Culture medium and culture method of roxburgh rose embryogenic stem cells and method for producing vitamin C at high yield by using roxburgh rose embryogenic stem cells

Abstract

The invention belongs to the technical field of plant tissue culture, and discloses a culture medium and a culture method for roxburgh rose embryonic stem cells and a method for producing vitamin C. The culture medium comprises a 1 / 2 MS culture medium, and NAA with the concentration being 0.05-0.20 mg / L, 6-BA with the concentration being 0.05-0.20 mg / L and KT with the concentration being 0.5-2.0 mg / L are added into the 1 / 2 MS culture medium; the method for producing vitamin C at high yield comprises the step of adding salicylic acid for induced culture. NAA, 6-BA and KT are added into a culture medium of the roxburgh rose embryogenic stem cells, and the growth of the roxburgh rose embryogenic stem cells can be better promoted by utilizing the synergistic effect of the three plant growth regulators on the growth of the roxburgh rose embryogenic stem cells and matching with a specific addition concentration range; salicylic acid is used for carrying out cell induction to generate an effective substance vitamin C of roxburgh rose, so that the content of vitamin C in the roxburgh rose embryonic stem cells is effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture, specifically, it relates to a culture medium, a culture method for prickly pear embryonic stem cells and a method for producing high levels of vitamin C. Background Art

[0002] The Rosaceae family is a family within the order Rosales of the class Dicotyledonous plants. It comprises herbaceous, shrubby, or tree plants, deciduous or evergreen. China has approximately 51 genera and over 1000 species, distributed throughout the country. Rosaceae plants include apples, pears, roses, and hybrid tea roses. Many varieties produce fruits rich in vitamins, sugars, and organic acids, which can be used as raw materials for processing fruit products such as dried fruit, preserves, jams, wines, cakes, juices, and fruit leather.

[0003] The prickly pear is a plant belonging to the genus Rosa in the family Rosaceae. Its fruit surface is covered with thorns, hence its common name, "thorny pineapple." It is a rare and nutritious fruit, valued for its health benefits. Prickly pears grow on sunny mountain slopes, in valleys, along roadsides, and in thickets at altitudes of 500–2500 meters. They are a natural wild fruit found in Guizhou, western Hubei, western Hunan, Liangshan, and Mianning mountain areas. Large-scale artificial cultivation exists in Guizhou Province and Kaifeng City, Henan Province.

[0004] The prickly pear fruit is rich in nutrients, including carbohydrates, proteins and amino acids, vitamins and tannins. Among these, it contains various vitamins such as vitamin C, vitamin P, B vitamins (B1, B2, etc.), vitamin E, folic acid, and carotene, with vitamin C being the most abundant. Specifically, every 100 grams of prickly pear pulp contains 2075–2725 mg of vitamin C, which is 800 times that of apples, 400 times that of bananas, 100 times that of tangerines, 22 times that of tomatoes, and 10 times that of kiwifruit. Therefore, the prickly pear is also known as the "King of Vitamin C."

[0005] In addition, prickly pear contains an internationally recognized anti-cancer active substance, superoxide dismutase, which has a powerful effect, helping the body improve its immunity and inhibiting carcinogens in cells. Besides this, prickly pear also contains many other anti-cancer substances, such as prickly pear triterpenes, prickly pear polysaccharides, protocatechuic acid, tannins, and selenium. The combined effect of these substances makes prickly pear effective in preventing cancer and controlling the metastasis of cancer cells.

[0006] However, the common sources of prickly pear are affected by many factors, such as adverse weather, environmental pollution, and changes in soil nutrients. Therefore, the rapid propagation of prickly pear is an urgent problem to be solved. In addition, the extraction and purification of active substances from growing plants is complex and expensive, making it of great research significance to find a way to rapidly and efficiently induce vitamin C production in prickly pear.

[0007] In view of this, the present invention is hereby proposed. Summary of the Invention

[0008] The technical problem to be solved by this invention is to overcome the shortcomings of the prior art and provide a culture medium, a culture method for prickly pear embryonic stem cells and a method for high-yield vitamin C production. By adding three plant growth regulators with synergistic effects, NAA, 6-BA and KT, to the culture medium, the culture of prickly pear embryonic stem cells is significantly promoted, and the growth rate of prickly pear embryonic stem cells can be significantly increased, thereby achieving efficient and rapid culture. Furthermore, the addition of salicylic acid to the culture medium can effectively increase the vitamin C content in the cultured prickly pear embryonic stem cells.

[0009] To solve the above-mentioned technical problems, the basic concept of the technical solution adopted by the present invention is as follows:

[0010] The first objective of this invention is to provide a culture medium for *Rhizopus rosaefolia* embryonic stem cells, the culture medium comprising: 1 / 2MS medium supplemented with NAA at a concentration of 0.05–0.20 mg / L, 6-BA at a concentration of 0.05–0.20 mg / L, and KT at a concentration of 0.5–2.0 mg / L.

[0011] The culture medium for prickly pear embryonic stem cells of the present invention contains NAA (naphthaleneacetic acid), 6-BA (6-benzylaminopurine), and KT (furanomethylaminopurine) in a certain mass concentration range. KT promotes cell division, while NAA and 6-BA promote differentiation. The three have a synergistic effect on the growth of prickly pear embryonic stem cells, thus significantly promoting the culture of prickly pear embryonic stem cells and significantly increasing the growth rate of embryonic stem cells, thereby achieving efficient and rapid culture.

[0012] Further solutions, as described 1 The 2MS medium was supplemented with NAA at a concentration of 0.10 mg / L, 6-BA at a concentration of 0.10 mg / L, and KT at a concentration of 1.0 mg / L.

[0013] Further solutions, as described 1 The mass ratio of NAA, 6-BA, and KT added to the 2MS medium is 1:1:10.

[0014] In this invention, experimental tests have shown that controlling the addition ratio of the above three plant growth regulators within a certain range can better promote the growth of prickly pear embryonic stem cells, resulting in better growth status of prickly pear embryonic stem cells and further increasing the culture yield of embryonic stem cells.

[0015] A second objective of this invention is to provide a method for culturing prickly pear embryonic stem cells, comprising the following steps:

[0016] (1) Pretreatment of explants: Take young prickly pear fruits, wash them, cut off the position containing embryonic stem cells at the top of the young fruit, and sterilize the obtained slices.

[0017] (2) Obtaining embryonic stem cells: Sterilized sections were inoculated onto solid culture medium for culture to generate embryonic stem cell clusters. The embryonic stem cell clusters were then detached from the solid culture medium. The obtained embryonic stem cell clusters were cut into pieces, placed in a culture container, and culture medium was added for suspension culture. The culture medium for suspension culture included... 1 The medium was prepared in 2MS, with NAA at a concentration of 0.05–0.20 mg / L, 6-BA at a concentration of 0.05–0.20 mg / L, and KT at a concentration of 0.5–2.0 mg / L. After culturing, the medium was filtered to obtain a filtrate containing embryonic stem cells.

[0018] (3) Expanded culture: Dilute the filtrate containing embryonic stem cells obtained in step (2) and transfer it for passage culture;

[0019] (4) Collection: Centrifuge the embryonic stem cell culture medium obtained by passage culture in step (3), discard the supernatant, and retain the precipitate to obtain prickly pear embryonic stem cells.

[0020] As one specific implementation method, the culture medium for suspension culture includes 1 / 2MS medium supplemented with 0.10 mg / L NAA, 0.10 mg / L 6-BA, and 1.0 mg / L KT.

[0021] In a further step, the cleaning process in step (1) includes: first cleaning the outer surface of the young fruit with dish soap, and then rinsing it with running water.

[0022] As one specific implementation, the cleaning in step (1) includes: first cleaning the outer surface of the young prickly pear fruit with dish soap, brushing off the surface dirt with a brush, and then soaking and rinsing with running water for 30 minutes.

[0023] In a further step, in step (1), the top 3-4 mm of the young prickly pear fruit containing embryonic stem cells is cut off and sliced ​​into thin slices, which are then placed in sterile water to prevent browning.

[0024] As one specific implementation method, the top 3-4 mm of the young prickly pear fruit containing embryonic stem cells is cut off with a scalpel blade in an ultra-clean workbench, cut into small thin slices, and quickly placed in sterile water to prevent browning.

[0025] In a further step, the sterilization process in step (1) includes: first disinfecting with 75% alcohol by volume, washing with sterile ultrapure water, then disinfecting the slices with a disinfectant with an effective chlorine content of 500 mg / L, and then washing with sterile ultrapure water.

[0026] As one specific implementation method, the slides are disinfected in 75% alcohol for 30 seconds, then immediately washed twice with sterile ultrapure water, then disinfected with a disinfectant with an effective chlorine content of 500 mg / L for 10 minutes, and then washed five times with sterile ultrapure water.

[0027] In this invention, the sterile ultrapure water refers to ultrapure water that has undergone sterilization treatment.

[0028] In a further step, in step (2), after the slices are inoculated onto a solid culture medium, they are cultured in the dark at 25°C for 20 days.

[0029] In a further embodiment, the solid culture medium comprises MS medium, wherein 2,4-D (2,4-dichlorophenoxyacetic acid) at a concentration of 2 mg / L, 6-BA at a concentration of 0.15 mg / L, and agar at a mass fraction of 0.7%.

[0030] As a specific implementation method, the sterilized slices in step (1) are inoculated onto a solid culture medium contained in the culture product. The inoculated culture flask is placed in the dark at 25°C. After 20 days, it is observed whether embryonic stem cell clusters are generated. Then, the culture flasks that have generated embryonic stem cell clusters are selected, placed in a biosafety cabinet, and the embryonic stem cell clusters are peeled off with sterile tweezers, washed twice in sterile water, and then the cell clusters are cut into small pieces as much as possible with a sterile scalpel blade, placed in a conical flask, and culture medium is added for suspension culture.

[0031] In a further step, in step (2), filtration is performed after the culture is completed, including: filtering the embryonic stem cell culture medium after the culture is completed using a No. 2 sieve for the first time, and filtering the resulting filtrate using an 80-mesh sieve for the second time.

[0032] In one specific implementation, the embryonic stem cell culture medium is filtered twice as described above in a biosafety cabinet to obtain a filtrate containing embryonic stem cells.

[0033] In the above scheme, since most stem cells are in the form of single cells or small cell clusters, large particles, or larger flakes and fragments in the culture medium are removed by the first filtration, and then the relatively smaller fragments are removed by the second filtration, so that the filtrate containing embryonic stem cells can be obtained.

[0034] In a further step, in step (2), the culture temperature is 22℃, the shaking speed is 110r / min, and the culture time is 48h.

[0035] In a further step, in step (3), the culture temperature is 22℃, the shaking speed is 110r / min, and the culture is repeated every 48h.

[0036] In a further embodiment, the culture medium used for subculture in step (3) is the same as the culture medium used for suspension culture in step (2).

[0037] In a further step, step (3) involves subculturing once or multiple times.

[0038] In one specific implementation, in step (3), the filtrate containing embryonic stem cells obtained in step (2) is diluted to a cell concentration of 10. 5 The cells were transferred to 1000 mL Erlenmeyer flasks for passage culture, using 300 mL of culture medium per flask. The cells were passaged every 48 hours until 12 flasks were reached. After 48 hours of culture, the embryonic stem cells were collected.

[0039] In step (4), the embryonic stem cell culture medium obtained by passage culture in step (3) is poured into a centrifuge bottle, the centrifugation speed is 2200 r / min, the centrifugation time is 10 minutes, after centrifugation, the supernatant is discarded and the precipitate is retained, thus obtaining prickly pear embryonic stem cells.

[0040] As a more specific implementation method, the culture method of prickly pear embryonic stem cells includes the following steps:

[0041] (1) Pretreatment of explants: Select young prickly pear fruits, first clean the outer surface of the young prickly pear fruits with detergent, gently brush off the surface dirt with a soft brush, and then soak and rinse with running water for 30 minutes. In a clean bench, use a scalpel to cut the position containing embryonic stem cells at the top 3-4 mm of the young prickly pear fruits, cut them into small thin slices, and quickly put them into sterile water to prevent browning. After cutting, disinfect the slices in 75% alcohol for 30 seconds, and then immediately wash them twice with sterile ultrapure water. Then disinfect the slices with a disinfectant with an effective chlorine content of 500 mg / L for 10 minutes, and then wash them five times with sterile ultrapure water.

[0042] (2) Obtaining embryonic stem cells: The sterilized slices from step (1) were inoculated onto solid culture medium in a culture flask. The inoculated culture flask was then placed in the dark at 25°C for 20 days to observe whether embryonic stem cell clusters were produced. Then, the culture flasks that produced embryonic stem cell clusters were selected and placed in a biosafety cabinet. The embryonic stem cell clusters were carefully detached from the solid culture medium with sterile forceps, washed twice in sterile water, and then the cell clusters were cut into small pieces as much as possible with a sterile scalpel. They were placed in conical flasks and culture medium was added for suspension culture. The culture medium for suspension culture included...1 The medium was prepared in 2MS, with NAA at a concentration of 0.05–0.20 mg / L, 6-BA at a concentration of 0.05–0.20 mg / L, and KT at a concentration of 0.5–2.0 mg / L. The conical flasks were placed in a shaker at 22°C and 110 rpm for 36–48 h. After the culture was completed, the culture medium was first filtered through a No. 2 sieve in a biosafety cabinet, and the filtrate was then filtered a second time through an 80-mesh sieve to obtain the filtrate containing embryonic stem cells. The cell concentration was then measured.

[0043] (3) Expanded culture: Dilute the filtrate containing embryonic stem cells obtained in step (2) to a cell concentration of 10. 5 Cells / mL were transferred to 1000mL Erlenmeyer flasks for subculture, using 300mL of culture medium per flask. The culture medium included... 1 / 2MS medium was prepared, with NAA at a concentration of 0.05–0.20 mg / L, 6-BA at a concentration of 0.05–0.20 mg / L, and KT at a concentration of 0.5–2.0 mg / L. The Erlenmeyer flasks were cultured in a shaker at 22°C and 110 rpm. The flasks were passaged every 48 hours until 12 flasks were cultured. After 48 hours of culture, the embryonic stem cells were collected.

[0044] (4) Collection: Pour the embryonic stem cell culture medium obtained by passage culture in step (3) into a centrifuge bottle, balance it, and centrifuge it with a centrifuge at a speed of 2200 r / min for 10 minutes. After centrifugation, discard the supernatant and keep the precipitate to obtain prickly pear embryonic stem cells.

[0045] The third objective of this invention is to provide a method for inducing high vitamin C production from prickly pear embryonic stem cells. The method involves seeding prickly pear embryonic stem cells into a culture medium and adding salicylic acid solution to make the final concentration of salicylic acid 2.50-3.75 μg / L for induction culture. After a certain period of culture, prickly pear embryonic stem cells with high vitamin C production are obtained.

[0046] A further approach involves using the prickly pear embryonic stem cell culture method provided in the second objective of this invention, to dilute the obtained prickly pear embryonic stem cells to a cell concentration of 10. 7 Cell culture medium of cells / mL;

[0047] Take 200 mL of the cell culture medium, add 0.2–0.3 mL of 5 mg / L salicylic acid solution, and add culture medium to a final volume of 400 mL, then perform induction culture.

[0048] A further proposed method involves induction culture at a temperature of 26°C, a shaking speed of 110 r / min, and a culture time of 48–72 h.

[0049] A further protocol, using culture media including 1 / 2MS medium supplemented with NAA at a concentration of 0.05–0.20 mg / L, 6-BA at a concentration of 0.05–0.20 mg / L, and KT at a concentration of 0.5–2.0 mg / L.

[0050] The present invention adds a certain concentration of salicylic acid to the culture medium as an inducer, which can significantly increase the vitamin C content in the culture of prickly pear embryonic stem cells.

[0051] As a specific implementation method, the method for inducing high vitamin C production from prickly pear embryonic stem cells is as follows:

[0052] The selected cell concentration was 10 in 200 mL of prickly pear embryonic stem cells. 7 Add 0.2–0.3 mL of 5 mg / L salicylic acid solution to a cell culture medium at a concentration of [number] cells / mL, and then use culture medium to bring the culture medium to a final volume of 400 mL. The culture medium includes [a specific culture medium]. 1 The prickly pear embryonic stem cells were cultured in 2MS medium supplemented with 0.05–0.20 mg / L NAA, 0.05–0.20 mg / L 6-BA, and 0.5–2.0 mg / L KT. The cells were then placed in a shaker at 26°C and 110 rpm for induction culture. After 48–72 h of culture, the high-vitamin C-producing prickly pear embryonic stem cells were collected.

[0053] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art.

[0054] 1. In this invention, the culture medium for culturing prickly pear embryonic stem cells is supplemented with NAA at a concentration of 0.05–0.20 mg / L, 6-BA at a concentration of 0.05–0.20 mg / L, and KT at a concentration of 0.5–2.0 mg / L. Experiments have shown that the above three plant growth regulators within this concentration range have a synergistic effect on the growth of prickly pear embryonic stem cells, which can significantly promote the culture of prickly pear embryonic stem cells and significantly increase the growth rate of prickly pear embryonic stem cells.

[0055] 2. In this invention, salicylic acid was added to a culture medium containing prickly pear embryonic stem cells. It was found that salicylic acid can induce prickly pear embryonic stem cells to produce high levels of vitamin C, thereby significantly increasing the vitamin C content in the cultured prickly pear embryonic stem cells.

[0056] The specific embodiments of the present invention will be described in further detail below. DETAILED DESCRIPTION

[0057] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0058] Example 1

[0059] This embodiment relates to a method for culturing prickly pear embryonic stem cells, the specific steps of which are as follows:

[0060] (1) Pretreatment of explants: Select 1 kg of young prickly pear fruit from Guizhou. First, wash the outer surface of the young prickly pear fruit with detergent and gently brush off the dirt with a soft brush. Then, soak and rinse with running water for 30 min. In a clean bench, use a scalpel to cut off the position containing embryonic stem cells at the top 3-4 mm of the young prickly pear fruit. Cut it into small thin slices and quickly put them into sterile water to prevent browning. After cutting, disinfect the slices in 75% alcohol for 30 s, and then immediately wash them twice with sterile ultrapure water. Then, disinfect the slices with a disinfectant with an effective chlorine content of 500 mg / L for 10 min, and then wash them five times with sterile ultrapure water.

[0061] (2) Obtaining embryonic stem cells: Sterilized sections were inoculated onto solid culture medium in culture flasks. The solid culture medium included MS medium, with 2,4-D at a concentration of 2 mg / L, 6-BA at a concentration of 0.15 mg / L, and agar at a mass fraction of 0.7%. The inoculated culture flasks were placed in the dark at 25°C for 20 days to observe whether embryonic stem cell clusters had formed. Then, the culture flasks that produced embryonic stem cell clusters were selected and placed in a biosafety cabinet. The embryonic stem cell clusters were carefully detached from the solid culture medium with sterile forceps, washed twice in sterile water, and then the cell clusters were cut into small pieces as much as possible with a sterile scalpel blade. The pieces were placed in conical flasks and cultured in suspension. The culture medium for suspension culture included... 1 The culture medium was prepared in 2MS medium, with NAA at a concentration of 0.10 mg / L, 6-BA at a concentration of 0.10 mg / L, and KT at a concentration of 1.0 mg / L. The conical flask was placed in a shaker at 22°C and 110 rpm for 48 h. After the culture was completed, the culture medium was first filtered through a No. 2 sieve in a biosafety cabinet, and the resulting filtrate was then filtered a second time through an 80-mesh sieve to obtain the filtrate containing embryonic stem cells. The cell concentration was then measured.

[0062] (3) Expanded culture: Dilute the filtrate containing embryonic stem cells obtained in step (2) to a cell concentration of 10. 5 Cells / mL were transferred to several 1000mL Erlenmeyer flasks for subculture, with 300mL of culture medium per flask. The culture medium included... 1 / 2MS medium was prepared, with NAA at a concentration of 0.10 mg / L, 6-BA at a concentration of 0.10 mg / L, and KT at a concentration of 1.0 mg / L. The Erlenmeyer flasks were placed in a shaker at 22°C and 110 r / min. The flasks were passaged every 48 hours until 12 flasks were reached. After 48 hours of culture, the embryonic stem cells were collected.

[0063] (4) Collection: Pour the embryonic stem cell culture medium obtained by passage culture in step (3) into a centrifuge bottle, balance it, and centrifuge it with a centrifuge at a speed of 2200 r / min for 10 minutes. After centrifugation, discard the supernatant and keep the precipitate to obtain prickly pear embryonic stem cells.

[0064] Example 2

[0065] This embodiment relates to a method for culturing prickly pear embryonic stem cells, the specific steps of which are as follows:

[0066] (1) Pretreatment of explants: Select 1 kg of young prickly pear fruit from Guizhou. First, wash the outer surface of the young prickly pear fruit with detergent and gently brush off the dirt with a soft brush. Then, soak and rinse with running water for 30 min. In a clean bench, use a scalpel to cut off the position containing embryonic stem cells at the top 3-4 mm of the young prickly pear fruit. Cut it into small thin slices and quickly put them into sterile water to prevent browning. After cutting, disinfect the slices in 75% alcohol for 30 s, and then immediately wash them twice with sterile ultrapure water. Then, disinfect the slices with a disinfectant with an effective chlorine content of 500 mg / L for 10 min, and then wash them five times with sterile ultrapure water.

[0067] (2) Obtaining embryonic stem cells: Sterilized sections were inoculated onto solid culture medium in culture flasks. The solid culture medium included MS medium, with 2,4-D at a concentration of 2 mg / L, 6-BA at a concentration of 0.15 mg / L, and agar at a mass fraction of 0.7%. The inoculated culture flasks were placed in the dark at 25°C for 20 days to observe whether embryonic stem cell clusters had formed. Then, the culture flasks that produced embryonic stem cell clusters were selected and placed in a biosafety cabinet. The embryonic stem cell clusters were carefully detached from the solid culture medium with sterile forceps, washed twice in sterile water, and then the cell clusters were cut into small pieces as much as possible with a sterile scalpel blade. The pieces were placed in conical flasks and cultured in suspension. The culture medium for suspension culture included... 1The medium was prepared in 2MS medium supplemented with 0.05 mg / L NAA, 0.05 mg / L 6-BA, and 0.5 mg / L KT. The conical flask was incubated at 22°C in a shaker at 110 rpm for 48 h. After incubation, the culture medium was first filtered through a No. 2 sieve in a biosafety cabinet, and the filtrate was then filtered a second time through an 80-mesh sieve to obtain the filtrate containing embryonic stem cells. The cell concentration was then measured.

[0068] (3) Expanded culture: Dilute the filtrate containing embryonic stem cells obtained in step (2) to a cell concentration of 10. 5 Cells / mL were transferred to several 1000mL Erlenmeyer flasks for subculture, with 300mL of culture medium per flask. The culture medium included... 1 / 2MS medium was prepared, with NAA, 6-BA, and KT added at a concentration of 0.05 mg / L. The Erlenmeyer flasks were placed in a shaker at 22°C and 110 r / min, and passaged every 48 h until 12 flasks were reached. After 48 h of culture, embryonic stem cells were collected.

[0069] (4) Collection: Pour the embryonic stem cell culture medium obtained by passage culture in step (3) into a centrifuge bottle, balance it, and centrifuge it with a centrifuge at a speed of 2200 r / min for 10 minutes. After centrifugation, discard the supernatant and keep the precipitate to obtain prickly pear embryonic stem cells.

[0070] Example 3

[0071] (1) Pretreatment of explants: Select 1 kg of young prickly pear fruit from Guizhou. First, wash the outer surface of the young prickly pear fruit with detergent and gently brush off the dirt with a soft brush. Then, soak and rinse with running water for 30 min. In a clean bench, use a scalpel to cut off the position containing embryonic stem cells at the top 3-4 mm of the young prickly pear fruit. Cut it into small thin slices and quickly put them into sterile water to prevent browning. After cutting, disinfect the slices in 75% alcohol for 30 s, and then immediately wash them twice with sterile ultrapure water. Then, disinfect the slices with a disinfectant with an effective chlorine content of 500 mg / L for 10 min, and then wash them five times with sterile ultrapure water.

[0072] (2) Obtaining embryonic stem cells: Sterilized sections were inoculated onto solid culture medium in culture flasks. The solid culture medium included MS medium, with 2,4-D at a concentration of 2 mg / L, 6-BA at a concentration of 0.15 mg / L, and agar at a mass fraction of 0.7%. The inoculated culture flasks were placed in the dark at 25°C for 20 days to observe whether embryonic stem cell clusters had formed. Then, the culture flasks that produced embryonic stem cell clusters were selected and placed in a biosafety cabinet. The embryonic stem cell clusters were carefully detached from the solid culture medium with sterile forceps, washed twice in sterile water, and then the cell clusters were cut into small pieces as much as possible with a sterile scalpel blade. The pieces were placed in conical flasks and cultured in suspension. The culture medium for suspension culture included... 1 The medium was prepared in 2MS medium supplemented with 0.20 mg / L NAA, 0.20 mg / L 6-BA, and 2.0 mg / L KT. The conical flask was placed in a shaker at 22°C and 110 rpm for 48 h. After the culture was completed, the culture medium was first filtered through a No. 2 sieve in a biosafety cabinet, and the filtrate was then filtered a second time through an 80-mesh sieve to obtain the filtrate containing embryonic stem cells. The cell concentration was then measured.

[0073] (3) Expanded culture: Dilute the filtrate containing embryonic stem cells obtained in step (2) to a cell concentration of 10. 5 Cells / mL were transferred to several 1000mL Erlenmeyer flasks for subculture, with 300mL of culture medium per flask. The culture medium included... 1 / 2MS medium was prepared, with NAA at a concentration of 0.20 mg / L, 6-BA at a concentration of 0.20 mg / L, and KT at a concentration of 2.0 mg / L. The Erlenmeyer flasks were placed in a shaker at 22°C and 110 r / min. The flasks were passaged every 48 h until 12 flasks were reached. After 48 h of culture, the embryonic stem cells were collected.

[0074] (4) Collection: Pour the embryonic stem cell culture medium obtained by passage culture in step (3) into a centrifuge bottle, balance it, and centrifuge it with a centrifuge at a speed of 2200 r / min for 10 minutes. After centrifugation, discard the supernatant and keep the precipitate to obtain prickly pear embryonic stem cells.

[0075] Comparative Example 1

[0076] This comparative example is based on Example 1, except that the culture medium used for suspension culture in step (2) and subculture in step (3) is replaced with a medium containing 0.10 mg / L NAA. 1 / 2MS medium.

[0077] Comparative Example 2

[0078] This comparative example is based on Example 1, except that the culture medium used for suspension culture in step (2) and subculture in step (3) is replaced with a medium containing 0.10 mg / L 6-BA. 1 / 2MS medium.

[0079] Comparative Example 3

[0080] This comparative example is based on Example 1, except that the culture medium used for suspension culture in step (2) and subculture in step (3) is replaced with a medium containing 1.0 mg / L KT. 1 / 2MS medium.

[0081] Comparative Example 4

[0082] This comparative example is based on Example 1, except that the culture medium used for suspension culture in step (2) and subculture in step (3) is replaced with a medium containing 0.10 mg / L NAA and 0.10 mg / L 6-BA. 1 / 2MS medium.

[0083] Comparative Example 5

[0084] This comparative example is based on Example 1, except that the culture medium used for suspension culture in step (2) and subculture in step (3) is replaced with a medium containing 0.10 mg / L NAA and 1.0 mg / L KT. 1 / 2MS medium.

[0085] Comparative Example 6

[0086] This comparative example is based on Example 1, except that the culture medium used for suspension culture in step (2) and subculture in step (3) is replaced with a medium containing 0.10 mg / L 6-BA and 1.0 mg / L KT. 1 / 2MS medium.

[0087] Comparative Example 7

[0088] This comparative example is based on Example 1, except that the culture medium used for suspension culture in step (2) and subculture in step (3) is replaced with a medium containing 0.30 mg / L NAA, 0.30 mg / L 6-BA and 3.0 mg / L KT. 1 / 2MS medium.

[0089] Comparative Example 8

[0090] This comparative example is based on Example 1, except that the culture medium used for suspension culture in step (2) and subculture in step (3) is replaced with a medium containing 0.50 mg / L NAA, 0.50 mg / L 6-BA and 5.0 mg / L KT. 1 / 2MS medium.

[0091] Comparative Example 9

[0092] This comparative example is based on Example 1, except that the culture medium used for suspension culture in step (2) and subculture in step (3) is replaced with a medium containing 0.05 mg / L NAA, 0.10 mg / L 6-BA and 2.0 mg / L KT. 1 / 2MS medium.

[0093] Comparative Example 10

[0094] This comparative example, based on Example 1, replaces the culture medium used for suspension culture in step (2) and subculture in step (3) with a medium containing 0.05 mg / L NAA, 0.10 mg / L 6-BA, and 1.0 mg / L KT. 1 / 2MS medium.

[0095] Comparative Example 11

[0096] This comparative example is based on Example 1, except that the culture medium used for suspension culture in step (2) and subculture in step (3) is replaced with a medium containing 0.05 mg / L NAA, 0.20 mg / L 6-BA and 1.0 mg / L KT. 1 / 2MS medium.

[0097] Comparative Example 12

[0098] This comparative example is based on Example 1, except that the culture medium used for suspension culture in step (2) and subculture in step (3) is replaced with a medium containing 0.20 mg / L NAA, 0.10 mg / L 6-BA and 1.0 mg / L KT. 1 / 2MS medium.

[0099] Experimental Example 1

[0100] In this experiment, the wet weight of the prickly pear embryonic stem cells obtained in Examples 1-3 and Comparative Examples 1-12 was measured, and the dry weight of the cells was measured after drying. The results are shown in Table 1 below.

[0101] Table 1. Statistics of cell dry and wet weight under different culture media conditions.

[0102]

[0103]

[0104] The results above show that the wet and dry weights of the embryonic stem cells cultured in Examples 1-3 were significantly higher than those in Comparative Examples 1-6, indicating that the simultaneous addition of three plant growth regulators, NAA, 6-BA, and KT, has a synergistic effect on the growth of *Pyracantha fortuneana* embryonic stem cells, significantly improving their growth rate. In Comparative Examples 9-12, the wet and dry weights of *Pyracantha fortuneana* embryonic stem cells were significantly lower than those in Examples 1-3, indicating that a mass ratio of NAA, 6-BA, and KT of 1:1:10 better promotes the growth of *Pyracantha fortuneana* embryonic stem cells, resulting in better growth status. In Comparative Examples 7 and 8, the wet and dry weights of *Pyracantha fortuneana* embryonic stem cells were significantly lower than those in Examples 1-3, indicating that adding plant growth regulators at concentrations above the specified range has no significant effect on promoting the growth of *Pyracantha fortuneana* embryonic stem cells. Furthermore, among Examples 1-3, the embryonic stem cells obtained in Example 1 had the highest wet and dry weights, indicating that the culture medium containing 0.10 mg / L NAA, 0.10 mg / L 6-BA, and 1.0 mg / L KT is most suitable for the growth of *Pyracantha fortuneana* embryonic stem cells.

[0105] Example 4

[0106] In this embodiment, uninduced prickly pear embryonic stem cells cultured in Example 1 were used, with a cell concentration of 10⁻¹⁰ cells per 200 mL. 7 Cell culture medium of cells / mL was prepared, with 0.2 mL of 5 mg / L salicylic acid solution added, using the culture medium from Example 1 (i.e., containing 0.10 mg / L NAA, 0.10 mg / L 6-BA, and 1.0 mg / L KT). 1 Add the culture medium (2MS medium) to a final volume of 400 mL (equivalent to a final salicylic acid concentration of 2.50 μg / L), place it in a shaker at 26℃ and a speed of 110 r / min for induction culture, and collect it after 48 h of culture.

[0107] Example 5

[0108] In this embodiment, uninduced prickly pear embryonic stem cells cultured in Example 1 were used, with a cell concentration of 10⁻¹⁰ cells per 200 mL. 7 Cell culture medium of cells / mL was prepared, with 0.2 mL of 5 mg / L salicylic acid solution added, using the culture medium from Example 1 (i.e., containing 0.10 mg / L NAA, 0.10 mg / L 6-BA, and 1.0 mg / L KT). 1 Add the culture medium (2MS medium) to a final volume of 400 mL (equivalent to a final salicylic acid concentration of 2.50 μg / L), place it in a shaker at 26℃ and a speed of 110 r / min for induction culture, and collect it after 72 h of culture.

[0109] Example 6

[0110] In this embodiment, uninduced prickly pear embryonic stem cells cultured in Example 1 were used, with a cell concentration of 10⁻¹⁰ cells per 200 mL. 7 Cell culture medium of cells / mL was prepared, with 0.3 mL of 5 mg / L salicylic acid solution added, using the culture medium from Example 1 (i.e., containing 0.10 mg / L NAA, 0.10 mg / L 6-BA, and 1.0 mg / L KT). 1 Add the culture medium (2MS medium) to a final volume of 400 mL (equivalent to a final salicylic acid concentration of 3.75 μg / L), place it in a shaker at 26 °C and a speed of 110 r / min for induction culture, and collect it after 48 h of culture.

[0111] Example 7

[0112] In this embodiment, uninduced prickly pear embryonic stem cells cultured in Example 1 were used, with a cell concentration of 10⁻¹⁰ cells per 200 mL. 7 Cell culture medium of cells / mL was prepared, with 0.3 mL of 5 mg / L salicylic acid solution added, using the culture medium from Example 1 (i.e., containing 0.10 mg / L NAA, 0.10 mg / L 6-BA, and 1.0 mg / L KT). 1 Add the culture medium (2MS medium) to a final volume of 400 mL (equivalent to a final salicylic acid concentration of 3.75 μg / L), place it in a shaker at 26 °C and a speed of 110 r / min for induction culture, and collect it after 72 h of culture.

[0113] Comparative Example 13

[0114] In this comparative example, uninduced prickly pear embryonic stem cells cultured in Example 1 were used, with a cell concentration of 10⁻⁶ cells per 200 mL. 7 Cell culture medium of cells / mL was prepared, with 0.4 mL of 5 mg / L salicylic acid solution added, using the culture medium from Example 1 (i.e., containing 0.10 mg / L NAA, 0.10 mg / L 6-BA, and 1.0 mg / L KT). 1 Add the culture medium (2MS medium) to a final volume of 400 mL (equivalent to a final salicylic acid concentration of 5.00 μg / L), place it in a shaker at 26℃ and a speed of 110 r / min for induction culture, and collect it after 48 h of culture.

[0115] Comparative Example 14

[0116] In this comparative example, uninduced prickly pear embryonic stem cells cultured in Example 1 were used, with a cell concentration of 10⁻⁶ cells per 200 mL. 7Cell culture medium of cells / mL was prepared, with 0.4 mL of 5 mg / L salicylic acid solution added, using the culture medium from Example 1 (i.e., containing 0.10 mg / L NAA, 0.10 mg / L 6-BA, and 1.0 mg / L KT). 1 Add the culture medium (2MS medium) to a final volume of 400 mL (equivalent to a final salicylic acid concentration of 5.00 μg / L), place it in a shaker at 26 °C and a speed of 110 r / min for induction culture, and collect it after 72 h of culture.

[0117] Comparative Example 15

[0118] In this comparative example, uninduced prickly pear embryonic stem cells cultured in Example 1 were used, with a cell concentration of 10⁻⁶ cells per 200 mL. 7 Cell culture medium at a concentration of cells / mL was used, employing the culture medium described in Example 1 (i.e., containing 0.10 mg / L NAA, 0.10 mg / L 6-BA, and 1.0 mg / L KT). 1 (2MS medium) The culture medium was added to a final volume of 400 mL and placed in a shaker at 26 °C and 110 r / min for induction culture. The culture was collected after 48 h.

[0119] Comparative Example 16

[0120] In this comparative example, uninduced prickly pear embryonic stem cells cultured in Example 1 were used, with a cell concentration of 10⁻⁶ cells per 200 mL. 7 Cell culture medium at a concentration of cells / mL was used, employing the culture medium described in Example 1 (i.e., containing 0.10 mg / L NAA, 0.10 mg / L 6-BA, and 1.0 mg / L KT). 1 (2MS medium) The culture medium was added to a final volume of 400 mL and placed in a shaker at 26 °C and 110 r / min for induction culture. The culture was collected after 72 h.

[0121] In Examples 4-7 and Comparative Examples 13-16 above, the method for collecting prickly pear embryonic stem cells after culture can refer to step (4) in Example 1.

[0122] Experimental Example 2

[0123] This experiment determined the vitamin C content of prickly pear embryonic stem cells extracted in Examples 4-7 and Comparative Examples 13-16. The specific determination method is as follows. It should be noted that all solution preparation and operations were carried out under light-protected conditions during the following determination process.

[0124] I. Instruments and Reagents

[0125] 1. Instruments: F-280 fluorescence spectrophotometer, LD-1L biological cell laboratory cell disruptor.

[0126] 2. Reagents:

[0127] (1) Metaphosphoric acid-acetic acid solution: Weigh 15g metaphosphoric acid, add 40mL glacial acetic acid and 250mL water, stir, let stand overnight to gradually dissolve, and add water to 500mL.

[0128] (2) 0.15mol / L sulfuric acid: Take 10mL of sulfuric acid, carefully add it to water, and then dilute it with water to 1200mL;

[0129] (3) Metaphosphoric acid-acetic acid-sulfuric acid solution: Weigh 15g metaphosphoric acid, add 40mL glacial acetic acid and 250mL 0.15mol / L sulfuric acid, stir, let stand overnight to gradually dissolve, and add 0.15mol / L sulfuric acid to 500mL;

[0130] (4) 50% sodium acetate solution: Weigh 500g of sodium acetate (CH3COONa·3H2O) and add water to 1000mL;

[0131] (5) Boric acid-sodium acetate solution: Weigh 3g of boric acid and dissolve it in 100mL of 50% sodium acetate solution. Prepare the solution immediately before use.

[0132] (6) o-phenylenediamine solution: Weigh 20 mg of o-phenylenediamine, dissolve it in water and dilute it to 100 mL before use;

[0133] (7) 0.04% thymol blue indicator solution: Weigh 0.1g of thymol blue, add 0.02mol / L sodium hydroxide solution, grind in a glass mortar until dissolved, the amount of sodium hydroxide used is about 10.75mL, after grinding and dissolving, dilute with water to 250mL; color change range: pH=1.2, red; pH=2.8, yellow; pH>4.0, blue;

[0134] (8) Activation of activated carbon: Add 200g of carbon powder to 1L of 1+9 hydrochloric acid, heat under reflux for 1-2 hours, filter, wash with water until no iron ions are present in the filtrate, dry in an oven at 110-120℃, and set aside for later use.

[0135] II. Solution Preparation

[0136] 1. Sample solution preparation: Weigh 100g of fresh prickly pear embryonic stem cells, add 100g of metaphosphoric acid-acetic acid solution, pour into a cell homogenizer in a biological cell laboratory and homogenize. Use 0.04% thymol blue indicator solution as an indicator, and adjust the pH to 1.2 with metaphosphoric acid-acetic acid solution and metaphosphoric acid-acetic acid-sulfuric acid solution. Take 20g of homogenate, dilute it to 100mL with metaphosphoric acid-acetic acid solution, filter, and the filtrate is the sample solution for later use.

[0137] 2. Ascorbic acid standard solution (1 mg / mL): Weigh 50 mg of ascorbic acid, dissolve it in metaphosphoric acid-acetic acid solution in a 50 mL volumetric flask, and dilute to the mark.

[0138] 3. Ascorbic acid standard working solution (100μg / mL): Take 10mL of ascorbic acid standard solution and dilute it to 100mL with metaphosphoric acid-acetic acid solution; adjust the pH value before making up to volume, and the adjustment method is the same as that used for the standard solution.

[0139] III. Sample Processing

[0140] 1. Take 100 mL of the sample solution and 100 mL of the ascorbic acid standard working solution into a capped Erlenmeyer flask, add 2 g of activated carbon, shake vigorously for 1 min, filter, discard the first few milliliters of filtrate, and collect all the remaining filtrates, namely the sample oxidation solution and the standard oxidation solution, for testing.

[0141] 2. Take 5 mL of standard oxidizing solution into two 50 mL volumetric flasks and label them "Standard" and "Standard Blank" respectively.

[0142] 3. Take 5 mL of the sample oxidation solution into two 50 mL volumetric flasks and label them "Sample" and "Sample Blank" respectively.

[0143] 4. Add 5 mL of boric acid-sodium acetate solution to both the "standard blank" and "sample blank" solutions, mix and shake for 15 min, dilute with water to 50 mL, place in a 4℃ refrigerator for 2 h, and then remove for use.

[0144] 5. Add 5 mL of 50% sodium acetate solution to both the "sample" and "standard" solutions, and dilute with water to 50 mL for later use.

[0145] 6. For the fluorescence reaction, take 2 mL each of the "standard blank" solution and "sample blank" solution from step 4, and the "sample" solution from step 5, and place them in 10 mL capped test tubes respectively. In a dark room, quickly add 5 mL of o-phenylenediamine solution to each tube, shake to mix, and react at room temperature for 35 min. Measure the fluorescence intensity using an excitation wavelength of 338 nm and an emission wavelength of 420 nm. Dilute the "standard" solution from step 5 to a series of concentration gradients, measure its fluorescence intensity in the same way as above, and subtract the fluorescence intensity of the standard blank solution from each. Plot a standard curve or perform relevant calculations with the obtained results as the ordinate and the corresponding ascorbic acid content as the abscissa. The linear regression equation is used for calculations.

[0146] IV. Result Calculation

[0147] The total content of ascorbic acid and dehydroascorbic acid (i.e., vitamin C content) in the sample is calculated using the formula X = (c × V / m) × F × (100 / 1000), where:

[0148] X — Total content of ascorbic acid and dehydroascorbic acid in the sample, unit: mg / 100g;

[0149] c—The concentration of the sample solution obtained from the standard curve or calculated from the regression equation, in μg / mL;

[0150] m—Sample mass, unit: g;

[0151] F—Dilution factor of the sample solution;

[0152] V – The volume of the sample used for the fluorescence reaction, in mL.

[0153] In this experiment, the vitamin C content determination data in the samples of Examples 4-7 and Comparative Examples 13-16 are shown in Table 2 below.

[0154] Table 2. Vitamin C Content Determination Table

[0155]

[0156] As can be seen from the data above, the vitamin C content of the prickly pear embryonic stem cells extracted in Examples 4-7 was 17.78 mg, 23.24 mg, 9.62 mg, and 12.12 mg, respectively, all significantly higher than the vitamin C content of the prickly pear embryonic stem cells in Comparative Examples 13-16. This indicates that adding 2.50–3.75 μg / L salicylic acid to the culture medium using the method of the present invention significantly increases the vitamin C content in the prickly pear embryonic stem cell culture. However, excessively high concentrations of salicylic acid have little effect on inducing vitamin C production from prickly pear embryonic stem cells. Meanwhile, the highest vitamin C content was observed in Example 5, indicating that the inducing agent concentration and culture time used in Example 5 can better induce vitamin C production from prickly pear embryonic stem cells.

[0157] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A culture medium for prickly pear embryonic stem cells, characterized in that, The culture medium includes 1 / 2MS medium, supplemented with NAA at a concentration of 0.05–0.20 mg / L, 6-BA at a concentration of 0.05–0.20 mg / L, and KT at a concentration of 0.5–2.0 mg / L.

2. The culture medium for *Prickly pear* embryonic stem cells according to claim 1, characterized in that, The aforementioned 1 The 2MS medium was supplemented with NAA at a concentration of 0.10 mg / L, 6-BA at a concentration of 0.10 mg / L, and KT at a concentration of 1.0 mg / L.

3. The culture medium for *Rhizopus rosae* embryonic stem cells according to claim 1 or 2, characterized in that, The aforementioned 1 The mass ratio of NAA, 6-BA, and KT added to the 2MS medium is 1:1:

10.

4. A method for culturing prickly pear embryonic stem cells, characterized in that, The following steps are involved: (1) Pretreatment of explants: Take young prickly pear fruits, wash them, cut off the position containing embryonic stem cells at the top of the young fruit, and sterilize the obtained slices. (2) Obtaining embryonic stem cells: Sterilized sections were inoculated onto solid culture medium for culture to generate embryonic stem cell clusters. The embryonic stem cell clusters were then detached from the solid culture medium. The obtained embryonic stem cell clusters were cut into pieces, placed in a culture container, and culture medium was added for suspension culture. The culture medium for suspension culture included... 1 The medium was prepared in 2MS, with NAA at a concentration of 0.05–0.20 mg / L, 6-BA at a concentration of 0.05–0.20 mg / L, and KT at a concentration of 0.5–2.0 mg / L. After culturing, the medium was filtered to obtain a filtrate containing embryonic stem cells. (3) Expanded culture: Dilute the filtrate containing embryonic stem cells obtained in step (2) and transfer it for passage culture; (4) Collection: Centrifuge the embryonic stem cell culture medium obtained by passage culture in step (3), discard the supernatant, and retain the precipitate to obtain prickly pear embryonic stem cells.

5. The method for culturing prickly pear embryonic stem cells according to claim 4, characterized in that, In step (2), the culture temperature is 22℃, the shaking speed is 110r / min, and the culture time is 48h.

6. The method for culturing *Rhizopus rosae* embryonic stem cells according to claim 4 or 5, characterized in that, In step (2), filtration is performed after the culture is completed, including: filtering the embryonic stem cell culture medium after the culture is completed using a No. 2 sieve for the first time, and filtering the resulting filtrate using an 80-mesh sieve for the second time.

7. The method for culturing *Rhizopus rosae* embryonic stem cells according to claim 4 or 5, characterized in that, In step (1), the cleaning includes: first washing the outer surface of the young fruit with dish soap, and then rinsing it with running water; And / or, in step (1), the sterilization process includes: first disinfecting with 75% alcohol by volume, washing with sterile ultrapure water, then disinfecting the slices with a disinfectant with an effective chlorine content of 500 mg / L, and then washing with sterile ultrapure water. And / or, in step (2), after the slices are inoculated onto solid culture medium, they are cultured in the dark at 25°C for 20 days.

8. A method for inducing high vitamin C production from prickly pear embryonic stem cells, characterized in that, Prickly pear embryonic stem cells were seeded into a culture medium and salicylic acid solution was added to make the final concentration of salicylic acid 2.50-3.75 μg / L for induction culture; after a certain period of culture, prickly pear embryonic stem cells with high vitamin C production were obtained.

9. The method for inducing high vitamin C production from prickly pear embryonic stem cells according to claim 8, characterized in that, Using the method for culturing prickly pear embryonic stem cells according to any one of claims 4-7, the obtained prickly pear embryonic stem cells are diluted to a cell concentration of 10. 7 Cell culture medium of cells / mL; Take 200 mL of the cell culture medium, add 0.2–0.3 mL of 5 mg / L salicylic acid solution, and add culture medium to a final volume of 400 mL, then perform induction culture.

10. The method for inducing high vitamin C production from prickly pear embryonic stem cells according to claim 8 or 9, characterized in that, The induction culture was carried out at a temperature of 26℃, a shaking speed of 110 r / min, and a culture time of 48–72 h.

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