CN109752226A - The method and application of pollen activity fast quantification under a kind of room temperature or high temperature - Google Patents
The method and application of pollen activity fast quantification under a kind of room temperature or high temperature Download PDFInfo
- Publication number
- CN109752226A CN109752226A CN201910010240.2A CN201910010240A CN109752226A CN 109752226 A CN109752226 A CN 109752226A CN 201910010240 A CN201910010240 A CN 201910010240A CN 109752226 A CN109752226 A CN 109752226A
- Authority
- CN
- China
- Prior art keywords
- pollen
- dyeing
- anther
- ttf
- activity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
The invention belongs to plant physiology and biochemistry technical applications.More particularly to the method and application of pollen activity fast quantification under a kind of room temperature or high temperature.The formula of pollen TTC dyeing liquor under room temperature and high temperature, dyeing concentration and most suitable dyeing time and the scope of application has been determined, has been prepared for compound staining solution formula: 1mol/L potassium dihydrogen phosphate 5.23g, dipotassium hydrogen phosphate 14.03g, the kaliumphosphate buffer that pH is 7；The 2,3, 5-Triphenyltertrazoliumchloride of 8g (TTC) is dissolved in resulting kaliumphosphate buffer again, is settled to 1L with distilled water, makes its final concentration of 8g/L.The present invention can be used for carrying out pollen activity quantitative detection to high-volume material, and the microscopic examination after dyeing is omitted simplifies the process of high-volume material pollen activity identification, be suitable for cotton, the identification of the various crops pollen activity such as rice.
Description
Technical field
The invention belongs to plant physiology and biochemistry technical applications.It is fast more particularly to pollen activity under a kind of room temperature or high temperature
The quantitative method of speed and dye and extract by dyeing liquor using the method, measure absorbance carry out quantitative scheme into
Row.One aspect of the present invention greatly simplifies operating process, on the other hand can also carry out accurate quantification to the vigor of pollen.
Background technique
With global warming, High Temperature Stress has become one of the Main Factors for influencing crop production.Plant organ hair
The variation educated to environment temperature is very sensitive, and high temperature will lead to the dysplasia of reproductive organs.Male reproductive organ is for environment
The sensitivity of temperature change is higher than female reproductive organ, especially stamen, and the extreme temperature of developmental stage can all lead to stamen
Or andro gamete (pollen) dysplasia causes male sterility.It is existing much to lead to male sterile report about high temperature stress, such as
Barley, arabidopsis, rice and cotton, among these due to pollen activity influenced by high temperature and abortion accounts for the overwhelming majority.Pollen
Activity be determine plant female organ can normal fertilization one of key factor, be directly related to plant can generate seed or
Great-hearted seed.
Influence of the high temperature to pollen activity is as there are difference for the time difference of stress, in the survey article of Zuo Dandan et al.
In, summarize common several pollen activity colouring methods, including I at this stage2- KI, fluorescent dye double-staining, acid fuchsin
Decoration method etc..The principle of these detection methods is substantially using stain, is contaminated a series of active contents in pollen
Color, carries out sediments microscope inspection, then by count the number of fertile pollen and pollen sterile in the visual field carry out further fertility and
Sterile degree determines.
It is to do germination of pollen tube test there are also a kind of detection method, pollen tube is placed on germination medium, carries out pollen
Tube germination induction identifies sterile degree by counting to sprout Average pollen number and do not sprout the number of pollen.
All there are some disadvantages in above method, such as qualitative to vigor progress can not only can be carried out quantitatively, not can be carried out large quantities of
The sample identification of amount is analyzed, and counting is affected by artificial subjective factor.Therefore, a kind of simple, accurate pollen is developed
Vigor qualitative and quantitative scheme plays a significant role accurate evaluation pollen activity.
2,3, 5-Triphenyltertrazoliumchloride (TTC) decoration method is existing frequently-used pollen activity identification method, and TTC is one
Kind redox pigment is colourless solution when being dissolved in water.TTC can enter plant cell, and by the dehydrogenase in plant cell
Reduction generates triphen methyl hydrazone (TTF) not soluble in water, and TTF is very stable at room temperature, is not easy to be oxidized, while the murder by poisoning to cell
Very little, so TTC passes through the coloring agent frequently as plant tissue, cell, including pollen activity identification.
TTC enters after pollen cell, TTF can be generated in pollen after being dehydrogenated enzyme reduction, and the TTF in pollen is not
It can enter in coloring agent through cell membrane, it is possible to the pollen after dyeing be carried out to microscopy under the microscope with the work to pollen
Property is counted.Active pollen can be red-dyed, under the microscope can be but each by easily discernable
The activity of a pollen be all it is different, the ability for generating TTF is also different.In be detected pollen, there are many
The medium pollen of activity.Under the microscope, such pollen activity very difficult discrimination of human eye.Simultaneously as be microscopy, it can not
Ensure to select the pollen activity of the pollen activity statistical result energy accurate representation material in the visual field, and to large batch of material
Carrying out can be fairly time consuming laborious when activity counts.Thus more problem is derived, causes TTC decoration method in pollen activity
Extensive use in identification is had too many difficulties to cope with.
TTC dyeing sizing technique of the present invention is the method based on microscopy after pollen TTC dyeing.It is tried by optimization
Proved recipe case, the present invention used in phosphate (preferably potassium phosphate) buffer TTC pollen staining can be made to carry out at normal temperature,
And it further determined the most suitable dyeing duration.The present invention is quantitative by comparison pollen simultaneously and anther is quantitative, specifies
Can accurately assess pollen activity by anther active level, both methods can satisfy large batch of pollen activity it is qualitative and
Quantitative detection.
Summary of the invention
Present invention aims to overcome that various decoration methods cannot be quick now, high-volume carries out lacking for pollen activity identification
It falls into, assesses pollen activity for quantitative method is carried out to extraction triphen methyl hydrazone (TTF) after pollen or the dyeing of entire anther, and
This method is used for large batch of pollen activity identification, to simplify quantitative work process, improves the accuracy of detection.
The present invention relates to the formula for the compound staining solution (reagent) that quantitative analysis uses, optimum dyeing time, dyeing liquors
Extracting process also relates to the application during a large amount of identification anther/pollen activities.Quantitative Treatment scheme of the invention can be used
In the qualitative and quantitative experiment of various plants anther/pollen activity.
It is described that technical scheme is as follows:
2,3, 5-Triphenyltertrazoliumchloride (TTC) dyeing of usual plant tissue needs to carry out under the conditions of 37 DEG C, Shen
It asks someone first at room temperature to grope optimal dyeing liquor osmotic pressure, 0mol/L, 0.05mol/L has been respectively adopted,
The kaliumphosphate buffer of 0.1mol/L, 0.2mol/L, 0.5mol/L, 1mol/L carry out pollen staining and settling test.Test knot
Fruit is as shown in Figures 2 and 3.By repeatedly attempting, it is determined that TTC can be under Yu Changwen or high temperature to plant pollen activity fast quantification
Method compound staining solution formula: i.e.: 1mol/L potassium dihydrogen phosphate (38.5mL, 5.23g), dipotassium hydrogen phosphate) 61.5mL,
14.03g), the kaliumphosphate buffer (normal osmotic pressure that the buffer can be used for keeping cell) of pH=7, by 8g 2,3,5- tri-
Tetraphenylphosphonium chloride tetrazole (TTC) is dissolved in above-mentioned resulting kaliumphosphate buffer, and is settled to 1L with distilled water, makes its final concentration
It (is kept in dark place) for 8g/L.
Applicant is using this compound staining solution to the crop genetic improvement country, Hua Zhong Agriculture University where the applicant
The cotton temperature sensing material of identification major test room early period is compareed in room temperature and the pollen in high-temperature process period has carried out pollen dye
Color (see Fig. 4).It is contaminated using pollen of the compound staining solution to dicotyledonous crops such as rape and monocot crops such as rice
Color determines that the compound staining solution is equally applicable to rape and rice, does not have great-hearted pollen that cannot be red-dyed, it is easy to
It is distinguished, there is certain extensive use potentiality (see Fig. 5).
TTF quantitative detection in the present invention is completed in microplate reader, and applicant is during standard curve is formulated
It was found that single hole sample TTF maximum applied sample amount does not exceed 30 μ g, the absorbance read more than microplate reader later is not with linear shape
Formula increases, as a result as shown in Figure 6.
Applicant carried out the comparative tests of two schemes at the same time: i.e. scheme 1: carrying out being protected from light dye merely to pollen
Color then carries out TTF and quantitatively dyes, it is found that the amount of TTF is linearly increased with the quality rising of pollen, and quantitative result is shown in Fig. 7, and 8
It is shown.Since in dye liquor, cotton anthers tissue will not be caught color (as shown in Figure 9), so further progress scheme 2:
Anther is taken into dyeing liquor, progress TTF is quantitative after being protected from light dyeing.The amount of TTF also linearly increases with the quality of anther (quantitative
Shown in the result is shown in Figure 10 and Figure 11).
Beneficial effects of the present invention:
By the analysis quantitative to the quantitative and whole anther of pollen, applicant is to test operation, including when dyeing continues
Between, sampling mode, quantitative approach etc. has carried out whole optimization, and simplification is easy out, accurately quantifies scheme.Advantages of the present invention
It is:
1: determined combination staining solution formula does not need to be placed in so that the TTC dyeing of pollen can carry out at room temperature
In incubator, but it need to be protected from light.
2: the present invention is 1h, more than 1 h to plant pollen or the most suitable dyeing time of anther, in plant pollen or anther
TTF does not just increase in linear form further according to the time.
3: in the unit time (time of the invention be 1h), with the increase of pollen or anther quality, the TTF's of generation
Amount is linear to be increased, while after high-temperature process, with the increase of anther and pollen quality, the TTF generated in 1h is still line
Character state, but high temperature can have an impact anther and pollen activity, and the yield of TTF in the unit time is caused to reduce.
4: the amount for the TTF that the anther of unit mass can be generated within the unit time is another as assessment pollen activity
A reliability index, reduce or eliminate due to shake off pollen not exclusively or during shaking off to pollen generate injury and produce
Raw error.
5: the composite can be widely applied to the different active identifications of plant pollen.
Detailed description of the invention
Fig. 1: quantitative technique route block diagram is dyed in the present invention.
Fig. 2: the plant pollen dyeing after dyeing 1h in the compound staining solution that the kaliumphosphate buffer of various concentration is prepared
Degree comparison.The effect of settling after different time sedimentation is illustrated simultaneously.It can be found that the compound staining of 0.1mol/L is molten
The dyeing of pollen is most deep in liquid, shows that the vigor of the pollen under the osmotic pressure is best, at the same time, is using 0.1mol/L
Kaliumphosphate buffer under, the sinking speed of plant pollen is faster than the pollen sinking speed under other several osmotic pressure.
Fig. 3: the pollen microscopic examination result after compound staining solution dyeing 1h under different osmotic.It was found that plant pollen exists
Vigor is kept best in the compound staining solution of 0.1mol/L.
Fig. 4: the microscopy of pollen staining under room temperature and high-temperature process is carried out to temperature sensitive cotton material.Under room temperature, substantially institute
Some pollen can dye red.But at high temperature, above-mentioned cotton temperature sensing material, which produces largely, does not have great-hearted pollen.
Fig. 5: the dye test result of the pollen of dicotyledonous crops rape and monocot crops rice.The quilt in microscopy picture
Being red-dyed is great-hearted pollen.
Fig. 6: being Specification Curve of Increasing process.Using 2,3,5 triphenyltetrazolium chlorid (TTC) compound staining of 8g/L
Solution is prepared: 0 μ g/L (only uses kaliumphosphate buffer), 5mg/L, 10mg/L, 25mg/L, 50mg/L, 100mg/L, 150mg/L,
The compound staining solution (i.e. dyeing composition staining solution) each 1 containing 2,3,5 triphenyltetrazolium chlorid (TTC) of 250mg/L
Milliliter, after the excessive sodium hydrosulfide of addition (5-10mg sodium hydrosulfide is added in 1mL compound staining solution) sufficiently reduction, the bodies such as addition
Product ethyl acetate is centrifuged after shaking extraction 5min, draws 200 μ L of supernatant, and upper microplate reader is measured, the extinction of each measurement
Value has corresponded to 0 μ g, 1 μ g, 2 μ g, 5 μ g, 10 μ g, 20 μ g, 30 μ g, the TTF amount of 50 μ g.As can be seen that being less than or equal in applied sample amount
When 30 μ g, absorbance be as the variation of the amount of TTF linearly increases, but more than 30 μ g after, just no longer with linear rule
Increase.
Fig. 7: being the pollen of phase homogenous quantities under room temperature, the amount of the TTF generated in different dyeing times, it can be seen that
When less than or equal to 60min, TTF is increased in linear form, but has been more than that 60min just no longer increases in linear form.
Fig. 8: being the plant pollen of phase homogenous quantities under high temperature, the amount of the TTF generated in different dyeing times.High temperature pair
Activity generates large effect, but equally when being less than or equal to 60min, TTF increases in linear form, but is more than
60min just no longer increases in linear form.
Fig. 9: being will to be dyed respectively after the anther tissue of different tests material and pollen separation as a result, can see
Cotton anthers group on the day of blooming out is woven in TTC and will not be red-dyed, and pollen can be red-dyed, and show to use
Entire anther is dyed.
Figure 10: being the anther of phase homogenous quantities under room temperature, amount and the pollen dye of the TTF generated in different dyeing times
Color result is similar, using anther dye it is quantitative by the way of can equally assess the activity of the material pollen.
Figure 11: being the plant anther of phase homogenous quantities under high temperature, the amount of the TTF generated in different dyeing times.In height
Wen Hou, the TTF amount that the amount of the TTF generated in the unit time will obviously be generated lower than anther under room temperature, within 60min TTF according to
So increase by linear forms.
Figure 12: the amount for the TTF that (60min) different quality plant anther generates in same time at normal temperature.The amount of TTF with
The linear trend growth of anther quality.
Figure 13: the amount for the TTF that (60min) different quality plant pollen generates in same time at normal temperature.The amount of TTF with
The increase of pollen quality linearly increases.
Figure 14: the amount for the TTF that (60min) different quality plant anther generates in same time at high temperature.The amount of TTF with
The linear trend growth of anther quality, while the TTF that the plant anther of unit mass generates is fewer than room temperature, shows its activity
It is impacted.
Figure 15: the amount for the TTF that (60min) different quality plant pollen generates in same time at high temperature.The amount of TTF with
The increase of pollen quality linearly increases, and the TTF that the plant pollen of unit mass generates is also fewer than room temperature, shows plant pollen
Activity receives the influence of high temperature really.
Figure 16: by Fig. 7-8 and Figure 10-Figure 15 test when TTF extracted amount.
Figure 17: the extraction results after being dyed to large batch of material, after directly broken plant anther extraction TTF.
Specific embodiment
Embodiment 1: optium concentration buffer and TTC dye liquor are prepared
2,3,5 triphenyltetrazolium chlorid (TTC) compound staining method used in the present invention is based on the China where applicant
Middle agriculture university's crop genetic National Key Laboratory it has been reported that method (Min, L., Zhu, L., Tu, L., Deng, F.,
Yuan,D.,and Zhang,X.(2013).Cotton GhCKI disrupts normal male reproduction by
delaying tapetum programmed cell death via inactivating starch synthase.The
Plant journal:for cell and molecular biology 75,823-835) improvement, in the publication, Shen
It asks someone to use the method that 37 DEG C of incubators are protected from light dyeing, it is contemplated that it is possible that the not condition of incubator, with reference to common slow
Fliud flushing catalogue has separately designed the phosphate buffer suspension pollen of various concentration, is 0mol/L, 0.05mol/L respectively,
0.1mol/L, 0.2mol/L, 0.5mol/L, 1mol/L.At the same time, with reference to the common TTC dyeing of root system of plant vital staining
Liquid concentration (reference: Wang Xuekui, Huang Jianliang, " plant physiology and biochemistry experimental principle and technology ", and the third edition, Higher Education Publishing House,
In January, 2015, Beijing), the present invention in using 8g/L TTC dye liquor concentration carry out dye test.As a result, it has been found that dense in 0.1M
It spends, in the kaliumphosphate buffer of pH=7, cotton pollen can hurtle down and sink to the bottom, and keep pollen cell normal condition, no
There is the phenomenon that rupture inactivation (see Fig. 2).After most suitable kaliumphosphate buffer has been determined, pass through the dye of this concentration combination staining solution
The activity of colour test, pollen can significantly be distinguished (such as Fig. 3, Fig. 4), the final kaliumphosphate buffer for using 0.1M,
The TTC dyeing liquor of configuration 8g/L is dyed.
Compound staining solution formula of the invention is as follows: potassium dihydrogen phosphate 5.23g；Dipotassium hydrogen phosphate 14.03g, addition are steamed
Distilled water is settled to 1L (adjusting pH to 7), adds TTC 8g；.
Embodiment 2: the dye test to Different Crop pollen
Using the compound staining solution in embodiment 1, to the flower of dicotyledonous crops such as rape and monocot crops such as rice
Powder has carried out dye test, carries out microscopy discovery to the pollen after dyeing, great-hearted pollen can be contaminated in two kinds of crops
Upper red shows that compound staining solution of the invention has certain broad applicability (as shown in Figure 5).
The most suitable ELISA Plate applied sample amount test of embodiment 3
When preparing the TTF standard items of various concentration, when discovery standard items TTF concentration is higher, visually cannot easily distinguish
Not Chu difference, in order to determine the accurate measurement range of microplate reader, using the compound staining solution in embodiment 1 in the present embodiment,
After diluting different multiples, make 0 μ g/L (only with kaliumphosphate buffer), 5mg/L, 10mg/L, 25mg/L, 50mg/L,
The compound staining solution of 100mg/L, 150mg/L, 250mg/L are each 1 milliliter (as shown in Figure 3), and excessive sodium hydrosulfide (1mL is added
Be added 5-10mg) sufficiently reduction after, be added isometric ethyl acetate shake extraction 5min after be centrifuged, draw 200 μ L of supernatant on enzyme
Mark instrument is measured, and the light absorption value of each measurement has corresponded to 0 μ g, 1 μ g, 2 μ g, 5 μ g, 10 μ g, 20 μ g, 30 μ g, the TTF of 50 μ g
Amount.As can be seen that absorbance is as the variation of the amount of TTF linearly increases, still when applied sample amount is less than or equal to 30 μ g
After 30 μ g, just no longer with linearly increasing.So the upper limit of the applied sample amount of final choice is 30 μ g (result is shown in Fig. 6).
Embodiment 4: most suitable dyeing time test
After most suitable kaliumphosphate buffer and TTC stin of thickness have been determined in embodiment 1, need to further confirm that most suitable
Dyeing time.Plant pollen dyeing time deficiency and the too long difficulty that can cause microscopy increase, and activity is difficult to distinguish, this master
If uncertain dyeing time causes.In the present embodiment, according to (Min, L etc., Sugar and auxin signaling
pathways respond to high-temperature stress during anther development as
revealed by transcript profiling analysis in cotton.(2014).Plant Physiology
164,1293-1308.) as a result, use report in high temperature sensitive material, and at normal temperature to most suitable dyeing time into
Determination is gone.It is devised in embodiment and 5min, 10min, 15min, 20min, 30min is carried out to the pollen of certain mass,
The test of quantitative TTF is crushed after 60min, 90min dyeing.The specific steps are first in 7 clean 2mL centrifuge tubes respectively
The pollen of phase homogenous quantities is added, the compound staining solution prepared in 1mL embodiment 1 is added, is protected from light dyeing and starts timing.When reaction
Between arrive after, the 2 diluted concentrated sulfuric acids of μ L (5mol/L) are added into each centrifuge tube and terminate reaction, by pollen it is broken after use 300-500 μ
The ethyl acetate of L extracts, and dilutes 2-3 times, draws ELISA Plate on 200 μ L and measures absorbance.It is tested by repetition more than three times
It was found that the TTF that pollen generates is linearly increased as time increases within 60min, it is just no longer linear more than 60min,
Therefore the dyeing time of final choice is 60min (result is shown in Fig. 7).
Embodiment 5: quantitative test is carried out to the pollen activity in the anther after high-temperature process
In the present embodiment, using quantitative method, examination quantitative after TTC is dyed has been carried out to the pollen after high-temperature process
It tests.As a result, it has been found that the activity of pollen receives apparent influence, but optimal dyeing time is still after high temperature stress
60min, after 60min, the yield of TTF no longer linearly increases (see Fig. 8).
Embodiment 6: anther active level is tested under room temperature and high temperature
Embodiment 2 the result shows that using certain mass Pollen Assemblage staining solution dyeing after carry out TTF quantitatively come body
Now activity is feasible, and the present invention considers that injury may be generated to pollen during obtaining pollen, and such injury can
Can generate certain influence to the activity of pollen, thus find entire anther is dyed after extraction TTF be quantitatively used for
Assessment activity.
Specific steps are as follows: first confirm that whether the presence of anther tissue can have an impact quantifying for pollen activity.Take 5
A different cotton variety separates pollen and anther tissue after the anther open to the same day carries out 1h dyeing.It can be seen that anther
Substantially it will not be red-dyed, pollen can then be red-dyed.In the present embodiment further using certain mass (such as
Anther 15mg) entirely dyed after without pollen collecting, directly carry out broken extraction, quantitative test carried out to TTF.Examination
It tests step and equally uses 5min, 10min, 15min, 20min, 30min are crushed progress TTF and quantitatively try after 60min, 90min dyeing
It tests.As a result, it has been found that the dyeing for using entire anther to carry out most preferably is tested as 60min, more than 60min, same test result is no longer
Linearly (see Fig. 1).
Embodiment 7: the pollen and anther of different quality carry out the quantitative test after room temperature and high temperature stress
Embodiment 4,5,6 has carried out qualification test using the anther or the pollen in anther of phase homogenous quantities, it is contemplated that no
The anther or pollen of homogenous quantities have different reducing powers, and the anther and different quality of different quality are used in the present embodiment
Pollen in anther has carried out the dye test of 60min, and two kinds of comparative tests being provided under room temperature and high temperature stress.This
Embodiment designs 1mg, 2mg, 5mg, 10mg, 15mg, the test group of 7 different qualities such as 20mg, 40mg, respectively to anther and flower
Powder carries out dyeing quantitative test.As a result, it has been found that the yield of TTF is to increase linear increase with the quality of anther or pollen
's.The ability that high temperature generates TTF to pollen or anther at the same time generates large effect, but the amount of the TTF generated according to
So and quality is linear (see Figure 12-Figure 15).The result shows that even if the anther or pollen of different quality still can be using these
Invention is identified and is assessed to activity.This can quantitatively be provided just for the anther under later period high-volume different materials different disposal
Benefit, can each one whole flower of quantitative all anther, and do not have to the quality of an entire anther for worrying that different materials are taken
Difference, so that being had an impact to quantitative result.
Embodiment 8: extraction test is carried out to the different materials after high-volume high-temperature process
It is according to the embodiment as a result, the quantitative result of anther and pollen can accurate evaluation under the dyeing time of 60min
The activity at ambient and elevated temperatures of material, at the same time, under the dyeing time of 60min, the anther or pollen of identical material are produced
The amount of raw TTF is (see the Figure 16) linearly increased with quality.It in the present embodiment, weighs, then is dyed using to anther
The scheme of extraction carries out quantitative test to high-volume material, is produced within the unit time by the anther or pollen of unit mass
The amount of raw TTF assesses (see Figure 17) activity under different materials room temperature and high temperature, different materials TTF at high temperature
Yield have very big difference, can visually identify, show that the present invention can be used for the activity identification of high-volume material.
Method of the invention is improved from pollen TTC decoration method, right although there are many TTC decoration method application of pollen
The pollen identification effect of extreme phenotype (vigor is extremely strong, and vigor is extremely weak) is fairly good, but high temperature is to the shadow of each pollen activity
For sound there are difference, the activity identification effect of the pollen by TTC dyeing to activity in intermediate state is less desirable.This
Invention improves traditional TTC decoration method, so that pollen still has preferable activity in compound staining solution, while can make
Dying operation carries out at room temperature, TTF is carried out it is quantitative rather than by way of carrying out microscopy to the pollen after dyeing, can be with
The activity of accurate evaluation respective material pollen under room temperature or high temperature stress.Meanwhile it is a discovery of the invention that by entire anther
Quantitative method is carried out, is still capable of the pollen activity of accurate evaluation respective material, greatly reduces operating process in this way, simultaneously
Decrease injury during obtaining pollen to pollen.Further, the flower that the present invention can be used by respective material
Medicine quality carries out quantitative method, large batch of carry out activity identification to anther after dyeing.Operation stream is greatly omitted as a result,
Journey quickly can be assessed accurately, and can be used for various crop except cotton pollen or anther it is active
Assessment.
Claims (6)
1. a kind of compound staining solution suitable for being dyed under plant anther/pollen room temperature or high temperature, which is characterized in that described
The formula of kaliumphosphate buffer in compound staining solution is the phosphoric acid hydrogen two of the potassium dihydrogen phosphate 5.23g, 1mol/L of 1mol/L
Potassium 14.03g is settled to 1L with distilled water, obtains kaliumphosphate buffer；Again by the 2,3,5 triphenyltetrazolium chlorid of 8g
(TTC) it is dissolved in the kaliumphosphate buffer of the 1L, makes its final concentration of 8g/L.
2. a kind of colouring method of plant anther/pollen, which is characterized in that formula preparation described in accordance with the claim 1 obtains
Compound staining solution, the optimum dyeing time in dyeing process is 1h at room temperature.
3. application of the compound staining solution described in claim 1 in plant pollen activity identification.
4. application of the method as claimed in claim 2 in plant pollen activity identification.
5. application as claimed in claim 3, wherein the plant includes cotton.
6. application as claimed in claim 4, wherein the plant includes cotton.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910010240.2A CN109752226B (en) | 2019-01-05 | 2019-01-05 | Method for quickly quantifying pollen activity at normal temperature or high temperature and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910010240.2A CN109752226B (en) | 2019-01-05 | 2019-01-05 | Method for quickly quantifying pollen activity at normal temperature or high temperature and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109752226A true CN109752226A (en) | 2019-05-14 |
| CN109752226B CN109752226B (en) | 2021-06-22 |
Family
ID=66405231
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910010240.2A Active CN109752226B (en) | 2019-01-05 | 2019-01-05 | Method for quickly quantifying pollen activity at normal temperature or high temperature and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109752226B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103940813A (en) * | 2014-04-16 | 2014-07-23 | 洽洽食品股份有限公司 | Method for quickly and qualitatively identifying new and old sunflower seeds based on TTC (triphenyltetrazolium chloride) dyeing process |
| CN104686011A (en) * | 2015-03-19 | 2015-06-10 | 北京市农林科学院 | Tetrazole dyeing method for measuring eggplant seed viability |
| CN104789632A (en) * | 2015-03-23 | 2015-07-22 | 中国农业科学院作物科学研究所 | Determination method of activity of stem tip cells of plant |
| CN105806839A (en) * | 2016-03-21 | 2016-07-27 | 北京北大明德科技发展有限公司 | Embryo-free rice freshness detecting reagent and method |
| CN106854620A (en) * | 2017-01-22 | 2017-06-16 | 贵州勤邦食品安全科学技术有限公司 | A kind of test piece of rapidly and efficiently detection total plate count and preparation method thereof |
-
2019
- 2019-01-05 CN CN201910010240.2A patent/CN109752226B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103940813A (en) * | 2014-04-16 | 2014-07-23 | 洽洽食品股份有限公司 | Method for quickly and qualitatively identifying new and old sunflower seeds based on TTC (triphenyltetrazolium chloride) dyeing process |
| CN104686011A (en) * | 2015-03-19 | 2015-06-10 | 北京市农林科学院 | Tetrazole dyeing method for measuring eggplant seed viability |
| CN104789632A (en) * | 2015-03-23 | 2015-07-22 | 中国农业科学院作物科学研究所 | Determination method of activity of stem tip cells of plant |
| CN105806839A (en) * | 2016-03-21 | 2016-07-27 | 北京北大明德科技发展有限公司 | Embryo-free rice freshness detecting reagent and method |
| CN106854620A (en) * | 2017-01-22 | 2017-06-16 | 贵州勤邦食品安全科学技术有限公司 | A kind of test piece of rapidly and efficiently detection total plate count and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109752226B (en) | 2021-06-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108398407B (en) | Human sperm motility rate and DNA damage double-parameter detection and analysis kit | |
| CN104198357B (en) | Flow Cytometry methods application in production of vaccine is monitored in real time | |
| Godman et al. | The composition of the LE and hematoxylin bodies of systemic lupus erythematosus | |
| CN104949946B (en) | A kind of application of fluorescence probe in hydrogen peroxide molecule detection | |
| CN108844931A (en) | LZQ fluorescence probe detects SO at the same time2With the application in HSA | |
| Tresch et al. | Probing mode of action in plant cell cycle by the herbicide endothall, a protein phosphatase inhibitor | |
| CN110055300A (en) | A kind of fungal infection detection kit and its application | |
| JP2002543851A (en) | Cell assays, methods and reagents | |
| CN101586145A (en) | Analyzing method for detecting activity of soil xylanase | |
| CN106525699A (en) | Peripheral blood lymphocyte micronucleus detection kit and detection method thereof | |
| CN101586146B (en) | Analyzing method for detecting activity of soil xylanase | |
| CN109752226A (en) | The method and application of pollen activity fast quantification under a kind of room temperature or high temperature | |
| Kępczyński et al. | Application of flow cytometry with a fluorescent dye to measurement of intracellular nitric oxide in plant cells | |
| Haskins et al. | Fluorometric Assay of Free and Bound Coumarin in Sweetclover 1 | |
| JP4694863B2 (en) | Evaluation method of microbial cell activity by flow cytometry analysis | |
| CN106198406A (en) | A kind of method of quick detection high whiteness flour wheat seed | |
| Diermeier‐Daucher et al. | Dynamic proliferation assessment in flow cytometry | |
| CN107290197A (en) | A kind of living cells mitochondria quantity dyes detection method | |
| CN108299402A (en) | A kind of preparation method and application of multi-functional hypersensitive Zn2+ two-photons detection fluorescent molecular probe | |
| Engasser et al. | Measurement of intracellular pH during the cultivation of hybridoma cells in batch and continuous cultures | |
| Kwaaitaal et al. | Fluorescence correlation spectroscopy and fluorescence recovery after photobleaching to study receptor kinase mobility in planta | |
| CN112782137B (en) | Flow cytometer detection method for living plankton living cells with 10-50 mu m | |
| CN101846638B (en) | Method for detecting injury of plasmalemmae of sperms of fishes | |
| CN105115950A (en) | Screening and evaluation method of salt tolerant peppermint | |
| Wongpatsa et al. | Effects of temperature on male sterility of two inbred lines of hybrid rice |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |