Application of strigolactone biosynthesis inhibitor in promoting gastrodia elata seed breeding

Abstract

The invention belongs to the technical field of traditional Chinese medicine planting, and particularly relates to application of strigolactone biosynthesis inhibitor in promoting gastrodia elata seed breeding. The strigolactones biosynthesis inhibitor has the function of specifically blocking SL synthesis key enzyme, and is used for treating gastrodia elata tubers, so that the content of superoxide anions in gastrodia elata and the activity of superoxide dismutase and catalase can be enhanced, the content of soluble solids and malondialdehyde in gastrodia elata and the activity of peroxidase are reduced, and the yield of gastrodia elata is increased. Therefore, the bud dormancy of the gastrodia elata is favorably broken, the germination of latent buds of the gastrodia elata is promoted, and efficient, simple and large-scale breeding of the gastrodia elata seeds is realized.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine planting technology, specifically involving the application of strigolactone biosynthesis inhibitors in promoting the propagation of Gastrodia elata seeds. Background Technology

[0002] Gastrodia elata is a traditional and precious Chinese medicinal herb with effects such as calming wind and stopping spasms, and suppressing liver yang. It has a wide range of applications. Its original plant, Gastrodia elata ( Gastrodia elata Blume is an orchid ( Orchidaceae A perennial herbaceous plant, without roots, unable to perform photosynthesis, and requires association with Agaricales (Agaricales order). Agaricales ) fungus Armillaria mellea ( Armillaria Gastrodia elata (ssp.) forms a mycorrhizal symbiotic relationship to complete its life activities. Cultivation and production of Gastrodia elata requires first propagating seed tubers, then inoculating the propagated seed tubers with Armillaria mellea for symbiotic cultivation to produce commercial Gastrodia elata. Seed tuber propagation is the primary step in Gastrodia elata cultivation and production. In recent years, the market demand for Gastrodia elata has increased dramatically, and its planting scale has expanded rapidly, placing higher demands on the efficiency of seed tuber propagation. Currently, there are two methods for propagating Gastrodia elata seed tubers: sexual reproduction and asexual reproduction. Sexual reproduction requires first harvesting seeds, then using germination fungi to induce seed germination to form protocorms, which then gradually develop into seed tubers (also called white tubers), which are directly used in production. Asexual reproduction involves inducing the development of several dormant buds from the seed tubers obtained through sexual reproduction. These dormant buds further develop into seed tubers (i.e., second-generation seed tubers), which are then used in production, essentially expanding the production of seed tubers.

[0003] However, the sexual reproduction method of Gastrodia elata seeds requires annual seed production and harvesting, which has limitations such as long time frame, complex operation, and high cost, seriously affecting the efficiency of Gastrodia elata seed propagation and greatly restricting the development of the Gastrodia elata cultivation industry. Therefore, there is an urgent need to develop more efficient Gastrodia elata seed propagation technology. The key to the asexual reproduction method of Gastrodia elata lies in the growth and development of dormant buds. The more and faster the dormant buds grow and develop, the more and faster the seed products will be obtained. However, no effective measures have yet been found to promote the growth and development of dormant buds. In view of this, how to accelerate the development process of dormant buds in Gastrodia elata, thereby improving the efficiency of Gastrodia elata seed propagation, has become an urgent problem to be solved in this field. Summary of the Invention

[0004] The purpose of this invention is to provide the application of strigolactone biosynthesis inhibitors in promoting the propagation of Gastrodia elata seeds. The strigolactone biosynthesis inhibitors can promote the propagation of Gastrodia elata seeds, achieving efficient, simple and large-scale propagation of Gastrodia elata seeds.

[0005] This invention provides the application of strigolactone biosynthesis inhibitors in promoting the propagation of Gastrodia elata seeds.

[0006] As a preferred embodiment, the strigolactone biosynthesis inhibitor is Tis108; the chemical formula of Tis108 is shown in Formula I: Formula I.

[0007] As a preferred embodiment, the effective concentration of Tis108 is 0.001~20 μM.

[0008] As a preferred embodiment, promoting the propagation of Gastrodia elata seeds includes increasing the number of latent buds that germinate and / or shortening the growth and development time of latent buds.

[0009] This invention also provides a method for promoting the propagation of Gastrodia elata seed tubers, comprising the following steps: soaking Gastrodia elata tubers in an aqueous solution of Tis108, a biosynthesis inhibitor of strigolactone, to obtain soaked Gastrodia elata tubers; and symbiotically culturing the soaked Gastrodia elata tubers with Armillaria mellea spawn to obtain Gastrodia elata seed tubers. The concentration of Tis108, a biosynthesis inhibitor of strigolactone, in the aqueous solution is 0.001~20 μM.

[0010] As a preferred embodiment, the soaking time is 2 to 10 minutes.

[0011] As a preferred embodiment, after obtaining the soaked Gastrodia elata tubers, the method further includes: placing the soaked Gastrodia elata tubers at room temperature for 2-3 hours.

[0012] As a preferred embodiment, the symbiotic culture time is 50-70 days.

[0013] As a preferred embodiment, the concentration of the strigolactone biosynthesis inhibitor Tis108 in the aqueous solution is 0.1~10 μM.

[0014] As a preferred embodiment, the Gastrodia elata tuber is white Gastrodia elata.

[0015] This invention provides the application of a strigolactone biosynthesis inhibitor in promoting the propagation of Gastrodia elata seed tubers. The strigolactone biosynthesis inhibitor described in this invention specifically blocks key enzymes involved in SL synthesis. Treatment of Gastrodia elata tubers with this inhibitor can enhance the production of superoxide anion (O2) in Gastrodia elata. 2- It reduces the content of soluble solids, malondialdehyde, and peroxidase (POD) in Gastrodia elata, thereby helping to break the dormancy of dormant buds and promote their germination.

[0016] This invention provides a method for promoting the propagation of Gastrodia elata seeds. The method is highly efficient, simple to operate, requires less time for seed propagation, and has lower costs, making it suitable for large-scale production. Results from examples show that treatment with a strigolactone biosynthesis inhibitor for 15 days increased the number of latent buds in white Gastrodia elata seeds by approximately 50% compared to natural germination, and the weight of newly formed Gastrodia elata seeds cultivated for 60 days increased by approximately 70% compared to natural growth. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 The structural diagram of Tis108, an inhibitor of strigolactone biosynthesis; Figure 2 Comparison of latent bud growth of Gastrodia elata white buds after 15 days of culture between Tis108-treated and untreated Gastrodia elata white buds. Figure 3 A comparison of the growth of new seed tubers of Gastrodia elata after 60 days of cultivation with Tis108-treated white tubers and untreated white tubers. Detailed Implementation

[0019] This invention provides the application of a strigolactone biosynthesis inhibitor in promoting the propagation of Gastrodia elata seeds. As one embodiment, the strigolactone biosynthesis inhibitor is Tis108; the chemical formula of Tis108 is shown in Formula I, see... Figure 1 : Formula I.

[0020] Strigolactone is a common plant hormone that can stimulate seed germination in parasitic plants and inhibit branching. Tis108 [6-phenoxy-1-phenyl-2-(1H-1,2,4-triazol-1-hexyl-1-one)] is a biosynthetic inhibitor of strigolactone, belonging to the triazole class of compounds. It specifically blocks key enzymes in strigolactone synthesis, inhibiting the biosynthesis of strigolactone in plants and exhibiting several important biological functions, including increasing branching and leaf area. Treatment of Gastrodia elata with Tis108 can enhance the absorption of superoxide anions (O2) in Gastrodia elata. 2- The content of soluble solids, malondialdehyde (MDA), and catalase (CAT) in Gastrodia elata is reduced, thereby helping to break the dormancy of dormant buds and promote their germination. As one implementation method, promoting the propagation of Gastrodia elata seeds includes increasing the number of dormant buds germinating and shortening the growth and development time of dormant buds.

[0021] As one implementation method, the effective concentration of Tis108 is 0.001~20 μM, or it can be 0.1~10 μM, or it can be 1~5 μM; in a specific embodiment of the present invention, the effective concentration of Tis108 can be any value in the range of 0.001~20 μM, for example, 0.001, 0.01, 0.05, 0.08, 0.5, 1, 1.5, 2, 2.5, 5, 7.5, 8, 10, 15, 18, 20 μM.

[0022] As one implementation method, promoting the propagation of Gastrodia elata seeds includes: increasing the number of latent buds that germinate and / or shortening the growth and development time of latent buds.

[0023] This invention also provides a method for promoting the propagation of Gastrodia elata seed tubers, comprising the following steps: soaking Gastrodia elata tubers in an aqueous solution of Tis108, a biosynthesis inhibitor of strigolactone, to obtain soaked Gastrodia elata tubers; and symbiotically culturing the soaked Gastrodia elata tubers with Armillaria mellea spawn to obtain Gastrodia elata seed tubers. The concentration of Tis108, a biosynthesis inhibitor of strigolactone, in the aqueous solution is 0.001~20 μM.

[0024] In one embodiment, the Gastrodia elata tuber is white gastrodia elata; the white gastrodia elata is fresh white gastrodia elata. In another embodiment, the present invention cleans the Gastrodia elata tuber to obtain cleaned Gastrodia elata tuber; the cleaning includes: rinsing the surface of the Gastrodia elata tuber with water to remove soil, placing it at room temperature in the dark, and draining the surface moisture; the time for placing it in the dark is 2-3 hours. This invention's method of placing the cleaned Gastrodia elata tuber in the dark mimics the light-free environment of the soil under natural conditions, which is beneficial to the growth of Gastrodia elata.

[0025] This invention involves soaking the cleaned Gastrodia elata tubers in an aqueous solution of Tis108, a biosynthesis inhibitor of strigolactone, to obtain soaked Gastrodia elata tubers. As one embodiment, the soaking time is 2-10 minutes, and can also be 5-8 minutes. In a specific embodiment of this invention, the soaking time can be any value within the range of 2-10 minutes, for example, 2, 3, 4, 5, 6, 7, or 8 minutes.

[0026] The concentration of Tis108, a strigolactone biosynthesis inhibitor, in the aqueous solution of the present invention is 0.001~20 μM. As one embodiment, the concentration of Tis108 in the aqueous solution is 0.1~10 μM, or 1~5 μM; in specific embodiments of the present invention, the effective concentration of Tis108 can be any value from 0.001~20 μM, for example, 0.001, 0.01, 0.05, 0.08, 0.5, 1, 1.5, 2, 2.5, 5, 7.5, 8, 10, 15, 18, 20 μM.

[0027] As one implementation method, after obtaining the soaked Gastrodia elata tubers, the method further includes: placing the soaked Gastrodia elata tubers at room temperature for 2-3 hours. This room temperature placement allows the surface moisture of the Gastrodia elata tubers to drain, thus avoiding the influence of surface moisture on subsequent inoculation with Armillaria mellea.

[0028] This invention involves symbiotic cultivation of soaked Gastrodia elata tubers with Armillaria mellea spawn to obtain Gastrodia elata seed tubers. In one embodiment, the ratio of Armillaria mellea spawn to Gastrodia elata tubers is 1:6, which satisfies the nutritional needs of Armillaria mellea for Gastrodia elata while ensuring effective seed tuber production. In another embodiment, the symbiotic cultivation includes placing the Gastrodia elata tubers and Armillaria mellea spawn in a moist substrate. In a specific embodiment of this invention, the moist substrate is a common horticultural seedling substrate with half its volume of water added; the common horticultural seedling substrate is a mixture of peat, perlite, and vermiculite in equal proportions.

[0029] In one implementation, the symbiotic culture time is 50-70 days. In a specific embodiment of the present invention, the symbiotic culture time can be any value within 50-70 days, such as 50, 51, 53, 55, 57, 58, 60, 62, 65, 67, or 70 days. In one implementation, the symbiotic culture is conducted at room temperature. The present invention has no special requirements for the temperature, humidity, light, etc. of the symbiotic culture; conventional settings in the art can be used. The strigolactone biosynthesis inhibitor used in the present invention is a plant growth regulator that is low in cost and environmentally friendly; the method used to promote the propagation of Gastrodia elata seeds is simple and easy to implement, requires no special treatment, and is suitable for large-scale application; the number of new Gastrodia elata seeds after treatment can be increased.

[0030] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1 In vitro culture experiment of Gastrodia elata tubers using Tis108, a biosynthesis inhibitor of strigolactone: (1) Cleaning of Gastrodia elata tubers: Freshly harvested white Gastrodia elata tubers were rinsed with tap water to remove surface soil and placed at room temperature for 2 hours. The tubers were randomly divided into two groups, one group was designated as Tis108 group and the other group was designated as control group. Each group had at least 20 tubers. Three groups were set up to repeat the experiment, and the average value of the results was taken.

[0032] (2) Preparation of Tis108 aqueous solution: Weigh 25 mg of strigolactone biosynthesis inhibitor Tis108 (S25193, Shanghai Yuanye Biotechnology Co., Ltd.), dissolve Tis108 in 745.4 μL of acetone, and dilute with sterile water to a molar concentration of 1 μM to obtain Tis108 aqueous solution for later use.

[0033] (3) Treatment of Gastrodia elata tubers: Tis108 group: Soak the white hemp tubers obtained in step (1) in a 1 μM Tis108 aqueous solution for 5 min, place at room temperature for 2 h, and drain the surface moisture. Control group: Gastrodia elata tubers were soaked in an acetone aqueous solution of equal concentration without Tis108 for 5 min, left at room temperature for 2 h, and then drained of surface moisture.

[0034] (4) Gastrodia elata cultivation: The Gastrodia elata tubers obtained in the two groups in step (3) were placed in a moist substrate and cultured at room temperature. The germination of dormant buds of white Gastrodia elata was recorded after 15 days. The number of dormant buds was counted. The formula is: Number of dormant buds = Total number of dormant buds in the treatment group / Total number of Gastrodia elata in the treatment group.

[0035] CAT activity (BC0205, Solarbio), POD activity (BC0095, Solarbio), and SOD activity (BC0170, Solarbio) were determined using the kit. Specific procedures were performed according to the kit instructions. Results are shown in Table 1 and... Figure 2 As shown.

[0036] Table 1 Comparison of phenotypic and physiological indicators of Gastrodia elata after Tis108 treatment

[0037] According to Table 1 and Figure 2 It can be seen that soaking white Gastrodia elata in 1 μM Tis108 solution increased the activity of CAT and SOD, the content of superoxide anion, and reduced the content of soluble solids and the activity of peroxidase (POD), thereby helping to break the dormancy of dormant buds and promote the germination of dormant buds.

[0038] Example 2 Symbiotic culture experiment using Tis108, a biosynthesis inhibitor of strigolactone, after treatment of Gastrodia elata tubers: (1) Cleaning of Gastrodia elata tubers: Freshly harvested white Gastrodia elata tubers were rinsed with tap water to remove surface soil and placed at room temperature for 2 hours. The tubers were randomly divided into two groups, one group was designated as Tis108 group and the other group was designated as control group. Each group had at least 30 tubers. Three groups were set up to repeat the experiment, and the average value of the results was taken.

[0039] (2) Preparation of Tis108 aqueous solution: Weigh 50 mg of strigolactone biosynthesis inhibitor Tis108 (S25193, Shanghai Yuanye Biotechnology Co., Ltd.) and dissolve it in 745.4 μL of acetone. Dilute it with sterile water to a molar concentration of 2 μM to obtain Tis108 aqueous solution for later use.

[0040] (3) Treatment of Gastrodia elata tubers: Tis108 group: Soak the white hemp tubers obtained in step (1) in a 2 μM Tis108 aqueous solution for 8 min, place at room temperature for 3 h, and drain the surface moisture. Control group: Gastrodia elata tubers were soaked in an acetone aqueous solution of equal concentration without Tis108 for 8 min, left at room temperature for 3 h, and then drained of surface moisture.

[0041] (4) Gastrodia elata cultivation: The Gastrodia elata tubers obtained in the two groups in step (3) were placed together with Armillaria mellea spawn bags in a moist substrate and cultured at room temperature. Each spawn bag was inoculated with 6 Gastrodia elata tubers. The symbiotic rate, number and quality of latent buds of Gastrodia elata and Armillaria mellea were observed after 60 days. The symbiotic rate of Gastrodia elata was calculated. The formula for the symbiotic rate of Gastrodia elata is: Gastrodia elata symbiotic rate = number of Gastrodia elata tubers symbiotic with Armillaria mellea / number of Gastrodia elata tubers inoculated in the treatment group. The results are shown in Table 2 and Figure 3 As shown.

[0042] Table 2. Growth of Gastrodia elata and Armillaria mellea symbiosis and newly formed Gastrodia elata seedlings after 60 days of Tis108 treatment.

[0043] According to Table 2 and Figure 3 It can be seen that soaking white hemp in 2 μM Tis108 solution can significantly promote the symbiosis between Gastrodia elata and Armillaria mellea, the formation of new seed hemp, and improve the quantity and quality of new seed hemp.

[0044] In summary, the method described in this invention can improve the activity of CAT and SOD, and the content of superoxide anion by soaking Gastrodia elata in Tis108, a biosynthesis inhibitor of strigolactone, while reducing the content of soluble solids and POD activity. This helps to break the dormancy of dormant buds in Gastrodia elata, promote the germination of dormant buds, and promote the symbiosis between Gastrodia elata and Armillaria mellea, as well as the formation of new seed tubers, thereby improving the quantity and quality of new seed tubers.

[0045] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of a jasmolin biosynthesis inhibitor in promoting the propagation of Gastrodia elata seedlings.

2. Use according to claim 1, characterized in that, The jasmolin biosynthesis inhibitor is Tis108; the chemical formula of the Tis108 is shown in Formula I: Formula I.

3. Use according to claim 2, characterized in that, The concentration of the Tis108 is 0.001-20 μM.

4. Use according to claim 1, characterized in that, The promoting the propagation of Gastrodia elata seedlings comprises increasing the number of Gastrodia elata latent bud germination and / or shortening the growth and development time of Gastrodia elata latent bud.

5. A method for promoting the propagation of Gastrodia elata Blume seeds, characterized by, The method comprises the following steps: immersing Gastrodia elata rhizomes in an aqueous solution of a jasmolin biosynthesis inhibitor Tis108 to obtain soaked Gastrodia elata rhizomes; and symbiotic culturing the soaked Gastrodia elata rhizomes with Armillaria mellea sticks to obtain Gastrodia elata seedlings; The concentration of the jasmolin biosynthesis inhibitor Tis108 in the aqueous solution is 0.001-20 μM.

6. The method of claim 5, wherein, The soaking time is 2-10 min.

7. The method of claim 5, wherein, After obtaining the soaked Gastrodia elata rhizomes, the method further comprises: placing the soaked Gastrodia elata rhizomes at room temperature for 2-3 h.

8. The method of claim 5, wherein, The symbiotic culturing time is 50-70 d.

9. The method of claim 5, wherein, The concentration of the jasmolin biosynthesis inhibitor Tis108 in the aqueous solution is 0.1-10 μM.

10. The method of any one of claims 5-9, wherein, The Gastrodia elata rhizomes are white Gastrodia elata rhizomes.

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