CN1136765C - Method for producing triploid of young Jiekong scallop - Google Patents
Method for producing triploid of young Jiekong scallop Download PDFInfo
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- CN1136765C CN1136765C CNB001113054A CN00111305A CN1136765C CN 1136765 C CN1136765 C CN 1136765C CN B001113054 A CNB001113054 A CN B001113054A CN 00111305 A CN00111305 A CN 00111305A CN 1136765 C CN1136765 C CN 1136765C
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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
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Abstract
The present invention relates to technique for producing a young triploid chlamys farreri. The regulation and control technique for influencing a major factor of the synchronous development of an ovum is mainly supplied, and an optimized induction parameter is mastered. The quality of the fine ovum is ensured so that the ovum is synchronously impregnated and develops. 6-DMAP is used as an inducing agent so that certain drug concentration is maintained, and starting time and persistent processing time are mastered. The technique can be used for producing the young triploid chlamys farreri at a large scale. Good varieties with rapid growth, big individuals, high yield and high disease resistance can be cultivated. According to a production breeding experiment, yield can be enhanced by 20 to 30%, and fresh chlamys farreri yield is enhanced by 60% or so.
Description
Technical field:
The invention belongs to the cell chromosome operating technology in the cellular biological technique, is under production scale chlamys farreri fertilized egg to be induced, and produces a kind of technical method of triploid seed.
Background technology:
Before the present invention makes, the polyploid shellfish breeding all has research in states such as U.S., day, English, polyploid shellfish yielding ability breeding has at present only obtained success in oyster, and in economic scallop kinds such as bay scallop, Chlamys nobilis, big scallop, though triploid induction is succeedd, but all be confined to experimental scale, do not realize industrialization.Because chlamys farreri is Chinese exclusive breed kind, therefore there is not this kind triploid induction Study on Technology abroad.
Domestic to the existing research of chlamys farreri triploid induction technology, adopt the temperature shock method to carry out the triploid induction of chlamys farreri as (1990) such as prince's ministers of Dalian aquatic product Academy, (1992) such as Lu Sui Fen of Qingdao Marine University adopt cytochalasin B to carry out that chlamys farreri is triploid induces, but above-mentioned research also is to only limit to experimental scale, does not see the research contents of the large-scale chlamys farreri triploid seedling growing process that is used to produce so far.
The method of artificial induction's polyploid, nothing more than physics, chemistry and biological method, use the more temperature shock method that mainly contains, hydrostatic pressing facture and cytochalasin B (hereinafter to be referred as CB), caffeine, 6-dimethylamino-purine chemicals revulsions such as (being called for short 6-DMAP).Though above-mentioned triploid induction technology is succeedd, and also can reach higher triploid rate, because test objective is to carry out scientific research, experimental scale is little, and the yielding ability problem is not taken in, and stock trick art content is difficult to promote the use of aborning.
Summary of the invention:
The objective of the invention is to propose a kind of new, utilize 6-DMAP as derivant, can be applied to the chlamys farreri triploid seedling growing process in the large-scale production, cultivate the triploid of young Jiekong scallop of fine qualities such as having growth is fast, individuality is big, adductor-somatic index is high, high-output stress-resistance.
The present invention finishes by following operating technology: in chlamys farreri triploid certain scale seed production process, solving close shellfish cultivates, long-pending ovum, fertilization and medication (6-DMAP) technology, grasp suitable inductive technology parameter, mainly comprise medicine initiated process time, drug concentration, medicine continues the processing time.
Ovum, fertilization and practical technique are cultivated, amassed to the parent shellfish: the effective accumulated temperature that close shellfish is supported temporarily should reach more than 200 ℃, and male and female parent shellfish should separately be supported temporarily in strictness; Adopt second order product ovum processing method, the ovum of output is collected in 0.3m earlier
3In the tank, when density reaches 2000/ml, add the seminal fluid fertilization, smart ovum ratio 100: 1, the time of fertilization requires to be no more than 2 hours after the ovum output, and sperm is no more than 1 hour; After fertilization is concentrated into ovum in the little tank of 16.6L, and ovum density is at 3,000 ten thousand/L, after carry out drug treating, processing finishes the back and directly moves in the pond and hatches.
The triploid induction technical parameter:
The medicine initiated process time: add medicine 6-DMAP when under light microscope, observing first polar body and 35-45% occurring.
The concentration of drug treating concentration: 6-DMAP is 10-70mg/L.
Medicine continues the processing time: 15-20 minute.
Embodiment:
Be described in detail extensive fry production process of the present invention below by embodiment:
Most preferred embodiment concrete operations technical process: when inducing the chlamys farreri triploid in the production, the general second order product ovum processing method that adopts, promptly earlier ovum is collected in the bigger container, 0.3 cubic metre of tank commonly used, ovum density reaches and begins fertilization about 2000/ml, the consumption of sperm is more than normal diploid production, the ratio that guarantees sperm and ovum is no less than 100: 1, after fertilization concentrates flushing again and goes into to indicate in the less tank of 16.6L scale, when 35-45% appears in the observation first polar body, 1 good gram 6-DMAP (concentration of 60mg/L) handles to add dissolving, the density of handling ovum is about 3,000 ten thousand/L, even for guaranteeing fertilization and drug effect, must constantly stir, handle and hatch directly into the pond after 15 minutes.
Finish the aforesaid operations process, at first will solve close shellfish and cultivate, long-pending ovum, fertilization technology and the inductive technology parameter of producing triploid of young Jiekong scallop comprise initiated process time, drug concentration, continue the processing time.
The parent shellfish is cultivated, long-pending ovum, fertilization technology: use 6-DMAP to induce the chlamys farreri triploid, obtain higher triploid induction rate, key issue is to guarantee that processed fertilized egg has the higher synchronous developmental character.The synchronism of development of fertilized ova is relevant with the quality of smart ovum, for chlamys farreri, prolongs close shellfish as far as possible and supports the time temporarily, and the effective accumulated temperature that close shellfish is supported temporarily should be more than 200 ℃.Should avoid artificial stimulation to lay eggs ahead of time, be the effective ways that obtain abundant mature egg under the present study condition as far as possible.Generally do not take the method for artificial stimulation to hasten parturition.In addition, should be fertilized as early as possible after whole process of production it should be noted that smart ovum output, be no more than 2 hours after the ovum output, sperm is no more than 1 hour, otherwise smart archiblast amount descends, and will influence fertilization rate.
Be also noted that in the fertilization process, as fertilization process whether synchronously, avoid sperm pollution etc.In actual production, because laying eggs and arranging essence of close shellfish all has the regular hour section, can not carry out simultaneously at one time, thereby the synchronism of the development of fertilized ova of natural insemination is relatively poor, in order to address this problem, the parent shellfish is during supporting temporarily, and it is separately strict that male and female are wanted, the smart laggard pedestrian worker's insemination of the row of laying eggs respectively.Sperm consumption during insemination is more than normal diploid production, should guarantee that the ratio of sperm and ovum is no less than 100: 1, must constantly stir after the insemination, so just can reach higher fertilization rate, makes fertilized egg that developmental character is synchronously arranged preferably simultaneously.
6-DMAP induces the shellfish triploid that temperature is not had dependence, does not influence the ability that reduction division recovers at a lower temperature.Under the normal condition, water temperature mainly has influence on the maiotic process of fertilized egg, and the synchronism of development of fertilized ova is higher under higher temperature, can obtain higher triploid rate.Therefore when inducing the shellfish triploid, should adopt higher water temperature.
Rational use of medicines technology because the 6-DMAP price is higher, in order to ensure certain drug treating concentration, reduces production costs again simultaneously as much as possible, and the method for taking the processing of second order product ovum is to keep the vigor of ovum.Earlier ovum is collected in the bigger container, 0.3 cubic metre of tank commonly used, ovum density reaches and begins insemination about 2000/ml.Ovum after the insemination is concentrated in the little tank of 16.6L, and the density of ovum is waited for drug treating about 3,000 ten thousand/L.
The inductive technology parameter:
1) the medicine initiated process time: the suitable parameter of inducing is the key that guarantees higher triploid rate and survival rate.Under 20 ℃ of conditions of water temperature, after fertilization the 20th minute, 25 minutes, 30 minutes, fertilized egg was handled 15 minutes with the 6-DMAP of 60mg/L respectively, and its triploid rate and survival rate see Table 1, table 2:
Triploid rate and incubation rate that the different zero-times of table 1. are handled
Zero-time (min) triploid rate (%) incubation rate (%)
20 71 68
30 66 60
The different initiated process times of table 2. are to the influence of triploid rate and D type larva survival rate
The initiated process time (min) 20 25 30
Triploid rate (%) 78.5 82.5 67.2
D type larva survival rate (%) 57.3 75.8 59.5
Result of the test shows; the selection on processing opportunity has appreciable impact to triploid rate and D type larva survival rate; the 25th minute begins to handle behind the ovum fertilization; can obtain higher triploid rate and D type larva survival rate; but; under the identical situation of experiment condition; adopt the identical initiated process time; in the inducing of different batches, also can obtain different triploid rate and survival rate; this mainly be the different batches ovum developmental process to some extent difference cause so determining of should not isolating of initiated process, and should be with the biological time of development of fertilized ova; the percentage calculating that is the first polar body appearance is comparatively accurate; through experiment the suitableeest initiated process time, 35-45% appears for well to observe first polar body, in large-scale production; also need to leave sufficient time with reference to general development time process.
2) handle the duration: behind the ovum fertilization 25 minutes (20 ℃ of water temperatures), first polar body accounted for 40% o'clock, used the 6-DMAP of 60mg/L to handle 10-20 minute, the results are shown in Table 3:
The different influences that continue the processing time to D type larva survival rate and triploid rate of table 3.
Processing time min D type larva survival rate triploid rate
10 80.5 66.6
15 75.0 83.0
20 50.2 85.0
Handled 10-20 minute under experimental condition, prolonging 6-DMAP processing time triploid rate increases to some extent, but rate of embryonic death also obviously rises.Continue 15 minutes processing times, can obtain higher inductivity and survival rate, can satisfy requirement of massive production.
3) drug treating concentration: the appropriate drug concentration of treatment is to obtain to improve the key of survival rate under the prerequisite of higher triploid rate.Behind the ovum fertilization 25 minutes (20 ℃ of water temperatures), first polar body accounted for 40% o'clock, used the 6-DMAP of 10-70mg/L to handle 15 minutes, the results are shown in Table 4:
Table 4. different pharmaceutical concentration influences drug concentration (mg/L) 10 20 30 40 50 60 70 triploid rate 25.8 30.2 41.8 53.8 68.2 83.0 84.8D type larva survival rates 95.0 90.4 91.0 85.5 80.2 75.0 43.2 to triploid rate and D type larva survival rate
As can be seen from the table, the 6-DMAP of 10-70mg/L can induce the chlamys farreri triploid, along with the increase triploid rate rising of drug concentration, but the decline of D type larva survival rate, the 6-DMAP concentration of 60mg/L can reach higher triploid rate and survival rate.
The technology of the present invention, the triploid induction rate of large-scale production can be stabilized in more than 80%, and the triploid rate of adult reaches about 70%.Bred the station at China Aquatic Science Research Institute's Long Island and carried out industrial experimentation in 1999 and 2000 2 years, 1999, seedling water 100 sides, coinduction ovum 2,000,000,000,2000, seedling water 700 sides, coprocessing ovum 15,000,000,000, the D-type young triploid rate in 2 years all reaches more than 80%, and the juvenile mollusk triploid rate reaches more than 70%.
Show that through 14 months culture experiments triploid is than the high average increase by 12.6% of dliploid shell, closed shell flesh is obviously greater than dliploid, and fresh meat post yield improves 66.3%, and scallop culture output improves 20-30% under existence conditions.
The present invention and prior art (is example with derivant cytochalasin B (CB)) contrast has following characteristics:
In the present triploid main method of artificial induction shellfish, physical method, no matter be thermal stimulus or pressure treatment, it is generally acknowledged and to reach 70% times change rate, and the chemicals revulsion, be the doubly change rate of maximum of CB and 6-DMAP, in the laboratory on a small scale under the situation, can reach or near 100%.Adopt the technology of the present invention, can under big condition of producing D-type young triploid rate be reached more than 80%, the juvenile mollusk triploid rate reaches more than 70%.
In chlamys farreri, 6-DMAP can obtain with CB same induce effect, and incubation rate greatly improves, and can reach more than 70%, is the first-selected derivant of triploid chlamys farreri large-scale production.
Compare with CB, 6-DMAP toxicity is little, and is little to operator's potential hazard.6-DMAP is water-soluble, and compounding pharmaceutical is easy, and processing does not need to wash ovum when finishing and can hatch directly into the pond, CB then needs to need rinse to remove residual medicine with dmso solution and after processing finishes, therefore, the use of 6-DMAP is easier than CB, is more suitable for being applied to large-scale production.
The triploid animal has sterility, in process of growth, have only less energy to be used for gonad development, more energy is used for growth, therefore the scallop triploid has fast, individual big, the characteristics such as product are of fine quality, high-output stress-resistance of growth, can obviously increase output, improve quality and reduce lethality, this is confirmed in the triploid research of multiple cultured scallop.The development and application of chlamys farreri triploid technology will make scallop culture output improve 20-30% under existence conditions, and fresh adductor-somatic index improves about 60%, and the problem that the present quality that exists of containment descends effectively, adductor-somatic index is low.
Claims (1)
1. the production method of a triploid of young Jiekong scallop is characterized in that in the seed rearing process, the cultivation of its close shellfish, and long-pending ovum, fertilization and practical technique are: the effective accumulated temperature that close shellfish is supported temporarily reaches more than 200 ℃, and male and female parent shellfish should strictly separately be supported temporarily; Adopt second order product ovum processing method, the ovum of output is collected in 0.3m earlier
3In the tank, when density reaches 2000/ml, add the seminal fluid fertilization, smart ovum ratio 100: 1, the time of fertilization requires to be no more than 2 hours after the ovum output, and sperm is no more than 1 hour; After fertilization is concentrated into ovum in the little tank of 16.6L, and ovum density is at 3,000 ten thousand/L, after carry out drug treating, processing finishes the back and directly moves in the pond and hatches; The triploid induction technical parameter: the medicine initiated process time is when observing first polar body 35-45% occurring under light microscope; Adding concentration is that the 6-DMAP of 10-70mg/L carries out drug treating; It is 15-20 minute that medicine continues the processing time.
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| CNB001113054A CN1136765C (en) | 2000-08-25 | 2000-08-25 | Method for producing triploid of young Jiekong scallop |
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| CNB001113054A CN1136765C (en) | 2000-08-25 | 2000-08-25 | Method for producing triploid of young Jiekong scallop |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1317945C (en) * | 2004-09-13 | 2007-05-30 | 大连水产学院 | Method for inducing stichopus japonicus triploid by static water pressure |
| CN101785437B (en) * | 2009-11-24 | 2011-12-07 | 广东海洋大学 | Chlamys nobilis offspring seed cultivation method |
| CN104855321A (en) * | 2015-06-10 | 2015-08-26 | 天津农学院 | Triploid chemical induction method suitable for Ruditapes philippinarum |
| CN105248328A (en) * | 2015-10-29 | 2016-01-20 | 苏州市美人半岛齐力生态农产品专业合作社 | Method for artificially breeding chlamys nobilis |
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