CN109511550B - Method for separating and culturing seaweed protoplast and regenerating plant - Google Patents
Method for separating and culturing seaweed protoplast and regenerating plant Download PDFInfo
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- 210000001938 protoplast Anatomy 0.000 title claims abstract description 104
- 241001474374 Blennius Species 0.000 title claims abstract description 49
- 238000000034 method Methods 0.000 title claims abstract description 43
- 238000012258 culturing Methods 0.000 title claims abstract description 41
- 230000001172 regenerating effect Effects 0.000 title claims abstract description 11
- 210000004027 cell Anatomy 0.000 claims abstract description 59
- 239000000243 solution Substances 0.000 claims abstract description 40
- 108090000790 Enzymes Proteins 0.000 claims abstract description 39
- 102000004190 Enzymes Human genes 0.000 claims abstract description 39
- 241000195493 Cryptophyta Species 0.000 claims abstract description 34
- 239000013535 sea water Substances 0.000 claims abstract description 22
- 210000001519 tissue Anatomy 0.000 claims abstract description 22
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims abstract description 20
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims abstract description 20
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims abstract description 20
- LJCNRYVRMXRIQR-OLXYHTOASA-L potassium sodium L-tartrate Chemical compound [Na+].[K+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O LJCNRYVRMXRIQR-OLXYHTOASA-L 0.000 claims abstract description 19
- 235000011006 sodium potassium tartrate Nutrition 0.000 claims abstract description 19
- 210000002421 cell wall Anatomy 0.000 claims abstract description 18
- 239000011259 mixed solution Substances 0.000 claims abstract description 14
- 238000004140 cleaning Methods 0.000 claims abstract description 12
- 239000002352 surface water Substances 0.000 claims abstract description 12
- 238000000227 grinding Methods 0.000 claims abstract description 10
- 239000001476 sodium potassium tartrate Substances 0.000 claims abstract description 10
- 238000005406 washing Methods 0.000 claims abstract description 10
- 238000002791 soaking Methods 0.000 claims abstract description 9
- 230000029087 digestion Effects 0.000 claims abstract description 7
- 210000000805 cytoplasm Anatomy 0.000 claims abstract description 6
- 239000012535 impurity Substances 0.000 claims abstract description 6
- 238000001556 precipitation Methods 0.000 claims abstract description 6
- 238000010008 shearing Methods 0.000 claims abstract description 6
- 239000010802 sludge Substances 0.000 claims abstract description 6
- 239000001963 growth medium Substances 0.000 claims description 39
- 229940088598 enzyme Drugs 0.000 claims description 38
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 24
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 15
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 15
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 claims description 15
- 235000010378 sodium ascorbate Nutrition 0.000 claims description 15
- 229960005055 sodium ascorbate Drugs 0.000 claims description 15
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 claims description 15
- 239000006228 supernatant Substances 0.000 claims description 15
- 230000003204 osmotic effect Effects 0.000 claims description 14
- 230000008569 process Effects 0.000 claims description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 12
- SODPIMGUZLOIPE-UHFFFAOYSA-N (4-chlorophenoxy)acetic acid Chemical compound OC(=O)COC1=CC=C(Cl)C=C1 SODPIMGUZLOIPE-UHFFFAOYSA-N 0.000 claims description 11
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 claims description 11
- 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 description 11
- 239000008103 glucose Substances 0.000 claims description 11
- 238000009630 liquid culture Methods 0.000 claims description 11
- 238000005286 illumination Methods 0.000 claims description 10
- 239000002244 precipitate Substances 0.000 claims description 10
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 9
- 238000002955 isolation Methods 0.000 claims description 9
- 229940074439 potassium sodium tartrate Drugs 0.000 claims description 9
- 210000001082 somatic cell Anatomy 0.000 claims description 9
- 108010059892 Cellulase Proteins 0.000 claims description 8
- 239000007853 buffer solution Substances 0.000 claims description 8
- 229940106157 cellulase Drugs 0.000 claims description 8
- 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 7
- 229930195725 Mannitol Natural products 0.000 claims description 7
- 239000000594 mannitol Substances 0.000 claims description 7
- 235000010355 mannitol Nutrition 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 239000003381 stabilizer Substances 0.000 claims description 7
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 6
- 239000000661 sodium alginate Substances 0.000 claims description 6
- 235000010413 sodium alginate Nutrition 0.000 claims description 6
- 229940005550 sodium alginate Drugs 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 5
- 239000012634 fragment Substances 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 5
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 4
- 210000000514 hepatopancreas Anatomy 0.000 claims description 4
- 239000001103 potassium chloride Substances 0.000 claims description 4
- 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 description 3
- 239000000600 sorbitol Substances 0.000 claims description 3
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 claims description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims 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 claims description 2
- 229930006000 Sucrose Natural products 0.000 claims description 2
- 150000001720 carbohydrates Chemical class 0.000 claims description 2
- 239000005720 sucrose Substances 0.000 claims description 2
- 230000008929 regeneration Effects 0.000 claims 8
- 238000011069 regeneration method Methods 0.000 claims 8
- 241000206572 Rhodophyta Species 0.000 claims 1
- 238000009395 breeding Methods 0.000 abstract description 8
- 230000001488 breeding effect Effects 0.000 abstract description 8
- 230000011681 asexual reproduction Effects 0.000 abstract description 3
- 238000013465 asexual reproduction Methods 0.000 abstract description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 12
- 238000011161 development Methods 0.000 description 7
- 239000000648 calcium alginate Substances 0.000 description 6
- 235000010410 calcium alginate Nutrition 0.000 description 6
- 229960002681 calcium alginate Drugs 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 230000009286 beneficial effect Effects 0.000 description 5
- 230000032823 cell division Effects 0.000 description 5
- 210000000170 cell membrane Anatomy 0.000 description 4
- 239000001913 cellulose Substances 0.000 description 4
- 229920002678 cellulose Polymers 0.000 description 4
- 238000004043 dyeing Methods 0.000 description 4
- 241001428166 Eucheuma Species 0.000 description 3
- 206010020649 Hyperkeratosis Diseases 0.000 description 3
- 239000013543 active substance Substances 0.000 description 3
- -1 alginate radical ions Chemical class 0.000 description 3
- 229920000615 alginic acid Polymers 0.000 description 3
- 239000001110 calcium chloride Substances 0.000 description 3
- 229910001628 calcium chloride Inorganic materials 0.000 description 3
- OKHHGHGGPDJQHR-YMOPUZKJSA-L calcium;(2s,3s,4s,5s,6r)-6-[(2r,3s,4r,5s,6r)-2-carboxy-6-[(2r,3s,4r,5s,6r)-2-carboxylato-4,5,6-trihydroxyoxan-3-yl]oxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylate Chemical compound [Ca+2].O[C@@H]1[C@H](O)[C@H](O)O[C@@H](C([O-])=O)[C@H]1O[C@H]1[C@@H](O)[C@@H](O)[C@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@H](O2)C([O-])=O)O)[C@H](C(O)=O)O1 OKHHGHGGPDJQHR-YMOPUZKJSA-L 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- CNGYZEMWVAWWOB-VAWYXSNFSA-N 5-[[4-anilino-6-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]-2-[(e)-2-[4-[[4-anilino-6-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]-2-sulfophenyl]ethenyl]benzenesulfonic acid Chemical compound N=1C(NC=2C=C(C(\C=C\C=3C(=CC(NC=4N=C(N=C(NC=5C=CC=CC=5)N=4)N(CCO)CCO)=CC=3)S(O)(=O)=O)=CC=2)S(O)(=O)=O)=NC(N(CCO)CCO)=NC=1NC1=CC=CC=C1 CNGYZEMWVAWWOB-VAWYXSNFSA-N 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 235000003642 hunger Nutrition 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 241000828356 Solieria <tachinid fly> Species 0.000 description 1
- 241000196294 Spirogyra Species 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention provides a method for separating and culturing seaweed protoplast and regenerating plants, belonging to the field of algae culture, which comprises the following steps: selecting algae with normal color, no rot and vigorous growth, cleaning sludge, miscellaneous algae and other auxiliary impurities on the surface of the algae with a brush, and usingHgCl2Disinfecting the solution, washing the solution by using disinfected seawater, cutting tender top parts of fresh alga bodies, sucking surface water by using filter paper, shearing or grinding the surface water into fine tissue blocks or small branches, soaking the fine tissue blocks or the small branches in a mixed solution of sodium potassium tartrate and polyvinylpyrrolidone, performing digestion and enzymolysis by using a seaweed tool enzyme solution, and performing centrifugal precipitation to obtain alga body cell protoplasm; and culturing the protoplast to obtain cells with regenerated cell walls, and further culturing to obtain regenerated plants. The invention provides a method for culturing algae with short seedling culture period and high seedling breeding efficiency, which can solve the problems of seed conservation and breeding of asexual reproduction type algae seedlings and can be used for productive seedling culture.
Description
Technical Field
The invention belongs to the field of algae culture methods, and particularly relates to a method for separating and culturing algae protoplasts and regenerating plants.
Background
Although the algae have no structures such as flowers, fruits, seeds and the like to propagate offspring, various reproduction modes are available to adapt to the environment. In the aspect of asexual reproduction, some cells can be directly divided into two parts, such as spirogyra, and can be broken into a plurality of sections, and each section can be grown into independent individuals; some algae can produce a plurality of spores with flagella, the spores can freely swim, and each spore grows into a new individual after being mature; some algae can produce thick-walled dormant spores when the environment is poor, and when the environment is proper, the algae germinate and grow into new individuals. In sexual reproduction, some algae produce male and female gametes, which are mated to form new individuals.
The invention discloses a method for separating, culturing and regenerating a plant by using eucheuma seaweed protoplast, which is mainly characterized in that according to totipotency of development of seaweed somatic cells, a seaweed tool enzyme is utilized to decompose eucheuma tissues to dissociate out the somatic cell protoplast to obtain somatic cells and cell lines of regenerated cell walls, and then the somatic cells and the cell lines are cultured to obtain a regenerated plant; however, the invention has low enzymolysis efficiency, and the protoplast culture condition is rough, which is not beneficial to improving the breeding efficiency of the seedlings.
Disclosure of Invention
The invention aims to provide a method for separating, culturing and regenerating a plant by using an alga protoplast, and provides a method for culturing alga with short seedling culture period and high seedling breeding efficiency, which can solve the problems of seed conservation and breeding of asexual reproduction alga seedlings and can be used for productive seedling culture.
The technical scheme adopted by the invention for realizing the purpose is as follows:
a method for separating and culturing the protoplast of seaweed and regenerating plant includes such steps as utilizing the tool enzyme of seaweed to decompose the tissue of seaweed and to dissociate the protoplast of seaweed to obtain the somatic cell and cell line of regenerated cell wall, and culturing to obtain regenerated plant. The method comprises the following specific steps:
firstly, selecting algae with normal color, no rot and vigorous growth, cleaning sludge, miscellaneous algae and other accessory impurities on the surface of the algae by using a hairbrush, and using 0.005-0.008% of HgCl2Disinfecting the solution for 5-10s, then washing the solution with disinfected seawater, cutting tender top parts of fresh alga bodies, sucking surface water with filter paper, shearing or grinding the surface water into fine tissue blocks or small branches, soaking the small tissue blocks or small branches in a mixed solution of potassium sodium tartrate and polyvinylpyrrolidone (PVP) for 2-3h, then carrying out digestion and enzymolysis by using a seaweed tool enzyme solution, and then carrying out centrifugal precipitation to obtain alga body cell protoplasm;
and step two, culturing the protoplast to obtain cells with regenerated cell walls, and further culturing to obtain regenerated plants.
Preferably, the potassium sodium tartrate and the PVP are respectively prepared into solutions with the concentration of 0.1-0.5M and 3-6M, and then the solutions are mixed according to the volume ratio of 1: 3-5; the sodium potassium tartrate and the PVP are mutually cooperated, so that cellulose on the surface of a cell wall can be expanded, the cell wall is accelerated to be dissolved or broken to cause cell plasmolysis, meanwhile, the toughness of a cell membrane can be improved, the enzymolysis efficiency of an alga body can be improved, and the integrity of an obtained protoplast can be ensured.
Preferably, the protoplast isolation is performed by: filtering the cell mixed solution by using a 250-400-mesh bolting silk to remove undigested cells, cell clusters and fragments; centrifuging the supernatant at 500-.
Preferably, the seaweed tool enzyme is a conch enzyme or a cellulase or a combination of both; more preferably, the conch enzyme is prepared by the following steps: cleaning and temporarily culturing conch in sterilized seawater for hungry for 2-3 days, shelling and taking digestive gland, grinding to homogenate, centrifuging at 10000-; when the raw enzyme solution is used, the raw enzyme solution is diluted to 15 to 25 percent by using seawater; the preparation method of the cellulase comprises the following steps: dissolving with 40-60mmol/L citric acid or acetic acid buffer solution (pH4.5-5.0) with concentration of 10-15%, wherein the acetic acid buffer solution is prepared from sterilized seawater.
Preferably, when the seaweed tool enzyme is used, an osmotic pressure stabilizer is added, and the osmotic pressure stabilizer is selected from one of mannitol, sorbitol, glucose, sucrose, KCl and NaCl; wherein, the suitable concentration of the alcohol and the saccharide is 1.2-2.6M, and the suitable concentration of the KCl and the NaCl is 0.5-0.8M.
Preferably, the conditions of enzymolysis are as follows: the pH value is 5.5-7.5, and the enzymolysis is carried out for 2-4.5h at 15-40 ℃.
Preferably, the protoplast culture method comprises: mixing and dripping equal amount of sodium alginate and protoplast in calcium chloride solution to form fixation, and inoculating to liquid culture medium for culture; dark culture is carried out for 5-7 days at the temperature of 22-26 ℃, then illumination culture is carried out, the illumination time is 8-10h/d, the light intensity is 2500-; wherein the liquid culture medium is PES culture medium containing naphthylacetic acid and 2, 4-chlorophenoxyacetic acid, and mannitol and glucose are used for maintaining osmotic pressure of the culture medium; the calcium chloride and alginate radical ions are chelated to form water-insoluble calcium alginate gel, cells are fixed, the protoplast can be effectively prevented from being adhered, the fixed-point positioning observation can be carried out on the protoplast, the development process of the protoplast can be tracked, and the cell division speed can be improved.
Preferably, the protoplast culture process comprises adding tryptophan and sodium ascorbate into the culture medium after the cells are divided for the first time, adjusting the pH of the culture medium to 5.5-5.8, and continuing to culture; the addition amount of tryptophan and sodium ascorbate is 1-5% and 0.5-2% of the weight of the culture medium; the special existence of tryptophan and sodium ascorbate is beneficial to improving the division frequency of protoplasts, accelerating the cell division to form filaments, fronds or calluses, and further differentiating to generate plants; secondly, the oxidation of phenolic substances and the like possibly generated in the process of culturing the protoplast can be effectively prevented, the stability of active substances in dividing cells is kept, the activity of the cells is improved, the differentiation and development of the protoplast are promoted, the seedling culture period is shortened, and meanwhile, the higher seedling breeding efficiency is kept.
The invention has the beneficial effects that:
1) the invention uses seaweed tool enzyme to carry out pretreatment on the alga before carrying out digestion and enzymolysis on the alga: soaking the algae in mixed solution of potassium sodium tartrate and PVP with the concentration of 0.1-0.5M and 3-6M respectively; the sodium potassium tartrate and the PVP are mutually cooperated, so that cellulose on the surface of a cell wall can be expanded, the cell wall is accelerated to be dissolved or broken to cause cell plasmolysis, meanwhile, the toughness of a cell membrane can be improved, the enzymolysis efficiency of an alga body can be improved, and the integrity of an obtained protoplast can be ensured;
2) the culture mode of the protoplast adopts the following steps: mixing and dripping equal amount of sodium alginate and protoplast in calcium chloride solution to form fixation, and inoculating to liquid culture medium for culture; the calcium chloride and alginate radical ions are chelated to form water-insoluble calcium alginate gel, cells are fixed, the protoplast can be effectively prevented from being adhered, the fixed-point positioning observation can be carried out on the protoplast, the development process of the protoplast can be tracked, and the cell division speed can be improved;
3) in the process of culturing the protoplast, when the cells are divided for the first time, tryptophan and sodium ascorbate are added into a culture medium, and the special existence of the tryptophan and the sodium ascorbate is beneficial to improving the division frequency of the protoplast and accelerating the division of the cells to form a filament, a thallus or a callus so as to differentiate to generate a plant; secondly, the oxidation of phenolic substances and the like possibly generated in the process of culturing the protoplast can be effectively prevented, the stability of active substances in dividing cells is kept, the activity of the cells is improved, the differentiation and development of the protoplast are promoted, the seedling culture period is shortened, and meanwhile, the higher seedling breeding efficiency is kept.
The invention adopts the technical scheme to provide the model essay, makes up the defects of the prior art, and has reasonable design and convenient operation.
Detailed Description
The present invention is further described in detail with reference to the following examples:
example 1:
the seaweed is Eucheuma Gelatinosum.
A method for separating and culturing the protoplast of seaweed and regenerating plant includes such steps as utilizing the tool enzyme of seaweed to decompose the tissue of seaweed and to dissociate the protoplast of seaweed to obtain the somatic cell and cell line of regenerated cell wall, and culturing to obtain regenerated plant. The method comprises the following specific steps:
(1) selecting algae with normal color, no rot and vigorous growth, cleaning sludge, miscellaneous algae and other auxiliary impurities on the surface of the algae with a brush, and adding 0.005% of HgCl2Disinfecting the solution for 5s, then washing the solution by using disinfected seawater, cutting tender parts at the top ends of fresh alga bodies, sucking surface water by using filter paper, shearing or grinding the surface water into fine tissue blocks or small branches, soaking the small tissue blocks or the small branches in a mixed solution of sodium potassium tartrate and PVP for 2h, then carrying out digestion and enzymolysis by using a seaweed tool enzyme solution, and then carrying out centrifugal precipitation to obtain alga body cell protoplasm;
wherein, the potassium sodium tartrate and the PVP are respectively prepared into solutions with the concentration of 0.1M and 3M and then mixed according to the volume ratio of 1: 3; the sodium potassium tartrate and the PVP are mutually cooperated, so that cellulose on the surface of a cell wall can be expanded, the cell wall is accelerated to be dissolved or broken to cause cell plasmolysis, meanwhile, the toughness of a cell membrane can be improved, the enzymolysis efficiency of an alga body can be improved, and the integrity of an obtained protoplast can be ensured;
the seaweed tool enzyme is conch enzyme, and the preparation method of the conch enzyme comprises the following steps: cleaning conch, temporarily culturing in sterilized seawater, starving for 2 days, removing shell, collecting digestive gland, grinding to homogenate, centrifuging at 10000rpm for 10min, discarding precipitate, adding acetone pre-cooled at-10 deg.C into supernatant, centrifuging, discarding precipitate, repeatedly adding acetone, centrifuging, collecting precipitate to obtain enzyme solution, and storing at-5 deg.C; when the raw enzyme solution is used, the raw enzyme solution is diluted to 15 percent by using seawater;
the enzymolysis conditions are as follows: 15ml of enzymolysis solution is adopted for every gram of algae; adding 2.2M sorbitol into the enzymolysis liquid as an osmotic pressure stabilizer; keeping the pH of the enzymolysis liquid at 6, and carrying out enzymolysis at 30 ℃ for 3.5 h;
the protoplast separation operation is as follows: filtering the cell mixture with 250 mesh silk to remove undigested cells, cell clusters and fragments; centrifuging the supernatant at 500rpm for 10min to allow single cell (protoplast) to sink, leaving cell debris in the supernatant, discarding the supernatant, washing with 1.0M glucose-containing sterilized seawater and centrifuging for 3 times, and collecting precipitate to obtain seaweed protoplast;
(2) culturing the protoplast to obtain cells with regenerated cell walls, and further culturing to obtain regenerated plants;
the protoplast culture mode is as follows: mixing sodium alginate and protoplast in equal amount, dropping into calcium chloride solution to form immobilization, inoculating into liquid culture medium, and culturing to obtain protoplast with initial plate density of 1 × 104Per mL; dark culture is carried out for 5 days at the temperature of 22 ℃, then illumination culture is carried out, the illumination time is 8h/d, the light intensity is 2500Lx, and half of the culture medium is replaced every 7 days; wherein the liquid culture medium is PES culture medium containing naphthylacetic acid and 2, 4-chlorophenoxyacetic acid, the concentrations of naphthylacetic acid and 2, 4-chlorophenoxyacetic acid are respectively 0.03mg/L and 1 mg/L10 days before culture, then the concentrations of the naphthylacetic acid and the 2, 4-chlorophenoxyacetic acid are adjusted to be 1mg/L and 0.1mg/L, and the osmotic pressure of the culture medium is maintained by 1.5M mannitol and 1.2M glucose; the calcium chloride and alginate radical ions are chelated to form water-insoluble calcium alginate gel, cells are fixed, the protoplast can be effectively prevented from being adhered, the fixed-point positioning observation can be carried out on the protoplast, the development process of the protoplast can be tracked, and the cell division speed can be improved;
a protoplast culture process, wherein after the cells are divided for the first time, tryptophan and sodium ascorbate are added into a culture medium, the pH value of the culture medium is adjusted to 5.5, and the culture is continued; the addition amount of tryptophan and sodium ascorbate is 1% and 0.5% of the weight of the culture medium; the special existence of tryptophan and sodium ascorbate is beneficial to improving the division frequency of protoplasts, accelerating the cell division to form filaments, fronds or calluses, and further differentiating to generate plants; secondly, the oxidation of phenolic substances and the like possibly generated in the process of culturing the protoplast can be effectively prevented, the stability of active substances in dividing cells is kept, the activity of the cells is improved, the differentiation and development of the protoplast are promoted, the seedling culture period is shortened, and meanwhile, the higher seedling breeding efficiency is kept.
Example 2:
the seaweed species is Solieria pellis.
A method for separating and culturing the protoplast of seaweed and regenerating plant includes such steps as utilizing the tool enzyme of seaweed to decompose the tissue of seaweed and to dissociate the protoplast of seaweed to obtain the somatic cell and cell line of regenerated cell wall, and culturing to obtain regenerated plant. The method comprises the following specific steps:
(1) selecting algae with normal color, no rot and vigorous growth, cleaning sludge, miscellaneous algae and other auxiliary impurities on the surface of the algae with a brush, and cleaning with 0.006% of HgCl2Disinfecting the solution for 6s, then washing the solution by using disinfected seawater, cutting tender parts at the top ends of fresh alga bodies, sucking surface water by using filter paper, shearing or grinding the surface water into fine tissue blocks or small branches, soaking the small tissue blocks or the small branches in a mixed solution of sodium potassium tartrate and PVP for 2h, then carrying out digestion and enzymolysis by using a seaweed tool enzyme solution, and then carrying out centrifugal precipitation to obtain alga body cell protoplasm;
wherein, the potassium sodium tartrate and the PVP are respectively prepared into solutions with the concentration of 0.3M and 4.5M and then are mixed according to the volume ratio of 1: 4;
the seaweed tool enzyme is prepared by mixing the same amount of conch enzyme and cellulase, and the preparation method of the conch enzyme comprises the following steps: cleaning conch, temporarily culturing in sterilized seawater, starving for 2 days, removing shell, collecting digestive gland, grinding to homogenate, centrifuging at 12000rpm for 6min, discarding precipitate, adding acetone pre-cooled at-15 deg.C into supernatant, centrifuging, discarding precipitate, repeatedly adding acetone, centrifuging, collecting precipitate to obtain enzyme solution, and storing at-10 deg.C; when the raw enzyme solution is used, the raw enzyme solution is diluted to 20% by using seawater; the preparation method of the cellulase comprises the following steps: dissolving with 50mmol/L citric acid or acetic acid buffer solution (pH of 4.8) at concentration of 12%, wherein the acetic acid buffer solution is prepared from sterilized seawater;
the enzymolysis conditions are as follows: 15ml of enzymolysis solution is adopted for every gram of algae; adding 0.7M NaCl serving as an osmotic pressure stabilizer into the enzymolysis liquid; keeping the pH of the enzymolysis liquid at 6, and carrying out enzymolysis at 30 ℃ for 3.5 h;
the protoplast separation operation is as follows: filtering the cell mixed solution by using 300-mesh bolting silk to remove undigested cells, cell clusters and fragments; centrifuging the supernatant at 700rpm for 9min to allow single cells (protoplasts) to sink, leaving cell debris in the supernatant, discarding the supernatant, washing with 1.0M glucose-containing sterilized seawater and centrifuging for 3 times, and collecting precipitate to obtain seaweed protoplasts;
(2) culturing the protoplast to obtain cells with regenerated cell walls, and further culturing to obtain regenerated plants;
the protoplast culture mode is as follows: mixing sodium alginate and protoplast in equal amount, dropping into calcium chloride solution to form immobilization, inoculating into liquid culture medium, and culturing to obtain protoplast with initial plate density of 5 × 105Per mL; dark culture is carried out for 6 days at the temperature of 24 ℃, then illumination culture is carried out, the illumination time is 10h/d, the light intensity is 2600Lx, and half of the culture medium is replaced every 7 days; wherein the liquid culture medium is PES culture medium containing naphthylacetic acid and 2, 4-chlorophenoxyacetic acid, the concentrations of the naphthylacetic acid and the 2, 4-chlorophenoxyacetic acid are respectively 0.03mg/L and 1 mg/L15 days before the culture, then the concentrations of the naphthylacetic acid and the 2, 4-chlorophenoxyacetic acid are adjusted to be 1mg/L and 0.1mg/L, and the osmotic pressure of the culture medium is maintained by 1.5M mannitol and 1.2M glucose;
a protoplast culture process, wherein after the cells are divided for the first time, tryptophan and sodium ascorbate are added into a culture medium, the pH value of the culture medium is adjusted to 5.6, and the culture is continued; tryptophan and sodium ascorbate were added in amounts of 3.2% and 1.1% by weight of the medium.
Example 3:
the seaweed is selected from thallus Porphyrae.
A method for separating and culturing the protoplast of seaweed and regenerating plant includes such steps as utilizing the tool enzyme of seaweed to decompose the tissue of seaweed and to dissociate the protoplast of seaweed to obtain the somatic cell and cell line of regenerated cell wall, and culturing to obtain regenerated plant. The method comprises the following specific steps:
(1) selecting algae with normal color, no rot and vigorous growth, cleaning sludge, miscellaneous algae and other auxiliary impurities on the surface of the algae with a brush, and cleaning with 0.008% of HgCl2Disinfecting the solution for 10s, then washing the solution by using disinfected seawater, cutting tender parts at the top ends of fresh alga bodies, sucking surface water by using filter paper, shearing or grinding the surface water into fine tissue blocks or small branches, soaking the small tissue blocks or the small branches in a mixed solution of sodium potassium tartrate and PVP for 3h, then carrying out digestion and enzymolysis by using a seaweed tool enzyme solution, and then carrying out centrifugal precipitation to obtain alga body cell protoplasm;
wherein, the potassium sodium tartrate and the PVP are respectively prepared into solutions with the concentration of 0.5M and 6M and then are mixed according to the volume ratio of 1: 5;
the seaweed tool enzyme is cellulase, and the preparation method of the cellulase comprises the following steps: dissolving with 60mmol/L citric acid or acetic acid buffer solution (pH 5.0) with concentration of 15%, wherein the acetic acid buffer solution is prepared from sterilized seawater;
the enzymolysis conditions are as follows: 15ml of enzymolysis solution is adopted for every gram of algae; adding 0.7M NaCl serving as an osmotic pressure stabilizer into the enzymolysis liquid; keeping the pH of the enzymolysis liquid at 6, and carrying out enzymolysis at 30 ℃ for 3.5 h;
the protoplast separation operation is as follows: filtering the cell mixed solution by using 400-mesh bolting silk to remove undigested cells, cell clusters and fragments; centrifuging the supernatant at 850rpm for 10min to allow single cell (protoplast) to sink, leaving cell debris in the supernatant, discarding the supernatant, washing with 1.2M glucose-containing sterilized seawater and centrifuging for 3 times, and collecting precipitate to obtain seaweed protoplast;
(2) culturing the protoplast to obtain cells with regenerated cell walls, and further culturing to obtain regenerated plants;
the protoplast culture mode is as follows: mixing sodium alginate and protoplast in equal amount, dropping into calcium chloride solution to form immobilization, inoculating into liquid culture medium, and culturing to obtain protoplast with initial plate density of 1 × 106Per mL; dark culture is carried out for 7 days at the temperature of 26 ℃,then, the culture is changed into illumination culture, the illumination time is 10h/d, the light intensity is 3000Lx, and half of the culture medium is replaced every 7 days; wherein the liquid culture medium is PES culture medium containing naphthylacetic acid and 2, 4-chlorophenoxyacetic acid, the concentrations of the naphthylacetic acid and the 2, 4-chlorophenoxyacetic acid are respectively 0.03mg/L and 1 mg/L15 days before the culture, then the concentrations of the naphthylacetic acid and the 2, 4-chlorophenoxyacetic acid are adjusted to be 1mg/L and 0.1mg/L, and the osmotic pressure of the culture medium is maintained by 1.5M mannitol and 1.2M glucose;
a protoplast culture process, wherein after the cells are divided for the first time, tryptophan and sodium ascorbate are added into a culture medium, the pH value of the culture medium is adjusted to 5.8, and the culture is continued; tryptophan and sodium ascorbate were added in amounts of 5% and 2% by weight of the medium.
Comparative example 1:
no pretreatment is carried out before the algae tissue is subjected to enzymolysis (namely, the algae tissue is not soaked in a mixed solution of sodium potassium tartrate and PVP); the remaining procedure was as in example 2.
Comparative example 2:
in the protoplast culture process, tryptophan and sodium ascorbate are not added into a culture medium; the remaining procedure was as in example 2.
Example 4:
identification of protoplasts:
identifying cells obtained by enzymolysis by adopting a fluorescent brightener dyeing method, preparing a VBL type fluorescent brightener into 0.1% dyeing solution by adopting the fluorescent dyeing method, dyeing the obtained sample for 5min, and observing the sample under a fluorescent microscope, wherein the excitation light wavelength is 370nm ultraviolet light; the mural cells have green fluorescence indicating that cellulose exists, and the protoplasts have no green fluorescence, are red fluorescence due to the existence of chloroplasts and indicate that no cell wall exists;
the density of the protoplast is measured by counting with a blood counting chamber; protoplast viability was determined using: putting the protoplast into a lower osmotic solution, wherein the volume of the protoplast can swell, and the volume of the protoplast can shrink when the protoplast is put into a higher osmotic solution, so that the protoplast is a living cell, and the protoplast is a dead cell when the volume of the protoplast is not changed;
counting the number of protoplasts in the enzymolysis process (enzymolysis for 3h) of the embodiment or the comparative example to obtain density data and survival rate data of the protoplasts, which are shown in table 1;
as can be seen from Table 1, the algal tissues were pretreated before enzymatic hydrolysis: the mixed solution of sodium potassium tartrate and PVP is adopted for soaking, so that the cell plasmolysis efficiency is improved, the separation density of the protoplast is increased in the same enzymolysis time, the strength and toughness of cell membranes can be improved, the integrity of the protoplast is maintained, and the survival rate of the protoplast is improved.
TABLE 1 isolation Density and survival data for protoplasts of the invention
Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.
The above embodiments are merely illustrative, and not restrictive, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the protection scope of the present invention should be defined by the claims.
Claims (8)
1. The method for separating and culturing the seaweed protoplast and regenerating the plant is characterized in that: decomposing seaweed tissues by using seaweed tool enzyme to obtain somatic cell protoplast, and culturing to obtain a regeneration plant;
before the seaweed tool enzyme carries out enzymolysis on seaweed tissues, soaking seaweed bodies in a mixed solution of sodium potassium tartrate and polyvinylpyrrolidone;
the concentrations of the potassium sodium tartrate and the polyvinylpyrrolidone are respectively 0.1-0.5M and 3-6M, and the potassium sodium tartrate and the polyvinylpyrrolidone are mixed according to the volume ratio of 1: 3-5;
the seaweed is of Rhodophyta.
2. The method for algal protoplast isolation culture and regeneration of a plant as claimed in claim 1, wherein: the method comprises the following steps:
firstly, selecting algae with normal color, no rot and vigorous growth, cleaning sludge, miscellaneous algae and other accessory impurities on the surface of the algae by using a hairbrush, and using 0.005-0.008% of HgCl2Disinfecting the solution for 5-10s, then washing the solution with disinfected seawater, cutting tender top parts of fresh alga bodies, sucking surface water with filter paper, shearing or grinding the surface water into fine tissue blocks or small branches, soaking the small tissue blocks or small branches in a mixed solution of potassium sodium tartrate and polyvinylpyrrolidone for 2-3h, then carrying out digestion and enzymolysis by using a seaweed tool enzyme solution, and then carrying out centrifugal precipitation to obtain alga body cell protoplasm;
and step two, culturing the protoplast to obtain cells with regenerated cell walls, and further culturing to obtain regenerated plants.
3. The method for algal protoplast isolation culture and regeneration of a plant as claimed in claim 1, wherein: the protoplast separation operation comprises the following steps: filtering the cell mixed solution by using a 250-400-mesh bolting silk to remove undigested cells, cell clusters and fragments; centrifuging the supernatant at 500-850rpm for 8-10min to allow the protoplast to sink, leaving cell debris in the supernatant, discarding the supernatant, washing and centrifuging with sterilized seawater containing 1.0-1.2M glucose for 2-3 times, and collecting the precipitate to obtain the algal protoplast.
4. The method for algal protoplast isolation culture and regeneration of a plant as claimed in claim 1, wherein: the seaweed tool enzyme is conch enzyme or cellulase or the combination of the two; the preparation method of the conch enzyme comprises the following steps: cleaning and temporarily culturing conch in sterilized seawater for hungry for 2-3 days, shelling and taking digestive gland, grinding to homogenate, centrifuging at 10000-; when the raw enzyme solution is used, the raw enzyme solution is diluted to 15 to 25 percent by using seawater; the preparation method of the cellulase comprises the following steps: dissolving with 40-60mmol/L citric acid or acetic acid buffer solution with concentration of 10-15%, wherein the acetic acid buffer solution is prepared from sterilized seawater.
5. The method for algal protoplast isolation culture and regeneration of a plant as claimed in claim 1, wherein: when the seaweed tool enzyme is used, an osmotic pressure stabilizer is added, and the osmotic pressure stabilizer is selected from one of mannitol, sorbitol, glucose, sucrose, KCl and NaCl; wherein, the suitable concentration of the alcohol and the saccharide is 1.2-2.6M, and the suitable concentration of the KCl and the NaCl is 0.5-0.8M.
6. The method for algal protoplast isolation culture and regeneration of a plant as claimed in claim 1, wherein: the enzymolysis conditions are as follows: the pH value is 6, and the enzymolysis is carried out for 3.5h at the temperature of 30 ℃.
7. The method for algal protoplast isolation culture and regeneration of a plant as claimed in claim 1, wherein: the protoplast culture mode is as follows: mixing and dripping equal amount of sodium alginate and protoplast in calcium chloride solution to form fixation, and inoculating to liquid culture medium for culture; dark culture is carried out for 5-7 days at the temperature of 22-26 ℃, then illumination culture is carried out, the illumination time is 8-10h/d, the light intensity is 2500-; wherein the liquid culture medium is PES culture medium containing naphthylacetic acid and 2, 4-chlorophenoxyacetic acid, and mannitol and glucose are used for maintaining osmotic pressure of the culture medium.
8. The method for algal protoplast isolation culture and regeneration of a plant as claimed in claim 1, wherein: in the protoplast culture process, after the cells are divided for the first time, tryptophan and sodium ascorbate are added into a culture medium, the pH value of the culture medium is adjusted to be 5.5-5.8, and the culture is continued; the addition amount of tryptophan and sodium ascorbate is 1-5% and 0.5-2% of the culture medium weight.
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