CN111238888A - Efficient sugarcane or sugarcane near-edge seed stem tip chromosome flaking method - Google Patents
Efficient sugarcane or sugarcane near-edge seed stem tip chromosome flaking method Download PDFInfo
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Abstract
The invention provides a high-efficiency sugarcane or sugarcane kindred seed stem tip chromosome flaking method, and belongs to the technical field of cell biology. The invention provides a high-efficiency sugarcane stem tip chromosome flaking method aiming at the problems that sugarcane chromosomes are large in number and small in shape and ideal mitotic phase metaphase cells are difficult to obtain by means of sugarcane stem tip meristem tissue of sugarcane and sugarcane kindred species in a vigorous growth period, has the advantages of convenience in material taking, large meristem sample size, vigorous division, multiple metaphase cells, clear and dispersed chromosome structures, and discloses the technical key points of material selection, pretreatment, fixation, dissociative staining, flaking and chromosome shape microscopic observation. The method is simple and convenient, accurate and reliable in result, good in repeatability, easy to operate and short in experimental period, and improves the efficiency of sugarcane chromosome genome analysis. The invention provides technical support for sugarcane karyotype research, germplasm resource classification identification and protection utilization.
Description
Technical Field
The invention belongs to the technical field of cell biology, and particularly relates to an efficient sugarcane or sugarcane kindred seed stem tip chromosome flaking method.
Background
Sugarcane is an important sugar crop and energy plant in the world, and the genus Saccharum sinensis comprises 6 species of thin-stem wild species, large-stem wild species, tropical species, Indian species, Chinese species and fleshy panicle species (also called cion species), and closely related plants of Saccharum officinarum, Cymbopogon yunnanensis, Heyawana and the like in the subfamily Saccharum officinarum. Modern sugarcane varieties mostly contain 2-3 relatives of sugarcane genera, have narrow genetic basis and serious sexual degeneration, and breeders perform intraspecific, interspecific and intergeneric hybridization utilization in order to cultivate new excellent sugarcane varieties. It is expected that resistance genes of wild species and closely related plants can be introduced into sugarcane, thereby enriching the genetic basis of sugarcane. The method relates to chromosome research in researches such as sugarcane germplasm resource classification, exogenous chromosome identification, gene chromosome positioning, genetic relationship identification, meiosis mechanism and the like.
The preparation of sugarcane chromosome slide is the basis for developing sugarcane cytogenetics, sugarcane molecular cytogenetics and related researches based on chromosomes and the like. However, sugarcane is the most complex heteropolyploid plant, has a large number of chromosomes (ranging from 64 to 128) and a small shape, and is difficult to obtain an ideal chromosome slide specimen of a cell in a mitotic phase. At present, sugarcane root tip meristem is mainly adopted at home and abroad for flaking, but the following problems still exist in the actual operation process: the root tip of the water culture sugarcane has viscous secretion, which influences the chromosome flaking effect. The root tips of the barrel planting or sand culture are not affected by sticky secretions, but the sediment needs to be cleaned during collection, so the operation is complicated; sugarcane roots are small, main roots are few, fibrous roots are many, a meristem area is small, and 1 to 2 root tips are generally used for preparing 1 slide specimen; the root tips of the sugarcane in the barrel culture, the sand culture or the water culture are greatly influenced by the nutritional environment factors, the root tips are different in thickness and tenderness, the division period is difficult to control, and only a few root tips in 1 batch of root tip samples are usually in the mitosis period; the fact that the metaphase cells can be observed in each slide cannot be guaranteed, root tips need to be collected repeatedly, and slide observation needs to be repeated, so that the workload is increased; the test and the experimental period are both long, and generally, the identification of the number of chromosomes of 1 material takes about 1-2 weeks.
Disclosure of Invention
In view of the above, the present invention aims to provide an efficient sugarcane or sugarcane near-edge seed stem tip chromosome flaking method, which can rapidly obtain metaphase cells with clean flaking background, clear chromosome morphological structure and good dispersion, and is convenient for chromosome number statistics and karyotype analysis.
The invention provides a high-efficiency sugarcane or sugarcane near-edge seed stem tip chromosome flaking method, which comprises the following steps:
1) collecting the shoot apical meristem of the vigorous sugarcane or the kindred species thereof and cutting the shoot apical meristem into 0.3-0.5 cm3In the form of small blocks, using precooled ddH2Soaking in O sterile water, and pretreating at 4 ℃ for 5-24 hours to obtain a pretreatment material;
2) fixing the pretreatment material under the fixing condition of 4 ℃ for 2-3 d by using ddH2O sterilized waterAfter cleaning, dissociating for 20-25 min by using a dissociation solution under the water bath condition of 50-65 ℃ to obtain a dissociation material;
the dissociation liquid is a mixed liquid formed by mixing 1mol/L HCl solution and 45% acetic acid aqueous solution with volume percentage concentration in an equal volume;
3) transferring the dissociation material onto a glass slide, removing surface moisture, dyeing with an improved phenol kappaucin dyeing solution, crushing the dissociation material, removing tissue residues, covering a cover glass, dyeing for 4-8 min, removing the dyeing solution around the cover glass, and tabletting to obtain a slide specimen with dispersed chromosomes.
Preferably, the shoot apical meristem area tissue in the step 1) comprises a basal meristem area tissue of a sugarcane shoot apical and/or a 2-3 layer of shoot meristem area tissue wrapping the shoot apical, and the 2-3 layer of shoot apical meristem area tissue wrapping the shoot apical is a shoot meristem area tissue within 3cm above the shoot apical.
Preferably, the harvesting period of the meristem of the shoot tip of the vigorous growing sugarcane or sugarcane kindred in step 1) includes at the stage of sugarcane jointing, elongation or late elongation.
Preferably, the collection time of the meristem of the shoot tip of the vigorous growing sugarcane or the kindred sugarcane in the step 1) is 9: 00-11: 00 in the morning of a sunny day.
Preferably, the fixing solution for fixing in the step 2) is Carnot fixing solution;
the Carnot stationary liquid is prepared from absolute ethyl alcohol and glacial acetic acid according to a volume ratio of 3: 1, and mixing the components in a ratio of 1.
Preferably, the tabletting method in step 3) is to strike the cover glass with one end of a pencil with a rubber eraser to disperse the chromosomes, and then press the cover glass with a thumb to evenly disperse the cells, wherein the chromosomes are on the same plane.
Preferably, the sugarcane varieties comprise Trita procumbens, 57NG208, Min sugar 70-611, RB72-454 and F1Cliff 96-48 or BC2And 07-86 parts of sugarcane.
Preferably, the sugarcane kindred species include sugarcane wild species and sugarcane kindred plants;
the sugarcane wild species comprise cleft hand density and big stem wild species; the sugarcane kindred plants include tall fescue, mango, Yunnan tall fescue and Heyawang.
The invention provides a sugarcane chromosome dispersed slide specimen prepared by the sugarcane or sugarcane kindred seed stem tip chromosome flaking method, the chromosome morphological structure is clear, non-overlapping division phase cells are dispersed, and the background is clean and has no impurities.
The invention provides application of the slide specimen with dispersed sugarcane or sugarcane allied species chromosomes in karyotype analysis or germplasm resource classification and protection utilization of sugarcane or sugarcane allied species.
Compared with the prior art, the efficient sugarcane or sugarcane kindred seed stem tip chromosome flaking method provided by the invention has the following beneficial effects:
1) in the material selection, the shoot apical meristem of sugarcane or sugarcane kindred species is selected for flaking in the vigorous growth period, the range of the meristem is large, the cell division cycle is concentrated, 30-50 chromosome slide specimens can be prepared in batches by a single shoot apex, more metaphase cells can be observed on each flaking, more than 20 cells which are convenient for chromosome number identification and karyotype analysis can be observed in 10 flaking generally, and compared with 50-100 flaking needing to be observed at the root apex, the flaking time and microscopic observation time are saved.
2) The tissue sample of the stem tip meristem of sugarcane or sugarcane kindred species has the characteristic of convenient collection, the growth period of the sugarcane is long (1-1.5 years), and the stem tips of the sugarcane in the jointing, extending and later vigorous growth periods are suitable for preparing chromosome slide specimens except the seedling period and the tillering period. The time of about 7-8 months in one year can be reached at any time, the sugarcane stem tip can be collected in the field to carry out related cytological study, compared with the root tip and stem segment germination chromosome flaking technology, repeated sampling is not needed, the complex steps of repeated collection and cleaning of the root tip are avoided, the workload is reduced, and the working efficiency is improved.
3) The method has the advantages that the meristem of the stem tip of sugarcane or sugarcane kindred species is pretreated at 4 ℃, the synchronous accumulation of metaphase cells is facilitated, the metaphase cells with clear chromosome morphological structures and good dispersion can be obtained in a short time through tabletting observation, observation is facilitated, the chromosome number identification of 1 sugarcane germplasm material can be completed within 2 days, and compared with the condition that 1-2 weeks are needed for the germination of the root tip and the stem segment, the experimental period is shortened, and the analysis efficiency of sugarcane chromosome genomes is improved.
4) The dissociation liquid is adopted for dissociation for 20-25 min, cells are easy to disperse, chromosomes are clear and overlap little, cells are difficult to disperse due to too short dissociation time (10-15 min), chromosomes overlap much, the dissociation time is too long (30min or more), cells are easy to break, and chromosomes are lost or overlap much.
5) The dyeing time is controlled within 4-8 min, the moderate integral dyeing degree can be ensured, and the condition of incomplete dyeing or excessive dyeing can not be generated.
The method provided by the invention is simple and convenient, and has accurate and reliable identification result, good repeatability, good stability and easy operation. The method can be used for preparing good sugarcane chromosome slices by people without cytological training. Meanwhile, the method provided by the invention is also suitable for the dripping method and the smear method. The chromosome slide specimen prepared by the invention provides technical support for sugarcane chromosome karyotype research, germplasm resource classification, protection and the like, and also provides help for sugarcane kindred species or other crop chromosome quantity and karyotype analysis.
Drawings
FIG. 1 is a flow chart of a sugar cane shoot tip chromosome flaking technique;
FIG. 2 is a photograph of a tissue sample of a meristem region of a sugarcane stem tip;
FIG. 3 is a microscopic view of the chromosomes of cells in the mitotic phase of sugarcane stem tips (magnification 400X, objective 40X, ocular 10X);
FIG. 4 is a microscopic photograph of the chromosomes of cells in the mitotic phase of sugarcane stem apex (1000 magnification, 100 objective, 10 eyepiece);
FIG. 5 is a photograph of a tissue sample of a meristematic region of a sugarcane root tip;
FIG. 6 is a chromosome effect observation picture of a meristem of a sugarcane root tip meristem;
FIG. 7 is a photograph of a tissue sample of a sprouting shoot of a sugarcane stem section;
FIG. 8 is a chromosome effect observation diagram of a sprouting tissue of a sugarcane stem segment;
FIG. 9 is a chromosome effect observation diagram of a 8-hydroxyquinoline solution treated at normal temperature for 4 hours;
FIG. 10 is a view showing the effect of chromosome staining at different staining times;
FIG. 11 is a graph showing the observation of the effect of chromosomes at different dissociation times;
FIG. 12 is a chromosome observation map of sugarcane parent and hybrid progeny germplasm materials;
FIG. 13 is an observation picture of chromosomes of sugarcane wild species and kindred plants
FIG. 14 is a photograph of meristematic cells of the shoot apical meristem region of sugarcane.
Detailed Description
The invention provides a high-efficiency sugarcane or sugarcane near-edge seed stem tip chromosome flaking method, which comprises the following steps:
1) collecting the shoot apical meristem of the vigorously growing sugarcane or sugarcane kindred species and cutting into 0.3-0.5 cm3In the form of small blocks, using precooled ddH2Soaking in O sterile water, and pretreating at 4 ℃ for 5-24 hours to obtain a pretreatment material;
2) fixing the pretreatment material under the fixing condition of 4 ℃ for 2-3 d by using ddH2After cleaning with O sterile water, dissociating for 20-25 min with a dissociation solution under the water bath condition of 50-65 ℃ to obtain a dissociation material;
the dissociation liquid is a mixed liquid formed by mixing 1mol/L HCl solution and 45% acetic acid aqueous solution with volume percentage concentration in an equal volume;
3) transferring the dissociation material onto a glass slide, removing surface moisture, dyeing with an improved phenol kappaucin dyeing solution, crushing the dissociation material, removing tissue residues, covering a cover glass, dyeing for 4-8 min, removing the dyeing solution around the cover glass, and tabletting to obtain a slide specimen with dispersed chromosomes.
The flow chart of the sugarcane or sugarcane kindred seed stem tip chromosome flaking method provided by the invention is shown in figure 1. The invention is obtained by the tabletting methodIn the slide specimen, the picture of the meristematic cells of the sugarcane stem tip is shown in figure 14, the meristematic tissue range is large, the division phase is concentrated, the proportion of mitotic cells is high, the picture of the chromosomes of the cells in the division phase required by research and analysis is easy to observe, and black arrows point to the cells in the division phase. The method collects the shoot apical meristem tissues of the vigorously growing sugarcane or sugarcane kindred species and cuts the shoot apical meristem tissues into 0.3-0.5 cm3In the form of small blocks, using precooled ddH2And (3) soaking in O sterile water, and pretreating for 5-24 h at 4 ℃, preferably for 12h to obtain the pretreatment material.
The method provided by the invention is suitable for all sugarcane varieties and plants with closer relativity to sugarcane. In order to show that the technical scheme of the invention can prepare and obtain the chromosome slide specimen with clear chromosome morphological structure, dispersed non-overlapping metaphase cells and clean background and no impurities, the embodiment of the invention uses the variety of Saccharum plant, namely, the variety of Geilala, 57NG208, Min sugar 70-611, RB72-454, F1Cliff 96-48 or BC2Examples of sugarcane varieties 07-86 are illustrated, but this should not be understood as applying only to the above mentioned varieties of sugarcane. Meanwhile, the method provided by the invention is also suitable for all the allied species of the sugarcane; the closely related species of sugarcane preferably include sugarcane wild species and sugarcane closely related plants; the present embodiment is described by taking as an example the case where the wild species of sugarcane has a dense cut hand and a large stem wild species and the closely related plants of sugarcane preferably include tall fescue, miscanthus, yunnan sugarcane, heyawang and the like, but this should not be construed as being limited to only the above-mentioned closely related plants of sugarcane.
In the invention, the shoot apical meristem comprises a sugarcane shoot apical basal meristem and/or 2-3 layers of shoot meristems wrapping the shoot apex, and the 2-3 layers of shoot apical meristems wrapping the shoot apex are shoot meristems within 3cm from the shoot apex. The meristem of the stem tip of the sugarcane which does not flower differentiates to form a meristem of a young leaf and a meristem of the base of the stem tip of the sugarcane, and the base of the meristem of the young leaf is connected with the base of the stem tip. The method selects the meristem at the base of the sugarcane stem tip and/or the meristem of 2-3 layers of young leaves wrapping the stem tip for flaking, has the advantages of large meristem range and concentrated cell division cycle, and is suitable for being used as a material for preparing chromosome disperse slides. In the present invention, the harvest period of the shoot apical meristem of a vigorously growing sugarcane or sugarcane kindred species is preferably included in the jointing stage, elongation stage or late elongation stage of sugarcane. In the invention, the collection time of the meristem of the shoot tip of the vigorously growing sugarcane or sugarcane kindred species is preferably 9: 00-11: 00 in the morning of a sunny day, and the time of the winter is delayed to 14: 00-16: 00 in the afternoon.
In the present invention, the volume of the small block is preferably 0.4cm3. Precooled ddH for sugarcane stem tip meristem tissue small block2And (3) soaking in O sterile water, and pretreating at 4 ℃ for 5-24 hours, so that the cells in the metaphase phase can be synchronously accumulated. Compared with the mitotic phase cells obtained by a conventional pretreatment scheme of treating 0.002mol/l 8-hydroxyquinoline solution for 4 hours at normal temperature, the mitotic phase cells have more cells and better effect; by adopting the pretreatment method, the chromosome number identification of 1 sugarcane germplasm material can be completed within 2 days, and compared with the condition that the germination of the root tip and the stem segment needs 1-2 weeks, the method shortens the experimental period and improves the efficiency of sugarcane chromosome genome analysis.
After the pretreatment material is obtained, the pretreatment material is fixed under the fixing condition of 4 ℃ for 2-3 d by ddH2After cleaning with O sterile water, dissociating for 20-25 min with a dissociation solution under the water bath condition of 50-65 ℃ to obtain a dissociation material; the dissociation liquid is a mixed liquid formed by mixing 1mol/L HCl solution and 45% acetic acid aqueous solution with volume percentage concentration in an equal volume. In the present invention, the fixing condition is preferably provided by a refrigerator.
In the present invention, the fixative solution for fixation is preferably carnot fixative solution; the Carnot stationary liquid is preferably absolute ethyl alcohol and glacial acetic acid according to the volume ratio of 3: 1, and mixing the components in a ratio of 1. After the fixation is finished, the section can be observed, or the sample is subjected to ddH2Rinsing with O sterile water twice, each time for 10min, storing in 70% ethanol, and storing at 4 deg.C for long term.
In the present invention, the washing is preferably performed twice to completely remove the fixing solution. The dissociation is preferably carried out in a water bath at 60 ℃; the time for dissociation is preferably 20 min. The dissociation helps to soften the immobilized material so that the cells are evenly spread on the slide during subsequent processing.
After obtaining the dissociation material, transferring the dissociation material onto a glass slide, removing surface moisture, dyeing with an improved phenol carbowax staining solution, crushing the dissociation material, removing tissue residues, covering the glass slide, dyeing for 4-8 min, removing the staining solution around the glass slide, and tabletting to obtain the slide specimen with dispersed chromosomes.
In the invention, the method for removing the surface moisture preferably adopts absorbent paper to absorb the surface moisture of the dissociation material, so as to eliminate the influence of the moisture on the dyeing effect. The modified phenol carbowax staining solution was purchased from Kunming cloud. The action is gentle when the dissociation material is crushed without air bubbles. The clamping of the fragmentation and dissociation material is more beneficial to obtaining the slide specimen with clear chromosome form and clean background than the direct beating without clamping of the fragmentation and dissociation material.
In the invention, the tabletting method is preferably that one end of a pencil with a rubber is used for knocking the cover glass to disperse the chromosomes, diffuse impurities and make the tabletting background clean, and then the cover glass is pressed by a thumb to make the cells evenly dispersed, wherein the chromosomes are positioned on the same plane.
In the present invention, microscopic examination is preferably performed in order to verify the effect of producing a slide specimen with dispersed chromosomes. The microscopic examination method preferably comprises the steps of observing by using a microscope, observing by using a low power lens (observing by using a 20X or 40X objective lens because the chromosome of the sugarcane is small), finding a proper visual field and a middle-stage mitotic phase Cell, transferring to a high power lens (100X objective lens), and photographing and counting the number by using olympus Cell Sens Standard software. Metaphase cells with clear chromosome morphological structure and good dispersion are selected, and photographing and karyotyping are carried out by using a Zeiss Metasystem automatic karyotyping system.
The invention provides a sugarcane chromosome dispersed slide specimen prepared by the sugarcane or related seed stem tip chromosome flaking method, the chromosome morphological structure is clear, non-overlapping division phase cells are dispersed, and the background is clean and has no impurities.
The invention provides application of the sugarcane or the slide specimen with chromosome dispersion of the allied species thereof in karyotype analysis or germplasm resource classification and protection utilization of the sugarcane or the allied species thereof.
The method for preparing sugarcane or its kindred shoot tip chromosome with high efficiency provided by the invention is explained in detail by the following examples, but the method is not to be construed as limiting the scope of the invention.
Example 1
Sugarcane stem tip chromosome flaking method
1. Sugarcane is taken as a test material, and the meristem of the sugarcane stem tip (a shape chart is shown in figure 2) in the field is collected and cut into 0.3cm in the morning (9: 00-11: 00) on sunny days3The small blocks are pretreated.
2. Pre-cooled ddH for pretreatment2Soaking in sterile water, pretreating at 4 deg.C for 12 hr, fixing with Carnot fixing solution (1 glacial acetic acid: 3 ethanol, prepared in situ) at 4 deg.C in refrigerator for 3 days, placing meristem in 70% ethanol, and storing at 4 deg.C for long term.
3. In the preparation of tablets, the stored meristematic tissue material is removed and treated with ddH2Rinsing with O sterile water for 10min for 2 times. Then, the mixture of 1mol/LHCl solution and 45 vol% acetic acid solution is dissolved in water bath at 60 ℃ for 20 min. By ddH2Rinsing with O sterile water for 1 time and 10 min. The shoot apical meristem was excised and placed on a clean glass slide, 20. mu.l of modified phenol Carbowax staining solution was added, the meristem was gently crushed with forceps, the tissue residue was removed, a cover slip was applied, and staining was performed at room temperature for 5 min.
4. After dyeing is finished, the tablet is lightly tapped by a pencil with an eraser, so that meristematic region tissues and cells of the sugarcane can be uniformly dispersed on the glass slide, chromosomes are dispersed, impurities are diffused, and the background of the tablet is clean. Finally, the cells were flattened by pressing with the thumb, and the chromosomes were on the same plane.
5. The observation was carried out using an optical microscope, and after finding a suitable visual field and cells in the mitotic phase, the cells were observed and photographed using a high power microscope (100 Xobjective).
Results
The tissue range of the sugarcane shoot apical meristem is large, the cell division cycle is concentrated, and an ideal division phase chromosome slide specimen is easily obtained, and the result is shown in a figure 3 and a figure 4. As can be seen from the chromosome distribution in FIGS. 3 and 4, the chromosome slide specimen prepared by the method has clear chromosome morphological structure, dispersed non-overlapping metaphase cells, and clean background without impurities. And the operation is simple and convenient, 30-50 chromosome slide specimens can be prepared in batches by a single stem tip, more metaphase cells can be observed on each slide, and more than 20 cells which are convenient for chromosome number identification and karyotype analysis can be observed in 10 slides generally.
Comparative example 1
1. Sugarcane is taken as a test material, and 0.8-1.0 cm of root tips (shown in a form chart of figure 5) of barrel-planted sugarcane are collected in the morning (9: 00-11: 00) of a fine day for pretreatment.
2. Pre-cooled ddH for pretreatment2Soaking in sterilized water, pretreating at 4 deg.C for 12 hr, fixing with Carnot fixing solution (1 glacial acetic acid: 3 ethanol, prepared in situ) at 4 deg.C in refrigerator for 3 days, placing root tip in 70% ethanol, and storing at 4 deg.C for long term.
3. During tabletting, the stored root tip material is taken out and ddH is used2Rinsing with O sterile water for 10min for 2 times. Then, the mixture of 1mol/L HCl solution and 45% acetic acid solution with volume concentration is dissolved in water bath at 60 ℃ for 20 min. By ddH2Rinsing with O sterile water for 1 time and 10 min. The meristem of the root tip was excised, 20. mu.l of modified phenol Carbowax staining solution was added, the meristem was gently crushed with forceps, the tissue residue was removed, and the tissue was covered with a cover slip and stained at room temperature for 5 min.
4. After dyeing is finished, the sheet is lightly tapped by a pencil with an eraser, so that root tip tissues and cells can be uniformly dispersed on the glass slide, chromosomes are dispersed, impurities are diffused, and the sheet making background is clean. Finally, the cells were flattened by pressing with the thumb, and the chromosomes were on the same plane.
5. The observation was carried out using an optical microscope, and after finding a suitable visual field and cells in the mitotic phase, the cells were observed and photographed using a high power microscope (100 Xobjective).
Results
The sugarcane needs about 6 months from planting to stably collecting the root tip, the plant is weak and small due to early collection, the plant is difficult to grow in the later period, and the tissue sample of the root tip is not good. When the root tip is collected, the sediment needs to be cleaned, and the operation is complicated; sugarcane roots are small, main roots are few, fibrous roots are many, a meristem area is small, and 1 to 2 root tips are generally used for preparing 1 slide specimen; the root tips of the sugarcane in the barrel culture, the sand culture or the water culture are greatly influenced by the nutritional environment factors, the root tips are different in thickness and tenderness, the division period is difficult to control, and only a few root tips in 1 batch of root tip samples are usually in the mitosis period; the method has the advantages that the method can not ensure that the metaphase cells can be observed in each slice, root tips are repeatedly collected and the slice is repeatedly observed, the sugarcane is re-planted into a barrel after the root tips are collected, new roots can grow out for collection after 3-4 weeks, and the workload is increased; the test and the experimental period are both long, and generally, the identification of the number of chromosomes of 1 material takes about 1-2 weeks. Through experimental analysis, the root tip needs to be prepared and observed for 50-100 slices to observe more than 20 cells for chromosome number identification and karyotype analysis, and a large amount of slice preparation time and microscopic observation time need to be consumed. Meanwhile, the root tip is taken as a material for sheet production and needs repeated sampling, so that the workload is greatly increased, and the working efficiency is reduced. FIG. 6 is an observation of the production of tablets using root tips.
Conclusion
Compared with the sugarcane root tip flaking technology, the sugarcane stem tip chromosome flaking technology has the characteristics of convenient sampling, concentrated splitting phases and easy obtaining of good metaphase splitting phases. The chromosome number identification of 1 material can be completed within 2 days, the workload of workers is reduced, and the efficiency is improved by more than 7-8 times compared with the root tip flaking efficiency.
Comparative example 2
Sugarcane is taken as a test material, stem segments are collected in a sand culture manner in the morning (9: 00-11: 00) of a sunny day, and sprouts are produced (a morphological picture is shown in figure 7, wherein A is a picture of the appearance of fresh sprouts taken off from the stem segments, and B is a picture of the tissue of dissociated sprout meristems according to the method in the embodiment 1.
Results
The results are shown in FIG. 8. The tissue range of the meristematic zone of the germinating bud of the sugarcane stem segment is small, and the splitting phase is not concentrated. The chromosome flaking effect is inferior to that of sugarcane stem tip and root tip.
Comparative example 3
Tableting was carried out by the method of example 1, with the only difference being the use of pre-cooled ddH2And (3) treating O at 4 ℃ for 5-24 h, and replacing 0.002mol/l 8-hydroxyquinoline solution to treat for 4h at normal temperature. The method treats the meristem of the stem tip of the sugarcane at the temperature of 4 ℃, is favorable for synchronously accumulating metaphase cells, can obtain metaphase cells with clear chromosome morphological structure and good dispersion in a short time through tabletting observation, and is convenient for observation. The result is better than that of the treatment for 4 hours at normal temperature by using 0.002mol/l 8-hydroxyquinoline solution.
The observation result of the chromosome effect of 4h of normal temperature treatment by using 0.002mol/l 8-hydroxyquinoline solution is shown in FIG. 9. The chromosome shape is not clear enough, the sticky phenomenon is easy to generate, the 8-hydroxyquinoline solution is easy to be influenced by environmental factors, and the high temperature has toxic action on cells and chromosomes.
Comparative example 4
In order to examine the influence of different dyeing times on the chromosome flaking effect, the method of the present invention was prepared by the method of example 1, but when room temperature dyeing was performed by using the modified phenol carbowax dyeing solution, dyeing was performed for 2min, 5min and 10min, respectively.
The results are shown in FIG. 10. When the slide is stained for 2min (A in FIG. 10), the color of the chromosome in the slide is light, which is inconvenient for microscopic observation, and when the slide is stained for 10min (C in FIG. 10), the color of the chromosome in the slide is too dark. While staining for 5min (B in FIG. 10), the chromosome color was moderate.
Comparative example 5
In order to examine the influence of different dissociation times on the chromosome flaking effect, the present invention was prepared by the method of example 1, but when dissociation was performed using a dissociation solution, the dissociation times were set to 10min, 15min, 20min, 25min, and 30 min.
The results are shown in FIG. 11. As can be seen from FIG. 11, dissociation for 10min and 15min results in less cell dispersion and more chromosome overlap; dissociation for 20min and 25min, cell is easy to disperse, chromosome is clear, and overlap is little; after 30min of dissociation, the cells are easy to break, and the chromosomes are lost or overlapped more. Therefore, the dissociation time is controlled to be 20-25 min, and the optimal preparation of the chromosome slide specimen is realized.
Example 2
Method for identifying chromosome number of sugarcane parent species and filial generation germplasm
1. The original sugarcane parent seed of the Tagladesh and 57NG208, the hybrid parent seed of the Min sugar of 70-611 and RB72-454 and the hybrid progeny germplasm F1Cliff 96-48 and BC2The sugarcane 07-86 is a test material, and is 9: 00-11: 00 collecting meristematic region tissues of the sugarcane stem tip for pretreatment.
2. Meristem tissue was placed in penicillin bottles with pre-cooled ddH for pretreatment2Soaking in sterile water, pretreating at 4 deg.C for 20 hr, fixing with instant Carnot fixing solution (glacial acetic acid: ethanol 1:3) in refrigerator at 4 deg.C for 2-3 days, placing meristem in 70% ethanol, and storing at 4 deg.C for a long time.
3. Taking out the stored meristematic tissue material and applying ddH2Rinsing with O sterile water for 10min for 2 times. Then, the mixture of 1mol/LHCl solution and 45% acetic acid solution with volume percentage concentration is used for dissociation for 22min in water bath at 60 ℃. By ddH2Rinsing with O sterile water for 1 time and 10 min. Cutting off the meristem tissue of the sugarcane stem tip, placing the meristem tissue on a clean glass slide, adding 20 mu.l of improved phenol carbopol fuchsin staining solution, slightly clipping the meristem tissue by using forceps, removing tissue residues, covering a cover glass, and staining for 5min at room temperature.
4. After dyeing is finished, the tablet is lightly tapped by a pencil with an eraser, so that meristematic region tissues and cells of the sugarcane are uniformly dispersed on the glass slide, chromosomes are dispersed, impurities are diffused, and the background of the tablet is clean. Finally, the cells were flattened by pressing with the thumb, and the chromosomes were on the same plane.
5. The observation is carried out by using an optical microscope, and the cells are observed by using a low power lens (the chromosomes of the sugarcane are very small, and 20X or 40X objective lenses are generally used), and then transferred to a high power lens (100X objective lenses) for observation and photographing after suitable visual fields and metaphase cells are found.
6. As a result, the invention can obtain good picture of the cells in the metaphase of the division phase, and the chromosome has clear morphological structure, good dispersion and convenient counting.
The chromosome number identification results of the 6 test materials in this example are shown by observation and statistics: both the number of chromosomes of the somatic cells of the geodesh and the 57NG208 are 2 n-80; the Fujian sugar 70-611 and the RB72-454 are respectively 2 n-105 and 2 n-112; filial generation germplasm material F1The number of cliff 96-48 chromosomes is 2 n-80; hybrid progeny germplasm material BC2The number of chromosomes of the sugarcane canes 07-86 is 2 n-114 (see fig. 12, the chromosomes of the sugarcane stem tips are complete, dispersed and not overlapped, the chromosome constriction is clear, and the method can be used for the number identification and karyotype analysis of the germplasm chromosomes of sugarcane parent species and filial generations).
Example 3
Identification of chromosome number of sugarcane wild species and allied plants
The sugarcane wild species are closely cut into hands and large-stem wild species, closely related plants such as the tall fescue, the mango, the Yunnan tall fescue and the heba king contain a plurality of new resistance genes which are not contained in the sugarcane, have the characteristics of strong ratoon and good clustering property, have greater value in the hybridization utilization and genetic improvement of the sugarcane, deeply research the chromosome ploidy and the cytogenetic behavior of the resources, and lay a foundation for the classification identification and mining utilization of the resources. The invention takes sugarcane wild species with dense cutting hands, big stem wild species and 3 sugarcane kindred plants as test materials, and the ratio of the sugarcane wild species to the big stem wild species is 9: 00-11: 00, from the national sugarcane germplasm resource garden, collecting meristematic region tissues of stem tips for pretreatment, and discussing whether the technical method is suitable for chromosome analysis of sugarcane wild species with dense hand cutting, big-stem wild species and kindred.
The results of pretreatment, fixation, dissociation staining, flaking and microscopic examination show that the invention is also suitable for flaking the stem tip chromosomes of sugarcane wild species with dense hand-clefts, large-stem wild species and kindred plants, the obtained chromosome has many metaphase cells, clear morphological structure and good dispersion, is convenient for the researches of number statistics, karyotype analysis and the like, and lays a foundation for the gene mining and chromosome genetic research of sugarcane wild species and kindred plants.
The identification results of the chromosome numbers of somatic cells of a sugarcane wild species such as a Vietnamese Henry (Vietnamese No. 3), a large-stem wild species (51NG3), and closely related plants such as a tall fescue (No. 1) and a mango (Jiangxi 2012-117) and a Yunnan tall fescue (Yunnan 2007-51) are respectively 2 n-80, 125, 60, 38 and 30 (see fig. 13, the chromosome of the stem tip of the sugarcane can be used for the classification and identification of sugarcane wild species and closely related plant resources).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. An efficient sugarcane or sugarcane near-edge seed stem tip chromosome flaking method is characterized by comprising the following steps:
1) collecting the shoot apical meristem of the vigorously growing sugarcane or sugarcane kindred species and cutting into 0.3-0.5 cm3In the form of small blocks, using precooled ddH2Soaking in O sterile water, and pretreating at 4 ℃ for 5-24 hours to obtain a pretreatment material;
2) fixing the pretreatment material under the fixing condition of 4 ℃ for 2-3 d by using ddH2After cleaning with O sterile water, dissociating for 20-25 min with a dissociation solution under the water bath condition of 50-65 ℃ to obtain a dissociation material;
the dissociation liquid is a mixed liquid formed by mixing 1mol/L HCl solution and 45% acetic acid aqueous solution with volume percentage concentration in an equal volume;
3) transferring the dissociation material onto a glass slide, removing surface moisture, dyeing with an improved phenol kappaucin dyeing solution, crushing the dissociation material, removing tissue residues, covering a cover glass, dyeing for 4-8 min, removing the dyeing solution around the cover glass, and tabletting to obtain a slide specimen with dispersed chromosomes.
2. The method for preparing sugarcane or sugarcane kindred seed stem tip chromosomes according to claim 1, wherein the stem tip meristem area tissue in the step 1) comprises a sugarcane stem tip basal meristem area tissue and/or 2-3 layers of young leaf meristematic area tissues wrapping the stem tip, and the 2-3 layers of young leaf meristematic area tissues wrapping the stem tip are young leaf meristematic area tissues within 3cm from the stem tip.
3. The method for chromosome production of the stem tip of sugarcane or sugarcane kindred species according to claim 1, wherein the harvesting period of the meristem of the stem tip of the vigorous-growing sugarcane or sugarcane kindred species in step 1) is included in the jointing stage, the elongation stage or the late elongation stage of sugarcane.
4. The sugarcane or sugarcane kindred stem tip chromosome slide-making method according to any one of claims 1 to 3, characterized in that the collection time of the meristematic region of the stem tip of the vigorous-growing sugarcane or sugarcane kindred is 9:00 to 11:00 in the morning of a sunny day in step 1).
5. The method for preparing sugarcane or sugarcane relative seed stem tip chromosomes according to claim 1, wherein the fixing stationary liquid in the step 2) is Carnot stationary liquid;
the Carnot stationary liquid is prepared from absolute ethyl alcohol and glacial acetic acid according to a volume ratio of 3: 1, and mixing the components in a ratio of 1.
6. The method for preparing sugarcane or sugarcane relative seed stem tip chromosomes according to claim 1, wherein the tabletting method in the step 3) is to knock an end of a pencil with a rubber on the cover glass to disperse the chromosomes, and then press the cover glass with a thumb to evenly disperse the cells, wherein the chromosomes are on the same plane.
7. The method for preparing sugarcane or sugarcane kindred seed stem tip chromosome according to any one of claims 1, 2, 3, 5 and 6, wherein the variety of sugarcane comprises Heilala, 57NG208, Min sugar 70-611, RB72-454,F1Cliff 96-48 or BC2And 07-86 parts of sugarcane.
8. The method for chromosome production of the stem tip of sugarcane or sugarcane kindred species according to any one of claims 1, 2, 3, 5 and 6, wherein the sugarcane kindred species comprises sugarcane wild species and sugarcane kindred plants;
the sugarcane wild species comprise cleft hand density and big stem wild species; the sugarcane kindred plants include tall fescue, mango, Yunnan tall fescue and Heyawang.
9. The sugarcane or sugarcane kindred species stem tip chromosome preparation method provided by any one of claims 1-8 is characterized in that chromosome morphological structures are clear, non-overlapping division phase cells are dispersed, and backgrounds are clean and free of impurities.
10. The use of the sugar cane or sugar cane relative species chromosome dispersion slide specimen of claim 9 in the chromosome karyotyping or germplasm resource classification and protection utilization of sugar cane or sugar cane relative species.
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| AU2020103466A AU2020103466A4 (en) | 2020-01-16 | 2020-11-16 | Efficient method for preparing chromosome from shoot tip of sugarcane or sugarcane related species |
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| 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 |
| CN112444436A (en) * | 2020-11-23 | 2021-03-05 | 浙江大学 | Pretreatment method for integral immunostaining of adult diaphragm |
| CN114938807A (en) * | 2022-05-06 | 2022-08-26 | 中国热带农业科学院海口实验站 | Method for preparing chromosome specimen of root tip meristematic region of passionfruit subgenus plant |
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| CN113188876B (en) * | 2021-04-29 | 2022-10-25 | 西北农林科技大学 | Preparation method of jujube root tip chromosome tablet |
| CN114062087A (en) * | 2021-11-07 | 2022-02-18 | 福建省热带作物科学研究所 | Method for preparing chromosome of melastoma plant based on FISH hybridization |
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| CN112444436A (en) * | 2020-11-23 | 2021-03-05 | 浙江大学 | Pretreatment method for integral immunostaining of adult diaphragm |
| CN114938807A (en) * | 2022-05-06 | 2022-08-26 | 中国热带农业科学院海口实验站 | Method for preparing chromosome specimen of root tip meristematic region of passionfruit subgenus plant |
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