Disease-resistant tissue culture seedling raising method for liriope latifolia

Abstract

The invention discloses a disease-resistant tissue culture seedling raising method for liriope latifolia. The method comprises the following steps: (1) accelerating germination; (2) cleaning and disinfecting the explants; (3) inducing callus culture; (4) multiplication culture; (5) rooting culture; (6) domesticating the tissue culture seedlings and the like. According to the method, the disease resistance of the liriope latifolia is remarkably improved through an immune induction procedure, occurrence and propagation of diseases are reduced, the cost and labor intensity of plant disease management are reduced, meanwhile, dependence on chemical pesticides is reduced, and the risk of pesticide residues is reduced.

Description

Technical Field

[0001] The invention relates to the field of agricultural production, in particular to a disease-resistant tissue culture seedling method for ophiopogon japonicus. Background Art

[0002] Liriope platyphylla FTWang & Tang is a plant of the Asparagaceae family. Its root tubers are used as medicine. Liriope platyphylla is native to China. It likes warm and humid climate conditions with abundant rainfall and a relatively shaded environment. It is cold-resistant and avoids strong light and high temperature. Traditional propagation methods mostly use division propagation. The propagation speed of Liriope platyphylla is not particularly fast, and it requires a certain amount of time and conditions.

[0003] At present, the planting of broad-leaved ophiopogon japonicus faces the problems of slow growth, poor stress resistance, and susceptibility to diseases. Traditional seedling raising methods usually adopt division propagation. However, the propagation speed of broad-leaved ophiopogon japonicus is not particularly fast, and it requires a certain amount of time and conditions. It is also more demanding on the environment, such as soil moisture, light intensity, temperature and other factors will affect its normal growth. In addition, the broad-leaved ophiopogon japonicus plant itself has weak resistance to a variety of pathogens, and is prone to black spot disease, root nematodes, mole crickets, etc., especially black spot disease. Once the disease occurs, it often causes serious economic losses. In order to increase yield and resist diseases, farmers often have to use a large amount of chemical fertilizers and pesticides, which not only increases production costs, but also may have adverse effects on the ecological environment and human health. Therefore, the present invention uses the stem segment of broad-leaved ophiopogon japonicus as an explant for tissue culture propagation, the method is simple, easy to operate, fast growth rate, can effectively resist diseases and insect pests, and the ability to adapt to adversity is significantly improved, which effectively promotes the sustainable development of the broad-leaved ophiopogon japonicus industry. Summary of the invention

[0004] The purpose of the present invention is to provide a disease-resistant tissue culture seedling method for broad-leaved ophiopogon japonicus, which can improve the growth rate, disease resistance and ability to adapt to adverse conditions of broad-leaved ophiopogon japonicus.

[0005] The technical solution of the present invention is a disease-resistant tissue culture seedling method of broad-leaved Liriope japonicus, which is carried out according to the following steps:

[0006] (1) Germination: Select the underground short stems of broad-leaved Ophiopogon japonicus that are free of rot and disease and are strong and placed in a dark room, maintain the temperature at 26-30°C and the humidity at 55-65%, and wait until the underground short stems sprout to 1-4 cm;

[0007] (2) Explant cleaning and disinfection: Take out the underground short stem segment explant of the bud, first soak it in 75% alcohol for 10-20 seconds, wash it with sterile water, and then soak it in 600-1000 times carbendazim solution for 2-4 times, each time for 1-5 minutes, and wash it with sterile water after each soaking to obtain a clean and disinfected explant;

[0008] (3) Inducing callus culture: The cleaned and disinfected explant stem tip is cut obliquely at the base, and the stem tip with the base cut obliquely is placed vertically on the callus culture medium for callus culture. The culture conditions are: temperature 20-30°C, humidity 75-85%, and culture under dim light conditions for 25-35 days to grow clustered adventitious buds;

[0009] The callus culture medium is 1 / 2MS+0.5-1.0mg / L 6-BA+0.1-0.2mg / L TDZ+10-15g / L agar+0.2-0.3mg / L brassinosteroid; the pH value is adjusted to 5.8;

[0010] (4) Proliferation culture: the clustered adventitious buds are cut off and transferred to a proliferation culture medium for proliferation culture. The culture conditions are: temperature 18-25°C, humidity 75-85%, light quality white light, photoperiod 10-14h / d, light intensity 1000-3000Lux. After culturing for 25-35 days, the proliferated broad-leaved Ophiopogon japonicus seedlings are obtained;

[0011] The proliferation medium is 1 / 8MS+0.5-1.5mg / L 6-BA+1-3mg / L TDZ+5-15g / L agar, and the pH value is adjusted to 5.8;

[0012] (5) Rooting culture: The proliferating broad-leaved Ophiopogon japonicus seedlings were vertically transferred to a rooting medium for rooting culture. The culture conditions were: temperature 18-25°C, humidity 75-85%, light quality white light, light intensity 1000-3000 Lux, photoperiod 10-14 h / d, culture for 10-20 days, and rooted broad-leaved Ophiopogon japonicus tissue culture seedlings were obtained.

[0013] The rooting medium is WPM+150-250 mg / L choline chloride+1-3 mg / L TDZ+5-15 g / L agar, and the pH value is adjusted to 5.8;

[0014] (6) Acclimation of tissue culture seedlings: Place the broad-leaved Ophiopogon japonicus tissue culture seedlings outdoors under shade and hardening for 5-10 days. Open the shed for ventilation every 2-4 days. After continuing to shade and harden for 3-5 days, remove the broad-leaved Ophiopogon japonicus tissue culture seedlings, rinse with sterile water, and transplant the broad-leaved Ophiopogon japonicus tissue culture seedlings to the soil substrate sprayed with 500-1000 times carbendazim solution for disinfection. Spray irrigation once a day until the transplanted broad-leaved Ophiopogon japonicus tissue culture seedlings grow new leaves. Then, remove the shade net and accept normal light to obtain broad-leaved Ophiopogon japonicus tissue culture seedlings.

[0015] In the above step (1), germination: select rot-free and disease-free, strong and robust broad-leaved Ophiopogon japonicus underground short stems and place them in a light-free room, maintain the temperature at 26-30°C and the humidity at 58-62%, and wait until the underground short stems sprout to 2-3 cm.

[0016] In the above step (2), the explants are cleaned and disinfected: stem segments are taken as explants, firstly immersed in 75% alcohol for 15 seconds for disinfection, then washed with sterile water, and then immersed in 800 times carbendazim solution for 3 times, each time for 3 minutes, and then washed with sterile water to clean and disinfect the explants.

[0017] In the above step (3), the culture conditions are 23-27° C., 78-82% humidity, and 30 days of culture under dim light conditions; the callus tissue culture medium is 1 / 2MS+0.5mg / L 6-BA+0.1mg / L TDZ+10g / L agar+0.2mg / L brassinosteroids; and the pH value is adjusted to 5.8.

[0018] In the aforementioned step (4), the culture conditions are: temperature of 18-22°C, humidity of 78-82%, light quality of white light, photoperiod of 12h / d, light intensity of 2000Lux, and proliferation culture for 30d; the proliferation culture medium is 1 / 8MS+1mg / L 6-BA+2mg / L TDZ+10g / L agar, and the pH value is adjusted to 5.8.

[0019] In the aforementioned step (5), the culture conditions are: temperature 20-22°C, humidity 78-82%, light quality white light, photoperiod 12h / d, light intensity 2000Lux, and culture for 15 days; the rooting medium is WPM+200mg / L choline chloride+2mg / L TDZ+10g / L agar, and the pH is adjusted to 5.8.

[0020] In the above step (6), the tissue culture seedlings are acclimated: the broad-leaved ophiopogon tissue culture seedlings are placed in an outdoor greenhouse for shade and hardening for 7 days, the greenhouse is opened for ventilation every 2 days, and after continuing the shade and hardening for 4 days, the broad-leaved ophiopogon tissue culture seedlings are removed, rinsed with sterile water, and transplanted into a soil matrix sprayed with 800 times carbendazim solution for disinfection. The broad-leaved ophiopogon tissue culture seedlings are sprayed once a day until the transplanted broad-leaved ophiopogon tissue culture seedlings grow new leaves, then the shade net is removed to receive normal light, and a complete broad-leaved ophiopogon tissue culture seedling plant is obtained.

[0021] In the aforementioned step (6), the soil matrix is: humus soil: coconut bran: sandy loam: activated carbon = 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.2-1.0.

[0022] Specifically, in the aforementioned step (6), the soil matrix is: humus soil: coconut bran: sandy loam: activated carbon = 1:1:1:0.5.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The present invention uses 75% alcohol and carbendazim liquid to sterilize the broad-leaved ophiopogon explants. The black spot disease that ophiopogon is susceptible to is caused by a variety of fungi. Carbendazim liquid can effectively prevent and treat a variety of crop diseases caused by fungi. At the same time, alcohol disinfection of explants can kill a variety of bacteria. The combination of 75% alcohol and carbendazim liquid can reduce the risk of broad-leaved ophiopogon explants being susceptible to diseases. In addition, by using 75% alcohol and carbendazim liquid to jointly disinfect and sterilize the underground short stems of broad-leaved ophiopogon in a dark room, the influence of light on the efficacy of the drug can be reduced, which is beneficial to improving the sterilization effect.

[0025] 2. Adding brassinosteroids to the callus medium is conducive to the cell division of broad-leaved Ophiopogon japonicus buds, and is conducive to the induced growth and differentiation of broad-leaved Ophiopogon japonicus buds, so that the induction rate reaches 100%, and the buds are more differentiated and grow well. At the same time, adding TDZ during the callus, proliferation and rooting stages can not only promote tissue differentiation, but also promote the formation of anti-septicemia, improve plant resistance, induce plant immunity to resist pathogens and viruses, enable broad-leaved Ophiopogon japonicus tissues to reach a normal growth state, and improve the defense of broad-leaved Ophiopogon japonicus against diseases to achieve immune resistance.

[0026] 3. In the rooting stage, adding choline chloride can promote the photosynthesis of broad-leaved ophiopogon seedlings, accelerate the rooting process of broad-leaved ophiopogon seedlings, and shorten the time required for rooting. In 15 days, the rooting rate reaches 100%, and the root length reaches 4.58cm.

[0027] 4. Use humus, coconut bran, sandy loam and activated carbon as the acclimatization soil matrix for broad-leaved Ophiopogon japonicus tissue culture seedlings, and spray the soil matrix with 800 times carbendazim solution for sterilization. At the same time, shade the seedlings to effectively prevent the tissue culture seedlings from being infected with pathogens, and the survival rate of the tissue culture seedlings can reach 100%.

[0028] In summary, the present invention significantly improves the disease resistance of broad-leaved Ophiopogon japonicus through the immune induction program, reduces the occurrence and spread of diseases, reduces the cost and labor intensity of plant disease management, and at the same time reduces dependence on chemical pesticides and reduces the risk of pesticide residues. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually in accordance with conventional experimental conditions. Unless otherwise specified, the reagents used in the present invention can be obtained commercially.

[0030] Embodiment 1:

[0031] (1) Germination: Select the underground short stems of broad-leaved Ophiopogon japonicus that are free of rot and disease and are strong and placed in a dark room, maintain the temperature at 28±2°C and the humidity at 60±5%, and wait until the underground short stems sprout to a size of 2-3 cm;

[0032] (2) Explant cleaning and disinfection: Take the stem segment as the explant, first soak it in 75% alcohol for 15 seconds, wash it with sterile water, then soak it in 800 times carbendazim solution for 3 times, each time for 3 minutes, and wash it with sterile water to clean and disinfect the explant;

[0033] (3) Inducing callus culture: The base of the cleaned and disinfected explant stem tip was cut obliquely, and the stem tip with the base cut obliquely was placed vertically on the callus culture medium for callus culture. The culture conditions were: the temperature was 25±2°C, the humidity was 80±2%, and the culture was carried out under dark light conditions for 30 days;

[0034] The callus culture medium was 1 / 2MS+0.5mg / L 6-BA+0.1mg / L TDZ+10g / L agar+0.2mg / L brassinosteroids; the pH value was adjusted to 5.8;

[0035] (4) Proliferation culture: The clustered adventitious buds were cut off and transferred to the proliferation medium for proliferation culture. The culture conditions were as follows: temperature 20±2°C, humidity 80±2%, light quality white light, photoperiod 12h / d, light intensity 2000Lux, and proliferation culture for 30 days;

[0036] The proliferation medium was 1 / 8MS + 1mg / L 6-BA + 2mg / L TDZ + 10g / L agar, and the pH was adjusted to 5.8;

[0037] (5) Rooting culture: The proliferating broad-leaved Ophiopogon japonicus seedlings were vertically transferred to the rooting medium for rooting culture. The culture conditions were: temperature 21±2°C, humidity 80±2%, light quality white light, photoperiod 12h / d, and light intensity 2000Lux. The culture was carried out for 15 days to obtain rooted broad-leaved Ophiopogon japonicus tissue culture seedlings.

[0038] The rooting medium was WPM + 200 mg / L choline chloride + 2 mg / L TDZ + 10 g / L agar, and the pH was adjusted to 5.8;

[0039] (6) Acclimation of tissue culture seedlings: Place the broad-leaved Ophiopogon japonicus tissue culture seedlings outdoors under shade and hardening for 7 days. Open the shed for ventilation every 2 days. After 4 days of shade and hardening, remove the broad-leaved Ophiopogon japonicus tissue culture seedlings, rinse with sterile water, and transplant the broad-leaved Ophiopogon japonicus tissue culture seedlings into a soil matrix (humus soil: coconut bran: sandy loam: activated carbon = 1:1:1:0.5) sprayed and disinfected with 800 times carbendazim solution. Spray irrigation once a day until the transplanted broad-leaved Ophiopogon japonicus tissue culture seedlings grow new leaves. Then, remove the shade net and accept normal light to obtain complete broad-leaved Ophiopogon japonicus tissue culture seedlings.

[0040] Embodiment 2:

[0041] (1) Germination: Select the underground short stems of broad-leaved Ophiopogon japonicus that are free of rot and disease and are strong and placed in a dark room, maintain the temperature at 28±2°C and the humidity at 60±5%, and wait until the underground short stems sprout to 1-2 cm;

[0042] (2) Explant cleaning and disinfection: Take out the underground short stem segment explant of the bud, soak it in 75% alcohol for 10 seconds, wash it with sterile water, and then soak it in 1000 times carbendazim solution twice, each time for 5 minutes. After each soaking, wash it with sterile water to obtain a clean and disinfected explant;

[0043] (3) Inducing callus culture: The cleaned and disinfected explant stem tip was cut obliquely at the base, and the stem tip with the base cut obliquely was placed vertically on the callus culture medium for callus culture. The culture conditions were: temperature 28±2°C, humidity 83±2%, and cultured in dim light for 35 days to grow clustered adventitious buds;

[0044] The callus culture medium is 1 / 2MS+0.5mg / L 6-BA+0.2mg / L TDZ+10g / L agar+0.2mg / L brassinosteroid; the pH value is adjusted to 5.8;

[0045] (4) Proliferation culture: The clustered adventitious buds were cut off and transferred to the proliferation medium for proliferation culture. The culture conditions were: temperature 20±2°C, humidity 77±2%, light quality white light, photoperiod 14h / d, light intensity 3000Lux. After culturing for 25 days, the proliferated broad-leaved Ophiopogon japonicus seedlings were obtained;

[0046] The proliferation medium is 1 / 8MS+1.5mg / L 6-BA+1mg / L TDZ+15g / L agar, and the pH value is adjusted to 5.8;

[0047] (5) Rooting culture: The proliferating broad-leaved Ophiopogon japonicus seedlings were vertically transferred to the rooting medium for rooting culture. The culture conditions were: temperature 22±2°C, humidity 80±2%, light quality white light, light intensity 1000 Lux, photoperiod 14 h / d, culture for 20 days, and rooted broad-leaved Ophiopogon japonicus tissue culture seedlings were obtained;

[0048] The rooting medium is WPM+250mg / L choline chloride+1mg / L TDZ+15g / L agar, and the pH value is adjusted to 5.8;

[0049] (6) Acclimation of tissue culture seedlings: Place the broad-leaved Ophiopogon japonicus tissue culture seedlings outdoors under shade and hardening for 10 days. Open the shed for ventilation every 4 days. After 3 days of shade and hardening, remove the broad-leaved Ophiopogon japonicus tissue culture seedlings, rinse with sterile water, and transplant the broad-leaved Ophiopogon japonicus tissue culture seedlings to the soil substrate sprayed with 1000 times carbendazim solution for disinfection. Spray irrigation once a day until the transplanted broad-leaved Ophiopogon japonicus tissue culture seedlings grow new leaves. Then, remove the shade net and accept normal light to obtain broad-leaved Ophiopogon japonicus tissue culture seedlings.

[0050] Embodiment 3:

[0051] (1) Germination: Select the underground short stems of broad-leaved Ophiopogon japonicus that are free of rot and disease and are strong and placed in a dark room, maintain the temperature at 28±2°C and the humidity at 60±5%, and wait until the underground short stems sprout to 3-4 cm;

[0052] (2) Explant cleaning and disinfection: Take out the underground short stem segment explant of the bud, soak it in 75% alcohol for 20 seconds, wash it with sterile water, and then soak it in 600 times carbendazim solution for 4 times, each time for 1 minute. After each soaking, wash it with sterile water to obtain a clean and disinfected explant;

[0053] (3) Inducing callus culture: The cleaned and disinfected explant stem tip was cut obliquely at the base, and the stem tip with the base cut obliquely was placed vertically on the callus culture medium for callus culture. The culture conditions were: temperature 22±2°C, humidity 75±5%, and cultured in dim light for 25 days to grow clustered adventitious buds;

[0054] The callus culture medium is 1 / 2MS+1.0mg / L 6-BA+0.1mg / L TDZ+15g / L agar+0.3mg / L brassinosteroid; the pH value is adjusted to 5.8;

[0055] (4) Proliferation culture: The clustered adventitious buds were cut off and transferred to the proliferation medium for proliferation culture. The culture conditions were: temperature 23±2°C, humidity 83±2%, light quality white light, photoperiod 10h / d, light intensity 1000Lux. After 35 days of culture, the proliferated broad-leaved Ophiopogon japonicus seedlings were obtained;

[0056] The proliferation medium is 1 / 8MS+0.5mg / L 6-BA+3mg / L TDZ+5g / L agar, and the pH value is adjusted to 5.8;

[0057] (5) Rooting culture: The proliferating broad-leaved Ophiopogon japonicus seedlings were vertically transferred to the rooting medium for rooting culture. The culture conditions were: temperature 20±2°C, humidity 77±2%, light quality white light, light intensity 3000 Lux, photoperiod 10 h / d, culture for 10 days, and rooted broad-leaved Ophiopogon japonicus tissue culture seedlings were obtained;

[0058] The rooting medium is WPM+150mg / L choline chloride+3mg / L TDZ+5g / L agar, and the pH value is adjusted to 5.8;

[0059] (6) Acclimation of tissue culture seedlings: Place the broad-leaved Ophiopogon japonicus tissue culture seedlings outdoors for 5 days of shade and hardening. Open the shed for ventilation every 2 days. After 5 days of shade and hardening, remove the broad-leaved Ophiopogon japonicus tissue culture seedlings, rinse with sterile water, and transplant the broad-leaved Ophiopogon japonicus tissue culture seedlings to the soil substrate sprayed with 500 times carbendazim solution for disinfection. Spray irrigation once a day until the transplanted broad-leaved Ophiopogon japonicus tissue culture seedlings grow new leaves. Then, remove the shade net and accept normal light to obtain broad-leaved Ophiopogon japonicus tissue culture seedlings.

[0060] Embodiment 4:

[0061] (1) Germination: Select the underground short stems of broad-leaved Ophiopogon japonicus that are free of rot and disease and are strong and placed in a dark room, maintain the temperature at 28±2°C and the humidity at 60±5%, and wait until the underground short stems sprout to a size of 2-3 cm;

[0062] (2) Explant cleaning and disinfection: Take out the underground short stem segment explant of the bud, soak it in 75% alcohol for 18 seconds, wash it with sterile water, and then soak it in 800 times carbendazim solution for 3 times, each time for 2 minutes. After each soaking, wash it with sterile water to obtain a clean and disinfected explant;

[0063] (3) Inducing callus culture: The cleaned and disinfected explant stem tip was cut obliquely at the base, and the stem tip with the base cut obliquely was placed vertically on the callus culture medium for callus culture. The culture conditions were: temperature 25±2°C, humidity 80±2%, and cultured in dim light for 28 days to grow clustered adventitious buds;

[0064] The callus culture medium is 1 / 2MS+01.0mg / L 6-BA+0.2mg / L TDZ+10g / L agar+0.2mg / L brassinosteroid; the pH value is adjusted to 5.8;

[0065] (4) Proliferation culture: The clustered adventitious buds were cut off and transferred to the proliferation medium for proliferation culture. The culture conditions were: temperature 22±2°C, humidity 80±2%, light quality white light, photoperiod 13h / d, light intensity 2500Lux. After 33 days of culture, the proliferated broad-leaved Ophiopogon japonicus seedlings were obtained;

[0066] The proliferation medium is 1 / 8MS+1.0mg / L 6-BA+2.5mg / L TDZ+10g / L agar, and the pH value is adjusted to 5.8;

[0067] (5) Rooting culture: The proliferating broad-leaved Ophiopogon japonicus seedlings were vertically transferred to the rooting medium for rooting culture. The culture conditions were: temperature 20±2°C, humidity 80±2%, light quality white light, light intensity 1500 Lux, photoperiod 11 h / d, culture for 18 days, and rooted broad-leaved Ophiopogon japonicus tissue culture seedlings were obtained;

[0068] The rooting medium is WPM+220mg / L choline chloride+1.5mg / L TDZ+10g / L agar, and the pH value is adjusted to 5.8;

[0069] (6) Acclimation of tissue culture seedlings: Place the broad-leaved Ophiopogon japonicus tissue culture seedlings outdoors under shade and hardening for 9 days. Open the shed for ventilation every 3 days. After 4 days of shade and hardening, remove the broad-leaved Ophiopogon japonicus tissue culture seedlings, rinse with sterile water, and transplant the broad-leaved Ophiopogon japonicus tissue culture seedlings to the soil substrate sprayed with 600 times carbendazim solution for disinfection. Spray irrigation once a day until the transplanted broad-leaved Ophiopogon japonicus tissue culture seedlings grow new leaves. Then, remove the shade net and accept normal light to obtain broad-leaved Ophiopogon japonicus tissue culture seedlings.

[0070] Embodiment 5:

[0071] (1) Germination: Select the underground short stems of broad-leaved Ophiopogon japonicus that are free of rot and disease and are strong and placed in a dark room, maintain the temperature at 28±2°C and the humidity at 60±5%, and wait until the underground short stems sprout to a size of 2-3 cm;

[0072] (2) Explant cleaning and disinfection: Take out the underground short stem segment explant of the bud, soak it in 75% alcohol for 12 seconds, wash it with sterile water, and then soak it in 800 times carbendazim solution twice, each time for 4 minutes. After each soaking, wash it with sterile water to obtain a clean and disinfected explant;

[0073] (3) Inducing callus culture: The base of the cleaned and disinfected explant shoot tip is cut obliquely, and the shoot tip with the base cut obliquely is placed vertically on the callus culture medium for callus culture. The culture conditions are: temperature 25±2°C, humidity 80±2%, and culture under dim light conditions for 32 days to grow clustered adventitious buds;

[0074] The callus culture medium is 1 / 2MS+0.5mg / L 6-BA+0.1mg / L TDZ+15g / L agar+0.3mg / L brassinosteroid; the pH value is adjusted to 5.8;

[0075] (4) Proliferation culture: The clustered adventitious buds were cut off and transferred to the proliferation medium for proliferation culture. The culture conditions were: temperature 22±2°C, humidity 80±2%, light quality white light, photoperiod 11h / d, light intensity 1500Lux. After 28 days of culture, the proliferated broad-leaved Ophiopogon japonicus seedlings were obtained;

[0076] The proliferation medium is 1 / 8MS+1.5mg / L 6-BA+3mg / L TDZ+5g / L agar, and the pH value is adjusted to 5.8;

[0077] (5) Rooting culture: The proliferating broad-leaved Ophiopogon japonicus seedlings were vertically transferred to the rooting medium for rooting culture. The culture conditions were: temperature 20±2°C, humidity 80±2%, light quality white light, light intensity 2500 Lux, photoperiod 13 h / d, culture for 12 days, and rooted broad-leaved Ophiopogon japonicus tissue culture seedlings were obtained;

[0078] The rooting medium is WPM+180mg / L choline chloride+2.5mg / L TDZ+10g / L agar, and the pH value is adjusted to 5.8;

[0079] (6) Acclimation of tissue culture seedlings: Place the broad-leaved Ophiopogon japonicus tissue culture seedlings outdoors under shade and hardening for 6 days. Open the shed for ventilation every 2 days. After 3 days of shade and hardening, remove the broad-leaved Ophiopogon japonicus tissue culture seedlings, rinse with sterile water, and transplant the broad-leaved Ophiopogon japonicus tissue culture seedlings to the soil substrate sprayed with 800 times carbendazim solution for disinfection. Spray irrigation once a day until the transplanted broad-leaved Ophiopogon japonicus tissue culture seedlings grow new leaves. Then, remove the shade net and accept normal light to obtain broad-leaved Ophiopogon japonicus tissue culture seedlings.

[0080] The inventors conducted the following experiments to verify the effects of the present invention:

[0081] Experimental Example 1

[0082] Germination: Select the underground short stems of radix ophiopogonis that are free of rot and disease and strong and place them in a dark room, maintain the temperature at 28±2℃ and the humidity at 60±5%, and wait until the underground short stems sprout to 1-3cm;

[0083] Explant cleaning and disinfection: Take out the underground short stem segment explant of the bud, first soak it in 75% alcohol for disinfection (10s, 15s, 20s), wash it with sterile water, and then soak it in (600 times, 800 times, 1000 times) carbendazim solution (2 times, 3 times, 4 times), soak it for 3 minutes each time, and wash it with sterile water after each soaking to obtain clean and disinfected explants;

[0084] Table 1 Contamination rate and survival rate of different disinfection schemes

[0085]

[0086]

[0087] The results are shown in Table 1. The best effect was achieved when the samples were disinfected with 75% alcohol for 15 seconds and immersed in 800 times carbendazim solution for 3 minutes each time. After 5 days, the contamination rate was 10% and the survival rate reached 85%.

[0088] Experimental Example 2

[0089] Induced callus culture: The cleaned and disinfected explant stem tip base was cut obliquely, and the stem tip with the base cut obliquely was placed vertically on the callus culture medium for callus culture. The culture conditions were as follows: the culture conditions were 25±2°C, 80±2% humidity, and cultured under dark light conditions. Different proportions of callus culture medium were used to culture clustered adventitious buds. Each group of culture medium cultured 30 plants, and the induced growth of clustered adventitious buds of each group of explants was recorded after 30 days of culture:

[0090] Table 2 Induction rate and growth of adventitious buds induced by different callus culture media and hormones

[0091]

[0092] The results are shown in Table 2. The induction rates of experimental groups 6 and 7 for induced callus culture were good, reaching more than 90%, especially in group 6, where the induction rate of stem segments in 1 / 2MS basal medium with 0.5mg / L 6-BA, 0.1mg / L TDZ, 10g / L agar and 0.2mg / L brassinosteroid was 100%. Different concentrations of hormones had a large induction rate for stem segments, and the addition of 6-BA, TDZ and brassinosteroid could accelerate the growth of axillary buds. The induction rates of stem segments in MS and WPM medium were far lower than those in 1 / 2MS medium.

[0093] Experimental Example 3

[0094] Proliferation culture: The clustered adventitious buds were cut off and transferred to the proliferation culture medium for proliferation culture. The culture conditions were as follows: temperature 25±2℃, humidity 80±2%, light quality white light, photoperiod 12h / d, light intensity 2000Lux. Different proportions of proliferation culture medium were selected for proliferation culture. Each group of culture medium cultured 30 plants. The proliferation growth of the clustered adventitious buds of each group of explants was recorded after 30 days of culture:

[0095] Table 3 Effects of different hormone concentrations on the proliferation and growth of adventitious buds

[0096]

[0097] The results are shown in Table 3. The proliferation coefficient of experimental group 3 in proliferation culture reached a maximum of 6.7, and the induction rate reached 100%. Different concentrations of hormones had a greater effect on the proliferation of clustered adventitious buds. Therefore, adding 1 mg / L 6-BA, 2 mg / L TDZ, and 10 g / L agar to 1 / 8 MS basal culture medium is the most suitable combination for inducing the proliferation of clustered adventitious buds.

[0098] Experimental Example 4

[0099] Rooting culture: The proliferating broad-leaved Ophiopogon japonicus seedlings were vertically transferred to the rooting medium for rooting culture. The culture conditions were as follows: temperature 21±1°C, humidity 80±2%, white light, light intensity 2000Lux, photoperiod 12h / d, culture for 15 days, and different proportions of proliferation culture medium were selected for proliferation culture. Each group of culture medium cultured 20 plants. After 30 days of culture, the number of roots and root length of the plants were counted, and the rooting rate and average root length were finally calculated:

[0100] Table 4 Effects of different concentrations of hormone combinations on rooting of seedlings

[0101]

[0102]

[0103] The results are shown in Table 4. The rooting effect of experimental group 3 of rooting culture was the best, with an average root length of 4.58 cm and a rooting rate of 100%. Different concentrations of hormones had a greater effect on the rooting of seedlings. Therefore, adding 200 mg / L choline chloride, 2 mg / L TDZ, and 10 g / L agar to the WPM basal culture medium was the most suitable combination for rooting seedlings.

[0104] Experimental Example 5

[0105] Acclimation of tissue culture seedlings: Place the broad-leaved Ophiopogon japonicus tissue culture seedlings outdoors for 7 days of shade and hardening. Open the shed for ventilation every 2 days. After continuing to shade and harden for 4 days, remove the broad-leaved Ophiopogon japonicus tissue culture seedlings, rinse with sterile water, and transplant the broad-leaved Ophiopogon japonicus tissue culture seedlings into soil substrates sprayed and disinfected with different concentrations (600 times, 800 times, 1000 times) of carbendazim. Spray irrigation once a day until the transplanted broad-leaved Ophiopogon japonicus tissue culture seedlings grow new leaves. Then, remove the shade net to receive normal light to obtain tissue culture seedlings. Observe the survival rate of the tissue culture seedlings to confirm the adaptability of the tissue culture seedlings.

[0106] The soil matrix selection scheme and results are shown in Table 5, with 30 tissue culture seedlings in each group:

[0107] Table 5 Disease resistance and survival rate of different soil matrices and carbendazim solution

[0108]

[0109] The results are shown in Table 5. There is no significant difference in the survival rate among the groups, and the survival rate is above 90%. Among them, the soil matrix humus soil: coconut bran: sandy loam: activated carbon = 1:1:1:0.5 sprayed with 800 times carbendazim solution can reach a survival rate of 100%, which is a soil matrix suitable for the growth of broad-leaved ophiopogon tissue culture seedlings.

[0110] Conclusion: The plant tissue culture seedling technology of the present invention has the following advantages:

[0111] The plants have strong differentiation ability, fast growth rate, strong disease resistance and good ability to adapt to adversity; obtaining complete virus-free plants can not only reduce costs and maintain variety advantages, but most importantly, it improves the anti-virus ability of broad-leaved Ophiopogon japonicus, fundamentally solving the hazards of black spot disease, root nematodes and mole crickets in broad-leaved Ophiopogon japonicus.

Claims

1. A disease-resistant tissue culture seedling method for broad-leaved ophiopogon japonicus, characterized in that: The tissue culture seedling method is carried out according to the following steps: (1) Germination: Select the underground short stems of broad-leaved Ophiopogon japonicus that are free of rot and disease and are strong and placed in a dark room, maintain the temperature at 26-30°C and the humidity at 55-65%, and wait until the underground short stems sprout to 1-4 cm; (2) Explant cleaning and disinfection: Take out the underground short stem segment explant of the bud, first soak it in 75% alcohol for 10-20 seconds, wash it with sterile water, and then soak it in 600-1000 times carbendazim solution for 2-4 times, each time for 1-5 minutes, and wash it with sterile water after each soaking to obtain a clean and disinfected explant; (3) Inducing callus culture: The cleaned and disinfected explant stem tip is cut obliquely at the base, and the stem tip with the base cut obliquely is placed vertically on the callus culture medium for callus culture. The culture conditions are: temperature 20-30°C, humidity 75-85%, and culture under dim light conditions for 25-35 days to grow clustered adventitious buds; The callus culture medium is 1 / 2MS+0.5-1.0mg / L 6-BA+0.1-0.2mg / LTDZ+10-15g / L agar+0.2-0.3mg / L brassinosteroids; the pH value is adjusted to 5.8; (4) Proliferation culture: the clustered adventitious buds are cut off and transferred to a proliferation culture medium for proliferation culture. The culture conditions are: temperature 18-25°C, humidity 75-85%, light quality white light, photoperiod 10-14h / d, light intensity 1000-3000Lux. After culturing for 25-35 days, the proliferated broad-leaved Ophiopogon japonicus seedlings are obtained; The proliferation medium is 1 / 8MS+0.5-1.5mg / L 6-BA+1-3mg / L TDZ+5-15g / L agar, and the pH value is adjusted to 5.8; (5) Rooting culture: The proliferating broad-leaved Ophiopogon japonicus seedlings were vertically transferred to a rooting medium for rooting culture. The culture conditions were: temperature 18-25°C, humidity 75-85%, light quality white light, light intensity 1000-3000 Lux, photoperiod 10-14 h / d, culture for 10-20 days, and rooted broad-leaved Ophiopogon japonicus tissue culture seedlings were obtained. The rooting medium is WPM+150-250 mg / L choline chloride+1-3 mg / L TDZ+5-15 g / L agar, and the pH value is adjusted to 5.8; (6) Acclimation of tissue culture seedlings: Place the broad-leaved Ophiopogon japonicus tissue culture seedlings outdoors under shade and hardening for 5-10 days. Open the shed for ventilation every 2-4 days. After continuing to shade and harden for 3-5 days, remove the broad-leaved Ophiopogon japonicus tissue culture seedlings, rinse with sterile water, and transplant the broad-leaved Ophiopogon japonicus tissue culture seedlings to the soil substrate sprayed with 500-1000 times carbendazim solution for disinfection. Spray irrigation once a day until the transplanted broad-leaved Ophiopogon japonicus tissue culture seedlings grow new leaves. Then, remove the shade net and accept normal light to obtain broad-leaved Ophiopogon japonicus tissue culture seedlings.

2. The disease-resistant tissue culture seedling method of broad-leaved Liriope japonicus according to claim 1, characterized in that: In the step (1), germination is performed by selecting rot-free and disease-free, strong and robust broad-leaved Ophiopogon japonicus underground short stems and placing them in a light-free room, maintaining a temperature of 26-30° C. and a humidity of 58-62%, until the underground short stems sprout to a length of 2-3 cm.

3. The disease-resistant tissue culture seedling method of broad-leaved Liriope japonicus according to claim 1, characterized in that: In the step (2), the explant is cleaned and disinfected: a stem segment is taken as an explant, firstly immersed in 75% alcohol for 15 seconds for disinfection, washed with sterile water, then immersed in 800 times carbendazim solution for 3 times, each time for 3 minutes, and washed with sterile water to clean and disinfect the explant.

4. The disease-resistant tissue culture seedling method of broad-leaved Liriope japonicus according to claim 1, characterized in that: In the step (3), the culture conditions are 23-27° C., 78-82% humidity, and 30 days of culture under dim light conditions; the callus culture medium is 1 / 2MS+0.5 mg / L 6-BA+0.1 mg / L TDZ+10 g / L agar+0.2 mg / L brassinosteroids; and the pH value is adjusted to 5.

8.

5. The disease-resistant tissue culture seedling method of broad-leaved Liriope japonicus according to claim 1, characterized in that: In the step (4), the culture conditions are: temperature of 18-22° C., humidity of 78-82%, light quality of white light, photoperiod of 12 h / d, light intensity of 2000 Lux, and proliferation culture for 30 days; the proliferation culture medium is 1 / 8MS+1 mg / L 6-BA+2 mg / L TDZ+10 g / L agar, and the pH value is adjusted to 5.

8.

6. The disease-resistant tissue culture seedling method of broad-leaved Liriope japonicus according to claim 1, characterized in that: In the step (5), the culture conditions are: temperature 20-22° C., humidity 78-82%, light quality white light, photoperiod 12 h / d, light intensity 2000 Lux, and culture for 15 days; the rooting medium is WPM+200 mg / L choline chloride+2 mg / L TDZ+10 g / L agar, and the pH is adjusted to 5.

8.

7. The disease-resistant tissue culture seedling method of broad-leaved Liriope japonicus according to claim 1, characterized in that: In the step (6), the tissue culture seedlings are acclimated: the broad-leaved ophiopogon tissue culture seedlings are placed in an outdoor greenhouse for shade and hardening for 7 days, the greenhouse is opened for ventilation every 2 days, and after continuing the shade and hardening for 4 days, the broad-leaved ophiopogon tissue culture seedlings are removed, rinsed with sterile water, and transplanted into a soil matrix sprayed with 800 times carbendazim solution for disinfection, and sprinkled once a day until the transplanted broad-leaved ophiopogon tissue culture seedlings grow new leaves, then the shade net is removed to receive normal light, thereby obtaining a complete broad-leaved ophiopogon tissue culture seedling plant.

8. The disease-resistant tissue culture seedling method of broad-leaved Liriope japonicus according to claim 1 or 7, characterized in that: In the step (6), the soil matrix is: humus: coconut bran: sandy loam: activated carbon = 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.2-1.

0.

9. The disease-resistant tissue culture seedling method of broad-leaved Liriope japonicus according to claim 8, characterized in that: In the step (6), the soil matrix is: humus: coconut bran: sandy loam: activated carbon = 1:1:1:0.5.

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