CN111504746A - Soybean cell nucleus dissociation liquid suitable for flow cytometer analysis and application thereof - Google Patents
Soybean cell nucleus dissociation liquid suitable for flow cytometer analysis and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of biology, and particularly relates to a soybean cell nucleus dissociation solution suitable for flow cytometry analysis and application thereof, wherein the soybean cell nucleus dissociation solution suitable for flow cytometry analysis comprises 45mM magnesium chloride, 20mM MOPS, 30mM sodium citrate and a nonionic surfactant; and lays a foundation for simply, conveniently, quickly and accurately researching the content, the cell cycle and the genetic ploidy of the soybean nuclear DNA by using a flow cytometer.
Description
Technical Field
The invention belongs to the field of biology, and particularly relates to soybean cell nucleus dissociation liquid suitable for flow cytometry analysis and application thereof.
Background
Flow cytometry is a method of multiparameter, rapid analytical quantitation and sorting of single cells or biological particles in a liquid stream using a flow cytometer. With the development of fluorescent molecular labeling technology, flow cytometry is widely applied in the field of life science: such as analyzing various metabolic characteristics of cells by flow cytometry, judging apoptosis and necrosis, evaluating differentiation and proliferation properties of cells, and DNA content analysis, determination of cell population proportion and sorting. The application of flow cytometry in the research of botany mainly has the following aspects: (1) plant cell nuclear assays, such as: determining the content of DNA, performing plant genetic ploidy analysis, cell cycle analysis, and determining genome size; (2) analyzing and sorting chromosomes and constructing a chromosome library; (3) analyzing and sorting protoplast and cell fusion products; (4) the application is in system biology and ecology.
However, unlike animal cells or the field of medical research, plant cells have tough cell walls, and cellular components such as polyphenols are abundantly enriched, making it difficult to isolate single cell suspensions for flow detection. Therefore, to perform qualitative and quantitative analysis of nuclear DNA in plant materials, two main strategies are currently adopted: one strategy is to harvest protoplasts by enzymatic removal of the cell wall of the plant cell, which are disrupted by water absorption in a hypotonic solution to further separate the cell nucleus; another strategy is to rupture the plant cells by physically and mechanically cutting the plant material in a suitable buffer, thereby releasing the nuclei into the buffer.
Soybean is an important grain and oil crop in China, however, in recent years, drought or flood disasters occur frequently, and the production development of the soybean is seriously influenced. The better the root system of the soybean grows, the less the degree of damage caused by water stress and the stronger the stress resistance. Therefore, the deep excavation of the regulation and control mechanism of soybean root system development under the adverse circumstances has important practical significance for alleviating the threat of drought or waterlogging and ensuring the safe production of soybeans. The growth of plant roots is independent of cell division and differentiation, whether initiation and division of root primordia, or formation and activation of meristems, involving two alternating cycles of cell division and cell growth in the cell cycle. In addition, some cells in plants, due to developmental and/or environmental factors, skip G2 after S phase and return directly to G1 phase, reinitiating DNA replication, so cycling to form polyploid cells. Therefore, the research on the mechanism of cell cycle regulation can not only clarify the molecular mechanism of plant growth and development, but also be beneficial to the clarification of the mechanism of the influence of stress on the growth of crops.
However, in the prior art, a cell nucleus dissociation solution suitable for leaf and root tissues of different soybean varieties does not exist, and the efficiency of separating cell nuclei by using enzymolysis and mechanical cutting in other species is low, so that the method is not suitable for large-scale sample preparation and detection.
Disclosure of Invention
In order to solve the technical problems, the invention provides a high-efficiency, simple and cheap preparation method of the cell nucleus suspension suitable for the young tissues (including root tips and leaves) of the soybeans, which can be used for high-throughput detection of a flow cytometer.
A soybean cell nucleus dissociation liquid suitable for flow cytometry analysis comprises 42-48 mM magnesium chloride, 18-22 mM MMOPS, 26-32 mM sodium citrate and a nonionic surfactant.
Further, the nonionic surfactant is Tween-20 or Triton X-100.
Further, the concentration of the Triton X-100 is 0.5% -1%.
The invention also provides a preparation method of the soybean cell nucleus dissociation liquid suitable for flow cytometry analysis, which comprises the steps of mixing the raw materials, adjusting the pH value to 7.0 by using 1M sodium hydroxide, filtering by 0.22-mum, subpackaging and storing at-20 ℃.
The invention also provides application of the soybean cell nucleus dissociation liquid suitable for flow cytometry analysis in cell nucleus dissociation of soybean leaf or root tissues.
Further, taking fresh tender soybean leaf or root tissues, precooling on ice, adding precooled cell nucleus dissociation liquid, immersing the plant materials in the dissociation liquid, and rapidly cutting the plant materials at one time by using a sharp blade; slightly blowing and sucking the cell nucleus dissociation liquid, but avoiding generating bubbles, filtering and centrifuging the cell nucleus dissociation liquid, re-suspending the obtained precipitate by the cell nucleus dissociation liquid, dyeing by DAPI to obtain a cell nucleus suspension, and detecting by using a flow cytometry.
Further, placing fresh tender soybean leaf or root tissue in a mortar, adding precooled cell nucleus dissociation liquid, grinding, filtering and centrifuging, resuspending the obtained precipitate by the cell nucleus dissociation liquid, dyeing by DAPI to obtain cell nucleus suspension, and detecting by a flow cytometry.
Further, the centrifugation time was 1min, the rotation speed was 1000rpm, and the temperature was 4 ℃.
Further, standing for 5min after filtering, standing for 2min after resuspension, and loading on a flow cytometry.
Further, the concentration of DAPI is 4 μ g/m L, and staining is carried out for 10-60 min.
Advantageous effects
Since flow cytometry requires analysis of large quantities of nuclear suspensions in a short time, the purity and concentration of the extracted nuclear suspension are high, with a concentration of 1 × 105-1×107The nuclear dissociation liquid provided by the invention has good applicability on different soybean varieties, has good nuclear dissociation effect on soybean leaf and root tissues, has reliable corresponding cell cycle analysis experiment results, provides technical support for further researching the influence of adverse environment on the copy level in the soybean nucleus, and lays a foundation for simply, quickly and accurately researching the content, cell cycle and genetic ploidy of soybean nuclear DNA by using a flow cytometer.
In addition, the best dissociation effect is obtained by adopting the concentration of the nonionic surfactant provided by the technical scheme of the invention; when the centrifugal strength is too large or the centrifugal time is too short, the cell nucleus still suspends in the nuclear dissociation liquid to achieve the effect of enrichment, and the redundant nuclear dissociation liquid is discarded and lost in the next step; if the centrifugation strength is too high or the centrifugation time is too long, impurities in the prepared cell nucleus suspension can be obviously increased and even interfere with detection. The centrifugal strength and time provided by the invention have the best dissociation effect. If a large amount of sample materials need to be detected, the mechanical cutting method is very time-consuming (about 20min for processing a sample), the invention adopts the method of grinding the soybean materials in the nuclear dissociation liquid, and can rapidly dissociate a large number of sample volumes (only 3min is needed for preparing each sample on average) by controlling the standing time, the centrifugation speed and the centrifugation time, thereby providing necessary conditions for analyzing a large amount of samples in a short time. Meanwhile, if the sample amount of the detection sample is less (the material is less), the sample can be fully dissociated by using a grinding mode, and the separation efficiency is higher than that of mechanical cutting.
Drawings
FIG. 1 shows the scattered light characteristics of the cell nucleus of the soybean tender leaf tissue obtained by different treatments of the nuclear dissociation liquid;
FIG. 2 shows the relative DNA content of soybean root cell nucleus obtained from different nuclear dissociation liquids;
FIG. 3 is the relative content of nuclear DNA of soybean root cell obtained from different nuclear dissociation liquids and morphological observation; (A-B) A nucleus dissociation liquid; (C-D) B nucleus dissociation liquid;
FIG. 4 is an analysis of the cell cycle of different soybean varieties before and after PEG simulated drought stress; (A, C, E, G) the relative DNA content of the root tip nuclei of different soybean varieties in normal growth conditions; (B, D, F, H) relative DNA content of root tip nuclei of different soybean varieties after 20% PEG6000 simulated drought stress treatment. (A and B) Holladay; (C and D) PI 567611; (E and F) PI 567651; (G and H) Fiskeyy III.
FIG. 5 shows that the method of grinding with mortar using the B-nucleus dissociation liquid results in a large number of fragments and a reduced number of effective nuclei if the steps of filtration, standing for 5min and pre-machine detection, standing for 2min are not adopted.
Detailed Description
Example 1
1 materials and methods
1.1 Experimental materials
The tested materials were soybean varieties including Williams 82, Holladay, PI567611, PI 567651, and Fiskeby III, and were stored by the soybean genetic breeding laboratory at the university of yangzhou.
1.2 methods
1.2.1 preparation of dissociation liquid for plant cell nucleus
The formula of 6 nuclear dissociation liquids used in the experiment is shown in table 1.
TABLE 1 Nuclear dissociation buffer formulation
The nuclear dissociation liquid avoids repeated freeze thawing, and can be stored for several days at 4 ℃ after thawing.
1.2.2PEG 6000 simulated drought treatment
The soybean seeds are planted in soil after water absorption and expansion, the soybean seeds germinate and grow for 6 days, soybean seedlings with the same growth state are selected, dust is cleaned on the premise of avoiding damaging the soybean root system, the root system is respectively immersed in 0% and 20% PEG6000 solution with concentration and stressed for 7 days, residual PEG6000 of the root system is cleaned, and young root tips of about 2cm are taken for preparing a cell nucleus suspension.
1.2.3 preparation of cell Nuclear suspensions
The method comprises the following steps of taking fresh tender soybean leaves or root tissues, washing dust on the surfaces with distilled water, sucking residual water with filter paper, placing a culture dish on ice for precooling, adding 5m L precooled cell nucleus dissociation liquid, immersing plant materials in the dissociation liquid, immediately cutting the plant materials rapidly on the ice with a sharp blade in one step, slightly blowing and sucking the cell nucleus dissociation liquid without generating air bubbles so as to reduce released cell nuclei to be adhered to the plant tissues, filtering the cell nucleus dissociation liquid through a 400-mesh cell filter, placing filtrate at 4 ℃ or incubating on the ice for 5min, centrifuging the filtrate at 4 ℃ and 1000rpm for 1min, carefully removing supernatant, re-suspending the obtained precipitate with a proper volume of fresh cell nucleus dissociation liquid, and staining the precipitate with DAPI with the final concentration of 4 mu g/m L for 10min to 60 min.
If a large number of different samples need to be prepared, plant tissues can be placed in a mortar, the mortar is placed on ice for precooling, a proper amount of precooled cell nucleus dissociation liquid is added, after light grinding on ice, all suspension is transferred and filtered through a 400-mesh cell filter to a 50m L centrifuge tube, the supernatant is transferred to a new 50m L centrifuge tube, the centrifuge is carried out at 4 ℃ and 1000rpm for 1min, the supernatant is carefully removed, the obtained precipitate is resuspended through a proper volume of fresh cell nucleus dissociation liquid, and the precipitate is stained with DAPI with the final concentration of 4 mu g/m L for 10min to 60 min.
1.2.4 flow cytometry analysis
Taking 500 mu L of the prepared cell nucleus suspension, detecting with a FACS L SRFortessa flow cytometry (BD company, USA) under the condition of low flow rate, at least absorbing 10000 DAPI stained particles, standing the suspension prepared in a grinding mode for 5min after filtering, standing the suspension for 2min before detecting, and obtaining a better effect (figure 5). analyzing the result with Modfit5.0 software, respectively drawing a relation graph of the number (count) of the absorbed particles and the fluorescence intensity (F L), wherein all the sample operation parameters are consistent, and the measured parameters mainly comprise relative fluorescence intensity, Coefficient of Variation (CV) and debris background (DF).
2 results
2.1 Effect of different Nuclear dissociation liquids on Nuclear dissociation
In the research, 6 different nuclear dissociation liquids are adopted to separate the cell nucleus of the soybean leaf cell. According to the detection signals of scattered light, clear 2-cluster organelle particle signal accumulation areas can be obtained by the A nuclear dissociation liquid (figure 1A), the B nuclear dissociation liquid (figure 1B) and the C nuclear dissociation liquid (figure 1C), and only one cluster of cell particle signal accumulation area is obtained in the D nuclear dissociation liquid (figure 1D), the E nuclear dissociation liquid (figure 1E) and the F nuclear dissociation liquid (figure 1F) through flow detection analysis. By comparing the scattered light signals of the cell nucleus in the soybean root, the signals in the selected area of the black frame are confirmed to correspond to the scattered light signals of the cell nucleus, and the signals enriched in the area outside the black frame are presumed to be chloroplast organelles.
2.2 Effect of different dissociation solutions on the relative content of DNA
In order to further determine the nuclear dissociation liquid suitable for the extraction of the soybean young leaf cell nucleus, the experimental result uses Modfit5.0 software, takes the fluorescence area signal as the horizontal axis and the cell nucleus particle number as the vertical axis, and displays the statistical result according to the selected signal area. The relative DNA content histogram (FIG. 2) shows that: the cell nucleus suspensions extracted from the A nucleus dissociation solution (FIG. 2A), the C nucleus dissociation solution (FIG. 2B) and the B nucleus dissociation solution (FIG. 2C) all formed normally distributed DNA peaks in G0/G1, and had coefficient of variation CVs of less than 5%, 2.78%, 4.24% and 2.96%, respectively. However, polyploidy is detected in a little amount in the A nucleus dissociation solution and the C nucleus dissociation solution, and no obvious peak is formed in the G2/M phase, which may cause certain errors in analysis of DNA ploidy, cell cycle and cell number in each phase of mitosis. The B nucleus dissociation liquid can detect a remarkable G2/M phase peak, and although the B nucleus dissociation liquid forms more fragments than the A nucleus dissociation liquid and the C nucleus dissociation liquid, the B nucleus dissociation liquid is relatively better in detecting cell cycle and genetic ploidy. In contrast, the E-nucleus dissociation solution (FIG. 2E) and the F-nucleus dissociation solution (FIG. 2F) had poor DNA peaks, many fragments, and large coefficient of variation. The D nuclear dissociation liquid (fig. 2D) can form a normally distributed DNA peak, but the DNA peak is significantly reduced and greatly deviated from the DNA peak obtained by the other 5 nuclear dissociation liquids in fluorescence intensity, indicating that the D nuclear dissociation liquid may have an influence on the distribution of chromatin in the nucleus.
We further performed dissociation of cell nuclei in Sophora subprostrata by using the A-nucleus dissociation solution (FIG. 3A) and the B-nucleus dissociation solution (FIG. 3B), and the research results show that the two nuclear dissociation solutions form normally distributed DNA peaks in the G0/G1 phase and the G2/M phase. In summary, the A and B nucleus dissociation solution is suitable for dissociation of nuclei in soybean leaves and roots.
2.3 comparison of the extraction efficiency of cell nuclei by different nuclear dissociation liquids
The morphological observations of the nuclei of the suspension cells extracted with two different dissociation solutions A and B are shown in FIG. 3. The result shows that the two nuclear dissociation liquids not only can obtain more single cell nucleuses, but also the cell nucleuses are suspended in the nuclear dissociation liquid in a full round or oval shape and are well recognized. Therefore, the two nuclear dissociation liquids can better ensure that the whole cell nucleus is released from the cell, and can reduce the liberation of secondary metabolites in the cell, although a small amount of a plurality of single-particle cell nuclei are adhered together or adhered with other impurities.
Comparing cell fragments generated by 6 nuclear dissociation liquids, it is found that there are more fragments in the F and E nuclear dissociation liquids, wherein the E nuclear dissociation liquid can not separate complete cell nuclei, and the morphology of the cell nuclei is incomplete, which results in that fluorescence signals can not be gathered. Therefore, these two nuclear dissociation liquids are not suitable for isolating soybean nuclei. Wherein the chloroplast in the D nucleus dissociation solution is less polluted, but the cell debris is more. F. The flow rates of the E and D nuclear dissociation liquids are low, and the number of particles in unit time is small, which also indicates that the 3 nuclear dissociation liquids can not separate the cell nucleus efficiently.
2.4 applicability and applicability of B nucleus dissociation liquid to different soybean varieties
To verify whether the B nucleus dissociation solution has wide adaptability, fresh and tender root tips of different soybean varieties Holladay, PI567611, PI 567651 and Fiskeyy III are taken respectively, mechanically cut in the B nucleus dissociation solution to release nuclei, and the DNA content of the nuclei is detected by a flow cytometer and the genetic ploidy is analyzed, and the result is shown in FIG. 4. The cell nucleus suspension forms normally distributed DNA peaks in the G0/G1 phase and the G2/M phase, and the coefficient of variation CV is less than 5 percent. Therefore, the B-nucleus dissociation liquid has good applicability to other soybean varieties. Although there are slight differences in the scattered light characteristics of the nuclei of different soybean varieties, it is likely that the nuclei have slight differences in particle size and complexity.
For further analysis of the application of the B dissociation solution and the flow cytometer in soybean cell cycle detection, the regulation and control of PEG6000 simulated drought treatment on the cell cycle of the root tips of different soybean varieties are simultaneously detected. The results showed that 20% PEG6000 significantly promoted nuclear replication (relative increase in the number of cells at G2 phase) (fig. 4). Meanwhile, the soybean of different varieties has different response degrees to the stress of PEG with the same concentration, for example, the cell cycle of the root tip of PI 567651 is remarkably blocked, but the response degree of the PI567611 to the stress of the PEG is lower than that of other varieties (FIGS. 4E and 4F). The research result not only shows that the cell cycle of the soybean root tip can be successfully detected by using the B-nucleus dissociation solution and the flow cytometry, but also shows that drought stress participates in regulating and controlling the cell cycle process of the soybean root tip.
Since flow cytometry requires analysis of large quantities of nuclear suspensions in a short time, the purity and concentration of the extracted nuclear suspension are high, with a concentration of 1 × 105-1×107High purity intact single-granule nuclei per m L are preferred in this experiment because chloroplast DNA binds to the dye DAPI and fluorescence appears during sorting to interfere with nuclear signaling, while the root tissue lacks chloroplasts, and thus, nuclei from which they were preparedThe purity and concentration of the nuclear suspension are higher. Thus, leaf tissue is a more suitable material for flow cytometry sorting than root tissue.
The nuclear dissociation liquid is added with nonionic surfactant (such as Tween-20 or Triton X-100) to promote release of nuclei and prevent sticky substances from sticking to nuclei. In this example, Triton X-100 was added to all the nuclear dissociation liquids, but the effect of extracting nuclei was different. Among all the cell nucleus dissociation solutions, the A dissociation solution has the best dissociation effect, the Triton X-100 concentration is 0.1%, while the F and E cell nucleus dissociation solutions have higher Triton X-100 concentration (0.5% -1%), and the Triton X-100 concentration in the cell nucleus dissociation solution can be one of the key factors. In addition, when the centrifugal strength is too high or the centrifugal time is too short, the cell nucleus still suspends in the nuclear dissociation liquid to achieve the enrichment effect, and the redundant nuclear dissociation liquid is discarded and lost in the next step; if the centrifugation strength is too high or the centrifugation time is too long, impurities in the prepared cell nucleus suspension can be obviously increased and even interfere with detection.
Intranuclear replication helps to maintain plant growth under stress conditions to accommodate adverse environmental factors. In many species, water stress can inhibit the mitotic cycle by reducing the activity of cyclin-dependent kinases, increasing the cycle of endoreduplication. The increase of the replication level in the nucleus can promote the expansion of cells, thereby relieving the influence of water deficiency on the size of the cells and helping the plants to relieve the stress injury. The embodiment further performs preliminary analysis on regulation and control of the cell cycle under soybean drought stress on the basis of screening the nuclear dissociation liquid suitable for soybean tissues, the variation coefficient of the cell nucleus suspension liquid extracted by the B nuclear dissociation liquid is less than 5%, the fragments are few, and the main peak is very obvious, so that the nuclear dissociation liquid has a good effect of separating the cell nucleus of the soybean root, the corresponding cell cycle analysis experiment result is reliable, and the technical support is provided for further researching the influence of adversity on the soybean nuclear replication level.
The research utilizes the flow cytometry to analyze and compare the separation effect of 6 cell nucleus dissociation liquids, and finds the cell nucleus dissociation liquid suitable for soybean leaf and root tissues. The A nucleus dissociation liquid has the best nucleus separation effect, the B nucleus dissociation liquid is next to the A nucleus dissociation liquid, but the B nucleus dissociation liquid can detect a remarkable G2/M phase peak and is relatively better in cell cycle and genetic ploidy. Further, by comparing the cell cycle of the root tip cell nucleus before and after PEG treatment in different soybean varieties, the B nucleus dissociation solution also has good applicability and applicability. The research lays a foundation for simply, conveniently, quickly and accurately researching the content, the cell cycle and the genetic ploidy of the soybean nuclear DNA by using a flow cytometer.
Claims (10)
1. The soybean cell nucleus dissociation liquid suitable for flow cytometry analysis is characterized by comprising 42-48 mM of magnesium chloride, 18-22 mM of MOPS, 26-32 mM of sodium citrate and a nonionic surfactant.
2. The soybean cell nucleus dissociation solution suitable for flow cytometry analysis of claim 1, wherein the non-ionic surfactant is Tween-20 or Triton X-100.
3. The soybean cell nucleus dissociation solution suitable for flow cytometry analysis as claimed in claim 1, wherein the concentration of Triton X-100 is 0.5-1%.
4. The method for preparing the soybean cell nucleus dissociation liquid suitable for flow cytometry analysis according to any one of claims 1 to 3, wherein the raw materials are mixed, adjusted to pH 7.0 with 1M sodium hydroxide, filtered at 0.22- μ M, and separately stored at-20 ℃.
5. Use of the soybean cell nuclear dissociation liquid suitable for flow cytometry analysis as described in any one of claims 1 to 3 for cell nuclear dissociation of soybean leaf or root tissue.
6. The use of the liquid for the nuclear dissociation of soybean leaves or root tissue according to claim 5, wherein fresh and tender soybean leaves or root tissue is taken, pre-cooled on ice, pre-cooled nuclear dissociation liquid is added, the plant material is immersed in the dissociation liquid, and then rapidly cut at one time with a sharp blade; slightly blowing and sucking the cell nucleus dissociation liquid, but avoiding generating bubbles, filtering and centrifuging the cell nucleus dissociation liquid, re-suspending the obtained precipitate by the cell nucleus dissociation liquid, dyeing by DAPI to obtain a cell nucleus suspension, and detecting by using a flow cytometry.
7. The application of the soybean cell nucleus dissociation solution suitable for flow cytometry analysis in cell nucleus dissociation of soybean leaf or root tissue as claimed in claim 5, wherein fresh and tender soybean leaf or root tissue is taken and placed in a mortar, precooled cell nucleus dissociation solution is added, after grinding, filtration and centrifugation are carried out, the obtained precipitate is re-suspended by the cell nucleus dissociation solution, cell nucleus suspension is obtained after DAPI staining, and the detection is carried out by adopting a flow cytometry.
8. The use of the liquid for the nuclear dissociation of soybean cells suitable for flow cytometry analysis as claimed in claim 6 or 7, wherein the centrifugation time is 1min, the rotation speed is 1000rpm, and the temperature is 4 ℃.
9. The use of the liquid for the nuclear dissociation of soybean suitable for flow cytometry analysis according to claim 7, wherein the liquid is left for 5min after filtration and is loaded on the flow cytometer after being left for 2min after being resuspended.
10. The use of the liquid for the nuclear dissociation of soybean leaves and root tissues as claimed in claim 6 or 7, wherein the DAPI concentration is 4 μ g/m L, and the staining time is 10-60 min.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104316373A (en) * | 2014-10-22 | 2015-01-28 | 江苏省农业科学院 | Extraction method for cell nucleuses of eggplant leaves suitable for flow cytometry |
| CN110520735A (en) * | 2017-04-14 | 2019-11-29 | 文塔纳医疗系统公司 | Dissociate the separation based on size of fixing organization |
| CN110595988A (en) * | 2019-10-14 | 2019-12-20 | 中国科学院昆明植物研究所 | Preparation method and application of cell nucleus suitable for detecting plant C value by flow cytometry |
| CN110823654A (en) * | 2019-11-06 | 2020-02-21 | 四川省自然资源科学研究院 | Method for identifying ploidy of kiwi fruits by adopting flow cytometer, dissociation liquid and preparation method |
-
2020
- 2020-04-29 CN CN202010357240.2A patent/CN111504746A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104316373A (en) * | 2014-10-22 | 2015-01-28 | 江苏省农业科学院 | Extraction method for cell nucleuses of eggplant leaves suitable for flow cytometry |
| CN110520735A (en) * | 2017-04-14 | 2019-11-29 | 文塔纳医疗系统公司 | Dissociate the separation based on size of fixing organization |
| CN110595988A (en) * | 2019-10-14 | 2019-12-20 | 中国科学院昆明植物研究所 | Preparation method and application of cell nucleus suitable for detecting plant C value by flow cytometry |
| CN110823654A (en) * | 2019-11-06 | 2020-02-21 | 四川省自然资源科学研究院 | Method for identifying ploidy of kiwi fruits by adopting flow cytometer, dissociation liquid and preparation method |
Non-Patent Citations (1)
| Title |
|---|
| 涂红艳等: "金姜花薄层细胞培养及再生植株倍性稳定性的检测", 《广东第二师范学院学报》 * |
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