Method for obtaining virus-free seedlings by using Chinese yam stem tip protocorm-like bodies

Abstract

The invention discloses a method for obtaining virus-free seedlings by using Chinese yam stem tip protocorm-like bodies, and belongs to the technical field of Chinese yam detoxification. The method comprises the following steps: (1) determining the types of viruses carried by Chinese yam tissue culture seedlings; (2) taking a 3-5 week-old Chinese yam tissue culture seedling carrying a target virus, cutting a stem segment with a stem tip and a length of 8-12 mm under a sterile condition, stripping stem segment tissues and scale leaves on the outer layer of an axillary bud, and inoculating the axillary bud with a growth point of 1-3 mm into a protocorm-like body induction culture medium for culture; (3) after inoculating for 4-6 weeks, picking primary Chinese yam protocorm-like bodies obtained by induction in the step (2), respectively carrying out RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection on target viruses, and screening to obtain detoxified protocorm-like bodies; (4) carrying out secondary subculture multiplication on the detoxified protocorm-like body; (5) carrying out subculture multiplication on the protocorm-like body for the third time; (6) carrying out target virus RT-PCR detection on the protocorm-like bodies subjected to secondary subculture and tertiary subculture in the steps (4) and (5), and counting the detoxification rate; (7) plant regeneration and rapid propagation of the detoxified protocorm-like bodies; and (8) transplanting regenerated virus-free seedlings. According to the method, the common problems of low survival rate and low detoxification rate of the current Chinese yam stem tips are solved, and the yield can be increased on the basis of keeping the characteristics of the original variety.

Description

Technical Field

[0001] This invention belongs to the field of yam detoxification technology, and particularly relates to a method for obtaining detoxified seedlings using yam stem tip protocorms. Background Technology

[0002] Yam (Dioscorea spp.) is an annual or perennial vine belonging to the Dioscoreaceae family and the Dioscorea genus. It is highly nutritious and has medicinal value, making it a popular food crop in West Africa and a highly efficient economic crop in my country, valued for both its medicinal and culinary uses. It has become a pillar industry in various producing regions. However, because yam production relies on asexual propagation through seed planting or using tuber cuttings as seedlings, viral diseases spread with the seed tubers and accumulate year by year, becoming a major factor restricting the development of the yam industry.

[0003] Yam virus disease occurs in all growing regions of the world, but the main pathogenic viruses differ from region to region. Currently, more than 20 types of yam viruses have been reported. Among the more widespread DNA viruses are *Dioscorea bacilliform* virus (DBV), *Dioscorea nummularia* associated virus (DNUaV), and *Yam yellow spot mosaic virus* (YYSMV). RNA viruses include *Yam mosaic virus* (YMV), *Yam mild mosaic virus* (YMMV), *Japanese yam mosaic virus* (JYMV), *Chinese yam necrotic mosaic virus* (CYNMV), *Yam chlorotic necrotic mosaic virus* (YCNMV), *Yam chlorotic necrosis virus* (YCNV), *Yam virus X* (YVX), *Cucumber mosaic virus* (CMV), and *Yam virus Y* (YVX). The study identified several viruses, including YVY, yam latent virus (YLV), broad bean wilt virus-2 (BBWV-2), and yam spherical virus (YSV). Previously, the research group used small RNA sequencing and RT-PCR to conduct preliminary virus identification on 56 yam samples from germplasm across the country in a yam germplasm resource nursery jointly established by Wenxian County and their institution. They found at least eight viruses infecting yams, mostly in co-infection.

[0004] Shoot tip virus elimination is a core technology for cultivating virus-free seedlings. Once a plant virus infects a plant, it spreads throughout the plant. However, the virus content in the root tip and shoot tip is very low, and the closer to the apical meristem, the lower the virus content. When the diameter is about 0.1 mm from the apical meristem, the virus is almost non-existent. Therefore, if this part of the shoot tip is cultured to be virus-free, virus-free seedlings can be obtained. Based on this principle, shoot tip virus elimination technology has emerged, and many plants have used this method for virus elimination, such as potatoes, sweet potatoes, orchids, and purple garlic. In addition, heat treatment for virus removal has been developed based on the virus's intolerance to high temperatures (most viruses die at 50°C); cryogenic treatment for virus removal has been developed based on the fact that only shoot tip meristems can survive at extremely low temperatures; antiviral chemical agents are used to inhibit viral proliferation for virus removal; and electrotherapy is used to denature viral nucleic acids in plant tissues using electrical pulses. These virus removal techniques, combined with shoot tip removal, have led to more combined virus removal methods, all of which have achieved good results in practical applications. Examples include heat treatment + shoot tip stripping (e.g., orchids, Rehmannia glutinosa, strawberries), shoot tip stripping + chemical treatment (e.g., Phalaenopsis orchids, Cymbidium goeringii), shoot tip stripping + electrotherapy (e.g., Rehmannia glutinosa), and shoot tip stripping + cryotherapy (e.g., grapes, potatoes). However, these combined virus removal techniques require the stripping of small shoot tip tissues, resulting in a high contamination rate and low shoot tip survival rate. Therefore, finding a simple and feasible solution to address this viral problem in yams is imperative.

[0005] Patent CN201310516558 employs a shoot tip detoxification technique. The explants are field-grown *Dioscorea opposita* plants, with shoot tips of 0.1–0.3 mm taken. The differentiation and induction medium used is MS basal medium + 1.0–3.5 mg·L⁻¹. -1 6-BA + α-naphthaleneacetic acid + 0.5~3.0 mg·L -1 NAA + 0.2~2.5mg·L -1 2.4-D + 0.1~0.3mg ZT + 100~200 mg·L -1 Coconut milk + 30g·L -1 Sucrose + 6.5g·L -1Agar, tested using serological methods, did not detect potato virus X (PVX), potato virus Y (PVY), potato leafroll virus (PLRV), potato virus M (PVM), potato virus S (PVS), and potato virus A (PVA), which are highly harmful to Huilou yam. However, problems encountered with the cut stem tips included difficult handling, slow growth, and high browning rates. The culture medium composition was complex, and plant hormones such as ZT were expensive, which may hinder its widespread application in the rapid propagation of other yam varieties. Patent CN202311750770 uses heat treatment combined with stem tip and antiviral agent detoxification. The treated seedlings were tissue culture seedlings of Anshun yam, the treatment temperature was 40℃ to 50℃, the heat treatment time was 8 to 24 h, and the cut stem tips were 0.3 to 0.5 mm. The key detoxification medium was MS basal medium supplemented with 30 to 70 mg·L⁻¹. -1 Ribavirin, after culturing for 2-3 months, can produce regenerated plants with 2-3 leaves. Testing has shown that this method effectively protects Anshun yam from Japanese yam mosaic virus (JYMV), potato virus Y (PVY), potato virus X (PVX), and potato leafroll virus (PLRV); 0.3-0.5 The extraction of shoot tip tissue requires a high level of operational experience, and the detoxification effect depends on the use of detoxification chemical agents. Ribavirin, commonly known as "virazole," mainly inhibits the replication of potato virus X (PVX), potato virus Y (PVY), potato leafroll virus (PLRV), YMMV, and JYMV. Its inhibitory effect on YYSMV and YLV has not been reported, and ribavirin has a significant inhibitory effect on the growth of yam plants. Furthermore, the absence of activated carbon in the culture medium means that the browning during the formation of yam seedlings causes continuous damage to the shoot tip tissue, which is detrimental to its survival and seedling formation. In contrast, protocorms are artificial seed morphological structures formed during plant tissue culture, composed of embryogenesis and organ development. They produce less browning during the induction of protocorms and have a higher proliferation coefficient. Cheng Yuan et al. (2024) found that cutting 2 mm of shoot tips infected with yam mottle virus (YMOV) and using 60 mol·L⁻¹... -1 Treatment with morpholine guanidine hydrochloride or high-temperature treatment at 40℃ for 24 h can effectively remove YMOV while ensuring a high survival rate and growth rate; Zhang Lei et al. (2014) found that morpholine guanidine hydrochloride treatment can also be used to remove YMMV from Chinese yam seeds. The aforementioned methods for yam detoxification, besides some inherent shortcomings, share two common problems: firstly, they do not conduct virus screening on the material to be detoxified, potentially leading to missed detections and the rise of secondary viruses to primary viruses; secondly, the culture method makes it difficult to guarantee the survival rate of shoot tips, and only one regenerated seedling can be obtained from one shoot tip. Summary of the Invention

[0006] To address the aforementioned issues, this invention first conducts a virus assessment of the materials to be detoxified, identifying the target virus species to be removed and detected. This resolves the common problem of missed detection of other viruses due to subjective identification of the target virus. The invention employs a combination of 1-3 mm shoot tips and protocorms for culture. Skilled shoot tip stripping techniques ensure both the effectiveness and survival rate of shoot tip detoxification. Furthermore, the protocorms induced from a single shoot tip have a high propagation coefficient. By utilizing the liquid rapid propagation medium for detoxified test-tube seedlings previously explored by the research group, the propagation coefficient is further improved, thereby solving the common problems of low survival rate and low detoxification rate of yam shoot tips.

[0007] This invention screens PLB lines that are completely free of the three viruses mentioned above during the induction culture of primary protocorms of yam. These lines are then subjected to a second and third subculture. RT-PCR detection shows that no virus recurrence was found in the screened virus-free PLB lines. The third subculture of protocorms to a certain number is then regenerated into virus-free test-tube seedlings, which are then transplanted into greenhouses, resulting in a yield increase of more than 30% while maintaining the original varietal characteristics.

[0008] This invention provides a method for obtaining virus-free seedlings using yam stem tip protocorms, comprising the following steps: (1) Determine the types of viruses carried by yam tissue culture seedlings; (2) Take 3-5 week old yam tissue culture seedlings carrying the target virus, cut stem segments with stem tips of 8-12 mm in length under aseptic conditions, peel off the stem segment tissue and the outer scale leaves of axillary buds, and inoculate the axillary buds with 1-3 mm growth points into the protocorm induction medium and culture in the dark at 23±2℃. The protocorm induction medium in step (2) is: MS + 0.8~1.2 mg·L⁻¹ -1 TDZ +28~32 g·L -1 Sucrose + 6~8 g·L -1 Carrageenan, pH 5.8~6.2; (3) When the inoculation period is 4-6 weeks, the primary yam protocorms obtained by induction in step (2) are selected and subjected to RT-PCR detection of the target virus to screen and obtain virus-free protocorms; (4) The virus-free yam protocorm lines obtained in step (3) are longitudinally cut into small segments with a diameter of 0.4~0.6 cm and inoculated onto protocorm proliferation medium for proliferation culture; the culture conditions for rapid proliferation of protocorms are: temperature 23±2℃, humidity 50~60%, dark culture, culture time 4~6 weeks, to complete the secondary subculture proliferation of virus-free protocorms; The protocorm proliferation medium in step (4) is: MS + 8~10 mg·L -1 6-BA + 28~32 g·L -1Sucrose + 6~8 g·L -1 Carrageenan, pH 5.8~6.2; (5) The proliferating protocorms obtained in step (4) are cut longitudinally into small segments with a diameter of 0.4~0.6 cm, and then inoculated again on the proliferation medium described in step (4) for subculture proliferation culture at a temperature of 23±2℃ and in the dark for 4~6 weeks to complete the three subculture proliferation of protocorms; (6) Take the second and third subculture protocorms described in steps (4) and (5), perform RT-PCR detection of the target virus, count the detoxification rate, and detect the stability of virus removal; (7) Regeneration and rapid propagation of virus-free protocorms: Select protocorms from the three subcultures in step (5) and inoculate them on the plant regeneration medium to promote differentiation. After differentiation, the regenerated plants are transferred to the rapid propagation medium and cultured until each line has 50 to 100 seedlings. The culture conditions for the regeneration of protocorms are: temperature 23±2℃, humidity 50 to 60%, light 1200 lx, and culture time 45 to 60 days. The plant regeneration medium in step (7) is: MS + 1~3 mg·L -1 KT+ 0.01~0.03 mg·L -1 NAA+ 28~32 g·L -1 Sucrose + 6~8 g·L -1 Carrageenan, pH 5.8~6.2; The rapid propagation culture medium in step (7) is: MS + 1~3 mg·L -1 KT + 0.01~0.03 mg·L -1 NAA + 0.008~0.012 mg·L -1 PP 333 + 28~32 g·L -1 Sucrose liquid culture medium.

[0009] In one embodiment of the present invention, the target virus is at least one of JYMV, YYSMV, YLV, YMMV, and YVX.

[0010] In one embodiment of the present invention, the protocorm induction medium in step (2) is: MS + 1 mg·L - 1 TDZ +30 g·L -1 Sucrose + 7 g·L -1 Carrageenan, pH 5.8~6.2.

[0011] In one embodiment of the present invention, the axillary buds in step (2) are cultured for 5 weeks after being inoculated into the protocorm induction medium.

[0012] In one embodiment of the present invention, the protocorm proliferation medium in step (4) is: MS + 9 mg·L - 1 6-BA + 30 g·L -1 Sucrose + 7 g·L -1 Carrageenan, pH 5.8~6.2.

[0013] In one embodiment of the present invention, step (5) involves dark culture for 5 weeks.

[0014] In one embodiment of the present invention, the plant regeneration culture medium in step (7) is: MS + 2 mg·L⁻¹ -1 KT+0.02 mg·L -1 NAA + 30 g·L -1 Sucrose + 7 g·L -1 Carrageenan, pH 5.8~6.2.

[0015] In one embodiment of the present invention, the rapid propagation culture medium in step (7) is: MS + 2 mg·L⁻¹ -1 KT + 0.02 mg·L -1 NAA + 0.01 mg·L -1 PP 333 + 30 g·L -1 Sucrose liquid culture medium.

[0016] In one embodiment of the present invention, step (7) is followed by step (8) to transplant the virus-free regenerated seedlings obtained in step (7).

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a technique for inducing the formation of virus-free seedlings from yam stem tip tissue into protocorms, which can solve the common problems of low survival rate and low virus-free rate of yam stem tips, and can increase yield while maintaining the original varietal characteristics. Attached Figure Description

[0018] Figure 1 The results show the detection of virus species in the test-tube seedlings of Dioscorea opposita in Example 1.

[0019] Figure 2The following are examples of virus-free yam plants induced and cultured using protocormoids from shoot tips in Example 1: (A) *Dioscorea opposita* test-tube seedlings; (B) shoot tip explants; (C) protocormoids formed from shoot tips after 4-6 weeks; (D) subculture propagation of protocormoids; (E) regeneration of protocormoid plants; (F) 4-week-old regenerated test-tube seedlings; (G) transfer of in vitro seedlings to substrate; (H) seedlings collected 3 months after transplanting. The scale bar for (A) and (C-H) is 1 cm, and the scale bar for (B) is 1 mm.

[0020] Figure 3 The JYMV, YYSMV, and YLV amplification products in PLBs of the S1-S3 generations induced by RT-PCR agarose gel electrophoresis in Example 1 are shown. Among them, 4-1, 4-2, 4-3, 20-1, 20-2, and 20-3 refer to different clones of subculture 4# and 20# in S1, respectively.

[0021] Figure 4 The following is a statistical analysis of the detoxification rate of PLBs induced by the shoot tips of Dioscorea opposita in Example 1 and an analysis of the growth indicators of the detoxified seedlings, including (A) the detoxification rate of the first generation PLBs; (B) the propagation coefficient; (C) the chlorophyll content; (D) the leaf size; (E) the planting length; and (F) the planting weight. Detailed Implementation

[0022] Example 1 This embodiment provides a method for obtaining virus-free yam seedlings through protocorm induction, including the following steps: (1) To investigate the viruses carried by tissue culture seedlings, randomly select test tube seedlings of Chinese yam to be detoxified and conduct RT-PCR detection of five common viruses in the field and greenhouse to confirm the types of viruses carried by the yam to be detoxified, and use them as the basis for detecting whether the regenerated seedlings of stem tip peeling combined with protocorm culture are detoxified. (2) Stem tip peeling and protocorm induction of test-tube seedlings: Take 3-5 week old yam tissue culture seedlings carrying the target virus, cut a stem segment about 10 mm long with the stem tip on a clean bench, peel off the stem segment tissue and the outer scale leaves of the axillary buds, and quickly inoculate the axillary buds with 1-3 mm growth points into the protocorm induction medium (i.e., PLB induction medium), and culture in the dark at 23±2℃. When peeling the axillary buds, sterile scalpels and forceps need to be replaced when peeling off the segment tissue and the outer young leaves of the axillary buds; the yam protocorm induction medium in step (2) is: MS + 1 mg·L -1 TDZ +30 g·L -1 Sucrose + 7 g·L -1 Carrageenan, pH 5.8~6.2; (3) Screening of virus-free protocorms: 4-6 weeks after inoculation, select the first generation of yam protocorms obtained by induction in step (2), and extract RNA from the tissues of each line number after cutting. After reverse transcription, RT-PCR detection of the target virus is performed. (4) Secondary subculturing of detoxified protocorms: The detoxified yam protocorms obtained in step (3) are longitudinally cut into small segments with a diameter of about 0.5 cm and inoculated onto protocorm proliferation medium (i.e., PLB proliferation medium) for proliferation culture; The culture conditions for rapid proliferation of protocorms are: temperature 23±2℃, humidity 50~60%, dark culture, culture time 4~6 weeks; The proliferation medium in step (4) is: MS + 9 mg·L -1 6-BA + 30 g·L -1 Sucrose + 7 g·L -1 Carrageenan, pH 5.8~6.2; (5) Three subcultures of protocorms: The proliferating protocorms obtained in step (4) above are cut longitudinally into small segments with a diameter of about 0.5 cm, and then inoculated again on the proliferation medium described in step (4) for subculture. The culture is carried out in a dark culture room at a temperature of 23±2℃ for 4~6 weeks. (6) Virus detection of yam protocorms from the second and third subcultures: Take the second and third subculture protocorms from steps (4) and (5) and perform RT-PCR detection of the target virus, count the detoxification rate, and detect the stability of virus removal. (7) Regeneration and rapid propagation of virus-free protocorms: Select protocorms from the three subcultures in step (5) and inoculate them onto the plant regeneration medium to promote differentiation. After differentiation, the regenerated plants are transferred to the rapid propagation medium and cultured until each line has 50-100 seedlings. The culture conditions for the regeneration of protocorms are: temperature 23±2℃, humidity 50-60%, light 1200 lx, and culture time 45-60 days. The regeneration medium for yam protocorms in step (7) is: MS + 2 mg·L⁻¹. -1 KT (furanomethylaminopurine) + 0.02 mg·L -1 NAA + 30 g·L -1 Sucrose + 7 g·L -1 Carrageenan, pH 5.8-6.2; the rapid propagation medium for virus-free yam seedlings in step (7) is: MS + 2 mg·L⁻¹ -1 KT + 0.02 mg·L -1 NAA + 0.01 mg·L -1 PP 333 (Paclobutrazol) + 30 g·L -1 Sucrose liquid culture medium; (8) Transplanting of virus-free regenerated seedlings: Take about 4-week-old virus-free plants cultured in the above-mentioned rapid propagation culture base, open the sealing film and move them to natural light. After 3-5 days, rinse the culture medium from the roots of the seedlings with tap water and transplant them into nutrient pots containing nutrient soil: vermiculite: perlite = 3:1:1 (volume ratio) and cultivate them in an artificial climate culture room. The culture conditions are: temperature 23±2℃, humidity 70~85%, light 2000 lx, and light duration 16 h / d.

[0023] result: 1. Identification of virus species in tissue culture seedlings of Dioscorea opposita To detect the types of pathogenic viruses infecting *Dioscorea opposita* tissue culture seedlings, leaves were randomly collected from the seedlings for testing for five common yam viruses (primers are shown in Table 1). The results showed that three viruses, JYMV, YYSMV, and YLV, were present in the *Dioscorea opposita* tissue culture seedling samples using RT-PCR. Figure 1 ).

[0024] Table 1. Primer information used for RT-PCR detection of virus in Huaihe River yam.

[0025] Note: JYMV, Japanese yam mosaic virus; YYSMV, yam yellow spot mosaic virus; YLV, yam latent virus; YMMV, yam mild mosaic virus; YVX, yam X virus.

[0026] 2. Virus-free seedlings obtained from the protocorm-like cultivation of the stem tips of Chinese yam. 2.1 Shoot tip stripping to induce protocorm-like formation and screening to obtain virus-free protocorm-like formations The source plants for providing shoot tip explants were tissue culture seedlings of Dioscorea opposita co-infected with the three viruses JYMV, YYSMV and YLV mentioned above. Figure 2 A). Thirty axillary buds separated from the source plant ( Figure 2 In B), 20 of them successfully formed primary protocorms (PLBs) after being cultured in PLB induction medium for 28-42 days. Figure 2 (C) The remaining explanted tissues turned brown, necrotized, failed to grow, and subsequently died within 7–14 days. The surviving protocorms were isolated individually and transferred to subculture media for further proliferation.

[0027] A total of 20 shoot tip amplified tissues / PLBs were obtained and isolated and cultured into independent plant lines. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect JYMV, YYSMV, and YLV viruses. Of the 20 PLB lines in the initial culture, 2 lines were negative for all three viruses by RT-PCR, 7 lines were free of two viruses, and 11 lines were free of only one virus. The devirulence rates for freeing all three viruses, freeing two viruses, and freeing only one virus were 10%, 35%, and 55%, respectively (Table 2). Gel electrophoresis showed that one or two amplicones representing JYMV, YYSMV, and YLV infection (603 bp, 446 bp, and 547 bp) were present in the 20 S1 generation PLB lines obtained by induction. No PLB lines contained amplicones representing all three viruses. Figure 3 The detoxification rates of JYMV, YYSMV, and YLV were 45%, 15%, and 85%, respectively. Figure 4 A). Discard PLB strains infected with one or two viruses as indicated by S1 test results.

[0028] Table 2. Results of virus detection using RT-PCR after protocorm culture induced by stem tip explants from Dioscorea opposita.

[0029] Note: S1~S3 represent the first to third generation protocorms.

[0030] 2.2 Proliferation of virus-free protocorms and virus identification The virus-free PLB lines PLB4# and PLB20# were further propagated on PLB proliferation medium. Figure 2 D). RT-PCR gel analysis showed that no viral amplicon was detected in any of the proliferating clones PLB4# and PLB20# that had reached the 2nd and 3rd generations, and the viral recurrence rate was 0%, indicating that the three viruses had been completely eliminated (Table 2). Figure 3 ).

[0031] 2.3 Regeneration, transplanting, and acquisition of virus-free seedlings These virus-free PLB lines were cultured into buds ( Figure 2 E), induced rooting and development into small plantlets in the same liquid culture medium ( Figure 2 F). From the time PLB was transferred to the regeneration medium, the time to obtain regenerated seedlings was between 50 and 80 days. The propagation coefficients of the in vitro seedlings during the 30-day culture period were calculated. The propagation coefficients of virus-free PLB4# and PLB20# in vitro seedlings were 5.90 and 5.47, respectively, significantly higher than those of the non-virus-free in vitro seedlings (CK). Figure 4 B).

[0032] The detached plantlets with well-developed leaves and roots were transferred to plastic pots filled with substrate. Figure 2 G), showing a near 100% survival rate. RT-PCR testing was performed on 50 regenerated plantlets, and all plants were found to be free of the three viruses (Table 2). Statistical analysis of chlorophyll content and leaf size in 30-day-old potted seedlings showed that the chlorophyll content of virus-free PLB4# and PLB20# potted seedlings was slightly higher than the control group, although the results were not statistically significant. However, the leaves of the non-virus-free control group (CK, i.e., plants obtained by directly planting yam stem segments) clearly showed mottled patterns and uneven leaf surfaces, and the leaf area of ​​the virus-free protocorm seedlings was significantly or extremely significantly higher than that of the control group (CK). Figure 4 (C and 4D) Overall, the growth status of the virus-free protocorm seedlings was higher than that of the CK control group.

[0033] Approximately 60 days later, the seedlings in pots were collected. The length and weight of the virus-free PLB4# and PLB20# potted seedlings were significantly higher than those of the virus-free control group (CK). The seedling length of PLB4# and PLB20# potted seedlings was 2.18 and 2.19 times that of the control group (CK), respectively, and the seedling weight was 2.04 and 1.77 times that of the control group (CK), respectively. Figure 4 E and 4F).

[0034] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for obtaining virus-free seedlings using stem tip protocorm-like bodies of Dioscorea japonica, characterized by, The method comprises the following steps: (1) determining the types of viruses carried by the yam tissue culture seedlings; (2) taking 3-5-week-old yam tissue culture seedlings carrying target viruses, cutting stem segments with stem tips of 8-12 mm in length under sterile conditions, peeling the outer scale leaves of the stem segment tissues and axillary buds, inoculating the axillary buds with 1-3 mm of growth points retained into protocorm-like body induction medium, and performing dark culture at 23±2℃; The protocorm-like body induction medium in the step (2) is: MS+0.8~1.2 mg·L -1 TDZ +28~32 g·L -1 Sucrose +6~8 g·L -1 Carrageenan, pH is 5.8~6.2; (3) picking the primary yam protocorm-like bodies obtained through step (2) when inoculation is performed for 4-6 weeks, and performing RT-PCR detection of the target viruses to obtain detoxified protocorm-like bodies; (4) cutting the detoxified yam protocorm-like bodies obtained through step (3) into small segments with a diameter of 0.4-0.6 cm in a longitudinal direction, and inoculating the small segments into protocorm-like body proliferation medium to perform proliferation culture; the culture conditions for rapid proliferation of the protocorm-like bodies are as follows: a temperature of 23±2℃, a humidity of 50-60%, dark culture, a culture time of 4-6 weeks, and completion of secondary subculture and proliferation of the detoxified protocorm-like bodies; The protocorm proliferation medium in the step (4) is: MS+8~10 mg·L -1 6-BA+28~32 g·L -1 Sucrose+6~8 g·L -1 Carrageenan, pH 5.8~6.2; (5) cutting the protocorm-like bodies obtained through step (4) into small segments with a diameter of 0.4-0.6 cm in a longitudinal direction again, inoculating the small segments into the proliferation medium in step (4) again to perform subculture and proliferation culture, and performing dark culture at a temperature of 23±2℃ for 4-6 weeks to complete tertiary subculture and proliferation of the protocorm-like bodies; (6) taking the secondary subculture and tertiary subculture protocorm-like bodies in steps (4) and (5), performing target virus RT-PCR detection, counting the detoxification rate, and detecting the stability of virus removal; (7) plant regeneration and rapid propagation of the detoxified protocorm-like bodies, picking the tertiary subculture protocorm-like bodies in step (5) and inoculating the protocorm-like bodies into plant regeneration medium to promote differentiation, and transferring the differentiated regenerated plants into rapid propagation medium to culture until 50-100 seedlings are obtained in each strain; the culture conditions for protocorm-like body plant regeneration are as follows: a temperature of 23±2℃, a humidity of 50-60%, illumination of 1200 lx, and a culture time of 45-60 days; The plant regeneration medium in the step (7) is: MS+1~3 mg·L -1 KT+ 0.01~0.03 mg·L -1 NAA+28~32 g·L -1 Sucrose+6~8 g·L -1 Carrageenan, pH is 5.8~6.2; The rapid propagation culture medium in the step (7) is: MS+1~3 mg·L -1 KT + 0.01~0.03 mg·L -1 NAA+0.008~0.012 mg·L -1 PP 333 + 28~32 g·L -1 Sucrose liquid culture medium.

2. The method of claim 1, wherein, The target virus is at least one of JYMV, YYSMV, YLV, YMMV and YVX.

3. The method of claim 2, wherein, The protocorm-like body induction medium in the step (2) is: MS+1 mg·L -1 TDZ +30 g·L -1 Sucrose+7 g·L -1 Carrageenan, pH 5.8~6.

2.

4. The method of claim 3, wherein, In step (2), the culture time after the axillary buds are inoculated into the protocorm-like body induction medium is 5 weeks.

5. The method of claim 4, wherein, The protocorm proliferation medium in the step (4) is: MS+9 mg·L -1 6-BA+30 g·L -1 Sucrose+7 g·L -1 Carrageenan, pH is 5.8~6.

2.

6. The method of claim 5, wherein, In step (5), the dark culture is performed for 5 weeks.

7. The method of claim 6, wherein, The plant regeneration medium in step (7) is: MS+2 mg·L -1 KT+ 0.02 mg·L -1 NAA+30 g·L -1 Sucrose+7 g·L -1 Carrageenan, pH 5.8~6.

2.

8. The method of claim 7, wherein, The rapid propagation culture medium in the step (7) is: MS+2 mg·L -1 KT + 0.02 mg·L -1 NAA+0.01 mg·L -1 PP 333 + 30 g·L -1 Sucrose liquid culture medium.

9. The method of claim 8, wherein, After step (7), step (8) of transplanting the detoxified regenerated seedlings obtained through step (7) is further included.

Images