1242405 A7 _ B7 五、發明説明（1 ) 爱』月領域 本發明係有關植物耕種、植物選殖（cloning)和園藝。特 定言之，本發明係有關玫瑰植物營養繁殖或組織培養及有 關衍生自該植物或組織培養物的玫瑰幼樹之試管内謗導開 花。更特別者，本發明係有關玫瑰和玫瑰植物試管内微型 繁殖、花蕾謗發和開花所用培養基配方和有效率方法。 I明背景 玫瑰植物屬於薔薇科（RosaceaeJuss family)植物。此科非 常龐大，有超過100屬和2000草本到木本種植物爲其成員。 許多重要的食用和裝飾用植物都在薔薇科内，包括例如草 莓’蘋果，杏仁，樓桃，桃子和黑莓。包括在現代玫瑰發 展中的正確物種已不得而知。大多數玫瑰品種都出現在北 半球的溫帶區，特別是從中國南部和遠東，到喜瑪拉雅和 孟加拉進入伊索匹亞且西進到北美從北極圈到新墨西哥。 在1 8世紀末從遠東傳到歐洲的重複或終年無間斷開花之玫 瑰植物已是在中國，印度，和日本無數代育種之結果。玫 瑰是全世界最普遍的花卉之一。玫瑰常以插木，盆栽植物 ’或在居家庭院中的樣品植物之形式生長。有許多插木玫 瑰類型’包括例如長莖標準，短莖標準，小花情人（small flowered sweetheart)和多花束等玫瑰。 由於大部分現行商業玫瑰變種都是雜交種，因此從種子 來繁殖不是一種可行的抉擇，主要係因爲在不同代之間有 屬性分離現象之故。雜交種插木玫瑰的繁殖通常係經由接 芽法、或將特別的嫩枝小枝，或切枝，插枝到既有的野生 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐) 1242405 A7 B7 五、發明説明（2 ) 玫瑰根莖上。對於小型盆栽玫瑰，在溫室中植物切枝的野 外無性繁殖爲常用的做法。上述諸方法的替代做法爲微型 繁殖（micro-propagation)，也稱爲腋芽繁殖或試管内無性繁 殖，其爲在無菌消毒狀況下從腋芽繁殖植物之方法。微型 繁殖具有將合意的植物快速繁殖成許多遺傳上一致的幼樹 之優點。微型繁殖所得幼樹也不會有細菌、眞菌和病毒等 感染且也不會有蟲害。微型繁殖程序包括從相關植物製備 活組織（explant)，在補充著植物激素的培養基上栽培該活 組織，培育，及收取與類型一致的嫩枝（或有根的嫩枝） [Douglas，"Methods in Molecular Biology，Vol. 6，W. Pollard， J. M. Walker, eds. (1990); George and Sherrington, Exegetics, Ltd. U. K·，p. 3 (1984);和Brown and Thorpe, In: Cell Culture and Somatic Cell Genetics of Plants，p49-65，I. K. Vasil，ed. (1986)]。微型繁殖提供某些植物大量繁殖的潛在成本效益 手段。此項技術的一項重要考慮事項爲對所繁殖的植物添 加明顯的經濟價値。相關文獻的探討揭露出現有的繁殖方 案在謗導幼樹試管内花栽培上通常很沒有效率。效率係決 定於所用的植物變種，不過即使對於最好的育種其效率也 仍然爲低者。 於最近數年中，高度爲7.5公分到20公分的盆栽玫瑰變 種因其小型尺寸及相對於插木玫瑰較長的貯存期而變得非 常受歡迎。此外，以合理價格產生多次花之能力更拓展盆 栽玫瑰的普遍性。對於迷你玫瑰雜交種的育種也在全世界 進行中。舉例而言，單在美國就有300種以上的迷你玫瑰雜 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐) 1242405 A7 B7 五、發明説明（3 ) 交種登記有案。對於商業迷你玫瑰雜交種最有名的育種者 爲丹麥的Poulsen Roser ApS(註册商標PARADE®)和荷蘭De Ruiter的New Rose International。迷你盆栽玫瑰係爲裝飾效 用顯示在花園、中庭或甚至於在家中。不過，迷你盆栽玫 瑰用爲室内裝飾上十分受到限制，主要是因爲對於盆内土 壤的要求之故。此外，在考慮1 5 - 3 0公分之間的總平均高 度（包括盆子）之下，即使是迷你盆栽植物的高度對於放置 在，例如，桌子上也仍然太高。另外，由於普世增加的都 市化，有愈來愈多的人花在例如家庭或辦公室中愈多的上 等品質户内時間。因此，目前對於乾淨小型，具有長貯存 期，開多次花的裝飾用玫瑰植物有其需求存在著。 玫瑰的商業繁殖通常是經由將特別的幼芽切枝接芽或插 枝到既有的野生玫瑰根莖上。於最近數年内，有許多文章 探討透過组織培養的玫瑰繁殖。文獻探討揭露出不同的變 種和類別顯示出大幅變異性營養與激素要求。大部分商業 玫瑰栽培者仍以傳統手段繁殖玫瑰植物。對於攀緣玫瑰 Improved Blaze所開發的培養基促成三種迷你玫瑰變種的明 顯生長但不是對於所有的雜交種茶香玫瑰都是如此 (Hasegawa, J. Amer. Soc. Hort. Sci. 1980，105:216-220) 〇 再 者，增殖雜交種玫瑰（R· hybrida)培育種Tropicana和Bridal Pink的植物生長調節劑之要求也與對兩種老世界性品種r damascena和R. canina的要求也有顯著第不同（Khosh-Khui and Sink, J. Hort. Sci. 1982, 57, 315-319; Khosh-Khui and1242405 A7 _ B7 V. Description of the Invention (1) Field of Love The present invention relates to plant cultivation, plant cloning and horticulture. In particular, the present invention relates to the vegetative propagation or tissue culture of a rose plant and to the blooming in a test tube of a young rose tree derived from the plant or tissue culture. More specifically, the present invention relates to the formulation and efficient methods of medium used for micropropagation, flower bud development and flowering in vitro of roses and rose plants. The bright background The rose plant belongs to the RosaceaeJuss family. This family is very large, with more than 100 genera and 2,000 herbs to woody plants as members. Many important edible and decorative plants are in the Rosaceae family, including, for example, raspberry 'apple, almond, floor peach, peach and blackberry. The correct species included in modern rose development are unknown. Most rose varieties are found in the temperate zones of the northern hemisphere, especially from southern China and the Far East, to Himalayas and Bangladesh into Isopia and west to North America from the Arctic Circle to New Mexico. The repetitive or endless flowering rose plant that spread from the Far East to Europe at the end of the 18th century has been the result of numerous generations of breeding in China, India, and Japan. Rose is one of the most common flowers in the world. Roses are often grown in the form of cut wood, potted plants, or sample plants in the home garden. There are many types of rosewood inserts ’including, for example, long stem standard, short stem standard, small flowered sweetheart, and multi-bouquet roses. Since most of the current commercial rose varieties are hybrids, breeding from seeds is not a viable option, mainly due to the phenomenon of attribute separation between different generations. The breeding of hybrid cutwood roses is usually through the budding method, or special tender branches or cut branches, and cut into the existing wild. The paper size is applicable to China National Standard (CNS) A4 (210 X 297). (Centi) 1242405 A7 B7 5. Description of the invention (2) Rose roots. For small potted roses, wild asexual propagation of cut plants in the greenhouse is common practice. An alternative to the above methods is micro-propagation, also known as axillary bud propagation or asexual propagation in a test tube, which is a method of propagating plants from axillary buds under sterile conditions. Micropropagation has the advantage of rapidly propagating a desired plant into many genetically consistent young trees. The young trees obtained from micro-propagation will not be infected by bacteria, maggots and viruses, and will not be infected by insects. Miniature propagation procedures include preparing living tissue from related plants, growing the living tissue on a medium supplemented with plant hormones, cultivating, and harvesting shoots (or rooted shoots) of the same type [Douglas, " Methods in Molecular Biology, Vol. 6, W. Pollard, JM Walker, eds. (1990); George and Sherrington, Exegetics, Ltd. U.K., p. 3 (1984); and Brown and Thorpe, In: Cell Culture and Somatic Cell Genetics of Plants, p49-65, IK Vasil, ed. (1986)]. Micropropagation provides a potential cost-effective means of mass reproduction of certain plants. An important consideration for this technology is the significant economic value added to the plants that are propagated. Discussion of relevant literature reveals that existing breeding schemes are often inefficient in inducing flower cultivation in vitro of young trees. Efficiency depends on the plant variety used, but it is still low even for the best breeding. In recent years, varieties of potted roses with a height of 7.5 cm to 20 cm have become very popular due to their small size and long shelf life compared to cut rose. In addition, the ability to produce multiple flowers at a reasonable price further expands the universality of potted roses. Breeding of mini rose hybrids is also ongoing worldwide. For example, in the United States alone, there are more than 300 types of mini rose miscellaneous paper sizes that are applicable to the Chinese National Standard (CNS) A4 (210 X 297 mm) 1242405 A7 B7 V. Description of the invention (3) Registration of the case . The most famous breeders of commercial mini rose hybrids are Poulsen Roser ApS (registered trademark of PARADE®) in Denmark and New Rose International in De Ruiter, Netherlands. Mini potted roses are displayed for decoration in the garden, atrium or even at home. However, mini potted roses are very limited for interior decoration, mainly because of the requirements for soil in the pot. In addition, taking into account the total average height between 15 and 30 cm (including pots), even the height of mini potted plants is still too high for placement on, for example, a table. In addition, due to the increasing urbanization of the world, more and more people are spending more and more high-quality indoor time in, for example, homes or offices. Therefore, there is currently a demand for decorative rose plants that are clean and small, have a long storage period, and bloom many times. Commercial reproduction of roses is usually done by cutting or cutting special young shoots into existing wild rose rhizomes. In recent years, there have been many articles discussing the propagation of roses through tissue culture. The literature review revealed that different variants and categories show significant variability in nutrition and hormone requirements. Most commercial rose growers still reproduce rose plants by traditional means. The medium developed for the climbing rose Improved Blaze promotes the apparent growth of three mini rose varieties but not for all hybrid tea roses (Hasegawa, J. Amer. Soc. Hort. Sci. 1980, 105: 216-220 ) 〇 Furthermore, the requirements for plant growth regulators of the R. hybrida breeding species Tropicana and Bridal Pink are also significantly different from those for the two old world varieties r damascena and R. canina (Khosh -Khui and Sink, J. Hort. Sci. 1982, 57, 315-319; Khosh-Khui and
Sink, Scentia Horticulturae 1982 17，371-376)。 -6- 本紙張尺度適用中國國家標準(CNS) A4規格(210X 297公釐） 1242405 A7 B7 五、發明説明（9 ) 本發明也有關，與其他一起者，開花玫瑰植物，其可以 保留在培養基中而無需給予養分或給水一段可使植物繼續 微升之時間。當玫瑰植物係處於封閉試管内之時，其典型 地可以保持超過一個月。 對於擬用於本發明中的封閉試管之唯一限制在於彼等必 須能夠維持住而不會洩漏出含有水分和養分例如礦物質、 鹽和維生素之固體培育培養基。於一特別具體實例中，該 培育培養基也可含有一或多種染料，其係經特別選擇以增 強在試管内生長的試管内栽培開花植物之吸引性。於一具 體實例中，係用一蓋和一底一起構成一封閉試管且該蓋和 底係經接著到使彼等在正常處置與傳遞中不會分離且使得 水分都保持在該試管之内。該蓋，且合意者整個試管，係 由高透光性，高清晰度材料所製成且該試管的内表面上視 情況塗覆著化學品以防止水份凝結在該試管的内表面上， 而對於内部的試管内花栽培幼樹產生清晰的景觀。適當的 材料包括聚碳酸酯、聚（甲基丙烯酸甲酯）和玻璃。可以耐 受高熱（如>160°C)和高壓大氣壓）或可以用放射同位素 照射殺菌的具有高清晰度之任何其他材料也可以使用。可 以用於此目的之防霧性化學品的例子有Sicanett®，爲義大 利 Anfora (SV)的產品，與 Siclair® (Nettoyant Universal)， 爲法國Si-International S. Α·所製造的抗-靜電劑（anti· statique)。防霧劑係經直接噴布在試管的内表面上。 本發明也有關上述組織培養物微型繁殖法所包括的兩個 步驟之每一個別步驟。 -12- 本纸張尺度適用中國國家標準(CNS) A4規格(210X 297公釐) 1242405 A7Sink, Scentia Horticulturae 1982 17, 371-376). -6- This paper size is in accordance with Chinese National Standard (CNS) A4 (210X 297 mm) 1242405 A7 B7 V. Description of the invention (9) The invention is also related to the others, the flowering rose plant, which can be retained in the culture medium Without giving nutrients or water for a period of time that allows the plant to continue to rise slightly. When the rose plant line is in a closed test tube, it can typically remain for more than a month. The only limitation on the closed test tubes intended for use in the present invention is that they must be able to be maintained without leaking out of a solid cultivation medium containing water and nutrients such as minerals, salts and vitamins. In a particular embodiment, the cultivation medium may also contain one or more dyes, which are specifically selected to enhance the attractiveness of flowering plants grown in test tubes grown in test tubes. In a specific example, a closed test tube is constructed with a lid and a bottom, and the lid and the bottom are attached so that they do not separate during normal handling and transfer and keep the moisture inside the test tube. The cover, and the entire test tube of the applicant, is made of a highly transparent and high-definition material and the inner surface of the test tube is optionally coated with chemicals to prevent water from condensing on the inner surface of the test tube. For young flowers growing inside the test tube, a clear landscape is produced. Suitable materials include polycarbonate, poly (methyl methacrylate), and glass. Any other material with high definition that can withstand high heat (such as > 160 ° C) and high atmospheric pressure or can be sterilized by irradiation with radioisotopes can also be used. Examples of anti-fogging chemicals that can be used for this purpose are Sicanett®, a product of Anfora (SV) in Italy, and Siclair® (Nettoyant Universal), an anti-static made by Si-International S. A ·, France (Anti · statique). The anti-fogging agent is sprayed directly on the inner surface of the test tube. The present invention also relates to each of the two steps included in the above-mentioned tissue culture micropropagation method. -12- This paper size applies to China National Standard (CNS) A4 (210X 297 mm) 1242405 A7
獨地使用或與一或多種其他植物生長激素組合地使用。 孩培養基可經改質使得可以針對特定的植物變種提供激 素組成分。.在謗導開花時，可以改變基礎培養基中的養分 水平。例如，提供組織培養植物細胞所需一般性營養和生 長要求所用的Murashige and Skoog (MS)培養基可以使用其 他習用的培育或生長培養基予以取代，如技藝中已知者。 熟看此技者可以輕易地了解每一種成分的正確用量係決 定於要栽培的玫瑰型。典型者，先以較低劑量使用諸化合 物，在視需要增加用量以達到合意的效果。 適當地，於加到花蕾謗導所用組成物和方法之中時，赛 達有龍的濃度係在〇 · 4 - 0.5毫克/升之間。 當爷基腺嘌呤係加到增殖和營養生長方法所用的培養基 之中時’其最適濃度爲介於1毫克/升與2毫克/升之間。當 宇基腺嘌呤係加到有謗導出的花蕾之幼樹的伸長所用培養 基中之時，其最適濃度爲約0.1毫克/升。 當莕乙酸爲加到本發明培養基内的植物生長激素時，其 最適濃度爲介於0.05毫克/升與0.1毫克/升之間。 當Θ丨嗓乙酸爲加到本發明培養基内的植物生長激素時， 其添加濃度爲介於0.05毫克/升與0.1毫克/升之間。最適者 ’用於幼樹增殖時吲哚乙酸的濃度爲約〇 · 1毫克/升。當丨 哚乙酸係加到有謗導出的花蕾之幼樹的伸長所用培養基中 之時，其最適濃度爲約1.0毫克/升。 對於微型繁殖條件的多種變更係諳於此技者所輕易明白 者且包括培育起始所用的供予組織，其係來自具有恰當基 -14- 本纸張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐） 1242405 A7 B7 五、發明説明（12 ) 因型與生理和發育狀態的植物。此外，活組織來源、植物 變種、和培養物生長所處物理環境等的變異也都涵蓋在本 發明之内。·例如，氣體環境、溫度和光照等條件可經變異 以提供給裝在封閉試管内的改良玫瑰。通常，花蕾謗導所 用的最適溫度爲S 2 5 °C。 任何適當的膠凝劑都可以使用，例如但不限於包括膠凝 素（gellan)的 PHYTAGEL.TM·;包括膠凝素的 GEL-RITE.TM.;包括膠凝素（gellan)的 AGARGEL.TM.;瓊脂例 如但不限於A，E和Μ型瓊脂，高凝膠強度，經純化的 Bacteriological Flake ;瓊脂與 PHYTAGEL. ΤΜ·的摻合物之 AGARGEL.TM.;瓊脂糖，例如但不限於VII型；海藻酸； 鹿角菜膠；轉移凝膠（transfergel)或經乙基纖維素；與類似 者。 玫瑰活組織係本發明一具體實例。玫瑰活組織可以裝在 内裝前述培育培養基的封閉試管内，且可以培育到產生玫 瑰植物，例如，開花玫瑰植物爲止。該活組織可以得自植 物，例如但不限於薔薇屬，包括但不限於雜交種玫瑰例如 培育植物 Orange PARADE®，Fiesta PARADE®，Scarlet PARADE®，Bianca PARADE®，和 Frosty PARADE®與類似 者；大馬士革玫瑰（Rosa damascena)，多花玫瑰（Rosa multiflora)，和法國薔薇（R〇sa gallica)，與類似者0 本發明組織培養技術包括在一封閉試管内培育玫瑰活組 織’例如培養皿、試管、燒瓶、eppend〇rf管、嬰兒食物瓶 、裝罐頭瓶、及能夠根據本發明方法支持本發明玫瑰植物 -15- 本紙張尺度適用中國國家襟準(CNS) A4規格(210 X 297公釐） 1242405 A7 B7Used alone or in combination with one or more other plant growth hormones. The culture medium can be modified to provide hormone components for specific plant varieties. When demonstrating flowering, the nutrient levels in the basal medium can be changed. For example, Murashige and Skoog (MS) media used to provide the general nutrition and growth requirements for tissue culture plant cells can be replaced with other conventional cultivation or growth media, as known in the art. Those skilled in the art can easily understand that the correct amount of each ingredient depends on the type of rose to be cultivated. Typically, the compounds are used at a lower dose first, and the amount is increased as needed to achieve the desired effect. Appropriately, when added to the composition and method used for flower bud demonstrating, the concentration of cedarone is between 0.4-0.5 mg / liter. When the hepatoadenosine is added to the medium used for the method of proliferation and vegetative growth, its optimum concentration is between 1 mg / liter and 2 mg / liter. When Ukid adenine is added to the medium used for elongation of young trees with flower buds derived from it, the optimum concentration is about 0.1 mg / liter. When acetic acid is a plant growth hormone added to the medium of the present invention, its optimum concentration is between 0.05 mg / liter and 0.1 mg / liter. When Θ 丨 acetic acid is a plant growth hormone added to the medium of the present invention, its added concentration is between 0.05 mg / L and 0.1 mg / L. Optimum 'when used for the growth of young trees, the concentration of indoleacetic acid is about 0.1 mg / liter. When indole acetic acid was added to the medium used for elongation of young trees with flower buds derived therefrom, the optimum concentration was about 1.0 mg / liter. The various changes to the micro-breeding conditions are those that are easily understood by the skilled person and include the donor organization used for the initiation of breeding. They are derived from those with appropriate bases. -14- This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1242405 A7 B7 V. Description of the invention (12) Plants with type and physiological and developmental status. In addition, variations in the source of living tissue, plant varieties, and the physical environment in which the culture is grown are also encompassed by the present invention. • For example, conditions such as gaseous environment, temperature, and light can be mutated to provide a modified rose in a closed test tube. Generally, the optimum temperature for flower buds is S 2 5 ° C. Any suitable gelling agent can be used, such as, but not limited to, PHYTAGEL.TM. Including gellan; GEL-RITE.TM. Including gellan; AGARGEL.TM including gellan .; Agar such as, but not limited to, A, E and M type agar, high gel strength, purified Bacteriological Flake; agar and PHYTAGEL. TM · Blend AGARGEL.TM .; agarose, such as but not limited to VII Type; alginic acid; carrageenan; transfergel or ethyl cellulose; and the like. Rose living tissue is a specific example of the present invention. Rose living tissue can be contained in a closed test tube containing the aforementioned cultivation medium, and can be grown to produce a rose plant, for example, a flowering rose plant. The living tissue can be obtained from plants such as, but not limited to, Rosa, including but not limited to hybrid roses such as cultivated plants Orange PARADE®, Fiesta PARADE®, Scarlet PARADE®, Bianca PARADE®, and Frosty PARADE® and the like; Damascus Rose (Rosa damascena), Rosa multiflora, and French rose (Rosa gallica), and the like0 The tissue culture technique of the present invention includes cultivating rose living tissue in a closed test tube, such as a petri dish, test tube, Flask, eppendor tube, baby food bottle, canned bottle, and rose plant capable of supporting the present invention according to the method of the present invention-15- This paper size is applicable to China National Standard (CNS) A4 (210 X 297 mm) 1242405 A7 B7
植物生長激素一詞指的是植物激素。於低濃度時，此等 有機物質可以促進植物嫩枝的伸長且可以控制其他特殊的 生長效應。植物生長激素包括3-(3 -4卜朵基）丙酸（ip八），脱 落酸（ΑΒΑ)，和2,4-D，莕乙酸和GA3 〇 培育植物（cultivar) ” 一詞指的是商業上可取得，園藝學 衍生的變種，以與天然發生的變種有所區別。 如本文中所用者，”微型繁殖"指的是植物的試管内無性 純種係繁殖，其中經由在含有恰當濃度的植物激素、礦物 質、維生素和醣類之培養基内培育可以在短時間内從母體 植物經謗導的芽得到許多新的嫩枝。 於本發明的實施中用到下列培養基： RMS〇 ··改質MS培養基，含有全強度Murashige Skoog無 基養分和Gamborg’s B5維生素（最後濃度1 〇亳克/升屢胺鹽 酸鹽，1毫克/升菸鹼酸，1毫克/升吡哆醇，1〇〇毫克/升肌 醇），pH 5.8，加上2%蔗糖且用0.30%植物凝膠予以膠凝。 在不同的發育階段，可對該培養基補充不同的植物激素。 RMSi :補充有2毫克/升6 -爷基腺嗓呤和0.05毫克/升蕃 乙酸的RMS0。 RMS2 ··含有3%蔗糖且補充有0.4-0.5毫克/升赛達有龍 、0.05毫克/升莕乙酸、0.1毫克/升激動素和400毫克/升肌 醇的RMS〇。 RMS3 :含有3%蔗糖且補充有0.5-1.0毫克/升玉米素、 0.05毫克/升苯乙酸、和400毫克/升肌醇的RMS〇。 RMS4 :含有3%蔗糖且補充有0.1毫克/升芊基腺嘌呤、 -17- 本紙張尺度適用中國國家標準(CNS) A4規格(210X 297公釐） 1242405 A7 ~_— _57______ 五、發明説明（15 ) " ^一" --- 1.0毫克/升啕哚乙酸和400毫克/升肌醇的汉％8。 ▲rms5:含有2%蔑糖且補充有2毫克/升爷基0腺料和i 〇 耄克/升吲哚乙酸的RMS〇。 您母枝得到活組織 於-較佳具體實射，使用有⑲芽的幼枝作爲活组織。 爲了確保該活組織不會使培養基遭細菌和眞菌感染（污染） ，於使用前將該活組織表面殺菌。技藝中有許^種滅菌技 術可以用於培育所用活組織的製備目的。此等技術包括將 活組織浸在含有至少一種滅菌劑的溶液中。此等滅菌劑包 括次氯酸鈉、次氣酸鈣、氣化汞、乙醇等。 於本發明較佳方法中，將取自成熟植物長1〇_12公分的 嫩枝剝除掉外面的葉子並先在水流下清洗，接著使用〇1% «8(：12予以滅菌5-1〇分鐘，然後用水流完全清洗3〇分鐘以 上。之後將該嫩枝浸在10_15% C1〇r〇xR(含有5 25%次氣酸 鈉）溶液中10-15分鐘。最後，使用無菌蒸餾水清洗該嫩枝 4-6次。另外，可以使用技藝中熟知的技術從玫瑰植物培育 物衍生出嫩枝。因此可以擬議使用得自組織培養物的嫩枝 取代從成熟植物製備的嫩枝而不違離本發明範圍。 幼樹形成 將活組織置於固體培養基上並在使用45〇…55〇〇 lux光強 度（由日光型光提供）的16-小時照光期下培育。當幼樹暴露 於上述照光條件之下時，溫度係經維持在2 3 t。另外，在 移除光源之後，幼樹係經保持在i 9 t。將完整嫩枝置於 RMS![補充有2毫克/升6_芊基腺嘌呤和〇〇5毫克/升莕乙酸 -18· 本紙張尺度適用中國國家標準297公釐) 1242405The term plant growth hormone refers to plant hormones. At low concentrations, these organic substances can promote the elongation of plant shoots and control other special growth effects. Phytohormones include 3- (3--4butoryl) propionic acid (ip eight), abscisic acid (ΑΒΑ), and 2,4-D, acetic acid, and GA3. The term "cultivar" refers to Commercially available, horticulturally-derived variants to distinguish them from naturally occurring variants. As used herein, "micropropagation" refers to asexual pure germline propagation of plants in vitro, which occurs via Cultivation in the proper concentration of plant hormones, minerals, vitamins and carbohydrates can produce many new shoots from the defamated buds of the mother plant in a short time. The following medium is used in the practice of the present invention: RMS ..... Modified MS medium containing full-strength Murashige Skoog baseless nutrients and Gamborg's B5 vitamins (final concentration 10 g / L of chloramine hydrochloride, 1 mg / Liter of nicotinic acid, 1 mg / liter of pyridoxine, 100 mg / liter of inositol), pH 5.8, plus 2% sucrose and gelled with 0.30% plant gel. The medium can be supplemented with different plant hormones at different developmental stages. RMSi: RM0 supplemented with 2 mg / L of 6-dye adenine and 0.05 mg / L of acetic acid. RMS2. RMS containing 3% sucrose and supplemented with 0.4-0.5 mg / L of Sedaronol, 0.05 mg / L of acetic acid, 0.1 mg / L of kinetin and 400 mg / L of inositol. RMS3: RMS containing 3% sucrose and supplemented with 0.5-1.0 mg / L zeatin, 0.05 mg / L phenylacetic acid, and 400 mg / L inositol. RMS4: Contains 3% sucrose and is supplemented with 0.1 mg / L of adenyl adenine, -17- This paper size applies Chinese National Standard (CNS) A4 specifications (210X 297 mm) 1242405 A7 ~ __ _57______ 5. Description of the invention ( 15) " ^ a " --- 1.0% / L indole acetic acid and 400 mg / L inositol Han% 8. ▲ rms5: RMS with 2% sucrose and supplemented with 2 mg / litre of 0 gland and i 0 g / litre of indole acetic acid. Your mother shoots get living tissue Yu-preferably specific shoots, using young shoots with ridge buds as living tissue. In order to ensure that the living tissue does not infect (contaminate) the culture medium with bacteria and fungi, the surface of the living tissue is sterilized before use. There are a number of sterilization techniques in the art that can be used for the purpose of preparing living tissue used for cultivation. These techniques include immersing living tissue in a solution containing at least one sterilant. Such sterilants include sodium hypochlorite, calcium hypochlorite, vaporized mercury, ethanol, and the like. In the preferred method of the present invention, the twigs taken from mature plants with a length of 10-12 cm are stripped of the outer leaves and washed under water, and then sterilized with 0% «8 (: 12 5-1 0 minutes, and then completely washed with water for more than 30 minutes. After that, the tender shoots were immersed in a 10_15% C10rOxR (containing 525% sodium hypooxygenate) solution for 10-15 minutes. Finally, washed with sterile distilled water The shoots are 4-6 times. In addition, the shoots can be derived from the rose plant culture using techniques well known in the art. It is therefore possible to replace the shoots prepared from mature plants with the shoots derived from the tissue culture. Out of the scope of the present invention. Sapling formation Place living tissue on solid medium and grow under a 16-hour light period using 450,000 ... 550,000 lux light intensity (provided by daylight). When the saplings are exposed to the above Under light conditions, the temperature was maintained at 2 3 t. In addition, after removing the light source, the young tree was maintained at i 9 t. Place the complete shoots at RMS! [Supplied with 2 mg / L 6_ Glucosyl adenine and 0.05 mg / L acetic acid-18 · Suitable for paper size Chinese National Standard 297 mm) 1242405
的RMS〇]之上培育。活組織逐漸變綠且通常在^◦至^^天之 後會形成新芽。於此製備時點，從嫩枝切取芽供幼樹增殖 所用。 增殖 幼樹增殖爲從小樹或復活生長植物取得，典型地係在根 尖或芽尖處正在快速分裂的細胞之生長點或部位，稱爲分 生組織者取得。將該枝材置於前面定義的激素與營養培養 基上以產生活組織，其可漸漸地形成具有新芽的幼樹。然 後此等活組織取下個別的芽並如上述置於前面定義的激素 與營養培養基中培育以從單一完整嫩枝產生眾多幼樹。此 種從單一母體的無性植物繁殖可用來選殖具有與該母體相 2遺傳特性的植物子代。將切芽轉移到汉“心培養基上並培 育約50天。利用上述程序，諸芽可生長成爲幼樹並從此等 幼樹長出新芽。該等新形成的芽於可順利切取時即可切取 並轉移到新的RMSi培養基以繁殖和增殖幼樹。增殖比例通 常在原有起始活組織數目的4到7倍。我們發現從莖段組織 培養物所得新形成芽在花謗導之前需要最少兩回合的增殖 以使新形成的幼樹時間一致並產生高品質的花及良好的花 誘導比例。對於謗導開花之前可以進行的幼樹增殖回合數 沒有限制。我們發現經繁殖超過兩年的幼樹仍然可以在謗 導之後產生高品質的花。 花蕾謗導 花蕾謗導爲商業規模試管内花栽培的關鍵。本發明所涵 蓋的花蕾謗導有兩種可替換的做法。 -19- 1242405 A7 ----—___ _ B7 五、發明説明------- 於具體實例中，在增殖之後，係經由將增殖後的幼樹 置於裝在封閉試管内的RMS2培養基上培育以謗導花蕾。該 謗導培育係持續約50天。於此步驟之後，將幼樹轉移到裝 有則4培養基的封閉試管内以進行伸長步驟，其持續約15 至30 1。於伸長之後，將幼樹轉移到有20毫克/升到50毫 升氨苄青黴素的RMSq培養基上。轉移經伸長的花材枝 最佳時點爲在經伸長莖上有花蕾形成之後。通常在10到20 天之内即可開花。 ，於另一但仍爲較佳具體實例中，於增殖之後係經由將經 增殖後的幼樹置於封閉試管内的纽、培養基上培育一段約 5〇天2期間以謗導花蕾。於謗導之後，將幼樹直接轉移到 有20愛克/升到50毫克/升氨芊青黴素的11撾％培養基上。通 常在1 5到2 0天之内即可開花。 實施例 本發明要在下面的實施例中進一步説明，該等實施例係 經提出作爲產明所用而無意對本發明有任何方式的限制。 其中利用技藝中熟知的標準技術或後文中特定地述及之技 術0 實施例1 本實施例展7F出在增補赛達有龍的培養基上之花蕾謗導 。使用取自成熟植物具有腋芽的嫩枝作爲活組織。將玫瑰 活組織置於固體培養基上使用4500-5500 lux光強度（由日光 型光提供）之1 6 -小時照光期，於有照光的2 3 〇c溫度和沒有 照光的19°C溫度下培育。將完整嫩枝置於RMSi之上培育。 -20· 本纸張尺度適用中國國家標準(CNS) A4規格(210X297公 1242405 A7 B7 五、發明説明（18 ) 活組織逐漸變綠且在1〇至丨5天之後形成新芽。從嫩枝切取 芽。在增殖之後，將增殖後的幼樹（>2次增殖）置於rmS2培 養基上培育·以謗導花蕾。於謗導花蕾之後，將幼樹轉移到 伸長培養基上進行試管内花栽培。幼樹在伸長培養基RMS4 上培育15-30天後，轉移到有20-50毫克/升濃度的氨苄青 徽素之RMS〇培養基中。轉移到有加氨芊青黴素之rmsg後， 於1 0 - 2 0天内即開花。有4 〇至5 〇 %的幼樹開花且有超過 8 0%得花具有正常外觀，正常的花係經定義爲具有與使用 相同的培育植物經由其他培育方法所長成的花所具者相同 之所有類型花器官的花。未開花的幼樹可以轉回進行繁酸 。所栽培的花枝在低於2 5 °C下之搁置期（shelf life)超過1個 月。 實施例2 本實施例展示出在增補玉米素的培養基上之花蕾謗導。 使用取自成熟植物具有腋芽的嫩枝作爲活組織。將玫瑰活 組織置於固體培養基上使用4500-5500 lux光強度（由日光型 光提供）之16·小時照光期，於有照光的23 °C溫度和沒有照 光的19 C溫度下培育。將增殖後的幼樹（>2次增殖）置於 RMS3培養基上培育。於花蕾謗導之後，將具有花蕾的幼樹 直接轉移到有20-50毫克/升氨芊青黴素tRMS()培養基中進 行花栽培。於1 5 - 2 0天内即開花。有4 〇至5 〇 %的幼樹開花 且有超過80%得花爲正常者。未開花的幼樹可以轉回進行 繁殖。所栽培的花枝在低於2 5。〇下之搁置期（shelf i丨f e )超 過1個月。 -21 - 本紙張尺度適財關¥料(_)域^(2胸97公着)— 1242405 A7 B7 五、發明説明（19 ) 實施例3 使用取自 Fiesta PARADE®，Scarlet PARADE®，Bianca PARADE®，和Frosty PARADE®諸培育植物的玫瑰植物作爲 活組織並使用上述組成物和方法予以生長。在RMS5培養基 上增殖，該培養基含有取代莕乙酸的，5丨哚乙酸而用以取代 RMSi。在將此等培育植物置於RMS5上增殖之後，將經增殖 的幼樹轉移到RMS24RMS3上進行花蕾謗導，如上面”實施 例1 ’’（赛達有龍-謗導花蕾）或”實施例2 ”（玉米素-謗導花蕾） 中所述者。有超過3 0 %的幼樹經用本發明試管内花栽培方 法增殖及處理後產生具有正常的花之幼樹。 必須了解者，本發明方法和組成物可以用多種具體實例 形式併入，本文只揭示出其中一些而已。熟諳者都明白有 其他具體實例存在且不違離本發明旨意。因此，所述及的 具體實例皆爲闡述性且不可視爲係限制性者。 -22- 本紙張尺度適用中國國家標準(CNS) A4規格(210X 297公釐)RMS0]. Living tissues gradually turn green and new shoots usually form after ^ ◦ to ^^ days. At this point of preparation, buds were cut from the young shoots for the young tree to multiply. Proliferation Saplings are proliferated from small trees or resurrected growing plants, typically at the growth point or location of cells that are rapidly dividing at the root tip or shoot tip, and are called meristems. This shoot is placed on the previously defined hormone and nutrient culture medium to produce living tissue, which can gradually form young trees with new shoots. These living tissues are then used to remove individual shoots and cultivated in the hormone and nutrient medium previously defined as described above to produce numerous young trees from a single intact shoot. Such asexual plant reproduction from a single parent can be used to breed plant progeny that have genetic characteristics that are similar to that of the parent. The cut buds are transferred to Han's heart medium and cultivated for about 50 days. Using the above procedure, the buds can grow into young trees and grow new shoots from these young trees. The newly formed buds can be cut when they can be successfully cut. And transferred to a new RMSi medium to propagate and propagate young trees. The proportion of proliferation is usually 4 to 7 times the original number of original living tissues. We found that newly formed buds obtained from stem segment tissue cultures require a minimum of two before flowering. Proliferation of rounds to make newly formed saplings consistent in time and produce high-quality flowers and good flower induction ratios. There is no limit to the number of saplings that can be propagated before flowering. We have found that saplings that have been reproduced for more than two years Trees can still produce high-quality flowers after being seduced. Flower buds are the key to flower cultivation in vitro on commercial scales. There are two alternative methods of flower buds covered by the present invention. -19- 1242405 A7 ----____ _ B7 V. Description of the invention ------- In the specific example, after the proliferation, the young tree after the proliferation is cultivated on the RMS2 medium in a closed test tube. Instigate flower buds. The insulting breeding line lasts about 50 days. After this step, the young tree is transferred to a closed test tube containing 4 medium for the elongation step, which lasts about 15 to 30 1. After elongation, Saplings are transferred to RMSq medium with 20 mg / L to 50 ml of ampicillin. The best time to transfer an elongated flowering branch is after flower buds have formed on the elongated stem. Usually within 10 to 20 days Can bloom. In another but still preferred embodiment, after propagation, the young plants are grown in a closed tube in a closed tube and cultured for a period of about 50 days. After defamation, the young trees were directly transferred to 11% Lao media with 20 gram / liter to 50 mg / liter ampicillin. Usually flowering within 15 to 20 days. The invention will be further explained in the following examples, which are proposed for use in the invention and are not intended to limit the invention in any way. Among them, standard techniques well known in the art or techniques specifically mentioned later are used. Example 1 Example 7F is a flower bud on a supplemented Saida dragon medium. A tender branch with axillary buds taken from a mature plant is used as living tissue. Rose living tissue is placed on a solid medium using a light intensity of 4500-5500 lux ( (Provided by daylight type light) during the 16-hour photoperiod, cultivated at a temperature of 230 ° C with light and 19 ° C without light. Intact shoots are grown on RMSi. -20 · This paper The Zhang scale applies the Chinese National Standard (CNS) A4 specification (210X297 male 1242405 A7 B7 V. Description of the invention (18) The living tissue gradually turns green and new shoots are formed after 10 to 5 days. The buds are cut from the tender branches. After proliferation The young trees after propagation (> secondary proliferation) were cultivated on rmS2 medium to induce flower buds. After inducing the flower buds, the young trees were transferred to an elongation medium for in-tube flower cultivation. After saplings are incubated on elongation medium RMS4 for 15-30 days, they are transferred to RMSO medium with ampicillin at a concentration of 20-50 mg / l. After transfer to rmsg with ammonium penicillin, flowering occurs within 10-20 days. 40 to 50% of the young trees bloom and more than 80% of the flowers have a normal appearance. A normal flower line is defined as having the same flowers as those grown by other cultivation methods using the same cultivated plants. Flowers of all types of flower organs. Unflowered saplings can be turned back for acid growth. The cultivated flowering branches have a shelf life of more than 1 month at temperatures below 25 ° C. Example 2 This example demonstrates flower buds on a medium supplemented with zeatin. Tender branches with axillary buds taken from mature plants were used as living tissue. The rose tissue was placed on a solid medium using a light intensity of 4500-5500 lux (provided by daylight type) for a 16-hour photoperiod, and incubated at 23 ° C with light and 19C without light. The proliferated saplings (> secondary proliferation) were grown on RMS3 medium. After the flower buds were seduced, the young trees with flower buds were directly transferred to a tRMS () medium containing 20-50 mg / L ampicillin for flower cultivation. Blossom within 15-20 days. 40 to 50% of the young trees are flowering, and more than 80% have to be normal. Unflowered saplings can be transferred back for reproduction. The cultivated flowering branches are below 25. The shelf life below (shelf if) is more than 1 month. -21-The paper size is suitable for financial matters. Material (_) field ^ (2 breasts and 97 books) — 1242405 A7 B7 V. Description of the invention (19) Example 3 Uses taken from Fiesta PARADE®, Scarlet PARADE®, Bianca PARADE ®, and Frosty PARADE® rose plants are cultivated as living tissue and grown using the composition and method described above. It was propagated on RMS5 medium, which contained acetic acid instead of acetic acid and replaced with RMSi. After these cultivated plants are propagated on RMS5, the propagated young trees are transferred to RMS24RMS3 for flower bud dissemination, as in the above-mentioned "Example 1" (Saida Youlong-Liao Bud) or "Example 2 "(zeatin-defying flower buds). More than 30% of the young trees have been propagated and treated with the in-tube flower cultivation method of the present invention to produce young trees with normal flowers. It must be understood that this The method and composition of the invention can be incorporated in a variety of specific examples, and only a few of them are disclosed herein. Those skilled in the art will recognize that other specific examples exist without departing from the spirit of the present invention. Therefore, the specific examples mentioned are all illustrative. -22- This paper size applies to China National Standard (CNS) A4 (210X 297 mm)