CN109845644B - Method for removing pseudostellaria heterophylla fava bean wilting virus by using micro-stem tip and ultralow temperature - Google Patents
Method for removing pseudostellaria heterophylla fava bean wilting virus by using micro-stem tip and ultralow temperature Download PDFInfo
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Abstract
The invention provides a method for removing pseudostellaria heterophylla fava bean wilting virus by utilizing micro-stem tips and ultralow temperature, which comprises the following steps: 1) taking field pseudostellaria stem sections as explants for tissue culture, sterilizing and culturing to obtain tissue culture seedlings, carrying out subculture to obtain a large number of tissue culture seedlings, carrying out micro-stem tip culture to obtain micro-stem tip virus-free seedlings, 3) pre-culturing and loading, 4) vitrifying, 5) treating with liquid nitrogen, 6) unfreezing, 7) inducing seedling formation, and 8) carrying out virus detection. The virus-free seedling obtained by removing the broad bean wilting virus BBMV of the radix pseudostellariae by combining the micro stem tip with the ultra-low temperature cryotherapy is thoroughly removed, the stem tip stripping is easy to operate, and the method has wide application value in the aspect of promoting the healthy and sustainable development of the radix pseudostellariae industry.
Description
Technical Field
The invention belongs to the field of plant tissue culture, and particularly relates to a method for removing pseudostellaria heterophylla fava bean wilting virus by utilizing micro-stem tips and ultralow temperature.
Background
Radix Pseudostellariae is root tuber of Pseudostellaria heterophylla of Caryophyllaceae. Collected in summer when most of the stem leaves wither, cleaned, removed of fibrous roots, placed in boiling water for slightly scalding and then dried in the sun or directly dried in the sun. Radix Pseudostellariae calendar "pharmacopoeia of the people's republic of China" is collected. Radix Pseudostellariae has effects of invigorating qi and spleen, promoting fluid production and moistening lung; the traditional Chinese medicine composition is clinically used for treating spleen deficiency and tiredness, inappetence, weakness after illness, deficiency of qi and yin, spontaneous perspiration and thirst, lung dryness and dry cough and other symptoms. The dosage of the radix pseudostellariae is increased year by year, and the market prospect is wide.
Due to the fact that the radix pseudostellariae root tuber is used for asexual propagation for a long time, the radix pseudostellariae virus diseases are commonly caused and serious diseases are caused. According to investigation, the incidence area of the floral leaf diseases of the radix pseudostellariae in the area is as high as 40-50%; the incidence of disease in part of growing areas is still as high as 60% or more, which leads to annual decline in yield. The main types of viruses for which the radix pseudostellariae is infected are reported at present: turnip mosaic virus (TuMV), Cucumber Mosaic Virus (CMV), Broad Bean Wilting Virus (BBWV), and Tobacco Mosaic Virus (TMV). At present, there are various methods for obtaining virus-free plants, and related technologies thereof have many patents, but the existing methods do not disclose the production process of virus-free radix pseudostellariae, have high inoculation pollution and low transplanting survival rate, and do not provide a way for solving the problem of virus re-infection and the like.
Disclosure of Invention
The invention aims to provide a method for removing pseudostellaria heterophylla fava bean wilting virus by utilizing micro-stem tips and ultralow temperature.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for removing pseudostellaria heterophylla fava bean wilting virus by utilizing micro-stem tips and ultralow temperature comprises the following steps:
1) selecting a radix pseudostellariae plant expressing broad bean wilting virus symptoms;
2) cutting a diseased plant into stem sections, inoculating the stem sections into an MS culture medium containing 0.2mg/L NAA after sterilization, inoculating the stem sections into the MS culture medium when the stem sections grow to 4 sections, inoculating 1-2mm bud tips into the MS culture medium when side buds grow to 1-1.5cm, and culturing the micro-stem tips to form seedlings;
3) cutting stem sections cultured by stem tips, connecting the stem sections to a primary culture medium, and culturing for 10 days at room temperature;
4) placing the stem segments of the radix pseudostellariae obtained in the step 2) on a subculture medium for room temperature culture for 1-6 weeks;
5) stripping 1-2mm of stem tips obtained in the step 3), reserving 1-3 sheets of leaf primordium, and placing the stem tips on a culture medium for dark culture for 1-3 days, wherein the culture medium is an MS culture medium added with 0.3mol/L sucrose and 7g/L agar, and the culture temperature is 4 ℃;
6) putting the stem tip pre-cultured in the step 4) into a loading liquid, wherein the loading liquid is an MS culture medium added with 2mol/L of glycerol and 1mol/L of sucrose, the pH is =5.8, and the stem tip is loaded for 20-40min at room temperature;
7) transferring the loaded stem tip into PVS2 solution, operating on ice, and treating for 30-50 min;
8) sucking PVS2 solution by a pipette gun, dripping the PVS2 solution in an aluminum foil box, putting the stem tip in the step 6) into the liquid drop, putting the aluminum foil box with the liquid drop into liquid nitrogen for processing for 30min, immediately carrying out water bath thawing processing at 40 ℃ for 0.2-1.5min, and then sending into an ice water mixture at 0 ℃ for processing for 10 min; quickly putting into unloading liquid for 20min, wherein the unloading liquid is MS culture medium added with 1.2mol/L sucrose;
9) taking out the stem tip, placing into a post-culture medium, dark-culturing at 0 deg.C for 5-7 days, and culturing under 200lux of light for 1 month to obtain radix Pseudostellariae regenerated plant;
10) and performing BBMV detection on the regenerated plant of the radix pseudostellariae by using an RT-PCR technology to obtain a strain with the BBMV removed from the radix pseudostellariae broad bean wilting virus.
The primary culture medium is: MS medium added 0.2mg/L naphthylacetic acid, 30g/L sucrose, 6.8g/L agar, pH = 5.8.
The subculture medium is MS medium + 30g/L sucrose +7g/L agar.
Step 7) PVS2 solution to MS medium was added glycerol to a final concentration of 30% (W/V), 15% (W/V) polyethylene glycol, 15% (W/V) dimethyl sulfoxide, and 0.4mol/L sucrose, pH = 5.8.
And 9) adding 0.05mol/L gibberellin, 7g/L agar and 30g/L sucrose into the MS culture medium as the post-culture medium.
The invention has the advantages that:
compared with the traditional detoxification method, the method has no strict requirement on the size of the taken stem tip, reduces the difficulty in operation, has the detoxification rate remarkably higher than that of the traditional stem tip tissue culture detoxification method, has simple and efficient detoxification operation, is easy to obtain virus-free plants, and opens up a new way for breeding the non-toxic seedlings of the radix pseudostellariae.
Drawings
FIG. 1 shows the BBMV detection result of the virus-free seedling treated at ultralow temperature, and the position of 1 has no band, which indicates that the plant does not carry the BBMV virus.
FIG. 2 shows BBMV detection results of stem-tip virus-free seedlings, and the position of 1 has a strip, which indicates that the plants still carry BBMV virus.
FIG. 3 shows the BBMV detection result of the virus-carrying test-tube plantlet obtained by explant culture, wherein 1 has a strip at the position, the strip is bright, and the plant carries a large amount of BBMV virus.
Detailed Description
Example 1
1. A method for removing pseudostellaria heterophylla fava bean wilting virus by utilizing micro-stem tips and ultralow temperature comprises the following steps:
1) the method takes the Pseudostellaria Cudrania tricuspidata No. 2 plant as a material, the plant material is represented by a mosaic, a mottle mosaic and a shrunken leaf, and the virus detection steps are as follows:
the material, the collection and the preservation of the radix pseudostellariae diseased plant sample come from Cudrania tricuspidata county of Ningde city in Fujian province. Fresh plant tissues are collected and rapidly transferred into liquid nitrogen for storage, and the liquid nitrogen is used for total RNA extraction.
A reagent total RNA extraction and purification Kit E.Z.N.A. Plant RNA Kit (R6827-01) was purchased from OMEGA; the reverse transcription kit TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix is purchased from Beijing all-purpose gold biotechnology (TransGen Biotech) Co.Ltd; r isTaqEnzymes were purchased from Takara Bio-engineering Ltd; a100 bp DNA Marker was purchased from Guangzhou Dongsheng Biotechnology Ltd.
Primer design according to GenBank registeredTuMV CPThe nucleotide sequence of the gene (accession number AJ 000690.1) was used to design TuMV primers, which were synthesized by Biotechnology (Shanghai) GmbH. An upstream primer F: 5'-AAGGCGTTGGTGCTGGTTA-3', respectively; a downstream primer R: 5'-CTCGTCAAGACTCCGCTGTA-3' are provided.
Extraction of viral RNA Total RNA extraction Plant RNA Kit (R6827-01) extraction Kit of OMEGA company for extraction, respectively using eppendorf BioPhotometer plus micro nucleic acid protein analyzer and agarose gel electrophoresis with mass fraction of 1.5% to detect concentration and purity, and using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix reverse transcription Kit to remove DNA and reverse to cDNA, and storing at-20 ℃ for later use.
RT-PCR detection virus PCR reaction system 50 mm3: 10 XPCR Buffer (containing Mg)2+）5 mm3，dNTP（2.5 mmol/dm3）4 mm3Forward and reverse primers (10. mu. mol/dm)3) Each 2mm3 ，Taq DNA Polymerase（5 U/ mm3）0.5 mm3The amount of cDNA template is about 2mm3Adding up double distilled water to total volume of 50 mm3. PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 40 cycles; extending for 10min at 72 ℃; stopping at 4 ℃. PCR products obtained by amplification are treated with 1.5% of agarAnalysis by lipolysis gel electrophoresis.
Finally, selecting the radix pseudostellariae plants expressing the symptoms of the broad bean wilting virus.
2) Cutting a diseased plant into stem segments, sterilizing, inoculating the stem segments into a 0.2mg/L NAA culture medium, inoculating the stem segments into an MS culture medium when the stem segments grow to 4 sections, inoculating the bud tips of about 1mm into the MS culture medium when the lateral buds grow to 1.5cm, and culturing the micro-stem tips to form seedlings.
3) Cutting stem segments cultured by stem tips, and connecting the stem segments to a primary culture medium, wherein the primary culture medium is as follows: MS add 0.2mg/L naphthylacetic acid, 30g/L sucrose, 6.8g/L agar, pH =5.8, incubate for 10d of one cycle.
4) The pseudostellaria root stem segments are taken and placed on a subculture medium ((30 g/L sucrose +7g/L agar + MS)) to be cultured for 3 weeks at room temperature.
5) Stripping 2mm of stem tip, leaving 2 pieces of leaf primordium, placing the stem tip on a culture medium for dark culture for 3 days, adding 0.3mol/L sucrose and 7g/L agar into the culture medium MS, and culturing at 4 ℃.
6) And (3) putting the pre-cultured stem tip into a loading liquid, wherein the loading liquid is MS, 2mol/L of glycerol and 1mol/L of cane sugar are added, the pH is =5.8, and the stem tip is loaded for 30min at room temperature.
7) The loaded stem tip is taken and transferred into a PVS2 solution, the PVS2 solution is MS, 30% (W/V) glycerol, 15% (W/V) polyethylene glycol, 15% (W/V) dimethyl sulfoxide and 0.4mol/L sucrose are added to the solution, the pH is =5.8, and the operation is carried out on ice for 40 min.
8) Sucking PVS2 solution by using a liquid transfer gun, dripping the PVS2 solution in an aluminum foil box, putting the stem tip in the step 6) into the liquid drop, putting the aluminum foil box with the liquid drop into liquid nitrogen for treating for 30min, immediately carrying out water bath thawing treatment at 40 ℃ for 1min, and then sending into an ice water mixture at 0 ℃ for treating for 10 min; taking out and rapidly putting into unloading liquid for 20min, wherein the unloading liquid is MS and added with 1.2mol/L sucrose.
9) Taking out the stem tip, placing the stem tip into a post-culture medium, adding 0.05mol/L gibberellin, 7g/L agar and 30g/L sucrose into the post-culture medium for MS, performing dark culture at 0 ℃ for 6 days, and performing weak light culture.
10) And (3) performing BBMV detection on the regenerated plant of the radix pseudostellariae by using an RT-PCR technology, wherein the specific method steps are as described in step 1, and obtaining a strain line from which the BBMV of the radix pseudostellariae broad bean wilting virus is removed.
The survival rate of the stem tip of the embodiment can reach 62 percent.
Example 2
1. A method for removing pseudostellaria heterophylla fava bean wilting virus by utilizing micro-stem tips and ultralow temperature comprises the following steps:
1) the method takes the Pseudostellaria Cudrania tricuspidata No. 2 plant as a material, the plant material is represented by a mosaic, a mottle mosaic and a shrunken leaf, and the virus detection steps are as follows:
the material, the collection and the preservation of the radix pseudostellariae diseased plant sample come from Cudrania tricuspidata county of Ningde city in Fujian province. Fresh plant tissues are collected and rapidly transferred into liquid nitrogen for storage, and the liquid nitrogen is used for total RNA extraction.
A reagent total RNA extraction and purification Kit E.Z.N.A. Plant RNA Kit (R6827-01) was purchased from OMEGA; the reverse transcription kit TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix is purchased from Beijing all-purpose gold biotechnology (TransGen Biotech) Co.Ltd; r isTaqEnzymes were purchased from Takara Bio-engineering Ltd; a100 bp DNA Marker was purchased from Guangzhou Dongsheng Biotechnology Ltd.
Primer design according to GenBank registeredTuMV CPThe nucleotide sequence of the gene (accession number AJ 000690.1) was used to design TuMV primers, which were synthesized by Biotechnology (Shanghai) GmbH. An upstream primer F: 5'-AAGGCGTTGGTGCTGGTTA-3', respectively; a downstream primer R: 5'-CTCGTCAAGACTCCGCTGTA-3' are provided.
Extraction of viral RNA Total RNA extraction Plant RNA Kit (R6827-01) extraction Kit of OMEGA company for extraction, respectively using eppendorf BioPhotometer plus micro nucleic acid protein analyzer and agarose gel electrophoresis with mass fraction of 1.5% to detect concentration and purity, and using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix reverse transcription Kit to remove DNA and reverse to cDNA, and storing at-20 ℃ for later use.
RT-PCR detection virus PCR reaction system 50 mm3: 10 XPCR Buffer (containing Mg)2+）5 mm3，dNTP（2.5 mmol/dm3）4 mm3Forward and reverse primers (10. mu. mol/dm)3) Each 2mm3 ，Taq DNA Polymerase（5U/ mm3）0.5 mm3The amount of cDNA template is about 2mm3Adding up double distilled water to total volume of 50 mm3. PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 40 cycles; extending for 10min at 72 ℃; stopping at 4 ℃. The PCR product obtained by amplification was analyzed by 1.5% agarose gel electrophoresis.
Finally, selecting the radix pseudostellariae plants expressing the symptoms of the broad bean wilting virus.
2) Cutting a diseased plant into stem sections, sterilizing, inoculating the stem sections into a 0.2mg/L NAA culture medium, inoculating the stem sections into an MS culture medium when the stem sections grow to 4 sections, inoculating the bud tips of about 1mm into the MS culture medium when the lateral buds grow to 1cm, and culturing the micro-stem tips to form seedlings.
3) Cutting stem segments cultured by stem tips, and connecting the stem segments to a primary culture medium, wherein the primary culture medium is as follows: MS add 0.2mg/L naphthylacetic acid, 30g/L sucrose, 6.8g/L agar, pH =5.8, incubate for 10d of one cycle.
4) The stem segments of the pseudostellaria heterophylla are taken and placed on a subculture medium ((30 g/L of sucrose +7g/L of agar + MS)) to be cultured for 2 weeks at room temperature.
5) Stripping 2mm of stem tip, leaving 1 leaf primordium, placing the stem tip on a culture medium for dark culture for 3 days, wherein the culture medium is MS, and adding 0.3mol/L sucrose and 7g/L agar, and the culture temperature is 4 ℃.
6) And (3) putting the pre-cultured stem tip into a loading liquid, wherein the loading liquid is MS, 2mol/L of glycerol and 1mol/L of cane sugar are added, the pH is =5.8, and the stem tip is loaded for 30min at room temperature.
7) The loaded stem tips were transferred to PVS2 solution, PVS2 solution was MS medium added with 30% (W/V) glycerol, 15% (W/V) polyethylene glycol, 15% (W/V) dimethyl sulfoxide and 0.4mol/L sucrose to final concentration, pH =5.8, and treated for 30min on ice.
8) Sucking PVS2 solution by using a liquid transfer gun, dripping the PVS2 solution in an aluminum foil box, putting the stem tip in the step 6) into the liquid drop, putting the aluminum foil box with the liquid drop into liquid nitrogen for treatment for 30min, immediately carrying out water bath thawing treatment at 40 ℃ for 30s, and then sending into an ice water mixture at 0 ℃ for treatment for 10 min; taking out, and rapidly putting into unloading liquid for 20min, wherein the unloading liquid is MS added with 1.2mol/L sucrose.
9) Taking out the bud, placing into a post-culture medium, adding 0.05mol/L gibberellin, 7g/L agar, 30g/L sucrose into MS, dark culturing at 0 deg.C for 6 days, and culturing with weak light.
10) And (3) performing BBMV detection on the regenerated plant of the radix pseudostellariae by using an RT-PCR technology, wherein the specific method steps are as described in step 1, and obtaining a strain line from which the BBMV of the radix pseudostellariae broad bean wilting virus is removed.
The survival rate of the stem tip of the embodiment can reach 68 percent.
Example 3
1. A method for removing pseudostellaria heterophylla fava bean wilting virus by utilizing micro-stem tips and ultralow temperature comprises the following steps:
1) the Cudrania tricuspidata radix pseudostellariae No. 2 plant is taken as a material, the plant material is represented as a mosaic, a mottle mosaic and a shrunken leaf, and the virus detection steps are as follows:
the material, the collection and the preservation of the radix pseudostellariae diseased plant sample come from Cudrania tricuspidata county of Ningde city in Fujian province. Fresh plant tissues are collected and rapidly transferred into liquid nitrogen for storage, and the liquid nitrogen is used for total RNA extraction.
A reagent total RNA extraction and purification Kit E.Z.N.A. Plant RNA Kit (R6827-01) was purchased from OMEGA; the reverse transcription kit TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix is purchased from Beijing all-purpose gold biotechnology (TransGen Biotech) Co.Ltd; r isTaqEnzymes were purchased from Takara Bio-engineering Ltd; a100 bp DNA Marker was purchased from Guangzhou Dongsheng Biotechnology Ltd.
Primer design according to GenBank registeredTuMV CPThe nucleotide sequence of the gene (accession number AJ 000690.1) was used to design TuMV primers, which were synthesized by Biotechnology (Shanghai) GmbH. An upstream primer F: 5'-AAGGCGTTGGTGCTGGTTA-3', respectively; a downstream primer R: 5'-CTCGTCAAGACTCCGCTGTA-3' are provided.
Extraction of viral RNA Total RNA extraction Plant RNA Kit (R6827-01) extraction Kit of OMEGA company for extraction, respectively using eppendorf BioPhotometer plus micro nucleic acid protein analyzer and agarose gel electrophoresis with mass fraction of 1.5% to detect concentration and purity, and using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix reverse transcription Kit to remove DNA and reverse to cDNA, and storing at-20 ℃ for later use.
RT-PCR detection virus PCR reaction system 50 mm3: 10 XPCR Buffer (containing Mg)2+）5 mm3，dNTP（2.5 mmol/dm3）4 mm3Forward and reverse primers (10. mu. mol/dm)3) Each 2mm3 ，Taq DNA Polymerase（5U/ mm3）0.5 mm3The amount of cDNA template is about 2mm3Adding up double distilled water to total volume of 50 mm3. PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 40 cycles; extending for 10min at 72 ℃; stopping at 4 ℃. The PCR product obtained by amplification was analyzed by 1.5% agarose gel electrophoresis, and bands were observed. Finally, selecting the radix pseudostellariae plants expressing the symptoms of the broad bean wilting virus.
2) Cutting a diseased plant into stem segments, sterilizing, inoculating the stem segments into a 0.2mg/L NAA culture medium, inoculating the stem segments into an MS culture medium when the stem segments grow to 4 sections, inoculating the bud tips of about 1mm into the MS culture medium when the lateral buds grow to 1.5cm, and culturing the micro-stem tips to form seedlings.
3) Cutting stem segments cultured by stem tips, and connecting the stem segments to a primary culture medium, wherein the primary culture medium is as follows: MS add 0.2mg/L naphthylacetic acid, 30g/L sucrose, 6.8g/L agar, pH =5.8, incubate for 10d of one cycle.
4) The pseudostellaria root stem segments are taken and placed on a subculture medium ((30 g/L sucrose +7g/L agar + MS)) to be cultured for 4 weeks at room temperature.
5) Stripping 1mm of stem tip, leaving 3 leaves of leaf primordium, placing the stem tip on a culture medium for dark culture for 2 days, adding 0.3mol/L sucrose and 7g/L agar into the culture medium MS, and culturing at 4 ℃.
6) And (3) putting the pre-cultured stem tip into a loading liquid, wherein the loading liquid is MS, 2mol/L of glycerol and 1mol/L of cane sugar are added, the pH is =5.8, and the stem tip is loaded for 40min at room temperature.
7) The loaded stem tip was transferred to PVS2 solution, PVS2 solution was MS medium added with 30% (W/V) glycerol, 15% (W/V) polyethylene glycol, 15% (W/V) dimethyl sulfoxide and 0.4mol/L sucrose to final concentration, pH =5.8, and treated for 50min by working on ice.
8) Sucking PVS2 solution by using a pipette gun, dripping the PVS2 solution in an aluminum foil box, putting the stem tip in the step 6) into the liquid drop, putting the aluminum foil box with the liquid drop into liquid nitrogen for treating for 30min, immediately carrying out water bath thawing treatment at 40 ℃ for 1.5min, and then sending into an ice water mixture at 0 ℃ for treating for 10 min; taking out and rapidly putting into unloading liquid for 20min, wherein the unloading liquid is MS and added with 1.2mol/L sucrose.
9) Taking out the bud, placing into a post-culture medium, adding 0.05mol/L gibberellin, 7g/L agar, 30g/L sucrose into MS, dark culturing at 0 deg.C for 6 days, and culturing with weak light.
10) And (3) performing BBMV detection on the regenerated plant of the radix pseudostellariae by using an RT-PCR technology, wherein the specific method steps are as described in step 1, and obtaining a strain line from which the BBMV of the radix pseudostellariae broad bean wilting virus is removed.
The survival rate of the stem tip of the embodiment can reach 60 percent.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> institute of agricultural biological resources of academy of agricultural sciences of Fujian province
<120> method for removing pseudostellaria root and broad bean wilting virus by using micro-stem tip and ultralow temperature
<130> 2
<160> 2
<170> PatentIn version 3.3
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<211> 19
<212> DNA
<213> Artificial sequence
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aaggcgttgg tgctggtta 19
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence
<400> 2
ctcgtcaaga ctccgctgta 20
Claims (4)
1. A method for removing pseudostellaria heterophylla fava bean wilting virus by utilizing micro-stem tips and ultralow temperature is characterized by comprising the following steps: the method comprises the following steps:
1) selecting a radix pseudostellariae plant expressing broad bean wilting virus symptoms;
2) cutting a diseased plant into stem sections, inoculating the stem sections into an MS culture medium containing 0.2mg/L NAA after sterilization and disinfection, inoculating the stem sections into the MS culture medium when the stem sections grow to 4 sections, inoculating 1-2mm bud tips into the MS culture medium when side buds grow to 1-1.5cm, and culturing the micro-stem tips to form seedlings;
3) cutting stem sections cultured by stem tips, connecting the stem sections to a primary culture medium, and culturing for 10 days at room temperature;
4) putting the stem segments of the radix pseudostellariae obtained in the step 3) on a subculture medium, and culturing for 1-6 weeks at room temperature;
5) stripping 1-2mm of stem tips obtained in the step 4), reserving 1-3 sheets of leaf primordium, and placing the stem tips on a culture medium for dark culture for 1-3 days, wherein the culture medium is an MS culture medium added with 0.3mol/L sucrose and 7g/L agar, and the culture temperature is 4 ℃;
6) putting the stem tip pre-cultured in the step 5) into a loading liquid, wherein the loading liquid is an MS culture medium added with 2mol/L of glycerol and 1mol/L of sucrose, the pH is =5.8, and the stem tip is loaded for 20-40min at room temperature;
7) transferring the loaded stem tip into PVS2 solution, operating on ice, and treating for 30-50 min;
8) sucking PVS2 solution by a pipette gun, dripping the PVS2 solution in an aluminum foil box, putting the stem tip in the step 7) into the liquid drop, putting the aluminum foil box with the liquid drop into liquid nitrogen for processing for 30min, immediately carrying out water bath thawing processing at 40 ℃ for 0.2-1.5min, and then sending into an ice water mixture at 0 ℃ for processing for 10 min; quickly putting into unloading liquid for 20min, wherein the unloading liquid is MS culture medium added with 1.2mol/L sucrose;
9) taking out the stem tip, placing into a post-culture medium, dark-culturing at 0 deg.C for 5-7 days, and culturing under 200lux of light for 1 month to obtain radix Pseudostellariae regenerated plant;
10) performing BBMV detection on the regenerated plant of the radix pseudostellariae by using an RT-PCR technology to obtain a strain with the BBMV removed from the radix pseudostellariae broad bean wilting virus;
wherein, the PVS2 solution is MS culture medium added with 30% W/V glycerol, 15% W/V polyethylene glycol, 15% W/V dimethyl sulfoxide and 0.4mol/L sucrose, and the pH is = 5.8.
2. The method for removing wilting virus of radix pseudostellariae and broad bean by using micro-stem tip and ultra-low temperature as claimed in claim 1, wherein: the primary culture medium is: MS medium added 0.2mg/L naphthylacetic acid, 30g/L sucrose, 6.8g/L agar, pH = 5.8.
3. The method for removing wilting virus of radix pseudostellariae and broad bean by using micro-stem tip and ultra-low temperature as claimed in claim 1, wherein: the subculture medium is MS medium + 30g/L sucrose +7g/L agar.
4. The method for removing wilting virus of radix pseudostellariae and broad bean by using micro-stem tip and ultra-low temperature as claimed in claim 1, wherein: and 9) adding 0.05mol/L gibberellin, 7g/L agar and 30g/L sucrose into the MS culture medium as the post-culture medium.
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