CN111264393B - Method for rapidly breeding epimedium test-tube plantlets - Google Patents
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Classifications
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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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
-
- 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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention provides a method for rapidly breeding epimedium test-tube plantlets, and relates to the technical field of traditional Chinese medicine cultivation. The method comprises the following steps: (1) inoculating the dormant epimedium seeds serving as explants to a buffer culture medium containing streptomycin or penicillin for culture to obtain epimedium seed embryos; (2) carrying out callus induction and proliferation, cluster bud differentiation, cluster bud proliferation, rooting culture and tissue culture seedling domestication on epimedium seed embryos, and then transplanting the embryos into a seedling culture substrate for culture to obtain epimedium test-tube seedlings; the culture device for the bearing culture medium for the cluster bud multiplication and rooting culture adopts an aerial fog tissue culture device. The method of the invention adopts aerial fog culture, reduces the contamination rate of the test-tube plantlet of the epimedium herb, obviously improves the transplanting survival rate of the test-tube plantlet of the epimedium herb, can reach 92 percent and is beneficial to realizing the large-scale production of the epimedium herb.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine cultivation, in particular to a method for quickly breeding epimedium test-tube plantlets.
Background
Herba Epimedii (EpinediumLinn.) is perennial herb of Epimedium of berberidaceae, and is called herba Epimedii, folium Epimedii, herba Adonidis, and herba Adonidis pestle. The rootstock is thick, short or transverse, hard, multi-fibrous root and brown; the stem is single or multiple, smooth, and has brown scale at the base, and the stem and leaf has effects of invigorating kidney, tonifying yang, strengthening tendons and bones, and dispelling pathogenic wind and removing dampness. Modern medical research shows that icariin, the main effective component in epimedium, can prevent and treat cardiovascular and cerebrovascular diseases, improve immunity, enhance bone metabolism and prevent osteoporosis. In addition, it has certain effects in resisting tumor, oxidation and aging. Besides the medicinal value, the epimedium also has peculiar posture, and leaves and flowers have higher ornamental value, so the epimedium also has great prospect as a novel garden plant to be vigorously developed. At present, epimedium is mainly used as a medicinal material by digging wild resources, so that the number of the medicinal materials in main producing areas is reduced year by year, and the damage to resources and ecology is large. The active development of the artificial cultivation of epimedium and the establishment of the rapid propagation technology can not only obtain better economic benefit, but also promote the ecological restoration.
Epimedium seeds have obvious dormancy phenomenon, can emerge in about 1 year under natural conditions, and the rate of emergence is low, mainly because the form of embryo is mature and the seeds contain germination inhibiting substances. The currently reported methods for breaking the dormancy of epimedium seeds mainly comprise sand stratification, temperature change stratification, hormone treatment and the like. However, when the epimedium is propagated asexually from underground rhizomes, the epimedium is easily restricted by factors such as bacteria, fungi, plant sources, growth environment and the like, so that the seed propagation and the rhizome propagation are difficult to realize large-scale production. In addition, in the plant tissue culture method, the liquid culture has higher nutrient element utilization rate compared with the solid culture, so that the plant can proliferate more quickly, grow fast and robustly, but the liquid culture medium has poor ventilation and needs to be shaken, so that vitrification is often serious, and although the immersion culture developed in recent years solves the gas exchange problem to a certain extent, the submerged culture still has the adverse effects on the culture due to low dissolved oxygen and large shearing force.
Disclosure of Invention
In view of the above, the invention aims to provide a method for rapidly breeding epimedium test-tube plantlets, which can improve the seed germination rate and the tissue culture propagation rate of epimedium and realize the large-scale production of epimedium.
In order to solve the technical problems, the invention provides the following technical scheme:
a method for rapidly breeding epimedium test-tube plantlets comprises the following steps:
(1) inoculating the dormant epimedium seeds serving as explants to a buffer culture medium containing streptomycin or penicillin for culture to obtain epimedium seed embryos;
(2) carrying out callus induction and proliferation, cluster bud differentiation, cluster bud proliferation, rooting culture and tissue culture seedling domestication on epimedium seed embryos, and then transplanting the embryos into a seedling culture substrate for culture to obtain epimedium test-tube seedlings; the culture device for the bearing culture medium for the cluster bud multiplication and rooting culture adopts an aerial fog tissue culture device.
Preferably, the dormancy breaking method specifically comprises the following steps: soaking seeds for 24-36h by using a solution containing 20mg/L of fluazinone and 500mg/L of gibberellin, and then carrying out sand stratification for 88-95 days.
Preferably, the concentration of the streptomycin or the penicillin is 0.1g/L of streptomycin or 400mg/L of penicillin.
Preferably, the formula of the buffer medium is as follows: MS + streptomycin 0.1g/L or penicillin 400mg/L + agar 7.0-7.5g/L + sucrose 25-30g/L, pH5.8-6.0.
Preferably, the callus induction medium formula is as follows: 1/2MS +1.2-2.0mg/L6-BA +0.8-1.5mg/L2,4-D +0.1-0.5mg/LIBA + agar 7.0-7.5g/L + sucrose 25-30g/L, pH 5.8-6.0.
Preferably, the formula of the culture medium for the propagation of the cluster buds is as follows: MS +6-BA 1.0 mg.L-1+NAA 0.5mg·L-1。
Preferably, the formula of the rooting culture medium is as follows: 1/2MS + NAA0.5mg/L + active carbon 1.0g/L + sucrose 25-30g/L, pH5.8-6.0.
Preferably, the conditions for the propagation of the cluster buds and the rooting culture are as follows: the temperature is 23-25 ℃, the humidity is 75-85%, the illumination is 14-16h/d, and the illumination intensity is 2000-2500 Lx.
Preferably, the seedling culture substrate is a mixture of grass carbon, perlite and vermiculite, and the volume ratio of the grass carbon, the perlite and the vermiculite is 2:1: 1.
Preferably, the conditions for transplanting into the seedling substrate for culturing are as follows: the illumination intensity is 1500-.
The invention has the beneficial effects that:
the method optimizes the tissue culture and rapid propagation system of the epimedium by adding streptomycin or penicillin into the culture medium, obviously reduces the pollution rate of test-tube plantlets at the initial stage of tissue culture of the epimedium, and improves the quality and the survival rate of the test-tube plantlets of the epimedium. The invention adopts the aerial fog tissue culture device to increase the dissolved oxygen, is beneficial to the growth and development of the culture, can coordinate the contradiction between liquid phase and gas phase, enhances the gas exchange, enables the cultivated epimedium plants to be stronger, is beneficial to the epimedium plants to adapt to the external environment more quickly, and ensures that the transplanting survival rate of the test-tube plantlets reaches 92 percent.
Detailed Description
The invention provides a method for rapidly breeding epimedium test-tube plantlets, which comprises the following steps:
(1) inoculating the dormant epimedium seeds serving as explants to a buffer culture medium containing streptomycin or penicillin for culture to obtain epimedium seed embryos;
(2) carrying out callus induction and proliferation, cluster bud differentiation, cluster bud proliferation, rooting culture and tissue culture seedling domestication on epimedium seed embryos, and then transplanting the embryos into a seedling culture substrate for culture to obtain epimedium test-tube seedlings; the culture device for the bearing culture medium for the cluster bud multiplication and rooting culture adopts an aerial fog tissue culture device.
In the invention, the dormancy breaking is preferably performed by sand stratification after seed soaking by using a solution containing the fluazinone and the gibberellin; the preferred content of the fluazinone and the gibberellin is 20mg/L fluazinone and 500mg/L gibberellin; the seed soaking time is preferably 24-36h, more preferably 28-32 h; the sand deposit is preferably layered for 88-95 days, more preferably for 90 days.
Before the dormant epimedium seeds are used as explants, the disinfection is preferably required. The disinfection in the invention is preferably primary disinfection and thorough disinfection; the preliminary disinfection is preferably to soak epimedium seeds in a bacteriostatic agent; wherein the bacteriostatic agent is preferably 2D-106 bacteriostatic agent or HgCl with the volume fraction of 0.1 percent2(ii) a The thorough disinfection is preferably carried out by soaking the seeds in alcohol and then placing the seeds in HgCl2Soaking in the solution; wherein the alcohol is preferably 75% alcohol by volume, HgCl2HgCl with a volume fraction of 0.1% is preferred2。
In the present invention, the explant is inoculated in a buffer medium containing streptomycin or penicillin for culture. The streptomycin or the penicillin is preferably 0.1g/L streptomycin or 400mg/L penicillin. The formula of the buffer culture medium is preferably as follows: MS + streptomycin 0.1g/L or penicillin 400mg/L + agar 7.0-7.5g/L + sucrose 25-30g/L, pH5.8-6.0. The buffer culture medium is added with streptomycin or penicillin, so that the test-tube plantlet pollution rate at the initial stage of herba epimedii tissue culture can be remarkably reduced, and the quality and the survival rate of herba epimedii are improved.
In order to determine the formula of the buffer culture medium, as an implementable mode, the invention respectively inoculates seeds which have broken dormancy into three buffer culture media with different formulas, namely, MS, agar 7.0-7.5g/L, sucrose 25-30g/L and pH 5.8-6.0; 0.1g/L of MS + streptomycin + 7.0-7.5g/L of agar + 25-30g/L of cane sugar, and the pH value is 5.8-6.0; ③ MS + penicillin 400mg/L + agar 7.0-7.5g/L + sucrose 25-30g/L, pH5.8-6.0, inoculating 50 seeds to each culture medium, counting the contamination rate after 7 days, the results are as follows:
TABLE 1 Effect of penicillin or streptomycin addition on Epimedium seed contamination rate
As can be seen from Table 1, the buffer culture media with different formulas have different pollution effects on epimedium seeds, and the addition of penicillin or streptomycin can obviously reduce the pollution rate of the epimedium seeds and is beneficial to improving the quality and survival rate of the epimedium.
In the invention, the epimedium seed embryo is used for callus induction and multiplication culture. The callus induction is preferably carried out in an induction medium, and the formula of the induction medium is preferably as follows: 1/2MS +1.2-2.0mg/L6-BA +0.8-1.5mg/L2,4-D +0.1-0.5mg/L IBA + agar 7.0-7.5g/L + sucrose 25-30g/L, pH5.8-6.0, more preferably 1/2MS +1.5mg/L6-BA +1.0mg/L2,4-D +0.3mg/LIBA + agar 7.3g/L + sucrose 28 g/L; the callus proliferation culture is preferably carried out in a proliferation culture medium, and the formula of the proliferation culture medium is preferably as follows: MS +2.0mg/L6-BA +0.2mg/L LNAA agar 7.0-7.5g/L + sucrose 25-30g/L, pH 5.8-6.0.
In the present invention, cluster bud differentiation is performed after callus is obtained by callus induction and proliferation. The cluster bud differentiation is carried out in a cluster bud differentiation culture medium; the formula of the cluster bud differentiation medium is preferably as follows: 1/2MS +1.0mg/L6-BA +0.5mg/L NAA; the differentiation culture time of the cluster buds is preferably 30-40d, and more preferably 35-38 d.
In the invention, the culture device of the bearing culture medium for the propagation and rooting culture of the cluster buds adopts an aerosol tissue culture device. The aerosol tissue culture device can increase dissolved oxygen, coordinate liquid phase and gas phase contradiction, enhance gas exchange, facilitate the growth and development of epimedium, make the cultivated plants stronger, enable the plants to adapt to the external environment more quickly, make the stomatal function of the plants more complete, and reduce vitrified plants. Meanwhile, the aerosol tissue culture device does not use agar or gel, so that the cost is saved. The source, the model and the structure of the aerosol tissue culture device are not specially limited, and the existing known aerosol tissue culture devices can be applied to the invention. In the specific embodiment of the invention, the aerosol tissue culture device is provided by Jiangsu agriculture and forestry occupational technology academy. The working conditions of the aerosol tissue culture device are reasonably set according to different models and working efficiency, and the aerosol tissue culture device is preferably electrified every 6 hours for 5 minutes.
In the invention, the culture medium for the propagation and rooting culture of the cluster buds is preferably a liquid culture medium carried by an air mist tissue culture device; the culture medium for the propagation of the cluster buds is preferably: MS +6-BA 1.0 mg.L-1+NAA 0.5mg·L-1pH5.8-6.0; the culture medium for rooting culture is preferably: 1/2MS + NAA0.5mg/L + active carbon 1.0g/L + sucrose 25-30g/L, pH5.8-6.0.
In order to determine the influence of different compositions of rooting culture media on epimedium test-tube plantlets, as an implementable mode, the tissue culture seedlings which grow to 3-5 cm through propagation of multiple buds are inoculated to two culture media with different formulas, namely 1/2MS, NAA0.5mg/L, sucrose 25-30g/L, agar 7.0-7.5g/L and pH5.8-6.0; 1/2MS + NAA0.5mg/L + active carbon 1.0g/L + sucrose 25-30g/L + agar 7.0-7.5g/L, pH5.8-6.0, culturing for 30d, taking 10 strains, and counting the plant height and root system number as follows:
TABLE 2 influence of different rooting media on the plant height and root number of Epimedium
It can be seen that the rooting culture media with different formulas have different influences on the height and the root number of the epimedium herb, and the addition of the activated carbon can obviously improve the plant height of the epimedium herb test-tube plantlet, increase the root number of the epimedium herb test-tube plantlet and be beneficial to improving the quality and the transplanting survival rate of the epimedium herb test-tube plantlet.
In the present invention, the culture conditions for the proliferation and rooting culture of the multiple shoots are preferably: the temperature is 23-25 ℃, the humidity is 75-85%, the illumination is 14-16h/d, the illumination intensity is 2000-: the temperature is 24 ℃, the humidity is 80%, the illumination is 15h/d, and the illumination intensity is 2300 Lx.
In the invention, the epimedium tissue culture seedlings after rooting culture and domestication are transplanted into a seedling culture substrate for culture. The seedling raising substrate is preferably a mixture of grass carbon, perlite and vermiculite, and the volume ratio of the grass carbon to the perlite to the vermiculite is preferably grass carbon: perlite: vermiculite 2:1: 1; the conditions for transplanting into the seedling substrate for culturing are preferably as follows: the illumination intensity is 1500-3000lx, the illumination time is 10-14h/d, and the temperature is 20-24 ℃; more preferably, the illumination intensity is 2500lx, the illumination time is 12h/d, and the temperature is 22 ℃.
Experimental results show that the epimedium tissue culture rapid propagation system is optimized, and the aerosol tissue culture device is adopted for aerosol culture, so that the test-tube seedling pollution rate at the initial stage of epimedium tissue culture is remarkably reduced, the quality and the survival rate of the test-tube seedlings of the epimedium are improved, the transplanted test-tube seedlings grow robustly, and the transplanting survival rate reaches 92%.
The present invention will be described in detail with reference to examples for better understanding the objects, technical solutions and advantages of the present invention, but they should not be construed as limiting the scope of the present invention.
In the following examples, epimedium is from Mincounty, Xinglong mountain area, Gansu province; the materials, reagents and the like used are commercially available unless otherwise specified.
Example 1
(1) Soaking seeds of epimedium in a dormant state with full seeds, no diseases and mature shedding in a solution containing 20mg/L of fluazinone and 500mg/L of gibberellin for 24 hours to break dormancy, and then carrying out outdoor sand accumulation for about 90 days.
(2) With dormancy releaseCleaning the seeds in the explant, sequentially washing the seeds with washing powder and distilled water, putting the seeds into a mesh bag, immersing the seeds in a 2D-106 bacteriostatic agent for 3 hours, taking out the seeds, washing the seeds with sterile water, and performing primary disinfection; putting the seeds in alcohol with volume fraction of 75% in a superclean workbench, oscillating the beaker to enable the alcohol to be fully contacted with the surface of the explant, and repeatedly cleaning for 3 times by using sterile water after 30 s; with a volume fraction of 0.1% HgCl2Thoroughly sterilizing the seeds for 6min, and repeatedly cleaning with sterile water for 3 times; and (3) placing the sterilized explant on sterile filter paper, after moisture is absorbed, inoculating the sterilized seeds into a buffer culture medium of MS + streptomycin 0.1g/L + agar 7.0g/L + sucrose 25g/L and pH5.8-6.0 by using sterilized tweezers to obtain epimedium seed embryos.
(3) Transferring the seed embryo obtained in the step (2) to a callus induction culture medium of 1/2MS +1.2mg/L6-BA +0.8mg/L2,4-D +0.1mg/L IBA + agar 7.0g/L + sucrose 25g/L and pH5.8-6.0 for culture to obtain a callus, and transferring the callus to a multiplication culture medium of MS +2.0mg/L6-BA +0.2mg/LNAA agar 7.0g/L + sucrose 25g/L and pH5.8-6.0 for callus multiplication; wherein, the culture conditions of induction and proliferation are as follows: the temperature is 23 ℃, the humidity is 75%, the illumination is 14h/d, and the illumination intensity is 2000 Lx.
(4) Cutting and transferring the green visible callus obtained in the step (3) into a cluster bud differentiation culture medium of 1/2MS +1.0mg/L6-BA +0.5mg/LNAA for culturing for 30d to obtain epimedium adventitious buds; when the cluster buds grow to 2cm, cutting the cluster buds into single plants under aseptic condition, inoculating to MS +6-BA 1.0 mg.L-1+NAA 0.5mg·L-1The aerosol device of the multiplication culture medium carries out multiplication on the net, and the tissue culture seedlings are subjected to multiplication culture again by the same method every 30 days according to the requirement on the number of the tissue culture seedlings.
(5) Inoculating the tissue culture seedling growing to 3cm to a bearing net of an aerosol device of 1/2MS, NAA0.5mg/L, active carbon 1.0g/L, cane sugar 25g/L and culture medium with pH of 5.8-6.0 to carry out rooting culture; the culture conditions were: the temperature is 23 ℃, the humidity is 75%, the illumination is 14h/d, and the illumination intensity is 2000 Lx.
(6) When the tissue culture seedlings grow to 5cm, domestication of the tissue culture seedlings is firstly carried out, and then the domesticated tissue culture seedlings are transplanted into a plant charcoal: perlite: culturing in a culture medium containing vermiculite 2:1:1 to obtain the epimedium test-tube plantlet.
Example 2
(1) Soaking seeds of epimedium in a dormant state with full seeds, no diseases and mature shedding in a solution containing 20mg/L of fluazinone and 500mg/L of gibberellin for 36h to break dormancy, and then carrying out outdoor sand accumulation for about 90 d.
(2) Cleaning the seed in dormancy-released state with washing powder and distilled water, placing the seed into a mesh bag, and soaking in HgCl with volume fraction of 0.1%2Soaking for 6min, taking out, washing with sterile water, and performing primary disinfection; placing the seeds in 75% alcohol by volume fraction in a superclean bench, oscillating the beaker to make the alcohol fully contact with the surface of the explant, and repeatedly cleaning with sterile water for 5 times after 30 s; with a volume fraction of 0.1% HgCl2Thoroughly sterilizing the seeds for 10min, and repeatedly cleaning with sterile water for 5 times; and (3) placing the sterilized explant on sterile filter paper, after moisture is absorbed, inoculating the sterilized seeds into a buffer culture medium of MS + streptomycin 0.1g/L + agar 7.5g/L + sucrose 30g/L and pH5.8-6.0 by using sterilized tweezers to obtain epimedium seed embryos.
(3) Transferring the seed embryo obtained in the step (2) to a callus induction culture medium of 1/2MS +2.0mg/L6-BA +1.5mg/L2,4-D +0.5mg/L IBA + agar 7.5g/L + sucrose 30g/L and pH5.8-6.0 for culture to obtain a callus, and transferring the callus to a multiplication culture medium of MS +2.0mg/L6-BA +0.2mg/LNAA agar 7.5g/L + sucrose 30g/L and pH5.8-6.0 for callus multiplication; wherein, the culture conditions of induction and proliferation are as follows: the temperature is 25 ℃, the humidity is 85%, the illumination is 16h/d, and the illumination intensity is 2500 Lx.
(4) Cutting and transferring the green visible callus obtained in the step (3) into a cluster bud differentiation culture medium of 1/2MS +1.0mg/L6-BA +0.5mg/LNAA for culturing for 40d to obtain epimedium adventitious buds; when the cluster buds grow to 3cm, cutting the cluster buds into single plants under aseptic condition, and inoculating to MS +6-BA 1.0 mg.L-1+NAA 0.5mg·L-1The aerosol device of the multiplication culture medium carries out multiplication on the net, and the tissue culture seedlings are subjected to multiplication culture again by the same method every 45 days according to the requirement on the number of the tissue culture seedlings.
(5) Inoculating the tissue culture seedling growing to 5cm to a bearing net of an aerosol device of 1/2MS, NAA0.5mg/L, active carbon 1.0g/L, sucrose 30g/L and a culture medium with pH of 5.8-6.0 to carry out rooting culture; the culture conditions were: the temperature is 25 ℃, the humidity is 85%, the illumination is 16h/d, and the illumination intensity is 2500 Lx.
(6) When the tissue culture seedlings grow to 8cm, domestication of the tissue culture seedlings is firstly carried out, and then the domesticated tissue culture seedlings are transplanted into a plant charcoal: perlite: culturing in a culture medium containing vermiculite 2:1:1 to obtain the epimedium test-tube plantlet.
Example 3
(1) Soaking seeds of epimedium in a dormant state with full seeds, no diseases and mature shedding in a solution containing 20mg/L of fluazinone and 500mg/L of gibberellin for 30h to break dormancy, and then carrying out outdoor sand accumulation for about 90 d.
(2) Cleaning the seeds in the dormancy-released state as explants with washing powder and distilled water in sequence, putting the seeds into a mesh bag, immersing the seeds in a 2D-106 bacteriostatic agent for 3 hours, taking out the seeds, washing the seeds with sterile water, and performing primary disinfection; putting the seeds in alcohol with volume fraction of 75% in a superclean workbench, oscillating the beaker to enable the alcohol to be fully contacted with the surface of the explant, and repeatedly cleaning for 4 times by using sterile water after 30 s; with a volume fraction of 0.1% HgCl2Thoroughly sterilizing the seeds for 8min, and repeatedly cleaning with sterile water for 4 times; and (3) placing the sterilized explant on sterile filter paper, after moisture is absorbed, inoculating the sterilized seeds into a buffer culture medium of MS + streptomycin 0.1g/L + agar 7.3g/L + sucrose 28g/L and pH5.8-6.0 by using sterilized tweezers to obtain epimedium seed embryos.
(3) Transferring the seed embryo obtained in the step (2) to a callus induction culture medium of 1/2MS +1.5mg/L6-BA +1.0mg/L2,4-D +0.3mg/L IBA + agar 7.3g/L + sucrose 28g/L and pH5.8-6.0 for culture to obtain a callus, and transferring the callus to a multiplication culture medium of MS +2.0mg/L6-BA +0.2mg/LNAA agar 7.3g/L + sucrose 28g/L and pH5.8-6.0 for callus multiplication; wherein, the culture conditions of induction and proliferation are as follows: the temperature is 24 ℃, the humidity is 80%, the illumination is 15h/d, and the illumination intensity is 2300 Lx.
(4) The green visible callus obtained in the step (3) is cut and transferred to 1/2MS +1.0mg/L6-BA +0.5mg/LNAA cluster bud differentiationCulturing in culture medium for 35d to obtain herba Epimedii adventitious bud; when the cluster buds grow to 3cm, cutting the cluster buds into single plants under aseptic condition, and inoculating to MS +6-BA 1.0 mg.L-1+NAA 0.5mg·L-1The aerosol device of the multiplication culture medium carries out multiplication on the net, and the tissue culture seedlings are subjected to multiplication culture again by the same method every 40 days according to the requirement on the number of the tissue culture seedlings.
(5) Inoculating the tissue culture seedling growing to 5cm to a bearing net of an aerosol device of 1/2MS, NAA0.5mg/L, active carbon 1.0g/L, sucrose 28g/L and a culture medium with pH of 5.8-6.0 to perform rooting culture; the culture conditions were: the temperature is 24 ℃, the humidity is 80%, the illumination is 15h/d, and the illumination intensity is 2300 Lx.
(6) When the tissue culture seedlings grow to 8cm, domestication of the tissue culture seedlings is firstly carried out, and then the domesticated tissue culture seedlings are transplanted into a plant charcoal: perlite: culturing in a culture medium containing vermiculite 2:1:1 to obtain the epimedium test-tube plantlet.
Comparative example 1
The procedure described in this comparative example 1 is similar to example 3, except that: in comparative example 1, the clump shoot enrichment medium in step (4) and the rooting medium in step (5) were both conventional agar media, and no aerosol tissue culture apparatus was used.
Respectively transferring the epimedium test-tube seedlings with the height of 5-8 cm and the roots of 5-6 obtained in the example 3 and the comparative example 1 into an outdoor shade place with the temperature of 10-15 ℃ and the humidity of 60-70% to open a cover for acclimatization and culture for 5-7 days, taking out, washing off residual culture medium on the seedlings by using clear water, then putting the seedlings into 400mg/L carbendazim solution for soaking for 20-30 min, and finally transplanting the seedlings into a container containing vermiculite in volume ratio: coconut husk: in a seedling raising plug tray with a matrix of 1:1:1, the test-tube seedlings of the epimedium are cultured for 28-35 days in a greenhouse with the temperature of 21-25 ℃, the illumination intensity of 2000-3000 lx and the humidity of 60-70% when the test-tube seedlings of the epimedium are transplanted, outdoor field transplanting is carried out when the plants grow to 10cm high, the transplanting time is selected to be between 5:00 and 6:00 in the afternoon, and the seedlings are watered thoroughly. The proliferation coefficient and survival rate of the test-tube plantlet of example 3 and comparative example 1 can be counted as follows:
TABLE 3 influence of different culture methods on the transplanting survival rate of test-tube plantlets of Epimedium herb
The method adopts the aerosol tissue culture device to rapidly breed the epimedium test-tube plantlet, obviously improves the multiplication coefficient compared with the existing epimedium test-tube plantlet breeding mode, and improves the transplanting survival rate by more than 20 percent and reaches 92 percent at most.
According to the embodiment, the method provided by the invention optimizes the herba epimedii tissue culture rapid propagation system, adopts the aerosol tissue culture device to carry out aerosol culture, obviously reduces the test-tube seedling pollution rate at the initial stage of herba epimedii tissue culture, improves the quality and survival rate of the test-tube seedlings of herba epimedii, ensures that the transplanted test-tube seedlings grow robustly and the transplanting survival rate reaches 92%.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (7)
1. A method for rapidly breeding epimedium test-tube plantlets is characterized by comprising the following steps:
inoculating the dormant epimedium seeds serving as explants to a buffer culture medium containing streptomycin or penicillin for culture to obtain epimedium seed embryos; the formula of the buffer culture medium is as follows: MS + streptomycin 0.1g/L or penicillin 400mg/L + agar 7.0-7.5g/L + sucrose 25-30g/L, pH5.8-6.0;
carrying out callus induction and proliferation, cluster bud differentiation, cluster bud proliferation, rooting culture and tissue culture seedling domestication on epimedium seed embryos, and then transplanting the embryos into a seedling culture substrate for culture to obtain epimedium test-tube seedlings; the culture device for the bearing culture medium for the propagation and rooting culture of the cluster buds adopts an aerial fog tissue culture device; the formula of the culture medium for proliferating the cluster buds is as follows: MS +6-BA 1.0 mg.L-1+NAA 0.5mg·L-1。
2. The method according to claim 1, wherein the breaking of dormancy is specifically: soaking seeds in a solution containing 20mg/L of fluazinone and 500mg/L of gibberellin for 24-36h, and then carrying out sand stratification for 88-95 days.
3. The method of claim 1, wherein the callus-inducing medium is formulated as: 1/2MS +1.2-2.0mg/L6-BA +0.8-1.5mg/L2,4-D +0.1-0.5mg/L IBA + agar 7.0-7.5g/L + sucrose 25-30g/L, pH 5.8-6.0.
4. The method of claim 1, wherein the rooting culture medium is formulated as: 1/2MS + NAA0.5mg/L + active carbon 1.0g/L + sucrose 25-30g/L, pH5.8-6.0.
5. The method of claim 1, wherein the conditions for the propagation of the multiple shoots and the rooting culture are as follows: the temperature is 23-25 ℃, the humidity is 75-85%, the illumination is 14-16h/d, and the illumination intensity is 2000-2500 Lx.
6. The method as claimed in claim 1, wherein the volume ratio of the seedling substrate is grass peat: perlite: vermiculite =2:1: 1.
7. The method as claimed in claim 1, wherein the conditions for the cultivation by transplanting into a seedling raising substrate are as follows: the illumination intensity is 1500-.
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