CN115354013B - Tobacco protoplast preparation method - Google Patents
Tobacco protoplast preparation method Download PDFInfo
- Publication number
- CN115354013B CN115354013B CN202210986948.3A CN202210986948A CN115354013B CN 115354013 B CN115354013 B CN 115354013B CN 202210986948 A CN202210986948 A CN 202210986948A CN 115354013 B CN115354013 B CN 115354013B
- Authority
- CN
- China
- Prior art keywords
- tobacco
- protoplast
- root
- preparation
- root tip
- 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.)
- Active
Links
- 210000001938 protoplast Anatomy 0.000 title claims abstract description 84
- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 69
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 244000061176 Nicotiana tabacum Species 0.000 title 1
- 241000208125 Nicotiana Species 0.000 claims abstract description 68
- 239000007788 liquid Substances 0.000 claims abstract description 28
- 239000000463 material Substances 0.000 claims abstract description 24
- 230000000694 effects Effects 0.000 claims abstract description 21
- 108090000790 Enzymes Proteins 0.000 claims abstract description 20
- 102000004190 Enzymes Human genes 0.000 claims abstract description 20
- 241001002356 Valeriana edulis Species 0.000 claims abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 13
- 238000000605 extraction Methods 0.000 claims abstract description 9
- 210000001519 tissue Anatomy 0.000 claims description 25
- 229940088598 enzyme Drugs 0.000 claims description 19
- 108010059892 Cellulase Proteins 0.000 claims description 18
- 229940106157 cellulase Drugs 0.000 claims description 18
- 230000002255 enzymatic effect Effects 0.000 claims description 14
- 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 10
- 229930195725 Mannitol Natural products 0.000 claims description 10
- 239000000594 mannitol Substances 0.000 claims description 10
- 235000010355 mannitol Nutrition 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 10
- 239000007987 MES buffer Substances 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 238000002955 isolation Methods 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
- 238000005520 cutting process Methods 0.000 claims description 5
- 239000000706 filtrate Substances 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 108010029182 Pectin lyase Proteins 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 210000000805 cytoplasm Anatomy 0.000 claims description 3
- 230000010355 oscillation Effects 0.000 claims description 3
- 239000008223 sterile water Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims 1
- 239000011780 sodium chloride Substances 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 28
- 230000000762 glandular Effects 0.000 abstract description 14
- 238000011160 research Methods 0.000 abstract description 14
- 210000004209 hair Anatomy 0.000 abstract description 11
- 241000196324 Embryophyta Species 0.000 abstract description 10
- 238000000926 separation method Methods 0.000 abstract description 6
- 238000002474 experimental method Methods 0.000 abstract description 3
- 210000000056 organ Anatomy 0.000 abstract description 3
- 238000013461 design Methods 0.000 abstract description 2
- 238000002224 dissection Methods 0.000 abstract description 2
- 238000005457 optimization Methods 0.000 abstract description 2
- 230000024053 secondary metabolic process Effects 0.000 abstract description 2
- 238000007781 pre-processing Methods 0.000 abstract 1
- 239000000872 buffer Substances 0.000 description 7
- 239000000413 hydrolysate Substances 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 4
- 210000002768 hair cell Anatomy 0.000 description 4
- 210000004907 gland Anatomy 0.000 description 3
- 206010016654 Fibrosis Diseases 0.000 description 2
- 241000207746 Nicotiana benthamiana Species 0.000 description 2
- 108010059820 Polygalacturonase Proteins 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 108010093305 exopolygalacturonase Proteins 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 230000004761 fibrosis Effects 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 108090000856 Lyases Proteins 0.000 description 1
- 102000004317 Lyases Human genes 0.000 description 1
- 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 1
- 150000001273 acylsugars Chemical class 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000004163 cytometry Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229930004069 diterpene Natural products 0.000 description 1
- 125000000567 diterpene group Chemical group 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000010297 mechanical methods and process Methods 0.000 description 1
- 210000000473 mesophyll cell Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 239000001814 pectin Substances 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 210000002706 plastid Anatomy 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
- -1 terpenoid compounds Chemical class 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000002268 wool Anatomy 0.000 description 1
- 235000020138 yakult Nutrition 0.000 description 1
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
-
- 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
- C12N2509/10—Mechanical dissociation
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 application belongs to the technical field of tobacco tissue organ dissection, and particularly relates to a preparation method of tobacco protoplast. The method aims at the preparation and separation application of tobacco root cells or protoplast of tobacco leaf cells, and comprises the following steps: preparing enzymolysis liquid, preprocessing materials, extracting by enzymolysis, separating protoplast and the like. Based on the research summary of the existing plant cell protoplast extraction method, the inventor combines the structural characteristics of tobacco root tissues and tobacco cells, combines the research requirement of metabonomics generated by secondary metabolism, carries out further optimization design on the type and the dosage of biological enzyme required in the process of preparing protoplast by an enzymolysis method, and constructs the protoplast preparation method suitable for tobacco root tip sources or tobacco glandular hair sources. Preliminary experiment results show that the protoplast obtained by the preparation method has a large quantity and high activity, and meanwhile, the related extraction method is simple, quick and short in time consumption, so that the application has good scientific research practical value.
Description
Technical Field
The application belongs to the technical field of tobacco tissue organ dissection, and particularly relates to a preparation method of tobacco protoplast.
Background
Tobacco is used as a special cash crop, is highly relevant to national economy development, and is relatively simple in cultivation management due to relatively short growth cycle, so that the tobacco is a common mode plant in botanic research.
In the plant study, when the study is carried out on various metabolites or cell levels, the preparation and the acquisition of protoplasts are the basis of relevant experiments. Plant protoplasts refer to naked cells surrounded by only the plasma membrane after removal of the cell wall.
In the prior art, the commonly used protoplast preparation and separation methods comprise a mechanical method, a chemical method, an enzymolysis method and the like, wherein the enzymolysis method is a mainstream plant protoplast preparation method at present because of the advantages of safety, effectiveness, relative controllability, rapidity and the like. However, in objective terms, since the differences in the structural composition of different plant cells are large and the research purposes are different, there is no general preparation method for preparing plant protoplasts, and only the optimal protoplast isolation preparation method can be respectively explored according to the differences among different plants, different plant tissues and organs and different cells.
Disclosure of Invention
The application aims at providing a protoplast preparation and separation method suitable for research of tobacco secondary metabolites, thereby laying a certain technical foundation for research of tobacco cell metabonomics.
The technical scheme adopted by the application is described in detail below.
The preparation method of the tobacco protoplast is suitable for the protoplast preparation and separation application aiming at root cells or tobacco leaf (glandular hair) cells in the study of tobacco metabonomics, and specifically comprises the following steps:
firstly, preparing enzymolysis liquid
Taking tobacco root tips or tobacco leaves (glandular hairs) as a protoplast preparation material (raw material) source;
when aiming at tobacco root tip materials, the enzymolysis liquid prepared by the protoplasm is as follows: MES buffer containing cellulase celllast, pectolyase (or Macerozyme); the specific formula comprises the following components:
MES、5 mM + CaC1 2 10 mM +nacl, 10 mM +mannitol Manmitol (pH=5.7), 0.35M +1.5% cellulase celllast+1.0-1.5% pectase (or educase Macerozyme) +0.1% BSA;
when aiming at tobacco leaf (glandular wool) materials, the enzymolysis liquid prepared by the protoplasm is as follows: CPW buffer containing cellulase cellust and Macerozyme; the specific formula comprises the following components:
buffer CPW (pH 5.8) +mannitol Manmitol (pH 5.7), 0.4 M+1% cellulase celllast+0.5% educase Macerozyme;
the buffer CPW (pH 5.8): KH (KH) 2 PO 4 、27.2 mg/L + KNO 3 、101.0 mg/L + CaCl 2 •2H 2 O、 1480.0 mg/L + MgSO 4 •7H 2 O、246.0 mg/L + KI、0.16 mg/L + CuSO 4 •5H 2 O、0.025 mg/L;
The cellulase is, for example, cellulase Onozuka R-10 with the enzyme activity of 10000U/g;
the pectase is, for example, pectase Y-23 with the enzyme activity of 1000U/g;
the isolated enzyme macerozyme is, for example, macerozyme R-10 with an enzyme activity of 3000U/g;
(2) Pretreatment of materials
Taking sterile cultured tobacco or fresh tobacco (glandular hair) as a protoplast preparation material (raw material) source; the pretreatment comprises the following steps:
taking root tip tissue of tobacco which germinates and grows for 7-10 days aiming at root tissue, cleaning with sterile water (not less than 3 times), sucking water by filter paper, and cutting with a blade along the growth direction of the root tip to obtain 0.5cm;
selecting tobacco leaves which are fully stretched in 4-6 leaf periods after tobacco sprouting and growing according to tobacco leaf tissues, placing the tobacco leaves on ice, and rapidly cutting the tobacco leaves into filaments with the width of about 1mm for later use;
such as in particular K326, burley or yellow tobacco;
(3) Enzymatic extraction
Placing the root tips or tobacco filaments pretreated in the step (2) into a container, adding the corresponding enzymolysis liquid prepared in the step (1), and performing vibration treatment for 1-5 h at 26-30 ℃ (for example, 80rpm vibration treatment for 3 h);
the specific dosage aspect is as follows:
every 100 root tips, the consumption of the enzymolysis liquid is 5mL; the consumption of the enzymolysis liquid is 10mL for each fresh tobacco leaf;
(4) Protoplast isolation
After the vibration treatment in the step (3) is finished, the mixed solution is filtered by a 250-mesh cell sieve;
centrifuging the root tip filtrate, retaining the precipitate, and re-suspending the precipitate with MES buffer solution (without lyase) to obtain tobacco root tip protoplast extractive solution;
and centrifuging the filtrate of the tobacco leaves at 500rpm for 5-10 min, and reserving supernatant to obtain tobacco gland hair cell protoplast extract with higher activity and purity.
Based on the research summary of the existing plant cell protoplast extraction method, the inventor combines the structural characteristics of tobacco root tissues and tobacco leaf cells, combines the research requirement of secondary metabolism generation metabonomics (the root is the synthesis part of tobacco alkaloid, and the tobacco glandular hair is the main synthesis part of terpenes, especially diterpenes), carries out further optimization design on the type and the dosage of biological enzyme required in the protoplast preparation process by an enzymolysis method, and constructs the protoplast preparation method suitable for tobacco root tip sources or tobacco glandular hair sources.
After the related extraction process parameters are optimized, the preliminary experimental result shows that the number of the prepared protoplast is large, the activity is high, wherein the number of the protoplast in the tobacco root extract can reach 1.0x10 6 The number of the protoplast in the tobacco gland hair cell leaf extract per ml can reach 5.3 multiplied by 10 5 The activity of the protoplast is higher per ml>85%). Meanwhile, the related extraction method is simple, quick and short in time consumption, so that the application has good scientific research practical value, and a good technical foundation can be laid for related cytology research and metabonomics research.
Drawings
FIG. 1 shows root tip protoplasts obtained from combinations of different enzymes of K326;
FIG. 2 shows root tip protoplasts obtained with different enzyme concentrations of K326;
FIG. 3 shows root tip protoplasts obtained by treatment at different treatment times of K326;
FIG. 4 shows root tip protoplasts obtained after treatment of root tips of different growth times of K326;
FIG. 5 shows the results of activity assays of root protoplasts of different tobacco varieties; in the figure: a: benshiyan; b: k326; under the color view, the color is bright yellow: living cells; blue (blue-green): dead cells or cell debris;
FIG. 6 shows tobacco leaf (glandular hair) protoplasts obtained by treatment at different times of K326;
FIG. 7 shows tobacco leaf (glandular hair) protoplast cells in supernatant and pellet after 3 hours of K326 treatment;
FIG. 8 shows tobacco leaf (glandular hair) protoplasts under different mannitol treatment conditions for K326;
FIG. 9 shows tobacco leaf (glandular hair) protoplasts obtained after cleavage of K326 and yellow flower tobacco leaves.
Detailed Description
The present application is further illustrated below with reference to examples. Before describing the specific embodiments, the following description will briefly explain some experimental contexts in the following embodiments.
Experimental materials:
cellulase cellurast: cellulase Onozuka R-10 (enzyme activity 10000U/g), educt enzyme macerozyme: macerozyme R-10 (enzyme activity 3000U/g), pectase pectolyase: pectolylase Y-23 (enzyme activity 1000U/g), all of the products of Yakult Japan company;
tobacco material:
culturing tobacco K326 and yellow flower tobacco in a greenhouse, picking leaves which are fully stretched in 4-6 leaf periods, and preparing gland Mao Yuansheng plastids;
root tissue materials adopt root tissues which germinate (K326, nicotiana benthamiana) under aseptic conditions and grow for 7-10 days;
the experimental method comprises the following steps:
when the number of protoplasts is read by cytometry: firstly, taking 20 mu L of protoplast suspension, and adding 20 mu L of trypan blue for uniformly mixing; then 10. Mu.L of the suspension was added to a cell counting plate, allowed to stand for 1 minute, and counted by an insertion cell counter; cell concentration N (number/mL) was read and cell number was calculated:
cell number = N x cell volume (mL).
Example 1
In this example, tobacco root tissue is taken as an example, and the procedure for preparing the relevant protoplasts is briefly described below.
Firstly, preparing enzymolysis liquid
In combination with the summary of the prior art and the analysis of the tissue structure of the tobacco root (compared with the tissue of the tobacco flake, the degree of fibrosis of the tobacco root is high, and the cellulose content is high), the inventor considers that the enzymolysis of cellulase and pectase is mainly dependent when preparing the root tissue protoplast, but in order to determine the proper biological enzyme dosage proportion, and in combination with the related pre-test result, the inventor performs further screening on a plurality of better biological enzyme liquid combinations, in particular:
enzymatic hydrolysate 1:
MES、5 mM + CaC1 2 10 mM +nacl, 10 mM +mannitol Manmitol (pH=5.7), 0.35M +1.5% cellulase celllast+1.5% educase Macerozyme+0.1% BSA;
enzymolysis liquid 2:
MES、5 mM + CaC1 2 10 mM +nacl, 10 mM +mannitol Manmitol (pH=5.7), 0.35M +1.5% cellulase celllast+1.5% pectinase+0.1% BSA;
enzymolysis liquid 3:
MES、5 mM + CaC1 2 10 mM +nacl, 10 mM +mannitol Manmitol (pH=5.7), 0.35M +1.5% cellulase celllast+1.0% pectinase+0.1% BSA;
(2) Pretreatment of materials
Taking tobacco root tip tissue, cleaning with sterile water (3 times), sucking water by filter paper, and cutting root tip with length of 0.5cm along the growth direction of root tip;
(3) Enzymatic extraction
Placing the root tip pretreated in the step (2) into a container, respectively adding the corresponding enzymolysis liquid prepared in the step (1), and carrying out oscillation treatment at 28 ℃;
the specific dosage aspect is as follows:
every 100 root tips, the consumption of the enzymolysis liquid is 5mL;
(4) Protoplast isolation
After the vibration treatment in the step (3) is finished, the mixed solution is filtered by a 250-mesh cell sieve;
centrifuging the filtrate at 700rpm for 10min, retaining the precipitate, and re-suspending the precipitate with 500 μl of buffer;
the buffer solution is the solution without biological enzyme in the step (I) (namely, the enzymolysis solution in the step (I) does not contain the rest components of cellulase, pectase and educing enzyme; when in resuspension, the corresponding buffer solution is adopted for resuspension, namely, protoplast obtained by the treatment group of the enzymolysis solution 1 adopts the buffer solution without the corresponding biological enzyme in the enzymolysis solution 1);
protoplasts obtained from different treatment groups were counted using a cytometer and activity was detected using a fluorescence microscope. The detection results of protoplasts obtained from different material sources and different enzymolysis parameters are shown in fig. 1-4. Specifically:
when different types of enzymolysis liquid are adopted for treatment (the materials are root tissues growing for 7 days, and the enzymolysis oscillation treatment is carried out for 3 hours), the fact that the enzymolysis liquid 2 obtains the largest number of protoplasts (the treatment group result of the enzymolysis liquid 2 in the figure) can be seen, and the best preparation effect is shown; the reason for this analysis is that: for the tender tobacco root tip tissue, the pectin content is relatively high, so that the combination of cellulase and pectase is more suitable.
In terms of the concentration of biological enzymes in the enzymatic hydrolysate (materials all grow into root tissue for 7 days, and the enzymatic shaking treatment is carried out for 3 hours), it can be seen (fig. 2): the use amount of pectase is properly increased, which is more favorable for dissociating cell walls, thereby obtaining more protoplasts.
As regards the enzymatic treatment time (materials all growing in root tissue for 7 days, treated with enzymatic hydrolysate 2), it can be seen (fig. 3): the enzymolysis treatment time is properly improved, which is beneficial to the separation of the prepared protoplast, thereby facilitating the subsequent research and application.
For the preparation of protoplast material (material is root tissue grown for 7 or 10 days, enzymatic hydrolysate 2 is treated for 3 h) it can be seen (fig. 4): root tip tissue grown for 7 days is more favorable for obtaining higher number of protoplasts, and analysis considers that the root tip tissue grown for 10 days has higher fibrosis degree, which is unfavorable for preparing and obtaining protoplasts (or, the relevant enzymolysis dosage proportion needs to be explored and adjusted again).
In addition, under the same lysis conditions (7 tissues are grown, 2h treatment with enzymatic hydrolysate) for different tobacco varieties, the results indicate that: the number of protoplasts was comparable (10 5 Orders of magnitude), but the root protoplasts of nicotiana benthamiana were more active and less fragmented (fig. 5A), while the root protoplasts of K326 were less active and more fragmented (fig. 5B).
Based on the optimized conditions of the experimental results, the inventor further prepares protoplast cells by taking root tip parts of root tissues (K326) growing for 7 days as materials, treats the protoplast cells for 3 hours by adopting enzymolysis liquid 2, and further separates and resuspents the protoplast cells, and the cell count result shows that the number of the protoplasts can reach 1.0x10 6 The cell activity is higher, and the preparation effect is better.
Example 2
In this example, protoplasts were prepared from tobacco leaf material, and the detailed procedure is outlined below.
Firstly, preparing enzymolysis liquid
The enzymolysis liquid is as follows: CPW buffer containing cellust and an educase Macerozyme; the specific formula comprises the following components:
enzymatic hydrolysate 1:
buffer CPW (pH 5.8) +Manmitol (pH 5.7), 0. M +1% celllast+0.5% Macerozyme;
enzymolysis liquid 2
Buffer CPW (pH 5.8) +Manmitol (pH 5.7), 0. M +1% celllast+0.5% Macerozyme;
the buffer CPW (pH 5.8): KH (KH) 2 PO 4 、27.2 mg/L + KNO 3 、101.0 mg/L + CaCl 2 •2H 2 O、 1480.0 mg/L + MgSO 4 •7H 2 O、246.0 mg/L + KI、0.16 mg/L + CuSO 4 •5H 2 O、0.025 mg/L。
(2) Pretreatment of materials
Selecting tobacco leaves which are fully stretched in 4-6 leaf periods after the tobacco germinates and grows, placing the tobacco leaves on ice, and rapidly cutting the tobacco leaves into filaments with the width of about 1mm for later use;
(3) Enzymatic extraction
Placing the root tips or tobacco filaments pretreated in the step (2) into a container, adding the corresponding enzymolysis liquid prepared in the step (1), and vibrating at 30 ℃ and 50 rpm;
the specific dosage aspect is as follows: the consumption of the enzymolysis liquid is 10mL for each fresh tobacco leaf;
(4) Protoplast isolation
After the vibration treatment in the step (3) is finished, the mixed solution is filtered by a 250-mesh cell sieve;
centrifuging the filtrate at 500rpm for 10min, and keeping supernatant to obtain tobacco leaf (glandular hair) cell protoplast extractive solution with high activity and purity.
Protoplasts obtained from different treatment groups were counted using a cytometer and activity was detected using a fluorescence microscope. The detection results of protoplasts obtained from different material sources and different enzymolysis parameters are shown in fig. 5-8. Specifically:
as for the enzymolysis treatment time (K326 tobacco leaf, enzymolysis liquid 1 treatment), it can be seen (fig. 6): the enzymolysis treatment time is properly prolonged, so that more protoplasts can be effectively obtained;
regarding the protoplast isolation method (K326 tobacco leaf, enzymolysis solution 1 treatment for 3 h), it can be seen (FIG. 7): different from the separation mode of root tip source protoplast, the number of protoplast in supernatant after centrifugation is more and more complete; analysis suggests that the main cause is: the glandular hair cells on the surface of the tobacco leaves contain a large amount of metabolites such as terpenoid compounds and acyl sugar, and the like, and are relatively light, so that the protoplasts after centrifugation are mainly distributed in the supernatant, namely the protoplasts prepared by the supernatant can be considered as the main glandular hair cell protoplasts of the tobacco leaves; in the precipitation, although a certain amount of protoplasts exist, the impurity cells and mesophyll cells are more, so that the influence of factors such as purity and the like is considered, and the method is not suitable for the subsequent metabonomics research and study;
as for mannitol concentration (K326 tobacco leaves, different enzymatic solutions were treated for 3 h), it can be seen (fig. 8): the protoplast prepared by 0.4M mannitol has more complete structure and more quantity, and the result shows that the osmotic pressure under the condition of 0.4M mannitol is more suitable for preparing the protoplast of the tobacco leaves;
as for the tobacco variety distinction (enzymatic hydrolysate 1 treatment for 3 h), it can be seen (fig. 9): the difference between the two is not great, which shows that the method has better universality, but compared with the method, when the K326 tobacco leaves are treated, the prepared protoplast has better dispersity and is more suitable.
Based on the optimized conditions of the experimental results, the inventor further uses K326 tobacco leaves as materials to prepare protoplast cells, adopts enzymolysis liquid 1 to treat for 3 hours, and further separates and resuspents, and the cell count result shows that the number of protoplasts in the supernatant can reach 5.3X10 5 The cell activity is higher, and the preparation effect is better.
Claims (2)
1. The preparation method of the tobacco protoplast is characterized by comprising the following steps:
(1) Preparing enzymolysis liquid
Taking tobacco root tips as a protoplast preparation material source; the tobacco is K326 or Benshi tobacco;
aiming at tobacco root tip materials, the enzymolysis liquid prepared by the protoplasm is as follows: MES buffer solution containing cellulase celllast and pectase; the formula comprises the following components:
MES、5 mM + CaC1 2 10 mM +NaCl, 10 mM +mannitol Manmitol, 0.35M +1.5% cellulase celllast+1.5% pectase+0.1% BSA;
the cellulase is cellulase Onozuka R-10 with the enzyme activity of 10000U/g;
the pectase is pectolyase Y-23 with the enzyme activity of 1000U/g;
(2) Pretreatment of materials
Taking root tip tissue of tobacco which germinates and grows for 7 days aiming at root tissue, and cutting the root tip tissue along the growth direction of the root tip after cleaning with sterile water;
(3) Enzymatic extraction
Placing the root tip pretreated in the step (2) into a container, adding the corresponding enzymolysis liquid prepared in the step (1), and carrying out oscillation treatment for 3 hours at the temperature of 28 ℃;
every 100 root tips, the consumption of the enzymolysis liquid is 5mL;
(4) Protoplast isolation
After the vibration treatment in the step (3) is finished, filtering the mixed solution;
and (3) centrifuging the filtrate of the root tip, reserving the precipitate, and re-suspending the precipitate with a buffer solution to obtain the tobacco root tip protoplast extract.
2. The method for preparing tobacco protoplasts according to claim 1, wherein in the step (4), the centrifugation is: and centrifuging at 500-700 rpm for 5-15 min.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210986948.3A CN115354013B (en) | 2022-08-17 | 2022-08-17 | Tobacco protoplast preparation method |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210986948.3A CN115354013B (en) | 2022-08-17 | 2022-08-17 | Tobacco protoplast preparation method |
Publications (2)
Publication Number | Publication Date |
---|---|
CN115354013A CN115354013A (en) | 2022-11-18 |
CN115354013B true CN115354013B (en) | 2024-02-02 |
Family
ID=84002952
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210986948.3A Active CN115354013B (en) | 2022-08-17 | 2022-08-17 | Tobacco protoplast preparation method |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN115354013B (en) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS61104781A (en) * | 1984-10-25 | 1986-05-23 | Nisshin Oil Mills Ltd:The | Preparation of protoplast of lipase-producing microorganism |
WO2001096581A1 (en) * | 2000-06-15 | 2001-12-20 | Kaneka Corporation | Method of inducing gene expression in plant and the plant |
CN109321536A (en) * | 2018-11-13 | 2019-02-12 | 中国烟草总公司郑州烟草研究院 | The extracting method of oxidizing ferment enzyme activity in a kind of raising leaf tobacco extract |
CN111979173A (en) * | 2020-08-27 | 2020-11-24 | 三峡大学 | Preparation method of sorghum protoplast and transient expression transformation method |
CN114276976A (en) * | 2021-11-30 | 2022-04-05 | 内蒙古农业大学 | Dissociation method of picea mongolica root tip cell protoplast |
-
2022
- 2022-08-17 CN CN202210986948.3A patent/CN115354013B/en active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS61104781A (en) * | 1984-10-25 | 1986-05-23 | Nisshin Oil Mills Ltd:The | Preparation of protoplast of lipase-producing microorganism |
WO2001096581A1 (en) * | 2000-06-15 | 2001-12-20 | Kaneka Corporation | Method of inducing gene expression in plant and the plant |
CN109321536A (en) * | 2018-11-13 | 2019-02-12 | 中国烟草总公司郑州烟草研究院 | The extracting method of oxidizing ferment enzyme activity in a kind of raising leaf tobacco extract |
CN111979173A (en) * | 2020-08-27 | 2020-11-24 | 三峡大学 | Preparation method of sorghum protoplast and transient expression transformation method |
CN114276976A (en) * | 2021-11-30 | 2022-04-05 | 内蒙古农业大学 | Dissociation method of picea mongolica root tip cell protoplast |
Non-Patent Citations (5)
Title |
---|
Promoter analysis of the Catharanthus roseus geraniol 10-hydroxylase gene involved in terpenoid indole alkaloid biosynthesis;Nitima Suttipanta et al.;Biochim Biophys Acta;第1769卷(第2期);139-148 * |
烟草原生质体的制备和分析;尚飞等;河南师范大学学报(自然科学版);41(03);130-132、137 * |
王俊丽.细胞工程原理与技术.中央民族大学出版社,2006,(第1版),第161-164页. * |
王涛等.绿色植物生长调节剂(GGR)的研究.中国科学技术出版社,2009,(第1版),第24页倒数第1段. * |
陈名红等.烟草K326叶肉原生质体培养再生植株及影响因素的研究.云南民族大学学报(自然科学版).2006,第15卷(第4期),第328页第1.2节,第329页第2.1节,第331页左栏第2段,表1. * |
Also Published As
Publication number | Publication date |
---|---|
CN115354013A (en) | 2022-11-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN101633934B (en) | Plant expression vector for expressing auxin synthetic related genes and application in improvement of cotton fiber traits | |
CN109652455B (en) | Magnetic nano-carrier mediated efficient genetic transformation method for non-heading Chinese cabbages and application thereof | |
Huhtinen et al. | Ornithine-and putrescine-supported divisions and cell colony formation in leaf protoplasts of alders (Alnus glutinosa and A. incana) | |
CN109554327B (en) | Method for separating and purifying amomum villosum mesophyll protoplast | |
JP2002233258A (en) | Method for mass propagation of adventitious root of panax ginseng c.a. meyer, panax ginseng c.a. meyer grown under completely natural condition and panax ginseng c.a. meyer wildly grown in mountain by tissue culture and method for improving saponin content | |
CN115261237B (en) | Strain for promoting germination of azalea seeds and application thereof | |
CN101352144B (en) | Cross breeding method of Ganoderma lucidum | |
CN106967670A (en) | A kind of preparation method of birch-leaf pear protoplast | |
EP0525914A1 (en) | Synthetic seed | |
CN115354013B (en) | Tobacco protoplast preparation method | |
CN108522281A (en) | A kind of breeding method using ethylmethane sulfonate Vitro Mutation D.farinosus | |
CN112980766A (en) | Method for separating cotton hypocotyl single cells | |
CN110669718A (en) | Method for separating and purifying root, stem and leaf protoplasm body of larch and performing instantaneous high-efficiency conversion | |
CN116925923A (en) | Fungus PH30W for promoting gastrodia elata seed germination and application thereof | |
CN114258858B (en) | Method for inducing embryogenic callus of sugar beet | |
CN114350546B (en) | Pseudomonas bacteria and their use in promoting plant growth, flowering and fruit setting | |
CN109880788A (en) | The cabbage type rape protoplast electrofusion and genetic transforming method and regenerating system used not limited by genotype | |
CN107760708B (en) | The method for improving Jatropha curcus fruit yield by being overexpressed JcARF19 gene | |
CN103789325A (en) | Cotton cell wall extensin gene GbEXPATR and application | |
CN103409362A (en) | Liquid medium and cultivation method for pollen in-vitro germination of arabidopsis thaliana | |
CN102499084B (en) | Quick-breeding method of directly inducing and mycorrhizal seedlings of ledum plant mycorrhizal in test tube | |
CN114258859B (en) | Method for inducing red beet embryogenic callus | |
CN114208671B (en) | Method for inducing embryogenic callus of fodder beet | |
CN115181735B (en) | Composite enzyme liquid and method for preparing poplar root protoplast | |
CN110724660B (en) | Preparation method and application of camphor pine needle protoplast |
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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant |