A method for improving the number of mitotic metaphase chromosomes in root tips of sugarcane or sugarcane relative species
By optimizing sugarcane stem treatment and root tip pretreatment methods, and combining enzymatic digestion and fixation techniques, the problems of long preparation time and poor quality of sugarcane root tip chromosome slides have been solved, enabling rapid and efficient chromosome observation, which is convenient for sugarcane cytogenetics research.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- GUANGXI ZHUANG AUTONOMOUS REGION ACAD OF AGRI SCI
- Filing Date
- 2023-03-30
- Publication Date
- 2026-04-21
AI Technical Summary
Preparing chromosome slides from sugarcane root tips is difficult, time-consuming, and of poor quality, making it hard to obtain ideal chromosome division phases and affecting sugarcane cytogenetics research.
Sugarcane stems were treated with indole-3-acetic acid solution, cultured in sand or peat substrate, and root tips were treated with dichlorobenzene aqueous solution at low temperature. Cellulase and pectinase were used for enzymatic digestion, and Carnoy's fixative was used for fixation. Phenol-fuchsin staining was modified, and the slide preparation process was optimized.
It significantly shortens the slide preparation cycle, increases the number and clarity of chromosome metaphases, facilitates karyotype analysis, and improves the efficiency of sugarcane cytogenetics research.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant chromosome preparation technology, and specifically relates to a method for preparing slides that increases the number of chromosomes in metaphase of root tip mitosis in sugarcane or closely related species. Background Technology
[0002] Sugarcane, belonging to the genus *Saccharum* of the family Poaceae, is the world's fifth largest agricultural crop. It is both a major sugar crop for sugar production and an important energy crop for fuel ethanol production. Conducting cytogenetic research on sugarcane is of great significance for the utilization of sugarcane germplasm resources and breeding research. The preparation and observation of sugarcane chromosome slides are fundamental to conducting cytogenetics and molecular cytogenetics studies on ploidy, classification, and phylogenetic relationships in sugarcane, playing a crucial role in sugarcane genetics and breeding. High-quality chromosome slide preparation is a prerequisite for the smooth progress of subsequent work such as karyotype analysis and in situ hybridization. However, because sugarcane is an allopolyploid plant, it has a large number of chromosomes, an inconsistent number, and small morphology, making it difficult to obtain ideal chromosome division phases, thus posing a significant challenge to chromosome slide preparation. Currently, chromosome number observation in sugarcane can be performed using root tips, shoot tips, young leaves, or pollen mother cells. Studies have found that root tips yield the best results. However, the following problems exist in actual operation: the root tips of hydroponically grown sugarcane have viscous secretions, which affect sample processing and slide preparation; when taking roots from sugarcane grown in buckets, the seedlings are small, with few and weak roots, and the roots are buried in the soil, making sampling inconvenient; after the tillering stage, the sugarcane roots wrap around the soil, and the soil sticks to the bucket walls, making it time-consuming and laborious to pull out the entire sugarcane plant for root extraction, and the roots vary in size and age, resulting in inconsistent quality and making it difficult to grasp the chromosome division cycle, so it cannot be guaranteed that all collected root tips will show the metaphase of chromosome division. After one sampling, it takes at least half a month for new roots to grow before sampling can be done again. From sugarcane planting and cultivation to obtaining root tips for chromosome observation, the cycle is long, inefficient, and the chromosome morphology and structure are not clear. Summary of the Invention
[0003] The purpose of this invention is to provide a method for preparing root tip chromosomes that increases the number of chromosomes in metaphase of mitosis in sugarcane or closely related species, overcoming the shortcomings of existing methods such as poor quality and long preparation time for sugarcane root tip chromosomes.
[0004] To achieve the above objectives, the present invention provides a method for preparing slides that increases the number of chromosomes in metaphase of root tip mitosis in sugarcane or closely related species, comprising the following steps:
[0005] (1) Sugarcane stem treatment: Soak the two-bud segments of sugarcane or closely related species in an indole-3-acetic acid solution with a concentration of 80~120mg / L, and then use sand or peat substrate for moist cultivation.
[0006] (2) Root tip pretreatment: After culturing for 2-3 days, when the roots are 1-2 cm long, select healthy young root tips and place them in a saturated dichlorobenzene aqueous solution at 8-12℃ for 3-5 hours.
[0007] (3) Fixation: Clean the root tips treated in step (2) with ddH2O, hypotonic with ddH2O for 25-35 min, and then transfer them to Carnoy fixative for fixation;
[0008] (4) Enzymatic hydrolysis: Wash the root tips with ddH2O, place the root tip growth point in a mixture of cellulase and pectinase for enzymatic hydrolysis, wash away the hydrolysate, and obtain the enzymatically hydrolyzed root tips;
[0009] (5) Slide preparation: The root tips obtained in step (4) are coated on a glass slide to prepare a slide, and the slide is dried.
[0010] (6) Staining treatment.
[0011] Preferably, in the above-described method for preparing slides to increase the number of chromosomes in metaphase of root tip mitosis in sugarcane or closely related species, the concentration of the indole-3-acetic acid solution is 100 mg / L.
[0012] Preferably, in the above-mentioned method for preparing slides to increase the number of chromosomes in metaphase of root tip mitosis in sugarcane or closely related species, step (1) involves culturing in a constant temperature incubator at 25~28℃.
[0013] Preferably, in the above-mentioned method for preparing slides to increase the number of chromosomes in metaphase of root tip mitosis in sugarcane or sugarcane closely related species, in step (1), a healthy mature sugarcane or the lower part of a seed stalk of a sugarcane closely related species is selected and cut into double-bud segments.
[0014] Preferably, in the above-described method for preparing slides to increase the number of chromosomes in metaphase of root tip mitosis in sugarcane or closely related species, fucoidan is added to the indole-3-acetic acid solution, and the amount of fucoidan added is 20-60 mg / L. Adding fucoidan to the indole-3-acetic acid solution can further regulate the number of chromosomes in metaphase of root tip mitosis.
[0015] Preferably, in the above-described method for preparing slides to increase the number of chromosomes in metaphase of root tip mitosis in sugarcane or closely related species, the amount of added brown algae oligosaccharide is 20-30 mg / L.
[0016] Preferably, in the above-mentioned method for preparing slides to increase the number of chromosomes in metaphase of root tip mitosis in sugarcane or closely related species, in step (2), healthy young root tips are selected at 9-10 am and placed in a saturated dichlorobenzene aqueous solution at 10°C for 3-5 hours.
[0017] Preferably, in the above-mentioned method for preparing root tips to increase the number of chromosomes in metaphase of mitosis in sugarcane or closely related species, in step (3), the root tips obtained in step (2) are washed with ddH2O 3-4 times, each time for 5-8 minutes; then transferred to Carnoy's fixative and fixed in the dark at 4°C for 20-24 hours. The Carnoy's fixative is composed of methanol and glacial acetic acid in a volume ratio of 3:1.
[0018] Preferably, in the above-mentioned method for preparing slides to increase the number of chromosomes in metaphase of root tip mitosis in sugarcane or closely related species, in step (4), the mass fraction of cellulase in the mixture of cellulase and pectinase is 3.5% and the mass fraction of pectinase is 1.75%; the enzymatic hydrolysis is carried out at 30~38℃ for 8~10h.
[0019] Preferably, in the above-mentioned method for preparing slides to increase the number of chromosomes in metaphase of root tip mitosis in sugarcane or closely related species, the staining treatment in step (6) is: staining with modified phenol fuchsin for 8-10 minutes.
[0020] Compared with existing technologies, the present invention has the following advantages:
[0021] 1. The present invention provides a method for preparing slides that increases the number of chromosomes in metaphase of root tip mitosis in sugarcane or closely related species. From root tip culture to chromosome observation, the process takes as little as 4 days, with a short cycle, high efficiency, convenient and rapid sampling, a large number of chromosomes in metaphase, clear morphology, and a clean slide background, which is convenient for chromosome counting and observation. This method is of great significance for conducting sugarcane cytogenetics research.
[0022] 2. The present invention provides a method for preparing slides that increases the number of chromosomes in metaphase of root tip mitosis in sugarcane or closely related species. This method involves treating the double bud segments of the stem of sugarcane or closely related species with indole-3-acetic acid to promote root tip cell differentiation and division, thereby increasing the number of chromosomes in metaphase of root tip mitosis. Low-temperature treatment with saturated dichlorobenzene aqueous solution at 8-10°C can make the chromosomes in the root tip morphology clear and dispersed, thus obtaining more chromosome images with good dispersion and clarity, which is convenient for karyotype analysis. Attached Figure Description
[0023] Figure 1 The image shows a microscopic observation of the root tip chromosomes of the F1 material of the *Imperata cylindrica* complex obtained by the method of Example 1 of this invention (1000X magnification, 100X objective lens, 10X eyepiece).
[0024] Figure 2 The image shows a microscopic observation of the root tip chromosomes of the F1 material of the *Imperata cylindrica* complex obtained by the method of Example 2 of this invention (1000X magnification, 100X objective lens, 10X eyepiece).
[0025] Figure 3 The image shows a microscopic observation of the root tip chromosomes of the F1 material of the *Imperata cylindrica* complex obtained by the method of Comparative Example 7 in this invention (1000X magnification, 100X objective lens, 10X eyepiece).
[0026] Figure 4 The images shown are microscopic observations of the root tip chromosomes of the *Gnaphalium affine* material obtained by different slide preparation methods in Experiment 3 of this invention (1000X magnification, 100X objective lens, 10X eyepiece). Among them, (a) is the slide preparation method of Example 1, (b) is the slide preparation method of Example 2, (c) is the slide preparation method of Comparative Example 7, and (d) is the slide preparation method of Comparative Example 8. Implementation
[0027] The specific embodiments of the present invention will be described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Example 1
[0028] A method for preparing slides to increase the number of chromosomes during metaphase of root tip mitosis in sugarcane or closely related species includes the following steps:
[0029] (1) Sugarcane stem treatment: Select the middle and lower part of the stem of healthy mature sugarcane or sugarcane closely related species, cut it into double-bud segments, soak it in a 100 mg / L indole-3-acetic acid solution at room temperature for 8 hours, and then keep it moist in a constant temperature incubator at 28℃ with sand; 1L of indole-3-acetic acid solution is prepared by first dissolving 100 mg of indole-3-acetic acid in 10 mL of anhydrous ethanol, and then adding water to make up to 1L;
[0030] (2) Root tip pretreatment: After culturing for 3 days, when the roots are 1-2 cm long, select healthy young root tips at 9:00-10:00 am and immerse them in a saturated dichlorobenzene aqueous solution at 10°C for 3 hours.
[0031] (3) Fixation: Wash the root tip three times with ddH2O for 5 min each time, and then transfer it to freshly prepared Carnoy fixative (methanol: glacial acetic acid volume ratio 3:1) for 20 h in the dark at 4℃ after 30 min of hypotonic ddH2O.
[0032] (4) Enzymatic hydrolysis: Wash the root tip with ddH2O 3 times for 5 minutes each time, remove the root tip, cut off the root cap, place the root tip growth point in a mixture of 3.5% cellulase and 1.75% pectinase at 37°C for 8 hours, the root tip epidermis will fall off, wash away the hydrolysate, and the enzymatically hydrolyzed root tip will be obtained.
[0033] (5) Slide preparation: Select the root tips obtained in the two steps (4) and spread them on a glass slide to prepare a slide. Dry the slide with an alcohol flame.
[0034] (6) Staining observation: After staining with modified phenol fuchsin for 8 minutes, the stain was photographed and observed under a microscope. Example 2
[0035] A method for preparing slides to increase the number of chromosomes during metaphase of root tip mitosis in sugarcane or closely related species includes the following steps:
[0036] (1) Sugarcane stem treatment: Select healthy mature sugarcane or the lower part of the stem of a closely related species of sugarcane, cut it into double-bud segments, and soak it in a mixed solution of indole-3-acetic acid and fucoidan for 8 hours at room temperature. The concentration of indole-3-acetic acid is 75 mg / L and the concentration of fucoidan is 25 mg / L. Then, it is kept moist in a constant temperature incubator at 28℃ with sand. The preparation of 1L of mixed solution of indole-3-acetic acid and fucoidan is as follows: first, dissolve 100 mg of indole-3-acetic acid in 10 mL of anhydrous ethanol, dissolve 25 mg of fucoidan in 10 mL of water, mix the two and then add water to make up to 1L.
[0037] (2) Root tip pretreatment: After culturing for 3 days, when the roots are 1-2 cm long, select healthy young root tips at 9:00-10:00 am and place them in a saturated dichlorobenzene aqueous solution at 10°C for 3 hours.
[0038] (3) Fixation: Wash the root tip three times with ddH2O for 5 min each time, and then transfer it to freshly prepared Carnoy fixative (methanol: glacial acetic acid volume ratio 3:1) for 20 h in the dark at 4℃ after 30 min of hypotonic ddH2O.
[0039] (4) Enzymatic hydrolysis: Wash the root tip with ddH2O 3 times for 5 minutes each time, remove the root tip, cut off the root cap, place the root tip growth point in a mixture of 3.5% cellulase and 1.75% pectinase at 37°C for 8 hours, the root tip epidermis will fall off, wash away the hydrolysate, and the enzymatically hydrolyzed root tip will be obtained.
[0040] (5) Slide preparation: Select the root tips obtained in the two steps (4) and spread them on a glass slide to prepare a slide. Dry the slide with an alcohol flame.
[0041] (6) Staining observation: After staining with modified phenol fuchsin for 8 minutes, the stain was photographed and observed under a microscope.
[0042] Comparative Example 1
[0043] The difference between this comparative example and Example 1 is that the concentration of indole-3-acetic acid solution in step (1) is 50 mg / L, while the other steps and parameters are the same as in Example 1.
[0044] Comparative Example 2
[0045] The difference between this comparative example and Example 1 is that the concentration of indole-3-acetic acid solution in step (1) is 150 mg / L, while the other steps and parameters are the same as in Example 1.
[0046] Comparative Example 3
[0047] The difference between this comparative example and Example 1 is that in step (1), an indolebutyric acid solution with a concentration of 100 mg / L was used for soaking, while other steps and parameters were the same as in Example 1.
[0048] Comparative Example 4
[0049] The difference between this comparative example and Example 1 is that: in step (1), a brown algae oligosaccharide solution with a concentration of 100 mg / L was used for soaking, while other steps and parameters were the same as in Example 1.
[0050] Comparative Example 5
[0051] The difference between this comparative example and Example 1 is that: in step (1), a brown algae oligosaccharide solution with a concentration of 200 mg / L was used for soaking, while other steps and parameters were the same as in Example 1.
[0052] Comparative Example 6
[0053] The difference between this comparative example and Example 1 is that: in step (1), a brown algae oligosaccharide solution with a concentration of 50 mg / L was used for soaking, while other steps and parameters are the same as in Example 1.
[0054] Comparative Example 7
[0055] The difference between this comparative example and Example 1 is that in step (2), the sample is placed in a saturated aqueous solution of p-dichlorobenzene and treated at 20°C for 3 hours. Other steps and parameters are the same as in Example 1.
[0056] Comparative Example 8
[0057] The difference between this comparative example and Example 1 is that in step (2), the sample is placed in a saturated aqueous solution of p-dichlorobenzene and treated at 30°C for 3 hours. Other steps and parameters are the same as in Example 1.
[0058] Experiment 1
[0059] The experimental subject was the F1 material of *Imperata cylindrica* var. *mongolica*. Examples 1-2 and Comparative Examples 1-5 were set up, and slides were prepared using the methods of Examples 1-2 and Comparative Examples 1-5. A control group was included, which was soaked in water, and other procedures were the same as in Example 1. Observation was performed using a Nikon Ni microscope, and images were taken using a NIS Elements imaging system. The number of cells in metaphase of the entire slide was counted under a 100X objective and a 10X eyepiece. Each group was repeated three times. For each group, five double-bud segments of sugarcane stalks were cultured, and ten slides were prepared for observation and statistical analysis. The average of the three replicates was taken as the statistical result, as shown in Table 1.
[0060] As shown in Table 1, treating sugarcane seed stalks with indole-3-acetic acid (IAA) and then culturing them, by optimizing the IAA concentration, can yield more metaphase chromosomes, improve the quality of sugarcane root tip slides, shorten the experimental cycle, and increase slide preparation efficiency, which is beneficial for sugarcane cytogenetics research. Adding fucoidan, in synergy with IAA, can further obtain more metaphase chromosomes, improve slide quality, and facilitate karyotype analysis.
[0061] Table 1. Number of cells in metaphase observed under different treatments
[0062] Group Number of cells in metaphase on each slide Example 1 24 Example 2 30 Comparative Example 1 12 Comparative Example 2 11 Comparative Example 3 17 Comparative Example 4 14 Comparative Example 5 12 Comparative Example 6 8 control group 7
[0063] Experiment 2
[0064] The test subject was the F1 material of the *Imperata cylindrica* var. *mongolica* composite. The slides were prepared and observed using the methods described in Example 1, Example 2, and Comparative Example 7. Figure 1 and Figure 2 The images shown are chromosome images observed using the methods of Example 1 and Example 2 (magnification 1000×, objective lens 100X, eyepiece 10X). As can be seen from the images, the chromosome morphology and structure are clear and well dispersed, which is beneficial for karyotype analysis. Figure 3 The chromosome images observed in the slide prepared using the method in Comparative Example 7 (1000× magnification, 100X objective lens, 10X eyepiece) show that the chromosomes are curved, structurally unclear, and poorly dispersed. In summary, it can be seen that treating the root tip with a saturated dichlorobenzene solution at 10℃ results in clearer and more dispersed morphology, facilitating karyotype analysis.
[0065] Experiment 3
[0066] The test subject was GXS79-9 material, and slides were prepared and observed using the methods described in Example 1, Example 2, Comparative Example 7, and Comparative Example 8. Figure 4 The images show chromosomes prepared using different methods (magnification 1000×, objective lens 100X, eyepiece 10X). As can be seen from the figures, the chromosomes prepared using the methods of Examples 1 and 2 of this invention have clear morphological structures and are well dispersed, which is beneficial for karyotype analysis. The chromosome images prepared using the methods of Comparative Examples 7 and 8 show chromosomes with curved morphology, unclear structure, and poor dispersion.
[0067] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be limited by the claims and their equivalents.
Claims
1. A method of increasing the number of mitotic metaphase chromosomes in root tips of sugarcane or a sugarcane relative, the method comprising, Includes the following steps: (1) Sugarcane stem treatment: Soak the double-bud segments of sugarcane or closely related species stems in a solution of indole-3-acetic acid with a concentration of 60~120mg / L, and add brown algae oligosaccharide to the indole-3-acetic acid solution, with an amount of brown algae oligosaccharide added of 20~60mg / L; and then use sand or peat substrate for moist cultivation. (2) Root tip pretreatment: After culturing for 2-3 days, when the roots are 1-2 cm long, select healthy young root tips and place them in a saturated dichlorobenzene aqueous solution at 8-12℃ for 3-5 hours. (3) Fixation: Clean the root tips treated in step (2) with ddH2O, hypotonic with ddH2O for 25-35 min, and then transfer them to Carnoy fixative for fixation; (4) Enzymatic hydrolysis: Wash the root tips with ddH2O, place the root tip growth point in a mixture of cellulase and pectinase for enzymatic hydrolysis, wash away the hydrolysate, and obtain the enzymatically hydrolyzed root tips; (5) Slide preparation: The root tips obtained in step (4) are coated on a glass slide to prepare a slide, and the slide is dried. (6) Staining treatment.
2. The squaring method for increasing the number of mitotic metaphase chromosomes in root tips of sugar cane or a sugar cane relative according to claim 1, characterised in that, The concentration of the indole-3-acetic acid solution is 100 mg / L.
3. The squaring method for increasing the number of mitotic metaphase chromosomes in root tips of sugar cane or a sugar cane relative according to claim 1, characterised in that, In step (1), the culture is carried out in a constant temperature incubator at 25~28℃.
4. The squaring method for increasing the number of mitotic metaphase chromosomes in root tips of sugar cane or a sugar cane relative according to claim 1, characterized in that, In step (1), select the lower part of a healthy, mature sugarcane or a closely related species of sugarcane and cut it into double-bud segments.
5. The method for preparing slides to increase the number of chromosomes in metaphase of root tip mitosis in sugarcane or closely related species according to claim 1, characterized in that, The amount of the added brown algae oligosaccharide is 20~30 mg / L.
6. The squaring method for increasing the number of mitotic metaphase chromosomes in root tips of sugar cane or a sugar cane relative according to claim 1, characterised in that, In step (2), healthy young root tips are selected at 9-10 am and placed in a saturated dichlorobenzene aqueous solution at 10°C for 3-5 hours.
7. The squaring method for increasing the number of mitotic metaphase chromosomes in root tips of sugar cane or a sugar cane relative according to claim 1, characterised in that, In step (3), the root tip obtained in step (2) is washed with ddH2O 3 to 4 times, each time for 5 to 8 minutes; then it is transferred to Carnoy's fixative and fixed in the dark at 4°C for 20 to 24 hours. The Carnoy's fixative is composed of methanol and glacial acetic acid in a volume ratio of 3:
1.
8. The squaring method of increasing the number of mitotic metaphase chromosomes in root tips of sugar cane or a sugar cane relative according to claim 1, characterised in that, In step (4), the mass fraction of cellulase in the mixture of cellulase and pectinase is 3.5% and the mass fraction of pectinase is 1.75%; the enzymatic hydrolysis is carried out at 30~38℃ for 8~10h.
9. The squaring method of increasing the number of mitotic metaphase chromosomes in root tips of sugar cane or a sugar cane relative according to claim 1, characterised in that, In step (6), the staining treatment is as follows: staining with modified phenol fuchsin for 8-10 minutes.