Establishment method of efficient fenugreek regeneration system
By using a specific culture medium and growth regulator in fenugreek tissue culture, the problems of low regeneration efficiency and poor stability of fenugreek were solved, achieving efficient adventitious bud differentiation, proliferation and rooting, providing a stable regeneration system and providing technical support for fenugreek germplasm breeding.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- XINJIANG UYGUR AUTONOMOUS REGION DRUG RESEARCH INSTITUTE
- Filing Date
- 2026-03-16
- Publication Date
- 2026-05-12
AI Technical Summary
Existing fenugreek tissue culture techniques suffer from low regeneration efficiency and poor stability, and the rooting and transplant survival rates need to be improved.
Using a primary induction medium, a proliferation medium, and a rooting medium with specific compositions, 5-azacytidine, spermidine, fluazinon, trehalose, paclobutrazol, uridine, sodium nitroprusside, and methyl jasmonate were added respectively. Through the synergistic effect of different culture stages, adventitious shoots were induced to form, expand, and root.
The study achieved an adventitious bud differentiation rate of 91.3% for fenugreek, a proliferation coefficient of 10.8, and a rooting rate of 97.5%, significantly improving the propagation speed and seedling quality, making it suitable for large-scale production.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of medicinal plant tissue culture technology, specifically relating to a method for establishing a high-efficiency regeneration system for fenugreek. Background Technology
[0003] Fenugreek ( Fenugreek L. (Trigonella foenum-graecum) is a core ingredient in the formula of Hanchuan Zupa Granules, exhibiting unique industrial value. Its seeds are rich in active ingredients such as trigonelline, diosgenin, and flavonoids, possessing various effects including lowering blood sugar, blood lipids, cholesterol, anti-oxidation, and anti-inflammation. It is used to treat bronchitis, cough and nasal congestion, cold hernia, abdominal distension, cold-damp beriberi, and kidney deficiency with lower back pain. The plant grows to about 60 cm tall, with a well-developed root system, erect stem, pinnate trifoliate leaves, and yellowish-white or pale yellow corollas. The pods average 7.5 cm in length, each containing an average of 29 seeds; it is commonly known as bitter bean, fragrant bean, or fenugreek. It is widely commercially cultivated in many countries. Fenugreek possesses drought-resistant, cold-resistant, and heat-resistant properties.
[0004] Plant tissue culture technology is an important foundation for achieving rapid in vitro propagation, germplasm resource preservation, and genetic transformation. Currently, there are some research reports on fenugreek tissue culture, but existing techniques still have many shortcomings, mainly in the following aspects: (1) low regeneration efficiency and poor stability; (2) the survival rate of rooting and transplanting needs to be improved. Therefore, developing an efficient and stable fenugreek regeneration system is very meaningful for accelerating the propagation of superior fenugreek germplasm. Summary of the Invention
[0005] In order to overcome the above-mentioned shortcomings of the existing technology, the purpose of this invention is to provide a method for establishing a high-efficiency regeneration system for fenugreek, which can realize the high-quality and efficient propagation of fenugreek seedlings.
[0006] The technical solution of this invention to solve the above-mentioned technical problems is as follows: A method for establishing a high-efficiency regeneration system for fenugreek is provided, comprising the following steps: (1) Primary induction culture: Fenugreek explants were inoculated into primary induction culture medium for induction culture to produce adventitious shoots; wherein, the primary induction culture medium includes: basal culture medium, growth regulator, 10 -9 ~10 -11 M 5-azacytidine, 0.1–0.5 mM spermidine and 3–8 μM fluazinon; (2) Proliferation culture: After the adventitious buds are divided, they are transferred to the proliferation culture medium to achieve the amplification of adventitious buds; wherein, the proliferation culture medium includes: basal culture medium, growth regulator, 1~3 wt% trehalose, 0.02~0.08 mg / L paclobutrazol and 8~12 μM uridine; (3) Rooting culture: The stem segments obtained from the proliferation culture in step (2) are transferred to the rooting culture medium for culture to induce rooting; wherein, the rooting culture medium includes: basal culture medium, growth regulator, 10~20 μM sodium nitroprusside and 0.1~10 μmol / L methyl jasmonate.
[0007] The beneficial effects of this invention are as follows: The basal culture medium in the fenugreek regeneration system provides basic substances such as carbon sources and inorganic salts, while specific active ingredients are added at each culture stage. For example, the primary induction medium mainly induces the production of adventitious shoots from fenugreek explants. This medium includes basal culture medium, growth regulators, and also contains 5-azacytidine, spermidine, and fluazinon. 5-azacytidine, by incorporating into the DNA chain, captures DNA methyltransferases, leading to DNA demethylation and making cells more responsive to signals from external growth regulators. In the primary induction stage, explants are under in vitro stress and are prone to endogenous accumulation of ABA (abscisic acid), inhibiting cell division and shoot differentiation. Fluazinon, by blocking ABA synthesis, reduces the ABA level in the explants, thereby relieving the inhibitory effect on cell division and shoot primordia formation. Speridine plays the role of a "cell division and morphogenesis regulator" during tissue culture, stabilizing cell state. In the initial induction medium, 5-azacytidine is responsible for "unlocking" genes, fluazindrone is responsible for "eliminating" inhibitory signals, and spermidine is responsible for "stabilizing" cell state and providing impetus for division. Combined with the direct induction effect of growth regulators, the four substances work synergistically, and together with the basal medium and growth regulators, significantly improve the ability of explants to transform into regenerated shoots.
[0008] The main focus of proliferation culture is to rapidly and efficiently amplify adventitious shoots, addressing issues such as vitrification and weak growth. The proliferation culture medium includes: basal medium, growth regulators, trehalose, paclobutrazol, and uridine. In the proliferation medium, trehalose not only serves as a slowly released carbon source, but more importantly, its high hydration capacity allows it to replace water molecules and bind to membrane phospholipids and proteins, forming a protective membrane that effectively prevents cell dehydration and membrane phase transition, reducing vitrification, a common phenomenon in tissue culture. Paclobutrazol makes adventitious shoots compact and sturdy, resulting in high-quality regenerated shoots for rooting. Urate is a prerequisite for RNA synthesis, specifically pyrimidine nucleotides. During rapid proliferation, cells need to synthesize large amounts of RNA to meet protein synthesis demands; exogenous uridine supplementation accelerates energy and nucleic acid metabolism, providing a sufficient material basis for rapid cell division. In the proliferation medium, trehalose provides physicochemical protection, ensuring cell structural stability; paclobutrazol optimizes shoot morphology and quantity; and uridine directly provides raw materials for nucleic acid synthesis. The synergistic effect of these three, combined with the basal medium and growth regulators, achieves high-quality, large-scale, and efficient proliferation of shoots.
[0009] Rooting culture induces stem segments to develop well-developed, healthy root systems, improving transplant survival rates. Rooting media include: basal medium, growth regulators, sodium nitroprusside, and methyl jasmonate. Exogenous sodium nitroprusside mimics and enhances endogenous NO signaling, initiating and accelerating root primordia formation. Methyl jasmonate induces the synthesis and deposition of lignin and suberin in the rooting sites, promoting vascular tissue differentiation and root development, resulting in more complete and mechanically strong roots. The combination of sodium nitroprusside and methyl jasmonate, along with the basal medium and growth regulators, regulates root primordia induction and root development, ultimately yielding regenerated plants with well-developed root systems and high vigor.
[0010] Based on the above technical solution, the present invention can be further improved as follows: Furthermore, the primary induction medium in step (1) includes: basal medium, growth regulator, 10 -9 M 5-azacytidine, 0.3 mM spermidine and 5 μM fluazinon.
[0011] Furthermore, the growth regulators in the primary induction medium in step (1) include 0.5~1.5 mg / L of α-naphthaleneacetic acid (NAA) and 0.1~0.8 mg / L of 6-benzylaminopurine (6-BA).
[0012] The technical effects of the above-mentioned further technical solutions are as follows: In the initial induction stage, the fenugreek stem segments are transformed into a meristematic state and eventually form adventitious buds. During this process, the main role of NAA is to initiate dedifferentiation and induce cells to form callus tissue or competent cells. That is, a specific concentration of NAA helps cells break the original differentiation state and regain the ability to divide. On the basis of NAA initiating multidifferentiation, 6-BA is responsible for promoting cells to enter the division cycle and guiding these dividing cells to develop in the direction of bud formation. Combined with 5-azacytidine to open the pluripotency potential of cells, spermidine to protect cells from oxidative damage, and fluazindrone to remove the inhibition of endogenous ABA, these substances work synergistically to break through the traditional fenugreek regeneration bottleneck and achieve a highly efficient initial induction effect.
[0013] Furthermore, the growth regulators in the primary induction medium in step (1) include 1.0 mg / L of α-naphthaleneacetic acid and 0.5 mg / L of 6-benzylaminopurine.
[0014] Furthermore, the proliferation culture medium in step (2) includes: basal culture medium, growth regulator, 1 wt% trehalose, 0.05 mg / L paclobutrazol and 10 μM uridine.
[0015] Furthermore, the growth regulators in the proliferation medium in step (2) include 0.2-0.8 mg / L of 6-benzylaminopurine and 0.1-0.5 mg / L of α-naphthaleneacetic acid.
[0016] The technical effects of the above-mentioned further technical solution are as follows: During the proliferation stage of adventitious buds, two major problems often exist: firstly, only callus tissue grows into buds; secondly, although there are many buds, they are thin and weak, prone to vitrification, and unable to root. In the proliferation culture process of this invention, 6-BA drives cell division and bud formation, NAA guides orderly bud differentiation, paclobutrazol inhibits excessive growth caused by GA, resulting in short, sturdy, and compact buds, trehalose prevents cell vitrification during rapid growth, and uridine provides sufficient energy for the entire process. In this stage of culture, this invention can obtain high-quality, highly vigorous adventitious buds without sacrificing the proliferation rate.
[0017] Furthermore, the growth regulators in the proliferation medium in step (2) include 0.6 mg / L of 6-benzylaminopurine and 0.2 mg / L of α-naphthaleneacetic acid.
[0018] Furthermore, the rooting medium in step (3) includes: basal medium, growth regulator, 15 μM sodium nitroprusside and 5 μmol / L methyl jasmonate.
[0019] Furthermore, the growth regulators in the rooting medium in step (3) include 0.5~1.5 mg / L of α-naphthaleneacetic acid and 0.2~0.8 mg / L of 6-benzylaminopurine.
[0020] The technical effects of adopting the above-mentioned further technical solutions are as follows: During the rooting culture stage, the main objective is to induce the differentiation of basal cells in the stem segment into root primordia, which then further develop into a complete root system. During the rooting culture process, NAA promotes the differentiation of basal cells in the stem segment and the formation of root primordia; 6-BA synergistically promotes the early differentiation of root primordia with NAA; sodium nitroprusside amplifies and stabilizes the rooting induction effect through NO signaling; and methyl jasmonate induces the synthesis of lignin and other substances, constructing a well-developed root system with complete structure and high mechanical strength. These substances work synergistically to achieve a well-developed root system and improve the transplant survival rate.
[0021] Furthermore, the growth regulators in the rooting medium in step (3) include 1.0 mg / L of α-naphthaleneacetic acid and 0.5 mg / L of 6-benzylaminopurine.
[0022] Furthermore, the basal culture medium in steps (1), (2) and (3) is MS medium.
[0023] The present invention has the following beneficial effects: This invention uses sterile stem segments of fenugreek seedlings as explants to construct a regeneration system. Adventitious buds were successfully induced using a primary induction medium, solving the problem of difficult regeneration of fenugreek adventitious buds. After adventitious bud proliferation culture and rooting culture, regenerated tissue culture seedlings were obtained, providing technical support for the construction of a stable genetic transformation system for fenugreek.
[0024] The efficient, sterile, and rapid propagation system for fenugreek established in this invention achieves an adventitious bud differentiation rate of 91.3%, a proliferation coefficient of 10.8, and a rooting rate of 97.5%. It has a fast propagation speed and can be used for large-scale production of high-quality seedlings, while improving both yield and quality. Detailed Implementation
[0025] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.
[0026] Example 1: A method for establishing a high-efficiency regeneration system for fenugreek includes the following steps: (1) Primary induction culture The hypocotyls of aseptic fenugreek seedlings (5 cm tall, 30 days old) were cut, and 2 cm long aseptic stem segments were inoculated into primary induction medium and cultured in a dark room at 24°C for 10 days, followed by 15 days of light culture at 24°C. The differentiation of adventitious buds on the explants was recorded (buds ≥0.5 mm in length were considered valid). While observing the presence of callus tissue on the stem segments, the presence of explant contamination, mold, or other abnormalities was also observed.
[0027] The primary induction medium described above includes: MS basal medium and the following components at final concentrations: 0.1 mg / L 6-benzylaminopurine (6-BA), 0.5 mg / L α-naphthaleneacetic acid (NAA), 10 -9 M 5-azacytidine, 0.1 mM spermidine and 3 μM fluazinon.
[0028] (2) Proliferation culture The adventitious buds formed in the initial induction culture were divided into 2 cm single buds and then transferred to the proliferation medium. They were first cultured in the dark at 24℃ for 10 days, and then transferred to normal light conditions at 24℃ for another 20 days. The total number of stem segments for each treatment was counted and the proliferation coefficient was calculated.
[0029] The above-mentioned proliferation medium includes: MS basal medium and the following components at final concentrations: 0.2 mg / L 6-benzylaminopurine (6-BA), 0.1 mg / L α-naphthaleneacetic acid (NAA), 1 wt% trehalose, 0.02 mg / L paclobutrazol and 8 μM uridine.
[0030] (3) Rooting culture Select robust single buds, 3-4 cm tall, obtained from propagation culture. Remove the callus tissue and 1-2 mm of aged tissue from the base. Inoculate the buds into rooting medium and culture at 24℃ under light for 20 days. After 20 days, record the number of rooted buds per bottle, the average root length, and calculate the rooting rate.
[0031] The above-mentioned rooting medium includes: MS basal medium and the following components at final concentrations: 0.5 mg / L α-naphthaleneacetic acid (NAA), 0.2 mg / L 6-benzylaminopurine (6-BA), 10 μM sodium nitroprusside and 0.1 μmol / L methyl jasmonate.
[0032] Example 2: A method for establishing a high-efficiency regeneration system for fenugreek includes the following steps: (1) Primary induction culture The hypocotyls of aseptic fenugreek seedlings (5 cm tall, 30 days old) were cut, and 2 cm long aseptic stem segments were inoculated into primary induction medium and cultured in a dark room at 24°C for 10 days, followed by 15 days of light culture at 24°C. The differentiation of adventitious buds on the explants was recorded (buds ≥0.5 mm in length were considered valid). While observing the presence of callus tissue on the stem segments, the presence of explant contamination, mold, or other abnormalities was also observed.
[0033] The primary induction medium described above included: MS basal medium and the following components at final concentrations: 0.8 mg / L 6-benzylaminopurine (6-BA), 1.5 mg / L α-naphthaleneacetic acid (NAA), and 10... -11 M 5-azacytidine, 0.5 mM spermidine and 8 μM fluazinon.
[0034] (2) Proliferation culture The adventitious buds formed in the initial induction culture were divided into 2 cm single buds and then transferred to the proliferation medium. They were first cultured in the dark at 24℃ for 10 days, and then transferred to normal light conditions at 24℃ for another 20 days. The total number of stem segments for each treatment was counted and the proliferation coefficient was calculated.
[0035] The above-mentioned proliferation medium includes: MS basal medium and the following components at final concentrations: 0.8 mg / L 6-benzylaminopurine (6-BA), 0.5 mg / L α-naphthaleneacetic acid (NAA), 3 wt% trehalose, 0.08 mg / L paclobutrazol and 12 μM uridine.
[0036] (3) Rooting culture Select robust single buds, 3-4 cm tall, obtained from propagation culture. Remove the callus tissue and 1-2 mm of aged tissue from the base. Inoculate the buds into rooting medium and culture at 24℃ under light for 20 days. After 20 days, record the number of rooted buds per bottle, the average root length, and calculate the rooting rate.
[0037] The above-mentioned rooting medium includes: MS basal medium and the following components at final concentrations: 1.5 mg / L α-naphthaleneacetic acid (NAA), 0.8 mg / L 6-benzylaminopurine (6-BA), 20 μM sodium nitroprusside and 10 μmol / L methyl jasmonate.
[0038] Example 3: A method for establishing a high-efficiency regeneration system for fenugreek includes the following steps: (1) Primary induction culture The hypocotyls of aseptic fenugreek seedlings (5 cm tall, 30 days old) were cut, and 2 cm long aseptic stem segments were inoculated into primary induction medium and cultured in a dark room at 24°C for 10 days, followed by 15 days of light culture at 24°C. The differentiation of adventitious buds on the explants was recorded (buds ≥0.5 mm in length were considered valid). While observing the presence of callus tissue on the stem segments, the presence of explant contamination, mold, or other abnormalities was also observed.
[0039] The primary induction medium described above includes: MS basal medium and the following components at final concentrations: 0.5 mg / L 6-benzylaminopurine (6-BA), 1.0 mg / L α-naphthaleneacetic acid (NAA), 10 -9 M 5-azacytidine, 0.3 mM spermidine and 5 μM fluazinon.
[0040] (2) Proliferation culture The adventitious buds formed in the initial induction culture were divided into 2 cm single buds and then transferred to the proliferation medium. They were first cultured in the dark at 24℃ for 10 days, and then transferred to normal light conditions at 24℃ for another 20 days. The total number of stem segments for each treatment was counted and the proliferation coefficient was calculated.
[0041] The above-mentioned proliferation medium includes: MS basal medium and the following components at final concentrations: 0.6 mg / L 6-benzylaminopurine (6-BA), 0.2 mg / L α-naphthaleneacetic acid (NAA), 1 wt% trehalose, 0.05 mg / L paclobutrazol and 10 μM uridine.
[0042] (3) Rooting culture Select robust single buds, 3-4 cm tall, obtained from propagation culture. Remove the callus tissue and 1-2 mm of aged tissue from the base. Inoculate the buds into rooting medium and culture at 24℃ under light for 20 days. After 20 days, record the number of rooted buds per bottle, the average root length, and calculate the rooting rate.
[0043] The above-mentioned rooting medium includes: MS basal medium and the following components at final concentrations: 1.0 mg / L α-naphthaleneacetic acid (NAA), 0.5 mg / L 6-benzylaminopurine (6-BA), 15 μM sodium nitroprusside and 5 μmol / L methyl jasmonate.
[0044] Comparative Example 1: Compared with Example 3, Comparative Example 1 did not contain 5-azacytidine, spermidine, and fluazinam in the primary induction medium; it did not contain trehalose, paclobutrazol, and uridine in the proliferation medium; and it did not contain sodium nitroprusside and methyl jasmonate in the rooting medium. The rest of the contents were the same as in Example 3.
[0045] Comparative Example 2: Compared with Example 3, Comparative Example 2 did not contain 5-azacytidine in the primary induction culture medium, but was otherwise the same as Example 3.
[0046] Comparative Example 3: Compared with Example 3, Comparative Example 3 did not contain spermidine in the primary induction culture medium, but was otherwise the same as Example 3.
[0047] Comparative Example 4: Compared with Example 3, Comparative Example 4 did not contain fluazinam in the primary induction medium, but was otherwise the same as Example 3.
[0048] Comparative Example 5: Compared with Example 3, Comparative Example 5 did not contain trehalose in its proliferation culture medium, but was otherwise the same as Example 3.
[0049] Comparative Example 6: Compared with Example 3, Comparative Example 6 did not contain paclobutrazol in its proliferation medium, but was otherwise identical to Example 3.
[0050] Comparative Example 7: Compared with Example 3, Comparative Example 7 did not contain uridine in its proliferation culture medium, but was otherwise the same as Example 3.
[0051] Comparative Example 8: Compared with Example 3, Comparative Example 8 did not contain sodium nitroprusside in its rooting medium, but was otherwise identical to Example 3.
[0052] Comparative Example 9: Compared with Example 3, Comparative Example 9 did not contain methyl jasmonic acid in its rooting medium, but was otherwise identical to Example 3.
[0053] Experimental Field Transplantation 1. Seedling hardening treatment The rooted tissue culture seedlings obtained in Examples 1-3 and Comparative Examples 1-9 were moved to room temperature and natural light conditions and hardened off for 3 days with the lids off.
[0054] 2. Substrate preparation Mix peat moss, perlite, and vermiculite in a volume ratio of 3:1:1, then spray with a 0.1% potassium permanganate solution, stir, cover with a film and leave for 24 hours, then fill into seedling trays and water thoroughly.
[0055] 3. Transplanting After hardening off, wash the seedlings with clean water to remove the culture medium attached to the roots, soak the roots in 0.1% carbendazim solution for 2 minutes, and then transplant them into the substrate for routine management. During this period, observe the seedling condition and root condition, and calculate the transplant survival rate.
[0056] The relevant index data results of Examples 1-3 and Comparative Examples 1-9 are shown in the table below:
[0057] As shown in the table above, during the initial induction stage, the absence of any one of 5-azacytidine, spermidine, or fluridone in the culture medium significantly affected the differentiation rate of adventitious shoots in fenugreek. Only when 5-azacytidine, spermidine, and fluridone were added to the culture medium did the three substances work synergistically to improve the differentiation rate of adventitious shoots, nearly doubling it compared to Comparative Example 1. During the proliferation culture, the absence of trehalose resulted in severe vitrification and impaired proliferation; the absence of paclobutrazol led to excessive vegetative growth, significantly affecting the proliferation coefficient; and the absence of uridine resulted in insufficient energy and impaired proliferation. Only the synergistic effect of all three substances significantly improved the proliferation coefficient, increasing it fourfold compared to Comparative Example 1. During the rooting stage, sodium nitroprusside provides NO signals to initiate rooting, while methyl jasmonate induces lignin to strengthen the root system. The two work synergistically to increase the rooting rate from 58.3% (Comparative Example 1) to 97.5%, an increase of 67%. The root lengths of Comparative Example 8 (deficient in sodium nitroprusside) and Comparative Example 9 (deficient in methyl jasmonate) were significantly lower than in Example 3, and root branching was also poor, demonstrating that sodium nitroprusside and methyl jasmonate have a synergistic promoting effect on root elongation and branching. Furthermore, compared with the comparative examples, the transplant survival rate of the embodiments of this invention increased from 45.2% to 96.2%, an increase of 1.1 times.
[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for establishing a high-efficiency regeneration system for fenugreek, characterized in that, Includes the following steps: (1) Primary induction culture: Fenugreek explants were inoculated into primary induction culture medium for induction culture to produce adventitious shoots; wherein, the primary induction culture medium includes: basal culture medium, growth regulator, 10 -9 ~10 -11 M 5-azacytidine, 0.1–0.5 mM spermidine and 3–8 μM fluazinon; (2) Proliferation culture: After the adventitious buds are divided, they are transferred to the proliferation culture medium to achieve the amplification of adventitious buds; wherein, the proliferation culture medium includes: basal culture medium, growth regulator, 1~3 wt% trehalose, 0.02~0.08 mg / L paclobutrazol and 8~12 μM uridine; (3) Rooting culture: The stem segments obtained from the proliferation culture in step (2) are transferred to the rooting culture medium for culture to induce rooting; wherein, the rooting culture medium includes: basal culture medium, growth regulator, 10~20 μM sodium nitroprusside and 0.1~10 μmol / L methyl jasmonate.
2. The method for establishing a high-efficiency regeneration system for fenugreek according to claim 1, characterized in that, The primary induction medium in step (1) includes: basal medium, growth regulator, 10 -9 M 5-azacytidine, 0.3 mM spermidine and 5 μM fluazinon.
3. The method for establishing a high-efficiency regeneration system for fenugreek according to claim 1 or 2, characterized in that, The growth regulators in the primary induction medium in step (1) include 0.5-1.5 mg / L of α-naphthaleneacetic acid and 0.1-0.8 mg / L of 6-benzylaminopurine.
4. The method for establishing a high-efficiency regeneration system of fenugreek according to claim 3, characterized in that, The growth regulators in the primary induction medium in step (1) include 1.0 mg / L of α-naphthaleneacetic acid and 0.5 mg / L of 6-benzylaminopurine.
5. The method for establishing a high-efficiency regeneration system for fenugreek according to claim 1, characterized in that, The proliferation medium in step (2) includes: basal medium, growth regulator, 1 wt% trehalose, 0.05 mg / L paclobutrazol and 10 μM uridine.
6. The method for establishing a high-efficiency regeneration system of fenugreek according to claim 1 or 5, characterized in that, The growth regulators in the proliferation medium in step (2) include 0.2-0.8 mg / L of 6-benzylaminopurine and 0.1-0.5 mg / L of α-naphthaleneacetic acid.
7. The method for establishing a high-efficiency regeneration system of fenugreek according to claim 6, characterized in that, The growth regulators in the proliferation medium in step (2) include 0.6 mg / L of 6-benzylaminopurine and 0.2 mg / L of α-naphthaleneacetic acid.
8. The method for establishing a high-efficiency regeneration system for fenugreek according to claim 1, characterized in that, The rooting medium in step (3) includes: basal medium, growth regulator, 15 μM sodium nitroprusside and 5 μmol / L methyl jasmonate.
9. The method for establishing a high-efficiency regeneration system of fenugreek according to claim 1 or 8, characterized in that, The growth regulators in the rooting medium in step (3) include 0.5-1.5 mg / L of α-naphthaleneacetic acid and 0.2-0.8 mg / L of 6-benzylaminopurine.
10. The method for establishing a high-efficiency regeneration system of fenugreek according to claim 9, characterized in that, The growth regulators in the rooting medium in step (3) include 1.0 mg / L of α-naphthaleneacetic acid and 0.5 mg / L of 6-benzylaminopurine.