Freezing positioning culture method of azaleaceae plant stem cells and application of freezing positioning culture method

Abstract

The invention discloses a freezing positioning culture method of azaleaceae plant stem cells and application of the freezing positioning culture method, and belongs to the technical field of plant stem cells. In order to solve the problems of inaccurate stem cell positioning, low induction efficiency and pollution in the prior art, paraffin embedding is combined with a liquid nitrogen freezing slicing technology, the multi-vacuole stem cells in young fruits are accurately positioned through neutral red staining, and the extraction success rate exceeds 85%. Inducing medium components (containing 80-120 mg / L of ascorbic acid, 1-3 mg / L of IAA and 2-6 mg / L of glutamic acid / arginine / cysteine combination) are optimized, so that the callus formation time is shortened to 2-7 days, and the efficiency is improved by 4 times compared with that of a traditional method. In the amplification stage, amphotericin B (2-6 mg / L) is added, liquid is supplemented in stages for culture, and the pollution rate is lower than 5%. The obtained stem cells are directly used for eye protection chewable tablets (containing 20-30% of stem cells) after being dried, and clinical tests show that the asthenopia score can be reduced by 19.3% (Plt; 0.01) after the eye protection chewable tablets are continuously taken for 2 weeks. The method is suitable for blueberries, rhododendron, bilberries and the like, breaks through the limitation of fresh fruit processing, and provides a new way for functional food development.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant stem cells, and in particular relates to a freezing positioning culture method of Ericaceae plant stem cells and an application thereof. Background Art

[0002] The Ericaceae family is an important woody plant group encompassing several subfamilies, including the Vaccinioideae and Rhododendroideae. These include species of significant economic and ecological value, such as blueberry (Vaccinium spp.), rhododendron (Rhododendron spp.), bilberry (Vaccinium vitis-idaea), and enkianthus (Enkianthus spp.). 810 Among these, Vaccinium species, represented by blueberry and bilberry, are known as the "king of berries" for their berries, which are rich in anthocyanins, vitamins, and minerals. They have recently attracted global attention as health foods. Rhododendron species are renowned for their ornamental and medicinal properties, with some species, such as Rhododendron simsii, widely used in landscaping. Enkianthus species, with their distinctive bell-shaped inflorescences and cold tolerance, have potential in horticulture and ecological restoration.

[0003] Blueberries (Vaccinium species. Cyanococcus) are perennial deciduous or evergreen shrubs with an optimal growth temperature of 13-30°C (13-30°C), and can tolerate short-term temperatures of 40-50°C (40-50°C). They are widely distributed in temperate to subtropical regions, including Asia, Europe, and North America. Wild blueberries in my country are primarily found in the Changbai Mountains and Greater and Lesser Khingan Mountains in Heilongjiang, Jilin, and Liaoning. Varieties include the lowbush blueberry (adapted to alpine climates) and the rabbiteye blueberry (adapted to subtropical climates). Artificial cultivation began in the 1980s, and large-scale cultivation has now been established in Shandong, Yunnan, and Guizhou provinces, with Shandong Province leading the country in industrialized area.

[0004] Blueberries are rich in anthocyanins, vitamins (VA, VE, and VB), minerals (potassium, zinc, manganese, etc.), SOD enzymes, and flavonoids. They possess antioxidant, anti-inflammatory, eye-protecting, and anti-cancer properties. 108 Clinical studies have shown that anthocyanins can promote retinal rhodopsin regeneration, improve visual fatigue, and prevent myopia. The Food and Agriculture Organization (FAO) has listed blueberries as one of the five healthiest foods.

[0005] Vaccinium vitis-idaea: Found in high-altitude acidic soils, its fruit contains tannins and organic acids, offering both edible and medicinal uses. It can be used to extract natural pigments or develop functional beverages. Some species of Rhododendron: Some species, such as the Rhododendron molle, contain flavonoids, which have analgesic and anti-inflammatory properties, but their toxicity should be considered. Campanula: It is cold-resistant and adaptable to acidic, poor soils. It can be used for ecological restoration and landscaping. Its unique floral structure offers potential ornamental value.

[0006] Currently, research on Ericaceae plants is primarily focused on blueberries, while development of species such as bilberry and bellflower is still in its early stages. Blueberry stem cell culture technology has yet to be reported, and efficient stem cell proliferation and targeted differentiation are key to achieving large-scale production. Furthermore, cross-species technology integration (such as the combined application of blueberry and apple stem cells) and the breeding of stress-resistant varieties (such as those with salt and alkali tolerance and disease and pest resistance) will be key areas of future research.

[0007] Despite their high nutritional value, blueberries face multiple challenges in their industrialization: environmental adaptability: Blueberries require acidic soil (pH 4.5-5.5), which limits suitable growing areas in my country. Furthermore, soil pollution exacerbates the difficulty of cultivation. Chinese blueberry breeding has long relied on imported varieties, such as the northern highbush blueberry "Blue Plenty" and the southern highbush blueberry "Emerald," resulting in a shortage of original varieties. While tissue culture technology has made progress (such as patent CN201610867326.3, which reduces the risk of browning by optimizing culture media), stem cell culture remains a niche area. Blueberry byproducts, such as peel and seeds, are rich in anthocyanins and oils, but existing technologies fail to efficiently extract them, resulting in a waste of resources.

[0008] Currently, research on Ericaceae plants is primarily focused on blueberries, while development of species such as bilberry and bellflower is still in its early stages. Blueberry stem cell culture technology has yet to be reported, and efficient stem cell proliferation and targeted differentiation are key to achieving large-scale production. Summary of the Invention

[0009] The present invention addresses the problems of the existing technology gap in Ericaceae plant stem cell culture technology, the low efficiency of traditional tissue culture and the limitations of blueberry industrial application. It proposes a high-efficiency stem cell culture method based on freezing positioning and its application, and develops eye-protection function products based on the method.

[0010] The present invention can be achieved through the following technical solutions: A method for cryopreservation culture of Ericaceae plant stem cells comprises the following steps: (1) Determine the location of stem cells: Take young fruits of Ericaceae plants, wash them, embed them in paraffin, and freeze them in liquid nitrogen for 5-10 minutes. Then slice them. After dewaxing the slices in 25-30℃ warm water, stain them with 10% neutral red to locate stem cells with more than 3 vacuoles. (2) Young fruit disinfection: Take another young fruit of Ericaceae plant, wash it, and then soak it in chlorine dioxide disinfectant for 2 minutes, then soak it in 75% ethanol for 5 minutes, rinse it with sterile water and then dry it; (3) Callus induction: According to the stem cell position determined in step (1), the non-stem cell tissue of the young fruit treated in step (2) is removed, and the stem cell tissue is cut into pieces of 1-3 mm, spread on the surface of the induction medium, and cultured in the dark at 25±1°C for 2-7 days to obtain callus-like tissue; the induction medium is an MS medium containing final concentrations of 3-6 mg / ml agar, 80-120 mg / L ascorbic acid, 1-3 mg / L IAA, 2-6 mg / L glutamic acid, 2-6 mg / L arginine, and 2-6 mg / L cysteine; (4) Stem cell expansion: Callus-like tissue was picked and spot-inoculated on the same induction medium as in step (3), cultured in the dark at 25±1°C for 6-12 days, and cell clusters were obtained and transferred to expansion medium for suspension culture; the expansion medium was MS or B5 medium containing final concentrations of 1-3 mg / L 6-BA, 1-3 mg / L NAA, and 2-6 mg / L amphotericin B, respectively.

[0011] In step (1), the liquid nitrogen freezing time is 5 minutes and the slice thickness is 10-20 μm.

[0012] The specific operation method of step (1) determining the location of stem cells is as follows: take 2-4 young fruits of Ericaceae plants, clean the surface and absorb the surface moisture with filter paper, then embed them with paraffin and freeze them in liquid nitrogen for about 5 minutes, take them out, fix them on a microtome and slice them, put the paraffin slices containing the young fruits into warm water at 25-30℃ to melt the paraffin, place the young fruit slices on a slide with a soft brush, and stain them with 10% neutral red. After staining, rinse off the excess dye with running water, and observe under a microscope to find stem cells. Cells containing more than 3 vacuoles are stem cells.

[0013] The propagation culture conditions of step (4) are: shaker speed 110 r / min, temperature 22-34°C, adding culture medium twice, and total culture time 96 hours.

[0014] The Ericaceae plant stem cells obtained by the method showed more than three vacuoles after 10% neutral red staining, had a moisture content of <5% after drying at room temperature, maintained activity for ≥12 months, and could stably proliferate. Disclosed is an eye protection chewable tablet comprising a blueberry stem cell culture. The tablet comprises, by weight, 20-30% of dried blueberry stem cells with a moisture content of less than 5%, 20-30% of sorbitol, 3-8% of lactose, 0.5-2% of citric acid, 8-12% of dextrin, 0.5-1.5% of magnesium stearate, and 30-35% of microcrystalline cellulose.

[0015] The preparation method of the eye-protecting chewable tablet comprises the following steps: (a) The stem cells were centrifuged and dried under reduced pressure until the moisture content was less than 5%, and then crushed through an 80-mesh sieve; (b) granulating with sorbitol, lactose, citric acid, dextrin, magnesium stearate, and microcrystalline cellulose; (c) Dry under reduced pressure at 40-50°C until the moisture content is less than 5%, and press into tablets with a weight of 2 ± 0.2 g per tablet.

[0016] The Ericaceae plant is selected from blueberry (Vaccinium spp.), azalea (Rhododendron simsii), bilberry (Vaccinium vitis-idaea) or bellflower (Enkianthus campanulatus).

[0017] The 75% ethanol is an ethanol aqueous solution with a volume percentage of 75%.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention, through innovative cryo-positioning, culture medium optimization, and contamination-resistant amplification systems, has achieved efficient culture of Ericaceae plant stem cells for the first time and expanded into the field of functional foods. This addresses the core pain points of the blueberry industry, which relies on fresh fruit processing and has low by-product utilization, and provides technical support for the development of high-value-added products, as follows: 1. Significantly improved stem cell culture efficiency Precise positioning: Cryosectioning technology has increased the success rate of stem cell extraction from less than 30% with traditional methods to over 85% (Examples 1-3); Short induction cycle: Optimized culture medium shortens callus formation time to 2-7 days, which is four times more efficient than the 15-30 days in patent CN201610867326.3; Controllable contamination: Amphotericin B inhibits fungal contamination, and the contamination rate in the amplification stage is <5%, which is significantly lower than the 15-20% under conventional aseptic operation.

[0019] 2. Functional advantages of stem cells Ingredient integrity: Stem cells retain the blueberry's native antioxidant complex (anthocyanin, SOD, vitamin group), and its free radical scavenging rate is 1.8 times higher than that of anthocyanin extract (determined by DPPH method); Enhanced stability: Dried stem cells maintain activity for 12 months at room temperature, which is longer than the 6 months of anthocyanin extract (accelerated test data).

[0020] 3. Breakthrough in industrial application Cost reduction: Directly using stem cells to replace fruit raw materials reduces fresh fruit consumption by 90% (Example 4); Verification of eye protection function: Clinical trials (experimental examples) showed that after the subjects took the chewable tablets for 2 weeks, the comprehensive score of visual fatigue decreased by 19.3% (P<0.01), which was significantly better than the 8.5% improvement rate of commercially available anthocyanin chewable tablets.

[0021] 4. Technology universality This method was successfully applied to species in the Ericaceae family, including Vaccinium (blueberry, lingonberry), Rhododendron (azalea), and Campanula (bellflower), with differences in stem cell proliferation rates <10%, demonstrating the strong cross-species applicability of the technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a graph of blueberry stem cell growth; Figure 2 This is a graph of the growth of azalea stem cells; Figure 3 This is a graph of the growth of bilberry stem cells; Figure 4 is a graph showing the average overall fatigue level of the subjects before and after using the eye protection composition; DETAILED DESCRIPTION

[0023] A method for cryopreservation culture of Ericaceae plant stem cells comprises the following steps: (1) Determine the location of stem cells: Take young fruits of Ericaceae plants, wash them, embed them in paraffin, and freeze them in liquid nitrogen for 5-10 minutes. Then slice them. After dewaxing the slices in 25-30℃ warm water, stain them with 10% neutral red to locate stem cells with more than 3 vacuoles. (2) Young fruit disinfection: Take another young fruit of Ericaceae plant, wash it, and then soak it in chlorine dioxide disinfectant for 2 minutes, then soak it in 75% ethanol for 5 minutes, rinse it with sterile water and then dry it; (3) Callus induction: According to the stem cell position determined in step (1), the non-stem cell tissue of the young fruit treated in step (2) is removed, and the stem cell tissue is cut into pieces of 1-3 mm and spread on the surface of the induction medium. The pieces are cultured in the dark at 25±1°C for 2-7 days to obtain callus-like tissue; the induction medium is an MS medium containing agar at a final concentration of 3-6 mg / ml, ascorbic acid at 80-120 mg / L, IAA at 1-3 mg / L, glutamic acid at 2-6 mg / L, arginine at 2-6 mg / L and cysteine ​​at 2-6 mg / L; (4) Stem cell expansion: The callus-like tissue is picked and dot-inoculated on the same induction medium as in step (3), cultured in the dark at 25±1°C for 6-12 days, and after obtaining cell clusters, the cells are transferred to the expansion medium for suspension culture; the expansion medium is an MS or B5 medium containing 6-BA at a final concentration of 1-3 mg / L, NAA at 1-3 mg / L and amphotericin B at 2-6 mg / L.

[0024] In step (1), the liquid nitrogen freezing time is 5 minutes and the slice thickness is 10-20 μm.

[0025] The specific operation method of step (1) determining the location of stem cells is as follows: take 2-4 young fruits of Ericaceae plants, clean the surface and absorb the surface moisture with filter paper, then embed them with paraffin and freeze them in liquid nitrogen for about 5 minutes, take them out, fix them on a microtome and slice them, put the paraffin slices containing the young fruits into warm water at 25-30℃ to melt the paraffin, place the young fruit slices on a slide with a soft brush, and stain them with 10% neutral red. After staining, rinse off the excess dye with running water, and observe under a microscope to find stem cells. Cells containing more than 3 vacuoles are stem cells.

[0026] The propagation culture conditions of step (4) are: shaker speed 110 r / min, temperature 22-34°C, adding culture medium twice, and total culture time 96 hours.

[0027] The stem cells showed more than three vacuoles after being stained with 10% neutral red, had a moisture content of <5% after being dried at room temperature, and maintained activity for ≥12 months.

[0028] An eye protection chewable tablet comprises a blueberry stem cell culture prepared by the method of the present application, and the formula, by weight percentage, is: 20-30% dried blueberry stem cells with a moisture content of <5%, 20-30% sorbitol, 3-8% lactose, 0.5-2% citric acid, 8-12% dextrin, 0.5-1.5% magnesium stearate, and 30-35% microcrystalline cellulose.

[0029] The preparation method of the eye protection chewable tablets includes: centrifugally drying the stem cells and then crushing them through an 80-mesh sieve, mixing them with the remaining ingredients to form granules, drying them under reduced pressure at 40-50° C. until the moisture content is less than 5%, and pressing them into tablets.

[0030] The Ericaceae plant is selected from blueberry (Vaccinium spp.), azalea (Rhododendron simsii), bilberry (Vaccinium vitis-idaea) or bellflower (Enkianthus campanulatus).

[0031] The specific components of the MS medium of the present invention are shown in Table 1, and the specific components of the BS medium are shown in Table 2.

[0032] Table 1 MS culture medium composition: Table 2 B5 culture medium composition: Example 1 This embodiment relates to a method for cryopreservation culture of blueberry stem cells, comprising the following steps: (1) Stem cell positioning: Take 3 blueberry young fruits, wash them, embed them in paraffin, freeze them in liquid nitrogen for 5 minutes, and cut them into 15 μm thick slices with a microtome. Immerse the slices in 28°C warm water for dewaxing, stain them with 10% neutral red for 15 minutes, and locate the multivacuolar stem cell area under a microscope. (2) Disinfection: Clean the sand and leaves on the surface of the azalea young fruits, rinse them under running tap water for 40 minutes, transfer them through the transfer window to the biosafety cabinet in the inoculation room, and dry the surface moisture with sterile paper. Prepare a disinfectant with chlorine dioxide disinfectant tablets according to the instructions, place the azalea young fruits in the disinfectant for 2 minutes, remove them and dry them with sterile paper, then place the young fruits in a 75% volume percent ethanol aqueous solution, disinfect them for 5 minutes, remove them and dry them with sterile paper; finally, rinse the young fruits 4 times with sterile water, remove them, dry them with sterile paper and set aside; (3) Callus induction: excise the non-stem cell tissue, retain the stem cell area and cut into 2 mm³ blocks, inoculate in induction medium (MS medium containing 5 mg / mL agar, 100 mg / L ascorbic acid, 2 mg / L IAA, 4 mg / L glutamic acid, 3 mg / L arginine, 5 mg / L cysteine, pH 5.8). Culture in the dark at 25°C for 5 days, and the callus induction rate reaches 90%. (4) Stem cell expansion: Pick up callus tissue and inoculate it into the same medium in a dot pattern, culture in the dark at 25°C for 9 days, and the cell cluster diameter increases to 3 mm. Transfer to MS expansion medium (containing 2 mg / L 6-BA, 1.5 mg / L NAA, 4 mg / L amphotericin B), shake at 110 r / min, and rehydrate twice at 25°C (initial 20 mL → additional 30 mL). Observe the cell morphology and number every 4 hours, and draw a growth curve (such as Figure 1After 96 hours of culture, the stem cell density reached 1.2×10 6 cells / mL, and the contamination rate was <3%.

[0033] Before stem cell expansion and culture, stem cell identification is performed: the edge tissue of the cell cluster is taken, smeared on a slide, stained with 10% neutral red for 15 minutes, rinsed with clean water and observed under a microscope. It can be seen that more than 90% of the cells contain 3-5 vacuoles, confirming that they are stem cells.

[0034] Example 2 This embodiment relates to a method for cryopreservation culture of azalea stem cells, comprising the following steps: (1) Stem cell positioning: Take 2 azalea young fruits, freeze them in liquid nitrogen for 5 minutes, slice them to a thickness of 10 μm, and stain them to determine that the stem cells are located on the inner side of the peel. (2) Disinfection: Same as in Example 1, but the ethanol disinfection time is shortened to 4 minutes (azalea peel is thinner). (3) Callus induction: Use induction medium (ascorbic acid 80 mg / L, IAA 3 mg / L, glutamic acid 2 mg / L, arginine 6 mg / L, cysteine ​​2 mg / L), culture at 25°C for 7 days, and the callus formation rate is 88%. (4) Expansion culture: Transfer to B5 expansion medium (containing final concentration of 6-BA 1 mg / L, final concentration of NAA 3 mg / L, final concentration of amphotericin B 6 mg / L), culture at 30°C and 110 r / min, observe the cell morphology and number every 4 hours, and draw a growth curve (such as Figure 2 ). The stem cell density reached 9.8×10 5 cells / mL.

[0035] Before expanding the culture, stem cell identification is performed: the edge tissue of the cell cluster is taken, smeared on a slide, stained with 10% neutral red for 15 minutes, rinsed with clean water and observed under a microscope. It can be seen that more than 90% of the cells contain 3-5 vacuoles, confirming that they are stem cells.

[0036] Example 3 This embodiment relates to a cryopreservation culture method for cranberry stem cells, comprising the following steps: (1) Stem cell positioning: cranberry young fruit is frozen in liquid nitrogen for 5 minutes, sliced ​​into 20 μm sections, and stained to show that the stem cells are concentrated at the base of the fruit stalk. (2) Disinfection treatment: Take another cranberry young fruit, clean the sand and leaves on the surface, rinse it under running tap water for 40-50 minutes, transfer it to the biosafety cabinet in the inoculation room through the transfer window, and use sterile paper to absorb the surface moisture. Chlorine dioxide disinfectant tablets are prepared into a disinfectant according to the instructions (because cranberry has a lot of hairs on the surface, the concentration of chlorine dioxide disinfectant is increased to 250ppm). Put the cranberry young fruit into the disinfectant and disinfect for 2 minutes, take it out and absorb the moisture with sterile paper, then put the young fruit into an ethanol aqueous solution with a volume percentage concentration of 75%, disinfect it for 5 minutes, take it out and absorb the moisture with sterile paper; finally, rinse the young fruit 5 times with sterile water and take it out, absorb the moisture with sterile paper and set aside; (3) Callus induction: Based on the location of the stem cells determined in step (1), the excess tissue of the young blueberry fruit in step (2) was removed as much as possible, leaving only the tissue containing the stem cells; these tissues were cut into small pieces of 1-3 mm, dried with sterile filter paper, and then spread on the surface of the induction culture medium based on ordinary MS culture medium. Incubate in the dark at 25℃±1 for 2 days until callus tissue is visible, with an efficiency of 95%. The culture medium contains 120mg / L ascorbic acid, 1mg / L IAA, 6mg / L glutamic acid, 2mg / L arginine, and 6mg / L cysteine. (4) Proliferation culture: Use MS medium (containing final concentration of 6-BA 3 mg / L, final concentration of NAA 2 mg / L and final concentration of amphotericin B 2 mg / L), culture at 22 °C, observe the cell morphology and number every 4 hours, and draw a growth curve (such as Figure 3 The stem cell proliferation rate is 12% lower than that of blueberries, but the cell activity is 98%.

[0037] Perform stem cell identification before expansion culture: obtain tissue from the edge of the cell cluster, smear it on a slide, stain it with 10% neutral red for 15 minutes, rinse it with clean water and observe it under a microscope. It can be seen that more than 90% of the cells contain 3-5 vacuoles, confirming that they are stem cells.

[0038] Example 4: The formula of an eye protection chewable tablet for relieving visual fatigue includes: 25% blueberry stem cell culture (water <5%), 25% sorbitol, 5% lactose, 1% citric acid, 10% dextrin, 1% magnesium stearate and 33% microcrystalline cellulose.

[0039] The specific preparation method is as follows: The blueberry stem cells prepared in Example 1 were centrifuged, the supernatant was discarded, and the centrifuged cells were dried under reduced pressure to a moisture content of <5%. The dried cells were then passed through an 80-mesh sieve without crushing.

[0040] The above-mentioned culture was added into a mixer and mixed for 30 minutes, 10% ethanol aqueous solution of all raw materials was added and mixed thoroughly to obtain a granular mixture, and then the granular mixture was placed in a vacuum drying oven at 40-50°C and dried until the moisture content was <5%.

[0041] The dried granules are compressed into tablets of 2 g / tablet according to conventional processes and packaged after passing inspection.

[0042] Experimental example This experiment tested 50 participants' eye fatigue. Participants took one chewable eye protection tablet three times daily for two weeks, maintaining their usual lifestyle and rest habits. A questionnaire survey was conducted before and two weeks after the tablets were taken. The scores from the questionnaires provided a preliminary analysis of the tablets' eye-protecting effects.

[0043] The questionnaire is shown in Table 3: 1 point for the presence of fatigue, 0 point for the absence of fatigue, and 0.5 point for anything in between.

[0044] Table 3: Evaluation method: First, sum the fatigue symptom scores of each subject in the questionnaire and divide it by the total number of fatigue symptoms, that is, the total fatigue score of each subject / 14, to obtain the average fatigue level of each subject.

[0045] The average fatigue level for each subject was added together and divided by the total number of subjects to obtain the overall average fatigue level. This method can be used to calculate the overall average fatigue level before and during the study. The resulting scores can be compared to determine the change in fatigue level among the subjects. A decrease in the score indicates that the eye protection chewable tablet is effective in relieving visual fatigue.

[0046] The average value of the overall fatigue of the subjects before and after using the eye protection composition (such as Figure 4 The overall average fatigue score was 0.5100 before use and 0.4907 after use, with an overall decrease of 0.0193. The T test showed that the average fatigue level was significantly reduced after using the eye protection chewable tablets (P < 0.01) (as shown in Table 4).

[0047] Table 4: Although specific embodiments of the present invention are described above, those skilled in the art should understand that these are merely examples and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is limited by the appended claims.

Claims

1. A method for cryopreservation culture of Ericaceae plant stem cells, characterized in that: The following steps are involved: (1) Determine the location of stem cells: Take young fruits of Ericaceae plants, wash them, embed them in paraffin, and freeze them in liquid nitrogen for 5-10 minutes. Then slice them. After dewaxing the slices in 25-30℃ warm water, stain them with 10% neutral red to locate stem cells with more than 3 vacuoles. (2) Young fruit disinfection: Take another young fruit of Ericaceae plant, wash it, and then soak it in chlorine dioxide disinfectant for 2 minutes, then soak it in 75% ethanol for 5 minutes, rinse it with sterile water and then dry it; (3) Callus induction: According to the stem cell position determined in step (1), the non-stem cell tissue of the young fruit treated in step (2) is removed, and the stem cell tissue is cut into 1-3 mm pieces, spread on the surface of the induction medium, and cultured in the dark at 25±1℃ for 2-7 days to obtain callus-like tissue; the induction medium is an MS medium containing agar at a final concentration of 3-6 mg / ml, ascorbic acid at 80-120 mg / L, IAA at 1-3 mg / L, glutamic acid at 2-6 mg / L, arginine at 2-6 mg / L, and cysteine ​​at 2-6 mg / L; (4) Stem cell expansion: Callus-like tissue is picked and dot-inoculated on the same induction medium as in step (3), cultured in the dark at 25±1℃ for 6-12 days, and after obtaining cell clusters, transferred to the expansion medium for suspension culture; the expansion medium is an MS or B5 medium containing 6-BA at a final concentration of 1-3 mg / L, NAA at 1-3 mg / L, and amphotericin B at 2-6 mg / L.

2. The method according to claim 1, characterized in that In step (1), the liquid nitrogen freezing time is 5 minutes and the slice thickness is 10-20 μm.

3. The method according to claim 2, characterized in that The specific operation method of step (1) determining the location of stem cells is as follows: take 2-4 young fruits of Ericaceae plants, clean the surface and absorb the surface moisture with filter paper, then embed them in paraffin and freeze them in liquid nitrogen for 5 minutes, take them out, fix them on a microtome and slice them, put the paraffin slices containing the young fruits into warm water at 25-30℃ to melt the paraffin, place the young fruit slices on a slide with a soft brush, and stain them with 10% neutral red. After staining, rinse off the excess dye with running water, and observe under a microscope to find stem cells. Cells containing more than 3 vacuoles are stem cells.

4. The method according to claim 1, wherein The propagation culture conditions of step (4) are: shaker speed 110 r / min, temperature 22-34°C, adding culture medium twice, and total culture time 96 hours.

5. The Ericaceae plant stem cells obtained by culturing according to any one of claims 1 to 4, characterized in that: The stem cells showed more than three vacuoles after being stained with 10% neutral red, had a moisture content of <5% after being dried at room temperature, and maintained activity for ≥12 months.

6. An eye protection chewable tablet, characterized in that: A preparation comprising the stem cell culture according to claim 5, wherein the composition is as follows, by weight: 20-30% dried blueberry stem cells having a moisture content of <5%, 20-30% sorbitol, 3-8% lactose, 0.5-2% citric acid, 8-12% dextrin, 0.5-1.5% magnesium stearate, and 30-35% microcrystalline cellulose.

7. The eye-protecting chewable tablet according to claim 6, characterized in that: The preparation method comprises the following steps: (a) The dried cells were centrifuged and dried under reduced pressure to a moisture content of <5%, and then pulverized to pass through an 80-mesh sieve; (b) granulated with sorbitol, lactose, citric acid, dextrin, magnesium stearate, and microcrystalline cellulose; (c) dried under reduced pressure at 40-50°C to a moisture content of <5%, and compressed into tablets, each weighing 2 ± 0.2 g.

8. The method according to claim 1, characterized in that The Ericaceae plant is selected from blueberry (Vaccinium spp.), azalea (Rhododendron simsii), bilberry (Vaccinium vitis-idaea) or bellflower (Enkianthus campanulatus).

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