JPH022577B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel method for collecting seaweed seedlings using seaweed protoplasts. The life history of seaweed was elucidated by Drieux of the University of Manchester in the United Kingdom in 1942, but later the filamentous structure (Conchoc) in the life history of seaweed was clarified.
-elis) By artificially managing the growing period (approximately 6 months), it is possible to make any fixed number of conchospores settle on the seaweed cultivation net, and as a result, it is possible to change the growth rate from natural collection to artificial cultivation. The path to aquaculture was opened. However, this is simply a way to artificially perform only the filamentous growth period, and it is necessary to keep the seaweed leaves in a net until they mature. The disadvantage was that the body often became unhealthy, and subsequent filamentous growth was uneven. Under these circumstances, seaweed seedlings are collected using free filaments obtained by selecting well-developed seaweed fronds and then growing filaments from the separated fruit spores using a microbial culture method. A technology has been developed, and seaweed seedlings are now widely harvested using this technology. However, although this method of collecting seaweed seedlings using free filaments has made it possible to preserve any species throughout the year at the free filament production stage, in actual industrial production scale, free filaments are harvested early in the spring. The equipment required to attach and grow spores on shells, form spores, release them in the sea in early autumn, and grow them on seaweed nets is expensive, and in addition, the work required to grow these spores is expensive. The drawback is that management is complicated. In other words, a seed center with large equipment was set up, and the free filaments stored there were cut with a blender, etc., and they were spread on a large number of oyster shells and grown to form spores. Currently, they are grown by releasing shell spores in the sea and attaching them to seaweed nets, which requires management such as water replacement and light irradiation. Furthermore, although the above-mentioned method of collecting seaweed seedlings using free filaments can be called a kind of artificial cultivation technique, it still follows the conventional life history of seaweed, so it is difficult to obtain seaweed fronds. It is inevitable that this will take a long time. In view of the current state of the seaweed seedling collection method as described above, the present inventor conducted research to develop a seaweed seedling collection technique that allows seaweed fronds to be cultivated in a relatively short period of time and with simple operations. We obtained the knowledge that the protoplasts of the seaweed thallus, which are obtained by converting the body into protoplasts through enzymatic treatment, attach themselves to the seaweed net and grow to the thallus, similar to the conchospores in the life history of seaweed.
The present invention has now been accomplished. That is, the present invention utilizes the protoplasts of the seaweed thallus as a seed to replace schistospores, thereby enabling the cultivation of seaweed in an extremely short period of time without going through the filamentous growth period in the life history of the seaweed. The purpose is to provide seedling methods. The present invention will be explained in detail below. The feature of the present invention is that seaweed protoplasts obtained by converting seaweed fronds into protoplasts by enzymatic treatment are grown by growing them on a seaweed cultivation net. In recent years, with the development of biological cell fusion technology as a genetic engineering method, preparation of protoplasts of various plants has become popular. Therefore, the polysaccharides that make up the cell walls of seaweeds, especially the laver, are different from the polysaccharides (mainly cellulose) in land plants, and are mainly indigestible polysaccharides such as xylan and mannan. It was considered difficult to convert it into protoplasts by enzymatic treatment. However, the present inventor first discovered that a specific microorganism belonging to the genus Pseudomonas,
By treating the seaweed thallus with an enzyme solution containing at least xylan hydrolase and mannan hydrolase prepared from the culture solution obtained by culturing in a specific medium, we succeeded in producing nori protoplasts. He invented a preparation method (Japanese Patent Application No. 149378-1983). That is, the feature of the above invention is that a microorganism (Feikoken Jokyo No. 330) that has the ability to hydrolyze indigestible polysaccharides (mainly xylan and mannan) belonging to the genus Pseudomonas is used in the production of seaweed thallus or seaweed leaf. The seaweed thallus is treated with an enzyme solution containing at least xylan hydrolase and mannan hydrolase prepared from a culture solution obtained by culturing in a medium containing body-derived polysaccharides as an inducer, and converted into protoplasts. There is a particular thing. Further, subsequent research revealed that, in addition to seaweed thallus or polysaccharides derived therefrom, β-1,3 xyloside bonds and β-
Plants containing polysaccharides having 1,4 mannoside bonds as a component (e.g. sardine fern, konnyaku)
Alternatively, it has been found that polysaccharides derived from the plant can also be used. First, the method for obtaining seaweed protoplasts used in the present invention will be explained in more detail. In order to prepare seaweed protoplasts, it is necessary to decompose the indigestible polysaccharides, mainly consisting of xylan and mannan, which constitute the cell walls of seaweed fronds. An enzyme solution containing a degrading enzyme and a mannan hydrolase is used. Preparation of enzyme solution: Belonging to the genus Pseudomonas,
Pseudomonas, a microorganism isolated from seawater
SP.PT-5 (Feikoken Jokyo No. 330) is made from seaweed or a polysaccharide having β-1,3 xyloside bonds and β-1,4 mannoside bonds, which are the constituent units of seaweed polysaccharides. At least xylan hydrolase and mannan hydrolase are obtained by culturing in a medium containing a plant body containing the plant as an inducer or a polysaccharide derived from seaweed or a polysaccharide derived from the above-mentioned plant body as an inducer. Obtain the enzyme solution. The amount of seaweed or the above plant and the polysaccharide derived therefrom added to the above medium is 1 as the amount of polysaccharide.
2 to 2% by weight is appropriate, and the medium uses the polysaccharide as the inducer as a carbon source, and a nitrogen source such as peptone or yeast extract, and furthermore,
It is preferable to use a solution prepared by adding an inorganic substance such as K ₂ HPO ₄ or FeCl ₃ dissolved in seawater or artificial seawater and adjusting the pH to around 7.5 with a buffer solution. The above Pseudomonas SP used here.
The mycological properties of PT-5 (Feikoken Joyori No. 330) are as follows. Mycological properties: (i) Morphology Regarding cells grown in ZoBELL 2216E medium, (a) The morphology of the cells is rod and the size is 0.5-1 μm ×
1.5-2.5μm (b) Motile, flagella are monopolar (c) Gram staining is negative (ii) Growth condition Growth condition in ZoBELL 2216E slant medium, (b) At a temperature of 20-27℃ Grows well (b) Deposit pale yellow pigment (c) Forms filiform colonies (iii) Physiological properties (a) Catalase test positive (b) Oxidase test positive (c) Acid from glucose Formation Positive (d) O-F test 0 (by Hugh Leifson method) (e) Vibro-Static Agent (0/129) Negative (f) Agar liquefaction ability + (g) Xylan resolution + (h) Mannan resolution + ( (li) Aerobicity Incidentally, the above microorganism has the ability to hydrolyze xylan, mannan, and porphyrane, which are the indigestible polysaccharides. The conditions for culturing the above-mentioned microorganisms in the medium described above are to carry out the cultivation at a temperature of about 25°C for about 4 days with stirring and aeration, and then to culture the culture solution obtained in this way at a low temperature of about 4°C. Centrifugation (10000r.p.
m) and collect the supernatant as an enzyme solution. The enzyme solution thus obtained contains xylan hydrolase, mannan hydrolase, and porphyrane hydrolase. Next, protoplast formation of seaweed fronds using the enzyme solution obtained as described above is carried out as follows. Protoplast formation of seaweed thallus: For 4 to 5 sheets of seaweed thallus of about 5 cm,
Nori protoplasts can be prepared by allowing about 10 ml of the above enzyme solution concentrated 5 to 10 times to react for 3 to 4 hours. The mechanism of action of the above enzyme solution on the seaweed thallus is that the mannan hydrolase in the enzyme solution acts on the granular mannan present on the surface layer of the seaweed thallus, forming large cuts in the thallus and damaging the cell walls. The purpose is to facilitate the action of the xylan hydrolase in the enzyme solution on the formed microfibrillar form of xylan. Furthermore, the porphyrane hydrolase contained in the enzyme solution acts on porphyrane, which is a cell-filling substance of the seaweed thallus, and further promotes the protoplast formation of the seaweed. In addition, in converting the seaweed into protoplasts,
When combined with proteases (e.g. papain),
It is even more effective because it can break down the thin layer of protein that coats the surface of the seaweed thallus. The seaweed protoplasts thus obtained are in a healthy state because they are prepared only by enzymatic treatment without physically cutting the seaweed. Next, in the present invention, the seaweed protoplasts obtained by converting the seaweed fronds into protoplasts as described above are attached to the seaweed net. To carry out this settlement, seaweed protoplasts are grown in artificial seawater (for algae).
Asp.12) may be suspended in the above suspension and brought into contact with the seaweed net.At this time, the seaweed net is shaken in the above suspension to improve the contact, and also to promote settlement on the seaweed net. In order to do this, light irradiation is performed for a certain period of time. The protoplasts attached to the seaweed net in this way undergo cell division and proliferate, and after about a month, the leaf length reaches 2.
More than 100 seaweed fronds of ~3 cm in length will grow per net. Therefore, according to the present invention, there is no need to go through the long filament phase (spring to summer) of about 6 months in the conventional life history of seaweed, and the formation of seaweed thallus directly from seaweed protoplasts is possible in an extremely short period of time. It becomes possible to cultivate. In addition, according to the conventional method based on the life history of seaweed, the chromosomes of seaweed are 2N from fertilization to the filament stage, and undergo meiosis at the stage of spore formation to become N-chromosomes. Since seaweed has three chromosomes, its traits are 2 ³ = 8. That is, excluding the case of sudden mutation, it will produce shell spores with eight different traits. In other words, seaweed with eight different traits is produced, and this is one reason why the quality of seaweed is not constant from year to year. However, in the present invention, since the n-body seaweed thallus is converted into protoplasts, the obtained protoplasts are also n-body, and these n-body protoplasts divide and proliferate as they are to become the seaweed thallus. This method has the great advantage of making it possible to grow seaweed fronds with their original characteristics, resulting in the production of seaweed with stable quality. As described above, the use of seaweed protoplasts as seedlings according to the present invention makes it possible to cultivate seaweed, which can be called an epoch-making method, and therefore it is believed that there will be great benefits in cultivation and production of seaweed. EXAMPLES The present invention will be explained in more detail with reference to Examples below. Examples Preparation of seaweed protoplasts Preparation of enzyme solution for protoplastization of seaweed thallus: Pseudomonas sp.PT-
5. Inoculate FEIKEN JYOYO BP-330 into a medium with the following composition, and aerate it while stirring at 300 rpm (aeration amount
Culture was carried out at 25°C for 4 days (2000ml/min). Medium composition: Seaweed or seaweed-derived polysaccharide 1.0 (wt%) Peptone 1.0 Yeast extract 0.1 K ₂ HPO ₄ 0.01 FeCl ₃ 0.6mg / Tris buffer 0.1 (wt%) Dissolve in seawater at the above ratio to adjust the pH to 7.5 adjust. To prepare an enzyme solution from the culture solution obtained by culturing as described above, the culture solution is incubated at a temperature of 4°C.
Centrifuge for 30 minutes (10,000 rpm) and use the supernatant as the enzyme solution. The results of examining the enzyme activity of the enzyme solution prepared in this manner against indigestible polysaccharides are shown in the table below.

[Table] (Note) …Very strong activity
...strongly active
+...Slightly strong activity As seen in the table, the enzyme solution used in the present invention has excellent activity against mannan and xylan. Preparation of seaweed protoplasts: Place 4 to 5 seaweed fronds of about 5 cm in an L-shaped test tube, and add 10ml of papain solution (dissolved in acetate buffer to pH 6.0). While the seaweed fronds were immersed in the papain solution, they were shaken at a temperature of 25° C. for 20 minutes (mono-type shaker 70 strokes/min). Place 4 to 5 5cm pieces of seaweed leaves treated as described above into an L-shaped test tube, add 10ml of the above enzyme solution, and heat at 20°C while immersing the seaweed leaves in the enzyme solution. using a mono-shape shaker at a temperature of 4
Nori protoplasts were obtained by shaking for hours (70 strokes/min). Growth of seaweed thallus using seaweed protoplasts The seaweed protoplasts prepared as described above were suspended in artificial seawater (Asp.12 for algae) with the following composition at a concentration of 1 x 10 ⁵ cells/ml. Ta. Composition of artificial seawater (Asp.12 for algae): NaCl 28g MgSO ₄・7H ₂ O 7g MgCl ₂・6H ₂ O 4g KCl 0.4g CaCl ₂・2H ₂ O 1.46g NaNO ₃ 0.1g K ₃ PO ₄ 10mg Glycerophosphate Sodium 10mg Na ₂ SiO ₃・9H ₂ O 150mg Vitamin B ₁₂ 0.2μg Biotin 1μg Thiamine 100μg P Metal 10ml S Metal 10ml Trisaminomethane 1g Nitrilotriacetic acid 0.1g Distilled water 1000ml Adjusted to PH8.0. The compositions of P Metal and S Metal in the above composition are as follows.

【table】

[Table] 20 ml of the protoplast suspension obtained as described above was placed in a 100 ml beaker, 50 cremonite nets (2-3 cm) were placed in it, and the mixture was heated at 15°C.
Irradiation of 6000 Lux (irradiation so that the light period is 9 hours/day) is carried out in a room with a temperature of
Shaking was performed using a type shaker at 40 strokes/min. After 2 days, it was confirmed by microscopic observation that about 70% of the protoplasts had settled on the mesh, and that they were dividing and proliferating into 2 to 3 cells. Furthermore, after using one net, it was observed with the naked eye that there were 100 leaves with a leaf length of 2 to 3 cm per net.
It was observed that more than one leaf had grown.

Claims (1)

[Scope of Claims] 1. A method for collecting seedlings using seaweed protoplasts, which is characterized by growing seaweed protoplasts obtained by converting seaweed thallus into protoplasts by enzymatic treatment and growing them on a seaweed net. 2. The method for collecting seedlings according to claim 1, in which seaweed protoplasts suspended in artificial seawater are attached to a seaweed net and allowed to grow.