CN103598100B - Cultural method of bulbus fritillariae cirrhosae polyploidy virus-free seedlings - Google Patents
Cultural method of bulbus fritillariae cirrhosae polyploidy virus-free seedlings Download PDFInfo
- Publication number
- CN103598100B CN103598100B CN201310616273.4A CN201310616273A CN103598100B CN 103598100 B CN103598100 B CN 103598100B CN 201310616273 A CN201310616273 A CN 201310616273A CN 103598100 B CN103598100 B CN 103598100B
- Authority
- CN
- China
- Prior art keywords
- polyploid
- bulbus fritillariae
- fritillariae cirrhosae
- illumination
- bulb
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Abstract
The invention discloses a cultural method of bulbus fritillariae cirrhosae polyploidy virus-free seedlings. The cultural method comprises the following steps: inducing a polyploidy callus on cells in a growing point area of pretreated adventitious roots, and performing induction culturing, rooting culturing, domestication and transplantation on bulb buds, so as to obtain the bulbus fritillariae cirrhosae polyploidy virus-free seedlings. According to the cultural method disclosed by the invention, a whole set of cultural method of the bulbus fritillariae cirrhosae polyploidy virus-free seedlings is established, and the cultural method has a good detoxification effect and is low in culturing cost, and a novel path is provided for rapid cultivating of good strains of bulbus fritillariae cirrhosae.
Description
Technical field
The present invention relates to the method for tissue culture of a kind of Bulbus Fritillariae Cirrhosae plant, particularly relate to a kind of cultural method of Bulbus Fritillariae Cirrhosae polyploid detoxic seedling.
Background technology
Bulbus Fritillariae Cirrhosae Fritillaria cirrhosa D.Don) be Liliaceae herbaceos perennial, grow the height at height above sea level 3500m-4500m, main product in Sichuan, Tibet, the provinces and regions such as Yunnan.Bulbus Fritillariae Cirrhosae has clearing heat and moistening lung, effect of preventing phlegm from forming and stopping coughing, and for lung-heat type cough, the few phlegm of dry cough, deficiency of Yin labor is coughed, and coughs up the symptoms such as sputum streaked with blood.Bulbus Fritillariae Cirrhosae is the good medicine of rare treatment cough etc., and because Bulbus Fritillariae Cirrhosae is expensive, in addition in recent years without plan excavating blindly, natural resources is increasingly exhausted, estimates also to be difficult to alleviate within nearly decades.
The breeding of Bulbus Fritillariae Cirrhosae plant often adopts the sexual reproduction carried out with seed and vegetative propagation two kinds of modes of carrying out with bulb.No matter but be the breeding adopting seed or bulb to carry out, all virus can be caught because of various environmental factor for perennial Bulbus Fritillariae Cirrhosae plant, and plant is once after being infected virus, deformity will be produced, and seriously can suppress its growth, thus the quality and yield of Bulbus Fritillariae Cirrhosae medicinal material is finally caused to decline widely.More seriously infected virus not only can be survived for a long time in plant organ, also can hand on the organ of plant is generation upon generation of, thus makes to have infected viral plant variety and there will be and degenerate significantly, even serious and endangered because infecting.
Polyploid plant has plant organ " gigantism " feature, and simultaneously polyploid plant strengthens with the otherness of gene activity and enzyme because number gene increases, therefore its annidation and higher to the resistance of adverse circumstance.The detoxic seedling Cultivating techniques of Bulbus Fritillariae Cirrhosae plant and multiploid induction technology are organically combined, can cultivate best in quality, fast growth, active principle content are high and the Bulbus Fritillariae Cirrhosae polyploid detoxic seedling of strong stress resistance.
Summary of the invention
The object of the present invention is to provide the cultural method of the Bulbus Fritillariae Cirrhosae polyploid detoxic seedling that a kind of detoxification efficiency is good, fast growth, active principle content are high.
The cultural method of Bulbus Fritillariae Cirrhosae polyploid detoxic seedling provided by the invention comprises Fiber differentiation step and the following step of Bulbus Fritillariae Cirrhosae group training bulb:
The acquisition of polyploid adventive root: Bulbus Fritillariae Cirrhosae group training bulb is first inoculated in 1/2MS+IBA0.1 ~ 0.3mgL
-1+ colchicine 200 ~ 700mgL
-1medium in, cultivate 2 ~ 5 days under the condition being 12 ~ 18 DEG C without light and temperature, proceed to not containing in the above-mentioned medium of colchicine again, cultivation temperature be 18 ~ 24 DEG C, illumination every day 4 ~ 8h and intensity of illumination continue to cultivate under being the condition of 800 ~ 1200lx, the polyploid adventive root having shape thick at the base portion of Bulbus Fritillariae Cirrhosae bulb produces;
The pretreatment of polyploid adventive root: polyploid adventive root is cut rear access MS+6-BA0.1 ~ 0.5mgL from foundation portion
-1+ glycerine 100 ~ 200mlL
-1medium in, under unglazed photograph and cultivation temperature are the condition of 35 ~ 40 DEG C, carry out the preculture process of 15 ~ 25 days;
The cultivation of polyploid adventive root growing point region cell: on superclean bench, cut the cell in growing point region on polyploid adventive root, accessed callus inducing medium B5+6-BA0.5 ~ 2.0mg.L
-1+ 2,4-D0.5 ~ 4.0mg.L
-1in, cultivation temperature be 15 ~ 22 DEG C, illumination every day 5 ~ 8h, intensity of illumination cultivate 40 ~ 60 days under being the condition of 400 ~ 1000lx, obtains polyploid callus;
The induction of Bulbus Fritillariae Cirrhosae polyploid bulb bud: choose quality comparatively closely, fast growth and color is yellow-white or flaxen polyploid callus, accessed polyploid bulb bud inducement medium MS+6-BA1.0 ~ 4.0mgL
-1+ IAA0.1 ~ 0.3mgL
-1in, cultivation temperature be 18 ~ 22 DEG C, illumination every day 12 ~ 20h, intensity of illumination cultivate 45 ~ 60 days under being the condition of 1000 ~ 3000lx, obtains Bulbus Fritillariae Cirrhosae polyploid bulb bud;
The culture of rootage of polyploid bulb bud: the polyploid bulb bud cutting growth 1.0 ~ 3.0cm on super-clean bench, as without offspring, is proceeded to root media 1/2MS+NAA0.1 ~ 0.3mgL
-1+ IBA0.1 ~ 0.3mgL
-1middle root induction, obtains Bulbus Fritillariae Cirrhosae polyploid detoxic seedling;
Hardening and transplanting: choose the Bulbus Fritillariae Cirrhosae polyploid detoxic seedling growing more than 2 adventive root, open bottle cap, after 3 ~ 5 days, it taken out from blake bottle in indoor hardening, after washing away the medium on adventive root, transplant in the mellow soil to sterilization and grow.
The Fiber differentiation step of described Bulbus Fritillariae Cirrhosae group training bulb is: choose the scaly leaf on the Bulbus Fritillariae Cirrhosae bulb of growth more than 2 years, disinfection after clean with running water, then accesses MS+6-BA1.0 ~ 3.0mgL
-1+ IAA0.1 ~ 0.5mgL
-1+ KT0.5 ~ 1.5mgL
-1in medium, cultivation temperature be 16 ~ 22 DEG C, illumination every day 6 ~ 10h, intensity of illumination induce under being the condition of 1000 ~ 1500lx Bulbus Fritillariae Cirrhosae group train bulb; Described disinfecting refers to mercuric chloride sterilization 4 ~ 8min that concentration is 0.1% ~ 0.2% on super-clean bench, then with the moisture with sterilized filter paper blotting material surface after aseptic washing 3 ~ 6 times.The Fiber differentiation of Bulbus Fritillariae Cirrhosae group training bulb also can adopt current published method.
Described in the incubation step of above-mentioned polyploid adventive root growing point region cell, the length in growing point region is 0.2 ~ 1.0mm.
In above-mentioned hardening and transplant step, the condition of culture of hardening is temperature 8 ~ 18 DEG C, relative moisture 80% ~ 90%, illumination every day 6 ~ 10h, intensity of illumination 400 ~ 800lx, transplant to the condition of culture in mellow soil be temperature 12 ~ 22 DEG C, relative moisture 75% ~ 80%, illumination every day 8 ~ 12h, intensity of illumination be 800 ~ 1000lx.
Above-mentioned all medium also comprise agar 4.0 ~ 7.0gL
-1with sucrose 15 ~ 50gL
-1, the pH value of medium is 5.5 ~ 6.5.
The present invention selects the growing point region cell induction on adventive root after pretreatment to go out polyploid callus, obtain Bulbus Fritillariae Cirrhosae polyploid detoxic seedling through the induction of polyploid bulb bud, the culture of rootage of polyploid bulb bud and hardening and transplanting and other steps again, the Bulbus Fritillariae Cirrhosae polyploid detoxic seedling cultivated has the advantages such as the high and strong stress resistance of fast growth, active principle content.The inventive method is that the quickly breeding of Bulbus Fritillariae Cirrhosae improved seeds provides a new way.
Below in conjunction with embodiment, the present invention is described in further detail.
Embodiment
Embodiment 1
(1) acquisition of Bulbus Fritillariae Cirrhosae group training bulb: choose the scaly leaf on the Bulbus Fritillariae Cirrhosae plant bulb of growth more than 2 years, after clean with running water, be placed on super-clean bench with concentration be 0.1% mercuric chloride sterilization 4min after, wash 3 times with sterile water concussion again, finally then access with the moisture on sterilized filter paper blotting material surface the MS+6-BA1.0mgL that pH value is 5.8
-1+ IAA0.1mgL
-1+ KT0.5mgL
-1+ sucrose 30gL
-1+ agar 6.0gL
-1medium in, Fiber differentiation 55 days under cultivation temperature 16 DEG C, illumination every day 6h, intensity of illumination are the condition of 1000lx, Bulbus Fritillariae Cirrhosae group training bulb inductivity be 77.65%;
(2) acquisition of Bulbus Fritillariae Cirrhosae polyploid adventive root: the Bulbus Fritillariae Cirrhosae group of acquisition training bulb is first inoculated in the 1/2MS+IBA0.1mgL that pH value is 6.2
-1+ colchicine 200mgL
-1+ sucrose 15gL
-1+ agar 4.5gL
-1medium in, Fiber differentiation 2 days under the condition being 12 DEG C without light and temperature, then proceed to not containing the above-mentioned medium of colchicine and 1/2MS+IBA0.1mgL
-1+ sucrose 15gL
-1+ agar 4.5gL
-1medium in, cultivation temperature be 18 DEG C, illumination every day 4h and intensity of illumination continue cultivation 60 days under being the condition of 800lx, the inductivity of its polyploid adventive root is 41.17%;
(3) pretreatment of polyploid adventive root: it is the MS+6-BA0.1mgL of 6.0 that polyploid adventive root (namely root-tip cells detects chromosome number is 2n=4x=48) is cut rear access pH value from its base portion
-1+ glycerine 100mlL
-1+ sucrose 45gL
-1+ agar 5gL
-1medium in, being placed in unglazed photograph and cultivation temperature is the preculture process that the incubator of 35 DEG C carries out 20 days, and the survival rate of adventive root is about 57.85%;
(4) cultivation of polyploid adventive root growing point region cell: on superclean bench, cuts the cell in 0.2mm region, polyploid adventive root growing point place, is accessed the callus inducing medium B5+6-BA0.5mg.L that pH value is 5.8
-1+ 2,4-D0.5mg.L
-1+ sucrose 50gL
-1+ agar 6.5gL
-1in, cultivation temperature be 15 DEG C, illumination every day 5h, intensity of illumination be the condition of 400lx under cultivate 55 days, polyploid callus induction rate is 42.16%;
(5) induction of Bulbus Fritillariae Cirrhosae polyploid bulb bud: choose quality comparatively closely, fast growth and color is the callus of yellow-white, be linked into the bulb bud inducement medium MS+6-BA1.0mgL that pH value is 5.5
-1+ IAA0.1mgL
-1+ sucrose 40gL
-1+ agar 6gL
-1in; cultivation temperature be 18 DEG C, illumination every day 12h, intensity of illumination be the condition of 1000lx under cultivate 45 days; the inductivity of polyploid bulb bud is 57.21%, and adopt double-antibodies sandwich ELISA to carry out Viral diagnosis to the Bulbus Fritillariae Cirrhosae polyploid bulb bud cultivated, its virus elimination rate is 92.86%;
(6) culture of rootage of polyploid bulb bud: the polyploid bulb bud cutting growth 1.0cm on super-clean bench as without offspring, transferred into pH value be 5.6 root media 1/2MS+NAA0.1mgL
-1+ IBA0.1mgL
-1+ sucrose 50gL
-1+ agar 6.5gL
-1middle root induction, cultivating and adding up rooting rate afterwards in 55 days is 83.14%;
(7) hardening and transplanting: choose the Bulbus Fritillariae Cirrhosae polyploid detoxic seedling growing more than 2 adventive root, open bottle cap, first temperature be 8 DEG C, hardening 3 days in the culturing room of relative moisture 80%, illumination every day 6h and intensity of illumination 400lx, again it is taken out from blake bottle, after washing away the medium on adventive root, transplant in the mellow soil to sterilization, temperature be 12 DEG C, relative moisture 75%, illumination every day 8h and intensity of illumination grow under being the condition of 800lx, the survival rate of Bulbus Fritillariae Cirrhosae polyploid detoxic seedling is 88.64%.
Embodiment 2
(1) acquisition of Bulbus Fritillariae Cirrhosae group training bulb: choose the scaly leaf on the Bulbus Fritillariae Cirrhosae plant bulb of growth more than 2 years, after clean with running water, be placed on super-clean bench with concentration be 0.1% mercuric chloride sterilization 6min after, wash 5 times with sterile water concussion again, finally then access with the moisture on sterilized filter paper blotting material surface the MS+6-BA2.0mgL that pH value is 5.8
-1+ IAA0.3mgL
-1+ KT1.0mgL
-1+ sucrose 30gL
-1+ agar 6.0gL
-1medium in, Fiber differentiation 55 days under cultivation temperature 20 DEG C, illumination every day 8h, intensity of illumination are the condition of 1200lx, Bulbus Fritillariae Cirrhosae group training bulb inductivity be 91.65%;
(2) acquisition of Bulbus Fritillariae Cirrhosae polyploid adventive root: the Bulbus Fritillariae Cirrhosae group of acquisition training bulb is first inoculated in the 1/2MS+IBA0.2mgL that pH value is 6.0
-1+ colchicine 400mgL
-1+ sucrose 15gL
-1+ agar 4.5gL
-1medium in, Fiber differentiation 4 days under the condition being 16 DEG C without light and temperature, then proceed to not containing the above-mentioned medium of colchicine and 1/2MS+IBA0.2mgL
-1+ sucrose 15gL
-1+ agar 4.5gL
-1medium in, cultivation temperature be 22 DEG C, illumination every day 6h and intensity of illumination continue cultivation 60 days under being the condition of 1000lx, the inductivity of its polyploid adventive root is 63.59%;
(3) pretreatment of polyploid adventive root: it is the MS+6-BA0.3mgL of 6.0 that polyploid adventive root (namely root-tip cells detects chromosome number is 2n=4x=48) is cut rear access pH value from its base portion
-1+ glycerine 150mlL
-1+ sucrose 45gL
-1+ agar 5gL
-1medium in, being placed in unglazed photograph and cultivation temperature is the preculture process that the incubator of 38 DEG C carries out 20 days, and the survival rate of adventive root is about 48.65%;
(4) cultivation of polyploid adventive root growing point region cell: on superclean bench, cuts the cell in 0.4mm region, polyploid adventive root growing point place, is accessed the callus inducing medium B5+6-BA1.0mg.L that pH value is 6.0
-1+ 2,4-D3.0mg.L
-1+ sucrose 50gL
-1+ agar 6.5gL
-1in, cultivation temperature be 20 DEG C, illumination every day 6h, intensity of illumination be the condition of 800lx under cultivate 55 days, polyploid callus induction rate is 85.6%;
(5) induction of Bulbus Fritillariae Cirrhosae polyploid bulb bud: choose quality comparatively closely, fast growth and color is the callus of yellow-white, be linked into the bulb bud inducement medium MS+6-BA2.0mgL that pH value is 5.5
-1+ IAA0.2mgL
-1+ sucrose 40gL
-1+ agar 6gL
-1in; cultivation temperature be 20 DEG C, illumination every day 16h, intensity of illumination be the condition of 2800lx under cultivate 50 days; the inductivity of polyploid bulb bud is 75.45%, and adopt double-antibodies sandwich ELISA to carry out Viral diagnosis to the Bulbus Fritillariae Cirrhosae polyploid bulb bud cultivated, its virus elimination rate is 95.16%;
(6) culture of rootage of polyploid bulb bud: the polyploid bulb bud cutting growth 2.5cm on super-clean bench, as without offspring, is proceeded to the root media 1/2MS+NAA0.2mgL that pH value is 5.8
-1+ IBA0.2mgL
-1+ sucrose 50gL
-1+ agar 6.5gL
-1middle root induction, cultivating and adding up rooting rate afterwards in 55 days is 89.17%, and the adventive root number of growth is more than 3 ~ 5;
(7) hardening and transplanting: choose the Bulbus Fritillariae Cirrhosae polyploid detoxic seedling growing more than 3 adventive root, open bottle cap, first temperature be 15 DEG C, hardening 4 days in the culturing room of relative moisture 85%, illumination every day 8h and intensity of illumination 600lx, again it is taken out from blake bottle, after washing away the medium on adventive root, transplant in the mellow soil to sterilization, temperature be 20 DEG C, relative moisture 75%, illumination every day 10h and intensity of illumination grow under being the condition of 1000lx, the survival rate of Bulbus Fritillariae Cirrhosae polyploid detoxic seedling is 90.4%.
Embodiment 3
(1) acquisition of Bulbus Fritillariae Cirrhosae group training bulb: choose the scaly leaf on the Bulbus Fritillariae Cirrhosae plant bulb of growth more than 2 years, after clean with running water, be placed on super-clean bench with concentration be 0.1% mercuric chloride sterilization 8min after, wash 6 times with sterile water concussion again, finally then access with the moisture on sterilized filter paper blotting material surface the MS+6-BA3.0mgL that pH value is 5.6
-1+ IAA0.5mgL
-1+ KT1.5mgL
-1+ sucrose 30gL
-1+ agar 6.0gL
-1medium in, Fiber differentiation 55 days under cultivation temperature 22 DEG C, illumination every day 10h, intensity of illumination are the condition of 1500lx, Bulbus Fritillariae Cirrhosae group training bulb inductivity be 73.85%;
(2) acquisition of Bulbus Fritillariae Cirrhosae polyploid adventive root: the Bulbus Fritillariae Cirrhosae group of acquisition training bulb is first inoculated in the 1/2MS+IBA0.3mgL that pH value is 6.2
-1+ colchicine 700mgL
-1+ sucrose 15gL
-1+ agar 4.5gL
-1medium in, Fiber differentiation 5 days under the condition being 18 DEG C without light and temperature, then proceed to not containing the above-mentioned medium of colchicine and 1/2MS+IBA0.3mgL
-1+ sucrose 15gL
-1+ agar 4.5gL
-1medium in, cultivation temperature be 24 DEG C, illumination every day 8h and intensity of illumination continue cultivation 60 days under being the condition of 1200lx, the inductivity of polyploid adventive root is 61.86%;
(3) pretreatment of polyploid adventive root: get polyploid adventive root (namely root-tip cells detects chromosome number is 2n=4x=48), cutting rear access pH value from its base portion is the MS+6-BA0.5mgL of 6.0
-1+ glycerine 200mlL
-1+ sucrose 45gL
-1+ agar 5.0gL
-1medium in, being placed in unglazed photograph and cultivation temperature is the preculture process that the incubator of 40 DEG C carries out 20 days, and the survival rate of adventive root is about 23.85%;
(4) cultivation of polyploid adventive root growing point region cell: on superclean bench, cuts the cell in 0.2mm region, growing point place on polyploid adventive root, is accessed the callus inducing medium B5+6-BA2.0mg.L that pH value is 5.8
-1+ 2,4-D4.0mg.L
-1+ sucrose 50gL
-1+ agar 6.5gL
-1in, cultivation temperature be 22 DEG C, illumination every day 8h, intensity of illumination be the condition of 1000lx under cultivate 55 days, polyploid callus induction rate is 92.6%;
(5) induction of Bulbus Fritillariae Cirrhosae polyploid bulb bud: choose quality comparatively closely, fast growth and color is the callus of yellow-white, be linked into the bulb bud inducement medium MS+6-BA4.0mgL that pH value is 5.7
-1+ IAA0.3mgL
-1+ sucrose 40gL
-1+ agar 6.0gL
-1in, cultivation temperature be 22 DEG C, illumination every day 20h, intensity of illumination be the condition of 3000lx under cultivate 60 days, the inductivity of polyploid bulb bud is 71.16%, and adopt double-antibodies sandwich ELISA to carry out Viral diagnosis to the Bulbus Fritillariae Cirrhosae polyploid bulb bud cultivated, its virus elimination rate is 70.96%;
(6) culture of rootage of polyploid bulb bud: the polyploid bulb bud cutting growth 3.0cm on super-clean bench as without offspring, transferred into pH value be 5.8 root media 1/2MS+NAA0.3mgL
-1+ IBA0.3mgL
-1+ sucrose 50gL
-1+ agar 6.5gL
-1middle root induction, cultivating and adding up rooting rate afterwards in 55 days is 84.97%;
(7) hardening and transplanting: choose the Bulbus Fritillariae Cirrhosae polyploid detoxic seedling growing more than 3 adventive root, open bottle cap, first temperature be 18 DEG C, hardening 5 days in the culturing room of relative moisture 90%, illumination every day 10h and intensity of illumination 800lx, again it is taken out from blake bottle, after washing away the medium on adventive root, transplant in the mellow soil to sterilization, temperature be 22 DEG C, relative moisture 80%, illumination every day 12h and intensity of illumination grow under being the condition of 1000lx, the survival rate of Bulbus Fritillariae Cirrhosae polyploid detoxic seedling is 88.17%.
Embodiment 4
The training of group described in embodiment 2 step (2) Bulbus Fritillariae Cirrhosae bulb reconfiguration is entered 1/2MS+IBA0.2mg.L
-1+ colchicine 700mg.L
-1+ agar 6.0gL+ sucrose 30gL
-1medium in cultivate 5 days, other step is with embodiment 2, and the inductivity of polyploid adventive root is 62.18%.
Embodiment 5
Change medium described in embodiment 2 step (3) into MS+6-BA0.1mgL
-1+ glycerine 100mlL
-1+ sucrose 20gL
-1+ agar 7gL
-1medium in, other step is with embodiment 2, and the survival rate of polyploid adventive root is about 43.64%.
Embodiment 6
The pretreatment of the polyploid adventive root in embodiment 2 step (3) being changed in temperature is preculture 25 days in the incubator of 40 DEG C, and other step is with embodiment 2, and the survival rate of its adventive root is about 27.12%.
Embodiment 7
The cell reconfiguration in polyploid adventive root growing point region in embodiment 2 step (4) is entered B5+6-BA2.0mg.L
-1+ 2,4-D1.0mg.L
-1+ sucrose 20gL
-1+ agar 6.0gL
-1in, cultivation temperature be 15 DEG C, illumination every day 5h and intensity of illumination cultivate under being the condition of 400lx, other step is with embodiment 2, and the inductivity of polyploid callus is 62.63%.
Embodiment 8
Callus reconfiguration in embodiment 2 step (5) is entered MS+6-BA1.0mgL
-1+ IAA0.3mgL
-1+ sucrose 20gL
-1+ agar 7gL
-1inducing culture in, be 18 DEG C in cultivation temperature, cultivate under the condition of illumination every day 12h and intensity of illumination 1000lx, other step is with embodiment 2, and the inductivity of polyploid bulb bud is 73.47%.
Embodiment 9
In embodiment 2 step (6), the polyploid bulb bud reconfiguration cutting growth 1.5cm enters 1/2MS+NAA0.3mgL
-1+ IBA0.3mgL
-1root media in, other step is with embodiment 2, and its rooting rate is 84.13%, and the adventive root number of growth is 1 ~ 3.
Comparing embodiment 1
By the training of Bulbus Fritillariae Cirrhosae group described in embodiment 2 step (2) bulb changing that cultivation temperature is 22 DEG C into containing the condition of culture in the medium of colchicine, illumination every day 6 hours and intensity of illumination be 1000lx, other step is with embodiment 2, and the inductivity of its polyploid adventive root is 32.52%.
Comparing embodiment 2
After being cut from foundation portion by polyploid adventive root described in embodiment 1 step (3), reconfiguration enters MS+6-BA0.3mg.L+ agar 7.0gL
-1medium in, the incubator being placed in 45 DEG C carries out pretreatment, other step with embodiment 2, the survival rate 0% of its adventive root.
Comparing embodiment 3
The pre-treatment step of the polyploid adventive root in embodiment 2 step (3) cancelled, other step is with embodiment 2, and the virus elimination rate of Bulbus Fritillariae Cirrhosae polyploid plantlet in vitro is 35.16%.
Comparing embodiment 4
In embodiment 2 step (4), change the length in the polyploid adventive root growing point region cut into 0.1mm, other step is with embodiment 2, and the inductivity of its polyploid callus is 14.16%.
Comparing embodiment 5
Changed into by the inducing culturing condition of bulb bud in embodiment 2 step (5) and carrying out under unglazed photograph, other step is with embodiment 2, and the inductivity of polyploid bulb bud is 32.16%.
Comparing embodiment 6
Change the root media of the polyploid bulb bud in embodiment 2 step (6) into MS+NAA0.2mgL
-1, other step is with embodiment 2, and cultivating and adding up rooting rate afterwards in 55 days is 34.42%.
Comparing embodiment 7
In embodiment 2 step (7), adopt the Bulbus Fritillariae Cirrhosae polyploid detoxic seedling of growth less than 2 adventive root to carry out hardening and transplanting, other step is with embodiment 2, and the survival rate of Bulbus Fritillariae Cirrhosae polyploid detoxic seedling is 47.12%.
Claims (5)
1. a cultural method for Bulbus Fritillariae Cirrhosae polyploid detoxic seedling, comprises the Fiber differentiation step of Bulbus Fritillariae Cirrhosae group training bulb, characterized by further comprising following steps:
The acquisition of Bulbus Fritillariae Cirrhosae polyploid adventive root: Bulbus Fritillariae Cirrhosae group training bulb is first inoculated in 1/2MS+IBA0.1 ~ 0.3mgL
-1+ colchicine 200 ~ 700mgL
-1medium in, cultivate 2 ~ 5 days under the condition being 12 ~ 18 DEG C without light and temperature, proceed to not containing in the above-mentioned medium of colchicine again, cultivation temperature be 18 ~ 24 DEG C, illumination every day 4 ~ 8h and intensity of illumination continue to cultivate under being the condition of 800 ~ 1200lx, the polyploid adventive root having shape thick at the base portion of Bulbus Fritillariae Cirrhosae bulb produces;
The pretreatment of polyploid adventive root: polyploid adventive root is cut rear access MS+6-BA 0.1 ~ 0.5mgL from foundation portion
-1+ glycerine 100 ~ 200mlL
-1medium in, under unglazed photograph and cultivation temperature are the condition of 35 ~ 40 DEG C, carry out the preculture process of 15 ~ 25 days;
The cultivation of polyploid adventive root growing point region cell: on superclean bench, cut the cell in growing point region on polyploid adventive root, is accessed callus inducing medium B5+6-BA 0.5 ~ 2.0mg.L
-1+ 2,4-D 0.5 ~ 4.0mg.L
-1in, cultivation temperature be 15 ~ 22 DEG C, illumination every day 5 ~ 8 hours, intensity of illumination cultivate 40 ~ 60 days under being the condition of 400 ~ 1000lx, obtains polyploid callus;
The induction of Bulbus Fritillariae Cirrhosae polyploid bulb bud: choose quality comparatively closely, fast growth and color is yellow-white or flaxen polyploid callus, accessed polyploid bulb bud inducement medium MS+6-BA 1.0 ~ 4.0mgL
-1+ IAA 0.1 ~ 0.3mgL
-1in, cultivation temperature be 18 ~ 22 DEG C, illumination every day 12 ~ 20h, intensity of illumination be the condition of 1000 ~ 3000lx under cultivate 45 ~ 60 days, obtain Bulbus Fritillariae Cirrhosae polyploid bulb bud, adopt double-antibodies sandwich ELISA to carry out Viral diagnosis to the Bulbus Fritillariae Cirrhosae polyploid bulb bud cultivated;
The culture of rootage of polyploid bulb bud: the polyploid bulb bud cutting growth 1.0 ~ 3.0cm on super-clean bench, as without offspring, is proceeded to root media 1/2MS+NAA0.1 ~ 0.3mgL
-1+ IBA0.1 ~ 0.3mgL
-1middle root induction, obtains Bulbus Fritillariae Cirrhosae polyploid detoxic seedling;
Hardening and transplanting: choose the Bulbus Fritillariae Cirrhosae polyploid detoxic seedling growing more than 2 adventive root, open bottle cap, after 3 ~ 5 days, it taken out from blake bottle in indoor hardening, after washing away the medium on adventive root, transplant in the mellow soil to sterilization and grow.
2. the cultural method of a kind of Bulbus Fritillariae Cirrhosae polyploid detoxic seedling according to claim 1, it is characterized in that the Fiber differentiation step of described Bulbus Fritillariae Cirrhosae group training bulb is: choose the scaly leaf on the Bulbus Fritillariae Cirrhosae bulb of growth more than 2 years, disinfection after clean with running water, then accesses MS+6-BA1.0 ~ 3.0mgL
-1+ IAA0.1 ~ 0.5mgL
-1+ KT0.5 ~ 1.5mgL
-1in medium, cultivation temperature be 16 ~ 22 DEG C, illumination every day 6 ~ 10h, intensity of illumination induce under being the condition of 1000 ~ 1500lx Bulbus Fritillariae Cirrhosae group train bulb.
3. the cultural method of a kind of Bulbus Fritillariae Cirrhosae polyploid detoxic seedling according to claim 2, it is characterized in that: described in disinfect and refer to mercuric chloride sterilization 4 ~ 8min that concentration is 0.1% ~ 0.2% on super-clean bench, then with the moisture with sterilized filter paper blotting material surface after aseptic washing 3 ~ 6 times.
4. the cultural method of a kind of Bulbus Fritillariae Cirrhosae polyploid detoxic seedling according to claim 1, is characterized in that: described in the incubation step of polyploid adventive root growing point region cell, the length in growing point region is 0.2 ~ 1.0mm.
5. the cultural method of a kind of Bulbus Fritillariae Cirrhosae polyploid detoxic seedling according to claim 1, it is characterized in that: in hardening and transplant step, the condition of culture of hardening is temperature 8 ~ 18 DEG C, relative moisture 80% ~ 90%, illumination every day 6 ~ 10h, intensity of illumination 400 ~ 800lx, transplant to the condition of culture in mellow soil be temperature 12 ~ 22 DEG C, relative moisture 75% ~ 80%, illumination every day 8 ~ 12h, intensity of illumination be 800 ~ 1000lx.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310616273.4A CN103598100B (en) | 2013-11-27 | 2013-11-27 | Cultural method of bulbus fritillariae cirrhosae polyploidy virus-free seedlings |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310616273.4A CN103598100B (en) | 2013-11-27 | 2013-11-27 | Cultural method of bulbus fritillariae cirrhosae polyploidy virus-free seedlings |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103598100A CN103598100A (en) | 2014-02-26 |
| CN103598100B true CN103598100B (en) | 2015-03-25 |
Family
ID=50116418
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310616273.4A Expired - Fee Related CN103598100B (en) | 2013-11-27 | 2013-11-27 | Cultural method of bulbus fritillariae cirrhosae polyploidy virus-free seedlings |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103598100B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105409765A (en) * | 2016-01-25 | 2016-03-23 | 东北农业大学 | Method for inducing tillered onion polyploidy plant |
| CN106900769A (en) * | 2017-03-02 | 2017-06-30 | 申翰昭 | The growth regulating composition and its application process of bulbus fritillariae cirrhosae |
| CN108522272A (en) * | 2018-02-02 | 2018-09-14 | 洪江市潘多拉中药材生态种植有限公司 | A kind of implantation methods of bulbus fritillariae cirrhosae |
| CN110226483B (en) * | 2019-07-24 | 2021-10-26 | 云南省林木种苗工作总站(云南省林木种苗花卉产品质量监督检验站) | Polygonatum kingianum seedling culture method |
-
2013
- 2013-11-27 CN CN201310616273.4A patent/CN103598100B/en not_active Expired - Fee Related
Non-Patent Citations (5)
| Title |
|---|
| 卷叶贝母多倍体鳞茎诱导方法研究;王跃华;《安徽农业科学》;20111231;第39卷(第7期);第3839-3841页 * |
| 川贝母组织培养的研究;陈敏等;《中国中医药杂志》;19951231;第20卷(第8期);第461-462，511页 * |
| 离体培养条件下伊犁贝母四倍体诱导及筛选;刘传军等;《时珍国医国药》;20081231;第19卷(第12期);第2849-2851页 * |
| 秋水仙素诱导川贝母(Fritillaria cirrhosa D.Don)愈伤组织多倍体的研究;王强等;《武汉植物学研究》;20021231;第20卷(第6期);第449-452页 * |
| 贝母组织培养研究进展;耿茂林等;《淮北煤炭师范学院学报（自然科学版）》;20070331;第28卷(第1期);第38-42页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103598100A (en) | 2014-02-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10624278B2 (en) | Cultivation method for the rapid propagation of Davidia involucrata winter buds | |
| CN109258460B (en) | Cultivation method for obtaining virus-free seedlings of Zea mays by combining micro-stem tip cultivation with heat treatment | |
| CN104855285B (en) | A kind of organic culture one-step-seedling formation abductive approach of polyploid Herba Anoectochili roxburghii | |
| CN107155898B (en) | A kind of dendrobium candidum carries out expanding numerous method using stem section thin slice | |
| CN104813939A (en) | Method for constructing lotus regeneration system | |
| CN103598100B (en) | Cultural method of bulbus fritillariae cirrhosae polyploidy virus-free seedlings | |
| CN109757373A (en) | A kind of Jing Banxia quick breeding method for tissue culture | |
| CN101933455A (en) | In vitro propagation method for cinnamomum japonicum | |
| CN110150152A (en) | A kind of method of tissue cultivating strawberry | |
| Bag et al. | An efficient method for acclimatization | |
| CN105145365A (en) | Green globular body tissue culture propagation method for Alsophila costularis | |
| CN100394845C (en) | In-bottle production method of detoxified small seed ball of east lily | |
| CN104719158A (en) | Method for rapidly establishing medium-sized Chinese pennisetum herb tissue culture regeneration system by taking seeds as explants | |
| CN107223566B (en) | A kind of Wulian poplar method for tissue culture | |
| CN103229721B (en) | Tissue culture propagation method of Gynura formosana | |
| CN103155869A (en) | Sweet cherry rootstock Colt tissue culture method | |
| CN115250922B (en) | Method for inducing new wheat straw epicotyl to form callus and regenerating plant | |
| CN114698549B (en) | Tissue culture medium and tissue culture method for rapid propagation of grape stock stem segments | |
| CN114600774B (en) | Atractylodes chinensis tissue culture and rapid propagation method | |
| CN107466864B (en) | A kind of method of dendrobium candidum vegetative propagation sprouting and rooting | |
| CN103141384A (en) | Rapid tissue culture propagation method of pot chrysanthemum cultivars | |
| CN109673514A (en) | A kind of method of south yampi type Chinese yam tissue-culturing quick-propagation | |
| CN106665358A (en) | Rapid shoot induction and tissue culture propagation method for platycodon grandiflorum leaves | |
| CN102630566A (en) | Method for cultivation and production of paclitaxel from induction of taxus chinensis var. mairei axillary bud in vitro | |
| CN105850735A (en) | Haloxylon ammodendron regenerated seedling and callus acquisition method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150325 Termination date: 20161127 |