A method for improving the germination rate of cistanche seed

Abstract

The application relates to the technical field of seed germination, in particular to a method for improving the germination rate of Cistanche seed. The method comprises the following steps: (1) preparing a seed culture solution; (2) seed disinfection; (3) seed pretreatment; (4) seed culture. The method can significantly improve the germination rate of Cistanche seed.

Description

Technical Field

[0001] This invention relates to the field of seed germination technology, and specifically to a method for improving the germination rate of Cistanche deserticola seeds. Background Technology

[0002] Cistanche deserticola seeds exhibit dormancy. Germination requires not only a lengthy after-ripening process but also a germination stimulus signal from the host root system. In recent years, the area under artificial cultivation of Cistanche deserticola has been expanding annually. However, due to the small, round, and delicate nature of the seeds, natural germination is difficult, and the seeds are expensive. This results in difficulty controlling the sowing rate, high planting costs, low seed utilization, and a low parasitism rate, hindering artificial inoculation and propagation. In their study, Sheng Jinhua et al. reported changes in endogenous hormone content during the post-ripening stage of Cistanche deserticola seeds [J]. They indicated that low-temperature stratification treatment could break seed dormancy and complete seed post-ripening within three months. After post-ripening, the endogenous hormone content in the seeds increased, with higher levels of indole-3-acetic acid (IAA), zeatin (ZR), and gibberellin (GA3), while the abscisic acid (ABA) content decreased. Niu Dongling et al., in their study on the dormancy and germination characteristics of Cistanche deserticola seeds [J], used room-temperature water treatment for 30 days and cultured seeds with GA3. Under constant temperature conditions of 25℃, the germination rate reached a maximum of 16.7%. In the above studies, the germination of Cistanche deserticola seeds was only induced by chemical substances or treated with plant hormones, resulting in limited improvement in germination rate. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention proposes a method for improving the germination rate of Cistanche deserticola seeds.

[0004] This invention is achieved through the following technical solution: A method for improving the germination rate of Cistanche deserticola seeds includes the following steps: (1) Preparation of seed culture medium: (11) Dry the oyster mushrooms to constant weight, pulverize them through a 60-80 mesh sieve to obtain oyster mushroom powder, add the oyster mushroom powder to an 80% ethanol solution, heat and reflux at 90°C twice for 3 hours each time, filter, dry the filter residue to obtain pretreated oyster mushroom powder. (12) Add the pretreated oyster mushroom powder obtained in step (11) to distilled water, sterilize at 121℃ for 20 min, and after cooling, use 10 7 Inoculate activated Lactobacillus paracasei at an inoculum size of CFU / mL and ferment at 37°C for 24 h. (13) After fermentation in step (12), sterilize at 100℃ for 10 min, sonicate at 300-400W for 10 min, water bath at 90℃ for 30 min, cool and centrifuge at 6000-7000 r / min for 10-15 min, take the supernatant, add 4 times the volume of ethanol, let stand at 4℃ overnight, centrifuge at 8000 rpm for 10-15 min, wash the precipitate with anhydrous ethanol, reconstitute with distilled water, freeze dry under vacuum to obtain oyster mushroom fermentation agent; (14) After washing and drying licorice, purslane, and poplar flowers, pulverize them separately and pass them through an 80-100 mesh sieve. Mix them in a mass ratio of 3:2:1 to obtain a mixed Chinese herbal powder. Add the mixed Chinese herbal powder to deionized water and mix well. Sterilize at 121℃ for 30 min, cool to room temperature, and then... 7 Inoculate activated Lactobacillus plantarum at an inoculation rate of CFU / mL; (15) After inoculation in step (14), ferment at 37℃ for 24 h, centrifuge at 10000 rpm for 8-12 min, filter the supernatant through a 0.22μm filter membrane, add gibberellin at a ratio of 100-150 mg / L, add the oyster mushroom fermentation agent obtained in step (13) at a ratio of 15-20 mg / mL, mix well, and obtain seed culture solution; (2) Seed disinfection: Take mature and plump Cistanche deserticola seeds, rinse them 3 times with distilled water, soak them in 70% ethanol for 2 minutes, rinse them 3 times with distilled water, soak them in 2% sodium hypochlorite solution for 10 minutes, rinse them 3 times with distilled water, and use sterile filter paper to dry the surface moisture of the seeds. (3) Seed pretreatment: The Cistanche deserticola seeds disinfected in step (2) were soaked in distilled water for 4 h in the dark at 30℃, then added to the seed culture solution and soaked in the dark at 25℃ for 20-24 h. The seeds were rinsed 3 times with sterile water and then air-dried. (4) Seed culture: 50 seeds of Cistanche deserticola after pretreatment in step (3) were evenly inoculated into a 9 cm diameter petri dish with two layers of filter paper, 4 mL of seed culture solution was added, the dish was sealed with sealing film, and cultured in the dark at 25℃. 1 mL of seed culture solution was added every 7 days.

[0005] Further, in step (11), the ratio of the oyster mushroom powder to the ethanol solution is 1 g: 15-20 mL.

[0006] Further, in step (12), the ratio of the oyster mushroom powder to distilled water is 1 g: 10-15 mL.

[0007] Furthermore, in step (12), the Lactobacillus paracasei was purchased from the Guangdong Provincial Microbial Culture Collection Center, with the number GDMCC NO.1.159.

[0008] Further, in step (14), the ratio of the mixed Chinese medicine powder to deionized water is 1 g: 15-20 mL.

[0009] Furthermore, in step (14), the Lactobacillus plantarum was purchased from Guangdong Provincial Microbial Culture Collection Center, with the number GDMCC NO.1.1516.

[0010] Furthermore, in step (2), the diameter of the Cistanche deserticola seeds is preferably 0.6-0.7 mm.

[0011] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method to improve the germination rate of Cistanche deserticola seeds. The method involves alcohol extraction and defatting of Pleurotus ostreatus, followed by fermentation of large molecules such as polysaccharides by Lactobacillus paracasei. After fermentation with probiotics, the large polysaccharides in Pleurotus ostreatus are degraded into low-molecular-weight active oligosaccharides, transforming the active polysaccharides into oligosaccharide fragments and specific signaling molecules that are more easily absorbed by the seeds. During fermentation, small-molecule organic acids such as lactic acid and acetic acid produced by Lactobacillus paracasei, synergistically with the active fungal oligosaccharides, can efficiently penetrate into the seed interior. The combination of Lactobacillus paracasei metabolites and Pleurotus ostreatus degradation products can mimic the germination stimulation signals secreted by the host root system, effectively initiating the germination of Cistanche deserticola seeds and inducing haustoria differentiation. This invention also employs Lactobacillus plantarum to ferment a traditional Chinese medicine composition of licorice, purslane, and poplar flowers. Lactobacillus plantarum fermentation not only gently extracts active ingredients such as flavonoids and organic acids from the traditional Chinese medicine but also produces new secondary metabolites through microbial transformation. This synergistic effect regulates the hormonal balance within the seed. The fermentation broth contains amino acids, small peptides, and oligosaccharides, providing nutrition for early seed germination. Simultaneously, the phenolic acids in the fermentation broth help maintain cell wall rigidity in the early haustorium structure. Lactobacillus plantarum fermentation enhances biological activity, degrading macromolecular polysaccharides from traditional Chinese medicine into easily absorbed oligosaccharides, and producing abundant amino acids and short-chain fatty acids. The free flavonoids and phenolic acids in the fermentation broth are natural antioxidants, scavenging reactive oxygen species produced during seed germination, increasing germination rate, and ensuring normal radicle growth after germination. The small amino acids, oligosaccharides, and organic acids produced during fermentation can be absorbed and utilized by the seeds, providing a continuous and sufficient energy and nutrient supply for seed germination, radicle elongation, and subsequent haustorium differentiation. Attached Figure Description

[0012] Figure 1 The germination rate of Cistanche deserticola seeds described in Examples 1-3 and Comparative Examples 1-4 of this invention; Figure 2 The haustorium formation rate of the seeds of Cistanche deserticola described in Examples 1-3 and Comparative Examples 1-4 of this invention. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. However, this invention is not limited to the following embodiments. It should be noted that, unless otherwise specified, all chemical reagents involved in this invention are purchased through commercial channels.

[0014] Example 1: A method for improving the germination rate of Cistanche deserticola seeds, comprising the following steps: (1) Preparation of seed culture medium: (11) Dry the oyster mushrooms to constant weight, pulverize them through a 60-80 mesh sieve to obtain oyster mushroom powder. Add 10 g of oyster mushroom powder to 200 mL of 80% ethanol solution, heat and reflux at 90°C twice for 3 h each time, filter, dry the filter residue to obtain pretreated oyster mushroom powder. (12) Add 6 g of the pretreated oyster mushroom powder obtained in step (11) to 90 mL of distilled water, sterilize at 121℃ for 20 min, and after cooling, use 10 7 Activated Lactobacillus paracasei was inoculated at a CFU / mL inoculum and fermented at 37℃ for 24 h; Lactobacillus paracasei was purchased from Guangdong Provincial Microbial Culture Collection Center, with the number GDMCC NO.1.159; (13) After fermentation in step (12), sterilize at 100℃ for 10 min, sonicate at 400W for 10 min, bathe in water at 90℃ for 30 min, cool and centrifuge at 7000 r / min for 15 min, take the supernatant, add 4 times the volume of ethanol, let stand at 4℃ overnight, centrifuge at 8000 rpm for 15 min, wash the precipitate with anhydrous ethanol, reconstitute with distilled water, freeze dry under vacuum to obtain oyster mushroom fermentation agent; (14) After washing and drying licorice, purslane, and poplar flowers, pulverize them separately and pass them through a 100-mesh sieve. Mix them in a mass ratio of 3:2:1 to obtain a mixed Chinese herbal powder. Add 10 g of the mixed Chinese herbal powder to 200 mL of deionized water and mix well. Sterilize at 121℃ for 30 min, cool to room temperature, and then sterilize at 4×10⁻⁶. 7 The activated Lactobacillus plantarum was inoculated at a concentration of CFU / mL; Lactobacillus plantarum was purchased from Guangdong Provincial Microbial Culture Collection Center, with the number GDMCC NO.1.1516. (15) After inoculation in step (14), ferment at 37℃ for 24 h, centrifuge at 10000 rpm for 12 min, filter the supernatant through a 0.22 μm filter membrane, add gibberellin at a ratio of 150 mg / L, add the oyster mushroom fermentation agent obtained in step (13) at a ratio of 20 mg / mL, mix well, and obtain seed culture solution. (2) Seed disinfection: Take mature and plump Cistanche deserticola seeds with a diameter of 0.7 mm, rinse them 3 times with distilled water, soak them in 70% ethanol for 2 min, rinse them 3 times with distilled water, soak them in 2% sodium hypochlorite solution for 10 min, rinse them 3 times with distilled water, and use sterile filter paper to dry the surface moisture of the seeds. (3) Seed pretreatment: The Cistanche deserticola seeds disinfected in step (2) were soaked in distilled water for 4 h in the dark at 30℃, then added to the seed culture solution and soaked in the dark at 25℃ for 24 h. The seeds were rinsed 3 times with sterile water and then air-dried. (4) Seed culture: 50 seeds of Cistanche deserticola after pretreatment in step (3) were evenly inoculated into a 9 cm diameter petri dish with two layers of filter paper, 4 mL of seed culture solution was added, the dish was sealed with sealing film, and cultured in the dark at 25℃. 1 mL of seed culture solution was added every 7 days.

[0015] Example 2: A method for improving the germination rate of Cistanche deserticola seeds, comprising the following steps: (1) Preparation of seed culture medium: (11) Dry the oyster mushrooms to constant weight, crush them through a 60-mesh sieve to obtain oyster mushroom powder, add 10 g of oyster mushroom powder to 150 mL of 80% ethanol solution, heat and reflux at 90°C twice for 3 h each time, filter, dry the filter residue to obtain pretreated oyster mushroom powder. (12) Add 6 g of the pretreated oyster mushroom powder obtained in step (11) to 60 mL of distilled water, sterilize at 121℃ for 20 min, and after cooling, use 10 7 Activated Lactobacillus paracasei was inoculated at a CFU / mL inoculum and fermented at 37℃ for 24 h; Lactobacillus paracasei was purchased from Guangdong Provincial Microbial Culture Collection Center, with the number GDMCC NO.1.159; (13) After fermentation in step (12), sterilize at 100℃ for 10 min, sonicate at 300W for 10 min, bathe in water at 90℃ for 30 min, cool and centrifuge at 6000 r / min for 10 min, take the supernatant, add 4 times the volume of ethanol, let stand at 4℃ overnight, centrifuge at 8000 rpm for 10 min, wash the precipitate with anhydrous ethanol, reconstitute with distilled water, freeze dry under vacuum to obtain oyster mushroom fermentation agent; (14) After washing and drying licorice, purslane, and poplar flowers, pulverize them separately and pass them through an 80-mesh sieve. Mix them in a mass ratio of 3:2:1 to obtain a mixed Chinese herbal powder. Add 10 g of the mixed Chinese herbal powder to 150 mL of deionized water and mix well. Sterilize at 121℃ for 30 min, cool to room temperature, and then sterilize at 4×10⁻⁶. 7 The activated Lactobacillus plantarum was inoculated at a concentration of CFU / mL; Lactobacillus plantarum was purchased from Guangdong Provincial Microbial Culture Collection Center, with the number GDMCC NO.1.1516. (15) After inoculation in step (14), ferment at 37℃ for 24 h, centrifuge at 10000 rpm for 8 min, filter the supernatant through a 0.22 μm filter membrane, add gibberellin at a ratio of 100 mg / L, add the oyster mushroom fermentation agent obtained in step (13) at a ratio of 15 mg / mL, mix well, and obtain seed culture solution. (2) Seed disinfection: Take mature and plump Cistanche deserticola seeds with a diameter of 0.6 mm, rinse them 3 times with distilled water, soak them in 70% ethanol for 2 min, rinse them 3 times with distilled water, soak them in 2% sodium hypochlorite solution for 10 min, rinse them 3 times with distilled water, and use sterile filter paper to dry the surface moisture of the seeds. (3) Seed pretreatment: The Cistanche deserticola seeds disinfected in step (2) were soaked in distilled water for 4 h in the dark at 30℃, then added to the seed culture solution and soaked in the dark at 25℃ for 20 h. The seeds were rinsed 3 times with sterile water and then air-dried. (4) Seed culture: 50 seeds of Cistanche deserticola after pretreatment in step (3) were evenly inoculated into a 9 cm diameter petri dish with two layers of filter paper, 4 mL of seed culture solution was added, the dish was sealed with sealing film, and cultured in the dark at 25℃. 1 mL of seed culture solution was added every 7 days.

[0016] Example 3: A method for improving the germination rate of Cistanche deserticola seeds, comprising the following steps: (1) Preparation of seed culture medium: (11) Dry the oyster mushrooms to constant weight, crush them through a 70-mesh sieve to obtain oyster mushroom powder, add 10 g of oyster mushroom powder to 180 mL of 80% ethanol solution, heat and reflux at 90°C twice for 3 h each time, filter, dry the filter residue to obtain pretreated oyster mushroom powder. (12) Add 6 g of the pretreated oyster mushroom powder obtained in step (11) to 80 mL of distilled water, sterilize at 121℃ for 20 min, and after cooling, use 10 7 Activated Lactobacillus paracasei was inoculated at a CFU / mL inoculum and fermented at 37℃ for 24 h; Lactobacillus paracasei was purchased from Guangdong Provincial Microbial Culture Collection Center, with the number GDMCC NO.1.159; (13) After fermentation in step (12), sterilize at 100℃ for 10 min, sonicate at 350W for 10 min, bathe in water at 90℃ for 30 min, cool and centrifuge at 6500 r / min for 12 min, take the supernatant, add 4 times the volume of ethanol, let stand at 4℃ overnight, centrifuge at 8000 rpm for 12 min, wash the precipitate with anhydrous ethanol, reconstitute with distilled water, freeze dry under vacuum to obtain oyster mushroom fermentation agent; (14) After washing and drying licorice, purslane, and poplar flowers, pulverize them separately and pass them through a 90-mesh sieve. Mix them in a mass ratio of 3:2:1 to obtain a mixed Chinese herbal powder. Add 10 g of the mixed Chinese herbal powder to 180 mL of deionized water and mix well. Sterilize at 121℃ for 30 min, cool to room temperature, and then sterilize at 4×10⁻⁶. 7 The activated Lactobacillus plantarum was inoculated at a concentration of CFU / mL; Lactobacillus plantarum was purchased from Guangdong Provincial Microbial Culture Collection Center, with the number GDMCC NO.1.1516. (15) After inoculation in step (14), ferment at 37℃ for 24 h, centrifuge at 10000 rpm for 10 min, filter the supernatant through a 0.22 μm filter membrane, add gibberellin at a ratio of 120 mg / L, add the oyster mushroom fermentation agent obtained in step (13) at a ratio of 18 mg / mL, mix well, and obtain seed culture solution. (2) Seed disinfection: Take mature and plump Cistanche deserticola seeds with a diameter of 0.65 mm, rinse them 3 times with distilled water, soak them in 70% ethanol for 2 min, rinse them 3 times with distilled water, soak them in 2% sodium hypochlorite solution for 10 min, rinse them 3 times with distilled water, and use sterile filter paper to dry the surface moisture of the seeds. (3) Seed pretreatment: The Cistanche deserticola seeds disinfected in step (2) were soaked in distilled water for 4 h in the dark at 30℃, then added to the seed culture solution and soaked in the dark at 25℃ for 22 h. The seeds were rinsed 3 times with sterile water and then air-dried. (4) Seed culture: 50 seeds of Cistanche deserticola after pretreatment in step (3) were evenly inoculated into a 9 cm diameter petri dish with two layers of filter paper, 4 mL of seed culture solution was added, the dish was sealed with sealing film, and cultured in the dark at 25℃. 1 mL of seed culture solution was added every 7 days.

[0017] The only difference between Comparative Example 1 and Example 1 is that no oyster mushroom fermentation agent was added.

[0018] The only difference between Comparative Example 2 and Example 1 is that step (14) is omitted. Distilled water is filtered through a 0.22 μm filter membrane, gibberellin is added at a ratio of 150 mg / L, and oyster mushroom fermentation agent is added at a ratio of 20 mg / mL. The mixture is then mixed to form a seed culture solution.

[0019] The only difference between Comparative Example 3 and Example 1 is that after adding the pretreated oyster mushroom powder to distilled water, Lactobacillus paracasei was not inoculated, fermentation was not carried out, and extraction was performed twice at 90°C for 2 hours each time. Four times the volume of ethanol was added to the extract, and the mixture was allowed to stand overnight at 4°C. After centrifugation at 8000 rpm for 15 minutes, the precipitate was washed with anhydrous ethanol, reconstituted with distilled water, and freeze-dried under vacuum. The resulting product was used to replace the oyster mushroom fermentation agent.

[0020] The only difference between Comparative Example 4 and Example 1 is that after adding the mixed Chinese herbal medicine powder to deionized water, it was not inoculated with Lactobacillus plantarum, and fermentation was not carried out. After soaking for 1 hour, it was decocted and boiled for another 30 minutes. The decoction was then filtered to obtain the extract. The filter residue was subjected to the same operation. The two extracts were combined and filtered through a 0.22 μm filter membrane. Gibberellin was added at a ratio of 150 mg / L and oyster mushroom fermentation agent was added at a ratio of 20 mg / mL. The mixture was then mixed to obtain the seed culture medium.

[0021] Experimental Example 1: Following the methods of Examples 1-3 and Comparative Examples 1-4, Cistanche deserticola seeds were cultured, observed every 7 days, and cultured for 30 days. The germination rate was then recorded, and the results are as follows: Figure 1 As shown.

[0022] Figure 1 The results showed that the germination rate of Cistanche deserticola seeds in Examples 1-3 was significantly higher than that in Comparative Examples 1-4. Specifically, Comparative Example 1 did not add oyster mushroom fermentation agent; Comparative Example 2 directly added gibberellin and oyster mushroom fermentation agent to distilled water; Comparative Example 3 did not ferment the oyster mushroom powder; and Comparative Example 4 did not ferment the mixed herbal powder. All of these operations reduced the germination rate of Cistanche deserticola seeds, indicating that the method of the present invention can significantly improve the germination rate of Cistanche deserticola seeds.

[0023] Experimental Example 2: Following the methods of Examples 1-3 and Comparative Examples 1-4, *Cistanche deserticola* seeds were cultured, with observations every 7 days for 70 days. The haustorium formation rate was calculated. The haustorium formation rate was calculated as: [(Number of seeds forming haustoriums / Number of germinating seeds)] × 100%. The results are as follows: Figure 2 As shown.

[0024] Figure 2 The results showed that the haustorium formation rate of *Cistanche deserticola* in Examples 1-3 was significantly higher than that in Comparative Examples 1-4. Specifically, Comparative Example 1 did not add *Pleurotus ostreatus* fermentation agent; Comparative Example 2 directly added gibberellin and *Pleurotus ostreatus* fermentation agent to distilled water; Comparative Example 3 did not ferment the *Pleurotus ostreatus* powder; and Comparative Example 4 did not ferment the mixed herbal powder. All of these operations reduced the haustorium formation rate of *Cistanche deserticola* seeds, indicating that the method of the present invention can significantly improve the haustorium formation rate of *Cistanche deserticola* seeds and increase seed utilization.

[0025] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

Claims

1. A method for improving the germination rate of Cistanche deserticola seeds, characterized in that, Includes the following steps: (1) Preparation of seed culture medium: (11) Dry and pulverize the oyster mushrooms to obtain oyster mushroom powder. Add the oyster mushroom powder to an ethanol solution, heat, filter, and dry to obtain pretreated oyster mushroom powder. (12) Add the pretreated oyster mushroom powder obtained in step (11) to distilled water, sterilize, cool and inoculate with Lactobacillus paracasei for fermentation; (13) After fermentation in step (12), sterilize, sonicate at 300-400W for 10 min, bathe in water at 90℃ for 30 min, cool and centrifuge, take the supernatant, add ethanol, let stand, centrifuge, wash the precipitate, reconstitute with distilled water, freeze dry under vacuum to obtain oyster mushroom fermentation agent. (14) After washing and drying the licorice, purslane and poplar flowers, pulverize them and mix them in a mass ratio of 3:2:1 to obtain mixed Chinese medicine powder. Add the mixed Chinese medicine powder to deionized water, mix well, sterilize, cool, and inoculate with Lactobacillus plantarum. (15) After inoculation in step (14), ferment, centrifuge, filter the supernatant, add gibberellin at a ratio of 100-150 mg / L, add the oyster mushroom fermentation agent obtained in step (13) at a ratio of 15-20 mg / mL, mix well, and obtain seed culture solution. (2) Disinfection of Cistanche deserticola seeds; (3) Seed pretreatment: The Cistanche deserticola seeds disinfected in step (2) were soaked in distilled water for 4 hours in the dark at 30°C, then added to the seed culture solution and soaked in the dark at 25°C for 20-24 hours. The seeds were rinsed three times with sterile water and then air-dried. (4) Seed culture: The pretreated Cistanche deserticola seeds from step (3) were inoculated into a petri dish and cultured with seed culture solution; In step (12), the Lactobacillus paracasei was purchased from Guangdong Provincial Microbial Culture Collection Center, with the number GDMCCNO.1.159; In step (14), the Lactobacillus plantarum was purchased from Guangdong Provincial Microbial Culture Collection Center, with the number GDMCCNO.1.1516.

2. The method for improving the germination rate of Cistanche deserticola seeds according to claim 1, characterized in that, In step (11), the ratio of oyster mushroom powder to ethanol solution is 1 g: 15-20 mL.

3. The method for improving the germination rate of Cistanche deserticola seeds according to claim 2, characterized in that, In step (12), the ratio of oyster mushroom powder to distilled water is 1 g: 10-15 mL.

4. The method for improving the germination rate of Cistanche deserticola seeds according to claim 3, characterized in that, In step (14), the ratio of the mixed Chinese medicine powder to deionized water is 1 g: 15-20 mL.

Images