In-vitro regeneration method of ferula sinkiangensis and application of in-vitro regeneration method

Abstract

The invention discloses an in-vitro regeneration method of ferula sinkiangensis and application of the in-vitro regeneration method. According to the method, the efficient in-vitro regeneration method for the ferula sinkiang from embryos to complete in-vitro regenerated plants is established through hormone regulation and optimization, so that the germination efficiency of the embryos is improved, the high callus induction rate and seedling rate of the ferula sinkiang are obtained, the in-vitro regeneration efficiency of the ferula sinkiang is greatly improved, and the in-vitro regeneration cost is reduced. The efficient in-vitro regeneration method of the ferula sinkiangensis not only lays a good foundation for variety improvement and biotechnology breeding of the ferula sinkiangensis, but also provides technical support for resource protection and recovery and industrial application of the ferula sinkiangensis.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue culture, and particularly relates to an in vitro regeneration method of Xinjiang Ferula and application thereof. Background Art

[0002] Xinjiang Ferula ( Ferula sinkiangensis KM Shen ) is a member of the Apiaceae family ( Umbelliferae ) Ferula ( Ferula L. ) is a perennial, once-fruiting herb, 0.2~1m tall, with a special onion and garlic smell throughout the plant. Xinjiang Ferula is rich in resources, and currently only Xinjiang Ferula and Fukang Ferula are included in the pharmacopoeia. As the base plant of medicinal Ferula, Xinjiang Ferula has the effects of eliminating accumulation, detoxifying, killing insects, promoting blood circulation and regulating menstruation, and can be used to treat stomach problems, blood stasis, abdominal pain caused by worms and other diseases. Xinjiang Ferula is mainly distributed in Nanshan, Shihezi, Yining, Yili, Nileke and other areas. In recent years, due to the sharp increase in the market purchase price of Xinjiang Ferula, Xinjiang Ferula has been severely illegally mined. In addition, factors such as farmland reclamation, grazing, and low seed germination rate have caused the distribution area of ​​Xinjiang Ferula to increase from 1400 hm2 to 2018. 2 Sharply reduced to less than 133 hm 2 , natural plant resources have been severely damaged and are on the verge of extinction. In 2021, Xinjiang Ferula was included in the "National List of Key Protected Wild Plants" and is a second-level protected plant. Therefore, it is urgent to protect the existing Xinjiang Ferula resources and rapidly expand Xinjiang Ferula plant resources through various means.

[0003] The scarcity of Xinjiang Ferula seedlings is a key factor contributing to its endangered status. Currently, tissue culture regeneration systems are the most effective technology for preserving endangered plant resources and expanding the seedling base. In recent years, gene editing has shown great potential in improving medicinal plants, particularly in regulating stress resistance and the synthesis of medicinal metabolites. However, due to the limitations of efficient regeneration systems, gene editing has not yet been applied to the conservation and research of Xinjiang Ferula germplasm resources. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to efficiently realize the in vitro regeneration of Xinjiang Ferula.

[0005] In order to solve the above technical problems, the present invention first provides an in vitro regeneration method of Xinjiang Ferula.

[0006] The in vitro regeneration method of Xinjiang Ferula provided by the present invention comprises the following steps: 1) culturing embryos of Xinjiang Ferula in an embryo germination medium to obtain germinated embryos; the embryo germination medium contains KT and 2,4-D; 2) culturing the hypocotyls obtained from the germinated embryos in a callus induction and proliferation differentiation medium to obtain callus tissue that has differentiated into somatic embryos; the callus induction and proliferation differentiation medium contains NAA and ZT; 3) culturing the callus tissue that has been differentiated to obtain somatic embryos in a seedling and rooting medium to obtain Xinjiang Ferula regenerated seedlings.

[0007] In the above method, the step 1) further includes the following steps: disinfecting the Xinjiang Ferula seeds, and obtaining Xinjiang Ferula embryos from the disinfected seeds.

[0008] In some embodiments, the disinfection method comprises the following steps: soaking the Xinjiang Ferula seeds in 75% alcohol for 30 seconds, then soaking them in 30% H2O2 for 5 minutes, then washing them with sterile water for 3-5 times, and finally soaking them in sterile water at 22°C in the dark for 36 hours.

[0009] In the above method, in step 1), the concentration of KT in the embryo germination medium may be 0.05-0.2 mg / L, 0.05-0.1 mg / L, or 0.1-0.2 mg / L, specifically 0.05 mg / L, 0.1 mg / L, or 0.2 mg / L.

[0010] In some embodiments, the concentration of KT in the embryo germination medium is 0.1 mg / L.

[0011] The concentration of 2,4-D in the embryo germination medium may be 0.05-0.2 mg / L, 0.05-0.1 mg / L, or 0.1-0.2 mg / L, specifically 0.05 mg / L, 0.1 mg / L, or 0.2 mg / L.

[0012] In some embodiments, the concentration of 2,4-D in the embryo germination medium is 0.1 mg / L.

[0013] In some specific embodiments, the embryo germination medium consists of MS medium, sucrose, KT, 2,4-D and plant gel, wherein the concentration of sucrose in the embryo germination medium is 25 g / L, the concentration of KT in the embryo germination medium is 0.1 mg / L, the concentration of 2,4-D in the embryo germination medium is 0.1 mg / L, and the concentration of plant gel in the embryo germination medium is 2.0 g / L.

[0014] In some specific embodiments, the pH value of the embryo germination medium is 5.6-5.8.

[0015] In the above method, in step 1), the culture conditions can be culturing at 22°C light for 16 h / 20°C dark for 8 h for 5-7 days, specifically culturing at 22°C light for 16 h / 20°C dark for 8 h for 5 days.

[0016] In the above method, in step 2), the length of the hypocotyl is 0.3-0.5 cm.

[0017] In the above method, in step 2), the concentration of NAA in the callus induction and proliferation differentiation medium may be 0.2-0.5 mg / L, specifically 0.2 mg / L or 0.5 mg / L.

[0018] In some embodiments, the concentration of NAA in the callus induction and proliferation differentiation medium is 0.5 mg / L.

[0019] The concentration of ZT in the callus induction and proliferation differentiation medium may be 0.5-2.0 mg / L, 0.5-1.0 mg / L, or 1.0-2.0 mg / L, specifically 0.5 mg / L, 1.0 mg / L, or 2.0 mg / L.

[0020] In some embodiments, the concentration of ZT in the callus induction and proliferation differentiation medium is 1.0 mg / L.

[0021] In some specific embodiments, the callus induction and proliferation differentiation medium consists of MS medium, sucrose, NAA, ZT and plant gel, wherein the concentration of sucrose in the callus induction and proliferation differentiation medium is 25 g / L, the concentration of NAA in the callus induction and proliferation differentiation medium is 0.5 mg / L, the concentration of ZT in the callus induction and proliferation differentiation medium is 1.0 mg / L, and the concentration of plant gel in the callus induction and proliferation differentiation medium is 2.0 g / L.

[0022] In some specific embodiments, the pH value of the callus induction and proliferation and differentiation medium is 5.6-5.8.

[0023] In the above method, in step 2), the culture conditions are culturing at 22°C light for 16 h / 20°C dark for 8 h for 10-18 days, specifically culturing at 22°C light for 16 h / 20°C dark for 8 h for 14 days.

[0024] In the above method, in step 3), the seedling and rooting medium includes sucrose.

[0025] The concentration of the sucrose in the seedling and rooting medium may be 20-30 g / L, specifically 20 g / L.

[0026] In some specific embodiments, the seedling and rooting medium consists of MS medium, sucrose and plant gel, wherein the concentration of sucrose in the seedling and rooting medium is 20 g / L, and the concentration of plant gel in the seedling and rooting medium is 2.0 g / L.

[0027] In some specific embodiments, the pH value of the seedling and rooting medium is 5.6-5.8.

[0028] In the above method, steps 1) to 3) are all performed in a tissue culture room.

[0029] In the above method, after step 3), the method further comprises the step of transplanting the Xinjiang Ferula regenerated seedlings into a substrate for cultivation to obtain Xinjiang Ferula regenerated plants.

[0030] In some embodiments, the transplanting method includes the following steps: half-opening the bottle cap of the culture bottle containing the Xinjiang Ferula regenerated seedlings, hardening the seedlings for 2 days, then completely opening the bottle cap, hardening the seedlings for 2 days, then washing the Xinjiang Ferula regenerated seedlings, soaking them in carbendazim, drying them, and then transplanting them into a substrate for cultivation to obtain Xinjiang Ferula regenerated plants.

[0031] In some embodiments, the substrate consists of peat moss, peat moss, and vermiculite.

[0032] In some specific embodiments, the volume ratio of the peat soil, the peat soil, and the vermiculite is 2:1:1.

[0033] In some embodiments, the transplanting process only involves shallow burying of the roots into the substrate.

[0034] In some embodiments, the culture conditions are as follows: culturing at a relative humidity of 65%, 22° C. light for 16 h / 20° C. dark for 8 h for 10 days.

[0035] In order to solve the above technical problems, the present invention also provides a set of reagents for in vitro regeneration of Xinjiang Ferula.

[0036] The complete set of reagents for in vitro regeneration of Xinjiang Ferula provided by the invention comprises the above-mentioned embryo germination medium, the above-mentioned callus induction and proliferation differentiation medium and the above-mentioned seedling formation and rooting medium.

[0037] Furthermore, the complete set of reagents also includes reagents for disinfecting Xinjiang Ferula seeds, such as 75% alcohol, 30% H2O2, and sterile water.

[0038] Furthermore, the set of reagents also includes a matrix for culturing Xinjiang Ferula regeneration seedlings, such as a matrix composed of peat soil, peat soil and vermiculite (the volume ratio of peat soil, peat soil and vermiculite is 2:1:1).

[0039] In order to solve the above technical problems, the present invention finally provides any one of the following applications a1) to a8): a1) Use of the above reagent set in the in vitro regeneration of Ferula xinjiangensis; a2) Use of the above reagent set in the preparation of a product for in vitro regeneration of Ferula dahurica; a3) Use of the above-mentioned embryo germination medium in improving the germination efficiency of Xinjiang Ferula embryos; a4) Use of the above-mentioned embryo germination medium in the preparation of a product for improving the germination efficiency of Xinjiang Ferula embryos; a5) Use of the above-mentioned callus induction and proliferation differentiation medium to improve the callus induction rate and / or differentiation rate of Ferula sinensis; a6) Use of the above-mentioned callus induction and proliferation and differentiation medium in the preparation of a product for improving the callus induction rate and / or differentiation rate of Xinjiang Ferula; a7) Use of the above-mentioned seedling and rooting medium to improve the seedling and / or rooting rate of Ferula sibiricum; a8) Use of the above-mentioned seedling and rooting culture medium in the preparation of a product that improves the seedling rate and / or rooting rate of Xinjiang Ferula.

[0040] The present invention establishes an efficient in vitro regeneration method for Xinjiang Ferula from seed embryos to complete in vitro regenerated plants through hormone regulation and optimization. This method not only improves the efficiency of seed embryo germination, but also achieves a high callus induction rate and seedling rate of Xinjiang Ferula, greatly improving the in vitro regeneration efficiency of Xinjiang Ferula and reducing the cost of in vitro regeneration. The efficient in vitro regeneration method for Xinjiang Ferula provided by the present invention not only lays a good foundation for Xinjiang Ferula variety improvement and biotechnology breeding, but also provides technical support for the protection and restoration of Xinjiang Ferula resources and their industrial application.

[0041] The beneficial effects of the present invention are as follows: (1) Efficient embryo germination: The existing technology uses gibberellins to activate embryo germination, which takes 60 days. The present invention uses a combination of KT and low-concentration 2,4-D hormones to break the dormancy of Xinjiang Ferula embryos. No low temperature and dark culture are required. The embryo can be efficiently germinated in just 7 days, which is shortened from 60 days of the traditional method to 7 days, greatly improving the efficiency of embryo germination.

[0042] (2) High-efficiency regeneration system: The present invention is the first to explore a tissue culture rapid propagation technology system for Xinjiang Ferula callus induction and somatic embryo differentiation to obtain regenerated plants. Callus tissue obtained by inducing cotyledons and hypocotyls can directly dedifferentiate to produce somatic embryos, without the need for multiple subcultures to induce somatic embryo differentiation. Embryonic callus differentiation takes only 14 days, and the callus induction rate and adventitious bud differentiation rate can reach 94.44% and 84.72%, respectively. Moreover, the seedling and rooting culture medium of the present invention does not add any hormones, which not only reduces the occurrence of deformed seedlings, but also achieves a seedling rate of 96.66% and a rooting rate of 90%. In addition, the transplant survival rate of regenerated plants can reach 43%, greatly improving the efficiency of obtaining regenerated plants.

[0043] (3) Short cycle and industrial application: The in vitro regeneration method of Xinjiang Ferula provided by the present invention has a short cycle. It only takes 59 days from the embryo to the transplantable surviving regenerated plant. The steps are simple, the number of subcultures is small, the adventitious bud proliferation multiples can reach more than 5 times, and the annual reproduction capacity can reach 100,000 plants. It can be directly used for large-scale rapid reproduction of the endangered medicinal plant Xinjiang Ferula, realizing factory-based seedling cultivation. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The flow chart of the in vitro regeneration method of Xinjiang Ferula of the present invention is shown. A is a photograph of an embryo after 7 days of culture in an embryo germination medium. B is a photograph of a hypocotyl and an embryo with existing callus tissue cultured in a callus induction and proliferation differentiation medium for 10 days, producing a large amount of embryonic callus tissue. C is a photograph of embryonic callus tissue cultured in a seedling formation and rooting medium for 14 days. D and E are photographs after 10 days of subculture. F is a photograph of a regenerated seedling. G is a photograph of a regenerated seedling that has not yet taken root and continues to be cultured for rooting. H is the root system of the regenerated seedling. I is a complete regenerated seedling.

[0045] Figure 2 This is a schematic diagram of the process for transplanting regenerated seedlings according to the present invention. A and B are Xinjiang Ferula regenerated seedlings. C is a complete regenerated seedling ready for transplanting. D and E are regenerated seedlings transplanted into a substrate. F is a surviving regenerated plant after transplanting. DETAILED DESCRIPTION

[0046] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0047] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0048] The MS basal medium in the following examples is a commonly used medium and can be purchased through commercial channels (such as Beijing Cool Laibo Biotechnology Co., Ltd.) or prepared by yourself according to the following composition and concentration: the solvent is water, and the solutes and their concentrations are as follows: macroelements: 1900 mg / L potassium nitrate (KNO3), 1650 mg / L ammonium nitrate (NH4NO3), 170 mg / L potassium dihydrogen phosphate (KH2PO4), 370 mg / L magnesium sulfate heptahydrate (MgSO4·7H2O), 440 mg / L calcium chloride dihydrate (CaCl2·2H2O); trace elements: 0.83 mg / L potassium iodide (KI), 6.2 mg / L boric acid (H3BO3), 22.3 mg / L manganese sulfate tetrahydrate (MnSO4·4H2O), 8.6 mg / L zinc sulfate heptahydrate (ZnSO4·7H2O), 0.25 mg / L sodium molybdate (Na2MoO4·2H2O), 0.025 mg / L Copper sulfate pentahydrate (CuSO4·5H2O), 0.025 mg / L cobalt chloride hexahydrate (CoCl2·6H2O); iron salt: 37.25 mg / L disodium ethylenediaminetetraacetic acid (Na2·EDTA), 27.85 mg / L ferrous sulfate heptahydrate (FeSO4·7H2O); organic components: 100 mg / L inositol, 2 mg / L glycine, 0.1 mg / L thiamine hydrochloride (VB1), 0.5 mg / L pyridoxine hydrochloride (VB6), 0.5 mg / L nicotinic acid (VB3) or nicotinamide (VPP).

[0049] In the following examples, 2,4-D (2,4-dichlorophenoxyacetic acid), KT (6-glycosylaminopurine), ZT (zeatin), NAA (naphthaleneacetic acid), and IBA (3-indolebutyric acid) were of analytical grade, and sucrose and Phytagel were products of Beijing Coolab Biotechnology Co., Ltd.

[0050] Example 1: A method for in vitro regeneration of Ferula xinjiangensis 1. Seed disinfection Select plump and complete Xinjiang Ferula seeds and place them in a 100 mL conical flask. Add 75% alcohol for sterilization for 30 seconds. After removing the alcohol, add 10% H2O2 and soak for 60 minutes. Then wash them with sterile water for 3-5 times. Then, soak the washed seeds in sterile water at 25°C in the dark for 36 hours to obtain sterilized soaked seeds.

[0051] 2. Germination of embryos (7 days) Use a dissecting needle to remove intact embryos from sterilized, soaked seeds and inoculate them into embryo germination medium, sowing 12 seeds per dish. After inoculation, the seeds were cultured at 22°C for 16 h in light and 20°C for 8 h in darkness. The seeds were divided into 10 groups based on the composition and concentration of the embryo germination medium. The formulation for each group is shown in Table 1. After 3 days of culture, continuous observation was performed to record the time when young embryos ceased germination. The final number of germinated explants was recorded, and the embryo germination rate was calculated. Embryo germination rate = (number of germinated explants / total number of explants) × 100%.

[0052] Table 1. Formula of embryo germination medium in each group

[0053] Experiments have shown that the germination rate of Xinjiang Ferula embryos in the embryo germination medium of groups A1 and A7 is relatively high, reaching over 80%. The germination rate of group A1 is even higher than that of group A7. After two days of culture in the embryo germination medium of group A1, the cotyledons of Xinjiang Ferula embryos begin to open and turn green. After another five days of culture, the cotyledons turn completely green. Therefore, the embryo germination medium of group A1 is determined to be the optimal embryo germination medium. Its formula is as follows: MS + 25 g / L sucrose + 0.1 mg / L KT + 0.1 mg / L 2,4-D + 2.0 g / L phytogel, with a pH of 5.6-5.8.

[0054] 3. Callus Induction and Differentiation (14 days) When the cotyledons grow to about 0.5 cm 2 When the hypocotyls reached 1-1.5 cm, the hypocotyls (0.3-0.5 cm in length) were cut and transferred to callus induction and proliferation differentiation medium for culture at 22°C with 16 h of light and 20°C with 8 h of darkness. The culture medium components and concentrations were used to divide the plants into 13 groups, as shown in Table 2. After some calli had formed at the base of the embryos, the plants were subcultured in the callus induction and proliferation differentiation medium. After 14 days of culture, extensive callus formed at both ends of the hypocotyls, and some calli had entered the somatic embryonic differentiation stage, with numerous cotyledonary embryos clearly visible. The callus induction rate was calculated daily after 5 days of culture in the callus induction and proliferation differentiation medium, and the adventitious bud differentiation rate was calculated daily after 7 days of culture in the callus induction and proliferation differentiation medium. The callus induction rate = (number of explants with observed callus / total number of explants) × 100%. Adventitious bud differentiation rate = (number of explants with observed adventitious bud differentiation / total number of explants) × 100%.

[0055] The experiments revealed that the explants in the B3 callus induction and proliferation differentiation medium exhibited the best induction and differentiation, with a callus induction rate of 94.44% and an adventitious bud differentiation rate of 84.72%. Therefore, the B3 callus induction and proliferation differentiation medium was determined to be the optimal medium for callus induction and proliferation differentiation. Its formula was as follows: MS + 0.5 mg / L NAA + 1.0 mg / L ZT + 25 g / L sucrose + 2.0 g / L phytogel, with a pH of 5.6-5.8.

[0056] Table 2. Formulas of callus induction and proliferation and differentiation culture media in each group

[0057] 4. Seedling formation and rooting culture (24 days) Calli that had differentiated into somatic embryos were transferred to seedling and rooting medium and cultured at 22°C with 16 h of light and 20°C with 8 h of darkness. Six groups were assigned based on the composition and concentration of the seedling and rooting medium. The formulations for each group are shown in Table 3. After 14 days of culture, the somatic embryos differentiated into seedlings (regenerated seedlings without roots) and were then transferred to seedling and rooting medium for further culture at 22°C with 16 h of light and 20°C with 8 h of darkness. After 10 days of culture, regenerated Xinjiang Ferula seedlings with robust root systems (roots 2-5 cm long) and intact plants were obtained. The seedling rate was continuously counted after 5 days of culture in the seedling and rooting medium, and the rooting rate was counted after 7 days of culture in the seedling and rooting medium. The seedling rate = (number of observed normal seedlings / total number of inoculated explants) × 100%. The rooting rate = (number of observed roots / total number of inoculated explants) × 100%.

[0058] Experiments revealed that callus tissue that had differentiated into somatic embryos exhibited the best seedling and rooting results when grown in the C1 seedling and rooting medium, achieving a seedling rate of 96.66% and a rooting rate of 90%, with robust and complete root systems. Therefore, the optimal seedling and rooting medium was determined to be C1, with the following formulation: MS + 20 g / L sucrose + 2.0 g / L phytogel, with a pH of 5.6-5.8.

[0059] Table 3. Formulas of seedling and rooting medium for each group

[0060] 5. Transplanting (14 days) Regenerated seedlings with roots extending to at least 2.5 cm and showing no vitrification or deformities should be transferred from the tissue culture room to the greenhouse. First, partially open the culture bottle cap and harden for two days. Then, fully open the bottle cap and harden for another two days. The seedlings should then be washed, soaked in carbendazim for 10-15 minutes, allowed to dry, and transplanted into a substrate (peat moss: vermiculite = 2:1:1, v / v). During transplanting, ensure only the roots are shallowly buried in the substrate, avoiding the remaining growth point. After transplanting, culture the seedlings in a transparent seedling box with a lid in a temperature range of 65% relative humidity, 22°C for 16 h light / 20°C for 8 h dark. Open the lid three to four times daily for short periods (a few minutes to ten minutes) of ventilation. Observe the seedlings daily. Once new leaves are clearly visible and the plants are standing upright and vigorous, gradually increase the ventilation time and eventually completely remove the lid. After 10 days of cultivation, the seedlings were observed to be healthy, without yellowing or wilting, and to have grown new leaves, indicating survival after transplantation. These regenerated plants were then obtained and their transplant survival rate was calculated. The transplant survival rate of the regenerated plants was calculated as (number of surviving seedlings after transplantation / total number of transplanted seedlings) × 100%. Experiments have shown that the transplant survival rate of the regenerated plants of the present invention can reach 43%.

[0061] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A method for in vitro regeneration of Xinjiang Ferula, comprising the following steps: 1) culturing embryos of Xinjiang Ferula in an embryo germination medium to obtain germinated embryos; the embryo germination medium contains KT and 2,4-D; 2) culturing the hypocotyls obtained from the germinated embryos in a callus induction and proliferation differentiation medium to obtain callus tissue that has differentiated into somatic embryos; the callus induction and proliferation differentiation medium contains NAA and ZT; 3) culturing the callus tissue that has been differentiated to obtain somatic embryos in a seedling and rooting medium to obtain Xinjiang Ferula regenerated seedlings.

2. The method according to claim 1, wherein: The method further comprises the following steps before step 1): sterilizing Xinjiang Ferula seeds and obtaining Xinjiang Ferula embryos from the sterilized seeds.

3. The method according to claim 1 or 2, characterized in that: In the step 1), the concentration of KT in the embryo germination medium is 0.05-0.2 mg / L; Alternatively, the concentration of 2,4-D in the embryo germination medium is 0.05-0.2 mg / L.

4. The method according to any one of claims 1 to 3, characterized in that: In the step 1), the culture conditions are culturing at 22° C. with 16 h of light and 20° C. with 8 h of darkness for 5-7 days.

5. The method according to any one of claims 1 to 4, characterized in that: In the step 2), the concentration of NAA in the callus induction and proliferation differentiation medium is 0.2-0.5 mg / L; Alternatively, the concentration of ZT in the callus induction and proliferation differentiation medium is 0.5-2.0 mg / L.

6. The method according to any one of claims 1 to 5, characterized in that: In the step 2), the culture conditions are culturing at 22° C. with 16 h of light and 20° C. with 8 h of darkness for 10-14 days.

7. The method according to any one of claims 1 to 6, characterized in that: In step 3), the seedling and rooting medium includes sucrose; Alternatively, the concentration of the sucrose in the seedling and rooting medium is 20-30 g / L.

8. The method according to any one of claims 1 to 7, characterized in that: After step 3), the method further comprises transplanting the Xinjiang Ferula regenerated seedlings into a substrate for cultivation to obtain Xinjiang Ferula regenerated plants.

9. A set of reagents for in vitro regeneration culture of Xinjiang Ferula, comprising the embryo germination medium according to any one of claims 1 to 8, the callus induction and proliferation differentiation medium according to any one of claims 1 to 8, and the seedling and rooting medium according to any one of claims 1 to 8.

10. Any application of a1) to a8) below: a1) Use of the reagent set according to claim 9 in in vitro regeneration of Ferula foetida; a2) Use of the reagent set according to claim 9 in the preparation of a product for in vitro regeneration of Ferula foetida; a3) Use of the embryo germination medium according to any one of claims 1 to 8 in improving the germination efficiency of Xinjiang Ferula embryos; a4) Use of the embryo germination medium according to any one of claims 1 to 8 in the preparation of a product for improving the germination efficiency of Xinjiang Ferula embryos; a5) Use of the callus induction and proliferation differentiation medium according to any one of claims 1 to 8 in improving the callus induction rate and / or differentiation rate of Ferula sinica; a6) Use of the callus induction and proliferation differentiation medium according to any one of claims 1 to 8 in the preparation of a product for improving the callus induction rate and / or differentiation rate of Ferula foetida; a7) Use of the seedling and rooting medium according to any one of claims 1 to 8 for improving the seedling and / or rooting rate of Ferula foetida; a8) Use of the seedling and rooting medium according to any one of claims 1 to 8 in the preparation of a product for improving the seedling and / or rooting rate of Xinjiang Ferula.

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