CN103149189A - Method for rapidly detecting wheat tissue callose - Google Patents
Method for rapidly detecting wheat tissue callose Download PDFInfo
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- CN103149189A CN103149189A CN2013100758486A CN201310075848A CN103149189A CN 103149189 A CN103149189 A CN 103149189A CN 2013100758486 A CN2013100758486 A CN 2013100758486A CN 201310075848 A CN201310075848 A CN 201310075848A CN 103149189 A CN103149189 A CN 103149189A
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Abstract
The invention discloses a method for dyeing and observing wheat tissue callose and belongs to the technical field of chemical plant tissue positioning. The method mainly comprises the following steps: (1) preparing a solution; (2) treating the material; (3) slicing; (4) dyeing materials; (5) producing sections; and (6) exciting through a laser excitation device, and observing and shooting through a fluorescence microscope. By adoption of a bare-handed slicing method, the wheat tissue slices which are manufactured in a bare-handed mode are dyed under the alkaline conditions by using the callose specifity fluorescent dye-water-soluble aniline blue, the sample slice manufacturing, dyeing and observing period is obviously shortened, and the callose is accurately positioned. The method is high in operability, the manufactured slices are conveniently observed, and the acquired image is clear.
Description
Technical field
The present invention relates to the chemical field of locating technology of plant tissue microsection and structural constituent, specifically a kind of method for quick of Wheat Tissue callose.
Background technology
Callose is a kind of with β-1, and the glucosan that the 3-bond is closed is being brought into play important regulating action, the normal growth metabolism process of its composition and decomposition direct relation plant in the vital movements such as the screen casing metabolism of plant, Development of Gametophytes.In addition, when being subject to extraneous poor environment, plant coerces or intense stimulus, during as adverse circumstances such as pathogen infection, Nutrient Stress, mechanical damage, high temperature, also physiological activity (the Wang of regulating plant cell is fed back in callose metabolism meeting immediately, McCallum, Fetch etc., 2012; Chapman etc., 2009; Saulnier etc., 2012).Under adverse circumstance, interior surface or the interior obstruction of sieve aperture that can synthesize rapidly callose and deposit to sieve plate of sieve tube element; In case environmental stimuli is removed, the callose that deposits in sieve plate surface or sieve aperture can rapidly disappear, and makes screen casing recover normal transportation function.Therefore, the deposition of callose is relevant with the resistance of clearing up with plant, the plant callose of resistance form less and slow (Cheng etc., 2012).In a word, the callose metabolism is one of important research contents of plant field.
Wheat is two annual crops, will suffer a series of natural adverse circumstances and biological adverse circumstance in the whole history of life.In adverse circumstance, in Wheat Tissue, particularly microtubule fasolculus, induce synthetic callose, affect plant to absorption and the transportation of moisture, nutrient.Under adverse circumstance, the pustulation period wheat affects transporting of plant reserve substance because callose stops up plasmodesmus, screen casing etc. in seed, have a strong impact on filling rate, finally causes production declining.Therefore, in the monitoring adverse circumstance in wheat microtubule fasolculus tissue the synthetic and content of callose be a key index of measurement improving yield of wheat stable yields, be the important evidence that the breeding scholar screens good wheat breed.
At present, still indefinite for callose growth and decline process control mechanism, strengthening research is necessary, and wherein the accurate mark of film-making and callose location is crucial fast.It is generally preparation paraffin section or freezing microtome section that conventional plant is organized the microexamination film-making.Yet, the preparation paraffin section, the material pre-treatment program is loaded down with trivial details, and the cycle is long, and its process comprises the series of steps such as FAA fixes, dehydration, waxdip, dimethylbenzene dewaxing, and easily produces the volatility toxic gas in operation, and personnel are damaged and environmental pollution.The more important thing is, incur loss through delay because its cycle is long vegetation growth state in growth is made Rapid identification and remedial measures.The freezing microtome section technology, although comparative maturity on animal material (there is no cell membrane because of its cell) microsection manufacture, and its technology is difficult to grasp on plant tissue, and instrument is expensive, the consumptive material cost is high, more seriously, because material is difficult to be fixed on base, easily cause the cutter that quivers when cutting the thicker material of the epidermis silicretes such as wheat, paddy rice, affect production effect.
Therefore, carrying out quick film-making, Fast Labeling, fast detecting for the plant tissue in growth is judgement vegetation growth state and the key that proposes emergent remedial measures.Aniline blue can produce the selectivity reaction to callose, can induce yellow-green fluorescence under ultraviolet excitation, thereby can be used to detect callose deposition position and relative content.
Summary of the invention
The object of the present invention is to provide a kind of method for quick of Wheat Tissue callose.The present invention carries out the free-hand slicing method film-making to wheat leaf blade tissue, stem tissue, and use water-soluble aniline blue under alkali condition to the callose of deposition in tissue carry out accurate mark, precise positioning with and the comparison of relative content, reduced to greatest extent the error that produces in the operating process.
The technical solution adopted in the present invention is:
A kind of method for quick of Wheat Tissue callose is characterized in that, comprises the following steps:
(1) preparation of solution:
The preparation of immobile liquid: with ethanol with methenyl choloride by 3:1(v/v) mix after as mixed solvent, take by above-mentioned mixed solvent as 0.15% trichloroacetic acid solution as solvent dose volume mark;
The preparation of dyeing load buffer: the K for preparing 50～150mmol/L of pH=8.5～9.5
2HPO
4Damping fluid or Tris-HCl damping fluid; Then the 0.01g water-soluble aniline blue is dissolved in and is dyeing load liquid in the above-mentioned damping fluid of 100ml;
(2) material processed: get the tissues such as wheat stalk, leaf, distilled water cleans up,
If enter at once laboratory operation, blot with thieving paper;
If can not at once enter experimental implementation, material is cut into the long tissue block of 3～5mm, fix with immobile liquid, during dyeing, material is taken out, clean with 50% ethanol, distilled water successively, then blot with thieving paper;
(3) free-hand section is made: use sharp cutter, it is 20～40 μ m sections that the tissues such as wheat stalk, leaf are cut into thickness, is placed on microslide;
(4) dyeing material: the sample dyeing load buffer of drawing 100～150 μ L with liquid-transfering gun (pipe) drips in the section that is placed in microslide, then microslide is inserted and vacuumize in vessel, after sealing with the vacuum pump 5min that bleeds, lucifuge is placed 5～10min under 25 ℃ of conditions subsequently, sop up dyeing load buffer on sample with liquid-transfering gun, draw section after distilled water rinsing dyeing with liquid-transfering gun afterwards;
(5) 100 μ L50% glycerine are dropped in material surface, then use the cover glass mounting;
(6) microscopic examination is taken pictures: will be placed on epifluorescence microscope through the section that above-mentioned steps is processed, excite with the laser excitation device, examine under a microscope, and take pictures with the ccd image acquisition system.
Preferably, described sharp cutter is razor blade or knife blade.
Preferably, described excitation apparatus excitation wavelength is 400～485nm, and emission wavelength is 490～560nm.
The present invention has advantages of:
(1) traditional paraffin section to the telltale mark of callose consuming timely grow (2～3 week), freezing method equipment needed thereby condition is higher.In the present invention, the free-hand section method is adopted in the plant tissue film-making, and this method is easy, is easy to grasp, and saves time fast; If draw materials in the laboratory or outdoor drawing materials can enter experimental implementation in 30 minutes, material need not fixing can dyeing.Avoided the distortion of callose content and position during sample long time treatment in the additive method, dyeing.The more important thing is, the fast detecting result can be made quick judgement and formulate remedial measures the wheat growth situation in growth.
(2) adopt meta-alkalescence damping fluid configuration dyeing load liquid, eliminated the interference of water-soluble aniline blue self color to sections observation, guarantee accurate positioning, relative content difference is obvious.
(3) material after above-mentioned processing and dyeing, can change alcohol over to as untimely observation and put refrigerator or use the glycerine mounting, puts refrigerator and can preserve several weeks, and the callose after dyeing is not made significant difference.
(4) the method is workable, and dyestuff and other reagent used in experiment are noiseless to the callose Fluirescence observation.
Description of drawings
Fig. 1 is that in embodiment 1, pustulation period wheat leaf blade histofluorescence excites rear result at the fluorescence microscopy Microscopic observation.
Fig. 2 is that in embodiment 2, pustulation period wheat stalk histofluorescence excites rear result at the fluorescence microscopy Microscopic observation.
Fig. 3 is that the wheat stalk histofluorescence in florescence of executing in right amount after in embodiment 3, immobile liquid is fixed under the nitrogen condition excites rear result at the fluorescence microscopy Microscopic observation.
Fig. 4 is that in embodiment 3, the wheat stalk histofluorescence in florescence under the fixing rear low amount of nitrogen condition of immobile liquid excites rear result at the fluorescence microscopy Microscopic observation.
Embodiment
For illustrating the understanding to feature of the present invention, below in conjunction with the embodiment of some indefinitenesses, the present invention is further elaborated.
Embodiment 1
A kind of method for quick of Wheat Tissue callose, the application in wheat breed Jimai 22 leaf tissues, concrete operations are as follows:
Obtain solution:
The preparation of dyeing load buffer: the Tris-HCl damping fluid (pH=8.8) of preparation 100mmol/L; Then the 0.01g water-soluble aniline blue is dissolved in the above-mentioned damping fluid of 100ml and is the dyeing load buffer.
Material processed: get grouting wheat leaf blade tissue in mid-term, take back immediately the laboratory, blot with thieving paper.
Free-hand section is made: with sharp cutter (knife blade), it is 20～40 μ m sections that the wheat leaf blade tissue is cut into thickness, is placed on microslide;
Dyeing material: the sample dyeing load buffer with liquid-transfering gun or pipette, extract 100～150 μ L drips in the section that is placed in microslide, then microslide is inserted and vacuumize in vessel, after sealing with the vacuum pump 5～8min that bleeds, to take out the bubble of material surface, impel load buffer to enter fast material internal, remove simultaneously bubble and also can improve observing effect; Lucifuge is placed 5～10min under 25 ℃ of conditions subsequently, sops up dyeing load buffer on sample with liquid-transfering gun, draws section after distilled water rinsing dyeing with liquid-transfering gun afterwards;
100 μ L50% glycerine are dropped in slice surface, then use the cover glass mounting;
Microscopic examination is taken pictures: will be placed on epifluorescence microscope through the section that above-mentioned steps is processed, excite with the laser excitation device, excitation wavelength is 400～485nm, and emission wavelength is 490～560nm, examine under a microscope, and take pictures with the ccd image acquisition system.
Result as shown in Figure 1, (in figure, the arrow indication is that callose produces position and relative content to contain the callose of high level on vascular bundle catheter wall, epidermal tissue, sclerenchyma cell wall, high brightness position explanation callose content is high, and low-light level position explanation callose content is relatively low) (scale=200 μ m).
Embodiment 2
A kind of method for quick of Wheat Tissue callose, the application in wheat breed Jimai 22 stem tissues, concrete operations are as follows:
Obtain solution:
The preparation of dyeing load buffer: the K2HPO4 damping fluid (pH=9.5) of preparation 150mmol/L; Then the 0.01g water-soluble aniline blue is dissolved in the above-mentioned damping fluid of 100ml and is the dyeing load buffer;
Material processed: get grouting wheat stalk tissue in mid-term, take back immediately the laboratory, blot with thieving paper.
Free-hand section is made: with sharp cutter (knife blade), it is 20～40 μ m sections that the wheat leaf blade tissue is cut into thickness, is placed on microslide;
Dyeing material: the sample dyeing load buffer with liquid-transfering gun or pipette, extract 100～150 μ L drips in the section that is placed in microslide, then microslide is inserted and vacuumize in vessel, after sealing with the vacuum pump 5～8min that bleeds, to take out the bubble of material surface, impel load buffer to enter fast material internal, remove simultaneously bubble and also can improve observing effect; Lucifuge is placed 5～10min under 25 ℃ of conditions subsequently, sops up dyeing load buffer on sample with liquid-transfering gun, draws section after distilled water rinsing dyeing with liquid-transfering gun afterwards;
100 μ L50% glycerine are dropped in slice surface, then use the cover glass mounting;
Microscopic examination is taken pictures: will be placed on epifluorescence microscope through the section that above-mentioned steps is processed, excite with the laser excitation device, excitation wavelength is 400～485nm, and emission wavelength is 490～560nm, examine under a microscope, and take pictures with the ccd image acquisition system.
Result as shown in Figure 2, callose is mainly synthetic in vascular bundle xylem and phloem cell wall, and micro-callose synthetic (in figure, the arrow indication is that callose produces the position) (scale=200 μ m) is arranged in prothenchyma (of wood).
Embodiment 3
A kind of method for quick of Wheat Tissue callose; fix 24 hours at immobile liquid, (the direct chopping and returning of its stalk after upper season crop harvest corn under the conservation tillage condition; wheat is sowed with no-tillage machine), the application in different amount of nitrogen Jimai in lower florescence 22 stem tissues, concrete operations are as follows:
Obtain solution:
The preparation of immobile liquid: with ethanol with methenyl choloride by 3:1(v/v) mix after as mixed solvent, take by above-mentioned mixed solvent as 0.15% trichloroacetic acid solution as solvent dose volume mark;
The preparation of dyeing load buffer: the K of preparation 67mmol/L
2HPO
4Damping fluid (pH=9.0); Then the 0.01g water-soluble aniline blue is dissolved in the above-mentioned damping fluid of 100ml and is the dyeing load buffer;
Material processed: get grouting wheat stalk tissue in mid-term, take back immediately the laboratory, blot with thieving paper.
Free-hand section is made: with sharp cutter (knife blade), it is 20～40 μ m sections that the wheat leaf blade tissue is cut into thickness, is placed on microslide;
Dyeing material: the sample dyeing load buffer with liquid-transfering gun or pipette, extract 100～150 μ L drips in the section that is placed in microslide, then microslide is inserted and vacuumize in vessel, after sealing with the vacuum pump 5～8min that bleeds, to take out the bubble of material surface, impel load buffer to enter fast material internal, remove simultaneously bubble and also can improve observing effect; Lucifuge is placed 5～10min under 25 ℃ of conditions subsequently, sops up dyeing load buffer on sample with liquid-transfering gun, draws section after distilled water rinsing dyeing with liquid-transfering gun afterwards;
100 μ L50% glycerine are dropped in slice surface, then use the cover glass mounting;
Microscopic examination is taken pictures: will be placed on epifluorescence microscope through the section that above-mentioned steps is processed, excite with the laser excitation device, excitation wavelength is 400～485nm, and emission wavelength is 490～560nm, examine under a microscope, and take pictures with the ccd image acquisition system.
Result is as shown in accompanying drawing 3 and accompanying drawing 4, callose is mainly synthetic in vascular bundle xylem and phloem cell wall, micro-callose synthetic (in figure, the arrow indication is that callose produces the position) is arranged in prothenchyma (of wood), simultaneously, the fluorescence labeling demonstration, as shown in Figure 3, under the proper N condition, callose content is lower, as shown in Figure 4, and callose deposition more (scale=200 μ m) in the wheat stalk vascular bundle under low amount of nitrogen.
Though above in conjunction with the embodiments the present invention is described in detail, still, described those skilled in the art can understand, and under the prerequisite that does not break away from aim of the present invention, in the scope of claim protection, can also change above-described embodiment.
Claims (3)
1. the method for quick of a Wheat Tissue callose, is characterized in that, comprises the following steps:
(1) preparation of solution:
The preparation of immobile liquid: with ethanol and methenyl choloride by volume for 3:1 mix after as mixed solvent, take by above-mentioned mixed solvent as 0.15% trichloroacetic acid solution as solvent dose volume mark;
The preparation of dyeing load buffer: the K for preparing 50～150mmol/L of pH=8.5～9.5
2HPO
4Damping fluid or Tris-HCl damping fluid; Then the 0.01g water-soluble aniline blue is dissolved in the above-mentioned damping fluid of 100ml, is the dyeing load buffer;
(2) material processed: get wheat stalk tissue or leaf texture, distilled water cleans up,
If enter at once laboratory operation, blot with thieving paper;
If can not at once enter experimental implementation, wheat stalk tissue or leaf texture are cut into the long tissue block of 3～5mm, fix with immobile liquid, during dyeing, material is taken out, clean with 50% ethanol, distilled water successively, then blot with thieving paper;
(3) free-hand section is made: use sharp cutter, will cut into thickness through the wheat stalk tissue of step (2) processing or leaf texture is that 20～40 μ m cut into slices, and is placed on microslide;
(4) dyeing material: the sample dyeing load buffer with liquid-transfering gun or pipette, extract 100～150 μ L drips in the section that is placed in microslide, then microslide is inserted and vacuumize in vessel, after sealing with the vacuum pump 5～8min that bleeds, lucifuge is placed 5～10min under 25 ℃ of conditions subsequently, sop up dyeing load buffer on sample with liquid-transfering gun, draw section after distilled water rinsing dyeing with liquid-transfering gun afterwards;
(5) 100 μ L50% glycerine are dropped in slice surface, then use the cover glass mounting;
(6) microscopic examination is taken pictures: will be placed on epifluorescence microscope through the section that step (5) is processed, excite with the laser excitation device, examine under a microscope, and take pictures with the ccd image acquisition system.
2. the method for quick of a kind of Wheat Tissue callose according to claim 1, is characterized in that, described sharp cutter is razor blade or knife blade.
3. the method for quick of a kind of Wheat Tissue callose according to claim 1, is characterized in that, described excitation apparatus excitation wavelength is 400～485nm, and emission wavelength is 490～560nm.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106198465A (en) * | 2016-06-10 | 2016-12-07 | 甘肃农业大学 | The method observing the deposition of creeping bentgrass leaf tissue callose based on paraffin section and aniline blue fluorescent staining method |
| CN106383047A (en) * | 2016-11-09 | 2017-02-08 | 北京林业大学 | Staining method for observing histopathologic process of fungus disease in leaf segment of plant |
| CN109459262A (en) * | 2018-12-21 | 2019-03-12 | 东北农业大学 | A kind of preparation method that maturation maize root system is temporarily sliced |
| CN113129281A (en) * | 2021-04-13 | 2021-07-16 | 广西大学 | Wheat stem section parameter detection method based on deep learning |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106198465A (en) * | 2016-06-10 | 2016-12-07 | 甘肃农业大学 | The method observing the deposition of creeping bentgrass leaf tissue callose based on paraffin section and aniline blue fluorescent staining method |
| CN106383047A (en) * | 2016-11-09 | 2017-02-08 | 北京林业大学 | Staining method for observing histopathologic process of fungus disease in leaf segment of plant |
| CN109459262A (en) * | 2018-12-21 | 2019-03-12 | 东北农业大学 | A kind of preparation method that maturation maize root system is temporarily sliced |
| CN113129281A (en) * | 2021-04-13 | 2021-07-16 | 广西大学 | Wheat stem section parameter detection method based on deep learning |
| CN113129281B (en) * | 2021-04-13 | 2022-06-21 | 广西大学 | Wheat stem section parameter detection method based on deep learning |
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Application publication date: 20130612 |