Sweet potato seedling detoxification rapid propagation method and application

Abstract

The invention belongs to the technical field of sweet potato seedling detoxification, and particularly relates to a sweet potato seedling detoxification rapid propagation method and application, and the sweet potato seedling detoxification rapid propagation method comprises the following steps: carrying out heat treatment culture on sweet potato seedlings at 36-38 DEG C, disinfecting, and taking stem segments with the length of 0.5-1.5 cm as explants; inoculating the explant into a primary culture medium for culturing; after multiple shoots are induced from the explants, inoculating the explants into a subculture medium for culturing; cutting the cultured cluster buds into single buds, inoculating the single buds into a rooting culture medium for culturing, and detecting the detoxification effect after bud seedlings root to obtain detoxified sweet potato tissue culture seedlings. By means of the method, virus detoxification of the sweet potato seedlings can be achieved, the detoxification rate reaches up to 95%, the survival rate reaches 96.7%, the seedling breeding period of 2-3 months is shortened to 45-50 days, the health level and breeding efficiency of the seedlings are improved, and the yield and quality of sweet potatoes are further improved.

Description

Technical Field

[0001] The invention belongs to the technical field of virus-free sweet potato seedlings, and particularly relates to a virus-free and rapid propagation method for sweet potato seedlings and its application. Background Art

[0002] Excellent sweet potato LPFS ( Ipomoea batatas Sweet potato (Sweet potato) is an important food and cash crop, but its production has long been severely threatened by viral diseases. Over 30 viruses are known to infect sweet potatoes. Among these, high-quality sweet potato LPFS is primarily infected with a combination of sweet potato feathery mottle virus (SPFMV), sweet potato latent virus (SPLV), and sweet potato chlorotic stunt virus (SPCSV), resulting in extremely high rates of infection in seedlings. Viruses accumulate through asexual reproduction in seedlings, causing leaf yellowing and deformity, stunted stems, reduced root yield, and deteriorated quality (e.g., reduced starch content and increased fiber). Furthermore, viruses can reduce sweet potato's resistance to stress and exacerbate outbreaks of fungal and bacterial diseases, severely hindering the sustainable development of the industry.

[0003] Currently, commonly used sweet potato virus-free methods include heat treatment, chemical treatment, and stem apex stripping. Heat treatment inhibits viral replication by maintaining high temperatures of 60°C to 70°C, but it can cause heat damage to sweet potato seedlings, affecting their survival rate. Chemical treatment is complex, requiring precise control of drug use, and is prone to phytotoxicity and environmental pollution. The stem apex stripping method, due to its small size, requires strict in vitro culture conditions, making it difficult to successfully induce seedlings. Furthermore, the virus migrates rapidly within the sweet potato, affecting the virus-free rate of the stem apex seedlings, the survival rate of transplanted virus-free tissue culture seedlings, and the accuracy of virus disease detection, thus hindering the production and application of virus-free sweet potato seedlings. Therefore, there is an urgent need to develop an efficient and stable virus-free rapid propagation technology to improve the success rate of virus-free, propagation efficiency, and seedling quality. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a method and application for the rapid propagation of sweet potato seedlings by virus removal. The method provided by the present invention can achieve virus removal of sweet potato seedlings, shorten the breeding cycle of seedlings, improve the health level and breeding efficiency of seedlings, thereby improving the yield and quality of sweet potatoes.

[0005] To achieve the above purpose, the specific technical solutions of the present invention are as follows: The invention provides a method for virus-free and rapid propagation of sweet potato seedlings, comprising the following steps: utilizing the characteristic that viruses are sensitive to high temperatures, subjecting the sweet potato seedlings to heat treatment and culturing at 36°C to 38°C, thereby inactivating the sweet potato virus and making the virus propagation speed in the sweet potato slower than the growth speed of the plant, thereby ensuring that the sweet potato stem tip growth cone region is virus-free, achieving preliminary virus-free, and reducing the contamination rate of tissue culture seedlings; at the same time, stimulating stem tip meristem cells to accelerate division; and the high temperature promotes the production of heat shock proteins in plant cells, thereby improving plant stress resistance. After disinfection, surface microorganisms are killed and a 0.5-1.5 cm long sterilized stem segment is taken as an explant. This part has a low degree of differentiation, strong division ability, and relatively low virus content. The explants are inoculated into primary culture medium to induce the shoot apical meristem to break through dormancy, grow and differentiate, and produce clustered buds; When the explants grow clustered buds, they are inoculated into subculture medium to promote the elongation of clustered buds, increase the number of buds, and provide more materials for subsequent proliferation and reproduction; The cultivated buds are cut into single buds and inoculated into subculture medium to further increase the number of plants while maintaining their virus-free status; cutting the cultured clustered buds into single buds, inoculating them into a rooting medium, and culturing them to allow the single buds to take root and form complete regenerated seedlings. After the regenerated seedlings are formed, testing the virus-free effect to obtain virus-free sweet potato tissue culture seedlings; Each liter of the primary culture medium consists of 0.9 mg to 1.1 mg of 6-benzylaminopurine, 0.09 mg to 1.11 mg of naphthaleneacetic acid and MS culture medium; each liter of the secondary culture medium consists of 0.9 mg to 1.1 mg of 6-benzylaminopurine, 0.09 mg to 0.11 mg of naphthaleneacetic acid, 0.2 mg to 1 mg of gibberellic acid and MS culture medium; each liter of the rooting culture medium consists of 0.9 mg to 1.1 mg of 6-benzylaminopurine, 0.09 mg to 1.11 mg of naphthaleneacetic acid and MS culture medium.

[0006] Furthermore, the thermal culture time is 42 days to 56 days, and the light exposure time is 11 hours / day to 13 hours / day.

[0007] Furthermore, the explant is cultured in the primary culture medium at a temperature of 23° C. to 27° C., a light intensity of 2300 lx to 2700 lx, and a light duration of 11 h / d to 15 h / d.

[0008] Furthermore, the clustered buds are cultured in the subculture medium at a temperature of 23° C. to 27° C., a light intensity of 2300 lx to 2700 lx, and a light duration of 11 h / d to 15 h / d.

[0009] Furthermore, the temperature of the single bud cultured in the rooting medium is 23° C. to 27° C., the light intensity is 2300 lx to 2700 lx, and the light duration is 11 h / d to 15 h / d.

[0010] Furthermore, the disinfection is specifically performed by soaking in 75% alcohol by volume for 20s to 50s, rinsing with sterile water, and then soaking in 1% sodium hypochlorite solution with an effective concentration of 1% for 13min to 17min, and rinsing with sterile water.

[0011] Furthermore, the method for detecting the detoxification effect is a PCR method, an antigen detection method or an antibody detection method.

[0012] Furthermore, the method for detecting the detoxification effect comprises the following steps: Using the regenerated seedling cDNA as a template, PCR amplification was performed on the template using a primer set for amplifying sweet potato feathery mottle virus, sweet potato latent virus, sweet potato chlorotic spot virus, sweet potato G virus or sweet potato chlorotic stunt virus, and the detoxification effect was determined based on the amplification results.

[0013] Furthermore, the primer set for amplifying the sweet potato feathery mottle virus comprises an upstream sequence such as the nucleotide sequence shown in SEQ ID NO.1 and a downstream sequence such as the nucleotide sequence shown in SEQ ID NO.2; the primer set for amplifying the sweet potato chlorotic stunt virus comprises an upstream sequence such as the nucleotide sequence shown in SEQ ID NO.3 and a downstream sequence such as the nucleotide sequence shown in SEQ ID NO.4; the primer set for amplifying the sweet potato latent virus comprises an upstream sequence such as the nucleotide sequence shown in SEQ ID NO.5 and a downstream sequence such as the nucleotide sequence shown in SEQ ID NO.6; the primer set for amplifying the sweet potato chlorotic spot virus comprises an upstream sequence such as the nucleotide sequence shown in SEQ ID NO.7 and a downstream sequence such as the nucleotide sequence shown in SEQ ID NO.8; the primer set for amplifying the sweet potato G virus comprises an upstream sequence such as the nucleotide sequence shown in SEQ ID NO.9 and a downstream sequence such as the nucleotide sequence shown in SEQ ID NO.10.

[0014] Furthermore, the variety of the sweet potato is the excellent sweet potato LPFS, Xushu No. 32, Longshu No. 9, Jishu No. 13 or Guishu No. 9.

[0015] The second aspect of the present invention provides an application of the above-mentioned method in sweet potato breeding.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The invention provides an efficient and rapid method for detoxifying and rapidly propagating sweet potato seedlings and its application, comprising the following steps: culturing and sterilizing the sweet potato seedlings at 36°C to 38°C, taking a stem segment with a length of 0.5 to 1.5 cm as an explant; placing the explant in a primary culture medium (MS culture medium containing 0.9 mg / L to 1.1 mg / L 6-benzylaminopurine and 0.09 mg / L to 1.11 mg / L naphthaleneacetic acid) for culture to induce clustered buds; and placing the explant in a secondary culture medium (MS culture medium containing 0.9 mg / L to 1.11 mg / L naphthaleneacetic acid) after the clustered buds grow out of the explant. The method comprises the following steps: culturing the virus-free sweet potato tissue culture seedlings in an MS medium containing 0.09 mg / L to 0.11 mg / L naphthaleneacetic acid and 0.2 mg / L to 1 mg / L gibberellic acid; cutting the cultured clustered buds into single buds, placing the buds in a rooting medium (MS medium containing 0.9 mg / L to 1.1 mg / L 6-benzylaminopurine and 0.09 mg / L to 1.11 mg / L naphthaleneacetic acid), detecting the virus-free effect after the buds take root, and obtaining virus-free sweet potato tissue culture seedlings; and hardening the virus-free seedlings at 15°C to 25°C and transplanting them. First, through heat treatment combined with specific stem tip selection, the virus removal rate was increased from the conventional 70%~85% to 95%, fundamentally solving the problem of high virus rate in seedlings; secondly, through specific culture medium and culture conditions, the breeding cycle was shortened from the traditional 2 months to 3 months to 45 days to 50 days, thereby improving production efficiency; at the same time, the improved culture system made the seedling survival rate as high as 96.7%.

[0017] The sweet potato seedling virus-free rapid propagation method provided by the present invention improves the health level and propagation efficiency of the seedlings, is easy to operate and has good repeatability, is particularly suitable for factory-based seedling cultivation of excellent sweet potato LPFS varieties, provides efficient and reliable technical support for the sweet potato industry, and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 This is a picture of the stem apex excision of excellent sweet potato LPFS under a 40x microscope. Figure A is a picture of the stem apex peeling under a 40x dissecting microscope. Figure B is a picture of the stem apex inoculation.

[0020] Figure 2Figures show the growth of sweet potato stem segments cultured in primary culture media with different formulations; Figure A (Example 2) shows the culture medium formulation of MS + 1.0 mg / L 6-BA + 0.09 mg / L NAA; Figure B (Example 1) shows the culture medium formulation of MS + 1.0 mg / L 6-BA + 0.11 mg / L NAA; Figure C (Example 5) shows the culture medium formulation of MS + 1.1 mg / L 6-BA + 0.10 mg / L NAA; Figure D (Example 3) shows the culture medium formulation of MS + 1.0 mg / L 6-BA + 0.10 mg / L NAA; and Figure E (Example 4) shows the culture medium formulation of MS + 0.9 mg / L 6-BA + 0.10 mg / L NAA.

[0021] Figure 3 Figure 3 is a comparison of the growth of sweet potato shoot tips cultured in subculture culture media with different formulations. In Figure a (Example 4), the culture medium formula is MS + 1.0 mg / L 6-BA + 0.11 mg / L NAA + 0.6 mg / L GA3; in Figure b (Example 1), the culture medium formula is MS + 1.0 mg / L 6-BA + 0.11 mg / L NAA + 0.8 mg / L GA3; in Figure c (Example 3), the culture medium formula is MS + 1.1 mg / L 6-BA + 0.10 mg / L NAA + 0.4 mg / L GA3; in Figure d (Example 5), the culture medium formula is MS + 1.0 mg / L 6-BA + 0.10 mg / L NAA + 1.0 mg / L GA3; in Figure e (Example 2), the culture medium formula is MS + 0.9 mg / L 6-BA + 0.10 mg / L NAA + 0.2 mg / L GA3.

[0022] Figure 4 The agarose gel electrophoresis diagram of RNA quality detection, lanes 1 to 5 are RNA extracted from fresh leaves of the untreated superior sweet potato LPFS (CK), the superior sweet potato LPFS obtained in Example 1, and the superior sweet potato LPFS obtained in Comparative Examples 1 to 3, respectively.

[0023] Figure 5This is the agarose gel electrophoresis diagram of the original virus detection in sweet potato. Lanes A1, A2, A3, A4 and A5 represent the amplification results of the SPFMV, SPCSV, SPLV, SPCFV and SPVG virus primers under 52℃ amplification conditions, respectively; Lanes B1, B2, B3, B4 and B5 represent the amplification results of the SPFMV, SPCSV, SPLV, SPCFV and SPVG virus primers under 54℃ amplification conditions, respectively; Lanes C1, C2, C3, C4 and C5 represent the amplification results of the SPFMV, SPCSV, SPLV, SPCFV and SPVG virus primers under 56℃ amplification conditions, respectively. The amplification results of the SPFMV, SPCSV, SPLV, SPCFV and SPVG virus primer groups under the amplification conditions; lanes D1, D2, D3, D4 and D5 respectively represent the amplification results of the SPFMV, SPCSV, SPLV, SPCFV and SPVG virus primer groups under the 58°C amplification conditions; lanes E1, E2, E3, E4 and E5 respectively represent the amplification results of the SPFMV, SPCSV, SPLV, SPCFV and SPVG virus primer groups under the 60°C amplification conditions.

[0024] Figure 6 The electrophoresis diagrams show the detoxification effects of different treatments, where M represents DNA Marker. Lanes 1 to 5 respectively represent the amplification results of the cDNA of the untreated superior sweet potato LPFS (CK), the superior sweet potato LPFS obtained in Example 1, and the superior sweet potato LPFS obtained in Comparative Examples 1 to 3 using the sweet potato feathery mottle virus (SPFMV) primer set at 52°C. Lanes 6 to 10 respectively represent the amplification results of the cDNA of the untreated superior sweet potato LPFS (CK), the superior sweet potato LPFS obtained in Example 1, and the superior sweet potato LPFS obtained in Comparative Examples 1 to 3 using the sweet potato G virus (SPVG) primer set at 60°C.

[0025] Figure 7 This is a diagram illustrating the process of rapid propagation of sweet potato virus-free. Figure A shows the primary culture of the sweet potato stem tip; Figure B shows the bud induction stage of the sweet potato stem tip; Figure C shows the rooting induction stage of the sweet potato stem tip; Figure D shows the sweet potato stem tip growing into a complete plant.

[0026] Figure 8 These are virus-free seedling transplanting pictures. Pictures A, B and C are all high-quality sweet potato LPFS virus-free seedlings transplanted after hardening. DETAILED DESCRIPTION

[0027] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0028] The excellent sweet potato LPFS variety seed potatoes in the present invention were donated by Shaanxi Subway Agricultural Technology Co., Ltd.

[0029] High-quality sweet potato LPFS are susceptible to viral diseases, leading to leaf yellowing, stunted stems, reduced tuber yield, and decreased quality. Current virus removal methods include heat treatment, chemical treatment, and shoot apex stripping, but all have drawbacks: heat treatment can easily damage seedlings, chemical treatment can cause phytotoxicity and contamination, and shoot apex stripping is difficult to perform and can cause rapid virus migration, resulting in low virus removal rates and unstable seedling success rates. Furthermore, the survival rate of virus-free seedlings after transplantation is low, and virus detection efficiency is insufficient, hindering the large-scale production of virus-free seedlings. Therefore, there is an urgent need to develop an efficient and stable virus removal and rapid propagation technology to improve virus removal success rates, reproductive efficiency, and seedling quality.

[0030] The invention provides a method for rapid propagation of sweet potato seedlings free of virus and its application, comprising the following steps: culturing and sterilizing the sweet potato seedlings at 36-38°C, taking a stem segment with a length of 0.5-1.5 cm as an explant; placing the explant in a primary culture medium (MS culture medium containing 0.9 mg / L-1.1 mg / L 6-benzylaminopurine and 0.09 mg / L-1.11 mg / L naphthaleneacetic acid) to induce clustered buds; and placing the explant in a secondary culture medium (MS culture medium containing 0.9 mg / L-1.1 mg / L 6-benzylaminopurine and 0.09 mg / L-1.11 mg / L naphthaleneacetic acid) after the clustered buds grow out of the explant. The method comprises culturing the seedlings in an MS medium containing 0.9 mg / L to 1.1 mg / L 6-benzylaminopurine, 0.09 mg / L to 0.11 mg / L naphthaleneacetic acid, and 0.2 mg / L to 1 mg / L gibberellic acid; cutting the cultivated buds into single buds, placing them in a rooting medium (MS medium containing 0.9 mg / L to 1.1 mg / L 6-benzylaminopurine and 0.09 mg / L to 1.11 mg / L naphthaleneacetic acid), and testing the virus-free effect after the seedlings take root to obtain virus-free sweet potato tissue culture seedlings. The method provided by the present invention can achieve virus-free sweet potato seedlings, shorten the seedling reproduction cycle, improve the seedling health and reproduction efficiency, and further improve the yield and quality of sweet potatoes.

[0031] Example 1: A method for rapid propagation of sweet potato seedlings free of virus 1. Peat, vermiculite, and perlite were mixed in a mass ratio of 3:2:1 to obtain a mixed matrix. Robust, bud-pointed, high-quality sweet potato LPFS variety seed potatoes were placed in the mixed matrix and watered thoroughly. The culture conditions were 28°C and 12 h / d of light. When the sweet potato sprouts were 2 cm long, the sweet potato seedlings were placed in an artificial intelligence climate incubator at 37°C and 12 h / d of light for 49 days.

[0032] 2. Take a 2cm~3cm sweet potato stem segment containing terminal buds or axillary buds after heat treatment, soak it in detergent water and shake it for 15 minutes to rinse off the detergent on the surface of the sweet potato stem segment, and rinse it under running tap water for 40 minutes to clean the surface of the sweet potato stem segment; after draining the water, place the washed sweet potato stem segment in a sterilized culture bottle, place it in a clean workbench, soak it in 75v / v% alcohol for 30 seconds, rinse it with sterile water 3 times, then soak it in 1v / v% sodium hypochlorite for 15 minutes, and rinse it with sterile water 7 times for disinfection.

[0033] 3. Use a scalpel to cut off the sterilized sweet potato stem segment, retain a 1 cm stem segment as an explant, and inoculate it into the primary culture medium of MS+1.0 mg / L 6-BA+0.11 mg / L NAA to induce clustered buds. The culture conditions are: 25℃, light intensity 2500 lx, and light time 13 h / d.

[0034] 4. When the stem tip grows clustered buds, peel off the young leaves at the stem tip under a 40x dissecting microscope, cut off the young leaves of the stem tip tissue with 1-2 leaf primordium growth points, and inoculate them into the subculture medium of MS+1.0mg / L 6-BA+0.11mg / L NAA+0.8mg / L GA3 (see Figure 1 ), the culture conditions were: 25℃, light intensity 2500 lx, and photoperiod 13 h / d.

[0035] 5. Cut the cultivated clustered buds into single buds and inoculate them into rooting medium of MS+1.0 mg / L 6-BA+0.11 mg / L NAA. The culture conditions are: 25℃, light intensity 2500 lx, and light time 13 h / d.

[0036] 6. Move the virus-free seedlings to a 25°C indoor environment for hardening. During this period, spray the virus-free seedlings with water frequently to prevent excessive evaporation of water and death of the virus-free seedlings. After hardening, transplant them. First, rinse the culture medium around the roots with tap water. If the roots are too long, cut them to 15 cm with scissors before transplanting. The transplanting medium is the mixed medium prepared in step 1. Pay attention to heat preservation and moisture retention within one week after transplanting, and avoid direct sunlight.

[0037] Example 2: A method for rapid propagation of sweet potato seedlings free of virus 1. Peat, vermiculite, and perlite were mixed in a mass ratio of 3:2:1 to obtain a mixed matrix. Robust, bud-forming, high-quality sweet potato LPFS variety seed potatoes were placed in the mixed matrix and watered thoroughly. The culture conditions were 28°C and 12 h / d of light. When the sweet potato sprouts were 2 cm long, the sweet potato seedlings were placed in an artificial intelligence climate incubator at 36°C and 13 h / d of light for 42 days.

[0038] 2. Take a 2cm~3cm sweet potato stem segment containing terminal buds or axillary buds after heat treatment, soak it in detergent water and shake it for 15 minutes to rinse off the detergent on the surface of the sweet potato stem segment, and rinse it under running tap water for 40 minutes to clean the surface of the sweet potato stem segment; after draining the water, place the washed sweet potato stem segment in a sterilized culture bottle, place it in a clean workbench, soak it in 75v / v% alcohol for 30 seconds, rinse it with sterile water 3 times, then soak it in 1v / v% sodium hypochlorite for 15 minutes, and rinse it with sterile water 7 times for disinfection.

[0039] 3. Use a scalpel to cut off the sterilized sweet potato stem segment, retain a 1 cm stem segment as an explant, and inoculate it into the primary culture medium of MS+1.0 mg / L 6-BA+0.09 mg / L NAA to induce clustered buds. The culture conditions are: 23℃, light intensity 2600 lx, and light time 12 h / d.

[0040] 4. When clustered buds grow out of the stem tip, peel off the young leaves at the stem tip under a 40x dissecting microscope, cut off the young leaves of the stem tip tissue with 1 to 2 leaf primordium growth points, and inoculate them into the subculture medium of MS + 1.0 mg / L 6-BA + 0.11 mg / L NAA + 0.2 mg / L GA3. The culture conditions are: 23℃, light intensity 2600 lx, and light time 12 h / d.

[0041] 5. Cut the cultivated clustered buds into single buds and inoculate them into rooting medium of MS+1.0 mg / L 6-BA+0.09 mg / L NAA. The culture conditions are: 23℃, light intensity 2600 lx, and light time 12 h / d.

[0042] 6. Move the virus-free seedlings to a 25°C indoor environment for hardening. During this period, spray the virus-free seedlings with water frequently to prevent excessive evaporation of water and death of the virus-free seedlings. After hardening, transplant them. First, rinse the culture medium around the roots with tap water. If the roots are too long, cut them to 15 cm with scissors before transplanting. The transplanting medium is the mixed medium prepared in step 1. Pay attention to heat preservation and moisture retention within one week after transplanting, and avoid direct sunlight.

[0043] Example 3: A method for rapid propagation of sweet potato seedlings free of virus 1. Peat, vermiculite, and perlite were mixed in a mass ratio of 3:2:1 to obtain a mixed matrix. Strong, bud-forming, high-quality sweet potato LPFS variety seed potatoes were placed in the mixed matrix and watered thoroughly. The culture conditions were 28°C and 12 h / d of light. When the sweet potato sprouts were 2 cm long, the sweet potato seedlings were placed in an artificial intelligence climate incubator at 38°C and 11 h / d of light for 55 days.

[0044] 2. Take a 2cm~3cm sweet potato stem segment containing terminal buds or axillary buds after heat treatment, soak it in detergent water and shake it for 15 minutes to rinse off the detergent on the surface of the sweet potato stem segment, and rinse it under running tap water for 40 minutes to clean the surface of the sweet potato stem segment; after draining the water, place the washed sweet potato stem segment in a sterilized culture bottle, place it in a clean workbench, soak it in 75v / v% alcohol for 30 seconds, rinse it with sterile water 3 times, then soak it in 1v / v% sodium hypochlorite for 15 minutes, and rinse it with sterile water 7 times for disinfection.

[0045] 3. Use a scalpel to cut off the sterilized sweet potato stem segment, retain a 1 cm stem segment as an explant, and inoculate it into the primary culture medium of MS+1.0 mg / L 6-BA+0.10 mg / L NAA for induction culture. The culture conditions are: 24℃, light intensity 2700 lx, and light time 11 h / d.

[0046] 4. When clustered buds grow out of the stem tip, peel off the young leaves at the stem tip under a 40x dissecting microscope, cut off the young leaves of the stem tip tissue with 1 to 2 leaf primordium growth points, and inoculate them into the subculture medium of MS + 1.0 mg / L 6-BA + 0.11 mg / L NAA + 0.4 mg / L GA3. The culture conditions are: 24℃, light intensity 2700 lx, and light time 11 h / d.

[0047] 5. Cut the cultivated clustered buds into single buds and inoculate them into rooting medium of MS+1.0mg / L 6-BA+0.10mg / L NAA. The culture conditions are: 24℃, light intensity 2700 lx, and light time 11 h / d.

[0048] 6. Acclimation and Transplantation: Move the virus-free seedlings to a 25°C indoor environment for hardening. During this period, spray the seedlings with water frequently to prevent excessive evaporation and death. After hardening, transplant them. First, rinse the culture medium around the roots with tap water. If the roots are too long, cut them to 15 cm with scissors before transplanting. The transplanting medium is the mixed medium prepared in step 1. Pay attention to heat preservation and moisture retention for one week after transplanting, and avoid direct sunlight.

[0049] Example 4: A method for rapid propagation of sweet potato seedlings free of virus 1. Peat, vermiculite, and perlite were mixed in a mass ratio of 3:2:1 to obtain a mixed matrix. Robust, bud-forming, high-quality sweet potato LPFS variety seed potatoes were placed in the mixed matrix and watered thoroughly. The culture conditions were 28°C and 12 h / d of light. When the sweet potato sprouts were 2 cm long, the sweet potato seedlings were placed in an artificial intelligence climate incubator at 37°C and 11 h / d of light for 45 days.

[0050] 2. Take a 2cm~3cm sweet potato stem segment containing terminal buds or axillary buds after heat treatment, soak it in detergent water and shake it for 15 minutes to rinse off the detergent on the surface of the sweet potato stem segment, and rinse it under running tap water for 40 minutes to clean the surface of the sweet potato stem segment; after draining the water, place the washed sweet potato stem segment in a sterilized culture bottle, place it in a clean workbench, soak it in 75v / v% alcohol for 30 seconds, rinse it with sterile water 3 times, then soak it in 1v / v% sodium hypochlorite for 15 minutes, and rinse it with sterile water 7 times for disinfection.

[0051] 3. Use a scalpel to cut off the sterilized sweet potato stem segment, retain a 1 cm stem segment as an explant, and inoculate it into the primary culture medium of MS+0.9 mg / L 6-BA+0.10 mg / L NAA to induce clustered buds. The culture conditions are: 26℃, light intensity 2400 lx, and light time 13 h / d.

[0052] 4. When clustered buds grow out of the stem tip, peel off the young leaves at the stem tip under a 40x dissecting microscope, cut off the young leaves of the stem tip tissue with 1 to 2 leaf primordium growth points, and inoculate them into the subculture medium of MS + 1.0 mg / L 6-BA + 0.11 mg / L NAA + 0.6 mg / L GA3. The culture conditions are: 26℃, light intensity 2400 lx, and light time 13 h / d.

[0053] 5. Cut the cultivated clustered buds into single buds and inoculate them into rooting medium of MS+0.9mg / L 6-BA+0.10mg / L NAA. The culture conditions are: 26℃, light intensity 2400 lx, and light time 13 h / d.

[0054] 6. Move the virus-free seedlings to a 25°C indoor environment for hardening. During this period, spray the virus-free seedlings with water frequently to prevent excessive evaporation of water and death of the virus-free seedlings. After hardening, transplant them. First, rinse the culture medium around the roots with tap water. If the roots are too long, cut them to 15 cm with scissors before transplanting. The transplanting medium is the mixed medium prepared in step 1. Pay attention to heat preservation and moisture retention within one week after transplanting, and avoid direct sunlight.

[0055] Example 5: A method for rapid propagation of sweet potato seedlings free of virus 1. Peat, vermiculite, and perlite were mixed in a mass ratio of 3:2:1 to obtain a mixed matrix. Robust, bud-pointed, high-quality sweet potato LPFS variety seed potatoes were placed in the mixed matrix and watered thoroughly. The culture conditions were 28°C and 12 h / d of light. When the sweet potato sprouts were 2 cm long, the sweet potato seedlings were placed in an artificial intelligence climate incubator at 37°C and 12 h / d of light for 49 days.

[0056] 2. Take a 2cm~3cm sweet potato stem segment containing terminal buds or axillary buds after heat treatment, soak it in detergent water and shake it for 15 minutes to rinse off the detergent on the surface of the sweet potato stem segment, and rinse it under running tap water for 40 minutes to clean the surface of the sweet potato stem segment; after draining the water, place the washed sweet potato stem segment in a sterilized culture bottle, place it in a clean workbench, soak it in 75v / v% alcohol for 30 seconds, rinse it with sterile water 3 times, then soak it in 1v / v% sodium hypochlorite for 15 minutes, and rinse it with sterile water 7 times for disinfection.

[0057] 3. Use a scalpel to cut off the sterilized sweet potato stem segment, retain a 1 cm stem segment as an explant, and inoculate it into the primary culture medium of MS+1.1 mg / L 6-BA+0.10 mg / L NAA to induce clustered buds. The culture conditions are: 27℃, light intensity 2300 lx, and light time 12 h / d.

[0058] 4. When clustered buds grow out of the stem tip, peel off the young leaves at the stem tip under a 40x dissecting microscope, cut off the young leaves of the stem tip tissue with 1 to 2 leaf primordium growth points, and inoculate them into the subculture medium of MS + 1.0 mg / L 6-BA + 0.11 mg / L NAA + 1.0 mg / L GA3. The culture conditions are: 27℃, light intensity 2300 lx, and light time 12 h / d.

[0059] 5. Cut the cultivated clustered buds into single buds and inoculate them into rooting medium of MS+1.1mg / L 6-BA+0.10mg / L NAA. The culture conditions are: 27℃, light intensity 2300 lx, and light time 12 h / d.

[0060] 6. Move the virus-free seedlings to a 25°C indoor environment for hardening. During this period, spray the virus-free seedlings with water frequently to prevent excessive evaporation of water and death of the virus-free seedlings. After hardening, transplant them. First, rinse the culture medium around the roots with tap water. If the roots are too long, cut them to 15 cm with scissors before transplanting. The transplanting medium is the mixed medium prepared in step 1. Pay attention to heat preservation and moisture retention within one week after transplanting, and avoid direct sunlight.

[0061] Comparative Example 1: A method for rapid propagation of sweet potato seedlings free of virus 1. Peat, vermiculite and perlite were mixed in a mass ratio of 3:2:1 to obtain a mixed matrix. Strong, bud-forming, high-quality sweet potato LPFS variety seed potatoes were placed in the mixed matrix and watered thoroughly. The culture conditions were 28°C and 12 h / d of light. When the sweet potato sprouts were 2 cm long, the sweet potato seedlings were cultured at 25°C and 12 h / d of light for 49 days.

[0062] 2. Take a 2cm~3cm sweet potato stem segment containing terminal buds or axillary buds after heat treatment, soak it in detergent water and shake it for 15 minutes to rinse off the detergent on the surface of the sweet potato stem segment, and rinse it under running tap water for 40 minutes to clean the surface of the sweet potato stem segment; after draining the water, place the washed sweet potato stem segment in a sterilized culture bottle, place it in a clean workbench, soak it in 75v / v% alcohol for 30 seconds, rinse it with sterile water 3 times, then soak it in 1v / v% sodium hypochlorite for 15 minutes, and rinse it with sterile water 7 times for disinfection.

[0063] 3. Place the buds of the sterilized stem segments on a culture dish with filter paper, peel off the young leaves under a 40x dissecting microscope, cut off the young leaves of the stem tip tissue with 1 to 2 leaf primordia at the growth point, and inoculate them into a culture medium containing MS + 1.0 mg / L 6-BA + 0.11 mg / L NAA + 0.8 mg / L GA3. The culture conditions are: 25°C, light intensity 2500 lx, and light time 13 h / d.

[0064] 4. The cultured seedlings were then inoculated into a rooting medium containing MS + 1.0 mg / L 6-BA + 0.11 mg / L NAA. The culture conditions were: 25°C, light intensity 2500 lx, and light time 13 h / d.

[0065] 5. Move the virus-free seedlings to a 25°C indoor environment for hardening. During this period, spray the virus-free seedlings with water frequently to prevent excessive evaporation of water and death of the virus-free seedlings. After hardening, transplant them. First, rinse the culture medium around the roots with tap water. If the roots are too long, cut them to 15 cm with scissors before transplanting. The transplanting medium is the mixed medium prepared in step 1. Pay attention to heat preservation and moisture retention within one week after transplanting, and avoid direct sunlight.

[0066] Comparative Example 2: A method for rapid propagation of sweet potato seedlings free of virus 1. Peat, vermiculite, and perlite were mixed in a mass ratio of 3:2:1 to obtain a mixed matrix. Robust, bud-pointed, high-quality sweet potato LPFS variety seed potatoes were placed in the mixed matrix and watered thoroughly. The culture conditions were 28°C and 12 h / d of light. When the sweet potato sprouts were 2 cm long, the sweet potato seedlings were placed in an artificial intelligence climate incubator at 37°C and 12 h / d of light for 49 days.

[0067] 2. Take a 2cm~3cm sweet potato stem segment containing terminal buds or axillary buds after heat treatment, soak it in detergent water and shake it for 15 minutes to rinse off the detergent on the surface of the sweet potato stem segment, and rinse it under running tap water for 40 minutes to clean the surface of the sweet potato stem segment; after draining the water, place the washed sweet potato stem segment in a sterilized culture bottle, place it in a clean workbench, soak it in 75v / v% alcohol for 30 seconds, rinse it with sterile water 3 times, then soak it in 1v / v% sodium hypochlorite for 15 minutes, and rinse it with sterile water 7 times for disinfection.

[0068] 3. Place the buds of the sterilized stem segments on a culture dish with filter paper, peel off the young leaves under a 40x dissecting microscope, cut off the young leaves of the stem tip tissue with 1 to 2 leaf primordia at the growth point, and inoculate them into a culture medium containing MS + 1.0 mg / L 6-BA + 0.11 mg / L NAA + 0.8 mg / L GA3. The culture conditions are: 25°C, light intensity 2500 lx, and light time 13 h / d.

[0069] 4. The cultured seedlings were then inoculated into a rooting medium containing MS + 1.0 mg / L 6-BA + 0.11 mg / L NAA. The culture conditions were: 25°C, light intensity 2500 lx, and light time 13 h / d.

[0070] 5. Move the virus-free seedlings to a 25°C indoor environment for hardening. During this period, spray the virus-free seedlings with water frequently to prevent excessive evaporation of water and death of the virus-free seedlings. After hardening, transplant them. First, rinse the culture medium around the roots with tap water. If the roots are too long, cut them to 15 cm with scissors before transplanting. The transplanting medium is the mixed medium prepared in step 1. Pay attention to heat preservation and moisture retention within one week after transplanting, and avoid direct sunlight.

[0071] Comparative Example 3: A method for rapid propagation of sweet potato seedlings free of virus 1. Peat, vermiculite and perlite were mixed in a mass ratio of 3:2:1 to obtain a mixed matrix. Strong, bud-forming, high-quality sweet potato LPFS variety seed potatoes were placed in the mixed matrix and watered thoroughly. The culture conditions were 28°C and 12 h / d of light. When the sweet potato sprouts were 2 cm long, the sweet potato seedlings were cultured at 25°C and 12 h / d of light for 49 days.

[0072] 2. Take a 2cm~3cm sweet potato stem segment containing terminal buds or axillary buds after heat treatment, soak it in detergent water and shake it for 15 minutes to rinse off the detergent on the surface of the sweet potato stem segment, and rinse it under running tap water for 40 minutes to clean the surface of the sweet potato stem segment; after draining the water, place the washed sweet potato stem segment in a sterilized culture bottle, place it in a clean workbench, soak it in 75v / v% alcohol for 30 seconds, rinse it with sterile water 3 times, then soak it in 1v / v% sodium hypochlorite for 15 minutes, and rinse it with sterile water 7 times for disinfection.

[0073] 3. Use a scalpel to cut off the sterilized sweet potato stem segment, retain a 1 cm stem segment as an explant, and inoculate it into the primary culture medium of MS+1.0 mg / L 6-BA+0.11 mg / L NAA to induce the culture of clustered buds. The culture conditions are: 25℃, light intensity 2500 lx, and light time 13 h / d.

[0074] 4. When clustered buds grow out of the stem tip, peel off the young leaves at the stem tip under a 40x dissecting microscope, cut off the young leaves of the stem tip tissue with 1 to 2 leaf primordium growth points, and inoculate them into the subculture medium of MS + 1.0 mg / L 6-BA + 0.11 mg / L NAA + 0.8 mg / L GA3. The culture conditions are: 25℃, light intensity 2500 lx, and light time 13 h / d.

[0075] 5. Cut the cultivated clustered buds into single buds and inoculate them into rooting medium of MS+1.0 mg / L 6-BA+0.11 mg / L NAA. The culture conditions are: 25℃, light intensity 2500 lx, and light time 13 h / d.

[0076] 6. Move the virus-free seedlings to a 25°C indoor environment for hardening. During this period, spray the virus-free seedlings with water frequently to prevent excessive evaporation of water and death of the virus-free seedlings. After hardening, transplant them. First, rinse the culture medium around the roots with tap water. If the roots are too long, cut them to 15 cm with scissors before transplanting. The transplanting medium is the mixed medium prepared in step 1. Pay attention to heat preservation and moisture retention within one week after transplanting, and avoid direct sunlight.

[0077] Experimental Example 1: Effects of different culture media on the growth of superior sweet potato LPFS.

[0078] like Figure 2 As shown, the culture media containing plant growth hormones used in Examples 1 to 5 can all promote the growth of sweet potato stem segments after 15 days of culture. However, by comparison, bottle B (Example 1) has the best growth, with a large number of roots and larger plants with more branches. Therefore, MS+1.0 mg / L 6-BA+0.11 mg / L NAA is the best formula for the primary generation (cluster bud induction) culture medium.

[0079] like Figure 3 As shown, the primary culture medium used in Examples 1 to 5 can promote the growth of sweet potato shoot tips after 30 days of culture. However, by comparison, bottle b (Example 1) has the best growth, with fast shoot tip growth and differentiation. Therefore, MS+1.0mg / L6-BA+0.11mg / L NAA++0.8mg / LGA3 is the best primary culture medium formula.

[0080] Experimental Example 2: Seedling Reproduction Cycle Statistics The present invention makes statistics on the seedling propagation cycle, and the results are shown in Table 1.

[0081] Table 1 Statistics of seedling propagation cycle According to statistics, the breeding cycle of this technical solution is 45 to 50 days, which is significantly shorter than the conventional seedling breeding cycle.

[0082] Experimental Example 2: Detoxification Effect Statistics The present invention used high-quality sweet potato LPFS that had not been treated in any way as a control group (CK) to explore the detoxification effects of the methods provided in Example 1 and Comparative Examples 1 to 3.

[0083] When the tissue culture seedlings grew five leaves, primer sets were designed to amplify viruses and test the virus-free efficacy of the different treatments for the superior sweet potato LPFS. The test viruses were: sweet potato feathery mottle virus (SPFMV); sweet potato latent virus (SPLV); sweet potato chlorotic spot virus (SPCFV); sweet potato virus G (SPVG); and sweet potato chlorotic stunt virus (SPCSV). Using sweet potato leaf cDNA as a template, polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR) techniques were used to amplify the SPFMV, SPLV, SPCFV, SPVG, and SPCSV virus-specific primers. The amplification results were analyzed as follows:

[0084] 1. RNA Extraction 100 mg of fresh leaves from each of the untreated superior sweet potato LPFS (CK), the superior sweet potato LPFS obtained in Example 1, and the superior sweet potato LPFS obtained in Comparative Examples 1-3 were placed in a pre-cooled mortar and thoroughly ground into powder using liquid nitrogen. RNA was extracted from each set of leaves using a plant total RNA extraction kit (purchased from Tiangen Biochemical Technology Co., Ltd.) according to the instructions, and the RNA quality was tested. Figure 4 As shown, the extracted RNA is of good quality and can be used in subsequent experiments.

[0085] 2. Obtaining cDNA RNA was reverse transcribed into cDNA using a reverse transcription extraction kit (purchased from Tiangen Biochemical Technology Co., Ltd.) according to the instructions, and the quality of the cDNA was tested (Table 2).

[0086] Table 2 cDNA quality detection 3. PCR amplification The primer set for amplifying SPFMV is D1, the primer set for amplifying SPCSV is D2, the primer set for amplifying SPLV is D3, the primer set for amplifying SPCFV is D4, and the primer set for amplifying SPVG is D5. The primer sequences are shown in Table 3.

[0087] Table 3 Primer sequences Using cDNA as a template, PCR amplification was performed according to the following RT-PCR reaction procedure and reaction system.

[0088] Reaction procedure: First, perform temperature gradient PCR amplification, that is, use five virus primer sets D1, D2, D3, D4, and D5 to amplify the CK group cDNA, and set the temperature gradient to 52℃, 54℃, 56℃, 58℃, and 60℃. The purpose is to find out the original viruses in the excellent sweet potato LPFS and the optimal temperature for PCR amplification.

[0089] Each 25 μL reaction system contains: 1 μL cDNA, 2 μL each of the upstream primer and downstream primer in the D1 primer set / D2 primer set / D3 primer set / D4 primer set / D5 primer set, 12.5 μL Taq enzyme, and ddH2O to make up.

[0090] After the PCR amplification reaction is completed, 8 μL of the amplified product is taken for agarose gel electrophoresis detection. After the electrophoresis detection is completed, a gel imager is used to analyze and record the results.

[0091] Figure 5 The figures are agarose gel electrophoresis diagrams of gradient amplification of five viral primer sets in the cDNA template of untreated high-quality sweet potato LPFS (CK group). In the figure, lanes A1, A2, A3, A4 and A5 represent the amplification results of the five viral primer sets under 52°C amplification conditions; lanes B1, B2, B3, B4 and B5 represent the amplification results of the five viral primer sets under 54°C amplification conditions; lanes C1, C2, C3, C4 and C5 represent the amplification results of the five viral primer sets under 56°C amplification conditions; lanes D1, D2, D3, D4 and D5 represent the amplification results of the five viral primer sets under 58°C amplification conditions; lanes E1, E2, E3, E4 and E5 represent the amplification results of the five viral primer sets under 60°C amplification conditions.

[0092] Figure 5 The results showed that the excellent sweet potato LPFS contained two of the five common sweet potato viruses: SPFMV and SPVG. The optimal amplification temperatures of SPFMV and SPVG primers were 52℃ and 60℃, respectively.

[0093] Figure 6The electrophoresis diagrams are of the detoxification effects of different treatments. The results show that SPFMV can be removed by the four treatment methods of Comparative Examples 1 to 3 and Example 1, but SPVG can be removed only by the treatment method of Example 1.

[0094] The virus-free rates of the virus-free rapid propagation methods provided in Examples 1 to 5 were statistically analyzed: 20 virus-free seedlings obtained by culture were tested for viruses using RT-PCR, and the results showed that 19 strains were completely free of both viruses, and the virus-free rate was calculated to be 95%.

[0095] Experimental Example 3: Survival Rate Statistics Figure 7 The process of sweet potato virus-free rapid propagation from primary culture of stem tip to complete plant formation is demonstrated. The virus-free seedlings obtained by the virus-free rapid propagation method provided in Examples 1 to 5 are hardened. Figure 8 This shows the growth of excellent sweet potato LPFS virus-free seedlings transplanted after hardening. It can be seen that all three sweet potato seedlings showed good growth momentum, the overall plant grew vigorously, the leaves were stretched and the leaf color was bright, no obvious disease symptoms or abnormal growth were found, and the virus-free treatment effectively improved its growth quality and stress resistance. According to statistics, 29 out of 30 virus-free seedlings can survive after transplanting, so the survival rate is 96.7%.

[0096] It should be noted that when numerical ranges are mentioned in the present invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints may be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes preferred embodiments. Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0097] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A method for rapid propagation of sweet potato seedlings free of virus, characterized in that: The following steps are involved: After the sweet potato seedlings were heat-treated and cultured at 36-38°C and sterilized, stem segments with a length of 0.5-1.5 cm were taken as explants; The explants are cultured in a primary culture medium, and when clustered buds grow out of the explants, they are cultured in a secondary culture medium; Cutting the cultured clustered buds into single buds, placing them in a rooting medium for cultivation, and detecting the virus-free effect after the buds take root to obtain virus-free sweet potato tissue culture seedlings; Each liter of the primary culture medium consists of 0.9 mg to 1.1 mg of 6-benzylaminopurine, 0.09 mg to 1.11 mg of naphthaleneacetic acid and MS culture medium; each liter of the secondary culture medium consists of 0.9 mg to 1.1 mg of 6-benzylaminopurine, 0.09 mg to 0.11 mg of naphthaleneacetic acid, 0.2 mg to 1 mg of gibberellic acid and MS culture medium; each liter of the rooting culture medium consists of 0.9 mg to 1.1 mg of 6-benzylaminopurine, 0.09 mg to 1.11 mg of naphthaleneacetic acid and MS culture medium.

2. The method according to claim 1, characterized in that The heat treatment culture time is 42 days to 55 days, and the light exposure time is 11 hours / day to 13 hours / day.

3. The method according to claim 1, characterized in that The explants are cultured in the primary culture medium at a temperature of 23° C. to 27° C., a light intensity of 2300 lx to 2700 lx, and a light duration of 11 h / d to 15 h / d.

4. The method according to claim 1, wherein The clustered buds are cultured in a subculture medium at a temperature of 23° C. to 27° C., a light intensity of 2300 lx to 2700 lx, and a light duration of 11 h / d to 15 h / d.

5. The method according to claim 1, characterized in that The temperature of culturing the single bud in the rooting medium is 23° C. to 27° C., the light intensity is 2300 lx to 2700 lx, and the light duration is 11 h / d to 15 h / d.

6. The method according to claim 1, characterized in that The disinfection step comprises soaking in 75% alcohol by volume for 20 seconds to 50 seconds, rinsing with sterile water, and then soaking in 1% sodium hypochlorite solution with an effective concentration of 1% for 13 minutes to 17 minutes, and rinsing with sterile water.

7. The method according to claim 1, characterized in that The method for detecting the detoxification effect is a PCR method, an antigen detection method or an antibody detection method.

8. The method according to claim 1, characterized in that The sweet potato varieties are the excellent sweet potato LPFS, Xushu No. 32, Longshu No. 9, Jishu No. 13 or Guishu No.

9.

9. Use of the method according to any one of claims 1 to 8 in sweet potato breeding.

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