CN106769298A - A kind of microscopy observation method of plant tissue - Google Patents
A kind of microscopy observation method of plant tissue Download PDFInfo
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Abstract
The invention discloses a kind of microscopy observation method of plant tissue.The microscopy observation method of the plant tissue that the present invention is provided, will reuse potassium hydroxide and is digested, then carry out transparent processing after fixing process, it is to avoid enzymolysis or other operations, save cost；Transparent effect is good, good with fluorescent staining and marking operation compatibility：The internal structure of plant deep tissues can be clearly observed under fluorescence microscope by fluorescent staining.Digestion process combination transparent technology carries out the method for microexamination to plant deep tissues for the further investigation of higher plant phenotypic evaluation and gene expression pattern is laid a good foundation, especially played a significant role in the research that plant underlying tissue structure is observed, actual application value is high.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of microscopy observation method of plant tissue.
Background technology
As functional genomics, molecular biology research deepen continuously, expression portion of the specific gene in plant
Position its got the attention in the function of specific developmental stage.Fluorescence microscopy is observed and 3 Dimension Image Technique is plant phenotype point
Analysis, plant and pathogen interaction and Gene Expression Profile Analysis provide important tool.Compared with zooblast, plant cell
Wall is different from cytoplasm refractive index, and the signal noise ratio (snr) of image of acquisition drops in the pigment and aromatic substance of rich content in cell
It is low.Therefore, the microexamination in the past to plant cell is only limitted to several confluent monolayer cells that depth is about 30 μm or so, greatly influences
The parsing of plant cell structures and function.
For the internal structure or subcellular structure of observation of plant body deeper, generally require, by specially treated, to make plant
Thing sample reduces thickness and volume, light is just carried out microexamination through sample.Material requirements after treatment is small and thin, complete
Whole, transparent, holding original structure, and easily recognized with color.For reach above-mentioned requirements, it is necessary to take different flaking method or
Person vegetable material is carried out before imaging it is transparent, with the plant sample for facilitating tissues observed thicker, such as tobacco and tomato leaf
Piece, mongolicum Regel etc..
The content of the invention
One purpose of the present invention is to provide a kind of method that plant tissue microscope detects pre-treatment.
The method that the present invention is provided, comprises the following steps A：
1) plant tissue is fixed with fixer, the plant tissue after being fixed；
2) plant tissue after the fixation is digested with potassium hydroxide, obtains postdigestive plant tissue；
3) postdigestive plant tissue is washed with PHEM solution, plant tissue after being washed；
4) by plant tissue Immersion treatment in transparent liquid after the washing, obtain it is transparent after plant tissue, realize plant
Thing tissue microscope detects pre-treatment；
The PHEM solution includes PIPES (piperazidine -1,4- two (2-ethanesulfonic acid)), HEPES (hydroxyethyl piperazine second
Thiosulfonic acid), EGTA and MgCl2；
The transparent liquid is by urea, glycerine, Triton X-100 and the PHEM solution compositions；
In the above method,
The PIPES, the HEPES, the EGTA and the MgCl2Mole amount ratio be 40-80：15-35：5-
15：1-3；
Or the PIPES, the HEPES, the EGTA and the MgCl2Mole amount ratio be 60：25：15：2；
Or the PIPES, the HEPES, the EGTA and the MgCl2Mole amount ratio be 60：25：10：2；
Or the PIPES, the HEPES, the EGTA and the MgCl2Mole amount ratio be 40：15：5：1；
Or the proportioning of the urea, the glycerine, the Triton X-10 is 4-8mol：20-40ml：0.05-
0.15g；
Or the proportioning of the urea, the glycerine, the Triton X-10 is 6mol：20ml：0.1g；
Or the proportioning of the urea, the glycerine, the Triton X-10 is 6mol：30ml：0.1g；
Or the proportioning of the urea, the glycerine, the Triton X-10 is 4mol：20ml：0.1g.
In the above method,
The PHEM solution is by final concentration of 40-80mM PIPES, final concentration of 15-35mM HEPES, final concentration of 5-
15mM EGTA, final concentration of 1-3mM MgCl2Constituted with water；
Or the PHEM solution is by final concentration of 60mM PIPES, final concentration of 25mM HEPES, final concentration of 15mM
EGTA, final concentration of 2mM MgCl2Constituted with water；
Or the PHEM solution is by final concentration of 60mM PIPES, final concentration of 25mM HEPES, final concentration of 10mM
EGTA, final concentration of 2mM MgCl2Constituted with water；
Or the PHEM solution is by final concentration of 40mM PIPES, final concentration of 15mM HEPES, final concentration of 5mM
EGTA, final concentration of 1mM MgCl2Constituted with water；
Or the transparent liquid is by final concentration of 4-8M urea, percent by volume 20-40% glycerine, weight/mass percentage composition
0.05-0.15%Triton X-100 and PHEM solution compositions；
Or the transparent liquid is by final concentration of 6M urea, the glycerine of percent by volume 20%, percent by volume 0.1%
Triton X-100 and PHEM solution compositions；
Or the transparent liquid is by final concentration of 6M urea, the glycerine of percent by volume 30%, percent by volume 0.1%
Triton X-100 and PHEM solution compositions；
Or the transparent liquid is by final concentration of 4M urea, the glycerine of percent by volume 20%, percent by volume 0.1%
Triton X-100 and PHEM solution compositions.
In the above method,
The pH value of the PHEM solution is 6.9；
In the above method,
The mode of the plant tissue potassium hydroxide digestion by after fixation be the plant tissue after fixation is immersed in it is dense
Spend in the potassium hydroxide aqueous solution for mass percent 10-15%；
Or described is by the postdigestive plant tissue with the mode that PHEM solution is washed by postdigestive plant tissue
It is immersed in the PHEM solution.
In the above method,
The fixed fixer for using by glutaraldehyde, paraformaldehyde and the PHEM solution compositions,
The proportioning of the glutaraldehyde and the paraformaldehyde is 4-8ml：4-8g；
Or the fixer by volumn concentration for 4-8% glutaraldehydes, mass percent are 4-8% paraformaldehydes and institute
State PHEM solution compositions.
In the above method,
The temperature of the fixation is 0-4 DEG C, and the regular time is 10-16 hours；
Or the time of the digestion is 6-8 hours；
It is each 5-10 minutes or the mode of the washing is washing 3-5 times；
Or the time of the Immersion treatment is 5-10, the mode of the Immersion treatment is to change once described every 1 day
Bright liquid.
In the above method,
The plant tissue is that length is 0.5-1cm2Plant leaf blade or 0.5-1cm plant tissues long.
Second purpose of the invention is to provide a kind of microscopy observation method of plant tissue.
The method that the present invention is provided, including above-mentioned steps A.
The 3rd purpose of the present invention is to provide a kind of kit of plant tissue microscope detection.
The kit that the present invention is provided, including above-mentioned step A the PHEM solution and/or the transparent liquid.
The microscopy observation method of the plant tissue that the present invention is provided, it has advantages below：1) it is simple to operate, quick：Contracting
Short enzymolysis and the time of the early stage treatment such as transparent；2) reuse potassium hydroxide after fixing process to be digested, it is to avoid enzymolysis
Or other operations, save cost；3) transparent effect is good, good with fluorescent staining and marking operation compatibility：Can be contaminated by fluorescence
Color clearly observes the internal structure of plant deep tissues under fluorescence microscope.
It is demonstrated experimentally that carrying out microexamination to plant tissue using the method that the present invention is provided, observation depth is significantly improved；
The consumption of cellulase etc. is reduced, and reaches cost-effective purpose；It is further investigation gene expression in plants pattern and deep tissues
Structure provides important tool.As can be seen here, digestion process combination transparent technology carries out microexamination to plant deep tissues
Method is laid a good foundation for the further investigation of higher plant phenotypic evaluation and gene expression pattern, especially in plant deep tissues
Played a significant role in the research that structure is observed, actual application value is high.
Brief description of the drawings
Fig. 1 is the distribution situation of Remorin1.2 albumen in the Arabidopsis leaf observed under fluorescence microscope.
Fig. 2 is the distribution situation of the mongolicum Regel Chloroplast observed under ordinary optical microscope.
Fig. 3 is the distribution situation of Remorin albumen in the tobacco leaf observed under fluorescence microscope.
Fig. 4 is the tobacco leaf situation that comparative example is observed under fluorescence microscope.
Specific embodiment
Experimental technique used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc. in following embodiments, unless otherwise specified, commercially obtain.
Method therefor is conventional method unless otherwise instructed in following embodiments, and the percentage composition is unless otherwise instructed
It is weight/mass percentage composition.
The transparent processing and microscopic of embodiment 1, Arabidopsis leaf
First, reagent needed for the transparent processing of blade
Fixer is 4% by glutaraldehyde, paraformaldehyde and PHEM solution compositions, the wherein percent by volume of glutaraldehyde, many
The mass percent of polyformaldehyde is 4%.
PHEM solution is by final concentration of 40mM PIPES, 15mM HEPES, 5mM EGTA, 1mM MgCl2With water composition, pH
Be worth is 6.9；
Transparent liquid is by final concentration of 4M urea, 20% glycerine (percent by volume), percent by volume 0.1%Triton
X-100 and PHEM solution compositions.
2nd, the transparent processing and microscopic of Arabidopsis leaf
1st, the transparent processing of Arabidopsis leaf
Arabidopsis (Columbia ecotype) seed is planted on 1/2MS culture mediums, 0-4 DEG C of vernalization two days later, puts 23
Sprouted in ± 2 DEG C of incubators, continuation is cultivated 20-30 days during compost is moved to after five days, the clip lotus throne leaf blade before Taiwan is taken out.
1) above-mentioned lotus throne leaf blade is cut into 0.5cm2The fritter of square, is washed with deionized to going the removal of impurity；
2) by step 1) obtain 0.5cm2The fritter of square is submerged in being transferred to fixer, is fixed rear arabidopsis leaf
Piece；Fixed temperature is 4 DEG C, and regular time is 12 hours；Fixed to use shaking speed 50rpm, radius of turn is 50mm.
3) Arabidopsis leaf after fixation is placed in the potassium hydroxide aqueous solution of mass percent 10% and is submerged, digestion disappears
The change time is 4 hours, obtains postdigestive Arabidopsis leaf；
4) postdigestive Arabidopsis leaf is submerged 5 minutes in above-mentioned PHEM solution, is repeated 3 times, the plan after being washed
Southern mustard blade；
5) Arabidopsis leaf after washing is submerged in transparent liquid carries out transparent processing 3 days, changes once saturating every 1 day
Bright liquid；Obtain it is transparent after Arabidopsis leaf.
2nd, microscopic
By above-mentioned 1 obtain it is transparent after Arabidopsis leaf excitation wavelength for 488nm fluorescence microscopy Microscopic observation, knot
Fruit is as shown in figure 1, it is observed that epidermal cell and mesophyll cell from Fig. 1, it can be seen that Remorin eggs in Arabidopsis leaf
It is mainly distributed on cytoplasma membrane in vain, it is seen that cell interior has a small amount of vesicular structure.
The distribution of embodiment 2, husky green onion mesophyll cell Chloroplast
First, reagent needed for the transparent processing of blade
Fixer is 4% by glutaraldehyde, paraformaldehyde and PHEM solution compositions, the wherein percent by volume of glutaraldehyde, many
The mass percent of polyformaldehyde is 8%.
PHEM solution is by final concentration of 60mM PIPES, 25mM HEPES, 10mM EGTA, 2mM MgCl2Constituted with water,
PH value is 6.9；
Transparent liquid is by final concentration of 6M urea, 30% glycerine (percent by volume), percent by volume 0.1%Triton
X-100 and PHEM solution compositions.
2nd, the transparent processing and microscopic of mongolicum Regel
Husky green onion is cultivated high to 20-30cm, the aerial part of seedling is taken；
1) above-mentioned mongolicum Regel is cut into 0.8cm segments long, be washed with deionized to removal blade on soil and its
His impurity；
2) by step 1) 20 sections of mongolicum Regels obtaining are transferred in fixer and submerge；It is fixed to use shaking speed 50rpm,
Radius of turn is 50mm.
Fixed temperature is 4 DEG C, and regular time is 12 hours.
3) Arabidopsis leaf after fixation is placed in the potassium hydroxide aqueous solution of mass percent 10% and is submerged, digestion disappears
The change time is 8 hours, obtains postdigestive mongolicum Regel；
4) postdigestive mongolicum Regel is submerged 5 minutes in above-mentioned PHEM solution, is repeated 3 times, the sand after being washed
Folium Allii fistulosi piece；
5) mongolicum Regel after washing is submerged in transparent liquid carries out transparent processing 5 days, changes once transparent every 1 day
Liquid, obtain it is transparent after mongolicum Regel.
2nd, microscopic
By above-mentioned 1 obtain it is transparent after mongolicum Regel excitation wavelength for 488nm fluorescence microscopy Microscopic observation, as a result
As shown in Fig. 2 being observed that epidermal cell and mesophyll cell from Fig. 2, husky green onion mesophyll cell Chloroplast arrangement is tight, its
Between a small amount of stomata is distributed with.
The distribution of REMORIN albumen in embodiment 3, tobacco mesophyll cell
First, reagent needed for the transparent processing of blade
Fixer is 5% by glutaraldehyde, paraformaldehyde and PHEM solution compositions, the wherein percent by volume of glutaraldehyde, many
The mass percent of polyformaldehyde is 5%.
PHEM solution is by final concentration of 60mM PIPES, 20mM HEPES, 10mM EGTA, 2mM MgCl2Constituted with water,
PH value is 6.9；
Transparent liquid is by final concentration of 6M urea, 20% glycerine (percent by volume), percent by volume 0.1%Triton
X-100 and PHEM solution compositions.
2nd, the transparent processing and microscopic of tobacco leaf
By tobacco seed in vernalization at 20-25 DEG C, after seed is sprouted, flower cultivating soil plantation in 25 ± 2 DEG C of greenhouses is put, culture is extremely
Plant height 5-10cm.
1) Agrobacterium strain shaken cultivation to the OD values that will carry REMORIN2 genes are 0.8 (Detection wavelength 660nm),
6000rpm room temperatures are centrifuged 3 minutes collects thallines, and the resuspended thalline of buffer solution is injected with tobacco leaf；
2) by step 1) obtain bacterium solution be injected in tobacco leaf with 1mL syringes, above-mentioned tobacco seedling is put 25 ± 2
Continue to cultivate 36 hours in DEG C greenhouse；
3) blade after above-mentioned injection is cut into 0.5cm2The fritter of square, is washed with deionized to going the removal of impurity；
4) by step 1) obtain 0.5cm2The fritter of square is submerged in being transferred to fixer, is fixed rear Tobacco Leaf
Piece；
Fixed temperature is 4 DEG C, and regular time is 12 hours, fixed to use shaking speed 50rpm, and radius of turn is
50mm；
5) tobacco leaf after fixation is placed in the potassium hydroxide aqueous solution of mass percent 10% and is submerged, digestion；Disappear
The change time is 6 hours, obtains postdigestive tobacco leaf；
6) postdigestive tobacco leaf is submerged 5 minutes in above-mentioned PHEM solution, is repeated 3 times, 5 minutes every time, washed
Tobacco leaf after washing；
7) tobacco leaf after washing is submerged in transparent liquid carries out transparent processing 5 days, changes once transparent every 1 day
Liquid；Obtain it is transparent after tobacco leaf.
2nd, microscopic
By above-mentioned 1 obtain it is transparent after tobacco leaf excitation wavelength for 488nm fluorescence microscopy Microscopic observation, as a result
As shown in figure 3, it is observed that the mesophyll cell of table subcutaneous deep, Remorin albumen is main in tobacco mesophyll cell from Fig. 3
It is distributed in cytoplasma membrane.
Comparative example：Conventional material clearing method is incompatible with fluorescent protein labeling, it is impossible to for observe observe expression,
Thus it is only capable of observing the relatively thin material of thickness of sample, the larger sample of thickness can not be illuminated, observation thickness is only 30 μm or so
Several confluent monolayer cells.Fig. 4 is that conventional method observation thickness is 100 μm of results of tobacco leaf deep layer mesophyll cell.
The method used in the present invention is mutually compatible with fluorescent protein labeling, and fluorescence signal can be kept for the several months, and can be with
It was observed that thickness is 100 μm or so of deep layer cells.
Claims (10)
1. a kind of method that plant tissue microscope detects pre-treatment, comprises the following steps A：
1) plant tissue is fixed with fixer, the plant tissue after being fixed；
2) plant tissue after the fixation is digested with potassium hydroxide, obtains postdigestive plant tissue；
3) postdigestive plant tissue is washed with PHEM solution, plant tissue after being washed；
4) by plant tissue Immersion treatment in transparent liquid after the washing, obtain it is transparent after plant tissue, realize plant group
Knit microscope detection pre-treatment；
The PHEM solution includes PIPES, HEPES, EGTA and MgCl2；
The transparent liquid is by urea, glycerine, Triton X-100 and the PHEM solution compositions.
2. method according to claim 1, it is characterised in that：
The PIPES, the HEPES, the EGTA and the MgCl2Mole amount ratio be 40-80：15-35：5-15：1-3；
Or the PIPES, the HEPES, the EGTA and the MgCl2Mole amount ratio be 60：25：15：2；
Or the PIPES, the HEPES, the EGTA and the MgCl2Mole amount ratio be 60：25：10：2；
Or the PIPES, the HEPES, the EGTA and the MgCl2Mole amount ratio be 40：15：5：1；
Or the proportioning of the urea, the glycerine, the Triton X-10 is 4-8mol：20-40ml：0.05-0.15g；
Or the proportioning of the urea, the glycerine, the Triton X-10 is 6mol：20ml：0.1g；
Or the proportioning of the urea, the glycerine, the Triton X-10 is 6mol：30ml：0.1g；
Or the proportioning of the urea, the glycerine, the Triton X-10 is 4mol：20ml：0.1g.
3. method according to claim 1 and 2, it is characterised in that：
The PHEM solution is by final concentration of 40-80mM PIPES, final concentration of 15-35mM HEPES, final concentration of 5-15mM
EGTA, final concentration of 1-3mM MgCl2Constituted with water；
Or the PHEM solution by final concentration of 60mM PIPES, final concentration of 25mM HEPES, final concentration of 15mM EGTA,
Final concentration of 2mM MgCl2Constituted with water；
Or the PHEM solution by final concentration of 60mM PIPES, final concentration of 25mM HEPES, final concentration of 10mM EGTA,
Final concentration of 2mM MgCl2Constituted with water；
Or the PHEM solution is by final concentration of 40mM PIPES, final concentration of 15mM HEPES, final concentration of 5mM EGTA, end
Concentration is 1mM MgCl2Constituted with water；
Or the transparent liquid is by final concentration of 4-8M urea, percent by volume 20-40% glycerine, weight/mass percentage composition 0.05-
0.15%Triton X-100 and PHEM solution compositions；
Or the transparent liquid is by final concentration of 6M urea, the glycerine of percent by volume 20%, percent by volume 0.1%Triton
X-100 and PHEM solution compositions；
Or the transparent liquid is by final concentration of 6M urea, the glycerine of percent by volume 30%, percent by volume 0.1%Triton
X-100 and PHEM solution compositions；
Or the transparent liquid is by final concentration of 4M urea, the glycerine of percent by volume 20%, percent by volume 0.1%Triton
X-100 and PHEM solution compositions.
4. according to any described method in claim 1-3, it is characterised in that：
The pH value of the PHEM solution is 6.9.
5. according to any described method in claim 1-4, it is characterised in that：
The mode of the plant tissue potassium hydroxide digestion by after fixation is that the plant tissue after fixation is immersed in into concentration to be
In the potassium hydroxide aqueous solution of mass percent 10-15%；
Or described is to submerge the postdigestive plant tissue with the mode that PHEM solution is washed by postdigestive plant tissue
In the PHEM solution.
6. according to any described method in claim 1-5, it is characterised in that：
The fixed fixer for using by glutaraldehyde, paraformaldehyde and the PHEM solution compositions,
The proportioning of the glutaraldehyde and the paraformaldehyde is 4-8ml：4-8g；
Or the fixer is that 4-8% glutaraldehydes, mass percent are 4-8% paraformaldehydes and described by volumn concentration
PHEM solution compositions.
7. according to any described method in claim 1-6, it is characterised in that：
The temperature of the fixation is 0-4 DEG C, and the regular time is 10-16 hours；
Or the time of the digestion is 6-8 hours；
It is each 5-10 minutes or the mode of the washing is washing 3-5 times；
Or the time of the Immersion treatment is 5-10, the mode of the Immersion treatment is to change the once transparent liquid every 1 day.
8. according to any described method in claim 1-7, it is characterised in that：The plant tissue is that length is 0.5-1cm2
Plant leaf blade or 0.5-1cm plant tissues long.
9. a kind of microscopy observation method of plant tissue, including any described step A in claim 1-8.
10. the kit of a kind of plant tissue microscope detection, including in claim 1-8 it is any described in the step of A institute
State PHEM solution and/or the transparent liquid.
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Cited By (1)
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| CN113008646A (en) * | 2021-02-03 | 2021-06-22 | 广西特色作物研究院 | Method for eliminating sediment in tissue slices of citrus fixed samples |
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