JPH031954B2 - - Google Patents

Description

[Detailed description of the invention]

FIELD OF INDUSTRIAL APPLICATION The present invention relates to a method of producing cultured seaweed of a variety that does not lose its color and has strong oligotrophic tolerance in aquaculture in oligotrophic fishing grounds using a cell fusion technique. Conventional technical background Seaweed is generally cultivated in fishing grounds with a somewhat nutrient-rich environment. However, recently, due to the large amount of plankton in the sea, nitrogen and phosphorus in the seawater have decreased, resulting in oligotrophic conditions in seaweed farming fishing grounds (a decrease in nutrients necessary for seaweed cultivation due to a decrease in nitrogen and phosphorus). It has become a problem that this causes color fading in cultivated seaweed. On the other hand, as the production of seaweed increases, intensive cultivation is carried out in limited fishing grounds, and moreover, seaweed is harvested dozens of times during the cultivation period from November to March. Additionally, as the fishing season progresses, the amount of nutrient salts in seawater decreases, causing the discoloration of cultivated seaweed. However, no drastic measures have been taken to address the problem of malnutrition in seaweed farming areas as described above, and nitrogen source fertilizers such as ammonium sulfate have been introduced in response to the occurrence of the discoloration phenomenon described above. All that is being done is spraying the fishing grounds. However, fertilization in such fishing grounds is not very effective in preventing discoloration because the fertilizer inevitably washes away, and in addition, clams and oysters are often farmed in the same fishing grounds. Fertilizer application to fishing grounds has recently been restricted due to the impact it has on aquaculture. Therefore, measures to prevent the discoloration of cultivated seaweed caused by oligotrophic conditions in fishing grounds have become an important issue. Problems to be Solved by the Invention In view of the above-mentioned problems in cultivating seaweed, the present inventor discovered, in the process of considering countermeasures, that natural varieties of seaweed possess oligotrophic tolerance traits. Based on the protoplastization technology of seaweed that the present invention had previously successfully developed, the present invention succeeded in introducing the above-mentioned trait into cultured seaweed that lacks it, using a cell fusion method. He came up with an invention. That is, the present invention solves the problem of discoloration of cultured seaweed caused by oligotrophic feeding in fishing grounds by creating cultured seaweed of a variety possessing oligotrophic tolerance. The purpose of this study is to provide a method for creating cultured seaweed varieties with strong oligotrophic tolerance using fusion techniques. The present invention will be explained in detail below. Structure of the Invention The present invention is characterized by preparing protoplasts of a natural variety of seaweed that has a large amount of pigment and is highly resistant to oligotrophy.
On the other hand, by preparing protoplasts of cultured seaweed, fusing both of the resulting protoplasts to form a cell fusion, and then cultivating the cell fusion, we plan to create a variety of cultured seaweed with strong oligotrophic tolerance. be. Means for Solving the Problems The natural variety of seaweed with strong oligotrophic tolerance used in the present invention is commonly called rock seaweed, and is a wild species that grows attached to rocks. It is possible. These seaweeds generally have the same dark brown to black color as cultured seaweed, but their leaf shapes are round-leaf or similar.
It is not used for aquaculture because it is densely fertilized, has a slow growth rate, and only grows to a certain size. However, as mentioned above, it has been found that these seaweeds are much more resistant to oligotrophy than conventionally cultivated seaweeds. Next, we will show the results of an experiment on the oligotrophic tolerance of the above-mentioned natural varieties of seaweed in comparison with cultured seaweed. Note that the term "cultured seaweed" as used herein refers to Asakusanori and Susabi-nori, which are mainly used among the many varieties of seaweed currently used for cultivation. Experimental method for testing oligotrophic tolerance of seaweed: Three sheets of seaweed thallus (leaf length approximately 10 to 20 mm) of each variety were used as samples, and the concentration of NaNO ₃ alone was 1/1000.
16℃ in Asp.12 culture solution with the following composition reduced to
Culture was carried out under conditions of shaking culture at 6000 lux (9 hours of light, 15 hours of darkness) and 30 strokes/minute of shaking, without changing the water, and the state of discoloration of each leaflet was observed with the naked eye over time. It was investigated by observation and absorption spectra of leaf bodies. In addition, as a control, cultivation in Asp.12 culture solution without reducing the concentration of NaNO ₃ was carried out in the same manner. The results are shown in Table 1. Composition of Asp.12 culture solution: Distilled water 100ml NaCl 2.8g MgSO ₄ 7H ₂ O 0.7g MgCl ₂・6H ₂ O 0.4g KCl 0.07g Ca (as Cl ^- ) 40mg NaNO ₃ 10μg K ₂ HPO ₄ 1mg Na ₂ - Glyceric acid 1mg Vitamin B ₁₂ 0.02μg Biotin 0.1μg Thiamine 10μg 1) P metal mixture 1ml 2) S metal mixture 1ml Tris-aminomethane 0.1g PH 7.8-8.0 1) 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 ^- ) 50μg Co (as Cl ^- ) 100μg 2) S metal mixture composition distilled water 100ml Mo (Na ₂ MoO ₄ ) 5mg Br (as k ⁺ ) 100mg Sr (as Cl ^- ) 20mg Rb (as Cl ^- ) 2mg Li (as Cl ^- ) 2mg I (as k ⁺ ) 100μg Va (as Cl ^- ) 10μg

[Table] As shown in Table 1, the onset of discoloration of natural varieties of seaweed is considerably later than that of cultured seaweed, which indicates that natural varieties of seaweed are more tolerant to oligotrophy. In the present invention, the oligotrophic tolerance trait possessed by natural seaweed varieties as described above is introduced into cultured seaweed using a cell fusion method. Prepare. The protoplasts of each of the above seaweeds can be prepared by the method previously developed by the present inventor (Japanese Patent Application No. 58-149378 (Japanese Patent Application Laid-Open No. 60-41485) or Japanese Patent Application No. 59-22415 (Japanese Patent Application Laid-Open No. 60-168381). This can be done by applying the following. To give an overview of these methods, the former method uses a microorganism (Pseudomonas SP No. Article No. BP-330)
, seaweed or seaweed-derived polysaccharides (obtained by hot water extraction of seaweed to remove soluble components; residue mainly consisting of polysaccharides such as mannan or xylan, or the residue further purified to reduce the polysaccharide content) The method consists of centrifuging the culture solution obtained by culturing in a medium containing an inducing substance (e.g., increased concentration of thallus) and treating the nori fronds using the supernatant as an enzyme solution. The above enzyme solution contains mannan hydrolase and chilacin hydrolase, so when the enzyme solution is applied to the seaweed leaflets, 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 hydrolase to act on xylan in the form of microfibrils that form the cell wall, and the cell wall of the leaf body is decomposed and removed to form protoplasts. become. 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, the seaweed thallus is treated with protease in advance, and then β-1,3-xylanase and β
-1,4-mannanase treatment 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 the natural variety Maruva laver are prepared by the method described above, and protoplasts of cultured seaweeds Asakusanori and Susabi nori are prepared in the same manner, and the protoplasts of Maruva laver and each protoplast of Asakusanori or Susabi nori are cell-fused. let This cell fusion is best carried out by applying a known method. After adding a polyethylene glycol solution and a High-PH-Ca solution to the precipitate formed by mixing each of the two types of protoplasts mentioned above and leaving it to stand, culture this. liquid (e.g. 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. For example, when selecting a fusion product obtained by cell fusion of the protoplasts of Prunus sp. In Asakusa nori, the leaf length is 0.2 to 1 mm, while in Malva nori, the leaf length is 0.2 to 1 mm.
This is done by taking advantage of the difference between the two, which is when the leaf length is 0.5 to 23 mm. That is, it is preferable to select the above-mentioned cell fusion that has a narrow leaf type and releases monospores when the leaf length is about 10 mm. In addition, as a direct selection method, as mentioned above, taking advantage of the fact that the oligotrophic tolerance of the two is significantly different, the cell fusion described above is cultured in a nitrogen-deficient medium, and after an appropriate number of days, the color changes. You may also select those with narrow leaves that do not show any shedding. The cell fusion of protoplasts of cultured seaweed and natural variety of seaweed obtained as described above has the oligotrophic tolerance trait possessed by the natural variety of seaweed introduced, so the fusion must be cultivated. According to
It becomes possible to create new varieties of cultured seaweed that are highly resistant to poor nutrition. Therefore, according to the present invention, by using cultured seaweed of the above-mentioned variety, it becomes possible to solve the problem of discoloration caused by oligotrophic fishing grounds. EXAMPLES AND EFFECTS OF THE INVENTION The present invention and its effects will be specifically explained below with reference to Examples. This example is an example of how to create a cultured seaweed with strong oligotrophic tolerance using Asakusa nori, which is a typical cultured seaweed, and Malva seaweed, which is a typical natural variety. is not limited to this. Example: Preparation of protoplasts of Prunus chinensis and Porphyra chinensis:
Place 10 leaves (leaf length 10-20 mm) in each L-shaped test tube, and add 0.2% papain solution (M/15 Tris-HCl buffer, PH 7.4) to the leaves of Asakusanori.
ml and shaken at 20°C (100 strokes/min).
10 ml of the above-mentioned papain solution with a concentration of 1% was added to the leaves of Lava laver for 10 minutes under the same conditions. Next, each of the obtained leaf bodies was thoroughly washed with seawater and then placed in another L-shaped test tube, and each one was preliminarily infected with Pseudomonas sp. No. PT-5 (Feikoken Joyori No. BP). -330) in a medium with Porphyra japonica powder as a substrate, add 10ml of the enzyme solution (added with 0.75M mannitol), and react at 20°C with shaking (70 strokes/min) for 60 minutes. I made it into a protoplast. Each enzyme-treated mixture obtained in this way was
Filter through a 40μ mesh nylon net, centrifuge the filtrate (150 rpm, 5 minutes) and remove the supernatant.
An appropriate amount of artificial seawater having the composition shown below (nutrient-added seawater from Provasoli) was added to the residue to prepare each protoplast suspension. Composition of artificial seawater: Prepared by adding 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 *1 P metal mixture composition Distilled water 100ml Na ₂ - EDTA 10mg Fe (as Cl ^- ) 1mg B (H ₃ BO ₃ ) 20mg Mn (as Cl ^- ) 4mg Zn (as Cl ^- ) 500μg Co (as Cl ^- ) 100μg Creation of cell fusion: Mix the protoplasts of Asakusa nori and Malva nori prepared as described above, drop 0.1ml (approximately 10 ⁶ protoplasts) of this protoplast mixture into a Petri dish with a Pasteur pipette, and The protoplasts were left to settle on the glass surface for 10 minutes. To this precipitate, 0.2 ml of a polyethylene glycol solution having the composition shown below was added and left to stand for 10 minutes, and then 0.5 ml of a High PH-Ca solution having the following composition was added and left to stand for 5 minutes. Composition of polyethylene glycol solution: 54% aqueous solution of polyethylene glycol (MW6000) with 10.5mM of CaCl ₂ 2H ₂ O, K ₂ PO ₄ H ₂ O
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-chrysin 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 consisting 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 then added to the above culture solution. 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 was carried out at a temperature of 15° C. under 6000 Lux illumination with a light period of 9 hours (dark period of 15 hours). Selection of cell fusions When the leaves have grown to approximately 10 mm in length, only the narrow-leaved type is placed in a microplate, one leaf at a time, and 5 ml of artificial seawater (Provasoli's nutrient-added seawater) is added to each plate, followed by monospore release treatment (the culture described above). The temperature was changed from 15°C to 20°C), and only those that released monospores were selected, and the monospores were cultured and grown under the above-mentioned culture conditions to obtain adult leaves. Next, the oligotrophic tolerance of the adult leaves obtained as described above was tested and determined in accordance with the experimental method described in the text above. The results are shown in Table 2. For comparison, similar experiments were conducted on adult leaves of Asakusa nori and Maruba nori that were not subjected to cell fusion treatment, and the results are also shown in the table.

[Table] As seen in Table 2, it can be seen that the oligotrophic tolerance of the adult leaves of the seaweed made of the cell fusion obtained according to the present inventors is rather stronger than that of the adult leaves of the natural variety Marva laver.

Claims (1)

[Scope of Claims] 1. Protoplasts of a natural variety of seaweed with a high pigment content and strong oligotrophic tolerance are prepared, and on the other hand, protoplasts of cultured seaweed are prepared, and both protoplasts obtained are subjected to cell fusion to obtain a cell fusion product. form,
A method for producing a cultivated variety of seaweed with strong oligotrophic tolerance, which comprises then cultivating the cell fusion. 2. The production method according to claim 1, wherein the natural variety of seaweed is Malva laver. 3. The production method according to claim 1, wherein the cultured seaweed is Asakusanori or Susabi-nori.