CN103461137B - Method for inducing generation of fritillaria cirrhosa embryoids - Google Patents
Method for inducing generation of fritillaria cirrhosa embryoids Download PDFInfo
- Publication number
- CN103461137B CN103461137B CN201310436775.9A CN201310436775A CN103461137B CN 103461137 B CN103461137 B CN 103461137B CN 201310436775 A CN201310436775 A CN 201310436775A CN 103461137 B CN103461137 B CN 103461137B
- Authority
- CN
- China
- Prior art keywords
- illumination
- embryoid
- bulbus fritillariae
- fritillariae cirrhosae
- cultivate
- 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.)
- Active
Links
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for inducing generation of fritillaria cirrhosa embryoids, belonging to the technical field of biologics. The method comprises the following steps of selecting tender leaf sheath of a fritillaria cirrhosa plant as an explant, sterilizing, subsequently carrying out callus induction, grafting the callus into an MS (Mass Spectrometry)+cefotaxime sodium culture medium, culturing for 50 days to grow embryoids on the surface of the callus, further cutting down the embryoids taking 1-3 embryoids as one group, grafting into a rapid growth culture medium with 0.1-1mg.L<-1> MS+6-BA+100-450mg.L<-1> cefotaxime sodium, culturing for 60 days under the condition that the culture temperature is 15-25 DEG C, the embryoids are shined for 6-20 hours every day, and the shining intensity is 800-2,500lx, so as to grow the embryoids into green small fritillaria cirrhosa plants. According to the method, the fritillaria cirrhosa embryoids can be induced by utilizing antibiotic chemical factors of a certain concentration, and a novel way is provided for breeding of the fritillaria cirrhosa embryoids.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of method of inducing Bulbus Fritillariae Cirrhosae embryoid to produce particularly.
Background technology
Bulbus Fritillariae Cirrhosae (Fritillaria cirrhosa D.Don) is Liliaceae herbaceos perennial, is used as medicine, grows the height at height above sea level 3500m ~ 4500m with bulb, main product in Sichuan, Tibet, the provinces and regions such as Yunnan.Bulbus Fritillariae Cirrhosae likes Cold and cool climate condition, has cold-resistant, to like wet, be afraid of high temperature, happiness concealment characteristic.Bulbus Fritillariae Cirrhosae is the good medicine of important treatment cough, 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 plant growth is in the alpine scrub or meadow of height above sea level 2800-4700m, main dependence seed carries out sexual propagation, but because of the characteristic of its seed existing forms and physiology after-ripening, the problem such as therefore have that Bulbus Fritillariae Cirrhosae seedling fostering difficulty is large, the plantation time is long and reproduction coefficient is low.
Tissue culture technique is an important biotechnology.Current Bulbus Fritillariae Cirrhosae tissue culture technology is all the histoorgan by utilizing Bulbus Fritillariae Cirrhosae, selects when different growth hormone and basic element of cell division proportioning, obtains regeneration plant through evoked callus, Bud polarization, Shoot propagation and multiple link such as to take root." induction of bulb of fritillary callus and plant regeneration " as people such as the research of shoot regeneration frequency " improve Fritillaria Cirrhosa D. Don " of the people such as Liu Fan and Gao Yan is studied.Not only there is the problem that the Fiber differentiation cycle is long, cost cost is high in this cultural method, and there is culture materials in long incubation, easily send out variation celliferous, thus be difficult to guarantee former constancy of species.And the embryoid utilizing tissue culture technique to cultivate have avette with plant fertilization become the similar structure of zygotic embryo, the regeneration plant of Bulbus Fritillariae Cirrhosae can not only be obtained fast, in large quantities, and due to embryoid be all generally originate from individual cells, therefore, the Bulbus Fritillariae Cirrhosae embryoid cultivated all has the uniformity in genetic background, for the change carrying out various Physiology and biochemistry in the Cell Differentiation of Bulbus Fritillariae Cirrhosae plant and morphogenesis process, the research of the relevant issues such as structure change and gene expression has great importance.The current report that yet there are no by adopting the mode of cultivating Bulbus Fritillariae Cirrhosae embryoid to obtain regeneration plant.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, provide a kind of method of inducing Bulbus Fritillariae Cirrhosae embryoid to produce, the method step is simple, and workable, process stabilizing, is applicable to the extensive use in industrialized production.
The object of the invention is to be achieved through the following technical solutions:
Induce the method that Bulbus Fritillariae Cirrhosae embryoid produces, it comprises the following steps:
S1: the young tender leaf sheath choosing Bulbus Fritillariae Cirrhosae plant is explant, after clean with running water, with alcohol disinfecting 30 ~ 60s that concentration is 70% ~ 75%, then be the mercuric chloride sterilization 4 ~ 10min of 0.1% ~ 0.15% by concentration, finally wash 3 ~ 6 times with sterile water concussion, wash the rear moisture with sterilized filter paper blotting material surface, treated leaf sheath is inoculated in medium MS+6-BA 0.5 ~ 2.0mgL
-1+ 2,4-D 1 ~ 2mgL
-1+ IAA 0.1 ~ 1mgL
-1+ sucrose 20 ~ 60gL
-1+ agar 5.0 ~ 7.0 gL
-1in cultivate, the cultivation temperature adopted 16 ~ 20 DEG C, illumination every day 6 ~ 14h, intensity of illumination are evoked callus under the condition of 1000 ~ 2000lx;
S2: the color chosen through S1 step process is white and flaxen callus, and being cut into size is 0.5 ~ 2.5cm
2, then access MS+ Cefotaxime Sodium 200 ~ 600mgL
-1cultivate in medium and obtain callus embryoid in 40 ~ 60 days;
S3: callus embryoid is 1 group with 1 ~ 3 and cuts, accesses its quick growth medium MS+6-BA 0.1 ~ 1mgL
-1+ Cefotaxime Sodium 100 ~ 450mgL
-1in, the cultivation temperature adopted is 15 ~ 25 DEG C, illumination every day 6 ~ 20 hours, intensity of illumination are cultivate after 50 ~ 60 days under the condition of culture of 800 ~ 2500lx, obtains Bulbus Fritillariae Cirrhosae plantlet.
Preferably, the explant described in S1 step is the young tender leaf sheath being positioned at basal part of stem end.
Preferably, the medium described in S1 step is MS+6-BA 0.58mgL
-1+ 2,4-D 1.5mgL
-1+ IAA 0.5 mgL
-1+ sucrose 40gL
-1+ agar 6.0gL
-1.
Preferably, the cultivation temperature described in S1 step is 20 DEG C, illumination every day 10h, intensity of illumination be 1500lx.
Preferably, the medium described in S2 step is MS+ Cefotaxime Sodium 200 ~ 600mgL
-1, condition of culture is temperature 15 ~ 22 DEG C, illumination every day 8 ~ 16h, intensity of illumination are 1500 ~ 2500lx.
Preferably, the medium described in S2 step is MS+ Cefotaxime Sodium 400mgL
-1, condition of culture is temperature 18 DEG C, illumination every day 12h, intensity of illumination are cultivate 50 days under the condition of 1800lx.
Preferably, the embryoid described in S3 step is 1 group by 2 and cuts, access embryoid quick growth medium MS+6-BA 0.5mgL
-1+ Cefotaxime Sodium 300mgL
-1in, adopt that cultivation temperature is 22 DEG C, illumination every day 16 hours, intensity of illumination be cultivate 60 days under the condition of culture of 2000lx.
Preferably, complete after S3 step cultivates, opening and cultivate bottle cap and after 2 ~ 4 days, Bulbus Fritillariae Cirrhosae plantlet is taken out from blake bottle in indoor hardening, after washing away the medium on plant, then be transplanted in soft soil and grow.
Beneficial effect of the present invention is:
Present invention utilizes certain density antibiotics chemokines and induce Bulbus Fritillariae Cirrhosae embryoid, further providing a new way for utilizing the biotechnology embryoid that cultivates plants now.This technology is workable, has that less investment, cost are low, efficiency is high a advantage, and has important practical significance to effectively solving Bulbus Fritillariae Cirrhosae seedling shortage problem.
Embodiment
Below in conjunction with embodiment, the present invention will be further described, and protection scope of the present invention is not limited to the following stated:
Embodiment 1: a kind of method of inducing Bulbus Fritillariae Cirrhosae embryoid to produce, it comprises the following steps:
S1: the young tender leaf sheath choosing Bulbus Fritillariae Cirrhosae plant is explant, after clean with running water, with alcohol disinfecting 30 ~ 60s that concentration is 70% ~ 75%, then be the mercuric chloride sterilization 4 ~ 10min of 0.1% ~ 0.15% by concentration, finally wash 3 ~ 6 times with sterile water concussion, wash the rear moisture with sterilized filter paper blotting material surface, treated leaf sheath is inoculated in medium MS+6-BA 0.5 ~ 2.0mgL
-1+ 2,4-D 1 ~ 2mgL
-1+ IAA 0.1 ~ 1mgL
-1+ sucrose 20 ~ 60gL
-1+ agar 5.0 ~ 7.0 gL
-1in cultivate, the cultivation temperature adopted 16 ~ 20 DEG C, illumination every day 6 ~ 14h, intensity of illumination are evoked callus under the condition of 1000 ~ 2000 lx;
S2: the color chosen through S1 step process is white and flaxen callus, and being cut into size is 0.5 ~ 2.5cm
2, then access MS+ Cefotaxime Sodium 200 ~ 600mgL
-1cultivate in medium and obtain callus embryoid in 40 ~ 60 days;
S3: callus embryoid is 1 group with 1 ~ 3 and cuts, accesses its quick growth medium MS+6-BA0.1 ~ 1mgL
-1+ Cefotaxime Sodium 100 ~ 450mgL
-1in, the cultivation temperature adopted is 15 ~ 25 DEG C, illumination every day 6 ~ 20 hours, intensity of illumination are cultivate after 50 ~ 60 days under the condition of culture of 800 ~ 2500lx, obtains Bulbus Fritillariae Cirrhosae plantlet.
Embodiment 2: a kind of method of inducing Bulbus Fritillariae Cirrhosae embryoid to produce, it comprises the following steps:
S1: the young tender leaf sheath choosing Bulbus Fritillariae Cirrhosae plant is explant, after clean with running water, with the alcohol disinfecting 30s that concentration is 70%, then be the mercuric chloride sterilization 4min of 0.1% by concentration, finally wash 3 times with sterile water concussion, wash the rear moisture with sterilized filter paper blotting material surface, treated leaf sheath is inoculated in medium MS+6-BA 0.5mgL
-1+ 2,4-D 1mgL
-1+ IAA 0.1mgL
-1+ sucrose 20gL
-1+ agar 5.0gL
-1in cultivate, the cultivation temperature adopted 16 DEG C, illumination every day 6h, intensity of illumination are evoked callus under the condition of 1000lx;
S2: the color chosen through S1 step process is white and flaxen callus, and being cut into size is 0.5 ~ 2.5cm
2, then access MS+ Cefotaxime Sodium 200mgL
-1cultivate in medium and obtain callus embryoid in 40 days;
S3: callus embryoid is 1 group with 2 and cuts, accesses its quick growth medium MS+6-BA 0.1mgL
-1+ Cefotaxime Sodium 100mgL
-1in, the cultivation temperature adopted is 15 DEG C, illumination every day 6h, intensity of illumination are cultivate after 50 days under the condition of culture of 800lx, obtains Bulbus Fritillariae Cirrhosae plantlet.
Embodiment 3: a kind of method of inducing Bulbus Fritillariae Cirrhosae embryoid to produce, it comprises the following steps:
S1: the young tender leaf sheath choosing Bulbus Fritillariae Cirrhosae plant is explant, after clean with running water, with the alcohol disinfecting 60s that concentration is 75%, then be the mercuric chloride sterilization 10min of 0.15% by concentration, finally wash 6 times with sterile water concussion, wash the rear moisture with sterilized filter paper blotting material surface, treated leaf sheath is inoculated in medium MS+6-BA 2.0 mgL
-1+ 2,4-D 2mgL
-1+ IAA 1mgL
-1+ sucrose 60gL
-1+ agar 7.0gL
-1in cultivate, the cultivation temperature adopted 20 DEG C, illumination every day 14h, intensity of illumination are evoked callus under the condition of 2000lx;
S2: the color chosen through S1 step process is white and flaxen callus, and being cut into size is 2.5cm
2, then access MS+ Cefotaxime Sodium 600mgL
-1cultivate in medium and obtain callus embryoid in 60 days;
S3: callus embryoid is 1 group with 3 and cuts, accesses its quick growth medium MS+6-BA 1mgL
-1+ Cefotaxime Sodium 450mgL
-1in, the cultivation temperature adopted is 25 DEG C, illumination every day 20h, intensity of illumination are cultivate after 60 days under the condition of culture of 2500lx, obtains Bulbus Fritillariae Cirrhosae plantlet.
Embodiment 4: a kind of method of inducing Bulbus Fritillariae Cirrhosae embryoid to produce, it comprises the following steps:
S1: the young tender leaf sheath choosing Bulbus Fritillariae Cirrhosae plant is explant, after clean with running water, with alcohol disinfecting 30 ~ 60s that concentration is 70% ~ 75%, then be the mercuric chloride sterilization 6min of 0.1% ~ 0.15% by concentration, finally wash 5 times with sterile water concussion, wash the rear moisture with sterilized filter paper blotting material surface, treated leaf sheath is inoculated in medium MS+6-BA 0.58 mgL
-1+ 2,4-D 1.5mgL
-1+ IAA 0.5mgL
-1+ sucrose 40gL
-1+ agar 6.0gL
-1in cultivate, the cultivation temperature adopted 20 DEG C, illumination every day 10h, intensity of illumination are evoked callus under the condition of 1500lx;
S2: the color chosen through S1 step process is white and flaxen callus, and being cut into size is 1.5cm
2, then access MS+ Cefotaxime Sodium 400mgL
-1cultivate in medium and obtain callus embryoid in 50 days, condition of culture is 18 DEG C, illumination every day 12h, intensity of illumination are 2000lx;
S3: callus embryoid is 1 group with 2 and cuts, accesses its quick growth medium MS+6-BA 0.5mgL
-1+ Cefotaxime Sodium 350mgL
-1in, the cultivation temperature adopted is 22 DEG C, illumination every day 16 hours, intensity of illumination are cultivate after 60 days under the condition of culture of 2000lx, obtains Bulbus Fritillariae Cirrhosae plantlet.
Complete after S3 step cultivates, opening and cultivate bottle cap and after 2 ~ 4 days, Bulbus Fritillariae Cirrhosae plantlet is taken out from blake bottle in indoor hardening, after washing away the medium on plant, then be transplanted in soft soil and grow.
Embodiment 5: a kind of method of inducing Bulbus Fritillariae Cirrhosae embryoid to produce, it comprises the following steps:
S1: choosing the young tender leaf sheath that Bulbus Fritillariae Cirrhosae plant is positioned at basal part of stem end is explant, after clean with running water, with the alcohol disinfecting 40s that concentration is 72%, then to sterilize 5 min with the mercuric chloride that concentration is 0.12% ~ 0.15%, finally wash 4 times with sterile water concussion, wash the rear moisture with sterilized filter paper blotting material surface, treated leaf sheath is inoculated in medium MS+6-BA 0.8 mgL
-1+ 2,4-D 1.2mgL
-1+ IAA 0.6mgL
-1+ sucrose 30gL
-1+ agar 5.5gL
-1in cultivate, the cultivation temperature adopted 18 DEG C, illumination every day 8h, intensity of illumination are evoked callus under the condition of 1200lx;
S2: the color chosen through S1 step process is white and flaxen callus, and being cut into size is 0.5 ~ 1.0cm
2, then access MS+ Cefotaxime Sodium 300mgL
-1cultivate in medium and obtain callus embryoid in 45 days;
S3: callus embryoid is 1 group with 2 and cuts, accesses its quick growth medium MS+6-BA 0.6mgL
-1+ Cefotaxime Sodium 250mgL
-1in, the cultivation temperature adopted is 18 DEG C, illumination every day 10 hours, intensity of illumination are cultivate after 55 days under the condition of culture of 1200lx, obtains Bulbus Fritillariae Cirrhosae plantlet.
Complete after S3 step cultivates, opening and cultivate bottle cap and after 3 days, Bulbus Fritillariae Cirrhosae plantlet is taken out from blake bottle in indoor hardening, after washing away the medium on plant, then be transplanted in soft soil and grow.
Embodiment 6: a kind of method of inducing Bulbus Fritillariae Cirrhosae embryoid to produce, it comprises the following steps:
S1: choosing the young tender leaf sheath that Bulbus Fritillariae Cirrhosae plant is positioned at basal part of stem end is explant, after clean with running water, with alcohol disinfecting 30 ~ 60s that concentration is 70% ~ 75%, then be the mercuric chloride sterilization 4 ~ 10min of 0.1% ~ 0.15% by concentration, finally wash 3 ~ 6 times with sterile water concussion, wash the rear moisture with sterilized filter paper blotting material surface, treated leaf sheath is inoculated in medium MS+6-BA 0.5 ~ 2.0mgL
-1+ 2,4-D 1 ~ 2mgL
-1+ IAA 0.1 ~ 1mgL
-1+ sucrose 20 ~ 60gL
-1+ agar 5.0 ~ 7.0gL
-1in cultivate, the cultivation temperature adopted 16 ~ 18 DEG C, illumination every day 10 ~ 12h, intensity of illumination are evoked callus under the condition of 1500 ~ 1800lx;
S2: the color chosen through S1 step process is white and flaxen callus, and being cut into size is 1.0 ~ 2.0cm
2, then access MS+ Cefotaxime Sodium 300 ~ 400mgL
-1cultivate in medium and obtain callus embryoid in 45 ~ 50 days;
S3: callus embryoid is 1 group with 3 and cuts, accesses its quick growth medium MS+6-BA 0.5 ~ 1mgL
-1+ Cefotaxime Sodium 300 ~ 400mgL
-1in, the cultivation temperature adopted is 18 ~ 20 DEG C, illumination every day 10 ~ 15 hours, intensity of illumination are cultivate after 50 ~ 55 days under the condition of culture of 1500 ~ 2000lx, obtains Bulbus Fritillariae Cirrhosae plantlet.
Complete after S3 step cultivates, opening and cultivate bottle cap and after 2 ~ 4 days, Bulbus Fritillariae Cirrhosae plantlet is taken out from blake bottle in indoor hardening, after washing away the medium on plant, then be transplanted in soft soil and grow.
Embodiment 7: a kind of method of inducing Bulbus Fritillariae Cirrhosae embryoid to produce, it comprises the following steps:
S1: the young tender leaf sheath choosing Bulbus Fritillariae Cirrhosae plant is explant, after clean with running water, with alcohol disinfecting 50 ~ 60s that concentration is 70% ~ 75%, then be the mercuric chloride sterilization 6 ~ 8min of 0.1% ~ 0.15% by concentration, finally wash 5 times with sterile water concussion, wash the rear moisture with sterilized filter paper blotting material surface, treated leaf sheath is inoculated in medium MS+6-BA 2.0 mgL
-1+ 2,4-D 2mgL
-1+ IAA 1mgL
-1+ sucrose 50gL
-1+ agar 6.0gL
-1in cultivate, the cultivation temperature adopted 16 ~ 18 DEG C, illumination every day 10 ~ 12h, intensity of illumination are evoked callus under the condition of 1800 ~ 2000 lx;
S2: the color chosen through S1 step process is white and flaxen callus, and being cut into size is 0.5 ~ 1.5cm
2, then access MS+ Cefotaxime Sodium 400 ~ 500mgL
-1cultivate in medium and obtain callus embryoid in 40 days;
S3: callus embryoid is 1 group with 3 and cuts, accesses its quick growth medium MS+6-BA 0.5 ~ 1mgL
-1+ Cefotaxime Sodium 400 ~ 450mgL
-1in, the cultivation temperature adopted is 20 ~ 25 DEG C, illumination every day 15 ~ 20h, intensity of illumination are cultivate after 50 days under the condition of culture of 2000 ~ 2500lx, obtains Bulbus Fritillariae Cirrhosae plantlet.
Complete after S3 step cultivates, opening and cultivate bottle cap and after 2 ~ 4 days, Bulbus Fritillariae Cirrhosae plantlet is taken out from blake bottle in indoor hardening, after washing away the medium on plant, then be transplanted in soft soil and grow.
Embodiment 8: a kind of method of inducing Bulbus Fritillariae Cirrhosae embryoid to produce, it comprises the following steps:
S1: choosing the young tender leaf sheath that Bulbus Fritillariae Cirrhosae plant is positioned at basal part of stem end is explant, after clean with running water, with alcohol disinfecting 30 ~ 60s that concentration is 70% ~ 75%, then be the mercuric chloride sterilization 4 ~ 10min of 0.1% ~ 0.15% by concentration, finally wash 3 ~ 6 times with sterile water concussion, wash the rear moisture with sterilized filter paper blotting material surface, treated leaf sheath is inoculated in medium MS+6-BA 0.58mgL
-1+ 2,4-D 1.5mgL
-1+ IAA 0.5mgL
-1+ sucrose 40gL
-1+ agar 6.0gL
-1in cultivate, the cultivation temperature adopted is 20 DEG C, illumination every day 10h, intensity of illumination are evoked callus under the condition of 1500lx;
S2: the color chosen through S1 step process is white and flaxen callus, and being cut into size is 0.5 ~ 2.5cm
2, then access MS+ Cefotaxime Sodium, 400mgL
-1cultivate in medium, condition of culture be temperature 18 DEG C, illumination every day 12h, intensity of illumination be under the condition of 1800lx cultivate 50 days, obtain callus embryoid;
S3: callus embryoid is 1 group with 1 ~ 3 and cuts, accesses its quick growth medium MS+6-BA 0.1 ~ 1mgL
-1+ Cefotaxime Sodium 100 ~ 450mgL
-1in, the cultivation temperature adopted is 18 DEG C, illumination every day 12 hours, intensity of illumination are cultivate after 50 days under the condition of culture of 1800lx, obtains Bulbus Fritillariae Cirrhosae plantlet.
Complete after S3 step cultivates, opening and cultivate bottle cap and after 3 days, Bulbus Fritillariae Cirrhosae plantlet is taken out from blake bottle in indoor hardening, after washing away the medium on plant, then be transplanted in soft soil and grow.
Embodiment 9: a kind of method of inducing Bulbus Fritillariae Cirrhosae embryoid to produce, it comprises the following steps:
S1: the young tender leaf sheath choosing Bulbus Fritillariae Cirrhosae plant is explant, after clean with running water, with alcohol disinfecting 40 ~ 50s that concentration is 70% ~ 75%, then be mercuric chloride sterilization 6 ~ 8 min of 0.1% ~ 0.15% by concentration, finally wash 5 times with sterile water concussion, wash the rear moisture with sterilized filter paper blotting material surface, treated leaf sheath is inoculated in medium MS+6-BA 0.5 ~ 2.0mgL
-1+ 2,4-D 1 ~ 2mgL
-1+ IAA 0.1 ~ 1mgL
-1+ sucrose 20 ~ 60gL
-1+ agar 5.0 ~ 7.0 gL
-1in cultivate, the cultivation temperature adopted 16 ~ 20 DEG C, illumination every day 6 ~ 14h, intensity of illumination are evoked callus under the condition of 1000 ~ 2000 lx;
S2: the color chosen through S1 step process is white and flaxen callus, and being cut into size is 0.5 ~ 2.5cm
2, then access MS+ Cefotaxime Sodium 200 ~ 600mgL
-1cultivate in medium, condition of culture be temperature 18 DEG C, illumination every day 12h, intensity of illumination be under the condition of 1800lx cultivate 50 days, obtain callus embryoid;
S3: callus embryoid is 1 group with 1 ~ 3 and cuts, accesses its quick growth medium MS+6-BA 0.1 ~ 1mgL
-1+ Cefotaxime Sodium 100 ~ 450mgL
-1in, the cultivation temperature adopted is 15 ~ 25 DEG C, illumination every day 6 ~ 20 hours, intensity of illumination are cultivate after 50 ~ 60 days under the condition of culture of 800 ~ 2500lx, obtains Bulbus Fritillariae Cirrhosae plantlet.
Complete after S3 step cultivates, opening and cultivate bottle cap and after 2 ~ 4 days, Bulbus Fritillariae Cirrhosae plantlet is taken out from blake bottle in indoor hardening, after washing away the medium on plant, then be transplanted in soft soil and grow.
Embodiment 10: a kind of method of inducing Bulbus Fritillariae Cirrhosae embryoid to produce, it comprises the following steps:
S1: choosing the young tender leaf sheath that Bulbus Fritillariae Cirrhosae plant is positioned at basal part of stem end is explant, after clean with running water, with alcohol disinfecting 30 ~ 60s that concentration is 70% ~ 75%, then be the mercuric chloride sterilization 4 ~ 10min of 0.1% ~ 0.15% by concentration, finally wash 3 ~ 6 times with sterile water concussion, wash the rear moisture with sterilized filter paper blotting material surface, treated leaf sheath is inoculated in medium MS+6-BA 0.58mgL
-1+ 2,4-D 1.5mgL
-1+ IAA 0.5mgL
-1+ sucrose 40gL
-1+ agar 6.0gL
-1in cultivate, the cultivation temperature adopted is 20 DEG C, illumination every day 10h, intensity of illumination are evoked callus under the condition of 1500lx;
S2: the color chosen through S1 step process is white and flaxen callus, and being cut into size is 0.5 ~ 2.5cm
2, then access MS+ Cefotaxime Sodium 400mgL
-1, condition of culture is temperature 18 DEG C, illumination every day 12h, intensity of illumination are cultivate 50 days under the condition of 1800lx, obtains callus embryoid;
S3: callus embryoid is 1 group with 3 and cuts, accesses its quick growth medium MS+ Cefotaxime Sodium 400mgL
-1, condition of culture is temperature 22 DEG C, illumination every day 16h, intensity of illumination are cultivate 60 days under the condition of 2000lx, obtains Bulbus Fritillariae Cirrhosae plantlet.
Complete after S3 step cultivates, opening and cultivate bottle cap and after 2 ~ 4 days, Bulbus Fritillariae Cirrhosae plantlet is taken out from blake bottle in indoor hardening, after washing away the medium on plant, then be transplanted in soft soil and grow.
Embodiment 11: a kind of method of inducing Bulbus Fritillariae Cirrhosae embryoid to produce, it comprises the following steps:
S1: the young tender leaf sheath choosing Bulbus Fritillariae Cirrhosae plants stems base end is explant, after clean with running water, be placed on super-clean bench with the alcohol disinfecting 40s that concentration is 70%, then be the mercuric chloride sterilization 8min of 0.1% by concentration, finally wash 5 times with sterile water concussion, wash the rear moisture with sterilized filter paper blotting material surface, be inoculated in MS+6-BA 0.8mgL
-1+ 2,4-D 1.5mgL
-1+ IAA 0.5mgL
-1+ sucrose 40gL
-1+ agar 6.0gL
-1in, evoked callus under cultivation temperature 20 DEG C, illumination every day 10h, intensity of illumination are the condition of 1500lx;
S2: get colors into white and faint yellow, quality comparatively closely and the callus of fast growth, being cut into size is 2cm
2size access MS+ Cefotaxime Sodium 400mgL
-1in medium, condition of culture be temperature 18 DEG C, illumination every day 12h, intensity of illumination cultivate after 50 days under being the condition of 1800lx, has about 3 ~ 6 whites or yellow-white, the spherical embryoid generation in garden that volume is larger as seen on the surface of callus;
S3: embryoid is 1 group by 2 and cuts, access embryoid quick growth medium MS+6-BA 0.5mgL
-1+ Cefotaxime Sodium 300mgL
-1in, cultivation temperature be 22 DEG C, illumination every day 16h, intensity of illumination cultivate after 60 days under being the condition of culture of 2000lx, visible embryoid is grown to the green plantlet of healthy and strong Bulbus Fritillariae Cirrhosae fast.
Choose the Bulbus Fritillariae Cirrhosae plantlet of growth at more than 2cm, open bottle cap, after 3 days Bulbus Fritillariae Cirrhosae plantlet to be taken out from blake bottle in indoor hardening, and after Bulbus Fritillariae Cirrhosae plantlet is divided into individual plant, wash away the medium on plant, then be transplanted in soft soil and grow.
Effect of the present invention is further illustrated below by experiment:
Experiment one: Bulbus Fritillariae Cirrhosae embryoid of the present invention produces experiment
S1: the young tender leaf sheath choosing Bulbus Fritillariae Cirrhosae plants stems base end is explant, after clean with running water, be placed on super-clean bench with the alcohol disinfecting 40s that concentration is 70%, then be the mercuric chloride sterilization 8min of 0.1% by concentration, finally wash 5 times with sterile water concussion, wash the rear moisture with sterilized filter paper blotting material surface, be inoculated in MS+6-BA 0.8mgL
-1+ 2,4-D 1.5mgL
-1+ IAA 0.5mgL
-1+ sucrose 40gL
-1+ agar 6.0gL
-1in, evoked callus under cultivation temperature 20 DEG C, illumination every day 10h, intensity of illumination are the condition of 1500lx;
S2: get colors into white and faint yellow, quality comparatively closely and the callus of fast growth, being cut into size is 2cm
2size access MS+ Cefotaxime Sodium 400mgL
-1in medium, condition of culture be temperature 18 DEG C, illumination every day 12h, intensity of illumination cultivate after 50 days under being the condition of 1800lx, has about 3 ~ 6 whites or yellow-white, the spherical embryoid generation in garden that volume is larger as seen on the surface of callus;
S3: embryoid is 1 group by 2 and cuts, access embryoid quick growth medium MS+6-BA 0.5mgL
-1+ Cefotaxime Sodium 300mgL
-1in, cultivation temperature be 22 DEG C, illumination every day 16h, intensity of illumination cultivate after 60 days under being the condition of culture of 2000lx, visible embryoid is grown to the green plantlet of healthy and strong Bulbus Fritillariae Cirrhosae fast.
Choose the Bulbus Fritillariae Cirrhosae plantlet of growth at more than 2cm, open bottle cap, after 3 days Bulbus Fritillariae Cirrhosae plantlet to be taken out from blake bottle in indoor hardening, and after Bulbus Fritillariae Cirrhosae plantlet is divided into individual plant, wash away the medium on plant, then be transplanted in soft soil and grow.
Experiment two: change the medium that inducing embryoid body produces into MS+ Cefotaxime Sodium 200mgL in experiment one S2 step
-1, other step is with experiment one; After cultivating through 50 days, the surface of callus produces embryoid negligible amounts, only has 1 ~ 3.
Experiment three: change the medium that inducing embryoid body produces into MS+ Cefotaxime Sodium 600mgL in experiment one S2 step
-1, other step is with experiment one; After cultivating through 50 days, the surface of callus produces embryoid quantity more, have about 6 ~ 10.But the quality producing embryoid is not good, and shape is elongated taper shape, color mostly also is yellow, and with a small amount of browning.
Experiment four: in experiment one S3 step, the condition of culture of somatic embryogenesis Bulbus Fritillariae Cirrhosae plantlet is changed into: cultivation temperature is 15 DEG C, illumination every day 6 hours and intensity of illumination are cultivate under the condition of culture of 800lx, other step is with experiment one; After cultivating through 60 days, somatic embryogenesis Bulbus Fritillariae Cirrhosae plantlet is green-yellow, shorter and smaller.
Experiment five: reelect in experiment one S2 step as yellow, open-textured callus are that explant induction embryoid produces, other step is with experiment one; After cultivating through 50 days, have shape to be that the embryoid of elongate tapered produces as seen on the surface of Lax callus, but the embryoid quality produced is not good, and with obvious vitrification phenomenon, the Bulbus Fritillariae Cirrhosae plantlet that the later stage is cultivated can not healthy growth.
Experiment six: by experiment one S2 step, the medium of inducing embryoid body changes into only selects minimal medium MS0, and does not add the antibiotic of any concentration, and other step is with embodiment 1; After cultivating through 50 days, find that callus only produces a small amount of propagation, but produce there are no embryoid.
Experiment seven: in experiment one S3 step, the medium of somatic embryogenesis Bulbus Fritillariae Cirrhosae plantlet changes MS+6-BA 2mgL into
-1+ Cefotaxime Sodium 600mgL
-1in, other step is with embodiment 1; After cultivating through 60 days, the Bulbus Fritillariae Cirrhosae plantlet poor growth of regeneration, form is short and small, and has part to occur brownization situation.
Experiment eight: the condition of culture of somatic embryogenesis Bulbus Fritillariae Cirrhosae plantlet changes into and cultivating under the condition of unglazed photograph in every day in experiment one S3 step, and other step is with embodiment 1; After cultivating through 60 days, embryoid shows as own vol and obviously increases, but the Bulbus Fritillariae Cirrhosae plant grown is short and small etiolated seedling.
Experiment nine: in experiment one S3 step, the condition of culture of somatic embryogenesis Bulbus Fritillariae Cirrhosae plantlet changes into, cultivation temperature be 26 DEG C, after illumination every day more than 24 hours and intensity of illumination cultivate 60 days under being the condition of culture of 3000lx, although it is very fast that embryoid takes out seedling, but the Bulbus Fritillariae Cirrhosae plant of extracting out mostly is thin-high shape, and color mostly also is yellow green, also produces with obvious vitrification phenomenon.
The present invention adopts tissue culture technique, with the callus of Bulbus Fritillariae Cirrhosae plant children tender leaf sheath induction for culture materials, establish the quick propagating technology being induced Bulbus Fritillariae Cirrhosae embryoid by antibiotics chemokines, this research is that the cultivation of Bulbus Fritillariae Cirrhosae improved seeds and a large amount of Fast-propagation provide new way.Implement the inventive method in addition, only simple Plant Tissue Breeding equipment need be had to carry out, therefore practical, toxigenic capacity falls, and feasibility is high, simultaneously also for utilizing the tissue culture technique embryoid that cultivates plants to further provide a new way.
Claims (6)
1. a method of inducing Bulbus Fritillariae Cirrhosae embryoid to produce, is characterized in that: it comprises the following steps:
S1: the young tender leaf sheath choosing Bulbus Fritillariae Cirrhosae plant is explant, after clean with running water, with alcohol disinfecting 30 ~ 60 s that concentration is 70 % ~ 75 %, then be mercuric chloride sterilization 4 ~ 10 min of 0.1% ~ 0.15% by concentration, finally wash 3 ~ 6 times with sterile water concussion, wash the rear moisture with sterilized filter paper blotting material surface, treated leaf sheath is inoculated in medium MS+6-BA0.5 ~ 2.0 mgL
-1+ 2,4-D 1 ~ 2mgL
-1+ IAA0.1 ~ 1 mgL
-1+ sucrose 20 ~ 60 gL
-1+ agar 5.0 ~ 7.0 gL
-1in cultivate, the cultivation temperature adopted 16 ~ 20 DEG C, illumination every day 6 ~ 14h, intensity of illumination are evoked callus under the condition of 1000 ~ 2000 lx;
S2: the color chosen through S1 step process is white and flaxen callus, and being cut into size is 0.5 ~ 2.5 cm
2, then access MS+ Cefotaxime Sodium 300 ~ 500 mgL
-1cultivate in medium and obtain callus embryoid in 40 ~ 60 days, condition of culture is temperature 15 ~ 22 DEG C, illumination every day 8 ~ 16h, intensity of illumination are 1500 ~ 2500 lx;
S3: callus embryoid is 1 group with 1 ~ 3 and cuts, accesses its quick growth medium MS+6-BA0.1 ~ 1mgL
-1+ Cefotaxime Sodium 100 ~ 450 mgL
-1in, the cultivation temperature adopted is 15 ~ 25 DEG C, illumination every day 6 ~ 20 hours, intensity of illumination are cultivate after 50 ~ 60 days under the condition of culture of 800 ~ 2500lx, obtains Bulbus Fritillariae Cirrhosae plantlet;
Complete after S3 step cultivates, opening and cultivate bottle cap and after 2 ~ 4 days, Bulbus Fritillariae Cirrhosae plantlet is taken out from blake bottle in indoor hardening, after washing away the medium on plant, then be transplanted in soft soil and grow.
2. a kind of method of inducing Bulbus Fritillariae Cirrhosae embryoid to produce according to claim 1, is characterized in that: the explant described in S1 step is the young tender leaf sheath being positioned at basal part of stem end.
3. a kind of method of inducing Bulbus Fritillariae Cirrhosae embryoid to produce according to claim 1, is characterized in that: the medium described in S1 step is MS+6-BA0.58 mgL
-1+ 2,4-D 1.5mgL
-1+ IAA0.5 mgL
-1+ sucrose 40 gL
-1+ agar 6.0 gL
-1.
4. a kind of method of inducing Bulbus Fritillariae Cirrhosae embryoid to produce according to claim 1, is characterized in that: the cultivation temperature described in S1 step is 20 DEG C, illumination every day 10h, intensity of illumination are 1500 lx.
5. a kind of method of inducing Bulbus Fritillariae Cirrhosae embryoid to produce according to claim 1, is characterized in that: the medium described in S2 step is MS+ Cefotaxime Sodium 400 mgL
-1, condition of culture is temperature 18 DEG C, illumination every day 12h, intensity of illumination are cultivate 50 days under the condition of 1800lx.
6. a kind of method of inducing Bulbus Fritillariae Cirrhosae embryoid to produce according to claim 1, is characterized in that: the embryoid described in S3 step is 1 group by 2 and cuts, the quick growth medium MS+6-BA0.5mgL of access embryoid
-1+ Cefotaxime Sodium 300 mgL
-1in, adopt that cultivation temperature is 22 DEG C, illumination every day 16 hours, intensity of illumination be cultivate 60 days under the condition of culture of 2000 lx.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310436775.9A CN103461137B (en) | 2013-09-24 | 2013-09-24 | Method for inducing generation of fritillaria cirrhosa embryoids |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310436775.9A CN103461137B (en) | 2013-09-24 | 2013-09-24 | Method for inducing generation of fritillaria cirrhosa embryoids |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103461137A CN103461137A (en) | 2013-12-25 |
| CN103461137B true CN103461137B (en) | 2015-05-27 |
Family
ID=49786382
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310436775.9A Active CN103461137B (en) | 2013-09-24 | 2013-09-24 | Method for inducing generation of fritillaria cirrhosa embryoids |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103461137B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114847159A (en) * | 2022-04-28 | 2022-08-05 | 云南龙藏生物科技有限公司 | Tissue culture regeneration method for bulbus fritillariae cirrhosae |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105638458B (en) * | 2015-12-29 | 2018-03-02 | 云南澈川生物科技有限公司 | A kind of method for tissue culture of bulbus fritillariae cirrhosae |
| CN112931210B (en) * | 2021-03-18 | 2022-06-21 | 四川迪菲特药业有限公司 | Bulb proliferation culture method taking fritillaria paniculata bulb discs as explants |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08112045A (en) * | 1994-10-17 | 1996-05-07 | Koa Oil Co Ltd | Method for mass production of seed and seedling of plant of genus fritillaria |
| CN102002474B (en) * | 2009-09-03 | 2012-08-08 | 薛永新 | Rapid bulb multiplication method by using fritillaria cirrhosa leaves as explant |
| CN101638669B (en) * | 2009-09-09 | 2011-11-02 | 成都大学 | Method for producing bulbus fritillariae cirrhosae total alkaloid by adopting cell mass suspension culture |
| CN102100177B (en) * | 2010-11-19 | 2012-08-29 | 成都大学 | Efficient degerming method used during rapid production of bulbus fritillariae cirrhosae compost |
-
2013
- 2013-09-24 CN CN201310436775.9A patent/CN103461137B/en active Active
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114847159A (en) * | 2022-04-28 | 2022-08-05 | 云南龙藏生物科技有限公司 | Tissue culture regeneration method for bulbus fritillariae cirrhosae |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103461137A (en) | 2013-12-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111758559B (en) | Sterile sowing and seedling raising method for distant hybrid seeds of phalaenopsis amabilis and rhynchophylla | |
| CN103283599B (en) | Method for culturing in vitro embryos and regenerating plants of ormosia hosiei in western Hubei province | |
| CN106613988B (en) | The quasi- small method for potted of encrinite category commodity of plastotype quickly breeding difficulty cutting propagation in bottle | |
| CN103960133A (en) | Method for tissue culture and rapid propagation of Rosa rugosa Thunb. | |
| CN105494100A (en) | Tissue culture and rapid propagation method of oriental cherries | |
| CN102487823B (en) | Rapid breeding method of Artemisia annua | |
| CN102217551B (en) | Tissue culture quick reproduction method for dendrobium chrysotoxum lindl bud tips | |
| CN103430842A (en) | Quick propagation method of hybrid orchid tissue culture | |
| CN106718926A (en) | A kind of leaf of plum Chinese ilex quick breeding method for tissue culture | |
| CN103461137B (en) | Method for inducing generation of fritillaria cirrhosa embryoids | |
| CN104145814A (en) | Method for obtaining regeneration plants by stem tissue culture of cerasus cerasoides (var. cerasoides) | |
| CN100394845C (en) | In-bottle production method of detoxified small seed ball of east lily | |
| CN104145813B (en) | Method for propagating stems of aristolochia fordiana | |
| CN103477988A (en) | Culture in vitro and rapid propagation method for syzygium grijsii | |
| CN102577972A (en) | Method for tissue culture of hoya kerrii | |
| CN101015280B (en) | Tissue culture method for fast propagation of primula denticulata ssp.sino-denticulata | |
| CN103155869A (en) | Sweet cherry rootstock Colt tissue culture method | |
| CN101015279B (en) | Tissue culture method for fast propagation of primula poissonii | |
| CN103430822B (en) | Aquaculture seed reproduction method for micro seed tubers of konjac | |
| CN100559935C (en) | The tissue culture and rapid propagation method that orange lamp stand is heralded spring | |
| CN105660398B (en) | A kind of tissue cultivation rapid breeding method of the big wood paint in mao of dam | |
| CN104221851A (en) | Method for in-vitro culture and rapid propagation of gunnera manlcata l | |
| CN111448985A (en) | Tissue culture method of rosa tenuifolia | |
| CN104488726A (en) | Outside-bottle rooting method of radix pseudostellariae test-tube plantlets | |
| CN105359968A (en) | Tissue culture method for cerasus campanulata |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |