KR100989384B1 - Method of producing a viable tetraploid oyster - Google Patents

Abstract

PURPOSE: A method of producing a viable tetraploid oyster is provided, which can produce tetraploid oyster efficiently by combination of cyto gallacine-B and 6-dimethylamino purine(DMAP). CONSTITUTION: A method of producing a viable tetraploid oyster comprises: a step of fertilizing an egg of a diploid male oyster and an egg of a triploid female oyster; a step of interrupting the emission of the first polar body from the fertilized egg; and a step of producing a tetraploid oyster by cultivating the fertilized egg. The step of interrupting the emission of the first polar body includes a step of bringing the fertilized egg into contact with cyto gallacine and 6-dimethylamino purine(DMAP).

Description

Method of producing tetraploid oysters {Method of producing a viable tetraploid oyster}

One embodiment of the present invention is directed to a method for efficiently producing viable tetraploid oysters.

Most sexual reproductions have two sets of chromosomes and are called diploids. Meiosis is the process of reducing the number of chromosomes by half to prevent doubling the number of chromosomes in each generation. This is a two step process, whereby one diploid cell becomes four haploid cells, each with one set of chromosomes. One or all four of these haploid cells can mature into functional eggs or sperm known as spouses.

Tetraploid oysters are important for a variety of purposes, including triploid, hybridization and other breeding programs. Triploid oysters have been known to have certain commercial advantages, such as better taste and improved growth rate, compared to normal diploid oysters during the normal reproduction of diploids. At present such triplet oysters are produced from normal diploid oysters using a specific chromosome set manipulation technique, whereby meiosis in oocytes causes the oocytes to release the second polar body during the second meiosis. Engineered to retain in oocytes.

Cytogalasin B, (CB) and 6-dimethylaminopurine (DMAP), on the other hand, have been known as compounds that inhibit the release of the first polar body to extracellular during meiosis in fertilized eggs.

However, even according to the above-described prior art, a method for efficiently producing tetraploid oysters using a combination of cytogalasin B, (CB) and 6-dimethylaminopurine (DMAP) is not known.

One embodiment of the present invention provides a method for efficiently producing a viable tetraploid oyster.

One embodiment of the present invention comprises the steps of fertilizing the eggs of the diploid female oyster with sperm of the diploid male oyster; Retarding the release of the second polar body from the fertilized egg; And culturing the fertilized egg to produce a viable tetraploid oyster; wherein the release of the second polar body comprises contacting the fertilized egg with cytogalasine and 6-dimethylaminopurine (DMAP). It provides a method for producing a viable tetraploid oyster comprising.

The method includes the step of fertilizing the eggs of triploid female oysters with the sperm of diploid male oysters. Triploid oysters are commercially available (Allen, Jr., S.K. (1988); Oceanus 31, 58-63). Triploid oysters use chromosomal set manipulation techniques to allow meiosis to be manipulated in oocytes to retain a second polar body in oocytes instead of releasing a second polar body during second meiosis. (diploid oyster) can be produced. Triploid oysters can be examined by flow cytometry prior to spawning to confirm their ploidy. Eggs of triploid oysters can be collected by strip spawning. The eggs can be washed with filtered seawater and held on a suitable screen such as a 25 μm screen. The triploid oyster used in one embodiment of the present invention may be conditioned by placing in an environment with high temperature and abundant food. The conditioning preferably begins at an early stage of gametogenesis immediately after winter dormancy.

The eggs are then fertilized with sperm obtained from normal diploid males. The amount of sperm used for fertilization may be about 10 or more sperm cells per egg. The modification can be made by known methods. For example, it can be made by contacting the eggs and sperm in seawater.

The method also includes retarding the release of the second polar body from the fertilized egg. The inhibition of release of the second polar body may comprise contacting the modified egg with cytogalasine and 6-dimethylaminopurine (DMAP).

The contact may be in the time range of 30% to 90% of the time the fertilized egg releases the second polar body. The contact may be, for example, made in 5 to 15 minutes after the correction.

The contact may be the simultaneous contact of the fertilized egg, cytogalasine and 6-dimethylaminopurine (DMAP). In addition, the contact may be to sequentially contact the fertilized egg, cytogalasine and 6-dimethylaminopurine (DMAP). For example, the contact may be contacting cytogalasine with the fertilized egg followed by contacting 6-dimethylaminopurine (DMAP) with the fertilized egg. The contact may, for example, contact the cytogalasine with the fertilized egg in a time range of 30% to 90% of the time when the fertilized egg releases the second polar body, followed by 6-dimethylaminopurine (DMAP) The fertilized egg may be contacted in a time range of 50% to 90% of the time for which the fertilized egg releases the second polar body. One embodiment of the contacting is contacting cytogalasine with the fertilized egg at 5 to 15 minutes after fertilization and then at 8.3 to 15 minutes after fertilizing the fertilized egg with 6-dimethylaminopurine (DMAP). May be in contact.

In addition, the contact may be the addition of the fertilized egg to the concentration of 0.25 to 1.0 ppm of cytogalasin in DMSO and 0.25 to 1.0 ppm of DMAP in DMSO. For example, the contact may be the addition of the fertilized egg to a concentration of 0.25 to 0.75 ppm of cytogalasin in DMSO and a DMAP of 0.25 to 0.75 ppm in DMSO.

The method also includes culturing the fertilized egg to produce a viable tetraploid oyster.

The culture may be cultured by a conventional method for culturing fertilized eggs known in the art. For example, the culture may be made at 18 ℃ to 30 ℃, for example, 23 ℃ to 28 ℃. The culture may be at a salinity of 17 ppt to 30 ppt, for example, about 20 ppt to 22 ppt.

In this method, the oysters may be of the genus Crassostrea . Crassostrea genus may be selected from the group consisting of Crassostrea gigas , Crassostrea sikamai , Crassostrea rivularis and Crassostrea virginica . The liquor may also be a genus of Pinctada . For example, the oyster may be Pinctada magaratifera .

By the method of the present invention, a tetraploid oyster can be produced that can grow and mature in natural conditions in which a conventional diploid oyster can naturally grow, and the tetraploid oyster is crossed with the diploid oyster to produce a triploid oyster. Can be generated.

According to one embodiment of the present invention, tetraploid oysters can be efficiently produced from triplex oysters and diploid oysters.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

The triploid Pacific oyster used in this example was 2 years old and made by arresting the release of PB2. Triploid oysters were individually identified by flow cytometry before spawning. Spouses were collected by strip spawning. The eggs were passed through an 85 μm screen to remove large tissue debris and washed on a 25 μm screen. All modifications and treatments were performed at 25 ° C. to 28 ° C. in 2 μm filtered seawater. The salinity of the seawater used in this example was about 20 ppt to 22 ppt.

Eggs derived from triploids were fertilized with haploid sperm derived from diploids. After fertilization, fertilized eggs were divided into the following groups: control group, control group, and experimental group.

Fertilized eggs were incubated without treatment with chemicals. In Comparative Groups 1 and 2, the fertilized eggs were treated with cytogalacin B to arrest the release of the second polar body. Cytogalacin B (hereinafter abbreviated CB) was prepared by dissolving in dimethylsulfoxide (DMSO), and fertilized eggs contained 0.25 mg / l containing 0.5% DMSO (Comparative Group 1) and 0.5 mg / containing 0.5% DMSO. to a final concentration of l (Comp. 2). In Comparative Groups 3 and 4, the fertilized eggs were treated with 6-dimethylaminopurine (abbreviated as DMAP) to retard the release of the second polar body. DMAP was prepared by dissolving in dimethyl sulfoxide (DMSO) and the final concentration of 0.25 mg / l (Comp. 3) with 0.5% DMSO and 0.5 mg / l (Comp. 4) with 0.5% DMSO in fertilized eggs. Was added.

In Experiments 1 and 2, the fertilized eggs were treated with CB and DMAP to arrest the release of the second polar body. CB and DMAP were prepared by dissolving in dimethylsulfoxide (DMSO) and fertilized eggs containing 0.25 mg CB / l and 0.5 mg DMAP / l (Experimental Group 1) and 0.5 mg CB / containing 0.5% DMSO. 1 and 0.5 mg DMAP / l (Experimental Group 2) at the final concentration.

In the control, comparative and experimental groups 1 and 2, each treatment started 5 minutes after fertilization and continued for 15 minutes. After each treatment, fertilized eggs were washed with 1% DMSO-sea water and incubated at a density of 65 eggs / ml.

In Experiment 3, the fertilized eggs were treated with CB and DMAP to arrest the release of the second polar body. CB and DMAP were prepared by dissolving in dimethyl sulfoxide (DMSO). The fertilized eggs were treated at the final concentration of 0.25 mg CB / l containing 0.5% DMSO at 5 minutes after fertilization and 0.25 mg at 10 minutes after fertilization. It was added at a final concentration of DMAP / l and lasted up to 15 minutes after fertilization (Experimental Group 3).

In Experiment 4, the fertilized eggs were treated with CB and DMAP to arrest the release of the second polar body. CB and DMAP were prepared by dissolving in dimethyl sulfoxide (DMSO). The fertilized eggs were treated at the final concentration of 0.5 mg CB / l containing 0.5% DMSO at 5 minutes after fertilization and 0.5 mg at 10 minutes after fertilization. It was added at a final concentration of DMAP / l and lasted up to 15 minutes after fertilization (Experimental Group 4). In experimental groups 3 and 4, after each treatment, fertilized eggs were washed with 1% DMSO-sea water and incubated at a density of 65 eggs / ml.

Table 1.

group Used eggs
(x1000) split(%) % Survival rate (%) 7 days survival rate (%) 35% survival rate Control 100 85.4 22.8 5.7 0.003 Comparative Group 1 100 83.5 6.4 3.1 0.073 Comparative Group 2 150 82.7 6.0 2.8 0.065 Comparative Group 3 100 80.5 8.5 4.0 0.085 Comparative Group 4 150 79.6 9.5 4.5 0.092 Experimental Group 1 100 90.5 50.5 30.5 0.51 Experimental Group 2 150 92.5 51.5 31.5 0.62 Experimental Group 3 180 95.5 55.0 34.0 0.80 Experimental Group 4 190 96.5 54.5 33.5 0.89

In Table 1, percentage cleavage was determined from 90 minutes to 120 minutes after fertilization. Survival rates of divided embryos were recorded 1, 7 and 35 days after fertilization (spat). As shown in Table 1, Experimental Groups 1 to 4 were significantly higher in percentage of cleavage and 35-day survival after fertilization compared to control and comparison groups. That is, in the step of inhibiting the release of the second polar body from the fertilized egg, larvae can be produced with high efficiency by using a combination of CB and DMAP.

In addition, the ploidy composition of surviving larvae on these sampling days was determined using flow cytometry. Three months after fertilization, live oysters were harvested to count body weight and chromosome number. For chromosome analysis, oysters were treated with colchicine (0.005%) for 12 hours with intensive feeding. Oysters were separated from the shells and weighed. Next, the whole body was cut and fixed in an acetic acid / methanol mixture (1: 3). Appropriate amount of fixed sample was poured onto the slides and air dried. Slides were Leishman's stain. At least 10 metaphase cells that did not show signs of chromosomal loss were counted for each oyster. Only individuals whose chromosome number was clearly determined were used for the analysis. Oysters with 20, 30 and 40 chromosomes were named diploid, triploid and tetraploid, respectively. Table 2 shows the results of measuring the weight and the number of stains of 50 oysters derived from the experimental group at 3 months after fertilization. Obviously only chromosome counts were included in the data, and oysters without measurable mid-term cells were excluded from the analysis.

Table 2.

Exudation Experimental Group 1 Experimental Group 2 Experimental Group 3 Experimental Group 4 Number(%) Body weight (mg) Number(%) Body weight (mg) Number(%) Body weight (mg) Number(%) Body weight (mg) Diploid 1 (2.0) 60 1 (2.1) 65 1 (2.0) 66 1 (2.0) 63 Triplicate 1 (2.0) 90 1 (2.1) 100 1 (2.0) 99 1 (2.0) 91 Tetraploid 39 (79.6) 300 38 (79.2) 312 38 (79.6) 320 39 (78.0) 323 Dimer 7 (14.2) 180 7 (14.6) 185 6 (14.2) 190 7 (14.0) 192 Mosaic 1 (2.0) 62 1 (2.1) 65 1 (2.0) 67 2 (4.1) 68 Sum 49 (100) 268.8 48 (100) 287.8 49 (100) 273.2 50 (100) 284.6

 As shown in Table 2, at three months after fertilization, tetraploid oysters occupied a high proportion in the range of 78% to 79.6% in oysters 1 to 4 cm in length.

In the step of inhibiting the release of the second polar body from the fertilized egg from the result of this example, it was possible to produce quadruple oysters with high efficiency by using a combination of CB and DMAP.

The produced tetraploid oysters were able to grow under the conditions of natural diploid oysters. In addition, the tetraploid oysters were able to produce triploid oysters by crossing with diploid oysters. In addition, the tetraploid oysters could produce other tetraploid oysters by crossing between the tetraploid oysters.

Claims (11)

Fertilizing eggs of triploid female oysters with sperm of diploid male oysters; Retarding the release of the first polar body from the fertilized egg; And Culturing the fertilized egg to produce a viable tetraploid oyster; wherein the release of the first polar body comprises contacting the fertilized egg with cytogalasine and 6-dimethylaminopurine (DMAP) How to produce a viable tetraploid oyster. The method of claim 1, Wherein said contacting said fertilized egg, cytogalasine, and 6-dimethylaminopurine (DMAP) simultaneously. The method of claim 1, Wherein said contacting sequentially contacts said fertilized eggs with cytogalasine and 6-dimethylaminopurine (DMAP). The method of claim 1, Wherein said contacting comprises contacting cytogalasine with said fertilized eggs followed by contacting 6-dimethylaminopurine (DMAP) with said fertilized eggs. The method of claim 1, The contacting comprises contacting cytogalasine and the fertilized egg in a time range of 30% to 90% of the time that the fertilized egg releases the second polar body, followed by contacting 6-dimethylaminopurine (DMAP) with the fertilized egg. And wherein the fertilized egg is contacted in a time range of 50% to 90% of the time to release the second polar body. The method of claim 1, Said contact is contacting cytogalasine with said fertilized egg at 5 to 15 minutes after fertilization and then contacting 6-dimethylaminopurine (DMAP) with fertilized egg at 8.3 to 15 minutes after fertilization. A method of producing viable tetraploid oysters. The method of claim 1, Wherein said contacting adds said fertilized eggs to cytogalasine at a concentration of 0.25-1.0 ppm in DMSO and DMAP at a concentration of 0.25-1.0 ppm in DMSO. The method of claim 7, wherein Wherein said contacting adds said fertilized egg to cytogalasin at a concentration of 0.25 to 0.75 ppm in DMSO and DMAP at a concentration of 0.25 to 0.75 ppm in DMSO. The method of claim 1, Wherein said contacting is in the time range of 30% to 90% of the time that said fertilized egg releases the second polar body. The method of claim 1, And wherein said contacting takes place between 5 and 15 minutes after fertilization. The method of claim 1, The culture is a method for producing a viable tetraploid oyster is made at a salinity of 17 ppt to 30 ppt at 18 ℃ to 30 ℃.