JP2023166645A - Method for culturing plant cells - Google Patents
Method for culturing plant cells Download PDFInfo
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- JP2023166645A JP2023166645A JP2020172481A JP2020172481A JP2023166645A JP 2023166645 A JP2023166645 A JP 2023166645A JP 2020172481 A JP2020172481 A JP 2020172481A JP 2020172481 A JP2020172481 A JP 2020172481A JP 2023166645 A JP2023166645 A JP 2023166645A
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- plant
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
Description
本発明は、植物細胞の培養方法及び植物体の再生方法に関する。 The present invention relates to a method for culturing plant cells and a method for regenerating a plant body.
（１）植物細胞の培養と植物体の再生について
イネ、トウモロコシ、コムギなどのイネ科植物において、未熟胚あるいは完熟胚を単離・培養し、植物体を再生した例が報告されているが、培養効率の種間差、品種間差は大きく、培養がほとんどできないものも多い。とりわけ、主要な作物では、コムギやトウモロコシにおいて培養困難な品種が多く知られている。現在までに種々の創意工夫によって、その効率は徐々に改善されてきた。その手段としては、主として、培養培地の改変と植物組織片への前処理の２つがある。
(1) About culturing plant cells and regenerating plants There have been reports of plants regenerating by isolating and culturing immature or mature embryos in gramineous plants such as rice, corn, and wheat. There are large differences in culture efficiency between species and cultivars, and there are many that can hardly be cultured. Among major crops, in particular, many varieties of wheat and corn are known to be difficult to cultivate. Until now, its efficiency has been gradually improved through various ingenuity. There are two main methods for this: modification of the culture medium and pretreatment of plant tissue pieces.
１）培養培地
アグロバクテリウムによるトウモロコシ形質転換において様々な培養培地の改善が試されている。Ｎ６基本培地での形質転換細胞の選抜（非特許文献１）、培地へのＡｇＮＯ３及びカルベニシリンの添加（非特許文献１、非特許文献２）、共存培地へのシステインの添加（非特許文献３）などによって、培養効率の向上がなされた。共存培養培地の組成及びゲル化剤を改変することにより、インディカイネでの形質転換効率（ｔｒａｎｓｆｏｒｍａｔｉｏｎ ｅｆｆｉｃｉｅｎｃｙ）が高まることも報告されている（非特許文献４）。
1) Culture medium Various improvements in culture media have been attempted in maize transformation using Agrobacterium. Selection of transformed cells in N6 basal medium (Non-patent document 1), addition of AgNO3 and carbenicillin to the medium (Non-patent document 1, Non-patent document 2), addition of cysteine to the coexisting medium (Non-patent document 3) The culture efficiency has been improved by these methods. It has also been reported that transformation efficiency in indica rice can be increased by modifying the composition of the co-culture medium and the gelling agent (Non-Patent Document 4).
２）植物組織片への前処理
Ｈｉｅｉ ｅｔ ａｌ．（２００６）（非特許文献５）はアグロバクテリウムを接種する前のイネ及びトウモロコシの未熟胚に熱処理や遠心処理をすることにより形質転換効率が高まること、及びこれまで形質転換できなかった品種でも形質転換体が得られることを報告した。また、Ｉｓｈｉｄａ ａｎｄ Ｈｉｅｉ（ＷＯ２０１１／０１３７６４）（特許文献１）は、アグロバクテリウムと共存培養したコムギ未熟胚から胚軸を取り除くことにより、高い効率で形質転換植物が得られることを見出した。
2) Pretreatment of plant tissue pieces Hiei et al. (2006) (Non-Patent Document 5) has shown that heat treatment and centrifugation treatment of immature embryos of rice and maize before inoculation with Agrobacterium increases transformation efficiency, and that even varieties that could not be transformed until now can be transformed. It was reported that transformants could be obtained. Furthermore, Ishida and Hiei (WO2011/013764) (Patent Document 1) found that transformed plants could be obtained with high efficiency by removing hypocotyls from immature wheat embryos co-cultured with Agrobacterium.
このように培地組成の改変や材料となる植物組織片への前処理などの工夫により、アグロバクテリウムによるイネ、トウモロコシ及びコムギの形質転換における培養法は、格段の効率の向上及び適応品種の拡大がなされている。 In this way, by changing the culture medium composition and pre-treating the plant tissue pieces used as materials, the cultivation method for the transformation of rice, corn, and wheat using Agrobacterium has significantly improved efficiency and expanded the range of applicable varieties. is being done.
その他の方法としては、近年では、形質転換体を作出する方法として、受精卵を用いる方法（非特許文献６）やインプランタパーティクルガン法（非特許文献７）がある。
（２）トレハロース
ネムリユスリカなどの微小動物、イワヒバなどの植物が砂漠などの極度に乾燥した環境の下で乾眠し、生存できるのは、トレハロースが生体内に高濃度で蓄積されるためであることが知られている。また、トレハロースは乾燥休眠時でも機能するほか、動植物に様々なストレス耐性機能性を付与することが報告されている（非特許文献８）。
In recent years, other methods for producing transformants include a method using fertilized eggs (Non-Patent Document 6) and an implanta particle gun method (Non-Patent Document 7).
(2) Trehalose The reason why microscopic animals such as midges and plants such as Selaginella can survive in extremely dry environments such as deserts is because trehalose accumulates in high concentrations within the body. It has been known. In addition, trehalose functions even during dry dormancy and has been reported to impart various stress tolerance functions to animals and plants (Non-Patent Document 8).
しかしながら、一般的な植物においては、極限環境で生存する前述の動植物ほどの高濃度のトレハロースを含有することはない。加えて、植物の一次代謝において、炭素源として、トレハロースが利用されることはない。事実、植物体内のスクロースと比較すると、トレハロースの含有濃度は低いことが明らかとなっている。例えば、シロイズナズナでは、葉中のスクロースの含有量は９ｍｇ／ｇ（乾燥重量）であるが、トレハロースの含有量は０ｍｇ／ｇ（乾燥重量）であり、茎中のスクロースの含有量は７．５ｍｇ／ｇ（乾燥重量）であるが、トレハロースの含有量は０．１ｍｇ／ｇ（乾燥重量）である（非特許文献９）。 However, common plants do not contain trehalose in concentrations as high as those of the above-mentioned plants and animals that live in extreme environments. In addition, trehalose is not used as a carbon source in the primary metabolism of plants. In fact, it has been revealed that the concentration of trehalose is lower than that of sucrose in plants. For example, in Arabidopsis thaliana, the sucrose content in leaves is 9 mg/g (dry weight), the trehalose content is 0 mg/g (dry weight), and the sucrose content in stems is 7.5 mg/g (dry weight). /g (dry weight), but the content of trehalose is 0.1 mg/g (dry weight) (Non-Patent Document 9).
人工的な植物組織の培養では、常法として、炭素源はスクロース及び／又はマルトースが使用されている。一方、トレハロースの植物組織培養における利用はほとんどない。しかしながら、例外的に、熱帯性着生種のシンビジウムであるＣｙｍｂｉｄｉｕｍ ｆｉｎｌａｙｓｏｎｉａｎｕｍのプロトコーム様球体（ＰＬＢ）形成では、トレハロースの添加が有効であることが示されている。ショ糖５～４０ｇ／ｌやトレハロース１０～８０ｇ／ｌの濃度で、ＰＬＢ形成は促進され、それぞれ２０ｇ／ｌの濃度が最も有効であった。シュートからの発根はトレハロース添加区のみで認められ、トレハロースはシュートからの発根に有効と報告されている（非特許文献１０）。 In the cultivation of artificial plant tissues, sucrose and/or maltose are commonly used as carbon sources. On the other hand, trehalose is rarely used in plant tissue culture. However, as an exception, the addition of trehalose has been shown to be effective in protocorm-like sphere (PLB) formation in the tropical epiphytic cymbidium Cymbidium finlaysonianum. PLB formation was promoted at concentrations of 5 to 40 g/l of sucrose and 10 to 80 g/l of trehalose, with each concentration of 20 g/l being the most effective. Rooting from shoots was observed only in the trehalose-added plot, and trehalose is reported to be effective for rooting from shoots (Non-Patent Document 10).
一方、コムギにおいては、トレハロース利用による小胞子の組織培養による植物体再生の試みがなされた研究があるが、トレハロースの効果はなかったと報告されている（非特許文献１１）。また、トレハロースは植物の代謝や生育を阻害することが知られている（非特許文献１２、非特許文献１３）。オオムギでは、トレハロースは炭水化物関連遺伝子の制御を妨げることが報告されている（非特許文献１４、非特許文献１５）。これらにより、トレハロースはスクロース及び／又はマルトースの代わりに、イネ科の植物の培養に利用されることはなかった。 On the other hand, in wheat, there is a study in which an attempt was made to regenerate a plant by tissue culture of microspores using trehalose, but it has been reported that trehalose was not effective (Non-Patent Document 11). Furthermore, trehalose is known to inhibit plant metabolism and growth (Non-Patent Document 12, Non-Patent Document 13). In barley, trehalose has been reported to interfere with the regulation of carbohydrate-related genes (Non-Patent Document 14, Non-Patent Document 15). For these reasons, trehalose has not been used in place of sucrose and/or maltose for culturing Poaceae plants.
本発明は、植物細胞の培養方法及び植物体の再生方法を提供することを目的とする。 An object of the present invention is to provide a method for culturing plant cells and a method for regenerating a plant body.
本発明者らは上記問題解決のために鋭意研究に努めた結果、コムギ未熟胚の培養・再生化において、常法で用いる炭素源（スクロースやマルトース）ではなく、トレハロースを含有する培地で培養することによって、常法培地と比較して、顕著に植物再生率が向上することを見出し、本発明を完成させた。本方法の利用によって、形質転換作物の作出やゲノム編集作物の作出が効率的に可能となった。 As a result of intensive research efforts by the present inventors to solve the above problem, we have developed a method for culturing and regenerating immature wheat embryos using a medium containing trehalose instead of the conventional carbon source (sucrose or maltose). The present inventors have discovered that this significantly improves the plant regeneration rate compared to a conventional culture medium, and has completed the present invention. By using this method, it has become possible to efficiently create transformed crops and genome-edited crops.
限定されるわけではないが、本発明は以下の態様を含む。
［態様１］
イネ科植物の細胞を、トレハロースを含有する培養培地を用いて培養することを含む、植物細胞の培養方法。
［態様２］
イネ科の植物細胞を培養し、植物細胞群及び／又は植物再生体を得る、ことを含む、態様１に記載の方法。
［態様３］
イネ科植物が、イネ、トウモロコシ、コムギ、オオムギ、ライムギ、及びソルガムからなる群から選択される、態様１又は２に記載の方法。
［態様４］
イネ科植物が、コムギである、態様１－３のいずれか１項に記載の方法。
［態様５］
イネ科植物の細胞が、未熟胚、完熟種子、受精卵、カルス、プロトプラスト、又はそれらに由来する細胞群である、態様１－４のいずれか１項に記載の方法。
［態様６］
イネ科植物の細胞が、未熟胚又はそれに由来する細胞群である、態様１－５のいずれか１項に記載の方法。
［態様７］
イネ科植物の細胞に、核酸、タンパク質及び核酸タンパク質複合体からなる群から選択される物質を導入する工程を含む、態様１－６のいずれか１項に記載の方法。
［態様８］
物質の導入により形質転換又はゲノム編集を行う、態様７に記載の方法。
［態様９］
パーティクルガン法又はアグロバクテリウム法によって物質を導入する、態様８又は９に記載の方法。
［態様１０］
植物細胞の培養効率が向上する、態様１－９のいずれか１項に記載の方法。
［態様１１］
物質の導入効率が向上する、態様７－９のいずれか１項に記載の方法。
［態様１２］
イネ科植物の細胞からの植物体の再生方法であって、
（１）イネ科植物の細胞を、トレハロースを含有する培養培地を用いて培養し、そして、
（２）再分化培地で培養する、
ことを含む、植物体の再生方法。
The present invention includes, but is not limited to, the following aspects.
[Aspect 1]
A method for culturing plant cells, the method comprising culturing the cells of a grass family plant using a culture medium containing trehalose.
[Aspect 2]
The method according to aspect 1, comprising culturing plant cells of the Poaceae family to obtain a plant cell group and/or a regenerated plant.
[Aspect 3]
The method according to aspect 1 or 2, wherein the gramineous plant is selected from the group consisting of rice, corn, wheat, barley, rye, and sorghum.
[Aspect 4]
The method according to any one of aspects 1 to 3, wherein the gramineous plant is wheat.
[Aspect 5]
The method according to any one of aspects 1 to 4, wherein the grass cell is an immature embryo, a ripe seed, a fertilized egg, a callus, a protoplast, or a group of cells derived therefrom.
[Aspect 6]
The method according to any one of aspects 1 to 5, wherein the grass cell is an immature embryo or a group of cells derived therefrom.
[Aspect 7]
The method according to any one of aspects 1 to 6, comprising the step of introducing a substance selected from the group consisting of a nucleic acid, a protein, and a nucleic acid protein complex into a cell of a grass family plant.
[Aspect 8]
The method according to aspect 7, wherein transformation or genome editing is performed by introducing a substance.
[Aspect 9]
The method according to aspect 8 or 9, wherein the substance is introduced by a particle gun method or an Agrobacterium method.
[Aspect 10]
The method according to any one of aspects 1-9, wherein the efficiency of culturing plant cells is improved.
[Aspect 11]
The method according to any one of aspects 7-9, wherein the introduction efficiency of the substance is improved.
[Aspect 12]
A method for regenerating a plant from cells of a grass family plant, the method comprising:
(1) Cultivate the cells of a grass family plant using a culture medium containing trehalose, and
(2) Cultivate in regeneration medium,
A method for regenerating a plant, including:
従来培養が困難であるといわれているイネ科植物の培養を容易とし、培養細胞から植物再生体を効率的に取得することが可能になった。 It has now become easier to culture Gramineae plants, which are conventionally said to be difficult to culture, and it has become possible to efficiently obtain regenerated plants from cultured cells.
１．植物細胞の培養方法
本発明は、植物の培養方法に関する。本発明の方法は、イネ科植物の細胞を、トレハロースを含有する培養培地を用いて培養することを含む。
1. TECHNICAL FIELD The present invention relates to a method for culturing plants. The method of the present invention includes culturing cells of a grass family plant using a culture medium containing trehalose.
植物
上記方法における「植物」は、イネ科の植物である。イネ科植物としては、イネ科（Ｐｏａｃｅａｅ）に属するいずれの植物でも用いることができる。一態様として、非限定的に、イネ科植物は、イネ、トウモロコシ、コムギ、オオムギ、ライムギ、及びソルガムからなる群から選択される。（本明細書において、「一態様」と言及した場合には、非限定的であることを意図する。）上記培養方法は、特に、「難培養」とされる植物あるいは品種に用いることが可能である。「難培養」とは、培養が困難、具体的には、例えば、植物体から単離された細胞や組織等の培養が困難、脱分化等の処理によるカルスの形成や、カルスからの植物体への再分化が困難である、ことを意味する。
Plant The "plant" in the above method is a plant of the Poaceae family. As the grass family plant, any plant belonging to the Poaceae family can be used. In one embodiment, and without limitation, the grass is selected from the group consisting of rice, corn, wheat, barley, rye, and sorghum. (In this specification, when "one embodiment" is referred to, it is intended to be non-limiting.) The above culture method can be used particularly for plants or varieties that are considered "difficult to culture." It is. "Difficult to culture" means that it is difficult to culture, specifically, for example, it is difficult to culture cells or tissues isolated from a plant, the formation of a callus through treatments such as dedifferentiation, or the formation of a plant from a callus. This means that it is difficult to redifferentiate into
一態様として、イネ科の植物はコムギ（Ｔｒｉｔｉｃｕｍ）属に属するものが好ましい。例えば、１粒系のコムギとして、Ｔ．ａｅｇｉｌｏｐｏｉｄｅｓ、Ｔ．ｔｈａｏｕｄａｒ、及び、Ｔ．ｍｏｎｏｃｏｃｃｕｍ（１粒コムギ）を、２粒系のコムギとして、Ｔ．ｄｉｃｏｃｃｏｉｄｅｓ、Ｔ．ｄｉｃｏｃｃｕｍ（２粒コムギ、エンマーコムギ）、Ｔ．ｐｙｒｏｍｉｄａｌｅ、Ｔ．ｏｒｉｅｎｔａｌｅ（コーラサンコムギ）、Ｔ．ｄｕｒｕｍ（デュラムコムギ、マカロニコムギ）、Ｔ．ｔｕｒｇｉｄｕｍ（リベットコムギ）、Ｔ．ｐｏｌｏｎｉｃｕｍ（ポーランドコムギ）、及びＴ．ｐｅｒｓｉｃｕｍ（ペルシャコムギ）を、並びに３粒系のコムギとして、Ｔ．ａｅｓｔｉｖｕｍ（普通コムギ、パンコムギ）、Ｔ．ｓｐｅｌｔａ（スペルトコムギ）、Ｔ．ｃｏｍｐａｃｔｕｍ（クラブコムギ、密穂コムギ）、Ｔ．ｓｐｈａｅｒｏｃｏｃｃｕｍ（インド矮性コムギ）、Ｔ．ｍａｈａ（マカコムギ）、及びＴ．ｖａｖｉｌｏｖｉｉ（バビロビコムギ）を挙げることができる。本発明においてパンコムギ（Ｔ．ａｅｓｔｉｖｕｍ）又はマカロニコムギ（Ｔ．ｄｕｒｕｍ）に属する品種が好適であり、特に、Ｔ．ａｅｓｔｉｖｕｍに属する品種が好適である。 In one embodiment, the grass family plants preferably belong to the genus Triticum. For example, as a single grain of wheat, T. aegilopoides, T. thaoudar, and T. monococcum (one-grain wheat), T. dicoccoides, T. dicoccum (two grain wheat, emmer wheat), T. pyromidale, T. orientale (Korasan wheat), T. durum (durum wheat, macaronic wheat), T. turgidum (rivet wheat), T. polonicum (Polish wheat), and T. polonicum. persicum (Persian wheat), as well as three-grain wheat, T. aestivum (common wheat, bread wheat), T. selta (spelt wheat), T. compactum (club wheat, dense ear wheat), T. sphaerococcum (Indian dwarf wheat), T. maha (maca wheat), and T. vavilovii (wheat). In the present invention, varieties belonging to bread wheat (T. aestivum) or macaronic wheat (T. durum) are suitable, and especially T. A variety belonging to the genus Aestivum is suitable.
イネ科植物の細胞
植物材料としては、限定されるものではないが、イネ科植物の未熟胚、完熟種子、受精卵、カルス、プロトプラスト、又はそれらに由来する細胞群が挙げられる。好ましくは、未熟胚又は完熟種子である。本明細書において「未熟胚」とは、受粉後の登熟過程にある未熟種子の胚をいう。本発明の方法に供される未熟胚のステージ（熟期）は特に限定されるものではなく、受粉後いかなる時期に採取されたものであってもよいが、受粉後７から２１日後のものが好ましい。「完熟種子」とは、受粉後の登熟過程が終了して種子として完熟しているものをいう。「受精卵」とは、精細胞と卵細胞とが融合した細胞を意味する。「植物受精卵」とは、植物の胚嚢を含む組織から単離される受精した卵細胞である、即ち、植物体の段階で受粉・受精させ、該植物体から単離した受精卵、であってもよい。あるいは、受粉・受精前の植物体から卵細胞及び精細胞を単離したのち、それらを融合させて受精卵細胞を作出及び取得してもよい。なお、電気的な融合により受精卵を作成及び取得してもよい。また、異種の植物同士の卵細胞と精細胞の融合により得られた受精卵、であってもよい。「プロトプラスト」とは、植物細胞から細胞壁を取り除いた細胞である。
Cellular plant materials of grasses include, but are not limited to, immature embryos, ripe seeds, fertilized eggs, callus, protoplasts, or cell groups derived therefrom of grasses. Preferably, they are immature embryos or ripe seeds. As used herein, the term "immature embryo" refers to an embryo of an immature seed that is in the process of ripening after pollination. The stage (mature stage) of immature embryos to be subjected to the method of the present invention is not particularly limited, and embryos may be collected at any time after pollination, but embryos collected 7 to 21 days after pollination are preferred. preferable. "Ripe seeds" refer to seeds that have completed the ripening process after pollination and are fully ripe as seeds. A "fertilized egg" means a cell in which a sperm cell and an egg cell are fused. A "fertilized plant egg" is a fertilized egg cell isolated from a tissue including an embryo sac of a plant, that is, a fertilized egg that has been pollinated and fertilized at the plant stage and then isolated from the plant. Good too. Alternatively, egg cells and sperm cells may be isolated from a plant before pollination and fertilization, and then fused to produce and obtain fertilized egg cells. Note that fertilized eggs may be created and obtained by electrical fusion. Alternatively, it may be a fertilized egg obtained by fusion of egg cells and sperm cells of different types of plants. A "protoplast" is a plant cell whose cell wall has been removed.
トレハロースを含有する培養培地
上記植物の培養方法は、イネ科植物の細胞を、トレハロースを含有する培養培地を用いて培養することを含む。限定的に、一態様において、植物細胞の培養は、未熟胚から植物体を再生させる工程において行う。
Culture medium containing trehalose The method for culturing a plant described above includes culturing cells of a grass family plant using a culture medium containing trehalose. In one embodiment, the culturing of plant cells is carried out in a step of regenerating a plant from an immature embryo.
以下、植物細胞の培養方法の態様の例を説明する。植物細胞の培養培地は、植物細胞を通常培養するために通常使用されるものでもよく、特に限定されない。非限定的に、例えばＷＬＳ培地、ＷＬＳ－ＲＥＳ培地、ＭＳ培地、ＬＳ培地、Ｎ６培地、Ｂ５培地、Ｒ２培地、ＣＣ培地を基本とする培地等が挙げられる。これらの培地は、基本の濃度から高濃度にされたものでも、希釈されたものであってもよい。好ましくは、１／１０倍から２倍量、より好ましくは、１／５倍から１倍量である。 Examples of aspects of the method for culturing plant cells will be described below. The culture medium for plant cells may be one commonly used for culturing plant cells, and is not particularly limited. Examples include, but are not limited to, media based on WLS medium, WLS-RES medium, MS medium, LS medium, N6 medium, B5 medium, R2 medium, CC medium, and the like. These media may be enriched or diluted from the basic concentration. Preferably, the amount is 1/10 to 2 times, more preferably 1/5 to 1 time.
一態様において、基本となる培地は、ＷＬＳ培地、ＷＬＳ－ＲＥＳ培地である。これらの培地は以下の組成を有する。
ＷＬＳ培地： １×ＬＳメジャー無機塩、１００μＭ ＦｅＥＤＴＡ、１×ＬＳマイナー無機塩、１×ＭＳビタミン、０．５ｍｇ／Ｌ ２，４－Ｄ、２．２ｍｇ／Ｌ ピクローラム、４％ マルトース一水和物、５００ｍｇ／Ｌ グルタミン、１００ｍｇ／Ｌ カゼイン加水分解物、３．７ｍＭ ＭｇＣｌ２、０．１９５％ ＭＥＳ、５７μＭ アスコルビン酸、５μＭ ＡｇＮＯ３、５ｇ／Ｌ アガロース、ｐＨ５．８
ＷＬＳ－ＲＥＳ培地（ＷＬＳ培地の２．２ｍｇ／ｌ ピクローラムを２．５ｍｇ／ｌ ダイカンバに置換した培地）： １×ＬＳメジャー無機塩、１００μＭ ＦｅＥＤＴＡ、１×ＬＳマイナー無機塩、１×ＭＳビタミン、０．５ｍｇ／Ｌ ２，４－Ｄ、２．５ｍｇ／Ｌ ダイカンバ、４％ マルトース一水和物、５００ｍｇ／Ｌ グルタミン、１００ｍｇ／Ｌ カゼイン加水分解物、３．７ｍＭ ＭｇＣｌ２、０．１９５％ ＭＥＳ、５７μＭ アスコルビン酸、５μＭ ＡｇＮＯ３、５ｇ／Ｌ アガロース、ｐＨ５．８
「トレハロース」は、グルコースが１，１－グリコシド結合している二糖の一種である。適宜調整された培養培地にトレハロースを添加する。培養培地に添加されるトレハロースの濃度は特に限定されない。一態様において、培養培地中のトレハロースの濃度は、２０ｍＭ以上、５０ｍＭ以上、７５ｍＭ以上、１００ｍＭ以上、１２５ｍＭ以上、１５０ｍＭ以上、１８０ｍＭ以上、２００ｍＭ以上、２２０ｍＭ以上、２４０ｍＭ以上、２５０ｍＭ以上である。一態様において、培養培地中のトレハロース濃度は、１０００ｍＭ以下、８００ｍＭ以下、６００ｍＭ以下、５００ｍＭ以下、４５０ｍＭ以下、４００ｍＭ以下、３５０ｍＭ以下、３００ｍＭ以下、２８０ｍＭ以下である。非限定的に、培養培地中のトレハロースの濃度は２０ｍＭ以上１０００ｍＭ以下、５０ｍＭ以上８００ｍＭ以下、１００ｍＭ以上６００ｍＭ以下、１５０ｍＭ以上４００ｍＭ以下である。
In one embodiment, the basic medium is WLS medium or WLS-RES medium. These media have the following composition.
WLS medium: 1x LS major inorganic salts, 100 μM FeEDTA, 1x LS minor inorganic salts, 1x MS vitamins, 0.5 mg/L 2,4-D, 2.2 mg/L picloram, 4% maltose monohydrate , 500mg/L glutamine, 100mg/L casein hydrolyzate, 3.7mM MgCl2 , 0.195% MES, 57μM ascorbic acid, 5μM AgNO3 , 5g/L agarose, pH 5.8
WLS-RES medium (medium in which 2.2 mg/l picloram in WLS medium was replaced with 2.5 mg/l dicamba): 1 x LS major inorganic salt, 100 μM FeEDTA, 1 x LS minor inorganic salt, 1 x MS vitamin, 0 .5mg/L 2,4-D, 2.5mg/L dicamba, 4% maltose monohydrate, 500mg/L glutamine, 100mg/L casein hydrolyzate, 3.7mM MgCl2 , 0.195% MES, 57 μM ascorbic acid, 5 μM AgNO 3 , 5 g/L agarose, pH 5.8
“Trehalose” is a type of disaccharide in which glucose is linked with a 1,1-glycosidic bond. Add trehalose to the appropriately adjusted culture medium. The concentration of trehalose added to the culture medium is not particularly limited. In one embodiment, the concentration of trehalose in the culture medium is 20mM or more, 50mM or more, 75mM or more, 100mM or more, 125mM or more, 150mM or more, 180mM or more, 200mM or more, 220mM or more, 240mM or more, 250mM or more. In one embodiment, the trehalose concentration in the culture medium is 1000 mM or less, 800 mM or less, 600 mM or less, 500 mM or less, 450 mM or less, 400 mM or less, 350 mM or less, 300 mM or less, 280 mM or less. Without limitation, the concentration of trehalose in the culture medium is 20 mM or more and 1000 mM or less, 50 mM or more and 800 mM or less, 100 mM or more and 600 mM or less, and 150 mM or more and 400 mM or less.
一態様において、上記トレハロースを含む培養培地は、トレハロースを含む代わりに、常法で用いる炭素源（例えば、スクロースやマルトース）を含まない、あるいは、含量を減じてもよい。一態様において、培養培地はトレハロース以外の炭素源を含まない。炭素源とは、スクロースや、マルトース、グルコース等を意味する。但し、培地中の糖がトレハロースのみである場合を長期間とすることはできない。言い換えると、トレハロース以外を含まない炭素源での長期培養は除く。 In one embodiment, instead of containing trehalose, the culture medium containing trehalose may not contain or have a reduced content of a carbon source (eg, sucrose or maltose) used in a conventional method. In one embodiment, the culture medium does not contain carbon sources other than trehalose. Carbon source means sucrose, maltose, glucose, etc. However, it is not possible to maintain the condition where the only sugar in the medium is trehalose for a long period of time. In other words, long-term cultivation with a carbon source containing nothing other than trehalose is excluded.
上記トレハロースの濃度は、例えば培地が固体のように、トレハロースが必ずしも培地全体に均一に含まれているとは限らない場合、培地単位体積当たりの含有量として把握することができる。 The concentration of trehalose can be understood as the content per unit volume of the medium, for example, when trehalose is not necessarily uniformly contained throughout the medium, such as when the medium is solid.
培養工程において、トレハロースを培地に添加する時期は特に限定されない。添加時期は、培養開始時もしくはそれ以降でもよい。非限定的に、一態様において、トレハロースを含む培養培地を、植物細胞が再生分化能を有する時期まで、植物細胞を培養する工程の、いずれかの時期に使用する。一態様において、トレハロースを含む培養培地を未熟胚が調製されてからの短期間、例えば、１６日以内、８日以内、５日以内、３日以内、２日以内使用する。一態様において、トレハロースを含む培養培地を未熟胚が調製されてから植物への物質の導入を行うまでの期間使用する。培養期間中、トレハロースを培養培地に、一度に添加してもよく、あるいは、（例えば数回に）分けて添加してもよい。一態様において、パーティクルガンで物質導入を伴う培養においては、パーティクルガン処理前の当日、１日前、あるいは２日前からトレハロースを含む培養培地上に未熟胚及びそれに由来する細胞群を置床し、培養するのが良い。また、一態様において、アグロバクテリウムで物質導入を伴う培養においては、未熟胚に由来する細胞群を材料とした場合、アグロバクテリウム接種処理前の当日、１日前、あるいは、接種後２日後までトレハロースを含む培養培地上にそれらを置床し、培養するのが良く、未熟胚を材料とした場合、アグロバクテリウム接種処理の当日、あるいは、接種後２日後までトレハロースを含む培養培地上にそれらを置床し、培養するのが良い。 In the culture process, the timing of adding trehalose to the medium is not particularly limited. The addition time may be at the start of culture or later. In one embodiment, without limitation, a culture medium containing trehalose is used at any stage of the process of culturing plant cells until the plant cells have regenerative differentiation potential. In one embodiment, the culture medium containing trehalose is used for a short period of time after the immature embryo is prepared, eg, within 16 days, within 8 days, within 5 days, within 3 days, within 2 days. In one embodiment, a culture medium containing trehalose is used from the time the immature embryo is prepared until the introduction of the substance into the plant. During the culture period, trehalose may be added to the culture medium all at once or in portions (eg, in several portions). In one embodiment, in culture involving substance introduction using a particle gun, immature embryos and a group of cells derived therefrom are placed on a culture medium containing trehalose on the day, one day, or two days before particle gun treatment, and cultured. It's good. In one embodiment, in culturing with Agrobacterium that involves substance introduction, when a cell group derived from an immature embryo is used as the material, on the day before Agrobacterium inoculation, one day before, or two days after inoculation. It is best to place them on a culture medium containing trehalose and culture them. If immature embryos are used as materials, they should be placed on a culture medium containing trehalose on the day of Agrobacterium inoculation treatment or until 2 days after inoculation. It is best to leave it in a bed and cultivate it.
非限定的に、一態様において、培地は、植物成長調整物質を含んでもよい。植物成長調整物質は、オーキシン類である。具体的には、例えば、オーキシン類として特にインドール－３－酢酸（ＩＡＡ）、２，４－ジクロロフェノキシ酢酸（２，４－Ｄ）、ピクローラム、ダイカンバ、ナフタレン酢酸（ＮＡＡ）、ナフトキシ酢酸、フェニル酢酸、２，４，５－トリクロロフェノキシ酢酸又は他のオーキシン類を添加することができる。一態様において、オーキシン類は、２，４－Ｄ、ＩＡＡ、ＮＡＡからなる群より選択される植物ホルモンである。あるいは、カイネチンや４ＰＵのようなサイトカイニンなど、他の植物成長調節物質を添加することもできる。これらの植物成長調節物質は、単独でも複数を異なる濃度で混合させてもよい。 In one embodiment, the medium may include, without limitation, a plant growth regulator. Plant growth regulators are auxins. Specifically, for example, auxins include indole-3-acetic acid (IAA), 2,4-dichlorophenoxyacetic acid (2,4-D), picloram, dicamba, naphthaleneacetic acid (NAA), naphthoxyacetic acid, and phenylacetic acid. , 2,4,5-trichlorophenoxyacetic acid or other auxins can be added. In one embodiment, the auxins are plant hormones selected from the group consisting of 2,4-D, IAA, NAA. Alternatively, other plant growth regulators can be added, such as kinetin or cytokinins such as 4PU. These plant growth regulators may be used alone or in combination at different concentrations.
一様態において、培養培地に、フィーダー（ナース）細胞を加えてもよい。フィーダー細胞の種類は特に限定されない。例えば、イネの液体培養細胞や、コムギの液体培養細胞などを用いることが可能である。公知のフィーダー細胞や植物外植片を用いることが可能である。例えば、イネ浮遊細胞培養物（Ｏｃ、理化学研究所バイオリソース研究センター）や、コムギ懸濁培養細胞（ＨＴ３－１）などが挙げられる。植物外植片としては、子房などが挙げられる。フィーダー細胞を添加する時期は限定されるものではなく、植物細胞の培養開始前、開始時、開始後であってもよい。 In one embodiment, feeder (nurse) cells may be added to the culture medium. The type of feeder cells is not particularly limited. For example, liquid cultured rice cells, liquid cultured wheat cells, etc. can be used. It is possible to use known feeder cells and plant explants. Examples include rice suspension cell culture (Oc, RIKEN BioResource Research Center) and wheat suspension culture cell culture (HT3-1). Examples of plant explants include ovaries and the like. The timing of adding feeder cells is not limited, and may be before, at the time of, or after the start of culturing of plant cells.
培養温度は、適宜選択可能であり、好ましくは１８℃－３５℃、さらに好ましくは２０－３０℃、最も好ましくは２３－２８℃で行われる。また、この工程の培養は好ましくは暗所、もしくは薄暗い所で行われるが、これに限定されない。 The culture temperature can be selected as appropriate, and is preferably carried out at 18-35°C, more preferably 20-30°C, and most preferably 23-28°C. Furthermore, the culturing in this step is preferably carried out in a dark or dimly lit place, but is not limited thereto.
一態様において、上記培養方法は、イネ科の植物細胞を培養し、植物細胞群及び／又は植物再生体を得る、ことを含む。
細胞群は、胚様体、球状様胚、カルスなどであってもよい。受精卵を分裂誘導し細胞増殖させ、胚様体、球状様胚、カルスなどを形成させる工程は、植物によって最適条件が異なるため特に限定されない。
In one embodiment, the culturing method includes culturing plant cells of the Poaceae family to obtain a plant cell group and/or a regenerated plant.
The cell group may be an embryoid body, a globoid-like embryo, a callus, or the like. The process of inducing division of fertilized eggs to cause cell proliferation to form embryoid bodies, globoid-like embryos, callus, etc. is not particularly limited, as optimal conditions differ depending on the plant.
「植物再生体を得る」ことについて、「２．植物体の再生方法」において詳述する。
本発明はまた、イネ科植物の細胞を培養するための、トレハロースを含有する培養培地、トレハロースの、イネ科植物の細胞の培養培地への使用、イネ科植物の細胞の培養培地に使用されるトレハロースも含む。
"Obtaining a regenerated plant" will be described in detail in "2. Method for regenerating a plant."
The present invention also relates to a culture medium containing trehalose for culturing cells of a grass family, the use of trehalose in a culture medium of a cell of a grass family, and a use of trehalose in a culture medium of cells of a grass family. Also includes trehalose.
一態様において、上記培養方法により、トレハロースを含まない培地で培養した場合と比較して、植物細胞の培養効率が向上する。植物細胞の培養効率は、非限定的に、例えば、培養におけるカルス化率等を意味する。 In one embodiment, the culture method described above improves the efficiency of culturing plant cells as compared to culturing in a medium that does not contain trehalose. The culturing efficiency of plant cells means, without limitation, for example, the callus formation rate in culturing.
非限定的に、培養効率（例えば、カルス化率）が、例えば、１．１倍以上、２倍以上、３倍以上、５倍以上、１０倍以上、３０倍以上に向上する。「培養効率が向上する」には、トレハロースを含まない培地で培養した場合にはカルスが形成されなかったイネ科の品種において、カルス形成が生じるようになる場合も含む。 Without limitation, the culture efficiency (for example, callus formation rate) is improved by, for example, 1.1 times or more, 2 times or more, 3 times or more, 5 times or more, 10 times or more, 30 times or more. "Culture efficiency is improved" includes cases where callus formation occurs in a grass variety that did not form callus when cultured in a medium that does not contain trehalose.
２．植物体の再生方法
本発明は、植物体の再生方法に関する。本発明の再生方法は、イネ科植物の細胞からの植物体の再生方法であって、
（１）イネ科植物の細胞を、トレハロースを含有する培養培地を用いて培養し、そして、
（２）再分化培地で培養する、
ことを含む。
2. TECHNICAL FIELD The present invention relates to a method for regenerating a plant. The regeneration method of the present invention is a method for regenerating a plant from the cells of a grass family, comprising:
(1) Culturing the cells of a grass family plant using a culture medium containing trehalose, and
(2) Cultivate in regeneration medium,
Including.
「植物」、「イネ科植物」、「（イネ科植物の）細胞」、「培養培地」、「トレハロース」等の用語の意義は、「１．植物細胞の培養方法」において記載した通りである。
再分化工程、すなわち、再分化培地での培養工程は特に限定されない。培養細胞群を、任意の培地、例えば、本明細書の実施例に記載したＬＳＦ培地（例えば、ＬＳＦＣｕＰ５（－Ｃ）培地）からなる再分化培地に移し、培養する。非限定的に、再分化工程は光を照射して行ってもよい。再分化培地には、例えば、ＬＳＦ培地、ＭＳ培地、Ｂ５培地、Ｎ６培地であり、アガロース、寒天やゲランガム、ゲルライト等固化材を使用した固体培地を含まない液体培地であってもよい。
The meanings of terms such as "plant", "grass family plant", "cell (of grass family plant)", "culture medium", "trehalose", etc. are as described in "1. Plant cell culture method". .
The regeneration step, ie, the culture step in a regeneration medium, is not particularly limited. The cultured cell population is transferred to an arbitrary medium, for example, a regeneration medium consisting of the LSF medium (eg, LSFCuP5(-C) medium) described in the Examples of this specification, and cultured. Without limitation, the redifferentiation step may be performed by irradiating light. Examples of the redifferentiation medium include LSF medium, MS medium, B5 medium, and N6 medium, and may be a liquid medium that does not contain a solid medium using a solidifying material such as agarose, agar, gellan gum, or gelite.
再分化培地には、オーキシン、サイトカイニンを含む植物成長調節物質を添加してもよい。一態様において、再分化培地は、オーキシン類を含む。「オーキシン」、「サイトカイニン」等の用語の意義は、「１．植物細胞の培養方法」において記載した通りである。 Plant growth regulators including auxin and cytokinin may be added to the regeneration medium. In one embodiment, the regeneration medium includes auxins. The meanings of terms such as "auxin" and "cytokinin" are as described in "1. Method for culturing plant cells."
再分化工程によって、根、シュートなどが再分化した個体を、土が入ったポットなどに移植すれば、正常な植物個体として育成させることができる。
上記植物再生法によって再生されたイネ科植物（再生植物体）も提供される。再生された植物とは、未熟胚の培養、再分化により再生された植物、該植物から得られた細胞、組織等や、上記植物再生法により得られた再分化当代である「Ｔ０世代」やＴ０世代の植物の自殖種子に由来するＴ１世代」などの後代植物、それらを片親にして交配した雑種植物やその後代植物を含む。
If an individual whose roots, shoots, etc. have been redifferentiated through the regeneration process is transplanted into a pot filled with soil, it can be grown as a normal plant individual.
A gramineous plant (regenerated plant body) regenerated by the above-mentioned plant regeneration method is also provided. Regenerated plants include plants regenerated by culturing immature embryos or redifferentiation, cells, tissues, etc. obtained from the plants, and "T0 generation" which is the redifferentiated generation obtained by the above-mentioned plant regeneration method. It includes progeny plants such as "T1 generation derived from self-fertilized seeds of T0 generation plants", hybrid plants hybridized with these plants as single parents, and progeny plants thereof.
本発明以前は、イネ科植物において特に「難培養」とされる種や品種について、効率よく培養することができず、再生された植物を得ることは困難或いは不可能であった。上記植物再生法により、このような植物、品種についても簡便な方法で効率よく培養、再生植物体を得ることが可能になった。 Prior to the present invention, it was not possible to efficiently culture grass species and varieties that were particularly difficult to cultivate, and it was difficult or impossible to obtain regenerated plants. The above-mentioned plant regeneration method has made it possible to efficiently culture such plants and varieties and obtain regenerated plants using a simple method.
３．植物への物質の導入
植物、特に単子葉植物の遺伝子組換え技術は、１９９０年代にアグロバクテリウムを利用した方法がイネ、トウモロコシで開発されたことを契機に急速に利用が普及した。現在までに様々な形質転換方法が開発されてきている。
3. Introduction of Substances into Plants Genetic modification techniques for plants, particularly monocots, have rapidly become popular since the 1990s, when a method using Agrobacterium was developed for rice and corn. Various transformation methods have been developed to date.
また、近年効率的にゲノム編集を行うことが可能になりつつあるが、これも作物種、品種ごとに組織培養の容易性が異なる点が、ゲノム編集効率（ｇｅｎｏｍｅ ｅｄｉｔｉｎｇ ｅｆｆｉｃｉｅｎｃｙ）に大きな影響を与えるため、実用化の妨げとなっている。 In addition, although it has become possible to perform genome editing efficiently in recent years, the ease of tissue culture differs depending on crop species and varieties, which has a large impact on genome editing efficiency. This is an obstacle to practical application.
本発明の、植物細胞の培養方法、植物再生方法により、イネ科植物の細胞を培養することによって得た細胞群から、安定して効率よく再生植物体を得ることが可能になった。上記植物細胞の培養方法、再生方法の工程におけるいずれかの段階において、植物細胞に物質を導入することにより、物質が導入された再生植物を安定して効率よく取得することができる。一態様において、未熟胚に形質転換物質を導入する。 According to the method for culturing plant cells and the method for regenerating plants of the present invention, it has become possible to stably and efficiently obtain regenerated plants from a cell group obtained by culturing cells of grasses. By introducing a substance into a plant cell at any stage in the above-mentioned plant cell culture method or regeneration method, a regenerated plant into which the substance has been introduced can be stably and efficiently obtained. In one embodiment, a transforming agent is introduced into an immature embryo.
一態様において、上記培養方法、植物再生方法の植物細胞は、物質が導入された植物細胞である。一態様において、発明の培養方法、植物再生方法の植物細胞は、イネ科植物の細胞に、核酸、タンパク質及び核酸タンパク質複合体からなる群から選択される物質を導入する工程を含む。 In one embodiment, the plant cells used in the above culture method and plant regeneration method are plant cells into which a substance has been introduced. In one aspect, the plant cell culture method and plant regeneration method of the invention includes the step of introducing a substance selected from the group consisting of nucleic acids, proteins, and nucleic acid-protein complexes into cells of a grass family plant.
導入する物質は、非限定的に、核酸、タンパク質及び核酸タンパク質複合体からなる群から選択される。核酸は特に限定されず、ＤＮＡ、ＲＮＡ、両者の結合体、混合物であってもよい。好ましくはベクターのような環状ＤＮＡ、直鎖ＤＮＡ、環状ＲＮＡ又は直鎖ＲＮＡである。使用される形質転換方法に応じた任意の長さのものを使用可能である。タンパク質は、決まった順番で様々なアミノ酸が、アミド結合（「ペプチド結合」ともいう）でつながった分子の総称である。タンパク質は、ゲノム編集のためのＣａｓ９ヌクレアーゼ等ヌクレアーゼや、修飾酵素、抗体等を含む。核酸タンパク質複合体は、核酸とタンパク質が複合体を形成したものである。例えば、デオキシリボ核タンパク質（ＤＮＡとＶｉｒＤ２タンパク質の複合体等）、リボ核タンパク質（ガイドＲＮＡとＣａｓ９タンパク質の複合体等）などが含まれる。 The substance introduced is selected from the group consisting of, but not limited to, nucleic acids, proteins, and nucleic acid protein complexes. The nucleic acid is not particularly limited, and may be DNA, RNA, a combination thereof, or a mixture thereof. Preferably, it is a circular DNA such as a vector, a linear DNA, a circular RNA, or a linear RNA. Any length can be used depending on the transformation method used. Protein is a general term for molecules in which various amino acids are connected in a fixed order by amide bonds (also called ``peptide bonds''). Proteins include nucleases such as Cas9 nuclease for genome editing, modification enzymes, antibodies, and the like. A nucleic acid protein complex is a complex formed by a nucleic acid and a protein. Examples include deoxyribonucleoproteins (complexes of DNA and VirD2 protein, etc.), ribonucleoproteins (complexes of guide RNA and Cas9 protein, etc.), and the like.
２種類以上の核酸、タンパク質及び核酸タンパク質複合体を導入してもよい。核酸は、２種類以上のＤＮＡ又はＲＮＡでも、ＤＮＡとＲＮＡの組み合わせでもよい。核酸とタンパク質など異なる種の物質を導入してもよい。 Two or more types of nucleic acids, proteins, and nucleic acid protein complexes may be introduced. The nucleic acid may be two or more types of DNA or RNA, or a combination of DNA and RNA. Different types of substances such as nucleic acids and proteins may be introduced.
物質を植物に導入する方法は、植物に所望の物質を導入することのできる公知の方法ならば特に限定されず、植物の種類に応じて適宜選択することができる。例えば、パーティクルガン法、ポリエチレングリコール法（ＰＥＧ法）、エレクトロポレーション法、マイクロインジェクション法、ウィスカー法などの物理化学的方法（ＤＮＡの直接導入法）あるいはアグロバクテリウム法などの生物学的方法（ＤＮＡの間接導入法）を好ましく用いることができる。一態様において、パーティクルガン法又はアグロバクテリウム法によって物質を導入する。 The method for introducing a substance into a plant is not particularly limited as long as it is a known method that can introduce a desired substance into a plant, and can be appropriately selected depending on the type of plant. For example, physicochemical methods (direct DNA introduction methods) such as the particle gun method, polyethylene glycol method (PEG method), electroporation method, microinjection method, and whisker method, or biological methods such as the Agrobacterium method ( Indirect DNA introduction method) can be preferably used. In one embodiment, the substance is introduced by particle gun or Agrobacterium methods.
植物への物質の導入は、非限定的に、例えば、国際公開ＷＯ２０１７／１７１０９２（特許文献２）、国際公開ＷＯ２０１８／１４３４８０（特許文献３）に記載の方法によって行うことができる。 Substances can be introduced into plants without limitation, for example, by the methods described in International Publication WO2017/171092 (Patent Document 2) and International Publication WO2018/143480 (Patent Document 3).
一態様において、物質の導入により形質転換又はゲノム編集を行ってもよい。
ゲノム編集とは、部位特異的ヌクレアーゼを利用して、思い通りに標的遺伝子を改変する技術である。部位特異的ヌクレアーゼとしては、２００５年以降に開発・発見された、ＺＦＮ（ズィーエフエヌ、又は、ジンクフィンガーヌクレアーゼ）、ＴＡＬＥＮ（タレン）、ＣＲＩＳＰＥＲ／Ｃａｓ９（クリスパー・キャスナイン）などが知られている。
In one embodiment, transformation or genome editing may be performed by introducing a substance.
Genome editing is a technology that uses site-specific nucleases to modify target genes as desired. As site-specific nucleases, ZFN (zinc finger nuclease), TALEN, CRISPER/Cas9, etc., which were developed and discovered after 2005, are known.
イネ科植物、例えば、コムギの組織培養と再生体作出は、コムギの品種開発においては、必要な技術である。とりわけ、遺伝子組換えコムギを作出する際やゲノム編集コムギを作出する際には、例えば、アグロバクテリウム法やパーティクルガン法で遺伝子導入した未熟胚や培養細胞を組織培養し、カルスから再生体を取得する方法が標準法となっている。なお、ゲノム編集作出では、ＤＮＡの導入に代わって、リボヌクレオタンパク質（ＲＮＰ）を導入した未熟胚や培養細胞を培養し、再生体を取得してもよい。 BACKGROUND OF THE INVENTION Tissue culture and reproduction of grasses, such as wheat, are necessary techniques for developing wheat varieties. In particular, when producing genetically modified wheat or genome-edited wheat, for example, immature embryos or cultured cells into which genes have been introduced using the Agrobacterium method or particle gun method are tissue cultured, and regenerated bodies are extracted from callus. The method of acquisition is the standard method. In addition, in genome editing production, instead of introducing DNA, immature embryos or cultured cells into which ribonucleoproteins (RNPs) have been introduced may be cultured to obtain regenerated cells.
一態様において、上記培養方法により、トレハロースを含まない培地で培養した場合と比較して、物質の導入効率が向上する。物質の導入効率は、非限定的に、例えば、培養におけるカルス化率、形質転換率（ｔｒａｎｓｆｏｒｍａｔｉｏｎ ｆｒｅｑｕｅｎｃｙ）、ゲノム編集率（ｇｅｎｏｍｅ ｅｄｉｔｉｎｇ ｆｒｅｑｕｅｎｃｙ）等を意味する。 In one embodiment, the above culture method improves the efficiency of substance introduction compared to the case of culturing in a medium that does not contain trehalose. The introduction efficiency of a substance means, without limitation, for example, callus formation rate, transformation frequency, genome editing frequency, etc. in culture.
本明細書中で、「物質の導入効率が高い」とは、高い効率で目的物質が植物細胞へ導入されること、未熟胚等から高い効率でカルスが誘導されること、形質転換カルスから高い効率で再分化が起こること、を包含する概念である。また、本明細書において「物質の導入効率が向上する」とは、目的物質の植物細胞への導入効率が向上すること、未熟胚等からのカルス誘導率が向上すること、形質転換カルスからの再分化効率が向上すること、を包含する概念である。 In this specification, "high substance introduction efficiency" refers to the introduction of a target substance into plant cells with high efficiency, the induction of callus from immature embryos etc. with high efficiency, and the introduction of high efficiency from transformed callus. This is a concept that encompasses the fact that redifferentiation occurs efficiently. In addition, in this specification, "the efficiency of introduction of a substance is improved" means that the efficiency of introduction of a target substance into plant cells is improved, the rate of callus induction from immature embryos, etc. is improved, and the efficiency of callus induction from transformed callus is improved. This is a concept that includes improving redifferentiation efficiency.
非限定的に、物質の導入効率（例えば、形質転換効率）が、例えば、１．３倍以上、１．５倍以上、２倍以上、３倍以上、４倍以上、１０倍以上、２５倍以上に向上する。「物質の導入効率が向上する」には、トレハロースを含まない培地で培養した場合には形質転換ができない、あるいは、ゲノム編集ができないイネ科の品種において、これらができるようになる場合も含む。 Non-limiting examples include cases where the introduction efficiency of the substance (for example, transformation efficiency) is, for example, 1.3 times or more, 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, 10 times or more, 25 times or more. Improve above. "Increasing the efficiency of introducing a substance" includes cases in which transformation is not possible when cultured in a medium that does not contain trehalose, or cases in which it becomes possible to perform these transformations in Poaceae cultivars in which genome editing is not possible.
以下、実施例に基づいて本発明を詳細に説明するが、本発明はこれらの実施例に限定されるものではない。当業者は本明細書の記載に基づいて容易に本発明に修飾・変更を加えることができ、それらは本発明の技術的範囲に含まれる。 EXAMPLES Hereinafter, the present invention will be explained in detail based on Examples, but the present invention is not limited to these Examples. Those skilled in the art can easily make modifications and changes to the present invention based on the description in this specification, and these are included within the technical scope of the present invention.
実施例１：コムギ未熟胚からのカルス誘導
（１）未熟胚の調製及び培養
ＷＯ２０１１／０１３７６４（特許文献１）及びＩｓｈｉｄａ（２０１５）（非特許文献１６）の方法の一部を改変してコムギの未熟胚の調製を行った。Ｆｉｅｌｄｅｒ、Ｃｌａｉｒｅ、Ｍａｃｅ、ＪＴＸ００１、Ｃａｄｅｎｚａ、Ｐａｒａｇｏｎ、ＪＴＸ００２、Ｃｒｏｎｏｘ及びＪａｇｇｅｒの９種類の品種を試験に供した。
Example 1: Callus induction from immature wheat embryos (1) Preparation and culture of immature embryos A part of the method of WO2011/013764 (Patent Document 1) and Ishida (2015) (Non-Patent Document 16) was modified to induce callus from wheat immature embryos. Immature embryos were prepared. Nine varieties were tested: Fielder, Claire, Mace, JTX001, Cadenza, Paragon, JTX002, Cronox, and Jagger.
開花後１４日前後のコムギの未熟種子を脱頴し、７０％エタノール中で４５秒間表面殺菌処理した後、Ｔｗｅｅｎ ２０を１滴含む１％次亜塩素酸ナトリウムで１０分間表面殺菌処理した。処理後の未熟種子を滅菌水で４回洗浄し、実体顕微鏡下で未熟胚を採取し、そして、ＷＬＳ－ｌｉｑ培地中に回収した。ＷＬＳ－ｌｉｑ培地で１度未熟胚を洗浄後、５，０００×ｇ、４℃、１０分間遠心処理した。 Immature wheat seeds about 14 days after flowering were demolded, surface sterilized in 70% ethanol for 45 seconds, and then surface sterilized with 1% sodium hypochlorite containing 1 drop of Tween 20 for 10 minutes. The immature seeds after the treatment were washed four times with sterile water, and the immature embryos were collected under a stereomicroscope and collected in WLS-liq medium. After washing the immature embryo once with WLS-liq medium, it was centrifuged at 5,000×g and 4° C. for 10 minutes.
２５個の未熟胚をシャーレに移し、胚軸をメスで除去した後、以下の「ＷＬＳ９０－ＲＥＳ培地」又は「ＷＬＳ０／９０－ＲＥＳ」上に胚盤側を上向きにして置床し、２５℃、暗黒下で、１日間培養した。得られた未熟胚を（３）のパーティクルガン処理に用いた。 After transferring 25 immature embryos to a Petri dish and removing the hypocotyls with a scalpel, place them on the following "WLS90-RES medium" or "WLS0/90-RES" with the scutellum side facing upward, and place them at 25°C. The cells were cultured in the dark for 1 day. The obtained immature embryo was used for particle gun treatment (3).
各培地の組成は以下の通りである。
ＷＬＳ－ｌｉｑ培地： ０．１×ＬＳメジャー無機塩、１０μＭ ＦｅＥＤＴＡ、０．１×ＬＳマイナー無機塩、０．１×ＭＳビタミン、１％グルコース、０．０５％ＭＥＳ、ｐＨ５．８
ＷＬＳ９０－ＲＥＳ培地： １×ＬＳメジャー無機塩、１００μＭ ＦｅＥＤＴＡ、１×ＬＳマイナー無機塩、１×ＭＳビタミン、０．５ｍｇ／Ｌ ２，４－Ｄ、２．５ｍｇ／Ｌ ダイカンバ、９％ マルトース一水和物（２５０ｍＭ）、５００ｍｇ／Ｌ グルタミン、１００ｍｇ／Ｌ カゼイン加水分解物、３．７ｍＭ ＭｇＣｌ２、０．１９５％ ＭＥＳ、５７μＭ アスコルビン酸、５μＭ ５ｇ／Ｌ アガロース、ｐＨ５．８
ＷＬＳ０／９０－ＲＥＳ培地：ＷＬＳ９０－ＲＥＳ培地のマルトース一水和物を、濃度９．４６％のトレハロース二水和物（２５０ｍＭ）に変更した培地。
The composition of each medium is as follows.
WLS-liq medium: 0.1x LS major inorganic salts, 10 μM FeEDTA, 0.1x LS minor inorganic salts, 0.1x MS vitamins, 1% glucose, 0.05% MES, pH 5.8
WLS90-RES medium: 1x LS major inorganic salts, 100 μM FeEDTA, 1x LS minor inorganic salts, 1x MS vitamins, 0.5 mg/L 2,4-D, 2.5 mg/L dicamba, 9% maltose monohydrate (250mM), 500mg/L glutamine, 100mg/L casein hydrolyzate, 3.7mM MgCl2 , 0.195% MES, 57μM ascorbic acid, 5μM 5g/L agarose, pH 5.8
WLS0/90-RES medium: A medium in which maltose monohydrate in WLS90-RES medium was replaced with trehalose dihydrate (250 mM) at a concentration of 9.46%.
（２）ｂａｒ遺伝子、Ｃａｓ９遺伝子及びｓｇＲＮＡ遺伝子のプラスミド構築及び調製
ｂａｒ遺伝子、Ｃａｓ９遺伝子及びｓｇＲＮＡ遺伝子を含むプラスミドを構築した。ｂａｒ遺伝子の塩基配列を配列番号１に示した。ｂａｒ遺伝子は３５Ｓプロモーターで制御し、ターミネーターにはＮｏｓを用いた。これらをｐＵＣ１９ベクター内に挿入した。
(2) Plasmid construction and preparation of bar gene, Cas9 gene, and sgRNA gene A plasmid containing the bar gene, Cas9 gene, and sgRNA gene was constructed. The base sequence of the bar gene is shown in SEQ ID NO: 1. The bar gene was controlled by a 35S promoter, and Nos was used as the terminator. These were inserted into the pUC19 vector.
ＳｐＣａｓ９（Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｐｙｏｇｅｎｅｓに由来するＣａｓ９）遺伝子の塩基配列を配列番号２に示した。配列番号２はＳｖｉｔａｓｈｅｖ ｅｔ ａｌ．（２０１５）の報告（非特許文献１７）より引用した。ＳｐＣａｓ９は、トウモロコシユビキチンの第１イントロンを有するトウモロコシユビキチンプロモーターとＮｏｓ及び３５Ｓターミネーターの間に挿入し、加えて、アミノ末端（Ｎ末端）側にＳＶ４０由来核移行シグナル（９アミノ酸）及びカルボキシル末端（Ｃ末端）側にｖｉｒＤ２由来核移行シグナル（１８アミノ酸）をコードする配列が含まれるように、ｐＵＣ１９ベクター内に挿入したプラスミドを構築した。ｓｇＲＮＡ転写ユニットの塩基配列を配列番号３に示した。コムギＵ６ポリメラーゼＩＩＩのプロモ―ター及びターミネーターを含むｓｇＲＮＡ遺伝子配列をｐＵＣ１８ベクター内に挿入したプラスミドを構築した。なお、ｓｇＲＮＡ遺伝子配列として、コムギＬＯＸ２遺伝子のターゲット配列を用いた（非特許文献１８）。 The base sequence of the SpCas9 (Cas9 derived from Streptococcus pyogenes) gene is shown in SEQ ID NO: 2. SEQ ID NO: 2 is as described by Svitashev et al. (2015) report (Non-Patent Document 17). SpCas9 is inserted between the maize ubiquitin promoter that has the first intron of maize ubiquitin and Nos and 35S terminators, and in addition, an SV40-derived nuclear localization signal (9 amino acids) and a carboxyl terminus (C A plasmid was constructed by inserting it into the pUC19 vector so that the sequence encoding the virD2-derived nuclear localization signal (18 amino acids) was included on the (terminal) side. The base sequence of the sgRNA transcription unit is shown in SEQ ID NO: 3. A plasmid was constructed in which the sgRNA gene sequence containing the wheat U6 polymerase III promoter and terminator was inserted into the pUC18 vector. Note that the target sequence of the wheat LOX2 gene was used as the sgRNA gene sequence (Non-Patent Document 18).
（３）パーティクルガンによるＤＮＡ導入及びその後の培養
コムギに対するバーティクルガンによるＤＮＡの導入する方法は、基本的にはＢａｒｃｅｌｏ ａｎｄ Ｌａｚｚｅｒｉ（１９９５）（非特許文献１９）及びＲａｓｃｏ－Ｇａｕｎｔ（２００１）（非特許文献２０）の方法を参考とした。
(3) DNA introduction using a particle gun and subsequent cultivation The method of introducing DNA into wheat using a particle gun is basically the method described in Barcelo and Lazzeri (1995) (Non-patent Document 19) and Rasco-Gaunt (2001) (Non-Patent Document 19). The method described in Reference 20) was used as reference.
１５ｍｇの金粒子（ＢＩＯ－ＲＡＤ製、粒径０．６μｍ）を１．５ｍｌチューブに取り分けた。前述のチューブに０．５ｍｌの７０％エタノールを加え、超音波を９０秒間行った後、１５分間振とうした。５秒間遠心し、上清を捨てた。０．５ｍｌの滅菌水に懸濁し、１分間ボルテックスし、遠心後上清を除去した。上記処理を３回繰り返した。０．３７５ｍｌの５０％グリセロールに懸濁（４０ｍｇ／ｍｌ）後、金粒子懸濁液として４℃で保存した。 15 mg of gold particles (manufactured by BIO-RAD, particle size 0.6 μm) was placed in a 1.5 ml tube. 0.5 ml of 70% ethanol was added to the tube described above, subjected to ultrasound for 90 seconds, and then shaken for 15 minutes. Centrifuge for 5 seconds and discard the supernatant. The suspension was suspended in 0.5 ml of sterile water, vortexed for 1 minute, and the supernatant was removed after centrifugation. The above process was repeated three times. After suspending (40 mg/ml) in 0.375 ml of 50% glycerol, the gold particle suspension was stored at 4°C.
調製した金粒子懸濁液（４０ｍｇ／ｍｌ）を均一になるまで５分間ボルテックスした。プラスミドＤＮＡの金粒子へのコーティングとマイクロキャリヤーへの接着を行った。金粒子２０μｌ（０．８ｍｇ）、（２）で調整したプラスミドＤＮＡ（ｂａｒ遺伝子６８０ｆｍｏｌ、Ｃａｓ９遺伝子２０４ｆｍｏｌ及びｓｇＲＮＡ遺伝子２０４ｆｍｏｌ）を混和した後、合計で４０μｌになるように水を加えた後、ＴｒａｎｓＩＴ－２０２０（登録商標）（０．８ μｌ）を加えた。その後、２、３分間続けてボルテックスした後、１分間チューブを静地した。フラッシュ（２ｓ）遠心後、上清を捨て、次に、５６μｌの７０％エタノールで洗浄した。フラッシュ（２ｓ）遠心後、上清を捨て、５６μｌの１００％エタノールで洗浄した。フラッシュ（２ｓ）遠心後、上清を捨てた後、３４μｌのエタノール（１００％）で再懸濁した。タッピングで穏やかに懸濁した後、ボルテックス処理した（２－３秒）。その後、超音波洗浄機で３０秒処理した。 The prepared gold particle suspension (40 mg/ml) was vortexed for 5 minutes until uniform. Plasmid DNA was coated onto gold particles and attached to microcarriers. After mixing 20 μl (0.8 mg) of gold particles and the plasmid DNA prepared in (2) (680 fmol of bar gene, 204 fmol of Cas9 gene, and 204 fmol of sgRNA gene), water was added to make a total of 40 μl, and TransIT- 2020 (registered trademark) (0.8 μl) was added. Thereafter, after vortexing was continued for 2 to 3 minutes, the tube was left still for 1 minute. After flash (2s) centrifugation, the supernatant was discarded and then washed with 56 μl of 70% ethanol. After flash (2 s) centrifugation, the supernatant was discarded and washed with 56 μl of 100% ethanol. After flash centrifugation (2 s), the supernatant was discarded, and the suspension was resuspended in 34 μl of ethanol (100%). After gently suspending the mixture by tapping, it was vortexed (2-3 seconds). Thereafter, it was treated with an ultrasonic cleaner for 30 seconds.
（１）に記載の通り調製した未熟胚２５個に対して、Ｂｉｏｌｉｓｔｉｃ ＰＤＳ－１０００／Ｈｅ Ｐａｒｔｉｃｌｅ Ｄｅｌｉｖｅｒｙ Ｓｙｓｔｅｍ取扱説明書（ＢＩＯ－ＲＡＤ）に従って、パーティクルガン（ＢＩＯＬＩＳＴＩＣ ＰＤＳ１０００／Ｈｅ，Ｂｉｏ－Ｒａｄ）で遺伝子導入した。１ショット当たり５μｌの金粒子懸濁液を使用した。ターゲット距離はストッピングプレートから５ｃｍで、撃ち込み圧は６５０ｐｓｉとした。パーティクルガン処理後、プレート培地上の未熟胚を２８℃で１日間（暗所）、続いて２５℃で培養した。 25 immature embryos prepared as described in (1) were transfected with genes using a particle gun (BIOLISTIC PDS1000/He, Bio-Rad) according to the Biolistic PDS-1000/He Particle Delivery System instruction manual (BIO-RAD). Introduced. 5 μl of gold particle suspension was used per shot. The target distance was 5 cm from the stopping plate, and the firing pressure was 650 psi. After particle gun treatment, the immature embryos on the plate medium were cultured at 28°C for 1 day (in the dark) and subsequently at 25°C.
以下の表は、未熟胚調性から葉サンプリングまでの各工程の日数、培地、実験内容（工程）をまとめたものである。 The table below summarizes the number of days, culture medium, and experimental details (steps) for each step from immature embryonic development to leaf sampling.
ＷＬＳ６０－Ｐ５の組成： １×ＬＳメジャー無機塩、１００μＭ ＦｅＥＤＴＡ、１×ＬＳマイナー無機塩、１×ＭＳビタミン、０．５ｍｇ／Ｌ ２，４－Ｄ、２．２ｍｇ／Ｌ ピクローラム、６％ マルトース一水和物、５００ｍｇ／Ｌ グルタミン、１００ｍｇ／Ｌ カゼイン加水分解物、３．７ｍＭ ＭｇＣｌ２、０．１９５％ ＭＥＳ、５７μＭ アスコルビン酸、５μＭ ＡｇＮＯ３、５ｍｇ／Ｌ ホスフィノトリシン、５ｇ／Ｌ アガロース、ｐＨ５．８
ＷＬＳ－Ｐ１０の組成： １×ＬＳメジャー無機塩、１００μＭ ＦｅＥＤＴＡ、１×ＬＳマイナー無機塩、１×ＭＳビタミン、０．５ｍｇ／Ｌ ２，４－Ｄ、２．２ｍｇ／Ｌ ピクローラム、４％ マルトース一水和物、５００ｍｇ／Ｌ グルタミン、１００ｍｇ／Ｌ カゼイン加水分解物、３．７ｍＭ ＭｇＣｌ２、０．１９５％ ＭＥＳ、５７μＭ アスコルビン酸、５μＭ ＡｇＮＯ３、１０ｍｇ／Ｌ ホスフィノトリシン、５ｇ／Ｌ アガロース、ｐＨ５．８
ＬＳＦＣｕＰ５（－Ｃ）の組成： １×ＬＳメジャー無機塩、１００μＭ ＦｅＥＤＴＡ、１×ＬＳマイナー無機塩、１×ＬＳビタミン、０．２ｍｇ／Ｌ ＩＢＡ、１０μＭ ＣｕＳＯ４、１．５％ スクロース、０．０５％ ＭＥＳ、５ｍｇ／Ｌ ホスフィノトリシン、３ｇ／Ｌ ゲライト、ｐＨ５．８
（４）結果
（ｉ）カルス化率及び形質転換率に対するトレハロース添加培地の効果
（３）においてパーティクルガンによるＤＮＡ導入を行った未熟胚について、カルス化率及び形質転換率を調べた。
Composition of WLS60-P5: 1xLS major inorganic salt, 100μM FeEDTA, 1xLS minor inorganic salt, 1xMS vitamin, 0.5mg/L 2,4-D, 2.2mg/L picloram, 6% maltose. Hydrate, 500mg/L glutamine, 100mg/L casein hydrolyzate, 3.7mM MgCl2 , 0.195% MES, 57μM ascorbic acid, 5μM AgNO3 , 5mg/L phosphinothricin, 5g/L agarose, pH 5 .8
Composition of WLS-P10: 1 x LS major inorganic salt, 100 μM FeEDTA, 1 x LS minor inorganic salt, 1 x MS vitamin, 0.5 mg/L 2,4-D, 2.2 mg/L picloram, 4% maltose. Hydrate, 500mg/L glutamine, 100mg/L casein hydrolyzate, 3.7mM MgCl2 , 0.195% MES, 57μM ascorbic acid, 5μM AgNO3 , 10mg/L phosphinothricin, 5g/L agarose, pH 5 .8
Composition of LSFCuP5(-C): 1x LS major inorganic salt, 100 μM FeEDTA, 1x LS minor inorganic salt, 1x LS vitamin, 0.2 mg/L IBA, 10 μM CuSO 4 , 1.5% sucrose, 0.05 % MES, 5mg/L phosphinothricin, 3g/L gelite, pH 5.8
(4) Results (i) Effect of trehalose-added medium on callus formation rate and transformation rate The callus formation rate and transformation rate of the immature embryos into which DNA was introduced using a particle gun in (3) were examined.
カルス化率は、表１の工程６において、（生じたカルス数／供試未熟胚数）×１００（％）により求めた。形質転換率は、表１の工程７において、選択マーカーｂａｒ遺伝子による選択を行うためのホスフィノトリシンを含有する培地にカルスを植え替えて培養し、（再生個体が生じた供試未熟胚数／供試未熟胚数）×１００（％）により求めた。結果を表２に示す。 The callus formation rate was determined in step 6 of Table 1 by (number of callus produced/number of immature embryos tested) x 100 (%). The transformation rate was determined by culturing the callus by replanting it in a medium containing phosphinothricin for selection using the selection marker bar gene in step 7 of Table 1. It was determined by (number of immature embryos tested) x 100 (%). The results are shown in Table 2.
表２に示す通り、（３）においてパーティクルガンによるＤＮＡ導入を行った、供試した９品種、Ｆｉｅｌｄｅｒ、Ｃｌａｉｒｅ、Ｍａｃｅ、ＪＴＸ００１、Ｃａｄｅｎｚａ、Ｐａｒａｇｏｎ、ＪＴＸ００２、Ｃｒｏｎｏｘ及びＪａｇｇｅｒにおいて、マルトース含有の標準培地よりも、トレハロース培地において、カルス化率（％）が顕著に向上した。さらに７品種、Ｃｌａｉｒｅ、Ｍａｃｅ、ＪＴＸ００１、Ｃａｄｅｎｚａ、Ｐａｒａｇｏｎ、ＪＴＸ００２及びＣｒｏｎｏｘにおいて、マルトース含有の標準培地よりも、トレハロース培地において、形質転換率（％）が顕著に向上した。 As shown in Table 2, in the nine varieties tested (Fielder, Claire, Mace, JTX001, Cadenza, Paragon, JTX002, Cronox, and Jagger) in which DNA was introduced using a particle gun in (3), the maltose-containing standard medium Also, in the trehalose medium, the callus formation rate (%) was significantly improved. Furthermore, in seven varieties, Claire, Mace, JTX001, Cadenza, Paragon, JTX002, and Cronox, the transformation rate (%) was significantly improved in the trehalose medium than in the maltose-containing standard medium.
（ｉｉ）ゲノム編集率に対するトレハロース添加培地の効果
（３）においてパーティクルガンによるＤＮＡ導入を行った未熟胚について、ゲノム編集率を調べた。ゲノム編集率は以下のように調べ、（ゲノム編集個体が生じた供試未熟胚数／供試未熟胚数）×１００（％）により求めた。
(ii) Effect of trehalose-supplemented medium on genome editing rate The genome editing rate was investigated for immature embryos into which DNA was introduced using a particle gun in (3). The genome editing rate was investigated as follows and was determined by (number of immature embryos tested/number of immature embryos tested) x 100 (%).
パーティクルガン処理約８５日後、プラントボックス内で１０ｃｍ～１５ｃｍ程度に伸びた葉５～１０ｍｇ程度を切り取り、サンプリングした。液体窒素で葉サンプルを凍結し、ＴｉｓｓｕｅＬｙｓｅｒ ＩＩ（ＱＩＡＧＥＮ）を用いて凍結した葉サンプルを粉砕した。その後、ＱＩＡｃｕｂｅＨＴ（ＱＩＡＧＥＮ）のプロトコールに従い、ＱＩＡｃｕｂｅＨＴでＤＮＡを抽出した。ゲノム編集の標的とした、Ａ、ＢおよびＤゲノムのＴａＬＯＸ２遺伝子について、ＰＣＲ／ＲＥ分析を行い、ゲノム編集による変異を検出した。 Approximately 85 days after the particle gun treatment, approximately 5 to 10 mg of leaves that had grown to approximately 10 to 15 cm in the plant box were cut and sampled. The leaf samples were frozen with liquid nitrogen and the frozen leaf samples were ground using a TissueLyser II (QIAGEN). Thereafter, DNA was extracted with QIAcubeHT according to the protocol of QIAcubeHT (QIAGEN). PCR/RE analysis was performed on the TaLOX2 genes of genomes A, B, and D, which were targeted for genome editing, to detect mutations caused by genome editing.
ＰＣＲ反応は、２０μｌ反応液中に、１０×ＥｘＴａｑ Ｂｕｆｆｅｒ ２μｌ、ｄＮＴＰ Ｍｉｘｔｕｒｅ（各２．５ｍＭ）１．６μｌ、プライマーＬＯＸ２－Ｆ（１０μＭ）０．４μｌ、プライマーＬＯＸ２－Ｒ（１０μＭ）０．４μｌ、ＥｘＴａｑＨＳ ０．１μｌ、ゲノムＤＮＡ４μｌを混和し、９８℃１０秒、６０℃３０秒、７２℃１分を３０回行った。ＰＣＲプライマーはＺｈａｎｇら（非特許文献１８）のプライマーを使用した。得られたＰＣＲ産物を５μｌとり、１０×ＮＥＢ ＣｕｔＳｍａｒｔ ｂｕｆｆｅｒ ２μｌ、制限酵素ＳａｃＩ－ＨＦ（２０Ｕ／μｌ）を１μｌ含む２０μｌの溶液を調製し、３７℃で３時間インキュベーションした後、１．５％ＴｙｐｅＩＩアガロースゲルを用いた電気泳動で分析した。なお、ネガティブコントロールにはＦｉｅｌｄｅｒのゲノムＤＮＡを用いた。制限酵素ＳａｃＩによって、ＰＣＲ産物が切断された場合はゲノム編集されていないと判断し、切断されない場合はゲノム編集されていると判断した。 For the PCR reaction, in a 20 μl reaction solution, 2 μl of 10×ExTaq Buffer, 1.6 μl of dNTP Mixture (2.5 mM each), 0.4 μl of primer LOX2-F (10 μM), 0.4 μl of primer LOX2-R (10 μM), 0.1 μl of ExTaqHS and 4 μl of genomic DNA were mixed and heated at 98°C for 10 seconds, 60°C for 30 seconds, and 72°C for 1 minute 30 times. The PCR primers used were those of Zhang et al. (Non-Patent Document 18). Take 5 μl of the obtained PCR product, prepare a 20 μl solution containing 2 μl of 10× NEB CutSmart buffer, and 1 μl of restriction enzyme SacI-HF (20 U/μl), incubate at 37°C for 3 hours, and add 1.5% Type II. It was analyzed by electrophoresis using agarose gel. Note that Fielder's genomic DNA was used as a negative control. If the PCR product was cleaved by the restriction enzyme SacI, it was determined that the genome had not been edited, and if it was not cleaved, it was determined that the genome had been edited.
結果を表３に示す。 The results are shown in Table 3.
表３の通り、供試した５品種、Ｃｌａｉｒｅ、Ｍａｃｅ、ＪＴＸ００１、Ｐａｒａｇｏｎ及びＪＴＸ００２において、マルトース含有の標準培地よりも、トレハロース培地において、ゲノム編集率が顕著に向上した。 As shown in Table 3, in the five tested varieties, Claire, Mace, JTX001, Paragon, and JTX002, the genome editing rate was significantly improved in the trehalose medium than in the maltose-containing standard medium.
本発明により、従来培養が困難であるといわれているイネ科植物の培養を容易とし、培養細胞から植物再生体を効率的に取得することが可能になった。上記植物細胞の培養方法、再生方法の工程におけるいずれかの段階において、植物細胞に物質を導入することにより、物質が導入された再生植物を安定して効率よく取得することもできる。本方法の利用によって、従来困難であったイネ科植物の形質転換作物の作出やゲノム編集作物の作出が効率的にできることとなる。 According to the present invention, it has become possible to easily culture gramineous plants, which are conventionally said to be difficult to culture, and to efficiently obtain regenerated plants from cultured cells. By introducing a substance into a plant cell at any stage in the process of the above plant cell culture method or regeneration method, it is also possible to stably and efficiently obtain a regenerated plant into which the substance has been introduced. By using this method, it is now possible to efficiently create transformed crops of gramineous plants and genome-edited crops, which have been difficult in the past.
配列番号１は、ｂａｒ遺伝子の塩基配列である。
配列番号２は、ＳｐＣａｓ９遺伝子の塩基配列である。
配列番号３は、ｓｇＲＮＡ発現ユニットの配列の塩基配列である。
SEQ ID NO: 1 is the base sequence of the bar gene.
SEQ ID NO: 2 is the base sequence of the SpCas9 gene.
SEQ ID NO: 3 is the base sequence of the sgRNA expression unit.
Claims (12)
（１）イネ科植物の細胞を、トレハロースを含有する培養培地を用いて培養し、そして、
（２）再分化培地で培養する、
ことを含む、植物体の再生方法。 A method for regenerating a plant from cells of a grass family plant, the method comprising:
(1) Culturing the cells of a grass family plant using a culture medium containing trehalose, and
(2) Cultivate in regeneration medium,
A method for regenerating a plant, including:
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