JP2925186B2 - Composition containing polymer mitogen activator in higher plants and its use - Google Patents
Composition containing polymer mitogen activator in higher plants and its useInfo
- Publication number
- JP2925186B2 JP2925186B2 JP1281934A JP28193489A JP2925186B2 JP 2925186 B2 JP2925186 B2 JP 2925186B2 JP 1281934 A JP1281934 A JP 1281934A JP 28193489 A JP28193489 A JP 28193489A JP 2925186 B2 JP2925186 B2 JP 2925186B2
- Authority
- JP
- Japan
- Prior art keywords
- plant
- medium
- protoplasts
- culture
- derived
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 241000196324 Embryophyta Species 0.000 title claims description 53
- 239000000203 mixture Substances 0.000 title claims description 29
- 239000003226 mitogen Substances 0.000 title description 8
- 229920000642 polymer Polymers 0.000 title description 7
- 239000012190 activator Substances 0.000 title 1
- 239000002609 medium Substances 0.000 claims description 25
- 239000001963 growth medium Substances 0.000 claims description 19
- 241000219095 Vitis Species 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 6
- 230000002297 mitogenic effect Effects 0.000 claims description 6
- 244000061458 Solanum melongena Species 0.000 claims description 5
- 235000009754 Vitis X bourquina Nutrition 0.000 claims description 5
- 235000012333 Vitis X labruscana Nutrition 0.000 claims description 5
- 235000014787 Vitis vinifera Nutrition 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 5
- 235000002597 Solanum melongena Nutrition 0.000 claims description 4
- 239000012228 culture supernatant Substances 0.000 claims description 3
- 210000001938 protoplast Anatomy 0.000 description 46
- 210000004027 cell Anatomy 0.000 description 15
- 230000000694 effects Effects 0.000 description 10
- 238000000108 ultra-filtration Methods 0.000 description 8
- 206010020649 Hyperkeratosis Diseases 0.000 description 7
- 241000208292 Solanaceae Species 0.000 description 7
- 230000000394 mitotic effect Effects 0.000 description 7
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 6
- 230000008929 regeneration Effects 0.000 description 6
- 238000011069 regeneration method Methods 0.000 description 6
- 239000000600 sorbitol Substances 0.000 description 6
- 239000007640 basal medium Substances 0.000 description 5
- 239000000306 component Substances 0.000 description 5
- 108010059892 Cellulase Proteins 0.000 description 3
- 241000219094 Vitaceae Species 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000012136 culture method Methods 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 235000021021 grapes Nutrition 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108010029182 Pectin lyase Proteins 0.000 description 2
- 240000007377 Petunia x hybrida Species 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229940106157 cellulase Drugs 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000004114 suspension culture Methods 0.000 description 2
- 241000208173 Apiaceae Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 235000009392 Vitis Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は、植物細胞の分裂促進活性を有する高分子分
裂促進活性因子、該因子を含有する高等植物由来プロト
プラスト用培養培地及び該培地を用いた細胞培養法に関
するものである。本発明はプロトプラストからの細胞、
カルス、植物体の再生及び細胞培養による物質生産等に
適用されうるものであり、また、プロトプラスト系を用
いた細胞融合、若しくは遺伝子組換え等のバイオテクノ
ロジーを応用した新植物の開発、細胞培養による物質生
産等にも利用されうるものである。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a macromolecular mitogenic factor having a plant cell mitogenic activity, a culture medium for higher plant-derived protoplasts containing the factor, and a culture medium containing the factor. Cell culture method. The invention relates to cells from protoplasts,
It can be applied to callus, regeneration of plant bodies, substance production by cell culture, etc., and development of new plants applying biotechnology such as cell fusion using a protoplast system or genetic recombination, cell culture It can also be used for material production and the like.
高等植物由来のプロトプラストの培養培地としては、
通常植物の組織培養に使用される培地（原田、駒嶺編・
植物細胞組織培養、理工学社、1979年）に、浸透圧調節
剤として糖もしくは糖アルコール、窒素源として無機の
アンモニウム塩、硝酸塩、その他アミノ酸を添加した培
養培地が用いられている。また、植物細胞の懸濁培地か
ら採取されるコンディションド・メディウム（conditio
ned medium、以下CDMと略す）を培地に添加することに
より、細胞の増殖が促進されることも知られている。As a culture medium for protoplasts derived from higher plants,
Medium usually used for tissue culture of plants (Harada, Komamine,
A culture medium containing a sugar or a sugar alcohol as an osmotic pressure regulator, an inorganic ammonium salt, a nitrate, or another amino acid as a nitrogen source is used in plant cell tissue culture, Riken Co., 1979). In addition, conditioned medium (conditio) collected from the suspension medium of plant cells
It is also known that the addition of ned medium (hereinafter abbreviated as CDM) to the medium promotes cell growth.
ところで、上記培養培地で培養した場合、植物によっ
てはプロトプラストは分裂増殖してカルスとなり植物体
を再生するものもあるが、プロトプラスト化すると何れ
の植物に於いても細胞のもつ生物活性は著しく低下する
ことがある。即ち、培養過程でプロトプラストの破壊や
死滅、細胞壁の生合成能や細胞の分裂能の消失等の障害
が起こる。By the way, when cultured in the above-mentioned culture medium, depending on plants, protoplasts may divide and proliferate into callus to regenerate plants, but when protoplasts are formed, the biological activity of cells in any plant is significantly reduced. Sometimes. That is, during the culture process, damages such as destruction or death of protoplasts, loss of cell wall biosynthesis ability and cell division ability occur.
ナス科の最も生物活性の高い植物、例えばダバコ、ペ
チュニアまたはその他の植物に於いても、あるいは分裂
増殖能を高くした継代カルスから単離されたプロトプラ
ストに於いても、２〜３割しか分裂増殖能を示さず、残
りの殆どの細胞は、プロトプラスト化によって分裂能が
消失する。Even in the most biologically active plants of the Solanaceae family, such as Davaco, Petunia or other plants, or in protoplasts isolated from passaged calli with increased mitotic growth potential, only 20 to 30% split Most of the remaining cells, which have no proliferative ability, lose their ability to divide by protoplastization.
他の植物に於いてはプロトプラストが全く分裂しない
か、分裂する場合に於いても最適条件下で数パーセント
しか分裂増殖能を示さない。In other plants, the protoplasts do not divide at all, or when they divide, they show only a few percent mitotic growth under optimal conditions.
また、植物細胞の懸濁培養から採取されるCDMを培地
に添加することによりプロトプラストの分裂増殖がある
程度促進されることが知られている〔例えば、Kitamura
Y.らのPlant Sci.,60（２）,245〜250ページ、1989及
びSomers D.A.らのPlant Sci.,56（３）,249〜256ペー
ジ、1987〕。しかしながら、CDMの添加で必ずしもプロ
トプラストの培養が可能になるほどの促進効果を示すと
は限らず、却って阻害作用をもたらす場合がある。即
ち、CDM中に残存する培地成分や糖、ホルモン、または
細胞から出される老廃物や未知の成分が原因であると言
われている。さらに、Birnberg P.R.らのJ.Plant Physi
ol.,132（３）,316〜321ページに記載されるように、一
般にCDM中の分裂促進因子は低分子（分子量約1000）で
あると言われており、同じく低分子である培地成分その
他と分離することは困難である。It is also known that the addition of CDM collected from a suspension culture of plant cells to the culture medium promotes the division and proliferation of protoplasts to some extent (for example, Kitamura
Y. et al., Plant Sci ., 60 (2), pp. 245-250, 1989 and Somers DA et al., Plant Sci ., 56 (3), 249-256, 1987]. However, the addition of CDM does not always show a promoting effect enough to allow protoplast culture, and may rather exert an inhibitory effect. That is, it is said that the cause is medium components, sugars, hormones, waste products and unknown components from cells remaining in CDM. Furthermore, Birnberg PR et al., J. Plant Physi
ol ., 132 (3), pp. 316-321, it is generally said that the mitogen in CDM is a small molecule (molecular weight: about 1000). And difficult to separate.
一方、特開平１−157376号公報によれば、ナス科及び
セリ科等に属する高等植物由来のプロトプラストの培養
培地が提案され一定の効果が確認されているが、その適
用範囲に制限があることが窺える。例えば、栽培植物と
して重要な位置を占めるブドウ科等の植物については、
そのプロトプラストの培養に成功していない。なお、ブ
ドウのプロトプラストの培養例については、K.G.M.Sken
eのVitis，14,177〜180,1975にあるごとくかなり早期か
ら研究が行われているが、その植物体再生の成功例はみ
られない。On the other hand, according to JP-A-1-157376, a culture medium for protoplasts derived from higher plants belonging to the Solanaceae family and the Umbelliferae family has been proposed and a certain effect has been confirmed, but its application range is limited. I can see. For example, for plants such as grape which occupy an important position as cultivated plants,
The protoplast has not been successfully cultured. For examples of grape protoplast culture, see KGMSken
Although research has been conducted from a very early stage as described in e Vitis , 14 , 177-180, 1975, there have been no successful cases of plant regeneration.
従来の技術には、一部の高等植物由来のプロトプラス
トの培養培地が提案されているものの、プロトプラスト
系を高等植物のバイオテクノロジーに利用するに当たっ
ては、その基本技術としてさらに多様なプロトプラスト
の効率的な培養方法の開発が当業技術分野に於いて強く
望まれるであろう。In the prior art, a culture medium for protoplasts derived from some higher plants has been proposed.However, in utilizing the protoplast system for biotechnology of higher plants, a more diversified protoplast efficient as a basic technology has been proposed. The development of culture methods would be highly desirable in the art.
本発明者等は、上記課題を解決すべく鋭意研究の結
果、ナス科植物由来のCDM中の分裂促進因子は高分子側
にあり、限外濾過により阻害の要因になる低分子成分を
除けること及び低分子成分（特に糖類）を除くことによ
り凍結乾燥が容易になり、CDM中の高分子分裂促進因子
の濃度を高くすることによりプロトプラストの培養が効
率的に行えることを見いだし、本発明を完成するに到っ
た。The present inventors have conducted intensive studies to solve the above problems, and as a result, the mitogen in CDM derived from Solanaceae plants is on the high molecular side, and ultrafiltration can remove low molecular components that are a cause of inhibition. It was found that lyophilization was facilitated by removing low molecular components (especially saccharides), and that protoplasts could be cultured efficiently by increasing the concentration of high molecular mitogen in CDM, and completed the present invention. I came to.
即ち、本発明の第一の発明は、ナス科植物由来の培養
物上清より、分子量10万未満の低分子を除いた植物細胞
の分裂促進活性を有する高分子分裂促進活性因子含有組
成物であり、第二の発明は、該組成物を添加した高等植
物由来プロトプラストの培養培地であり、第三の発明
は、高等植物由来プロトプラストの培養において、該培
養培地で高等植物由来プロトプラストを培養することを
特徴とする培養法である。That is, the first invention of the present invention is a composition containing a high-molecular mitogen-activating factor having a mitogenic activity of a plant cell obtained by removing a low-molecular compound having a molecular weight of less than 100,000 from a culture supernatant of a Solanaceae plant. Yes, the second invention is a culture medium for higher plant-derived protoplasts to which the composition is added, and the third invention is to culture higher plant-derived protoplasts in the culture medium in culturing higher plant-derived protoplasts. A culture method characterized by the following.
以下、本発明を詳述する。 Hereinafter, the present invention will be described in detail.
先ず本発明の第一の発明にいうナス科植物由来の培養
物上清とは、ナス科に属する植物、例えばナス、タバ
コ、ペチュニアまたはハシリドコロのいずれかの組織に
ついて、それ自体公知の懸濁培地で培養した懸濁培養物
を継代３〜４日目の対数増殖期に遠心分離し、必要によ
り上澄みを濾過処理して細胞残渣を除いたもの（CDM）
をいう。なお、こうして調製したCDMは非常に変異しや
すいので、長時間放置しないことが肝要である。所定の
分離促進活性を保存する観点からはこのもののpHを5.6
付近に維持することが好ましい。First, a culture supernatant derived from a Solanaceae plant according to the first invention of the present invention is a suspension medium known per se for a plant belonging to the Solanaceae family, for example, any tissue of eggplant, tobacco, petunia, or hasidoro. The suspension culture cultured in step 3 was centrifuged during the logarithmic growth phase on day 3 to 4 of the passage, and the supernatant was filtered, if necessary, to remove cell debris (CDM).
Say. The CDM thus prepared is very susceptible to mutation, so it is important not to leave it for a long time. From the viewpoint of preserving the predetermined separation promoting activity, the pH of this is set at 5.6.
It is preferable to keep it near.
このようなCDMから分子量10万未満の低分子を除く手
段としては、当該技術分野で使用される各種の分子量に
基づく植物成分の分画方法を適用することができるが、
常用されている限外濾過膜を使用する方法が特に好まし
い。As a means for removing low-molecular weight less than 100,000 from such CDM, a method for fractionating plant components based on various molecular weights used in the art can be applied.
A method using a commonly used ultrafiltration membrane is particularly preferred.
かかる手段を施すことにより得られる組成物は、それ
自体この発明の高分子分裂促進活性因子含有組成物たり
うるが、取り扱いの容易さを考慮するとさらに次の処理
を行うことが好ましい。すなわち、前記の分子量10万未
満の低分子除去物を、密閉してない小型の容器に移し、
イソプロピルアルコール等の溶媒を用いて短時間に冷凍
庫で冷凍したのち、凍結乾燥機に持続して凍結乾燥を行
うことによりさらに高活性で安定な本発明の組成物とす
ることができる。Although the composition obtained by applying such means may be the composition containing the polymer mitogen-activating factor of the present invention itself, it is preferable to further perform the following treatment in consideration of ease of handling. That is, the low-molecular-weight-removed product having a molecular weight of less than 100,000 is transferred to an unsealed small container,
After freezing in a freezer for a short time using a solvent such as isopropyl alcohol, the composition of the present invention can be made more active and stable by freeze-drying continuously in a freeze dryer.
次に、本発明の第二の発明は、該組成物を添加した高
等植物由来プロトプラストの培養培地である。即ち、培
地中に、分子量10万の限外濾過によって低分子を除き、
凍結乾燥によって濃縮したナスのCDM由来の高分子分裂
促進活性因子組成物を通常のプロトプラスト培養に用い
られる基本培地に添加して調製される高等植物由来プロ
トプラストの培養培地である。Next, a second invention of the present invention is a culture medium for protoplasts derived from higher plants to which the composition is added. That is, in the medium, low molecular weight is removed by ultrafiltration with a molecular weight of 100,000,
This is a culture medium for higher plant-derived protoplasts prepared by adding a composition of a macromolecular mitogen-activating factor derived from eggplant CDM concentrated by freeze-drying to a basic medium used for ordinary protoplast culture.
本発明に使用される基本培地としては、通常のプロト
プラスト培養に用いられる培地ならばいずれを用いても
良く、例えばムラシゲ・スクーグ培地（Murashige and
Skoog Physiol.Plant 15,473,1962）、B5培地（Gembor
g,Miller and Ojima,Exp.Cell Res.50,151,1968）、ナ
ガタ・タケベ培地（Nagata and Takebe.Planta,99,12,1
971）等が挙げられる。基本培地への上記組成物の添加
量は、組成物中の分裂促進活性、基本培地組成及び培養
の対象である高等植物種によって変動するので臨界的で
ない。当業者であれば各植物種について簡単な実験を行
うことによりその最適量を決定しうるであろう。As a basal medium used in the present invention, any medium may be used as long as it is a medium used for ordinary protoplast culture. For example, Murashige and Skoog medium (Murashige and
Skoog Physiol.Plant 15,473,1962), B5 medium (Gembor
g, Miller and Ojima, Exp. Cell Res. 50, 151, 1968), Nagata and Takebe medium (Nagata and Takebe. Planta, 99, 12, 1)
971). The amount of the composition to be added to the basal medium is not critical because it varies depending on the mitogenic activity in the composition, the basal medium composition and the higher plant species to be cultured. Those skilled in the art will be able to determine the optimal amount by performing simple experiments on each plant species.
基本培地への上組成物の添加は、限外濾過処理した直
後の組成物をそのまま添加するか、またはその凍結乾燥
物を基本培地中に溶解することにより高等植物由来プロ
トプラスト用培養培地が得られる。また、添加時期はプ
ロトプラストの培養初期に添加するのが好ましいが、一
定のコロニーを形成した後に添加してもよい。Addition of the upper composition to the basal medium can be performed by directly adding the composition immediately after the ultrafiltration treatment, or by dissolving the lyophilized product in the basal medium to obtain a culture medium for higher plant-derived protoplasts. . The addition is preferably performed at the beginning of the protoplast culture, but may be performed after certain colonies are formed.
本発明の第三の発明は、高等植物由来のプロトプラス
トの培養法において、上記培養培地で高等植物由来プロ
トプラストを培養することを特徴とする培養法である。The third invention of the present invention is a method for culturing protoplasts derived from higher plants, wherein the protoplasts derived from higher plants are cultured in the above-mentioned culture medium.
本発明に使用される高等植物とは、上記組成物の調製
に用いたナス科植物はもとより、異なる科に属する植物
（例えば、ブドウ等）も包含する。また、そのプロトプ
ラストとしては、特に限定されずいずれのプロトプラス
トでも用いることができる。即ち、プロトプラストの大
部分は体細胞（特に、葉肉）由来プロトプラスト又はカ
ルス由来プロトプラストの何れかに大別することができ
るが、いずれについても用いることができる。The higher plants used in the present invention include not only solanaceous plants used for preparing the above composition, but also plants belonging to different families (for example, grapes). The protoplast is not particularly limited, and any protoplast can be used. That is, most of the protoplasts can be roughly classified into either protoplasts derived from somatic cells (particularly, mesophyll) or protoplasts derived from callus, and any of them can be used.
高植物由来の細胞は、いずれの場合もプロトプラスト
化により生物活性が低下するが、このとき上記培養培地
でプロトプラストを培養すると、無添加の培地で培養す
るときに比べて分裂増殖が増加するようになる。無添加
の培地ではプロトプラストが分裂能を示さない場合も、
この分裂促進活性因子を含む培地で培養すると分裂を開
始することができる。また、コロニーに至る前に分裂を
停止した場合でも、本発明の高分子分裂促進活性因子含
有組成物を含む培養培地を頻繁に植え換えることにより
分裂を継続し、コロニーを形成し、さらに植物体を再生
することができる。In any case, the cells derived from high plants have reduced biological activity due to protoplast formation.At this time, when protoplasts are cultured in the above culture medium, mitotic growth is increased as compared to when cultured in an additive-free medium. Become. When protoplasts do not show division ability in the medium without addition,
When cultured in a medium containing this mitogen, mitosis can be initiated. In addition, even when the division is stopped before reaching the colony, the division is continued by frequently replacing the culture medium containing the composition containing the macromolecular mitogen-activating factor of the present invention, the colony is formed, and further the plant body is formed. Can be played.
なお、培養方法は、培地に上記組成物を添加する以外
はそれ自体公知方法が使用できる。As a culture method, a method known per se can be used except that the above composition is added to the medium.
以下、実施例により本発明の効果を具体的に説明す
る。なお、実施例の培養では高等植物であるブドウを例
にして説明するが、高等植物はこれに限定されるもので
はない。Hereinafter, the effects of the present invention will be specifically described with reference to examples. In the cultivation of Examples, grapes, which are higher plants, will be described as an example, but higher plants are not limited thereto.
実施例１ 高分子分裂促進活性因子組成物の製造 先ず分裂促進活性を有するCDMの検討を行い、種々のC
DMの効果を３日後の分裂率を指標として調べた結果、ナ
ス科ナスが最も適していることが判った。その結果を第
１表に示した。Example 1 Production of Polymer Mitogen-Activating Factor Composition First, CDM having mitogenic activity was examined and various C
As a result of examining the effect of DM using the division rate after 3 days as an index, it was found that Solanaceae was most suitable. The results are shown in Table 1.
次に、特に分裂活性の強いナスのCDM中にみられる分
裂促進活性因子の分子量を検討するため、３万及び10万
の限外濾過を行い、各画分の分裂活性を測定した。その
結果、分子量10万以上においてもCDM自体と同様の活性
を保持していることが判り、阻害作用を有しないCDMが
得られた。結果を第２表に示す。 Next, 30,000 and 100,000 ultrafiltrations were performed to determine the molecular weight of the mitogenic factor found in the CDM of eggplant with particularly strong mitotic activity, and the mitotic activity of each fraction was measured. As a result, even at a molecular weight of 100,000 or more, it was found that the same activity as CDM itself was retained, and CDM having no inhibitory action was obtained. The results are shown in Table 2.
以上の効果は、CDMとして、１週間毎に継代している
ナス科ナスの液体培養物を継代３〜４日目の対数増殖期
に遠心分離（100rpm、10分間）し、その上澄みを濾紙に
よって細胞残渣を除いたものを用いて得た。この時点で
は非常に変異しやすいので、長時間放置しないことが必
要である。このときCDMのpHは、5.6付近でないと分裂促
進活性を示さない。次に以上のようにして採取したCDM
を分子量10万の限外濾過膜を用いて限外濾過を行った。
高分子側のCDMが２〜3mlになったら蒸留水を100ml程足
して、再び限外濾過を行った。この作業を４〜５回繰り
返すことにより低分子成分を除いた。更に、このCDMを
密閉してない小型の容器に移し、イソプロピルアルコー
ル等の溶媒で短時間に凍結させた。−80℃の冷凍庫で１
時間冷凍したのち、凍結乾燥機に接続して凍結乾燥を行
うことにより本発明の高分子分裂促進活性因子含有組成
物が得られた。 The above effects were obtained by centrifuging (100 rpm, 10 minutes) the liquid culture of Solanaceae eggplant that had been subcultured every week as CDM during the logarithmic growth phase on the third and fourth passages. It was obtained using a filter paper from which cell debris had been removed. At this point, it is very easy to mutate, so it is necessary not to leave it for a long time. At this time, the pH of CDM does not show mitogenic activity unless it is around 5.6. Next, the CDM collected as above
Was subjected to ultrafiltration using an ultrafiltration membrane having a molecular weight of 100,000.
When the CDM on the polymer side became 2-3 ml, about 100 ml of distilled water was added, and ultrafiltration was performed again. This operation was repeated 4 to 5 times to remove low molecular components. Further, the CDM was transferred to an unsealed small container, and was frozen in a short time with a solvent such as isopropyl alcohol. 1 in a freezer at -80 ℃
After freezing for a period of time, the composition was connected to a freeze dryer and freeze-dried, whereby a composition containing a polymer mitogen-activating factor of the present invention was obtained.
実施例２ 高分子分裂促進活性因子組成物の活性測定 ブドウの葯由来のカルスで再分化能を消失し、分裂増
殖能の比較的高いものを選択して該活性を維持している
ものを1.5％cellulase onozuka R−10,1.5％cellulase
onozuka RS,0.3％pectolyase Y−23を含む0.4Mソルビト
ール溶液中で90分酵素処理を行い、プロトプラストを得
た。メッシュで濾過したのち、0.4Mのソルビトール液で
洗浄した。Example 2 Measurement of Activity of Macromolecular Mitogen-Activating Factor Composition The callus derived from grape anthers whose regeneration ability was lost and whose relatively high mitotic proliferation ability was selected to maintain the activity was 1.5%. % Cellulase onozuka R-10,1.5% cellulase
Enzyme treatment was performed in a 0.4 M sorbitol solution containing onozuka RS, 0.3% pectolyase Y-23 for 90 minutes to obtain protoplasts. After filtration through a mesh, the resultant was washed with a 0.4 M sorbitol solution.
こうして得られたプロトプラストを５×102,1×103,5
×103,1×104cell/mlの密度で、0.1mg/NAA、1.0mg/
BA、0.05Mシュークロース、0.35Mソルビトールを含むNa
gata＆Takebe培地（基本培地）を用い25℃暗条件下で培
養した。この培地に実施例１で得られた組成物を添加し
て培養し、３日後に分裂率を測定した。その結果を第３
表に示した。この結果より、本発明の高分子分裂促進活
性因子含有組成物は、濃縮によって阻害作用が強くなら
ず、さらに濃縮してその効果が高められる可能性を有す
ることがわかる。The protoplasts obtained in this way were 5 × 10 2 , 1 × 10 3 , 5
0.1mg / NAA, 1.0mg / NA at a density of × 10 3 , 1 × 10 4 cell / ml
Na with BA, 0.05M sucrose, 0.35M sorbitol
The culture was performed using a gata & Takebe medium (basic medium) at 25 ° C in the dark. The composition obtained in Example 1 was added to this medium and cultured, and the division rate was measured after 3 days. The result is the third
It is shown in the table. From these results, it can be seen that the polymer mitogen-activating factor-containing composition of the present invention does not have a strong inhibitory effect due to concentration, and has a possibility of being further concentrated to enhance the effect.
実施例３ ブドウ由来プロトプラストからの植物体の再
生 ブドウの葯由来カルスで、再分化能はあるが分裂活性
の低いカルスを1.5％cellulase onozuka R−10,1.5％ce
llulase onozuka RS,0.3％pectolyase Y−23を含む0.4M
ソルビトール溶液中で90分酵素処理を行い、プロトプラ
ストを得た。メッシュで濾過したのち、0.4Mのソルビト
ール液で洗浄した。 Example 3 Regeneration of Plant from Grape-Derived Protoplasts Callus derived from grape anthers, which has a regeneration ability but has low mitotic activity, is reduced to 1.5% cellulase onozuka R-10, 1.5% ce
0.4M containing llulase onozuka RS, 0.3% pectolyase Y-23
Enzyme treatment was performed for 90 minutes in a sorbitol solution to obtain protoplasts. After filtration through a mesh, the resultant was washed with a 0.4 M sorbitol solution.
こうして得られたプロトプラストを５×104cell/mlの
密度で、0.1mg/NAA,1.0mg/BA、0.05Mシュークロー
ス、0.35Mソルビトールを含むNagata＆Takebe培地で培
養した。この培地の実施例１で得られた組成物を30％添
加して培養した。本発明の組成物の添加と移植の効果を
第４表に示す。The protoplasts thus obtained were cultured at a density of 5 × 10 4 cells / ml in a Nagata & Takebe medium containing 0.1 mg / NAA, 1.0 mg / BA, 0.05 M sucrose, and 0.35 M sorbitol. The medium was added with the composition obtained in Example 1 at 30% and cultured. Table 4 shows the effects of the addition of the composition of the present invention and the transplantation.
無添加のものは第１分裂をおこなったところで枯死し
たが、高分子分裂促進活性因子含有組成物を含む培地で
培養したものは分裂を繰り返し、新しい培地に頻繁に植
え換えることによりカルスを再生した。このカルスは再
分化し、植物体を再生した。ブドウでは、植物を再生で
きるプロトプラストの培養例は今まで無かったが、実施
例２で得られた培養培地を用いることにより培養が可能
になった。The non-added one died at the time of the first division, but the one cultured in the medium containing the composition containing the macromolecular mitogen activated factor repeatedly divided and regenerated the callus by frequently replacing the medium with a new medium. . This callus redifferentiated and regenerated the plant. In the case of grapes, there have been no examples of culturing protoplasts that can regenerate plants, but culturing has become possible by using the culture medium obtained in Example 2.
〔発明の効果〕 本発明の高分子分裂促進活性因子含有組成物を含む培
養培地で培養すれば、プロトプラストの生物活性が上昇
することにより、高等植物のプロトプラストの効率のよ
い培養が可能となり、そのプロトプラストの培養から植
物体の再生も可能となる。 (Effect of the Invention) When cultured in a culture medium containing the polymer mitogen-activating factor-containing composition of the present invention, the biological activity of protoplasts is increased, thereby enabling efficient culture of protoplasts of higher plants. Regeneration of a plant from culture of protoplasts is also possible.
フロントページの続き (58)調査した分野(Int.Cl.6，ＤＢ名) C12N 1/00 - 7/08 A01G 1/00 - 1/02 A01G 1/06 - 1/12 A01G 5/00 - 7/06 A01G 16/00 A01G 17/00 - 17/02 A01G 17/18 A01H 1/00 - 17/00 ＢＩＯＳＩＳ（ＤＩＡＬＯＧ) ＷＰＩ（ＤＩＡＬＯＧ)Continuation of the front page (58) Field surveyed (Int.Cl. 6 , DB name) C12N 1/00-7/08 A01G 1/00-1/02 A01G 1/06-1/12 A01G 5/00-7 / 06 A01G 16/00 A01G 17/00-17/02 A01G 17/18 A01H 1/00-17/00 BIOSIS (DIALOG) WPI (DIALOG)
Claims (4)
万未満の低分子を除いて得られる、ブドウ科植物由来植
物細胞の分裂促進活性を有する高分子分裂促進活性因子
含有組成物。The present invention relates to a solubilized eggplant-derived culture supernatant, which has a molecular weight of 10
A composition containing a macromolecular mitogen-activating factor having a mitogenic activity of grape plant-derived plant cells, which is obtained by removing less than 10,000 low molecules.
１に記載の組成物。2. The composition according to claim 1, wherein said vine is a grape.
ブドウ科植物由来植物細胞用培養培地。3. A culture medium for plant cells derived from a grape plant, comprising the composition according to claim 1 or 2.
いて、請求項３に記載の培地により該植物細胞を培養す
ることを特徴とする方法。4. A method for cultivating a plant cell derived from a grape plant, which comprises culturing the plant cell with the medium according to claim 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1281934A JP2925186B2 (en) | 1989-10-31 | 1989-10-31 | Composition containing polymer mitogen activator in higher plants and its use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1281934A JP2925186B2 (en) | 1989-10-31 | 1989-10-31 | Composition containing polymer mitogen activator in higher plants and its use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03147781A JPH03147781A (en) | 1991-06-24 |
| JP2925186B2 true JP2925186B2 (en) | 1999-07-28 |
Family
ID=17645971
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1281934A Expired - Lifetime JP2925186B2 (en) | 1989-10-31 | 1989-10-31 | Composition containing polymer mitogen activator in higher plants and its use |
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| Country | Link |
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| Title |
|---|
| Ｐｌａｎｔ Ｓｃｉｅｎｃｅ，１９８７，Ｖｏｌ．５３，Ｎｏ．３，ｐ．２４９−２５６ |
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