Method for preparing tobacco metaphase chromosome from tender tobacco plant and application of tobacco metaphase chromosome

Abstract

The invention discloses a method for preparing tobacco metaphase chromosomes by utilizing young tobacco plants and application, and belongs to the technical field of preparation of tobacco chromosome specimens. The method comprises the following steps: germinating tobacco seeds at room temperature for 5-7 days, stripping seed shells when the seeds germinate and radicles grow to about 2 mm, sequentially performing N2O treatment on seedlings with radicles, fixing with a stationary liquid, performing enzymolysis in an enzymatic hydrolysate, lightly grinding plant tissues by using a sterile grinding rod until cells are dispersed, centrifuging, airing, adding glacial acetic acid, performing uniform vortex mixing, and performing freeze drying to obtain the tobacco seed suspension. And dripping a small amount of suspension on a clean glass slide to prepare the tobacco metaphase chromosome specimen. According to the method, materials are easy to obtain, the period is extremely short, operation is safe, the chromosome quality is high, and a reliable and efficient standardized scheme is provided for tobacco cytogenetics, molecular cytology and breeding research.

Description

Technical Field

[0001] This invention relates to the field of tobacco chromosome specimen preparation technology, specifically to a method and application for preparing tobacco metaphase chromosomes using young tobacco plants. Background Technology

[0002] tobacco( Nicotiana tabacum Tobacco mono (SST) is one of my country's important economic crops, and its yield and quality directly affect the development of my country's agricultural economy. Over the long term, common tobacco varieties have faced the dual threats of pests and diseases and environmental stress. Some varieties have experienced a decline in resistance and quality, making the need for genetic improvement of common tobacco increasingly urgent.

[0003] Wild tobacco resources are abundant (approximately 70 species), and through long-term natural selection, they have evolved adaptability to harsh environments, accumulating a large number of superior genes for stress resistance and disease resistance. This serves as a natural "gene bank" for broadening the genetic base of common tobacco and exploring key breeding genes. Distant hybridization is a core technical pathway for introducing superior genes from wild tobacco into common tobacco. Currently, some disease-resistant genes have been introduced into common tobacco through distant hybridization. In the process of tobacco distant hybridization breeding, the preparation of tobacco chromosome specimens is of great significance: 1) It can determine the number and composition of chromosomes, providing "visible, countable, and distinguishable" intuitive materials for chromosome engineering, facilitating karyotype analysis, identification of chromosome structural variations, and precise identification and tracking of exogenous chromosomes, thereby improving the efficiency and reliability of distant hybridization and resistance breeding. 2) Chromosome preparation is a prerequisite for molecular cytogenetics techniques such as fluorescence in situ hybridization (FISH), and can be used to locate molecular markers such as rDNA and analyze chromosome structure and evolution. 3) It enhances the usability of tobacco as a model plant in cytogenetics.

[0004] However, the preparation of metaphase chromosomes in tobacco remains a bottleneck. Existing techniques for identifying tobacco chromosomes are not perfect, limiting the accurate identification of chromosome structural variations and exogenous chromosomes, thus hindering the in-depth development of tobacco distant hybridization breeding. Current methods mainly include: 1) The root tip method for preparing metaphase chromosomes using root tips: This method requires culturing tobacco seeds for 30-45 days until the seedlings reach the "three leaves and one heart" stage. After transplanting, once new roots have grown, 1-2 cm of slender root tips (only 0.3-0.5 mm in diameter) are cut and prepared into slides. The initial material preparation period is as long as 40-60 days, resulting in a long experimental cycle; 2) The ovary method for preparing metaphase chromosomes using ovaries: This method selects tobacco plants at a specific developmental stage, waits for the plants to grow to the flowering and budding stage, and then screens out young ovaries with a corolla length less than or equal to half the calyx length. The preparation of ovaries for the early stages of seedling cultivation and flowering takes several weeks to months, and only a portion of the flower buds are harvested. The operation is cumbersome and the entire process is time-consuming, requiring multiple steps such as ovary screening, 24-hour low-temperature pretreatment, overnight fixation, and enzymatic digestion. It takes more than 2 days from material collection to obtaining chromosome specimens. The process is complex and time-consuming. The experimental cycle is long and the adaptability is poor, depending on the flowering and budding of the plants. The early culture period is long. For tobacco varieties that grow slowly or are sensitive to the environment, abnormal flower bud development can easily prolong the cycle or lead to failure in slide preparation. The pretreatment process uses 8-hydroxyquinoline solution, which is carcinogenic. Long-term operation can pose a potential threat to the health of experimental personnel through contact or inhalation.

[0005] Therefore, there is an urgent need for a method to shorten the preparation time of metaphase chromosomes in tobacco and obtain high-quality chromosome specimens. Summary of the Invention

[0006] This invention provides a method and application for preparing tobacco metaphase chromosomes using young tobacco plants. The method provided by this invention can shorten the preparation time of tobacco metaphase chromosomes, solving the problems of long preparation time and low efficiency in existing technologies.

[0007] To achieve the above-mentioned objectives, the technical solution adopted by this invention is: a method for preparing metaphase chromosomes of tobacco using young tobacco plants, comprising the following steps: Seed germination and pretreatment: Select plump tobacco seeds, place them in a petri dish, add an appropriate amount of distilled water to moisten the seeds, and germinate at 20℃-26℃ for 5-7 days, keeping the tobacco seeds moist and clean throughout the period; when the seeds germinate and the radicle grows to 1.5mm-2.5mm, peel off the seed coat, and place the seedlings with radicles into perforated centrifuge tubes moistened with ddH2O, and keep them on ice for 1-5 minutes; then place the centrifuge tubes in a sealed container filled with N2O for further treatment; after treatment, add fixative to completely submerge the seedlings, and fix in an ice bath for 10-15 minutes; after fixation, rinse with ddH2O, and finally add 70% ethanol, and store in a sealed container at -15℃ to -25℃; Metaphase chromosome preparation: Remove the seedlings from the refrigerator and allow them to return to room temperature. Rinse with ddH2O to remove any residual ethanol, blot dry the surface moisture, and transfer them to a centrifuge tube containing enzymatic hydrolysate. Centrifuge briefly for 10-15 seconds to ensure the tissue is completely submerged in the hydrolysate for enzymatic hydrolysis. After hydrolysis, place the centrifuge tube on ice to stop the reaction. Rinse with 70% ethanol, leaving a small amount of ethanol. Gently grind the plant tissue with a sterile grinding rod until the cells are dispersed. Centrifuge, discard the supernatant, and air dry. Add glacial acetic acid, vortex to mix, and then drop a small amount of the suspension onto a clean glass slide. Allow it to air dry naturally to obtain a slide containing tobacco metaphase chromosomes.

[0008] Furthermore, the method also includes a preservation step: after the slide has been air-dried, it is observed under an optical microscope to select fields with clear metaphase division and mark the positions on the side of the slide with a pencil; the treated slide is then packaged in a sealed bag and placed in a 4°C refrigerator for short-term preservation or placed in a -20°C refrigerator for long-term preservation.

[0009] Furthermore, in the seed germination and pretreatment steps, plump tobacco seeds were selected, placed in a petri dish lined with two layers of sterile filter paper, and an appropriate amount of distilled water was added to moisten the filter paper. Germination was carried out at 20℃-26℃ for 5-7 days.

[0010] Furthermore, in the seed germination and pretreatment step, the centrifuge tubes were placed in a sealed container filled with N2O for 2-3 hours.

[0011] Furthermore, in the seed germination and pretreatment steps, the fixative is 90%-95% glacial acetic acid pre-cooled at 3℃-5℃.

[0012] Furthermore, during the seed germination and pretreatment steps, 70% ethanol is added, ensuring that the ethanol completely covers the seedling root tips.

[0013] Furthermore, in the metaphase chromosome preparation step, the enzymatic digest consists of 1%-3% cellulase, 0.5%-1.5% pectinase, and the remainder water by mass percentage.

[0014] Furthermore, in the metaphase chromosome preparation step, the enzymatic digestion conditions are: 36℃-38℃ water bath digestion for 70-75 minutes.

[0015] Furthermore, in the preservation step, screening for clear metaphases means screening for well-dispersed and morphologically intact chromosomes.

[0016] The application of the method provided by this invention in the preparation of tobacco metaphase chromosome specimens.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: The method provided by this invention utilizes whole tobacco plants that have germinated for 5-7 days to prepare metaphase chromosomes, shortening the preparation time and improving the efficiency of metaphase chromosome preparation in terms of material collection time. Microscopic observation of the optimized tobacco chromosome preparation method provided by this invention reveals that a large number of high-quality metaphase cells can be stably obtained from the prepared specimens. Under the microscope, chromosomes are of uniform and moderate length, with clear and unobstructed edges, and are well-dispersed (without overlap or adhesion), allowing for clear differentiation of the morphological characteristics of individual chromosomes. This lays a reliable cytological foundation for subsequent chromosome counting, karyotype analysis, and ND-FISH experiments. This invention offers readily available materials, a very short cycle time, safe operation, and high-quality chromosomes, providing a reliable and efficient standardized solution for tobacco cytogenetics, molecular cytology, and breeding research. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a microscopic image of the chromosomes (scale bar 10 μm) of a tobacco mid-stage chromosome specimen prepared using the method described in this application from young plants of Yunyan 85.

[0020] Figure 2 This is a microscopic image of a tobacco mid-stage chromosome specimen (scale bar: 10 μm) prepared using the method described in this application from young tobacco plants of Yunyan 87.

[0021] Figure 3 This is a microscopic image (scale bar 10 μm) of a tobacco metaphase chromosome specimen prepared using the method described in this application from young tobacco TN90 plants. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0023] A first aspect of this invention provides a method for preparing tobacco metaphase chromosomes using young tobacco plants, comprising the following steps: Seed germination and pretreatment: Select plump tobacco seeds, place them in a petri dish, add an appropriate amount of distilled water to moisten the seeds, and germinate at 20℃-26℃ for 5-7 days, keeping the tobacco seeds moist and clean throughout the period; when the seeds germinate and the radicle grows to 1.5mm-2.5mm, peel off the seed coat, and place the seedlings with radicles into perforated centrifuge tubes moistened with ddH2O, and keep them on ice for 1-5 minutes; then place the centrifuge tubes in a sealed container filled with N2O for further treatment; after treatment, add fixative to completely submerge the seedlings, and fix in an ice bath for 10-15 minutes; after fixation, rinse with ddH2O, and finally add 70% ethanol, and store in a sealed container at -15℃ to -25℃; Metaphase chromosome preparation: Remove the seedlings from the refrigerator and allow them to return to room temperature. Rinse with ddH2O to remove any residual ethanol, blot dry the surface moisture, and transfer them to a centrifuge tube containing enzymatic hydrolysate. Centrifuge briefly for 10-15 seconds to ensure the tissue is completely submerged in the hydrolysate for enzymatic hydrolysis. After hydrolysis, place the centrifuge tube on ice to stop the reaction. Rinse with 70% ethanol, leaving a small amount of ethanol. Gently grind the plant tissue with a sterile grinding rod until the cells are dispersed. Centrifuge, discard the supernatant, and air dry. Add glacial acetic acid, vortex to mix, and then drop a small amount of the suspension onto a clean glass slide. Allow it to air dry naturally to obtain a slide containing tobacco metaphase chromosomes.

[0024] Specifically, this invention utilizes whole tobacco plants that have germinated for 5-7 days to prepare metaphase chromosomes, significantly shortening the preparation time and improving the efficiency. The prepared tobacco metaphase chromosome specimens can stably yield a large number of high-quality metaphase cells. The method of this application can completely replace the traditional "root tip method" and "ovary method," shortening the preparation cycle by more than 85% compared to the root tip method (40-60 days) and the ovary method (several weeks to months). Seedlings are treated with N2O in a sealed environment for 2 hours, replacing the carcinogenic 8-hydroxyquinoline and eliminating the potential threat to the health of laboratory personnel.

[0025] In some feasible implementations, the method also includes a preservation step: after the slide has been air-dried, it is observed under an optical microscope to select fields of view with clear metaphases and marks the positions on the side of the slide with a pencil; the treated slide is then packaged in a sealed bag and placed in a 4°C refrigerator for short-term preservation or in a -20°C refrigerator for long-term preservation.

[0026] Specifically, under an optical microscope, fields of view showing clear metaphase are selected, and the locations are marked on the side of the slide with a pencil to facilitate quick and accurate location of target cells during observation under a fluorescence microscope. Depending on the subsequent experimental cycle, cells are stored either for short periods at 4°C or long periods at -20°C to avoid repeated freeze-thaw cycles affecting chromosome morphology, ensuring safety and long-term storage.

[0027] In some feasible implementations, during the seed germination and pretreatment steps, plump tobacco seeds are selected, placed in a petri dish lined with double-layered sterile filter paper, and moistened with an appropriate amount of distilled water. The seeds are then germinated at 20℃-26℃ for 5-7 days.

[0028] Specifically, tobacco seeds are placed between two layers of sterile filter paper, ensuring that the seeds have enough moisture to germinate while preventing them from being submerged and unable to germinate.

[0029] In some feasible implementations, during the seed germination and pretreatment steps, centrifuge tubes are placed in a sealed container filled with N2O for 2-3 hours.

[0030] Specifically, the pretreatment stage of this invention uses safe nitrous oxide (N2O), avoiding the health risks posed by carcinogenic agents. Through a 2-3 hour treatment, more nitrous oxide can penetrate the material cells and act on the microtubules, causing more cells to arrest in metaphase of mitosis.

[0031] In some feasible implementations, the fixative in the seed germination and pretreatment steps is 90%-95% glacial acetic acid pre-cooled at 3°C-5°C.

[0032] Specifically, the purpose of fixation is to allow the treated material cells to remain in metaphase II of mitosis for the next division. 90%-95% glacial acetic acid can rapidly penetrate the cells of the material. Pre-cooling the 90%-95% glacial acetic acid is to prevent the rapid evaporation of the high concentration of glacial acetic acid.

[0033] In some feasible implementations, 70% ethanol is added during the seed germination and pretreatment steps, such that the ethanol covers the seedling root tips.

[0034] Specifically, using 70% ethanol to preserve materials can prevent material spoilage and also prevent excessive dehydration of tissues.

[0035] In some feasible implementations, during the metaphase chromosome preparation step, the enzymatic digest consists of 1%-3% cellulase, 0.5%-1.5% pectinase, and the remainder water by weight percentage.

[0036] Specifically, 1%-3% cellulase and 0.5%-1.5% pectinase can effectively digest the fiber and pectin substances in plant cell walls, and can effectively break down cell walls, thereby releasing metaphase chromosomes in the cells.

[0037] In some feasible implementations, the enzymatic digestion conditions in the metaphase chromosome preparation step are: 36℃-38℃ water bath digestion for 70-75 minutes.

[0038] Specifically, cellulase and pectinase exhibit the best activity at 36℃-38℃, and treatment for 70-75 minutes ensures cell wall degradation while avoiding excessive cell degradation.

[0039] In some feasible implementations, in the preservation step, screening for a clear field of view of metaphases means screening for well-dispersed and morphologically intact chromosomes.

[0040] Specifically, well-dispersed and morphologically intact chromosomes are beneficial for chromosome counting and accurate observation of chromosome structure.

[0041] A second aspect of the present invention provides the application of the above method in the preparation of tobacco metaphase chromosome specimens.

[0042] Specifically, this invention addresses the shortcomings of existing technologies: the root tip method requires culturing seeds for 30-45 days until they reach the "three leaves and one bud" stage, followed by transplanting and harvesting root tips after new roots have grown, taking 40-60 days in the early stages; and the ovary method requires waiting for the plant to grow to the flowering and budding stage, resulting in a material preparation cycle of several weeks to months from seedling cultivation to flowering, and only a portion of the flower buds are harvested to obtain the ovary, leading to a long experimental cycle. This invention uses short-germinating seedlings with radicles as the core material for preparing tobacco mid-phase chromosomes. It only requires germinating seeds on filter paper moistened with distilled water at 25℃ and pH 7.0 for 5-7 days, and then removing the seed coat when the radicle is about 2mm long. The material acquisition cycle is only 5-7 days, significantly shortening the experimental cycle.

[0043] Example 1 In this embodiment, young plants of different tobacco materials, namely Yunyan 85, Yunyan 87 and TN90, were used to prepare mid-term chromosomes of tobacco.

[0044] (I) Method for preparing metaphase chromosomes of tobacco using young tobacco plants The method includes the following steps: 1. Seed germination and pretreatment Plump seeds of tobacco varieties Yunyan 85, Yunyan 87, and TN90 were selected and placed in petri dishes lined with double-layered sterile filter paper. The seeds were positioned between the filter papers, and the filter paper was moistened with a suitable amount of distilled water (pH 7.0). Germination was carried out at 25℃ for 5-7 days, during which time the filter paper was kept moist and clean. On the 6th day, the seeds germinated, and the radicle grew to approximately 2 mm. The seed coat was removed, and the seedlings with radicles were placed in perforated 0.5 mL centrifuge tubes moistened with ddH2O. The tubes were then placed on ice for 2 minutes. The centrifuge tubes were then placed in a sealed container filled with N2O for 2 hours. After treatment, 90% glacial acetic acid fixative (pre-cooled to 4℃, completely submerging the seedlings) was added, and the seedlings were fixed in an ice bath for 10 minutes. After fixation, the seedlings were rinsed 3-5 times with ddH2O to remove residual fixative. Finally, 70% ethanol (enough to cover the root tips) was added, and the seedlings were sealed and stored at -20℃ for later use.

[0045] 2. Metaphase chromosome preparation Remove the seedlings stored at -20℃ from the freezer, thaw them at room temperature, and rinse them three times with ddH2O to remove any residual ethanol. Remove the seedlings with tweezers, blot off the surface moisture with filter paper, and transfer them to a 0.5mL centrifuge tube containing 20μL of enzymatic hydrolysate (2wt% cellulase + 1wt% pectinase + the remainder water). Centrifuge briefly for 10 seconds to ensure the tissue is completely submerged in the hydrolysate, and then incubate at 37℃ for 70 minutes. After enzymatic digestion, the centrifuge tube was placed on ice to stop the reaction. It was then rinsed 3-5 times with 70% ethanol, retaining approximately 100 μL of ethanol. The plant tissue was gently ground with a sterile grinding rod until the cells were dispersed. The tube was centrifuged at 4500 rpm for 3 minutes, the supernatant was discarded, and the centrifuge tube was inverted to air dry any remaining ethanol. 100 μL of 100% glacial acetic acid was added, and the mixture was vortexed. 8 μL of the suspension was then dropped onto a clean glass slide and placed in a slide container pre-sprayed with ddH2O to air dry naturally (until no visible water droplets remain). The dried slide was then observed under an Olympus BX51 optical microscope. Fields of view showing clear metaphase (well-dispersed chromosomes with intact morphology) were selected, and the positions were marked on the side of the slide with a pencil. This completed the preparation of a slide containing tobacco metaphase chromosomes, serving as a tobacco metaphase chromosome specimen. The results of the Olympus BX51 optical microscope observation are as follows: Figure 1 , Figure 2 and Figure 3 .

[0046] 3. Save High-quality slides can be used directly in subsequent experiments after being treated with an ultraviolet crosslinker. Slides requiring short-term storage should be sealed in bags and stored at 4°C, while slides requiring long-term storage should be sealed in bags and stored at -20°C to avoid repeated freeze-thaw cycles that could affect chromosome morphology.

[0047] (II) Results Figure 1Metaphase chromosomes obtained from young plants of Yunyan 85. Figure 2 Metaphase chromosomes obtained from young plants of Yunyan 87. Figure 3 Metaphase chromosomes obtained from young TN90 plants. Figures 1-3 As can be seen, chromosome specimens prepared using the method described in this application, when examined under an Olympus BX51 optical microscope, yielded stable, abundant, and high-quality metaphase cells. The obtained chromosomes were of uniform and moderate length, with clear and unambiguous edges, and were well-dispersed among each other (without overlap or adhesion). The morphological characteristics of individual chromosomes could be clearly distinguished, laying a reliable cytological foundation for subsequent chromosome counting, karyotype analysis, and ND-FISH experiments.

[0048] It will be readily understood by those skilled in the art that the above-described advantageous methods can be freely combined and superimposed without conflict. The above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application. The above are merely preferred embodiments of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of this application, and these improvements and modifications should also be considered within the protection scope of this application.

Claims

1. A method for preparing metaphase chromosomes of tobacco using young tobacco plants, characterized in that, The method includes the following steps: Seed germination and pretreatment: Select plump tobacco seeds, place them in a petri dish, add an appropriate amount of distilled water to moisten the seeds, and germinate at 20℃-26℃ for 5-7 days, keeping the tobacco seeds moist and clean throughout the period; when the seeds germinate and the radicle grows to 1.5mm-2.5mm, peel off the seed coat, and place the seedlings with radicles into perforated centrifuge tubes moistened with ddH2O, and keep them on ice for 1-5 minutes; then place the centrifuge tubes in a sealed container filled with N2O for further treatment; after treatment, add fixative to completely submerge the seedlings, and fix in an ice bath for 10-15 minutes; after fixation, rinse with ddH2O, and finally add 70% ethanol, and store in a sealed container at -15℃ to -25℃; Metaphase chromosome preparation: Remove the seedlings from the refrigerator and allow them to return to room temperature. Rinse with ddH2O to remove any residual ethanol, blot dry the surface moisture, and transfer them to a centrifuge tube containing enzymatic hydrolysate. Centrifuge briefly for 10-15 seconds to ensure the tissue is completely submerged in the hydrolysate for enzymatic hydrolysis. After hydrolysis, place the centrifuge tube on ice to stop the reaction. Rinse with 70% ethanol, leaving a small amount of ethanol. Gently grind the plant tissue with a sterile grinding rod until the cells are dispersed. Centrifuge, discard the supernatant, and air dry. Add glacial acetic acid, vortex to mix, and then drop a small amount of the suspension onto a clean glass slide. Allow it to air dry naturally to obtain a slide containing tobacco metaphase chromosomes.

2. The method for preparing tobacco metaphase chromosomes using young tobacco plants according to claim 1, characterized in that, The preservation steps include: after the slides have been air-dried, observe them under an optical microscope, select fields with clear metaphase divisions, and mark the positions on the side of the slides with a pencil; after the slides have been treated, pack them in a sealed bag and store them in a 4°C refrigerator for short-term storage or in a -20°C refrigerator for long-term storage.

3. A method for preparing tobacco metaphase chromosomes using young tobacco plants according to claim 1 or 2, characterized in that, In the seed germination and pretreatment steps, plump tobacco seeds were selected, placed in a petri dish lined with double-layer sterile filter paper, and moistened with an appropriate amount of distilled water. Germination was carried out at 20℃-26℃ for 5-7 days.

4. A method for preparing tobacco metaphase chromosomes using young tobacco plants according to claim 1 or 2, characterized in that, In the seed germination and pretreatment steps, centrifuge tubes were placed in a sealed container filled with N2O for 2-3 hours.

5. A method for preparing tobacco metaphase chromosomes using young tobacco plants according to claim 1 or 2, characterized in that, In the seed germination and pretreatment steps, the fixative is 90%-95% glacial acetic acid pre-cooled at 3℃-5℃.

6. A method for preparing tobacco metaphase chromosomes using young tobacco plants according to claim 1 or 2, characterized in that, During the seed germination and pretreatment steps, 70% ethanol is added, ensuring that the ethanol completely covers the root tips of the seedlings.

7. A method for preparing tobacco metaphase chromosomes using young tobacco plants according to claim 1 or 2, characterized in that, In the metaphase chromosome preparation step, the enzymatic digest consists of 1%-3% cellulase, 0.5%-1.5% pectinase, and the remainder water by weight percentage.

8. A method for preparing tobacco metaphase chromosomes using young tobacco plants according to claim 1 or 2, characterized in that, In the metaphase chromosome preparation process, the enzymatic digestion conditions are: 36℃-38℃ water bath digestion for 70-75 minutes.

9. A method for preparing tobacco metaphase chromosomes using young tobacco plants according to claim 2, characterized in that, In the preservation step, screening for clear metaphases means screening for well-dispersed and morphologically intact chromosomes.

10. The use of the method according to any one of claims 1-9 in the preparation of tobacco metaphase chromosome specimens.

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