A method for ultra-low temperature preservation and detoxification of Pinellia ternata plants

Abstract

The invention discloses a method for ultra-low temperature preservation and detoxification of pinellia ternata, comprising material culture, loading treatment, vitrification treatment, ultra-low temperature treatment, thawing and warming treatment, unloading treatment and regeneration plant culture. The pinellia ternata vitrification ultra-low temperature preservation technology system established by the invention can be applied to the preservation of pinellia ternata germplasm resources. Compared with field preservation and delayed growth preservation technology, the technology can greatly save manpower and material resources, is easy to operate, is not easily attacked by natural disasters, does not need to transfer plant materials regularly, occupies a small space, has low preservation cost, long preservation time, is genetically stable, and can preserve pinellia ternata germplasm for a long time; the technology can also be applied to the detoxification of pinellia ternata plants. After detoxification of test tube tubers of second-generation diseased plants of pinellia ternata, it was found through experimental verification that the detoxification rate of soybean mosaic virus (SMV) was 85.7%, and the detoxification rate of cucumber mosaic virus (CWV) was 57.1%, and the detoxification effect was better than that of commonly used heat treatment and stem tip detoxification.

Description

Technical Field

[0001] The invention belongs to the technical field of crop breeding, and in particular relates to a method for ultra-low temperature preservation and detoxification of pinellia ternata plants. Background Art

[0002] Pinellia Pinellia ternate (Thunb.) Beri. is a plant of the Araceae family and a perennial herb of the genus Pinellia. Its tubers are used as medicine. It has the effects of drying dampness and resolving phlegm, lowering adverse reactions and stopping vomiting, and eliminating lumps and dispersing knots. It is a commonly used bulk Chinese medicinal material in my country and has a history of more than 2,000 years of medicinal use. In recent years, the market demand for Pinellia in my country is about 6,000 tons. Nearly 800 Chinese medicine companies in my country use Pinellia in nearly 558 prescriptions, and the frequency of use of Pinellia ranks 22nd. In recent years, studies have found that Pinellia has many important functions such as anti-tumor, anti-inflammatory, antibacterial, and anti-epileptic. With the continuous development of the medicinal value of Pinellia, its demand is increasing, and the wild resources of Pinellia are decreasing. Germplasm resources are the basic guarantee for the stable development of crop breeding. Germplasm resources of asexually propagated crops are mainly preserved in the field and in vitro. Field preservation requires a lot of land and human resources, is costly, and is easily attacked by various natural disasters. In vitro preservation can effectively save land and labor, the preservation method is relatively simple, and it can be protected from the harm of microorganisms such as viruses during the preservation process. Ultra-low temperature preservation is to store the in vitro plant materials in liquid nitrogen (-196℃). Theoretically, the materials can be stored indefinitely and their genetic stability can be maintained to the greatest extent. At present, the main source of Pinellia ternata medicinal materials is field cultivation. Pinellia ternata mainly relies on bulbils for asexual reproduction, which leads to the serious problems of cultivated germplasm degradation and resource mixing. In addition, people's uncontrolled excavation of wild resources has caused the wild germplasm to almost disappear. In addition, long-term asexual reproduction has caused a large accumulation of viruses in the plants, inhibiting the normal growth of the plants. The germplasm has declined year by year, which has seriously affected the improvement of the yield and quality of Pinellia ternata. Ultra-low temperature preservation technology can not only be used to preserve plant germplasm resources but also to detoxify plants. Its detoxification mechanism has been revealed by the localization of viruses in plant stem tips, histology and cell ultrastructure observations. The cells of the apical meristem of the stem tip are closely arranged, have a large nuclear-cytoplasmic ratio, and the vacuole volume is also small. Therefore, it is not easy to freeze in the cells after ultra-low temperature treatment, and it is easier to survive. According to the hypothesis of uneven distribution of viruses on plants, the apical meristem region usually does not carry or carries a small amount of pathogens, and the surviving cells may be non-toxic after ultra-low temperature freezing. As the distance from the apical meristem increases, the cell and vacuole volume of cells in other parts increases, and the nuclear-cytoplasmic ratio decreases. Usually, cells with large vacuoles and low nuclear-cytoplasmic ratio have a lower survival rate after ultra-low temperature freezing. Since the apical meristem cells have a strong ability to divide, they can form plant organs, while the cells with viruses are killed. Therefore, the development of complete plants from cells without viruses is also non-toxic. There are no reports on the ultra-low temperature preservation and detoxification of Pinellia ternata. Summary of the invention

[0003] The purpose of the present invention is to provide a method for ultra-low temperature preservation of regenerated plants and detoxification of Pinellia ternata, so as to achieve safe preservation of Pinellia ternata germplasm resources through ultra-low temperature preservation of Pinellia ternata and provide a technical method for detoxification of Pinellia ternata.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is:

[0005] Step 1: Material cultivation:

[0006] Healthy or diseased Pinellia ternata seed stems are sown in a flower pot to germinate and grow to obtain Pinellia ternata plants, petioles are sterilized and cut into 0.5-2 cm pieces, and small pieces are inoculated into 1 / 2MS culture medium to obtain an independent and complete small tuber and germinate into a seedling, the petioles of the test tube seedlings are used as materials to induce culture again to obtain small tubers and germinate into seedlings, to obtain first-generation tubers and first-generation seedlings, the stems and leaves are cut off, the first-generation tubers are transferred to a fresh culture medium, the tubers germinate into seedlings again and grow up, to obtain second-generation tubers and second-generation seedlings, and second-generation healthy test tube tubers or second-generation diseased test tube tubers are selected from the second-generation tubers, and when the stems and leaves are withered and in a dormant state, the tubers and culture bottles are pretreated at 4°C in the dark for 1-2 weeks;

[0007] Step 2: Loading process:

[0008] Peel off a 0.2-1 mm-sized stem tip from the second-generation healthy test tube tuber or the second-generation diseased test tube tuber obtained in step 1, and place the stem tip in a sterile cryopreservation tube containing 1 mL of filtered and sterilized loading solution, wherein the loading solution is MS+2 mol / L glycerol+0.4 mol / L sucrose+1 μmol / L glutathione, pH 5.7, and treat at room temperature for 20 min before removing the loading solution with a pipette;

[0009] Step 3: Vitrification treatment:

[0010] Add 1.0-2 mL of filtered and sterilized PVS2 vitrification protection solution to the sterile cryopreservation tube obtained in step 2. The formula of PVS2 vitrification protection solution is MS + 30% glycerol + 15% ethylene glycol + 15% dimethyl sulfoxide + 0.4 M sucrose + glutathione 1 μmol / L, pH 5.7, and treat the shoot tip at room temperature for 20-120 minutes;

[0011] Step 4: Ultra-low temperature treatment:

[0012] The sterile cryopreservation tube obtained in step 3 is placed in liquid nitrogen for storage for no less than 120 minutes;

[0013] Step 5: Thawing and reheating:

[0014] Take out the sterile cryopreservation tube obtained in step 4 from liquid nitrogen and thaw it in a 38°C water bath for 2 minutes;

[0015] Step 6: Uninstallation process:

[0016] After thawing, use a pipette to remove the PVS2 vitrification protection solution in the sterile cryopreservation tube, and then add 1.0-2mL of filtered sterilized unloading solution. The formula of the unloading solution is 1 / 2MS+1.2mol / L sucrose, pH 5.7, and treat the shoot tip for 20 minutes.

[0017] Step 7: Regeneration plant culture:

[0018] Take out the shoot tip treated in step six from the sterile cryopreservation tube, absorb the unloading liquid on the surface with sterile filter paper, and inoculate it into 1 / 2MS + 6-KT0.5mg / L + NAA0.2mg / L + glutathione1μmol / L + gibberellin0.1mg / L + acid hydrolyzed casein200mg / L + 0.7% agar, pH5.8 culture medium, first culture in the dark for 7 days, and then transfer to 500-1000LUX weak light conditions to culture the regenerated plants.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The pinellia vitrification ultra-low temperature preservation technology system established by the present invention can be applied to the preservation of pinellia germplasm resources. Compared with field preservation and delayed growth preservation technology, this technology can greatly save manpower and material resources, is easy to operate, is not easily attacked by natural disasters, does not require regular transfer of plant materials, has many advantages such as small space occupation, low preservation cost, long preservation time, genetic stability, etc., and can preserve pinellia germplasm for a long time.

[0021] Many scholars agree that no matter which cryopreservation method is used, the type of material and the selection of physiological state are key factors in the success of cryopreservation. It is not only related to the material's resistance to drying, dehydration and freezing, but also has a great impact on the selection of suitable conditions for subsequent procedures. Therefore, selecting suitable materials and physiological states, and establishing a preservation system adapted to the material based on the characteristics of the material can not only simplify the cryopreservation procedure, but also improve the practicality of this method, which is one of the feasible ways to solve the low survival rate of cryopreservation. This study directly uses the buds of the growth period of Pinellia as the preservation material, and the survival rate after freezing is very low. The present invention uses the dormant buds of the second-generation healthy test tube tubers as the preservation material combined with low-temperature training to carry out cryopreservation research on Pinellia germplasm, and a very high regeneration rate has been achieved.

[0022] During the vitrification cryopreservation process, due to the cryoprotectant and extremely low temperature treatment, plant cells and tissues may suffer various stresses, including dehydration, osmotic pressure and toxic damage, which may lead to the production of reactive oxygen species and cause oxidative damage to cells. Antioxidant enzymes and antioxidants can remove the excessive ROS (reactive oxygen species) produced by plants under adverse conditions to ensure that cells will not suffer severe oxidative damage. The present invention adds the antioxidant glutathione during dormant bud loading, PVS2 vitrification treatment and recovery growth. Experiments have confirmed that the above treatment can make the plant regeneration rate after cryopreservation as high as 75%.

[0023] In addition, this technology can also be used to detoxify Pinellia plants. After using this ultra-low temperature preservation technology system to detoxify the second-generation diseased test tube tubers of Pinellia, experimental verification showed that the detoxification rate of soybean mosaic virus (SMV) was 85.7%, and the detoxification rate of cucumber mosaic virus (CWV) was 57.1%. The detoxification effect is better than the commonly used heat treatment and stem tip detoxification. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The stem tips are from different sources, where A is mature tubers; B is first-generation test tube tubers and test tube seedlings; C is second-generation test tube tubers and test tube seedlings; D is in vitro stem tips;

[0025] Figure 2 The effects of different sizes of shoot tips on cryopreservation of shoot tips;

[0026] Figure 3 The effect of low temperature acclimation time on the cryopreservation of shoot tips;

[0027] Figure 4 The effect of different PVS2 treatment time on the cryopreservation of shoot apex;

[0028] Figure 5 The effects of different physiological ages and states on the cryopreservation of shoot tips;

[0029] Figure 6 The survival of shoot tips preserved at ultra-low temperatures and plant regeneration, where A is the greening of the shoot tips; B is the growth of the shoot tips; C is the ultra-low temperature regenerated plants;

[0030] Figure 7 To investigate the effects of unloading on cryopreservation of shoot apex;

[0031] Figure 8 The phenotypes of the ultra-low temperature regenerated plants from the shoot tips, where A is the ultra-low temperature regenerated plant that survived transplantation; B. Two different leaf types;

[0032] Fig. 9The amplification diagram of ultra-low temperature regenerated plants with different primer pairs, where A and B are primer 835, C and D are primer 880, E and F are primer 881; Lanes 1-5 in A, C and E are 40-1, and lanes 6-10 are 40-4; Lanes 1-5 in B, D and F are 40-3, and lanes 6-10 are 40-6;

[0033] Fig.10 Figures of regenerated plants after detoxification by different detoxification methods, where A is a regenerated plant after secondary detoxification by stem tip removal, B is a regenerated plant after ultra-low temperature detoxification, C is a regenerated plant after 2 weeks of detoxification at 38°C, D is a regenerated plant after 2 weeks of detoxification at 35°C, and E is a regenerated plant after 4 weeks of detoxification at 35°C.

[0034] Fig.11 The leaves show different degrees of mosaic after virus removal, where A is a normal leaf, B is a leaf with no obvious mosaic symptoms, C is a slight mosaic, and D is a severe mosaic.

[0035] Fig.12 These are the pictures of DAS-ELISA detection, where A is SMV detection and B is CMV detection. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0037] A method for ultra-low temperature preservation of regenerated plants and detoxification of Pinellia ternata comprises the following steps:

[0038] Step 1: Material cultivation:

[0039] The pinellia seed stems are sown in a flower pot to germinate and grow to obtain pinellia plants, the petioles are sterilized, cut into small sections of 0.5-2 cm, inoculated into 1 / 2MS culture medium to obtain an independent and complete small tuber and germinate into a seedling, the petioles of the test tube seedlings are used as materials to induce small tubers again and germinate into seedlings to obtain first-generation tubers and first-generation seedlings, the stems and leaves are cut off, the first-generation tubers are transferred to a fresh culture medium, the tubers are germinated into seedlings again and grow up to obtain second-generation tubers and second-generation seedlings, and the second-generation healthy test tube tubers or the second-generation diseased plant test tube tubers are selected from the second-generation tubers, and when the stems and leaves are withered and in a dormant state, the tubers are pretreated at 4°C in the dark for 1-2 weeks;

[0040] Step 2: Loading process:

[0041] Peel off a 0.2-1 mm-sized stem tip from the second-generation healthy test tube tuber or the second-generation diseased test tube tuber obtained in step 1, and place the stem tip in a sterile cryopreservation tube containing 1 mL of filtered and sterilized loading solution, wherein the loading solution is MS+2 mol / L glycerol+0.4 mol / L sucrose+1 μmol / L glutathione, pH 5.7, and treat at room temperature for 20 min before removing the loading solution with a pipette;

[0042] Step 3: Vitrification treatment:

[0043] Add 1.0-2.0 mL of filtered and sterilized PVS2 vitrification protection solution to the sterile cryopreservation tube obtained in step 2. The formula of PVS2 vitrification protection solution is MS + 30% glycerol + 15% ethylene glycol + 15% dimethyl sulfoxide + 0.4 M sucrose + glutathione 1 μmol / L, pH 5.7, and treat the shoot tip at room temperature for 20-120 minutes;

[0044] Step 4: Ultra-low temperature treatment:

[0045] The sterile cryopreservation tube obtained in step 3 is placed in liquid nitrogen for storage for no less than 120 minutes;

[0046] Step 5: Thawing and reheating:

[0047] Take out the sterile cryopreservation tube obtained in step 4 from liquid nitrogen and thaw it in a 38°C water bath for 2 minutes;

[0048] Step 6: Uninstallation process:

[0049] After thawing, use a pipette to remove the PVS2 vitrification protection solution in the sterile cryopreservation tube, and then add 1.0-2mL of filtered sterilized unloading solution. The formula of the unloading solution is 1 / 2MS+1.2mol / L sucrose, pH 5.7, and treat the shoot tip for 20 minutes.

[0050] Step 7: Regeneration plant culture:

[0051] Take out the shoot tip treated in step six from the sterile cryopreservation tube, absorb the unloading liquid on the surface with sterile filter paper, and inoculate it into 1 / 2MS + 6-KT0.5mg / L + NAA0.2mg / L + glutathione1μmol / L + gibberellin0.1mg / L + acid hydrolyzed casein200mg / L + 0.7% agar, pH5.8 culture medium, first culture in the dark for 7 days, and then transfer to 500-1000LUX weak light conditions to culture the regenerated plants.

[0052] Embodiment 1: A method for cryopreserving and regenerating pinellia plants, comprising the following steps:

[0053] Step 1: Material cultivation:

[0054] The seed stems of Pinellia ternata are sown in a flower pot to germinate and grow to obtain Pinellia ternata plants, the petioles are sterilized and cut into 0.5 cm pieces, and the small pieces are inoculated into 1 / 2MS culture medium to obtain an independent and complete small tuber and germinate into a seedling, the petioles of the test tube seedlings are used as materials to induce small tubers again and germinate into seedlings to obtain first-generation tubers and first-generation seedlings, the stems and leaves are cut off, the first-generation tubers are transferred to a fresh culture medium, the tubers germinate into seedlings again and grow up to obtain second-generation tubers and second-generation seedlings, and the second-generation healthy test tube tubers among the second-generation tubers are selected, and when the stems and leaves are withered and in a dormant state, the tubers are pretreated at 4°C in the dark for 1 week;

[0055] Step 2: Loading process:

[0056] Peel off a 0.2 mm stem tip from the second-generation healthy test tube tuber obtained in step 1, and place the stem tip in a sterile cryopreservation tube containing 1 mL of filtered and sterilized loading solution, wherein the loading solution is MS + 2 mol / L glycerol + 0.4 mol / L sucrose + 1 μmol / L glutathione, pH 5.7, and treat at room temperature for 20 min. Then remove the loading solution with a pipette;

[0057] Step 3: Vitrification treatment:

[0058] Add 1.0 mL of filtered and sterilized PVS2 vitrification protection solution to the sterile cryopreservation tube obtained in step 2. The formula of PVS2 vitrification protection solution is MS + 30% glycerol + 15% ethylene glycol + 15% dimethyl sulfoxide + 0.4 M sucrose + glutathione 1 μmol / L, pH 5.7, and treat the shoot tip at room temperature for 20 minutes;

[0059] Step 4: Ultra-low temperature treatment:

[0060] The sterile cryopreservation tube obtained in step 3 is placed in liquid nitrogen for storage for 120 minutes;

[0061] Step 5: Thawing and reheating:

[0062] Take out the sterile cryopreservation tube obtained in step 4 from liquid nitrogen and thaw it in a 38°C water bath for 2 minutes;

[0063] Step 6: Uninstallation process:

[0064] After thawing, use a pipette to remove the PVS2 vitrification protection solution in the sterile cryopreservation tube, and then add 1.0 mL of filtered sterilized unloading solution. The formula of the unloading solution is 1 / 2MS + 1.2 mol / L sucrose, pH 5.7, and treat the shoot tip for 20 minutes.

[0065] Step 7: Regeneration plant culture:

[0066] Take out the shoot tip treated in step six from the sterile cryopreservation tube, absorb the unloading liquid on the surface with sterile filter paper, and inoculate it into 1 / 2MS + 6-KT0.5mg / L + NAA0.2mg / L + glutathione1μmol / L + gibberellin0.1mg / L + acid hydrolyzed casein200mg / L + 0.7% agar, pH5.8 culture medium, first culture in the dark for 7 days, and then transfer to 500LUX weak light conditions to culture the regenerated plants.

[0067] Embodiment 2: A method for cryopreserving and regenerating pinellia plants, comprising the following steps:

[0068] Step 1: Material cultivation:

[0069] The seed stems of Pinellia ternata are sown in a flower pot to germinate and grow to obtain Pinellia ternata plants, the petioles are sterilized and cut into 2 cm, and the small segments are inoculated into 1 / 2MS culture medium to obtain an independent and complete small tuber and germinate into a seedling, the petioles of the test tube seedlings are used as materials to induce small tubers again and germinate into seedlings to obtain first-generation tubers and first-generation seedlings, the stems and leaves are cut off, the first-generation tubers are transferred to a fresh culture medium, the tubers germinate into seedlings again and grow up to obtain second-generation tubers and second-generation seedlings, and the second-generation healthy test tube tubers among the second-generation tubers are selected, and when the stems and leaves are withered and in a dormant state, the tubers are pretreated at 4°C in the dark for 2 weeks;

[0070] Step 2: Loading process:

[0071] Peel off a 1 mm stem tip from the second-generation healthy test tube tuber obtained in step 1, and place the stem tip in a sterile cryopreservation tube containing 1 mL of filtered and sterilized loading solution. The loading solution is MS + 2 mol / L glycerol + 0.4 mol / L sucrose + 1 μmol / L glutathione, pH 5.7. After treatment at room temperature for 20 min, remove the loading solution with a pipette;

[0072] Step 3: Vitrification treatment:

[0073] Add 2 mL of filtered and sterilized PVS2 vitrification protection solution to the sterile cryopreservation tube obtained in step 2. The formula of PVS2 vitrification protection solution is MS + 30% glycerol + 15% ethylene glycol + 15% dimethyl sulfoxide + 0.4 M sucrose + glutathione 1 μmol / L, pH 5.7, and treat the shoot tip at room temperature for 120 minutes;

[0074] Step 4: Ultra-low temperature treatment:

[0075] The sterile cryopreservation tube obtained in step 3 is placed in liquid nitrogen for storage for no less than 120 minutes;

[0076] Step 5: Thawing and reheating:

[0077] Take out the sterile cryopreservation tube obtained in step 4 from liquid nitrogen and thaw it in a 38°C water bath for 2 minutes;

[0078] Step 6: Uninstallation process:

[0079] After thawing, use a pipette to remove the PVS2 vitrification protection solution in the sterile cryopreservation tube, and then add 2 mL of filtered sterilized unloading solution. The formula of the unloading solution is 1 / 2MS + 1.2 mol / L sucrose, pH 5.7, and treat the shoot tip for 20 minutes.

[0080] Step 7: Regeneration plant culture:

[0081] Take out the shoot tip treated in step six from the sterile cryopreservation tube, absorb the unloading liquid on the surface with sterile filter paper, and inoculate it into 1 / 2MS + 6-KT0.5mg / L + NAA0.2mg / L + glutathione1μmol / L + gibberellin0.1mg / L + acid hydrolyzed casein200mg / L + 0.7% agar, pH5.8 culture medium, first culture in the dark for 7 days, and then transfer to 1000LUX weak light conditions to culture regenerated plants.

[0082] Embodiment 3: A method for cryopreserving and regenerating pinellia plants, comprising the following steps:

[0083] Step 1: Material cultivation:

[0084] The pinellia seed stems were sown in a flower pot to germinate and grow to obtain pinellia plants, the petioles were disinfected and cut into 1 cm, and the small segments were inoculated into 1 / 2MS culture medium to obtain an independent and complete small tuber and germinate into seedlings, the petioles of the test tube seedlings were used as materials to induce small tubers again and germinate into seedlings to obtain first-generation tubers and first-generation seedlings, the stems and leaves were cut off, the first-generation tubers were transferred to fresh culture medium, the tubers germinated into seedlings again and grew up to obtain second-generation tubers and second-generation seedlings, and the second-generation healthy test tube tuber 1 was selected from the second-generation tubers, and when its stems and leaves withered and were in a dormant state, the tubers were pretreated at 4°C in the dark for 1.5 weeks;

[0085] Step 2: Loading process:

[0086] Peel off a 0.8 mm shoot tip from the second-generation healthy test tube tuber obtained in step 1, and place the shoot tip in a sterile cryopreservation tube containing 1 mL of filtered and sterilized loading solution. The loading solution is MS + 2 mol / L glycerol + 0.4 mol / L sucrose + 1 μmol / L glutathione, pH 5.7. After treating at room temperature for 20 min, remove the loading solution with a pipette;

[0087] Step 3: Vitrification treatment:

[0088] Add 1.6 mL of filtered and sterilized PVS2 vitrification protection solution to the sterile cryopreservation tube obtained in step 2. The formula of PVS2 vitrification protection solution is MS + 30% glycerol + 15% ethylene glycol + 15% dimethyl sulfoxide + 0.4 M sucrose + glutathione 1 μmol / L, pH 5.7, and treat the shoot tip at room temperature for 80 minutes;

[0089] Step 4: Ultra-low temperature treatment:

[0090] The sterile cryopreservation tube obtained in step 3 is placed in liquid nitrogen for storage for no less than 120 minutes;

[0091] Step 5: Thawing and reheating:

[0092] Take out the sterile cryopreservation tube obtained in step 4 from liquid nitrogen and thaw it in a 38°C water bath for 2 minutes;

[0093] Step 6: Uninstallation process:

[0094] After thawing, use a pipette to remove the PVS2 vitrification protection solution in the sterile cryopreservation tube, and then add 1.6 mL of filtered sterilized unloading solution. The formula of the unloading solution is 1 / 2MS + 1.2 mol / L sucrose, pH 5.7, and treat the shoot tip for 20 minutes.

[0095] Step 7: Regeneration plant culture:

[0096] Take out the shoot tip treated in step six from the sterile cryopreservation tube, absorb the unloading liquid on the surface with sterile filter paper, and inoculate it into 1 / 2MS + 6-KT0.5mg / L + NAA0.2mg / L + glutathione1μmol / L + gibberellin0.1mg / L + acid hydrolyzed casein200mg / L + 0.7% agar, pH5.8 culture medium, first culture in the dark for 7 days, and then transfer to 800LUX weak light conditions to culture the regenerated plants.

[0097] Embodiment 4, a method for ultra-low temperature detoxification of Pinellia tuber, comprising the following steps:

[0098] Step 1: Material cultivation:

[0099] The seed stems of Pinellia ternata were sown in a flower pot to germinate and grow to obtain Pinellia ternata plants, the petioles were sterilized and cut into 0.5 cm, and the small segments were inoculated into 1 / 2MS culture medium to obtain an independent and complete small tuber and germinate into a seedling, the petioles of the test tube seedlings were used as materials to induce small tubers again and germinate into seedlings to obtain first-generation tubers and first-generation seedlings, the stems and leaves were cut off, the first-generation tubers were transferred to a fresh culture medium, the tubers germinated into seedlings again and grew up to obtain second-generation tubers and second-generation seedlings, the test tube tubers of the second-generation diseased plants were selected, and when the stems and leaves were withered and in a dormant state, the tubers were pretreated at 4°C in the dark for 1 week;

[0100] Step 2: Loading process:

[0101] Peel off a 0.2 mm stem tip from the second-generation diseased plant test tube tuber obtained in step 1, place the stem tip in a sterile cryopreservation tube containing 1 mL of filtered sterilized loading solution, the loading solution being MS + 2 mol / L glycerol + 0.4 mol / L sucrose + 1 μmol / L glutathione, pH 5.7, and treat at room temperature for 20 min before removing the loading solution with a pipette;

[0102] Step 3: Vitrification treatment:

[0103] Add 1.0 mL of filtered and sterilized PVS2 vitrification protection solution to the sterile cryopreservation tube obtained in step 2. The formula of PVS2 vitrification protection solution is MS + 30% glycerol + 15% ethylene glycol + 15% dimethyl sulfoxide + 0.4 M sucrose + glutathione 1 μmol / L, pH 5.7, and treat the shoot tip at room temperature for 20 minutes;

[0104] Step 4: Ultra-low temperature treatment:

[0105] The sterile cryopreservation tube obtained in step 3 is placed in liquid nitrogen for storage for 120 minutes;

[0106] Step 5: Thawing and reheating:

[0107] Take out the sterile cryopreservation tube obtained in step 4 from liquid nitrogen and thaw it in a 38°C water bath for 2 minutes;

[0108] Step 6: Uninstallation process:

[0109] After thawing, use a pipette to remove the PVS2 vitrification protection solution in the sterile cryopreservation tube, and then add 1.0 mL of filtered sterilized unloading solution. The formula of the unloading solution is 1 / 2MS + 1.2 mol / L sucrose, pH 5.7, and treat the shoot tip for 20 minutes.

[0110] Step 7: Regeneration plant culture:

[0111] Take out the shoot tip treated in step six from the sterile cryopreservation tube, absorb the unloading liquid on the surface with sterile filter paper, and inoculate it into 1 / 2MS + 6-KT0.5mg / L + NAA0.2mg / L + glutathione1μmol / L + gibberellin0.1mg / L + acid hydrolyzed casein200mg / L + 0.7% agar, pH5.8 culture medium, first culture in the dark for 7 days, and then transfer to 500LUX weak light conditions to culture the regenerated plants.

[0112] Embodiment 5, a method for ultra-low temperature detoxification of Pinellia tuber, comprising the following steps:

[0113] Step 1: Material cultivation:

[0114] The seed stems of Pinellia ternata are sown in a flower pot to germinate and grow to obtain Pinellia ternata plants, the petioles are sterilized and cut into 2 cm, and the small segments are inoculated into 1 / 2MS culture medium to obtain an independent and complete small tuber and germinate into a seedling, the petioles of the test tube seedlings are used as materials to induce small tubers again and germinate into seedlings to obtain first-generation tubers and first-generation seedlings, the stems and leaves are cut off, the first-generation tubers are transferred to a fresh culture medium, the tubers germinate into seedlings again and grow up to obtain second-generation tubers and second-generation seedlings, the test tube tubers of the second-generation diseased plants are selected, and when the stems and leaves are withered and in a dormant state, the tubers are pretreated at 4°C in the dark for 2 weeks;

[0115] Step 2: Loading process:

[0116] Peel off a 1 mm stem tip from the second-generation diseased plant test tube tuber obtained in step 1, and place the stem tip in a sterile cryopreservation tube containing 1 mL of filtered sterilized loading solution. The loading solution is formulated as MS + 2 mol / L glycerol + 0.4 mol / L sucrose + 1 μmol / L glutathione, pH 5.7. After treating at room temperature for 20 min, remove the loading solution with a pipette;

[0117] Step 3: Vitrification treatment:

[0118] Add 2.0 mL of filtered and sterilized PVS2 vitrification protection solution to the sterile cryopreservation tube obtained in step 2. The formula of PVS2 vitrification protection solution is MS + 30% glycerol + 15% ethylene glycol + 15% dimethyl sulfoxide + 0.4 M sucrose + glutathione 1 μmol / L, pH 5.7, and treat the shoot tip at room temperature for 120 minutes;

[0119] Step 4: Ultra-low temperature treatment:

[0120] The sterile cryopreservation tube obtained in step 3 is placed in liquid nitrogen for storage for 120 minutes;

[0121] Step 5: Thawing and reheating:

[0122] Take out the sterile cryopreservation tube obtained in step 4 from liquid nitrogen and thaw it in a 38°C water bath for 2 minutes;

[0123] Step 6: Uninstallation process:

[0124] After thawing, use a pipette to remove the PVS2 vitrification protection solution in the sterile cryopreservation tube, and then add 2.0 mL of filtered sterilized unloading solution. The formula of the unloading solution is 1 / 2MS + 1.2 mol / L sucrose, pH 5.7, and treat the shoot tip for 20 minutes.

[0125] Step 7: Regeneration plant culture:

[0126] Take out the shoot tip treated in step six from the sterile cryopreservation tube, absorb the unloading liquid on the surface with sterile filter paper, and inoculate it into 1 / 2MS + 6-KT0.5mg / L + NAA0.2mg / L + glutathione1μmol / L + gibberellin0.1mg / L + acid hydrolyzed casein200mg / L + 0.7% agar, pH5.8 culture medium, first culture in the dark for 7 days, and then transfer to 1000LUX weak light conditions to culture regenerated plants.

[0127] Embodiment 6, a method for ultra-low temperature detoxification of Pinellia tuber, comprising the following steps:

[0128] Step 1: Material cultivation:

[0129] The seed stems of Pinellia ternata were sown in a flower pot to germinate and grow to obtain Pinellia ternata plants, the petioles were sterilized and cut into 1 cm, and the small segments were inoculated into 1 / 2MS culture medium to obtain an independent and complete small tuber and germinate into a seedling, the petioles of the test tube seedlings were used as materials to induce small tubers again and germinate into seedlings to obtain first-generation tubers and first-generation seedlings, the stems and leaves were cut off, the first-generation tubers were transferred to a fresh culture medium, the tubers germinated into seedlings again and grew up to obtain second-generation tubers and second-generation seedlings, and the test tube tubes of the second-generation diseased plants were selected, and when the stems and leaves were withered and in a dormant state, the tubers were pretreated at 4°C in the dark for 1.5 weeks;

[0130] Step 2: Loading process:

[0131] Peel off a 0.4 mm-sized stem tip from the second-generation diseased plant test tube tuber obtained in step 1, and place the stem tip in a sterile cryopreservation tube containing 1 mL of filtered and sterilized loading solution. The loading solution is formulated as MS + 2 mol / L glycerol + 0.4 mol / L sucrose + 1 μmol / L glutathione, pH 5.7. After treating at room temperature for 20 min, remove the loading solution with a pipette;

[0132] Step 3: Vitrification treatment:

[0133] Add 1.5 mL of filtered and sterilized PVS2 vitrification protection solution to the sterile cryopreservation tube obtained in step 2. The formula of PVS2 vitrification protection solution is MS + 30% glycerol + 15% ethylene glycol + 15% dimethyl sulfoxide + 0.4 M sucrose + glutathione 1 μmol / L, pH 5.7, and treat the shoot tip at room temperature for 100 minutes;

[0134] Step 4: Ultra-low temperature treatment:

[0135] The sterile cryopreservation tube obtained in step 3 is placed in liquid nitrogen for storage for 120 minutes;

[0136] Step 5: Thawing and reheating:

[0137] Take out the sterile cryopreservation tube obtained in step 4 from liquid nitrogen and thaw it in a 38°C water bath for 2 minutes;

[0138] Step 6: Uninstallation process:

[0139] After thawing, use a pipette to remove the PVS2 vitrification protection solution in the sterile cryopreservation tube, and then add 2 mL of filtered sterilized unloading solution. The formula of the unloading solution is 1 / 2MS + 1.2 mol / L sucrose, pH 5.7, and treat the shoot tip for 20 minutes.

[0140] Step 7: Regeneration plant culture:

[0141] Take out the shoot tip treated in step six from the sterile cryopreservation tube, absorb the unloading liquid on the surface with sterile filter paper, and inoculate it into 1 / 2MS + 6-KT0.5mg / L + NAA0.2mg / L + glutathione1μmol / L + gibberellin0.1mg / L + acid hydrolyzed casein200mg / L + 0.7% agar, pH5.8 culture medium, first culture in the dark for 7 days, and then transfer to 800LUX weak light conditions to culture the regenerated plants.

[0142] Experimental Example 1: Method for ultra-low temperature preservation of regenerated plants of Pinellia ternata:

[0143] 1. Test Materials

[0144] The pinellia seeds and stems used in the present invention were purchased from Heze City, Shandong Province. After the seedlings were potted in the laboratory, the healthy plants were selected, the petioles were cut off and disinfected with 75% alcohol for 10 seconds, and then with 0.1% HgCl 2 (containing Tween 80) for 5 minutes, and finally disinfected with 2% NaClO for 8 minutes, and finally rinsed three times with sterile water. Use sterile filter paper to absorb the surface moisture, and cut the petioles into petiole segments of about 1 cm, inoculate them in culture medium (1 / 2MS+3% sucrose+7% agar), and place them in an artificial climate box at 25°C for light-proof culture. After the test tube tubers are formed, they are cultured in the light (lighting time: 12h / d; light intensity is about 2000lx). After the stems and leaves of the tubers are cut off, the stems and leaves can be pulled out many times. The number of times the stems and leaves are pulled out is regarded as several generations of test tube seedlings and tubers. The stem tips used for ultra-low temperature preservation come from mature tubers, first-generation tubers, second-generation fresh tubers (the culture medium has not shrunk), and second-generation dormant tubers (the culture medium has shrunk) (see Figure 1 ).

[0145] 2. Shoot apex vitrification cryopreservation technology

[0146] 1. Effects of different sizes of stem tips on cryopreservation of stem tips

[0147] First-generation tube tubers were selected, and stem tips of different sizes were dissected: large (0.8-1mm), medium (0.5-0.7mm), and small (0.2-0.4mm). The stem tips were then placed in a sterile cryovial containing 1 mL of filter-sterilized loading solution (MS+2mol / L glycerol+0.4mol / L sucrose+glutathione 1μmol / L, pH5.7), loaded at room temperature for 20 minutes, the loading solution was discarded, and then 2 mL of filter-sterilized PVS2 vitrification protection solution (MS+30% glycerol+15% ethylene glycol+15% dimethyl sulfoxide+0.4M sucrose+glutathione 1μmol / L) was added, treated at room temperature for 40 minutes, and then the cryovial was put into liquid nitrogen for no less than 120 minutes. When thawing, the cryovial was taken out of the liquid nitrogen and thawed in a 38°C water bath for 2 minutes. After thawing, discard the PVS2 vitrification protection solution, then add 2 mL of filtered sterilized unloading solution, unload for 20 min, finally take the shoot tip out of the cryopreservation tube, absorb the unloading solution on the surface with sterile filter paper, inoculate into 1 / 2MS + glutathione 1μmol / L + gibberellin 0.1mg / L + acid hydrolyzed casein 200mg / L + 3% sucrose + 0.7% agar, pH 5.8 culture medium, and culture in an artificial climate box at 25℃ in the dark. After the test tube tubers are formed, culture in the light, with a lighting time of 12h / d and a light intensity of about 2000lx.

[0148] like Figure 2 As shown in the figure, as the size of the shoot tip gradually decreases, the survival rate and regeneration rate after cryopreservation also gradually decrease significantly. The survival rate of the large shoot tip is 61.1%, and the regeneration rate is 58.3%, while the survival rate and regeneration rate of the small shoot tip are significantly reduced, which are 25% and 21.9%, respectively. The reason may be that as the size of the shoot tip decreases, its own stress resistance gradually weakens, and the cytotoxicity of dimethyl sulfoxide after PVS2 treatment is too toxic to the shoot tip. The large shoot tip itself has strong stress resistance and is easier to operate when dissecting the shoot tip. Therefore, the optimal size of the Pinellia shoot tip for cryopreservation is 0.8-1mm.

[0149] 3. Effect of low temperature acclimation time on cryopreservation of shoot tips

[0150] The first generation tubers together with the culture medium were placed in a 4°C refrigerator for cryogenic acclimation for one and two weeks, and then the stem tips were peeled and vitrified for ultra-low temperature preservation. The rest of the operations were the same as those in the article "The Effect of Different Sizes of Shoot Tips on Shoot Tip Ultra-low Temperature Preservation".

[0151] like Figure 3As shown in the figure, the survival rate and regeneration rate of the materials treated with low temperature for 0 weeks, one week, and two weeks after ultra-low temperature preservation were significantly different. The survival rate and regeneration rate of the materials treated for one week were the highest, both 69.4%, and the survival rate and regeneration rate of the shoot tips without low temperature training were both 53.3%, while the survival rate and regeneration rate were the lowest after two weeks of low temperature treatment, both 26.8%. The reason may be that low temperature training improves the stress resistance of the shoot tips, but the treatment time is too long, which causes certain damage to the vitality of the shoot tips, thus reducing the survival rate after ultra-low temperature treatment.

[0152] 4. Effects of different PVS2 treatment times on cryopreservation of shoot tips

[0153] First generation tube tubers were selected, and shoot tips of 0.5-0.7 mm in size were dissected out in an ultra-clean workbench. PVS2 was treated at room temperature for 0, 20, 40, 90, and 120 min. The rest of the operations were the same as those in the study on the effects of different sizes of shoot tips on cryopreservation of shoot tips.

[0154] PVS treatment is a key step in vitrification cryopreservation, and PVS2 is a commonly used vitrification protective solution. When PVS2 is used to treat materials, the cells can be dehydrated while the cryoprotectant solution penetrates into the cells, increasing the viscosity of the cytoplasm, thereby reducing the damage to the cells during cryopreservation. However, the dimethyl sulfoxide in PVS2 is cytotoxic and may interfere with cell metabolism, enzyme activity, cell cycle, cell growth and function, and may even cause cell death. Therefore, it is crucial to choose an appropriate PVS2 treatment time. The 0.5-0.7mm pinellia stem apex was dissected as a preservation material to study the effects of PVS2 vitrification protective solution treatment for 20, 40, 60, 90, and 120 minutes on the survival rate and regeneration rate of the stem apex.

[0155] The results are as follows Figure 4 As shown in the data, there was no significant difference in the survival rate and regeneration rate of the shoot apex when treated with PVS2 for 20-60 minutes, but the survival rate and regeneration rate were the highest at 40 minutes, both of which were 55.7%. As the PVS2 treatment time increased, the survival rate and regeneration rate of the shoot apex decreased significantly, but when the treatment time was 120 minutes, the survival rate and regeneration rate were only 23.7%. The reason may be that the long treatment time of PVS2 produced a certain toxic effect on the shoot apex.

[0156] 5. Effects of different physiological ages and states on cryopreservation of shoot tips

[0157] Take tubers of different physiological ages and states: first-generation tubers, second-generation fresh tubers, second-generation dormant tubers, dissect the stem tips, and then perform the same operations as in the effect of different sizes of stem tips on cryopreservation of stem tips.

[0158] Depend on Figure 5It can be seen that there is no significant difference in the survival rate and regeneration rate of the first-generation tubers and the second-generation fresh tubers. The survival rates are 48.6% and 51.2%, and the regeneration rates are 43.2% and 46.1%, respectively. The survival rate and regeneration rate of the second-generation dormant tuber shoot tips are significantly improved, both of which are 75%. The reason may be that the first-generation tubers and the second-generation fresh tubers are fresh tubers, and their shoot tips have a higher water content. When they are frozen, ice crystals are easily formed, which damages the cells and reduces the survival rate. The water content of the shoot tips of the second-generation dormant tubers is low, which is more suitable for ultra-low temperature storage. The shoot tips will turn green 2 weeks after the recovery culture, start to grow after 4 weeks, and can regenerate into plants after 6 weeks, and the plants grow well (see Figure 6 ).

[0159] 5. Effect of unloading on cryopreservation of shoot tips

[0160] The second generation dormant tubers were taken, the stem tips were separated and vitrified for ultra-low temperature preservation. The unloading treatment was the same as the operation method in the Effect of stem tips of different sizes on ultra-low temperature preservation of stem tips. The non-unloading treatment meant omitting the unloading step, and other operations were the same as those in the unloading treatment.

[0161] Both the unloading and non-unloading treatments had significant effects on the survival and regeneration rates of shoot tips cryopreserved (see Figure 7 ). The survival rate and regeneration rate of the shoot tips with the unloading procedure were both as high as 60%, while the survival rate and regeneration rate of the shoot tips without the unloading procedure were only 27.8%. This shows that unloading is an essential technical link for the cryopreservation of the shoot tips of Pinellia ternata.

[0162] 6. Genetic stability test of ultra-low temperature regenerated plants

[0163] (1) Morphological detection

[0164] The 44 ultra-low temperature regenerated plants cultured in Example 3 were taken out from the artificial climate box, first cultured under natural light for 3 days, then the sealing film was removed and the seedlings were exposed for 3 days for transplanting, and the leaf shape, leaf color, tuber color and root color of the regenerated plants were observed and counted.

[0165] It was found that there was no difference in leaf shape, leaf color, tuber color and root color among plants in the same strain. However, two leaf shapes were found between different strains: one was heart-shaped (length-width ratio was about 1:1), and the other was elliptical (length-width ratio was about 2:1), indicating that the genetic background of the stem tips of Pinellia ternata from different stem sources was different (see Figure 8 ).

[0166] (2) Genetic stability testing at the molecular level

[0167] 1-8, 8 lines (a group of plants regenerated after ultra-low temperature preservation of one stem tip is called a line), with a total of 44 regenerated plants, were randomly selected, and the ISSR-PCR technology was used to detect the genetic stability of the ultra-low temperature regenerated plants.

[0168] ①Extract DNA using the modified CTAB method

[0169] ② Primer screening

[0170] According to the theoretical annealing temperature, different temperature gradients were automatically generated on the PCR amplification instrument for amplification. Five primers were selected from 18 primers and the optimal annealing temperature was determined.

[0171] ③ISSR-PCR reaction system and amplification procedure

[0172] The best reaction system for ISSR-PCR reaction was the optimized 25 μL mixed system: 12.5 μL 2× Taq PCR Master MixⅡ (containing dye solution), 9.5 μL ddHO 2 O, 1 μL primer, and 2 μL DNA were mixed evenly, and amplified on a PCR amplification instrument. The amplification program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 49.5°C (taking primer 835 as an example) for 45 s, extension at 72°C for 2 min, 35 cycles; complete extension at 72°C for 7 min, amplification ended, and stored at 4°C.

[0173] ④ISSR-PCR product detection

[0174] The genomic DNA of 44 ultra-low temperature regenerated plants of Pinellia ternata was amplified by PCR using the five selected primers (880, 881, 835, 554, IRRS-77) at the optimal annealing temperature. After the amplification, 5 μL of the amplified product was electrophoresed on a 1.5% agarose gel (1 μL of green nucleic acid dye was added to every 50 mL), using 0.5TBE as the electrophoresis buffer, the voltage was 110V, the electrophoresis was performed for about 90 minutes, and the image was taken and photographed on a gel imager.

[0175] (3) Data statistics and analysis

[0176] This study adopted a single-factor experimental design, with 5 replicates per treatment and 6 stem tips per replicate. The ISSR-PCR amplified bands were statistically analyzed by manual band reading, with "1" for bands present in the electrophoresis diagram and "0" for bands absent. Inconsistencies between the same strains were considered variant bands (including deletions and additions). Data were analyzed using Excel software, and variance analysis was performed using IBM SPSS 26.0 Duncan's new multiple range method.

[0177] Five primers were used to amplify the DNA of 44 cryopreserved plants, and a total of 1575 identifiable bands were obtained. Comparative analysis showed that the amplified band patterns between cryopreserved plants of the same strain were highly consistent, with only one variant band found in 40-5, with a variation rate of 0.063% (see Table 1 and Fig. 9 ). However, there were some differences in the banding patterns of ultra-low temperature regenerated plants between different strains, which was consistent with the phenotypic identification results, indicating that there were genetic differences in the stem tips from different tuber sources.

[0178]

[0179] Experimental Example 2: Method for ultra-low temperature detoxification of Pinellia tuber:

[0180] 1. Test Materials

[0181] In July 2021, diseased plants of Pinellia ternata were collected from the Hezheng Medicinal Botanical Garden of Gansu University of Chinese Medicine, transplanted in the laboratory, and propagated using tissue culture methods.

[0182] 2. Test methods

[0183] 1. Propagation of diseased plants

[0184] The petioles were cut and sterilized with 75% alcohol for 10 s, and then with 0.1% HgCl 2 (containing 1-2 drops of Tween 80) solution for 5 minutes, then disinfect with 2% NaClO for 8 minutes, and finally rinse with sterile water for 3 times. Use sterile filter paper to absorb the surface moisture, cut the petiole into small pieces of about 1 cm, inoculate in culture medium (1 / 2MS+3% sucrose+0.7% agar, pH5.8), place in an artificial climate box at 25℃ for light-proof culture, obtain test tube seedlings of diseased plants and use DAS-ELISA to detect whether they contain viruses.

[0185] 2. Take the stem tip for multiple times to remove the virus

[0186] Take the tuber of the first generation of diseased Pinellia ternata plants and take the stem tip of 0.5-0.7 mm under the dissecting microscope, inoculate it in the medium of 1 / 2MS+0.5mg / LKT+0.2 mg / L NAA+3% sucrose+0.7% agar, count the survival rate of the stem tip after 15 days, count the seedling rate of the stem tip after 30 days, and use DAS-ELISA method to detect viruses on the leaves of the test tube seedlings. Then peel off the stem tip of 0.5-0.7 mm for the second time for culture, observe the statistical indicators and virus detection methods in the same way as 1.

[0187] 3. Heat treatment combined with stem tip culture detoxification

[0188] The first generation diseased plant test tube seedlings (with tubers) in the vigorous growth period were placed in a light incubator with an initial temperature of 32°C and raised to 38°C at a rate of 1°C / d for 2 weeks; and cultured at 35°C for 2 and 4 weeks. After the culture was completed, 0.5-0.7 mm stem tips were peeled off under a dissecting microscope and inoculated into a culture medium of 1 / 2MS + 0.5 mg / L KT + 0.2 mg / L NAA + 3% sucrose + 0.7% agar. The statistical indicators and virus detection methods were the same as those in 1.

[0189] 4. Ultra-low temperature therapy

[0190] The ultra-low temperature virus-free plants were cultured using the method of Example 6. The survival rate of the stem tips was counted after 30 days, and the seedling rate of the stem tips was counted after 60 days. The virus was identified by morphological observation, and the virus was detected on the leaves of the test tube seedlings using the DAS-ELISA method.

[0191] 3. Data Statistics and Analysis

[0192] This study adopted a single-factor experimental design, with 5 replicates for each treatment and 6 shoot tips for each replicate. Excel software was used for data statistics, and IBM SPSS26.0 Duncan's new multiple range method was used for variance analysis.

[0193] 4. Results and Analysis

[0194] 1. Phenotypic symptoms of diseased plants and detection of virus infection in diseased plants by DAS-ELISA

[0195] The leaves of the Pinellia test tube seedlings obtained by tissue culture still had mosaic phenomenon, and a few leaves were slightly wrinkled. According to the instructions of the SMV and CMV antibody kits (purchased from Jiangsu Jingmei Biotechnology Co., Ltd.), virus detection was performed on the leaf tissues of the Pinellia diseased test tube seedlings (see Table 2). The positive control used virus standards; the negative control used normal virus-free Pinellia test tube seedlings; the blank control used PBST buffer. The specific operations were performed according to the instructions. The OD of the control well 405 Quality requirements for blank control wells, negative control wells, and positive control wells: OD values ​​of blank control wells and negative control wells 405 <0.20; positive OD 405 / Negative control OD 405 >5-10. Result judgment: When all control reactions are normal, the sample OD 405 / Negative control OD 405 Value ≥ 2, judged as positive; sample OD 405 / Negative control OD 405 A value < 2 was considered negative.

[0196]

[0197] As shown in Table 2, the test tube seedlings of Pinellia ternata produced after tissue culture propagation of diseased Pinellia ternata plants showed positive results in ELISA test, indicating that the test tube seedlings are still diseased plants and can be used for subsequent detoxification tests.

[0198] 2. Comparison of the effects of different detoxification methods

[0199] Different virus-free methods were used to culture the diseased Pinellia ternata plants and observe the phenotype of the test tube seedlings (see Fig.10 and 11 ), and the leaves of the test tube seedlings were cut and tested by ELISA method (see Fig.12 ), as shown in Table 3-5, different detoxification methods have different detoxification effects. The regeneration rate of the shoot tip after the second extraction was 100%. Although the detoxification rate for CMV was 40%, the detoxification rate for SMV was lower than the other four methods, only 80%; although the regeneration rate of the shoot tip was the lowest at only 70% in ultra-low temperature therapy, the detoxification rate for SMV was 85.7%, and the detoxification rate for CWV was the highest among all detoxification treatments at 57.1%, so this method is a suitable detoxification technology for Pinellia ternata; compared with the heat treatment at 35°C for 2 weeks, the test tube seedlings were all dry after the treatment. Compared with the two methods, although the regeneration rate of 2 weeks of heat treatment at 38℃ was lower than that of 2 weeks of heat treatment at 35℃, the virus-free rates of SMV and CWV were higher than those of 2 weeks of heat treatment at 35℃, which were 88% and 28% respectively; the test tube seedlings were also dried after 4 weeks of heat treatment at 35℃ and 2 weeks of heat treatment at 35℃, but the regeneration rate decreased with the extension of heat treatment time, but the virus-free rates of SMV and CWV at 4 weeks of heat treatment at 35℃ were higher than those of 2 weeks of heat treatment at 35℃, which were 88% and 48% respectively. Therefore, combined with the analysis of the two indicators of regeneration rate and virus-free rate, the appropriate virus-free technology system for Pinellia ternata is 4 weeks of heat treatment at 35℃ and ultra-low temperature therapy.

[0200]

[0201]

[0202] .

Claims

1. A method for ultra-low temperature preservation of regenerated plants and detoxification of Pinellia ternata, characterized in that The steps include: Step 1: Material cultivation: Healthy or diseased Pinellia ternata seed stems are sown in a flower pot to germinate and grow to obtain Pinellia ternata plants, petioles are sterilized, cut into small sections of 0.5-2 cm, and inoculated into 1 / 2MS culture medium to obtain an independent and complete small tuber and germinate into a seedling, the petioles of the test tube seedlings are used as materials to induce culture again to obtain small tubers and germinate into seedlings, to obtain first-generation tubers and first-generation seedlings, the stems and leaves are cut off, the first-generation tubers are transferred to a fresh culture medium, the tubers germinate into seedlings again and grow up, to obtain second-generation tubers and second-generation seedlings, and second-generation healthy test tube tubers or second-generation diseased test tube tubers are selected from the second-generation tubers, and when the stems and leaves are withered and in a dormant state, the tubers and culture bottles are pretreated at 4°C in the dark for 1 week; Step 2: Loading process: Peel off a 0.2-1 mm-sized stem tip from the second-generation healthy test tube tuber or the second-generation diseased test tube tuber obtained in step 1, and place the stem tip in a sterile cryopreservation tube containing 1 mL of filtered and sterilized loading solution, wherein the loading solution is MS+2 mol / L glycerol+0.4 mol / L sucrose+1 μmol / L glutathione, pH 5.7, and treat at room temperature for 20 min before removing the loading solution with a pipette; Step 3: Vitrification treatment: Add 1.0-2 mL of filtered and sterilized PVS2 vitrification protection solution to the sterile cryopreservation tube obtained in step 2. The formula of PVS2 vitrification protection solution is MS + 30% glycerol + 15% ethylene glycol + 15% dimethyl sulfoxide + 0.4 M sucrose + glutathione 1 μmol / L, pH 5.7, and treat the shoot tip at room temperature for 20-120 minutes; Step 4: Ultra-low temperature treatment: The sterile cryopreservation tube obtained in step 3 is placed in liquid nitrogen for storage for no less than 120 minutes; Step 5: Thawing and reheating: Take out the sterile cryopreservation tube obtained in step 4 from liquid nitrogen and thaw it in a 38°C water bath for 2 minutes; Step 6: Uninstallation process: After thawing, use a pipette to remove the PVS2 vitrification protection solution in the sterile cryopreservation tube, and then add 1.0-2mL of filtered sterilized unloading solution. The formula of the unloading solution is 1 / 2MS+1.2mol / L sucrose, pH 5.7, and treat the shoot tip for 20 minutes. Step 7: Regeneration plant culture: Take out the shoot tip treated in step six from the sterile cryopreservation tube, absorb the unloading liquid on the surface with sterile filter paper, and inoculate it into 1 / 2MS + 6-KT0.5mg / L + NAA0.2mg / L + glutathione1μmol / L + gibberellin0.1mg / L + acid hydrolyzed casein200mg / L + 0.7% agar, pH5.8 culture medium, first culture in the dark for 7 days, and then transfer to 500-1000LUX weak light conditions to culture the regenerated plants.

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