CN101326906A - Low temperature storage method of Porphyra haitanensis filament germplasm by encapsulation - Google Patents
Low temperature storage method of Porphyra haitanensis filament germplasm by encapsulation Download PDFInfo
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- CN101326906A CN101326906A CNA2008100715259A CN200810071525A CN101326906A CN 101326906 A CN101326906 A CN 101326906A CN A2008100715259 A CNA2008100715259 A CN A2008100715259A CN 200810071525 A CN200810071525 A CN 200810071525A CN 101326906 A CN101326906 A CN 101326906A
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- glueballs
- germplasm
- filament
- porphyra haitanensis
- low temperature
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Abstract
The invention relates to a method for preservation of Porphyra haihanensis protonema germ plasm by encapsulation under low-temperature, relating to Porphyra haihanensis, particularly relating to a method for preservation of Porphyra haihanensis protonema germ plasm by encapsulation under low-temperature. The Porphyra haihanensis protonema is cultured; the protonema culture solution is filtered and removed, and the moisture is sucked up; the protonema is placed in the sodium alginate solution for suspension, and suspending alga solution is achieved after even mixture; glue balls are prepared; the glue balls are dehydrated; and the glue balls are frozen for preservation. After encapsulated embedding, the Porphyra haihanensis protonema can be placed in a general refrigerator for preserving Porphyra haihanensis protonema germ plasm for long-term, and the survival rate of the long preserved protonema does not have obvious change; only a general refrigerator is required, rather than sophisticated ultra-low temperature equipment; the operation is simple, no complicated operating procedure is needed; contamination to the protonema germ plasm can be reduced to the largest extent.
Description
Technical field
The present invention relates to a kind of Porphyra haitanensis, especially relate to a kind of encapsulated low temperature store method of Porphyra haitanensis filament germplasm.
Background technology
Porphyra haitanensis (Porphyra haitanenis) is the distinctive warm temperate zone property kind of China, is the important economic red algae that Fujian, Zhejiang and Guangdong Coastal are propagated artificially, and its output accounts for 80% of national laver gross yield.In recent years, continuous expansion along with the Porphyra haitanensis area under cultivation, it is too intensive to culture the sea area, adopt the conchospore seedling and unrestrictedly do sth. in advance, add that most breed sea area is generally all adopted once to introduce wild planting vegetables, reserve the production model of autotrophy for a long time for one's own use, not only cause porphyra haitanensis germ plasma degeneration, seedling to come off in a large number, and make blade lose original gloss and elasticity gradually, and growth cycle shortens, and output of former algae and quality descend.Simultaneously because selfing, the inbreeding probability of laver are higher; culturing the genetic diversity of germplasm loses gradually; the germ plasm resource homogeneity; gene pureization inferior; therefore in protection Porphyra haitanensis nature population germ plasm resource; to existing Porphyra haitanensis elite germplasm by the in addition selection sort and preserved protection of genetic character rule; avoid the decline of breeding proterties; and in culturing room, the breeding of having preserved is increased and use the filamentous cell engineering and carry out factorial seedling growth, caused algologist's great attention.
Porphyra haitanensis has thallus in life and two of filamentouss are from generation to generation special-shaped, and the thallus preservation is carried out the method for deepfreeze after can adopting and drying in the shade, but is difficult to permanent preservation.Because the nuclear phase of filamentous is an amphiploid, in preserving cultivation mitosis only taking place grows, can diffuse conchospore again after its maturation simultaneously and grow up to thallus again, therefore filamentous can be preserved as germplasm.The preservation of filamentous has fluid preservation and ultralow temperature liquid nitrogen to preserve two kinds of methods at present.The fluid preservation method relatively is suitable for the amplification cultivation of secondary preservation and algal filament, but needs often to change culture fluid, big and easy pollution of workload.Keep blastogenesis stability easily though the ultralow temperature liquid nitrogen is preserved, be convenient to long preservation, time saving and energy saving, need to use freezeproof protectant, and survival rate is low, therefore use and be subjected to certain influence.
The nineties in 20th century, encapsulated evaporation begins to be used for the stored frozen of vegetable material, and this method is not only simple to operate, does not need complex device, and without freezeproof protectant, is a significant improvement to the freezing preservation technology of ultralow temperature.But in the low temperature of marine alga germplasm is preserved, do not see relevant report as yet.
Summary of the invention
The encapsulated low temperature store method that the purpose of this invention is to provide a kind of Porphyra haitanensis filament germplasm.
Technical scheme of the present invention is by the Porphyra haitanensis filament germplasm is carried out encapsulated embedding, forms glueballs, and then the method that directly places-20 ℃ refrigerator to preserve of the glueballs after will dewatering.
The present invention includes following steps:
1) the Porphyra haitanensis filamentous is cultivated;
2) filter removal filamentous culture fluid, and suck dry moisture;
3) filamentous is dropped in the sodium alginate solution, suspend, and the mixing algae liquid that must suspend;
4) preparation glueballs;
5) glueballs is carried out dehydration processing;
6) the freezing preservation of glueballs.
During use, glueballs is recovered.
Porphyra haitanensis filamentous culture condition is: culture fluid is preferably the sterilization seawater, and cultivation temperature is preferably 20 ± 1 ℃, and intensity of illumination is preferably 1000～1500lx, and the photoperiod is preferably 12h (light): 12h (secretly).
Described filtration can be adopted the bolting silk net filtration, and described blotting can adopt blotting paper to blot.
By mass percentage, the concentration of described sodium alginate solution is preferably 3%, adopts the preparation of filamentous culture fluid.
The described method for preparing glueballs is with the syringe of band syringe needle suspension algae drop to be gone into to contain the CaCl of 0.1mol/L
2Concentration be in the NaCl solution of (by mass percentage) 3%, shake, can make algae liquid sinking hardening, form glueballs.
The described method that glueballs is carried out dehydration processing is to remove by filter CaCl with finishing immobilized glueballs
2Solution is put into culture dish after blotting glueballs surface raffinate, culture dish is put into the big culture dish that the bottom is placed with the silica gel of having dried again, covers the culture dish lid, dewaters after with the periphery sealing with adhesive tape.The diameter of big culture dish is chosen as 15cm, covers the culture dish lid, and the insulating box that can put into 25 ℃ that dewaters after with adhesive tape periphery being sealed dewaters.
Glueballs can take out from culture dish after dehydration, puts into the freeze-drying pipe, stored frozen.Glueballs can take out from culture dish behind the best 10h that dewaters, and puts into the freeze-drying pipe, directly drops into stored frozen in-20 ℃ of refrigerators.
The glueballs of freezing preservation is taken out, put into the filamentous culture fluid, glueballs is dissolved, centrifugal results material, and suspend again with fresh medium, recover to cultivate.The filamentous culture fluid can be the filamentous culture fluid that 20mL contains the 0.05mol/L sodium citrate.
The Porphyra haitanensis filamentous of encapsulated low temperature long preservation, survival rate can reach more than 70%, and with the holding time prolongation, its survival rate does not have significant change.
Compare with existing method, the present invention has following outstanding advantage: after (1) carries out encapsulated embedding to the Porphyra haitanensis filamentous, can place general refrigerator that the Porphyra haitanensis filament germplasm is carried out long preservation, marked change can not take place in the filamentous survival rate of long preservation; (2) need not complicated ultralow temperature equipment, only need general refrigerator to get final product; (3) simple to operate, need not the complicated operations program; (4) can farthest reduce the pollution of filament germplasm.
Embodiment
(1) cultivate the Porphyra haitanensis filamentous, condition of culture is: 20 ± 1 ℃ of cultivation temperature, and intensity of illumination 1000～1500lx, the photoperiod is 12h (light): 12h (secretly).
(2) remove the filamentous culture fluid with the bolting silk net filtration, and blot with blotting paper.
(3) filamentous that blots is dropped in 3% sodium alginate solution, suspend again, behind the mixing, with the CaCl that with the syringe of No. 8 syringe needles the algae drop is gone into to contain 0.1mol/L
23%NaCl solution in, shake gently, make the hardening of slowly sinking of algae liquid, form glueballs.
(4) will finish immobilized glueballs and remove CaCl with the bolting silk net filtration
2Solution, blotting paper blots the little culture dish of putting into diameter 6cm behind the surperficial raffinate, again 3 little culture dishes is put into diameter 15cm, and the bottom is placed with in the big culture dish of the silica gel that one deck dried, cover the culture dish lid, the insulating box of putting into 25 ℃ after with adhesive tape periphery being sealed dewaters.
(5) glueballs can take out from culture dish behind dehydration processing 10h, puts into the freeze-drying pipe, directly drops into stored frozen in-20 ℃ of refrigerators.
(6) when will recovering when cultivating to filamentous, the glueballs of freezing preservation is taken out, put into the sodium citrate filamentous culture fluid that 20mL contains 0.05mol/L, glueballs is dissolved, centrifugal results material, and suspend again with fresh medium, recover to cultivate.
Claims (10)
1. the encapsulated low temperature store method of Porphyra haitanensis filament germplasm is characterized in that may further comprise the steps:
1) the Porphyra haitanensis filamentous is cultivated;
2) filter removal filamentous culture fluid, and suck dry moisture;
3) filamentous is dropped in the sodium alginate solution, suspend, and the mixing algae liquid that must suspend;
4) preparation glueballs;
5) glueballs is carried out dehydration processing;
6) the freezing preservation of glueballs.
2. the encapsulated low temperature store method of Porphyra haitanensis filament germplasm as claimed in claim 1, it is characterized in that Porphyra haitanensis filamentous culture condition is: culture fluid is the sterilization seawater, cultivation temperature is 20 ± 1 ℃, and intensity of illumination is 1000～1500lx, and the photoperiod is 12h (light): 12h (secretly).
3. the encapsulated low temperature store method of Porphyra haitanensis filament germplasm as claimed in claim 1 is characterized in that the described bolting silk net filtration that is filtered into.
4. the encapsulated low temperature store method of Porphyra haitanensis filament germplasm as claimed in claim 1 is characterized in that described blotting to blotting paper blot.
5. the encapsulated low temperature store method of Porphyra haitanensis filament germplasm as claimed in claim 1 is characterized in that by mass percentage the concentration of described sodium alginate solution is 3%, adopts the preparation of filamentous culture fluid.
6. the encapsulated low temperature store method of Porphyra haitanensis filament germplasm as claimed in claim 1 is characterized in that the described method for preparing glueballs is with the syringe of band syringe needle suspension algae drop to be gone into to contain by mass percentage the CaCl of 0.1mol/L
2Concentration be in 3% the NaCl solution, to shake, can make algae liquid sinking hardening, form glueballs.
7. the encapsulated low temperature store method of Porphyra haitanensis filament germplasm as claimed in claim 1 is characterized in that the described method that glueballs is carried out dehydration processing is to remove by filter CaCl with finishing immobilized glueballs
2Solution is put into culture dish after blotting glueballs surface raffinate, culture dish is put into the big culture dish that the bottom is placed with the silica gel of having dried again, covers the culture dish lid, dewaters after with the periphery sealing with adhesive tape.
8. the encapsulated low temperature store method of Porphyra haitanensis filament germplasm as claimed in claim 7 is characterized in that the diameter of big culture dish is elected 15cm as, covers the culture dish lid, and the insulating box of putting into 25 ℃ that dewaters after with adhesive tape periphery being sealed dewaters.
9. the encapsulated low temperature store method of Porphyra haitanensis filament germplasm as claimed in claim 1 is characterized in that glueballs after dehydration, promptly takes out from culture dish, put into the freeze-drying pipe, stored frozen.
10. the encapsulated low temperature store method of Porphyra haitanensis filament germplasm as claimed in claim 1 is characterized in that the glueballs of freezing preservation is taken out, and puts into the filamentous culture fluid, glueballs is dissolved, centrifugal results material, and suspend again with fresh medium, recover to cultivate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100715259A CN101326906A (en) | 2008-08-01 | 2008-08-01 | Low temperature storage method of Porphyra haitanensis filament germplasm by encapsulation |
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2008100715259A CN101326906A (en) | 2008-08-01 | 2008-08-01 | Low temperature storage method of Porphyra haitanensis filament germplasm by encapsulation |
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| CN101326906A true CN101326906A (en) | 2008-12-24 |
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| CNA2008100715259A Pending CN101326906A (en) | 2008-08-01 | 2008-08-01 | Low temperature storage method of Porphyra haitanensis filament germplasm by encapsulation |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102696580A (en) * | 2012-06-21 | 2012-10-03 | 中国农业科学院作物科学研究所 | Ultralow-temperature storage and regenerative-culture method for embedding and drying of isolated stem tips of test-tube horseradish plantlets |
| CN103114071A (en) * | 2013-01-30 | 2013-05-22 | 汕头大学 | Method for rapidly and concentratedly preparing porphyra haitanensis protoplast |
| CN103621395A (en) * | 2013-12-16 | 2014-03-12 | 中国海洋大学 | Embedding-dehydrating ultralow temperature storage method for scytosiphon lomentaria mitoplast |
| CN110100718A (en) * | 2019-05-10 | 2019-08-09 | 山东省海洋生物研究院 | A kind of porphyra haitanensis cold net cultural method suitable for northern sea area |
-
2008
- 2008-08-01 CN CNA2008100715259A patent/CN101326906A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102696580A (en) * | 2012-06-21 | 2012-10-03 | 中国农业科学院作物科学研究所 | Ultralow-temperature storage and regenerative-culture method for embedding and drying of isolated stem tips of test-tube horseradish plantlets |
| CN102696580B (en) * | 2012-06-21 | 2014-04-02 | 中国农业科学院作物科学研究所 | Ultralow-temperature storage and regenerative-culture method for embedding and drying of isolated stem tips of test-tube horseradish plantlets |
| CN103114071A (en) * | 2013-01-30 | 2013-05-22 | 汕头大学 | Method for rapidly and concentratedly preparing porphyra haitanensis protoplast |
| CN103114071B (en) * | 2013-01-30 | 2014-07-30 | 汕头大学 | Method for rapidly and concentratedly preparing porphyra haitanensis protoplast |
| CN103621395A (en) * | 2013-12-16 | 2014-03-12 | 中国海洋大学 | Embedding-dehydrating ultralow temperature storage method for scytosiphon lomentaria mitoplast |
| CN103621395B (en) * | 2013-12-16 | 2015-04-08 | 中国海洋大学 | Embedding-dehydrating ultralow temperature storage method for scytosiphon lomentaria mitoplast |
| CN110100718A (en) * | 2019-05-10 | 2019-08-09 | 山东省海洋生物研究院 | A kind of porphyra haitanensis cold net cultural method suitable for northern sea area |
| CN110100718B (en) * | 2019-05-10 | 2021-06-25 | 山东省海洋生物研究院 | Porphyra haitanensis cold storage net culture method suitable for northern sea area |
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Open date: 20081224 |