JPS61212280A - Production of cultivated laver of new type having high resistance to nutrition deficiency - Google Patents

Abstract

PURPOSE:To obtain cultivated laver not causing fading even if nutrition is insufficient in a fishing ground, by subjecting protoplast of laver of naturally occurring kind which has a large amount of dyestuff and strong resistance to deficiency of nutrition and protoplast of cultivated laver to cell fusion. CONSTITUTION:Protoplast of laver (Porphyra suborbiculata Kjellman, Porphyra okamurarai Ueda, Porphyra deutate Kjellman, etc.) of naturally occurring type which has a large amount of dyestuff and strong resistance to deficiency of nutrition is prepared and protoplast of cultivated laver is prepared. Both the protoplasts are subjected to cell fusion, the prepared fused cell is grown, to give cultivated laver which will not fade even in raising in a fishing ground deficient in nutrition.

Description

[Detailed Description of the Invention] The present invention utilizes a cell fusion technique to create a variety of cultured seaweed that does not fade in color and is highly resistant to poor nutrition in aquaculture in poor nutrition fishing grounds. Regarding the method.

Traditional standing technique blood plate! Seaweed is generally cultivated in fishing grounds with a somewhat nutrient-rich environment. However, recently, due to the large amount of plankton in the ocean, nitrogen and phosphorus in the seawater have decreased.
As a result, it has become a problem that the seaweed cultivation and fishing grounds are becoming oligotrophic (a decrease in the nutritional salts necessary for cultivation of seaweed due to a decrease in nitrogen and phosphorus), and the phenomenon of discoloration of cultivated seaweed is caused.

On the other hand, with the increase in seaweed production, intensive cultivation has been carried out in limited fishing grounds, and 1)
As the seaweed is harvested dozens of times during the cultivation period from March to March, the amount of nutrients in the seawater decreases as the fishing season progresses, causing the discoloration of cultivated seaweed. There is.

However, no drastic countermeasures have been taken to combat 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 discoloration of cultivated seaweed caused by oligotrophic conditions in fishing grounds have become an important issue.

In view of the above-mentioned problems in seaweed cultivation, the present inventor discovered that natural varieties of seaweed possess oligotrophic tolerance traits in the process of considering countermeasures. Under the heading, the present inventors have succeeded in introducing the above-mentioned trait into cultured seaweed that lacks it by using a cell fusion method based on the seaweed protoplastization technology that they had previously successfully developed. He came up with an invention.

In other words, the present invention solves the problem of discoloration of cultured seaweed caused by oligotrophic fishing in fishing grounds by creating a variety of cultured seaweed that has oligotrophic tolerance. The purpose of this study is to provide a method for creating cultured seaweed that is highly resistant to oligotrophic conditions using this method.

The present invention will be explained in detail below.

The feature of the present invention is that protoplasts of a natural variety of seaweed with a large amount of color and strong resistance to oligotrophy are prepared, and on the other hand, protoplasts of cultured seaweed are prepared, and both of the obtained protoplasts are subjected to cell fusion. The object of the present invention is to form a cell fusion and then grow the cell fusion to create a cultivated variety of laver with strong oligotrophic tolerance.

B. A method for fortune-telling 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 shape is close to the round leaf type, and they are densely fertilized and grow at a slow rate, only growing to a certain size. These seaweeds are not used for aquaculture, however, as mentioned above, it has been found that their oligotrophic tolerance is much stronger than that of conventionally cultivated seaweeds.

Next, we will show the results of an experiment on the oligotrophic tolerance of the above 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.

° About life.

Experimental method: As samples, three sheets of seaweed thallus (leaf length approximately 10 to 20 mm) of each variety were incubated at 16°C in an Asp.
Culture was carried out under shaking culture conditions of 6,000 lux (9 hours of light, 15 hours of clear light) and 30 strokes/minute of shaking without changing the water, and the state of color fading of each leaflet was observed with the naked eye over time. It was determined by the absorption spectrum of the leaf.

As a control, we also used As without reducing the concentration of NaN01.
p, 12 culture in culture medium was carried out in the same manner. The results are shown in Table 1.

As, 12 ゛: Distilled water 100 tall NaCl
2.8 gMgSO, 7H
200,7g MgCl26thO0,4g KCI O,07gC
a (as C1~) 40 m
gNaNOl 10 1)
gKJPk 1 m
gNa2-glycerolin a 1 mg vitamin B12 0.02 μg biotin
0. I 1) gthiamine 1
0 1! g1) pH metal mixture 1 ml
2) SII metal mixture 1 tel trisaminomethane 0.1 g pH 7,8-8.0 1) Pn metal mixture composition Distilled water 100 mj! Na2-[! D
TA 100 mgFe (as C1-
) 1 mgB (HiBOi)
2OragMn(as C1″″)
4 a+gZn(as C1-) 50
μgGo(as C1-) 100
8g2) SI [metal mixture composition Distilled water 100 vllIMo (Na
Suri o04) 5 l1) g8r (as
k") 100 mgSr(as C1-
) 20 tagRb(as C1-)
2 mgLi (as C1-)
2 sgl (ask”) 100 1)
gVa (as C1-) 10 μg As shown in Table 1, the time of discoloration of natural varieties of seaweed is considerably later than that of cultured seaweed, which indicates that natural varieties of seaweed are highly tolerant to oligotrophy. .

In the present invention, the oligotrophic tolerance trait possessed by the above-mentioned natural varieties of seaweed is introduced into cultured seaweed using a cell fusion method. Prepare.

The protoplasts of each of the above-mentioned seaweeds can be prepared using the method previously developed by the present inventor (Japanese Patent Application No. 149378/1982 or 22415/1982).

To give an overview of these methods, the former method uses microorganisms (Pseudomonas sp. PT.
-5. BP-330), seaweed or seaweed-derived polysaccharides (residues mainly consisting of polysaccharides such as mannan or xylan obtained by hot water extraction of seaweed to remove soluble components) The resulting culture solution is centrifuged by culturing in a medium containing as an inducer substance (the residue has been further purified to increase the content of polytidines), and the supernatant is used as an enzyme solution to incubate 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 hydrolase to act on xylan in the form of microfibrils that form the cell wall, allowing the cell wall of the leaf body to be degraded and removed and converted into protoplasts. Become. Furthermore, since the enzyme solution contains borphyranase, it also acts on and decomposes porphyran, which is an intercellular material of the seaweed thallus, thereby 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 the latter method, the seaweed thallus is treated with protease in advance and then treated with β-1,3-xylanase and β-1°4-mannanase, or the seaweed thallus is treated with β-1,3-xylanase and β-1°4-mannanase. It consists of treatment with 1,4-mannanase and volufilanase, and in a very short time,
Furthermore, healthy seaweed thallus protoplasts can be prepared.

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 preferably carried out by applying a known method. After adding a polyethylene glycol solution and a former gh-pH-Ca solution to the precipitate formed by mixing the two types of protoplasts mentioned above and leaving it to stand, Culture medium (e.g. artificial seawater Asp, 12 or Provaso
li (nutrient-added seawater) is added and cultured to form a fusion. In addition, the culture is carried out at a temperature of 15°C and 6000 Lux.
It is best to carry out the test under the conditions of 9 hours of brightness and 15 hours of light.

The cell fusion product (fused cells) obtained as described above is identified (selected) by the following procedure. This identification means that when two types of protoplasts, A and B, are fused, in addition to the fusion of the two types of cells (AXB), there are also fusions of the same type of cells (BXB and AXA) and unfused cells. Therefore, from these, a fusion of two types of cells (A
This is done to select XB). The method for the above-mentioned identification is preferably carried out using trait markers of both cells, and can be carried out according to the combination of varieties as described below, but is not limited thereto.

For example, when selecting a fusion product obtained by cell fusion of the protoplasts of Prunus sp. This is done by taking advantage of the difference between the two, that in the case of Asakusa nori, the leaf length is 0.2 to 1 cm, while in the case of Maruba laver, the leaf length is 0.5 to 231 cm. That is, it is preferable to select the above-mentioned cell fusion that has 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 select those with narrow leaves that do not show any shedding.

The cell fusion of protoplasts of cultured seaweed and natural seaweed obtained as described above has the oligotrophic tolerance trait possessed by natural seaweed, so the fusion is cultivated. By doing so, it becomes possible to create new varieties of cultured seaweed with strong oligotrophic tolerance. Therefore, according to the present invention, by using the cultured seaweed of the above-mentioned variety, it becomes possible to solve the problem of discoloration caused by oligotrophication of fishing grounds.

The present invention and its effects will be specifically explained with reference to Examples below. This example is an example of how to create cultured seaweed with strong oligotrophic tolerance using Asakusanori, a typical cultured seaweed, and Malva seaweed, a typical natural variety. is not limited to this.

1 Plate Preparation of each protoplast of Asakusanori and Maruba amanori: 10 pieces of each leaf of Asakusanori and Maruba amanori (
Place leaves (10-20 mm in length) in L-shaped test tubes, and add 0.2% papain solution (
M/15 ToIJS hydrochloric acid buffer, pH 7,400ml
and shaken (100 strokes/min) at 20°C for 5 minutes.
Treat for 1 minute, and add 1% 1 degree (7) of the above papain solution to 10nj! and under similar conditions 1
Processed for 0 minutes.

Next, each leaflet obtained was thoroughly washed with seawater and then placed in another rhombus-shaped test tube.
Pseudomonas) sp, NaPT-5(ii
Enzyme solution (0,7
Add 5M mannitol) and react at 20'C with shaking (70 strokes/min) for 60 minutes.
Protoplastization was performed.

Each of the enzyme-treated mixtures thus obtained was filtered through a 40 μm mesh nylon net, and the filtrate was centrifuged (150 μm).
0 rpm for 5 minutes), the supernatant was removed, and an appropriate amount of artificial seawater having the following composition (nutrient-added seawater from Provasol i) was added to the residue to prepare each protoplast suspension.

Composition of artificial seawater: Filtered seawater 1.0On+j! For that, the following nutritional supplements 2mj2
Prepared by adding.

Strawberry distilled water 100ml! NaN0q
350 mg Na2-glycerophosphate 50 mgFe (as EDTA:1
: 1 mole) 2.5 mg! lp ■Metal mixture
25m7! Vitamin B12 10
8g thiamine o, smg biotin
5μg "TRl5" (Sigma Co,) 500
mgpH7,8 XIp [Metal mixture composition Distilled water 100 taINa2-E
DT^ 100 mgFe (as C
1-) 1 mgB (HiBO3)
20 lagMn(as C1”-
) 4 tsgZn(as C1”-
) 500 μgCo (as C1-)
Mix the protoplasts of Asakusanori and Maruba Nori prepared as described above, and transfer 0.1 mJ (approximately 106 protoplasts) of this protoplast mixture into Vetri I using a Pasteur pipette. It was added dropwise and left for 5 to 10 minutes to precipitate protoplasts on the glass surface. Add 0.2 mj of a polyethylene glycol solution of the following composition to this precipitate! After adding and leaving it for 10 minutes, add High
0.5n+1 of pH-Ca solution was added and left for 5 minutes.

CaCl22H2010.5mM, KtPO+H was added to a 54% aqueous solution of polyethylene glycol α' and polyethylene glycol (H6,000).
Add 200,7+++M and glucose 0.1M.

Former h■-Ca and H20 were dissolved in distilled water to respective concentrations of 100 mM CaCl22H20 and 0.4M glucose.

■ 100mM NaOH-glycine buffer (pH
10,5) Dissolve glucose to a concentration of 0.4M.

Mix the above solutions ① and ① in a ratio of 1:1 before use.

Next, 0.3 tml of a culture solution consisting of artificial seawater (Provasol i nutrient-added seawater) shown below was added to the mixture left as described above, and after 5 minutes, 0.3 tml of the culture solution was added.
1 from the Petri dish, and then added the above culture solution to it for 5 minutes and then 0.3a+j! After repeating this operation five times, the above culture solution was newly added and cultured. Cultivation was carried out at a temperature of 15° C. under 6.000 Lux illuminance with 9 hours of light (15 hours of light).

When the leaf length of Mitsukidenkin Ikumi Sata grew to about 10 m+a, only the narrow-leaved type was placed in a microplate, one leaf at a time, and treated with artificial seawater (Provas).

! Add 5 ml of each nutrient-added seawater (I), perform monospore release treatment (change only the temperature from 15°C to 20"C under the above culture conditions), and select only those that released monospores. The spores 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.

As shown 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 Malva laver.

Claims (3)

[Claims] (1) Prepare protoplasts from a natural variety of seaweed that has a large amount of pigment and is highly resistant to oligotrophy, and on the other hand, prepare protoplasts from cultured seaweed, fuse both of the resulting protoplasts to form a cell fusion, and then A method for producing a cultivated variety of seaweed with strong oligotrophic tolerance, which comprises 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.