CN107494358B - A kind of preparation method of Hong Kong oyster tetraploid children shellfish - Google Patents
A kind of preparation method of Hong Kong oyster tetraploid children shellfish Download PDFInfo
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- CN107494358B CN107494358B CN201710900628.0A CN201710900628A CN107494358B CN 107494358 B CN107494358 B CN 107494358B CN 201710900628 A CN201710900628 A CN 201710900628A CN 107494358 B CN107494358 B CN 107494358B
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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
- 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
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- 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
The invention discloses a kind of preparation methods of Hong Kong oyster tetraploid children shellfish.The present invention passes through the sport technique segments such as preferred triploid ovum, selected diploid sperm, inhibition fertilized eggs second polar body, acquisition tetraploid larva, cultivation tetraploid progeny, part triploid ovum, which is utilized, can exercise the characteristic of normal reductive division, create a kind of breeding method of inhibition fertilized eggs (three times ovum+diploid sperm) second polar body preparation tetraploid.The present invention is different from the method by inhibiting fertilized eggs first polar body to obtain tetraploid, traditional production of hybrid seeds mode is overturned, monopolization of the external force such as the U.S., France, South Korea to oyster tetraploid Cultivating techniques is broken, the Hong Kong oyster children shellfish that can be survived is obtained for the first time, feasibility is provided to use biological method to produce 100% Hong Kong oyster triploid from now on, it also is the industry stable development of the South China coastal Hong Kong oyster, industry improved variety provides possibility.The present invention have many advantages, such as it is implementable, repeatable, can be using.
Description
Technical field:
The invention belongs to shellfish Biotechnology in Genetic Breeding fields in Marine agriculture, and in particular to a kind of Hong Kong oyster tetraploid children
The preparation method of shellfish.
Background technique:
Tetraploid refers to that cell or organism have four sets of chromosomes, and it only includes two resisdyes that it, which is different from diplont,
Colour solid includes three sets of chromosomes also different from triploid organism.In nature, almost there is not been reported for natural tetraploid shellfish,
The tetraploid of artificial induction is mainly concentrated on the species such as oyster, scallop, mussel.Under normal conditions, the production method packet of tetraploid
It includes and inhibits the release of fertilized eggs polar body, inhibit First cleavage, promote normal ploidy to be fertilized by cell fusion or gynogenesis
Ovum chromosome doubling forms tetraploid by embryonic development.By scholars for many years the study found that using triploid ovum with
Diploid sperm fertilization can obtain survival shellfish tetraploid adult, other methods by inhibiting the release of fertilized eggs first polar body
Not applicable at present, although most methods can obtain tetraploid larva, its larva abnormal can not form tetraploid children
Shellfish causes follow-up work research that can not carry out.
For oyster, Guo Ximing etc. is fertilized using triploid ovum and diploid sperm, is inhibited using chemicals
Fertilized eggs first polar body produces long oyster tetraploid in the world for the first time, and then, it is male to cultivate nearly river using same procedure
The tetraploids such as oyster, American oyster, Kumamoto oyster.Guo Xi penetrating judgment is awarded to be thought in patent CN1144454A, triploid ovum and two
The fertilized eggs formed after times body sperm fertilization can generate double monocentric divisions (in meiosis by inhibiting first polar body release
In the process, 1 first polar body forms one group of 3N in conjunction with egg cell, and 1 first polar body and second polar body form one group of 3N, and only the
During secondary meiosis, to the phenomenon that the separation of the two poles of the earth), that is to say, that after triploid ovum first polar body is suppressed, shape
At (+1 1.5N first polar body of 1.5N ovum) of 3N and (the 1 1.5N first polar body+1.5N second polar body) of 3N, then this
The sperm of 3N (+1 1.5N first polar body of 1.5N ovum) and 1N can be retained after the fertilized eggs inhibition first polar body of sample, thus
Tetraploid is formed, is cultivated by carefulness, obtains the long oyster of the tetraploid that can be survived.
By being fertilized using triploid ovum and diploid sperm repeatedly the study found that the method awarded using Guo Xi penetrating judgment,
Inhibit first polar body, a certain number of tetraploid larvas can be obtained, and this larva can survive, forms tetraploid children
Shellfish and adult.The inventor of CN102573453B patent has also continued the method that Guo Xi penetrating judgment is awarded, and changes and inhibits making for drug
With method, by a kind of inhibitor development is used alone as two kinds of inhibitor are used in combination, four times of long oyster of survival are also obtained
Body.But inventors have found that inhibiting the first polar body method of fertilized eggs in addition to using triploid ovum and diploid sperm after fertilization
It can obtain outside survival tetraploid, its second polar body can also be inhibited to discharge, obtain survival oyster tetraploid.Trace it to its cause be because
Normal meiosis function can be executed for part triploid ovum, it is possible to obtain 4N filial generation (1.5N ovum+1.5N
Diode+1N sperm), and the larva can survive to metamorphosis, form young shellfish, grow up to adult.
Summary of the invention
The object of the present invention is to provide a kind of preparation methods of Hong Kong oyster tetraploid children shellfish, are obtained using the preparation method
The Hong Kong oyster tetraploid children shellfish that can survive, to be produced from now on using the sub- biological method of diplont ovum+tetraploid production of sperm
100% triploid has established solid foundation.
In order to solve the problems, such as that the South China coastal Hong Kong oyster Wheat summer sowing phase is excessively thin, the present invention is being applied
On the basis of CN104255586B patented technology successfully obtains 100% Hong Kong oyster triploid, triploid ovum and diploid are utilized
Sperm fertilization obtains the characteristics of discharging by inhibition second polar body, normal reductive division function can be performed using part three times ovum
The Hong Kong oyster tetraploid children shellfish that can be survived was obtained, to pass through diplont ovum+tetraploid sperm mode of manufacture 100% 3 from now on
Times body has established solid foundation, and new excellent variety is also provided for the oyster culture of south China area.
The preparation method of Hong Kong oyster tetraploid of the invention, which comprises the following steps:
A, preferred triploid ovum: in Hong Kong oyster mating period, flow cytomery Hong Kong oyster triploid is utilized
Individual selects 3N individual, dissects triploid later, select sexual gland part, carries out sediments microscope inspection gender, screens female
Body, selects that egg development is neat, yolk color is relatively deep, ovum amount >=10,000,000 individuals are as female parent, weeds out ovum
Quantity is few, sexual gland is not developed and individual males；The ovum of female parent is extruded, is filtered, is put using 200 mesh sieve tulles
It is placed in salinity 15-20ppt seawater and activates 30-60min, wash ovum with 400 mesh sieve tulles later, repeat 2-3 times, remove impurity,
It is rounded completely to ovum, then with FCM analysis ovum ploidy is can using for 6N；
B, selected diploid sperm: dissecting conventional Hong Kong oyster diploid, takes part sexual gland, sediments microscope inspection gender, choosing
Sexual gland is full, sperm motility is strong individual is selected as male parent, weeds out sperm motility difference or debility individual；To step
When 90% or more of triploid ovum quantity is rounded in a, the sperm for dissecting male parent, which is put into 15-20ppt seawater, activates 10-
15min, sperm >=90% is froliced happily under microscope, and utilizing flow cytomery sperm ploidy is can using for 1N；
C, inhibit fertilized eggs second polar body: monomer sperm is added in monomer triploid ovum, and controlling around each ovum has 6-9
A sperm, triploidy rate control form fertilized eggs in≤3 ten thousand/ml；During this period, temperature control is at 28-30 DEG C, salinity
Control is in 15-20ppt；Later, with micro- sem observation fertilized eggs the first diode release time, when the quantity accounting of first polar body
When occurring 50%, start to inhibit fertilized eggs using cytochalasin B, until 10% untreated fertilized eggs of hair on-hand quantity
When into two-cell stage, drug-treated terminates, and washes away the cytochalasin in ovum liquid, is placed in cement pit and hatches；
D, it obtains tetraploid larva: hatching by 24-30h, polyploid D-shaped larva can be obtained, by flow cytometer
Detection, discovery larva composition is only 4N and 2.5N, and no other ploidy larvas generate, wherein tetraploid ratio is in 30-90%.
E, it cultivates tetraploid progeny: tetraploid larva is cultivated using conventional method, when larva grows to 15d, survival
Rate is 3.2-6.5%, is tetraploid after testing, the aneuploid of 2.5N is all dead；It cultivates, obtains by 30d
The tetraploid juvenile mollusk of a height of 3-5mm of shell, larval metamorphosis rate are 10-20%；It is cultivated by 90d, obtains the high 2-3cm's of shell
Tetraploid children shellfish.
The cytochalasin be using concentration 0.5mg/L cytochalasin B.
It is described to select that egg development is neat, yolk color is relatively deep, ovum amount >=10,000,000 individuals are as female parent,
The ovum diameter of its ovum is preferably 70 μm or so.
The present invention is different from traditional oyster tetraploid preparation method, and there is following essential differences: 1. core technology method
Entirely different: conventional method is to inhibit fertilized eggs (triploid ovum+diploid sperm) first polar body, because of partial fertilization ovum
It will appear double monocentric division phenomenons, to obtain tetraploid (3N monocentric division+1N sperm) filial generation；And the present invention is to inhibit fertilization
Ovum (triploid ovum+diploid sperm) second polar body, because partial fertilization ovum can execute normal meiosis, from obtaining
Tetraploid (1.5N ovum+1.5N second polar body+1N sperm) filial generation；2. the opportunity of processing is different: conventional method is selected in the first pole
Body starts to process, and the present invention selection first polar body occurs 50% and starts to process；Conventional method selective fertilization ovum occurs
50% ends processing, and present invention selection is ended processing when the 10% of control group fertilized eggs quantity enters two-cell stage；3. locating
It is different to manage the time: conventional method is mostly 12-15min using the inhibitor processing fertilized eggs time, and the processing time of the invention
For 30-40min；4. treatment effect is different: conventional process acquisition D-shaped larva composition is extremely complex, includes 2N, 3N, 4N
And aneuploid, and the D-shaped larva obtained after present invention processing forms fairly simple, only 2.5N and 4N；5. filial generation is cultivated difficult
Degree is different: after conventional method obtains larva, survival rate of larvae is lower than 0.1%, and the larvae development that the present invention obtains is young to eyespot
The survival rate of worm is 1.2% or more；Conventional method is lower than 1% from eyebot larvae to abnormal distortion ratio, and the present invention obtains eye
Point larva is to metamorphosis is completed, and distortion ratio is 10% or more.
The present invention passes through preferred triploid ovum, selected diploid sperm, inhibition fertilized eggs second polar body, acquisition tetraploid
Larva cultivates the sport technique segments such as tetraploid progeny, and part triploid ovum, which is utilized, can exercise the characteristic of normal reductive division,
Create a kind of breeding method of inhibition fertilized eggs (three times ovum+diploid sperm) second polar body preparation tetraploid.
The present invention is different from the method by inhibiting fertilized eggs first polar body to obtain tetraploid, has overturned traditional production of hybrid seeds mould
Formula has broken monopolization of the external force such as the U.S., France, South Korea to oyster tetraploid Cultivating techniques, and obtaining can survive for the first time
Hong Kong oyster children shellfish, for from now on use biological method produce 100% Hong Kong oyster triploid provide feasibility, be also China
The coastal Hong Kong oyster industry stable development in south, industry improved variety provide possibility.The present invention have it is implementable, repeatable, can answer
The advantages that using.
Detailed description of the invention:
Fig. 1 is conventional method, wherein 1- triploid ovum；2- fertilization, 3- inhibit pb1, and the bis- monopoles of 4- separate, and tetra- times of 5-
Body；
Conventional method Technology Roadmap is fertilized using triploid ovum and diploid sperm, inhibits fertilized eggs first polar body,
Partial fertilization ovum produces double monopole segregation phenomenons, the second polar body of 3N is released, to obtain tetraploid；
Fig. 2 is Technology Roadmap of the invention, wherein 1- triploid ovum；2- fertilization, 3- discharge pb1, and 4- inhibits pb2,
5- tetraploid；
The technology of the present invention route schematic diagram.It is fertilized using triploid ovum and diploid sperm, inhibits the second pole of fertilized eggs
Body releases the first polar body of 3N, restrains the second polar body of 1.5N since partial fertilization ovum travels normal reductive division function
+ 1.5N egg cell+1N sperm=4N, forms 4 times of bodies, and filial generation can survive；
Note: pb1 indicates first polar body；Pb2 indicates second polar body；N indicates a set of chromosome.Wherein,Indicate a set of dyeing
Body (10 chromosome), andIndicate half set chromosome (5 chromosome).
Specific embodiment:
The following examples are further illustrations of the invention, rather than limiting the invention.
Embodiment 1
A, on May 30th, 2017, in Hong Kong oyster mating period, 63 shell height preferred triploid ovum: are randomly choosed
In the two age Hong Kong oyster triploids of 12-15cm, using flow cytomery triploid individual, discovery has 57 3N individuals,
Dissect triploid later, select sexual gland part using toothpick, carry out sediments microscope inspection and identify gender, find 46 female individuals,
10 sexual glands do not develop, 1 male；In 46 female individuals, it is fuller to screen 7 sexual glands, ovum quality compared with
Good individual, egg development is neat, and yolk color is deeper, and ovum diameter extrudes its ovum at 70 μm or so, utilizes 200 mesh thin,tough silk
Net filtration, ovum are placed in salinity 15ppt seawater and activate 30min, wash ovum with 400 mesh sieve tulles later, be repeated 3 times, go to clean
Matter, by calculating ovum amount between 1000-2500 ten thousand, discovery has 3 individual ovums that can be rounded completely, other 4 individuals
Ovum is oval or pyriform, is weeded out, is the 6N of standard with flow cytomery 3 individual ovum ploidies, will
It is spare as triploid parent's ovum liquid；
B, selected diploid sperm: the two age diploid Hong Kong oysters of 18 a height of 10-13cm of shell of dissection are taken using toothpick
Part sexual gland, sediments microscope inspection identify gender, and discovery has 5 males, select sexual gland is full, sperm motility is strong 3
Body weeds out 2 individuals of sperm motility difference as male parent；It is sea-water activated by 48min salinity 15ppt, three in step a
Times body ovum 90% is rounded, and the sperm for dissecting 3 male parent's individuals at this time, which is put into 20ppt seawater, activates 10min, and 3
Sperm bounce ratio is respectively 90%, 96%, 99% to body under the microscope, is 1N using flow cytomery sperm ploidy,
It is spare as diploid sperm；
C, inhibit fertilized eggs second polar body: according to one-to-one principle, 3 monomer sperms are added in step a in optional step b
Artificial insemination is carried out in the triploid ovum of 3 monomers, controlling has 6-9 sperm around each ovum, triploidy rate control exists
≤ 3 ten thousand/ml, keep stirring fertilized eggs, it is ensured that each ovum has sperm entrance, forms 3 groups of family fertilized eggs；In this phase
Between, at 28.2 DEG C, salinity is controlled in 15ppt for temperature control；Later, when being discharged with micro- the first diode of sem observation fertilized eggs
Between, when after fertilization 25min, fertilized eggs first polar body, which occurs 50%, (i.e. in all fertilized eggs, has 50% ratio of quantity to go out
Handled when existing first polar body), start to inhibit fertilized eggs using the cytochalasin B of 0.5mg/L, until after fertilization 60min,
It was found that there is the untreated fertilized eggs of 10% quantity to enter two-cell stage, drug-treated terminates, and washes away ovum using 400 mesh sieve tulles
Cytochalasin in liquid, each one-to-one family are respectively placed in 1m3Hatch in cement pit；
D, it obtains tetraploid larva: hatching by 30h, polyploid D-shaped larva, quantity difference are obtained in 3 cement pits
It is 1,000,000,800,000,500,000；By flow cytomery, it is found that larva composition is only 4N and 2.5N, without other times
Property larva generate, wherein tetraploid ratio is respectively 37.5%, 54.6%, 87.3%, remaining is the aneuploid of 2.5N
Larva；
E, it cultivates tetraploid progeny: cultivating tetraploid progeny, when larva grows to 15d, 3 groups of family survival rate difference
It is 3.2%, 4.5%, 6.5%, is tetraploid after testing, the aneuploid of 2.5N is all dead；It is trained by 30d
It educates, obtains the tetraploid juvenile mollusk of a height of 3-5mm of shell, 3 groups of family difference juvenile mollusk quantity are 3840,5400,6500, children
Worm distortion ratio is respectively 12%, 15%, 20%；It is cultivated by 90d, obtains the tetraploid children shellfish of the high 2-3cm of shell, 3 groups of familys
From juvenile mollusk to the survival rate in young shellfish stage close to 100%, there is not yet dead occur.
Embodiment 2
A, preferred triploid ovum: on June 3rd, 2017, in Hong Kong oyster mating period, 35 shell height is randomly choosed and are existed
The two age Hong Kong oyster triploids of 12-15cm, using flow cytomery triploid individual, discovery has 28 3N individuals, it
After dissect triploid, select sexual gland part using toothpick, carry out sediments microscope inspection and identify gender, find 23 female individuals, 5
Sexual gland is not developed；In 23 female individuals, screen that 5 sexual glands are fuller, and ovum quality is preferably individual, and egg development is whole
Together, yolk color is deeper, and ovum diameter extrudes its ovum at 70 μm or so, is filtered using 200 mesh sieve tulles, and ovum is placed in salt
60min is activated in degree 20ppt seawater, ovum is washed with 400 mesh sieve tulles later, is repeated 2 times, removes impurity, is existed by calculating ovum amount
Between 1000-2000 ten thousand, discovery has 2 individual ovums that can be rounded completely, and other 3 individual ovums are oval or pears
Shape is weeded out, and is the 6N of standard with flow cytomery 2 individual ovum ploidies, as triploid parent
Ovum liquid is spare；
B, selected diploid sperm: the two age diploid Hong Kong oysters of 10 a height of 11-13cm of shell of dissection are taken using toothpick
Part sexual gland, sediments microscope inspection identify gender, and discovery has 3 males, select sexual gland is full, sperm motility is strong 2
Body weeds out 1 individual of sperm motility difference as male parent；It is sea-water activated by 45min salinity 20ppt, three in step a
Times body ovum 90% is rounded, and the sperm for dissecting 2 male parent's individuals at this time, which is put into 15ppt seawater, activates 15min, and 3
Sperm bounce ratio is respectively 95%, 99% to body under the microscope, is 1N using flow cytomery sperm ploidy, as two
Times body sperm is spare；
C, inhibit fertilized eggs second polar body: according to one-to-one principle, 2 monomer sperms are added in step a in optional step b
Artificial insemination is carried out in the triploid ovum of 2 monomers, controlling has 6-9 sperm around each ovum, triploidy rate control exists
≤ 3 ten thousand/ml, keep stirring fertilized eggs, it is ensured that each ovum has sperm entrance, forms 2 groups of family fertilized eggs；In this phase
Between, at 29.6 DEG C, salinity is controlled in 20ppt for temperature control；Later, when being discharged with micro- the first diode of sem observation fertilized eggs
Between, when after fertilization 23min, fertilized eggs first polar body, which occurs 50%, (i.e. in all fertilized eggs, has 50% ratio of quantity to go out
Handled when existing first polar body), start to inhibit fertilized eggs using the cytochalasin B of 0.5mg/L, until after fertilization 52min,
It was found that there is the untreated fertilized eggs of 10% quantity to enter two-cell stage, drug-treated terminates, and washes away ovum using 400 mesh sieve tulles
Cytochalasin in liquid, each one-to-one family are respectively placed in 1m3Hatch in cement pit；
D, it obtains tetraploid larva: by hatching for 24 hours, polyploid D-shaped larva, quantity difference is obtained in 2 cement pits
It is 800,000,900,000；By flow cytomery, it is found that larva composition is only 4N and 2.5N, no other ploidy larvas produce
It is raw, wherein tetraploid ratio is respectively 60.9%, 90.0%, remaining is the aneuploid larva of 2.5N；
E, it cultivates tetraploid progeny: tetraploid larva, when larva grows to 15d, 2 groups of families is cultivated using conventional method
Be survival rate it is respectively 3.9%, 4.8%, is tetraploid after testing, the aneuploid of 2.5N is all dead；By
30d is cultivated, and obtains the tetraploid juvenile mollusk of a height of 3-5mm of shell, 3 groups of family difference juvenile mollusk quantity are 3120,5616, children
Worm distortion ratio is respectively 10%, 13%；It is cultivated by 90d, obtains the tetraploid children shellfish of the high 2-3cm of shell, 3 groups of familys are from juvenile mollusk
To the survival rate in young shellfish stage close to 100%, there is not yet dead occur.
Claims (3)
1. a kind of preparation method of Hong Kong oyster tetraploid, which comprises the following steps:
A, preferred triploid ovum: in Hong Kong oyster mating period, using flow cytomery Hong Kong oyster triploid individual,
3N individual is selected, dissects triploid later, selects sexual gland part, sediments microscope inspection gender is carried out, screens female individuals, is selected
Egg development is neat, yolk color is relatively deep, ovum amount >=10,000,000 individuals are as female parent, weed out ovum quantity it is few,
Sexual gland is not developed and individual males；The ovum of female parent is extruded, is filtered using 200 mesh sieve tulles, is placed in salt
30-60min is activated in degree 15-20ppt seawater, washes ovum with 400 mesh sieve tulles later, is repeated 2-3 times, impurity is removed, to ovum
It is rounded completely, then with FCM analysis ovum ploidy is can using for 6N；
B, selected diploid sperm: dissecting conventional Hong Kong oyster diploid, takes part sexual gland, sediments microscope inspection gender, selectivity
The individual that gland is full, sperm motility is strong weeds out sperm motility difference or debility individual as male parent；To in step a
When 90% or more of triploid ovum quantity is rounded, the sperm for dissecting male parent, which is put into 15-20ppt seawater, activates 10-
15min, sperm >=90% is froliced happily under microscope, and utilizing flow cytomery sperm ploidy is can using for 1N；
C, inhibit fertilized eggs second polar body: monomer sperm is added in monomer triploid ovum, and controlling has 6-9 essence around each ovum
Son, triploidy rate control form fertilized eggs in≤3 ten thousand/ml；During this period, at 28-30 DEG C, salinity is controlled for temperature control
In 15-20ppt；Later, with micro- sem observation fertilized eggs the first diode release time, when the quantity accounting of first polar body occurs
When 50%, start to inhibit fertilized eggs using cytochalasin B, until 10% untreated fertilized eggs of hair on-hand quantity enter
When two-cell stage, drug-treated terminates, and washes away the cytochalasin in ovum liquid, is placed in cement pit and hatches；
D, it obtains tetraploid larva: by hatching, obtaining polyploid D-shaped larva；
E, it cultivates tetraploid progeny: tetraploid larva being cultivated using conventional method, obtains tetraploid children shellfish.
2. the preparation method of Hong Kong oyster tetraploid according to claim 1, which is characterized in that the cytochalasin
Be using concentration 0.5mg/L cytochalasin B.
3. the preparation method of Hong Kong oyster tetraploid according to claim 1 or 2, which is characterized in that the selection ovum
Son development is neat, yolk color is relatively deep, ovum amount >=10,000,000 individuals are as female parent, and the ovum diameter of ovum is 70 μm.
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| CN108124801B (en) * | 2018-03-13 | 2020-09-18 | 中国海洋大学 | Induction method of novel oyster variety Haoda No. 2 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 |
| CN112931323A (en) * | 2021-03-05 | 2021-06-11 | 中国海洋大学 | Induction method of high heterozygosity tetraploid of novel variety Haoda No. 3 of crassostrea gigas |
| CN113749059B (en) * | 2021-10-29 | 2023-03-24 | 中国海洋大学 | Cultivation method for rapidly growing triploid crassostrea gigas' Hayao No. 1 |
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| CN100415085C (en) * | 2004-08-13 | 2008-09-03 | 中国海洋大学 | Method of cutturing polyploid seed breedign using diploid/tetraploid oclimera abalone |
| CN100486426C (en) * | 2005-11-25 | 2009-05-13 | 李思发 | Method for constructing allotetraploid of bluntsnout bream |
| FR2913982B1 (en) * | 2007-03-23 | 2009-05-29 | Ifremer | OBTAINING TETRAPLOID BIVALVE MOLLUSCS FROM DIPLOID GENERATORS |
| CN102573453B (en) * | 2009-08-14 | 2014-05-07 | 西亚益维株式会社 | Method for producing viable tetraploid oysters |
| CN102090360B (en) * | 2010-12-17 | 2012-02-29 | 中国水产科学研究院黄海水产研究所 | High-efficient induction method of tetraploid cynoglossus semilaevis fish fries |
| CN106106280A (en) * | 2016-06-30 | 2016-11-16 | 湖南文理学院 | A kind of improve crucian carp, the method for Cyprinus carpio male tetraploid incubation rate |
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