JP6357313B2 - Method for selecting cells capable of somatic embryogenesis and method for producing plant body - Google Patents
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Description
本発明は、植物組織（外植片）から不定胚形成能のある細胞を誘導し選抜する方法に関する。
本発明はまた、上記の選抜された不定胚形成能のある細胞から不定胚を誘導し植物体を作製する方法に関する。
The present invention relates to a method for inducing and selecting cells capable of forming somatic embryos from plant tissues (explants).
The present invention also relates to a method for producing a plant body by inducing a somatic embryo from the selected cells having the ability to form somatic embryos.
植物の栄養体は、挿し木、株分け、塊茎、りん茎などを利用し増殖されるが、植物種によってはインビトロ培養による増殖法が利用されている。現在用いられているインビトロ培養法は、無菌環境下での挿し木、株分け、塊茎誘導、りん茎誘導のほか、植物体再生能を有する細胞から不定芽や不定胚を経由し植物体を再生する方法などである。 Plant nutrients are proliferated using cuttings, stockings, tubers, stalks, etc., but depending on the plant species, a growth method by in vitro culture is used. In-vitro culture methods currently used include cuttings, stocking, tuber induction, and phosphorus stem induction under aseptic conditions, as well as methods for regenerating plant bodies from cells with plant regeneration ability via adventitious buds and adventitious embryos. Etc.
植物細胞から不定胚を経由して植物体を再生する方法は、大量の植物体が効率よく得られるので期待される手法である。しかし、すべての細胞から植物体が再生するわけではなく、植物体再生効率を最大化できてはいない。特に、不定胚形成能が高い細胞と不定胚形成能がないもしくは低い細胞が同時に存在する状態で細胞の増殖を行っていることに課題がある。不定胚形成能が高い細胞を形態等から経験的に選別することはある程度可能ではあるが、選別された細胞から再度不定胚形成能がないもしくは低い細胞が生じてしまう。また、一般的に不定胚形成能がある細胞の増殖はその他の細胞より劣るので、選別を伴わない増殖により不定胚形成能がある細胞の割合が時間とともに減ってしまい、最終的に不定胚形成能がないもしくは低い細胞が大半をしめることになってしまう。このように植物体再生が期待されない細胞が存在する状態で細胞を増殖することは、細胞増殖時に無駄が生じるとともに植物体再生時の効率を大きく減じてしまう。 A method of regenerating a plant body from a plant cell via a somatic embryo is an expected technique because a large number of plant bodies can be obtained efficiently. However, the plant body does not regenerate from all cells, and the plant body regeneration efficiency cannot be maximized. In particular, there is a problem in that cells are proliferated in a state in which cells having high somatic embryogenic ability and cells having no somatic embryogenic ability or low cells exist simultaneously. Although it is possible to select cells with high somatic embryogenic ability empirically to some extent from the morphology or the like, cells that have no or low somatic embryogenic ability are generated again from the sorted cells. In addition, since the proliferation of cells with somatic embryogenic ability is generally inferior to other cells, the proportion of cells with somatic embryogenic ability decreases with time due to proliferation without selection, and eventually somatic embryogenesis is formed. The cells that are not capable or low will be the majority. Propagating cells in the presence of cells that are not expected to regenerate plants in this way results in waste during cell growth and greatly reduces the efficiency during plant regeneration.
本出願人による特許文献１には、不定胚を経由する植物再生のための、植物組織からの不定胚形成能を有する細胞の誘導および増殖工程、あるいは該不定胚形成能を有する細胞 からの不定胚の誘導工程において、ヒストン脱アセチル化酵素阻害剤を含む培地で植物細胞を培養することを含むことを特徴とする、不定胚形成能のないあるいは低い細胞の生成を抑制する方法が開示されている。
ところで、本発明と関係する植物のアセチル-CoAカルボキシラーゼは、色素体や細胞質に存在し、脂肪酸合成、フラボノイド合成などに関わっており、多くの植物では、色素体にみられる４つの各サブユニットの２量体からなるヘテロメリックな酵素と、細胞質にみられる単一のポリペプチドの２量体からなるホモメリックな酵素が存在することが知られている（非特許文献１）。しかし、例外があるもののイネ科植物の色素体アセチル-CoAカルボキシラーゼはホモメリックな酵素である。この相違を利用してホモメリックな酵素の阻害剤がイネ科雑草の選択的除草剤として利用されている（非特許文献２）。 By the way, plant acetyl-CoA carboxylase related to the present invention is present in plastids and cytoplasm, and is involved in fatty acid synthesis, flavonoid synthesis, etc. In many plants, each of the four subunits found in plastids. It is known that there exists a heteromeric enzyme composed of a dimer and a homomeric enzyme composed of a dimer of a single polypeptide found in the cytoplasm (Non-patent Document 1). However, with exceptions, the plastid acetyl-CoA carboxylase of the grass family is a homomeric enzyme. By utilizing this difference, homomeric enzyme inhibitors have been used as selective herbicides for gramineous weeds (Non-patent Document 2).
簡便に不定胚形成能のないもしくは低い細胞の増殖を抑制することにより、植物体再生能が高い細胞の割合を高める方法を提供し、従来法に比してより効率的な不定胚による植物体の作製（すなわち、生産）を可能とする。 Providing a method to increase the proportion of cells with high plant regeneration ability by simply suppressing the growth of cells with no or low somatic embryogenic ability, and more efficient somatic embryo plants compared to conventional methods Can be produced (ie, produced).
特許文献１には、不定胚の誘導工程において、ヒストン脱アセチル化酵素阻害剤を含む培地で植物細胞を培養することを含む、不定胚形成能のないもしくは低い細胞の生成を抑制する方法が開示されているが、ヒストン脱アセチル化酵素阻害剤はその効果が不十分な場合があった。また、植物二次代謝阻害剤に、不定胚形成能のないもしくは低い細胞の生成を抑制する作用効果があることは、これまで知られていなかった。
鋭意検討の結果、アセチル-CoAカルボキシラーゼ阻害剤やチオカーバメイト刑除草剤などの植物二次代謝阻害剤を含む培地で植物組織培養することにより、不定胚形成能のないもしくは低い細胞の増殖を特異的に抑制できることを見出し、これにより、植物体再生能が高い細胞の割合を高めることが可能となり、不定胚からの植物体再生の効率を改善することを見出した。これにより本発明を完成した。 As a result of intensive studies, the growth of cells with no or low somatic embryogenic ability is specifically identified by culturing plant tissues in a medium containing plant secondary metabolism inhibitors such as acetyl-CoA carboxylase inhibitor and thiocarbamate herbicide. It has been found that the ratio of cells having high plant regeneration ability can be increased, thereby improving the efficiency of plant regeneration from somatic embryos. This completed the present invention.
本発明は以下の特徴を有する。
（１）下記の工程（ａ）〜（ｃ）：
（ａ）植物組織を、１種又は複数種の植物二次代謝阻害剤を含む培地で培養して、該植物組織から不定胚形成能を有する植物細胞を誘導し及び選抜する工程、又は、不定胚形成能を有する植物細胞を含む植物細胞塊を、1種又は複数種の植物二次代謝阻害剤を含む培地で培養して、不定胚形成能を有する植物細胞を選抜する工程、
（ｂ）工程（ａ）で選抜された不定胚形成能を有する植物細胞から不定胚を誘導する工程、
（ｃ）工程（ｂ）で誘導された不定胚から植物体を再生する工程、
を含む、植物体の作製方法。
The present invention has the following features.
(1) The following steps (a) to (c):
(A) a step of culturing a plant tissue in a medium containing one or more kinds of plant secondary metabolism inhibitors to induce and select plant cells having somatic embryogenic ability from the plant tissue, or indefinite Culturing a plant cell mass containing plant cells having embryogenic ability in a medium containing one or more kinds of plant secondary metabolism inhibitors, and selecting plant cells having somatic embryogenic ability,
(B) a step of inducing somatic embryos from plant cells having the ability to form somatic embryos selected in step (a),
(C) a step of regenerating a plant from the somatic embryo induced in step (b),
A method for producing a plant body, comprising:
（２）工程（ａ）において、選抜された不定胚形成能を有する植物細胞を維持及び/又は増殖することをさらに含む、上記（１）に記載の方法。 (2) The method according to (1), further comprising maintaining and / or proliferating selected plant cells having somatic embryogenic ability in the step (a).
（３）植物組織を１種又は複数種の植物二次代謝阻害剤を含む培地で培養して不定胚形成能のある植物細胞を選抜することを特徴とする、不定胚形成能のある植物細胞の選抜方法。 (3) Plant cells capable of somatic embryogenesis characterized by selecting plant cells capable of somatic embryogenesis by culturing plant tissues in a medium containing one or more kinds of plant secondary metabolism inhibitors. Selection method.
（４）不定胚形成能のある植物細胞を、１種又は複数種の植物二次代謝阻害剤を含む培地で維持することを特徴とする、不定胚形成能のある植物細胞の維持方法。 (4) A method for maintaining plant cells capable of somatic embryogenesis, comprising maintaining plant cells capable of somatic embryogenesis in a medium containing one or more kinds of plant secondary metabolism inhibitors.
（５）植物組織を１種又は複数種の植物二次代謝阻害剤を含む培地で培養することを含むことを特徴とする、不定胚形成能のないもしくは低い細胞の生成を抑制する方法。 (5) A method for suppressing the generation of cells having no or low somatic embryogenic ability, comprising culturing a plant tissue in a medium containing one or more kinds of plant secondary metabolism inhibitors.
（６）植物二次代謝阻害剤が、アセチル-CoAカルボキシラーゼ阻害剤である、上記（１）〜（５）のいずれかに記載の方法。 (6) The method according to any one of (1) to (5) above, wherein the plant secondary metabolism inhibitor is an acetyl-CoA carboxylase inhibitor.
（７）アセチル-CoAカルボキシラーゼ阻害剤が、シクロヘキサンジオン、アリルオキシプロピオン酸エステル及びフェニルピラゾリンからなる群から選択される、上記（６）に記載の方法。 (7) The method according to (6) above, wherein the acetyl-CoA carboxylase inhibitor is selected from the group consisting of cyclohexanedione, allyloxypropionate and phenylpyrazolin.
（８）シクロヘキサンジオンが、セトキシジムである、上記（７）に記載の方法。
（９）アリルオキシプロピオン酸エステルが、フルアジホップである、上記（７）に記載の方法。
(8) The method according to (7) above, wherein the cyclohexanedione is cetoxydim.
(9) The method according to (7) above, wherein the allyloxypropionic acid ester is fluazifop.
（１０）植物二次代謝阻害剤が、チオカーバメイト刑除草剤である、上記（１）〜（５）のいずれかに記載の方法。
（１１）チオカーバメイト刑除草剤が、ピリブチカルブである、上記（１０）に記載の方法。
(10) The method according to any one of (1) to (5) above, wherein the plant secondary metabolism inhibitor is a thiocarbamate herbicide.
(11) The method according to (10) above, wherein the thiocarbamate herbicide is pilibutycarb.
本発明は、不定胚からの植物体の再生において、植物組織を１種又は複数種の植物二次代謝阻害剤を含む培地で培養して、該植物組織から不定胚形成能を有する植物細胞を誘導し及び選抜すること、あるいは、不定胚形成能を有する植物細胞を含む植物細胞塊を、1種又は複数種の植物二次代謝阻害剤を含む培地で培養して、不定胚形成能を有する植物細胞を選抜することを特徴とする、植物体の効率的な作製を可能にする。 The present invention relates to regeneration of a plant body from an somatic embryo, by culturing a plant tissue in a medium containing one or more kinds of plant secondary metabolism inhibitors, so that plant cells having the ability to form somatic embryos from the plant tissue are obtained. Induced and selected, or plant cell mass containing plant cells having somatic embryogenic ability is cultured in a medium containing one or more kinds of plant secondary metabolism inhibitors, and has somatic embryogenic ability It enables efficient production of plants characterized by selecting plant cells.
以下に、本発明をさらに具体的に説明する。
本発明は、第１の態様により、下記の工程（ａ）〜（ｃ）：
（ａ）植物組織を、１種又は複数種の植物二次代謝阻害剤を含む培地で培養して、該植物組織から不定胚形成能を有する植物細胞を誘導し及び選抜する工程、又は、不定胚形成能を有する植物細胞を含む植物細胞塊を、1種又は複数種の植物二次代謝阻害剤を含む培地で培養して、不定胚形成能を有する植物細胞を選抜する工程、
（ｂ）工程（ａ）で選抜された不定胚形成能を有する植物細胞から不定胚を誘導する工程、
（ｃ）工程（ｂ）で誘導された不定胚から植物体を再生する工程、
を含む、植物体の作製方法を提供する。
上記方法の工程（ａ）は、選抜された不定胚形成能を有する植物細胞を維持及び/又は増殖することをさらに含むことができる。
Hereinafter, the present invention will be described more specifically.
According to the first aspect of the present invention, the following steps (a) to (c):
(A) a step of culturing a plant tissue in a medium containing one or more kinds of plant secondary metabolism inhibitors to induce and select plant cells having somatic embryogenic ability from the plant tissue, or indefinite Culturing a plant cell mass containing plant cells having embryogenic ability in a medium containing one or more kinds of plant secondary metabolism inhibitors, and selecting plant cells having somatic embryogenic ability,
(B) a step of inducing somatic embryos from plant cells having the ability to form somatic embryos selected in step (a),
(C) a step of regenerating a plant from the somatic embryo induced in step (b),
A method for producing a plant body is provided.
Step (a) of the above method may further comprise maintaining and / or growing selected plant cells having somatic embryogenic ability.
本発明における重要な特徴は、工程（ａ）、すなわち、植物組織を、１種又は複数種の植物二次代謝阻害剤を含む培地で培養して、該植物組織から不定胚形成能を有する植物細胞を誘導し及び選抜すること、又は、不定胚形成能を有する植物細胞を含む植物細胞塊を、1種又は複数種の植物二次代謝阻害剤を含む培地で培養して、不定胚形成能を有する植物細胞を選抜すること、である。 An important feature of the present invention is that the plant having the ability to form somatic embryos from the plant tissue by culturing the plant tissue in a medium containing one or more kinds of plant secondary metabolism inhibitors. Inducing and selecting cells, or culturing a plant cell mass containing plant cells having somatic embryogenic ability in a medium containing one or more kinds of plant secondary metabolism inhibitors to produce somatic embryogenic ability Selecting plant cells having
＜植物二次代謝阻害剤＞
植物の代謝は、エネルギー獲得のための生命維持の基本物質としての炭水化物、タンパク質、脂肪及び核酸の合成と、二次代謝と呼ばれる、生命維持にとって必ずしも必要でない多数の物質の合成に分けられる。二次代謝で合成される物質には、薬剤上の有用物質が多く含まれており、例えば、アルカロイド、テルペノイド、イソプレノイド、フラボノイドなどが挙げられる。
<Plant secondary metabolism inhibitor>
Plant metabolism is divided into the synthesis of carbohydrates, proteins, fats and nucleic acids as life sustaining fundamentals for energy acquisition, and the synthesis of a number of substances not necessarily necessary for life sustaining, called secondary metabolism. Substances synthesized by secondary metabolism contain many useful substances on drugs, and examples include alkaloids, terpenoids, isoprenoids, and flavonoids.
本発明者らが見出した、植物二次代謝阻害剤が不定胚形成能のないもしくは低い細胞の増殖を特異的に抑制できるという知見は、不定胚形成能のないもしくは低い細胞から、不定胚形成能を有する植物細胞を選抜することを可能にした。このような知見は、これまで知られていなかった。 The finding that the plant secondary metabolism inhibitor found by the present inventors can specifically suppress the proliferation of cells having no or low somatic embryogenesis is the formation of somatic embryos from cells having no or low somatic embryogenic ability. It was possible to select plant cells having the ability. Such knowledge has not been known so far.
すなわち、本発明によれば、植物の細胞培養において、植物二次代謝阻害剤、例えばアセチル-CoAカルボキシラーゼ阻害剤及び/又はチオカーバメート刑除草剤の１種又は２種以上の二次代謝阻害剤を含む培地で培養することにより、不定胚形成能のない又は低い細胞の増殖を抑制することが可能である。 That is, according to the present invention, in plant cell culture, a plant secondary metabolism inhibitor, for example, an acetyl-CoA carboxylase inhibitor and / or a thiocarbamate herbicide, one or more secondary metabolism inhibitors are added. By culturing in the medium containing it, it is possible to suppress the growth of cells having no or low somatic embryogenic ability.
本明細書に記載される「不定胚形成能のないもしくは低い細胞」とは、不定胚形成能のある細胞と比べて不定胚形成能が低いもしくは不定胚形成能がない細胞を意味する。 The “cells having no or low somatic embryogenic ability” described in the present specification mean cells having low somatic embryogenic ability or no somatic embryogenic ability compared to cells having somatic embryogenic ability.
植物のアセチル-CoAカルボキシラーゼは、アセチルCoAのアセチル基にCO2を結合させてマロニルCoAを合成する酵素であり、色素体や細胞質に存在し、脂肪酸合成、フラボノイド合成などに関っている。多くの植物では、色素体にみられる４つの各サブユニットの2量体からなるヘテロメリックな酵素と、細胞質にみられる単一のポリペプチドの2量体からなるホモメリックな酵素が存在することが知られている。しかし、例外があるもののイネ科植物の色素体アセチル-CoAカルボキシラーゼはホモメリックな酵素である。この相違を利用してホモメリックな酵素の阻害剤がイネ科雑草の選択的除草剤として利用されている。 Plant acetyl-CoA carboxylase is an enzyme that synthesizes malonyl-CoA by binding CO 2 to the acetyl group of acetyl-CoA. It exists in the plastids and cytoplasm, and is involved in fatty acid synthesis and flavonoid synthesis. In many plants, there is a heteromeric enzyme consisting of a dimer of each of the four subunits found in plastids, and a homomeric enzyme consisting of a single polypeptide dimer found in the cytoplasm. Are known. However, with exceptions, the plastid acetyl-CoA carboxylase of the grass family is a homomeric enzyme. Utilizing this difference, inhibitors of homomeric enzymes are used as selective herbicides for gramineous weeds.
アセチル-CoAカルボキシラーゼ阻害剤として使用しうる化合物は、除草剤活性を有する以下のもの、例えば、（１）シクロヘキサンジオン、（２）アリルオキシプロピオン酸エステル、（３）フェニルピラゾリンなどの化学グループが知られる。 Compounds that can be used as acetyl-CoA carboxylase inhibitors include the following compounds having herbicidal activity, such as (1) cyclohexanedione, (2) allyloxypropionate, (3) phenylpyrazoline, etc. known.
上記（１）の除草剤としてアロキシジム、ブトロキシジム、クレトジム、シクロキシジム、プロホキシジム、セトキシジム、テプラロキシジム、トラルコキシジム、上記（２）の除草剤としてクロジナホップ プロパルギル、シハロホップ ブチル、ジクロホップメチル、フェノキサプロップ P エチル、フルアジホップ 、ハロキシホップ R メチル、プロパキザホップ、キザロホップ P エチル、上記（３）の除草剤としてピノキサデンなど、が知られている。本発明に利用できるアセチル-CoAカルボキシラーゼ阻害剤は、阻害剤としての機能を有する限りこれらの物質に限定されるものではない。 As the herbicide of (1) above, alloxydim, butroxidim, cretodim, cycloxydim, proxoxime, cetoxydim, tepraloxydim, tralcoxidim, and as the herbicide of (2) above, clodinahop propargyl, cihalohop butyl, diclohop methyl, phenoxaprop P ethyl, Known as fluazifop, haloxyhop R methyl, propaxahop, quizalofop p-ethyl, and pinoxaden as the herbicide of the above (3). The acetyl-CoA carboxylase inhibitor that can be used in the present invention is not limited to these substances as long as it has a function as an inhibitor.
本発明で使用可能なチオカーバメイト刑除草剤は、非限定的に、例えば、ピリブチカルブ（正式名称：Ｏ−３−ｔｅｒｔ−ブチルフェニル−６−メトキシ−２−ピリジル（メチル）チオカーバメート）、エスプロカルブ（正式名称：S-ベンジル (RS)-1,2-ジメチルプロピル (エチル)チオカーバメート）、モリネート（Ｓ−エチル＝ヘキサヒドロ−１Ｈ−アゼピン−１−カルボチオアート）、ブチレート（N,N-ジイソブチルチオカルバミド酸S-エチル）、ベンチオカーブ（S-(4-クロロベンジル) N,N-ジエチルチオカーバメート）、プロスルホカルブ（S−ベンジル−ジプロピルチオカルバマート）、などである。 Thiocarbamate herbicides that can be used in the present invention include, but are not limited to, for example, pyributicarb (formal name: O-3-tert-butylphenyl-6-methoxy-2-pyridyl (methyl) thiocarbamate), esprocarb ( Official names: S-benzyl (RS) -1,2-dimethylpropyl (ethyl) thiocarbamate), molinate (S-ethyl = hexahydro-1H-azepine-1-carbothioate), butyrate (N, N-diisobutylthio) S-ethyl carbamate), bencho curve (S- (4-chlorobenzyl) N, N-diethylthiocarbamate), prosulfocarb (S-benzyl-dipropylthiocarbamate), and the like.
一般に、不定胚を経由する植物体再生の工程は、（ａ）植物組織から不定胚形成能を有する細胞を誘導・維持・増殖する工程、（ｂ）不定胚形成能を有する細胞から不定胚を誘導する工程、（ｃ）不定胚からの植物体再生工程を含む。これらの中で、（ａ）の工程に、上記アセチル-CoAカルボキシラーゼ阻害剤及びチオカーバメイト刑除草剤のうち１種あるいは複数種を添加した培地で植物組織を培養することにより不定胚形成能のある細胞の選択的増殖が可能となる。それぞれのアセチル-CoAカルボキシラーゼ阻害剤及び/又はチオカーバメイト刑除草剤の添加濃度は、5〜500μMの範囲が好ましいが、植物種により最適濃度が異なるため、不定胚形成能のある細胞の増殖を著しく抑制することなく、不定胚形成能のないもしくは低い細胞の増殖を抑制する最適濃度を事前に測定・確認することが望ましい。以下の濃度に限定されるものではないが、例えばヤツガタケトウヒでは本発明の実施例で用いているセトキシジムの場合、50〜500μM、好ましくは70〜300μM、さらに好ましくは80〜250μMの範囲で用いることができる。例えばテーダマツでは本発明の実施例で用いているセトキシジムの場合、10〜100μM、好ましくは15〜80μM、さらに好ましくは20〜75μMの範囲で用いることができる。例えばニンジンでは本発明の実施例で用いているセトキシジムの場合、10〜100μM、好ましくは30〜70μM、さらに好ましくは40〜60μMの範囲で用いることができる。テーダマツについて、フルアジホップの場合、7〜150μM、好ましくは8〜100μM、さらに好ましくは10〜60μMの範囲で用いることができるし、また、ピイブチカルブの場合、10〜200μM、好ましくは15〜150μM、さらに好ましくは20〜130μMの範囲で用いることができる。 In general, the plant regeneration process via somatic embryos includes (a) a step of inducing, maintaining, and proliferating cells having somatic embryogenic ability from plant tissues, and (b) a somatic embryo from cells having somatic embryogenic ability. A step of inducing, and (c) a plant regeneration step from somatic embryos. Among these, the plant tissue is cultured in a medium supplemented with one or more of the above-mentioned acetyl-CoA carboxylase inhibitor and thiocarbamate herbicide in the step (a), and somatic embryo-forming ability is obtained. Allows selective growth of cells. The addition concentration of each acetyl-CoA carboxylase inhibitor and / or thiocarbamate herbicide is preferably in the range of 5 to 500 μM, but the optimum concentration differs depending on the plant species, so that the proliferation of cells having somatic embryogenic ability is remarkably increased. It is desirable to measure and confirm in advance the optimal concentration that suppresses the growth of cells with no or low somatic embryogenesis without suppression. Although it is not limited to the following concentrations, for example, in the case of cetoxydim used in the examples of the present invention, in the case of Spodoptera, it is used in the range of 50 to 500 μM, preferably 70 to 300 μM, more preferably 80 to 250 μM. be able to. For example, in the case of cetixium used in Examples of the present invention for Teida pine, it can be used in the range of 10-100 μM, preferably 15-80 μM, more preferably 20-75 μM. For example, in the case of cetoxydim used in the examples of the present invention for carrot, it can be used in the range of 10 to 100 μM, preferably 30 to 70 μM, more preferably 40 to 60 μM. For Teeda pine, in the case of fluazifop, it can be used in the range of 7 to 150 μM, preferably 8 to 100 μM, more preferably 10 to 60 μM, and in the case of Pibuticalbu, 10 to 200 μM, preferably 15 to 150 μM, more preferably Can be used in the range of 20 to 130 μM.
さらに、上述したアセチル-CoAカルボキシラーゼ阻害剤又はチオカーバメイト刑除草剤の施用に用い得る植物、用い得る培養工程を例示する。 Furthermore, plants that can be used for application of the above-described acetyl-CoA carboxylase inhibitor or thiocarbamate herbicide, and culture processes that can be used are exemplified.
＜植物＞
本発明は、不定胚を提供する植物に適用可能である。植物としては、以下のものに限定されないが、好ましくはマンサク科、ヒノキ科、マツ科、マメ科、フトモモ科、ヤナギ科、クワ科、セリ科、サトイモ科、ユリ科及びヒルガオ科に属する、木本植物、双子葉植物、単子葉植物などの植物が挙げられる。
<Plant>
The present invention is applicable to plants that provide somatic embryos. The plant is not limited to the following, but is preferably a tree belonging to the witch hazel family, cypress family, pine family, legume family, myrtaceae family, willow family, mulberry family, serie family, taro family, lily family, and myrtaceae family. Examples of the plant include dicotyledonous plants and monocotyledonous plants.
上記植物の例は、以下のとおりである。マンサク科（スイートガム等）、ヒノキ科（ヒノキ等）、マツ科（マツやトウヒ等）、マメ科（アルファルファ、アカシア等）、フトモモ科（ユーカリ等）、ヤナギ科（ポプラ等）、クワ科（ゴムノキ等）、セリ科（ニンジン、セロリ等）、サトイモ科（スパティフィラム等）、ユリ科（アスパラガス等）、ヒルガオ科（サツマイモ等）。 Examples of the plant are as follows. Witch hazel (sweet gum, etc.), cypress (hinoki), pine (pine, spruce, etc.), legume (alfalfa, acacia, etc.), myrtaceae (eucalyptus, etc.), willow (poplar, etc.), mulberry ( Rubber tree, etc.), celery family (carrot, celery etc.), taro family (spatiphyllum etc.), lily family (asparagus etc.), convolvulaceae family (sweet potato etc.).
＜植物組織から不定胚形成能を有する細胞を誘導・維持・増殖する工程＞
本発明に用いる各種植物における未熟胚、葉、葉柄、根等の各種植物組織からの細胞の誘導、維持および増殖条件は特に限定はなく、公知の情報を使用することができる。例えば、農林水産研究文献解題No.１７ 植物バイオテクノロジー 平成３年、国際公開WO2007/064028等に記載されている条件を用いることができる。具体的には、例えば、ショ糖１〜６％(w/v%)、２,４-ジクロロフェノキシ酢酸(2,4-D)１〜８ppm、６-ベンジルアデニン0.05〜２ppmを含み、pHを５〜７ に調整したMurashige-Skoog(MS)培地（寒天0.8〜1.2％による固体培地）で、暗所、20〜30℃で培養するという条件を挙げることができる。ここで言う細胞とは、少なくとも不定胚形成能を有する細胞（エンブリオジェニックカルス、E細胞）を含む細胞を指し、他に不定胚形成能を有しない細胞（ノンエンブリオジェニックカルス、NE細胞）も適宜含み得る。両者の割合は植物種別、培養条件等の要因によって変化し得る。本工程の培地にアセチル-CoAカルボキシラーゼ阻害剤及びチオカーバメイト刑除草剤からなる群から選択された1種又は2種以上の植物二次代謝阻害剤を、対象植物ごとに前もって決定された適切な量を添加し培養することで、不定胚形成能のある細胞を優先的に増殖することが可能となる。
<Inducing, maintaining, and proliferating cells having somatic embryogenic ability from plant tissues>
Conditions for inducing, maintaining, and growing cells from various plant tissues such as immature embryos, leaves, petioles, roots, etc. in various plants used in the present invention are not particularly limited, and known information can be used. For example, the conditions described in Agriculture, Forestry and Fisheries Research Literature Solution No. 17 Plant Biotechnology 1991, International Publication WO2007 / 064028, etc. can be used. Specifically, for example, it contains 1 to 6% (w / v%) sucrose, 1 to 8 ppm of 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.05 to 2 ppm of 6-benzyladenine. An example is a Murashige-Skoog (MS) medium (solid medium with agar 0.8 to 1.2%) adjusted to 5 to 7 and cultured at 20 to 30 ° C. in the dark. The cell mentioned here refers to a cell containing at least cells having somatic embryogenesis (embryogenic callus, E cell), and other cells having no somatic embryogenic ability (non-embryogenic callus, NE cell) are also appropriately used. May be included. The ratio of both can vary depending on factors such as plant type and culture conditions. Appropriate amount determined in advance for each target plant, one or more plant secondary metabolism inhibitors selected from the group consisting of acetyl-CoA carboxylase inhibitor and thiocarbamate herbicide in the medium of this step It is possible to preferentially proliferate cells having the ability to form somatic embryos.
＜不定胚形成能を有する細胞から不定胚を誘導する工程＞
本発明に用いる上記で得られた細胞からの不定胚の誘導条件は特に限定はなく、公知の情報を使用することができる。例えば、農林水産研究文献解題No.１７ 植物バイオテクノロジー 平成３年、国際公開WO2007/064028等に記載されている条件を用いることができる。具体的には、一例として、グルタミン酸及び／又はプロリンを各々１〜10mM、ショ糖 0.5〜４％(w/v%)添加し、pHを５〜７に調整したMS培地に、0.05〜５g／lとなるようカルスを置床し、明所、20〜30℃で培養するという条件を挙げることができる。なお、固体培地と液体培地の何れの条件も適用可能である。また、光条件にも特に限定はない。
<Step of inducing somatic embryos from cells having somatic embryogenic ability>
The conditions for inducing somatic embryos from the cells obtained above used in the present invention are not particularly limited, and publicly known information can be used. For example, the conditions described in Agriculture, Forestry and Fisheries Research Literature Solution No. 17 Plant Biotechnology 1991, International Publication WO2007 / 064028, etc. can be used. Specifically, as an example, 1 to 10 mM of glutamic acid and / or proline and 0.5 to 4% (w / v%) of sucrose are added, and 0.05 to 5 g / The condition of placing the callus so that it becomes l and culturing at 20-30 ° C. in a bright place can be mentioned. In addition, any conditions of a solid culture medium and a liquid culture medium are applicable. Also, the light conditions are not particularly limited.
＜不定胚の脱水工程＞
不定胚はそのまま発芽工程に供試することができる。ただし、不定胚を脱水や乾燥することによりその後の植物体再生率が向上する場合があることが知られている。また、保存が必要な場合は、不定胚に脱水や乾燥処理を施すことで一定期間の保存が可能となる。脱水を行う容器は特に限定されないが、少量を処理する場合はシャーレ（直径９cm、高さ1.5cm、など）、多量の場合は透明なプラスティック製の箱型容器（例えば22cm×17cm×7cm程度のサイズ）を用いることができる。何れの場合も底部にペーパータオルを敷いて不定胚を適量入れる（９cmシャーレの場合は 1〜20g程度、上記箱型容器の場合は30〜100g程度）。光環境は、12〜16時間日長、光合成光量子束密度1〜80μmole/m2/sec、 の明条件がよいが、暗条件でも可能である。温度は20℃〜30℃、好ましくは23℃〜27℃とすることができる。期間は1日〜20日、好ましくは2日〜10日とすることができる。
脱水を終えた不定胚は、ペーパータオルを敷いた9cmシャーレに入れた状態にて、４℃程度の低温、暗所にて保存することが可能であり、植物種別にもよるが １〜3ヶ月程度であれば保存後の不定胚の発芽に障害を及ぼすことはない。
<Dehydration process of somatic embryo>
Somatic embryos can be used in the germination process as they are. However, it is known that the regeneration rate of subsequent plant bodies may be improved by dehydrating or drying somatic embryos. Moreover, when preservation is required, the somatic embryo can be preserved for a certain period of time by subjecting it to dehydration and drying. The container for dehydration is not particularly limited, but a petri dish (diameter 9 cm, height 1.5 cm, etc.) is used for processing a small amount, and a transparent plastic box-type container (for example, about 22 cm x 17 cm x 7 cm) is used for a large quantity. Size) can be used. In either case, place an appropriate amount of somatic embryos by placing a paper towel on the bottom (about 1 to 20 g for a 9 cm petri dish, about 30 to 100 g for the box type container). The light environment has good light conditions of 12 to 16 hours in day length and photosynthetic photon flux density of 1 to 80 μmole / m 2 / sec, but it can also be in dark conditions. The temperature can be 20 ° C to 30 ° C, preferably 23 ° C to 27 ° C. The period can be from 1 day to 20 days, preferably from 2 days to 10 days.
The somatic embryos that have been dehydrated can be stored in a 9cm petri dish with paper towels in a dark place at a low temperature of about 4 ° C. Depending on the type of plant, about 1-3 months Therefore, germination of somatic embryos after storage will not be impaired.
＜不定胚からの幼植物体再生工程＞
上記のようにして得られた不定胚又は脱水不定胚は固体培地上でも液体培地中でも高効率で発芽する。発芽に用いる培地はMS培地などを基本培地とし、糖源として例えばショ糖を1〜6％、好ましくは2〜4％添加する。発根などが激しい場合は、その他の糖類としてソルビトール又はマンニトールを1〜6％、好ましくは2〜4％を添加し、根の伸長を抑制することも可能である。植物生長調節物質は特に添加する必要はないが、ジベレリン類、オーキシン類、サイトカイニン類からなる群から選択した少なくとも1種の植物ホルモンを添加して発芽を促進させてもよい。培地のpHは通常5〜7 とすることができる。光環境は明条件、12〜16時間日長、光合成光量子束密度1〜80μmole/m2/secとすることができる。温度は 20℃〜35℃、好ましくは25℃〜30℃とすることができる。固体培地の場合は、例えば寒天（0.8〜1.2 w/v％）又はゲルライト（0.1〜0.5 w/v％）を用いて培地を固形化する。容器は植物組織培養に用いられるものであれば特に限定されるものではない。発芽した不定胚は発根も伴い、そのまま幼植物体へと生育する。
<Regeneration process of seedlings from somatic embryos>
The somatic embryos or dehydrated somatic embryos obtained as described above germinate with high efficiency in both solid media and liquid media. As a medium used for germination, an MS medium or the like is used as a basic medium, and, for example, sucrose is added as a sugar source in an amount of 1 to 6%, preferably 2 to 4%. When rooting is severe, it is possible to add 1 to 6%, preferably 2 to 4% of sorbitol or mannitol as other saccharides to suppress root elongation. A plant growth regulator is not particularly required to be added, but germination may be promoted by adding at least one plant hormone selected from the group consisting of gibberellins, auxins and cytokinins. The pH of the medium can usually be 5-7. The light environment can be a light condition, 12 to 16 hours day length, and a photosynthetic photon flux density of 1 to 80 μmole / m 2 / sec. The temperature can be 20 ° C to 35 ° C, preferably 25 ° C to 30 ° C. In the case of a solid medium, the medium is solidified using, for example, agar (0.8 to 1.2 w / v%) or gellite (0.1 to 0.5 w / v%). The container is not particularly limited as long as it is used for plant tissue culture. Germinated somatic embryos are rooted and grow into young plants as they are.
＜温室への移植工程＞
不定胚から発芽し再分化した幼植物体は、培養容器から取出し温室にて正常に生育する。移植に用いる培養土は特に限定されるものではなく、育苗用に市販されている培養土でよい。幼植物体を移植した後、1〜3週間程度、適度の加湿と遮光を行うことが好ましい。
<Transplantation process to greenhouse>
Seedlings germinated from somatic embryos and redifferentiated are taken out of the culture vessel and grow normally in a greenhouse. The culture soil used for transplantation is not particularly limited, and may be a commercially available soil for raising seedlings. After transplanting the young plant body, it is preferable to perform appropriate humidification and shading for about 1 to 3 weeks.
本発明はさらに、第２の態様により、植物組織を１種又は複数種の植物二次代謝阻害剤を含む培地で培養して不定胚形成能のある植物細胞を選抜することを特徴とする、不定胚形成能のある植物細胞の選抜方法を提供する。 According to the second aspect of the present invention, the plant tissue is cultured in a medium containing one or more kinds of plant secondary metabolism inhibitors, and plant cells capable of somatic embryogenesis are selected. A method of selecting plant cells capable of somatic embryogenesis is provided.
本発明はさらに、第３の態様により、不定胚形成能のある植物細胞を、１種又は複数種の植物二次代謝阻害剤を含む培地で維持することを特徴とする、不定胚形成能のある植物細胞の維持方法を提供する。 According to the third aspect of the present invention, the plant cell capable of somatic embryogenesis is maintained in a medium containing one or more kinds of plant secondary metabolism inhibitors. A method for maintaining certain plant cells is provided.
本発明はさらに、第４の態様により、植物組織を１種又は複数種の植物二次代謝阻害剤を含む培地で培養することを含むことを特徴とする、不定胚形成能のないもしくは低い細胞塊の生成を抑制する方法を提供する。 According to the fourth aspect, the present invention further includes culturing a plant tissue in a medium containing one or more types of plant secondary metabolism inhibitors, or cells having no or low somatic embryogenic ability A method for suppressing the formation of lumps is provided.
これらのいずれの発明も、植物組織を１種又は複数種の植物二次代謝阻害剤を含む培地で培養することを共通の特徴としている。具体的な説明は、上記と同様であり、ここで繰り返されるべきである。 Any of these inventions has a common feature that the plant tissue is cultured in a medium containing one or more kinds of plant secondary metabolism inhibitors. The specific description is similar to that described above and should be repeated here.
以下、実施例により本発明を更に詳細に説明するが、これらの実施例は本発明の範囲を何ら制限するものではないことに留意するべきである。
本実施例で使用する培地の組成を以下の表１に示す。
Hereinafter, the present invention will be described in more detail by way of examples. However, it should be noted that these examples do not limit the scope of the present invention in any way.
The composition of the medium used in this example is shown in Table 1 below.
＜実施例1＞
ヤツガタケトウヒの未熟種子から常法に従って誘導し（丸山ら、” 絶滅危惧種ヤツガタケトウヒの不定胚形成”、 日本森林学会大会学術講演集（2007）118巻）、固体増殖培地（EM）で2〜3週間毎に継代培養されている培養細胞（森林総合研究所保有）について、まず、不定胚誘導能から以下の様に細胞の分類を行った。不定胚誘導培地（MP）を20ml添加した直帰9cmシャーレに約1gの細胞塊をシャーレに置床し、5週間培養後にシャーレあたりの不定胚数を算出した（9回の試験の平均）。その結果により、良好な不定胚誘導能を有する培養細胞（表2の(1)の細胞、E細胞）と、不定胚誘導能を有しないもしくは低い細胞（表2の(2)の細胞、NE細胞）に分類した。
<Example 1>
Induced from immature seeds of Spodoptera spp. (Maruyama et al., “Sterile embryo formation of endangered Spruce spp.”, Annual Conference of the Japanese Forest Society (2007) 118) For cultured cells subcultured every 2 to 3 weeks (owned by Forest Research Institute), first, the cells were classified as follows based on the ability to induce somatic embryos. Approximately 1 g of a cell mass was placed in a petri dish with 20 ml of somatic embryo induction medium (MP) added, and the number of somatic embryos per petri dish was calculated after culturing for 5 weeks (average of 9 tests). As a result, cultured cells having a good ability to induce somatic embryos (cells in Table 2 (1), E cells) and cells having no or low somatic embryo-inducing ability (cells in (2) in Table 2, NE). Cell).
それらE細胞及びNE細胞を以下の試験に供試した。セトキシジムを100、250、500μMの濃度で添加した固体増殖培地（EM培地、20ml/シャーレ、セトキシジムは99％エタノールに溶解して添加）に、E細胞及びNE細胞の細胞塊（約100mg）をシャーレあたり9個ずつ置床した。25℃、暗所条件にて3週間培養した後、各細胞塊の生重量を計測し、細胞塊あたりに平均増加重量を算出した。 These E cells and NE cells were subjected to the following test. E and NE cell masses (approx. 100 mg) were added to a solid growth medium (EM medium, 20 ml / Petri dish, cetoxydim dissolved in 99% ethanol) supplemented with 100, 250, or 500 μM cetoxydim. Nine were placed per one. After culturing at 25 ° C. in a dark place for 3 weeks, the raw weight of each cell mass was measured, and the average increased weight per cell mass was calculated.
セトキシジムを添加した培地上での細胞の増殖は、E細胞については100μM区では何ら影響を受けず、250μM区では大きく抑制は受けたものの継続した増殖が認められた。一方NE細胞については、全ての濃度区においてほぼ完全に増殖が阻害され、E細胞の結果と顕著な差異となった（図1）。この結果から、セトキシジムは不定胚形成能を失ったヤツガタケトウヒ細胞（NE細胞）の増殖を特異的に抑制することが示された。 The proliferation of cells on a medium supplemented with cetoxidim was not affected at all in the 100 μM section for E cells, and continued growth was observed in the 250 μM section although it was greatly suppressed. On the other hand, for NE cells, proliferation was almost completely inhibited in all concentration groups, which was markedly different from that of E cells (Fig. 1). From these results, it was shown that cetoxidim specifically suppresses the growth of Spodoptera spp. Cells (NE cells) that have lost the ability to form somatic embryos.
＜実施例2＞
テーダマツの未熟種子から常法に従って誘導し（GS. Pullmanら、”Improving loblolly pine somatic embryo maturation : comparison of somatic and zygotic embryo morphology, germination and gene expression” Plant Cell Rep. (2003) 21 : 747-758）、固体増殖培地（LP培地にゲルライトを0.25％濃度で添加した培地）で2〜3週間毎に継代培養されている培養細胞について、まず、不定胚誘導能から以下の様に細胞の分類を行った。不定胚誘導培地（LPSA培地）を20ml添加した直帰9cmシャーレに約1gの細胞塊をシャーレに置床し、5週間培養後にシャーレあたりの不定胚数を算出した（５回の試験の平均）。その結果により、良好な不定胚誘導能を有する培養細胞（表3の(1)の細胞、E細胞）と、不定胚誘導能を有しない或いは低い細胞（表3の(2)の細胞、NE細胞）に分類した。
<Example 2>
It is derived from immature seeds of Teida pine according to a conventional method (GS. Pullman et al., “Improving loblolly pine somatic embryo maturation: comparison of somatic and zygotic embryo morphology, germination and gene expression” Plant Cell Rep. (2003) 21: 747-758) For cultured cells that have been subcultured every 2 to 3 weeks in a solid growth medium (medium supplemented with 0.25% gellite in LP medium), first classify the cells according to the ability to induce somatic embryos as follows: went. Approximately 1 g of a cell mass was placed in a petri dish with 20 ml of somatic embryo induction medium (LPSA medium) added, and the number of somatic embryos per petri dish was calculated after culturing for 5 weeks (average of 5 tests). As a result, cultured cells with good somatic embryo-inducing ability (cells in Table 3 (1), E cells) and cells with no or low somatic embryo-inducing ability (cells in (3) in Table 3, NE) Cell).
それらE細胞及びNE細胞を以下の試験に供試した。セトキシジムを25、50、100μMの濃度で添加した固体増殖培地（LP培地にゲルライトを0.25％濃度で添加した培地、20ml/シャーレ）に、E細胞及びNE細胞の細胞塊（約100mg）をシャーレあたり9個ずつ置床した。25℃、暗所条件にて3週間培養した後、各細胞塊の生重量を計測し、細胞塊あたりの平均増加重量を算出した。 These E cells and NE cells were subjected to the following test. E and NE cell masses (about 100 mg) per petri dish in solid growth medium (20 ml / dish medium containing 0.25% gellite in LP medium) supplemented with cetoxidim at a concentration of 25, 50, 100 μM Nine pieces were placed. After culturing at 25 ° C. under dark conditions for 3 weeks, the raw weight of each cell mass was measured, and the average weight increase per cell mass was calculated.
セトキシジムを添加した培地上での細胞の増殖は、E細胞については25μM区では殆ど影響を受けず、50μM区では抑制は受けたものの継続した増殖が認められた。一方NE細胞については、全ての濃度区においてほぼ完全に増殖が阻害され、E細胞の結果と顕著な差異となった（図2）。この結果から、セトキシジムは不定胚形成能を失ったテーダマツ細胞（NE細胞）の増殖を特異的に抑制することが示された。 The proliferation of cells on the medium supplemented with cetoxidim was hardly affected in the 25 μM section for E cells, but continued growth was observed although it was suppressed in the 50 μM section. On the other hand, for NE cells, proliferation was almost completely inhibited in all concentration groups, which was markedly different from that of E cells (Fig. 2). From these results, it was shown that cetoxidim specifically inhibits the growth of Teida pine cells (NE cells) that have lost somatic embryogenic ability.
＜実施例3＞
ニンジンの苗の葉柄部分から常法に従って誘導し（大川博志ら、「不定胚形成能のないあるいは低い細胞の生成抑制方法」、特許公開2010 - 220493）、固体増殖培地（0.1ppm 2.4-D、ショ糖2%、0.2%ゲルライトを含むMS培地）で3〜4週間毎に継代培養されている培養細胞について、不定胚誘導能からE細胞とNE細胞に分類した。
<Example 3>
It is derived from the petioles of carrot seedlings according to a conventional method (Hiroshi Okawa et al., “Method for suppressing the generation of cells with no or low somatic embryogenesis”, Patent Publication 2010-220493), solid growth medium (0.1 ppm 2.4-D, Cultured cells subcultured every 3 to 4 weeks in MS medium containing 2% sucrose and 0.2% gellite) were classified into E cells and NE cells based on their ability to induce somatic embryos.
それらE細胞及びNE細胞を以下の試験に供試した。セトキシジムを50μMの濃度で添加した固体増殖培地（0.1ppm 2.4-D、ショ糖2%、0.2%ゲルライトを含むMS培地、20ml/シャーレ）に、E細胞及びNE細胞の細胞塊（約100mg）をシャーレあたり4-5個ずつ置床した。22℃、暗所条件にて3週間培養した後、各細胞塊の増殖状態を観察した。 These E cells and NE cells were subjected to the following test. E and NE cell masses (about 100 mg) were added to a solid growth medium (0.1 ppm 2.4-D, MS medium containing 2% sucrose, 0.2% gellite, 20 ml / dishlet) supplemented with 50 µM cetoxydim. 4-5 pieces were placed per petri dish. After culturing at 22 ° C. under dark conditions for 3 weeks, the growth state of each cell mass was observed.
セトキシジムを添加した培地上での細胞の増殖は、E細胞について若干抑制は受けたものの継続した増殖が認められた。一方NE細胞については、ほぼ完全に増殖が阻害され、E細胞の結果と顕著な差異となった（図3）。この結果から、セトキシジムは不定胚形成能を失ったニンジン細胞（NE細胞）の増殖を特異的に抑制することが示された。 The growth of cells on the medium supplemented with cetoxydim was observed to be continued although the E cells were slightly suppressed. On the other hand, the proliferation of NE cells was almost completely inhibited from the results of E cells (Fig. 3). From these results, it was shown that cetoxydim specifically inhibits the proliferation of carrot cells (NE cells) that have lost the ability to form somatic embryos.
＜実施例4＞
実施例2と同じテーダマツのE細胞及びNE細胞を用いて以下の試験を実施した。フルアジホップを5、12.5、25、50、100μMの濃度で添加した固体増殖培地（LP培地にゲルライトを0.25％濃度で添加した培地、20ml/シャーレ、フルアジホップはDMSOで溶解）に、E細胞及びNE細胞の細胞塊（約100mg）をシャーレあたり9個ずつ置床した。25℃、暗所条件にて3週間培養した後、各細胞塊の生重量を計測し、細胞塊あたりの平均増加重量を算出した。
<Example 4>
The following test was performed using E cells and NE cells of the same Teida pine as in Example 2. E cells and NE cells in solid growth medium with fluazihop added at concentrations of 5, 12.5, 25, 50, 100 μM (medium with 0.25% gellite added to LP medium, petri dish, fluazihop dissolved in DMSO) Nine cell masses (about 100 mg) were placed per petri dish. After culturing at 25 ° C. under dark conditions for 3 weeks, the raw weight of each cell mass was measured, and the average weight increase per cell mass was calculated.
フルアジホップを添加した培地上での細胞の増殖は、E細胞については12.5μM区までは何ら影響を受けず、25μM以上の区ではかなりの抑制は受け増殖スピードは著しく低下したものの、継続した増殖が認められた。一方NE細胞については、5μM区では増殖抑制は受けなかったが、12.5μM以上の区では完全に増殖が阻害され、E細胞の結果と顕著な差異となった（図4）。この結果から、フルアジホップは不定胚形成能を失ったテーダマツ細胞（NE細胞）の増殖を特異的に抑制することが示された。 Cell growth on media supplemented with fluazifop is not affected at all up to 12.5 μM for E cells. Admitted. On the other hand, the growth of NE cells was not suppressed in the 5 μM group, but the growth was completely inhibited in the group of 12.5 μM or more, which was markedly different from the result of E cells (FIG. 4). From this result, it was shown that fluazifop specifically suppresses the proliferation of Teida pine cells (NE cells) that have lost somatic embryogenic ability.
＜実施例5＞
実施例2と同じテーダマツのE細胞及びNE細胞を用いて以下の試験を実施した。ピリブチカルブを25、50、100μMの濃度で添加した固体増殖培地（LP培地にゲルライトを0.25％濃度で添加した培地、20ml/シャーレ、ピリブチカルブはDMSOで溶解）に、E細胞及びNE細胞の細胞塊（約100mg）をシャーレあたり9個ずつ置床した。25℃、暗所条件にて3週間培養した後、各細胞塊の生重量を計測し、細胞塊あたりの平均増加重量を算出した。
<Example 5>
The following test was performed using E cells and NE cells of the same Teida pine as in Example 2. E-cell and NE-cell masses (solid media with a concentration of 0.25% gellite added to LP medium, 20 ml / dish, pyributicalbu dissolved in DMSO) with a concentration of 25, 50, and 100 μM pilibutycarb. About 100 mg) was placed 9 pieces per petri dish. After culturing at 25 ° C. under dark conditions for 3 weeks, the raw weight of each cell mass was measured, and the average weight increase per cell mass was calculated.
ピリブチカルブを添加した培地上での細胞の増殖は、E細胞については25μM区から緩やかな抑制は見られたものの継続した増殖が認められた。一方NE細胞については、全ての区は完全に増殖が阻害され、E細胞の結果と顕著な差異となった（図5）。この結果から、ピリブチカルブ不定胚形成能を失ったテーダマツ細胞（NE細胞）の増殖を特異的に抑制することが示された。 Regarding the growth of cells on the medium supplemented with pyributicarb, although E-cells were moderately suppressed from the 25 μM section, continuous growth was observed. On the other hand, in the case of NE cells, proliferation was completely inhibited in all sections, which was markedly different from that of E cells (Fig. 5). From this result, it was shown that the proliferation of Teida pine cells (NE cells) that lost the ability to form somatic embryos was specifically suppressed.
＜実施例6＞
実施例3と同様の条件にてニンジンのカルスを誘導する際に、カルス誘導培地にセトキシジムを50μM添加した培地と無添加の培地を用いた。夫々の培地で誘導されたカルスを、特に選別することなく無作為に同じ培地（セトキシジム添加培地、無添加培地）に継代培養した。その後同様の継代培養を3〜4週間毎に約2年間繰り返した後、夫々のカルスを植物体再生培地（ショ糖1％、マンニトール1％、0.8%寒天を含むMS培地、20ml／シャーレ、pH5.8）に置床し、1ヶ月後に植物体再分化の状況を観察した。
<Example 6>
When inducing carrot callus under the same conditions as in Example 3, a medium in which 50 μM cetoxydim was added to the callus induction medium and a medium without addition were used. The callus induced in each medium was randomly subcultured to the same medium (cetoxydim-added medium, non-added medium) without any particular selection. Thereafter, the same subculture was repeated every 3 to 4 weeks for about 2 years, and then each callus was regenerated into a plant regeneration medium (MS medium containing 1% sucrose, 1% mannitol, 0.8% agar, 20 ml / dish, The plant was placed at pH 5.8) and the state of plant regeneration was observed after one month.
何れのカルスについても明確な植物体の再分化はまだ認められなかったものの、顕微鏡観察を行ったところ、セトキシジム添加地で誘導及び維持されたカルスについては植物体再生に繋がり得る形態形成の兆候を認めた。一方、セトキシジム無添加培地で誘導及び維持されたカルスは何ら形態変化を示さず、カルス細胞のままであった。この結果より、セトキシジムをカルス誘導及び維持時に用いることにより、継代培養時の肉眼的な特徴による選抜作業無しに、植物体再生能のある、または高いカルスを誘導しうる可能性が把握できた。 Although no clear regeneration of plant bodies was observed for any callus, microscopic observations showed that callus induced and maintained in the place where cetoxydim was added showed signs of morphogenesis that could lead to plant regeneration. recognized. On the other hand, the callus induced and maintained in the cetoxydim-free medium did not show any morphological changes and remained as callus cells. From this result, it was possible to grasp the possibility of inducing callus having plant regeneration ability or high callus without using a selection operation based on macroscopic characteristics during subculture by using cetoxydim during callus induction and maintenance. .
本発明は、不定胚形成能のないもしくは低い細胞（NE細胞）の増殖を抑制することによって、従来法に比してより効率的に不定胚形成能のある細胞（E細胞）を選抜及び維持し、効率的に植物体を再生する方法を提供することを可能にしたことにより、農業分野にとり利用価値が大きい。 The present invention selects and maintains cells with the ability to form somatic embryos (E cells) more efficiently than conventional methods by suppressing the growth of cells with or without somatic embryogenesis (NE cells). In addition, since it has become possible to provide a method for efficiently regenerating a plant body, the utility value is great for the agricultural field.
Claims (9)
（ａ）植物組織を、アセチル-CoAカルボキシラーゼ阻害剤及びチオカーバメイト系除草剤から選択される１種又は複数種の植物二次代謝阻害剤を含む培地で培養して、該植物組織から不定胚形成能を有する植物細胞を誘導し及び選抜する工程、又は、不定胚形成能を有する植物細胞を含む植物細胞塊を、アセチル-CoAカルボキシラーゼ阻害剤及びチオカーバメイト系除草剤から選択される1種又は複数種の植物二次代謝阻害剤を含む培地で培養して、不定胚形成能を有する植物細胞を選抜する工程、
（ｂ）工程（ａ）で選抜された不定胚形成能を有する植物細胞から不定胚を誘導する工程、
（ｃ）工程（ｂ）で誘導された不定胚から植物体を再生する工程、
を含む、植物体の作製方法。 The following steps (a) to (c):
(A) Plant tissue is cultured in a medium containing one or more kinds of plant secondary metabolism inhibitors selected from acetyl-CoA carboxylase inhibitors and thiocarbamate herbicides, and somatic embryo formation from the plant tissues One or more selected from an acetyl-CoA carboxylase inhibitor and a thiocarbamate herbicide , wherein the plant cell mass comprising a plant cell having an ability to form somatic embryos or a plant cell mass having an ability to form somatic embryos is selected. cultured in medium containing species plant secondary metabolism inhibitor, a step of screening the plant cells with embryogenic potential,
(B) a step of inducing somatic embryos from plant cells having the ability to form somatic embryos selected in step (a),
(C) a step of regenerating a plant from the somatic embryo induced in step (b),
A method for producing a plant body, comprising:
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