JPS60176523A - Seedling method utilizing protoplast of laver - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel method for seeding and seeding seaweed using seaweed protoplasts.

The life history of seaweed was elucidated by Drew of the University of Manchester in England in 1945, but later, the life history of seaweed was clarified by the filament (Conchoc-e'li).
s) By artificially managing the growing period (approximately 6 months), it is possible to attach a given number of conch spores to the seaweed cultivation net, resulting in a transition from natural collection of seaweed to artificial cultivation. The road was opened.

However, this is simply a way to artificially perform only the filamentous growth period, and it is necessary to keep the nori thallus in a net until it matures. The disadvantage was that the filamentous bodies often became unhealthy and the subsequent growth of filamentous bodies was uneven.

Under these circumstances, we developed a technique for seeding and seeding of seaweed using free filaments obtained by selecting well-developed seaweed thallus and then growing filaments from the separated fruit spores using a microbial culture method. was developed, and this technology is now widely used for seeding and seeding of seaweed.

However, the method of seeding and seeding seaweed using free filaments is
At the production stage of free filaments, it has become possible to preserve any species throughout the year, but in actual industrial production, free filaments are grown by growing them on shells in the spring.
In order to form spores, the equipment required to release the chospores in the sea in early autumn and grow them on seaweed nets is costly, and in addition, the management of these growing operations is complicated. It will be done.

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 seaweed seedling method using free filaments described above 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 it will take a long time.

In view of the current state of the seaweed seedling method as described above, the present inventor conducted research to develop a seaweed seedling technology that allows the cultivation of seaweed fronds in a relatively short period of time and with simple operations. The present invention was made based on the knowledge that protoplasts of seaweed thallus, which are obtained by converting them into protoplasts through enzyme treatment, attach to seaweed nets and grow to the thallus, similar to the chospores in the life history of seaweed. Ta.

That is, the present invention provides seaweed seeds and seedlings that can grow seaweed thallus in an extremely short period of time without going through the filamentous growth period in the life history of seaweed, by using protoplasts of seaweed thallus as seeds in place of conchospores. The purpose is to provide law.

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 from various plants has become popular.

However, in seaweeds, especially in seaweeds, the polysaccharides that make up their cell walls are different from polysaccharides (mainly cellulose) in land plants, and are mainly indigestible polysaccharides such as xylan and mannan. Therefore, its formation into protoplasts through enzymatic treatment was considered to be a regeneration process.

However, the present inventor previously discovered that the genus Pseudomonas (Ps.
By treating the seaweed fronds with an enzyme solution containing at least xylan hydrolase and mannan hydrolase prepared from a culture solution obtained by culturing a specific microorganism belonging to the genus Eudomonas in a specific medium. ,
He succeeded in producing seaweed protoplasts and invented a method for their preparation (Japanese Patent Application No. 149378/1982).

That is, the feature of the above invention is that the genus Pseudomonas (Ps.
A culture medium containing a microorganism (Feikoken Jokyo No. 330) that has the ability to hydrolyze indigestible polysaccharides (mainly xylan and mannan) belonging to S. eudomonas) and a seaweed thallus or a polysaccharide derived from a seaweed thallus as an inducer. The purpose is to treat each moss thallus with an enzyme solution containing at least xylan hydrolase and mannan hydrolase prepared from a culture solution obtained by culturing the moss in a medium to convert it into protoplasts.

Further, subsequent research revealed that, in addition to seaweed thallus or its derived polysaccharide, β-
It has been found that plants containing polysaccharides having 1,3 xyloside bonds and β-1,4 mannoside bonds as components (for example, sardine fern, konnyaku) or polysaccharides derived from these plants can also be used.

Therefore, 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.

Ill production of enzyme solution: Pseudomon, a microorganism isolated from seawater belonging to the genus Pseudomonas
as SP.

PT-5 (Feikoken Jokyo No. 330) is combined with β-L3 xyloside bonds and β
- By culturing in a medium containing a plant containing a polysaccharide having a 1,4 mannoside bond as an inducer, or a medium containing a seaweed-derived polysaccharide or a polysaccharide derived from the above-mentioned plants as an inducer, An enzyme solution containing xylan hydrolase and mannan hydrolase is obtained.

The appropriate amount of the seaweed or the plant or the polysaccharide derived therefrom to the medium is 1 to 2% by weight as the amount of polysaccharide, and the medium uses the polysaccharide as the inducer as a carbon source, To this, nitrogen sources such as peptone and yeast extract, and inorganic substances such as 1(21(PO, Pe, CIi) dissolved in seawater or artificial seawater are added, and the pH is adjusted to around 7.5 with a buffer solution. It is recommended to use

In addition, the above-mentioned Pseudomonas S used here
The mycological properties of P, PT-5 (Feikoken Joyori No. 330) are as follows.

Mycological properties: i) Morphology Regarding the cells grown in ZoBELL 2216B medium, (a) The morphology of the cells is rod and the size is 0.5-1 μm×
1.5-2.5 μm (b) Motile, with unipolar flagella (c) Durham staining is negative ii) Growth status Growth status in ZoBELL '2216E slant medium, (b) At 20-27°C Grows well at certain temperatures (mouth) Deposit pale yellow pigment (c) Forms filiform colonies iii
) Physiological properties (a) Catalase test Positive (b) Oxidase test Positive (c) Production of acid from glucose Positive (d) 0-F test 0 (by lugh Leifson method) (e) Vi
bro-3tatic Agent (0/129)
Negative test) Agar liquefaction ability + (g) Xylan decomposition ability + (h) Mannan decomposition ability + (S) .

The culture conditions for the above microorganism in the medium as described above are 25°
The process was carried out at a temperature of about C for about 4 days under stirring and aeration, and then,
The culture solution obtained by culturing in this manner is centrifuged at a low temperature of about 4° C. (10.0 OOr, p, m), and the supernatant is collected as an enzyme solution.

The enzyme solution obtained in this manner contains xylan hydrolase, mannan hydrolase, and borphyrane 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, convert 3 to 4 approximately 10 ml of the above enzyme solution concentrated 5 to 10 times.
By aging, seaweed protoplasts can be prepared. The mechanism of action of the above enzyme solution on the seaweed thallus is as follows:
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 to form microfibrillar xylan, which forms the cell wall. The purpose is to facilitate the action of xylan hydrolase in the liquid. Furthermore, the porphyran hydrolase contained in the enzyme solution acts on volphyrane, which is a cell-filling substance of the seaweed thallus, and thus promotes the formation of seaweed into protoplasts.

In addition, when converting the seaweed into protoplasts, it is more effective to use protease (for example, papain) in combination (1) because it can decompose the thin layer of protein covering the surface layer 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. In order to carry out this settlement, it is recommended to suspend the seaweed protoplasts in artificial seawater (Asp for algae, 12) and bring them into contact with the seaweed net. The material is shaken in a turbid liquid and irradiated with light for a certain period of time to promote attachment to the seaweed net.

The protoplasts that have settled on the seaweed net in this way undergo cell division and proliferate, and after about a month, more than 100 seaweed fronds with a body length of 2 to 3 cm will grow per net.

Therefore, according to the present invention, the filament phase (spring to summer), which lasts for about 6 months, in the conventional 1Jij moss life history.
It is possible to grow seaweed fronds directly from seaweed protoplasts in an extremely short period of time, without the need for a process.

Furthermore, according to the conventional method based on the life history of seaweed, seaweed has 2N chromosomes from fertilization to the filamentous stage, and undergoes meiosis to become N chromosomes at the stage of spore formation. Since seaweed has three chromosomes, its traits are 23-
8, that is, excluding the case of sudden transition, it will produce chospores with eight different traits. That is,
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 that retain their original characteristics, resulting in the production of seaweed with stable quality.

As mentioned above, by using seaweed protoplasts as seeds according to the present invention, it becomes possible to cultivate seaweed, which can be called an epoch-making method, and we believe that there will be great benefits in terms of breeding and production of seaweed.

EXAMPLES The present invention will be explained in more detail with reference to Examples below.

Example 1 Preparation of enzyme solution for protoplastization of seaweed thallus: Pseudomonas sp.
, PT-5, and BP-330 from Kaikoken were inoculated into a medium with the following composition, and the mixture was stirred at 300 r, p, m, and aerated at 25°C (aeration rate: 2000 ml/min).
Culture was performed for 4 days.

Medium composition: Polysaccharide derived from seaweed or seaweed 1.0 (wt%) Peptone 1.0 Yeast extract 0.1 tl PO+ 0, 01 FeCI: + 0.6 mg/(1 Tris buffer 0 .1 (wt%) Dissolve in seawater at the above ratio to adjust pl+ to 7.5.

To prepare an enzyme solution from the culture solution obtained by culturing as described above, the culture solution is centrifuged for 30 minutes at a temperature of 4° C. (10,000 r, p, m). Use the clear liquid as the enzyme solution.

The results of examining the enzyme activity of the enzyme solution thus prepared against indigestible polysaccharides are shown in the table below.

Table (Note) To 10+ −−−−−−−−−Activity is very strong→−
+ - - - - Strong activity + - - - - 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: (1) Place 4 to 5 sheets of seaweed thallus of about 5 cm in a molded test tube, and add papain solution (dissolved in acetate buffer to ρ116.0) lOmff. While immersing the nori fronds in the papain solution, shake at a temperature of 25°C for 20 minutes (Mono-type shaker 70 strokes/min).
I let it happen.

Place 4 to 5 pieces of seaweed leaves, each about 5 cm long, treated as described above, into an L-shaped test tube, and add 10 ml of the above enzyme solution to this.
The seaweed fronds were immersed in the enzyme solution and shaken for 4 hours (70 strokes/min) using a mono-type shaker at a temperature of 20°C to obtain seaweed protoplasts.

The protoplasts of seaweed prepared as described above were added to artificial seawater (Asp for algae, 12) with the following composition.
m1. It was suspended so that

1.11 composition of artificial seawater (Asp for algae, 12): NaC
l 28 g MgSOgin7H207g MgCI261120 4 g MCI 0.4 g CaCI22t120], 46 gNaNOm 0
．． 1 g K*PO++ 10 mg Sodium glycerophosphate 10 mg Na2SiOB 91120 150 mg Vitamin B
12 0.2 μg Biotin 1 μg Thiamine 100 μg PIIMetal 10 rnl S HMetal 10 mA' Trisaminomethane 1 g Nitrilotriacetic acid 0.1 g Distilled water 1000 m7! Adjust to pH 13.0.

The compositions of pHMetal and SrIMetal in the above composition are as follows.

Suspensions 2 of Protoplus 1 to 2 obtained as described above
0 mN was placed in a 100 m j2 capacity beaker, and 50 cremonite nets (2 to 3 cm) were placed there.
Irradiation was carried out at 6000 Lux (irradiation so that the brightness was 9 hours and 7 days) in a room at a temperature of 0.degree. C., and shaking was carried out at 40 strokes/minute using a mono shaker.

After 2 days, it was confirmed by microscopic observation that about 70% of the protoplasts had settled on the omentum, and that they were dividing and proliferating into 2 to 3 cells. After another month, the body length was 2 to 3 years old when observed with the naked eye.
It was observed that more than 100 leaves per centimeter of the net were grown.

Applicant Koyoshi Shoji Co., Ltd. Agent Miya 1) Yutaka Hiro

Claims (1)

[Scope of Claims] (]) A seeding method using seaweed protoplasts, which is characterized in that seaweed protoplasts obtained by converting seaweed bodies into protoplasts by enzymatic treatment are grown by growing them on a seaweed net. (2) Claim No. 1 (Seed method according to Claims 1 and 1), in which seaweed protoplasts suspended in artificial seawater are attached to a seaweed net and grown.