CN102577959A - Tissue culture and rapid propagation method of epimedium wushanense and propagated epimedium wushanense - Google Patents
Tissue culture and rapid propagation method of epimedium wushanense and propagated epimedium wushanense Download PDFInfo
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Abstract
The invention relates to a tissue culture and rapid propagation method of epimedium wushanense and propagated epimedium wushanense. The method comprises the following steps of: A) performing stratification on epimedium wushanense seeds at low temperature of 0-10 DEG C after disinfection; B) performing disinfection treatment on the seeds after stratification, and stripping out seed embryos with sterile tweezers for later use; C) inoculating the seed embryos after disinfection into an induction culture medium for culture, and enabling the seed embryos to induce calli and further differentiate adventitious buds or enabling the seed embryos to directly induce buds; D) transferring the buds obtained in the step C) into a propagation culture medium, and generating tissue culture cluster buds; and E) transferring small plants grown from the cluster buds obtained by induction and differentiation in the step D) into a rooting culture medium, culturing to form white fibrous roots, and further forming complete plants. According to the method disclosed by the invention, the propagation coefficient of the epimedium wushanense can be improved, the propagation period can be shortened, and the method further has important values for protecting wild epimedium wushanense resources and promoting sustainable utilization and industrialization of the epimedium wushanense.
Description
Technical field
The invention belongs to traditional Chinese medicine culture technique field, relate to the Epimedium wushanense mating system, particularly the tissue culture and rapid propagation method of Epimedium wushanense.
Background technology
Epimedium wushanense Epimedium wushanense T.S.Ying is the perennial perennial root draft of a Berberidaceae Epimedium medicinal plant, is contained kind of Pharmacopoeia of the People's Republic of China version in 2010.Be used as medicine with its dry leaf, kidney-replenishing is arranged, strengthening the bones and muscles, the effect of wind-damp dispelling, and have the cardiovascular system of improvement, regulate physiologically actives such as internal secretion and anti-osteoporosis.The Epimedium wushanense medicinal raw material mainly relies on wild resource, and to the continuous increase of its demand, the Epimedium wushanense wild resource is seriously damaged, and can not satisfy the demand of domestic and international market along with both at home and abroad.The modes of reproduction of barrenwort mainly contains at present: through the vegetative propagation and the seminal propagation (sexual propagation) of underground rhizome, there is tangible dormancy phenomenon in the barrenwort seed, and the breeding germination rate is low, and poor growth, adopts division propagation in the production mostly.
People expectation has a kind ofly can breed and cultivate Epimedium wushanense with low-cost, high reproductive rate and/or short method of breeding cycle, satisfying the medicinal demand of increasing Epimedium wushanense, and protects wild medicine resource.
Summary of the invention
The purpose of this invention is to provide and a kind ofly can breed and cultivate Epimedium wushanense with low-cost, high reproductive rate and/or short method of breeding cycle.The inventor finds with the Epimedium wushanense seed to use specific condition of culture for breeding material, can advantageously realize above-mentioned requirement about the one or more aspects of Epimedium wushanense in breeding.The present invention is based on this discovery and be accomplished.
For this reason, first aspect present invention relates to a kind of tissue culture and rapid propagation method of Epimedium wushanense, may further comprise the steps:
A. the explant lamination is handled: with handling in the husky lamination of hiding of 0～10 ℃ of low temperature after the Epimedium wushanense seed disinfection;
B. explant sterilization: with the disinfection of lamination seeds treated, with aseptic nipper embryo is stripped out again, subsequent use;
C. just generation induces: the embryo after will sterilizing is inoculated in the inducing culture to be cultivated, and makes the embryo evoked callus and differentiates indefinite bud, embryo is directly induced sprout;
D. shoot proliferation: gained bud among the step C is changed in the proliferated culture medium over to generation group training clump bud;
E. regeneration plant root induction: the plantlet of inducing the bud clump of the long health of 1～4cm of differentiation to grow up to step D changes in the root media, cultivates forming white fibrous root, and then forms whole plant.
According to the method for first aspect present invention, wherein in the steps A, said sterilization is that following mode is handled: seed is washed with flowing water, in alcohol, soak, again at HgCl
2The middle immersion used aseptic water washing again.In one embodiment; In the steps A; Said sterilization is that following mode is handled: flowing water flushing 15-30min (for example about 30min), soak 10-60s (for example about 30s), again at the HgCl of 0.1-0.5% (for example about 0.5%) in 45-75% (for example about 75%) alcohol
2Middle 2-8min (for example about 6min), aseptic water washing 2-6 time (for example about 5 times) of soaking.
According to the method for first aspect present invention, wherein in the steps A, said low temperature is husky, and to hide that lamination handles be that following mode is handled: make seed and river sand by 1: 2～5 mixed, keep water content 50～70%, hide laminations processing 75～120 days through 3～8 ℃ of low temperature are husky.The seed of handling thus can be broken Seed Dormancy.In one embodiment, said river sand is the river sand that behind high-temperature sterilization 1-5h (for example about 2h), is cooled to room temperature again.In one embodiment, said seed and river sand are by 1: (1-5), and for example about 1: 3 mixed.In one embodiment, said seed and river sand are pressed 1: 3 mixed, keep water content 40-70% (for example about 60%).In one embodiment, said seed and river sand are pressed 1: 3 mixed, keep water content 60%, handle 60-100 days (for example about 90 days) through the husky lamination of hiding of 2-5 ℃ of low temperature.
According to the method for first aspect present invention, wherein among the step B, said sterilization is to disinfect in alcohol earlier, then uses HgCl
2Sterilization.In one embodiment; Among the step B, the seed flush away river sand with after the lamination processing end soaks 10-60s (for example about 30s) in 45-75% (for example about 75%) alcohol; Aseptic water washing 2-6 time (for example about 5 times) is used the HgCl of 0.1-0.5% (for example about 0.5%) again
2Sterilization 2-8min (for example about 6min), aseptic water washing 2-6 time (for example about 5 times).In one embodiment, among the step B, embryo is stripped out gently, behind the aseptic filter paper suck dry moisture, be ready for use on next treatment step with cooled tweezers of calcination and scalpel.
Method according to first aspect present invention; Wherein among the step C; Said inducing culture is to comprise MS+2; The medium of 4-D2mg/L+IBA 2mg/L+NAA 0.5mg/L+ sucrose 30g/L+0.6% agar, or comprise the medium of MS+6-BA 1mg/L+NAA 0.5mg/L+IBA 1mg/L+ sucrose 30g/L+0.6% agar, or comprise the medium of MS+IBA 2mg/L+6-BA 0.5mg/L+ sucrose 30g/L+0.6% agar.In one embodiment; Among the step C; Said inducing culture is to comprise MS+2; The medium of 4-D 2mg/L+IBA 2mg/L+NAA 0.5mg/L+ sucrose 30g/L+0.6% agar, and the medium that comprises MS+6-BA 1mg/L+NAA 0.5mg/L+IBA 1mg/L+ sucrose 30g/L+0.6% agar; Or comprise the medium of MS+IBA 2mg/L+6-BA 0.5mg/L+ sucrose 30g/L+0.6% agar.
Method according to first aspect present invention; Wherein to induce be following the processing the first generation of step C: the embryo after will sterilizing is inoculated in and comprises MS+2; Cultivate in the inducing culture of 4-D 2mg/L+IBA 2mg/L+NAA 0.5mg/L+ sucrose 30g/L+0.6% agar; Produce callus, then in the inducing culture that comprises MS+6-BA 1mg/L+NAA 0.5mg/L+IBA1mg/L+ sucrose 30g/L+0.6% agar, cultivate, differentiate indefinite bud; Perhaps, the embryo after the sterilization is inoculated in the inducing culture that comprises MS+IBA 2mg/L+6-BA 0.5mg/L+ sucrose 30g/L+0.6% agar and cultivates, directly induce and sprout.
According to the method for first aspect present invention, wherein among the step D, said proliferated culture medium is the medium that comprises MS+6-BA1.0mg/L+NAA 0.5mg/L+ sucrose 30g/L+0.6% agar.In one embodiment, the time of in proliferated culture medium, cultivating is 20～60 days, preferred 25～45 days, and for example about 30 days.
According to the method for first aspect present invention, wherein in the step e, said root media is the medium that comprises 1/2MS+0.1NAAmg/L.
According to the method for first aspect present invention, the pH value of the medium that wherein uses in each step is pH5.0～7.0, preferred pH5.5～6.5, more preferably pH5.8.
According to the method for first aspect present invention, add 30g/L sucrose and 0.6% agar in the MS medium that wherein uses in each step independently of one another.
According to the method for first aspect present invention, the medium that wherein uses in each step is through 100-130 ℃ (for example about 121 ℃), and 0.1-0.5Mpa (for example about 0.1Mpa) high-temperature sterilization 15-25min (for example about 25min) handles.
According to the method for first aspect present invention, wherein using the condition of medium culture in each step is 20～30 ℃ of temperature, intensity of illumination 1500～3000lx, illumination 8～15h/d.According to the method for first aspect present invention, wherein using the condition of medium culture in each step is 25 ± 2 ℃ of temperature, intensity of illumination 2000lx, illumination 12h/d.
According to the method for first aspect present invention, it may further comprise the steps:
A. the explant lamination is handled: with the Epimedium wushanense seed, and the flowing water flushing, handle in the husky lamination of hiding of 0～10 ℃ of low temperature the sterilization back;
B. explant sterilization: with the lamination seeds treated, earlier with alcohol-pickled, soak with mercuric chloride, aseptic water washing is clean, with aseptic nipper embryo is stripped out, and is subsequent use again;
C. just generation induces: the embryo after will sterilizing is inoculated in and comprises MS+2; Cultivate in the inducing culture of 4-D 2mg/L+IBA 2mg/L+NAA0.5mg/L+ sucrose 30g/L+0.6% agar; Produce callus; Then in the inducing culture that comprises MS+6-BA 1mg/L+NAA 0.5mg/L+IBA 1mg/L+ sucrose 30g/L+0.6% agar, cultivate, differentiate indefinite bud; Perhaps, the embryo after the sterilization is inoculated in the inducing culture that comprises MS+IBA 2mg/L+6-BA 0.5mg/L+ sucrose 30g/L+0.6% agar and cultivates, directly induce and sprout;
D. shoot proliferation: gained bud among the step C is changed in the proliferated culture medium that comprises MS+6-BA 1.0mg/L+NAA 0.5mg/L+ sucrose 30g/L+0.6% agar over to generation group training clump bud;
E. regeneration plant root induction: the plantlet that will induce the bud clump of the long health of 1～4cm of differentiation to grow up to changes in the root media, forms white fibrous root after 1 month, and then forms whole plant.
According to the method for first aspect present invention, wherein in each step, when cultivating, the time of its cultivation and the degree of cultivation can be confirmed according to those skilled in the art's the technical experience and the actual conditions of operation, and not do special qualification.For example, in step C of the present invention, embryo is inoculated in and comprises MS+2, cultivates about 1 month in the inducing culture of 4-D 2mg/L+IBA 2mg/L+NAA0.5mg/L+ sucrose 30g/L+0.6% agar, will produce callus; Then cultivation can differentiate indefinite bud in about 1 month in the medium that comprises MS+6-BA 1mg/L+NAA 0.5mg/L+IBA 1mg/L+ sucrose 30g/L+0.6% agar.
Second aspect present invention relates to a kind of Epimedium wushanense strain plants, and it is obtained by the tissue culture and rapid propagation method breeding.
Strain according to the Epimedium wushanense of second aspect present invention is planted, and it is obtained by the said tissue culture and rapid propagation method breeding of the arbitrary embodiment of first aspect present invention.In one embodiment, to plant be to be obtained through the tissue culture and rapid propagation method breeding by the processing procedure of the said steps A of the arbitrary embodiment of first aspect present invention, B, C, D and E in the strain of said Epimedium wushanense.
Third aspect present invention relates to a kind of bud of Epimedium wushanense, and it is obtained by the tissue culture and rapid propagation method breeding.
According to the bud of the Epimedium wushanense of third aspect present invention, it is obtained by the said tissue culture and rapid propagation method breeding of the arbitrary embodiment of first aspect present invention.In one embodiment, to plant be to be obtained through the tissue culture and rapid propagation method breeding by the processing procedure of the said steps A of the arbitrary embodiment of first aspect present invention, B, C and D in the strain of said Epimedium wushanense.
Fourth aspect present invention relates to a kind of callus of Epimedium wushanense, and it is obtained by the tissue culture and rapid propagation method breeding.
According to the callus of the Epimedium wushanense of fourth aspect present invention, it is obtained by the said tissue culture and rapid propagation method breeding of the arbitrary embodiment of first aspect present invention.In one embodiment, to plant be to be obtained through the tissue culture and rapid propagation method breeding by the processing procedure of the said steps A of the arbitrary embodiment of first aspect present invention, B and C in the strain of said Epimedium wushanense.
Arbitrary technical characterictic in the arbitrary embodiment of first aspect present invention is equally applicable to arbitrary other embodiment, as long as this being suitable for contradiction can not occur.Describe in further detail helping in the face of the inventive method down understanding of the present invention.
In the present invention, MS or MS medium are Murashige and Skoog cultivated design for tobacco cell in 1962 (and therefore abbreviating the MS medium as); Being characterized in that mineral salt and ion concentration are higher, is more stable ionic equilibrium solution, and its nitrate content is high; The quantity and the ratio of its nutrient are suitable; Can satisfy the nutrition and the physiological requirements of plant cell, thereby the scope of application is wider, most Plant Tissue Breeding are bred with its minimal medium as medium fast.Medium that the present invention uses or the additive that wherein adds are that those skilled in the art can obtain according to existing knowledge, perhaps can directly buy from market.For example the MS medium can directly be buied with commodity basis.In the present invention; As not explanation in addition; Mention that the MS medium is the MS medium that has added 6g/L agar and 30g/L sucrose, if, also be meant the MS medium that has added 6g/L agar and 30g/L sucrose with the medium that MS+6g/L agar+similar fashion such as 30g/L sucrose are mentioned.
In the present invention, 2,4-D refers to 2,4 dichlorophenoxyacetic acid
In the present invention, 6-BA refers to 6-benzyladenine
In the present invention, IBA refers to indolebutyric acid
In the present invention, NAA refers to NAA
The invention provides a kind of tissue culture and rapid propagation method of Epimedium wushanense.The inventive method can shorten incubation time, and the expanding propagation coefficient guarantees the seedling quality and quantity, finally solves the production and the supply problem of seedling, and is significant to the sustainable use that realizes Epimedium wushanense.
The object of the present invention is to provide a kind of tissue culture and rapid propagation method of Epimedium wushanense, make the embryo of Epimedium wushanense mature seed after on the MS medium 1 month, induce callus, differentiation is sprouted after two months, and taking root after 4 months grows up to whole plant.
The tissue culture and rapid propagation method of Epimedium wushanense provided by the present invention comprises:
A. explant is selected and handled: with the Epimedium wushanense seed that collects, remove impurity and not full particle, flowing water flushing 30min soaks 30s, again at 0.1%HgCl in 75% alcohol
2In soak 6min, with the mixed of the river sand that is cooled to room temperature through high-temperature sterilization 2h, keep water content 60% behind the aseptic water washing 5 times by 1: 3, handle through 5 ℃ of low temperature laminations and broke seed dormancy in 90 days.
B. explant sterilization: the seed flush away river sand with after the lamination processing end, in 75% alcohol, soak 30s, aseptic water washing 5 times is used 0.1%HgCl again
2Sterilization 6min, aseptic water washing 5 times strips out embryo with cooled tweezers of calcination and scalpel gently, and is behind the aseptic filter paper suck dry moisture, subsequent use.
C. just generation induces: will handle the gained embryo through step B and insert just for inducing culture, condition of culture is the additional respectively 6g/L agar of MS, 30g/L sucrose, pH5.8, temperature (25 ± 2) ℃, light intensity 2000lx, illumination 12h/d.At MS+2, cultivate on the medium of 4-D 2mg/L+IBA 2mg/L+NAA 0.5mg/L that callus induction rate reaches 63.3% after 1 month; The callus differentiation rate can reach 73.8% after continuing to cultivate January on the medium of MS+6-BA 1mg/L+NAA 0.5mg/L+IBA 1mg/L.Perhaps, will through step B handle the gained embryo be linked into cultivate for 1～February on the MS+IBA 2mg/L+6-BA0.5mg/L medium after bud induction rate can reach 77.8%.
D. shoot proliferation: divide bottle with the indefinite bud of callus differentiation among the step C or the bud clump of directly inducing, be transferred in the shoot proliferation medium, after 1 month on the proliferated culture medium of MS+6-BA 1.0mg/L+NAA 0.5mg/L the growth coefficient of bud can reach 2.17.
E. regeneration plant root induction: the plantlet that will induce the bud clump of the long health of 1～4cm of differentiation to grow up to changes among the root media 1/2MS+0.1NAAmg/L, forms white fibrous root after 1 month, and the root system number is generally at 2～5.
The application also asks for protection Epimedium wushanense callus, bud and the plant according to the tissue culture and rapid propagation method acquisition of above-mentioned Epimedium wushanense; It is characterized in that: selecting the embryo of Epimedium wushanense mature seed is explant; After cultivating 1 month on the MS medium, induce callus, differentiated indefinite bud after 1 month, also can directly induce and sprout without the callus approach; Be transferred to then on the root media, root induction forms whole plant.
The application also asks for protection the Epimedium wushanense plant that a kind of tissue culture and rapid propagation method of Epimedium wushanense obtains; It is characterized in that: induce white or green fine and close, solid and eugonic callus differentiation and bud formation more earlier; Or directly induce and sprout, root induction forms whole plant then.
The invention has the advantages that: the present invention induces embryo to produce callus and differentiates indefinite bud; The process of callus differentiation indefinite bud need not changed culture medium prescription, and differentiation rate reaches 73.8%; And embryo can directly induce indefinite bud, has shortened the process of inducing of indefinite bud greatly.The present invention can improve the reproduction coefficient of Epimedium wushanense, shorten the breeding cycle, and method is simple, and cost is lower, is suitable for production application.
Description of drawings
Fig. 1 has shown the photo of the kind embryo explants after the mature seed rip cutting, and scale is represented 0.5mm among the figure.
Fig. 2 has shown and has cultivated the photo that explant edge after 10 days grows the warty tissue of green, white that scale is represented 1mm among the figure.
Fig. 3 has shown and has cultivated white smooth that explant after 1 month forms, the photo of the callus of densification, and scale is represented 1mm among the figure.
Fig. 4 has shown and has cultivated the photo that callus after 2 months differentiates indefinite bud that scale is represented 5mm among the figure.
Fig. 5 has shown the photo of the bud of growing thickly that bud propagation back forms.
Fig. 6 has shown the photo of the formation whole plant of taking root.
Embodiment
Embodiment 1: the induction experiment of Epimedium wushanense seed treatment and embryo
One, explant is selected and handled: with the Epimedium wushanense seed that collects, remove impurity and not full particle, flowing water flushing 30min soaks 30s, again at 0.1%HgCl in 75% alcohol
2In soak 6min, aseptic water washing 5 times, even by 1: 3 mixed behind the aseptic filter paper suck dry moisture with the river sand that behind 121 ℃, 0.1Mpa high-temperature sterilization 2h, is cooled to room temperature, keep water content 60%, broke seed dormancy in 90 days through 5 ℃ of low temperature laminations processing.
Two, explant sterilization: the seed flush away river sand with after the lamination processing end, in 75% alcohol, soak 30s, aseptic water washing 5 times is used 0.1%HgCl again
2Sterilization 6min, aseptic water washing 5～6 times behind the aseptic filter paper suck dry moisture, strips out embryo with cooled tweezers of calcination and scalpel gently, and is behind the aseptic filter paper suck dry moisture, subsequent use.
Three, just in generation, induce
1. callus induces and breaks up
Step 2 gained embryo is inoculated in the MS medium that adds 6g/L agar and 30g/L sucrose; Also randomly added 2 in this medium, one of 4-D, 6-BA, NAA, IBA or any two kinds or the more kinds of combination and the additive of their variable concentrations.Each medium pH 5.8, and through 121 ℃, 0.1Mpa high-temperature sterilization 25min processing.Cultivation temperature (25 ± 2) ℃, intensity of illumination 2000lx, illumination 12h/d.The inventor finds that the best medium of inducing the Epimedium wushanense callus to produce is MS+2,4-D 2mg/L+IBA 2mg/L+NAA 0.5mg/L, and inductivity reaches 63.3%; The optimum medium of callus differentiation is MS+6-BA 1mg/L+NAA 0.5mg/L+IBA 1mg/L, and differentiation rate reaches 73.8%.The medium of other composition in callus induction rate and callus differentiation rate all below 40%.
2. bud induces
Step 2 gained embryo is inoculated in variable concentrations 2,4-D (1,2,4,6mg/L), IBA (0.5,1,2,4mg/L) respectively with the MS medium of 6-BA (0.5,1mg/L) independent assortment on.The inventor finds that the inductivity of bud is the highest on MS+IBA2mg/L+6-BA 0.5mg/L medium, is 77.8%, and on the medium of other combination the inductivity of bud all below 45%.
Four. shoot proliferation
The step 3 gained bud of growing thickly is grown to the bud clump that is divided into 2～3 buds about 2cm again and is inoculated into the propagation of carrying out bud in the medium that 6-BA (0.1,1,10mg/L) that MS adds variable concentrations and NAA (0.5mg/L) make up; Not add HORMONE TREATMENT as contrast, the result shows that MS+6-BA 1.0mg/L+NAA 0.5mg/L is the proliferated culture medium of bud; Both can make blastogenesis long healthy and strong; Growth rate is suitable, and keeps than the high proliferation multiple, and growth coefficient reaches 2.17.The medium of other combination aspect growth coefficient far below the above result who comprises the proliferated culture medium gained of MS+6-BA 1.0mg/L+NAA 0.5mg/L.
Five. the regeneration plant root induction
The no offspring that the bud clump of the health that the 1～4cm that induces differentiation is long grows up to changes over to and carries out culture of rootage among the root media 1/2MS+0.1NAAmg/L, and the base portion of bud grows the fibrous root of white gradually after 1 month, and radical is generally at 2～5.
Above result shows; The optimum medium of Epimedium wushanense embryo callus induction and differentiation is respectively MS+2; 4-D 2mg/L+IBA 2mg/L+NAA 0.5mg/L, MS+6-BA 1mg/L+NAA 0.5mg/L+IBA1mg/L, inductivity and differentiation rate reach 63.3%, 73.8% respectively; The optimum medium that bud is induced is MS+IBA2mg/L+6-BA 0.5mg/L, and inductivity reaches 77.8%; The bud proliferated culture medium is MS+6-BA 1.0mg/L+NAA0.5mg/L, and growth coefficient is 2.17; Root media is 1/2MS+0.1NAAmg/L.
Embodiment 2: tissue culture and rapid propagation method is cultivated Epimedium wushanense
A. explant is selected and handled: with the Epimedium wushanense seed that collects, remove impurity and not full particle, flowing water flushing 30min soaks 30s, again at 0.1%HgCl in 75% alcohol
2In soak 6min, aseptic water washing 5 times, behind the aseptic filter paper suck dry moisture with through 121 ℃, it is even by 1: 3 mixed that 0.1Mpa high-temperature sterilization 2h is cooled to the river sand of room temperature, keeps water content 60%, handles through 5 ℃ of low temperature laminations and broke seed dormancy in 90 days.
B. explant sterilization: the seed flush away river sand with after the lamination processing end, in 75% alcohol, soak 30s, aseptic water washing 5 times is used 0.1%HgCl again
2Sterilization 6min, aseptic water washing 5～6 times behind the aseptic filter paper suck dry moisture, strips out embryo with cooled tweezers of calcination and scalpel gently, and is behind the aseptic filter paper suck dry moisture, subsequent use.
C. just in generation, induce: will handle the gained embryo through step B and insert the initial culture base, and MS+2 after 1 month, callus induction rate reaches 63.3% on the 4-D2mg/L+IBA 2mg/L+NAA 0.5mg/L medium; The callus differentiation rate reaches 73.8% on the MS+6-BA1mg/L+NAA 0.5mg/L+IBA 1mg/L medium; Bud induction rate reaches 77.8% on the MS+IBA2mg/L+6-BA 0.5mg/L medium.
D. shoot proliferation: divide bottle with the indefinite bud of callus differentiation among the step C and the bud clump of directly inducing, be transferred in the shoot proliferation medium, growth coefficient is 2.17 after 1 month.
E. regeneration plant root induction: the plantlet that will induce the bud clump of the long health of 1～4cm of differentiation to grow up to changes in the root media, forms white fibrous root after 1 month, and the root system number is generally at 2～5.
Claims (10)
1. the tissue culture and rapid propagation method of Epimedium wushanense may further comprise the steps:
A. the explant lamination is handled: with handling in the husky lamination of hiding of 0～10 ℃ of low temperature after the Epimedium wushanense seed disinfection;
B. explant sterilization: with the disinfection of lamination seeds treated, with aseptic nipper embryo is stripped out again, subsequent use;
C. just generation induces: the embryo after will sterilizing is inoculated in the inducing culture to be cultivated, and makes the embryo evoked callus and differentiates indefinite bud, embryo is directly induced sprout;
D. shoot proliferation: gained bud among the step C is changed in the proliferated culture medium over to generation group training clump bud;
E. regeneration plant root induction: the plantlet of inducing the bud clump of the long health of 1～4cm of differentiation to grow up to step D changes in the root media, cultivates forming white fibrous root, and then forms whole plant.
2. according to the process of claim 1 wherein that in the steps A, said sterilization is that following mode is handled: flowing water flushing 15-30min, soak 30s, again at 0.1%-0.5%HgCl in 45-75% alcohol
2Middle 2-8min, aseptic water washing 2-6 time of soaking.
3. according to the process of claim 1 wherein in the steps A, said low temperature is husky, and to hide that lamination handles be that following mode is handled: make seed and river sand by 1: 2～5 mixed, keep water content 50～70%, hide laminations processing 75～120 days through 3～8 ℃ of low temperature are husky.
4. according to the process of claim 1 wherein that among the step B, said sterilization is to disinfect in alcohol earlier, then uses HgCl
2Sterilization.
5. according to the method for claim 1, it is characterized in that:
Among the step C; Said inducing culture is to comprise MS+2; The medium of 4-D 2mg/L+IBA 2mg/L+NAA 0.5mg/L+ sucrose 30g/L+0.6% agar; Or comprise the medium of MS+6-BA 1mg/L+NAA 0.5mg/L+IBA 1mg/L+ sucrose 30g/L+0.6% agar, or comprise the medium of MS+IBA 2mg/L+6-BA 0.5mg/L+ sucrose 30g/L+0.6% agar;
Among the step D, said proliferated culture medium is the medium that comprises MS+6-BA 1.0mg/L+NAA 0.5mg/L+ sucrose 30g/L+0.6% agar; And/or
In the step e, said root media is the medium that comprises 1/2MS+0.1NAAmg/L.
6. according to the method for claim 1; Wherein among the step C; Be following the processing for inducing just: the embryo after will sterilizing is inoculated in and comprises MS+2, cultivates in the inducing culture of 4-D 2mg/L+IBA 2mg/L+NAA 0.5mg/L+ sucrose 30g/L+0.6% agar, produces callus; Then in the inducing culture that comprises MS+6-BA 1mg/L+NAA 0.5mg/L+IBA1mg/L+ sucrose 30g/L+0.6% agar, cultivate, differentiate indefinite bud; Perhaps, the embryo after the sterilization is inoculated in the inducing culture that comprises MS+IBA 2mg/L+6-BA 0.5mg/L+ sucrose 30g/L+0.6% agar and cultivates, directly induce and sprout.
7. according to each described method of claim 1 to 6, it may further comprise the steps:
A. the explant lamination is handled: with the Epimedium wushanense seed, and the flowing water flushing, handle in the husky lamination of hiding of 0～10 ℃ of low temperature the sterilization back;
B. explant sterilization: with the lamination seeds treated, earlier with alcohol-pickled, soak with mercuric chloride, aseptic water washing is clean, with aseptic nipper embryo is stripped out, and is subsequent use again;
C. just generation induces: the embryo after will sterilizing is inoculated in and comprises MS+2; Cultivate in the inducing culture of 4-D 2mg/L+IBA 2mg/L+NAA0.5mg/L+ sucrose 30g/L+0.6% agar; Produce callus; Then in the inducing culture that comprises MS+6-BA 1mg/L+NAA 0.5mg/L+IBA 1mg/L+ sucrose 30g/L+0.6% agar, cultivate, differentiate indefinite bud; Perhaps, the embryo after the sterilization is inoculated in the inducing culture that comprises MS+IBA 2mg/L+6-BA 0.5mg/L+ sucrose 30g/L+0.6% agar and cultivates, directly induce and sprout;
D. shoot proliferation: gained bud among the step C is changed in the proliferated culture medium that comprises MS+6-BA 1.0mg/L+NAA 0.5mg/L+ sucrose 30g/L+0.6% agar over to generation group training clump bud;
E. regeneration plant root induction: the plantlet that will induce the bud clump of the long health of 1～4cm of differentiation to grow up to changes in the root media, forms white fibrous root after 1 month, and then forms whole plant.
8. an Epimedium wushanense strain is planted, and it is obtained by each described method breeding of claim 1 to 7.
9. the bud of an Epimedium wushanense, it is obtained by each described method breeding of claim 1 to 7.
10. the callus of an Epimedium wushanense, it is obtained by each described method breeding of claim 1 to 7.
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