A method for increasing the content of glabrene by hairy root culture

Abstract

The application discloses a method for improving the content of glabridin by hairy root culture, and the method comprises the following steps: culturing seeds of Glycyrrhiza glabra to obtain aseptic seedlings, cutting cotyledon and hypocotyl of the aseptic seedlings, and inducing the tissues to generate hairy roots by Agrobacterium rhizogenes R1600; after the hairy roots are obtained by subculture, an exogenous substrate, daidzein, required for glabridin generation is introduced in the subsequent subculture, and the culture condition is periodically controlled by temperature change, so that the generation of glabridin is promoted. The content of glabridin in the hairy roots obtained by the culture method reaches 0.20-0.32% of the dry weight of the hairy roots, the time period is short, and the content of glabridin of the Glycyrrhiza glabra which is planted or wild for 3-5 years can be reached.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for increasing the content of glycyrrhizin through hairy root culture. Background Technology

[0002] Glabrdin is an isopentenyl isoflavone compound unique to *Glycyrrhiza glabra* L., possessing a wide range of biological properties, such as antioxidant, anti-inflammatory, anti-atherosclerotic, energy metabolism regulator, estrogen regulator, neuroprotective, anti-osteoporosis, and skin whitening effects. The content of glabrdin in *Glycyrrhiza glabra* roots is 0.10%-0.25%, and in the bark it is 0.15%-0.35%. The raw material sources of glabrdin in my country are very limited. Furthermore, licorice is a perennial herb, and glabrdin, as a secondary metabolite, requires a long growth period to accumulate; wild *Glycyrrhiza glabra* generally requires 5-6 years or more to achieve a high glabrdin content.

[0003] Currently, methods for producing glycyrrhizin include extraction and chemical synthesis. Synthetic methods offer certain advantages in alleviating the shortage of *Glycyrrhiza glabra* resources in my country. Patent CN118290435A discloses a method for synthesizing optically pure glycyrrhizin, using 2,2-dimethyl-2H-chromen-5-ol and levo-epoxychloropropane as raw materials. Through substitution and cyclization, a benzodihydropyran structure is obtained, followed by sulfonation, aryl substitution, and deprotection to obtain glycyrrhizin. However, the synthetic method described in this invention has not yet been mass-produced. Furthermore, some other synthetic methods suffer from high synthesis costs, hindering large-scale production. Therefore, the current production of glycyrrhizin in my country mainly relies on extraction from *Glycyrrhiza glabra*.

[0004] Hairy roots possess the characteristics, secondary metabolic pathways, and genetic stability of the parent plants. They also grow rapidly and produce higher levels of products than normal plants and suspension culture cells, overcoming the dependence on exogenous plant hormones in plant cell culture. This allows for the extraction of high levels of target secondary metabolites from hairy root cultures. Many scholars and experts in my country have conducted extensive research on this topic. In the literature "Three-Dimensional Regeneration and Hairy Root Induction Research of Licorice," Lei Cheng et al. established a technical platform for hairy root induction in licorice and obtained hairy roots. They systematically studied the factors affecting hairy root induction (different strains, different explants, different bacterial suspension concentrations, and different infection times), finding that strain LBA9402 easily induces hairy roots and is a sensitive strain for Ural licorice. Infecting Ural hypocotyls with a two-fold dilution of LBA9402 Agrobacterium rhizogenes suspension yielded optimal results in approximately 5-10 minutes. In the literature "Research on Licorice Hairy Root Culture System", Xie Liqiong et al. used hairy roots induced by Ural licorice and Glycyrrhiza glabra as materials, and employed different solid and liquid culture methods to provide seed sources. The experiments analyzed the effects of seed source, culture method, culture temperature, rotation speed, and aeration on the growth of licorice hairy roots. They studied shake-flask culture, 5L fermentation culture, and optimized 10L stirred-fluid culture conditions. The results showed that under the conditions of hairy root inoculum of 1‰-3‰, culture temperature of 25℃, rotation speed of 40r / min, and aeration rate of 0.5vvm, the biomass of licorice hairy roots cultured in 10L stirred-fluid culture could increase 55-60 times in 60 days, which was no less than that of shake-flask culture. High-performance liquid chromatography (HPLC) detected a glycyrrhizic acid content of 0.1% in licorice hairy roots. In the literature "Study on the Induction Culture and Flavonoid Content Detection of Licorice Hairy Roots", Lu Hongyu et al. studied the induction and in vitro culture of licorice (Glycyrrhiza uralensis) hairy roots and the production of flavonoids. The results showed that among different Agrobacterium rhizogenes strains, strain A4 had the best infection effect, with approximately 96% of cotyledonary explants producing hairy roots. Among different explants, cotyledonary nodes showed the highest transformation efficiency, with hairy root production occurring in just 3-4 days. During liquid culture of hairy roots, the fastest growth rate was observed at an inoculum size of 0.3 g and a culture volume of 500 mL, with the hairy root wet weight increasing 41-fold. Hairy roots can produce the medicinal component glycyrrhizin, with the highest flavonoid content in the roots exceeding that of commercial licorice, reaching 2.042% of the dry weight, approximately 4.3 times that of untransformed plant roots. Flavonoids from the hairy roots were also secreted into the culture medium, with a maximum amount of 1.36 mg per 100 mL of culture medium. In the literature "Cultivation of Hairy Roots of Licorice", Zhang Yinlin et al. induced hairy roots by infecting the hypocotyl or cotyledons of sterile licorice seedlings with Agrobacterium rhizogenes strain 15834. During 3 weeks of liquid culture, the hairy root proliferation rate was 43.6-46.9 times, and the roots grew well under 10L roller bottle culture conditions. It was found that the content of licorice flavonoids in the hairy roots was higher than that in 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 licorice”, Gao Jingxiu found that the growth and development of hairy roots and the content of glycyrrhizin were different from those of licorice from different sources. Chen Shirong et al. [7] induced hairy root clones in licorice cotyledons and hypocotyls using Agrobacterium tumefaciens strain 15834. In the literature “Transformation of licorice cotyledons and hypocotyls by Agrobacterium tumefaciens”, Chen Shirong et al. induced hairy root clones in licorice cotyledons and hypocotyls using Agrobacterium tumefaciens strain 15834. In the literature “Establishment of hairy root culture system and analysis of chemical composition of licorice”, Du Min et al. induced hairy roots in sterile seedlings of Ural licorice using Agrobacterium tumefaciens strains 15834, A4, R1000, 1601, LBA91-8, etc., and detected five common flavonoids such as glycyrrhizin chalcone in the hairy roots. Patent CN118147219A discloses an efficient method for inducing hairy roots of Glycyrrhiza uralensis, and discloses the GurFAZ3 gene of Glycyrrhiza uralensis and its application in regulating the synthesis of flavonoids in hairy roots of Glycyrrhiza uralensis.

[0005] However, there are currently no studies on increasing the glycyrrhizin content in the hairy roots of Glycyrrhiza glabra. Summary of the Invention

[0006] To address the growing consumer demand for glycyrrhizin in cosmetics, health foods, and special medical foods, as well as the scarcity of wild licorice root resources in my country and the short growth cycle and low glycyrrhizin content of artificially cultivated licorice root, this invention focuses on finding a way to stimulate glycyrrhizin synthesis. It proposes and implements for the first time a method to increase glycyrrhizin content through hairy root culture. This method combines the basic characteristics of hairy roots with exogenous substrates required for glycyrrhizin biosynthesis and periodic high and low temperature control stimulation, achieving an effective increase in glycyrrhizin content within a short period.

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

[0008] This invention provides a method for increasing the content of glycyrrhizin through hairy root culture. After culturing sterile seedlings from 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 and stable growth. In subsequent subculture, daidzein is introduced and the final product is obtained by periodic temperature-controlled culture.

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

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

[0011] (2) Activation of bacterial strain: The activated Agrobacterium rhizogenes R1601 single colony was cultured on YEB medium, and then transferred to YEB liquid medium. It was cultured in the dark, at 25±1℃ and with shaking until the logarithmic growth phase. The bacterial solution in the logarithmic growth phase was diluted to obtain the activated bacterial solution.

[0012] (3) Explant inoculation and co-culture: Cut the licorice explants from step (1) into small segments or pieces, immerse them in the activated bacterial solution obtained in step (2) for 10-15 min, then blot off the excess liquid on the surface with sterile filter paper, and place them on MS medium for co-culture at 25±1℃ in the dark for 3-5 days.

[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 MS liquid medium and shake them, then transfer them to MS solid medium for culture. After the hairy roots are induced, take the hairy roots and transfer them to MS solid medium for culture. Subculture once every 5 days, and repeat the subculture to remove bacteria until sterile plaques appear on the medium.

[0014] (5) Inducing the increase of glycyrrhizin content in hairy roots: Take the hairy roots cultured in step (4), transfer them to MS liquid medium containing daidzein, and culture them at 25±1℃, 110-120r / min and in the dark for 5-7 days. Then, carry out periodic temperature variation culture to obtain the final product.

[0015] The daidzein used in this invention is derived from soybean, a legume that is easy to grow, has a high content of daidzein, and is low in cost.

[0016] The technical solution of this invention combines the basic characteristics of hairy roots with exogenous substrates required for the biosynthesis of glycyrrhizin and periodic high and low temperature control stimulation to achieve the goal of effectively increasing the content of glycyrrhizin in a short period of time.

[0017] In some embodiments, after the seeds germinate in step (1), they are cultured for 20-25 days under conditions of temperature 25±2℃, light intensity 2000-4000Lux, and light duration 15-17h / d until the hypocotyl reaches 2-3cm in length.

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

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

[0020] In some embodiments, the OD600 of the activated bacterial solution 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 explants cultured in step (3), rinse them with sterile water 3-5 times, place them in MS liquid medium containing 250-350 mg / L Kan and shake for 10-20 min, then transfer them to MS solid medium containing 250-350 mg / L Kan and culture them until hairy roots are induced. Then take the hairy roots and transfer them to MS solid medium containing 50-100 mg / L Kan and culture them. Subculture them every 5 days and repeat the subculture to remove bacteria until sterile plaques 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℃ and culture for 5-10 days, then culture at 10-15℃ for 3-5 days, continue to raise the temperature to 28-32℃ and culture for 5-10 days, then culture at 10-15℃ for 3-5 days, and so on, repeating the high temperature and low temperature periodic culture.

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

[0024] In some embodiments, the glycyrrhizin content in the final product obtained from the culture is ≥0.20%.

[0025] This invention is the first to utilize the characteristics of hairy roots, secondary metabolic pathways, and genetic stability of parent plants, and uses daidzein from soybean as an exogenous substrate to supplement the essential intermediate for the synthesis of glycyrrhizin in plants. The introduction of daidzein greatly promotes the corresponding biochemical reactions. Also, for the first time, the culture of licorice hairy roots is subjected to periodic temperature-controlled stimulation to jointly promote the biosynthesis of glycyrrhizin, greatly promoting the production of glycyrrhizin in the hairy roots of Glycyrrhiza glabra.

[0026] The method of this invention produces glycyrrhizin content in hairy roots that reaches 0.20-0.32% of the cell stem cell value, while cultivated or wild-grown licorice requires at least 3-5 years of growth to achieve the same content. In comparison, the method of increasing glycyrrhizin content through hairy root culture in this invention has advantages such as a short growth cycle, no geographical, time, or climatic limitations, and ease of industrialization, making the industrial production of glycyrrhizin through the co-culture of hairy roots and exogenous substrates a possibility.

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

[0028] The method of increasing glycyrrhizin content through hairy root culture in this invention can significantly increase glycyrrhizin content in a short time. It has the advantages of short growth cycle, no regional, time and climate restrictions, and easy industrialization, making it possible to industrialize the production of glycyrrhizin by co-culturing hairy roots with exogenous substrates. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of the invention. Various modifications and variations to the specific embodiments described in this specification are apparent to those skilled in the art without departing from the scope or spirit of the invention. Other embodiments derived from this specification will be apparent to those skilled in the art. This application specification and embodiments are merely exemplary.

[0032] The terms “include,” “including,” “have,” or “contain” used in this article are all open-ended, meaning they include but are not limited to.

[0033] The sulfuric acid used in this invention is AR grade.

[0034] Example 1

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

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

[0037] (2) Activation of the strain: Agrobacterium rhizogenes R1601 was activated three times on YEB medium containing 50 mg / L Kan and 1.5% agar powder. After the strain was revived and formed single colonies, single colonies were cultured on YEB medium containing 50 mg / L Kan and 1.5% agar powder for 24 h. Then, they were transferred to YEB liquid medium and cultured in the dark at 24 °C and 100 r / min with shaking. The bacterial solution in the logarithmic growth phase with OD600 of 0.8 was diluted with sterile water to OD600 of 0.5 and pH of 5.8 to obtain the activated bacterial solution.

[0038] (3) Explant inoculation and co-culture: Cut the licorice explants from step (1) into 0.5cm segments, immerse them in the activated bacterial solution from step (2) for 10min, remove them and blot off the excess bacterial solution with sterile filter paper, and place them on MS medium for co-culture at 24℃ in the dark for 3d.

[0039] (4) Induction and culture of hairy roots: Take out the explants from step (3), rinse them three times with sterile water, shake them for 10 min in MS liquid medium containing 300 mg / L Kan, and then transfer them to MS solid medium containing 300 mg / L Kan for culture. After hairy roots are induced, cut the hairy roots and transfer them to MS medium with a Kan concentration of 100 mg / L for culture. Subculture every 5 days, and repeat the subculture to remove bacteria until sterile plaques appear on the medium.

[0040] (5) Inducing the increase of glycyrrhizin content in hairy roots: Take the hairy roots cultured in step (4) and transfer them to MS liquid medium supplemented with 10 mg / L daidzein. Culture them at 24℃, 110 r / min and in the dark. After 5 days, carry out periodic temperature-changing culture: raise the culture temperature to 32℃ and culture for 10 days, then culture at 10℃ for 5 days, continue to raise the temperature to 32℃ and culture for 10 days, then culture at 10℃ for 5 days. Repeat this high temperature and low temperature periodic culture.

[0041] The content of glycyrrhizin in hairy roots was measured on day 60 and day 90 after the start of variable temperature culture.

[0042] Example 2

[0043] A method for increasing the content of glycyrrhizin through hairy root culture, the specific implementation method is roughly the same as that in Example 1, the difference being step (5):

[0044] Inducing the increase of glycyrrhizin content in hairy roots: Take the hairy roots cultured in step (4) and transfer them to MS liquid medium supplemented with 8 mg / L daidzein. Culture them at 25℃, 115 r / min and in the dark. After 5 days, carry out periodic temperature-changing culture: raise the culture temperature to 32℃ and culture for 10 days, then culture at 10℃ for 5 days, continue to raise the temperature to 32℃ and culture for 10 days, then culture at 10℃ for 5 days, and so on, repeating the high temperature and low temperature periodic culture.

[0045] The content of glycyrrhizin in hairy roots was measured on day 60 and day 90 after the start of variable temperature culture.

[0046] Example 3

[0047] A method for increasing the content of glycyrrhizin through hairy root culture, the specific implementation method is roughly the same as that in Example 1, the difference being step (5):

[0048] Inducing the increase of glycyrrhizin content in hairy roots: Take the hairy roots cultured in step (4) and transfer them to MS liquid medium supplemented with 5 mg / L daidzein. Culture them at 26℃, 120 r / min and in the dark. After 7 days, carry out periodic temperature-changing culture: raise the culture temperature to 32℃ and culture for 10 days, then culture at 10℃ for 5 days, continue to raise the temperature to 32℃ and culture for 10 days, then culture at 10℃ for 5 days, and so on, repeating the high temperature and low temperature periodic culture.

[0049] The content of glycyrrhizin in hairy roots was measured on day 60 and day 90 after the start of variable temperature culture.

[0050] Example 4

[0051] A method for increasing the content of glycyrrhizin through hairy root culture, the specific implementation method is roughly the same as that in Example 1, the difference being step (5):

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

[0053] The content of glycyrrhizin in hairy roots was measured on day 60 and day 90 after the start of variable temperature culture.

[0054] Example 5

[0055] A method for increasing the content of glycyrrhizin through hairy root culture, the specific implementation method is roughly the same as that in Example 1, the difference being step (5):

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

[0057] The content of glycyrrhizin in hairy roots was measured on day 60 and day 90 after the start of variable temperature culture.

[0058] Example 6

[0059] A method for increasing the content of glycyrrhizin through hairy root culture, the specific implementation method is roughly the same as that in Example 1, the difference being step (5):

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

[0061] The content of glycyrrhizin in hairy roots was measured on day 60 and day 90 after the start of variable temperature culture.

[0062] Example 7

[0063] A method for increasing the content of glycyrrhizin through hairy root culture, the specific implementation method is roughly the same as that in Example 1, the difference being step (5):

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

[0065] The content of glycyrrhizin in hairy roots was measured on day 60 and day 90 after the start of variable temperature culture.

[0066] Compare with Example 1

[0067] A method for increasing the content of glycyrrhizin through hairy root culture, the specific implementation method is roughly the same as that in Example 1, the difference being step (5):

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

[0069] The content of glycyrrhizin in the hairy roots was detected on day 60 and day 90 after the start of cultivation.

[0070] Compare with Example 2

[0071] A method for increasing the content of glycyrrhizin through hairy root culture, the specific implementation method is roughly the same as that in Example 1, the difference being step (5):

[0072] Inducing the increase of glycyrrhizin content in hairy roots: Take the hairy roots cultured in step (4) and transfer them to MS liquid medium supplemented with 8 mg / L daidzein. Culture them at 25℃, 115 r / min and in the dark. The glycyrrhizin content in the hairy roots was detected on the 60th and 90th day after the start of culture.

[0073] Compare with Example 3

[0074] A method for increasing the content of glycyrrhizin through hairy root culture, the specific implementation method is roughly the same as that in Example 1, the difference being step (5):

[0075] Inducing the increase of glycyrrhizin content in hairy roots: Take the hairy roots cultured in step (4), transfer them to MS liquid medium, and culture them at 25℃, 115r / min, and in the dark. After 5 days, carry out periodic temperature-changing culture, raise the culture temperature to 32℃ for 10 days, culture at 10℃ for 5 days, continue to raise the temperature to 32℃ for 10 days, and then continue to lower the culture temperature to 10℃ for 5 days. Repeat this high-temperature and low-temperature periodic culture.

[0076] The content of glycyrrhizin in hairy roots was measured on day 60 and day 90 after the start of variable temperature culture.

[0077] Performance testing

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

[0079] Table 1

[0080]

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

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

[0083] Examples 1-3 show that a daidzein concentration of 8 mg / L is better than 5 mg / L and 10 mg / L; Examples 3-5 show that a temperature range of 30-12℃ is better than a temperature range of 32℃-10℃ and 28-15℃; Examples 2 and 6 show that a temperature cycle of 10 days of high temperature and 5 days of low temperature is better than a temperature cycle of 5 days of high temperature and 3 days of low temperature.

[0084] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present application in any way. Although the present application discloses the preferred embodiment as described above, it is not intended to limit the present application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of the present application using the disclosed technical content are equivalent to equivalent implementation cases. Any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the technical solution of the present invention are still within the scope of the technical solution.

Claims

1. A method for increasing the content of glycyrrhizin through hairy root culture, characterized in that, After culturing sterile seedlings from seeds, the cotyledons and / or hypocotyl tissues of the sterile seedlings were cut off and hairy roots were induced using Agrobacterium rhizogenes to obtain hairy roots with good and stable growth. In subsequent subcultures, daidzein was introduced and the final product was obtained by periodic temperature-controlled culture. The specific steps are as follows: (1) Obtaining explants: Select healthy and plump seeds from wild Glycyrrhiza glabra collected in Xinjiang, treat with sulfuric acid for 35-45 min, rinse clean, then treat with mercuric chloride solution, rinse with sterile water 3-5 times, inoculate onto MS basal medium, adjust the pH to 5.8-6.0, and culture in the dark at 25±2℃ for 5-7 days. After the seeds germinate, culture until the hypocotyl is 2-3 cm long and the cotyledons unfold to obtain sterile Glycyrrhiza glabra seedlings. Take the hypocotyl and / or cotyledons of the sterile seedlings as explants of Glycyrrhiza glabra. (2) Activation of bacterial strain: The activated Agrobacterium rhizogenes R1601 single colony was inoculated on YEB medium and cultured, then transferred to YEB liquid medium and cultured in the dark, at 25±1℃ with shaking until the logarithmic growth phase. The bacterial solution in the logarithmic growth phase was diluted to obtain the activated bacterial solution. (3) Explant inoculation and co-culture: Cut the licorice explants from step (1) into small segments or pieces, immerse them in the activated bacterial solution obtained in step (2) for 10-15 min, then blot off the excess liquid on the surface with sterile filter paper, and place them on MS medium for co-culture at 25±1℃ in the dark for 3-5 days. (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 MS liquid medium and shake them, then transfer them to MS solid medium for culture. After the hairy roots are induced, take the hairy roots and transfer them to MS solid medium for culture. Subculture once every 5 days, and repeat the subculture to remove bacteria until sterile plaques appear on the medium. (5) Inducing the increase of glycyrrhizin content in hairy roots: Take the hairy roots cultured in step (4), transfer them to MS liquid medium containing daidzein, and culture them at 25±1℃, 110-120 r / min and in the dark for 5-7 days. Then, carry out periodic temperature variation culture to obtain the final product. The concentration of daidzein in the MS liquid culture medium used in step (5) is 5-10 mg / L; The specific operation of the periodic temperature change in step (5) is as follows: raise the culture temperature to 28-32℃ and culture for 5-10 days, then culture at 10-15℃ for 3-5 days, continue to raise the temperature to 28-32℃ and culture for 5-10 days, then culture at 10-15℃ for 3-5 days, and so on, repeating the high temperature and low temperature periodic cycle culture.

2. The method for increasing glycyrrhizin content through hairy root culture according to claim 1, characterized in that, In step (1), after the seeds germinate, they are cultured for 20-25 days under the conditions of temperature 25±2℃, light intensity 2000-4000Lux, and light duration 15-17h / d until the hypocotyl is 2-3cm long.

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

4. The method for increasing glycyrrhizin content through hairy root culture according to claim 1, characterized in that, The OD600 of the bacterial solution in the logarithmic growth phase in step (2) is 0.8-1.2.

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