CN101402938B - Method for producing protoplast - Google Patents
Method for producing protoplast Download PDFInfo
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- CN101402938B CN101402938B CN2008102266112A CN200810226611A CN101402938B CN 101402938 B CN101402938 B CN 101402938B CN 2008102266112 A CN2008102266112 A CN 2008102266112A CN 200810226611 A CN200810226611 A CN 200810226611A CN 101402938 B CN101402938 B CN 101402938B
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- enzymolysis
- protoplastis
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- 210000001938 protoplast Anatomy 0.000 title claims abstract description 15
- 238000004519 manufacturing process Methods 0.000 title description 2
- 210000004027 cell Anatomy 0.000 claims abstract description 68
- 239000000243 solution Substances 0.000 claims abstract description 56
- 239000000725 suspension Substances 0.000 claims abstract description 32
- 238000000034 method Methods 0.000 claims abstract description 29
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims abstract description 20
- 235000010355 mannitol Nutrition 0.000 claims abstract description 19
- 239000000463 material Substances 0.000 claims abstract description 10
- 241000196324 Embryophyta Species 0.000 claims abstract description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 8
- 239000007864 aqueous solution Substances 0.000 claims abstract description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 5
- 239000008103 glucose Substances 0.000 claims abstract description 5
- 239000011780 sodium chloride Substances 0.000 claims abstract description 4
- 229930195725 Mannitol Natural products 0.000 claims description 18
- 239000000594 mannitol Substances 0.000 claims description 18
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 12
- 108010059892 Cellulase Proteins 0.000 claims description 7
- 238000001556 precipitation Methods 0.000 claims description 7
- 235000013311 vegetables Nutrition 0.000 claims description 7
- 229940106157 cellulase Drugs 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 5
- 241000208125 Nicotiana Species 0.000 claims description 5
- 235000002637 Nicotiana tabacum Nutrition 0.000 claims description 5
- 229940098773 bovine serum albumin Drugs 0.000 claims description 5
- 241000219194 Arabidopsis Species 0.000 claims description 4
- 241001233957 eudicotyledons Species 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 abstract description 10
- 238000006243 chemical reaction Methods 0.000 abstract description 7
- 239000007788 liquid Substances 0.000 abstract description 5
- 108090000623 proteins and genes Proteins 0.000 abstract description 2
- 241000219195 Arabidopsis thaliana Species 0.000 abstract 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 abstract 1
- JXLHNMVSKXFWAO-UHFFFAOYSA-N azane;7-fluoro-2,1,3-benzoxadiazole-4-sulfonic acid Chemical compound N.OS(=O)(=O)C1=CC=C(F)C2=NON=C12 JXLHNMVSKXFWAO-UHFFFAOYSA-N 0.000 abstract 1
- 239000001110 calcium chloride Substances 0.000 abstract 1
- 229910001628 calcium chloride Inorganic materials 0.000 abstract 1
- 235000011148 calcium chloride Nutrition 0.000 abstract 1
- 230000010261 cell growth Effects 0.000 abstract 1
- 238000005119 centrifugation Methods 0.000 abstract 1
- 238000000749 co-immunoprecipitation Methods 0.000 abstract 1
- 230000002380 cytological effect Effects 0.000 abstract 1
- 235000019387 fatty acid methyl ester Nutrition 0.000 abstract 1
- 238000009396 hybridization Methods 0.000 abstract 1
- 238000013507 mapping Methods 0.000 abstract 1
- 239000013049 sediment Substances 0.000 abstract 1
- 238000001890 transfection Methods 0.000 description 10
- 229920001223 polyethylene glycol Polymers 0.000 description 9
- 239000001963 growth medium Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 3
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 150000001447 alkali salts Chemical class 0.000 description 2
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- MURGITYSBWUQTI-UHFFFAOYSA-N fluorescin Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC=C(O)C=C2OC2=CC(O)=CC=C21 MURGITYSBWUQTI-UHFFFAOYSA-N 0.000 description 2
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- 235000020138 yakult Nutrition 0.000 description 2
- 108010084185 Cellulases Proteins 0.000 description 1
- 102000005575 Cellulases Human genes 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
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- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 239000000819 hypertonic solution Substances 0.000 description 1
- 229940021223 hypertonic solution Drugs 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for conducting efficient preparation of plant suspension cell protoplasts. The method comprises the following steps: (1) plant cells are treated with manna sugar solution which is 0.5m to 1m and then collected; (2) the cells collected in step (1) are enzymolysized by an enzymolysis liquid; (3) an A solution is added into the system treated by step (2), a centrifugation is conducted after removing the unenzymolysized cell clots and then the sediment is collected, thus obtaining the protoplasts; and the A solution is aqueous solution and the solute thereof is the following final concentration materials: NaCl: 100mm to 200mm, CaCl2: 100mm to 150mm, KCL: 2mm to 10mm, glucose: 5mm to 10mm, and fatty acid methyl ester sulphonate: 1mm to 3mm; and the pH value of the A solution is 5.5 to 5.8. The method is mainly applicable to the preparation of BY2 suspension cell and arabidopsis thaliana suspension cell protoplasts. The suspension cell protoplasts separated by the method has high yield and good cell growth state which can meet the purposes of conducting a cytological mapping to a target gene, co-immunoprecipitation, Western hybridization and the like when being combined with the instantaneous conversion of a PEG mediate.
Description
Technical field
The present invention relates to a kind of method for preparing protoplastis.
Background technology
BY2 cell (Bright Yellow-2) is the suspension cell system that the mesophyll cell by tobacco prepares.This cell line growth speed is fast, can carry out mass-producing and cultivate, and growth conditions is controlled easily, is used for carrying out plant cell biology research at present widely, and also being described as is " Hela " cell of higher plant.Because the BY2 cell does not have the autofluorescence of chloroplast(id), therefore can be used for proteic Subcellular Localization analysis easily simultaneously.
Utilize fluorescin to merge to carry out proteic Subcellular Localization and performance analysis is most important for the proteic biological function of research.Utilize the method for stable conversion to transform BY2 suspension cell line or Arabidopis thaliana plant at present, need the time of some months usually.Carry out relevant research by the instantaneous conversion of preparation protoplastis and PEG mediation and then can shorten institute greatly and take time, raise the efficiency.
Summary of the invention
The purpose of this invention is to provide a kind of efficient production plant protoplast, and the short-cut method of good reproducibility.
The method for preparing plant protoplast provided by the present invention may further comprise the steps:
(1) handles vegetable cell, collecting cell then with the mannitol solution of 0.5-1M;
(2) cell of collecting with enzymolysis solution enzymolysis step (1); Described enzymolysis solution is the aqueous solution, and solute is the material of following final concentration: the quality percentage composition is the cellulase of 1%-2%; The quality percentage composition is the macerozyme of 0.2%-0.5%; The methyl sodiosul foaliphatate of 1-3mg/ml; The bovine serum albumin of 1-5mg/ml; 400-500mM N.F,USP MANNITOL; 1-5mM CaCl
2The pH of described enzymolysis solution is 5.5-5.8.
(3) add A solution in the system of handling through step (2), carry out centrifugal treating after removing the cell clot of enzymolysis not, collecting precipitation promptly obtains protoplastis; Described A solution is the aqueous solution, and solute is the material of following final concentration: 100-200mM NaCl, 100-150mM CaCl
2, 2-10mM KCl, 5-10mM glucose, 1-3mM methyl sodiosul foaliphatate; The pH of described A solution is 5.5-5.8.
Described method also comprises with the resulting protoplastis of described A solution washing, to remove the step of enzymolysis solution.
The concentration of N.F,USP MANNITOL is preferably 0.8-1M in the mannitol solution of described step 1).The temperature that mannitol solution is handled vegetable cell is 23-28 ℃, and the time is 10-20min.
Described vegetable cell is the dicotyledons cell; Described dicotyledons cell is preferably tobacco cell or arabidopsis cell.Wherein, described tobacco cell specifically can be the BY2 suspension cell, and described arabidopsis cell is specially the Arabidopis thaliana suspension cell.
Described enzymolysis solution is preferably the material that solute is following final concentration: the quality percentage composition is 1.5% cellulase; The quality percentage composition is 0.5% macerozyme; The methyl sodiosul foaliphatate of 2mg/ml; The bovine serum albumin of 1mg/ml; 500mM N.F,USP MANNITOL; 1mM CaCl
2The pH of described enzymolysis solution is preferably 5.7.Enzymolysis solution among the present invention can re-use after the membrane filtration degerming of 0.45 μ m.
The temperature of described enzymolysis can be 23-28 ℃, and the time of enzymolysis can be 3-5 hour.
Described A solution is preferably the material that solute is following final concentration: 154mM NaCl, 125mM CaCl
2, 5mMKCl, 5mM glucose, 1.5mM methyl sodiosul foaliphatate; The pH of described A solution is preferably 5.7.Above-mentioned A solution can be described as W5 solution.
Method of the present invention is applicable to preparation BY2 suspension cell protoplastis and Arabidopis thaliana suspension cell protoplastis.
When adopting method of the present invention to prepare BY2 suspension cell protoplastis, the cell of selecting for use is 2-3 days BY2 suspension cell of cultivation.Because the growth conditions of cell is most important to the separation efficiency of protoplastis, for guaranteeing the growth conditions unanimity of suspension cell, BY2 suspension cell switching ratio is preferably 1:5, transfers twice weekly, and in 24 ℃, the 130rpm lucifuge is cultivated.
The protoplastis that the inventive method is obtained carries out the PEG mediated transformation, and concrete grammar is as follows:
The protoplastis that separation obtains is resuspended with MaMG solution earlier, carry out instantaneous conversion with PEG solution then, stop transfection process with the W5 solution washing, finally be resuspended in the protoplast culture medium.
Consisting of of MaMG solution: 0.4M N.F,USP MANNITOL, 15mM MgCl
2, 4mM methyl sodiosul foaliphatate, pH are 5.7.
Consisting of of PEG-Ca transfection liquid: the quality percentage composition is 40% PEG4000 (Fluka), 0.2M N.F,USP MANNITOL, 100mM CaCl
2, use preceding preparation, dissolve fully behind the PEG and just can carry out transfection at least 1 hour, PEG solution can be in room temperature preservation, and uses in 5 days.This solution can not autoclaving.For reaching transfection effect preferably, recommend to use PEG4000 (Fluka, cat.No.81240)
Protoplast culture medium consists of: the basic salt of 4.3g/L MS (Sigma M5524); 0.4M sucrose (13.7%); The 500mg/L methyl sodiosul foaliphatate; 750mg/L CaCl
22H
2O; 250mg/L NH
4NO
3PH is 5.7.
Used cellulase can be commercially available various cellulases among the present invention, especially with cellulase R-10 (YakultHonsha Co.Ltd) best results, used macerozyme can be commercially available various macerozymes, especially with macerozymeR-10 (Yakult Honsha Co.Ltd) best results.
Present method is at first to utilize the N.F,USP MANNITOL of high sepage that vegetable cell is carried out the plasmolysis processing, with osmotic pressure is that the enzymolysis solution of 0.5M carries out enzymolysis, after enzymolysis is finished, add and the isopyknic hypotonic medium A of enzymolysis solution solution, centrifugal collection protoplastis, use the A solution washing once more, and make protoplastis be in swelling state, help the importing of next step foreign gene under the PEG mediation.This method is equally applicable to the preparation of Arabidopis thaliana suspension cell protoplastis.The present invention prepares the method for protoplastis, easy, efficient and good reproducibility.Can satisfy the needs of the experiments such as instantaneous conversion of PEG mediation with the protoplastis of this method preparation.
Description of drawings
Fig. 1 is the microscope picture of the BY2 suspension cell protoplastis of embodiment preparation.
Fig. 2 is the picture under fluorescent microscope after the protoplastis instantaneous conversion that PEG mediates among the embodiment, and the lower right corner is corresponding light field photo.
Embodiment
The preparation of embodiment 1, BY2 suspension cell protoplastis
1, collecting cell: in super clean bench, get 2-3 days BY2 suspension cell of 10ml cultivation in centrifuge tube.With the aperture is the filter filtration of 40 μ m, collecting cell.
2, plasmolysis: the cell of collecting is joined in the N.F,USP MANNITOL hypertonic solution of 10ml1M, handle 10-20min, filter collecting cell once more after making cell plasmolysis at 25 ℃.
3, enzymolysis: the cell precipitation of collecting is transferred in the enzymolysis solution of 10ml (the corresponding 10ml enzymolysis solution of original volume 10ml suspension cell), at room temperature 60rpm vibration lucifuge enzymolysis 3 hours on shaking table; Described enzymolysis solution is the aqueous solution, contains the various materials of following final concentration: the quality percentage composition is 1.5% cellulase cellulase R-10 (Yakult Honsha Co.Ltd); The quality percentage composition is 0.5% macerozyme macerozyme R-10 (YakultHonsha Co.Ltd); The methyl sodiosul foaliphatate of 2mg/ml; The bovine serum albumin of 1mg/ml; 500mM N.F,USP MANNITOL; 1mM CaCl
2, the pH of described enzymolysis solution is 5.7.
4, remove not enzymolysis cell mass: after enzymolysis finishes, in above-mentioned enzymolysis solution, add the W5 solution of 10ml, continue to leave standstill 10-20min, use the strainer filtering of 40 μ m then, remove the not cell mass of enzymolysis, collect filtrate, the BY2 suspension cell protoplastis that enzymolysis is good is arranged in filtrate.
5, collect protoplastis: the filtrate that step 4 obtains is transferred in the centrifuge tube, under the room temperature with the centrifugal 15min of the rotating speed of 1000rpm, collecting precipitation (BY2 suspension cell protoplastis), the microscope picture is seen Fig. 1.As shown in Figure 1, the protoplastis number that enzymolysis obtains is more, cell state good (protoplastis out of order may break and be fragment, and shape is non-just round, and tangible vacuolization etc. is arranged in the cell).
6, liquid is dezymotized in washing: the protoplastis with 10ml W5 solution washing obtains, leave standstill on ice, and make the protoplastis natural subsidence, careful absorption supernatant liquor behind about 15-30min blots clean supernatant as far as possible and does not touch protoplastis and precipitate; Use 10ml W5 solution suspension protoplastis once more, left standstill on ice at least two hours, make protoplastis natural subsidence once more.
Two, the protoplast transformation of PEG mediation
7, remove supernatant, eliminate supernatant as far as possible and do not touch the protoplastis precipitation; Adding MaMG solution in protoplastis precipitation, to make the concentration of BY2 suspension cell protoplastis be 5 * 10
6Individual/ml (calculating protoplastis concentration) with blood cell counting plate.Wherein, consisting of of MaMG solution: 0.4M N.F,USP MANNITOL, 15mM MgCl
2, 4mM methyl sodiosul foaliphatate, pH are 5.7.
8, (Arabidopis thaliana Biological resources center (ABRC), DNA CD3-987) (concentration 〉=1 μ g/ μ l) obtains suspension a to the MT-GK of interpolation 10-20 μ g in the protoplastis suspension of 300 μ l steps 7 preparation.
9, in suspension a, add the PEG-Ca transfection liquid (i.e. " (300+DNA) μ l ") that equates with suspension a volume, softly put upside down 10 times and make abundant mixing, incubated at room 30min.Wherein, the consisting of of PEG-Ca transfection liquid: the quality percentage composition be 40% PEG4000 (Fluka, cat.No.81240), 0.2M N.F,USP MANNITOL, 100mM CaCl
2
10, in the system of step 9, add the W5 solution (i.e. " 4 * (300+DNA) μ l ") that is equivalent to four times of suspension a volumes, put upside down mixing, stop transfection process, centrifugal 1000rpm * 3min; Collecting precipitation (protoplastis after the transfection), use then protoplast culture medium resuspended (protoplast culture medium consist of the basic salt of 4.3g/L MS (Sigma M5524); 0.4M sucrose (13.7%); The 500mg/L methyl sodiosul foaliphatate; 750mg/LCaCl
22H
2O; 250mg/L NH
4NO
3PH is 5.7), hatch after 12-18 hour with fluorescence microscope transfection effect (see figure 2) for 25-27 ℃.Fig. 2 is the BY2 protoplastis of the successful conversion of taking under fluorescent microscope, green fluorescence is the plastosome of green fluorescent protein mark among the figure; Fig. 2 lower right is corresponding light field picture, illustrates that the protoplasm somatocyte growth conditions of successful transfection is normal.
Claims (9)
1. method for preparing plant protoplast may further comprise the steps:
(1) handles vegetable cell, collecting cell then with the mannitol solution of 0.5-1M;
(2) cell of collecting with enzymolysis solution enzymolysis step (1); Described enzymolysis solution is the aqueous solution, and solute is the material of following final concentration: the quality percentage composition is the cellulase of 1%-2%; The quality percentage composition is the macerozyme of 0.2%-0.5%; The methyl sodiosul foaliphatate of 1-3mg/ml; The bovine serum albumin of 1-5mg/ml; 400-500mM N.F,USP MANNITOL; 1-5mM CaCl
2The pH value of described enzymolysis solution is 5.5-5.8;
(3) add A solution in the system of handling through step (2), carry out centrifugal treating after removing the cell clot of enzymolysis not, collecting precipitation promptly obtains plant protoplast; Described A solution is the aqueous solution, and solute is the material of following final concentration: 100-200mM NaCl, 100-150mM CaCl
2, 2-10mM KCl, 5-10mM glucose, 1-3mM methyl sodiosul foaliphatate; The pH value of described A solution is 5.5-5.8;
The temperature of handling vegetable cell in the described step 1) is 23-28 ℃, and the time is 10-20min;
Described step 2) temperature of enzymolysis described in is 23-28 ℃, and the time of enzymolysis is 3-5 hour.
2. method according to claim 1 is characterized in that: described method also comprises the step of the protoplastis that obtains with described A solution washing step (3).
3. method according to claim 1 and 2 is characterized in that: the concentration of N.F,USP MANNITOL is 0.8-1M in the mannitol solution of described step 1).
4. method according to claim 3 is characterized in that: described vegetable cell is the dicotyledons cell.
5. method according to claim 4 is characterized in that: described dicotyledons cell is tobacco cell or arabidopsis cell.
6. method according to claim 5 is characterized in that: described tobacco cell is the BY2 suspension cell, and described arabidopsis cell is the Arabidopis thaliana suspension cell.
7. method according to claim 6 is characterized in that: the solute in the described enzymolysis solution is the material of following final concentration: the quality percentage composition is 1.5% cellulase; The quality percentage composition is 0.5% macerozyme; The methyl sodiosul foaliphatate of 2mg/ml; The bovine serum albumin of 1mg/ml; 500mM N.F,USP MANNITOL; 1mMCaCl
2The pH of described enzymolysis solution is 5.7.
8. method according to claim 7 is characterized in that: the solute in the described A solution is the material of following final concentration: 154mMNaCl, 125mM CaCl
2, 5mMKCl, 5mM glucose, 1.5mM methyl sodiosul foaliphatate; The pH value of described A solution is 5.7.
9. method according to claim 8 is characterized in that: described plant protoplast is BY2 suspension cell protoplastis or Arabidopis thaliana suspension cell protoplastis.
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| CN2008102266112A CN101402938B (en) | 2008-11-17 | 2008-11-17 | Method for producing protoplast |
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|---|---|---|---|
| CN2008102266112A CN101402938B (en) | 2008-11-17 | 2008-11-17 | Method for producing protoplast |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106701658A (en) * | 2016-12-12 | 2017-05-24 | 中国科学院植物研究所 | Method for separating protoplast of tomato fruit |
| CN107488675A (en) * | 2017-09-29 | 2017-12-19 | 中国农业科学院油料作物研究所 | The separation of Rapeseed Protoplast and method for transformation |
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| CA2835253C (en) * | 2011-06-07 | 2018-06-26 | Janina KIRCHHOFF | Method for the generation of a monoclonal plant cell line |
| CN105018412B (en) * | 2015-07-27 | 2018-10-09 | 中国人民解放军第二军医大学 | A kind of datura method for preparing protoplast rapidly and efficiently |
| CN106167787B (en) * | 2016-08-23 | 2020-01-14 | 浙江农林大学 | Method for preparing xylem protoplast of betula luminifera and transient transformation |
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2008
- 2008-11-17 CN CN2008102266112A patent/CN101402938B/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106701658A (en) * | 2016-12-12 | 2017-05-24 | 中国科学院植物研究所 | Method for separating protoplast of tomato fruit |
| CN106701658B (en) * | 2016-12-12 | 2019-09-06 | 中国科学院植物研究所 | A method of separation tamato fruit protoplast |
| CN107488675A (en) * | 2017-09-29 | 2017-12-19 | 中国农业科学院油料作物研究所 | The separation of Rapeseed Protoplast and method for transformation |
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| CN101402938A (en) | 2009-04-08 |
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