JPS6260076B2 - - Google Patents

Description

[Detailed Description of the Invention] The present invention provides a method for cell fusion of seaweed, more specifically,
The present invention relates to a method for fusing cell membranes of shell spores released from seaweed filaments.

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.

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 investigated the problem of protoplasts that are released from filaments during the life history of seaweed. As a result of our research, we focused on the shell spores, which are squishy amoeboid cells with a diameter of approximately 10 to 13 microns, and obtained the knowledge that cell membrane fusion occurs when the shell spores are treated with a fusion-inducing substance.This led us to the present invention. I arrived at the eggplant.

Therefore, an object of the present invention is to provide a method for carrying out cell fusion of seaweed using the above-mentioned conchospores.

The present invention will be explained in detail below.

The present invention is characterized in that the shell spores released from seaweed filaments are treated with a fusion-inducing substance to cause cell membrane fusion.

During the life history of the seaweed, the chospores used for cell fusion in the present invention attach to the seaweed web, germinate, produce rhizoids, form a cell membrane, and divide and multiply to become the seaweed thallus. The shell spore, which is the stage before the formation of a seaweed thallus, is thought to have a similar morphology to a protoplast, which is obtained by removing the cell wall from the seaweed thallus.

Generally, according to reports on cell fusion in land plants, protoplasts obtained by treatment with enzymes such as cellulase burst unless treated in a hypertonic solution, so 0.4-0.7M mannitol or sorbitol was added. Treatment with hypertonic solutions is required.

However, when regenerating the fused cells, the culture medium is made hypertonic and the osmotic pressure of the external fluid of the protoplasts increases, causing some damage to the cell membrane on the surface of the protoplasts. It also becomes a factor that makes it impossible to grow under normal conditions.

However, as can be understood from the fact that the chospores used for cell fusion in the present invention are released from filaments in their natural state and then float in seawater and attach to the seaweed, culture without the addition of mantonyl or sorbitol is necessary. Even liquid will not explode by itself, so
It has the characteristic that it does not cause any growth problems as described above.

To artificially obtain the cholaspores released from seaweed filaments, mature filaments are cultured in artificial seawater with aeration and agitation to release the chospores (they are released in 3 to 4 days). It is preferable to centrifuge the culture solution containing the conciliar spores, collect the residue, and add an appropriate amount of sterilized artificial seawater to the residue to prepare a suspension of the conciliar spores.

Next, in order to perform cell fusion using the suspension of conchospores obtained in this way, the suspension is dropped onto a hole slide glass, and a solution of a fusion inducer, such as polyethylene glycol, is added to the suspension. (15~
20% wt/wt), polymer dextran sulfate, high concentration sodium nitrate or High PH-High Ca solution (50mM CaCl ₂ , PH10.5) etc. at room temperature for 40-50 min.
It is advisable to leave the cells for about a minute, and it is confirmed by observation under a microscope that cell fusion has occurred by treatment with a fusion-inducing substance such as polyethylene glycol.
The cell fusion was confirmed based on a schematic diagram showing the process of cell fusion published in "Cell Fusion in Plants, Cells" 12(8) 1980 by Hoshita et al. In fact, cell membrane fusion is virtually not observed unless treated with a fusion inducer such as polyethylene glycol.

When the chospores with cell membrane fusion as described above are cultured in artificial seawater, more than 90% of them develop rhizoids, form cell walls, and grow by dividing and multiplying.
This leads to the formation of a thallus.

Therefore, according to the present invention, using the chelaspores released from the filaments of seaweed, it becomes possible to perform cell fusion in the same manner as protoplasts.

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

Example: Preparation of schistospore suspension Mature filaments of seaweed were placed in human-strength seawater (Asp12) with the following composition, which is commonly used in algae research.
Culture was carried out at a temperature of 17-18° C. and an illuminance of 4000 lux (light period 8 hours, dark period 16 hours) with aeration and agitation.

After 3 to 4 days of the above culture, the release of conchospores was observed, so the culture solution was centrifuged (1000 rpm for 5 minutes), the resulting residue was collected, and an appropriate amount of the above sterilized artificial seawater was added to this. was added to obtain a schistospore suspension.

Composition of artificial seawater (Asp12): NaCl 28g MgSO ₄・7H ₂ O 7g MgCl ₂・6H ₂ O 4g KCl 400mg CaCl ₂・2H ₂ O 1.46g NaNO ₃ 100mg K ₂ HPO ₄ 10mg Sodium glycerophosphate 10mg Vitamin B ₁₂ 0.02 μg Biotin 0.1μg Thiamine 10μg PMetal 10ml SMetal 10ml Trisaminomethane 1g Distilled water 100ml 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 .01 mg CoCl ₂・6H ₂ O 4μg CuSO ₄・5H ₂ O 0.5μg Distilled water 1ml SMetal composition NaBr 1.2mg AlCl ₃・6H ₂ O 1.2mg SrCl ₂・6H ₂ O 0.6mg NaMoO ₄・2H ₂ O 0.12mg PbCl 0.03 mg KI 1.5 μg Distilled water 1 ml Cell fusion of conspores 150 mg of the conspore suspension prepared as described above
Drop 1 μl onto a hole slide glass, and add 450 μl of polyethylene glycol solution with the following composition.
Gradually add l as a fusion inducer and incubate at room temperature for 40 minutes.
Let stand for ~50 minutes.

Composition of polyethylene glycol solution: Polyethylene glycol 45g CaCl _{2.2H 2} O 735mg Distilled water 100ml To the polyethylene glycol treated material as described above, 500 μl of the above sterilized artificial seawater was gradually added, left at room temperature for 10 minutes, and then After slowly washing with sterilized artificial seawater, the whole slide glass was placed in a shear dish with an inner diameter of 9 cm.
Slowly inject 20ml of sterilized artificial seawater into this.
Temperature of 18℃ and illuminance of 7000 lux (8 hours light period,
Culture was performed in a dark period of 16 hours). In addition, the water of the culture solution was changed every two days.

After 10 days of culture, the leaves grew to a length of about 100μ, and the culture
After 20 days, it was confirmed that the leaves had grown to about 1 to 1.5 mm in length.

Claims (1)

[Scope of Claims] 1. A method for cell fusion of seaweed, which comprises treating shell spores released from filaments of seaweed with a fusion-inducing substance to cause cell membrane fusion. 2. The method according to claim 1, wherein the fusion inducer is polyethylene glycol.