CN102573453B - Method for producing viable tetraploid oysters - Google Patents
Method for producing viable tetraploid oysters Download PDFInfo
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- CN102573453B CN102573453B CN201080046057.5A CN201080046057A CN102573453B CN 102573453 B CN102573453 B CN 102573453B CN 201080046057 A CN201080046057 A CN 201080046057A CN 102573453 B CN102573453 B CN 102573453B
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- fertilized egg
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
- A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
- A01K67/0334—Genetically modified Molluscs
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/50—Culture of aquatic animals of shellfish
- A01K61/54—Culture of aquatic animals of shellfish of bivalves, e.g. oysters or mussels
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/03—Animals modified by random mutagenesis, e.g. using ENU, chemicals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/70—Invertebrates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/02—Animal zootechnically ameliorated
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
One detailed example of the present invention provides a method for efficiently producing tetraploid oysters using a combination of cytochalasin B (CB) and 6-dimethylaminopurine (DMAP).
Description
Technical field
Embodiments of the present invention relate to the method for the tetraploid oyster living with high efficiency production.
Background technology
The animal of most of sexual propagations has two group chromosomes, and they are called as dliploid.Reduction division is a kind of process that chromosome quantity is reduced by half, and with the chromosome quantity of avoiding each generation, doubles.This process has two steps, and by described step, a diploid cell becomes four haploid cells, and each haploid cell has a group chromosome.One or all four haploid cells can be ripe for having ovum or the sperm of function, and it is called as gamete.
Tetraploid oyster is important for realizing multiple object, comprises triploid, hybridization and other breeding system.Be known that, during the normal breeding period of dliploid oyster, compared with dliploid oyster, triploid oyster has the commercial benefit of the growth rate of better taste and Geng Gao.Current, utilized special chromosome set change technology from normal dliploid oyster production triploid oyster, wherein the reduction division in egg mother cell is changed, thereby between meiosis stage, in egg mother cell, is retaining second polar body, rather than discharges from egg mother cell.
Cytochalasin B (CB) and the known conduct of 6-dimethylaminopurine (DMAP) prevent the compound that first polar body (PB1) is discharged from cell during the reduction division of fertilized egg.
But, even according to above-mentioned routine techniques, the method for the known combinations produce tetraploid oyster that does not also utilize cytochalasin B (CB) and 6-dimethylaminopurine (DMAP).
Summary of the invention
Thereby, the object of this invention is to provide the method with the tetraploid oyster of high efficiency production work.
To achieve these goals, according to an aspect of the present invention, provide the method for producing the tetraploid oyster living, comprised step: with the sperm of diploid males oyster, make the feritilization of ovum of the female oyster of triploid; Stop first polar body to be discharged from described fertilized egg; And produce tetraploid oyster alive by cultivating described fertilized egg, the step that wherein stops first polar body to be discharged from described fertilized egg comprises makes described fertilized egg exposing cell relaxin B (CB) and 6-dimethylaminopurine (DMAP).
Described method comprises the step that makes the feritilization of ovum of triploid oyster with the sperm of diploid males oyster.The female oyster of triploid can be commercially available (Allen, Jr., S.K. (1988); Oceanus 31,58-63).Can utilize special chromosome set change technology from dliploid oyster production triploid oyster, wherein the reduction division in egg mother cell is changed, thereby between meiosis stage, in egg mother cell, retaining second polar body, rather than discharging from egg mother cell.Can before laying eggs, utilize the ploidy of flow cytometry checking triploid oyster.The ovum of triploid oyster can gather by cutting the female oyster of triploid (peeling off lay eggs (strip spawning)).The ovum gathering can clean and remain on suitable screen cloth with seawater, for example 25 μ m screen clothes.The triploid oyster of using in embodiments of the present invention can regulate with the environment that is rich in feed by they being placed in to high temperature.This adjusting preferably gametogenic initial period after hibernation and then starts.
Next, use available from the sperm of normal diploid males oyster and make the described feritilization of ovum.For the sperm of being fertilized, can comprise approximately 10, each ovum or more than 10 spermatids.Fertilization can be carried out according to any method known in the art, for example, makes ovum contact with sperm in seawater.
Described method comprises the step that stops first polar body to be discharged from fertilized egg.This step can comprise makes fertilized egg exposing cell relaxin B (CB) and 6-dimethylaminopurine (DMAP).
Described contact can be carried out in fertilized egg is discharged 30%～90% the time range of time of first polar body.In addition, described contact can be carried out 5～15 minutes after the feritilization of ovum.
The step that stops first polar body to be discharged from fertilized egg can comprise makes fertilized egg exposing cell relaxin B (CB) and 6-dimethylaminopurine (DMAP) simultaneously.In addition the step that, stops first polar body to be discharged from fertilized egg can comprise makes fertilized egg exposing cell relaxin B (CB) and 6-dimethylaminopurine (DMAP) in succession.For example, the step that stops first polar body to be discharged from fertilized egg can comprise to be made fertilized egg exposing cell relaxin B (CB) and then makes fertilized egg contact 6-dimethylaminopurine (DMAP).For example, stop the first polar body step of discharging from fertilized egg can be included in fertilized egg and discharge in 30%～90% the time range of time of first polar body and make fertilized egg exposing cell relaxin B (CB), then in fertilized egg is discharged 50%～90% the time range of time of first polar body, make fertilized egg contact 6-dimethylaminopurine (DMAP).More specifically, the step that stops first polar body to be discharged from fertilized egg makes fertilized egg exposing cell relaxin B (CB) 5～15 minutes after can being included in the feritilization of ovum, then after the feritilization of ovum, makes fertilized egg contact 6-dimethylaminopurine (DMAP) 8.3～15 minutes.
In addition, stoping the first polar body step of discharging from fertilized egg to comprise joins fertilized egg among the 6-dimethylaminopurine (DMAP) of 0.25～1.0ppm concentration in the cytochalasin B (CB) of 0.25～1.0ppm concentration in methyl-sulfoxide (DMSO) and methyl-sulfoxide (DMSO).For example, stoping the first polar body step of discharging from fertilized egg to comprise joins fertilized egg among the 6-dimethylaminopurine (DMAP) of 0.25～0.75ppm concentration in the cytochalasin B (CB) of 0.25～0.75ppm concentration in methyl-sulfoxide (DMSO) and methyl-sulfoxide (DMSO).
Described method further comprises the step of producing tetraploid oyster alive by cultivating described fertilized egg.
Described cultivation can be carried out according to any fertilized egg cultural method known in the art.For example, cultivation can, at 18 ℃～30 ℃, be carried out under the temperature condition of preferably 23 ℃～28 ℃.In addition, cultivation can be at 17ppt～33ppt, under the Variation of Salinity Condition of preferred about 20ppt～22ppt, carries out.
In described method, the oyster of use can belong to huge oyster (crassostrea) and belong to.The oyster that huge oyster belongs to can be one of to be selected from long oyster (crassostrea gigas), crassostrea sikamai, Crassostrea rivularis (crassostrea revularis) and American oyster (crassostrea virginica).In addition, the oyster of use can belong to nacre (pinctada) genus.For example, oyster can be black butterfly nacre (pinctada margaritifera).
The method according to this invention, can produce tetraploid oyster, and it can grow and be ripe under the natural environment of natural cultivation normal diploid oyster, and can be by the hybridization production triploid oyster between tetraploid oyster and dliploid oyster.
According to the embodiment of the present invention, can from triploid oyster and dliploid oyster, produce tetraploid oyster with high efficiency.
Embodiment
To describe hereinafter the preferred embodiment of the present invention in detail.But it being understood that in illustrative mode rather than in restrictive mode this embodiment is provided, to explain spirit of the present invention.
This embodiment utilizes the Crassostrea gigas triploidy of Second Year, and it is by stoping second polar body (PB2) to eject production.Before laying eggs, by flow cytometry, verify respectively triploid oyster.By peeling off the mode of laying eggs, gather their gamete.Make ovum remove large fragment of tissue by the screen cloth of 85 μ m, on 25 μ m screen clothes, clean.Fertilization and processing are all carried out in the seawater of 25 ℃～28 ℃ by 2 μ m screen filtrations.The salinity of the seawater using in this embodiment is about 20ppt～22ppt.
Use from the monoploid sperm of dliploid oyster and make the feritilization of ovum from triploid oyster.Fertilized egg is divided into control group, comparative group and experimental group.
The fertilized egg of control group is cultivated without chemical substance treatment.The fertilized egg of comparative group 1 and 2 is processed the discharge to stop first polar body with cytochalasin B (CB).CB is dissolved in methyl-sulfoxide (DMSO), at CB final concentration 0.25mg/l, contain 0.5%DMSO in the situation that (comparative group 1) and (comparative group 2) adds fertilized egg at CB final concentration 0.5mg/l, contain 0.5%DMSO in the situation that.The fertilized egg of comparative group 3 and 4 is processed the discharge to stop first polar body with 6-dimethylaminopurine (DMAP).DMAP is dissolved in methyl-sulfoxide (DMSO), at DMAP final concentration 0.25mg/l, contain 0.5%DMSO in the situation that (comparative group 3) and (comparative group 4) adds fertilized egg at DMAP final concentration 0.5mg/l, contain 0.5%DMSO in the situation that.
The fertilized egg of experimental group 1 and 2 processes to stop the discharge of first polar body with CB and DMAP.CB and DMAP are dissolved in DMSO, at final concentration 0.25mg CB/l and 0.25mg DMAP/l, contain 0.5%DMSO in the situation that (experimental group 1) and (experimental group 2) adds fertilized egg at final concentration 0.5mg CB/l and 0.5mgDMAP/l, contain 0.5%DMSO in the situation that.
Control group, comparative group 1 and 2 and experimental group 1 and 2 in, process from after fertilization and within five minutes, start to continue 15 minutes.After processing, fertilized egg is cleaned with 1%DMSO-seawater, with the density of 65 ovum/ml, cultivates.
The fertilized egg of experimental group 3 processes to stop the discharge of first polar body with CB and DMAP.CB and DMAP are dissolved in DMSO, at final concentration 0.25mg CB/l, contain 0.5%DMSO in the situation that, add fertilized egg, at after fertilization, within five minutes, start to process.At after fertilization, through 10 minutes, add fertilized egg in the situation that final concentration is 0.25mg DMAP/l, after fertilization is processed 15 minutes (experimental group 3) continuously.
The fertilized egg of experimental group 4 processes to stop the discharge of first polar body with CB and DMAP.CB and DMAP are dissolved in DMSO, at final concentration 0.5mg CB/l, contain 0.5%DMSO in the situation that, add fertilized egg, at after fertilization, within five minutes, start to process.At after fertilization, through 10 minutes, add fertilized egg in the situation that final concentration is 0.5mg DMAP/l, after fertilization is processed 15 minutes (experimental group 4) continuously.In experimental group 3 and 4, after processing, fertilized egg is cleaned with 1%DMSA-seawater, with the density of 65 ovum/ml, cultivates.
[table 1]
In table 1, at after fertilization, within 90～120 minutes, measure division percentage.The survival rate of the embryo of division in after fertilization recorded (oyster ovum) through 1 day, 7 days and 35 days.As shown in table 1, experimental group 1～4 has significantly higher division percentage and the after fertilization survival rate of 35 days.In the step that stops first polar body to be discharged from fertilized egg, the combination results larva that can use CB and DMAP.
In addition, utilize flow cytometry at the date collected of larva, to measure the ploidy composition of survival larva.Measurement is weight and the chromosome number through the survival oyster of collection in 3 months at after fertilization.For chromosome analysis, the survival oyster of collection is processed 12 hours with the intensive experience colchicin (0.005%) of feeding.From shell, separate the internal of oyster, measure the weight of oyster.Next, cut the complete health of oyster, be fixed in the mixture of acetic acid/carbinol mixture (1: 3).Appropriate fixed sample is poured on slide, at air drying.This slide is carried out to Leishman ' s dyeing.To each oyster do not show chromosome loss sign at least 10 metaphase cell count.Only those oysters of having measured chromosome quantity are used to chromosome analysis.There are 20,30 and 40 chromosomal oysters and be called dliploid, triploid and tetraploid.Table 2 has shown at after fertilization through 3 months from the weight of 50 oysters of experimental group and the measurement result of chromosome quantity.Only those chromosome quantity are known that the data of the oyster of counting are included in measurement result, and do not have the data of the oyster of measurable metaphase of cell to get rid of from chromosome analysis.
[table 2]
As shown in Table 2, in the oyster at after fertilization through 1 to the 4cm size of 3 months, tetraploid oyster accounts for 78%～79.6% percentage.
From the result of this embodiment, can find out, by the combination that prevents from using in the step that first polar body is discharged from fertilized egg CB and DMAP, can be with high efficiency production tetraploid oyster.
The tetraploid oyster producing can be cultivated under the breeding condition identical with natural dliploid oyster.In addition, triploid oyster can be by making tetraploid oyster and dliploid oyster hybridize to produce.In addition, between the tetraploid oyster producing, mating of the same race is cultivated and can be produced various types of tetraploid oysters.
Claims (9)
1. a method of producing tetraploid oyster alive, comprises step:
With the sperm of diploid males oyster, make the feritilization of ovum of the female oyster of triploid;
Stop first polar body to be discharged from fertilized egg; With
By cultivating described fertilized egg, produce tetraploid oyster alive, the step that wherein stops first polar body to be discharged from fertilized egg comprises makes described fertilized egg exposing cell relaxin B(CB) and the step of 6-dimethylaminopurine (DMAP),
In the step that prevention first polar body is discharged from fertilized egg, make described fertilized egg exposing cell relaxin B(CB simultaneously) and 6-dimethylaminopurine (DMAP),
Wherein, stoping the first polar body step of discharging from fertilized egg to comprise joins fertilized egg among the 6-dimethylaminopurine (DMAP) of 0.25～1.0ppm concentration in the cytochalasin B (CB) of 0.25～1.0ppm concentration in methyl-sulfoxide (DMSO) and methyl-sulfoxide (DMSO).
2. a method of producing tetraploid oyster alive, comprises step:
With the sperm of diploid males oyster, make the feritilization of ovum of the female oyster of triploid;
Stop first polar body to be discharged from fertilized egg; With
By cultivating described fertilized egg, produce tetraploid oyster alive, the step that wherein stops first polar body to be discharged from fertilized egg comprises makes described fertilized egg exposing cell relaxin B(CB) and the step of 6-dimethylaminopurine (DMAP),
In the step that prevention first polar body is discharged from fertilized egg, make described fertilized egg exposing cell relaxin B(CB in succession) and 6-dimethylaminopurine (DMAP),
Wherein, stoping the first polar body step of discharging from fertilized egg to comprise joins fertilized egg among the 6-dimethylaminopurine (DMAP) of 0.25～1.0ppm concentration in the cytochalasin B (CB) of 0.25～1.0ppm concentration in methyl-sulfoxide (DMSO) and methyl-sulfoxide (DMSO).
3. a method of producing tetraploid oyster alive, comprises step:
With the sperm of diploid males oyster, make the feritilization of ovum of the female oyster of triploid;
Stop first polar body to be discharged from fertilized egg; With
By cultivating described fertilized egg, produce tetraploid oyster alive, the step that wherein stops first polar body to be discharged from fertilized egg comprises makes described fertilized egg exposing cell relaxin B(CB) and the step of 6-dimethylaminopurine (DMAP),
In the step that prevention first polar body is discharged from fertilized egg, make described fertilized egg exposing cell relaxin B(CB), then make described fertilized egg contact 6-dimethylaminopurine (DMAP),
Wherein, stoping the first polar body step of discharging from fertilized egg to comprise joins fertilized egg among the 6-dimethylaminopurine (DMAP) of 0.25～1.0ppm concentration in the cytochalasin B (CB) of 0.25～1.0ppm concentration in methyl-sulfoxide (DMSO) and methyl-sulfoxide (DMSO).
4. according to the method for claim 3, wherein, stop the first polar body step of discharging from fertilized egg to comprise: in fertilized egg is discharged 30%～90% the time range of time of first polar body, to make fertilized egg exposing cell relaxin B(CB), then in discharging 50%～90% the time range of time of first polar body, fertilized egg makes fertilized egg contact 6-dimethylaminopurine (DMAP).
5. according to the method for claim 3, wherein, stop the first polar body step of discharging from fertilized egg to comprise: the feritilization of ovum after, to make fertilized egg exposing cell relaxin B(CB) 5～15 minutes, then after the feritilization of ovum, make fertilized egg contact 6-dimethylaminopurine (DMAP) 8.3～15 minutes.
6. according to the method described in any one in claim 1-3, wherein, stoping the first polar body step of discharging from fertilized egg to comprise joins fertilized egg among the 6-dimethylaminopurine (DMAP) of 0.25～0.75ppm concentration in the cytochalasin B (CB) of 0.25～0.75ppm concentration in methyl-sulfoxide (DMSO) and methyl-sulfoxide (DMSO).
7. according to the method described in any one in claim 1-3, wherein, described contact is carried out in fertilized egg is discharged 30%～90% the time range of time of first polar body.
8. according to the method described in any one in claim 1-3, wherein, described contact is carried out 5～15 minutes after the feritilization of ovum.
9. according to the method described in any one in claim 1-3, wherein, described cultivation is carried out under the condition of the 18 ℃～temperature of 30 ℃ and the salinity of 17ppt～33ppt.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020090075404A KR100989384B1 (en) | 2009-08-14 | 2009-08-14 | Method of producing a viable tetraploid oyster |
| KR10-2009-0075404 | 2009-08-14 | ||
| KR1020100075514A KR20120013512A (en) | 2010-08-05 | 2010-08-05 | Method of producing a viable tetraploid oyster |
| KR10-2010-0075514 | 2010-08-05 | ||
| PCT/KR2010/005169 WO2011019166A2 (en) | 2009-08-14 | 2010-08-06 | Method for producing viable tetraploid oysters |
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| CN102573453A CN102573453A (en) | 2012-07-11 |
| CN102573453B true CN102573453B (en) | 2014-05-07 |
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| CN201080046057.5A Active CN102573453B (en) | 2009-08-14 | 2010-08-06 | Method for producing viable tetraploid oysters |
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| JP (1) | JP5613240B2 (en) |
| CN (1) | CN102573453B (en) |
| CO (1) | CO6511223A2 (en) |
| WO (1) | WO2011019166A2 (en) |
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| US9385308B2 (en) * | 2010-03-26 | 2016-07-05 | Qualcomm Incorporated | Perpendicular magnetic tunnel junction structure |
| CN104255586B (en) * | 2014-08-15 | 2016-01-20 | 中国科学院南海海洋研究所 | A kind of time point quantitative Treatment method of producing Hong Kong oyster all-triploid |
| CN107494358B (en) * | 2017-09-28 | 2019-11-01 | 中国科学院南海海洋研究所 | A kind of preparation method of Hong Kong oyster tetraploid children shellfish |
| CN109730008B (en) * | 2019-03-11 | 2021-01-01 | 中国海洋大学 | Method for cultivating grapevine oyster tetraploid |
| CN112535128A (en) * | 2020-07-14 | 2021-03-23 | 厦门世倍海洋科技有限公司 | Method for producing allotetraploid by hybridizing diploid of Crassostrea ampelopsis Grossdentata and triploid of Crassostrea pacifica |
| CN112535127A (en) * | 2020-07-14 | 2021-03-23 | 厦门世倍海洋科技有限公司 | Method for producing allotetraploid by hybridizing diploid of Crassostrea ampelopsis Grossdentata and triploid of Crassostrea hongkongensis |
| CN113678764B (en) * | 2020-12-31 | 2023-08-04 | 青岛前沿海洋种业有限公司 | Method for producing tetraploid oyster and interspecific hybridization triploid oyster |
| CN113951194B (en) * | 2021-10-20 | 2022-12-13 | 青岛前沿海洋种业有限公司 | Method for producing triploid oysters by interspecific hybridization of Fujian oysters and sikauri oysters |
| CN114208735B (en) * | 2021-12-22 | 2023-02-28 | 中国科学院南海海洋研究所 | Method for cultivating rapid-growth new strain of hong Kong oyster triploid by backcross breeding technology |
| CN114600807A (en) * | 2022-04-02 | 2022-06-10 | 厦门大学 | Method for inducing Haliotis discus hannai tetraploid |
| CN116406649B (en) * | 2023-02-22 | 2024-03-19 | 中国科学院南海海洋研究所 | Method for improving genetic diversity of oyster tetraploid and constructing tetraploid stable group line |
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| JP3839842B2 (en) * | 1994-01-21 | 2006-11-01 | ラトガーズ， ザ ステイト ユニバーシティ オブ ニュー ジャージー | Tetraploid shellfish |
| US20050149997A1 (en) * | 2003-11-04 | 2005-07-07 | Wolozin Benjamin L. | Sterilization of marine organisms by manipulation of DNA content |
| CN1545873A (en) * | 2003-12-16 | 2004-11-17 | 中国海洋大学 | Method for inducting oyster tetraploid using 6-dimethylin purine |
| FR2913982B1 (en) * | 2007-03-23 | 2009-05-29 | Ifremer | OBTAINING TETRAPLOID BIVALVE MOLLUSCS FROM DIPLOID GENERATORS |
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2010
- 2010-08-06 JP JP2012524635A patent/JP5613240B2/en active Active
- 2010-08-06 CN CN201080046057.5A patent/CN102573453B/en active Active
- 2010-08-06 WO PCT/KR2010/005169 patent/WO2011019166A2/en active Application Filing
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2012
- 2012-03-09 CO CO12041581A patent/CO6511223A2/en unknown
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Also Published As
| Publication number | Publication date |
|---|---|
| CO6511223A2 (en) | 2012-08-31 |
| JP2013501518A (en) | 2013-01-17 |
| CN102573453A (en) | 2012-07-11 |
| WO2011019166A2 (en) | 2011-02-17 |
| JP5613240B2 (en) | 2014-10-22 |
| WO2011019166A3 (en) | 2011-07-14 |
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