A virus detoxification method for dioscorea opposita

Abstract

The present application provides a virus detoxification method of Chinese yam. The present application can effectively prevent Japanese yam mosaic virus (JYMV), potato virus Y (PVY), potato virus X (PVX) and potato leaf roll virus (PLRV) by peeling the stem tip of Chinese yam and using heat treatment and ribavirin detoxification. The optimal virus removal method is to peel the stem tip to 0.3-0.5mm, heat treatment at 40℃ for 8h, and ribavirin 50mg / L.

Description

Technical Field

[0001] This invention belongs to the field of plant virus removal technology, specifically relating to a method for virus removal from yam. Background Technology

[0002] Yam (Dioscorea oppositifolia L.) is a plant belonging to the genus Dioscorea in the family Dioscoreaceae. Its fleshy tubers are rich in nutrients such as starch, protein, amino acids, and vitamins. It has the effects of strengthening the spleen, nourishing the lungs, and benefiting the kidneys, possessing both edible and health-promoting value, and has broad application prospects. Anshun yam, as a specialty economic crop of Anshun region in Guizhou Province, has become an important tool for Guizhou's decisive victory in poverty alleviation and rural revitalization in recent years. Anshun yam has significantly higher water content, protein, and total amino acid content than yams produced in other provinces and cities in China, giving it higher edible and medicinal value. In production, yam tubers are usually used for asexual reproduction, which makes it easy for viruses to accumulate over generations, exacerbating the occurrence of viral diseases and seriously affecting yield and quality. Currently, 17 types of yam viruses have been reported both domestically and internationally. Among them, 11 viruses reported in my country mainly include Potatovirus Y (PVY), Potato Leaffoll Rolero Virus (PLRV), Potato Virus A (PVA), Potato Virus S (PVS), Potato Virus X (PVX), Potato Virus M (PVM), Yammild Mosaic Virus (YMMV), and Japanese Yam Mosaic Virus (JYMV). The application of biochemical agents cannot completely prevent the accumulation and spread of viruses at the source, and viral diseases still occur frequently in major yam-producing areas.

[0003] Producing high-quality seedlings using virus-free technology is an important means to improve the quality and efficiency of agricultural production. This technology has achieved good results in the production of various asexually propagated crops such as lilies, strawberries, pinellia, and potatoes. Previous studies have shown that shoot tip culture can effectively eliminate JYMV, PVY, and potato spindle tuber viroids in Nancheng yams, and virus-free yams obtained using this technology can increase yield by 38%. Currently, there is limited research on yam virus-free technology, and the commonly used single virus-free methods result in low efficiency. Therefore, it is essential to study the combined benefits of virus-free methods to improve the efficiency of yam virus-free production.

[0004] To improve the virus removal efficiency of yam, this study used Anshun yam as the test material. Based on the investigation of the distribution of virus types in Anshun area, and combined various methods such as shoot tip culture, heat treatment and chemical treatment, we explored the most suitable virus removal strategy for yam, in order to provide a reference for the virus removal production of yam. Summary of the Invention

[0005] The purpose of this invention is to provide a method for virus removal from yam. This invention, through heat treatment and ribavirin detoxification after peeling the yam stem tips, effectively prevents infection by Japanese yam mosaic virus (JYMV), potato virus Y (PVY), potato virus X (PVX), and potato leafroll virus (PLRV).

[0006] The technical solution of the present invention is a method for virus detoxification of yam. The method involves peeling the stem tip of the yam, followed by heat treatment and ribavirin detoxification to effectively remove Japanese yam mosaic virus, potato virus Y, potato virus X, and potato leafroll virus.

[0007] The aforementioned method for virus removal from yam includes the following steps:

[0008] (1) Heat treatment: Take potted yam plants and place them in an artificial climate chamber for cultivation. Under light conditions of 1500-2500 lux, heat treatment is carried out at 40-50℃ for 8-24 hours to obtain heat-treated yam for later use.

[0009] (2) Stem tip peeling: Take healthy and robust stem tips of yam after heat treatment, rinse with running water, disinfect with 75% ethanol (the amount of 75% ethanol is enough to submerge the explant), then disinfect with 0.1% mercuric chloride (the amount of 0.1% mercuric chloride is enough to submerge the explant), wash with sterile water, and peel the stem tips to 0.1-0.7 mm under a dissecting microscope in a clean bench to obtain peeled yam stem tips for later use;

[0010] (3) Preparation of ribavirin stock solution: Dissolve ribavirin in distilled water, shake well, and then filter and sterilize using a 0.22μm sterile filter membrane to obtain ribavirin stock solution for later use;

[0011] (4) Preparation of ribavirin medium: Take MS + agar 5-7g / L + sucrose 25-35g / L as the basic medium. The pH of the basic medium is 5.4-6.0. After sterilizing in an autoclave at 115-125℃ for 15-25min, cool to 35-65℃ and add ribavirin stock solution and mix well. The concentration of ribavirin is 30-70mg / L. Dispense and condense to obtain ribavirin medium for later use.

[0012] (5) Detoxification: Place the peeled yam stem tips in ribavirin culture medium and culture for 2-3 months. When the plant grows to 2-3 leaves, it is ready.

[0013] In step (1) above, heat treatment: take potted yam plants and place them in an artificial climate chamber for cultivation. Under 2000 lux light conditions, heat treatment at 40℃ for 8 hours is carried out to obtain heat-treated yam for later use.

[0014] In step (2) above, stem tip peeling: Take healthy and robust stem tips of yam after heat treatment, rinse with running water for 25-35 minutes, first disinfect with 75% ethanol for 15-25 seconds, the amount of 75% ethanol is enough to submerge the explant, then disinfect with 0.1% mercuric chloride for 5-10 minutes, the amount of 0.1% mercuric chloride is enough to submerge the explant, wash with sterile water 3-5 times, and then peel the stem tip to 0.3-0.5 mm under a dissecting microscope in a clean bench to obtain the peeled yam stem tip for later use.

[0015] Specifically, in step (2) above, stem tip peeling: take healthy and robust stem tips of yam after heat treatment, rinse with running water for 30 minutes, disinfect with 75% ethanol for 20 seconds, the amount of 75% ethanol is enough to submerge the explant, then disinfect with 0.1% mercuric chloride for 8 minutes, the amount of 0.1% mercuric chloride is enough to submerge the explant, wash with sterile water 4 times, and then peel the stem tip to 0.3-0.5 mm under a dissecting microscope in a clean bench to obtain the peeled yam stem tip for later use.

[0016] In step (3) above, the ribavirin mother liquor is prepared by dissolving 7.0g of ribavirin in distilled water and making up to 10mL. After shaking well, it is filtered and sterilized using a 0.22μm sterile filter membrane to obtain the ribavirin mother liquor for later use.

[0017] In step (4) above, the ribavirin culture medium is prepared as follows: MS + agar 6g / L + sucrose 30g / L is taken as the basic culture medium. The pH of the basic culture medium is 5.6-5.8. After sterilizing in an autoclave at 121℃ for 20min, the ribavirin stock solution is added when cooled to 40-60℃ and mixed well. The ribavirin concentration is 50mg / L. The mixture is dispensed and condensed to obtain the ribavirin culture medium for later use.

[0018] In step (5) above, the cultivation conditions are 20-25℃, light intensity is 1500-2500 lux, and light duration is 10-14h / day.

[0019] Specifically, in the aforementioned step (5), the cultivation conditions are 21-23℃, light intensity is 2000 lux, and light duration is 12h / day.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] The method for detoxifying yams according to this invention effectively prevents infection by Japanese yam mosaic virus (JYMV), potato virus Y (PVY), potato virus X (PVX), and potato leafroll virus (PLRV), resulting in a yam survival rate of over 85%. Attached Figure Description

[0022] Figure 1 : Yam stem tips from treatment group 5, cultured for 40 days in the orthogonal experiment for yam virus elimination;

[0023] Figure 2 : Yam stem tips cultured for 72 days in treatment group 5 of the orthogonal experiment for yam virus elimination;

[0024] Figure 3 : Yam stem tips cultured for 97 days in the 5th treatment group of the orthogonal experiment for yam detoxification.

[0025] Specific implementation methods

[0026] Example 1: Virus detoxification method for yam

[0027] (1) Place the potted yam in an artificial climate chamber for cultivation. Under 2000 lux light conditions, heat-treat at 40℃ for 8 hours to obtain heat-treated yam for later use.

[0028] (2) Stem tip peeling: Take healthy and robust stem tips of yam after heat treatment, rinse with running water for 30 minutes, disinfect with 75% ethanol for 20 seconds, the amount of 75% ethanol is enough to submerge the explant, then disinfect with 0.1% mercuric chloride for 8 minutes, the amount of 0.1% mercuric chloride is enough to submerge the explant, rinse with sterile water 4 times, peel the stem tip to 0.3-0.5 mm under a dissecting microscope in a clean bench to obtain the peeled yam stem tip for later use;

[0029] (3) Preparation of ribavirin stock solution: Dissolve 7.0g of ribavirin in distilled water and make up to 10mL. Shake well and then filter and sterilize using a 0.22μm sterile filter membrane to obtain ribavirin stock solution for later use.

[0030] (4) Preparation of ribavirin medium: MS + agar 6g / L + sucrose 30g / L is taken as the basic medium. The pH of the basic medium is 5.6-5.8. After sterilizing in an autoclave at 121℃ for 20min, the ribavirin stock solution is added when cooled to 40-60℃ and mixed well. The concentration of the ribavirin stock solution is 50mg / L. The solution is dispensed and condensed to obtain ribavirin medium for later use.

[0031] (5) Detoxification: Place the peeled yam stem tips in ribavirin medium and culture for 2-3 months. The culture conditions are 21-23℃, light intensity of 2000 lux, and light duration of 12h / day. Detoxification can be completed when the plant grows to 2-3 leaves.

[0032] Example 2: Virus detoxification method for yam

[0033] (1) Place the potted yam in an artificial climate chamber for cultivation. Under 2000 lux light conditions, heat-treat at 40℃ for 24 hours to obtain heat-treated yam for later use.

[0034] (2) Stem tip peeling: Take healthy and robust stem tips of yam after heat treatment, rinse with running water for 30 minutes, disinfect with 75% ethanol for 20 seconds, the amount of 75% ethanol is enough to submerge the explant, then disinfect with 0.1% mercuric chloride for 8 minutes, the amount of 0.1% mercuric chloride is enough to submerge the explant, rinse with sterile water 4 times, peel the stem tip to 0.3-0.5 mm under a dissecting microscope in a clean bench to obtain the peeled yam stem tip for later use;

[0035] (3) Preparation of ribavirin stock solution: Dissolve 7.0g of ribavirin in distilled water and make up to 10mL. Shake well and then filter and sterilize using a 0.22μm sterile filter membrane to obtain ribavirin stock solution for later use.

[0036] (4) Preparation of ribavirin medium: MS + agar 6g / L + sucrose 30g / L is taken as the basic medium. The pH of the basic medium is 5.6-5.8. After sterilizing in an autoclave at 121℃ for 20min, the ribavirin stock solution is added when cooled to 40-60℃ and mixed well. The concentration of the ribavirin stock solution is 50mg / L. The solution is dispensed and condensed to obtain ribavirin medium for later use.

[0037] (5) Detoxification: Place the peeled yam stem tips in ribavirin medium and culture for 2-3 months. The culture conditions are 21-23℃, light intensity of 2000 lux, and light duration of 12h / day. Detoxification can be completed when the plant grows to 2-3 leaves.

[0038] Example 3: Virus detoxification method for yam

[0039] (1) Heat treatment: Take potted yam plants and place them in an artificial climate chamber for cultivation. Under 1500 lux light conditions, heat treatment is carried out at 40℃ for 8 hours to obtain heat-treated yam for later use.

[0040] (2) Stem tip peeling: Take healthy and robust stem tips of yam after heat treatment, rinse with running water for 25 minutes, disinfect with 75% ethanol for 15 seconds, the amount of 75% ethanol is enough to submerge the explant, then disinfect with 0.1% mercuric chloride for 5 minutes, the amount of 0.1% mercuric chloride is enough to submerge the explant, rinse with sterile water 3 times, peel the stem tip to 0.1-0.3 mm under a dissecting microscope in a clean bench to obtain the peeled yam stem tip for later use;

[0041] (3) Preparation of ribavirin stock solution: Dissolve 7.0g of ribavirin in distilled water and make up to 10mL. Shake well and then filter and sterilize using a 0.22μm sterile filter membrane to obtain ribavirin stock solution for later use.

[0042] (4) Preparation of ribavirin medium: Take MS + agar 5g / L + sucrose 25g / L as the basic medium. The pH of the basic medium is 5.4-5.6. After sterilizing in an autoclave at 115℃ for 15min, add ribavirin stock solution when cooled to 35-45℃ and mix well. The concentration of ribavirin stock solution is 30mg / L. Dispense and condense to obtain ribavirin medium for later use.

[0043] (5) Detoxification: Place the peeled yam stem tips in ribavirin medium and culture for 2-3 months. The culture conditions are 20-22℃, light intensity of 1500 lux, and light duration of 10h / day. Detoxification can be completed when the plant grows to 2-3 leaves.

[0044] Example 4: Virus detoxification method for yam

[0045] (1) Heat treatment: Take potted yam plants and place them in an artificial climate chamber for cultivation. Under 2500 lux light conditions, heat treatment is carried out at 50℃ for 24 hours to obtain heat-treated yam for later use.

[0046] (2) Stem tip peeling: Take healthy and robust stem tips of yam after heat treatment, rinse with running water for 35 minutes, disinfect with 75% ethanol for 25 seconds, the amount of 75% ethanol is enough to submerge the explant, then disinfect with 0.1% mercuric chloride for 10 minutes, the amount of 0.1% mercuric chloride is enough to submerge the explant, wash with sterile water 5 times, peel the stem tip to 0.5-0.7 mm under a dissecting microscope in a clean bench to obtain the peeled yam stem tip for later use;

[0047] (3) Preparation of ribavirin stock solution: Dissolve 7.0g of ribavirin in distilled water and make up to 10mL. Shake well and then filter and sterilize using a 0.22μm sterile filter membrane to obtain ribavirin stock solution for later use.

[0048] (4) Preparation of ribavirin medium: MS + agar 7g / L + sucrose 35g / L is taken as the basic medium. The pH of the basic medium is 5.7-5.9. After sterilizing in an autoclave at 125℃ for 25min, the ribavirin stock solution is added when cooled to 45-65℃ and mixed well. The concentration of the ribavirin stock solution is 70mg / L. The solution is dispensed and condensed to obtain ribavirin medium for later use.

[0049] (5) Detoxification: Place the peeled yam stem tips in ribavirin medium and culture for 2-3 months. The culture conditions are 23-25℃, light intensity of 2500 lux, and light duration of 14h / day. Detoxification can be completed when the plant grows to 2-3 leaves.

[0050] Example 5: Virus detoxification method for yam

[0051] (1) Heat treatment: Take potted yam plants and place them in an artificial climate chamber for cultivation. Under 2000 lux light conditions, heat treatment is carried out at 45℃ for 16 hours to obtain heat-treated yam for later use.

[0052] (2) Stem tip peeling: Take healthy and robust stem tips of yam after heat treatment, rinse with running water for 30 minutes, disinfect with 75% ethanol for 20 seconds, the amount of 75% ethanol is enough to submerge the explant, then disinfect with 0.1% mercuric chloride for 9 minutes, the amount of 0.1% mercuric chloride is enough to submerge the explant, rinse with sterile water 4 times, peel the stem tip to 0.3-0.5 mm under a dissecting microscope in a clean bench to obtain the peeled yam stem tip for later use;

[0053] (3) Preparation of ribavirin stock solution: Dissolve 7.0g of ribavirin in distilled water and make up to 10mL. Shake well and then filter and sterilize using a 0.22μm sterile filter membrane to obtain ribavirin stock solution for later use.

[0054] (4) Preparation of ribavirin medium: MS + agar 6g / L + sucrose 30g / L is taken as the basic medium. The pH of the basic medium is 5.8-6.0. After sterilizing in an autoclave at 122℃ for 20min, the ribavirin stock solution is added when cooled to 40-60℃ and mixed well. The concentration of the ribavirin stock solution is 50mg / L. The solution is dispensed and condensed to obtain ribavirin medium for later use.

[0055] (5) Detoxification: Place the peeled yam stem tips in ribavirin medium and culture for 2-3 months. The culture conditions are 20-22℃, light intensity of 2000 lux, and light duration of 13h / day. Detoxification can be completed when the plant grows to 2-3 leaves.

[0056] The inventors conducted the following experiments to verify the effectiveness of the invention:

[0057] 1. Materials and Methods

[0058] 1.1 Test Materials

[0059] The twin yam samples used for virus detection in this study were collected in Pingba, Anshun, Guizhou Province, and the yam plants used for virus detoxification were grown in greenhouses at the Guizhou Academy of Agricultural Sciences.

[0060] 1.2 Detoxification Culture

[0061] This study used the size of the peeled stem tips, heat treatment time, heat treatment temperature, and ribavirin concentration as experimental factors, with three experimental levels for each (Table 1), to explore the detoxification efficiency of yam under different conditions.

[0062] Table 1. Levels of Experimental Factors

[0063]

[0064]

[0065] A four-factor, three-level orthogonal experimental design was used with IBM SPSS Statistics 21 (Table 2), and the yam detoxification experiment was carried out according to the designed orthogonal table.

[0066] Table 2 Orthogonal Experimental Design Table

[0067]

[0068] Heat treatment: Potted Anshun yam plants were placed in an artificial climate chamber for cultivation. The cultivation temperature and time were determined according to the orthogonal experimental design table. The light intensity was 2000 lux and the light duration was 12 h / d.

[0069] Stem tip peeling: Take healthy and robust stem tips of Anshun yam, rinse with running water for 30 minutes, disinfect with 75% ethanol for 20 seconds, disinfect with 0.1% mercuric chloride for 8 minutes, rinse with sterile water 4 times, peel off the corresponding size of stem tips under a dissecting microscope in a clean bench, and place them in a culture medium for culture.

[0070] Ribavirin addition: Dissolve 7.0g of ribavirin in distilled water and bring the volume to 10mL to prepare a 0.7g / mL stock solution. After mixing, filter and sterilize using a 0.22μm sterile filter membrane, and add it to the sterilized but non-coagulated basal culture medium.

[0071] Basic culture medium: MS medium + 6 g / L agar + 30 g / L sucrose, pH 5.6-5.8.

[0072] Culture conditions: The light culture conditions are (22±1)℃, light intensity 2000 lux, and light duration 12h / day.

[0073] 1.3 RNA extraction and virus detection

[0074] The RNA extraction kit was purchased from BIOREBA, Switzerland, and the young leaves of the tested yam were tested according to the kit's instructions. The c-DNA reverse transcription kit was purchased from ABI, and reverse transcription was performed according to the kit's instructions.

[0075] The viruses detected were: Potato virus Y (PVY), Potato leafroll virus (PLRV), Potato virus A (PVA), Potato virus M (PVM), Potato virus S (PVS), Potato virus X (PVX), Chinese yam mild mosaic virus (YMMV), and Japanese yam mosaic virus (JYMV). The ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit gene (rbcL) of *Dioscorea opposita* was used as an internal control gene. Gene amplification was performed using a 10 μL system with 1UEx-Taq (Takara) under the following reaction conditions: 95℃ for 3 min, 94℃ for 30 s, 53℃ for 30 s, 72℃ for 40 s, and 72℃ for 10 min, for a total of 30 cycles. Primer sequences and amplified fragment sizes are shown in Table 3. 3 μL of PCR amplification product was subjected to gel electrophoresis, with DL2000 as a reference.

[0076] Table 3. Primer sequence information for virus detection

[0077]

[0078] 1.4 Data Analysis

[0079] Data analysis was performed using IBM SPSS Statistics 21 and WPS 2023 software.

[0080] 2 Results and Analysis

[0081] 2.1 Virus detection in yam from Guizhou region

[0082] Virus detection was performed on collected yam samples using designed virus-specific primers. The results showed that yams from Guizhou Province were mainly infected by Japanese yam mosaic virus, potato virus X, potato leafroll virus, and potato virus Y. Other types of viruses were not detected (Table 4). Therefore, subsequent virus elimination experiments mainly targeted the elimination of these four viruses: Japanese yam mosaic virus, potato virus X, potato leafroll virus, and potato virus Y.

[0083] Table 4. Virus detection in yam in Guizhou Province

[0084]

[0085] 2.2 Effects of different detoxification methods on survival rate

[0086] The results of the range analysis (Table 5) show that among the four factors, the heat treatment temperature has the greatest impact on the survival rate of virus removal, followed by the size of the stem tip, while the concentration of ribavirin has the least impact on the survival rate. Overall, the best combination for virus removal with the highest survival rate is to peel the stem tip to 0.3-0.5 mm, heat treat at 40℃ for 8 h, and use ribavirin at 50 mg / L.

[0087] Table 5. Effects of different factor levels on survival rate

[0088]

[0089] The results of the analysis of variance (Table 6) show that all four factors had a highly significant impact on the survival rate. Based on the combined results of the range analysis and analysis of variance, the combination of stem tip peeling to 0.3-0.5 mm, heat treatment at 40℃ for 8 hours, and ribavirin at 50 mg / L for detoxification resulted in the best survival rate.

[0090] Table 6. Analysis of Variance of Survival Rate

[0091]

[0092] Note: In the significance column, an asterisk (*) in the upper right corner indicates p < 0.5, and a double-headed arrow (**) indicates p < 0.01. The same applies below.

[0093] 2.3 PVY1 virus removal efficiency

[0094] The results of the PVY1 virus removal range analysis (Table 7) show that among the four factors, heat treatment temperature has the greatest impact on the PVY1 virus removal rate, followed by stem tip size, while heat treatment time has the least impact. Overall, the optimal combination of methods for PVY1 virus removal is stem tip peeling to 0.3-0.5 mm, heat treatment at 40℃ for 8 h, and ribavirin 50 mg / L.

[0095] Table 7. Range Analysis of PVY1 Virus Elimination Rate

[0096]

[0097] The results of the ANOVA on PVY1 virus removal (Table 8) showed that all four factors had a highly significant impact on PVY1 virus removal. Combining the results of the range and variance analyses, the optimal removal method for PVY1 virus was achieved using a stem tip size of 0.3-0.5 mm, heat treatment at 40℃ for 8 hours, and ribavirin at 50 mg / L.

[0098] Table 8. Analysis of Variance on PVY1 Virus Elimination Rate

[0099]

[0100] 2.4 PVX virus removal rate

[0101] The results of the PVX virus removal range analysis (Table 9) show that, among the four factors, heat treatment temperature has the greatest impact on the PVX virus removal rate, followed by shoot tip size, while ribavirin concentration has the least impact. Overall, the optimal combination of methods for PVX virus removal is shoot tip peeling to 0.3-0.5 mm, heat treatment at 40℃ for 24 h, and ribavirin at 50 mg / L.

[0102] Table 9. Range Analysis of PVX Virus Elimination Rate

[0103]

[0104] The results of the ANOVA analysis on PVX virus removal (Table 10) showed that all four factors had a highly significant impact on PVX virus removal. Combining the results of the range analysis and ANOVA, the optimal removal method for PVX virus was achieved using a stem tip size of 0.3-0.5 mm, heat treatment at 40℃ for 24 h, and ribavirin at 50 mg / L.

[0105] Table 10. Analysis of Variance on PVX Virus Elimination Rate

[0106]

[0107] 2.5 PLRV virus removal rate

[0108] The results of the PLRV virus removal range analysis (Table 11) show that among the four factors, heat treatment temperature has the greatest impact on the PLRV virus removal rate, followed by stem tip size, while heat treatment time has the least impact. Overall, the optimal combination of methods for PLRV virus removal is stem tip peeling to 0.3-0.5 mm, heat treatment at 40℃ for 8 h, and ribavirin 50 mg / L.

[0109] Table 11 Range Analysis of PLRV Virus Elimination Rate

[0110]

[0111] Analysis of variance (ANOVA) results (Table 12) showed that all four factors had highly significant effects on PLRV virus eradication. Combining the results of range analysis and ANOVA, the optimal method for PLRV virus eradication was achieved using a stem tip size of 0.3-0.5 mm, heat treatment at 40℃ for 8 hours, and ribavirin at 50 mg / L.

[0112] Table 12 Analysis of Variance of PLRV Virus Elimination Rate

[0113]

[0114] 2.6 JYMV virus removal rate

[0115] According to the range analysis results of JYMV virus removal (Table 13), among the four factors, heat treatment temperature has the greatest impact on the JYMV virus removal rate, followed by stem tip size, and heat treatment time has the least impact. Overall, the optimal combination of methods for JYMV virus removal is stem tip peeling to 0.3-0.5 mm, heat treatment at 40℃ for 8 h, and ribavirin 50 mg / L.

[0116] Table 13. Range Analysis of JYMV Virus Elimination Rate

[0117]

[0118]

[0119] Analysis of variance (ANOVA) results (Table 14) showed that all four factors had highly significant effects on JYMV virus eradication. Combining the results of range analysis and ANOVA, the optimal method for JYMV virus eradication was achieved using a stem tip size of 0.3-0.5 mm, heat treatment at 40℃ for 8 hours, and ribavirin at 50 mg / L.

[0120] Table 14. Analysis of Variance on JYMV Virus Elimination Rate

[0121]

[0122] 2.7 Overall Removal Efficiency of Four Viruses

[0123] Analysis of the range of removal results for four viruses (PVY1, PVX, PLRV, and JYMV) (Table 15) shows that among the four factors, heat treatment temperature has the greatest impact on the removal rate of the four viruses, followed by stem tip size, while heat treatment time has the least impact. Overall, the optimal combination of methods for the simultaneous removal of the four viruses is stem tip peeling to 0.3-0.5 mm, heat treatment at 40℃ for 8 h, and ribavirin 50 mg / L.

[0124] Table 15 Analysis of the poor elimination efficiency of four types of viruses

[0125]

[0126]

[0127] Analysis of variance (ANOVA) results (Table 16) showed that all four factors had highly significant effects on the elimination of the four viruses. Combining the results of range analysis and ANOVA, the optimal elimination method for the four viruses was achieved using a stem tip size of 0.3-0.5 mm, heat treatment at 40℃ for 8 hours, and a ribavirin concentration of 50 mg / L.

[0128] Table 16. Analysis of Variance on Elimination Efficiency of Four Viruses

[0129]

[0130] 2.8 Discussion

[0131] Shoot tip stripping is the most commonly used method for plant tissue detoxification. The smaller the stripped tissue, the more significant the detoxification effect, but the survival rate also decreases. In the single shoot tip stripping method, the size of the stripped shoot tip is usually maintained between 0.1-0.3 mm. The results of this study show that the survival rate and detoxification rate are the highest when the stripped tissue is in the range of 0.3-0.5 mm. This may be related to the use of multiple detoxification methods in this study, which enhances the detoxification efficiency.

[0132] Heat treatment is generally used for the removal of temperature-sensitive viruses. By inactivating the virus at high temperatures, combined with shoot tip stripping, virus removal is achieved. In other crops, appropriate heat treatment combined with shoot tip stripping can effectively remove PVY, PVX, and PLRV from the shoot tip. The results of this study indicate that heat treatment temperature has the greatest impact on the removal of four viruses: JYMV, PVY, PVX, and PLRV. Treatment at 40℃ yielded the best virus removal effect, and the experimental results are basically consistent with previous studies.

[0133] Ribavirin primarily works by inactivating virus particles, combined with stem tip stripping to achieve detoxification. Studies have shown that stem tip stripping combined with ribavirin is effective in eliminating PVY. Our results indicate that a ribavirin concentration of 50 mg / L resulted in the best detoxification of the four viruses, which is largely consistent with previous findings.

[0134] The results showed that yam in Guizhou Province is mainly infected by four viruses: JYMV, PVY, PVX, and PLRV. The optimal combination of methods for removing JYMV, PVY, and PLRV viruses was: peeling the stem tip to 0.3-0.5 mm, heat treatment at 40℃ for 8 hours, and applying ribavirin at 50 mg / L. For PVX virus, the optimal removal method was achieved by peeling the stem tip to 0.3-0.5 mm, heat treatment at 40℃ for 24 hours, and applying ribavirin at 50 mg / L. This study provides a reference for optimizing the combination of methods for yam virus removal.

Claims

1. A method for virus-free breeding of Dioscorea opposita Thunb. comprising the steps of: The method is a method for effectively removing Japanese Dioscorea japonica mosaic virus, potato Y virus, potato X virus and potato leaf roll virus by peeling the stem tip of Dioscorea japonica and adopting heat treatment and ribavirin detoxification after the peeling; ​ The method comprises the following steps: (1) heat treatment: taking Dioscorea japonica potted plants and placing them in an artificial climate box for culture, under the condition of 1500-2500 lux illumination, heat treatment at 40°C for 8h to obtain heat-treated Dioscorea japonica, for standby; (2) stem tip peeling: taking the healthy and strong stem tips of the heat-treated Dioscorea japonica, washing with running water, first disinfecting with 75% ethanol, the amount of 75% ethanol being to immerse the explants, then disinfecting with 0.1% mercury, the amount of 0.1% mercury being to immerse the explants, washing with sterile water, and then peeling the stem tips to 0.3-0.5mm under a microscope on an ultraclean bench to obtain peeled Dioscorea japonica stem tips, for standby; (3) preparation of ribavirin mother liquor: dissolving ribavirin in distilled water, shaking uniformly, filtering and sterilizing with a 0.22μm sterile filter membrane to obtain ribavirin mother liquor, for standby; (4) preparation of ribavirin culture medium: taking MS+agar 5-7g / L+cane sugar 25-35g / L as a basic culture medium, the basic culture medium having a pH of 5.4-6.0, after high-pressure sterilization pot 115-125°C sterilization for 15-25min, cooling to 35-65°C, adding ribavirin mother liquor and mixing uniformly, the concentration of ribavirin being 50mg / L, condensing and dispensing to obtain ribavirin culture medium, for standby; (5) detoxification: placing the peeled Dioscorea japonica stem tips in the ribavirin culture medium for culture for 2-3 months, and when the plant growth reaches 2-3 leaves, it is ready.

2. The method of virus-free Dioscorea japonica according to claim 1, wherein: In the step (1), heat treatment: taking Dioscorea japonica potted plants and placing them in an artificial climate box for culture, under the condition of 2000 lux illumination, heat treatment at 40°C for 8h to obtain heat-treated Dioscorea japonica, for standby.

3. The method for virus detoxification of yam according to claim 1, characterized in that: In the step (2), stem tip peeling: taking the healthy and strong stem tips of the heat-treated Dioscorea japonica, washing with running water for 25-35min, first disinfecting with 75% ethanol for 15-25s, the amount of 75% ethanol being to immerse the explants, then disinfecting with 0.1% mercury for 5-10min, the amount of 0.1% mercury being to immerse the explants, washing with sterile water for 3-5 times, and then peeling the stem tips to 0.3-0.5mm under a microscope on an ultraclean bench to obtain peeled Dioscorea japonica stem tips, for standby.

4. The method for virus detoxification of yam according to claim 3, characterized in that: In the step (2), stem tip peeling: taking the healthy and strong stem tips of the heat-treated Dioscorea japonica, washing with running water for 30min, first disinfecting with 75% ethanol for 20s, the amount of 75% ethanol being to immerse the explants, then disinfecting with 0.1% mercury for 8min, the amount of 0.1% mercury being to immerse the explants, washing with sterile water for 4 times, and then peeling the stem tips to 0.3-0.5mm under a microscope on an ultraclean bench to obtain peeled Dioscorea japonica stem tips, for standby.

5. The method of virus-free Dioscorea japonica according to claim 1, wherein: In the step (3), preparation of ribavirin mother liquor: taking 7.0g ribavirin, dissolving in distilled water and constant volume to 10mL, shaking uniformly, filtering and sterilizing with a 0.22μm sterile filter membrane to obtain ribavirin mother liquor, for standby.

6. The method of virus-free Dioscorea japonica according to claim 1, wherein: In the step (4), the ribavirin medium is prepared: taking MS+agar 6 g / L+sucrose 30 g / L as a basic medium, the basic medium has a pH of 5.6-5.8, after sterilization in a high-pressure sterilization pot at 121 ℃ for 20 min, the ribavirin mother liquor is added when the medium is cooled to 40-60 ℃, the ribavirin concentration is 50 mg / L, the medium is condensed and divided, and the ribavirin medium is obtained and stored.

7. The method of claim 1, wherein the virus-free Dioscorea opposita Thunb. is obtained by the steps of: In the step (5), the culture condition is 20-25 ℃, the illumination is 1500-2500 lux, and the illumination time is 10-14 h / day. ​ 8. The method of claim 7, wherein the virus-free Dioscorea opposita Thunb. is obtained by the steps of: In the step (5), the culture condition is 21-23 ℃, the illumination is 2000 lux, and the illumination time is 12 h / day. ​

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