CN100334202C - Method for preserving pure line of protoplast of purple laver in ultra low temperature - Google Patents
Method for preserving pure line of protoplast of purple laver in ultra low temperature Download PDFInfo
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- CN100334202C CN100334202C CNB2004100239451A CN200410023945A CN100334202C CN 100334202 C CN100334202 C CN 100334202C CN B2004100239451 A CNB2004100239451 A CN B2004100239451A CN 200410023945 A CN200410023945 A CN 200410023945A CN 100334202 C CN100334202 C CN 100334202C
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- laver
- protoplastis
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- dissociation solution
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Abstract
The present invention relates to a method for storing laver protoplasts at ultralow temperature. The present invention is characterized in that the laver is sheared, and dissociation solution is used for carrying out enzymolysis for single piece; the weight ratio of the laver to the dissociation solution is 1: 1.2 to 1.5; the protoplasts are washed and are centrifugally precipitated after the enzymolysis; the protoplasts of different biotypes are filtered and collected by a bolting silk, are loaded in a freezing and storing pipe, are transited and dehydrated by a glass freezing and storing agent, and are immediately loaded in liquid nitrogen to be frozen and stored. The present invention has the advantages that germplasm of the laver biotype in the single cell level is stored, and thus, the present invention ensures the germplasm heredity to be stable, effectively prevents the genetic gene from variating or losing, and is favorable to utilize the laver protoplasma to carry out basic research on various experimental biologics of genetic breeding operation, physiology and biochemistry, etc.
Description
Technical field
The present invention relates to a kind of cryopreservation method of laver protoplastis pure lines of aquaculture field.
Background technology
Laver is important economical alga.China's main cultispecies class has the yezoensis laver (P.yezoensis) in the southern porphyra haitanensis (Porphyrahaitanensis) and the north.Comprise miniature thread sporophyte (diploid) and large-scale lobate gametophyte (monoploid) life history of laver.Different types of laver has different reproduction characteristics.The gametophyte of porphyra haitanensis is telianthus and dioecism, does not diffuse monospore, is generally sexual propagation.The gametophyte of yezoensis laver is a telianthus, except that sexual propagation, also can diffuse the capable vegetative propagation of monospore.Utilize sexual propagation to cultivate the gametophyte pure lines and need dioecious mating, selfing or hermaphroditic selfing again.Select just to obtain pure lines through continuous selfing, the time of cultivation is long, trivial operations.Yezoensis laver can diffuse monospore and carry out vegetative propagation cultivation pure lines, but these pure lines all are the many cells individualities.Existing report is preserved seedling with-20 ℃ temperature short period of time, is used for culturing in the sea.The also thread sporophyte of useful yezoensis laver and preserved by the lobate gametophytic very low temperature of pure lines that monospore produces, these are preserved and all belong to the many cells individuality.But the super cryopreservation method of the laver protoplastis of unicellular level pure lines yet there are no report.
Summary of the invention
The purpose of this invention is to provide the cryopreservation method of main laver protoplastis pure lines, it can remedy the above-mentioned deficiency of prior art.
The cryopreservation method of one main laver protoplastis, it is characterized in that laver is sheared, carry out single enzymolysis with dissociation solution, the weight ratio of laver and dissociation solution is 1: 1.2-1.5, with the washing of the protoplastis behind enzymolysis centrifugation, collect the protoplastis of different pure lines with silk cover filtering, put into frozen pipe, after glass frozen preservation agent transition and processed, it is frozen to put into liquid nitrogen.
The protoplastis that enzymolysis obtains among the present invention because it does not have cell walls, can be got rid of the tension force that cell produces between pool period.Replace thallus to carry out freezing with this protoplastis, intracellular matter and liquid can be solidified in shorter time, can more be formed uniformly vitrification substance, its advantage has been to realize the germplasm preservation of the pure laver line of unicellular level, thereby guaranteed the stable of blastogenesis, prevent genetic variation effectively or lose, help using the laver protoplasma to carry out the fundamental research of various experimental biologies such as genetic breeding operation and Physiology and biochemistry.
Specific embodiments
(1) will propagate artificially or the porphyra haitanensis or the yezoensis laver of self-sow dry in the shade, its water content is sealed in the plastics bag 30～40%, and quick-frozen is kept in-20～-30 ℃ the refrigerator-freezer.
(2) porphyra haitanensis of freezing preservation or yezoensis laver are put in the aseptic seawater recovered, cultivated 1～2 day.
(3) laver or the existing new fresh laver 2g that gathers with recovery is cut into 2mm
2Size adds dissociation solution 2.5-3.0g, at 20～25 ℃ of enzymolysis 3 hours of placing an order, with the washing of the protoplastis behind enzymolysis centrifugation, collects the protoplastiss of different pure lines with the silk cover filtering of 40 μ m, and with sterilizing seawer washing three times.Described dissociation solution is by 1-3% (concentration expressed in percentage by weight, marine alga toolenzyme down together) (is produced by Chinese Marine University, for many years commercially available), the cellulase (0nozuka R-10) of 0.8-1.2% and 25～30% glucose and deionized water form, wherein glucose plays permeate agent.
(4) get the 0.3g protoplastis, adding 1ML, to be diluted to concentration expressed in percentage by weight with seawater be that (this VS6 is by the dimethyl sulfoxide (DMSO) (DMSO) of 9-11%, 25～30% glycerine, the sucrose of 9-11% for 25% glass frozen preservation agent VS6, formulated with seawater) 0 ℃ of transition processing be after 5 minutes, centrifugal collection protoplastis, add 1ML in the VS6 of 0 ℃ of precooling, be transferred in the frozen pipe of 2ML, dewatered 3 minutes, it is frozen to put into liquid nitrogen container immediately.
(5) during recovery, place 38-42 ℃ of water-bath to thaw frozen pipe, in case disappearing, takes out immediately ice cube, the sterilization seawater that in 30 minutes, adds 7 times of volume precoolings, centrifugal collection protoplastis is cultivated with the sterilization seawer washing again, with diacetate esters fluorescein (fluorescein diacetate, FDA) active coloring method detects the frozen survival rate of protoplastis afterwards, reaches as high as 66.5%.
The present invention utilizes enzyme process to separate the laver protoplastis, makes can to cultivate the protoplastis pure lines by vegetative porphyra haitanensis; Also can overcome simultaneously yezoensis laver transfers the throwaway spore in state of nature and can only cultivate the thallophytic shortcoming of pure lines; And make the germplasm on the individual level preserve the germplasm preservation that becomes on the cell levels.Set up the cell strain storehouse of laver thus, make the germ plasm resource of cell strain systems such as the laver breeding clone in different places, various resistance clone, the following rareness kind clone of growing of extreme environments and other laver species, the stability that can both keep heredity for a long time is so that provide valuable germ plasm resource for fundamental research and production application.
Claims (1)
1, the cryopreservation method of a main laver protoplastis, it is characterized in that laver is sheared, carry out single enzymolysis with dissociation solution, the weight ratio of laver and dissociation solution is 1: 1.2-1.5, with the washing of the protoplastis behind enzymolysis centrifugation, collect the protoplastis of different pure lines with silk cover filtering, put into frozen pipe, after glass frozen preservation agent transition and processed, it is frozen to put into liquid nitrogen;
Described dissociation solution is made up of the marine alga toolenzyme of 1-3%, the cellulase of 0.8-1.2%, 25～30% glucose and deionized water, described glass frozen preservation agent is by the dimethyl sulfoxide (DMSO) of 9-11%, 25～30% glycerine, 9-11% sucrose, and is formulated with seawater.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100239451A CN100334202C (en) | 2004-04-20 | 2004-04-20 | Method for preserving pure line of protoplast of purple laver in ultra low temperature |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100239451A CN100334202C (en) | 2004-04-20 | 2004-04-20 | Method for preserving pure line of protoplast of purple laver in ultra low temperature |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1563358A CN1563358A (en) | 2005-01-12 |
| CN100334202C true CN100334202C (en) | 2007-08-29 |
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| CNB2004100239451A Expired - Fee Related CN100334202C (en) | 2004-04-20 | 2004-04-20 | Method for preserving pure line of protoplast of purple laver in ultra low temperature |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101418350B (en) * | 2008-10-28 | 2011-06-15 | 南京农业大学 | Method for removing strawberry light yellow edge virus by ultra low temperature technique |
| CN103013905B (en) * | 2013-01-19 | 2014-03-26 | 安徽科技学院 | Technique for preparing Sudan grass protoplast by using vacuum enzymolysis method |
| CN106508519A (en) * | 2016-11-24 | 2017-03-22 | 江苏省海洋水产研究所 | Green alga control method for porphyra yezoensis raft frame facility |
| CN114731943A (en) * | 2022-04-20 | 2022-07-12 | 常熟理工学院 | Method for preparing porphyra yezoensis fruit spores |
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2004
- 2004-04-20 CN CNB2004100239451A patent/CN100334202C/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| 条斑紫菜冷藏网试验及其产品质量分析 马家海 等，水产学报，第22卷 1998 * |
| 海藻工具酶 韩宝芹 等，海洋学报，第20卷第2期 1998 * |
| 海藻工具酶 韩宝芹 等，海洋学报，第20卷第2期 1998;条斑紫菜冷藏网试验及其产品质量分析 马家海 等，水产学报，第22卷 1998 * |
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| CN1563358A (en) | 2005-01-12 |
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