JPH03103183A - Cell culture of adventitious gene transduction, nutritive solution culture device and plant acclimation device - Google Patents

Cell culture of adventitious gene transduction, nutritive solution culture device and plant acclimation device

Info

Publication number
JPH03103183A
JPH03103183A JP1240730A JP24073089A JPH03103183A JP H03103183 A JPH03103183 A JP H03103183A JP 1240730 A JP1240730 A JP 1240730A JP 24073089 A JP24073089 A JP 24073089A JP H03103183 A JPH03103183 A JP H03103183A
Authority
JP
Japan
Prior art keywords
plant
nutrient solution
culture
air
chamber
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.)
Granted
Application number
JP1240730A
Other languages
Japanese (ja)
Other versions
JP2638217B2 (en
Inventor
Noriyasu Saito
斉藤 典保
Haruki Otsuki
大月 晴樹
Teruyoshi Nagao
長尾 照義
Fumiyuki Goto
文之 後藤
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Iseki and Co Ltd
Iseki Agricultural Machinery Mfg Co Ltd
Original Assignee
Iseki and Co Ltd
Iseki Agricultural Machinery Mfg Co Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Iseki and Co Ltd, Iseki Agricultural Machinery Mfg Co Ltd filed Critical Iseki and Co Ltd
Priority to JP1240730A priority Critical patent/JP2638217B2/en
Publication of JPH03103183A publication Critical patent/JPH03103183A/en
Application granted granted Critical
Publication of JP2638217B2 publication Critical patent/JP2638217B2/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

Links

Landscapes

  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

PURPOSE:To readily obtain a cell derived callus to which adventitious genes are transduced by tranducing adventitious genes to guard cells in epidermal tissue of plant by micro injection method, culturing the cells and selecting only green part containing chloroplast. CONSTITUTION:A leaf is immersed in a hypertonic solution, subjected to plasmolysis to be readily micro injected, adventitious genes (a) are transduced to guard cells 3 of epidermal tissue 1 of leaf by micro injection method using a micro pipette below a microscope attached to a manipulator, the epidermal tissue is released from other tissue by tweezers, placed on an agar medium 7 in a flask 6 and subjected to callus culture 8. Since the epidermal cell 2 in the epidermal tissue 1 does not contain chloroplast, green part containing chloroplast is selected to give guard cell derived callus 8a to which adventitious genus are transduced.

Description

【発明の詳細な説明】 (産業上の利用分野) 本発明は,植物の細胞に外来遺伝子をマイクロインジェ
クトする方法に関する. また本発明は,植物組織の養液培養装置並びに植物順化
装置に関する. (従来の技術) 植物の細胞に外来遺伝子をマイクロインジェクトする場
合、従来はブロトプラスト状態で行う。
DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for microinjecting foreign genes into plant cells. The present invention also relates to a nutrient solution culture device for plant tissue and a plant acclimation device. (Prior Art) When microinjecting a foreign gene into plant cells, it is conventionally carried out in a brotoplast state.

例えば,カリフラワーモザイクウイルスDNAを所定の
濃度に調整し、コマツナのプロトプラストと混合しプリ
ッキング(針で刺す)すると前記DNAが導入され、そ
の発現が蛍光塊により確認できる. またカル.スの細胞に,選択的に外来遺伝子をマイクロ
インジェクトする方法も開発されている.次に養液培養
装置の場合、従来(例えば特開昭63−44813号公
報)は、培養室底部に養液供給口と排出口を相対して開
口した構造で、養液を一方から供給し他方へ流下排出す
る..さらに従来の植物順化装置は、順化室に供給する
空気の温度湿度及び清浄度が一定であった.(発明が解
決しようとする問題点) プロトプラスト状態の細胞に外来遺伝子をマイクロイン
ジェクト法により導入する場合、導入後のプロトプラス
トの培養再生が困難で再生効率が悪いという問題点があ
った. カルスにマイクロインジェクトする方法は、ブロトブラ
スト状態の場合に比較して再生率は良いが,導入後の形
質転換細胞由来のカルスの選抜が困難で、抗生物質耐性
を調べるのに時間がかかり作業性が悪いという問題点が
残る. 次に従来の養液培養装置は養液の水位維持制御が困難で
、培養室の養液が過剰になり易いという問題点があった
. さらに従来の植物順化装置においては、植物が外部環境
に充分順化できなかった. そこで本発明は、第1に,外来遺伝子の導入を容易にし
、かつ導入後の細胞及び細胞由来のカルスを簡単に識別
することを目的とする.また第2に、養液培養装置の養
液水位が高〈なりすぎるのを防止することを目的とする
.また第3に、植物順化装置において順化室内の照明及
び空調を経時的に調整し、確実に植物を順化することを
目的とする. さらに第4に、順化室に供給する空気の清浄度を、順化
期間の経過に伴い徐々に外部環境に近付け、植物の順化
を容易にすることを目的とする。
For example, when cauliflower mosaic virus DNA is adjusted to a predetermined concentration and mixed with Komatsuna protoplasts and pricked (pricked with a needle), the DNA is introduced and its expression can be confirmed by fluorescent clusters. Cal again. Methods have also been developed to selectively microinject foreign genes into cells in other countries. Next, in the case of a nutrient solution culture device, conventionally (for example, Japanese Patent Application Laid-Open No. 63-44813) has a structure in which a nutrient solution supply port and a discharge port are opened facing each other at the bottom of the culture chamber, and the nutrient solution is supplied from one side. It is discharged downstream to the other side. .. Furthermore, in conventional plant acclimation devices, the temperature, humidity, and cleanliness of the air supplied to the acclimatization chamber are constant. (Problems to be Solved by the Invention) When foreign genes are introduced into protoplast cells by microinjection, there is a problem in that it is difficult to culture and regenerate the protoplasts after the introduction, and the regeneration efficiency is low. The method of microinjecting calli has a better regeneration rate than the brotoblast state, but it is difficult to select calli derived from transformed cells after introduction, and it takes time and labor to examine antibiotic resistance. The problem of poor quality remains. Next, conventional nutrient solution culture equipment has the problem that it is difficult to maintain and control the nutrient solution water level, and the nutrient solution in the culture room tends to become excessive. Furthermore, with conventional plant acclimation devices, plants were not able to acclimatize sufficiently to the external environment. Therefore, the first object of the present invention is to facilitate the introduction of a foreign gene and to easily identify cells and cell-derived callus after the introduction. The second purpose is to prevent the water level of the nutrient solution in the nutrient solution culture device from becoming too high. Third, the purpose of the plant acclimation device is to adjust the lighting and air conditioning in the acclimatization room over time to ensure acclimatization of the plants. A fourth purpose is to gradually bring the cleanliness of the air supplied to the acclimatization chamber closer to the external environment as the acclimatization period progresses, thereby facilitating the acclimatization of plants.

(問題点を解決するための手段) 上記問題点を解決するため,木発明のうち第1発明は、
植物の表皮組織中の孔辺細胞に,マイクロインジェクシ
ョン法により外来遺伝子を導入した後、これを培養する
ことを特徴とする.次に第2発明の養液培養装置は、植
物の培養室の排気口を養液面上限付近に開口して成る.
また第3発明の植物順化装置は、植物の順化室の室内の
温度、湿度、照度及び空気清浄度をそれぞれシーケンス
制御する. さらに第4発明の植物順化装置は、フィルタを備える清
浄空気供給路と外気供給路とにそれぞれ流量調節弁を介
装して両供給路の送出端を合流管に接続すると共に、こ
の合流管の出口側をパーティカルカウンタを経て植物の
順化室に接続して成る. (作用と効果) E81発明により外来遺伝子を導入した表皮組織のうち
表皮細胞には葉緑体がないから、葉緑体を有する緑色の
孔辺細胞を容易に識別できる.従って、培養中、生長し
た細胞及びカルスのうち緑色部分を選抜しこれを培養す
れば、外来遺伝子を導入した孔辺細胞及び細胞由来のカ
ルスが得られる. 第2発明によれば、培養室内の養液水位が上限に達する
と上限付近に開口する排気口より養液が室外に流出する
ので、培養室の養液が過剰になるおそれがない. 第3発明においては、順化室の温度、湿度、照度及び空
気清浄度をそれぞれシーケンス制御するから,植物が確
実に外部環境に順化する.第4発明においては、清浄空
気供給路と外気供給路にそれぞれ備える流量調整弁を作
動して,清浄空気と外気の混合比を調整し、これにより
順化室に供給する混合空気の清浄度を制御する。清浄度
ノ制御は、パーティカルカウンタの検知出力を内流量7
JJ整弁にフィードバックし、これらの弁開度を自動的
に変更することにより、清浄度を予め設定した値に合わ
せる.従って、第4発明によれば、順化室の植物は次第
に外部環境の空気清浄度に順化される. (実施例) 次に第1乃至第4発明の実施例を図面に示して説明する
. 第1〜6図は第1発明の第1実施例を示している。
(Means for solving the problem) In order to solve the above problem, the first invention among the wooden inventions is as follows:
It is characterized by introducing a foreign gene into guard cells in the epidermal tissue of a plant by microinjection, and then culturing it. Next, the nutrient solution culture device of the second invention is constructed by opening the exhaust port of the plant culture chamber near the upper limit of the nutrient solution level.
Further, the plant acclimation device of the third invention sequentially controls the temperature, humidity, illuminance, and air cleanliness of the plant acclimation chamber. Furthermore, in the plant acclimation device of the fourth invention, a flow rate control valve is interposed in each of the clean air supply path provided with a filter and the outside air supply path, and the delivery ends of both supply paths are connected to a confluence pipe, and the confluence pipe The outlet side of the plant is connected to the plant acclimation chamber via a particle counter. (Actions and Effects) Among the epidermal tissues into which foreign genes have been introduced according to the E81 invention, epidermal cells do not have chloroplasts, so green guard cells having chloroplasts can be easily identified. Therefore, by selecting the green parts of the grown cells and callus during culture and culturing them, guard cells and cell-derived callus into which foreign genes have been introduced can be obtained. According to the second invention, when the water level of the nutrient solution in the culture chamber reaches the upper limit, the nutrient solution flows out of the room through the exhaust port that opens near the upper limit, so there is no fear that the nutrient solution in the culture chamber becomes excessive. In the third invention, since the temperature, humidity, illuminance, and air cleanliness of the acclimatization chamber are controlled in sequence, the plants are reliably acclimated to the external environment. In the fourth invention, the flow rate adjustment valves provided in the clean air supply path and the outside air supply path are operated to adjust the mixing ratio of clean air and outside air, thereby increasing the cleanliness of the mixed air supplied to the acclimatization chamber. Control. Cleanliness control uses the detection output of the particle counter to control the internal flow rate of 7.
The cleanliness level is adjusted to the preset value by feeding back to the JJ valve control and automatically changing the opening degrees of these valves. Therefore, according to the fourth invention, the plants in the acclimatization chamber are gradually acclimated to the air cleanliness of the external environment. (Embodiments) Next, embodiments of the first to fourth inventions will be explained with reference to the drawings. 1 to 6 show a first embodiment of the first invention.

第1図は葉の断面構造を示し、l、1は表皮組織で、2
は表皮細胞、3は孔辺細胞である.表皮組織1、l間は
棚状組織4と海綿状組織5が上下に層をなし、これらの
組織4、5と孔辺細胞3には葉緑体が存在する. はじめに葉を高張液(0 .8Mレよ糖液なと)に浸漬
して,マイクロインジェクトしやすいように原形質分離
を起こさせておく. そしてマニュピュレータを装着した顕微鏡下で、孔辺細
胞に外来遺伝子aをマイクロピペットにより導入する(
第2図). 次に、表皮組織をビンセットで葉の他の組織より剥離し
(第3図)、フラスコ6内のカンテン培地7に置きカル
ス培養する(第4図).培養中、生長した細胞及びカル
ス8のうち(第5図)、緑色部分8a即ち葉緑体を持つ
部分を選抜して継体培養をする(第6図).緑色部分以
外の部分8bは葉緑体を持たない表皮組織で外来遺伝子
aがない. 次に第1発明の第2実施例を第7〜10図に示す. この実施例においては、先づ葉の表皮lOをメスで軽く
切り込みピンセットで剥離する(第7図A,B).この
表皮lOをマニュピュレータを装着した顕微鏡下に置き
,その孔辺細胞3にマイクロインジェクション法により
外来遺伝子aを注入した後(第8図A.B).表皮10
をフラスコ中の酵素l3に入れプロトブラストを単離す
る.(第9図A). 次にこのプロトブラストをパコールで密度勾配をつけた
遠心チューブ11に移しロータl2で遠心分離する(第
9図B,C), 遠心チューブ11の中のプロトブラストは、細胞の大き
さにより孔辺細胞3と表皮細胞2とに層間分離する(第
10図A). そこでパスツールピペット14を用いて目的の層より孔
辺細胞3を取り出し培養器l5に移して培養する(第l
O図B,C). このように遠心分離できない例外的植物の場合は、培養
後、葉緑体の有無によりカルスの色で識別して選別する
. 第2実施例は、表皮組織中の細胞の形態の違いに着目し
て孔辺細胞に外来遺伝子をマイクロインジェクションす
るから、従来のプロトプラスト状態のマイクロインジェ
クション法に比較して導入操作の効率が格段によい. また、表皮細胞と孔辺細胞は密度勾配遠心分離法により
容易に分離できるから,薬剤耐性に限界のある従来法に
比較し細胞選抜操作の効率が著し〈向上する. 次に第2発明の養液培養装置の実施例を第11図に示す
. この養液培養装置は培養室2lの下方に養液タンク22
を組立分解自在に連設した構造で、両者の境界にはシー
ル材23を介装して気密性を保つ。培養室21は透明で
、高温減菌処理が可能なポリカーポネートのようなプラ
スチック材により形成する. 培養室2lの底板に養液供給管24を貫通し,その下端
を養液タンク22の底部に開口する.25は多数の噴気
孔26を下向きに開口した噴気管で、養液タンク22の
底部に横架する.噴気管25の先端は閉鎖し、基端は開
口してある.培養室21内には、ポリエステル!a維若
しくはセラミック繊維のような吸水性に富む培地支持体
27を収容し、これに植物Pを植付ける。
Figure 1 shows the cross-sectional structure of a leaf, where l, 1 is the epidermal tissue, and 2
are epidermal cells, and 3 are guard cells. Between the epidermal tissues 1 and 1, a shelf tissue 4 and a spongy tissue 5 are layered one above the other, and chloroplasts are present in these tissues 4 and 5 and in the guard cells 3. First, the leaves are immersed in a hypertonic solution (0.8M sugar solution) to allow plasmolysis to occur to facilitate microinjection. Then, under a microscope equipped with a manipulator, foreign gene a is introduced into guard cells using a micropipette (
Figure 2). Next, the epidermal tissue is peeled off from the other tissues of the leaf using a bottle set (Fig. 3), and placed on an agar medium 7 in a flask 6 for callus culture (Fig. 4). During the culture, the green part 8a, that is, the part having chloroplasts, is selected from the grown cells and callus 8 (Fig. 5) and subcultured (Fig. 6). Part 8b other than the green part is an epidermal tissue that does not have chloroplasts and does not contain foreign gene a. Next, a second embodiment of the first invention is shown in FIGS. 7 to 10. In this example, the epidermis lO of the first leaf is lightly incised with a scalpel and peeled off with tweezers (Fig. 7A, B). This epidermis 1O was placed under a microscope equipped with a manipulator, and foreign gene a was injected into the guard cells 3 by the microinjection method (Fig. 8A and B). epidermis 10
into enzyme 13 in a flask to isolate protoblasts. (Figure 9A). Next, the protoblasts are transferred to a centrifuge tube 11 containing a density gradient with Pacol and centrifuged in a rotor 12 (Fig. 9B, C). There is interlayer separation into cell 3 and epidermal cell 2 (Figure 10A). Then, using a Pasteur pipette 14, guard cells 3 are taken out from the target layer and transferred to an incubator 15 to be cultured.
Figure O B, C). In the case of exceptional plants that cannot be centrifuged in this way, after culturing, they are identified and sorted by the color of their callus, depending on the presence or absence of chloroplasts. In the second example, foreign genes are microinjected into guard cells by focusing on differences in the morphology of cells in the epidermal tissue, so the efficiency of the introduction operation is significantly higher than in the conventional microinjection method for protoplasts. good. Furthermore, since epidermal cells and guard cells can be easily separated by density gradient centrifugation, the efficiency of cell selection operations is significantly improved compared to conventional methods that have limited drug resistance. Next, FIG. 11 shows an embodiment of the nutrient solution culture device of the second invention. This nutrient solution culture device has a nutrient solution tank 22 located below 2L of the culture chamber.
A sealing material 23 is interposed at the boundary between the two to maintain airtightness. The culture chamber 21 is transparent and made of a plastic material such as polycarbonate that can be sterilized at high temperatures. A nutrient solution supply pipe 24 is passed through the bottom plate of the culture chamber 2L, and its lower end is opened at the bottom of the nutrient solution tank 22. A fumarole pipe 25 has a number of fumarole holes 26 opening downward, and is horizontally suspended at the bottom of the nutrient solution tank 22. The tip of the fumarole pipe 25 is closed and the base end is open. Inside the culture room 21, there is polyester! A culture medium support 27 with high water absorption such as a-fiber or ceramic fiber is housed, and plants P are planted therein.

29は多数の通気孔30を穿つ空気供給管で、培養室2
lの上部に横架する. 空気供給管29の先端は閉鎖してあり,基端開口部は中
途を絞った通気管3lを.介して養液タンク22の上部
に導通する.32は温湿度計である. しかして培養室2lの側壁の養液水位上限付近に排気口
33を穿設する. 34は空気ポンプで、その吐出側を炭酸ガスタンク38
と共に混合器37に接続し、混合器37の出口側を薄膜
フィルタ41を経て前記の噴気管25と、養液タンク2
2の上部の送気管42とにそれぞれ接続する. 35、36はエアフィルタ、39、40は調節弁をそれ
ぞれ示す.43は炭酸ガスメータ、44は流量計である
. この実施例の装置は,初めにあらかじめ所定の養液を養
液タンク22に入れ,この養液タンク22と培養室2l
を組付け、両者の塊界をシール材23で密封後、オート
クレープ(高温高圧減菌器)により装置全体を滅菌,す
る。
29 is an air supply pipe that has many ventilation holes 30, and is connected to the culture chamber 2.
It is placed horizontally on top of l. The tip of the air supply pipe 29 is closed, and the opening at the base end is a vent pipe 3l with a narrowed midway. It is electrically connected to the upper part of the nutrient solution tank 22 through the nutrient solution tank 22. 32 is a thermohygrometer. Therefore, an exhaust port 33 is bored near the upper limit of the nutrient solution water level on the side wall of the culture chamber 2L. 34 is an air pump, and its discharge side is connected to a carbon dioxide tank 38
The outlet side of the mixer 37 is connected to the above-mentioned fumarole pipe 25 and the nutrient solution tank 2 through a thin film filter 41.
2 and the upper air pipe 42 respectively. 35 and 36 are air filters, and 39 and 40 are control valves, respectively. 43 is a carbon dioxide gas meter, and 44 is a flow meter. In the apparatus of this embodiment, a predetermined nutrient solution is first put into the nutrient solution tank 22, and then the nutrient solution tank 22 and the culture chamber 2l.
After assembling and sealing the boundary between the two with a sealant 23, the entire device is sterilized using an autoclave (high temperature and high pressure sterilizer).

滅菌後は、従来のように組立てるべき部品がないから、
雑菌汚染の危険がない. 次に炭酸ガスメータ43で炭酸ガス濃度が最適値(例え
ば1000ppm)になるようにiAm弁39、40を
調節しながら,空気ボンブ34の空気と炭酸ガスタンク
38の炭酸ガスを混合し、養液タンク22に給送する。
After sterilization, there are no parts to assemble like in the past.
There is no risk of bacterial contamination. Next, while adjusting the iAm valves 39 and 40 so that the carbon dioxide concentration reaches the optimal value (for example, 1000 ppm) with the carbon dioxide gas meter 43, the air in the air bomb 34 and the carbon dioxide in the carbon dioxide tank 38 are mixed, and the nutrient solution tank 22 is to be sent to.

培養初期は噴気管25にのみ空気(炭酸ガスを含む)を
送り、タンク22の上部に高湿度の空気を溜める。通気
管31の途中は絞ってあるからタンク22の内圧は次第
に高まり,この圧力に押されてタンク22の養液が養液
供給管24を上昇して上部の培養室21に流入する.培
養室21内の養液の水位が上限に達すると排気口33よ
り室外に排出され、養液の水位が上限を越えるのを防ぐ
At the initial stage of culture, air (including carbon dioxide gas) is sent only to the fumarole pipe 25, and highly humid air is stored in the upper part of the tank 22. Since the vent pipe 31 is constricted in the middle, the internal pressure of the tank 22 gradually increases, and this pressure causes the nutrient solution in the tank 22 to rise up the nutrient solution supply pipe 24 and flow into the culture chamber 21 in the upper part. When the water level of the nutrient solution in the culture chamber 21 reaches the upper limit, it is discharged outside from the exhaust port 33 to prevent the water level of the nutrient solution from exceeding the upper limit.

一方、タンク22の上部に溜った空気はその圧力で通気
管31を経て空気供給管29の通気孔30より培養室2
1に流入する.培養室21の空気は排気バイブ45より
室外に排気する.培養後期の1lll′i化の際は、空
気を専ら送気管42より養液タンク22に供給する。こ
れによりタンク22内の空気の湿度を低くでき、培養室
2lに乾いた空気を供給できる。
On the other hand, the air accumulated in the upper part of the tank 22 passes through the ventilation pipe 31 due to its pressure, and enters the culture chamber 2 through the ventilation hole 30 of the air supply pipe 29.
Flows into 1. The air in the culture chamber 21 is exhausted to the outside through an exhaust vibrator 45. During the latter stage of cultivation, air is exclusively supplied to the nutrient solution tank 22 through the air supply pipe 42. Thereby, the humidity of the air inside the tank 22 can be lowered, and dry air can be supplied to the culture chamber 2L.

このように図面の実施例では、養液タンク22の空気圧
を利用して養液を培養室21に送るから、養液送出専用
のボンブが不要となり装置が簡略化できる. 空気ボンプ34に貯蔵チャンバ(図示しない)を付設し
、この貯蔵チャンバより空気を養液タンク22に送るよ
うにすれば、空気圧は一定の範囲で変動するから、これ
に伴い養液タンク22の空気圧変動し、培養室21と養
液タンク22の養液が入れ換り養液の循環が可能となる
. 培養中,養液タンクの養液が蒸発等により減少しても,
培養室の液面は養液タンク22の空気圧により決定され
るため、常に維持できる.また,この実施例では、空気
を養液タイク22の噴気管25より供給するから、気泡
によりタンク22の養液が攪拌され溶存酸素量が増大し
,また培養室2lに湿度の高い空気を供給できるという
利点がある.高湿度の空気を作るのに,この実施例によ
れば,従来の加湿器を省略できる.次に第3発明と第4
発明の実施例の配管図を第12図に併せて示す. 50は順化装置である.そしてフィルタ51を備える清
浄空気供給路52とフィルタのない外気供給路53とに
それぞれ流量調整弁54.55を介装して両供給路52
、53の送出端を合流管56に接続する.合流管56の
出口側は、パーティカルカウンタ57を経て順化装置5
0の順化室に接続する.58は排出管で、59、60は
エアポンプをそれぞれ示す. 第13図は第3発明により、タマネギの培養苗を順化す
る場合の順化室内の空気の温度T、湿度H、照度L,及
び空気清浄度Pの関係を示すグラフである. しかしてタマネギの培養苗を培養器に入れて閉蓋し,こ
れを順化装置50に収容する.そして図示しない加湿器
により湿度を調整した外気と、フィルタ5lを経た清浄
空気との混合空気を順化室に供給し,所定プログラムに
従い、第13図のグラフのように混合空気の温度、及び
湿度を変化させる. 順化室は図示しない光源を備え,照度を24時間周期で
第13図に示す如〈徐々に明る〈なるように変動し,さ
らにlfit.量調節弁54、55を作動して外気と清
浄空気の混合比を変えることにより空気清浄度も第13
図の如く徐々に外部環境に近付ける. 第14図は第4発明の実施例による空気清浄度と清浄空
気量比との関係グラフで、フィルタ51を経由する清浄
空気の混合気に占める比率をEl [1!?の経過と共
に低下することにより、空気清浄度を外部環境まで下げ
続ける。
As described above, in the embodiment shown in the drawings, the air pressure of the nutrient solution tank 22 is used to send the nutrient solution to the culture chamber 21, so that a bomb dedicated to sending out the nutrient solution is not required, and the apparatus can be simplified. If a storage chamber (not shown) is attached to the air pump 34 and air is sent from this storage chamber to the nutrient solution tank 22, the air pressure in the nutrient solution tank 22 will change accordingly, since the air pressure will fluctuate within a certain range. The nutrient solution in the culture chamber 21 and the nutrient solution tank 22 are exchanged, allowing circulation of the nutrient solution. Even if the nutrient solution in the nutrient solution tank decreases due to evaporation during culturing,
Since the liquid level in the culture chamber is determined by the air pressure in the nutrient solution tank 22, it can be maintained at all times. In addition, in this embodiment, air is supplied from the fumarole pipe 25 of the nutrient solution tank 22, so the nutrient solution in the tank 22 is agitated by air bubbles, increasing the amount of dissolved oxygen, and supplying highly humid air to the culture chamber 2L. The advantage is that it can be done. According to this embodiment, a conventional humidifier can be omitted to create highly humid air. Next, the third invention and the fourth invention
A piping diagram of an embodiment of the invention is also shown in Fig. 12. 50 is an acclimatization device. Flow rate regulating valves 54 and 55 are respectively interposed in the clean air supply path 52 equipped with a filter 51 and the outside air supply path 53 without a filter, so that both supply paths 52
, 53 are connected to the confluence pipe 56. The outlet side of the merging pipe 56 passes through a particle counter 57 to the acclimation device 5.
Connect to acclimatization room 0. 58 is a discharge pipe, and 59 and 60 are air pumps, respectively. FIG. 13 is a graph showing the relationship among the temperature T, humidity H, illuminance L, and air cleanliness P of the air in the acclimatization chamber when culturing onion seedlings are acclimatized according to the third invention. Then, the cultured onion seedlings are placed in an incubator, the lid is closed, and this is placed in the acclimatization device 50. Then, a mixture of outside air whose humidity has been adjusted using a humidifier (not shown) and clean air that has passed through a filter 5L is supplied to the acclimatization chamber, and the temperature and humidity of the mixed air are adjusted as shown in the graph of Figure 13 according to a predetermined program. change. The acclimatization room is equipped with a light source (not shown), and the illuminance is varied in a 24-hour period so that it becomes "gradually brighter" as shown in FIG. 13, and lfit. By operating the volume control valves 54 and 55 to change the mixing ratio of outside air and clean air, the air cleanliness can be adjusted to the 13th level.
As shown in the figure, gradually approach the external environment. FIG. 14 is a graph showing the relationship between air cleanliness and clean air amount ratio according to the embodiment of the fourth invention, in which the ratio of clean air passing through the filter 51 to the mixture is expressed as El [1! ? As the air quality decreases over time, the air cleanliness continues to decrease to the outside environment.

これにより、培養苗は外部環境に適確に順化することが
できる。
This allows the cultured seedlings to acclimatize appropriately to the external environment.

【図面の簡単な説明】[Brief explanation of drawings]

第1〜第6図は第1発明の第1実施例を図解的に示す. 第1図は葉の組織の断面図、第2図はその要部拡大図で
、外来遺伝子を導入する工程を示す.第3図は葉の表皮
組織を剥離する工程を示す.第4図は表皮組織をフラス
コ内の培養地に置く工程を示す。第5図は培養中のフラ
スコを示す。第6図は培養したカルスのうち葉緑体を持
つ緑色部分のカルスを選抜し、別のフラスコへ移して継
体培養する工程を示す. 第7〜10図は第1発明の第2実施例を示す.第7図は
、葉の表皮剥離工程を示し、そのうち(A)は葉の表皮
にメスで切込み線を刻む工程を,また(B)は切込み線
で囲んだ表皮をビンセットで剥離する工程をそれぞれ示
す.第8図は,外来遺伝子導入工程を示し,そのうち(
A)はマニュビュレータで外来遺伝子を導入する様子を
表わし、(B)はその顕微鏡像を表わす.第9図はプロ
トプラストの単離工程を示し,(A)はフラスコ中の酵
素に表皮を入れるところを表わし、CB)はフラスコか
らプロトプラストを遠心チューブに移し、パコールで密
度勾配をつけるところを表わし、(C)は遠心チューブ
をロータに設置し回転するところを表わす.第10図は
細胞選抜工程を示し、(A)は孔辺細胞と表皮細胞が遠
心分離した状態を示す。CB)は遠心分離した孔辺細胞
をパスツールピペットで取り出す様子を示す。(C)は
孔辺細胞を培養器へ移す様子を示す。 第11図は第2発明の実施例で、養掖培養装置の断面と
その配管を示す. 第12図は第3及び第4発明の実施例の植物順化装置の
配管を示す。 第13図は第3発明の実施例により,タマネギの培養苗
を順化する場合の順化室の空気の温度、湿度,照度、及
び空気清浄度の関係グラフである。 第14図は、第4発明の実施例による順化室の空気清浄
度と清浄空気量比との関係グラフである. lは表皮組織、2は表皮細胞、3は孔辺細胞、6はフラ
スコ、7は培地、8はカルス、8aは孔辺細胞由来のカ
ルス、aは外来遺伝子,10は表皮、1lは遠心チュー
ブ、l3は酵素、12ロータ,22は培養室、23は養
液タンク,24は養液供給管、25は噴気管,27は培
地支持体、29は空気供給管,33は排気口、34は空
気ボンブ、38は炭酸ガスタンク、35、36及び41
はフィルタ、43は炭酸ガスメータ,50は順化装置、
59及び60はエアポンプ、54及び55は流量調整弁
、56は合流管、57はパーティカルカウンタ。 弔 図 第2図
1 to 6 schematically show a first embodiment of the first invention. Figure 1 is a cross-sectional view of the leaf tissue, and Figure 2 is an enlarged view of its main parts, showing the process of introducing a foreign gene. Figure 3 shows the process of peeling off the leaf epidermis. FIG. 4 shows the step of placing epidermal tissue in a culture medium in a flask. Figure 5 shows the flask during cultivation. Figure 6 shows the process of selecting the green part of the cultured callus that has chloroplasts, transferring it to another flask, and subculturing it. Figures 7 to 10 show a second embodiment of the first invention. Figure 7 shows the process of peeling off the epidermis of a leaf, of which (A) shows the process of making a score line on the leaf epidermis with a scalpel, and (B) shows the process of peeling off the epidermis surrounded by the score line with a bottle set. Each is shown below. Figure 8 shows the foreign gene introduction process, of which (
A) shows the introduction of a foreign gene with a manipulator, and (B) shows the microscopic image. Figure 9 shows the protoplast isolation process, (A) shows the epidermis being placed in the enzyme in the flask, CB) shows the process of transferring the protoplasts from the flask to a centrifuge tube and applying a density gradient with Pacol; (C) shows the centrifuge tube installed on the rotor and rotated. FIG. 10 shows the cell selection process, and (A) shows the state in which guard cells and epidermal cells have been centrifuged. CB) shows how centrifuged guard cells are removed with a Pasteur pipette. (C) shows how guard cells are transferred to an incubator. FIG. 11 shows an embodiment of the second invention, showing a cross section of the culture device and its piping. FIG. 12 shows the piping of the plant acclimation device according to the third and fourth embodiments of the invention. FIG. 13 is a graph showing the relationship between the temperature, humidity, illuminance, and air cleanliness of the air in the acclimatization chamber when culturing onion seedlings are acclimatized according to the third embodiment of the invention. FIG. 14 is a graph showing the relationship between the air cleanliness of the acclimatization chamber and the clean air amount ratio according to the embodiment of the fourth invention. l is epidermal tissue, 2 is epidermal cell, 3 is guard cell, 6 is flask, 7 is medium, 8 is callus, 8a is callus derived from guard cell, a is foreign gene, 10 is epidermis, 1l is centrifuge tube , l3 is an enzyme, 12 is a rotor, 22 is a culture chamber, 23 is a nutrient solution tank, 24 is a nutrient solution supply pipe, 25 is a fumarole pipe, 27 is a culture medium support, 29 is an air supply pipe, 33 is an exhaust port, 34 is a Air bomb, 38 is carbon dioxide tank, 35, 36 and 41
is a filter, 43 is a carbon dioxide gas meter, 50 is an acclimation device,
59 and 60 are air pumps, 54 and 55 are flow control valves, 56 is a merging pipe, and 57 is a particle counter. Funeral map 2nd figure

Claims (4)

【特許請求の範囲】[Claims] (1)植物の表皮組織中の孔辺細胞に、マイクロインジ
ェクション法により外来遺伝子を導入した後、これを培
養することを特徴とする外来遺伝子導入細胞培養法。
(1) A foreign gene-introduced cell culture method, which comprises introducing a foreign gene into guard cells in the epidermal tissue of a plant by a microinjection method, and then culturing the resultant.
(2)植物の培養室の排気口を養液面上限付近に開口し
て成る養液培養装置。
(2) A nutrient solution culture device in which the exhaust port of the plant culture chamber is opened near the upper limit of the nutrient solution level.
(3)植物の順化室の室内の温度、湿度、照度及び空気
清浄度をそれぞれシーケンス制御することを特徴とする
植物順化装置。
(3) A plant acclimation device characterized by sequentially controlling the indoor temperature, humidity, illuminance, and air cleanliness of a plant acclimation chamber.
(4)フィルタを備える清浄空気供給路と外気供給路と
にそれぞれ流量調節弁を介装して両供給路の送出端を合
流管に接続すると共に、この合流管の出口側をパーティ
カルカウンタを経て植物の順化室に接続して成る植物順
化装置。
(4) A flow rate control valve is interposed in each of the clean air supply path equipped with a filter and the outside air supply path, and the delivery ends of both supply paths are connected to a merging pipe, and a particle counter is connected to the outlet side of this merging pipe. A plant acclimatization device that is connected to a plant acclimatization chamber through a cable.
JP1240730A 1989-09-19 1989-09-19 Foreign gene transfer cell culture method Expired - Fee Related JP2638217B2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP1240730A JP2638217B2 (en) 1989-09-19 1989-09-19 Foreign gene transfer cell culture method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP1240730A JP2638217B2 (en) 1989-09-19 1989-09-19 Foreign gene transfer cell culture method

Related Child Applications (1)

Application Number Title Priority Date Filing Date
JP1192497A Division JP2891222B2 (en) 1997-01-07 1997-01-07 Plant acclimation equipment

Publications (2)

Publication Number Publication Date
JPH03103183A true JPH03103183A (en) 1991-04-30
JP2638217B2 JP2638217B2 (en) 1997-08-06

Family

ID=17063851

Family Applications (1)

Application Number Title Priority Date Filing Date
JP1240730A Expired - Fee Related JP2638217B2 (en) 1989-09-19 1989-09-19 Foreign gene transfer cell culture method

Country Status (1)

Country Link
JP (1) JP2638217B2 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5969215A (en) * 1993-10-14 1999-10-19 Zeneca Limited Method of plant tissue culture and regeneration

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5969215A (en) * 1993-10-14 1999-10-19 Zeneca Limited Method of plant tissue culture and regeneration

Also Published As

Publication number Publication date
JP2638217B2 (en) 1997-08-06

Similar Documents

Publication Publication Date Title
EP2832847B1 (en) Culture vessel and automated culture apparatus
US5707868A (en) Variable-volume reactor-type device and process for culturing cellular material
WO2013183121A1 (en) Culturing vessel and automatic culturing device
JP2002153260A (en) Device for culturing and observing one cell with microscope for long period
CN109810900A (en) Totally-enclosed cell culture gas control system
EP0776358A1 (en) Cell culture device
JP5960256B2 (en) Culture container and automatic culture device
US20100009443A1 (en) Laboratory Apparatus for a Controlled Environment
CN113755331B (en) Cell incubator of simulation altitude environment
JPS6231908B2 (en)
WO2012084212A1 (en) A method of examining tissue growth and conditioning of cells on a scaffold and a perfusion bioreactor
JPH03103183A (en) Cell culture of adventitious gene transduction, nutritive solution culture device and plant acclimation device
CN101897254A (en) Oscillatory seed liquor initiation device and method
DE202018004857U1 (en) Meander perfusion bioreactor for the differentiation, activation, stimulation and separation of cells
JPH01174373A (en) Laboratory dish, laboratory dish system, cell culture apparatus using thereof and culturing method
US20080206860A1 (en) Vessel and Culture System Including
Hale et al. Bioreactor development for continual-flow, liquid plant tissue culture
CN111849778B (en) Cell culture device and system
CN109385369B (en) Filtering system capable of preventing cell rupture
CN216237091U (en) Auxiliary device for appearance inspection of oocyte and embryo incubator
Bhosale et al. In vitro development of the embryo in a microfluidic device for automatic embryo trapping and co-culture with endometrial cells
Aguilera-Castrejon et al. Highly conducive ex utero mouse embryogenesis from pre-gastrulation to late organogenesis
Ibrahim et al. Construction and application of a microprojectile system for the transfection of organotypic brain slices
US20040132174A1 (en) Perfusion incubator
WO2004027016A1 (en) Improved perfusion incubator

Legal Events

Date Code Title Description
LAPS Cancellation because of no payment of annual fees