Preparation method of paraffin section for analyzing pinus massoniana stem xylem characteristics

Abstract

The invention belongs to the technical field of plant paraffin sections, and discloses a preparation method of a paraffin section for analyzing the characteristics of xylem of a masson pine stem. The method comprises the following steps: (1) softening; (2) segmenting; (3) dehydrating; (4) transparentizing; (5) waxing; (6) embedding; (7) slicing, spreading, adhering and baking; (8) dewaxing and rehydrating; (9) dyeing and decolorizing; and (10) sheet sealing. According to the method, the preparation steps of the paraffin section are improved, specifically, the method comprises the steps of softener selection, wood part segmentation, dyeing time adjustment and the like, by adopting the technical scheme, the paraffin section with complete masson pine stem tissue and clear structure can be obtained, and the anatomical characteristics of the masson pine stem xylem are easy to observe and measure.

Description

Technical Field

[0001] This invention belongs to the field of plant paraffin sectioning technology, specifically relating to a method for preparing paraffin sections for analyzing the xylem characteristics of Masson pine stems. Background Technology

[0002] Masson pine is one of the most important tree species for plantation afforestation due to its strong adaptability, rapid growth, high yield, and drought resistance. Its wood is widely used in papermaking, construction, furniture and other fields. Observing the xylem characteristics of Masson pine can quantify the radial growth process at the cellular level, providing a reference for studying the physiological mechanisms of Masson pine.

[0003] Paraffin sectioning is a common technique for studying plant tissue structure, typically involving steps such as fixation, softening, dehydration, clearing, wax impregnation, embedding, sectioning, mounting, dewaxing, and staining. For example, the invention patent CN114235536B discloses a method for preparing sections for observing lignin deposition in the stems of two-year-old Masson pine. However, Masson pine stems are relatively hard, and conventional sectioning methods result in tissue breakage, complex operations, and long processing times. Therefore, it is necessary to improve the traditional paraffin sectioning method and establish a paraffin sectioning method suitable for Masson pine stems, laying the foundation for exploring the annual growth process of Masson pine. Summary of the Invention

[0004] The present invention aims to provide a method for preparing paraffin sections for analyzing the xylem characteristics of Masson pine stems, in order to solve the problems of hard material, easy breakage of sections, and difficulty in observing the anatomical features of xylem in Masson pine stems.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing paraffin sections for analyzing the xylem characteristics of Masson pine stems, characterized by comprising the following steps: (1) Softening: Place the obtained pine core sample in a constant temperature water bath, add deionized water to immerse it, cover and steam at 75-85℃ for 8 hours, and after natural cooling, soak it at room temperature for 24-48 hours. (2) Segmentation: After removing the core sample, divide it into 5-10 mm stem segments and mark the side of each stem segment near the bark. (3) Dehydration: The softened stem segments were placed in 50% and 70% ethanol solutions for 1 hour each time, then placed in 90% ethanol solution twice for 50 minutes each time, and then placed in 100% ethanol solution twice for 40 minutes each time. (4) Transparency: The stem segments were treated with a 1:1 volume mixture of 100% ethanol and xylene for 40 min, and then treated with xylene twice for 40 min each time. (5) Wax impregnation: After the stem segments have become transparent, they are immersed in liquid paraffin for two treatments, each lasting 40 minutes. (6) Embedding: The sample was embedded at 60℃; (7) Slicing, spreading, gluing and baking: Cut the embedded sample into 8μm thick wax slices using a slicer, spread the wax slices in a 42℃ water bath, spread the gluing agent evenly on a glass slide, lift the spread wax slices with a glass slide, and then dry them in a 65℃ baking machine for 30min. (8) Dewaxing and rehydration: Place the dried sections in a staining tank and treat them with xylene four times, each time for 5-10 minutes. Then place the treated sections in 100%, 95%, and 70% ethanol solutions and distilled water for 20 seconds each time. (9) Staining and decolorization: Place the treated sections in a staining tank and treat with 1% safranin aqueous solution for 15 min. Place the stained sections in distilled water for 10 s, and then treat with 70% ethanol four times for 10 s each time. (10) Mounting: Add two drops of neutral resin to the prepared glass slide and mount it.

[0006] Preferably, in step (1), the core sample should be obtained from a healthy mature forest, with a diameter of 5-6 mm and a length of 20-50 cm, and softened by boiling in deionized water at 80°C for 8 hours and soaked for 24 hours.

[0007] Preferably, in step (2), the core sample is divided into segments from the bark to the core, and a neutral pen is used to mark the side of each stem segment near the bark along the vascular bundle, which is the bark direction.

[0008] Preferably, the paraffin wax used in step (5) is pure wax with a melting point of 58-60℃ and a melting point of 60-62℃.

[0009] Preferably, in step (6), during embedding, a portion of paraffin wax is first attached to the mold, the core is adjusted to be perpendicular to the conduit line and placed into the paraffin wax, the bark direction is uniformly to the left or right, the embedding box is placed on top, and then an appropriate amount of paraffin wax is attached. The mold and embedding box are then placed on a cooling table to cool.

[0010] Preferably, in step (7), the adhesive tablet is a mixture of egg white and glycerin in a volume ratio of 1:1.

[0011] Preferably, as described in step (7), the wax slides should be removed promptly after spreading to avoid affecting the observation of tissues due to the expansion of the wax slides.

[0012] The beneficial effects of this invention are as follows: In the process of preparing paraffin sections of Masson pine stems, this invention adopts a constant-temperature water bath softening method, reducing the use of chemical reagents and minimizing the environmental impact of waste liquid. Simultaneously, the time for dehydration, clearing, wax impregnation, and staining is adjusted, achieving the effects of shortening the time and improving the efficiency of sectioning while ensuring the sectioning effect. This invention can obtain paraffin sections with clear and complete tissue structure of Masson pine stems, facilitating the observation of the anatomical characteristics of the xylem in Masson pine stems, and is beneficial for conducting research on the annual growth process of Masson pine stems. Attached Figure Description

[0013] Figure 1 This is a micrograph of the stem tissue structure of *Pinus massoniana* in Example 1 of the present invention; Figure 2 For the observation of this invention Figure 1 Micrographs of the anatomical features of the xylem in a section; Figure 3 The slices obtained by softening treatment by heating and steaming in an 80℃ water bath for 6 hours and soaking for 24 hours. Detailed Implementation

[0014] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments, but this does not constitute any limitation on the present invention.

[0015] Example 1 As attached Figure 1 , 2 As shown in this embodiment, a preparation method for analyzing the xylem characteristics of Masson pine stems is provided. The method includes the following steps: (1) Sampling: Tree rings of Masson pine were sampled at breast height (about 1.3m) using a growth cone with an inner diameter of 5.15mm. The length of the obtained core sample was 38cm. (2) Softening: Place the pine core sample in an 80℃ constant temperature water bath and steam for 8 hours, then soak for 24 hours; (3) Segmentation: Divide the core sample into 50 stem segments of 5-10 mm from the bark towards the core. Mark the side of each stem segment near the bark with a ballpoint pen along the vascular bundle (this is the bark direction). (4) Dehydration: The softened stem segments were placed in 50%, 70%, 90%, 90%, 100%, and 100% ethanol solutions in sequence for 1 h, 1 h, 50 min, 50 min, 40 min, and 40 min, respectively. (5) Transparency: The stem segments were treated with a 1:1 volume mixture of 100% ethanol and xylene for 40 min, and then treated with xylene twice for 40 min each time. (6) Waxing: The transparent stem segments are immersed in liquid paraffin twice, each time for 40 minutes; the immersion temperature is 60-62℃; (7) Embedding: First, attach some paraffin wax to the mold, adjust the core to be perpendicular to the conduit line and immerse it in the paraffin wax, and make sure the bark is facing left or right. Place the embedding box on top, then attach an appropriate amount of paraffin wax. Place the mold and embedding box on a cold table to cool. After the bottom layer of wax solidifies, remove the mold to obtain a paraffin block with the embedding box tightly connected. (8) Slicing, spreading, gluing and baking: Cut the embedded paraffin block into 8μm wax slices using a rotary microtome. Spread the wax slices in a 42℃ water bath. Spread the gluing agent (a mixture of egg white and glycerin in a 1:1 volume ratio) evenly on the glass slide. Then, use the glass slide to lift the spread wax slices and place them on a 65℃ baking machine to dry for 30 minutes. (9) Dewaxing and rehydration: Place the dried sections in a staining tank and treat them with xylene four times, each time for 5-10 minutes. Then place the treated sections in 100%, 95%, and 70% ethanol solutions and distilled water for 20 seconds each time. (10) Staining and decolorization: Place the treated sections in a staining tank and treat with 1% safranin aqueous solution for 15 min. Place the stained sections in distilled water for 10 s, and then treat with 70% ethanol four times for 10 s each time. (11) Sealing: Add two drops of neutral resin to the prepared glass slide and cover it with a coverslip to seal the slide.

[0016] Softening treatment method: The softening treatment was set as follows: no softening, 80℃ water bath heating and cooking for 6 hours and soaking for 24 hours, 80℃ water bath heating and cooking for 8 hours and soaking for 24 hours, and 80℃ water bath heating and cooking for 8 hours and soaking for 48 hours. Other methods and steps were the same as in Example 1. The effects of different softening times on the preparation of paraffin sections of Pinus massoniana were compared, and the results are shown in Table 1.

[0017] Table 1. Effects of different softening times on the preparation of paraffin sections from Pinus massoniana stems. As shown in Table 1, the unsoftened core samples were too hard to be sectioned; the slide preparation results after heating and boiling in an 80℃ water bath for 6 hours and soaking for 24 hours were as follows. Figure 3 As shown, the core sample is hard and the sections are easily broken; when heated in a water bath at 80℃ for 8 hours and soaked for 24 hours, the slide preparation results are as follows. Figure 1 , 2 As shown, the sections are intact and have clear structures; the slide preparation results after heating and cooking in a water bath at 80℃ for 8 hours and soaking for 48 hours are similar to those obtained by traditional methods. Figure 1 , 2 Similarities; in summary, this invention preferentially selects 80℃ water bath heating and steaming for 8 hours and soaking for 24 hours as the softening treatment in the preparation of paraffin sections of Masson pine stems.

[0018] See Figure 1 , Figure 2 This is a paraffin section of a Masson pine stem according to an embodiment of the present invention. In the figure, LW represents latewood and EW represents earlywood. It can be seen that this method can obtain paraffin sections of Masson pine stems with intact xylem tissue structure, clear cell boundaries, and clear and beautiful staining. The process of preparing Masson pine stem paraffin sections using this method is simpler and more efficient than traditional paraffin sectioning techniques.

[0019] All aspects of this invention not described in detail are well-known to those skilled in the art. The embodiments described above are merely preferred embodiments of the invention and not limitations. Various modifications and refinements made to the technical solutions of this invention by those skilled in the art without departing from the scope of the claims should be covered within the scope of the claims of this invention.

Claims

1. A method for preparing paraffin sections for analyzing the xylem characteristics of Masson pine stems, characterized in that, Includes the following steps: (1) Softening: Place the pine core sample in a constant temperature water bath at 75-85℃ and steam for 8 hours, then soak for 24-48 hours; (2) Segmentation: After removing the core sample, divide it into 5-10 mm stem segments and mark the side of each stem segment near the bark. (3) Dehydration: The softened stem segments were placed in 50% and 70% ethanol solutions for 1 hour each time, then placed in 90% ethanol solution twice for 50 minutes each time, and then placed in 100% ethanol solution twice for 40 minutes each time. (4) Transparency: The stem segments were treated with a 1:1 volume mixture of 100% ethanol and xylene for 40 min, and then treated with xylene twice for 40 min each time. (5) Wax impregnation: After the stem segments have become transparent, they are immersed in liquid paraffin for two treatments, each lasting 40 minutes. (6) Embedding: The sample was embedded at 60℃; (7) Slicing, spreading, gluing and baking: Cut the embedded sample into 8μm thick wax slices using a slicer, spread the wax slices in a 42℃ water bath, spread the gluing agent evenly on a glass slide, lift the spread wax slices with a glass slide, and then dry them in a 65℃ baking machine for 30min. (8) Dewaxing and rehydration: Place the dried sections in a staining tank and treat them with xylene four times, each time for 5-10 minutes. Then place the treated sections in 100%, 95%, and 70% ethanol solutions and distilled water for 20 seconds each time. (9) Staining and decolorization: Place the treated sections in a staining tank and treat with 1% safranin aqueous solution for 15 min. Place the stained sections in distilled water for 10 s, and then treat with 70% ethanol four times for 10 s each time. (10) Mounting: Add two drops of neutral resin to the prepared glass slide and mount it.

2. The method for preparing paraffin sections for analyzing the xylem characteristics of Pinus massoniana stems according to claim 1, characterized in that, As described in step (1), the core samples should be obtained from healthy, mature forests, with a diameter of 5-6 mm and a length of 20-50 cm.

3. The method for preparing paraffin sections for analyzing the xylem characteristics of Pinus massoniana stems according to claim 1, characterized in that, In step (2), the core sample is divided into segments from the bark to the core. A neutral pen is used to mark the side of each stem segment near the bark along the vascular bundle, which is the bark direction.

4. The method for preparing paraffin sections for analyzing the xylem characteristics of Pinus massoniana stems according to claim 1, characterized in that, Step (6) When embedding, first attach some paraffin wax to the mold, adjust the core to be perpendicular to the conduit line and put in the paraffin wax, and make sure the bark is facing left or right. Place the embedding box on top, then attach an appropriate amount of paraffin wax, and place the mold and embedding box on a cooling table to cool.

5. The method for preparing paraffin sections for analyzing the xylem characteristics of Pinus massoniana stems according to claim 1, characterized in that, The adhesive tablet in step (7) is a mixture of egg white and glycerin in a volume ratio of 1:1.

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