CN104770296A - Method for rapidly propagating through adoption of aristolochia fordiana leaves - Google Patents
Method for rapidly propagating through adoption of aristolochia fordiana leaves Download PDFInfo
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- CN104770296A CN104770296A CN201510157533.5A CN201510157533A CN104770296A CN 104770296 A CN104770296 A CN 104770296A CN 201510157533 A CN201510157533 A CN 201510157533A CN 104770296 A CN104770296 A CN 104770296A
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention provides a method for rapidly propagating through adoption of aristolochia fordiana leaves. The method comprises the following steps: preparing an aristolochia fordiana sterile explant; performing callus inducing, namely removing veins of the sterile explant, dicing, inoculating an induction culture medium with the diced sterile explants, performing induction culture to obtain a callus; performing callus differentiating, namely inoculating a differential culture medium with the callus to obtain multiple shoots; hardening seedlings, namely inoculating a seedling culture medium with the multiple shoots, performing the seedling culture to obtain a test-tube plantlet; rooting, namely transplanting the test-tube plantlet into a rooting culture medium to obtain a test-tube plantlet with the root; and hardening the plantlet and transplanting, namely hardening the plantlet to obtain a complete plant, and then transplanting into a matrix to culture. The leaves are used as the explant, the material taking is convenient; a stem is saved so that the whole strain collecting is avoided, an aristolochia fordiana resource is saved, and the demands of the market to the aristolochia fordiana can be satisfied.
Description
Technical field
The present invention relates to field of plant tissue culture technique.More particularly, the present invention relates to and a kind ofly apply the method that ford dutchmanspipe root or herb blade carries out Fast-propagation.
Background technology
Ford dutchmanspipe root or herb is aristolochiaceae plant, herbaceous species, and root is cylindrical, elongated, and stem, without hair, often has vertical rib after dry.Have another name called five brave Tongcheng, centering grass, natural grass, wither, blood rattan secretly disappears, Wilson Begonia Rhizome.Nature and flavor are bitter, pungent, warm; Mild toxicity, has dispeling the wind, eases pain, subsides a swelling, detoxifies.Cure mainly treating rheumatic ostealgia, epigastric pain, stomachache, sphagitis, traumatic injury, child convulsion, venomous snake bite.Originate in Guangxi (Luchuan, Cangwu, Zhaoping), Guangdong (Boluo, Deqing, spring etc.), Jiangxi (Dexing), Zhejiang and Fujian, wild ford dutchmanspipe root or herb is often born in the underbrush of mountain valley with in the stone gap of mountain region, habitat requires comparatively strict, therefore Regional Distribution scope is quite limited, in recent years along with the continuous increase of market demand, increase gradually the collection capacity of wild resource, wild ford dutchmanspipe root or herb resource is on the verge of exhaustion.Utilize tissue culture rapid propagating technology; its reproduction coefficient can be increased fast and effectively; tissue cultures report at present about ford dutchmanspipe root or herb is less; not yet there is the relevant report adopting blade to cultivate as explant; therefore the present invention adopts blade to be explant, not only draws materials conveniently, and has saved stem section and avoid whole strain collection; be conducive to the protection of ford dutchmanspipe root or herb resource, also can meet the demand of market to ford dutchmanspipe root or herb medicinal material.
Summary of the invention
As the result of various extensive and careful research and experiment, the present inventor has been found that, common MS medium and common Plant Tissue Breeding condition can not meet the needs of ford dutchmanspipe root or herb Plant Tissue Breeding at all, therefore, the present inventor with the addition of different plant hormones or nutrient component to meet the nutritional need of ford dutchmanspipe root or herb blade Plant Tissue Breeding in common MS medium, and has carried out improving the requirement with satisfied application ford dutchmanspipe root or herb blade Fast-propagation targetedly to Plant Tissue Breeding condition.
An object of the present invention is to solve at least the problems referred to above, and the advantage will illustrated at least is below provided.
A further object of the invention is to provide a kind ofly applies the method that ford dutchmanspipe root or herb blade carries out Fast-propagation, and it can realize the Fast-propagation of ford dutchmanspipe root or herb, and can ensure quality and the medical value of ford dutchmanspipe root or herb.
A further object of the invention is for ford dutchmanspipe root or herb blade carries out culture medium prescription that Fast-propagation provides each stage preferably and condition of culture, improve the efficiency of ford dutchmanspipe root or herb tissue cultures, ensure the survival rate of the test-tube plantlet of tissue cultures, to obtain the complete test-tube plantlet of maximum ford dutchmanspipe root or herb for target within the shortest time.
In order to realize according to these objects of the present invention and other advantage, provide and a kind ofly applying the method that ford dutchmanspipe root or herb blade carries out Fast-propagation, comprising the following steps:
Step one, prepare ford dutchmanspipe root or herb aseptic explant;
Step 2, callus induction: aseptic explant is removed vein and is inoculated into after stripping and slicing process on inducing culture and carry out Fiber differentiation, obtain callus, wherein, MS+6-BA 1.0-1.5mg/L+2,4-D 2.0-4.0mg/L+NAA 0.5-1.5mg/L is contained in described inducing culture;
Step 3, Calli Differentiation: described callus is inoculated in differential medium and obtains Multiple Buds, wherein, the TDZ containing MS+1.0-2.0mg/L in described differential medium;
Step 4, strong sprout: be inoculated into by Multiple Buds in strong seedling culture base and carry out strong seedling culture, obtain test-tube plantlet, wherein, the powdered activated carbon containing MS+6-BA 0.1-0.5mg/L+IBA 0.1-0.5mg/L and 0.5-1.5g/L in described strong seedling culture base;
Step 5, to take root: test-tube plantlet is implanted into the test-tube plantlet obtaining band root in root media, wherein, the powdered activated carbon containing 1/2MS+IBA 0.1-0.5mg/L+NAA 0.5-1.0mg/L and 0.1-0.5g/L in root media; Step 6, hardening and transplanting: obtain whole plant after hardening, be then transplanted in matrix and cultivate.
In such scheme, owing to adopting the aseptic explant of blade as Plant Tissue Breeding of ford dutchmanspipe root or herb, blade, compared with stem section, has been saved stem section and has been avoided whole strain collection, be conducive to the protection of ford dutchmanspipe root or herb resource, also can meet the demand of market to ford dutchmanspipe root or herb medicinal material;
The interpolation concentration that powdered activated carbon is suitable is provided in step 5 of the present invention, not only provide a dark surrounds to rooting of vitro seedling, but also adsorbable a certain amount of harmful substance, but, can't benign species be adsorbed, the concentration of the nutriment of rooting of vitro seedling can not be reduced.
In such scheme, for the different phase of ford dutchmanspipe root or herb blade Plant Tissue Breeding provides different medium, make the aseptic explant of ford dutchmanspipe root or herb under the continuous action of various suitable medium, at utmost improve the planting percent of explant, reach the object of quick, effective and undamaged breeding ford dutchmanspipe root or herb.
Preferably, wherein, MS+6-BA 1.0mg/L+2,4-D 3.0mg/L+NAA 1.0mg/L is contained in described inducing culture; TDZ containing MS+2.0mg/L in described differential medium; Powdered activated carbon containing MS+6-BA0.1mg/L+IBA0.5mg/L and 1.5g/L in described strong seedling culture base; Powdered activated carbon containing 1/2MS+IBA 0.5mg/L+NAA 0.5mg/L and 0.5g/L in described root media.
Preferably, wherein, the ford dutchmanspipe root or herb leaf extract also containing 5-10mL/L in described inducing culture; Ford dutchmanspipe root or herb leaf extract also containing 15-20mL/L in described strong seedling culture base.
Preferably, wherein, described ford dutchmanspipe root or herb leaf extract is juice ford dutchmanspipe root or herb mature leaf being blended acquisition.
Preferably, wherein, matrix described in described step 6 is that the humus soil of 5 ~ 3: 3 ~ 2: 1, dry liver moss and perlite form by volume ratio.
In such scheme, first dry liver moss is in use manually ground into 40-60 object particle, after humus soil, dry liver moss and perlite being mixed in proportion afterwards, place 5-7d at normal temperatures, spray a water every day and carry out a convention stir simultaneously, injection flow rate is 50-100ml/kg matrix, become the matrix of cultivating complete test-tube plantlet afterwards, wherein, matrix of the present invention can provide necessary nutrition for test-tube plantlet, make ford dutchmanspipe root or herb test-tube plantlet growth good, survival rate is high.
Wild ford dutchmanspipe root or herb is often born in the underbrush of mountain valley with in the stone gap of mountain region, habitat requires comparatively strict, liver moss has more in the growing environment of present ford dutchmanspipe root or herb, for the growth of ford dutchmanspipe root or herb and climbing provide certain nutrition, and improve the soil environment of its growth, add certain dry liver moss in matrix provided by the present invention and do some process in early stage before use, make the better stripping of the nutrient component in dry liver moss in humus soil, growth for the test-tube plantlet of ford dutchmanspipe root or herb provides closer to natural growing environment, make the test-tube plantlet growth of ford dutchmanspipe root or herb vigorous, improve survival rate, and better keep the trophic component of wild ford dutchmanspipe root or herb, keep its medical value.
Preferably, wherein, matrix described in described step 6 is that the humus soil of 4: 2: 1, dry liver moss and perlite form by volume ratio.
Preferably, wherein, the young leaflet tablet applying ford dutchmanspipe root or herb stem apex top in described step one prepares described ford dutchmanspipe root or herb aseptic explant.
Preferably, wherein, the agar all containing 4.5g/L in described inducing culture, described differential medium, described strong seedling culture base and described root media and 25g/L sucrose, and pH value is 5.8.
Preferably, wherein, described step 4 is further comprising the steps of: be inoculated in subculture medium by described test-tube plantlet and carry out squamous subculture, wherein, agar containing MS+KT 0.1-0.5mg/L+6-BA 0.2-1.0mg/L+NAA 0.2-1.0mg/L, 4.5g/L in described subculture medium and 25g/L sucrose, condition of culture is: temperature 24-26 DEG C, intensity of illumination 2000lux, and the pH value of subculture medium is 5.8.
In such scheme, test-tube plantlet is inoculated in subculture medium and carries out squamous subculture, at the needs of the simultaneous adaptation large-scale production of guarantee ford dutchmanspipe root or herb quality.
Preferably, wherein, the sterilization of explant selection and explant is also comprised in described step one, wherein, the detailed process of the selection of explant is: get the young leaflet tablet near ford dutchmanspipe root or herb stem apex top, soak 30min, then under flowing water, scrub blade surface and the back side with capillary brush gently in running water, then spread out on blotting paper, natural air drying; The detailed process of the sterilization of explant is: by the 1% liquid detergent aqueous solution soaking 5-10min of the blade after natural air drying, wire tap water 10-15min, with through autoclaved sterile water rinse once on transfer room super-clean bench, then the alcohol-pickled 30-50s of 75% is used, be 0.1% mercuric chloride sterilization 4-6min by the 150ml volume fraction that with the addition of 2-3 and drip Tween-20 again, aseptic water washing 3-5 time, finally to spread out on sterilized filter paper to remove surface moisture, obtains aseptic explant;
In described step 2, the detailed process of callus induction is: aseptic explant is removed nerve structure, be cut into the square leaf block of 1.0-2.0cm, edge blade scratches, then be inoculated in described inducing culture, be 24-26 DEG C in cultivation temperature, under light culture condition, cultivate 7d, then at intensity of illumination 1500lux, light application time is cultivate 21d under the condition of 10-12h/d, obtains the callus of peak green;
In described step 3, the detailed process of Calli Differentiation is: by described callus, be cut into the square leaf block of 2.0cm, be inoculated in described differential medium, be 24-26 DEG C in cultivation temperature, intensity of illumination 1500lux, light application time is cultivate 21d under the condition of 10-12h/d, obtains Multiple Buds;
In described step 4, strong seedling culture detailed process is: described Multiple Buds is placed in described strong seedling culture base, at cultivation temperature 24-26 DEG C, and intensity of illumination 2000lux, light application time is cultivate 21d under the condition of 10-12h/d to obtain healthy and strong test-tube plantlet;
In described step 5, the detailed process of culture of rootage is: described healthy and strong test-tube plantlet is placed in described root media, cultivation temperature 24-26 DEG C, intensity of illumination 2000lux, and light application time is cultivate the complete test-tube plantlet that 21d obtains being with root under the condition of 10-12h/d;
The detailed process of hardening and transplanting in described step 6 is: after the complete test-tube plantlet that the band 2-4cm root obtained in described step 5 is long, the complete test-tube plantlet of having taken root is shifted out culturing room together with blake bottle, proceed to booth, under temperature is 25-27 °, place 7d, then bottle cap is opened, a small amount of running water is added in bottle, hardening 3-4d, take out, clean root medium, be transplanted in matrix, 7-10d plastic film moisturizing after transplanting, cover one deck newspaper more above, intensity of illumination is kept to be that 60-70% is the most suitable with shading rate, air humidity 85-90%, every 3d sprays 1 time.
The present invention at least comprises following beneficial effect:
The present invention adopts the aseptic explant of blade as Plant Tissue Breeding of ford dutchmanspipe root or herb, and blade, compared with stem section, has been saved stem section and avoided whole strain collection, be conducive to the protection of ford dutchmanspipe root or herb resource, also can meet the demand of market to ford dutchmanspipe root or herb medicinal material;
The present invention provides different medium for the different phase of ford dutchmanspipe root or herb blade Plant Tissue Breeding, make the aseptic explant of ford dutchmanspipe root or herb under the continuous action of various suitable medium, at utmost improve the planting percent of explant, reach the object of quick, effective and undamaged breeding ford dutchmanspipe root or herb;
First dry liver moss is in use manually ground into 40-60 object particle, after afterwards humus soil, dry liver moss and perlite being mixed in proportion, place 5-7d at normal temperatures, spray a water every day and carry out a convention stir simultaneously, injection flow rate is 50-100ml/kg matrix, becomes the matrix of cultivating complete test-tube plantlet afterwards, wherein, matrix of the present invention can provide necessary nutrition for test-tube plantlet, and make ford dutchmanspipe root or herb test-tube plantlet growth good, survival rate is high;
Wild ford dutchmanspipe root or herb is often born in the underbrush of mountain valley with in the stone gap of mountain region, habitat requires comparatively strict, liver moss has more in the growing environment of present ford dutchmanspipe root or herb, for the growth of ford dutchmanspipe root or herb and climbing provide certain nutrition, and improve the soil environment of its growth, add certain dry liver moss in matrix provided by the present invention and do some process in early stage before use, make the better stripping of the nutrient component in dry liver moss in humus soil, growth for the test-tube plantlet of ford dutchmanspipe root or herb provides closer to natural growing environment, make the test-tube plantlet growth of ford dutchmanspipe root or herb vigorous, improve survival rate, and better keep the trophic component of wild ford dutchmanspipe root or herb, keep its medical value,
The method by the restriction in season, thus is set up a set of being easy to and is grasped, easy saving, regenerating system rapidly and efficiently.
Part is embodied by explanation below by other advantage of the present invention, target and feature, part also will by research and practice of the present invention by those skilled in the art is understood.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, can implement according to this with reference to specification word to make those skilled in the art.
< example 1>
Apply the method that ford dutchmanspipe root or herb blade carries out Fast-propagation, comprise the following steps:
Step one, prepare ford dutchmanspipe root or herb aseptic explant, the sterilization of explant selection and explant, wherein, the detailed process of the selection of explant is: get the young leaflet tablet near ford dutchmanspipe root or herb stem apex top, 30min is soaked in running water, then under flowing water, scrub blade surface and the back side with capillary brush gently, then spread out on blotting paper, natural air drying; The detailed process of the sterilization of explant is: by the 1% liquid detergent aqueous solution soaking 5min of the blade after natural air drying, wire tap water 10min, with through autoclaved sterile water rinse once on transfer room super-clean bench, then the alcohol-pickled 30s of 75% is used, again with the 150m1 volume fraction that with the addition of 2 Tween-20s be 0.1% mercuric chloride sterilization 4min, aseptic water washing 3 times, finally to spread out on sterilized filter paper to remove surface moisture, obtains aseptic explant;
Step 2, callus induction: aseptic explant is removed vein and is inoculated into after stripping and slicing process on inducing culture and carry out Fiber differentiation, obtain callus, wherein, MS+6-BA 1.0mg/L+2,4-D 2.0mg/L+NAA 0.5mg/L is contained in described inducing culture; The detailed process of callus induction is: aseptic explant is removed nerve structure, be cut into the square leaf block of 1.0-2.0cm, edge blade scratches, then be inoculated in described inducing culture, be 24-26 DEG C in cultivation temperature, under light culture condition, cultivate 7d, then at intensity of illumination 1500lux, light application time is cultivate 21d under the condition of 10-12h/d, obtains the callus of peak green;
Step 3, Calli Differentiation: described callus is inoculated in differential medium and obtains Multiple Buds, wherein, the TDZ containing MS+1.0mg/L in described differential medium; The detailed process of Calli Differentiation is: by described callus, is cut into the square leaf block of 2.0cm, is inoculated in described differential medium, be 24-26 DEG C in cultivation temperature, intensity of illumination 1500lux, light application time is cultivate 21d under the condition of 10-12h/d, obtains Multiple Buds;
Step 4, strong sprout: be inoculated into by Multiple Buds in strong seedling culture base and carry out strong seedling culture, obtain test-tube plantlet, wherein, the powdered activated carbon containing MS+6-BA 0.1mg/L+IBA 0.1mg/L and 0.5g/L in described strong seedling culture base; Strong seedling culture detailed process is: described Multiple Buds is placed in described strong seedling culture base, at cultivation temperature 24-26 DEG C, and intensity of illumination 2000lux, light application time is cultivate 21d under the condition of 10-12h/d to obtain healthy and strong test-tube plantlet;
Step 5, to take root: test-tube plantlet is implanted into the test-tube plantlet obtaining band root in root media, wherein, the powdered activated carbon containing 1/2MS+IBA 0.1mg/L+NAA 0.5mg/L and 0.1g/L in root media; The detailed process of culture of rootage is: described healthy and strong test-tube plantlet is placed in described root media, cultivation temperature 24-26 DEG C, intensity of illumination 2000lux, and light application time is cultivate the complete test-tube plantlet that 21d obtains being with root under the condition of 10-12h/d;
Step 6, hardening and transplanting: hardening obtains whole plant, is transplanted in matrix afterwards and cultivates; The detailed process of hardening and transplanting is: after the complete test-tube plantlet that the band 2-4cm root obtained in described step 5 is long, the complete test-tube plantlet of having taken root is shifted out culturing room together with blake bottle, proceed to booth, under temperature is 25-27 °, place 7d, then open bottle cap, in bottle, add a small amount of running water, hardening 3-4d, take out, clean root medium, be transplanted in matrix, 7-10d plastic film moisturizing after transplanting, cover one deck newspaper more above, keep intensity of illumination to be that 60-70% is the most suitable with shading rate, air humidity 85-90%, every 3d spray 1 time.
In such scheme, owing to adopting the aseptic explant of blade as Plant Tissue Breeding of ford dutchmanspipe root or herb, blade, compared with stem section, has been saved stem section and has been avoided whole strain collection, be conducive to the protection of ford dutchmanspipe root or herb resource, also can meet the demand of market to ford dutchmanspipe root or herb medicinal material;
In such scheme, for the different phase of ford dutchmanspipe root or herb blade Plant Tissue Breeding provides different medium, make the aseptic explant of ford dutchmanspipe root or herb under the continuous action of various suitable medium, at utmost improve the planting percent of explant, reach the object of quick, effective and undamaged breeding ford dutchmanspipe root or herb.
Ford dutchmanspipe root or herb leaf extract also containing 5mL/L in described inducing culture; Ford dutchmanspipe root or herb leaf extract also containing 15mL/L in described strong seedling culture base.
Described ford dutchmanspipe root or herb leaf extract is juice ford dutchmanspipe root or herb mature leaf being blended acquisition.
Matrix described in described step 6 is that the humus soil of 5: 3: 1, dry liver moss and perlite form by volume ratio.
In such scheme, first dry liver moss is in use manually ground into 40-60 object particle, after afterwards humus soil, dry liver moss and perlite being mixed in proportion, place 5-7 days at normal temperatures, spray a water every day and carry out a convention stir simultaneously, injection flow rate is 50-100ml/kg matrix, become the matrix of cultivating complete test-tube plantlet afterwards, wherein, matrix of the present invention can provide necessary nutrition for test-tube plantlet, make ford dutchmanspipe root or herb test-tube plantlet growth good, survival rate is high.
Wild ford dutchmanspipe root or herb is often born in the underbrush of mountain valley with in the stone gap of mountain region, habitat requires comparatively strict, liver moss has more in the growing environment of present ford dutchmanspipe root or herb, for the growth of ford dutchmanspipe root or herb and climbing provide certain nutrition, and improve the soil environment of its growth, add certain dry liver moss in matrix provided by the present invention and do some process in early stage before use, make the better stripping of the nutrient component in dry liver moss in humus soil, growth for the test-tube plantlet of ford dutchmanspipe root or herb provides closer to natural growing environment, make the test-tube plantlet growth of ford dutchmanspipe root or herb vigorous, improve survival rate, and better keep the trophic component of wild ford dutchmanspipe root or herb, keep its medical value.
Agar all containing 4.5g/L in described inducing culture, described differential medium, described strong seedling culture base and described root media and 25g/L sucrose, and pH value is 5.8.
< example 2>
Apply the method that ford dutchmanspipe root or herb blade carries out Fast-propagation, comprise the following steps:
Step one, prepare ford dutchmanspipe root or herb aseptic explant, the sterilization of explant selection and explant, wherein, the detailed process of the selection of explant is: get the young leaflet tablet near ford dutchmanspipe root or herb stem apex top, 30min is soaked in running water, then under flowing water, scrub blade surface and the back side with capillary brush gently, then spread out on blotting paper, natural air drying; The detailed process of the sterilization of explant is: by the 1% liquid detergent aqueous solution soaking 5-10min of the blade after natural air drying, wire tap water 10-15min, with through autoclaved sterile water rinse once on transfer room super-clean bench, then the alcohol-pickled 30-50s of 75% is used, be 0.1% mercuric chloride sterilization 4-6min by the 150ml volume fraction that with the addition of 2-3 and drip Tween-20 again, aseptic water washing 3-5 time, finally to spread out on sterilized filter paper to remove surface moisture, obtains aseptic explant;
Step 2, callus induction: aseptic explant is removed vein and is inoculated into after stripping and slicing process on inducing culture and carry out Fiber differentiation, obtain callus, wherein, MS+6-BA 1.5mg/L+2,4-D 4.0mg/L+NAA 1.5mg/L is contained in described inducing culture; The detailed process of callus induction is: aseptic explant is removed nerve structure, be cut into the square leaf block of 1.0-2.0cm, edge blade scratches, then be inoculated in described inducing culture, be 24-26 DEG C in cultivation temperature, under light culture condition, cultivate 7d, then at intensity of illumination 1500lux, light application time is cultivate 21d under the condition of 10-12h/d, obtains the callus of peak green;
Step 3, Calli Differentiation: described callus is inoculated in differential medium and obtains Multiple Buds, wherein, the TDZ containing MS+2.0mg/L in described differential medium; The detailed process of Calli Differentiation is: by described callus, is cut into the square leaf block of 2.0cm, is inoculated in described differential medium, be 24-26 DEG C in cultivation temperature, intensity of illumination 1500lux, light application time is cultivate 21d under the condition of 10-12h/d, obtains Multiple Buds;
Step 4, strong sprout: be inoculated into by Multiple Buds in strong seedling culture base and carry out strong seedling culture, obtain test-tube plantlet, wherein, the powdered activated carbon containing MS+6-BA 0.5mg/L+IBA 0.5mg/L and 1.5g/L in described strong seedling culture base; Strong seedling culture detailed process is: described Multiple Buds is placed in described strong seedling culture base, at cultivation temperature 24-26 DEG C, and intensity of illumination 2000lux, light application time is cultivate 21d under the condition of 10-12h/d to obtain healthy and strong test-tube plantlet;
Step 5, to take root: test-tube plantlet is implanted into the test-tube plantlet obtaining band root in root media, wherein, the powdered activated carbon containing 1/2MS+IBA 0.5mg/L+NAA 1.0mg/L and 0.5g/L in root media; The detailed process of culture of rootage is: described healthy and strong test-tube plantlet is placed in described root media, cultivation temperature 24-26 DEG C, intensity of illumination 2000lux, and light application time is cultivate the complete test-tube plantlet that 21d obtains being with root under the condition of 10-12h/d;
Step 6, hardening and transplanting: hardening obtains whole plant, is transplanted in matrix afterwards and cultivates; The detailed process of hardening and transplanting is: after the complete test-tube plantlet that the band 2-4cm root obtained in described step 5 is long, the complete test-tube plantlet of having taken root is shifted out culturing room together with blake bottle, proceed to booth, under temperature is 25-27 °, place 7d, then open bottle cap, in bottle, add a small amount of running water, hardening 3-4d, take out, clean root medium, be transplanted in matrix, 7-10d plastic film moisturizing after transplanting, cover one deck newspaper more above, keep intensity of illumination to be that 60-70% is the most suitable with shading rate, air humidity 85-90%, every 3d spray 1 time.
In such scheme, owing to adopting the aseptic explant of blade as Plant Tissue Breeding of ford dutchmanspipe root or herb, blade, compared with stem section, has been saved stem section and has been avoided whole strain collection, be conducive to the protection of ford dutchmanspipe root or herb resource, also can meet the demand of market to ford dutchmanspipe root or herb medicinal material;
In such scheme, for the different phase of ford dutchmanspipe root or herb blade Plant Tissue Breeding provides different medium, make the aseptic explant of ford dutchmanspipe root or herb under the continuous action of various suitable medium, at utmost improve the planting percent of explant, reach the object of quick, effective and undamaged breeding ford dutchmanspipe root or herb.
Ford dutchmanspipe root or herb leaf extract also containing 10mL/L in described inducing culture; Ford dutchmanspipe root or herb leaf extract also containing 20mL/L in described strong seedling culture base.
Described ford dutchmanspipe root or herb leaf extract is juice ford dutchmanspipe root or herb mature leaf being blended acquisition.
Matrix described in described step 6 is that the humus soil of 4: 2: 1, dry liver moss and perlite form by volume ratio.
In such scheme, first dry liver moss is in use manually ground into 40-60 object particle, after humus soil, dry liver moss and perlite being mixed in proportion afterwards, place 5-7d at normal temperatures, spray a water every day and carry out a convention stir simultaneously, injection flow rate is 50-100ml/kg matrix, become the matrix of cultivating complete test-tube plantlet afterwards, wherein, matrix of the present invention can provide necessary nutrition for test-tube plantlet, make ford dutchmanspipe root or herb test-tube plantlet growth good, survival rate is high.
Wild ford dutchmanspipe root or herb is often born in the underbrush of mountain valley with in the stone gap of mountain region, habitat requires comparatively strict, liver moss has more in the growing environment of present ford dutchmanspipe root or herb, for the growth of ford dutchmanspipe root or herb and climbing provide certain nutrition, and improve the soil environment of its growth, add certain dry liver moss in matrix provided by the present invention and do some process in early stage before use, make the better stripping of the nutrient component in dry liver moss in humus soil, growth for the test-tube plantlet of ford dutchmanspipe root or herb provides closer to natural growing environment, make the test-tube plantlet growth of ford dutchmanspipe root or herb vigorous, improve survival rate, and better keep the trophic component of wild ford dutchmanspipe root or herb, keep its medical value.
Agar all containing 4.5g/L in described inducing culture, described differential medium, described strong seedling culture base and described root media and 25g/L sucrose, and pH value is 5.8.
< example 3>
Apply the method that ford dutchmanspipe root or herb blade carries out Fast-propagation, comprise the following steps:
Step one, prepare ford dutchmanspipe root or herb aseptic explant, the sterilization of explant selection and explant, wherein, the detailed process of the selection of explant is: get the young leaflet tablet near ford dutchmanspipe root or herb stem apex top, 30min is soaked in running water, then under flowing water, scrub blade surface and the back side with capillary brush gently, then spread out on blotting paper, natural air drying; The detailed process of the sterilization of explant is: by the 1% liquid detergent aqueous solution soaking 5-10min of the blade after natural air drying, wire tap water 10-15min, with through autoclaved sterile water rinse once on transfer room super-clean bench, then the alcohol-pickled 30-50s of 75% is used, be 0.1% mercuric chloride sterilization 4-6min by the 150ml volume fraction that with the addition of 2-3 and drip Tween-20 again, aseptic water washing 3-5 time, finally to spread out on sterilized filter paper to remove surface moisture, obtains aseptic explant;
Step 2, callus induction: aseptic explant is removed vein and is inoculated into after stripping and slicing process on inducing culture and carry out Fiber differentiation, obtain callus, wherein, MS+6-BA 1.5mg/L+2,4-D 3.0mg/L+NAA 1.0mg/L is contained in described inducing culture; The detailed process of callus induction is: aseptic explant is removed nerve structure, be cut into the square leaf block of 1.0-2.0cm, edge blade scratches, then be inoculated in described inducing culture, be 24-26 DEG C in cultivation temperature, under light culture condition, cultivate 7d, then at intensity of illumination 1500lux, light application time is cultivate 21d under the condition of 10-12h/d, obtains the callus of peak green;
Step 3, Calli Differentiation: described callus is inoculated in differential medium and obtains Multiple Buds, wherein, the TDZ containing MS+1.5mg/L in described differential medium; The detailed process of Calli Differentiation is: by described callus, is cut into the square leaf block of 2.0cm, is inoculated in described differential medium, be 24-26 DEG C in cultivation temperature, intensity of illumination 1500lux, light application time is cultivate 21d under the condition of 10-12h/d, obtains Multiple Buds;
Step 4, strong sprout: be inoculated into by Multiple Buds in strong seedling culture base and carry out strong seedling culture, obtain test-tube plantlet, wherein, the powdered activated carbon containing MS+6-BA 0.3mg/L+IBA 0.3mg/L and 1.0g/l in described strong seedling culture base; Strong seedling culture detailed process is: described Multiple Buds is placed in described strong seedling culture base, at cultivation temperature 24-26 DEG C, and intensity of illumination 2000lux, light application time is cultivate 21d under the condition of 10-12h/d to obtain healthy and strong test-tube plantlet;
Step 5, to take root: test-tube plantlet is implanted into the test-tube plantlet obtaining band root in root media, wherein, the powdered activated carbon containing 1/2MS+IBA 0.3mg/L+NAA 0.7mg/L and 0.3g/L in root media; The detailed process of culture of rootage is: described healthy and strong test-tube plantlet is placed in described root media, cultivation temperature 24-26 DEG C, intensity of illumination 2000lux, and light application time is cultivate the complete test-tube plantlet that 21d obtains being with root under the condition of 10-12h/d;
Step 6, hardening and transplanting: hardening obtains whole plant, is transplanted in matrix afterwards and cultivates; The detailed process of hardening and transplanting is: after the complete test-tube plantlet that the band 2-4cm root obtained in described step 5 is long, the complete test-tube plantlet of having taken root is shifted out culturing room together with blake bottle, proceed to booth, under temperature is 25-27 °, place 7d, then open bottle cap, in bottle, add a small amount of running water, hardening 3-4d, take out, clean root medium, be transplanted in matrix, 7-10d plastic film moisturizing after transplanting, cover one deck newspaper more above, keep intensity of illumination to be that 60-70% is the most suitable with shading rate, air humidity 85-90%, every 3d spray 1 time.
In such scheme, owing to adopting the aseptic explant of blade as Plant Tissue Breeding of ford dutchmanspipe root or herb, blade, compared with stem section, has been saved stem section and has been avoided whole strain collection, be conducive to the protection of ford dutchmanspipe root or herb resource, also can meet the demand of market to ford dutchmanspipe root or herb medicinal material;
In such scheme, for the different phase of ford dutchmanspipe root or herb blade Plant Tissue Breeding provides different medium, make the aseptic explant of ford dutchmanspipe root or herb under the continuous action of various suitable medium, at utmost improve the planting percent of explant, reach the object of quick, effective and undamaged breeding ford dutchmanspipe root or herb.
Ford dutchmanspipe root or herb leaf extract also containing 10mL/L in described inducing culture; Ford dutchmanspipe root or herb leaf extract also containing 15mL/L in described strong seedling culture base.Described ford dutchmanspipe root or herb leaf extract is juice ford dutchmanspipe root or herb mature leaf being blended acquisition.
Matrix described in described step 6 is that the humus soil of 3: 3: 1, dry liver moss and perlite form by volume ratio.
In such scheme, first dry liver moss is in use manually ground into 40-60 object particle, after afterwards humus soil, dry liver moss and perlite being mixed in proportion, place 5-7 days at normal temperatures, spray a water every day and carry out a convention stir simultaneously, injection flow rate is 50-100ml/kg matrix, become the matrix of cultivating complete test-tube plantlet afterwards, wherein, matrix of the present invention can provide necessary nutrition for test-tube plantlet, make ford dutchmanspipe root or herb test-tube plantlet growth good, survival rate is high.
Wild ford dutchmanspipe root or herb is often born in the underbrush of mountain valley with in the stone gap of mountain region, habitat requires comparatively strict, liver moss has more in the growing environment of present ford dutchmanspipe root or herb, for the growth of ford dutchmanspipe root or herb and climbing provide certain nutrition, and improve the soil environment of its growth, add certain dry liver moss in matrix provided by the present invention and do some process in early stage before use, make the better stripping of the nutrient component in dry liver moss in humus soil, growth for the test-tube plantlet of ford dutchmanspipe root or herb provides closer to natural growing environment, make the test-tube plantlet growth of ford dutchmanspipe root or herb vigorous, improve survival rate, and better keep the trophic component of wild ford dutchmanspipe root or herb, keep its medical value.Agar all containing 4.5g/L in described inducing culture, described differential medium, described strong seedling culture base and described root media and 25g/L sucrose, and pH value is 5.8.
In the large-scale production of reality, general meeting is in above-mentioned example 1-3, step 4 can also comprise the following steps: be inoculated in subculture medium by described test-tube plantlet and carry out squamous subculture, wherein, agar containing MS+KT 0.1-0.5mg/L+6-BA 0.2-1.0mg/L+NAA 0.2-1.0mg/L, 4.5g/L in described subculture medium and 25g/L sucrose, condition of culture is: temperature 24-26 DEG C, intensity of illumination 2000lux, and the pH value of subculture medium is 5.8.
In such scheme, test-tube plantlet is inoculated in subculture medium and carries out squamous subculture, at the needs of the simultaneous adaptation large-scale production of guarantee ford dutchmanspipe root or herb quality.
Following table 1 is the related data of above-mentioned example 1-3
The correlation computations formula related in upper table 1 is:
Explant number/inoculation explant number × 100% of the inductivity=production callus of callus;
The callus number of somatic embryo generation rate=occur somatic embryo/inoculate total callus number × 100%;
Callus number × 100% of the callus number of embryo differentiate rate=embryo differentiate/containing somatic embryo.
Although embodiment of the present invention are open as above, it is not restricted to listed in specification and embodiment utilization.It can be applied to various applicable the field of the invention completely.For those skilled in the art, can easily realize other amendment.Therefore do not deviating under the universal that claim and equivalency range limit, the present invention is not limited to specific details and illustrates here and the embodiment described.
Claims (10)
1. apply the method that ford dutchmanspipe root or herb blade carries out Fast-propagation, comprise the following steps:
Step one, prepare ford dutchmanspipe root or herb aseptic explant;
Step 2, callus induction: aseptic explant is removed vein and is inoculated into after stripping and slicing process on inducing culture and carry out Fiber differentiation, obtain callus, wherein, MS+6-BA1.0-1.5mg/L+2,4-D2.0-4.0mg/L+NAA0.5-1.5mg/L is contained in described inducing culture;
Step 3, Calli Differentiation: described callus is inoculated in differential medium and obtains Multiple Buds, wherein, the TDZ containing MS+1.0-2.0mg/L in described differential medium;
Step 4, strong sprout: be inoculated into by Multiple Buds in strong seedling culture base and carry out strong seedling culture, obtain test-tube plantlet, wherein, the powdered activated carbon containing MS+6-BA0.1-0.5mg/L+IBA0.1-0.5mg/L and 0.5-1.5g/L in described strong seedling culture base;
Step 5, to take root: test-tube plantlet is implanted into the test-tube plantlet obtaining band root in root media, wherein, the powdered activated carbon containing 1/2MS+IBA0.1-0.5mg/L+NAA0.5-1.0mg/L and 0.1-0.5g/L in root media;
Step 6, hardening and transplanting: hardening is transplanted in matrix after obtaining whole plant and cultivates.
2. application ford dutchmanspipe root or herb blade as claimed in claim 1 carries out the method for Fast-propagation, it is characterized in that,
Containing MS+6-BA1.0mg/L+2,4-D3.0mg/L+NAA1.0mg/L in described inducing culture; TDZ containing MS+2.0mg/L in described differential medium; Powdered activated carbon containing MS+6-BA0.1mg/L+IBA0.5mg/L and 1.5g/L in described strong seedling culture base; Powdered activated carbon containing 1/2MS+IBA0.5mg/L+NAA0.5mg/L and 0.5g/L in described root media.
3. application ford dutchmanspipe root or herb blade as claimed in claim 1 carries out the method for Fast-propagation, it is characterized in that, the ford dutchmanspipe root or herb leaf extract also containing 5-10mL/L in described inducing culture; Ford dutchmanspipe root or herb leaf extract also containing 15-20mL/L in described strong seedling culture base.
4. application ford dutchmanspipe root or herb blade as claimed in claim 3 carries out the method for Fast-propagation, and it is characterized in that, described ford dutchmanspipe root or herb leaf extract is juice ford dutchmanspipe root or herb mature leaf being blended acquisition.
5. application ford dutchmanspipe root or herb blade as claimed in claim 1 carries out the method for Fast-propagation, and it is characterized in that, matrix described in described step 6 is that the humus soil of 5 ~ 3: 3 ~ 2: 1, dry liver moss and perlite form by volume ratio.
6. application ford dutchmanspipe root or herb blade as claimed in claim 1 carries out the method for Fast-propagation, and it is characterized in that, matrix described in described step 6 is that the humus soil of 4: 2: 1, dry liver moss and perlite form by volume ratio.
7. application ford dutchmanspipe root or herb blade as claimed in claim 1 carries out the method for Fast-propagation, and it is characterized in that, the young leaflet tablet applying ford dutchmanspipe root or herb stem apex top in described step one prepares described ford dutchmanspipe root or herb aseptic explant.
8. application ford dutchmanspipe root or herb blade as claimed in claim 1 carries out the method for Fast-propagation, it is characterized in that, agar all containing 4.5g/L in described inducing culture, described differential medium, described strong seedling culture base and described root media and 25g/L sucrose, and pH value is 5.8.
9. application ford dutchmanspipe root or herb blade as claimed in claim 1 carries out the method for Fast-propagation, it is characterized in that, described step 4 is further comprising the steps of: be inoculated in subculture medium by described test-tube plantlet and carry out squamous subculture, wherein, agar containing MS+KT0.1-0.5mg/L+6-BA0.2-1.0mg/L+NAA0.2-1.0mg/L, 4.5g/L in described subculture medium and 25g/L sucrose, condition of culture is: temperature 24-26 DEG C, intensity of illumination 2000lux, and the pH value of subculture medium is 5.8.
10. application ford dutchmanspipe root or herb blade as claimed in claim 1 carries out the method for Fast-propagation, it is characterized in that, the sterilization of explant selection and explant is also comprised in described step one, wherein, the detailed process of the selection of explant is: get the young leaflet tablet near ford dutchmanspipe root or herb stem apex top, soak 30min, then under flowing water, scrub blade surface and the back side with capillary brush gently in running water, then spread out on blotting paper, natural air drying; The detailed process of the sterilization of explant is: by the 1% liquid detergent aqueous solution soaking 5-10min of the blade after natural air drying, wire tap water 10-15min, with through autoclaved sterile water rinse once on transfer room super-clean bench, then the alcohol-pickled 30-50s of 75% is used, be 0.1% mercuric chloride sterilization 4-6min by the 150ml volume fraction that with the addition of 2-3 and drip Tween-20 again, aseptic water washing 3-5 time, finally to spread out on sterilized filter paper to remove surface moisture, obtains aseptic explant;
In described step 2, the detailed process of callus induction is: aseptic explant is removed nerve structure, be cut into the square leaf block of 1.0-2.0cm, edge blade scratches, then be inoculated in described inducing culture, be 24-26 DEG C in cultivation temperature, under light culture condition, cultivate 7d, then at intensity of illumination 1500lux, light application time is cultivate 21d under the condition of 10-12h/d, obtains the callus of peak green;
In described step 3, the detailed process of Calli Differentiation is: by described callus, be cut into the square leaf block of 2.0cm, be inoculated in described differential medium, be 24-26 DEG C in cultivation temperature, intensity of illumination 1500lux, light application time is cultivate 21d under the condition of 10-12h/d, obtains Multiple Buds;
In described step 4, strong seedling culture detailed process is: described Multiple Buds is placed in described strong seedling culture base, at cultivation temperature 24-26 DEG C, and intensity of illumination 2000lux, light application time is cultivate 21d under the condition of 10-12h/d to obtain healthy and strong test-tube plantlet;
In described step 5, the detailed process of culture of rootage is: described healthy and strong test-tube plantlet is placed in described root media, cultivation temperature 24-26 DEG C, intensity of illumination 2000lux, and light application time is cultivate the complete test-tube plantlet that 21d obtains being with root under the condition of 10-12h/d;
The detailed process of hardening and transplanting in described step 6 is: after the complete test-tube plantlet that the band 2-4cm root obtained in described step 5 is long, the complete test-tube plantlet of having taken root is shifted out culturing room together with blake bottle, proceed to booth, under temperature is 25-27 °, place 7d, then bottle cap is opened, a small amount of running water is added in bottle, hardening 3-4d, take out, clean root medium, be transplanted in matrix, 7-10d plastic film moisturizing after transplanting, cover one deck newspaper more above, intensity of illumination is kept to be that 60-70% is the most suitable with shading rate, air humidity 85-90%, every 3d sprays 1 time, wherein, the concrete preparation method of described matrix is: first dry liver moss is in use manually ground into 40-60 object particle, after afterwards humus soil, dry liver moss and perlite being mixed in proportion, lucifuge places 5-7d at normal temperatures, spray a water every day and carry out a convention stir simultaneously, injection flow rate is 50-100ml/kg matrix.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108064698A (en) * | 2018-02-05 | 2018-05-25 | 遵义市龙驰生物科技有限公司 | A kind of natural crude drugs Ciliatenerve Knotweed Root tissue culture and rapid propagation method |
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Application publication date: 20150715 |