JP4708003B2 - Shoot tip culture method - Google Patents
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Description
本発明は、茎頂培養方法に関する。 The present invention relates to a shoot apical culture method.
植物の大量培養方法の一つとして、植物の茎頂培養方法が知られており、例えばイチゴ、ジャガイモ、ワサビ、ダイコン、サツマイモ、ショウガ、ニンニク、スイセン、ユリ、カーネーション、ナデシコ、ペチュニア、キク、カスミソウ、シュッコンカスミソウ、クルクマ、コチョウラン、シンビジューム等の種々の植物の大量培養に応用されている（例えば特許文献１、特許文献２、非特許文献１および非特許文献２参照。）。このような茎頂培養方法においては、より短期間で、クローン苗を製造することが望まれている。 Plant shoot apex culture methods are known as one of the mass cultivation methods of plants, such as strawberry, potato, wasabi, radish, sweet potato, ginger, garlic, narcissus, lily, carnation, radish, petunia, chrysanthemum, gypsophila. It is applied to large-scale culture of various plants such as Gypsophila, Curcuma, moth orchid, and Symbidium (see, for example, Patent Document 1, Patent Document 2, Non-Patent Document 1, and Non-Patent Document 2). In such a shoot tip culture method, it is desired to produce a cloned seedling in a shorter period of time.
このような状況のもと、本発明者らは、より短期間で植物を茎頂培養できる方法を開発すべく検討したところ、植物を茎頂培養する培地として、ハイドロキシアパタイト等のアパタイト化合物やトレハロースを含有する培養培地を用いることにより、茎頂、多芽体、シュート等の生長促進効果が見られ、クローン苗をより短期間で得ることを見出し、本発明に至った。 Under such circumstances, the present inventors have studied to develop a method capable of cultivating a plant at a shoot apex in a shorter period of time. As a medium for culturing a plant shoot apex, an apatite compound such as hydroxyapatite or trehalose can be used. It has been found that the growth promoting effect of shoot tips, multi-buds, shoots and the like is obtained by using a culture medium containing, and that clone seedlings can be obtained in a shorter period of time, leading to the present invention.
すなわち、本発明は、
１．アパタイト化合物およびトレハロースからなる群から選ばれる少なくとも一種を含有する培養培地を用い、植物の茎頂、多芽体またはシュートを培養することを特徴とする植物の茎頂培養方法；
２．アパタイト化合物およびトレハロースからなる群から選ばれる少なくとも一種を含有することを特徴とする茎頂培養用培養培地；
３．アパタイト化合物およびトレハロースからなる群から選ばれる少なくとも一種を有効成分として含有することを特徴とする茎頂培養用生長促進剤；
等を提供するものである。
That is, the present invention
1. A plant shoot apex culture method comprising culturing a plant shoot apex, multi-bud or shoot using a culture medium containing at least one selected from the group consisting of an apatite compound and trehalose;
2. A culture medium for shoot tip culture, comprising at least one selected from the group consisting of an apatite compound and trehalose;
3. A growth promoter for shoot tip cultivation, comprising as an active ingredient at least one selected from the group consisting of an apatite compound and trehalose;
Etc. are provided.
本発明によれば、より短期間で、植物を茎頂培養して、クローン苗を得ることができるため、より効率的な植物の培養増殖が可能となる。 According to the present invention, a plant can be cultured at a shoot apex in a shorter period of time to obtain a cloned seedling. Therefore, more efficient plant growth and growth is possible.
本発明の茎頂培養方法に用いられる植物としては、茎頂培養が可能な植物であればよく、例えばイチゴ、ジャガイモ、ワサビ、ダイコン、サツマイモ、ショウガ、ニンニク、スイセン、ユリ、カーネーション、ナデシコ、ペチュニア、キク、カスミソウ、シュッコンカスミソウ、クルクマ、コチョウラン、シンビジューム等が挙げられる。 The plant used in the shoot apical culture method of the present invention may be any plant that can be cultivated, for example, strawberry, potato, wasabi, radish, sweet potato, ginger, garlic, narcissus, lily, carnation, dianthus, petunia. , Chrysanthemum, gypsophila, gypsophila, curcuma, moth orchid, cymbidium and the like.
本発明の茎頂培養方法は、アパタイト化合物およびトレハロースからなる群から選ばれる少なくとも一種を含有する培地を用いて、植物の茎頂、多芽体またはシュートを培養することを特徴とするものであり、これにより、茎頂、多芽体またはシュートの生長を促進させることができ、より短期間で、クローン苗を得ることができる。 The shoot tip culture method of the present invention is characterized by culturing plant shoot tips, multi-buds or shoots using a medium containing at least one selected from the group consisting of an apatite compound and trehalose. As a result, the growth of shoot apex, multi-buds or shoots can be promoted, and a clonal seedling can be obtained in a shorter period of time.
本発明の茎頂培養方法を用いて、茎頂を培養する場合に用いられる茎頂としては、例えば対象となる植物の各茎から摘出した茎頂、塊茎から発芽・伸長した芽等から摘出したもの等が挙げられる。かかる茎頂は、通常植物から、茎頂を含む０．５〜５ｃｍ程度の長さの茎を採取し、採取した茎を水で洗浄処理し、例えばエタノール水、次亜塩素酸ナトリウム水溶液等の殺菌液で殺菌処理し、さらに滅菌水で洗浄処理した後、水分を取り除いた茎から、実体顕微鏡等で摘出したものが用いられる。かかる茎頂の大きさは、通常０．１〜０．７ｍｍ程度である。また、多芽体を培養する場合には、公知の培養培地で茎頂を培養して得られる多芽体を用いてもよいし、本発明の茎頂培養方法を用いて茎頂を培養して得られる多芽体を用いてもよい。また、シュートを培養する場合は、公知の培養培地で茎頂や多芽体を培養して得られるシュートをそのままもしくは分割して用いてもよいし、本発明の茎頂培養方法を用いて茎頂や多芽体を培養して得られるシュートをそのままもしくは分割して用いてもよい。 As the shoot apex used when cultivating the shoot apex using the shoot apex culture method of the present invention, for example, the shoot apex extracted from each stalk of the target plant, extracted from the buds germinated and elongated from the tuber, etc. And the like. Such shoot apex is usually collected from a plant with a length of about 0.5 to 5 cm including the shoot apex, and the collected stalk is washed with water, for example, ethanol water, sodium hypochlorite aqueous solution, etc. After sterilizing with a sterilizing solution and further with a sterilizing water, a material extracted from a stem from which water has been removed with a stereomicroscope or the like is used. The size of the shoot apex is usually about 0.1 to 0.7 mm. In addition, when cultivating multi-buds, multi-buds obtained by culturing the shoot apex in a known culture medium may be used, or the shoot apex is cultured using the shoot apex culture method of the present invention. You may use the multibud obtained by this. When shoots are cultured, the shoots obtained by culturing shoot apex and multi-buds in a known culture medium may be used as they are or divided, or the shoots may be used using the shoot apex culture method of the present invention. A shoot obtained by cultivating the apex and multi-buds may be used as it is or after being divided.
採取した茎頂、多芽体またはシュートを、アパタイト化合物およびトレハロースからなる群から選ばれる少なくとも一種を含有する培地（以下、茎頂培養用培養培地と略記する。）に置床せしめ、通常１５〜３０℃の培養温度で、例えば５００〜５００００ルクスで、１日当たり１０〜２４時間程度の照明下で培養が行われる。 The collected shoot apex, multibud or shoot is placed on a medium containing at least one selected from the group consisting of an apatite compound and trehalose (hereinafter abbreviated as a culture medium for shoot apex culture), usually 15-30. The culture is performed at a culture temperature of 0 ° C., for example, at 500 to 50000 lux, under illumination for about 10 to 24 hours per day.
本発明の茎頂培養方法に用いられる茎頂培養用培養培地は、アパタイト化合物およびトレハロースからなる群から選ばれる少なくとも一種（以下、有効成分と略記する。）を含有する培養培地であり、液体培地や該液体培地にゲル化剤を加えて固化させた固形培地等の公知の茎頂培養に用いられる培養培地に前記有効成分を加えた培養培地である。公知の茎頂培養に用いられる培養培地としては、例えばムラシゲ・スクーグ培地（ＭＳ培地）、ホワイト培地（Ｗ培地）、リンスマイヤー・スクーグ培地（ＬＳ培地）、ハイポネックス培地等が挙げられる。また、かかる培養培地には、オーキシン、サイトカニン、ジベリン等の植物生長調節剤を含んでいてもよい。 The culture medium for shoot tip culture used in the shoot tip culture method of the present invention is a culture medium containing at least one selected from the group consisting of an apatite compound and trehalose (hereinafter abbreviated as an active ingredient), and is a liquid medium. Or a culture medium in which the active ingredient is added to a known culture medium such as a solid medium obtained by adding a gelling agent to the liquid medium and solidifying the medium. Examples of the culture medium used for known shoot apical culture include Murashige-Skoog medium (MS medium), White medium (W medium), Rinsmeier-Skoog medium (LS medium), Hyponex medium, and the like. In addition, the culture medium may contain plant growth regulators such as auxin, cytocanin, and gibberine.
アパタイト化合物としては、例えばハイドロキシアパタイト、ハイドロキシアパタイトを構成する水酸基の一部もしくは全部が、例えばフッ素原子、塩素原子等のハロゲン原子で置換された、フッ化アパタイト、塩化アパタイト等のハロゲン化アパタイト等の単独または混合物が挙げられる。かかるアパタイト化合物としては、例えば市販されているものを用いてもよいし、例えば水の存在下に、リン酸カルシウムやリン分含有排液をアルカリ性カルシウム化合物で処理して得られるリン酸カルシウムを含むスラッジを７００〜１２００℃で加熱処理する方法（特開２０００−２８１３２２号公報参照。）等の公知の方法に準じて製造したものを用いてもよい。かかるアパタイト化合物の形状は、特に制限されない。また、トレハロースとしては、通常市販されているものが用いられる。本発明の茎頂培養方法には、アパタイト化合物またはトレハロースのいずれか一方を用いてもよいし、アパタイト化合物とトレハロースを混合して用いてもよい。 As the apatite compound, for example, hydroxyapatite, a part or all of the hydroxyl group constituting hydroxyapatite is substituted with a halogen atom such as a fluorine atom or a chlorine atom, and a halogenated apatite such as fluorinated apatite or chloroapatite. These may be used alone or as a mixture. As such an apatite compound, for example, a commercially available one may be used. For example, a sludge containing calcium phosphate obtained by treating calcium phosphate or a phosphorus-containing drainage solution with an alkaline calcium compound in the presence of water is 700 to 700. You may use what was manufactured according to well-known methods, such as the method of heat-processing at 1200 degreeC (refer Unexamined-Japanese-Patent No. 2000-281322). The shape of the apatite compound is not particularly limited. Moreover, as trehalose, what is marketed normally is used. In the shoot tip culture method of the present invention, either an apatite compound or trehalose may be used, or an apatite compound and trehalose may be mixed and used.
茎頂培養用培養培地中のかかる有効成分の含有量としては、通常０．０００５〜１重量％である。 The content of such active ingredients in the culture medium for shoot tip culture is usually 0.0005 to 1% by weight.
本発明の茎頂培養用培養培地は、前記有効成分を含有することを特徴とするものであり、本発明の茎頂培養用培養培地を用いることにより、植物の茎頂、多芽体またはシュートの生長を促進させることができる。本発明の茎頂培養用培養培地は、液体培地や該液体培地にゲル化剤を加えて固化させた固形培地等の公知の茎頂培養に用いられる培養培地に、前記有効成分を加えた培地であり、公知の茎頂培養に用いられる培養培地としては、例えばムラシゲ・スクーグ培地（ＭＳ培地）、ホワイト培地（Ｗ培地）、リンスマイヤー・スクーグ培地（ＬＳ培地）、ハイポネックス培地等が挙げられ、また、オーキシン、サイトカニン、ジベリン等の植物生長調節剤を含んでいてもよい。本発明の茎頂培養用培養培地中の有効成分の含有量としては、通常０．０００５〜１重量％である。本発明の茎頂培養用培養培地は、アパタイト化合物またはトレハロースのいずれか一方を含んでいてもよいし、アパタイト化合物およびトレハロースの両方を含んでいてもよい。 The culture medium for shoot apex culture of the present invention is characterized by containing the above-mentioned active ingredient. By using the culture medium for shoot apex culture of the present invention, the shoot apex, multi-bud or shoot of the plant is used. Can be promoted. The culture medium for shoot apex culture of the present invention is a medium obtained by adding the above active ingredient to a culture medium used for known shoot apex culture such as a liquid medium or a solid medium solidified by adding a gelling agent to the liquid medium. Examples of the culture medium used for known shoot apical culture include Murashige-Skoog medium (MS medium), White medium (W medium), Rinsmeier-Skoog medium (LS medium), Hyponex medium, and the like. In addition, plant growth regulators such as auxin, cytocanin, and gibberine may be included. The content of the active ingredient in the culture medium for shoot tip culture of the present invention is usually 0.0005 to 1% by weight. The culture medium for shoot apex culture of the present invention may contain either an apatite compound or trehalose, or may contain both an apatite compound and trehalose.
本発明の茎頂培養用生長促進剤は、アパタイト化合物およびトレハロースからなる群から選ばれる少なくとも一種を有効成分として含有するものであり、かかる茎頂培養用生長促進剤を、培養培地に加え、植物を茎頂培養することにより、前記培地に置床せしめられた植物の茎頂、多芽体またはシュートの生長を促進させることができる。茎頂培養用生長促進剤を培養培地に加える場合、培養培地中で、有効成分が、通常０．０００５〜１重量％となる量の茎頂培養用生長促進剤が用いられる。本発明の茎頂培養用生長促進剤の形態は、水溶液または水懸濁液であってもよいし、固体であってもよい。かかる茎頂培養用生長促進剤中の有効成分の含有量は特に制限されず、アパタイト化合物またはトレハロースのいずれか一方を含んでいてもよいし、アパタイト化合物およびトレハロースの両方を含んでいてもよい。また、かかる茎頂培養用生長促進剤には、例えば植物生長調節剤等を含んでいてもよい。 The growth promoter for shoot apex culture of the present invention contains at least one selected from the group consisting of an apatite compound and trehalose as an active ingredient, and the growth promoter for shoot apex culture is added to the culture medium, By culturing the shoot apex, the growth of the shoot apex, multi-bud or shoot of the plant placed on the medium can be promoted. When the growth promoter for shoot tip culture is added to the culture medium, the growth promoter for shoot tip culture is used in an amount such that the active ingredient is usually 0.0005 to 1% by weight in the culture medium. The form of the growth promoter for shoot apex culture of the present invention may be an aqueous solution, a water suspension, or a solid. The content of the active ingredient in the growth promoter for shoot apex culture is not particularly limited, and may contain either an apatite compound or trehalose, or may contain both an apatite compound and trehalose. In addition, the growth promoter for shoot apex culture may contain, for example, a plant growth regulator.
以下、実施例により本発明をさらに詳細に説明するが、本発明はこれら実施例に限定されない。 EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples.
実施例１
ハイポネックス培地（市販品）２００ｍＬに、１−ナフタレン酢酸ナトリウム水溶液（濃度：０．０１ｍｏｌ／Ｌ）１０μＬを加え、さらに、ハイドロキシアパタイト（和光純薬品；以下、ＨＡｐと略記する。）を、０．１重量％または０．０１重量％となる量、および寒天を、０．８重量％となる量それぞれ加え、ＨＡｐを含有する茎頂培養用培養培地を調製した。また、対照区として、ＨＡｐを加えない以外は前記と同様にして、培養培地を調製した。かかるＨＡｐを含有する茎頂培養用培養培地とＨＡｐを含まない培養培地（対照区）のそれぞれに、殺菌処理済みのシュッコンカスミソウ３品種（品種名：雪ん子、ブランシーおよび雪景色）の茎頂の上皮を１枚剥ぎ、植え付けた（植付け株数：雪ん子およびブランシーは５０株、雪景色は４０株）。なお、殺菌処理は、図解花のバイオ技術 増殖・育種とその関連技術，誠文堂新光社，１９９２年発行，第６２頁に記載の方法に準じて実施した。
Example 1
To 200 mL of Hyponex medium (commercial product), 10 μL of 1-naphthalene sodium acetate aqueous solution (concentration: 0.01 mol / L) was added, and hydroxyapatite (Wako Pure Chemical Industries; hereinafter abbreviated as HAp) was added to 0.1. A culture medium for shoot apical culture containing HAp was prepared by adding an amount of 0.1% by weight or agar to an amount of 0.8% by weight. As a control, a culture medium was prepared in the same manner as described above except that HAp was not added. Stem of three varieties of Gypsophila varieties (variety name: snow child, Blancy and snow landscape) that have been sterilized in each of the culture medium for shoot apex culture containing HAp and the culture medium not containing HAp (control group) One epithelium of the apex was peeled off and planted (number of planted strains: 50 strains of Snow and Blancy, 40 strains of snow). The sterilization treatment was carried out in accordance with the method described in illustrated flower biotechnology propagation / breeding and related technologies, published by Seikodo Shinkosha, 1992, page 62.
植付け後のＨＡｐを含有する茎頂培養用培地およびＨＡｐを含まない培養培地（対照区）を、人工気象装置（日本医科機械製；バイオトロンＬＨ２００）に入れ、培養温度約１９〜２３℃で、約７０００〜８０００ルクスの照明下、培養を行った。培養開始後６週間目において、草丈１．５ｃｍ以上のシュートに生長した株の数を調査した。結果を表１に示した。なお、表１中、草丈１．５ｃｍ以上のシュートに生長した株の割合は、植付け株数に対する草丈１．５ｃｍ以上のシュートに生長した株数の割合を表わす。 A shoot apical culture medium containing HAp after planting and a culture medium not containing HAp (control group) are placed in an artificial meteorological apparatus (manufactured by Nippon Medical Machinery; Biotron LH200) at a culture temperature of about 19 to 23 ° C. The culture was performed under illumination of about 7000 to 8000 lux. Six weeks after the start of culture, the number of strains grown on shoots having a plant height of 1.5 cm or more was examined. The results are shown in Table 1. In Table 1, the ratio of strains grown on shoots having a plant height of 1.5 cm or higher represents the ratio of the number of strains grown on shoots having a plant height of 1.5 cm or more to the number of planted strains.
実施例２
ハイポネックス培地（市販品）２００ｍＬに、１−ナフタレン酢酸ナトリウム水溶液（濃度：０．０１ｍｏｌ／Ｌ）１０μＬを加え、さらに、トレハロース（和光純薬品）を、０．０１重量％または０．００１重量％となる量、および寒天を、０．８重量％となる量それぞれ加え、トレハロースを含有する茎頂培養用培養培地を調製した。かかるトレハロースを含有する茎頂培養用培養培地に、殺菌処理済みのシュッコンカスミソウ（品種名：雪景色）の茎頂の上皮を１枚剥ぎ、植え付けた（植付け株数：４０株）。なお、殺菌処理は、図解花のバイオ技術 増殖・育種とその関連技術，誠文堂新光社，１９９２年発行，第６２頁に記載の方法に準じて実施した。
Example 2
Add 200 μL of 1-naphthalene sodium acetate aqueous solution (concentration: 0.01 mol / L) to 200 mL of Hyponex medium (commercial product), and add trehalose (Wako Pure Chemical Industries) to 0.01 wt% or 0.001 wt%. And agar were added in amounts of 0.8% by weight, respectively, to prepare a culture medium for shoot tip culture containing trehalose. One epithelium of the shoot apex of sterilized red gypsophila (variety name: snow scene) was peeled and planted on the shoot apical culture medium containing trehalose (number of planted strains: 40). The sterilization treatment was carried out in accordance with the method described in illustrated flower biotechnology propagation / breeding and related technologies, published by Seikodo Shinkosha, 1992, page 62.
植付け後のトレハロースを含有する茎頂培養用培地を、人工気象装置（日本医科機械製；バイオトロンＬＨ２００）に入れ、培養温度約１９〜２３℃で、約７０００〜８０００ルクスの照明下、培養を行った。培養開始後６週間目において、草丈１．５ｃｍ以上のシュートに生長した株の数を調査した。結果を表２に示した。なお、表２中、草丈１．５ｃｍ以上のシュートに生長した株の割合は、植付け株数に対する草丈１．５ｃｍ以上のシュートに生長した株数の割合を表わす。 The shoot apical culture medium containing trehalose after planting is placed in an artificial meteorological apparatus (manufactured by Nippon Medical Machinery; Biotron LH200) and cultured at a culture temperature of about 19-23 ° C. under illumination of about 7000-8000 lux. went. Six weeks after the start of culture, the number of strains grown on shoots having a plant height of 1.5 cm or more was examined. The results are shown in Table 2. In Table 2, the ratio of strains grown on shoots having a plant height of 1.5 cm or higher represents the ratio of the number of strains grown on shoots having a plant height of 1.5 cm or more to the number of planted strains.
実施例３
ハイポネックス培地（市販品）２００ｍＬに、１−ナフタレン酢酸ナトリウム水溶液（濃度：０．０１ｍｏｌ／Ｌ）１０μＬを加え、さらに、ＨＡｐ（和光純薬品）を０．１重量％となる量、トレハロース（和光純薬品）を、０．１重量％となる量、および寒天を、０．８重量％となる量それぞれ加え、ＨＡｐおよびトレハロースを含有する茎頂培養用培養培地を調製した。かかるＨＡｐおよびトレハロースを含有する茎頂培養用培養培地に、殺菌処理済みのシュッコンカスミソウ３品種（品種名：雪ん子、ブランシーおよび雪景色）の茎頂の上皮を１枚剥ぎ、植え付けた（植付け株数：雪ん子およびブランシーは５０株、雪景色は４０株）。なお、殺菌処理は、図解花のバイオ技術 増殖・育種とその関連技術，誠文堂新光社，１９９２年発行，第６２頁に記載の方法に準じて実施した。
Example 3
To 200 mL of Hyponex medium (commercially available product), 10 μL of 1-naphthalene sodium acetate aqueous solution (concentration: 0.01 mol / L) is added, and further HAp (Wako Pure Chemical Industries) is 0.1% by weight, trehalose (Wako Pure) A chemical) was added in an amount of 0.1% by weight, and agar was added in an amount of 0.8% by weight to prepare a culture medium for shoot apical culture containing HAp and trehalose. One epithelium of the shoot apex of three varieties of sterilized red gypsophila varieties (variety name: snow, Blancy, and Snowscape) was peeled off and planted on the shoot apical culture medium containing HAp and trehalose. Number of planted strains: 50 for Snow and Blancy, 40 for Snowscape). The sterilization treatment was carried out in accordance with the method described in illustrated flower biotechnology propagation / breeding and related technologies, published by Seikodo Shinkosha, 1992, page 62.
植付け後のＨＡｐおよびトレハロースを含有する茎頂培養用培地を、人工気象装置（日本医科機械製；バイオトロンＬＨ２００）に入れ、培養温度約１９〜２３℃で、約７０００〜８０００ルクスの照明下、培養を行った。培養開始後６週間目において、草丈１．５ｃｍ以上のシュートに生長した株の数を調査した。結果を表３に示した。なお、表３中、草丈１．５ｃｍ以上のシュートに生長した株の割合は、植付け株数に対する草丈１．５ｃｍ以上のシュートに生長した株数の割合を表わす。 A shoot apical culture medium containing HAp and trehalose after planting is placed in an artificial weather device (manufactured by Nippon Medical Machinery; Biotron LH200) at a culture temperature of about 19 to 23 ° C. under illumination of about 7000 to 8000 lux. Culture was performed. Six weeks after the start of culture, the number of strains grown on shoots having a plant height of 1.5 cm or more was examined. The results are shown in Table 3. In Table 3, the ratio of strains grown on shoots having a plant height of 1.5 cm or higher represents the ratio of the number of strains grown on shoots having a plant height of 1.5 cm or more to the number of planted strains.
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| CN103975854A (en) * | 2014-04-29 | 2014-08-13 | 卞佳林 | Culture medium for culturing strawberry stem tips and inducing plant differentiation and culture method thereof |
| CN104186351A (en) * | 2014-09-24 | 2014-12-10 | 江苏农林职业技术学院 | Tissue culture method of strawberries |
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