CN115404197A - Preparation method of cyperus esculentus protoplast - Google Patents
Preparation method of cyperus esculentus protoplast Download PDFInfo
- Publication number
- CN115404197A CN115404197A CN202211130820.3A CN202211130820A CN115404197A CN 115404197 A CN115404197 A CN 115404197A CN 202211130820 A CN202211130820 A CN 202211130820A CN 115404197 A CN115404197 A CN 115404197A
- Authority
- CN
- China
- Prior art keywords
- cyperus esculentus
- protoplast
- filtering
- protoplasts
- preparing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000001938 protoplast Anatomy 0.000 title claims abstract description 105
- 235000005853 Cyperus esculentus Nutrition 0.000 title claims abstract description 100
- 238000002360 preparation method Methods 0.000 title claims abstract description 32
- 241000103725 Cyperus esculentus var. esculentus Species 0.000 title 1
- 244000285774 Cyperus esculentus Species 0.000 claims abstract description 102
- 238000001914 filtration Methods 0.000 claims abstract description 23
- 239000000706 filtrate Substances 0.000 claims abstract description 12
- 239000004677 Nylon Substances 0.000 claims abstract description 8
- 238000004113 cell culture Methods 0.000 claims abstract description 8
- 229920001778 nylon Polymers 0.000 claims abstract description 8
- 239000002244 precipitate Substances 0.000 claims abstract description 7
- 239000006228 supernatant Substances 0.000 claims abstract description 7
- 238000007865 diluting Methods 0.000 claims abstract description 4
- 238000005406 washing Methods 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 23
- 230000002255 enzymatic effect Effects 0.000 claims description 20
- 108010059892 Cellulase Proteins 0.000 claims description 18
- 229940106157 cellulase Drugs 0.000 claims description 18
- 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 description 16
- 229930195725 Mannitol Natural products 0.000 claims description 16
- 239000000413 hydrolysate Substances 0.000 claims description 16
- 239000000594 mannitol Substances 0.000 claims description 16
- 235000010355 mannitol Nutrition 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 9
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 7
- 239000001110 calcium chloride Substances 0.000 claims description 7
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 7
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- 239000002689 soil Substances 0.000 claims description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 4
- 229940098773 bovine serum albumin Drugs 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 4
- 238000002791 soaking Methods 0.000 claims description 4
- 239000008223 sterile water Substances 0.000 claims description 4
- 230000001954 sterilising effect Effects 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 3
- 230000000249 desinfective effect Effects 0.000 claims description 3
- 239000001103 potassium chloride Substances 0.000 claims description 3
- 235000011164 potassium chloride Nutrition 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 239000007788 liquid Substances 0.000 abstract description 15
- 108090000790 Enzymes Proteins 0.000 description 12
- 102000004190 Enzymes Human genes 0.000 description 12
- 229940088598 enzyme Drugs 0.000 description 12
- 230000000694 effects Effects 0.000 description 10
- 239000000463 material Substances 0.000 description 9
- 241000196324 Embryophyta Species 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- CHADEQDQBURGHL-UHFFFAOYSA-N (6'-acetyloxy-3-oxospiro[2-benzofuran-1,9'-xanthene]-3'-yl) acetate Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(OC(C)=O)C=C1OC1=CC(OC(=O)C)=CC=C21 CHADEQDQBURGHL-UHFFFAOYSA-N 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 241000219195 Arabidopsis thaliana Species 0.000 description 3
- 240000008042 Zea mays Species 0.000 description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 3
- 210000002421 cell wall Anatomy 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 235000005822 corn Nutrition 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 230000003204 osmotic effect Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 229920002488 Hemicellulose Polymers 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 108010009736 Protein Hydrolysates Proteins 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000001814 pectin Substances 0.000 description 2
- 229920001277 pectin Polymers 0.000 description 2
- 235000010987 pectin Nutrition 0.000 description 2
- 238000005204 segregation Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 241001391944 Commicarpus scandens Species 0.000 description 1
- 241000234646 Cyperaceae Species 0.000 description 1
- 241000234653 Cyperus Species 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 235000003283 Pachira macrocarpa Nutrition 0.000 description 1
- 240000001085 Trapa natans Species 0.000 description 1
- 235000014364 Trapa natans Nutrition 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000008723 osmotic stress Effects 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 235000009165 saligot Nutrition 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 230000004960 subcellular localization Effects 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Botany (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a preparation method of cyperus esculentus protoplast, which comprises the following steps: (1) 6 different tissues of young roots, buds, leaves, stems, stolons and tillering nodes of the cyperus esculentus are respectively adopted; (2) Rapidly cutting the tissue into strips or slices, and rapidly transferring the tissue to a cell culture plate containing the enzymolysis liquid to ensure that the tissue is completely immersed in the enzymolysis liquid; (3) placing the cell culture plate on a shaking table, and carrying out enzymolysis in a dark place; (4) Filtering the enzymolysis liquid with a nylon mesh screen, clamping the residue into a protoplast container after filtering, washing with a W5 solution, filtering again until the residue is fully washed, and repeatedly filtering the filtrate with a nylon mesh to obtain the final filtrate; (5) And centrifuging the final filtrate, pouring the supernatant, diluting with a W5 solution, and re-suspending the protoplast precipitate to obtain the cyperus esculentus protoplast. The preparation method effectively solves the problems of low preparation efficiency and poor integrity of the cyperus esculentus protoplast.
Description
Technical Field
The invention belongs to the technical field of plant protoplast preparation, and particularly relates to a preparation method of cyperus esculentus protoplasts.
Background
Cyperus esculentus L is a plant of Cyperus genus of Cyperaceae family, also named as Tiger nut, iron water chestnut and Cyperus esculentus, and is a non-traditional underground tuber crop, and its round or oval bean-shaped tuber can accumulate a large amount of oil, and its yield and oil yield are high, and its stress resistance and adaptability are strong, and it can be planted in the soil of desertification, desert and salinization, etc., and does not compete with grain, so that it is a special oil crop with huge development potential and high comprehensive utilization value.
Protoplasts are naked cells that are enveloped by a plasma membrane after removal of the cell wall. Since protoplasts do not have cell walls, they are more susceptible to uptake of foreign genetic material and are ideal receptors for gene function studies and genetic transformation. Meanwhile, the protoplast has cell totipotency and can be regenerated and differentiated into a complete plant, so that the preparation and the culture of the protoplast are important ways for plant propagation and regeneration. The protoplast fusion is used for somatic cell hybridization, so that the breeding problem that the sexual hybridization of the cyperus esculentus is difficult can be solved, in addition, the protoplast is also an important material for genetic basic research, and the protoplast can be used for researching subcellular localization, protein interaction and the like of the cyperus esculentus gene, thereby laying a foundation for disclosing the gene function of the cyperus esculentus. Therefore, the high-quality and high-efficiency preparation of cyperus esculentus protoplasts is a prerequisite for the successful development of these studies. At present, the protoplast preparation technology of many model plants and food crops such as tobacco, arabidopsis thaliana, corn, rice and the like is very complete, but the research on the cyperus esculentus protoplast preparation at home and abroad is not reported yet.
The cyperus esculentus has long and hard blades, a thick wax layer covers the surface of the cyperus esculentus, and the cyperus esculentus has a cavity structure. At present, arabidopsis thaliana and corn are prepared into protoplasts by adopting leaves, and referring to the preparation methods of arabidopsis thaliana and corn protoplasts, when cyperus esculentus leaves are used for preparing the protoplasts, the facts that the number of cells of the cyperus esculentus leaves is small, barrier tissues are difficult to crack, the release amount of the protoplasts is small, the integrity is poor, and the requirements of follow-up tests cannot be met are found.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a preparation method of cyperus esculentus protoplasts. The preparation method is combined with the morphological structure and physiological growth characteristics of the cyperus esculentus, the optimal tissue material suitable for preparing the cyperus esculentus protoplast is explored, and a certain technology and material support are provided for cyperus esculentus gene function research, variety genetic improvement and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of cyperus esculentus protoplasts comprises the following steps:
(1) Selecting 6 different tissues of tender roots, buds, leaves, stems, stolons and tillering nodes of the cyperus esculentus which grow well, robust and consistent respectively;
(2) Selecting a sharp blade to rapidly cut the tissue obtained in the step (1) into thin strips or thin slices with the width of 0.5-1mm, and then rapidly transferring the thin strips or thin slices into a 12-hole cell culture plate containing enzymatic hydrolysate to be completely immersed in the enzymatic hydrolysate;
(3) Placing the cell culture plate in the step (2) on a shaking table, and carrying out enzymolysis in a dark place;
(4) Filtering the enzymatic hydrolysate obtained in the step (3) by using a 800-mesh nylon mesh screen soaked by a W5 solution, clamping the residue into a protoplast container after filtering, washing by using a W5 solution with a proper volume, filtering again, repeating for 3-5 times until the residue is fully washed, completely filtering the protoplast, and finally filtering the filtrate for 2-3 times by using a 1000-mesh nylon mesh to obtain the final filtrate;
(5) And (4) centrifuging the final filtrate obtained in the step (4) to obtain a supernatant and a cyperus esculentus protoplast precipitate, pouring off the supernatant, diluting with a W5 solution, and re-suspending the cyperus esculentus protoplast precipitate to obtain the cyperus esculentus protoplast.
Further, the specific method for selecting 6 different tissues in the step (1) comprises the following steps: soaking Cyperus esculentus tubers in 30 deg.C sterile water for 48 hr, transferring to sandy soil, culturing in a light incubator, and collecting young and tender roots and buds when the Cyperus esculentus buds grow to 0.5-1.0 cm; taking young and tender leaves and stems from cyperus esculentus seedlings which germinate for 7-10 days; the tillering section is taken from cyperus esculentus seedlings which sprout for 15-18 days; in the initial stage of elongation of stolons of the cyperus esculentus, tender stolons with the length of 0.5-1.0cm are taken.
Further, the tissue selected in the step (2) is a tillering node.
Further, the method for cutting the tissue by the blade in the step (2) comprises the following steps: firstly, disinfecting a blade by using 75% ethanol; and then removing the old leaves and leaf sheaths outside the cyperus esculentus plants before cutting, and cutting the exposed tender tillering nodes by using the sterilized blade.
Further, the preparation method of the enzymolysis solution in the step (2) comprises the following steps: firstly, a container added with 0.5 to 2.0 percent of cellulase, 0.3 to 1.2 percent of eductase, 100mM 2- (N-morpholine) ethanesulfonic acid and 8 to 12 percent of mannitol in mass volume ratio is heated in water bath for 8 to 10min; then cooled to room temperature and added with 0.1% bovine serum albumin and 1mM calcium chloride, followed by ddH 2 Regulating the pH value to 5.2-6.6 after the volume of O is constant; and finally filtering and sterilizing by using a 0.45-micron filter membrane to obtain the enzymatic hydrolysate.
Further, the mass volume ratio of the cellulase is 2%, the mass volume ratio of the eductase is 0.9%, and the mass volume ratio of the mannitol is 11%; the pH was 6.0.
Further, the working condition of the shaker in the step (3) is 20-30 ℃, and the rotating speed is 30-50rpm.
Furthermore, the time of the enzymolysis in the dark in the step (3) is 2-12h.
Furthermore, the time of the enzymolysis in dark is 6h.
Further, the preparation method of the W5 solution in the step (4) comprises the following steps: firstly, 154mM sodium chloride, 125mM calcium chloride, 5mM potassium chloride and 2mM 2- (N-morpholine) ethanesulfonic acid are dissolved in water to obtain a mixed solution, and then the pH value of the mixed solution is adjusted to 5.8 by potassium hydroxide to obtain the W5 solution.
Further, the rotation speed of the centrifugation in the step (5) is 600-900rpm, and the centrifugation time is 5-6min.
Compared with the prior art, the invention has the following positive beneficial effects:
(1) Different tissue materials have different cell structures and numbers, and protoplasts have different release difficulty and quantity during enzymolysis. The plant cell wall mainly comprises cellulose, hemicellulose, pectin and other components, but the proportion of each component is different among different plants. The cellulase can carry out enzymolysis on cellulose; the eductase is a complex enzyme, and can carry out enzymolysis on hemicellulose and pectin. When the cellulase and the isolation enzyme are used for carrying out enzymolysis separation on the protoplast, the enzyme combination concentration ratio can influence the release amount of the protoplast, and tests in the embodiment of the invention can prove that the cyperus esculentus protoplast has the best preparation effect when the cellulase with the mass-volume ratio of 2% and the isolation enzyme with the mass-volume ratio of 0.9% are combined. The proper enzymolysis time is very critical to the preparation of the protoplast, the enzymolysis is incomplete if the enzymolysis time is short, the protoplast is damaged if the enzymolysis time is long, and the protoplast is easy to break, and experiments in the embodiment of the invention prove that the effect of the cyperus esculentus protoplast preparation is optimal when the enzymolysis time is 6 hours. The pH value of the enzymolysis liquid plays an important role in maintaining the stability of a plasma membrane and the activity of enzyme, when the pH value is too high, the protoplast is dehydrated and shrunk, and when the pH value is too low, the protoplast is broken and inactivated, and tests in the embodiment of the invention can prove that when the pH value is 6.0, the effect of preparing the cyperus esculentus protoplast is optimal. The mannitol is used as an osmotic pressure stabilizer of the enzymatic hydrolysate, the osmotic pressure is influenced by the concentration, and the protoplast is poisoned by osmotic stress due to too low or too high osmotic pressure, so that the yield of the protoplast is influenced.
(2) The invention establishes a set of efficient cyperus esculentus protoplast preparation system, overcomes the problems of low cyperus esculentus leaf protoplast preparation efficiency and poor integrity, can prepare a large amount of high-quality cyperus esculentus protoplasts, and provides technical support for the research fields of cyperus esculentus later-stage protoplast culture, fusion, establishment of an instant transformation system, genetic character improvement, molecular breeding and the like.
Drawings
FIG. 1 is a Cyperus esculentus protoplast prepared from the tissue of Cyperus esculentus tillering nodes according to example 1 of the present invention;
FIG. 2 is a graph showing the effect of different tissue materials on the yield of Cyperus esculentus protoplasts in example 2 of the invention;
FIG. 3 is a graph of the effect of different enzyme combinations on the yield of Cyperus esculentus protoplasts according to example 3 of the invention;
FIG. 4 is a graph showing the effect of different enzymatic hydrolysis times on the yield of Cyperus esculentus protoplasts in example 4 of the present invention;
FIG. 5 is a graph showing the effect of different mannitol concentrations on the protoplast yield of Cyperus esculentus in example 5 of the present invention;
FIG. 6 is a graph showing the effect of different pH of the enzymatic hydrolysates on the yield of Cyperus esculentus protoplasts in example 6 of the invention.
Detailed Description
The following examples facilitate a better understanding of the invention, but do not limit it. Unless otherwise specified, the technical means used in the examples are conventional methods well known to those skilled in the art, and the reagents, consumables and the like used are commercially available without specific description.
Example 1
(1) Soaking tubers of the cyperus esculentus which are good in growth vigor and strong and consistent in sterile water at 30 ℃ for 48 hours, transferring the tubers to sandy soil, culturing in a light incubator, taking tillering nodes after the cyperus esculentus germinates for 15-18 days, and taking the tubers at present to ensure cell viability;
(2) Selecting a sharp blade, disinfecting with 75% ethanol, removing old leaves and leaf sheaths outside cyperus esculentus plants to expose young and tender tillering nodes, quickly cutting the cyperus esculentus into thin slices with the thickness of 0.5-1mm, weighing 0.2g of the cut thin slices, quickly transferring the thin slices into a 12-hole cell culture plate containing 4mL of enzymatic hydrolysate, and completely immersing the thin slices in the enzymatic hydrolysate;
the preparation method of the enzymolysis liquid in the embodiment is as follows: firstly, cellulase and cellulose with the mass volume ratio of 2.0 percentAdding 0.9% of segregation enzyme, 100mM 2- (N-morpholine) ethanesulfonic acid and 11% of mannitol in mass-volume ratio into a container, heating the container added with the reagents in a water bath at 40-50 ℃ for 8-10min, cooling to room temperature, adding 0.1% bovine serum albumin and 1mM calcium chloride, and finally, adding ddH 2 Adjusting pH to 6.0 after O constant volume, and filtering and sterilizing with 0.45 μm filter membrane to obtain the enzymolysis solution.
(3) Placing the cell culture plate in the step (2) on a shaking table at 25 ℃ and 40rpm, and performing enzymolysis for 6 hours in a dark place;
(4) Filtering the enzymatic hydrolysate obtained in the step (3) by using a nylon mesh screen of 800 meshes soaked by a W5 solution, clamping the residues into a protoplast container after filtering, washing the residues by using a W5 solution with a proper volume, filtering the residues again, repeating the filtering for 3 times until the residues are fully washed and the protoplasts are completely filtered, and finally repeating the filtering of the filtrate for 2 times by using a nylon mesh of 1000 meshes so as to better remove cell fragments to obtain the final filtrate;
the preparation method of the W5 solution in the embodiment is as follows: firstly, dissolving 154mM sodium chloride, 125mM calcium chloride, 5mM potassium chloride and 2mM 2- (N-morpholine) ethanesulfonic acid in water to obtain a mixed solution, and then adjusting the pH of the mixed solution to 5.8 by using potassium hydroxide to obtain the W5 solution.
(5) And (4) centrifuging the final filtrate obtained in the step (4) at the rotating speed of 800rpm for 5-6min to obtain a supernatant and a cyperus esculentus protoplast precipitate, carefully pouring off the supernatant, diluting with a W5 solution and re-suspending the cyperus esculentus protoplast precipitate to obtain the cyperus esculentus protoplast.
Firstly, calculating the yield of the cyperus esculentus protoplast by adopting a blood counting plate method, wherein the calculation formula is as follows: the yield of protoplasts (one/g) =25 middle squares (number of middle squares in hemacytometer, same as in example below) total number of protoplasts/0.1 × 1000 × dilution factor/mass of enzymatic material, and the yield of protoplasts obtained in this example was 22.91 × 10 5 Per gram; then, the protoplast viability is determined by adopting fluorescent dye Fluorescein Diacetate (FDA), and the specific determination method is as follows: preparing 1mg/mL working solution from FDA with acetone solution, storing at-20 deg.C, collecting 10 μ L zymolyzed protoplast, adding 1 μ L FDA stock solution (final concentration of 0.01%), mixing well, standing for 5min, flaking stained protoplast with a hemacytometer, observing under a fluorescence microscope that viable Cyperus esculentus protoplast is white fluorescent spot, as shown in FIG. 1.
Example 2
This example differs from example 1 only in that: the cyperus esculentus tissues selected in the step (1) are different, the concentration of the eductase, the concentration of mannitol and the pH value in the enzymolysis liquid in the step (2) are different, and other steps are completely the same as those in the example 1. Step (1) of this example: soaking tuber of Cyperus esculentus in sterile water at 30 deg.C for 48h, transferring to sandy soil, culturing in illumination incubator, selecting 6 tissues of tender root, bud, leaf, stem, stolon and tillering node of Cyperus esculentus with good growth vigor and uniform health, and taking tender root and bud when the length of Cyperus esculentus bud is 0.5-1.0 cm; taking young and tender leaves and stems from cyperus esculentus seedlings which germinate for 7-10 days; the tillering section is taken from cyperus esculentus seedlings which sprout for 15-18 days; in the initial stage of elongation of stolons of the cyperus esculentus, tender stolons with the length of 0.5-1.0cm are taken. All materials are available at the moment. The preparation method of the enzymolysis liquid in the step (2) is as follows: cellulase with the mass volume ratio of 1.5 percent, macerase with the mass volume ratio of 0.9 percent, 100mM 2- (N-morpholine) ethanesulfonic acid and mannitol with the mass volume ratio of 10 percent are added into the above reagent, the mixture is heated in water bath at 40-50 ℃ for 8-10min, and after the mixture is cooled to room temperature, 0.1 percent bovine serum albumin, 1mM calcium chloride and ddH are added 2 Adjusting pH to 5.8 after O constant volume, filtering and sterilizing by a 0.45 mu m filter membrane, and preparing the solution as it is. The preparation of protoplasts was performed according to steps (2) to (5) of example 1 for 6 different cyperus esculentus tissues, respectively.
The results of this example show that different tissue types have different effects on the preparation of cyperus esculentus protoplasts, and specifically as shown in fig. 2, the yield of protoplasts prepared from 6 different tissues is as follows: the tillering node is the best material for preparing the cyperus esculentus protoplast.
Example 3
This example only differs from example 1 in that: the mass volume ratio of the cellulase and the eductase in the enzymolysis liquid in the step (2) is different, the mass volume ratio of the mannitol is 10 percent, the pH value of the enzymolysis liquid is 5.8, and other steps are completely the same as the steps in the embodiment 1. The combination of cellulase and macerozyme in the enzymatic hydrolysate of step (2) of this example at different concentrations is specifically shown in table 1.
TABLE 1 combination of cellulase and Segregation enzymes at different concentrations in the enzymatic hydrolysate
| Numbering of enzyme combinations | Cellulase mass to volume ratio (%) | Isolation enzyme mass to volume ratio (%) |
| 1 | 0.5 | 0.3 |
| 2 | 0.5 | 0.6 |
| 3 | 0.5 | 0.9 |
| 4 | 0.5 | 1.2 |
| 5 | 1.0 | 0.3 |
| 6 | 1.0 | 0.6 |
| 7 | 1.0 | 0.9 |
| 8 | 1.0 | 1.2 |
| 9 | 1.5 | 0.3 |
| 10 | 1.5 | 0.6 |
| 11 | 1.5 | 0.9 |
| 12 | 1.5 | 1.2 |
| 13 | 2.0 | 0.3 |
| 14 | 2.0 | 0.6 |
| 15 | 2.0 | 0.9 |
| 16 | 2.0 | 1.2 |
When the cyperus esculentus protoplast is prepared by adopting the enzymatic hydrolysate of the combination of the cellulase and the eductase with different mass-to-volume ratios in the table 1, different influences can be generated on the yield of the cyperus esculentus protoplast, and the specific result is shown in fig. 3, which indicates that the cellulase plays a main role in the preparation process of the cyperus esculentus protoplast, and the yield of the protoplast is obviously increased along with the increase of the mass-to-volume ratio of the cellulase; the mass-to-volume ratio of the eductase has a certain influence on the yield of the protoplast, the yield of the protoplast tends to increase and decrease with the increase of the concentration of the eductase, and the enzyme combination which is most suitable for preparing the cyperus esculentus protoplast is cellulase with the mass-to-volume ratio of 2.0 percent and the eductase with the mass-to-volume ratio of 0.9 percent.
Example 4
This example only differs from example 1 in that: the mass volume ratio of mannitol in the enzymolysis liquid in the step (2) is 10%, the pH value is 5.8, the light-proof enzymolysis time in the step (3) is different, and other steps are completely the same as those in the embodiment 1. In the embodiment, the time for the dark enzymolysis in step (3) is divided into the following 6 gradients, which are 2h, 4h, 6h, 8h, 10h and 12h respectively. When the cyperus esculentus protoplast is prepared by adopting different light-proof enzymolysis time, different influences are caused on the yield of the cyperus esculentus protoplast, and the specific result is shown in fig. 4, and the result shows that within 6h of the light-proof enzymolysis time, the yield of the cyperus esculentus tillering section protoplast is gradually increased along with the increase of the enzymolysis time, and then gradually decreases, so that the 6h is the optimal light-proof enzymolysis time for preparing the cyperus esculentus protoplast.
Example 5
This example differs from example 1 only in that: the mass volume ratio of mannitol in the enzymolysis liquid in the step (2) is different, the pH of the enzymolysis liquid is 5.8, and other steps are completely the same as those in the embodiment 1. In this example, the mannitol concentration in step (2) was divided into the following 5 gradients, 8%,9%,10%,11% and 12%, respectively. When the cyperus esculentus protoplast is prepared by adopting the enzymolysis solutions with different concentrations of mannitol, different influences are caused on the yield of the cyperus esculentus protoplast, and the specific result is shown in fig. 5. Thus, 11% is the optimal mannitol concentration for the preparation of cyperus esculentus protoplasts.
Example 6
This example only differs from example 1 in that: the pH of the enzymatic hydrolysate in the step (2) is different, and other steps are completely the same as those in the example 1. In this example, the pH of the enzymolysis solution in step (2) is divided into the following 8 gradients, which are 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4 and 6.6, respectively. The pH enzymolysis liquid is adopted to prepare the cyperus esculentus protoplast, and the specific result is shown in fig. 6, and the result shows that the yield of the cyperus esculentus tillering section protoplast is increased and then decreased along with the increase of the pH of the enzymolysis liquid, and when the pH of the enzymolysis liquid is 6.0, the yield of the protoplast reaches the highest value, and then the yield is decreased gradually. Thus, 6.0 is the optimum enzymatic hydrolysate pH for the preparation of cyperus esculentus protoplasts.
In conclusion, the cyperus esculentus tillering section is used as a protoplast preparation material, the enzyme solution is composed of 2.0% of cellulase in a mass-volume ratio and 0.9% of eductase in a mass-volume ratio, the enzymolysis time is 6 hours, the mannitol in a mass-volume ratio is 11%, the pH value of the enzymolysis solution is 6.0, and the yield of the cyperus esculentus protoplast is highest.
The invention has been described in detail with reference to specific embodiments and general illustrations. It will be apparent to those skilled in the art that the invention can be practiced within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. Accordingly, such modifications and improvements are intended to be within the scope of this invention without departing from the invention.
Claims (10)
1. A preparation method of cyperus esculentus protoplasts is characterized by comprising the following steps:
(1) Selecting 6 different tissues of tender roots, buds, leaves, stems, stolons and tillering nodes of the cyperus esculentus which grow well, robust and consistent respectively;
(2) Selecting a sharp blade to rapidly cut the tissue obtained in the step (1) into thin strips or thin slices with the width of 0.5-1mm, and then rapidly transferring the thin strips or thin slices into a 12-hole cell culture plate containing enzymatic hydrolysate to be completely immersed in the enzymatic hydrolysate;
(3) Placing the cell culture plate in the step (2) on a shaking table, and carrying out enzymolysis in a dark place;
(4) Filtering the enzymatic hydrolysate obtained in the step (3) by using a 800-mesh nylon mesh screen soaked by a W5 solution, clamping the residue into a protoplast container after filtering, washing by using a W5 solution with a proper volume, filtering again, repeating for 3-5 times until the residue is fully washed, completely filtering the protoplast, and finally filtering the filtrate for 2-3 times by using a 1000-mesh nylon mesh to obtain the final filtrate;
(5) And (4) centrifuging the final filtrate obtained in the step (4) to obtain a supernatant and a cyperus esculentus protoplast precipitate, pouring off the supernatant, diluting with a W5 solution, and re-suspending the cyperus esculentus protoplast precipitate to obtain the cyperus esculentus protoplast.
2. The method for preparing cyperus esculentus protoplasts according to claim 1, wherein the specific method for selecting 6 different tissues in step (1) is as follows: soaking Cyperus esculentus tubers in 30 deg.C sterile water for 48h, transferring to sandy soil, culturing in a light incubator, and collecting tender roots and buds when the length of Cyperus esculentus bud is 0.5-1.0 cm; taking young and tender leaves and stems from cyperus esculentus seedlings which germinate for 7-10 days; the tillering section is taken from cyperus esculentus seedlings which sprout for 15-18 days; in the initial stage of elongation of stolons of the cyperus esculentus, tender stolons with the length of 0.5-1.0cm are taken.
3. The method for preparing cyperus esculentus protoplasts according to claim 1, wherein the tissue selected in step (2) is tillering nodes.
4. The method for preparing cyperus esculentus protoplasts according to claim 1, wherein the method for cutting the tissues by the blade in the step (2) comprises the following steps: firstly, disinfecting a blade by using 75% ethanol; and then removing the old leaves and leaf sheaths outside the cyperus esculentus plants before cutting, and cutting the exposed tender tillering nodes by using the sterilized blade.
5. The method for preparing cyperus esculentus protoplasts according to claim 1, wherein the method for preparing the enzymatic hydrolysate in the step (2) comprises the following steps: firstly, a container added with 0.5 to 2.0 percent of cellulase, 0.3 to 1.2 percent of eductase, 100mM 2- (N-morpholine) ethanesulfonic acid and 8 to 12 percent of mannitol in mass volume ratio is heated in water bath for 8 to 10min; then cooled to room temperature and added with 0.1% bovine serum albumin and 1mM calcium chloride, followed by ddH 2 After O constant volume, regulating the pH value to 5.2-6.6; and finally filtering and sterilizing by using a 0.45-micrometer filter membrane to obtain the enzymatic hydrolysate.
6. The method of claim 5, wherein the cellulase is 2% by mass/volume, the macerase is 0.9% by mass/volume, and the mannitol is 11% by mass/volume; the pH was 6.0.
7. The method for preparing protoplasts of cyperus esculentus according to claim 1, wherein the shaker in step (3) is operated at 20-30 ℃ and 30-50rpm.
8. The method for preparing cyperus esculentus protoplasts according to claim 1, wherein the time for enzymolysis in step (3) is 2-12h without light.
9. The method for preparing cyperus esculentus protoplasts according to claim 1, wherein the W5 solution in step (4) is prepared by: firstly, 154mM sodium chloride, 125mM calcium chloride, 5mM potassium chloride and 2mM 2- (N-morpholine) ethanesulfonic acid are dissolved in water to obtain a mixed solution, and then the pH value of the mixed solution is adjusted to 5.8 by potassium hydroxide to obtain the W5 solution.
10. The method for preparing protoplasts of cyperus esculentus according to claim 1, wherein the centrifugation in step (5) is performed at 600-900rpm for 5-6min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211130820.3A CN115404197A (en) | 2022-09-16 | 2022-09-16 | Preparation method of cyperus esculentus protoplast |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211130820.3A CN115404197A (en) | 2022-09-16 | 2022-09-16 | Preparation method of cyperus esculentus protoplast |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115404197A true CN115404197A (en) | 2022-11-29 |
Family
ID=84166845
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211130820.3A Pending CN115404197A (en) | 2022-09-16 | 2022-09-16 | Preparation method of cyperus esculentus protoplast |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115404197A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105724139A (en) * | 2016-03-22 | 2016-07-06 | 北京大北农科技集团股份有限公司 | Application of herbicide tolerance protein |
| 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 |
| CN106318896A (en) * | 2016-08-23 | 2017-01-11 | 浙江农林大学 | Method for preparing and purifying cedarwood protoplast |
| AU2020103127A4 (en) * | 2020-10-30 | 2021-01-07 | Anhui Science And Technology University | A soybean leaf protoplast separation method for subcellular localization |
-
2022
- 2022-09-16 CN CN202211130820.3A patent/CN115404197A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105724139A (en) * | 2016-03-22 | 2016-07-06 | 北京大北农科技集团股份有限公司 | Application of herbicide tolerance protein |
| 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 |
| CN106318896A (en) * | 2016-08-23 | 2017-01-11 | 浙江农林大学 | Method for preparing and purifying cedarwood protoplast |
| AU2020103127A4 (en) * | 2020-10-30 | 2021-01-07 | Anhui Science And Technology University | A soybean leaf protoplast separation method for subcellular localization |
Non-Patent Citations (2)
| Title |
|---|
| 彭章;童华荣;梁国鲁;石艺琦;袁连玉;: "茶树叶片和胚根原生质体的分离及PEG诱导融合", 作物学报, no. 03 * |
| 苏彤;姚陆铭;张鑫;王彪;武天龙;: "大豆愈伤原生质体的制备和培养方式探究", 大豆科学, no. 05 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Baillie et al. | In vitro culture of isolated microspores and regeneration of plants in Brassica campestris | |
| Gangopadhyay et al. | Enhanced rate of multiplication and rooting through the use of coir in aseptic liquid culture media | |
| CN111374048B (en) | Chromosome doubling method of pepper or eggplant haploid plant | |
| CN109757373A (en) | A kind of Jing Banxia quick breeding method for tissue culture | |
| CN101243776A (en) | Cultivation method for cabbage type rape Isolated microspore plant strain | |
| CN111280065A (en) | Method for regenerating larch somatic embryos | |
| CN104521756A (en) | Method for producing trichosanthes tissue culture seedlings | |
| CN1869202A (en) | Free small spore culturing technology of non heading cabbage | |
| CN111543325A (en) | Induction culture method and induction culture medium for slash pine embryonic callus | |
| CN115623984B (en) | Apricot plant distant hybridization high-affinity backbone parent selection method and cotyledon abortive hybrid embryo rescue method based on genome heterozygosity | |
| CN101218893A (en) | Method for evoking sorghum mature embryo callus and establishing regeneration system | |
| CN114258858B (en) | Method for inducing embryogenic callus of sugar beet | |
| CN115404197A (en) | Preparation method of cyperus esculentus protoplast | |
| CN115896000A (en) | Separation and instantaneous transformation method of pepper protoplast | |
| CN116058281A (en) | Method for rapid propagation of polygonum mongolicum tissue | |
| JPS59224688A (en) | Breeding of plant cell | |
| CN117730780B (en) | Ningxia wolfberry pulp callus induction culture medium and induced proliferation culture method thereof | |
| Li et al. | Regeneration of plants from protoplasts of Arachis species (peanut) | |
| KR100302206B1 (en) | In-flight mass production and forge transplanting method | |
| CN118207150A (en) | Efficient separation method for camphor tree protoplasm | |
| KR101152111B1 (en) | Method for mass production of Phragmites communis by tissue culture | |
| CN115836647B (en) | Sterile induction plant regeneration method for young embryo of catalpa bungei | |
| CN115181735B (en) | Composite enzyme liquid and method for preparing poplar root protoplast | |
| Chabane et al. | Importance of protoplast culture in the genetic improvement of date palm (Phoenix dactylifera L.) | |
| Marsolais et al. | Progress in wheat and barley haploid induction using anther culture |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20221129 |