CN110878281A - Method for extracting submerged plant agave protoplast - Google Patents
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- 210000001938 protoplast Anatomy 0.000 title claims abstract description 68
- 238000000034 method Methods 0.000 title claims abstract description 24
- 240000004246 Agave americana Species 0.000 title abstract 2
- 239000000243 solution Substances 0.000 claims abstract description 37
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims abstract description 15
- 239000000600 sorbitol Substances 0.000 claims abstract description 15
- 102000038379 digestive enzymes Human genes 0.000 claims abstract description 10
- 108091007734 digestive enzymes Proteins 0.000 claims abstract description 10
- 210000002421 cell wall Anatomy 0.000 claims abstract description 9
- 239000012452 mother liquor Substances 0.000 claims abstract description 7
- 239000006228 supernatant Substances 0.000 claims abstract description 7
- 239000010413 mother solution Substances 0.000 claims abstract description 6
- 239000002244 precipitate Substances 0.000 claims abstract description 6
- 239000012224 working solution Substances 0.000 claims abstract description 4
- 238000002791 soaking Methods 0.000 claims abstract description 3
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 22
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- 239000012154 double-distilled water Substances 0.000 claims description 18
- 244000193174 agave Species 0.000 claims description 15
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 claims description 14
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 11
- 239000001110 calcium chloride Substances 0.000 claims description 11
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 11
- 239000001103 potassium chloride Substances 0.000 claims description 11
- 235000011164 potassium chloride Nutrition 0.000 claims description 11
- 239000011780 sodium chloride Substances 0.000 claims description 10
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 8
- 230000001133 acceleration Effects 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 4
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 230000001954 sterilising effect Effects 0.000 claims description 4
- 238000011049 filling Methods 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims 3
- 230000002255 enzymatic effect Effects 0.000 claims 3
- 108010009736 Protein Hydrolysates Proteins 0.000 claims 2
- 241000271309 Aquilaria crassna Species 0.000 claims 1
- 239000000413 hydrolysate Substances 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 239000008188 pellet Substances 0.000 claims 1
- 238000004321 preservation Methods 0.000 claims 1
- 241000196324 Embryophyta Species 0.000 abstract description 23
- 238000000746 purification Methods 0.000 abstract description 3
- 239000003761 preservation solution Substances 0.000 abstract description 2
- 230000001376 precipitating effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 5
- 108010059892 Cellulase Proteins 0.000 description 4
- 229940106157 cellulase Drugs 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 244000307697 Agrimonia eupatoria Species 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 230000029553 photosynthesis Effects 0.000 description 3
- 238000010672 photosynthesis Methods 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 230000009261 transgenic effect Effects 0.000 description 3
- 241001278836 Agrimonia pilosa Species 0.000 description 2
- 235000000641 Agrimonia pilosa Nutrition 0.000 description 2
- 244000020477 Ottelia alismoides Species 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 235000000125 common agrimony Nutrition 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 239000000819 hypertonic solution Substances 0.000 description 2
- 229940021223 hypertonic solution Drugs 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000000149 penetrating effect Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 235000016626 Agrimonia eupatoria Nutrition 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 241000220284 Crassulaceae Species 0.000 description 1
- 241000748095 Hymenopappus filifolius Species 0.000 description 1
- 241000544010 Ottelia Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 210000003484 anatomy Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 230000035425 carbon utilization Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000000243 photosynthetic effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The invention discloses a method for extracting a submerged plant agave protoplast, which comprises the following steps: A. preparing a mother solution: B. preparing a working solution: (1) preparing 10mL of digestive enzyme mother liquor by using the digestive enzyme solution; (2) preparing 100mL of mother solution by using the W5 solution; (3) preparing 100mL of mother solution by using the MMG solution; C. preparing an appliance: cutting off a blue gun head, two sets of clean culture dishes, a rectangular blade and a 2mL round-bottom centrifuge tube at the head part; D. cell wall enzymolysis: soaking herba Otteliae Alismoidis leaf in culture dish containing sorbitol solution, and precipitating with enzymolysis solution; E. enriching protoplasts; F. and (3) preserving the protoplast: removing supernatant, transferring the protoplast precipitate into a tube, injecting MMG solution, and storing to obtain a protoplast. The method is easy to implement, simple and convenient to operate, simple and quick, and relatively pure protoplasts can be obtained without complex purification. And (4) resuspending the collected protoplast in a preservation solution, and stably preserving for more than 24 h. Is suitable for extracting the protoplast of the submerged plant.
Description
Technical Field
The invention belongs to the field of biotechnology and cell biology, and particularly relates to a method for extracting a submerged plant agave protoplast, which is suitable for extracting the protoplast from a submerged plant leaf.
Background
The submerged plant, Ottelia alismoides, is a plant of the genus Ottelia of the family Amydaceae and widely distributed in various countries of east Asia. Although the leaves of the agave tongue are simple in anatomical structure and only have two or three layers of cells, the leaves can perform C4 metabolism and are unicellular C4 metabolic plants. Meanwhile, the Crassulaceae acid metabolic pathway (CAM) can be induced, and the bicarbonate radical in the water body can be used as an inorganic carbon source for photosynthesis, so that the variable inorganic carbon environment in the water body can be effectively dealt with. The research on the coexistence mechanism of different photosynthetic carbon utilization ways in the monocells of the agrimony is beneficial to deepening the understanding of the photosynthesis way, and the theoretical basis is possibly provided for the modification of the photosynthesis way of the crops, the enhancement of the carbon assimilation ability and the increase of the crop yield. However, the agave is not a model plant in biological research, a transgenic system of the agave is not established, and single cell culture is an effective way to establish the transgenic system, so that the separation and extraction of the protoplast of the agave becomes one of the key steps for establishing the transgenic system. The invention is obtained by optimally adjusting part of reagent components aiming at the agave tongue grass on the basis of the conventional technology of plant protoplast separation.
Disclosure of Invention
The invention aims to provide a method for extracting the protoplast of the submerged plant of the agave, which is easy to implement and simple and convenient to operate. The method has the advantages of simplicity, rapidness and capability of obtaining relatively pure protoplasts without complex purification.
In order to achieve the purpose, the invention adopts the following technical measures:
the technical conception is as follows: cellulase and macerozyme were used to degrade the cell wall and release protoplasts. Sorbitol is used as a penetrating substance, enzymolysis liquid of cellulase and eductase with certain concentration is prepared, and cell walls are degraded under the condition of 25 to 30 ℃. And (3) adding a slightly hypertonic solution into the enzymolysis solution to stop enzymolysis, and releasing the protoplast into the protoplast. Filtering to remove undegraded debris, collecting the filtrate containing protoplast, centrifuging, and enriching the protoplast. Resuspend with slightly hypertonic solution, centrifuge, collect protoplasts. And (4) resuspending the collected protoplast in a preservation solution, and stably preserving for more than 24 h. The invention mainly aims at a simple method for quickly extracting the protoplast of the submerged plant, which is developed by the submerged plant of the Ottelia alismoides and is suitable for extracting the protoplast of the submerged plant. The invention utilizes cellulase and eductase to degrade cell walls, and uses sorbitol as a penetrating substance to keep the integrity of protoplast.
A method for extracting a submerged plant agave protoplast comprises the following steps:
① mother liquor preparation:
dissolving 18.2g of 1M sorbitol in double distilled water, and metering to 100mL (at normal temperature and normal pressure);
0.2M Potassium chloride: dissolving 1.49g in double distilled water, and fixing the volume to 100mL (autoclaving at 121 ℃);
11.1g of 1M calcium chloride is dissolved in double distilled water, and the volume is determined to be 100mL (autoclaved at 121 ℃);
4.76g of 0.5M magnesium chloride is dissolved in double distilled water, and the volume is determined to be 100mL (autoclaved at 121 ℃);
8.99g of 1.54M sodium chloride is dissolved in double distilled water, and the volume is determined to be 100mL (autoclaved at 121 ℃);
0.1M 2- (N-morpholino) ethanesulfonic acid (MES):1.95g was dissolved in double distilled water, and the volume was adjusted to 100mL, and the pH was adjusted to 5.7(121 deg.C)
Autoclaving).
Note that: sorbitol is easy to grow bacteria, and can be filtered and sterilized with 0.21 μm filter membrane (the general use of 121 deg.C high temperature and high pressure sterilization can damage its components), and the total dosage can be reduced in each use, and it is used first. Other reagents can be used for many times (3-5 times) after high-temperature sterilization (121 ℃).
② preparing working solution:
(1) digestive enzyme solution (for use as prepared, the amount of mother liquor required to prepare 10mL of digestive enzyme is as follows)
The fully dissolved digestive enzymes should be brown, clear.
(2) W5 solution (amount of mother liquor required to make 100mL is as follows):
(3) MMG solution (amount of mother liquor required to make 100mL below):
③ appliance preparation-cutting the head of a blue tip, two sets of clean petri dishes (60mm format), a sharp rectangular blade (e.g., of the type used by boys shaving), a 2mL round-bottom centrifuge tube;
④ enzymolysis of cell wall, cutting the agave leaf into small pieces of 2cm wide and 4cm long, soaking in a culture dish containing 0.3M sorbitol solution, cutting into 1mm wide strips with a blade, changing a cutting edge if the leaf feels slightly retarded, and adding in 0.3M sorbitol solution to prevent drying at any time, picking the cut leaf into a conical flask containing 20mL of enzymolysis solution with a dissecting needle, ensuring that the leaf can be completely suspended in the enzymolysis solution, vacuumizing the conical flask for 3-5 min, and allowing the leaf not to float out and completely sink into the enzymolysis solution, considering that the higher enzyme activity can damage the protoplast at 40 deg.C, and the protoplast is sensitive to light, and performing enzymolysis in dark at 25-30 deg.C for 3-4 h.
⑤ enriching protoplast, injecting 20mL precooled W5(154mM sodium chloride, 125mM calcium chloride, 5mM potassium chloride and 2mM 2- (N-morpholino) ethanesulfonic acid) solution after enzymolysis to stop enzymolysis, filtering with 100-mesh gauze, loading the filtrate into four 10mL centrifuge tubes, centrifuging at 4 deg.C and 100g acceleration and deceleration for 8 steps for 5min, removing supernatant, transferring the precipitate into two 2mL centrifuge tubes, filling with precooled W5(154mM sodium chloride, 125mM calcium chloride, 5mM potassium chloride and 2mM 2- (N-morpholino) ethanesulfonic acid), ice-cooling for 30min, centrifuging at 4 deg.C and 100g acceleration and deceleration for 8 steps for 5min, and repeating the steps two to three times.
⑥ preserving the protoplast by removing the supernatant, transferring the protoplast precipitate into a tube, injecting MMG solution, preserving to obtain a protoplast, counting under a microscope, and calculating the protoplast concentration.
Through the technical measures of the six steps: the most important point is to prepare a digestive enzyme solution with proper concentration, maintain proper osmotic pressure, solve the problem of protoplast rupture after cell wall digestion and achieve the purpose of extracting the complete protoplast of the submerged plant leaves.
Compared with the prior art, the invention has the following advantages and effects:
plant protoplast extraction has become a routine method in plant cell biology engineering, mainly for terrestrial plants, especially food crops. According to the current mastered data, only a few works are carried out to extract protoplasts of aquatic plants, especially submerged plants, and the gradient centrifugation technology necessary for extracting protoplasts of terrestrial plants is adopted.
Compared with the limited work, the invention is mainly embodied in that compared with the prior art, the invention is convenient and quick, and complete and purer protoplast can be obtained without special purification methods (such as a density gradient centrifugation method). The method saves the steps of purifying the protoplast by other methods, and is convenient and quick.
Drawings
FIG. 1 is a schematic diagram of a method for extracting protoplasts of a submerged plant, Erythrophloe odorata, counting under a microscope of 100 times.
The protoplasts of the submerged plant, Agrimonia eupatoria, extracted by the method of the invention are counted under a 100-fold microscope, and the number is about 1.6 multiplied by 105Cells per ml.
FIG. 2 is a schematic view of the method for extracting the agave tongue protoplast of the submerged plant.
The protoplast of the agrimony extracted by the invention is observed under a 400-fold microscope, and the cell membrane of the protoplast is complete and spherical.
Detailed Description
The following detailed description is made with reference to the accompanying drawings and examples:
example 1:
a method for rapidly extracting a submerged plant agave protoplast comprises the following steps:
A. solution preparation:
(1) preparing a mother solution:
1M sorbitol 18.2g dissolved in double distilled water, constant volume to 100mL (normal temperature and pressure, using 0.21 μ M filter membrane sterilization);
0.2M Potassium chloride: dissolving 1.49g in double distilled water, and fixing the volume to 100mL (autoclaving at 121 ℃);
11.1g of 1M calcium chloride is dissolved in double distilled water, and the volume is determined to be 100mL (autoclaved at 121 ℃);
4.76g of 0.5M magnesium chloride is dissolved in double distilled water, and the volume is determined to be 100mL (autoclaved at 121 ℃);
8.99g of 1.54M sodium chloride is dissolved in double distilled water, and the volume is determined to be 100mL (autoclaved at 121 ℃);
0.1M 2- (N-morpholino) ethanesulfonic acid (MES):1.95g was dissolved in double distilled water, and the volume was adjusted to 100mL, and the pH was adjusted to 5.7 (autoclaving at 121 ℃).
(2) Preparing a working solution:
(A) digestive enzyme solution (ready for use): 1.5% (in terms of mass/volume) of cellulase R10, 0.4% (in terms of mass/volume) of macerase R10, 20mM of potassium chloride, 20mM of 2- (N-morpholino) ethanesulfonic acid, 10mM of calcium chloride and 0.1% (in terms of mass/volume) of bovine serum albumin were added to 0.3M sorbitol, respectively, and dissolved completely.
(B) W5 solution containing 154mM sodium chloride, 125mM calcium chloride, 5mM potassium chloride and 2mM 2- (N-morpholino) ethanesulfonic acid
(C) MMG solution containing 0.3M sorbitol, 15mM magnesium chloride and 4mM 2- (N-morpholino) ethanesulfonic acid;
through the above steps, a sterile digestive enzyme solution for cell wall digestion, a sterile W5 solution for eluting protoplasts, and a sterile MMG solution for preserving protoplasts were obtained.
B. The experimental process comprises the following steps:
(1) cell wall enzymolysis: the leaves of Agrimonia pilosa are cut into pieces of 2cm in width and 4cm in length and immersed in a petri dish containing 0.3M sorbitol solution. The silk with the width of about 1mm is cut by a blade, if the hand feels slightly retarded, a cutting edge is replaced, the whole process is carried out in 0.3M sorbitol solution, and the silk is added at any time to prevent drying. The cut leaves are picked into a conical flask with 20mL of enzymolysis liquid by using a dissecting needle, and the leaves can be completely suspended in the enzymolysis liquid. Vacuumizing the conical flask for 3-5 minutes, wherein the leaves can not float out and completely sink into the enzymolysis liquid after vacuumizing. Considering that the protoplast is damaged by the higher enzyme activity at 40 ℃ and the protoplast is sensitive to light, the enzyme is enzymolyzed for 3-4h at 25-30 ℃ in a dark place.
(2) And (3) enrichment of protoplasts: after enzymolysis, 20mL of precooled W5(154mM sodium chloride, 125mM calcium chloride, 5mM potassium chloride and 2mM 2- (N-morpholino) ethanesulfonic acid) solution is injected to stop enzymolysis, and gauze with 100 meshes is used for filtering; the filtered liquid is respectively put into four 10mL centrifuge tubes to be centrifuged for 5min at 4 ℃ and 100g at an acceleration and deceleration speed of 8 steps; removing supernatant, transferring the precipitate into two 2mL centrifuge tubes, filling with pre-cooled W5(154mM sodium chloride, 125mM calcium chloride, 5mM potassium chloride and 2mM 2- (N-morpholino) ethanesulfonic acid), ice-cooling for 30min, and centrifuging at 4 deg.C under 100g acceleration and deceleration for 8 steps for 5 min; this step can be repeated two to three times.
(3) And (3) preserving the protoplast: and removing the supernatant, transferring the protoplast precipitate into a tube, injecting the MMG solution into the tube, and preserving to finish the preparation of the protoplast. A protoplast is obtained, counted under a microscope, and the protoplast concentration is calculated.
C. The experimental results are as follows:
the method can be used for simply and quickly extracting the protoplast of the aquatic plant of the agave.
FIG. 1 shows the count of protoplasts of the submerged plant, Erythrophloe odorata, extracted by the present invention under 100-fold microscope at about 1.6^5 cells per ml, meeting the requirements for later stage genetic transformation and single cell culture.
FIG. 2 shows that when the protoplast of Agrimonia pilosa extracted by the present invention is observed under 400 times microscope, the cell membrane of the protoplast is intact and spherical, and the cytoplasmic circulation can be observed, which indicates that the cell has activity, and also proves that the method of the present invention is effective.
Claims (1)
1. A method for extracting a submerged plant agave protoplast is characterized by comprising the following steps:
A. preparing a mother solution:
dissolving 18.2g of 1M sorbitol in double distilled water, and carrying out constant volume treatment to 100mL at normal temperature and normal pressure;
0.2M Potassium chloride: dissolving 1.49g of the mixture in double distilled water, fixing the volume to 100mL, and sterilizing at 121 ℃ under high pressure;
dissolving 11.1g of 1M calcium chloride in double distilled water, diluting to 100mL, and autoclaving at 121 ℃;
dissolving 4.76g of 0.5M magnesium chloride in double distilled water, diluting to 100mL, and autoclaving at 121 ℃;
8.99g of 1.54M sodium chloride is dissolved in double distilled water, the volume is determined to be 100mL, and autoclaving is carried out at 121 ℃;
dissolving 0.1M 2- (N-morpholino) ethanesulfonic acid 1.95g in double distilled water, diluting to 100mL, adjusting pH to 5.7121 deg.C, and autoclaving;
B. preparing a working solution:
(1) preparing 10mL of digestive enzyme mother liquor by using the digestive enzyme solution:
(2) amount of 100mL of mother liquor prepared from W5 solution:
(3) the amount of the MMG solution for preparing 100mL of mother solution is as follows:
C. preparing an appliance: cutting off a blue gun head, two sets of clean culture dishes, a rectangular blade and a 2mL round-bottom centrifuge tube at the head part;
D. cell wall enzymolysis: cutting the agave eaglewood leaves into small pieces with the width of 2cm and the length of 4cm, soaking the small pieces in a culture dish filled with 0.3M sorbitol solution, cutting the small pieces into threads with the width of 1mm by a blade, changing one cutting edge when the blade feels blocked, carrying out the whole process in the 0.3M sorbitol solution, picking the cut leaves into a conical flask with 20mL of enzymatic hydrolysate by using a dissecting needle, suspending the leaves in the enzymatic hydrolysate, vacuumizing the conical flask for 3-5 minutes, sinking the conical flask into the enzymatic hydrolysate, and carrying out enzymolysis for 3-4 hours at the temperature of 25-30 ℃ in a dark place;
E. and (3) enrichment of protoplasts: injecting 20mL of precooled W5 after enzymolysis: 154mM sodium chloride, 125mM calcium chloride, 5mM potassium chloride and 2mM 2- (N-morpholino) ethanesulfonic acid, terminating the enzymolysis of the solution, and filtering the solution by using gauze of 100 meshes; the filtered liquid is respectively put into four 10mL centrifuge tubes to be centrifuged for 5min at 4 ℃ and 100g at an acceleration and deceleration speed of 8 steps; the supernatant was removed and the pellet was transferred to two 2mL centrifuge tubes and washed with precooled W5: 154mM sodium chloride, 125mM calcium chloride, 5mM potassium chloride and 2mM 2- (N-morpholino) ethanesulfonic acid, fully filling, carrying out ice bath for 30min, and then centrifuging for 5min at 4 ℃ and 100g acceleration and deceleration by 8 grades; this step is repeated two to three times;
F. and (3) preserving the protoplast: and removing the supernatant, transferring the protoplast precipitate into a tube, injecting the tube into an MMG solution for preservation, completing the preparation of the protoplast, obtaining a protoplast, counting under a microscope, and calculating the concentration of the protoplast.
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| CN115340971A (en) * | 2022-08-12 | 2022-11-15 | 中国科学院水生生物研究所 | Enzymolysis liquid for preparing auricularia auriculata protoplast, preparation method of auricularia auriculata protoplast and instantaneous conversion method |
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| CN109136167A (en) * | 2018-07-12 | 2019-01-04 | 北京林业大学 | The preparation method of lily mesophyll protoplast |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020173037A1 (en) * | 2001-03-29 | 2002-11-21 | Pati Pratap Kumar | Efficient method of protoplast culture |
| CN105695391A (en) * | 2016-03-25 | 2016-06-22 | 中国农业科学院茶叶研究所 | Extraction method of tea tree protoplast |
| CN106244516A (en) * | 2016-08-09 | 2016-12-21 | 湖南省农业生物技术研究中心 | A kind of extracting method of barnyard grass protoplast |
| CN108949665A (en) * | 2018-07-12 | 2018-12-07 | 北京林业大学 | The preparation method of lily petal protoplast |
| CN109136167A (en) * | 2018-07-12 | 2019-01-04 | 北京林业大学 | The preparation method of lily mesophyll protoplast |
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