Auriculariaceae strain PL03 and application thereof in promoting pleione bulbocodioides seeds to germinate and form seedlings
By using the Ceropegiaceae strain PL03 to symbiotically germinate with the seeds of the Cymbidium spp. in agar oatmeal culture medium and optimizing the culture conditions, the problem of the decreasing population of the Cymbidium spp. was solved, efficient seed germination and seedling formation were achieved, and the large-scale production of the Cymbidium spp. was promoted.
Patent Information
- Application Number
- CN202510620237.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-14
- Publication Date
- 2025-10-10
AI Technical Summary
Existing technology makes it difficult to effectively utilize the symbiotic mycorrhizal fungi of Cymbidium orchid for large-scale reproduction, resulting in a decrease in the population of Cymbidium orchid. The tissue culture method is time-consuming and has a low survival rate, which limits its industrial development.
The strain PL03 of Ceropegiaceae was used to promote seed germination and seedling formation of Cymbidium spp. The bacterial blocks were inoculated into agar oatmeal medium for symbiotic germination culture with seed suspension, and the culture conditions such as temperature, humidity and photoperiod were optimized.
The germination rate and seedling formation rate of the garlic orchid were significantly improved, the problems of high cost and complicated operation were solved, and the foundation was laid for large-scale production.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of microorganisms, and in particular to a Ceropegiaceae strain PL03 and an application thereof in promoting the germination of Orchidaceae seeds to form seedlings. Background Art
[0002] Pleione bulbocodioides, a semi-epiphytic orchid, is commonly known as "ice ball" due to its pseudobulb, which resembles a single garlic clove. It grows in evergreen broadleaf forests or shrublands at altitudes between 900 and 3,600 meters. Currently, due to overexploitation of its wild resources, its population has declined dramatically, putting it on the brink of extinction. Therefore, protecting wild Pleione resources is urgent.
[0003] The seeds of the garlic orchid are as fine as dust, and the natural reproduction coefficient is extremely low. The main method of artificial cultivation to protect the germplasm resources of the garlic orchid is tissue culture, which uses sterile germination of seeds and relies on pseudobulbs to induce germination. Although tissue culture can make the garlic orchid proliferate 3 to 4 times each year, this tissue culture method is time-consuming, and the survival rate of tissue culture seedlings is low. When the tissue culture seedlings are transplanted into the natural environment, their survival rate is also low. Therefore, the imperfection of the garlic orchid technology has greatly restricted the large-scale development of the garlic orchid industry.
[0004] During its germination phase, the orchid is completely dependent on mycorrhizal fungi for nutrition. The key to using symbiotic bacteria to promote the growth of orchids lies in finding the right fungi. Currently, there are few cases of effective fungi being put into practical use, and there are no reports of effective fungi isolated from the orchid itself.
[0005] Therefore, how to screen and utilize effective symbiotic fungi of Orchidaceae to achieve large-scale reproduction of Orchidaceae is an urgent problem to be solved in this field. Summary of the Invention
[0006] The object of the present invention is to provide a strain of the family Ceropegiaceae PL03, which can effectively promote the germination of seeds of Cyperus uniflorus and the formation of seedlings.
[0007] The present invention specifically includes the following technical solutions:
[0008] The invention discloses a strain PL03 of the family Ceropegiaceae. The strain PL03 of the family Ceropegiaceae is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2024751.
[0009] The present invention also provides the use of the above-mentioned Ceropegiaceae strain PL03 in promoting seed germination and / or seedling formation of Cymbidium spp.
[0010] The present invention also provides a bacterial agent containing the Otaceae strain PL03 according to claim 1.
[0011] Preferably, the bacterial agent includes one or more of bacterial blocks, culture supernatant and bacterial suspension.
[0012] The present invention also provides a method for promoting seed germination and seedling formation of Orchidaceae, which uses the above-mentioned Ceropegiaceae strain PL03 to promote seed germination and seedling formation, and specifically comprises the following steps:
[0013] Prepare seed suspension from the seeds of the Orchidaceae family;
[0014] An agar oatmeal culture medium is inoculated with a bacterial mass of the Otaceae strain PL03, a seed suspension is added to the surface of the agar oatmeal culture medium, the culture dish is sealed, and the seed suspension and bacterial mass in the agar oatmeal culture medium are subjected to symbiotic germination culture until the seeds germinate and / or seedlings are formed.
[0015] Preferably, the step of preparing the seeds of the Cymbidium monnieri into a seed suspension comprises: sterilizing the seeds of the Cymbidium monnieri to obtain sterile seeds; and mixing the sterile seeds, sterile distilled water and 0.1% agar solution to prepare a seed suspension.
[0016] Preferably, the specific operation of disinfecting the seeds of the Orchidaceae includes: soaking and disinfecting them in 1% sodium hypochlorite solution for 5 minutes, and then rinsing them with sterile water for 3 to 5 times to obtain sterile seeds.
[0017] Preferably, the method for preparing the mushroom block comprises:
[0018] The Ceropegiaceae strain PL03 was inoculated into a PDA culture medium for expansion culture until the culture medium was full of hyphae, thereby obtaining a fungus block of the Ceropegiaceae strain PL03.
[0019] Preferably, the conditions for the symbiotic germination culture are: temperature of 23-27° C., humidity of 55-65%, photoperiod L / D=12h / 12h, and light intensity of 4500-5500Lx.
[0020] Preferably, the inoculation ratio of the bacterial block to the seed suspension is 0.5 cm 3 : 30 tablets.
[0021] The beneficial effects of the above technical solution are:
[0022] The application provides a Serendipitaceae strain PL03, which is preserved in the China Center for Type Culture Collection (CCTCC) and has a preservation number of CCTCC NO: M 2024751. The Serendipitaceae strain PL03 provided by the application is isolated by a method of ex situ seed symbiotic germination, has strong specificity, and can effectively promote seed germination of a Ypsilandra to form seedlings. The PL03 strain of the application can be used for field direct seeding of Ypsilandra, and solves the problems of high cost, low yield and complicated operation of artificial culture of Ypsilandra, and lays a foundation for large-scale production of Ypsilandra.
[0023] Biological preservation instructions
[0024] The PL03 strain is classified as a Serendipitaceae species, and is preserved in the China Center for Type Culture Collection (CCTCC) on April 24, 2024, has a preservation address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Center for Type Culture Collection of Wuhan University, and a preservation number of CCTCC NO: M 2024751. DETAILED DESCRIPTION
[0025] The application provides a Serendipitaceae strain PL03. The Serendipitaceae strain PL03 is preserved in the China Center for Type Culture Collection (CCTCC) and has a preservation number of CCTCC NO: M 2024571.
[0026] The application also provides a bacterial agent containing the Serendipitaceae strain PL03 as described above.
[0027] As an embodiment, the bacterial agent includes one, two or three of a bacterial block, a culture supernatant and a bacterial suspension.
[0028] The application also provides application of the Serendipitaceae strain PL03 as described above in promoting seed germination and / or forming seedlings of Ypsilandra.
[0029] The application also provides a method for promoting seed germination and / or forming seedlings of Ypsilandra by using the Serendipitaceae strain PL03, which includes the following steps: preparing a seed suspension of Ypsilandra; inoculating a bacterial block of the Serendipitaceae strain PL03 in an agar oat medium, adding the seed suspension on the surface of the agar oat medium, sealing the culture dish, and then performing symbiotic germination culture on the seed suspension and the bacterial block in the agar oat medium until the seed germinates.
[0030] As an embodiment, the present invention further includes the step of continuing to cultivate the germinated Orchidaceae seeds after the Orchidaceae seeds germinate until seedlings are formed; as an embodiment, the conditions for continuing to cultivate the germinated seeds are: temperature of 23°C to 27°C, humidity of 60%, photoperiod L / D = 12h / 12h, and light intensity of 5000Lx; in a specific embodiment, after the Orchidaceae seeds germinate, the germinated Orchidaceae seeds are placed in an environment of 25°C, humidity of 60%, and photoperiod L / D = 12h / 12h for continued cultivation until seedlings are formed.
[0031] As an embodiment, the present invention prepares a seed suspension from Orchidaceae seeds. As an embodiment, the preparation of the seed suspension from Orchidaceae seeds of the present invention specifically comprises the following steps: sterilizing Orchidaceae seeds to obtain sterile seeds, and mixing the sterile seeds with a sterile agar solution to prepare a seed suspension. As an embodiment, the specific operation of sterilizing Orchidaceae seeds comprises: soaking and disinfecting Orchidaceae seeds in a 1% sodium hypochlorite solution for 5 minutes, and then rinsing with sterile water 3 to 5 times. As an embodiment, the specific preparation steps of the seed suspension comprise: mixing sterile seeds, sterile water, and 0.1% agar solution to suspend the seeds to prepare a seed suspension; in a specific embodiment, the present invention soaks Orchidaceae seeds in a 1% sodium hypochlorite solution for 5 minutes to disinfect, and then rinses with sterile water 5 times to obtain sterile seeds; then, the sterile seeds, sterile water, and 0.1% agar are mixed to suspend the seeds to prepare a seed suspension.
[0032] As an embodiment, the present invention inoculates a bacterial block of the Otaceae strain PL03 in an agar oatmeal medium. As an embodiment, the present invention inoculates the bacterial block in the middle of the agar oatmeal medium.
[0033] The present invention also provides a method for preparing the aforementioned bacterial block, comprising the following steps: inoculating the Otaceae strain PL03 into a PDA medium for strain culture to obtain an activated Otaceae strain PL03; and inoculating the activated Otaceae strain PL03 into a new PDA medium for expansion culture until the medium is fully grown with mycelium, thereby obtaining a bacterial block of the Otaceae strain PL03. In one embodiment, the strain culture is performed under the following conditions: a dark environment, a temperature of 23°C to 27°C, and a humidity of 50% to 60%. In another embodiment, the expansion culture is performed under the following conditions: a dark environment, a temperature of 23°C to 27°C, and a humidity of 50% to 60%. In a specific embodiment, the present invention takes out the Otaceae strain PL03 from a 4°C refrigerator, transfers the fungus to a new PDA culture medium using a sterilized inoculation loop, and then places it in a dark environment at a temperature of 25°C and a humidity of 55% for strain culture to obtain the Otaceae strain PL03; the Otaceae strain PL03 is inoculated into a new culture medium, placed in a dark environment at a temperature of 25°C and a humidity of 55% for expansion culture until the culture medium is full of mycelium, thereby obtaining a fungal block of the Otaceae strain PL03.
[0034] After adding the bacterial mass of the Otaceae strain PL03 to the agar oatmeal culture medium, as an embodiment, the present invention adds the seed suspension to the surface of the agar oatmeal culture medium, seals the culture dish, and then performs a symbiotic germination culture on the seed suspension and bacterial mass in the agar oatmeal culture medium until the seeds germinate. As an embodiment, based on the number of seeds in the seed suspension, the inoculation ratio of the bacterial mass to the seed suspension of the present invention is 0.5 cm 3 : 30 grains. As an embodiment, the sealing of the culture dish described in the present invention specifically includes the following steps: wrapping the culture dish with a sealing film, and then wrapping it with a plastic wrap to seal it, so as to moisturize, isolate the external environment, and avoid pollution. As an embodiment, the culture conditions for symbiotic germination described in the present invention are: temperature of 23℃~27℃, humidity of 55%~65%, photoperiod L / D=12h / 12h, and light intensity of 4500Lx~5500Lx; in a specific embodiment, after adding the bacterial block of the Otaceae strain PL03 to the agar oatmeal culture medium, the present invention mixes the seed suspension according to the same temperature as the bacterial block according to 0.5cm 3 The seeds and the bacterial blocks in the agar oatmeal culture medium were cultured at a ratio of 30:1 until the seeds germinated. The culture dish was wrapped with a sealing film and then sealed with a plastic wrap. The seeds and the bacterial blocks in the agar oatmeal culture medium were cultured in a symbiotic manner at a temperature of 25° C., a humidity of 60%, a photoperiod of L / D of 12 h / 12 h, and a light intensity of 5000 Lx.
[0035] In order to further illustrate the present invention, the embodiments provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present invention.
[0036] Example 1 Seed ex situ symbiotic germination
[0037] In March 2023, the native habitat matrix of the orchid was collected at the Lijiang Alpine Botanical Garden in Yunnan Province, brought back to the laboratory and divided into 18 plastic square boxes with the specifications of 20.5 cm long, 12.5 cm wide, and 6.0 cm high. A layer of nylon cloth with a mesh of 45 μm was covered on the native habitat matrix in the plastic square box, and sterile water was sprayed to ensure full contact between the nylon cloth and the native habitat matrix.
[0038] Seeds of the genus Echinops were purchased from a private nursery and stored in a -20°C freezer. Before the experiment, the seeds were removed from the freezer and placed in a disposable syringe. The contact surface between the syringe and the needle was blocked with a 2-cm-diameter nonwoven fabric. A 1% NaClO solution was then drawn into the syringe until the solution completely soaked the seeds and allowed enough room for shaking. The syringe was then shaken to allow the NaClO solution to fully contact the seeds for 5 minutes to disinfect them. The syringe was then rinsed with sterile distilled water, shaken, and then expelled five times to obtain sterilized seeds.
[0039] The sterilized Cymbidium orchid seeds, sterile distilled water and 0.1% agar suspension were mixed to suspend the Cymbidium orchid seeds to prepare a seed suspension.
[0040] Use a pipette to evenly transfer the seed suspension to the nylon gauze in the plastic square box, then evenly sprinkle 0.3 cm thick native habitat matrix on the surface of the nylon gauze to cover the nylon cloth, spray sterile water on the surface of the native habitat matrix to keep it moist, and place it in a constant temperature incubator with a culture temperature of 25°C and a photoperiod of L / D = 12 / 12h. Observe the seed germination every two weeks and maintain the humidity of the matrix in the plastic box.
[0041] After 45 days, protocorms were found in the box one after another. Symbiotic protocorms and symbiotic seedlings were picked out at different time periods for fungal isolation.
[0042] The standards for different stages of seed germination are as follows:
[0043] 1) Ungerminated seeds;
[0044] 2) The seeds absorb water and swell, producing root-like structures (germination stage);
[0045] 3) The seed continues to swell, breaking through the seed coat and the primary meristem appears (protocorm development stage);
[0046] 4) The first leaf emerged and roots emerged (seedling development and growth stage).
[0047] Isolation of the strain of the family Sebacina PL03
[0048] The symbiotic protocorms at the protocorm development stage of the symbiotic protocorms of the plant of the genus Aglaia isolated in Example 1 were washed with clean water for 3 times to remove the original habitat substrate on the surface of the symbiotic protocorms, and then the symbiotic protocorms were placed in a sterile culture dish containing 2% NaClO solution for 3 min and gently shaken to disinfect, and then washed with sterile water for 4 times.
[0049] The symbiotic protocorms after disinfection were cut into 0.5 mm size pieces with a sterile scalpel, and then the pieces were inserted into new PDA medium with tweezers, and placed in a 25°C incubator for dark culture. After new mycelium grew on the edge of the plant tissue pieces, the mycelium was cut with a sterile scalpel and transferred to new PDA medium, and purified for 4 times to obtain a purified strain, which was recorded as strain PL03.
[0050] The physiological study was carried out on the purified strain, which had the following microbial characteristics: the colony was yellow-brown and had white flocculent on the surface after 7 days of culture on PDA plate, and grew regularly and circularly.
[0051] The micro-morphological characteristics of the strain were observed by cover glass insertion piece culture method: the insertion pieces were prepared by conventional sectioning method after 10 days of culture at 25°C in dark inverted PDA medium. Under optical microscope, it was observed that the mycelium of the strain of the family Sebacina PL03 had septa, the diameter was 0.86 μm-2.72 μm, the growth rate of the mycelium was 0.108±0.005 mm / h, the branches were nearly at right angles, the septa were present near the branches, the colony edge was neat, and the old mycelium intertwined together to form spiral coils.
[0052] The DNA of the isolated strain was extracted by CTAB method, and the primers for PCR amplification were ITS1 and ITS4, and the sequences were shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively; the characteristics of the nrDNA ITS sequence in the nucleotide sequence of the strain were shown in SEQ ID NO. 3.
[0053] SEQ ID NO. 1: 5'-TCCGTAGGTGAACCTGCGG-3';
[0054] SEQ ID NO. 2: 5'-TCCTCCGCTTATTGATATGC-3';
[0055] SEQ ID NO. 3:
[0056] 5'-TCTTCCGTAGGGTGAACCTGCGGAAGGATCATTAATGAATCAAATTC -3';
[0057] The PCR reaction system (25 μl) included: 2.5 μl 10× PCR buffer, 0.4 μl dNTP, 1.5 μl Mg 2+ , 1.5μl ITS1, 1.5μl ITS4, 0.2μl Taq enzyme, 15.4μl ddH2O, 2μl DNA template;
[0058] Amplification reactions were performed on a Perkin Elmer PCR instrument with the following PCR cycles: 1 cycle of initial denaturation at 94°C for 3 min, 30 cycles of denaturation at 94°C for 1 min, annealing at 51°C for 1 min, and extension at 72°C for 1 min, and a final extension at 72°C for 10 min.
[0059] The PCR amplification products were sequenced by Shanghai Sangon Biotechnology Co., Ltd. The sequences were submitted to the National Center for Biotechnology Information database for comparison, and the classification status of the strain was preliminarily confirmed.
[0060] A BLAST comparison of the strain of the present invention showed that the strain was most similar to the uncultured Serendipitaceae fungus MG707393.1, with a maximum similarity of 98%. Based on the physiological studies of the purified strain and the BLAST comparison results, strain PL03 belongs to the family Ceroplastidae.
[0061] Purified strains were preserved using the conventional test tube slant method. The procedure is as follows: Pour an appropriate amount of prepared PDA medium into an 18 mm x 20 mm glass test tube, filling approximately one-third of the tube's volume. After silicone stoppering, sterilize the tube in an autoclave at 121°C for 20 minutes. After sterilization, place the tube in a clean bench and arrange it as a slant for later use. On the clean bench, inoculate the purified strain onto the PDA slant using a sterile inoculating needle, picking hyphae from the edge. The slant is labeled with the strain, serial number, and date. The inoculated tube is placed in a 25°C, dark incubator containing PDA, wrapped with parafilm, then wrapped with plastic wrap. The humidity in the incubator is maintained at approximately 50%. When hyphae begin to grow all over the PDA slant, remove the tube and store it in a 4°C refrigerator in the laboratory. The isolated strains are deposited with the China Type Culture Collection.
[0062] The Otaceae strain PL03 was deposited in the fungal bank of the Plant Germplasm Resources Conservation Group of Yunnan University in December 2023, and was deposited in the China Center for Type Culture Collection for biological preservation in April 2024, with the deposit number: CCTCC NO: M 2024751.
[0063] Example 3 Experiment on promoting symbiotic germination of Orchidaceae seeds by using the strain PL03 of the family Ceropegiaceae
[0064] The PL03 strain isolated in Example 2 was subjected to a symbiotic germination test with seeds in a symbiotic culture medium to test the effectiveness of the obtained fungus on the germination of Orchidaceae seeds. The steps are as follows:
[0065] Preparation of symbiotic culture medium: The symbiotic germination medium is oatmeal agar (OMA) medium, including 4g·L -1 Oatmeal powder and 15g L -1Agar, pH 5.7 of the culture medium. Prepare 3L of culture medium, prepare 120 culture dishes, a total of 4 treatment groups, respectively, test group, control group 1, control group 2 and blank control group, each treatment set 30 replicates.
[0066] Seed disinfection: Take the preserved seed of D. candidum from the-20℃ refrigerator, and place it at room temperature for 10h. Weigh 0.0083g of seed into the weighing paper, fold it well, and pour the seed into a disposable syringe on the clean bench. Plug the connection between the needle and the needle cylinder with sterilized non-woven fabric, and suck 1% sodium hypochlorite solution into the seed to make full contact, and oscillate the needle cylinder. After 5min, discharge the waste liquid, and then suck sterile water to wash 5 times to obtain sterile seeds.
[0067] Inoculation and sowing: inoculate the mycelium on new PDA medium to obtain a culture medium covered with mycelium, take a piece of culture medium containing mycelium to obtain a mycelial block; inoculate the sterile D. candidum seed with 1g·L -1 Mix the sterile agar solution to make a seed suspension.
[0068] Test group: inoculate 0.5cm 3 PL03 strain mycelial block, according to the ratio of mycelial block:D. candidum seed=0.5cm 3 :30 seeds, add seed suspension;
[0069] Control group 1: inoculate 0.5cm 3 Indian pear-shaped SI strain mycelial block, according to the ratio of mycelial block:D. candidum seed=0.5cm 3 :30 seeds, add seed suspension; Indian pear-shaped SI strain can be purchased through conventional market channels.
[0070] Control group 2: inoculate 0.5cm 3 Gelatin film Agp-1 strain mycelial block, according to the ratio of mycelial block:D. candidum seed=0.5cm 3 :30 seeds, add seed suspension; GenBank accession number of Agp-1 strain is: MK651837, which is disclosed in the literature "Are fungi from adult orchid roots the best symbionts at germination? A case study".
[0071] Blank control group: inoculate 0.5cm 3 PDA medium without mycelium, add 30 D. candidum seeds.
[0072] After the above-mentioned configuration is completed, the culture dish is wrapped with a sealing film and then with a plastic wrap for symbiotic germination culture; the seeded culture dish is placed in an artificial climate chamber and cultured at a constant temperature of 25°C, with a photoperiod of L / D = 12h / 12h and a light intensity of 5000Lx.
[0073] Seed germination was counted 30 days, 60 days, and 90 days after symbiotic culture. Germinated protocorms or seedlings were randomly selected according to different treatments at each stage for observation under a stereomicroscope. The germinated protocorms and seedlings were stained and observed to confirm the fungal infection of the seeds.
[0074] Seed germination and seedling formation are divided into the following four stages:
[0075] 1) Ungerminated seeds;
[0076] 2) The seeds absorb water and swell, producing root-like structures (germination stage);
[0077] 3) The seed continues to swell, breaking through the seed coat and the primary meristem appears (protocorm development stage);
[0078] 4) The first leaf grows and further grows, and roots grow (seedling development and growth stage).
[0079] Based on the above criteria, the seed germination rate (G), protocorm formation rate (P), and seedling development rate (S) were calculated. t represents the total number of seeds, g represents the number of seeds that germinated, p represents the number of seeds that formed protocorms, and s represents the number of seeds that formed seedlings. The calculation formulas are as follows: G = (g + p + s) / t, P = (p + s) / t, and S = s / t. All data are summarized as mean ± standard error (SE).
[0080] The experiment was terminated after 90 days of symbiotic culture. The statistical results at the 90th day and the non-parametric test methods Kruskal-Wallis test (KW) and Mann-Whitney U-test (U) were used to perform significance tests on the seed germination rate, protocorm formation rate, and seedling development rate of different treatments, with α = 0.05; the results are shown in Table 1.
[0081] Table 1 Effects of different fungal treatments on seed germination of Cymbidium spp. (90-day statistics)
[0082]
[0083]
[0084] As shown in Table 1, although the non-inoculated treatment can also make the seeds of the single garlic orchid germinate to the protocorm stage, no seedlings appear after 90 days of culture; the Agp-1 strain isolated from the protocorm of the bamboo leaf orchid did not produce seedlings after 90 days of symbiotic germination with the seeds of the single garlic orchid, and the germination rate and protocorm formation rate were also low, indicating that it had no obvious promoting effect on its germination; Indian pear-shaped spore (Serendipita indica) can promote the germination of the seeds of the single garlic orchid to the seedling stage, and the seedling rate reached 27.59±3.45% after 90 days of germination; the wax shell ear strain PL03 isolated from the protocorm of the single garlic orchid can significantly promote the formation of the protocorm and seedling development, and the seedling quality is significantly better than that of the other treatment groups. Although the SI strain can also promote the germination of the seeds of the single garlic orchid to the seedling stage, the quality of the seedlings in the later stage cannot keep up, and some seeds and seedlings begin to brown. The germination rate, protocorm formation rate and seedling development rate of the Ceropegiaceae strain PL03 were significantly higher than those of the SI treatment, indicating that the Ceropegiaceae strain PL03 is an effective fungus in promoting seed germination and seedling growth of Orchidaceae.
[0085] In summary, different fungal treatments have different effects on the germination of Orchidaceae seeds. All treatments can promote germination in the early stages of symbiotic germination, and in particular, the non-inoculated treatment can form a small amount of protocorms. However, this only represents the seed absorbing water and swelling, the embryo breaking through the seed coat and growing, and the beginning of meristem formation. It cannot be used as a standard for practical application. The more important indicator is the seedling development rate. In the later stage of symbiotic culture, that is, the seedling formation stage, although the SI strain can promote the formation of seedlings from Orchidaceae seeds to a certain extent, the seedling quality is far lower than that of the treatment with the Ceropegiaceae strain PL03, and there is also a certain degree of seed and seedling mortality. The Ceropegiaceae strain PL03, obtained from seedlings symbiotically germinated from Orchidaceae seeds ex situ, has the best effect on promoting the germination of Orchidaceae seeds. It can be used to symbiotically germinate Orchidaceae seeds using the Ceropegiaceae strain PL03 and Orchidaceae seeds to obtain high-quality symbiotic seedlings.
[0086] The strain of the present invention is carried out through a symbiotic germination experiment, in which sterile Cymbidium uniflorum seeds, different fungi and a blank control are cultured on an oat culture medium respectively. By comparing the seed germination rate, the protocorm formation rate and the seedling development rate, an effective strain that promotes the development of Cymbidium uniflorum seeds into seedlings is successfully screened, thereby opening up a new path for the efficient seedling cultivation, protection practice and population reconstruction of Cymbidium uniflorum, and providing an effective way to realize the actual breeding and production work of Cymbidium uniflorum and its return to the wild.
[0087] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements can be made without departing from the principles of the present invention. These improvements should also be regarded as the scope of protection of the present invention.
Claims
1. A Serendipitaceae strain PL03, characterized in that The Otaceae strain PL03 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M2024751.
2. Use of the Otaceae strain PL03 according to claim 1 in promoting seed germination and / or seedling formation of Orchidaceae.
3. A bacterial agent, characterized in that Contains the Otaceae strain PL03 as claimed in claim 1.
4. The bacterial agent according to claim 3, characterized in that The bacterial agent includes one or more of bacterial blocks, culture supernatant and bacterial suspension.
5. A method for promoting seed germination and seedling formation of Orchidaceae, characterized in that: The method of promoting seed germination and seedling formation by using the Otaceae strain PL03 according to claim 1 specifically comprises the following steps: Prepare seed suspension from the seeds of the Orchidaceae family; An agar oatmeal culture medium is inoculated with a bacterial mass of the Otaceae strain PL03, a seed suspension is added to the surface of the agar oatmeal culture medium, the culture dish is sealed, and the seed suspension and bacterial mass in the agar oatmeal culture medium are subjected to symbiotic germination culture until the seeds germinate and / or seedlings are formed.
6. The method according to claim 5, wherein The steps of preparing the seeds of the Cyperus rotundus into a seed suspension include: sterilizing the seeds of the Cyperus rotundus to obtain sterile seeds; and mixing the sterile seeds, sterile distilled water and 0.1% agar solution to prepare the seed suspension.
7. The method according to claim 6, wherein The specific operation of disinfecting the seeds of the orchid comprises: soaking and disinfecting them in a 1% sodium hypochlorite solution for 5 minutes, and then washing them with sterile water for 3 to 5 times to obtain sterile seeds.
8. The method according to claim 5, wherein The preparation method of the mushroom block comprises: The Otaceae strain PL03 was inoculated into a PDA culture medium for expansion culture until the culture medium was full of hyphae, thereby obtaining a fungus block of the Otaceae strain PL03.
9. The method according to claim 5, wherein The conditions for the symbiotic germination culture are: temperature of 23-27° C., humidity of 55-65%, photoperiod L / D=12h / 12h, and light intensity of 4500-5500Lx.
10. The method according to claim 5, wherein The inoculation ratio of the bacterial block to the seed suspension is 0.5 cm 3 : 30 tablets.