A lilyturf japonicus disease-resistant tissue culture seedling raising method

Abstract

The application discloses a disease-resistant tissue culture seedling raising method of Liriope platyphylla. The method comprises the following steps: (1) germination promotion; (2) cleaning and disinfection of explants; (3) induction of callus culture; (4) proliferation culture; (5) rooting culture; and (6) domestication of tissue culture seedlings. The disease resistance of Liriope platyphylla is significantly improved through the immune induction procedure, the occurrence and spread of diseases are reduced, the cost and labor intensity of plant disease management are reduced, the dependence on chemical pesticides is reduced, and the risk of pesticide residues is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural production, in particular to a disease-resistant tissue culture seedling raising method of Ophiopogon japonicus. BACKGROUND

[0002] Liriope platyphylla F.T.Wang & Tang is a plant of Liriope platyphylla F.T.Wang & Tang in Liliaceae. The rhizome is used as medicine. Liriope platyphylla F.T.Wang & Tang is native to China, and prefers warm and humid climate conditions and relatively shaded environment, and is cold-resistant, and dislikes strong light and high temperature. The traditional propagation method is mostly using division propagation. The propagation speed of Liriope platyphylla F.T.Wang & Tang is not particularly fast, and requires certain time and conditions.

[0003] At present, the planting of Liriope platyphylla F.T.Wang & Tang is faced with problems such as slow growth speed, poor stress resistance, and easy to be damaged by diseases. The traditional seedling raising method usually uses division propagation. The propagation speed of Liriope platyphylla F.T.Wang & Tang is not particularly fast, and requires certain time and conditions. The environmental requirements are also relatively harsh, such as soil humidity, light intensity, temperature and other factors will affect its normal growth. In addition, the Liriope platyphylla F.T.Wang & Tang plant itself has weak resistance to multiple pathogens, and is prone to black spot disease, root nematode, mole cricket and the like, especially black spot disease, once the disease occurs, it often causes serious economic losses. In order to improve the yield and resist diseases, farmers often have to use a large amount of chemical fertilizers and pesticides, which not only increases the production cost, but also may cause adverse effects on the ecological environment and human health. Therefore, the present application uses the stem segment of Liriope platyphylla F.T.Wang & Tang as an explant for tissue culture propagation, which is simple, easy to operate, fast in growth speed, can effectively resist diseases and pests, and the stress adaptation ability is significantly improved, effectively promoting the sustainable development of Liriope platyphylla F.T.Wang & Tang industry. SUMMARY

[0004] The purpose of the present application is to provide a disease-resistant tissue culture seedling raising method of Liriope platyphylla F.T.Wang & Tang. The growth speed, disease resistance and stress adaptation ability of Liriope platyphylla F.T.Wang & Tang can be improved.

[0005] The technical scheme of the present application is a disease-resistant tissue culture seedling raising method of Liriope platyphylla F.T.Wang & Tang, which is carried out according to the following steps:

[0006] (1) Germination: select healthy Liriope platyphylla F.T.Wang & Tang underground short stems without rot and disease, and place them in a lightless room, maintain the temperature at 26-30℃ and the humidity at 55-65%, and wait until the underground short stems germinate to 1-4cm;

[0007] (2) Clean and disinfect the explants: take the underground short stem segments of the buds as explants, first immerse and disinfect them with 75% alcohol for 10-20s, then wash them with sterile water, and then immerse them with 600-1000 times of carbendazim solution for 2-4 times, each time for 1-5min, and then wash them with sterile water after each time, to obtain clean and disinfected explants;

[0008] (3) Induce callus culture: obliquely cut the base of the clean and disinfected explants, and then vertically place the obliquely cut stem tips on a callus culture medium to culture callus, and the culture conditions are as follows: temperature 20-30℃, humidity 75-85%, and dark culture for 25-35d, to grow out the 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, and the pH value is adjusted to 5.8;

[0010] (4) Proliferation culture: cut off the clustered adventitious buds, and then transfer them to a proliferation culture medium to culture, and the culture conditions are as follows: temperature 18-25℃, humidity 75-85%, white light, light period 10-14h / d, light intensity 1000-3000Lux, and culture for 25-35d, to obtain the proliferated broadleaf smilacina bud seedlings;

[0011] The proliferation culture 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: vertically transfer the proliferated broadleaf smilacina bud seedlings to a rooting culture medium to culture, and the culture conditions are as follows: temperature 18-25℃, humidity 75-85%, white light, light intensity 1000-3000Lux, light period 10-14h / d, and culture for 10-20d, to obtain the rooted broadleaf smilacina tissue culture seedlings;

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

[0014] (6) acclimatization of the tissue culture seedlings: the tissue culture seedlings of L. latifolius are placed in the outdoor shade for 5-10 days, and the shed is ventilated once every 2-4 days. After 3-5 days of continued shade acclimation, the tissue culture seedlings of L. latifolius are removed, rinsed with sterile water, and transplanted into a soil matrix that has been disinfected with 500-1000 times of benomyl liquid. The transplanted tissue culture seedlings of L. latifolius are sprayed once a day until new leaves grow, and then the tissue culture seedlings are removed from the shade net to receive normal light, thereby obtaining the tissue culture seedlings of L. latifolius.

[0015] In the foregoing step (1), the seeds are soaked: healthy and disease-free L. latifolius underground short stems are selected and placed in a lightless room, and the temperature is maintained at 26-30℃ and the humidity is maintained at 58-62%. When the underground short stems sprout to a length of 2-3 cm, the soaking is completed.

[0016] In the foregoing step (2), the explant is cleaned and disinfected: the stem segments are used as explants, first immersed in 75% alcohol for 15 seconds, then washed with sterile water, and then immersed in 800 times benomyl liquid for 3 times, each time for 3 minutes, and then washed with sterile water to obtain the cleaned and disinfected explants.

[0017] In the foregoing step (3), the culture conditions are a temperature of 23-27℃, a humidity of 78-82%, and dark light culture for 30 days. The callus culture medium is 1 / 2MS+0.5mg / L 6-BA+0.1mg / L TDZ+10g / L agar+0.2mg / L brassinolide, and the pH value is adjusted to 5.8.

[0018] In the foregoing step (4), the culture conditions are a temperature of 18-22℃, a humidity of 78-82%, white light, a light period of 12h / d, a light intensity of 2000Lux, and proliferation culture for 30 days. 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 foregoing step (5), the culture conditions are a temperature of 20-22℃, a humidity of 78-82%, white light, a light period of 12h / d, a light intensity of 2000Lux, and culture for 15 days. The rooting culture medium is WPM+200mg / L choline chloride+2mg / L TDZ+10g / L agar, and the pH value is adjusted to 5.8.

[0020] In the aforementioned step (6), the tissue culture seedlings are acclimatized: the Liriope platyphylla tissue culture seedlings are placed in an outdoor greenhouse for 7 days of shade acclimation, with ventilation once every 2 days, and after 4 days of continued shade acclimation, the Liriope platyphylla tissue culture seedlings are removed, rinsed with sterile water, and transplanted into soil medium that has been disinfected with 800 times of carbendazim liquid, with irrigation once a day until new leaves grow on the transplanted Liriope platyphylla tissue culture seedlings, at which point the transplanted Liriope platyphylla tissue culture seedlings are removed from the shade net and subjected to normal light, and complete Liriope platyphylla tissue culture seedling plants are obtained.

[0021] In the aforementioned step (6), the soil medium is: humus soil: coconut coir: sandy loam soil: 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 medium is: humus soil: coconut coir: sandy loam soil: activated carbon = 1: 1: 1: 0.5.

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

[0024] 1. The present application uses 75% alcohol and carbendazim liquid to sterilize the Liriope platyphylla explants, and the black spot disease that Liriope platyphylla is prone to is caused by multiple fungi, and carbendazim liquid can effectively prevent and control various crop diseases caused by fungi, and alcohol disinfection of explants can kill various bacteria, and the combination of 75% alcohol and carbendazim liquid can reduce the risk of disease of Liriope platyphylla explants, and the use of 75% alcohol and carbendazim liquid to disinfect and sterilize Liriope platyphylla underground short stems in a dark room can reduce the influence of light on drug efficacy, which is conducive to improving the sterilization effect.

[0025] 2. The addition of brassinosteroids in the callus culture medium is conducive to cell division of Liriope platyphylla buds, and is conducive to the induction and differentiation of Liriope platyphylla sprouts, so that the induction rate reaches 100%, and there are more bud differentiations and good growth. At the same time, the addition of TDZ at the callus, proliferation and rooting stages can not only promote tissue differentiation, but also promote the formation of septicemia resistance and improve the plant resistance, induce the immunity of the plant to resist pathogen and virus invasion, so that the Liriope platyphylla tissue reaches a normal growth state, and the defense ability of Liriope platyphylla to diseases is improved to achieve immune disease resistance.

[0026] 3. In the rooting stage, the addition of choline chloride can promote the photosynthesis of Liriope platyphylla bud seedlings, accelerate the rooting process of Liriope platyphylla bud seedlings, and shorten the time required for rooting, and the rooting rate reaches 100% in 15 days, and the root length reaches 4.58 cm.

[0027] 4. Humus, coconut coir, sandy loam and activated carbon are used as the acclimatization soil matrix of the broad-leaved Liriope muscari tissue culture seedlings, 800 times of carbendazim liquid is sprayed on the soil matrix for sterilization, and the seedlings are shaded, so that the tissue culture seedlings can effectively avoid infection of pathogenic bacteria and the survival rate of the tissue culture seedlings reaches 100%.

[0028] In summary, the disease resistance of the broad-leaved Liriope muscari is significantly improved by the immune induction procedure, the occurrence and spread of diseases are reduced, the cost and labor intensity of plant disease management are reduced, the dependence on chemical pesticides is reduced, and the risk of pesticide residues is reduced. DETAILED DESCRIPTION

[0029] The application will be further described in detail below with reference to the examples, but the embodiments of the application are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the application are conventional reagents, methods and equipment in the technical field. The test methods in the following examples are generally carried out under conventional experimental conditions. Unless otherwise specified, the reagents used in the application are commercially available.

[0030] Example 1:

[0031] (1) Germination: Select healthy broad-leaved Liriope muscari underground short stems without rot and disease, place them in a lightless room, maintain the temperature at 28±2℃ and the humidity at 60±5%, and wait until the underground short stems germinate to 2-3cm;

[0032] (2) Clean and disinfect the explants: Take the stem segments as explants, first immerse and disinfect them with 75% alcohol for 15s, then wash them with sterile water, then immerse them with 800 times of carbendazim liquid for 3 times, each time for 3min, then wash them with sterile water, and clean and disinfect the explants;

[0033] (3) Induce callus culture: obliquely cut the base of the clean and disinfected explant stem tips, vertically place the obliquely cut stem tips on the callus culture medium for callus culture, and the culture conditions are as follows: temperature 25±2℃, humidity 80±2%, and dark culture for 30d;

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

[0035] (4) Proliferation culture: cut off the cluster adventitious buds, and transfer them to the proliferation culture medium for proliferation culture, and the culture conditions are as follows: temperature 20±2℃, humidity 80±2%, white light, light cycle 12h / d, light intensity 2000Lux, and proliferation culture for 30d;

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

[0037] (5) rooting culture: the proliferated bud seedlings of L. latifolius are vertically transferred to the rooting medium for rooting culture, and the culture conditions are as follows: the temperature is 21±2℃, the humidity is 80±2%, the light quality is white light, the light period is 12h / d, the light intensity is 2000Lux, the culture environment is 15d, and the rooted L. latifolius tissue culture seedlings are obtained;

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

[0039] (6) acclimatization of tissue culture seedlings: the L. latifolius tissue culture seedlings are placed outdoors for 7d of shading and seedling raising, and the shed is ventilated once every 2d; after 4d of continuous shading and seedling raising, the L. latifolius tissue culture seedlings are removed, rinsed with sterile water, and transplanted into soil matrix (humus soil: coconut husk: sandy soil: activated carbon = 1:1:1:0.5) sprayed with 800 times benomyl solution for disinfection; the transplanted L. latifolius tissue culture seedlings are sprayed once a day until new leaves grow, and then the shading net is removed to receive normal light, and the complete L. latifolius tissue culture seedling plants are obtained.

[0040] Example 2:

[0041] (1) Germination: select healthy L. latifolius underground short stems without rot and disease, place them in a lightless room, maintain the temperature at 28±2℃ and the humidity at 60±5%, and wait until the underground short stems sprout to 1-2cm;

[0042] (2) cleaning and disinfection of explants: take the explants of the sprouted underground short stems, first immerse them in 75% alcohol for 10s of disinfection, then rinse them with sterile water, and then immerse them in 1000 times benomyl solution for 2 times, each time for 5min, and rinse them with sterile water after each immersion to obtain clean and disinfected explants;

[0043] (3) induction of callus culture: the base of the clean and disinfected explants is obliquely cut, and the obliquely cut stem tips are vertically placed on the callus culture medium for callus culture, and the culture conditions are as follows: the temperature is 28±2℃, the humidity is 83±2%, and the culture is carried out in dark light for 35d to grow into multiple 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, and the pH value is adjusted to 5.8;

[0045] (4) proliferation culture: the adventitious buds are cut off and transferred to the proliferation culture medium for proliferation culture, the culture conditions are as follows: temperature 20±2℃, humidity 77±2%, light quality white light, light period 14h / d, light intensity 3000Lux, after 25d of culture, the broad-leaved Liriope tuberose bud seedlings are obtained;

[0046] The proliferation culture 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 proliferation-grown broad-leaved Liriope tuberose bud seedlings are vertically transferred to the rooting culture medium for rooting culture, the culture conditions are as follows: temperature 22±2℃, humidity 80±2%, light quality white light, light intensity 1000Lux, light period 14h / d, after 20d of culture, the broad-leaved Liriope tuberose tissue culture seedlings are obtained;

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

[0049] (6) tissue culture seedling domestication: the broad-leaved Liriope tuberose tissue culture seedlings are placed outdoors for 10d of shade acclimation, the shed is ventilated once every 4 days, after 3d of continued shade acclimation, the broad-leaved Liriope tuberose tissue culture seedlings are removed, rinsed with sterile water, and transplanted into soil matrix disinfected by spraying 1000 times benomyl solution, irrigated once a day until the transplanted broad-leaved Liriope tuberose tissue culture seedlings grow new leaves, then the shade net is removed to receive normal light, and the broad-leaved Liriope tuberose tissue culture seedling plants are obtained.

[0050] Example 3:

[0051] (1) germination: select healthy underground short stems of broad-leaved Liriope tuberose without rot and disease, place them in a lightless room, maintain the temperature at 28±2℃ and the humidity at 60±5%, and wait until the underground short stems sprout to 3-4cm;

[0052] (2) external cleaning and disinfection: take the sprouted underground short stem segments as the external explants, first immerse and disinfect them with 75% alcohol for 20s, then rinse them with sterile water, and then immerse them in 600 times benomyl solution for 4 times, each time for 1min, and rinse them with sterile water after each immersion, to obtain the cleaned and disinfected external explants;

[0053] (3) induction of callus culture: the cleaned and disinfected external explants are obliquely cut at the base of the stem tips, the obliquely cut stem tips are vertically placed on the callus culture medium for callus culture, the culture conditions are as follows: temperature 22±2℃, humidity 75±5%, dark light culture for 25d, and then the adventitious buds are grown;

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

[0055] (4) proliferation culture: the cluster adventitious buds are cut off and transferred to the proliferation culture medium for proliferation culture, the culture conditions are as follows: temperature 23±2℃, humidity 83±2%, light quality white light, light period 10h / d, light intensity 1000Lux, after 35d of culture, the proliferated L. platyphyllus bud seedlings are obtained;

[0056] The proliferation culture 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 proliferated L. platyphyllus bud seedlings are vertically transferred to the rooting culture medium for rooting culture, the culture conditions are as follows: temperature 20±2℃, humidity 77±2%, light quality white light, light intensity 3000Lux, light period 10h / d, after 10d of culture, the rooted L. platyphyllus tissue culture seedlings are obtained;

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

[0059] (6) acclimatization of the tissue culture seedlings: the L. platyphyllus tissue culture seedlings are placed outdoors for 5d of shading and seedling raising, the shed is ventilated once every 2 days, after 5d of continued shading and seedling raising, the L. platyphyllus tissue culture seedlings are removed, rinsed with sterile water, transplanted into soil matrix disinfected by spraying 500 times of benomyl solution, sprayed once a day until the transplanted L. platyphyllus tissue culture seedlings grow new leaves, then the L. platyphyllus tissue culture seedlings are removed from the shading net to receive normal light, and the L. platyphyllus tissue culture seedling plants are obtained.

[0060] Example 4:

[0061] (1) germination: select healthy L. platyphyllus underground short stems without rot and disease, place them in a lightless room, maintain the temperature at 28±2℃ and the humidity at 60±5%, and wait until the underground short stems sprout to 2-3cm;

[0062] (2) cleaning and disinfection of the explants: take the explants of the underground short stems with sprouts, first immerse and disinfect them with 75% alcohol for 18s, then rinse them with sterile water, and then immerse them with 800 times of benomyl solution for 3 times, each time for 2min, and rinse them with sterile water after each immersion, to obtain the cleaned and disinfected explants;

[0063] (3) Inducing callus culture: the cleaned and disinfected explant stem tip base is obliquely cut, the obliquely cut stem tip base is vertically placed on the callus culture medium for callus culture, the culture condition is: temperature 25±2℃, humidity 80±2%, culture under dark light for 28d, and the sprouted multiple adventitious buds are obtained;

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

[0065] (4) Proliferation culture: the multiple adventitious buds are cut off and transferred to the proliferation culture medium for proliferation culture, the culture condition is: temperature 22±2℃, humidity 80±2%, white light, light period 13h / d, light intensity 2500Lux, and the proliferated broadleaf Liriope tuberose bud seedlings are obtained after 33d of culture;

[0066] The proliferation culture 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 proliferated broadleaf Liriope tuberose bud seedlings are vertically transferred to the rooting culture medium for rooting culture, the culture condition is: temperature 20±2℃, humidity 80±2%, white light, light intensity 1500Lux, light period 11h / d, and the rooted broadleaf Liriope tuberose tissue culture seedlings are obtained after 18d of culture;

[0068] The rooting culture 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) Tissue culture seedling domestication: the broadleaf Liriope tuberose tissue culture seedlings are placed outdoors for 9d of shading seedling raising, the shed is ventilated once every 3 days, the broadleaf Liriope tuberose tissue culture seedlings are removed after 4d of continued shading seedling raising, rinsed with sterile water, transplanted into the soil matrix disinfected by spraying 600 times benomyl liquid, sprayed once a day until the transplanted broadleaf Liriope tuberose tissue culture seedlings grow new leaves, and then removed from the shading net to receive normal light to obtain the broadleaf Liriope tuberose tissue culture seedling plants.

[0070] Example 5:

[0071] (1) Germination: select healthy broadleaf Liriope tuberose underground short stems without rot and disease, place them in a lightless room, maintain the temperature at 28±2℃ and the humidity at 60±5%, and then wait for the underground short stems to sprout to 2-3cm;

[0072] (2) Clean and disinfect the explants: take the underground short stem segments of the buds as explants, first immerse and disinfect with 75% alcohol for 12s, then wash with sterile water, and then immerse twice with 800 times of carbendazim solution, each time for 4min, and wash with sterile water after each time, to obtain clean and disinfected explants;

[0073] (3) Induce callus culture: obliquely cut the base of the clean and disinfected explants, and vertically place the obliquely cut stem tips on a callus culture medium to culture callus, and the culture conditions are: temperature 25±2℃, humidity 80±2%, and dark culture for 32d, to grow out the 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 brassinolide, and the pH value is adjusted to 5.8;

[0075] (4) Proliferation culture: cut off the clustered adventitious buds, and transfer them to a proliferation culture medium for proliferation culture, and the culture conditions are: temperature 22±2℃, humidity 80±2%, white light, light cycle 11h / d, and light intensity 1500Lux, and after 28d of culture, the broad-leaved Liriope tuberose bud seedlings in proliferation growth are obtained;

[0076] The proliferation culture 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: vertically transfer the broad-leaved Liriope tuberose bud seedlings in proliferation growth to a rooting culture medium for rooting culture, and the culture conditions are: temperature 20±2℃, humidity 80±2%, white light, light intensity 2500Lux, and light cycle 13h / d, and after 12d of culture, the broad-leaved Liriope tuberose tissue culture seedlings in rooting are obtained;

[0078] The rooting culture 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) Acclimatization of the tissue culture seedlings: place the broad-leaved Liriope tuberose tissue culture seedlings outdoors for 6d of shade acclimatization, ventilate once every 2 days, continue the shade acclimatization for another 3d, then remove the broad-leaved Liriope tuberose tissue culture seedlings, rinse them with sterile water, and transplant them into the soil medium disinfected by spraying with 800 times of carbendazim solution, and spray once a day until the transplanted broad-leaved Liriope tuberose tissue culture seedlings grow new leaves, then remove the shade net to receive normal light, and obtain the broad-leaved Liriope tuberose tissue culture seedling plants.

[0080] The inventor verified the effect of the present application by performing the following experiment:

[0081] Experimental Example 1

[0082] Germination: select no rot no disease, healthy broadleaf mountain yam underground short stems in the dark room, maintain temperature 28±2℃, humidity 60±5%, wait for the underground short stems to sprout to 1-3cm, can;

[0083] Clean and disinfect the explant: take the underground short stem explant of the sprouted bud, first soak and disinfect with 75% alcohol (10s, 15s, 20s), wash with sterile water, then soak with (600-fold, 800-fold, 1000-fold) carbendazim solution (2 times, 3 times, 4 times), 3min each time, and wash 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 is achieved under the conditions of 75% alcohol disinfection for 15s, 800-fold carbendazim solution soaking for 3 times, 3min each time, the contamination rate is 10% after 5d, and the survival rate reaches 85%.

[0088] Experimental example 2

[0089] Induction of callus culture: the stem apex of the clean and disinfected explant is obliquely cut, the obliquely cut stem apex is vertically placed on the callus culture medium for callus culture, the culture conditions are temperature 25±2℃, humidity 80±2%, and dark culture conditions, different ratios of callus culture medium are selected for culture of multiple adventitious buds, 30 explants are cultured in each medium, and the induction and growth of multiple adventitious buds of each group of explants after 30d are recorded:

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

[0091]

[0092] The results are shown in Table 2. The induction rate of experimental groups 6 and 7 is better, reaching more than 90%, especially group 6, the induction rate of the stem segment in the 1 / 2MS basic medium added with 0.5mg / L 6-BA, 0.1mg / L TDZ, 10g / L agar and 0.2mg / L brassinolide is 100%. Different concentrations of hormones have a greater effect on the induction rate of the stem segment, and the addition of 6-BA, TDZ and brassinolide can accelerate the emergence of axillary buds. The induction rate of the stem segment in MS and WPM medium is far lower than that in 1 / 2MS medium.

[0093] Experimental example 3

[0094] Proliferation culture: the cluster adventitious buds were cut off and transferred to the proliferation medium for proliferation culture, the culture conditions were as follows: temperature 25±2℃, humidity 80±2%, white light, light period 12h / d, light intensity 2000Lux, different proportions of proliferation medium were selected for proliferation culture, 30 plants were cultured in each medium, the proliferation and growth of cluster adventitious buds of each group were recorded after 30 days of culture:

[0095] Table 3 Effect of different hormone combinations on the proliferation and growth of cluster adventitious buds

[0096]

[0097] As shown in Table 3, the proliferation coefficient of experimental group 3 was the highest, reaching 6.7, and the induction rate reached 100%, different concentrations of hormones had a greater effect on the proliferation of cluster adventitious buds, therefore, 1mg / L 6-BA, 2mg / L TDZ and 10g / L agar added to 1 / 8MS basic medium were the most suitable combination for the induction and proliferation of cluster adventitious buds.

[0098] Experimental Example 4

[0099] Rooting culture: the proliferated broad-leaved Liriope muscari bud seedlings were vertically transferred to the rooting medium for rooting culture, the culture conditions were as follows: temperature 21±1℃, humidity 80±2%, white light, light intensity 2000Lux, light period 12h / d, culture for 15d, different proportions of proliferation medium were selected for proliferation culture, 20 plants were cultured in each medium, the number and length of roots of the plants were recorded after 30 days of culture, and the rooting rate and average root length were finally calculated:

[0100] Table 4 Effect of different hormone combinations on the rooting of bud seedlings

[0101]

[0102]

[0103] As shown in Table 4, the rooting effect of experimental group 3 was the best, with an average root length of 4.58cm and a rooting rate of 100%, different concentrations of hormones had a greater effect on the rooting of bud seedlings, therefore, 200mg / L choline chloride, 2mg / L TDZ and 10g / L agar added to WPM basic medium were the most suitable combination for the rooting of bud seedlings.

[0104] Experimental Example 5

[0105] The tissue culture seedlings are acclimatized: the tissue culture seedlings of Liriope platyphylla are placed in the outdoor shade for 7 days, the shed is ventilated once every 2 days, the tissue culture seedlings of Liriope platyphylla are removed after being shaded for 4 days, the tissue culture seedlings of Liriope platyphylla are rinsed with sterile water, and the tissue culture seedlings of Liriope platyphylla are transplanted into soil substrates disinfected by spraying different concentrations (600 times, 800 times, 1000 times) of carbendazim solution, the transplanted tissue culture seedlings of Liriope platyphylla are sprayed once a day until the transplanted tissue culture seedlings of Liriope platyphylla grow new leaves, and then the tissue culture seedlings are removed from the shade net to receive normal light to obtain the tissue culture seedling plants, and the survival rate of the tissue culture seedling plants is observed to confirm the adaptability of the tissue culture seedlings.

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

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

[0108]

[0109] The results are shown in Table 5, and there is no significant difference in the survival rate among the groups, and the survival rate is more than 90%. Among them, the soil substrate disinfected by spraying 800 times of carbendazim solution humus: coconut husk: sandy soil: activated carbon = 1:1:1:0.5, the survival rate can reach 100%, which is a suitable soil substrate for the growth of Liriope platyphylla tissue culture seedlings.

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

[0111] The plant has strong differentiation ability, fast growth rate, strong disease resistance and good adversity adaptation ability; the complete plant without virus is obtained, which not only reduces the cost, maintains the variety advantage, and most importantly, improves the disease resistance of Liriope platyphylla, and fundamentally solves the harm of Liriope platyphylla to black spot disease, root nematode and mole cricket.

Claims

1. A method for cultivating disease-resistant seedlings of broadleaf liriope muscari, characterized in that: The tissue culture seedling method is carried out according to the following steps: (1) Germination: select no rot no disease, healthy broad-leaved lilyturf underground short stems in the dark room, maintain temperature 26-30 DEG C, humidity 55-65%, until the underground short stems sprout to 1-4 cm, can; (2) Clean and disinfect the explant: take the stem section as the explant, first soak and disinfect with 75% alcohol for 15s, wash with sterile water, then soak with 800 times carbendazim liquid for 3 times, each time for 3min, wash with sterile water, clean and disinfect the explant; (3) Induce callus culture: the clean and disinfected explant stem tip is obliquely cut at the base, the obliquely cut stem tip is vertically placed on the callus culture medium for callus culture, the culture conditions are temperature 23-27 DEG C, humidity 78-82%, dark culture for 30d; The callus culture medium is 1 / 2MS+0.5mg / L 6-BA+0.1mg / L TDZ+10g / L agar+0.2mg / L brassinolide; adjust pH value to 5.8; (4) Proliferation culture: the cluster adventitious buds are cut off and transferred to the proliferation culture medium for proliferation culture, the culture conditions are: temperature 18-22 DEG C, humidity 78-82%, white light, light cycle 12h / d, light intensity 2000Lux, proliferation culture for 30d; The proliferation culture medium is 1 / 8MS+1mg / L 6-BA+2mg / L TDZ+10g / L agar, adjust pH value to 5.8; (5) Rooting culture: the proliferated broad-leaved lilyturf bud seedlings are vertically transferred to the rooting culture medium for rooting culture, the culture conditions are: temperature 20-22 DEG C, humidity 78-82%, white light, light cycle 12h / d, light intensity 2000Lux, culture for 15d; The rooting culture medium is WPM+200mg / L choline chloride+2mg / L TDZ+10g / L agar, adjust pH value to 5.8; (6) Tissue culture seedling acclimation: the broad-leaved lilyturf tissue culture seedlings are placed in the outdoor greenhouse for 7d, ventilate once every 2d, continue to shade for 4d, then remove the broad-leaved lilyturf tissue culture seedlings, rinse with sterile water, transplant the broad-leaved lilyturf tissue culture seedlings into the soil matrix disinfected by spraying 800 times carbendazim liquid, spray once a day, until the transplanted broad-leaved lilyturf tissue culture seedlings grow new leaves, then remove the shade net to receive normal light, obtain the complete broad-leaved lilyturf tissue culture seedling plant; The soil matrix is: humus soil: coconut husk: sandy soil: activated carbon = 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.2-1.

0.

2. The disease-resistant tissue culture seedling raising method of L. japonicus according to claim 1, characterized in that: In step (1), germination: select no rot no disease, healthy broad-leaved lilyturf underground short stems in the dark room, maintain temperature 26-30 DEG C, humidity 58-62%, until the underground short stems sprout to 2-3 cm, can.

3. The disease-resistant tissue culture seedling raising method of L. japonicus according to claim 1, characterized in that: In step (6), the soil matrix is: humus soil: coconut husk: sandy soil: activated carbon = 1:1:1:0.5.

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