Disinfection method of oil-spot lily tissue culture explant
The combined disinfection method using sodium hypochlorite and thiazomycin solution solved the problem of microbial removal from Lilium oilsum explants, achieving a non-toxic and highly efficient disinfection effect and improving the success rate of Lilium oilsum tissue culture.
Patent Information
- Application Number
- CN202511932634.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-19
- Publication Date
- 2026-02-27
AI Technical Summary
Existing technologies are insufficient to effectively remove microorganisms from the explants of Lilium oilseedum. Traditional disinfectants are toxic and easily contaminate the explants and the environment, and there is a lack of disinfection methods specifically for Lilium oilseedum.
A combined disinfection method using sodium hypochlorite solution and thiazolyl spore solution was employed, including multiple shaking treatments with low-concentration sodium hypochlorite solution and soaking in thiazolyl spore solution, combined with sterile water rinsing and the addition of thiazolyl spore to the induction culture medium, while avoiding the use of mercury-containing substances.
It achieves efficient disinfection that is non-toxic and pollution-free, significantly reduces the contamination rate of explants, and improves the survival rate and propagation rate of Lilium lancifolium tissue culture.
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Figure BDA0005750454450000101
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of plant tissue culture, and relates to a disinfection method for tissue culture explants, in particular to a disinfection method for Ledebouria socialis (Baker) Jessop tissue culture explants. BACKGROUND
[0002] Ledebouria socialis (Baker) Jessop is a plant of Ledebouria of Asparagaceae, and is unique in morphology and strong in shade tolerance, and can be used as an ornamental plant for indoor potted plants and landscape gardening. As a ground cover plant, the root system of Ledebouria socialis can help to maintain water and soil and reduce surface runoff; the flower buds of Ledebouria socialis are edible. Ledebouria socialis can also be called golden lily, leopard lily and fresh flower lily according to different varieties and parts.
[0003] At present, there is little research on Ledebouria socialis, and the propagation method of Ledebouria socialis has not been reported. In the field of plant propagation, tissue culture is an effective way of high efficiency and industrial production, and has the advantages of short propagation cycle and high propagation coefficient. Specifically, tissue culture refers to the separation of tissues, organs or cells, protoplasts and the like from a plant body, which are inoculated on a culture medium containing various nutrients and plant hormones through aseptic operation under aseptic conditions to obtain a regenerated whole plant or produce other products with economic value. In the process of plant tissue culture, the disinfection treatment of explants is a key step for the success of tissue culture. There are often some microorganisms in the explants, and these microorganisms living in the plant tissues are difficult to be effectively removed by conventional alcohol disinfection. In addition, the traditional explant disinfection reagent also uses mercury-containing substances, which are toxic and easy to contaminate the explants and the environment. At the same time, there is no report on the disinfection method of Ledebouria socialis explants.
[0004] Therefore, it is necessary to provide a disinfection method for Ledebouria socialis tissue culture explants to improve the success rate of Ledebouria socialis tissue culture. SUMMARY
[0005] Therefore, the technical problem to be solved by the present application is that conventional disinfection methods cannot effectively remove microorganisms from explants, traditional disinfection reagents are toxic and easy to contaminate explants and the environment, and there is no disinfection method for Ledebouria socialis explants at present, so that a non-toxic, non-polluting and effective disinfection method for Ledebouria socialis tissue culture explants is provided.
[0006] To solve the above technical problems, the technical scheme of the present application is as follows:
[0007] The present application provides a disinfection method for Ledebouria socialis tissue culture explants, which comprises the following steps:
[0008] S1. After spraying alcohol on the surface of the leaves of the oil-spotted lily, place the oil-spotted lily in a sterile container, add sodium hypochlorite solution a with a mass concentration of 0.1%-0.3% to the sterile container, immerse the oil-spotted lily in the sodium hypochlorite solution a, and remove the liquid from the sterile container after shaking disinfection.
[0009] S2. Add a sodium hypochlorite solution b with a mass concentration of 0.7%-1% to the sterile container, immerse the oil-spotted lily in the sodium hypochlorite solution b, and remove the liquid from the sterile container after shaking and disinfection.
[0010] S3. Add a thiazomycin solution with a concentration of 300-500 mg / L to the sterile container, immerse the oil-spotted lily in the thiazomycin solution, and remove the liquid from the sterile container after shaking and sterilization.
[0011] S4. Rinse the oil-spotted lily leaves after step S3 with sterile water.
[0012] Preferably, after step S4, the method further includes: dividing the oil-spotted lily leaves, inoculating the divided oil-spotted lily leaves into an induction culture medium, and adding a thiazomycin solution with a concentration of 300-500 mg / L to the induction culture medium.
[0013] Preferably, before step S4, the procedure of repeating step S3 is included.
[0014] Preferably, in step S1, the oscillation speed during the oscillation disinfection process is 150-170 rpm, and the processing time is 40-60 min.
[0015] Preferably, in step S2, the oscillation speed during the oscillation disinfection process is 150-170 rpm, and the processing time is 40-60 min.
[0016] Preferably, in step S3, the oscillation speed during the oscillation disinfection process is 150-170 rpm, and the processing time is 15-30 min.
[0017] Preferably, the concentration of the alcohol is 70-80%.
[0018] Preferably, the induction medium is based on MS culture medium.
[0019] Preferably, step S1 includes the steps of picking the leaves of the oil-spotted lily and rinsing the leaves with running water.
[0020] The technical solution of the present invention has the following advantages compared with the prior art:
[0021] (1) The disinfection method for explants of *Lilium lancifolium* tissue culture provided by this invention includes the following steps: S1, spraying alcohol onto the surface of *Lilium lancifolium* leaves and then disinfecting with a 0.1%-0.3% sodium hypochlorite solution a by shaking; S2, disinfecting again with a 0.7%-1% sodium hypochlorite solution b by shaking; S3, disinfecting with a 300-500 mg / L thiazolyl solution by shaking; S4, rinsing with sterile water. The disinfectant used in this method does not contain toxic substances such as mercury, solving the problems of toxic damage to explants and environmental pollution caused by traditional disinfectants. By using multiple concentrations of sodium hypochlorite and thiazolyl in combination, *Lilium lancifolium* treated with this method exhibits low contamination rate and high explant survival rate, providing a foundation for the tissue culture of *Lilium lancifolium*.
[0022] (2) The disinfection method for explants of *Lilium lancifolium* tissue culture provided by the present invention further includes, after step S4: dividing *Lilium lancifolium* leaves, inoculating the divided leaves into an induction medium, and adding a 300-500 mg / L thiazomycin solution to the induction medium. By adding a low concentration of thiazomycin solution to the culture medium, disinfection and antibacterial effects can be further achieved during *Lilium lancifolium* tissue culture, effectively inhibiting bacterial contamination inside the *Lilium lancifolium* and further improving the survival rate of the *Lilium lancifolium*. Detailed Implementation
[0023] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which this invention pertains. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.
[0024] The present invention will now be described in further detail according to specific embodiments thereof.
[0025] Example 1
[0026] This embodiment provides a method for disinfecting explants from *Lilium lancifolium* tissue culture, filling a gap in current knowledge regarding *Lilium lancifolium* tissue culture and explant disinfection methods during the process. Specifically, the disinfection method for *Lilium lancifolium* tissue culture explants provided in this embodiment includes the following steps performed sequentially:
[0027] S0. Harvesting leaves of Lilium stropharia: Collect the 2nd-3rd leaves from the top of the Lilium stropharia plant. The collected leaves should have a petiole of 0.5-1.5cm. In this embodiment, the petiole length is 1cm. After cleaning the soil from the surface and crevices of the Lilium stropharia with a clean brush, rinse with running water for 30 minutes.
[0028] S1. After spraying the cleaned oil-spot lily leaves with 75% alcohol, place the oil-spot lily leaves in a sterile container in a sterile workbench. Add 0.2% sodium hypochlorite solution a to the sterile container, immersing the oil-spot lily leaves in sodium hypochlorite solution a. Place the sterile container on a shaker and shake at 160 rpm for 50 minutes to disinfect. Remove the disinfectant liquid from the sterile container.
[0029] S2. Add a 0.9% sodium hypochlorite solution b to the sterile container, immerse the oil-spotted lily leaves in the sodium hypochlorite solution b, place the sterile container on a shaker, and sterilize by shaking at 160 rpm for 50 minutes to remove the sterilizing liquid from the sterile container.
[0030] S3. Add a 400 mg / L thiazomycin solution to the sterile container, immerse the oil-spotted lily leaves in the thiazomycin solution, place the sterile container on a shaker, and sterilize by shaking at 160 rpm for 20 minutes to remove the sterilizing liquid from the sterile container.
[0031] Repeat step S3 once.
[0032] S4. Rinse the oil-spotted lily leaves after two disinfection treatments in step S3 with sterile water. In this embodiment, the sterile water rinsing is performed twice.
[0033] S5. Place the *Lilium lancifolium* leaf explants, which have undergone the above disinfection steps, on sterile filter paper. After absorbing excess moisture with the sterile filter paper, use sterile forceps and scissors to divide the *Lilium lancifolium* leaf explants into 1cm*1cm pieces. Inoculate the divided *Lilium lancifolium* leaf explants onto the induction medium. In this embodiment, the induction medium is based on MS medium, which also includes additional components such as cytokinins and auxins. Thiazolam at a concentration of 400mg / L is added to the induction medium, which serves as a disinfectant and antibacterial agent for plant tissue culture.
[0034] This disinfection method uses sodium hypochlorite solution and thiazolyl salvia solution as disinfectants, neither of which contains toxic substances such as mercury. This solves the problems of toxic damage to explants and environmental pollution caused by traditional disinfectants. By using multiple concentrations of sodium hypochlorite and thiazolyl salvia in combination, their synergistic effect achieves excellent disinfection and sterilization. Oil-spotted lilies treated with this method exhibit low contamination rates and high explant survival rates, providing a foundation for their tissue culture. Simultaneously, adding a low concentration of thiazolyl salvia solution to the induction medium further enhances disinfection and antibacterial effects during tissue culture, effectively inhibiting bacterial contamination within the oil-spotted lily and thus further improving the tissue culture survival rate.
[0035] Example 2
[0036] This embodiment provides a method for disinfecting explants from *Lilium lancifolium* tissue culture. The method for disinfecting explants from *Lilium lancifolium* tissue culture provided in this embodiment includes the following steps performed sequentially:
[0037] S0. Harvesting leaves of Lilium spp.: Harvest the 2nd-3rd leaves from the top of the Lilium spp. plant. The harvested leaves should have a petiole of 0.5-1.5cm. In this embodiment, the petiole length is 0.5cm. After cleaning the soil from the surface and crevices of the Lilium spp. with a clean brush, rinse with running water for 20 minutes.
[0038] S1. After spraying the cleaned oil-spot lily leaves with 70% alcohol, place the oil-spot lily leaves in a sterile container in a sterile workbench. Add 0.1% sodium hypochlorite solution a to the sterile container, immersing the oil-spot lily leaves in sodium hypochlorite solution a. Place the sterile container on a shaker and shake at 150 rpm for 40 minutes to disinfect. Remove the disinfectant liquid from the sterile container.
[0039] S2. Add a 0.7% sodium hypochlorite solution b to the sterile container, immerse the oil-spotted lily leaves in the sodium hypochlorite solution b, place the sterile container on a shaker, and sterilize by shaking at 150 rpm for 40 minutes to remove the sterilizing liquid from the sterile container.
[0040] S3. Add a 300 mg / L thiazomycin solution to the sterile container, immerse the oil-spotted lily leaves in the thiazomycin solution, place the sterile container on a shaker, and sterilize it by shaking at 150 rpm for 15 minutes to remove the sterilizing liquid from the sterile container.
[0041] Repeat step S3 once.
[0042] S4. Rinse the oil-spotted lily leaves after two disinfection treatments in step S3 with sterile water. In this embodiment, the sterile water rinsing is performed once.
[0043] S5. Place the *Lilium lancifolium* leaf explants, which have undergone the above disinfection steps, on sterile filter paper. After absorbing excess moisture with the sterile filter paper, use sterile forceps and sterile scissors to divide the *Lilium lancifolium* leaf explants into 0.8cm*0.8cm pieces. Inoculate the divided *Lilium lancifolium* leaf explants onto the induction medium. In this embodiment, MS medium is used as the basal medium for induction. Add 300mg / L thiazolyl to the induction medium, which serves as a disinfectant and antibacterial agent for plant tissue culture.
[0044] Example 3
[0045] This embodiment provides a method for disinfecting explants from *Lilium lancifolium* tissue culture. The method for disinfecting explants from *Lilium lancifolium* tissue culture provided in this embodiment includes the following steps performed sequentially:
[0046] S0. Harvesting leaves of Lilium 'Oildrop': Collect the 2nd-3rd leaves from the top of the Lilium 'Oildrop' plant. The collected leaves should have a petiole of 0.5-1.5cm. In this embodiment, the petiole length is 1.5cm. After cleaning the soil from the surface and crevices of the Lilium 'Oildrop' with a clean brush, rinse with running water for 40 minutes.
[0047] S1. After spraying the cleaned oil-spot lily leaves with 75% alcohol, place the oil-spot lily leaves in a sterile container in a sterile workbench. Add 0.3% sodium hypochlorite solution a to the sterile container, immersing the oil-spot lily leaves in sodium hypochlorite solution a. Place the sterile container on a shaker and shake at 170 rpm for 60 minutes to disinfect. Remove the disinfectant liquid from the sterile container.
[0048] S2. Add a 1% sodium hypochlorite solution b to the sterile container, immerse the oil-spotted lily leaves in the sodium hypochlorite solution b, place the sterile container on a shaker, and sterilize by shaking at 170 rpm for 60 minutes to remove the sterilizing liquid from the sterile container.
[0049] S3. Add a 500 mg / L thiazomycin solution to the sterile container, immerse the oil-spotted lily leaves in the thiazomycin solution, place the sterile container on a shaker, and sterilize by shaking at 170 rpm for 30 minutes to remove the sterilizing liquid from the sterile container.
[0050] Repeat step S3 once.
[0051] S4. Rinse the oil-spotted lily leaves after two disinfection treatments in step S3 with sterile water. In this embodiment, the sterile water rinsing is performed 3 times.
[0052] S5. Place the *Lilium lancifolium* leaf explants, which have undergone the above disinfection steps, on sterile filter paper. After absorbing excess moisture with the sterile filter paper, use sterile forceps and sterile scissors to divide the *Lilium lancifolium* leaf explants into 1.2cm*1.2cm pieces. Inoculate the divided *Lilium lancifolium* leaf explants onto the induction medium. In this embodiment, MS medium is used as the basal medium for induction. Add 500mg / L thiazolyl to the induction medium, which serves as a disinfectant and antibacterial agent for plant tissue culture.
[0053] Comparative Example 1
[0054] This comparative example provides a method for disinfecting explants from *Lilium lancifolium* tissue culture, comprising the following steps performed sequentially:
[0055] S0. Harvesting leaves of Lilium stropharia: Collect the 2nd-3rd leaves from the top of the Lilium stropharia plant. The collected leaves should have a petiole of 0.5-1.5cm. In this embodiment, the petiole length is 1cm. After cleaning the soil from the surface and crevices of the Lilium stropharia with a clean brush, rinse with running water for 30 minutes.
[0056] S1. After spraying the cleaned oil-spot lily leaves with 75% alcohol, place the oil-spot lily leaves in a sterile container in a sterile workbench. Add a 1% sodium hypochlorite solution to the sterile container, immersing the oil-spot lily leaves in the sodium hypochlorite solution. Place the sterile container on a shaker and shake at 160 rpm for 5-15 minutes to disinfect. Remove the disinfectant liquid from the sterile container.
[0057] S2. The oil-spotted lily leaves are rinsed and disinfected with sterile water. In this embodiment, the sterile water rinsing is performed twice.
[0058] Comparative Example 2
[0059] This comparative example provides a method for disinfecting explants from *Lilium lancifolium* tissue culture, comprising the following steps performed sequentially:
[0060] S0. Harvesting leaves of Lilium stropharia: Collect the 2nd-3rd leaves from the top of the Lilium stropharia plant. The collected leaves should have a petiole of 0.5-1.5cm. In this embodiment, the petiole length is 1cm. After cleaning the soil from the surface and crevices of the Lilium stropharia with a clean brush, rinse with running water for 30 minutes.
[0061] S1. After spraying the cleaned oil-spot lily leaves with 75% alcohol, place the oil-spot lily leaves in a sterile container in a sterile workbench. Add a 2% sodium hypochlorite solution to the sterile container, immersing the oil-spot lily leaves in the sodium hypochlorite solution. Place the sterile container on a shaker and shake at 160 rpm for 5-15 minutes to disinfect. Remove the disinfectant liquid from the sterile container.
[0062] S2. The oil-spotted lily leaves are rinsed and disinfected with sterile water. In this embodiment, the sterile water rinsing is performed twice.
[0063] Comparative Example 3
[0064] This comparative example provides a method for disinfecting explants from *Lilium lancifolium* tissue culture, comprising the following steps performed sequentially:
[0065] S0. Harvesting leaves of Lilium stropharia: Collect the 2nd-3rd leaves from the top of the Lilium stropharia plant. The collected leaves should have a petiole of 0.5-1.5cm. In this embodiment, the petiole length is 1cm. After cleaning the soil from the surface and crevices of the Lilium stropharia with a clean brush, rinse with running water for 30 minutes.
[0066] S1. After spraying the cleaned oil-spot lily leaves with 75% alcohol, place the oil-spot lily leaves in a sterile container in a sterile workbench. Add a 3% sodium hypochlorite solution to the sterile container, immersing the oil-spot lily leaves in the sodium hypochlorite solution. Place the sterile container on a shaker and shake at 160 rpm for 5-15 minutes to disinfect. Remove the disinfectant liquid from the sterile container.
[0067] S2. The oil-spotted lily leaves are rinsed and disinfected with sterile water. In this embodiment, the sterile water rinsing is performed twice.
[0068] Comparative Example 4
[0069] This comparative example provides a method for disinfecting explants from *Lilium lancifolium* tissue culture, comprising the following steps performed sequentially:
[0070] S0. Harvesting leaves of Lilium stropharia: Collect the 2nd-3rd leaves from the top of the Lilium stropharia plant. The collected leaves should have a petiole of 0.5-1.5cm. In this embodiment, the petiole length is 1cm. After cleaning the soil from the surface and crevices of the Lilium stropharia with a clean brush, rinse with running water for 30 minutes.
[0071] S1. After spraying the cleaned oil-spot lily leaves with 75% alcohol, place the oil-spot lily leaves in a sterile container in a sterile workbench. Add a 0.8% sodium hypochlorite solution containing 400 mg / L thiazomycin to the sterile container, immersing the oil-spot lily leaves in the above mixed disinfection solution. Place the sterile container on a shaker and shake at 160 rpm for 90 minutes to disinfect. Remove the disinfectant liquid from the sterile container.
[0072] S2. The oil-spotted lily leaves are rinsed and disinfected with sterile water. In this embodiment, the sterile water rinsing is performed twice.
[0073] Comparative Example 5
[0074] This comparative example provides a method for disinfecting explants from *Lilium lancifolium* tissue culture. This method is basically the same as that in Example 1, except that in step S1, the mass concentration of sodium hypochlorite solution a is 1%, and in step S2, the mass concentration of sodium hypochlorite solution b is 2%.
[0075] Comparative Example 6
[0076] This comparative example provides a method for disinfecting explants from Lilium lancifolium tissue culture. The method is basically the same as that in Example 1, except that in step S1, the mass concentration of sodium hypochlorite solution a is 2%.
[0077] Comparative Example 7
[0078] This comparative example provides a method for disinfecting explants from *Lilium lancifolium* tissue culture. This method is essentially the same as in Example 1, except that in step S3, the concentration of the thiazolycin solution is 500 mg / L, and step S3 is not repeated.
[0079] Comparative Example 8
[0080] This comparative example provides a method for disinfecting explants from Lilium lancifolium tissue culture. The method is basically the same as that in Example 1, except that in step S3, the concentration of thiazomycin solution is 700 mg / L.
[0081] Experimental Example
[0082] The disinfection and sterilization effects of the disinfection methods provided in Example 1 and Comparative Examples 1-8 on the explants of *Lilium lancifolium* tissue culture were tested respectively. The test results are shown in Table 1.
[0083] Table 1
[0084]
[0085] Experimental results showed that using sodium hypochlorite alone as a disinfectant for *Lilium lancifolium* explants resulted in an extremely high contamination rate. This is because the main contaminants on *Lilium lancifolium* explants are bacteria. Sodium hypochlorite solution is a broad-spectrum disinfectant that can destroy the cell walls, proteins, and nucleic acids of microorganisms through oxidation. Higher sodium hypochlorite concentrations caused greater damage to the *Lilium lancifolium* leaf explants, but did not significantly enhance the bactericidal effect. In contrast, thiazolyl spp. is an antibiotic with strong bactericidal activity. Furthermore, further increasing the concentration of thiazolyl spp. solution did not significantly change the contamination rate, indicating that higher concentrations of thiazolyl spp. solution did not provide a substantial enhancement effect.
[0086] When thiazomycin was added to sodium hypochlorite disinfectant before disinfecting the explants of *Lilium lancifolium* leaves, the contamination rate decreased by approximately 46% compared to sodium hypochlorite disinfectant alone, but the proportion of fungal contamination increased. The reasons are as follows: Thiazomycin contains a β-lactam ring, a structure fundamental to its antibacterial activity. This ring inhibits cell wall peptidoglycan synthases, a class of penicillin-binding proteins (PBPs), thereby hindering cell wall peptidoglycan synthesis, causing bacterial cell wall defects, swelling, and lysis. Furthermore, the β-lactam ring can trigger bacterial autolysis, leading to a lethal effect. However, the β-lactam ring is chemically unstable and easily degraded by acids, alkalis, and certain types of heavy metal ions (oxidants). Sodium hypochlorite disinfection works through the hypochlorous acid produced after hydrolysis. Hypochlorous acid has extremely strong oxidizing properties, capable of denaturing bacterial proteins, thus killing pathogenic microorganisms. Therefore, when sodium hypochlorite and thiazomycin are used together, on the one hand, sodium hypochlorite will oxidize and decompose thiazomycin, and on the other hand, the effective content of sodium hypochlorite will be reduced, thus losing its intended disinfection effect.
[0087] The disinfection method for *Lilium lancifolium* tissue culture explants provided in Example 1 of this application was adopted. Through multiple applications of a combined low-concentration sodium hypochlorite solution and thiazolyl salvia miltiorrhiza solution for disinfection, a highly efficient disinfection effect was achieved while significantly reducing damage to the *Lilium lancifolium* explants from the disinfectant. As mentioned earlier, this is because thiazolyl salvia miltiorrhiza is an antibiotic effective against bacteria, thus eliminating bacteria on the explants. Furthermore, the sequential use of sodium hypochlorite and thiazolyl salvia miltiorrhiza, applied individually, avoids the problem of reduced efficacy of either solution. Specifically, the technical effect is that the contamination rate of *Lilium lancifolium* leaf explants treated with multiple applications of a combined low-concentration sodium hypochlorite solution and thiazolyl salvia miltiorrhiza solution was reduced by 40.1%-86.1% compared to *Lilium lancifolium* leaf explants disinfected only with sodium hypochlorite solution.
[0088] Meanwhile, thiazomycin can effectively eliminate bacteria carried by *Lilium oleraceum* explants. Adding thiazomycin to the induction medium can effectively inhibit bacterial contamination within the *Lilium oleraceum* while simultaneously killing and eliminating bacteria, reducing the consumption of explants and culture medium, and improving the success rate of tissue culture. Low concentrations of thiazomycin do not produce side effects on *Lilium oleraceum*. To reduce costs and prevent the accumulation of thiazomycin in the plant that could cause plant mutations, the use of thiazomycin can be discontinued after one subculture of *Lilium oleraceum* leaf induction culture. The method provided in this application for disinfecting *Lilium oleraceum* leaf explants uses a disinfectant with low toxicity, is easy to handle, has simple disinfection procedures, low explant mortality, and low contamination rate, significantly improving the propagation rate of *Lilium oleraceum* tissue culture.
[0089] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.
Claims
1. A method for disinfecting explants from *Lilium arvense* tissue culture, characterized in that, Includes the following steps: S1. After spraying alcohol on the surface of the leaves of the oil-spotted lily, place the oil-spotted lily in a sterile container, add sodium hypochlorite solution a with a mass concentration of 0.1%-0.3% to the sterile container, immerse the oil-spotted lily in the sodium hypochlorite solution a, and remove the liquid from the sterile container after shaking disinfection. S2. Add a sodium hypochlorite solution b with a mass concentration of 0.7%-1% to the sterile container, immerse the oil-spotted lily in the sodium hypochlorite solution b, and remove the liquid from the sterile container after shaking and disinfection. S3. Add a thiazomycin solution with a concentration of 300-500 mg / L to the sterile container, immerse the oil-spotted lily in the thiazomycin solution, and remove the liquid from the sterile container after shaking and sterilization. S4. Rinse the oil-spotted lily leaves after step S3 with sterile water.
2. The method for disinfecting explants from *Lilium arvense* tissue culture according to claim 1, characterized in that, After step S4, the method further includes: dividing the oil-spotted lily leaves, inoculating the divided oil-spotted lily leaves into an induction culture medium, and adding a thiazomycin solution with a concentration of 300-500 mg / L to the induction culture medium.
3. The method for disinfecting explants from *Lilium lancifolium* tissue culture according to claim 1 or 2, characterized in that, Before step S4, the procedure includes repeating step S3.
4. The method for disinfecting explants from *Lilium arvense* tissue culture according to claim 3, characterized in that, In step S1, during the oscillation disinfection process, the oscillation speed is 150-170 rpm and the processing time is 40-60 min.
5. The method for disinfecting explants from *Lilium arvense* tissue culture according to claim 4, characterized in that, In step S2, during the oscillation disinfection process, the oscillation speed is 150-170 rpm and the processing time is 40-60 min.
6. The method for disinfecting explants from *Lilium arvense* tissue culture according to claim 5, characterized in that, In step S3, during the oscillation disinfection process, the oscillation speed is 150-170 rpm and the processing time is 15-30 min.
7. The method for disinfecting explants from *Lilium arvense* tissue culture according to claim 6, characterized in that, The alcohol concentration is 70-75%.
8. The method for disinfecting explants from *Lilium arvense* tissue culture according to claim 2, characterized in that, The induction medium is based on MS culture.
9. The method for disinfecting explants from *Lilium arvense* tissue culture according to claim 1, characterized in that, Before step S1, the steps of picking the leaves of the oil-spotted lily and rinsing the leaves with running water are also included.