CN103710439A - Method for quickly identifying chromosome number of rosa plant - Google Patents
Method for quickly identifying chromosome number of rosa plant Download PDFInfo
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Abstract
The invention provides a method for quickly identifying chromosome number of a rosa plant. The method comprises the steps of drawing materials, pretreatment, fixing, disaggregating, dyeing, tabletting, microscopic examination and the like. The tabletting process is digitalized in the tabletting step, and cover glass on the material is vertically knocked on by 28-34N of force for 160-200 times for the best. In preferable embodiments, the stem tip and/or blossom bud are adopted as materials for counting and analyzing the chromosomes. In preferable embodiments, the materials are obtained between nine and eleven in the morning or between three and four in the afternoon. In embodiments, growth points and positions of the materials are confirmed by an electron microscope in a scanning manner. The method disclosed by the invention has the advantages of diversified materials, accurate tabletting and the like, and is quick, accurate and efficient, and is especially applicable to quick and accurate identification of the chromosome number of the rosa plants on a large scale. Thus, the method is applied to quick and accurate identification of the chromosome number of the rosa plants on a large scale.
Description
Technical field
The present invention relates to the especially chromosome number object method of gul of plant identification, belong to plant cell biology and cytogenetics field.
Technical background
Karyomit(e) is the carrier of gene, and preparing chromosome specimen is cytogenetic basis, good Chromosome Technique, the prerequisite of carrying out especially chromosome banding, group type analysis, in situ hybridization etc.The observation and analysis of plant cell chromosome, has great importance for the analysis of the genetic phenomenons such as chromosome behavior and cytometaplasia in chromosome behavior analysis in dyeing karyotyping, reduction division process, cytogamy culturing process.In addition, the variation that genetic engineering breeding produces on gene level, also can carry out the assignment of genes gene mapping as basic Chromosomes Banding, in situ hybridization, fluorescence in situ hybridization by take dyeing compressing tablet.
The chromosome number of plant identification is a kind of very important analysis of chromosome analysis, and the material structure of general frequent employing active growth carries out.Stem apex and the tip of a root are considered to the tissue of active growth conventionally, are mitotic districts occurred frequently, thereby become the material that chromosome counting is conventional.For the tip of a root and stem apex, the material that bud is seldom analyzed as chromosome counting, reason may be that the materials such as pigment that exist in bud disturb, and needs the special pre-treatment more taking time and effort, and the time of drawing materials be subject to more restriction, as destroyed the growth conditions of plant.
Rose (Rosa) plant is world-renowned ornamental plant, comprises rose, Chinese rose and rose etc., and this genus has more than 200 to plant, and is distributed widely in cool temperature zone to subtropical zone.As one of global ornamental plant, the Cultivar of gul has more than 30000, and genetic background is very complicated, and chromosome number and ploidy differ greatly, at present known have 2,3,4,5,6,8 and 10 times of bodies and aneuploid etc., this is to cause the major cause that distant hybirdization is not affine and hybrid is highly sterile, is also that people obtain the major obstacle of good objective trait by cross-breeding.Therefore, necessary before cross-breeding parent material is carried out to Methods of Ploidy Identification.But, because gul is woody, and genetic background complicacy, karyomit(e) is middle-size and small-size, causes chromosome counting to analyze difficulty large, the evaluation herbaceous plant for example chromosome number object method of Dianthus caryophyllus L. cannot be effectively applied to gul.In addition, the histocyte that gul is difficult for getting suitable material, material is difficult to dissociate, cause that chromosome counting result is inaccurate, unstable even cannot be counted, and gate time is long, is not suitable for carrying out rapidly and accurately the Observation on Chromosome Number of kind in enormous quantities.
The chromosomal material of plant identification being usually used in is the tip of a root, but the seed germination of gul is more difficult, and the offspring of growing directly from seeds has variation, especially Chinese rose (Rosa Hybrida).Rose is a variety of in addition or kind cuttage root-taking difficulty, and the cuttage cycle is long, wastes time and energy, the easy lignifying of adventive root producing, difficult assurance is drawn materials the time, and when doing microscopy, in field of microscope, divide phase cell few, Chromosome spread is bad, is difficult to carry out chromosome counting.Though reporting with the tender stem apex of children of gul, someone does material, but because the division phase cell number of shoot tip meristem is limited, the difficulty of accurately drawing materials, and exist that histocyte is difficult for dissociating and cell content or secretory product disturb the problems such as compressing tablet effect.On Dianthus caryophyllus L., available bud is identified chromosome number, but there is not yet, utilizes the bud of gul as the report of the material of chromosome counting analysis.
Although the chromosome number analysis of accounts difficulty of gul is very large, extremely important.Especially need to develop the chromosomal method of accurate Rapid identification gul.
Summary of the invention
The object of the invention is for one in addressing the above problem or whole issue.For this reason, the present invention adopts following technical scheme:
1. a chromosome number object method of identifying gul, described method in turn includes the following steps:
(1) step of drawing materials: obtain the material from gul;
(2) pre-treatment step: use pretreatment fluid to process described material;
(3) fixing step: use stationary liquid to be fixed described material;
(4) dissociation steps: use the solution that dissociates to dissociate to described material;
(5) staining procedure: use dyeing solution to dye to described material;
(6) compressing tablet step: described material is carried out to compressing tablet; With
(7) microscopy step: by described material being detected with microscope the chromosomal number in the cell of determining described material;
Wherein, in described compressing tablet step, the power with 28 to 34N is vertically knocked 160 to 200 times the cover glass on described material.
2, the method as described in technical scheme 1, wherein, described knocking is divided into the first pass carrying out successively and knocks with second time and knock, and use and do not carry out with sharpening a pencil of rubber end, described first pass knocks with the power of 28N to 34N and vertically knocks cover glass on described material 80-100 time, described knocking for second time with identical dynamics carried out vertical knocking with number of times, and described first pass knocks and uses the rubber end of described pencil to carry out, described knocking for second time adopts another end of described pencil to carry out.
3, the method as described in technical scheme 1 or 2, wherein, described compressing tablet step is carried out in the following way: dyed described material is placed on slide glass, then drip the acetum of 45 volume %, covered, siphons away unnecessary solution with water-absorbing material, cover one deck filter paper, left hand is pinned one end of cover glass, and the right hand is taken pencil to carry out successively described first pass and knocked with described second time and knock, and finally cover glass is flattened and removes filter paper.
4, the method as described in any one in technical scheme 1 to 3, wherein, the material of described material for confirming to there is the material of vegetative point through electron-microscope scanning pre-detection and/or having confirmed vegetative point position through electron-microscope scanning pre-detection.
5, the method as described in any one in technical scheme 1 to 4, wherein, described material is the material that selects the free tip of a root, stem apex or bud to form, preferably, described material is stem apex and/or bud; More preferably, described material is bud.
6, the method as described in any one in technical scheme 1 to 5, wherein, described material the morning 9:00-11:00 or afternoon 3:00 to 4:00 time obtain, and be stem apex and/or bud, more preferably bud.
7, the method as described in any one in technical scheme 1 to 6, wherein, described stem apex is taken from annual major branch bar and/or 2 years raw side shoot bars; Preferably, described stem apex is that length is the terminal bud thing of 1.5mm to 3mm, and described bud is to be highly the bud top tissue of 1.5mm to 2.5mm.
8, the method as described in any one in technical scheme 1 to 7, wherein, the serve as reasons solution of the pretreating agent preparation of selecting the group that free santochlor, oxine, colchicine and α-bromonaphthalene etc. form of described pretreatment fluid, preferably, described pretreatment fluid is santochlor saturated aqueous solution.
9, the method as described in any one in technical scheme 1 to 8, wherein, described pre-treatment step is carried out in the following way: with scalper, described material is cut into two parts, put into the container that santochlor saturated aqueous solution is housed, with sealed membrane by described container sealing, use syringe to be pumped to described material and sink to treatment solution bottom, then 20 ℃ to 25 ℃ pre-treatment 2 hours to 3 hours, then with deionized water, clean.
10, the method as described in any one in technical scheme 1 to 9, wherein, described fixing step is carried out in the following way: described material is placed in the container that Kano stationary liquid is housed and this container is placed in ice chest and fixes 4 hours to 6 hours, then water cleans, and dehydrated alcohol and Glacial acetic acid that described Kano stationary liquid is 3:1 by volume ratio form.
11, the method as described in any one in technical scheme 1 to 10, wherein, described dissociation steps is carried out in the following way: use 0.8M to 1.2M aqueous hydrochloric acid in 55 ℃ to 65 ℃ water-baths, described material to be dissociated 8 minutes to 10 minutes, then water cleans.
12, the method as described in any one in technical scheme 1 to 11, wherein, described staining procedure carries out in the following way: described material is placed in the container that dye liquor is housed and dyes 10 minutes to 30 minutes, described dye liquor is carbol fuchsin dye liquor, and then water cleans; Preferably, the consumption of described dye liquor was not for just having described material.
13, the method as described in any one in technical scheme 1 to 12, wherein, described microscopy step is carried out in the following way: use 100 power microscopes to find the object observing scope of described material, then use 400 times of mirrors to find the cell in division stage, recycle 1000 times of oily mirrors and observe chromosomal number; Preferably, first utilize 1000 times of oily mirror pictures taken, and then according to picture, karyomit(e) is counted.
The present invention carries out electron-microscope scanning by material first before drawing materials, and make to draw materials size and position are more accurately and fast with efficient; Using the especially young tender bud of bud as material, make the variation of drawing materials simultaneously.This in advance to draw materials that to carry out electron-microscope scanning visual, and using the tender bud of children as the method for processing material, be applicable to more cuttage root-takings and be difficult for, the xylophyta somatic chromosome of blooming in many seasons is observed.
Compared with prior art, the present invention has for example following advantage: draw materials accurately (1), is necessary that Shi Kexian carries out electron-microscope scanning to material, makes the accurate positioning of drawing materials, intuitively, solved the difficult problem of accurately drawing materials existing in gul chromosome observation all the time.(2) variation of drawing materials, comprises bud and stem apex, can break away from the restriction of the time of drawing materials.(3) evaluation speed is fast, and each step time of whole process, easily control, can complete and identifies and/or can in 24 o'clock, obtain qualification result within a working days.(4) cost of expert testimony is low, and pretreatment agent is less expensive, identifies that used tool easily obtains, and can reuse.(5) pretreating effect is good, with syringe, pretreating containers is vacuumized, and the pretreatment time of material is shortened, and effect is better.(6) pressed disc technique is accurate, during compressing tablet knock dynamics and number of taps is accurate, make Chromosome spread even, counting accurately, repeatable high.
Accompanying drawing explanation
Fig. 1 is stem apex electron-microscope scanning figure.A: the stem apex (sweetbrier polyantha, rose class) that vegetative point is less; B: the more stem apex of apical growing point (month in and month out powder, Flos Rosae Chinensis); C: bud (that is, the small bud that inflorescence meristem includes young tender bud and growing) (month in and month out powder, Flos Rosae Chinensis).
Fig. 2 is the cell number statistic curve figure of the different time observable division phases under 400 times of drawing materials, and the material that wherein used is sweetbrier polyantha; The position of drawing materials is stem apex; At interval of 1hr, draw materials once.To counting in the cell of division phase in a visual field.Result show draw materials preferably the morning 9:00-11:00 or afternoon 3:00 to 4:00 carry out, especially best with the effect that the morning, 9:00-11:00 drew materials.
Fig. 3: A: pink blossom bud (2n=2x=14) month in and month out; B: powder stem apex (2n=2x=14) month in and month out; C: sweetbrier polyantha (2n=3x=21); D: Sa Mansha (4n=28); E: white jade hall disappearance two staining cells (2n=12) H: white jade hall (2n=2x=14); F: compressing tablet dynamics and to beat number of times inadequate, karyomit(e) and cell disperse inhomogeneous; G: dog rose (2n=5x=35); I: compressing tablet dynamics, strike sheet number of times and the time of drawing materials while all adopting method of the present invention to carry out under 400 times of light microscopics cell situation.
Embodiment
As mentioned above, gul is because genetic background is complicated, and plant tissue chaff interference is many, and karyomit(e) is little, analysis of accounts exist draw materials undesirable, analytical cycle long, repeatability is poor, result is inaccurate, is not suitable for carrying out the problems such as chromosome counting of material in enormous quantities.On the other hand, the chromosome analysis of gul is to very important and necessary again of breeding for example.In order to solve gul chromosome counting in prior art, analyze existing for example above-mentioned problems, the inventor has carried out long-term observation and research in this respect, has finally developed a kind of chromosome number object method of quick and precisely identifying gul.
The invention provides a kind of chromosome number object method of quick and precisely identifying gul, described method comprises the steps:
(1) step of drawing materials: obtain the material from gul;
(2) pre-treatment step: use pretreatment fluid to process described material;
(3) fixing step: use stationary liquid to be fixed described material;
(4) dissociation steps: use the solution that dissociates to dissociate to the described material through fixing;
(5) staining procedure: use dyeing solution to dye to the described material through dissociating;
(6) compressing tablet step: dyed described material is carried out to compressing tablet; With
(7) microscopy step: by the described material through compressing tablet being detected to the chromosomal number in the cell of determining described material with microscope;
Wherein, in described compressing tablet step, take 28 to 34N(for example can as 28,29,30,31,32,33 or 34N(newton)) power vertically knock the cover glass 160 to 200 times (for example 160,165,170,175,180,185,190,195 or 200 times) on described material.
Some preferred embodiment in, described knocking is divided into the first pass carrying out successively and knocks with second time and knock, and use and do not carry out with sharpening a pencil of rubber end, described first pass knocks that to take 28N to 34N(can be for example 28, 29, 30, 31, 32, 33 or 34N(newton)) the cover glass that vertically knocks on described material of power (can be for example 80 80-100 time, 85, 90, 95 or 100 times), described knocking for second time preferably carried out vertical knocking with identical dynamics with number of times, and described first pass knocks and uses the rubber end of described pencil to carry out, described knocking for second time adopts another end that there is no rubber of described pencil to carry out.Certainly, also can adopt other instrument replace with rubber do not sharpen a pencil knock, but the instrument that preferably knocks use has elasticity position and rigidity position, described first pass knocks and adopts elasticity position to carry out, drawn materials bulk tissue can be cushioned and disperse to be split into cell one by one, and each cell can be flattened; Described knocking for second time adopts rigidity position to carry out, and makes first pass knock scattered cell and spreads in a plane, is beneficial to later stage microscopy and observes and take pictures.
In some embodiments, described compressing tablet step for example can be carried out in the following way: dyed described material is placed on slide glass, then drip the acetum of 45 volume %, covered, for example, with water-absorbing material (filter paper), siphon away unnecessary solution, cover one deck filter paper, left hand is pinned one end of cover glass, fixed cap slide, prevents its slip; The right hand is taken pencil to carry out successively described first pass and is knocked with described second time and knock, and finally for example with thumb, cover glass is flattened and removes filter paper.
The inventor finds, whether a crucial part of the chromosome counting analysis of gul is can be in the situation that guarantee that the integrity of cell can fully be dissociated material structure and karyomit(e) can fully disperse, and the compressing tablet step in this point and the film-making process especially relation of compressing tablet dynamics is especially tight.Compressing tablet dynamics is not enough, and the weak effect that histocyte launches, causes difficult microscopic even cannot obtain result accurately; Compression force is spent greatly, equally may be because the integrity of cell is destroyed and cannot obtain the chromosome number of individual cells.Therefore, the inventor is by being decomposed into the required total dynamics of compressing tablet the combination that adopts specific dynamics and number of taps.Due to the dynamics of compressing tablet and number of times are carried out to digitizing, therefore can in the situation that guaranteeing that cell is complete, make compressing tablet effect better, the repeatability of acquired results is better, is also convenient to the inventive method to carry out mechanized operation.
In addition, the present invention also finds, identifies that another crucial part of chromosome number object of gul is obtaining of material.If sampling error, post-processed may need long time, and what for example in prior art, have even needs to be placed in frozen water more than 20 hours, sometimes even possibly cannot obtain needed result.The inventor finds, adopts electron-microscope scanning to carry out pre-detection to confirm vegetative point situation or the location of growing point of this material to material, so can play a multiplier effect.Afterwards, from a large amount of electron-microscope scannings, found that, stem apex (referring to Figure 1A and 1B) and bud (as Fig. 1 C), if drawn materials suitably, compressing tablet is reasonable, are applicable to for analyzing chromosome number very much.Especially for bud, it is generally acknowledged that bud contains the interfering substances such as pigment many, be not suitable for the analysis of chromosome number object, although also have in other plant afterwards, adopt bud to carry out the report of Chromosome Analysis, and adopted the method for reporting but not succeed to the karyomit(e) data analysis of gul.The present invention has afterwards carried out continuous trial, finally finds and can take bud as the chromosomal method of material analysis, and obtained ideal result.Therefore, in some embodiments, described material is preferably stem apex and/or bud; More preferably, described material is bud.
In some embodiments, described stem apex and/or bud are taken from annual major branch bar and/or 2 years raw side shoot bars; Preferably, described stem apex is that length is the terminal bud thing (as plate 1A, B) of 1.5mm to 3mm, and described bud is to be highly the bud inflorescence top tissue (as plate 1C) of 1.5mm to 2.5mm.For example, can get terminal bud (containing lateral bud) and the mellow and full length of growing that the vigorous top mellow and full length of parcel consolidation of outdoor cultivating and growing is 1.5-3mm is the tender bud of children of 1.5-2.5mm.While drawing materials, preferably choose the mellow and full stem apex of stem top parcel consolidation, shoot apical meristem is more.Also can be simultaneously by means of the means of electron-microscope scanning by formalness and microscopic pattern are combined, the more precision that makes to draw materials, can dwindle drawn materials volume like this, shortens pretreatment time, compressing tablet better effects if.
In addition, inventor's discovery, reasonably the time of drawing materials can make material be more suitable for identifying in chromosome number object.In some embodiments, described material the morning 9:00-11:00 or afternoon 3:00 to 4:00 time obtain, more preferably at 10:00 in the morning draw materials (referring to Fig. 2).In addition preferably, described material is stem apex and/or bud, more preferably bud.
The present invention has no particular limits pretreatment fluid, can use the conventional pretreating agent in this area, such as the pretreating agent that can use the group of compositions such as selecting free santochlor, oxine, colchicine and α-bromonaphthalene.Wherein, colchicine typical concentrations is: 0.05%-0.2%, and applied widely, the effect that pre-treatment has all had to large, medium and small type plant chromosome.But this reagent severe toxicity, is shown in that light easily decomposes, and expensive.Oxine, typical concentrations is 0.002mol/L, be applicable to middle-size and small-size chromosomal plant, but its effect is gentleer, use this reagent to carry out pretreated time, the pretreatment time needing is long, is generally 4-6 hour, and the number of accumulative total Metaphase Chromosome not as other pretreating agents high.α-bromonaphthalene is relatively applicable to the pre-treatment of Gramineae and waterplant, and other plant application is fewer.The action effect of santochlor saturated aqueous solution is stronger, use range is wider, and be applicable to large, medium and small type plant chromosome and observe, and low price, pretreatment time is short, is more suitable for the Rapid identification for the chromosomal ploidy of gul various in style.So, some preferred embodiment in, described pretreatment fluid is santochlor saturated aqueous solution.For example in some embodiments, described pre-treatment step can be carried out in the following way: with scalper, described material is cut into two parts, put into the container that santochlor saturated aqueous solution is housed, with sealed membrane by described container sealing, use syringe to be pumped to the bottom that described material sinks to treatment solution, 20 ℃ to 30 ℃ pre-treatment 2 hours to 3 hours, then water cleaned again.Pretreated temperature for example can be 20,21,22,23,24,25,26,27,28,29 or 30 ℃, and the treatment time can be for example 2,2.5 or 3 hours.More preferably, pretreated temperature is 25 ℃, and pretreatment time is 2.5 hours.If treatment temp is too high, may be easy to bring out karyomit(e) and sticks together; If treatment temp is too low, possibly cannot make karyomit(e) concentrated fast.In addition, if the treatment time is long, be easy to induced chromosome adhesion; If the treatment time is short, karyomit(e) concentrating degree is unfavorable for separated counting not.
The present invention has no particular limits stationary liquid, but some preferred embodiment in, described material is placed in the container that Kano stationary liquid is housed and this container is placed in ice chest and fixes 4 hours to 6 hours, then water cleans, and dehydrated alcohol and Glacial acetic acid that described Kano stationary liquid is 3:1 by volume ratio form.
The present invention has no particular limits the used solution that dissociates that dissociates, but preferably use 0.8M to 1.2M aqueous hydrochloric acid, for example 0.8,0.9,1.0,1.1 or the aqueous hydrochloric acid of 1.2M, in 55 ℃ to 65 ℃ water-baths, described material is dissociated 8 minutes to 10 minutes, then water cleans.Preferably, can be by the solution that dissociates in 50 ℃ to 70 ℃ preheatings for example two minutes, so that material can enter dissociated state more rapidly.The temperature of water-bath for example can be 55,60 or 65 ℃, more preferably 60 ℃.If excess Temperature or treatment time are long, may cause DNA molecular on karyomit(e) to degrade seriously, karyomit(e) is painted more shallow, is unfavorable for observing; If temperature is too low or the treatment time is too short, may make cellular segregation insufficient, it is little that nucleus and tenuigenin color contrast, is not easy to compressing tablet and observation.
The present invention has no particular limits dyeing solution, but preferably use carbol fuchsin (also to claim PHENOL 99.8 MIN ((CARBOLIC ACID)) pinkish red, it is conventional a kind of chromosome dyeing agent eighties, also be current domestic application the most a kind of plant chromosome staining agent, (refer to Li Maoxue, " introducing a kind of core and chromosomal good staining agent ", biology circular, 1982,5:53) dye liquor.For example can carry out in the following way described staining procedure: described material is placed in the container of the carbol fuchsin dye liquor of dye liquor, dye 10 minutes to 30 minutes (for example can 10,15,20,25 or 30 minutes) be housed, and then water cleans.Carbol fuchsin dye liquor can be configured in the following manner: be first made into three kinds of stostes, then be made into staining fluid, stoste A:3g magenta is dissolved in 100ml70% alcohol; Stoste B: get stoste A 10ml and join in 90ml 5% aqua carbolisata solution; Stoste C: get stoste B 55ml, add 6ml Glacial acetic acid and 6ml formalin (38% formaldehyde).Wherein, stoste A and stoste C can preserve for a long time, and stoste B limit was used in two weeks.The preparation of dye liquor: get stoste C10-20m1, add 45% Glacial acetic acid 80-90ml, then add sorbyl alcohol 1.8g, be made into the carbol fuchsin liquid of 10%-20% concentration, preferably place after two weeks and use, effect is remarkable, if use immediately after preparation, tinting strength is slightly poor.
Some preferred embodiment in, the consumption of described dye liquor was not for just having described material.The inventive method dyeing is abundant, simple to operate, health, and dye liquor can reuse.
The instrument that the present invention uses microscopy step has no particular limits, as long as can clearly observe the chromosome number of cell, but preferably this instrument has shoot function, make to utilize instrument comparison film to carry out autoscan observation and analyze the number dyeing and become possibility, thereby can exempt, adopt artificial mode to carry out the needs of chromosome counting, make thus method of the present invention be more suitable for large batch of chromosome number analysis of accounts.For example, in some embodiments, can use object observing scope that 100 power microscopes find described material (for example, the region that cell disperses, core district is colored), then use 400 times of mirrors to find the cell in division stage, recycling 1000 times of (eyepiece 10x, object lens 100x) oily mirrors counts chromosome number.Or utilize 1000 times of oily mirror pictures taken, finally according to captured picture, karyomit(e) is counted again.
In some embodiments of the inventive method, can before drawing materials, to stem top or bud, carry out electron-microscope scanning, fast and easy is accurately drawn materials; The tender bud of the children of usining can be so that draw materials variation as material.Drawing materials is in this technique the plant stem apex being grown under state of nature, when drawing materials, preferably controls the treatment time of each step, so just can obtain detected result the same day.In operating process, can use the medical vials with rubber plug that volume is 20ml as container, and insert in bottle and bleed with the syringe needle position of disposable needle tubing, make the pretreating effect of material better.In addition, the accurate number of times (80-100 time) that strikes the dynamics (28-34N) of sheet and strike sheet during compressing tablet, production effect is better, and Chromosome spread is easy counting evenly, can not cause chromosome elimination again, the accuracy of impact counting.Generally, if when the dynamics of compressing tablet surpasses 34N and/or beats number of times higher than 100 times, cell integrity is bad, karyomit(e) can lack and affect the accuracy of counting; When the dynamics of compressing tablet is lower than 28N and/or while beaing number of times lower than 80 times, Chromosome spread is bad, is difficult to counting.
In some embodiments, the present invention can carry out in the following way:
When pre-detection, before drawing materials, first detect the tender annual growth branch of the children raw side shoot sprouting brachyplast bar of (or being called major branch bar), 2 years of plant and the top of bud and carry out electron-microscope scanning, understand vegetative point distributing position and quantity, determine that this platymiscium open country growth is vigorous, the tender long shoot bar of annual children, length is that terminal bud thing and the length of 1.5-3mm is that the tender growth of flower bud of the mellow and full full children of 1.5-2.5mm profile is the most vigorous, and vegetative point is maximum, the most applicable chromosome counting of doing.
Draw materials the morning 9:00 to 10:00 or afternoon 3:00 to 4:00 carry out, getting terminal bud (can contain lateral bud) and the mellow and full length of growing that the vigorous top of the outdoor cultivating and growing mellow and full length of parcel consolidation is 1.5-3mm is the tender bud of children of 1.5-2.5mm.While drawing materials, will choose the mellow and full stem apex of stem top parcel consolidation, such shoot apical meristem is more.
During pre-treatment, can be by terminal bud and/or lateral bud or bud with scalper crosscut into two, then put in santochlor saturated aqueous solution, with sealed membrane, medical bottleneck is sealed, syringe is pumped to material and sinks to treatment solution bottom, make the abundant combination of pretreatment fluid and material, improve pretreated effect.25 ℃ of left and right pre-treatment 2.5h, water cleans.
In the time of fixedly, can use Kano stationary liquid (dehydrated alcohol: Glacial acetic acid=3:1), preferably matching while using is fixed 4 hours to 6 hours (can be for example 4 hours, 5 hours or 6 hours) in ice chest, and cell is fully killed, then water cleans.
While dissociating, can be by 1M aqueous hydrochloric acid preheating for example 2 minutes, then in for example dissociating for example 8 minutes to 10 minutes with it in 60 ℃ of water-baths, then water cleans.
During dyeing, can use carbol fuchsin dye liquor dyeing for example 10 minutes to 30 minutes.For example, material unification can be placed in the discarded medical vials that dye liquor is housed, the amount of staining agent be take and there just was not material as good.So not only save staining agent, also convenient and healthy, quick, compressing tablet better effects if.
During compressing tablet, can get the material in dye liquor, with 45% acetic acid with the pencil compressing tablet of rubber.Drip acetum, careful covered, with thieving paper siphon away more than solution.Cover one deck filter paper, left hand is pinned one end of cover glass, fixing.The right hand is taken pencil, first use the rubber end with the pencil of rubber, by the power of 28-34N, vertically beat the cover glass about 80-100 time of target location, material place, with cutting the identical dynamics same number in written one end, do not strike sheet again, finally with thumb, press flat, thereby cell is uniformly dispersed, and karyomit(e) can be distributed in same plane.Generalized case, compressing tablet required time is every slice, thin piece of 4-6min, each material presses 5-8 to open slice, thin piece, the material in 30min left and right.
During microscopy, can find object observing scope at 100 times of Microscopic observations, find the cell in division stage under 400 times of mirrors, 1000 times of oily mirrors (dripping pine and cypress oil) are taken pictures.Every slice, thin piece pressing, observing and taking pictures needs 8-15min, each material require 1h left and right.
If adopt bud as material, preferably using the tender bud of children, for example, is bud (referring to Fig. 1 C) after differentiation bud and before reduction division, and the plant in this stage is in the florescence, profile be can't see the form of bud and inflorescence, but can observe full mellow and full top.
When cleaning, preferably all adopt deionized water rinsing 3-5 time, every all over cleaning 5min left and right, at the bottom of implement can adopt glue head dropper to be close to bottle, to prevent that material is sucked out with liquid.
Those skilled in the art should know the saturated santochlor aqueous solution and the Ka Bao product solution using in the present invention.For example, can reference example as the compound method in the < < crop karyomit(e)s such as Li Maoxue and investigative technique > > (the 1st edition, in August, 1996) thereof.
Adopting terminal bud to carry out chromosome counting technology with traditional gul compares, the present invention draws materials precedent as carried out electron-microscope scanning to stem apex and the tender bud top of children, visual and precision makes to draw materials, solved the stem apex difficulty of drawing materials, non-meristematic cell disturbs, and tip of a root degree of lignification is high, the seed difficulty of taking root, the problems such as the cuttage root-taking cycle is long, and root length and rootage duration are wayward.Adopt young tender bud to carry out karyomit(e) compressing tablet, draw materials conveniently, can not destroy the growth conditions of plant, many in the cell number of metacinesis phase, solved gul stem apex vascular tissue and disturbed, karyomit(e) is little, counts a difficult difficult problem.In addition, in the present invention, drawing materials can be eugonic terminal bud and the bud under state of nature.If 9:00 in the morning starts to draw materials, the time of suitably control pre-treatment, fix, dissociate, dyeing, and adopt above-mentioned tabletting method, can in 12h, identify fast, efficiently and accurately chromosomal number.In addition, if first carry out electron-microscope scanning before drawing materials, make the precision of drawing materials, variation, saves time, and film-making efficiency is high, and Chromosome spread is even, easily counting.Certainly, the present invention is also applicable to other material degrees of lignification forest tree high and that bloom in many seasons and carries out chromosome counting rapidly and efficiently.
Below the mode by embodiment is further detailed the present invention, but these embodiment only provide with way of example, these embodiment should not be understood to be limitation of the present invention.
Embodiment
The instrument using in embodiment and medicine all can be commercially available, do not use special equipment or special medicine.All reagent is all purchased from Beijing chemical reagents corporation, and the stereoscope using in the embodiment of the present invention is Nikon(Nikon) smz800, the microscope model that dyeing observation post is used is Olympus(Olympus) BX51.
The material of using in embodiment, before drawing materials, has all carried out electron-microscope scanning.Wherein case study on implementation 3,4, and sweetbrier polyantha, white jade hall and dog rose in 6 belong to rose class, and scanning result is Figure 1A shown type; In case study on implementation 2,5, powder and Sa Mansha stem apex are Chinese rose class month in and month out, and scanning electron microscope is defined as the type of Figure 1B; The bud of the pink blossom month in and month out scanning electron microscope result of embodiment 1 is Fig. 1 C shown type.
(the young tender bud of R.chinensis ' Old Pink Daily ' .) carries out as material to using the powder month in and month out of 2 times of bodies.
A, the morning, 9:00, got the branch top in the florescence of outdoor cropping, the tender bud of children of the circular consolidation of formalness, and under stereoscope, stripping is rapidly highly the material about 2.0mm.
B, use santochlor, process 3h for 25 ℃.The top of the tender bud of children is the calyx sheet not launching, above close by fine hair, before processing, use scalper truncation 0.5mm under stereoscope, put into the saturated santochlor solution that 20ml is housed, with the syringe with syringe needle, be evacuated to material and sink to pretreatment fluid bottom, then with washed with de-ionized water 5 times.
C, use Kano stationary liquid are fixed 5 hours in ice chest, and then washed with de-ionized water is 5 times.
D, at 60 ℃ with 1M salt acid dissociation 10min, then washed with de-ionized water is 5 times.
E, dyeing: use carbol fuchsin dye liquor dyeing 20 minutes.Material unification is placed on is equipped with in the discarded medicine bottle that the volume of dye liquor is 20ml, and the amount of staining agent there just be not material.
F, get the material in dye liquor, drip 45% acetum, careful covered, siphons away unnecessary solution with thieving paper.Cover one deck filter paper, left hand is pinned one end of cover glass, fixing.The right hand is taken pencil, first with the pencil end with rubber, the cover glass 90 times that vertically beats target location, material place by the power of 30N (, first pass knocks), with cutting the identical dynamics same number in written one end, do not strike sheet (that is, knocking for second time) again, finally with thumb, flatten, cell is uniformly dispersed, and karyomit(e) is distributed in same plane.
G, microscopy: 100 times of mirrors (Olympus, BX51, lower with) lower observation, find object observing scope, at 400 times of mirrors (Olympus, BX51, lower with) the lower cell of finding in division stage, 1000 times of oily mirrors (dripping pine and cypress oil) take pictures (the results are shown in Figure 3A).
Embodiment 2
Using 2 times of bodies month in and month out powder (R.chinensis ' Old Pink Daily ' .) stem apex is as material.
The morning, 9:00, got the eugonic annotinous branch top of outdoor cropping, and under stereoscope, stripping rapidly length is the stem of 1.5mm left and right.In santochlor saturated solution, at 25 ℃, process 2.5h, washed with de-ionized water 5 times; Salt acid dissociation 8min with 1M.First pass knocks with knocking all for second time and carries out with the dynamics of 28N and 80 number of times; Described in the same case of all the other steps (1), result refers to Fig. 3 B.
Embodiment 3
Using 2 times of body white jade halls (R.multiflora var.albo-plena) stem apex as material.
The morning, 9:00, got the annual young shoot top 2cm of outdoor cropping, in layer peelled off blade around under stereoscope, and position, emergent stem top cuts with scalper the apical growing point position that front end length is 2.5mm left and right.25 ℃ of processing 3.0h of santochlor saturated solution.The white fine hair of close quilt above the blade of the tender terminal bud of children that this material newly grows.Kano stationary liquid is fixed 4 hours, the 1M aqueous hydrochloric acid 9min that dissociates.First pass knocks with knocking all for second time and carries out with the dynamics of 34N and 100 number of times.Described in the same case of all the other steps (1), result refers to Fig. 3 E and 3H.From result, can see, there is disappearance problem in the white jade hall karyomit(e) of 2 times of bodies, in Fig. 3 E, and 2n=2x=12; 2n=2x=14 in Fig. 3 H.These cells are from the identical position of drawing materials.This shows, use the inventive method the phenomenon of chromosome deletion in cell can be detected.
Embodiment 4
Using 3 times of body sweetbrier polyanthas (R.multiflora.sp) stem apex as material.
The morning 9:00, the degree of lignification of getting outdoor cropping is not high, color is emerald green, the tender stem apex of 2cm children of top parcel consolidation, cuts with scalper the apical growing point position that top length is 2.5mm left and right at stereoscope.25 ℃ of processing 3h of santochlor saturated solution.This material tender leaf is the tiny fine hair of adularescent around.Kano stationary liquid is 3h fixedly, the 1M aqueous hydrochloric acid 10min that dissociates.When first pass knocks with knocking all for second time, with the dynamics of 29N and 95 number of times, carry out, in the time of described in the same case of all the other steps (1), result refers to Fig. 3 C.When first pass knocks with knocking all for second time, with the dynamics of 25N and 80 number of times, carry out, in the time of described in the same case of all the other steps (1), result refers to Fig. 3 F.
Using 4 times of body Sa Mansha (R.hybrid cv.Samantha) stem apex as material.
The morning, 9:00, got the annual children of outdoor cropping tender, the top 2cm of the rubescent branch of blade, and under stereoscope, stripping rapidly length is the stem apex of 2.8mm left and right.25 ℃ of processing 2.5h of santochlor saturated aqueous solution, washed with de-ionized water 5 times; Kano stationary liquid 4h, the 1M aqueous hydrochloric acid 10min that dissociates.First pass knocks with knocking all for second time and carries out with the dynamics of 28N and 98 number of times.Described in the same case of all the other steps (1), result refers to figure D.When observing with 400 power microscopes, the result of observing, as shown in Fig. 3 I, can be seen from Fig. 3 I, many in the cell number of division stage.This also illustrates, adopts method of the present invention, before drawing materials, uses scanning electron microscope to determine the position of drawing materials, to identifying that this platymiscium ploidy is very effective.
Embodiment 6
Using dog rose (R.canina) stem apex of 5 times of bodies as material.
The morning, 10:00, got the top 2cm of the annual young shoot of outdoor cropping, and under stereoscope, stripping rapidly length is the stem apex of 3.0mm left and right.25 ℃ of processing 3h of santochlor saturated aqueous solution, washed with de-ionized water 5 times; Kano stationary liquid is 4h fixedly, the 1M aqueous hydrochloric acid 10min that dissociates, and described in the same case of all the other steps (1), result is as Fig. 3 G.
As shown in Figure 3, adopt the inventive method 2 times of body white jade hall chromosome deletion 2n=2x=12(plate 3E to be detected), powder stem apex (2n=2x=14) month in and month out, the sweetbrier polyantha stem apex (2n=3x=21) of 3 times of bodies; The dog rose stem apex (2n=5x=35) (Fig. 3 G) of the Sa Mansha stem apex (2n=4x=28) of 4 times of bodies and 5 times of bodies.
In addition, in conjunction with the result of scanning electron microscope, when drawing materials, also find, the Sa Mansha of 4 times of bodies is large compared with 2 times of body Fols Rosae shoot apical meristems, while drawing materials, can amplify scope of selecting material.Villous above the rose class tender leaf that single cropping is bloomed, terminal bud is larger, can preferably be controlled at 3mm left and right while drawing materials.Plant in the florescence, the formalness of young tender bud is mellow and full full, while drawing materials, easily identifies, and scanning electron microscope result please refer to Fig. 1 C.
The present invention before drawing materials to draw materials and carry out electron-microscope scanning, the use of combination visor, by drawn materials visual, can accurately represent size and the position of drawing materials, solve the difficult problem of drawing materials always existing in cytology research, this method is also applicable to other will carry out the floristics of cytology research.While drawing materials, young tender bud, as pretreated material, also can apply it in the xylophyta blooming in other many seasons and carry out cell dyeing observation, and variation is drawn materials.That when pre-treatment, for example uses is discarded
Cheap cheap, can reuse, syringe vacuumizes better processing effect.This technological invention is precisely arrived 28-34N by the dynamics of compressing tablet, and compressing tablet number of times precisely arrives 80-100 time, makes compressing tablet dynamics and time digitizing, is applicable to existing cell chromosome pressed disc technique.During dyeing, unified carrying out, both convenient and healthy, saves again staining agent, makes Color better.
Claims (10)
1. a chromosome number object method of identifying gul, described method in turn includes the following steps:
(1) step of drawing materials: obtain the material from gul;
(2) pre-treatment step: use pretreatment fluid to process described material;
(3) fixing step: use stationary liquid to be fixed described material;
(4) dissociation steps: use the solution that dissociates to dissociate to described material;
(5) staining procedure: use dyeing solution to dye to described material;
(6) compressing tablet step: described material is carried out to compressing tablet;
(7) microscopy step: by described material being detected with microscope the chromosomal number in the cell of determining described material;
Wherein, in described compressing tablet step, the power with 28 to 34N is vertically knocked 160 to 200 times the cover glass on described material.
2. the method for claim 1, wherein, described knocking is divided into the first pass carrying out successively and knocks with second time and knock, and use and do not carry out with sharpening a pencil of rubber end, described first pass knocks with the power of 28N to 34N and vertically knocks cover glass on described material 80-100 time, described knocking for second time with identical dynamics carried out vertical knocking with number of times, and described first pass knocks and uses the rubber end of described pencil to carry out, described knocking for second time adopts another end of described pencil to carry out.
3. method as claimed in claim 1 or 2, wherein, described compressing tablet step is carried out in the following way: dyed described material is placed on slide glass, then drip the acetum of 45 volume %, covered, siphons away unnecessary solution with water-absorbing material, cover one deck filter paper, left hand is pinned one end of cover glass, and the right hand is taken pencil to carry out successively described first pass and knocked with described second time and knock, and finally cover glass is flattened and removes filter paper.
4. method as claimed any one in claims 1 to 3, wherein, the material of described material for confirming to there is the material of vegetative point through electron-microscope scanning pre-detection and/or having confirmed vegetative point position through electron-microscope scanning pre-detection; Preferably, described material is the material that selects the free tip of a root, stem apex or bud to form; More preferably, described material is stem apex and/or bud; Further preferably, described material is bud; In addition preferably, described stem apex is taken from annotinous branch; More preferably, described stem apex is that length is the top tissue of the terminal bud of 1.5mm to 3mm, and described bud is that length is the inflorescence top tissue of 1.5mm to 2.5mm.
5. the method as described in any one in claim 1 to 4, wherein, described material the morning 9:00-11:00 or afternoon 3:00 to 4:00 time obtain, and be stem apex and/or bud, bud more preferably.
6. the method as described in any one in claim 1 to 5, wherein, described pre-treatment step is carried out in the following way: with scalper, described material is cut into two parts, put into the container that santochlor saturated aqueous solution is housed, with sealed membrane by described container sealing, use syringe to be pumped to the bottom that described material sinks to treatment solution, then 20 ℃ to 30 ℃ pre-treatment 2 hours to 3 hours, then water cleaned.
7. the method as described in any one in claim 1 to 6, wherein, described fixing step is carried out in the following way: described material is placed in the container that Kano stationary liquid is housed and this container is placed in ice chest and fixes 4 hours to 6 hours, then water cleans, and dehydrated alcohol and Glacial acetic acid that described Kano stationary liquid is 3:1 by volume ratio form.
8. the method as described in any one in claim 1 to 7, wherein, described dissociation steps is carried out in the following way: use 0.8M to 1.2M aqueous hydrochloric acid in 55 ℃ to 65 ℃ water-baths, described material to be dissociated 8 minutes to 10 minutes, then water cleans.
9. the method as described in any one in claim 1 to 8, wherein, described staining procedure carries out in the following way: described material is placed in the container that dye liquor is housed and dyes 10 minutes to 30 minutes, described dye liquor is carbol fuchsin dye liquor, and then water cleans; Preferably, the consumption of described dye liquor was not for just having described material.
10. method as claimed in any one of claims 1-9 wherein, wherein, described microscopy step is carried out in the following way: use 100 power microscopes to find the object observing scope of described material, then use 400 times of mirrors to find the cell in division stage, recycle 1000 times of oily mirrors and observe chromosomal number; Preferably, first utilize 1000 times of oily mirror pictures taken, and then according to picture, karyomit(e) is counted.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016179814A1 (en) * | 2015-05-13 | 2016-11-17 | 广州赫尔氏药物开发有限公司 | Gene related to papillary thyroid cancer |
| CN110672389A (en) * | 2019-10-12 | 2020-01-10 | 广州甘蔗糖业研究所湛江甘蔗研究中心 | Young-leaf-based Cinnamomum longepaniculatum chromosome karyotype analysis method |
| CN110849686A (en) * | 2019-11-28 | 2020-02-28 | 江苏省农业科学院 | Tabletting method for rapidly obtaining strawberry root tip chromosome and prepared tablet |
| CN112067410A (en) * | 2020-09-16 | 2020-12-11 | 山东农业大学 | Fringe-based chromosome karyotype analysis method for Chinese fringetree |
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| CN102492767A (en) * | 2011-11-15 | 2012-06-13 | 四川农业大学 | Preparation method of chromosome suitable for karyotype analysis of rubus plants |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016179814A1 (en) * | 2015-05-13 | 2016-11-17 | 广州赫尔氏药物开发有限公司 | Gene related to papillary thyroid cancer |
| CN110672389A (en) * | 2019-10-12 | 2020-01-10 | 广州甘蔗糖业研究所湛江甘蔗研究中心 | Young-leaf-based Cinnamomum longepaniculatum chromosome karyotype analysis method |
| CN110849686A (en) * | 2019-11-28 | 2020-02-28 | 江苏省农业科学院 | Tabletting method for rapidly obtaining strawberry root tip chromosome and prepared tablet |
| CN112067410A (en) * | 2020-09-16 | 2020-12-11 | 山东农业大学 | Fringe-based chromosome karyotype analysis method for Chinese fringetree |
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