CN112665941B - Detection method for chromosome number of cyperus esculentus - Google Patents
Detection method for chromosome number of cyperus esculentus Download PDFInfo
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Abstract
The application discloses a method for detecting the chromosome number of cyperus esculentus, which comprises the following steps: the method comprises the steps of rooting of cyperus esculentus seeds, sampling part treatment, laughing gas treatment, split phase fixation, enzymolysis, crushing, dripping, tabletting, dyeing and microscopic examination. According to the application, meristematic tissues with highest cell division index of the root tip of the cyperus esculentus are cut off by 8:50-10:20 a day, so that a good foundation is laid for the subsequent research of the karyotype of the cyperus esculentus; the chromosome number detection method can obtain clear cyperus esculentus chromosome number slices, so that the accurate number of the cyperus esculentus chromosomes can be accurately detected in a short time.
Description
Technical Field
The application belongs to the fields of plant cell biology and genetics, and particularly relates to a method for detecting the chromosome number of cyperus esculentus.
Background
Cyperus esculentus, latin name: cyperus esculentus L. Var. Satovus; english name: cyperus esculentus; generic name: tiger nuts (tigernut), also known as tigernut sedge (tigernut sedge), iron water chestnut shikim, underground walnuts, and the like. The stem and leaf of Cyperus plant of Cyperaceae of Cyperus can be grown on the ground, and the creeping stem in the ground can be swelled into bean for oil extraction or pulverizing. The cyperus esculentus is cold-resistant and drought-resistant, is suitable for being planted in marginal lands such as Sha Rang, has more than 500 kg per mu of land, contains more than 20% of oil, has about 75% of edible oil, is equivalent to high-oleic peanut oil, has oil quality comparable with olive oil, can be ground into powder, fed and the like, has high economic value, and is an ideal soybean substitute crop; the novel commercial crop which is only known at present and accumulates a large amount of grease in tubers and integrates grain, oil and feeding is quite wide in market prospect. The edible vegetable oil in China has huge consumption requirement, and the external dependence degree is 70%, wherein the external dependence degree of soybean is more than 90%. In order to ensure the safety of national edible vegetable oil supply, the scientific and technological department of China has listed 'Cyperus esculentus' as soybean substitute and compensation crops.
The chromosome is a vector of a gene, the preparation of a chromosome specimen is a basis of cytogenetics, and the preparation of a chromosome specimen is an excellent chromosome slide technology, and is a prerequisite for chromosome banding, group analysis, in situ hybridization and the like, and the observation and analysis of plant cell chromosomes are of great significance to chromosome behavior analysis in a chromosome karyotype analysis process, a meiosis process, analysis of chromosome behaviors and genetic phenomena such as cell mutation in a cell fusion culture process and the like. In addition, genetic engineering breeding can also be used for gene localization by chromosome banding, in situ hybridization and fluorescence in situ hybridization based on staining and tabletting.
The length of the cyperus esculentus chromosome is 0.5-1.2 mu m, and the cyperus esculentus chromosome belongs to a small chromosome type, and because the cyperus esculentus chromosome has a large number and belongs to a small chromosome, the tabletting difficulty is high, so that a material with a high cell division index needs to be searched for chromosome counting. The root tip is the most commonly used material for chromosome counting, particularly the seed root tip cells are the most reliable material for researching the chromosome, and the research of the optimal period and the treatment condition of the division of the root tip cells of the cyperus esculentus is the basis and the key for researching the chromosome number of the cyperus esculentus. At present, the method for detecting the chromosome number of the cyperus esculentus still belongs to a blank, so that a rapid detection method for the chromosome number of the cyperus esculentus is urgently needed.
Disclosure of Invention
In order to solve the defects in the prior art, the application aims to provide a method for detecting the chromosome number of the cyperus esculentus.
In order to achieve the above purpose, the present application adopts the following technical scheme:
a method for detecting the chromosome number of cyperus esculentus comprises the following steps:
(1) Root development of cyperus esculentus seeds: selecting 7-10 full-grain cyperus esculentus sample stem beans, treating, and sampling and tabletting when white water absorbing roots are generated at the base parts of stem bean sprouts;
(2) Sampling part and processing: removing the top root crown when the root length of the sample in the step (1) reaches 2-3mm, cutting off root tip meristematic tissue at the peak of root tip cell division, and then placing the removed root tip meristematic tissue in an ice-water mixture for 24 hours;
(3) Laughing gas treatment: taking out the root tip meristem treated in the step (2), placing the root tip meristem into a prepared centrifuge tube with a pricked top end of 0.5ml, wetting the centrifuge tube with water before an experiment, placing the centrifuge tube with the root tip meristem into a laughing gas tank, and standing for 3-5h;
(4) And fixing a split phase: immediately fixing a splitting phase under ice water bath condition after laughing gas treatment is finished, and cleaning root tip meristems in a centrifuge tube for later use after the splitting phase is fixed;
(5) Enzymolysis: cutting off the white root tip part of the root tip meristem with the length smaller than 2mm and treated in the step (4) by a blade, and placing the white root tip part into a centrifuge tube filled with 40 mu l of enzyme solution for enzymolysis;
(6) Crushing: after enzymolysis, washing the root tip subjected to enzymolysis for 3 times by using 70% alcohol by volume fraction, then fully crushing and vibrating the washed root tip in residual alcohol by using a dissecting needle, centrifuging cells to the bottom of a tube, and removing the residual alcohol from a centrifuge tube by inversion and airing; observing the material in the crushing process to prevent uneven crushing;
(7) Dropping tablets: treating the root tip meristem cells subjected to centrifugation in the step (6) to prepare a cell suspension, placing a clean glass slide in a pre-prepared wet box at the room temperature of about 27 ℃, taking out 15 mu L of the cell suspension from the centrifugal tube, dripping the cell suspension over a certain distance from the glass slide, immediately covering the box until the cell solution is dispersed, airing the glass slide, and taking out the glass slide for later use;
(8) And (3) tabletting: performing microscopic observation on the glass slide dried in the step (7) under an optical microscope or a phase contrast microscope to find out a cell area with good mitotic metaphase, and then performing chromosome flaking, preservation and standby;
(9) Dyeing: naturally air-drying the chromosome flaking in the step (8), dropwise adding fluorescent dye DAPI, adding a cover glass, and sealing the periphery of the cover glass with nail polish for later use;
(10) And (5) microscopic examination: the number of chromosomes and the karyotype of the chromosome slides stained in step (9) were observed with a fluorescence microscope.
Further, the specific method for treating the cyperus esculentus sample in the step (1) is as follows: airing the selected cyperus esculentus sample for 1-2 days, fully soaking the cyperus esculentus sample for 24 hours with warm water at 30-35 ℃, changing water every 6 hours, then placing the cyperus esculentus sample in a sterilized glass culture dish, paving double-layer filter paper at the bottom of the glass culture dish, keeping the filter paper moist, and placing the filter paper in an incubator with the relative humidity of 70% and the temperature of 25 ℃ for 48-72 hours in a shading manner, wherein the white cyperus esculentus stem beans are observed to begin to be illuminated for 8 hours every day.
Further, the time for shearing the root tip meristem in the step (2) is 8:50-10:20 a.m.
Further, the pressure in the laughing gas tank in the step (3) is 0.9-1.0MPa.
Further, the method for fixing the split phases in the step (4) comprises the following steps: adding glacial acetic acid with the volume fraction of 90% after the pre-cooling treatment at the temperature of 4 ℃ into the centrifuge tube in the step (3), and standing for 30-40min; the cleaning method after the fixation of the split phase is completed comprises the following steps: sucking glacial acetic acid from the centrifuge tube by using a rubber head dropper, and washing root tip meristems in the centrifuge tube twice by using double distilled water; if the root tip is to be preserved, 75% ethanol is added after washing, and double distilled water ddH2O is used for washing twice before preservation and use at-40 ℃.
Further, the enzymolysis liquid in the step (5) is a mixture of cellulase and pectase according to a mass ratio of 3:1, and the enzymolysis is carried out for 1h under the water bath condition of 37 ℃.
Further, the centrifugal rotating speed in the step (6) is 5000r/min, and the centrifugal rotating speed is 3min.
Further, the preparation method of the cell suspension of the root tip meristem in the step (7) comprises the following steps: adding 30-50 mu L of glacial acetic acid into the centrifugal tube filled with the cells of the root tip meristem in the step (6) according to the number of root tips, centrifuging, dissolving for 10-12min, and then swirling for 2-3 times to uniformly mix to obtain the cell suspension.
Further, the cyperus esculentus is classified into a round cyperus esculentus and a long cyperus esculentus.
Compared with the prior art, the application has the following advantages: (1) By the detection method, the highest time of the cell division index of the root tip of the cyperus esculentus is found to be 8:50-10:20 a day, and a good foundation is laid for the subsequent study of the chromosome number of the cyperus esculentus; (2) The chromosome number detection method can obtain clear cyperus esculentus chromosome flaking, so that the number of the cyperus esculentus chromosomes can be accurately detected in a short time.
Drawings
FIG. 1 is a graph of field growth, seed harvest and chromosome flaking of round-grained cyperus esculentus.
FIG. 2 is a plot of field growth, seed harvest and chromosome flaking of long grain cyperus esculentus.
FIG. 3 is a graph of chromosome tabletting of a non-ideal isolated Cyperus esculentus.
FIG. 4 is a graph of a background-interfering cyperus esculentus chromosome slide.
Detailed Description
The present application will be described in further detail with reference to examples, but it should be understood that the scope of the present application is not limited to the scope of these examples.
Examples
A method for detecting the chromosome number of cyperus esculentus comprises the following steps:
(1) Root development of cyperus esculentus seeds: selecting full-grain type cyperus esculentus sample stem beans and long-grain type cyperus esculentus sample stem beans, wherein 7-10 grains of the two grain type cyperus esculentus stem beans are respectively obtained, airing the selected two grain type cyperus esculentus sample stem beans for 1-2 days, fully soaking the selected two grain type cyperus esculentus sample stem beans in warm water at 30-35 ℃ for 24 hours, changing water every 6 hours, then placing the selected two grain type cyperus esculentus sample stem beans in a sterilized glass culture dish, paving double-layer filter paper at the bottom of the glass culture dish, keeping the filter paper moist, placing the filter paper in an incubator with relative humidity of 70% and temperature of 25 ℃ for 48-72 hours, observing that the cyperus esculentus stem beans begin to light for 8 hours every day, and sampling and tabletting when white water absorbing roots are generated at the base of stem bean sprouts;
(2) Sampling part and processing: removing the top root crown when the root length of the sample in the step (1) reaches 2-3mm, shearing root tip meristematic tissue in the period of 8:50-10:20 of the morning at the peak of cell division of the root tip, and then placing the removed root tip meristematic tissue in an ice-water mixture for 24 hours;
(3) Laughing gas treatment: taking out the root tip meristem treated in the step (2), putting the root tip meristem into a pre-prepared centrifuge tube with a pricked top end of 0.5ml, wetting the centrifuge tube with water before an experiment, putting the centrifuge tube with the root tip meristem into a laughing gas tank with the pressure of 0.9-1.0MP, and standing for 3-5h;
(4) And fixing a split phase: adding glacial acetic acid with the volume fraction of 90% after the laughing gas treatment and the precooling treatment at 4 ℃ into the centrifuge tube in the step (3), standing for 30-40min to finish the fixation of split phase, sucking the glacial acetic acid out of the centrifuge tube by using a rubber head dropper, and using double distilled water ddH 2 O, cleaning root tip meristems in the centrifuge tube twice for standby; if preservation of root tip is desired, 75% ethanol is added after cleaning, and double distilled water ddH is used before preservation at-40deg.C 2 O is washed twice;
(5) Enzymolysis: cutting off the root tip white part of the root tip meristem with the length less than 2mm and treated in the step (4) by a blade, putting the root tip white part into a centrifuge tube filled with 40 mu l of enzyme liquid, wherein the enzyme liquid is a mixture of cellulase and pectase according to the mass ratio of 3:1, and carrying out enzymolysis for 1h under the water bath condition of 37 ℃;
(6) Crushing: after enzymolysis, washing the root tip subjected to enzymolysis for 3 times by using 70% alcohol by volume fraction, then fully crushing and vibrating the washed root tip in the residual alcohol by using a dissecting needle, centrifuging for 3min at the rotating speed of 5000r/min, centrifuging cells to the bottom of a tube, and removing the residual alcohol from the centrifuge tube by inversion and airing; observing the form of the material in the crushing process to prevent uneven crushing;
(7) Dropping tablets: according to the quantity of root tips, adding 30-50 mu L of glacial acetic acid into the centrifuge tube filled with the cells of the root tip meristem in the step (6) for centrifugation, dissolving for 10-12min, then swirling for 2-3 times for uniform mixing to obtain the cell suspension, placing a clean glass slide into a pre-prepared moist box at the room temperature at about 27 ℃, taking out 15 mu L of the cell suspension from the centrifuge tube, dripping the cell suspension over a certain distance from the glass slide, immediately covering the box until the cell solution is dispersed, airing the glass slide, and taking out the glass slide for later use;
(8) And (3) tabletting: performing microscopic observation on the glass slide dried in the step (7) under an optical microscope or a phase contrast microscope to find out a cell area with good mitotic metaphase, and then performing chromosome flaking, preservation and standby;
(9) Dyeing: naturally air-drying the chromosome flaking in the step (8), dropwise adding fluorescent dye DAPI, adding a cover glass, and sealing the periphery of the cover glass with nail polish for later use;
(10) And (5) microscopic examination: the chromosome number and the karyotype of the chromosome flaking obtained in the step (9) are observed by a fluorescence microscope, the two granular chromosome flaking conditions of the cyperus esculentus observed under the fluorescence microscope are respectively shown in fig. 1 (round granule type) and fig. 2 (long granule type), the chromosomes of the two granular cells are well dispersed and easy to count, the chromosome number of the cyperus esculentus according to the two granular types obtained by observation is 156, and the chromosome length is 0.5-1.2 mu m, and the chromosome is the centromere chromosome near the middle part and the end part.
Comparative example
A method for detecting the chromosome number of cyperus esculentus comprises the following steps:
(1) Drawing materials: obtaining meristematic cells of the root tip of the cyperus esculentus, and shearing the meristematic cells of the root tip during the period of 3-4 pm;
(2) Pretreatment: putting the root tip meristematic cells obtained in the step (1) into an ice-water mixture for pretreatment for 22-24h;
(3) Fixing: immersing the pretreated meristematic cells of the root tips of the cyperus esculentus in glacial acetic acid for 30-40min to finish fixation;
(4) Dissociation: double steaming ddH for root tip meristem after fixation treatment 2 O is cleaned, immersed into a mixed dissociation solution of cellulase and pectase according to the mass ratio of 3:1 for dissociation for 10-20min at room temperature;
(5) Dyeing: crushing the dissociated root tip meristem, placing the crushed root tip meristem on a glass slide, dripping 3 drops of fluorescent dye DAPI, and dyeing for 5-10min;
(6) Covering a cover slip on the dyed meristem of the root tip of the cyperus esculentus, and covering water-absorbing paper until cells are dispersed;
(7) And (5) microscopic examination: four sides of the cover slip were mounted with nail polish, the number of chromosomes of the stained chromosome slides was observed with a fluorescence microscope, and the observed chromosome slides of the cyperus esculentus under the fluorescence microscope are shown in fig. 3 and 4, respectively.
Conclusion: compared with the comparative example, the chromosome in the chromosome slide of the application is observed under a fluorescence microscope to be uniformly dispersed, the slide background is clear, no interference is caused, and the number of the cyperus esculentus chromosomes is easy to observe; by the detection method of the number of the cyperus esculentus chromosomes in the comparative example, the non-uniform chromosome dispersion state in the chromosome slide is observed under a fluorescence microscope, the slide background is interfered and fuzzy, and the number of the cyperus esculentus chromosomes is difficult to observe. Therefore, according to the application, root tip meristems are cut for the cyperus esculentus seeds at a specific time (8:50-10:20 a.m.) and laughing gas treatment is performed, so that the root tip meristem cells with the highest cell division index are obtained, the preparation of clear cyperus esculentus chromosomes obtained in a later stage is facilitated, and the number of the cyperus esculentus chromosomes is more easily observed.
While the above-described embodiments of the present application have been described in detail, it should be understood that the above-described embodiments are illustrative and not to be construed as limiting the application, and that variations, modifications, alternatives, and variations may be made to the above-described embodiments by those skilled in the art within the scope of the application, which is also defined by the appended claims.
Claims (9)
1. The method for detecting the chromosome number of the cyperus esculentus is characterized by comprising the following steps of:
(1) Root development of cyperus esculentus seeds: selecting 7-10 full-grain cyperus esculentus sample stem beans, treating, and sampling and tabletting when white water absorbing roots are generated at the base parts of stem bean sprouts;
(2) Sampling part and processing: removing the top root crown when the root length of the sample in the step (1) reaches 2-3mm, cutting off root tip meristematic tissue at the peak of root tip cell division, and then placing the removed root tip meristematic tissue in an ice-water mixture for 24 hours;
(3) Laughing gas treatment: taking out the root tip meristem treated in the step (2), placing the root tip meristem into a prepared centrifuge tube with a pricked top end of 0.5ml, wetting the centrifuge tube with water before an experiment, placing the centrifuge tube with the root tip meristem into a laughing gas tank, and standing for 3-5h;
(4) And fixing a split phase: immediately fixing a splitting phase under ice water bath condition after laughing gas treatment is finished, and cleaning root tip meristems in a centrifuge tube for later use after the splitting phase is fixed;
(5) Enzymolysis: cutting off the white root tip part of the root tip meristem with the length smaller than 2mm and treated in the step (4) by a blade, and placing the white root tip part into a centrifuge tube filled with 40 mu l of enzyme solution for enzymolysis;
(6) Crushing: after enzymolysis, washing the root tip subjected to enzymolysis for 3 times by using 70% alcohol by volume fraction, then fully crushing and vibrating the washed root tip in residual alcohol by using a dissecting needle, centrifuging cells to the bottom of a tube, and removing the residual alcohol from a centrifuge tube by inversion and airing;
(7) Dropping tablets: treating the root tip meristem cells subjected to centrifugation in the step (6) to prepare a cell suspension, placing a clean glass slide in a pre-prepared wet box at the room temperature of about 27 ℃, taking out 15 mu L of the cell suspension from the centrifugal tube, dripping the cell suspension over a certain distance from the glass slide, immediately covering the box until the cell solution is dispersed, airing the glass slide, and taking out the glass slide for later use;
(8) And (3) tabletting: performing microscopic observation on the glass slide dried in the step (7) under an optical microscope or a phase contrast microscope to find out a cell area with good mitotic metaphase, and then performing chromosome flaking, preservation and standby;
(9) Dyeing: naturally air-drying the chromosome flaking in the step (8), dropwise adding fluorescent dye DAPI, adding a cover glass, and sealing the periphery of the cover glass with nail polish for later use;
(10) And (5) microscopic examination: the number of chromosomes and the karyotype of the chromosome slides stained in step (9) were observed with a fluorescence microscope.
2. The method for detecting the chromosome number of the cyperus esculentus according to claim 1, wherein the specific method for treating the cyperus esculentus sample in the step (1) is as follows: airing the selected cyperus esculentus sample for 1-2 days, fully soaking the cyperus esculentus sample for 24 hours with warm water at 30-35 ℃, changing water every 6 hours, then placing the cyperus esculentus sample in a sterilized glass culture dish, paving double-layer filter paper at the bottom of the glass culture dish, keeping the filter paper moist, and placing the filter paper in an incubator with the relative humidity of 70% and the temperature of 25 ℃ for 48-72 hours in a shading manner, wherein the white cyperus esculentus stem beans are observed to begin to be illuminated for 8 hours every day.
3. The method for detecting the chromosome number of the cyperus esculentus according to claim 1, wherein the time for shearing the meristem of the root tip in the step (2) is 8:50-10:20 a.m.
4. The method for detecting the chromosome number of the cyperus esculentus according to claim 1, wherein the pressure in the laughing gas tank in the step (3) is 0.9-1.0MPa.
5. The method for detecting the chromosome number of the cyperus esculentus according to claim 1, wherein the method for fixing the split phase in the step (4) is as follows: adding glacial acetic acid with the volume fraction of 90% after the pre-cooling treatment at the temperature of 4 ℃ into the centrifuge tube in the step (3), and standing for 30-40min; the cleaning method after the fixation of the split phase is completed comprises the following steps: sucking glacial acetic acid out of the centrifuge tube by using a rubber head dropper, and washing root tip meristems in the centrifuge tube twice by using double distilled water.
6. The method for detecting the chromosome number of the cyperus esculentus according to claim 1, wherein the enzymolysis liquid in the step (5) is a mixture of cellulase and pectase according to a mass ratio of 3:1, and the enzymolysis is performed for 1h under the water bath condition of 37 ℃.
7. The method for detecting the chromosome number of the cyperus esculentus according to claim 1, wherein the centrifugal rotation speed in the step (6) is 5000r/min, and the centrifugation is carried out for 3min.
8. The method for detecting the chromosome number of the cyperus esculentus according to claim 1, wherein the preparation method of the cell suspension of the root tip meristem in the step (7) comprises the following steps: adding 30-50 mu L of glacial acetic acid into the centrifugal tube filled with the cells of the root tip meristem in the step (6) according to the number of root tips, centrifuging, dissolving for 10-12min, and then swirling for 2-3 times to uniformly mix to obtain the cell suspension.
9. A method for detecting chromosome number of cyperus esculentus according to any one of claims 1 to 8, wherein said cyperus esculentus is classified into cyperus esculentus and cyperus longifolia.
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| AU2020100210A4 (en) * | 2020-02-13 | 2020-03-19 | Heilongjiang University | Slide Preparing Method Applied for Chromosome Identification of Distant Hybrid Beet |
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