A method for rapid propagation of licorice adventitious roots and licorice prepared therefrom

Abstract

The present application relates to the technical field of plant tissue culture, and specifically to a method for rapid propagation of licorice adventitious roots and licorice prepared therefrom. A method for rapid propagation of licorice adventitious roots, comprising the following steps: preparation and subpackaging: preparing a licorice adventitious root culture medium and subpackaging it into tissue culture bottles; inoculation: inoculating rhizobia into the licorice adventitious root culture medium; plugging: cutting licorice sterile seedlings into explants, plugging the explants into the licorice adventitious root culture medium inoculated with rhizobia; and cultivation: cultivating the explants. Compared with the prior art, the method for rapid propagation of licorice adventitious roots of the present application actively inoculates rhizobia on the licorice adventitious root culture medium, utilizes rhizobia to stimulate licorice to produce adventitious roots, and utilizes the symbiotic relationship between rhizobia and licorice adventitious roots to promote the continuous development of licorice adventitious roots and increase the production of glycyrrhizic acid.

Description

Technical Field

[0001] The present application relates to the technical field of plant tissue culture, and in particular to a method for rapid propagation of adventitious roots of licorice and licorice prepared therefrom. Background Art

[0002] Licorice is one of the most widely used drugs in traditional Chinese medicine. It has the effects of tonifying the spleen and replenishing qi, clearing away heat and detoxifying, removing phlegm and relieving cough, relieving acute pain, and harmonizing various medicines. According to incomplete statistics, the average annual demand for licorice in the world is more than 100,000 tons, of which up to 90% of licorice raw materials are provided by my country, and the demand is very large. The growth and development of licorice root system is closely related to the production and quality of licorice. The development of adventitious roots is an important part of the development of the entire root system. Usually, licorice with more adventitious roots and high root activity grows more vigorously.

[0003] Tissue culture can not only maintain the excellent traits of licorice, but also provide a large number of seedlings for the market, playing a positive role in the breeding of licorice. In tissue culture technology, the rapid propagation of licorice adventitious roots has always been the focus of researchers, and the number of roots and root activity of licorice adventitious roots can be increased by improving the formula of the culture medium.

[0004] In the prior art, most of the formulas of licorice culture medium are based on MS culture medium, and auxin and auxin analogs are added to promote rooting, such as IAA, IBA, NAA, etc., and the optimal culture medium formula is determined by adjusting their content. At present, the rapid propagation effect of licorice adventitious roots by the culture medium of licorice tissue culture is difficult to meet the higher requirements of technicians, and a new rapid propagation method of licorice adventitious roots is needed to further improve the rooting effect of licorice adventitious roots. Summary of the invention

[0005] In order to solve the problem that the culture medium in licorice tissue culture is difficult to meet the technicians' higher requirements for the rapid propagation effect of licorice adventitious roots, the present application provides a method for rapid propagation of licorice adventitious roots and licorice prepared therefrom. Compared with the existing technology, the method for rapid propagation of licorice adventitious roots has better induction effect and can also increase the glycyrrhizic acid content of licorice.

[0006] In the first aspect, the present application provides a method for rapid propagation of licorice adventitious roots, using the following technical solution:

[0007] A rapid propagation method for licorice adventitious roots comprises the following steps:

[0008] Preparation and packaging: prepare licorice adventitious root culture medium and dispense it into tissue culture bottles;

[0009] Inoculation: inoculating rhizobia onto the licorice adventitious root culture medium;

[0010] Insertion: cutting the sterile licorice seedlings into explants, and inserting the explants into the licorice adventitious root culture medium inoculated with rhizobia;

[0011] Cultivation: The explants are cultured.

[0012] By adopting the above technical scheme, rhizobia is a beneficial microorganism, and licorice is a leguminous plant. Rhizobia can increase the absorption of water and nutrients in the culture medium by licorice explants, and stimulate the differentiation of licorice adventitious roots; when licorice adventitious roots appear, rhizobia will infect licorice root hairs, and at the same time produce secretions to stimulate the root hairs, and then the rhizobia will enter the root hairs to form infection threads, continue to infect the inside of the root, and after entering the cortex, the rhizobia will rapidly multiply, and the rhizobia will continuously secrete nitrogenase to promote the conversion of nitrogen in the air into ammonia nitrogen; ammonia nitrogen is beneficial to the absorption and synthesis of biological macromolecules such as protein by the licorice root system, and promote the growth and development of adventitious roots;

[0013] After rhizobia infect licorice adventitious roots, nodules will be formed on the epidermis of the adventitious roots. The nodules are rich in a large number of rhizobia and hydrogen roots, which enable the licorice adventitious roots to better absorb and utilize nitrogen sources. Rhizobia can also produce nodule factors, which can combine with the cell membrane of licorice adventitious roots, so that rhizobia can attach to the licorice adventitious roots more firmly. In addition, after the rhizobia and licorice adventitious roots coexist, they will also stimulate the enzymes related to the glycyrrhizic acid synthesis pathway, thereby increasing the production of glycyrrhizic acid.

[0014] Compared with the prior art, the rapid propagation method of licorice adventitious roots of the present application actively inoculates rhizobia on the licorice adventitious root culture medium, uses rhizobia to stimulate licorice to produce adventitious roots, and uses the symbiotic relationship between rhizobia and licorice adventitious roots to promote the continuous development of licorice adventitious roots and increase the production of glycyrrhizic acid.

[0015] Preferably, the licorice adventitious root culture medium comprises the following components by weight per liter: MS culture medium dry powder 1.58-4.74 g, IBA 0.5-1 mg, sucrose 25-30 g, and agar 7.5-8 g.

[0016] By adopting the above technical solution, the MS culture medium dry powder provides inorganic salts for the tissue culture system. When the licorice adventitious roots are not generated, the rhizobia will directly use the inorganic salts in the licorice adventitious root culture medium for proliferation and development. Therefore, the content of the MS culture medium dry powder is appropriately reduced to reduce the proliferation and development rate of the rhizobia, so that more inorganic salts can be provided for the formation and growth of the licorice adventitious roots.

[0017] Preferably, the rhizobium includes Rhizobium leguminosarum, Rhizobium meliloti and Rhizobium loti.

[0018] By adopting the above technical scheme, pea rhizobium, meliloti rhizobium and lotus root rhizobium all belong to the genus Rhizobium, and are suitable for growing on the licorice adventitious root culture medium. In addition, pea rhizobium, meliloti rhizobium and lotus root rhizobium secrete acidic substances during their growth and development, which can well maintain the acidic environment of the licorice adventitious root culture medium. Therefore, the above three rhizobia are suitable for promoting the formation and growth and development of licorice adventitious roots.

[0019] Preferably, the inoculation amount of the rhizobium is 1000-5000 CFU / bottle.

[0020] By adopting the above technical scheme, if the inoculation amount of rhizobia is too low, the promoting effect of rhizobia on licorice explants is low, thereby reducing the number and length of adventitious roots of licorice explants; if the inoculation amount of rhizobia is too high, the rhizobia will multiply in large quantities in the tissue culture bottle, reducing the nutrients for the formation and growth of adventitious roots of licorice, thereby reducing the number and length of adventitious roots of licorice explants; for this reason, the applicant finally determined after a lot of research and experimental verification that the inoculation amount of rhizobia in this application is preferably the above.

[0021] Preferably, the ratio of the inoculum amount of the rhizobium in each tissue culture bottle to the mass of the MS culture medium dry powder in each liter of the licorice adventitious root culture medium is 3000 CFU: 2-3 g.

[0022] Through the above technical scheme, if the mass of MS culture medium dry powder in each liter of licorice adventitious root culture medium is too low, the inorganic salts required for the formation and growth of licorice adventitious roots will be insufficient, thereby reducing the number and length of the adventitious roots of the licorice explants; if the mass of MS culture medium dry powder in each liter of licorice adventitious root culture medium is too high, rhizobia will multiply in large quantities in the tissue culture bottle, which will also lead to insufficient inorganic salts required for the formation and growth of licorice adventitious roots, thereby reducing the number and length of the adventitious roots of the licorice explants; for this reason, the applicant finally determined after a lot of research and experimental verification that the ratio of the inoculation amount of rhizobia in each tissue culture bottle of the present application and the mass of MS culture medium dry powder in each liter of the licorice adventitious root culture medium should be as above.

[0023] Preferably, the licorice adventitious root culture medium further comprises cyclodextrin.

[0024] By adopting the above technical scheme, the cyclodextrin molecule presents a hollow cylindrical three-dimensional ring structure, has the characteristics of hydrophilic outer edge and hydrophobic inner cavity, and can adsorb many organic molecules. When cyclodextrin is added to the licorice adventitious root culture medium, it can adsorb the metabolites of rhizobia and licorice adventitious roots, reduce the feedback inhibition of the metabolic pathways of rhizobia and licorice adventitious roots by the metabolites of rhizobia and licorice adventitious roots, and contribute to the growth and development of rhizobia and licorice adventitious roots.

[0025] Preferably, the ratio of the inoculum amount of the rhizobium in each tissue culture bottle to the mass of cyclodextrin in each liter of the licorice adventitious root culture medium is 3000 CFU: 5-40 mg.

[0026] By adopting the above technical scheme, if the mass of cyclodextrin is too high, the metabolites of rhizobia and licorice adventitious roots will be absorbed too much by the other phase, which is not conducive to the accumulation of nutrients in rhizobia and licorice adventitious roots; if the mass of cyclodextrin is too low, the two-phase boundary formed by the culture system is not obvious, and the effect of promoting the growth and development of rhizobia and licorice adventitious roots is not obvious; for this reason, the applicant finally determined after a lot of research and experimental verification that the ratio of the inoculation amount of rhizobia in each tissue culture bottle of the present application and the mass of cyclodextrin in each liter of the licorice adventitious root culture medium should be as above.

[0027] Preferably, the explants are stem segments with leaves and stem segments with roots.

[0028] By adopting the above technical scheme, stem segments with leaves and stem segments with roots can generate a high number of adventitious roots, and are suitable as explants for this application. Stem segments without leaves and roots generate a low amount of adventitious roots, and are not suitable as explants for this application.

[0029] Preferably, in the culturing step, after the explants are cultured for 15-20 days, the explants are inserted into the licorice adventitious root culture medium which is not inoculated with the rhizobia for further culturing.

[0030] By adopting the above technical solution, after the explant grows for 15-20 days, the nutrients in the licorice adventitious root culture medium are consumed by rhizobia and licorice adventitious roots, and the licorice adventitious roots are difficult to continue to grow and develop. It is necessary to replace the tissue culture bottle for continued growth. At this time, the explant is already in a symbiotic state with the rhizobia. In order to supply the nutrients in the new licorice adventitious root culture medium to the growth and development of the licorice adventitious roots as much as possible, there is no need to inoculate rhizobia.

[0031] In a second aspect, the present application provides a licorice root prepared by any of the above-mentioned methods for rapid propagation of licorice adventitious roots.

[0032] The advantages of the licorice and the above-mentioned rapid propagation method of licorice adventitious roots over the prior art are the same, which will not be repeated here.

[0033] In summary, this application has the following beneficial effects:

[0034] 1. Compared with the prior art, the rapid propagation method of licorice adventitious roots of the present application actively inoculates rhizobia on the licorice adventitious root culture medium, uses rhizobia to stimulate licorice to produce adventitious roots, and uses the symbiotic relationship between rhizobia and licorice adventitious roots to promote the continuous development of licorice adventitious roots and increase the production of glycyrrhizic acid;

[0035] 2. The present application adds cyclodextrin to the licorice adventitious root culture medium to form a two-phase culture system, thereby reducing the feedback inhibition of the metabolic pathways of rhizobia and licorice adventitious roots by the metabolites of rhizobia and licorice adventitious roots, and facilitating the growth and development of rhizobia and licorice adventitious roots. DETAILED DESCRIPTION

[0036] The raw materials in this application include the following parts:

[0037] The MS culture medium dry powder adopts a commercially available product that does not contain sucrose and agar; the IBA adopts a commercially available product with a CAS number of 133-32-4 and an effective content ≥99%; the sucrose adopts a commercially available product with a CAS number of 57-50-1 and an effective content ≥99%; the agar adopts a commercially available product with an effective content ≥99%; and the cyclodextrin adopts a commercially available product with a CAS number of 7585-39-9 and an effective content ≥99%.

[0038] Pea Rhizobium, alfalfa Rhizobium, Lotus japonicum and soybean Bradyrhizobium are all products from Shanghai Preservation Biotechnology Center.

[0039] The present application is further described in detail below with reference to examples and comparative examples.

[0040] Example 1

[0041] Preparation of culture medium: 2.37 g MS culture medium powder, 0.8 mg IBA, 28 g sucrose and 8 g agar were heated and dissolved in 980 mL deionized water, the pH of the solution was adjusted to 5.8 with 1 mol / L HCl solution and 1 mol / L KOH solution, and then the volume was fixed to 1 L, and then sterilized at 121°C for 20 min to obtain licorice adventitious root culture medium;

[0042] Dispensing: Dispense the culture medium into tissue culture bottles, with 50 mL of licorice adventitious root culture medium in each tissue culture bottle;

[0043] Inoculation: 3000 CFU of Rhizobium leguminosarum were inoculated into the licorice adventitious root medium on each tissue culture bottle;

[0044] Insertion: Cut 5 cm stem segments with roots from the same batch of sterile licorice seedlings, and insert 2 stem segments with roots into each tissue culture bottle;

[0045] Cultivation: The stem segments with roots were cultured under the following conditions: temperature 25°C, light intensity 2000 lx, and photoperiod 14 h / day;

[0046] Transfer: After 18 days of culture, the stem segments with roots were inserted into new tissue culture bottles for further culture. The licorice adventitious root culture medium in the new tissue culture bottles was not inoculated with pea rhizobia.

[0047] Example 2-3

[0048] Example 2-3 Based on the preparation method of Example 1, the component content of the licorice adventitious root culture medium was adjusted, and the specific adjustments are shown in Table 1.

[0049] Comparative Example 1-2

[0050] Comparative Example 1-2 Based on the preparation method of Example 1, the component contents of the licorice adventitious root culture medium were adjusted, and the specific adjustments are shown in Table 1.

[0051] Comparative Example 3

[0052] Comparative Example 3 is based on the preparation method of Example 1, but without inoculating pea rhizobium, and other conditions remain unchanged.

[0053] Table 1 Data table of component contents of licorice adventitious root culture medium and pea rhizobium inoculation amount in Examples 1-3 and Comparative Examples 1-3

[0054]

[0055] Performance testing

[0056] 1. Number and length of adventitious roots

[0057] The inserted licorice explants of Examples 1-3 and Comparative Examples 1-3 of the present application were cultured for 30 days, and randomly sampled from the stem segments with roots of the same Example or Comparative Example, with a sampling rate of 5%. The sampled stem segments with roots were taken out to measure the number of adventitious roots and the length of adventitious roots, and the total number of roots, the number of explants and the total root length were counted;

[0058] Wherein, the total number of roots is the number of adventitious roots of all explants extracted from the same embodiment or comparative example; the number of explants is the number of stem segments with roots extracted from the same embodiment or comparative example; the total root length is the length of adventitious roots of all stem segments with roots extracted from the same embodiment or comparative example;

[0059] The number of roots and root length were calculated, where the number of roots = total number of roots / number of explants, and the root length = total root length / total number of roots. The calculation results are shown in Table 2.

[0060] 2. Glycyrrhizic acid The licorice explants inserted in Examples 1-3 and Comparative Examples 1-3 of the present application were cultured for 30 days, and glycyrrhizic acid was extracted from the adventitious roots of licorice according to the method of the Chinese Pharmacopoeia (2020 edition). The content of glycyrrhizic acid was determined by HLPC instrument, the detection wavelength was 254nm, the mobile phase was acetonitrile-0.01% phosphoric acid water, the flow rate was 1.0mL / min; the injection volume was 10μL, and the test results are shown in Table 2.

[0061] Table 2 Performance test data of Examples 1-3 and Comparative Examples 1-3

[0062]

[0063] Referring to Table 2, by comparing Examples 1-3 and Comparative Examples 1-3, it can be seen that compared with Example 1, the number of roots, root length and glycyrrhizic acid content of the adventitious roots of Examples 2-3 are less than those of Example 1, and the number of roots, root length and glycyrrhizic acid content of the adventitious roots of Comparative Examples 1-3 are significantly lower than those of Example 1; this indicates that the growth of licorice adventitious roots is better under the licorice adventitious root culture medium and culture conditions of Example 1.

[0064] Embodiment 4-5

[0065] Example 4-5 Based on the preparation method of Example 1, the type of rhizobium was adjusted, and the specific adjustment is shown in Table 3.

[0066] Comparative Example 4

[0067] Comparative Example 4 Based on the preparation method of Example 1, the type of rhizobium was adjusted, and the specific adjustment is shown in Table 3.

[0068] The licorice stem segments with roots prepared in Examples 4-5 and Comparative Example 4 were subjected to the above performance tests, and the test results are shown in Table 3.

[0069] Table 3 Rhizobium species and performance test data of Example 1, Examples 4-5 and Comparative Example 4

[0070]

[0071]

[0072] Referring to Table 3, by comparing Example 1, Examples 4-5 and Comparative Example 4, it can be seen that compared with Example 1, the number of roots, root length and glycyrrhizic acid content of the adventitious roots of Examples 4-5 are less than those of Example 1, and the number of roots, root length and glycyrrhizic acid content of the adventitious roots of Comparative Example 4 are significantly lower than those of Example 1; this indicates that the growth of licorice adventitious roots is better when inoculated with pea rhizobia.

[0073] Example 6

[0074] Example 6 Based on the preparation method of Example 1, the stem segment with roots is replaced with a stem segment with leaves, and the specific adjustments are shown in Table 4.

[0075] Comparative Example 5

[0076] Comparative Example 5 is based on the preparation method of Example 1, and the stem segment with roots is replaced with a blank stem segment (referring to a stem segment with neither roots nor leaves), and the specific adjustments are shown in Table 4.

[0077] The licorice prepared in Example 6 and Comparative Example 5 was subjected to the above performance tests, and the test results are shown in Table 4.

[0078] Table 4 Explant form and performance test data of Example 1, Example 6 and Comparative Example 5

[0079]

[0080] Referring to Table 4, by comparing Example 1, Example 6 and Comparative Example 5, it can be seen that compared with Example 1, the number of roots, root length and glycyrrhizic acid content of the adventitious roots of Example 6 are less than those of Example 1, and the number of roots, root length and glycyrrhizic acid content of the adventitious roots of Comparative Example 5 are significantly lower than those of Example 1; this indicates that the growth of licorice adventitious roots is better when the stem segments with roots are inserted.

[0081] Embodiment 7-8

[0082] In Example 7-8, based on the preparation method of Example 1, the inoculation amount of rhizobium in each bottle was adjusted, and the specific adjustment is shown in Table 5.

[0083] Comparative Examples 6-7

[0084] Comparative Example 6-7 is based on the preparation method of Example 1, and the inoculation amount of rhizobium in each bottle is adjusted. The specific adjustment is shown in Table 5.

[0085] The licorice prepared in Examples 7-8 and Comparative Examples 6-7 were subjected to the above performance tests, and the test results are shown in Table 5.

[0086] Table 5 Inoculation amount and performance test data of each bottle of rhizobium in Example 1, Examples 7-8 and Comparative Examples 6-7

[0087]

[0088]

[0089] Referring to Table 5, by comparing Example 1, Examples 7-8 and Comparative Examples 6-7, it can be seen that as the inoculation amount of rhizobia continues to increase, the number of roots, root length and glycyrrhizic acid content of licorice adventitious roots show a trend of first increasing and then decreasing. This may be because within a certain range, as the inoculation amount of rhizobia continues to increase, the rhizobia continuously promote the continuous formation and growth of licorice adventitious roots, thereby increasing the rooting effect of licorice adventitious roots; when it exceeds a certain range, the rhizobia reproduce too fast, reducing the nutrients for the formation and growth of licorice adventitious roots, thereby reducing the number of roots, root length and glycyrrhizic acid content of licorice adventitious roots.

[0090] Examples 9-10

[0091] In Examples 9-10, based on the preparation method of Example 1, the content of MS culture medium dry powder in each liter of licorice adventitious root culture medium was adjusted, and the specific adjustments are shown in Table 6.

[0092] Comparative Examples 8-9

[0093] Comparative Example 8-9 Based on the preparation method of Example 1, the content of MS culture medium dry powder in each liter of licorice adventitious root culture medium was adjusted, and the specific adjustment is shown in Table 6.

[0094] The licorice prepared in Examples 9-10 and Comparative Examples 8-9 were subjected to the above performance tests, and the test results are shown in Table 6.

[0095] Table 6 MS culture medium dry powder content and performance test data table of Example 1, Examples 9-10 and Comparative Examples 8-9

[0096]

[0097] Referring to Table 6, by comparing Example 1, Examples 9-10 and Comparative Examples 8-9, it can be seen that as the content of MS culture medium dry powder in each liter of licorice adventitious root culture medium continues to increase, the number of roots, root length and glycyrrhizic acid content of the adventitious roots of licorice show a trend of first increasing and then decreasing. This may be because as the content of MS culture medium dry powder in each liter of licorice adventitious root culture medium continues to increase, the content of inorganic salts in the culture medium continues to increase, which is beneficial to the growth and development of licorice adventitious roots; when the content of MS culture medium dry powder exceeds a certain range, the rhizobia on the culture medium grow too fast, reducing the number of roots, root length and glycyrrhizic acid content of the adventitious roots of licorice.

[0098] Examples 11-13

[0099] Example 11 Based on the preparation method of Example 1, 20 mg of cyclodextrin was added to each liter of licorice adventitious root culture medium.

[0100] In Examples 12-13, based on the preparation method of Example 11, the content of cyclodextrin was adjusted, and the specific adjustments are shown in Table 7.

[0101] Comparative Examples 10-11

[0102] Comparative Examples 10-11 are based on the preparation method of Example 11, and the content of cyclodextrin is adjusted. The specific adjustments are shown in Table 7.

[0103] The licorice prepared in Examples 11-13 and Comparative Examples 10-11 were subjected to the above performance tests, and the test results are shown in Table 7.

[0104] Table 7 Cyclodextrin content and performance test data of Example 1, Examples 11-13 and Comparative Examples 10-11

[0105]

[0106] Referring to Table 7, by comparing Example 1, Examples 11-13 and Comparative Examples 10-11, it can be seen that cyclodextrin is added to the licorice adventitious root culture medium to further improve the root number, root length and glycyrrhizic acid content of the licorice adventitious roots; as the content of cyclodextrin continues to increase, the root number, root length and glycyrrhizic acid content of the licorice adventitious roots show a trend of first increasing and then decreasing. This may be because within a certain range, as the content of cyclodextrin continues to increase, the licorice adventitious root culture medium forms a two-phase culture system, in which one phase continuously absorbs the metabolites of rhizobia and licorice adventitious roots, which reduces the feedback inhibition of the metabolic pathways of rhizobia and licorice adventitious roots by the metabolites of rhizobia and licorice adventitious roots, and contributes to the growth and development of rhizobia and licorice adventitious roots; when it exceeds a certain range, the metabolites generated by the licorice adventitious roots are absorbed too much by the other phase, and the accumulated metabolite content is reduced, thereby reducing the root number, root length and glycyrrhizic acid content of the licorice adventitious roots.

[0107] Examples 14-15

[0108] In Examples 14-15, based on the preparation method of Example 11, the time for replacing the stem segments with roots with new tissue culture bottles was adjusted. The specific adjustments are shown in Table 8.

[0109] Comparative Examples 12-13

[0110] Comparative Examples 12-13 are based on the preparation method of Example 11, and the time for replacing the stem segments with roots with new tissue culture bottles is adjusted. The specific adjustments are shown in Table 8.

[0111] The licorice prepared in Examples 14-15 and Comparative Examples 12-13 were subjected to the above performance tests, and the test results are shown in Table 8.

[0112] Table 8 Bottle changing time and performance test data of Example 11, Examples 14-15 and Comparative Examples 12-13

[0113]

[0114] Referring to Table 8, by comparing Example 11, Examples 14-15 and Comparative Examples 12-13, it can be seen that as the time for replacing the new tissue culture bottle with the rooted stem segment continues to increase, the number of roots, root length and glycyrrhizic acid content of the adventitious roots of licorice show a trend of first increasing and then decreasing. This may be because within a certain range, as the time for replacing the new tissue culture bottle with the rooted stem segment continues to increase, the rhizobia in the original tissue culture bottle continue to proliferate and continuously infect the adventitious roots of licorice, causing the number of roots, root length and glycyrrhizic acid content of the adventitious roots of licorice to continue to increase; after exceeding a certain range, the nutrients in the original tissue culture bottle are exhausted, causing the adventitious roots of licorice to stop growing, and the tissue culture bottle needs to be replaced before it can continue to grow.

[0115] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for rapid propagation of licorice adventitious roots, characterized in that: The following steps are involved: Preparation and packaging: prepare licorice adventitious root culture medium and dispense it into tissue culture bottles; Inoculation: inoculating rhizobia onto the licorice adventitious root culture medium; Insertion: cutting the sterile licorice seedlings into explants, and inserting the explants into the licorice adventitious root culture medium inoculated with rhizobia; Cultivating: culturing the explant; The inoculation amount of the rhizobium is 1000-5000 CFU / bottle; The licorice adventitious root culture medium is composed of the following components by weight per liter: MS culture medium dry powder 1.58-4.74 g, IBA 0.5-1 mg, sucrose 25-30 g, agar 7.5-8 g, cyclodextrin 5-40 mg; The rhizobium is selected from Rhizobium leguminosarum, Rhizobium luciliae, or Rhizobium lotifolium; The explants are stem segments with leaves and stem segments with roots.

2. The method for rapid propagation of licorice adventitious roots according to claim 1, characterized in that: The ratio of the inoculation amount of the rhizobium in each tissue culture bottle to the mass of the MS culture medium dry powder in each liter of the licorice adventitious root culture medium is 3000 CFU: 2-3 g.

3. The method for rapid propagation of licorice adventitious roots according to claim 1, characterized in that: The ratio of the inoculation amount of the rhizobium in each tissue culture bottle to the mass of cyclodextrin in each liter of the licorice adventitious root culture medium is 3000 CFU: 5-40 mg.

4. The method for rapid propagation of licorice adventitious roots according to claim 1, characterized in that: In the culturing step, after the explants are cultured for 15-20 days, the explants are inserted into the licorice adventitious root culture medium which is not inoculated with the rhizobia for further culturing.

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