CN111280059A - Efficient lily detoxification method - Google Patents
Efficient lily detoxification method Download PDFInfo
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- CN111280059A CN111280059A CN202010222990.9A CN202010222990A CN111280059A CN 111280059 A CN111280059 A CN 111280059A CN 202010222990 A CN202010222990 A CN 202010222990A CN 111280059 A CN111280059 A CN 111280059A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
Abstract
The invention discloses a high-efficiency lily detoxification method, which comprises the following steps: 1) selecting healthy and non-rotten lily scales to extract RNA, and performing virus detection through reverse transcription; 2) selecting healthy lily scales in the middle and inner layers for explant disinfection, and culturing to obtain a large number of aseptic seedlings with good growth vigor; 3) cutting the aseptic seedling small scale, and inducing to obtain compact callus; 4) inducing the callus into seedlings through differentiation culture; 5) cutting the leaves in the step 4) for virus detection, and obtaining lily virus-free seedlings after the detection is qualified. The lily detoxification method provided by the invention can be used for rapidly obtaining nontoxic seedlings by inducing the callus, solves the problems of complex lily detoxification process, high technical requirement and long time consumption at present, has the characteristics of high detoxification efficiency, simple process and low production equipment requirement, and can meet the requirement of industrial production.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture and detoxification, relates to a lily detoxification method, and particularly relates to a lily detoxification method for detoxification through callus induction.
Background
Lily plays a very important role in the flower family as a bulbous flower. However, the vegetative propagation of bulbs, scales, bulblets and the like is adopted for a long time, so that virus infection is easy to occur, bulb degeneration is often accompanied, and the quality and the yield of the lily are seriously influenced. Among them, viral diseases are one of the major diseases occurring worldwide in recent years. With the rapid increase of the import, introduction and planting area of the lilies in China and the non-standard planting and self-propagation of the seed balls, virus diseases begin to spread in the lily planting areas in China. The common incidence rate is 40% -50%, and the disease rate of the second generation seedball is more than 90%. Of the ten reported viruses infecting lily, the most prevalent and most harmful viruses occur in 3: cucumber Mosaic Virus (CMV), Lily Symptomless Virus (LSV) and Lily mottle virus (LMoV), which often occur in the form of multiple infections, mottle or deform the stems, leaves and flowers of plants, seriously resulting in dwarfing, degeneration and even death of plants, and seriously affecting the yield and quality of lilies. These viruses are mainly transmitted through insect vectors such as aphids, and the transmission speed is high, so that the viruses are difficult to be directly and effectively controlled by chemical agents or biological agents.
At present, the study on lily virus-free technology mainly focuses on the comprehensive application of technologies such as stem tip peeling, heat treatment and chemical treatment, and the like, and although the methods can effectively remove lily viruses. But the problems of more required detoxification materials, high pollution rate, difficult technical operation, complex working procedures, long period and the like exist, and the requirement of large-scale production is difficult to meet. For example, in chinese patent No. CN201410420844.1, "a lily detoxification method based on embryogenic callus specific induction", scale of lilium martagon was sterilized, then callus was induced, heat-treated after seedling formation, stem tips were peeled off, callus was induced again, and then seedling formation was induced. The detoxification method has the following problems: the demand of detoxification materials is very high, stem tips are not easy to strip, the survival rate is low, the process is complex, the consumed time is long, the technical requirement is high, and the method is difficult to apply to industrial production. In addition, chinese patent No. CN201410586353.4, "a comprehensive detoxification method of lily bulbs", which comprises performing heat treatment detoxification under the greenhouse environment conditions of 36-38 ℃ and 70-86% humidity, and then performing explant sterilization and growth point collection induction culture, and has the following problems: the equipment cost is high, the growth point is not easy to strip, the technical difficulty is high, the formed virus-free bulb is seriously degraded, although a part of viruses can be removed, the virus is easy to carry out secondary infection, and the practical application value is low. In addition, the virus detection of the lily bulbs before treatment is not carried out basically in the prior patent so as to confirm whether the raw materials are infected by the virus, so that the statistics of the virus removal rate is scientific.
Disclosure of Invention
The invention aims to overcome the defects of the existing lily detoxification technology, virus detection is carried out before detoxification treatment, and the detoxification method which has high sustainability, is easy to operate, has a short period and can be applied to large-scale production of lily is provided.
The purpose of the invention is realized by the following technical scheme:
a high-efficiency lily detoxification method comprises the following steps:
1) selecting healthy and non-rotten lily scales to extract RNA, and performing virus detection through reverse transcription;
2) selecting healthy lily scales in the middle and inner layers for explant disinfection, and culturing to obtain a large number of aseptic seedlings with good growth vigor;
3) cutting the aseptic seedling small scale, and inducing to obtain compact callus;
4) inducing the callus into seedlings through differentiation culture;
5) cutting the leaves in the step 4) for virus detection, and obtaining lily virus-free seedlings after the detection is qualified.
As a preferred technical scheme:
the detailed process of virus detection in step 1) is as follows: selecting healthy lily scales without rot, freezing the lily scales into liquid nitrogen, and extracting RNA by adopting a Trizol method; then carrying out reverse transcription by using a takara reverse transcription kit; designing primers of cucumber mosaic virus, lily symptomless virus and lily mottle virus for PCR amplification, and detecting whether the three viruses are contained; an important reagent for RNA extraction is Trizol. Random 6mers of reagents important in reverse transcription.
The detailed process of culturing to obtain the sterile seedlings in the step 2) comprises the following steps: selecting seed balls with less scabs, peeling the healthy scales at the middle and inner layers, washing the seeds under running water, sequentially disinfecting the seeds with 75% ethanol and 1% sodium hypochlorite, and sterilizing ddH2Washing with O for multiple times, and placing on filter paper for air drying; cutting the scales into squares, placing the squares with the concave surfaces upward in a bud inducing culture medium, and placing the squares in a tissue culture room for growth;
step 3) cutting sterile seedling small scales, and obtaining compact callus by induction in a detailed process: cutting off leaves of plants growing to 3-8cm high, peeling off the small scales with the concave surfaces facing upwards, and placing the small scales in an induction callus culture medium; placing the seeds in a tissue culture room for growth;
the detailed process of inducing the callus to be seedlings through differential culture in the step 4) comprises the following steps: cutting the compact callus to obtain mother body, subculturing for 1-2 times, and transferring to differentiation culture medium; placing the seeds in a tissue culture room for growth;
step 5) the detailed process of cutting the leaves in the step 4) for virus detection is as follows: randomly extracting 10-20% of the lily bulblets subjected to detoxification treatment in the step 4), carrying out RT-PCR (reverse transcription-polymerase chain reaction) retrovirus detection, and confirming that the lily bulblets do not contain cucumber mosaic virus, lily symptomless virus and lily mottle virus through detection, namely the lily detoxified seedlings.
Further preferred is: washing the middle and inner layer healthy scales stripped in the step 2) under running water for 2 hours, sterilizing with 75% ethanol for 30 seconds and 1% sodium hypochlorite for 15-20 minutes, and sterilizing with ddH2Washing with O for 5-8 times, and air drying.
The bud induction culture medium in the step 2) is as follows: MS +2.0mg/L6-BA +0.2mg/LNAA +7g/L agar +15g/L sucrose.
The callus induction culture medium in the step 3) is as follows: MS +1.0mg/LTDZ +2.0mg/L PIC +7g/L agar +15g/L sucrose.
The light culture condition of the small scale induction callus in the step 3) is dark and 20 ℃.
The differentiation medium in the step 4) is as follows: MS +0.4g/L active carbon +0.2mg/L NAA +9g/L agar +40g/L sucrose.
The transfer interval time of the callus subculture in the step 4) is 10-15 days. The culture conditions of the callus induction seedling in the step 4) are as follows: the light intensity was 1500Lx and the temperature was 20 ℃.
The detoxification principle of the high-efficiency lily detoxification method provided by the invention is as follows:
1) and possibly removing a part of viruses in the step 2) in the process of establishing the sterile seedlings by using the field seedlings: lily scales are used as explants, after induction and budding, the bud parts are cut for proliferation and growth. The growth rate of the buds is faster than the amplification rate of the virus, and the virus-containing mother bodies are discarded, so that a part of the virus can be removed.
2) And 3) taking the scales of the aseptic seedlings as explants to induce callus, wherein part of viruses are removed in the step 3): taking the scales of the aseptic seedlings as explants, inducing callus, cutting compact callus out of a matrix, and placing the matrix into a differentiation culture medium. The dense callus grew at a much greater rate than the virus amplification and rejected the virus-containing maternal portion, thus eliminating the virus.
The research of the invention shows that the process of establishing and culturing the sterile seedling by using the field seedling has no way of completely and simultaneously removing the three viruses, and the three viruses can be removed simultaneously only by combining two steps.
The innovation point of the technical scheme of the invention is as follows:
1) compared with the content of the existing invention, most of the methods adopted by the invention mostly take the stem tip as an explant, the stem tip is cut again after heat treatment, and the like, so that the operation difficulty is high, the steps are complicated, the detoxified seedling is successfully obtained by utilizing the scale of the aseptic seedling through one-time callus culture, the actual operation is easy, and the steps are few. The scale is used as an explant for callus induction, the callus induction rate is high, and the obtained callus has a good state. Therefore, the research has obvious innovation.
2) The callus induction medium used in this study was initiated, MS +1.0mg/LTDZ +2.0mg/LPIC +15g/L sucrose +7g/L agar. In the conventional callus induction medium, PIC or 2, 4-D is usually added, and the TDZ and PIC are used together in the present invention. Detoxification is carried out by using callus induction, the state of callus is the key for successful detoxification, and under the induction of the culture medium, compact and fast-growing embryogenic callus is obtained (as shown in figure 1).
3) After more than two years of research, the research finds that the detoxification effect of the scale-induced callus induced by grafting for 10-15 days is the best, and the growth speed of the callus is far faster than the propagation speed of the virus in the culture time. And inducing the differentiation of the plants after 10 to 15 days, and quickly forming seedlings.
4) In the research, virus detection is firstly carried out on the original seed ball, the fact that the virus-free material is infected with virus is determined (as shown in a virus detection glue picture before virus removal shown in figure 2), and finally, virus detection is carried out on the material subjected to virus removal treatment (as shown in a virus detection result after callus induction virus removal treatment shown in figure 3), so that the effectiveness of the virus removal method is more fully explained. Other inventions only detect the material that has finally been detoxified.
5) The virus-free seedlings could be obtained around 14 weeks by operating with the method of this study: 5 weeks are needed for establishing the sterile seedling system, the time for inducing callus and subculturing the callus by taking the sterile seedling scales as explants is 5 weeks, and the time for inducing the callus to be seedlings is 4 weeks.
Therefore, the research is innovative in combination of detoxification methods, innovative in study of callus-induced culture medium, innovative in consideration of effectiveness of detoxification methods, and capable of obtaining detoxified seedlings in a short time.
The invention relates to a high-efficiency lily detoxification method, which is a result obtained by the inventor through long-time exploration and research on the basis of a large number of experiments, and compared with the prior art, the method has the beneficial effects that:
by adopting the callus induction method, the invention effectively overcomes the problems of more detoxification materials, long detoxification steps, long detoxification time, and high required technology and equipment conditions, and can efficiently and quickly obtain the detoxification tissue culture seedlings.
Different from other researches which directly carry out detoxification on each part of the field seedling, the method adopts the technical scheme that the field seedball is firstly utilized to establish a sterile seedling system, so that the pollution rate and required materials are greatly reduced, and the detoxification efficiency is accelerated.
The invention adopts one generation of aseptic seedling scales to induce the callus, can remove cucumber mosaic virus, lily symptomless virus and lily mottle virus at one time, and obtains virus-free seedlings in 14 weeks. If aseptic seedlings are directly used as experimental materials, the detoxification time is only 9 weeks.
After the virus-free seedlings are obtained, a proper amount of activated carbon is added into a conventional culture medium, and a large amount of virus-free seedlings are quickly obtained at a very high propagation rate in a tissue culture mode, so that the virus-free seedlings can be widely applied to industrial production.
Drawings
FIG. 1 shows the state of lily callus under a stereomicroscope.
Wherein, A: healing at the initial stage; b: and (5) callus at the later stage.
FIG. 2 shows the results of detection of stock coccidiosis virus.
Wherein, M: DNA Marker DL 2000; 1: a CMV; 2: LSV; 3: LMoV).
FIG. 3 shows the virus detection results after callus induction detoxification treatment.
Wherein, a) CMV virus detection condition; b) LSV virus detection; c) LMoV virus detection;
m: 2000 marker; 1, positive control; 2-4: treating the seedlings; 5: and (5) negative control.
Detailed Description
The present invention will be further described with reference to the following examples.
A high-efficiency lily detoxification method comprises the following detailed steps:
1) selecting healthy and non-rotten lily scales to extract RNA, and performing virus detection through reverse transcription: selecting 0.5g of non-rotten healthy lily scales, freezing the lily scales into liquid nitrogen, and extracting RNA by adopting a trizol method; then carrying out reverse transcription by using a takara reverse transcription kit; primers (shown in table 1) of cucumber mosaic virus, lily symptomless virus and lily mottle virus are designed, a 20 microliter system is adopted for PCR amplification, and whether the three viruses are contained or not is detected;
TABLE 1 RT-PCR specific primers
2) Selecting healthy lily scales in the middle and inner layers for explant disinfection, and culturing to obtain a large number of aseptic seedlings with good growth vigor: selecting seed balls with less disease spots, stripping the healthy scales at the middle and inner layers, washing for 2 hours under running water, sterilizing for 30 seconds by using 75% ethanol, sterilizing for 15-20 minutes by using 1% sodium hypochlorite, and sterilizing ddH2Flushing for 5-8 times with O, and air drying by placing on filter paper; the scale is cut into squares with the size of 1 multiplied by 1cm, the concave surface is upwards placed in a bud induction culture medium, and the bud induction culture medium comprises the following components: MS +2.0mg/L6-BA +0.2mg/L NAA +15g/L sucrose +7g/L agar, pH of the culture medium is 5.8, and each culture medium is put into 2-3 pieces; growing in tissue culture room at illumination intensity of 1500Lx and temperature of 20 deg.C;
3) cutting aseptic seedling small scales, and inducing to obtain compact callus: cutting off leaves of a plant growing to 3-8cm high, peeling off the small scale, and placing the small scale with the concave surface facing upwards into an induction callus culture medium, wherein the induction callus culture medium comprises: MS +1.0mg/LTDZ +2.0mg/L PIC +15g/L sucrose +7g/L agar; placing in the dark, and growing at 20 ℃;
4) inducing the callus into seedlings through differentiation culture: cutting the compact callus to obtain the mother body, and subculturing for 1-2 times. Wherein, the transfer interval of the callus subculture is 10-15 days, and the callus subculture is transferred into a differentiation medium which is: MS +0.4g/L active carbon +0.2mg/L NAA +9g/L agar +40g/L sucrose; growing in tissue culture room at illumination intensity of 1500Lx and temperature of 20 deg.C;
5) cutting the leaves in the step 4) for virus detection, wherein the qualified detection is the lily virus-free seedling: randomly extracting 10-20% of the lily bulblets subjected to detoxification treatment in the step 4), carrying out RT-PCR (reverse transcription-polymerase chain reaction) retrovirus detection, and confirming that the lily bulblets do not contain cucumber mosaic virus, lily symptomless virus and lily mottle virus through detection, namely the lily detoxified seedlings. Callus is a mass of undifferentiated tissue formed from the interior or cut surface of an explant, which has the ability to redifferentiate. The invention obtains the detoxified seedling by means of the characteristics of the callus, and does not comprise other meanings.
The RT-PCR retrovirus detection method comprises the following specific steps:
1) reverse transcription
cDNA synthesis was described with reference to the takara kit.
The sampling system is as follows:
keeping the temperature at 65 ℃ for 5min, quickly cooling on ice, and continuously adding samples.
The sampling system is as follows:
slowly mixing the mixture evenly, and carrying out reverse transcription according to the following conditions:
10min at 30 ℃; 30-60 min at 42 ℃; 5min at 95 ℃; and (5) cooling on ice.
2) PCR amplification was performed using the synthesized 5 'primer and 3' primer using the synthesized first strand of cDNA as a template.
The PCR amplification system is as follows:
the reaction procedure is as follows:
the reaction was performed for 36 cycles on a PCR amplificator; extension was carried out at 72 ℃ for 2min and the reaction was stopped at 4 ℃. 10. mu.L of the amplification product was subjected to 1% agarose gel electrophoresis, and the results were observed using a BioRad gel imaging system.
Example 1 a highly efficient method for detoxifying lily, the method comprises the following steps:
1) selecting healthy and non-rotten lily scales to extract RNA, and performing virus detection through reverse transcription: selecting 0.5g of non-rotten healthy lily scales, freezing the lily scales into liquid nitrogen, and extracting RNA by adopting a Trizol method; then carrying out reverse transcription by using a takara reverse transcription kit; primers (shown in table 1) of cucumber mosaic virus, lily symptomless virus and lily mottle virus are designed, a 20 microliter system is adopted for PCR amplification, and whether the three viruses are contained or not is detected;
2) selecting healthy lily scales in the middle and inner layers for explant disinfection, and culturing to obtain a large number of aseptic seedlings with good growth vigor: selecting seed balls with less scabs, stripping the healthy scales at the middle and inner layers, washing for 2 hours under running water, sterilizing for 30 seconds by using 75% ethanol, sterilizing for 15 minutes by using 1% sodium hypochlorite, and sterilizing ddH2Washing with O for 5 times, and air drying by placing on filter paper; the scale is cut into squares with the size of 1 multiplied by 1cm, the concave surface is upwards placed in a bud induction culture medium, and the bud induction culture medium comprises the following components: MS +2.0mg/L6-BA +0.2mg/L NAA +15g/L sucrose +7g/L agar, pH of 5.8, put to 3 pieces per medium; growing in tissue culture room at illumination intensity of 1500Lx and temperature of 20 deg.C;
3) cutting the aseptic seedling small scale, and inducing to obtain compact callus; cutting off leaves of plants growing to 3cm high, peeling small scales, and placing the small scales with concave faces upwards into an induction callus culture medium, wherein the induction callus culture medium comprises: MS +1.0mg/LTDZ +2.0mg/LPIC +15g/L sucrose +7g/L agar; placing in the dark, and growing at 20 ℃;
4) inducing the callus into seedlings through differentiation culture; the callus grown to compact state was excised from the parent and subcultured 2 times. Wherein, the transfer interval of the callus subculture is 10 days, and the callus subculture is transferred to a differentiation medium which is: MS +0.4g/L active carbon +0.2mg/L NAA +9g/L agar +40g/L sucrose; growing in tissue culture room at illumination intensity of 1500Lx and temperature of 20 deg.C;
5) cutting the leaves in the step 4) for virus detection, wherein the qualified detection is the lily virus-free seedling: randomly extracting 10-20% of the lily bulblets subjected to detoxification treatment in the step 4), performing RT-PCR (reverse transcription-polymerase chain reaction) retrovirus detection (the specific steps are as described above), and determining that the lily bulblets do not contain cucumber mosaic virus, lily symptomless virus and lily mottle virus through detection, namely the lily detoxified seedlings.
Example 2 a highly effective method for detoxifying lily, the method comprises the following steps:
1) selecting healthy and non-rotten lily scales to extract RNA, and performing virus detection through reverse transcription: selecting 0.5g of non-rotten healthy lily scales, freezing the lily scales into liquid nitrogen, and extracting RNA by adopting a Trizol method; then carrying out reverse transcription by using a takara reverse transcription kit; primers (shown in table 1) of cucumber mosaic virus, lily symptomless virus and lily mottle virus are designed, a 20 microliter system is adopted for PCR amplification, and whether the three viruses are contained or not is detected;
2) selecting healthy lily scales in the middle and inner layers for explant disinfection, and culturing to obtain a large number of aseptic seedlings with good growth vigor: selecting seed balls with less scabs, stripping the healthy scales at the middle and inner layers, washing for 2 hours under running water, sterilizing for 30 seconds by using 75% ethanol, sterilizing for 15 minutes by using 1% sodium hypochlorite, and sterilizing ddH2Washing with O for 5 times, and air drying by placing on filter paper; the scale is cut into squares with the size of 1 multiplied by 1cm, the concave surface is upwards placed in a bud induction culture medium, and the bud induction culture medium comprises the following components: MS +2.0mg/L6-BA +0.2mg/LNAA +15g/L sucrose +7g/L agar, pH 5.8, 3 pieces of each medium; growing in tissue culture room at illumination intensity of 1500Lx and temperature of 20 deg.C;
3) cutting the aseptic seedling small scale, and inducing to obtain compact callus; cutting off leaves of a plant growing to 5cm high, peeling the small scale, and placing the plant with the concave surface facing upwards into an induction callus culture medium, wherein the induction callus culture medium comprises: MS +1.0mg/LTDZ +2.0mg/LPIC +15g/L sucrose +7g/L agar; placing in the dark, and growing at 20 ℃;
4) inducing the callus into seedlings through differentiation culture; the callus grown to compact state was excised from the parent and subcultured 1 time. Wherein the transfer interval of the callus subculture is 13 days, and transferring the callus subculture into a differentiation medium which comprises the following components: MS +0.4g/L active carbon +0.2mg/L NAA +9g/L agar +40g/L sucrose; growing in tissue culture room at illumination intensity of 1500Lx and temperature of 20 deg.C;
5) cutting the leaves in the step 4) for virus detection, wherein the qualified detection is the lily virus-free seedling: randomly extracting 10-20% of the lily bulblets subjected to detoxification treatment in the step 4), performing RT-PCR (reverse transcription-polymerase chain reaction) retrovirus detection (the specific steps are as described above), and determining that the lily bulblets do not contain cucumber mosaic virus, lily symptomless virus and lily mottle virus through detection, namely the lily detoxified seedlings.
Example 3 a highly efficient method for detoxifying lily, the method comprises the following steps:
1) selecting healthy and non-rotten lily scales to extract RNA, and performing virus detection through reverse transcription: selecting 0.5g of non-rotten healthy lily scales, freezing the lily scales into liquid nitrogen, and extracting RNA by adopting a Trizol method; then carrying out reverse transcription by using a takara reverse transcription kit; primers (shown in table 1) of cucumber mosaic virus, lily symptomless virus and lily mottle virus are designed, a 20 microliter system is adopted for PCR amplification, and whether the three viruses are contained or not is detected;
2) selecting healthy lily scales in the middle and inner layers for explant disinfection, and culturing to obtain a large number of aseptic seedlings with good growth vigor: selecting seed balls with less scabs, stripping the healthy scales at the middle and inner layers, washing for 2 hours under running water, sterilizing for 30 seconds by using 75% ethanol, sterilizing for 20 minutes by using 1% sodium hypochlorite, and sterilizing ddH2Washing with O for 8 times, and air drying by placing on filter paper; the scale is cut into squares with the size of 1 multiplied by 1cm, the concave surface is upwards placed in a bud induction culture medium, and the bud induction culture medium comprises the following components: MS +2.0mg/L6-BA +0.2mg/L NAA +15g/L sucrose +7g/L agar, pH of 5.8, put to 3 pieces per medium; growing in tissue culture room at illumination intensity of 1500Lx and temperature of 20 deg.C;
3) cutting the aseptic seedling small scale, and inducing to obtain compact callus; cutting off leaves of a plant growing to 8cm high, peeling the small scale, and placing the plant with the concave surface facing upwards into an induction callus culture medium, wherein the induction callus culture medium comprises: MS +1.0mg/LTDZ +2.0mg/LPIC +15g/L sucrose +7g/L agar; placing in the dark, and growing at 20 ℃;
4) inducing the callus into seedlings through differentiation culture; the callus grown to compact state was excised from the parent and subcultured 2 times. Wherein the transfer interval of the callus subculture is 15 days, and transferring the callus subculture into a differentiation medium which comprises the following components: MS +0.4g/L active carbon +0.2mg/L NAA +9g/L agar +40g/L sucrose; growing in tissue culture room at illumination intensity of 1500Lx and temperature of 20 deg.C;
5) cutting the leaves in the step 4) for virus detection, wherein the qualified detection is the lily virus-free seedling: randomly extracting 10-20% of the lily bulblets subjected to detoxification treatment in the step 4), performing RT-PCR (reverse transcription-polymerase chain reaction) retrovirus detection (the specific steps are as described above), and determining that the lily bulblets do not contain cucumber mosaic virus, lily symptomless virus and lily mottle virus through detection, namely the lily detoxified seedlings.
By adopting the callus induction method, the invention effectively overcomes the problems of more detoxification materials, long detoxification steps, long detoxification time, and high required technology and equipment conditions, and can efficiently and quickly obtain the detoxification tissue culture seedlings. In the embodiment, the callus is induced by using the first generation of sterile seedling scales, cucumber mosaic virus, lily symptomless virus and lily mottle virus can be removed at one time, and the virus-free seedlings are obtained in 14 weeks. After the detoxified seedlings are obtained, a proper amount of activated carbon (added in a differentiation culture medium to assist the growth of tissue culture seedlings) is added in a conventional culture medium, and a large amount of detoxified seedlings are quickly obtained at a very high propagation rate in a tissue culture mode, so that the method can be widely applied to industrial production.
The invention establishes a first generation sterile seedling system, takes the small scales to perform callus induction, and utilizes the extremely strong redifferentiation capability of the callus to induce seedling formation, thereby effectively overcoming the problems of complicated detoxification steps, high environmental condition requirement, high detoxification cost and long time existing in the prior lily detoxification technology. According to the invention, a generation of sterile seedling system is established, and then the small scales of the sterile seedling system are cut for callus induction, so that the tissue culture seedling without cucumber mosaic virus, lily symptomless virus and lily mottle virus is obtained at one time, and the tissue culture seedling has the advantages of high detoxification efficiency, simplicity in operation, short time, low environmental requirement, low cost and the like, and is suitable for large-scale industrial production.
The parts not involved in the present invention are the same as or can be implemented using the prior art. The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A high-efficiency lily detoxification method is characterized by comprising the following steps: the method comprises the following steps:
1) selecting healthy and non-rotten lily scales to extract RNA, and performing virus detection through reverse transcription;
2) selecting healthy lily scales in the middle and inner layers for explant disinfection, and culturing to obtain a large number of aseptic seedlings with good growth vigor;
3) cutting the aseptic seedling small scale, and inducing to obtain compact callus;
4) inducing the callus into seedlings through differentiation culture;
5) cutting the leaves in the step 4) for virus detection, and obtaining lily virus-free seedlings after the detection is qualified.
2. The method of claim 1, wherein:
the detailed process of virus detection in step 1) is as follows: selecting healthy lily scales without rot, freezing the lily scales into liquid nitrogen, and extracting RNA by adopting a Trizol method; then carrying out reverse transcription by using a takara reverse transcription kit; designing primers of cucumber mosaic virus, lily symptomless virus and lily mottle virus for PCR amplification, and detecting whether the three viruses are contained;
the detailed process of culturing to obtain the sterile seedlings in the step 2) comprises the following steps: selecting seed balls with less scabs, peeling the healthy scales at the middle and inner layers, washing the seeds under running water, sequentially disinfecting the seeds with 75% ethanol and 1% sodium hypochlorite, and sterilizing ddH2Washing with O for multiple times, and placing on filter paper for air drying; cutting the scales into squares, placing the squares with the concave surfaces upward in a bud inducing culture medium, and placing the squares in a tissue culture room for growth;
step 3) cutting sterile seedling small scales, and obtaining compact callus by induction in a detailed process: cutting off leaves of plants growing to 3-8cm high, peeling off the small scales with the concave surfaces facing upwards, and placing the small scales in an induction callus culture medium; placing the seeds in a tissue culture room for growth;
the detailed process of inducing the callus to be seedlings through differential culture in the step 4) comprises the following steps: cutting the compact callus to obtain mother body, subculturing for 1-2 times, and transferring to differentiation culture medium; placing the seeds in a tissue culture room for growth;
step 5) the detailed process of cutting the leaves in the step 4) for virus detection is as follows: randomly extracting 10-20% of the lily bulblets subjected to detoxification treatment in the step 4), carrying out RT-PCR (reverse transcription-polymerase chain reaction) retrovirus detection, and confirming that the lily bulblets do not contain cucumber mosaic virus, lily symptomless virus and lily mottle virus through detection, namely the lily detoxified seedlings.
3. The method of claim 2, wherein: washing the middle and inner layer healthy scales stripped in the step 2) under running water for 2 hours, sterilizing with 75% ethanol for 30 seconds and 1% sodium hypochlorite for 15-20 minutes, and sterilizing with ddH2Washing with O for 5-8 times, and air drying.
4. The method of claim 2, wherein: the bud induction culture medium in the step 2) is as follows: MS +2.0mg/L6-BA +0.2mg/L NAA +7g/L agar +15g/L sucrose.
5. The method of claim 2, wherein: the callus induction culture medium in the step 3) is as follows: MS +1.0mg/LTDZ +2.0mg/L PIC +7g/L agar +15g/L sucrose.
6. The method of claim 2, wherein: and 3) the light culture condition for inducing the callus by the small scales is dark.
7. The method of claim 2, wherein: the differentiation medium in the step 4) is as follows: MS +0.4g/L active carbon +0.2mg/L NAA +9g/L agar +40g/L sucrose.
8. The method of claim 2, wherein: the transfer interval time of the callus subculture in the step 4) is 10-15 days.
9. The method according to claim 1 or 2, characterized in that: the culture conditions of the callus induction seedling in the step 4) are as follows: the light intensity was 1500Lx and the temperature was 20 ℃.
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