CN108444956A - With the method for laser confocal microscope and fluorescent dye observation AM fungi clump branch structures - Google Patents
With the method for laser confocal microscope and fluorescent dye observation AM fungi clump branch structures Download PDFInfo
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- CN108444956A CN108444956A CN201810075725.5A CN201810075725A CN108444956A CN 108444956 A CN108444956 A CN 108444956A CN 201810075725 A CN201810075725 A CN 201810075725A CN 108444956 A CN108444956 A CN 108444956A
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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Abstract
The present invention relates to the methods with laser confocal microscope and fluorescent dye observation AM fungi clump branch structures.It includes the following steps：AM fungies establish homobium with aulophyte root system；AM fungi root tissues are handled using fluorescent dye solution, segmentation is placed on glass slide, and coverslip mounting is used in combination；Image is collected using confocal laser scanning microscope and carries out image procossing.Sensitive analysis and observation and can judge that AM fungies and other infect whether plant establishes symbiosis through the invention, can as identify AM fungies whether the sensitive method with plant symbiosis；It is easy to the development of 3 D stereo observation and analysis root system mycorhiza clump branch structure through the invention；Distribution situation of the mycorrhizal fungi clump branch structure arrived according to the observation in plant root, the enlargement of present invention research simultaneously plant root cells plasma membrane corresponding with clump branch structure can be used, it is called the biological function for enclosing clump branch film, as nutriment nitrogen ﹑ phosphorus transports the comparative studies that protein gene is expressed on film.
Description
Technical field
Present invention relates particularly to the methods with laser confocal microscope and fluorescent dye observation AM fungi clump branch structures.
Background technology
Mycorhiza is root system of plant and the homobium that mycorrhizal fungi is formed, and the mycorhiza in nature is divided into exotrophic mycorrhiza and Nei Sheng
Mycorhiza.Arbuscular mycorrhiza (Arbuscular Mycorrhizal, AM) fungi is a kind of endo-mycorrhiza of generally existing in nature
Fungi, about 90% vascular plant can formed arbuscular mycorrhiza on the earth.AM fungies and host plant mutualistic symbiosis：Very
The outer mycelium of the root of bacterium（Extraradical Mycelium,ERM）Phosphorus, nitrogen and other nutrients is absorbed from soil to supply
To host plant, absorbability of the plant to mineral element is improved, promotes plant growth, enhances the resistance etc. of plant, place
Main plant then supplies carbon aquatic products necessary to fungi growth.The structure of AM fungies is broadly divided into clump branch, mycelia and spore, in addition to
Most of AM fungies also have vesicle structure other than Gigaspora and the mould category of shield sporangiocyst.Clump branch is that AM fungus breeding bodies infect plant
Mycelia in the root of object root, the mycelia dendritic morphology that continuous bifurcated branch is formed in cortical cell（arbuscule）；It is corresponding to it
Be plant root cells plasma membrane enlargement, be called enclose clump branch film（PAM, periarbuscular membrane）.Enclose clump branch
Film and the gap surrounded with clump branch structure, which are called, encloses clump branch space（PAS, periarbuscular space）, enclose clump branch space
PAS is the main place that fungi carries out nutrition and information exchange with plant symbiosis body.
With the formation of AM fungi clump branch structures, great variety occurs for root cortical cell, this needs to reorganize cell
Plasma membrane includes the huge increase of the reconstruct of cytoskeleton and organelle, especially membrane area.The change of some of which cell is
To preceding traditional antibody and fluorescin fusion labelling technique detection, but these mycorhiza infected cells still have many biology
The function of aspect is that current traditional technology research is not known.
Further, since traditional light microscope can only observe the clump dendritic morphology after dyeing, and it can only be one flat
Image on face, thus be unable to complete observation understand clump dendritic morphology, affect to its biological function it is further research with
Solution.Then there is an urgent need to it is a kind of can 3 D stereo, multi-angle observation microscopic method.
Invention content
In view of the problems of the existing technology, it is an object of the invention to design, offer is a kind of to use laser confocal microscope
With the technical solution of the method for fluorescent dye observation AM fungi clump branch structures.
The method with laser confocal microscope and fluorescent dye observation AM fungi clump branch structures, it is characterised in that
Include the following steps：
1）AM fungies establish homobium with aulophyte root system；
2）Using fluorescent dye WGA-Alexa fluor488 solution treatment AM fungi root tissues, segmentation is placed on glass slide,
Coverslip mounting is used in combination；
3）Image is collected using confocal laser scanning microscope and carries out image procossing.
The method with laser confocal microscope and fluorescent dye observation AM fungi clump branch structures, it is characterised in that
The step 1）Specially：By the aulophyte in 3 week that takes root, takes out, be transferred to containing MS from MS agar mediums
On the culture dish of culture medium, while it being inoculated with arbuscular mycorrhizal fungi spore or mycelia that upper and Carrot Roots infect, carries out symbiosis training
It supports.
The method with laser confocal microscope and fluorescent dye observation AM fungi clump branch structures, it is characterised in that
The step 2）Specially：
A, by step 1）The plant roots of obtained AM fungal infections are soaked in 50% ethyl alcohol 1 hour；
B, it after washing with water, is put into 20%KOH solution and keeps the temperature 2 hours for 90 DEG C, then washed respectively with distilled water and PBS buffer solutions
It washs；
C, by the plant roots culture of the AM fungal infections after washing in the WGA-Alexa fluor488 containing a concentration of 5ug/ml
In the culture dish of solution overnight；
D, the root containing WGA-Alexa fluor488 is cut into 4 millimeters of long segments, then 4 millimeters of root segment tissues is positioned over thickness
It spends between 0.8~1.2mm on glass slide, with thickness in 0.17mm coverslip mountings.
The method with laser confocal microscope and fluorescent dye observation AM fungi clump branch structures, it is characterised in that
The step 3）Specially：Heel piece section is seen with Leica TCS-SP5 Laser Scanning Confocal Microscopes with 20 times or 63 times of immersion type eyepieces
Imaging is examined, while image DIC, laser 488nm, photo counting 610, scanning are compared according to phosphor collection disturbance
35.18 seconds time, Pixel Dwell 38.49 seconds, Pixel size 0.21um, Frame mode, scanning area image size
139.3 × 104.6um, optics sectional drawing are collected and are superimposed with the intervals 0.08um, and image is at Leica LAS-AF softwares 2.6.0
Reason.
Compared with prior art, the invention has the advantages that：
1. aulophyte and arbuscular mycorrhizal fungi are established homobium by the present invention using MS culture mediums, easily and effectively；
2. sensitive analysis and observation and can judge that AM fungies infect whether plant establishes symbiosis with other through the invention,
Can as identification AM fungies whether the sensitive method with plant symbiosis；
3. being easy to the development of 3 D stereo observation and analysis root system mycorhiza clump branch structure through the invention（Fig. 2）；
4. arrive according to the observation mycorrhizal fungi clump branch structure plant root distribution situation, can use the present invention simultaneously research with
The enlargement of the corresponding plant root cells plasma membrane of clump branch structure is called and encloses clump branch film（PAM, periarbuscular
membrane）Biological function, as nutriment nitrogen ﹑ phosphorus transports the comparative studies that protein gene is expressed on film.
Description of the drawings
Fig. 1 is the poplar root observed with conventional optical microscope and arbuscular mycorrhizal fungi symbiosis figure, Tu1AZhong
For poplar root and arbuscular mycorrhizal fungiRhizophagus irregularisEstablish symbiosis；Figure 1B is to be contaminated with methylene blue
The poplar root and arbuscular mycorrhizal fungi observed after colorR.irregularisClump branch structure.
Fig. 2 be observe through the invention host plant willow (Populus trichocarpa）With AM fungiesR.irregularisThe clump branch structure that symbiosis is established.
Specific implementation mode
It further illustrates the present invention with reference to embodiments.
Embodiment 1
1, material requested：Plant with willow (P.trichocarpa) body outer clone plant, arbuscular mycorrhizal fungi useR.irregularis。
2, experiment material, the special small culture dishes of Confocal or other small circular culture dishes.
3, the preparation of MS culture mediums；50 milliliters of macroelement in every liter of culture medium, 100 milliliters of trace element, glucose 1
Gram, 10 milliliters of vitamin.These elements are mixed, pH to 5.5 is adjusted, add 10 grams of agar powder, are sterilized 121 °C, 20 minutes.It is cooling
Afterwards, culture dish is poured into, it is spare.
4, poplar root and arbuscular mycorrhizal fungiR.irregularisEstablish homobium
Be careful the willow in 3 week that takes root to avoid with agar, transfer when them are taken out from MS agar mediums
Onto MS culture mediums, while being inoculated with the arbuscular mycorrhizal fungi that upper and Ri T-DNA conversions carrot root infectR.irregularisSpore or mycelia are conducive to establish homobium with poplar root.After co-culturing 3 months, with tweezers picking poplar
Tree root, removal and washing root agar, to wait for next operation.
5, AM fungi mycorhiza tissues are handled using dyestuff WGA-Alexa fluor488
It is first that the root of AM fungal infections is soaked in 50% ethyl alcohol 1 hour in order to observe clump branch microstructure, after then washing with water,
It being put into 90 DEG C of 20%KOH solution and keeps the temperature 2 hours, be then washed with distilled water 5 times, washed root is washed with PBS buffer solutions, then
Culture is placed to stay overnight in the special small culture dishes of Confocal and containing WGA-Alexa fluor488 solution.Then, in order to
Root containing WGA-Alexa fluor488, is cut into 4 by fluorescence microscope and confocal laser scanning microscope clump dendritic morphology
The long segment of millimeter.Finally, 4 millimeters of root segment tissues thickness is positioned between 0.8~1.2mm on glass slide, with thickness to be existed
0.17mm coverslip mountings.
6, it collects image using confocal laser scanning microscope and carries out image procossing
By heel piece section Leica TCS-SP5 Laser Scanning Confocal Microscopes 20 times or 63 times of immersion type eyepiece observation imagings.Root simultaneously
Image is compared according to phosphor collection disturbance（DIC）, laser 488nm, photo counting 610 (gain), sweep time
35.18 seconds, Pixel Dwell 38.49 seconds, Pixel size 0.21um.Frame mode, scanning area image size
139.3 × 104.6um. optics sectional drawing is collected and is superimposed with the intervals 0.08um.Image is at Leica LAS-AF softwares 2.6.0
Reason.
The results are shown in Figure 2 for observation.As Fig. 2 observes result compared with traditional method（Fig. 1）Advantage, advantage exists
In：1. clump branch can be clearly observed（arbuscular）Three-dimensional structure, be easy to 3 D stereo observation and analysis root system bacterium
The development of network of roots branch structure；2. can further judge the host plant whether with AM fungies formed symbiosis；3. according to sight
The mycorrhizal fungi clump branch structure observed is in the distribution situation of plant root, and can using the present invention, research is corresponding with clump branch structure simultaneously
Plant root cells plasma membrane enlargement, be called enclose clump branch film（PAM, periarbuscular membrane）Biology
Transport of substances function, as nitrogen ﹑ phosphorus transports the comparative studies that protein gene is expressed on film.
Claims (4)
1. with the method for laser confocal microscope and fluorescent dye observation AM fungi clump branch structures, it is characterised in that including following
Step：
1）AM fungies establish homobium with aulophyte root system；
2）Using fluorescent dye WGA-Alexa fluor488 solution treatment AM fungi root tissues, segmentation is placed on glass slide,
Coverslip mounting is used in combination；
3）Image is collected using confocal laser scanning microscope and carries out image procossing.
2. the method for observing AM fungi clump branch structures with laser confocal microscope and fluorescent dye as described in claim 1,
It is characterized in that the step 1）Specially：It by the aulophyte in 3 week that takes root, takes out, turns from MS agar mediums
It moves on on the culture dish containing MS culture mediums, while being inoculated with arbuscular mycorrhizal fungi spore or mycelia that upper and Carrot Roots infect, into
Row symbiosis culture.
3. the method for observing AM fungi clump branch structures with laser confocal microscope and fluorescent dye as described in claim 1,
It is characterized in that the step 2）Specially：
A, by step 1）The plant roots of obtained AM fungal infections are soaked in 50% ethyl alcohol 1 hour；
B, it after washing with water, is put into 20%KOH solution and keeps the temperature 2 hours for 90 DEG C, then washed respectively with distilled water and PBS buffer solutions
It washs；
C, by the plant roots culture of the AM fungal infections after washing in the WGA-Alexa fluor488 containing a concentration of 5ug/ml
In the culture dish of solution overnight；
D, the root containing WGA-Alexa fluor488 is cut into 4 millimeters of long segments, then 4 millimeters of root segment tissues is positioned over thickness
It spends between 0.8~1.2mm on glass slide, with thickness in 0.17mm coverslip mountings.
4. the method for observing AM fungi clump branch structures with laser confocal microscope and fluorescent dye as described in claim 1,
It is characterized in that the step 3）Specially：Heel piece section Leica TCS-SP5 Laser Scanning Confocal Microscopes 20 times or 63 times of water
The observation imaging of immersion eyepiece, while image DIC, laser 488nm, photo counting are compared according to phosphor collection disturbance
610,35.18 seconds sweep times, Pixel Dwell 38.49 seconds, Pixel size 0.21um, Frame mode, scanning area
Image 139.3 × 104.6um of size, optics sectional drawing are collected and are superimposed with the intervals 0.08um, image Leica LAS-AF softwares
2.6.0 processing.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113406050A (en) * | 2021-06-16 | 2021-09-17 | 广东省科学院生物工程研究所 | Method for identifying bacterial infection type based on duckweed |
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| US20030052280A1 (en) * | 2001-07-23 | 2003-03-20 | Foster Thomas H. | Method for operating a laser scanning confocal microscope system and a system thereof |
| CN1682584A (en) * | 2004-04-15 | 2005-10-19 | 广东省微生物研究所 | Method for establishing symboitic relationship for arbuscular nycorrhizal fungi and tomato hairy root |
| CN102770544A (en) * | 2010-01-25 | 2012-11-07 | 拜尔作物科学公司 | Methods for manufacturing plant cell walls comprising chitin |
| CN104371932A (en) * | 2014-10-21 | 2015-02-25 | 宁夏农林科学院 | Method for producing AM fungal inoculant |
| CN104531824A (en) * | 2014-12-16 | 2015-04-22 | 北京农学院 | Labeling method of apple pollen tube microfilament |
-
2018
- 2018-01-26 CN CN201810075725.5A patent/CN108444956A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030052280A1 (en) * | 2001-07-23 | 2003-03-20 | Foster Thomas H. | Method for operating a laser scanning confocal microscope system and a system thereof |
| CN1682584A (en) * | 2004-04-15 | 2005-10-19 | 广东省微生物研究所 | Method for establishing symboitic relationship for arbuscular nycorrhizal fungi and tomato hairy root |
| CN102770544A (en) * | 2010-01-25 | 2012-11-07 | 拜尔作物科学公司 | Methods for manufacturing plant cell walls comprising chitin |
| CN104371932A (en) * | 2014-10-21 | 2015-02-25 | 宁夏农林科学院 | Method for producing AM fungal inoculant |
| CN104531824A (en) * | 2014-12-16 | 2015-04-22 | 北京农学院 | Labeling method of apple pollen tube microfilament |
Non-Patent Citations (1)
| Title |
|---|
| 马继芳: "丛枝菌根（AM）真菌高效繁殖条件及应用的研究", 《中国优秀硕士学位论文全文数据库 农业科学辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113406050A (en) * | 2021-06-16 | 2021-09-17 | 广东省科学院生物工程研究所 | Method for identifying bacterial infection type based on duckweed |
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Application publication date: 20180824 |