A fluorescent probe for plant suberized cells, a staining method and application thereof

Abstract

The application provides a fluorescent probe for plant suberized cells, a staining method and application. The application first provides a fluorescent probe for plant suberized cells, wherein the fluorescent probe is a fluorescein solution or a fluorescein salt solution under alkaline conditions; and the application also provides a specific staining method for plant suberized cells, which comprises the following steps: mixing and staining a plant sample and a fluorescent probe staining solution, then washing, and observing imaging. The method only needs to use the fluorescent probe to directly perform immersion and washing in two steps, reduces the work of replacing and washing different reagents, adjusting temperature, and restaining, and compared with a traditional chemical staining method, greatly improves experimental efficiency; meanwhile, the fluorescent probe used in the application is more resistant to quenching than the most commonly used traditional chemical dye, and the stability is increased by about 4 times.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a fluorescent probe, staining method, and application for plant corky cells. Background Technology

[0002] In plants, the suberin lamellae of suberized cells are mainly composed of aliphatic and aromatic high-molecular-weight biopolyesters, deposited primarily in the cell walls of the endodermis, exodermis, and periderm of plant roots, distinguishing these cells from those in other locations. Suberized cells have many different functions: first, barrier protection, resisting pathogen invasion, drought, heavy metals, and other biotic and abiotic stresses; second, structural reinforcement, forming a hydrophobic layer on the outer side of the cell membrane, enhancing mechanical stability; and third, metabolic response, as external stress can induce suberin deposition, serving as a marker of plant stress response.

[0003] However, when using the most commonly used staining methods for cork cells—Fluorol Yellow 088 (also called Fluorescent Yellow 088 or FY 088), Sudan III staining, or Nile Red staining—for in situ observation of cork cells inside plants, the following three problems exist: First, poor specificity. In particular, Sudan III and Nile Red staining can also bind to other lipids or waxes, making it difficult to specifically distinguish cork lamellae. This has led to Fluorol Yellow 088 staining gradually becoming the mainstream detection method in this field. Second, it is time-consuming, cumbersome, and labor-intensive. Previous methods required 0.5 to 3.5 hours, and the traditional Fluorol Yellow 088 staining process is extremely cumbersome (requiring 20 minutes of aniline blue counterstaining, ethanol washing, glycerol preservation, or heating or clearing, etc.). Third, fluorescence is easily quenched, especially with Fluorol Yellow 088 staining, which easily leads to poor fluorescence stability and limited observation time, making it unsuitable for long-term and high-throughput batch observation.

[0004] The CAS number of fluorescein is 2321-07-5, and its structural formula is shown in Formula 1 below. Fluorescein is a classic yellow-green fluorescent small molecule dye and a common fluorescent probe. Due to its high molar absorptivity, high fluorescence quantum yield, low cost and high detection sensitivity, it is widely used in bioimaging.

[0005] Formula 1.

[0006] Luciferin exists in aqueous solutions in cationic, neutral, and anionic forms, making its absorption and fluorescence properties highly pH-dependent. Its fluorescence intensity is primarily influenced by the protonation state and the local microenvironment; under neutral to alkaline conditions, luciferin exists mainly in anionic form, exhibiting strong green fluorescence; while pH, polarity, hydrogen bonding, and matrix interactions significantly affect its absorption and emission behavior. Due to these physicochemical properties, luciferin and sodium luciferin have been widely used in animal tissues and clinical diagnostics, including assessing corneal epithelial defects, tear film integrity, and ocular surface barrier function, as well as observing retinal perfusion, leakage, and barrier disruption through luciferin angiography.

[0007] In plant biology, fluorescein-based tracers—5(6)-carboxyfluorescein (chemical formula C...) 21 H 12 O7 (CAS No. 94595-55-8) is transported into the plant from the cotyledon phloem through damaged areas on the surface of Arabidopsis thaliana cotyledons, and then transported to the root tip via the phloem, thus achieving real-time imaging of the phloem. This tracing method utilizes the transport capacity of the phloem and the strong fluorescence of fluorescein, but does not involve the specificity of fluorescein. During subsequent unloading of the phloem, 5(6)-carboxyfluorescein fluoresces in the endoderm, cortex, epidermis, and resting center, and cannot distinguish the suberized cells of the endodermis.

[0008] Furthermore, fluorescein diacetate (FDA), a luminescent agent based on fluorescein, does not emit light itself, but it can freely penetrate the cell membrane into living cells. Inside the cell, it is hydrolyzed into fluorescein by esterases and accumulates in the cell, emitting green fluorescence to indicate cell viability, such as in pollen, cultured cells, and protoplasts. It also does not have a specific effect on corky cells.

[0009] In summary, there are currently no reports on the use of fluorescein probes for specific staining and imaging of suberized cells in plants. Therefore, developing a simple staining method for suberized cells using fluorescein probes is of considerable value. Summary of the Invention

[0010] The purpose of this invention is to provide a staining method for plant suberized cells that is simple to operate, not easily quenched, and has broad species applicability.

[0011] This invention first provides a fluorescent probe for plant suberized cells. The fluorescent probe consists of fluorescein, fluorescein salt, or fluorescein derivative at a concentration of not less than 0.001 mg / mL under conditions of pH 12 ≤ pH ≤ 13.5. The pH range of conventional aqueous solutions is typically between 0 and 14. When pH 12 ≤ pH ≤ 13.5, staining with the fluorescent probe can clearly distinguish suberized cells, achieving specific staining of suberized cells.

[0012] Preferably, the fluorescein salt is sodium fluorescein or potassium fluorescein.

[0013] The fluorescein, fluorescein salt, or fluorescein derivative is prepared into a solution with water. Preferably, the concentration of the fluorescein is 0.001~100 mg / mL.

[0014] More preferably, the concentration of fluorescein is 0.1~1 mg / mL. Within this concentration range, there are obvious fluorescent staining results with low background fluorescence, which is more conducive to the observation and quantitative analysis of fluorescence results.

[0015] The present invention also provides the application of the fluorescent probe in staining plant corky cells or in preparing plant corky cell staining products.

[0016] Preferably, the plant cork cell staining product is a dye, a fluorescent probe, or a kit.

[0017] The present invention also provides a staining method for plant corky cells, which involves mixing and staining a plant sample with the fluorescent probe to obtain a stained plant sample.

[0018] Preferably, the plant sample is a plant root, bark, or in vitro corky cells obtained from an in vitro sample.

[0019] More preferably, the plant root is Arabidopsis root, cucumber root, Sedum aizoon root, rice root, tomato root, tobacco root, or willow root; the isolated in vitro corky cells are cork cells from the bark of Quercus variabilis.

[0020] Preferably, the time for mixing and staining plant samples and fluorescent probes is 10s to 24h.

[0021] In a preferred embodiment, the staining time for Arabidopsis thaliana roots is 1 minute; the staining time for cucumber and wheat roots is 4 minutes; the staining time for Sedum aizoon roots is 2 minutes; and the staining time for rice, tomato, tobacco, and willow roots is 8 minutes. Because the root thickness and length vary among different plants, the soaking and staining times also differ.

[0022] The fluorescent probe described in this invention can be used in conjunction with chemical dyes. That is, before or after mixing and staining the plant sample with the fluorescent probe, chemical dyes are used to stain the plant sample to achieve staining and localization of different cell wall structures in the plant sample.

[0023] Preferably, the chemical dye is propidium iodide staining solution or Karl Coflurrel fluorescent whitening agent.

[0024] Preferably, after staining, the stained plant samples are washed to facilitate imaging. The washing solvent is deionized water or an alkaline aqueous solution with the same pH as the fluorescein solution used for staining. Specifically, the alkaline aqueous solution is water with the same alkaline solution used to prepare the fluorescein solution adjusted to pH. The washing is performed 2 to 8 times to remove excess probe.

[0025] The staining method for plant corky cells provided by this invention only requires two steps: direct staining with fluorescein solution and washing. It can also be combined with other staining solutions or observed after sectioning, which greatly reduces the workload of changing different reagents and adjusting temperature.

[0026] In summary, the specific beneficial effects of the present invention are as follows: 1. Compared with previous staining methods such as fluorophenol yellow 088, which require 0.5 to 3.5 hours, the staining method provided by this invention can observe fluorescence after incubation with fluorescein for 10 seconds, and the soaking and rinsing operations can be completed in as little as 2 to 3 minutes, which greatly improves the staining efficiency.

[0027] 2. The fluorescence of the fluorescein used in this invention is more resistant to quenching than that of the most commonly used traditional chemical dye, fluorophenol yellow 088, and its stability time is increased by about 4 times.

[0028] 3. The method of the present invention has universality with different plants when observing cork cells in plant roots, and can also perform in vitro staining and observation of bark cork cells and their surrounding tissues.

[0029] In summary, the staining method of the present invention has stronger fluorescence that is not easily quenched, is very simple to operate without the need for counterstaining, has strong species universality, and can complete the soaking and rinsing operation in as little as 2-3 minutes for imaging observation. The fluorescence stability time is about 4 times longer than that of traditional methods, and if timely observation is required, there is no need for light-protected incubation during the staining process. Attached Figure Description

[0030] Figure 1 Fluorescence characteristics of fluorescein solution before and after UV light excitation (scale bar is 1 cm).

[0031] Figure 2 The staining results of 1 mg / mL sodium fluorescein on the roots of wild-type Arabidopsis thaliana Col-0 at different pH values ​​(scale bar is 100 μm).

[0032] Figure 3 The image shows the root-specific staining results of different luciferin concentrations on wild-type Arabidopsis thaliana Col-0 at pH 13 (scale bar is 50 μm).

[0033] Figure 4The image shows the staining results of 1 mg / mL fluorescein on the roots of wild-type Arabidopsis thaliana Col-0 at pH 13 at different time points (scale bar is 50 μm).

[0034] Figure 5 The image shows the root-specific staining results of wild-type Arabidopsis thaliana Col-0 at pH 13 using 1 mg / mL aqueous solutions of fluorescein, sodium fluorescein, and potassium fluorescein (scale bar: 50 μm).

[0035] Figure 6 Images of cork lamellae before and after staining under a transmission electron microscope (white arrows indicate cork lamellae; the subscript bar for 100k (100,000x) magnification is 1μm; the subscript bar for 300k (300,000x) magnification is 0.5μm).

[0036] Figure 7 A contrastive staining image of wild-type Arabidopsis thaliana (Col-0) roots and transgenic Arabidopsis thaliana roots with altered corky cells (scale bar 50 μm). Figure 7 In the diagram, 'a' represents the fluorescein staining pattern. Figure 7 b in the image represents the fluorophenol yellow 088 staining pattern.

[0037] Figure 8 Comparative images of quenching by fluorescein and fluorophenol yellow 088 (FY 088) staining in the roots of wild-type Arabidopsis thaliana Columbia Col-0. Figure 8 In the image, 'a' represents the quenched fluorescence image of a single root of wild-type Arabidopsis thaliana stained with fluorescein and fluorophenol yellow 088 (scale bar is 50 μm). Figure 8 In the figure, b is the quantitative fluorescence curve of the quenching results of staining with fluorescein and fluorophenol yellow 088 (3 roots for each).

[0038] Figure 9 This is a fluorescein staining image of cork cells in the bark of *Quercus variabilis*. Among them, Figure 9 In the image, 'a' represents a fluorescence comparison before and after fluorescein staining (the scale bar for the image before magnification is 100 μm, and the scale bar for the image after magnification is 20 μm). Figure 9 b in the diagram represents the quantitative fluorescence staining map (7 cells were quantified in both the control group and the fluorescein group).

[0039] Figure 10 This image shows the results of co-staining wild-type Arabidopsis thaliana (Col-0) roots with fluorescein and propidium iodide (PI). Figure 10 In the image, 'a' represents the stained fluorescence pattern (scale bar is 50 μm). Figure 10 In the diagram, b represents the fluorescence intensity of the root cross section after co-staining (using...). Figure 10 The white line passing through the root in the middle of the merged diagram (a) is the cross section.

[0040] Figure 11This image shows the results of co-staining wild-type Arabidopsis thaliana (Col-0) roots with fluorescein and Calcofluor White. Figure 11 In the image, 'a' represents the stained fluorescence pattern (scale bar is 50 μm). Figure 11 In the diagram, b represents the fluorescence intensity of the root cross section after co-staining (using...). Figure 11 The white line passing through the root in the middle of the merged diagram (a) is the cross section.

[0041] Figure 12 Three-dimensional reconstruction of roots of wild-type Arabidopsis thaliana Col-0 stained with fluorescein and fluorophenol yellow 088 (FY 088) (scale bar 50 μm, each image 1 μm thick).

[0042] Figure 13 The image shows the fluorescence quenching results of wild-type Arabidopsis roots stained with fluorescein and fluorophenol yellow 088 (FY 088) for 5 minutes. Figure 13 In the figure, 'a' represents the fluorescence contrast diagram (scale bar is 100 μm). Figure 13 In the figure, b represents the quantitative graph of relative fluorescence intensity at the root cross section.

[0043] Figure 14 This image shows the staining results of corky cells in cucumber roots. Figure 14 In the image, 'a' represents a cucumber plant (scale bar is 10cm). Figure 14 In the diagram, b represents the staining and root section construction diagram (scale bar is 100μm).

[0044] Figure 15 This image shows the staining results of corky cells on the roots of *Sedum aizoon*. Figure 15 'a' in the image is a picture of a Sedum australis plant (scale bar is 10cm). Figure 15 In the diagram, b represents the staining and root section construction diagram (scale bar is 100μm).

[0045] Figure 16 This image shows the staining results of corky cells in willow roots. Figure 16 In the image, 'a' represents a willow tree (scale bar is 20cm). Figure 16 In the diagram, b represents the staining and root section construction diagram (scale bar is 100μm).

[0046] Figure 17 This image shows the staining results of cork cells in tobacco roots. Figure 17 In the image, 'a' represents a tobacco plant (scale bar is 10cm). Figure 17 In the diagram, b represents the staining and root section construction diagram (scale bar is 100μm).

[0047] Figure 18 This image shows the staining results of corky cells in tomato roots. Figure 18 In the image, 'a' represents a tomato plant (scale bar is 10cm). Figure 18 In the diagram, b represents the staining and root section construction diagram (scale bar is 100μm).

[0048] Figure 19 This image shows the staining results of suberized cells in rice roots. Figure 19 In the image, 'a' represents a rice plant (scale bar is 10cm). Figure 19 In the diagram, b represents the staining and root section construction diagram (scale bar is 100μm).

[0049] Figure 20 This image shows the staining results of suberized cells in wheat roots. Figure 20 In the image, 'a' represents a wheat plant (scale bar is 10cm). Figure 20 In the diagram, b represents the staining and root section construction diagram (scale bar is 100μm). Detailed Implementation

[0050] The fluorescent probes used in the examples were fluorescein (CAS No.: 2321-07-5), sodium fluorescein (CAS No.: 518-47-8), or potassium fluorescein (CAS No.: 115144-35-9). The fluorescence characteristics of the fluorescent probe solutions under natural light and 365nm handheld UV lamp excitation are as follows: Figure 1 As shown.

[0051] The cucumber, sedum, willow, tobacco, tomato, rice, and wheat used in the examples were from Zhejiang University.

[0052] The wild-type Arabidopsis thaliana material and the transgenic material of cork cells used in the examples were cultured as follows: wild-type Arabidopsis thaliana Columbia Col-0 and endodermal transgenic material expressing the cork degradation gene were cultured. proCASP1::CDEF1 cork lamellar deletion mutant, cork lamellar enhancement mutant esb1-1 cork-enhanced mutant lcs2-1 The seeds were surface-sterilized with 10% (v / v) sodium hypochlorite for 5 min, washed 4 times with sterile water, and treated in a refrigerator at 4℃ for 2 days. Subsequently, the seeds were sown on 1 / 2 MS solid medium (a solid medium with half the nutrient concentration of standard MS medium) and placed vertically in a growth chamber. They were grown under light:dark cycle conditions of 16h:8h and 22℃:20℃. The experiment was conducted when the seedlings were 5-6 days old.

[0053] Endothelial-specific expression genes CASP1 Its full name is Casparian strip membrane domain protein 1, gene number AT2G36100; it acts as a degrading enzyme. CDEF1The full name of the gene is CUTICLE DESTRUCTING FACTOR 1, and its gene number is AT4G30140; it is a cork-enhancing mutant. esb1-1 The full name of the gene is ENHANCED SUBERIN1, and its gene number is AT2G28670; it is a cork-enhancing mutant. lcs2-1 The gene is also called LOTR1, and its full name is LORD OF THE RINGS 1, with the gene number AT5G50150.

[0054] In this embodiment, the Olympus FV3000 laser confocal imaging conditions are as follows: 561nm for bright field signal; 488nm exciter for green fluorescence signal, with an emission light acquisition range of 500~550nm; 561nm exciter for red fluorescence signal, with an emission light acquisition range of 590~650nm and 405nm for bright field signal; 405nm exciter for blue fluorescence signal, with an emission light acquisition range of 450~510nm and 561nm for bright field signal; fluorescence intensity analysis of the fluorescence images is performed using Fiji software (v.2.14.0).

[0055] Example 1: Staining of corky cells from Arabidopsis thaliana roots 1. pH screening To clarify the staining effect of fluorescein on suberized cells in the roots of wild-type Arabidopsis thaliana Col-0 seedlings at different pH levels, hydrochloric acid or sodium hydroxide was used to adjust the pH of the solution to establish a gradient experiment with different pH values. The concentration of fluorescein sodium used was 0.1 mg / mL (because the solubility of fluorescein under acidic conditions is much lower than that of its salt solution, fluorescein sodium salt was used in this example). The specific steps are as follows: (1) The whole plant of wild-type Arabidopsis thaliana Columbia Col-0 seedlings was placed in water (CK, control group) and sodium fluorescein aqueous solution with a concentration of 0.1 mg / mL at pH 1, 4, 7, 10, 12, 13 and 13.5 for 1-2 min respectively; (2) After staining, root samples of wild-type Arabidopsis thaliana Columbia Col-0 seedlings were taken with tweezers, placed in a 6-well plate, rinsed with deionized water 6-8 times, and then pressed into a slide for observation using Olympus FV3000 laser confocal imaging.

[0056] The results are as follows Figure 2 As shown, when pH=1, 4, 7 and 10, sodium fluorescein stains cells in root hairs, epidermis and cortex, but has poor staining effect on suberized cells, indicating that sodium fluorescein does not have a specific pattern for distinguishing different cell characteristics under the above pH conditions; when 12≤pH≤13.5, sodium fluorescein specifically labels suberized cells in the endodermis.

[0057] 2. Concentration Screening (1) The whole plant of wild-type Arabidopsis thaliana Columbia Col-0 seedlings was placed in water (CK, control group) and fluorescein aqueous solution with pH=13 at concentrations of 0.001, 0.01, 0.1, 1, 10, and 100 mg / mL for 1-2 min; (2) After staining, root samples of wild-type Arabidopsis thaliana Columbia Col-0 seedlings were taken with tweezers, placed in a 6-well plate, rinsed with deionized water 6-8 times, and then pressed into a slide for observation using Olympus FV3000 laser confocal imaging.

[0058] The results are as follows Figure 3 As shown (scale bar is 50 μm), fluorescein staining at a low concentration (0.001 mg / mL) after dilution with deionized water can still provide good specific imaging of corky cells, and the specific imaging effect is better when the concentration reaches 1 mg / mL.

[0059] 3. Staining time selection (1) The whole plant of wild-type Arabidopsis thaliana Columbia Col-0 seedlings was immersed in a 1 mg / mL fluorescein aqueous solution at pH=13 for 10s, 5min, 2h, 6h, 12h and 24h respectively for staining. (2) After staining, root samples of wild-type Arabidopsis thaliana Columbia Col-0 seedlings were taken with tweezers, placed in a 6-well plate, rinsed with deionized water 6-8 times, and then pressed into a slide for observation using Olympus FV3000 laser confocal imaging.

[0060] The results are as follows Figure 4 (Scale bar is 50μm) The fastest soaking staining time is 10s to complete the specific staining of suberized cells, and the longest soaking staining time is 24h to still observe the specific staining fluorescence of suberized cells.

[0061] 4. Observation of the staining effects of fluorescein, sodium fluorescein, and potassium fluorescein. (1) The whole plant of wild-type Arabidopsis thaliana Columbia Col-0 seedlings was immersed in a 1 mg / mL aqueous solution of fluorescein, sodium fluorescein and potassium fluorescein at pH=13 for 1-2 min. (2) After staining, root samples of wild-type Arabidopsis thaliana Columbia Col-0 seedlings were taken with tweezers, placed in a 6-well plate, rinsed with deionized water 6-8 times, and then pressed into a slide for observation using Olympus FV3000 laser confocal imaging.

[0062] The results are as follows Figure 5 As shown (scale bar is 50 nm), solutions of fluorescein, sodium fluorescein, and potassium fluorescein have the same cork cell-specific staining effect.

[0063] The cork lamellae of wild-type Arabidopsis thaliana Columbia Col-0 roots (fluorescein solution concentration 1 mg / mL, pH=13) were observed under a transmission electron microscope. Figure 6 As indicated by the white arrows, there is significant fluorescein enrichment in the cork lamellae after staining, demonstrating the specific staining effect of fluorescein on cork cells.

[0064] Example 2: Staining of transgenic genetic material from wild-type Arabidopsis thaliana and cork cells (1) Wild-type Arabidopsis thaliana Columbia Col-0 and endodermal expression of cork degradation genes were respectively introduced. proCASP1:: CDEF1 Transgenic plants lacking cork lamellae and transgenic mutants with enhanced cork lamellae esb1-1 cork-enhanced mutant lcs2-1 The entire plant was immersed in a 1 mg / mL pH 13 fluorescein aqueous solution for 1 min; (2) After staining, use tweezers to place the sample in a 6-well plate and rinse it with deionized water 6-8 times. After pressing it into a pellet, use Olympus FV3000 laser confocal imaging to observe the roots.

[0065] The results are as follows Figure 7 As shown (scale bar is 20μm), the early stage is the tender part of the root, which has no cork; the middle stage is the middle section of the root, which produces flaky cork; and the late stage is the old part of the root, which produces continuous cork.

[0066] The results showed that wild-type Arabidopsis thaliana Columbia Col-0 exhibited a distinct classical pattern of corky cells. proCASP1::CDEF1 The staining pattern of corky cells in transgenic plants was also similar to that of cork stained with fluorophenol yellow 088 in existing techniques (Li B, et al. Role of LOTR1 in Nutrient Transport through Organization of Spatial Distribution of Root Endodermal Barriers. Curr Biol. 2017 Mar 6;27(5):758-765. doi:10.1016 / j.cub.2017.01.030. Epub 2017 Feb 23. PMID:28238658.), with enhanced corky cell mutants. esb1-1 and lcs2-1 The staining pattern is similar to that of suberin stained with fluorophenol yellow 088 in existing techniques. Therefore, this method can effectively stain suberized cells.

[0067] Example 3: Determination of fluorescein staining performance of wild-type Arabidopsis thaliana materials To verify the fluorescence stability and persistence of the staining method during long-term observation, wild-type Arabidopsis thaliana Columbia Col-0 was stained using the same method as in Example 2. During fluorescence observation, an Olympus FV3000 laser confocal imaging system was used in preview mode, i.e., the laser was continuously excited. Images were taken at 0, 60, 120, 180, 240, 300, 360, 420, and 480 seconds, respectively. The fluorescence intensity was compared using Fiji software (v.2.14.0) for microscopic analysis, with the traditional chemical dye fluorophenol yellow 088 used as a control.

[0068] The method for staining with fluorophenol yellow 088 (FY 088) is as follows: A working solution of 0.01% (w / v) FY 088 in ethanol was prepared using a 1% (w / v) FY 088 stock solution in dimethyl sulfoxide (DMSO). The entire wild-type Arabidopsis thaliana (Columbia Col-0) plant was immersed in the fluorophenol yellow 088 staining working solution and incubated at room temperature in the dark for 30 min. The fluorophenol yellow 088 staining solution was then discarded. The roots were then counterstained with 0.5% (w / v) aniline blue solution in the dark for 20 min. After counterstaining, the staining solution was discarded, and the plant was quickly washed once in 50% (v / v) ethanol, then washed twice in ddH2O (double-distilled water), and stored in 50% (v / v) glycerol in the dark. Slides were prepared in 50% (v / v) glycerol and observed using an Olympus FV3000 laser confocal imaging system.

[0069] Each staining method was used in triplicate, and the fluorescence quenching was recorded in each group. Results are as follows: Figure 8 As shown, fluorophenol yellow 088 staining is almost invisible at 2-3 minutes of imaging, while fluorescein staining still shows visible fluorescence at 10-11 minutes of imaging. This indicates that the fluorescein probe has stronger fluorescence resistance to quenching than the traditional chemical dye fluorophenol yellow 088, and the fluorescence stability time is increased by about 4 times.

[0070] Example 4: Staining of bark cork cells (1) Mix 5 μL of fluorescein solution with a concentration of 1 mg / mL and pH=13 with 5 μL of cork cells with a concentration of 3 mg / mL, vortex to mix and stain for 2 min (the cork cells were given by the inventor of the invention patent application with publication number CN119662516A). (2) After staining, a washing step was performed: centrifuge at 13000 rpm for 30 s, discard the supernatant, add 1 mL of deionized water, vortex mix for 30 s, centrifuge at 13000 rpm for 2 min, and repeat the process of discarding the supernatant, adding 1 mL of water, vortexing for 30 s, and centrifuging for 2 min twice. Finally, discard the supernatant, add 100 μL of deionized water, vortex mix for 30 s, prepare the slide, and observe using an Olympus FV3000 laser confocal imaging system. The slide before staining was used as the control group. The results are as follows: Figure 9 As shown.

[0071] The results showed that the relative gray value of the fluorescence after staining was 29.48±3.046, the background value was 0.336±0.269, and the fluorescence intensity increased by 88.83 times; therefore, this method can efficiently stain and isolate bark cork cells from plants.

[0072] Example 5: Co-dyeing with fluorescein and conventional chemical dyes Propidium iodide (PI) and Calcofluor White are commonly used for staining and localizing the primary cell walls of plants. In this example, wild-type Arabidopsis thaliana Columbia Col-0 was used as the test material to detect the combined use of fluorescein with propidium iodide and Calcofluor White.

[0073] (1) The entire plant was immersed in 10 mg / L propidium iodide for 10 min, rinsed 4 times in deionized water, and then fluorescein stained on the propidium iodide-stained sample using the same method as in Example 2. Confocal imaging was performed using an Olympus FV3000 laser confocal microscope. The results are as follows: Figure 10 As shown (scale bar is 50μm); then use Fiji software to determine the position of the horizontal cross-section (e.g.) Figure 10 The relative fluorescence intensity of the white horizontal line in the combined figure is used for quantification.

[0074] (2) The entire plant was immersed in a 0.1% (w / v) Carcoflur fluorescent whitening agent solution for staining for 1 hour, rinsed 4 times in deionized water, and then stained with fluorescein using the same method as in Example 2. Confocal imaging was performed using an FV3000 laser confocal microscope. The results are as follows: Figure 11 As shown (scale bar is 50μm); then use Fiji software to determine the position of the horizontal cross-section (e.g.) Figure 11 The relative fluorescence intensity of the white horizontal line in the combined figure is used for quantification.

[0075] The results show that the dyeing method of the present invention can be used in combination with conventional chemical dyes.

[0076] Example 6: Three-dimensional reconstruction and performance determination of Arabidopsis wild-type materials after fluorescein staining To verify the three-dimensional reconstruction effect and fluorescence properties after staining, wild-type Arabidopsis thaliana Columbia Col-0 was stained with fluorescein and then observed and measured. The fluorescein staining method was the same as in Example 2; the fluorophenol yellow 088 staining method was the same as in Example 3.

[0077] For 3D reconstruction fluorescence imaging, an Olympus FV3000 laser confocal imaging system was used, capturing 45 images at 1μm intervals. A 20x microscope was used, with a scanning zoom of 4.0x. 3D reconstruction was then performed using Fiji software. The results are as follows: Figure 12 As shown.

[0078] After 5 minutes of imaging, the fluorescence quenching performance was captured at a magnification of 1.5x. Then, Fiji software was used to quantify the relative fluorescence intensity at the transverse section, quantifying three roots for each. The results are as follows: Figure 13 As shown. Figure 13 Taking the cross-sections represented by pink and black horizontal lines in section 'a' as examples, their respective cross-sections are compared with... Figure 13 In the diagram, lines of the same color in b correspond to the quantitative results of the relative fluorescence intensity at that position in the root (similarly, quantitative results of the relative fluorescence intensity of different roots are obtained; three roots are selected from both the fluorescein staining group and the fluorophenol yellow 088 staining group for quantification, and lines of different colors represent different roots).

[0079] The results showed that fluorescein staining revealed a complete three-dimensional reconstruction interface of suberized cells, especially the reconstruction of suberized cell assemblages in the X / Z axis sections. Furthermore, after 5 minutes of three-dimensional imaging, the relative fluorescence intensity of the control fluorophenol yellow 088 dye was basically reduced to the background value, while the fluorescence intensity of fluorescein was thousands of times higher. Therefore, fluorescein staining can effectively perform three-dimensional reconstruction and fluorescence analysis of suberized cells.

[0080] Example 7: Staining of corky cells in plant roots (1) The whole roots or cut root segments of cucumber, sedum, willow, tobacco, tomato, rice and wheat were soaked in a fluorescein solution of pH=13 at 1 mg / mL; cucumber and wheat roots were soaked for 4 min, sedum roots for 2 min, and willow, tobacco, tomato and rice roots for 8 min. (2) After staining, the root samples were placed in a 6-well plate with tweezers and rinsed 8 times with deionized water. After being pressed into slides, they were observed using Olympus FV3000 laser confocal imaging. The results are as follows: Figures 14-20 As shown (the scale bar for the plant image is 2cm, and the scale bar for the imaging image is 20μm).

[0081] The results showed that staining fluorescence could be observed in different plants, therefore the staining method of the present invention is universally applicable to different plants.

Claims

1. A fluorescent probe for plant corky cells, characterized in that, The fluorescent probe is composed of fluorescein, fluorescein salt, or fluorescein derivative at a concentration of not less than 0.001 mg / mL under conditions of 12 ≤ pH ≤ 13.

5.

2. The fluorescent probe according to claim 1, characterized in that, The concentration of the fluorescein is 0.001~100 mg / mL.

3. The application of the fluorescent probe according to claim 1 or 2, characterized in that, Applications in staining plant corky cells or preparing plant corky cell staining products.

4. The application according to claim 3, characterized in that, The plant suberized cell staining product is a dye, fluorescent probe, or kit.

5. A staining method for plant corky cells, characterized in that, Using the fluorescent probe according to any one of claims 1 or 2, the plant sample and the fluorescent probe are mixed and stained to obtain the stained plant sample.

6. The staining method according to claim 5, characterized in that, The plant sample is the root, bark, or isolated in vitro corky cells of a plant.

7. The staining method according to claim 6, characterized in that, The plant samples were derived from Arabidopsis thaliana, cucumber, Sedum aizoon, wheat, rice, tomato, tobacco, willow, or cork oak.

8. The staining method according to claim 5, characterized in that, The time for mixing and staining plant samples with fluorescent probes is 10s to 24h.

9. The staining method according to claim 5, characterized in that, Before or after mixing and staining plant samples with fluorescent probes, chemical dyes are used to stain the plant samples, thus achieving the combined use of fluorescent probes and chemical dyes.

10. The staining method according to claim 9, characterized in that, The chemical dye is propidium iodide staining solution or Karl Coflurrel fluorescent whitening agent.

Images