Anthurium andraeanum detoxification culture method

Abstract

The invention provides an anthurium andraeanum virus-free culture method. The anthurium andraeanum virus-free culture method comprises the following steps: step 1, pretreatment of explants; step 2, pretreatment of the composite detoxification induction liquid; step 3, photosensitive detoxification; step 4, starting culture of cluster buds; 5, proliferation and rooting culture; and step 6, seedling hardening and transplanting. According to the method, multiple mild and efficient technologies are organically integrated, so that multi-dimensional accurate removal of viruses is realized, and meanwhile, the life vitality of the explant is protected and excited to the greatest extent, so that the method has remarkable advantages in the aspects of detoxification rate, survival rate, seedling quality, stability and the like.

Description

Technical Field

[0001] This invention relates to the field of detoxification culture technology, specifically to a method for detoxification culture of Anthurium. Background Technology

[0002] Anthurium, a flower with high ornamental value, suffers from severe impacts from viral diseases, making virus-free cultivation crucial. This stems from two main reasons: First, Anthuriums easily accumulate and spread various viruses, especially Anthurium mosaic virus and latent virus, through asexual reproduction (such as division and tissue culture). These viruses systemically infect plants, causing mosaic, chlorosis, and necrotic spots on leaves, and deformed, smaller, and unevenly colored flowers, severely reducing their commercial value. Second, virus-infected plants exhibit weakened growth, poor resistance, and reduced productivity, ultimately affecting production efficiency and economic benefits. Therefore, obtaining virus-free seedlings through virus-free technology is the cornerstone of ensuring the healthy and efficient development of the Anthurium industry. However, existing virus-free cultivation techniques for Anthurium still present some unresolved technical challenges. The most significant is the contradiction between virus-free efficiency and survival rate. For example, while existing methods combining shoot tip culture with heat treatment are effective, high temperatures can damage the delicate shoot tip tissue, leading to a lower survival rate. Summary of the Invention

[0003] In view of this, the present invention proposes a method for detoxifying and culturing Anthurium to solve the above problems.

[0004] The technical solution of this invention is implemented as follows:

[0005] A method for virus-free culture of Anthurium includes the following steps:

[0006] Step 1: Explant pretreatment: Select healthy and pure Anthurium mother plants, cut off the terminal or lateral buds, gently brush off the surface dust with a soft brush under running water, then soak in alcohol, then soak in mercuric chloride solution for disinfection, then rinse with sterile water, and then peel off the stem tip meristem under a microscope on a clean bench to serve as the explant.

[0007] Step 2, Pretreatment with compound detoxification induction solution: The explants are inoculated into a pretreatment medium containing compound detoxification induction solution and cultured statically in the dark; the pretreatment medium is based on MS medium, and the compound detoxification induction solution contains the following components: ribavirin + 2-thiouracil + Schizophyllum commune extract + Isatis indigotica chloroform extract.

[0008] Step 3, photosensitization detoxification: The pretreated explants are inoculated into photosensitizer and then irradiated with laser. After irradiation, they are washed with sterile water.

[0009] Step 4: Start-up culture of clustered shoots: The explants after photosensitive virus-free treatment are inoculated onto the start-up culture medium and cultured under light conditions to obtain clustered shoots;

[0010] Step 5, Proliferation and Rooting Culture: The clustered buds are divided and transferred to the proliferation medium for propagation. They are cultured under light conditions and then subcultured for 2-3 generations. The healthy, rootless seedlings that have grown to 2-3 cm in height are transferred to the rooting medium to induce the formation of a complete root system and obtain seedlings.

[0011] Step 6, hardening off and transplanting: Harden off the seedlings in a greenhouse, then transplant them into a mixed substrate for conventional cultivation and management.

[0012] Furthermore, the lateral or terminal buds cut in step one are 3-5 cm long, soaked in 75 wt% alcohol for 30-60 seconds, disinfected by soaking in 0.1-0.2 wt% mercuric chloride solution for 10-15 minutes, and rinsed with sterile water 4-5 times. The size of the stem tip meristem is 0.3-0.5 mm and has 1-2 leaf primordia.

[0013] Furthermore, in step two, the bacteria are incubated statically in the dark at 23-27°C for 22-26 hours, with the following concentrations: ribavirin 18-22 mg / L, 2-thiouracil 4-6 mg / L, Schizophyllum commune extract 40-60 mg / L, and Isatis indigotica chloroform extract 8-12 mg / L.

[0014] Furthermore, the *Schizophyllum commune* extract was prepared by the following method: A standard strain of *Schizophyllum commune* was inoculated onto PDA agar plates under aseptic conditions and cultured at 25-28°C in the dark for 5-7 days to obtain activated mycelial blocks. Healthy activated mycelial blocks were picked and transferred to Erlenmeyer flasks containing PDB liquid medium. The flasks were then shaken and cultured on a shaker at 25-28°C and 100-200 rpm for 5-7 days to obtain a seed culture. The seed culture was then transferred at an inoculation rate of 9-11% to Erlenmeyer flasks containing fermentation medium, which consisted of the following components: 20-30 g / L glucose + 8-12 g / L soybean meal + 4-6 g / L yeast extract + 0.8-1.2 g / L potassium dihydrogen phosphate + 0.4-0.6 g / L magnesium sulfate. The culture was shaken and cultured at 25-28°C and 100-200 rpm for 7-10 days. The mycelium was collected by centrifugation, washed 2-3 times with deionized water, and drained. Prepare the mycelium for later use; mix the collected mycelium with deionized water at a mass ratio of 1:20-30, and break the mixture using an ultrasonic cell disruptor. Transfer the disrupted slurry to a round-bottom flask and reflux it in a 90-95℃ water bath for 2-3 hours. Repeat the extraction once and combine the two extracts. First, coarsely filter the combined extract through gauze, then centrifuge the filtrate at 4000-5000 rpm for 15-20 minutes and collect the supernatant. Cool the supernatant in an ice-water bath, and slowly add 3-4 times the volume of anhydrous ethanol pre-cooled to -10 to -20℃ under magnetic stirring. Let it stand at 3-5℃ for 10-15 hours, then centrifuge at 4000-6000 rpm for 10-15 minutes and collect the precipitate. Wash the precipitate twice with anhydrous ethanol and acetone, respectively, and then dry it in a vacuum drying oven at 40-50℃ until constant weight to obtain the Schizophyllum commune extract.

[0015] Furthermore, the chloroform extract of Isatis root was prepared by the following method: dried Isatis root was pulverized using a pulverizer and passed through a 40-60 mesh sieve to obtain Isatis root powder; the Isatis root powder was mixed with chloroform at a mass ratio of 1:12-15, added to an extractor, and heated in a water bath at 70-75℃ for continuous reflux extraction for 6-8 hours. After extraction, the extract was transferred to a rotary evaporator and evaporated at a water bath temperature of 40-45℃ and a pressure of -0.08 to -0.10 MPa. After 1.0-1.5 hours, crude chloroform extract of Isatis indigotica was obtained. 3-5 times its weight of chloroform was added to the crude chloroform extract, and the mixture was dissolved by shaking at 100-150 rpm. The solution was filtered through a funnel containing anhydrous sodium sulfate. The filtered solution was then rotary evaporated at a water bath temperature of 40-45℃ and a pressure of -0.08 to -0.10 MPa for 0.5-1.0 hours. Finally, it was placed in a vacuum drying oven and dried at 35-40℃ to constant weight to obtain the chloroform extract of Isatis indigotica.

[0016] Furthermore, in step three, the photosensitizer consists of 9-11 μmol / L phycocyanin, 14-16 μmol / L pheophytic acid a, and the remainder sterile water. After soaking for 25-35 minutes, the sample is vertically irradiated with a laser at a wavelength of 600-700 nm and a power density of 45-55 mW / cm² for 12-18 minutes, and then washed 3-4 times with sterile water.

[0017] Furthermore, in step four, the starting medium is MS + 0.8-1.2 mg / L 6-BA + 0.15-0.25 mg / L NAA, and cultured for 20-30 days under conditions of 23-27℃, light intensity of 1500-2000 Lux and photoperiod of 12h / d.

[0018] Furthermore, the proliferation medium in step five is: MS + 1.2-1.8 mg / L 6-BA + 0.15-0.25 mg / L NAA + 4-6 mg / L seaweed polysaccharide, and the rooting medium is: 1 / 2 MS + 0.45-0.55 mg / L IBA + 0.1-0.2 g / L activated carbon + 35-45 mL / L elderberry extract, cultured for 25-35 days under the conditions of temperature 23-27℃, light intensity 1500-2000 Lux and photoperiod 12 h / d.

[0019] Furthermore, in step six, the seedlings are hardened off for 7-14 days. The mixed substrate is composed of peat moss, perlite, and vermiculite in a mass ratio of (5-6):(2-3):(1.8-2.2).

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] 1. This invention constructs a multi-level detoxification system that combines chemical inhibition, immune induction, biological clearance and physical targeting through the synergistic effect of pretreatment with composite detoxification induction solution and photosensitive detoxification. It comprehensively intervenes in different stages of virus replication, infection and survival, and significantly improves the detoxification rate.

[0022] 2. The method of this invention achieves efficient detoxification while taking into account the physiological tolerance of explants. The polysaccharides of Schizophyllum commune and the extract of Isatis indigotica in the compound detoxification induction solution have immune activation and cell protection functions. The photosensitive detoxification process has the characteristics of strong targeting and short action time, which together reduce the stress damage to plants caused by traditional high temperature or chemical detoxification, thereby maintaining a high explant survival rate while ensuring a high detoxification rate.

[0023] 3. The virus-free seedlings cultivated by the method of this invention show excellent field adaptability after transplanting, with low virus incidence and strong plant growth. They are significantly better than non-virus-free or conventionally virus-free seedlings in key morphological indicators such as plant height and crown width. Detailed Implementation

[0024] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0025] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0026] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0027] Example 1

[0028] A method for virus-free culture of Anthurium includes the following steps:

[0029] Step 1: Explant pretreatment: Select healthy, purebred Anthurium mother plants, cut 3cm long terminal buds, gently brush off surface dust with a soft brush under running water, then soak in 75wt% alcohol for 30s, then soak in 0.1wt% mercuric chloride solution for 15min for disinfection, then rinse 4 times with sterile water. Finally, under a microscope on a clean bench, peel off the shoot tip meristem. The shoot tip meristem is 0.3mm in size and has one leaf primordium, and is used as the explant.

[0030] Step 2: Pretreatment with compound detoxification induction solution: The explants were inoculated into a pretreatment medium containing the compound detoxification induction solution and incubated statically at 23°C in the dark for 26 hours. The pretreatment medium was based on MS medium, and the compound detoxification induction solution contained the following components: 18 mg / L ribavirin + 4 mg / L 2-thiouracil + 40 mg / L Schizophyllum commune extract + 8 mg / L Isatis indigotica chloroform extract.

[0031] Step 3, photosensitization detoxification: The pretreated explants were inoculated into a photosensitizer consisting of 9 μmol / L phycocyanin, 14 μmol / L pheophytic acid a, and the remainder sterile water. After soaking for 35 min, they were vertically irradiated with a laser with a wavelength of 600 nm and a power density of 45 mW / cm² for 18 min. After irradiation, they were washed three times with sterile water.

[0032] Step 4: Initiation culture of clustered shoots: Explants treated with photosensitizers and detoxified were inoculated onto initiation medium. The culture dishes were placed in a 30-day environment with a temperature of 23℃, a light intensity of 1500 Lux, and a photoperiod of 12 h / d to obtain clustered shoots. The initiation medium consisted of MS medium supplemented with 0.8 mg / L 6-BA and 0.15 mg / L NAA.

[0033] Step 5: Proliferation and Rooting Culture: The clustered shoots were divided and transferred to a proliferation medium for propagation. They were cultured for 35 days under conditions of 23℃, 2000 Lux light intensity, and a 12h / d photoperiod. After two subcultures, robust, rootless seedlings reaching 2cm in height were transferred to a rooting medium to induce the formation of complete root systems, yielding seedlings. The proliferation medium consisted of MS + 1.2mg / L 6-BA + 0.15-0.25mg / L NAA + 4mg / L seaweed polysaccharide. The rooting medium consisted of 1 / 2 MS + 0.45mg / L IBA + 0.1g / L activated charcoal + 35mL / L elderberry extract.

[0034] Step 6, hardening off and transplanting: Harden off the seedlings in a greenhouse for 7 days, and then transplant them into a mixed substrate composed of peat moss, perlite and vermiculite in a mass ratio of 5:2:1.8 for conventional cultivation and management.

[0035] The above-mentioned *Schizophyllum commune* extract was prepared by the following method: *Schizophyllum commune* standard strain was inoculated onto PDA agar plates under aseptic conditions and cultured at 25°C in the dark for 7 days to obtain activated mycelial blocks; healthy activated mycelial blocks were picked and transferred to Erlenmeyer flasks containing PDB liquid medium, and cultured on a shaker at 25°C and 100 rpm for 7 days to obtain seed culture; the seed culture was transferred at an inoculation rate of 9% to Erlenmeyer flasks containing fermentation medium, which included the following components: 20 g / L glucose + 8 g / L soybean meal + 4 g / L yeast extract + 0.8 g / L potassium dihydrogen phosphate + 0.4 g / L magnesium sulfate, and cultured at 25°C and 100 rpm for 10 days. The mycelium was collected by centrifugation, rinsed twice with deionized water, and drained for later use. The collected mycelia were mixed with deionized water at a mass ratio of 1:20. The mixture was then disrupted using an ultrasonic cell disruptor. The disrupted slurry was transferred to a round-bottom flask and refluxed in a 90°C water bath for 3 hours. This extraction was repeated once, and the two extracts were combined. The combined extract was first coarsely filtered through gauze, and then the filtrate was centrifuged at 4000 rpm for 20 minutes. The supernatant was collected and cooled in an ice-water bath. Under magnetic stirring, three volumes of anhydrous ethanol pre-cooled to -10°C were slowly added. The mixture was allowed to stand at 3°C ​​for 15 hours, and then centrifuged at 4000 rpm for 15 minutes to collect the precipitate. The precipitate was washed twice with anhydrous ethanol and acetone, respectively, and then dried in a 40°C vacuum drying oven to constant weight to obtain the Schizophyllum commune extract.

[0036] The above-mentioned chloroform extract of Isatis indigotica was prepared by the following method: dried Isatis indigotica was pulverized by a pulverizer and passed through a 40-mesh sieve to obtain Isatis indigotica powder; the Isatis indigotica powder and chloroform were mixed at a mass ratio of 1:12, added to an extractor, and heated in a 70℃ water bath for continuous reflux extraction for 8 hours. After extraction, the extract was transferred to a rotary evaporator and evaporated at a 40℃ water bath temperature and -0.08MPa for 1.5 hours to obtain crude chloroform extract of Isatis indigotica; chloroform with a mass of 3 times its weight was added to the crude chloroform extract of Isatis indigotica and dissolved by shaking at 100 rpm. The solution was filtered through a funnel containing anhydrous sodium sulfate, and then the filtered solution was evaporated at a 40℃ water bath temperature and -0.08MPa for 1.0 hour. Subsequently, it was placed in a vacuum drying oven and dried at 35℃ to constant weight to obtain chloroform extract of Isatis indigotica.

[0037] Example 2

[0038] A method for virus-free culture of Anthurium includes the following steps:

[0039] Step 1: Explant pretreatment: Select healthy, purebred Anthurium mother plants, cut 4cm long terminal buds, gently brush off surface dust with a soft brush under running water, then soak in 75wt% alcohol for 45s, then soak in 0.15wt% mercuric chloride solution for 12min for disinfection, then rinse 4 times with sterile water. Finally, under a microscope on a clean bench, peel off the shoot tip meristem. The shoot tip meristem is 0.4mm in size and has 2 leaf primordia, and is used as the explant.

[0040] Step 2: Pretreatment with Compound Detoxification Induction Solution: The explants were inoculated into a pretreatment medium containing the compound detoxification induction solution and incubated statically at 25°C in the dark for 24 hours. The pretreatment medium was based on MS medium, and the compound detoxification induction solution contained the following components: 20 mg / L ribavirin + 5 mg / L 2-thiouracil + 50 mg / L Schizophyllum commune extract + 10 mg / L Isatis indigotica chloroform extract.

[0041] Step 3, Photosensitive Detoxification: The pretreated explants were inoculated into a photosensitizer consisting of 10 μmol / L phycocyanin, 15 μmol / L pheophytic chlorophyll a, and the remainder sterile water, and soaked for 30 min. Then, they were vertically irradiated with a laser at a wavelength of 650 nm and a power density of 50 mW / cm² for 15 min. After irradiation, they were washed four times with sterile water.

[0042] Step 4: Initiation culture of clustered shoots: Explants treated with photosensitizers and detoxified were inoculated onto initiation medium. The culture dishes were then placed in a 25°C environment with a light intensity of 1750 Lux and a photoperiod of 12 h / d for 25 days to obtain clustered shoots. The initiation medium consisted of MS medium supplemented with 1.0 mg / L 6-BA and 0.20 mg / L NAA.

[0043] Step 5: Proliferation and Rooting Culture: The clustered shoots were divided and transferred to a proliferation medium for propagation. The seedlings were cultured for 30 days under conditions of 25℃, 1750 Lux light intensity, and a 12h / d photoperiod. After three generations of subculturing, robust, rootless seedlings that had grown to 2cm in height were transferred to a rooting medium to induce the formation of complete root systems, thus obtaining seedlings. The proliferation medium consisted of MS + 1.6mg / L 6-BA + 0.20mg / L NAA + 5mg / L seaweed polysaccharide. The rooting medium consisted of 1 / 2 MS + 0.50mg / L 6-BA + 0.15g / L activated carbon + 40mL / L elderberry extract.

[0044] Step 6, hardening off and transplanting: Harden off the seedlings in a greenhouse for 10 days, and then transplant them into a mixed substrate composed of peat moss, perlite and vermiculite in a mass ratio of 5.5:2.5:2.0 for conventional cultivation and management.

[0045] The above-mentioned *Schizophyllum commune* extract was prepared by the following method: *Schizophyllum commune* standard strain was inoculated onto PDA agar plates under aseptic conditions and cultured at 27°C in the dark for 6 days to obtain activated mycelial blocks; healthy activated mycelial blocks were picked and transferred to Erlenmeyer flasks containing PDB liquid medium, and cultured on a shaker at 27°C and 150 rpm for 6 days to obtain seed culture; the seed culture was transferred at an inoculation rate of 10% to Erlenmeyer flasks containing fermentation medium, which included the following components: 25 g / L glucose + 10 g / L soybean meal + 5 g / L yeast extract + 1.0 g / L potassium dihydrogen phosphate + 0.5 g / L magnesium sulfate, and cultured at 26°C and 150 rpm for 8 days; the mycelium was collected by centrifugation, rinsed three times with deionized water, and drained for later use; the collected... The mycelium of the fungus was mixed with deionized water at a mass ratio of 1:25. The mixture was then disrupted using an ultrasonic cell disruptor. The disrupted slurry was transferred to a round-bottom flask and refluxed in a 92°C water bath for 2.5 h. The extraction was repeated once, and the extracts from both extractions were combined. The combined extract was first coarsely filtered through gauze, and then the filtrate was centrifuged at 4500 rpm for 18 min. The supernatant was collected and cooled in an ice-water bath. Under magnetic stirring, 3.5 times the volume of anhydrous ethanol pre-cooled to -15°C was slowly added. The mixture was allowed to stand at 4°C for 12.5 h, and then centrifuged at 5000 rpm for 12.5 min. The precipitate was collected and washed twice with anhydrous ethanol and acetone, respectively. The precipitate was then dried in a 45°C vacuum drying oven to constant weight to obtain the *Schizophyllum commune* extract.

[0046] The above-mentioned chloroform extract of Isatis indigotica was prepared by the following method: dried Isatis indigotica was pulverized by a pulverizer and passed through a 50-mesh sieve to obtain Isatis indigotica powder; the Isatis indigotica powder and chloroform were mixed at a mass ratio of 1:13.5 and added to an extractor, heated in a water bath at 72℃ for continuous reflux extraction for 7 hours; after extraction, the extract was transferred to a rotary evaporator and evaporated at a water bath temperature of 42℃ and a pressure of -0.09MPa for 1.2 hours to obtain crude chloroform extract of Isatis indigotica; chloroform with a mass of 4 times its weight was added to the crude chloroform extract of Isatis indigotica and dissolved by shaking at 130 rpm; the solution was filtered through a funnel containing anhydrous sodium sulfate, and then the filtered solution was evaporated at a water bath temperature of 42℃ and a pressure of -0.09MPa for 0.8 hours, and then placed in a vacuum drying oven and dried at 38℃ to constant weight to obtain chloroform extract of Isatis indigotica.

[0047] Example 3

[0048] A method for virus-free culture of Anthurium includes the following steps:

[0049] Step 1: Explant pretreatment: Select healthy, purebred Anthurium mother plants, cut lateral buds 5cm long, gently brush off surface dust with a soft brush under running water, then soak in 75wt% alcohol for 60s, then disinfect by soaking in 0.2wt% mercuric chloride solution for 10min, then rinse 5 times with sterile water. Finally, under a microscope on a clean bench, peel off the shoot tip meristem. The shoot tip meristem is 0.5mm in size and has 2 leaf primordia, and is used as the explant.

[0050] Step 2: Pretreatment with compound detoxification induction solution: The above explants were inoculated into a pretreatment medium containing compound detoxification induction solution and cultured statically at 27°C in the dark for 22 hours. The pretreatment medium was based on MS medium, and the compound detoxification induction solution contained the following components: 22 mg / L ribavirin + 6 mg / L 2-thiouracil + 60 mg / L Schizophyllum commune extract + 12 mg / L Isatis indigotica chloroform extract.

[0051] Step 3, Photosensitive Detoxification: The pretreated explants were inoculated into a photosensitizer consisting of 11 μmol / L phycocyanin, 16 μmol / L pheophytic chlorophyll a, and the remainder sterile water, and soaked for 25 min. Then, they were vertically irradiated with a laser at a wavelength of 700 nm and a power density of 55 mW / cm² for 12 min. After irradiation, they were washed four times with sterile water.

[0052] Step 4: Initiation culture of clustered shoots: Explants treated with photosensitizers and detoxified were inoculated onto initiation medium. The culture dishes were then placed under conditions of 27°C, 2000 Lux light intensity, and a 12-hour / day photoperiod for 20 days to obtain clustered shoots. The initiation medium consisted of MS medium supplemented with 1.2 mg / L 6-BA and 0.25 mg / L NAA.

[0053] Step 5: Proliferation and Rooting Culture: The clustered shoots were divided and transferred to a proliferation medium for propagation. They were cultured for 25 days under conditions of 27℃, 2000 Lux light intensity, and a 12h / d photoperiod. This was followed by three subcultures. Healthy, rootless seedlings reaching 3cm in height were then transferred to a rooting medium to induce the formation of complete root systems, yielding seedlings. The proliferation medium consisted of MS + 1.8 mg / L 6-BA + 0.25 mg / L NAA + 6 mg / L seaweed polysaccharide. The rooting medium consisted of 1 / 2 MS + 0.55 mg / L 6-BA + 0.2 g / L activated charcoal + 45 mL / L elderberry extract.

[0054] Step Six: Hardening Off and Transplanting: Harden off the seedlings in a greenhouse for 14 days. Then transplant them into a mixed substrate consisting of peat moss, perlite, and vermiculite in a mass ratio of 6:3:2.2, and carry out conventional cultivation management.

[0055] The above-mentioned *Schizophyllum commune* extract was prepared by the following method: *Schizophyllum commune* standard strain was inoculated onto PDA agar plates under aseptic conditions and cultured at 28°C in the dark for 5 days to obtain activated mycelial blocks; healthy activated mycelial blocks were picked and transferred to Erlenmeyer flasks containing PDB liquid medium, and cultured on a shaker at 28°C and 200 rpm for 5 days to obtain seed culture; the seed culture was transferred at an inoculation rate of 11% to Erlenmeyer flasks containing fermentation medium, which included the following components: 30 g / L glucose + 8-12 g / L soybean meal + 6 g / L yeast extract + 1.2 g / L potassium dihydrogen phosphate + 0.6 g / L magnesium sulfate, and cultured at 28°C and 200 rpm for 7 days. The mycelium was collected by centrifugation, rinsed three times with deionized water, and drained. The collected mycelium was mixed with deionized water at a mass ratio of 1:30. The mixture was then disrupted using an ultrasonic cell disruptor. The disrupted slurry was transferred to a round-bottom flask and refluxed in a 95°C water bath for 2 hours. This extraction was repeated once, and the two extracts were combined. The combined extract was first coarsely filtered through gauze, and then the filtrate was centrifuged at 5000 rpm for 15 minutes. The supernatant was collected and cooled in an ice-water bath. Under magnetic stirring, four volumes of anhydrous ethanol pre-cooled to -20°C were slowly added. The mixture was allowed to stand at 5°C for 10 hours, and then centrifuged at 6000 rpm for 10 minutes to collect the precipitate. The precipitate was washed twice with anhydrous ethanol and acetone, respectively, and then dried in a 50°C vacuum drying oven to constant weight to obtain the Schizophyllum commune extract.

[0056] The above-mentioned chloroform extract of Isatis indigotica was prepared by the following method: dried Isatis indigotica was pulverized by a pulverizer and passed through a 60-mesh sieve to obtain Isatis indigotica powder; the Isatis indigotica powder and chloroform were mixed at a mass ratio of 1:15, added to an extractor, and heated in a 75°C water bath for continuous reflux extraction for 6 hours. After extraction, the extract was transferred to a rotary evaporator and evaporated at a 45°C water bath temperature and -0.10 MPa for 1.0 hour to obtain crude chloroform extract of Isatis indigotica; chloroform at 5 times its mass was added to the crude chloroform extract of Isatis indigotica and dissolved by shaking at 150 rpm. The solution was filtered through a funnel containing anhydrous sodium sulfate, and then the filtered solution was evaporated at a 45°C water bath temperature and -0.10 MPa for 1.0 hour. Subsequently, it was placed in a vacuum drying oven and dried at 40°C to constant weight to obtain chloroform extract of Isatis indigotica.

[0057] Comparative Example 1

[0058] The difference between this comparative example and Example 2 is that the composite detoxification induction solution in step two contains only 20 mg / L ribavirin.

[0059] Comparative Example 2

[0060] Compared with Example 2, the difference in this comparative example is that the compound detoxification induction solution in step two does not contain Schizophyllum commune extract or Isatis indigotica chloroform extract, but contains the following components: 20 mg / L ribavirin + 5 mg / L 2-thiouracil.

[0061] Comparative Example 3

[0062] Compared with Example 2, the difference in this comparative example is that the compound detoxification induction solution does not contain Isatis indigotica chloroform extract, but contains the following components: 20 mg / L ribavirin + 5 mg / L 2-thiouracil + 50 mg / L Schizophyllum commune extract.

[0063] Comparative Example 4

[0064] The difference between this comparative example and Example 2 is that the photosensitive detoxification step in step three is omitted.

[0065] Comparative Example 5

[0066] Compared with Example 2, the difference in this comparative example is that the explants obtained in step one are not subjected to steps two and three. Instead, the explants obtained in step one are inoculated into heat-treated medium and cultured in a constant temperature incubator at 37°C for 25 days, and then inoculated into starter medium for subsequent culture. The heat-treated medium contains the following components: 1 / 2 MS + 20 g / L sucrose + 0.1 mg / L 6-BA + 6.5 g / L agar + 1.0 g / L activated carbon.

[0067] Survival rate and detoxification rate testing

[0068] Anthurium terminal buds or lateral buds of consistent condition were obtained from the same mother plant and cultured according to the methods described in Examples 1-3 and Comparative Examples 1-5, with 50 explants in each group. During the culture period, the explant survival rate of each group was calculated after the initiation of the shoot cluster culture. The explant survival rate (%) = (number of surviving explants / 50) × 100%. After the entire culture was completed, 50 seedlings were randomly selected from each group, and RT-PCR was used to specifically detect Anthurium mosaic virus and latent virus. The virus elimination rate of each group was calculated. The virus elimination rate (%) = (number of seedlings with negative virus test / total number of seedlings tested) × 100%. The test results are shown in Table 1.

[0069] Table 1

[0070] Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Survival rate % 78 80 78 64 64 70 84 48 Detoxification rate % 90 94 92 56 68 72 72 80

[0071] As can be seen from Table 1, the anthurium detoxification culture methods of Examples 1-3 of the present invention can balance survival rate and detoxification rate.

[0072] Comparing Example 2 with Comparative Example 3, the tryptophan and benzoxazinone components in the chloroform extract of Isatis indigotica in Example 2 can scavenge free radicals, help alleviate oxidative stress caused by chemical agents, and protect the fragile shoot tip meristem cells, thereby indirectly improving the survival rate. Furthermore, the indirubin and indoleone contained in the chloroform extract of Isatis indigotica have antiviral activity, acting on viral capsid proteins to inactivate them, thus further improving the detoxification rate. Therefore, compared to Comparative Example 3, Example 2, containing the chloroform extract of Isatis indigotica, has a higher survival rate and detoxification rate.

[0073] Comparing Example 2 with Comparative Example 2, the *Schizophyllum commune* polysaccharide in Example 2 activated the salicylic acid signaling pathway, inducing the expression of antiviral substances such as disease-related proteins. This provided a sustained, broad-spectrum internal antiviral environment for the explants, complementing the target-specific effects of cyclovir and other chemical inhibitors. Simultaneously, the immune activation process enhanced the activity of the antioxidant enzyme system in *Anthurium stenoptera* cells, further avoiding oxidative stress on *Anthurium stenoptera* cells during detoxification treatment, thus maintaining a high survival rate of the explants. Furthermore, *Schizophyllum commune* polysaccharide and *Isatis indigotica* chloroform extract function through a synergistic, combined attack and defense approach. *Schizophyllum commune* polysaccharide immune activation activates the plant's broad-spectrum antiviral capacity from within and enhances its resistance to oxidative damage; while *Isatis indigotica* extract directly inactivates virus particles from the outside and blocks their invasion. The two work synergistically to construct a three-dimensional defense network of "immunoprophylaxis + direct clearance," effectively clearing viruses while significantly reducing damage to *Anthurium stenoptera* cells during the detoxification process, thus simultaneously improving both the detoxification rate and survival rate. Therefore, compared with Comparative Example 2, Example 2, which contains Schizophyllum commune polysaccharide and Isatis indigotica chloroform extract, has a higher survival rate and detoxification rate.

[0074] Comparing Comparative Example 1 and Comparative Example 2, Comparative Example 2, with the addition of 2-thiouracil to Comparative Example 1, shows that 2-thiouracil competitively interferes with the normal metabolism of viral nucleic acids, inhibiting viral RNA synthesis. It is also incorporated into viral RNA, affecting its function as messenger RNA, thereby inhibiting the translation and assembly of viral capsid proteins. Ribavirin primarily attacks the viral replication polymerase, while 2-thiouracil simultaneously interferes with the nucleotide metabolism involved in viral replication. The combined use of these two drugs forms a multi-target attack network, further enhancing the genetic barrier of the detoxification treatment, resulting in a significantly better inhibitory effect on the virus than ribavirin alone.

[0075] Comparing Example 2 and Comparative Example 4, in Example 2, after the explants were soaked in a photosensitizer, the photosensitizing molecules specifically adsorbed and bound to the capsid protein of the virus particles. Under laser irradiation of a specific wavelength, the photosensitizing molecules absorbed light energy and transitioned from the ground state to the excited state. Subsequently, the excited-state photosensitizing molecules converted the surrounding ground-state oxygen molecules into highly reactive singlet oxygen through energy transfer. Singlet oxygen can rapidly oxidize the viral capsid protein, causing its structural denaturation and rupture, and further damaging the viral nucleic acid, resulting in gene chain breaks and base damage, thereby causing it to lose its replication ability. Therefore, compared with Comparative Example 4, the detoxification rate of Example 2 was significantly improved. Although the photosensitizing detoxification treatment led to a certain degree of reduction in the survival rate of explants, this negative impact was controllable and the decrease was relatively limited. In summary, the photosensitizing detoxification technology still has significant advantages in overall performance.

[0076] Comparing Example 2 with Comparative Example 5, Comparative Example 5, by placing the explants at high temperatures, was able to inhibit the replication of heat-sensitive viruses, thus achieving a better detoxification effect. However, high temperatures interfere with the normal metabolic activities of Anthurium cells, leading to protein denaturation, membrane system damage, nutrient depletion, and a large accumulation of reactive oxygen species, causing profound damage to the Anthurium cells themselves. This invention combines pretreatment with a compound detoxification induction solution with photosensitized detoxification, forming a multi-target, precise synergistic effect. Specifically, ribavirin and 2-thiouracil inhibit viral replication, Schizophyllum commune polysaccharide activates the plant's own antiviral immunity, Isatis indigotica extract destroys virus particles, and photosensitized detoxification utilizes photosensitizers to target the virus, generating reactive oxygen species in situ through photostimulation to destroy viral nucleic acids and capsids. Through gentle compound induction and relatively rapid photodynamic action, the damage to the explants is controlled within a manageable and recoverable range. This invention successfully breaks the contradiction between "high detoxification rate" and "low survival rate" in traditional high-temperature detoxification, achieving excellent detoxification effect while ensuring high survival rate of explants and robust subsequent growth and development.

[0077] field trials

[0078] One hundred seedlings each from Examples 1-3 were taken, and a control group of 100 seedlings (conventional tissue culture seedlings without any virus elimination treatment) was set up. Conventional cultivation management, including water and fertilizer management and pest and disease control, was carried out. After 6 months of planting, plant height and crown diameter were measured, and the average values ​​were taken. The incidence of viral diseases was then calculated as follows: Incidence rate (%) = (Number of symptomatic plants / 100) × 100%. The results are shown in Table 2.

[0079] Table 2

[0080] Height / cm Crown diameter / cm Incidence of viral diseases / % Example 1 26 30 7 Example 2 28 33 5 Example 3 27 31 8 control group 15 18 65

[0081] As shown in Table 2, the seedlings cultured using the virus-free culture method of Examples 1-3 of this invention exhibited significant growth advantages when planted in the field. Because the virus-free treatment effectively controlled the occurrence of the virus, the plants were protected from systemic viral infection, resulting in significant improvements in morphological indicators such as plant height and crown width. The results in Table 2 indicate that this virus-free culture method is stable and reliable, and has good prospects for widespread application.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for virus-free culture of Anthurium, characterized in that, Includes the following steps: Step 1: Explant pretreatment: Select healthy and pure Anthurium mother plants, cut off the terminal or lateral buds, gently brush off the surface dust with a soft brush under running water, then soak in alcohol, then soak in mercuric chloride solution for disinfection, then rinse with sterile water, and then peel off the stem tip meristem under a microscope on a clean bench to serve as the explant. Step 2, Pretreatment with compound detoxification induction solution: The explants are inoculated into a pretreatment medium containing compound detoxification induction solution and cultured statically in the dark; the pretreatment medium is based on MS medium, and the compound detoxification induction solution contains the following components: ribavirin + 2-thiouracil + Schizophyllum commune extract + Isatis indigotica chloroform extract. Step 3, photosensitization detoxification: The pretreated explants are inoculated into photosensitizer and then irradiated with laser. After irradiation, they are washed with sterile water. Step 4: Start-up culture of clustered shoots: The explants after photosensitive virus-free treatment are inoculated onto the start-up culture medium and cultured under light conditions to obtain clustered shoots; Step 5, Proliferation and Rooting Culture: The clustered buds are divided and transferred to the proliferation medium for propagation. They are cultured under light conditions and then subcultured for 2-3 generations. The healthy, rootless seedlings that have grown to 2-3 cm in height are transferred to the rooting medium to induce the formation of a complete root system and obtain seedlings. Step Six: Hardening off and Transplanting: Harden off the seedlings in a greenhouse, then transplant them into a mixed substrate for conventional cultivation and management.

2. The method for virus-free culture of Anthurium as described in claim 1, characterized in that, The lateral or terminal buds cut in step one are 3-5 cm long. They are soaked in 75 wt% alcohol for 30-60 seconds, disinfected by soaking in 0.1-0.2 wt% mercuric chloride solution for 10-15 minutes, and rinsed with sterile water 4-5 times. The size of the shoot tip meristem is 0.3-0.5 mm and has 1-2 leaf primordia.

3. The method for virus-free culture of Anthurium as described in claim 1, characterized in that, In step two, the bacteria are incubated statically in the dark at 23-27°C for 22-26 hours. The ribavirin content is 18-22 mg / L, the 2-thiouracil concentration is 4-6 mg / L, the Schizophyllum commune extract concentration is 40-60 mg / L, and the Isatis indigotica chloroform extract concentration is 8-12 mg / L.

4. The method for virus-free culture of Anthurium as described in claim 3, characterized in that, The *Schizophyllum commune* extract was prepared by the following method: a standard strain of *Schizophyllum commune* was inoculated onto a PDA plate medium under aseptic conditions and cultured at 25-28°C in the dark for 5-7 days to obtain activated mycelial blocks; the vigorous activated mycelial blocks were picked and transferred to an Erlenmeyer flask containing PDB liquid medium and placed in a shaker at 25-28°C and 100-200 rpm for 5-7 days to obtain seed culture; Transfer the seed culture at an inoculation rate of 9-11% to an Erlenmeyer flask containing fermentation medium, which comprises the following components: 20-30 g / L glucose + 8-12 g / L soybean meal + 4-6 g / L yeast extract + 0.8-1.2 g / L potassium dihydrogen phosphate + 0.4-0.6 g / L magnesium sulfate. Incubate at 25-28℃ and 100-200 rpm with shaking for 7-10 days. Collect the mycelium by centrifugation, rinse 2-3 times with deionized water, and drain. Mix the collected mycelium with deionized water at a mass ratio of 1:20-30, and disrupt the mixture using an ultrasonic cell disruptor. Transfer the disrupted slurry to a round-bottom flask and incubate at 90℃. Extract by reflux in a 95℃ water bath for 2-3 hours, repeat once, and combine the two extracts. First, coarsely filter the combined extract through gauze, then centrifuge the filtrate at 4000-5000 rpm for 15-20 minutes and collect the supernatant. Cool the supernatant in an ice-water bath, and under magnetic stirring, slowly add 3-4 times the volume of anhydrous ethanol pre-cooled to -10 to -20℃. Let it stand at 3-5℃ for 10-15 hours, then centrifuge at 4000-6000 rpm for 10-15 minutes and collect the precipitate. Wash the precipitate twice with anhydrous ethanol and acetone, respectively, then dry it in a vacuum drying oven at 40-50℃ until constant weight to obtain the *Schizophyllum commune* extract.

5. The method for virus-free culture of Anthurium as described in claim 3, characterized in that, The chloroform extract of Isatis indigotica root is prepared by the following method: dried Isatis indigotica root is pulverized using a pulverizer and passed through a 40-60 mesh sieve to obtain Isatis indigotica root powder; the Isatis indigotica root powder is mixed with chloroform at a mass ratio of 1:12-15, added to an extractor, and heated in a water bath at 70-75℃ for continuous reflux extraction for 6-8 hours. After extraction, the extract is transferred to a rotary evaporator and rotary evaporated at a water bath temperature of 40-45℃ and a pressure of -0.08 to -0.10 MPa for 1 hour. The crude chloroform extract of Isatis root was obtained by stirring for 0-1.5 hours. 3-5 times its weight of chloroform was added to the crude chloroform extract, and the mixture was dissolved by shaking at 100-150 rpm. The solution was filtered through a funnel containing anhydrous sodium sulfate. The filtered solution was then rotary evaporated at a water bath temperature of 40-45℃ and a pressure of -0.08 to -0.10 MPa for 0.5-1.0 hours. Finally, it was dried in a vacuum drying oven at 35-40℃ to constant weight to obtain the chloroform extract of Isatis root.

6. The method for virus-free culture of Anthurium as described in claim 1, characterized in that, In step three, the photosensitizer consists of 9-11 μmol / L phycocyanin, 14-16 μmol / L pheophytic acid a, and the remainder sterile water. The sample is soaked for 25-35 minutes, then vertically irradiated with a laser at a wavelength of 600-700 nm and a power density of 45-55 mW / cm² for 12-18 minutes, and washed 3-4 times with sterile water.

7. The method for virus-free culture of Anthurium as described in claim 1, characterized in that, In step four, the starting medium is MS + 0.8-1.2 mg / L 6-BA + 0.15-0.25 mg / L NAA, and cultured for 20-30 days under conditions of 23-27℃, light intensity of 1500-2000 Lux and photoperiod of 12h / d.

8. The method for virus-free culture of Anthurium as described in claim 1, characterized in that, The proliferation medium in step five is: MS + 1.2-1.8 mg / L 6-BA + 0.15-0.25 mg / L NAA + 4-6 mg / L seaweed polysaccharide. The rooting medium is: 1 / 2 MS + 0.45-0.55 mg / L IBA + 0.1-0.2 g / L activated carbon + 35-45 mL / L elderberry extract. The culture is carried out for 25-35 days under the conditions of temperature 23-27℃, light intensity 1500-2000 Lux and photoperiod 12h / d.

9. The method for virus-free culture of Anthurium as described in claim 1, characterized in that, In step six, the seedlings are hardened off for 7-14 days. The mixed substrate is composed of peat moss, perlite, and vermiculite in a mass ratio of (5-6):(2-3):(1.8-2.2).