Tissue culture method of lily bulbs

Abstract

The invention discloses a tissue culture method of lily bulbs, and relates to the technical field of garden plant cultivation. According to the method, a large amount of high-quality tissue culture seedlings are proliferated in a short time by adopting a technology of combining scales as explants with subculture; by optimizing the components of the culture medium and adopting a solid-semisolid combined culture method, the survival rate and differentiation quality of the explants are improved; different plant growth regulators are added in different stages, and vitamin C is added in the explant culture stage, so that tissue browning is prevented, cell proliferation is stimulated, and metabolic activity is maintained; by optimizing a transplanting matrix and adding peat soil, the water retention capacity of the matrix can be enhanced, and water accumulation is avoided; perlite and vermiculite are added to improve the drainage property of the substrate and oxygen supply of the root system, and balance water retention and air permeability; a small amount of humus soil is added, organic matter of the substrate is increased, and long-acting nutrients needed by the lily seedlings are guaranteed.

Description

Technical Field

[0001] The invention relates to the technical field of garden plant cultivation, in particular to a tissue culture method for lily seed bulbs. Background Art

[0002] With the improvement of people's living standards and the diversification of flower consumption demand, the market's requirements for lily varieties, colors, and shapes are becoming increasingly higher. Through tissue culture technology, lilies can be genetically improved and new varieties can be cultivated, creating varieties with unique ornamental value, enriching market supply, and meeting the needs of different consumers. Traditional lily propagation methods, such as bulb division, are slow and have a limited number of bulbs, while seed propagation has problems such as offspring trait separation and long growth cycles. Tissue culture technology can mass-produce high-quality, genetically consistent lily bulbs in a short period of time, meeting the market's high demand for seedlings and improving production efficiency. Lilies are easily infected by pathogens such as viruses and bacteria during the production process, resulting in poor plant growth and reduced flower quality. Tissue culture technology can remove viruses through methods such as stem tip culture, obtain virus-free seedlings, and improve yield and quality.

[0003] With existing tissue culture techniques, lily bulbs are prone to browning and vitrification during cultivation. Browning is caused by the oxidation of phenolic substances, which can inhibit growth and even cause death. Vitrification, however, results from unsuitable culture conditions, resulting in transparent tissue and brittle texture, which affects survival and transplantation. Addressing these issues requires comprehensive control of multiple factors, including culture medium composition, environmental conditions, and plant growth regulators. Therefore, the present invention provides a tissue culture method for lily bulb seeds to address these technical issues. Summary of the Invention

[0004] The purpose of the present invention is to provide a tissue culture method for lily seed bulbs to solve the problems raised in the prior art.

[0005] To achieve the above object, the present invention provides the following technical solution: a tissue culture method for lily seed bulbs, comprising the following steps: S1: Take lily seed balls, remove scales, remove rotten and brown scales, clean and disinfect to obtain explants; S2: After sterilizing the culture medium A and inoculation tools, place them in a clean bench, pick up the explants with tweezers, and inoculate them into culture bottles containing culture medium A. Cover the bottles, inoculating 2 to 3 explants per culture bottle to obtain explant culture bottles; S3: placing the explant culture bottle in a culture room for 2 to 4 weeks to obtain budding lily balls; S4: When the buds grow to 2-3 cm, they are removed from the explant culture bottle, divided into small segments with buds, and inoculated onto medium B for subculture for 3-4 weeks to obtain subcultured lily flower buds; S5: When the subculture lily buds grow to 3-5 cm, they are transferred to medium C and cultured for rooting for 2-3 weeks to obtain lily seedlings with root systems; S6: When the root length of the rooted lily seedlings reaches 2-3 cm, open the cover and place them in a culture room for 7-14 days to harden the seedlings. Then take them out, clean the culture medium C on the roots, and transplant them into the substrate to obtain lily plants.

[0006] Furthermore, culture medium A, culture medium B, and culture medium C are composed of culture medium and plant growth regulator.

[0007] Furthermore, the culture medium was MS (Murashige and Skoog) medium.

[0008] Furthermore, the plant growth regulator is any one of indolebutyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminoadenine (6-BA), and naphthaleneacetic acid (NAA), or a mixture of multiple thereof.

[0009] Furthermore, the components of culture medium A include: MS medium, 1.2-1.5 mg / L 6-benzyladenine (6-BA), 0.6-0.8 mg / L indolebutyric acid (IBA), 10-15 mg / L vitamin C (Vc), and 12-15 g / L agar; The components of medium B include: MS medium, 2.5-5.5 mg / L 6-BA, 0.05-0.15 mg / L IBA, 10-15 mg / L vitamin C, and 4-6 g / L agar. The ingredients of culture medium C include: 1 / 2 MS medium, 0.1~0.2 mg / L 6-BA, 0.5~2.0 mg / L IBA, and 12g / L~15g / L agar.

[0010] In the above technical solution, a solid medium with an agar concentration of 12g / L to 15g / L is used. After the scales are inoculated into the medium for initial culture, differentiation into adventitious buds, callus tissue, and bulblets is induced. Using a solid medium at this stage provides stable support for the explants, allowing them to attach and grow, facilitating tissue formation and differentiation, preventing drift or floating, and creating a relatively stable microenvironment for cell and tissue division and differentiation. A semi-solid medium with an agar concentration of 4g / L to 6g / L is used. When the callus tissue begins to differentiate into buds, the fluidity of the semi-solid medium helps the buds to expand and grow better in the medium, preventing growth restriction caused by overly tight fixation. The semi-solid medium also better maintains the even distribution of nutrients in the medium, providing a more stable nutritional environment for bud differentiation and promoting normal bud differentiation and growth.

[0011] Furthermore, the matrix is ​​a mixture of peat soil, perlite, vermiculite and humus soil.

[0012] Furthermore, the mass ratio of peat soil, perlite, vermiculite, and humus soil is 3: (1~2): (1~2): (0.1~0.2).

[0013] Among the above technical effects, adding peat soil can enhance the water retention capacity of the matrix and avoid water accumulation. Perlite and vermiculite can improve the drainage of the matrix and the oxygen supply to the roots, balance water retention and air permeability, reduce watering frequency, add a small amount of humus soil, increase the organic matter of the matrix, and ensure long-term nutrients.

[0014] Furthermore, the size of the scales is: 1.8~5cm in length and 0.5~2cm in width.

[0015] Furthermore, in step S1, the process conditions for cleaning and disinfection are: first soak in 70-75v% alcohol for 30-60s, take out and soak in 1-2v% sodium hypochlorite for 3-5min, and then rinse with sterile water 2-3 times.

[0016] Furthermore, in step S2, the sterilization process conditions are: pressure 103.2~103.5kPa, temperature 121.3~121.5℃, sterilization 1~2h; the clean bench is a Class A clean area.

[0017] Furthermore, in step S3, the environmental conditions of the culture chamber are: temperature 22-27°C, illumination 1000-5000 Lux, relative humidity 60-80%, and carbon dioxide concentration 350-450 ppm.

[0018] Furthermore, in step S6, the seedling hardening process is: starting from opening the lid for 1 to 2 hours every day, gradually increasing the opening time until the lid is fully opened every day; the cleaning process is: carefully separating the roots from the agar with tweezers, and then washing the roots with sterile water 2 to 3 times.

[0019] Furthermore, the transplanting process includes the following steps: keeping the substrate moist, shading, controlling the temperature at 20-25°C, gradually increasing the light to natural light 5000-10000 Lux, reducing shading, stretching the rooted lily seedlings into the substrate, burying the bulbs to a depth of 1 / 2-1 / 3, and watering after transplanting.

[0020] In the above technical solution, seedling hardening is carried out before transplanting to allow tissue culture seedlings to adapt to the external environment and improve the transplant survival rate. The roots are cleaned to remove residual culture medium to avoid contamination and salt accumulation, prevent damage to the root system, and the transplanting medium is kept moist but not waterlogged, which is conducive to root attachment. During the transplanting process, the depth of the bulb is controlled to avoid rotting due to excessive burial.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. The tissue culture method of lily bulbs of the present invention achieves the proliferation of high-quality tissue culture seedlings in a short period of time by combining scales as explants with subculture technology, breaking through the limitations of traditional bulb division, which are slow in speed and long in seed propagation cycle, and meeting the market demand for large-scale seedlings.

[0022] 2. The present invention provides a tissue culture method for lily bulbs. By optimizing the culture medium components and adding different plant growth regulators at different stages, the scales are inoculated into the culture medium for initial culture, inducing differentiation into adventitious buds, callus tissue, and bulblets. The use of a solid culture medium during this stage provides stable support for the explants, allowing them to adhere and grow, facilitating tissue formation and differentiation and preventing them from drifting or floating. When the callus tissue begins to differentiate into buds, the fluidity of the semi-solid culture medium helps the buds to expand and grow better in the culture medium, avoiding growth restriction caused by overly tight fixation. The semi-solid culture medium also better maintains a uniform distribution of nutrients in the culture medium, providing a more stable nutritional environment for bud differentiation and promoting normal bud differentiation and growth. This combined solid-semi-solid culture method significantly reduces browning and vitrification of lily bulbs during tissue culture, improving explant survival rate and differentiation quality.

[0023] 3. The tissue culture method of lily bulb seeds of the present invention: vitamin compounds participate in various metabolic activities in plant cells mainly in the form of various coenzymes. By adding vitamin C during the explant culture stage, the browning of tissues can be effectively prevented, cell proliferation can be stimulated, and metabolic activities can be maintained.

[0024] 4. The tissue culture method of lily bulbs of the present invention optimizes the transplanting matrix. The addition of peat soil can enhance the water retention capacity of the matrix and avoid water accumulation. Perlite and vermiculite can improve the drainage of the matrix and the oxygen supply to the roots, balance water retention and air permeability, reduce watering frequency, and add a small amount of humus soil to increase the organic matter of the matrix, thereby improving soil structure and increasing soil fertility, thereby ensuring the long-term nutrients required by the lily seedlings. DETAILED DESCRIPTION

[0025] 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.

[0026] In the following specific embodiments, Lily seed bulbs: White fragrant lily seed bulbs, average circumference 8cm; Scales: average length 3 cm, average width 1 cm; MS medium: Premium Pure GR, average pH 5.7, containing the following components by mass: 1900 mg / L potassium nitrate, 170 mg / L potassium dihydrogen phosphate, 440 mg / L calcium chloride, 6.2 mg / L boric acid, 8.6 mg / L zinc sulfate, 0.025 mg / L copper sulfate, 27.8 mg / L ferrous sulfate, 2 mg / L glycine, 0.5 mg / L pyridoxine hydrochloride, 37.3 mg / L disodium edetate, 1650 mg / L ammonium nitrate, 370 mg / L magnesium sulfate, 0.83 mg / L potassium iodide, 22.3 mg / L manganese sulfate, 0.25 mg / L sodium molybdate, 0.025 mg / L cobalt chloride, 100 mg / L inositol, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L niacin; Culture flask: Soda lime glass, 250 mL tissue culture flask, 9.2 cm high, 63 sealing cap, transparent; Peat soil: water absorption 99%, average pH 7.0; Clay: black, particle size 200 mesh; Perlite: density 2.4g / cm 3 , SiO2 content 70%, average particle size 4.5mm; Vermiculite: density 1.6g / cm 3 , SiO2 content 68%, average particle size 6.5mm; Humus soil: water absorption 65%, average pH 6.5, average organic matter content 30%; Example 1: A tissue culture method for lily bulbs, comprising the following steps: S1: Take lily seed balls, remove scales, remove rotten and brown scales, soak them in 75% alcohol for 30 seconds, take them out and soak them in 2% sodium hypochlorite for 3 minutes, and then rinse them twice with sterile water to obtain explants; S2: Sterilize culture medium A and inoculation tools at a pressure of 103.2 kPa and a temperature of 121.3°C for 1 hour, place them in a Class A clean bench, pick up explants with tweezers, and inoculate them into culture bottles containing culture medium A. Cover the bottles with caps, inoculating two explants per culture bottle to obtain explant culture bottles; S3: The explant culture bottles were placed in a culture room at a temperature of 22°C, a light intensity of 1000 Lux, a relative humidity of 60%, and a carbon dioxide concentration of 350 ppm for 2 weeks to obtain budding lily bulbs; S4: When the buds grow to 2 cm, they are removed from the explant culture bottle, divided into small segments with buds, and inoculated onto medium B for subculture for 3 weeks to obtain subcultured lily flower buds; S5: When the subculture lily buds grow to 3 cm, they are transferred to medium C and cultured for rooting for 2 weeks to obtain lily seedlings with root systems; S6: When the root length of the rooted lily seedlings reaches 2 cm, open the lid, place them in the culture room for 7 days of hardening, take them out, carefully separate the roots from the agar in the culture medium C with tweezers, then rinse the roots twice with sterile water, transplant them into the substrate, keep the substrate moist, shade them, control the temperature at 21°C, gradually increase the light to 5000 Lux of natural light, reduce the shade, stretch the rooted lily seedlings into the substrate, bury the bulbs to 1 / 2 depth, water them, and obtain lily plants; the hardening process is: start with opening the lid for 1 hour a day, gradually increase the opening time until it is completely open every day.

[0027] In step S2, the components of culture medium A are MS medium, 1.25 mg / L 6-BA, 0.6 mg / L IBA, 10 mg / L Vc, and the agar concentration is 12 g / L.

[0028] In step S4, the components of culture medium B include: MS culture medium, 2.5 mg / L 6-BA, 0.05 mg / L IBA, 10 mg / L Vc, and the agar concentration is 4 g / L.

[0029] In step S5, the components of culture medium C include: 1 / 2 MS culture medium, 0.1 mg / L 6-BA, 0.5 mg / L IBA, and an agar concentration of 12 g / L.

[0030] In step S6, the components of the matrix include peat soil, perlite, vermiculite, and humus soil; the mass ratio of peat soil, perlite, vermiculite, and humus soil is 3:1.5:1.5:0.1.

[0031] Example 2: A tissue culture method for lily bulbs, comprising the following steps: S1: Take lily seed balls, remove scales, remove rotten and brown scales, soak them in 75% alcohol for 40 seconds, take them out and soak them in 2% sodium hypochlorite for 4 minutes, and then rinse them twice with sterile water to obtain explants; S2: Sterilize culture medium A and inoculation tools at a pressure of 103.3 kPa and a temperature of 121.4°C for 1.5 h, place them in a Class A clean bench, pick up explants with tweezers, and inoculate them into culture bottles containing culture medium A. Cover the bottles with caps, inoculating two explants per culture bottle to obtain explant culture bottles; S3: The explant culture bottles were placed in a culture room at a temperature of 25°C, a light intensity of 2000 Lux, a relative humidity of 70%, and a carbon dioxide concentration of 400 ppm for 3 weeks to obtain budding lily bulbs; S4: When the buds grow to 2.5 cm, they are removed from the explant culture bottle, divided into small segments with buds, and inoculated onto medium B for subculture for 3 weeks to obtain subcultured lily flower buds; S5: When the subculture lily buds grow to 3 cm, they are transferred to medium C and cultured for rooting for 2 weeks to obtain lily seedlings with root systems; S6: When the root length of the rooted lily seedlings reaches 2 cm, open the lid, place them in the culture room for 10 days of hardening, take them out, carefully separate the roots from the agar in the culture medium C with tweezers, then rinse the roots twice with sterile water, transplant them into the substrate, keep the substrate moist, shade them, control the temperature at 22°C, gradually increase the light to 7000 Lux of natural light, reduce the shade, stretch the rooted lily seedlings into the substrate, bury the bulbs to 1 / 2 depth, water them, and obtain lily plants; the hardening process is: start with opening the lid for 1 hour a day, gradually increase the opening time until it is completely open every day.

[0032] In step S2, the components of culture medium A are MS medium, 1.3 mg / L 6-BA, 0.7 mg / L IBA, 12 mg / L Vc, and the agar concentration is 13 g / L.

[0033] In step S4, the components of culture medium B include: MS medium, 4 mg / L 6-BA, 0.12 mg / L IBA, 13 mg / L Vc, and the agar concentration is 5 g / L.

[0034] In step S5, the components of culture medium C include: 1 / 2 MS culture medium, 0.15 mg / L 6-BA, 1.5 mg / L IBA, and an agar concentration of 13 g / L.

[0035] In step S6, the components of the matrix include peat soil, perlite, vermiculite, and humus soil; the mass ratio of peat soil, perlite, vermiculite, and humus soil is 3:1.2:1.2:0.15.

[0036] Example 3: A tissue culture method for lily seed bulbs, comprising the following steps: S1: Take lily seed balls, remove scales, remove rotten and brown scales, soak them in 75% alcohol for 50 seconds, take them out and soak them in 2% sodium hypochlorite for 5 minutes, and then rinse them with sterile water three times to obtain explants; S2: Sterilize culture medium A and inoculation tools at a pressure of 103.5 kPa and a temperature of 121.5°C for 2 h, place them in a Class A clean bench, pick up explants with tweezers, and inoculate them into culture bottles containing culture medium A. Cover the bottles with caps, inoculating three explants per culture bottle to obtain explant culture bottles; S3: The explant culture bottles were placed in a culture room at a temperature of 27°C, a light intensity of 5000 Lux, a relative humidity of 80%, and a carbon dioxide concentration of 450 ppm for 4 weeks to obtain budding lily bulbs; S4: When the buds grow to 3 cm, they are removed from the explant culture bottle, divided into small segments with buds, and inoculated onto medium B for subculture for 4 weeks to obtain subcultured lily flower buds; S5: When the subculture lily buds grow to 5 cm, they are transferred to medium C and cultured for rooting for 3 weeks to obtain lily seedlings with root systems; S6: When the root length of the rooted lily seedlings reaches 3 cm, open the lid, place them in the culture room for 14 days of hardening, take them out, carefully separate the roots from the agar in the culture medium C with tweezers, then rinse the roots 3 times with sterile water, transplant them into the substrate, keep the substrate moist, shade them, control the temperature at 25°C, gradually increase the light to 10,000 Lux of natural light, reduce the shade, stretch the rooted lily seedlings into the substrate, bury the bulbs 1 / 3 of the depth, water them, and obtain lily plants; the hardening process is: start with opening the lid for 2 hours a day, gradually increase the opening time until it is completely open every day.

[0037] In step S2, the components of culture medium A are MS medium, 1.5 mg / L 6-BA, 0.8 mg / L IBA, 15 mg / L Vc, and the agar concentration is 15 g / L.

[0038] In step S4, the components of culture medium B include: MS culture medium, 5.5 mg / L 6-BA, 0.15 mg / L IBA, 15 mg / L Vc, and the agar concentration is 6 g / L.

[0039] In step S5, the components of culture medium C include: 1 / 2 MS culture medium, 0.2 mg / L 6-BA, 2.0 mg / L IBA, and an agar concentration of 15 g / L.

[0040] In step S6, the components of the matrix include peat soil, perlite, vermiculite, and humus soil; the mass ratio of peat soil, perlite, vermiculite, and humus soil is 3:2:2:0.2.

[0041] Comparative Example 1: A tissue culture method for lily bulbs, comprising the following steps: S1: Take lily seed balls, remove scales, remove rotten and brown scales, soak them in 75% alcohol for 30 seconds, take them out and soak them in 2% sodium hypochlorite for 3 minutes, and then rinse them twice with sterile water to obtain explants; S2: Sterilize culture medium A and inoculation tools at a pressure of 103.2 kPa and a temperature of 121.3°C for 1.5 h, place them in a Class A clean bench, pick up explants with tweezers, and inoculate them into culture bottles containing culture medium A. Cover the bottles, inoculating two explants per culture bottle to obtain explant culture bottles; S3: The explant culture bottles were placed in a culture room at a temperature of 22°C, a light intensity of 1000 Lux, a relative humidity of 60%, and a carbon dioxide concentration of 350 ppm for 2 weeks to obtain budding lily bulbs; S4: When the buds grow to 2 cm, they are removed from the explant culture bottle, divided into small segments with buds, and inoculated onto medium A for subculture for 3 weeks to obtain subcultured lily flower buds; S5: When the subculture lily buds grow to 3 cm, continue rooting culture for 2 weeks to obtain lily seedlings with root systems; S6: When the root length of the rooted lily seedlings reaches 2 cm, open the lid, place them in the culture room for 7 days of hardening, take them out, carefully separate the roots from the agar in the culture medium A with tweezers, then rinse the roots twice with sterile water, transplant them into the substrate, keep the substrate moist, shade them, control the temperature at 20°C, gradually increase the light to 5000 Lux of natural light, reduce the shade, stretch the rooted lily seedlings into the substrate, bury the bulbs to 1 / 2 depth, water them, and obtain lily plants; the hardening process is: start with opening the lid for 1 hour a day, gradually increase the opening time until it is completely open every day.

[0042] In step S2, the components of culture medium A are MS medium, 1.3 mg / L 6-BA, 0.7 mg / L IBA, 12 mg / L Vc, and the agar concentration is 13 g / L.

[0043] In step S6, the components of the matrix include peat soil, perlite, vermiculite, and humus soil; the mass ratio of peat soil, perlite, vermiculite, and humus soil is 3:1:1:0.2.

[0044] Comparative Example 2: A tissue culture method for lily bulbs, comprising the following steps: S1: Take lily seed balls, remove scales, remove rotten and brown scales, soak them in 75% alcohol for 45 seconds, take them out and soak them in 2% sodium hypochlorite for 3 minutes, and then rinse them twice with sterile water to obtain explants; S2: Sterilize culture medium B and inoculation tools at a pressure of 103.2 kPa and a temperature of 121.3°C for 1 hour, place them in a Class A clean bench, pick up explants with tweezers, and inoculate them into culture bottles containing culture medium B. Cover the bottles with caps, inoculating two explants per culture bottle to obtain explant culture bottles; S3: The explant culture bottles were placed in a culture room at a temperature of 22°C, a light intensity of 2000 Lux, a relative humidity of 60%, and a carbon dioxide concentration of 350 ppm for 2 weeks to obtain budding lily bulbs; S4: When the buds grow to 2 cm, they are removed from the explant culture bottle, divided into small segments with buds, and inoculated onto medium B for subculture for 3 weeks to obtain subcultured lily flower buds; S5: When the subculture lily buds grow to 3 cm, continue rooting culture for 2 weeks to obtain lily seedlings with root systems; S6: When the root length of the rooted lily seedlings reaches 2 cm, open the lid, place them in the culture room for 9 days of hardening, take them out, carefully separate the roots from the agar in the culture medium B with tweezers, then rinse the roots twice with sterile water, transplant them into the substrate, keep the substrate moist, shade them, control the temperature at 20°C, gradually increase the light to 8000 Lux of natural light, reduce the shade, stretch the rooted lily seedlings into the substrate, bury the bulbs to 1 / 2 depth, water them, and obtain lily plants; the hardening process is: start with opening the lid for 2 hours a day, gradually increase the opening time until it is completely open every day.

[0045] In step S4, the components of culture medium B include: MS culture medium, 2.5 mg / L 6-BA, 0.05 mg / L IBA, 10 mg / L Vc, and the agar concentration is 4 g / L.

[0046] In step S6, the components of the matrix include clay, perlite, vermiculite, and humus; the mass ratio of clay, perlite, vermiculite, and humus is 3:1:1:0.2.

[0047] Comparative Example 3: A tissue culture method for lily bulbs, comprising the following steps: S1: Take lily seed balls, remove scales, remove rotten and brown scales, soak in 75% alcohol for 50 seconds, take out and soak in 2% sodium hypochlorite for 3 minutes, and then rinse twice with sterile water to obtain explants; S2: Sterilize culture medium A and inoculation tools at a pressure of 103.4 kPa and a temperature of 121.4°C for 2 h, place them in a Class A clean bench, pick up explants with tweezers, and inoculate them into culture bottles containing culture medium A. Cover the bottles, inoculating three explants per culture bottle to obtain explant culture bottles; S3: The explant culture bottles were placed in a culture room at a temperature of 27°C, a light intensity of 3000 Lux, a relative humidity of 70%, and a carbon dioxide concentration of 450 ppm for 4 weeks to obtain budding lily bulbs; S4: When the buds grow to 3 cm, they are removed from the explant culture bottle, divided into small segments with buds, and inoculated onto medium B for subculture for 4 weeks to obtain subcultured lily flower buds; S5: When the subculture lily buds grow to 5 cm, they are transferred to medium C and cultured for rooting for 3 weeks to obtain lily seedlings with root systems; S6: When the root length of the rooted lily seedlings reaches 3 cm, open the lid, place them in the culture room for 14 days of hardening, take them out, carefully separate the roots from the agar in the culture medium C with tweezers, then rinse the roots 3 times with sterile water, transplant them into the substrate, keep the substrate moist, shade them, control the temperature at 20°C, gradually increase the light to 10,000 Lux of natural light, reduce the shade, stretch the rooted lily seedlings into the substrate, bury the bulbs 1 / 3 of the depth, water them, and obtain lily plants; the hardening process is: start with opening the lid for 2 hours a day, gradually increase the opening time until it is completely open every day.

[0048] In step S2, the components of culture medium A are MS medium, 1.5 mg / L 6-BA, 0.8 mg / L IBA, 15 mg / L Vc, and the agar concentration is 12 g / L.

[0049] In step S4, the components of culture medium B include: MS culture medium, 5.5 mg / L 6-BA, 0.15 mg / L IBA, 15 mg / L Vc, and the agar concentration is 6 g / L.

[0050] In step S5, the components of culture medium C include: 1 / 2 MS culture medium, 0.2 mg / L 6-BA, 2.0 mg / L IBA, and an agar concentration of 12 g / L.

[0051] In step S6, the components of the matrix include peat soil, perlite, and humus soil; the mass ratio of peat soil, perlite, and humus soil is 3:2:0.2.

[0052] Comparative Example 4: A tissue culture method for lily bulbs, comprising the following steps: S1: Take lily seed balls, remove scales, remove rotten and brown scales, soak them in 75% alcohol for 50 seconds, take them out and soak them in 2% sodium hypochlorite for 4 minutes, and then rinse them with sterile water three times to obtain explants; S2: Sterilize culture medium A and inoculation tools at a pressure of 103.2 kPa and a temperature of 121.3°C for 1 hour, place them in a Class A clean bench, pick up explants with tweezers, and inoculate them into culture bottles containing culture medium A. Cover the bottles with caps, inoculating two explants per culture bottle to obtain explant culture bottles; S3: The explant culture bottles were placed in a culture room at a temperature of 22°C, a light intensity of 1500 Lux, a relative humidity of 60%, and a carbon dioxide concentration of 350 ppm for 3 weeks to obtain budding lily bulbs; S4: When the buds grow to 2 cm, they are removed from the explant culture bottle, divided into small segments with buds, and inoculated onto medium B for subculture for 3 weeks to obtain subcultured lily flower buds; S5: When the subculture lily buds grow to 4 cm, they are transferred to medium C and cultured for rooting for 3 weeks to obtain lily seedlings with root systems; S6: When the root length of the rooted lily seedlings reaches 3 cm, open the lid, place them in the culture room for 12 days of hardening, take them out, carefully separate the roots from the agar in the culture medium C with tweezers, then rinse the roots 3 times with sterile water, transplant them into the substrate, keep the substrate moist, shade them, control the temperature at 25°C, gradually increase the light to 10,000 Lux of natural light, reduce the shade, stretch the rooted lily seedlings into the substrate, bury the bulbs 1 / 3 of the depth, water them, and obtain lily plants; the hardening process is: start with opening the lid for 2 hours a day, gradually increase the opening time until it is completely open every day.

[0053] In step S2, the components of culture medium A are MS medium, 1.5 mg / L 6-BA, 0.7 mg / L IBA, 15 mg / L Vc, and the agar concentration is 12 g / L.

[0054] In step S4, the components of culture medium B include: MS medium, 2.5 mg / L 6-BA, 0.15 mg / L IBA, 15 mg / L Vc, and the agar concentration is 4 g / L.

[0055] In step S5, the components of culture medium C include: 1 / 2 MS culture medium, 0.2 mg / L 6-BA, 2.0 mg / L IBA, and an agar concentration of 12 g / L.

[0056] In step S6, the components of the matrix include peat soil, perlite, and vermiculite; the mass ratio of peat soil, perlite, and vermiculite is 3:2:2.

[0057] The growth process of the lily seed bulbs of Examples 1-3 and Comparative Examples 1-4 was observed and recorded, and their growth conditions were detected and recorded, and the following test results were calculated: Test of proliferation coefficient: Based on the observed growth conditions, the proliferation coefficient is calculated by calculating the ratio of the number of newly induced adventitious buds to the number of buds in the inoculated stem segment.

[0058] Rooting rate test: Based on the observed growth conditions, the rooting rate is calculated as the ratio of the number of roots to the number of inoculations.

[0059] Comparison of lily plant growth: Using DB51 / T 2746-2021 as a reference standard, the growth of lily plants was observed.

[0060] Growth record sheet

[0061] According to the data in the above table, we can clearly draw the following conclusions: The lily plants obtained in Examples 1-3 were compared with the lily plants obtained in Comparative Examples 1-4. The test results showed that: Compared with the comparative example, the lily plants obtained in Examples 1-3 had a large proliferation coefficient, a high rooting rate, and a better growth condition.

[0062] Compared with Example 1, the lily plants obtained in Comparative Example 1 were tissue cultured using only a single culture medium, culture medium A. The proliferation coefficient of the lily plants was small, the rooting rate was low, and the growth was average. It can be seen that the use of a single culture medium is not as effective as the optimized culture medium composition in the present invention.

[0063] Compared with Example 1, the lily plants obtained in Comparative Example 2 were tissue cultured using culture medium B alone, and clay was used instead of peat soil in the transplanting medium. The proliferation coefficient of the lilies was small, the rooting rate was low, and the growth was average. It can be seen that the use of semi-solid culture medium B and clay in the transplanting medium cannot meet the high-quality growth requirements of lily bulbs. The use of peat soil can better retain moisture in the soil and promote the growth and development of lilies.

[0064] Compared with Example 1, the lily plants obtained in Comparative Example 3 did not add vermiculite to the transplanting medium, and the lily had a small proliferation coefficient, a low rooting rate, and good growth. It can be seen that without adding vermiculite, the nutrients in the medium cannot be slowly released, the viscosity is relatively high, and the requirements of water retention and drainage cannot be balanced. The lily plants did not grow as well as the lily plants with vermiculite added in the present invention.

[0065] Compared with Example 1, the lily plants obtained in Comparative Example 4 did not add humus to the transplanting medium, and the lilies had a small proliferation coefficient, a low rooting rate, and good growth. It can be seen that without adding humus, the nutritional needs of the soil cannot be met, which is not conducive to long-term guarantee of the growth and development needs of the lily plants. Adding a small amount of humus can increase the organic matter of the matrix, improve the soil structure, increase soil fertility, and ensure long-term nutrients for the lily plants.

[0066] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

Claims

1. A tissue culture method for lily bulbs, characterized by: The following steps are involved: S1: Take lily seed balls, remove scales, remove rotten and brown scales, clean and disinfect to obtain explants; S2: After sterilizing the culture medium A and inoculation tools, place them in a clean bench, pick up the explants with tweezers, and inoculate them into culture bottles containing culture medium A. Cover the bottles, inoculating 2 to 3 explants per culture bottle to obtain explant culture bottles; S3: placing the explant culture bottle in a culture room for 2 to 4 weeks to obtain budding lily balls; S4: When the buds grow to 2-3 cm, they are removed from the explant culture bottle, divided into small segments with buds, and inoculated onto medium B for subculture for 3-4 weeks to obtain subcultured lily flower buds; S5: When the subculture lily buds grow to 3-5 cm, they are transferred to medium C and cultured for rooting for 2-3 weeks to obtain lily seedlings with root systems; S6: When the root length of the rooted lily seedlings reaches 2-3 cm, open the cover and place them in a culture room for 7-14 days to harden the seedlings. Then take them out, clean the culture medium C on the roots, and transplant them into the substrate to obtain lily plants.

2. The tissue culture method for lily bulbs according to claim 1, characterized in that: The components of medium A include: MS medium, 1.2~1.5 mg / L 6-BA, 0.6~0.8 mg / L IBA, 10~15 mg / L vitamin C, and 12g / L~15g / L agar; The components of medium B include: MS medium, 2.5-5.5 mg / L 6-BA, 0.05-0.15 mg / L IBA, 10-15 mg / L vitamin C, and 4-6 g / L agar. The ingredients of culture medium C include: 1 / 2 MS medium, 0.1~0.2 mg / L 6-BA, 0.5~2.0 mg / L IBA, and 12g / L~15g / L agar.

3. The tissue culture method for lily bulbs according to claim 1, characterized in that: In step S6, the matrix is ​​a mixture of peat soil, perlite, vermiculite and humus soil.

4. The tissue culture method for lily bulbs according to claim 3, characterized in that: The mass ratio of the peat soil, perlite, vermiculite and humus soil is 3: (1-2): (1-2): (0.1-0.2).

5. The tissue culture method for lily bulbs according to claim 1, characterized in that: In step S1, the process conditions for cleaning and disinfection are: first soak in 70-75v% alcohol for 30-60s, take out and soak in 1-2v% sodium hypochlorite for 3-5min, and then rinse with sterile water 2-3 times.

6. The tissue culture method for lily bulbs according to claim 1, characterized in that: In step S2, the sterilization process conditions are: pressure 103.2~103.5kPa, temperature 121.3~121.5℃, sterilization 1~2h; the clean bench is a Class A clean area.

7. The tissue culture method for lily bulbs according to claim 1, characterized in that: In step S3, the environmental conditions of the culture chamber are: temperature 22-27°C, illumination 1000-5000 Lux, relative humidity 60-80%, and carbon dioxide concentration 350-450 ppm.

8. The tissue culture method for lily bulbs according to claim 1, characterized in that: In step S6, the seedling hardening process is: starting from opening the lid for 1 to 2 hours every day, gradually increasing the opening time until the lid is fully opened every day; the cleaning process is: carefully separating the roots from the agar with tweezers, and then washing the roots with sterile water 2 to 3 times.

9. The tissue culture method for lily bulbs according to claim 1, characterized in that: In step S6, the transplanting process The method comprises the following steps: keeping the substrate moist, shading, controlling the temperature at 20-25 degrees Celsius, gradually increasing the light to natural light 5000-10000 Lux, reducing shading, stretching the rooted lily seedlings into the substrate, burying the bulbs to a depth of 1 / 2-1 / 3, and watering after transplanting.

Images