Setaria italica root tip chromosome slide preparation method

Abstract

The invention discloses a foxtail millet root tip chromosome slide preparation method which mainly comprises the following steps: after seeds germinate, taking young roots of the seeds, pretreating the young roots with Amiprophos-methyl (APM), treating the young roots with N2O, fixing the young roots with pre-cooled 90% glacial acetic acid (4 DEG C), performing an enzymolysis drop method on the treated root tip growing points, and finally performing microscopic examination under a phase contrast microscope. The chromosome slide preparation method is convenient and simple to operate, a large number of mitosis metaphase can be obtained, chromosome dispersion is good, the background is shallow, counting is easy, and the mitosis phase is not prone to being lost.

Description

Technical Field

[0001] The invention relates to the field of plant cell genetics, and in particular to a method for preparing chromosomes of millet root tips. Background Art

[0002] The preparation of millet chromosome preparation is the basis for conducting millet cytogenetics, millet molecular cytogenetics and other related research based on chromosomes. In the preparation process of millet chromosome preparation, pretreatment is a very important link. Reasonable pretreatment can obtain a large number of mitotic phases and metaphase chromosomes with good morphology. The mechanism of pretreatment is to inhibit the assembly of microtubules into spindle fibers, but it does not inhibit the cell division in the early stage. Therefore, all cells entering the metaphase are blocked and cannot enter the late stage, accumulating a large number of metaphase mitotic phases; it can induce the chromosomes to further shorten and straighten, which is convenient for chromosome dispersion and makes the chromosome morphology clearer. The commonly used methods in chromosome preparation are physical methods and chemical methods. The physical method is to pretreat the plant material with low temperature, the treatment temperature is generally 0-8℃, or to pretreat the material directly with an ice-water mixture. This method has no destructive effect on chromosomes, and the chromosomes are shortened evenly. It is simple and easy to operate, and is applicable to all kinds of crops, but not all metaphases can be obtained at any time. The commonly used reagents for chemical pretreatment are: colchicine, p-dichlorobenzene and 8-hydroxyquinoline. Colchicine is commonly used in aqueous solution with a concentration of 0.05-0.2%, which is suitable for large, medium and small chromosomes, especially for chromosome counting, making chromosomes easier to disperse, but showing constrictions is poor, and colchicine is highly toxic. The effect of pretreatment with p-dichlorobenzene is equivalent to that of colchicine, and it is also highly toxic. 8-Hydroxyquinoline is suitable for medium and small chromosomes and is good for in vitro treatment. The advantage is that it shows clear constrictions, but the disadvantage is that the metaphase is not as many as others. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a method for preparing chromosomes of millet root tips in view of the deficiencies in the above-mentioned prior art.

[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for preparing chromosomes from millet root tips, comprising the following steps: S1, seed rooting; S2, pretreatment: pretreatment by immersing the roots in APM solution; S3, laughing gas treatment; S4. Fixation: fix the roots after nitrous oxide treatment with glacial acetic acid; Add 90% glacial acetic acid precooled at 4°C and fix for 7 minutes; S5. Preparation: Take out the roots, rinse with distilled water, cut off the root cap, and heat the root tip meristem hydrolyzate in a water bath; after the hydrolysis, add TE buffer to terminate the reaction; dry the TE buffer, add alcohol to wash, and finally centrifuge to dry the alcohol; add the suspension, stir with a dissecting needle to suspend the cells; mix the suspension by pipetting, then pipette the suspension, drop it in mid-air on a glass slide, and wait until the suspension on the slide is completely dry to complete the preparation of millet root tip chromosomes.

[0005] Preferably, step S1 specifically comprises: Place the dry seeds in a culture dish with filter paper to retain water. Add water to the dish to cover the seeds and germinate at 28°C. Pour out excess water after the seeds turn white. Treat at 4°C for 24 hours and then grow at 28°C away from light.

[0006] Preferably, step S2 specifically comprises: When the root length is 1-1.3 cm, add 20 μmol / L APM solution to the culture dish until the roots are submerged, and place it in a 28°C incubator for growth pretreatment for 2.5 hours.

[0007] Preferably, the preparation method of the 20 μmol / L APM solution in step S2 is: adding 100 μL of 0.2 mol / L APM mother solution to 1000 mL of distilled water to prepare a 20 μmol / L APM solution; the preparation method of the 0.2 mol / L APM mother solution is: dissolving 60.86 mg APM in 10 mL of -20°C precooled acetone to obtain a 0.2 mol / L APM mother solution, which is stored at -20°C.

[0008] Preferably, step S3 specifically comprises: The pretreated root tips were washed in a culture dish filled with distilled water, the roots were cut with scissors, and treated with nitrous oxide at a pressure of 0.5-2 MPa for 15-60 minutes at room temperature.

[0009] Preferably, step S3 specifically comprises: The pretreated root tips were washed twice in a culture dish filled with distilled water for 4-5 minutes each time, the roots were cut with scissors, and the roots were placed in a 2 mL centrifuge tube with holes. An appropriate amount of deionized water was added to the centrifuge tube in advance to keep it moist. At room temperature, the roots were treated with nitrous oxide at a pressure of 1 MPa for 0.5 hours.

[0010] Preferably, step S4 is specifically: After being treated with nitrous oxide, the roots were fixed with 90% glacial acetic acid precooled at 4°C for 7 minutes.

[0011] Preferably, step S5 is specifically: Take out the root, rinse it with distilled water for 2-3 times, remove excess water with absorbent paper, cut off the root cap, put it in a centrifuge tube containing 20μL root apical meristem enzymatic solution, and enzymolyze it in a water bath at 37℃ for 50-60min; after the enzymolysis is completed, add 200μL 1×TE buffer to terminate the reaction; use a pipette to suck up the TE buffer, add 70% alcohol by mass to wash twice, and finally centrifuge to completely dry the alcohol, add 30μL of the freshly prepared suspension, stir with a dissecting needle to suspend the cells; place a clean glass slide in a moist foam box, use a pipette to mix the suspension, then suck up 3μL of the suspension, drop it on the glass slide in mid-air, and wait until the suspension on the glass slide is completely dry to complete the preparation of millet root tip chromosomes; The suspension is obtained by mixing acetic acid and methanol in a volume ratio of 5:1.

[0012] Preferably, the preparation method of 1×TE buffer in step S5 is: Add 100 mL of 10× TE buffer to 1000 mL of distilled water to prepare 1× TE buffer; The preparation method of 10×TE buffer is as follows: add 100 mL 1M Tris-HCl and 20 mL 0.5M EDTA Na2 to distilled water to make up to 1000 mL, adjust the pH to 8.0, filter with a 0.45 μm filter membrane, sterilize, and store at 4°C; The preparation method of 0.5M EDTA Na2 is as follows: 186.1g EDTA Na2 and 16g NaOH are added to ddH2O to dissolve, and then the volume is adjusted to 1000mL with ddH2O, mixed, the pH is adjusted to 8, filtered with a 0.45μm filter membrane, sterilized, and 0.5M EDTA Na2 is obtained, and stored at 4°C; The preparation method of 1M Tris-HCl is as follows: 121.1g Tris-Base and 49mL concentrated HCl with a mass concentration of 37% are added to ddH2O to dissolve, and then the volume is adjusted to 1000mL with ddH2O, mixed, the pH is adjusted to 7.5, filtered with a 0.45μm filter membrane, sterilized, and 1M Tris-HCl is obtained, and stored at 4°C; The preparation method of the root apical meristem hydrolysate is as follows: 0.4 g of cellulase, 0.1 g of pectinase and 9.5 g of KCL buffer are mixed, completely dissolved on ice, and then dispensed into sterilized 0.5 ml centrifuge tubes, 20 μl per tube, and stored at -20°C.

[0013] Preferably, the method for preparing millet root tip chromosomes comprises the following steps: S1. Seed rooting: dry seeds were placed in a culture dish with filter paper to retain water. Water was added to the culture dish to cover the seeds and the seeds were germinated at 28°C. After the seeds turned white, excess water in the culture dish was poured out, leaving only the filter paper moist. The seeds were moved to a 4°C refrigerator for 24 hours, and then the culture dish was moved to a 28°C incubator to grow in the dark. S2. Pretreatment: When the roots are 1-1.3 cm long, add 20 μmol / L APM solution to the culture dish until the roots are submerged, and place in a 28°C incubator for 2.5 hours of growth pretreatment; S3, nitrous oxide treatment: the pretreated root tips were washed twice in a culture dish filled with distilled water for 5 minutes each time, the roots were cut with scissors, and the roots were placed in a 2 ml centrifuge tube with holes, and an appropriate amount of deionized water was added to the centrifuge tube in advance to keep it moist. At room temperature, the roots were treated with nitrous oxide at a pressure of 1 MPa for 0.5 hours; S4, fixation: place the 2 ml centrifuge tube containing the roots after being treated with nitrous oxide on crushed ice, add 90 wt% glacial acetic acid precooled at 4°C into the centrifuge tube, and fix for 7 minutes; S5, preparation: take out the root, rinse it with distilled water for 2-3 times, remove excess water with absorbent paper, cut off the root crown, put it in a centrifuge tube containing 20μL enzyme solution, and perform enzymolysis in a water bath at 37℃ for 50-60min; after the enzymolysis, add 200μL 1×TE buffer to terminate the reaction; use a pipette to dry the TE buffer, add 70% alcohol to wash twice, and finally centrifuge to completely dry the alcohol, add 30μL of the freshly prepared suspension, stir with a dissecting needle to suspend the cells; place a clean glass slide in a moist foam box, use a pipette to mix the suspension, then aspirate 3μL of the suspension, drop it in mid-air on the glass slide, wait for the suspension on the glass slide to dry completely, and the preparation of millet root tip chromosomes is completed; The suspension is obtained by mixing acetic acid and methanol in a volume ratio of 5:1.

[0014] The slides prepared according to the above method are placed under a phase contrast microscope for observation, and cells with good chromosome morphology, no overlap or little overlap, and clear appearance are selected and marked with material names. They can be directly photographed for karyotype analysis, or used for chromosome banding or fluorescence in situ hybridization experiments.

[0015] The beneficial effects of the present invention are: The invention provides a method for preparing chromosome slices of millet root tips, the main steps of which are: after the seeds germinate, taking the young roots, pre-treating them with amiprophos-methyl (APM), treating them with nitrous oxide (N2O), fixing them with pre-cooled 90% glacial acetic acid (4°C), performing an enzymatic hydrolysis drop-slice method on the treated root tip growth points, and finally inspecting them under a phase contrast microscope; The present invention uses amiprophos-methyl (APM) in the pretreatment stage to replace traditional colchicine, p-dichlorobenzene and 8-hydroxyquinoline; APM is a specific drug that directly interferes with plant microtubule synthesis, and its effect is similar to colchicine, and it plays a role in collecting metaphase chromosomes. Different APM treatment times have a certain effect on the metaphase index of mitosis; APM has a high affinity for microtubule protein, and at a lower concentration, it has a stronger depolymerization ability for microtubule protein, a higher doubling frequency, and a smaller toxic effect on plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a fluorescence microscopic image of the sample sheet prepared in Example 1; Figure 2 This is a fluorescent microscopic image of the sample piece prepared in Example 1 after staining; Figure 3 is a fluorescence microscopic image of the sample sheet prepared in Example 2; Figure 4 This is a fluorescent microscopic image of the sample piece prepared in Example 2 after staining. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0018] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0019] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0020] Example 1 Chromosome preparation of Yugu No. 1 (2n=2x=18) S1. Seed rooting: Place about 50 seeds of Yugu No. 1 millet in a petri dish lined with moist filter paper, add enough distilled water to fully soak the seeds in distilled water, and place in a 28℃ constant temperature box for germination. After the seeds turn white after 24 hours, pour out the excess water in the petri dish, keep only the filter paper moist, move to a 4℃ refrigerator for 24 hours, and then move the petri dish to a 28℃ incubator for growth away from light.

[0021] S2. Pretreatment: When the millet seed roots are about 1-1.3 cm long, add 20 μmol / L APM solution to the culture dish until the roots are submerged, and place in a 30°C incubator for growth pretreatment for 2.5 hours.

[0022] S3. Nitrous oxide treatment: wash the pretreated root tips in a culture dish filled with distilled water twice for 5 minutes each time, and cut the young roots with scissors; put the roots into a 2.0 ml centrifuge tube with holes, and add an appropriate amount of deionized water to the centrifuge tube in advance to keep it moist; treat with nitrous oxide at a pressure of 1 MPa for 0.5 hours at room temperature.

[0023] S4. Fixation: Place the 2.0 ml centrifuge tube containing the roots after being treated with nitrous oxide on crushed ice, add pre-cooled 90 wt % glacial acetic acid (4°C) into the centrifuge tube, and fix for 7 minutes.

[0024] S5. Preparation: Take out the roots, rinse them with distilled water for 3 times, remove excess water with absorbent paper, cut off the root cap, and place it in a centrifuge tube containing 20μL root apical meristem enzymatic solution; enzymolysis in a 37℃ water bath for 50-60min; after the enzymolysis, quickly add 200μl 1×TE buffer to terminate the reaction; use a pipette to dry the TE buffer, add 70% alcohol to wash twice, and finally centrifuge to thoroughly dry the alcohol; dry the alcohol, add 30μl of the freshly prepared suspension (acetic acid: methanol = 5:1), stir with a dissecting needle to suspend the cells; place a clean glass slide in a moist foam box, use a pipette to mix the suspension, then aspirate 3μl of the suspension, drop it in mid-air on the glass slide, and wait for the suspension on the slide to dry completely.

[0025] The preparation method of 1×TE buffer is as follows: Add 100 mL of 10× TE buffer to 1000 mL of distilled water to prepare 1× TE buffer; The preparation method of 10×TE buffer is as follows: add 100 mL 1M Tris-HCl and 20 mL 0.5M EDTA Na2 to distilled water to make up to 1000 mL, adjust the pH to 8.0, filter with a 0.45 μm filter membrane, sterilize, and store at 4°C; The preparation method of 0.5M EDTA Na2 is as follows: 186.1g EDTA Na2 and 16g NaOH are added to ddH2O to dissolve, and then the volume is adjusted to 1000mL with ddH2O, mixed, the pH is adjusted to 8, filtered with a 0.45μm filter membrane, sterilized, and 0.5M EDTA Na2 is obtained, and stored at 4°C; The preparation method of 1M Tris-HCl is as follows: 121.1g Tris-Base and 49mL concentrated HCl with a mass concentration of 7% are added to ddH2O to dissolve, and then the volume is adjusted to 1000mL with ddH2O, mixed, the pH is adjusted to 7.5, filtered with a 0.45μm filter membrane, sterilized, and 1M Tris-HCl is obtained, and stored at 4°C; The preparation method of the root apical meristem hydrolysate is as follows: 0.4 g of cellulase, 0.1 g of pectinase and 9.5 g of KCL buffer are mixed, completely dissolved on ice, and then dispensed into sterilized 0.5 ml centrifuge tubes, 20 μl per tube, and stored at -20°C.

[0026] Microscopic examination: Place the slide under a 20x objective lens of a fluorescent microscope and observe the results. Figure 1 As shown in the figure, A and B are metaphases that appear randomly in different regions under a fluorescence microscope; from Figure (1), it can be seen that the number of mitotic metaphases of the material pretreated with APM has increased significantly. After DAPI staining, the photo was taken under a fluorescence microscope with a 100x oil lens. The staining results are shown in Figure (2). The scale bar in the figure is shown in Figure (2). It can be seen from the figure that the number of chromosomes in the root tip cells of the millet is 18, the metaphases are numerous, the chromosomes are well dispersed, and there is little overlap.

[0027] Example 2 Chromosome preparation of Jingu 21 (2n=2x=18) Jinzhong No. 21 is similar to Yugu No. 1 in terms of seed rooting, pretreatment, nitrous oxide treatment, and film making, as detailed in Example 1.

[0028] Microscopic examination: Place the slide under a 20x objective lens of a fluorescent microscope and observe the results. Figure 3 As shown in the figure, A and B are metaphases that appear in random different areas under a fluorescence microscope; from Figure (3), it can be seen that the number of mitotic metaphases in the material pretreated with APM has increased significantly. After DAPI staining, the photo was taken under a 100x oil lens under a fluorescence microscope. The staining results are shown in Figure (4). The scale bar in the figure is as shown in Figure (4). It can be seen from the figure that the number of chromosomes in the root tip cells of Jingu No. 21 is 18, the metaphases are many, the chromosomes are well dispersed, and there is little overlap.

[0029] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A method for preparing chromosome sections from millet root tips, characterized in that: The following steps are involved: S1, seed rooting; S2, pretreatment: pretreatment by immersing the roots in APM solution; S3, laughing gas treatment; S4. Fixation: fix the roots after nitrous oxide treatment with glacial acetic acid; Add 90% glacial acetic acid precooled at 4°C and fix for 7 minutes; S5. Preparation: Take out the roots, rinse with distilled water, cut off the root cap, and heat the root tip meristem hydrolyzate in a water bath; after the hydrolysis, add TE buffer to terminate the reaction; dry the TE buffer, add alcohol to wash, and finally centrifuge to dry the alcohol; add the suspension, stir with a dissecting needle to suspend the cells; mix the suspension by pipetting, then pipette the suspension, drop it in mid-air on a glass slide, and wait until the suspension on the slide is completely dry to complete the preparation of millet root tip chromosomes.

2. The method for preparing millet root tip chromosomes according to claim 1, characterized in that: Step S1 is specifically as follows: Place the dry seeds in a culture dish with filter paper to retain water. Add water to the dish to cover the seeds and germinate at 28°C. Pour out excess water after the seeds turn white. Treat at 4°C for 24 hours and then grow at 28°C away from light.

3. The method for preparing millet root tip chromosomes according to claim 1, characterized in that: Step S2 is specifically as follows: When the root length is 1-1.3 cm, add 20 μmol / L APM solution to the culture dish until the roots are submerged, and place it in a 28°C incubator for growth pretreatment for 2.5 hours.

4. The method for preparing millet root tip chromosomes according to claim 3, characterized in that: The preparation method of the 20 μmol / L APM solution in step S2 is: add 100 μL of 0.2 mol / L APM mother solution to 1000 mL of distilled water to prepare a 20 μmol / L APM solution; the preparation method of the 0.2 mol / L APM mother solution is: dissolve 60.86 mg APM in 10 mL of -20°C precooled acetone to obtain a 0.2 mol / L APM mother solution, which is stored at -20°C.

5. The method for preparing millet root tip chromosomes according to claim 1, characterized in that: Step S3 is specifically as follows: The pretreated root tips were washed in a culture dish filled with distilled water, the roots were cut with scissors, and treated with nitrous oxide at a pressure of 0.5-2 MPa for 15-60 minutes at room temperature.

6. The method for preparing millet root tip chromosomes according to claim 5, characterized in that: Step S3 is specifically as follows: The pretreated root tips were washed twice in a culture dish filled with distilled water for 4-5 minutes each time, the roots were cut with scissors, and the roots were placed in a 2 mL centrifuge tube with holes. An appropriate amount of deionized water was added to the centrifuge tube in advance to keep it moist. At room temperature, the roots were treated with nitrous oxide at a pressure of 1 MPa for 0.5 hours.

7. The method for preparing chromosomes from millet root tips according to claim 1, characterized in that: Step S4 is specifically as follows: After being treated with nitrous oxide, the roots were fixed with 90% glacial acetic acid precooled at 4°C for 7 minutes.

8. The method for preparing millet root tip chromosomes according to claim 7, characterized in that: Step S5 is specifically as follows: The roots were taken out and rinsed with distilled water for 2-3 times. Excess water was removed with absorbent paper. The root cap was cut off and placed in a centrifuge tube containing 20 μL of the root apical meristem enzymatic solution. The enzymatic solution was hydrolyzed in a water bath at 37°C for 50-60 min. After the enzymatic solution was completed, 200 μL of 1×TE buffer was added to terminate the reaction. The TE buffer was drained with a pipette, and 70% alcohol was added to wash twice. Finally, the alcohol was completely drained by centrifugation. 30 μL of the prepared suspension was added, and the cells were suspended by stirring with a dissecting needle. Place a clean glass slide in a moist foam box, use a pipette to mix the suspension, then aspirate 3 μL of the suspension and drop it onto the glass slide in mid-air. Wait until the suspension on the glass slide is completely dry, and the millet root tip chromosome preparation is complete. The suspension is obtained by mixing acetic acid and methanol in a volume ratio of 5:1; The root apical meristem hydrolysate was obtained by mixing cellulase, pectinase and KCL buffer.

9. The method for preparing millet root tip chromosomes according to claim 8, characterized in that: The preparation method of 1×TE buffer in step S5 is: Add 100 mL of 10× TE buffer to 1000 mL of distilled water to prepare 1× TE buffer; The preparation method of 10×TE buffer is as follows: add 100 mL 1M Tris-HCl and 20 mL 0.5M EDTA Na2 to distilled water to make up to 1000 mL, adjust the pH to 8.0, filter with a 0.45 μm filter membrane, sterilize, and store at 4°C; The preparation method of 0.5M EDTA Na2 is as follows: 186.1g EDTA Na2 and 16g NaOH are added to ddH2O to dissolve, and then the volume is adjusted to 1000mL with ddH2O, mixed, the pH is adjusted to 8, filtered with a 0.45μm filter membrane, sterilized, and 0.5M EDTA Na2 is obtained, and stored at 4°C; The preparation method of 1M Tris-HCl is as follows: 121.1g Tris-Base and 49mL concentrated HCl with a mass concentration of 7% are added to ddH2O to dissolve, and then the volume is adjusted to 1000mL with ddH2O, mixed, the pH is adjusted to 7.5, filtered with a 0.45μm filter membrane, sterilized, and 1M Tris-HCl is obtained, and stored at 4°C; The preparation method of the root apical meristem hydrolysate is as follows: 0.4 g of cellulase, 0.1 g of pectinase and 9.5 g of KCL buffer are mixed, completely dissolved on ice, and then dispensed into sterilized 0.5 ml centrifuge tubes, 20 μl per tube, and stored at -20°C.

10. The method for preparing chromosome sections of millet root tips according to any one of claims 1 to 9, characterized in that: The following steps are involved: S1. Seed rooting: dry seeds were placed in a culture dish with filter paper to retain water. Water was added to the culture dish to cover the seeds and the seeds were germinated at 28°C. After the seeds turned white, excess water in the culture dish was poured out, leaving only the filter paper moist. The seeds were moved to a 4°C refrigerator for 24 hours, and then the culture dish was moved to a 28°C incubator to grow in the dark. S2. Pretreatment: When the roots are 1-1.3 cm long, add 20 μmol / L APM solution to the culture dish until the roots are submerged, and place in a 28°C incubator for 2.5 hours of growth pretreatment; S3, nitrous oxide treatment: the pretreated root tips were washed twice in a culture dish filled with distilled water for 5 minutes each time, the roots were cut with scissors, and the roots were placed in a 2 ml centrifuge tube with holes, and an appropriate amount of deionized water was added to the centrifuge tube in advance to keep it moist. At room temperature, the roots were treated with nitrous oxide at a pressure of 1 MPa for 0.5 hours; S4, fixation: place the 2 ml centrifuge tube containing the roots after being treated with nitrous oxide on crushed ice, add 90 wt% glacial acetic acid precooled at 4°C into the centrifuge tube, and fix for 7 minutes; S5, preparation: take out the root, rinse it with distilled water for 2-3 times, remove excess water with absorbent paper, cut off the root crown, put it in a centrifuge tube containing 20μL enzyme solution, and perform enzymolysis in a water bath at 37℃ for 50-60min; after the enzymolysis, add 200μL 1×TE buffer to terminate the reaction; use a pipette to dry the TE buffer, add 70% alcohol to wash twice, and finally centrifuge to completely dry the alcohol, add 30μL of the freshly prepared suspension, stir with a dissecting needle to suspend the cells; place a clean glass slide in a moist foam box, use a pipette to mix the suspension, then aspirate 3μL of the suspension, drop it in mid-air on the glass slide, wait for the suspension on the glass slide to dry completely, and the preparation of millet root tip chromosomes is completed; The suspension is obtained by mixing acetic acid and methanol in a volume ratio of 5:1.

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