CN107246987A - A kind of method of Helminthosporium sativum chromosome sectioning - Google Patents
A kind of method of Helminthosporium sativum chromosome sectioning Download PDFInfo
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- CN107246987A CN107246987A CN201710418560.2A CN201710418560A CN107246987A CN 107246987 A CN107246987 A CN 107246987A CN 201710418560 A CN201710418560 A CN 201710418560A CN 107246987 A CN107246987 A CN 107246987A
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- 210000000349 chromosome Anatomy 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 42
- 241000190150 Bipolaris sorokiniana Species 0.000 title claims abstract description 12
- GQPLMRYTRLFLPF-UHFFFAOYSA-N Nitrous Oxide Chemical compound [O-][N+]#N GQPLMRYTRLFLPF-UHFFFAOYSA-N 0.000 claims abstract description 52
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 46
- 235000013842 nitrous oxide Nutrition 0.000 claims abstract description 26
- 229960000583 acetic acid Drugs 0.000 claims abstract description 18
- 239000012362 glacial acetic acid Substances 0.000 claims abstract description 8
- 238000012545 processing Methods 0.000 claims abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 30
- 210000004027 cell Anatomy 0.000 claims description 16
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- 239000000243 solution Substances 0.000 claims description 9
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- 230000012010 growth Effects 0.000 claims description 6
- 239000010813 municipal solid waste Substances 0.000 claims description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 2
- 239000012224 working solution Substances 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims 1
- 230000002786 root growth Effects 0.000 claims 1
- 239000000052 vinegar Substances 0.000 claims 1
- 235000021419 vinegar Nutrition 0.000 claims 1
- 230000031864 metaphase Effects 0.000 abstract description 8
- 238000000386 microscopy Methods 0.000 abstract description 7
- 230000011278 mitosis Effects 0.000 abstract description 6
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 abstract description 4
- XYFCBTPGUUZFHI-UHFFFAOYSA-N Phosphine Chemical compound P XYFCBTPGUUZFHI-UHFFFAOYSA-N 0.000 abstract description 4
- 229910000073 phosphorus hydride Inorganic materials 0.000 abstract description 2
- 230000035784 germination Effects 0.000 abstract 1
- 241000209140 Triticum Species 0.000 description 18
- 235000021307 Triticum Nutrition 0.000 description 18
- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 description 10
- 238000004043 dyeing Methods 0.000 description 10
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 9
- 241000196324 Embryophyta Species 0.000 description 7
- 244000098338 Triticum aestivum Species 0.000 description 7
- 235000007264 Triticum durum Nutrition 0.000 description 5
- 241000209143 Triticum turgidum subsp. durum Species 0.000 description 5
- 229960001338 colchicine Drugs 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 241000122204 Dasypyrum villosum Species 0.000 description 4
- 208000035199 Tetraploidy Diseases 0.000 description 4
- OCJBOOLMMGQPQU-UHFFFAOYSA-N 1,4-dichlorobenzene Chemical compound ClC1=CC=C(Cl)C=C1 OCJBOOLMMGQPQU-UHFFFAOYSA-N 0.000 description 3
- 239000005725 8-Hydroxyquinoline Substances 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 239000006059 cover glass Substances 0.000 description 3
- 230000002559 cytogenic effect Effects 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 229960003540 oxyquinoline Drugs 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- MCJGNVYPOGVAJF-UHFFFAOYSA-N quinolin-8-ol Chemical compound C1=CN=C2C(O)=CC=CC2=C1 MCJGNVYPOGVAJF-UHFFFAOYSA-N 0.000 description 3
- 238000013456 study Methods 0.000 description 3
- 210000002262 tip cell Anatomy 0.000 description 3
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- 231100000419 toxicity Toxicity 0.000 description 2
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- 244000025254 Cannabis sativa Species 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 241000723375 Colchicum Species 0.000 description 1
- 102000029749 Microtubule Human genes 0.000 description 1
- 108091022875 Microtubule Proteins 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- IAOZJIPTCAWIRG-QWRGUYRKSA-N aspartame Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)OC)CC1=CC=CC=C1 IAOZJIPTCAWIRG-QWRGUYRKSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 238000003200 chromosome mapping Methods 0.000 description 1
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- 230000007613 environmental effect Effects 0.000 description 1
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- 230000002068 genetic effect Effects 0.000 description 1
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- 238000009396 hybridization Methods 0.000 description 1
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- 238000004519 manufacturing process Methods 0.000 description 1
- 230000021121 meiosis Effects 0.000 description 1
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- 239000000203 mixture Substances 0.000 description 1
- VHEWQRWLIDWRMR-UHFFFAOYSA-N n-[methoxy-(4-methyl-2-nitrophenoxy)phosphinothioyl]propan-2-amine Chemical group CC(C)NP(=S)(OC)OC1=CC=C(C)C=C1[N+]([O-])=O VHEWQRWLIDWRMR-UHFFFAOYSA-N 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
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Classifications
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- 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
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
The present invention discloses a kind of method of Helminthosporium sativum chromosome sectioning, it is concretely comprised the following steps after germination, take its young root, after the careless phosphine pretreatment of methyl amine, handled with laughing gas, (4 DEG C) fixations of 90% glacial acetic acid of precooling are used again, the tip of a root growing point after processing are subjected to pressed disc method, finally the microscopy under phase contrast microscope.The chromosome flaking method of the present invention is easy to operate simple, can obtain substantial amounts of mitosis metaphase phase, Chromosome spread is good and background is shallower, it is easy to count, and split coil method is not easy to lose.
Description
Technical field
The invention belongs to plant cytogenetics field, it is related to a kind of method of Helminthosporium sativum chromosome sectioning.
Background technology
Domestic and international breeding practice proves that hereditary basis is narrow to have turned into " bottleneck " that wheat breeding is difficult to make a breakthrough.For
Wheat safety in production is ensured, Agro-ecology and Environmental security is ensured, in the urgent need to the new genetic resources of development and utilization, cultivates comprehensive
Close better resistance, adaptable new variety of wheat.Wheat wild kindred plant is lived in natural environment for a long time, is subjected to various
The test of adverse circumstances, the favorable genes for not possessing with a large amount of common wheats or having been lost under the conditions of artificial selection, such as
Disease-resistant, degeneration-resistant, pest-resistant, big many of fringe, high-quality etc..The excellent genes near isogenic wheat line are imported by distant hybridization mode
The new germ plasm of common wheat initiative, can both enrich wheat genetic variation, and widen the germ plasm resource of wheat, and be not related to and turn base
It is the effective way (Friebe etc., 1996) for promoting wheat breeding to make a breakthrough because of safety issue.In rising for wheat
Source Study on Evolution, the identification of alien chromatin, the chromosome mapping of gene, physical map structure, meiosis mechanism etc. are studied
In, it is directed to the correlative study of chromosome.
The preparation of chromosome of wheat film-making, is to carry out Wheat cytogenetics, wheat molecular cytogenetics and be based on
The basis of the correlative studys such as chromosome.In the set-up procedure of chromosome of wheat film-making, pretreatment is a very important ring
Section, rational pretreatment can obtain substantial amounts of split coil method and the good metaphase chromosome of form.The mechanism of pretreatment is to suppress micro-pipe
Albumen is assembled into spindle fiber, but does not suppress the cell division of early stage, therefore, the cell of all entrance mid-terms be blocked and can not
Into the later stage, a large amount of division phases are accumulated；Can induce chromosome, further shortening is straightened, and is easy to Chromosome spread and is made dyeing
Volume morphing is apparent.The method commonly used in practical operation has physical method and chemical method.Physical method is to plant with low temperature
Material is pre-processed, and treatment temperature is directly pre-processed typically at 0~8 DEG C, or with mixture of ice and water to material.This method pair
Chromosome is without destruction, and the uniform of chromosome shortening is simple and easy to do, and various crops are all suitable for, but to be whenever all not
More mid-term phase can be obtained.The conventional reagent of chemical method pretreatment has：Colchicine, paracide and 8-hydroxyquinoline.
The colchicine typical concentrations 0.05-0.2% aqueous solution, it is adaptable to large, medium and small chromosome, is especially suitable for chromosome counting,
Chromosome is set to be easily dispersed preferably, but display contriction is poor, and also colchicine toxicity is stronger.The effect of paracide pretreatment
It is suitable with colchicine, it may have stronger toxicity；8-hydroxyquinoline, it is suitable in, microchromosome, ex vivo treatment is good.Advantage exists
Clear in display contriction, shortcoming is mid-term mutually not as other many.
The content of the invention
The purpose of the present invention is that not enough there is provided a kind of side of Helminthosporium sativum chromosome sectioning for prior art above-mentioned
Method.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of method of Helminthosporium sativum chromosome sectioning, is comprised the steps of：
1) seed root of hair；
2) pre-process：When root a length of 1.8-3cm, 20 μm of ol/LAPM solution are added into culture dish to root is submerged, are placed in
25 DEG C of incubator growths are pre-processed 2 hours；
3) laughing gas is handled：By the tip of a root of pretreatment, cleaned in the culture dish equipped with distilled water, root is cut with scissors, in room
Under the conditions of temperature, the laughing gas for being 1MPa with pressure is handled 1-2 hours；
4) it is fixed：Root to after having been handled with laughing gas adds 90% glacial acetic acid of 4 DEG C of precoolings, fixed 5-10min；
5) film-making：The tip of a root is first taken out, a moment is dissociated in 45% acetic acid, root cap is first cut away, then cuts tip of a root growing point
It is placed on slide, dripping gently to tap after 45% acetic acid dissociated cell, covered disperses cell, outside alcolhol burner
Direct tablet compressing after flame slightly dries.
It is used as preferred, the step 1 of Helminthosporium sativum chromosome flaking method of the present invention) method of seed root of hair is：By seed
It is positioned over to be lined with the culture dish of moistening filter paper and is germinateed in 25 DEG C of insulating boxs, after seed shows money or valuables one carries unintentionally, by water unnecessary in culture dish
Pour in separately, only keep filter paper moistening, move in 4 DEG C of refrigerators and handle 20-24h, then again move to culture dish in 25 DEG C of incubators
Lucifuge grows to 1.8-3.0cm.
Be used as preferred, the step 2 of Helminthosporium sativum chromosome flaking method of the present invention) in 20 μm of ol/L APM solution preparation
Method is：100 μ l 0.2mol/L APM mother liquors are added into 1000ml distilled water, 20 μm of ol/L working solution is made into；It is described
The compound methods of 0.2mol/L APM mother liquors be：60.86mg APM are dissolved in the acetone of 10ml precoolings, are put into -20 DEG C of preservations.
Be used as preferred, the step 3 of Helminthosporium sativum chromosome flaking method of the present invention) described in laughing gas processing method be：
By the tip of a root of pretreatment, cleaned 2 times in the culture dish equipped with distilled water, it is each 4-5 minutes, root is cut with scissors, root is put into
In eppendorf centrifuge tubes with holes, appropriate deionized water is previously added in centrifuge tube to keep moistening, in room temperature condition
Under, the laughing gas for being 1MPa with pressure is handled 1-2 hours.
Be used as preferred, the step 4 of Helminthosporium sativum chromosome flaking method of the present invention) described in the method for fixation be：Use
The eppendorf centrifuge tubes for filling root after laughing gas has been handled are placed on trash ice, and the 90% of 4 DEG C of precoolings is added into centrifuge tube
Glacial acetic acid, fixed 5-10min.
The method of Helminthosporium sativum chromosome sectioning of the present invention is further preferably comprised the following steps：
1) seed root of hair：Seed is positioned over to be lined with the culture dish of moistening filter paper and germinateed in 25 DEG C of insulating boxs, seed
After showing money or valuables one carries unintentionally, moisture unnecessary in culture dish is outwelled, filter paper moistening is only kept, moves in 4 DEG C of refrigerators and handle 24h, then again will
Culture dish moves to lucifuge in 25 DEG C of incubators and grown；
2) pre-process：When root long 1.8-3cm, 20 μm of ol/L APM solution are added into culture dish to root is submerged, are placed in
25 DEG C of incubator growths are pre-processed 2 hours；
3) laughing gas is handled：By the tip of a root of pretreatment, clean 2 times, 5 minutes every time, use in the culture dish equipped with distilled water
Scissors cuts root, root is put into eppendorf centrifuge tubes with holes, appropriate deionized water is previously added in centrifuge tube to keep
Moistening, at ambient temperature, the laughing gas for being 1MPa with pressure are handled 1-2 hours；
4) it is fixed：The eppendorf centrifuge tubes for filling root after having been handled with laughing gas are placed on trash ice, to centrifuge tube
Middle 90% glacial acetic acid for adding 4 DEG C of precoolings, fixed 5-10min；
5) film-making：The tip of a root is first taken out, a moment is dissociated in 45% acetic acid, root cap is first cut away with blade, the tip of a root is then cut
Growing point is placed on slide, and dripping gently to tap after 45% acetic acid dissociated cell, covered disperses cell, in wine
Direct tablet compressing after smart lamp flame envelope slightly dries.
Observation under phase contrast microscope will be placed in by obtained slide according to the method described above, selective staining volume morphing is well, no
It is overlapping or it is overlapping less, clearly cell, can directly take a picture carry out karyotyping, can be used for Chromosomes Banding or fluorescence is former
Position hybrid experiment.
The present invention replaces colchicum in pretreatment stage using methyl amine grass phosphine (Amiprophos-methyl, APM) APM
Element, paracide and 8-hydroxyquinoline, APM are a kind of specific drugs of direct interference Microtubules in plants synthesis, and its effect is similar
In colchicine, play a part of collecting metaphase chromosome, different APM processing times have certain to mitosis metaphase index
Influence.APM has very high affinity to tubulin, in low concentration, and the depolymerization ability to tubulin is stronger, to plant
Toxic action it is also small.
Compared with prior art, beneficial effects of the present invention are embodied in simple to operate, it is not necessary to special installation；It is convenient easy
Learn, the personnel not trained by cytology, according to the inventive method, can obtain substantial amounts of mitosis metaphase phase,
Good chromosome of wheat film-making can be prepared.
Brief description of the drawings
Fig. 1：Amplify 100 times of (microscopes after hexaploid common wheat China spring chromosome sectioning DAPI dyeing：10 times of eyepiece
10 times of × object lens), A, B are the distribution situation for the mid-term phase seen under the different visuals field；
Fig. 2：Amplify 1000 times of (microscopes after hexaploid common wheat China spring chromosome sectioning DAPI dyeing：Eyepiece 10
Times × 100 times of object lens), it is microscopy picture (2n=42) of the cell under fluorescence microscope
Fig. 3：Amplify 100 times of (microscopes after tetraploid durum wheat chromosome sectioning DAPI dyeing：10 times × object lens of eyepiece
10 times), A, B are the distribution situation for the mid-term phase seen under the different visuals field；
Fig. 4：Amplify 1000 times of (microscopes after tetraploid durum wheat chromosome sectioning DAPI dyeing：10 times × thing of eyepiece
100 times of mirror), it is microscopy picture (2n=28) of the cell under fluorescence microscope；
Fig. 5：Amplify 100 times of (microscopes after Dasypyrum villosum chromosome sectioning DAPI dyeing：10 times × object lens of eyepiece 10
Times), A, B are the distribution situation for the mid-term phase seen under the different visuals field；
Fig. 6：Amplify 1000 times of (microscopes after Dasypyrum villosum chromosome sectioning DAPI dyeing：10 times × object lens of eyepiece
100 times), it is microscopy picture (2n=14) of the cell under fluorescence microscope
Embodiment
Embodiment 1：The chromosome sectioning citing of common wheat China spring (2n=42, AABBDD)：
Seed root of hair:15 common wheat China spring seeds are placed on and are lined with the culture dish of moistening filter paper, are added enough
Distilled water, seed is fully soaked in distilled water, be put into 25 DEG C of insulating boxs germinate.After 24 hours seeds of placement show money or valuables one carries unintentionally,
Moisture unnecessary in culture dish is outwelled, filter paper moistening is only kept, moves in 4 DEG C of refrigerators and handle 24h, then again move culture dish
Into 25 DEG C of incubators, lucifuge grows.
Pretreatment:When the root of wheat seed is about 2cm, 20 μm of ol/L APM solution are added into culture dish to submergence
Root, is placed in 25 DEG C of incubator growths and pre-processes 2 hours.
Laughing gas processing：By the tip of a root of pretreatment, 2 times are cleaned in the culture dish equipped with distilled water, 5 minutes every time, with cutting
Knife clip young root (if needing to obtain normal plant in experimentation, it is necessary to retain a complete root).Root is put into band
In the eppendorf2.0ml centrifuge tubes in hole, appropriate deionized water is previously added in centrifuge tube to keep moistening.In room temperature bar
Under part, the laughing gas for being 1MPa with pressure is handled 1.5 hours.
It is fixed：The eppendorf centrifuge tubes for filling root after having been handled with laughing gas are placed on trash ice, into centrifuge tube
Add 90% glacial acetic acid (4 DEG C) of precooling, fixed 5-10min.
Film-making：The tip of a root is first taken out, tip of a root part is cut with blade, a moment is dissociated in 45% acetic acid, is first cut away with blade
Root cap, then cuts tip of a root growing point 2mm (in practical operation, 1.5-2.5mm tips of a root part can be cut as needed) and is placed in load
On slide, 45% acetic acid dissociated cell is dripped, covered, the side pad of cover glass is behind the edge of blade, with dissection
Pin, which gently taps cover glass, disperses root-tip cells, disperses cell, is slightly dried in alcolhol burner flame envelope, slide is placed on
Between two layers of filter paper, with thumb by direct tablet compressing after tabletting of being exerted oneself in the position of cover glass.
Microscopy：Slide is placed in 20 times of thing Microscopic observations of fluorescence microscope, as can be seen from Figure 1 located in advance by APM
Material after reason, the quantity of mitosis metaphase phase is significantly improved.After DAPI dyeing, in 100 times of oil mirrors of fluorescence microscope
Lower photograph, coloration result such as Fig. 2, figure medium scale is shown in i.e., and as seen from the figure, the Wheat root tip cells chromosome number is
42, division phases are more, and Chromosome spread is good, overlapping few.
Embodiment 2：The chromosome sectioning citing of tetraploid durum wheat (2n=28, AABB)：
Tetraploid durum wheat is handled in seed root of hair, pretreatment, laughing gas, with hexaploid common wheat in terms of film-making, in detail
See that example 1 is shown in implementation.
Microscopy：Slide is placed in 20 times of thing Microscopic observations of fluorescence microscope, as can be seen from Figure 3 located in advance by APM
Material after reason, the quantity of mitosis metaphase phase is significantly improved.After DAPI dyeing, in 100 times of oil mirrors of fluorescence microscope
Lower photograph, coloration result such as Fig. 4, figure medium scale is shown in i.e., as seen from the figure, the durum wheat root tip cell chromosome number
For 28, division phases are more, and Chromosome spread is good, overlapping few.
Embodiment 3：The chromosome sectioning citing of Dasypyrum villosum (2n=14, VV)：
Seed root of hair:20 tuft Wheat Seeds are placed on and are lined with the culture dish of moistening filter paper, enough distilled water is added,
Seed is fully soaked in distilled water, be put into 25 DEG C of insulating boxs and germinate.After 24 hours seeds of placement show money or valuables one carries unintentionally, by culture dish
Unnecessary moisture is outwelled, and is only kept filter paper moistening, is moved in 4 DEG C of refrigerators and handle 24h, culture dish then is moved into 25 DEG C of cultures again
Lucifuge grows in case.
Pretreatment:When the root of tuft Wheat Seeds is about 2cm-3cm, 20 μm of ol/L APM solution are added extremely into culture dish
Root is submerged, 25 DEG C of incubator growths is placed in and pre-processes 2 hours.
Laughing gas processing：By the tip of a root of pretreatment, 2 times are cleaned in the culture dish equipped with distilled water, 5 minutes every time, with cutting
Knife clip young root (if needing to obtain normal plant in experimentation, it is necessary to retain a complete root).Root is put into band
In the eppendorf2.0ml centrifuge tubes in hole, appropriate deionized water is previously added in centrifuge tube to keep moistening.In room temperature bar
Under part, the laughing gas for being 1MPa with pressure is handled 1 hour.
It is fixed：The eppendorf centrifuge tubes for filling root after having been handled with laughing gas are placed on trash ice, into centrifuge tube
Add 90% glacial acetic acid (4 DEG C) of precooling, fixed 5-10min.
Film-making：With example 1.
Microscopy：Slide is placed in 20 times of thing Microscopic observations of fluorescence microscope, as can be seen from Figure 5 located in advance by APM
Material after reason, the quantity of mitosis metaphase phase is significantly improved.After DAPI dyeing, in 100 times of oil mirrors of fluorescence microscope
Lower photograph, coloration result such as Fig. 6, figure medium scale is shown in i.e., and as seen from the figure, the haynaldia villosa root tip cell chromosome number is
14, division phases are more, and Chromosome spread is good, overlapping few.
Claims (6)
1. a kind of method of Helminthosporium sativum chromosome sectioning, it is characterised in that comprise the steps of：
1) seed root of hair；
2) pre-process：When root a length of 1.8-3cm, 20 μm of ol/LAPM solution are added into culture dish to root is submerged, 25 DEG C are placed in
Incubator growth pretreatment 2 hours；
3) laughing gas is handled：By the tip of a root of pretreatment, cleaned in the culture dish equipped with distilled water, root is cut with scissors, in room temperature bar
Under part, the laughing gas for being 1MPa with pressure is handled 1-2 hours；
4) it is fixed：Root to after having been handled with laughing gas adds 90% glacial acetic acid of 4 DEG C of precoolings, fixed 5-10min；
5) film-making：The tip of a root is first taken out, a moment is dissociated in 45% acetic acid, root cap is first cut away, then cuts tip of a root growing point and be placed in
On slide, dripping gently to tap after 45% acetic acid dissociated cell, covered disperses cell, light in alcolhol burner flame envelope
It is micro- to dry rear direct tablet compressing.
2. the method for seed root of hair is according to the method described in claim 1, it is characterised in that step 1)：Seed is positioned over pad
Have in the culture dish of moistening filter paper and germinateed in 25 DEG C of insulating boxs, after seed shows money or valuables one carries unintentionally, moisture unnecessary in culture dish is outwelled, only
Filter paper moistening is kept, moves in 4 DEG C of refrigerators and handles 20-24h, culture dish then is moved into lucifuge in 25 DEG C of incubators again grows to
1.8-3cm。
3. the compound method of 20 μm of ol/L APM solution is according to the method described in claim 1, it is characterised in that step 2)：
100 μ l 0.2mol/L APM mother liquors are added into 1000ml distilled water, 20 μm of ol/L working solution is made into；Described
The compound method of 0.2mol/L APM mother liquors is：60.86mg APM are dissolved in the acetone of 10ml precoolings, are put into -20 DEG C of preservations.
4. the method for the laughing gas processing described according to the method described in claim 1, it is characterised in that step 3) is：Will pretreatment
The tip of a root, in the culture dish equipped with distilled water clean 2 times, it is each 4-5 minutes, cut root with scissors, root be put into holes
Appropriate deionized water is previously added in eppendorf centrifuge tubes, in centrifuge tube to keep moistening, at ambient temperature, with pressure
The strong laughing gas for 1MPa is handled 1-2 hours.
5. the method for the fixation described according to the method described in claim 1, it is characterised in that step 4) is：Being handled with laughing gas
The eppendorf centrifuge tubes for filling root after complete are placed on trash ice, and 90% ice vinegar of 4 DEG C of precoolings is added into centrifuge tube
Acid, fixed 5-10min.
6. the method according to any one of claim 1-5, it is characterised in that comprise the following steps：
1) seed root of hair：Seed is positioned over to be lined with the culture dish of moistening filter paper and germinateed in 25 DEG C of insulating boxs, seed shows money or valuables one carries unintentionally
Afterwards, moisture unnecessary in culture dish is outwelled, only keeps filter paper moistening, move in 4 DEG C of refrigerators and handle 24h, then again will culture
Ware moves to lucifuge in 25 DEG C of incubators and grown；
2) pre-process：When root long 1.8-3cm, 20 μm of ol/L APM solution are added into culture dish to root is submerged, 25 DEG C are placed in
Incubator growth pretreatment 2 hours；
3) laughing gas is handled：By the tip of a root of pretreatment, cleaned 2 times in the culture dish equipped with distilled water, 5 minutes every time, use scissors
Root is cut, root is put into eppendorf centrifuge tubes with holes, appropriate deionized water is previously added in centrifuge tube to keep wet
Profit, at ambient temperature, the laughing gas for being 1MPa with pressure are handled 1-2 hours；
4) it is fixed：The eppendorf centrifuge tubes for filling root after having been handled with laughing gas are placed on trash ice, added into centrifuge tube
Enter 90% glacial acetic acid of 4 DEG C of precoolings, fixed 5-10min；
5) film-making：The tip of a root is first taken out, a moment is dissociated in 45% acetic acid, root cap is first cut away with blade, tip of a root growth is then cut
Point is placed on slide, and dripping gently to tap after 45% acetic acid dissociated cell, covered disperses cell, in alcolhol burner
Direct tablet compressing after flame envelope slightly dries.
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