Virus-free culture method of lily bulbs

Abstract

The invention discloses a virus-free culture method of lily bulbs, and relates to the technical field of lily bulb breeding, stem tips are adopted to culture lily bulbs, the stem tips have no vascular bundles, so that the lily bulbs contain few viruses, and an antioxidant is added to inhibit browning during stem tip culture; the preparation method comprises the following steps: preparing an antioxidant microcapsule by taking polyvinylpyrrolidone as a capsule wall and vitamin C, tert-butylhydroquinone and butylated hydroxyanisole as a capsule core, loading the antioxidant microcapsule on the surface of activated carbon, and adding a culture medium, so that the browning phenomenon is effectively inhibited; according to the method, detoxification propagation is carried out by utilizing a heat treatment method, the detoxification rate is effectively improved, detoxification is carried out by utilizing chemical treatment, and virus infection can be effectively inhibited by adding virazole, actinomycin D and 5-dihydrouracil into a culture medium, so that detoxified lily bulbs are cultured.

Description

TECHNICAL FIELD

[0001] The present application relates to lily seed ball breeding technology field, and particularly to a lily seed ball detoxification culture method. BACKGROUND

[0002] Lily is an important ornamental flower, which is very popular in the market. Lily has strong adaptability and wide application, and is deeply loved by people. The planting method of lily is mainly cuttage and ball division for seed ball propagation. Multi-generation cuttage and ball division leads to lily virus accumulation, which affects lily flower quality. After lily is infected with virus, the plant will have virus for life, and cannot be directly prevented and treated with drugs.

[0003] In order to solve the problem that lily is infected with virus, the present application provides a lily seed ball detoxification culture method. SUMMARY

[0004] The present application aims to provide a lily seed ball detoxification culture method to solve the problems in the prior art.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical scheme: A lily seed ball detoxification culture method, comprising the following steps: Step one: planting lily seed ball, culturing for 4 weeks, taking the sprouting bulb, cutting from the stem node, removing all external scales, retaining the ball core, disinfecting, inoculating into the culture medium containing antioxidant, culturing, and obtaining leaf from; Step two: cutting the leaf from, inoculating into the culture medium containing chemical agent, culturing, transferring to seed ball induction culture medium, the light intensity being 1600-1800Lx, culturing, and obtaining a detoxified lily seed ball.

[0006] More preferably, the disinfection step is: washing with neutral soap water for 5-7min, washing with tap water for 24-26h, mercury sterilization for 10-12min, soaking in alcohol for 15-17s, collecting lily stem tip, peeling off all leaf blades, heat treating for 20-22 days, cutting into stem tip with a diameter of 0.2-0.4mm, and washing with sterilized water for 5-7min.

[0007] More preferably, the culture medium containing antioxidant comprises the following substances: MS culture medium, 2.0-3.0mg / L 6-benzyladenine, 0.5-1.5mg / L naphthalene acetic acid, 30-32g / L sucrose, 5-8g / L agar powder and 7.5-8.5g / L modified activated carbon, adjusting pH to 5.8, high-pressure sterilization, and obtaining the culture medium containing antioxidant.

[0008] The optimized culture medium containing chemical agents comprises the following components: MS culture medium, 0.5-1.5 mg / L naphthalene acetic acid, 30-32 g / L sucrose, 10-12 mg / L virus bithionol, 10-12 mg / L actinomycin D, 10-12 mg / L 5-dihydro-uracil, 5-8 g / L agar powder, 3.5-4.5 g / L activated carbon, and dropwise addition of sodium hydroxide to adjust pH to 6.

[0009] The optimized seed ball induction culture medium comprises the following components: MS culture medium, 5-7 mg / L kinetin, 0.5-1.5 mg / L naphthalene acetic acid, 30-32 g / L sucrose, 5-8 g / L agar powder, 3.5-4.5 g / L activated carbon, and the pH of the seed ball induction culture medium is 5.8.

[0010] The optimized preparation method of the modified activated carbon comprises the following steps: taking antioxidant microcapsules and activated carbon, adding sodium hydroxide solution, stirring uniformly, ultrasonic dispersion for 14-16 h, reaction for 12-14 h, washing, drying, and obtaining the modified activated carbon.

[0011] The optimized preparation method of the antioxidant microcapsules comprises the following steps: taking polyvinylpyrrolidone and adding into deionized water, adding antioxidant composite solution, stirring uniformly, heating at a rotation speed of 400-600 rad / min, dropwise addition of sodium hydroxide solution, adding sodium sulfite solution, reaction for 1-3 h, dropwise addition of sodium chloride solution, reaction for 1-3 h, centrifugation, washing, suction filtration, vacuum drying, and obtaining the antioxidant microcapsules.

[0012] The optimized preparation method of the antioxidant composite solution comprises the following steps: taking vitamin C and adding into deionized water, adding tert-butyl hydroquinone, butylated hydroxyanisole and dibutyl hydroxytoluene, and adding polyoxyethylene sorbitan monooleate to prepare the antioxidant composite solution.

[0013] Compared with the prior art, the present application has the following beneficial effects: 1. After virus invades lily plants, generally through vascular bundle transfer, the stem tip has no vascular bundle, so the stem tip contains less virus, and therefore the stem tip is used to culture lily seed balls, virus in the body can be removed, and virus-free lily flower seed balls are obtained; during stem tip detoxification culture, reducing browning phenomenon is an important factor for the survival rate of stem tip culture, in order to inhibit the browning phenomenon, antioxidant is added during stem tip culture to inhibit the browning phenomenon.

[0014] 2. After cutting, the stem tip will secrete phenolic substances and polyphenol oxidase. Phenolic substances are oxidized by polyphenol oxidase, resulting in browning. Vitamin C, tert-butylhydroquinone and butylated hydroxyanisole are selected as antioxidants. The three act synergistically to enhance the antioxidant performance. Polyvinylpyrrolidone can react with phenolic and quinone substances, thereby effectively preventing the browning phenomenon induced by phenolic and quinone substances. Antioxidant microcapsules are prepared with polyvinylpyrrolidone as the capsule wall and vitamin C, tert-butylhydroquinone and butylated hydroxyanisole as the capsule core. The capsules are loaded onto the surface of activated carbon and added to the culture medium. The antioxidants are slowly released and absorbed by the stem tip, effectively curbing the occurrence of browning.

[0015] 3. Activated carbon has a large specific surface area, a developed pore structure, and a strong adsorption capacity, and can carry more antioxidant microcapsules; the surface of activated carbon contains hydroxyl and carboxyl groups, which can react with phenolic substances secreted by the stem tip and effectively curb the browning phenomenon.

[0016] 4. Using heat treatment to carry out virus-free reproduction effectively improves the virus-free rate. Heat treatment is because viruses and plant cells have different tolerance to temperature. Viruses will denature at higher temperatures, thereby inhibiting the spread and reproduction of viruses; higher temperatures can also promote plant growth, increase the virus-free area of ​​the plant growth point, and facilitate cutting larger stem tips for cultivation, thereby improving the survival rate of stem tips.

[0017] 5. Use chemical treatment to detoxify. Add antiviral agents to the culture medium to inhibit the reproduction of viruses. Adding ribavirin, actinomycin D, and 5-dihydrouracil to the culture medium can effectively inhibit the infection of viruses such as lily symptomless virus and lily mottle virus, thereby cultivating detoxified lily seed bulbs. DETAILED DESCRIPTION

[0018] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0019] The source and model of the material are not specially limited, and exemplarily include: vitamin C with a product number of 255564-5G, provided by Jiaxing Yigao Medical Instrument Co., Ltd.; tert-butyl hydroquinone with a product number of 53035ES76, provided by Yisheng Biotech (Shanghai) Co., Ltd.; butylated hydroxyanisole with a product number of T0787, provided by Shanghai Taoshu Biological Technology Co., Ltd.; dibutylated hydroxytoluene with a product number of 08, provided by Xi'an Jinxing Pharmaceutical Auxiliary Co., Ltd.; polyvinylpyrrolidone with a product number of HBWS221, provided by Hubei Weisheng Chemical Reagent Co., Ltd.; activated carbon with a product number of 4158, provided by Shanghai Ehe Trade Co., Ltd.; 6-benzyladenine with a product number of CP003352, provided by Hubei Weisheng Chemical Reagent Co., Ltd.; naphthalene acetic acid with a product number of ZY86, provided by Shanghai Zeye Biological Technology Co., Ltd.; agar powder with a product number of A109143-500g, provided by Shanghai Yunguan Mechanical and Electrical Equipment Co., Ltd.; sucrose with a product number of S432966-100g, provided by Beijing Naizan Biological Technology Co., Ltd.; virus azole with a product number of DH85746, provided by Zibo Dehai Chemical Co., Ltd.; actinomycin D with a product number of KL17008, provided by Shanghai Kanglang Biological Technology Co., Ltd.; 5-dihydrouracil with a product number of B17868-5g, provided by Shandong Xiya Chemical Co., Ltd.

[0020] Example 1: A detoxification culture method of lily seed balls Step 1: (1) 6g of vitamin C is added to 100mL of deionized water, 3g of tert-butyl hydroquinone, 5g of butylated hydroxyanisole and 5g of dibutylated hydroxytoluene are added, 0.3g of polyoxyethylene sorbitan monooleate is added as a surfactant to prepare an antioxidant composite solution; (2) 5g of polyvinylpyrrolidone is added to 150mL of deionized water, 20mL of the antioxidant composite solution is added, stirred uniformly, heated to 80℃ at a speed of 400rad / min, 30mL of 20% sodium hydroxide solution is added as a dispersant, 15mL of sodium sulfite solution is added, reacted for 1h, 30mL of 25% sodium chloride solution is added, reacted for 1h, centrifuged, washed, filtered, vacuum dried to obtain antioxidant microcapsules; (3) 5g of the antioxidant microcapsules and 5g of activated carbon are added to 100mL of sodium hydroxide solution, stirred uniformly, ultrasonically dispersed for 14h, reacted at 80℃ for 12h, washed, dried to obtain modified activated carbon; (4) Take MS medium, 2.0 mg / L 6-benzyladenine 2.1 mg, 0.5 mg / L naphthalene acetic acid 0.6 mg, 30 g / L sucrose 30 g, 5 g / L agar powder 8 g and 7.5 g / L modified activated carbon 7.5 g, adjust pH to 5.8, autoclave, obtain the medium containing antioxidants; Step 2: First, plant lily bulbs, cultivate at 35°C for 4 weeks, take the sprouting bulbs, the bud length is 8 cm, cut off from the stem node, remove all external scales, keep the bulb core with a length of 3 cm, wash with neutral soap water for 5 min, flush with tap water for 24 h, 0.1% mercury sterilization for 10 min, soak in alcohol for 15 seconds, collect lily stem tips, peel off all the leaves, heat treatment at 38°C for 20 days, cut into stem tips with a diameter of 0.2 mm, wash with sterilized water for 5 min, inoculate into the medium containing antioxidants, cultivate for 70 days, obtain leaf from; Step 3: Cut the leaf from into 0.5 cm small pieces, inoculate into the medium containing chemical agents (MS medium+0.5 mg / L naphthalene acetic acid 0.5 mg+30 g / L sucrose 30 g+10 mg / L virus 10 mg+10 mg / L actinomycin D 10 mg+10 mg / L 5-dihydro-uracil 10 mg+5 g / L agar powder 8 g+3.5 g / L activated carbon 3.75 g), drop sodium hydroxide, adjust the medium pH=6, cultivate for 70 days, transfer to the bulb induction medium (MS medium+5 mg / L kinetin 5 mg+0.5 mg / L naphthalene acetic acid 0.5 mg+30 g / L sucrose 30 g+5 g / L agar powder 8 g+3.5 g / L activated carbon 3.75 g), set the bulb induction medium pH=5.8, cultivate at 25°C, light intensity is 1600 Lx, for 70 days, obtain a detoxified lily flower bulb.

[0021] Example 2: A detoxification culture method of lily flower bulb: Step 1: (1) Take 6 g of vitamin C into 100 mL of deionized water, add 3 g of tert-butyl hydroquinone, 5 g of butylated hydroxyanisole and 5 g of dibutyl hydroxytoluene, add 0.3 g of polyoxyethylene sorbitan monooleate as a surfactant, prepare an antioxidant composite solution; (2) Take 5 g of polyvinylpyrrolidone into 150 mL of deionized water, add 20 mL of antioxidant composite solution, stir uniformly, heat to 100°C at a speed of 600 rad / min, drop 30 mL of 20% sodium hydroxide solution as a dispersant, add 15 mL of sodium sulfite solution, react for 3 h, drop 30 mL of 25% sodium chloride solution, react for 3 h, centrifuge, wash, suction filter, vacuum dry, obtain antioxidant microcapsules; (3) Take 5 g of antioxidant microcapsules and 5 g of activated carbon, add 100 mL of sodium hydroxide solution, stir uniformly, ultrasonic dispersion for 16 h, react at 100°C for 14 h, wash, dry, and obtain modified activated carbon; (4) Take MS medium, 3.0 mg / L 6-benzyladenine 2.1 mg, 1.5 mg / L naphthalene acetic acid 0.6 mg, 32 g / L sucrose 30 g, 8 g / L agar powder 8 g, and 8.5 g / L modified activated carbon 7.5 g, adjust pH to 5.8, autoclave, and obtain a culture medium containing an antioxidant; Step 2: First, plant lily bulbs, culture at 40°C for 6 weeks, take the sprouting bulbs, the bud length is 8 cm, cut off from the stem node, remove all external scales, retain the bulb core with a length of 5 cm, wash with neutral soap water for 7 min, flush with tap water for 26 h, 0.1% mercury sterilization for 12 min, soak in alcohol for 17 seconds, collect lily stem tips, peel off all the leaves, heat treatment at 40°C for 25 days, cut into stem tips with a diameter of 0.4 mm, wash with sterilized water for 7 min, inoculate into the culture medium containing the antioxidant, and culture for 80 days to obtain leaf from; Step 3: Cut the leaf from into small pieces of 0.7 cm, inoculate into the culture medium containing chemical agents (MS medium + 1.5 mg / L naphthalene acetic acid 0.5 mg + 32 g / L sucrose 30 g + 12 mg / L virus zole 10 mg + 12 mg / L actinomycin D 10 mg + 12 mg / L 5-dihydroxy uracil 10 mg + 8 g / L agar powder 8 g + 4.5 g / L activated carbon 3.75 g), add sodium hydroxide dropwise, adjust the pH of the culture medium to 6, culture for 80 days, transfer to the seed ball induction medium (MS medium + 7 mg / L kinetin 5 mg + 1.5 mg / L naphthalene acetic acid 0.5 mg + 32 g / L sucrose 30 g + 8 g / L agar powder 8 g + 4.5 g / L activated carbon 3.75 g), set the pH of the seed ball induction medium to 5.8, culture at 30°C, light intensity is 1800 Lx, culture for 90 days, and obtain a detoxified lily flower seed ball.

[0022] Example 3: A detoxification culture method of lily flower seed ball: Step 1: (1) Take 6 g of vitamin C and add it to 100 mL of deionized water, add 3 g of tert-butyl hydroquinone, 5 g of butylated hydroxyanisole, and 5 g of dibutyl hydroxytoluene, add 0.3 g of polyoxyethylene sorbitan monooleate as a surfactant, and prepare an antioxidant composite solution; (2) Take 5 g of polyvinylpyrrolidone and add it to 150 mL of deionized water, add 20 mL of antioxidant composite solution, stir until uniform, heat to 90°C at a speed of 500 rad / min, add 30 mL of 20% sodium hydroxide solution as a dispersant, add 15 mL of sodium sulfite solution, react for 2 h, add 30 mL of 25% sodium chloride solution, react for 2 h, centrifuge, wash, filter, and vacuum dry to obtain antioxidant microcapsules; (3) Take 5 g of antioxidant microcapsules and 5 g of activated carbon, add them to 100 mL of sodium hydroxide solution, stir until uniform, ultrasonic dispersion for 15 h, react at 90°C for 14 h, wash, dry, and obtain modified activated carbon; (4) Take MS medium, 2.1 mg of 6-benzyladenine, 0.6 mg of 1-methoxyacetic acid, 30 g of 31 g / L sucrose, 8 g of 6 g / L agar powder, and 7.5 g of 8 g / L modified activated carbon, adjust the pH to 5.8, and autoclave to obtain a culture medium containing an antioxidant; Step 2: First, plant lily bulbs at 37°C for 5 weeks, take the sprouted bulbs, cut off the stems at the joint, remove all external scales, retain the core with a length of 4 cm, wash with neutral soap water for 6 min, rinse with tap water for 25 h, sterilize with 0.1% mercury for 11 min, soak in alcohol for 16 seconds, collect lily stem tips, remove all leaves, heat treat at 39°C for 21 days, cut the stem tips into 0.3 mm in diameter, rinse with sterilized water for 6 min, inoculate into the culture medium containing the antioxidant, and culture for 75 days to obtain leaf from; Step 3: Cut the leaf from into 0.6 cm pieces, inoculate into the culture medium containing the chemical agent (MS medium + 0.5 mg of 1 mg / L naphthalene acetic acid + 30 g of 31 g / L sucrose + 10 mg of 11 mg / L virus zole + 10 mg of 11 mg / L actinomycin D + 10 mg of 11 mg / L 5-dihydroxyuracil + 8 g of 6 g / L agar powder + 3.75 g of 4 g / L activated carbon), add sodium hydroxide, adjust the pH of the culture medium to 6, culture for 75 days, transfer to the seed ball induction medium (MS medium + 5 mg of 6 mg / L kinetin + 0.5 mg of 1 mg / L naphthalene acetic acid + 30 g of 31 g / L sucrose + 8 g of 6 g / L agar powder + 3.75 g of 4 g / L activated carbon), set the pH of the seed ball induction medium to 5.8, culture at 28°C with a light intensity of 1700 Lx for 80 days to obtain a detoxified lily flower seed ball.

[0023] Comparative Example 1: Do not add an antioxidant, and follow the rest of the procedures of Reference Example 1 as follows: Step 1: Take MS medium, 2.0 mg / L 6-benzyladenine 2.1 mg, 0.5 mg / L naphthalene acetic acid 0.6 mg, 30 g / L sucrose 30 g, 5 g / L agar powder 8 g and 3.5 g / L activated carbon 7.5 g, adjust pH to 5.8, autoclave, to obtain the medium; Step 2: First, plant lily bulbs, cultivate at 35°C for 4 weeks, take the sprouting bulbs, the bud length is 8 cm, cut off from the stem node, remove all external scales, retain the bulb core with a length of 3 cm, wash with neutral soap water for 5 min, flush with tap water for 24 h, 0.1% mercury sterilization for 10 min, soak in alcohol for 15 seconds, collect lily stem tips, peel off all the leaves, heat treatment at 38°C for 20 days, cut into stem tips with a diameter of 0.2 mm, wash with sterilized water for 5 min, inoculate into the culture medium, cultivate for 70 days, to obtain leaves.

[0024] Comparative Example 2: No activated carbon is added, the rest is according to Example 1, the specific operation is as follows: Step 1: (1) Take 6 g of vitamin C and add it to 100 mL of deionized water, add 3 g of tert-butyl hydroquinone, 5 g of butylated hydroxyanisole and 5 g of dibutyl hydroxytoluene, add 0.3 g of polyoxyethylene sorbitan monooleate as a surfactant to prepare an antioxidant composite solution; (2) Take 5 g of polyvinylpyrrolidone and add it to 150 mL of deionized water, add 20 mL of the antioxidant composite solution, stir uniformly, heat to 80°C at a speed of 400 rad / min, add 30 mL of 20% sodium hydroxide solution as a dispersant, add 15 mL of sodium sulfite solution, react for 1 h, add 30 mL of 25% sodium chloride solution, react for 1 h, centrifuge, wash, suction filter, vacuum dry to obtain antioxidant microcapsules; (3) Take MS medium, 2.0 mg / L 6-benzyladenine 2.1 mg, 0.5 mg / L naphthalene acetic acid 0.6 mg, 30 g / L sucrose 30 g, 5 g / L agar powder 8 g and antioxidant microcapsules 7.5 g, adjust pH to 5.8, autoclave, to obtain the medium containing antioxidants; Comparative Example 3: Change the capsule wall of the antioxidant microcapsule to polyacrylate, the rest is according to Example 1, the specific operation is as follows: Step 1: (1) Take 6 g of vitamin C and add it to 100 mL of deionized water, add 3 g of tert-butyl hydroquinone, 5 g of butylated hydroxyanisole and 5 g of dibutyl hydroxytoluene, add 0.3 g of polyoxyethylene sorbitan monooleate as a surfactant to prepare an antioxidant composite solution; (2) Take 200 mL of deionized water, add 10 g of acrylic acid and 10 g of acrylamide, pass nitrogen, add 3.2 g of sodium thiosulfate and 2.8 g of sodium sulfite, heat to 35°C, stir evenly, react for 3 h, obtain polyacrylate, cool to 20°C, add 20 mL of oxidant composite solution, add 2 g of dipotassium hydrogen phosphate as dispersant, add 4 g of glutaraldehyde, stir evenly, react for 3 h, vacuum filtration, dry, obtain antioxidant microcapsule; (3) Take 5 g of antioxidant microcapsule and 5 g of activated carbon, add 100 mL of sodium hydroxide solution, stir evenly, ultrasonic dispersion for 14 h, react at 80°C for 12 h, wash, dry, obtain modified activated carbon; (4) Take MS medium, 2.0 mg / L 6-benzyladenine 2.1 mg, 0.5 mg / L naphthalene acetic acid 0.6 mg, 30 g / L sucrose 30 g, 5 g / L agar powder 8 g and 7.5 g / L modified activated carbon 7.5 g, adjust pH to 5.8, autoclave, obtain culture medium containing antioxidant.

[0025] Comparative Example 4: no chemical treatment, the rest of Reference Example 1, the specific operation is as follows: Step 3: The leaves are cut into 0.5 cm pieces, inoculated into the culture medium (MS medium + 0.5 mg / L naphthalene acetic acid 0.5 mg + 30 g / L sucrose 30 g + 5 g / L agar powder 8 g + 3.5 g / L activated carbon 3.75 g), drop sodium hydroxide, adjust the pH of the culture medium to 6, culture for 70 days, transfer to the seed ball induction medium (MS medium + 5 mg / L kinetin 5 mg + 0.5 mg / L naphthalene acetic acid 0.5 mg + 30 g / L sucrose 30 g + 5 g / L agar powder 8 g + 3.5 g / L activated carbon 3.75 g), set the pH of the seed ball induction medium to 5.8, culture for 70 days at 25°C, light intensity of 1600 Lx, obtain a detoxified lily flower seed ball.

[0026] Comparative Example 5: no heat treatment, the rest of Reference Example 1, the specific operation is as follows: Step 2: First, plant lily bulbs, culture for 4 weeks, take the sprouting bulbs, the bud length is 8 cm, cut off from the stem node, remove all external scales, retain the ball core with a length of 3 cm, wash with neutral soap water for 5 min, flush with tap water for 24 h, 0.1% mercury sterilization for 10 min, soak in alcohol for 15 seconds, collect lily stem tips, peel off all the leaves, cut into stem tips with a diameter of 0.2 mm, wash with sterilized water for 5 min, inoculate into the culture medium containing antioxidant, culture for 70 days, obtain leaf from.

[0027] Experiment: 1. Measure the browning phenomenon: The stem tips in Examples 1-3 and Comparative Examples 1-5 were selected and cultured in a culture medium for a period of time. The browning of the selected stem tips was observed and the data was recorded. The results are shown in Table 1 below. 2. Measure the detoxification rate of the lily seed balls: The detoxified lily seed balls prepared in Examples 1-3 and Comparative Examples 1-5 were taken and added to 10 mL of a phosphate buffer solution with a pH of 7.4. The mixture was ground in an ice bath, centrifuged, and the supernatant was taken for double-antigen sandwich enzyme-linked immunoassay detection. The results are recorded in Table 2 below.

[0028]

[0029]

[0030] Conclusion: From the above experimental data, it can be seen that the lily seed balls prepared in the examples have a high detoxification rate, good antiviral performance, and less browning. The lilies grow well.

[0031] Comparing Comparative Example 1, which does not add an antioxidant, with Example 1, it can be seen that the lily seed balls in Comparative Example 1 grow slowly and have severe browning, but have a high detoxification rate. Selecting vitamin C, tert-butyl hydroquinone, and butylated hydroxyanisole as antioxidants enhances the antioxidant performance through synergistic action. Polyvinylpyrrolidone can control the production of phenolic substances, and can react with phenolic and quinone substances, thereby effectively preventing browning induced by phenolic and quinone substances.

[0032] Comparing Comparative Example 2, which does not add activated carbon, with Example 1, it can be seen that the lily seed balls in Comparative Example 2 grow slowly and have severe browning. Activated carbon has a large specific surface area, a developed pore structure, and strong adsorption capacity, and can load more antioxidant microcapsules. The surface of activated carbon contains hydroxyl and carboxyl groups, which can react with phenolic substances secreted by the stem tips, effectively suppressing browning.

[0033] Comparing Comparative Example 3, which changes the capsule wall of the antioxidant microcapsules to polyacrylate, with Example 1, it can be seen that the lily seed balls in Comparative Example 3 grow slowly and have severe browning. Antioxidant microcapsules were prepared using polyvinylpyrrolidone as the capsule wall and vitamin C, tert-butyl hydroquinone, and butylated hydroxyanisole as the capsule core, and were loaded onto the surface of activated carbon. The antioxidant was slowly released and absorbed by the stem tips when added to the culture medium, effectively suppressing the occurrence of browning.

[0034] The lily seed balls in Comparative Example 4 are grown slowly, and the browning is slight, and the detoxification rate is lower than that of the lily seed balls prepared in Example 1. In Comparative Example 4, no chemical treatment is performed, and the lily seed balls carry viruses, which affect the growth of lily. Chemical treatment is used for detoxification, and an anti-virus agent is added to the culture medium to inhibit the propagation of viruses. Virkon, actinomycin D and 5-dihydro-uracil are added to the culture medium to effectively inhibit the infection of lily symptomless virus and lily mottle virus, thereby culturing detoxified lily seed balls.

[0035] The lily seed balls in Comparative Example 5 are grown slowly, and the browning is slight, and the detoxification rate is lower than that of the lily seed balls prepared in Example 1. Heat treatment is used for detoxification and propagation, and the detoxification rate is effectively improved. Heat treatment is because the tolerance of viruses and plant cells to temperature is different. Viruses are denatured at a higher temperature, thereby inhibiting the spread and propagation of viruses. Higher temperature can also promote the growth of plants, and the virus-free area of the growth point of the plants is increased, which is beneficial to the cutting of larger stem tips for culture and the improvement of the survival rate of the stem tips.

[0036] It is apparent for those skilled in the art that the present application is not limited to the details of the above exemplary embodiments, but can be implemented in other concrete forms without departing from the spirit or essential characteristics of the present application. Therefore, the embodiments should be considered in all aspects as illustrative and not restrictive, and the scope of the present application is defined by the appended claims rather than the above description, and it is intended to embrace all changes falling within the meaning and range of equivalents of the claims.

Claims

1. A method for virus-free cultivation of lily bulbs, characterized by: The following steps are involved: Step 1: Plant lily bulbs, culture them for 4 weeks, take the sprouted bulbs, cut them from the stem nodes, remove all the external scales, keep the bulb core, disinfect it, inoculate it into a culture medium containing antioxidants, culture it, and obtain leaves; Step 2: Cut the leaves into pieces, inoculate them into a culture medium containing chemical agents, culture them, transfer them to a bulb induction culture medium, culture them under a light intensity of 1600-1800 Lx, and obtain a virus-free lily bulb.

2. The method for virus-free cultivation of lily bulbs according to claim 1, characterized in that: The disinfection steps are: washing with neutral soapy water for 5 to 7 minutes, rinsing with tap water for 24 to 26 hours, sterilizing with mercuric chloride for 10 to 12 minutes, soaking in alcohol for 15 to 17 seconds, collecting lily stem tips, peeling off all leaves, heat treating for 20 to 22 days, cutting into stem tips with a diameter of 0.2 to 0.4 mm, and rinsing with sterilized water for 5 to 7 minutes.

3. The method for virus-free cultivation of lily bulbs according to claim 1, characterized in that: The culture medium containing the antioxidant comprises the following substances: MS culture medium, 2.0-3.0 mg / L 6-benzyladenine, 0.5-1.5 mg / L naphthaleneacetic acid, 30-32 g / L sucrose, 5-8 g / L agar powder and 7.5-8.5 g / L modified activated carbon. The pH value is adjusted to 5.8 and the culture medium is sterilized by high pressure to obtain the culture medium containing the antioxidant.

4. The method for virus-free cultivation of lily bulbs according to claim 1, characterized in that: The culture medium containing chemical agents includes the following substances: MS culture medium, 0.5-1.5 mg / L naphthaleneacetic acid, 30-32 g / L sucrose, 10-12 mg / L ribavirin, 10-12 mg / L actinomycin D, 10-12 mg / L 5-dihydrouracil, 5-8 g / L agar powder, 3.5-4.5 g / L activated carbon, and sodium hydroxide is added dropwise to adjust the pH to 6.

5. The method for virus-free cultivation of lily bulbs according to claim 1, characterized in that: The seed ball induction culture medium comprises the following substances: MS culture medium, 5-7 mg / L kinetin, 0.5-1.5 mg / L naphthaleneacetic acid, 30-32 g / L sucrose, 5-8 g / L agar powder, and 3.5-4.5 g / L activated carbon. The pH value of the seed ball induction culture medium is 5.

8.

6. The method for virus-free cultivation of lily bulbs according to claim 3, characterized in that: The preparation method of the modified activated carbon comprises the following steps: taking antioxidant microcapsules and activated carbon, adding them to a sodium hydroxide solution, stirring them evenly, ultrasonically dispersing them for 14 to 16 hours, reacting them for 12 to 14 hours, washing them, and drying them to obtain the modified activated carbon.

7. The method for virus-free cultivation of lily bulbs according to claim 6, characterized in that: The preparation method of the antioxidant microcapsules comprises the following steps: adding polyvinyl pyrrolidone to deionized water, adding an antioxidant composite solution, stirring uniformly, heating at a speed of 400 to 600 rad / min, dropping a sodium hydroxide solution, adding a sodium sulfite solution, reacting for 1 to 3 hours, dropping a sodium chloride solution, reacting for 1 to 3 hours, centrifuging, washing, filtering, and vacuum drying to obtain the antioxidant microcapsules.

8. The method for virus-free cultivation of lily bulbs according to claim 7, characterized in that: The preparation method of the antioxidant composite solution comprises the following steps: adding vitamin C to deionized water, adding tert-butylhydroquinone, butylated hydroxyanisole and butylated hydroxytoluene, and adding polyoxyethylene sorbitan monooleate to prepare the antioxidant composite solution.

9. The virus-free lily seed bulbs prepared according to the virus-free culture method of lily seed bulbs according to any one of claims 1 to 8.

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