JPS60256381A - Cell fusion in laver - Google Patents
Cell fusion in laverInfo
- Publication number
- JPS60256381A JPS60256381A JP59109667A JP10966784A JPS60256381A JP S60256381 A JPS60256381 A JP S60256381A JP 59109667 A JP59109667 A JP 59109667A JP 10966784 A JP10966784 A JP 10966784A JP S60256381 A JPS60256381 A JP S60256381A
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- JP
- Japan
- Prior art keywords
- seaweed
- cell fusion
- monospores
- fusion
- cell
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/14—Plant cells
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- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Botany (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Cultivation Of Seaweed (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
皮粟上匹程且分界
本発明は、海苔の葉状体から放出される単胞子または果
胞子を細胞融合手法を適用して細胞質融合させる方法に
関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for cytoplasmic fusion of monospores or carospores released from seaweed fronds by applying a cell fusion technique.
すなわち、本発明は、海苔葉体のプロトプラストを用い
ることなく、海苔を細胞融合させる方法に関する。That is, the present invention relates to a method for cell fusion of seaweed without using protoplasts of seaweed thallus.
従来■及否
近年、遺伝子工学的手法としての細胞融合に関する研究
開発が盛んに行われるようになり、陸上植物においては
既に実用化の段階にまで成功した例もしられている。In recent years, research and development on cell fusion as a genetic engineering method has been actively conducted, and there have already been cases where it has reached the stage of practical application in land plants.
しかし、−１海藻類（アマノリ類を含む）においては現
在までのところ細胞融合についての成功例は未だ報告さ
れていない。However, to date, no successful case of cell fusion has been reported in -1 seaweeds (including laver).
このような事情は、陸上植物においては市販の酵素剤（
例えばセルラーゼ・オノヅカ、ペクトリアーゼ、マセロ
チーム）を用いて簡単にプロトプラストを調製できるが
海藻類、特にアマノリ類ではそれの組織を構成している
骨格成分である多糖類が上述したような酵素剤で処理し
ても健全な状態でプロトプラスト化されないことに基因
している。This situation is due to the fact that commercially available enzyme preparations (
For example, protoplasts can be easily prepared using cellulase, pectolyase, macerozyme), but in seaweeds, especially seaweed, the polysaccharides that are the skeletal components of their tissues are treated with the enzymes mentioned above. This is due to the fact that they do not convert into protoplasts in a healthy state.
すなわち、アマノリ類の組織を構成している多糖類はキ
シラン、マンナン及びポルノイランから成っており、特
に細胞壁は強固な繊維状に形成されたキシランから成っ
ているので上記酵素剤によっては加水分解されないから
である。因に、現在のところ上記３種の多糖類に対して
加水分解能を合わせ持つ酵素剤についても報告はみられ
ない。In other words, the polysaccharides that make up the tissues of laver are composed of xylan, mannan, and porno-ylang, and the cell walls in particular are made of xylan formed into strong fibers, so they cannot be hydrolyzed by the enzymes mentioned above. It is from. Incidentally, there are currently no reports on enzyme agents that have the ability to hydrolyze the three types of polysaccharides mentioned above.
■ （゛ しよ゛と ４肌用点
上述したように、海苔の組織を酵素で処理することによ
り健全なプロトプラストを調製することが非常に困難で
ある現状に鑑み、本発明者は海苔の生活史において、海
苔の葉状体から放出されて間もない直径１０〜１３ミク
ロン程度のグニャグニャしたアメーバ−状の細胞である
単胞子並びに果胞子に着目し、研究した結果、これらの
単胞子または果胞子を直接融合処理すると細胞質融合が
行なわれることの知見を得て、本発明をなすに至った。(4 points for skin use) As mentioned above, in view of the current situation where it is extremely difficult to prepare healthy protoplasts by treating seaweed tissue with enzymes, the present inventor decided to improve the life of seaweed. Historically, research has focused on monospores and carospores, which are squishy amoeboid cells with a diameter of about 10 to 13 microns that have just been released from seaweed fronds. The present invention was accomplished based on the finding that cytoplasmic fusion occurs when directly fused.
すなわち、本発明は、海苔のプロトプラストを用いるこ
となく、海苔の細胞融合を行なうことに成功したもので
あって、上記単胞子または果胞子を用いて海苔の細胞融
合を行ない得る方法を提供することを目的とする。That is, the present invention has succeeded in performing seaweed cell fusion without using seaweed protoplasts, and provides a method that can perform seaweed cell fusion using the monospores or caropspores described above. With the goal.
以下本発明の詳細な説明する。The present invention will be explained in detail below.
光肌夏援戊
本発明の構成上の特徴は、海苔の葉状体から放出される
単胞子または果胞子を細胞融合手法を適用して細胞質融
合させることにある。A structural feature of the present invention resides in that monospores or fruit spores released from seaweed fronds are subjected to cytoplasmic fusion by applying a cell fusion technique.
本発明において細胞融合に用いる単胞子は、海苔の生活
史において海苔網に付着して発芽し、仮根を生じて細胞
壁を形成し、***増殖して海苔葉体になるものであって
、この生活史において細胞壁を形成する前の段階である
単胞子は、海苔葉体からその細胞壁を除去して得られる
プロトプラストと同様な形態にあるものと解される。The monospores used for cell fusion in the present invention attach to the seaweed network during the life history of seaweed, germinate, produce rhizoids, form cell walls, and divide and proliferate to become seaweed thallus. Monospores, which are a stage in life history before cell wall formation, are understood to have a similar morphology to protoplasts obtained by removing the cell wall from seaweed thallus.
また、果胞子も海苔の生活史において海苔葉体から離脱
して貝殻などに付着し、やがて潜入して ）糸状体にな
るものであって、細胞壁が薄く、上記単胞子と同様に、
海苔をプロトプラスト化したものと同様な形態にあるも
のと解される。In addition, during the life history of seaweed, caropspores also detach from the seaweed thallus, attach themselves to shells, etc., and eventually infiltrate into filamentous bodies (), which have thin cell walls and, like the monospores mentioned above,
It is thought to have a similar form to protoplasts of seaweed.
これらの単胞子並びに果胞子を人工的に得るには、単胞
子ではアサクサノリの場合０．２〜１．０ｍｎ＋程度の
幼葉を、スサビノリの場合１．０〜１０ｃｍ程度の葉体
を人工海水中で培養し、葉体縁辺部がら細胞が離脱する
のを確認した後、多数の葉体がら細胞が離脱した段階で
、それを含有する培養液を遠心分離して得られる残渣を
採取するとよく、また、果胞子では成熟した海苔葉体を
人工海水中に静置し、水温１５℃、１５００Ｌｕｘの照
度下での明朗１２時間および晴朗１２時間の周期で培養
して果胞子を放出させ、ついでこの果胞子を含有する培
養液を遠心分離して得られる残渣を分離するとよい。To obtain these monospores and caropspores artificially, for monospores, young leaves of about 0.2 to 1.0 mm+ in the case of Asakusa nori, and leaves of about 1.0 to 10 cm in the case of Asakusanori, are placed in artificial seawater. After culturing at the thallus edge and confirming that cells have detached from the thallus margin, at the stage where a large number of thallus cells have detached, it is best to centrifuge the culture solution containing the cells and collect the resulting residue. In addition, for caropspores, mature seaweed fronds are placed in artificial seawater and cultured under a water temperature of 15°C and an illuminance of 1500 Lux with a cycle of 12 hours of light and 12 hours of clear light to release caropspores. It is preferable to separate the residue obtained by centrifuging the culture solution containing fruit spores.
本発明では上述のようにして人工的に得られた単胞子ま
たは果胞子から成る残渣に適量の人工海水を加えてそれ
ぞれの懸濁液を調製したものを用い、下記手段により細
胞融合を行なう。In the present invention, cell fusion is performed by the following means using a suspension prepared by adding an appropriate amount of artificial seawater to the residue consisting of monospores or carospores artificially obtained as described above.
細胞融合の手法：
上述のようにして調製した単胞子並びに果胞子の各懸濁
液をペトリ皿などに滴下し、放置してガラス表面上に沈
澱を生成させ、この沈澱に融合誘導物質として、ポリエ
チレングリコール溶液（５４％）を加え放置後、更にＨ
ｉｇｈ ｐＨ−カルシウム溶・液（１００ｍＭ ＣａＣ
＋２、ｐＨ１０，５）を加えて室温下に放置する。なお
、この場合、Ｈｉｇｈ ｐＨ−カルシウム溶液の添加を
２回繰返して行なうことが好ましい。Cell fusion method: Each suspension of monospores and carospores prepared as described above is dropped into a Petri dish, etc., and left to stand to form a precipitate on the glass surface. After adding polyethylene glycol solution (54%) and leaving it to stand, further H
igh pH-Calcium solution/liquid (100mM CaC
+2, pH 10.5) and left at room temperature. In this case, it is preferable to repeat the addition of the High pH calcium solution twice.
次に、上述のように処理したものに人工海水から成る培
養液を加えて培養すると、単胞子並びに果胞子の細胞質
融合体がそれぞれ顕微鏡下での観察により確認される。Next, when a culture solution consisting of artificial seawater is added to the treated product as described above and cultured, cytoplasmic fusions of monospores and carpospores are confirmed by observation under a microscope.
因に、上記細胞融合の処理を行なわない場合には単胞子
および果胞子のいずれについても細胞質融合体は実質上
確認されない。Incidentally, when the above-mentioned cell fusion treatment is not performed, virtually no cytoplasmic fusions are observed in either monospores or caropspores.
血貫勿羨果
叙上のとおり、本発明によると、海苔の幼葉を培養する
ことにより得られる単胞子、もしくは海苔の葉体を培養
して得られる果胞子を細胞融合させることにより、その
調製が困難とされる海苔のプロトプラストを用いなくと
も、海苔の細胞融合を行ない得るので、海苔の細胞融合
技術上益するところが大きく、したがって、細胞融合体
の海苔の生産面における利用上有用である。As described above, according to the present invention, monospores obtained by culturing young leaves of seaweed or caropspores obtained by culturing thallus of seaweed are cell-fused. Since seaweed cell fusion can be performed without using seaweed protoplasts, which are difficult to prepare, the technology has great benefits for seaweed cell fusion technology, and therefore, the cell fusion product is useful in the production of seaweed. .
以下実施例を示して本発明を更に具体的に説明する。The present invention will be explained in more detail below with reference to Examples.
次１１０＝
単胞子の懸濁液の調製：
アサクサノリの０．２〜１．０ｍｎ＋サイズの幼葉を下
記に示す組成の人工海水（Ａｓｐ １２）中で１８℃の
温度において４０００Ｌｕｘの照度下（明期８時間、晴
朗１６時間の周期）で培養し、単胞子の放出が認められ
９ だ段階で培養液を遠心分離しく１１０００ｒｐ、
５分間）、得られた残渣を採取し、これに上記人工海水
から成る培養液の適量を加えて′単胞子懸濁液とした。Next 110 = Preparation of suspension of monospores: Young leaves of 0.2 to 1.0 mm+ size of Aspergillus chinensis were grown in artificial seawater (Asp 12) with the composition shown below at a temperature of 18° C. under an illuminance of 4000 Lux (bright light). The culture solution was cultured at a cycle of 8 hours in the morning and 16 hours in the clear, and when the release of monospores was observed, the culture solution was centrifuged at 11,000 rp.
5 minutes), the resulting residue was collected, and an appropriate amount of the above-mentioned culture solution consisting of artificial seawater was added to it to prepare a monospore suspension.
人工海水（ＡＳＰ １２）の組成：
ＮａＣｌ ２８ ｇ
ＭＢＳＯ４７Ｈ２０７ｇ
ＭｇＣｈ’６Ｈ２０４ｇ
ＫＣＩ ７００ ｍｇ
ＣａＣ１２２Ｈ２０１，４７ｇ
ＮａＮＯ３１００ｍｇ
ＫＪＰＯＩ４１０ ｍｇ
グリセロ燐酸ナトリウム １０ ｍｇ
ビタミンＢ１２ ０．２ｐｇ
ビオチン １８ｇ
チアミン １００ μｇ
Ｐ ＩＩ Ｍｅｔａｌ １０ ｍ１ｌ
Ｓ Ｕ Ｍｅｔａｌ １０ ｍｊ！
トリスアミノメタン １ｇ
蒸留水 １０００１０
０Ｏ８，０〜８．１
上工」虹紅勿組爪 旦工」１１辺皿威
ＥＤＴＡ １ ｍｇ ＮａＢｒ １．２ ｍｇ）（ヨＢ
Ｏ＋ １ ｍｇ ＡｌＣｌ３ ６Ｈ２０１，２ｍｇＭｎ
Ｃ１２４Ｈ２００，Ｍ ｍｇ ５ｒＣＩ２６Ｈ２００，
６ｍｇＦｅＣ１２６Ｈ２００，０５ｍｇ ＮａＭｏ１％
・２Ｈ２００，１２ｍｇＺｎＣｌ２０．０１ ｍｇ
ＰｂＣｌ ０．０３ｍｇＣｏＣｌ２６Ｈ２０４Ｉｇ Ｋ
Ｉ １．５／’ｇＣｕＳＯ４５）１２００．５ ／ｊｇ
蒸留水 １ ｍｌ！蒸留水 １ ｍｌ
単胞子の細胞質融合：
上述のようにして調製した単胞子懸濁液の０．１ｍｌを
ピペットでペトリ皿内に滴下し、５〜１ｏ分放置してガ
ラス表面上に沈澱を生成させた。この沈澱に下記組成の
ポリエチレングリコール溶液０．２１Ｉｌβを加えて３
０分放置後、これに下記組成のＨｉｇｈ ｐＨ−カルシ
ウム溶液０．５ｍｌ１を加えて１０分放置後、更にｌ（
ｉｇｈ ｐＨ−カルシウム溶液０．５ｍＩ２を加えて５
分間放置した。Composition of artificial seawater (ASP 12): NaCl 28 g MBSO47H207g MgCh'6H204g KCI 700 mg CaC122H201,47g NaNO3100mg KJPOI410 mg Sodium glycerophosphate 10 mg Vitamin B12 0.2p g Biotin 18g Thiamine 100 μg P II Metal 10 ml S U Metal 10 mj ! Tris-aminomethane 1g Distilled water 100010 0O8,0~8.1 11 sides EDTA 1 mg NaBr 1.2 mg) (YoB
O+ 1 mg AlCl3 6H201, 2 mgMn
C124H200, M mg 5rCI26H200,
6mgFeC126H200.05mg NaMo1%
・2H200, 12mgZnCl20.01mg
PbCl 0.03mgCoCl26H204Ig K
I 1.5/'gCuSO45) 1200.5/jg
1 ml of distilled water! Distilled water 1 ml Cytoplasmic fusion of monospores: Pipette 0.1 ml of the monospore suspension prepared above into a Petri dish and leave to stand for 5 to 10 minutes to form a precipitate on the glass surface. I let it happen. To this precipitate was added 0.21 Ilβ of a polyethylene glycol solution with the following composition.
After leaving it for 0 minutes, add 0.5 ml of a High pH calcium solution with the following composition, and after leaving it for 10 minutes, add an additional 1 (
igh pH - Add 0.5ml of calcium solution to 5
Leave it for a minute.
ポリエチレングリコール溶液の組成：
ポリエチレングリコール（ＭＷ ６，０００）の５４％
水溶液にＣａＣｌ２２１１２０１０．５ｍＭ、ＫＨ２Ｐ
Ｏ＋Ｈ２００，７ｍＭおよびグルコース０． ＩＭの濃
度になるように添加したもの。Composition of polyethylene glycol solution: 54% of polyethylene glycol (MW 6,000)
CaCl22112010.5mM, KH2P in aqueous solution
O+H200, 7mM and glucose 0. Added to the concentration of IM.
Ｈｉｇｈ ｐＨ−カルシウム溶液の組成：■ ＣａＣｌ
２２１１２０１００ｍＭ、グルコース０．４Ｍを蒸留水
に熔解した溶液と、■ ＮａＯＨ−グリシンバッファー
（ｐＨ１０，５）にグルコース０．４Ｍを熔解した溶
液を使用に際し、１：１の割合に混合したもの。High pH-calcium solution composition: ■ CaCl
221120 A solution of 100mM glucose dissolved in distilled water and 1) A solution of 0.4M glucose dissolved in NaOH-glycine buffer (pH 10,5) were mixed at a ratio of 1:1.
次に、上述のように処理したものに、０．３ｎ＋４２の
培養液（人工海水Ａｓｐ １２）を加え５分放置後、０
．３ｍｌをシャーレから吸い取った。これらの操作を５
回反復して行なった後、新しい培養液（人工海水Ａｓρ
１２）を加えて培養を行なった。この培養液中に単胞子
の細胞質融合体の生成が確認された。Next, 0.3n+42 culture solution (artificial seawater Asp 12) was added to the treated material as described above, and after leaving it for 5 minutes,
．． 3 ml was sucked out from the petri dish. These operations 5
After repeating the experiment several times, use a new culture medium (artificial seawater Asρ).
12) was added and cultured. Production of a monospore cytoplasmic fusion was confirmed in this culture solution.
なお、アサクサノリの幼葉に代えてスサビノリの１．０
〜１０ｃｌ１１の葉体を用いても同様に細胞質融合体の
生成が確認された。In addition, instead of the young leaves of Asakusanori, 1.0 of Asakusanori is used.
Generation of cytoplasmic fusions was similarly confirmed using ~10cl11 leaflets.
実施桝ｌ
果胞子の懸濁液の調製：
成熟した海苔葉体を、実施例１で用いたと同様の人工海
水Ａｓｐ １２中で、１５℃の温度において１５００
Ｌｕｘの照度下（明期１２時間、晴朗１２時間の周期）
で培養した。培養から３〜４日目に果胞子の゛放出がみ
られたので、培養液を遠心分離しく１１０００ｒｐ 、
５分間）、得られた残渣を採取し、これに上記人工海水
Ａｓｐ １２を培養液として適量加えて果胞子懸濁液と
した。Example 1: Preparation of a suspension of fruit spores: Mature seaweed thallus were incubated at 1500 °C in artificial seawater Asp 12 similar to that used in Example 1 at a temperature of 15 °C.
Under Lux illumination (12 hours of light, 12 hours of clear weather)
It was cultured in Since the release of fruit spores was observed on the 3rd to 4th day of culture, the culture solution was centrifuged at 11,000 rpm.
5 minutes), the resulting residue was collected, and an appropriate amount of the above-mentioned artificial seawater Asp 12 was added thereto as a culture medium to prepare a fruit spore suspension.
果胞子の細胞質融合：
実施例１において単胞子の懸濁液に代えて果胞子の懸濁
液を用いるほかは、実施例１に記載と同様の手順で細胞
融合を行なった。Cytoplasmic Fusion of Carospores: Cell fusion was carried out in the same manner as described in Example 1, except that a suspension of carospores was used in place of the monospore suspension in Example 1.
培養後の培養液中に果胞子の細胞質融合体の生成が顕微
鏡下に確認された。After culturing, the formation of cytoplasmic fusions of fruit spores was confirmed under a microscope in the culture solution.
Claims (2)
を細胞融合手法を適用して細胞質融合させることを特徴
とする海苔の細胞融合方法。(1) A method for cell fusion of seaweed, characterized in that monospores or caropspores released from seaweed thallus are subjected to cytoplasmic fusion by applying a cell fusion method.
Ｈ−カルシウム溶液を融合誘導物質として用いる細胞融
合手法を適用するものである特許請求の範囲第（１）項
記載の方法。(2) Polyethylene glycol solution and High p
The method according to claim (1), which applies a cell fusion technique using H-calcium solution as a fusion inducer.
Priority Applications (9)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59109667A JPS60256381A (en) | 1984-05-31 | 1984-05-31 | Cell fusion in laver |
| AT84112071T ATE40568T1 (en) | 1983-10-18 | 1984-10-09 | METHOD OF CELL MERGING OF LETTUCE. |
| NZ209829A NZ209829A (en) | 1983-10-18 | 1984-10-09 | Method for the cell fusion of seaweed |
| EP84112071A EP0141304B1 (en) | 1983-10-18 | 1984-10-09 | Method for the cell fusion of laver |
| DE8484112071T DE3476558D1 (en) | 1983-10-18 | 1984-10-09 | Method for the cell fusion of laver |
| AU34135/84A AU585486B2 (en) | 1983-10-18 | 1984-10-11 | Method for the cell fusion of laver |
| CA000465577A CA1225607A (en) | 1983-10-18 | 1984-10-16 | Method for the cell fusion of laver |
| ES536826A ES8600388A1 (en) | 1983-10-18 | 1984-10-17 | Method for the cell fusion of laver. |
| KR1019840006432A KR920007397B1 (en) | 1983-10-18 | 1984-10-17 | Method for the cell fusion of laver |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59109667A JPS60256381A (en) | 1984-05-31 | 1984-05-31 | Cell fusion in laver |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS60256381A true JPS60256381A (en) | 1985-12-18 |
| JPS6260077B2 JPS6260077B2 (en) | 1987-12-14 |
Family
ID=14516107
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP59109667A Granted JPS60256381A (en) | 1983-10-18 | 1984-05-31 | Cell fusion in laver |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS60256381A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114008124B (en) | 2019-04-25 | 2023-09-29 | 东洋制罐集团控股株式会社 | Cellulose nanocrystal complex and method for producing same |
-
1984
- 1984-05-31 JP JP59109667A patent/JPS60256381A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6260077B2 (en) | 1987-12-14 |
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