Celery leaf nano-silver disinfectant and its application in primary culture of Malus robusta

Abstract

The present application relates to a kind of celery leaf nano silver disinfectant and its application in malus halliana primary culture, belong to woody plant tissue culture technical field, using celery leaf extract and AgNO3 solution are mixed, irradiation is carried out under direct sunlight until the solution produces the golden yellow of nano silver characteristics, the celery leaf nano silver disinfectant is used in explant disinfection in malus halliana primary culture, good effect is obtained, finally effectively kill the pathogenic bacteria on the surface of malus halliana explant, and browning rate is greatly reduced, and the tissue culture of large-scale malus halliana explant is successfully realized.

Description

Technical Field

[0001] This invention belongs to the field of woody plant tissue culture technology, and in particular relates to a celery leaf nano-silver disinfectant and its application in the primary culture of *Cathaya argyrophylla*. Background Technology

[0002] The Chinese catalpa (Catalpa bungei CAMey.) is a widely distributed and excellent native tree species in my country. With its well-developed root system, upright trunk, lush foliage, and strong adaptability, it is an ideal economic and landscaping tree species for soil and water conservation, agroforestry, and urban greening. It plays a vital role in forestry and greening projects and enjoys the reputation of "King of Trees." The golden catalpa (Catalpa bungei var.) is an excellent geographical variety of the Chinese catalpa, with distinct regional characteristics and high recognition. The golden catalpa is highly adaptable and grows vigorously. Its wood is also of excellent quality, possessing advantages such as toughness, water and rot resistance, resistance to insect infestation, beautiful grain, low shrinkage, resistance to warping and cracking, and ease of processing, giving it extremely high ecological and economic value.

[0003] However, the catalpa tree is a cross-pollinated tree, exhibiting self-incompatibility, resulting in low fruit yields and a low seed germination rate. Asexual propagation methods such as cuttings, grafting, and layering are also inefficient, making natural reproduction difficult. This hinders the large-scale and industrialized production of *Cathaya argyrophylla*, leading to a scarcity of germplasm resources. Currently, the natural distribution areas of *Cathaya argyrophylla* possess abundant planting resources and genetic diversity, urgently requiring preservation and promotion. Modern tissue culture technology, employing asexual reproduction, ensures the genetic stability of germplasm resources, preventing the loss or mutation of superior genes due to hybridization or pollination. It effectively preserves excellent plant germplasm resources and allows for rapid, large-scale propagation without natural limitations, making it an excellent technology system for the breeding of high-quality *Cathaya argyrophylla* seedlings.

[0004] Primary culture is the first step in plant tissue culture. It involves treating the plant explants with disinfectants to eliminate microorganisms adhering to their surfaces, followed by in vitro culture in a sterile environment. Currently, commonly used disinfectants for primary plant culture include mercuric chloride, sodium hypochlorite (bleaching powder), ethanol, and peracetic acid. Among these, mercuric chloride has the best disinfection effect and is the most widely used, but it is water-soluble, highly toxic, and poses a significant hazard to personnel and the environment. Furthermore, while existing disinfectants can kill most microorganisms on plant surfaces, factors such as plant species, variety, growth environment, tissue type, and the surface microbial community can all affect the disinfection effect. In actual production, we have found significant differences in the effectiveness of primary disinfection between different varieties of Catalpa macrocarpa and between different genotypes of the same variety. This is particularly pronounced in *Cathaya argyrophylla*, which is prone to oxidation during tissue culture. In the process of preserving superior wild germplasm of *Sorbus acutus*, sterilization often fails during primary culture. This is mainly due to the following contradictions in primary sterilization: using mild disinfectants and small doses often fails to achieve the desired sterilization effect, failing to completely kill various microorganisms and resulting in a high contamination rate; while using strong disinfectants and large doses can damage plants, affecting cell growth and differentiation, making explants prone to browning and ultimately death. Many wild cultivated *Sorbus acutus* resources often suffer total loss during primary culture, making it impossible to obtain sterile seedlings and leading to preservation failure. Therefore, primary culture has become a bottleneck for the rapid propagation of *Sorbus acutus* through tissue culture. To achieve successful tissue culture of superior *Sorbus acutus* germplasm resources, it is necessary to explore and research more suitable disinfectants and treatment methods based on specific germplasm, develop new sterilization strategies, and establish a more complete primary culture technology system.

[0005] Silver nanoparticles (AgNPs) are nanoscale metallic silver particles prepared using nanoparticle technology. They possess advantages such as significant bactericidal effects, a broad antibacterial spectrum, and low likelihood of inducing drug resistance, earning them the reputation of a new generation of antibacterial agents. Silver nanoparticles can achieve excellent bactericidal effects at extremely low concentrations, such as ppm, and exhibit good biocompatibility and low toxicity. They have been widely used in clinical, catering, chemical, aquaculture, and cosmetic fields. Biosynthetic silver nanoparticle technology is a new technique that utilizes active substances from bacteria, fungi, plants, and algae as reducing agents and stabilizers to prepare silver nanoparticles. Biosynthesized silver nanoparticles typically exhibit higher biocompatibility and antibacterial activity, as well as good persistence and stability. However, different biological raw materials can lead to variations in the size, shape, stability, and surface properties of silver nanoparticles, resulting in significant differences in their interaction with target microorganisms and their bactericidal effects. Summary of the Invention

[0006] This invention provides a celery leaf nano-silver disinfectant and its application in the primary culture of *Cathaya argyrophylla*. After extensive screening, celery leaves were successfully selected as the raw material for the photocatalytic biosynthesis of the nano-silver disinfectant, which achieved excellent results in the primary disinfection of *Cathaya argyrophylla* explants. This effectively killed pathogens on the surface of *Cathaya argyrophylla* explants and significantly reduced the browning rate, successfully achieving large-scale tissue culture of *Cathaya argyrophylla* explants.

[0007] This invention is achieved through the following technical solution:

[0008] A celery leaf nano-silver disinfectant, the preparation method of the disinfectant is as follows: celery leaves are washed, dried, and pulverized. Ethanol with a volume ratio of 10-90% is added at a material-to-liquid ratio of 1:10 to 1:100 (g / mL). The mixture is stirred evenly, and then subjected to ultrasonic vibration at 100-500W for 5-10 minutes. After heating and reflux at 60℃ for 10-60 minutes, the mixture is naturally cooled, centrifuged, and the supernatant is collected.

[0009] The above celery leaf extract was mixed with AgNO3 solution to achieve a final AgNO3 concentration of 0.1-10 mM. The celery leaf extract was diluted 2-100 times with double-distilled water as the dilution solvent. The mixture was vortexed and irradiated under direct sunlight (10,000-100,000 lx) until the solution produced a golden yellow color characteristic of nano-silver. The solution was then stored at 4°C in the dark for later use.

[0010] A method for primary culture of *Acer buergerianum* using the aforementioned celery leaf nano-silver disinfectant, the method comprising the following steps:

[0011] (1) Collection of explants: When the dormant buds of the golden catalpa tree grow to 1-2 cm, collect branches containing terminal and axillary buds before the bracts open. Use pruning shears to cut the branches into 15-20 cm stem segments as explants, bring them back indoors for hydroponics, change the water once every 3 days, and when the dormant buds begin to sprout, prune the branches into 2-4 cm segments with axillary or terminal buds for primary culture.

[0012] (2) Cleaning of explants: Soak the stem segments in dish soap and rinse them clean;

[0013] (3) Stem segment disinfection: Transfer the cleaned explants to a sterile workbench and disinfect with 70% alcohol for 30 seconds, then rinse once with sterile water; add celery leaf biosynthetic nano-silver disinfectant diluted 100 to 10000 times, immerse the explants, shake well, disinfect for 8 to 10 minutes, rinse with sterile water, absorb the surface moisture, and inoculate the stem segments into the primary culture medium; culture temperature 25±2℃, light intensity 1500 to 3000 lx, and light duration 16 h / d.

[0014] As one of the preferred embodiments, the stem segments are soaked in 0.05% dish soap for 10 minutes.

[0015] As one of the preferred embodiments, the primary culture medium is: MS basal medium + 6-BA 2.0 mg / L + NAA 0.05 mg / L + KT 0.1 mg / L + sucrose 30 g / L + agar 7.0 g / L, pH 5.8.

[0016] As one of the preferred embodiments, the celery leaf nano-silver disinfectant is used after being diluted 100-10000 times.

[0017] The beneficial effects of this invention compared to the prior art are as follows:

[0018] 1) The raw material used in this invention is vegetable processing waste, turning waste into treasure at a low cost. The synthesis method is simple and easy to implement, and the reaction does not require special equipment. It can be completed by short-term light exposure, resulting in low energy consumption and easy promotion.

[0019] 2) The disinfectant of this invention is used in extremely low amounts, leaves little residue, and has good biocompatibility, and will not cause significant toxicity to the explants of *Cathaya argyrophylla*.

[0020] 3) The disinfectant of this invention has strong antibacterial properties and a broad antibacterial spectrum, and is suitable for the disinfection of a variety of primary surface microorganisms, with a high survival rate of explant germination.

[0021] 4) The nano-silver disinfectant prepared by this invention is suitable for disinfecting explants of *Sorbus acutus*. It can completely kill pathogens and avoid browning, thus improving the survival rate of primary culture. It has great practical significance for the rapid propagation and large-scale production of superior *Sorbus acutus* strains and provides technical support for the large-scale and rapid production of *Sorbus acutus*. Attached Figure Description

[0022] Figure 1 The ultraviolet-visible absorption spectrum of the celery biosynthesized silver nanoparticles prepared by the method of this invention.

[0023] Figure 2 Transmission electron microscopy image of the celery biosynthesized silver nanoparticles prepared in this invention.

[0024] Figure 3 Explants of *Cathaya argyrophylla* that successfully germinated after treatment with the celery biosynthesized nano-silver disinfectant of this invention. Detailed Implementation

[0025] The technical solution of the present invention will be further explained below with reference to the embodiments and accompanying drawings, but the scope of protection of the present invention is not limited in any way by the embodiments.

[0026] Example 1: Preparation of celery leaf biosynthesized nano-silver disinfectant:

[0027] Preparation of celery leaf extract: Celery leaves were washed with running water, dried in a drying oven at 60℃ for 24 hours, pulverized, and then mixed with 40% ethanol (by volume) at a material-to-liquid ratio of 1:10 (g / mL). The mixture was stirred thoroughly. After ultrasonic agitation at 100W for 10 minutes, the mixture was refluxed at 60℃ for 30 minutes. After natural cooling, the mixture was centrifuged at 12000rpm for 10 minutes, and the supernatant was collected and stored at 4℃ for later use.

[0028] Biosynthesis of nano-silver disinfectant: The above celery leaf extract was mixed with AgNO3 solution to make the final concentration of AgNO3 1mM. The dilution factor of celery leaf extract was 20 times, and the dilution solvent was double-distilled water. The mixture was vortexed and irradiated under direct sunlight (10000-100000lx) for 10 min. The solution produced the characteristic golden yellow color of nano-silver. It was stored at 4℃ in the dark for later use.

[0029] The celery leaf biosynthesized nano-silver disinfectant exhibits a distinct characteristic absorption peak of nano-silver at 450 nm, and the peak shape is symmetrical, indicating that the nano-silver particle size distribution is uniform. Figure 1 From the transmission electron microscope image ( Figure 2 As can be seen from the above, the nano-silver particles prepared by this invention have uniform size, are spherical or ellipsoidal, with a particle size range of 4–40 nm. The small particle size provides excellent penetrability, which is beneficial for achieving a bactericidal effect. Figure 2 It can also be seen that biomaterials are attached to the surface of the silver nanoparticles, which can improve the stability and biocompatibility of the silver nanoparticles. The method for biosynthesizing silver nanoparticles disclosed in this invention does not rely on special equipment or energy consumption, and can achieve synthesis within 10 minutes, which is beneficial to environmental protection and energy conservation. Furthermore, shortening the synthesis time can reduce the aggregation of silver nanoparticles, thereby obtaining smaller and more uniform silver nanoparticles and improving product quality. The above physicochemical characterization also shows that the prepared product has achieved this objective.

[0030] Example 2: Preparation of biosynthesized nano-silver disinfectant from Ligustrum lucidum fruit

[0031] Preparation of Ligustrum lucidum fruit extract: Mature Ligustrum lucidum fruits were collected, washed with running water, dried in a drying oven at 60℃ for 24 hours, pulverized, and then mixed with ultrapure water at a ratio of 1:10 (g / mL). The mixture was stirred evenly. After ultrasonic vibration at 100W for 10 min, it was refluxed at 90℃ for 30 min. After natural cooling, it was centrifuged at 12000 rpm for 10 min, and the supernatant was collected and stored at 4℃ for later use.

[0032] Biosynthesis of nano-silver disinfectant: The above-mentioned privet fruit extract was mixed with AgNO3 solution to make the final concentration of AgNO3 1mM. The privet fruit extract was diluted 20 times with double-distilled water as the dilution solvent. The mixture was vortexed and irradiated under direct sunlight (10000-100000lx) for 10 min. The solution produced the characteristic golden yellow color of nano-silver. It was then stored at 4℃ in the dark for later use.

[0033] Example 3: Primary culture methods of *Cathaya argyrophylla*, *Cathaya yunnanensis*, and *Cathaya argyrophylla*.

[0034] Explant collection: Wild *Cathaya argyrophylla* species from the Weizishan Provincial Nature Reserve in Yantai City, Shandong Province, were used as experimental materials, while locally introduced *Cathaya argyrophylla* and *Cathaya yunnanensis* species were used as control materials for primary culture. When the dormant buds of *Cathaya argyrophylla*, *Cathaya argyrophylla*, and *Cathaya argyrophylla* reached 1–2 cm in length, branches containing terminal and axillary buds were collected before the bracts opened. The branches were then cut into 15–20 cm stem segments using pruning shears to serve as explants. These segments were brought back indoors for hydroponic culture, with the water changed every 3 days. When the dormant buds began to sprout, the branches were pruned into 2–4 cm segments with axillary or terminal buds for primary culture.

[0035] Cleaning of explants: Soak the stem segments in 0.05% detergent for 10 minutes, then rinse them with running tap water and prepare for disinfection.

[0036] Stem segment disinfection: The explants were transferred to a sterile workbench. The pruned catalpa stem segments were disinfected in 70% alcohol for 30 seconds and rinsed once with sterile water. The explants were then submerged in a solution of celery leaf biosynthetic nano-silver prepared in Example 1 (diluted 1000 times), privet biosynthetic nano-silver disinfectant prepared in Example 2, and 0.1% HgCl2. The solution was shaken thoroughly and disinfected for 8 minutes. To further compare the disinfection effect of different HgCl2 solution soaking times on *Cathaya argyrophylla*, treatments of soaking in 0.1% HgCl2 solution for 6 minutes and 10 minutes were also set up. After disinfection, the stem segments were rinsed 5 times with sterile water, and finally, the surface moisture was absorbed with sterile absorbent paper. The stem segments were then inoculated into the primary culture medium. The culture temperature was 25±2℃, the light intensity was 1500~3000lx, and the photoperiod was 16h / d.

[0037] The primary culture medium was: MS basal medium + 6-BA 2.0 mg / L + NAA 0.05 mg / L + KT 0.1 mg / L + sucrose 30 g / L + agar 7.0 g / L, pH 5.8.

[0038] One month later, calculate the browning rate, contamination rate, and germination rate using the following formula:

[0039] Browning rate = (Total number of browned explants / Total number of inoculated explants) × 100%

[0040] Contamination rate = (Total number of contaminated explants / Total number of inoculated explants) × 100%

[0041] Germination rate = (Total number of germinating explants / Total number of inoculated explants) × 100%

[0042] The germination standard is that the terminal or axillary buds of the golden catalpa tree gradually sprout, swell, and gradually unfold leaves under the induction of the primary culture medium.

[0043] Table 1 shows the effects of different disinfection treatments on Catalpa explants. As can be seen from Table 1, the celery leaf biosynthetic nano-silver disinfectant prepared in Example 1, when used to disinfect three different Catalpa varieties (Yu Catalpa, Dian Catalpa, and Golden Silk Catalpa), exhibits low toxicity and minimal residue from the biosynthetic nano-silver disinfectant. Therefore, it is beneficial for improving the germination survival rate and reducing the browning rate of the three Catalpa explants. Figure 3The germinating shoots obtained after successful explanting of *Cathaya argyrophylla* were robust, vigorous, and showed no browning. Therefore, the celery leaf biosynthesized nano-silver disinfectant has good bactericidal effects against common bacteria, stubborn bacteria, drug-resistant bacteria, and deep tissue infections caused by fungi, greatly improving the browning of explants, and also has advantages such as being environmentally friendly, safe, and long-lasting. In Example 2, biosynthesized nano-silver from *Ligustrum lucidum* fruit was used to treat *Cathaya argyrophylla*, *Cathaya yunnanensis*, and *Cathaya argyrophylla* in primary culture. Statistical results showed that although it had a good antibacterial effect, it could not reduce the browning rate of *Cathaya argyrophylla*, resulting in a low final germination rate. Using different biological materials to prepare nano-silver disinfectants results in differences in the size, shape, surface charge, and surface attachments of the synthesized nano-silver particles due to the different active substances and properties contained in the biological materials. This directly affects their interaction with plants and microorganisms, producing different disinfection effects. Furthermore, biosynthesized nano-silver contains different bioactive molecules, which may synergistically affect microorganisms, increasing or decreasing the disinfection effect. Using conventional methods in existing technologies, disinfection with 0.1% HgCl2 for 6, 8, and 10 minutes resulted in significantly higher contamination and browning rates for *Cathaya argyrophylla* compared to the use of biosynthesized nano-silver disinfectants. While extending the HgCl2 disinfection time reduced the contamination rate, it also significantly increased the browning rate, ultimately preventing successful primary culture of *Cathaya argyrophylla*. Comparisons between different experimental groups in the examples also revealed significant differences in the ease of primary culture of different *Cathaya argyrophylla* varieties. Different varieties or genotypes of *Cathaya argyrophylla* may exhibit varying sensitivities to nano-silver disinfectants. Differences in physiological structure, epidermal characteristics, or natural antibacterial abilities among different *Cathaya argyrophylla* varieties may lead to varying responses to nano-silver disinfectants. Furthermore, differences in genotype among different plant varieties result in variations in polyphenol oxidase activity, antioxidant capacity, and cell differentiation ability, ultimately leading to different browning rates. Among the three catalpa varieties, *Cathaya argyrophylla* is more sensitive to disinfection and prone to browning. Traditional disinfectants and other biosynthetic nano-silver methods make it difficult to obtain sterile seedlings. However, the method described in this invention balances the antibacterial properties and browning prevention of *Cathaya argyrophylla*, ultimately achieving a higher germination rate. Therefore, the technical solution of this invention is suitable for the primary culture of *Cathaya argyrophylla*, significantly reducing both contamination and browning rates while greatly improving germination and survival rates. This plays a decisive role in overcoming the bottleneck in the primary culture of *Cathaya argyrophylla*.

[0044] Table 1. Effects of different disinfection treatments on Catalpa tree explants

[0045]

[0046] Example 4

[0047] A primary culture method suitable for *Acer buergerianum*, comprising the following steps:

[0048] (1) Preparation of celery leaf extract: Celery leaves were washed with running water, dried, and pulverized. Ethanol with a volume ratio of 10% was added at a material-to-liquid ratio of 1:10 (g / mL). The mixture was stirred evenly and then subjected to ultrasonic vibration at 100W for 5 min. After reflux at 60℃ for 10 min, the mixture was allowed to cool naturally and then centrifuged to collect the supernatant.

[0049] Biosynthesis of nano-silver disinfectant: The above celery leaf extract was mixed with AgNO3 solution to make the final concentration of AgNO3 10mM. The dilution factor of celery leaf extract was 2 times, and the dilution solvent was double-distilled water. The mixture was vortexed and irradiated under direct sunlight (10000-100000lx) for 10 min. The solution produced the characteristic golden yellow color of nano-silver. It was stored at 4℃ in the dark for later use.

[0050] (2) Explant disinfection treatment, including the following steps:

[0051] Explant collection: Collect explants of Catalpa macrophylla. When the dormant buds of Catalpa macrophylla grow to 1-2 cm, collect branches containing terminal and axillary buds before the bracts open. Use pruning shears to cut the branches into 15-20 cm stem segments for explants. Bring them back indoors for hydroponic culture, changing the water every 3 days. When the dormant buds begin to sprout, prune the branches into 2-4 cm segments with axillary or terminal buds for primary culture.

[0052] Cleaning of explants: Soak the stem segments in 0.05% detergent for 10 minutes, then rinse them with running tap water and prepare for disinfection.

[0053] Stem segment disinfection: Transfer the explants to a sterile workbench. Disinfect the pruned catalpa stem segments with 70% alcohol for 30 seconds, then rinse once with sterile water. Add the prepared nano-silver disinfectant diluted 1000 times, immerse the explants completely, shake thoroughly, disinfect for 8 minutes, rinse five times with sterile water, and finally absorb surface moisture with sterile absorbent paper. Inoculate the stem segments into the primary culture medium. The culture temperature is 25±2℃, the light intensity is 1500~3000lx, and the photoperiod is 16h / d.

[0054] The primary culture medium was: MS basal medium + 6-BA 2.0 mg / L + NAA 0.05 mg / L + KT 0.1 mg / L + sucrose 30 g / L + agar 7.0 g / L, pH 5.8.

[0055] One month later, the browning rate, contamination rate, and germination rate were calculated to be 16.7%, 0%, and 82.3%, respectively.

[0056] Example 5

[0057] A primary culture method suitable for *Acer buergerianum*, comprising the following steps:

[0058] (1) Preparation of celery leaf extract: Celery leaves were washed with running water, dried in a drying oven at 60℃ for 24 hours, pulverized, and then mixed with 90% ethanol at a ratio of 1:100 (g / mL). The mixture was stirred evenly, ultrasonically vibrated at 500W for 10 minutes, and then refluxed at 60℃ for 60 minutes. After natural cooling, the mixture was centrifuged at 12000rpm for 10 minutes, and the supernatant was collected and stored at 4℃ for later use.

[0059] Biosynthesis of nano-silver disinfectant: The above celery leaf extract was mixed with AgNO3 solution to make the final concentration of AgNO3 0.1 mM. The dilution factor of the celery leaf extract was 100 times, and the dilution solvent was double-distilled water. The mixture was vortexed and irradiated under direct sunlight (10,000-100,000 lx) for 10 min. The solution produced the characteristic golden yellow color of nano-silver. It was stored at 4℃ in the dark for later use.

[0060] (2) Disinfection treatment of *Acer buergerianum* explants, including the following steps:

[0061] Explant collection: In early April, explants were collected from branches of Catalpa bungei. When the dormant buds of the Catalpa bungei grew to 2cm, branches containing terminal and axillary buds were collected before the bracts opened. The branches were cut into 20cm stem segments using pruning shears to serve as explants. These segments were brought indoors for hydroponic culture, with the water changed every 3 days. When the dormant buds began to sprout, the branches were pruned into 4cm segments with axillary or terminal buds for primary culture.

[0062] Cleaning of explants: Soak the stem segments in 0.05% detergent for 10 minutes, then rinse them with running tap water and prepare for disinfection.

[0063] Stem segment disinfection: Transfer the explants to a sterile workbench. Disinfect the trimmed *Cathaya argyrophylla* stem segments in 70% alcohol for 30 seconds, then rinse once with sterile water. Add a 10,000-fold diluted nano-silver disinfectant to completely submerge the explants, shake thoroughly, and disinfect for 8 minutes. Rinse three times with sterile water. Finally, absorb surface moisture with sterile absorbent paper and inoculate the stem segments into the primary culture medium. Culture temperature: 25±2℃; light intensity: 1500–3000 lx; ​​photoperiod: 16 h / d.

[0064] The primary culture medium was: MS basal medium + 6-BA 2.0 mg / L + NAA 0.05 mg / L + KT 0.1 mg / L + sucrose 30 g / L + agar 7.0 g / L, pH 5.8.

[0065] One month later, the browning rate, contamination rate, and germination rate were calculated to be 10.0%, 10.0%, and 80.0%, respectively.

[0066] Example 6

[0067] Preparation of celery leaf extract: Celery leaves were washed with running water, dried in a drying oven at 60℃ for 24 hours, pulverized, and then mixed with 50% ethanol at a ratio of 1:50 (g / mL). The mixture was stirred evenly, ultrasonically vibrated at 250W for 8 minutes, and then refluxed at 60℃ for 35 minutes. After natural cooling, the mixture was centrifuged at 12000rpm for 10 minutes, and the supernatant was collected and stored at 4℃ for later use.

[0068] Biosynthesis of nano-silver disinfectant: The above celery leaf extract was mixed with AgNO3 solution to make the final concentration of AgNO3 1mM. The dilution factor of the celery leaf extract was 50 times, and the dilution solvent was double-distilled water. The mixture was vortexed and irradiated under direct sunlight (10000-100000lx) for 10 min. The solution produced the characteristic golden yellow color of nano-silver. It was stored at 4℃ in the dark for later use.

[0069] (2) Disinfection treatment of *Acer buergerianum* explants, including the following steps:

[0070] Explant collection: In early April, explants were collected from branches of Catalpa bungei. When the dormant buds of the Catalpa bungei grew to 1 cm, branches containing terminal and axillary buds were collected before the bracts opened. The branches were cut into 18 cm stem segments with pruning shears to serve as explants. These segments were brought back indoors for hydroponic culture, with the water changed every 3 days. When the dormant buds began to sprout, the branches were pruned into 3 cm segments with axillary or terminal buds for primary culture.

[0071] Cleaning of explants: Soak the stem segments in 0.05% detergent for 10 minutes, then rinse them with running tap water and prepare for disinfection.

[0072] Stem segment disinfection: Transfer the explants to a sterile workbench. Disinfect the trimmed *Cathaya argyrophylla* stem segments in 70% alcohol for 30 seconds, then rinse once with sterile water. Add a 100-fold diluted nano-silver disinfectant to completely submerge the explants, shake thoroughly, disinfect for 9 minutes, rinse four times with sterile water, and finally absorb surface moisture with sterile absorbent paper. Inoculate the stem segments into the primary culture medium. Culture temperature: 25±2℃; light intensity: 1500–3000 lx; ​​photoperiod: 16 h / d.

[0073] The primary culture medium was: MS basal medium + 6-BA 2.0 mg / L + NAA 0.05 mg / L + KT 0.1 mg / L + sucrose 30 g / L + agar 7.0 g / L, pH 5.8.

[0074] One month later, the browning rate, contamination rate, and germination rate were calculated to be 6.7%, 6.7%, and 86.7%, respectively.

Claims

1. A method for primary culture of *Acer palmatum* using celery leaf nano-silver disinfectant, characterized in that, The method includes the following steps: (1) Collection of explants: When the dormant buds of the golden catalpa tree grow to 1-2 cm, collect branches containing terminal and axillary buds before the bracts open. Use pruning shears to cut the branches into 15-20 cm stem segments as explants. Bring them back indoors for hydroponics and change the water every 3 days. When the dormant buds begin to sprout, prune the branches into 2-4 cm segments with axillary or terminal buds for primary culture. (2) Cleaning of explants: Soak the stem segments in dish soap and rinse them clean; (3) Stem segment disinfection: Transfer the cleaned explants to a sterile workbench and disinfect with 70% alcohol for 30 seconds, then rinse once with sterile water; add celery leaf biosynthetic nano-silver disinfectant diluted 100 to 10000 times, immerse the explants, shake well, disinfect for 8 to 10 minutes, rinse with sterile water, absorb the surface moisture, and inoculate the stem segments into the primary culture medium; culture temperature 25±2 ℃, light intensity 1500 to 3000 lx, light duration 16 h / d; The preparation method of the celery leaf nano-silver disinfectant is as follows: Preparation of celery leaf extract: Celery leaves were washed, dried, and pulverized. Ethanol with a volume ratio of 10-90% was added at a material-to-liquid ratio of 1:10~1:100 (g / mL). The mixture was stirred evenly and then subjected to ultrasonic vibration at 100-500 W for 5-10 min. The mixture was then heated under reflux at 60 ℃ for 10-60 min. After natural cooling, the mixture was centrifuged and the supernatant was collected. The above celery leaf extract was mixed with AgNO3 solution to make the final concentration of AgNO3 0.1-10 mM. The dilution ratio of the celery leaf extract was 2-100 times, and the dilution solvent was double-distilled water. The mixture was vortexed and irradiated under direct sunlight until the solution produced a golden yellow color characteristic of nano-silver. It was then stored at 4°C in the dark for later use.

2. The method according to claim 1, characterized in that, In step (2), the stem segments are soaked in 0.05% by mass detergent for 10 minutes.

3. The method according to claim 1, characterized in that, The primary culture medium was: MS basal medium + 6-BA 2.0 mg / L + NAA 0.05 mg / L + KT 0.1 mg / L + sucrose 30 g / L + agar 7.0 g / L, pH 5.8.

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