Restricted fixation and embedding method for plant tissues

Abstract

The present invention provides a restrictive fixation and embedding method for plant tissues. The method is achieved through chemical fixation, resin infiltration, and positioning technology within an embedding block. The method is suitable for restrictive fixation and embedding of special positions of irregular samples. The method can obtain accurate cell spatial positions while maintaining the original cell morphology and structure of the plant. The method is suitable for positioning and sectioning of rice caryopsis embryonic cells and observing the cell morphology and structure at special positions of plant samples with similar irregular morphologies, thereby laying the foundation for subsequent acquisition of high-quality sections.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue embedding, in particular to a restrictive fixed embedding method of plant tissue. Background Art

[0002] The development of conventional electron microscopy in life sciences has made it possible to observe the ultrastructure of tissues and cells. Before observing the ultrastructure of plant tissues and cells, a semi-thin section is made from the embedded tissue block to determine its position. This serves as a preliminary test of the sample's condition to determine its suitability for further ultrathin sectioning. More importantly, it facilitates spatial localization of the plant tissue. Because plant tissue growth exhibits distinct directional characteristics, and the distribution and arrangement of cells exhibit strong spatial relationships, accurate spatial localization is a crucial step before ultrastructural observation.

[0003] For plant tissues with regular shapes, clear cell alignment, and ease of cutting, restricted cutting is performed during sampling based on the direction of tissue growth. During embedding, the desired tissue section is aligned with the sectioning direction to ensure accurate visualization of the target cells during tissue sectioning. However, for smaller or more irregularly shaped plant tissues, such as the rice caryopsis and similar tissues used in the present invention, where restricted cutting is not feasible, a method for fixing their orientation within the embedding block is highly desirable.

[0004] Currently, for tissues with orientation deviation during embedding, the common approach is to first cut the resin block from the embedded specimen, attach this block to a blank resin block according to the sectioning direction, and then section on a microtome. Alternatively, sectioning can be performed directly on the microtome by adjusting the blade angle and the specimen holder angle. However, both of these methods present certain challenges. The first method, which involves attaching the resin block to the blank resin block, requires first cutting the original resin block according to the desired sectioning direction, polishing the cut surface, and then attaching it to the blank resin block according to the adjusted direction. Because the sectioning direction is relatively microscopic relative to the entire resin block, cutting the resin block in this direction can be unstable. Furthermore, if the two resin blocks are not firmly attached, they can break apart during trimming, resulting in sample loss. The second method, which involves adjusting the orientation directly on the microtome, is suitable for samples with minimal left-right deviation. However, samples with significant left-right deviation or that have flipped over will exceed the microtome's adjustment range. In these cases, the first method is still necessary to adjust the general orientation before adjusting the microtome, which increases instability and time. Summary of the Invention

[0005] The present invention aims to provide a restrictive fixed embedding method for plant tissues, specifically, to provide a positioning embedding method that can maintain the position and direction of plant samples during embedding, and can provide precise directional positioning. Semi-thin sections with accurate positions can be obtained without cutting, gluing embedding blocks, or changing the knife edge or sample rod angle. Ultra-thin sections can be performed on the basis of the completed positioning of the semi-thin sections, and ultrastructural observation and analysis can be performed using a transmission electron microscope.

[0006] To achieve the purpose of the present invention, in a first aspect, the present invention provides a method for restrictive fixation and embedding of plant tissues, comprising the following steps:

[0007] (1) Chemical fixation of plant tissues;

[0008] (2) Dehydration treatment;

[0009] (3) resin penetration;

[0010] (4) Sample embedding.

[0011] Wherein, step (4) includes the following sub-steps:

[0012] A. Preparation of mold

[0013] Cut off a 5-10mm length of the 200μL pipette tip from the tip to form the mold. The length of the mold should be the same as the width of the embedding hole in the embedding plate. Cut off 1 / 4 of the area on one side of the mold, so that the mold is 3 / 4 of a circle when viewed from both ends. The cut 1 / 4 is the opening of the mold, with the open side being the front and the opposite side being the back. The end of the mold closest to the tip of the pipette tip is the top end, and the end away from the tip of the pipette tip is the bottom end. The two long sides of the opening are the left and right sides. The opening of the mold runs from the top to the bottom end, so that the mold is 3 / 4 of a circle when viewed from both ends.

[0014] B. Loading plant tissue into the mold

[0015] Before loading, a small amount of resin is added to the mold to remove the air in the mold; then, according to the morphology of the plant tissue, the resin-infiltrated plant tissue is inserted into the mold from the bottom end in a specific direction;

[0016] C. Encapsulation and polymerization

[0017] The mold containing the plant tissue is placed in the embedding hole, with the front of the mold facing the inside of the embedding hole and the back of the mold facing the top of the embedding hole. The mold is fixed by the elasticity of the embedding plate. Resin is then dripped into the embedding hole so that the resin fully wraps the mold and merges with the resin inside the mold through the mold opening. The embedding plate is oven-polymerized at 60-65°C for 4-5 hours, and then at 70-72°C for 40-48 hours (preferably, the embedding plate is oven-polymerized at 60°C for 5 hours, and then at 72°C for 48 hours).

[0018] D. After polymerization is completed, remove the mold from the embedding hole, remove excess resin around the mold, and use tweezers to gently push the mold from the bottom to the top to separate the mold from the resin inside the mold and expose the plant tissue.

[0019] The plant tissue is rice caryopsis, preferably rice caryopsis 3 to 5 days after rice flowering, more preferably caryopsis tissue 3 days after rice flowering.

[0020] The fixation and embedding technology of the present invention is also applicable to irregular plant tissue organs similar to rice caryopsis but with special observation positions.

[0021] This method is particularly suitable for rice caryopsis samples and similar plant tissues with irregular shapes and appearances in the early stages of development.

[0022] Furthermore, step B includes: taking the tissue near the embryo end of the rice caryopsis, inserting the rice caryopsis from the lower end of the mold with the stigma end facing upward and the near embryo end facing downward; after half of the rice caryopsis tissue is inserted into the mold, lightly touching the rice caryopsis tissue near the embryo end, and adjusting the direction of the rice caryopsis so that the back tissue of the rice caryopsis is parallel to the right side of the mold opening.

[0023] Furthermore, step (1) includes glutaraldehyde fixation and washing: after cutting off the tissue of 1 / 4 of the rice caryopsis near the stigma end, placing it in a centrifuge tube filled with 2.5% glutaraldehyde, and fixing it under a vacuum degree of 0.080-0.085 MPa for 4-5 hours (preferably, fixing it under a vacuum degree of 0.085 MPa for 4 hours); then taking out the centrifuge tube, fixing it in the dark at 4°C for 10-12 hours; aspirating the glutaraldehyde in the centrifuge tube, and adding 0.1M PB buffer to wash the rice caryopsis, preferably washing it 6 times.

[0024] Furthermore, step (2) comprises: after the rice caryopsis is chemically fixed, dehydrating it with 30% ethanol, 50% ethanol, 70% ethanol, and 90% ethanol in sequence, with each dehydration time being 10 minutes; then dehydrating it with 100% ethanol twice, each time for 10 minutes; and finally dehydrating it with 100% acetone twice, each time for 10 minutes.

[0025] Furthermore, step (3) comprises: after the rice caryopsis is dehydrated, gradient infiltration is performed in sequence with 20-30% resin acetone solution, 45-55% resin acetone solution, 70-80% resin acetone solution, and finally infiltration with 100% resin three times;

[0026] Preferably, gradient infiltration is performed using 25% resin acetone solution, 50% resin acetone solution, and 75% resin acetone solution in sequence.

[0027] Furthermore, step (3) is specifically as follows: under the condition of a vacuum degree of 0.080-0.085 MPa, treating the rice caryopsis with a 25% resin acetone solution for 2.5-3.5 hours, treating the rice caryopsis with a 50% resin acetone solution for 13.5-14.5 hours, treating the rice caryopsis with a 75% resin acetone solution for 7.5-8.5 hours, treating the rice caryopsis with a 100% resin for 13.5-14.5 hours, treating the rice caryopsis with a 100% resin for 7.5-8.5 hours, and finally treating the rice caryopsis with a 100% resin for 13.5-14.5 hours;

[0028] Preferably, under a vacuum degree of 0.080-0.085 MPa, the rice caryopsis is treated with 25% resin acetone solution for 3 hours, the rice caryopsis is treated with 50% resin acetone solution for 14 hours, the rice caryopsis is treated with 75% resin acetone solution for 8 hours, the rice caryopsis is treated with 100% resin for 14 hours, the rice caryopsis is treated with 100% resin for 8 hours, and finally the rice caryopsis is treated with 100% resin for 14 hours.

[0029] The resin used in the present invention can be SPURR, which is prepared from 4221, 736, NSA, and DMAE (commercially available) according to a hard formula, wherein 4221 10g, 736 4g, NSA 26g, and DMAE 0.3g.

[0030] Step (4) Before making the mold, place the pipette tip and embedding plate in an oven for drying. Specifically, place the 200 μL pipette tip and embedding plate in a 72°C oven and bake for 15 hours to remove the water droplets adsorbed on the surface.

[0031] In a second aspect, the present invention provides application of the method in preparing plant tissue slices.

[0032] The present invention also provides application of the method in preparing caryopsis embedding slices and fixing plant tissue organs similar to rice caryopsis.

[0033] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0034] The present invention provides a method suitable for restrictive fixation and embedding of special positions of irregular samples. It can obtain accurate cell spatial positions while maintaining the original cell morphology and structure of the plant. It is suitable for positioning sectioning of rice caryopsis embryonic cells and observing the cell morphology and structure at special positions of plant samples with similar irregular shapes, thus laying the foundation for subsequent acquisition of high-quality sections. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The structure of the mold of the present invention is shown in FIG. 1 , which is the upper end; 2 , which is the lower end; 3 , which is the left side; 4 , which is the right side; 5 , which is the front side; and 6 , which is the back side.

[0036] Figure 2 Figure 1 shows the rice caryopsis used in a preferred embodiment of the present invention. Figure A shows the caryopsis morphology as seen from a vertical perspective. Figure B shows the caryopsis morphology as seen from the side. In both figures, the upper left corner of the caryopsis represents the region where the embryonic cells are located near the embryonic end.

[0037] Figure 3 This is the mold used in the preferred embodiment of the present invention. Figure A shows a comparison of cutout locations of a 200μL pipette tip. The top tip is the original, uncut tip, the middle tip is the tip with 5mm removed from the tip, and the bottom tip is the tip with 10mm removed from the tip. The mold of the present invention was cutout 5-10mm from the tip, i.e., the middle tip is longer than the bottom tip. Figures B and C show the topography of the mold when viewed from above, representing 3 / 4 of a circle.

[0038] Figure 4 Figure 1 shows the assembly of a rice caryopsis and mold and their placement into an embedding well in a preferred embodiment of the present invention. Figure A shows the assembled rice caryopsis and mold. The stigma end of the caryopsis is near the upper end of the mold, while the subembryonic end is near the lower end. The dorsal tissue of the subembryonic end of the caryopsis is aligned parallel to the right edge of the mold. Figure B shows the assembled mold and caryopsis placed into the embedding well. The right side of the image represents the inside of the embedding well, while the left side represents the top of the well. The mold opening is positioned toward the inside of the well. The yellow liquid is resin.

[0039] Figure 5 Figures 1 and 2 illustrate the resin block after mold embedding in a preferred embodiment of the present invention. A is a front view of the resin block. B is a side view of the resin block, with the mold opening facing inward. C is a view of the caryopsis without the mold, showing the caryopsis position shifted and the orientation of the embryonic cell tissue region near the embryo end altered.

[0040] Figure 6 Figure 1 shows the removal of the resin surrounding the mold and the removal of the mold in a preferred embodiment of the present invention. Figure A shows the removal of the resin surrounding the mold, and Figure B is an enlarged view of Figure A. Figure C shows the exposed caryopsis structure after mold removal.

[0041] Figure 7 These are semi-thin sections of caryopsis after mold embedding in a preferred embodiment of the present invention. A and B show the embryonic cell structure of a caryopsis 3 days after anthesis, with the nearly oval tissue representing the embryonic cells. C and D show the embryonic cell structure of a caryopsis 5 days after anthesis, with the pear-shaped tissue representing the embryonic cells.

[0042] Figure 8 Figures A and B are transmission electron micrographs of rice caryopsis embryonic cells from a 3-day-old rice caryopsis, and Figure B is a transmission electron micrograph of rice caryopsis embryonic cells from a 5-day-old rice caryopsis.

[0043] Figure 9 The diagrams are the cell structures of rice caryopsis embryos observed under a microscope in Comparative Example 1 of the present invention. A is a transmission electron micrograph of 3-day-old rice caryopsis embryos, and B is a transmission electron micrograph of 5-day-old rice caryopsis embryos.

[0044] Figure 10 The diagrams are diagrams of the embryonic cell structure of rice caryopsis observed under a microscope in Comparative Example 2 of the present invention. A is a diagram of the embryonic cell of a 3-day-old rice caryopsis, and B is a diagram of the embryonic cell of a 5-day-old rice caryopsis. DETAILED DESCRIPTION

[0045] The present invention provides a method for positioning and embedding rice caryopsis in an embedding block, which is achieved by chemical fixation, resin infiltration and positioning and embedding in an embedding block. The resin infiltration and positioning in the embedding block are performed according to the following procedures:

[0046] (1) Rice caryopsis were fixed with 2.5% glutaraldehyde for 4 h, washed with 0.1 M PB, and dehydrated with gradient ethanol concentrations;

[0047] (2) Acetone dehydration and resin infiltration. Specific conditions are shown in Table 1:

[0048] Table 1

[0049]

[0050] Before using acetone, 10g of anhydrous sodium sulfate was added to absorb the residual water in the acetone to ensure complete dehydration. The resins of various concentrations were prepared in acetone.

[0051] (3) Prepare the mold. Take a 200 μL pipette tip and cut an opening on one side with a width of 1.5 mm and a length equal to the mold.

[0052] (4) Assemble the mold and rice caryopsis, insert the rice caryopsis from the bottom of the mold with the stigma end facing up and the embryo end facing down; adjust the direction of the caryopsis so that the back tissue is parallel to the right side of the mold opening;

[0053] (5) Place the assembly horizontally in the embedding plate. Use the elasticity of the embedding plate to fix the two ends of the mold in the embedding hole, with the front of the mold facing the inside of the embedding hole and the back of the mold facing the top of the embedding plate. Add 0.7 ml of resin into the embedding hole.

[0054] (6) Resin polymerization: 60°C oven polymerization for 5 h, 72°C oven polymerization for 48 h;

[0055] (7) Trim and slice the blocks, remove the excess resin wrapped around the mold, remove the mold, and expose the caryopsis; use 300 μm semi-thin sections to locate and observe cell arrangement, and use 90 nm ultra-thin sections to observe ultrastructure.

[0056] The embryonic cells of rice caryopsis are located in the dorsal tissue near the embryo end, presenting a spherical structure and composed of multiple cells densely arranged. As the rice caryopsis develops, the shape of the embryonic cells also changes. Only the cell sections produced by slicing perpendicular to the embryonic cells can reflect their most authentic developmental characteristics and morphological features. Through practical tests, it was found that controlling the direction and position of the rice caryopsis during embedding and polymerization can accurately fix the direction of the embryonic cells perpendicular to the slicing direction and obtain accurate positioning. At the same time, since the embryonic cells are arranged more densely, the present invention has achieved high-quality embedding and slicing results by exploring a large number of conditions and continuously adjusting a series of conditions from fixation, dehydration to resin infiltration.

[0057] In the present invention, when the rice caryopsis is infiltrated during the resin infiltration embedding, the resin is infiltrated in a gradient manner, and the infiltration gradient is 20-30%, 45-55%, 70-80%, 100%, 100%, and 100% in sequence.

[0058] Preferably, when the rice caryopsis is infiltrated during the resin infiltration embedding, the resin is infiltrated in a gradient manner, and the infiltration gradient is 25%, 50%, 75%, 100%, 100%, and 100% in sequence.

[0059] In the present invention, the gradient infiltration is specifically as follows: treating rice caryopsis with a 25% resin-acetone mixture for 2.5-3.5 hours, treating rice caryopsis with a 50% resin-acetone mixture for 13.5-14.5 hours, treating rice caryopsis with a 75% resin-acetone mixture for 7.5-8.5 hours, treating rice caryopsis with a 100% resin for 13.5-14.5 hours, treating rice caryopsis with a 100% resin for 7.5-8.5 hours, and then treating rice caryopsis with a 100% resin for 13.5-14.5 hours.

[0060] Preferably, the rice caryopsis is treated with a 25% resin-acetone mixture for 3 hours, the rice caryopsis is treated with a 50% resin-acetone mixture for 14 hours, the rice caryopsis is treated with a 75% resin-acetone mixture for 8 hours, the rice caryopsis is treated with 100% resin for 14 hours, the rice caryopsis is treated with 100% resin for 8 hours, and the rice caryopsis is treated with 100% resin for 14 hours.

[0061] According to the characteristics of rice caryopsis embryonic cells, the present invention sets the resin infiltration to the above-mentioned specific gradient mode through a large number of trial and error experiments to achieve a good embedding effect, so that subsequent ultrathin section observation can obtain high-quality, clear and complete ultrastructure images.

[0062] In the present invention, the chemical fixation and dehydration steps before the resin embedding and infiltration are performed under a vacuum condition of 0.085 MPa; 0.1M PB is washed 6 times, each time for 10 minutes; the gradient concentration of ethanol is 30%, 50%, 70%, 90%, 100%, and 100%, and the dehydration time for each gradient is 10 minutes.

[0063] In the present invention, after resin infiltration, a 200 μL gun tip is used at a distance of 5-10 mm from the tip as a fixed mold for embedding, and its length is consistent with the width of the embedding plate hole; the opening side of the mold is the front side, and the corresponding side is the back side; the front side of the mold close to the right direction is the lower end, and the front side close to the left direction is the upper end; the two long sides of the opening are the left and right sides.

[0064] In the present invention, the mold is baked in a 72°C oven for 15 hours before use; 0.1 ml of resin is added to the mold before assembly; during assembly, a rice caryopsis is inserted from the bottom of the mold with the stigma end facing upward and the embryo-proximal end facing downward; and the direction of the caryopsis is adjusted so that the back tissue is parallel to the right side of the mold opening.

[0065] Oven baking can effectively remove residual moisture droplets on the surface of the gun tip and embedding plate, which can cause the polymerized resin to break into pieces during slicing. Adding 0.1 ml of resin to the mold before assembly can remove air from the mold space and prevent the formation of bubbles and cavities during polymerization. During embedding, the front of the mold faces the inside of the embedding hole and the back of the mold faces the top of the embedding plate. This allows the resin inside and outside the mold to merge through the opening, facilitating the formation of a complete resin block during polymerization.

[0066] The mold is placed in the embedding hole with its front side facing the inside of the embedding hole and its back side facing the top of the embedding plate; the resin in the mold polymerizes with the resin in the embedding hole outside the mold through the opening.

[0067] The specifications of the gun tip and the cutting position selected by the mold are adjusted according to the size of the caryopsis, and the length is consistent with the width of the embedding hole.

[0068] In the present invention, the excess resin around the back of the mold is trimmed off, and the remaining resin wrapped around the mold is cut off with a double-sided blade; the mold is gently pushed from the lower end to the upper end using tweezers to separate the mold and the caryopsis.

[0069] In this invention, the resin and mold are made of different materials. The resin surrounding and within the mold does not coalesce into a uniform mass, resulting in a dielectric difference between the mold and the resin. For this reason, after removing the excess resin, the restrictive factors surrounding the mold disappear, allowing the mold to be separated by applying a force from the bottom to the top with tweezers.

[0070] The caryopsis may be fresh caryopsis of rice 3-5 days after flowering. Preferably, the caryopsis tissue may be fresh caryopsis of rice 3 days after flowering.

[0071] This method can also be applied to plant tissue organs that are irregular like rice caryopsis but have special observation positions.

[0072] The present invention also provides application of the method in preparing caryopsis embedding slices and fixing plant tissue organs similar to rice caryopsis.

[0073] The method of the present invention specifically comprises the following steps:

[0074] (1) Chemical fixation;

[0075] (2) Dehydration at room temperature;

[0076] (3) resin penetration;

[0077] (4) Caryopsis embedding.

[0078] The reagent used for chemical fixation was 2.5% glutaraldehyde; the dehydration reagents at room temperature were 30%, 50%, 70%, 90%, 100% ethanol and 100% acetone; the resin was SPURR, prepared according to a hardening formula of 4221, 736, NSA, and DMAE, including 10g of 4221, 4g of 736, 26g of NSA, and 0.3g of DMAE.

[0079] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0080] The structure of the mold used in the following examples is shown in Figure 1 Among them, 1-top, 2-bottom, 3-left side, 4-right side, 5-front, 6-back.

[0081] The appearance of the rice caryopsis used in the following examples is shown in Figure 2 Among them, A shows the morphology of rice caryopsis taken from a vertical perspective. B shows the morphology of rice caryopsis taken from the side. In both pictures, the upper left corner of the rice caryopsis is organized as the area where the embryonic cells near the embryo end are located.

[0082] The material location of the mold used in the following examples is shown in Figure 3 Figure A shows a comparison of the cutout positions of a 200μL tip. The top tip is the original, uncut tip, the middle tip is the tip with 5mm removed from the tip, and the bottom tip is the tip with 10mm removed from the tip. The mold of the present invention was cutout 5-10mm from the tip, i.e., the middle tip is longer than the bottom tip. Figures B and C show the topography of the mold as viewed from above, representing 3 / 4 of a circle.

[0083] The schematic diagram of assembling rice caryopsis and mold and placing them into embedding hole is shown in Figure 4 Figure A shows the assembly of a rice caryopsis and a mold. The stigma end of the caryopsis is near the top of the mold, and the subembryonic end is near the bottom. The dorsal tissue of the subembryonic end of the caryopsis is aligned parallel to the right side of the mold. Figure B shows the assembled mold and caryopsis placed in the embedding hole. The right side of the image represents the inside of the embedding hole, while the left side represents the top of the hole. The mold is placed with the opening facing the inside of the hole. The yellow liquid is resin.

[0084] The schematic diagram of the resin polymerizing into a block after mold embedding is shown in Figure 5 A is a front view of the resin block. B is a side view of the resin block, with the mold opening facing inward. C is a photo of a caryopsis aggregate without a mold, showing that the caryopsis is offset and the direction of the embryonic cell tissue region near the embryo end has changed.

[0085] See the schematic diagram of removing the resin wrapped around the mold and removing the mold. Figure 6 A is the image after the resin around the mold is removed, B is the enlarged image of A, and C is the exposed caryopsis tissue after the mold is removed.

[0086] Diagram of rice caryopsis embryonic cell structure. A is a transmission electron micrograph of a 3-day-old rice caryopsis embryonic cell, and B is a transmission electron micrograph of a 5-day-old rice caryopsis embryonic cell.

[0087] Example 1 Fixation and embedding of rice caryopsis 3 days after flowering (mold)

[0088] This embodiment provides a method for positioning and embedding rice caryopsis in an embedding plate, which is specifically as follows:

[0089] 1. Glutaraldehyde fixation and washing

[0090] (1) Take rice caryopsis 3 days after anthesis, cut off 1 / 4 of the tissue near the stigma end of the rice caryopsis to facilitate the penetration of the fixative, and place it in a centrifuge tube containing 2 ml of 2.5% glutaraldehyde; vacuum pump to 0.085 MPa to accelerate the penetration of the fixative, and fix for 4 hours;

[0091] (2) Remove the centrifuge tube and fix it in the dark at -4℃ for 12 hours.

[0092] (3) Aspirate the glutaraldehyde in the centrifuge tube and add 2 ml of 0.1 M PB buffer to wash the rice caryopsis to remove any residual glutaraldehyde after fixation. Wash six times, 5 min each time.

[0093] 2. Ethanol gradient dehydration

[0094] (1) Dehydrate by adding 2 ml of 30% ethanol, 50% ethanol, 70% ethanol, and 90% ethanol in sequence, with each dehydration time of 10 min;

[0095] (2) Dehydrate by adding 2 ml of 100% ethanol twice, each time for 10 minutes.

[0096] 3. Acetone dehydration and resin penetration

[0097] Resin is incompatible with ethanol, so dehydration with 100% acetone is performed twice before resin infiltration to fully replace the ethanol in the sample. See Table 1 for the detailed process.

[0098] 4. Prepare the mold

[0099] Before the last resin infiltration, place the 200μL gun tip and the embedding plate in a 72℃ oven for 15 hours to remove the water droplets adsorbed on the surface. After the resin infiltration is completed, the mold is prepared. According to the width of the embedding hole in the embedding plate, the corresponding length of the gun tip is cut off. The width of the embedding hole in this embodiment is 5mm, so the length of 5-10mm from the tip of the 200μL gun tip is cut off as the mold. Use a blade to cut off 1 / 4 of the area on one side of the mold, which is a 3 / 4 circle when viewed from both ends of the mold; the cut 1 / 4 is the opening of the mold, and the opening side is the front, and the corresponding one is the back; the front of the mold close to the right direction is the lower end, and close to the left direction is the upper end; the two long sides of the opening are the left and right sides; the opening of the mold runs through from the upper end to the lower end, and the mold is a 3 / 4 circle when viewed from both ends.

[0100] 5. Assemble the mold and rice caryopsis

[0101] Before assembly, add 0.1 ml of resin to the mold to remove air from the mold. During assembly, use tweezers to gently grasp the tissue near the embryonic end of the caryopsis and insert the rice caryopsis from the bottom of the mold with the stigma end facing up and the embryonic end facing down. After half of the caryopsis tissue is inserted into the mold, gently touch the caryopsis tissue near the embryonic end and adjust the direction of the caryopsis so that the back tissue is parallel to the right side of the mold opening.

[0102] 6. Mold and sample embedding and polymerization

[0103] Place the assembled mold and sample horizontally within the embedding well, with the front of the mold facing the inside of the well and the back facing the top. Use the elasticity of the embedding plate to secure the mold. Add 0.7ml of resin to the embedding well, ensuring the resin fully envelops the mold and fuses with the resin inside through the mold opening. Oven-polymerize at 60°C for 5 hours and 72°C for 48 hours.

[0104] 7. Remove the mold to expose the caryopsis tissue

[0105] Use a trimmer to trim the resin around the back of the mold, and then use a double-sided blade to trim the resin wrapped around both sides of the mold; use tweezers to gently push the mold from the bottom to the top. Due to the medium difference between the mold and the resin, the mold is separated from the resin, and the caryopsis tissue is exposed.

[0106] 8. Trimming, sectioning and staining

[0107] The sample was clamped on the microtome. Without adjusting the direction of the sample rod and the angle of the blade, the tissue was trimmed directly to the position of the embryonic cells of the caryopsis with a glass knife. The slices were sliced ​​300 nm and placed on a glass slide. After drying at 60°C, the slices were stained with toluidine blue. The cell morphology and position structure were observed under an optical microscope. Figure 7 (AB) It can be seen that the cell tissue morphology of the caryopsis embryo is complete and the relative position is accurate. After determining the cell position information, a diamond knife was used to slice 90 nm and the slice was scooped out and stained on a copper grid for electron microscopy observation, which revealed a clear cell structure.

[0108] Example 2 Fixation and embedding of rice caryopsis 5 days after flowering (mold)

[0109] This embodiment provides a method for embedding endosperm tissue, which is as follows (parts not described in detail are the same as those in Example 1):

[0110] 1. Glutaraldehyde fixation and washing

[0111] (1) Take rice caryopsis 5 days after anthesis, cut off 1 / 4 of the tissue near the stigma end of the rice caryopsis to facilitate the penetration of the fixative, and place it in a centrifuge tube containing 2 ml of 2.5% glutaraldehyde; vacuum pump to 0.085 MPa to accelerate the penetration of the fixative, and fix for 4 hours;

[0112] (2) Remove the centrifuge tube and fix it in the dark at -4℃ for 12 hours.

[0113] (3) Aspirate the glutaraldehyde in the centrifuge tube and add 2 ml of 0.1 M PB buffer to wash the rice caryopsis to remove any residual glutaraldehyde after fixation. Wash six times, 5 min each time.

[0114] 2. Ethanol gradient dehydration

[0115] (1) Dehydrate by adding 2 ml of 30% ethanol, 50% ethanol, 70% ethanol, and 90% ethanol in sequence, with each dehydration time of 10 min;

[0116] (2) Dehydrate by adding 2 ml of 100% ethanol twice, each time for 10 minutes.

[0117] 3. Acetone dehydration and resin penetration

[0118] Resin is incompatible with ethanol, so dehydration with 100% acetone is performed twice before resin infiltration to fully replace the ethanol in the sample. See Table 1 for the detailed process.

[0119] 4. Prepare the mold

[0120] Before the last resin infiltration, place the 200μL gun tip and the embedding plate in a 72℃ oven for 15 hours to remove the water droplets adsorbed on the surface. After the resin infiltration is completed, the mold is prepared. According to the width of the embedding hole in the embedding plate, the corresponding length of the gun tip is cut off. The width of the embedding hole in this embodiment is 5mm, so the length of 5-10mm from the tip of the 200μL gun tip is cut off as the mold. Use a blade to cut off 1 / 4 of the area on one side of the mold, which is a 3 / 4 circle when viewed from both ends of the mold; the cut 1 / 4 is the opening of the mold, and the opening side is the front, and the corresponding one is the back; the front of the mold close to the right direction is the lower end, and close to the left direction is the upper end; the two long sides of the opening are the left and right sides; the opening of the mold runs through from the upper end to the lower end, and the mold is a 3 / 4 circle when viewed from both ends.

[0121] 5. Assemble the mold and rice caryopsis

[0122] Before assembly, add 0.1 ml of resin to the mold to remove air from the mold. During assembly, use tweezers to gently grasp the tissue near the embryonic end of the caryopsis and insert the rice caryopsis from the bottom of the mold with the stigma end facing up and the embryonic end facing down. After half of the caryopsis tissue is inserted into the mold, gently touch the caryopsis tissue near the embryonic end and adjust the direction of the caryopsis so that the back tissue is parallel to the right side of the mold opening.

[0123] 6. Mold and sample embedding and polymerization

[0124] Place the assembled mold and sample horizontally within the embedding well, with the front of the mold facing the inside of the well and the back facing the top. Use the elasticity of the embedding plate to secure the mold. Add 0.7ml of resin to the embedding well, ensuring the resin fully envelops the mold and fuses with the resin inside through the mold opening. Oven-polymerize at 60°C for 5 hours and 72°C for 48 hours.

[0125] 7. Remove the mold to expose the caryopsis tissue

[0126] Use a trimmer to trim the resin around the back of the mold, and then use a double-sided blade to trim the resin wrapped around both sides of the mold; use tweezers to gently push the mold from the bottom to the top. Due to the medium difference between the mold and the resin, the mold is separated from the resin, and the caryopsis tissue is exposed.

[0127] 8. Trimming, sectioning and staining

[0128] The sample was clamped on the microtome. Without adjusting the direction of the sample rod and the angle of the blade, the tissue was trimmed directly to the position of the embryonic cells of the caryopsis with a glass knife. The slices were sliced ​​300 nm and placed on a glass slide. After drying at 60°C, the slices were stained with toluidine blue. The cell morphology and position structure were observed under an optical microscope. Figure 7(CD), it can be seen that the cell tissue morphology of the caryopsis embryo is intact and the relative position is accurate; after determining the cell position information, a diamond knife was used to slice 90nm, and the slice was scooped out and stained on a copper grid, and electron microscopy was performed, and it was found that the cell structure was clear.

[0129] Comparative Example 1:

[0130] This comparative example provides a method for embedding endosperm tissue, which is the same as the method in Example 1, except that no mold is used during embedding. Instead, the rice caryopsis is directly placed into the embedding hole for embedding. During the resin polymerization process, the position of the rice caryopsis is shifted, the direction of the embryonic cell organization is changed, and the spatial position and morphological characteristics of the embryonic cells produced by direct sectioning are changed, such as Figure 8 As shown in Figure 2, the embryonic cell position relative to the caryopsis is greatly displaced, and the morphological structure also changes accordingly. The space between cells increases, and the cell structure near the embryo end of the rice caryopsis is deformed as a whole. Figure 9 Among them, A is a transmission electron micrograph of 3-day-old rice caryopsis embryonic cells, and B is a transmission electron micrograph of 5-day-old rice caryopsis embryonic cells.

[0131] In summary, the rice caryopsis embedding method provided by the present invention is achieved through chemical fixation, resin infiltration, and positioning within the embedding block. By exploring the resin infiltration gradient, time, and positioning and embedding within the embedding block, this method can obtain rice caryopsis tissue samples that maintain the original plant cell structure and morphology and are precisely positioned. This method is suitable for rice caryopsis samples with irregular shapes and appearances during early development and similar plant tissues. Tissue samples embedded using the present method can subsequently be quickly and precisely sectioned into semi-thin sections, as well as high-quality ultrathin sections, suitable for ultrastructural observation using transmission electron microscopy.

[0132] Comparative Example 2:

[0133] This comparative example provides a method for embedding rice caryopsis tissue, which is the same as the methods of Examples 1 and 2, except that the resin infiltration procedure is as shown in Table 2:

[0134] Table 2

[0135]

[0136] The obtained slice results are shown in Figure 10 .from Figure 10 It can be seen that the rice caryopsis slices are wrinkled and there are a large number of cavities inside the cells, which affects the observation of cell structure.

[0137] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for restrictive fixation and embedding of plant tissues, characterized in that: The following steps are involved: (1) Chemical fixation of plant tissues; (2) Dehydration treatment; (3) Resin penetration; (4) Sample embedding; Wherein, step (4) includes the following sub-steps: A. Making mold Cut off a 5-10mm length of the 200mL pipette tip from the tip to form the mold. The length of the mold should be the same as the width of the embedding hole in the embedding plate. Cut off 1 / 4 of the area on one side of the mold, so that the mold is a 3 / 4 circle when viewed from both ends. The cut-off 1 / 4 is the mold opening, with the open side being the front and the opposite side being the back. The end of the mold closest to the pipette tip is the top end, and the end away from the pipette tip is the bottom end. The two long sides of the opening are the left and right sides. The mold opening runs from the top end to the bottom end. B. Loading plant tissue into the mold Before loading, a small amount of resin is added to the mold to remove the air in the mold; then, according to the morphology of the plant tissue, the resin-infiltrated plant tissue is inserted into the mold from the bottom end in a specific direction; C. Encapsulation and polymerization Place the mold containing the plant tissue in the embedding hole, with the front of the mold facing the inside of the embedding hole and the back facing the top of the embedding hole. Use the elasticity of the embedding plate to fix the mold. Then, add resin to the embedding hole so that the resin fully wraps the mold and merges with the resin inside the mold through the mold opening. Polymerize the embedding plate in an oven at 60-65°C for 4-5 hours, and then in an oven at 70-72°C for 40-48 hours. D. After polymerization is completed, remove the mold from the embedding hole, remove excess resin around the mold, and use tweezers to gently push the mold from the bottom to the top to separate the mold from the resin inside the mold and expose the plant tissue.

2. The method according to claim 1, characterized in that The plant tissue is rice caryopsis.

3. The method according to claim 2, characterized in that The plant tissue is rice caryopsis 3 to 5 days after rice flowering.

4. The method according to claim 2, characterized in that Step B includes: taking tissue near the embryonic end of a rice caryopsis, inserting the rice caryopsis from the lower end of a mold with the stigma end facing upward and the embryonic end facing downward; after half of the rice caryopsis tissue is inserted into the mold, lightly touching the rice caryopsis tissue near the embryonic end, and adjusting the direction of the rice caryopsis so that the back tissue of the rice caryopsis is parallel to the right side of the mold opening.

5. The method according to claim 2, characterized in that Step (1) includes glutaraldehyde fixation and washing: after cutting off 1 / 4 of the tissue near the stigma end of the rice caryopsis, placing it in a centrifuge tube filled with 2.5% glutaraldehyde, and fixing it under a vacuum degree of 0.080-0.085 MPa for 4-5 hours; then taking out the centrifuge tube, fixing it in the dark at 4°C for 10-12 hours; aspirating the glutaraldehyde in the centrifuge tube, and adding 0.1M PB buffer to wash the rice caryopsis.

6. The method according to claim 5, characterized in that The rice caryopsis was washed 6 times with 0.1 M PB buffer.

7. The method according to claim 5, characterized in that Step (2) comprises: after the rice caryopsis is chemically fixed, dehydrating it with 30% ethanol, 50% ethanol, 70% ethanol and 90% ethanol in sequence, with each dehydration time being 10 minutes; then dehydrating it with 100% ethanol twice, with each dehydration time being 10 minutes; and finally dehydrating it with 100% acetone twice, with each dehydration time being 10 minutes.

8. The method according to claim 7, characterized in that Step (3) comprises: after the rice caryopsis is dehydrated, gradient infiltration is performed with 20-30% resin acetone solution, 45-55% resin acetone solution, 70-80% resin acetone solution in sequence, and finally infiltration is performed with 100% resin three times.

9. The method according to claim 8, characterized in that After dehydration, rice caryopsis were infiltrated with 25% resin acetone solution, 50% resin acetone solution, and 75% resin acetone solution in a gradient manner.

10. The method according to claim 8, characterized in that Step (3) is specifically as follows: under the condition of a vacuum degree of 0.80-0.85 MPa, treating the rice caryopsis with a 25% resin acetone solution for 2.5-3.5 h, treating the rice caryopsis with a 50% resin acetone solution for 13.5-14.5 h, treating the rice caryopsis with a 75% resin acetone solution for 7.5-8.5 h, treating the rice caryopsis with a 100% resin for 13.5-14.5 h, treating the rice caryopsis with a 100% resin for 7.5-8.5 h, and finally treating the rice caryopsis with a 100% resin for 13.5-14.5 h.

11. The method according to claim 10, characterized in that Under vacuum conditions of 0.080-0.085 MPa, rice caryopsis were treated with 25% resin acetone solution for 3 h, 50% resin acetone solution for 14 h, 75% resin acetone solution for 8 h, 100% resin for 14 h, 100% resin for 8 h, and finally 100% resin for 14 h.

12. The method according to any one of claims 1 to 11, characterized in that The resin is SPURR.

13. The method according to any one of claims 1 to 11, characterized in that Step (4) Before making the mold, place the gun tip and embedding plate in an oven for drying.

14. Use of the method according to any one of claims 1 to 13 in the preparation of plant tissue slices.

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