AU2020103466A4

AU2020103466A4 - Efficient method for preparing chromosome from shoot tip of sugarcane or sugarcane related species

Info

Publication number: AU2020103466A4
Application number: AU2020103466A
Authority: AU
Inventors: Qing Cai; Chunyan KONG; Chunjia Li; Xujuan LI; Xiuqin LIN; Hongbo Liu; Xinlong Liu; Xin Lu; Jun MAO; Zhuandi WU; Chaohua XU; Jiafang ZHOU; Qiuyan ZI
Original assignee: Sugarcane Research Institute of Yunnan Academy of Agricultural Sciences
Current assignee: Sugarcane Research Institute of Yunnan Academy of Agricultural Sciences
Priority date: 2020-01-16
Filing date: 2020-11-16
Publication date: 2021-01-28
Anticipated expiration: 2028-11-16
Also published as: CN111238888A

Abstract

The disclosure provides an efficient method for preparing chromosomes from a shoot tip of sugarcane or sugarcane related species, and belongs to the technical field of cell biology. The disclosure provides an efficient method for preparing chromosomes from a shoot tip of sugarcane in view of the problems that sugarcane chromosomes are numerous and small and ideal metaphase cells are difficult to be obtained by root tip preparation. Chromosome preparation is performed by utilizing a shoot apical meristem of vigorously growing sugarcane or sugarcane related species. The method has the advantages of convenient material taking, large sample size of meristem, vigorous division, many metaphase cells and clear and dispersed chromosome structure; technical points of material selection, pretreatment, fixation, dissociation and staining, preparation, and microscopic observation of chromosome morphology are disclosed. The method features convenience, accurate and reliable result, excellent repeatability, easy operation, and short experimental period, and improves the efficiency of sugarcane karyotyping analysis. The disclosure provides technical support for the investigation of sugarcane chromosome karyotype and the classification, identification, protection and utilization of germplasm resources. DRAWINGS Material Selection: Sugarcane shoot apical meristem Pretreatment: Treat with sterile ddH20 for 5-24 h at 4°C Fixation: Treat with Carnoy's fluid (ethanol:glacial acetic acid = 3:1, freshly prepared) for 2 3 days 4°C Dissociation and Staining: In the presence of a mixture of isometric 1 N HCl and 45% acetic acid aqueous solution, break wall and dissociate in a water bath at 50-65°C for 20-25 min; stain with modified carbopol fuchsin stain for 4-8 min at room temperature Slide Preparation: Tap the slide with a pencil to make cells spread, so that a clean background of the slide is obtained Microscopic Examination: Observe, photograph, and count under an optical microscope FIG. 1 FIG. 2

Description

DRAWINGS

Material Selection: Sugarcane shoot apical meristem

Pretreatment: Treat with sterile ddH 20 for 5-24 h at 4°C

Fixation: Treat with Carnoy's fluid (ethanol:glacial acetic acid = 3:1, freshly prepared) for 2 3 days 4°C

Dissociation and Staining: In the presence of a mixture of isometric 1 N HCl and 45% acetic acid aqueous solution, break wall and dissociate in a water bath at 50-65°C for 20-25 min; stain with modified carbopol fuchsin stain for 4-8 min at room temperature

Slide Preparation: Tap the slide with a pencil to make cells spread, so that a clean background of the slide is obtained

Microscopic Examination: Observe, photograph, and count under an optical microscope

FIG. 1

FIG. 2

EFFICIENT METHOD FOR PREPARING CHROMOSOME FROM SHOOT TIP OF SUGARCANE OR SUGARCANE RELATED SPECIES TECHNICAL FIELD The disclosure relates to the technical field of cell biology, and in particular to an efficient method for preparing chromosomes from a shoot tip of sugarcane or sugarcane related species. BACKGROUND Sugarcane is an important sugar crop and energy plant in the world. The genus Saccharum L. includes 6 species: Saccharum spontaneum, Saccharum robustum, Saccharum officinarum, Saccharum barberi, Saccharum sinense, and Saccharum spontaneum var. edulis (also known as Saccharum edule). In Saccharinae Griseb, Saccharum arundinaceum, Erianthus rockii and Saccharum narenga are all relatives of sugarcane. Most of the modem sugarcane cultivars contain 2-3 related species in the genus Saccharum L., with a narrow genetic basis and serious degradation of species. In order to cultivate new and excellent sugarcane cultivars, breeders have utilized intraspecific, interspecific and intergeneric hybridization. It is expected that resistance genes of wild species and relative plants can be introduced into the sugarcane to enrich the genetic basis of the sugarcane. Research on classification and utilization of sugarcane germplasm resources, identification of alien chromosomes, chromosomal location of genes, relationship identification, and mechanism of meiosis is related to chromosome research. Preparation of sugarcane chromosome is the basis for the development of sugarcane cytogenetics, sugarcane molecular cytogenetics and related research based on chromosomes. However, sugarcane is the most complex allopolyploid plant, which has a large number of chromosomes (range 64-128), is small in size, and is difficult to obtain ideal chromosomal slide specimens of metaphase cells. At present, sugarcane root-tip meristems are mainly used in China and overseas to prepare slides, but there are still some problems in the actual operation: the hydroponic sugarcane root tips have sticky secretions, which affect the effect of chromosome slices. Barrel-grown or sand-cultured root tips have no effect of sticky secretions, but the sand needs to be cleaned during collection, which is cumbersome; sugarcane roots are fine, and there are few main roots and more fibrous roots; the meristem area is very small, and generally, one slide specimen can be prepared with 1-2 root tips; the root tips of sugarcane planted in barrel, sand or water is substantially affected by nutritional and environmental factors, and the thickness and age of the root tips are different; division cycle is difficult to grasp; only a few root tips in the mitotic phase are often present in a batch of root tip samples; there is no guarantee that the metaphase cells can be observed on each slide, so that it is necessary to repeatedly collect the root tips and prepare another slide to observe, leading to increased workload; both test and experimental cycles are long, and in general, it takes about 1 or 2 weeks to identify the number of chromosomes in one material. SUMMARY In view of this, an objective of the disclosure is to provide an efficient method for preparing chromosomes from a shoot tip of sugarcane or sugarcane related species. The method can quickly obtain a clean background, chromosomes are clear in morphology and structure, and well-spread metaphase cells are observed, being convenient to count chromosomes and conduct karyotype analysis. An efficient method for preparing chromosomes from a shoot tip of sugarcane or sugarcane related species includes the following steps: 1) collecting a shoot apical meristem of vigorously growing sugarcane or sugarcane related species and cutting into 0.3-0.5 cm3 small pieces, soaking the shoot apical meristem in pre-cooled sterile ddH 20, and pretreating the shoot apical meristem for 5-24 h at 4°C to obtain a pretreatment material; 2) fixing the pretreatment material at 4°C for 2-3 days; after washing with sterile ddH2 0, dissociating the pretreatment material with a dissociation solution in a water bath at 50-65°C for -25 min to obtain a dissociated material; where the dissociation solution is a mixture formed by isovolumic mixing of 1 mol/L HCl solution and 45% (v/v) acetic acid aqueous solution; and 3) transferring the dissociated material onto a glass slide, removing water from the surface, staining the dissociated material with modified carbopol fuchsin stain, crushing the dissociated material, removing tissue residues, covering with a coverslip, and staining for 4-8 min; removing the stain around the coverslip, and pressing the slide to prepare a slide specimen of chromosomal spread. Preferably, the shoot apical meristem in step 1) includes meristems at the base of the shoot tip of the sugarcane and/or 2-3 layers of spire meristems enveloping the shoot tip, and the 2-3 layers of spire meristems enveloping the shoot tip are spire meristems within 3 cm above the shoot tip. Preferably, the shoot apical meristem of the vigorously growing sugarcane or sugarcane related species in step 1) is collected at the jointing stage, elongation stage, or late elongation stage. Preferably, the shoot apical meristem of the vigorously growing sugarcane or sugarcane related species in step 1) is collected from 9:00 a.m. to 11:00 a.m. on a sunny day. Preferably, Camoy's fluid is a fixative for the fixation in step 2); the Carnoy's fluid is made by mixing absolute ethanol and glacial acetic acid in a volume ratio of 3:1.

Preferably, a method for pressing the slide in step 3) is to tap the coverslip with one end of a pencil with an eraser, disperse the chromosomes, and then press the coverslip with a thumb to make cells spread evenly, so that the chromosomes are on the same plane. Preferably, the cultivars of the sugarcane includes Saccharum officinarum cv. Badila, Saccharum cv. 57NG208, Saccharum cv. Mintang 70-611, Saccharum cv. RB72-454, F1 generation Saccharum cv. Yacheng 96-48, or BC 2 generation Saccharumcv. Yunzhe 07-86. Preferably, the sugarcane related species includes wild species of sugarcane and species of sugarcane related genera; the wild species of sugarcane includes Saccharum spontaneum and Saccharum robustum; the species of sugarcane related genera includes Saccharum arundinaceum, Miscanthus sinensis, Erianthusrockii and Saccharum narenga. The disclosure further provides a slide specimen of sugarcane chromosomal spread prepared by the above method for preparing chromosomes from a shoot tip of sugarcane or sugarcane related species, where the chromosomes are clear in morphology and structure, metaphase cells are dispersed and non-overlapping, and background is clean without impurities. The disclosure further provides use of the above slide specimen of chromosomal spread of the sugarcane or sugarcane related species in the chromosome karyotype analysis of the sugarcane or sugarcane related species or the classification, identification, protection and utilization of germplasm resources. Compared with the prior art, the efficient method for preparing chromosomes from a shoot tip of sugarcane or sugarcane related species provided by the disclosure has the following beneficial effects: 1) In material selection, shoot apical meristems of vigorously growing sugarcane or sugarcane related species are selected for preparation. Thus, the meristems have a wide range; cell division cycle is concentrated; a batch of 30-50 sheets of chromosome slide specimens can be prepared from a single shoot tip, and more metaphase cells can be observed on each slide. Generally, more than 20 cells convenient for chromosome number identification and karyotype analysis can be observed within 10 slides, saving preparation time and microscopic observation time compared with 50-100 slides for root tip observation. 2) Shoot apical meristem samples of sugarcane or sugarcane related species are characteristic of convenient collection and long growth cycle of sugarcane (1-1.5 years). Shoot tips of grows vigorously sugarcane at the jointing, elongation and later stages, except for the seedling and tillering stages, are suitable for chromosome slide specimen preparation. Sugarcane shoot tips can be collected in the field at any time for related cytological studies in approximately 7-8 months a year. Compared with root tips and axillary bud chromosome preparation techniques, there is no need to repeat sampling, avoiding cumbersome steps of repeated root tip collection and cleaning, reducing the workload and improving work efficiency. 3) Pretreatment of the shoot apical meristems of sugarcane or sugarcane related species at 4°C is conducive to the simultaneous accumulation of metaphase cells. By compressing the slide, well-spread metaphase cells with clear chromosome morphology and structure can be obtained for observation in a short time. The chromosome number of a sugarcane germplasm material can be identified within two days compared with 1 or 2 weeks required for root tips and axillary buds, shortening the experimental period and improving the efficiency of sugarcane karyotyping analysis. 4) Through dissociation with dissociation solution for 20-25 min, the cells are easy to disperse, the chromosomes are clear, and there is little overlap; too short dissociation time (10-15 min) leads to difficult cell dispersing and more chromosome overlaps; too long dissociation time (30 min and above) easily leads to cell fragmentation, loss of chromosomes, or more overlap. 5) The staining time is controlled within 4-8 min, which can ensure that the overall staining degree is moderate, and there will be no insufficient or excessive staining. The method provided by the disclosure is simple, and features accurate and reliable identification result, excellent repeatability and stability, and easy operation. Personnel without cytology training may also prepare good sugarcane chromosome slides by using the method of the disclosure. Meanwhile, the method provided by the disclosure is further applicable to the drop method and the smear method. The chromosome slide specimen prepared by the disclosure will provide technical support for sugarcane chromosome karyotype research, germplasm resource classification and protection, etc., as well as help for chromosome number identification and karyotype analysis of sugarcane related species or other crops. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flow chart of a technical method for preparing chromosomes from a shoot tip of sugarcane; FIG. 2 illustrates samples of shoot apical meristems of sugarcane; FIG. 3 is a microscopic view of chromosomes of metaphase cells of a shoot tip of sugarcane (400x magnification, 40x objective lens, 1Ox eyepiece); FIG. 4 is a microscopic view of chromosomes of metaphase cells of a shoot tip of sugarcane (1,000x magnification, 100x objective lens, 1Ox eyepiece); FIG. 5 illustrates samples of root apical meristems of sugarcane; FIG. 6 illustrates the effect of chromosomes of root apical meristems of sugarcane; FIG. 7 illustrates samples of axillary buds of sugarcane; FIG. 8 illustrates the effect of chromosomes of axillary buds of sugarcane;

FIG. 9 illustrates the effect of chromosomes treated with 8-hydroxyquinoline solution for 4 h at room temperature; FIG. 10 illustrates the effect of chromosomes at different staining time; FIG. 11 illustrates the effect of chromosomes at different dissociation time; FIG. 12 illustrates chromosomes of germplasm materials of sugarcane parents and hybrid progenies; FIG. 13 illustrates chromosomes of wild species and relative plants of sugarcane; FIG. 14 illustrates shoot apical meristematic cells of sugarcane. DETAILED DESCRIPTION The disclosure provides an efficient method for preparing chromosomes from a shoot tip of sugarcane or sugarcane related species, including the following steps: 1) collecting a shoot apical meristem of vigorously growing sugarcane or sugarcane related species and cutting into 0.3-0.5 cm3 small pieces, soaking the shoot apical meristem in pre-cooled sterile ddH 20, and pretreating the shoot apical meristem for 5-24 h at 4°C to obtain a pretreatment material; 2) fixing the pretreatment material at 4°C for 2-3 days; after washing with sterile ddH2 0, dissociating the pretreatment material with a dissociation solution in a water bath at 50-65°C for -25 min to obtain a dissociated material; where the dissociation solution is a mixture formed by isovolumic mixing of 1 mol/L HCl solution and 45% (v/v) acetic acid aqueous solution; and 3) transferring the dissociated material onto a glass slide, removing water from the surface, staining the dissociated material with modified carbopol fuchsin stain, crushing the dissociated material, removing tissue residues, covering with a coverslip, and staining for 4-8 min; removing the stain around the coverslip, and pressing the slide to prepare a slide specimen of chromosomal spread. The flow chart of the method for preparing chromosomes from a shoot tip of sugarcane or sugarcane related species provided by the disclosure is shown in FIG. 1. In a slide specimens obtained by the method of the disclosure, shoot apical meristematic cells of sugarcane are shown in FIG. 14. The meristem has a wide range, division phases are concentrated, and the proportion of mitotic cells is relatively high. It is easy to observe chromosomes of metaphase cells desired in the research and analysis. Black arrows point to cells at the division phase. The disclosure collects and cuts the shoot apical meristem of the vigorously growing sugarcane or sugarcane related species into 0.3-0.5 cm3 small pieces, soaks in pre-cooled sterile ddH20, and pretreats the shoot apical meristem for 5-24 h and more preferably 12 h at 4°C to obtain the pretreated material.

The method provided by the disclosure is suitable for all sugarcane cultivars and plants with a close genetic relationship with sugarcane. In order to show that the technical solution of the disclosure can prepare chromosomal slide specimens with clear chromosome morphology and structure, dispersed and non-overlapping metaphase cells, and a clean background without impurities, the examples of the disclosure will be described in conjunction with sugarcane cultivars Saccharum officinarum cv. Badila, Saccharum cv. 57NG208, Saccharumcv. Mintang -611, Saccharum cv. RB72-454, F1 generation Saccharum cv. Yacheng 96-48, or BC 2 generation Saccharum cv. Yunzhe 07-86, but this should not be construed as only applicable to the above sugarcane cultivars. Meanwhile, the method provided by the disclosure is further applicable to all sugarcane related species; the sugarcane related species preferably include wild species of sugarcane and species of sugarcane related genera; the examples of the disclosure will be described in conjunction with the wild species of sugarcane Saccharum spontaneum and Saccharum robustum and preferred species of sugarcane related genera Saccharum arundinaceum,Miscanthus sinensis, Erianthusrockii and Saccharum narenga, but this should not be construed as only limiting to the above-mentioned species of sugarcane related genera. In the disclosure, the shoot apical meristem includes meristems at the base of the shoot tip of the sugarcane and/or 2-3 layers of spire meristems enveloping the shoot tip, and the 2-3 layers of spire meristems enveloping the shoot tip are spire meristems within 3 cm above the shoot tip. A shoot apical meristem of unflowered sugarcane differentiates into the spire meristems and the meristems at the base of the shoot tip of the sugarcane, and the base of the spire meristems is connected with the meristems at the base of the shoot tip. The disclosure selects meristems at the base of the shoot tip of the sugarcane and/or 2-3 layers of spire meristems enveloping the shoot tip for slide preparation. Thus, the meristems have a wide range, and cell division cycle is concentrated, which is suitable to be a material for preparing a slide of chromosomal spread. In the disclosure, the shoot apical meristem of the vigorously growing sugarcane or sugarcane related species is preferably collected at the jointing stage, elongation stage, or late elongation stage. In the disclosure, the shoot apical meristem of the vigorously growing sugarcane or sugarcane related species is preferably collected from 9:00 a.m. to 11:00 a.m. on a sunny day, and is delayed to 14:00 p.m. to 16:00 p.m. in winter. In the disclosure, the small piece is preferably 0.4 cm3 in volume. The small pieces of the shoot apical meristem of sugarcane are soaked in pre-cooled sterile ddH 20 and pretreated for -24 h at 4°C, which is beneficial to the simultaneous accumulation of metaphase cells. Compared with the conventional pretreatment protocol of 0.002 mol/L 8-hydroxyquinoline solution for 4 h at room temperature, the method obtains more metaphase cells and has a better effect; using this pretreatment method, the chromosome number of a sugarcane germplasm material can be identified within two days compared with 1 or 2 weeks required for root tips and axillary buds, shortening the experimental period and improving the efficiency of sugarcane karyotyping analysis. After the pretreatment material is obtained, the disclosure fixes the pretreatment material at 4°C for 2-3 days. After washing with sterile ddH 20, the pretreatment material is dissociated with a dissociation solution in a water bath at 50-65°C for 20-25 min to obtain a dissociated material; the dissociation solution is a mixture formed by isovolumic mixing of 1 mol/L HCl solution and % (v/v) acetic acid aqueous solution. In the disclosure, the fixing condition is preferably provided by a refrigerator. In the disclosure, Carnoy's fluid is preferably a fixative for the fixation; the Carnoy's fluid is preferably made by mixing absolute ethanol and glacial acetic acid in a volume ratio of 3:1. After the fixation, a slide can be observed; alternatively, after rinsing a sample twice with sterile ddH 20 for 10 min each time, the slide is stored in 70% alcohol at 4°C for long-term storage and later use. In the disclosure, the washing is preferably performed twice in order to completely remove the fixative. The dissociation is preferably carried out in a 60°C water bath; the dissociation preferably lasts for 20 min. The dissociation helps to make a fixed material soft, so that the cells are evenly spread on a glass slide during the subsequent slide pressing process. After the dissociated material is obtained, the disclosure transfers the dissociated material onto a glass slide, and stains the dissociated material with a modified carbopol fuchsin stain after removing water from the surface; the dissociated material is crushed, tissue residues are removed, and a coverslip is covered to stain for 4-8 min; the stain around the coverslip is removed, and the slide is pressed to prepare a glass slide specimen with chromosomes dispersed. In the disclosure, a method for removing the water from the surface is preferably to adopt absorbent paper to absorb the water on the surface of the dissociated material to eliminate the influence of the water on staining effect. The modified carbopol fuchsin stain was purchased from Kunming Yunke Biotechnology Co., Ltd. When crushing the dissociated material, action should be gentle without producing bubbles. Crushing the dissociated material is more conducive to obtaining a slide specimen with clear chromosome morphology and a clean background than directly tapping the slide without crushing the dissociated material. In the disclosure, a method for pressing the slide is preferably to tap the coverslip with one end of a pencil with an eraser, disperse the chromosomes, and make impurities diffuse to obtain a clean background of the slide; then press the coverslip with a thumb to make cells spread evenly, so that the chromosomes are on the same plane. In the disclosure, in order to verify the effect of the slide specimen of chromosomal spread prepared, it is preferable to perform microscopic examination. The microscopic examination preferably includes the following steps: observing microscopically, first under the low power lens (because sugarcane chromosomes are small, 20x or 40x objective lens is often used), and then under a high power lens (100x objective lens) after finding a proper field and metaphase cells, and then using Olympus Cell Sens Standard software to take pictures and count the numbers. Metaphase cells with clear chromosome morphology and structure are selected, and pictures are taken and karyotype analysis is performed by Zeiss Metasystem automatic karyotype analysis system. The disclosure provides a slide specimen of sugarcane chromosomal spread prepared by the method for preparing chromosomes from a shoot tip of sugarcane or sugarcane related species. The chromosomes are clear in morphology and structure, metaphase cells are dispersed and non-overlapping, and the background is clean without impurities. The disclosure provides use of the slide specimen of chromosomal spread of the sugarcane or sugarcane related species in the chromosome karyotype analysis of the sugarcane or sugarcane related species or the classification, identification, protection and utilization of germplasm resources. The efficient method for preparing chromosomes from a shoot tip of sugarcane or sugarcane related species provided by the disclosure will be described in detail below with reference to examples, but they should not be construed as limiting the protection scope of the disclosure. Example 1 Method for preparing chromosomes from a shoot tip of sugarcane 1. Using sugarcane as a test material, shoot apical meristems (see FIG. 2 for the morphology) of sugarcane collected in the field in the morning (9:00 a.m. to 11:00 a.m.) on a sunny day were cut into 0.3 cm 3 small pieces for pretreatment. 2. Pre-treatment: The shoot apical meristems were soaked in pre-cooled sterile ddH2 0, pre-treated for 12 h at 4°C, and then fixed with Carnoy's fluid (glacial acetic acid: ethanol = 1:3, freshly prepared) in a refrigerator at 4°C for three days. The meristems were placed in 70% ethanol and stored at 4°C for a long time. 3. When making slides, the stored meristematic materials were taken out and rinsed with sterile ddH 20 twice for 10 min each time. Then, a mixture of isometric 1 mol/L HCl solution and % (v/v) acetic acid aqueous solution was used for dissociation in a water bath at 60°C for 20 min. The meristematic materials were rinsed once with sterile ddH2 0 for 10 min. A shoot apical meristem was cut off and placed on a clean glass slide, 20 1 of modified carbopol fuchsin stain was added, the meristem was gently crushed with tweezers, tissue residues were removed, and a coverslip was covered to stain for 5 min at room temperature. 4. After staining, the slide was tapped with a pencil with an eraser to disperse the sugarcane meristem and cells on the slide evenly, so that the chromosomes were spread, impurities were diffused, and a clean background of the slide was obtained. Finally, the slide was pressed with a thumb to make the cells spread evenly, so that the chromosomes were on the same plane. 5. Observation was performed under an optical microscope, first under a low power lens, and then under a high power lens (100x objective lens) after finding a proper field and metaphase cells, for observation and photographing. Results Chromosome slide specimens with a wide range of sugarcane shoot apical meristem, concentrated cell division cycle, and readily available ideal metaphase were used. The results are shown in FIGS. 3 and 4. It can be seen from the chromosome distributions in FIGS. 3 and 4 that, using the chromosome slide specimen prepared by the above method, the chromosomes are clear in morphology and structure, metaphase cells are dispersed and non-overlapping, and the background is clean without impurities. Moreover, the operation is simple; a batch of 30-50 sheets of chromosome slide specimens can be prepared from a single shoot tip, and more metaphase cells can be observed on each slide. Generally, more than 20 cells convenient for chromosome number identification and karyotype analysis can be observed within 10 slides. Comparative Example 1 1. Using sugarcane as a test material, 0.8-1.0 cm root tips of barrel-grown sugarcane (see FIG. 5 for the morphology) were collected in the morning on a sunny day (9:00 a.m. to 11:00 a.m.) for pretreatment. 2. Pre-treatment: The root tips were soaked in pre-cooled sterile ddH2 0, pre-treated for 12 h at 4°C, and then fixed with Camoy's fluid (glacial acetic acid: ethanol = 1:3, freshly prepared) in a refrigerator at 4°C for three days. The root tips were placed in 70% ethanol and stored at 4°C for a long time. 3. When making slides, the stored root tip materials were taken out and rinsed with sterile ddH2 0 twice for 10 min each time. Then, a mixture of isometric 1 mol/L HCl solution and 45% (v/v) acetic acid aqueous solution was used for dissociation in a water bath at 60°C for 20 min. The meristematic materials were rinsed once with sterile ddH 20 for 10 min. A root apical meristem was cut off, 20 1 of modified carbopol fuchsin stain was added, the meristem was gently crushed with tweezers, tissue residues were removed, and a coverslip was covered to stain for 5 min at room temperature. 4. After staining, the slide was tapped with a pencil with an eraser to disperse the root tip tissue and cells on the slide evenly, so that the chromosomes were spread, impurities were diffused, and a clean background of the slide was obtained. Finally, the slide was pressed with a thumb to make the cells spread evenly, so that the chromosomes were on the same plane.

5. Observation was performed under an optical microscope, first under a low power lens, and then under a high power lens (100x objective lens) after finding a proper field and metaphase cells, for observation and photographing. Results It takes about six months for sugarcane to be able to collect the root tips from planting. Early harvesting leads to weak plants, so that it is difficult for the plants to grow later, and the root tip tissue samples are even worse. When collecting root tips, it is necessary to clean the sand and the operation is cumbersome; the sugarcane roots are fine, and there are few main roots and more fibrous roots; the meristem area is very small, and generally, one slide specimen can be prepared with 1-2 root tips; the root tips of sugarcane planted in barrel, sand or water is substantially affected by nutritional and environmental factors, and the thickness and age of the root tips are different; division cycle is difficult to grasp; only a few root tips in the mitotic phase are often present in a batch of root tip samples. There is no guarantee that the metaphase cells can be observed on each slide, so that it is necessary to repeatedly collect the root tips and prepare another slide to observe; after the sugarcane root tips are collected, the sugarcane has to be planted back into the barrel, and it takes 3-4 weeks to grow new roots to collect, which leads to an increase in workload; both test and experiment cycles are long, and in general, it usually takes about 1 or 2 weeks to identify the number of chromosomes in one material. According to experimental analysis, it is necessary to prepare and observe 50-100 slides of root tips to observe more than 20 cells for chromosome number identification and karyotype analysis, which requires a lot of time for slide preparation and microscopic observation. At the same time, preparing a slide with the root tip as a material requires repeated sampling, which substantially increases the workload and reduces the work efficiency. FIG. 6 is the observation result of a slide prepared with a root tip. Conclusion Compared with sugarcane root tip preparation technology, the use of sugarcane shoot tip chromosome preparation technology is characteristic of convenient sampling, concentrated division phases and readily available good metaphase. The identification of the chromosome number of one material can be completed within two days, so as to reduce the workload of the staff, and the efficiency increase by more than 7-8 times compared with that of root tip slide preparation. Comparative Example 2 Using sugarcane as a test material, sand-cultured axillary buds (see FIG. 7 for the morphology, where A illustrates the appearance of fresh bourgeoning buds removed from the stem segment, and B illustrates meristems of dissociated bourgeoning buds) were collected in the morning (9:00 a.m. to 11:00 a.m.) on a sunny day, slides were prepared according to the method of Example 1. Results The result is shown in FIG. 8. The meristem area of the axillary bud of sugarcane is small and division phases are not concentrated. The chromosome preparation effect is not as good as that of sugarcane shoot and root tips. Comparative Example 3 Slides were prepared by the method of Example 1, and the only difference was a step of treatment with pre-cooled ddH 20 for 5-24 h at 4°C, instead of 0.002 mol/L 8-hydroxyquinoline solution for 4 h at room temperature. In the disclosure, sugarcane shoot apical meristems are treated at 4°C, which is conducive to the simultaneous accumulation of metaphase cells. Through the slide pressing and observation, well-dispersed metaphase cells with clear chromosome morphology and structure were obtained in a short time, which were convenient for observation. The result was better than that obtained by the treatment with 0.002 mol/L 8-hydroxyquinoline solution for 4 h at room temperature. FIG. 9 illustrates the effect of chromosomes treated with 0.002 mol/L 8-hydroxyquinoline solution for 4 h at room temperature. The chromosome is not clear enough morphologically, and stickiness occurs readily. The 8-hydroxyquinoline solution is susceptible to environmental factors, and high temperature has a toxic effect on cells and chromosomes. Comparative Example 4 In order to examine the influence of different staining time on the effect of chromosome preparation, the disclosure adopts the method of Example 1 for preparation. However, when staining with modified 1 carbopol fuchsin stain at room temperature, staining was performed for 2, 5, and 10 min, respectively. The results are shown in FIG. 10. When staining for 2 min (FIG. 10A), the chromosomes in the slide is relatively light in color, which are not convenient for microscopic observation. When staining for 10 min (FIG. 10C), the chromosomes in the slide are too dark in color. When staining for 5 min (FIG. 1OB), the chromosomes show a moderate color. Comparative Example 5 In order to examine the influence of different dissociation time on the effect of chromosome preparation, the disclosure adopts the method of Example 1 for preparation. However, when dissociating with dissociation solution, dissociation time was set to 10, 15, 20, 25, and 30 min, respectively. The results are shown in FIG. 11. From FIG. 11, after dissociation for 10 and 15 min, cells are difficult to disperse with more overlapped chromosomes; after dissociation for 20 and 25 min, the cells are easy to disperse, with clear chromosomes with less overlaps; after dissociation for 30 min, the cells are easily broken, and chromosomes are lost or there are more overlaps. It can be seen that controlling the dissociation time between 20 and 25 min is the best for preparing chromosome slide specimens. Example 2 Identification of chromosome number of sugarcane parental species and germplasms of hybrid progenies 1. Original parent species of sugarcane Saccharum officinarum cv. Badila and Saccharum cv. 57NG208, hybrid parental species Saccharum cv. Mintang 70-611 and Saccharumcv. RB72-454, and germplasms of hybrid progenies Fi generation Saccharum cv. Yacheng 96-48, or BC 2 generation Saccharum cv. Yunzhe 07-86 were used as test materials. Sugarcane shoot apical meristems were collected for pretreatment on a sunny day from 9:00 a.m. to 11:00 a.m. 2. Meristems were put into a penicillin bottle, soaked in pre-cooled sterile ddH2 0, pre-treated for 20 h at 4°C, and fixed with freshly prepared Carnoy's fluid (glacial acetic acid: ethanol = 1:3) in a refrigerator at 4°C for 2-3 days. The meristems were placed in 70% ethanol and stored in the refrigerator at 4°C for later use. 3. The stored meristematic materials were taken out and rinsed twice with sterile ddH 20 for min each time. Then, a mixture of isometric 1 mol/L HCl solution and 45% (v/v) acetic acid aqueous solution was used for dissociation in a water bath at 60°C for 22 min. The meristematic materials were rinsed once with sterile ddH 20 for 10 min. A sugarcane root apical meristem was cut off, 20 1 of modified carbopol fuchsin stain was added, the meristem was gently crushed with tweezers, tissue residues were removed, and a coverslip was covered to stain for 5 min at room temperature. 4. After staining, the slide was tapped with a pencil with an eraser to disperse the sugarcane meristems and cells on the slide evenly, so that the chromosomes were spread, impurities were diffused, and a clean background of the slide was obtained. Finally, the slide was pressed with a thumb to make the cells spread evenly, so that the chromosomes were on the same plane. 5. Observation was performed under an optical microscope, first under a low power lens (because sugarcane chromosomes are very small, a 20x or 40x objective lens is generally used), and then under a high power lens (100x objective lens) after finding a proper field and metaphase cells, for observation and photographing. 6. As a result, a good picture of metaphase cells at the division phase was obtained by using the disclosure; the chromosomes were clear in morphology and structure and well-spread, which were convenient for counting. After observation and counting, the chromosome number identification results of the six test materials in this example were as follows: the somatic chromosome numbers of both Saccharum officinarum cv. Badila and Saccharum cv. 57NG208 are 2n = 80; those of Saccharumcv. Mintang -611 and Saccharum cv. RB72-454 were 2n = 105 and 2n = 112, respectively; the chromosome number of the germplasm material of hybrid progeny F generation Saccharumcv. Yacheng 96-48 was 2n = 80; the chromosome number of the germplasm material of hybrid progeny BC 2 generation Saccharum cv. Yunzhe 07-86 was 2n = 114 (see FIG. 12; the chromosomes the sugarcane shoot tips are complete, well-spread, and not overlapping, and the chromosome constriction is clear; the method of the disclosure can be used for the chromosome number identification and karyotype analysis of sugarcane parental species and germplasms of hybrid progenies). Example 3 Identification of chromosome numbers of wild species of sugarcane and species of sugarcane related genera Wild species of sugarcane, Saccharum spontaneum and Saccharum robustum, and species of sugarcane related genera, Saccharum arundinaceum, Miscanthus sinensis, Erianthus rockii and Saccharum narenga, contain many new resistance genes not found in sugarcane. With strong ratooning and excellent clumping ability, these characteristics are of great value in the cross utilization and genetic improvement of sugarcane. In-depth study of the chromosome ploidy and cytological genetic behavior of these resources will lay the foundation for the classification, identification, excavation and utilization of resources. In the disclosure, using wild species of sugarcane Saccharum spontaneum and Saccharum robustum and three sugarcane related species as test materials, shoot apical meristems were collected in the national sugarcane germplasm resource nursery from 9:00 a.m. to 11:00 a.m. on a sunny day to be pretreated. The objective was to explore whether the technical method of the disclosure was suitable for the chromosome analysis of Saccharum spontaneum and Saccharum robustum, as well as sugarcane related genera. After pretreatment, fixation, dissociation and staining, slice preparation, and microscopic examination, the results show that the disclosure is also suitable for the preparation of chromosomes of the shoot tips of wild species of sugarcane Saccharum spontaneum and Saccharum robustum, as well as species of sugarcane related genera. Many metaphase cells are obtained; the chromosomes are clear in morphology and structure and well-spread, which are convenient for chromosome number identification and karyotype analysis, laying the foundation for the gene mining and chromosomal genetic research of wild species and relative plants of sugarcane. The somatic chromosome number identification results of wild species of sugarcane

Saccharum spontaneum (Saccharum spontaneum cv. Vietnam No. 3) and Saccharum robustum (Saccharum robustum cv. 51NG3), and species of sugarcane related genera Saccharum arundinaceum (Saccharum arundinaceum cv. Yacheng No. 1), Miscanthus sinensis (Miscanthus sinensis cv. Jiangxi 2012-117), and Erianthus rockii (Erianthusrockiicv. Yunnan 2007-51) were 2n = 80, 125, 60, 38, and 30, respectively (see FIG. 13; sugarcane shoot tip chromosome preparation can be used for taxonomic identification of wild species of sugarcane and species of sugarcane related genera). The foregoing descriptions is merely preferred examples of the disclosure; it should be noted that various modifications and variations can also be made by one of ordinary skill in the art without departing from the principles of the disclosure and fall within the scope of the disclosure.

Claims

What is claimed is: 1. An efficient method for preparing chromosomes from a shoot tip of sugarcane or sugarcane related species, comprising the following steps: 1) collecting a shoot apical meristem of vigorously growing sugarcane or sugarcane related species and cutting into 0.3-0.5 cm3 small pieces, soaking the shoot apical meristem in pre-cooled sterile ddH 20, and pretreating the shoot apical meristem for 5-24 h at 4°C to obtain a pretreatment material; 2) fixing the pretreatment material at 4°C for 2-3 days; after washing with sterile ddH2 0, dissociating the pretreatment material with a dissociation solution in a water bath at 50-65°C for -25 min to obtain a dissociated material; wherein the dissociation solution is a mixture formed by isovolumic mixing of 1 mol/L HCl solution and 45% (v/v) acetic acid aqueous solution; and 3) transferring the dissociated material onto a glass slide, removing water from the surface, staining the dissociated material with modified carbol fuchsin stain, crushing the dissociated material, removing tissue residues, covering with a coverslip, and staining for 4-8 min; removing the stain around the coverslip, and pressing the slide to prepare a slide specimen of chromosomal spread.
2. The method for preparing chromosomes from a shoot tip of sugarcane or sugarcane related species according to claim 1, wherein the shoot apical meristem in step 1) comprises meristems at the base of the shoot tip of the sugarcane and/or 2-3 layers of spire meristems enveloping the shoot tip, and the 2-3 layers of spire meristems enveloping the shoot tip are spire meristems within 3 cm above the shoot tip.
3. The method for preparing chromosomes from a shoot tip of sugarcane or sugarcane related species according to claim 1, ; wherein the shoot apical meristem of the vigorously growing sugarcane or sugarcane related species in step 1) is collected at the jointing stage, elongation stage, or late elongation stage.
4. The method for preparing chromosomes from a shoot tip of sugarcane or sugarcane related species according to any one of claims 1 to 3, wherein the shoot apical meristem of the vigorously growing sugarcane or sugarcane related species in step 1) is collected from 9:00 a.m. to 11:00 a.m. on a sunny day.
5. The method for preparing chromosomes from a shoot tip of sugarcane or sugarcane related species according to claim 1, wherein Carnoy's fluid is a fixative for the fixation in step 2); the Carnoy's fluid is made by mixing absolute ethanol and glacial acetic acid in a volume ratio of 3:1.
6. The method for preparing chromosomes from a shoot tip of sugarcane or sugarcane related species according to claim 1, wherein a method for pressing the slide in step 3) is to tap the coverslip with one end of a pencil with an eraser, disperse the chromosomes, and then press the coverslip with a thumb to make cells spread evenly, so that the chromosomes are on the same plane.
7. The method for preparing chromosomes from a shoot tip of sugarcane or sugarcane related species according to any one of claims 1, 2, 3, 5 and 6, wherein the cultivars of the sugarcane comprises Saccharum officinarum cv. Badila, Saccharum cv. 57NG208, Saccharumcv. Mintang -611, Saccharum cv. RB72-454, F1 generation Saccharum cv. Yacheng 96-48, or BC 2 generation Saccharumcv. Yunzhe 07-86.
8. The method for preparing chromosomes from a shoot tip of sugarcane or sugarcane related species according to any one of claims 1, 2, 3, 5 and 6, wherein the sugarcane related species comprises wild species of sugarcane and species of sugarcane related genera; the wild species of sugarcane comprises Saccharum spontaneum and Saccharum robustum; the species of sugarcane related genera comprises Saccharum arundinaceum, Miscanthus sinensis, Erianthusrockii and Saccharum narenga.
9. A slide specimen of sugarcane chromosomal spread prepared by the method for preparing chromosomes from a shoot tip of sugarcane or sugarcane related species according to any one of claims 1 to 8, wherein the chromosomes are clear in morphology and structure, metaphase cells are dispersed and non-overlapping, and background is clean without impurities.
10. Use of the slide specimen of chromosomal spread of the sugarcane or sugarcane related species according to claim 9 in the chromosome karyotype analysis of the sugarcane or sugarcane related species or the classification, identification, protection and utilization of germplasm resources.