Method for increasing glabridin content through hairy root culture

Abstract

The invention discloses a method for improving glabridin content through hairy root culture, which comprises the following steps of: culturing sterile seedlings by glycyrrhiza glabra seeds, cutting out tissues such as cotyledons and hypocotyls of the sterile seedlings, performing hairy root induction by using agrobacterium rhizogenes R1600, performing subculture to obtain hairy roots, and culturing the hairy roots to obtain the glabridin content. An exogenous substrate, namely daidzein, required by glabridin generation is introduced into subsequent subculture, and periodic temperature change control is performed on culture conditions, so that glabridin generation is promoted. The content of glabridin in the hairy roots obtained through the culture method reaches 0.20-0.32% of the dry value of the hairy roots, the time period is short, and the content of glabridin in glycyrrhiza glabra which is planted or grows for 3-5 years in a wild mode can reach all the content of glabridin in glycyrrhiza glabra which is planted or grows for 3-5 years.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for increasing the content of glabridin by hairy root culture. Background Art

[0002] Glabridin is a unique prenylated isoflavone compound in Glycyrrhiza glabra L., and has a wide range of biological properties, such as antioxidant, anti-inflammatory, anti-atherosclerotic, energy metabolism regulation, estrogen regulation, neuroprotection, anti-osteoporosis, whitening, etc. The content of glabridin in the roots of Glycyrrhiza glabra L. is 0.10%-0.25%, and the content in the bark of Glycyrrhiza glabra L. is 0.15-0.35%. Only a small part of Xinjiang in China has wild Glycyrrhiza glabra L., so the raw material source of glabridin in China is very limited. In addition, licorice is a perennial herb, and glabridin, as a secondary metabolite, requires a long growth time to accumulate. Wild Glycyrrhiza glabra L. generally needs more than 5-6 years to have a high content of glabridin.

[0003] At present, the methods for producing glabridin include extraction method, chemical synthesis method, etc. The synthesis method has certain advantages in alleviating the tense situation of Glycyrrhiza glabra L. resources in China. Patent CN118290435A discloses a synthesis method of optically pure glabridin. Using 2,2-dimethyl-2H-chromen-5-ol and (-)-epichlorohydrin as raw materials, a benzodihydropyran structure is obtained through substitution and cyclization, and then glabridin is prepared through sulfonylation, aryl substitution and deprotection in sequence. However, the synthesis method involved in the present invention has not been mass-produced yet. In addition, some other synthesis methods have the disadvantages of high synthesis cost and being not conducive to large-scale production. Therefore, at present, the production of glabridin in China mainly relies on extraction from Glycyrrhiza glabra L.

[0004] Hairy roots possess the characteristics, secondary metabolic pathways, and genetic stability of the parent plant. Moreover, they grow rapidly, and the product content is higher than that of normal plants and suspension-cultured cells. They overcome the dependence on exogenous phytohormones in plant cell culture, enabling the acquisition of high levels of the target secondary metabolites from hairy root cultures. Many scholars and experts in China have conducted a lot of research on this: In the literature "Study on the Stereo Regeneration and Hairy Root Induction of Glycyrrhiza uralensis Fisch.", Lei Cheng et al. established a technical platform for the induction of Glycyrrhiza uralensis Fisch. hairy roots and obtained Glycyrrhiza uralensis Fisch. hairy roots. They systematically studied the factors affecting hairy root induction (different strains, different explants, different bacterial suspension concentrations, different infection times) and found that the strain LBA9402 is easy to induce hairy roots and is a sensitive strain for Glycyrrhiza uralensis Fisch. Infecting the hypocotyls of Glycyrrhiza uralensis Fisch. with a two-fold dilution of the LBA9402 Agrobacterium rhizogenes suspension is optimal at around 5 - 10 minutes. In the literature "Study on the Hairy Root Culture System of Glycyrrhiza uralensis Fisch.", Xie Liqiong et al. used the hairy roots induced from Glycyrrhiza uralensis Fisch. and Glycyrrhiza glabra L. as materials and provided seed sources using different solid and liquid culture methods. They experimentally analyzed the effects of factors such as the source of hairy root seeds, culture method, culture temperature, rotation speed, and aeration volume on the growth of Glycyrrhiza uralensis Fisch. hairy roots, studied the shake-flask culture and 5L fermentation culture of Glycyrrhiza uralensis Fisch. hairy roots, and completed the optimization of the 10L stirred-tank culture conditions. The results showed that under the conditions of a hairy root inoculation amount of 1‰ - 3‰, a culture temperature of 25 °C, a rotation speed of 40 r / min, and an aeration volume of 0.5 vvm, the biomass of Glycyrrhiza uralensis Fisch. hairy roots in 10L stirred-tank culture could increase by 55 - 60 times in 60 days, not lower than that of shake-flask culture. The content of glycyrrhizic acid in Glycyrrhiza uralensis Fisch. hairy roots detected by high-performance liquid chromatography was 0.1%. In the literature "Study on the Induction Culture of Glycyrrhiza uralensis Fisch. Hairy Roots and the Detection of Their Flavonoid Content", Lu Hongyu et al. studied the induction and in vitro culture of Glycyrrhiza uralensis Fisch. hairy roots and the production of their flavonoids. Among different Agrobacterium rhizogenes strains, the A4 strain had the best infection effect, and about 96% of the cotyledon node explants produced hairy roots. Among different explants, the cotyledon node had the highest transformation efficiency, and hairy roots could be produced in only 3 - 4 days. During the liquid culture of hairy roots, when the inoculation amount was 0.3 g and the culture volume was 500 mL, the growth rate was the fastest, and the wet weight of hairy roots increased by 41 times. Hairy roots could produce the medicinal component glycyrrhizoflavone, and the highest flavonoid content in the roots was higher than that of commercial Glycyrrhiza uralensis Fisch., reaching 2.042% of the dry weight, about 4.3 times that of the roots of untransformed plants. The flavonoids in hairy roots were also secreted into the culture medium, with the highest amount being 1.36 mg per 100 mL of the culture medium. In the literature "The Culture of Glycyrrhiza uralensis Fisch. Hairy Roots", Zhang Yinlin et al. induced hairy roots after infecting the hypocotyls or cotyledons of Glycyrrhiza uralensis Fisch. sterile seedlings with the 15834 strain of Agrobacterium rhizogenes. During the 3-week liquid culture period, the proliferation rate of hairy roots was 43.6 - 46.9 times, and they grew well under the 10L rotary flask culture conditions. It was found that the content of glycyrrhizoflavonoids in hairy roots was higher than that of normal root cultures, but the synthesis of glycyrrhizic acid was not detected in the experimental results.In the literature "Production of Glycyrrhizin in Hairy Roots of Glycyrrhiza uralensis Fisch.", Gao Jingxiu found that the growth and development of hairy roots produced by Glycyrrhiza uralensis Fisch. from different sources and the content of glycyrrhizin were different. Chen Shirong et al. [7] induced the cotyledons and hypocotyls of Glycyrrhiza uralensis Fisch. with Agrobacterium rhizogenes strain 15834, and both could induce the production of hairy root clones. In the literature "Transformation of Glycyrrhiza uralensis Fisch. Cotyledons and Hypocotyls by Agrobacterium rhizogenes", Chen Shirong et al. induced the cotyledons and hypocotyls of Glycyrrhiza uralensis Fisch. with Agrobacterium rhizogenes strain 15834, and both could induce the production of hairy root clones. In the literature "Establishment of a Hairy Root Culture System of Glycyrrhiza uralensis Fisch. and Analysis of Chemical Constituents", Du Min et al. induced hairy roots from aseptic seedlings of Glycyrrhiza uralensis Fisch. with Agrobacterium rhizogenes 15834, A4, R1000, 1601, LBA91-8, etc., and detected five common flavonoid compounds such as licorice chalcone in the hairy roots. Patent CN118147219A discloses an efficient induction method for hairy roots of Glycyrrhiza pallidiflora Maxim., and discloses the Glycyrrhiza uralensis Fisch. GurFAZ3 gene and its application in regulating the synthesis of flavonoids in Glycyrrhiza uralensis Fisch. hairy roots.

[0005] However, there is currently no relevant research on increasing the content of glabridin in the hairy roots of Glycyrrhiza glabra L. Summary of the Invention

[0006] In order to solve the problems of the increasing consumption demand for glabridin in cosmetics, health foods, and special medical foods, and the scarcity of wild Glycyrrhiza glabra L. resources in China, as well as the short growth cycle and low glabridin content of artificially cultivated Glycyrrhiza glabra L., the present invention focuses on seeking a way to stimulate the synthesis of glabridin, and for the first time proposes and implements a method for increasing the content of glabridin through hairy root culture. This method combines the basic characteristics of hairy roots with exogenous substrates, the basic substances required for the biosynthesis of glabridin, and periodic high and low temperature control stimulation to achieve the purpose of effectively increasing the content of glabridin in a short time.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] The present invention provides a method for increasing the content of glabridin through hairy root culture. After cultivating aseptic seedlings with seeds, the cotyledons and / or hypocotyl tissues of the aseptic seedlings are intercepted and used for hairy root induction with Agrobacterium rhizogenes to obtain hairy roots with good growth conditions and stability. In the subsequent subculture, daidzein is introduced, and at the same time, periodic temperature change control culture is carried out to obtain the final product.

[0009] In some embodiments, the specific steps of the method for increasing the content of glabridin through hairy root culture are as follows:

[0010] (1) Obtaining explants: Select healthy and plump seeds collected from wild Glycyrrhiza glabra var. glabra, treat them with sulfuric acid for 35 - 45 min, rinse them thoroughly, then treat them with mercuric chloride solution, rinse with sterile water 3 - 5 times, inoculate them onto the MS basal medium, adjust the pH value to 5.8 - 6.0, and culture them in the dark at 25 ± 2 °C for 5 - 7 d. After the seeds germinate, continue to culture until the hypocotyl grows to 2 - 3 cm and the cotyledons unfold to obtain sterile seedlings of Glycyrrhiza glabra var. glabra. Take the hypocotyl and / or cotyledons of the sterile seedlings as the explants of Glycyrrhiza glabra var. glabra;

[0011] (2) Activation of the strain: Culture a single colony of the activated Agrobacterium rhizogenes R1601 on the YEB medium, then transfer it to the YEB liquid medium, and culture it in the dark at 25 ± 1 °C with shaking until the logarithmic growth phase. Dilute the bacterial liquid in the logarithmic growth phase to obtain the activated bacterial liquid;

[0012] (3) Infection and co - culture of explants: Cut the explants of Glycyrrhiza glabra var. glabra in step (1) into small segments or pieces, immerse them in the activated bacterial liquid obtained in step (2) for 10 - 15 min, then blot the excess liquid on the surface with sterile filter paper, and place them on the MS medium for co - culture at 25 ± 1 °C in the dark for 3 - 5 d;

[0013] (4) Induction and culture of hairy roots: Take out the explants cultured in step (3), rinse them with sterile water 3 - 5 times, put them in the MS liquid medium and shake, then transfer them to the MS solid medium for culture. After inducing hairy roots, transfer the hairy roots to the MS solid medium for culture, and subculture every 5 d, repeatedly subculture and sterilize until no sterile spots appear on the medium;

[0014] (5) Inducing and increasing the content of glabridin in hairy roots: Take the hairy roots cultured in step (4), transfer them to the MS liquid medium containing daidzein, and culture them at 25 ± 1 °C, 110 - 120 r / min in the dark for 5 - 7 d, and then carry out periodic variable - temperature culture to obtain the final product.

[0015] In the present invention, the daidzein used is sourced from the leguminous plant Glycine max which is easy to grow, has a high content, and low cost.

[0016] The technical solution of the present invention combines the basic characteristics of hairy roots with the exogenous substrate of the basic substances required for glabridin biosynthesis and the control and stimulation of periodic high - low temperature changes, achieving the purpose of effectively increasing the content of glabridin in a short time.

[0017] In some embodiments, after the seeds germinate in step (1), they are further cultured at 25 ± 2 °C, with a light intensity of 2000 - 4000 Lux and a light time of 15 - 17 h / d for 20 - 25 d until the hypocotyl grows to 2 - 3 cm.

[0018] In some embodiments, the YEB medium in step (2) contains 50-100 mg / L of Kan and 1-2% of agar powder.

[0019] In some embodiments, the OD600 of the bacterial liquid in the logarithmic growth phase in step (2) is 0.8-1.2.

[0020] In some embodiments, the OD600 of the activated bacterial liquid in step (2) is 0.5-0.6, and the pH is 5.8-6.0.

[0021] In some embodiments, the specific operation of step (4) is as follows: Take out the explant cultured in step (3), rinse it 3-5 times with sterile water, place it in an MS liquid medium containing 250-350 mg / L of Kan and shake it for 10-20 min, then transfer it to an MS solid medium containing 250-350 mg / L of Kan for culture. After inducing hairy roots, take the hairy roots and transfer them to an MS solid medium containing 50-100 mg / L of Kan for culture, and subculture every 5 days, and repeatedly subculture to remove bacteria until no bacterial spots appear on the medium.

[0022] In some embodiments, the specific operation of the periodic temperature change in step (5) is as follows: Raise the culture temperature to 28-32 °C and culture for 5-10 days, then culture at 10-15 °C for 3-5 days, continue to raise the temperature to 28-32 °C and culture for 5-10 days, and then culture at 10-15 °C for 3-5 days, and so on for repeated high-temperature and low-temperature periodic cyclic culture.

[0023] In some embodiments, the concentration of daidzein in the MS liquid medium used in step (5) is 5-10 mg / L.

[0024] In some embodiments, the content of glabridin in the finally obtained culture product is ≥0.20%.

[0025] The present invention first utilizes the characteristics of hairy roots having the characteristics, secondary metabolic pathways and genetic stability of the parental plants, and uses daidzein in soybeans as an exogenous substrate supplement for the essential intermediate in the synthesis of glabridin in plants. The introduction of daidzein greatly promotes the corresponding biochemical reactions; similarly, for the first time, the periodic temperature change control stimulation is carried out on the culture of Glycyrrhiza glabra hairy roots, which jointly promotes the biosynthesis of glabridin and greatly promotes the production of glabridin in Glycyrrhiza glabra hairy roots.

[0026] The glabridin content in the hairy roots cultured by the method of the present invention reaches 0.20-0.32% of the dry cell weight, while it takes at least about 3-5 years of growth period for cultivated or wild Glycyrrhiza glabra to reach this content. In comparison, the method for increasing the glabridin content by culturing hairy roots in the present invention has the advantages of short growth cycle, being unrestricted by region, time and climate, and being easy for industrialization, making it possible to industrialize the production of glabridin by co-culturing hairy roots with exogenous substrates.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] The method for increasing the glabridin content by culturing hairy roots in the present invention can significantly increase the glabridin content in a short time, has the advantages of short growth cycle, being unrestricted by region, time and climate, and being easy for industrialization, making it possible to industrialize the production of glabridin by co-culturing hairy roots with exogenous substrates. Detailed implementation manners

[0029] The various exemplary implementation manners of the present invention will be described in detail below. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention description, which are obvious to those skilled in the art. Other implementation manners obtained from the description of the present invention are obvious to those skilled in the art. The description and examples of the present application are only exemplary.

[0032] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0033] The sulfuric acid used in the present invention is of AR grade.

[0034] Example 1

[0035] A method for increasing the content of glabridin by hairy root culture, the specific steps are as follows:

[0036] (1) Obtaining explants: Select plump seeds collected from wild Glycyrrhiza glabra, treat them with sulfuric acid for 40 min, rinse them thoroughly with clean water, then treat them with 0.1% mercuric chloride solution for 10 min, rinse them 3 times with sterile water, inoculate them onto the MS basal medium supplemented with 30 g / L sucrose and 6 g / L agar, adjust the pH value to 5.8, and culture them in the dark at 23 °C for 5 d. After the seeds germinate, transfer them to a light condition with a temperature of 23 °C, a light intensity of 2000 Lux, and a light duration of 15 h / d for 20 d. When the hypocotyl grows to 2 - 3 cm and the cotyledons unfold, sterile seedlings of Glycyrrhiza glabra can be obtained. Take the hypocotyls and cotyledons of the sterile seedlings as explants;

[0037] (2) Activation of the strain: Agrobacterium rhizogenes R1601 is activated 3 times on the YEB medium containing 50 mg / L Kan and 1.5% agar powder. After the strain recovers and forms single colonies, take a single colony and culture it on the YEB medium containing 50 mg / L Kan and 1.5% agar powder for 24 h, and then transfer it to the YEB liquid medium. Culture it under dark conditions at 24 °C and 100 r / min with shaking. Dilute the bacterial liquid in the logarithmic growth phase with an OD600 of 0.8 to an OD600 of 0.5 and a pH of 5.8 with sterile water to obtain the activated bacterial liquid;

[0038] (3) Infection and co - culture of explants: Cut the Glycyrrhiza glabra explants in step (1) into 0.5 cm segments, immerse them in the activated bacterial liquid in step (2) for 10 min, take them out and blot dry the excess bacterial liquid with sterile filter paper, and place them on the MS medium for co - culture at 24 °C under dark conditions for 3 d;

[0039] (4) Induction and culture of hairy roots: Take out the explants in step (3), rinse them 3 times with sterile water, shake them in the MS liquid medium containing 300 mg / L Kan for 10 min, and then transfer them to the MS solid medium containing 300 mg / L Kan for culture. After inducing hairy roots, cut the hairy roots and transfer them to the MS medium with a Kan concentration of 100 mg / L for culture, and subculture them every 5 d. Repeatedly subculture and sterilize until no sterile spots appear on the medium;

[0040] (5) Inducing and increasing the content of glabridin in hairy roots: Take the hairy roots cultured in step (4), transfer them to an MS liquid medium supplemented with 10 mg / L daidzein, and culture them at 24 °C, 110 r / min under dark conditions. After 5 days, perform periodic temperature-changing culture: raise the culture temperature to 32 °C and culture for 10 days, then culture at 10 °C for 5 days, continue to raise the temperature to 32 °C and culture for 10 days, and then culture at 10 °C for 5 days. Repeat this high-temperature and low-temperature periodic cycle culture.

[0041] Detect the content of glabridin in the hairy roots on the 60th day and 90th day respectively after the start of the temperature-changing culture.

[0042] Example 2

[0043] A method for increasing the content of glabridin by culturing hairy roots, the specific implementation method is generally the same as that of Example 1, except for step (5):

[0044] Inducing and increasing the content of glabridin in hairy roots: Take the hairy roots cultured in step (4), transfer them to an MS liquid medium supplemented with 8 mg / L daidzein, and culture them at 25 °C, 115 r / min under dark conditions. After 5 days, perform periodic temperature-changing culture: raise the culture temperature to 32 °C and culture for 10 days, then culture at 10 °C for 5 days, continue to raise the temperature to 32 °C and culture for 10 days, and then culture at 10 °C for 5 days. Repeat this high-temperature and low-temperature periodic cycle culture.

[0045] Detect the content of glabridin in the hairy roots on the 60th day and 90th day respectively after the start of the temperature-changing culture.

[0046] Example 3

[0047] A method for increasing the content of glabridin by culturing hairy roots, the specific implementation method is generally the same as that of Example 1, except for step (5):

[0048] Inducing and increasing the content of glabridin in hairy roots: Take the hairy roots cultured in step (4), transfer them to an MS liquid medium supplemented with 5 mg / L daidzein, and culture them at 26 °C, 120 r / min under dark conditions. After 7 days, perform periodic temperature-changing culture: raise the culture temperature to 32 °C and culture for 10 days, then culture at 10 °C for 5 days, continue to raise the temperature to 32 °C and culture for 10 days, and then culture at 10 °C for 5 days. Repeat this high-temperature and low-temperature periodic cycle culture.

[0049] Detect the content of glabridin in the hairy roots on the 60th day and 90th day respectively after the start of the temperature-changing culture.

[0050] Example 4

[0051] A method for increasing the content of glabridin by hairy root culture, the specific implementation is generally the same as that of Example 1, except for step (5):

[0052] Inducing and increasing the content of glabridin in hairy roots: Take the hairy roots cultured in step (4), transfer them to an MS liquid medium supplemented with 5 mg / L daidzein, and culture them at 25 °C, 115 r / min, and in the dark. After 7 days, perform periodic temperature-changing culture: raise the culture temperature to 30 °C and culture for 10 days, then culture at 12 °C for 5 days, continue to raise the temperature to 32 °C and culture for 10 days, and then culture at 10 °C for 5 days. Repeat the high-temperature and low-temperature periodic cycle culture in this way.

[0053] Detect the content of glabridin in the hairy roots on the 60th day and 90th day after the start of the temperature-changing culture respectively.

[0054] Example 5

[0055] A method for increasing the content of glabridin by hairy root culture, the specific implementation is generally the same as that of Example 1, except for step (5):

[0056] Inducing and increasing the content of glabridin in hairy roots: Take the hairy roots cultured in step (4), transfer them to an MS liquid medium supplemented with 5 mg / L daidzein, and culture them at 25 °C, 115 r / min, and in the dark. After 7 days, perform periodic temperature-changing culture: raise the culture temperature to 28 °C and culture for 10 days, then culture at 15 °C for 5 days, continue to raise the temperature to 28 °C and culture for 10 days, and then culture at 15 °C for 5 days. Repeat the high-temperature and low-temperature periodic cycle culture in this way.

[0057] Detect the content of glabridin in the hairy roots on the 60th day and 90th day after the start of the temperature-changing culture respectively.

[0058] Example 6

[0059] A method for increasing the content of glabridin by hairy root culture, the specific implementation is generally the same as that of Example 1, except for step (5):

[0060] Inducing and increasing the content of glabridin in hairy roots: Take the hairy roots cultured in step (4), transfer them to an MS liquid medium supplemented with 8 mg / L daidzein, and culture them at 25 °C, 115 r / min, and in the dark. After 7 days, perform periodic temperature-changing culture: raise the culture temperature to 32 °C and culture for 5 days, then culture at 10 °C for 3 days, continue to raise the temperature to 32 °C and culture for 5 days, and then culture at 10 °C for 3 days. Repeat the high-temperature and low-temperature periodic cycle culture in this way.

[0061] Detect the content of glabridin in the hairy roots on the 60th day and 90th day after the start of the temperature-changing culture respectively.

[0062] Example 7

[0063] A method for increasing the content of glabridin by hairy root culture, the specific implementation is generally the same as that of Example 1, except for step (5):

[0064] Inducing and increasing the content of glabridin in hairy roots: Take the hairy roots cultured in step (4), transfer them to an MS liquid medium supplemented with 8 mg / L daidzein, and culture them at 25 °C, 115 r / min, and in the dark. After 5 days, perform periodic temperature-changing culture: raise the culture temperature to 30 °C and culture for 10 days, then culture at 12 °C for 5 days, continue to raise the temperature to 30 °C and culture for 10 days, and then culture at 10 °C for 5 days, and so on, repeating the high-temperature and low-temperature periodic cyclic culture.

[0065] Detect the content of glabridin in the hairy roots on the 60th day and 90th day after the start of the temperature-changing culture respectively.

[0066] Control Example 1

[0067] A method for increasing the content of glabridin by hairy root culture, the specific implementation is generally the same as that of Example 1, except for step (5):

[0068] Inducing and increasing the content of glabridin in hairy roots: Take the hairy roots cultured in step (4), transfer them to an MS liquid medium, and culture them at 25 °C, 115 r / min, and in the dark.

[0069] Detect the content of glabridin in the hairy roots on the 60th day and 90th day after the start of the culture respectively.

[0070] Control Example 2

[0071] A method for increasing the content of glabridin by hairy root culture, the specific implementation is generally the same as that of Example 1, except for step (5):

[0072] Inducing and increasing the content of glabridin in hairy roots: Take the hairy roots cultured in step (4), transfer them to an MS liquid medium supplemented with 8 mg / L daidzein, and culture them at 25 °C, 115 r / min, and in the dark. Detect the content of glabridin in the hairy roots on the 60th day and 90th day after the start of the culture respectively.

[0073] Control Example 3

[0074] A method for increasing the content of glabridin by hairy root culture, the specific implementation is generally the same as that of Example 1, except for step (5):

[0075] Inducing and increasing the content of glabridin in hairy roots: Take the hairy roots cultured in step (4), transfer them to MS liquid medium, and culture them at 25°C, 115 r / min, and in the dark. After 5 days, perform periodic temperature variation culture. Raise the culture temperature to 32°C and culture for 10 days, then culture at 10°C for 5 days. Continue to raise the temperature to 32°C and culture for 10 days, and then lower the culture temperature to 10°C and culture for 5 days. Repeat the high-temperature and low-temperature periodic cycle culture in this way.

[0076] Detect the content of glabridin in the hairy roots on the 60th day and 90th day respectively after the start of the temperature variation culture.

[0077] Performance test

[0078] The experimental parameters of each example and control example, and the content of glabridin detected in the hairy roots on the 60th day and 90th day are shown in Table 1.

[0079] Table 1

[0080]

[0081] From the detection results of the glabridin content in the hairy roots of Glycyrrhiza glabra in Example 1 and Control Examples 1-3, it can be seen that the culture of glabridin hairy roots has the characteristics, secondary metabolic pathways and genetic stability of its parent plants. The introduction of exogenous substrate daidzein can effectively promote the synthesis of glabridin, and the stimulating effect of temperature variation control can also effectively increase the content of glabridin.

[0082] Compared with Control Examples 1-3, the coexistence of the introduction of exogenous substrate daidzein and periodic temperature variation control stimulation in Examples 1-5 and 7 can effectively increase the content of glabridin in the hairy roots of Glycyrrhiza glabra.

[0083] It can be seen from Examples 1-3 that a daidzein concentration of 8 mg / L is better than 5 mg / L and 10 mg / L; it can be seen from Examples 3-5 that the temperature variation control of 30-12°C is better than the temperature variation ranges of 32°C-10°C and 28-15°C; it can be seen from Examples 2 and 6 that culturing at high temperature for 10 days and low temperature for 5 days is better than the temperature variation cycle of high temperature for 5 days and low temperature for 3 days.

[0084] The above is only a preferred embodiment of the present invention, and it does not impose any form of limitation on this application. Although this application is disclosed as above with preferred embodiments, it is not used to limit this application. Any person skilled in the art, without departing from the scope of the technical solution of this application, using the technical content disclosed above to make some changes or modifications is equivalent to equivalent implementation cases. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution.

Claims

1. A method for increasing the content of glabridin by hairy root culture, characterized in that: After culturing sterile seedlings with seeds, the cotyledons and / or hypocotyl tissues of the sterile seedlings are cut off and hairy roots are induced using Agrobacterium rhizogenes to obtain hairy roots with good growth conditions and stability. In the subsequent subculture, soybean aglycone is introduced, and periodic temperature-controlled culture is performed to obtain the final product.

2. The method for increasing the content of glabridin by hairy root culture according to claim 1, characterized in that: The specific steps are as follows: (1) Obtaining explants: selecting healthy and full seeds collected from wild Glycyrrhiza glabra, treating them with sulfuric acid for 35-45 minutes, rinsing them, treating them with mercuric chloride solution, rinsing them with sterile water for 3-5 times, inoculating them on MS basal medium, adjusting the pH value to 5.8-6.0, culturing them at 25±2°C in the dark for 5-7 days, culturing them until the hypocotyl grows to 2-3 cm after the seeds germinate, and the cotyledons unfold to obtain sterile seedlings of Glycyrrhiza glabra, and taking the hypocotyl and / or cotyledon of the sterile seedlings as Glycyrrhiza glabra explants; (2) Activation of bacterial strains: A single colony of the activated Agrobacterium rhizogenes R1601 was inoculated on a YEB medium for culture, and then transferred to a YEB liquid medium for culture in a dark environment at 25±1°C with shaking until the logarithmic growth phase. The bacterial solution in the logarithmic growth phase was diluted to obtain an activated bacterial solution; (3) Explant infection and co-cultivation: Cut the Glycyrrhiza glabra explants obtained in step (1) into small pieces or fragments, immerse them in the activated bacterial solution obtained in step (2) for 10-15 minutes, dry the excess liquid on the surface with sterile filter paper, and place them on MS culture medium for co-cultivation at 25±1°C in the dark for 3-5 days; (4) Induction and cultivation of hairy roots: Take out the explants after cultivation in step (3), rinse them with sterile water for 3-5 times, put them into MS liquid culture medium and shake them, then transfer them to MS solid culture medium for cultivation. After the hairy roots are induced, take out the hairy roots and transfer them to MS solid culture medium for cultivation. Subculture them every 5 days, and sterilize them repeatedly until no bacterial plaque appears on the culture medium. (5) Inducing and increasing the content of glabridin in hairy roots: The hairy roots cultured in step (4) are transferred to MS liquid culture medium containing daidzein, cultured at 25±1°C, 110-120 r / min, and dark conditions for 5-7 days, and then cultured periodically at variable temperatures to obtain the final product.

3. The method for increasing the content of glabridin by hairy root culture according to claim 2, characterized in that: After the seeds germinate in step (1), they are cultured for 20-25 days at a temperature of 25±2° C., a light intensity of 2000-4000 Lux, and a light duration of 15-17 h / d, until the hypocotyl grows to 2-3 cm.

4. The method for increasing the content of glabridin by hairy root culture according to claim 2, characterized in that: In the step (2), the YEB culture medium contains 50-100 mg / L of Kan and 1-2% of agar powder.

5. The method for increasing the content of glabridin by hairy root culture according to claim 2, characterized in that: The OD600 of the bacterial solution in the logarithmic growth phase in step (2) is 0.8-1.

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6. The method for increasing the content of glabridin by hairy root culture according to claim 2, characterized in that: The specific operation of the periodic temperature change in step (5) is: raising the culture temperature to 28-32°C for 5-10 days, then culturing at 10-15°C for 3-5 days, continuing to raise the temperature to 28-32°C for 5-10 days, then culturing at 10-15°C for 3-5 days, and repeating the high temperature and low temperature periodic cycle culture.

7. The method for increasing the content of glabridin by hairy root culture according to claim 2, characterized in that: The concentration of daidzein in the MS liquid culture medium used in step (5) is 5-10 mg / L.

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