JPH0229313B2 - - Google Patents

Description

[Detailed description of the invention]

Industrial Application Field The present invention relates to a method for transforming seaweed by cell fusion;
More specifically, the present invention relates to a transformation method for obtaining a seaweed possessing excellent desired traits by fusing protoplasts of two types of seaweed belonging to the genus Porphyra to form so-called hybrid cells. Conventional technology In recent years, research into so-called cell fusion, the fusion between cells of different organisms, has become very active as a genetic engineering method, and some methods have already reached the stage of practical application in land plants. There is. However, there are few research reports on cell fusion in seaweeds, especially in seaweeds, and no successful example has yet been found. Incidentally, although protoplasts of land plants such as wheat, barley, and soybeans can be easily prepared using commercially available enzymes (e.g., cellulase, macerozyme, etc.), enzymes that can convert linseed plants into protoplasts are not currently available. This is thought to be one of the reasons why cell fusion technology in laver species lags behind that of land plants. So far, as an attempt to convert laver to protoplasts, Fujita et al. reported on ``Isolation of protoplasts from laver and laver by enzyme treatment and their generation'' (Japan Fisheries Society, 1981). Autumn Lecture Abstracts, p. 32, 211)
The only thing that can be seen is that In this method by Fujita et al., about 40% of the crude enzyme solution obtained from the culture solution of Pseudomonas SP strain (strain P-1) is added to the leaf pieces obtained by physically shredding the seaweed thallus.
Protoplasts are prepared by reacting the seaweed for several hours, but this method requires a long time for the enzyme treatment to obtain the protoplasts, and in addition, the protoplasts obtained are There is a high possibility that the protoplasts will become unhealthy, and therefore cell fusion using these protoplasts may be hindered. However, in the cell fusion method, the health of the protoplasts used is a very important factor; if the protoplasts are unhealthy, the fusion rate during cell fusion will be low, and the subsequent growth in culture will be affected. This is because it is thought that it will become inferior. In addition, in order to cut the seaweed thallus using the above method, it is recognized that the enzyme produced by the Pseudomonas SP strain does not act on the surface layer of the seaweed, but acts from the cut part, breaking down the cell wall of the seaweed and forming protoplasts. It is thought that it is based on. According to a report by Preston et al., mannan exists in the form of granules on the surface of seaweed, the cell wall of seaweed is composed of xylan in the form of microfibrils, and porphyranes are thought to exist as a cell filling substance. Ru. Also, according to LAHanic et al., there is a thin coating of protein on the surface of seaweed. In other words, from the viewpoint of the structure of seaweed, it seems that it was thought that seaweed could not be transformed into protoplasts unless it was cut. The present inventor investigated the conversion of seaweed into protoplasts from the viewpoint that in order to obtain healthy seaweed protoplasts suitable for cell fusion, it is necessary to disintegrate the seaweed from its surface layer without physically cutting it. As a result, by treating the seaweed with an enzyme solution containing at least mannan hydrolase and xylan hydrolase, or further with an enzyme solution containing porphyrane hydrolase,
We obtained the knowledge that seaweed protoplasts can be prepared in a healthy state and developed a technology for converting seaweed into protoplasts. [Unexamined Patent Publication No. 41485/1983 (Patent Application No. 1983-
149378)] Problems to be Solved by the Invention With the above-mentioned success in converting seaweed into protoplasts, the present inventor prepared protoplasts from two species of seaweed belonging to the genus Porphyra, and applied a cell fusion technique. The present inventors have discovered that by creating a cytoplasmic fusion product, a seaweed that combines the excellent traits possessed by the two types of seaweed described above can be obtained, leading to the present invention. That is, an object of the present invention is to provide a transformation method for obtaining a seaweed possessing excellent desired traits by fusing protoplasts of two types of seaweed belonging to the genus Porphyra to create a cytoplasmic fusion product. It's about doing. The present invention will be explained in detail below. Structure of the Invention The present invention is based on cell fusion of seaweed, which involves preparing protoplasts of two types of seaweed belonging to the genus Porphyra, fusing these protoplasts to create a cytoplasmic fusion, and cultivating the resulting cytoplasmic fusion. The present invention relates to a transformation method characterized in that protoplasts of the seaweed are prepared by treating seaweed thallus with mannan hydrolase, xylan hydrolase and protease. Currently, there are many varieties of seaweed used for aquaculture, but the two most commonly used are Susabinori and Asakusanori. In particular, Japanese sagebrush is the most widely cultivated species in Japan, and has a strong reproductive capacity and an excellent appearance such as color and luster. The disadvantage is that the structure that is not present becomes coarse and stiff. On the other hand, although the leaves of Asakusanori are soft, fragrant, and delicious in nutrient-rich fishing grounds, they suffer from severe discoloration and poor quality in nutrient-poor fishing grounds. In addition, the wild strain of Lava laver is small and has a round leaf shape, and its growth rate is low, so it is not suitable for aquaculture, but it has the ability to withstand relatively little discoloration even in oligotrophic fishing grounds. Therefore, in the present invention, by combining the protoplasts of two of the three types of seaweed, the above-mentioned seaweeds, and combining them with cells, we can combine the excellent characteristics originally possessed by each of these seaweeds. The goal is to cultivate new varieties of seaweed with the following characteristics. In the present invention, protoplasts of each of the seaweeds described above are first prepared by treating the seaweed thallus with mannan hydrolase, xylan hydrolase, and protease. As mannan hydrolase and xylan hydrolase,
For example, microorganisms (Pseudomonas sp. PT-5,
Nori or polysaccharides derived from seaweed (residue mainly consisting of polysaccharides such as mannan or xylan obtained by hot water extraction of seaweed to remove soluble components) The resulting culture solution may be centrifuged by culturing in a medium containing as an inducer (a product obtained by further purification to increase the polysaccharide content), and the supernatant may be used as an enzyme solution. 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 that form the cell wall to be acted upon by xylan hydrolase, 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 also acts on and decomposes porphyrane as a cell filler substance of the seaweed thallus, thereby further promoting protoplast formation. In addition, during the above-mentioned protoplast formation, it is necessary to previously treat the seaweed thallus with a protease such as papain, or to allow the protease to act in parallel with the above-mentioned enzyme solution. In the present invention, cell fusion is carried out for each of two types of protoplasts obtained by producing protoplasts from Porphyra japonica, Porphyra chinensis, and P. elegans as described above. Cell fusion is preferably carried out by applying a known method, and the precipitate formed by mixing the above two types of protoplasts is mixed with a polyethylene glycol solution and
After adding the -PH-Ca solution and leaving it to stand, a culture medium (artificial seawater Asp. 12) is added thereto and cultured to create a cytoplasmic fusion. The culture is preferably carried out at a temperature of 15 degrees Celsius, an illumination intensity of 6000 Lux, a light period of 9 hours, and a dark period of 15 hours. The cytoplasmic fusions obtained as described above can be identified in the cell fusion of protoplasts of Noriflora japonica and Noriflora japonica, as well as the cell fusion of protoplasts in Noriflora japonica and Amanori japonica, as the color of each cell is different as shown below. However, in the case of cell fusion of protoplasts of P. chinensis and P. chinensis, the cells have the same color, so identification is carried out by staining the protoplasts in advance as described below. Colors of cells of each seaweed: Susabi nori (Susabi green) Green nori Nori Brown nori Brown Identification by brown staining: One of the protoplasts of Asakusa nori and Nori nori is stained in advance with neutral red having the composition below and used for cell fusion. Neutral Red 10mg Artificial seawater (Asp12) 100ml Prepare by adding and dissolving mannitol to 0.75M in a solution with the above composition. Identification of the above cytoplasmic fusion was performed under a microscope,
The fusions are selected and sucked up using a capillary tube such as a Pasteur pipette, cultured under the conditions described above, and the traits of the resulting adult leaves are evaluated. Nuclear fusion is confirmed by staining a portion of the adult leaf obtained as described above with DAPI and observing it under a fluorescence microscope. Effects of the Invention Next, Table 1 shows the results of determining the characteristics of adult leaves obtained by culturing the identified cytoplasmic fusions obtained by cell fusion according to the present invention.
The results are shown in Table 3. In addition, Tables 1 to 3 also show the characteristics originally possessed by each variety of seaweed used for cell fusion.
are shown respectively.

【table】

【table】

[Table] As shown in Table 1 above, the cytoplasmic fusion resulting from the combination of Asakusanori x Malva laver possesses the good taste inherent in Asakusanori and exhibits a trait that combines oligotrophic tolerance, which Asakusanori lacks. are doing. In other words, in the past, Asakusanori was considered to be a particularly excellent variety in terms of taste, but it had the disadvantage of causing so-called "discoloration" in malnutritive fishing grounds for cultivation, but because it is a wild species, it has not been cultivated. By cell fusion with the unsuitable Malva laver, it becomes possible to obtain a variety suitable for aquaculture that has oligotrophic tolerance. In addition, during the cultivation of Asakusanori, outbreaks of Akagushi disease are often observed, and there are currently no measures to prevent it. The fusion product obtained by cell fusion with Green exhibits the color tone of the leaves originally possessed by Asakusanori, and also exhibits a trait that is resistant to Akagusanori disease. Furthermore, as shown in Table 3, the cytoplasmic fusion resulting from the combination of Susabi Green, a mutant strain of Susabi nori, and the wild type Susabi nori, exhibits a preferable brown leaf color and a preferable leaf shape. A trait with a broad linear shape is obtained. Incidentally, the leaf shape of the Maruba laver is circular and the growth rate is poor, so it is not suitable for aquaculture. EXAMPLES The present invention will be explained in more detail with reference to Examples below. Example 1 Preparation of protoplasts of seaweed: Five leaves of Asakusa nori, approximately 1 cm in size, were immersed in a 0.2% papain solution (a solution of 20 mg of papain dissolved in 10 ml of Tris-HCl buffer with pH 7.4) at 20°C. Shake for 5 minutes at a temperature of (70 strokes/
minutes). Next, the papain-treated leaves were preliminarily treated with Pseudomonas sp.
No.) was immersed in an enzyme solution (added with 0.75M mannitol) obtained by cultivating the Porphyra spp.
minutes) to convert into protoplasts. The resulting enzyme-treated mixture was filtered through a 40μ mesh nylon net, and the filtrate was centrifuged (1500 rpm, 5 minutes).
Protoplasts were obtained in the residue. Table 4 shows the number of protoplasts produced. The number of protoplasts produced was determined by using a Thoma hemocytometer for protoplasts obtained from five leaves of about 1 cm in size. In addition, the health of the protoplasts was evaluated by determining the regeneration rate of the protoplasts obtained in this manner. The regeneration rate was determined by suspending protoplasts in artificial seawater ASP12, which will be described later, at a temperature of 15°C.
It was determined by static culture for 7 days under the conditions of 9 hours of light and 15 hours of darkness with an illuminance of 6000 Lux. The results are shown in Table 4. Protoplasts of Susabi green and Maruba laver were prepared by the same procedure as above. Cell fusion: Using a Pasteur pipette, drop 0.1 ml (10 ⁶ pieces) of the protoplast mixture, which is a mixture of two types of protoplasts, Malva laver and Susabi green, prepared as described above, into a Petri dish. The protoplasts were left to settle on the glass surface for a minute. To this precipitate, 0.2 ml of a polyethylene glycol solution having the following composition 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 A 54% aqueous solution of polyethylene glycol (MW6000) contains CaCl ₂ 2H ₂ O 10.5mM, K ₂ PO ₄ .
Add 0.7mM _H2O and 0.1M glucose. 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, add 0.3 ml of a culture solution consisting of artificial seawater Asp.12 shown below to the mixture left as described above.
After 5 minutes, suck up 0.3 ml from the Petri dish and then add the above culture solution to it. After 5 minutes, suck up that 0.3 mL 5 times, then add the above culture solution and start culturing. Ta. 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). 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 Tris-aminomethane 1g Distilled water 1000ml PH 8.0-8.1

[Table] Selection of cytoplasmic fusions: Since the protoplasts of Malva laver and Susabi green are brown in the former and green in the latter, it is possible to identify the fusions, so identify the fusions under a microscope. It was selected by sucking it up with a Pasteur pipette. The cytoplasmic fusion thus selected was cultivated and grown in the artificial seawater Asp12 at a temperature of 15° C. under 6000 Lux illumination with a light period of 9 hours (dark period of 15 hours) to obtain adult leaves. When we examined the traits of these adult leaves, we found that they possessed the traits of Maruba laver x Susabi green shown in Table 3 above. Example 2 Since the protoplasts of Marva laver and Aspergillus laver prepared by the same procedure as described in Example 1 exhibit the same brown color, the protoplasts of Aspergillus laver were pre-stained with a neutral red of the above composition, and then the protoplasts of both were dyed. Cell fusion was carried out in the same manner as described in Example 1, and the resulting cytoplasmic fusion was selected, and then cultured and grown to obtain adult leaves. When the traits of this adult leaf were examined, it was found to possess the traits of Lava laver x Asakusanori in Table 1. Comparative Example 1 Protoplasts of Aspergillus chinensis were prepared in the same manner as in Example 1, except that the culture solution shown below was used instead of the 0.2% papain solution. Regarding the obtained protoplasts, the number of produced protoplasts and the reproduction rate were determined in the same manner as in Example 1. The results are shown in Table 4. Composition of culture solution Filter sterilized seawater 1000ml NaNO ₃ 169.98mg Na ₂ HPO ₄・12H ₂ O 35.80mg NaEDTA 11.16mg FeCl ₃ 0.54mg PImetal solution 2ml PH 8.0 Composition of PImetal solution Distilled water 1000ml H ₃ BO ₃ 6.1840g MnCl ₂・4H ₂ O 0.6925g ZnCl ₂ 0.0545g CoCl ₂・6H ₂ O 2.3795mg CuCl ₂・2H ₂ O 0.0170mg

[Table] Evaluation As is clear from the results shown in Table 4, when protease treatment is performed, compared to untreated cases,
The number of protoplasts produced is much larger, and the obtained protoplasts are extremely healthy, as shown by their reproduction rate.

Claims (1)

[Scope of Claims] 1. Cell fusion of seaweed in which protoplasts of two types of seaweed belonging to the genus Porphyra are prepared, the protoplasts are fused to create a cytoplasmic fusion, and the resulting cytoplasmic fusion is grown. In the transformation method, preparation of protoplasts of the seaweed,
A transformation method characterized by treating seaweed thallus with mannan hydrolase, xylan hydrolase and protease. 2. The transformation method according to claim 1, wherein the seaweed thallus is treated with protease prior to treatment with mannan hydrolase and xylan hydrolase. 3. The transformation method according to claim 1, wherein the seaweed thallus is treated with protease in parallel with the treatment with mannan hydrolase and xylan hydrolase. 4. The transformation method according to claim 1, wherein the two types of seaweed are Susabi nori and Asakusanori. 5. The transformation method according to claim 1, wherein the two types of seaweed are Japanese laver and Japanese seaweed. 6. The transformation method according to claim 1, wherein the two types of seaweed are Asakusanori and Maruba Nori.