Method and system for identifying bougainvillea glabra karyotype chromosome number
Through a method including pretreatment, fixation, dissociation, water absorption expansion and staining, the number of chromosomes of bougainvillea was successfully identified, solving the problem of identification in the prior art and improving the breeding success rate.
Patent Information
- Application Number
- CN202510256621.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-05
- Publication Date
- 2025-06-06
AI Technical Summary
The prior art is difficult to effectively identify and identify the number of chromosomes of bougainvillea, resulting in difficulties and uncertainties in breeding.
A method including pretreatment, fixation, dissociation, water absorption and expansion and staining is adopted to prepare cell chromosome preparations of bougainaceae by using chemical agents such as saturated p-dichlorobenzene solution, α-bromonaphthalene, cycloheximide, paraformaldehyde fixation solution, HCl solution, KCl solution and Kabao solution, and microscopic examination and photographing are performed to determine the number of chromosomes.
It has achieved efficient identification of the number of chromosomes of bougainvillea, which can clarify the cardinality of chromosomes, and provides important guidance for traditional hybrid breeding of bougainvillea to improve the breeding success rate.
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Figure CN120102254A_ABST
Abstract
Description
Technical Field
[0001] The invention provides a method and system for identifying the number of chromosomes of bougainvillea karyotype. Background Art
[0002] Bougainvillea (Bougainvillea spp.), also known as bougainvillea, bougainvillea, scarf flower, triangle flower, and rhododendron, is an evergreen climbing shrub belonging to the Caryophyllales family. Because of its colorful bracts, it has become one of the most popular garden, greening and landscaping flower crops in the world.
[0003] At present, most of the bougainvillea varieties in my country are introduced, and there are few bougainvillea varieties cultivated independently, which has a great impact on the profits of the bougainvillea industry (Yang Guangsui et al., 2018). Therefore, improving the breeding efficiency of bougainvillea and creating new varieties of bougainvillea are effective means to promote the development of the bougainvillea industry. Karyotype analysis refers to the analysis and research of the number, morphology, length, banding type and centromere position of cell chromosomes. It is an effective means to solve the breeding difficulties caused by unclear chromosome number in hybrid breeding.
[0004] At present, there are some problems in the breeding of Bougainvillea: (1) The existing Bougainvillea variety resources are mixed and the relationship is unclear, which brings great difficulties to hybrid breeding; (2) The chromosome karyotype of organisms is quite fixed. Therefore, it can be used as an important means of botanical classification and genetic research, so that scholars have more reference materials when studying plants with controversial classification to reach a consensus. However, according to existing literature reports, the karyotype of Bougainvillea is diverse, namely 2n=20, 2n=28, 2n=32, 2n=34 and 2n=48 (Vaidya & Manandhar, 2013; Data SK2021; Sun Lina et al., 2019), so there is controversy about the chromosome base number of Bougainvillea. (3) It has been reported that there are more than 1,000 varieties of Bougainvillea, and the number of chromosomes is indeed very rich, such as 2n = 28, 2n = 34, 2n = 32, 2n = 20, 2n = 48, 3n = 51 and 4n = 68 (Vaidya & Manandhar, 2013; Data SK2021; Sun Lina et al., 2019). The main influencing factor of Bougainvillea's self-incompatibility is the mixed chromosome number and unclear ploidy.
[0005] In summary, only by understanding the number of chromosomes of each variety can we further understand the relationship of Bougainvillea, provide important guiding strategies for hybrid breeding, and improve the success rate of hybrid breeding. Only by conducting a detailed investigation of the number of chromosomes of each variety of Bougainvillea and understanding the number of chromosomes of existing varieties can we obtain better varieties through hybrid breeding. Therefore, studying the number of chromosomes of Bougainvillea and clarifying whether the karyotype of Bougainvillea is the same has become the most basic and most urgent problem at present. Summary of the invention
[0006] The purpose of the present invention is to provide a method and system for identifying the number of chromosomes in Bougainvillea karyotype, so as to achieve efficient analysis.
[0007] In order to achieve the above object, one of the objects of the present invention is to provide a method for identifying the number of chromosomes of Bougainvillea karyotype, comprising the following steps:
[0008] S1. Pre-treat the root tip of Bougainvillea in a mixed solution containing saturated p-dichlorobenzene solution, 5-20 μL α-bromonaphthalene and 0.008% 15-30 μL cycloheximide for 3-5 h;
[0009] S2, place the root tip obtained in S1 in paraformaldehyde fixative for 12-24h;
[0010] S3, take out the root apex obtained in S2, clean it, put it in HCl solution, and place it in a water bath for 8-10 minutes;
[0011] S4. Soak the root tip obtained in S3 in 0.075 mol / L KCl solution for 120-180 s;
[0012] S5. Place the root cap and meristem of the root tip into Carbosol solution and stain for 20-40 minutes;
[0013] S6. Take the root cap area and meristem area of the root tip and place them on a clean glass slide to obtain cell chromosome preparations, and take photos under a microscope.
[0014] In the identification method of the present invention, S1 is used to fix cells in the cell division stage. If only bromonaphthalene is added, the chromosome shrinking effect is poor. When cycloheximide is added, the chromosome shrinking effect is significantly improved. However, when α-bromonaphthalene is less than 5 μL and the pretreatment is less than 3 hours, the chromosome shrinking effect is poor. When α-bromonaphthalene is greater than 20 μL and the pretreatment is greater than 5 hours, the fixation effect cannot be achieved and the chromosome degradation cannot be seen.
[0015] In the identification method of the present invention, S2 is used to fix the cells treated with the above pretreatment solution. If the fixation time is greater than 24 hours or less than 12 hours, the cells will be damaged, thereby causing the loss of chromosomes in the cells. When the fixation time is 12-24 hours, the fixation effect is best.
[0016] In the identification method of the present invention, S3 is used for dissociation of the cell wall. When the dissociation time is greater than 10 minutes, the young leaves may have cell damage problems. When the dissociation time is less than 8 minutes, the cell wall of the young leaves may not be completely dissociated, which affects the staining of the cells and the observation of chromosomes. When the dissociation time is 8-10 minutes, the dissociation effect is best.
[0017] In the identification method of the present invention, S4 is used for water absorption and swelling of cells, which promotes easy dispersion of chromosomes during tableting. If the immersion time is greater than 180s, the material will have cell damage, causing the loss of chromosomes in the cells. If the immersion time is less than 120s, the cell water absorption of the material cannot reach the saturated state, which affects the poor dispersion of cells during tableting. When soaked for 120-180s, the water absorption and swelling effect of the material is good. Washing with 0.075mol / L KCl solution can swell the cells well, promote the good dispersion of chromosomes after tableting, and make it easier to clearly identify the number and morphology of chromosomes.
[0018] In the identification method of the present invention, S5 is used to dye the chromosomes to facilitate the observation of the number of chromosomes. When the staining time is greater than 40 minutes, the color of the cytoplasm of the material will also deepen, increasing the color of the background, making it difficult to distinguish the chromosomes well. When the staining time is less than 20 minutes, the chromosomes of the cells cannot be stained, and thus the chromosomes are difficult to observe. When the staining is for 20-40 minutes, the chromosome staining and the background of the cells can be easily distinguished, and the chromosomes are clearly visible, with a good effect.
[0019] Preferably, in S2, the preparation method of the paraformaldehyde fixative is: 2 O, 1M NaOH, paraformaldehyde, 1M Na-P Buffer, and 50% glutaraldehyde were mixed and the volume was adjusted to 100 mL.
[0020] Preferably, in S4, the solvent of the KCl solution is ddH 2 O or 1 M Tris (pH = 8.0).
[0021] Preferably, in S4, the soaking times are 2-3 times.
[0022] Preferably, in S3, the water bath temperature is 60°C.
[0023] Preferably, in S6, the root cap region and meristem of the root tip are placed on a clean glass slide, the Carbo solution is absorbed by filter paper, and a small drop of distilled water is dripped, a clean cover glass is clamped with tweezers, and the central part of the cover glass is used to cover the root tip, and then the cover glass is knocked with the distal end of the tweezers to flatten the cells. Then press vertically with the thumb, and the filter paper absorbs the excess distilled water between the slide and the cover glass. The cover glass can be gently tapped with a dissecting needle to disperse the cells. The force should not be too great, and the cells must be kept in a complete morphology. The cover glass is pressed vertically downward with the thumb to obtain a cell chromosome preparation.
[0024] Preferably, in S6, a low-power microscope is used for observation first, and after finding a cell area with better cell dispersion, a high-power microscope (oil microscope) is used for careful observation to find cells at different stages, and after finding cells with better chromosome dispersion and clear numbers, photographs are taken.
[0025] The second object of the present invention is to provide a karyotype chromosome number identification system for Bougainvillea, comprising a saturated p-dichlorobenzene solution, 5-20 μL α-bromonaphthalene, 0.008% 15-30 μL cycloheximide, a paraformaldehyde fixative, an HCl solution, a 0.075 mol / L KCl solution, a Carbo solution
[0026] The third object of the present invention is to provide a cell chromosome preparation obtained by a method for identifying the number of chromosomes in the karyotype of Bougainvillea.
[0027] The present invention explores the karyotype analysis experiment of Bougainvillea and finally establishes an effective karyotype analysis method. The karyotype method of Bougainvillea provided by the present invention is used to prepare the cell chromosome preparation, and the chromosomes are dispersed without overlapping, twisting, or breaking. The main constriction and satellite are clear, which is conducive to the karyotype analysis of Bougainvillea chromosomes. It can not only clarify the cardinality of the chromosomes of Bougainvillea, but also indicate the direction for the analysis of traditional hybrid breeding of Bougainvillea. The karyotype analysis method provided by the present invention is helpful to clarify the cardinality of the chromosomes of Bougainvillea and can provide guidance for the traditional hybrid breeding of Bougainvillea. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 The figure is the staining result of Example 1-4. DETAILED DESCRIPTION
[0029] The following is further described in detail through specific implementation methods:
[0030] The method for identifying the number of chromosomes of Bougainvillea karyotype comprises the following steps:
[0031] S1. Take the root tip of Bougainvillea 1-3 cm long, remove it and put it into an EP tube filled with 1 mL saturated p-dichlorobenzene solution, 5 μL α-bromonaphthalene and 15 μL mixed solution of 80 ppm 15-30 μL cycloheximide for pretreatment for 3 hours.
[0032] The preparation method of saturated p-dichlorobenzene solution is shown in Table 1. The saturated p-dichlorobenzene solution is dissolved in a 60°C water bath overnight and stored in a 4°C refrigerator for later use. The preparation method of cycloheximide is shown in Table 2.
[0033] Table 1: Preparation method of saturated p-dichlorobenzene solution
[0034] Reagent name Dosage Paradichlorobenzene 3g <![CDATA[ddH 2 O(distilled water)]]> 200mL
[0035] Table 2: Cycloheximide preparation method
[0036] Reagent name Dosage Cycloheximide 8mg <![CDATA[ddH 2 O(distilled water)]]> 100mL
[0037] S2. Fix the root tip obtained in S1 in paraformaldehyde (PFA) fixative for 12 hours.
[0038] The preparation method of PFA fixative is shown in Table 3 below:
[0039] Table 3: Preparation method of PFA fixative
[0040] Reagent name Dosage <![CDATA[ddH 2 The]]> 80mL 1MNaOH 80μL PFA 4g 1MNa-PBuffer 5mL 50% Glutaraldehyde 0.5mL <![CDATA[ddH 2 The]]> Make up to 100mL
[0041] In S2, the preparation method of paraformaldehyde fixative is as follows: add ddH 2 O, 1 M NaOH, paraformaldehyde, 1 M Na-P Buffer, and 50% glutaraldehyde were mixed and the volume was adjusted to 100 mL.
[0042] S3. Take out the root tip obtained in S2, wash it with water 2-3 times, put it into 1mL 1N HCl solution, and place it in a 60℃ water bath for 8 minutes for dissociation. The preparation method of 1N HCl solution is shown in Table 4 below:
[0043] Table 4: Preparation method of 1N HCl solution
[0044] Reagent name Dosage HCl(conc.) 8.3mL <![CDATA[ddH 2 O(distilled water)]]> Up to 100mL
[0045] S4. Place the root tip obtained in S3 in an EP tube filled with 0.075 mol / L KCl (solvent is ddH2O or 1 M Tris (pH=8.0) solution and soak for 2-3 times, each time for 150 seconds, to allow the cells to absorb water and swell.
[0046] Using distilled water as the hypotonic fluid has the best effect (wash twice with distilled water, 2 min 30 s each time), which can swell the cells well and promote the chromosomes to be well dispersed after pressing, making it easier to clearly identify the number and morphology of chromosomes.
[0047] S5. Place the root cap and meristem of the root tip in a clean culture dish containing Carbosolution (Solabo Biotechnology Co., Ltd.) and stain for 20 min.
[0048] S6. Take the root cap and meristem of the root tip and place them on a clean glass slide. Use filter paper to absorb the Carbo solution, and then add a small drop of distilled water. Use tweezers to pick up a clean cover glass and cover the root tip with the center of the cover glass. Then use the distal end of the tweezers to knock the cover glass to flatten the cells. Then press vertically with your thumb and use the filter paper to absorb the excess distilled water between the slide and the cover glass. You can use a dissecting needle to gently knock the cover glass to disperse the cells. Do not use too much force to make sure that the cells remain intact. Use your thumb to press the cover glass vertically downward to obtain a cell chromosome preparation.
[0049] Use Nikon ECLIPSE Ci upright fluorescence microscope to examine and take pictures. First use low power microscope to observe, find the cell area with good cell dispersion, then switch to high power microscope (oil microscope) to observe carefully, find cells at different stages, and take pictures after finding cells with good chromosome dispersion and clear number.
[0050] Examples 1-4
[0051] The main differences between Examples 1-4 are shown in Table 5.
[0052] Table 5: Main differences between Examples 1-4
[0053]
[0054]
[0055] The bougainvillea variety of Example 1 is Bougainvillea glabra'Marlu' (Chinese name is "Snow Purple"); the bougainvillea variety of Example 2 is Bougainvillea RedVelve (Chinese name is "Red Fox Tail"); the bougainvillea variety of Example 3 is Bougainvillea buttiana'Blondie' (Chinese name is "Golden Girl"); the bougainvillea variety of Example 4 is Bougainvillea×buttiana'Scarlet Queen' (Chinese name is "Scarlet Queen"). Figure 1 The figure is the staining result of Example 1-4. Figure 1 As shown, the constriction effect of Bougainvillea chromosomes is good, the chromosomes are not overlapped, twisted, or broken, and the main constriction and satellites are clearly visible.
[0056] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.
Claims
1. A method for identifying the number of chromosomes in Bougainvillea karyotype, characterized in that: The following steps are involved: S1. Pre-treat the root tip of Bougainvillea in a mixed solution containing saturated p-dichlorobenzene solution, 5-20 μL α-bromonaphthalene and 0.008% 15-30 μL cycloheximide for 3-5 h; S2, place the root tip obtained in S1 in paraformaldehyde fixative for 12-24h; S3, take out the root apex obtained in S2, clean it, put it in HCl solution, and place it in a water bath for 8-10 minutes; S4. Soak the root tip obtained in S3 in 0.075 mol / L KCl solution for 120-180 s; S5. Place the root cap and meristem of the root tip into Carbosol solution and stain for 20-40 minutes; S6. Take the root cap area and meristem area of the root tip and place them on a clean glass slide to obtain cell chromosome preparations, and take photos under a microscope.
2. The method for identifying the number of chromosomes of Bougainvillea karyotype according to claim 1, characterized in that: In S2, the paraformaldehyde fixative solution is prepared by mixing ddH2O, 1M NaOH, paraformaldehyde, 1M Na-P Buffer, and 50% glutaraldehyde, and then adjusting the volume to 100 mL.
3. The method for identifying the number of chromosomes of Bougainvillea karyotype according to claim 1, characterized in that: In S4, the solvent of the KCl solution is ddH2O or 1M Tris (pH=8.0).
4. The method for identifying the number of chromosomes of Bougainvillea karyotype according to claim 1, characterized in that: In S4, the number of immersions is 2-3 times.
5. The method for identifying the number of chromosomes of Bougainvillea karyotype according to claim 1, characterized in that: In S3, the water bath temperature was 60°C.
6. The method for identifying the number of chromosomes of Bougainvillea karyotype according to claim 1, characterized in that: In S6, take the root cap and meristem of the root tip and place them on a clean glass slide. Use filter paper to absorb the Carbo solution, and then drop a small drop of distilled water. Use tweezers to pick up a clean cover glass and cover the root tip with the center of the cover glass. Then use the distal end of the tweezers to knock the cover glass to flatten the cells. Then press vertically with your thumb and use the filter paper to absorb the excess distilled water between the slide and the cover glass. You can use a dissecting needle to gently knock the cover glass to disperse the cells. The force should not be too great. Make sure that the cells maintain their complete morphology. Use your thumb to press the cover glass vertically downward to obtain a cell chromosome preparation.
7. The method for identifying the number of chromosomes of Bougainvillea karyotype according to claim 1, characterized in that: In S6, first use a low-power microscope to observe, and after finding the cell area with better cell dispersion, switch to a high-power microscope (oil microscope) for careful observation, looking for cells at different stages. After finding cells with better chromosome dispersion and clear numbers, take pictures.
8. A karyotype chromosome number identification system of Bougainvillea, comprising saturated p-dichlorobenzene solution, 5-20 μL α-bromonaphthalene, 15-30 μL 0.008% cycloheximide, paraformaldehyde fixative, HCl solution, 0.075 mol / L KCl solution, and Carbosolution.
9. Cell chromosome preparation obtained according to the method for identifying the number of karyotype chromosomes of Bougainvillea according to any one of claims 1 to 7.