CN113170731B - High-temperature detoxification method based on lily bulb scale leaf tips and application thereof - Google Patents
High-temperature detoxification method based on lily bulb scale leaf tips and application thereof Download PDFInfo
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Abstract
The invention provides a high-temperature detoxification method based on lily bulb leaf tips and application thereof, wherein lily bulb flakes or bulblets are used as materials, aseptic bulblets are cultured, variable-temperature alternate treatment at 37 ℃ and 23-25 ℃ is adopted, and then the high-temperature treatment at 95 ℃ and accurate control of treatment time are combined, so that the detoxification efficiency of the lily bulb virus CMV, LSV and LMoV by the lily bulb leaf tips is greatly improved, the stripped leaf tips are 1.0-2.0 mm in length, instrument equipment such as dissecting mirrors and the like are not needed for the leaf tips with the length, and generally 6-10 or more leaf tips exist in 1 bulblet, so that the lily bulb leaf tips are easier to obtain materials, simple and convenient to operate and low in cost. Compared with the traditional heat treatment, the heat treatment of the invention has short time consumption and high detoxification rate. The invention breaks through the defect that stem tip culture is often used as a main part in the prior art, provides a new way for culturing lily non-toxic seedlings, has good application prospect and lays a foundation for industrial seedling culture.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture and detoxification, and particularly relates to a high-temperature detoxification method based on lily bulb scale leaf tips and application thereof.
Background
The lily is perennial bulbous flower, has many varieties and strains, long flowering phase, large flower, rich color and beautiful flower appearance; has excellent ornamental value, medicinal value and edible value. Siberian (Lilium 'siberia') is one of the main cut-flower lilies which are well sold in the market, belongs to oriental lily, has large flower and pure white color, and has the market price of about 15 yuan at present. In recent years, along with the enlargement of lily production scale, seed balls propagate automatically and circulate rapidly, so that lily virus disease accumulation becomes more and more serious. In lily production, seed balls or scales are mostly adopted for asexual propagation, but the asexual propagation has the defects of small propagation coefficient, continuous accumulation of viruses and the like, so that the plants are easy to generate the viruses. Up to 19 or more types of viruses infecting Lily have been reported, and the most prominent viruses among them are Lily mottle virus (LMoV), cucumber Mosaic Virus (CMV), lily Symptomless Virus (LSV), and the like. After lily is infected with virus, the color of lily is dim, the leaves are deformed, even the plant withers, the normal physiological function of the plant is damaged, the yield is reduced, the quality is degraded, and the yield and the quality of 'Siberian' lily are seriously damaged (Mapingxia, 2007, 'Siberian' lily detoxification and virus detection. Master academic thesis. Lanzhou: northwest university.) so as to cause serious problems for lily production (Fengxihong, lijia, luxianobu. Lily virus removal technical research progress [ J ]. Agronomy report, 2015,5 (9): 91-95.). The effective prevention and treatment of virus diseases is difficult by using chemical agents or biological agents, and the direct use of lily virus-free seedlings has become an important measure for controlling virus diseases (Shenshulin. Lily virus disease and its inspection [ J ]. Plant quarantine, 1996,10 (4): 223-226.). Therefore, the establishment of a lily high-efficiency virus-free rapid propagation technology has important significance for the industrial development thereof.
At present, lily detoxification technology mainly uses lily micro-stem tip culture as a main method and combines other treatment modes. For example, wang super et al take lily plant cultured at variable temperature (illumination treatment at 38 ℃ for 169h and darkness treatment at 25 ℃ for 8 hours every day), cut stem tip with size of 0.5-0.8 mm, and inoculate the stem tip into culture medium containing ribavirin with concentration of 10mg/L, CMV detoxication rate is less than 65%, LSV is less than 50%, and survival rate is 57.62% (Wang super, wang Wen He, zhao Xiang Yun, etc.. Lily Virus removal technical research [ J ]. Proceedings of Beijing academy of agriculture, 2012,27 (1): 25-28.). The tissue culture seedlings of lily are pretreated at the high temperature of 65 ℃ for 30min by the GaoHuiqing and the like, and then the stem tips of 0.2-0.5 mm are peeled off for carrying out detoxification culture, wherein the detoxification rates of CMV and LSV are respectively 46.88% and 74.65%, but the average survival rate is less than 60% (GaoHuiQing, liang Lang, wangxihong, and the like; application research of stem tip culture and heat treatment technology in lily detoxification [ J ]. Shanxi university Committee, 2010,30 (6): 528-532.). Juanbo et al treated the bulbil of Lilium tigrinum at 38 deg.C for 20 days, then stripped 0.4-0.6 mm of stem tip, inoculated in bud induction medium with 5-10 mg/L ribavirin, and found that the detoxification rate is 90% but the survival rate is only about 60% (Zhouxiabo, wu Yi, ding Liu, etc. study on 3 major virus detoxification methods of Lilium tigrinum, 2016 (10): 7-10.). However, the stem tips stripped by the method are small and less than 0.8mm, the stem tips are not easy to strip, instrument equipment such as a dissecting mirror is also needed, the cost is high, only 1 stem tip is usually required for 1 small bulb, the material is difficult to obtain, and the heat treatment time is long. Researches show that plant viruses can be effectively killed under the condition that the heat treatment temperature is higher than 70 ℃, the higher the temperature is, the higher the detoxification rate is, but the survival rate of seedlings is reduced, and therefore, the key of the plant detoxification production is to regulate and control the heat treatment temperature and enable the seedlings to survive.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a high-temperature detoxification method based on lily scale leaf tips and application thereof, and solves the problems of difficult stem tip material taking, high cost and long heat treatment time of the conventional lily detoxification method.
In order to realize the purpose, the invention adopts the following scheme: a high-temperature detoxification method based on lily bulb scale leaf tips comprises the following steps:
1) Using lily bulbs or bulblets as materials, performing conventional surface disinfection treatment, then placing the materials on a primary culture medium for culturing for 2-3 weeks to obtain lily aseptic bulblet materials, then using the lily aseptic bulblets as explants, and culturing in a secondary culture medium for 3-4 weeks to obtain a large number of aseptic bulblets with normal growth;
2) The aseptic bulblets obtained in the step 1) are placed at 37 ℃ for pretreatment for 12h, then placed at 23-25 ℃ and the normal temperature for treatment for 12h, and the temperature-changing treatment is repeated for 3-5 times;
3) Treating the material subjected to heat treatment in the step 2) at 95 ℃ for 7min, then stripping off the leaf tips of the scales with the length of 1-2mm, inoculating the leaf tips to a bud differentiation culture medium, culturing for 2-3 weeks, inducing to generate cluster buds, then inoculating the adventitious buds to a proliferation culture medium for 1-2 weeks, inducing to generate more adventitious buds, and then inoculating the adventitious buds to a rooting culture medium, culturing for 1-2 weeks to obtain rooted seedlings, namely the detoxified seedlings of lily;
4) Taking the scales of the detoxified seedlings in the step 3) as materials, inoculating the scales to an adventitious bud induction culture medium, performing adventitious bud induction culture to generate adventitious buds, inoculating the adventitious buds to a proliferation culture medium, performing subculture proliferation to obtain proliferated seedlings, transferring the proliferated seedlings to a rooting culture medium, and performing rooting culture to obtain rooted seedlings, thus obtaining a large number of detoxified tissue culture seedlings.
Preferably, the disinfection is to soak the fabric in a washing powder solution for 5-15 min, wash the fabric under running water for 1-2 h, place the fabric on a sterile operating platform and put the fabric into a sterile container, soak the fabric in 75% ethanol for 40s, treat the fabric for 15min by using 0.1% mercuric chloride solution, and finally wash the fabric for 6-8 times by using sterile water.
Preferably, the adventitious bud induction culture medium is MS +6-BA 1.5mg/L + NAA 0.2mg/L + sucrose 30g/L + agar 7g/L, and the pH value of the culture medium is 5.6-6.0.
Preferably, the subculture multiplication medium is MS +6-BA 2.0mg/L + NAA 0.2mg/L + sucrose 30g/L + agar 7g/L, and the pH value of the medium is 5.6-6.0.
Preferably, the rooting medium is MS + NAA 1mg/L + sucrose 30g/L + agar 7g/L, and the pH value of the medium is 5.6-6.0.
Preferably, the conditions of the adventitious bud induction culture, the proliferation culture and the rooting culture are as follows: the culture temperature is 25 +/-2 ℃, the humidity is 60-70%, the illumination intensity is 3000lx-5000lx, and the sunshine time is 12-14 h/d.
The invention also provides application of the method in breeding non-toxic lily seedlings.
Compared with the prior art, the invention has the following beneficial effects:
1. the Lily bulblets are alternately treated at the temperature of 38 ℃ and the temperature of 25 ℃, so that the high-temperature resistance of the leaf tips of the scales is improved, the virus content of the leaf tips of the scales is remarkably reduced and controlled, and the virus removal efficiency of the leaf tips on Lily mottle virus (LMoV), cucumber Mosaic Virus (CMV) and Lily Symptomless Virus (LSV) is greatly improved by combining the high-temperature treatment at the temperature of 95 ℃ and the accurate control of the treatment time, the virus removal rate reaches over 83.3 percent (wherein the LSV is 83.3 percent, and the rest is 100 percent), the survival rate of Lily seedlings can reach 13.8 percent, and the later-stage propagation development is ensured. The invention directly strips the leaf tips with the length of 1.0-2.0 mm by naked eyes without using instruments and equipment such as a dissecting mirror, and generally 1 bulblet has 6-10 leaf tips, so the invention has the advantages of easier material taking, simple and convenient operation and low cost. Compared with the traditional heat treatment, the high-temperature short-time treatment at 95 ℃ is utilized, the virus can be effectively killed, the time consumption is short, the detoxification rate is obviously improved, the treatment temperature is too high or too low, and the treatment time is too long or too short, so that the detoxification effect of the invention cannot be achieved. The invention breaks through the problem that the prior art usually focuses on stem tip culture, and simultaneously solves the problems of high stem tip stripping difficulty, high cost and low shedding rate.
2. The treated scaly leaf tips grow well on the culture medium, the tissue culture seedling propagation coefficient is high, a large number of high-quality virus-free seedlings can be effectively obtained, reliable seedling guarantee is brought to large-area planting, yield increase and quality improvement are ensured, the virus-free rapid propagation method is low in cost and high in efficiency, a new way is provided for cultivating lily virus-free seedlings, the application prospect is good, and a foundation is laid for industrial seedling culture.
Drawings
FIG. 1 is a photograph of the tips of the scales of the tissue-cultured seedlings in example 1.
FIG. 2 is a graph showing the growth of the control squash tips; a.1-2 mm small scale leaf tip culture; b. culturing the growth condition for 20 d; c. culturing the growth condition of 40 d; d. the growth condition was cultured for 60 days.
FIG. 3 is a diagram showing the growth of the tips of the small flakes after high temperature treatment at 95 ℃ for various periods of time; a, treating for 3min; e-h, processing for 5min; i-l. treatment for 7min.
FIG. 4 is a diagram showing the growth of the tips of the small flakes after being treated at a high temperature of 85 ℃ for various periods of time; a, treating for 3min; e-h, processing for 5min; i-l. treatment for 7min.
FIG. 5 is a graph of electrophoresis of detoxification validation after high temperature treatment at 85 ℃ for various periods of time; m is 2000Marker, lane 1 internal reference 18s; lane 2 is CMV in the test sample; lane 3 is LMoV in the test sample; lane 4 is LSV in the test sample; a.85 ℃ treatment for 3min to obtain a detoxification verification chart; b.processing the detoxication verification chart for 5min at 85 ℃; c, treating the detoxification verification chart for 7min at 85 ℃;
FIG. 6 is a graph of electrophoresis of detoxification validation after high temperature treatment at 95 ℃ for various periods of time; m is 2000Marker, lane 1 is internal reference 18s; lane 2 is detection of CMV in the sample; lane 3 is LMoV detected in the sample; lane 4 is LSV in the test sample; a.95 ℃ treatment for 3min; b.95 deg.C treating for 5min to obtain detoxication verification chart; and c.95 ℃ treatment for 7min.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The experimental material used in the examples below was lilium siberia.
The following examples refer to media
Primary culture medium: MS +6-BA 0.5mg/L + NAA 0.05mg/L + sucrose 30g/L + agar 7g/L, pH5.6-6.0.
Subculture medium: MS +6-BA 0.5mg/L + NAA 0.2mg/L + sucrose 30g/L + agar 7g/L, pH5.6-6.0.
Adventitious bud induction medium: MS +6-BA 1.5mg/L + NAA 0.2mg/L + sucrose 30g/L + agar 7g/L, pH5.6-6.0.
Subculture multiplication medium: MS +6-BA 2.0mg/L + NAA 0.2mg/L + sucrose 30g/L + agar 7g/L, pH5.6-6.0.
Rooting culture medium: MS + NAA 1mg/L + sucrose 30g/L + agar 7g/L, pH5.6-6.0.
Example 1 Lily high-temperature detoxification method based on scale and leaf tips
1) Primary culture: washing Bulbus Lilii of "Siberian" with washing powder, washing in running water for more than 30min, placing the bulb in a sterile container on a clean bench, soaking in 75% ethanol for 40s, rinsing with sterile water, and placing into 0.1% HgCl 2 Sterilizing for 12min, and washing with sterile water for 6-8 times. And then, the inner side of the cleaned scale is horizontally placed on a primary culture medium upwards for induction culture for 2-3 weeks to obtain a sterile bud.
2) Subculturing: and (3) taking the aseptic bulblets of the lilies as explants, inoculating the aseptic bulblets of the lilies into a subculture medium, and culturing for 3-4 weeks to obtain the aseptic bulblets with normal growth.
3) Thermal exercise treatment: placing the lily tissue culture seedling in an illumination incubator, treating for 12h at the high temperature of 37 ℃, then standing for 12h at the temperature of 25 ℃, wherein the photoperiod is 12h/12h, and the illumination intensity is about 5000lx; the temperature-changing treatment was repeated 3 times.
4) High-temperature treatment: respectively treating the tissue culture seedlings subjected to heat treatment in the step 3) at a high temperature of 95 ℃ for 3min, 5min and 7min, taking out, directly stripping 1-2mm leaf apex meristems (shown as figure 1) on an ultra-clean workbench with naked eyes, inoculating the leaf apex meristems to a bud differentiation culture medium for culturing for 2-3 weeks to generate cluster buds, inoculating the adventitious buds to a bud differentiation and proliferation culture medium for culturing for 1-2 weeks to induce generation of more adventitious buds, and then inoculating the adventitious buds to a rooting culture medium for culturing for 1-2 weeks to obtain rooted seedlings, namely the detoxified seedlings of lily. Of these, those not treated at high temperature were used as a control group. Each group was subjected to 3 replicates.
The results show that: the growth of the leaf tips of the lily bulblets directly peeled from the control group without high temperature treatment is shown in Table 1. The leaf apex growth change of the surviving lilium siberia is shown in figure 2, and after the leaf apex is accessed into the culture medium, the leaf apex expands about 20 days and begins to form adventitious buds; the adventitious bud grows leaves and the root system grows about 40 days, and the adventitious bud grows 60 days to form a test-tube seedling.
TABLE 1 control of the growth of the leaf tips of the lilium siberia
Survival = number surviving/total number inoculated
The leaf tip growth conditions of the 'Siberian' lily small scale after high temperature treatment at 95 ℃ are shown in table 2, the leaf tip growth changes of the surviving 'Siberian' lily are shown in figure 3, and after the leaf tips are accessed into the culture medium, the leaf tips expand about 20 days and begin to form adventitious buds; the adventitious bud grows leaves and the root system grows about 40 days, and the adventitious bud grows about 60 days to form a test-tube seedling.
TABLE 2 growth of the small scale tips of Siberian lily treated at 95 deg.C
Survival = number of normal survivors/total number of inoculations
As can be seen from table 2, the survival rate of the test-tube plantlets decreased with the increase of the treatment time, and was extremely low when the treatment time was more than 7min, and no data was provided.
Example 2
The high temperature treatment temperature was 85 ℃ and the other steps were the same as in example 1.
After the high-temperature treatment at 85 ℃, the growth condition of the leaf tips of the lilium siberia small scales is shown in table 3, the growth change of the living lilium siberia leaf tips is shown in figure 4, and after the leaf tips are accessed into the culture medium, the leaf tips expand and begin to form adventitious buds after about 20 days; the adventitious bud grows leaves and the root system grows about 40 days, and the adventitious bud grows 60 days to form a test-tube seedling.
TABLE 3 growth of the leaf tips of the lilium siberia treated at 85 ℃
Survival = number of normal survivors/total number of inoculations
Example 3 detection of viruses in Lily Material
Extracting lily RNA: to test the detoxification effect, a certain number of seedlings were randomly extracted from the lily test-tube seedlings prepared in examples 1-2, and total RNA from leaves was extracted using a small amount extraction kit (R4151) for TIANGEN (TIANGEN) plant RNA, and stored at-20 ℃ for use.
cDNA Synthesis: cDNA Synthesis of eachThe total volume of the reaction was 20. Mu.l, first strand cDNA was synthesized using TIANGEN plant cDNA reverse transcription kit (KR 116), and the reaction reagents included 2. Mu.l total RNA, 2. Mu.l 5 Xg DNA Buffer, RNase-Free ddH 2 O6. Mu.l, incubating at 42 ℃ for 5min, and standing on ice; 10 XKing RT Buffer 2, fastKing RT Enzyme Mix 1. Mu.l, FQ-RT Primer Mix 2. Mu.l, RNase-Free ddH were added 2 O5. Mu.l, reaction program 30min for 42 ℃ incubation and 5min for 70 ℃ inactivation.
And (3) PCR amplification: the PCR reaction reagent adopts a Dalianbao biological product (RR 001A), the total volume of the PCR reaction is 20 mul, and the PCR reaction reagent specifically comprises: mu.l of reverse transcribed synthetic cDNA,10 XBuffer (20 mM Mg) 2+ ) Mu.l of 2. Mu.l, 2.5mM/l dNTP 2. Mu.l, 0.5. Mu.l of each of 10. Mu.M/l forward and reverse primers, 0.2. Mu.l of 5U/. Mu.l Taq DNA polymerase, 13.8. Mu.l ddH 2 O; the amplification procedure was: pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃ for 30s, annealing at 54 ℃ for 30s, and extension at 72 ℃ for 1min for 38 cycles; extending for 5min at 72 ℃; storing at 12 deg.C. The results are shown in FIGS. 5 to 6.
Specific primer pairs of LMoV, LSV and CMV are designed through a lily virus CP (coat protein) gene sequence, and the sizes of PCR products amplified respectively are 623bp, 439bp and 255bp. The lily 18S primer is used as an internal reference, and the specific sequence is as follows:
LMoV-F：5’-GTTCCAGGCAAATGAGACACTC-3’
LMoV-R：5’-GTGCTAGATTGAAGTCGGTGAGA-3’
LSV-F：5’-ATGAAGGTTGGCGTCGTAT-3’
LSV-R：5’-CCTCAGCAGAAGTGGGTC-3’
CMV-F：5’-TTGCGTTTCGTCTACTGGATCT-3’
CMV-R：5’-CAAAGGTTGGGTGGTTAATGG-3’
18S-F：5’-CGCAAGGCTGAAACTTAAAGG-3’
18S-R：5’-CAGACAAATCGCTCCACCAAC-3’
the results show that the test-tube plantlets after being treated at a high temperature of 85 ℃ for 3-7 min contain 1 or 2 of 3 viruses (CMV, LSV and LMoV). The test-tube plantlet after being treated at the high temperature of 95 ℃ for 3-5 min still contains 1 of 3 viruses. And the test-tube plantlet treated at the high temperature of 95 ℃ for 7min can not amplify target bands of CMV, LSV or LMoV, namely CMV, LSV and LMoV can be simultaneously detoxified, and the statistical results are shown in tables 4 and 5.
TABLE 4 detoxification rate of lily bulb scale leaf tips treated at 85 deg.C
TABLE 5 detoxification rate of lily bulb scale leaf tips treated at 95 DEG C
The results show that: in the test-tube plantlet cultured after the high-temperature treatment at 95 ℃ for 7min, the detoxification rate of CMV is 100%, the detoxification rate of LSV is 83.3%, the detoxification rate of LMoV is 100%, and the test-tube plantlet without virus CMV, LSV or LMOV is the detoxified plantlet. It can be seen that the detoxification effect of the present invention cannot be achieved either by too low a treatment temperature (e.g. 85 ℃) or by too short/long a treatment time.
Example 4 tissue culture Rapid propagation of detoxified seedlings
1) Adventitious bud induction
Taking the detoxified seedling in the embodiment 3 to continue to culture for 15-30 days, taking the scale of the outer layer of the detoxified seedling to inoculate in an adventitious bud induction culture medium with MS +6-BA 1.5mg/L + NAA 0.2mg/L + sucrose 30g/L and pH of 5.6-6.0, wherein the induction culture condition is that the culture temperature is 25 +/-2 ℃, the humidity is 60-70%, the illumination intensity is 3000lx-5000lx, and the sunshine time is 12-14 h/d for culture for 30-45 days.
2) Multiplication subculture by using adventitious buds of virus-free seedlings
Respectively inoculating the adventitious buds induced in the step 1) into MS +6-BA 2.0mg/L + NAA 0.2mg/L + sucrose 30g/L and pH5.6-6.0 subculture multiplication culture media, and inducing culture conditions: culturing for 30-45 days under the illumination conditions that the culture temperature is 25 +/-2 ℃, the humidity is 60-70%, the illumination intensity is 3000lx-5000lx and the sunshine time is 12-14 h/d to obtain the proliferated seedlings.
3) Inducing rooting by using proliferated seedling
Inoculating the proliferated seedling obtained in the step 2) into a rooting culture medium comprising MS, NAA 1mg/L and sucrose 30g/L and having pH of 5.6-6.0 for culture, wherein the induction culture conditions are as follows: culturing for 30-45 days under the illumination conditions that the culture temperature is 25 +/-2 ℃, the humidity is 60-70%, the illumination intensity is 3000lx-5000lx and the sunshine time is 12-14 h/d until the roots are rooted, thus obtaining the detoxified tissue culture seedling.
Finally, the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting, although the applicant has described the present invention in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention and shall be covered by the claims of the present invention.
Claims (7)
1. A high-temperature detoxification method based on lily bulb scale leaf tips is characterized by comprising the following steps:
1) Using lily bulbs or bulblets as materials, performing conventional surface disinfection treatment, then placing the materials on a primary culture medium for culturing for 2 to 3 weeks to obtain a lily aseptic bud material, and then using the lily aseptic buds as explants to culture in a secondary culture medium for 3 to 4 weeks to obtain a large number of aseptic bulbs with normal growth;
2) Placing the sterile bulblet obtained in the step 1) at 37 ℃ for pretreatment for 12h, then placing the aseptic bulblet at the normal temperature of 23-25 ℃ for treatment for 12h, and repeating the temperature change treatment for 3-5 times;
3) Treating the material subjected to heat treatment in the step 2) at 95 ℃ for 7min, then stripping off the leaf tips of the scales with the length of 1-2mm, inoculating the leaf tips to a bud differentiation culture medium, culturing for 2-3 weeks, inducing to generate cluster buds, then inoculating the cluster buds to a proliferation culture medium for 1-2 weeks, inducing to generate more adventitious buds, and then inoculating to a rooting culture medium, culturing for 1-2 weeks to obtain rooted seedlings, thus obtaining the lily detoxified seedlings;
4) Inoculating the scales of the detoxified seedlings in the step 3) to an adventitious bud induction culture medium for adventitious bud induction culture to generate adventitious buds, inoculating the adventitious buds to a subculture multiplication culture medium for subculture multiplication to obtain multiplied seedlings, and transferring the multiplied seedlings to a rooting culture medium for rooting culture to obtain rooted seedlings, namely obtaining a large number of detoxified tissue culture seedlings.
2. The high-temperature detoxification method based on lily bulb scale leaf tips as claimed in claim 1, wherein the disinfection is carried out by soaking in a washing powder solution for 5 to 15min, flushing under running water for 1 to 2h, placing the lily bulb scale leaf tips in an aseptic container, soaking in 75% by volume ethanol for 40s, treating with 0.1% by mass mercuric solution for 15min, and flushing with sterile water for 6 to 8 times.
3. The high-temperature detoxification method based on lily scales leaf tips as claimed in claim 1, wherein the adventitious bud induction culture medium is MS +6-BA 1.5mg/L + NAA 0.2mg/L + sucrose 30g/L + agar 7g/L, and the pH value of the culture medium is 5.6-6.0.
4. The high-temperature detoxification method based on lily scales as claimed in claim 1, wherein the subculture multiplication medium is MS +6-BA 2.0mg/L + NAA 0.2mg/L + sucrose 30g/L + agar 7g/L, and the pH value of the medium is 5.6-6.0.
5. The high-temperature detoxification method based on lily scales leaf tips as claimed in claim 1, wherein the rooting medium is MS + NAA 1mg/L + sucrose 30g/L + agar 7g/L, and the pH value of the medium is 5.6-6.0.
6. The high-temperature detoxification method of lily scale leaf tips according to claim 1, wherein the conditions of adventitious bud induction culture, subculture multiplication culture and rooting culture are as follows: the culture temperature is 25 +/-2 ℃, the humidity is 60-70%, the illumination intensity is 3000lx to 5000lx, and the sunshine time is 12-14 h/d.
7. Use of the method according to any one of claims 1 to 6 for breeding non-toxic lily seedlings.
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