JPS6260077B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for cytoplasmically fusion of monospores or carospores released from laver fronds by applying a cell fusion technique.

That is, the present invention relates to a method for cell fusion of seaweed without using protoplasts of seaweed thallus.

Conventional Technology 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.

However, to date, no successful case of cell fusion has been reported in seaweeds (including laver).

This is because for land plants, protoplasts can be easily prepared using commercially available enzymes (e.g., cellulase, pectoliase, macerozyme), but for seaweeds, especially seaweeds, the skeleton that makes up their tissues is difficult to prepare. This is because the component polysaccharides are not converted into protoplasts in a healthy state even when treated with the enzymes mentioned above.

In other words, the polysaccharides that make up the tissues of laver are composed of xylan, mannan, and porphyrane, and the cell walls in particular are composed of xylan formed into strong fibers, so they cannot be hydrolyzed by the enzymes mentioned above. This is because it is not done. Incidentally, there are currently no reports on enzyme agents that have the ability to hydrolyze the three types of polysaccharides mentioned above.

Problems to be Solved by the Invention As mentioned above, in view of the current situation where it is extremely difficult to prepare healthy protoplasts by treating seaweed tissues with enzymes, the present inventors have solved the following problems in the life history of seaweed: We 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, and as a result of our research, we developed a direct fusion treatment for these monospores and carospores. This led to the discovery that cytoplasmic fusion occurs, leading to the present invention.

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.

The present invention will be explained in detail below.

Structure of the Invention The structural feature of the present invention is that monospores or caropspores released from seaweed thallus are cytoplasmically fused by applying a cell fusion technique.

The monospores used for cell fusion in the present invention are those that attach to the seaweed network during the life history of seaweed, germinate, produce rhizoids, form cell walls, and divide and multiply 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.

Also, during the life history of seaweed, caropspores detach from the seaweed thallus, attach themselves to shells, etc., and eventually infiltrate and become filamentous.They have thin cell walls, and like the monospores mentioned above, they can be used to transform seaweed into protoplasts. It is understood that it is in a form similar to that of the original.

In order to obtain these monospores and caropspores artificially, young leaves of about 0.2 to 1.0 mm in the case of Monospores, and leaves of about 1.0 to 10 cm in the case of Noriflora, are cultured in artificial seawater, and the leaves are grown in artificial seawater. After confirming that cells have detached from the body margin, at the stage where many cells have detached from the leaflets, it is best to centrifuge the culture solution containing the cells and collect the resulting residue;
For caropospores, 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 darkness to release caropspores. It is preferable to separate the residue obtained by centrifuging the culture solution containing fruit spores.

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.

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. Polyethylene glycol solution (54
%) and leave to stand, then add High PH-calcium solution (100mM CaCl ₂ , PH10.5) and leave to stand at room temperature. In this case, it is preferable to repeat the addition of the High PH-calcium solution twice.

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.

Incidentally, when the above-mentioned cell fusion treatment is not performed, virtually no cytoplasmic fusions are observed in either monospores or caropspores.

Effects of the Invention As described above, according to the present invention, monospores obtained by culturing young leaves of seaweed or fruit spores obtained by culturing thallus of seaweed can be prepared by cell fusion. Since seaweed cell fusion can be carried out without using seaweed protoplasts, which are considered difficult, the technology has great benefits for seaweed cell fusion technology, and therefore, the cell fusion product is useful in the production of seaweed. .

The present invention will be explained in more detail below with reference to Examples.

Example 1 Preparation of a suspension of monospores: Young leaves of Aspergillus orientalis with a size of 0.2 to 1.0 mm were grown in artificial seawater (Asp12) with the composition shown below at a temperature of 18°C under an illuminance of 4000 Lux (8 hours light period, dark period 16
When the release of monospores was observed, the culture solution was centrifuged (1000 rpm, 5 minutes), the resulting residue was collected, and an appropriate amount of the culture solution consisting of the above artificial seawater was added to it. was added to make a monospore suspension.

Composition of artificial seawater (ASP12): NaCl 28g MgSO ₄・7H ₂ O 7g MgCl ₂・6H ₂ O 4g KCl 700mg CaCl ₂・2H ₂ O 1.47g NaNO ₃ 100mg K ₂ HPO ₄ 10mg Sodium glycerophosphate 10mg Vitamin B ₁₂ 0.2 μg Biotin 1μg Thiamin 100μg PMetal 10ml SMetal 10ml Tris-aminomethane 1g Distilled water 1000ml PH 8.0-8.1 Composition of PMetal EDTA 1mg H ₃ BO ₃ 1mg MnCl ₂・4H ₂ O 0.14mg FeCl ₂・6H ₂ O 0.05mg ZnCl ₂ 0. 01mg CoCl ₂・6H ₂ O 4μg CuSO ₄・5H ₂ O 0.5μg Distilled water 1ml SMetal composition NaBr 1.2mg SrCl ₂・6H ₂ O 0.6mg AlCl ₃・6H ₂ O 1.2mg NaMoO ₄・2H ₂ O 0.12mg PbCl 0.03 mg KI 1.5 μg Distilled water 1 ml Cytoplasmic fusion of monospores: 0.1 μg of the monospore suspension prepared as described above
ml was pipetted into a Petri dish and left for 5-10 minutes to form a precipitate on the glass surface. Add 0.2% of a polyethylene glycol solution of the following composition to this precipitate.
ml and leave it for 30 minutes, then add High of the following composition to this.
After 0.5 ml of PH-calcium solution was added and left for 10 minutes, 0.5 ml of High PH-calcium solution was added and left for 5 minutes.

Composition of polyethylene glycol solution: 54% aqueous solution of polyethylene glycol (MW6000) with _{10.5mM of CaCl2.2H2O , KH2PO4.H2O}
Added to a concentration of 0.7mM and glycose 0.1M.

Composition of High PH-calcium solution: When using a solution of 100mM of CaCl ₂ 2H ₂ O and 0.4M of glycose dissolved in distilled water and a solution of 0.4M of glycose dissolved in NaOH-glycine buffer (PH10.5), : Mixed at a ratio of 1.

Next, 0.3 ml of culture solution (artificial seawater Asp12) was added to the mixture treated as described above, and after leaving it for 5 minutes,
I sucked 0.3 ml out of the siere. After repeating these operations five times, a new culture medium (artificial seawater Asp12) was added and cultured. Production of a monospore cytoplasmic fusion was confirmed in this culture solution.

In addition, the production of cytoplasmic fusions was similarly confirmed even when 1.0 to 10 cm leaves of Asakusanori were used instead of young leaves of Asakusanori.

Example 2 Preparation of a suspension of caropspores: Mature seaweed thallus was grown in artificial seawater Asp12 similar to that used in Example 1 at a temperature of 15°C.
Culture was performed under an illuminance of 1500 Lux (12-hour light period, 12-hour dark period). The release of fruit spores was observed on the 3rd to 4th day of culture, so the culture solution was centrifuged (1000 rpm, 5 minutes), the resulting residue was collected, and the above artificial seawater Asp12 was added to the culture solution. An appropriate amount was added to make a fruit spore suspension.

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 instead of the suspension of monospores in Example 1.

After culturing, the formation of cytoplasmic fusions of caropspores was confirmed under a microscope in the culture solution.

Claims (1)

[Scope of Claims] 1. A method for cell fusion of seaweed, which comprises performing cytoplasmic fusion of monospores or caropspores released from seaweed fronds by applying a cell fusion technique. 2 Polyethylene glycol solution and High
The method according to claim 1, which applies a cell fusion technique using a PH-calcium solution as a fusion inducer.