A general method for preparing a nuclear suspension for flow cytometry of rosaceous plants

Abstract

This invention discloses a universal method for preparing nuclear suspensions suitable for flow cytometry of Rosa species, belonging to the field of plant biotechnology. The method uses WPB dissociation buffer and follows a standardized procedure to process Rosa leaves in different storage states through sample dissociation, filtration, staining, and incubation steps: leaf tissue is dissociated using WPB dissociation buffer pre-cooled to 4°C, allowed to stand on ice, filtered through a 40µm cell filter, and brought to volume. Propidium iodide and ribonuclease A are added for staining and incubation before flow cytometry analysis. This invention is directly applicable to Rosa leaves in three storage states: fresh, silica gel dried, and -80°C frozen. It is simple to operate, highly versatile, and the prepared nuclear suspension yields high-quality peak histograms with low coefficient of variation when analyzed by flow cytometry, providing an efficient and standardized technical solution for large-scale ploidy analysis and genome size determination of Rosa species.

Description

Technical Field

[0001] This invention relates to the field of plant biotechnology, specifically to a method for preparing a flow cytometry cell nuclear suspension for plant ploidy analysis and genome size determination, and particularly to a universal method for preparing a cell nuclear suspension compatible with three different storage conditions: fresh, silica gel dried, and -80°C frozen leaves of the genus Rosa. Background Technology

[0002] As a key technology for rapid identification of plant ploidy and determination of genome size, the accuracy and reproducibility of flow cytometry results are highly dependent on the quality of the obtained cell nuclear suspension, which usually requires symmetry of the G1 peak in the peak histogram and a low coefficient of variation.

[0003] Systematic surveys of Rosa germplasm resources often face challenges such as remote sampling locations, high cold chain transportation costs, and long sample preservation periods. Therefore, a large number of field-collected samples require rapid silica gel drying or -80°C freezing for storage before analysis. However, current technologies typically require adjustments to the dissociation solution composition (such as reducing agent and detergent concentration) or processing procedures for plant samples in different storage states, making it difficult to achieve a universally applicable process. This not only increases the complexity of experimental operations but also leads to poor comparability between results from different batches, severely hindering large-scale, high-throughput ploidy analysis and genome size determination of Rosa germplasm resources.

[0004] Commercially available WPB dissociation buffer (Shanghai Yuanye Biotechnology Co., Ltd., product number R30048) is a known plant cell nucleus extraction buffer, but existing reports are mostly limited to its application in processing a small number of fresh plant samples. Currently, there are no publicly available reports, either domestically or internationally, on a universal method for preparing nuclei from Rosa species leaves that can be stably adapted to fresh, silica gel-dried, and -80℃ frozen storage using the same buffer system and standardized operating procedures. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a versatile, standardized, and stable method for preparing Rosa genus cell nuclear suspensions. This invention discovers that by using specific operating parameters in conjunction with a WPB dissociation solution system, a single process can stably process Rosa genus leaves in different storage states. The prepared cell nuclear suspensions can be directly used for plant ploidy analysis and genome size determination by flow cytometry.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A universal method for preparing nuclear suspensions suitable for flow cytometry of Rosa species, employing the same standardized process to treat fresh, silica gel-dried, or -80°C frozen Rosa leaves, includes the following steps: (1) Sample dissociation and filtration: Weigh 0.05 g of Rosa leaf tissue (samples in different storage states were pretreated using conventional methods: fresh leaves were used directly; silica gel-dried leaves had their surface silica gel particles removed with a soft brush; -80℃ frozen leaves were used directly without thawing), place them in a petri dish, and add 2 mL of WPB dissociation buffer pre-cooled to 4℃; quickly and vertically cut the leaf tissue into fine fragments for 1 min with a sharp single-edged blade, ensuring that the leaf tissue remained submerged in the WPB dissociation buffer throughout the cutting process; let the mixture containing the fine fragments stand on ice for 10 min; then filter the mixture through a 40 µm cell filter into a 2 mL centrifuge tube, collect 1 mL of filtrate. If the filtrate is less than 1 mL, add 1×PBS buffer pre-cooled to 4℃ to bring the total volume of the filtrate to 1 mL.

[0007] (2) Staining and incubation: Add 50 µL each of propidium iodide (PI) dye and ribonuclease A (RNase A) solution diluted to 50 µg / mL with 1×PBS buffer to the filtrate obtained in step (1); mix by inverting and incubate at room temperature in the dark for 15 min to obtain a cell nuclear suspension sample that can be detected by flow cytometry.

[0008] Furthermore, the filtrate obtained in step (1) can be stored in a 4°C refrigerator, and step (2) must be completed within 6 hours, without affecting the integrity of the cell nucleus or the detection results.

[0009] The beneficial effects of this invention are: it provides a unified and standardized process and parameters, effectively overcoming the limitations of existing methods that require adjustments to the pretreatment scheme based on the storage state of plant leaves. This method is simple to operate, highly versatile, and can stably prepare suspensions with low impurities and high cell nucleus integrity from Rosa leaves stored under different conditions. Flow cytometry analysis yields peak histograms with sharp peak shapes and low coefficient of variation (CV), thus significantly improving the efficiency of plant ploidy analysis and genome size determination. Attached Figure Description

[0010] Figure 1 The peak histogram of flow cytometry values ​​for the nuclear suspension of fresh single-petaled rose leaf cells prepared according to Example 1 of this invention; Figure 2 The flow cytometry peak histogram of the silica gel-dried Rosa polyphylla leaf cell nucleus suspension prepared according to Example 2 of the present invention; Figure 3 The peak histogram of flow cytometry for the -80℃ frozen fragrant rose leaf cell nucleus suspension prepared according to Example 3 of the present invention; Figure labeling: The horizontal axis in the figure represents PI fluorescence intensity (Comp-B9-A), which represents the nuclear fluorescence intensity value; the vertical axis represents the number of cell events, which represents the number of cell nuclei corresponding to the fluorescence intensity; the Mean labeled in the figure is the average fluorescence intensity, and CV is the coefficient of variation. Detailed Implementation

[0011] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0012] The WPB dissociation solutions used in all embodiments of the present invention include, but are not limited to, the WPB dissociation solution (product number R30048) produced by Shanghai Yuanye Biotechnology Co., Ltd., which should be pre-cooled to 4°C before use.

[0013] Example 1: Preparation of cell nuclear suspension from fresh Rosa genus leaves (1) Material preparation: Take single-petaled pink rose ( Rosa multiflora var. cathayensis Healthy, fresh young leaves. Prepare WPB dissociation buffer, 1×PBS buffer, propidium iodide (PI) dye (1 mg / mL), and ribonuclease A (RNase A) solution (10 mg / mL). All reagents should be pre-cooled to 4°C. Prepare a Cytek® Aurora full-spectrum flow cytometer, analytical balance, ice box, culture dishes, 40 µm filter, 2 mL centrifuge tubes, and 5 mL flow cytometry tubes.

[0014] (2) Sample preparation: 1) Dissociation: Weigh 0.05 g of fresh leaf using an analytical balance and place it in a culture dish. Add 2 mL of WPB dissociation solution pre-cooled to 4°C, and immediately use a sharp single-edged blade to quickly and vertically cut the leaf for 1 min until the tissue is finely fragmented. During the cutting process, ensure that the leaf is always immersed in the dissociation solution. Place the culture dish on ice and let it stand for 10 min. Then filter all the liquid and debris in the culture dish through a 40 µm cell filter into a 2 mL centrifuge tube, and add 1×PBS buffer pre-cooled to 4°C to bring the total volume of the filtrate to 1 mL. Discard the cell filter and tissue residue. 2) Staining and incubation: Add 50 µL of PI working solution diluted to 50 µg / mL with 1×PBS buffer and 50 µL of RNase A working solution at a concentration of 50 µg / mL to the 1 mL filtrate. Mix by inverting, wrap the centrifuge tube with aluminum foil to protect it from light, and incubate at room temperature for 15 min.

[0015] (3) Flow Cytometry: All incubated samples were transferred to 5 ml flow cytometry tubes and analyzed using a Cytek® Aurora full-spectrum flow cytometer. PI fluorescence signals were acquired using 488 nm laser excitation. The FSC voltage was set to 2 V, the SSC voltage to 4 V, and the FSC Area Scaling Factor was adjusted to 0.58. 10,000 cell events were collected for each sample. Results are as follows: Figure 1 The results show that the main peak of the peak histogram is obvious, and the CV value is less than 5%.

[0016] Example 2: Preparation of nuclear suspension of Rosa leaves by silica gel drying and storage (1) Material preparation: Prepare the Poly Rose ( Rosa × paulii Immediately after harvesting, place the Rehder leaves in a resealable bag containing sufficient silica gel and store at room temperature for 30 days. Before use, remove silica gel particles from the leaf surface with a soft brush.

[0017] (2) Sample preparation: Except that the initial state of the sample is a silica gel dried leaf, the other steps are exactly the same as in Example 1.

[0018] (3) Detection and Results: The flow cytometry detection conditions were the same as in Example 1. The results are as follows: Figure 2 The results show that the main peak of the peak histogram is obvious, and the CV value is less than 5%.

[0019] Example 3: Preparation of cell nuclear suspension from Rosa genus leaves frozen at -80℃ (1) Material preparation: Prepare the fragrant roses ( Rosa odorata After wrapping the leaves in aluminum foil, they were quickly placed in dry ice and then transferred to an ultra-low temperature freezer at -80°C for storage for one month.

[0020] (2) Sample processing: Except that the initial state of the sample is a frozen leaf at -80℃, the other steps are exactly the same as in Example 1.

[0021] (3) Detection and Results: The flow cytometry detection conditions were the same as in Example 1. The results are as follows: Figure 3 The results show that the main peak of the peak histogram is obvious, and the CV value is less than 5%.

[0022] The above embodiments demonstrate that the standardized method provided by the present invention can directly and stably process Rosa leaves stored in three different ways: fresh, silica gel dried, and -80℃ frozen. The prepared cell nuclear suspensions can all meet the requirements for flow cytometry ploidy analysis and genome size determination.

Claims

1. A universal method for preparing a nuclear suspension suitable for flow cytometry of Rosa species, characterized in that, Fresh, silica gel-dried, or -80°C frozen Rosa leaves are processed using the same standardized procedure, which includes the following steps: (1) Sample dissociation and filtration: Weigh 0.05 g of Rosa genus leaf tissue (fresh leaves can be used directly; silica gel dried leaves can be cleaned with a soft brush to remove silica gel particles from the surface; -80℃ frozen leaves can be used directly without thawing), place them in a petri dish, add 2 mL of WPB dissociation solution pre-cooled to 4℃; use a sharp single-edged blade to quickly cut the tissue vertically for 1 min to fine fragments, and keep the leaf tissue immersed in WPB dissociation solution throughout the cutting process. Let the mixture stand on ice for 10 min, and then filter it through a 40 µm cell filter into a 2 mL centrifuge tube. Collect 1 mL of filtrate. If the filtrate is less than 1 mL, add 1×PBS buffer pre-cooled to 4℃ to make up to 1 mL. (2) Staining and incubation: Add 50 μL each of propidium iodide (PI) dye and ribonuclease A (RNase A) solution with a working concentration of 50 µg / mL to the filtrate obtained in step (1); mix by inverting and incubate at room temperature in the dark for 15 min to obtain a cell nuclear suspension that can be detected by flow cytometry.

2. The method according to claim 1, characterized in that, In step (1), the WPB dissociation solution is a product of Shanghai Yuanye Biotechnology Co., Ltd. with product number R30048, or a plant buffer solution with the same components.

3. The method according to claim 1, characterized in that, Before proceeding to step (2), the filtrate after volume adjustment in step (1) should be stored at 4°C for no more than 6 hours.

4. The application of the method according to any one of claims 1 to 3 in the identification of ploidy in Rosa species, screening of ploidy in hybrid offspring, detection of polyploid breeding, or determination of genome size.

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