A method for preparing chromosomes of root meristems of buckwheat plants

Abstract

The invention discloses a method for preparing chromosomes of the root meristem of a plant belonging to the genus Fagopyrum, and relates to the technical fields of cytogenetics and genomics. The preparation method comprises the following steps: germinating and culturing buckwheat seeds until the seed roots grow to 1 to 4 cm, then cutting the root tips, pre-treating with N2O, and fixing with glacial acetic acid to obtain the fixed root tips; cutting the root tip meristems on the fixed root tips, performing enzymatic hydrolysis, and then preparing and staining to obtain the chromosomes of the root meristems of the plant belonging to the genus Fagopyrum. The buckwheat chromosome preparation obtained by the preparation method of the invention has many metaphase division phases, dispersed chromosomes, and good morphology, and can be used for chromosome genetic research such as chromosome karyotype analysis or downstream experiments, providing an effective technical reserve for chromosome genetic research of buckwheat germplasm.

Description

Technical Field

[0001] The invention relates to the technical field of cytogenetics and genomics, and in particular to a method for preparing chromosomes of root meristems of buckwheat plants. Background Art

[0002] Buckwheat is an annual or perennial, herbaceous or semi-shrub dicotyledonous plant belonging to the genus Fagopyrum of the Polygonaceae family. There are 21 species of the genus Fagopyrum, with a chromosome base number of 8 and two ploidy types: diploid and tetraploid. Buckwheat resources are abundant in southwest China, which is the origin of buckwheat and the diversity center of germplasm resources (Luthar et al., 2020; Gao et al., 2022). my country is a major buckwheat-growing country, with the second largest buckwheat planting area in the world. It has a short growth period, is cold-resistant and drought-resistant, and has obvious production advantages (Shi Jianqiang, 2016; Maria et al., 2020). The main cultivated species are sweet buckwheat (Fagopyrum esculentum) and bitter buckwheat (Fagopyrum tataricum) (Zhou Meiliang, 2021). Buckwheat is a specialty crop that can be used as both medicine and food. It can effectively prevent, control and assist in the treatment of diabetes, hypertension, etc. Its rich bioactive ingredients such as rutin also have antioxidant and anti-tumor effects (Vollmannová et al., 2021).

[0003] Cytogenetics is the foundation of plant genetic breeding research and an important theoretical basis for genetic breeding work. The morphological and structural analysis of chromosomes is a basic requirement for molecular cytogenetics research, among which obtaining high-quality mitotic metaphase chromosomes is a prerequisite for cytogenetics research. Chromosome preparation can be used to determine the number of chromosomes, ploidy, chromosome morphology, identify specific chromosome characteristics, and the relationship between species (Fei Min, 2013; Shang et al., 2020; Liu et al., 2021). Techniques such as karyotype analysis, chromosome banding, and in situ hybridization are all based on mitotic metaphase chromosomes with a clear background, complete and undamaged, single layer, and uniform distribution. Karyotype analysis is to determine the number of chromosomes and the presence of satellites by observing the metaphase of mitosis in root tip cells, combined with chromosome length measurement, centromere position, and arm ratio, and then establish a preliminary karyotype based on chromosome length (Liu Yuhong, 1985; Cai Lianbing and Feng Haisheng, 1997). Although there has been a certain research foundation for the preparation of root tip chromosomes in the past, there are different degrees of differences in tissue hardness, cell wall thickness and toughness, cell size and chromosome number between different species and even different hybrids of the same species (Li Le et al., 2023). In the relevant research on buckwheat chromosome preparation, previous researchers did not propose detailed measures and plans for how to obtain the most suitable materials for chromosome preparation.

[0004] Chromosome specimen preparation is chromosome preparation, which takes the metaphase chromosomes of somatic cells as the research object. Since the metaphase of cell mitosis lasts for a short time, the frequency of metaphase chromosomes is low. In order to make more cells stay in metaphase, chemical or physical methods are often used to pre-treat the materials, so that metaphase cells stop dividing and enter the late stage, and at the same time, a large number of cells in the early stage of mitosis chromatin condense and shorten to present the metaphase morphology. Whether the pre-treatment drugs and time are appropriate directly affect the success or failure of mitotic metaphase chromosome preparation. Chemical reagents include colchicine solution, p-dichlorobenzene, 8-hydroxyquinoline, saturated α-bromonaphthalene solution, etc. The treatment time is 5 to 27 hours and the temperature conditions are strictly required. The above-mentioned chemical drugs can prevent or destroy the formation of spindles, are highly toxic, and pose a risk of harm to the body of experimental personnel. The physical method mainly refers to the low temperature method, which takes 1 to 3 days for pretreatment, takes a long time and has unsatisfactory results (Huang Yunhua, 2009; Liu Jianlin et al., 2009; Chen Qingfu, 2010; Sheng Maoyin, 2013). Previous researchers used Carnoy's fixative to fix meristems, and used KCl and HCl to soften tissues. The operation was complicated and time-consuming. The reagents and staining times used for chromosome staining were different, such as 10 minutes of staining with modified phenol fuchsin staining solution, 1 hour of staining with 3% Giemsa staining solution, and 15 minutes of staining with 10% Giemsa staining solution.

[0005] The preparation of chromosomes in metaphase of mitosis is carried out by using the pressing method, smear method, wall-removing hypotonic method, flame drying method, etc.; based on the results of past studies, most of the buckwheat chromosomes have residual non-meristem tissue, uneven or overlapping chromosome distribution, local mechanical stretching of chromosomes, or even chromosome damage, which affects the judgment of chromosome ploidy level and cytogenetic karyotype research.

[0006] Buckwheat chromosomes are 1 to 3 μm long and have similar morphology, belonging to the category of small chromosomes. From the current reports on buckwheat chromosomes, there is a lack of research on the causes of differences in chromosome morphology and structure between buckwheat and related species or cultivars. It is necessary to compare the chromosome morphology of different buckwheat materials through efficient chromosome identification methods. Different buckwheat chromosomes have similar morphology, and individual characteristics are difficult to identify. Without good chromosome pieces, it is difficult to clearly identify chromosome arms and domains such as chromosome ends and centromeres on these small chromosomes.

[0007] In summary, the preparation of traditional buckwheat root tip cell mitotic chromosomes requires many chemicals, is time-consuming, expensive, has high temperature requirements and complicated steps. Therefore, how to provide a method for preparing chromosomes from the root meristem of buckwheat plants to obtain a large number of high-quality mitotic metaphase chromosomes is a problem that technicians in this field urgently need to solve. Summary of the invention

[0008] The purpose of the present invention is to provide a method for preparing chromosomes of root meristems of buckwheat plants to solve the problems existing in the above-mentioned prior art. The buckwheat chromosome preparation obtained by the method has many metaphase division phases, dispersed chromosomes and good morphology, and can be used for chromosome genetic research such as chromosome karyotype analysis or downstream experiments, thereby providing an effective technical reserve for buckwheat germplasm chromosome genetic research.

[0009] To achieve the above object, the present invention provides the following solutions:

[0010] The present invention provides a method for preparing chromosomes of root meristems of fagopyrum plants, comprising the following steps:

[0011] Buckwheat seeds were germinated and cultured until the seed roots grew to 1-4 cm, then the root tips were cut and the roots were cultured with N 2 O was used for pretreatment, and then fixed with glacial acetic acid to obtain the fixed root apex;

[0012] The root tip meristem is cut off from the fixed root tip, subjected to enzymatic hydrolysis, and then sliced ​​and stained to obtain the root meristem chromosomes of the fagopyrum plant.

[0013] Furthermore, the buckwheat seeds are germinated and cultured under dark conditions.

[0014] Furthermore, the temperature of the germination culture is 21° C. and the air humidity is 70-80%.

[0015] Furthermore, the pretreatment time is 1 hour.

[0016] Furthermore, the enzymes used in the enzymatic treatment are cellulase and pectinase.

[0017] Furthermore, the temperature of the enzymatic hydrolysis treatment is 37°C.

[0018] Furthermore, the enzymatic treatment time is 93 to 100 minutes.

[0019] Furthermore, the film preparation adopts a hanging drop film method.

[0020] Furthermore, the staining adopts DAPI staining method.

[0021] The present invention discloses the following technical effects:

[0022] 1. The present invention provides a method for preparing chromosome specimens for karyotype analysis of buckwheat plants with low cost, simpler and faster operation process, and more ideal buckwheat chromosome specimens. Buckwheat chromosome preparations obtained by the method of the present invention are clean, with many metaphases, dispersed chromosomes and good morphology, and can be used for chromosome karyotype analysis or downstream experiments (such as chromosome fluorescence in situ hybridization) and other chromosome genetic research, providing an effective technical reserve for buckwheat germplasm chromosome genetic research.

[0023] 2. The present invention adopts the method of seed germination in dark to carry out root apex culture, the root apex activity is strong and the sampling time is flexible; N 2 O pretreatment of the root apex and enzymatic hydrolysis methods are used to soften the tissue, and the effect is stable; it has the advantages of saving time, experimental materials and labor, and improving experimental efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1The chromosome preparation results of Xiqiao No. 2 of Example 1 and Comparative Example 1; wherein A and C are seeds of Xiqiao No. 2 treated with light for 0d and 2d, respectively; B and D are seeds of Xiqiao No. 2 treated with light for 16h / darkness for 0d and 2d, respectively; EG are the chromosome preparation results of seed roots treated with light; Figure HK is the chromosome preparation results of seed roots treated with light for 16h / darkness for 8h; the scale bars in DK are all 2μm; in AC, 1 represents Example 1, and 2 represents Comparative Example 1;

[0026] Figure 2 The results of chromosome preparation of bitter buckwheat cultivars using the method of Example 1; wherein AL are Xiqiao No. 1, Xiqiao No. 2, Xiqiao No. 4, Xiqiao No. 5, Xiqiao No. 6, Xiqiao No. 7, Xiqiao No. 9, Yunqiao No. 2, Qianku No. 2, Pinku No. 1, Chuanqiao No. 3 and Jiujiang bitter buckwheat; the scale bar in AL is 2 μm;

[0027] Figure 3 The results of chromosome preparation of sweet buckwheat cultivars using the method of Example 1; AD are white sweet buckwheat, red sweet buckwheat, sweet 2 and sweet 2 respectively; the scale bars in AD are all 2 μm;

[0028] Figure 4 The results of chromosome preparation of wild related species and golden buckwheat using the method of Example 1; wherein AC are respectively wild buckwheat, wild buckwheat and golden buckwheat; the scale bars in AC are 2 μm;

[0029] Figure 5 The results of chromosome preparation of local varieties of bitter buckwheat using the method of Example 1; wherein AC are E'er, Etumuler and Equ, respectively; and the scale bars in AC are all 2 μm. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The experimental materials used in the present invention are shown in Table 1, the main experimental reagents are shown in Table 2, and the main instruments are shown in Table 3.

[0036] Table 1 Experimental materials

[0037]

[0038]

[0039] Table 2 Experimental reagents

[0040]

[0041] Table 3 List of the names, models and manufacturers of the main instruments used in the experiment

[0042]

[0043] Example 1

[0044] Chromosome preparation was performed on the experimental materials shown in Table 1 using the following method:

[0045] (1) Material cultivation

[0046] Place buckwheat seeds in a petri dish containing moist filter paper, wrap the petri dish with aluminum foil to keep the seeds in the dark, and place them in a constant temperature incubator at 21°C and 70% to 80% humidity. Observe the growth of the seed roots after 72 hours. When the seed roots grow to 1 to 4 cm, proceed to the next step.

[0047] (2) Root apex sampling and fixation

[0048] At 9-10 am (or 15-16 pm), cut the root tip of buckwheat 1 cm (up to 4 cm in length) and put it in a moist 2 μL centrifuge tube. 2 O pretreatment for 1 hour, then the root apex was taken out and fixed with 90% glacial acetic acid for 5 minutes, and then the root apex was placed in 75% alcohol and stored in a 4°C refrigerator.

[0049] (3) Root apex enzymatic hydrolysis

[0050] The fixed root tips were taken out from the 4°C refrigerator and washed with ddH 2 O rinse twice, 5 minutes each time, to ensure that there is no alcohol residue on the root apex. After washing off the alcohol, use filter paper to absorb the water on the surface of the root apex. Place the root apex on a slide, cut off 1mm of the tip with a blade, and place it in the enzymatic solution, making sure that the root apex is at the bottom of the enzymatic solution. Place the enzymatic solution with the root apex in a constant temperature incubator at 37°C for 93 minutes (up to 100 minutes).

[0051] (4) Chromosome preparation

[0052] After the enzymatic hydrolysis of the root tip is completed, the enzymatic hydrolyzate is taken out and 75% alcohol is quickly added to terminate the enzyme reaction. The enzymatic hydrolyzate is replaced with 75% alcohol 3 times. When the enzymatic hydrolyzate is replaced for the third time, about 20μL of alcohol is retained in the centrifuge tube, centrifuged at 6000r / min for 2min, the supernatant is discarded, and the alcohol is completely evaporated. Add 20μL of 90% glacial acetic acid, blow and beat evenly with a pipette to obtain the root tip meristem suspension. Then use a pipette to absorb 10μL of the root tip meristem suspension, raise it to 10cm vertically above the clean slide for hanging drop film, and place the slide in a dark and humid environment to dry naturally (5-10min can be completed). Hanging drop film and natural drying allow the root tip cell suspension to fall on the slide and be evenly distributed. When 90% glacial acetic acid is extended on the slide, the cells are naturally pushed apart and spread, and the split phase is dispersed and not easy to overlap.

[0053] Enzyme solution formula:

[0054] Prepare 0.01 M citrate buffer (pH 4.5): 6 H 8 O 7 )1.47g, hydrated citric acid (C 6 H 8 O 7 ·H 2 O) 1.05 g, ddH 2 O and make up to 500 mL.

[0055] Weigh 400 mg of cellulase and 200 mg of pectinase, dissolve them in 10 mL of 0.01 M citric acid buffer, and then dispense them into 0.5 mL centrifuge tubes, 20 μL per tube (the above operations are performed on ice to prevent degradation of the enzymatic solution), and store them in a -20°C refrigerator for later use.

[0056] (5) Microscopic examination

[0057] 10 μL DAPI (diluted in 1×PBS: DAPI=2:1 ratio) was dropped onto the dried slide for staining and then covered with a coverslip. The chromosome image was observed under a 100x microscope using an Olympus BX-63 microscope (Olympus, Japan) equipped with a DP-70CCD camera. The mitotic phases with good chromosome morphology and dispersion were selected and photographed. After the microscopic examination, the coverslip was removed and the slides were washed with gradient alcohol of 70%, 85%, and 100% for 1 min each, and finally stored at -20°C for later use.

[0058] The chromosome preparation results of each experimental material are as follows Figure 2-5 As shown, the results show that all experimental materials in Table 1 were prepared by this method, and the chromosome division phases obtained were scattered, non-overlapping, and had good morphology.

[0059] Comparative Example 1

[0060] Chromosome preparation was performed on Xiqiao 2 using the same method as in Example 1, except that the dark condition in step (1) was replaced with 16 h light / 8 h dark.

[0061] The chromosome preparation results of Xiqiao No. 2 in Example 1 and Comparative Example 1 are as follows Figure 1 As shown, the results showed that under the same growth conditions, the seed roots were longer in the dark than in the light 16h / dark 8h condition.

[0062] like Figure 1 As shown in the figure, the chromosome preparation effect of the root tip grown in the dark is better than that of the root tip treated with 16h light / 8h dark conditions. The root tip chromosome preparation period under the dark condition has more correct division phases, and the sampling time is not affected by the light and dark conditions, which breaks the inherent limitation of sampling time and greatly increases the convenience of buckwheat preparation.

[0063] Experimental Example 1

[0064] Sampling time optimization:

[0065] Chromosome preparation was performed on Xiqiao No. 2 using the same method as in Example 1, except that the root tip sampling time in step (2) was replaced with 9-10 a.m., 11-12 a.m., 15-16 p.m., 17-18 p.m., and 20-21 p.m., respectively.

[0066] The results showed that chromosome preparations could be obtained at the above times, and there were more cells in the middle and late stages of cell division in the root apical meristem cells between 9 and 10 a.m. and 15 and 16 p.m.

[0067] Experimental Example 2

[0068] N 2 OProcessing time optimization:

[0069] Chromosome preparation of Xiqiao No. 2 was carried out in the same manner as in Example 1, except that the N 2 O pretreatment time was adjusted to 0.5h, 1h, 1.5h, 2h, and 2.5h, respectively.

[0070] N 2 O treatment time control is a key technology, the purpose is to make a large number of cells stay in the metaphase of mitosis. 2 It was found that too long a treatment time would cause the chromosomes to be too short, which is not conducive to cytogenetic analysis; too short a treatment time would be not conducive to chromosome counting and karyotype analysis. 2 O The experiment of treatment time determined that the optimal treatment time is 1h.

[0071] Experimental Example 3

[0072] Optimization of root apical enzymatic hydrolysis time:

[0073] Chromosome preparation was performed on Xiqiao No. 2 using the same method as in Example 1, except that the enzymatic hydrolysis time in step (3) was adjusted to 90 min, 91 min, 92 min, 93 min, 94 min, 95 min, 96 min, 97 min, 98 min, 99 min, and 100 min, respectively.

[0074] The present invention finds by adjusting different root tip enzymolysis times that the root tip tissue is not completely digested by the enzymolysis solution when the enzymolysis time is short; the root tip tissue is over-digested by the enzymolysis solution when the treatment time is long, and the root tip becomes too soft or broken, resulting in the overflow of intracellular lysates, which is not conducive to subsequent dissection and crushing. At an enzymolysis time of 93 to 100 minutes, mitotic buckwheat metaphase chromosomes with a clear background, complete and undamaged, single-layer and evenly spread can be observed. Finally, the present invention determines that the root tip enzymolysis time is 93 to 100 minutes.

[0075] In summary, the present invention provides a method for preparing chromosomes from the root meristem of plants of the genus Fagopyrum. Using the method of the present invention, the root culture time can be reduced, the root tip treatment can be performed at any time of the day, the sampling time can be flexibly selected, and the influence of the time of cutting the root on the film-making effect is reduced. The pretreatment time can be reduced from 5 to 27 hours to 1 hour, and the enzymatic hydrolysis time of buckwheat is clearly 93 to 100 minutes. The method of the present invention greatly reduces the film-making time, and can efficiently and massively obtain metaphase chromosomes with uniform distribution and good morphology. The present invention concretizes the reagent ratio and time parameters by improving and optimizing the root tip cell culture conditions, enzymatic hydrolysis time and observation method, and is particularly suitable for the preparation of chromosomes from the root meristem of plants of the genus Fagopyrum, which is convenient for technology promotion and application.

[0076] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing chromosomes from root meristems of Fagopyrum spp., characterized in that: The following steps are involved: The buckwheat seeds were germinated and cultured until the seed roots grew to 1-4 cm, and then the root tips were cut, pretreated with N2O, and then fixed with glacial acetic acid to obtain fixed root tips; cutting off the root apex meristem from the fixed root apex, placing the root apex in an enzymatic solution for enzymatic treatment, and obtaining an enzymatic solution; Rapidly add 75% alcohol to the enzymatic hydrolysate to terminate the enzyme reaction, replace the enzymatic hydrolysate with 75% alcohol for 3 times, centrifuge for the last time, discard the supernatant, let it air dry until the alcohol is completely volatilized, add 90% glacial acetic acid, blow and beat evenly with a pipette to obtain a root apical meristem suspension; Taking the root tip meristem suspension, preparing slices and staining them by the hanging drop method, and obtaining the root meristem chromosomes of the fagopyrum plant; The germination culture is carried out under dark conditions; The enzymes used in the enzymatic treatment are cellulase and pectinase; the concentrations of the cellulase and pectinase in the enzymatic solution are 40 mg / mL and 20 mg / mL, respectively; The temperature of the enzymatic hydrolysis treatment is 37°C and the time is 93 to 100 minutes; The staining adopts DAPI staining method.

2. The preparation method according to claim 1, characterized in that: The temperature of the germination culture is 21° C., and the air humidity is 70-80%.

3. The preparation method according to claim 1, characterized in that: The pretreatment time is 1 h.

Images