CN105699141A - Tabletting method of eucalyptus chromosome - Google Patents
Tabletting method of eucalyptus chromosome Download PDFInfo
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- CN105699141A CN105699141A CN201610087721.XA CN201610087721A CN105699141A CN 105699141 A CN105699141 A CN 105699141A CN 201610087721 A CN201610087721 A CN 201610087721A CN 105699141 A CN105699141 A CN 105699141A
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- 238000000034 method Methods 0.000 title claims abstract description 21
- 244000166124 Eucalyptus globulus Species 0.000 title claims abstract 7
- 210000000349 chromosome Anatomy 0.000 title abstract description 27
- 238000004043 dyeing Methods 0.000 claims abstract description 13
- 230000031864 metaphase Effects 0.000 claims abstract description 12
- 239000000463 material Substances 0.000 claims abstract description 9
- 230000011278 mitosis Effects 0.000 claims abstract description 4
- 230000002759 chromosomal effect Effects 0.000 claims description 13
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 12
- 239000005864 Sulphur Substances 0.000 claims description 12
- 150000001412 amines Chemical class 0.000 claims description 12
- 239000012153 distilled water Substances 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 241000006109 Eucalyptus delegatensis Species 0.000 claims description 8
- 239000000834 fixative Substances 0.000 claims description 6
- 238000007654 immersion Methods 0.000 claims description 6
- 108010059892 Cellulase Proteins 0.000 claims description 5
- 229940106157 cellulase Drugs 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 208000035199 Tetraploidy Diseases 0.000 claims description 4
- 239000006185 dispersion Substances 0.000 claims description 4
- 229940088598 enzyme Drugs 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
- 239000002250 absorbent Substances 0.000 claims description 3
- 230000002745 absorbent Effects 0.000 claims description 3
- 238000000386 microscopy Methods 0.000 claims description 2
- 238000010186 staining Methods 0.000 claims description 2
- 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 26
- 210000004027 cell Anatomy 0.000 abstract description 22
- 229960001338 colchicine Drugs 0.000 abstract description 13
- 239000003795 chemical substances by application Substances 0.000 abstract description 7
- 230000008569 process Effects 0.000 abstract description 4
- 230000001988 toxicity Effects 0.000 abstract description 3
- 231100000419 toxicity Toxicity 0.000 abstract description 3
- 241000196324 Embryophyta Species 0.000 abstract description 2
- 239000005587 Oryzalin Substances 0.000 abstract 2
- UNAHYJYOSSSJHH-UHFFFAOYSA-N oryzalin Chemical compound CCCN(CCC)C1=C([N+]([O-])=O)C=C(S(N)(=O)=O)C=C1[N+]([O-])=O UNAHYJYOSSSJHH-UHFFFAOYSA-N 0.000 abstract 2
- 231100000433 cytotoxic Toxicity 0.000 abstract 1
- 230000001472 cytotoxic effect Effects 0.000 abstract 1
- 241000219927 Eucalyptus Species 0.000 description 12
- 230000032823 cell division Effects 0.000 description 9
- 241000894007 species Species 0.000 description 9
- 238000005516 engineering process Methods 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 3
- 238000012549 training Methods 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 239000003226 mitogen Substances 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 238000012545 processing 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
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change 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
- 238000009402 cross-breeding Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 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
- 210000004940 nucleus Anatomy 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000005464 sample preparation method Methods 0.000 description 1
- 238000007493 shaping process Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 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
- 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
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention belongs to the technical field of plant identification and discloses a tabletting method of eucalyptus chromosome.The tabletting method includes following steps: taking materials, pretreating, fixing, dissociating, dyeing, tabletting and performing microscopic examination.In the tabletting method, oryzalin is adopted as a pretreatment agent, so that cytotoxic effect is reduced obviously when cell mitosis is blocked; cells can be in normal mitosis, so that during fixing, number of accumulated metaphase cells is large, chromosome tabletting success probability is increased, and stability is improved.Oryzalin is low in cost and far lower than colchicine in toxicity; in the tabletting process, environment pollution can be reduced and degree and risk of causing damage to people are lowered.
Description
Technical field
The invention belongs to plant identification technical field, particularly to a kind of chromosomal tabletting method of Eucalyptus。
Background technology
Chromosome is hereditary material at the carrier of the visible aspect of naked eyes and material base, and chromosomal number, form are the significant heritability features of species, can be used for identifying or assisting in property and differentiates species, sibship between species and the degree of variation of species。Grasp Chromosome Pressing Technology, carry out chromosomal quantity and morphologic observation is a basic and basic RESEARCH ON CELL-BIOLOGY technology and means, the research such as this technology is determined at species identification and discriminating, thing Interspecific relationship, species Ploidy Identification and cross-breeding is widely used。
Eucalyptus is the general designation that Myrtaceae (Mytraceae) cup fruit tree belongs to (Angophora), umbrella room genus (Corymbia) and Eucalyptus (Eucalyptus) seeds。Eucalyptus is a kind of exotic tree species, it is the important artificial forest reproducting tree species of south China, now its research is concentrated mainly on introducing and planting and timber processing, genetic improvement, genetic map construction and molecular mark, Efficient Cultivation and ecological effect correlational study, the research of its cell aspect is little。External once someone did the chromosomal observational study of Eucalyptus not of the same race, but existence and stability is poor, low success rate of deficiency；Accordingly, it sometimes is necessary to a large amount of sample preparations, apparent chromosome karyotype analysis image could be obtained；Because Colchicine is expensive, cause that tabletting is relatively costly。Eucalyptus Chromosome Pressing Technology only has fragmentary research at home, from research report, still without grasping Chromosome Pressing Technology ripe, stable and high-quality。If the Chromosome Pressing Technology that Eucalyptus is ripe, stable can be developed at home, then can for carry out differentiate between Eucalyptus kind, Ploidy Identification and Interspecific relationship determine the support that provides the necessary technical。This valuable R&D direction of pressed-disc technique another one is under the premise not reducing tablet quality, searches out succedaneum more less than Colchicine toxicity and price。
Summary of the invention
In order to overcome shortcoming and the deficiency of above-mentioned prior art, the primary and foremost purpose of the present invention is in that to provide a kind of chromosomal tabletting method of Eucalyptus。
It is an object of the invention to be realized by following proposal:
A kind of chromosomal tabletting method of Eucalyptus, including step in detail below:
(1) draw materials: the diploid or the tetraploid group that take tail alpine ash clone (Eucalyptusurophylla × Eucalyptusgrandis) DH32-29 train the seedling rooting tip of a root of 5～10 days of taking root, from part within most advanced place 0.5cm；
(2) pretreatment: cleaned up by the tip of a root distilled water taken, puts into immersion treatment 3h～7h in the amine sulphur spirit of 0.002～0.02g/L；
(3) fixing: pretreated tip of a root distilled water to be cleaned up, puts in Ka Nuoshi fixative and fix 24h under 4 DEG C of conditions；
(4) dissociate: from fixative, take out the tip of a root, rinse with distilled water, put into the 30～40min that dissociates in the mixed enzyme solution of 37 DEG C；
(5) dyeing and tabletting: after the tip of a root distilled water rinsing after dissociating, it is placed on microscope slide, with blade partially cut-away to root cap and region of elongation, drip 1 carbolfuchsin dyeing liquor, dyeing 10min, then, covered, then beat coverslip with erasing rubber gently, with absorbent paper, unnecessary dyeing liquor is blotted；
(6) microscopy: tabletting is placed in basis of microscopic observation, the dispersion of selective staining body, fully shrinks, in bar-shaped, clear and be in the cell of split coil method mitosis metaphase and carry out counting and taking 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 drawn materials described in step (1) is 8～18 points。
Preferably, the time drawn materials described in step (1) is point in the morning 8～10。
It is furthermore preferred that the time drawn materials described in step (1) is point in the morning 9。
Mixed enzyme solution described in step (4) refers to the mixed liquor of 10g/L cellulase that volume ratio is 1:1 and 10g/L pectase。
Preferably, the finger that dissociates described in step (4) dissociates 35min。
The mechanism of the present invention is:
In the present invention, the purpose of pretreatment is to accumulate phase cell more metaphase of cell division when fixing, thus improving the success rate of tabletting, promotes tabletting effect, it is thus achieved that a high proportion of as far as possible metaphase of cell division phase cell。When Colchicine processes, blocking cell mitogen process, it is thus achieved that during phase cell metaphase of cell division, because desired concn is higher, make Colchicine that cell to create bigger toxic action, cause that phase cell number metaphase of cell division of accumulation reduces, the successful ratio of chromosome tabletting reduces, and stability reduces。And desired concn during amine sulphur spirit pretreatment, it is about the 10% of Colchicine, it is when blocking cell mitogen, deleterious cellular effects is substantially alleviated, and cell can carry out positive eumitosis, thus, when fixing, phase cell number metaphase of cell division of accumulation is many, and the successful ratio of chromosome tabletting increases, and stability improves。Fixing purpose is to kill rapidly living cells, stablizes the effect of pretreatment；The purpose of acidolysis is softening cell wall and degradation of cell matter, in order to chromosomal dispersion and flattening；The purpose of dyeing is specific to make chromosome (or chromatin) painted, it is simple to examines under a microscope and presents chromosome image。
The present invention, relative to prior art, has such advantages as and beneficial effect:
(1) spirit of amine sulphur is used can to improve stability and the success rate of tabletting, it is thus achieved that higher proportion of chromosome separation image in mid-term；
(2) Colchicine price is 600～1000 yuan/g, and the amine sulphur spirit of this institute is extremely cheap, is about the 2% of Colchicine price, can greatly reduce experimental cost；
(3) Colchicine toxicity is much larger than the spirit of amine sulphur, therefore, after using amine sulphur spirit to substitute Colchicine, in tableting processes, can reduce the pollution to environment, substantially reduce degree and risk that people produces poison。
Accompanying drawing explanation
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。
Fig. 5 is phase cell proportion comparison diagram metaphase of cell division adopting different pretreatments agent to obtain when processing in embodiment 3 and comparative example 1。
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this。
The diploid group training of tail alpine ash clone DH32-29 used in embodiment is taken root, and (this seedling is national high quality tree species to Seedling, breeding accession designation number is: state S-SC-EU-001-2011), purchased from forest farm, state-owned east gate, Guangxi Zhuang Autonomous Region, the tetraploid group training of the tail alpine ash clone DH32-29 used Seedling of taking root is induced obtained by the diploid group training of tail alpine ash clone DH32-29 Seedling of taking root。Cellulase and pectase are purchased from Sangon Biotech (Shanghai) Co., Ltd.。Other reagent used all can be buied from market routine。
Embodiment 1
The diploid group taking tail alpine ash clone DH32-29 trains the tip of a root of Seedling of taking root, put into immersion treatment 4h in the amine sulphur spirit of 0.01g/L, then by the tip of a root distilled water wash after immersion, put in Ka Nuoshi fixative and fix 24h under 4 DEG C of conditions, the tip of a root is taken out from fixative, rinse with distilled water, put into the 30min that dissociates in the 10g/L cellulase that volume ratio is 1:1 of 37 DEG C and the mixed liquor of 10g/L pectase, take out the tip of a root, clean with distilled water, it is placed on clean microscope slide, with blade partially cut-away to root cap and region of elongation, drip 1 carbolfuchsin dyeing liquor, dyeing 10min, covered, coverslip is beaten gently with erasing rubber, with absorbent paper, unnecessary dyeing liquor is blotted, then tabletting is placed in basis of microscopic observation。Chromosome observation figure is as shown in Figure 1。
Embodiment 2
By 30min, the enzymolysis time in embodiment 1 is increased to 40min, and all the other operations are all identical with embodiment 1。Chromosome observation figure is as shown in Figure 2。
Embodiment 3
By 30min, the enzymolysis time in embodiment 1 is increased to 35min, and all the other operations are all identical with embodiment 1。The chromosome observation figure of gained is as shown in Figure 3。
From Fig. 1～3 it can be seen that as enzymolysis 30min (such as Fig. 1), enzymolysis is not enough, and chromosome is still strapped in nucleus, does not disperse；As enzymolysis 40min (such as Fig. 2), dissociating excessively, not only cell membrane is decomposed, and chromosome is also decomposed, it is more difficult to observe the chromosome of shaping。Only when Dissociation time is suitable for, desirable dissociation effect could be obtained。Therefore, from the facility of operation and cost consideration, it is preferred (such as Fig. 3) with cellulase and pectase mixed solution in 37 DEG C of constant temperature 35min that dissociates。
Embodiment 4
The seedling diploid that taken root by tail alpine ash clone DH32-29 in embodiment 3 replaces with tail alpine ash clone DH32-29 and takes root seedling tetraploid, and all the other conditions are all identical with embodiment 3。
Fig. 4 is the chromosome design sketch of different ploidy cells in embodiment 3～4, even 4 times of bodies, when having 44 chromosomes, remain to realize the dispersion that chromosome shrinks fully and is distributed, chromosome karyotype analysis software can be used to carry out counting and karyotyping, this reliability also demonstrating the method that this research is found out and maturity。
Comparative example 1
Pretreating agent in embodiment 3 is changed into the Colchicine of 0.1g/L by the amine sulphur spirit of 0.01g/L, and all the other conditions are all identical with embodiment 3。
Fig. 5 adopts phase cell proportion comparison diagram metaphase of cell division obtained during different pretreatments agent in embodiment 3 and comparative example 1, when being pretreating agent when adopting Colchicine, phase cell metaphase of cell division reaching requirement of 10%～20% can be obtained, and in the present invention, adopt the spirit of amine sulphur as pretreating agent, can obtaining phase cell metaphase of cell division reaching requirement of 15%～30%, with Colchicine as compared with pretreating agent, success rate significantly improves。
Above-described embodiment is the present invention preferably embodiment; but embodiments of the present invention are also not restricted to the described embodiments; the change made under other any spirit without departing from the present invention and principle, modification, replacement, combination, simplification; all should be the substitute mode of equivalence, be included within protection scope of the present invention。
Claims (5)
1. the chromosomal tabletting method of Eucalyptus, it is characterised in that include step in detail below:
(1) draw materials: the diploid or the tetraploid group that take tail alpine ash clone DH32-29 train the seedling rooting tip of a root of 5～10 days of taking root, from part within most advanced place 0.5cm；
(2) pretreatment: cleaned up by the tip of a root distilled water taken, puts into immersion treatment 3h～7h in the amine sulphur spirit of 0.002～0.02g/L；
(3) fixing: pretreated tip of a root distilled water to be cleaned up, puts in Ka Nuoshi fixative and fix 24h under 4 DEG C of conditions；
(4) dissociate: from fixative, take out the tip of a root, rinse with distilled water, put into the 30～40min that dissociates in the mixed enzyme solution of 37 DEG C；
(5) tabletting and dyeing: after the tip of a root distilled water rinsing after dissociating, it is placed on microscope slide, with blade partially cut-away to root cap and region of elongation, drip 1 carbolfuchsin dyeing liquor, dyeing 10min, then, covered, and coverslip is beaten gently with erasing rubber, with absorbent paper, unnecessary dyeing liquor is blotted；
(6) microscopy: tabletting is placed in basis of microscopic observation, the dispersion of selective staining body, fully shrinks, in bar-shaped, clear and be in the cell of split coil method mitosis metaphase and carry out counting and taking pictures。
2. the chromosomal tabletting method of Eucalyptus according to claim 1, it is characterised in that:
The concentration of the amine sulphur spirit described in step (2) is 0.01g/L, and the time of described immersion treatment is 4h。
3. the chromosomal tabletting method of Eucalyptus according to claim 1, it is characterised in that:
The time drawn materials described in step (1) is point in the morning 9。
4. the chromosomal tabletting method of Eucalyptus according to claim 1, it is characterised in that:
Mixed enzyme solution described in step (4) is volume ratio is the mixed liquor of the 10g/L cellulase of 1:1 and 10g/L pectase。
5. the chromosomal tabletting method of Eucalyptus according to claim 1, it is characterised in that:
The finger that dissociates described in step (4) dissociates 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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Cited By (3)
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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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| US20110122492A1 (en) * | 2009-07-10 | 2011-05-26 | Ikonisys, Inc. | Custom filtration slide and filtration apparatus and method |
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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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| CN105699141B (en) | 2017-12-19 |
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