Paraffin section manufacturing method for analyzing photosynthetic characteristics of bamboo reed leaves
By adding softening steps and optimizing other steps during paraffin sectioning, the problem of unclear tissue structure of bamboo leaves was solved, and high-quality sections suitable for photosynthetic characteristics were prepared.
Patent Information
- Application Number
- CN202510687276.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-27
- Publication Date
- 2025-08-15
AI Technical Summary
The leaves of reed bamboo are hard, and it is difficult to prepare sections with clear tissue structures in existing paraffin sectioning technology, which affects the study of photosynthetic characteristics.
The softening steps were added during the paraffin sectioning process, and treated with 15% HF solution for 4 days, the section thickness was 10 μm, the crocus dyed for 70 min, and the solid green dyed for 10 s were optimized, and the dehydration, transparency, wax permeability and dyeing steps were optimized.
Sections with complete and clear tissue structure and clear staining were prepared, which is suitable for the study of photosynthetic characteristics of bamboo leaves, improving the accuracy and efficiency of the study.
Smart Images
Figure CN120489675A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of paraffin sections, and in particular relates to a method for preparing paraffin sections for analyzing photosynthetic characteristics of Arundo donax leaves. Background Art
[0002] Arundo donax is a perennial rhizome plant belonging to the genus Arundo donax in the Poaceae family, widely distributed in tropical and temperate regions. Its leaves are high in protein, adaptable to a wide range of conditions, and highly productive. Its excellent resistance to biotic and abiotic stresses makes it a viable resource and forage plant, as well as a valuable resource for saline-alkali land remediation and wastewater purification.
[0003] Arundo donax leaves are the primary photosynthetic organ and are directly exposed to the external environment, making them extremely sensitive to environmental changes. Their structural characteristics can reflect the plant's growth state and the impact of environmental changes. Therefore, in-depth research on the anatomical structure and photosynthetic characteristics of Arundo donax leaves will contribute to its comprehensive utilization.
[0004] Paraffin sectioning is a common technique for studying morphological changes in plant cell tissues. However, the rigid structure of Arundo donax leaves makes them difficult to wax and section. Therefore, strengthening the application of paraffin sectioning in the study of photosynthetic characteristics of Arundo donax leaves can provide technical support for the structural and process-based study of Arundo donax leaves. Revealing the photosynthetic characteristics of Arundo donax leaves at a microscopic level is of great significance for the establishment of key anatomical databases and will also facilitate the development and application of paraffin sectioning technology. Summary of the Invention
[0005] (1) Sampling: Select fresh Arundo donax leaves, clean them, take a 20-30 mm wide leaf with the midrib as the center, and then trim it into 10 mm long sections;
[0006] (2) Fixation: Fix the sample with a fixative for 24 to 48 hours;
[0007] (3) Softening: The sample was immersed in 15% HF for 4 days;
[0008] (4) Dehydration: Dehydrate the softened samples by treating them with 70%, 80%, 95%, 100%, and 100% ethanol-water solutions for 1 h each.
[0009] (5) Transparency: Treat the sample with anhydrous ethanol and xylene in a volume ratio of 1:1 for 1 h, and then treat the sample with xylene twice, each for 1 h;
[0010] (6) Paraffinization and embedding: Treat the transparent sample with a saturated paraffin xylene solution at 37°C for 48 hours; then raise the oven temperature to 65°C, replace the saturated paraffin xylene solution in the wax cup with preheated molten paraffin, and treat for 4 to 8 hours; immerse the sample vertically in the liquid paraffin on the bottom film of the embedding box, place the embedding box lid with holes in the bottom film, and then place the embedding box on a condenser plate for solidification to complete the embedding;
[0011] (7) Sectioning, spreading, attaching, and baking: Fix the cover of the embedding box on the microtome and perform continuous sectioning at a thickness of 10 μm. Connect the small wax pieces into a long strip slightly shorter than the slide. Place the wax strip in a 38-42°C water bath to unfold it. Then pick it up with a slide and attach it to the slide. Dry it thoroughly on a 42°C baking machine for 20 min.
[0012] (8) Dewaxing: Place the fully dried sections in a staining jar and treat with xylene twice, 5 minutes each time;
[0013] (9) Staining and decolorization: The treated sections were placed in anhydrous ethanol and xylene solution with a volume ratio of 1:1 for 3 min, then treated with 100%, 100%, 95%, 85%, and 70% ethanol for 2 min each, treated in safranin staining solution for 70 min, rinsed the staining solution slowly with water for 30 s, and then treated with 70%, 85%, 95%, and 95% ethanol for 2 min each;
[0014] (10) Restaining, decolorization, and dehydration: stain with 0.1% fast green for 10 seconds, rinse the stain slowly with water for 30 seconds, then treat with 95%, 100%, and 100% ethanol by volume for 2 minutes each, then treat with a solution of anhydrous ethanol and xylene in a volume ratio of 1:1 for 3 minutes, and finally treat with xylene twice for 3 minutes;
[0015] (11) Sealing: Add an appropriate amount of neutral gum to the treated slide and place it in a ventilated place to dry for 5 minutes.
[0016] Preferably, in step (1), the leaf selected is the first leaf that is fully expanded from the top downward, and the middle part of the leaf is sliced for observation. The present invention mainly selects leaves that are mature leaves that have been cultured in soil for 5 months after rooting in tissue culture.
[0017] Preferably, the FAA fixative in step (2) comprises 70% ethanol solution, 40% formaldehyde solution and glacial acetic acid in a volume ratio of 90:5:5; and the volume of the FAA fixative is more than 20 times the volume of the Phragmites australis rhizome and leaf samples.
[0018] Preferably, in step (5), the paraffin wax is kept in liquid state during the paraffinization process, and the temperature is kept at 56-60°C at a constant temperature.
[0019] Preferably, a hand-cranked slicer is used for slicing in step (7), and the blade needs to be replaced frequently to ensure that it is sharp and undamaged to avoid damaging the plant material during the slicing process.
[0020] Preferably, in step (7), when the plant material is placed on the drying machine for full drying, the drying time should not be too long, and the next step can be carried out after the water evaporates, so as to avoid placing the plant material on the drying machine for too long and affecting the integrity of the plant material.
[0021] Preferably, in step (9), the treated slices are placed in a safranin staining solution for treatment for at least 70 minutes, and the treatment time should not be too long to avoid damaging the leaves.
[0022] Preferably, in step (11), the amount of neutral gum used for sealing should not be too much, and after placing the cover slip, press gently to prevent the cover slip from moving and causing sample displacement or damage, which would affect the observation effect.
[0023] The beneficial effects of the present invention include at least:
[0024] The present invention adds a softening step to the paraffin sectioning process of Arundo donax leaves and defines methods for dehydration, softening, transparency, wax penetration, staining, and decolorization. This method is suitable for studying the photosynthetic characteristics of Arundo donax leaves. One objective of the present invention is to address the problems of blank slices and unclear tissue structure in Arundo donax leaf sections prepared using conventional paraffin sectioning techniques. The paraffin sections of Arundo donax leaves produced in this patent have complete cellular tissue and clear structure, facilitating in-depth research on the morphological structure and photosynthetic characteristics of Arundo donax leaves. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 This is a paraffin section image obtained by slicing the leaf at 2 months old selected in the present invention.
[0026] Figure 2 This is a paraffin section obtained from leaf slices cultured in soil for 5 months in the present invention.
[0027] Figure 3 This is a paraffin section obtained after softening treatment for 8 days.
[0028] Figure 4 This is a paraffin section image obtained when the section thickness is set to 6 μm.
[0029] Figure 5 This is a paraffin section stained with safranin for 70 minutes and fast green for 30 seconds.
[0030] Figure 6This is a paraffin section stained with safranin for 150 minutes and fast green for 10 seconds. Specific implementation methods
[0031] The following will be combined with specific embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.
[0032] Example 1
[0033] This embodiment provides a paraffin section method for analyzing the photosynthetic characteristics of Phragmites australis leaves, comprising the following steps:
[0034] (1) Sampling: Select fresh Arundo donax leaves, clean them, take leaves 20-30 mm wide with the midrib as the center, and then trim them into 10 mm long sections;
[0035] (2) Fixation: Fix the sample with fixative for 24 hours;
[0036] (3) Softening: The sample was immersed in 15% HF for 4 days;
[0037] (4) Dehydration: Dehydrate the softened samples by treating them with 70%, 80%, 95%, 100%, and 100% ethanol-water solutions for 1 h each.
[0038] (5) Transparency: Treat the sample with anhydrous ethanol and xylene in a volume ratio of 1:1 for 1 h, and then treat the sample with xylene twice, each for 1 h;
[0039] (6) Paraffinization and embedding: Treat the transparent sample with a saturated solution of paraffin and xylene at 37°C for 48 hours; then raise the oven temperature to 65°C, replace the saturated solution of paraffin and xylene in the wax cup with preheated molten paraffin, and treat for 4-8 hours; finally, embed; then immerse the sample vertically in the liquid paraffin on the bottom film of the embedding box, place the embedding box lid with holes in the bottom film, and then place the embedding box on a condenser plate for solidification;
[0040] (7) Sectioning, spreading, attaching, and baking: Fix the cover of the embedding box on the microtome and perform continuous sectioning with a thickness of 10-15 μm. Connect the small wax pieces into a long strip slightly shorter than the slide. Place the wax strip in a 38-42°C water bath and unfold it. Then pick it up with a slide and attach it to the slide. Dry it in a 42°C baking machine for 20 minutes.
[0041] (8) Dewaxing: Place the fully dried sections in a staining jar and treat with xylene twice, 3 minutes each time;
[0042] (9) Staining and decolorization: The treated sections were placed in a solution of anhydrous ethanol and xylene in a volume ratio of 1:1 for 3 minutes, then treated with 100%, 100%, 95%, 85%, and 70% ethanol for 2 minutes each, treated with safranin staining solution for 70 minutes, rinsed the staining solution slowly with clean water for 30 seconds, and then treated with 70%, 85%, 95%, and 95% ethanol for 2 minutes each.
[0043] (10) Restaining, decolorization, and dehydration: stain with 0.1% fast green for 10 seconds, rinse the stain slowly with water for 30 seconds, then treat with 95%, 100%, and 100% ethanol by volume for 2 minutes each, then treat with a solution of anhydrous ethanol and xylene in a volume ratio of 1:1 for 3 minutes, and finally treat with xylene twice for 3 minutes;
[0044] (11) Sealing: Add neutral gum to the treated slide and place it in a ventilated place to dry for 5 minutes.
[0045] About the selection of Arundo donax leaves:
[0046] The leaves of Arundo donax were taken at different stages, and the stages were set as after tissue culture rooting, soil culture for 2 months, and soil culture for 5 months. The first leaf fully expanded from the top was taken. The other method steps were the same as in Example 1. The effects of different growth stages on the paraffin section preparation effect of Arundo donax leaves were compared. The results are shown in Table 1.
[0047] Table 1 Effects of different growth stages on the preparation of paraffin sections of Phragmites australis leaves
[0048] serial number Collection period Slice effect 1 Phragmites australis leaves cultured in soil for two months The epidermis, assimilated tissue, and vascular bundle structures are clear. 2 Phragmites australis leaves cultivated in soil for five months The epidermis, assimilated tissue, and vascular bundle structures are clear
[0049] As shown in Table 1, the effect of leaf slices after 2 months of soil culture of Phragmites australis tissue culture seedlings is as follows: Figure 1 As shown in the figure, the epidermis, assimilation tissue and vascular bundle structure are clear in the slices, and the cell tissue phase is relatively small; the leaf slice effect of Phragmites australis tissue culture seedlings after 5 months of soil culture is as follows Figure 2 As shown in the figure, the epidermis, assimilated tissue and vascular bundle structure are clear in the slices, and the leaves of the 5-month-old Arundo donax are larger than those of the 2-month-old. Comprehensive comparison shows that the present invention is applicable to the paraffin sectioning of Arundo donax leaves at different growth stages.
[0050] Softening treatment method:
[0051] The softening treatment was set to no softening, 15% HF softening treatment for 1 day, 15% HF softening treatment for 4 days, and 15% HF softening treatment for 8 days. The other method steps were the same as Example 1. The effects of different softening times on the paraffin section preparation effect of Arundo donax leaves were compared. The results are shown in Table 1.
[0052] Table 2 Effects of different softening times on the preparation of paraffin sections of Phragmites australis leaves
[0053]
[0054] As shown in Table 1, when the leaves were not softened or softened with 15% HF for 1 day, they were too hard to slice and the slices were empty. When softened with 15% HF for 4 days, the slices were Figure 2 As shown, the leaf epidermis, assimilation tissue, and vascular bundle structure are clear; when softened with 15% HF for 8 days, the preparation results are as follows Figure 3 As shown, the leaf epidermis, assimilation tissue, and vascular bundle structure are clear; when softened with 15% HF for 8 days, the preparation results are as follows Figure 3 As shown, the leaf epidermis, assimilation tissue, and vascular bundle structure are clear; when softened with 15% HF for 15 days, the preparation results are the same as those of Figure 3 The leaf epidermis, assimilated tissue and vascular bundle structure are clear; when softened with 15% HF for 30 days, the preparation results are similar to those of Figure 3 The leaf epidermis, assimilated tissue, and vascular bundle structure were clear; when softened with 15% HF for 90 days, the leaf color was abnormal, the texture was brittle, and microscopic observation was impossible. After comprehensive comparison, the present invention selected 15% HF softening for 4 days for the softening treatment of Arundo donax leaf paraffin sections.
[0055] Selection of different slice thicknesses:
[0056] The paraffin sections were prepared with thicknesses of 6, 10, and 15 μm. Other steps were the same as in Example 1. The effects of different section thicknesses on the preparation of paraffin sections of Phragmites australis leaves were compared. The results are shown in Table 3.
[0057] Table 3 Effects of different slice thicknesses on paraffin sections of Arundo donax leaves
[0058] serial number thickness Slice effect 1 6μm Tissue damage and incomplete vascular structure 2 10μm The tissue structure is complete and clear, and the vascular bundle structure is easy to distinguish 3 15μm The tissue structure is complete and clear, and the vascular bundle structure is easy to distinguish
[0059] As shown in Table 3, when the slice thickness is set to 6 μm, the preparation results are as follows Figure 4 As shown, the tissue is wrinkled and folded, the vascular bundle structure is incomplete, and the tissue structure cannot be clearly observed; when the slice thickness is set to 10μm, the preparation results are as follows Figure 2 As shown in Figure 2, the tissue structure is complete and clear, and the vascular bundle structure is easy to distinguish. When the slice thickness is set to 15μm, the preparation results are the same as those of Figure 2The tissue structure is complete and clear, and the vascular bundle structure is easy to distinguish. Comprehensive comparison, the present invention sets the slice thickness to 10 μm for paraffin section preparation of Arundo donax leaves.
[0060] Dyeing treatment:
[0061] The paraffin sections were subjected to three treatments: safranin staining for 70 min / fast green staining for 10 s, safranin staining for 70 min / fast green staining for 30 s, and safranin staining for 150 min / fast green staining for 10 s. The other method steps were the same as in Example 1. The effects of different staining times on the preparation effect of paraffin sections of Phragmites australis leaves were compared. The results are shown in Table 4.
[0062] Table 4 Effects of different staining times on the preparation of paraffin sections of Phragmites australis leaves
[0063]
[0064]
[0065] As shown in Table 4, the results of staining with safranin dye for 70 minutes and fast green dye for 10 seconds are as follows: Figure 2 As shown, the leaf epidermis and vascular bundle structures are clear, the red and green staining effects are good, and the structures are easy to distinguish; when stained with safranin dye for 70 minutes and fast green dye for 30 seconds, the preparation results are as follows Figure 5 As shown in the figure, the epidermal structure of the leaf is relatively clear, but the color separation of the vascular bundles is not obvious, and the whole leaf is green. When the leaf is stained with safranin dye for 150 minutes and fast green dye for 10 seconds, the preparation results are as follows: Figure 6 As shown, the leaf epidermal structure is relatively clear, but the color separation of safranin and fast green is not obvious, with more red and less green, and the phloem and xylem structures of the vascular bundle are difficult to distinguish. Comprehensive comparison, the present invention selects to use safranin dye for 70 minutes and fast green dye for 10 seconds to prepare paraffin sections of Arundo donax leaves.
[0066] In summary, the present invention improves the preparation steps for paraffin sections of Arundo donax leaves, including softening with a 15% HF solution for 4 days, selecting a paraffin section thickness of 10 μm, staining with safranin for 70 minutes, and staining with Fast Green for 10 seconds. This method fully preserves the cellular arrangement and tissue structure characteristics of Arundo donax leaves, producing sections with intact and clear tissue structure and distinct staining. This method is an excellent paraffin section preparation method for analyzing the photosynthetic characteristics of Arundo donax leaves.
[0067] The above describes the preferred embodiments of the present invention. It should be understood that the present invention is not limited to the specific embodiments described above. A person skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for preparing paraffin sections for analyzing the photosynthetic characteristics of Phragmites australis leaves, characterized in that: The following steps are involved: (1) Sampling: Select fresh Arundo donax leaves, clean them, take a 20-30 mm wide leaf with the midrib as the center, and then trim it into 10 mm long sections; (2) Fixation: Fix the sample with FAA fixative for 24 to 48 hours; (3) Softening: The sample was immersed in 15% HF for 4 days; (4) Dehydration: Dehydrate the softened samples by treating them with 70%, 80%, 95%, 100%, and 100% ethanol-water solutions for 1 h each. (5) Transparency: Treat the sample with anhydrous ethanol and xylene in a volume ratio of 1:1 for 1 h, and then treat the sample with xylene twice, each for 1 h; (6) Paraffinization and embedding: Treat the transparent sample with a saturated paraffin xylene solution at 37°C for 48 hours; then raise the oven temperature to 65°C, replace the saturated paraffin xylene solution in the wax cup with preheated molten paraffin, and treat for 4 to 8 hours; immerse the sample vertically in the liquid paraffin on the bottom film of the embedding box, place the embedding box lid with holes in the bottom film, and then place the embedding box on a condenser plate for solidification to complete the embedding; (7) Sectioning, spreading, attaching, and baking: Fix the cover of the embedding box on the microtome and perform continuous sectioning at a thickness of 10 μm. Connect the small wax pieces into a long strip slightly shorter than the slide. Place the wax strip in a 38-42°C water bath to unfold it. Then pick it up with a slide and attach it to the slide. Dry it thoroughly on a 42°C baking machine for 20 min. (8) Dewaxing: Place the fully dried sections in a staining jar and treat with xylene twice, 3 minutes each time; (9) Staining and decolorization: The treated sections were placed in a solution of anhydrous ethanol and xylene in a volume ratio of 1:1 for 3 min, then treated with 100%, 100%, 95%, 85%, and 70% ethanol for 2 min each, and treated in safranin staining solution for 70 min. The staining solution was slowly rinsed with water for 30 s, and then treated with 70%, 85%, 95%, and 95% ethanol for 2 min each; (10) Restaining, decolorization, and dehydration: stain with 0.1% fast green for 10 seconds, rinse the stain slowly with water for 30 seconds, then treat with 95%, 100%, and 100% ethanol by volume for 2 minutes each, then treat with a solution of anhydrous ethanol and xylene in a volume ratio of 1:1 for 3 minutes, and finally treat with xylene twice for 3 minutes; (11) Sealing: Add an appropriate amount of neutral gum to the treated slide and place it in a ventilated place to dry for 5 minutes.
2. The method for preparing paraffin sections for analyzing photosynthetic characteristics of Phragmites australis leaves according to claim 1, wherein: As described in step (1), the leaf selected is the first leaf that is fully expanded from the top downward, and the middle part of the leaf is sliced for observation. The present invention mainly selects leaves that are mature leaves that have been cultured in soil for 5 months after rooting in tissue culture.
3. The method for preparing paraffin sections for analyzing photosynthetic characteristics of Phragmites australis leaves according to claim 2, wherein: The FAA fixative in step (2) comprises 70% ethanol solution, 40% formaldehyde solution and glacial acetic acid in a volume ratio of 90:5:5; and the volume of the FAA fixative is more than 20 times the volume of the Phragmites australis rhizome sample.
4. The method for preparing paraffin sections for analyzing photosynthetic characteristics of Phragmites australis leaves according to claim 3, wherein: Step (5) keeps the paraffin wax in liquid state during the paraffinization process and keeps the temperature at 56-60°C at a constant temperature.
5. The method for preparing paraffin sections for analyzing photosynthetic characteristics of Phragmites australis leaves according to claim 4, wherein: When slicing in step (7), a hand-cranked slicer is used, and the blade needs to be replaced frequently to ensure that it is sharp and undamaged to avoid damaging the plant material during the slicing process.
6. The method for preparing paraffin sections for analyzing photosynthetic characteristics of Phragmites australis leaves according to claim 5, characterized in that: When the plant material is placed on the drying machine to be fully dried in step (7), the drying time should not be too long. The next step can be carried out after the moisture evaporates to avoid placing the plant material on the drying machine for too long to affect the integrity of the plant material.
7. The method for preparing paraffin sections for analyzing photosynthetic characteristics of Phragmites australis leaves according to claim 6, characterized in that: In step (9), the treated slices are placed in safranin staining solution for 70 minutes. The treatment time should not be too long to avoid damaging the leaves.
8. A method for preparing paraffin sections for analyzing photosynthetic characteristics of Phragmites australis leaves according to any one of claims 1 to 7, characterized in that: In step (11), do not use too much neutral gum when sealing the slide. After placing the cover slip, press gently to prevent the cover slip from moving and causing sample displacement or damage, which would affect the observation effect.