KR20130001479A - A method for mass production of undariopsis peterseniana using regeneration and maturation induction of free-living gametophytes of undariopsis peterseniana - Google Patents

Abstract

PURPOSE: A undaria peterseniana mass production method by inducing the regeneration and aging of undaria peterseniana free-living gametophytes is provided by securing the optimal condition for the regeneration and aging of the Undaria peterseniana free-living gametophytes. CONSTITUTION: Zoospores are released from an apothecium site of Undaria peterseniana. The released zoospores are cultured, and form gametophytes. Separate the gametophytes formed into female and male gametophytes. Culture the separated female and male gametophytes until the gametophytes reach the respective diameter of 3-10mm. Cut the cultured female and male gametophytes in the respective size of 50-400 micrometers. Culture the excised female and male gametophytes under the cell count of 3-25 cell/ind., the temperature condition of 5-20deg. C , the illumination of 5-40micromolm-2s-1, and the photoperiod of 10:14(Light:Dark)-14:10(L:D). Cross-fertilize the cultivated female and male gametophytes. Culture the gametophytes in a liquid culture medium of PESI(Provasoli`s enriched seawater media). Culture the female and male gametophytes until the gametophytes reach the diameter size of 5-6mm. Excise the female gametophytes in the size of 50-100 micrometers. Excise the male gametophytes in the size of 60-150 micrometers. Culture the female gametophytes under the cell count of 3-8 cell/ind., the temperature condition of 10-20 deg. C, the illumination of 15-30 micromolm-2s-1, and the photoperiod of 10:14(Light:Dark). Culture the male gametophytes under the cell count of 8-12 cell/ind., the temperature condition of 5-20 deg. C, the illumination of 15-25 micromolm-2s-1, and the photoperiod of 14:10(Light:Dark). [Reference numerals] (AA) Matured thallus of Undaria peterseniana; (BB) Rinsing the matured thallus, cut after washing, with sterilized seawater and ABM solution; (CC) Releasing zoospores at 10°C, 20umolm^-2s^-1, and 10:14h(L:D); (DD) 0.1ml zoospore solution; (EE) Separating the zoospores by dilution; (FF) Female gametophyte; (GG) Male gametophyte; (HH) Mass-producing clone gametophyte; (I1,I2) Cutting and regenerating every 20 days at 15°C, 20umolm^-2s^-1, and 10:14h(L:D); (JJ) Mixing the cut female and male gametophyte at the ratio of 1:1; (KK) Forming gamete for at least 15 days at 15°C, 20umolm^-2s^-1, and 14:10h(L:D); (LL) Forming young sporophyte for 40 days at 15°C, 60umolm^-2s^-1, and 14:10h(L:D)

Description

A method for mass production of Undariopsis peterseniana using regeneration and maturation induction of free--living gametophytes of Undariopsis peterseniana}

The present invention relates to a method for mass production of broad seaweed, a large brown algae which is a kind of seaweed.

Large brown algae [ Undariopsis peterseniana ( Kjellman) Miyabe et Okamura] has been reported to be distributed in the narrow waterway between Gapdal- ri and Udo in Jeju Island, Gapado and Cheongsando (Kang JW., Bull Pusan Fish Coll 7, 1-125, 1966; Lee YP. , Algae 13, 419-426, 1998; Lee KW et al., Cheju MBC, 235-269, 1991; Lee IK et al., Korean J Phycol 6, 131- 143, 1991)). Known to distribute (Hwang EK et al., J Appl Phycol DOI 10.1007 / s10811-010-9598-3, 2010; Hwang EK et al., J Kor Fish Soc 43, 231-238, 2010). The area of the broad-sea basin is directly affected by the Tsushima turbulence, and the habitat is predominantly located in the 15 m depth area of low algae (Lee, 1998) (Lee KW et al., Cheju MBC, 235-269, 1991; Hwang EK et al., J Kor Fish Soc 43, 231-238, 2010).

Traditionally, it has been used in the Jeju area for sashimi, but due to the sharp decline in natural resources, it has been the subject of setting the harvest prohibition period as a useful fishery resource under the Enforcement Decree of the Fisheries Resource Management Act (Hwang EK et al., J Kor Fish Soc 43). , 231-238, 2010). Recently, Hwang et al. Revealed the growth and maturation cycles in natural communities through ecological studies of protected broad-sea bream (Hwang EK et al., J Kor Fish Soc 43, 231-238, 2010). By introducing broad-season in Wando, Jeollanam-do, Korea, the test was successful, and the proper conditions for growth were investigated (Hwang EK et al., J Appl Phycol DOI 10.1007 / s10811-010-9598-3, 2010).

In the case of large brown algae, the generational alternation of spores and spores is mostly composed of heterogeneous generational alternations, and therefore, techniques such as separation, culture, and induction of male and female spores are required in order to facilitate breeding and hybridization. Thus, hybridization between seaweed genus has been previously performed using the physiological and ecological characteristics of seaweed (Migita S., Bull Fac Fish Nagasaki Univ 15, 24-48, 1963; Kang JW., Bull Pusan Fish). Coll 7, 1-125, 1966; Saito Y., Jpn Soc Phycol 117-132, 1972), due to the characteristics of these large brown algae, have not led to continuous breeding or industrial use.

It is known that brown algae, which carry out heterogeneous generation alternation, can separately grow male and female spouses from spouse generation and grow asexually, and induce or inhibit spouse formation through environmental control (Luning K., J Phycol , 16, 1-15, 1980). Recently, Westermeir et al. Demonstrated successful mass culture using glass embryo cultures by successfully cultivating glass embryos of Lessonia trabeculata and Macrocystis pyrifera. (Westermeier R, et al., Aqua Res, 37, 164-171, 2006). The establishment of a free embryo culture system means that the production of seedlings is possible throughout the year independent of the age of leaf maturation in nature (Wi MY, et al., J Kor Fish Soc, 41, 381-388, 2008).

The growth method by culturing glass embryos can secure a large amount of spores with only a small amount of mature leaves, and the acquired spores are not only capable of continuous subculture by mass growth but also by the induction of cloned spores. It is a useful way to lay the groundwork for mating and starter sarcomas (Wi MY, et al., J Kor Fish Soc, 41, 381-388, 2008). Regeneration and maturation induction conditions for mass cultivation of these free embryos are E. coli (Wi MY, et al., J Kor Fish Soc, 41, 381-388, 2008) and Gompi (Hwang EK et al., J Kor Fish Soc 43). , 231-238, 2010), but in the case of laver, no conditions for inducing separation, regeneration and maturation of glass embryos have been identified.

In Korea, regeneration of E. coli (Wi MY, et al., J Kor Fish Soc, 41, 381-388, 2008) and Gompi (Hwang EK et al., J Kor Fish Soc 43, 231-238, 2010) Although maturation induction conditions have been investigated, it has not been reached until now by culturing glass embryos (Wi MY, et al., J Kor Fish Soc, 41, 381-388, 2008). In particular, if the range of habitat is very limited or protected algae, such as broad seaweed, artificial seedling production method that can produce large amounts of spores stably by using small amount of imitation in artificial farming, secures the most efficient seedlings to conserve natural resources. It can be called a method.

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Accordingly, the present invention recognizes such a problem, and establishes the conditions for mass culture of glass embryos through the separation, regeneration and maturation of the broad-breasted glass embryos, thereby contributing to the study of breeding and hybridization in the seaweed genus, and further protecting. The present invention has been completed by contributing to the provision of a stable seedling supply plan for the broad species, the target species, and an effective protection plan for the natural population.

It is an object of the present invention to establish the optimum conditions for the regeneration and maturation of large brown algae larvae glass embryos, to provide a method for mass production of larvae.

In order to achieve the above object,

1) releasing the citron from the pericardial plaque of Undariopsis peterseniana ;

2) culturing the released strainer to form a spheroid;

3) separating the formed spouses into male and female spouses;

4) culturing the separated male and female spores until reaching a size of 3 mm to 10 mm in diameter, respectively;

5) cutting the cultured male and female spores into a size of 50 μm to 400 μm, respectively;

6) The cleaved female and male spores were 3 to 25 cell / individual (ind.), A temperature of 10 to 20 ° C., an illuminance of 5 to 40 μmol ⁻² s ⁻¹ , and a photoperiod of 10:14 ( Light: Dark) to 14:10 (L: D), culturing under the conditions of; And

7) Provides a mass production method of the sea bass, comprising the step of mating the cultured male and female spores.

In addition,

1) releasing the citron from the pericardial plaque of Undariopsis peterseniana ;

2) culturing the released strainer to form a spheroid;

3) separating the formed spouses into male and female spouses;

4) culturing the separated male and female spores until reaching a size of 3 mm to 10 mm in diameter, respectively;

5) cutting the cultured male and female spores into a size of 50 μm to 400 μm, respectively; And

6) The cleaved female and male acousoids were each cell number 3 to 25 cell / ind., Temperature 5 to 20 ° C., roughness 15 to 30 μmol ⁻² s ⁻¹ , and photoperiod 10:14 (Light: Dark). ) To 14:10 (L: D) provides a method for growth and growth of the stratum corneum spores.

In addition, the present invention is the Undariopsis peterseniana ) Induction of maturation of broad-leaved cancer embryos comprising culturing cancer embryos at a temperature of 10-20 ° C., roughness 15-25 μmol ⁻² s ⁻¹ , and photoperiod at 14:10 (L: D) Provide a method.

In addition, the present invention is Undariopsis peterseniana ) method of inducing maturation of stratum corneum embryos comprising culturing the embryos at a temperature of 5 to 15 ° C., roughness of 5 to 15 μmol ⁻² s ⁻¹ , and photoperiod at 10:14 (L: D) To provide.

In this study, the artificial cultivation technique was implemented by realizing the optimal culture conditions for the production of broad seaweed by optimizing the method of separating the spores, separating the glass embryos, and inducing the maturation method during the separation and culture of the broad-seasoned female and male spores. It can be developed and used for mass production. Through this, it is possible to mass-produce sea bass and to make artificial cultivation, thereby contributing to the protection of the environment and the increase of income of fishers.

Figure 1 is a schematic diagram showing a large scale sea basal method according to the present invention.
Figure 2 is a view showing a photomicrograph at each step of separating and incubating the glass embryos of stratum corneum.
a: mature sporophyte sporophyte;
b: cross section of scapula sporangia;
c: engrafted resident;
d: cancer spheroid cultured for 5 days;
e: male spheroid cultured for 5 days;
f: Cancer embryo clusters cultured for 30 days;
g: doubling clusters incubated for 30 days;
h: section of cancer embryo;
i: intercept of diploid;
j: clone cluster of dark glass embryo formed with organ; And
k: Clone cluster of a glass embryo formed with a long period.
Figure 3 is a graph showing the relative growth rate according to the initial cut length of the kaleidoscope glass embryos at 15 ° C., 20 μmol ⁻² s ⁻¹ and 14:10 (L: D) culture conditions:
a: cancer embryo; And
B: Amate.
Figure 4 is a graph showing the relative growth rate according to the cell number conditions when cleavage of lamellae glass embryos at 15 ℃, 20μmol- ² s ^-1 and 14:10 (L: D) culture conditions:
a: cancer embryo; And
B: Amate.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for mass production of kaleidoscope spores comprising the following steps:

1) releasing the citron from the pericardial plaque of Undariopsis peterseniana ;

2) culturing the released strainer to form a spheroid;

3) separating the formed spouses into male and female spouses;

4) culturing the separated male and female spores until reaching a size of 3 mm to 10 mm in diameter, respectively;

5) cutting the cultured male and female spores into a size of 50 μm to 400 μm, respectively;

6) The cleaved female and male acousoids were each cell number 3 to 25 cell / ind., Temperature 10 to 20 ° C., illuminance 5 to 40 μmol ⁻² s ⁻¹ , and photoperiod 10:14 (Light: Dark). Culturing at a condition of ˜14: 10 (L: D); And

7) crossing the cultured male and female spores.

In this method, removal of the miscellaneous material after cutting the scapula plaques of the stratum corneum of step 1) may be performed by brushing 2-3 times with a brush.

In addition, it is preferable that the user release of step 1) is performed through the shade in the room, and the shade is preferably performed for 1 to 4 hours at a temperature of 10 ° C., but is not limited thereto. Light amount 5 to 40 μmol ⁻² s ⁻¹ and photoperiod 10:14 (Light: Dark) to 14:10 (L: D) are preferred, but are not limited thereto. At this time, it is preferable to remove the miscellaneous by filtering the discharged strainer through an additional 50 ~ 200 ㎛ mesh, but is not limited thereto.

In the above method, the medium used for culturing in step 2) is preferably a liquid medium or a solid medium, more preferably a liquid medium, but is not limited thereto. The liquid medium may be used as long as it is a medium used for cultivating algae, but is preferably an inorganic medium, and more preferably PESI (Tatewaki, 1966), but is not limited thereto.

In the method, it is preferable to use a microtubule having a pore diameter of 50 to 150 µm prepared by melting the glass tube tip under microscope, but not limited thereto.

In the above method, it is preferable that the size of the female and male mater of step 4) is incubated in the size of 3 mm to 10 mm, more preferably in the size of 5 mm to 6 mm, and incubated in the size of 5 mm. Most preferred, but not limited to.

In the above method, the spores of step 5) are preferably pulverized for 1 minute at 8,000 rpm using a homogenizer homogenizer, and the female and male spores are preferably cut to a size of 50 μm to 400 μm, but not limited thereto. It doesn't work. More specifically, the female embryo is preferably cut to a size of 50 to 100 μm, and the male embryo is preferably cut to a size of 60 to 150 μm, but is not limited thereto.

In the above method, the medium used for culturing in step 6) is preferably a liquid medium or a solid medium, more preferably a liquid medium, but is not limited thereto. The liquid medium may be used as long as it is a medium used for cultivating algae, but is preferably an inorganic medium, and more preferably PESI (Tatewaki, 1966), but is not limited thereto.

In the above method, the cancer embryo in step 6) has a cell number of 3 to 8 cell / ind., A temperature of 15 ° C., an illuminance of 15 to 30 μmol ⁻² s ^−1, and a photoperiod 10:14 (L: D). It is preferable to culture in, but is not limited thereto. In addition, in step 6), the embryos are cultured under the conditions of cell number 8-12 cell / ind., Temperature 15 ° C., roughness 15-30 μmolm- ² s ⁻¹ and photoperiod 14:10 (L: D). Preferably, but not limited to.

In the above method, the female embryo of step 6) is preferably matured by incubating at a temperature of 10 to 20 ° C., a roughness of 15 to 25 μmol ⁻² s ^−1, and a photoperiod 14:10 (L: D). It is not limited to this. In addition, it is preferable to further incubate the male embryo of step 6) at a temperature of 5 to 15 ° C., a roughness of 5 to 15 μmolm ⁻² s ^−1, and a photoperiod 10:14 (L: D), but not limited thereto. It doesn't work.

Through a specific embodiment of the present invention, by culturing the shedder released from the kaleidoscope according to the present invention to form a spouse, optimal cell number, temperature, roughness and photoperiod to proliferate the growth of the formed male and female The condition was established, and when it was used, it was confirmed that both male and female spouses enter the reproductive or adulthood phase.

Therefore, if mated mated mates using the maturation method of male and female spouts according to the present invention, it will be possible to mass production of the bast.

In addition,

1) releasing the citron from the pericardial plaque of Undariopsis peterseniana ;

2) culturing the released strainer to form a spheroid;

3) separating the formed spouses into male and female spouses;

4) culturing the separated female and male spores until reaching a size of 3 mm to 7 mm in diameter, respectively;

5) cutting the cultured male and female spores into a size of 50 μm to 150 μm, respectively;

6) The cleaved female and male acinar bodies were 3 to 12 cell / ind, respectively, the temperature was 10 to 20 ° C., the illuminance was 15 to 30 μmol ⁻² s ⁻¹ , and the photoperiod was 10:14 (Light: Dark). ) To 14:10 (L: D) provides a method for growth and growth of the stratum corneum spores.

In this method, removal of the miscellaneous material after cutting the scapula plaques of the stratum corneum of step 1) may be performed by brushing 2-3 times with a brush.

In addition, it is preferable that the user release of step 1) is performed through the shade in the room, and the shade is preferably performed for 1 to 4 hours at a temperature of 10 ° C., but is not limited thereto. Light amount 5 to 40 μmol ⁻² s ⁻¹ and photoperiod 10:14 (Light: Dark) to 14:10 (L: D) are preferred, but are not limited thereto. At this time, it is preferable to remove the miscellaneous by filtering the discharged strainer through an additional 50 ~ 200 ㎛ mesh, but is not limited thereto.

In the above method, the medium used for culturing in step 2) is preferably a liquid medium or a solid medium, more preferably a liquid medium, but is not limited thereto. The liquid medium may be used as long as it is a medium used for cultivating algae, but is preferably an inorganic medium, and more preferably PESI (Tatewaki, 1966), but is not limited thereto.

In the method, it is preferable to use a microtubule having a pore diameter of 50 to 150 µm prepared by melting the glass tube tip under microscope, but not limited thereto.

In the above method, it is preferable that the size of the female and male mater of step 4) is incubated in the size of 3 mm to 10 mm, more preferably in the size of 5 mm to 6 mm, and incubated in the size of 5 mm. Most preferred, but not limited to.

In the above method, the spores of step 5) are preferably pulverized for 1 minute at 8,000 rpm using a homogenizer homogenizer, and the female and male spores are preferably cut to a size of 50 μm to 400 μm, but not limited thereto. It doesn't work. More specifically, the female embryo is preferably cut to a size of 50 to 100 μm, and the male embryo is preferably cut to a size of 60 to 150 μm, but is not limited thereto.

In the above method, the medium used for culturing in step 6) is preferably a liquid medium or a solid medium, more preferably a liquid medium, but is not limited thereto. The liquid medium may be used as long as it is a medium used for cultivating algae, but is preferably an inorganic medium, and more preferably PESI (Tatewaki, 1966), but is not limited thereto.

In the method, the cancer embryo in step 6) has a cell number of 3 to 8 cell / ind., A temperature of 10 to 20 ° C., an illuminance of 15 to 25 μmol ⁻² s ^−1, and a photoperiod 10:14 (L: D ) Is preferably cultured in, but not limited to. Further, in step 6), the diploids were prepared under conditions of 8 to 12 cell / ind., Temperature of 10 to 20 ° C., roughness of 15 to 20 μmol ^-2 s ^-1, and photoperiod 14:10 (L: D). Preferably, the culture is not limited thereto.

In addition, the present invention is the Undariopsis peterseniana ) Induction of maturation of broad-leaved cancer embryos comprising culturing cancer embryos at a temperature of 10-20 ° C., roughness 15-25 μmol ⁻² s ⁻¹ , and photoperiod at 14:10 (L: D) Provide a method.

In the method of inducing maturation of broad-leaved cancerous embryos, the broad-leaved cancerous embryos are cultured under conditions of a temperature of 10 to 20 ° C., a roughness of 15 to 25 μmol ⁻² s ⁻¹ , and a photoperiod of 14:10 (L: D). It is preferable to induce maturation by incubating at a temperature of 15 ° C., roughness 20 μmolm ⁻² s ⁻¹ , and photoperiod at 14:10 (L: D) to induce maturation, but is not limited thereto. It doesn't work.

Skeletons do not form spores simply by the presence of female and male mates, which are free actors. To form a spore, the male and female spores must mature to form cancer and male germ cells.

Through specific examples of the present invention, in order to establish the optimal culture conditions for maturation of cancer embryos, maturation of the broad-leaved cancer embryos was observed according to temperature, illuminance and photoperiod. As a result, the most optimal conditions for inducing the maturation of the cancer mater (see k in Fig. 2) are the conditions of temperature 15 ° C., illuminance 20 μmol ⁻² s ⁻¹ , and photoperiod 14:10 (L: D). I could confirm that.

Therefore, using these culture conditions, it is possible to more effectively mature the rapeseed cancer embryos.

In addition, the present invention is Undariopsis peterseniana ) method of inducing maturation of stratum corneum embryos comprising culturing the embryos at a temperature of 5 to 15 ° C., roughness of 5 to 15 μmol ⁻² s ⁻¹ , and photoperiod at 10:14 (L: D) To provide.

In the method of inducing maturation of the broad-habited male embryos, the broad-leaved male embryos are cultured under conditions of temperature of 5 to 15 ° C., roughness of 5 to 15 μmol ⁻² s ⁻¹ , and photoperiod at 10:14 (L: D). It is preferable to induce maturation, it is more preferable to induce maturation by incubating at a temperature of 10 ° C., roughness 10 μmolm ⁻² s ⁻¹ , and photoperiod at 10:14 (L: D), but not limited thereto. .

Through specific examples of the present invention, in order to establish the optimum culture conditions for maturing the embryo, the maturation of the larvae in accordance with temperature, roughness and photoperiod was observed. As a result, it was confirmed that the maturation of the broad-habited acinar was most effectively induced when cultured at a temperature of 10 ° C., a roughness of 10 μmol ⁻² s ⁻¹ , and a photoperiod at 10:14 (L: D).

Thus, by using these culture conditions, it is possible to more effectively mature the rapeseed aploids.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples and Experimental Examples.

< Example  1> zoospore  Release and spheroid separation

The maturity imitations used in this study were collected in May 2009 at 12-15 m depth in Udo-myeon, Bukjeju-gun, Jeju Island. The collected mature imitation is placed in an ice box and immediately transported to the laboratory. The cut part of the sleeping bag is washed several times with sterile seawater, and then put into the 500 ml beaker filled with 200 ml of sterile seawater, and the incubator (EYELA MTI 202B, Japan) was used to induce dribbler release for 4 hours at 10 ° C., 20 μmol ⁻² s ⁻¹ and 10:14 h (L: D) conditions. 0.1 mL of the released distilled liquor was taken, and the dilution using a 12 multi well-plate was used to dispense the distilled liquor sequentially as shown in FIG. 1 to lower the density. After 4-5 days, if the spouse was identified as male or female, it was divided into female and female embryos under an inverted microscope (Axio Observer A1, Carl zeiss, Germany), and placed in sterile Petri dishes with a diameter of 5 cm. The culture was returned at 2-3 days intervals while incubating. According to the growth of male and female spores, the culture vessel was transferred to 250 ml, and PESI medium was added thereto, followed by aeration for 50 to 60 days until each of the female and male spore clusters reached a diameter of 5 mm.

As a result, as shown in Fig. 2, it was observed that the resident (Fig. 2c) released from the follicular plaque (Fig. 2b) of the stratum corneum leaf (Fig. 2a) immediately attached to the substrate and spherical engraftment. After 5 days of culture at a temperature of 15 ° C. and roughness of 20 μmol ⁻² s ⁻¹ and 14:10 h (L: D) photoperiod, the resident grew into a female embryo (FIG. 2D) or a female (FIG. 2E), respectively. . Each male and female spores proliferated into spore clusters as shown in Fig. 2f-g after about 30 days under aeration conditions. Sections of male and female mates cleaved using a homogenizer (FIGS. 2H-I) are then grown to ventricular clusters under aeration conditions or cultured to induce the formation of organs and organs as shown in FIG. 2J-K after 20 days. It was.

< Experimental Example  1> Growth comparison of free cosine fragments by cleavage size

In order to determine the regeneration by size of the glass embryo fragments, 0.1 g-fresh wt. Of asexually cultured male and female spore clusters were placed in a 250 ml beaker with 100 ml of sterile seawater and each homogenizer (DI 25 basic, GMBH & Co. , Germany) for 60 seconds at intervals of rotational speed (8,000, 9,500, 13,500, 20,500 and 24,000 rpm) and then at 15 ° C and 20 μmol ^-2 s ^-1 and 14: 10h (L: D). After incubation for 20 days using 12 multi-well plates, the length and cell number of the spores were measured under an inverted microscope. The growth of the length and the number of cells by the size group of the cleaved spheroids was calculated by comparing the average length and average cell number of the spores at the start of the culture with those at the end of the culture.

Length growth by fragment size of the glass embryos showed a difference in each cutting size and cell number conditions at the beginning of the experiment as shown in FIGS. 3 and 4.

In the case of female embryos, the growth rate of the female embryos was the highest as 3.5 ± 0.2% day ⁻¹ after 20 days of culture in the condition of 77.0 ± 23.3 μm where the cut size of the spores was the smallest as shown in FIG. 3A. The growth rate was the lowest as 1.4 ± 0.4% day ⁻¹ after 20 days of culture at 163.4 ± 21.4 ㎛. In the case of male embryos, as shown in FIG. 3B, the growth rate was the highest at 4.1 ± 0.5% day ⁻¹ after 20 days of culture in the condition of 95.4 ± 29.9 μm of the smallest cutting size, and 375.4 ± 17.1 μm having the largest cutting size. The growth rate was the lowest as 0.1 ± 0.06% day ⁻¹ after 20 days of culture under the conditions (FIG. 3).

In addition, according to the cell number conditions at the cutting of the spheroid, in the case of cancer embryos, as shown in FIG. At 20 days, the highest growth rate was 2.0 ± 0.2% day ^-1 and the highest number of cells was 12.0 ± 0.5 cell / ind. After 20 days of incubation, the growth rate was the lowest at 1.1 ± 0.2% day ⁻¹ . In the case of the male embryo, the smallest number of cells at the beginning of the experiment as shown in Figure 4b 9.7 ± 0.5 cell / ind. At 20 days, the highest growth rate was 4.8 ± 0.5% day ^-1 and the highest cell number was 21.4 ± 0.4 cell / ind. After 20 days of culture under the conditions, the growth rate was the lowest as 3.1 ± 0.4% day ⁻¹ (FIG. 4).

Thus, the sedan of glass embryos grows smaller in size (about 100 μm) and in fewer cells (about 5 cells for cancer embryos, about 10 cells for male embryos) in terms of length growth or increase in cell number. This tended to be high.

< Experimental Example  2> Growth and maturation of glass embryo fragments by culture conditions

In order to determine the optimal environmental conditions for the growth and maturation of glass fragments after cutting the glass medium, the male and female glass mediums grown in chunks were crushed for 1 minute at a speed of 8,000 rpm, which was the best cutting condition with a homogenizer. In 12 multi well-plates, 1 ml of PESI medium was filled, and one male and female parent was collected per well. Cultivation conditions were 4 temperature sections (5, 10, 15, 20) and 4 crude tools (5, 10, 20, 40 μmol ^-2 s ^-1 ) and 3 photoperiod sections [10:14, 12 : 12, 14: 10h (L: D)] using a multi room incubator (DS-14MCLP), the growth and maturation of the mating spleen was observed by an inverted microscope. The illuminance was measured using LI-1400 (LI-COR, USA), and the adjustment of the illuminance section was controlled using the Leee filter ND 209, 210, and 211. Length growth was measured by screening the spheroids that were cut after grinding with a homogenizer, and the PESI medium was changed every 7 days. The relative growth rate (RGR) of glass embryos was determined using the methods of Wi et al. (2009) and Hwang et al. (2010) (Wi MY, et al., J Kor Fish Soc, 41, 381-388, 2008; Hwang EK et al., J Kor Fish Soc 43, 231-238, 2010).

In addition, the significance analysis on the temperature, roughness and photoperiod condition of the growth rate of the spore fragments obtained in the indoor culture experiment was carried out using one-way ANOVA (Zar, 1984). ver. 8.0 and SYSTAT ver. 9.0 was used at the significance level of 0.05. Growth rate data was arcsine transformed prior to statistical analysis (Parker RE., Introductory statistics for biology. 2nd edition. Edward Arnold, London, 122pp, 1979).

<2-1> Growth rate of glass embryo fragments by culture conditions

The fragments of the glass embryos were cultured under different culture conditions, and the differences were shown in Table 1 below.

The growth rate of female embryos was 2.5 ± 0.2% day ^-1 at 15 ℃ after 20 days of culture and the lowest as 0.2 ± 0.1% day ^-1 at 5 ℃ (p < 0.05). By illumination period was 20 μ㏖m ^-2 s ^-1 conditions at highest, ^{2.5 ± 0.2% day -1, 5} μ㏖m -2 s -1 was the lowest in the condition to ^{0.6 ± 0.2% day -1 (p} < 0.05). The photoperiod condition was highest as 2.6 ± 0.2% day ^-1 at 10: 14h (L: D) and lowest as 2.1 ± 0.3% day ^-1 at 14: 10h (L: D) (p <0.05).

The growth rate of the embryos was highest as 1.6 ± 0.3% day ^-1 at 15 ℃ after 20 days of culture and lowest as 0.5 ± 0.2% day ^-1 at 20 ℃ (p <0.05). By illumination period was 20 μ㏖m ^-2 s ^-1 conditions the highest at a ^{3.6 ± 0.3% day -1, 5} μ㏖m -2 s -1 it was the lowest in the condition to ^{0.9 ± 0.2% day -1 (p} < 0.05). The photoperiod condition was highest as 2.7 ± 0.2% day ^-1 at 14: 10h (L: D) and lowest as 1.0 ± 0.2% day ^-1 at 10: 14h (L: D) (p> 0.05 ).

<2-2> Comparison of Maturation of Glass Embryos by Culture Conditions

The maturation of the female embryo showed a difference according to temperature, illuminance and photoperiod conditions as shown in Table 2 below. According to the temperature conditions, the ovulatory phase begins to form first after 10 days of culture at 15-20 ° C., and after 15 days, more than 30% of the female embryos form ovules. After 20 days of culture, about 50% of the female embryos form ovulatory phases. Indicated. However, in 5-10 ° C., no ovulation was observed until 20 days of culture. According to the roughness conditions, about 10% of the female embryos formed ovulatory phase after 10 days of culture at 20 μmolm ^-2 s ^-1 condition, and form of ovulatory phase after 20 days of culture at 40 μmolm ^-2 s ^-1 condition. , 5-10 μmol ^-2 s ^-1 did not form an egg stage until 20 days of culture. In the photoperiod condition, the formation of the janggi period was observed first after 10 days of culture at 14: 10h (L: D) condition, and the formation of the janggi period was observed after 15 days of culture at 12: 12h and 10: 14h (L: D) condition. It became.

The maturation of the embryos was shown by temperature, illuminance and photoperiod conditions as shown in Table 3 below. According to the temperature conditions, the formation of the long-term organ began to appear at 10 ° C. after 5 days of culture, and the formation of the long-term period was observed after 10 days of culture at 5 ° C. and 20 days of culture at 15-20 ° C. conditions. According to the roughness conditions, the formation of the long-term organ was observed first at 10 μmolm ^-2 s ^-1 after 5 days of culture, and the formation of the long-term period was observed after 15 days of culture at 5 μmolm ^-2 s ^-1 . In the 40 μmol ⁻² s ⁻¹ condition, no organs were formed until 20 days of culture. In the photoperiod condition, the first period was formed after 10 days of culture at 10: 14h (L: D), and the formation of the period was observed after 10 days of culture at 12: 12h (L: D). In L: D), the formation of the longest period was observed after 20 days of culture.

The present invention relates to a method for mass propagation of larvae spleen and maturation method of spouses. The method of the present invention can be used not only for the ecological restoration of the extinct species of endangered species through mass propagation of spores, which can be usefully used for breeding of basalt, but also contribute to the income increase of fishers. Furthermore, the present invention can be actively used to supply cosmetics, pharmaceuticals and health food raw materials that create high value added through the mass production of sea bass.

Claims (18)

1) releasing the citron from the pericardial plaque of Undariopsis peterseniana ;
2) culturing the released strainer to form a spheroid;
3) separating the formed spouses into male and female spouses;
4) culturing the separated male and female spores until reaching a size of 3 mm to 10 mm in diameter, respectively;
5) cutting the cultured male and female spores into a size of 50 μm to 400 μm, respectively;
6) The cleaved female and male acousoids were each cell number 3 to 25 cell / ind., Temperature 5 to 20 ° C., roughness 5 to 40 μmol ⁻² s ⁻¹ , and photoperiod 10:14 (Light: Dark). Culturing at a condition of ˜14: 10 (L: D); And
7) Mass production method of the rice flakes, comprising the step of mating the cultured male and female spores.
The method of claim 1, wherein the culture of step 2) or step 4) is performed in Provasoli's enriched seawater media (PESI) liquid medium.
The method for mass production of basalica according to claim 1, wherein the male and female spores of step 4) are cultured until they reach a size of 5 to 6 mm in diameter.
According to claim 1, wherein the female embryo of step 5) is a mass production method of the basalica, characterized in that to cut to a size of 50 ~ 100 ㎛.
The method of mass production of rice bran according to claim 1, wherein the male embryo of step 5) is cut to a size of 60 to 150 µm.
The method of claim 1, wherein the cancer embryo in step 6) has a cell number of 3 to 8 cell / ind., A temperature of 10 to 20 ° C., an illuminance of 15 to 30 μmol ⁻² s ^−1, and a photoperiod 10:14 (L: A method for mass production of kaleidoscope, characterized by culturing in D).
The method of claim 6, wherein the female embryo is further incubated at a temperature of 10 ~ 20 ℃, roughness 15 ~ 30 μmol ^-2 s ^-1 and photoperiod 14:10 (L: D) to induce maturation Mass production method of sea bass.
The method of claim 1, wherein in step 6) the embryos have a cell number of 8 to 12 cell / ind., A temperature of 5 to 20 ° C., an illuminance of 15 to 25 μmol ⁻² s ^−1, and a photoperiod 14:10 (L: D) A method for mass production of basaltic sieves, which is cultured in D).
The method of claim 8, wherein the embryo is further incubated at a temperature of 5 ~ 15 ℃, roughness 5 ~ 15 μmolm- ² s- ¹ and photoperiod 10: 14 (L: D) to induce maturation Mass production method of sea bass.
1) releasing the citron from the pericardial plaque of Undariopsis peterseniana ;
2) culturing the released strainer to form a spheroid;
3) separating the formed spouses into male and female spouses;
4) culturing the separated male and female spores until reaching a size of 3 mm to 10 mm in diameter, respectively;
5) cutting the cultured male and female spores into a size of 50 μm to 400 μm, respectively; And
6) The cleaved female and male acousoids were each cell number 3 to 25 cell / ind., Temperature 5 to 20 ° C., roughness 15 to 30 μmol ⁻² s ⁻¹ , and photoperiod 10:14 (Light: Dark). ) 14 to 14:10 (L: D), comprising the step of cultivating growth of the stratum corneum spores.
The method according to claim 10, wherein the culture of step 2) or 4) is performed in Provasoli's enriched seawater media (PESI) liquid medium.
The growth and propagation method of the basaltic spores of claim 10, wherein the female and male spores of step 4) are cultured until they reach a size of 5 to 6 mm in diameter.
11. The method for growing and propagating the stratum corneum spores according to claim 10, wherein the female embryo of step 5) is cut into a size of 50 to 100 µm.
According to claim 1, wherein the growth of the stratum corneum spheroidal body characterized in that the step of 5) cutting the embryo in the size of 60 ~ 150 ㎛.
The method of claim 10, wherein the cancer embryo in step 6) has a cell number of 3 to 8 cell / ind., A temperature of 10 to 20 ° C., an illuminance of 15 to 25 μmol ⁻² s ^−1, and a photoperiod 10:14 (L: D) growth and growth method of the stratum corneum spores, characterized by culturing in D).
The method of claim 10, wherein in step 6) the embryos have a cell number of 8 to 12 cell / ind., A temperature of 10 to 20 ° C., an illuminance of 15 to 30 μmol ⁻² s ^−1, and a photoperiod 14:10 (L: D) growth and growth method of the stratum corneum spores, characterized by culturing in D).
Undariopsis peterseniana ) Induction of maturation of broad-leaved cancer embryos comprising culturing cancer embryos at a temperature of 10-20 ° C., roughness 15-25 μmol ⁻² s ⁻¹ , and photoperiod at 14:10 (L: D) Way.
Undariopsis peterseniana ) method of inducing maturation of stratum corneum embryos comprising culturing the embryos at a temperature of 5 to 15 ° C., roughness of 5 to 15 μmol ⁻² s ⁻¹ , and photoperiod at 10:14 (L: D) .

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