CN102329878B - Orychophragmus violaceus chromosome acetic acid orcein banding method - Google Patents
Orychophragmus violaceus chromosome acetic acid orcein banding method Download PDFInfo
- Publication number
- CN102329878B CN102329878B CN 201110311523 CN201110311523A CN102329878B CN 102329878 B CN102329878 B CN 102329878B CN 201110311523 CN201110311523 CN 201110311523 CN 201110311523 A CN201110311523 A CN 201110311523A CN 102329878 B CN102329878 B CN 102329878B
- Authority
- CN
- China
- Prior art keywords
- chromosome
- acetic acid
- banding
- orcein
- root
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses an orychophragmus violaceus chromosome acetic acid orcein banding method for solving the problems that the traditional chromosome separation banding method is not suitable for small chromosome plants such as orychophragmus violaceus, the procedure is complex, the operating conditions are not easily mastered and the banding success rate is low. Cell chromosomes can be easily separated by adopting acetic acid orcein for dyeing and combining a chromosome preparation method, the chromosomes have good shape after being lightly pressed and are clear in banding display, and identification of target chromosomes is facilitated. The plant chromosome banding method is very simple and convenient and easy in operation, is particularly suitable for banding pattern analysis of plants with small chromosomes, and is clear in displayed banding patterns, high in success rate and convenient for genetic analysis of plants.
Description
Technical field:
The invention belongs to the cytogenetics field, be specifically related to Orychophragmus violaceus chromosome banding method.
Background technology:
Chromosome banding technique is the routine of differential staining body and research chromosome structure variation and classical technology.It is with the processing of karyomit(e) through certain procedures, and with behind the specific dyeing, light and shade replaces or the different band of the depth to make karyomit(e) demonstrate one by one at its major axis, and such band just is called chromosomal band.Banding technique can disclose chromosomal " anatomy " feature, can demonstrate the chromosomal special banding pattern of each bar of eukaryote, the abundant chromosomal special marking that can be used for identifying is provided, this is for identification and analyze every karyomit(e) necessary condition is provided, thereby can be than karyotype analytical procedure differential staining body more accurately.The diversity of chromosome banding, heterozygosity etc. have reflected the complicacy of this body structure of karyomit(e), and the genetic mechanism of planting lower variation, species differentiation and forming.By to aobvious further investigation with mechanism, can carry out more deep understanding to chromosomal composition, structure, function and behavior etc., thereby find out the rule that biological stain body banding pattern changes, this is significant for the Genetic Mechanisms that really discloses organic evolution from cell and molecular level, and on this basis and then reach Artificial Control and transform the purpose of biological heredity, variation.
Chromosome banding technique is the method for karyomit(e) being carried out differential stain with dyestuff, with karyomit(e) after acid, alkali, temperature etc. are processed again with dyeing, or alone some fluorescent dyeing just can be observed the different or bright secretly different vertical structure with line of the depth.Plant chromosome aobvious with on the most frequently used be the Giemsa banding technique, wherein commonly used have C-band, G-band and a N-band etc.The C-band is distributed more widely in plant, all may occur at end of chromosome, middle-end, both sides, kinetochore, nucleolar constriction place, and accuracy is higher, the main method that plant is divided band, the research of the aspects such as widespread use and plant cytology, cytogenetics, plant taxonomy, the origin of species, chromosome engineering, plant breeding.
Method commonly used is as follows at present:
1. material is prepared to treat about the long 2cm of onion bulb stem root of hair, and the Broad Bean Seeds seed soaking is germinateed, and treats that young root grows to about 3cm, cuts the tip of a root and carries out pre-treatment.The broad bean main root tip of a root cuts the rear secondary root that continues to grow, and can cut the secondary root tip of a root again and carry out pre-treatment.Barley seed germinates to the long 1cm of young root, and the young root that cuts white carries out pre-treatment.
2. the pre-treatment tip of a root is 0.Pre-treatment 2～3h in 05% colchicine solution.Treatment temp is generally 25 ℃.Must be with flushing with clean water repeatedly after the pre-treatment, the flush away liquid.
3. fixing above each material is put into the Ka Nuoshi stationary liquid and is fixed 0.5～24h after pretreatment, is transformed into 70% alcohol, places refrigerator to save backup.
4. the tip of a root that dissociates places in the 0.1N hydrochloric acid soln under 60 ℃ of constant temperature processes 10～15min.The barley tip of a root is processed 30min with 1% polygalacturonase under 37 ℃, then place 60 ℃ of lower 5min of processing in the 0.1N hydrochloric acid soln.
Above-mentioned materials with acid treatment after, must with distilled water flushing repeatedly remove residual acid solution, otherwise will affect chromosomal aobvious band effect.
5. compressing tablet is identical with conventional plant chromosome tabletting method.Compressing tablet in 45% acetic acid is made white.Check the Chromosome spread degree under phase microscope, it is many mutually to pick out division, the uniform slice, thin piece of Chromosome spread.The slide of selecting freezes through liquid nitrogen, CO2 dry ice or semiconductor cooler, opens cover glass with blade.Put under the room temperature dry.
6. the chromosome specimen after the dry air dehydration generally needs the dry air through 4～7 days, divides tape handling again.The time of the different required dryings of material is different.Onion requires air dried time tighter, and without the not aobvious band of air dried karyomit(e), a dry week shows terminal band by aobvious tape handling, can show simultaneously terminal band and centromeric band behind the dry two weeks.Broad bean, rye, barley then require time of drying very not strict.
7. the chromosome specimen after the aobvious tape handling dry air can show tape handling.Treatment process is different, can show different banding patterns.
(1) C band HSG method (Hydrochloric acid～Saline～Giemsa method): the chromosome specimen after the dry air is immersed 0.2N hydrochloric acid (about 25 ℃) process respectively 30 and 60min.With distilled water flushing repeatedly after, in 2 * SSC solution of 60 ℃, be incubated 30min, with distilled water flushing for several times, room temperature is air-dry, can dye again.
BSG method (Barium～Saline～Giesa method): the chromosome specimen after the dry air is immersed in the staining jar of the 5% hydrated barta saturated solution that fills new preparation, process at ambient temperature 5～10min, then after repeatedly washing scum carefully with distilled water, in 2 * SSC solution of 60 ℃, be incubated 60min, again with distilled water flushing for several times, room temperature is air-dry, can dye.
2) the N band will cut pre-treatment 24h in 0 ℃ of frozen water about rye, barley seed root of hair 1cm.In the Ka Nuoshi stationary liquid fixedly more than half an hour, the 2h that dyes in 1% acetic acid magenta, compressing tablet in 45% acetic acid then, freezing method is opened.Thereafter the 10min that under 60 ℃ of conditions of 45% acetic acid, decolours, the 10min that under 95% alcohol room temperature, dewaters again, gas is dried to spend the night.In 1M NaH2PO4 solution, be incubated 2min under 95 ℃ of constant temperature at last, distilled water flushing, gas can dye after doing.
8. Giemsa staining Jim Sa mother liquor dilutes according to a certain percentage with the 1/15M phosphoric acid buffer.For example, 10 parts of phosphoric acid buffers add 1 part of Jim Sa mother liquor dilution and are 10:1, generally all adopt button to dye method dyeing.On a clean sheet glass, symmetrical two toothpicks or the match stick placed, distance equates with material ranges on the slide glass.Will be downward with the slide upset of material, be placed on the toothpick, then in the space between slide glass and the sheet glass, slowly splash into staining fluid on one side along slide glass, at room temperature dyeing.Dyeing time is different because of material, and Yin Jimusa dyestuff lot number is different, variant qualitatively, so its stin of thickness and dyeing time need appropriately adjust.
9. the slide sample after the dyeing of microscopy and mounting is used distilled water flush away excess dyestuff, the decolouring of hyperchromasia phosphoric acid damping fluid.Get final product microscopy after air-dry under the room temperature, select clearly slice, thin piece of chromosome banding pattern, use gummy pad.
The above-mentioned method Orychophragmus violaceus that step is used for microchromosome of drawing materials can not satisfy aobvious band effect, and aobvious band success ratio is low, and colchicine is used in pre-treatment, and is poisonous, contaminate environment.Then the rear compressing tablet that dissociates shows tape handling, and could dye presents the band line, and operation steps is a lot, uncertain increasing, and aobvious band success ratio is low.
Summary of the invention:
It is simple to the purpose of this invention is to provide a kind of program, and step is few, and agents useful for same is few, easy and simple to handle, and aobvious band success ratio is high, is applicable to the chromosomal aceto-orcein Banded method of microchromosome plant Orychophragmus violaceus.
The present invention is achieved in that
Orychophragmus violaceus chromosome acetic acid orcein banding method of the present invention may further comprise the steps:
(1) draws materials: with seed warm water soaking 6～12h, then change in the Gibberellins solution of 600mg/L and put into 4 ℃ of refrigerators 20-25 hours, then with seed 22-23 ℃ of germinations in culture dish, lucifuge. behind the Seed sprouting, seed was put into 4 ℃ of refrigerators 20-25 hours again, taking-up is put back to 22-23 ℃ of greenhouses and is germinateed, when treating that root grows to 1-2cm, and the intercepting Root apical meristem;
(2) pre-treatment: Root apical meristem was processed 23-24 hours through 0-1 ℃ of frozen water;
(3) fixing: as with Ka Nuoshi liquid I Root apical meristem to be fixed 23-25 hours at 4 ℃, Ka Nuoshi liquid I prescription: raw spirit: Glacial acetic acid=3:1, volume ratio;
(4) dissociate: Root apical meristem is put into the 1mol/L HCl that is preheating to 60 ℃ dissociate, Dissociation time is 8 minutes,
(5) dyeing is put into the aceto-orcein staining fluid with Root apical meristem, and the mass percentage concentration of orcein in acetic acid solution is 0.1%, and 20-26 ℃ kept 12-24 hours;
(6) with Root apical meristem with the acetic acid compressing tablet that after the distillation washing with concentration expressed in percentage by volume is 45%.
Drawing materials and pre-treatment step in the inventive method; Combine the advantage of various seed-soaking methods, make it to be suitable for the seed germination of Orychophragmus violaceus, and but free from environmental pollution, chromosome banding is processed and is carried out in the lump in dyeing course, without separate treatment, acetic acid can play aobvious tape handling effect, and also simultaneously dyeing of orcein dye, program is simplified, and has reduced a lot of steps in operation; Therefore avoided many influence factors, such as the operate miss in each step, probabilistic impacts such as the temperature of each step, time, thereby aobvious band success ratio improves.Such as the dry air in the traditional method, time of drying, length was very large on aobvious band impact, and some plant is very strict to requiring time of drying, bad grasp.And for example freezing when taking off cover plate behind compressing tablet, easily karyomit(e) is together taken off with cover plate, cause chromosome elimination.In aobvious tape handling, the treatment time of the reagent such as used acid or alkali also is difficult to support and holds, and is long, lacked all to show band.Thereby traditional method is too many because of step, and agents useful for same is also many, and is strict, and influence factor is numerous, and success ratio is too low.Program of the present invention is simple, and step and agents useful for same are few, is operated with the impact of reagent littlely, thereby aobvious band success ratio is high.
Traditional method is also different because of material with time of Giemsa staining, and the dyestuff lot number is different, variant qualitatively, so its stin of thickness and the bad support of dyeing time are held.Grasp easily with the aceto-orcein dyeing time, simple to operate, easy dyeing.
The used hydrochloric acid of the present invention and background technology hydrochloric acid indifference, the difference of Dissociation time just, because floristics is different, Dissociation time is different, the present invention's 8 minutes time kind that dissociates is only suitable to Orychophragmus violaceus, is that the best dissociates the time.
Plant microchromosome Banded method disclosed by the invention is applicable to the microchromosome plants such as Orychophragmus violaceus.Drawing materials of seed is optimized in traditional method with pretreatment process in the present invention, with warm water soaking, GA3(Plant hormones regulators,gibberellins) vernalization, then vernalization in 4 degree refrigerators, vernalization is once again when Seed sprouting shows money or valuables one carries unintentionally, when the length seminal root grows to 1～2cm, the intercepting tip of a root, too short or long then tip of a root meristematic cell is less.The tip of a root of intercepting was processed 23-24 hours with frozen water, and the treatment time is long, and then the karyomit(e) concentrating degree is high, was not easy aobvious band, the too short less and most chromosome forms that also do not form suitable aobvious band of karyomit(e) that then are in mitosis metaphase.Experiment shows, by of the present invention draw materials and pretreatment process then the germinating time of seed is more neat, and the quality of sprouting is good, the root division is vigorous, the cell that is in mitosis metaphase is more, chromosome form is better, is suitable for aobvious band operation.
In plant microchromosome Banded method of the present invention, the Orychophragmus violaceus root-tip cells separates the cell compressing tablet by eight minutes dissociate among 60 ℃ the 1mol/L HCl easily, and the Chromosome spread degree is good, zero lap easy to identify.Too short such as Dissociation time, then cell is not easy to press off, the chromosome repeat None-identified; Long such as Dissociation time, cell rupture then, karyomit(e) is easily lost.Root-tip cells is dissociated and is spent the night with normal temperature dyeing in the aceto-orcein afterwards, and method is simple, and is effective, and the program of the so much complexity of traditional method useless has been avoided the impact of factors on chromosome banding.With the conventional compressing tablet of 45% acetic acid, namely can be observed mauve band line with the ordinary optical microscope microscopy after the dyeing.
Banded method of the present invention, processing ease, easy, efficient, practical, be a kind of plant microchromosome Banded method of novelty, and be suitable for equally the aobvious band analysis of other macrochromosome plants to have important scientific research and be worth.The present invention compares with existing method, and its advantage and effect can further be summarised as:
1, plant microchromosome aceto-orcein Banded method provided by the invention, it is drawn materials and pretreatment process makes the germinating time of seed more neat, and the quality of sprouting is good, the root division is vigorous, the cell that is in mitosis metaphase is more, chromosome form is better, is suitable for the aobvious band operation of the plants such as Orychophragmus violaceus.Traditional method is undesirable for the effect of Orychophragmus violaceus.
2, adopt plant microchromosome aceto-orcein Banded method provided by the invention, its Dissociation time separates its cell compressing tablet for the just right grasp of Orychophragmus violaceus easily, and the Chromosome spread degree is good, zero lap easy to identify.
3, plant microchromosome aceto-orcein Banded method provided by the invention spends the night with the dyeing of aceto-orcein normal temperature, through the conventional compressing tablet of acetic acid, namely can be observed mauve band line with the ordinary optical microscope microscopy.This dyeing process is simple, and success ratio is high, aobviously is with effectively, and the program of the so much complexity of traditional method useless has been avoided the impact of factors on chromosome banding.
4, plant microchromosome aceto-orcein Banded method provided by the invention, suitability is wide, both applicable to the microchromosome plant, such as Orychophragmus violaceus, rape, also be applicable to the macrochromosome plant, only need change Dissociation time and method of drawing material to get final product, dyeing and other method are constant.Used medicine all is conventional reagent, and low price is easy to get.
Embodiment:
By the following examples the present invention is further described specifically.It is important to point out; following embodiment is only for the present invention is described further; can not be interpreted as limiting the scope of the invention; affiliated art skilled staff is according to the foregoing invention content; the present invention is made some nonessential improvement and adjustment, should still belong to protection scope of the present invention.
The contriver has obtained good result at present with the experiment that is used for the Orychophragmus violaceus chromosome banding of Orychophragmus violaceus chromosome banding of the present invention.
Specific implementation method
1) draws materials.Get seed and in warm water, soak 8h, then change in the Gibberellins solution of 600mg/L and be put in 24h in 4 ℃ of refrigerators.Then with seed 22 ℃ of germinations in culture dish, be put in and be lined with on the wetting filter paper of distilled water lucifuge.When Seed sprouting, again seed is put into 4 ℃ of refrigerator 24h, and then taken out, put back to 22 ℃ of greenhouses.When treating that root grows to 1～2cm, the intercepting Root apical meristem.2) pre-treatment.The plant root tip meristematic tissue is after 0-1 ℃ of frozen water is processed 23h, with Ka Nuoshi liquid I(raw spirit: Glacial acetic acid=3: 1) to 4 ℃ of materials fixedly about 24h.3) dissociate.The 1mol/L HCl that material is put into 60 ℃ of preheatings dissociates, and Dissociation time is eight minutes.4) dyeing.It is 0.1% aceto-orcein staining fluid normal temperature dyeing 24h that material is put into mass percentage concentration.5) karyomit(e) compressing tablet.With the conventional compressing tablet of the rear acetic acid with 45% of distilled water washing, microscopy.
2, aobvious band effect
The Orychophragmus violaceus Chromosome spread is good, and the band line is clear, and karyological character is easy to identify, is conducive to further genetic analysis.
Claims (1)
1. Orychophragmus violaceus chromosome acetic acid orcein banding method is characterized in that, may further comprise the steps:
(1) draws materials: seed with warm water soaking 6～12h, is then changed in the Gibberellins solution of 600mg/L and put into 4 ℃ of refrigerators 20-25 hours, then with seed 22-23 ℃ of germinations in culture dish, lucifuge; Behind the Seed sprouting, seed was put into 4 ℃ of refrigerators 20-25 hours again, taken out and put back to 22-23 ℃ of greenhouses germinations, when treating that root grows to 1-2cm, cut Root apical meristem;
(2) pre-treatment: Root apical meristem was processed 23-24 hours through 0-1 ℃ of frozen water;
(3) fixing: as with Ka Nuoshi liquid I Root apical meristem to be fixed 23-25 hours at 4 ℃, Ka Nuoshi liquid I prescription: raw spirit: Glacial acetic acid=3:1, volume ratio;
(4) dissociate: Root apical meristem is put into the 1mol/L HCl that is preheating to 60 ℃ dissociate, Dissociation time is 8 minutes;
(5) dyeing: Root apical meristem is put into the aceto-orcein staining fluid, and the mass percentage concentration of orcein in acetic acid solution is 0.1%, and 20-26 ℃ kept 12-24 hours;
(6) with Root apical meristem with the acetic acid compressing tablet that after the distillation washing with concentration expressed in percentage by volume is 45%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110311523 CN102329878B (en) | 2011-10-14 | 2011-10-14 | Orychophragmus violaceus chromosome acetic acid orcein banding method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110311523 CN102329878B (en) | 2011-10-14 | 2011-10-14 | Orychophragmus violaceus chromosome acetic acid orcein banding method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102329878A CN102329878A (en) | 2012-01-25 |
| CN102329878B true CN102329878B (en) | 2013-04-17 |
Family
ID=45481839
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110311523 Expired - Fee Related CN102329878B (en) | 2011-10-14 | 2011-10-14 | Orychophragmus violaceus chromosome acetic acid orcein banding method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102329878B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111323288A (en) * | 2020-04-03 | 2020-06-23 | 贵州省烟草科学研究院 | Winged tobacco monosome identification method |
| CN115372554A (en) * | 2022-07-29 | 2022-11-22 | 江西农业大学 | Research method for influence of different treatment methods on number of pueraria lobata chromosomes |
-
2011
- 2011-10-14 CN CN 201110311523 patent/CN102329878B/en not_active Expired - Fee Related
Non-Patent Citations (9)
| Title |
|---|
| Karyotype variation and evolution in populations of the Chinese endemic Orychophragmusviolaceus complex (Brassicaceae);Lirong Zhou et al.;《Nordic Journal of Botany》;20081231;第26卷(第5-6期);376页右栏第2段 * |
| Lirong Zhou et al..Karyotype variation and evolution in populations of the Chinese endemic Orychophragmusviolaceus complex (Brassicaceae).《Nordic Journal of Botany》.2008,第26卷(第5-6期),375-383. |
| 应用有机溶剂提高种子活力;徐本美 等;《植物生理学通讯》;19871231(第4期);17右栏第1段至18页左栏第2段，18页右栏第3段，表3 * |
| 应用赤霉素有机溶剂渗入法打破花卉种子休眠和促进萌发;顾增辉 等;《种子》;19921231(第1期);55页右栏第2段至56页左栏第1段，表1，图1 * |
| 徐本美 等.应用有机溶剂提高种子活力.《植物生理学通讯》.1987,(第4期),17-20. |
| 甜菜染色体组型制备技术;白庆武;《中国甜菜糖业》;19821231(第3期);50-54 * |
| 白庆武.甜菜染色体组型制备技术.《中国甜菜糖业》.1982,(第3期),50-54. |
| 陈景长 等.蔬菜育苗方式与技术.《蔬菜育苗手册》.中国农业大学出版社,2000,59. * |
| 顾增辉 等.应用赤霉素有机溶剂渗入法打破花卉种子休眠和促进萌发.《种子》.1992,(第1期),55-58. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102329878A (en) | 2012-01-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| VonAbrams et al. | Seed dormancy in Rosa as a function of climate | |
| Gialvalis et al. | Plant hormones alter fiber initiation in unfertilized, cultured ovules of Gossypium hirsutum | |
| CN102589943B (en) | Method for sheeting peanut root tip cell chromosome in mitosis metaphase | |
| CN101482515A (en) | Tabletting method for locust stem tip chromosome | |
| CN111238888A (en) | Efficient sugarcane or sugarcane near-edge seed stem tip chromosome flaking method | |
| Orkwiszewski et al. | Phase identity of the maize leaf is determined after leaf initiation | |
| CN102564821B (en) | Tabletting method of plum blossom shoot tip chromosome | |
| CN102329878B (en) | Orychophragmus violaceus chromosome acetic acid orcein banding method | |
| CN108849498B (en) | Method for promoting accumulation of flavonoid substances in leaf tissues of ornamental crabapple | |
| CN103695557A (en) | Gingko karyotype analysis method based on stem tips | |
| CN104297034A (en) | Method applied to chromosome production of single tobacco plant by tender ovary | |
| CN110564821A (en) | RNA in-situ hybridization optimization method for cucumber seedling gene localization research | |
| CN106350593A (en) | Dripping piece preparation method of plant chromosome | |
| CN104614211A (en) | Paraffin sectioning method of Magnolia sieboldii K.Koch seeds | |
| CN111175102A (en) | Method for preparing slices of root tip chromosomes of Paeonia plants | |
| CN109943619B (en) | In-situ observation method for plant root hair | |
| CN106644636A (en) | Microdissection technology of tobacco chromosomes | |
| CN101613755B (en) | Method for identifying carnation chromosome number by bud | |
| CN116465702A (en) | Method for preparing chromosome of tulip tree material | |
| CN112697556B (en) | Method for preparing citrus plant root tip chromosome tablet | |
| Schuster | Cytogenetics in fruit breeding. Preparation methods for mitotic chromosomes | |
| Ruffoni et al. | The temporary immersion system (TIS) for the improvement of micropropagation of ornamental plants | |
| Corsi et al. | Embryo and endosperm response to ageing in Triticum durum seeds as revealed by chromosomal damage in the root meristem | |
| CN112067410A (en) | Fringe-based chromosome karyotype analysis method for Chinese fringetree | |
| Seagull | Cytoskeletal stability affects cotton fiber initiation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130417 Termination date: 20141014 |
|
| EXPY | Termination of patent right or utility model |