KR100540071B1 - Method of producing Grateloupia filicina seedlings from spores - Google Patents

Abstract

The present invention relates to a method of producing seedlings from the spores of the genus Noir, more specifically, 1) attaching to the engaged in large quantities by releasing the spores in the shade for efficient spore release of the mature leaf of the genus Noir; 2) culturing the spores attached to the hook to form the half root of the spores; And 3) growing seedlings by culturing the wedges attached to the rhizome. Seedling production method of the sesame noir of the present invention can be usefully used for the mass production of the sesame noir, which relies on natural production only and the amount of resources is rapidly decreasing due to overfishing.

Description

Method of producing Grateloupia filicina seedlings from spores             

Figure 1 shows the genuari (A) and the genuari (B) in the habitat, tetraspores in tissue cutting and tetraspores in trophoblasts (C and D), supersporangia in tissue cutting and immediately before release. E and F) pictures,

2 is a graph showing the monthly growth of the genus Noir leaf with a supersporangia,

Figure 3 is a graph showing the monthly growth of sesame noir spores of mature maturity,

Figure 4 is a graph showing the monthly growth of sesame noir tetrasporus itself of sexual maturity,

5 is a graph showing the comparison of the size of the leaf of the genus Noir tetrasporae and superspores of mature maturity,

6 is a comparative photograph observing the appearance of the real Zinosaur tetraspores and superspores,

Figure 7 is a graph showing the maturity of maturity and maturity type of the Charm Noir

8A and 8B show the release of superspores (A), tetrasporangia (B), cell migration (C), cell migration and rhizome formation (C), and early root germination (E) F), sphincter development (G), spore development and leaf germination leading to rhizome and ectomus (H) and leaf germination (I and J),

9 is a graph comparing the discharge of the Chinois spores according to the emission induction conditions,

FIG. 10 is a photograph showing an experiment in which the Ginari spores attached to Guraron yarn are grown in Guraron, mass cultivation of true Zinari seedlings through growth (A), outdoor culture (B) and constant temperature mass culture (C).

The present invention relates to a method for producing seedlings of faucets, and more particularly, to a method for producing seedlings of faucets by attaching induced and released sesame seeds to spores.

The genus is widely distributed all over the world, and 6 genera and 21 species in Korea are known to inhabit the east and south coasts. The body is somewhat flattened in the rectus muscle and is a flat linear form with short stems. It is 20 to 30 cm in height, 2 to 3 mm in width, and has a slightly distinct main axis, branching from both edges to the upper right. The upper part of the branch of the main axis usually extends long so that there are no small branches, and sometimes a small branch may appear on the surface.

The fronds of the genus are gathered from the ectomus and have one distinct main axis, branched one or two times on both sides of the main axis, and the lower part of the stem is biased and filled with mucus. Main axis is wider and thicker than branch. As it goes up, it becomes circumferential and becomes long and sharp. The color of fronds is bright red to pink, the lower part is cartilaginous, soft and slippery, and the inner part of the frond is divided into cortex and inner layer and cell rows reach 10 to 11 rows. Cortical layers of right-branched leaflets are 6-8 cell layers, cells grow larger toward the inside, cells are 5-7 μm, and length is 7-15 μm.

Charcoal noir has a change in body shape, and the plants belonging to it are G. prolongata , G. divaricata , G. okamurai , G. turuturu , etc. It is used as an edible or flavor.

At present, the savory noir is collected from early October to mid-March and sold at high prices. However, since it relies only on natural production, the fishery's income is not only limited. Therefore, there is an urgent need to develop a mass production method of the genius using artificial seedling production.

On the other hand, conventional techniques related to seaweed culture include artificial farming methods of spp. Red algae using spores (Korean Patent Application No. 1986-5332), and recently produced seedlings of red grateloupia acuminate by tissue culture. The method has been patented (Patent Publication 2002-82233). The patent relates to tissue culture of the cousin of Chinoisuri, using the method of harvesting the genuaris cousin from the sea to obtain tetraspores themselves, cutting the tissues to obtain regenerated tissues, and then obtaining tetraspores. There is a way to increase after fitting it to the ropes grown for a month in a tissue culture lab yeopche pieces reported in jinu Ari (Grateloupia filicina) in nearby Japan (Seiji Migita, 1988, Nippon Suisan Gakkaishi). In the above method, the superspores and tetraspores are differentiated into mature leaflets to make Jinari leaves, and the leaflets are cut into many sections, and then each tissue section is cultured in a petri-dish to redifferentiate the new leaflets. Get After 35 days of incubation, the leaflets grow to about 1.2 mm, and every year the tissue sections are re-differentiated to obtain a number of leaflets, which are then sprinkled on oyster shells or laver synthetic twine for a month. Raised in the laboratory. Here, many leaves grow, and after about two months of about 15 cm, they are transferred to the sea and cultured. However, the method of culturing the genius leaf body tissues and cultivating them in the sea has a disadvantage that imitation is difficult to grow properly in the sea and thus requires a more improved method of growing the genius.

In order to produce seaweed seedlings, seedlings must be grown in the field of employment, and as described above, the method of planting the leaf tissue sections after incubating the leaf tissue is difficult to plant the tissue sections in the field and is more difficult to plant densely. . As such, it is difficult to plant tissue sections densely, and since the planted tissues do not have roots yet, the proportion of seedlings that are lost in seawater and settled in the field before the roots grow is very low, and the culture of Ginari is still practical. Did not succeed.

Thus, the present inventors can devise a method for releasing a large amount of spores derived from the mature leaf of the genus Noir can attach a large amount of spores to the entrepreneurs, the roots by culturing the spores attached to the spores in the medium The present invention has been completed by developing a method for mass production of seedlings of sesame seeds by establishing conditions that can be lowered.

It is an object of the present invention to provide a method for producing seedlings of sesame noir in large quantities by attaching induced and released sesame noir spores to the entrails.

In order to achieve the above object, the present invention comprises the steps of: 1) attaching to the engaged by releasing a large amount of spores of the genus Noir; 2) culturing the spores attached to the hook to form the half root of the spores; And 3) provides a method of producing seedlings from the spores of the savanna Noir comprising the step of culturing the haptic attached to the half root.

Hereinafter, the present invention will be described in detail.

The present invention

1) releasing a large amount of spores of mature leaflets of the sesame noir to attach to the engaged;

2) culturing the spores attached to the hook to form the half root of the spores; And

3) It provides a method for producing seedlings from the spores of sesame noir including the step of cultivating the working muscles attached to the half-roots.

In step 1, the mature leaves are preferably leaves that have been shaded for 10 to 12 hours, and more preferably those that have been shaded for 12 hours for effective spore release.

In step 2, the spores may be superspores or tetrasporers, more preferably spores. Engagement may be selected from the group consisting of guraron yarn, light shielding film, palm yarn and nylon, more preferably guraron yarn. In addition, the medium is preferably PES medium.

In addition, the culture conditions are preferably a temperature of 15 to 20 ℃, the roughness is preferably 3,000 to 3500 lux, photoperiod is preferably 10 to 12L: 14 to 12D. Therefore, the optimum culture conditions are most preferably cultured in 20 ℃, 3000 lux and 12L: 12D photoperiod environment. In the photoperiod, L and D represent light and dark, respectively.

The indoor seedling production of sesame noir can be produced by incubating indoors of the semi-roots and spores attached to the fabricated through the above method.

In the present invention, the spores of the genus chinensis or the tetraspores were spores attached to Guronon, followed by mass culture. In the experimental zone incubated in the superspores, half-rats were formed on the 20th day of culture (see FIGS. 8A and 8B).

As a result of investigating the distribution and environment of Jinari on the east coast of Korea, we confirmed the survival condition of Jinari. The tetraspores and superspores appeared from September to February, and as a result of spore culture test, cytoplasmic migration began in one day at 20 ℃, 3000 lux, 12L: 12D condition, and rooting began to germinate two days later. After this time, the root length was developed to 100 μm, and the superspores and tetraspores showed the same developmental shape (see FIGS. 8A and 8B). The tetraspores and superspores developed in the form of ectomus muscle after about 30 days, and the size was about 80-120 μm (97 μm on average), and the upright lobes germinated around 40 days (see Table 2).

The present inventors examined the distribution and habitat environment of Jinari at two months interval from February to November in Goseong-gun, Daeseong, Yangyang-gun, Hosan, Samcheok-si, Hosan, Pohang, Guryongpo, Gangwon-do. Water temperature, salinity, dissolved oxygen, pH, and Chl-a (in units of chlorophyll as the unit of chlorophyll) in the Jianuri habitat, YSI 6000 (Yellow Spring Incorporated, USA) and a spectrophotometer ( Varian australia PTY LTD, Australia). The environment of the East Coast Zinari distribution region is summarized in Table 1 below.

Environment of the East Coast Zinari Area (February to November) area Water temperature (℃) Salinity ^_(0/00) Dissolved Oxygen (mg / ℓ) pH Chl-a (μg / L) Goseong-gun Daejin 6.4 to 25.5 32.2 to 33.9 5.8 to 8.3 7.6 to 8.5 0.8 to 1.9 Yangyang-gun Garden 6.8-25.8 32.5 to 33.2 6.8 to 8.5 7.7-8.3 0.7 to 1.8 Samcheok-si Hosan 6.6 to 25.7 18.6-25.1 6.5-8.6 7.5-8.0 0.9 to 2.0 Pohang Guryongpo 7.0-26.8 32.5 ~ 32.9 6.3 to 8.2 7.4 to 8.1 0.7 to 1.5

As can be seen in Table 1, the water temperature of the habitat environment of the genuari ranged from 6.4 to 26.9 ° C and the salinity ranged from 18.6 to 33.9. Samcheok-si had a low salinity of 18.6 to 25.1 due to freshwater only. In addition, dissolved oxygen was found to be 5.8 to 8.6 mg / l.

In order to confirm the life history of the Jinuaari, we collected and observed the True Jinua each month from Daejin, Gangseong-do, Daejin, Sokcho-si, Jangsa-dong, and Samsan-si Hosan.

As a result, the sperm nodule was made of fertilized cells by fertilization between sperm made in testes and eggs made in pottery, and fertilized hairs had fertilized hair and fertilization was made through conjunctiva. The crude cells were developed into superspores with the development of hematopoietic stem cells, and the size of the supersporangia was 70 to 98 μm (87 μm on average) (FIG. 1E). The released spores were round to 15-23 μm (18 μm on average) (FIG. 1F).

The leaves with supersporangia through fertilization appeared around September, and the leaf size was 12.4 ± 4.45 cm (Fig. 2). The leafs with supersporangia persisted until January of the following year, and the leaf size was 7.6 ± 5.4 cm when the release was completed and the end recording began from April. Leafs were not found in June and July, and appeared in August and showed a size of 8.8 ± 2.3 cm.

The leaflets with tetrasporangia also appeared from September, the same period as the leafs with supersporangia. At this time, the size of tetraspores ranged from 16 to 22 µm (average 17 µm) and 15 to 23 µm (18 µm average). Its shape and color are similar to, making it difficult to distinguish between them. At the same time as the superspores, end-recording phenomena and emergence appeared to be simultaneously occurring in the leaf formation by the superspores and the leaf formation by the tetraspores (FIG. 3 and FIG. 4).

As a result of investigating the leaves of the tetrasporangia and superspores of mature maturity, the tetraspores were 18.95 ± 6.1 ㎝ and the superspores were 15.61 ± 4.8 ㎝ (Fig. 5). Although slightly protruding and rough leaf model was able to distinguish somewhat in appearance, it was possible to observe the microscope by tissue cutting for accurate classification (Fig. 6). The distribution ratio of tetraspores and superspores of adult maturity was examined by month. In September, when sexual maturity began, 30% of tetraspores, 20% of superspores, and 50% of the leaves with immature vegetative cells. In October, tetraspores were 60% and superspores were 40%. In February, tetraspores were 42% and superspores were 23%.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Experimental Example 1 Spore Release from Mature Nosy Fructus

The sampled leaves were washed three times with a brush in filtered sterile seawater, and then subjected to one-time disinfection treatment in 10% Povidone-iodine solution for 1 minute to determine maturity due to tissue cutting. . PES (Provasoli's Enriched Seawater) media stock was diluted in filtered sterilized seawater (121 ° C., 1.2 kg / cm 2) in a plate (12 × 3 cm) for incubation using the obtained spores, and cultured at 20 ° C., 3,000 lux, 12L. : 12D environment was set.

The preparation method of PES medium is as follows.

First, a stock solution was prepared by dissolving 1.00 g of Na ₂ glycerophosphate, 7.00 g of NaNO ₃ , 8.14 g of Tris HCl, and 1.86 g of Tris Base or 10 g (pH 7.8) in 970 ml of distilled water. It was. 20 ml of a sterilized B vitamin mixture, 500 ml of sterile Fe (as EDTA 1: 1 molar solution), 500 ml of sterile PII trace metal mixture and 10 ml of KI solution (0.25 mg / ml) were mixed with the stock solution. PES medium stock was then prepared by adding sterile 4 ml GeO ₂ stock solution. 20 ml of PES medium stock was diluted in 1,000 ml of sterile seawater and used for cultivation of Jinari leaves.

The composition of the B vitamin mixed solution, Fe (as EDTA 1: 1 molar solution), PII trace metal mixed solution and GeO ₂ stock solution is shown below.

① B vitamin mixture Vitamin B ₁₂ 1.0 mg Thiamine 50.0 mg Biotin 0.5 mg ------------------------- Distilled water 100 ml

② Fe (as EDTA 1: 1 molar solution) Fe (NH ₄ ) ₂ (SO ₄ ) * 6H ₂ O 1.40 g (alternative) Fe ₂ (NH ₄ ) ₂ (SO ₄ ) * 24H ₂ O 2.12 g Na ₂ EDTA 1.32 g ------------------------------- 2,000 ml of distilled water

③ PⅡ trace metal mixture Boric acid (H ₃ BO ₃ ) 2.24 g Fe (FeCl ₃ ) * 6H ₂ O 0.096 g Mn (MnSO ₄ ) 0.24 g Zn (ZnSO ₄ ) * 7H ₂ O 0.044 g Co (CoSO ₄ ) * 7H ₂ O 0.0096 g Na ₂ EDTA 2.00 g ------------------------------------ Distilled water 2,000 ml

④ GeO ₂ stock solution (optional) GeO ₂ 0.25 g -------------------------------------- 1,000 ml of distilled water

The superspores and tetradistributors induced by the above method (A and B in Fig. 8a) began to move cytoplasm in a day at 20 ℃, 3000 lux, 12L: 12D culture conditions (Fig. 8a C) After 2 days, roots began to germinate (D and E in FIG. 8A), and after 13 days, the length of the roots was 100 μm, showing the same developmental shape (F in FIG. 8A). Tetraspores and superspores developed in the form of ectomus after about 30 days of results (G and H in FIG. 8B) and the size was about 80-120 μm (97 μm on average) and developed into lobes (I in FIG. 8B). And J).

Experimental Example 2 Comparative Experiment of Spore Development According to Temperature

To determine the proper incubation temperature of the spores, set the temperature at 5 ° C, 10 ° C, 15 ° C, 20 ° C, 25 ° C and 30 ° C, and put the slide glass together with the leaves on the plate (12 × 3cm) every week. The PES medium was replaced with a microscope. Spore release, cell migration, rhizome germination, rhizome disappearance, hepatic muscle and lobe germination were compared according to temperature (Table 2).

Development time (days) of spores by temperature Development environment Spore release Cell migration Root germination Extinction Normal root 60㎛ Normal root 100㎛ Leaf germination 5 ℃ 2:20 minutes 1.5 4 28 Test _ _ 10 ℃ 2:00 min 1.3 2.5 25 36 45 Half head 15 ℃ 1:30 min 20:00 minutes 2.2 23 30 38 Half head 20 ℃ 1:10 minutes 15:00 minutes 1.6 20 25 30 40 25 ℃ 1:00 min 10:00 min One 15 60% death 80% test _ 30 ℃ 0:30 minutes 7:00 minutes 23:00 minutes Test - _ _

As a result, the developmental speed was very slow at 5 ℃ compared to other test groups, spore release was about 2 hours 20 minutes and cell migration took 1.5 days. The root germination took place after 4 days, and the extinction took 28 days. Spore release in 2 hours and cell migration took 1.3 days. It took 2.5 days for rooting germination and 36 days for sputum muscle formation and 45 days for sputum muscle size of 100 ㎛. Spore release occurred at 15 ° C in 1 hour 30 minutes, cell migration in 20 hours, and leaf germination did not occur after 40 days. The spores were released at about 1 hour and 10 minutes, cell migration was only 15 hours, and the formation of ectomus was only 25 days. Spore release occurred after 1 hour at 25 ℃, and cell migration was started after 10 hours, and root germination started after 1 day, but 60% of them were dyed by the formation of apex. The spores were released in 30 minutes and spores were released in 30 minutes, and the cell development was rapid in 7 hours. From the results, it was confirmed that the proper incubation temperature of the spores is 20 ℃.

Experimental Example 3 Comparison of Spore Release Rate According to the Condition of Matured Leaves

To determine the proper culture environment of spores, the amount of spores released according to the conditions was compared. Mature spores (approximately 20 g of wet weight, around 12 ± 3 cm in leaves) were placed on the plate and immediately spores released after sampling at 2 h, 4 h and 6 h at 20 ° C, 3000 lux, 12L: 12D Induced, the spores were compared to 30 minutes sunlight exposure, 12 hours refrigerated (4 ℃) and 12 hours in the room shade treatment was compared to each other.

As a result, 30 ± 4 test specimens induced immediate spore release, 33 ± 5 test specimens exposed to sunlight for 30 minutes, 42 ± 3 test specimens refrigerated for 12 hours, and 78 ± test specimens treated with 12 hours indoor shade. Five spores were released (FIG. 9). From the above results, it was confirmed that the amount of spores released after 12 hours of indoor shade treatment was the highest, and only a small amount of spores were released from the test data for 6 hours.

Example 1 Production of Sesame Noir Seedlings Using Spores

For mass production of spores, 50 g of mature superspores and tetraspores were separated and cultured using a UV-filtered seawater (1 μm filter) in a 5 L acrylic bath. After putting the Guraron company and the slide glass engaged in the 5 l acrylic tank and incubated for 12 hours by setting the environment to 20 ℃, 12L: 12D, 3,000 lux (Fig. 10 A to C). As a result, it was observed that the eccentric muscle was formed in the Guralon company on the 20th day of culture. When observed under a microscope, five to seven testicular cultivars and three to five tetraspores were cultured in the superspores, and it was confirmed that more spores were produced when cultured in the superspores.

As described above, the seedling production method of sesame noir of the present invention can be usefully used for mass production of seedlings of the genuari, whose resources are rapidly decreasing due to overfishing and the like.

Claims (10)

1) releasing a large amount of spores by dry-drying the mature leaf of the sesame noir and attaching it to a working place; 2) culturing the spores attached to the hook to form the half root of the spores; And 3) A method of producing seedlings from the spores of tuna chinensis, comprising the step of cultivating the stratum muscles attached to the half-roots. The method of claim 1, wherein the shade treatment of step 1 is performed for 12 hours. The method of claim 1, wherein said spores of step 2 are superspores or tetradistributors. The method of claim 1, wherein the engagement of step 2 is gouron. The method of claim 1, wherein the medium of step 2 is Provasoli's Enriched Seawater (PES) medium. The method of claim 5, wherein the PES medium is Na ₂ glycerophosphate, NaNO ₃ , Tris HCl, Tris Base, B vitamin mixture, Fe solution (as EDTA 1: 1 molar solution), PII trace metal mixture, KI solution and GeO ₂ A stock solution. The method of claim 1, wherein the culture conditions of step 2 is characterized in that the temperature range of 15 to 20 ℃. The method of claim 1, wherein the culture conditions of step 2 is characterized in that the roughness range of 3,000 to 3,500 lux. The method of claim 1, wherein the culture conditions of step 2 is characterized in that 10 to 12L: 14 to 12D photoperiod. The method of claim 7, wherein the culture conditions are 20 ° C., 3,000 lux and 12L: 12D photoperiod.

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