CN104359738A - Dry plant tissue treatment method applied to flow cytometry - Google Patents
Dry plant tissue treatment method applied to flow cytometry Download PDFInfo
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Abstract
The invention relates to a dry plant tissue treatment method applied to flow cytometry, belonging to the technical field of biological analysis and aiming at solving the problem that only a fresh sample can be analyzed in the prior art. The treatment method comprises the following steps: performing cell nucleus extraction, staining and analyzing, wherein a cell nucleus extracting solution used by cell nucleus extraction comprises the following components: 12-18mM of Tris, 1-3Mm of Na2EDTA, 0.2-0.3mM of spermine tetrahydrochloride, 70-90mM of KCl, 10-25mM of NaCl, 10-20mM of beta-mercaptoethanol and 0.2-0.5 percent of Triton X-100, and the pH value is 6.5-7.0. The method has the effects of preventing cell nucleus DNA degradation, improving the stability of cell nucleus and lowering probability of oxidation of cell nucleus, and can be applied to various dry plant tissues, and the application range is widened.
Description
Technical field
The present invention relates to a kind of disposal route being applicable to flow cytometry plant drying tissue, belong to biological analysis technical field.
Background technology
Flow cytometry (flow cytometry) is that a kind of flow cytometer that utilizes carries out the technology of fast quantitative analysis and sorting to the individual cells be in liquid stream or other biologic grain etc.In botany research, then can be used for plant Ploidy Identification, nucleus DNA quantitative measurement, reproductive way qualification etc.
At present, conventional flow cytometry plant chromosome ploidy all adopts the tissue of the tip of a root of fresh plant, blade or delicacy, because it can avoid starch, polysaccharide and other metabolins containing higher concentration in old tissue, and these species distribution are in tenuigenin, tenuigenin closely sticked together with nucleus, have impact on nuclear purity, simultaneously, also accelerate nuclear aging, increase the viscosity of sample, make it can not detect the ploidy of testing sample exactly.But, on the other hand, if adopt fresh or young tender tissue, just require that vegetable material will complete mensuration at short notice, otherwise nucleus DNA can be degraded, the accuracy of detection can be affected equally.So just limit and can not carry out flow cytometry from the plant sample of field acquisition, significantly limit the application of flow cytometry in plant ecology, population biology, polant systematics.As Chinese patent application (publication number: CN102243150A) discloses a kind of nucleus extraction method being applicable to the mature leaves of fruit trees of flow cytometry, by improving the component of nucleus extraction liquid, described nucleus extraction fluid component is:
The Na of the NaCl of the KCl of 15-20mmol/L Tris, 40-60mmol/L, 10-20mmol/L, 2-5mmol/L
2eDTA, 10-20mmol/L MgSO
4, 40-50mmol/L sucrose and 0.5-1.0%Triton X-100, volume adds ddH2O to 200ml, adjusts pH to be after 7.5-8, adds the 15mmol/L mercaptoethanol of 200 μ l.Make be conducive to maintaining nuclear integrality and prevent them from assembling by adding sucrose, and by improving the concentration for the treatment of of Triton-X-100 to 1.0%, impel tenuigenin disperse and optionally dissolve chloroplast membranes, make to be conducive to punching on nuclear membrane, so that fluorescent dye enters smoothly.Although, the disposal route being mentioned to mature leaf is had in prior art, but still do not relate to the method that process detects dried plant tissue in prior art, and be through processed due to dried plant tissue, plant cell tissue is adhered together with nucleus substantially, is difficult to be separated, and the plant cell membrane after dehydration is difficult to broken wall, be difficult to extract nucleus and ensure Color, thus affect the accuracy of flow cytometry; On the other hand, due to dried plant, tissue storage is of long duration, and endonuclear DNA exists the problem of easy fracture and degraded.
Summary of the invention
The present invention is directed to above problems of the prior art, provide a kind of disposal route being applicable to flow cytometry plant drying tissue, problem to be solved by this invention is how Realization analysis plant drying tissue, improves the scope of application of flow cytometry.
The object of the invention is to be achieved by the following technical programs, a kind of disposal route being applicable to flow cytometry plant drying tissue, this disposal route comprises nucleus extraction, dyeing and analysis, and the component of described nucleus extraction nucleus extraction liquid used is:
Tris:12mM ~ 18mM; Na
2eDTA:1mM ~ 3mM; Spermine hydrochloride: 0.2mM ~ 0.3mM; KCl:70mM ~ 90mM, NaCl:10mM ~ 25mM; Beta-mercaptoethanol: 10mM ~ 20mM; Triton X-100:0.2% (vol/vol) ~ 0.5% (vol/vol); PH value is 6.5 ~ 7.0.
The present invention is applicable to the disposal route of flow cytometry plant drying tissue, because plant tissue is as after the dryings such as blade, root or stem, be substantially be in completely dehydration after, plant tissue can adhere to nucleus surface, be difficult to reach separating effect, and plant cell membrane is difficult to broken wall, also have impact on nuclear extraction and achromatic problem; Meanwhile, dry plant tissue due to period of storage comparatively of a specified duration, be easy to cause DNA break and degraded.In order to solve the problem, the present invention is effect in order to stabilized cell core DNA by adding spermine hydrochloride Main Function in nucleus extraction liquid, effectively can prevent its fracture and degraded, improve extraction efficiency and the purity requirement of DNA, ensure nuclear integrality and concrete dispersion effect preferably, be conducive to carrying out flow cytometry, and by adding Triton X-100 and improving its concentration, solve the problem that the cell membrane caused because dewatering is difficult to broken wall, thus reach good treatment effect, on the other hand, due to the plant tissue through super-dry, plant tissue is harder, need longer time chopping, and the overlong time of cutting, easily cause polyphenols oxidized, therefore, the present invention is by adding beta-mercaptoethanol to reach the effect preventing polyphenols oxidized.By the pH value of regulation system 6.5 ~ 7.0, brownization of vegetable material can be reduced, be conducive to the rupture of membranes of dried plant cell, thus be easy to nuclear release.Unit mM described above represents mmol/L, volume ratio vol/vol represents the volumetric usage of Triton X-100 and the percent by volume of whole cell core extract, percent value under the condition that both volume unit is identical, preferably, in described nucleus extraction liquid, Triton X-100 is 0.25% ~ 0.35% (vol/vol).
Be applicable in the disposal route of flow cytometry plant drying tissue above-mentioned, as preferably, this disposal route specifically comprises the following steps:
A, nucleus extraction: the nucleus extraction liquid adding precooling in the plant tissue of drying, then carry out chopping process, cross and filter residue, collect containing nucleolate filtrate;
B, dyeing and analysis: in above-mentioned filtrate, add RNaseA and iodate third ingot carry out dyeing process, the concentration of described iodate third ingot is 0.15mg/mL ~ 0.25mg/mL, after dyeing process, carries out flow cytometry.
Because the nucleus surface of dried plant tissue has the impurity such as a small amount of cell membrane, plant tissue, the process difficult dyeing in dyeing can be caused, thus the precision of the accuracy of impact analysis, the present invention finds that the concentration by improving iodate third ingot added well solves achromatic problem, make DNA more easily by Dye Adsorption, reach good Color.In addition, by adopting the nucleus extraction liquid of precooling to extract, be more conducive to preventing nuclear degraded and ensureing nuclear integrality.Described dried plant is organized preferably to be stored in advance in ice bag or under freezing conditions and is preserved.
Be applicable in the disposal route of flow cytometry plant drying tissue above-mentioned, as preferably, the detailed process of shredding process described in steps A is:
The plant tissue of the drying of precooling is put into the container of precooling, add the nucleus extraction liquid of precooling, subsequently with blade, plant tissue is carried out first time and shred process, filter, collect containing nucleolate filtrate, adopt blade that remaining plant tissue is carried out second time again and shred process, until whole plant tissues is chopped into pasty state.Preferably, the time of each chopping process was preferably over more than 10 minutes.After first time chopping, substantially be cut into granule, but because the plant tissue of drying is comparatively hard, granule swims in extract, is difficult to reach chop up effect further, therefore, the present invention, by secondary chopping process, first by the extracting liquid filtering after first time chopping process out, then carries out secondary chopping, thus well reach the effect of chopping, be also more conducive to achieving nuclear Extraction and separation.As further preferred, the thickness of described blade is 0.1mm ~ 0.3mm.Fundamental purpose is conducive to smudge cells, contributes to the effect making nucleus free; In addition, the thickness of blade is less, and blade will be sharper, but the hardness of blade can weaken; Blade thickness is higher, and blade sharper degree is poor, and plant tissue is just difficult to chopping, the just more difficult chopping discharge nucleus of especially dry plant tissue.Select the blade in above-mentioned thickness range, an equilibrium point can be selected on blade stiffness and sharpness, be adapted to the chopping of dry tissue.
Be applicable in the disposal route of flow cytometry plant drying tissue above-mentioned, as preferably, the amount ratio that the weight of plant tissue dry described in steps A and nucleus extraction liquid amass is: 50:1 ~ 45:1, the unit of weight of the plant tissue of described drying is milligram, and the volume unit of described nucleus extraction liquid is milliliter.The weight of plant tissue will within the specific limits, and plant tissue ratio that is very few or extract consumption is too high, and plant tissue can be caused to swim on liquid level, and in unit volume liquid, nucleus amount is very few, is difficult to be detected; And plant tissue consumption is too much or extract usage ratio is very few, cause again the cell separated to be difficult to dispersion, tissue is difficult to shred, and the nucleus amount in unit volume can be made equally very few.
Be applicable in the disposal route of flow cytometry plant drying tissue above-mentioned, as preferably, the concentration of the ingot of iodate third described in step B is 0.18mg/mL ~ 0.22mg/mL.Object is the Color in order to improve dyestuff.
Be applicable in the disposal route of flow cytometry plant drying tissue above-mentioned, as preferably, plant tissue dry described in steps A is selected from silica dehydrator, natural air drying or press dry the plant drying tissue of sample.By adopting disposal route of the present invention, thus dry plant tissue can be applicable to, improve the scope of application greatly.
Be applicable in the disposal route of flow cytometry plant drying tissue above-mentioned, as preferably, the flow type analyzer that flow cytometry described in step B adopts is that Attune sound wave focuses on stream type cell analyzer.As further preferred, the optimum configurations that the sound wave of Attune described in step B focuses on stream type cell analyzer is:
| Relevant parameter | Voltage range (mV) |
| FSC | 2300～2400 |
| BL1 | 1500～1800 |
| SSC | 2500～2600 |
| BL2 | 1800～1900 |
| LB3 | 1300～1400 |
| VL1 | 1200～1300 |
| VL2 | 1900～2000 |
| VL3 | 2100～2400 |
Above-mentioned parameter is eight optical filter passages in flow cytometer, and its numerical value refers to the magnitude of voltage of optical filter passage, by the voltage-regulation of FSC and SSC to optimum range can be found corresponding cell or nucleus monoid.The voltage that when the present invention measures dry sample, voltage used measures higher than fresh sample, object is the discrimination in order to improve the fluorescence peak between nucleus and cell fragment, is conducive to the determination of ploidy.
In sum, the present invention compared with prior art has the following advantages:
1. the present invention is applicable to the disposal route of flow cytometry plant drying tissue, realize preventing the degraded of nucleus DNA by adding spermine hydrochloride and improving Triton X-100 and improve its stability, and solve by adding beta-mercaptoethanol the oxidized problem caused because dried plant organizes chopping process better, the present invention is improved nucleus extraction liquid by above-mentioned, thus enable method of the present invention be applicable to dry plant tissue, and various different plant drying tissue can also be applicable to, substantially increase the scope of application.
2. the present invention is applicable to the disposal route of flow cytometry plant drying tissue, by improving the concentration of iodate third ingot, solve because the nucleus surface impurity that easily adhesion is a small amount of of dried plant tissue is as cell membrane and plant tissue fragment, realization makes DNA be easier to, by Dye Adsorption, reach coloring effect.
Accompanying drawing explanation
Fig. 1 is the flowcytometric results figure of the dry leaf sample of Solidago Canadensis that method of the present invention obtains.
Fig. 2 is the flowcytometric results figure of the dry leaf sample of oriole that method of the present invention obtains.
Fig. 3 is the flowcytometric results figure of the dry leaf sample of fraises des bois that method of the present invention obtains.
Fig. 4 is the flowcytometric results figure of the dry leaf sample of planting strawberry that method of the present invention obtains.
Fig. 5 is the flowcytometric results figure of the dry leaf sample of planting strawberry that method of the present invention obtains.
Fig. 6 is the flowcytometric results figure of the dry petiole sample of planting strawberry that method of the present invention obtains.
Fig. 7 is the flowcytometric results figure of the dry stolon sample of planting strawberry that method of the present invention obtains.
Fig. 8 is the flowcytometric results figure of the dry tip of a root sample of planting strawberry that method of the present invention obtains.
Fig. 9 is the flowcytometric results figure of the dry leaf sample of clover that method of the present invention obtains.
Figure 10 is the flowcytometric results figure of the dry leaf sample of throatroot that method of the present invention obtains.
Figure 11 is the flowcytometric results figure of the dry leaf sample of Alternanthera philoxeroides that method of the present invention obtains.
Figure 12 is the flowcytometric results figure of the dry leaf sample of Horseweed Herb that method of the present invention obtains.
Figure 13 is the flowcytometric results figure of the dry leaf sample of black nightshade that method of the present invention obtains.
Figure 14 is the flowcytometric results figure of the dry leaf sample of orange that method of the present invention obtains.
Figure 15 is the flowcytometric results figure of the Soybean drying leaf sample that method of the present invention obtains.
Figure 16 is the flowcytometric results figure of the dry leaf sample of safflower Loropetalum wood that method of the present invention obtains.
Figure 17 is the flowcytometric results figure of the dry leaf sample of paper mulberry that method of the present invention obtains.
Figure 18 is the flowcytometric results figure of the dry leaf sample of bidens pilosa that method of the present invention obtains.
Embodiment
Below by specific embodiments and the drawings, technical scheme of the present invention is described in further detail, but the present invention is not limited to these embodiments.
Embodiment 1
The present embodiment is that the ploidy of Solidago Canadensis measures, and the component of nucleus extraction liquid used in the present embodiment is:
Tris:12mMmM; Na
2eDTA:1mM; Spermine hydrochloride: 0.2mM; KCl:70mM, NaCl:10mM; Beta-mercaptoethanol: 10mM; Triton X-100:0.3% (vol/vol); PH value is 7.0, and pH value can adopt the NaOH solution of 1mM to regulate, and described nucleus extraction liquid is stored in the refrigerator of-20 DEG C for subsequent use in advance;
Solidago Canadensis mature leaf (blade through silica dehydrator) 50mg is put into precooled plastic culture dish, add in the nucleus extraction liquid 1mL of precooling again, then double-edged razor blade chopper blade is adopted, chopping process lasts more than 10 minutes, first use 30 micrometer nylon membrane filtrations, collect containing nucleolate filtrate, again secondary chopping process is carried out to remaining blade, namely double-edged razor blade is adopted to continue chopper blade, chopping process lasts more than 10 minutes, until whole blades is cut into pasty state, rinse with nucleus extraction liquid again, filter, collect containing nucleolate extract, after the extract of twice collection is merged, add 10 microlitre RNaseAs (concentration is 1mg/mL), place more than 10 minutes under the condition of 4 DEG C after mixing, in extract, add concentration be again the ultimate density of iodate third ingot in the iodate third ingot solution to system of 1mg/mL is 0.2mg/mL, under lucifuge condition, dyeing is carried out 30 minutes to nucleus DNA, after having dyeed, carry out flow cytometry, preferred described flow cytometry adopts Attune sound wave to focus on stream type cell analyzer, and the optimum configurations that described Attune sound wave focuses on stream type cell analyzer is:
| Relevant parameter | Magnitude of voltage mV (millivolt) |
| FSC | 2350 |
| BL1 | 1600 |
| SSC | 2560 |
| BL2 | 1850 |
| LB3 | 1340 |
| VL1 | 1250 |
| VL2 | 1950 |
| VL3 | 2200 |
As shown in Figure 1, the fluorescent value of the dry leaf sample of Solidago Canadensis is 3259937 to concrete analysis result, and the nuclear DNA content peak surveyed is very thin and concentrated, its CV value 4.54%, and as calculated, Solidago Canadensis is 6 times of bodies, identical with bibliographical information.
Embodiment 2
The present embodiment is that the ploidy of oriole measures, and the component of nucleus extraction liquid used in the present embodiment is:
Tris:14mM; Na
2eDTA:1mM; Spermine hydrochloride: 0.2mM; KCl:70mM, NaCl:15mM; Beta-mercaptoethanol: 20mM; Triton X-100:0.5% (vol/vol); PH value is 7.0, and pH value can adopt the NaOH solution of 1mM to regulate, and described nucleus extraction liquid is stored in the refrigerator of-20 DEG C for subsequent use in advance;
Oriole mature leaf (blade through silica dehydrator) 50mg is put into precooled plastic culture dish, add in the nucleus extraction liquid 1mL of precooling again, then double-edged razor blade chopper blade is adopted, chopping process lasts more than 10 minutes, first use 30 micrometer nylon membrane filtrations, collect containing nucleolate filtrate, again secondary chopping process is carried out to remaining blade, namely double-edged razor blade is adopted to continue chopper blade, chopping process lasts more than 10 minutes, until whole blades is cut into pasty state, rinse with nucleus extraction liquid again, filter, collect containing nucleolate extract, after the extract of twice collection is merged, add 10 microlitre RNaseAs (concentration is 1mg/mL), place more than 10 minutes under the condition of 4 DEG C after mixing, in extract, add concentration be again the ultimate density of iodate third ingot in the iodate third ingot solution to system of 1mg/mL is 0.21mg/mL, under lucifuge condition, dyeing is carried out 30 minutes to nucleus DNA, after having dyeed, carry out flow cytometry, preferred described flow cytometry adopts Attune sound wave to focus on stream type cell analyzer, and the optimum configurations that described Attune sound wave focuses on stream type cell analyzer is:
| Relevant parameter | Magnitude of voltage mV (millivolt) |
| FSC | 2300 |
| BL1 | 1700 |
| SSC | 2600 |
| BL2 | 1800 |
| LB3 | 1400 |
| VL1 | 1300 |
| VL2 | 1900 |
| VL3 | 2300 |
Concrete analysis result is illustrated in fig. 2 shown below, and the fluorescent value of the dry leaf sample of oriole is 986872, and the nuclear DNA content peak surveyed is very thin and concentrated, its CV value 3.24%, and as calculated, oriole is 2 times of bodies, identical with bibliographical information.
Embodiment 3
The present embodiment is that the ploidy of Solidago Canadensis measures, and the component of nucleus extraction liquid used in the present embodiment is:
Tris:18mM; Na
2eDTA:3mM; Spermine hydrochloride: 0.2mM; KCl:70mM, NaCl:25mM; Beta-mercaptoethanol: 20mM; Triton X-100:0.2% (vol/vol); PH value is 7.0, and pH value can adopt the NaOH solution of 1mM to regulate, and described nucleus extraction liquid is stored in the refrigerator of-20 DEG C for subsequent use in advance;
Solidago Canadensis mature leaf (blade through silica dehydrator) 45mg is put into precooled plastic culture dish, add in the nucleus extraction liquid 1mL of precooling again, then employing thickness is the double-edged razor blade chopper blade of 0.1mm, chopping process lasts more than 10 minutes, first use 30 micrometer nylon membrane filtrations, collect containing nucleolate filtrate, again secondary chopping process is carried out to remaining blade, namely employing thickness is the double-edged razor blade continuation chopper blade of 0.1mm, chopping process lasts more than 10 minutes, until whole blades is cut into pasty state, rinse with nucleus extraction liquid again, filter, collect containing nucleolate extract, after the extract of twice collection is merged, add 10 microlitre RNaseAs (concentration is 1mg/mL), place more than 10 minutes under the condition of 4 DEG C after mixing, in extract, add concentration be again the ultimate density of iodate third ingot in the iodate third ingot solution to system of 1mg/mL is 0.22mg/mL, under lucifuge condition, dyeing is carried out 30 minutes to nucleus DNA, after having dyeed, carry out flow cytometry.Preferred flow cytometry described above adopts Attune sound wave to focus on stream type cell analyzer, and the optimum configurations that described Attune sound wave focuses on stream type cell analyzer is:
| Relevant parameter | Magnitude of voltage mV (millivolt) |
| FSC | 2300 |
| BL1 | 1500 |
| SSC | 2600 |
| BL2 | 1900 |
| LB3 | 1400 |
| VL1 | 1200 |
| VL2 | 2000 |
| VL3 | 2400 |
The concrete analysis result of above-mentioned flow cytometry is consistent with the accordingly result in embodiment 1.
Embodiment 4
The present embodiment is that the ploidy of Solidago Canadensis measures, and the component of nucleus extraction liquid used in the present embodiment is:
Tris:15mM; Na
2eDTA:2mM; Spermine hydrochloride: 0.25mM; KCl:80mM, NaCl:20mM; Beta-mercaptoethanol: 15mM; Triton X-100:0.5% (vol/vol); PH value is 7.0, and pH value can adopt the NaOH solution of 1mM to regulate, and described nucleus extraction liquid is stored in the refrigerator of-20 DEG C for subsequent use in advance;
Solidago Canadensis mature leaf (the sample blade through pressing dry) 48mg is put into precooled plastic culture dish, add in the nucleus extraction liquid 1mL of precooling again, then employing thickness is the double-edged razor blade chopper blade of 0.3mm, chopping process lasts more than 10 minutes, first use 30 micrometer nylon membrane filtrations, collect containing nucleolate filtrate, again secondary chopping process is carried out to remaining blade, namely employing thickness is the double-edged razor blade continuation chopper blade of 0.3mm, chopping process lasts more than 10 minutes, until whole blades is cut into pasty state, rinse with nucleus extraction liquid again, filter, collect containing nucleolate extract, after the extract of twice collection is merged, add 10 microlitre RNaseAs (concentration is 1mg/mL), place more than 10 minutes under the condition of 4 DEG C after mixing, in extract, add concentration be again the ultimate density of iodate third ingot in the iodate third ingot solution to system of 1mg/mL is 0.18mg/mL, under lucifuge condition, dyeing is carried out 30 minutes to nucleus DNA, after having dyeed, carry out flow cytometry, , preferred described flow cytometry adopts Attune sound wave to focus on stream type cell analyzer, and the optimum configurations that described Attune sound wave focuses on stream type cell analyzer is:
| Relevant parameter | Magnitude of voltage mV (millivolt) |
| FSC | 2400 |
| BL1 | 1800 |
| SSC | 2500 |
| BL2 | 1800 |
| LB3 | 1300 |
| VL1 | 1300 |
| VL2 | 1900 |
| VL3 | 2100 |
The concrete analysis result of above-mentioned flow cytometry is consistent with the accordingly result in embodiment 1.
Embodiment 5
The present embodiment is that the ploidy of oriole measures, and the component of nucleus extraction liquid used in the present embodiment is:
Tris:15mM; Na
2eDTA:2mM; Spermine hydrochloride: 0.2mM; KCl:70mM, NaCl:20mM; Beta-mercaptoethanol: 10mM; Triton X-100:0.4% (vol/vol); PH value is 7.0, and pH value can adopt the NaOH solution of 1mM to regulate, and described nucleus extraction liquid is stored in the refrigerator of-20 DEG C for subsequent use in advance;
Oriole mature leaf (blade through natural air drying) 48mg is put into precooled plastic culture dish, add in the nucleus extraction liquid 1mL of precooling again, then employing thickness is the double-edged razor blade chopper blade of 0.2mm, chopping process lasts more than 10 minutes, first use 30 micrometer nylon membrane filtrations, collect containing nucleolate filtrate, again secondary chopping process is carried out to remaining blade, namely employing thickness is the double-edged razor blade continuation chopper blade of 0.2mm, chopping process lasts more than 10 minutes, until whole blades is cut into pasty state, rinse with nucleus extraction liquid again, filter, collect containing nucleolate extract, after the extract of twice collection is merged, add 10 microlitre RNaseAs (concentration is 1mg/mL), place more than 10 minutes under the condition of 4 DEG C after mixing, in extract, add concentration be again the ultimate density of iodate third ingot in the iodate third ingot solution to system of 1mg/mL is 0.20mg/mL, under lucifuge condition, dyeing is carried out 30 minutes to nucleus DNA, after having dyeed, carry out flow cytometry, preferred described flow cytometry adopts Attune sound wave to focus on stream type cell analyzer, and the optimum configurations that described Attune sound wave focuses on stream type cell analyzer is:
| Relevant parameter | Magnitude of voltage mV (millivolt) |
| FSC | 2350 |
| BL1 | 1700 |
| SSC | 2560 |
| BL2 | 1840 |
| LB3 | 1360 |
| VL1 | 1250 |
| VL2 | 1950 |
| VL3 | 2300 |
The concrete analysis result of flow cytometry is consistent with the corresponding analysis result in embodiment 2.
Embodiment 6
The present embodiment is the mensuration of fraises des bois ploidy, and described fraises des bois picks up from Tibet autonomous region of Tibetan Nyingchi County, and the component of nucleus extraction liquid used in the present embodiment is:
Tris:15mM; Na
2eDTA:2mM; Spermine hydrochloride: 0.2mM; KCl:70mM, NaCl:20mM; Beta-mercaptoethanol: 10mM; Triton X-100:0.4% (vol/vol); PH value is 7.0, and pH value can adopt the NaOH solution of 1mM to regulate, and described nucleus extraction liquid is stored in the refrigerator of-20 DEG C for subsequent use in advance;
Fraises des bois mature leaf (blade through natural air drying) 50mg is put into precooled plastic culture dish, add in the nucleus extraction liquid 1mL of precooling again, then employing thickness is the double-edged razor blade chopper blade of 0.3mm, chopping process lasts more than 10 minutes, first use 30 micrometer nylon membrane filtrations, collect containing nucleolate filtrate, again secondary chopping process is carried out to remaining blade, namely employing thickness is the double-edged razor blade continuation chopper blade of 0.2mm, chopping process lasts more than 10 minutes, until whole blades is cut into pasty state, rinse with nucleus extraction liquid again, filter, collect containing nucleolate extract, after the extract of twice collection is merged, add 10 microlitre RNaseAs (concentration is 1mg/mL), place more than 10 minutes under the condition of 4 DEG C after mixing, in extract, add concentration be again the ultimate density of iodate third ingot in the iodate third ingot solution to system of 1mg/mL is 0.22mg/mL, under lucifuge condition, dyeing is carried out 30 minutes to nucleus DNA, after having dyeed, carry out flow cytometry, preferred described flow cytometry adopts Attune sound wave to focus on stream type cell analyzer, and the optimum configurations that described Attune sound wave focuses on stream type cell analyzer is:
| Relevant parameter | Magnitude of voltage mV (millivolt) |
| FSC | 2350 |
| BL1 | 1700 |
| SSC | 2540 |
| BL2 | 1850 |
| LB3 | 1340 |
| VL1 | 1250 |
| VL2 | 1950 |
| VL3 | 2250 |
Concrete analysis result is illustrated in fig. 3 shown below, and the dry leaf sample of fraises des bois mainly demonstrates two peaks, and the fluorescent value at top is 282600, its CV value 4.67%, and as calculated, fraises des bois is 2 times of bodies, identical with bibliographical information.
Embodiment 6
The present embodiment is the mensuration of planting strawberry (beauty strawberry) ploidy, and concrete method wind embodiment 1 is consistent, repeats no more here.
Concrete analysis result is illustrated in fig. 4 shown below, and the fluorescent value of the dry leaf sample of planting strawberry is 918891, its CV value 2.71%, and as calculated, planting strawberry is 8 times of bodies, identical with bibliographical information.
Embodiment 7
The present embodiment is to verify that method of the present invention is generally applicable to all kinds of dry tissue test of plant, the present embodiment measures with the different tissues of planting strawberry (beauty strawberry), concrete disposal route is consistent with embodiment 1, difference is all kinds of dry setup action test sample selecting planting strawberry, and concrete test result is as follows.
As shown in Figure 5, be select the dry leaf sample of planting strawberry, it be 954729, CV value is 2.17% that its flow cytometric analysis results shows its fluorescent value.
As shown in Figure 6, be select the dry petiole sample of planting strawberry, it be 892773, CV value is 2.60% that its flow cytometric analysis results shows its fluorescent value.
As shown in Figure 7, be select the dry stolon sample of planting strawberry, it be 872748, CV value is 3.03% that its flow cytometric analysis results shows its fluorescent value.
As shown in Figure 8, be select the dry tip of a root sample of planting strawberry, it be 882152, CV value is 1.84% that its flow cytometric analysis results shows its fluorescent value.
Embodiment 8
The present embodiment is to verify that method of the present invention is generally applicable to all kinds of plant drying tissue test, concrete disposal route is consistent with embodiment 1, difference is all kinds of dry setup action test sample selecting planting strawberry, and concrete test result is as follows.
As shown in Figure 9, be select trefoil dry blade (blade through silica dehydrator) sample, it be 1009494, CV value is 3.30% that its flow cytometric analysis results shows its fluorescent value.
As shown in Figure 10, be dry blade (blade through the silica dehydrator) sample selecting throatroot, it be 410217, CV value is 3.94% that its flow cytometric analysis results shows its fluorescent value.
As shown in figure 11, be dry blade (blade through the silica dehydrator) sample selecting Alternanthera philoxeroides, it be 1622145, CV value is 4.74% that its flow cytometric analysis results shows its fluorescent value.
As shown in figure 12, be dry blade (blade through the silica dehydrator) sample selecting Horseweed Herb, it be 307825, CV value is 3.61% that its flow cytometric analysis results shows its fluorescent value.
As shown in figure 13, be dry blade (blade through the silica dehydrator) sample selecting black nightshade, it be 959260, CV value is 4.79% that its flow cytometric analysis results shows its fluorescent value.
As shown in figure 14, be dry blade (blade through the silica dehydrator) sample selecting orange, it be 375486, CV value is 4.46% that its flow cytometric analysis results shows its fluorescent value.
As shown in figure 15, be dry blade (blade through the silica dehydrator) sample selecting soybean, it be 1186217, CV value is 3.97% that its flow cytometric analysis results shows its fluorescent value.
As shown in figure 16, be dry blade (blade through the silica dehydrator) sample selecting safflower Loropetalum wood, it be 1385057, CV value is 4.67% that its flow cytometric analysis results shows its fluorescent value.
As shown in figure 17, be dry blade (blade through the silica dehydrator) sample selecting paper mulberry, it be 360781, CV value is 4.29% that its flow cytometric analysis results shows its fluorescent value.
As shown in figure 18, be dry blade (blade through the silica dehydrator) sample selecting bidens pilosa, it be 1074118, CV value is 3.02% that its flow cytometric analysis results shows its fluorescent value.
Specific embodiment described in the present invention is only to the explanation for example of the present invention's spirit.Those skilled in the art can make various amendment or supplement or adopt similar mode to substitute to described specific embodiment, but can't depart from spirit of the present invention or surmount the scope that appended claims defines.
Although made a detailed description the present invention and quoted some specific embodiments as proof, to those skilled in the art, only otherwise it is obvious for leaving that the spirit and scope of the present invention can make various changes or revise.
Claims (9)
1. be applicable to a disposal route for flow cytometry plant drying tissue, this disposal route comprises nucleus extraction, dyeing and analysis, it is characterized in that, the component of described nucleus extraction nucleus extraction liquid used is:
Tris:12mM ~ 18mM; Na
2eDTA:1mM ~ 3mM; Spermine hydrochloride: 0.2mM ~ 0.3mM; KCl:70mM ~ 90mM, NaCl:10mM ~ 25mM; Beta-mercaptoethanol: 10mM ~ 20mM; Triton X-100:0.2% (vol/vol) ~ 0.5% (vol/vol); PH value is 6.5 ~ 7.0.
2. be applicable to the disposal route of flow cytometry plant drying tissue according to claim 1, it is characterized in that, this disposal route specifically comprises the following steps:
A, nucleus extraction: the nucleus extraction liquid adding precooling in the plant tissue of drying, then carry out chopping process, cross and filter residue, collect containing nucleolate filtrate;
B, dyeing and analysis: in above-mentioned filtrate, add RNaseA and iodate third ingot carry out dyeing process, the concentration of described iodate third ingot is 0.15mg/mL ~ 0.25mg/mL, after dyeing process, carries out flow cytometry.
3. be applicable to the disposal route of flow cytometry plant tissue according to claim 2, it is characterized in that, the detailed process of shredding process described in steps A is:
The plant tissue of the drying of precooling is put into the container of precooling, add the nucleus extraction liquid of precooling, subsequently with blade, plant tissue is carried out first time and shred process, filter, collect containing nucleolate filtrate, adopt blade that remaining plant tissue is carried out second time again and shred process, until whole plant tissues is chopped into pasty state.
4. be applicable to the disposal route of flow cytometry plant tissue according to claim 3, it is characterized in that, the amount ratio that the weight of plant tissue dry described in steps A and nucleus extraction liquid amass is: 50:1 ~ 45:1, the unit of weight of the plant tissue of described drying is milligram, and the volume unit of described nucleus extraction liquid is milliliter.
5. according to claim 3 or 4, be applicable to the disposal route of flow cytometry plant tissue, it is characterized in that, the thickness of described blade is 0.1mm ~ 0.3mm.
6. according to claim 2-4 any one for the disposal route of flow cytometry plant tissue, it is characterized in that, the concentration of the ingot of iodate third described in step B is 0.18mg/mL ~ 0.22mg/mL.
7. according to claim 2-4 any one, be applicable to the disposal route of flow cytometry plant tissue, it is characterized in that, plant tissue dry described in steps A is selected from through silica dehydrator, natural air drying or the plant drying tissue pressing dry sample.
8. according to claim 2-4 any one, be applicable to the disposal route of flow cytometry plant tissue, it is characterized in that, the flow type analyzer that flow cytometry described in step B adopts is that Attune sound wave focuses on stream type cell analyzer.
9. be applicable to the disposal route of flow cytometry plant tissue according to claim 8, it is characterized in that, the optimum configurations that the sound wave of Attune described in step B focuses on stream type cell analyzer is:
。
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