Application and methods of using *Gastromyzolium galactosomum* NBG001 to promote the germination or seedling growth of Isatis indigotica seeds.

Abstract

This invention provides an application and method for using *Gastrodia galactosum* NBG001 to promote the germination of *Isatis indigotica* seeds or the growth of seedlings, belonging to the field of plant growth promotion technology. The *Gastrodia galactosum* NBG001 was deposited at the China Center for Type Culture Collection on July 7, 2025, with accession number CCTCC NO: M 20251547. After treating *Isatis indigotica* seeds with *Gastrodia galactosum* NBG001, the germination rate of *Isatis indigotica* increased to 40.6%, which is 1.6 times that of the control group. After transplanting, the fresh weight, number of leaves, and number of roots of *Isatis indigotica* seedlings treated with *Gastrodia galactosum* NBG001 were significantly higher than those of the control group, increasing by 1.6, 1.5, and 1.2 times, respectively.

Description

Technical Field

[0001] This invention relates to the field of plant growth promotion technology, specifically to the application and method of using *Gastromyxonomycetes NBG001* to promote the germination of *Isatis indigotica* seeds or the growth of seedlings. Background Technology

[0002] Isatis indigotica ( Isatis indigotica Fort. Isatis indigotica (Isatis tinctoria) is a biennial herbaceous plant belonging to the genus Isatis indigotica in the Brassicaceae family. Its dried roots and leaves are considered traditional Chinese medicinal materials. However, the seeds of Isatis indigotica are enclosed in pods, resulting in low germination rates and slow growth. Therefore, plant growth regulators are widely used in cultivation to improve yield and promote seed germination, plant growth, and development. Plant growth regulators, such as sodium nitrophenolate, mepiquat chloride, and paclobutrazol, have been detected at high rates in Isatis indigotica root and leaves, seriously affecting the quality and safety of the medicinal materials.

[0003] Plant growth-promoting fungi can promote seed germination, plant growth, and root development. Furthermore, they offer better ecological safety compared to chemical growth regulators. Summary of the Invention

[0004] In order to overcome the above-mentioned defects and deficiencies in the prior art, the present invention provides an application and method of Galactobacillus gnatus NBG001 in promoting the germination of Isatis indigotica seeds or the growth of seedlings, which can effectively promote the germination of Isatis indigotica seeds or the growth of seedlings.

[0005] To solve the above technical problems:

[0006] The first objective of this invention is to provide a *Gastrozyme galactosomalidone* NBG001 strain, which was deposited at the China Center for Type Culture Collection on July 7, 2025, with accession number CCTCC NO: M 20251547. Soaking in a *Gastrozyme galactosomalidone* NBG001 solution significantly promotes the germination of *Isatis indigotica* seeds and the growth of seedlings.

[0007] The second objective of this invention is to provide an application of Galactobacillus NBG001 in promoting the germination of Isatis indigotica seeds and the growth of seedlings.

[0008] A third objective of this invention is to provide a method for promoting the germination of Isatis indigotica seeds or the growth of seedlings, comprising the following steps:

[0009] Isolation and identification of S1 Galactobacillus NBG001;

[0010] S2 preparation of Galactys NBG001 bacterial culture;

[0011] S3 Isatis indigotica seed surface sterilization;

[0012] S4 germination treatment: Sterilized woad seeds were soaked in a solution of *Gastrodia elata* galactosomal yeast NBG001.

[0013] S5 Place the treated Isatis indigotica seeds from step S4 into a petri dish containing double-layered sterile filter paper, seal the dish, and place it in a light incubator for germination;

[0014] After S6 germinates, it is transplanted into the cultivation substrate.

[0015] Galactobacillus NBG001 was isolated from Lycoris plants in the Lycoris radiata cultivation area of ​​Nanjing Zhongshan Botanical Garden. The isolation process is as follows:

[0016] Lycoris plants were collected from the Lycoris radiata planting area of ​​Nanjing Zhongshan Botanical Garden (32°03'N, 118°50'E). Roots were cut with a sterile blade and surface-sterilized in a laminar flow hood. The specific procedure involved immersion in 75% ethanol for 1 minute, immersion in 5% sodium hypochlorite for 5 minutes, immersion in 75% ethanol again for 1 minute, and rinsing three times with sterile double-distilled water. After blotting dry on sterile filter paper, the roots were cut into 1 cm segments with sterile scissors and cultured on PDA medium for 5 days. Mycelia growing from the cuts were picked, transferred to new PDA medium, streaked for purification, and single colonies were obtained. Single colonies were picked and cultured in PDA medium at 200 rpm and 30°C for 2 days. Total DNA was extracted, and the 18S rRNA region of the fungus was amplified using primers SSU0817F and 1196R. Sequencing and BLAST comparison with known sequences in GenBank identified it as *Galactomyces galactosoma*. Galactomycesgeotrichum Next, a single colony was picked and cultured on a new PDA medium for 5 days. After observing the colony morphology, it was confirmed to be *Gastromyces galactosomalid*, and named NBG001.

[0017] The specific process of step S2 is as follows:

[0018] Pick a single colony formed on PDA medium and place it in 50 mL of PDB medium. Incubate in a shake flask at 200 rpm and 30°C for 2 days. Then, [the colony is then incubated at 4000 ml / mL]. g Collect bacterial cells by centrifugation for 10 min. After washing the bacterial cells twice with 10 mM MgSO4 buffer, resuspend them in 10 mM MgSO4 buffer and adjust the OD600 to 0.1 for later use.

[0019] The specific process of step S3 is as follows: Isodon seeds are soaked in sterile double-distilled water to absorb water, then transferred to 75% ethanol for soaking, then washed with double-distilled water, and the water is absorbed by sterile filter paper and set aside for use.

[0020] The specific process of step S4 is as follows: the sterilized Isatis indigotica seeds from step S3 are soaked in the Gastrodia elata NBG001 bacterial solution for 1 hour.

[0021] In step S5, the illuminance is 1500 lx and the illumination time is 16 h•d. -1 The culture temperature was 22°C.

[0022] In step S6, the cultivation substrate consists of peat moss and vermiculite in a volume ratio of 1:1.

[0023] The beneficial technical effects achieved by this invention: This invention provides a type of Galactobacillus galactosidase (Gastromycin galactosidase). Galactomycesgeotrichum Application and methods of NBG001 in promoting the germination of Isatis indigotica seeds and the growth of seedlings: After treating Isatis indigotica seeds with Galactomyces galactosum NBG001, the germination rate of Isatis indigotica increased to 40.6%, which was 1.6 times that of the control group. After transplanting, the Isatis indigotica seedlings treated with Galactomyces galactosum NBG001 had significantly higher fresh weight, number of leaves, and number of roots than the control group seedlings, increasing by 1.6, 1.5, and 1.2 times, respectively. Attached Figure Description

[0024] Figure 1 The colony morphology and phylogenetic tree of Galactobacillus NBG001 are shown; (A) is a photograph of the colony morphology of NBG001; (B) is a phylogenetic tree constructed based on the 18S rRNA sequence.

[0025] Figure 2 This is a statistical analysis of the germination rate of Isatis indigotica seeds after treatment with Galactobacillus NBG001 bacterial solution in an embodiment of the present invention.

[0026] Figure 3 This is a photograph of the morphology of Isatis indigotica germinating after being treated with Galactobacillus NBG001 bacterial solution for 10 days, according to an embodiment of the present invention.

[0027] Figure 4 This is a statistical analysis of the growth of Isatis indigotica seedlings after treatment with Galactobacillus gnatus NBG001 bacterial solution in an embodiment of the present invention; wherein, (A) is a photograph of Isatis indigotica seedlings; (B) is a statistical analysis of the fresh weight of Isatis indigotica seedlings; (C) is a statistical analysis of the number of leaves of Isatis indigotica seedlings; and (D) is a statistical analysis of the number of roots of Isatis indigotica seedlings.

[0028] Figure 5 This is an example of gene expression in Isatis indigotica seeds and seedlings after treatment with Galactobacillus NBG001 bacterial solution according to an embodiment of the present invention.

[0029] Figure 6 Gene Ontology (GO) enrichment of the woad-specific expression gene induced by *Gastromyxonomycetes NBG001* bacterial culture in this embodiment of the invention;

[0030] Figure 7This invention relates to the expression of genes related to seed germination and growth in Isatis indigotica seeds and seedlings after treatment with Galactobacillus NBG001 bacterial solution in an embodiment of the present invention.

[0031] The preservation information of the *Gastrodinium galactosomalidase* of this invention is as follows:

[0032] Galactobacillus cereus, classified and named Galactomycesgeotrichum NBG001 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20251547, on July 7, 2025. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments. These embodiments are only used to more clearly illustrate the technical solutions of the present invention and should not be construed as limiting the scope of protection of the present invention.

[0034] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0035] Example 1: Isolation and Identification Method of Galactobacillus NBG001

[0036] Galactobacillus NBG001 was isolated from Lycoris plants at Nanjing Zhongshan Botanical Garden. The specific isolation process is as follows:

[0037] Lycoris plants were collected from the Lycoris radiata planting area of ​​Nanjing Zhongshan Botanical Garden (32°03'N, 118°50'E). The roots were cut off with a sterile blade and surface sterilized in a laminar flow hood. The specific procedure was as follows: soaking in 75% ethanol for 1 minute, soaking in 5% sodium hypochlorite for 5 minutes, soaking again in 75% ethanol for 1 minute, and rinsing three times with sterile double-distilled water. After blotting dry on sterile filter paper, the roots were cut into 1 cm long segments with sterile scissors and cultured on potato dextrose agar (PDA) medium (200 g potato, 20 g glucose, 20 g agar, 1 L water) for 5 days. Mycelia growing from the cuts were then transferred to fresh potato dextrose agar PDA medium for streaking purification, yielding single colonies. Single colonies were picked and cultured in PDB medium at 200 rpm and 30°C for 2 days. Total DNA was extracted, and the 18S rRNA region of the fungus was amplified using primers SSU0817F and 1196R. Sequencing was performed, and the result was compared with a known sequence in GenBank via BLAST, confirming the fungal *Galactomyces cerevisiae*. Next, single colonies were picked and cultured on fresh PDB medium for 5 days. Colony morphology was observed, confirming the *Galactomyces cerevisiae* genus, and named NBG001. The colony morphology of NBG001 and the phylogenetic tree constructed based on 18S rRNA are shown below. Figure 1 As shown, A is a photograph of colony morphology, and B is a growth tree.

[0038] Example 2: Method for promoting the germination of Isatis indigotica seeds or the growth of seedlings using Galactyspora NBG001

[0039] 1. Preparation of *Gastromycosis galactosomalidone* NBG001 suspension: A single colony formed on PDA medium was picked and placed in 50 mL of PDB medium. After incubation in a shake flask at 200 rpm and 30°C for 2 days, the suspension was further incubated at 4000 mL / min. g Collect bacterial cells by centrifugation for 10 min. After washing the bacterial cells twice with 10 mM MgSO4 buffer, resuspend them in 10 mM MgSO4 buffer and adjust the OD600 to 0.1 for later use.

[0040] 2. Sterilization treatment of Isatis indigotica seeds: Take about 1000 Isatis indigotica seeds and randomly place them into two sterile 50 mL centrifuge tubes, with about 500 seeds in each tube. Then, add 30 mL of sterile double-distilled water and let them swell at 30°C for 5 hours. After that, transfer the Isatis indigotica seeds to 75% ethanol and soak them for 30 minutes. Then, wash them three times with sterile double-distilled water and blot dry with sterile filter paper. Set aside for use.

[0041] 3. Germination Treatment: After sterilization, the Isatis indigotica seeds were soaked for 1 hour. Then, they were placed in square petri dishes (11 cm × 11 cm) lined with double-layered sterile filter paper, with 25 seeds per dish, for a total of 20 dishes (500 seeds). 25 mL of sterile double-distilled water was added to each dish. The dishes were then sealed with sealing film and placed in a light incubator at a light intensity of 1500 lx for 16 h·d. -1 The culture temperature was 22°C.

[0042] 4. Seedling Transplanting: Ten days after germination, transplant the seedlings into a 1:1 (v / v) mixture of peat moss and vermiculite. First, fill the pot with the substrate. Then, dig a hole in the center and place the roots of the woad seedling into the hole. Finally, completely cover the roots with the substrate, water thoroughly, and cover the pot with plastic wrap to prevent the seedlings from drying out and wilting. Remove the plastic wrap two days after transplanting.

[0043] Example 3: Effect of *Gastromyzolium galactosomalidum* NBG001 on the germination rate of Isatis indigotica seeds.

[0044] Sterilized Isatis indigotica seeds were randomly divided into two groups: one group was soaked in a culture of Galactobacillus NBG001 (treatment group), and the other group was soaked in 10 mM MgSO4 buffer (control group). After soaking for 1 h, the seeds of the treatment and control groups were placed in square Petri dishes (11 cm × 11 cm) lined with double-layered sterile filter paper, with 25 seeds per dish. Each treatment and control group consisted of 20 dishes, totaling 500 seeds. 25 mL of sterile double-distilled water was added to each dish. The square Petri dishes were then sealed with sealing film and placed in a light incubator at an illuminance of 1500 lx for 16 h·d. -1 The cultivation temperature was 22°C. The germination rate of each group of seeds was observed and recorded daily. Isatis indigotica seeds with a radicle length exceeding half the seed diameter were considered to have germinated. The germination rate was calculated using the following formula:

[0045] Germination rate = (Number of germinating seeds / Number of seeds tested) × 100%;

[0046] The germination rate of Isatis indigotica seeds was as follows: Figure 2 As shown: After 4 days of treatment, the germination rate of Isatis indigotica seeds in the group soaked in the Galactobacillus NBG001 bacterial solution began to be higher than that in the control group. After 6 days of treatment, the germination rate of Isatis indigotica seeds in the group soaked in the bacterial solution began to be significantly higher than that in the control group. This significant promotion continued until 10 days of treatment. At this time, the germination rate of the control group seeds was 25.4%, while the germination rate of the treated group seeds reached 40.6%. Galactobacillus NBG001 can increase the germination rate of Isatis indigotica seeds by 1.6 times.

[0047] Ten days after treatment with Isatis tinctoria seeds, two randomly selected plates from each of the control and treatment groups were used to photograph the germinated seeds. The results are as follows: Figure 3 As shown, after soaking in the solution of Galactobacillus NBG001, not only was the germination rate of Isatis indigotica seeds significantly higher than that of the control group, but the growth of the seedlings after germination was also significantly better than that of the control group.

[0048] Example 4: Effects of *Gastromyzolium galactosomalidum* NBG001 on the growth of *Isatis indigotica* seedlings

[0049] Ten days after germination, seedlings of similar growth from both the control and treatment groups were transplanted into a 1:1 (v / v) mixture of peat moss and vermiculite. Ten seedlings were transplanted from each group. First, the pots were filled with the cultivation substrate. Then, a hole was dug in the center, and the roots of the Isatis indigotica seedlings were placed in the hole. Finally, the roots were completely covered with the cultivation substrate, thoroughly watered, and the pots were covered with plastic wrap to prevent dehydration and wilting. The plastic wrap was removed two days after transplanting. Ten days after transplanting, i.e., 20 days after seed germination, the Isatis indigotica seedlings were harvested, weighed, and the number of leaves and roots was counted. The results are as follows: Figure 4 As shown.

[0050] from Figure 4 It can be seen that the fresh weight, leaf number, and root number of Isatis indigotica seedlings in the immersion group in the Galactobacillus NBG001 bacterial solution were significantly higher than those in the control group, increasing by 1.6, 1.5, and 1.2 times, respectively.

[0051] Example 5: Effect of *Gastromycosis galactosidase* NBG001 treatment on *Isatis indigotica* gene expression.

[0052] Isatis tinctoria seeds treated with *Gastrodia elata* NBG001 for 3 and 6 days were harvested, seed pods were removed, and the seeds were flash-frozen in liquid nitrogen. Total RNA was extracted, and after quality control, mRNA was enriched. The mRNA was randomly fragmented into small fragments of approximately 300 bp, reverse-engineered into cDNA using random primers, adapters were ligated, and PCR amplification was performed to obtain libraries, which were then sequenced. After quality control of the sequencing data, the data were compared with the Isatis tinctoria genome for gene annotation. Simultaneously, the expression levels of different genes were quantitatively analyzed to obtain the FPKM values ​​of each sample in the control and treatment groups. Further analysis was performed using R software. The treatment method for total RNA extraction from Isatis tinctoria seedlings was the same.

[0053] It should be noted that since the woad seeds had not yet germinated on day 3, the total RNA extracted on day 3 was from the woad seeds, while on day 6, since the seeds had already germinated, the total RNA extracted was from the woad seedlings. The results are as follows. Figure 5 – Figure 7 As shown. Among them, Figure 5 Gene expression of Isatis indigotica seeds after 3 days (3 DAT) and 6 days (6 DAT) of treatment with Galactobacillus gnatus NBG001 inoculum; Figure 6 The GO enrichment of specific expression genes in Isatis tinctoria seeds after treatment with Galactobacillus NBG001 bacterial culture. Figure 7 This study statistically analyzed the expression of genes related to seed germination and growth in Isatis indigotica seeds and seedlings treated with Galactobacillus NBG001 bacterial culture.

[0054] from Figure 5 The results show that after 3 and 6 days of treatment, the gene expression of Isatis indigotica in the *Gastrodia elata* NBG001 inoculum group differed from that in the control group. Specifically, the samples treated with *Gastrodia elata* NBG001 inoculum for 3 and 6 days showed significant segregation on the first principal component (PC1), with a variance contribution rate of 69.88%; while the samples in the control and treatment groups showed significant segregation on the second principal component (PC2), with a variance contribution rate of 7.21%. Therefore, compared with the control group, the gene expression of Isatis indigotica seeds and seedlings showed significant differences after treatment with *Gastrodia elata* NBG001 inoculum.

[0055] from Figure 6 The results show that after 3 days of treatment with Galactobacillus galactosidase NBG001, genes related to seed germination were enriched in Isatis indigotica seeds; after 6 days of treatment, genes related to cell cycle, seedling development, seed germination, and cytokinin-activated signaling were enriched in Isatis indigotica seedlings; this demonstrates that NBG001 promotes seed germination and seedling growth at the gene expression level.

[0056] from Figure 7 The results show that on day 3 after treatment with *Gastrodia galactosum* NBG001 inoculum, the expression of genes related to *Isatis tinctoria* germination showed significant differences compared to the control group, especially lin15055.t2 and lin27175.t2. The differences were even more pronounced on day 6, particularly lin04558.t1, lin08249.t1, lin15055.t2, lin23218.t5, and lin24252.t3. Therefore, treatment with *Gastrodia galactosum* NBG001 inoculum altered gene expression in *Isatis tinctoria* seeds and seedlings, further promoting seed germination and seedling growth.

[0057] The present invention has been disclosed above with reference to preferred embodiments, but it is not intended to limit the present invention. All technical solutions obtained by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the present invention.

Claims

1. A type of *Geotrichum galactosidase* that promotes the germination of *Isatis indigotica* seeds or the growth of seedlings (… Galactomyces geotrichum NBG001, characterized in that: The aforementioned Galactylogyrus NBG001 was deposited at the China Center for Type Culture Collection on July 7, 2025, with accession number CCTCC NO: M 20251547.

2. The Galactobacillus galactosidase according to claim 1 ( Galactomyces geotrichum NBG001, characterized in that: Treating Isatis tinctoria seeds with Galactyspora NBG001 bacterial solution promotes seed germination and seedling growth.

3. A method for promoting the germination of Isatis indigotica seeds or the growth of seedlings, characterized in that... Includes the following steps: S1 prepares the bacterial culture of Galactobacillus NBG001 as described in claim 1; S2 Isatis indigotica seed surface sterilization; S3 germination treatment: Sterilized woad seeds were soaked in a solution of *Gastrodia elata* galactosomal yeast NBG001. S4 Place the treated Isatis indigotica seeds from step S3 into a petri dish containing double-layered sterile filter paper, seal the dish, and place it in a light incubator for germination; After S5 germinates, it is transplanted into the cultivation substrate.

4. The method for promoting the germination of Isatis indigotica seeds or the growth of seedlings according to claim 3, characterized in that, The specific process of step S1 is as follows: Pick a single colony formed on PDA medium and place it in 50 mL of PDB medium. Incubate in a shake flask at 200 rpm and 30°C for 2 days. Then, [the colony is then incubated at 4000 ml / mL]. g Collect bacterial cells by centrifugation for 10 min. After washing the bacterial cells twice with 10 mM MgSO4 buffer, resuspend them in 10 mM MgSO4 buffer and adjust the OD600 to 0.1 for later use.

5. The method for promoting the germination of Isatis indigotica seeds or the growth of seedlings according to claim 3, characterized in that, The specific process of step S2 is as follows: Isodon seeds are soaked in sterile double-distilled water to absorb water, then transferred to 75% ethanol for soaking, washed with double-distilled water, and dried with sterile filter paper before use.

6. The method for promoting the germination of Isatis indigotica seeds or the growth of seedlings according to claim 3, characterized in that, The specific process of step S3 is as follows: The sterilized Isatis indigotica seeds in step S2 are soaked in the bacterial solution of Galactobacillus NBG001 for 1 hour.

7. The method for promoting the germination of Isatis indigotica seeds or the growth of seedlings according to claim 3, characterized in that, In step S4, the illuminance is 1500 lx and the illumination time is 16 h•d. -1 The culture temperature was 22°C.

8. The method for promoting the germination of Isatis indigotica seeds or the growth of seedlings according to claim 3, characterized in that, In step S5, the cultivation substrate consists of peat moss and vermiculite in a volume ratio of 1:1.

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