A method of extending the range of action of germinants
By adding GR24 or KAR1/KAR2 hormones to the germination medium, the problem of decreasing germination rate with increasing distance in the symbiotic germination of Gastrodia elata seeds was solved, and the germination rate was significantly improved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- KUNMING INST OF BOTANY CHINESE ACAD OF SCI
- Filing Date
- 2026-04-30
- Publication Date
- 2026-06-16
AI Technical Summary
In the existing technology, during the symbiotic germination process of Gastrodia elata seeds, the range of action of the germination fungus is limited, resulting in a decrease in the germination rate as the distance between the seed and the fungal block increases.
Adding 0.01-0.1 μmol/L of GR24, 0.01-0.1 μmol/L of KAR1, or 0.01 μmol/L of KAR2 to the germination medium can promote the germination of Gastrodia elata seeds at a distance of 10 mm-15 mm from the germination block.
It significantly improved the germination rate of Gastrodia elata seeds located 10-15 mm away from the germination fungal block, and expanded the effective range of the germination fungus.
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Abstract
Description
Technical Field
[0001] This application relates to the field of seed germination technology, and more particularly to a method for expanding the range of action of germination bacteria. Background Technology
[0002] In the symbiotic germination process of Gastrodia elata seeds, in order to objectively reflect the true vigor of the seeds, facilitate the observation and statistical analysis of germination rates, and reduce dependence on fungal strains, the prior art, CN118489330A, discloses a method for germinating Gastrodia elata seeds. This method involves sowing Gastrodia elata seeds at a distance of 5-15 mm from the germination fungal block in the germination medium. In most cases, the germination rate is not significantly different from the seed vigor measured by TTC staining, thus reflecting the true vigor of Gastrodia elata seeds well. Furthermore, experimental results show that this method reduces dependence on the type and quality of the fungal strain to a certain extent. The patent text also states that "the germination rate at 5 mm from the germination fungus is 68.1% ± 4.0% (mean ± standard error, the same below); the germination rate at 10 mm from the germination fungus is 45.0% ± 3.6%; and the germination rate at 15 mm from the germination fungus is 8.8% ± 3.9%."
[0003] Clearly, the experimental data from the aforementioned patent text shows that the germination rate at a distance of 5 mm from the germinating fungus is significantly higher than that at 10 mm and 15 mm. That is, the symbiotic germination rate decreases with increasing distance between the seed and the fungal block. Therefore, the effective range of the germinating fungus in this method is essentially limited.
[0004] Improving the germination effect of symbiotic germination of Gastrodia elata seeds has always been a pursuit of those skilled in the art. Summary of the Invention
[0005] To address or partially address the problems existing in related technologies, this application provides a method for expanding the range of action of germination bacteria. By adding 0.01-0.1 μmol / L of GR24, 0.01-0.1 μmol / L of KAR1, or 0.01 μmol / L of KAR2 to the germination medium, the germination rate of Gastrodia elata seeds located 10 mm-15 mm away from the germination bacteria can be significantly improved, thus expanding the range of action of the germination bacteria.
[0006] A method for expanding the range of action of germination fungi in this application includes the following steps: (1) disinfection treatment of Gastrodia elata seeds; (2) Propagation of germination bacteria; (3) Preparation of germination culture medium; (4) Germination of Gastrodia elata seeds: Take the germination fungus block with germination fungus hyphae after propagation to the predetermined size in step (2) and place it on the germination medium. Sow the detoxified Gastrodia elata seeds within a range of 5-15 mm away from the germination fungus block in the germination medium. Place the germination medium in an incubator for dark culture. The germination medium in step (3) contains 0.01-1 μmol / L GR24, 0.01-1 μmol / L KAR1, or 0.01-0.1 μmol / L KAR2. Furthermore, the germination medium in step (3) contains 0.01 μmol / L GR24 or 0.01 μmol / L KAR1.
[0007] Furthermore, the germination medium is a 1% agar / water medium.
[0008] Furthermore, in step (4), the detoxified Gastrodia elata seeds are sown within a range of 10-15 mm from the germination fungal blocks in the germination medium.
[0009] Furthermore, the germinating fungal blocks in step (4) are round or rectangular.
[0010] Furthermore, the germinating bacterial block is round and has a diameter of 8 mm.
[0011] The beneficial effects of this application are: 1. This invention solves the problem that the germination rate of Gastrodia elata seeds decreases as the distance between the seeds and the fungal block increases during symbiotic germination. This invention expands the range of action of the germination fungus, especially promoting the germination rate at locations farther away from the germination fungal block (10mm and 15mm).
[0012] For the germination rate of 8mm diameter circular mycelial blocks of LSY strain at a distance of 10mm from the germination block, the ordinary culture medium was 45.0%. 0.01-1μmol / L GR24, 0.1μmol / L KAR1, and 0.01μmol / L KAR2 all significantly or extremely significantly promoted germination, increasing the germination rate to 72.5-77.5%. At a distance of 15mm from the germination block, the germination rate of ordinary culture medium was 8.8%. The addition of 0.1μmol / L GR24 significantly promoted germination, with a germination rate of 31.9%, which was much higher than the control. For 5mm×5mm square mycelial blocks of LSY strain, the germination rate at 10mm from the block on ordinary medium was 25%, while the germination rates on 0.1 and 1μmol / L GR24 medium were 62.50% and 50.00%, respectively. The germination rates on 0.1 and 1μmol / L KAR1 medium were 58.75% and 65.00%, respectively. The germination rate on 0.1μmol / L KAR2 medium was 60.00%, all significantly higher than the control. At 15mm from the block, the germination rate on ordinary medium was 3.75%, while the germination rates on 0.1 and 1μmol / L GR24 medium were 30.00% and 27.50%, respectively. The germination rate on 0.1μmol / L KAR2 medium was 37.50%, all significantly higher than the control. Detailed Implementation
[0013] The embodiments of this application will now be described in more detail with reference to the examples. While embodiments of this application are shown in the examples, it should be understood that this application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to make this application more thorough and complete, and to fully convey the scope of this application to those skilled in the art.
[0014] Example 1 This embodiment examines and compares the effects of distance from the germinating bacteria and the effects of different added hormones, and includes the following steps: Step 1: Seed (Capsule) Acquisition: On July 19, 2024, capsules of *Gastrodia elata* harvested by a farmer in Yiliang County were purchased. The capsules were collected approximately 23 days after the farmer's self-pollination, and were slightly cracked. The capsules were placed in an envelope and brought back to the Southwest Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, the next day. A saturated saline solution was prepared and placed in a sealed container. After removing the *Gastrodia elata* seeds from the fruits, they were placed in a small aluminum dish and placed in the sealed container, but not in contact with the saturated saline solution. They were dried at 15°C to approximately 75% relative humidity. The dried seeds were placed in a sealed container (such as a cryovial) and stored at -20°C. After 6 months of liquid nitrogen storage, the seeds were removed, heated in a 42°C water bath for 2 minutes, and the container surface was wiped dry before opening the container to remove the seeds.
[0015] Step 2, Seed (Capsule) Disinfection: Take an appropriate amount of Gastrodia elata seeds, place them in a 2ml centrifuge tube, add 1.5ml of a mixture of 0.5% sodium dichloroisocyanurate (NaDCC) and 1% Tween 20, and disinfect for 3 minutes; after 3 minutes, replace with fresh disinfectant and repeat 3 times. After disinfection, rinse the seeds 5 times with sterile water.
[0016] Step 3, Pre-culture of germination fungus: A 1cm section of the germination fungus log (self-numbered LSY) purchased in Yiliang in April 2022 and stored at 4℃ was taken. 2 Small, sheet-like bacterial blocks were inoculated onto potato dextrose agar (PDA) medium and pre-cultured at 23°C in the dark for 3 weeks.
[0017] Step 4, prepare germination medium and sow seeds: Prepare 1% agar / water medium or 1% agar / water medium containing 0.01-1 μmol / L styracin (GR24) or 0.01-1 μmol / L Karrikin (KAR1 or KAR2).
[0018] Step 5, Sowing: Using a punch, take a circular germination block (8 mm in diameter) from PDA medium, pre-cultured at 23°C in the dark for 3 weeks, and place it in the center of the germination medium. Sow the seeds at distances of 5 mm, 10 mm, and 15 mm from the block. Perform four replicates (in petri dishes), with 40 seeds sown in each replicate. Place the petri dishes in a 23°C incubator for cultivation under dark conditions.
[0019] Step 6, Germination Count: Observe every 7 days after sowing and count the number of bulbs. Count the germination rate after 6-8 weeks.
[0020] Step 7: Perform one-way ANOVA on the germination data using R software, and compare the significance of differences between each treatment and the control using the least significant place (LSD) method. The experimental results are shown in Table 1. Table 1. Germination rate of Gastrodia elata seeds at distances of 5 mm, 10 mm, and 15 mm from the germination block on different culture media. In Table 1, * indicates a significant difference compared to the control (P < 0.05), and ** indicates an extremely significant difference compared to the control (P < 0.01). Table 1 shows that the germination rate at 5 mm from the mycelium block was 68.1% on ordinary medium. The germination rate was improved by adding GR24, KAR1, and KAR2, but there was no significant difference compared with the control. At 10 mm from the mycelium block, the germination rate was 45.0% on ordinary medium. GR24 (0.01-1 μmol / L), KAR1 (0.1 μmol / L), and KAR2 (0.01 μmol / L) all significantly or extremely significantly promoted germination, increasing the germination rate to 66.3-77.5%. At 15 mm from the mycelium block, the germination rate was 8.8% on ordinary medium. GR24 (0.1 μmol / L) significantly promoted germination, with a germination rate of 31.9%, which was much higher than the control germination rate of 8.8%.
[0021] Example 2 Step 1: Seed (Capsule) Acquisition: On June 29, 2022, capsules of *Gastrodia elata* harvested by a farmer in Yiliang County were purchased. The capsules were collected approximately 23 days after the farmer's self-pollination, and were slightly cracked. The capsules were placed in an envelope and brought back to the Southwest Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, the next day. A saturated saline solution was prepared and placed in a sealed container. After removing the *Gastrodia elata* seeds from the capsules, they were placed in a small aluminum dish and placed in the sealed container, but not in contact with the saturated saline solution. The seeds were dried at 15°C to approximately 75% relative humidity. The dried seeds were then placed in a sealed container (such as a cryovial), and the sealed container containing the seeds was placed in liquid nitrogen for preservation. After 4 months of liquid nitrogen preservation, the seeds were removed, heated in a 42°C water bath for 2 minutes, and the surface of the container was wiped dry before opening the container and removing the seeds.
[0022] Step 2, Seed (Capsule) Disinfection: Take an appropriate amount of Gastrodia elata seeds, place them in a 2ml centrifuge tube, add 1.5ml of a mixture of 0.5% sodium dichloroisocyanurate (NaDCC) and 1% Tween 20, and disinfect for 3 minutes; after 3 minutes, replace with fresh disinfectant and repeat 3 times. After disinfection, rinse the seeds 5 times with sterile water.
[0023] Step 3, Pre-culture of germination fungus: A 1cm section of the germination fungus log (self-numbered LSY) purchased in Yiliang in April 2022 and stored at 4℃ was taken. 2 Small, sheet-like bacterial blocks were inoculated onto potato dextrose agar (PDA) medium and pre-cultured at 23°C in the dark for 3 weeks.
[0024] Step 4: Prepare germination medium and sow seeds: Prepare 1% agar / water medium or 1% agar / water medium containing 0.1-1 μmol / L styracin (GR24) or 0.1-1 μmol / L Karrikin (KAR1 or KAR2).
[0025] Step 5, Sowing: Using a scalpel, cut a 5mm × 5mm square germination block from PDA medium, pre-cultured at 23°C in the dark for 3 weeks, and place it in the center of the germination medium; sow the seeds at distances of 5mm, 10mm, and 15mm from the germination block. There are four replicates (petitions), with 40 seeds sown in each replicate. Place the petri dishes in a 23°C incubator for cultivation under dark conditions.
[0026] Step 6, Germination Count: Observe every 7 days after sowing and count the number of bulbs. Generally, all seeds will germinate in 6-8 weeks.
[0027] Step 7: Data analysis. One-way ANOVA was performed on the germination data using R software, and the least significant digit (LSD) method was used to compare the significance of differences between each treatment and the control. The results are shown in Table 2.
[0028] Table 2 Germination rate of Gastrodia elata seeds at distances of 5 mm, 10 mm, and 15 mm from the germination block on different culture media. In Table 2, * indicates a significant difference compared to the control (P < 0.05), and ** indicates an extremely significant difference compared to the control (P < 0.01). Table 2 shows that the germination rate at a distance of 5 mm from the mycelial block was 36.3% on ordinary medium. The germination rate was significantly increased by mediums with added GR24, KAR1, and KAR2, ranging from 65.0% to 93.8%. At a distance of 10 mm from the mycelial block, the germination rate was 25.0% on ordinary medium. GR24 (0.01-1 μmol / L), KAR1 (0.1 μmol / L), and KAR2 (0.01 μmol / L) all significantly or significantly promoted germination, increasing the germination rate to 50.0%-65.0%. At a distance of 15 mm from the mycelial block, the germination rate was 3.8% on ordinary medium. The addition of GR24 (0.1 μmol / L and 1 μmol / L) significantly promoted germination, with germination rates of 30.00% and 27.5%, respectively, far exceeding the control. The addition of KAR2 (0.1 μmol / L) also significantly promoted germination, with a germination rate of 37.5%.
[0029] Comparative Example 1 Comparative Example 1 used the same seeds and added hormones and their concentrations as Example 1, the difference being that aseptic germination was performed on 1 / 2 MS medium. Because no mycelial blocks were added, the seeds were randomly sown onto the medium. The results showed that none of the seeds in this comparative example germinated within 8 weeks. This indicates that GR24 and KAR1 and KAR2 are only effective in symbiotic germination.
[0030] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.
Claims
1. A method for expanding the scope of action of germination bacteria, comprising the following steps: (1) Disinfection treatment of Gastrodia elata seeds; (2) Propagation of germination bacteria; (3) Preparation of germination culture medium; (4) Germination of Gastrodia elata seeds: Take the germination fungus block with germination fungus hyphae after propagation to the predetermined size in step (2) and place it on the germination medium. Sow the detoxified Gastrodia elata seeds within a range of 5-15 mm away from the germination fungus block in the germination medium. Place the germination medium in an incubator for dark culture. The characteristic feature is that the germination medium in step (3) contains 0.01-1 μmol / L of GR24, 0.01-0.1 μmol / L of KAR1, or 0.01 μmol / L of KAR2.
2. The method for expanding the effective range of germination bacteria according to claim 1, characterized in that, The germination medium in step (3) contains 0.01 μmol / L GR24 or 0.01 μmol / L KAR1.
3. A method for expanding the effective range of germination bacteria according to claim 1 or 2, characterized in that, The germination medium is 1% agar / water medium.
4. The method for expanding the effective range of germination bacteria according to claim 3, characterized in that, In step (4), the detoxified Gastrodia elata seeds are sown within a range of 10-15 mm from the germination fungal blocks in the germination medium.
5. The method for expanding the effective range of germination bacteria according to claim 1, characterized in that, The germination fungal blocks in step (4) are round or rectangular.
6. The method for expanding the effective range of germination bacteria according to claim 5, characterized in that, The germinating bacterial block is round and has a diameter of 8 mm.