A method for expanding the range of action of germination bacteria using smoke water
By adding tobacco water to the germination medium of Gastrodia elata seeds, the range of action of the germination bacteria was expanded, the problem of the symbiotic germination rate of Gastrodia elata seeds decreasing with distance was solved, the germination rate was improved and the dependence on the strain was reduced.
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
- 2026-07-14
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 significant decrease in the germination rate as the distance between the seed and the fungal block increases.
The use of tobacco water to expand the range of action of germination bacteria was investigated. By adding 0.1%-5% tobacco water, especially 0.2% and 2% concentrations, to the germination medium, the germination rate of Gastrodia elata seeds located 10mm-15mm away from the germination bacteria was significantly improved.
It significantly expanded the range of action of germination bacteria, improved the germination rate at locations farther from the germination block, and reduced dependence on the type and quality of the bacteria.
Smart Images
Figure CN122375296A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of seed germination technology, and in particular to a method for expanding the range of action of germination bacteria using tobacco water. 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 effective range of germination bacteria using tobacco water, which can significantly improve the germination rate of Gastrodia elata seeds located 10mm-15mm away from the germination bacteria and expand the effective range of the germination bacteria.
[0006] A method for expanding the effective range of germinating bacteria using flue gas as described in this application includes the following steps: (1) Disinfection treatment of Gastrodia elata seeds; (2) Propagation of germination bacteria; (3) Prepare the germination 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 in 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. Specifically, the germination culture medium in step (3) is a 1% agar / water culture medium containing tobacco water; wherein the tobacco water is obtained by dissolving the flue gas generated after heating the plant material into water; wherein the plant material is square bamboo or yellow grass.
[0007] Furthermore, the tobacco water is prepared by the following method: Take 55g of dried and pulverized plant material and heat it 11 times. Each time, take 5g of plant material and spread it evenly in a metal support plate with a diameter of 10cm. The metal support plate is placed on the heating surface of the heating device, and a gas collecting funnel is set above the metal support plate. The gas collecting funnel has its outlet facing upward and is connected to the inner cavity of the first conical flask near the bottom through a gas guide pipe. Three conical flasks are provided in series, each containing 1L of distilled water. The air inlet of the first conical flask is connected to the gas collecting funnel, and the air outlet of the third conical flask is connected to a negative pressure device through a pipe. The heating device is activated to raise the temperature to 300±30℃. Each heating treatment lasts for 10 minutes. The heat is conducted to the metal support plate through the heating surface, heating the plant material and generating smoke. Under the negative pressure generated by the negative pressure device, the flue gas is collected by the gas collecting funnel and then enters three conical flasks connected in series along the gas guide pipe. The flue gas is dissolved by distilled water in each conical flask in the form of bubbles, and the resulting solution after cooling is the flue gas solution.
[0008] Furthermore, the concentration of tobacco water in the germination culture medium is 0.1%-5%.
[0009] Furthermore, the concentration of tobacco water in the germination medium is 0.2%.
[0010] Furthermore, the concentration of tobacco water in the germination culture medium is 2%.
[0011] Furthermore, the predetermined diameter is 8mm.
[0012] Furthermore, the metal support disk is an aluminum disk.
[0013] 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 and promotes the germination rate at positions (10mm and 15mm) farther away from the germination fungal block.
[0014] 2. This invention has demonstrated through verification experiments using different germination bacteria purchased from the market that adding tobacco water to the culture medium can significantly expand the range of action of the germination bacteria. This method effectively reduces the dependence on the type and quality of bacteria during the symbiotic germination of Gastrodia elata to a certain extent. Attached Figure Description
[0015] The above and other objects, features and advantages of this application will become more apparent from the more detailed description of exemplary embodiments thereof in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments thereof.
[0016] Figure 1 The tobacco water preparation apparatus of this application. Detailed Implementation
[0017] 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.
[0018] Example 1 This embodiment of a method for expanding the effective range of germinating bacteria using flue gas includes the following steps: Step 1: Seed (capsule) Acquisition: In July 2024, seeds (capsules) of *Gastrodia elata* were purchased from a farmer in Yiliang County. The capsules were collected approximately 23 days after the farmer had self-pollinated them, 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.
[0019] 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.
[0020] 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.
[0021] Step 4, Prepare the tobacco water: Dry the square bamboo ( ) in an oven at 107°C. Chimonobambusa quadrangularis (Franceschi) Makino) dried and crushed using a DFY-200C swing-type high-speed mill. Utilizing the attached... Figure 1 The device shown, Figure 1 The apparatus shown in this embodiment for preparing flue gas mother liquor includes: a heating device, an aluminum pan, a gas collecting device, conical flasks, and a negative pressure device (vacuum pump). The aluminum pan is used to hold plant materials. A heating device is provided below the aluminum pan for heating it, and a gas collecting device (funnel) is provided above the aluminum pan for collecting flue gas. Three conical flasks are connected in series between the gas collecting device and the negative pressure device via pipes. The conical flasks contain 1L of distilled water for dissolving the flue gas.
[0022] Specifically, during operation, the electric furnace is heated to approximately 300±30°C. Under the negative pressure of approximately 80Kpa from the vacuum pump, the flue gas generated by heating is passed through three conical flasks containing 1L of distilled water. Each heating session lasts approximately 10 minutes. After all materials have been heated, the flue gas and water in the conical flasks are removed.
[0023] Step 5: Prepare germination medium: Prepare 1% agar / water medium containing 2-50 ml / L of tobacco water (tobacco water concentration 0.2%-5%).
[0024] Step 6, Sowing: Using a punch, take an 8mm diameter 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. Perform four replicates (petitions), with 40 seeds sown in each replicate. Place the petri dishes in a 23°C incubator for cultivation under dark conditions.
[0025] Step 7, Germination Count: Observe every 7 days after sowing and count the number of bulbs. Count the germination rate after 6-8 weeks.
[0026] Step 8: 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 results are shown in Table 1.
[0027] Table 1 shows the germination rates of Gastrodia elata seeds at distances of 5 mm, 10 mm, and 15 mm from the LSY germination block on culture media with different concentrations of tobacco water. The letters indicate significant differences between treatments (P < 0.05). As shown in Table 1, the germination rate at a distance of 10 mm from the mycelium block on ordinary culture medium was 31.5% ± 12.5%, while the germination rates on culture media with 2% and 0.2% concentrations of tobacco water were 88.5% ± 5.5% and 85.3% ± 5.0%, respectively, which were significantly higher than the control. The germination rate at a distance of 15 mm from the mycelium block was 1.0% ± 0.9%, while the germination rates on culture media with 2% and 0.2% concentrations of tobacco water were 67.0% ± 12.7% and 86.3% ± 5.0%, respectively, which were also significantly higher than the control.
[0028] The above results indicate that the addition of tobacco water to the culture medium can significantly expand the range of action of commercially available germination bacteria LSY.
[0029] Example 2 This embodiment of a method for expanding the effective range of germinating bacteria using flue gas includes the following steps: Step 1: Seed (capsule) Acquisition: In July 2024, seeds (capsules) of *Gastrodia elata* were purchased from a farmer in Yiliang County. The capsules were collected approximately 23 days after the farmer had self-pollinated them, 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.
[0030] 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.
[0031] Step 3, Pre-culture of germination bacteria: Germination bacteria logs (self-numbered GZJS) purchased in Yiliang in April 2023 and stored at 4℃ were inoculated into 1cm2 pieces on potato dextrose agar (PDA medium) and pre-cultured at 23°C in the dark for 3 weeks.
[0032] Step 4, Prepare the tobacco water: Dry the square bamboo ( ) in an oven at 107°C. Chimonobambusa quadrangularis (Franceschi) Makino) dried and crushed using a DFY-200C swing-type high-speed mill. Similarly, using an attached... Figure 1 The apparatus shown is used to prepare flue gas mother liquor. Specifically, 55g of material is taken and divided into 10 portions, each 5g portion is placed in an aluminum pan with a diameter of 10cm. The pan is heated to about 300°C in an electric furnace. Under the negative pressure of about 80Kpa from a vacuum pump, the flue gas generated by heating is passed through three conical flasks containing 1L of distilled water. Each heating lasts about 10 minutes. After all the material has been heated, the flue gas mother liquor in the conical flasks is removed.
[0033] Step 5: Prepare germination medium: Prepare 1% agar / water medium or 1% agar / water medium containing 2-50 ml / L of flue gas (flue gas concentration 0.2%-5%).
[0034] Step 6, Sowing: Using a punch, take an 8mm diameter 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. Perform four replicates (petitions), with 40 seeds sown in each replicate. Place the petri dishes in a 23°C incubator for cultivation under dark conditions.
[0035] Step 7, Germination Count: Observe every 7 days after sowing and count the number of bulbs. Count the germination rate after 6-8 weeks.
[0036] Step 8: 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 results are shown in Table 2.
[0037] Table 2 shows the germination rates of Gastrodia elata seeds at distances of 5 mm, 10 mm, and 15 mm from the GZJS germination block on culture media with different concentrations of tobacco water. The letters indicate significant differences between the treatments (P < 0.05). As shown in Table 2, the germination rate at a distance of 10 mm from the mycelial block was 4.0% ± 3.6%. After adding different concentrations of tobacco water, the highest germination rate was 89.0% ± 6.7% (5% tobacco water), followed by 86.0% ± 4.6% on the 0.5% tobacco water culture medium, both significantly higher than the control. The germination rate at a distance of 15 mm from the mycelial block was 0. After adding 0.5% tobacco water, the germination rate was 68.3% ± 12.4%, significantly higher than the control. These results indicate that the addition of tobacco water to the culture medium can significantly expand the effective range of the commercially available germination bacterium GZJS.
[0038] Example 3 This embodiment of a method for expanding the effective range of germinating bacteria using flue gas includes the following steps: Step 1: Seed (capsule) Acquisition: In July 2024, seeds (capsules) of *Gastrodia elata* were purchased from a farmer in Yiliang County. The capsules were collected approximately 23 days after the farmer had self-pollinated them, 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.
[0039] 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.
[0040] Step 3, Pre-culture of germination bacteria: Germination bacteria logs (self-numbered GZJS) purchased in Yiliang in April 2023 and stored at 4℃ were inoculated into 1cm2 pieces on potato dextrose agar (PDA medium) and pre-cultured at 23°C in the dark for 3 weeks.
[0041] Step 4, Prepare the tobacco water: Add yellow reeds ( Heteropogon contortus (L.) P. Beauv. ex Roemer & Schult.) was dried at 15°C and 15% relative humidity until the moisture content was below 10%, and then ground using a DFY-200C swing-type high-speed mill. Similarly, using an attached... Figure 1 The apparatus shown is used to prepare flue gas. Specifically, 55g of material is taken and divided into 10 portions, each 5g portion is placed in an aluminum pan with a diameter of 10cm. The pan is heated to about 300°C in an electric furnace. Under the negative pressure of about 80Kpa from a vacuum pump, the flue gas generated by heating is passed through three conical flasks containing 1L of distilled water. Each heating lasts about 10 minutes. After all the material has been heated, the flue gas in the conical flasks is removed.
[0042] Step 5: Prepare germination medium: Prepare 1% agar / water medium or 1% agar / water medium containing 1-20 ml / L of flue gas (flue gas concentration 0.1%-2%).
[0043] Step 6, Sowing: Using a punch, take an 8mm diameter 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. Perform five replicates (petitions), with 20 seeds sown in each replicate. Place the petri dishes in a 23°C incubator for cultivation under dark conditions.
[0044] Step 7, Germination Count: Observe every 7 days after sowing and count the number of bulbs. Count the germination rate after 6-8 weeks.
[0045] Step 8: Perform one-way ANOVA on the germination data using R software, and use Ducan's method to compare the significance of differences between each treatment and the control. The results are shown in Table 3.
[0046] Table 3. Germination rates of Gastrodia elata seeds at distances of 5 mm, 10 mm, and 15 mm from the GZJS germination block on culture media with different concentrations of tobacco water. Different letters indicate significant differences between treatments (P < 0.05). As shown in Table 3, the germination rate at a distance of 15 mm from the mycelial block was 9.0% ± 5.57%. After adding different concentrations of tobacco water, the highest germination rate was 58.0% ± 3.4% (0.1% concentration tobacco water), followed by 45.0% ± 15.3% on the 0.2% concentration tobacco water culture medium, both significantly higher than the control. These results indicate that adding tobacco water prepared from Imperata cylindrica to the culture medium can significantly expand the effective range of the commercially available germination bacterium GZJS.
Claims
1. A method for expanding the effective range of germination bacteria using tobacco water, comprising the following steps: (1) Disinfection treatment of Gastrodia elata seeds; (2) Propagation of germination bacteria; (3) Prepare the germination 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 in 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 culture medium in step (3) is a 1% agar / water culture medium containing tobacco water; wherein the tobacco water is obtained by dissolving the flue gas generated after heating the plant material into water; wherein the plant material is square bamboo or yellow grass.
2. The method for expanding the effective range of germinating bacteria using tobacco water according to claim 1, characterized in that, The tobacco water is prepared by the following method: Take 55g of dried and pulverized plant material and heat it 11 times. Each time, take 5g of plant material and spread it evenly in a metal support plate with a diameter of 10cm. The metal support plate is placed on the heating surface of the heating device, and a gas collecting funnel is set above the metal support plate. The gas collecting funnel has its outlet facing upward and is connected to the inner cavity of the first conical flask near the bottom through a gas guide pipe. Three conical flasks are provided in series, each containing 1L of distilled water. The air inlet of the first conical flask is connected to the gas collecting funnel, and the air outlet of the third conical flask is connected to a negative pressure device through a pipe. The heating device is activated to raise the temperature to 300±30℃. Each heating treatment lasts for 10 minutes. The heat is conducted to the metal support plate through the heating surface, heating the plant material and generating smoke. Under the negative pressure generated by the negative pressure device, the flue gas is collected by the gas collecting funnel and then enters three conical flasks connected in series along the gas guide pipe. The flue gas is dissolved by distilled water in each conical flask in the form of bubbles, and the resulting solution after cooling is the flue gas solution.
3. The method for expanding the effective range of germination bacteria using tobacco water according to claim 1, characterized in that, The concentration of tobacco water in the germination medium is 0.1%-5%.
4. The method for expanding the effective range of germination bacteria using tobacco water according to claim 3, characterized in that, The concentration of tobacco water in the germination medium is 0.2%.
5. The method for expanding the effective range of germination bacteria using tobacco water according to claim 3, characterized in that, The concentration of tobacco water in the germination medium is 2%.
6. A method for expanding the effective range of germinating bacteria using tobacco water according to any one of claims 1 to 5, characterized in that, The predetermined diameter is 8mm.
7. The method for expanding the effective range of germinating bacteria using tobacco water according to claim 1, characterized in that, The metal support plate is an aluminum plate.