WO2020027002A1 - Method for producing seaweed cells - Google Patents

Method for producing seaweed cells Download PDF

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WO2020027002A1
WO2020027002A1 PCT/JP2019/029542 JP2019029542W WO2020027002A1 WO 2020027002 A1 WO2020027002 A1 WO 2020027002A1 JP 2019029542 W JP2019029542 W JP 2019029542W WO 2020027002 A1 WO2020027002 A1 WO 2020027002A1
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seaweed
cells
culture
medium
spores
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PCT/JP2019/029542
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French (fr)
Japanese (ja)
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平岡　雅規
幸記 田中
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国立大学法人高知大学
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Priority to CN201980051157.8A priority Critical patent/CN112534042A/en
Priority to JP2020533507A priority patent/JP7353653B2/en
Publication of WO2020027002A1 publication Critical patent/WO2020027002A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G33/00—Cultivation of seaweed or algae
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H13/00—Algae
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management

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  • the present invention relates to a method for producing seaweed cells and the like.
  • Seaweed cultivation is generally performed by growing mother alga bodies of seaweed and producing seeds and seedlings from spores obtained therefrom.
  • Patent Literature 1 discloses a seaweed cultivation method in which spores of seaweed are seeded at high density on a flat plate, spores are attached to each other, and the resulting clumps are cultured. According to this method, the seaweed can be grown in a state of being suspended in the medium, and therefore, can be easily harvested.
  • An object of the present invention is to provide a technique for easily and efficiently producing seaweed.
  • the present inventor has conducted intensive studies in view of the above-mentioned problems, and as a result, seaweed spores, single cells derived from the spores, and at least one selected from the group consisting of the spores and / or the cell mass of the single cells, It has been found that culturing under stirring conditions in a medium 1 substantially containing no morphogenesis-inducing factor enables simple and efficient production of seaweed cells usable as a raw material for producing seaweed. As a result of further research based on this finding, the present invention has been completed.
  • the present invention includes the following embodiments.
  • Item 1 At least one selected from the group consisting of seaweed spores, single cells derived from the spores, and cell masses of the spores and / or the single cells, in a medium 1 substantially free of seaweed morphogenesis-inducing factors;
  • a method for producing a seaweed cell comprising:
  • Item 4. ⁇ The method according to any one of Items 1 to 3, wherein the seaweed cells include single cells.
  • Item 5 (A1) a step of culturing spores of seaweed in the medium 1, and (A2) a step of culturing the culture of step A1 in the medium 1 while stirring. Item 5.
  • Item 7. ⁇ The method according to any one of Items 1 to 6, wherein the medium 1 is an artificial seawater medium.
  • Item 8. ⁇ The method according to any one of Items 1 to 7, wherein the seaweed is an edible seaweed.
  • Item 9 The production method according to any one of Items 1 to 8, wherein the culture under the stirring condition is aeration culture.
  • Item 10 A step of culturing the seaweed cells obtained by the production method according to any one of Items 1 to 9 in a medium 2 containing a seaweed morphogenesis factor.
  • a method for producing seaweed comprising:
  • seaweed cells that can be used as a raw material for producing seaweed can be simply and efficiently produced, and thus seaweed can be simply and efficiently produced.
  • FIG. 1 is a photograph of the cultured cells of Macchitoe after 32 days of culture in the culture test of Macchitoe in Example 1.
  • FIG. 2 is a photograph of the cultured cells of Macchitoe after 32 days of culturing when a morphogen was added, in a culture performed in parallel with the test for cultivation of Macchitoe in Example 1.
  • FIG. 3 is a growth curve of a single cell group of A. chinensis cultured in a medium without a morphogenesis-inducing factor.
  • FIG. 4 is a logarithmic transformation curve of the absorbance data of FIG. It shows a linear increase from the 2nd day to the 7th day of the culture and can be regarded as a logarithmic phase.
  • FIG. 5 is a logarithmic growth curve of a single cell group of Hilohano human exa cultivated in a medium containing no morphogenesis inducer of Example 2.
  • FIG. 6 is a photograph of the thallus obtained after the culture of Example 4.
  • FIG. 7 is a photograph obtained by spreading and photographing the leaf-like body obtained after the culture in Example 4.
  • FIG. 8 is a photograph of the thallus of a normal human euple grown from spores. Arrows indicate attached roots.
  • a method for producing seaweed Cell (A) seaweed spores, single cells derived from the spores, and at least one selected from the group consisting of the spores and / or the cell mass of the single cells, A method for producing seaweed cells (hereinafter referred to as “the method for producing seaweed cells of the present invention”), which comprises culturing under stirring conditions in a medium 1 substantially containing no seaweed morphogenesis-inducing factor. There is also.) Hereinafter, this will be described.
  • Seaweeds are not particularly limited, and are usually green algae.
  • a seaweed belonging to the order Hihibimidoro is preferable from the viewpoint that the production efficiency of seaweed cells can be further improved.
  • Examples of seaweeds belonging to the order Hibimidori include, for example, seaweeds belonging to the family Echinaceae, Echinacea spp., Capsa anonnaceae, Lansinaceae, Echinacea spp., And more preferably seaweeds belonging to the Echinacea spp., Echinacea spp., And the like.
  • a seaweed belonging to the family Iridaceae is mentioned.
  • seaweeds belonging to the family Amidori include the seaweeds belonging to the genus Aesculus, the genus Aesculus, and the like, and preferably the seaweeds belonging to the genus Aesculus.
  • seaweeds belonging to the genus Humanexa include, for example, Hirohanohitoexa, humanexa, Usuhitoexa, Ezohitoexa, Shinkaihitoexa, Atsubahitoe, Atsukawahitoe, and the like. Seaweeds may be used alone or in combination of two or more.
  • Spores are spores obtained from seaweed and are not particularly limited. Spores are germ cells.
  • the state of the spores is not particularly limited, and examples of the spores include zoospores, gametes, zygotes, tetraspores, fruit spores, monospore zoospores, and neutral zoospores.
  • the spores may be used alone or in combination of two or more.
  • Spore-derived single cells are not particularly limited as long as they are spore-derived undifferentiated single cells (cells constituting differentiated thallus are not included in the “single cells” here).
  • Single cells are usually approximately spherical. The diameter of a single cell is, for example, 3 to 20 ⁇ m and 5 to 15 ⁇ m. Single cells may be used alone or in combination of two or more.
  • the cell mass is formed by assembling at least one selected from the group consisting of the spores and the single cells, and is not particularly limited as long as it does not include the cell mass constituting the differentiated thallus.
  • the number of cells constituting the cell mass is, for example, about 2 to 300, 5 to 200, and about 10 to 100.
  • the cells that make up the cell mass are usually approximately spherical.
  • the diameter of the cell mass is, for example, 10 to 500 ⁇ m, 20 to 300 ⁇ m, or 30 to 200 ⁇ m.
  • the cell mass may be a single type or a combination of two or more types.
  • the method for producing seaweed cells of the present invention uses, as a starting material, at least one selected from the group consisting of the spores, single cells, and cell clusters described above.
  • seaweed morphogenesis-inducing factors have been reported so far and are not particularly limited.
  • seaweeds such as green algae were cultured in a synthetic medium without using seawater
  • the phenomenon that the algae bodies collapsed without being able to form or maintain the form was known.
  • the microorganism include strains belonging to Cytophaga-Flavobacterium-Bacteriodes @ complex such as genus Flavobacterium, Zoberia, and Tenashibaculum, and mutants derived from these strains.
  • the seaweed morphogenesis inducer include salusin.
  • the medium (medium 1) used in the method for producing seaweed cells of the present invention does not substantially contain this seaweed morphogenesis factor.
  • “substantially does not contain” considers the case where the seaweed morphogenesis factor is inevitably mixed, and in such a case, “substantially does not contain” the seaweed morphogenesis inducer. It can be said that.
  • a medium that does not substantially contain a seaweed morphogenesis inducer does not contain a seaweed morphogenesis inducer and / or a microorganism that produces the same (for example, natural seawater or natural seawater components are not added). Not) medium.
  • an artificial seawater medium containing no such can be used as the medium 1.
  • the artificial seawater medium is not particularly limited, and examples thereof include artificial seawater itself, and a medium formed by appropriately adding nutrient components (excluding seaweed morphogenesis inducers and microorganisms producing the same) to artificial seawater as necessary.
  • the culture is performed under stirring conditions.
  • the culture under the stirring condition is not particularly limited as long as a part or the whole of the medium is stirred.
  • the medium is aerated by an aeration pump or the like (aeration culture), or the medium is pumped or the like. It is performed by passing a liquid (for example, a culture medium), moving a stirrer (for example, rotating), or shaking a culture vessel. During culture, the cells grow in suspension.
  • the culture temperature is not particularly limited as long as it is a temperature at which seaweeds can grow, and is, for example, 5 to 35 ° C, preferably 10 to 30 ° C, and more preferably 15 to 25 ° C.
  • the light conditions during the culture are not particularly limited as long as the light conditions allow seaweeds to grow, and include, for example, natural light light-dark cycle conditions, artificial light conditions, artificial light light-dark cycle conditions, and the like.
  • the culture period is not particularly limited, but is, for example, one to several tens of days, and two to seven days.
  • the method for producing seaweed cells of the present invention includes, as one embodiment, the following two steps. (A1) a step of culturing spores of seaweed in the medium 1, and (A2) a step of culturing the culture of step A1 in the medium 1 with stirring.
  • the culture in step A1 is preferably a static culture.
  • the culture period in step A1 is, for example, 10 days to several months, 20 days to 1.5 months.
  • a culture containing single cells or cell clusters derived from spores is obtained.
  • the definitions of other terms are the same as above.
  • seaweed cells (mainly containing single cells derived from spores) usable as a raw material for producing seaweed can be proliferated and easily and efficiently obtained in large quantities.
  • obtaining a raw material (spore) for producing seaweed requires a complicated operation of growing mother alga bodies and obtaining spores therefrom.
  • the obtained seaweed cells can be used (as seaweed-producing cells) for the production of seaweed described below.
  • it can be used, for example, as a raw material for food or biofuel.
  • a method for producing a seaweed provides, in one aspect, a method for producing a seaweed, comprising the step of culturing the seaweed cells obtained by the method of the present invention in a medium 2 containing a seaweed morphogenesis factor (In this specification, it may be referred to as “the method for producing seaweed of the present invention”). Hereinafter, this will be described.
  • the medium 2 containing the seaweed morphogenesis-inducing factor is not particularly limited, and examples thereof include natural seawater itself and a medium obtained by appropriately adding nutrients to natural seawater as needed.
  • a medium obtained by adding a seaweed morphogenesis-inducing factor, a microorganism producing the same to the medium 1, and the like can be used.
  • the mode of culture in the method for producing seaweed of the present invention is not particularly limited.
  • seaweed cells may be seeded on a plate at high density, seaweed cells adhere to each other, and the resulting clumps may be subjected to suspension culture to produce seaweed.
  • seaweeds may be produced by disseminating seaweed cells at a relatively low density, attaching them to a solid phase such as a thread or a flat plate, and culturing in this state, as in the conventional method.
  • the seaweed cells may be cultured in a floating state (preferably under stirring conditions).
  • the culture temperature, light conditions, and the like are the same as in the above step (A).
  • the obtained seaweed can be used, for example, as food or as a raw material for biofuels.
  • Example 1 Spores (zoospores) capable of swimming with flagella from the mature alga body of the multicellular green alga Pseudocystis were released in a glass dish filled with sterile seawater. When light is irradiated from one direction, the zoospores having negative phototacticity gather in the direction opposite to the light irradiation side. Utilizing this phototaxis, zoospores were aseptically isolated by swimming in sterile seawater.
  • the isolated zoospores (200 to 300) were seeded on a sterilized plastic petri dish (diameter 6 cm) filled with nutrient-fortified sterilized seawater, and allowed to stand in a culture chamber for culturing.
  • Nutrition-enriched sterilized seawater was prepared by adding 20 mL of nutrient for ES medium to 1 L of artificial seawater.
  • the nutrient for the ES medium was prepared according to the formula described on pages 501-502 of Algal culturing techniques (edited by RA Andersen, published in 2005, Elsevier Academic Press).
  • the artificial seawater was prepared by dissolving an artificial seawater producing powder (for Marine Art SF-1 ⁇ 25 L, Tomita Pharmaceutical Co., Ltd.) in 25 L of distilled water and steam sterilizing at 121 ° C. and 1 atm for 5 minutes.
  • the culture conditions were set to 100 ⁇ mol s ⁇ 1 m 2 of light with a white fluorescent lamp as a light source, a light / dark cycle of 12 hours / 12 hours, and a temperature of 20 ° C.
  • the spores of Macchitoe formed a group of several tens of cells in about one month of culture, as shown in FIG.
  • the spores of Machitoe showed a leaf-like morphology.
  • the cell group in FIG. 1 is easily divided into individual cells or a cell mass in which a small number of cells are loosely connected to form a cell suspension by stirring the inside of a glass petri dish with a glass pipette. 10 mL of this cell suspension was transferred to a 1-L flask filled with nutrient-fortified sterilized seawater, and cultured under the same water temperature and light conditions as described above, with aeration and agitation. In order to measure the change in the number of cells with the number of culture days, the culture seawater was withdrawn daily from the flask, and the absorbance at a wavelength of 730 nm was measured using an absorbance meter.
  • Example 2 A leaf-like multicellular green alga, Hirohanohitoexa, which is widely cultivated in various parts of Japan for food use, was cultured and tested in the same manner as in Example 1. Since the phyllophora has a life cycle in which a large, leafy gametophyte generation and a small sporophyte generation of about 0.05 mm in diameter alternate, the microspores were first cultured and matured. The zoospores were released from the mature microspores and used for culture tests.
  • FIG. 5 shows the logarithmic growth data.
  • 10 mL of the cell suspension of Hiraginophora extensa in log phase was subcultured into another 1 L flask and cultured under the same conditions, the cells grew at the same specific growth rate.
  • Example 3 20 cm of Cremona thread having a diameter of 2 mm was added to the cell suspension of Hirohanohitoegusa prepared in Example 2 to adsorb cells well.
  • the Cremona thread with the cells adsorbed was cultured in a 500 mL beaker filled with natural filtered seawater to which a nutrient was added.
  • Porphyranconco Daiichi Seimitsu
  • 0.5 mL was added to 1 L of natural filtered seawater.
  • Light and temperature were set to the same conditions as described in Example 1, and the natural seawater medium was changed daily.
  • leaf-shaped Hirohanohitoegusa extended about 1 cm from the Cremona thread. This is considered to be due to the induction of morphogenic factors present in natural seawater to induce leaf-like multicellular bodies from cells in a single cell state.
  • natural seawater it was shown that the population of single-celled Hirohano human Exa grown in large amounts on a sterile medium had the ability to develop into a multicellular body.
  • Example 4 Each single cell in the cell suspension of Hilo-Hano human Exa prepared in Example 2 was multicellularized with salusin. Specifically, the procedure was performed as follows. 5 mL (1 to 5,000 cells) of the cell suspension of Hironohitoexa prepared in Example 2 was transferred to a 500 mL beaker, and the concentration of salusin was adjusted to 1000 fmol / L in the fortified and sterilized seawater used in Example 1. The culture was carried out with 500 mL of the medium added to, while stirring by aeration. Light and temperature conditions were set the same as in Example 1. After 10 days in culture, multicellular thallus developed.
  • FIG. 6 shows a photograph of the thallus obtained after the culture.
  • FIG. 7 shows a photograph obtained by spreading the leaf-shaped body on a slide glass.
  • FIG. 8 shows a photograph of the thallus of a normal human eegusa grown from spores.
  • the foliates obtained by the ordinary method develop fibrous attachment roots (arrows in FIG. 8) for adhering to rocks and the like, while the foliates obtained in the present example have It was found that such attached roots were not formed.

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Abstract

The problem addressed by the present invention is to provide a technique for producing seaweed easily and efficiently. The problem is solved by a method for producing seaweed cells that includes a step for culturing under stirred conditions at least one selected from the group consisting of seaweed spores, single cells derived from the spores, and a cell mass of the spores and/or the single cells in a medium 1 substantially free of seaweed morphogens.

Description

海藻細胞の製造方法Method for producing seaweed cells
　本発明は、海藻細胞の製造方法等に関する。 The present invention relates to a method for producing seaweed cells and the like.
　海藻の養殖は、一般的には、海藻の母藻体を育て、そこから得られた胞子から種苗を作製することによって行われる。 殖 Seaweed cultivation is generally performed by growing mother alga bodies of seaweed and producing seeds and seedlings from spores obtained therefrom.
　種苗は、通常、胞子を糸や平板等の固相に付着させることによって作製される。しかしながら、この方法では、海藻は固相に付着した状態で生育するので、収穫時に海藻を固相から取外す作業が必要になり、煩雑である。一方で、特許文献１では、海藻の胞子を平板に高密度で播種して胞子同士を互いに付着させ、得られた集塊を培養することによる海藻養殖方法が開示されている。この方法であれば、海藻を培地中に浮遊させた状態で生育させることができるので、簡便に収穫することができる。 Seedlings are usually produced by attaching spores to a solid phase such as thread or plate. However, in this method, since the seaweed grows in a state of being attached to the solid phase, it is necessary to remove the seaweed from the solid phase at the time of harvesting, which is complicated. On the other hand, Patent Literature 1 discloses a seaweed cultivation method in which spores of seaweed are seeded at high density on a flat plate, spores are attached to each other, and the resulting clumps are cultured. According to this method, the seaweed can be grown in a state of being suspended in the medium, and therefore, can be easily harvested.
　しかし、いずれの方法でも、海藻製造原料である胞子を得るために、母藻体を育て、そこから胞子を得るという煩雑な作業を要してしまう。 However, in either method, in order to obtain spores as a raw material for producing seaweed, a complicated operation of growing mother alga bodies and obtaining spores therefrom is required.
特許第３８２８３５９号Patent No. 3828359
　本発明は、簡便且つ効率的に海藻を製造するための技術を提供することを課題とする。 An object of the present invention is to provide a technique for easily and efficiently producing seaweed.
　本発明者は上記課題に鑑みて鋭意研究を進めた結果、海藻の胞子、該胞子由来の単細胞、並びに該胞子及び／又は該単細胞の細胞塊からなる群より選択される少なくとも１種を、海藻形態形成誘導因子を実質的に含有しない培地１中で、攪拌条件下で培養することにより、海藻製造原料として利用可能な海藻細胞を簡便且つ効率的に製造できることを見出した。この知見に基づいてさらに研究を進めた結果、本発明が完成した。 The present inventor has conducted intensive studies in view of the above-mentioned problems, and as a result, seaweed spores, single cells derived from the spores, and at least one selected from the group consisting of the spores and / or the cell mass of the single cells, It has been found that culturing under stirring conditions in a medium 1 substantially containing no morphogenesis-inducing factor enables simple and efficient production of seaweed cells usable as a raw material for producing seaweed. As a result of further research based on this finding, the present invention has been completed.
　即ち、本発明は、下記の態様を包含する。 That is, the present invention includes the following embodiments.
　項１．　（Ａ）海藻の胞子、該胞子由来の単細胞、並びに該胞子及び／又は該単細胞の細胞塊からなる群より選択される少なくとも１種を、海藻形態形成誘導因子を実質的に含有しない培地１中で、攪拌条件下で培養する工程、
を含む、海藻細胞の製造方法。 Item 1. (A) at least one selected from the group consisting of seaweed spores, single cells derived from the spores, and cell masses of the spores and / or the single cells, in a medium 1 substantially free of seaweed morphogenesis-inducing factors; In, a step of culturing under stirring conditions,
A method for producing a seaweed cell, comprising:
　項２．　前記海藻がヒビミドロ目に属する海藻である、項１に記載の製造方法。 Item 2. <4> The production method according to item 1, wherein the seaweed is a seaweed belonging to the order of the order Hibismidori.
　項３．　前記海藻がヒトエグサ属に属する海藻である、項１又は２に記載の製造方法。 Item 3. <4> The production method according to item 1 or 2, wherein the seaweed is a seaweed belonging to the genus Humane.
　項４．　前記海藻細胞が単細胞を含む、項１～３のいずれかに記載の製造方法。 Item 4.製造 The method according to any one of Items 1 to 3, wherein the seaweed cells include single cells.
　項５．　（Ａ１）海藻の胞子を前記培地１中で培養する工程、及び
（Ａ２）工程Ａ１の培養物を、前記培地１中で、攪拌しながら培養する工程、
を含む、項１～４のいずれかに記載の製造方法。 Item 5. (A1) a step of culturing spores of seaweed in the medium 1, and (A2) a step of culturing the culture of step A1 in the medium 1 while stirring.
Item 5. The production method according to any one of Items 1 to 4, comprising:
　項６．　前記工程Ａ１における培養が静置培養である、項５に記載の製造方法。 Item 6. <6> The production method according to item 5, wherein the culture in the step A1 is static culture.
　項７．　前記培地１が人工海水培地である、項１～６のいずれかに記載の製造方法。 Item 7.製造 The method according to any one of Items 1 to 6, wherein the medium 1 is an artificial seawater medium.
　項８．　前記海藻が食用海藻である、項１～７のいずれかに記載の製造方法。 Item 8.製造 The method according to any one of Items 1 to 7, wherein the seaweed is an edible seaweed.
　項９．　前記攪拌条件下での培養が通気培養である、項１～８のいずれかに記載の製造方法。 Item 9. Item 10. The production method according to any one of Items 1 to 8, wherein the culture under the stirring condition is aeration culture.
　項１０．　項１～９のいずれかに記載の製造方法で得られた海藻細胞を、海藻形態形成誘導因子を含有する培地２中で培養する工程、
を含む、海藻の製造方法。 Item 10. Item 10. A step of culturing the seaweed cells obtained by the production method according to any one of Items 1 to 9 in a medium 2 containing a seaweed morphogenesis factor.
A method for producing seaweed, comprising:
　項１１．　付着根を有しない、海藻葉状体又は海藻。 Item 11.葉 Seaweed foliate or seaweed having no attached root.
　本発明によれば、海藻製造原料として利用可能な海藻細胞を簡便且つ効率的に製造することができ、ひいては海藻を簡便且つ効率的に製造することができる。 According to the present invention, seaweed cells that can be used as a raw material for producing seaweed can be simply and efficiently produced, and thus seaweed can be simply and efficiently produced.
図１は、実施例１のマキヒトエ培養実験における培養32日後のマキヒトエ培養細胞の写真である。FIG. 1 is a photograph of the cultured cells of Macchitoe after 32 days of culture in the culture test of Macchitoe in Example 1. 図２は、実施例１のマキヒトエ培養実験と同時並行して実施した培養で、形態形成物質を添加した場合の培養32日後のマキヒトエ培養細胞の写真である。FIG. 2 is a photograph of the cultured cells of Macchitoe after 32 days of culturing when a morphogen was added, in a culture performed in parallel with the test for cultivation of Macchitoe in Example 1. 図３は形態形成誘導因子が存在しない培地で培養したマキヒトエの単細胞群の増殖曲線である。FIG. 3 is a growth curve of a single cell group of A. chinensis cultured in a medium without a morphogenesis-inducing factor. 図４は図３の吸光度データを対数変換した増殖曲線である。培養2日目から7日目まで直線増加を示しており対数期とみなせる。一方、0-2日目と8-9日目は直線から外れ、それぞれ誘導期、定常期への移行期とみなされる。FIG. 4 is a logarithmic transformation curve of the absorbance data of FIG. It shows a linear increase from the 2nd day to the 7th day of the culture and can be regarded as a logarithmic phase. On the other hand, days 0-2 and days 8-9 deviate from the straight line, and are considered to be the induction period and the transition period to the stationary period, respectively. 図５は実施例２の形態形成誘導因子が存在しない培地で培養したヒロハノヒトエグサの単細胞群の対数期の増殖曲線である。FIG. 5 is a logarithmic growth curve of a single cell group of Hilohano human exa cultivated in a medium containing no morphogenesis inducer of Example 2. 図６は実施例４の培養後に得られた葉状体の写真である。FIG. 6 is a photograph of the thallus obtained after the culture of Example 4. 図７は実施例４の培養後に得られた葉状体をスライドガラス上で広げて撮影した写真である。FIG. 7 is a photograph obtained by spreading and photographing the leaf-like body obtained after the culture in Example 4. 図８は胞子から生長した通常のヒトエグサの葉状体の写真である。矢印は付着根を示す。FIG. 8 is a photograph of the thallus of a normal human euple grown from spores. Arrows indicate attached roots.
　本明細書中において、「含有」及び「含む」なる表現については、「含有」、「含む」、「実質的にからなる」及び「のみからなる」という概念を含む。 に お い て In this specification, the expressions “contain” and “contain” include the concepts of “contain”, “contain”, “consisting essentially of” and “consisting only of”.
　１．海藻細胞の製造方法
　本発明は、その一態様において、（Ａ）海藻の胞子、該胞子由来の単細胞、並びに該胞子及び／又は該単細胞の細胞塊からなる群より選択される少なくとも１種を、海藻形態形成誘導因子を実質的に含有しない培地１中で、攪拌条件下で培養する工程、を含む、海藻細胞の製造方法（本明細書において、「本発明の海藻細胞製造方法」と示すこともある。）に関する。以下、これについて説明する。 1. Method for Producing Seaweed Cell In one aspect of the present invention, (A) seaweed spores, single cells derived from the spores, and at least one selected from the group consisting of the spores and / or the cell mass of the single cells, A method for producing seaweed cells (hereinafter referred to as “the method for producing seaweed cells of the present invention”), which comprises culturing under stirring conditions in a medium 1 substantially containing no seaweed morphogenesis-inducing factor. There is also.) Hereinafter, this will be described.
　海藻は、特に制限されるものではなく、通常、緑藻である。海藻としては、海藻細胞の製造効率をより高めることができるという観点から、ヒビミドロ目に属する海藻が好ましい。ヒビミドロ目に属する海藻としては、例えばカイミドリ科、マキヒトエグサ科、カプサアオノリ科、ランソウモドキ科、ヒビミドロ科等に属する海藻が挙げられ、好ましくはカイミドリ科、マキヒトエグサ科等に属する海藻が挙げられ、より好ましくはカイミドリ科に属する海藻が挙げられる。カイミドリ科に属する海藻としては、例えばヒトエグサ属、カイミドリ属等に属する海藻が挙げられ、好ましくはヒトエグサ属に属する海藻が挙げられる。ヒトエグサ属に属する海藻としては、例えばヒロハノヒトエグサ、ヒトエグサ、ウスヒトエグサ、エゾヒトエグサ、シンカイヒトエグサ、アツバヒトエ、アツカワヒトエ等が挙げられ、好ましくはヒロハノヒトエグサ等の食用の海藻が挙げられる。海藻は、１種単独であっても、２種以上の組合せであってもよい。 Seaweeds are not particularly limited, and are usually green algae. As the seaweed, a seaweed belonging to the order Hihibimidoro is preferable from the viewpoint that the production efficiency of seaweed cells can be further improved. Examples of seaweeds belonging to the order Hibimidori include, for example, seaweeds belonging to the family Echinaceae, Echinacea spp., Capsa anonnaceae, Lansinaceae, Echinacea spp., And more preferably seaweeds belonging to the Echinacea spp., Echinacea spp., And the like. Preferably, a seaweed belonging to the family Iridaceae is mentioned. Examples of the seaweeds belonging to the family Amidori include the seaweeds belonging to the genus Aesculus, the genus Aesculus, and the like, and preferably the seaweeds belonging to the genus Aesculus. Examples of the seaweeds belonging to the genus Humanexa include, for example, Hirohanohitoexa, humanexa, Usuhitoexa, Ezohitoexa, Shinkaihitoexa, Atsubahitoe, Atsukawahitoe, and the like. Seaweeds may be used alone or in combination of two or more.
　胞子は、海藻から得られる胞子であるものであり、特に制限されない。胞子とは、生殖細胞のことである。胞子の状態は特に制限されず、胞子としては例えば遊走子、配偶子、接合子、四胞子、果胞子、単子嚢遊走子、中性遊走子等が挙げられる。胞子は、１種単独であっても、２種以上の組合せであってもよい。 Spores are spores obtained from seaweed and are not particularly limited. Spores are germ cells. The state of the spores is not particularly limited, and examples of the spores include zoospores, gametes, zygotes, tetraspores, fruit spores, monospore zoospores, and neutral zoospores. The spores may be used alone or in combination of two or more.
　胞子由来の単細胞は、胞子に由来する未分化の単細胞（分化した葉状体を構成する細胞は、ここでいう「単細胞」に包含されない。）である限り特に制限されない。単細胞は、通常、ほぼ球形である。単細胞の直径は、例えば３～２０μｍ、５～１５μｍである。単細胞は、１種単独であっても、２種以上の組み合わせであってもよい。 単 Spore-derived single cells are not particularly limited as long as they are spore-derived undifferentiated single cells (cells constituting differentiated thallus are not included in the “single cells” here). Single cells are usually approximately spherical. The diameter of a single cell is, for example, 3 to 20 μm and 5 to 15 μm. Single cells may be used alone or in combination of two or more.
　細胞塊は、上記胞子及び上記単細胞からなる群より選択される少なくとも１種が集合して形成されたものであり、分化した葉状体を構成する細胞塊を包含しない限り、特に制限されない。細胞塊を構成する細胞数は、例えば２～３００個、５～２００個、１０～１００個程度である。細胞塊を構成する細胞は、通常、ほぼ球形である。細胞塊の直径は、例えば１０～５００μｍ、２０～３００μｍ、３０～２００μｍである。細胞塊は１種単独であっても、２種以上の組合せであってもよい。 The cell mass is formed by assembling at least one selected from the group consisting of the spores and the single cells, and is not particularly limited as long as it does not include the cell mass constituting the differentiated thallus. The number of cells constituting the cell mass is, for example, about 2 to 300, 5 to 200, and about 10 to 100. The cells that make up the cell mass are usually approximately spherical. The diameter of the cell mass is, for example, 10 to 500 μm, 20 to 300 μm, or 30 to 200 μm. The cell mass may be a single type or a combination of two or more types.
　本発明の海藻細胞製造方法は、上記した胞子、単細胞、及び細胞塊からなる群より選択される少なくとも１種を、出発材料として使用する。 海 The method for producing seaweed cells of the present invention uses, as a starting material, at least one selected from the group consisting of the spores, single cells, and cell clusters described above.
　海藻形態形成誘導因子は、これまで各種報告されており、特に制限されない。緑藻類等の海藻を、海水を使用せずに合成培地で培養すると形態形成ができずに或いは形態を維持できずに藻体が崩れるという現象が知られていたところ、この原因が、海水中に存在する微生物が生産する物質（海藻形態形成誘導因子）であることが分かっている（国際公開第２００４／００７５１０号、特開第２００３－１８９８４５号公報等）。この微生物としては、例えばフラボバクテリウム属、ゾベリア属、テナシバキュラム属等のＣｙｔｏｐｈａｇａ－Ｆｌａｖｏｂａｃｔｅｒｉｕｍ－Ｂａｃｔｅｒｉｏｄｅｓ  ｃｏｍｐｌｅｘに属する菌株や、これらの菌株に由来する変異株を挙げることができる。海藻形態形成誘導因子としては、例えばサルーシン等が挙げられる。 A variety of seaweed morphogenesis-inducing factors have been reported so far and are not particularly limited. When seaweeds such as green algae were cultured in a synthetic medium without using seawater, the phenomenon that the algae bodies collapsed without being able to form or maintain the form was known. It is known to be a substance (seaweed morphogenesis inducer) produced by existing microorganisms (WO 2004/007510, JP-A-2003-189845, etc.). Examples of the microorganism include strains belonging to Cytophaga-Flavobacterium-Bacteriodes @ complex such as genus Flavobacterium, Zoberia, and Tenashibaculum, and mutants derived from these strains. Examples of the seaweed morphogenesis inducer include salusin.
　本発明の海藻細胞製造方法で使用する培地（培地１）は、この海藻形態形成誘導因子を実質的に含有しない。ここで、「実質的に含有しない」とは、不可避的に海藻形態形成誘導因子が混入する場合を考慮したものであり、このような場合は海藻形態形成誘導因子を「実質的に含有しない」といえる。換言すれば、海藻形態形成誘導因子を実質的に含有しない培地とは、海藻形態形成誘導因子及び／又はそれを産生する微生物が添加されていない（例えば、天然海水や天然海水の成分が添加されていない）培地であるといえる。海藻形態形成誘導因子及びそれを産生する微生物は海水中に含まれるため、培地１としては、例えば、これらを含有しない人工海水培地を使用することができる。人工海水培地としては、特に制限されず、例えば人工海水そのもの、人工海水に必要に応じて栄養成分（海藻形態形成誘導因子及びそれを産生する微生物は除く）を適宜添加してなる培地等が挙げられる。 培 地 The medium (medium 1) used in the method for producing seaweed cells of the present invention does not substantially contain this seaweed morphogenesis factor. Here, “substantially does not contain” considers the case where the seaweed morphogenesis factor is inevitably mixed, and in such a case, “substantially does not contain” the seaweed morphogenesis inducer. It can be said that. In other words, a medium that does not substantially contain a seaweed morphogenesis inducer does not contain a seaweed morphogenesis inducer and / or a microorganism that produces the same (for example, natural seawater or natural seawater components are not added). Not) medium. Since the seaweed morphogenesis-inducing factor and the microorganisms that produce it are contained in seawater, for example, an artificial seawater medium containing no such can be used as the medium 1. The artificial seawater medium is not particularly limited, and examples thereof include artificial seawater itself, and a medium formed by appropriately adding nutrient components (excluding seaweed morphogenesis inducers and microorganisms producing the same) to artificial seawater as necessary. Can be
　培養は、攪拌条件下で行われる。攪拌条件下での培養は、培地の一部又は全体が攪拌される態様である限り特に制限されないが、例えば、エアレーションポンプ等で培地中に通気すること（通気培養）、ポンプ等で培地中に液体（例えば、培地等）を通液すること、攪拌子を運動（例えば、回転等）させること、培養容器を振とうすること等によって行われる。培養中、細胞は、浮遊した状態で生育する。 The culture is performed under stirring conditions. The culture under the stirring condition is not particularly limited as long as a part or the whole of the medium is stirred. For example, the medium is aerated by an aeration pump or the like (aeration culture), or the medium is pumped or the like. It is performed by passing a liquid (for example, a culture medium), moving a stirrer (for example, rotating), or shaking a culture vessel. During culture, the cells grow in suspension.
　培養温度は、海藻が生育可能な温度である限り特に制限されず、例えば５～３５℃、好ましくは１０～３０℃、より好ましくは１５～２５℃である。 The culture temperature is not particularly limited as long as it is a temperature at which seaweeds can grow, and is, for example, 5 to 35 ° C, preferably 10 to 30 ° C, and more preferably 15 to 25 ° C.
　培養時の光条件としては、海藻が生育可能な光条件である限り特に制限されず、例えば自然光の明暗周期条件、人工光条件、人工光明暗周期条件等が挙げられる。 光 The light conditions during the culture are not particularly limited as long as the light conditions allow seaweeds to grow, and include, for example, natural light light-dark cycle conditions, artificial light conditions, artificial light light-dark cycle conditions, and the like.
　培養期間は、特に制限されるものではないが、例えば１～数十日間、２～７日間である。 The culture period is not particularly limited, but is, for example, one to several tens of days, and two to seven days.
　本発明の海藻細胞製造方法は、一態様として、以下の２工程を含む。
（Ａ１）海藻の胞子を前記培地１中で培養する工程、及び
（Ａ２）工程Ａ１の培養物を、前記培地１中で、攪拌しながら培養する工程。 The method for producing seaweed cells of the present invention includes, as one embodiment, the following two steps.
(A1) a step of culturing spores of seaweed in the medium 1, and (A2) a step of culturing the culture of step A1 in the medium 1 with stirring.
　工程Ａ１における培養は、好ましくは静置培養である。また、工程Ａ１における培養期間は、例えば１０日間～数ヶ月間、２０日間～１．５ヶ月間である。工程Ａ１により、胞子由来の単細胞や細胞塊を含む培養物が得られる。その他の用語の定義等については、上記と同様である。 培養 The culture in step A1 is preferably a static culture. The culture period in step A1 is, for example, 10 days to several months, 20 days to 1.5 months. By the step A1, a culture containing single cells or cell clusters derived from spores is obtained. The definitions of other terms are the same as above.
　斯かる本発明の海藻細胞製造方法によって、海藻製造原料として利用可能な海藻細胞（主に、胞子由来の単細胞を含む）を増殖させ、簡便且つ効率的に大量に得ることが可能である。 According to the method for producing seaweed cells of the present invention, seaweed cells (mainly containing single cells derived from spores) usable as a raw material for producing seaweed can be proliferated and easily and efficiently obtained in large quantities.
　従来、海藻製造原料（胞子）を得るには、母藻体を育て、そこから胞子を得るという煩雑な作業を要していた。一方、本発明によれば、少量の胞子から、海藻製造原料となる海藻細胞を大量に調製することが可能である。また、得られた海藻細胞を少量用意して、そこから大量の海藻製造原料を調製することが可能である。すなわち、本発明によれば、海藻製造原料（胞子）を得るには、母藻体を育て、そこから胞子を得るという煩雑な作業を経ずに、或いは該作業を必要最小限に留め、大量の海藻製造原料を調製することが可能である。 Conventionally, obtaining a raw material (spore) for producing seaweed requires a complicated operation of growing mother alga bodies and obtaining spores therefrom. On the other hand, according to the present invention, it is possible to prepare a large amount of seaweed cells as a raw material for producing seaweed from a small amount of spores. Moreover, it is possible to prepare a small amount of the obtained seaweed cells and prepare a large amount of seaweed production material therefrom. That is, according to the present invention, in order to obtain seaweed production raw materials (spores), a complicated process of growing mother alga bodies and obtaining spores therefrom is not necessary, or the operation is minimized, and a large amount of Can be prepared.
　得られた海藻細胞は、後述の海藻の製造に（海藻製造用細胞として）使用することができる。他にも、例えば食用、バイオ燃料の原材料等として利用することも可能である。 海 The obtained seaweed cells can be used (as seaweed-producing cells) for the production of seaweed described below. In addition, it can be used, for example, as a raw material for food or biofuel.
　２．海藻の製造方法
　本発明は、その一態様において、本発明の製造方法で得られた海藻細胞を、海藻形態形成誘導因子を含有する培地２中で培養する工程、を含む、海藻の製造方法（本明細書において、「本発明の海藻製造方法」と示すこともある。）に関する。以下、これについて説明する。 2. A method for producing a seaweed The present invention provides, in one aspect, a method for producing a seaweed, comprising the step of culturing the seaweed cells obtained by the method of the present invention in a medium 2 containing a seaweed morphogenesis factor ( In this specification, it may be referred to as “the method for producing seaweed of the present invention”). Hereinafter, this will be described.
　海藻形態形成誘導因子を含有する培地２としては、特に制限されないが、例えば天然海水そのものや、天然海水に必要に応じて栄養成分を適宜添加してなる培地等が挙げられる。或いは、培地２としては、培地１に対して、海藻形態形成誘導因子、それを産生する微生物等を添加してなる培地を使用することもできる。 The medium 2 containing the seaweed morphogenesis-inducing factor is not particularly limited, and examples thereof include natural seawater itself and a medium obtained by appropriately adding nutrients to natural seawater as needed. Alternatively, as the medium 2, a medium obtained by adding a seaweed morphogenesis-inducing factor, a microorganism producing the same to the medium 1, and the like can be used.
　本発明の海藻製造法における培養の態様は、特に制限されない。例えば、特許文献１に記載のように、海藻細胞を平板に高密度で播種して海藻細胞同士を互いに付着させ、得られた集塊を浮遊培養することによって、海藻を製造してもよい。或いは、従来法のように、海藻細胞を比較的低密度で播種して、糸や平板等の固相に付着させ、その状態で培養することによって、海藻を製造してもよい。また、海藻細胞を浮遊状態で（好ましくは攪拌条件下で）培養してもよい。培養温度、光条件等については、上記工程（Ａ）と同様である。 培養 The mode of culture in the method for producing seaweed of the present invention is not particularly limited. For example, as described in Patent Literature 1, seaweed cells may be seeded on a plate at high density, seaweed cells adhere to each other, and the resulting clumps may be subjected to suspension culture to produce seaweed. Alternatively, seaweeds may be produced by disseminating seaweed cells at a relatively low density, attaching them to a solid phase such as a thread or a flat plate, and culturing in this state, as in the conventional method. Further, the seaweed cells may be cultured in a floating state (preferably under stirring conditions). The culture temperature, light conditions, and the like are the same as in the above step (A).
　本発明の一態様においては、本発明の海藻製造方法により、付着根を有さない葉状体や海藻を得ることも可能である。 の 一 In one embodiment of the present invention, it is also possible to obtain a leaf-like body or seaweed having no attached root by the method for producing seaweed of the present invention.
　得られた海藻は、例えば食用、バイオ燃料の原材料等として利用することが可能である。 海 The obtained seaweed can be used, for example, as food or as a raw material for biofuels.
　以下に、実施例に基づいて本発明を詳細に説明するが、本発明はこれらの実施例によって限定されるものではない。 Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.
　実施例１
　多細胞緑藻マキヒトエの成熟藻体から鞭毛によって遊泳できる胞子（遊走子）を、滅菌海水で満たしたガラスシャーレ内で放出させた。一方向から光を照射すると、負の走光性をもつ遊走子は光照射側とは反対の方向に集合する。この走光性を利用し、無菌海水中を泳がせて遊走子を無菌的に単離した。 Example 1
Spores (zoospores) capable of swimming with flagella from the mature alga body of the multicellular green alga Pseudocystis were released in a glass dish filled with sterile seawater. When light is irradiated from one direction, the zoospores having negative phototacticity gather in the direction opposite to the light irradiation side. Utilizing this phototaxis, zoospores were aseptically isolated by swimming in sterile seawater.
　単離した遊走子（200-300個）を、栄養補強滅菌海水で満たした滅菌プラスチックシャーレ（直径6cm）に播種し、培養庫内に静置して培養した。栄養補強滅菌海水は、人工海水1Lに対しES培地用栄養剤を20mL添加して作製した。ES培地用栄養剤は、Algal culturing techniques （R.A. Andersen編集、2005年出版、Elsevier Academic Press）の501-502ページに記載の処方で作製した。人工海水は、人工海水作製用粉末（マリンアートSF-1・25L用、富田製薬）を蒸留水25Lに溶解し、121℃、1気圧で5分間蒸気滅菌して作製した。培養条件は、白色蛍光灯を光源として光量100μmol s-1 m2、明暗周期12時間/12時間、温度20℃に設定した。 The isolated zoospores (200 to 300) were seeded on a sterilized plastic petri dish (diameter 6 cm) filled with nutrient-fortified sterilized seawater, and allowed to stand in a culture chamber for culturing. Nutrition-enriched sterilized seawater was prepared by adding 20 mL of nutrient for ES medium to 1 L of artificial seawater. The nutrient for the ES medium was prepared according to the formula described on pages 501-502 of Algal culturing techniques (edited by RA Andersen, published in 2005, Elsevier Academic Press). The artificial seawater was prepared by dissolving an artificial seawater producing powder (for Marine Art SF-1 · 25 L, Tomita Pharmaceutical Co., Ltd.) in 25 L of distilled water and steam sterilizing at 121 ° C. and 1 atm for 5 minutes. The culture conditions were set to 100 μmol s −1 m 2 of light with a white fluorescent lamp as a light source, a light / dark cycle of 12 hours / 12 hours, and a temperature of 20 ° C.
　この培養条件でマキヒトエの胞子は図１に示すように培養約1ヶ月で数10個の細胞が集まった細胞群を形成した。ちなみに、本培養試験と同時並行して実施された形態形成物質を添加した培養試験では、図２に示すようにマキヒトエの胞子は葉状の形態に発生した。 で Under these culture conditions, the spores of Macchitoe formed a group of several tens of cells in about one month of culture, as shown in FIG. By the way, in the culture test to which the morphogen was added, which was carried out concurrently with the main culture test, as shown in FIG. 2, the spores of Machitoe showed a leaf-like morphology.
　図１の細胞群は、ガラスシャーレ内をガラスピペットでかき混ぜると容易に個別の細胞もしくは少数の細胞が緩やかに繋がった細胞塊に分かれて細胞懸濁液となる。この細胞懸濁液10mLを、栄養補強滅菌海水で満たした1Lフラスコに移して、上述と同じ水温、光条件で、通気により攪拌しながら培養した。培養日数に伴う細胞数の変化を計測するために、フラスコから毎日培養海水を抜き取り吸光度計で波長730nmの吸光度を計測した。 る と The cell group in FIG. 1 is easily divided into individual cells or a cell mass in which a small number of cells are loosely connected to form a cell suspension by stirring the inside of a glass petri dish with a glass pipette. 10 mL of this cell suspension was transferred to a 1-L flask filled with nutrient-fortified sterilized seawater, and cultured under the same water temperature and light conditions as described above, with aeration and agitation. In order to measure the change in the number of cells with the number of culture days, the culture seawater was withdrawn daily from the flask, and the absorbance at a wavelength of 730 nm was measured using an absorbance meter.
　その結果、図３に示すようにマキヒトエ細胞は指数関数的に増殖した。さらに図４のように対数変換してデータを解析すると、単細胞性の微細藻類や細菌でみられる増殖様式と同様に誘導期、対数期、定常期への移行がみられた。すなわち、フラスコ培養2日目まではゆっくりと増殖し（誘導期）、それ以降から7日目までは安定した対数増殖に入り（対数期）、8日目からは成長速度が低下し始めた（定常期への移行期）。 As a result, as shown in FIG. 3, Macchitoe cells grew exponentially. Further, when the data was analyzed by logarithmic transformation as shown in FIG. 4, the transition to the lag phase, logarithmic phase, and stationary phase was observed, similar to the growth pattern seen in unicellular microalgae and bacteria. That is, the cells grew slowly until the second day of the flask culture (induction phase), then entered a stable logarithmic growth period (log phase) from day 7 until the growth rate began to decrease from day 8 ( Transition to stationary phase).
　培養2日目から7日目までの対数期の日間比増殖速度は0.55（R2=0.999）であり、毎日1.7倍で増殖した。また、吸光度と細胞密度の関係は血球計算盤を用いた計測により、細胞密度（細胞数/mL）＝1830×吸光度の関係式が得られた。この式により培養2日目から7日目の5日間で1Lフラスコ中の細胞数は1.5万個から23万個に増えたと見積もられた。 The daily specific growth rate in the log phase from day 2 to day 7 of culture was 0.55 (R 2 = 0.999), and the cells grew 1.7 times daily. The relationship between the absorbance and the cell density was determined by measurement using a hemocytometer, and a relational expression of cell density (number of cells / mL) = 1830 × absorbance was obtained. According to this formula, it was estimated that the number of cells in the 1-L flask increased from 15,000 to 230,000 in 5 days from the second day to the seventh day of the culture.
　以上のように、マキヒトエを形態形成誘導因子が存在しない無菌培地で攪拌しながら培養することで、多細胞の体を発達させずに単細胞状態の細胞群を大量増殖させることができた。 As described above, by cultivating Macchitoe in a sterile medium containing no morphogenesis-inducing factor while stirring, a large number of cells in a single cell state could be grown without developing a multicellular body.
　実施例２
　食用として日本各地で広く養殖されている葉状の多細胞緑藻ヒロハノヒトエグサを実施例１と同じ方法で培養試験した。ヒロハノヒトエグサは、大型で葉状の配偶体世代と直径約0.05mmの微小な胞子体世代が交代する生活環をもつので、まず微小胞子体を培養して成熟させた。そして成熟した微小胞子体から遊走子を放出させ、培養試験に使用した。 Example 2
A leaf-like multicellular green alga, Hirohanohitoexa, which is widely cultivated in various parts of Japan for food use, was cultured and tested in the same manner as in Example 1. Since the phyllophora has a life cycle in which a large, leafy gametophyte generation and a small sporophyte generation of about 0.05 mm in diameter alternate, the microspores were first cultured and matured. The zoospores were released from the mature microspores and used for culture tests.
　その結果、1Lフラスコの培養においてヒロハノヒトエグサの増殖様式はマキヒトエと同様に、誘導期・対数期・定常期への移行期を示した。そのうちの対数期の増殖データを図５に示す。日間比増殖速度は0.67（R2=0.989）であり、毎日2.0倍で増殖した。対数期にあるヒロハノヒトエグサの細胞懸濁液10mLを別の1Lフラスコに植え継いで同条件で培養すると同様の比増殖速度で増殖した。 As a result, in the culture of the 1-L flask, the growth pattern of Hilo-Hano-hito-exa showed a transition period to an induction period, a logarithmic period, and a stationary period, as in Macchitoe. FIG. 5 shows the logarithmic growth data. The specific growth rate per day was 0.67 (R 2 = 0.989), and the cells grew 2.0 times daily. When 10 mL of the cell suspension of Hiraginophora extensa in log phase was subcultured into another 1 L flask and cultured under the same conditions, the cells grew at the same specific growth rate.
　すなわち、ヒロハノヒトエグサを形態形成誘導因子が存在しない無菌培地で攪拌しながら培養することで、多細胞の体を発達させずに単細胞状態の細胞群を大量増殖させることができた。また、この細胞群を植え継いで培養することで毎日2倍の高い増殖速度で継続して増殖させることができた。 That is, by culturing Hirohano human exa in a sterile medium containing no morphogenesis-inducing factors while stirring, a large number of cells in a single cell state could be grown without developing a multicellular body. Further, by subculturing and culturing this cell group, it was possible to continuously grow at twice the growth rate every day.
　実施例３
　実施例２で作製したヒロハノヒトエグサの細胞懸濁液に直径2mmのクレモナ糸20cmを入れて細胞群をよく吸着させた。細胞を吸着したクレモナ糸を、栄養剤を添加した天然濾過海水で満たした500mLビーカーに入れて培養を行った。栄養剤はポルフィランコンコ（第一製網）を使用し、天然濾過海水1Lに対し0.5mLを添加した。光と温度は実施例１の記載と同じ条件に設定し、天然海水培地は毎日交換した。 Example 3
20 cm of Cremona thread having a diameter of 2 mm was added to the cell suspension of Hirohanohitoegusa prepared in Example 2 to adsorb cells well. The Cremona thread with the cells adsorbed was cultured in a 500 mL beaker filled with natural filtered seawater to which a nutrient was added. As a nutrient, Porphyranconco (Daiichi Seimitsu) was used, and 0.5 mL was added to 1 L of natural filtered seawater. Light and temperature were set to the same conditions as described in Example 1, and the natural seawater medium was changed daily.
　培養1ヶ月後、クレモナ糸から葉状のヒロハノヒトエグサが1cm程度伸長した。これは天然海水中に存在する形態形成誘導因子によって、単細胞状態の細胞から葉状の多細胞体が誘導されたとみなされる。天然海水を使用することで、無菌培地で大量増殖させたヒロハノヒトエグサ単細胞群は、多細胞の体に発達できる能力をもつことが示された。 (1) After one month of culture, leaf-shaped Hirohanohitoegusa extended about 1 cm from the Cremona thread. This is considered to be due to the induction of morphogenic factors present in natural seawater to induce leaf-like multicellular bodies from cells in a single cell state. By using natural seawater, it was shown that the population of single-celled Hirohano human Exa grown in large amounts on a sterile medium had the ability to develop into a multicellular body.
　実施例４
　実施例２で作製したヒロハノヒトエグサの細胞懸濁液中の各単細胞をサルーシンによって多細胞化させた。具体的には、次のようにして行った。実施例２で作製したヒロハノヒトエグサの細胞懸濁液5mL(細胞数1～5千個)を500mLビーカーに移し、実施例１で使用した栄養補強滅菌海水にサルーシンを1000fmol/L濃度になるように添加した培地500mLで、通気により撹拌しながら培養を行った。光と温度の条件は実施例１と同じに設定した。培養10日後、多細胞から成る葉状体が発生した。 Example 4
Each single cell in the cell suspension of Hilo-Hano human Exa prepared in Example 2 was multicellularized with salusin. Specifically, the procedure was performed as follows. 5 mL (1 to 5,000 cells) of the cell suspension of Hironohitoexa prepared in Example 2 was transferred to a 500 mL beaker, and the concentration of salusin was adjusted to 1000 fmol / L in the fortified and sterilized seawater used in Example 1. The culture was carried out with 500 mL of the medium added to, while stirring by aeration. Light and temperature conditions were set the same as in Example 1. After 10 days in culture, multicellular thallus developed.
　培養後に得られた葉状体の写真を図６に示す。また、葉状体をスライドガラス上で広げて撮影した写真を図７に示す。一方で、胞子から生長した通常のヒトエグサの葉状体の写真を図８に示す。 写真 FIG. 6 shows a photograph of the thallus obtained after the culture. In addition, FIG. 7 shows a photograph obtained by spreading the leaf-shaped body on a slide glass. On the other hand, FIG. 8 shows a photograph of the thallus of a normal human eegusa grown from spores.
　通常の方法で得られた葉状体は、岩などに付着するための繊維状の付着根（図８中、矢印）が発達するのに対して、本実施例で得られた葉状体にはこのような付着根が形成されないことが分かった。 The foliates obtained by the ordinary method develop fibrous attachment roots (arrows in FIG. 8) for adhering to rocks and the like, while the foliates obtained in the present example have It was found that such attached roots were not formed.

Claims (11)

  1. （Ａ）海藻の胞子、該胞子由来の単細胞、並びに該胞子及び／又は該単細胞の細胞塊からなる群より選択される少なくとも１種を、海藻形態形成誘導因子を実質的に含有しない培地１中で、攪拌条件下で培養する工程、
    を含む、海藻細胞の製造方法。     (A) at least one selected from the group consisting of seaweed spores, single cells derived from the spores, and cell masses of the spores and / or the single cells, in a medium 1 substantially free of seaweed morphogenesis-inducing factors; In, a step of culturing under stirring conditions,
    A method for producing a seaweed cell, comprising:
  2. 前記海藻がヒビミドロ目に属する海藻である、請求項１に記載の製造方法。 The production method according to claim 1, wherein the seaweed is a seaweed belonging to the order Hibimidori.
  3. 前記海藻がヒトエグサ属に属する海藻である、請求項１又は２に記載の製造方法。 The production method according to claim 1, wherein the seaweed is a seaweed belonging to the genus Humane.
  4. 前記海藻細胞が単細胞を含む、請求項１～３のいずれかに記載の製造方法。 4. The production method according to claim 1, wherein the seaweed cells include single cells.
  5. （Ａ１）海藻の胞子を前記培地１中で培養する工程、及び
    （Ａ２）工程Ａ１の培養物を、前記培地１中で、攪拌しながら培養する工程、
    を含む、請求項１～４のいずれかに記載の製造方法。     (A1) a step of culturing spores of seaweed in the medium 1, and (A2) a step of culturing the culture of step A1 in the medium 1 while stirring.
    The production method according to any one of claims 1 to 4, comprising:
  6. 前記工程Ａ１における培養が静置培養である、請求項５に記載の製造方法。 The production method according to claim 5, wherein the culture in the step A1 is a static culture.
  7. 前記培地１が人工海水培地である、請求項１～６のいずれかに記載の製造方法。 The method according to any one of claims 1 to 6, wherein the medium 1 is an artificial seawater medium.
  8. 前記海藻が食用海藻である、請求項１～７のいずれかに記載の製造方法。 The production method according to any one of claims 1 to 7, wherein the seaweed is an edible seaweed.
  9. 前記攪拌条件下での培養が通気培養である、請求項１～８のいずれかに記載の製造方法。 The production method according to any one of claims 1 to 8, wherein the culture under stirring conditions is aeration culture.
  10. 請求項１～９のいずれかに記載の製造方法で得られた海藻細胞を、海藻形態形成誘導因子を含有する培地２中で培養する工程、
    を含む、海藻の製造方法。     A step of culturing the seaweed cells obtained by the production method according to any one of claims 1 to 9 in a medium 2 containing a seaweed morphogenesis factor,
    A method for producing seaweed, comprising:
  11. 付着根を有しない、海藻葉状体又は海藻。 A seaweed foliate or seaweed having no attached root.
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