CN114600807A - Method for inducing Haliotis discus hannai tetraploid - Google Patents
Method for inducing Haliotis discus hannai tetraploid Download PDFInfo
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Classifications
-
- 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
-
- 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
An inducing method of Haliotis discus hannai tetraploid, relates to a cultivation method of shellfish tetraploid. The method comprises the following steps: 1) accelerating the maturing of the breeding abalone: placing the parent abalones in a parent abalones cultivation pool for synchronous ripening; 2) selection and artificial spawning induction of parent abalones: selecting female triploid individuals with well-developed gonads and male diploid individuals with mature gonads, which are determined by ploidy, to hasten parturition until male and female breeding abalones lay eggs and discharge sperms; 3) selection and mixed fertilization of sperm and egg: respectively filtering impurities in the sperm and eggs in the step 2), adding diploid sperms into triploid egg liquid for mixing and fertilization to obtain fertilized eggs; 4) inhibiting the first polar body of a fertilized egg with a drug; 5) and (5) removing the medicine and hatching. The high-efficiency and stable obtaining of the haliotis discus hannai tetraploid is realized by ensuring the gonad maturity of the parent abalones (female triploid and male diploid) and optimizing the induction conditions (the drug treatment time and the drug concentration).
Description
Technical Field
The invention relates to a method for cultivating tetraploid shellfish, in particular to a method for inducing tetraploid haliotis discus hannai.
Background
Chromosome manipulation (Chromosome manipulation) is widely used as an important genetic improvement means in various shellfish such as oyster, scallop, clam, mussel, pearl oyster and abalone. The triploid oyster has the characteristics of rapid growth, excellent meat quality, annual harvest and the like, so that the triploid-tetraploid breeding technology is commercially applied in the oyster cultivation industry all over the world; the breeding species mainly comprise Pacific oyster (Crassostragigas) and American oyster (Crassostreae virginica), and the breeding range is wide in the United states, France, China, Australia and Mexico. Unlike the direct induction of triploid by chemical or physical methods, commercial holotriploid oysters are obtained by biological hybridization (2N × 4N). The method is simple and convenient to operate, has stable ploidy rate, does not have adverse effects on embryos possibly caused by drug residues, and is highly dependent on feasible tetraploid breeding technology and stably established tetraploid populations.
Tetraploid production is mainly through three routes: the first is directly adopting a chemical or physical method to inhibit the first polar body (PB1) or the first Mitosis (Mitosis I) of the diploid ovum; secondly, methods such as gynogenesis or cell fusion are used; the two direct methods are adopted for inducing tetraploids on Pacific oyster, chlamys farreri, Pinctada fucata, mussels and the like, and the fact that tetraploid embryos or larvae are difficult to cultivate into adults, the number of individuals capable of surviving to juvenile mollusks or adults is limited, and tetraploid populations are difficult to establish is found. And thirdly, tetraploids are indirectly obtained by inhibiting PB1 of fertilized eggs after crossing of female triploid individuals and male diploids, in 1994, Guo and Allen establish the method for the first time and obtain a viable Pacific oyster tetraploid adult. Subsequently, this indirect method is further extended to shells such as pacific oysters, crassostrea gigas (crassostrea keensis), bay scallops (argopectenradians), and the like. The research work on tetraploid of Haliotis discus hannai (Haliotis disco hannai) starts in 1995, and reports that Cytochalasin B (CB) at 1mg/L inhibits the release of fertilized egg (2 Nx 2N) PB1 of Haliotis discus hannai (2 Nx 2N) by using a direct method, and the tetraploid rate is only 21.9% at most; 0.05g/L colchicine is used for inhibiting the first cleavage of fertilized eggs, and the highest tetraploid induction rate is 13.0 percent. Journal of shellfish research (vol. 19 of 2000) reported that induction of tetraploids by PB1 in fertilized eggs of Haliotis discus hannai (2 Nx 2N) was suppressed by using 0.8mg/L of CB or 175 to 225. mu. mol/L of 6-dimethylaminopurine (6-DMAP), and the induction rate was 20% to 35.9%. In addition to Haliotis discus hannai et Schott, in the biotechnological Notification (supplement of 2008), CB is used for respectively inhibiting diploid ovum PB1, inhibiting diploid ovum PB1, second polar body (PB2) and inhibiting triploid ovum PB1 to induce the tetraploid of Haliotis diversicoloraylis (Haliotis diversicolorayliis), wherein the tetraploid induction rate of the latter (inhibiting triploid ovum PB1) is 15-40%, and the instability of induced drug toxicity and doubling rate can influence the number of subsequent adults.
At present, the direct method is mainly used for inducing the haliotis discus hannai tetraploid, and the indirect method, namely the work of inhibiting the triploid ovum polar body from inducing the tetraploid is not reported. Meanwhile, the abalone tetraploid seed production technology has the problems of insufficient number of female triploid mature oocytes, immature induction conditions, low induction rate, unstable induction effect, low larva survival rate and the like, so that the mature and stable haliotis discus hannai tetraploid seed production technology cannot be formed.
Disclosure of Invention
The invention aims to provide the inducing method of the Haliotis discus hannai tetraploid, which can solve the problems of low inductivity, poor stability and the like of the existing tetraploid inducing technology, effectively improve the tetraploid doubling rate of the Haliotis discus hannai, obtain tetraploid adults in batches and lay the foundation for producing the full triploid Haliotis discus hannai by using a female diploid and a male tetraploid.
The invention comprises the following steps:
1) accelerating the maturing of the breeding abalone: placing the parent abalones in a parent abalones cultivation pool for synchronous ripening;
2) selection and artificial spawning induction of parent abalones: selecting female triploid individuals with well-developed gonads and male diploid individuals with mature gonads, which are determined by ploidy, to hasten parturition until male and female breeding abalones lay eggs and discharge sperms;
3) selection and mixed fertilization of sperm and egg: respectively filtering impurities in the sperm and eggs in the step 2), adding diploid sperms into triploid egg liquid for mixing and fertilization to obtain fertilized eggs;
4) inhibiting the first polar body of a fertilized egg with a drug;
5) and (5) removing the medicine and hatching.
In the step 1), the specific method for accelerating the maturing of the cultured abalone can be as follows: taking the haliotis discus hannai triploid and the male haliotis discus hannai diploid which have good individual vitality and do not have external damage as seed haliotis discus hannai, and respectively placing the seed haliotis discus hannai triploid and the male haliotis discus hannai diploid in parent haliotis discus hannai cultivation pools for synchronous ripening; the abalone can be grown for 2-3 years, and the shell length is 60-120 mm; the water temperature of the parent abalone culture pond is 22-24 ℃.
In step 2), the specific method for selection and artificial spawning of the breeding abalones can be as follows: carrying out induced spawning on a female triploid individual with good ploidy of gonad development and a male diploid individual with mature gonad development by adopting a method of drying in the shade and irradiating seawater by combining ultraviolet rays until female and male parent abalones lay eggs and discharge sperms; the gonads of the female triploid individuals with well-developed gonads are close to dark green, and the gonad plumpness reaches over 2/3 of the diploid with the same period of maturity; the ploidy determination method is to cut out the upper tentacles of the abalone parts for flow cytometry detection.
In step 3), the specific method for selecting sperm and egg and mixed fertilization can be as follows: respectively filtering impurities in the sperm and eggs in the step 2) by using a 300-micron bolting silk, adding the diploid sperm with good vitality determined by microscopic examination into triploid egg liquid with high quality, mixing and fertilizing, and dividing into a treatment group and a control group; the diploid sperm with good activity means that the sperm has good mobility and dispersity; the triploid ovum with high quality is that the vitelline membrane of the ovum is complete and has regular shape and no accidental fertilization; the mass ratio of the sperms to the ova can be (20-15): 1; the fertilization temperature can be 17-24 ℃, and the salinity is 30-35.
In step 4), the specific method for inhibiting the first polar body of the fertilized egg by the drug can be as follows: and (3) taking the development condition of the fertilized eggs of the control group in the step 3) as a reference, and when the first polar body of the fertilized eggs of the control group begins to release (when the first polar body appears in the visual field), quickly placing the fertilized eggs of the treatment group into 6-DMAP induction liquid with the final concentration of 20-40 mg/L for treatment for 10-20 min.
In step 5), the specific method for removing the drug and incubating can be as follows: after the drug treatment is finished, the fertilized eggs are placed in seawater which is obtained by fresh filtration of 25 mu m bolting silk, the seawater is cleaned twice and managed according to the conventional incubation and later-stage cultivation method, and the tetraploid rate is measured by using a flow cytometer at the period of facial disc larvae.
Compared with the existing abalone tetraploid preparation method, the invention realizes the efficient and stable obtaining of the haliotis discus hannai tetraploid by ensuring the gonad maturity of parent abalones (female triploid and male diploid) and optimizing induction conditions (drug treatment time and drug concentration) on the basis of successfully obtaining 100% or nearly 100% haliotis discus hannai tetraploid by the CN110178799B patent technology.
The invention has the following outstanding advantages:
1. preferably, triploid species of abalone: under the natural culture condition, the gonad maturity of the triploid of the haliotis discus hannai is low. The reasonable maturation promoting abalone triploid is favorable for gonad development and selection of breeding abalone. And selecting well-developed female triploid abalone species, and inducing spawning after ploidy identification, thereby providing guarantee for the subsequent ovum quality and ovum amount.
2. The improved induction method comprises the following steps: compared with the traditional indirect method for preparing the shellfish tetraploid, which usually takes fixed time as the processing opportunity to inhibit PB1, the method disclosed by the invention takes the real-time observed PB1 release condition of a control group as a biological index to indicate the processing time. Meanwhile, a low-toxicity inducer 6-DMAP is adopted to induce tetraploid within an effective induction concentration range. The improved induction method can ensure that PB1 is effectively inhibited, avoid the problems of low doubling rate, unstable doubling rate and the like caused by the influence of uncertainty of environmental factors on the development of fertilized eggs, and reduce the adverse effects of the toxic and side effects of the original use of CB on the survival and the environment of subsequent embryos and larvae.
3. Stable and efficient inductivity: in the prior direct induction method, the inductivity of the abalone tetraploid is changed between 13 percent and 80 percent, but the method can produce the tetraploid abalone by the haliotis discus hannai triploid and the haliotis discus hannai diploid with high efficiency (the inductivity is more than 95 percent).
Detailed Description
The following examples further illustrate the invention.
The method for inducing Haliotis discus hannai tetraploid comprises the following steps:
1) accelerating the maturing of the breeding abalone: taking the haliotis discus hannai triploid and the male haliotis discus hannai diploid which have good individual vitality and do not have external damage as seed haliotis discus hannai, and respectively placing the seed haliotis discus hannai triploid and the male haliotis discus hannai diploid in parent haliotis discus hannai cultivation pools for synchronous ripening; the abalone is grown for 2-3 years, and the shell length is 60-120 mm; the temperature of the parent abalone culture pond is 22-24 ℃.
2) Selection and artificial spawning induction of parent abalones: carrying out induced spawning on a female triploid individual with good ploidy of gonad development and a male diploid individual with mature gonad development by adopting a method of drying in the shade and irradiating seawater by combining ultraviolet rays until female and male parent abalones lay eggs and discharge sperms; the gonads of the female haliotis discus hannai triploid individuals with good gonad development are close to dark green, and the gonad plumpness reaches over 2/3 of the mature diploid in the same period; the ploidy determination method is to cut out the upper tentacles of the abalone parts for flow cytometry detection.
3) Selection and mixed fertilization of sperm and egg: respectively filtering impurities in the sperm and eggs in the step 2) by using a 300-micron bolting silk, adding the diploid sperm with good vitality determined by microscopic examination into triploid egg liquid with high quality, mixing and fertilizing, and dividing into a treatment group and a control group; the sperm with good activity means that the sperm has good mobility and dispersity; the high-quality ovum means that the yolk membrane of the ovum is complete and has a regular shape and no accidental fertilization; the mass ratio of the sperms to the ova can be (20-15): 1; the fertilization temperature is 17-24 ℃, and the salinity is 30-35.
4) Drug inhibition of the first polar body of fertilized egg: and (3) taking the development condition of the fertilized eggs of the control group in the step 3) as a reference, and when the first polar body of the fertilized eggs of the control group begins to release (when the first polar body appears in the visual field), quickly placing the fertilized eggs of the treatment group into 6-DMAP induction liquid with the final concentration of 20-40 mg/L for treatment for 10-20 min.
5) Removing the medicines and hatching: after the drug treatment is finished, the fertilized eggs are placed in seawater which is obtained by fresh filtration of 25 mu m bolting silk, the fertilized eggs are washed twice and managed according to a conventional hatching and later-stage culture method, and the tetraploid rate is measured by using a flow cytometer in the period of facial disc larvae.
Specific examples are given below.
Example 1
1) Taking 2-3-year-old Haliotis discus hannai triploid and male Haliotis discus hannai diploid without external damage as seed Haliotis discus hannai, and respectively placing in parent Haliotis discus hannai cultivation ponds at 22-24 ℃ for synchronous ripening promotion.
2) After the gonads of the parent abalones mature, 16 female triploids with good gonad development and with gonad color close to dark green are selected, partial upper feet are cut to touch hands for ploidy determination, and non-triploid individuals (5) are excluded. And (3) respectively carrying out induced spawning on the remaining female triploid individuals and the 4 male diploids with mature gonads by adopting a mode of drying in the shade and irradiating seawater by ultraviolet rays until sperm and eggs are discharged.
3) The impurities in the sperm and egg were filtered off using 300 μm silk. And performing microscopic examination to determine the quality and the activity of the sperms, performing mixed fertilization, dividing fertilized eggs into two parts, and respectively marking the fertilized eggs as a control group and an experimental group, wherein the number ratio of the sperms to the sperms in the microscopic examination is (20-15): 1.
4) And (3) observing the development condition of the fertilized eggs of the control group in real time, and quickly placing the fertilized eggs of the experimental group into 6-DMAP induction liquid with the final concentration of 30mg/L for treatment for 15min when PB1 appears in a microscope field.
5) After the timing is finished, the fertilized eggs of the experimental group are washed twice in seawater filtered by 25 mu m bolting silk, and the induction is stopped.
6) Obtaining tetraploid seedlings of Haliotis discus hannai according to conventional hatching and late-stage culture method. The ploidy rate of the faceplates larvae (20-24 h) is determined by a flow cytometer, and the tetraploid induction rate of the experimental group is 95.01%.
8) The temperature of seawater in the fertilization and larva incubation processes is 17.5-20.3 ℃.
Example 2
1) Taking 2-3-year-old Haliotis discus hannai triploid and male Haliotis discus hannai diploid without external damage as seed Haliotis discus hannai, and respectively placing in parent Haliotis discus hannai cultivation ponds at 22-24 ℃ for synchronous ripening promotion.
2) After the gonads of the parent abalones mature, 33 female triploids with well-developed gonads and with gonad color close to dark green are selected, partial upper feet are cut to tentacles for ploidy determination, and non-triploid individuals (0) are excluded. And (3) respectively carrying out induced spawning on the remaining female triploid individuals and the 6 male diploids with mature gonads by adopting a mode of drying in the shade and irradiating seawater by ultraviolet rays until sperm and eggs are discharged.
3) The impurities in the sperm and egg were filtered off using 300 μm silk. And performing microscopy to determine the egg quality and sperm motility, then performing mixed fertilization, dividing fertilized eggs into two parts, and marking the fertilized eggs as a control group and an experimental group respectively, wherein the number ratio of the sperm to the eggs in the microscopy is (20-15): 1.
4) And (3) observing the development condition of the fertilized eggs of the control group in real time, and when the release of the first PB1 is observed in the visual field, quickly placing the fertilized eggs of the experimental group in 6-DMAP induction solution with the final concentration of 40mg/L for treatment for 10 min.
5) After the timing is finished, the fertilized eggs of the experimental group are washed twice in seawater filtered by 25 mu m bolting silk, and the induction is stopped.
6) Obtaining tetraploid seedlings of Haliotis discus hannai according to conventional hatching and late-stage culture method. The ploidy rate of the faceplate larvae (20-24 h) is determined by a flow cytometer, and the tetraploid induction rate of the experimental group is 96.68%.
8) The temperature of seawater in the fertilization and larva incubation processes is 20-22.3 ℃.
Example 3
1) Taking 2-3-year-old Haliotis discus hannai triploid and male Haliotis discus hannai diploid without external damage as seed Haliotis discus hannai, and respectively placing in parent Haliotis discus hannai cultivation ponds at 22-24 ℃ for synchronous ripening promotion.
2) After the gonads of the parent abalones mature, 31 female triploids with good gonad development and gonad color close to dark green are selected, partial upper feet tentacles are cut to carry out ploidy determination, and non-triploid individuals (1) are excluded. And (3) respectively carrying out induced spawning on the remaining female triploid individuals and 8 male diploids with mature gonads by adopting a mode of drying in the shade and irradiating seawater by ultraviolet rays until sperm and eggs are discharged.
3) The impurities in the sperm and egg were filtered off using 300 μm silk. And performing microscopic examination to determine the quality and the activity of the sperms, performing mixed fertilization, dividing fertilized eggs into two parts, and respectively marking the fertilized eggs as a control group and an experimental group, wherein the number ratio of the sperms to the sperms in the microscopic examination is (20-15): 1.
4) And (3) observing the development condition of the fertilized eggs of the control group in real time, and when the release of the first PB1 is observed in the visual field, quickly placing the fertilized eggs of the experimental group in 6-DMAP induction solution with the final concentration of 35mg/L for treatment for 10 min.
5) After the timing is finished, the fertilized eggs of the experimental group are washed twice in seawater filtered by 25 mu m bolting silk, and the induction is stopped.
6) Obtaining tetraploid seedlings of Haliotis discus hannai according to conventional hatching and late-stage culture method. The ploidy rate of the faceplates larvae (20-24 h) is determined by a flow cytometer, and the tetraploid induction rate of the experimental group is 95.30%.
8) The temperature of seawater in the fertilization and larva incubation processes is 20.3-21 ℃.
Table 1 shows the results of different batches of induced haliotis discus hannai tetraploid test.
TABLE 1
The above embodiments are only used to further detail the technical solution of the present invention, and are not limited thereto. Those skilled in the art will readily modify and/or replace some of the features of the embodiments described in the examples without departing from the spirit and scope of the present invention as defined by the appended claims.
Claims (10)
1. The method for inducing the Haliotis discus hannai tetraploid is characterized by comprising the following steps:
1) accelerating the maturing of the breeding abalone: placing the parent abalones in a parent abalones cultivation pool for synchronous ripening;
2) selection and artificial spawning induction of parent abalones: selecting female triploid individuals with well-developed gonads and male diploid individuals with mature gonads, which are determined by ploidy, to hasten parturition until female and male seed abalones lay eggs and discharge sperms;
3) selection and mixed fertilization of sperm and egg: respectively filtering impurities in the sperm and eggs in the step 2), adding diploid sperms into triploid egg liquid for mixing and fertilization to obtain fertilized eggs;
4) inhibiting the first polar body of a fertilized egg with a drug;
5) and (5) removing the medicine and hatching.
2. The method for inducing tetraploid of Haliotis discus hannai as claimed in claim 1, wherein in step 1), the specific method for accelerating maturation of the Haliotis discus hannai is as follows: taking the haliotis discus hannai triploid and the male haliotis discus hannai diploid which have good individual vitality and have no external damage as seed haliotis discus hannai, and respectively placing the seed haliotis discus hannai triploid and the male haliotis discus hannai diploid in a parent haliotis discus hannai cultivation pool to synchronously promote maturity.
3. The method for inducing Haliotis discus hannai tetraploid as claimed in claim 1, wherein in step 1), the seed abalone is 2-3 years old, and the shell length is 60-120 mm; the water temperature of the parent abalone culture pond is 22-24 ℃.
4. The method for inducing Haliotis discus hannai tetraploid as claimed in claim 1, wherein in step 2), the selection and artificial induced spawning of the seed abalone are specifically: the method of drying in the shade and irradiating the seawater by ultraviolet rays is adopted to hasten the parturition of the female triploid individuals with good ploidy of gonad development and the male diploid individuals with mature gonad development until the male and female abalone spawns and discharges sperm.
5. The method for inducing Haliotis discus hannai tetraploid as claimed in claim 1, wherein in step 2), the gonad of said female triploid individual with well-developed gonad is near dark green, and the gonad fullness reaches above 2/3 of the mature diploid; the ploidy determination method is to cut out the upper tentacles of the abalone parts for flow cytometry detection.
6. The method for inducing Haliotis discus hannai tetraploid as claimed in claim 1, wherein in step 3), the specific method of selecting sperm and ovum and mixed fertilization is: respectively filtering impurities in the sperm and eggs in the step 2) by using a 300-micron bolting silk, adding the diploid sperm with good vitality determined by microscopic examination into triploid egg liquid with high quality, mixing and fertilizing, and dividing into a treatment group and a control group; the diploid sperm with good activity means that the sperm has good mobility and dispersity; the triploid ovum with high quality means that the vitelline membrane of the ovum is complete, the shape is regular and no accidental fertilization exists.
7. The method for inducing Haliotis discus hannai tetraploid as claimed in claim 6, wherein the mass ratio of sperm to ovum is (20-15): 1.
8. The method for inducing Haliotis discus hannai tetraploid as claimed in claim 6, wherein the fertilization temperature is 17-24 deg.C, and salinity is 30-35.
9. The method for inducing Haliotis discus hannai tetraploid as claimed in claim 1, wherein in step 4), the method for inhibiting the first polar body of fertilized egg by said drug comprises: and taking the development condition of the fertilized eggs of the control group in the step 3) as a reference, and quickly placing the fertilized eggs of the treatment group in 6-DMAP induction liquid with the final concentration of 20-40 mg/L for treatment for 10-20 min when the first polar body of the fertilized eggs of the control group starts to release.
10. The method for inducing Haliotis discus hannai tetraploid as claimed in claim 1, wherein in step 5), the specific method of drug removal and incubation is: after the drug treatment is finished, the fertilized eggs are placed in seawater which is obtained by fresh filtration of 25 mu m bolting silk, the seawater is cleaned twice and managed according to the conventional incubation and later-stage cultivation method, and the tetraploid rate is measured by using a flow cytometer at the period of facial disc larvae.
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| CN114208734A (en) * | 2021-12-22 | 2022-03-22 | 中国科学院南海海洋研究所 | Method for improving production performance of portuguese oyster triploid by integrating multiple breeding technologies |
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| CN109730008A (en) * | 2019-03-11 | 2019-05-10 | 中国海洋大学 | A method of cultivating Portuguese oyster tetraploid |
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