CN100521922C - Super-low temp. quick-freezing preservation method for kelp swarm spore - Google Patents
Super-low temp. quick-freezing preservation method for kelp swarm spore Download PDFInfo
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- CN100521922C CN100521922C CNB2005100442031A CN200510044203A CN100521922C CN 100521922 C CN100521922 C CN 100521922C CN B2005100442031 A CNB2005100442031 A CN B2005100442031A CN 200510044203 A CN200510044203 A CN 200510044203A CN 100521922 C CN100521922 C CN 100521922C
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Abstract
The present invention discloses an ultra-low temperature quick freeze storage method of sea-tangle swarm (embryo) spore. Said method includes the following steps: material treatment, equilibration, quick freeze-storing and anabiosis. It can store lots of germplasm material, and its survival rate is high.
Description
One, technical field
The invention belongs to the marine biotechnology field, relate to a kind of large-scale economic algae-kelp germplasm preservation technology.
Two, background technology
Sea-tangle (Laminaria japonica Aresch) is the important economic algae of China; cultured output and scale occupy first place in the world for years; abundant germ plasm resource is the important substance basis of fish production; protecting and make good use of the kelp germplasm resource will be to improve the sea-tangle varietal level; realize that kelp variety optimization production provides important assurance; sea-tangle is divided two tangible stages from generation to generation history of life: sporophyte generation and gametophytic generation; gametophytic generation is the monoploid stage of sea-tangle; the trip spore is the incipient stage of gametophytic generation; after the main laminaria maturation; diffuse the trip spore; trip spore moving about through about 2 hours; or adhere to or the formation sporoblast that suspends, sporoblast is sprouted into gametophyte gradually.Traditional guarantor's kind technology be exactly this stage with egagametophyte (female gametophyte) and the single separation and Culture of male gametophyte (male gametophyte), form clone (clone), preserve germplasm by the clonal method of successive transfer culture.But this traditional preservation technology has tangible deficiency: (1) liquid culture is unique preservation approach, and means are single, has limited the scale and the efficient of preserving; (2) liquid culture is preserved and generally need be upgraded one time culture fluid in first quarter moon, and workload is big, and complex operation, frequent operation have also increased the chance that germplasm pollutes and mixes; (3) can't carry out aseptic process to main laminaria before adopting spore, whole preservation process all under nonsterile conditions, can not thoroughly be stopped the pollution of the especially assorted algae of microorganism; (4) cultivate in phenomenon such as unusual, the unisexual reproduction of cellular morphology happen occasionally, cultivate the factor and remain further to be optimized.Therefore, present guarantor's technology of planting can't accomplish that germplasm forever preserves, and can't get rid of the possibility that mixes, pollutes, makes a variation fully.
The research that algae germplasm ultralow temperature is preserved starts from the sixties in 20th century, and development speed is very fast after the eighties, to the seawater algae, from the microalgae to the macro, has all given more concern from the non-economy algae to economic algae from fresh water algae.The method that the algae superfreeze is preserved mainly is two step freezings (two-step freezing), this method at first adds freezeproof protectant in preserving material, carry out pre-freeze, the material of again pre-freeze being crossed drops into snap frozen in the liquid nitrogen, has become the conventional method of the freezing preservation of algae germplasm at present; The main cause that superfreeze preservation The Application of Technology is restricted is the foundation of refrigeration operation program mainly by experimental experimental result, and the preservation technology that is fit to a kind of algae may be inapplicable fully to another kind of algae.The frozen kind of using in large-scale economic algae of ultralow temperature two-step method has laver and undaria pinnitafida, and the method that relevant sea-tangle trip (embryo) spore ultralow temperature is preserved still is a blank at present.
Three, summary of the invention
The objective of the invention is to improve existing successive transfer culture and preserve the deficiency of clone technology, and provide a kind of be used for kelp spore preserve, simple and easy to do, conserving species material, the higher sea-tangle of survival rate are swum the ultralow temperature quick-freezing preservation method of (embryo) spore in a large number.
The object of the present invention is achieved like this, and the ultralow temperature quick-freezing preservation method of sea-tangle trip (embryo) spore is characterized in that this method may further comprise the steps: material processed, balance, frozen and recovery fast.
Material processed is to select the sporangium color and luster to be the sea-tangle that film blackish green, surperficial adularescent begins to come off, clip has sporangial blade, with take off the cotton and sterilization seawater of paper put on the skin repeatedly wash ten surplus time, place beaker, pour 5-10 ℃ of cold sterilization seawater into, diffuse spore, spore concentration reaches 50/160 * when above, leaches impurity and mucus obtains spore suspension with 300 mesh sieve thin,tough silk.
Balance is that spore suspension is mixed balance 15min in 0-4 ℃ of refrigerator with the 20%DMSO of sterilization seawater preparation in the 1:1 ratio.
Fast cooling is frozen to be that the spore mixed liquor after the balance is packed in the frozen pipe of 1.5ml, and frozen pipe directly drops into liquid nitrogen.
After recovery is superfreeze, frozen pipe is taken out from liquid nitrogen, move on to concussion fast in 39 ℃ of waters bath with thermostatic control rapidly, disappear to pour in the culture fluid to last ice crystal and cultivate.
Compared with the prior art the present invention has following distinguishing feature and good effect: the present invention adopts the ultralow temperature snap frozen to preserve sea-tangle trip (embryo) spore, change vessel and can only preserve the conventional method that a strain is, efficient has improved in a plurality of strains system in can preserving product (kind) be in vessel; The metabolism under ultralow temperature (196 ℃) state of sea-tangle trip (embryo) spore stops fully, and life can recover its original biologically active and hereditary capacity again with static form long preservation in this state after thawing.The ultralow temperature quick-freezing preservation method of sea-tangle trip (embryo) spore is simple, has solved the problem of polluting, mixing, make a variation during kelp germplasm is preserved effectively and keeps inheritance stability, is expected to realize the permanent preservation of kelp germplasm.The maximum characteristics one of the inventive method are the inheritance stabilities that can keep kind, the 2nd, and simple and easy to do, the 3rd, conserving species material in a large number: compare inheritance stability with traditional store method; Compare simple and easy to doly with trip (embryo) spore progressive cryopreservation method, refrigeration operation has only a step, promptly drops into liquid nitrogen, does not have intermediate link; With the frozen conserving species material in a large number of comparing of clone ultralow temperature, once-through operation can be preserved a plurality of strains system of product (kind) in being, clone ultralow temperature is preserved once-through operation can only preserve a strain system.The acquisition of material of the present invention is subject to seasonal restrictions, and only could obtain in the sea-tangle mature period.Therefore each is valuable, simultaneous necessity is arranged for sea-tangle trip (embryo) spore ultralow temperature quick-freezing preservation method, sea-tangle trip (embryo) spore ultralow temperature progressive cryopreservation method, sea-tangle filamentous programmed cooling freeze preservation and four kinds of store methods of sea-tangle filamentous progressive cryopreservation method.
Four, specific embodiments
Below in conjunction with embodiment the present invention is described in further detail.
Embodiment, the ultralow temperature quick-freezing preservation method of sea-tangle trip (embryo) spore, by material processed, balance, fast cooling is freezing and the recovery realize, material processed is to select the sporangium color and luster to be the sea-tangle that film blackish green, surperficial adularescent begins to come off, clip 5x5cm has sporangial blade, with take off the cotton and sterilization seawater of paper put on the skin repeatedly wash ten surplus time, place the 500ml beaker, pour 8 ℃ of cold sterilization seawater into, diffuse spore.Spore concentration reaches 80-100/160 * time, leaches impurity and mucus acquisition spore suspension with 300 mesh sieve thin,tough silk; Balance is that spore suspension is mixed balance 15min in 0-4 ℃ of refrigerator with the 20%DMSO of sterilization seawater preparation in the 1:1 ratio; Frozen is that the spore after the balance and protectant mixed liquor are packed in the frozen pipe of 1.5ml (cryotubes), and frozen pipe directly drops into liquid nitrogen; After recovery is superfreeze, frozen pipe is taken out from liquid nitrogen, move on to concussion fast in 39 ℃ of waters bath with thermostatic control rapidly, disappear to pour in the culture fluid to last ice crystal and cultivate, the frozen time of ultralow temperature can be chosen as the case may be, in the needs recovery, finish frozen getting final product, do not have certain restriction.
Sea-tangle trip (embryo) spore that adopts the inventive method to preserve, and fluorescein(e) diacetate (fiuorescein diacetate, FDA) the vital staining method is measured survival rate, and survival rate reaches about 30%.
Claims (1)
- The ultralow temperature quick-freezing preservation method of 1, sea-tangle trip, sporoblast, this method may further comprise the steps: material processed, balance, frozen and recovery fast is characterized in that:Material processed is to select the sporangium color and luster to be the sea-tangle that film blackish green, surperficial adularescent begins to come off, clip has sporangial blade, with absorbent cotton and the sterilization seawater put on the skin repeatedly wash ten surplus time, place beaker, pour 5-10 ℃ of cold sterilization seawater into, diffuse spore, spore concentration reaches 80-100/160 * time, leaches impurity and mucus acquisition spore suspension with 300 mesh sieve thin,tough silk;Balance is that spore suspension is mixed balance 15min in 0-4 ℃ of refrigerator with the 20%DMSO of sterilization seawater preparation in the 1:1 ratio;Fast frozen is that the spore mixed liquor after the balance is packed in the frozen pipe of 1.5ml, and frozen pipe directly drops into liquid nitrogen;After recovery is superfreeze, frozen pipe is taken out from liquid nitrogen, move on to concussion fast in 39 ℃ of waters bath with thermostatic control rapidly, disappear to pour in the culture fluid to last ice crystal and cultivate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100442031A CN100521922C (en) | 2005-08-04 | 2005-08-04 | Super-low temp. quick-freezing preservation method for kelp swarm spore |
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2005100442031A CN100521922C (en) | 2005-08-04 | 2005-08-04 | Super-low temp. quick-freezing preservation method for kelp swarm spore |
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| Publication Number | Publication Date |
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| CN1742553A CN1742553A (en) | 2006-03-08 |
| CN100521922C true CN100521922C (en) | 2009-08-05 |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101019503B (en) * | 2007-02-02 | 2010-04-14 | 宁波大学 | Germ plasm preserving method for kelp |
| CN108271680B (en) * | 2018-01-12 | 2020-10-09 | 中国水产科学研究院南海水产研究所 | Cryopreservation seedling culture method for gloiopeltis algae holdfast |
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