Method for promoting efficient germination of agriophyllum squarrosum seeds
By using mechanical grinding and gradient ethanol disinfection, the problems of slow and long germination cycles of *Potentilla anserina* seeds have been solved, achieving efficient and sterile seed germination, improving germination rate and seedling uniformity, and simplifying the operation process.
Patent Information
- Application Number
- CN202510529713.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-25
- Publication Date
- 2025-11-14
AI Technical Summary
The seeds of *Potentilla anserina* exhibit delayed germination, a long germination cycle, low germination rate, and poor seedling uniformity, problems that are difficult to effectively address with existing technologies.
Seeds were mechanically polished and then disinfected using a gradient ethanol method, which included initial disinfection with 75% ethanol followed by gradually increasing the ethanol concentration to 100%, along with rinsing with sterile water and specific culture conditions to promote seed germination.
It significantly improved the aseptic germination rate of *Potentilla anserina* seeds to over 95%, shortened the germination initiation time to within 24 hours, reduced the germination cycle to within 5 days, improved seedling uniformity, and simplified the processing procedure.
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Figure CN120937568A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant tissue culture and genetic transformation technology, and more specifically, to a method for promoting efficient germination of *Potentilla anserina* seeds. Background Technology
[0002] *Agriophyllum squarrosum*, commonly known as sand rice, is an annual herbaceous plant belonging to the Amaranthaceae family in the Caryophyllales order. It is widely distributed on shifting and semi-shifting sand dunes in my country, has a long history of use as food, and exhibits strong adaptability to adverse conditions, making it a promising crop. Its seeds are rich in protein, essential amino acids, and unsaturated fatty acids, possessing significant nutritional value and medicinal development potential. The establishment of *Agriophyllum squarrosum* populations creates an adaptive environment for the migration and colonization of other plants, and is a crucial driving factor in the early vegetation succession of shifting sand dunes. However, a genetic transformation system for *Agriophyllum squarrosum* has not yet been established, severely restricting research into the functional analysis of its stress-resistance genes, CRISPR / Cas9 gene editing, and molecular design breeding—requiring the application of modern biotechnology. Tissue culture of sterile seedlings is a prerequisite for constructing a genetic transformation system, and seed sterilization and homogeneous germination, as the primary steps in aseptic treatment, are even more important.
[0003] In aseptic culture research of plant seeds, commonly used disinfectants include ethanol, sodium hypochlorite, mercuric chloride, hydrogen peroxide, and tissue culture antibacterial agents such as PPM. However, due to differences in morphology, seed coat permeability, and endophytic flora composition, different plant seeds exhibit significant differences in their sensitivity to disinfectants and the effectiveness of treatment. *Potentilla anserina* seeds have a thick seed coat, forming a natural barrier to disinfectant penetration. Simultaneously, these seeds exhibit a unimodal continuous germination pattern and varying degrees of dormancy. Under natural conditions, their germination process is constrained by multiple environmental factors, including sand burial depth, diurnal temperature variation, and microbial infection, resulting in slow germination rates (starting 5-6 days after sowing), long germination cycles (lasting 10-15 days), and poor uniformity of germination. Current techniques using 10% sodium hypochlorite immersion followed by 24-hour immersion in a 2g / L GA3 (gibberellin) solution under dark conditions can partially overcome seed dormancy barriers, increasing the germination rate to 84%, but technical bottlenecks such as delayed germination initiation and long germination cycles still exist.
[0004] In view of this, the present invention is proposed. Summary of the Invention
[0005] The purpose of this invention is to provide a method for promoting the efficient germination of *Polygonum multiflorum* seeds to solve the problems of delayed germination initiation, long germination cycle, low germination rate, and poor seedling uniformity.
[0006] This invention is implemented as follows:
[0007] In a first aspect, the present invention provides a method for promoting efficient germination of *Potentilla chinensis* seeds, comprising the following steps:
[0008] (1) Mix the seeds to be germinated with the polishing medium so that the polishing medium polishes the seeds;
[0009] (2) Disinfect the polished seeds with ethanol.
[0010] The present invention has the following beneficial effects:
[0011] This invention reveals that mechanical abrasion of *Potentilla anserina* seeds can effectively reduce the seed coat's resistance to water absorption and gas exchange by physically abrading the seed coat's cuticle, thereby mitigating the physical dormancy effect induced by the seed coat. The microcracks formed after seed abrasion can synergistically enhance the penetration efficiency of subsequent disinfectants. The germination method provided by this invention not only overcomes the inhibition of seed germination by physical dormancy of the seed coat but also inhibits microbial infection, exhibiting a synergistic inhibitory effect. This results in a sterile germination rate exceeding 95%, representing a 13.2 percentage point increase in seed germination rate compared to existing sodium hypochlorite disinfection combined with gibberellin treatment.
[0012] This invention uses non-toxic ethanol to replace traditional mercuric chloride disinfectant, which can eliminate the interference of heavy metal residues on subsequent genetic transformation and control the pollution rate to within 2.43%.
[0013] This invention uses mechanical polishing to shorten the germination initiation time to within 24 hours and reduce the germination cycle to within 5 days, effectively solving the bottleneck problem of poor uniformity of sterile seedling materials.
[0014] The method provided by this invention reduces the entire processing time to 3-5 minutes, has a high degree of standardization, and provides a stable and reliable source of sterile seedlings for the construction of the *Polygonum cuspidatum* genetic transformation system.
[0015] This method also fills the gap in the aseptic germination technology system for desert plants, and provides key technical support for gene function analysis and molecular breeding of desert plants. Attached Figure Description
[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.
[0017] Figure 1 A graph showing the statistical results of the trend of bacterial contamination rate in different treatment groups with seed germination culture time;
[0018] Figure 2 A graph showing the statistical results of the change in seed germination rate of different treatment groups with seed germination culture time;
[0019] Figure 3 A schematic diagram showing the second day of germination of *Polygonum multiflorum* seeds treated with different disinfection methods on a culture medium. Detailed Implementation
[0020] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.
[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.
[0022] In a first aspect, the present invention provides a method for promoting efficient germination of *Potentilla chinensis* seeds, comprising the following steps:
[0023] (1) Mix the seeds to be germinated with the polishing medium so that the polishing medium polishes the seeds;
[0024] (2) Disinfect the polished seeds with ethanol.
[0025] This invention reveals that mechanical abrasion of *Potentilla anserina* seeds can effectively reduce the seed coat's resistance to water absorption and gas exchange by physically abrading the seed coat's cuticle, thereby mitigating the physical dormancy effect induced by the seed coat. The microcracks formed after seed abrasion can synergistically enhance the penetration efficiency of subsequent disinfectants. The germination method provided by this invention not only overcomes the inhibition of seed germination by physical dormancy of the seed coat but also inhibits microbial infection, exhibiting a synergistic inhibitory effect. This results in a sterile germination rate exceeding 95%, representing a 13.2 percentage point increase in seed germination rate compared to existing sodium hypochlorite disinfection combined with gibberellin treatment.
[0026] The method provided by this invention can eliminate the interference of heavy metal residues on subsequent genetic transformation, shorten the seed germination cycle, improve the uniformity of sterile seedlings, simplify the seed process, shorten the processing time, and provide a stable and reliable source of sterile seedlings for the construction of the *Potentilla anserina* genetic transformation system.
[0027] In a preferred embodiment of the present invention, disinfection is performed using a gradient ethanol system.
[0028] In a preferred embodiment of the present invention, disinfection using a gradient ethanol system refers to: first disinfecting the polished seeds with 75% ethanol, and then contacting the seeds with 80%-100% ethanol. In an optional manner, after disinfection with 75% ethanol, the seeds are then contacted with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% ethanol.
[0029] 75% ethanol can inactivate microorganisms on the seed surface, while 80%-100% ethanol can quickly rinse, enhance penetration, and remove residual impurities.
[0030] In one implementation, the polished seeds are first disinfected with 75% ethanol, and then the seeds are brought into contact with 95%-100% ethanol. Experiments show that this gradient treatment increases the disinfection success rate to over 98% and reduces the contamination rate by 40% compared to traditional sodium hypochlorite treatment.
[0031] In one implementation, the polished seeds are first disinfected with 75% ethanol for 30-50 seconds, and then contacted with 95%-100% ethanol for 5-10 seconds.
[0032] In a preferred embodiment of the present invention, the volume ratio of the seeds to be germinated to the abrasive medium is 1:1 to 1:1.2; or the mass ratio is 1.8:1 to 2.0:1. For example, the volume ratio of the seeds to be germinated to the abrasive medium is 1:1, 1:1.1, or 1:1.2. For example, the mass ratio is 1.8:1, 1.9:1, or 2.0:1.
[0033] In a preferred embodiment of the present invention, the abrasive medium refers to hard particles or materials that can remove the abrasive cuticle layer of the seed coat of the object to be abraded (seed) by means of friction or impact.
[0034] Abrasive media include, but are not limited to, natural abrasives such as quartz sand, diamond, and corundum (e.g., garnet). Synthetic abrasives include silicon carbide, alumina (synthetic corundum), and cubic boron nitride (CBN). Abrasive media are selected from quartz sand or micro / nano particles. Micro / nano particles refer to particles with an average particle size of micrometers or nanometers.
[0035] In one embodiment, the average particle size of the quartz sand is 1-2 mm.
[0036] In a preferred embodiment of the present invention, the polishing time is 30-65 seconds, and the frequency of the polisher is set to 30-40 Hz. For example, the frequency of the polisher is 30 Hz, 31 Hz, 32 Hz, 33 Hz, 34 Hz, 35 Hz, 38 Hz, or 40 Hz.
[0037] In a preferred embodiment of the present invention, the polished seeds are rinsed with sterile water and then disinfected with ethanol.
[0038] In a preferred embodiment of the present invention, the method further includes: removing the ethanol from the ethanol-sterilized seeds, and then placing the ethanol-removed seeds in a culture medium for cultivation. Ethanol removal includes, but is not limited to, methods such as air drying, oven drying, and blow-drying.
[0039] In a preferred embodiment of the present invention, the culture is conducted under dark conditions at a temperature cycle of 20-22℃ (8-9h) / 30-32℃ (15-16h). Under these culture conditions, precise synchronization of the germination process can be achieved in conjunction with mechanical polishing.
[0040] In a preferred embodiment of the present invention, the culture medium is MS solid medium, such as 1 / 2 MS solid medium.
[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.
[0042] The features and performance of the present invention will be further described in detail below with reference to embodiments.
[0043] Example 1
[0044] This embodiment provides a method for promoting efficient germination of *Potentilla anserina* seeds.
[0045] Mature seeds of *Potentilla anserina* collected from Wuwei, Gansu Province, were selected, and healthy seeds of uniform size were chosen for later use. The seeds were mixed with quartz sand of approximately 1-2 mm in diameter at a volume ratio of 1:1. The mixture was placed in a grinder and ground at 30 Hz for 60 ± 5 seconds at room temperature. The mixture was then separated through a 24-mesh stainless steel filter and rinsed three times with distilled water sterilized at 121℃ for 20 minutes.
[0046] On a clean bench, place the seeds into 2ml centrifuge tubes, filling the tubes no more than 1 / 3 full. Add 75% ethanol (v / v) for 30-50 seconds to sterilize, then invert to mix. Discard the 75% ethanol and rinse quickly with 95% ethanol (v / v) for 5 seconds. Discard the 95% ethanol and spread the seeds evenly on 90mm diameter sterile filter paper to air dry for 30 minutes. Prepare 1 / 2 MS solid medium (pH 5.8±0.1), aliquot into 100mm×100mm petri dishes, and sow 36 seeds per dish. Set up 8 biological replicates (n=8) and incubate in a climate chamber at 20℃ (8h) / 30℃ (16h) in the dark.
[0047] Starting from day 5 of cultivation, record the germination status daily from 09:00 to 10:00. Germination is defined as the radicle breaking through the seed coat. The experiment is terminated on day 11, and the following calculations are made: Contamination rate = (Number of contaminated seeds / Total number of seeds) × 100%, Germination rate = (Number of germinated seeds / Total number of seeds) × 100%. Seeds are considered contaminated if they have mycelial patches around them or white mycelium on their surface.
[0048] Comparative Example 1
[0049] Collect mature seeds of *Potentilla anserina* from Wuwei, Gansu Province, and select healthy seeds of uniform size for later use. Place the seeds in a gauze bag and rinse them continuously under running tap water for five minutes. Spread them out on filter paper and dry at room temperature for 30 minutes.
[0050] On a clean bench, place the seeds into 2ml centrifuge tubes, filling the tubes no more than 1 / 3 full. Add 75% ethanol (v / v) for 30-50 seconds to sterilize, then invert to mix. Discard the 75% ethanol and rinse quickly with 95% ethanol (v / v) for 5 seconds. Discard the 95% ethanol and spread the mixture evenly on 90mm diameter sterile filter paper to air dry for 30 minutes.
[0051] Prepare 1 / 2 MS solid medium (pH 5.8±0.1), dispense into 100mm×100mm petri dishes, and sow 36 seeds per dish. Set up 8 biological replicates (n=8), place in an artificial climate chamber, and incubate under dark conditions with temperature cycles of 20℃ (8h) / 30℃ (16h). From day 5, record the germination status daily from 09:00 to 10:00; germination is defined as the radicle breaking through the seed coat. The experiment is terminated on day 11, and the following calculations are made: contamination rate = (number of contaminated seeds / total number of seeds) × 100%, germination rate = (number of germinated seeds / total number of seeds) × 100%.
[0052] Comparative Example 2
[0053] Collect mature seeds of *Potentilla anserina* from Wuwei, Gansu Province, and select healthy seeds of uniform size for later use. Place the seeds in a gauze bag and rinse them continuously under running tap water for five minutes. Spread them out on filter paper and dry at room temperature for 30 minutes.
[0054] On a clean bench, place the seeds into 2ml centrifuge tubes, filling the tubes no more than 1 / 3 full. Add 1% NaClO (by volume) for 5 minutes to sterilize, inverting to mix. Discard the NaClO, then add 75% ethanol for 30-50 seconds, inverting to mix. Discard the ethanol, then rinse three times with sterile distilled water containing 0.1% (w / v) carbendazim, inverting to mix for 5 minutes each time. After the final rinse, spread the seeds evenly on 90mm diameter sterile filter paper and air dry for 1 hour.
[0055] Prepare 1 / 2 MS solid medium (pH 5.8±0.1), dispense into 100mm×100mm petri dishes, and sow 36 seeds per dish. Set up 8 biological replicates (n=8), place in an artificial climate chamber, and incubate under dark conditions with temperature cycles of 20℃ (8h) / 30℃ (16h). From day 5, record the germination status daily from 09:00 to 10:00; germination is defined as the radicle breaking through the seed coat. The experiment is terminated on day 11, and the following calculations are made: contamination rate = (number of contaminated seeds / total number of seeds) × 100%, germination rate = (number of germinated seeds / total number of seeds) × 100%.
[0056] Comparative Example 3
[0057] Mature seeds of *Potentilla anserina* collected from Wuwei, Gansu Province, were selected, and healthy seeds of uniform size were chosen for later use. The seeds were placed in a gauze bag and rinsed continuously under running tap water for five minutes. They were then spread flat on filter paper and dried at room temperature for 30 minutes. On a clean bench, the seeds were placed into 2ml centrifuge tubes, filling the tubes to no more than 1 / 3 full. A 0.1% HgCl2 solution was added for 5 minutes to sterilize the solution, and the tubes were inverted to mix thoroughly. The HgCl2 solution was discarded, and the tubes were then rinsed three times with sterile distilled water containing 0.1% (w / v) carbendazim, each time for 5 minutes, inverting to mix thoroughly. After the final rinse, the tubes were spread flat on 90mm diameter sterile filter paper and air-dried for 1 hour.
[0058] Prepare 1 / 2 MS solid medium (pH 5.8±0.1), dispense into 100mm×100mm petri dishes, and sow 36 seeds per dish. Set up 8 biological replicates (n=8), place in an artificial climate chamber, and incubate under dark conditions with temperature cycles of 20℃ (8h) / 30℃ (16h). From day 5, record the germination status daily from 09:00 to 10:00; germination is defined as the radicle breaking through the seed coat. The experiment is terminated on day 11, and the following calculations are made: contamination rate = (number of contaminated seeds / total number of seeds) × 100%, germination rate = (number of germinated seeds / total number of seeds) × 100%.
[0059] Comparative Example 4
[0060] Mature seeds of *Potentilla anserina* collected from Wuwei, Gansu Province, were selected, and healthy seeds of uniform size were chosen for later use. The seeds were placed in a gauze bag and rinsed continuously under running tap water for five minutes. They were then spread flat on filter paper and dried at room temperature for 30 minutes. On a clean bench, the seeds were placed into 2ml centrifuge tubes, filling the tubes to no more than one-third full. The tubes were then soaked in sterile distilled water containing 0.1% (w / v) carbendazim for 4 hours, and then spread flat on 90mm diameter sterile filter paper to air dry for 1 hour.
[0061] Prepare 1 / 2 MS solid medium (pH 5.8±0.1), dispense into 100mm×100mm petri dishes, and sow 36 seeds per dish. Set up 8 biological replicates (n=8), place in an artificial climate chamber, and incubate under dark conditions with temperature cycles of 20℃ (8h) / 30℃ (16h). From day 5, record the germination status daily from 09:00 to 10:00; germination is defined as the radicle breaking through the seed coat. The experiment is terminated on day 11, and the following calculations are made: contamination rate = (number of contaminated seeds / total number of seeds) × 100%, germination rate = (number of germinated seeds / total number of seeds) × 100%.
[0062] Comparative Example 5
[0063] Mature seeds of *Potentilla anserina* collected from Wuwei, Gansu Province, were selected, and healthy seeds of uniform size were chosen for later use. The seeds were placed in a gauze bag and rinsed continuously under running tap water for five minutes. They were then spread flat on filter paper and dried at room temperature for 30 minutes. On a clean bench, the seeds were placed into 2ml centrifuge tubes, filling the tubes to no more than 1 / 3 full. A 3% (v / v) H2O2 aqueous solution was added, and the tubes were allowed to soak for 4 hours. Afterward, they were spread flat on 90mm diameter sterile filter paper and air-dried for 1 hour.
[0064] Prepare 1 / 2 MS solid medium (pH 5.8±0.1), dispense into 100mm×100mm petri dishes, and sow 36 seeds per dish. Set up 8 biological replicates (n=8), place in an artificial climate chamber, and incubate under dark conditions with temperature cycles of 20℃ (8h) / 30℃ (16h). From day 5, record the germination status daily from 09:00 to 10:00; germination is defined as the radicle breaking through the seed coat. The experiment is terminated on day 11, and the following calculations are made: contamination rate = (number of contaminated seeds / total number of seeds) × 100%, germination rate = (number of germinated seeds / total number of seeds) × 100%.
[0065] Comparative Example 6
[0066] Collect mature seeds of *Potentilla anserina* from Wuwei, Gansu Province, and select healthy seeds of uniform size for later use. Place the seeds in a gauze bag and rinse them continuously under running tap water for five minutes. Spread them out on filter paper and dry at room temperature for 30 minutes.
[0067] On a clean bench, seeds were placed into 2ml centrifuge tubes, filling the tubes to no more than 1 / 3 full. 1 / 2MS culture medium containing 5% PPM was added, and the tubes were allowed to soak for 4 hours. Then, the seeds were spread evenly on 90mm diameter sterile filter paper and air-dried for 1 hour. 1 / 2MS solid culture medium (pH 5.8±0.1) containing 0.1% PPM was prepared and dispensed into 100mm×100mm petri dishes, with 36 seeds sown in each dish. Eight biological replicates (n=8) were set up and placed in an artificial climate chamber with a temperature cycle of 20℃ (8h) / 30℃ (16h) under dark conditions. From day 5, germination status was recorded daily from 09:00 to 10:00; germination was defined as the radicle breaking through the seed coat. The experiment was terminated on day 11, and the following calculations were made: contamination rate = (number of contaminated seeds / total number of seeds) × 100%, germination rate = (number of germinated seeds / total number of seeds) × 100%.
[0068] PPM is a broad-spectrum antifungal agent that targets fungi in bacterial and plant tissue culture media, as well as contaminated tissues. The active ingredient in PPM can penetrate the cell walls of fungi or bacteria.
[0069] Comparative Example 7
[0070] Mature seeds of *Potentilla anserina* collected from Wuwei, Gansu Province, were selected, and healthy seeds of uniform size were chosen for later use. The seeds were placed in a gauze bag and rinsed continuously under running tap water for five minutes. They were then spread flat on filter paper and dried at room temperature for 30 minutes. On a clean bench, the seeds were placed into 2ml centrifuge tubes, filling no more than 1 / 3 of the tube's volume. A 0.1% Triton X-100 aqueous solution was added for rinsing and soaking for 5 minutes. Two more rinsing sessions were then conducted using sterile distilled water containing 0.1% (w / v) carbendazim, each lasting 5 minutes with inverted mixing. A 5-minute rinsing session was then conducted using 3% NaClO, with inverted mixing. The NaClO was discarded, and the seeds were then rinsed with 75% ethanol for 30-50 seconds with inverted mixing. The ethanol was discarded, and three rinsing sessions were conducted using sterile distilled water containing 0.1% (w / v) carbendazim, each lasting 5 minutes with inverted mixing. After the final rinse, the seeds were spread flat on 90mm diameter sterile filter paper and air-dried for 1 hour.
[0071] Prepare half MS solid medium (pH 5.8±0.1), dispense into 100 mm × 100 mm petri dishes, and sow 36 seeds per dish. Set up 8 biological replicates (n=8), place in an artificial climate chamber, set the temperature cycle to 20℃ (8 h) / 30℃ (16 h), and culture under dark conditions. From day 5 of culture, record the germination status daily from 09:00 to 10:00. Germination is defined as the radicle breaking through the seed coat. The experiment is terminated on day 11, and the following calculations are made: contamination rate = (number of contaminated seeds / total number of seeds) × 100%, germination rate = (number of germinated seeds / total number of seeds) × 100%.
[0072] Experimental Example 1
[0073] Experiment on the effects of different disinfection (germination) methods on seed germination and contamination.
[0074] Figure 1 This is a graph showing the trend of bacterial contamination rate as a function of culture time for Examples 1 and Comparative Examples 1-7. Figure 2 This is a graph showing the trend of seed germination rate as a function of culture time for Example 1 and Comparative Examples 1-7. Figure 3 This is a schematic diagram showing the second day of germination of *Potentilla anserina* seeds treated in Example 1 and Comparative Examples 1-7 on the culture medium. The results show that the method provided by this invention increases the aseptic germination rate to 95.14% (Example 1), an improvement of 13.2 percentage points compared to existing technologies. The use of non-toxic ethanol instead of traditional mercuric chloride disinfectant eliminates the interference of heavy metal residues on subsequent genetic transformation, controlling the contamination rate to within 2.43% (Example 1).
[0075] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A method for promoting efficient germination of *Potentilla anserina* seeds, characterized in that, It includes the following steps: (1) Mix the seeds to be germinated with a polishing medium, so that the polishing medium polishes the seeds; (2) Disinfect the polished seeds with ethanol.
2. The method according to claim 1, characterized in that, The disinfection is performed using a gradient ethanol system.
3. The method according to claim 2, characterized in that, The aforementioned gradient ethanol system for disinfection refers to: first disinfecting the polished seeds with 75% ethanol, and then contacting the seeds with 80%-100% ethanol. Preferably, the polished seeds are first disinfected with 75% ethanol, and then contacted with 95%-100% ethanol. Preferably, the polished seeds are first disinfected with 75% ethanol for 30-50 seconds, and then contacted with 95%-100% ethanol for 5-10 seconds.
4. The method according to claim 1, characterized in that, The volume ratio of the seeds to be germinated to the polishing medium is 1:1 to 1:1.2; or the mass ratio is 1.8:1 to 2.0:
1.
5. The method according to claim 4, characterized in that, The polishing medium is selected from quartz sand or micro / nano particles; Preferably, the average particle size of the quartz sand is 1-2 mm.
6. The method according to claim 5, characterized in that, The polishing time is 30-65 seconds, and the frequency of the polisher is set to 30-40 Hz.
7. The method according to claim 6, characterized in that, After polishing, the seeds are rinsed with sterile water and then disinfected with ethanol.
8. The method according to any one of claims 1-7, characterized in that, The method further includes: removing the ethanol from the ethanol-sterilized seeds, and then placing the ethanol-removed seeds in a culture medium for cultivation.
9. The method according to claim 8, characterized in that, The culture was conducted under dark conditions with a temperature cycle of 20-22℃ (8-9h) / 30-32℃ (15-16h).
10. The method according to claim 9, characterized in that, The culture medium is MS solid culture medium.