Application of strigolactones in improving symbiotic germination of Gastrodia elata seeds after heat stress

Abstract

The application relates to the application of strigolactone in improving the symbiotic germination effect of degraded germination fungi after heat stress and Gastrodia elata seeds, and relates to the technical field of seed germination. Specifically, the degraded germination fungi after heat stress refers to germination fungi strains after 5h of high-temperature treatment at 35 DEG C; the application comprises the following steps: adding strigolactone in a germination medium to improve the symbiotic germination rate of degraded germination fungi and Gastrodia elata seeds after heat stress; and the concentration of strigolactone in the germination medium is 0.01 micromol / L. By adding 0.01 micromol / L of GR24 in 1% agar / water medium, the symbiotic germination rate of degraded germination fungi and Gastrodia elata seeds after heat stress can be significantly improved.

Description

Technical Field

[0001] This application relates to the field of seed germination technology, and in particular to the application of styrax lactone in improving the symbiotic germination effect of heat-stressed degenerative germination bacteria and Gastrodia elata seeds. Background Technology

[0002] Gastrodia elata, a precious traditional Chinese medicine, has tiny seeds without endosperm. Under natural conditions, it must rely on germination fungi (such as Osmunda japonica and Dendrobium nobile) to provide nutrients for germination. Therefore, the activity of germination fungi is one of the key factors for the successful symbiotic germination of Gastrodia elata seeds.

[0003] Chinese patent CN118489330A discloses a method for germinating Gastrodia elata seeds. This method involves sowing Gastrodia elata seeds 5-15 mm away from germination blocks in the germination medium to induce germination. This method can better reflect the true viability of Gastrodia elata seeds and reduces dependence on the type and quality of the fungal strain to some extent. However, this method still cannot completely solve the problem of germination fungal degradation caused by environmental factors.

[0004] In actual production and transportation, germination fungi often face the risk of high-temperature stress. Especially under conditions of improper transportation or storage in summer, germination fungi may be exposed to high-temperature environments above 35°C, leading to a decrease in fungal activity. Studies have shown that high-temperature stress significantly reduces the ability of germination fungi to promote symbiotic germination of Gastrodia elata seeds, resulting in economic losses.

[0005] Strigolactones are a class of plant hormones, and their synthetic analog GR24 has been shown to promote seed germination in parasitic plants and participate in regulating seed germination and heat tolerance in model plants such as Arabidopsis thaliana. However, there are no reports on the application of strigolactones in restoring the activity of germinating bacteria after heat stress and improving their symbiotic germination effect with Gastrodia elata seeds.

[0006] The elimination or mitigation of the decline in symbiotic germination of Gastrodia elata seeds caused by degenerate germination fungi has always been a pursuit of those skilled in the art. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art in which the activity of germinating bacteria decreases after high temperature stress and the symbiotic germination rate with Gastrodia elata seeds decreases, and to provide a new use for styrax lactone (GR24), namely, to use styrax lactone to quickly restore the symbiotic germination ability of germinating bacteria after heat stress.

[0008] This application provides the application of styracil lactone in improving the symbiotic germination effect of heat-stressed degenerative germination bacteria and Gastrodia elata seeds. In particular, the heat-stressed degenerative germination bacteria refers to germination bacteria strains that have been treated at 35°C for 5 hours. The application includes: adding strigolactone to a germination medium, culturing heat-stressed germinating bacteria in a medium containing strigolactone to obtain germinating bacteria with restored activity; the concentration of strigolactone in the germination medium is 0.01 μmol / L.

[0009] Furthermore, the application specifically includes the following steps: (1) Preparation of germination medium containing stromalactone: Add stromalactone to the germination medium and mix well to make the final concentration of stromalactone 0.01 μmol / L. (2) Inoculation of germinating bacteria after heat stress: The germinating bacteria treated at 35℃ were inoculated onto the germination medium prepared in step (1), and the disinfected Gastrodia elata seeds were sown within 5mm of the fungal block for symbiotic germination culture. The culture was carried out in the dark at 20-25℃ for 30-60 days, and the germination rate was counted.

[0010] The beneficial effects of this application are: Simulated storage or transportation at 35°C for 5 hours significantly reduced the effectiveness of germinating bacteria, causing the symbiotic germination rate to drop dramatically from 64.4% to 45.0%. This invention, by adding 0.01 μmol / L GR24 to the germination medium, significantly improved the symbiotic effect of the degenerating germinating bacteria after 35°C treatment for 5 hours, increasing the germination rate from 45.0% to 63.8%, restoring it to the level before heat stress. Detailed Implementation

[0011] 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.

[0012] Example 1 During the storage and transportation of germinating bacteria, improper storage can cause the strains to be subjected to high-temperature stress, leading to a decline in strain quality. This embodiment simulates the decline in strain quality after heat exposure and examines the effect of hormones on germinating bacteria. It includes the following steps: 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 fruits, 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 14 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.

[0013] 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.

[0014] Step 3: Pre-culture of germinating bacteria: CGMCC5.894 strain from the China General Microbiological Culture Collection Center was used. 8 mm diameter mycelial blocks were inoculated onto potato dextrose agar (PDA) medium and pre-cultured at 23°C in the dark for 3 weeks.

[0015] Step 4, Preparation of germination medium and sowing: Prepare 1% agar / water medium containing 0.01 μmol / L styracin (GR24) or Karrikin (KAR1 or KAR2), and 1% agar / water medium without GR24. Step 5: Simulate high temperature to weaken the bacterial strain: Use a punch to take germination blocks of PDA medium with a diameter of 8 mm that have been pre-cultured at 23°C in the dark for 3 weeks, and place them at 23, 30 and 35°C for 5 hours respectively.

[0016] Step 5, Sowing: Place the untreated or weakened inoculum in the center of the germination medium; sow the seeds 5 mm away from the inoculum block, for a total of four replicates (petitions), with 40 seeds sown in each replicate. Place the petri dishes in a 23°C incubator and incubate under dark conditions.

[0017] 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.

[0018] 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 1.

[0019] The results showed that the germination rate of the germinating bacteria treated at 35°C for 5 hours (45.0%) was significantly lower than that of the control (64.4%); 0.01 μmol / L GR24 significantly increased the germination rate of the germinating bacteria treated at 35°C for 5 hours (63.8%).

[0020] Table 1 shows the co-germination rates (mean ± standard error) of different treatments at different temperatures (23, 30, 35°C) for 5 hours on ordinary medium (i.e., control, GR24 concentration of 0 μmol / L) and on mediums supplemented with 0.01 μmol / L KAR1, KAR2 and GR24. Different letters indicate significant differences between different treatments (P < 0.05). 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. Application of a strigolactone in improving the symbiotic germination effect of a degraded germination fungus after heat stress on Gastrodia elata seeds, characterized in that, The heat-stress-induced degenerative germination bacteria refer to germination bacteria strains that have been treated at 35°C for 5 hours. The application includes: adding strigolactone to the germination medium, using the strigolactone-added medium to promote the symbiotic germination of heat-stressed degenerating germination bacteria and Gastrodia elata seeds, thereby increasing the symbiotic germination rate of the seeds; the concentration of strigolactone in the germination medium is 0.01 μmol / L.

2. The application according to claim 1, characterized in that, The application specifically includes the following steps: (1) preparing a germination medium containing styrax lactone: add styrax lactone to the germination medium, mix well, and make the final concentration of styrax lactone 0.01 μmol / L; (2) inoculating germinating bacteria after heat stress: inoculate the germinating bacteria after high temperature treatment at 35℃ into the germination medium prepared in step (1), sow the detoxified Gastrodia elata seeds within 5 mm away from the fungal block, and culture them in the dark at 20-25℃ for 30-60 days, and count the germination rate.

3. The application of styracil lactone in the preparation of a culture medium for restoring the symbiotic germination ability of germinating bacteria and Gastrodia elata seeds after heat stress, characterized in that, The germination medium contains 0.01 μmol / L of strigolactone.

4. The application according to claim 1 or 4, characterized in that, The germination medium is 1% agar / water medium.

5. A method for restoring the symbiotic germination ability of germinating fungi and Gastrodia elata seeds after heat stress, characterized in that, Includes the following steps: (1) The germination bacteria treated at 35℃ for 5 hours were inoculated onto a germination medium containing 0.01 μmol / L styrax lactone, and the sterilized Gastrodia elata seeds were sown around the germination bacteria block for symbiotic germination.

6. The method according to claim 6, characterized in that, The symbiotic germination culture described in step (1) includes: sowing Gastrodia elata seeds within 5 mm of the mycelium block and incubating them in the dark at 20-25℃ for 30-60 days.

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