A method for rapidly breaking dormancy of sand whip seeds and sand whip seeds with broken dormancy

Abstract

This invention provides a method for rapidly breaking the dormancy of *Trichoderma harzianum* seeds and the method for breaking the dormancy of *Trichoderma harzianum* seeds, which relates to the field of plant seed treatment technology, including the following steps: (1) Physical initiation treatment: soaking the disinfected *Trichoderma harzianum* seeds in warm water at 45-55℃ for 10-14h; (2) Chemical initiation treatment: soaking the seeds treated in step (1) in a gibberellin solution with a mass concentration of 0.03%-0.07% for 20-28h; (3) Biological seed dressing treatment: dressing the seeds treated in step (2) with *Trichoderma harzianum* MCBC3, wherein the mass of the inoculant is 0.05%-0.15% of the seed mass; This invention can effectively break the dormancy mechanism of *Trichoderma harzianum* seeds and promote seed germination. The germination rate of *Trichoderma harzianum* seeds treated by the preferred method of this invention can reach 87.00% on the 21st day, and can also make the seed germination synchronization higher, solving the problem of uneven natural germination.

Description

Technical Field

[0001] This invention relates to the field of plant seed treatment technology, specifically to a method for rapidly breaking dormancy in *Sargassum fusiforme* seeds and the method for breaking dormancy in *Sargassum fusiforme* seeds. Background Technology

[0002] *Psammochloa villosa* (Trin.) Bor is a perennial herbaceous plant belonging to the Poaceae family. It is a typical psammophyte, mainly distributed on mobile and semi-fixed sand dunes in arid and semi-arid regions of Inner Mongolia, Shaanxi, Ningxia, and Gansu in my country. *Psammochloa villosa* possesses extremely strong drought, cold, and poor soil tolerance, with a well-developed root system and strong sand-fixing ability. It is an important native plant germplasm resource for ecological restoration and windbreak and sand fixation in desertified areas of northern my country.

[0003] However, *Salvia splendens* seeds possess inherent physiological dormancy characteristics. Under natural conditions, *Salvia splendens* seeds have a dense and hard seed coat, poor water and air permeability, and contain certain endogenous inhibitory substances, leading to difficulty in germination, low germination rates, and uneven germination. Existing research indicates that untreated *Salvia splendens* seeds typically have low germination rates under conventional culture conditions, severely limiting their large-scale application in ecological restoration projects.

[0004] Therefore, developing a comprehensive treatment method that can quickly and efficiently break the dormancy of *Salvia splendens* seeds and significantly improve the germination rate is of great practical significance and application value in meeting the urgent need for high-quality *Salvia splendens* seedlings in desertification control. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for rapidly breaking the dormancy of *Sargassum fusiforme* seeds and the method for breaking the dormancy of *Sargassum fusiforme* seeds, as detailed below: A method for rapidly breaking dormancy in seeds of *Sargassum fusiforme* includes the following steps: (1) Physical initiation treatment: Soak the disinfected seeds of *Sargassum fusiforme* in warm water at 45-55℃ for 10-14 hours; (2) Chemical initiation treatment: The seeds treated in step (1) were soaked in a gibberellin (GA3) solution with a mass concentration of 0.03%-0.07% for 20-28 hours; (3) Biological seed dressing treatment: The seeds treated in step (2) are dressed with Trichoderma harzianum MCBC3, and the mass of the inoculant is 0.05%-0.15% of the seed mass; the strain preservation number of Trichoderma harzianum MCBC3 is CGMCC No.41745, which was deposited at the China General Microbiological Culture Collection Center on January 6, 2025.

[0006] Furthermore, in step (1), the water temperature is 50℃ and the soaking time is 12h.

[0007] Furthermore, in step (2), the mass concentration of the gibberellin (GA3) solution is 0.05%.

[0008] Furthermore, the mass of Trichoderma harzianum MCBC3 mentioned in step (3) is 1% of the seed mass.

[0009] Furthermore, before step (1), seed disinfection treatment is also included: soak the seeds of *Sargassum fusiforme* in 75% alcohol for 1-3 minutes, then rinse them with distilled water 4-6 times, and dry them with filter paper for later use.

[0010] Furthermore, after the chemical initiation treatment in step (2), the seeds are washed with distilled water 2-4 times.

[0011] Further, the seed dressing method described in step (3) is as follows: place the seeds of Trichoderma harzianum and Trichoderma harzianum MCBC3 in a sealed bag, add 10%-20% of the seed weight of distilled water, seal the bag and shake it vigorously to make the fungicide evenly adhere to the seed surface, and then open the sealed bag to dry.

[0012] On the other hand, the dormancy-breaking seeds of *Sargassum fusiforme* prepared by the method provided by the present invention have a germination rate of over 87.00% on the 21st day.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The seeds of *Trichoderma harzianum* possess inherent physiological dormancy characteristics. Their dense, hard seed coats have poor water and air permeability and contain endogenous inhibitory substances, leading to germination difficulties. This invention combines physical initiation treatment (warm water soaking), chemical initiation treatment (gibberellin soaking), and biological seed dressing treatment (*Trichoderma harzianum*) to effectively break the dormancy mechanism of *Trichoderma harzianum* seeds and promote germination. Experimental results show that *Trichoderma harzianum* seeds treated with the preferred method of this invention (50℃ warm water soaking for 12h + 0.05% gibberellin soaking for 24h + 1% *Trichoderma harzianum* dressing) achieved a germination rate of 87.00% on day 21, significantly higher than the untreated control group and superior to single treatment methods. This also improves seed germination synchronization and solves the problem of uneven natural germination.

[0014] 2. This invention provides a method for rapidly breaking dormancy in seeds of *Trichoderma harzianum*. This invention combines physical initiation treatment (warm water soaking), chemical initiation treatment (gibberellin soaking), and biological seed dressing treatment (*Trichoderma harzianum*). Physical initiation treatment, through warm water soaking, improves the water and air permeability of the seed coat, facilitating water absorption, swelling, and the initiation of internal physiological activities. Gibberellin in the chemical initiation treatment is a plant growth regulator that promotes the synthesis and signal transduction of endogenous hormones in the seed, thereby enhancing the seed's germination ability. *Trichoderma harzianum* in the biological seed dressing treatment may improve the microenvironment around the seed by producing beneficial metabolites or competitively inhibiting pathogens, further promoting healthy germination and growth.

[0015] 3. The physical, chemical and biological treatment methods used in this invention are all environmentally friendly technologies that will not cause pollution or damage to environmental elements such as soil and water. At the same time, Trichoderma harzianum, as a biological control agent, can also help reduce the amount of chemical pesticides used and protect the safety and health of the ecological environment. Attached Figure Description

[0016] Figure 1 The effects of different physical factor treatments on the germination of *Fragaria spp.* seeds; Figure 2 The improvement rate of seed germination rate of Fritillaria cirrhosa under different physical factor treatments compared with the CK treatment; Figure 3 The effects of different chemical treatments on the germination of seeds of Fritillaria cirrhosa; Figure 4 The increase in germination rate of Fritillaria cirrhosa seeds under different chemical treatments compared to the control (CK) treatment; Figure 5 The effects of different biological factor treatments on the germination of Fritillaria cirrhosa seeds; Figure 6 The improvement rate of seed germination rate of Fritillaria cirrhosa under different biological factor treatments compared with the CK treatment; Figure 7 The effects of different combined factor treatments on the germination of seeds of Fritillaria cirrhosa; Figure 8 The improvement rate of seed germination rate of Fritillaria cirrhosa under different combined factor treatments compared with the CK treatment. Detailed Implementation

[0017] Example 1 A method for rapidly breaking dormancy in seeds of *Sargassum fusiforme* includes the following steps: (1) Physical initiation treatment: Soak the disinfected seeds of *Sargassum fusiforme* in warm water at 45℃ for 10 hours; (2) Chemical initiation treatment: The seeds treated in step (1) were soaked in a 0.03% gibberellin (GA3) solution for 20 hours; (3) Biological seed dressing treatment: The seeds treated in step (2) are dressed with Trichoderma harzianum MCBC3, and the mass of the inoculant is 0.1% of the seed mass; the strain preservation number of Trichoderma harzianum MCBC3 is CGMCC No.41745, which was deposited at the China General Microbiological Culture Collection Center on January 6, 2025.

[0018] Furthermore, before step (1), seed disinfection treatment is also included: soak the seeds of *Sargassum fusiforme* in 75% alcohol for 1 minute, then rinse them 4 times with distilled water, and dry them with filter paper for later use.

[0019] Furthermore, after the chemical initiation treatment in step (2), the seeds are washed twice with distilled water.

[0020] Further, the seed mixing method described in step (3) is as follows: place the seeds of Trichoderma harzianum and Trichoderma harzianum MCBC3 in a sealed bag, add 20% of the seed weight of distilled water, seal the bag and shake it vigorously to make the inoculant evenly adhere to the seed surface, and then open the sealed bag to dry.

[0021] Example 2 A method for rapidly breaking dormancy in seeds of *Sargassum fusiforme* includes the following steps: (1) Physical initiation treatment: Soak the disinfected seeds of *Sargassum fusiforme* in warm water at 55℃ for 14 hours; (2) Chemical initiation treatment: The seeds treated in step (1) were soaked in a 0.07% gibberellin (GA3) solution for 28 hours; (3) Biological seed dressing treatment: The seeds treated in step (2) are dressed with Trichoderma harzianum MCBC3, and the mass of the inoculant is 0.15% of the seed mass; the strain preservation number of Trichoderma harzianum MCBC3 is CGMCC No.41745, which was deposited at the China General Microbiological Culture Collection Center on January 6, 2025.

[0022] Furthermore, before step (1), seed disinfection treatment is also included: soak the seeds of *Sargassum fusiforme* in 75% alcohol for 3 minutes, then rinse them 6 times with distilled water, and dry them with filter paper for later use.

[0023] Furthermore, after the chemical initiation treatment in step (2), the seeds are washed four times with distilled water.

[0024] Further, the seed mixing method described in step (3) is as follows: place the seeds of Trichoderma harzianum and Trichoderma harzianum MCBC3 in a sealed bag, add 10% of the seed weight of distilled water, seal the bag and shake it vigorously to make the inoculant evenly adhere to the seed surface, and then open the sealed bag to dry.

[0025] Example 3 A method for rapidly breaking dormancy in seeds of *Sargassum fusiforme* includes the following steps: (1) Physical initiation treatment: Soak the disinfected seeds of *Sargassum fusiforme* in warm water at 50°C for 12 hours; (2) Chemical initiation treatment: The seeds treated in step (1) were soaked in a 0.05% gibberellin (GA3) solution for 24 hours; (3) Biological seed dressing treatment: The seeds treated in step (2) are dressed with Trichoderma harzianum MCBC3, and the mass of the inoculant is 0.05% of the seed mass; the strain preservation number of Trichoderma harzianum MCBC3 is CGMCC No.41745, which was deposited at the China General Microbiological Culture Collection Center on January 6, 2025.

[0026] Furthermore, before step (1), seed disinfection treatment is also included: soak the seeds of *Sargassum fusiforme* in 75% alcohol for 2 minutes, then rinse them 5 times with distilled water, and dry them with filter paper for later use.

[0027] Furthermore, after the chemical initiation treatment in step (2), the seeds are washed three times with distilled water.

[0028] Further, the seed mixing method described in step (3) is as follows: place the seeds of Trichoderma harzianum and Trichoderma harzianum MCBC3 in a sealed bag, add 15% of the seed weight of distilled water, seal the bag and shake it vigorously to make the inoculant evenly adhere to the seed surface, and then open the sealed bag to dry.

[0029] Experimental Section I. Experimental Objective The study investigated the effects of different physical, chemical, and biological factors, as well as the combination of these three factors, on the germination of *Sargassum fusiforme* seeds, and screened out the factors most conducive to the germination of *Sargassum fusiforme* seeds.

[0030] II. Experimental Materials 1. Test seeds: The seeds of *Rhizophora stylosa* were collected in the wild in Ordos.

[0031] 2. Test reagents and bacterial strains: Sodium hydroxide (granules) (purity ≥96%) was purchased from Tianjin Fengchuan Chemical Reagent Technology Co., Ltd., potassium permanganate was purchased from Harbin Qianhe Animal Pharmaceutical Manufacturing Co., Ltd., hydrogen peroxide and gibberellin (GA3) were purchased from Beijing Solarbio Technology Co., Ltd., and cytokinin (6-BA) was purchased from Tianjin Zhonglian Chemical Reagent Co., Ltd.

[0032] Trichoderma harzianum MCBC3, with the culture accession number CGMCC No. 41745, was deposited on January 6, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0033] Bacillus sp. MCYP-Z2-13, with accession number CGMCC No.33162, was deposited on December 23, 2024, at the China General Microbiological Culture Collection Center, located at No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0034] Arbuscular mycorrhizal fungi: Rhizophagus intraradices, purchased from Sichuan Geological and Mineral Resources Yimida Environmental Protection Technology Co., Ltd., trade name: Endophytic mycorrhizal fungi agent, implementation standard GB20287-2006, registration certificate number: Microbial Fertilizer (2018) Approval No. (3500).

[0035] III. Experimental Design Processing group settings: A control group (CK) was set up. There were 12 physical factor treatments (water temperature was the initial water temperature), 16 chemical factor treatments, 12 biological factor treatments, and 6 combined factor treatments. The biological factor treatment was seed dressing. Each treatment had three replicates. Detailed treatment groups and their numbers are shown in Tables 1 and 2.

[0036] Table 1 Single-factor treatment

[0037] Table 2 Treatment of composite factors

[0038] IV. Experimental Methods Weigh out 48 portions of sand whip seeds, each 20g, disinfect by soaking in 75% alcohol for 2 minutes, rinse 5 times with distilled water, and dry with filter paper for later use.

[0039] 1. Treatment of physical factors Seed soaking treatment: (1) Use a graduated cylinder to collect 100 mL of distilled water at temperatures of 30℃, 40℃ and 50℃ respectively, collect 3 times at each temperature, and pour them into different petri dishes; (2) Place the sterilized sand whip into a petri dish and cover it with a filter paper to ensure that the sand whip seeds are fully soaked; (3) After soaking the seeds of *Sarcandra glabra* for the appropriate time, pour out the water, use filter paper to absorb the remaining water, and arrange the seeds on a plate after they have dried.

[0040] Polishing process: Take three portions of sand whip seeds, place them between two sheets of sandpaper (CC-360), press them down with your palms, and gently rub them 4, 12, and 20 times respectively before arranging them on a plate.

[0041] The germination rate of *Sargassum fusiforme* seeds treated with different physical factors was analyzed, and the results were as follows ( Figure 1 , Figure 2 The results showed that, except for W9, W10, W11, and W12, all other treatments improved the germination rate of *Sargassum fusiforme* seeds on day 21. Treatments W5 and W8 showed the best effect in improving the germination rate on day 21, with W8 being the treatment that achieved the best germination rate at 58.89% on day 21.

[0042] 2. Chemical Factor Treatment (1) Prepare 100 mL of each of the corresponding solutions and pour them into a petri dish; (2) Place the sterilized sand whip into a petri dish and cover it with a filter paper to ensure that the sand whip seeds are fully soaked; (3) After soaking the seeds of *Sarcandra glabra* for the appropriate time, pour out the water, rinse them three times with distilled water, absorb the remaining water with filter paper, and arrange them on a plate after they have dried.

[0043] The germination rate of *Sargassum fusiforme* seeds treated with different chemical factors was analyzed, and the results were as follows ( Figure 3 , Figure 4 The results showed that, except for treatments H5 and H13, all other treatments increased the germination rate of *Vaccaria segetalis* seeds. Treatments with gibberellin concentrations H1, H2, and H3, as well as treatment with sodium hydroxide soaking (H9), all showed good effects, with germination rates of 63.7%, 63.93%, 61.79%, and 65.33% on day 21, respectively. Among these, treatments with gibberellin concentrations H1, H2, and H3 showed the best overall effect, while H9 showed the best effect.

[0044] 3. Treatment with biological factors (1) Pour the sterilized seeds of *Sargassum fusiforme* into a sealed bag; (2) Use an electronic balance to weigh the corresponding mass of inoculant (the mass of the inoculant is a specified percentage of the seed mass) and pour it into a sealed bag; (3) Pour 5 mL of distilled water into the sealed bag; (4) After sealing the bag, shake it vigorously to ensure that the inoculant is mixed evenly; (5) Open the sealed bag and arrange the seeds on a plate after they have dried.

[0045] The germination rate of *Vigna arvense* seeds treated with different biological factors was analyzed, and the results were as follows ( Figure 5 , Figure 6The results showed that the germination rate of *Sargassum fusiforme* seeds treated with inoculant was lower than that of the control (CK) group. The least affected group was S1, with a germination rate of 38.67% on day 21.

[0046] 4. Treatment of complex factors First, seeds were soaked in warm water, then soaked in a chemical reagent solution, and finally treated with a biological agent (Table 2). The germination rate of *Sargassum fusiforme* seeds under different combined treatments was analyzed, and the results were as follows (…). Figure 7 , Figure 8 The results showed that the F1, F2, F4, and F5 treatments all increased the germination rate of *Sargassum fusiforme* seeds by more than 75%, with germination rates of 78.67%, 81.33%, 87.00%, and 80.67% on day 21, respectively. Among them, the F4 treatment had the best effect, with a germination rate of 87.00%.

[0047] It should be noted that when biological factors were used alone, Trichoderma harzianum seed treatment failed to directly increase the germination rate of *Trichoderma harzianum* seeds, and even slightly decreased it. This is mainly because the inoculant itself does not have the physiological effect of directly breaking seed dormancy. Furthermore, under sterile laboratory conditions, the main limiting factor for seed germination remains dormancy, not pathogenic factors. At this stage, the dormancy characteristics of the seeds cause them to exhibit rejection or competitive inhibition of microorganisms, resulting in a slightly lower germination rate compared to the control. However, *Trichoderma harzianum* was still included in the combined treatment, primarily based on its potential function as a biocontrol agent. *Trichoderma harzianum* can inhibit the infection of seeds by soil pathogens through mechanisms such as competition, antagonism, and induced resistance, thereby providing a healthier microecological environment for seed germination in the soil. Therefore, although its improvement on germination rate under sterile conditions was not significant, in actual soil sowing, *Trichoderma harzianum* helps reduce the incidence of seedling diseases and improve seedling uniformity and seedling survival rate. This invention combines physical and chemical dormancy-breaking treatments with biological protection methods, which not only effectively breaks the dormancy of sand whip seeds, but also improves their adaptability and survival ability in the natural environment, thereby optimizing the entire process from germination to seedling emergence.

Claims

1. A method for rapidly breaking dormancy in seeds of *Sargassum fusiforme*, characterized in that, Includes the following steps: (1) Physical initiation treatment: Soak the disinfected seeds of *Sargassum fusiforme* in warm water at 45-55℃ for 10-14 hours; (2) Chemical initiation treatment: The seeds treated in step (1) were soaked in a gibberellin solution with a mass concentration of 0.03%-0.07% for 20-28 hours; (3) Biological seed dressing treatment: The seeds treated in step (2) are dressed with Trichoderma harzianum MCBC3, wherein the mass of Trichoderma harzianum MCBC3 is 0.05%-0.15% of the seed mass; the strain preservation number of Trichoderma harzianum MCBC3 is CGMCCNo.41745.

2. The method for rapidly breaking dormancy in seeds of *Sargassum fusiforme* according to claim 1, characterized in that, In step (1), the temperature of the warm water is 50°C and the soaking time is 12 hours.

3. The method for rapidly breaking dormancy in seeds of *Sargassum fusiforme* according to claim 1, characterized in that, In step (2), the mass concentration of the gibberellin solution is 0.05%.

4. The method for rapidly breaking dormancy in seeds of *Sargassum fusiforme* according to claim 1, characterized in that, In step (3), the mass of Trichoderma harzianum MCBC3 is 1% of the seed mass.

5. The method for rapidly breaking dormancy in seeds of *Sargassum fusiforme* according to claim 1, characterized in that, Before step (1), seed disinfection is also included: soak the seeds of *Sargassum fusiforme* in 75% alcohol for 1-3 minutes, then rinse them with distilled water 4-6 times, and dry them with filter paper for later use.

6. The method for rapidly breaking dormancy in seeds of *Sargassum fusiforme* according to claim 1, characterized in that, After the chemical initiation treatment in step (2), the seeds are also washed with distilled water 2-4 times.

7. The method for rapidly breaking dormancy in seeds of *Sargassum fusiforme* according to claim 1, characterized in that, In step (3), the specific method of seed dressing is as follows: place the seeds of Trichoderma harzianum and Trichoderma harzianum MCBC3 in a sealed bag, add distilled water, seal the bag and shake it vigorously to make the inoculant evenly adhere to the seed surface, and then open the sealed bag to dry.

8. The method for rapidly breaking dormancy in seeds of *Sargassum fusiforme* according to claim 7, characterized in that, The amount of distilled water added should be 10%-20% of the seed weight.

9. Dormant seeds of *Sargassum fusiforme* prepared by the method according to any one of claims 1-8.

10. The dormancy-breaking seed of *Sargassum fusiforme* according to claim 9, characterized in that, The germination rate of the *Sargassum fusiforme* seeds reached over 87.00% on day 21.

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