CN111480577B - Tissue culture rapid propagation method for epimedium wushanense inflorescence - Google Patents
Tissue culture rapid propagation method for epimedium wushanense inflorescence Download PDFInfo
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- CN111480577B CN111480577B CN202010434889.XA CN202010434889A CN111480577B CN 111480577 B CN111480577 B CN 111480577B CN 202010434889 A CN202010434889 A CN 202010434889A CN 111480577 B CN111480577 B CN 111480577B
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- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention discloses a wushanense inflorescence tissue culture rapid propagation method, which is a complete process of inducing explants to generate callus and differentiating adventitious buds or cluster buds by disinfecting inflorescences, further inducing the buds to form fibrous roots, and finally forming healthy and complete plants through seedling hardening and raising. The differentiation rate of callus differentiation bud is as high as 83.7%, and the multiplication coefficient of cluster bud is as high as 5.1. The method is easy to operate, has the advantages of high reproduction rate, low cost, short period, robust seedlings and the like, provides basis for the scale production of the epimedium, and has certain promotion effect on the protection and utilization of epimedium resources.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a tissue culture rapid propagation method taking epimedium wushanense inflorescence as an explant.
Background
Epimedium wushanense (Epimedium wushanense Ying) is a Berberidaceae (Berberidaceae) Epimedium (Epimedium) plant, and the part of the plant used as a medicine is a dry leaf, so that the Epimedium wushanense (Epimedium wushanense Ying) is one of the most widely and long-standing Chinese medicinal materials in China. China is a main supply country of epimedium medicinal materials and extracts in the world. Modern pharmacology indicates that with the development of new medicine varieties such as anti-cancer and nerve protection and the like which take epimedium as a raw material, the demand of epimedium medicinal materials is further increased, but the epimedium medicinal materials mainly depend on wild resources for a long time, and the reserve of the wild epimedium resources is sharply reduced due to the large mining, so that the current contradiction between supply and demand of the epimedium medicinal materials is serious. Obviously, the production requirements cannot be met by relying on wild herba epimedii medicinal materials, and the industrialization of artificial planting of the herba epimedii medicinal materials is urgent. The main breeding modes of the epimedium herb are underground rhizome breeding and seed breeding, however, underground rhizome needs a large amount of basic seedlings and easily causes generation-by-generation accumulation of viruses and the like, so that the quality and the yield are reduced; the seed germination rate of epimedium is low, the seed seedling grows slowly, and the production of the epimedium seedling is severely restricted. By adopting the tissue culture technology, a large number of epimedium seedlings can be rapidly obtained while the excellent characters of the female parent are kept, the development of the artificial planting industry of the epimedium is effectively promoted, and the wild resources of the epimedium in China are protected.
The plant tissue culture technology is a vegetative propagation technology which utilizes organs, tissues and the like of plants as explants, and differentiates the explants into complete plants through induction, so that a great amount of seedlings can be quickly obtained while excellent characters of female parents are kept. At present, the tissue culture and rapid propagation of epimedium is reported less, and the tissue culture and rapid propagation by taking inflorescence as explant is more rarely reported. Wushan epimedium is a species recorded in Chinese pharmacopoeia, and is not only an excellent traditional Chinese medicine but also an excellent gardening ornamental plant.
The invention provides a tissue culture rapid propagation method taking epimedium wushanense inflorescence as explant, the differentiation rate of callus differentiation bud can reach as high as 83.7%, the multiplication coefficient of cluster bud can reach 5.1, a large number of aseptic seedlings with high survival rate can be obtained in a short time, and the tissue culture rapid propagation method has positive effects on protecting wild resources of epimedium wushanense and realizing sustainable utilization of epimedium wushanense resources.
Disclosure of Invention
The invention aims to provide a tissue culture and rapid propagation method of epimedium wushanense inflorescence, which takes epimedium inflorescence as an explant to carry out tissue culture propagation of epimedium, has simple operation, low cost and high survival rate, and provides reference for scale production of epimedium.
In order to further achieve the purpose, the invention adopts the following technical scheme: a tissue culture rapid propagation method of epimedium wushanense inflorescence comprises the following steps:
(1) and (3) inflorescence disinfection: collecting fresh and disease-free inflorescences with the soil surface of 10-20cm from early spring germination of epimedium wushanense, putting explant materials on a 500ml big beaker, covering gauze, putting the beaker under tap water, washing for 1 hour, draining, transferring the beaker into an ultraclean workbench, shaking and sterilizing the beaker with 75% alcohol solution at 120rpm for 30-45s, taking out the beaker, and rinsing the beaker with sterile water for 3-4 times to remove residual alcohol; then sterilized with 0.1% mercuric chloride for 5 minutes, rinsed 4-5 times with sterile water, and placed on sterilized filter paper to remove residual water for use.
(2) Inducing the explant to generate callus: cutting the sterilized inflorescence into 1-1.5cm sections in a super clean bench, and then inoculating the sections onto a callus induction culture medium; culturing under the conditions of light intensity of 1000-; changing the culture medium once every 20 days, wherein the formula of the changed culture medium is consistent with that of the original culture medium, inducing to generate callus, and counting the callus growth rate after 20 days.
(3) Inducing the callus to differentiate adventitious buds or cluster buds: transferring the callus generated by induction to an adventitious bud induction culture medium, culturing under the conditions of light intensity of 1000-1500lux and illumination of 13 h/day, wherein the culture temperature is 24-26 ℃, inducing the formation of adventitious buds, and counting the induction rate of the adventitious buds after 30 days.
Propagation culture of buds: cutting a single bud, inoculating the bud to a bud multiplication culture medium, carrying out subculture, carrying out culture under the conditions of light intensity of 2000 plus 2500lux and illumination for 13 h/day, wherein the culture temperature is 24-26 ℃, inducing to form cluster buds, and counting the multiplication coefficient of the cluster buds after 30 days.
(4) And (3) inducing buds to form fibrous roots: cutting a single thick bud under aseptic condition, inoculating the bud in a rooting culture medium, culturing under the illumination conditions of 2000 and 2500lux light intensity and 13 h/day light intensity, wherein the culture temperature is 24-26 ℃, white fibrous roots are formed, and the rooting rate is counted after 30 days.
(5) Hardening and raising seedlings: selecting tissue culture seedlings with good growth vigor and relatively developed root systems, removing the covers of tissue culture bottles in a seedling exercising preparation room, and exercising the seedlings for 3-4 days under the conditions of light intensity of 2000 plus 2500lux and light intensity of 13 h/day, the culture temperature is 24-26 ℃, and the air humidity is 65-80%, and water spraying and moisture preservation are carried out in the period; cleaning the culture medium, planting the culture medium in the garden soil: vermiculite: peat soil: hardening the seedling for 40-60 days in the matrix mixed with the humus soil in a ratio of 2:1:2: 1; after hardening off, transplanting to a nursery garden, sterilizing nursery garden soil by adopting chlorothalonil 75% wettable powder according to 700 times of liquid of 500 plus materials before transplanting, applying a proper amount of organic fertilizer, raking, planting at a planting distance of 15-20cm, covering soil, compacting, watering fully, meanwhile, erecting a sunshade net with the sunshade rate of 60-70%, paying attention to insect prevention and drought and waterlogging prevention, and counting the survival rate after 60 days.
In the invention, the inflorescence disinfection process adopted in the step (1) can fully wash away residual dust soil powder and most of bacterial fungi, so that the inflorescence is kept sterile to the maximum extent.
In the invention, the callus is formed at the wound by cutting in the step (2) to a certain extent, and the inflorescence belongs to the tender part of the plant, so that the totipotency is high, and the callus is more easily induced.
In the invention, the vermiculite in the substrate in the step (5) is used for loosening and ventilating soil to prevent soil hardening, and as the growth environment of the epimedium herb is under-forest humus soil which is fertile and porous in soil, the amount of the soil in the field is large to ensure that water is not excessively lost; the key point of the substrate adopted by the invention is that the soil is not bonded under the condition of keeping certain humidity and nutrition, and the growth condition of the epimedium under the natural environment is simulated to the maximum extent.
In the invention, a proper amount of organic fertilizer is applied to the nursery garden before transplanting in the step (5), and the organic fertilizer has high nutrient content, the use amount is not too much, and the organic fertilizer is easy to burn seedlings if the use amount is too much, and the organic fertilizer is reasonably matched with the traditional compound fertilizer for use.
In the invention, the sunshade net in the step (5) is used for simulating that epimedium wushanense is born in forests and other places with higher sunshade in a natural state so as to prevent sunburn.
In the invention, the seedling exercising in the step (5) is based on a buffering process from completely sterile tissue culture seedlings to underground transplanting, and the seedlings need to absorb nutrients from soil in the process, so that the seedlings are better adapted to the natural environment and grow more robustly.
In the invention, the step (5) of preventing insects and drought and waterlogging refers to that 1: 200075% of chlorothalonil liquid medicine needs to be regularly weeded and sprayed, manual sprinkling irrigation is adopted when raining does not occur for many days in summer, the humidity of the chlorothalonil liquid medicine reaches 65% -80%, and regular drainage is paid attention to.
The steps (1), (2) and (3) are key points of the invention, firstly, inflorescences which are rarely related up to now are selected as explants, a new disinfection mode is applied, and differentiation and proliferation culture mediums of buds are integrated, so that time and labor are saved and material cost is reduced on the basis of ensuring differentiation and proliferation coefficients; the combination mode of different hormones also effectively improves the induction rate and the proliferation coefficient of the callus, well verifies the totipotency of the cells, and lays a good foundation for the industrial production of epimedium wushanense.
The callus induction culture medium in the step (2) is as follows: MS4.74g/L +2,4-D (1mg/mL)2.0-4.0mL/L + IBA (1mg/mL)0.5-1.0mL/L + sucrose 30g/L + carrageenan 7.5g/L, pH is adjusted to 6.5.
The adventitious bud induction culture medium or bud proliferation culture medium in the step (3) is as follows: MS4.74g/L +6-BA (1mg/mL)0.5-1.0mL/L + IBA (1mg/mL)0.5-1.0mL/L + sucrose 30g/L + carrageenan 7.5g/L, and pH is adjusted to 6.5.
In the invention, the culture medium adopted by the culture of the buds and the multiplication of the buds in the step (3) is the same, so that the experimental operation process is simplified under the condition of ensuring the bud emergence and the multiplication coefficient.
The rooting medium in the step (4) is as follows: 1/2MS 4.74g/L + NAA (1mg/mL)0.5-1.0mL/L + activated carbon 0.2-0.4g/L + sucrose 30g/L + agar 6.0g/L, pH adjusted to 6.5.
In the invention, the rooting medium adopted in the step (4) contains 6-BA with a certain concentration, so that the growth of roots is promoted to a certain extent, and the grown root system is thicker and is favorable for seedling exercising growth.
In the present invention, MS refers to the basic formulation of a medium for tissue culture of general crops.
In the present invention, 6-BA means 6-benzylaminoadenine.
In the present invention, NAA means alpha-naphthylacetic acid.
In the present invention, IBA refers to indolebutyric acid.
In the present invention, 2,4-D is 2, 4-dichlorophenoxyacetic acid.
In the present invention, the callus growth rate is (fresh mass of callus at the time of statistics-fresh mass of callus at the time of inoculation)/fresh mass of callus at the time of inoculation × 100%.
In the present invention, the adventitious bud induction rate is the number of callus pieces differentiated into adventitious buds/the number of callus pieces inoculated × 100%.
In the present invention, the multiple shoot growth coefficient is the number of shoots grown/the number of shoots at the time of inoculation.
In the present invention, the rooting rate is the number of rooted seedlings/number of inoculated seedlings × 100%.
Compared with the prior art, the invention has the advantages and beneficial effects that: the invention establishes the rapid tissue propagation system of the epimedium, the formed tissue culture seedling keeps the excellent characters of the female parent, the inheritance is stable, the generation-by-generation accumulation of viruses is effectively avoided, the propagation rate is high, the test operation is simple, the production cost is low, and the technical support is provided for the large-scale planting of the epimedium.
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FIG. 1 shows a fresh, disease-free inflorescence collected by the present invention;
FIG. 2 shows callus induced by inflorescence according to example 1 of the present invention;
FIG. 3 shows a plantlet differentiated from an inflorescence callus according to example 2 of the present invention;
FIG. 4 shows the induction of root generation in clumped seedlings according to example 1 of the present invention.
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and together with the description, serve to explain the principles of the application.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention.
Example 1: a tissue culture rapid propagation method of epimedium wushanense inflorescence comprises the following steps:
(1) and (3) inflorescence disinfection: collecting fresh and disease-free inflorescences with the soil surface of 10 cm, 12 cm, 14 cm, 16 cm, 18 cm or 20cm, which are emitted by epimedium wushanense in early spring, placing the explant material on a 500ml big beaker, covering gauze, placing the beaker under tap water, washing the beaker for 1 hour, draining the explant material, transferring the beaker into an ultraclean workbench, shaking and disinfecting the beaker for 30 seconds by using 75% alcohol solution at 120rpm, taking out the beaker, and rinsing the beaker with sterile water for 3 times to remove residual alcohol; then sterilized with 0.1% mercuric chloride for 5 minutes, rinsed 4 times with sterile water, and placed on sterilized filter paper to remove residual water for use.
(2) Inducing the explant to generate callus: cutting the sterilized inflorescence into small sections with the length of 1.5cm in a super clean bench, and then inoculating the small sections onto a callus induction culture medium, wherein the callus induction culture medium comprises the following components in percentage by weight: 4mL/L of MS4.74g/L +2,4-D (1mg/mL), 1mL/L of IBA (1mg/mL), 30g/L of cane sugar and 7.5g/L of carrageenan, and the pH is adjusted to 6.5; culturing under the conditions of light intensity of 1000lux and illumination of 13 h/day, wherein the culture temperature is 24 ℃; changing the culture medium once every 20 days, wherein the formula of the changed culture medium is consistent with that of the original culture medium, inducing to generate callus, and counting the callus growth rate after 20 days, wherein the callus growth rate is 163.27%.
(3) Inducing the callus to differentiate adventitious buds or cluster buds: transferring the callus generated by induction into an adventitious bud induction culture medium, wherein the adventitious bud induction culture medium comprises the following formula: MS4.74g/L +6-BA (1mg/mL)1mL/L + IBA (1mg/mL)1mL/L + sucrose 30g/L + carrageenan 7.5g/L, pH adjusted to 6.5. Culturing under the conditions of light intensity of 1000lux and illumination of 13 h/day, culturing at 24 ℃, inducing the formation of adventitious buds, and counting the induction rate of the adventitious buds after 30 days, wherein the induction rate is 83.7%.
Propagation culture of buds: cutting a single bud and inoculating the bud into a bud multiplication medium, wherein the formula of the bud multiplication medium is as follows: MS4.74g/L +6-BA 1ml/L + IBA 1ml/L + sucrose 30g/L + carrageenan 7.5g/L, pH is adjusted to 6.5, subculture is carried out, culture is carried out under the conditions of 2000lux of light intensity and 13 h/day of illumination, the culture temperature is 24 ℃, the multiplication coefficient of the cluster buds is counted after 30 days, and the multiplication coefficient is 5.1.
(4) And (3) inducing buds to form fibrous roots: cutting a single stout bud under aseptic condition, inoculating the bud to a rooting culture medium, wherein the formula of the rooting culture medium is as follows: 1/2MS 4.74g/L + NAA (1mg/mL)1mL/L + activated carbon 0.2g/L + sucrose 30g/L + agar 6.0g/L, pH adjusted to 6.5. Culturing under the conditions of 2000lux light intensity and 13 h/day illumination, wherein the culture temperature is 24 ℃, white fibrous roots are formed, and the rooting rate is counted after 30 days and is 68.14%.
(5) Hardening and raising seedlings: selecting tissue culture seedlings with good growth vigor and relatively developed root systems, removing covers of tissue culture bottles in a seedling hardening preparation room, culturing under the conditions of light intensity of 2000lux and illumination of 13 h/day, hardening seedlings for 3 days at the culture temperature of 24 ℃ and the air humidity of 65%, and keeping moisture by spraying water in the period; cleaning the culture medium, planting the culture medium in the garden soil: vermiculite: peat soil: hardening the seedling for 40 days in the matrix mixed by 2:1:2:1 humus soil; transplanting to nursery garden after hardening, sterilizing nursery garden soil with 75% wettable chlorothalonil powder in 500, 560, 600, 650 or 700 times of liquid, applying proper amount of organic fertilizer, and harrowing2O5-K2O is 15-15-1530 kg/mu), planting is carried out at a planting distance of 20cm, sufficient water is poured after soil covering and compaction, meanwhile, a sunshade net with the sunshade rate of 60% or 65% or 70% is erected, insect prevention and drought and waterlogging prevention are noticed, the survival rate is counted after 60 days, and the survival rate is 86%.
Example 2: a tissue culture rapid propagation method of epimedium wushanense inflorescence comprises the following steps:
(1) and (3) inflorescence disinfection: collecting fresh and disease-free inflorescences with the soil surface of 10 cm, 12 cm, 14 cm, 16 cm, 18 cm or 20cm, which are emitted by epimedium wushanense in early spring, placing the explant material on a 500ml big beaker, covering gauze, placing the beaker under tap water, washing the beaker for 1 hour, draining the washed explant material, transferring the washed explant material into an ultraclean workbench, shaking and sterilizing the washed explant material for 45 seconds by using 75% alcohol solution at 120rpm, taking out the washed explant material, and rinsing the washed explant material with sterile water for 4 times to remove residual alcohol; then sterilized with 0.1% mercuric chloride for 5 minutes, rinsed 5 times with sterile water, and placed on sterilized filter paper to remove residual water for use.
(2) Inducing the explant to generate callus: cutting the sterilized inflorescence into small sections with the length of 1cm in a super clean bench, and then inoculating the small sections onto a callus induction culture medium, wherein the callus induction culture medium comprises the following formula: MS4.74g/L +2,4-D (1mg/mL)3mL/L + IBA (1mg/mL)1mL/L + sucrose 30g/L + carrageenan 7.5g/L, pH is adjusted to 6.5; culturing under the conditions of light intensity of 1500lux and illumination of 13 h/day, wherein the culture temperature is 26 ℃; the culture medium is replaced once every 20 days, the formula of the replaced culture medium is consistent with that of the original culture medium, callus is induced to generate, and the growth rate is counted after 20 days, and the growth rate of the callus is 158.1%.
(3) Inducing the callus to differentiate adventitious buds or cluster buds: transferring the callus generated by induction to an adventitious bud induction culture medium, wherein the adventitious bud induction culture medium comprises the following formula: MS4.74g/L +6-BA (1mg/mL)0.5mL/L + IBA (1mg/mL)1mL/L + sucrose 30g/L + carrageenan 7.5g/L, pH is adjusted to 6.5; culturing under the conditions of light intensity of 1500lux and illumination of 13 h/day, culturing at 26 ℃, inducing the formation of adventitious buds, and counting the induction rate of the adventitious buds after 30 days, wherein the induction rate is 80.15%.
Propagation culture of buds: cutting a single bud and inoculating the bud into a bud multiplication medium, wherein the formula of the bud multiplication medium is as follows: MS4.74g/L +6-BA (1mg/mL)1mL/L + IBA (1mg/mL)1mL/L + sucrose 30g/L + carrageenan 7.5g/L, adjusting pH to 6.5, carrying out subculture, carrying out culture under the conditions of 2500lux of light intensity and 13 h/day of illumination, wherein the culture temperature is 26 ℃, inducing to form cluster buds, and counting the proliferation coefficient of the cluster buds after 30 days, wherein the proliferation coefficient is 5.1.
(4) And (3) inducing buds to form fibrous roots: cutting a single stout bud under aseptic condition, inoculating the bud to a rooting culture medium, wherein the formula of the culture medium is as follows: 1/2MS 4.74g/L + NAA (1mg/mL)1mL/L + active carbon 0.3g/L + sucrose 30g/L + agar 6.0g/L, pH adjusted to 6.5; culturing under the conditions of light intensity of 2500lux and illumination of 13 h/day, wherein the culture temperature is 26 ℃, white fibrous roots are formed, and the rooting rate is counted after 30 days and is 72.3%.
(5) Hardening and raising seedlings: selecting tissue culture seedling with good growth and relatively developed root system, removing the cover of the tissue culture bottle in seedling hardening preparation room under the light intensity of 2500lux, 1Culturing under illumination for 3 h/day, hardening the seedlings for 4 days at the culture temperature of 26 ℃ and the air humidity of 80%, and spraying water for moisturizing; cleaning the culture medium, planting the culture medium in the garden soil: vermiculite: peat soil: hardening the seedling for 50 days in the matrix mixed by 2:1:2:1 humus soil; transplanting to nursery garden after hardening, sterilizing nursery garden soil with 75% wettable chlorothalonil powder in 500, 560, 600, 650 or 700 times of liquid, applying proper amount of organic fertilizer, and harrowing2O5-K2O is 15-15-1530 kg/mu), planting is carried out at a planting distance of 17cm, sufficient water is poured after covering soil and compacting, meanwhile, a sunshade net with the sunshade rate of 60 percent or 65 percent or 70 percent is erected, insect prevention and drought and waterlogging prevention are noticed, the survival rate of seedlings is counted after 60 days, and the survival rate is 86 percent.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
Claims (2)
1. A tissue culture and rapid propagation method of epimedium wushanense inflorescence is characterized by comprising the following steps:
(1) and (3) inflorescence disinfection: collecting fresh and disease-free inflorescences with the soil surface of 10-20cm from early spring germination of epimedium wushanense, putting explant materials into a large beaker of 500ml, covering a gauze, putting the beaker into tap water, washing the beaker for 1 hour, draining the washed beaker, transferring the beaker into an ultraclean workbench, shaking and sterilizing the beaker with 75% alcohol solution at 120rpm for 30-45s, taking out the beaker, and rinsing the beaker with sterile water for 3-4 times to remove residual alcohol; sterilizing with 0.1% mercuric chloride for 5 min, rinsing with sterile water for 4-5 times, and placing on sterilized filter paper to remove residual water;
(2) inducing the explant to generate callus: cutting the sterilized inflorescence into small blocks of 1-1.5cm in a super clean bench, and then inoculating the small blocks onto a callus induction culture medium; culturing under the conditions of light intensity of 1000-; replacing the culture medium once every 20 days, wherein the formula of the replaced culture medium is consistent with that of the original culture medium, and inducing to generate callus;
the callus induction culture medium comprises: MS4.74g/L +2, 4-D2.0-4.0 mg/L + IBA 0.5-1.0mg/L + sucrose 30g/L + carrageenan 7.5g/L, pH is adjusted to 6.5;
(3) inducing the callus to differentiate adventitious buds or cluster buds: transferring the callus generated by induction to an adventitious bud induction culture medium, and culturing under the conditions of light intensity of 1000-;
the adventitious bud induction culture medium comprises: MS4.74g/L +6-BA 0.5-1.0mg/L + IBA 0.5-1.0mg/L + sucrose 30g/L + carrageenan 7.5g/L, pH is adjusted to 6.5;
propagation culture of buds: cutting a single bud, inoculating the bud to a bud multiplication culture medium, carrying out subculture, and carrying out culture under the conditions of light intensity of 2000 plus 2500lux and illumination for 13 h/day, wherein the culture temperature is 24-26 ℃, and inducing to form cluster buds;
the bud multiplication culture medium comprises: MS4.74g/L +6-BA 1mg/L + IBA 1mg/L + sucrose 30g/L + carrageenan 7.5g/L, pH is adjusted to 6.5;
(4) and (3) inducing buds to form fibrous roots: cutting a single thick bud under aseptic condition, inoculating the bud in a rooting culture medium, and culturing under the illumination conditions of 2000 and 2500lux light intensity and 13 h/day light intensity, wherein the culture temperature is 24-26 ℃ to form white fibrous roots;
the rooting culture medium comprises: 1/2MS 4.74g/L + NAA 0.5-1.0mg/L + active carbon 0.2-0.4g/L + sucrose 30g/L + agar 6.0g/L, pH adjusted to 6.5;
(5) hardening and raising seedlings: selecting tissue culture seedlings with good growth vigor and relatively developed root systems, removing the covers of tissue culture bottles in a seedling exercising preparation room, and carrying out culture under the illumination conditions of 2000-2500lux light intensity and 13 h/day, wherein the culture temperature is 24-26 ℃, the air humidity is 65-80%, and the exercising is carried out for 3-4 days, and water spraying and moisture preservation are carried out in the period; cleaning the culture medium, planting the culture medium in the garden soil: vermiculite: peat soil: hardening seedlings in a seedling hardening room for 40-60 days in a matrix mixed by humus soil in a ratio of 2:1:2: 1; transplanting the seedlings to a nursery garden after hardening, disinfecting soil of the nursery garden before transplanting, applying a proper amount of organic fertilizer, raking the nursery garden, planting the seedlings at a distance of 15-20cm, covering soil, compacting, watering, and meanwhile erecting a sunshade net with the sunshade rate of 60-70%, and paying attention to insect prevention and drought and waterlogging prevention.
2. The tissue culture rapid propagation method of epimedium wushanense inflorescence according to claim 1, characterized in that the nursery soil is disinfected by using 500-fold and 700-fold diluted solution of 75% chlorothalonil wettable powder before transplantation in the step (5).
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