CN103881960A - Method and special reagent for separation and purification of Chishui dendrobium nobile protoplasts - Google Patents
Method and special reagent for separation and purification of Chishui dendrobium nobile protoplasts Download PDFInfo
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- CN103881960A CN103881960A CN201410153412.9A CN201410153412A CN103881960A CN 103881960 A CN103881960 A CN 103881960A CN 201410153412 A CN201410153412 A CN 201410153412A CN 103881960 A CN103881960 A CN 103881960A
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Abstract
The invention discloses a method and a special reagent for separation and purification of Chishui dendrobium nobile protoplasts. The special reagent comprises a reagent A and a reagent, wherein the reagent A is composed of MES, KCl, Mannitol, CellulaseR10, PectolyaseY-23, BSA, CaCl2 and ddH2O, and the reagent B (10 ml) is composed of MES, KCl, Mannitol, BSA, CaCl2 and ddH2O in a ratio. A method for preparing the protoplasts by use of the reagent comprises the following steps: 1) treating dendrobium nobile leaf chips on a shaking table in the dark by use of a solution A for about 4 hours to release the protoplasts in the leaves; 2) filtering the protoplast solution obtained in the step 1) by use of a 200-mesh cell sieve, collecting the filtrate, centrifuging for 3 minutes, dissolving the precipitate by use of a solution B, and repeatedly centrifuging and purifying the protoplasts; 3) carrying out activity detection on the purified protoplasts by use of FDA (fluorescein diacetate).
Description
Technical field
The present invention relates to plant cell engineering field, be specifically related to obtain with the tissue cultured seedling blade of Chishui dendrobium stem the isolation and purification method of protoplastis.
Background technology
Dendrobium stem (
dendrobiumnobilelindl.) there is remarkable efficacy at aspects such as treatment cataract, reinforcing stomach reg fluid, antitumor, anti-ageing, Cardiovarscular, raising body's immunity, antifatigues, there is wide market at home and abroad, simultaneously become countries in the world diseases prevention, cured the disease and the superfine product good medicine of health care.In the multiple regional strains of dendrobium stem of Crack In Guizhou Chishui, the medicinal index composition such as polysaccharide, alkaloid, luxuriant and rich with fragrance class and bibenzyl is high compared with other place; pharmaceutical use is also larger; and carried out geography symbol product protection in March, 2006 by State Administration for Quality Supervision and Inspection and Quarantine's approval; thereby become Guizhou Province, first obtains the Chinese medicinal materials product that Products of Local Geographical Indication is protected, and is also the stem of noble dendrobium kind that first obtains " protection of national geography famous special product ".By cell engineering method, dendrobium stem is carried out to genetic improvement and there is important researching value and market potential.
Plant protoplast refers to the cell of having removed plasma membrane parcel after whole cell wallss, is the ideal material of carrying out fundamental research.The protoplastis of separation and purification, can be for carrying out the structure of the transmission of vegetable cell information, energy transformation, Cell wall synthesis mechanism, film and the mechanism of action of function, nucleocytoplasmic relation, mutation induction, hybridization or self-incompatible mechanism, intercellular interaction, plant hormone, separate various organoids, introduce organoid, research that protoplast transformation imports the aspects such as foreign gene, bioreactor for constructing.
First the present invention has collected the germ plasm resource of multiple dendrobium stems, after evaluating, with the seed of the dendrobium stem provenance that is derived from Chishui be explant, carry out seed germination by plant tissue culture technology, protocorm induction and tissue culture regeneration seedling are cultivated, system made the plant tissue culture fast breeding technique system of Chishui dendrobium stem, further taking the blade of tissue culture regeneration seedling as material, obtain protoplastis with enzymolysis process, after purifying, obtain purity high, energetic protoplastis, for the germ plasm resource of Chishui dendrobium stem is expanded, commercialization genetic breeding, certain basic substance has been established in the follow-up studies such as improvement of genes.
Summary of the invention
The object of the present invention is to provide a kind of simple method of obtaining the protoplastis that Crack In Guizhou Chishui dendrobium stem purity is high, energetic, density is large.
For realizing object of the present invention, the present invention includes two steps of isolation and purification of Chishui dendrobium stem protoplastis, contain the separation of protoplastis, the purifying of protoplastis, four aspects such as mensuration, the qualification of protoplastis vigor of protoplast yield.Particular content is as follows:
1, the separation of protoplastis: get the aseptic regrowth of dendrobium stem or Wild plant, choose large and full blade, it in Bechtop, is the fragment of 1mm left and right by its transverse cutting, put into little culture dish, solution A is poured in culture dish, draw materials is repeatedly dipped repeatedly in solution A with tweezers, object makes material fully contact with solution A, make hydrolysis result more abundant, decide the usage quantity of solution A according to the number of material, just flooding blade segment with solution A is advisable, then seal with sealing compound, be placed on constant-temperature table and shake, 26-28 DEG C, 60rpm, dark processing 4h.After protoplastis fully separates from material, (4 h left and right), stops enzymolysis.Blow and beat gently blade with suction nozzle, to discharge whole protoplastiss.
Described culture dish is diameter 60 mm, high 15 mm glass culture dishs.
Described special agent solution A composition is as table 1.
The each constituent concentration of described special agent solution A is: Mannitol 0.8 mmol/ml, MES 0.2 mmol/ml, KCl 0.08 mmol/ml, CaCl
20. 25 mmol/ml, BSA 10 mg/ml.
2, the purifying of protoplastis: enzymolysis 4h left and right protoplastis mixed solution is filtered with 200 object cell sieves, again filtrate is placed in to 2ml centrifuge tube, centrifugal 3min under 100g/min, make protoplastis sedimentation, then abandon supernatant, sediment is slowly added in solution B, under the rotating speed of 100 g/min centrifugal 3-5 time, each 3min, finally obtains the protoplastis of purifying.
Described special agent solution B composition is as table 2.
Table 2 solution B composition and content (taking 10ml as example):
The each constituent concentration of described special agent solution B is: Mannitol 0.8 mmol/ml, MES 0.2 mmol/ml, KCl 0.08 mmol/ml, CaCl
20. 25 mmol/ml, BSA 10 mg/ml
3, the mensuration of protoplast yield: the protoplastis after purifying is proceeded in 2ml solution B, and piping and druming, is evenly distributed protoplastis gently, draw one with liquid-transfering gun and drop on blood counting chamber, covered, is placed under inverted microscope and counts, continuous counter three times, averages; Calculate the output of the protoplastis that every gram of blade obtains according to the number that adds solution A Leaf, calculation result with every gram of blade cell number represent (individual/g).
4, the qualification of protoplastis vigor: utilize FDA(fluorescein diacetate) see through after protoplastis, under the effect of esterase, decompose rapidly, discharging fluorescence element, thus make great-hearted protoplastis send yellowish green fluorescence to identify the vigor of protoplastis.While calculating protoplastis vigor, need under bright field, observe protoplastis sum, and then forward under fluorescence and record great-hearted protoplastis number, finally calculate protoplastis vigor rate.
Described protoplastis vigor method of calculation are: the percentage ratio that accounts for whole observation protoplastiss with great-hearted protoplastis represents, i.e. the vigor rate of protoplastis=(the protoplastis number/protoplastis sum being colored) × 100%.Count measurement three times, averages.
The present invention provides effective technique means for obtaining the Crack In Guizhou Chishui dendrobium stem protoplastis that purity is high, energetic, density is large, for basic substance has been established in the follow-up studies such as the germ plasm resource expansion of Chishui dendrobium stem, business breeding, improvement of genes.
Described special agent is successively used in conjunction with by reagent solution A and reagent solution B, and ingredients listed and proportioning are not only only confined to Chishui dendrobium stem protoplastis isolation and purification, are also applicable to the stem of noble dendrobium protoplastis isolation and purification of other kinds.
The present invention compared with prior art tool has the following advantages and beneficial effect:
1). dendrobium stem protoplastis induction in Chishui provided by the invention is simple with purification technique system, and experimental cost is low, repeatable high;
2). it is material that the present invention selects Crack In Guizhou Chishui genuineness Chinese medicinal materials dendrobium stem first, protoplastis after separation and purification, has established certain material, technical foundation by follow-up studies such as the germ plasm resource expansion for Chishui dendrobium stem, genetic breeding, genetically engineereds;
3). the protoplast yield that the present invention utilizes Chishui dendrobium stem blade to obtain can reach 3.72 × 10
7individual/g, vigor rate is 86.31%, can be the biotechnology breedings such as dendrobium stem somatic hybridization and genetic transformation good receptor system is provided.
Embodiment:
Following instance is used for illustrating the present invention, but is not used for limiting the scope of the invention.
A kind of Chishui dendrobium stem protoplastis isolation and purification method and special agent have been invented in this research, and implementation step is as follows:
1, the separation of protoplastis: tissue culture regeneration seedling or the Wild plant of getting dendrobium stem, choose large and full blade, after mercuric chloride sterilizing, tear epidermis off with tweezers, it is the fragment of 1mm left and right by its transverse cutting, put into clean culture dish, ready solution A is poured in culture dish, got blade is repeatedly dipped repeatedly in solution A with tweezers, blade two sides is fully contacted with solution A, make hydrolysis result more abundant, according to material number decide and need the usage quantity of how many solution A, drawn materials and be advisable just to flood, then seal with sealing compound, be placed in dark processing 4 h on constant-temperature table, concussion condition is 26-28 DEG C, 60rmp.Described special agent solution A composition is table 1.
The each constituent concentration of described special agent solution A is: Mannitol 0.16 mol/ml, MES 0.2 mol/ml, KCl 0.032 mol/ml, CaCl
20.625 mol/ml.After enzymolysis stops, blowing and beating gently blade with suction pipe, to discharge whole protoplastiss.
2, the purifying of protoplastis: protoplastis mixed solution is filtered with 200 object cell sieves, to remove the residual pieces staying after enzymolysis, again the filtrate of having filtered is placed in to 2ml centrifuge tube, centrifugal 3min under 100g/min, make protoplastis sedimentation, add solution B, centrifugal 3min under 100g/min after abandoning supernatant liquor, rinsing 3-5 time, finally obtains the protoplastis of purifying.Described special agent solution B composition is table 2.
The each constituent concentration of described special agent solution B is: Mannitol 0.8 mmol/ml, MES 0.2 mmol/ml, KCl 0.08 mmol/ml, CaCl
20. 25 mmol/ml, BSA 10 mg/ml.
3, the mensuration of protoplast yield: add in 2ml solution B in the protoplastis after purifying, piping and druming gently, protoplastis is evenly distributed, cover glass is covered above the nucleonics on blood counting chamber, drop in a lateral edges of cover glass by solution B of liquid-transfering gun absorption, it is slowly goed deep in nucleonics, until solution B is full of nucleonics along the gap between cover glass and tally; Then tally is placed under inverted microscope and is counted, keep microscope stage level when counting, continuous counter three times, averages; Calculate the output of the protoplastis that every gram of blade obtains according to the number that adds solution A Leaf, calculation result with every gram of blade cell number represent (individual/g).
4, the qualification of protoplastis vigor: utilize FDA(fluorescein diacetate) see through after protoplastis, under the effect of esterase, decompose rapidly, discharging fluorescence element, thus make great-hearted protoplastis send yellowish green fluorescence to identify the vigor of protoplastis.While calculating protoplastis vigor, first under bright field, observe protoplastis sum, and then forward record under fluorescence to and send the protoplastis number of yellow-green fluorescence, finally calculate protoplastis vigor rate.
Described protoplastis vigor method of calculation are: the percentage ratio that accounts for whole observation protoplastiss with great-hearted protoplastis represents, i.e. the vigor rate of protoplastis=(the protoplastis number/protoplastis sum being colored) × 100%.Count measurement three times, averages.
Although mentioned above, the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, it made some modifications or improvements, and can make this invention have more operability.Therefore,, not departing from these modifications or improvements on basis of the present invention, all belong to the scope of protection of present invention.Described special agent is successively used in conjunction with by reagent solution A and reagent solution B, and ingredients listed and proportioning are not only only confined to Chishui dendrobium stem protoplastis isolation and purification, are also applicable to the stem of noble dendrobium protoplastis isolation and purification of other kinds.
Claims (2)
1. a Chishui dendrobium stem protoplastis isolation and purification method and special agent, it is characterized in that: two steps of isolation and purification of Chishui dendrobium stem protoplastis, contain the separation of protoplastis, the purifying of protoplastis, the mensuration of protoplast yield, qualification four aspects of protoplastis vigor; Method is as follows:
1), the separation of protoplastis: get the aseptic regrowth of dendrobium stem or Wild plant, choose large and full blade, it in Bechtop, is the fragment of 1mm left and right by its transverse cutting, put into little culture dish, solution A is poured in culture dish, draw materials is repeatedly dipped repeatedly in solution A with tweezers, material is fully contacted with solution A, make hydrolysis result more abundant, decide the usage quantity of solution A according to the number of material, just flooding blade segment with solution A is advisable, then seal with sealing compound, be placed on constant-temperature table and shake, 26-28 DEG C, 60rpm, dark processing 4h, after protoplastis fully separates from material, stop enzymolysis, blow and beat gently blade with suction nozzle, to discharge whole protoplastiss,
Described special agent solution A composition is table 1;
Table 1 solution A composition and content are taking 10 ml as example:
The each constituent concentration of described special agent solution A is: Mannitol 0.8 mmol/ml, MES 0.2 mmol/ml, KCl 0.08 mmol/ml, CaCl
20. 25 mmol/ml, BSA 10 mg/ml;
2), the purifying of protoplastis: enzymolysis 4h left and right protoplastis mixed solution is filtered with 200 object cell sieves, again filtrate is placed in to 2ml centrifuge tube, centrifugal 3min under 100g/min, make protoplastis sedimentation, then abandon supernatant, sediment is slowly added in solution B, under the rotating speed of 100 g/min centrifugal 3-5 time, each 3min, finally obtains the protoplastis of purifying;
Described special agent solution B composition is table 2;
Table 2 solution B composition and content are taking 10ml as example:
The each constituent concentration of described special agent solution B is: Mannitol 0.8 mmol/ml, MES 0.2 mmol/ml, KCl 0.08 mmol/ml, CaCl
20. 25 mmol/ml, BSA 10 mg/ml;
3), the mensuration of protoplast yield: the protoplastis after purifying is proceeded in 2ml solution B, and piping and druming, is evenly distributed protoplastis gently, draw one with liquid-transfering gun and drop on blood counting chamber, covered, is placed under inverted microscope and counts, continuous counter three times, averages; Calculate the output of the protoplastis that every gram of blade obtains according to the number that adds solution A Leaf, calculation result with every gram of blade cell number represent (individual/g);
4), the qualification of protoplastis vigor: utilizing FDA is that fluorescein diacetate sees through after protoplastis, under the effect of esterase, decompose rapidly, discharging fluorescence element, thus make great-hearted protoplastis send yellowish green fluorescence to identify the vigor of protoplastis.While calculating protoplastis vigor, need under bright field, observe protoplastis sum, and then forward under fluorescence and record great-hearted protoplastis number, finally calculate protoplastis vigor rate.
2. a kind of Chishui dendrobium stem protoplastis isolation and purification method according to claim 1 and special agent, it is characterized in that: described special agent is successively used in conjunction with by reagent solution A and reagent solution B, ingredients listed and proportioning are not only only confined to Chishui dendrobium stem protoplastis isolation and purification, are also applicable to the stem of noble dendrobium protoplastis isolation and purification of other kinds.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104130970A (en) * | 2014-07-31 | 2014-11-05 | 遵义医学院 | Method for preparing bletilla striata and protoplast and special reagent thereof |
| CN104357376A (en) * | 2014-11-18 | 2015-02-18 | 张敏跃 | Method for preparing and purifying yam protoplast |
| CN104357377A (en) * | 2014-11-21 | 2015-02-18 | 遵义医学院 | Method for separating and purifying dendrobium nobile protoplast and formula of special reagent |
| CN105316276A (en) * | 2015-11-27 | 2016-02-10 | 甘肃农业大学 | Method for rapidly obtaining halogeton glomeratus leaf protoplasts |
| CN108795839A (en) * | 2018-07-02 | 2018-11-13 | 中国科学院成都生物研究所 | A kind of method of stem of noble dendrobium single-cell suspension culture |
Citations (1)
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|---|---|---|---|---|
| CN101358181A (en) * | 2008-09-09 | 2009-02-04 | 南京大学 | Separation method of xylem parenchyma cell protoplast of rice root |
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2014
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Patent Citations (1)
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| CN101358181A (en) * | 2008-09-09 | 2009-02-04 | 南京大学 | Separation method of xylem parenchyma cell protoplast of rice root |
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| KHENTRY Y ET AL.: "Protoplast isolation and culture of Dendrobium Sonia "Bom 17"", 《KASETSART JOURNAL,NATURAL SCIENCES》 * |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104130970A (en) * | 2014-07-31 | 2014-11-05 | 遵义医学院 | Method for preparing bletilla striata and protoplast and special reagent thereof |
| CN104357376A (en) * | 2014-11-18 | 2015-02-18 | 张敏跃 | Method for preparing and purifying yam protoplast |
| CN104357377A (en) * | 2014-11-21 | 2015-02-18 | 遵义医学院 | Method for separating and purifying dendrobium nobile protoplast and formula of special reagent |
| CN105316276A (en) * | 2015-11-27 | 2016-02-10 | 甘肃农业大学 | Method for rapidly obtaining halogeton glomeratus leaf protoplasts |
| CN105316276B (en) * | 2015-11-27 | 2018-08-03 | 甘肃农业大学 | The method for being quickly obtained salt sward Leaves Protoplast |
| CN108795839A (en) * | 2018-07-02 | 2018-11-13 | 中国科学院成都生物研究所 | A kind of method of stem of noble dendrobium single-cell suspension culture |
| CN108795839B (en) * | 2018-07-02 | 2022-02-11 | 中国科学院成都生物研究所 | Method for suspension culture of dendrobium single cells |
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