CN106769298B - 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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- 238000000034 method Methods 0.000 title claims abstract description 41
- 238000000386 microscopy Methods 0.000 title claims abstract description 8
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims abstract description 33
- 230000029087 digestion Effects 0.000 claims abstract description 4
- PBJBVIHLBRYRQC-UHFFFAOYSA-N 1-o-[2-(diethylamino)ethyl] 3-o-ethyl 2-methyl-2-phenylpropanedioate Chemical compound CCN(CC)CCOC(=O)C(C)(C(=O)OCC)C1=CC=CC=C1 PBJBVIHLBRYRQC-UHFFFAOYSA-N 0.000 claims description 45
- 239000000243 solution Substances 0.000 claims description 45
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 42
- 239000007788 liquid Substances 0.000 claims description 25
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 23
- IHPYMWDTONKSCO-UHFFFAOYSA-N 2,2'-piperazine-1,4-diylbisethanesulfonic acid Chemical compound OS(=O)(=O)CCN1CCN(CCS(O)(=O)=O)CC1 IHPYMWDTONKSCO-UHFFFAOYSA-N 0.000 claims description 21
- 239000007990 PIPES buffer Substances 0.000 claims description 21
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 21
- 239000004202 carbamide Substances 0.000 claims description 21
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 claims description 21
- 235000011187 glycerol Nutrition 0.000 claims description 21
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 21
- 239000000203 mixture Substances 0.000 claims description 21
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 12
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 229920004890 Triton X-100 Polymers 0.000 claims description 10
- 239000013504 Triton X-100 Substances 0.000 claims description 10
- 238000005406 washing Methods 0.000 claims description 10
- 229930040373 Paraformaldehyde Natural products 0.000 claims description 9
- 229920002866 paraformaldehyde Polymers 0.000 claims description 9
- 238000007654 immersion Methods 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 5
- 238000002203 pretreatment Methods 0.000 claims description 4
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims 9
- 239000007995 HEPES buffer Substances 0.000 claims 9
- 241000196324 Embryophyta Species 0.000 abstract description 44
- 238000012545 processing Methods 0.000 abstract description 12
- 230000014509 gene expression Effects 0.000 abstract description 6
- 230000007071 enzymatic hydrolysis Effects 0.000 abstract description 3
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 abstract description 3
- 239000007850 fluorescent dye Substances 0.000 abstract description 3
- 238000011835 investigation Methods 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 2
- 238000011156 evaluation Methods 0.000 abstract description 2
- 210000001519 tissue Anatomy 0.000 description 27
- 241000208125 Nicotiana Species 0.000 description 18
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 18
- 241000219194 Arabidopsis Species 0.000 description 14
- 210000000473 mesophyll cell Anatomy 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000000799 fluorescence microscopy Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 108010001384 remorin Proteins 0.000 description 4
- 244000291564 Allium cepa Species 0.000 description 3
- 235000002732 Allium cepa var. cepa Nutrition 0.000 description 3
- 210000003763 chloroplast Anatomy 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 102000034287 fluorescent proteins Human genes 0.000 description 3
- 108091006047 fluorescent proteins Proteins 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 229920006324 polyoxymethylene Polymers 0.000 description 3
- 240000002853 Nelumbo nucifera Species 0.000 description 2
- 235000006508 Nelumbo nucifera Nutrition 0.000 description 2
- 235000006510 Nelumbo pentapetala Nutrition 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 210000001339 epidermal cell Anatomy 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000002356 single layer Substances 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- WFCSWCVEJLETKA-UHFFFAOYSA-N 2-piperazin-1-ylethanol Chemical compound OCCN1CCNCC1 WFCSWCVEJLETKA-UHFFFAOYSA-N 0.000 description 1
- 241000589158 Agrobacterium Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 244000056139 Brassica cretica Species 0.000 description 1
- 235000003351 Brassica cretica Nutrition 0.000 description 1
- 235000003343 Brassica rupestris Nutrition 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- QKSKPIVNLNLAAV-UHFFFAOYSA-N bis(2-chloroethyl) sulfide Chemical compound ClCCSCCCl QKSKPIVNLNLAAV-UHFFFAOYSA-N 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 239000002361 compost Substances 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- CCIVGXIOQKPBKL-UHFFFAOYSA-N ethanesulfonic acid Chemical compound CCS(O)(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 235000010460 mustard Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 238000009738 saturating Methods 0.000 description 1
- 210000004895 subcellular structure Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005418 vegetable material Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
The invention discloses a kind of microscopy observation methods of plant tissue.The microscopy observation method of plant tissue provided by the invention will reuse potassium hydroxide and digest, then carries out transparent processing after fixing process, avoid enzymatic hydrolysis or other operations, save cost；Transparent effect is good, with fluorescent staining and marking operation good compatibility: can clearly observe the internal structure of plant deep tissues under fluorescence microscope by fluorescent staining.Digestion process combination transparent technology carries out the method for microexamination to plant deep tissues and lays a good foundation for the further investigation of higher plant phenotypic evaluation and gene expression pattern, it especially plays a significant role in the research that plant underlying tissue structure is observed, practical application value is high.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to a kind of microscopy observation method of plant tissue.
Background technique
As functional genomics, molecular biology research deepen continuously, specific gene is in the intracorporal expression portion of plant
Position its got the attention in the function of specific developmental stage.Fluorescence microscopy observation and 3 dimension imaging technology are 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 pigment of rich content and aromatic substance make the signal noise ratio (snr) of image obtained drop in cell
It is low.Therefore, several confluent monolayer cells that depth is about 30 μm or so are only limitted to the microexamination of plant cell in the past, greatly influenced
The parsings of plant cell structures and function.
For the internal structure or subcellular structure of observation of plant body deeper, generally require to make to plant by specially treated
Object sample reduced thickness and volume make light just can be carried out microexamination through sample.Material requirements that treated is small and thin, complete
Whole, transparent, holding original structure, and there is color to be easy identification.To reach above-mentioned requirements, need to take different flaking method or
Person is transparent to vegetable material progress before imaging, with the plant sample for facilitating observation tissue thicker, such as tobacco and tomato leaf
Piece, mongolicum Regel etc..
Summary of the invention
A purpose of the invention is to provide a kind of method of plant tissue microscope detection pre-treatment.
Method provided by the invention, includes 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 and plant
Object 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 composition；
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 proportion of the urea, the glycerine, the Triton X-10 are 4-8mol:20-40ml:0.05-
0.15g；
Or the proportion of the urea, the glycerine, the Triton X-10 are 6mol:20ml:0.1g；
Or the proportion of the urea, the glycerine, the Triton X-10 are 6mol:30ml:0.1g；
Or the proportion of the urea, the glycerine, the Triton X-10 are 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 MgCl2It is formed 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 MgCl2It is formed 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 MgCl2It is formed 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 MgCl2It is formed with water；
Or the transparent liquid is by final concentration of 4-8M urea, percent by volume 20-40% glycerine, mass percentage
0.05-0.15%Triton X-100 and PHEM solution composition；
Or the transparent liquid is by final concentration of 6M urea, 20% glycerine of percent by volume, percent by volume 0.1%
Triton X-100 and PHEM solution composition；
Or the transparent liquid is by final concentration of 6M urea, 30% glycerine of percent by volume, percent by volume 0.1%
Triton X-100 and PHEM solution composition；
Or the transparent liquid is by final concentration of 4M urea, 20% glycerine of percent by volume, percent by volume 0.1%
Triton X-100 and PHEM solution composition.
In the above method,
The pH value of the PHEM solution is 6.9；
In the above method,
The plant tissue by after fixation with the mode that potassium hydroxide digests be the plant tissue after fixing is immersed in it is dense
Degree is in the potassium hydroxide aqueous solution of mass percent 10-15%；
Or described by postdigestive plant tissue is by the postdigestive plant tissue with the mode that PHEM solution washs
It is immersed in the PHEM solution.
In the above method,
The fixed fixer used by glutaraldehyde, paraformaldehyde and the PHEM solution composition,
The proportion of the glutaraldehyde and the paraformaldehyde is 4-8ml:4-8g；
Or the fixer by volumn concentration is 4-8% glutaraldehyde, mass percent is 4-8% paraformaldehyde and institute
State PHEM solution composition.
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；
Or the mode of the washing is washing 3-5 times, it is 5-10 minutes each；
Or the time of the Immersion treatment is 5-10, the mode of the Immersion treatment is primary described every replacement in 1 day
Bright liquid.
In the above method,
The plant tissue is that length is 0.5-1cm2Plant leaf blade or 0.5-1cm long plant tissue.
Second purpose of the invention is to provide a kind of microscopy observation method of plant tissue.
Method provided by the invention, including above-mentioned steps A.
Third purpose of the present invention is to provide a kind of kit of plant tissue microscope detection.
Kit provided by the invention, include the steps that above-mentioned A the PHEM solution and/or the transparent liquid.
The microscopy observation method of plant tissue provided by the invention has the advantage that 1) easy to operate, quick: contracting
The time of short enzymatic hydrolysis and the pre-processings such as transparent；2) potassium hydroxide is reused after fixing process to be digested, avoid enzymatic hydrolysis
Or other operations, save cost；3) transparent effect is good, with fluorescent staining and marking operation good 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 method provided by the invention, observation depth is significantly improved；
The dosage of cellulase etc. is reduced, and achievees the purpose that save cost；For further investigation gene expression in plants mode and deep tissues
Structure provides important tool.It can be seen that 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
It plays a significant role in the research that structure is observed, practical application value is high.
Detailed description of the invention
Fig. 1 is the distribution situation of Remorin1.2 albumen in the Arabidopsis leaf observed under fluorescence microscope.
Fig. 2 is the distribution situation for 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 method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Method therefor is conventional method unless otherwise instructed in following embodiments, and the percentage composition is unless otherwise instructed
It is mass percentage.
Embodiment 1, the transparent processing of Arabidopsis leaf and microscopic
One, reagent needed for the transparent processing of blade
For fixer by glutaraldehyde, paraformaldehyde and PHEM solution composition, wherein the percent by volume of glutaraldehyde is 4%, more
The mass percent of polyformaldehyde is 4%.
PHEM solution is by final concentration of 40mM PIPES, 15mM HEPES, 5mM EGTA, 1mM MgCl2It is formed with water, pH
Value 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 composition.
Two, the transparent processing and microscopic of Arabidopsis leaf
1, the transparent processing of Arabidopsis leaf
Arabidopsis (Columbia ecotype) seed is planted on 1/2MS culture medium, 0-4 DEG C of vernalization two days later, sets 23
It is sprouted in ± 2 DEG C of incubators, moves back within five days and continue culture 20-30 days, the clip lotus throne leaf blade before taking out platform into compost.
1) above-mentioned lotus throne leaf blade is cut into 0.5cm2The fritter of square is washed with deionized to removal impurity；
2) 0.5cm for obtaining step 1)2The fritter of square, which is transferred in fixer, to be submerged, arabidopsis leaf after being fixed
Piece；Fixed temperature is 4 DEG C, and regular time is 12 hours；It is fixed to use shaking speed 50rpm, radius of turn 50mm.
3) Arabidopsis leaf after fixation is placed in the potassium hydroxide aqueous solution of mass percent 10% and is submerged, digested, disappear
Changing the 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, it is quasi- after being washed
Southern mustard blade；
5) Arabidopsis leaf after washing is submerged to progress transparent processing 3 days in transparent liquid, it is primary saturating every replacement in 1 day
Bright liquid；Obtain it is transparent after Arabidopsis leaf.
2, microscopic
By above-mentioned 1 obtain it is transparent after Arabidopsis leaf excitation wavelength be 488nm fluorescence microscopy under the microscope, knot
Fruit is as shown in Figure 1, it can be observed that epidermal cell and mesophyll cell from Fig. 1, it can be seen that Remorin egg in Arabidopsis leaf
It is white to be mainly distributed on cytoplasma membrane, it is seen that there are a small amount of vesicular structures for cell interior.
The distribution of embodiment 2, husky green onion mesophyll cell Chloroplast
One, reagent needed for the transparent processing of blade
For fixer by glutaraldehyde, paraformaldehyde and PHEM solution composition, wherein the percent by volume of glutaraldehyde is 4%, more
The mass percent of polyformaldehyde is 8%.
PHEM solution is by final concentration of 60mM PIPES, 25mM HEPES, 10mM EGTA, 2mM MgCl2It is formed 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 composition.
Two, the transparent processing and microscopic of mongolicum Regel
Husky green onion is cultivated to 20-30cm high, the aerial part of seedling is taken；
1) above-mentioned mongolicum Regel is cut into the segment of 0.8cm long, is washed with deionized to the soil removed on blade and its
His impurity；
2) 20 sections of mongolicum Regels that step 1) obtains are transferred in fixer and are submerged；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, digested, disappear
Changing the 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 to progress transparent processing 5 days in transparent liquid, it is primary transparent every replacement in 1 day
Liquid, obtain it is transparent after mongolicum Regel.
2, microscopic
By above-mentioned 1 obtain it is transparent after mongolicum Regel excitation wavelength be 488nm fluorescence microscopy under the microscope, 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 close,
Between a small amount of stomata is distributed with.
The distribution of REMORIN albumen in embodiment 3, tobacco mesophyll cell
One, reagent needed for the transparent processing of blade
For fixer by glutaraldehyde, paraformaldehyde and PHEM solution composition, wherein the percent by volume of glutaraldehyde is 5%, more
The mass percent of polyformaldehyde is 5%.
PHEM solution is by final concentration of 60mM PIPES, 20mM HEPES, 10mM EGTA, 2mM MgCl2It is formed 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 composition.
Two, the transparent processing and microscopic of tobacco leaf
By tobacco seed vernalization at 20-25 DEG C, after seed is sprouted, sets flower cultivating soil in 25 ± 2 DEG C of greenhouses and plant, culture is extremely
Plant height 5-10cm.
It 1) is 0.8 (Detection wavelength 660nm) by the Agrobacterium strain shaken cultivation for carrying REMORIN2 gene to OD value,
6000rpm room temperature is centrifuged 3 minutes collection thallus, and thallus is resuspended with tobacco leaf injection buffer；
2) bacterium solution that step 1) obtains is injected in tobacco leaf with 1mL syringe, above-mentioned tobacco seedling is set 25 ± 2
Continue culture 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 removal impurity；
4) 0.5cm for obtaining step 1)2The fritter of square, which is transferred in fixer, to be submerged, Tobacco Leaf after being fixed
Piece；
Fixed temperature is 4 DEG C, and regular time is 12 hours, and fixed to use shaking speed 50rpm, 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, digested；Disappear
Changing the 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, is washed
Tobacco leaf after washing；
7) tobacco leaf after washing is submerged to progress transparent processing 5 days in transparent liquid, it is primary transparent every replacement in 1 day
Liquid；Obtain it is transparent after tobacco leaf.
2, microscopic
By above-mentioned 1 obtain it is transparent after tobacco leaf excitation wavelength be 488nm fluorescence microscopy under the microscope, as a result
As shown in figure 3, being observed that the mesophyll cell of table subcutaneous deep from Fig. 3, Remorin albumen is main in tobacco mesophyll cell
It is distributed in cytoplasma membrane.
Comparative example: conventional material clearing method and fluorescent protein labeling are incompatible, it is impossible to be used in observation fluorescent protein expression,
It is thus only capable of the relatively thin material of observation thickness of sample, the biggish sample of thickness cannot be illuminated, and observation thickness is only 30 μm or so
Several confluent monolayer cells.Fig. 4 is result of the conventional method observation with a thickness of 100 μm of tobacco leaf deep layer mesophyll cells.
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
Observe the deep layer cells with a thickness of 100 μm or so.
Claims (12)
1. a kind of method of plant tissue microscope detection pre-treatment, includes 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 composition.
2. according to the method described in claim 1, it is characterized by:
The PIPES, the HEPES, the EGTA and the MgCl2Mole amount ratio be 40-80:15-35:5-15:1-3
；
The urea, the glycerine, the Triton X-10 proportion be 4-8mol:20-40ml:0.05-0.15g.
3. according to the method described in claim 1, it is characterized by:
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；
The urea, the glycerine, the Triton X-10 proportion be 6mol:20ml:0.1g；
Or the proportion of the urea, the glycerine, the Triton X-10 are 6mol:30ml:0.1g；
Or the proportion of the urea, the glycerine, the Triton X-10 are 4mol:20ml:0.1g.
4. method according to claim 1 to 3, it is characterised in that:
The PHEM solution is by final concentration of 40-80 mM PIPES, final concentration of 15-35 mM HEPES, final concentration of 5-
15mM EGTA, final concentration of 1-3 mM MgCl2It is formed with water；The transparent liquid is by final concentration of 4-8 M urea, volume basis
Than 20-40% glycerine, mass percentage 0.05-0.15% Triton X-100 and PHEM solution composition.
5. method according to claim 1 to 3, it is characterised in that: the PHEM solution is by final concentration of 60 mM
PIPES, final concentration of 25 mM HEPES, final concentration of 15mM EGTA, final concentration of 2mM MgCl2It is formed with water；
Or the PHEM solution is by final concentration of 60 mM PIPES, final concentration of 25 mM HEPES, final concentration of 10 mM
EGTA, final concentration of 2 mM MgCl2It is formed with water；
Or the PHEM solution is by final concentration of 40 mM PIPES, final concentration of 15 mM HEPES, final concentration of 5 mM
EGTA, final concentration of 1 mM MgCl2It is formed with water；
The transparent liquid is by final concentration of 6 M urea, 20% glycerine of percent by volume, 0.1% Triton X- of percent by volume
100 and PHEM solution composition；
Or the transparent liquid is by final concentration of 6M urea, 30% glycerine of percent by volume, 0.1% Triton X- of percent by volume
100 and PHEM solution composition；
Or the transparent liquid is by final concentration of 4 M urea, 20% glycerine of percent by volume, 0.1% Triton of percent by volume
X-100 and PHEM solution composition.
6. method according to claim 1 or 2, it is characterised in that:
The pH value of the PHEM solution is 6.9.
7. method according to claim 1 or 2, it is characterised in that:
The plant tissue by after fixation is that the plant tissue after fixing is immersed in concentration to be with the mode that potassium hydroxide digests
In the potassium hydroxide aqueous solution of mass percent 10-15%；
Described by postdigestive plant tissue is to be immersed in the postdigestive plant tissue with the mode that PHEM solution washs
In the PHEM solution.
8. method according to claim 1 or 2, it is characterised in that:
The fixed fixer used by glutaraldehyde, paraformaldehyde and the PHEM solution composition,
The proportion of the glutaraldehyde and the paraformaldehyde is 4-8 ml:4-8 g.
9. method according to claim 1 or 2, it is characterised in that:
The fixer by volumn concentration is 4-8% glutaraldehyde, mass percent is that 4-8% paraformaldehyde and the PHEM are molten
Liquid composition.
10. method according to claim 1 or 2, it is characterised in that:
The temperature of the fixation is 0-4 DEG C, and the regular time is 10-16 hours；
The time of the digestion is 6-8 hours；
The mode of the washing is washing 3-5 times, 5-10 minutes each；
The time of the Immersion treatment is 5-10, and the mode of the Immersion treatment is that the primary transparent liquid was replaced every 1 day.
11. method according to claim 1 or 2, it is characterised in that: the plant tissue is that length is 0.5-1cm's long
Plant tissue.
12. a kind of microscopy observation method of plant tissue includes the steps that any A in claim 1-11.
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