishes gas source 170 and first medium volume Fluid communication between the devices 130. Controller 190 also fluidly isolates second culture vessel 150 from growth vessel 110. At 308, the controller enters a first mode of operation and drives a gas source 170 to deliver pressurized gas to the first medium container 130 to displace the first volume of liquid contained therein into the growth vessel 110. At 310, the controller then enters a third connected mode and allows at least a portion of the first volume of liquid in the growing vessel 110 to return to the first medium container 130 by releasing the gas source 170.

在312，如果第一培養次序未完成，所述控制器190返回到308，並再次進入第一操作模式。如果第一培養次序是完成的，所述控制器在314開始第二培養次序。在316，所述控制器建立第二培養基容器150和生長器皿110之間的流體連通。所述控制器還建立氣體源170和第二培養基容器150之間的流體連通。所述控制器還將第一培養基容器130與生長器皿110流體隔離。在318，所述控制器進入第二操作模式，並驅動氣體源170將加壓氣體輸送到第二培養基容器150，以將其中含有的第二體積的液體置換到生長器皿110中。在320，所述控制器隨後進入第三連通模式，並且藉由解除氣體源170允許生長器皿110中至少一部分第二體積的液體返回到第二培養基容器150。 At 312, if the first culture sequence is not complete, the controller 190 returns to 308 and enters the first mode of operation again. If the first culture sequence is completed, the controller begins a second culture sequence at 314. At 316, the controller establishes fluid communication between the second medium container 150 and the growth vessel 110. The controller also establishes fluid communication between the gas source 170 and the second medium container 150. The controller also fluidly isolates the first medium container 130 from the growth vessel 110. At 318, the controller enters a second mode of operation and drives a gas source 170 to deliver pressurized gas to the second medium reservoir 150 to displace the second volume of liquid contained therein into the growth vessel 110. At 320, the controller then enters a third connected mode and allows at least a portion of the second volume of liquid in the growing vessel 110 to return to the second medium container 150 by releasing the gas source 170.

在322，如果第二培養次序未完成，所述控制器190返回到318，並再次進入第二操作模式。如果第二培養次序是完成的但(由324確定)植物繁殖次序未完成，所述控制器190 返回到步驟304並再次開始第一培養次序。如果植物繁殖次序是完成的，控制器190在326退出植物繁殖過程。在一個具體例中，所述控制器190包括用於信號通知植物繁殖次序結束的視覺及/或音訊指示器。 At 322, if the second culture sequence is not complete, the controller 190 returns to 318 and enters the second mode of operation again. If the second culture sequence is completed but (determined by 324) the plant reproduction order is not completed, the controller 190 Returning to step 304 and starting the first culture sequence again. If the plant breeding sequence is complete, controller 190 exits the plant propagation process at 326. In one embodiment, the controller 190 includes a visual and/or audio indicator for signaling the end of the plant breeding sequence.

因此本發明之態樣在提供對於獨立控制、多個培養基、脈動時間(即啟動/解除時間)和培養時間、以及整組可用液體培養基的總培養迴圈數的控制完全可程式設計的半自動或全自動的、封閉的植物繁殖系統方面是有利的。期望地，該系統的所有元件是可高溫滅菌的，因此是可再度使用的。藉由降低勞動力、疏忽和污染的損失而達成顯著的成本節約。 Thus aspects of the present invention provide fully programmable semi-automatic or programmable control for independent control, multiple media, pulsation time (ie, start/release time) and incubation time, and total number of culture loops for the entire set of available liquid media. A fully automated, closed plant propagation system aspect is advantageous. Desirably, all of the components of the system are autoclavable and therefore reusable. Significant cost savings are achieved by reducing the loss of labor, neglect and pollution.

植物微體繁殖系統的非限制性實例包括在美國專利號3,578,431；4,320,594；4,669,217；4,908,315；4,934,096；5,049,505；5,088,231；5,104,527；5,119,588；5,139,956；5,171,683；5,184,420；5,186,895；5,212,906；5,225,342；5,558,984；5,597,731；和US 6,753,178中所描述者。在Etienne等人(Bioreactors in coffee micropropagation,Braz.J.Plant Physiol.,18(1)：45-54,2006)；Ziv(Bioreactor Technology for Plant Micropropagation Horticultural Reviews,Volume 24,Jules Janick編輯ISBN 0-471-33374-3)；和Paek等人(Application of bioreactors for large-scale micropropagation systems of plants,In vitro Cell.Dev.Biol.-Plant 37：149-157,March-April 2001)中可發現植物微體繁殖系統的更多實例。 Non-limiting examples of plant micro-propagation systems include those in U.S. Patent Nos. 3,578,431; 4,320,594; 4,669,217; 4,908,315; 4,934,096; 5,049,505; 5,088,231; 5,104,527; 5,119,588; 5,139,956; 5,171,683; 5,184,420; 5,186,895; 5,212,906; 5,225,342; 5,558,984; 5,597,731; As described in US 6,753,178. In Etienne et al. ( Bioreactors in coffee micropropagation , Braz. J. Plant Physiol., 18(1): 45-54, 2006); Ziv ( Bioreactor Technology for Plant Micropropagation Horticultural Reviews , Volume 24, Jules Janick, editor ISBN 0-471 -33374-3); and plant micro-organisms can be found in Paek et al. ( Application of bioreactors for large-scale micropropagation systems of plants , In vitro Cell. Dev. Biol.-Plant 37: 149-157, March-April 2001) More examples of reproductive systems.

可理解的是，本發明的植物繁殖系統還包括藉由添加本領域技術人員已知的系統的一個或多個部分/特徵而從本文中描述的示例性系統衍生的系統。 It will be appreciated that the plant propagation system of the present invention also includes systems derived from the exemplary systems described herein by the addition of one or more portions/features of systems known to those skilled in the art.

(用於植物微體繁殖的架) (frame for plant micropropagation)

許多植物之物種具有熟知的微體繁殖過程。在一些情況下，例如，期望間歇地將生長培養基中培養的植物組織曝露於液體營養溶液。一些已知設計為實施該功能的系統並不適當且有破損及/或機械故障的傾向。此外，已知系統對於大規模的應用不夠健全。因此，需要改進的裝置以間歇地將組織培養小植株的微環境曝露於液體營養溶液。 Many plant species have well-known micropropagation processes. In some cases, for example, it is desirable to intermittently expose plant tissue cultured in a growth medium to a liquid nutrient solution. Some systems known to be designed to perform this function are not suitable and have a tendency to break and/or mechanically fail. Furthermore, known systems are not sufficiently robust for large-scale applications. Therefore, there is a need for an improved device for intermittently exposing the microenvironment of tissue culture plantlets to a liquid nutrient solution.

在一些具體例中，裝置包括框架、由框架支持的架組件，和與架組件連接的驅動組件。在這類具體例中，可配置該驅動組件以給予架組件相對於框架的擺動運動(oscillating motion)，而使繁殖器皿中和由架組件支援的組織培養小植株間歇地曝露於液體營養溶液。 In some embodiments, the device includes a frame, a frame assembly supported by the frame, and a drive assembly coupled to the frame assembly. In such embodiments, the drive assembly can be configured to impart oscillating motion to the frame assembly relative to the frame, while the tissue culture plantlets in the breeding vessel and supported by the shelf assembly are intermittently exposed to the liquid nutrient solution.

圖8和9是依據一個具體例的擺動架100的立體圖。擺動架100包括框架110、架組件140和驅動組件170。框架100包括支柱111、上跨構件120和底125。可以從任何合適的材料形成框架100的元件。例如，在一些具體例中，框架100可以從鋁形成。在其他的具體例中，框架100可從鋁合金、鋼和/或鋼合金形成，並且可以具有任何合適的規格(gauge)或厚度。 8 and 9 are perspective views of the swing frame 100 according to a specific example. The swing frame 100 includes a frame 110, a frame assembly 140, and a drive assembly 170. The frame 100 includes a strut 111, an upper span member 120, and a bottom 125. The components of the frame 100 can be formed from any suitable material. For example, in some embodiments, the frame 100 can be formed from aluminum. In other specific examples, the frame 100 can be formed from aluminum alloys, steel, and/or steel alloys, and can have any suitable gauge or thickness.

支柱111可以是任何合適的配置且從底125向上延伸，如本文中以進一步的細節描述。上跨構件120可以具有任何合適的大小、形狀或配置。例如，在一些具體例中，上跨構件120可以是塑造(formed)(如機械彎曲)或壓出(extruded)C-溝槽。在另外的具體例中，上跨構件120可以是基本上閉合或實心結構，例如，如箱型管(box tubing)或棒材(bar stock)。配置上跨構件120以將其連接到支柱111的上面部分。以該方式，上跨構件120能增加擺動架100的上面部分的剛度及/或強度。 The struts 111 can be of any suitable configuration and extend upward from the base 125 as described in further detail herein. The upper cross member 120 can have any suitable size, shape, or configuration. For example, in some embodiments, the upper span member 120 can be formed (eg, mechanically bent) or extruded C-trenched. In further embodiments, the upper span member 120 can be a substantially closed or solid structure, such as, for example, a box tubing or a bar stock. The upper span member 120 is configured to connect it to the upper portion of the strut 111. In this manner, the upper span member 120 can increase the stiffness and/or strength of the upper portion of the swing frame 100.

底125可以是任何合適的平臺或結構。例如，儘管圖8-10所示的是基本I-型(如包括橫跨構件，其連接於與橫跨構件垂直排列的兩個支撐構件)，在其他的具體例中，底125可具有任何形狀或配置。在一些具體例中，例如，底125可具有基本為矩形的結構。在其他的具體例中，底125可包括硬化(stiffening)構件及/或類似物。例如，在一些具體例中，底125可包括與底125的上表面連接(如用螺絲旋緊、焊接、用鉚釘釘牢或以其他方式扣緊)之片狀金屬部分，其配置以增加底125的剛度和/或強度。此外，在圖8-10顯示的具體例中，底125包括一組腳輪(caster wheel)而可移動或重置擺動架100。 The base 125 can be any suitable platform or structure. For example, although Figures 8-10 show a basic I-shape (eg, including a cross member that is attached to two support members that are vertically aligned with the cross member), in other embodiments, the base 125 can have any Shape or configuration. In some embodiments, for example, the base 125 can have a substantially rectangular configuration. In other embodiments, the base 125 can include a stiffening member and/or the like. For example, in some embodiments, the base 125 can include a sheet metal portion that is coupled to the upper surface of the bottom 125 (eg, screwed, welded, riveted, or otherwise fastened), configured to increase the bottom The stiffness and/or strength of 125. Moreover, in the particular example shown in Figures 8-10, the base 125 includes a set of caster wheels to move or reset the swing frame 100.

如圖10所示，底125包括從底125的上表面延伸的支撐構件126。更具體地，該支撐構件126被固定地連接(如焊 接)到底125的上表面並至少承受一個支柱111的一部分。此外，支撐構件126之一包括配置以分別承受驅動組件170的驅動軸(drive shaft)172和搖軸(rocker shaft)182的驅動軸開口127和搖軸開口128。 As shown in FIG. 10, the base 125 includes a support member 126 that extends from the upper surface of the base 125. More specifically, the support member 126 is fixedly connected (eg, welded The upper surface of the bottom portion 125 is received and at least one portion of the pillar 111 is received. Additionally, one of the support members 126 includes a drive shaft opening 127 and a rocker opening 128 that are configured to withstand a drive shaft 172 and a rocker shaft 182 of the drive assembly 170, respectively.

支柱111包括限定基本為C-型的橫截面(圖11)和蓋112(圖10)的一組壁(walls)。如圖8-10中所示，配置支柱111以從底125的上表面延伸。更具體地，將支柱111置於支撐構件126周圍，由此使支柱111從底125的上表面延伸出去。類似而言，支撐構件126被置於由限定C型橫截面的一組壁限定的容積115內。以此種方式，支柱111可以與底125及/或支撐構件126連接(如焊接及/或扣緊)。支柱111可進一步限定配置任意數量的接受擺動架100的部分的孔。例如，至少一個支柱111可包括驅動軸開口113和搖軸開口114，其被配置為分別接受驅動軸172和搖軸182，分別納入驅動組件170，且在支撐構件126中被分別安置為與驅動軸開口127和搖軸開口128對齊。 The struts 111 include a set of walls defining a substantially C-shaped cross section (Fig. 11) and a cover 112 (Fig. 10). As shown in Figures 8-10, the struts 111 are configured to extend from the upper surface of the bottom 125. More specifically, the strut 111 is placed around the support member 126, thereby extending the strut 111 from the upper surface of the bottom 125. Similarly, the support member 126 is placed within a volume 115 defined by a set of walls defining a C-shaped cross section. In this manner, the post 111 can be coupled (eg, welded and/or fastened) to the base 125 and/or the support member 126. The post 111 can further define an aperture configured with any number of portions that receive the swing frame 100. For example, at least one of the struts 111 can include a drive shaft opening 113 and a rocker opening 114 that are configured to receive the drive shaft 172 and the rocker shaft 182, respectively, into the drive assembly 170, and are respectively disposed and driven in the support member 126 The shaft opening 127 is aligned with the rocker opening 128.

在一些具體例中，支柱111可配置為限定任何數量的孔和/或突起以參與照明系統(未顯示)及/或控制系統(未顯示)的一部分。在一些具體例中，每個蓋112可與各自的支柱111連接以使蓋112和支柱111將一組電子元件(未顯示)儲存於容積115內。例如，在一些具體例中，所述容積115可含有電線、開關、繼電器、電子裝置(如可程式設計邏輯控制器 (PLC)包括，例如，至少一個處理器、一個記憶體和一個網路介面)及/或類似物。在一些具體例中，至少一個支柱111可包括感測器支架121。在這類具體例中，可將感測器置於感測器支架121上，並且指示及/或監控架組件140相對於框架110的位置，如本文中進一步所描述。 In some embodiments, the struts 111 can be configured to define any number of holes and/or protrusions to participate in a portion of the illumination system (not shown) and/or control system (not shown). In some embodiments, each cover 112 can be coupled to a respective post 111 such that the cover 112 and post 111 store a set of electronic components (not shown) within the volume 115. For example, in some embodiments, the volume 115 can contain wires, switches, relays, electronic devices (eg, programmable logic controllers) (PLC) includes, for example, at least one processor, one memory and one network interface) and/or the like. In some embodiments, the at least one post 111 can include a sensor bracket 121. In such a specific example, the sensor can be placed on the sensor mount 121 and the position of the rack assembly 140 relative to the frame 110 can be indicated and/or monitored, as further described herein.

架組件140與支柱111(參見如圖8)可旋轉地連接，且包括一組架141、一組外部軸襯(outer bushings)150、一組內部軸襯(inner bushings)155和一組連桿145。例如，如圖12所示，架組件140可包括任何適合數量的配置為垂直堆疊的架141。此外，架141藉由連桿145可操作地連接在一起(例如，連桿145傳遞至少一部分力以引起架組件140的每個架141同時轉動，如本文中進一步所描述)。儘管在圖12中顯示為包括兩個連桿145，在一些具體例中，架組件可包括任何適合數量的連桿141。例如，在一些具體例中，架組件可包括一組的四個連桿141以將第一組的兩個連桿145置於架141的一個側面而第二組的兩個連桿145置於架141的第二側面。 The frame assembly 140 is rotatably coupled to the post 111 (see FIG. 8) and includes a set of brackets 141, a set of outer bushings 150, a set of inner bushings 155, and a set of links. 145. For example, as shown in FIG. 12, the shelf assembly 140 can include any suitable number of shelves 141 that are configured to be stacked vertically. In addition, the shelves 141 are operatively coupled together by links 145 (eg, the links 145 transmit at least a portion of the force to cause each frame 141 of the frame assembly 140 to rotate simultaneously, as further described herein). Although shown in FIG. 12 as including two links 145, in some embodiments, the frame assembly can include any suitable number of links 141. For example, in some embodiments, the shelf assembly can include a set of four links 141 to place the first set of two links 145 on one side of the frame 141 and the second set of two links 145 on The second side of the frame 141.

如圖13顯示的，每個架141包括一組由支撐管144連接在一起的平臺142(例如，將一個支撐管144置於架141的一個側面且第二支撐管144置於架141的第二側面)。如圖14所顯示，平臺142限定了限定為雙回路(double return)的橫截面形狀，由此增加平臺142的強度和剛度。此外，架 141的支撐管144中的至少一個是配置為與連桿145連接。 As shown in Figure 13, each frame 141 includes a plurality of platforms 142 joined together by a support tube 144 (e.g., one support tube 144 is placed on one side of the frame 141 and the second support tube 144 is placed on the frame 141). Two sides). As shown in Figure 14, the platform 142 defines a cross-sectional shape defined as a double return, thereby increasing the strength and stiffness of the platform 142. In addition, the shelf At least one of the support tubes 144 of 141 is configured to be coupled to the link 145.

配置外部軸襯150和內部軸襯155(圖15)以將架組件140可旋轉地連接到支柱111。更具體的，將內部軸襯155與架141的支撐管144剛性連接且將外部軸襯150與支柱111剛性連接。以該種方式，可將內部軸襯155可旋轉地置於由外部軸襯150限定的開口151之內。因此，架141可繞著置於外部軸襯150的開口151之內的內部軸襯155轉動，以回應由驅動組件170施加的至少一部分力。 The outer bushing 150 and the inner bushing 155 (FIG. 15) are configured to rotatably couple the frame assembly 140 to the strut 111. More specifically, the inner bushing 155 is rigidly coupled to the support tube 144 of the frame 141 and the outer bushing 150 is rigidly coupled to the post 111. In this manner, the inner bushing 155 can be rotatably disposed within the opening 151 defined by the outer bushing 150. Accordingly, the frame 141 can be rotated about the inner bushing 155 disposed within the opening 151 of the outer bushing 150 in response to at least a portion of the force applied by the drive assembly 170.

現參見圖16，驅動組件包括發動機171、傳動齒輪(drive gear)173和搖動組件180。發動機171可以是限定任何適合的扭矩及/或輸出速率的任何適合發動機。例如，在一些具體例中，所述發動機171是Bison 650AC。如圖10顯示，將發動機171配置為與至少一個支柱111連接，以使驅動軸172從發動機171延伸，經過支柱111的驅動軸開口113和支撐構件126的驅動軸開口127。將傳動齒輪173配置為置於驅動軸172周圍，且容納於由支柱111限定的容積115中。 Referring now to Figure 16, the drive assembly includes an engine 171, a drive gear 173, and a rocking assembly 180. Engine 171 may be any suitable engine that defines any suitable torque and/or output rate. For example, in some specific examples, the engine 171 is a Bison 650 AC. As shown in FIG. 10, the engine 171 is configured to be coupled to at least one of the struts 111 such that the drive shaft 172 extends from the engine 171 through the drive shaft opening 113 of the strut 111 and the drive shaft opening 127 of the support member 126. The drive gear 173 is configured to be placed around the drive shaft 172 and housed in a volume 115 defined by the struts 111.

搖動組件180包括搖動齒輪181、搖軸182、軸承183、安裝托架184和搖動軸襯186。搖動齒輪181可以是任何適合的尺寸及/或限定任何適合數量的齒。此外，將搖動齒輪181置於容積115之內，且可操作地連接於傳動齒輪173，例如藉由鏈(未顯示)。可以如此安排傳動齒輪173和搖動齒輪181以限定傳動齒輪173和搖動齒輪181之間的期望的齒 輪比率。 The rocking assembly 180 includes a rocking gear 181, a rocker shaft 182, a bearing 183, a mounting bracket 184, and a rocking bushing 186. The rocking gear 181 can be any suitable size and/or define any suitable number of teeth. In addition, the rocking gear 181 is placed within the volume 115 and is operatively coupled to the transmission gear 173, such as by a chain (not shown). The transmission gear 173 and the rocking gear 181 may be arranged to define desired teeth between the transmission gear 173 and the rocking gear 181 Round ratio.

配置搖軸182以***搖動齒輪181和軸承183中，並且經由支撐構件126的搖軸開口128和支柱111的搖軸開口114延伸。軸承183可用於促進搖軸182的轉動及/或降低搖動組件180上的磨損。進一步將搖軸182配置為經由搖動軸襯186***且固定連接(如焊接)於安裝托架184。隨著搖軸182與安裝托架184的連接，可將安裝托架184與第一架141的支撐管144連接。因此，在安裝托架184與第一架141和搖軸182連接的情況下，架組件140可操作地連接於發動機171。 The rocker shaft 182 is configured to be inserted into the rocking gear 181 and the bearing 183, and extends through the rocking shaft opening 128 of the support member 126 and the rocking shaft opening 114 of the strut 111. Bearing 183 can be used to facilitate rotation of rocker shaft 182 and/or reduce wear on rocking assembly 180. The rocker shaft 182 is further configured to be inserted via a rocking bushing 186 and fixedly coupled (eg, welded) to the mounting bracket 184. As the rocker shaft 182 is coupled to the mounting bracket 184, the mounting bracket 184 can be coupled to the support tube 144 of the first frame 141. Thus, with the mounting bracket 184 coupled to the first frame 141 and the rocker 182, the frame assembly 140 is operatively coupled to the engine 171.

例如，圖17-19分別說明了第一種配置、第二種配置、第三種配置中擺動架100的一部分。如圖17中可見的，擺動架100可為第一種配置，而使架141的平臺142與水準軸基本平行(例如，架141與地面平行)。在一些具體例中，架141可以與連桿145基本垂直，而擺動架100處於第一種配置。 For example, Figures 17-19 illustrate a portion of the swing frame 100 in the first configuration, the second configuration, and the third configuration, respectively. As can be seen in Figure 17, the swing frame 100 can be of the first configuration such that the platform 142 of the frame 141 is substantially parallel to the level axis (e.g., the frame 141 is parallel to the ground). In some embodiments, the frame 141 can be substantially perpendicular to the link 145 and the swing frame 100 is in the first configuration.

如圖18顯示的，可將搖動齒輪181以箭頭AA的方向旋轉將擺動架100移向第二種配置。更具體地，發動機171(圖18中未顯示)可以是電子連接(例如，如控制台處於「開」位置)，而使發動機171旋轉驅動軸172和傳動齒輪173。可以將發動機171配置為以任何給定的輸出速率旋轉驅動軸172。例如，在一些具體例中，可以將發動機171配置為以0.5 RPM和1 RPM之間的速率旋轉驅動軸172。 As shown in Figure 18, the rocking gear 181 can be rotated in the direction of arrow AA to move the swing frame 100 to the second configuration. More specifically, the engine 171 (not shown in FIG. 18) may be an electronic connection (eg, such as when the console is in the "on" position), causing the engine 171 to rotate the drive shaft 172 and the transmission gear 173. Engine 171 can be configured to rotate drive shaft 172 at any given output rate. For example, in some embodiments, engine 171 can be configured to rotate drive shaft 172 at a rate between 0.5 RPM and 1 RPM.

如上描述的，搖動齒輪181藉由一種鏈可操作地連接於傳動齒輪173。因此，該鏈將由發動機171產生的旋轉力的一部分傳遞到搖動齒輪181，而使搖動齒輪181以箭頭AA的方向旋轉。隨著安裝托架184與第一架141連接(如上所述)，由發動機171產生的轉動力的一部分被施加到第一架141上。以該種方式，推動第一架141的一端以箭頭BB的方向移動而推動第一架141的另一端以箭頭CC的方向移動。此外，由於連桿145與架141的每一個連接，連桿145將由發動機171產生的轉動力的一部分傳遞到每一個架141上。因此，每個架141被配置為與第一架141同時移動，以回應由發動機171產生的轉動力的至少一部分。此外，當與如培養的植物組織一起使用時，架141的繞軸旋轉動作可以使置於平臺142的表面上的一組植物組織的部分，例如根，間歇地傾斜，而使所述部分(如根)交替地浸沒和脫離器皿中含有的液體營養物。進一步擴展的，架141的繞軸轉動動作可以將架141以相對於水準軸的某角度放置，由此，液體營養物以箭頭DD的方向流動。 As described above, the rocking gear 181 is operatively coupled to the transmission gear 173 by a chain. Therefore, the chain transmits a part of the rotational force generated by the engine 171 to the rocking gear 181, and the rocking gear 181 is rotated in the direction of the arrow AA. As the mounting bracket 184 is coupled to the first frame 141 (as described above), a portion of the rotational force generated by the engine 171 is applied to the first frame 141. In this manner, one end of the first frame 141 is pushed to move in the direction of the arrow BB to push the other end of the first frame 141 to move in the direction of the arrow CC. Further, since the link 145 is connected to each of the frames 141, the link 145 transmits a part of the rotational force generated by the engine 171 to each of the frames 141. Accordingly, each of the shelves 141 is configured to move simultaneously with the first frame 141 in response to at least a portion of the rotational force generated by the engine 171. Furthermore, when used with plant tissue as cultured, the pivoting action of the frame 141 can cause portions of a set of plant tissue, such as roots, placed on the surface of the platform 142 to be intermittently tilted while the portion is The root nutrient is alternately immersed and detached from the liquid nutrients contained in the vessel. Further expanded, the pivoting action of the frame 141 can place the frame 141 at an angle relative to the level axis, whereby liquid nutrients flow in the direction of arrow DD.

如圖19顯示，可將搖動齒輪181以箭頭EE的方向(基本和方向AA相反)旋轉將擺動架100從第二種配置移至第三種配置。隨著搖動齒輪181以箭頭EE的方向移動，推動第一架141的一端以箭頭FF的方向移動(基本與方向BB相反)且推動第一架141的另一端的部分以箭頭GG的方向移動 (基本與方向CC相反)。此外，連桿145推動架組件140的每一個架141與第一架141同時移動。由此，當與如培養的植物組織一起使用時，架141以方向EE繞軸轉動的動作可以推動液體營養物以箭頭HH的方向流動，而使培養的植物組織(如根)交替的浸沒和脫離器皿中含有的液體營養物。 As shown in Figure 19, the swinging gear 181 can be rotated in the direction of the arrow EE (substantially opposite to the direction AA) to move the swing frame 100 from the second configuration to the third configuration. As the rocking gear 181 moves in the direction of the arrow EE, one end of the first frame 141 is pushed to move in the direction of the arrow FF (substantially opposite to the direction BB) and the portion pushing the other end of the first frame 141 is moved in the direction of the arrow GG. (basically contrary to the direction CC). In addition, the link 145 pushes each of the frames 141 of the frame assembly 140 to move simultaneously with the first frame 141. Thus, when used with plant tissue as cultured, the action of the frame 141 pivoting in the direction EE can push the liquid nutrients to flow in the direction of the arrow HH, thereby alternately immersing the cultured plant tissues (such as roots). Remove the liquid nutrients contained in the vessel.

在使用時，可以將擺動架100配置為在第二種配置和第三種配置之間擺動。在一些具體例中，擺動架100可以給定的迴圈時間在第二種配置和第三種配置之間擺動。例如，在一些具體例中，所述迴圈時間可以是25秒(例如，擺動時間為15秒且在進入相反方向之前在第二種配置或第三種配置中的保留時間為10秒)。在其他的具體例中，所述迴圈時間可以是任何其他的適合的時間長度。在一些具體例中，所述擺動架100可包括感測器(上述)。在這類具體例中，可將該感測器，例如一種磁性感測器配置為感知架組件140相對於框架100的位置。可將該感測器配置為與例如可程式設計邏輯控制器處於電子通信的。所述可程式設計邏輯控制器和感測器可檢測系統故障。例如，在一些具體例中，可將所述可程式設計邏輯控制器配置為如果所述感測器未以預定的時段(例如35秒)感知架組件140的位置的話，向輸出裝置發送電子信號以產生適合的輸出。所述輸出可以是可聽見的警報、閃光、電話、電子郵件及/或任何其他適合的通知。 In use, the swing frame 100 can be configured to swing between a second configuration and a third configuration. In some embodiments, the swing frame 100 can swing between the second configuration and the third configuration for a given loop time. For example, in some embodiments, the loop time may be 25 seconds (eg, the swing time is 15 seconds and the retention time in the second configuration or the third configuration is 10 seconds before entering the opposite direction). In other embodiments, the loop time can be any other suitable length of time. In some embodiments, the swing frame 100 can include a sensor (described above). In such a specific example, the sensor, such as a magnetic sensor, can be configured to sense the position of the shelf assembly 140 relative to the frame 100. The sensor can be configured to be in electronic communication with, for example, a programmable logic controller. The programmable logic controller and sensor can detect system faults. For example, in some embodiments, the programmable logic controller can be configured to send an electrical signal to an output device if the sensor does not sense the position of the shelf assembly 140 for a predetermined period of time (eg, 35 seconds). To produce a suitable output. The output can be an audible alert, flash, phone, email, and/or any other suitable notification.

可使用任何適合的製造技術產生本文中描述的元件。例 如，在一些具體例中，一些元件可以是模壓的。在一些具體例中，所述元件可以是塑造的(如彎曲的)。在這類具體例中，所述元件可包括任何適合的特徵以使該元件限定特定的材料屬性。例如，平臺142在上面描述為包括雙回路的，該雙回路被配置為增加平臺142的強度及/或剛度。在一些具體例中，其他元件可包括類似的特徵。例如，在一些具體例中，支柱111可包括雙回路。在其他的具體例中，所述連桿145可包括雙回路。 The elements described herein can be produced using any suitable fabrication technique. example For example, in some embodiments, some of the components may be molded. In some embodiments, the elements can be shaped (e.g., curved). In such specific embodiments, the element can include any suitable feature to cause the element to define a particular material property. For example, platform 142 is described above as including a dual loop configured to increase the strength and/or stiffness of platform 142. In some embodiments, other elements may include similar features. For example, in some embodiments, the strut 111 can include a dual loop. In other specific examples, the link 145 can include a dual circuit.

可以任何適合的方式組合本文中描述的元件。例如，在一些具體例中，元件可以是焊接的。在其他的具體例中，所述元件的至少一部分可以是機械扣緊的。例如，在一些具體例中，可以藉由螺栓和螺帽、螺絲釘、插腳及/或類似物將本文中描述的元件的部分組合(如連接)。在一些具體例中，可以使用自扣螺帽(如PEM螺帽)和螺栓或螺絲釘一起組合所述元件的部分。 The elements described herein can be combined in any suitable manner. For example, in some embodiments, the components can be soldered. In other embodiments, at least a portion of the elements can be mechanically fastened. For example, in some embodiments, portions of the elements described herein can be combined (e.g., joined) by bolts and nuts, screws, pins, and/or the like. In some embodiments, a portion of the component can be assembled using a self-fastening nut (such as a PEM nut) and a bolt or screw.

儘管已在上面描述了各種具體例，需要理解的是其僅被作為實例呈現，且無限制性。在上述圖表及/或具體例指示以某些定位或位置安排的某些元件的情況下，可以修改元件的安排。類似地，在上述方法及/或事件指示以某種順序發生的某些事件及/或步驟的情況下，可以修改該事件及/或步驟的排序。雖然已經特別顯示和描述了具體例，需要理解的是可以進行各種形式和細節上的改變。 Although various specific examples have been described above, it is to be understood that they are presented by way of example only and not limitation. Where the above figures and/or specific examples indicate certain elements arranged in certain positions or positions, the arrangement of the elements may be modified. Similarly, where the above methods and/or events indicate certain events and/or steps that occur in a certain order, the ordering of the events and/or steps can be modified. While the specific examples have been particularly shown and described, it is understood that various changes in form and detail may be made.

儘管已經將各種具體例描述為具有特定的特徵及/或元件的組合，具有來自上述任何具體例的任何特徵及/或元件的組合的其他具體例是可能的。 Although various specific examples have been described as having specific features and/or combinations of elements, other specific examples having any features and/or combinations of elements from any of the specific examples described above are possible.

(植物微體繁殖的方法) (Method of plant micropropagation)

微體繁殖的植物可起始於選擇的植物組織塊，稱為「培植體」或「母體植物」。該培植體是將在組織培養過程中發育的細胞的來源。例如，所述培植體可以是來自如下的任何部分或細胞的集合：頂端***組織、頂芽、腋芽、不定芽、副芽、假頂芽、形成層(cambium)、側生分生組織、側芽、營養芽(vegetative buds)、繁殖芽(reproductive buds)、混合芽(mixed buds)、苗段(shoot segments)、苗尖(shoot apices)、莖段(stem segments)、莖的未成熟節段、側苗、幼苗、種子、開始從地面出現的苗、未成熟的花芽、花序、花冠段、葉段，或它們的任何部分。在一個具體例中，從約1週齡、約2週齡、約3週齡、約1月齡、約2月齡、約3月齡、約半年齡、約1年齡、約2年齡、約3年齡、約5年齡或更老的植物中選取培植體。獲得所述培植體的植物可在任何適合的條件下生長，包括但不限於，在生長室(chamber)中生長、在溫室中生長、在田間生長或在組織培養容器中(培養皿、margenta箱等)。在一些具體例中，所述培植體是從作為生長培養基上的原種維持的叢生苗獲得的組織培養物。在一些具體例中，所述培植體是具有一個或多個腋芽的節段，其可 以是休眠的或有活性的。在一些其他的具體例中，所述培植體是種子或其部分。 Micropropagated plants can be initiated from selected plant tissue blocks, known as "plants" or "parent plants." The culture is the source of cells that will develop during tissue culture. For example, the culture may be any part or collection of cells from the apical dividing tissue, apical bud, axillary bud, adventitious bud, accessory bud, pseudotop bud, cambium, lateral meristem, lateral bud , vegetative buds, reproductive buds, mixed buds, shoot segments, shoot apices, stem segments, immature segments of stems, Side seedlings, seedlings, seeds, seedlings that begin to appear from the ground, immature flower buds, inflorescences, corolla segments, leaf segments, or any part thereof. In one specific example, from about 1 week old, about 2 weeks old, about 3 weeks old, about 1 month old, about 2 months old, about 3 months old, about half age, about 1 age, about 2 years old, about Plants were selected from plants of 3 years old, about 5 years old or older. The plants from which the culture is obtained can be grown under any suitable conditions, including, but not limited to, growth in a chamber, growth in a greenhouse, growth in a field, or in a tissue culture vessel (dish, margenta box) Wait). In some embodiments, the culture is a tissue culture obtained from a seedling maintained as a stock on a growth medium. In some embodiments, the implant is a segment having one or more axillary buds, which may It is dormant or active. In some other specific embodiments, the implant is a seed or a portion thereof.

本發明提供了用於植物的體外微體繁殖的方法，所述植物如裸子植物、被子植物、單子葉植物、雙子葉植物、作物、農業/經濟/環境上重要的植物等。 The present invention provides methods for in vitro micropropagation of plants, such as gymnosperms, angiosperms, monocots, dicots, crops, agricultural/economic/environmentally important plants, and the like.

在一些具體例中，本文揭示的方法可用於裸子植物的體外微細繁殖。例如，所述方法可用於下列科/目的植物的體外微體繁殖：蘇鐵科(Cycadaceae)、澤米鐵科(Zamiaceae)、銀杏科(Ginkgoaceae)、千歲蘭科(Welwitschiaceae)、買麻藤科(Gnetaceae)、麻黃科(Ephedraceae)、松科(Pinaceae)、南洋杉科(Araucariaceae)、羅漢松科(Podocarpaceae)、金松科(Sciadopityaceae)、柏科(Cupressaceae)或紫杉科(Taxaceae)。 In some embodiments, the methods disclosed herein can be used for in vitro micropropagation of gymnosperms. For example, the method may be used in vitro following families MICROFOSSILS / target plant breeding: cycads (Cycadaceae), zamiaceae (Zamiaceae), Ginkgo (Ginkgoaceae), Chitose Orchidaceae (Welwitschiaceae), Gnetum Section ( Gnetaceae), ephedra (Ephedraceae), Pinaceae (Pinaceae), Araucariaceae (Araucariaceae), Podocarpaceae (Podocarpaceae), Jinsong Ke (Sciadopityaceae), Cupressaceae (Cupressaceae) or Taxaceae (Taxaceae).

在一些具體例中，本文揭示的方法可用於被子植物的體外微體繁殖。例如，所述方法可用於金魚藻科(Ceratophyllum)、金粟蘭科(Chloranthaceae)、真雙子葉(eudicots)、木蘭類(magnoliids)或單子葉植物的科/目的植物的體外微體繁殖。 In some embodiments, the methods disclosed herein can be used for in vitro micropropagation of angiosperms. For example, the method can be used for in vitro micropropagation of families of plants of the family Ceratophyllum , Chloranthaceae , eudicots, magnoliids or monocots.

在一些具體例中，本文揭示的方法可用於雙子葉植物的體外微體繁殖。例如，所述方法可用於下列科/目中的植物的體外微體繁殖：黃楊科(Buxaceae)、雙頰果科(Didymelaceae)、清風藤科(Sabiaceae)、昆欄樹科(Trochodendraceae)、水青樹科(Tetracentraceae)、毛茛目 (Ranunculales)、山龍眼目(Proteales)、鱗枝樹科(Aextoxicaceae)、智利藤科(Berberidopsidaceae)、五椏果科(Dilleniaceae)、洋二仙草目(Gunnerales)、石竹目(Caryophyllales)、虎耳草目(Saxifragales)、檀香目(Santalales)、薔薇類(rosids)、單果樹科(Aphloiaceae)、四棱果科(Geissolomataceae)、西蘭木科(Ixerbaceae)、美洲苦木科(Picramniaceae)、栓皮果科(Strassburgeriaceae)、葡萄科(Vitaceae)、燧體木目(Crossosomatales)、牻牛兒枝目(Geraniales)、桃金娘目(Myrtales)、蒺藜科(Zygophyllaceae)、刺毬果科(Krameriaceae)、蒜樹科(Huaceae)、衛矛目(Celastrales)、金虎尾目(Malpighiales)、酢漿草目(Oxalidales)、豆目(Fabales)、薔薇目(Rosales)、葫蘆目(Cucurbitales)、殼鬥目(Fagales)、癭椒樹科(Tapisciaceae)、白花菜目(Brassicales)、錦葵目(Malvales)、無患子目(Sapindales)、菊類(asterids)、山茱萸目(Cornales)、杜鵑花目(Ericales)、紫草科(Boraginaceae)、茶茱萸科(Icacinaceae)、五蕊茶科(Oncothecaceae)、二歧草科(Vahliaceae)、絞木目(Garryales)、茄目(Solanales)、龍膽目(Gentianales)、唇形目(Lamiales)、鱗葉樹科(Bruniaceae)、彎藥樹科(Columelliaceae)、離水花科(Desfontainiaceae)、寄奴花科(Eremosynaceae)、鼠刺科(Escalloniaceae)、盔瓣花科(Paracryphiaceae)、多香木科(Polyosmaceae)、楔蕊花科 (Sphenostemonacae)、智利木科(Tribelaceae)、冬青目(Aquifoliales)、傘形目(Apiales)、川續斷目(Dipsacales)或菊目(Asterales)。 In some embodiments, the methods disclosed herein can be used for in vitro micropropagation of dicots. For example, the method can be used for in vitro micropropagation of plants in the following families/heads: Buxaceae , Didymelaceae , Sabiaceae , Trochodendraceae , water Evergreen Branch (Tetracentraceae), Ranunculales (Ranunculales), proteales (Proteales), aextoxicon Branch (Aextoxicaceae), Chile vine Branch (Berberidopsidaceae), dilleniaceae (dilleniaceae), gunnerales (Gunnerales), Caryophyllales , Saxifragales , Santalales , rosids , Aphloiaceae , Geissolomataceae , Ixerbaceae , Picramniaceae , Strassburgeriaceae , Vitaceae , Crossosomatales , Geraniales , Myrtales , Zygophyllaceae thorn cones Branch (Krameriaceae), huaceae (Huaceae), celastrales (Celastrales), malpighiales (Malpighiales), oxalidales (Oxalidales), fabales (Fabales) Rosales (Rosales), cucurbitales (Cucurbitales), fagales (Fagales), tapisciaceae (Tapisciaceae), Capparales (Brassicales), Malvales (Malvales), Sapindales (Sapindales), chrysanthemum class ( asterids), cornales (cornales), Ericales (Ericales), comfrey (Boraginaceae), icacinaceae (icacinaceae), five Camellia Branch (Oncothecaceae), dichotomous Caoke (Vahliaceae), garryales (garryales ), Solanales , Gentianales , Lamiales , Bruniaceae , Columelliaceae , Desfontainiaceae , Eremosynaceae , Escalloniaceae , Paracryphiaceae , Polyosmaceae , Sphenostemonacae , Tribelaceae , Aquifoliales , Apiales , Dipsacales or Asterales .

在一些具體例中，本文中所述方法可用於單子葉植物的體外微體繁殖。例如，所述方法可用於下列科/目中的植物的體外微體繁殖：菖蒲目(Acorales)、澤瀉目(Alismatales)、天門冬目(Asparagales)、薯蕷目(Dioscoreales)、百合目(Liliales)、露兜樹目(Pandanales)、無葉蓮目(Petrosaviales)、多須草科(Dasypogonaceae)、檳榔目(Arecales)、鴨蹠草目(Commelinales)、禾本目(Poales)或薑目(Zingiberales)。 In some embodiments, the methods described herein can be used for in vitro micropropagation of monocots. For example, the method can be used for in vitro micropropagation of plants in the following families/ elements : Acorales , Alismatales , Asparagales , Dioscoreales , Liliales ), pandanales (Pandanales), no lotus leaf mesh (Petrosaviales), dasypogonaceae (dasypogonaceae), betel nut head (Arecales), commelinales (commelinales), poales (Poales) or ginger head (Zingiberales ).

在一些具體例中，本文中所述方法可用於竹物種的體外微體繁殖，例如毛竹(如Phyllostachys edulisi‘Moso’)、蓉城竹、缺苞箭竹、菲白竹、維氏熊竹、菲黃竹、筱竹、丘斯誇竹(Chusquea Culeo“Cana Prieta”)、白綠竹、楠竹、烏芽竹、馬來甜龍竹或瓜多竹。在一些具體例中，所述竹物種是毛竹，Moso。 In some embodiments, the methods described herein can be used for in vitro micropropagation of bamboo species, such as Phyllostachys pubescens (such as Phyllostachys edulisi 'Moso'), Rongcheng Bamboo, Arrowhead Bamboo, Philippine White Bamboo, Vicki 's Bear Bamboo, and Yellow Bamboo , Chusquea Culeo "Cana Prieta" , white green bamboo, bamboo, black bud bamboo, Malay sweet dragon bamboo or melon bamboo. In some embodiments, the bamboo species is Phyllostachys pubescens, Moso.

在一些具體例中，所述方法用於快速的竹體外微體繁殖。在本文中公開的方法可以實現高的苗增殖率。如本文使用的術語「增殖率」指微體繁殖過程中起始於單個培植體獲得的植物苗的增殖倍數。例如，在培植體是包含單個芽的節段且一個微體繁殖迴圈後獲得3個苗的情況下，增殖率是3X。在一些具體例中，使用芽誘導培養基和苗伸長/維持培養 基，可以在微體繁殖後實現至少約2X到約30X的增殖率。例如，可以在約5天、6天、7天、8天、9天、10天、11天、12天、13天、14天、15天、16天、17天、18天、19天、20天、21天、22天、23天、24天、25天、26天、27天、或約28天或更多天之內實現約2X、3X、4X、5X、6X、7X、8X、9X、10X、11X、12X、13X、14X、15X、16X、17X、18X、19X、20X、21X、22X、23X、24X、25X、26X、27X、28X、29X、約30X、或更高。 In some embodiments, the method is used for rapid microbial propagation in vitro. The methods disclosed herein can achieve high seedling growth rates. The term "proliferation rate" as used herein refers to the multiplication ratio of plant seedlings obtained from a single culture body during micropropagation. For example, in the case where the culture body is a segment containing a single bud and three seedlings are obtained after one micro-propagation loop, the proliferation rate is 3X. In some specific examples, bud induction medium and shoot elongation/maintenance culture are used. The base can achieve a proliferation rate of at least about 2X to about 30X after micropropagation. For example, it can be about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, Approximately 2X, 3X, 4X, 5X, 6X, 7X, 8X, within 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or about 28 days or more, 9X, 10X, 11X, 12X, 13X, 14X, 15X, 16X, 17X, 18X, 19X, 20X, 21X, 22X, 23X, 24X, 25X, 26X, 27X, 28X, 29X, about 30X, or higher.

在一些具體例中，本發明是基於意想不到的發現，即培植體在含有強細胞***素如TDZ的第一培養基上的脈動處理，隨後該培植體在含有一種或多種除TDZ之外的細胞***素的第二培養基上的處理，可提供具有意想不到的高增殖率的快速體外微體繁殖，所述除TDZ之外的細胞***素如相對弱於TDZ的細胞***素，例如間拓樸林、激動素、異戊烯腺嘌呤(iP，例如2ip)、玉米素、反玉米素、玉米素核苷、二氫玉米素、苄胺基嘌呤(BAP)或苄基腺苷([9R]BAP)。 In some embodiments, the present invention is based on the unexpected discovery that the implant is pulsed on a first medium containing a strong cytokinin such as TDZ, and then the culture is in a cell containing one or more cells other than TDZ. Treatment on a second medium of mitogens provides rapid in vitro micropropagation with an unexpectedly high rate of proliferation, such as cytokinins other than TDZ, such as cytokinins that are relatively weaker than TDZ, such as inter-topography Lin, kinetin, isopentenyl adenine (iP, eg 2ip), zeatin, anti-zein, zeatin nucleoside, dihydro zeatin, benzylaminopurine (BAP) or benzyl adenosine ([9R] BAP).

在一些具體例中，方法包括使用芽誘導培養基和苗伸長/維持培養基，其中所述芽誘導培養基包含強細胞***素，例如TDZ，而且苗伸長/維持培養基包含相對弱的細胞***素，例如間拓樸林、激動素、異戊烯腺嘌呤(iP，例如2ip)、玉米素、反玉米素、玉米素核苷、二氫玉米素、苄胺基嘌呤(BAP)或苄基腺苷([9R]BAP)。 In some embodiments, the method comprises using a shoot induction medium and a shoot elongation/maintenance medium, wherein the shoot induction medium comprises a strong cytokinin, such as TDZ, and the shoot elongation/maintenance medium comprises a relatively weak cytokinin, such as Topolin, kinetin, isopentenyl adenine (iP, eg 2ip), zeatin, anti-zein, zeatin nucleoside, dihydro zeatin, benzylaminopurine (BAP) or benzyl adenosine ([ 9R] BAP).

本文中描述了芽誘導培養基的實例。在一些具體例中，芽誘導培養基包含一種或多種強細胞***素或其類似物。在一些具體例中，芽誘導培養基僅包含一種強細胞***素，其中所述細胞***素是TDZ或其類似物。在一些具體例中，芽誘導培養基中強細胞***素(例如TDZ)的濃度是約0.25 mg/L到約100 mg/L，例如，約0.5 mg/L到約2 mg/L。 Examples of shoot induction media are described herein. In some embodiments, the shoot induction medium comprises one or more strong mitogens or analogs thereof. In some embodiments, the shoot induction medium comprises only one strong cytokinin, wherein the cytokinin is TDZ or an analog thereof. In some embodiments, the concentration of strong cytokinin (e.g., TDZ) in the shoot induction medium is from about 0.25 mg/L to about 100 mg/L, for example, from about 0.5 mg/L to about 2 mg/L.

本文中描述了苗伸長/維持培養基的實例。在一些具體例中，苗伸長/維持培養基包含一種或多種相對弱的細胞***素，例如除TDZ以外的細胞***素。在一些具體例中，苗伸長/維持培養基僅包含一種相對弱的細胞***素，例如BAP、間拓樸林、ip(例如2ip)、玉米素、玉米素核苷或其組合。在一些具體例中，苗伸長/維持培養基包含多於一種細胞***素。在一些具體例中，苗伸長/維持培養基中細胞***素的濃度是約0.01 mg/L到約100 mg/L，例如0.25 mg/L到約5 mg/L。 Examples of shoot elongation/maintenance media are described herein. In some embodiments, the shoot elongation/maintenance medium comprises one or more relatively weak cytokinins, such as cytokinins other than TDZ. In some embodiments, the shoot elongation/sustainment medium comprises only a relatively weak cytokinin, such as BAP, metatopolin, ip (eg, 2ip), zeatin, zeatin nucleoside, or a combination thereof. In some embodiments, the shoot elongation/maintenance medium comprises more than one cytokinin. In some embodiments, the concentration of cytokinin in the shoot elongation/maintenance medium is from about 0.01 mg/L to about 100 mg/L, such as from 0.25 mg/L to about 5 mg/L.

在一些具體例中，芽誘導培養基及/或苗伸長/維持培養基包含一種或多種生長素，例如β-萘氧乙酸(NAA)、2,4-二氯苯氧乙酸(2,4-D)、吲哚-3-丁酸(IBA)、吲哚-3-乙酸(IAA)、毒莠定或其類似物。在一些具體例中，芽誘導培養基及/或苗伸長/維持培養基包含NAA。在一些具體例中，所述培養基中生長素的濃度是0.01 mg/L到約50 mg/L，例如約0.25 mg/L到約0.5 mg/L。 In some embodiments, the shoot induction medium and/or shoot elongation/maintenance medium comprises one or more auxins, such as beta-naphthyloxyacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D). , indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), chlorpyrifos or an analogue thereof. In some embodiments, the shoot induction medium and/or shoot elongation/maintenance medium comprises NAA. In some embodiments, the concentration of auxin in the medium is from 0.01 mg/L to about 50 mg/L, such as from about 0.25 mg/L to about 0.5 mg/L.

在一些具體例中，所述方法包括(a)在芽誘導培養基中培養組織培養物、培植體或種子/種子的部分以誘導苗芽形成；(b)在苗伸長/維持培養基中培養從步驟(a)獲得的苗芽。 In some embodiments, the method comprises (a) cultivating a portion of a tissue culture, culture or seed/seed in a shoot induction medium to induce shoot formation; (b) culturing in a shoot elongation/maintenance medium from the step (a) Seedlings obtained.

所述方法可進一步包括(c)在芽誘導培養基中培養從步驟(b)獲得的苗以誘導苗芽形成；並(d)在苗伸長/維持培養基中培養從步驟(c)獲得的苗芽。 The method may further comprise (c) cultivating the shoot obtained from the step (b) in a shoot induction medium to induce shoot formation; and (d) cultivating the shoot obtained from the step (c) in the shoot elongation/maintenance medium .

在一些具體例中，所述方法進一步包括(e)將培養步驟(c)和步驟(d)重複至少一次。 In some embodiments, the method further comprises (e) repeating the culturing step (c) and step (d) at least once.

在一些具體例中，所述芽誘導培養基和苗伸長/維持培養基都是液體培養基。液體培養基的優點在於可以迅速和容易地將老的培養基用新鮮的培養基替換，或將一種類型的培養基用另一種類型的培養基替換，而不用將植物的幼苗從一個容器轉移到另一個容器中。因此，在一些具體例中，整個微體繁殖過程是在單個容器中完成，例如，在一個生物反應器中。 In some embodiments, the shoot induction medium and the shoot elongation/maintenance medium are both liquid media. An advantage of the liquid medium is that the old medium can be quickly and easily replaced with fresh medium, or one type of medium can be replaced with another type of medium without transferring the seedlings of the plant from one container to another. Thus, in some embodiments, the entire micropropagation process is performed in a single container, for example, in a bioreactor.

在一些具體例中，所述芽誘導培養基及/或苗伸長/維持培養基是半固體或固體培養基。在一些具體例中，可以任何期望的順序連續使用液體培養基和半固體培養基或固體培養基。例如，步驟(a)及/或(c)中的芽誘導培養基是液體、半固體或固體；步驟(b)及/或步驟(d)中的苗伸長/維持培養基是液體、半固體或固體。 In some embodiments, the shoot induction medium and/or shoot elongation/maintenance medium is a semi-solid or solid medium. In some embodiments, the liquid medium and the semi-solid medium or solid medium can be used continuously in any desired order. For example, the shoot induction medium in steps (a) and/or (c) is liquid, semi-solid or solid; the shoot elongation/maintenance medium in step (b) and/or step (d) is liquid, semi-solid or solid. .

在一些具體例中，步驟(a)或步驟(c)的培養時間持續約1 小時到約3週。例如，步驟(a)或步驟(c)的培養時間持續約1小時、約2小時、約3小時、約4小時、約6小時、約7小時、約8小時、約9小時、約10小時、約11小時、約12小時、約14小時、約16小時、約18小時、約20小時、約22小時、約24小時、約30小時、約36小時、約42小時、約48小時、約54小時、約60小時、約1週、約1.5週、約2週、約2.5週、約3週、約3.5週、約4週或更長。 In some embodiments, the incubation time of step (a) or step (c) lasts about 1 Hours to about 3 weeks. For example, the incubation time of step (a) or step (c) lasts for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours. About 11 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, about 1 week, about 1.5 weeks, about 2 weeks, about 2.5 weeks, about 3 weeks, about 3.5 weeks, about 4 weeks or longer.

在一些具體例中，步驟(b)或步驟(d)的培養時間持續約0.5週、約1週、約1.5週、約2週、約2.5週、約3週、約3.5週、約4週、約4.5週、約5週、約5.5週、約6週、約7週、約8週或更長。 In some embodiments, the incubation time of step (b) or step (d) lasts for about 0.5 weeks, about 1 week, about 1.5 weeks, about 2 weeks, about 2.5 weeks, about 3 weeks, about 3.5 weeks, about 4 weeks. , about 4.5 weeks, about 5 weeks, about 5.5 weeks, about 6 weeks, about 7 weeks, about 8 weeks or longer.

步驟中的培養時間可以根據植物的物種、培植體的類型、期望的增殖率調整。不希望受理論束縛的，在一些具體例中，對於竹物種，步驟(a)或步驟(c)的培養時間可以是約1小時到約3週，例如，約24小時到約60小時，且步驟(b)或步驟(d)的培養時間可以是約24小時到約4週，例如，約3天到約5天。 The culture time in the step can be adjusted depending on the species of the plant, the type of the plant, and the desired proliferation rate. Without wishing to be bound by theory, in some embodiments, for bamboo species, the incubation time of step (a) or step (c) may be from about 1 hour to about 3 weeks, for example, from about 24 hours to about 60 hours, and The incubation time of step (b) or step (d) may be from about 24 hours to about 4 weeks, for example, from about 3 days to about 5 days.

重複步驟(c)和步驟(d)可進一步改進苗的增殖率。例如，步驟(a)的處理和步驟(b)的處理之後產生的多個苗可經步驟(c)和步驟(d)的一輪或多輪處理。在一些具體例中，將步驟(c)的處理和步驟(d)的處理進行至少1次、至少2次、至少3次、至少4次、至少5次、至少6次、至少7次、至少8 次或更多次。由於所有步驟中的處理是短時間的，需要達到非常高的苗增殖率的總時間非常短。 Repeating steps (c) and (d) can further improve the proliferation rate of the shoots. For example, the plurality of seedlings produced after the processing of step (a) and the processing of step (b) may be subjected to one or more rounds of processing of steps (c) and (d). In some embodiments, the treatment of step (c) and the treatment of step (d) are performed at least once, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 One or more times. Since the treatment in all steps is short-lived, the total time required to reach a very high seedling proliferation rate is very short.

在一些具體例中，在進行下一個步驟之前可將老的培養基用新鮮的培養基替換1次、2次、3次或更多次，重複(a)到(e)的每個步驟。 In some embodiments, the old medium can be replaced with fresh medium for one, two, three or more times before the next step, and each step of (a) to (e) is repeated.

在一些具體例中，起始於單個培植體，本方法可在約3週內提供約10X到約30X的苗的增殖率。此外，可以在約6週、約10週、約2個月、約2.5個月、約3個月、約4個月、約5個月、約6個月內獲得至少約500、至少約1,000、至少約2,000、至少約3,000、至少約4,000、至少約5,000、至少約6,000、至少約7,000、至少約8,000、至少約9,000、至少約10,000、至少約20,000、至少約30,000、至少約40,000、至少約50,000、至少約60,000、至少約70,000、至少約80,000、至少約90,000、至少約100,000、或更多個植物的苗。 In some embodiments, starting from a single implant, the method provides a proliferation rate of about 10X to about 30X in about 3 weeks. In addition, at least about 500, at least about 1,000 may be obtained within about 6 weeks, about 10 weeks, about 2 months, about 2.5 months, about 3 months, about 4 months, about 5 months, about 6 months. At least about 2,000, at least about 3,000, at least about 4,000, at least about 5,000, at least about 6,000, at least about 7,000, at least about 8,000, at least about 9,000, at least about 10,000, at least about 20,000, at least about 30,000, at least about 40,000, at least Seedlings of about 50,000, at least about 60,000, at least about 70,000, at least about 80,000, at least about 90,000, at least about 100,000, or more plants.

為了進一步改進苗的增殖率，可以在從步驟(a)、(b)、(c)和(d)中選擇一個或多個步驟期間或緊接其後加入分離步驟。例如，步驟(a)及/或步驟(c)中產生的多個苗芽，或步驟(c)及/或步驟(c)中產生的多個苗可以被分為單個的部分，且可將每個分開的部分置於包含新鮮培養基的單個容器中。例如，芽誘導培養基中產生的多個苗芽可以被分成單個的部分，並且或者置於新鮮的芽誘導培養基上，或者置於新鮮的 苗伸長/維持培養基上；苗伸長/維持培養基中產生的多個苗可以被分成單個的部分，並且或者置於新鮮的苗伸長/維持培養基上，或者置於新鮮的芽誘導培養基上。每個分開的部分可包含1、2、3、4、5、6、7、8、9、10或更多個苗芽或苗。 In order to further improve the proliferation rate of the shoot, the separation step may be added during or immediately after one or more steps selected from steps (a), (b), (c) and (d). For example, the plurality of shoots produced in step (a) and/or step (c), or the plurality of shoots produced in step (c) and/or step (c) may be divided into individual parts, and may be Each separate portion is placed in a single container containing fresh medium. For example, multiple shoots produced in shoot induction medium can be divided into individual parts and either placed on fresh shoot induction medium or placed fresh Seedling elongation/sustainment medium; multiple shoots produced in shoot elongation/maintenance medium can be divided into individual sections and either placed on fresh shoot elongation/maintenance medium or placed on fresh shoot induction medium. Each separate portion may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sprouts or shoots.

本發明還提供了使用本文中描述的植物生長系統的用於植物微體繁殖的方法。 The invention also provides methods for plant micropropagation using the plant growth systems described herein.

在一些具體例中，本發明的植物生長系統用於植物的微體繁殖。在一些具體例中，其用於竹的微體繁殖。在一些具體例中，其用於下列的微體繁殖：毛竹、蓉城竹、缺苞箭竹、菲白竹、維氏熊竹、菲黃竹、筱竹、丘斯誇竹、白綠竹、楠竹、烏芽竹、馬來甜龍竹、或瓜多竹、毛金竹(Nigra Henon)、青川箭竹(Rufa)或紫竹(Nigra)。 In some embodiments, the plant growth system of the invention is used for micropropagation of plants. In some embodiments, it is used for micropropagation of bamboo. In some specific examples, it is used for the following micro-propagation: Phyllostachys pubescens, Phyllostachys pubescens, Phyllostachys pubescens, Philippine white bamboo, Vicki's bear bamboo, Philippine yellow bamboo, 筱 bamboo, 丘斯夸竹, white green bamboo, bamboo , Ubud Bamboo, Malay Sweet Dragon Bamboo, or Guado Bamboo, Nigra Henon, Rukawa or Nigra.

再次參見圖1中的系統100，在用於植物的微體繁殖中，植物繁殖次序起始於將培植體置入生長器皿110。在一些具體例中，第一培養基容器130包含如本文中描述的芽誘導培養基，而第二培養基容器150包含苗伸長/維持培養基。 Referring again to system 100 in FIG. 1, in the micropropagation for plants, the order of plant propagation begins with placing the implants into growth vessel 110. In some embodiments, the first medium container 130 comprises a shoot induction medium as described herein, and the second medium container 150 comprises a shoot elongation/maintenance medium.

在一些具體例中，所述芽誘導培養基包含有效量的噻苯隆(TDZ)或其類似物，並且其中所述苗伸長/維持培養基包含有效量的一種或多種除TDZ或其類似物以外的細胞***素。在一些具體例中，所述芽誘導培養基中TDZ或其類似物的濃度是約0.25 mg/L到約100 mg/L，例如，從0.5 mg/L到 約2 mg/L。在一些具體例中，苗伸長/維持培養中一種或多種除TDZ或其類似物以外的細胞***素選自：N⁶-苄胺基嘌呤(BAP)、間拓樸林(mT)、玉米素、激動素、2-異戊烯腺嘌呤(2ip)、腺嘌呤半硫酸鹽、二甲基烯丙基腺嘌呤、N-(2-氯-4-吡啶基)-N'-苯基脲(4-CPPU)、及其各自的類似物所組成之群。在一些具體例中，一種或多種除TDZ或其類似物以外的細胞***素的濃度為從約0.01 mg/L到約100 mg/L，例如，從約0.25 mg/L到約5 mg/L。在一些具體例中，所述芽誘導培養基及/或苗伸長/維持培養基進一步包含一種或多種生長素，例如β-萘氧乙酸(NAA)、2,4-二氯苯氧乙酸(2,4-D)、吲哚-3-丁酸(IBA)、吲哚-3-乙酸(IAA)、毒莠定、及其各自的類似物。 In some embodiments, the shoot induction medium comprises an effective amount of thiazolone (TDZ) or an analog thereof, and wherein the shoot elongation/sustainment medium comprises an effective amount of one or more other than TDZ or an analog thereof Cytokinin. In some embodiments, the concentration of TDZ or an analog thereof in the shoot induction medium is from about 0.25 mg/L to about 100 mg/L, for example, from 0.5 mg/L to about 2 mg/L. In some embodiments, one or more cytokinins other than TDZ or an analog thereof in the shoot elongation/maintenance culture are selected from the group consisting of: N ⁶ -benzylaminopurine (BAP), metatopia (mT), zeatin , kinetin, 2-isopentene adenine (2ip), adenine hemisulfate, dimethylallyl adenine, N-(2-chloro-4-pyridyl)-N'-phenylurea ( 4-CPPU), a group of its respective analogs. In some embodiments, the concentration of one or more cytokinins other than TDZ or an analog thereof is from about 0.01 mg/L to about 100 mg/L, for example, from about 0.25 mg/L to about 5 mg/L. . In some embodiments, the shoot induction medium and/or shoot elongation/maintenance medium further comprises one or more auxins, such as beta-naphthyloxyacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4). -D), indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), chlorpyrifos, and their respective analogs.

在一些具體例中，第一培養次序304持續約半小時到約3週，例如，約24小時到約60小時，而第二培養次序314持續約24小時到約4週，例如，約3天到約5天。 In some embodiments, the first culture sequence 304 lasts from about half an hour to about three weeks, for example, from about 24 hours to about 60 hours, while the second culture sequence 314 lasts from about 24 hours to about 4 weeks, for example, about 3 days. It will take about 5 days.

在一些具體例中，植物繁殖次序的長度由達到的增殖率決定。在一些具體例中，增殖率為在約3週到約6個月內從至少約1,000到至少約100,000。 In some embodiments, the length of the plant propagation order is determined by the rate of proliferation achieved. In some embodiments, the proliferation rate is from at least about 1,000 to at least about 100,000 over a period of from about 3 weeks to about 6 months.

就發明者所知，此為首次將這類系統用於植物的微體繁殖，特別是竹的微體繁殖。使用生物反應器的方法是獨特的，至少由於下列原因： To the best of the inventors' knowledge, this is the first time that such systems have been used for the micropropagation of plants, especially the micropropagation of bamboo. The method of using a bioreactor is unique for at least the following reasons:

1.該方法既適用於小規模(例如實驗室)也適用於大規模(例如工業)的植物微體繁殖。 1. The method is applicable to both small scale (e.g., laboratory) and large scale (e.g., industrial) plant micropropagation.

2.該方法允許以更有效的方式使用本文中描述的脈動微體繁殖技術(例如，迴圈的培養基；更少的勞動；更精確的控制；更少的污染等)。 2. The method allows for the use of the pulsatile micropropagation techniques described herein (eg, looped media; less labor; more precise control; less contamination, etc.) in a more efficient manner.

3.該方法為較以前的方法能有極大改進的苗/植物的繁殖(例如，使用固體培養基的微體繁殖，和不在生物反應器使用液體培養基的微體繁殖)。 3. This method is a breeding of seedlings/plants which can be greatly improved over the previous methods (for example, micropropagation using a solid medium, and micropropagation using a liquid medium in a bioreactor).

4.該方法為較之前的方法能有更高的植物存活率，特別是對於某些植物物種，如毛竹。 4. This method has a higher plant viability than the previous method, especially for certain plant species, such as bamboo.

5.竹釋放酚類化合物，當其在培養基/環境中積累時，對苗/植物是有害的，這一直都是使用固體培養基的一個問題。從固體生長環境(例如，組織培養管/箱中的植物微體繁殖)轉向液體環境並將脈動方法和生物反應器組合，本發明在獲得的苗/植物的數量上實現了較大的改進，並實現了改進作為結果產生的植物的健康和產生完整尺寸植物的能力。不希望受任何理論束縛的，發明人相信這些成果是利用本發明的生物反應器系統，控制/降低苗/小植株對曝露於生長組合物中毒性成分(例如，某些植物激素，如TDZ)及/或植物產生的副產物(例如酚類化合物)的結果。 5. Bamboo releases phenolic compounds which are harmful to seedlings/plants when they accumulate in the medium/environment, which has always been a problem with solid media. The invention achieves a significant improvement in the number of seedlings/plants obtained by diverting from a solid growth environment (eg, propagation of plant microsomes in tissue culture tubes/tanks) to a liquid environment and combining the pulsation method with the bioreactor. And the ability to improve the health of the resulting plants and to produce fully sized plants is achieved. Without wishing to be bound by any theory, the inventors believe that these efforts are to utilize the bioreactor system of the present invention to control/reduce the toxic components of the seedlings/plantlets exposed to the growing composition (eg, certain phytohormones such as TDZ). And/or the result of a by-product produced by the plant, such as a phenolic compound.

除了上述基於使用「芽誘導培養基」和「苗伸長/維持培養基」組合的方法以外，本發明還提供了基於使用「階段1 培養基」、「階段2培養基」、「階段3培養基」及/或更多培養基的選擇性植物微體繁殖方法。 In addition to the above-described methods based on the combination of "bud induction medium" and "emergence elongation/maintenance medium", the present invention also provides the use based on "stage 1 Selective plant micropropagation methods for medium, "stage 2 medium", "stage 3 medium" and/or more media.

在具體例中，所述方法包括使用至少一種「階段1培養基」和至少一種「階段2培養基」，和培植體。可以依照本公開使用多種適當的培植體。在依據本公開的某些具體例中，可將莖的不成熟節段作為培植體材料使用。在一個具體例中，所述培植體可以是新的生長有節莖(growth cane)，其側苗剛剛破開節段上的鞘。新的生長有節莖包括那些從當前的季節或年度內的植物獲得的，其中這類新的生長有節莖可以從植物上的任何節獲得。在一個具體的具體例中，培植體材料包括或限於從有節莖的底部起第三個節。 In a specific example, the method comprises the use of at least one "stage 1 medium" and at least one "stage 2 medium", and a culture body. A variety of suitable implants can be used in accordance with the present disclosure. In some embodiments according to the present disclosure, the immature segments of the stem can be used as a culture material. In one embodiment, the implant may be a new growth cane with the lateral seedling just breaking the sheath on the segment. New growth stalks include those obtained from plants in the current season or year, where such new growth stalks can be obtained from any section of the plant. In a specific embodiment, the implant material includes or is limited to a third section from the bottom of the stalk.

在一些具體例中，所述植物是竹。在WO/2011/100762中，其藉由引用方式全部併入本文，其係描述收集和初始地消毒竹的培植體的詳細方法。在一些具體例中，可以使用消毒劑例如二氯異三聚氰酸、二氯異氰尿酸、三氯三三酮(trichlorotriazinetriona)、氯化汞、過氧化氫、FungiGoneTM(bioWorld，Inc.，Dublin，OH)、植物防腐劑。在一些具體例中，在起始的消毒之後，可以將竹的外鞘(outer sheaths)剝去並丟棄，並將剩餘部分放置到商業漂白劑或類似的消毒溶液的約1%至約50%的溶液中。在一些具體例中，可將漂白劑加熱到約20-60℃，例如23-50℃。在一些具體例中，還可將組織的超音波處理(sonication)和真空滲透(vacuum infiltration)與描述的消毒步驟一起使用。 In some embodiments, the plant is bamboo. In WO/2011/100762, which is hereby incorporated by reference in its entirety, it is incorporated herein in its entirety in its entirety in its entirety in the the the the the the In some embodiments, a disinfectant such as dichloroisocyano cyanide, dichloroisocyanuric acid, trichlorotrile can be used. Trichlorotriazine triona, mercuric chloride, hydrogen peroxide, FungiGoneTM (bioWorld, Inc., Dublin, OH), plant preservative. In some embodiments, after the initial sterilization, the outer sheaths of the bamboo can be stripped and discarded, and the remainder placed to about 1% to about 50% of the commercial bleach or similar disinfecting solution. In the solution. In some embodiments, the bleaching agent can be heated to about 20-60 ° C, such as 23-50 ° C. In some embodiments, sonication and vacuum infiltration of the tissue can also be used with the described sterilization steps.

在一些具體例中，增殖過程可藉由連續分離和增殖苗基本上無限地持續。在一些具體例中，可不引起新的培植體而重複增殖迴圈至少1個月、至少3個月、至少6個月、至少12個月、至少24個月、至少36個月、或更長。在一些具體例中，增殖迴圈包括每迴圈1-10天、每迴圈2-9天、每迴圈3-6天、每迴圈0.5-3天、每迴圈4-5天、每迴圈0.5-1天、每迴圈10-120天等。 In some embodiments, the proliferation process can be substantially infinitely sustained by continuous separation and proliferation of the shoots. In some embodiments, the proliferative loop may be repeated for at least 1 month, at least 3 months, at least 6 months, at least 12 months, at least 24 months, at least 36 months, or longer without causing a new implant. . In some embodiments, the proliferation loop includes 1-10 days per loop, 2-9 days per loop, 3-6 days per loop, 0.5-3 days per loop, 4-5 days per loop, 0.5-1 days per loop, 10-120 days per loop, etc.

本發明具有許多優點。不希望受任何理論束縛的，本文揭示的方法不需要使用種子或花序以起始植物，或選擇患病的起始植物，或使用抗生素、體細胞胚胎發生、假小穗(pseudospiklets)，或誘導及/或逆轉開花。為了組織培養之後的成功生長，產生的植物不需要直接在盆上灑水而用頂部(overhead)灑水就可保持健康，而且不需要在轉移到溫室或其他生長條件之前對光強度或濕度條件的多種調整。此外，培養基可以不含聚天冬胺酸、海藻濃縮物及/或表面活性劑。這些對於以前方法的改進提供了有關於產生植物的健康以及生長和處理效率之更額外的優點。 The invention has many advantages. Without wishing to be bound by any theory, the methods disclosed herein do not require the use of seeds or inflorescences to initiate plants, or to select diseased starting plants, or to use antibiotics, somatic embryogenesis, pseudospiklets, or induction. And/or reverse flowering. For successful growth after tissue culture, the resulting plants do not need to be sprinkled directly on the basin and sprinkled with overhead to maintain health, and do not require light intensity or humidity conditions prior to transfer to the greenhouse or other growing conditions. A variety of adjustments. Furthermore, the medium may be free of polyaspartic acid, seaweed concentrates and/or surfactants. These improvements to previous methods provide additional advantages regarding the health of the plants and the efficiency of growth and processing.

在一些具體例中，本發明可用於草的繁殖。在一些具體例中，所述微體繁殖的植物未被遺傳修飾過。其他特定具體例排除了微體繁殖步驟中特美汀(timentin)及/或卡那黴素的使用。 In some embodiments, the invention is useful for the propagation of grass. In some embodiments, the micropropagated plants are not genetically modified. Other specific examples preclude the use of timetin and/or kanamycin in the micropropagation step.

在一些具體例中，當本方法用於竹的繁殖時，使用年齡在3個月和3年之間的竹植物的培植體。在一些具體例中，可將具有剛破鞘的側苗的有節莖的節作為培植體使用。在一些具體例中，可將每個節段切成苗完整的3-5毫米的段。在一些具體例中，可將外鞘剝去並丟棄，而且將剩餘的節段部分置於具有終濃度為0.6%的氫氧化鈉的10%的漂白溶液中。在一些具體例中，可將漂白溶液中的培植體在可調速的Lab Rotators，Barnstead/Lab line orbital Shaker(模型號KS 260)搖動臺上以每分鐘6-9轉放置1小時。然後可將該培植體置於具有終濃度為0.06%的氫氧化鈉的1%的漂白溶液中，並放回到搖動臺上30分鐘。可以重複該1%的漂白溶液的步驟。 In some embodiments, when the method is used for the propagation of bamboo, a plant of bamboo plants between the ages of 3 months and 3 years is used. In some embodiments, a stalked section having a newly-sheathed side seedling can be used as a culture. In some embodiments, each segment can be cut into a complete 3-5 mm segment of the seedling. In some embodiments, the outer sheath can be stripped and discarded, and the remaining segment portions are placed in a 10% bleach solution having a final concentration of 0.6% sodium hydroxide. In some embodiments, the cultures in the bleach solution can be placed on a variable speed Lab Rotators, Barnstead/Lab line orbital Shaker (Model No. KS 260) shaking table for 1 hour at 6-9 revolutions per minute. The culture can then be placed in a 1% bleach solution with a final concentration of 0.06% sodium hydroxide and returned to the shaking table for 30 minutes. This step of 1% bleaching solution can be repeated.

在一些具體例中，可隨後將單個的培植體置於管中階段1培養基上(15-25 mL)，並且將該管置於處在從65℉-70℉的溫度和36-54 μmole/m²/s²的全光譜光度的受調控的清潔的生長室。起始的階段1培養基可以是pH 5.7的b-12c-iv。隨後可以每10-120天(通常每21天)將培植體轉移到新鮮的b-12c-iv培養基，而丟棄污染的管。 In some embodiments, a single culture body can then be placed on the stage 1 medium (15-25 mL) in the tube and placed at a temperature from 65 °F to 70 °F and 36-54 μmole/ A full-spectrum photometrically controlled clean growth chamber of m ² /s ² . The initial Stage 1 medium can be b-12c-iv at pH 5.7. The cultures can then be transferred to fresh b-12c-iv medium every 10 to 120 days (typically every 21 days) and the contaminated tubes discarded.

在一些具體例中，如果利用b-12c-iv培養基的強化版本，在轉移到本文中公開的「標準」培養基或含有實質降低或沒有細胞***素的培養基(本文中使用的「減料」培養基)以進行10-120天的週期的剩餘部分之前，可以將培植體在強化 培養基中放置0.25、0.5、0.75、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47或48小時。放置於強化培養基中的另外的時段包括任意的0.1和240小時之間，且可包括但不限於，0.1-0.5小時、0.3-2.5小時、2.5-6小時、1-10小時、5-15小時、10-20小時、15-25小時、20-30小時、25-35小時、30-40小時、35-45小時、40-50小時、45-55小時、50-60小時、55-65小時、60-70小時、65-75小時、70-80小時、75-85小時、80-90小時、85-95小時、90-100小時、95-105小時、100-110小時、105-115小時、110-120小時、115-125小時、120-130小時、125-135小時、130-140小時、135-145小時、140-150小時、145-155小時、150-160小時、155-165小時、160-170小時、165-175小時、170-180小時、175-185小時、180-190小時、185-195小時、190-200小時、195-205小時、200-210小時、205-215小時、210-220小時、215-225小時、220-230小時、225-235小時、230-240小時、235-245小時、240-250小時、3-6小時、7-17小時、12-22小時、17-27小時、22-32小時、27-37小時、32-42小時、37-47小時、42-52小時、47-57小時、52-62小時、57-67小時、62-72小時、67-77小時、72-82小時、77-87小時、82-92小時、87-97小時、 92-102小時、97-107小時、102-112小時、107-117小時、112-122小時、117-127小時、122-132小時、127-137小時、132-142小時、137-147小時、142-152小時、147-157小時、152-162小時、157-167小時、162-172小時、167-177小時、172-182小時、177-187小時、162-172小時、167-177小時、182-192小時、187-197小時、192-202小時、197-207小時、202-212小時、207-217小時、212-222小時、217-227小時、222-232小時、227-237小時、232-242小時、237-247小時或242-252小時。強化培養基中的放置還可比標準或減料培養基中的週期少0.5小時，比標準或減料培養基中的週期少1小時，和比標準或減料培養基中的週期少1和240小時之間的全部時段。或者，代替在標準或減料培養基中進行週期的剩餘部分，可將培植體在強化培養基上放置一段時間然後在標準或減料培養基上培養完整的週期時間(即10-120天，未因花在強化培養基的時間而減少)。 In some embodiments, if an enhanced version of the b-12c-iv medium is utilized, transfer to the "standard" medium disclosed herein or a medium containing substantially reduced or no cytokinin ("reduce" medium used herein) ) before the remainder of the 10-120 day cycle, the implant can be strengthened Place 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 in the medium. , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47 or 48 hours. Additional periods of time placed in the fortified medium include between any of 0.1 and 240 hours, and may include, but are not limited to, 0.1-0.5 hours, 0.3-2.5 hours, 2.5-6 hours, 1-10 hours, 5-15 hours. 10-20 hours, 15-25 hours, 20-30 hours, 25-35 hours, 30-40 hours, 35-45 hours, 40-50 hours, 45-55 hours, 50-60 hours, 55-65 hours 60-70 hours, 65-75 hours, 70-80 hours, 75-85 hours, 80-90 hours, 85-95 hours, 90-100 hours, 95-105 hours, 100-110 hours, 105-115 hours , 110-120 hours, 115-125 hours, 120-130 hours, 125-135 hours, 130-140 hours, 135-145 hours, 140-150 hours, 145-155 hours, 150-160 hours, 155-165 hours , 160-170 hours, 165-175 hours, 170-180 hours, 175-185 hours, 180-190 hours, 185-195 hours, 190-200 hours, 195-205 hours, 200-210 hours, 205-215 hours 210-220 hours, 215-225 hours, 220-230 hours, 225-235 hours, 230-240 hours, 235-245 hours, 240-250 hours, 3-6 hours, 7-17 hours, 12-22 hours , 17-27 hours, 22-32 hours, 27-37 hours, 32-42 hours, 37-4 7 hours, 42-52 hours, 47-57 hours, 52-62 hours, 57-67 hours, 62-72 hours, 67-77 hours, 72-82 hours, 77-87 hours, 82-92 hours, 87- 97 hours, 92-102 hours, 97-107 hours, 102-112 hours, 107-117 hours, 112-122 hours, 117-127 hours, 122-132 hours, 127-137 hours, 132-142 hours, 137-147 hours, 142-152 hours, 147-157 hours, 152-162 hours, 157-167 hours, 162-172 hours, 167-177 hours, 172-182 hours, 177-187 hours, 162-172 hours, 167-177 hours, 182-192 hours, 187-197 hours, 192-202 hours, 197-207 hours, 202-212 hours, 207-217 hours, 212-222 hours, 217-227 hours, 222-232 hours, 227-237 hours, 232-242 hours, 237-247 hours or 242-252 hours. Placement in the fortified medium may also be 0.5 hours less than in standard or reduced medium, 1 hour less than in standard or reduced medium, and 1 and 240 hours less than in standard or reduced medium. All time slots. Alternatively, instead of performing the remainder of the cycle in standard or reduced medium, the culture can be placed on the fortified medium for a period of time and then cultured on standard or reduced medium for a complete cycle time (ie, 10-120 days, not due to flowers) Reduced in time to strengthen the medium).

不含有細胞***素或含有實質性降低的細胞***素的培養基可以是減料b-9培養基、減料CW2培養基、減料b-10培養基、減料b-11培養基、減料b-12c培養基、減料b-1培養基、減料b-4培養基、減料b-6培養基、減料CW1培養基、減料CW3培養基、減料CW4培養基、減料CW5培養基、減料CW6培養基、減料B-9N2培養基、減料B-12C CPPU培養基、減料B-12C DPU培養基，其或者除去所有 的細胞***素及/或生長素，或者至少一種細胞***素及/或生長素的量可降低1%、5%、10%、15%、20%、25%、30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、98%、99%、100%、1-10%、5-15%、10-20%、15-25%、20-30%、25-35%、30-40%、35-45%、40-50%、45-55%、50-60%、55-65%、60-70%、65-75%、70-80%、75-85%、80-90%、85-95%、90-100%、3-6%、7-17%、12-22%、17-27%、22-32%、27-37%、32-42%、37-47%、42-52%、47-57%、52-62%、57-67%、62-72%、67-77%、72-82%、77-87%、82-92%或87-97%。減料培養基的非限制性實例包括(表格未顯示無細胞***素或生長素的具體例)：培養基B-9N2 The medium containing no cytokinin or containing substantially reduced cytokinin may be reduced b-9 medium, reduced CW2 medium, reduced b-10 medium, reduced b-11 medium, reduced b-12c medium , b-1 medium, b-4 medium, b-6 medium, reduced CW1 medium, reduced CW3 medium, reduced CW4 medium, reduced CW5 medium, reduced CW6 medium, reduced material B -9N2 medium, reduced B-12C CPPU medium, reduced B-12C DPU medium, or all cytokinins and/or auxins removed, or at least one cytokinin and/or auxin can be reduced by 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, 1-10%, 5-15%, 10-20%, 15-25%, 20-30%, 25-35%, 30- 40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60-70%, 65-75%, 70-80%, 75-85%, 80- 90%, 85-95%, 90-100%, 3-6%, 7-17%, 12-22%, 17-27%, 22-32%, 27-37%, 32-42%, 37- 47%, 42-52%, 47-57%, 52-62%, 57-67%, 62-72%, 67-77%, 72-82% , 77-87%, 82-92% or 87-97%. Non-limiting examples of the reduced medium include (the table does not show specific examples of no cytokinin or auxin): medium B-9N2

培養基B-12C CPPU Medium B-12C CPPU

培養基B-12C DPU Medium B-12C DPU

或者，可以將上面提到的細胞***素用類似或更高程度的較弱細胞***素替換。例示性的較弱細胞***素包括玉米素和激動素。 Alternatively, the cytokinin mentioned above can be replaced with a similar or higher degree of weaker cytokinin. Exemplary weaker cytokinins include zeatin and kinetin.

可以藉由瓊脂的細菌性變色或可見的表面污染鑑別污染的管。這些培植體可在選擇的b-12c-iv培養基上保留3-4個10-120天的週期(通常21天的週期)或者如在強化步驟中修改的(強化培養基的時段為0.25、0.5、0.75、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47或48小時，在轉移到標準的或減料的培養基以進行10-120天的週期的剩餘部分或進行完整的10-120天的週期之前)。置於強化培養基中的另外的時段包括在0.1和240小時之間的任意時間，且可包括但不限於：0.1-0.5小時、0.3-2.5小時、2.5-6小時、1-10小時、5-15小時、10-20小時、15-25小時、20-30小時、25-35小時、30-40小時、35-45小時、40-50小時、45-55小時、50-60小時、55-65小時、 60-70小時、65-75小時、70-80小時、75-85小時、80-90小時、85-95小時、90-100小時、95-105小時、100-110小時、105-115小時、110-120小時、115-125小時、120-130小時、125-135小時、130-140小時、135-145小時、140-150小時、145-155小時、150-160小時、155-165小時、160-170小時、165-175小時、170-180小時、175-185小時、180-190小時、185-195小時、190-200小時、195-205小時、200-210小時、205-215小時、210-220小時、215-225小時、220-230小時、225-235小時、230-240小時、235-245小時、240-250小時、3-6小時、7-17小時、12-22小時、17-27小時、22-32小時、27-37小時、32-42小時、37-47小時、42-52小時、47-57小時、52-62小時、57-67小時、62-72小時、67-77小時、72-82小時、77-87小時、82-92小時、87-97小時、92-102小時、97-107小時、102-112小時、107-117小時、112-122小時、117-127小時、122-132小時、127-137小時、132-142小時、137-147小時、142-152小時、147-157小時、152-162小時、157-167小時、162-172小時、167-177小時、172-182小時、177-187小時、162-172小時、167-177小時、182-192小時、187-197小時、192-202小時、197-207小時、202-212小時、207-217小時、212-222小時、217-227小時、222-232小時、227-237小時、232-242小時、237-247小時或242-252小時。強化培養基中的放置還可比標準或減料培 養基中的週期少0.5小時，比標準或減料培養基中的週期少1小時，以及比標準或減料培養基中的週期少1和240小時之間的任意的時段。如果培植體在特定的強化培養基類型(例如，b-12c)上培養，當轉移到標準或減料培養基時，所述標準或減料培養基可具有相同的類型(例如，標準或減料b-12c)或不同的類型(例如，標準或減料CW1、CW2、CW6、b6、b9等)。 Contaminated tubes can be identified by bacterial discoloration of agar or visible surface contamination. These cultures can retain 3-4 10-120 day cycles (usually 21 day cycles) on the selected b-12c-iv medium or as modified in the fortification step (the incubation medium is 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 hours, at Transfer to standard or reduced media for a remainder of the 10-120 day cycle or for a full 10-120 day cycle). Additional time periods placed in the fortified medium include any time between 0.1 and 240 hours, and may include, but are not limited to, 0.1-0.5 hours, 0.3-2.5 hours, 2.5-6 hours, 1-10 hours, 5- 15 hours, 10-20 hours, 15-25 hours, 20-30 hours, 25-35 hours, 30-40 hours, 35-45 hours, 40-50 hours, 45-55 hours, 50-60 hours, 55- 65 hours, 60-70 hours, 65-75 hours, 70-80 hours, 75-85 hours, 80-90 hours, 85-95 hours, 90-100 hours, 95-105 hours, 100-110 hours, 105-115 hours, 110-120 hours, 115-125 hours, 120-130 hours, 125-135 hours, 130-140 hours, 135-145 hours, 140-150 hours, 145-155 hours, 150-160 hours, 155-165 hours, 160-170 hours, 165-175 hours, 170-180 hours, 175-185 hours, 180-190 hours, 185-195 hours, 190-200 hours, 195-205 hours, 200-210 hours, 205-215 hours, 210-220 hours, 215-225 hours, 220-230 hours, 225-235 hours, 230-240 hours, 235-245 hours, 240-250 hours, 3-6 hours, 7-17 hours, 12-22 hours, 17-27 hours, 22-32 hours, 27-37 hours, 32-42 hours, 37-47 hours, 42-52 hours, 47-57 hours, 52-62 hours, 57-67 hours, 62-72 hours, 67-77 hours, 72-82 hours, 77-87 hours, 82-92 hours, 87-97 hours, 92-102 hours, 97-107 hours, 102-112 hours, 107-117 hours, 112-122 hours, 117-127 hours, 122-132 hours, 127-137 hours, 132-142 hours, 137-147 hours, 142-152 hours 147-157 hours, 152-162 hours, 157-167 hours, 162-172 hours, 167-177 hours, 172-182 hours, 177-187 hours, 162-172 hours, 167-177 hours, 182-192 hours, 187-197 hours, 192-202 hours, 197-207 hours, 202-212 hours, 207-217 hours, 212-222 hours, 217-227 hours, 222-232 hours, 227-237 hours, 232-242 hours, 237-247 hours or 242-252 hours. Placement in the fortified medium can also be compared to standard or reduced feed The cycle in the nutrient base is 0.5 hours less, one hour less than the cycle in standard or reduced media, and any period between 1 and 240 hours less than the cycle in standard or reduced media. If the culture is cultured on a particular enhanced medium type (eg, b-12c), the standard or reduced medium may be of the same type (eg, standard or reduced b- when transferred to standard or reduced medium). 12c) or a different type (for example, standard or reduced material CW1, CW2, CW6, b6, b9, etc.).

在本文揭示的特定具體例中，培養時段為小於12週、小於9週或小於6週。 In a particular embodiment disclosed herein, the incubation period is less than 12 weeks, less than 9 weeks, or less than 6 weeks.

如果增殖產生，可將培植體在第三個週期之後從培養基上取出。如果增殖未產生或者未以顯著程度產生，可將培植體保留在培養基進行第四個週期。 If proliferation occurs, the culture body can be removed from the culture medium after the third cycle. If proliferation is not produced or is not produced to a significant extent, the culture can be retained in the medium for the fourth cycle.

接下來可將活的苗轉移到階段2培養基(如果在前面的步驟中使用標準b-12c，或者如果使用基本的強化步驟，則轉移到階段3培養基)，例如pH為5.7的b-9、CW1、CW2、CW3、CW4、CW5、CW6、b-6、B-9N2、B-12C CPPU或B-12C DPU。可將該培養物保留在該階段2培養基上，直到分離到新管和進一步擴展獲得期望數量的苗。一般地，時間的範圍包括10-120天的週期(通常14-21天的週期)，在該時間內，將培養物安排為經過另一輪增殖或轉移到階段3或階段4培養基，例如，pH為5.7的b-10-iv或b-11-iv以進一步增殖。 The live shoots can then be transferred to Stage 2 medium (if standard b-12c is used in the previous step, or if the basic boost step is used, transfer to Stage 3 medium), such as b-9 with a pH of 5.7, CW1, CW2, CW3, CW4, CW5, CW6, b-6, B-9N2, B-12C CPPU or B-12C DPU. The culture can be retained on this stage 2 medium until a new tube is isolated and further expanded to obtain the desired number of shoots. Generally, the range of time includes a period of 10-120 days (typically a period of 14-21 days) during which time the culture is arranged to undergo another round of propagation or transfer to Stage 3 or Stage 4 medium, for example, pH. It is b-10-iv or b-11-iv of 5.7 for further proliferation.

在本文中公開的具體的具體例中，培養時間段為小於12週、小於9週或小於6週。 In a specific embodiment disclosed herein, the culture period is less than 12 weeks, less than 9 weeks, or less than 6 weeks.

或者，在轉移到相同或不同類型的標準或減料培養基以進行10-120天的週期的剩餘部分或完整的10-120天的週期之前，還可將活的苗在強化b-9、CW1、CW2、CW3、CW4、CW5、CW6、b-6、B-9N2、B-12C CPPU、B-12C DPU培養基上放置0.25、0.5、0.75、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47或48小時的時段。置於強化培養基中的另外的時段包括0.1和240小時之間的任意時段，且可包括但不限於，0.1-0.5小時、0.3-2.5小時、2.5-6小時、1-10小時、5-15小時、10-20小時、15-25小時、20-30小時、25-35小時、30-40小時、35-45小時、40-50小時、45-55小時、50-60小時、55-65小時、60-70小時、65-75小時、70-80小時、75-85小時、80-90小時、85-95小時、90-100小時、95-105小時、100-110小時、105-115小時、110-120小時、115-125小時、120-130小時、125-135小時、130-140小時、135-145小時、140-150小時、145-155小時、150-160小時、155-165小時、160-170小時、165-175小時、170-180小時、175-185小時、180-190小時、185-195小時、190-200小時、195-205小時、200-210 小時、205-215小時、210-220小時、215-225小時、220-230小時、225-235小時、230-240小時、235-245小時、240-250小時、3-6小時、7-17小時、12-22小時、17-27小時、22-32小時、27-37小時、32-42小時、37-47小時、42-52小時、47-57小時、52-62小時、57-67小時、62-72小時、67-77小時、72-82小時、77-87小時、82-92小時、87-97小時、92-102小時、97-107小時、102-112小時、107-117小時、112-122小時、117-127小時、122-132小時、127-137小時、132-142小時、137-147小時、142-152小時、147-157小時、152-162小時、157-167小時、162-172小時、167-177小時、172-182小時、177-187小時、162-172小時、167-177小時、182-192小時、187-197小時、192-202小時、197-207小時、202-212小時、207-217小時、212-222小時、217-227小時、222-232小時、227-237小時、232-242小時、237-247小時或242-252小時。強化培養基中的放置還可比標準或減料培養基中的週期少0.5小時，比標準或減料培養基中的週期少1小時，以及比標準或減料培養基中的週期少1和240小時之間的任意時段。 Alternatively, live seedlings may be intensified b-9, CW1 prior to transfer to the same or different types of standard or subtractive medium for a remainder of the 10-120 day cycle or a complete 10-120 day cycle. , CW2, CW3, CW4, CW5, CW6, b-6, B-9N2, B-12C CPPU, B-12C DPU medium placed on 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 , 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 hours. Additional time periods placed in the fortified medium include any period between 0.1 and 240 hours, and may include, but are not limited to, 0.1-0.5 hours, 0.3-2.5 hours, 2.5-6 hours, 1-10 hours, 5-15 Hours, 10-20 hours, 15-25 hours, 20-30 hours, 25-35 hours, 30-40 hours, 35-45 hours, 40-50 hours, 45-55 hours, 50-60 hours, 55-65 Hours, 60-70 hours, 65-75 hours, 70-80 hours, 75-85 hours, 80-90 hours, 85-95 hours, 90-100 hours, 95-105 hours, 100-110 hours, 105-115 Hours, 110-120 hours, 115-125 hours, 120-130 hours, 125-135 hours, 130-140 hours, 135-145 hours, 140-150 hours, 145-155 hours, 150-160 hours, 155-165 Hours, 160-170 hours, 165-175 hours, 170-180 hours, 175-185 hours, 180-190 hours, 185-195 hours, 190-200 hours, 195-205 hours, 200-210 Hours, 205-215 hours, 210-220 hours, 215-225 hours, 220-230 hours, 225-235 hours, 230-240 hours, 235-245 hours, 240-250 hours, 3-6 hours, 7-17 Hours, 12-22 hours, 17-27 hours, 22-32 hours, 27-37 hours, 32-42 hours, 37-47 hours, 42-52 hours, 47-57 hours, 52-62 hours, 57-67 Hours, 62-72 hours, 67-77 hours, 72-82 hours, 77-87 hours, 82-92 hours, 87-97 hours, 92-102 hours, 97-107 hours, 102-112 hours, 107-117 Hour, 112-122 hours, 117-127 hours, 122-132 hours, 127-137 hours, 132-142 hours, 137-147 hours, 142-152 hours, 147-157 hours, 152-162 hours, 157-167 Hours, 162-172 hours, 167-177 hours, 172-182 hours, 177-187 hours, 162-172 hours, 167-177 hours, 182-192 hours, 187-197 hours, 192-202 hours, 197-207 Hours, 202-212 hours, 207-217 hours, 212-222 hours, 217-227 hours, 222-232 hours, 227-237 hours, 232-242 hours, 237-247 hours or 242-252 hours. Placement in the fortified medium may also be 0.5 hours less than in standard or subtractive medium, 1 hour less than in standard or reduced medium, and 1 and 240 hours less than in standard or reduced medium. Any time.

一般地，每個增殖週期可獲得每管1-10個苗。 Generally, from 1 to 10 seedlings per tube can be obtained per proliferation cycle.

在從增殖過程移出後，可將苗轉移到含有階段3、階段4或階段5培養基的小組織培養箱(稱為「magenta箱」)中10-120天(通常14-21天)，在該實例中，在pH為5.7的BR-2 進行10-120天(通常14-21天)或在pH為5.7的Amel進行10-120天(通常14-21天)。如上，可將苗在強化培養基中放置較短的時間隨後在標準或減料培養基放置10-120天的週期的剩餘部分或完整的10-120天的週期。 After removal from the proliferation process, the shoots can be transferred to a small tissue culture incubator (called "magenta box") containing Stage 3, Stage 4 or Stage 5 medium for 10-120 days (usually 14-21 days). In the example, BR-2 at pH 5.7 It is carried out for 10-120 days (usually 14-21 days) or at Amel pH 5.7 for 10-120 days (usually 14-21 days). As above, the shoots can be placed in the fortified medium for a shorter period of time and then placed in the standard or reduced medium for the remainder of the 10-120 day cycle or a complete 10-120 day cycle.

在本文中揭示的特定具體例中，培養時間為小於12週、小於9週或小於6週。 In a particular embodiment disclosed herein, the incubation time is less than 12 weeks, less than 9 weeks, or less than 6 weeks.

如可以被本領域的通常技術人員理解的，當使用強化培養基時，由於之後標準或減料培養基的使用，強化培養基的使用提高了特定過程中的培養基階段的數量。如果僅在一個階段使用強化培養基，該過程一般擴大1個培養基階段。如果在兩個階段使用強化培養基，該過程一般擴大2個培養基階段。如果在三個階段使用強化培養基，該過程一般擴大3個培養基階段等。 As can be appreciated by one of ordinary skill in the art, when a fortified medium is used, the use of the fortified medium increases the number of media stages in a particular process due to subsequent use of standard or reduced feed media. If the fortification medium is used only in one stage, the process generally expands one medium stage. If the fortification medium is used in two stages, the process generally expands the two medium stages. If the fortification medium is used in three stages, the process generally expands the three medium stages and the like.

或者，也可以使用下列步驟(階段1、階段2、階段3等，培養基係如文中別處所定義)：隨後可將各個培植體置於管中的階段1培養基(15-25 mL)上，並將管置於處在溫度從65℉-70℉和全光譜光度36-90 μmole/m²/s²的受調控清潔生長室。階段1培養基可以是pH為5.7的標準b-12c-iv或強化b-12c-iv培養基。如果置於標準b-12c-iv，每10-120天(一般每21天)可將培植體轉移到新鮮的b-12c-iv培養基，而將污染的管丟棄。如果置於強化b-12c-iv培養基，可將培植體在強化培養基上保留0.25、 0.5、0.75、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47或48小時，並且隨後轉移到沒有強化組分的培養基(標準或減料)以進行10-120天的週期的剩餘部分或完整的10-120天的週期。置於強化培養基中的另外時段包括0.1和240小時之間的任意時段，且可包括但不限於，0.1-0.5小時、0.3-2.5小時、2.5-6小時、1-10小時、5-15小時、10-20小時、15-25小時、20-30小時、25-35小時、30-40小時、35-45小時、40-50小時、45-55小時、50-60小時、55-65小時、60-70小時、65-75小時、70-80小時、75-85小時、80-90小時、85-95小時、90-100小時、95-105小時、100-110小時、105-115小時、110-120小時、115-125小時、120-130小時、125-135小時、130-140小時、135-145小時、140-150小時、145-155小時、150-160小時、155-165小時、160-170小時、165-175小時、170-180小時、175-185小時、180-190小時、185-195小時、190-200小時、195-205小時、200-210小時、205-215小時、210-220小時、215-225小時、220-230小時、225-235小時、230-240小時、235-245小時、240-250小時、3-6小時、7-17小時、12-22小時、17-27小時、22-32小時、27-37小時、32-42小時、37-47小時、42-52小時、47-57小時、52-62小時、 57-67小時、62-72小時、67-77小時、72-82小時、77-87小時、82-92小時、87-97小時、92-102小時、97-107小時、102-112小時、107-117小時、112-122小時、117-127小時、122-132小時、127-137小時、132-142小時、137-147小時、142-152小時、147-157小時、152-162小時、157-167小時、162-172小時、167-177小時、172-182小時、177-187小時、162-172小時、167-177小時、182-192小時、187-197小時、192-202小時、197-207小時、202-212小時、207-217小時、212-222小時、217-227小時、222-232小時、227-237小時、232-242小時、237-247小時或242-252小時。強化培養基中的放置還可比標準或減料培養基中的週期少0.5小時，比標準或減料培養基中的週期少1小時，以及比標準或減料培養基中的週期少1和240小時之間的任意時段。這些培植體可在b-12c-iv培養基或強化b-12c-iv培養基上保留2個10-120天的週期(通常21天的週期)。在週期之間，可以移除多餘的鞘。在轉移到第三個週期時，可將培植體轉移到階段2培養基或階段3培養基(根據是否使用強化步驟)，在本實例中，用7 g/L而非上面提供的5.5 g/L的角叉膠補充的標準b-12c-iv或者用7 g/L而非上面提供的5.5 g/L的角叉膠補充的強化b-12c-iv進行0.25、0.5、0.75、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、 34、35、36、37、38、39、40、41、42、43、44、45、46、47或48小時，隨後轉移到標準或減料b-12c-iv。置於強化培養基中的另外的時段包括在0.1和240小時之間的任意時段，且可包括但不限於，0.1-0.5小時、0.3-2.5小時、2.5-6小時、1-10小時、5-15小時、10-20小時、15-25小時、20-30小時、25-35小時、30-40小時、35-45小時、40-50小時、45-55小時、50-60小時、55-65小時、60-70小時、65-75小時、70-80小時、75-85小時、80-90小時、85-95小時、90-100小時、95-105小時、100-110小時、105-115小時、110-120小時、115-125小時、120-130小時、125-135小時、130-140小時、135-145小時、140-150小時、145-155小時、150-160小時、155-165小時、160-170小時、165-175小時、170-180小時、175-185小時、180-190小時、185-195小時、190-200小時、195-205小時、200-210小時、205-215小時、210-220小時、215-225小時、220-230小時、225-235小時、230-240小時、235-245小時、240-250小時、3-6小時、7-17小時、12-22小時、17-27小時、22-32小時、27-37小時、32-42小時、37-47小時、42-52小時、47-57小時、52-62小時、57-67小時、62-72小時、67-77小時、72-82小時、77-87小時、82-92小時、87-97小時、92-102小時、97-107小時、102-112小時、107-117小時、112-122小時、117-127小時、122-132小時、127-137小時、132-142小時、137-147 小時、142-152小時、147-157小時、152-162小時、157-167小時、162-172小時、167-177小時、172-182小時、177-187小時、162-172小時、167-177小時、182-192小時、187-197小時、192-202小時、197-207小時、202-212小時、207-217小時、212-222小時、217-227小時、222-232小時、227-237小時、232-242小時、237-247小時或242-252小時。強化培養基中的放置還可比標準或減料培養基中的週期少0.5小時，比標準或減料培養基中的週期少1小時，以及比標準或減料培養基中的週期少1和240小時之間的任意時段。第三個週期之後，可以清理培植體。可將培植體在用7 g/L而非上面提供的5.5 g/L的角叉膠補充的b-12c-iv上保留10-120天的週期(通常21天的週期)，直到觀察到多個苗。可以在3-15個月內觀察到多個苗。當使用多個週期時，可將外置體在標準的培養基上培養所有週期，在強化培養基隨後在標準培養基上培養所有週期或者在強化培養基隨後在減料培養基上培養所有週期。或者，可使培植體在整個週期中接受任意組合和順序的一種或多種處理。 Alternatively, the following steps (Stage 1, Stage 2, Stage 3, etc., medium as defined elsewhere) can also be used: each individual can then be placed in Stage 1 medium (15-25 mL) in a tube and The tube was placed in a regulated clean growth chamber at a temperature ranging from 65 °F to 70 °F and a full spectrum of luminosity of 36-90 μmole/m ² /s ² . The Stage 1 medium can be a standard b-12c-iv or fortified b-12c-iv medium having a pH of 5.7. If placed in standard b-12c-iv, the cultures can be transferred to fresh b-12c-iv medium every 10 to 120 days (typically every 21 days) and the contaminated tubes discarded. If placed in the fortified b-12c-iv medium, the culture can be retained on the fortified medium at 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 hours, and then transferred to the medium without the fortified component (standard or reduced) for the remainder of the 10-120 day cycle Or a full 10-120 day period. Additional time periods placed in the fortified medium include any period between 0.1 and 240 hours, and may include, but are not limited to, 0.1-0.5 hours, 0.3-2.5 hours, 2.5-6 hours, 1-10 hours, 5-15 hours. 10-20 hours, 15-25 hours, 20-30 hours, 25-35 hours, 30-40 hours, 35-45 hours, 40-50 hours, 45-55 hours, 50-60 hours, 55-65 hours 60-70 hours, 65-75 hours, 70-80 hours, 75-85 hours, 80-90 hours, 85-95 hours, 90-100 hours, 95-105 hours, 100-110 hours, 105-115 hours , 110-120 hours, 115-125 hours, 120-130 hours, 125-135 hours, 130-140 hours, 135-145 hours, 140-150 hours, 145-155 hours, 150-160 hours, 155-165 hours , 160-170 hours, 165-175 hours, 170-180 hours, 175-185 hours, 180-190 hours, 185-195 hours, 190-200 hours, 195-205 hours, 200-210 hours, 205-215 hours 210-220 hours, 215-225 hours, 220-230 hours, 225-235 hours, 230-240 hours, 235-245 hours, 240-250 hours, 3-6 hours, 7-17 hours, 12-22 hours , 17-27 hours, 22-32 hours, 27-37 hours, 32-42 hours, 37-4 7 hours, 42-52 hours, 47-57 hours, 52-62 hours, 57-67 hours, 62-72 hours, 67-77 hours, 72-82 hours, 77-87 hours, 82-92 hours, 87- 97 hours, 92-102 hours, 97-107 hours, 102-112 hours, 107-117 hours, 112-122 hours, 117-127 hours, 122-132 hours, 127-137 hours, 132-142 hours, 137- 147 hours, 142-152 hours, 147-157 hours, 152-162 hours, 157-167 hours, 162-172 hours, 167-177 hours, 172-182 hours, 177-187 hours, 162-172 hours, 167- 177 hours, 182-192 hours, 187-197 hours, 192-202 hours, 197-207 hours, 202-212 hours, 207-217 hours, 212-222 hours, 217-227 hours, 222-232 hours, 227- 237 hours, 232-242 hours, 237-247 hours or 242-252 hours. Placement in the fortified medium may also be 0.5 hours less than in standard or subtractive medium, 1 hour less than in standard or reduced medium, and 1 and 240 hours less than in standard or reduced medium. Any time. These cultures can be maintained for two 10-120 day cycles (usually 21 day cycles) on b-12c-iv medium or fortified b-12c-iv medium. The excess sheath can be removed between cycles. Upon transfer to the third cycle, the culture can be transferred to Stage 2 medium or Stage 3 medium (depending on whether the intensive step is used), in this example, 7 g/L instead of the 5.5 g/L provided above Carrageenan-added standard b-12c-iv or reinforced reinforced b-12c-iv supplemented with 7 g/L instead of the 5.5 g/L carrageenan provided above for 0.25, 0.5, 0.75, 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 hours, then transferred to standard or reduced material b -12c-iv. Additional time periods placed in the fortified medium include any period between 0.1 and 240 hours, and may include, but are not limited to, 0.1-0.5 hours, 0.3-2.5 hours, 2.5-6 hours, 1-10 hours, 5- 15 hours, 10-20 hours, 15-25 hours, 20-30 hours, 25-35 hours, 30-40 hours, 35-45 hours, 40-50 hours, 45-55 hours, 50-60 hours, 55- 65 hours, 60-70 hours, 65-75 hours, 70-80 hours, 75-85 hours, 80-90 hours, 85-95 hours, 90-100 hours, 95-105 hours, 100-110 hours, 105- 115 hours, 110-120 hours, 115-125 hours, 120-130 hours, 125-135 hours, 130-140 hours, 135-145 hours, 140-150 hours, 145-155 hours, 150-160 hours, 155- 165 hours, 160-170 hours, 165-175 hours, 170-180 hours, 175-185 hours, 180-190 hours, 185-195 hours, 190-200 hours, 195-205 hours, 200-210 hours, 205- 215 hours, 210-220 hours, 215-225 hours, 220-230 hours, 225-235 hours, 230-240 hours, 235-245 hours, 240-250 hours, 3-6 hours, 7-17 hours, 12- 22 hours, 17-27 hours, 22-32 hours, 27-37 hours, 32-42 hours 37-47 hours, 42-52 hours, 47-57 hours, 52-62 hours, 57-67 hours, 62-72 hours, 67-77 hours, 72-82 hours, 77-87 hours, 82-92 hours, 87-97 hours, 92-102 hours, 97-107 hours, 102-112 hours, 107-117 hours, 112-122 hours, 117-127 hours, 122-132 hours, 127-137 hours, 132-142 hours, 137-147 hours, 142-152 hours, 147-157 hours, 152-162 hours, 157-167 hours, 162-172 hours, 167-177 hours, 172-182 hours, 177-187 hours, 162-172 hours, 167-177 hours, 182-192 hours, 187-197 hours, 192-202 hours, 197-207 hours, 202-212 hours, 207-217 hours, 212-222 hours, 217-227 hours, 222-232 hours, 227-237 hours, 232-242 hours, 237-247 hours or 242-252 hours. Placement in the fortified medium may also be 0.5 hours less than in standard or subtractive medium, 1 hour less than in standard or reduced medium, and 1 and 240 hours less than in standard or reduced medium. Any time. After the third cycle, the implants can be cleaned. The culture can be kept on a b-12c-iv supplemented with 7 g/L instead of the 5.5 g/L carrageenan provided above for a period of 10-120 days (usually a 21 day period) until more is observed Seedlings. Multiple shoots can be observed within 3-15 months. When multiple cycles are used, the external body can be cultured on standard medium for all cycles, followed by incubation of all media on a culture medium or on a medium of consolidation and subsequent incubation on a medium of reduction. Alternatively, the implant can be subjected to one or more treatments in any combination and sequence throughout the cycle.

在本文揭示的特定具體例中，培養時間為小於12週、小於9週或小於6週。 In a particular embodiment disclosed herein, the incubation time is less than 12 weeks, less than 9 weeks, or less than 6 weeks.

一旦培植體顯示出多個苗，可以將其或者維持在產生苗時的當前的培養基上(每10-120天轉移到新鮮的培養基)或者轉移到後續的培養基。非限制性的後續培養基包括但不限 於，pH為5.7的b-9培養基、CW1培養基、CW2培養基、CW3培養基、CW4培養基、CW5培養基、CW6培養基或b-6培養基，或它們的強化版本，隨後轉移到標準或減料培養基。可以將培養物保留在當前的或後續的培養基上，直到分離到新管和進一步擴展獲得期望數量的苗。一般地，時間範圍包括10-120天的週期(通常21天的週期)，在該週期之間可以安排培養物進行另一輪的增殖或轉移到下一個階段的培養基，例如「magenta箱」中pH為5.7的BR-2培養基10-120天(通常21天)或pH為5.7的Amel培養基10-120天(通常14-21天)。 Once the culture exhibits multiple shoots, it can be maintained either on the current medium at the time the seed is produced (transferred to fresh medium every 10 to 120 days) or transferred to subsequent media. Non-limiting follow-up media include but are not limited The b-9 medium, CW1 medium, CW2 medium, CW3 medium, CW4 medium, CW5 medium, CW6 medium or b-6 medium having a pH of 5.7, or an enhanced version thereof, is then transferred to a standard or reduced medium. The culture can be retained on the current or subsequent medium until a new tube is isolated and further expanded to obtain the desired number of shoots. Typically, the time frame includes a 10-120 day cycle (typically a 21 day cycle) during which the culture can be arranged for another round of proliferation or transfer to the next stage of the culture medium, such as the pH in the "magenta box". The A-2 medium of 5.7 BR-2 medium is 10-120 days (usually 21 days) or pH 5.7 for 10-120 days (usually 14-21 days).

本文揭示的特定具體例中，培養時間為小於12週、小於9週或小於6週。 In a particular embodiment disclosed herein, the incubation time is less than 12 weeks, less than 9 weeks, or less than 6 weeks.

在甚至更具體的非限制性具體例中，可以依據連續的段落[000198]-[0002-1]中描述的步驟在下列培養基(pH為5.5-5.7)中微體繁殖下列物種：大青籬竹(Arundinaria gigantea)：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；巴苦竹(Bambusa balcoa)：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；龍頭竹(Bambusa vulgaris)：b-9-v、CW1-v、CW3-v、 CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；黃金間碧竹(Bambusa vulgaris‘Vitatta’)：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；白綠竹：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；馬甲竹(Bambusa tulda)：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；勃氏甜龍竹(Dendrocalamus brandesii)：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；馬來甜龍竹：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；版納甜龍竹(Dendrocalamus hamiltoni)：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；龍竹(Dendrocalamus giganteus)：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；黃竹(Dendrocalamus membranaceus)：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；印度實竹(Dendrocalamus strictus)：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；菲律賓巨草竹(Gigantochloa aspera)：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；小黑竹(Gigantochloa scortechini)：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；瓜多竹(Guadua culeata)：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；尼加拉瓜瓜多竹(Guadua aculeata‘Nicaragua’)：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；抱葉瓜多竹(Guadua amplexifolia)：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本； 狹葉瓜多竹(Guadua angustifolia)：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；雙色狹葉瓜多竹(Guadua angustofolia bi-color)：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；圓錐瓜多竹(Guadua paniculata)：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；梨竹(Melocanna bambusoides)：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；李海竹(Neohouzeaua dullooa(Teinostachyum))：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；蘆葦竹(Ochlandra travancorica)：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；毛竹：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；紫竹：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、 B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；毛金竹(Phyllostachys nigra‘Henon’)：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本；思勞竹(Schizostachyum lumampao)：b-9-v、CW1-v、CW3-v、CW4-v、CW5-v、CW6-v、B-9N2-v、B-12C CPPU-v、B-12C DPU-v或其強化或減料版本。 In an even more specific, non-limiting embodiment, the following species can be micropropagated in the following medium (pH 5.5-5.7) according to the procedure described in consecutive paragraphs [000198]-[0002-1]: Large Green Fence Arundinaria gigantea: b-9-v, CW1-v, CW3-v, CW4-v, CW5-v, CW6-v, B-9N2-v, B-12C CPPU-v, B-12C DPU- v or its enhanced or reduced version; Bambusa balcoa: b-9-v, CW1-v, CW3-v, CW4-v, CW5-v, CW6-v, B-9N2-v, B- 12C CPPU-v, B-12C DPU-v or its enhanced or reduced version; Bambusa vulgaris: b-9-v, CW1-v, CW3-v, CW4-v, CW5-v, CW6-v, B-9N2-v, B-12C CPPU-v, B-12C DPU-v or its enhanced or reduced version; Bambusa vulgaris 'Vitatta' :b-9-v, CW1-v, CW3-v, CW4-v, CW5-v, CW6-v, B-9N2-v, B-12C CPPU-v, B-12C DPU-v or its strengthening or Reduced version; white green bamboo: b-9-v, CW1-v, CW3-v, CW4-v, CW5-v, CW6-v, B-9N2-v, B-12C CPPU-v, B-12C DPU-v or its enhanced or reduced version; Bambusa tulda: b-9-v, CW1-v, CW3-v, CW4-v, CW5-v, CW6-v, B-9N2-v, B-12C CPPU-v, B-12C DPU-v or its enhanced or reduced version; Dendrocalamus brandesii: b-9-v, CW1-v, CW3-v, CW4-v, CW5 -v, CW6-v, B-9N2-v, B-12C CPPU-v, B-12C DPU-v or its enhanced or reduced version; Malay Sweet Dragon Bamboo: b-9-v, CW1-v, CW3-v, CW4-v, CW5-v, CW6-v, B-9N2-v, B-12C CPPU-v, B-12C DPU-v or its enhanced or reduced version; Dendrocalamus hamiltoni ): b-9-v, CW1-v, CW3-v, CW4-v, CW5-v, CW6-v, B-9N2-v, B-12C CPPU-v, B-12C DPU-v or its enhancement Or reduced version; Dragon Bamboo (Dendrocalamus giganteus): B-9-v, CW1-v, CW3-v, CW4-v, CW5-v, CW6-v, B-9N2-v, B-12C CPPU-v, B-12C DPU-v or its enhanced or reduced version; Dendrocalamus membranaceus: b-9-v, CW1-v, CW3-v, CW4-v, CW5-v, CW6-v, B-9N2-v, B-12C CPPU-v, B-12C DPU-v or its enhanced or reduced version; Dendrocalamus strictus: b-9-v, CW1-v, CW3-v, CW4-v, CW5-v , CW6-v, B-9N2-v, B-12C CPPU-v, B-12C DPU-v or its enhanced or reduced version; Gigantochloa aspera: b-9-v, CW1-v , CW3-v, CW4-v, CW5-v, CW6-v, B-9N2-v, B-12C CPPU-v, B-12C DPU-v or its enhanced or reduced version; small black bamboo (Gigantochloa scortechini ): b-9-v, CW1-v, CW3-v, CW4-v, CW5-v, CW6-v, B-9N2-v, B-12C CPPU-v, B-12C DPU-v or its enhancement Or reduced version; Guadua culeata: b-9-v, CW1-v, CW3-v, CW4-v, CW5-v, CW6-v, B-9N2-v, B-12C CPPU- v, B-12C DPU-v or its enhanced or reduced version; Guadua aculeata 'Nicaragua': b-9-v, CW1-v, CW3-v, CW4-v, CW5-v, CW6-v, B-9N2-v, B-12C CPPU-v, B-12C DPU-v or its enhanced or reduced version; Guadua amplexifolia: b- 9-v, CW1-v, CW3-v, CW4-v, CW5-v, CW6-v, B-9N2-v, B-12C CPPU-v, B-12C DPU-v or its enhanced or reduced version ; Guadua angustifolia: b-9-v, CW1-v, CW3-v, CW4-v, CW5-v, CW6-v, B-9N2-v, B-12C CPPU-v, B -12C DPU-v or its enhanced or reduced version; Guadua angustofolia bi-color: b-9-v, CW1-v, CW3-v, CW4-v, CW5-v, CW6 -v, B-9N2-v, B-12C CPPU-v, B-12C DPU-v or its enhanced or reduced version; Guadua paniculata: b-9-v, CW1-v, CW3 -v, CW4-v, CW5-v, CW6-v, B-9N2-v, B-12C CPPU-v, B-12C DPU-v or its fortified or reduced version; Melocanna bambusoides: b -9-v, CW1-v, CW3-v, CW4-v, CW5-v, CW6-v, B-9N2-v, B-12C CPPU-v, B-12C DPU-v or its strengthening or subtractive Version; Neohouzeaua dullooa (Teinostachyum): b-9-v, CW1-v, CW3-v, CW4-v, CW5-v, CW6-v, B-9N2-v, B-12C CPPU-v, B-12C DPU-v or its enhanced or reduced version; Ochlandra travancorica: b-9-v, CW1-v, CW3-v, CW4-v, CW5-v, CW6-v, B-9N2 -v, B-12C CPPU-v, B-12C DPU-v or its enhanced or reduced version; Bamboo: b-9-v, CW1-v, CW3-v, CW4-v, CW5-v, CW6- v, B-9N2-v, B-1 2C CPPU-v, B-12C DPU-v or its enhanced or reduced version; Zizhu: b-9-v, CW1-v, CW3-v, CW4-v, CW5-v, CW6-v, B-9N2-v, B-12C CPPU-v, B-12C DPU-v or its enhanced or reduced version; Phyllostachys nigra'Henon': b-9-v, CW1-v, CW3-v , CW4-v, CW5-v, CW6-v, B-9N2-v, B-12C CPPU-v, B-12C DPU-v or its enhanced or reduced version; Schizostachyum lumampao: b- 9-v, CW1-v, CW3-v, CW4-v, CW5-v, CW6-v, B-9N2-v, B-12C CPPU-v, B-12C DPU-v or its enhanced or reduced version .

在一些具體例中，所述方法包括使用本發明的生物反應器。在一些具體例中，所述方法包括使用本發明的架。在一些具體例中，所述方法包括使用本發明的生物反應器和架。在一些具體例中，當在微體繁殖中交替使用兩種或更多種上述培養基時，可以使用本文中描述的生物反應器，例如，當交替使用階段1和階段2培養基時，當交替使用階段3和階段4培養基時，和/或當交替使用階段1、階段2和階段3培養基時等。 In some embodiments, the method comprises using a bioreactor of the invention. In some embodiments, the method includes the use of a rack of the present invention. In some embodiments, the method includes the use of the bioreactor and rack of the present invention. In some embodiments, when two or more of the above mediums are alternately used in micropropagation, the bioreactor described herein may be used, for example, when alternating phase 1 and phase 2 media are used alternately. Stage 3 and Stage 4 media, and/or when Phase 1, Stage 2 and Stage 3 media are used alternately.

(套組) (set)

本發明還提供用於植物繁殖的套組。在一些具體例中，所述套組包括一種或多種本發明的培養基。在一些具體例中，所述套組包括植物物種的一種或多種培植體。 The invention also provides kits for plant propagation. In some embodiments, the kit comprises one or more media of the invention. In some embodiments, the kit comprises one or more implants of a plant species.

例如，套組可包括一種或多種芽誘導培養基、苗伸長/維持培養基、b-9-i培養基、b-9-ii培養基、b-9-iii培養基、b-9-iv 培養基、b-9-v培養基、強化b-9-i培養基、強化b-9-ii培養基、強化b-9-iii培養基、強化b-9-iv培養基、強化b-9-v培養基、減料b-9-i培養基(下述減料培養基)、減料b-9-ii培養基、減料b-9-iii培養基、減料b-9-iv培養基、減料b-9-v培養基、CW2-i培養基、CW2-ii培養基、CW2-iii培養基、CW2-iv培養基、CW2-v培養基、強化CW2-i培養基、強化CW2-ii培養基、強化CW2-iii培養基、強化CW2-iv培養基、強化CW2-v培養基、減料CW2-i培養基、減料CW2-ii培養基、減料CW2-iii培養基、減料CW2-iv培養基、減料CW2-v培養基、b-10-i培養基、b-10-ii培養基、b-10-iii培養基、b-10-iv培養基、b-10-v培養基、強化b-10-i培養基、強化b-10-ii培養基、強化b-10-iii培養基、強化b-10-iv培養基、強化b-10-v培養基、減料b-10-i培養基、減料b-10-ii培養基、減料b-10-iii培養基、減料b-10-iv培養基、減料b-10-v培養基、b-11-i培養基、b-11-ii培養基、b-11-iii培養基、b-11-iv培養基、b-11-v培養基、強化b-11-i培養基、強化b-11-ii培養基、強化b-11-iii培養基、強化b-11-iv培養基、強化b-11-v培養基、減料b-11-i培養基、減料b-11-ii培養基、減料b-11-iii培養基、減料b-11-iv培養基、減料b-11-v培養基、b-12c-i培養基、b-12c-ii培養基、b-12c-iii培養基、b-12c-iv培養基、b-12c-v培養基、強化b-12c-i培養基、強化b-12c-ii培養基、強化b-12c-iii培養基、強化b-12c-iv培 養基、強化b-12c-v培養基、減料b-12c-i培養基、減料b-12c-ii培養基、減料b-12c-iii培養基、減料b-12c-iv培養基、減料b-12c-v培養基、b-1-i培養基、b-1-ii培養基、b-1-iii培養基、b-1-iv培養基、b-1-v培養基、強化b-1-i培養基、強化b-1-ii培養基、強化b-1-iii培養基、強化b-1-iv培養基、強化b-1-v培養基、減料b-1-i培養基、減料b-1-ii培養基、減料b-1-iii培養基、減料b-1-iv培養基、減料b-1-v培養基、b-4-i培養基、b-4-ii培養基、b-4-iii培養基、b-4-iv培養基、b-4-v培養基、強化b-4-i培養基、強化b-4-ii培養基、強化b-4-iii培養基、強化b-4-iv培養基、強化b-4-v培養基、減料b-4-i培養基、減料b-4-ii培養基、減料b-4-iii培養基、減料b-4-iv培養基、減料b-4-v培養基、b-6-i培養基、b-6-ii培養基、b-6-iii培養基、b-6-iv培養基、b-6-v培養基、強化b-6-i培養基、強化b-6-ii培養基、強化b-6-iii培養基、強化b-6-iv培養基、強化b-6-v培養基、減料b-6-i培養基、減料b-6-ii培養基、減料b-6-iii培養基、減料b-6-iv培養基、減料b-6-v培養基、CW1-i培養基、CW1-ii培養基、CW1-iii培養基、CW1-iv培養基、CW1-v培養基、強化CW1-i培養基、強化CW1-ii培養基、強化CW1-iii培養基、強化CW1-iv培養基、強化CW1-v培養基、減料CW1-i培養基、減料CW1-ii培養基、減料CW1-iii培養基、減料CW1-iv培養基、減料CW1-v培養基、CW3-i培養基、CW3-ii培養基、CW3-iii培養基、 CW3-iv培養基、CW3-v培養基、強化CW3-i培養基、強化CW3-ii培養基、強化CW3-iii培養基、強化CW3-iv培養基、強化CW3-v培養基、減料CW3-i培養基、減料CW3-ii培養基、減料CW3-iii培養基、減料CW3-iv培養基、減料CW3-v培養基、CW4-i培養基、CW4-ii培養基、CW4-iii培養基、CW4-iv培養基、CW4-v培養基、強化CW4-i培養基、強化CW4-ii培養基、強化CW4-iii培養基、強化CW4-iv培養基、強化CW4-v培養基、減料CW4-i培養基、減料CW4-ii培養基、減料CW4-iii培養基、減料CW4-iv培養基、減料CW4-v培養基、CW5-i培養基、CW5-ii培養基、CW5-iii培養基、CW5-iv培養基、CW5-v培養基、強化CW5-i培養基、強化CW5-ii培養基、強化CW5-iii培養基、強化CW5-iv培養基、強化CW5-v培養基、減料CW5-i培養基、減料CW5-ii培養基、減料CW5-iii培養基、減料CW5-iv培養基、減料CW5-v培養基、CW6-i培養基、CW6-ii培養基、CW6-iii培養基、CW6-iv培養基、CW6-v培養基、強化CW6-i培養基、強化CW6-ii培養基、強化CW6-iii培養基、強化CW6-iv培養基、強化CW6-v培養基、減料CW6-i培養基、減料CW6-ii培養基、減料CW6-iii培養基、減料CW6-iv培養基、減料CW6-v培養基、B-9N2-i培養基、B-9N2-ii培養基、B-9N2-iii培養基、B-9N2-iv培養基、B-9N2-v培養基、強化B-9N2-i培養基、強化B-9N2-ii培養基、強化B-9N2-iii培養基、強化 B-9N2-iv培養基、強化B-9N2-v培養基、減料B-9N2-i培養基、減料B-9N2-ii培養基、減料的B-9N2-iii培養基、減料B-9N2-iv培養基、減料B-9N2-v培養基、B-12C CPPU-i培養基、B-12C CPPU-ii培養基、B-12C CPPU-iii培養基、B-12C CPPU-iv培養基、B-12C CPPU-v培養基、強化B-12C CPPU-i培養基、強化B-12C CPPU-ii培養基、強化B-12C CPPU-iii培養基、強化B-12C CPPU-iv培養基、強化B-12C CPPU-v培養基、減料B-12C CPPU-i培養基、減料B-12C CPPU-ii培養基、減料B-12C CPPU-iii培養基、減料B-12C CPPU-iv培養基、減料B-12C CPPU-v培養基、B-12C DPU-i培養基、B-12C DPU-ii培養基、B-12C DPU-iii培養基、B-12C DPU-iv培養基、B-12C DPU-v培養基、強化B-12C DPU-i培養基、強化B-12C DPU-ii培養基、強化B-12C DPU-iii培養基、強化B-12C DPU-iv培養基、強化B-12C DPU-v培養基、減料B-12C DPU-i培養基、減料B-12C DPU-ii培養基、減料B-12C DPU-iii培養基、減料B-12C DPU-iv培養基、減料B-12C DPU-v培養基、Br-2-i培養基、Br-2-ii培養基、Br-2-iii培養基、Br-2-iv培養基、Br-2-v培養基、強化Br-2-i培養基、強化Br-2-ii培養基、強化Br-2-iii培養基、強化Br-2-iv培養基、強化Br-2-v培養基、減料Br-2-i培養基、減料Br-2-ii培養基、減料Br-2-iii培養基、減料Br-2-iv培養基、減料Br-2-v培養基、Ech-i培養基、Ech-ii培養基、Ech-iii培養 基、Ech-iv培養基、Ech-v培養基、強化Ech-i培養基、強化Ech-ii培養基、強化Ech-iii培養基、強化Ech-iv培養基、強化Ech-v培養基、減料Ech-i培養基、減料Ech-ii培養基、減料Ech-iii培養基、減料Ech-iv培養基、減料Ech-v培養基、Amel-i培養基、Amel-ii培養基、Amel-iii培養基、Amel-iv培養基、Amel-v培養基、強化Amel-i培養基、強化Amel-ii培養基、強化Amel-iii培養基、強化Amel-iv培養基、強化Amel-v培養基、減料Amel-i培養基、減料Amel-ii培養基、減料Amel-iii培養基、減料Amel-iv培養基、及/或減料Amel-v培養基。在另外的具體例中，所述套組可包含一種或多種用於組織培養過程的容器，其包括但不限於，管、罐、箱、壺、杯、無菌袋技術、生物反應器、暫時的浸漬器皿等。在另一個具體例中，所述套組可包含用於竹的組織培養的說明書。在另一個具體例中，所述套組包含前述的組合。在相同或不同的容器中，可以發現各種套組的元件。此外，當供應套組時，可將培養基的不同組分包裝在分別的容器中並在使用前立即混合。如此分別包裝組分可允許長期的儲存，而不損失活性組分的功能。或者，可以預先混合地提供培養基。 For example, the kit can include one or more shoot induction medium, shoot elongation/maintenance medium, b-9-i medium, b-9-ii medium, b-9-iii medium, b-9-iv Medium, b-9-v medium, fortified b-9-i medium, fortified b-9-ii medium, fortified b-9-iii medium, fortified b-9-iv medium, fortified b-9-v medium, reduced Material b-9-i medium (reduced medium below), reduced b-9-ii medium, reduced b-9-iii medium, reduced b-9-iv medium, reduced b-9-v medium , CW2-i medium, CW2-ii medium, CW2-iii medium, CW2-iv medium, CW2-v medium, fortified CW2-i medium, fortified CW2-ii medium, fortified CW2-iii medium, fortified CW2-iv medium, Strengthen CW2-v medium, reduce CW2-i medium, reduce CW2-ii medium, reduce CW2-iii medium, reduce CW2-iv medium, reduce CW2-v medium, b-10-i medium, b- 10-ii medium, b-10-iii medium, b-10-iv medium, b-10-v medium, fortified b-10-i medium, fortified b-10-ii medium, fortified b-10-iii medium, Strengthen b-10-iv medium, strengthen b-10-v medium, reduce b-10-i medium, reduce b-10-ii medium, reduce b-10-iii medium, reduce b-10-iv Medium, reduced b-10-v medium, b-11-i medium, b-11-ii medium, b-11 -iii medium, b-11-iv medium, b-11-v medium, fortified b-11-i medium, fortified b-11-ii medium, fortified b-11-iii medium, fortified b-11-iv medium, Strengthen b-11-v medium, reduce b-11-i medium, reduce b-11-ii medium, reduce b-11-iii medium, reduce b-11-iv medium, reduce b-11- v medium, b-12c-i medium, b-12c-ii medium, b-12c-iii medium, b-12c-iv medium, b-12c-v medium, fortified b-12c-i medium, fortified b-12c -ii medium, fortified b-12c-iii medium, fortified b-12c-iv culture Nutrient, fortified b-12c-v medium, reduced b-12c-i medium, reduced b-12c-ii medium, reduced b-12c-iii medium, reduced b-12c-iv medium, reduced b -12c-v medium, b-1-i medium, b-1-ii medium, b-1-iii medium, b-1-iv medium, b-1-v medium, enhanced b-1-i medium, fortification B-1-ii medium, fortified b-1-iii medium, fortified b-1-iv medium, fortified b-1-v medium, reduced b-1-i medium, reduced b-1-ii medium, reduced Feed b-1-iii medium, reduced b-1-iv medium, reduced b-1-v medium, b-4-i medium, b-4-ii medium, b-4-iii medium, b-4 -iv medium, b-4-v medium, fortified b-4-i medium, fortified b-4-ii medium, fortified b-4-iii medium, fortified b-4-iv medium, fortified b-4-v medium , reduced b-4-i medium, reduced b-4-ii medium, reduced b-4-iii medium, reduced b-4-iv medium, reduced b-4-v medium, b-6- i medium, b-6-ii medium, b-6-iii medium, b-6-iv medium, b-6-v medium, fortified b-6-i medium, fortified b-6-ii medium, fortified b- 6-iii medium, fortified b-6-iv medium Strengthen b-6-v medium, reduce b-6-i medium, reduce b-6-ii medium, reduce b-6-iii medium, reduce b-6-iv medium, reduce b-6- v medium, CW1-i medium, CW1-ii medium, CW1-iii medium, CW1-iv medium, CW1-v medium, fortified CW1-i medium, fortified CW1-ii medium, fortified CW1-iii medium, fortified CW1-iv Medium, fortified CW1-v medium, reduced CW1-i medium, reduced CW1-ii medium, reduced CW1-iii medium, reduced CW1-iv medium, reduced CW1-v medium, CW3-i medium, CW3- Ii medium, CW3-iii medium, CW3-iv medium, CW3-v medium, fortified CW3-i medium, fortified CW3-ii medium, fortified CW3-iii medium, fortified CW3-iv medium, fortified CW3-v medium, reduced CW3-i medium, reduced material CW3 -ii medium, reduced CW3-iii medium, reduced CW3-iv medium, reduced CW3-v medium, CW4-i medium, CW4-ii medium, CW4-iii medium, CW4-iv medium, CW4-v medium, Strengthen CW4-i medium, strengthen CW4-ii medium, strengthen CW4-iii medium, strengthen CW4-iv medium, strengthen CW4-v medium, reduce CW4-i medium, reduce CW4-ii medium, reduce CW4-iii medium , reduced CW4-iv medium, reduced CW4-v medium, CW5-i medium, CW5-ii medium, CW5-iii medium, CW5-iv medium, CW5-v medium, fortified CW5-i medium, fortified CW5-ii Medium, fortified CW5-iii medium, fortified CW5-iv medium, fortified CW5-v medium, reduced CW5-i medium, reduced CW5-ii medium, reduced CW5-iii medium, reduced CW5-iv medium, reduced material CW5-v medium, CW6-i medium, CW6-ii medium, CW6-iii medium, CW6-iv medium, C W6-v medium, fortified CW6-i medium, fortified CW6-ii medium, fortified CW6-iii medium, fortified CW6-iv medium, fortified CW6-v medium, reduced CW6-i medium, reduced material CW6-ii medium, reduced CW6-iii medium, reduced CW6-iv medium, reduced CW6-v medium, B-9N2-i medium, B-9N2-ii medium, B-9N2-iii medium, B-9N2-iv medium, B- 9N2-v medium, fortified B-9N2-i medium, fortified B-9N2-ii medium, fortified B-9N2-iii medium, fortified B-9N2-iv medium, fortified B-9N2-v medium, reduced B-9N2-i medium, reduced B-9N2-ii medium, reduced B-9N2-iii medium, reduced material B-9N2-iv Medium, reduced B-9N2-v medium, B-12C CPPU-i medium, B-12C CPPU-ii medium, B-12C CPPU-iii medium, B-12C CPPU-iv medium, B-12C CPPU-v medium , strengthen B-12C CPPU-i medium, strengthen B-12C CPPU-ii medium, strengthen B-12C CPPU-iii medium, strengthen B-12C CPPU-iv medium, strengthen B-12C CPPU-v medium, reduce material B- 12C CPPU-i medium, reduced material B-12C CPPU-ii medium, reduced material B-12C CPPU-iii medium, reduced material B-12C CPPU-iv medium, reduced material B-12C CPPU-v medium, B-12C DPU -i medium, B-12C DPU-ii medium, B-12C DPU-iii medium, B-12C DPU-iv medium, B-12C DPU-v medium, fortified B-12C DPU-i medium, fortified B-12C DPU -ii medium, fortified B-12C DPU-iii medium, fortified B-12C DPU-iv medium, fortified B-12C DPU-v medium, reduced B-12C DPU-i medium, reduced material B-12C DPU-ii medium , reduce B-12C DPU-iii medium, reduce B-12C DPU-iv medium, reduce B-12C DPU-v medium, Br-2-i medium, Br-2-ii medium, Br-2-iii medium, Br-2-iv medium, Br-2-v medium, enhanced Br-2-i Medium, fortified Br-2-ii medium, fortified Br-2-iii medium, fortified Br-2-iv medium, fortified Br-2-v medium, reduced Br-2-i medium, reduced material Br-2-ii Medium, reduced Br-2-iii medium, reduced Br-2-iv medium, reduced Br-2-v medium, Ech-i medium, Ech-ii medium, Ech-iii culture Base, Ech-iv medium, Ech-v medium, fortified Ech-i medium, fortified Ech-ii medium, fortified Ech-iii medium, fortified Ech-iv medium, fortified Ech-v medium, for reduced Ech-i medium, minus Ech-ii medium, reduced Ech-iii medium, reduced Ech-iv medium, reduced Ech-v medium, Amel-i medium, Amel-ii medium, Amel-iii medium, Amel-iv medium, Amel-v Medium, fortified Amel-i medium, fortified Amel-ii medium, fortified Amel-iii medium, fortified Amel-iv medium, fortified Amel-v medium, reduced Amel-i medium, reduced Amel-ii medium, reduced Amel- Iii medium, reduced Amel-iv medium, and/or reduced Amel-v medium. In further embodiments, the kit can include one or more containers for a tissue culture process including, but not limited to, tubes, cans, boxes, jugs, cups, sterile bag technology, bioreactors, temporary Impregnation vessels, etc. In another embodiment, the kit can include instructions for tissue culture of bamboo. In another embodiment, the kit comprises the aforementioned combination. In the same or different containers, the components of the various sets can be found. In addition, when the kit is supplied, the different components of the medium can be packaged in separate containers and mixed immediately prior to use. Such separate packaging components allow for long term storage without loss of function of the active component. Alternatively, the medium may be supplied in advance.

藉由下列不應理解為限制的實例進一步說明本發明。所有本應用中引用的所有參考文獻、專利和公開專利申請的內容以及附圖，以引用方式全部併入本文以用於所有目的。 The invention is further illustrated by the following examples which are not to be construed as limiting. The contents of all of the references, patents and published patent applications cited in this application, and the drawings are hereby incorporated by reference in its entirety for all purposes.

[實施例] [Examples] (實施例1) (Example 1) 毛竹以階段1至階段5培養基的微體繁殖-IMicro-propagation of Phyllostachys pubescens L. with Stage 1 to Stage 5 medium-I

以約3月齡和約3年齡之間的竹植物開始，將來自側苗剛破鞘的有節莖的節用作培植體。將每個節段切成3-5毫米的段及完整的苗。將一些培植體，包括取自1年或更長時間的有節莖的培植體，在70%的異丙醇的罐中搖動3秒以預先沖洗，隨後在流動的自來水中沖洗1分鐘。其他的培植體不預先沖洗。 Starting from a bamboo plant between about 3 months old and about 3 years old, the stalked knot from the side seedling has been used as a culture body. Cut each segment into 3-5 mm segments and complete seedlings. Some of the cultures, including stems from 1 year or longer, were shaken in a 70% isopropanol jar for 3 seconds to pre-rinse and then rinsed in running tap water for 1 minute. Other implants are not rinsed in advance.

將外鞘剝去並丟棄，而且將剩餘的節段部分置於10%的漂白溶液中。將漂白溶液中的培植體在可調速Lab Rotators，Barnstead/Lab line orbital Shaker(模型號KS 260)搖動臺上以每分鐘6-9轉放置1小時。對於一些培植體，包括取自從1年齡或更長的有節莖者，修改步驟而於10%的漂白溶液加入數滴Tween 20並將該培植體浸漬45分鐘而非1小時。隨後將所述培植體放置於1%的漂白溶液中，並放回到搖動臺上30分鐘。隨後重複該1%的漂白溶液步驟並可隨後用無菌的蒸餾水沖洗。 The outer sheath was peeled off and discarded, and the remaining segment was placed in a 10% bleach solution. The cultures in the bleach solution were placed on a rocking table at a variable speed Lab Rotators, Barnstead/Lab line orbital Shaker (model number KS 260) for 6 hours at 6-9 revolutions per minute. For some implants, including those with stems from 1 year or longer, the procedure was modified to add a few drops of Tween 20 to 10% of the bleach solution and the implant was immersed for 45 minutes instead of 1 hour. The culture was then placed in a 1% bleach solution and returned to the shaking table for 30 minutes. This 1% bleach solution step is then repeated and can then be rinsed with sterile distilled water.

隨後將單個的培植體置於管中階段1培養基上(15-25 mL)，並且將該管置於溫度從65℉-70℉和全光譜光度36-90 μmole/m²/s²的受調控清潔生長室。在本實施例中，階段1培養基是強化b-12c-iv或如實施例10中描述。這些外植體在強化b-12c-iv培養基上保留2個8-118天的週期(通常12 天的週期)。在週期之間將多餘的鞘移除。當轉移到第三個週期時，培植體被轉移到階段2培養基，在本實施例中，是用0.5-3 g/L(最佳是2 g/L)的酪蛋白羥基化物補充的強化b-12c-iv。在第三個週期之後，清理該培植體。將培植體保留在用0.5-3 g/L的酪蛋白羥基化物補充的強化b-12c-iv上進行8-118天的週期(通常12天的週期)，直到觀察到多個苗。一般在3-15個月內觀察到多個苗。 The individual cultures were then placed on stage 1 medium (15-25 mL) in the tube and placed at a temperature of 65-F to 70 °F and a full-spectrum luminosity of 36-90 μmole/m ² /s ² Regulate the clean growth chamber. In this example, the Stage 1 medium is fortified b-12c-iv or as described in Example 10. These explants retained two 8-118 day cycles (usually a 12-day cycle) on the fortified b-12c-iv medium. The excess sheath is removed between cycles. When transferred to the third cycle, the culture body is transferred to the Stage 2 medium, in this example, with a 0.5-3 g/L (preferably 2 g/L) casein hydroxylate supplementation b. -12c-iv. After the third cycle, the culture is cleaned. The cultures were retained on the fortified b-12c-iv supplemented with 0.5-3 g/L of casein hydroxylate for a period of 8-118 days (typically a 12 day cycle) until multiple shoots were observed. Multiple shoots are generally observed within 3-15 months.

一旦所述培植體顯示多個苗，將其轉移到在本實施例中是pH為5.7的減料B-9N2-iv的階段3培養基進行10-120天的週期(通常21天的週期)。將苗在該階段3培養基和在本實施例中為減料B-9N2-iv液體(除去瓊脂)的階段4培養基之間交替以用於連續交替的10-120天的週期(通常21天的週期)，直到通過分離到新管和進一步的擴展獲得期望數量的苗。一般每個繁殖週期每管獲得1-10個苗。隨後將該苗置於階段5培養基，在本實施例中是pH 5.7標準Amel-iv進行10-120天(通常14-21天)。 Once the culture showed multiple shoots, it was transferred to Stage 3 medium, which was a reduced material B-9N2-iv having a pH of 5.7 in this example, for a period of 10-120 days (typically a 21 day cycle). The shoots are alternated between the Stage 3 medium and the Stage 4 medium, which in this example is a reduced B-9N2-iv liquid (removed agar) for a continuous alternating 10-120 day cycle (usually 21 days) Cycle) until the desired number of shoots are obtained by separation into new tubes and further expansion. Generally, 1-10 seedlings are obtained per tube per breeding cycle. The shoot is then placed in Stage 5 medium, in this example pH 5.7 standard Amel-iv for 10-120 days (typically 14-21 days).

(實施例2) (Example 2) 毛竹以階段1至階段5培養基微體繁殖-IIPhyllostachys pubescens with medium 1 to stage 5 medium micropropagation - II

如實施例1選擇培植體並對其消毒。隨後將單個培植體置於管中階段1培養基上(15-25 mL)，並且將該管置於溫度從65℉-70℉和全光譜光度36-90 μmole/m²/s²的受調控清潔生長室。在本實施例中，階段1培養基是強化b-12c-iii或如實 施例10中描述。這些培植體在強化b-12c-iii培養基上保留2個10-120天的週期(通常14天的週期)(注意：在本實施例中，儘管培養基強化，時間並未縮短，因為b-12c-iii被視為弱強化培養基)。在週期之間移除多餘的鞘。當轉移到第三個週期時，將培植體轉移到階段2培養基，在本實施例中是減料B-9N2-iii。在1或2個10-120天的週期(一般14天的週期)之後，清理該培植體並將其放回到階段1培養基以進行額外的1或2個10-120天的週期(通常14天的週期)。將該培植體保留在交替的階段1和階段2培養基上，直到觀察到多個苗。一般在3-15個月之內觀察到多個苗。 The culture body was selected and sterilized as in Example 1. Subsequently, a single culture body was placed on the stage 1 medium (15-25 mL) in the tube, and the tube was placed at a temperature ranging from 65 °F to 70 °F and the full spectrum luminosity was 36-90 μmole/m ² /s ² . Clean the growth chamber. In this example, the Stage 1 medium is fortified b-12c-iii or as described in Example 10. These cultures retained two 10-120 day cycles (usually a 14-day cycle) on the fortified b-12c-iii medium (note: in this example, although the medium was fortified, the time was not shortened because b-12c -iii is considered a weak booster medium). Remove excess sheath between cycles. When transferred to the third cycle, the culture was transferred to Stage 2 medium, which in this example was reduced B-9N2-iii. After 1 or 2 10-120 day cycles (typically a 14-day cycle), the culture is cleared and returned to Stage 1 medium for an additional 1 or 2 10-120 day cycles (usually 14 The cycle of the day). The cultures were kept on alternating Phase 1 and Stage 2 media until multiple shoots were observed. Multiple shoots are generally observed within 3-15 months.

一旦培植體顯示多個苗，其被轉移到階段3培養基，在本實施例中是pH為5.7的減料B-9N2-iii進行10-120天的週期(通常21天的週期)。在該階段3培養基和在本實施例中為減料B-9N2-iii液體(除去瓊脂)的階段4培養基之間，以連續交替的10-120天週期(通常21天的週期)交替苗，直到分離到新管並進一步擴展觀察到期望數量的苗。一般每個增殖週期每管獲得1-10個苗。隨後將苗置於階段5培養基，在本實施例中是pH為5.7的標準Amel-iv進行10-120天(通常14-21天)。 Once the culture shows multiple shoots, it is transferred to Stage 3 medium, in this example a reduced feed B-9N2-iii with a pH of 5.7 for a period of 10-120 days (typically a 21 day period). Between the stage 3 medium and the stage 4 medium which in this example is a reduced B-9N2-iii liquid (removed agar), the seedlings are alternated in successively alternating 10-120 day cycles (usually 21 day cycles). Until the new tube is separated and further expanded to observe the desired number of seedlings. Generally, 1-10 seedlings are obtained per tube per proliferation cycle. The shoots are then placed in stage 5 medium, in this example a standard Amel-iv having a pH of 5.7 for 10-120 days (typically 14-21 days).

(實施例3) (Example 3) 白綠竹以階段1、階段2和階段3培養基微體繁殖White-green bamboo with medium 1, stage 2 and stage 3 medium micro-propagation

如實施例1選擇培植體並對其消毒。隨後將單個培植體置於管中階段1培養基上(15-25 mL)，並且將該管置於溫度從 65℉-70℉和全光譜光度36-90 μmole/m²/s²的受調控清潔生長室。在本實施例中，階段1培養基是強化b-12c-ii或如實施例10中描述。這些培植體在強化b-12c-ii培養基上保留2個8-118天的週期(通常12天的週期)。在週期之間除去多餘的鞘。將培植體保留在迴圈的強化b-12c-ii培養基上，直到觀察到多個苗。一般3-15個月之內觀察到多個苗。 The culture body was selected and sterilized as in Example 1. Subsequently, a single culture body was placed on the stage 1 medium (15-25 mL) in the tube, and the tube was placed at a temperature ranging from 65 °F to 70 °F and the full spectrum luminosity was 36-90 μmole/m ² /s ² . Clean the growth chamber. In this example, the Stage 1 medium is fortified b-12c-ii or as described in Example 10. These cultures retained two 8-118 day cycles (usually a 12-day cycle) on the fortified b-12c-ii medium. Remove excess sheath between cycles. The cultures were kept on the looped fortified b-12c-ii medium until multiple shoots were observed. Multiple seedlings are generally observed within 3-15 months.

一旦培植體顯示多個苗，其被轉移到階段2培養基，在本實施例中為pH為5.7的減料b-10-iv，10-120天的週期(通常21天的週期)。在減料b-10-iv培養基上以連續交替的10-120天的週期(通常21天的週期)保留苗，直到分離到新管並進一步擴展觀察到期望數量的苗。一般每個增殖週期每管獲得1-10個苗。隨後將苗置於階段3培養基，在本實施例中，是pH為5.7的標準Amel-iv進行10-120天(通常14-21天)。 Once the culture shows multiple shoots, it is transferred to Stage 2 medium, in this example a b-10-iv with a pH of 5.7, a 10-120 day cycle (typically a 21 day cycle). Seedlings were maintained on reduced b-10-iv medium in successively alternating 10-120 day cycles (typically 21 day cycles) until new tubes were isolated and further expanded to observe the desired number of shoots. Generally, 1-10 seedlings are obtained per tube per proliferation cycle. The shoots are then placed in Stage 3 medium, in this example, a standard Amel-iv having a pH of 5.7 for 10-120 days (typically 14-21 days).

(實施例4) (Example 4) 毛竹以階段1至階段4培養基微體繁殖Phyllostachys pubescens with medium 1 to stage 4 medium micropropagation

如實施例1選擇培植體並對其消毒。隨後將單個培植體置於管中階段1培養基上(15-25 mL)，並且將該管置於溫度從65℉-70℉和全光譜光度200-500 μmole/m²/s²的受調控清潔生長室。在本實施例中，階段1培養基是強化b-12c-iv或如實施例10中描述。這些培植體在強化b-12c-iv培養基上保留2個8-118天的週期(通常12天的週期)。在週期之間除去 多餘的鞘。將培植體保留在迴圈的強化b-12c-ii培養基上，直到觀察到多個苗。一般3-15個月之內觀察到多個苗。 The culture body was selected and sterilized as in Example 1. Subsequently, a single culture body was placed on the stage 1 medium (15-25 mL) in the tube, and the tube was placed at a temperature ranging from 65 °F to 70 °F and the full spectrum luminosity was 200-500 μmole/m ² /s ² . Clean the growth chamber. In this example, the Stage 1 medium is fortified b-12c-iv or as described in Example 10. These cultures retained two 8-118 day cycles (usually a 12 day cycle) on the fortified b-12c-iv medium. Remove excess sheath between cycles. The cultures were kept on the looped fortified b-12c-ii medium until multiple shoots were observed. Multiple seedlings are generally observed within 3-15 months.

一旦培植體顯示多個苗，其被轉移到階段2培養基，在本實施例中為pH為5.7的減料B-9N2-iv，10-120天的週期(通常21天的週期)。在該階段2培養基和在本實施例中為強化b-11-iv的階段3培養基之間，以連續交替的10-120天的週期(通常21天的週期)交替苗，直到分離到新管並進一步擴展獲得期望數量的苗。一般每個增殖週期每管獲得1-10個苗。隨後將苗置於階段4培養基，在本實施例中，是pH為5.7的標準Amel-iv進行10-120天(通常14-21天)。 Once the seedlings showed multiple shoots, they were transferred to Stage 2 medium, in this example a reduced feed B-9N2-iv with a pH of 5.7, a 10-120 day cycle (typically a 21 day cycle). Between the stage 2 medium and the stage 3 medium which intensified b-11-iv in this example, the seedlings were alternated in successively alternating 10-120 day cycles (usually 21 day cycles) until a new tube was isolated And further expand to obtain the desired number of seedlings. Generally, 1-10 seedlings are obtained per tube per proliferation cycle. The shoots are then placed in Stage 4 medium, in this example, a standard Amel-iv having a pH of 5.7 for 10-120 days (typically 14-21 days).

(實施例5) (Example 5) 竹植物以階段1、階段2、階段3、階段4、階段5及/或階段6培養基微體繁殖Bamboo plants are propagated in stage 1, stage 2, stage 3, stage 4, stage 5 and/or stage 6 medium.

以3月齡和3年齡之間的竹植物開始，使用來自側苗剛破鞘的有節莖的節作為培植體。將每個節段切成3-5毫米的段及完整的苗。將外鞘剝去並丟棄，剩餘節段部分的塊置入終濃度為0.6%氫氧化鈉的10%漂白溶液中。將該漂白溶液中的培植體在可調速Lab Rotators，Barnstead/Lab line orbital Shaker(模型號KS 260)搖動臺上以每分鐘6-9轉放置1小時。隨後將培植體置於終濃度為0.06%氫氧化鈉的1%漂白溶液中，並放回到搖動臺上30分鐘。隨後重複該1%的漂白溶液步驟。 Starting from bamboo plants between 3 months old and 3 years old, the stalked knots from the stalk of the side seedlings were used as the culture body. Cut each segment into 3-5 mm segments and complete seedlings. The outer sheath was peeled off and discarded, and the remaining segments were placed in a 10% bleach solution with a final concentration of 0.6% sodium hydroxide. The cultures in the bleach solution were placed on a rocking table of a variable speed Lab Rotators, Barnstead/Lab line orbital Shaker (model number KS 260) at 6-9 revolutions per minute for 1 hour. The cultures were then placed in a 1% bleach solution at a final concentration of 0.06% sodium hydroxide and returned to the shaking table for 30 minutes. This 1% bleach solution step is then repeated.

隨後將單個培植體置於管中階段1培養基上(15-25 mL)，並且將該管置於溫度從65℉-70℉和全光譜光度36-90 μmole/m²/s²的受調控清潔生長室。本實施例中，起始的階段1培養基是pH為5.7的強化b-12c-iv。將培植體在強化b-12c-iv培養基上保留1-36小時，之後轉移到在本實施例中為減料b-12c-iv培養基的階段2培養基，進行10-120天(通常21天)週期的剩餘部分。 Subsequently, a single culture body was placed on the stage 1 medium (15-25 mL) in the tube, and the tube was placed at a temperature ranging from 65 °F to 70 °F and the full spectrum luminosity was 36-90 μmole/m ² /s ² . Clean the growth chamber. In this example, the initial Stage 1 medium was a fortified b-12c-iv having a pH of 5.7. The culture body was retained on the fortified b-12c-iv medium for 1-36 hours, and then transferred to the stage 2 medium which was reduced in the b-12c-iv medium in this example for 10-120 days (usually 21 days). The remainder of the cycle.

將污染的管丟棄。可以瓊脂的細菌性變色或可見的表面污染鑑別污染的管。這些培植體經歷3-4次階段1/2培養基的交替，每次包括10-120天的週期(通常21天的週期)。對每個週期(階段1和階段2培養基之間的交替)，培植體的亞組在階段1強化b-12c-iv培養基上1-36小時，隨後轉移到階段2減料b-12c-iv培養基進行剩餘的週期。其他的培植體在強化/減料方案的培養和標準b-12c-iv培養基的培養之間交替(階段1、階段2和階段3培養基之間的交替)。或者，培植體的第一10-120天的週期可以在標準b-12c-iv培養基中開始該過程，並隨後轉移到強化/減料方案進行下面的一個或多個週期。 Discard contaminated tubes. Contaminated tubes can be identified by bacterial discoloration of agar or visible surface contamination. These cultures were subjected to 3-4 stages of 1/2 medium exchange, each including a 10-120 day cycle (usually a 21 day cycle). For each cycle (alternation between Stage 1 and Stage 2 media), a subgroup of cultures was incubated on stage 1 fortified b-12c-iv medium for 1-36 hours, followed by transfer to stage 2 reduction b-12c-iv The medium is subjected to the remaining cycle. Other cultures were alternated between cultures of the booster/narrowing protocol and culture of standard b-12c-iv media (alternation between phase 1, phase 2 and phase 3 media). Alternatively, the first 10-120 day cycle of the culture can be initiated in standard b-12c-iv medium and subsequently transferred to the boost/suppression protocol for one or more of the following cycles.

如果增殖發生，將培植體在第三個週期之後從培養基取出。如果未發生增殖或未以顯著程度發生增殖，將培植體保留在培養基上進行第四個週期。基於特定培植體之前處理和處理參數(在強化/減料上或在強化/減料和標準之間交替的 進行所有週期)，選擇強化/減料或標準培養基。 If proliferation occurs, the culture is removed from the culture medium after the third cycle. If proliferation did not occur or proliferation did not occur to a significant extent, the cultures were retained on the medium for the fourth cycle. Based on prior treatment and processing parameters of specific implants (on strengthening/reducing or alternating between strengthening/reducing and standards) For all cycles), select fortification/reduction or standard medium.

接下來將活的苗轉移到階段3或階段4培養基(根據之前的處理)，在本實施例中是pH為5.7的標準b-9-iv。將培養物保留在b-9-iv培養基上，直到分離到新管並進一步擴展獲得期望數量的苗。一般地，時間的範圍包括10-120天的週期(通常14-21天的週期)，在週期之間將培養物安排為經過另一輪的階段3或階段4培養基中的增殖，或轉移到階段4或階段5培養基，在本實施例中是pH為5.7的標準b-10-iv，以進一步增殖。每個增殖週期每管可獲得1-10個苗。 The live shoots are next transferred to Stage 3 or Stage 4 media (according to previous treatment), in this example standard b-9-iv with a pH of 5.7. The culture was retained on b-9-iv medium until a new tube was isolated and further expanded to obtain the desired number of shoots. Generally, the range of time includes a period of 10-120 days (typically a period of 14-21 days), and the culture is scheduled to undergo proliferation through another round of Stage 3 or Stage 4 medium, or to a stage. 4 or stage 5 medium, in this example standard b-10-iv having a pH of 5.7, for further proliferation. 1-10 seedlings are obtained per tube per proliferation cycle.

在從增殖過程移出之後，將苗轉移到含有階段5或階段6培養基，在本實施例中是pH為5.7的標準BR-2-iv的小組織培養箱(稱為「magenta箱」)進行10-120天(通常14-21天)。 After removal from the proliferation process, the shoots are transferred to a small tissue culture incubator (referred to as "magenta box") containing Stage 5 or Stage 6 medium, in this example a standard BR-2-iv having a pH of 5.7. -120 days (usually 14-21 days).

(實施例6) (Example 6)

如實施例1選擇培植體並對其消毒。隨後將培植體轉移到含有階段1培養基的罐，在本實施例中，階段1培養基是pH為5.7的標準b-12c-iv(液體；30-40 mL)，在溫度從65℉-70℉和全光譜光度36-90 μmole/m²/s²的受調控清潔生長室中。每10-120天(通常每21天)將該培植體轉移到新鮮的標準的b-12c-iv培養基，而丟棄污染的管。藉由瓊脂的細菌性變色或可見的表面污染鑑別污染的管。這些培植體在標準b-12c-iv培養基上保留3-4個10-120天的週期(通常21天的週期)。 The culture body was selected and sterilized as in Example 1. The culture is then transferred to a tank containing Stage 1 medium. In this example, Stage 1 medium is standard b-12c-iv (liquid; 30-40 mL) at pH 5.7 at temperatures from 65 °F to 70 °F. And a full-spectrum luminosity of 36-90 μmole/m ² /s ² in a regulated clean growth chamber. The cultures were transferred to fresh standard b-12c-iv medium every 10 to 120 days (usually every 21 days) and the contaminated tubes were discarded. Contaminated tubes are identified by bacterial discoloration of agar or visible surface contamination. These cultures were maintained on standard b-12c-iv medium for 3-4 10-120 day cycles (typically 21 day cycles).

隨後將培養物轉移到階段2培養基上，在本實施例中，是提供每分鐘6-9轉旋轉架上罐中的強化b-11-iv(液體)。將該培養物在pH為5.7的強化b-11-iv培養基上保留0.5-12小時，並隨後轉移到階段3的減料b-12c-iv培養基上進行10-120天的週期(通常14天的週期)的剩餘部分，直到分離到新罐並進一步擴展獲得期望數量的苗。每個增殖週期每罐可獲得1-15個苗。隨後將苗置於在本實施例中是pH為6的標準Ech-iv的階段4培養基上10-120天(通常14-21天)。 The culture is then transferred to Stage 2 medium, in this example, to provide enhanced b-11-iv (liquid) in a can on a 6-9 rotation per minute. The culture was retained on reinforced b-11-iv medium at pH 5.7 for 0.5-12 hours and then transferred to stage 3 reduced b-12c-iv medium for a period of 10-120 days (typically 14 days) The remainder of the cycle) until the new canister is separated and further expanded to obtain the desired number of shoots. 1-15 shoots per pot can be obtained per fermentation cycle. The shoots were then placed on Stage 4 medium, which is a standard Ech-iv pH 6 in this example, for 10-120 days (typically 14-21 days).

(實施例7) (Example 7)

如實施例1中選擇培植體並對其消毒。隨後將該培植體轉移到含有階段1培養基的管中，在本實施例中，所述階段1培養基是強化b-12c-iv，放置24-48小時，隨後將培養物轉移到階段2的減料b-12c-iv培養基10-21天(一般為14)。 The culture body was selected and sterilized as in Example 1. The culture is then transferred to a tube containing Stage 1 medium, in this example, the Stage 1 medium is fortified b-12c-iv, placed for 24-48 hours, and then the culture is transferred to Stage 2 minus Feed b-12c-iv medium for 10-21 days (typically 14).

隨後將苗轉移到階段3培養基，在本實施例中，是magenta箱中的強化b-9-iv(40-50 mL)。在強化b-9-iv培養基上保留12-36小時，隨後轉移到階段4減料b-9-iv培養基或減料CW-2-iv培養基進行10-120天的週期(通常14天的週期)的剩餘部分，直到分離到新的箱並進一步擴展獲得期望數量的苗。每個增殖週期每箱可獲得1-20個苗。隨後將苗置於在本實施例中是強化BR-2-iv的階段5培養基上5-10小時，接著轉移到階段6減料BR-2-iv進行10-120天(通常14-21天)週期的剩餘部分。 The shoots were then transferred to Stage 3 medium, in this example, the fortified b-9-iv (40-50 mL) in the magenta box. Retained on fortified b-9-iv medium for 12-36 hours, then transferred to stage 4 reduced b-9-iv medium or reduced CW-2-iv medium for a period of 10-120 days (usually 14 days period) The rest of the) until the new box is separated and further expanded to obtain the desired number of seedlings. 1-20 seedlings per box are available for each proliferation cycle. The shoots are then placed on stage 5 medium in this example for enhanced BR-2-iv for 5-10 hours, followed by transfer to stage 6 minus BR-2-iv for 10-120 days (usually 14-21 days) The rest of the cycle.

(實施例8) (Example 8)

如實施例1選擇培植體並對其消毒。隨後將該培植體轉移到含有在本實施例中是pH為5.7的強化b-12c-ii的階段1培養基的管。將培植體在強化b-12c-ii培養基上保留3-18小時，隨後轉移到標準b-12c-iv培養基上10-21天(通常14天)。在強化b-12c-ii中3-18小時，隨後在標準b-12c-iv中10-21天，重複該迴圈直到培植體開始增殖。隨後將苗轉移到在本實施例中是pH為5.5的標準b-1-iv的階段3培養基進行10-120天的週期(通常21天的週期)，直到分離到新管並進一步擴展獲得期望數量的苗。每個增殖週期每管可獲得1-10個苗。隨後將苗置於在本實施例中pH為5.7的強化Br-2-iv的階段4培養基中1-10小時，隨後在標準Br-2-iv中14-21天。 The culture body was selected and sterilized as in Example 1. The culture was then transferred to a tube containing Stage 1 medium of enhanced b-12c-ii having a pH of 5.7 in this example. The cultures were retained on the fortified b-12c-ii medium for 3-18 hours and subsequently transferred to standard b-12c-iv medium for 10-21 days (typically 14 days). The loop was repeated 3-12 hours in the boosted b-12c-ii followed by 10-21 days in the standard b-12c-iv until the culture began to proliferate. The shoots are then transferred to stage 3 medium, which is a standard b-1-iv having a pH of 5.5 in this example, for a period of 10-120 days (usually a 21 day cycle) until a new tube is separated and further expanded to obtain the desired The number of seedlings. 1-10 seedlings are obtained per tube per proliferation cycle. The shoots were then placed in Stage 4 medium of fortified Br-2-iv having a pH of 5.7 in this example for 1-10 hours, followed by 14-21 days in standard Br-2-iv.

(實施例9) (Example 9)

如實施例1選擇培植體並對其消毒。隨後將培植體置於含有在本實施例中是強化b-12c-iv的階段1培養基的管中1-24小時。隨後將苗轉移到在本實施例中是標準b-10-iv培養基的階段2培養基中10-120天(一般為14)。持續階段1到階段2培養基的交替直到培植體開始增殖。一旦開始增殖，將苗轉移到pH為5.5的標準b-4-iv培養基中進行10-120天的週期(通常21天的週期)，直到分離到新管並進一步擴展獲得期望數量的苗。每個增殖週期每管可獲得1-10個苗。隨 後將苗置於在本實施例中是pH為5.7的強化Br-2-iv的階段4培養基中1-10小時，隨後在階段5培養基，標準Amel-iv中進行10-120天(通常14-21天)。 The culture body was selected and sterilized as in Example 1. The culture body was then placed in a tube containing Stage 1 medium which was enhanced b-12c-iv in this example for 1-24 hours. The shoots are then transferred to Stage 2 medium, which is standard b-10-iv medium in this example, for 10-120 days (typically 14). The mixing of the medium from stage 1 to stage 2 is continued until the culture begins to proliferate. Once proliferation is initiated, the shoots are transferred to standard b-4-iv medium at pH 5.5 for a period of 10-120 days (typically a 21 day cycle) until a new tube is isolated and further expanded to obtain the desired number of shoots. 1-10 seedlings are obtained per tube per proliferation cycle. With The seedlings are then placed in Stage 4 medium of intensive Br-2-iv having a pH of 5.7 in this example for 1-10 hours, followed by 10-120 days in Stage 5 medium, standard Amel-iv (usually 14 -21 days).

(實施例10) (Embodiment 10)

如實施例1選擇培植體並對其消毒。隨後將培植體轉移至含有階段1培養基的管中，在本實施例中是如實施例1中描述的標準b-12c-iv培養基。隨後將苗轉移到在本實施例中是pH為5.5的強化b-9-iv的階段2培養基中4-24小時。在強化b-9-iv培養基中培養4-24小時之後，將苗置於階段3標準b-9-iv培養基中10-120天(通常21天)。重複強化和標準b-9-iv培養基之間的交替，直到分離到新管並進一步擴展獲得期望數量的苗。每個增殖週期每管可獲得1-10個苗。隨後將苗置於在本實施例中是pH為5.7的Amel-iv的階段4培養基中10-120天(通常14-21天)。 The culture body was selected and sterilized as in Example 1. The culture is then transferred to a tube containing Stage 1 medium, in this example a standard b-12c-iv medium as described in Example 1. The shoots were then transferred to stage 2 medium of enhanced b-9-iv having a pH of 5.5 in this example for 4-24 hours. After 4-24 hours of incubation in fortified b-9-iv medium, the shoots were placed in stage 3 standard b-9-iv medium for 10-120 days (typically 21 days). The alternation between the intensification and standard b-9-iv medium was repeated until a new tube was isolated and further expanded to obtain the desired number of shoots. 1-10 seedlings are obtained per tube per proliferation cycle. The shoots were then placed in Stage 4 medium, Amel-iv, pH 5.7 in this example for 10-120 days (typically 14-21 days).

(實施例11) (Example 11)

如實施例1選擇培植體並對其消毒。隨後將培植體轉移至含有階段1培養基的箱中，在本實施例中是標準b-10-iv(40-50 mL)。將苗保留在交替的標準b-10-iv培養基(10-120天的週期(通常21天的週期))、階段2強化b-10-iv培養基(1-10小時)和階段3減料b-10-iv培養基(10-120天的週期(通常10天的週期))，直到分離到新的箱並進一步擴展獲得期望數量的苗。每個增殖週期每箱可獲得1-20個苗。 隨後將苗置於在本實施例中是pH為5.7的Amel-iv的階段4培養基中10-120天(通常14-21天)。 The culture body was selected and sterilized as in Example 1. The cultures were then transferred to a tank containing Stage 1 medium, in this example standard b-10-iv (40-50 mL). Retain the shoots in alternating standard b-10-iv medium (10-120 day cycle (usually 21 day cycle)), stage 2 fortified b-10-iv medium (1-10 hours) and stage 3 subtraction b -10-iv medium (10-120 day cycle (usually a 10-day cycle)) until a new bin is isolated and further expanded to obtain the desired number of shoots. 1-20 seedlings per box are available for each proliferation cycle. The shoots were then placed in Stage 4 medium, Amel-iv, pH 5.7 in this example for 10-120 days (typically 14-21 days).

(實施例12) (Embodiment 12)

如實施例1選擇培植體並對其消毒。隨後將培植體轉移至含有在本實施例中是強化b-12c-i的階段1培養基的管中1-24小時，之後在階段2減料b-10-i中以10-21天(一般為14)進行3-4個週期。然後將苗轉移到在本實施例中是pH為5.5的強化b-9-iv的階段3培養基中1-5小時，之後轉移到階段4的標準b-9-iv培養基進行10-120天的週期(通常21天的週期)，直到分離到新管並進一步擴展獲得期望數量的苗。還可使用pH為5.5的交替的強化和標準或減料B-6培養基。每個增殖週期每管可獲得1-10個苗。隨後將苗置於在本實施例中是pH為5.7的Amel-iv的階段5培養基中10-120天(通常14-21天)。 The culture body was selected and sterilized as in Example 1. The transplant is then transferred to a tube containing Stage 1 medium that is enhanced b-12c-i in this example for 1-24 hours, followed by 10-21 days in Stage 2 reduction b-10-i (general For 3-4 cycles of 14). The shoots were then transferred to stage 3 medium of enhanced b-9-iv having a pH of 5.5 in this example for 1-5 hours, after which they were transferred to stage 4 standard b-9-iv medium for 10-120 days. Cycle (usually a 21-day cycle) until a new tube is separated and further expanded to obtain the desired number of shoots. Alternate fortification and standard or reduced B-6 media at pH 5.5 can also be used. 1-10 seedlings are obtained per tube per proliferation cycle. The shoots were then placed in Stage 5 medium, Amel-iv, pH 5.7 in this example, for 10-120 days (typically 14-21 days).

(實施例13) (Example 13)

如實施例1選擇培植體並對其消毒。隨後將培植體轉移至含有階段1培養基的管中，在本實施例中，階段1培養基是如實施例1中描述的標準的b-12c-iv。隨後將培植體轉移至在本實施例中是pH為5.5的強化b-10-iv的階段2培養基0.5-3小時，之後在作為階段3培養基的標準b-10-iv培養基中進行10-120天的週期(通常21天的週期)。重複該強化到標準b-10-iv週期，直到分離到新管並進一步擴展獲得期望 數量的苗。每個增殖週期每管可獲得1-10個苗。隨後將苗置於在本實施例中是pH為5.7的標準的Amel-iv的階段3培養基中10-120天(通常14-21天)。 The culture body was selected and sterilized as in Example 1. The culture is then transferred to a tube containing Stage 1 medium, which in this example is the standard b-12c-iv as described in Example 1. The culture body is then transferred to the phase 2 medium of the enhanced b-10-iv having a pH of 5.5 in this example for 0.5 to 3 hours, followed by 10-120 in the standard b-10-iv medium as the stage 3 medium. The period of the day (usually a 21-day cycle). Repeat this enhancement to the standard b-10-iv cycle until the new tube is separated and further expanded to achieve the desired The number of seedlings. 1-10 seedlings are obtained per tube per proliferation cycle. The shoots were then placed in Stage 3 medium of standard Amel-iv pH 5.7 in this example for 10-120 days (typically 14-21 days).

(實施例14) (Example 14)

以3月齡和3年齡之間的竹植物作為開始，使用來自側苗剛破鞘的有節莖的節作為培植體。將每個節段切成3-5毫米的段及完整的苗。將一些培植體，包括取自1年或更長時間的有節莖的培植體，在70%的異丙醇的罐中搖動3秒以預先沖洗，隨後將它們在流動的自來水中沖洗1分鐘。不預先沖洗其他的培植體。 Starting from a bamboo plant between 3 months old and 3 years old, a stalked knot from a side seedling has been used as a culture body. Cut each segment into 3-5 mm segments and complete seedlings. Some cultures, including stems from 1 year or longer, were shaken in a 70% isopropanol jar for 3 seconds to pre-rinse, then rinsed in running tap water for 1 minute. . Do not rinse other implants beforehand.

將外鞘剝去並丟棄，且將剩餘節段部分置入10%的漂白溶液中。將該漂白溶液中的培植體在可調速Lab Rotators，Barnstead/Lab line orbital Shaker(模型號KS 260)搖動臺上以每分鐘6-9轉放置1小時。對於包括取自1年齡或更大齡有節莖的培植體，於10%的漂白溶液加入數滴Tween 20，並修改所述步驟將該培植體浸漬45分鐘而非1小時。隨後將所述培植體放置於1%的漂白溶液中，並放回到搖動臺上30分鐘。隨後重複該1%的漂白溶液步驟。 The outer sheath was peeled off and discarded, and the remaining segment was placed in a 10% bleach solution. The cultures in the bleach solution were placed on a rocking table of a variable speed Lab Rotators, Barnstead/Lab line orbital Shaker (model number KS 260) at 6-9 revolutions per minute for 1 hour. For inclusions including stems from 1 year or older, a few drops of Tween 20 were added to 10% of the bleach solution and the procedure was modified to impregnate the plants for 45 minutes instead of 1 hour. The culture was then placed in a 1% bleach solution and returned to the shaking table for 30 minutes. This 1% bleach solution step is then repeated.

隨後將單個培植體置於管中階段1培養基上(15-25 mL)，並且將該管置於溫度從65℉-70℉和全光譜光度36-90 μmole/m2/s2的受調控清潔生長室。在本實施例中，階段1培養基是pH為5.7的標準b-12c-iv。每10-120天(通常每 21天)將該外植體轉移到新鮮的b-12c-iv培養基，而丟棄污染的管。這些培植體在b-12c-iv培養基上保留2個10-120天的週期(通常21天的週期)。在週期之間除去多餘的鞘。在轉移到第三個週期時，將該培植體轉移到在本實施例中為用7 g/L的角叉膠(而非上面提供的5.5 g/L)補充的強化b-12c-i的階段2培養基中0.5-10小時，之後在標準b-12c-iv中放置10-120天(通常為21)。在第三個週期之後，清理該培植體。將該培植體保留在這些交替的階段2和階段3培養基上一定的時間，直到觀察到多個苗。可以在3-15個月之內觀察到多個苗。 A single culture body was then placed on the Stage 1 medium (15-25 mL) in the tube and placed in a regulated clean growth at a temperature from 65 °F to 70 °F and a full spectrum of luminosity of 36-90 μmole/m2/s2. room. In this example, the Stage 1 medium was standard b-12c-iv having a pH of 5.7. Every 10 to 120 days (usually every The explants were transferred to fresh b-12c-iv medium for 21 days and the contaminated tubes were discarded. These cultures were maintained on b-12c-iv medium for two 10-120 day cycles (typically a 21 day cycle). Remove excess sheath between cycles. Upon transfer to the third cycle, the culture was transferred to a fortified b-12c-i supplemented with 7 g/L carrageenan (instead of 5.5 g/L provided above) in this example. Stage 2 medium is 0.5-10 hours and then placed in standard b-12c-iv for 10-120 days (typically 21). After the third cycle, the culture is cleaned. The culture was retained on these alternating Phase 2 and Stage 3 media for a certain period of time until multiple shoots were observed. Multiple shoots can be observed within 3-15 months.

一旦培植體顯示多個苗，或者將其維持在其交替的階段2培養基/階段3培養基上，或者將其轉移到在本實施例中pH為5.7時使用的標準b-9-iv的階段4培養基。除了使用b-9培養基之一之外，還可以使用pH為5.7的CW1培養基。在階段2/3培養基或階段4培養基上保持該培養物，直到分離到新管並進一步擴展獲得期望數量的苗。一般地，時間的範圍包括10-120天的週期(通常21天的週期)，在週期之間將培養物安排為經過另一輪的增殖，或者轉移到階段4或在本實施例中是pH為5.7的標準BR-2-iv的階段5培養基中以在「magenta箱」中進行10-120天(通常21天)。 Once the culture shows multiple shoots, or maintain it on its alternate Phase 2 medium/stage 3 medium, or transfer it to stage 4 of standard b-9-iv used at pH 5.7 in this example. Medium. In addition to using one of the b-9 media, a CW1 medium having a pH of 5.7 can also be used. The culture is maintained on Stage 2/3 medium or Stage 4 medium until a new tube is isolated and further expanded to obtain the desired number of shoots. Generally, the range of time includes a period of 10-120 days (typically a 21 day period), the culture is scheduled to undergo another round of proliferation between cycles, or transferred to Stage 4 or in this embodiment is pH The stage 5 medium of the standard BR-2-iv of 5.7 was carried out in the "magenta box" for 10-120 days (usually 21 days).

(實施例15) (Example 15)

如實施例10選擇培植體並對其消毒。隨後將培植體轉移 到含有在本實施例中為強化b-12c-iv(液體；30-40 mL)的階段1培養基的罐中。這些培植體在強化b-12c-iv培養基上保留0.5-24小時，隨後在階段2減料b-12c-i中放置10-120天(通常為21)。將該過程重複2次。在週期之間除去多餘的鞘。 The culture body was selected and sterilized as in Example 10. Subsequent transfer of the implant To a tank containing Stage 1 medium which was reinforced b-12c-iv (liquid; 30-40 mL) in this example. These cultures were maintained on fortified b-12c-iv medium for 0.5-24 hours and then placed in stage 2 reduction b-12c-i for 10-120 days (typically 21). This process was repeated twice. Remove excess sheath between cycles.

在轉移到第三個週期時，將培植體轉移到在本實施例中為用7 g/L的角叉膠(而非5.5 g/L)補充的標準b-12c-iv的階段3培養基中10-120天的週期(通常21天的週期)。在第三個週期之後，清理該培植體。將該培植體在用7 g/L的角叉膠補充的階段3標準的b-12c-iv中保持10-120天的週期(通常21天的週期)，直到觀察的多個苗。可以在3-15個月之內觀察到多個苗。 Upon transfer to the third cycle, the cultures were transferred to Stage 3 medium in standard b-12c-iv supplemented with 7 g/L carrageenan (instead of 5.5 g/L) in this example. 10-120 day period (usually 21 day period). After the third cycle, the culture is cleaned. The culture was maintained in a phase 3 standard b-12c-iv supplemented with 7 g/L carrageenan for a period of 10-120 days (typically a 21 day cycle) until multiple shoots were observed. Multiple shoots can be observed within 3-15 months.

一旦培植體顯示多個苗，或者將其維持在其階段3培養基上，或者轉移到階段4培養基，在本實施例中，是提供每分鐘6-9轉的旋轉架上的罐中的pH為5.7的強化b-11-iv(液體)。苗在強化b-11-iv上保留0.5-24小時，之後轉移到階段5標準b-11-iv。該培養物在階段3或階段4/5培養基上保留10-120天的週期(通常14天的週期)，直到分離到新罐並進一步擴展獲得期望數量的苗。每個增殖週期每罐獲得1-15個苗。隨後將該苗置於階段4或階段6培養基中，在本實施例中是pH為6的強化Ech-iv，1-24小時。然後將苗轉移到階段5或階段7培養基(即，標準Ech-iv)中10-120天(通常21天)。 Once the culture exhibits multiple shoots, or maintains it on its Stage 3 medium, or transfers to Stage 4 medium, in this example, the pH in the tank on a rotating rack providing 6-9 revolutions per minute is Enhanced 5.7 b-11-iv (liquid). The shoots were retained on the fortified b-11-iv for 0.5-24 hours before being transferred to stage 5 standard b-11-iv. The culture is maintained on Stage 3 or Stage 4/5 medium for a period of 10-120 days (typically a 14 day period) until a new tank is isolated and further expanded to obtain the desired number of shoots. 1-15 shoots per pot were obtained per fermentation cycle. The shoot is then placed in Stage 4 or Stage 6 medium, in this example a fortified Ech-iv at pH 6, 1-24 hours. The shoots are then transferred to Stage 5 or Stage 7 medium (i.e., standard Ech-iv) for 10-120 days (typically 21 days).

(實施例16) (Embodiment 16)

如實施例10選擇培植體並對其消毒。隨後將該培植體轉移到含有在本實施例中為強化b-12c-iv的階段1培養基的管中。這些培植體在強化b-12c-iv培養基上保留0.5-24小時，之後轉移到階段2標準b-12c-iv培養基進行10-120天的週期(通常21天的週期)。將此交替重複3次。在週期之間除去多餘的鞘。當轉移到第四個週期時，將培植體轉移到在本實施例中是用7 g/L的角叉膠(而非5.5 g/L)補充的強化b-12c-iv的階段3培養基中1-24小時，之後轉移到階段4減料b-12c-ii培養基。在第四個週期之後，清理培植體。將該培植體保留在交替的階段3/4培養基上進行10-120天的週期(通常21天的週期)，直到觀察到多個苗。可以在3-15個月之內觀察到多個苗。 The culture body was selected and sterilized as in Example 10. The culture was then transferred to a tube containing Stage 1 medium, which in this example was reinforced b-12c-iv. These cultures were maintained on fortified b-12c-iv medium for 0.5-24 hours before being transferred to stage 2 standard b-12c-iv medium for a period of 10-120 days (typically a 21 day cycle). This is repeated three times. Remove excess sheath between cycles. When transferred to the fourth cycle, the cultures were transferred to phase 3 medium in the enhanced b-12c-iv supplemented with 7 g/L carrageenan (instead of 5.5 g/L) in this example. After 1-24 hours, transfer to stage 4 reduced b-12c-ii medium. After the fourth cycle, the culture body is cleaned. The cultures were kept on alternating stage 3/4 medium for a period of 10-120 days (typically a 21 day period) until multiple shoots were observed. Multiple shoots can be observed within 3-15 months.

一旦培植體顯示多個苗，或者將其維持在其階段3/4培養基，或者轉移到階段5培養基，在本實施例中，為magenta箱中的標準b-9-iv(40-50 mL)(也可使用CW1培養基或其強化和減料和/或標準版本)。在b-9-iv培養基上保留10-120天的週期(通常14天的週期)，直到分離到新的箱並進一步擴展獲得期望數量的苗。每個增殖週期每箱可獲得1-20個苗。隨後將該苗置於階段5或階段6培養基，在本實施例中為BR-2-iv，10-120天(通常14-21天)。 Once the culture shows multiple shoots, or maintain it in its stage 3/4 medium, or transfer to Stage 5 medium, in this example, the standard b-9-iv (40-50 mL) in the magenta box (CW1 medium or its fortified and reduced material and/or standard versions can also be used). A 10-120 day cycle (usually a 14 day cycle) is maintained on the b-9-iv medium until a new box is isolated and further expanded to obtain the desired number of shoots. 1-20 seedlings per box are available for each proliferation cycle. The shoot is then placed in Stage 5 or Stage 6 medium, in this example BR-2-iv, 10-120 days (usually 14-21 days).

(實施例17) (Example 17)

如實施例10選擇培植體並對其消毒。隨後將該培植體轉移到含有階段1培養基的管中，在本實施例中階段1培養基為標準b-12c-iv，如實施例28所描述。這些培植體在標準b-12c-iv培養基上保留2個10-120天的週期(通常21天的週期)。在週期之間，除去多餘的鞘。當轉移到第三個週期時，將培植體轉移到階段2培養基，在本實施例是用7 g/L(而非5.5 g/L)的角叉膠補充的標準的b-12c-iv。在第三個週期之後，清理培植體。將所述培植體在用7 g/L的角叉膠補充的標準b-12c-iv上保留10-120天的週期(通常21天的週期)，直到觀察到多個苗。可以在3-15個月之內觀察到多個苗。 The culture body was selected and sterilized as in Example 10. The culture was then transferred to a tube containing Stage 1 medium, which in this example was standard b-12c-iv as described in Example 28. These cultures were maintained on standard b-12c-iv medium for two 10-120 day cycles (typically 21 day cycles). Remove excess sheath between cycles. When transferred to the third cycle, the cultures were transferred to Stage 2 medium, in this example a standard b-12c-iv supplemented with 7 g/L (rather than 5.5 g/L) carrageenan. After the third cycle, the implants are cleared. The cultures were maintained on a standard b-12c-iv supplemented with 7 g/L carrageenan for a period of 10-120 days (typically a 21 day cycle) until multiple shoots were observed. Multiple shoots can be observed within 3-15 months.

一旦外植體顯示多個苗，或者將其維持在其階段2培養基，或者轉移到在本實施例中是pH為5.5的強化b-1-iv的階段3培養基5-10小時，隨後轉移到階段4的無細胞***素的b-1培養基進行10-120天的週期(通常21天的週期)。重複階段3/4培養基的交替，直到分離到新管並進一步擴展獲得期望數量的苗。每個增殖週期每管可獲得1-10個苗。隨後將苗置於階段3或階段5培養基，在本實施例中是pH為5.7的強化Br-2-iv中1-10小時，接著在減料Br-2-i中10-120天(通常14-21天)。 Once the explants display multiple shoots, either maintain them in their Stage 2 medium, or transfer to Stage 3 medium with enhanced b-1-iv pH of 5.5 in this example for 5-10 hours, then transfer to Stage 4 cytokinin-free b-1 medium is subjected to a 10-120 day cycle (typically a 21 day cycle). The alternating phase 3/4 medium was repeated until a new tube was isolated and further expanded to obtain the desired number of shoots. 1-10 seedlings are obtained per tube per proliferation cycle. The shoots are then placed in Stage 3 or Stage 5 medium, in this example 1-10 hours in fortified Br-2-iv with a pH of 5.7, followed by 10-120 days in the reduction of Br-2-i (usually 14-21 days).

(實施例18) (Embodiment 18)

如實施例10選擇培植體並對其消毒。隨後將培植體轉移到含有階段1培養基的管中，在本實施例中，階段1培養基 為強化b-12c-iv。這些培植體在強化b-12c-iv培養基上保留0.5-10小時，隨後轉移到階段2的無細胞***素的b-12c培養基中進行2個10-120天的週期(通常21天的週期)。在週期之間，除去多餘的鞘。當轉移到第三個週期時，將培植體轉移到在本實施例中是用7 g/L的角叉膠(而非5.5 g/L)補充的強化b-12c-iii的階段3培養基中0.5-10小時，之後轉移回階段2無細胞***素的b-12c培養基。第三個週期之後，清理培植體。將該培植體在交替的階段3/2培養基上保留10-120天的週期(通常21天的週期)，直到觀察到多個苗。可以在3-15個月之內觀察到多個苗。 The culture body was selected and sterilized as in Example 10. The transplant is then transferred to a tube containing Stage 1 medium, in this example, Stage 1 medium. To strengthen b-12c-iv. These cultures were retained on fortified b-12c-iv medium for 0.5-10 hours and subsequently transferred to stage 2 cytokinin-free b-12c medium for 2 10-120 day cycles (usually 21 day cycles) . Remove excess sheath between cycles. When transferred to the third cycle, the cultures were transferred to Stage 3 medium of fortified b-12c-iii supplemented with 7 g/L carrageenan (instead of 5.5 g/L) in this example. After 0.5-10 hours, transfer back to stage 2 cytokinin-free b-12c medium. After the third cycle, the culture body is cleaned. The cultures were maintained on alternating stage 3/2 media for a period of 10-120 days (typically a 21 day cycle) until multiple shoots were observed. Multiple shoots can be observed within 3-15 months.

一旦培植體顯示多個苗，或者將其維持在其交替的階段3/2培養基，或者轉移到在本實施例中是pH為5.5的強化b-4-iv的階段4培養基12-36小時，隨後是階段5的無細胞***素的b-4-iv 10-120天(通常21天的週期)，直到分離到新管並進一步擴展獲得期望數量的苗。每個增殖週期每管可獲得1-10個苗。隨後將苗置於階段4或階段6培養基，在本實施例中是pH為5.7的Br-2-iv中10-120天(通常14-21天)。 Once the culture shows multiple shoots, or maintain it in its alternating stage 3/2 medium, or transfer to stage 4 medium in the enhanced b-4-iv pH of 5.5 in this example for 12-36 hours, This is followed by stage 5 cytokinin-free b-4-iv for 10-120 days (usually a 21-day cycle) until a new tube is isolated and further expanded to obtain the desired number of shoots. 1-10 seedlings are obtained per tube per proliferation cycle. The shoots are then placed in Stage 4 or Stage 6 medium, in this example, in a Br-2-iv pH 5.7 for 10-120 days (typically 14-21 days).

(實施例19) (Embodiment 19)

如實施例10選擇培植體並對其消毒。隨後將該培植體轉移到含有階段1培養基的管，在本實施例中，階段1培養基為標準b-12c-iv，亦如實施例28所描述。這些培植體在 b-12c-iv培養基上保留2個10-120天的週期(通常21天的週期)。在週期之間，除去多餘的鞘。當轉移到第三個週期時，將培植體轉移到階段2培養基，在本實施例中，為用7 g/L的角叉膠而非上面提供的5.5 g/L補充的標準b-12c-iv。在第三個週期之後，清理該培植體。將該培植體在用7 g/L的角叉膠補充的標準b-12c-iv上保留10-120天的週期(通常21天的週期)，直到觀察到多個苗。可以在3-15個月之內觀察到多個苗。 The culture body was selected and sterilized as in Example 10. The culture is then transferred to a tube containing Stage 1 medium. In this example, Stage 1 medium is standard b-12c-iv, as also described in Example 28. These implants are in Two 10-120 day cycles (usually 21 day cycles) are retained on the b-12c-iv medium. Remove excess sheath between cycles. When transferred to the third cycle, the culture was transferred to Stage 2 medium, in this example, with 7 g/L of carrageenan instead of the standard b-12c- supplemented with 5.5 g/L provided above. Iv. After the third cycle, the culture is cleaned. The cultures were maintained on a standard b-12c-iv supplemented with 7 g/L carrageenan for a period of 10-120 days (typically a 21 day cycle) until multiple shoots were observed. Multiple shoots can be observed within 3-15 months.

一旦該培植體顯示多個苗，或者將其維持在其階段2培養基上，或者轉移到在本實施例中是pH為5.5的標準b-9-iv的階段3培養基進行10-120天的週期(通常21天)，直到分離到新管並進一步擴展獲得期望數量的苗。還可以使用pH為5.5的b-6培養基。每個增殖週期每管可獲得1-10個苗。隨後將苗置於階段3或階段4培養基，在本實施例中是pH為5.7的強化Amel-iv培養基中0.5-24小時，接著轉移到階段4或5無細胞***素的Amel培養基中進行10-120天(通常14-21天)。 Once the culture exhibits multiple shoots, or maintain it on its Stage 2 medium, or transfer to Stage 3 medium, which is a standard b-9-iv pH of 5.5 in this example, for a period of 10-120 days. (usually 21 days) until a new tube is separated and further expanded to obtain the desired number of shoots. It is also possible to use b-6 medium having a pH of 5.5. 1-10 seedlings are obtained per tube per proliferation cycle. The shoots are then placed in Stage 3 or Stage 4 medium, in this example in a fortified Amel-iv medium at pH 5.7 for 0.5-24 hours, followed by transfer to Stage 4 or 5 cytokinin-free Amel medium for 10 -120 days (usually 14-21 days).

(實施例20) (Embodiment 20)

如實施例10選擇培植體並對其消毒。隨後將該培植體轉移到含有階段1培養基的箱，在本實施例中，階段1培養基為強化b-10-iv(40-50 mL)。培植體在強化b-10-iv培養基上保留1-5小時，並隨後轉移到階段2無細胞***素的b-10 培養基1-5天，然後轉移到階段3減料b-10-ii培養基5-115天(通常18天)。在週期之間除去多餘的鞘，並將該交替重複兩次。當轉移到第三次交替時，培植體被轉移到在本實施例中為用7 g/L的角叉膠(而非5.5 g/L)補充的強化b-10c-i的階段4培養基中1-5小時，隨後轉移到階段5無細胞***素的b-10c 1-5天和階段6的減料b-12c中5-115天(通常18天)。在第三個週期之後清理培植體。將該培植體保留在交替的階段4/5/6培養基上直到觀察到多個苗。可以在3-15個月之內觀察到多個苗。將培養物保留在交替的階段4/5/6培養基直到獲得期望數量的苗。每個增殖週期每箱可獲得1-20個苗。隨後將該苗在本實施例中是pH為5.7的標準的Amel-iv的階段7培養基上放置10-120天(通常14-21天)。 The culture body was selected and sterilized as in Example 10. The culture was then transferred to a tank containing Stage 1 medium. In this example, Stage 1 medium was fortified b-10-iv (40-50 mL). The cultures were retained on enhanced b-10-iv medium for 1-5 hours and subsequently transferred to stage 2 cytokinin-free b-10 The medium was 1-5 days and then transferred to stage 3 reduced b-10-ii medium for 5-115 days (usually 18 days). The excess sheath is removed between cycles and the alternation is repeated twice. When transferred to the third alternation, the culture was transferred to Stage 4 medium in the enhanced b-10c-i supplemented with 7 g/L carrageenan (instead of 5.5 g/L) in this example. 1-5 hours, then transferred to stage 5 cytokinin-free b-10c 1-5 days and stage 6 reduction b-12c 5-115 days (usually 18 days). The culture body is cleaned after the third cycle. The cultures were kept on alternating stage 4/5/6 medium until multiple shoots were observed. Multiple shoots can be observed within 3-15 months. Cultures were kept in alternating stages of 4/5/6 medium until the desired number of shoots were obtained. 1-20 seedlings per box are available for each proliferation cycle. The shoot is then placed on stage 7 medium of standard Amel-iv pH 5.7 in this example for 10-120 days (typically 14-21 days).

(實施例21) (Example 21)

如實施例10選擇培植體並對其消毒。隨後將該培植體轉移到含有階段1培養基的管，在本實施例中，階段1培養基為標準b-12c-iv，如實施例28所描述。這些培植體在標準b-12c-iv培養基上保留2個10-120天的週期(通常21天的週期)。在週期之間，除去多餘的鞘。當轉移到第三個週期時，將該培植體轉移到階段2培養基，在本實施例中，為用7 g/L的角叉膠而非上面提供的5.5 g/L補充的標準b-12c-iv。在第三個週期之後，清理培植體。將該培植體保留在用7 g/L的角叉膠補充的標準b-12c-iv上10-120天的週期(通常21 天的週期)，直到觀察到多個苗。可以在3-15個月之內觀察到多個苗。 The culture body was selected and sterilized as in Example 10. The culture is then transferred to a tube containing Stage 1 medium, in this example, Stage 1 medium is standard b-12c-iv, as described in Example 28. These cultures were maintained on standard b-12c-iv medium for two 10-120 day cycles (typically 21 day cycles). Remove excess sheath between cycles. When transferred to the third cycle, the culture was transferred to Stage 2 medium, in this example, a standard b-12c supplemented with 7 g/L of carrageenan instead of the 5.5 g/L supplied above. -iv. After the third cycle, the implants are cleared. The culture was kept on a standard b-12c-iv supplemented with 7 g/L carrageenan for a period of 10-120 days (usually 21 Day cycle) until multiple seedlings were observed. Multiple shoots can be observed within 3-15 months.

一旦培植體顯示多個苗，或者將其維持在其階段2培養基上，或者轉移到在本實施例中是pH為5.5的強化b-9-iv的階段3培養基0.5-24小時，隨後轉移到階段4無細胞***素的b-4培養基10-120天。持續該階段2或階段3/4培養基的交替週期(通常21天的週期)，直到分離到新管並進一步擴展獲得期望數量的苗。還可以使用pH為5.5的強化或標準b-6培養基。每個增殖週期每管可獲得1-10個苗。隨後將該苗在階段3或階段5培養基，在本實施例中是pH為5.7的強化Amel-iv中24小時，接著轉移到標準Amel-iv進行10-120天(通常14-21天)。 Once the culture body shows multiple shoots, or maintain it on its Stage 2 medium, or transfer to Stage 3 medium of enhanced b-9-iv at pH 5.5 in this example for 0.5-24 hours, then transfer to Stage 4 cytokinin-free b-4 medium for 10-120 days. The alternating cycle of this stage 2 or stage 3/4 medium (usually a 21 day cycle) is continued until a new tube is separated and further expanded to obtain the desired number of shoots. A fortified or standard b-6 medium with a pH of 5.5 can also be used. 1-10 seedlings are obtained per tube per proliferation cycle. The shoot is then seeded in Stage 3 or Stage 5 medium, in this example a reinforced Amel-iv pH 5.7 for 24 hours, followed by transfer to standard Amel-iv for 10-120 days (typically 14-21 days).

如可被通常技術人員從提供的實施例理解的，單個物種的組織培養方法包括培養基、時間和生長條件的輕微的變化。這些對於單個物種的變化需要基於包括位置、期望的產量、起始材料等因素而最佳化。 As can be understood by one of ordinary skill in the examples provided, tissue culture methods for a single species include slight changes in culture medium, time, and growth conditions. These changes to individual species need to be optimized based on factors including location, desired yield, starting materials, and the like.

對於上列實施例中提供的各個物種，特別是具體例，其各可起始及/或增殖於：b-9-i培養基、b-9-ii培養基、b-9-iii培養基、b-9-iv培養基、b-9-v培養基、強化b-9-i培養基、強化b-9-ii培養基、強化b-9-iii培養基、強化b-9-iv培養基、強化b-9-v培養基、減料b-9-i培養基(以下描述減料培養基)、減料b-9-ii培養基、減料b-9-iii培養基、減料b-9-iv 培養基、減料b-9-v培養基、CW2-i培養基、CW2-ii培養基、CW2-iii培養基、CW2-iv培養基、CW2-v培養基、強化CW2-i培養基、強化CW2-ii培養基、強化CW2-iii培養基、強化CW2-iv培養基、強化CW2-v培養基、減料CW2-i培養基、減料CW2-ii培養基、減料CW2-iii培養基、減料CW2-iv培養基、減料CW2-v培養基、b-10-i培養基、b-10-ii培養基、b-10-iii培養基、b-10-iv培養基、b-10-v培養基、強化b-10-i培養基、強化b-10-ii培養基、強化b-10-iii培養基、強化b-10-iv培養基、強化b-10-v培養基、減料b-10-i培養基、減料b-10-ii培養基、減料b-10-iii培養基、減料b-10-iv培養基、減料b-10-v培養基、b-11-i培養基、b-11-ii培養基、b-11-iii培養基、b-11-iv培養基、b-11-v培養基、強化b-11-i培養基、強化b-11-ii培養基、強化b-11-iii培養基、強化b-11-iv培養基、強化b-11-v培養基、減料b-11-i培養基、減料b-11-ii培養基、減料b-11-iii培養基、減料b-11-iv培養基、減料b-11-v培養基、b-12c-i培養基、b-12c-ii培養基、b-12c-iii培養基、b-12c-iv培養基、b-12c-v培養基、強化b-12c-i培養基、強化b-12c-ii培養基、強化b-12c-iii培養基、強化b-12c-iv培養基、強化b-12c-v培養基、減料b-12c-i培養基、減料b-12c-ii培養基、減料b-12c-iii培養基、減料b-12c-iv培養基、減料b-12c-v培養基、b-1-i培養基、b-1-ii培養基、b-1-iii培養基、b-1-iv培養基、b-1-v培養基、強化 b-1-i培養基、強化b-1-ii培養基、強化b-1-iii培養基、強化b-1-iv培養基、強化b-1-v培養基、減料b-1-i培養基、減料b-1-ii培養基、減料b-1-iii培養基、減料b-1-iv培養基、減料b-1-v培養基、b-4-i培養基、b-4-ii培養基、b-4-iii培養基、b-4-iv培養基、b-4-v培養基、強化b-4-i培養基、強化b-4-ii培養基、強化b-4-iii培養基、強化b-4-iv培養基、強化b-4-v培養基、減料b-4-i培養基、減料b-4-ii培養基、減料b-4-iii培養基、減料b-4-iv培養基、減料b-4-v培養基、b-6-i培養基、b-6-ii培養基、b-6-iii培養基、b-6-iv培養基、b-6-v培養基、強化b-6-i培養基、強化b-6-ii培養基、強化b-6-iii培養基、強化b-6-iv培養基、強化b-6-v培養基、減料b-6-i培養基、減料b-6-ii培養基、減料b-6-iii培養基、減料b-6-iv培養基、減料b-6-v培養基、CW1-i培養基、CW1-ii培養基、CW1-iii培養基、CW1-iv培養基、CW1-v培養基、強化CW1-i培養基、強化CW1-ii培養基、強化CW1-iii培養基、強化CW1-iv培養基、強化CW1-v培養基、減料CW1-i培養基、減料CW1-ii培養基、減料CW1-iii培養基、減料CW1-iv培養基、減料CW1-v培養基、CW3-i培養基、CW3-ii培養基、CW3-iii培養基、CW3-iv培養基、CW3-v培養基、強化CW3-i培養基、強化CW3-ii培養基、強化CW3-iii培養基、強化CW3-iv培養基、強化CW3-v培養基、減料CW3-i培養基、減料CW3-ii培養基、減料CW3-iii培養基、減料 CW3-iv培養基、減料CW3-v培養基、CW4-i培養基、CW4-ii培養基、CW4-iii培養基、CW4-iv培養基、CW4-v培養基、強化CW4-i培養基、強化CW4-ii培養基、強化CW4-iii培養基、強化CW4-iv培養基、強化CW4-v培養基、減料CW4-i培養基、減料CW4-ii培養基、減料CW4-iii培養基、減料CW4-iv培養基、減料CW4-v培養基、CW5-i培養基、CW5-ii培養基、CW5-iii培養基、CW5-iv培養基、CW5-v培養基、強化CW5-i培養基、強化CW5-ii培養基、強化CW5-iii培養基、強化CW5-iv培養基、強化CW5-v培養基、減料CW5-i培養基、減料CW5-ii培養基、減料CW5-iii培養基、減料CW5-iv培養基、減料CW5-v培養基、CW6-i培養基、CW6-ii培養基、CW6-iii培養基、CW6-iv培養基、CW6-v培養基、強化CW6-i培養基、強化CW6-ii培養基、強化CW6-iii培養基、強化CW6-iv培養基、強化CW6-v培養基、減料CW6-i培養基、減料CW6-ii培養基、減料CW6-iii培養基、減料CW6-iv培養基、減料CW6-v培養基、B-9N2-i培養基、B-9N2-ii培養基、B-9N2-iii培養基、B-9N2-iv培養基、B-9N2-v培養基、強化B-9N2-i培養基、強化B-9N2-ii培養基、強化B-9N2-iii培養基、強化B-9N2-iv培養基、強化B-9N2-v培養基、減料B-9N2-i培養基、減料B-9N2-ii培養基、減料B-9N2-iii培養基、減料B-9N2-iv培養基、減料B-9N2-v培養基、B-12C CPPU-i培養基、B-12C CPPU-ii培 養基、B-12C CPPU-iii培養基、B-12C CPPU-iv培養基、B-12C CPPU-v培養基、強化B-12C CPPU-i培養基、強化B-12C CPPU-ii培養基、強化B-12C CPPU-iii培養基、強化B-12C CPPU-iv培養基、強化B-12C CPPU-v培養基、減料B-12C CPPU-i培養基、減料B-12C CPPU-ii培養基、減料B-12C CPPU-iii培養基、減料B-12C CPPU-iv培養基、減料B-12C CPPU-v培養基、B-12C DPU-i培養基、B-12C DPU-ii培養基、B-12C DPU-iii培養基、B-12C DPU-iv培養基、B-12C DPU-v培養基、強化B-12C DPU-i培養基、強化B-12C DPU-ii培養基、強化B-12C DPU-iii培養基、強化B-12C DPU-iv培養基、強化B-12C DPU-v培養基、減料B-12C DPU-i培養基、減料B-12C DPU-ii培養基、減料B-12C DPU-iii培養基、減料B-12C DPU-iv培養基、減料B-12C DPU-v培養基、或其依據本文中描述各種過程之組合。 For each of the species provided in the above examples, particularly specific examples, each can be initiated and/or propagated in: b-9-i medium, b-9-ii medium, b-9-iii medium, b- 9-iv medium, b-9-v medium, fortified b-9-i medium, fortified b-9-ii medium, fortified b-9-iii medium, fortified b-9-iv medium, fortified b-9-v Medium, reduced b-9-i medium (hereinafter described as reduction medium), reduced b-9-ii medium, reduced b-9-iii medium, reduced material b-9-iv Medium, reduced b-9-v medium, CW2-i medium, CW2-ii medium, CW2-iii medium, CW2-iv medium, CW2-v medium, fortified CW2-i medium, fortified CW2-ii medium, fortified CW2 -iii medium, fortified CW2-iv medium, fortified CW2-v medium, reduced CW2-i medium, reduced CW2-ii medium, reduced CW2-iii medium, reduced CW2-iv medium, reduced CW2-v medium , b-10-i medium, b-10-ii medium, b-10-iii medium, b-10-iv medium, b-10-v medium, fortified b-10-i medium, fortified b-10-ii Medium, fortified b-10-iii medium, fortified b-10-iv medium, fortified b-10-v medium, reduced b-10-i medium, reduced b-10-ii medium, reduced material b-10- Iii medium, reduced b-10-iv medium, reduced b-10-v medium, b-11-i medium, b-11-ii medium, b-11-iii medium, b-11-iv medium, b -11-v medium, fortified b-11-i medium, fortified b-11-ii medium, fortified b-11-iii medium, fortified b-11-iv medium, fortified b-11-v medium, reduced b- 11-i medium, reduced b-11-ii medium, reduced b-11-iii medium, reduced Material b-11-iv medium, reduced b-11-v medium, b-12c-i medium, b-12c-ii medium, b-12c-iii medium, b-12c-iv medium, b-12c-v Medium, fortified b-12c-i medium, fortified b-12c-ii medium, fortified b-12c-iii medium, fortified b-12c-iv medium, fortified b-12c-v medium, reduced b-12c-i medium , reduced b-12c-ii medium, reduced b-12c-iii medium, reduced b-12c-iv medium, reduced b-12c-v medium, b-1-i medium, b-1-ii medium , b-1-iii medium, b-1-iv medium, b-1-v medium, fortification B-1-i medium, fortified b-1-ii medium, fortified b-1-iii medium, fortified b-1-iv medium, fortified b-1-v medium, reduced b-1-i medium, reduced material B-1-ii medium, reduced b-1-iii medium, reduced b-1-iv medium, reduced b-1-v medium, b-4-i medium, b-4-ii medium, b- 4-iii medium, b-4-iv medium, b-4-v medium, fortified b-4-i medium, fortified b-4-ii medium, fortified b-4-iii medium, fortified b-4-iv medium , strengthen b-4-v medium, reduce b-4-i medium, reduce b-4-ii medium, reduce b-4-iii medium, reduce b-4-iv medium, reduce b-4 -v medium, b-6-i medium, b-6-ii medium, b-6-iii medium, b-6-iv medium, b-6-v medium, fortified b-6-i medium, fortified b- 6-ii medium, fortified b-6-iii medium, fortified b-6-iv medium, fortified b-6-v medium, reduced b-6-i medium, reduced b-6-ii medium, reduced b -6-iii medium, reduced b-6-iv medium, reduced b-6-v medium, CW1-i medium, CW1-ii medium, CW1-iii medium, CW1-iv medium, CW1-v medium, fortification CW1-i medium, CW1-ii medium, fortified CW1-iii medium, fortified CW1-iv medium, fortified CW1-v medium, reduced CW1-i medium, reduced CW1-ii medium, reduced CW1-iii medium, reduced material CW1-iv Medium, reduced material CW1-v medium, CW3-i medium, CW3-ii medium, CW3-iii medium, CW3-iv medium, CW3-v medium, fortified CW3-i medium, fortified CW3-ii medium, fortified CW3-iii Medium, fortified CW3-iv medium, fortified CW3-v medium, reduced CW3-i medium, reduced CW3-ii medium, reduced CW3-iii medium, reduced material CW3-iv medium, reduced CW3-v medium, CW4-i medium, CW4-ii medium, CW4-iii medium, CW4-iv medium, CW4-v medium, fortified CW4-i medium, fortified CW4-ii medium, fortified CW4-iii medium, fortified CW4-iv medium, fortified CW4-v medium, reduced CW4-i medium, reduced material CW4-ii medium, reduced material CW4-iii medium, reduced material CW4-iv medium, reduced material CW4-v Medium, CW5-i medium, CW5-ii medium, CW5-iii medium, CW5-iv medium, CW5-v medium, fortified CW5-i medium, fortified CW5-ii medium, fortified CW5-iii medium, fortified CW5-iv medium , Strengthen CW5-v medium, reduce CW5-i medium, reduce CW5-ii medium, reduce CW5-iii medium, reduce CW5-iv medium, reduce CW5-v medium, CW6-i medium, CW6-ii Medium, CW6-iii medium, CW6-iv medium, CW6-v medium, fortified CW6-i medium, fortified CW6-ii medium, fortified CW6-iii medium, fortified CW6-iv medium, fortified CW6-v medium, reduced material CW6 -i medium, reduced CW6-ii medium, reduced CW6-iii medium, reduced CW6-iv medium, CW6-v medium, B-9N2-i medium, B-9N2-ii medium, B-9N2-iii medium, B-9N2-iv medium, B-9N2-v medium, fortified B-9N2-i medium, fortification B-9N2-ii medium, fortified B-9N2-iii medium, fortified B-9N2-iv medium, fortified B-9N2-v medium, reduced B-9N2-i medium, reduced material B-9N2-ii medium, reduced Material B-9N2-iii medium, reduced material B-9N2-iv medium, reduced material B-9N2-v medium, B-12C CPPU-i medium, B-12C CPPU-ii culture Nutrient, B-12C CPPU-iii medium, B-12C CPPU-iv medium, B-12C CPPU-v medium, fortified B-12C CPPU-i medium, fortified B-12C CPPU-ii medium, fortified B-12C CPPU -iii medium, fortified B-12C CPPU-iv medium, fortified B-12C CPPU-v medium, reduced material B-12C CPPU-i medium, reduced material B-12C CPPU-ii medium, reduced material B-12C CPPU-iii Medium, reduced material B-12C CPPU-iv medium, reduced material B-12C CPPU-v medium, B-12C DPU-i medium, B-12C DPU-ii medium, B-12C DPU-iii medium, B-12C DPU -iv medium, B-12C DPU-v medium, fortified B-12C DPU-i medium, fortified B-12C DPU-ii medium, fortified B-12C DPU-iii medium, fortified B-12C DPU-iv medium, fortified B -12C DPU-v medium, reduced material B-12C DPU-i medium, reduced material B-12C DPU-ii medium, reduced material B-12C DPU-iii medium, reduced material B-12C DPU-iv medium, reduced material B -12C DPU-v medium, or a combination thereof according to various processes described herein.

(實施例22) (Example 22) 用於脈動方法的方法及材料Method and material for pulsation method

國際專利申請公告號WO2011100762係以引用方式全部併入本文中，其係描述用於竹組織培養的一般過程。 International Patent Application Publication No. WO2011100762, which is incorporated herein by reference in its entirety, is incorporated herein by reference in its entirety herein in its entirety herein in its entirety herein in its entirety

下表描述實施例1-3中使用的芽誘導培養基和苗伸長培養基。 The following table describes the bud induction medium and the shoot elongation medium used in Examples 1-3.

(實施例23) (Example 23) 藉由脈動方法的竹微體繁殖-IBamboo micropropagation by pulsating method - I

在本實施例中，將毛竹的組織培養物接種到生物反應器中。將芽誘導培養基接種到生物反應器中並將該培養物在生長室中培養48小時。48小時之後，將芽誘導培養基用苗伸 長和維持培養基替換，並將竹植物額外培養5天的時間。將上面的週期再重複兩次而總共有三次芽誘導處理和三次苗伸長和維持處理。 In this example, a tissue culture of Phyllostachys pubescens is inoculated into a bioreactor. The shoot induction medium was inoculated into a bioreactor and the culture was cultured in a growth chamber for 48 hours. After 48 hours, shoot the medium for shoot induction Long and maintenance medium was replaced and bamboo plants were incubated for an additional 5 days. The above cycle was repeated twice more for a total of three shoot induction treatments and three shoot elongation and maintenance treatments.

在處理時間結束時，收集資料以確定培養物的增殖率。與膠狀培養基中增殖率為2X的對照處理相比，達成28X的增殖率。 At the end of the treatment time, data were collected to determine the rate of proliferation of the culture. A 28X proliferation rate was achieved compared to the control treatment in the gelatinous medium with a proliferation rate of 2X.

(實施例24) (Example 24) 藉由脈動方法的竹微體繁殖-IIBamboo micropropagation by pulsating method - II

在本實施例中，從溫室中收集毛竹的有節莖片段並在將節片段放置到芽誘導培養基之前表面滅菌。該莖片段具有單一個芽，其可以是休眠的或有活性的。在生長室中將培養物培養1小時到3週的時間，而以2週為最佳培養物培養時間。 In this example, the stalk segments of Phyllostachys pubescens were collected from the greenhouse and surface sterilized prior to placing the segment fragments into the shoot induction medium. The stem segment has a single bud which can be dormant or active. The culture was incubated in the growth chamber for a period of 1 hour to 3 weeks, and 2 weeks was the optimal culture incubation time.

該休眠的或有活性的腋芽在芽誘導階段中產生苗。在芽誘導階段結束時分離該苗芽並在芽伸長及維持培養基上培養1到3週的時間。 The dormant or active axillary buds produce shoots during the shoot induction phase. The shoot is isolated at the end of the shoot induction phase and incubated on shoot elongation and maintenance medium for a period of 1 to 3 weeks.

在此過程中，該單一個的腋芽發展成為多個苗。重複芽誘導過程和苗伸長/維持過程數次以得到足以從單一個腋芽產生100,000個植物的苗增殖率。 In this process, the single axillary bud develops into multiple seedlings. The shoot induction process and the shoot elongation/maintenance process were repeated several times to obtain a shoot growth rate sufficient to produce 100,000 plants from a single axillary shoot.

(實施例25) (Embodiment 25) 藉由脈動方法的竹微體繁殖-IIIBamboo micropropagation by pulsation method - III

在本實施例中，將毛竹的種子表面滅菌並置於芽誘導培養基。將該培養物在生長室中培養1小時到3週的時間。 In this example, the seeds of the bamboo were sterilized and placed in a bud induction medium. The culture is incubated in a growth chamber for a period of from 1 hour to 3 weeks.

種子在芽誘導培養基中發芽並產生多個苗。隨後將由種子衍生的多個苗在苗伸長/維持培養基中放置1到3週的時間。 The seeds germinate in the shoot induction medium and produce multiple shoots. Multiple seedlings derived from seeds are then placed in shoot elongation/maintenance medium for a period of 1 to 3 weeks.

將芽誘導和芽伸長/維持過程重複數次以達成足以從單一個種子培植體產生100,000個植物的苗增殖率。 The shoot induction and shoot elongation/maintenance processes were repeated several times to achieve a shoot growth rate sufficient to produce 100,000 plants from a single seed culture.

(實施例26) (Example 26) 藉由脈動方法的竹微體繁殖-VIBamboo micropropagation by pulsating method - VI

在本實施例中，將蓉城竹、缺苞箭竹、菲白竹、維氏熊竹、菲黃竹、筱竹、丘斯誇竹、白綠竹、楠竹、烏芽竹、馬來甜龍竹或狹葉瓜多竹的組織培養物接種到生物反應器中。將芽誘導培養基接種到生物反應器並將培養物在生長室中培養48小時。48小時之後，將芽誘導培養基用苗伸長和維持培養基替換，並將竹植物額外培養5天的時間。將上面的週期再重複2次，而總共有三次芽誘導處理和三次苗伸長和維持處理。 In this embodiment, Rongcheng bamboo, 苞Baozhu, Philippine white bamboo, Vickers bear bamboo, Philippine yellow bamboo, 筱 bamboo, 丘斯夸竹, white green bamboo, bamboo, bud bamboo, Malay sweet dragon bamboo Or a tissue culture of the Phyllostachys pubescens is inoculated into the bioreactor. The shoot induction medium was inoculated into the bioreactor and the culture was cultured in the growth chamber for 48 hours. After 48 hours, the shoot induction medium was replaced with shoot elongation and maintenance medium, and the bamboo plants were additionally cultured for a period of 5 days. The above cycle was repeated twice more, and there were a total of three shoot induction treatments and three shoot elongation and maintenance treatments.

在處理時間結束時，收集資料以確定培養物的增殖率。與膠狀培養基中增殖率為2X的對照處理相比，每個竹物種達成約10X到約28X的增殖率。 At the end of the treatment time, data were collected to determine the rate of proliferation of the culture. Each bamboo species achieved a proliferation rate of about 10X to about 28X compared to a control treatment with a proliferation rate of 2X in the gelatinous medium.

(實施例27) (Example 27) 藉由脈動方法的竹微體繁殖-VBamboo micro-propagation by pulsating method -V

從溫室中收集蓉城竹、缺苞箭竹、菲白竹、維氏熊竹、菲黃竹、筱竹、丘斯誇竹、白綠竹、楠竹、烏芽竹、馬來甜龍竹或狹葉瓜多竹的有節莖的片段，並在將節部分放置到芽誘 導培養基之前表面滅菌。該節的片段具有一個單一的芽，其可以是休眠的或有活性的。將該培養物在生長室中培養1小時到3週的時間，而以2週為最佳培養時間。 Collecting Rongcheng bamboo, 苞Baozhu, Philippine white bamboo, Vicki's bear bamboo, Philippine yellow bamboo, 筱 bamboo, 丘斯夸竹, white green bamboo, bamboo, bud bamboo, Malay sweet dragon bamboo or narrow leaf from the greenhouse a segment of the stalk of the cucurbit bamboo, and placing the section of the bud into the bud The surface is sterilized before the medium is introduced. Fragments of this section have a single bud which can be dormant or active. The culture was cultured in a growth chamber for a period of 1 hour to 3 weeks, and 2 weeks was the optimum culture time.

該休眠的或活性的腋芽在芽誘導階段中產生苗。在芽誘導階段結束時分離該苗芽並在芽伸長和維持培養基上培養1到3週的時間。 The dormant or active axillary buds produce shoots during the shoot induction phase. The shoot is isolated at the end of the shoot induction phase and incubated on shoot elongation and maintenance medium for a period of 1 to 3 weeks.

在此時段內，該單一個的腋芽發展成為多個苗。重複芽誘導和苗伸長/維持過程數次以得到足以從單一個腋芽產生100,000個植物的苗增殖率。 During this time period, the single axillary bud developed into multiple seedlings. The shoot induction and shoot elongation/maintenance processes were repeated several times to obtain a shoot growth rate sufficient to produce 100,000 plants from a single axillary bud.

(實施例28) (Embodiment 28) 藉由脈動方法的竹微體繁殖-VIBamboo micropropagation by pulsating method - VI

將蓉城竹、缺苞箭竹、菲白竹、維氏熊竹、菲黃竹、筱竹、丘斯誇竹、白綠竹、楠竹、烏芽竹、馬來甜龍竹或狹葉瓜多竹的種子表面消毒並置於芽誘導培養基上。將該培養物在生長室中培養1小時到3週的時間。 Will be Rongcheng bamboo, 苞Baozhu, Philippine white bamboo, Vickers bear bamboo, Philippine yellow bamboo, 筱 bamboo, 丘斯夸竹, white green bamboo, bamboo, bud bamboo, Malay sweet dragon bamboo or narrow leaf melon The seed surface is sterilized and placed on a bud induction medium. The culture is incubated in a growth chamber for a period of from 1 hour to 3 weeks.

該種子在芽誘導培養基中發芽並產生多個苗。隨後將由種子衍生的多個苗在苗伸長/維持培養基中放置1到3週的時間。 The seed germinates in the shoot induction medium and produces a plurality of shoots. Multiple seedlings derived from seeds are then placed in shoot elongation/maintenance medium for a period of 1 to 3 weeks.

將芽誘導和芽伸長/維持過程重複數次以實現足以從單一個種子培植體產生100,000個植物的苗增殖率。 The shoot induction and shoot elongation/maintenance processes were repeated several times to achieve a shoot growth rate sufficient to produce 100,000 plants from a single seed culture.

(實施例29) (Example 29) 使用生物反應器的竹微體繁殖Bamboo micropropagation using bioreactor

˙材料和方法 Materials and Method

將毛竹、毛金竹(Nigra Henon)、青川箭竹(Rufa)和紫竹(Nigra)植物的苗培養物在間歇浸沒式生物反應器中生長，如圖中描述的。使用液體的增殖培養基B11和Dic 25/30 ST。 Seedling cultures of Phyllostachys pubescens, Nigra Henon, Rufa and Nigra plants were grown in an intermittent immersion bioreactor as depicted in the figure. Liquid proliferating media B11 and Dic 25/30 ST were used.

步驟step

將約100到300個不同竹物種的微體苗(micro shoot)使用無菌技術接種到間歇浸沒式生物反應器中。將該生物反應器密封並在生長室中培養(25℃且照相週期(photo period)為16/8)。將該培養物在Dic 25/30 ST液體培養基中總共曝露5天且在B11培養基中總共2天。將培養基迴圈(脈動)重複3到6週的時間。每3週一次將培養基完全轉變為新鮮的培養基。 Micro shoots of about 100 to 300 different bamboo species were inoculated into the batch immersion bioreactor using aseptic techniques. The bioreactor was sealed and incubated in a growth chamber (25 ° C and a photo period of 16/8). The culture was exposed for a total of 5 days in Dic 25/30 ST liquid medium and 2 days in B11 medium. The medium was circulated (pulsating) for a period of 3 to 6 weeks. The medium was completely converted to fresh medium every 3 weeks.

˙結果 result

該微體苗對培養基的脈動處理有反應並快速增殖。在3到6週的培養時間內，從每個生物反應器產生了估計的數量為1000到10000的植物。 The micro-plants respond to the pulsation treatment of the medium and rapidly proliferate. An estimated number of plants from 1000 to 10,000 were produced from each bioreactor during the 3 to 6 week incubation period.

(實施例30) (Embodiment 30) 使用生物反應器的竹微體繁殖-IIBamboo micro-propagation using bioreactor-II

˙材料和方法 Materials and Method

將毛竹、毛金竹、青川箭竹、華西箭竹、秋竹(Chusquea culeou)、伯齡達竹、神農箭竹、「九寨溝」華西箭竹、菲白竹、丘斯誇竹、缺苞箭竹(Fargesia denudate)、「平武」拐棍竹、「臥龍」拐棍竹、紫竹、麻竹屬和斑葉白綠竹(Variagated Old Hamii)之植物苗培養物在間歇浸沒式生物反應器中生長，如圖式中描述。下表列出了各個竹物種的液體增殖培養基。 Bamboo, Maojinzhu, Qingchuan Jianzhu, Huaxi Jianzhu, Qiushu (Chusquea culeou), Burunda Bamboo, Shennong Jianzhu, "Jiuzhaigou" Huaxi Jianzhu, Feibaizhu, Qiuscu bamboo, Fargesia denudate, "flat" "Wu" stick bamboo, "Wolong" stick bamboo, Zizhu, Maju and variegated white green bamboo (Variagated The plant culture of Old Hamii) is grown in an intermittent immersion bioreactor as depicted in the scheme. The following table lists the liquid proliferation media for each bamboo species.

圖7顯示了B11、DIC 25/30 ST和DIC 25/30 DT的配方。 Figure 7 shows the formulation of B11, DIC 25/30 ST and DIC 25/30 DT.

˙步驟 step

將不同竹物種的微體苗使用無菌技術接種到間歇浸沒式生物反應器中。將該生物反應器密封並在生長室中培養 (25℃且照相週期是16/8)。依據表4指定的條件，將該培養物曝露於Dic 25/30 ST液體培養基和B11培養基。將培養基迴圈(脈動)重複3-6週的時間。每3週一次將該培養基完全轉變為新鮮的培養基。 Micro-plants of different bamboo species were inoculated into an intermittent immersion bioreactor using aseptic techniques. Sealing the bioreactor and culturing it in a growth chamber (25 ° C and the photo cycle is 16/8). The culture was exposed to Dic 25/30 ST liquid medium and B11 medium according to the conditions specified in Table 4. The medium was circulated (pulsating) for a period of 3-6 weeks. The medium was completely converted to fresh medium every 3 weeks.

˙結果 result

該微體苗對培養基的脈動處理有反應並快速增殖。在3到6週的培養時間內，從每個生物反應器產生了估計的數量為1000到10000的植物。 The micro-plants respond to the pulsation treatment of the medium and rapidly proliferate. An estimated number of plants from 1000 to 10,000 were produced from each bioreactor during the 3 to 6 week incubation period.

(實施例31) (Example 31) 其他植物的微體繁殖Micropropagation of other plants

˙材料和方法 Materials and Method

將天竺葵(Geranium rozanne)、金色知風草(Hakonechloa macra‘Aureola’)、金葉知風草(Hakonechloa macra‘All gold’)、聖誕玫瑰(Helleborus Ivory Prince)、紐西蘭麻(Phormium)、山葵(Wasabi C2)、大青籬竹(Arundinaria gigantea)、馬鈴薯和馬鈴薯植物的苗培養物在間歇浸沒式生物反應器中培養，如附圖式所描述。在下表中列出了每個竹物種的液體增殖培養基。 Geranium rozanne , Hakonechloa macra' Aureola ' , Hakonechloa macra 'All gold', Helleborus Ivory Prince, Phormium, Wasabi Seed cultures of C2), Arundinaria gigantea , potato and potato plants are grown in an intermittent immersion bioreactor as described in the accompanying drawings. The liquid proliferation medium for each bamboo species is listed in the table below.

B11、DKW、DIC 25/30 ST、Wasabi、DIC 25/30、Lilly light和Lilly dark的配方係如圖7所示。 The formulations of B11, DKW, DIC 25/30 ST, Wasabi, DIC 25/30, Lilly light and Lilly dark are shown in Figure 7.

˙步驟 step

將表5中列出的不同植物物種的微體苗使用無菌技術接種到間歇浸沒式生物反應器中。將該生物反應器密封並在生長室中培養(25℃且照相周期是16/8)。在依據表5的條件下，將該培養物曝露於適合的培養基。將培養基迴圈(脈動)重複3-6週的時間。每3週一次將該培養基完全轉變為新鮮的培養基。 The micro-plants of the different plant species listed in Table 5 were inoculated into the batch immersion bioreactor using aseptic technique. The bioreactor was sealed and incubated in a growth chamber (25 ° C and the photographic cycle was 16/8). The culture was exposed to a suitable medium under the conditions according to Table 5. The medium was circulated (pulsating) for a period of 3-6 weeks. The medium was completely converted to fresh medium every 3 weeks.

˙結果 result

該微體苗對培養基的脈動處理有反應並快速增殖。在3到6週的培養時間內，從每個生物反應器產生了估計的數量為1000到10000的植物。 The micro-plants respond to the pulsation treatment of the medium and rapidly proliferate. An estimated number of plants from 1000 to 10,000 were produced from each bioreactor during the 3 to 6 week incubation period.

本案所揭示發明的原理可應用到許多可能的具體例中，舉例說明的具體例應被認為僅是實例，且不應作為限制本發明的範圍。 The principles of the invention disclosed herein may be applied to a number of possible specific examples, and the specific examples are to be considered as illustrative only and are not intended to limit the scope of the invention.

除另外定義，本文中所有的技術和科學術語具有和本發明所屬的技術領域的通常技術人員一般所理解的相同的含義。儘管可在實施或本發明的測試中使用任何與本文所述類似或等同的方法和材料，本文係敘述較佳的方法和材料。 Unless otherwise defined, all technical and scientific terms used herein have the same meaning meaning meaning Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.

所有引用的公開刊物、專利和專利公開案係以引用方式全部併入本文以用於所有目的。又，說明書所引用之存在GenBank中的核酸序列和多肽序列係以引用方式併入本文。 All of the cited publications, patents, and patent publications are hereby incorporated by reference in their entirety for all purposes. Further, the presence of nucleic acid sequences and polypeptide sequences in GenBank as cited in the specification is incorporated herein by reference.

本文中討論的公開刊物僅以其所提供揭示內容在本案申請日之前者。本文中的所有內容都不應理解為以先前的發明認定本發明並非早於該公開刊物。 The publications discussed herein are solely for their disclosure prior to the filing date of the present application. Nothing in this document should be construed as a prior invention that the invention is not prior to the publication.

儘管本發明係連結其特定具體例所描述，應理解的是其能進一步修改，而且本案意圖涵蓋大致上依據本發明的原理和包括從本案揭示內容以本發明所屬領域的已知或慣例而衍伸的任何變化、用途或修改，且可應用於上文提出的基本特徵和如下附加的申請專利範圍中。 Although the present invention has been described in connection with the specific embodiments thereof, it is understood that it can be further modified, and the present invention is intended to cover the principles of the present invention and the disclosure of the present disclosure. Any variations, uses or modifications of the extensions are applicable to the basic features set forth above and in the scope of the appended claims below.

100‧‧‧植物繁殖系統，擺動架 100‧‧‧Plant breeding system, swing frame

110‧‧‧生長器皿，框架 110‧‧‧ growing vessels, frame

111‧‧‧支柱 111‧‧‧ pillar

112‧‧‧蓋 112‧‧‧ Cover

113‧‧‧驅動軸開口 113‧‧‧Drive shaft opening

114‧‧‧搖軸開口 114‧‧‧Shaft opening

115‧‧‧容積 115‧‧‧ volume

120‧‧‧上跨構件 120‧‧‧Upper components

121‧‧‧感測器支架 121‧‧‧Sensor bracket

125‧‧‧底 125‧‧‧ bottom

126‧‧‧支撐構件 126‧‧‧Support members

127‧‧‧驅動軸開口 127‧‧‧Drive shaft opening

128‧‧‧搖軸開口 128‧‧‧Shaft opening

130‧‧‧第一培養基容器 130‧‧‧First medium container

140‧‧‧架組件 140‧‧‧ components

141‧‧‧架 141‧‧‧

142‧‧‧平臺 142‧‧‧ platform

144‧‧‧支撐管 144‧‧‧Support tube

145‧‧‧連桿 145‧‧‧ Connecting rod

150‧‧‧第二培養基容器，外部軸襯 150‧‧‧Second medium container, external bushing

151‧‧‧開口 151‧‧‧ openings

155‧‧‧內部軸襯 155‧‧‧Internal bushing

170‧‧‧氣體源，驅動組件 170‧‧‧Gas source, drive assembly

171‧‧‧發動機 171‧‧‧ engine

172‧‧‧驅動軸 172‧‧‧ drive shaft

173‧‧‧傳動齒輪和 173‧‧‧Transmission gears and

180‧‧‧搖動組件 180‧‧‧Shake components

181‧‧‧搖動齒輪 181‧‧‧Shake gear

182‧‧‧搖軸 182‧‧‧Shaft axis

183‧‧‧軸承 183‧‧‧ bearing

184‧‧‧安裝托架 184‧‧‧ mounting bracket

186‧‧‧搖動軸襯 186‧‧‧Shake bushing

190‧‧‧控制器 190‧‧‧ Controller

200‧‧‧植物繁殖系統 200‧‧‧Plant reproduction system

210‧‧‧生長器皿 210‧‧‧ growing utensils

212‧‧‧閉合物 212‧‧‧ Closed

216‧‧‧柄 216‧‧‧ handle

220‧‧‧氣體交換口 220‧‧‧ gas exchange port

222‧‧‧流體交換口 222‧‧‧ fluid exchange port

226‧‧‧流體導管 226‧‧‧ Fluid conduit

230‧‧‧第一培養基容器 230‧‧‧First medium container

232‧‧‧第一流體口 232‧‧‧First fluid port

236‧‧‧第二流體口 236‧‧‧Second fluid port

238‧‧‧接合器 238‧‧‧ Adapter

240‧‧‧支管 240‧‧‧ branch

242‧‧‧管 242‧‧‧ tube

244‧‧‧管 244‧‧‧ tube

246‧‧‧閥 246‧‧‧ valve

248‧‧‧閥 248‧‧‧Valve

250‧‧‧第二培養基容器 250‧‧‧Second medium container

252‧‧‧濾器 252‧‧‧ filter

270‧‧‧氣體泵(氣體源) 270‧‧‧ gas pump (gas source)

290‧‧‧計時器控制電路 290‧‧‧Timer control circuit

300‧‧‧植物繁殖次序 300‧‧‧Plant reproduction order

302‧‧‧開始植物繁殖次序 302‧‧‧Starting plant reproduction order

304‧‧‧開始第一培養次序 304‧‧‧Starting the first training sequence

306‧‧‧建立第一培養基容器和生長器皿之間的流體連通 306‧‧‧ Establish fluid communication between the first medium container and the growing vessel

308‧‧‧執行第一操作模式 308‧‧‧Executing the first mode of operation

310‧‧‧執行第三操作模式 310‧‧‧Executing the third mode of operation

312‧‧‧第一培養次序是否完成？ 312‧‧ Is the first training order completed?

314‧‧‧開始第二培養次序 314‧‧‧Starting the second training sequence

316‧‧‧建立第二培養基容器和生長器皿之間的流體連通 316‧‧‧ Establish fluid communication between the second medium container and the growing vessel

318‧‧‧執行第二操作模式 318‧‧‧Executing the second mode of operation

320‧‧‧執行第三操作模式 320‧‧‧Executing the third mode of operation

322‧‧‧第二培養次序是否完成？ 322‧‧ Is the second training order completed?

324‧‧‧植物繁殖次序是否完成？ 324‧‧ Is the plant reproduction order completed?

326‧‧‧結束植物繁殖次序 326‧‧‧End of plant reproduction order

圖1為本發明系統之方塊圖。 Figure 1 is a block diagram of the system of the present invention.

圖2為圖1系統的非限制性具體例之示意圖。 2 is a schematic diagram of a non-limiting embodiment of the system of FIG. 1.

圖3為圖2系統的培養基容器之示意圖。 Figure 3 is a schematic illustration of the medium container of the system of Figure 2.

圖4為圖2系統的支管之示意圖。 4 is a schematic view of a branch pipe of the system of FIG. 2.

圖5A為圖2系統的生長器皿之前視圖。 Figure 5A is a front elevational view of the growth vessel of the system of Figure 2.

圖5B為圖5A的生長器皿之側視圖。 Figure 5B is a side elevational view of the growth vessel of Figure 5A.

圖5C為圖5A的生長器皿之俯視圖。 Figure 5C is a top plan view of the growth vessel of Figure 5A.

圖6為本發明的植物繁殖次序之流程圖。 Figure 6 is a flow chart showing the order of propagation of plants of the present invention.

圖7為顯示B-11、DIC 25/30 ST、DIC 25/30 DT、DKW、Wasabi、DIC 25/30、Lilly light和Lilly dark培養基配方之表，除了以克/L代表糖以外，所有的成分都以毫克/L代表。 Figure 7 is a table showing the formulation of B-11, DIC 25/30 ST, DIC 25/30 DT, DKW, Wasabi, DIC 25/30, Lilly light, and Lilly dark medium, except for gram/L for sugar, all The ingredients are expressed in mg/L.

圖8為依據一個具體例的擺動架之正面立體圖。 Fig. 8 is a front perspective view of a swing frame according to a specific example.

圖9為圖8的擺動架之側面立體圖。 Figure 9 is a side perspective view of the swing frame of Figure 8.

圖10為圖9擺動架標示為區域Z部分的放大分解圖。 Figure 10 is an enlarged exploded view of the portion of the swing frame of Figure 9 labeled as zone Z.

圖11為圖8擺動架中所包括支柱(upright)沿圖8中線4-4之橫截面圖。 Figure 11 is a cross-sectional view of the upright included in the swing frame of Figure 8 taken along line 4-4 of Figure 8.

圖12為圖8擺動架中所包括架組件之立體圖。 Figure 12 is a perspective view of the frame assembly included in the swing frame of Figure 8.

圖13為圖12架組件的部分的立體圖。 Figure 13 is a perspective view of a portion of the frame assembly of Figure 12.

圖14為圖6的架組件的部分中包括的平臺(platform)沿圖13中線7-7之橫截面圖。 Figure 14 is a cross-sectional view of the platform included in the portion of the shelf assembly of Figure 6 taken along line 7-7 of Figure 13.

圖15為圖11架組件中所包括軸襯(bushings)的立體圖。 Figure 15 is a perspective view of the bushings included in the frame assembly of Figure 11.

圖16為圖8擺動架中所包括驅動組件的分解圖。 Figure 16 is an exploded view of the drive assembly included in the swing frame of Figure 8.

圖17為圖8擺動架的部分在第一種配置下之側視圖。 Figure 17 is a side elevational view of the portion of the swing frame of Figure 8 in a first configuration.

圖18為圖8擺動架的部分在第二種配置下之側視圖。 Figure 18 is a side elevational view of the portion of the swing frame of Figure 8 in a second configuration.

圖19為圖8擺動架的部分在第三種配置下之側視圖。 Figure 19 is a side elevational view of the portion of the swing frame of Figure 8 in a third configuration.

100‧‧‧植物繁殖系統 100‧‧‧Plant reproduction system

110‧‧‧生長器皿 110‧‧‧ growing utensils

130‧‧‧第一培養基容器 130‧‧‧First medium container

150‧‧‧第二培養基容器 150‧‧‧Second medium container

170‧‧‧氣體源 170‧‧‧ gas source

190‧‧‧控制器 190‧‧‧ Controller

Claims (20)

一種體外微體繁殖竹的方法，其包含：(a)在芽誘導培養基中培養竹的組織培養物、培植體或種子以誘導苗芽的形成；(b)在苗伸長/維持培養基中培養從步驟(a)中獲得的苗芽；(c)在芽誘導培養基中培養來自步驟(b)的苗以誘導苗芽的形成；(d)在苗伸長/維持培養基中培養從步驟(c)中獲得的苗芽；及(e)重複培養步驟(c)及步驟(d)至少一個額外的迴圈；其中，所述芽誘導培養基包含有效量的噻苯隆(TDZ)或其類似物，且其中所述苗伸長/維持培養基，除了TDZ或其類似物之外，包含有效量的一種或多種細胞***素；其中，可選擇性地，步驟(a)及/或步驟(c)中之芽誘導培養基是液體培養基及/或固體培養基，且步驟(b)及/或步驟(d)中之苗伸長/維持培養基是液體培養基及/或固體培養基。 A method for microbial propagation of bamboo in vitro, comprising: (a) cultivating a tissue culture, a plant or a seed of bamboo in a shoot induction medium to induce formation of a shoot; (b) cultivating in a shoot elongation/maintenance medium The shoot bud obtained in the step (a); (c) cultivating the shoot from the step (b) in the shoot induction medium to induce the formation of the shoot; (d) cultivating in the shoot elongation/maintenance medium from the step (c) The obtained shoots; and (e) repeating the culture step (c) and the step (d) at least one additional loop; wherein the shoot induction medium comprises an effective amount of thidiazuron (TDZ) or an analogue thereof, and Wherein the shoot elongation/maintenance medium comprises, in addition to TDZ or an analogue thereof, an effective amount of one or more cytokinins; wherein, optionally, the buds in step (a) and/or step (c) The induction medium is a liquid medium and/or a solid medium, and the seedling elongation/maintenance medium in step (b) and/or step (d) is a liquid medium and/or a solid medium. 如申請專利範圍第1項之方法，其中，步驟(e)係重複至少一次。 The method of claim 1, wherein the step (e) is repeated at least once. 如申請專利範圍第1項之方法，其中，所述之竹係為毛竹(Phyllostachys edulisi‘Moso’)、蓉城竹(Phyllostachys bissetti)、缺苞箭竹(Fargesia denudata)、菲白竹(Pleioblastus fortunei)、維氏熊竹(Sasa Veitchii)、菲黃竹(Pleioblastus viridistriatus)、筱竹(Thamnocalamus crassinodus)、丘斯誇竹(Chusquea Culeo“Cana Prieta”)、白綠竹(Bambusa Old Hamii)、楠竹(Phyllostachys Moso)、烏芽竹(Phyllostachys Atrovaginata)、馬來甜龍竹(Dendrocalamus Asper)或狹葉瓜多竹(Guadua Angustifolia)、紫竹(Phylostachys Nigra)、青川箭竹(Fargesia rufa)、華西箭竹(Fargesia nitida)、伯齡達竹(Borinda Boliana)、神農箭竹(Fargesia murielae)、菲白竹(Pleioblastus fortune)、拐棍竹(Fargesia robusta)及白綠竹(Bambusa Oldhamii)。 The method of claim 1, wherein the bamboo system is Phyllostachys edulisi 'Moso', Phyllostachys bissetti , Fargesia denudata , Pleioblastus fortunei , and Dimensions Sasa Veitchii , Pleioblastus viridistriatus , Thamnocalamus crassinodus , Chusquea Culeo “Cana Prieta” , Bambusa Old Hamii , Phyllostachys Moso ), Phyllostachys Atrovaginata , Dendrocalamus Asper or Guadua Angustifolia , Phylostachys Nigra , Fargesia rufa , Fargesia nitida , Borinda Boliana , Fargesia murielae , Pleioblastus fortune , Fargesia robusta , and Bambusa Oldhamii . 如申請專利範圍第1項之方法，其中，所述之竹培植體是竹莖的節，其中該竹莖的節係可選擇性地包含節間。 The method of claim 1, wherein the bamboo plant is a knot of a bamboo stem, wherein the knot of the bamboo stem optionally comprises an internode. 如申請專利範圍第1項之方法，其中，該芽誘導培養基中TDZ或其類似物的濃度是約0.5 mg/L至約2 mg/L。 The method of claim 1, wherein the concentration of TDZ or an analog thereof in the bud induction medium is from about 0.5 mg/L to about 2 mg/L. 如申請專利範圍第1項之方法，其中，該苗伸長/維持培養基中除了TDZ或其類似物之外，所述之一種或多種細胞***素係選自由N⁶-苄胺基嘌呤(BAP)、間拓樸林(mT)、玉米素、激動素、2-異戊烯腺嘌呤(2ip)、腺嘌呤半硫酸鹽、二甲基烯丙基腺嘌呤、N-(2-氯-4-吡啶基)-N'-苯基脲(4-CPPU)以及其各自的類似物所組成之群，其中，可選擇性地，該苗伸長/維持培養基中除了TDZ或其類似物之外的一種或多種細胞***素的濃度是從約0.25 mg/L至約5 mg/L。 The method of claim 1, wherein the one or more cytokinins of the seedling elongation/maintenance medium are selected from the group consisting of N ⁶ -benzylaminopurine (BAP), in addition to TDZ or an analog thereof. , meta-forest (mT), zeatin, kinetin, 2-isopentene adenine (2ip), adenine hemisulfate, dimethylallyl adenine, N-(2-chloro-4- a group consisting of pyridyl)-N'-phenylurea (4-CPPU) and its respective analogs, wherein, optionally, one of the seedling elongation/maintenance medium other than TDZ or the like The concentration of the plurality of cytokinins is from about 0.25 mg/L to about 5 mg/L. 如申請專利範圍第1項之方法，其中，該芽誘導培養基及/或苗伸長/維持培養基進一步包含一種或多種生長素，其中，可選擇性地，該一種或多種生長素係選自由β-萘氧乙酸(NAA)、2,4-二氯苯氧乙酸(2,4-D)、吲哚-3-丁酸(IBA)、吲哚-3-乙酸(IAA)、毒莠定(picloram)及其各自的類似物所組成之群。 The method of claim 1, wherein the bud induction medium and/or the shoot elongation/maintenance medium further comprises one or more auxins, wherein, optionally, the one or more auxins are selected from the group consisting of β- Naphthoxyacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), picoram a group of their respective analogs. 一種系統，其包含：用於在無菌或基本上無菌的環境中培養植物組織的生長器皿；具有第一流體口及第二流體口的第一培養基容器，該第一流體口與該生長器皿是流體可連接的；具有第一流體口和第二流體口的第二培養基容器，該第一流體口與該生長器皿是流體可連接的；氣體源，其與該第一培養基容器的第二流體口及該第二培養基容器的第二流體口是流體可連接的；以及控制器，其係可以第一操作模式和第二操作模式操作，該第一操作模式係將加壓氣體從氣體源輸送到第一培養基容器以將其中含有的第一體積的液體置換到生長器皿，以及該第二操作模式係將加壓氣體從氣體源輸送到第二培養基容器，以將其中含有的第二體積的液體置換到生長器皿。 A system comprising: a growth vessel for cultivating plant tissue in a sterile or substantially sterile environment; a first culture vessel having a first fluid port and a second fluid port, the first fluid port and the growth vessel being a fluid connectable; a second medium container having a first fluid port and a second fluid port, the first fluid port being fluidly connectable to the growth vessel; a gas source, the second fluid of the first medium container a second fluid port of the port and the second medium container is fluidly connectable; and a controller operable in the first mode of operation and the second mode of operation, the first mode of operation is to deliver pressurized gas from the source of gas Passing to the first medium container to displace the first volume of liquid contained therein to the growth vessel, and the second mode of operation transporting pressurized gas from the gas source to the second medium container to contain the second volume contained therein The liquid is displaced into the growing vessel. 如申請專利範圍第8項之系統，其中，該控制器係可以第三操作模式操作，該第三操作模式係允許生長器皿中含有 的液體從生長器皿流至第一培養基容器及第二培養基容器中至少一者。 The system of claim 8, wherein the controller is operable in a third mode of operation, the third mode of operation is allowed to be contained in the growth vessel The liquid flows from the growth vessel to at least one of the first medium container and the second medium container. 如申請專利範圍第9項之系統，其中，該控制器係可以第一培養次序操作，該第一培養次序係在第一操作模式之後執行該第三操作模式。 A system of claim 9, wherein the controller is operable in a first culture sequence that is performed after the first mode of operation. 如申請專利範圍第10項之系統，其中，該控制器係可以第二培養次序操作，該第二培養次序係在第二操作模式之後執行該第三操作模式。 A system of claim 10, wherein the controller is operable in a second culture sequence that is performed after the second mode of operation. 如申請專利範圍第11項之系統，其中，該控制器係進一步可以植物繁殖模式操作，該植物繁殖模式係執行該第一培養次序及該第二培養次序。 The system of claim 11, wherein the controller is further operable in a plant propagation mode, the plant propagation mode performing the first culture sequence and the second culture sequence. 如申請專利範圍第8項之系統，其進一步包含一支管，其係與該生長器皿、該第一培養基容器及該第二培養基容器流體可連接，其中，該支管可操作以控制該生長器皿及該第一培養基容器之間，以及該生長器皿及該第二培養基容器之間的液體流動。 The system of claim 8 further comprising a tube fluidly connectable to the growth vessel, the first medium container, and the second medium container, wherein the branch is operable to control the growth vessel and Liquid flow between the first medium container and between the growth vessel and the second medium container. 一種用於植物組織微體繁殖於生物反應器中交換液體培養基之方法，該生物反應器包含用於培養植物組織的生長器皿、與生長器皿流體可連接的第一培養基容器、與生長器皿流體可連接的第二培養基容器、以及與第一培養基容器及第二培養基容器流體可連接的氣體源，所述方法包括： 建立第一培養基容器及生長器皿之間的流體連通；將第二培養基容器與生長器皿流體分離；建立氣體源及第一培養基容器之間的流體連通；將壓縮氣體輸送到第一培養基容器以將第一體積液體從第一培養基容器置換到生長器皿；允許至少一部分的第一體積液體從生長器皿流回到第一培養基容器中；建立第二培養基容器及生長器皿之間的流體連通；將第一培養基容器與生長器皿流體分離；建立氣體源及第二培養基容器之間的流體連通；將壓縮氣體輸送到第二培養基容器以將第二體積液體從第一培養基容器置換到生長器皿；及允許至少一部分的第二體積液體從生長器皿流回到第二培養基容器中。 A method for exchanging a liquid medium in a bioreactor for plant tissue micro-propagation, the bioreactor comprising a growth vessel for cultivating plant tissue, a first medium container connectable to the growth vessel fluid, and a fluid for growing the vessel a second medium container connected, and a gas source connectable to the first medium container and the second medium container, the method comprising: Establishing fluid communication between the first medium container and the growth vessel; separating the second medium container from the growth vessel fluid; establishing fluid communication between the gas source and the first medium container; delivering the compressed gas to the first medium container to Displacement of the first volume of liquid from the first medium container to the growth vessel; allowing at least a portion of the first volume of liquid to flow from the growth vessel back into the first medium container; establishing fluid communication between the second medium container and the growth vessel; Dissolving a medium container fluidly with the growth vessel; establishing fluid communication between the gas source and the second medium container; delivering the compressed gas to the second medium container to displace the second volume of liquid from the first medium container to the growth vessel; and allowing At least a portion of the second volume of liquid flows from the growth vessel back into the second medium container. 一種用於微體繁殖竹之培養基，其中，該培養基係包含間拓樸林或其類似物及至少一種或至少兩種其他細胞***素，其中該培養基係維持至少6個月的增殖週期。 A medium for micropropagating bamboo, wherein the medium comprises metatopia or an analogue thereof and at least one or at least two other cytokinins, wherein the medium maintains a proliferation cycle of at least 6 months. 如申請專利範圍第15項之培養基，其中，該培養基包含至少一種生長素或至少兩種生長素，其中，可選擇性地，該培養基係選自下列所組成之群：(1)包含NAA和BAP或其類似物的培養基；(2)包含NAA和噻苯隆或其類似物的培養基； (3)包含NAA和IBA或其類似物的培養基；(4)包含NAA和2ip或其類似物的培養基；(5)包含BAP和噻苯隆或其類似物的培養基；(6)包含BAP和IBA或其類似物的培養基；(7)包含BAP和2ip或其類似物的培養基；(8)包含IBA和噻苯隆或其類似物的培養基；(9)包含IBA和2ip或其類似物的培養基；(10)包含噻苯隆和2ip或其類似物的培養基；(11)包含NAA、BAP和間拓樸林或其類似物的培養基，如標準或強化CW2培養基、標準或強化CW1培養基、或標準或強化b-10培養基；(12)包含NAA、BAP、間拓樸林和噻苯隆或其類似物的培養基，如標準或強化b-12c培養基、標準或強化b-9培養基、標準或強化b-11培養基、或標準或強化B-9N2培養基；(13)包含NAA、BAP、間拓樸林和噻苯隆和CCPU及/或DPU或其類似物的培養基，如標準或強化B-12C CPPU培養基或標準或強化B-12C CPU培養基；(14)包含NAA、BAP、間拓樸林和IBA或其類似物的培養基，如標準或強化CW3或標準或強化CW5培養基；(15)包含NAA、BAP、間拓樸林、噻苯隆和IBA或其類似物的培養基，如標準或強化CW4或標準或強化CW6培養基； (16)包含NAA、BAP、噻苯隆和2ip或其類似物的培養基，如標準或強化b-1或標準或強化b-4培養基；(17)包含NAA、噻苯隆和2ip或其類似物的培養基，如標準或強化b-6培養基；或(18)包含以下、基本上由以下組成或由以下所組成的培養基：強化b-9-i培養基、強化b-9-ii培養基、強化b-9-iii培養基、強化b-9-iv培養基、強化b-9-v培養基、減料b-9-i培養基、減料b-9-ii培養基、減料b-9-iii培養基、減料b-9-iv培養基、減料b-9-v培養基、強化CW2-ii培養基、強化CW2-iii培養基、強化CW2-iv培養基、強化CW2-v培養基、減料CW2-i培養基、減料CW2-ii培養基、減料CW2-iii培養基、減料CW2-iv培養基、減料CW2-v培養基、強化b-10-i培養基、強化b-10-ii培養基、強化b-10-iii培養基、強化b-10-iv培養基、強化b-10-v培養基、減料b-10-i培養基、減料b-10-ii培養基、減料b-10-iii培養基、減料b-10-iv培養基、減料b-10-v培養基、強化b-11-i培養基、強化b-11-ii培養基、強化b-11-iii培養基、強化b-11-iv培養基、強化b-11-v培養基、減料b-11-i培養基、減料b-11-ii培養基、減料b-11-iii培養基、減料b-11-iv培養基、減料b-11-v培養基、強化b-12c-i培養基、強化b-12c-ii培養基、強化b-12c-iii培養基、強化b-12c-iv培養基、強化b-12c-v培養基、減料b-12c-i培養基、減料b-12c-ii培養基、減料b-12c-iii 培養基、減料b-12c-iv培養基、減料b-12c-v培養基、強化b-1-i培養基、強化b-1-ii培養基、強化b-1-iii培養基、強化b-1-iv培養基、強化b-1-v培養基、減料b-1-i培養基、減料b-1-ii培養基、減料b-1-iii培養基、減料b-1-iv培養基、減料b-1-v培養基、強化b-4-i培養基、強化b-4-ii培養基、強化b-4-iii培養基、強化b-4-iv培養基、強化b-4-v培養基、減料b-4-i培養基、減料b-4-ii培養基、減料b-4-iii培養基、減料b-4-iv培養基、減料b-4-v培養基、強化b-6-i培養基、強化b-6-ii培養基、強化b-6-iii培養基、強化b-6-iv培養基、強化b-6-v培養基、減料b-6-i培養基、減料b-6-ii培養基、減料b-6-iii培養基、減料b-6-iv培養基、減料b-6-v培養基、強化CW1-i培養基、強化CW1-ii培養基、強化CW1-iii培養基、強化CW1-iv培養基、強化CW1-v培養基、減料CW1-i培養基、減料CW1-ii培養基、減料CW1-iii培養基、減料CW1-iv培養基、減料CW1-v培養基、強化CW3-i培養基、強化CW3-ii培養基、強化CW3-iii培養基、強化CW3-iv培養基、強化CW3-v培養基、減料CW3-i培養基、減料CW3-ii培養基、減料CW3-iii培養基、減料CW3-iv培養基、減料CW3-v培養基、強化CW4-i培養基、強化CW4-ii培養基、強化CW4-iii培養基、強化CW4-iv培養基、強化CW4-v培養基、減料CW4-i培養基、減料CW4-ii培養基、減料CW4-iii培養基、減料CW4-iv培養基、減料CW4-v培養基、 強化CW5-i培養基、強化CW5-ii培養基、強化CW5-iii培養基、強化CW5-iv培養基、強化CW5-v培養基、減料CW5-i培養基、減料CW5-ii培養基、減料CW5-iii培養基、減料CW5-iv培養基、減料CW5-v培養基、強化CW6-i培養基、強化CW6-ii培養基、強化CW6-iii培養基、強化CW6-iv培養基、強化CW6-v培養基、減料CW6-i培養基、減料CW6-ii培養基、減料CW6-iii培養基、減料CW6-iv培養基、減料CW6-v培養基、B-9N2-i培養基、B-9N2-ii培養基、B-9N2-iii培養基、B-9N2-iv培養基、B-9N2-v培養基、強化B-9N2-i培養基、強化B-9N2-ii培養基、強化B-9N2-iii培養基、強化B-9N2-iv培養基、強化B-9N2-v培養基、減料B-9N2-i培養基、減料B-9N2-ii培養基、減料B-9N2-iii培養基、減料B-9N2-iv培養基、減料B-9N2-v培養基、B-12C CPPU-i培養基、B-12C CPPU-ii培養基、B-12C CPPU-iii培養基、B-12C CPPU-iv培養基、B-12C CPPU-v培養基、強化B-12C CPPU-i培養基、強化B-12C CPPU-ii培養基、強化B-12C CPPU-iii培養基、強化B-12C CPPU-iv培養基、強化B-12C CPPU-v培養基、減料B-12C CPPU-i培養基、減料B-12C CPPU-ii培養基、減料B-12C CPPU-iii培養基、減料B-12C CPPU-iv培養基、減料B-12C CPPU-v培養基、B-12C DPU-i培養基、B-12C DPU-ii培養基、B-12C DPU-iii培養基、B-12C DPU-iv培養基、B-12C DPU-v培養基、強 化B-12C DPU-i培養基、強化B-12C DPU-ii培養基、強化B-12C DPU-iii培養基、強化B-12C DPU-iv培養基、強化B-12C DPU-v培養基、減料B-12C DPU-i培養基、減料B-12C DPU-ii培養基、減料B-12C DPU-iii培養基、減料B-12C DPU-iv培養基或減料B-12C DPU-v培養基。 The medium according to claim 15, wherein the medium comprises at least one auxin or at least two auxins, wherein, optionally, the medium is selected from the group consisting of: (1) comprising NAA and a medium for BAP or an analog thereof; (2) a medium comprising NAA and thidiazuron or an analogue thereof; (3) a medium comprising NAA and IBA or an analogue thereof; (4) a medium comprising NAA and 2ip or an analogue thereof; (5) a medium comprising BAP and thidiazuron or an analogue thereof; (6) comprising BAP and a medium for IBA or an analog thereof; (7) a medium comprising BAP and 2ip or an analogue thereof; (8) a medium comprising IBA and thidiazuron or an analogue thereof; (9) comprising IBA and 2ip or an analogue thereof a medium; (10) a medium comprising thidiazuron and 2ip or an analogue thereof; (11) a medium comprising NAA, BAP and metatopia or an analogue thereof, such as a standard or fortified CW2 medium, a standard or fortified CW1 medium, Or standard or fortified b-10 medium; (12) medium containing NAA, BAP, metatopolin and thidiadiam or analogues thereof, such as standard or fortified b-12c medium, standard or fortified b-9 medium, standard Or fortified b-11 medium, or standard or fortified B-9N2 medium; (13) medium containing NAA, BAP, metatopolin and thiabendazole and CCPU and/or DPU or analogues thereof, such as standard or fortified B -12C CPPU medium or standard or fortified B-12C CPU medium; (14) contains NAA, BAP, metatopia and IBA or analogues thereof Nutrients such as standard or fortified CW3 or standard or fortified CW5 media; (15) media containing NAA, BAP, metatopolin, thiabendone and IBA or analogues thereof, such as standard or fortified CW4 or standard or fortified CW6 Medium (16) A medium comprising NAA, BAP, thidiazuron and 2ip or an analogue thereof, such as standard or fortified b-1 or standard or fortified b-4 medium; (17) comprising NAA, thidiazuron and 2ip or the like a medium, such as a standard or fortified b-6 medium; or (18) a medium comprising, consisting essentially of, or consisting of: fortified b-9-i medium, fortified b-9-ii medium, fortified B-9-iii medium, fortified b-9-iv medium, fortified b-9-v medium, reduced b-9-i medium, reduced b-9-ii medium, reduced b-9-iii medium, Reduce b-9-iv medium, reduce b-9-v medium, strengthen CW2-ii medium, strengthen CW2-iii medium, strengthen CW2-iv medium, strengthen CW2-v medium, reduce CW2-i medium, reduce CW2-ii medium, reduced CW2-iii medium, reduced CW2-iv medium, reduced CW2-v medium, fortified b-10-i medium, fortified b-10-ii medium, fortified b-10-iii medium , strengthen b-10-iv medium, strengthen b-10-v medium, reduce b-10-i medium, reduce b-10-ii medium, reduce b-10-iii medium, reduce b-10- Iv medium, reduced b-10-v medium, B-11-i medium, fortified b-11-ii medium, fortified b-11-iii medium, fortified b-11-iv medium, fortified b-11-v medium, reduced b-11-i medium, reduced Material b-11-ii medium, reduced b-11-iii medium, reduced b-11-iv medium, reduced b-11-v medium, fortified b-12c-i medium, fortified b-12c-ii medium , strengthen b-12c-iii medium, strengthen b-12c-iv medium, strengthen b-12c-v medium, reduce b-12c-i medium, reduce b-12c-ii medium, reduce b-12c-iii Medium, reduced b-12c-iv medium, reduced b-12c-v medium, fortified b-1-i medium, fortified b-1-ii medium, fortified b-1-iii medium, fortified b-1-iv Medium, fortified b-1-v medium, reduced b-1-i medium, reduced b-1-ii medium, reduced b-1-iii medium, reduced b-1-iv medium, reduced b- 1-v medium, fortified b-4-i medium, fortified b-4-ii medium, fortified b-4-iii medium, fortified b-4-iv medium, fortified b-4-v medium, reduced b-4 -i medium, reduced b-4-ii medium, reduced b-4-iii medium, reduced b-4-iv medium, reduced b-4-v medium, fortified b-6-i medium, fortified b -6-ii medium, fortified b-6-iii medium, fortified b-6-iv medium, fortified b-6-v medium, reduced b-6-i medium, reduced b-6-ii medium, reduced material B-6-iii medium, reduced b-6-iv medium, reduced b-6-v medium, fortified CW1-i medium, fortified CW1-ii medium, fortified CW1-iii medium, fortified CW1-iv medium, fortified CW1-v medium, reduced material CW1-i medium, reduced material CW1-ii medium, reduced material CW1-iii medium, reduced material CW1-iv medium, reduced material CW 1-v medium, fortified CW3-i medium, fortified CW3-ii medium, fortified CW3-iii medium, fortified CW3-iv medium, fortified CW3-v medium, reduced CW3-i medium, reduced CW3-ii medium, reduced CW3-iii medium, reduced CW3-iv medium, reduced CW3-v medium, fortified CW4-i medium, fortified CW4-ii medium, fortified CW4-iii medium, fortified CW4-iv medium, fortified CW4-v medium, Reduced CW4-i medium, reduced CW4-ii medium, reduced CW4-iii medium, reduced CW4-iv medium, reduced CW4-v medium, Strengthen CW5-i medium, strengthen CW5-ii medium, strengthen CW5-iii medium, strengthen CW5-iv medium, strengthen CW5-v medium, reduce CW5-i medium, reduce CW5-ii medium, reduce CW5-iii medium , reducing CW5-iv medium, reducing CW5-v medium, strengthening CW6-i medium, strengthening CW6-ii medium, strengthening CW6-iii medium, strengthening CW6-iv medium, strengthening CW6-v medium, reducing CW6-i Medium, reduced material CW6-ii medium, reduced material CW6-iii medium, reduced material CW6-iv medium, reduced material CW6-v medium, B-9N2-i medium, B-9N2-ii medium, B-9N2-iii medium , B-9N2-iv medium, B-9N2-v medium, fortified B-9N2-i medium, fortified B-9N2-ii medium, fortified B-9N2-iii medium, fortified B-9N2-iv medium, fortified B- 9N2-v medium, reduced material B-9N2-i medium, reduced material B-9N2-ii medium, reduced material B-9N2-iii medium, reduced material B-9N2-iv medium, reduced material B-9N2-v medium, B-12C CPPU-i medium, B-12C CPPU-ii medium, B-12C CPPU-iii medium, B-12C CPPU-iv medium, B-12C CPPU-v medium, fortified B-12C CPPU-i medium, fortified B-12C C PPU-ii medium, B-12C CPPU-iii medium, B-12C CPPU-iv medium, B-12C CPPU-v medium, B-12C CPPU-i medium, B-12C CPPU-ii Medium, reduced material B-12C CPPU-iii medium, reduced material B-12C CPPU-iv medium, reduced material B-12C CPPU-v medium, B-12C DPU-i medium, B-12C DPU-ii medium, B- 12C DPU-iii medium, B-12C DPU-iv medium, B-12C DPU-v medium, strong B-12C DPU-i medium, B-12C DPU-ii medium, B-12C DPU-iii medium, B-12C DPU-iv medium, B-12C DPU-v medium, B-12C DPU-i medium, reduced B-12C DPU-ii medium, reduced B-12C DPU-iii medium, reduced B-12C DPU-iv medium or reduced B-12C DPU-v medium. 一種微體繁殖竹之方法，其包含在申請專利範圍第15及16項中任一項的培養基中培養竹培植體及/或苗。 A method of micropropagating bamboo, which comprises cultivating a bamboo plant and/or seedling in a medium of any one of claims 15 and 16. 如申請專利範圍第17項之方法，其中，所述之竹是蓉城竹(Phyllostachys bissetti)；缺苞箭竹(Fargesia denudata)；菲白竹(Pleioblastus fortunei)；維氏熊竹(Sasa Veitchii)；菲黃竹(Pleioblastus viridistriatus)；筱竹(Thamnocalamus crassinodus)；丘斯誇竹(Chusquea Culeo“Cana Prieta”)；白綠竹(Bambusa Old Hamii)；楠竹(Phyllostachys Moso)；烏芽竹(Phyllostachys Atrovaginata)；馬來甜龍竹(Dendrocalamus Asper)；狹葉瓜多竹(Guadua Angustifolia)，大青籬竹(Arundinaria gigantea)；巴苦竹(Bambusa balcoa)；龍頭竹(Bambusa vulgaris)；黃金間碧竹(Bambusa vulgaris ‘Vitatta’)；白綠竹(Bambusa Oldhamii)；馬甲竹(Bambusa tulda)；勃氏甜龍竹(Dendrocalamus brandesii)；馬來甜龍竹(Dendrocalamus asper)；版納甜龍竹(Dendrocalamus hamiltoni)；龍竹(Dendrocalamus giganteus)；黃竹(Dendrocalamus membranaceus)；印度實竹(Dendrocalamus strictus)；菲律賓巨草竹(Gigantochloa aspera)；小黑竹(Gigantochloa scortechini)；瓜多竹(Guadua culeata)；尼加拉瓜瓜多竹(Guadua aculeata ‘Nicaragua’)；抱葉瓜多竹(Guadua amplexifolia)；狹葉瓜多竹(Guadua angustifolia)；雙色狹葉瓜多竹(Guadua angustofolia bi-color)；圓錐瓜多竹(Guadua paniculata)；梨竹(Melocanna bambusoides)；李海竹(Neohouzeaua dullooa(Teinostachyum))；蘆葦竹(Ochlandra travancorica)；毛竹(Phyllostachys edulis ‘Moso’)；紫竹(Phyllostachys nigra)；毛金竹(Phyllostachys nigra‘Henon’)；或思勞竹(Schizostachyum lumampao)。 The method of claim 17, wherein the bamboo is Phyllostachys bissetti ; Fargesia denudata ; Pleioblastus fortunei ; Sasa Veitchii ; Bamboo ( Pleioblastus viridistriatus ); Thamnocalamus crassinodus ; Chusquea Culeo "Cana Prieta" ; Bambusa Old Hamii ; Phyllostachys Moso ; Phyllostachys Atrovaginata ; Dendrocalamus Asper ; Guadua Angustifolia , Arundinaria gigantea ; Bambusa balcoa ; Bambusa vulgaris ; Bambusa vulgaris 'Vitatta' ); Bambusa Oldhamii ; Bambusa tulda ; Dendrocalamus brandesii ; Dendrocalamus asper ; Dendrocalamus hamiltoni ; bamboo (Dendrocalamus giganteus); Wong Chuk (Dendrocalamus membranaceus); India is bamboo (Dendrocalamus strictus); Philippines giant grass (Gigantochloa aspera); black bamboo (Gigantochloa scortechini); melon multi bamboo (Guadua culeata); Nicaragua melon and more bamboo (Guadua aculeata 'Nicaragua'); hold multi-leaf melon bamboo (Guadua amplexifolia); narrow melon and more bamboo leaves (Guadua Angustifolia ); Guadua angustofolia bi-color ; Guadua paniculata ; Melocanna bambusoides ; Neohouzeaua dullooa (Teinostachyum ); Reed bamboo ( Ochlandra travancorica ); ( Phyllostachys edulis 'Moso' ); Phyllostachys nigra ; Phyllostachys nigra'Henon' ; or Schizostachyum lumampao . 如申請專利範圍第17項之方法，其中，該方法包括將竹培植體及/或苗依序地在強化培養基及減料培養基中培養，或依序地在減料培養基及強化培養基中培養。 The method of claim 17, wherein the method comprises sequentially cultivating the bamboo plant and/or the seedling in the fortification medium and the medium, or sequentially culturing the medium and the medium. 一種用於植物繁殖之架，其如說明書實質地描述及顯示者。 A rack for plant reproduction, which is substantially described and displayed as described in the specification.