CN102620960A - Leaf sheath tissue section manufacturing method for enabling users to observe callose of rice leaf sheath tissues - Google Patents
Leaf sheath tissue section manufacturing method for enabling users to observe callose of rice leaf sheath tissues Download PDFInfo
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- CN102620960A CN102620960A CN201210048297XA CN201210048297A CN102620960A CN 102620960 A CN102620960 A CN 102620960A CN 201210048297X A CN201210048297X A CN 201210048297XA CN 201210048297 A CN201210048297 A CN 201210048297A CN 102620960 A CN102620960 A CN 102620960A
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Abstract
A leaf sheath tissue section manufacturing method for enabling users to observe callose of rice lead sheath tissues belongs to the technical field of micro-structural slicing and includes: (1) preprocessing of materials; (2) sample fixing; (3) section freezing; (4) section unfolding; (5) dying; and (6) microscopic observation and photographing. The leaf sheath tissue section manufacturing method remarkably shortens time for sample fixing, slicing and dying, basically the whole process from preprocessing of samples to observation and photographing under a fluorescence microscope can be finished in about 12 hours, the section obtained through the lead sheath tissue section manufacturing method is complete in tissue structure, the images are clear, and simultaneously the leaf sheath tissue section manufacturing method effectively solves the problem that slicing with hands can lead the tissues of the materials to be fragile, thickness of the sections to be uneven, a paraffin slicing process is complex, the period is long and the like. Working efficiency is remarkably improved.
Description
Technical field
The invention belongs to microstructure microtomy field, be specifically related to a kind ofly be used to observe rice leaf sheath and organize the leaf sheath of callose to organize flaking method.
Background technology
The plant vasular beam system is made up of bast and xylem, is the main conducting tissue that runs through in the whole plant body, is bearing the long-distance transportation in the plant body, is the passage that plant is carried photosynthate, moisture and mineral nutrition.The bast tissue is made up of screen casing, companion cell and parenchyma cell, contains multiple materials such as callose, callose synzyme, tough hide collagen in the phloem sieve tube, in the metabolism of screen casing, plays important regulating action.Callose (callous) (principal ingredient is β-1, the 3-glucosan) synthetic, decompose the normal growth metabolism process of plant that is directly connected to, in vital movements such as the screen casing metabolism of plant, gametophyte growth, bringing into play important regulatory role.Research shows, the synthetic callose (Jaffemet al, 1984) of extraneous environment stress inducing plant cell but mechanical damage, pathogen infection, insect cause harm etc., and the accumulation of callose and plant disease-resistant, pest-resistant characteristic relevant (Tian Guozhong etc., 1994; Ecaleet al., 1995,1999; Serranoet al., 1998).
Plant tissue slice method kind is many, and each microtomy all respectively has characteristics and limitation, and using always has free-hand slicing method, paraffin method, freezing method etc.Free-hand slicing method is promptly used the shaver cutting material, is difficult to cut very thinly, is prone to produce the gage distortion phenomenon, is difficult to obtain complete section; Though paraffin method is the most frequently used method, owing to need before the material section, be prone to make that material becomes fragile, hardening and distortion through series of steps such as fixing, dehydration, transparent and embeddings, process is loaded down with trivial details, the cycle is long; Freezing method is a kind of fast method of cutting into slices after utilizing freezing-microtome to make material freezing.
At present, the detection of callose aniline blue decoration method (Li Shi father-in-law etc., 1990 commonly used in the plant tissue; Adrienne et al., 2005; Penet et al., 2005; Shen Ye etc., 2006; Ding Xinlun etc., 2008), also useful improvement phenol magenta-aniline blue pressed disc method (Liu Xiaorui etc., 2008), main observation of plant leaf tissue receive behind the environment stress such as disease and pest with plant microsporocyte meiosis process in the dynamic change of callose.Ding Xinlun etc. (2008) adopt free-hand slicing method and aniline blue fluorescent dye technical research rice stripe virus (RSV) coerce down the influence that callose deposits in anti-, the sense rice varieties blade.Method therefor is: the rice seedling leaf tissue is done free-hand section; Soaked overnight in 96% ethanol again, place phosphate buffer (0.07mol/L, pH=9) in 30 min; With 0.005% aniline blue (0.07mol/L; PH=9) dyeing 60 min, 20% glycerine mounting is just being put microscopically at Olympus fluorescence at last and is being observed with ultraviolet excitation.But free-hand section is difficult to cut very thinly, be prone to produce the gage distortion phenomenon.Because water cut is different in rice leaf tissue and the leaf sheath tissue, and leaf sheath cells of tissues chamber is big and wall is thin, often has bubble to exist in the leaf sheath tissue of sampling back, can't use free-hand section, and the easy fragmentation of cutting into slices is imperfect, is difficult to obtain complete institutional framework.
Summary of the invention
To the problem that prior art exists, the objective of the invention is to design provides a kind of and is used to observe leaf sheath and organizes the leaf sheath of callose to organize the technical scheme of flaking method.
Describedly a kind ofly be used to observe rice leaf sheath and organize the leaf sheath of callose to organize flaking method, it is characterized in that comprising following processing step:
1) material pre-treatment: get the rice leaf sheath tissue in tillering stage, clean and to dry the back is cut into 0.3～0.5 cm rapidly with blade segment, put into 10% glycerite then immediately, and with vacuum pump 10～20 min that bleed, till the sinking of leaf sheath tissue;
2) sample is fixed: add 1 earlier at cold base and organize frozen rubber; On the cold platform that the leaf sheath that again step 1) is obtained is organized segment vertically to place to keep flat; Adding 2～5 again organizes frozen rubber to organize segment to wrap fully in leaf sheath; And be placed on the sample stage in the freezing-microtome, behind placement 100～140 min, promptly can be used for freezing microtome section;
3) freezing microtome section: will be stained with leaf sheath and organize on cold of segment cold the pickup groove that is fixed on freezing-microtome; Tighten fixed screw; And the slice thickness adjusting knob transferred to slice thickness 10 μ m; The minimum control temperature of casing is-40 ℃, and a section minimum control temperature is-50 ℃, shakes rotation hand wheel and cuts into slices;
4) exhibition sheet: elder generation near slicer, whenever cuts a section with microslide, lightly section is moved on on the microslide with writing brush, and every microslide sticks 15～20 sections;
5) dyeing: after posting section; At room temperature let frozen rubber dry slightly; Put into 96% after alcohol-pickled 6～10 hours, take out and alcohol is dried, add 1 ml concentration again and be 1/15 mol/L, pH value and be 7.0 phosphate buffer on cutting into slices; Adding 1 ml concentration behind 45 min again is that 0.1 mol, pH value are 7.0 aniline blue dyeing liquor, behind 60 min that dye microslide is slowly tilted to outwell dyeing liquor;
6) microexamination with take pictures: when slice is just dry, is just putting microscopically at Olympus BX51 fluorescence immediately and observing the callose in the leaf sheath tissue with ultraviolet excitation, and taking pictures with the camera of this microscope special use.
Describedly a kind ofly be used to observe rice leaf sheath and organize the leaf sheath of callose to organize flaking method, it is characterized in that described step 1) intermediate pump is that SHZ-CA type circulation ability of swimming is used vacuum pump more.
Describedly a kind ofly be used to observe rice leaf sheath and organize the leaf sheath of callose to organize flaking method, it is characterized in that described step 2) in before the leaf sheath cold platform organizing segment vertically to place to keep flat, with lens wiping paper its lip-deep residual liquid is blotted earlier.
Describedly a kind ofly be used to observe rice leaf sheath and organize the leaf sheath of callose to organize flaking method, when it is characterized in that freezing microtome section in the described step 3) such as the frost layer on the cryoprobe thicker, should in time start the defrosting program to remove frost layer on the slicer in case roll film.
The present invention has significantly shortened the time that sample is fixed, cut into slices and dyes; Basically can accomplish sample pre-treatments about 12 hours and observe the whole process of taking pictures down to fluorescent microscope; And the present invention the biopsy tissues structural integrity, the clear picture that obtain; Also efficiently solve simultaneously frangible, the gage distortion of cutting into slices of free-hand section material structure, the paraffin section process is loaded down with trivial details, long problems such as (needing 2 time-of-weeks usually) of cycle, and the work efficiency utmost point significantly improves.
Description of drawings
Observed rice leaf sheath is organized cross section ultrastructure (210 times) under Fig. 1 fluorescent microscope;
Under Fig. 2 fluorescent microscope in the fibrovascular system leaf sheath organize fascicular ultrastructure (amplifying 820 times);
The ultrastructure of callose (amplifying 820 times) in the vascular bundle screen casing in the fibrovascular system under Fig. 3 fluorescent microscope.
Embodiment
Below further specify the present invention through specific embodiment.
Embodiment
Rice varieties and breeding time: TN1, seedling stage
Test period: the 5-12 month in 2011
Test place: Zhejiang Academy of Agricultural Science plant protection and institute of microbiology plant protection engineering research chamber
Test method: material pre-treatment, fixed sample, freezing microtome section, exhibition sheet, dyeing and observation process such as take pictures.
1) material pre-treatment: the rice leaf sheath tissue of getting seedling stage; Dry the back is cut into 0.3～0.5 cm rapidly with blade segment after cleaning gently; Put into 10% glycerite immediately; Many with SHZ-CA type circulation ability of swimming with vacuum pump 10～20 min that bleed, be preferably 15 min, till the sinking of leaf sheath tissue.
2) sample is fixed: the leaf sheath tissue is taken out, be placed on the lens wiping paper its lip-deep residual liquid is blotted, add a freezing-microtome (Leica CM 1900 Cryostat earlier at cold base; Leica Microsystems Nussloch GmbH; German) the special-purpose frozen rubber (Jung Tissue Freezing Medium) of organizing on the cold platform of again leaf sheath that cuts being organized segment vertically to place to keep flat, adds 2～5 again and organizes frozen rubber that leaf sheath is organized fully to wrap; Be placed on the sample stage in the freezing-microtome; After placing 100～140 min, be preferably 120 min, promptly can be used for freezing microtome section.
3) freezing microtome section: cold that will be stained with the leaf sheath tissue is fixed on freezing-microtome (Leica CM 1900 Cryostat; Leica Microsystems Nussloch GmbH on cold pickup groove German), tightens fixed screw, and the slice thickness adjusting knob is transferred to slice thickness 10 μ m, and the minimum control temperature of casing is-40 ℃, and a section minimum control temperature is-50 ℃.Shaking rotation hand wheel cuts into slices.Thicker like the frost layer on the cryoprobe therebetween, should in time start the defrosting program to remove frost layer on the slicer in case roll film.
4) exhibition sheet: earlier with microslide with suitable angle near slicer, whenever cut a section, lightly section is moved on on the microslide with writing brush, every microslide sticks 15～20 sections.
5) dyeing: after posting section; At room temperature let frozen rubber dry slightly; Put into 96% after alcohol-pickled 8～10 hours, take out alcohol is dried, add 1 ml concentration and be 1/15 mol/L, pH value and be 7.0 phosphate buffer on cutting into slices; The submergence section gets final product; Add the aniline blue dyeing liquor that 1 ml concentration is 0.1 mol/L (it is 1/15 mol/L that the aniline blue dyeing liquor uses concentration, and the phosphate buffer of pH=7.0 is formulated) behind 45 min again, behind 60 min that dye microslide is slowly tilted to outwell dyeing liquor.
6) microexamination with take pictures: when slice is just dry; Just put microscopically at Olympus BX51 fluorescence immediately and observing the callose (yellow-greenish phosphorescent light part) in the leaf sheath tissue with ultraviolet excitation; And with the special-purpose camera of this microscope (QIMAGING Micro Publisher 5.0 RTV Canada) take pictures.
Test findings: Fig. 1 shows that complete rice seedling leaf sheath organizes cross section ultrastructure; Can clear view to a leaf sheath organize and have five fibrovascular systems on the square section at least; Each fibrovascular system is made up of bast and xylem; Callose (yellow-greenish phosphorescent light part) is arranged in the phloem sieve tube, do not have callose in the xylem vessel.The deposition situation of callose in callose and the epidermal tissue in ultrastructure in Fig. 2～fibrovascular system of 3 demonstrations and the vascular bundle screen casing.
Claims (4)
1. one kind is used to observe rice leaf sheath and organizes the leaf sheath of callose to organize flaking method, it is characterized in that comprising following processing step:
1) material pre-treatment: get the rice leaf sheath tissue in tillering stage, clean and to dry the back is cut into 0.3～0.5 cm rapidly with blade segment, put into 10% glycerite then immediately, and with vacuum pump 10～20 min that bleed, till the sinking of leaf sheath tissue;
2) sample is fixed: add 1 earlier at cold base and organize frozen rubber; On the cold platform that the leaf sheath that again step 1) is obtained is organized segment vertically to place to keep flat; Adding 2～5 again organizes frozen rubber to organize segment to wrap fully in leaf sheath; And be placed on the sample stage in the freezing-microtome, behind placement 100～140 min, promptly can be used for freezing microtome section;
3) freezing microtome section: will be stained with on cold the pickup groove that cold of leaf sheath tissue be fixed on freezing-microtome; Tighten fixed screw; And the slice thickness adjusting knob transferred to slice thickness 10 μ m; The minimum control temperature of casing is-40 ℃, and a section minimum control temperature is-50 ℃, shakes rotation hand wheel and cuts into slices;
4) exhibition sheet: elder generation near slicer, whenever cuts a section with microslide, lightly section is moved on on the microslide with writing brush, and every microslide sticks 15～20 sections;
5) dyeing: after posting section; At room temperature let frozen rubber dry slightly; Put into 96% after alcohol-pickled 6～10 hours, take out and alcohol is dried, add 1 ml concentration again and be 1/15 mol/L, pH value and be 7.0 phosphate buffer on cutting into slices; Adding 1ml concentration behind 45 min again is that 0.1 mol, pH value are 7.0 aniline blue dyeing liquor, behind 60 min that dye microslide is slowly tilted to outwell dyeing liquor;
6) microexamination with take pictures: when slice is just dry, is just putting microscopically at Olympus BX51 fluorescence immediately and observing the callose in the leaf sheath tissue with ultraviolet excitation, and taking pictures with the camera of this microscope special use.
2. as claimed in claim 1ly a kind ofly be used to observe rice leaf sheath and organize the leaf sheath of callose to organize flaking method, it is characterized in that described step 1) intermediate pump is that SHZ-CA type circulation ability of swimming is used vacuum pump more.
3. as claimed in claim 1ly a kind ofly be used to observe rice leaf sheath and organize the leaf sheath of callose to organize flaking method; It is characterized in that described step 2) in before the leaf sheath cold platform organizing segment vertically to place to keep flat, with lens wiping paper its lip-deep residual liquid is blotted earlier.
4. as claimed in claim 1ly a kind ofly be used to observe rice leaf sheath and organize the leaf sheath of callose to organize flaking method; Frost layer when it is characterized in that freezing microtome section in the described step 3) such as on the cryoprobe is thicker, should in time start the defrosting program to remove frost layer on the slicer in case roll film.
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Cited By (8)
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| CN102967497A (en) * | 2012-12-05 | 2013-03-13 | 沈阳农业大学 | Method for observing in a plant blade microstructure in an oriented manner by treating blade |
| CN103149189A (en) * | 2013-03-11 | 2013-06-12 | 山东省农业科学院作物研究所 | Method for rapidly detecting wheat tissue callose |
| CN105547793A (en) * | 2016-01-13 | 2016-05-04 | 扬州大学 | Method for manufacturing complete section of corn mature seed farinaceous albumen with assistance of nail polish |
| 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 |
| CN106770354A (en) * | 2016-12-26 | 2017-05-31 | 东莞百电子有限公司 | Cut analytic approach in a kind of top of BGA package weld failure |
| CN108572105A (en) * | 2018-04-13 | 2018-09-25 | 山东中医药大学 | A kind of preparation method of brain tissue frozen section |
| CN109374373A (en) * | 2018-10-09 | 2019-02-22 | 黑龙江省农业科学院牡丹江分院 | A kind of Apple paraffin section fast method for preparing |
| CN111238892A (en) * | 2020-01-19 | 2020-06-05 | 宁夏大学 | Permanent flaking method of lichen sporophore microstructure |
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2012
- 2012-02-29 CN CN201210048297XA patent/CN102620960A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102967497A (en) * | 2012-12-05 | 2013-03-13 | 沈阳农业大学 | Method for observing in a plant blade microstructure in an oriented manner by treating blade |
| CN103149189A (en) * | 2013-03-11 | 2013-06-12 | 山东省农业科学院作物研究所 | Method for rapidly detecting wheat tissue callose |
| CN105547793A (en) * | 2016-01-13 | 2016-05-04 | 扬州大学 | Method for manufacturing complete section of corn mature seed farinaceous albumen with assistance of nail polish |
| CN105547793B (en) * | 2016-01-13 | 2018-01-30 | 扬州大学 | A kind of corn mature seed farinaceous albumen nail polish aids in whole slices preparation method |
| 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 |
| CN106770354A (en) * | 2016-12-26 | 2017-05-31 | 东莞百电子有限公司 | Cut analytic approach in a kind of top of BGA package weld failure |
| CN108572105A (en) * | 2018-04-13 | 2018-09-25 | 山东中医药大学 | A kind of preparation method of brain tissue frozen section |
| CN109374373A (en) * | 2018-10-09 | 2019-02-22 | 黑龙江省农业科学院牡丹江分院 | A kind of Apple paraffin section fast method for preparing |
| CN111238892A (en) * | 2020-01-19 | 2020-06-05 | 宁夏大学 | Permanent flaking method of lichen sporophore microstructure |
| CN111238892B (en) * | 2020-01-19 | 2023-02-24 | 宁夏大学 | Permanent flaking method of lichen sporophore microstructure |
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Application publication date: 20120801 |