JPH031953B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a method for producing a variety of cultured seaweed that is highly resistant to akagusa disease, which often occurs in cultured seaweed. Conventional technical background Akagsare disease that occurs in cultivated seaweed is caused by Akagsare fungi that belong to the Pythium genus of the algae, as described in "Seaweed Disease" (published by Seiseisha Genseikaku) edited by the Japan Fisheries Society. It is one of the diseases that occurs widely in seaweed fishing grounds in various parts of Japan, reducing the quality and yield of seaweed, and sometimes causing significant damage. Symptoms of akagusare disease are small, faded spots that are initially seen here and there on the seaweed fronds, and then these spots rapidly spread to show rust-red circular spots about 5 to 20 mm in size. This lesion then changes from greenish-yellow to light yellow, and gradually fades from the center of the lesion.As the disease progresses, the diseased area extends to the entire surface of the nori thallus, and the bleached lesion gradually rots. It ends up falling off. Therefore, if there is a diseased area at the base of the leaf, the leaf will fall off through the cracks and be washed away, resulting in
If the disease progresses quickly, from the beginning of the disease outbreak to
The cracks may turn into empty nets within about 10 days. On the other hand, there are research reports on the mycological properties of the pathogenic bacteria of Akagusare disease, its growth environment factors, and nutritional requirements, and there are also studies on the effects of antibiotics, pesticides, and surfactants on the growth inhibition of the hyphae of the pathogenic bacteria. Although some research has been done on this, appropriate prevention and treatment methods for akagusare disease have not yet been established, as it is considered undesirable to use the above-mentioned chemicals on the sea surface of cultivated seaweed. is the current situation. Incidentally, as the only research report, Noda reported on the preventive and therapeutic effects of akagusare disease using amino acids such as histidine, methionine, and tyrosine (Japan Suisan Gakushu, Vol. 45, No. 9, 1155--
1162). In light of the current situation described above, countermeasures against akagusare disease in seaweed farming are an important issue. Problems to be Solved by the Invention In view of the above-mentioned current situation, the present inventor investigated the creation of a variety of cultivated seaweed that is essentially resistant to Akagusare disease, and as a result, he succeeded in turning seaweed into protoplasts. In line with this, the present invention was accomplished by utilizing a cell fusion technique. The present inventors discovered that the natural varieties of Maruba laver, which are not suitable for cultivation, and Susabi green, a mutant of Susabi nori, have strong resistance to akagase disease, and prepared protoplasts of these natural varieties. By fusing each of these protoplasts with protoplasts from cultured seaweed, they succeeded in introducing traits that are highly resistant to Akagushi disease into cultured seaweed. In addition, by preparing protoplasts of the above-mentioned Maruba laver and Susabi green and fusing them together, we succeeded in obtaining a new variety of seaweed that is resistant to Akagushi disease and suitable for aquaculture. That is, an object of the present invention is to provide a method for producing a variety of cultured seaweed that is highly resistant to Akagushi disease using a cell fusion technique. The present invention will be explained in detail below. Structure of the Invention The feature of the present invention is to prepare protoplasts of Susabi Green or Malva Nori, which are natural varieties with strong resistance to Akagushi disease, to prepare protoplasts of cultured seaweed, and to cell-fuse both of the obtained protoplasts to form cells. forming a fusion, then cultivating the cell fusion, preparing the protoplasts of Susabi green and Maruba laver, fusing both of the obtained protoplasts to form a cell fusion, and then cultivating the cell fusion. It is about cultivating a fusion body. Means for Solving the Problems The natural variety (wild strain) used in the present invention, Malva lamanori, is commonly called rock laver and grows by attaching to rocks, but its leaves exhibit a round-leaf shape. (The thallus of cultured seaweed exhibits a narrow-leaf shape) and has a low growth rate, making it unsuitable for aquaculture and therefore not used. Furthermore, Susabi Green, which is also used in the present invention, is a mutant variety of Susabi Nori, but it is not used for aquaculture because it is a strain lacking phycoerythrin pigment (red) and has green leaves. However, in the process of examining the properties of Maruba seaweed and Susabi green, it was discovered that these two varieties possess traits that are more resistant to Akagusare disease than native farmed seaweed. This was discovered based on the experimental results shown in . Experimental method: Pythium porphyrae was infected with Arasaki B.
Cultivate in a medium at 20°C for 7 days, cut out the green part of the colony that has formed, place it on a shear dish, crush it lightly, and then add sterile semi-seawater to release zoospores. These 4 x 10 ⁴ zoospores (counted with a Thoma hemocytometer) were inoculated into 20 ml of artificial seawater in a Schare.
In addition, 5 sheets of seaweed leaves (about 1.0 to 5.0 cm in size) of each variety of Susabi Green, Maruba Amanoori, and Asakusanori and Susabi Nori as cultured seaweed are added.
Soak the leaves at 20℃, 4000 Lux (9 hours light period,
The cells were cultured stationary under a 15-hour dark period), and the number of infected sites and the number of penetrating cells in each leaflet were observed using a microscope over a period of 10 days. Infection here is based on the observation of penetrating cells by the hyphae of the fungus, and the number of penetrating cells indicates the rate at which the hyphae grow. The above experimental results are shown in Tables 1 to 3.

[Table] Shows the number of days that have elapsed since the above infected areas were confirmed.

【table】

[Table] As shown in Tables 1 to 3, it can be seen that Susabi Green and Maruba Nori have significantly higher resistance to Akagusare disease than the cultivated seaweeds Asakusanori and Susabi Nori. In the present invention, we focused on Susabi green and Maruba seaweed, which are not suitable for conventionally cultured seaweed as described above, but are highly resistant to Akagushi disease. By using this method and introducing it into cultured seaweed, we can create a variety of cultured seaweed that is highly resistant to Akagushi blight.For this purpose, we first prepare protoplasts of Susabi green and Maruba seaweed. As mentioned above, the protoplasts are prepared by the method previously developed by the present inventor (Japanese Patent Application No.
149378 (Unexamined Japanese Patent Publication No. 1983-41485) or Patent Application No. 1983-
22415 (Japanese Unexamined Patent Publication No. 168381/1983)). Next, to give an overview of these methods, the former method uses microorganisms (Pseudomonas SP No. PT-5, Pseudomonas SP No. PT-5,
(Feikoken Joyori No. BP-330), seaweed or seaweed-derived polysaccharides (residue mainly consisting of polysaccharides such as mannan or xylan obtained by extracting seaweed with hot water to remove soluble components) The culture solution obtained by culturing the residue in a medium containing as an inducer (the residue was further purified to increase the polysaccharide content) was centrifuged, and the supernatant was used as an enzyme solution to extract the seaweed fronds. It consists of processing. The above enzyme solution contains mannan hydrolase and xylan hydrolase, so when the enzyme solution is applied to the seaweed thallus, the mannan hydrolase acts on the granular mannan present on the surface of the seaweed. A large cut is formed in the leaf body, which makes it easier for xylan in the form of microfibrils forming the cell wall to be acted upon by the xylan hydrolase solution, and the cell wall of the leaf body is decomposed and removed to form protoplasts. It becomes like this. Furthermore, since the enzyme solution contains a porphyranase degrading enzyme, it acts on and decomposes porphyrane as a cell-filling substance of the seaweed thallus, thereby further promoting protoplast formation. In addition, during the above-mentioned protoplast formation, it is more effective if the seaweed thallus is treated with a protease such as papain in advance, or if the protease is allowed to act in parallel with the above-mentioned enzyme solution. In addition, in the latter method, after pre-treating the seaweed thallus with protease, β-1,3-xylanase and β-
treatment with 1,4-mannanase or β-
It consists of treatment with 1,3-xylanase, β-1,4-mannanase and porphyranase, and can prepare healthy seaweed thallus protoplasts in a very short time. In the present invention, protoplasts of Susabi green and Maruva laver are prepared respectively by the method described above, and protoplasts of Asakusanori and Susabi nori, which are cultivated seaweeds, are prepared in the same way. Cell fusion is performed with each protoplast. This cell fusion is preferably carried out by applying a known method, and the precipitate formed by mixing each of the above two types of protoplasts is mixed with a polyethylene glycol solution.
After adding the High-PH-Ca solution and leaving it to stand, a culture solution (for example, artificial seawater Asp. 12 or Provasoli's nutrient-added seawater) is added and cultured to form a fusion. The culture is preferably carried out at a temperature of 15° C. with an illuminance of 6000 Lux, a light period of 9 hours, and a dark period of 15 hours. The cell fusion product (fused cells) obtained as described above is identified (selected) by the following procedure. This distinction is made when two types of protoplasts, A and B, are fused together, in addition to a fusion of the two types of cells (A x B), a fusion of the same type of cells (B x
B and A×A) and non-fused cells are mixed, so from these, a fusion of two types of cells (A×
This is done to select B). In addition,
As a method for the above-mentioned discrimination, it is preferable to use trait markers of both cells, and this can be done by cultivar combination as described below.
It is not limited to this. Identification of cell fusions: a) Selection of cell fusions between protoplasts of Asakusanori or Susabi-nori and protoplasts of Malva laver is a round-leafed type, and the leaf length at the time of release of monospores is 0.2 to 1 mm for Asakusanori;
10 to 70 mm of Susabi nori and Maruba laver
It is 0.5 to 23 mm, and each takes advantage of the fact that they are different. In other words, it is preferable to select a cell fusion of Asakusa nori and Maruva laver that releases monospores when the leaves are narrow and about 10 mm in length. In addition, as a direct selection method, as is clear from the above experiment, by taking advantage of the remarkable difference in resistance to Akagusare disease between the Maruba laver and cultured seaweed, we infected the Akagusare disease bacteria and selected fine leaves without infected areas. You may. b) Selection of cell fusions of protoplasts of Asakusanori or Susabi nori and protoplasts of Susabi green In this case, all three types of fusions are narrow-lobed, and this marker cannot be used, so the thallus Selection is made using differences in color tone. That is,
While Asakusanori and Susabi nori both have black-brown leaves, Susabi Green has green leaves, so select those with black-brown leaves. Furthermore, since the release timing of monospores of Susabi-nori and Susabi-green is the same, this trait marker cannot be used for the fusion of Susabi-nori and Susabi-green; can be used as in (a) above, and it is best to select those that are dark brown and release monospores on leaves several tens of mm long. In addition, in the combination of the two, it is also possible to directly select by utilizing resistance to Akagase disease as in the case of (a). Furthermore, as a method for selecting cell fusions, in addition to methods a) and b) above, immediately after the cell fusion treatment, fusions containing a mixture of colors of the two types of protoplasts used for the fusion were collected under a microscope in capillary tubes. The selection may be made by For example, in Susabi Green and the above-mentioned cultured seaweed, the protoplasts in the former are green and the latter are blackish-brown in color, so it is best to select protoplasts at the moment when the green and blackish-brown colors are mixed during the cell fusion process. However, in the case of Maruba laver and the above-mentioned cultured seaweed, both have black-brown leaves, so one protoplast was stained with neutral red with the composition below and the cells were fused, and the cells were fused in the same manner as above. Try to select protoplasts at the moment when they are mixed with neutral red color. Composition of Neutral Red: Neutral Red 10 mg Artificial seawater (Asp.12) 100 ml Add and dissolve mannitol to a concentration of 0.75 M to the solution with the above composition. As mentioned above, by introducing traits that are highly resistant to Akagushi disease possessed by the wild species Maruva seaweed or Susabi green into the original cultivated seaweed, Asakusanori and Susabi nori, we have made them resistant to Akagushi disease. New varieties of cultivated seaweed can be created. In addition, in the present invention, by fusing the protoplasts of the above-mentioned Maruba laver and Susabi green, which are originally resistant to seaweed but are not suitable for cultured seaweed, we have created a seaweed that is resistant to seaweed and is suitable for aquaculture. new varieties of cultivated seaweed can be created. That is, protoplasts of Malva laver and Susabi green were prepared as described above,
Both protoplasts are subjected to cell fusion using the procedure described above, and the resulting cell fusion is selected as described below. Selection of cell fusion As mentioned above, the leaves of Maruba laver are blackish-brown and the leaves of Susabi green are green, so after performing the above cell fusion treatment,
After leaving the cells at a temperature of 12 hours, select cells that are a mixture of black, brown and green, and culture them in the same manner as described above. The cell fusion product of Maruba seaweed and Susabi green obtained in this way erases the unsuitable traits for cultured seaweed that each possessed, and possesses traits suitable for aquaculture. Because it has strong disease resistance, it will be possible to use it as a new variety of cultivated seaweed. EXAMPLES AND EFFECTS OF THE INVENTION The present invention will be described in more detail below with reference to Examples, and the effects of the present invention will also be explained. Example 1 This example shows how to introduce the akagusanori disease-resistant trait possessed by Maruba laver into cultured seaweeds Asakusanori and Susabi nori to create a variety of cultured seaweed with strong akagusa nori resistance. be. Preparation of protoplasts of laver laver, laver laver, and laver laver. Ten leaves of each of the above seaweed (leaf length 10 to 20 mm) were placed in L-shaped test tubes according to variety, and each was treated with a dilute solution of papain (M/ 10 ml of 15 Tris-HCl buffer, pH 7.4) was added and treated at 20°C with shaking (100 strokes/min). For Maruba manori and Asakusanori, use 1% papain solution for 10 minutes, and for Asakusanori,
It was treated with a 0.2% papain solution for 5 minutes. After thoroughly washing each leaf body treated as described above with seawater, it was placed in another L-shaped test tube, and Pseudomonas SP No. PT-5 (Feikoken Joyori No. BP-330) was added to it in advance. ) was cultured in a medium containing Porphyra japonica powder as a substrate. Add 10 ml of the enzyme solution (added with 0.75 M mannitol) and shake at 20°C for 60 minutes.
470 strokes/min) to produce protoplasts. Each of the enzyme-treated mixtures obtained was filtered through a 40μ mesh nylon net, the filtrate was centrifuged (1500 rpm, 5 minutes) to remove the supernatant, and the residue was mixed with an appropriate amount of artificial seawater with the following composition (with the addition of nutrients from Provasoli). seawater) to prepare protoplast suspensions. Composition of artificial seawater: Add 2ml of the following nutrients to 100ml of filtered seawater. Distilled water 100ml NaNO ₃ 350mg Na ₂ - Glycerophosphoric acid 50mg Fe (as EDTA; 1:1 mol) 2.5mg *1 P metal mixture 25ml Vitamin B ₁₂ 10μg Thiamine 0.5mg Biotin 5μg “TRIS” (Sigma Co.) 500mg PH 7.8 *2 P metal mixture composition Distilled water 100ml Na ₂ - EDTA 100mg Fe (as Cl ^- ) 1mg B (H ₃ BO ₃ ) 20mg Mn (as Cl ^- ) 4mg Zn (as Cl ^- ) 100μg Creation of cell fusion As described above Combine and mix the protoplasts of Maruba laver prepared in the above manner and protoplasts of Asakusa nori and Susabi nori, respectively, and drop 0.1 ml (approximately 10 ⁶ pieces) of each of the obtained mixtures into a Petri dish using a Pasteur pipette. 5-10
The protoplasts were left to settle on the glass surface for a minute. After adding 0.2 ml of a polyethylene glycol solution with the following composition to this precipitate and leaving it for 10 minutes,
Furthermore, 0.5 ml of High PH-Ca solution having the following composition 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 , K2PO4.H2O}
Add 0.7mM and glucose 0.1M. Composition of High PH-Ca solution: 100mM CaCl ₂ 2H ₂ O and glucose
Dissolve in distilled water to each concentration of 0.4M. 100mM NaOH-glycine buffer (PH
10.5) Dissolve glucose to a concentration of 0.4M. Mix the above solutions in a 1:1 ratio before use. Next, 0.3 ml of a culture solution made of artificial seawater (nutrient-added seawater from Provasoli) shown below was added to the solution left as described above, and after 5 minutes, 0.3 ml of the culture solution was sucked up from the Petri dish, and the above culture solution was added to it. After 5 minutes, the operation of sucking up 0.3 ml was repeated 5 times, and then the above culture solution was newly added and cultured. Cultivation is carried out at a temperature of 15℃ under 6000 Lux illumination during the light period 9.
(dark period 15 hours). Selection of cell fusions Asakusanori is narrow-leaved type;
Malva laver is a round-leaf type, and the leaf length is 0.2 to 1 mm, 10 to 70 mm, and 10 to 70 mm, respectively, at the time of release of monospores.
0.5 to 23 mm, so during cultivation under the above culture conditions, in the case of a combination of Asakusa nori and Maruba no. Add 5 ml of seawater (Provasoli's nutrient-added seawater) and monospore release treatment (under the above-mentioned culture conditions, only change the temperature from 15℃).
The temperature was changed to 20°C) and those that released monospores were selected. In addition, in the case of a combination of Porphyra spp. and P. spp., we also selected those with narrow leaves, several millimeters in length, and the ability to release monospores. The monospores released from each of the cell fusions selected in this way are cultured under the above-mentioned culture conditions, and grown to produce two new varieties of cultured seaweed that are resistant to Akagusare disease, as shown below. I got the kind. Example 2 Protoplasts of Asakusa nori, Susabi nori, and Susabi green were prepared in the same manner as described in Example 1. The papain treatment of Susabi Green was carried out at a papain concentration of 1% for 10 minutes.
Then, in the same manner as described in Example 1, protoplasts of Asakusanori and Susabi Green and Susabi Green and Susabi Green were respectively fused and cultured. Leaflets that were not infected in the test were selected. The infection test was carried out according to the method described in the text above, and on the 6th day, each leaflet was observed under a microscope and leaflets with no infection were selected. Wash the leaves thoroughly with sterile seawater, spread them out on a filter paper, place another filter paper on top, and press down.
Remove moisture and hold at 5℃ for 12 hours, then -25℃
It was held for 24 hours. Incidentally, since the Akagusare fungus has no drought resistance or freeze resistance, the leaves are kept frozen as described above. When the selected leaflets are grown as described above,
As will be described later, two new varieties of cultured seaweed possessing resistance to akagusare disease were obtained. Example 3 This example shows how to create a new variety of cultivated seaweed that is resistant to Akagushi disease and suitable for aquaculture by fusing protoplasts of the natural varieties Maruba seaweed and Susabi green. It is. According to the same procedure as described in Example 1, protoplasts of Susabi green and Maruba laver were prepared, and these were subjected to cell fusion. After the fusion treatment, the cells were left at 5° C. for 12 hours, and cell fusion was selected by picking up the mixed green and black-brown cells using an Olympus injectoscope (capillary tip diameter: 30 μm). This was cultured under the culture conditions described in the text above, to obtain seaweed suitable for aquaculture that is resistant to rust disease, as described later. Resistance test against Akagusare disease Tables 4 and 5 show the results of a resistance test against Akagusare disease on the seaweed thallus consisting of each cell fusion obtained in Examples 1 to 3. As a control, the results of a similar test on each seaweed thallus before cell fusion are also shown. Test method: The test was conducted in accordance with the experimental method for Akagusare disease described in the text above.

【table】

[Table] represents.
As shown in Tables 4 and 5, all of the cultured seaweeds made from cell fusions produced according to the present invention exhibit excellent resistance to oxidation disease, and are conventionally unsuitable for culture due to their leaf shape or leaf color. Seaweed made from a cell fusion of Susabi Green and Marva laver can now be used as cultured seaweed.

Claims (1)

[Scope of Claims] 1. Protoplasts of Susabi Green or Malva Nori, which are natural varieties with strong resistance to Akagushi disease, are prepared, and on the other hand, protoplasts of cultured seaweed are prepared, and both of the obtained protoplasts are cell-fused to obtain a cell fusion product. 1. A method for producing a cultivated type of seaweed with strong resistance to Akagusai disease, which comprises forming a cell fusion product, and then cultivating the cell fusion. 2. The method according to claim 1, wherein the cultivated seaweed is Asakusanori or Susabi-nori. 3. Prepare protoplasts of Susabi Green and Malva Nori, which are natural varieties with strong resistance to Akagushi disease, fuse both of the resulting protoplasts to form a cell fusion, and cultivate the cell fusion. A method for producing cultivated seaweed of a variety with strong resistance to akagusare disease.