CN114934007B - Preparation method of sweet potato protoplast - Google Patents
Preparation method of sweet potato protoplast Download PDFInfo
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- CN114934007B CN114934007B CN202210773463.6A CN202210773463A CN114934007B CN 114934007 B CN114934007 B CN 114934007B CN 202210773463 A CN202210773463 A CN 202210773463A CN 114934007 B CN114934007 B CN 114934007B
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- 244000017020 Ipomoea batatas Species 0.000 title claims abstract description 62
- 235000002678 Ipomoea batatas Nutrition 0.000 title claims abstract description 62
- 210000001938 protoplast Anatomy 0.000 title claims abstract description 56
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 239000000463 material Substances 0.000 claims abstract description 46
- 239000007788 liquid Substances 0.000 claims abstract description 31
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims abstract description 27
- 229930195725 Mannitol Natural products 0.000 claims abstract description 25
- 239000000594 mannitol Substances 0.000 claims abstract description 25
- 235000010355 mannitol Nutrition 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 19
- 229940059442 hemicellulase Drugs 0.000 claims abstract description 18
- 108010002430 hemicellulase Proteins 0.000 claims abstract description 18
- 108010059892 Cellulase Proteins 0.000 claims abstract description 15
- 229940106157 cellulase Drugs 0.000 claims abstract description 15
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 claims abstract description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 6
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 6
- 238000002156 mixing Methods 0.000 claims abstract description 4
- 238000002791 soaking Methods 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 25
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 12
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 11
- 230000002255 enzymatic effect Effects 0.000 claims description 10
- 239000000413 hydrolysate Substances 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 9
- 206010020649 Hyperkeratosis Diseases 0.000 claims description 8
- 239000011534 wash buffer Substances 0.000 claims description 8
- 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
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 239000001103 potassium chloride Substances 0.000 claims description 6
- 235000011164 potassium chloride Nutrition 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 5
- 239000000706 filtrate Substances 0.000 claims description 5
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 4
- 239000002244 precipitate Substances 0.000 claims description 3
- 230000000408 embryogenic effect Effects 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 14
- 241000196324 Embryophyta Species 0.000 abstract description 6
- 210000004027 cell Anatomy 0.000 abstract description 6
- 108090000790 Enzymes Proteins 0.000 description 11
- 102000004190 Enzymes Human genes 0.000 description 11
- 229940088598 enzyme Drugs 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 8
- 210000002421 cell wall Anatomy 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 230000007910 cell fusion Effects 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 238000009396 hybridization Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000003381 stabilizer Substances 0.000 description 3
- 235000020138 yakult Nutrition 0.000 description 3
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000003204 osmotic effect Effects 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 241000228245 Aspergillus niger Species 0.000 description 1
- 241000207782 Convolvulaceae Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000237858 Gastropoda Species 0.000 description 1
- 206010034972 Photosensitivity reaction Diseases 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000036211 photosensitivity Effects 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 230000004960 subcellular localization Effects 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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Abstract
The invention provides a preparation method of a sweet potato protoplast, belonging to the technical field of plant cell biology. Comprising the following steps: soaking sweet potato material in mannitol solution for dark culture; obtaining a dark culture material; mixing the obtained dark culture material with enzymolysis liquid, and performing enzymolysis to obtain protoplast; the enzymolysis liquid comprises the following components: cellulase RS, educase R10, hemicellulase, bovine serum albumin, morpholinoethanesulfonic acid, and the like. The method provided by the invention can be used for obtaining the sweet potato protoplast with high yield and strong activity.
Description
Technical Field
The invention belongs to the technical field of plant cell biology, and particularly relates to a preparation method of a sweet potato protoplast.
Background
Sweet potato (Ipomoea batatas [ L. ] lam.), annual herbaceous plant, convolvulaceae, sweet potato is a high-yield and highly adaptable food crop. The root tuber is not only used as staple food, but also an important raw material for food processing and starch and alcohol manufacturing industries, and the root, stem and leaf are excellent feeds. Sweet potato originates in tropical america, is an important grain, feed, energy and industrial raw crop in the world, and China is the largest sweet potato production and consumption country in the world.
Plant protoplast is a living cell only wrapped by a plasma membrane, is often used as an ideal material for research such as cell fusion, somatic cell hybridization, genetic transformation, subcellular localization and the like because of no cell wall, and has important application value in many physiological and biochemical researches and theoretical researches on plant cell totipotency. Sweet potato has self-copulation and same group hybridization incompatibility, and cell fusion can overcome the problem of incompatibility among remote cells, so as to realize hybridization and gene recombination. Up to now, a complete set of sweet potato protoplast preparation method is still lacking.
Disclosure of Invention
Therefore, the invention aims to provide a preparation method of the sweet potato protoplast, and the sweet potato protoplast with high yield and strong activity can be obtained by adopting the method provided by the invention.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of a sweet potato protoplast, which comprises the following steps:
1) Soaking sweet potato material in mannitol solution for dark culture; obtaining a dark culture material;
2) Mixing the dark culture material obtained in the step 1) with an enzymolysis solution, and carrying out enzymolysis to obtain protoplasts;
the enzymolysis liquid takes water as a solvent and comprises the following components: 1.0 to 3.0 percent of cellulase RS, 1.5 percent of eduction enzyme R10, 0.5 to 1.5 percent of hemicellulase, 0.1 percent of bovine serum albumin, 80mM of morpholinoethanesulfonic acid, 0.4 to 0.6M of mannitol, 100mM of potassium chloride, 20mM of calcium chloride, 20mM of magnesium chloride and pH value of 5.5.
Preferably, the enzymolysis liquid in the step 2) further comprises snailase with the mass percentage of 1 percent.
Preferably, the sweet potato material of step 1) comprises one or more of root tip, leaf blade, leaf stalk, shoot tip meristem and callus of sweet potato.
Preferably, the length and width of the sweet potato material are respectively 0.5-1 mm.
Preferably, the molar concentration of the mannitol solution in the step 1) is 0.6M, and the dark culture time is 30min.
Preferably, the volume ratio of the mass of the dark culture material to the enzymolysis liquid in the step 2) is 1g to 10ml.
Preferably, the enzymolysis conditions in the step 2) include: the temperature is 28 ℃, the oscillating speed is 80rpm, and the enzymolysis time is 2-4 hours.
Preferably, when the sweet potato material is a leaf, the enzymolysis time is 3 hours;
when the sweet potato material is a petiole, the enzymolysis time is 2 hours;
when the sweet potato material is a stem tip meristem, the enzymolysis time is 4 hours;
when the sweet potato material is callus, the enzymolysis time is 2h;
when the sweet potato material is root tip, the enzymolysis time is 3.5h.
Preferably, after the enzymolysis in the step 2), the method further includes: and filtering the zymolyte, centrifuging the obtained filtrate, and washing the obtained precipitate to obtain protoplast.
Preferably, the centrifugation conditions include: the centrifugal force is 250g, and the centrifugal time is 10min;
the washing buffer used for the washing was a mannitol solution having a molar concentration of 0.4M.
According to the invention, the composition and concentration of the enzymolysis liquid are continuously adjusted, and the optimal enzymolysis liquid formula is selected, so that the protoplast yield is improved, and the reduction of the activity of the protoplast caused by the overhigh enzyme content is avoided as much as possible. The optimized enzymolysis time length can avoid the phenomenon that protoplast is broken and imagined caused by overlong enzymolysis time of the protoplast in enzymolysis liquid, thereby obtaining the protoplast with high yield and strong activity. In addition, protoplast with higher activity is obtained by selecting sweet potato experimental materials with good physiological state.
The beneficial effects of the invention are as follows:
1) The invention provides a set of complete sweet potato protoplast preparation technology through researches on the physiological state, enzymolysis liquid composition, enzymolysis time and the like of sweet potato materials.
2) The invention greatly shortens the time required by preparing the protoplast of the sweet potato and is beneficial to ensuring the activity of the protoplast;
3) The sweet potato protoplast obtained by the technology has high yield and strong activity, can be applied to protoplast culture, cell fusion, genetic transformation and other aspects, and has remarkable economic and social benefits.
Drawings
FIG. 1 is a sweetpotato root tip protoplast.
Detailed Description
The invention provides a preparation method of a sweet potato protoplast, which comprises the following steps:
1) Soaking sweet potato material in mannitol solution for dark culture; obtaining a dark culture material;
2) Mixing the dark culture material obtained in the step 1) with an enzymolysis solution, and carrying out enzymolysis to obtain protoplasts;
the enzymolysis liquid takes water as a solvent and comprises the following components: 1.0 to 3.0 percent of cellulase RS, 1.5 percent of eduction enzyme R10, 0.5 to 1.5 percent of hemicellulase, 0.1 percent of bovine serum albumin, 80mM of morpholinoethanesulfonic acid, 0.4 to 0.6M of mannitol, 100mM of potassium chloride, 20mM of calcium chloride, 20mM of magnesium chloride and pH value of 5.5.
The sweet potato material is soaked in mannitol solution for dark culture; dark culture material was obtained.
In the present invention, the sweet potato material is preferably fresh sweet potato material. In the present invention, the sweet potato material preferably includes one or more of root tip, leaf blade, leaf stalk, shoot tip meristem and callus of sweet potato. The method for obtaining the shoot tip meristem and the callus is not particularly limited, and the shoot tip meristem and the callus can be obtained by adopting a conventional culture method. In the present invention, the width and length of the sweet potato material are preferably 0.5 to 1mm, respectively. In the present invention, the root tip of the sweet potato is preferably the root tip of the hydroponic 5 d.
In the present invention, the molar concentration of the mannitol solution is preferably 0.6M, and the time of the dark culture is preferably 30min. In the invention, the mannitol solution has the function of separating the material from the wall, facilitating enzymolysis, and the function of the dark culture is to reduce the problem of low activity caused by the photosensitivity of the protoplast. In the present invention, the mannitol solution preferably has a pH of 5.5.
The invention mixes the obtained dark culture material with enzymolysis liquid for enzymolysis to obtain protoplast.
In the invention, the volume ratio of the mass of the dark culture material to the enzymolysis liquid is preferably 1g to 10ml. In the present invention, the conditions for the enzymolysis preferably include: the temperature is 28 ℃, the oscillating speed is 80rpm, and the enzymolysis time is 2-4 hours. In the invention, when the sweet potato material is a leaf, the enzymolysis time is preferably 3 hours; when the sweet potato material is a petiole, the enzymolysis time is preferably 2 hours; when the sweet potato material is a shoot tip meristem, the enzymolysis time is preferably 4 hours; when the sweet potato material is callus, the enzymolysis time is preferably 2h; when the sweet potato material is root tip, the enzymolysis time is preferably 3.5h.
In the invention, the enzymolysis liquid takes water as a solvent and comprises the following components: 1.0 to 3.0 percent of cellulase RS, 1.5 percent of eduction enzyme R10, 0.5 to 1.5 percent of hemicellulase, 0.1 percent of bovine serum albumin, 80mM of morpholinoethanesulfonic acid, 0.4 to 0.6M of mannitol, 100mM of potassium chloride, 20mM of calcium chloride, 20mM of magnesium chloride and pH value of 5.5. In the present invention, the CELLULASE RS acts to cleave the cell wall and is preferably derived from CELLULASE RS (Yakult, CAS No. 9012-54-8). In the present invention, the function of the eductase R10 is to separate plant tissue into individual cells, preferably from Macerozyme R-10 (Yakult, CAS No. 9032-75-1). In the present invention, the hemicellulase acts to hydrolyze the cell wall, and the source is preferably a hemicellulase derived from Aspergillus niger (Macklin, CAS No. 9025-56-3). In the present invention, the bovine serum albumin is an enzyme activity stabilizer. In the invention, the morpholinoethanesulfonic acid acts as a surfactant to enhance the enzymatic activity. In the present invention, the mannitol functions as a stabilizer for maintaining the osmotic pressure balance inside and outside the cell membrane. In the present invention, the potassium chloride, calcium chloride and magnesium chloride function to activate enzyme activity. The pH value of the enzymolysis liquid is preferably adjusted to 5.5 by using 0.1MTris HCl.
In the invention, the enzymolysis liquid also preferably comprises snailase with the mass percentage of 1 percent. In the present invention, the Snailase enzyme acts to hydrolyze the cell wall, and the source is preferably Snailase (Solarbio, CAS No. 9032-75-1).
After the enzymolysis, the obtained enzymolysis product preferably further comprises: and filtering the zymolyte, centrifuging the obtained filtrate, and washing the obtained precipitate to obtain protoplast.
In the present invention, the filtration uses a frontal filter membrane with a pore size of preferably 70. Mu.m.
In the present invention, the centrifugation conditions preferably include: the centrifugal force was 250g and the centrifugal time was 10min. In the present invention, the washing buffer used for the washing is a mannitol solution having a molar concentration of 0.4M, and the mannitol functions as a stabilizer for maintaining the osmotic pressure balance inside and outside the cell membrane. In the present invention, the number of times of washing is preferably 3, taking the volume ratio of the mass of the material to the enzymatic hydrolysate of 1g:10ml as an example, the operation preferably comprises: the first time of adding the same volume of washing buffer as the enzymolysis liquid, the second time of adding 5ml of washing buffer and the third time of adding 2ml of washing buffer.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
A preparation method of a sweet potato protoplast comprises the following steps:
(1) Cutting adventitious root tip of sweet potato of water culture 5d into small sections with width and length of about 1mm respectively with blade, and culturing in pretreatment liquid for 30min in dark condition. Wherein the pretreatment liquid comprises the following components: 0.6M mannitol solution, pH 5.5;
(2) Enzymolysis: taking out the root tip tissue in the step (1), placing the root tip tissue in a centrifuge tube, adding a proper amount of enzymolysis liquid (10 ml of enzymolysis liquid is added for every 1g of material), and then placing the mixture on a shaking table at 80rpm, and carrying out enzymolysis for 3.5 hours at 28 ℃ under dark conditions to obtain enzymolysis reaction liquid containing protoplasts.
TABLE 1 enzymolysis liquid Components
| Cellulase RS | 1.5％ |
| Educt enzyme R10 | 1.5％ |
| Hemicellulase(s) | 1.5％ |
| BSA | 0.1％ |
| MES | 80mM |
| Mannitol (mannitol) | 0.4M |
| Potassium chloride | 100mM |
| Calcium chloride | 20mM |
| Magnesium chloride | 20mM |
| 0.1MTrisHCl | Adjusting pH to 5.5 |
| The solvent of the enzymolysis liquid is deionized water |
Note that: is the mass percentage
(3) Washing and purifying: after the enzymolysis is finished, the enzymolysis reaction solution is filtered (the aperture of a filter membrane is 70 mu M), the filtrate is placed in a centrifuge tube, supernatant is discarded after centrifugation (250 Xg and 10 min), and washing buffer solution (0.4M mannitol solution) is used for 3 times to obtain protoplast heavy suspension.
Example 2
The same procedure as in example 1 was followed except that 1.0% snail enzyme (mass%) was added to the enzymatic hydrolysate.
Example 3
The same method as in example 2 is distinguished in that the material is sweet potato leaves and the enzymolysis time is 3 hours.
Example 4
The same method as in example 2 is distinguished in that the material is sweet potato petiole and the enzymolysis time is 2h.
Example 5
The same method as in example 2 is used, except that the material is the meristem of the sweet potato stem tip, and the enzymolysis time is 4 hours.
Example 6
The same method as in example 2 is used, except that the material is stem tip embryogenic callus of sweet potato, and the enzymolysis time is 4h.
Comparative example 1
The same method as in example 1 is distinguished in that 1.5% of the hemicellulase in the enzymatic hydrolysate is replaced by 1.0% of pectase, all in mass percent.
Comparative example 2
The same method as in example 1 is distinguished in that 1.5% of hemicellulase in the enzymatic hydrolysate is replaced by 1.0% of crashing enzyme, which is the mass percentage content.
Comparative example 3
The same method as in example 1 is distinguished in that 1.5% of hemicellulase in the enzymatic hydrolysate is replaced by 0.5% of hemicellulase, and the content is in mass percent.
Comparative example 4
The same method as in example 1 is distinguished in that 1.5% of hemicellulase in the enzymatic hydrolysate is replaced by 0.5% of hemicellulase, 1.5% of cellulase RS is replaced by 3.0% of cellulase RS, and the content is in percentage by mass.
Comparative example 5
The same method as in example 1 is distinguished in that 1.5% of hemicellulase in the enzymatic hydrolysate is replaced by 1.0% of hemicellulase, and the content is in mass percent.
Comparative example 6
The same method as in example 1 is distinguished in that 1.5% of hemicellulase in the enzymatic hydrolysate is replaced by 1.0% of hemicellulase, 1.5% of cellulase RS is replaced by 1.0% of cellulase RS, and the content is in percentage by mass.
Comparative example 7
A preparation method of a sweet potato protoplast comprises the following steps:
(1) Cutting sweet potato leaf stalks into small sections with the width and the length of about 1mm respectively by a blade, and culturing in pretreatment liquid for 30min under dark conditions. Wherein the pretreatment liquid comprises the following components: 0.6M mannitol solution, pH 5.5;
(2) Enzymolysis: taking out the petiole tissue in the step (1), placing the petiole tissue in a centrifuge tube, adding a proper amount of enzymolysis liquid (10 ml of enzymolysis liquid is added for every 1g of material), and then placing the petiole tissue on a shaking table at 80rpm, and carrying out enzymolysis for 16 hours at 28 ℃ in dark condition to obtain enzymolysis reaction liquid containing protoplast.
TABLE 2 enzymolysis liquid components (all are mass percent content)
| Cellulase R10 | 0.4％ |
| Educt enzyme R10 | 0.2％ |
| MES | 5mM |
| Mannitol (mannitol) | 0.6M |
| Calcium chloride | 0.5％ |
| 0.1MTrisHCl | Adjusting pH to 5.5 |
| The solvent of the enzymolysis liquid is deionized water |
Note that: is the mass percentage
Wherein the CELLULASE R10 is CELLULASE R10 (Yakult, CAS No. 9012-54-8).
(3) Washing and purifying: after the enzymolysis is finished, the enzymolysis reaction solution is filtered (the aperture of a filter membrane is 70 mu M), the filtrate is placed in a centrifuge tube, supernatant is discarded after centrifugation (250 Xg and 10 min), and washing buffer solution (0.4M mannitol solution) is used for 3 times to obtain protoplast heavy suspension.
The sweet potato protoplasts obtained by the sweet potato protoplast preparation methods in examples 1 to 6 and comparative examples 1 to 7 were collected, and the number of protoplasts in 25 middle squares in the central large square was counted with a hemocytometer and repeated 3 times. Protoplast yield was calculated according to the following formula:
protoplast yield (number/g) = (number of protoplasts in the central large square×10) 4 X total volume of suspension)/fresh mass of material used to separate protoplasts
The activity of the protoplast of the sweet potato was detected by trypan blue staining and repeated 3 times. And taking 20 mu L of purified protoplast suspension on a glass slide, adding 2 mu L of 0.4% trypan blue staining solution, standing at room temperature, incubating for 3min, and microscopic examination, wherein the undyed protoplast has activity and is blue inactive. Protoplast viability was counted using the following formula:
protoplast viability (%) = number of unstained protoplasts/total number of protoplasts x 100
The specific data are shown in Table 3.
The data in Table 3 show that the sweet potato protoplast prepared by the enzymolysis liquid formula in the last example 2 has high yield and strong activity and can be applied to different tissue parts of sweet potato by adjusting the content of enzymolysis liquid system and the like.
TABLE 3 yield and Activity of sweet potato protoplasts
Note that: the protoplast yield and the vitality values are all averaged
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (5)
1. The preparation method of the sweet potato protoplast is characterized by comprising the following steps:
1) Soaking stem tip embryogenic callus of sweet potato in mannitol solution for dark culture; obtaining a dark culture material; the molar concentration of the mannitol solution is 0.6M, and the dark culture time is 30min;
2) Mixing the dark culture material obtained in the step 1) with an enzymolysis solution, and carrying out enzymolysis to obtain protoplasts;
the enzymolysis liquid takes water as a solvent and comprises the following components: cellulase RS with the mass percentage of 1.5%, educase R10 with the mass percentage of 1.5%, hemicellulase with the mass percentage of 1.5%, snailase with the mass percentage of 1%, bovine serum albumin with the mass percentage of 0.1%, morpholinoethanesulfonic acid with the molar concentration of 80mM, mannitol with the molar concentration of 0.4M, potassium chloride with the molar concentration of 100mM, calcium chloride with the molar concentration of 20mM, magnesium chloride with the molar concentration of 20mM and the pH value of 5.5;
the enzymolysis conditions comprise: the temperature is 28 ℃, the oscillating speed is 80rpm, and the enzymolysis time is 4 hours.
2. The preparation method according to claim 1, wherein the sweet potato material has a length and a width of 0.5 to 1mm, respectively.
3. The method according to claim 1, wherein the ratio of the mass of the dark culture material to the volume of the enzymatic hydrolysate in step 2) is 1 g/10 ml.
4. The method according to claim 1, wherein after the enzymolysis in the step 2), the method further comprises: and filtering the zymolyte, centrifuging the obtained filtrate, and washing the obtained precipitate to obtain protoplast.
5. The method of claim 4, wherein the centrifugation conditions comprise: the centrifugal force is 250g, and the centrifugal time is 10min;
the washing buffer used for the washing was a mannitol solution having a molar concentration of 0.4M.
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| WO2011035422A1 (en) * | 2009-09-22 | 2011-03-31 | Medicago Inc. | Method of preparing plant-derived vlps |
| CN114058564A (en) * | 2021-10-27 | 2022-02-18 | 上海欧易生物医学科技有限公司 | Enzymolysis liquid suitable for carrying out enzymolysis on wormwood leaf tissue, preparation method of wormwood leaf tissue protoplast and application of wormwood leaf tissue protoplast |
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