CN104130970A - Method for preparing bletilla striata and protoplast and special reagent thereof - Google Patents
Method for preparing bletilla striata and protoplast and special reagent thereof Download PDFInfo
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- CN104130970A CN104130970A CN201410371645.6A CN201410371645A CN104130970A CN 104130970 A CN104130970 A CN 104130970A CN 201410371645 A CN201410371645 A CN 201410371645A CN 104130970 A CN104130970 A CN 104130970A
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Abstract
A method for preparing bletilla striata and protoplast comprises the following steps: separation of protoplast, purification of protoplast, determining on yield of protoplast, and determining on activity ratio of protoplast. A special reagent applied to the method comprises an enzyme solution A and enzyme solution B; the enzyme solution A comprises MES, KCl, Mannitol, CellulaseR10, PectolyaseY-23, BSA, CaCl2 and ddH 2O; and the enzyme solution B comprises MES, KCl, Mannitol, BSA, CaCl2 and ddH2O. The method is simple and practicable, low in experiment cost and high in repeatability. The yield of protoplast obtained by utilizing bletilla striata and leaves thereof can reach 4.8*10<6> per gram, and the activity ratio is 92.26%. The method provides good material basis for bletilla striata and somatic hybridization, mutant induction, organelle separation, protoplast, protoplast transformation and bioreactor construction.
Description
Technical field
The present invention relates to a kind of method and special agent thereof of preparing pale reddish brown bletilla protoplastis, be specially the method for obtaining protoplastis with the tissue cultured seedling blade of pale reddish brown bletilla, belong to field of plant cell engineering technology.
Background technology
Pale reddish brown bletilla
bletilla striata(Thunb.) Reichb.f. has the effect such as astringing to arrest bleeding, detumescence and promoting granulation, be used for the treatment of the diseases such as spitting of blood, haematemesis, traumatic hemorrhage, sore swollen toxin, chapped skin, also there is antibacterial analgesia, anti-tumor activity, improve the health, increase immunity of organisms, there is medicinal function and health-care effect.Meanwhile, pale reddish brown bletilla is also the important raw and processed materials of industry, makeup and gardens gardening, all has wide market at home and abroad.By cell engineering method, pale reddish brown bletilla is carried out to genetic improvement and there is important economic worth and social effect.
Plant protoplast refers to the cell of having removed plasma membrane parcel after whole cell wallss, is the ideal material as bio-reactor and 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 inventor has collected the germ plasm resource of national multiple pale reddish brown bletillas provenance, be that research material carries out enzymolysis with blade, release obtains protoplastis, after purifying, obtained high, the energetic protoplastis of purity, for pale reddish brown bletilla germ plasm resource expansion, commercialization genetic breeding, improvement of genes, bio-reactor prepare the follow-up studies such as medicinal ingredients and established certain basic substance.
Summary of the invention
Technical problem to be solved by this invention is: utilize the blade of pale reddish brown bletilla to obtain the method for the protoplastis that purity is high, energetic, density is large and the special agent for the method.
For solving the problems of the technologies described above, the present invention adopts following technical scheme:
Prepare a method for pale reddish brown bletilla protoplastis, comprise the steps: the separation of protoplastis, the purifying of protoplastis, the mensuration of protoplast yield, the mensuration of protoplastis vigor rate;
(1) separation of protoplastis: choose pale reddish brown bletilla tissue culture regeneration seedling or the large and full blade of Wild plant, the fragment that is 1mm by its transverse cutting in Bechtop, put into 60mm culture dish, enzyme solution A is poured in culture dish, with tweezers by draw materials and be soaked in enzyme solution A, pH value is 5.7, decide the usage quantity of enzyme solution A according to the number of material, enzyme solution A and material proportion are 10ml:1g, then seal with sealing compound, be placed in dark processing 4 h on constant-temperature table, concussion condition is 26-28 DEG C, 120rpm, stop enzymolysis, now protoplastis is fully separated, blow and beat gently blade with suction nozzle, to discharge whole protoplastiss, obtain protoplastis enzyme solution,
(2) purifying of protoplastis: protoplastis enzyme 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 enzyme solution B, under the rotating speed of 100 g/min centrifugal 3-5 time, each 3min, finally obtains the protoplastis of purifying;
(3) mensuration of protoplast yield: the protoplastis of purifying is proceeded in 2ml enzyme 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 enzyme solution A Leaf, calculation result with every gram of Leaves Protoplast number represent (individual/g);
(4) mensuration of protoplastis vigor rate: 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 culture dish is the glass culture dish of diameter 60 mm, high 15 mm.
For the preparation of a special agent for the method for pale reddish brown bletilla protoplastis, comprise enzyme solution A and enzyme solution B;
Described enzyme solution A proportioning is as follows:
Composition add-on
MES 1.0ml（200 mmol）
KCl 2.5ml（80 mmol）
Mannitol 5.0ml（0.8 mol）
Cellulase R10 150.0mg
Pectolyase Y-23 50.0mg
BSA 1.0ml（10 mg）
CaCl
2 400.0ul（250 mmol）
ddH
2O 100.0ul
In described enzyme solution A, constituent concentration is: Mannitol 0.16 mol/ml, MES 0.2 mol/ml, KCl 0.032 mol/ml, CaCl
20.625 mol/ml;
Described enzyme solution B proportioning is as follows:
Composition add-on
MES 1.0ml（200 mmol）
KCl 2.5ml（80 mmol）
Mannitol 5.0ml（0.8 mol）
BSA 1.0ml（10 mg）
CaCl
2 400.0ul（250 mmol）
ddH
2O 100.0ul
In described enzyme solution B, constituent concentration is: Mannitol 0.16 mol/ml, MES 0.2 mol/ml, KCl 0.032 mol/ml, CaCl
20.625 mol/ml.
Compared with prior art, tool of the present invention has the following advantages and beneficial effect:
1. pale reddish brown bletilla protoplastis induction provided by the invention is simple with purification technique system, and experimental cost is low, repeatable high;
2. the present invention carries out after the separation and purification of protoplastis pale reddish brown bletilla first, and the follow-up studies such as the germ plasm resource expansion for pale reddish brown bletilla, genetic breeding, genetically engineered are established to certain basic substance;
3. the protoplast yield that the present invention utilizes pale reddish brown bletilla blade to obtain can reach 4.8 × 10
6individual/g, vigor rate is 92.26%, can be the biotechnology breedings such as pale reddish brown bletilla somatic hybridization and genetic transformation good receptor system is provided.
Brief description of the drawings:
Fig. 1 is pale reddish brown bletilla protoplastis of the present invention (10 × 40);
Fig. 2 is pale reddish brown bletilla of the present invention unpurified protoplastis (10 × 20) after 4 hours enzymolysis;
Fig. 3 is the pale reddish brown bletilla protoplastis (10 × 20) after purifying of the present invention;
Fig. 4 is the pale reddish brown bletilla protoplastis (10 × 20) after FDA dyeing after purifying of the present invention.
Embodiment:
Following instance is used for illustrating the present invention, but is not used for limiting the scope of the invention.
Prepare a method for pale reddish brown bletilla protoplastis, comprise the steps: the separation of protoplastis, the purifying of protoplastis, the mensuration of protoplast yield, the mensuration of protoplastis vigor rate;
(1) separation of protoplastis: tissue culture regeneration seedling or the Wild plant of getting pale reddish brown bletilla, choose large and full blade, after mercuric chloride sterilizing, tear epidermis off with tweezers, the fragment that is 1mm by its transverse cutting, put into clean culture dish, ready enzyme solution A is poured in culture dish, mix, decide according to drawn materials number the usage quantity that needs how many enzyme solution A, drawn materials and be advisable just to flood, then with sealing compound sealing, be placed in dark processing 4 h on constant-temperature table, concussion condition is 26-28 DEG C, 120 rmp.After enzymolysis stops, beating blade with suction pipe featheriness, to discharge whole protoplastiss (accompanying drawing 1,2), obtain protoplastis enzyme solution;
(2) purifying of protoplastis: protoplastis enzyme 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 enzyme solution B, centrifugal 3min under 100g/min after abandoning supernatant liquor, rinsing 3-5 time, finally obtains the protoplastis (accompanying drawing 3) of purifying.
(3) mensuration of protoplast yield: add in 2ml enzyme solution B in the protoplastis of 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 with enzyme solution B of liquid-transfering gun absorption, it is slowly goed deep in nucleonics, until enzyme 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 enzyme solution A Leaf, calculation result with every gram of blade cell number represent (individual/g).
(4) mensuration of protoplastis vigor rate: utilize FDA(fluorescein diacetate) see through after protoplastis, under the effect of esterase, decompose rapidly, discharging fluorescence element, identifies the vigor of protoplastis (accompanying drawing 4) thereby make great-hearted protoplastis send yellowish green fluorescence.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 culture dish is diameter 60 mm, high 15 mm glass culture dishs.
For the preparation of a special agent for pale reddish brown bletilla protoplastis method, comprise enzyme solution A and enzyme solution B;
Described enzyme solution A proportioning is as follows:
Composition add-on
MES 1.0ml（200 mmol）
KCl 2.5ml（80 mmol）
Mannitol 5.0ml（0.8 mol）
Cellulase R10 150.0mg
Pectolyase Y-23 50.0mg
BSA 1.0ml（10 mg）
CaCl
2 400.0ul（250 mmol）
ddH
2O 100.0ul
In described enzyme solution A, constituent concentration is: Mannitol 0.16 mol/ml, MES 0.2 mol/ml, KCl 0.032 mol/ml, CaCl
20.625 mol/ml.
Described enzyme solution B proportioning is as follows:
Composition add-on
MES 1.0ml（200 mmol）
KCl 2.5ml（80 mmol）
Mannitol 5.0ml（0.8 mol）
BSA 1.0ml（10 mg）
CaCl
2 400.0ul（250 mmol）
ddH
2O 100.0ul
In described enzyme solution B, constituent concentration is: Mannitol 0.16 mol/ml, MES 0.2 mol/ml, KCl 0.032 mol/ml, CaCl
20.625 mol/ml.
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 pale reddish brown bletilla protoplastis that purity is high, energetic, density is large, for follow-up studies such as the germ plasm resource expansion of bletilla, business breeding, improvement of genes and established basic substance using this protoplastis as bio-reactor.
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.
Claims (3)
1. a method of preparing pale reddish brown bletilla protoplastis, is characterized in that, the method comprises the steps: the separation of protoplastis, the purifying of protoplastis, the mensuration of protoplast yield, the mensuration of protoplastis vigor rate;
(1) separation of protoplastis: choose pale reddish brown bletilla tissue culture regeneration seedling or the large and full blade of Wild plant, the fragment that is 1mm by its transverse cutting in Bechtop, put into 60mm culture dish, enzyme solution A is poured in culture dish, with tweezers by draw materials and be soaked in enzyme solution A, pH value is 5.7, decide the usage quantity of enzyme solution A according to the number of material, enzyme solution A and material proportion are 10ml:1g, then seal with sealing compound, be placed in dark processing 4 h on constant-temperature table, concussion condition is 26-28 DEG C, 120rpm, stop enzymolysis, now protoplastis is fully separated, blow and beat gently blade with suction nozzle, to discharge whole protoplastiss, obtain protoplastis enzyme solution,
(2) purifying of protoplastis: protoplastis enzyme 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 enzyme solution B, under the rotating speed of 100 g/min centrifugal 3-5 time, each 3min, finally obtains the protoplastis of purifying;
(3) mensuration of protoplast yield: the protoplastis of purifying is proceeded in 2ml enzyme 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 enzyme solution A Leaf, calculation result with every gram of Leaves Protoplast number represent (individual/g);
(4) mensuration of protoplastis vigor rate: 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.
2. the method for the pale reddish brown bletilla protoplastis of preparation according to claim 1, is characterized in that: described culture dish is the glass culture dish of diameter 60 mm, high 15 mm.
3. for a special agent for the method for the pale reddish brown bletilla protoplastis of preparation claimed in claim 1, comprise enzyme solution A and enzyme solution B;
Described enzyme solution A proportioning is as follows:
Composition add-on
MES 1.0ml（200 mmol）
KCl 2.5ml（80 mmol）
Mannitol 5.0ml（0.8 mol）
Cellulase R10 150.0mg
Pectolyase Y-23 50.0mg
BSA 1.0ml（10 mg）
CaCl
2 400.0ul（250 mmol）
ddH
2O 100.0ul
In described enzyme solution A, constituent concentration is: Mannitol 0.16 mol/ml, MES 0.2 mol/ml, KCl 0.032 mol/ml, CaCl
20.625 mol/ml;
Described enzyme solution B proportioning is as follows:
Composition add-on
MES 1.0ml（200 mmol）
KCl 2.5ml（80 mmol）
Mannitol 5.0ml（0.8 mol）
BSA 1.0ml（10 mg）
CaCl
2 400.0ul（250 mmol）
ddH
2O 100.0ul
In described enzyme solution B, constituent concentration is: Mannitol 0.16 mol/ml, MES 0.2 mol/ml, KCl 0.032 mol/ml, CaCl
20.625 mol/ml.
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| CN201410371645.6A CN104130970B (en) | 2014-07-31 | 2014-07-31 | A kind of method preparing pale reddish brown Pseudobulbus Bletillae (Rhizoma Bletillae) protoplast and special agent thereof |
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| CN201410371645.6A CN104130970B (en) | 2014-07-31 | 2014-07-31 | A kind of method preparing pale reddish brown Pseudobulbus Bletillae (Rhizoma Bletillae) protoplast and special agent thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110283775A (en) * | 2019-07-12 | 2019-09-27 | 遵义医科大学 | Secondary metabolite synthetic method in a kind of promotion bletilla suspended culture cell |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103881960A (en) * | 2014-04-17 | 2014-06-25 | 遵义医学院 | Method and special reagent for separation and purification of Chishui dendrobium nobile protoplasts |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103881960A (en) * | 2014-04-17 | 2014-06-25 | 遵义医学院 | Method and special reagent for separation and purification of Chishui dendrobium nobile protoplasts |
Non-Patent Citations (2)
| Title |
|---|
| KHENTRY Y ET AL.: "Protoplast isolation and culture of Dendrobium Sonia "Bom 17"", 《KASETSART JOURNAL,NATURAL SCIENCES》, 31 December 2006 (2006-12-31), pages 361 - 369 * |
| 严寒等: "马蹄金子叶原生质体的分离技术", 《植物生理学通讯》, 28 February 2005 (2005-02-28), pages 76 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110283775A (en) * | 2019-07-12 | 2019-09-27 | 遵义医科大学 | Secondary metabolite synthetic method in a kind of promotion bletilla suspended culture cell |
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