JPH03285625A - Cultivation of plant cell - Google Patents
Cultivation of plant cellInfo
- Publication number
- JPH03285625A JPH03285625A JP8863290A JP8863290A JPH03285625A JP H03285625 A JPH03285625 A JP H03285625A JP 8863290 A JP8863290 A JP 8863290A JP 8863290 A JP8863290 A JP 8863290A JP H03285625 A JPH03285625 A JP H03285625A
- Authority
- JP
- Japan
- Prior art keywords
- culture
- horseradish
- pod
- light irradiation
- culturing
- 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.)
- Pending
Links
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 18
- 240000003291 Armoracia rusticana Species 0.000 claims abstract description 16
- 235000011330 Armoracia rusticana Nutrition 0.000 claims abstract description 16
- 238000012258 culturing Methods 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 9
- 239000011780 sodium chloride Substances 0.000 claims abstract description 9
- 241000196324 Embryophyta Species 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 8
- 102000003992 Peroxidases Human genes 0.000 abstract description 4
- 230000028327 secretion Effects 0.000 abstract description 4
- 102000004190 Enzymes Human genes 0.000 abstract 2
- 108090000790 Enzymes Proteins 0.000 abstract 2
- 239000007788 liquid Substances 0.000 abstract 2
- 108040007629 peroxidase activity proteins Proteins 0.000 abstract 2
- 230000035755 proliferation Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 27
- 230000000694 effects Effects 0.000 description 8
- 230000004663 cell proliferation Effects 0.000 description 4
- 238000012136 culture method Methods 0.000 description 3
- 241000390128 Eutrema Species 0.000 description 2
- 206010021033 Hypomenorrhoea Diseases 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000006870 ms-medium Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 241000589156 Agrobacterium rhizogenes Species 0.000 description 1
- 241000219196 Armoracia Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 241000275031 Nica Species 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 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 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 241001478887 unidentified soil bacteria Species 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は、西洋ワサビの毛状根細胞を培養し。[Detailed description of the invention] [Industrial application field] The present invention involves culturing hairy root cells of horseradish.
有用物質である酵素パーオキシダーゼを効率的に生産す
る方法に関する。This invention relates to a method for efficiently producing the enzyme peroxidase, which is a useful substance.
西洋ワサビは、酵素パーオキシダーゼを多量に生産する
。Horseradish produces large amounts of the enzyme peroxidase.
ここに、西洋ワサビは、ＡｒｍｏｒａｃｉａＬａ ａ
ｔｈｉｆｏｌｉａ Ｇ１１ｉｂと称されるもので２日
本由来のミズワサビ（Ｗａｓａｂｉａ Ｊａ ｏｎ
ｉｃａ）、ハタワサビ（Ｗａｓａｂｉａ Ｊａ ｏ
ｎｉｃａ）などとは異なるものである。Here, the horseradish is Armoracia La a
It is called thifolia G11ib, and it is called Wasabia thifolia.
ica), Wasabia Ja o
nica), etc.
また、上記酸素酵素オキシダーゼ（以下、 ＰＯＤと
いう）は、臨床検査用、或いは特定遺伝子を検出するた
めの発色用プローブなどに汎用される有用物質である。Further, the oxygen enzyme oxidase (hereinafter referred to as POD) is a useful substance commonly used for clinical tests or coloring probes for detecting specific genes.
また、上記ＰＯＤは５西洋ワサビ、特にその毛状根細胞
中に生成される。ここに毛状根細胞とは土壌細菌（バク
テリア）のアグロバクテリウム・リゾゲネス（Ａ ｒ
ｏｂａｃｔｅｒｉｕｍ ｒｈｉｚｏ ｅｎｅｓ）の
細胞中に存在するＲｉプラスミドが、植物細胞中に入る
ことによって誘発される細い不定根細胞のことである。The above POD is also produced in horseradish, especially in its hairy root cells. Hairy root cells are soil bacteria Agrobacterium rhizogenes (A r
This refers to thin adventitious root cells induced by the Ri plasmid present in the cells of Bacterium rhizo enes entering the plant cells.
そして、−旦毛状根細胞に形質転換された細胞は、極め
て速い速度で増殖し、以後もずっとその形態を保持する
（「植物組織培養」第６巻第１号、１９８９年発行参照
）。The cells transformed into hairy root cells proliferate at an extremely high rate and maintain this form even afterward (see "Plant Tissue Culture", Vol. 6, No. 1, published in 1989).
また、従来、西洋ワサビ毛状根細胞の基本的な培養は５
次のようである。即ち、培養液としてはホルモン無添加
のムラシゲ・スクーグ（Ｍｕｒａｓｈｉｇｅ ａｎｄ
Ｓｋｏｏｇ）培地（以下ＭＳ培地という）を用い、
ＰＨ５，８〜６．０糖源にはシコ糖（シュクロース）を
３重量％添加したものを用いている。In addition, conventionally, the basic culture of horseradish hairy root cells is
It looks like this: That is, the culture solution was a hormone-free Murashige and Skoog (Murashige and Skoog).
Skoog) medium (hereinafter referred to as MS medium),
PH5.8 to 6.0 The sugar source used was one to which 3% by weight of sucrose was added.
また、培養に当たっては２三角フラスコ（１００〜５０
０ｍｐ、）を用い、２５°Ｃ２暗黒条件下で旋回振とう
培養（１００ｒｐｍ）を行う。In addition, when culturing, two Erlenmeyer flasks (100 to 50
Perform orbital shaking culture (100 rpm) at 25° C. in the dark using 0mp, ).
そして、上記培養を行った後、その毛状根細胞破砕物中
より目的とするＰＯＤを採取する。After carrying out the above culture, the desired POD is collected from the hairy root cell fragments.
しかしながら、上記従来の培養方法では、ＰＯＤを連続
大量生産することができない。However, the conventional culture methods described above do not allow continuous mass production of POD.
即ち７上記西洋ワサビの毛状根細胞は、動物細胞とは異
なり２個々の細胞が固い細胞壁に囲まれている。そのた
め１毛状根細胞を培養液中で懸濁培養しても１毛状根細
胞によって生産されたＰＯＤは、殆ど細胞内にとどまっ
たままで、培養液中に分泌される量は少ない。That is, in the hairy root cells of horseradish described above, unlike animal cells, each individual cell is surrounded by a hard cell wall. Therefore, even if one hairy root cell is cultured in suspension in a culture solution, most of the POD produced by one hairy root cell remains within the cell, and only a small amount is secreted into the culture solution.
なお、ＰＯＤ生産植物に関しては、東南アジアの水生野
菜であるパックブンがあるが、その生産効率は西洋ワサ
ビより劣る。また、該バソクブンの毛状根細胞培養に関
しては、ｒＰＩａｎｔ ｔｉｓｓｕｅ ｃｕｌｔｕ
ｒｅ ｐ、ｅｔｔｅｒｓ６．１５９−１６１ （１９
８９）Ｊに記載されている。Regarding POD-producing plants, there is Pakbun, which is an aquatic vegetable from Southeast Asia, but its production efficiency is inferior to that of horseradish. In addition, regarding the hairy root cell culture of the rPIant tissue culture,
rep, etters6.159-161 (19
89) Described in J.
本発明はかかる従来の問題点に鑑み、培養液中にＰＯＤ
を効率良く分泌させ、ＰＯＤの生産量を向上させること
ができる。西洋ワサビ毛状根細胞の培養方法を提供しよ
うとするものである。In view of such conventional problems, the present invention provides POD in the culture solution.
can be secreted efficiently and the production amount of POD can be improved. The present invention aims to provide a method for culturing horseradish hairy root cells.
〔課題の解決手段］
本発明は、西洋ワサビの毛状根細胞を培養する方法にお
いて、その培養液中に塩化すＩ・リウムを添加すると共
に光照射下において培養することを特徴とする植物細胞
の培養方法にある。[Means for Solving the Problems] The present invention provides a method for culturing hairy root cells of horseradish, which comprises adding I.rium chloride to the culture solution and culturing under light irradiation. This is in the culture method.
本発明において、上記塩化ナトリウム（ＮａＣりは、培
養液中に２０〜２００ｍＭ（ミリモル）添加することが
好ましい。２０ｍＭ未満では細胞の増殖速度は大きいが
ＰＯＤの分泌量が少ない。また、ＮａＣ１濃度が高くな
るとＰＯＤ分泌量は大きくなるが、２００ｍＭを越える
と細胞の増殖速度が小さくなる。In the present invention, it is preferable to add 20 to 200 mM (mmol) of the above sodium chloride (NaC) into the culture medium. If it is less than 20 mM, the cell proliferation rate is high but the amount of POD secreted is small. As the concentration increases, the amount of POD secreted increases, but when the concentration exceeds 200 mM, the cell proliferation rate decreases.
また、上記光照射は、蛍光灯、白熱灯、太陽光等により
行う。光照射の照度は５例えば３０００〜２００００ル
ツクスを用いる。また、培養中の光照射は、明期（光照
射時期）と暗！ＩＪＩ （光照射なしの時期）を繰り返
すことが望ましい。Further, the light irradiation is performed using a fluorescent lamp, an incandescent lamp, sunlight, or the like. The illuminance of the light irradiation is 5, for example, 3,000 to 20,000 lux. Also, the light irradiation during cultivation is during the light period (light irradiation period) and the dark period! It is desirable to repeat IJI (period without light irradiation).
本発明においては、培養液中に塩化ナトリウムを添加す
ると共に光照射下に培養を行う。そのため、光照射によ
って西洋ワサビ毛状根細胞の増殖速度が向上し、また塩
化ナトリウムと光照射の相乗効果によって培養液中への
ＰＯＤの分泌が促進される。そのため、ＰＯＤの生産速
度が向上する。In the present invention, sodium chloride is added to the culture solution and the culture is carried out under light irradiation. Therefore, light irradiation improves the growth rate of horseradish hairy root cells, and the synergistic effect of sodium chloride and light irradiation promotes the secretion of POD into the culture solution. Therefore, the production speed of POD is improved.
したがって８本発明によれば３毛状根細胞より培養液中
へＰＯＤを効率良く分泌させることができ、またＰＯＤ
の生産量を向上させることができる。西洋ワサビ毛状根
細胞の培養方法を提供することができる。Therefore, according to the present invention, POD can be efficiently secreted from hairy root cells into the culture medium, and POD
production can be improved. A method for culturing horseradish hairy root cells can be provided.
〔実施例］
本発明の実施例にかかる西洋ワサビ毛状根細胞の培養方
法につき、第１Ａ図〜第２Ｂ図を用いて説明する。[Example] A method for culturing horseradish hairy root cells according to an example of the present invention will be explained using FIGS. 1A to 2B.
即ち、本例の培養においては、まず１．００ ｍ ｌ三
角フラスコ中に４０ｍｊ２の前記ＭＳ培地を入れ。That is, in the culture of this example, first, 40 mj2 of the above MS medium was placed in a 1.00 ml Erlenmeyer flask.
更に下記のごとくＮａＣ１を添加して、４種類の培養液
を調整した。Furthermore, NaCl was added as described below to prepare four types of culture solutions.
ＮａＣ１濃度は、０（無添加）、 １．０． ５０゜
１００ｍＭとした。光照射は、光源として蛍光灯を用い
、照度３５００ルツクス（Ｌｕχ）、明暗サイクルは明
期１４時間、時期１０時間とした。NaCl concentration is 0 (no addition), 1.0. 50° and 100mM. For light irradiation, a fluorescent lamp was used as a light source, the illuminance was 3500 lux (Luχ), and the light/dark cycle was 14 hours in the light period and 10 hours in the period.
また、培養は、ｐＨ５゜９，２５°Ｃ１振とう１１００
ｒｐで、４０日間行った。In addition, the culture was carried out at pH 5°9, 25°C, shaking at 1100°C.
rp for 40 days.
また、培養期間中ば、１週間（７日）毎に細胞を三角フ
ラスコから取り出しクリーンへフチ下で細胞重量を電子
テンビンで測定し、培養液中における細胞重量を測定し
た。また、ＰＯＤはサンプリングした培養液より測定し
、養液中におけるＰＯＤ活性（ユニット／ｍρ）を測定
した。ＰＯＤ活性の測定は、オルＩ−・アミノフェノー
ル法により行った。During the culture period, the cells were taken out from the Erlenmeyer flask every week (7 days) and placed in a clean room under the rim, and the cell weight was measured using an electronic scale to measure the cell weight in the culture solution. In addition, POD was measured from the sampled culture solution, and POD activity (unit/mρ) in the nutrient solution was measured. Measurement of POD activity was performed by the ol-I-aminophenol method.
また、比較のため、光照射なしの暗黒上培養も行った。For comparison, culture was also performed in the dark without light irradiation.
実験の結果を、第１Ａ図〜第２Ｂ図に示した。The results of the experiment are shown in Figures 1A to 2B.
第１Ａ図及び第１Ｂ図は、比較例としての暗黒上培養に
おける細胞重量とＰＯＤ活性を示す。また第２Ａ図及び
第２Ｂ図は２本発明の培養法における細胞重量とＰＯＤ
活性を示す。Figures 1A and 1B show cell weight and POD activity in dark culture as a comparative example. In addition, Figures 2A and 2B show the cell weight and POD in the culture method of the present invention.
Shows activity.
同図より知られるごとく、細胞増殖に関しては暗黒上培
養（第１Ａ図）、光照射下培養（第２Ａ図）とも、Ｎａ
Ｃｆｆの添加量が増すと細胞増殖が阻害される。As can be seen from the figure, regarding cell proliferation, both dark culture (Figure 1A) and light irradiation culture (Figure 2A)
As the amount of Cff added increases, cell proliferation is inhibited.
しかし、目的とするＰＯＤ生産、つまり培養液中へのＰ
ＯＤの分泌に関しては、第１Ｂ図と第２Ｂ図とを比較し
て分かるように、暗黒上培養に比して光照射上培養はＰ
ＯＤ活性の増大、つまりＰＯＤ分泌量の向上が大きいこ
とが分る。そしてＮａ Ｃｆｆ添加量の増加と共に培養
液中のＰＯＤ活性も増大している（第２Ｂ図）。However, the desired POD production, that is, the POD production in the culture solution.
Regarding the secretion of OD, as can be seen by comparing Figures 1B and 2B, culture under light irradiation has a lower P
It can be seen that there is a large increase in OD activity, that is, a large improvement in the amount of POD secretion. As the amount of Na Cff added increased, the POD activity in the culture solution also increased (Figure 2B).
上記のごと＜、ＮａＣｆｆ１添加で、かつ光照射上培養
により、ＰＯＤを効率良く分泌させることができる。As described above, by adding NaCff1 and culturing under light irradiation, POD can be secreted efficiently.
図は実施例を示し、第１Ａ図及び第１Ｂ図は暗黒上培養
における。第２Ａ図及び第２Ｂ図は光照射上培養におけ
る。培養時間と細胞重量（ｇｄｗ／ｊ２）及びＰＯＤ活
性との関係を示す線図である。The figure shows an example; Figures 1A and 1B are incubation in the dark. FIGS. 2A and 2B show the culture under light irradiation. It is a diagram showing the relationship between culture time, cell weight (gdw/j2), and POD activity.
Claims (2)
、その培養液中に塩化ナトリウムを添加すると共に光照
射下において培養することを特徴とする植物細胞の培養
方法。(1) A method for culturing hairy root cells of horseradish, which comprises adding sodium chloride to the culture solution and culturing under light irradiation.
対して２０〜２００ミリモル添加することを特徴とする
植物細胞の培養方法。(2) The method for culturing plant cells according to claim 1, characterized in that 20 to 200 mmol of sodium chloride is added to the culture solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8863290A JPH03285625A (en) | 1990-04-03 | 1990-04-03 | Cultivation of plant cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8863290A JPH03285625A (en) | 1990-04-03 | 1990-04-03 | Cultivation of plant cell |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH03285625A true JPH03285625A (en) | 1991-12-16 |
Family
ID=13948185
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP8863290A Pending JPH03285625A (en) | 1990-04-03 | 1990-04-03 | Cultivation of plant cell |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH03285625A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007000057A (en) * | 2005-06-22 | 2007-01-11 | Nippon Paper Industries Co Ltd | Method for culturing tissue of eucalyputus plant and method for producing clone seedling |
-
1990
- 1990-04-03 JP JP8863290A patent/JPH03285625A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007000057A (en) * | 2005-06-22 | 2007-01-11 | Nippon Paper Industries Co Ltd | Method for culturing tissue of eucalyputus plant and method for producing clone seedling |
| JP4701019B2 (en) * | 2005-06-22 | 2011-06-15 | 日本製紙株式会社 | Eucalyptus plant tissue culture method and clone seedling production method |
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