CN108680418A - A kind of rapid fluorescence colouring method of crop in cruciferae pollen - Google Patents
A kind of rapid fluorescence colouring method of crop in cruciferae pollen Download PDFInfo
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- 239000000975 dye Substances 0.000 claims abstract description 18
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- INAAIJLSXJJHOZ-UHFFFAOYSA-N pibenzimol Chemical compound C1CN(C)CCN1C1=CC=C(N=C(N2)C=3C=C4NC(=NC4=CC=3)C=3C=CC(O)=CC=3)C2=C1 INAAIJLSXJJHOZ-UHFFFAOYSA-N 0.000 claims abstract description 16
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 14
- 229930006000 Sucrose Natural products 0.000 claims abstract description 14
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 14
- 239000005720 sucrose Substances 0.000 claims abstract description 14
- 235000019441 ethanol Nutrition 0.000 claims abstract description 9
- 239000008363 phosphate buffer Substances 0.000 claims abstract description 9
- 125000004494 ethyl ester group Chemical group 0.000 claims abstract description 7
- 235000011187 glycerol Nutrition 0.000 claims abstract description 7
- 230000008569 process Effects 0.000 claims description 5
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims 1
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- 229910052698 phosphorus Inorganic materials 0.000 claims 1
- 239000011574 phosphorus Substances 0.000 claims 1
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 claims 1
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- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
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- General Health & Medical Sciences (AREA)
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
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Abstract
The invention discloses a kind of rapid fluorescence colouring methods of crop in cruciferae pollen, include the following steps：S1 dyeing liquors are prepared：Hoechst33258 fluorescent dyes and sucrose are dissolved in phosphate buffer PBS first, it is made into the solution one of 20 40 μ g/L of Hoechst33258 concentration, 8 10wt% of sucrose concentration, then the solution two containing ethyl alcohol 60 70% (v/v), ethyl ester acetic acid 10% (v/v), glycerine 30 20% (v/v) is prepared, by solution one and solution two by volume 1:1 mixing shakes up to obtain dyeing liquor, and keeps in dark place at low temperature；S2 is dyed：Take in step S1 dyeing liquor obtained on glass slide, then the opening flower or bud from being won on inflorescence and needing to dye, it is won with tweezers in the dyeing liquor that anther is directly placed on glass slide, gently pinching anther, shed pollen, sundries is taken out, smearing makes pollen be uniformly mixed with dyeing liquor, then covered.The present invention can solve the problems, such as cumbersome, the time-consuming and work consuming present in current pollen fluorescence colour, while improve the stability and reproducibility of fluorescence colour.
Description
Technical field
The present invention relates to a kind of pollen fluorescent staining methods, and in particular to a kind of rapid fluorescence of crop in cruciferae pollen
Colouring method.
Background technology
Plant pollen wall is one layer of special cells wall being mainly made of sporopollenin and cellulose, by two layers of outer wall and inner wall
Composition.Wherein, it is inside and outside two layers that outer wall, which is divided into, and sexine is in cellular or netted, and intexine is then flat structures, outer wall
Main component be sporopollenin substance.And interior wall construction is relatively easy, mainly contains cellulose, pectin and protein etc..By
More complicated than general plant cell wall in structure in plant pollen wall and close thick and solid, especially mature flower powder is penetrating
Property and translucidus it is very poor, therefore do not combined after most plants pollen staining transparent technology be difficult to observe under the microscope it is clear thin
Karyon.
Currently, plant pollen, which dyes common method, acetic red dyeing, phenol magenta method, the dyeing of siderotil hematoxylin
Method and fluorescence colour etc., the general operation step using " fixed (Ka Nuoshi fixers) → washing → dyeing → decoloration → microscopy "
Suddenly, but for most of plant maturation pollen, it is also necessary to it could be observed after alcohol grading dehydration and wintergreen are transparent,
Its is complex for operation step, time-consuming, completes whole operation process and needs several hours or more, even up to 2-3 days.
In terms of the specificity of coloring agent, acetic red dyeing, phenol magenta method and siderotil hematoxylin staining are used
Coloring agent compared with fluorescent dye used in fluorescent staining (such as Hoechst33258, DAPI), specificity relatively
Difference, confrontation core can dye, and only dye levels are different, therefore dyeing time and decoloration influence greatly coloration result,
In addition the difference of the temperature condition of its coloring also because of crop species, Pollen stage and when dyeing etc. has very big difference
It is different, thus these methods in practical operation often because dyeing time is long too short or decoloration it is improper due to cannot get clearly,
And the result of matter nuclear staining contrast distinctness.Fluorescence colour uses the fluorescent dye with DNA specific bonds, with DNA chain
It is exceedingly fast in conjunction with speed, can reach saturation in more than ten minutes, be not in cause cytoplasm to dye because dyeing time is long
Deep the problem of influencing result observation, and coloration result is stablized, therefore fluorescence colour is in the experiment for having fluorescence microscope
Under the conditions of have become plant pollen dyeing observation prefered method.
In recent years, free pollen (microspore) culture of crop in cruciferae is widely applied in breeding, is being spent
It needs accurately to conclude its Pollen stage when powder culture, since fluorescent dye is to the specificity and stability of DNA,
Using fluorescer dye observation determine Pollen stage researcher tend to increase, but be all made of it is above-mentioned it is cumbersome, take
Operating procedure, it is difficult to accomplish to complete the judgement of Pollen stage in the short time, and mature flower powder is using conventional Chinese ilex
Oily transparent technology can make the boundary of pollen wall profile and background become unclear, and the fluorescent staining intensity of trophonucleus also can be because of dehydration step
Suddenly it decreases, to make the effect of fluorescence colour have a greatly reduced quality.
Invention content
In view of the deficiencies of the prior art, the present invention is intended to provide a kind of rapid fluorescence dyeing of crop in cruciferae pollen
Method solves the problems, such as cumbersome, the time-consuming and work consuming present in current pollen fluorescence colour, while improving the steady of fluorescence colour
Qualitative and reproducibility.
To achieve the goals above, the present invention adopts the following technical scheme that：
A kind of rapid fluorescence colouring method of crop in cruciferae pollen, includes the following steps：
S1 dyeing liquors are prepared：Hoechst33258 fluorescent dyes and sucrose are dissolved in phosphate buffer PBS first, are made into
Then the solution one of Hoechst33258 concentration 20-40 μ g/L, sucrose concentration 8-10wt% prepare the (v/ of 60-70% containing ethyl alcohol
V), ethyl ester acetic acid 10% (v/v), glycerine 30-20% (v/v) solution two, by solution one and solution two by volume 1:1 mixing
It shakes up to obtain dyeing liquor, and keeps in dark place at low temperature；
S2 is dyed：Dyeing liquor obtained is taken in step S1 on glass slide, then from being won on inflorescence and needing to dye
Opening flower or bud, win in the dyeing liquor that anther is directly placed on glass slide, gently pinching anther, shed pollen, takes
Go out sundries, smearing makes pollen be uniformly mixed with dyeing liquor, then covered.
Further, the pH of the phosphate buffer PBS is 7.4.
Further, in step S1, dyeing liquor is kept in dark place at a temperature of 4 DEG C.
Further, step S2 detailed processes are：
Dyeing liquor obtained is taken in 15-20 μ L steps S1 on glass slide, then from being won on inflorescence and needing to dye
Opening flower or bud, won with tweezers in the dyeing liquors that anther 1-2 are directly placed on glass slide, gently pinching anther,
Shed pollen, takes out sundries, and smearing 5-10 seconds makes pollen be uniformly mixed with dyeing liquor, then covered, waits for 0-5 points
Clock, dyeing are completed.
The beneficial effects of the present invention are：
1, the rapid fluorescence colouring method of crop in cruciferae pollen of the invention only needs a step that traditional flower can be completed
The series of operation steps of powder decoration method are not only simple, fast, and entire dying operation process can be completed for 2-5 minutes, Er Qieran
Color effect stability, under room temperature dark condition at least 2 weeks it is constant, matter nuclear staining contrast is apparent, pollen it is clear-cut as it can be seen that repeat
Property is good.
2, the present invention may be implemented once to only use dyeing liquor 15-20 μ L, also few to the requirement of sample number, can be significantly
It saves experimental study cost and reduces environmental pollution.
The present invention can be used for the reality of Pollen stage when the research of crop in cruciferae pollen fertility, microspore-isolated culture
When determining and pollen cultures during the cytological observation etc. of microspore nuclear fission and embryoid development.
Description of the drawings
Fig. 1 is to carry out the fluorescent staining microscopic examination result schematic diagram that rape opens flower mature pollen using the method for the present invention
(400x)；
Fig. 2 is the fluorescent staining microscopic examination result schematic diagram (400x) that Flower Bud of Chinese Cabbage pollen is carried out using the method for the present invention
Fig. 3 is the fluorescent staining microscopic examination result schematic diagram (400x) that cabbage heart bud pollen is carried out using the method for the present invention；
Fig. 4 is the fluorescent staining microscopy that microspore division during the free pollen cultures of cabbage mustard is carried out using the method for the present invention
Result schematic diagram (400x)；
Fig. 5 is the fluorescent staining microscopic examination result schematic diagram (400x) that rape bud pollen is carried out using conventional method.
Specific implementation mode
Below with reference to attached drawing, the invention will be further described, it should be noted that following embodiment is with this technology
Premised on scheme, detailed embodiment and specific operating process are given, but protection scope of the present invention is not limited to this
Embodiment.
Embodiment 1：Rape opens the fluorescent staining method of flower mature pollen
1, dyeing liquor is prepared：Hoechst33258 fluorescent dyes and sucrose are dissolved in phosphate buffer (PBS, pH first
=7.4) it, is made into the solution one of 20 μ g/L of Hoechst33258 concentration, sucrose concentration 10wt%, then prepares and contains ethyl alcohol 60%
(v/v), the solution two of ethyl ester acetic acid 10% (v/v), glycerine 30% (v/v), by solution one and solution two by volume 1:1 mixing
Dyeing liquor is obtained after shaking up, and is placed in 4 DEG C of refrigerators and is kept in dark place.
2, it dyes：The obtained dyeing drop of 15 μ L steps 1 is drawn on glass slide, then from inflorescence with micropipettor
The opening flower for needing to dye observation is won, is won in 1 dyeing liquor being directly placed on glass slide of anther with tweezers, is gently pinched
Anther is squeezed, shed appropriate pollen, takes out and is crushed the sundries such as anther wall, and smearing for a moment (5-10 seconds) keeps pollen mixed with the dyeing liquor
It closes uniformly, then covered.
3, it observes and photographs to record：After covered, it is placed under fluorescence microscope (Olympus BX51) and observes, object lens
40 times of amplification factor, 10 times of eyepiece amplification factor.It is photographed to record using MicroPublisher 5.0RTV cameras.
The results are shown in Figure 1, and mature flower powder is clear-cut, after trophonucleus and two sperm nucleus are colored, in ultraviolet light
Blue-fluorescence is sent out under irradiation, matter core contrast is apparent, high-visible.
Embodiment 2：The fluorescent staining method of Flower Bud of Chinese Cabbage pollen
1, dyeing liquor is prepared：Hoechst33258 fluorescent dyes and sucrose are dissolved in phosphate buffer (PBS, pH first
=7.4) it, is made into the solution one of 30 μ g/L of Hoechst33258 concentration, sucrose concentration 8wt%, then prepares the 70% (v/ containing ethyl alcohol
V), the solution two of ethyl ester acetic acid 10% (v/v), glycerine 20% (v/v), by solution one and solution two by volume 1:1 mixing is shaken
Dyeing liquor is obtained after even, is placed in 4 DEG C of refrigerators and is kept in dark place.
2, it dyes：Draw in 17 μ L steps 1 that dyeing drop obtained is on glass slide with micropipettor, then from inflorescence
On win need dye observation bud, stripped in 1 dyeing liquor being directly placed on glass slide of anther with tweezers, gently pinching
Anther, shed appropriate pollen, takes out and is crushed the sundries such as anther wall, and smear makes pollen be mixed with the dyeing liquor for a moment (5-10 seconds)
Uniformly, then covered.
3, it observes and photographs to record：After covered 2 minutes, it is placed under fluorescence microscope (Olympus BX51) and sees
It examines, 40 times of object lens magnification, 10 times of eyepiece amplification factor.It is photographed to record using MicroPublisher 5.0RTV cameras.
The results are shown in Figure 2, and pollen grain is clear-cut, after trophonucleus and two sperm nucleus are colored, is irradiated in ultraviolet light
Under send out blue-fluorescence, matter core contrast is apparent, high-visible.
Embodiment 3：The fluorescent staining method of cabbage heart bud pollen
1, dyeing liquor is prepared：Hoechst33258 fluorescent dyes and sucrose are dissolved in phosphate buffer (PBS, pH first
=7.4) it, is made into the solution one of 40 μ g/L of Hoechst33258 concentration, sucrose concentration 9wt%, then prepares the 65% (v/ containing ethyl alcohol
V), the solution two of ethyl ester acetic acid 10% (v/v), glycerine 25% (v/v), by solution one and solution two by volume 1:1 mixing is shaken
Dyeing liquor is obtained after even, is placed in 4 DEG C of refrigerators and is kept in dark place.
2, it dyes：Obtained dyeing drop is drawn in 20 μ L steps 1 on glass slide, then from inflorescence with micropipettor
On win need dye observation bud, won in 2 dyeing liquors being directly placed on glass slide of anther with tweezers, gently pinching
Anther, shed appropriate pollen, takes out and is crushed the sundries such as anther wall, and smear makes pollen be mixed with the dyeing liquor for a moment (5-10 seconds)
Uniformly, then covered.
3, it observes and photographs to record：After covered 3 minutes, it is placed under fluorescence microscope (Olympus BX51) and sees
It examines, 40 times of object lens magnification, 10 times of eyepiece amplification factor.It is photographed to record using MicroPublisher 5.0RTV cameras.
The results are shown in Figure 3, and mature flower powder is clear-cut, after trophonucleus and two sperm nucleus are colored, in ultraviolet light
Blue-fluorescence is sent out under irradiation, matter core contrast is apparent, high-visible.
Embodiment 4：The fluorescent staining method that microspore is divided during the free pollen cultures of cabbage mustard
1, dyeing liquor is prepared：Hoechst33258 fluorescent dyes and sucrose are dissolved in phosphate buffer (PBS, pH first
=7.4) it, is made into the solution one of 40 μ g/L of Hoechst33258 concentration, sucrose concentration 10wt%, then prepares and contains ethyl alcohol 70%
(v/v), the solution two of ethyl ester acetic acid 10% (v/v), glycerine 20% (v/v), by solution one and solution two by volume 1:1 mixing
It shakes up to obtain dyeing liquor, is placed in 4 DEG C of refrigerators and is kept in dark place.
2, it dyes：20 μ L steps 1 dyeing drop obtained is drawn on glass slide with micropipettor, then is drawn through centrifugation
Culture pollen (32 DEG C heat shock culture 48 hours) 1 μ L drops of precipitation gently smear a moment (5-10 with tweezers in the dyeing liquor
Second), then covered.
3, it observes and photographs to record：Covered after five minutes, is placed under fluorescence microscope (Olympus BX51) and sees
It examines, 40 times of object lens magnification, 10 times of eyepiece amplification factor.It is photographed to record using MicroPublisher 5.0RTV cameras.
The results are shown in Figure 4, and in 32 DEG C after heat shock culture 48 hours, pollen grain obviously expands mid-late uninucleate stage pollen,
It can be seen that the pollen of trophonucleus homeokinesis 1 time at 2 cores, and the pollen of 8 cores are formed by 3 divisions, matter core contrast is apparent, clearly
It is clear visible.
Comparative example：Traditional fluorescent staining method
10, anther is stripped from the rape bud that next day opens to be placed in 25X25mm measuring cups, with Ka Nuoshi fixers
(absolute ethyl alcohol：Glacial acetic acid=3：1, v/v) 48 hours are fixed, then with 95%, 70%, 50%, 25% ethyl alcohol and distilled water
Transition is washed, each 30 minutes of the time, later with the Hoechst33258 phosphate buffers (PBS, pH=7.4) of a concentration of 20 μ g/L
Dye 3 hours under room temperature dark condition, then with 30%, 50%, 70%, 95% Gradient elution using ethanol, 30 minutes at different levels, most
Soaked in absolute ethyl alcohol 1 hour or more is used afterwards, is then used absolute ethyl alcohol with after wintergreen mixed liquor (1: 1, v/v) displacement, then is used the winter
Green oil changes liquid 3 times, 1-2 hours each.The reagent and distilled water that above step uses are 2mL, after wintergreen is transparent, take 1
Anther and a small amount of wintergreens with tweezers gently pinching anther, allow pollen to dissociate, then covered is glimmering on glass slide
It is observed under light microscope (Olympus BX51), 40 times of object lens magnification, 10 times of eyepiece amplification factor photographs to record use
MicroPublisher 5.0RTV cameras.
The results are shown in Figure 5, and pollen essences nuclear staining is clear, but trophonucleus fluorescence intensity is not as good as the method for embodiment 1-4, and
And the boundary of pollen wall and background is unclear, influences result judgement.
For those skilled in the art, it can be provided various corresponding according to above technical solution and design
Change and distortion, and all these change and distortions, should be construed as being included within the protection domain of the claims in the present invention.
Claims (4)
1. a kind of rapid fluorescence colouring method of crop in cruciferae pollen, which is characterized in that include the following steps：
S1 dyeing liquors are prepared：Hoechst33258 fluorescent dyes and sucrose are dissolved in phosphate buffer PBS first, are made into
The solution one of Hoechst33258 concentration 20-40 μ g/L, sucrose concentration 8-10wt%, then dose volume percentage is containing ethyl alcohol
The solution two of 60-70%, ethyl ester acetic acid 10%, glycerine 30-20%, by solution one and solution two by volume 1:1 mixing shakes up
Dyeing liquor is obtained, and is kept in dark place at low temperature；
S2 is dyed：Take in step S1 dyeing liquor obtained on glass slide, then opening from being won on inflorescence and needing to dye
It lets off fireworks piece or bud, wins in the dyeing liquor that anther is directly placed on glass slide, gently pinching anther, shed pollen, takes out miscellaneous
Object, smearing make pollen be uniformly mixed with dyeing liquor, then covered.
2. the rapid fluorescence colouring method of crop in cruciferae pollen according to claim 1, which is characterized in that the phosphorus
The pH of phthalate buffer PBS is 7.4.
3. the rapid fluorescence colouring method of crop in cruciferae pollen according to claim 1, which is characterized in that step S1
In, dyeing liquor is kept in dark place at a temperature of 4 DEG C.
4. the rapid fluorescence colouring method of crop in cruciferae pollen according to claim 1, which is characterized in that step S2
Detailed process is：
Take in 15-20 μ L steps S1 dyeing liquor obtained on glass slide, then opening from being won on inflorescence and needing to dye
It lets off fireworks piece or bud, is won in 1-2 dyeing liquors being directly placed on glass slide of anther with tweezers, gently pinching anther, sheds
Pollen takes out sundries, and smearing 5-10 seconds makes pollen be uniformly mixed with dyeing liquor, then covered, waits for 0-5 minutes, dye
Color is completed.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810555491.4A CN108680418B (en) | 2018-06-01 | 2018-06-01 | Dyeing liquid and method for quickly dyeing cruciferous crop pollen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810555491.4A CN108680418B (en) | 2018-06-01 | 2018-06-01 | Dyeing liquid and method for quickly dyeing cruciferous crop pollen |
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| Publication Number | Publication Date |
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| CN108680418A true CN108680418A (en) | 2018-10-19 |
| CN108680418B CN108680418B (en) | 2021-03-23 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2717539C1 (en) * | 2019-04-10 | 2020-03-23 | Федеральное государственное бюджетное учреждение науки Сибирский федеральный научный центр агробиотехнологий Российской академии наук (СФНЦА РАН) | Method for determining botanical origin of honey |
| CN112903636A (en) * | 2021-01-15 | 2021-06-04 | 中国农业科学院蜜蜂研究所 | Method for rapidly determining pollination strength of crops |
| CN112924259A (en) * | 2021-02-01 | 2021-06-08 | 上海师范大学 | Observation method for specifically identifying different parts of pollen, corresponding dye and application |
| CN114018919A (en) * | 2021-10-27 | 2022-02-08 | 中国热带农业科学院海口实验站 | Method for detecting diploid banana pollen activity |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2717539C1 (en) * | 2019-04-10 | 2020-03-23 | Федеральное государственное бюджетное учреждение науки Сибирский федеральный научный центр агробиотехнологий Российской академии наук (СФНЦА РАН) | Method for determining botanical origin of honey |
| CN112903636A (en) * | 2021-01-15 | 2021-06-04 | 中国农业科学院蜜蜂研究所 | Method for rapidly determining pollination strength of crops |
| CN112903636B (en) * | 2021-01-15 | 2023-03-10 | 中国农业科学院蜜蜂研究所 | Method for rapidly determining pollination strength of crops |
| CN112924259A (en) * | 2021-02-01 | 2021-06-08 | 上海师范大学 | Observation method for specifically identifying different parts of pollen, corresponding dye and application |
| CN114018919A (en) * | 2021-10-27 | 2022-02-08 | 中国热带农业科学院海口实验站 | Method for detecting diploid banana pollen activity |
| CN114018919B (en) * | 2021-10-27 | 2023-06-16 | 中国热带农业科学院海口实验站 | Method for detecting activity of diploid banana pollen |
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