A method for callus induction and large-scale suspension culture of high-flavonoid-producing alpine edelweiss

Abstract

The present invention discloses a method for inducing callus tissue of high-flavonoid-yielding alpine edelweiss and culturing it in reactor suspension. The present invention mainly aims at the technical defects of the prior art, and provides a method for stably obtaining callus tissue of high-flavonoid-yielding alpine edelweiss and conducting large-scale reactor culture, so as to solve the problem that there is currently no alpine edelweiss cell line with high flavonoid content. The present invention establishes a method for inducing callus of alpine edelweiss by conducting a large number of screening and comparison of basic elements and hormones in the culture medium and exploring other experimental conditions, inducing callus tissue with the stems and leaves of sterile seedlings, and establishing a tissue culture scheme for culturing alpine edelweiss cell lines that can produce high-flavonoid compounds, and can use disposable bioreactors to amplify step by step to mass-produce high-flavonoid-yielding alpine edelweiss cells.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture and relates to a method for inducing callus and large-scale suspension culture of high-flavonoid-yielding alpine edelweiss. Background Art

[0002] Alpine edelweiss (Leontopodium alpinum) is a plant of the genus Edelweiss in the Asteraceae family, also known as "Edelweiss", a perennial herb that grows mostly in limestone soil, gravel, etc. at altitudes of 1,000 to 3,400 meters. It is a native plant in Europe and is even known as the national flower of Switzerland.

[0003] There are many chemical components in the alpine edelweiss plant. So far, terpenes, phenylpropanoids (phenolic acid, flavonoids, coumarins, lignans), fatty acids and polyacetylenes have been isolated. Alpine edelweiss has good pharmacological activity in the cardiovascular and nervous systems, and also has anti-inflammatory, antibacterial, antioxidant, diuretic and chemical control effects. Therefore, alpine edelweiss is widely used in food, medicine and cosmetics. However, due to excessive mining, the number of alpine edelweiss has decreased sharply, and it has been listed as an endangered species by the International Union for Conservation of Nature. Many countries such as Austria, Germany, Italy and Liechtenstein have introduced laws to protect wild alpine edelweiss. Alpine edelweiss has almost no synthesis of flavonoids and other substances in the early growth stage. After flowering, various metabolites begin to accumulate until the peak flowering period. The flowering time of wild alpine edelweiss is different (throughout the growing season of edelweiss). In order to facilitate the collection of growers and purchasers, the domesticated artificially cultivated varieties can bloom in the same time period. After being raised in seedbeds, alpine edelweiss is cultivated by growers in the Swiss Alps until it matures. However, due to the steep mountain walls, mechanical harvesting is impossible, and the scale of alpine edelweiss cultivation is limited. Through plant cell culture technology, not only can the wild resources of alpine edelweiss be protected from destruction, but also alpine edelweiss can be produced on a large scale without being restricted by natural conditions such as seasons, climate, and geographical location to meet market demand.

[0004] Plant tissue culture technology, specifically involving alpine edelweiss, uses the stems and leaves of sterile seedlings as explants to induce cell dedifferentiation to form callus tissue, and uses suspension culture and large-scale culture of callus tissue to efficiently cultivate alpine edelweiss cells with high flavonoid production.

[0005] There is no patent on the tissue culture of alpine edelweiss, the tissue culture system is not yet mature, and there are no public literature reports on the induction of alpine edelweiss callus and large-scale suspension culture. At present, the research on alpine edelweiss is mostly focused on edelweiss acid, and there is no report on the establishment and culture method of alpine edelweiss cell line with high production of flavonoid compounds. Summary of the invention

[0006] This patent aims to address the technical defects of the prior art and provide a method for stably obtaining high-flavonoid callus tissue of alpine edelweiss and conducting large-scale culture in a reactor, so as to solve the problem that there is currently no alpine edelweiss cell line with high flavonoid content. The present invention has established a method for inducing alpine edelweiss callus by conducting a large number of screening and comparisons of basic elements and hormones in the culture medium and exploring other experimental conditions, inducing callus tissue with the stems and leaves of sterile seedlings, and establishing an induction and tissue culture scheme for an alpine edelweiss cell line with high flavonoid production, and can use disposable bioreactors to scale up step by step to mass-produce high-flavonoid-producing alpine edelweiss cells.

[0007] To achieve the above purpose, the present invention discloses the following technical contents:

[0008] A method for inducing callus tissue of high-flavonoid-yielding alpine edelweiss and culturing it in a reactor scale suspension culture, characterized by the following steps:

[0009] (1) Disinfection of alpine edelweiss seeds: Wash the alpine edelweiss seeds with tap water to remove dust, disinfect with 75% ethanol for 30-60 seconds, disinfect with 4% sodium hypochlorite solution for 8-10 minutes, wash with sterile water 4 times, place on the basic culture medium BM, culture at 20-25 ℃, light intensity 500-1000 lux. It will germinate in 7-21 days; the basic culture medium BM refers to: 1 / 2MS or MS + 30 g / L sucrose, agar 0.7-1.0%, pH 5.8-6.0;

[0010] (2) Callus induction of alpine edelweiss: Cut the sterile seedling leaves (or stems) into square pieces (or stem segments) with a side length of 0.5-1 cm according to aseptic operation, place them in callus induction medium CIM, culture at 20-25 ℃, light intensity <1000 lux, and yellow loose callus will form at the edge of the cut after 14-21 days. Divide the callus into small pieces of about 0.5 cm in size, place them in callus subculture medium CM1, culture at 20-25 ℃, light intensity <1000 lux, and subculture every 10-15 days.

[0011] The callus induction medium CIM refers to: B5 + 2-4 mg / L 2,4-D + 30 g / L sucrose + 0.1 mg / L L-glutamine + 0.2 mg / L hydrolyzed milk protein or B5 + 2-4 mg / L 2,4-D + 0.1-0.5 mg / L 6-BA + 30 g / L sucrose + 0.1 mg / L L-glutamine + 0.2 mg / L hydrolyzed milk protein, agar 0.7-1.0%, pH 5.8-6.0;

[0012] The callus subculture medium CM1 refers to: B5 + 1-2.5 mg / L 2,4-D + 30 g / L sucrose + 0.1 mg / L L-glutamine + 0.2 mg / L hydrolyzed milk protein, agar 0.7-1.0%, pH 5.8-6.0;

[0013] (3) Suspension culture of alpine edelweiss callus: Select alpine edelweiss callus with loose texture, light yellow to yellow color, and fast growth rate, inoculate it into suspension culture medium CM2 at a ratio of 0.1-1 g / 10 mL, place it in a shaker at 100-115 rpm, maintain the temperature at 20-25 °C, and subculture it every 7-10 days at a ratio of 5-15 g / 100 mL (wet weight). Growth rate 2-3 times / 7 d; the suspension medium CM2 refers to B5 + 1-2 mg / L 2,4-D + 30 g / L sucrose + 0.1 mg / L L-glutamine + 0.2 mg / L hydrolyzed milk protein or B5 + 1-2 mg / L 2,4-D + 0.1-0.2 mg / L 6-BA +30 g / L sucrose + 0.1 mg / L L-glutamine + 0.2 mg / L hydrolyzed milk protein, pH 5.8-6.0;

[0014] (4) Disposable bioreactor culture of alpine edelweiss callus: Select suspension culture materials that have been cultured for 7 days and are in the same state, and inoculate 1500-2500 mL of culture and 2500-3500 mL of sterilized suspension culture medium CM2 into a 5 L disposable bioreactor to make the final volume 5000 mL. The vibration frequency is 9.5 times / min, and the air flow rate is 0.1 m³ / h. Cultivate for 7 days. When subculturing, inoculate the culture into a new disposable bioreactor, add 2-4 times the volume of suspension culture medium CM2, and oscillate and aerate. The oscillation frequency ranges from 2-15 times / min, the ventilation volume is 0.05-0.3 m³ / h, and the vibration frequency of the reactor during the culture process is 5-20 times / min. The culture is carried out in the bioreactor disclosed in the Chinese patent literature with publication number CN103224882A or CN204385208U, and the vibration frequency of the reactor during the culture process is 5-20 times / min.

[0015] The present invention further discloses the application of the reactor-scale suspension culture method in rapidly obtaining high-yield alpine edelweiss, wherein the flavonoid content is ≥9%. Experimental results show that the scheme of the present invention can efficiently obtain alpine edelweiss tissue culture rich in flavonoid compounds (total flavonoids ≥9%), and the culture strategy of the present invention can enable the alpine edelweiss cell line to maintain stable growth and expression of flavonoid secondary metabolites at a scale of 0.1-200L.

[0016] The present invention is described in more detail as follows:

[0017] The key points of the high-flavonoid-yielding alpine edelweiss callus induction and reactor-scale suspension culture method disclosed in the present invention are different from the existing schemes in that:

[0018] At present, there are no reports on the induction and large-scale cultivation methods of alpine edelweiss callus in the world. The flavonoid content of alpine edelweiss and its tissue culture reported so far is extremely low (≤1.5%). This patent obtains a callus induction scheme for alpine edelweiss, which can obtain well-dedifferentiated callus (total flavonoids ≥9%). Under the culture conditions described in this patent, it is possible to use a disposable bioreactor to cultivate high-yield flavonoid callus on a large scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure is a process flow chart of the callus induction and large-scale suspension culture method of high-flavonoid-yielding alpine edelweiss.

[0020] Figure 2 Figure 1: Diagram of the callus induction process of alpine edelweiss;

[0021] Figure 3 This is the growth curve of suspension culture of alpine edelweiss. DETAILED DESCRIPTION

[0022] The present invention is described below through specific implementation schemes. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the implementation scheme should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these implementation schemes, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention. Alpine edelweiss seeds are commercially available, and the raw materials and reagents used in the present invention are commercially available.

[0023] Example 1: Aseptic seedling culture and callus induction of alpine edelweiss

[0024] Seed germination of alpine edelweiss: wash the alpine edelweiss seeds with tap water to remove dust, disinfect them with 75% ethanol for 30 s, wash them three times with sterile water; disinfect them with 4% sodium hypochlorite solution for 10 min, wash them four times with sterile water, place them on culture medium BM (1 / 2MS + 30 g / L sucrose, 0.7% agar, pH 5.8), culture them at 25 °C, light intensity 1000 lux; after 17 days of culture, the seeds germinated and sterile seedlings of alpine edelweiss were obtained.

[0025] Callus induction of alpine edelweiss: Aseptic seedlings of alpine edelweiss were grown to about 3 cm in height. The leaves (or stems) were cut into square pieces (or stem segments) with a side length of 0.5-1 cm according to sterile operation and placed in callus induction medium CIM (B5 + 4 mg / L 2,4-D + 0.5 mg / L 6-BA + 30 g / L sucrose + 0.1 mg / L L-glutamine + 0.2 mg / L hydrolyzed milk protein, agar 0.7%, pH 5.8); cultured at 25 ℃, light intensity <1000 lux, and yellow loose callus tissue was formed on the edge of the cut after 21 days of culture; the callus tissue was divided into small pieces of about 0.5 cm and placed in callus subculture medium CM1 (B5 + 2.0 mg / L 2,4-D + 30 g / L sucrose + 0.1 mg / L L-glutamine + 0.2 mg / L Hydrolyzed milk protein, agar 0.7%, pH 5.8); cultured at 25 ℃, light intensity <1000 lux, subcultured every 15 days.

[0026] The 10th generation solid callus tissue was collected and dried at 65 °C. The total flavonoid content was detected as follows: the dried alpine edelweiss cells were ground and crushed, and the dry powder was obtained by 40 mesh sieve; 0.1000 g of the dry powder was weighed in a 50 mL centrifuge tube, 25 mL of 80% ethanol was added, and the suspension was vortexed; ultrasonic cleaning was performed for 30 min (power 500 W, temperature 60 °C); centrifuged at 5000 rpm / min for 10 min, and the supernatant was used for total flavonoid content determination. Take 6 mL of the solution to be tested, place it in a 25 mL volumetric flask, add 1 mL of 5% sodium nitrite solution, shake well, and place it for 6 minutes, add 1 mL of 10% aluminum nitrate solution, shake well, and place it for 6 minutes, add 10 mL of 4% sodium hydroxide test solution, add pure water to the scale, shake well, and place it for 15 minutes, and use the corresponding reagent as a blank, and measure the absorbance at a wavelength of 500 nm according to the spectrophotometric method (Appendix IV A of Part II of the Chinese Pharmacopoeia 2010). Using rutin as the reference substance, the linear regression equation y=kx+b parameters were calculated based on the absorbance and its corresponding mass. The total flavonoid content (in terms of rutin) of the sample was calculated using the formula C=(Ab)×V0 / (k×m×V1), where: C--the total flavonoid content in the sample, mg / g; A--the absorbance value of the sample; k, b--the slope and intercept of the standard curve; m--the sample weight; V0--the total volume of the sample extract after constant volume; V1--the volume of the sample for measurement. The test results are shown in Table 1

[0027] Table 1 Flavonoid content of solid callus of Edelweiss alpine

[0028]

[0029] Example 2: Suspension culture and growth curve of alpine edelweiss

[0030] Select the callus tissue of Alpine Leontopodium with loose texture, light yellow to yellow color and fast growth rate, inoculate it into suspension medium CM2 (B5 + 2.0 mg / L 2,4-D + 30 g / L sucrose + 0.1 mg / L L-glutamine + 0.2 mg / L hydrolyzed milk protein, pH 5.8) at a ratio of 1 g / 10 mL, and culture it in a shaker at 115 rpm / min and keep the temperature at 25 ℃; when the biomass reaches 12 g, inoculate it into a 500 mL shake flask (medium volume is 100 mL) at a ratio of 12 g / 100 mL (wet weight), and subculture it every 7 days. After 5 generations of subculture, a stable Alpine Leontopodium suspension cell line was obtained;

[0031] Prepare suspension culture medium CM2, and dispense into 500 ml triangular shake flasks with a liquid volume of 100 ml; use the above suspension cell line as the seed source, inoculate 15 g of alpine edelweiss cells in each bottle (filter the wet cells of the culture medium with a sieve), and culture at 25 ℃ and 106 rpm / min; take samples from each bottle on the 0th, 2nd, 4th, 6th, 8th, 10th, and 11th day of culture to test the wet weight, fresh weight, dry weight of cells in each bottle, as well as the pH, conductivity, refractive index and other indicators of the culture medium, and take 3 bottles each time, take the average value to make the following curve, indicating that the biomass of alpine edelweiss cells can reach about 3 times in one growth cycle (8 days), and one production cycle should be set at 8-10 days:

[0032] Table 2 Growth of alpine edelweiss in suspension culture

[0033]

[0034] The samples were collected on the 8th and 10th days and dried at 65°C. The test results are shown in Table 3 (the test method is the same as in Example 1).

[0035] Table 3 Flavonoid content in suspension culture cells of Edelweiss

[0036]

[0037] Example 3 5-200L expansion culture of alpine edelweiss

[0038] 5-L disposable bioreactor culture: After 7 generations of shake flask culture, 1500 mL of suspension culture and 2000 mL of sterilized suspension culture medium CM2 were inoculated into a 5-L disposable bioreactor, and culture was carried out in a bioreactor disclosed in Chinese patent literature with publication number CN103224882A or CN204385208U, with a vibration frequency of 9.5 times / min and an air flow of 0.1 m³ / h for 7 days.

[0039] Subculture in a 20-liter disposable bioreactor: 5000 mL of suspension culture was inoculated into a 20-liter disposable bioreactor, and 7000 mL of sterilized suspension culture medium CM2 was added. Culture was performed in a bioreactor disclosed in Chinese patent literature with publication number CN103224882A or CN204385208U, with a vibration frequency of 9.5 times / min and an air flow of 0.2 m³ / h for 7 days.

[0040] Subculture in a 100-200 L disposable reactor: 40 L of suspension culture was inoculated into a 100-200 L disposable bioreactor, and 60 L of sterilized suspension culture medium CM2 was added. The culture was carried out in a bioreactor disclosed in the Chinese patent literature with publication number CN103224882A or CN204385208U, with a vibration frequency of 5.5 times / min, air flow of 0.3 m³ / h, and oxygen flow (95% purity) of 0.06 m³ / h. After culturing for 10 days, 10.35 kg of fresh cells were harvested, and part of the fresh cells were dried at 65 ° C. The flavonoid content was detected to be 11.20% (the detection method was the same as in Example 1).

[0041] Example 4 Comparative Experiment

[0042] Literature reports that the flavonoid content in alpine edelweiss plants and tissue cultures is relatively low, less than 1.5%. The flavonoid content of the alpine edelweiss callus obtained by the method of the present invention is ≥9%.

[0043]

[0044] Conclusion: The method of the present invention can efficiently obtain alpine edelweiss tissue culture rich in flavonoids (total flavonoids ≥ 9%). The culture strategy described in this patent can enable the alpine edelweiss cell line to maintain stable growth and expression of flavonoid secondary metabolites at a scale of 0.1-200 L.

Claims

1. A method for inducing callus tissue and reactor suspension culture of high-flavonoid-producing alpine edelweiss, Features Proceed as follows: (1) Disinfection of alpine edelweiss seeds: Wash the alpine edelweiss seeds with tap water to remove dust, disinfect them with 75% ethanol for 30-60 seconds, disinfect them with 4% sodium hypochlorite solution for 8-10 minutes, wash them with sterile water 4 times, place them on the basic culture medium BM, culture them at 20-25℃, and light intensity of 500-1000 lux; they can germinate in 7-21 days; the basic culture medium BM refers to: 1 / 2MS + 30 g / L sucrose, 0.7-1.0% agar, pH 5.8-6.0; (2) Callus induction of alpine edelweiss: Take the leaves or stems of sterile seedlings and cut them into square pieces or stem segments with a side length of 0.5-1 cm. Place them in callus induction medium CIM and culture them at 20-25 ℃ with a light intensity of <1000 lux. After 14-21 days, loose yellow callus will form at the edge of the cut. Divide the callus into small pieces of about 0.5 cm and place them in callus subculture medium CM1 at 20-25 ℃ with a light intensity of <1000 lux. Subculture them every 10-15 days. The callus induction medium CIM refers to: B5 + 2-4 mg / L 2,4-D + 0.1-0.5 mg / L 6-BA + 30g / L sucrose + 0.1 mg / L L-glutamine + 0.2 mg / L hydrolyzed milk protein, agar 0.7-1.0%, pH 5.8-6.0; The callus subculture medium CM1 refers to: B5 + 1-2.5 mg / L 2,4-D + 30 g / L sucrose + 0.1 mg / L L-glutamine + 0.2 mg / L hydrolyzed milk protein, agar 0.7-1.0%, pH 5.8-6.0; (3) Suspension culture of alpine edelweiss callus: Select alpine edelweiss callus with loose texture, light yellow to yellow color, and fast growth rate, inoculate it into suspension culture medium CM2 at a ratio of 1 g / 10 mL, place it in a shaker at 100-115 rpm, maintain the temperature at 20-25 °C, and subculture once every 7 days at a ratio of 5-15 g / 100 mL wet weight; the growth rate is 2-3 times / 7 days; The suspension medium CM2 refers to B5 + 1-2 mg / L 2,4-D + 30 g / L sucrose + 0.1 mg / L L-glutamine + 0.2 mg / L hydrolyzed milk protein, pH 5.8-6.0; (4) Disposable bioreactor culture of callus tissue of alpine edelweiss: Select suspension culture materials that have been cultured for 7 days and are in the same state. Inoculate 1500-2500 mL of culture and 2500-3500 mL of sterilized suspension culture medium CM2 into a 5 L disposable bioreactor to make the final volume 5000 mL. The vibration frequency is 9.5 times / min and the air flow rate is 0.1 m³ / h. Cultivate for 7 days. When subculturing, inoculate the culture into a new disposable bioreactor, add 2-4 times the volume of suspension culture medium CM2, and culture with shaking and ventilation. The flavonoid content of the alpine edelweiss callus tissue induced in step (2) and cultured in steps (3) and (4) is ≥ 9%.

2. Application of the cultivation method according to claim 1 in rapidly obtaining a high-yield alpine edelweiss culture of flavonoids, wherein the flavonoid content is ≥9%.

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