A method for isolating and culturing a formation layer stem cell of burdock

Abstract

This invention discloses a method for inducing and culturing burdock stem cells and stem cells, belonging to the field of plant biotechnology. Using burdock roots as explants, the cambium is determined by staining, and the cambium is obtained using enzymatic digestion. The cambium is then inoculated into a specific ratio of induction solid medium and cultured in the dark to efficiently induce primary stem cells. These primary stem cells are then transferred to a proliferation solid medium for subculture and selection to obtain and maintain a vigorous, soft stem cell line. The solid stem cell line is further transferred to a liquid medium for shaking culture to establish a suspension stem cell line. This invention provides high induction efficiency, stable stem cell characteristics, and good reproducibility, offering a high-quality starting material and technical platform for in vitro preservation of burdock germplasm, large-scale production of secondary metabolites, and genetic transformation.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, specifically relating to a method for isolating and culturing burdock cambium stem cells. Background Technology

[0002] Lignans in burdock, particularly arctiin and arctigenin, possess unique biosynthetic pathways and a wide range of biological activities. Traditional breeding and cultivation methods suffer from long cycles and low efficiency. Utilizing plant tissue culture technology, especially by establishing stable stem cell and stem cell culture systems, is a crucial prerequisite for the preservation of burdock germplasm resources, large-scale production of bioactive substances, and genetic engineering improvement. Currently, there are few publicly available reports on burdock tissue culture, and the existing methods suffer from low induction rates, easy browning of stem cells, uneven growth states, and difficulty in maintaining an undifferentiated state, thus limiting its subsequent applications.

[0003] Prior art, CN109576209A, discloses a method for isolating and culturing ginseng cambium stem cells, the method comprising the step of treating tissue containing ginseng cambium with a compound of formula I. It also relates to ginseng cambium stem cells obtained according to the method of the present invention and their use in the preparation of products for ginseng suspension culture. However, the method heavily relies on specific compounds and is only applicable to ginseng, lacking broad applicability. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for isolating and culturing burdock cambium stem cells, which has a high induction success rate, fast proliferation rate, and can maintain stem cell characteristics for a long time.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A method for isolating and culturing burdock cambium stem cells, comprising the following steps: S1. Fresh burdock root was selected as the explant, and the location of the cambium was determined by sectioning and staining the explant. S2. After surface disinfection of fresh burdock root, enzymatic hydrolysis is performed, and the burdock cambium is obtained by differential dissociation method. S3. Seed the cambium layer onto an induction solid medium and culture it under low light to induce the generation of primary stem cells; S4. The induced primary stem cells are transferred to a proliferating solid culture medium and cultured in the dark to establish and maintain the stem cell line.

[0006] Further, select fresh, uniformly sized burdock root midsections, and cut thin slices with a thickness of 1-2 cell layers perpendicular to the long axis of the root. Place the slices on a glass slide and stain them with phloroglucinol-hydrochloric acid. In this way, find neatly arranged, radially flattened cells with dense cytoplasm in the colorless bands of the slices, which are the cambium cell layers.

[0007] Furthermore, the explant surface disinfection procedure was as follows: rinse with running water for 30 minutes, place in a sterile conical flask and rinse three times with sterile water, soak in 75% ethanol for 45 seconds and then rinse three times with sterile water, then disinfect with a solution of 6%-14% sodium hypochlorite solution and sterile water at a mass ratio of 1:3 for 20 minutes, and finally rinse five times with sterile water; after surface disinfection, place in 0.1 mol / L phosphate buffered PBS pre-cooled to 4°C and soak for 10 minutes to wash away mucus and impurities on the tissue surface, while allowing the tissue to pre-cool.

[0008] Further, after surface sterilization of fresh burdock roots, enzymatic hydrolysis was performed to prepare a 100mL enzymatic hydrolysate containing: 1.0-1.5g pectinase, 0.2-0.3g cellulase, 7.29g mannitol, and 0.02g sodium azide. The solution was then diluted to 100mL with 0.1mol / L PBS (pH 7.0-7.2). The pH was adjusted to 7.0 by adding 1mol / L NaOH. The concentration and time of enzymatic hydrolysis were adjusted according to the different growth stages of the burdock roots: for seedlings with low root lignification, 1.0g of pectinase was added to the hydrolysate, and the hydrolysis time was 3-4 hours; for mature plants with high root lignification, 1.5g of pectinase was added to the hydrolysate, and the hydrolysis time was 5-6 hours.

[0009] Furthermore, the enzymatic hydrolysis method is a gradient low-temperature enzymatic hydrolysis treatment: the pretreated central column structure is placed in the enzymatic hydrolysis solution, the volume of which should cover the burdock root tissue, and placed in a constant temperature shaker at 4℃, with low-speed shaking at 50-60 rpm for staged enzymatic hydrolysis, all in the dark. Specific stages:

[0010] Phase 1: Enzymatic hydrolysis for 2 hours to degrade the intercellular adhesions between the outer phloem and the cambium;

[0011] Second stage: Remove burdock root tissue, wash once with pre-cooled PBS, replace with fresh enzymatic hydrolysate, and continue enzymatic hydrolysis, focusing on degrading the intercellular adhesions between the cambium and xylem; Determining the endpoint of enzymatic hydrolysis: Gently pinch the burdock root tissue with tweezers. The xylem column can be gently separated from the phloem and cambium. The cambium should still be a continuous thin film without falling off or breaking. The entire enzymatic hydrolysis process should not exceed 6 hours to avoid excessive enzymatic hydrolysis that could damage the cambium cells.

[0012] Furthermore, the enzymatic hydrolysis was terminated immediately upon completion. Gradient osmotic pressure was used for elution to gradually restore tissue osmotic pressure and simultaneously wash away residual enzyme solution, preventing continued enzymatic hydrolysis. Specific procedures included: soaking burdock root tissue in pre-cooled 0.6 mol / L mannitol-PBS buffer for 15 min to wash away residual enzyme solution using hypertonic elution, maintaining cell contraction and preventing cell wall rupture; then switching to 0.4 mol / L mannitol-PBS buffer and soaking for 10 min for moderate osmotic pressure transition; finally, switching to 0.2 mol / L mannitol-PBS buffer and soaking for 10 min for hypotonic elution to restore normal cell morphology, keeping the cambium cell layer relaxed and continuous; the entire process was performed at 4°C, with gentle shaking of the test tube during elution to avoid vigorous shaking that could cause the cambium layer to detach.

[0013] Furthermore, the enzymatically hydrolyzed burdock explants were sterilely rinsed to obtain sterile burdock cambium cells: the tissue was soaked in pre-cooled 4°C water for 5 minutes, then removed and gently dried with absorbent paper. The adhesion between the burdock cambium and the xylem and phloem had been significantly loosened. After being cut into appropriate sizes, the tissue was rinsed with sterile water to obtain sterile burdock cambium cells.

[0014] Furthermore, the induction solid medium used MS as the basal medium, which contained 30 g / L sucrose, 7 g / L agar, 2.0 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.5 mg / L 6-benzylaminopurine (6-BA), with a pH of 5.8. For the preparation of the induction solid medium in sterile Erlenmeyer flasks: 32 g of MS basal medium was weighed, water was added to adjust the pH to 5.8, and the volume was brought to 800 mL. This was then placed in a 1 L Erlenmeyer flask, and 2,4-D and 6-BA were added. The Erlenmeyer flasks were then sterilized in an autoclave. After sterilization, the induction solid medium was dispensed into the sterilized Erlenmeyer flasks, 40 mL per flask, and allowed to cool and solidify. The cambium layer was inoculated into the induction solid medium using sterilized and cooled forceps, with the cambium layer facing upwards.

[0015] Further, primary stem cells were detached from the explant and crushed into pieces approximately 0.5 cm in size using tweezers. 3 Small pieces of primary stem cells were transferred to a proliferation solid medium and cultured in a completely dark environment at 25°C. The cells were subcultured every 14 days. After three subcultures, a burdock stem cell line with uniform growth, rapid proliferation, and a consistently soft state was obtained.

[0016] Furthermore, the proliferation solid medium used MS as the basal medium, which contained 30 g / L sucrose and 7 g / L agar. 2.0 mg / L 6-benzylaminopurine (6-BA) and 0.5 mg / L naphthaleneacetic acid (NAA) were added to adjust the pH to 5.8. Then, 0.5 mg / L gibberellin, 100 mg / L ascorbic acid, and 150 mg / L citric acid were added to promote growth and prevent browning of burdock stem cells. For the preparation of the proliferation solid medium in sterile Erlenmeyer flasks: 32 g of MS basal culture was weighed, water was added to adjust the pH to 5.8, and the volume was brought to 800 mL. This was then placed in a 1 L Erlenmeyer flask, and 6-BA and NAA were added. The flask was then placed in an autoclave for sterilization. After sterilization, the proliferation solid medium was dispensed into sterile Erlenmeyer flasks, 40 mL per flask, and allowed to cool and solidify.

[0017] Beneficial effects: 1. High induction efficiency: By optimizing the specific concentration ratio of 6-BA and NAA, the induction rate of burdock stem cell tissue was significantly improved to over 85%, and the induction time was shortened; 2. Excellent stem cell characteristics: The use of a specific subculture proliferation solid culture medium (NAA and 6-BA combination) can effectively maintain the ideal state of loose and fragile stem cells, allowing them to maintain high division potential and undifferentiated characteristics, facilitating long-term culture; 3. Stable and controllable system: A complete technical system from solid induction to solid proliferation and then to liquid suspension culture has been established, allowing for flexible selection of culture methods according to different application purposes (such as germplasm preservation and metabolite production), facilitating large-scale application; 4. Good reproducibility: The method steps are clear, the conditions are well-defined, and it is easy to standardize operation and replicate verification. Attached Figure Description

[0018] Figure 1 This is a photograph of a longitudinal section of the burdock root tip stained with phloroglucinol-hydrochloric acid in an embodiment of the present invention; the red stained part in the figure is the xylem, and the outer layer of the xylem is the cambium.

[0019] Figure 2 This is a photograph of the state of stem cells generated after 10 days of burdock explant induction culture in an embodiment of the present invention.

[0020] Figure 3 This is a photograph of burdock stem cells in a soft state after proliferation, as shown in an embodiment of the present invention.

[0021] Figure 4 This is a microscopic photograph of stem cells stained with neutral red burdock root in an embodiment of the present invention. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments.

[0023] This invention discloses a method for isolating and culturing burdock cambium stem cells, the steps of which are as follows:

[0024] Step 1. Disinfection and preparation of explants: Fresh burdock roots were selected as explants. The root tips were peeled and then surface-sterilized. They were cut into appropriately sized segments or pieces using a sterile knife. The cambium location was determined by staining the xylem with phloroglucinol-hydrochloric acid. Figure 1 As shown.

[0025] Step 2. Isolation and Culture of Cambium Stem Cells: Take 2-3 cm long main root segments from fresh burdock roots. Use a scalpel to remove the epidermis and outer cortex, retaining the stele structure containing the cambium, xylem, and phloem. Perform surface sterilization. After sterilization, immerse the tissue in 0.1 mol / L phosphate-buffered saline (PBS, pH 7.2) pre-cooled to 4°C for 10 min to wash away surface mucus and impurities. Simultaneously, allow the tissue to pre-cool to prepare for subsequent low-temperature enzymatic hydrolysis. Place the pre-treated stele structure in the enzymatic hydrolysis solution and hydrolyze for 2 hours to degrade the intercellular adhesions between the outer phloem and cambium. After the first hydrolysis, remove the tissue, wash gently once with pre-cooled PBS, replace with fresh enzymatic hydrolysis solution, and continue hydrolysis for 3-4 hours. Immediately terminate the hydrolysis after completion and elute using a gradient osmotic pressure to gradually restore tissue osmotic pressure while washing away residual enzyme solution to prevent continued enzymatic action. Maintain the entire process at 4°C. After osmotic pressure regulation, the tissue is immersed in pre-cooled water (4°C) for 5 minutes. It is then removed and gently blotted dry with absorbent paper before separation. At this point, the adhesion between the cambium and the xylem and phloem has significantly loosened, and the cambium cell layer is less easily torn by mechanical force, thus obtaining cambium cells. These cells are then cut into appropriate sizes and rinsed 3-5 times with sterile water to obtain sterile cambium cells. They are then inoculated onto an induction solid medium and cultured in the dark or under low light conditions. The induction solid medium is based on MS medium (containing 30 g / L sucrose and 7 g / L agar), supplemented with a combination of plant growth regulators: 2.0 mg / L 6-BA and 0.5 mg / L NAA, with a pH of 5.8.

[0026] Step 3. Subculture and proliferation of cambium stem cells and establishment of stem cell lines: The soft, granular tissue obtained from culture was peeled off, divided, and transferred to a proliferation solid medium for subculture. Subculture was performed every 2-3 weeks to screen and expand cell lines with vigorous growth and uniform condition. The proliferation solid medium was MS medium (containing 30 g / L sucrose and 7 g / L agar) and a combination of plant growth regulators: 2.0 mg / L 6-BA and 0.5 mg / L NAA, with a pH of 5.8. Additionally, gibberellin 0.5 mg / L, ascorbic acid 100 mg / L, and citric acid 150 mg / L were added to promote growth and prevent browning of burdock stem cells.

[0027] Step 4. Establishment of suspension stem cell line: The excellent soft stem cell tissue obtained in step 3 is inoculated into a liquid proliferation solid culture medium without agar, and cultured in a shaker with shaking suspension. The cells are subcultured regularly to establish a fast-growing and uniform burdock suspension stem cell line.

[0028] Example 1: I. Determination of the cambium of burdock: The lignified cell walls (mainly vessels and fibers in the secondary xylem) were specifically stained (cherry red) using phloroglucinol-hydrochloric acid, which contrasted with the cambium cells and phloem cells (negative staining, appearing in their natural color) that were not lignified or had very low lignification.

[0029] Material preparation: Select fresh, uniformly sized middle section of burdock root (the area of ​​vigorous secondary growth).

[0030] Hand cutting: Using a sharp double-edged blade or razor blade, cut thin slices (ideally 1-2 cell layers thick) perpendicular to the long axis of the root.

[0031] Slide selection and mounting: Select the thinnest and most intact burdock root slices from clean water and cut them horizontally. Use tweezers to transfer them to the center of a clean glass slide. Use absorbent paper to blot away excess moisture around the slices, but keep the material moist.

[0032] Staining: Add 1-2 drops of 1% phloroglucinol ethanol solution, ensuring complete immersion of the material, and let it stand for 1-2 minutes. Pay special attention to adding 1-2 drops of concentrated hydrochloric acid to the side of the material while the staining solution is still wet. Quickly and gently guide the acid flow with the tip of a toothpick to mix it with the phloroglucinol solution on the material, or tilt the slide to mix the two solutions.

[0033] Observe immediately: the staining is obvious and can be distinguished with the naked eye.

[0034] Locating the cambium ring: In the field of view, the stained red vessel groups / bundles form a discontinuous ring-shaped area, which is the secondary xylem. Outside this red ring, immediately adjacent to a band of cells of varying width with little or no red color or only a very pale background. Within this colorless band, carefully search for a layer of neatly arranged, radially flattened (rectangular) cells with relatively dense cytoplasm. This is the cambium cell layer, specifically as follows... Figure 1 As shown.

[0035] II. Acquisition of Cambium Cells: In burdock roots, cambium cells are young meristematic tissues with thin cell walls and the middle layer mainly composed of pectin. Adjacent xylem cells are highly lignified, and phloem cells contain more cellulose. The key to intercellular adhesion among these three tissues lies in the pectin in the middle layer and a small amount of transcellular cellulose filaments. Differential dissociation method targets and degrades only the intercellular adhesion substances between the cambium and the xylem and phloem, without disrupting the intercellular connections of the cambium cells themselves. Simultaneously, through low temperature and osmotic pressure regulation, the young cambium cells are prevented from swelling or rupturing due to water absorption or loss, thus achieving cambium acquisition.

[0036] 1) Material selection and pretreatment: Fresh burdock roots were collected, and 2-3 cm long segments of the main root were cut. The epidermis and outer cortical tissue were removed using a scalpel, preserving the stele structure containing the cambium, xylem, and phloem. The root tip was then desquamated and surface-sterilized. The surface sterilization procedure for the burdock root tip was as follows: rinsing with running water for 30 minutes, rinsing three times with sterile water in a sterile conical flask, soaking in 75% ethanol for 45 seconds, rinsing three times with sterile water, then sterilizing with a solution of 6-14% sodium hypochlorite and sterile water in a 1:3 ratio for 20 minutes, and finally rinsing five times with sterile water. After surface sterilization, the root tip was immersed in 0.1 mol / L phosphate-buffered saline (PBS) (pH 7.2) pre-cooled to 4°C for 10 minutes to wash away mucus and impurities on the tissue surface, while simultaneously pre-cooling the tissue in preparation for subsequent low-temperature enzymatic hydrolysis.

[0037] 2) Preparation of low-temperature enzymatic hydrolysate: The enzymatic hydrolysate is a complex enzyme system that balances pectin degradation efficiency and tissue protection. It is prepared and used immediately after being refrigerated at 4°C throughout the process. The formula is as follows (for a 100mL system): Add 1.0-1.5g pectinase, 0.2-0.3g cellulase, 7.29g mannitol, and 0.02g sodium azide to 100mL with 0.1mol / L PBS (pH 7.0-7.2). Adjust the pH to 7.0 by adding 1mol / L NaOH dropwise.

[0038] 3) Gradient low-temperature enzymatic hydrolysis treatment: The pretreated column structure was placed in the enzymatic hydrolysis solution, ensuring the solution completely covered the tissue. It was then placed in a 4°C constant-temperature shaker and subjected to low-speed shaking (50-60 rpm) for staged enzymatic hydrolysis, completely protected from light. Specific stages: Phase 1: Enzymatic hydrolysis for 2 hours to degrade the intercellular adhesions between the outer phloem and the cambium; Second stage: Remove the tissue, wash it once with pre-cooled PBS, replace with fresh enzymatic hydrolysate, and continue enzymatic hydrolysis for 3-4 hours, focusing on degrading the intercellular adhesions between the cambium and xylem. Determining the endpoint of enzymatic hydrolysis: Gently pinch the tissue with tweezers. The xylem pillars can be gently separated from the phloem and cambium. The cambium should still be a continuous thin film without peeling or breaking. The total enzymatic hydrolysis time should not exceed 6 hours to avoid excessive enzymatic hydrolysis that could damage the cambium cells.

[0039] 4) Gradient osmotic pressure regulation: After enzymatic hydrolysis is complete, immediately terminate the hydrolysis and gradually restore the tissue osmotic pressure by eluting with a gradient osmotic pressure. Simultaneously, wash away any residual enzyme solution to prevent continued enzymatic hydrolysis. Specific procedures are as follows: Soak the tissue in pre-cooled 0.6 mol / L mannitol-PBS buffer (pH 7.2) for 15 min to wash away residual enzymes with hypertonic osmotic pressure, maintain cell contraction, and prevent cell wall rupture. Then, soak in 0.4 mol / L mannitol-PBS buffer for 10 min to transition to a moderate osmotic pressure. Finally, soak in 0.2 mol / L mannitol-PBS buffer for 10 min to restore normal cell morphology with hypotonic osmotic pressure, keeping the cambium cell layer relaxed and continuous. The entire process is carried out at 4°C. Gently shake the test tube during elution to avoid vigorous shaking that could cause the cambium layer to detach.

[0040] 5) Acquisition of sterile cambium: After osmotic pressure regulation, the tissue was immersed in pre-cooled water (4°C) for 5 minutes, then removed and gently dried with absorbent paper. At this point, the adhesion between the cambium and the xylem and phloem had loosened considerably. After being cut into appropriate sizes with a sterile scalpel, the tissue was rinsed 35 times with sterile water to obtain sterile burdock cambium cells.

[0041] III. Induction of Burdock Stem Cells: The obtained cambium layer was seeded onto an induction solid medium, ensuring the cambium layer faced upwards. Medium formulation: MS medium (containing 30 g / L sucrose and 7 g / L agar) was used as the basal medium, supplemented with 2.0 mg / L 2,4-D and 0.5 mg / L 6-BA, pH 5.8. To prepare the medium, a 100 mL Erlenmeyer flask was first washed and rinsed with deionized water, wrapped with sealing film and newspaper, and then placed in an autoclave to prepare a sterile Erlenmeyer flask. 32 g of MS medium was weighed, and a small amount of water was added to adjust the pH to 5.8, then the volume was adjusted to 800 mL. This volume was then transferred to a 1 L Erlenmeyer flask, and 2,4-D and 6-BA were added. The flask was then placed in an autoclave for sterilization. After sterilization, the medium was poured into the sterilized 100 mL Erlenmeyer flasks, approximately 40 mL per flask. After cooling and solidification, the cambium layer was transferred to the medium using sterilized and cooled forceps, with the cambium layer facing upwards. The burdock stem cells were cultured at 25°C under low light conditions (12h / 12h photoperiod). Because burdock has abundant endophytic bacteria, the cultured burdock stem cells needed to be observed daily. If contamination was found in a flask, the uncontaminated burdock roots should be promptly transferred to a new culture medium. After two weeks of culture, over 90% of the explant incision sites successfully induced primary stem cells that were milky white to pale yellow and soft in texture. Figure 2 As shown.

[0042] IV. Subculture and proliferation of burdock stem cell lines: 1) Obtain primary stem cells that are milky white to pale yellow and have a soft texture. Use sterilized forceps to detach them from the explant and then crush them into pieces approximately 0.5 cm in size. 3 Small pieces.

[0043] 2) Transfer to proliferation solid medium. The medium formula is: MS as the basic medium (containing 30 g / L sucrose and 7 g / L agar) with 2.0 mg / L 6-BA and 0.5 mg / L NAA added, pH 5.8. In addition, gibberellin 0.5 mg / L, ascorbic acid 100 mg / L and citric acid 150 mg / L are added to promote growth and prevent browning of burdock stem cells. To prepare the culture medium, first wash a 100mL Erlenmeyer flask and rinse it with deionized water. Wrap it with sealing film and newspaper and place it in an autoclave to prepare a sterile Erlenmeyer flask. Weigh 32g of MS culture medium, add a small amount of water to adjust the pH to 5.8, and then make up to 800mL. Put the mixture into a 1L Erlenmeyer flask and add 2.0mg / L 6-BA and 0.5mg / L NAA. Then put the flask into an autoclave for sterilization. After sterilization, pour the culture medium into the sterilized 100mL Erlenmeyer flasks, about 40mL per flask, and let it cool and solidify.

[0044] 3) Incubate in a completely dark environment at 25°C.

[0045] 4) Subculture every 14 days. After three subcultures and selections, burdock stem cell lines with uniform growth, rapid proliferation, and consistently soft texture are obtained, such as... Figure 3 As shown.

[0046] V. Establishment of burdock suspension stem cell line: 1) Weigh 2.0g of the soft burdock stem cells cultured in Example 2 and put them into a 250mL Erlenmeyer flask containing 100mL of liquid proliferation solid culture medium (the composition is the same as the proliferation solid culture medium in Example 2, but without the addition of agar).

[0047] 2) Place the Erlenmeyer flask on a shaker at 110 rpm and culture it under shaking conditions at 25°C in the dark.

[0048] 3) Subculture every 7 days: Let the culture stand for a while, aspirate the bottom cell clusters, and inoculate them into fresh liquid culture medium at a ratio of 1:3 (volume ratio).

[0049] 4) After 4-5 subcultures, a well-dispersed, finely granulated, and stably growing burdock suspension stem cell line was successfully established. This cell line has a high biomass multiplication rate.

[0050] VI. Verification of Burdock Stem Cells: Plant stem cells and callus cells differ significantly in cell morphology, vacuolar characteristics, and cell arrangement. These differences stem from their different differentiation states and functional characteristics. Plant stem cells, being in an undifferentiated state, have relatively small and few vacuoles, rich in vacuolar contents, and appear a deep red after neutral red staining. During dedifferentiation, callus cells gradually enlarge and increase in number, with relatively fewer vacuolar contents, and appear a lighter or pale red after neutral red staining. In some highly vacuolated callus cells, the vacuoles may occupy most of the cell volume, causing the nucleus to be squeezed to the cell edge. Therefore, we used neutral red staining to verify whether the cells obtained in Example 1 were indeed burdock stem cells.

[0051] Cell preparation: Seed burdock stem cells in good logarithmic growth state into a culture dish. Use tweezers to take an appropriate size of stem cell block and place it on a clean glass slide. If the cells are too large, use tweezers to break them into a state suitable for microscopic observation.

[0052] Staining solution preparation: Weigh 0.1g of neutral red powder and dissolve it in 100mL of physiological saline (or PBS buffer, pH 7.2-7.4). After complete dissolution, filter with filter paper and store at 4℃ protected from light for later use.

[0053] Staining procedure: Place two drops of pre-warmed working solution containing neutral red onto a glass slide containing stem cells, ensuring complete coverage of the cells. Return to the incubator and incubate at 37°C for 10-15 minutes (the exact time may need to be adjusted depending on the situation, ensuring sufficient but not excessive dye uptake).

[0054] Washing and observation: Carefully aspirate the staining solution. Gently rinse the cells 1-2 times with pre-warmed PBS or physiological saline to remove any unabsorbed stain. Observe immediately under a light microscope.

[0055] Typical positive results: Highly viable stem cells show numerous bright red / orange-red granular deposits in their cytoplasm; the cell nucleus is unstained, while the lysosomes and vacuolar structures of stained cells appear red. Figure 4 As shown.

Claims

1. A method for isolating and culturing burdock cambium stem cells, characterized in that, Includes the following steps: S1. Fresh burdock root was selected as the explant, and the location of the cambium was determined by sectioning and staining the explant. S2. After surface disinfection of fresh burdock root, enzymatic hydrolysis is performed, and the burdock cambium is obtained by differential dissociation method. S3. Seed the cambium layer onto an induction solid medium and culture it under low light to induce the generation of primary stem cells; S4. The induced primary stem cells are transferred to a proliferation solid culture medium and cultured in the dark to establish and maintain the stem cell line.

2. The method for isolating and culturing burdock cambium stem cells according to claim 1, characterized in that, Select fresh, uniformly sized burdock root midsections and cut thin slices with a thickness of 1-2 cell layers perpendicular to the long axis of the root. Place the slices on a glass slide and stain them with phloroglucinol-hydrochloric acid. In the colorless bands of the slices, find neatly arranged, radially flattened cells with dense cytoplasm, which are the cambium cell layers.

3. The method for isolating and culturing burdock cambium stem cells according to claim 3, characterized in that, The explant surface disinfection procedure was as follows: rinse with running water for 30 minutes, place in a sterile conical flask and rinse three times with sterile water, soak in 75% ethanol for 45 seconds and then rinse three times with sterile water, then disinfect with a solution of 6%-14% sodium hypochlorite and sterile water at a mass ratio of 1:3 for 20 minutes, and finally rinse five times with sterile water; after surface disinfection, place in 0.1 mol / L phosphate buffered PBS pre-cooled to 4°C and soak for 10 minutes to wash away mucus and impurities on the tissue surface, while allowing the tissue to pre-cool.

4. The method for isolating and culturing burdock cambium stem cells according to claim 1, characterized in that, After surface sterilization of fresh burdock roots, enzymatic hydrolysis was performed. A 100mL hydrolysate was prepared with the following ingredients: 1.0-1.5g pectinase, 0.2-0.3g cellulase, 7.29g mannitol, and 0.02g sodium azide. The solution was brought to 100mL with 0.1mol / L PBS (pH 7.0-7.2). The pH was adjusted to 7.0 by adding 1mol / L NaOH. The hydrolysis concentration and time were adjusted according to the different growth stages of the burdock roots: for seedlings with low root lignification, 1.0g of pectinase was added to the hydrolysate for 3-4 hours; for mature plants with high root lignification, 1.5g of pectinase was added to the hydrolysate for 5-6 hours.

5. The method for isolating and culturing burdock cambium stem cells according to claim 4, characterized in that, The enzymatic hydrolysis method is a gradient low-temperature enzymatic hydrolysis treatment: the pretreated central column structure is placed in the enzymatic hydrolysis solution, the volume of which should cover the burdock root tissue. It is then placed in a 4℃ constant-temperature shaker and subjected to low-speed shaking at 50-60 rpm for staged enzymatic hydrolysis, all in the dark. Specific stages are as follows: Phase 1: Enzymatic hydrolysis for 2 hours to degrade the intercellular adhesions between the outer phloem and the cambium; Second stage: Remove burdock root tissue, wash once with pre-cooled PBS, replace with fresh enzymatic hydrolysate, and continue enzymatic hydrolysis, focusing on degrading the intercellular adhesions between the cambium and xylem; Determining the endpoint of enzymatic hydrolysis: Gently pinch the burdock root tissue with tweezers. The xylem column can be gently separated from the phloem and cambium. The cambium should still be a continuous thin film without falling off or breaking. The entire enzymatic hydrolysis process should not exceed 6 hours to avoid excessive enzymatic hydrolysis that could damage the cambium cells.

6. The method for isolating and culturing burdock cambium stem cells according to claim 5, characterized in that, The enzymatic hydrolysis was terminated immediately upon completion. Gradient osmotic pressure was used for elution to gradually restore tissue osmotic pressure and simultaneously wash away residual enzyme solution, preventing continued enzymatic hydrolysis. Specific procedures were as follows: Burdock root tissue was soaked in pre-cooled 0.6 mol / L mannitol-PBS buffer for 15 min to wash away residual enzyme solution using hypertonic elution, maintaining cell contraction and preventing cell wall rupture; then, 0.4 mol / L mannitol-PBS buffer was used for soaking for 10 min to transition to moderate osmotic pressure; finally, 0.2 mol / L mannitol-PBS buffer was used for soaking for 10 min to restore normal cell morphology using hypotonic elution, keeping the cambium cell layer relaxed and continuous; the entire process was performed at 4°C, and the test tube was gently shaken during elution to avoid vigorous shaking that could cause the cambium layer to detach.

7. The method for isolating and culturing burdock cambium stem cells according to claim 6, characterized in that, The enzymatically hydrolyzed burdock explants were sterilely rinsed to obtain sterile burdock cambium cells. The tissues were soaked in pre-cooled 4°C water for 5 minutes, then removed and gently dried with absorbent paper. The adhesion between the burdock cambium and the xylem and phloem had been significantly loosened. After being cut into appropriate sizes, the tissues were rinsed with sterile water to obtain sterile burdock cambium cells.

8. The method for isolating and culturing burdock cambium stem cells according to claim 1, characterized in that, The induction solid medium was based on MS medium, which contained 30 g / L sucrose, 7 g / L agar, 2.0 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.5 mg / L 6-benzylaminopurine (6-BA), with a pH of 5.

8. The induction solid medium was prepared using sterile Erlenmeyer flasks: 32 g of MS medium was weighed, the pH was adjusted to 5.8 with water, and the volume was brought to 800 mL. This was then placed in a 1 L Erlenmeyer flask, and 2,4-D and 6-BA were added. The flask was then sterilized in an autoclave. After sterilization, the induction solid medium was dispensed into the sterilized Erlenmeyer flasks, 40 mL per flask, and allowed to cool and solidify. The cambium layer was inoculated into the induction solid medium using sterilized and cooled forceps, with the cambium layer facing upwards.

9. The method for isolating and culturing burdock cambium stem cells according to claim 8, characterized in that, Primary stem cells were detached from the explant and crushed into pieces approximately 0.5 cm in size using tweezers. 3 Small pieces of primary stem cells were transferred to a proliferation solid medium and cultured in a completely dark environment at 25°C. The cells were subcultured every 14 days. After three subcultures, a burdock stem cell line with uniform growth, rapid proliferation, and a consistently soft state was obtained.

10. The method for isolating and culturing burdock cambium stem cells according to claim 1, characterized in that, The proliferation solid medium used MS as the basal medium, which contained 30 g / L sucrose and 7 g / L agar. 2.0 mg / L 6-benzylaminopurine (6-BA) and 0.5 mg / L naphthaleneacetic acid (NAA) were added to adjust the pH to 5.

8. Then, 0.5 mg / L gibberellin, 100 mg / L ascorbic acid, and 150 mg / L citric acid were added to promote growth and prevent browning of burdock stem cells. The proliferation solid medium was prepared in sterile Erlenmeyer flasks: 32 g of MS basal medium was weighed, water was added to adjust the pH to 5.8, and the volume was brought to 800 mL. This was then placed in a 1 L Erlenmeyer flask, and 6-BA and NAA were added. The flask was then sterilized in an autoclave. After sterilization, the proliferation solid medium was dispensed into sterile Erlenmeyer flasks, 40 mL per flask, and allowed to cool and solidify.

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