CN105699141B - A kind of tabletting method of eucalyptus chromosome - Google Patents
A kind of tabletting method of eucalyptus chromosome Download PDFInfo
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- CN105699141B CN105699141B CN201610087721.XA CN201610087721A CN105699141B CN 105699141 B CN105699141 B CN 105699141B CN 201610087721 A CN201610087721 A CN 201610087721A CN 105699141 B CN105699141 B CN 105699141B
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- 210000000349 chromosome Anatomy 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 23
- 244000166124 Eucalyptus globulus Species 0.000 title claims abstract 7
- 210000004027 cell Anatomy 0.000 claims abstract description 23
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims abstract description 13
- 239000005864 Sulphur Substances 0.000 claims abstract description 13
- 150000001412 amines Chemical class 0.000 claims abstract description 13
- 238000010494 dissociation reaction Methods 0.000 claims abstract description 13
- 230000005593 dissociations Effects 0.000 claims abstract description 13
- 238000004043 dyeing Methods 0.000 claims abstract description 12
- 230000031864 metaphase Effects 0.000 claims abstract description 12
- 239000000463 material Substances 0.000 claims abstract description 9
- 230000008569 process Effects 0.000 claims abstract description 5
- 238000000386 microscopy Methods 0.000 claims abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 239000012153 distilled water Substances 0.000 claims description 10
- 241000006109 Eucalyptus delegatensis Species 0.000 claims description 8
- 210000001519 tissue Anatomy 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 208000035199 Tetraploidy Diseases 0.000 claims description 4
- 238000007654 immersion Methods 0.000 claims description 4
- HZLHRDBTVSZCBS-UVJJDBRNSA-N 4-[(e)-(4-aminophenyl)-(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-2-methylaniline;hydrochloride Chemical compound Cl.C1=CC(=N)C(C)=C\C1=C(C=1C=C(C)C(N)=CC=1)/C1=CC=C(N)C=C1 HZLHRDBTVSZCBS-UVJJDBRNSA-N 0.000 claims description 3
- 108010084185 Cellulases Proteins 0.000 claims description 3
- 102000005575 Cellulases Human genes 0.000 claims description 3
- 239000006059 cover glass Substances 0.000 claims description 2
- 230000011278 mitosis Effects 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- 238000010186 staining Methods 0.000 claims description 2
- 239000000975 dye Substances 0.000 claims 1
- 230000002035 prolonged effect Effects 0.000 claims 1
- 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 abstract description 24
- 229960001338 colchicine Drugs 0.000 abstract description 12
- 239000003795 chemical substances by application Substances 0.000 abstract description 7
- 238000009825 accumulation Methods 0.000 abstract description 3
- 239000003226 mitogen Substances 0.000 abstract description 3
- 231100000419 toxicity Toxicity 0.000 abstract description 3
- 230000001988 toxicity Effects 0.000 abstract description 3
- 241000196324 Embryophyta Species 0.000 abstract description 2
- 230000000903 blocking effect Effects 0.000 abstract description 2
- 230000001413 cellular effect Effects 0.000 abstract description 2
- 230000002939 deleterious effect Effects 0.000 abstract description 2
- 230000000116 mitigating effect Effects 0.000 abstract 1
- 241000219927 Eucalyptus Species 0.000 description 12
- 241000894007 species Species 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 3
- 230000007812 deficiency Effects 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 108010059892 Cellulase Proteins 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 229940106157 cellulase Drugs 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 238000003825 pressing Methods 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 241001083082 Angophora Species 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 241000006100 Corymbia <angiosperm> Species 0.000 description 1
- 241001233195 Eucalyptus grandis Species 0.000 description 1
- 241000404037 Eucalyptus urophylla Species 0.000 description 1
- 241000219926 Myrtaceae Species 0.000 description 1
- 241000234479 Narcissus Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 230000005770 chromosome separation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 239000000686 essence Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000005464 sample preparation method Methods 0.000 description 1
- 238000007493 shaping process Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to plant identification technical field, discloses a kind of tabletting method of eucalyptus chromosome.This method comprises the following steps：Materials, pretreatment, fixed, dissociation, dyeing and tabletting and microscopy.This method is used as pretreating agent using the spirit of amine sulphur, when blocking cell mitogen, to the obvious mitigation of deleterious cellular effects, cell can carry out positive eumitosis, so as to which when fixing, the metaphase phase cell number of accumulation is more, therefore, the successful ratio increase of chromosome tabletting is made, stability improves.And amine sulphur spirit is cheap, toxicity is far smaller than colchicine, in tableting processes, can reduce the pollution to environment, reduces and produces the degree and risk poisoned to people.
Description
Technical field
The invention belongs to plant identification technical field, more particularly to a kind of tabletting method of eucalyptus chromosome.
Background technology
Chromosome is carrier and material base of the inhereditary material in the visible aspect of naked eyes, and number, the form of chromosome are things
Kind significant heredity feature, differentiate species available for identifying or assisting in property, the change DRS of affiliation and species between species
Degree.Chromosome Pressing Technology is grasped, it is a basic and basic cell life to carry out the quantity of chromosome and morphologic observation
Thing investigative technique and means, this technology is in species identification and discriminating, thing Interspecific relationship determines, species Ploidy Identification and
It is widely used in the research such as crossbreeding.
Eucalyptus is Myrtaceae (Mytraceae) cup fruit tree category (Angophora), umbrella room category (Corymbia) and eucalyptus category
(Eucalyptus) general designation of seeds.Eucalyptus is a kind of exotic tree species, is the important artificial forest reproducting tree species of south China, now
It is studied and is concentrated mainly on introducing and planting and timber processing, genetic improvement, genetic map construction and molecular mark,
Efficient Cultivation and ecological effect correlative study, the research to its cell aspect are seldom.External once someone did eucalyptus dye not of the same race
The observational study of colour solid, but existence and stability is poor, low success rate of deficiency；Accordingly, it sometimes is necessary to a large amount of sample preparations, could be obtained clear
Clear visible chromosome karyotype analysis image；Because colchicine is expensive, cause tabletting cost higher.Eucalyptus chromosome tabletting
Technology only has fragmentary research at home, in terms of research report, still without the chromosome tabletting for grasping ripe, stabilization and high quality
Technology.Can be to carry out eucalyptus inter-species mirror if the Chromosome Pressing Technology that eucalyptus is ripe, stable can be developed at home
Not, Ploidy Identification and Interspecific relationship determine the support that provides the necessary technical.The pressed-disc technique another valuable grind
Originating party searches out the substitute lower than colchicine toxicity and price to being on the premise of tablet quality is not reduced.
The content of the invention
In order to overcome the shortcomings and deficiencies of the prior art described above, primary and foremost purpose of the invention is to provide a kind of eucalyptus dyeing
The tabletting method of body.
The purpose of the present invention is realized by following proposal：
A kind of tabletting method of eucalyptus chromosome, including step in detail below：
(1) draw materials：Take tail alpine ash clone (Eucalyptus urophylla × Eucalyptus grandis) DH32-
29 diploid or tetraploid tissue culture are taken root the seedling rooting tip of a root of 5~10 days, away from most advanced place 0.5cm with inside points；
(2) pre-process：The tip of a root taken is cleaned up with distilled water, is put into 0.002~0.02g/L amine sulphur spirit and soaks
Bubble processing 3h~7h；
(3) it is fixed：The pretreated tip of a root is cleaned up with distilled water, is put into Ka Nuoshi fixers in 4 DEG C of conditions
Lower fixed 24h；
(4) dissociate：The tip of a root is taken out from fixer, is rinsed with distilled water, be put into 37 DEG C of mixed enzyme solution dissociation 30~
40min；
(5) dyeing and tabletting：After the tip of a root after dissociation is rinsed with distilled water, it is placed on slide, with blade root cap
And region of elongation is partially cut-away, the drop carbolfuchsin dyeing liquor of drop 1,10min is dyed, then, covered is then light with erasing rubber
Rap and make cover glass, unnecessary dyeing liquor is blotted with blotting paper；
(6) microscopy：Tabletting is placed in micro- Microscopic observation, selective staining body is scattered, fully shrinks, in bar-shaped, it is clear and
Cell in mitosis metaphase split coil method is counted and taken pictures.
Preferably, the concentration of the amine sulphur spirit described in step (2) is 0.01g/L, and the time of described immersion treatment is 4h.
The time of materials described in step (1) is 8~18 points.
Preferably, the time of the materials described in step (1) is the morning 8~10 point.
It is furthermore preferred that the time of the materials described in step (1) is at 9 points in the morning.
Mixed enzyme solution described in step (4) refers to that volume ratio is 1：1 10g/L cellulases and 10g/L pectases
Mixed liquor.
Preferably, the dissociation described in step (4) refers to dissociation 35min.
The present invention mechanism be：
In the present invention, the purpose of pretreatment is to accumulate more metaphase phase cells when fixing, so as to improve pressure
The success rate of piece, tabletting effect is lifted, obtain metaphase phase cell as a high proportion of as possible.During colchicine processing, hindering
Disconnected cell mitogen process, when obtaining metaphase phase cell, concentration is higher because needed for, colchicine is generated cell
Larger toxic action, the metaphase phase cell number of accumulation is caused to reduce, the successful ratio of chromosome tabletting reduces, stability
Reduce.And 10% of the required concentration, about colchicine during the spirit pretreatment of amine sulphur, it is right when blocking cell mitogen
Deleterious cellular effects substantially mitigate, and cell can carry out positive eumitosis, so as to which when fixing, the metaphase of accumulation is mutually thin
Born of the same parents' number is more, and the successful ratio increase of chromosome tabletting, stability improves.Fixed purpose is rapid kill living cells, stable
The effect of pretreatment；The purpose of acidolysis is softening cell membrane and degradation of cell matter, in order to the scattered and flattening of chromosome；Dyeing
Purpose be it is specific make chromosome (or chromatin) colour, be easy to observe and present chromosome image under the microscope.
The present invention is had the following advantages and beneficial effect relative to prior art：
(1) stability and success rate of tabletting can be improved using the spirit of amine sulphur, obtains higher proportion of chromosome separation mid-term
Image；
(2) colchicine price is 600~1000 yuan/g, and the amine sulphur spirit of this research institute is extremely cheap, is the autumn
2% or so of narcissus element price, can greatly reduce experimental cost；
(3) colchicine toxicity is much larger than the spirit of amine sulphur, therefore, after substituting colchicine using the spirit of amine sulphur, in tableting processes
In, the pollution to environment can be reduced, substantially reduces and the degree and risk poisoned is produced to people.
Brief description of the drawings
Fig. 1 is the chromosome observation figure in embodiment 1.
Fig. 2 is the chromosome observation figure in embodiment 2.
Fig. 3 is the chromosome observation figure in embodiment 3.
Fig. 4 is the chromosome design sketch of different ploidy cells in embodiment 3~4.
The metaphase phase cell that Fig. 5 is obtained when being and being handled in embodiment 3 and comparative example 1 using different pretreatments agent
Ratio comparison diagram.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are unlimited
In this.
(seedling is national woods to tail alpine ash clone DH32-29 used diploid tissue culture rooted seedling in embodiment
Wooden breeding, breeding accession designation number are：State S-SC-EU-001-2011), purchased from the state-owned east gate forest farm in Guangxi Zhuang Autonomous Region, institute
The tail alpine ash clone DH32-29 used tetraploid tissue culture rooted seedling by tail alpine ash clone DH32-29 diploid tissue culture
Rooted seedling induces to obtain.Cellulase and pectase are purchased from Sangon Biotech (Shanghai) Co., Ltd..Used is other
Reagent can routinely be bought from market.
Embodiment 1
The tip of a root of tail alpine ash clone DH32-29 diploid tissue culture rooted seedling is taken, is put into 0.01g/L amine sulphur spirit and soaks
Bubble processing 4h, then by the distillation water washing of the tip of a root after immersion, it is put into Ka Nuoshi fixers and fixes 24h under the conditions of 4 DEG C,
The tip of a root is taken out from fixer, is rinsed with distilled water, the volume ratio for being put into 37 DEG C is 1：1 10g/L cellulases and 10g/L fruits
30min is dissociated in the mixed liquor of glue enzyme, the tip of a root is taken out, is cleaned, be placed on clean slide with distilled water, with blade root cap
And region of elongation is partially cut-away, the drop carbolfuchsin dyeing liquor of drop 1,10min is dyed, covered, lid is gently beaten with erasing rubber
Slide, unnecessary dyeing liquor is blotted with blotting paper, tabletting is then placed in micro- Microscopic observation.Chromosome observation figure such as Fig. 1
It is shown.
Embodiment 2
Enzymolysis time in embodiment 1 is increased into 40min by 30min, remaining operation is same as Example 1.Dyeing
Body observation figure is as shown in Figure 2.
Embodiment 3
It is 35min that enzymolysis time in embodiment 1 is increased by 30min, and remaining operation is same as Example 1.Gained
Chromosome observation figure it is as shown in Figure 3.
From Fig. 1~3 as can be seen that when digesting 30min (such as Fig. 1), enzymolysis deficiency, chromosome is still strapped in cell
In core, do not disperse；When digesting 40min (such as Fig. 2), dissociation is excessive, and not only cell membrane is decomposed, and chromosome is also decomposed, compared with
Hardly possible observes the chromosome of shaping.Only when Dissociation time is suitable, preferable dissociation effect could be obtained.Therefore, from operation
Convenient and cost consideration, it is preferred (such as Fig. 3) in 37 DEG C of constant temperature dissociation 35min with cellulase and pectase mixed solution.
Embodiment 4
Tail alpine ash clone DH32-29 in the embodiment 3 seedling diploids that take root are replaced with into tail alpine ash clone DH32-
29 take root seedling tetraploid, and remaining condition is same as Example 3.
Fig. 4 is the chromosome design sketch of different ploidy cells in embodiment 3~4, even 4 times of bodies, there is 44 chromosomes
When, remain to realize that chromosome is sufficiently scattered in contraction and distribution, can be counted using chromosome karyotype analysis software
And karyotyping, this also demonstrates the reliability and maturity for the method that this research is found out.
Comparative example 1
Pretreating agent in embodiment 3 is changed to 0.1g/L colchicine by 0.01g/L amine sulphur spirit into, remaining condition is equal
It is same as Example 3.
The metaphase phase cell proportion that Fig. 5 is obtained when being and using different pretreatments agent in embodiment 3 and comparative example 1
Comparison diagram, when using colchicine for pretreating agent, 10%~20% metaphase phase cell for reaching requirement can be obtained,
And in the present invention, using amine sulphur spirit be used as pretreating agent, can obtain 15%~30% the metaphase for reaching requirement it is mutually thin
Born of the same parents, compared with colchicine is as pretreating agent, success rate significantly improves.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (5)
1. a kind of tabletting method of eucalyptus chromosome, it is characterised in that including step in detail below：
(1) draw materials：Take tail alpine ash clone DH32-29 diploid or tetraploid tissue culture taken root the seedling rooting tip of a root of 5~10 days,
Away from most advanced place 0.5cm with inside points；
(2) pre-process：The tip of a root taken is cleaned up with distilled water, be put into immersion treatment 3h in 0.01g/L amine sulphur spirit~
7h；
(3) it is fixed：The pretreated tip of a root is cleaned up with distilled water, is put into Ka Nuoshi fixers under the conditions of 4 DEG C solid
Determine 24h；
(4) dissociate：The tip of a root is taken out from fixer, is rinsed with distilled water, be put into 37 DEG C of mixed enzyme solution dissociation 30~
40min；
(5) tabletting and dyeing：After the tip of a root after dissociation is rinsed with distilled water, it is placed on slide, root cap and is prolonged with blade
Long area is partially cut-away, the drop carbolfuchsin dyeing liquor of drop 1, dyes 10min, then, covered, and gently beaten with erasing rubber
Cover glass, unnecessary dyeing liquor is blotted with blotting paper；
(6) microscopy：Tabletting is placed in micro- Microscopic observation, selective staining body is scattered, fully shrinks, clear and be in bar-shaped
The cell of mitosis metaphase split coil method is counted and taken pictures.
2. the tabletting method of eucalyptus chromosome according to claim 1, it is characterised in that：
The time of described immersion treatment is 4h.
3. the tabletting method of eucalyptus chromosome according to claim 1, it is characterised in that：
The time of materials described in step (1) is at 9 points in the morning.
4. the tabletting method of eucalyptus chromosome according to claim 1, it is characterised in that：
Mixed enzyme solution described in step (4) is that volume ratio is 1：1 10g/L cellulases and the mixed liquor of 10g/L pectases.
5. the tabletting method of eucalyptus chromosome according to claim 1, it is characterised in that：
Dissociation described in step (4) refers to dissociation 35min.
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| CN201610087721.XA CN105699141B (en) | 2016-02-16 | 2016-02-16 | A kind of tabletting method of eucalyptus chromosome |
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| CN201610087721.XA CN105699141B (en) | 2016-02-16 | 2016-02-16 | A kind of tabletting method of eucalyptus chromosome |
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| CN109387509A (en) * | 2017-08-03 | 2019-02-26 | 北京林业大学 | A kind of tulip chromosome and STUDY ON THE KARYOTYPE technology |
| CN109708935A (en) * | 2018-12-15 | 2019-05-03 | 济南艾迪康医学检验中心有限公司 | A kind of production method of high-resolution peripheral blood chromosome G band |
| CN112255069A (en) * | 2020-10-22 | 2021-01-22 | 南京农业大学 | Dendrobium huoshanense root tip specimen tablet and preparation method and application thereof |
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| CN103270948B (en) * | 2013-04-17 | 2015-04-08 | 桂林百科农业科技有限公司 | Two polyploid centella asiatica varieties and cultivation method thereof |
| CN105021433A (en) * | 2015-06-05 | 2015-11-04 | 中国农业科学院草原研究所 | Squash slide preparation method for root tip chromosome of Elymus plant |
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