Preparation method of macadamia leaf bud paraffin sections

Abstract

The present invention provides a method for preparing paraffin sections of macadamia leaf buds, belonging to the technical field of plant paraffin sections. The present invention performs concentrated hydrochloric acid softening before dehydration. The softening concentration and softening time are selected, which directly affect the integrity of the sections. The above-mentioned solution of the present invention can produce complete and clear sections, and has great application potential for materials with hirsute characteristics. The method of the present invention can produce complete and continuous wax strips with a thickness of between 15 and 20 μm and an integrity of more than 73.3%, which can meet the needs of anatomical structure analysis. The tissue in the middle of the section is intact, and the tissue structure is clear after staining, with bright colors.

Description

Technical Field

[0001] The invention relates to the technical field of plant paraffin sections, and in particular to a method for preparing macadamia leaf bud paraffin sections. Background Art

[0002] Macadamia (Macadamia integrifolia) is an evergreen tree in the Proteaceae family. Its kernels are rich in unsaturated fatty acids, protein, and sugars, boasting high nutritional value and a unique flavor. my country accounts for 60% of the world's cultivated area as a commercial tree. Despite this significant planting area, my country accounts for only 14% of the total production, creating an urgent need for high-yield cultivation technologies.

[0003] Buds are the fundamental units of plant growth and development, and their internal structural changes require anatomical analysis. Paraffin sectioning is the most widely used and cost-effective method. Typical paraffin sectioning techniques include fixation, dehydration, transparency, wax impregnation, embedding, sectioning, mounting, baking, and observation. Macadamia leaf buds are located in the axils of leaves and are covered with a dense layer of gray-brown hairs. Their hard texture makes it difficult for reagents to penetrate the material during sectioning, making it difficult to obtain complete images of macadamia paraffin sections using conventional paraffin sectioning techniques.

[0004] In the prior art, for this type of hairy plant material, researchers have added a softening step before the paraffin section dehydration step:

[0005] The more commonly used methods include boiling in water for 30 minutes (see Shuai Huanli, Yang Tuxi, Wei Anzhi, et al. Improvement of paraffin section method for apricot flower buds [J]. Journal of Fruit Science, 2011, 28(3): 536-539; Chen Lina, Xue Hui, Li Haoxian, et al. Improvement of paraffin section method for pomegranate flower buds [J]. Anhui Agricultural Sciences, 2017, 45(2): 4);

[0006] Soak in 20% hydrochloric acid for 24 hours (see Zhang Ledao, Cao Shiying, Wang Guoze, et al. Improved paraffin section technology and its application in drying of Chinese yam [J]. Food and Fermentation Industries, 2023, 49(1): 238-243; Li Zehua. Study on flower bud differentiation and embryology of Feijoa [D]. Southwest University of Science and Technology [2024-05-30]);

[0007] Ethanol:glycerol (1:1) immersion method (Xu Junliang, Zhang Yiping, Su Wei, et al. A paraffin section method for tree stem tissue: CN 201610965041[P].CN 106525530 A[2024-05-30].), etc.

[0008] However, the slices prepared by the above methods are incomplete. How to further optimize the preparation method of paraffin slices to obtain complete and clear microscopic images of macadamia leaf buds is a problem that needs to be solved urgently. Summary of the Invention

[0009] In view of this, the present invention provides a method for preparing paraffin sections of macadamia leaf buds, which solves the problem that it is difficult to obtain complete paraffin sections due to the hardness and high hairiness of macadamia leaf buds. The obtained paraffin sections have high clarity, high integrity and good staining effect.

[0010] The method for preparing macadamia leaf bud paraffin sections of the present invention comprises the following steps:

[0011] (1) Fixation: Immerse the entire bud in FAA solution for more than 24 hours;

[0012] (2) Softening: Transfer the fixed material to a 25-35% hydrochloric acid solution and soften it for 2-4 days;

[0013] (3) Dehydration: Rinse the softened buds with distilled water and then soak them in ethanol for dehydration;

[0014] (4) Transparency: After dehydration, the buds were immersed in a xylene-anhydrous ethanol mixed solution, pure xylene, and pure xylene until transparent;

[0015] (5) Wax dipping: Place the transparent buds in a crushed wax solution and place them in an oven at 50-60°C for 30 min. Take out the buds and place them in pure paraffin wax, and place them in an oven at 50-60°C for 3 days to obtain the wax-dipping buds.

[0016] (6) Sectioning: Add new wax to the embedding box, place the wax-soaked bud, and solidify it at room temperature overnight. After trimming the wax block until the surface is flat, slice it with a microtome. Place the complete section in a constant temperature water bath filled with distilled water at 40-45°C and flatten it.

[0017] (7) Adhesive and spreading: Dip a small amount of adhesive tablet and spread it evenly on the glass slide, flatten the slice on the glass slide, and bake for 2 to 3 hours;

[0018] (8) staining to obtain the macadamia leaf bud paraffin section.

[0019] Preferably, the FAA solution in step (1) is more than 10 times the volume of the leaf bud.

[0020] Preferably, the dehydration in step (3) is to rinse the softened buds with distilled water and then soak them in 70%, 85%, 95%, 100% and 100% ethanol in sequence for 20 minutes each.

[0021] Preferably, the volume ratio of xylene to anhydrous ethanol in the xylene-anhydrous ethanol mixed solution in step (4) is 1:1.

[0022] Preferably, the crushed wax solution in step (5) is prepared by adding crushed wax to xylene until saturated; and the pure paraffin wax in step (5) is replaced 3 to 4 times during the heating period.

[0023] Preferably, the slice thickness in step (6) is 10 to 20 μm.

[0024] Preferably, the flattening process in step (6) is performed with the smooth side facing downward.

[0025] Preferably, the adhesive tablet in step (7) is composed of glycerol and egg white in a mass ratio of 1:1; and the temperature of the baking tablet is 40-45°C.

[0026] Preferably, the dyeing in step (8) comprises the following steps:

[0027] (a) Place the slide containing the leaf bud in xylene twice for 15 min each time;

[0028] (b) Place in a mixed solution of xylene and anhydrous ethanol at a volume ratio of 1:1 for 1 to 2 minutes;

[0029] (c) The mixture was then placed in anhydrous ethanol, 85% ethanol solution, 70% ethanol solution, 50% ethanol solution, and distilled water, for 1-2 minutes each.

[0030] (d) After removal, stain with 0.5% toluidine blue solution for 10 seconds, and gently remove excess stain with distilled water.

[0031] (e) sequentially transferring the sample to a 50% ethanol solution, a 70% ethanol solution, an 85% ethanol solution, anhydrous ethanol, a mixed solution of xylene and anhydrous ethanol in a volume ratio of 1:1, and xylene for 1-2 minutes each, adding a small amount of neutral gum, and covering with a coverslip to obtain the macadamia leaf bud paraffin section.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention provides a method for preparing paraffin sections of macadamia nut buds. The fixed macadamia nut buds are softened with concentrated hydrochloric acid. The softening and slight corrosion of the hydrochloric acid can dissolve part of the villi on the surface of the buds, reduce their water-proof performance, soften the epidermal tissue, and allow solutions such as ethanol, xylene, and dye, as well as paraffin, to better penetrate into the interior of the buds and better blend with the paraffin, thereby improving the integrity of the sections and achieving rich colors after dyeing.

[0034] The present invention adopts a hydrochloric acid softening step and selects the softening concentration and softening time, which directly affect the integrity of the slices. The above scheme of the present invention can obtain complete and clear slices and has great application potential on hairy materials.

[0035] The method of the present invention can produce complete and continuous wax strips with a thickness of 15-20 μm and an integrity of over 73.3%, which can meet the needs of anatomical structure analysis. The tissue in the middle of the section is intact, and after staining, the tissue structure is clear and the color is bright. Moreover, this method can be used to prepare paraffin sections not only of macadamia leaf buds but also of macadamia branches. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is an optical microscope image of the bud tissue in Example 1;

[0037] Figure 2 This is an optical microscope image of the bud tissue in Example 2;

[0038] Figure 3 is an optical microscope image of the stem tissue in Example 3;

[0039] Figure 4 The strip diagram of paraffin sections with different softening treatments;

[0040] Figure 5 The microstructure of leaf bud slices with different softening treatments;

[0041] Figure 6 The strip diagram of paraffin sections after being softened with hydrochloric acid of different concentrations;

[0042] Figure 7 The microscopic structure of leaf bud slices after being softened by hydrochloric acid at different concentrations;

[0043] Figure 8 The band diagrams of paraffin sections at different treatment times. DETAILED DESCRIPTION

[0044] The present invention will be further described below with reference to the embodiments.

[0045] Example 1

[0046] A method for preparing paraffin sections of macadamia leaf buds, comprising the following steps:

[0047] (1) Fixation: Use a scalpel to cut the hairy and hard leaf buds at the leaf axils, gently scrape off part of the hairs on the bud surface with a scalpel until part of the epidermal tissue is exposed, and then immerse the entire bud in FAA (70% ethanol: glacial acetic acid: formalin = 18:1:1) solution for 24 h, with the FAA reagent being 10 times the volume of the material;

[0048] (2) Softening: The fixed material was transferred to a 30% hydrochloric acid solution and softened for 3 days.

[0049] (3) Dehydration: After the softened buds were rinsed with distilled water, they were immersed in gradient concentrations of ethanol (70%, 85%, 95%, 100%, and 100%) for 20 min each to complete the dehydration.

[0050] (4) Transparency: After dehydration, the buds were transferred to a mixed solution of xylene and anhydrous ethanol with a volume ratio of 1:1, pure xylene, and pure xylene, and immersed for 20 minutes each, and the transparency was completed;

[0051] (5) Wax dipping: Add crushed wax to xylene until saturated, transfer the buds to the saturated solution, and place them in a 60°C oven for 30 minutes; then replace the saturated solution with pure paraffin wax, and place them in a 60°C oven for 3 days, replacing the pure paraffin wax once a day during the storage period;

[0052] (6) Embedding: Add new wax to the embedding box and place the wax-soaked bud. After solidifying overnight at room temperature, trim the wax block until the surface is smooth and stick it to a small wooden block. Slice the bud with a microtome to a thickness of 10-20 μm. Place the complete slice in a constant temperature water bath filled with 40°C distilled water and flatten it with the shiny side facing down.

[0053] (7) Adhesive and spreading: Take a small amount of adhesive (glycerol: egg white volume ratio of 1:1) and spread it evenly on the slide, and select qualified slices and flatten them on the slide; place the slide on a 40℃ slicing table and bake for 3 hours;

[0054] (8) Dyeing: The specific steps are as follows:

[0055] (a) Place the slide containing the leaf bud in xylene twice, each time for 15 min.

[0056] (b) Place in a mixed solution of xylene and anhydrous ethanol with a volume ratio of 1:1 for 1 min;

[0057] (c) The cells were then placed in anhydrous ethanol, 85% ethanol solution, 70% ethanol solution, 50% ethanol solution, and distilled water, for 1 min each.

[0058] (d) After removal, stain with 0.5% toluidine blue solution for 10 seconds, and gently remove excess stain with distilled water.

[0059] (e) The samples were sequentially transferred to 50% ethanol solution, 70% ethanol solution, 85% ethanol solution, anhydrous ethanol, a mixed solution of xylene and anhydrous ethanol with a volume ratio of 1:1, and xylene for 1 min each, and a small amount of neutral gum (neutral gum purchased from Solebold) was added dropwise. The samples were covered with a coverslip to obtain the paraffin sections of macadamia leaf buds.

[0060] The bud tissue structure was observed using an optical microscope. Figure 1 . Figure 1 The undifferentiated growth point of the bud and the sepals on both sides are clearly visible.

[0061] Example 2

[0062] A method for preparing macadamia leaf bud paraffin sections, comprising the same steps as in Example 1.

[0063] The difference is that the raw material of Example 2 is the leaf buds that have already sprouted in the leaf axils.

[0064] The bud tissue structure was observed using an optical microscope. Figure 2 . Figure 2 The central growth point of the differentiated leaf bud, the leaf primordia on both sides and the two sepals are clearly visible.

[0065] Example 3

[0066] A method for preparing paraffin sections of macadamia nut branches, with the same steps as in Example 1.

[0067] The difference is that the raw materials of Example 3 are macadamia nut branches, which are cut into small segments with a horizontal diameter × vertical diameter × height of less than 5×5×3 mm using pruning shears.

[0068] The tissue structure of the stem was observed using an optical microscope. Figure 3 . Figure 3 It can be seen in the figure that this method is used on the stem, and the stem's epidermis, vascular bundles, phloem and xylem and other tissue structures are clearly visible.

[0069] The following control test was carried out on the treatment method of the present invention:

[0070] Experiment 1: Exploring the effects of different softening methods on paraffin sections of macadamia leaf buds

[0071] The method of Example 1 was used to prepare macadamia leaf bud paraffin sections, except that the softening method was different:

[0072] Treatment 1: No softening treatment.

[0073] Treatment 2: Wrap the buds in gauze and boil them in water for 30 minutes to soften them before dehydration.

[0074] Treatment 3: Before dehydration, the buds were softened by soaking in ethanol:glycerol (1:1) for 3 days.

[0075] Treatment 4: Same as Example 1.

[0076] The integrity of the wax strips obtained after different softening treatments is shown in Table 1:

[0077] Table 1

[0078]

[0079] As can be seen from Table 1, treatments 1 to 3 are unable to form a complete wax band (10-15 μm).

[0080] The paraffin sections strips of different softening treatments in treatments 1 to 4 are as follows Figure 4 The microstructure of leaf bud slices in treatment 3 and treatment 4 with different softening treatments is shown in Figure 2. Figure 5 .

[0081] from Figure 4 and Figure 5 It can be seen that in Comparative Examples 1 to 3, the wax bands are hollow in the middle, the wax strips are broken, the tissue is broken, and the leaf primordium tissue of the leaf bud cannot be observed under a microscope; while the method of Treatment 4 (the present invention) can form a complete wax band and obtain a clear microstructure image.

[0082] Experiment 2: Comparison of softening effects of different hydrochloric acid concentrations

[0083] A method for preparing macadamia leaf bud paraffin sections was prepared using the method of Example 1, except that:

[0084] Treatment 5: The hydrochloric acid concentration of the softening process is 10%, and the softening time is 2 days.

[0085] Treatment 6: The hydrochloric acid concentration of the softening process is 20%, and the softening time is 2 days.

[0086] Treatment 7: The hydrochloric acid concentration of the softening process is 30%, and the softening time is 2 days.

[0087] Treatment 8: The hydrochloric acid concentration of the softening process is 40%, and the softening time is 2 days.

[0088] The integrity of the wax strips after softening with hydrochloric acid at different concentrations in treatments 5 to 6 is shown in Table 2:

[0089] Table 2

[0090]

[0091] As can be seen from Table 2, the integrity of the sections treated with 30% and 40% concentrated hydrochloric acid was relatively high, and the wax band integrity was between 46.6% and 53.3% at a thickness of 15-20 μm.

[0092] The paraffin sections after treatments 5 to 8 with different concentrations of hydrochloric acid softening were as follows: Figure 6 ; The microstructure of leaf bud slices after softening with different concentrations of hydrochloric acid Figure 7 .from Figure 6 and Figure 7As can be seen, treatments with 10% and 20% hydrochloric acid showed poor results, with the tissue in the middle of the slice easily damaged. After staining, toluidine blue stained well with 10%-30% hydrochloric acid, while the color faded after treatment with 40% hydrochloric acid. Therefore, 30% hydrochloric acid is the optimal concentration for treatment.

[0093] Experiment 3: Comparison of treatment effects with different softening times

[0094] A method for preparing macadamia leaf bud paraffin sections (hydrochloric acid concentration 30%) was prepared using the method of Example 1, with the following differences:

[0095] Treatment 9: Softening time is 0d.

[0096] Treatment 10: Softening time is 1 day.

[0097] Treatment 11: Softening time is 2d.

[0098] Treatment 12: Softening time is 3d.

[0099] Treatment 13: Softening time is 4 days.

[0100] The wax strip integrity under different softening time treatments is shown in Table 3:

[0101] Table 3

[0102]

[0103] As can be seen from Table 3, the integrity of the wax strips treated for 3d and 4d is very good, reaching more than 73.3% at a thickness of 15-20μm.

[0104] Paraffin sections at different treatment times Figure 8 ;from Figure 8 It can be seen that the middle tissue is not damaged, so 3-4 days are the best treatment days.

[0105] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing paraffin sections of macadamia leaf buds, characterized in that: The following steps are involved: (1) Fixation: Immerse the entire bud in FAA solution for more than 24 hours; (2) Softening: Transfer the fixed material to a 25-35% hydrochloric acid solution and soften it for 2-4 days; (3) Dehydration: Rinse the softened buds with distilled water and then soak them in ethanol for dehydration; (4) Transparency: After dehydration, the buds were immersed in a xylene-anhydrous ethanol mixed solution, pure xylene, and pure xylene until transparent; (5) Wax dipping: Place the transparent buds in a crushed wax solution and place them in an oven at 50-60°C for 30 min. Take out the buds and place them in pure paraffin wax, and place them in an oven at 50-60°C for 3 days to obtain the wax-dipping buds. (6) Sectioning: Add new wax to the embedding box, place the wax-soaked bud, and solidify it at room temperature overnight. After trimming the wax block until the surface is flat, slice it with a microtome. Place the complete section in a constant temperature water bath filled with distilled water at 40-45°C and flatten it. (7) Adhesive and spreading: Dip a small amount of adhesive tablet and spread it evenly on the glass slide, flatten the slice on the glass slide, and bake for 2 to 3 hours; (8) staining to obtain the macadamia leaf bud paraffin section.

2. The method for preparing the macadamia leaf bud paraffin sections according to claim 1, wherein The FAA solution in step (1) is more than 10 times the volume of the leaf bud.

3. The method for preparing the macadamia leaf bud paraffin sections according to claim 1, wherein The dehydration in step (3) is to rinse the softened buds with distilled water and then soak them in 70%, 85%, 95%, 100% and 100% ethanol in sequence for 20 minutes each.

4. The method for preparing the macadamia leaf bud paraffin sections according to claim 1, wherein The volume ratio of xylene to anhydrous ethanol in the xylene-anhydrous ethanol mixed solution of step (4) is 1:

1.

5. The method for preparing the macadamia leaf bud paraffin sections according to claim 1, wherein: The crushed wax solution described in step (5) is prepared by adding crushed wax to xylene until saturated; the pure paraffin described in step (5) is replaced 3 to 4 times during the heating period.

6. The method for preparing macadamia leaf bud paraffin sections according to claim 1, wherein: The slice thickness in step (6) is 10 to 20 μm.

7. The method for preparing macadamia leaf bud paraffin sections according to claim 1, wherein: The adhesive tablet in step (7) is composed of glycerol and egg white in a mass ratio of 1:1; the temperature of the baking tablet is 40-45°C.

8. The method for preparing macadamia leaf bud paraffin sections according to claim 1, wherein: The dyeing described in step (8) comprises the following steps: (a) Place the slide containing the leaf bud in xylene twice for 15 min each time; (b) Place in a mixed solution of xylene and anhydrous ethanol at a volume ratio of 1:1 for 1 to 2 minutes; (c) The mixture was then placed in anhydrous ethanol, 85% ethanol solution, 70% ethanol solution, 50% ethanol solution, and distilled water, for 1-2 minutes each. (d) After removal, stain with 0.5% toluidine blue solution for 10 seconds, and gently remove excess stain with distilled water. (e) sequentially transferring the sample to a 50% ethanol solution, a 70% ethanol solution, an 85% ethanol solution, anhydrous ethanol, a mixed solution of xylene and anhydrous ethanol in a volume ratio of 1:1, and xylene for 1-2 minutes each, adding a small amount of neutral gum, and covering with a coverslip to obtain the macadamia leaf bud paraffin section.

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