Multipurpose lily detoxification method and application thereof

Abstract

The invention discloses a multi-purpose lily detoxification method and application thereof, and the method comprises the following steps: (1) establishing an aseptic seedling system: selecting third-layer scales from outside to inside of lily bulbs' Flying Shaohua 'and' Flying Tianorange 'as explants, disinfecting the explants, and carrying out tissue culture to establish the aseptic seedling system; (2) virus detection and detoxification: taking leaves from aseptic seedlings to detect viruses, and performing detoxification treatment on positive samples; the detoxification treatment method comprises at least one of induced callus detoxification, stem tip combined heat treatment detoxification, stem tip combined virazole detoxification, stem tip combined virazole and heat treatment detoxification. According to the method disclosed by the invention, a high-efficiency virus detoxification technology is established specially aiming at the problem that novel multi-purpose lily varieties' Fangzhou orange 'and' Fangzhou Shaohua 'which have comprehensive characters and are popular with the market are easily infected with CMV and LSV, and a technical support is provided for large-scale production of the two novel multi-purpose lily detoxification seedballs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant detoxification, and relates to a multi-purpose lily detoxification method and application thereof, in particular to a detoxification method of two new varieties of multi-purpose lilies 'Feixiang Shaohua' and 'Feixiang Tiancheng' which are independently bred by a lily research team of Nanjing Agricultural University, have excellent comprehensive traits, are popular in the market, and have multiple purposes such as ornamental, edible and tea use. BACKGROUND

[0002] Lily (Lilium spp.) is an important ornamental flower and economic crop, which occupies an important position in the global flower market due to its rich flower colors, elegant morphology and cultural connotation symbolizing purity, and is one of the world's five famous fresh-cut flowers. Because lily often adopts asexual reproduction methods such as bulb and bud, it is easy to be infected by viruses, resulting in phenomena such as bulb degeneration, growth weakening, flower malformation and yield reduction. Virus disease is different from fungal and bacterial diseases, and cannot be prevented by fungicides or antibiotics. At present, there is no cure in production, and the application of biological technology for detoxification is the most simple and effective way. Multi-purpose lily has high ornamental, edible and drinking values, and there are few reports on the detoxification of multi-purpose lily at present. Meanwhile, different lily varieties have different genotypes, and their detoxification methods and efficiency also have great differences. Like other lily varieties, the new varieties 'Feixiang Shaohua' and 'Feixiang Tiancheng' are easy to be infected by viruses, and cucumber mosaic virus (CMV) and lily symptomless virus (LSV) are the main viruses infecting the two new varieties. Therefore, cultivating lily bulbs without CMV and LSV infection is an important prerequisite for large-scale industrialization of the two new varieties. Therefore, the present application takes 'Feixiang Shaohua' and 'Feixiang Tiancheng' as research materials, compares a plurality of detoxification treatment methods, and obtains a high-efficiency detoxification method for the two new lily varieties, which has important significance for cultivating virus-free lily bulbs and promoting industrialization of new varieties. SUMMARY

[0003] The purpose of the present application is to establish a high-efficiency detoxification technology for the new lily varieties 'Feixiang Shaohua' and 'Feixiang Tiancheng', and to provide scientific and technological support for large-scale industrialization thereof.

[0004] The technical solution adopted by the present application to solve the technical problems is as follows:

[0005] A multi-purpose lily detoxification method, which comprises the following steps:

[0006] (1) Establishing a sterile seedling system: selecting 'Feixiang Shaohua' and 'Feixiang Tiancheng' lily bulbs to take the third layer of scales from the outside as explants for disinfection and then used for tissue culture to establish a sterile seedling system;

[0007] (2) Virus detection and detoxification: leaf samples are taken from the virus-free seedlings for virus detection, and positive samples are subjected to detoxification treatment; the detoxification treatment method is at least one of induced callus detoxification, stem tip combined with heat treatment detoxification, stem tip combined with virus zide detoxification, and stem tip combined with virus zide and heat treatment detoxification.

[0008] Further: the detoxification treatment method is preferably at least one of induced callus detoxification, stem tip combined with heat treatment detoxification, and stem tip combined with virus zide and heat treatment detoxification.

[0009] Further: the virus removed in the detoxification treatment is cucumber mosaic virus (CMV) or / and lily symptomless virus (LSV).

[0010] Further: when the virus is cucumber mosaic virus (CMV), the detoxification treatment method is induced callus detoxification.

[0011] Further: when the virus is lily symptomless virus (LSV), the detoxification treatment method is at least one of induced callus detoxification, stem tip combined with heat treatment detoxification, and stem tip combined with virus zide and heat treatment detoxification.

[0012] Further: the concentration of virus zide in the stem tip combined with virus zide detoxification or the stem tip combined with virus zide and heat treatment detoxification is 1-20 mg / L; preferably 10-20 mg / L; further preferably 15-20 mg / L; most preferably 20 mg / L.

[0013] Further: the culture conditions of the virus-free seedlings are an average temperature of (23 ± 2) ℃, a light intensity of 2000 Lx, a light cycle of 12 h light / 12 h darkness, and a culture time of 40-50 d.

[0014] Further: in the detoxification treatment of induced callus detoxification, the culture medium for inducing callus is MS + 1.0 mg / L TDZ + 2.0 mg / L PIC + 15 g / L sucrose + 7 g / L agar.

[0015] Further: in the detoxification treatment of stem tip combined with heat treatment detoxification or stem tip combined with virus zide and heat treatment detoxification, the heat treatment conditions are constant temperature treatment in a 30℃ light incubator for 7 d, then the temperature is adjusted to 40℃ light for 14 h / 28℃ darkness for 10 h, and the treatment is performed for 30 d.

[0016] Further preferably:

[0017] Induce callus detoxification: Peel off small scales of virus-infected lily sterile tissue culture seedlings and place them in callus induction medium (MS + 1.0 mg / L TDZ + 2.0 mg / L PIC + 15 g / L sucrose + 7 g / L agar) for culture. After successful induction of callus tissue, culture it for another 15-25 days and transfer the successfully induced buds to the medium of MS + 0.5 mg / L 6-BA +0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar.

[0018] Shoot tip detoxification combined with heat treatment: Sterile lily tissue culture seedlings were placed in a 30°C light incubator for 7 days. The temperature was then adjusted to 40°C for 14 hours of light and 28°C for 10 hours of darkness for 30 days. After heat treatment, 0.3-0.8 mm stem tips were cut from tissue culture seedlings in good condition and inoculated into a medium containing MS, 0.5 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sucrose, and 7 g / L agar for stem tip culture.

[0019] Detoxification of stem tip combined with ribavirin: 0.3-0.8 mm stem tips of the obtained lily sterile tissue culture seedlings were cut and inoculated into a culture medium containing 1-20 mg / L ribavirin in MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar for cultivation.

[0020] Shoot tip binding with ribavirin and heat treatment for detoxification: First, sterile tissue culture seedlings of lily were heat-treated and placed in a 30°C light incubator for 7 days. The temperature was then adjusted to 40°C for 14 hours of light and 28°C for 10 hours of darkness for 30 days. After heat treatment, 0.3-0.8 mm stem tips of tissue culture seedlings in good condition were excised and inoculated into a medium containing 1-20 mg / L ribavirin in MS, 0.5 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sucrose, and 7 g / L agar.

[0021] The screening process of the multi-purpose lily detoxification method of the present invention comprises the following steps:

[0022] (1) Establish a sterile seedling system

[0023] Strong, plump bulbs of the new multipurpose lily cultivars 'Fei Xiang Shao Hua' and 'Fei Xiang Tian Cheng', both infected with cucumber mosaic virus (CMV) and lily symptomless virus (LSV), were selected. The roots were removed, and damaged outer scales were peeled. The third innermost scale from the outside, which was well-hydrated and plump, was used as an explant. After disinfection, plant tissue culture was performed to establish a sterile seedling system. Sterile seedlings were cultured under an average temperature of (23 ± 2)°C, a light intensity of 2000 lux, and a photoperiod of 12 h light / 12 h dark for 40-50 days.

[0024] (2) Virus testing of sterile vaccines

[0025] When the leaves of the sterile tissue culture seedlings of lily to be obtained grow to 8-10 cm, the leaves are taken and tested for viruses. The tissue culture seedlings that are confirmed to still contain CMV and LSV viruses in the test results are detoxified.

[0026] (3) Detoxification

[0027] The virus-infected tissue culture seedlings of 'Fei Xiang Shao Hua' and 'Fei Xiang Tian Cheng' were detoxified by induced callus tissue, detoxified by shoot tip combined with heat treatment, detoxified by shoot tip combined with ribavirin, and detoxified by shoot tip combined with ribavirin and heat treatment.

[0028] To induce callus detoxification: Small scales from virus-infected tissue culture seedlings were removed and cultured in a callus induction medium consisting of MS + 1.0 mg / L TDZ + 2.0 mg / L PIC + 15 g / L sucrose + 7 g / L agar. After successful callus induction, culture for approximately 20 days (15-25 days). Transfer the successfully induced shoots to a medium consisting of MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar. Once seedlings have established, the virus-free rate was determined.

[0029] Shoot tip detoxification combined with heat treatment: Sterile lily tissue culture seedlings were placed in a 30°C light incubator for 7 days. The temperature was then adjusted to 40°C for 14 hours of light and 28°C for 10 hours of darkness for 30 days. After heat treatment, 0.3-0.8 mm stem tips were cut from tissue culture seedlings in good condition and inoculated into a medium containing MS, 0.5 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sucrose, and 7 g / L agar. After the seedlings emerged, the detoxification rate was calculated.

[0030] Detoxification of stem tip combined with ribavirin: 0.3-0.8 mm stem tips were cut from the sterile tissue culture seedlings of lily, and inoculated into the culture medium containing MS + 0.5 mg / L 6-BA +0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar with concentrations of 0 mg / L, 10 mg / L, 15 mg / L, and 20 mg / L ribavirin, respectively. After the seedlings were formed, the detoxification rate was calculated.

[0031] Detoxification of stem tips by combining ribavirin with heat treatment: Sterile tissue culture seedlings of lily were first heat-treated and placed in a 30°C light incubator for 7 days. The temperature was then adjusted to 40°C for 14 hours of light and 28°C for 10 hours of darkness for 30 days. After heat treatment, 0.3-0.8 mm stem tips of tissue culture seedlings in good condition were selected and inoculated into ribavirin-containing MS (0 mg / L, 10 mg / L, 15 mg / L, or 20 mg / L) medium, supplemented with 0.5 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sucrose, and 7 g / L agar. After seedlings emerged, the detoxification rate was calculated.

[0032] (4) Data analysis and comparison to obtain the optimal detoxification solution

[0033] After detoxification using the above methods, 30 lily tissue culture seedlings from each treatment that had successfully induced regeneration were randomly selected. Leaves from these seedlings were used as material for virus testing, and the detoxification rate was calculated. Lily seedlings that showed no positive bands for either CMV or LSV were considered detoxified. By comparing and analyzing the detoxification effects of each detoxification treatment, a detoxification method optimal for multi-purpose lilies was developed.

[0034] In a second aspect, the present invention seeks to protect the application of the above method in detoxification of multi-purpose lilies.

[0035] Beneficial effects of the present invention:

[0036] This invention establishes a virus-free rapid propagation technology system for two new multi-purpose lily varieties: Four methods (including 10 detoxification treatments) were used for detoxification of the two new multi-purpose lily varieties, 'Fei Xiang Shao Hua' and 'Fei Xiang Tian Cheng'. These methods included callus induction, shoot apex heat treatment, shoot apex ribavirin, and shoot apex ribavirin combined with heat treatment. The best overall detoxification treatment for both lilies was callus induction, with total detoxification rates reaching 55.56% and 51.11%, respectively. CMV detoxification rates reached 84.45% and 78.89%, respectively, and LSV detoxification rates reached 66.67% and 63.33%, respectively. Furthermore, the best treatment for LSV detoxification was ribavirin combined with shoot apex and heat treatment at a concentration of 20 mg / L, achieving detoxification rates of 71.67% and 83.33%, respectively. The present invention establishes a virus-free and rapid propagation technology system for multi-purpose lilies, which has the characteristics of strong specificity, simple operation, low cost, etc., and lays a good foundation for the industrial development of these two new lily varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Comparison of the virus-free rates of 'Fei Xiang Shao Hua' under 10 treatments.

[0038] 1: callus induction treatment; 2: shoot apex-incorporated heat treatment; 3-6: shoot apex-incorporated ribavirin treatments, with ribavirin concentrations of 0 mg / L, 10 mg / L, 15 mg / L, and 20 mg / L, respectively; 7-10: shoot apex-incorporated ribavirin and heat treatments, with ribavirin concentrations of 0 mg / L, 10 mg / L, 15 mg / L, and 20 mg / L, respectively. Different letters indicate significant differences (Student-Newman-Keuls test, P < 0.05).

[0039] Figure 2 Comparison of the virus-free rates of 'Feixiang Tiancheng' under 10 treatments.

[0040] 1: callus induction treatment; 2: shoot apex-incorporated heat treatment; 3-6: shoot apex-incorporated ribavirin treatments, with ribavirin concentrations of 0 mg / L, 10 mg / L, 15 mg / L, and 20 mg / L, respectively; 7-10: shoot apex-incorporated ribavirin and heat treatments, with ribavirin concentrations of 0 mg / L, 10 mg / L, 15 mg / L, and 20 mg / L, respectively. Different letters indicate significant differences (Student-Newman-Keuls test, P < 0.05). DETAILED DESCRIPTION

[0041] Below in conjunction with the figures and examples, the specific embodiments of the present invention are further described in detail. The following examples are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the reagents and equipment used in the following examples are all commercially available or commonly used in the art, and the methods used in the following examples are all commonly used methods in the art.

[0042] Example 1

[0043] (1) Establish a sterile seedling system

[0044] Select the strong, plump and spot-less bulbs of 'Feixiang Shaohua' and 'Feixiang Tiancheng' that are confirmed to be infected with both CMV and LSV. Remove the roots and peel off the damaged scales on the outer layer. Use the third layer of bulbs from the outside to the inside that are well-hydrated and have no obvious diseases as explants for plant tissue culture to establish a sterile seedling system.

[0045] The inner healthy scales of the peeled lily were disinfected as explants. First, the dirt on the surface of the scales was washed off with clean water, and then they were placed under running water for 1.5-2 hours, dried, placed in a clean conical flask and transferred to a clean workbench. They were soaked and shaken for 1 minute in 75% ethanol solution for disinfection, rinsed twice with sterile water, and then soaked in 1% sodium hypochlorite solution for disinfection for 20 minutes. They were soaked and rinsed 3-5 times with sterile water, placed on sterile filter paper to dry, and the disinfected lily scales were cut into small squares of 1 cm × 1 cm. They were inoculated into the induction differentiation medium with the concave side facing up. About 3 pieces were placed in each culture dish. After inoculation, they were cultured upside down in the tissue culture room. The setting conditions were an average temperature of (23 ± 2) °C, a light intensity of 2000 Lx, and a photoperiod of 12 h light / 12 h dark. When the induced adventitious buds grow to 1-2.5 cm, select the vigorously growing sterile tissue culture seedlings, cut their adventitious buds from the scales to form single buds, and transfer them to the subculture proliferation medium. After culturing for 50 days, the uninfected tissue culture seedlings are used for subsequent processing.

[0046] Differentiation induction medium: 'Fei Xiang Shao Hua': MS + 2.0 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 7 g / L agar; 'Fei Xiang Tian Cheng': MS + 0.5 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 7 g / L agar. Subculture proliferation medium: Both consisted of MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar.

[0047] (2) Virus testing of sterile vaccines

[0048] When the leaves of the aseptic seedlings obtained in step (1) grow to 8-10 cm, the leaves are taken, and virus detection is performed on the leaves; the aseptic seedlings still containing CMV and LSV in the detection results are subjected to detoxification treatment.

[0049] (3) Detoxification treatment

[0050] ① Inducing callus detoxification

[0051] The obtained lily aseptic seedlings are subjected to callus induction detoxification; small scales of the aseptic seedlings are peeled and placed in a culture medium for callus induction, and the culture medium is MS + 1.0 mg / L TDZ + 2.0 mg / L PIC + 15 g / L sucrose + 7 g / L agar. After successful induction of callus, the successfully induced callus is transferred to a culture medium of MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar after about 20 days of culture, and part of the buds successfully induced are transferred to the culture medium. When the seedlings grow to 8-10 cm, 30 randomly selected seedlings are subjected to virus detection, and the detoxification rate is calculated by repeating the detection for 3 times.

[0052] Callus induction rate (%) = (number of explants inducing callus / total number of inoculated explants) x 100.

[0053] Browning rate (%) = (number of browning callus / total number of induced callus) x 100.

[0054] Detoxification rate (%) = (number of virus-free plants detected / total number of detected plants) x 100.

[0055] ② Stem tip combined with heat treatment detoxification

[0056] The obtained lily aseptic seedlings are subjected to heat treatment, and the aseptic seedlings are placed in a 30℃ light incubator for constant temperature treatment for 7 days, and then the temperature is adjusted to 40℃ light for 14 hours and 28℃ dark for 10 hours, and the treatment is performed for 30 days. After the heat treatment, 30 surviving and better state aseptic seedlings are selected, and 0.3-0.8 mm stem tips are cut and inoculated into a culture medium of MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar for culture, and the survival rate of the stem tips is calculated. After the stem tips induce seedlings and successfully grow leaves, 20 randomly selected seedlings are subjected to virus detection, and the detoxification rate is calculated by repeating the detection for 3 times.

[0057] Stem tip survival rate (%) = (number of surviving stem tip plants / total number of inoculated stem tips) x 100.

[0058] ③ Stem tip combined with virus zole treatment detoxification

[0059] Thirty lily seedlings were sterilely cultured and then treated with stem apex treatment. 0.3-0.8 mm of their stem tips were excised and inoculated with 0 mg / L, 10 mg / L, 15 mg / L, or 20 mg / L of ribavirin in MS, 0.5 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sucrose, and 7 g / L agar, respectively. The survival rates of the stem apex were then calculated. When the leaves of the induced shoot apex seedlings reached 8-10 cm, 20 plants were randomly selected for virus testing, repeated three times, and the virus-free rates were calculated.

[0060] ④Stem tip combined with ribavirin and heat treatment for detoxification

[0061] Sterile lily tissue culture seedlings were first heat-treated and placed in a 30°C light incubator for 7 days. The temperature was then adjusted to 40°C with 14 h of light and 28°C with 10 h of darkness for 30 days. After the heat treatment, 0.3-0.8 mm shoot tips were excised from 30 surviving and well-maintained tissue culture seedlings. These shoot tips were then inoculated into a culture medium containing 0 mg / L, 10 mg / L, 15 mg / L, or 20 mg / L of ribavirin in MS, 0.5 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sucrose, and 7 g / L agar. The survival rate of the shoot tips was then calculated. After the shoot tips had established themselves as seedlings and successfully grown leaves, 20 randomly selected plants were tested for virus infection three times, and the virus-free efficiency was calculated.

[0062] (4) Comparison and analysis of detoxification effects among different detoxification treatment methods

[0063] After virus-free treatment using each of the above treatment methods, 30 lily tissue culture seedlings from each treatment that had successfully induced regeneration were randomly selected. Leaves were collected and total RNA was extracted for virus testing. The virus-free rates were calculated. Lily seedlings were considered virus-free if no positive bands were detected for either CMV or LSV. The statistical results were analyzed by variance analysis using SPSS software to determine the most suitable virus-free methods for these two versatile lilies.

[0064] Result Analysis

[0065] (1) Detoxification effect of induced callus treatment

[0066] Table 1 Virus-free effect of callus induced by 'Fei Xiang Shao Hua'

[0067]

[0068] Table 2 Virus-free effect of callus induction in 'Feixiang Tiancheng'

[0069]

[0070] Virus testing was performed on tissue culture seedlings obtained from callus induction in 'Fei Xiang Shao Hua' and 'Fei Xiang Tian Cheng'. The virus-free rates for CMV, LSV, and overall are shown in Tables 1 and 2. The average virus-free rates for CMV and LSV in 'Fei Xiang Shao Hua' were 84.45%, 66.67%, and 55.56% respectively, while the average virus-free rates for CMV and LSV in 'Fei Xiang Tian Cheng' were 78.89%, 63.33%, and 51.11% respectively. Overall, the CMV virus-free rates for both lilies after callus induction were very high. While the virus-free rate for LSV was slightly lower, it still exceeded 60%. The overall virus-free rates for both lilies reached over 50%, meeting the requirements for virus-free lily bulb propagation. It can be seen that inducing callus tissue can effectively remove the two major harmful viruses in 'Fei Xiang Shao Hua' and 'Fei Xiang Tian Cheng', which is an effective detoxification method.

[0071] (2) Detoxification effect of stem tip combined with heat treatment

[0072] Table 3 Effects of heat treatment on shoot tip survival rate

[0073]

[0074] Note: Different letters indicate significant differences (Student-Newman-Keuls test, P < 0.05).

[0075] Table 3 shows that heat treatment had no significant effect on the shoot apex survival rate of the two versatile lily varieties, 'Fei Xiang Shao Hua' and 'Fei Xiang Tian Cheng'. Furthermore, there was no significant difference in shoot apex survival rate between heat-treated and unheat-treated lilies. Furthermore, heat treatment of the shoot apex had no significant effect on seedling establishment time, delaying seedling establishment by an average of 1-2 days, but with little effect on the growth status of the shoot apex. In summary, heat treatment did not significantly affect the shoot apex survival rate or seedling establishment time of the two functional lilies.

[0076] Table 4 Effect of heat treatment combined with detoxification of shoot tips of 'Fei Xiang Shao Hua'

[0077]

[0078] Table 5 Virus removal effect of heat treatment combined with shoot tip treatment of 'Feixiang Tiancheng'

[0079]

[0080] The virus detection results for 'Fei Xiang Shao Hua' and 'Fei Xiang Tian Cheng' after stem apex combined heat treatment are shown in Tables 4 and 5. The average CMV and LSV elimination rates for 'Fei Xiang Shao Hua' were 28.33%, 63.33%, and 16.67% for the total. The average CMV and LSV elimination rates for 'Fei Xiang Tian Cheng' were 35.00%, 73.33%, and 18.33% for the total. The LSV elimination rates for both lilies were higher in the stem apex combined heat treatment, but the total elimination rates were lower.

[0081] (3) Detoxification effect of stem tip-bound ribavirin

[0082] Table 6 Effect of ribavirin concentration on shoot tip survival rate

[0083]

[0084] Note: Different letters indicate significant differences (Student-Newman-Keuls test, P < 0.05).

[0085] Table 6 shows the effects of different ribavirin concentrations on the shoot apex survival rate and seedling establishment time of these two multipurpose lilies. At ribavirin concentrations of 10 mg / L, 15 mg / L, and 20 mg / L, there were significant differences in shoot apex survival rate between 'Fei Xiang Shao Hua' and 'Fei Xiang Tian Cheng', but no significant difference in seedling establishment time between the two varieties. Ribavirin treatment had no significant effect on the shoot apex survival rate or seedling establishment time of either 'Fei Xiang Shao Hua' or 'Fei Xiang Tian Cheng', and there were no significant differences among the different concentrations.

[0086] Table 7 Detoxification effect of ribavirin treatment on the stem tips of 'Feixiang Shaohua' and 'Feixiang Tiancheng'

[0087]

[0088] Note: Different letters indicate significant differences (Student-Newman-Keuls test, P < 0.05).

[0089] Table 7 shows the virus-free efficacy of shoot-apex-treated ribavirin for 'Fei Xiang Shao Hua' and 'Fei Xiang Tian Cheng'. When treated with the same concentration of ribavirin, there were no significant differences in CMV, LSV, and overall virus-free rates. Significant differences in CMV, LSV, and overall virus-free rates were observed between 'Fei Xiang Shao Hua' treated with and without ribavirin, indicating that shoot-apex-treated ribavirin is more effective than shoot-apex treatment alone in the virus-free treatment of 'Fei Xiang Shao Hua'. For CMV, the virus-free rate of 'Fei Xiang Tian Cheng' was significantly different from that of the 'untreated' ribavirin treatment only when the ribavirin concentration was 15 mg / L and 20 mg / L. Similarly, for LSV, the significant difference was only produced when the ribavirin concentration was 20 mg / L. The total virus-free rate was also significantly different when the ribavirin concentration was 15 mg / L and 20 mg / L. This shows that for 'Fei Xiang Tian Cheng', the virus-free effect was not obvious when the ribavirin concentration was lower than 15 mg / L. The overall virus-free effect was the best when the ribavirin concentration was 20 mg / L combined with shoot tip treatment. For 'Fei Xiang Shao Hua', the detoxification efficiency was the highest when the ribavirin concentration was 20 mg / L, with CMV, LSV and total detoxification rates reaching 36.67%, 58.33% and 26.67% respectively. There was no significant difference in CMV, LSV and total detoxification rates when the ribavirin concentration was 15 mg / L and 20 mg / L. Therefore, combined with the above shoot tip survival rate and seedling time, a ribavirin concentration between 15-20 mg / L can be selected for shoot tip-bound ribavirin detoxification treatment of 'Fei Xiang Tian Cheng'.

[0090] (4) Detoxification effect of stem tip combined with ribavirin and heat treatment

[0091] Table 8 Effect of ribavirin concentration on the survival rate of heat-treated shoot tips

[0092]

[0093] Note: Different letters indicate significant differences (Student-Newman-Keuls test, P < 0.05).

[0094] Table 8 shows the effects of different ribavirin concentrations on the shoot tip survival rate and seedling establishment time after heat treatment. There were no significant differences in shoot tip survival rate and seedling establishment time between 'Fei Xiang Shao Hua' and 'Fei Xiang Tian Cheng' at the four ribavirin concentration gradients. Furthermore, there were no significant differences in shoot tip survival rate and seedling establishment time between 'Fei Xiang Shao Hua' and 'Fei Xiang Tian Cheng' at the same ribavirin concentration, indicating that different ribavirin concentrations did not significantly affect shoot tip growth after heat treatment.

[0095] Table 9 Detoxification effects of ribavirin combined with heat treatment on the stem tips of 'Feixiang Shaohua' and 'Feixiang Tiancheng'

[0096]

[0097] Note: Different letters indicate significant differences (Student-Newman-Keuls test, P < 0.05).

[0098] Table 9 shows the CMV, LSV, and total virus-free rates of 'Fei Xiang Shao Hua' and 'Fei Xiang Tian Cheng' after shoot-apex-bound ribavirin and heat treatment. When treated with the same ribavirin concentration, there were no significant differences in CMV, LSV, and total virus-free rates between 'Fei Xiang Shao Hua' and 'Fei Xiang Tian Cheng'. With respect to CMV virus-free rates, 'Fei Xiang Shao Hua' achieved the best virus-free rate at a ribavirin concentration of 20 mg / L, reaching 41.67%, with significant differences observed between the two concentrations. 'Fei Xiang Shao Hua' also achieved the best virus-free rate at a ribavirin concentration of 20 mg / L, with a virus-free rate of 48.33%. With respect to LSV virus-free rates, 'Fei Xiang Shao Hua' and 'Fei Xiang Tian Cheng' achieved the best virus-free rates at a ribavirin concentration of 20 mg / L, with virus-free rates of 71.67% and 83.33%, respectively. The overall virus-free rates of 'Fei Xiang Shao Hua' and 'Fei Xiang Tian Cheng' were highest at a ribavirin concentration of 20 mg / L, reaching 31.67% and 38.33%, respectively. 'Fei Xiang Shao Hua' showed significant differences in overall virus-free rates at 15 and 20 mg / L of ribavirin; 'Fei Xiang Shao Hua' showed significant differences at 20 mg / L of ribavirin compared to other concentrations. Based on the CMV, LSV, and overall virus-free rates of the two varieties, a ribavirin concentration of 15-20 mg / L is recommended for 'Fei Xiang Shao Hua' when combined with ribavirin and heat treatment at the shoot apex, while 20 mg / L is the most suitable concentration for 'Fei Xiang Tian Cheng'.

[0099] (5) Comparison of detoxification effects among different detoxification treatment methods

[0100] Table 10 Detoxification effects of 10 treatments on 'Fei Xiang Shao Hua'

[0101]

[0102] Note: Different letters indicate significant differences (Student-Newman-Keuls test, P < 0.05).

[0103] Table 11 Detoxification effects of 10 treatments on 'Fei Xiang Tian Cheng'

[0104]

[0105] Note: Different letters indicate significant differences (Student-Newman-Keuls test, P < 0.05).

[0106] The CMV, LSV, and total virus-free efficacy of the multipurpose lilies 'Fei Xiang Shao Hua' and 'Fei Xiang Tian Cheng' under these 10 virus-free treatments are shown in Tables 10 and 11. The data for the shoot apex-bound heat-based virus-free treatment in the table are the same as those for the shoot apex-bound ribavirin and heat-based virus-free treatments at a ribavirin concentration of 0 mg / L in Treatment 7, indicating that the treatments were the same.

[0107] According to Table 10 and Figure 1 'Fei Xiang Shao Hua' achieved the best overall detoxification effect when inducing callus detoxification, with the highest CMV, LSV, and total detoxification rates reaching 84.45%, 66.67%, and 55.56%, respectively. Among the 10 treatments, the total detoxification rate for induced callus detoxification was significantly different from that of the other treatments. Secondly, shoot apex-bound ribavirin and heat treatment ranked second overall in terms of detoxification effectiveness, with the highest efficacy observed when the ribavirin concentration was above 15 mg / L. Regarding the single virus, CMV, the 'Fei Xiang Shao Hua' callus-induced method achieved the highest detoxification rate, reaching 84.45%, which was significantly different from the other treatments. The next best treatments were treatment 6, which achieved detoxification with shoot apex-bound ribavirin at a ribavirin concentration of 20 mg / L, and treatments 9 and 10, which achieved detoxification with shoot apex-bound ribavirin and heat treatment at ribavirin concentrations of 15 mg / L and 20 mg / L, respectively. With regard to LSV, except for the shoot apex-bound ribavirin treatment, the other six treatments showed similar efficacy in detoxifying 'Fei Xiang Shao Hua' and were relatively effective. Among these detoxification methods, heat treatment significantly improved LSV removal. Comprehensive comparative analysis suggests that callus-induced detoxification is the most appropriate method for detoxifying 'Fei Xiang Shao Hua' plants co-infected with CMV and LSV.

[0108] From Table 11 and Figure 2As shown, among the ten detoxification treatments, the highest total and CMV detoxification rates were achieved in the induced callus treatment for 'Fei Xiang Tian Cheng, at 78.89% and 51.11%, respectively. LSV achieved a detoxification rate of 63.33%, indicating that induced callus had the highest detoxification efficiency. For CMV, the highest detoxification rate was achieved in 'Fei Xiang Tian Cheng' using induced callus, followed by treatments 6, 9, and 10, all exceeding 40%. This suggests that ribavirin concentrations below 15 mg / L are ineffective against CMV. For LSV, shoot-apex-bound ribavirin and heat treatment were more effective in detoxifying 'Fei Xiang Tian Cheng', achieving the highest detoxification rate at a ribavirin concentration of 20 mg / L, reaching 83.33%. Shoot-apex-bound heat treatment was second, followed by induced callus treatment, and shoot-apex-bound ribavirin was relatively ineffective. Based on the overall detoxification effect of 'Fei Xiang Tian Cheng', although the LSV detoxification efficiency in treatment 10 was the highest when the concentration of ribavirin was 20 mg / L, the CMV and total detoxification rates showed a significant downward trend at this time. Therefore, for 'Fei Xiang Tian Cheng' plants infected with both CMV and LSV viruses, it is more appropriate to choose the method of inducing callus detoxification for detoxification.

[0109] In summary, the results of the above examples indicate that the overall virus-free rate of 'Fei Xiang Shao Hua' is slightly higher than that of 'Fei Xiang Tian Cheng', but the difference is not significant. The optimal overall detoxification treatment for both 'Fei Xiang Shao Hua' and 'Fei Xiang Tian Cheng' is callus induction. Ribavirin at a concentration of 20 mg / L combined with shoot apex and heat treatment is most effective for detoxifying lily plants infected solely with LSV.

Claims

1. A multi-purpose lily detoxification method, characterized by: The method comprises the following steps: (1) Establishment of sterile seedling system: Select the third scale from the outside to the inside of the bulbs of lilies 'Fei Xiang Shao Hua' and 'Fei Xiang Tian Cheng' as explants and sterilize them for tissue culture to establish a sterile seedling system; (2) Virus detection and detoxification: Take leaves from sterile seedlings to detect viruses, and detoxify positive samples; the detoxification method is at least one of induced callus detoxification, stem tip combined with heat treatment detoxification, stem tip combined with viraviril detoxification, stem tip combined with viraviril and heat treatment detoxification.

2. The method according to claim 1, wherein: The detoxification treatment method is at least one of inducing callus tissue detoxification, detoxification by combining stem tip with heat treatment, detoxification by combining stem tip with ribavirin and heat treatment.

3. The method according to claim 1, wherein: The viruses removed in the detoxification treatment are cucumber mosaic virus and / or lily symptom-free virus.

4. The method according to claim 1, 2 or 3, characterized in that: When the virus is cucumber mosaic virus, the detoxification method is to induce callus tissue detoxification.

5. The method according to claim 1, 2 or 3, characterized in that: When the virus is lily symptomless virus, the detoxification method is at least one of inducing callus detoxification, stem tip combined with heat treatment detoxification, stem tip combined with ribavirin and heat treatment detoxification.

6. The method according to claim 1, 2 or 5, characterized in that: The concentration of ribavirin in the shoot tip-bound ribavirin detoxification or the shoot tip-bound ribavirin and heat-treatment detoxification is 1-20 mg / L, preferably 10-20 mg / L, and further preferably 15-20 mg / L.

7. The method according to claim 1, wherein: The culture conditions of the sterile seedlings are an average temperature of 23 ± 2 ° C, a light intensity of 2000 Lx, a photoperiod of 12 h light / 12 h dark, and a culture period of 40-50 days.

8. The method according to claim 1, 2, 4 or 5, characterized in that: In the detoxification treatment for inducing callus detoxification, the culture medium for inducing callus is: MS + 1.0 mg / L TDZ + 2.0 mg / L PIC + 15 g / L sucrose + 7 g / L agar.

9. The method according to claim 1, 2 or 5, characterized in that: The heat treatment conditions in the detoxification treatment of the shoot tip combined with heat treatment or the shoot tip combined with ribavirin and heat treatment are: constant temperature treatment at 30°C in a light incubator for 7 days, then adjusting the temperature to 40°C for 14 hours of light / 28°C for 10 hours of darkness, and treating for 30 days.

10. Use of the method according to any one of claims 1 to 9 in detoxification of multi-purpose lilies.

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