CN103749307A - Tissue culture and propagation method for brachystemma calycinum - Google Patents
Tissue culture and propagation method for brachystemma calycinum Download PDFInfo
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- CN103749307A CN103749307A CN201410036700.6A CN201410036700A CN103749307A CN 103749307 A CN103749307 A CN 103749307A CN 201410036700 A CN201410036700 A CN 201410036700A CN 103749307 A CN103749307 A CN 103749307A
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Abstract
The invention discloses a tissue culture and propagation method for brachystemma calycinum, and develops a culture medium used together with the same. By using stems with buds as explants, the method has the advantages of large number of raw materials, and convenience in material accessing. By tissue culture propagation, the culture cycle can be shortened and high-quality seedlings of brachystemma calycinum can be rapidly propagated. The propagation coefficient of 30d buds cultured by the method can reach 6.1 times, the rooting rate of tissue culture seedlings can reach more than 92.6% through expanding propagation, strong seedlings and rooting culture, and the survival rate after transplanting matrix can reach more than 89.5%. Therefore,the method solves the scale seedling problem of brachystemma calycinum, is beneficial to mass production of seedlings, meets market demands, and has a high application value.
Description
Technical field
The invention belongs to Plant Tissue Breeding propagation technique field, relate in particular to the tissue culture propagation of a kind of short lobe China pink.
Background technology
Short lobe China pink, derives from the short lobe of the short lobe carnation of Caryophyllaceae (flower Brachystemma calycinum D.Don), is Caryophyllaceae Mono-species genus; Its root or all herbal medicine claim achyranthes aspera (strengthening), the grass (precious jade) etc. that knots, and taste is sweet, light, and property is flat; Have clearing heat and detoxicating, stimulate the circulation of the blood and cause the muscles and joints to relax, dispel rheumatism, the effect of inducing diuresis to reduce edema, synthetism myogenic, for diseases such as arthralgia due to wind-dampness, traumatic injury, synthetism, nephritic dropsy, osteomyelitis, sore furuncle, scrofulas.Short lobe China pink is distributed in the provinces and regions such as China Guangxi, Sichuan, Yunnan, main product in Long Lin, the Tianlin County in west, osmanthus, reach the clouds, the ground such as Napo County, Jingxi, Tiane, Nandan, Donglan.
Short lobe China pink is to commonly use simply the strong precious jade medicinal material of tradition.In the strong precious jade area in west, osmanthus, conventional this medicine of resident and pig cylinder bone, with pot, are healthy and strong to the strong muscle of children mostly, take good care of health to the people of body void after being ill; Doctor among the people be take this medicine as main ingredient, and treatment lumbar muscle strain, rheumatic arthritis, carpopedal spasm, the disease such as weak has special effect after being ill.In view of its definite effect, Guangxi Wei Jian pharmaceutcal corporation, Ltd is developed to exclusive herbal species " except stasis of blood restoring and treating injured soft tissues falls apart ", situation of selling well Hongkong and Macro and south east asia; Meanwhile, big strong medicine company is also liked the custom of Baoshang according to the usage of Baoshang among the people and Guangdong,Hongkong and Macao resident, plans it and develops into and resemble Canton love-pea vine, smilax, the such soup stock of Niu great Li.Along with continually developing of its series of products, production-scale continuous expansion, raw materials requirement increases to 5 tons of left and right from the hundreds of kilogram of 2007, make its resource day by day in short supply, Long Lin from Tianlin County, native country to periphery of medicinal material purchase, the county such as reach the clouds are again to neighbouring province Yunnan, Guizhou and other places, and existing wild resource has been difficult to meet the production-scale needs of expanding day.For strengthening the protection of short lobe China pink and exploitation, need widely popularize the cultivating and growing of short lobe China pink, realize its artificial cultivation, the primary problem solving is exactly breeding of seedling.The main mode of reproduction of current short lobe China pink is that cuttage and plant division are bred, but this modes of reproduction is difficult to realize amount reproduction seedling in a short time, and after too much for plant division, plant vivotoxin increases, and the stalwartness that affects plant is grown up; And adopt biotechnology tissue culture technique, and can accelerate its reproduction speed and obtain a large amount of test-tube plantlets, can meet the needs in seedling market.
Summary of the invention
The technical problem to be solved in the present invention be to provide a kind of easy to operation, cultivation cycle is short, the tissue culture propagation of breeding the short lobe China pink that quick survival rate is high.
For solving the problems of the technologies described above, the present invention by the following technical solutions: the tissue culture propagation of short lobe China pink, comprises the following steps:
(1) the drawing materials and sterilizing of explant: get the stem with bud that short lobe China pink cuts into 2-3cm and carry out disinfection as explant;
(2) obtaining of in vitro cuttings: the explant disinfecting is placed in to inducing culture and carries out adventitious bud inducing and germinate and to obtain in vitro cuttings;
(3) test-tube plantlet breeding is cultivated: in vitro cuttings is placed in to proliferated culture medium and cultivates a large amount of indefinite buds of acquisition;
(4) the growth of plants is cultivated: the indefinite bud after breeding is placed in to strong seedling culture base and carries out strong seedling culture and obtain healthy and strong plant;
(5) rooting of vitro seedling is cultivated: healthy and strong plant is placed in to root media cultivation and obtains complete band offspring;
(6) test-tube plantlet acclimatization and transplants: screen complete band offspring and carry out transplanting in matrix and cultivating after hardening, then transplant to land for growing field crops;
Inducing culture be take MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, Medium's PH Value is 5.8, and adds 1.0mgL
-16-benzyladenine (6-BA), 0.1mgL
-1methyl α-naphthyl acetate (NAA);
Proliferated culture medium be take MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, 1gL
-1active carbon, Medium's PH Value is 5.8, and adds 0.1-2.0mgL
-16-benzyladenine (6-BA), 0.01-1.0mgL
-1methyl α-naphthyl acetate (NAA), 0.1-0.5mgL
-1heteroauxin (IAA);
Strong seedling culture base be take MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, 1gL
-1active carbon, Medium's PH Value is 5.8, and adds 0.1-1.5mgL
-16-benzyladenine (6-BA), 0.1-2mgL
-1methyl α-naphthyl acetate (NAA);
Root media be take 1/2MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, 1gL
-1active carbon, Medium's PH Value is 5.8, and adds 0.05-1.0mgL
-1indolebutyric acid (IBA), 0.5-2.0mgL
-1methyl α-naphthyl acetate (NAA).
Sterilization in step (1) is undertaken by following operation: first 2-3 is dripped to liquid detergent and splash in the beaker of dress 50ml running water, explant is put into beaker and stir gently 2min, then clean explant surface dirt with cotton, the slight flowing water of running water rinses 8-10min; Dislocation superclean bench, with 75% alcohol immersion 30S, sterile water (distilled water after autoclaving) rinses one time, and then be 0.1% mercuric chloride sterilization 6-8min, aseptic water washing 3-5 time by the 50ml concentration of having added 1-2 and drip Tween-20, finally place the stainless steel plate disinfecting, cut into the long stem with bud of 1.0-1.5cm, obtain aseptic explant.
The condition of culture of step (2) to (5) is: temperature is 23-25 ℃, intensity of illumination 1500-2000lux, and light application time is 10-12h/d.
The incubation time of step (2) to (5) is respectively: 15-20d, 30d, 30d, 25d.
Acclimatization and transplants in step (6) is undertaken by following operation: through step (5), obtain the whole plant with root, treat that seedling grows to 5-6cm, when root grows to 2-4cm, selecting well-grown, neat, healthy and strong bottle seedling, is the indoor hardening of carrying out of 23-25 ℃ in room temperature, and in bottle, add a small amount of running water to flood medium, hardening 5-7d, takes out test-tube plantlet with tweezers, cleans root medium, be transplanted to growth 40-50d in matrix (, draining ventilative to loosen is well advisable), then transplant to land for growing field crops.
Matrix is that perlite, sandstone and silt mix in the ratio of 1:2:1.
The medium that short lobe China pink tissue is cultivated, comprises inducing culture, proliferated culture medium, strong seedling culture base, root media,
Inducing culture be take MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, Medium's PH Value is 5.8, and adds 1.0mgL
-16-benzyladenine (6-BA), 0.1mgL
-1methyl α-naphthyl acetate (NAA);
Proliferated culture medium be take MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, 1gL
-1active carbon, Medium's PH Value is 5.8, and adds 0.1-2.0mgL
-16-benzyladenine (6-BA), 0.01-1.0mgL
-1methyl α-naphthyl acetate (NAA), 0.1-0.5mgL
-1heteroauxin (IAA);
Strong seedling culture base be take MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, 1gL
-1active carbon, Medium's PH Value is 5.8, and adds 0.1-1.5mgL
-16-benzyladenine (6-BA), 0.1-2mgL
-1methyl α-naphthyl acetate (NAA);
Root media be take 1/2MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, 1gL
-1active carbon, Medium's PH Value is 5.8, and adds 0.05-1.0mgL
-1indolebutyric acid (IBA), 0.5-2.0mgL
-1methyl α-naphthyl acetate (NAA).
The problem of breeding existence for current short lobe China pink, inventor has set up the tissue culture propagation of a kind of short lobe China pink, and has developed matching used medium.This method is usingd stem with bud as explant, has that raw material quantity is many, the advantage easily of drawing materials, and passes through tissue culture propagating; can shorten cultivation cycle Fast-propagation and go out short lobe China pink high quality seedling; be conducive to large-scale production seedling, thereby meet the need of market, there is higher using value.
Outstanding advantages of the present invention is:
<1> adopts the short lobe China pink of biological technique method to carry out tissue culture propagating, by induction and the breeding of short lobe China pink indefinite bud, the proterties that can keep original kind, can breed in a short time and obtain the plant that a large amount of proterties are consistent, meet need of production, and for providing the short lobe China pink factorial seedling growth of merit that reference is provided.
<2> MS used and 1/2MS solid culture medium comprise macro-and microelements, the chemical agent lower costs such as 6-benzyladenine, methyl α-naphthyl acetate, heteroauxin and indolebutyric acid, the concentration of adding are suitable, concrete effect is as follows: 6-BA and NAA in inducing culture, can sprout by evoking adventive bud; In proliferated culture medium, 6-BA, NAA and IAA can promote the propagation of bud; In strong seedling culture base, 6-BA and NAA can promote the propagation again of the strong and bud of seedling; In 1/2MS root media, IBA and NAA can promote seedling rooting, after hardening, transplant to the nutrition cup that fills matrix.
This method of <3> is cultivated 30d bud reproduction coefficient can reach 6.1 times;, strong sprout numerous through expanding and culture of rootage; group training seedling rooting rate reaches more than 92.6%, and after transplanting medium, survival rate, more than 89.5%, efficiently solves the scale breeding problem of short lobe China pink.
Embodiment
Embodiment 1
(1) the drawing materials and sterilizing of explant: get the stem with bud that short lobe China pink cuts into 2-3cm and carry out disinfection as explant; First 2-3 is dripped to liquid detergent and splash in the beaker of dress 50ml running water, explant is put into beaker and stir gently 2min, then clean explant surface dirt with cotton, the slight flowing water of running water rinses 8-10min; Dislocation superclean bench, with 75% alcohol immersion 30S, sterile water (distilled water after autoclaving) rinses one time, and then be 0.1% mercuric chloride sterilization 6-8min, aseptic water washing 3-5 time by the 50ml concentration of having added 1-2 and drip Tween-20, finally place the stainless steel plate disinfecting, cut into the long stem with bud of 1.0-1.5cm, obtain aseptic explant.
(2) obtaining of in vitro cuttings: the explant disinfecting is placed in to inducing culture and carries out adventitious bud inducing germination (temperature is 23-25 ℃, intensity of illumination 1500-2000lux, light application time is 10-12h/d) 15-20d, obtain in vitro cuttings;
(3) test-tube plantlet propagation is cultivated: in vitro cuttings is placed in to proliferated culture medium and cultivates (temperature is 23-25 ℃, intensity of illumination 1500-2000lux, light application time is 10-12h/d) 30d, obtain a large amount of indefinite buds, adventitious bud proliferation coefficient is 3.8;
(4) the growth of plants is cultivated: the indefinite bud after breeding is placed in to strong seedling culture base and carries out strong seedling culture (temperature is 23-25 ℃, intensity of illumination 1500-2000lux, light application time is 10-12h/d) 30d, obtain healthy and strong plant, indefinite bud secondary growth coefficient is 2.4;
(5) rooting of vitro seedling is cultivated: healthy and strong plant is placed in to MS root media, and to cultivate 25d(temperature be 23-25 ℃, intensity of illumination 1500-2000lux, light application time is 10-12h/d), obtaining complete band offspring, rooting rate is 92.6%;
(6) test-tube plantlet acclimatization and transplants: obtain the whole plant with root through step (5), treat that seedling grows to 5-6cm, when root grows to 2-4cm, select well-grown, neat, healthy and strong bottle seedling, it in room temperature, is the indoor hardening of carrying out of 23-25 ℃, and in bottle, add a small amount of running water to flood medium, hardening 5-7d, with tweezers, take out test-tube plantlet, clean root medium, be transplanted to growth 40-50d in matrix (perlite, sandstone and silt mix in the ratio of 1:2:1, and, draining ventilative to loosen is well advisable), transplant to land for growing field crops, survival rate is 92% again.
Wherein, the culture medium prescription of each stage use is as follows:
Inducing culture be take MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, Medium's PH Value is 5.8, and adds 1.0mgL
-16-benzyladenine (6-BA), 0.1mgL
-1methyl α-naphthyl acetate (NAA);
Proliferated culture medium be take MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, 1gL
-1active carbon, Medium's PH Value is 5.8, and adds 0.1mgL
-16-benzyladenine (6-BA), 0.05mgL
-1methyl α-naphthyl acetate (NAA), 0.1mgL
-1heteroauxin (IAA);
Strong seedling culture base be take MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, 1gL
-1active carbon, Medium's PH Value is 5.8, and adds 0.1mgL
-16-benzyladenine (6-BA), 0.1mgL
-1methyl α-naphthyl acetate (NAA);
Root media be take 1/2MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, 1gL
-1active carbon, Medium's PH Value is 5.8, and adds 0.05mgL
-1indolebutyric acid (IBA), 0.5mgL
-1methyl α-naphthyl acetate (NAA).
Embodiment 2
Other are substantially with embodiment 1, and only following content is different:
In step (3), proliferated culture medium be take MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, 1gL
-1active carbon, Medium's PH Value is 5.8, and adds 1.0mgL
-16-benzyladenine (6-BA), 0.5mgL
-1methyl α-naphthyl acetate (NAA), 0.3mgL
-1heteroauxin (IAA); Adventitious bud proliferation coefficient is 6.1.
In step (4), strong seedling culture base be take MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, 1gL
-1active carbon, Medium's PH Value is 5.8, and adds 0.8mgL
-16-benzyladenine (6-BA), 1.0mgL
-1methyl α-naphthyl acetate (NAA), indefinite bud secondary growth coefficient is 2.9;
In step (5), root media be take 1/2MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, 1gL
-1active carbon, Medium's PH Value is 5.8, and adds 0.1mgL
-1indolebutyric acid (IBA), 1.0mgL
-1methyl α-naphthyl acetate (NAA), rooting rate is 99.2%.
In step (6), survival rate is 90%.
Embodiment 3
Other are substantially with embodiment 1, and only following content is different:
In step (3), proliferated culture medium be take MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, 1gL
-1active carbon, Medium's PH Value is 5.8, and adds 2.0mgL
-16-benzyladenine (6-BA), 1.0mgL
-1methyl α-naphthyl acetate (NAA), 0.5mgL
-1heteroauxin (IAA); Adventitious bud proliferation coefficient is 4.3.
In step (4), strong seedling culture base be take MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, 1gL
-1active carbon, Medium's PH Value is 5.8, and adds 1.5mgL
-16-benzyladenine (6-BA), 2.0mgL
-1methyl α-naphthyl acetate (NAA), indefinite bud secondary growth coefficient is 3.2;
In step (5), root media be take 1/2MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, 1gL
-1active carbon, Medium's PH Value is 5.8, and adds 1.0mgL
-1indolebutyric acid (IBA), 2.0mgL
-1methyl α-naphthyl acetate (NAA), rooting rate is 94.7%.
In step (6), survival rate is 88%.
Claims (7)
1. a tissue culture propagation for short lobe China pink, is characterized in that comprising the following steps:
(1) the drawing materials and sterilizing of explant: get the stem with bud that short lobe China pink cuts into 2-3cm and carry out disinfection as explant;
(2) obtaining of in vitro cuttings: the explant disinfecting is placed in to inducing culture and carries out adventitious bud inducing and germinate and to obtain in vitro cuttings;
(3) test-tube plantlet propagation is cultivated: in vitro cuttings is placed in to proliferated culture medium and cultivates a large amount of indefinite buds of acquisition;
(4) the growth of plants is cultivated: the indefinite bud after breeding is placed in to strong seedling culture base and carries out strong seedling culture and obtain healthy and strong plant;
(5) rooting of vitro seedling is cultivated: healthy and strong plant is placed in to root media cultivation and obtains complete band offspring;
(6) test-tube plantlet acclimatization and transplants: screen complete band offspring and carry out transplanting in matrix and cultivating after hardening, then transplant to land for growing field crops; Described inducing culture be take MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, Medium's PH Value is 5.8, and adds 1.0mgL
-16-benzyladenine, 0.1mgL
-1methyl α-naphthyl acetate;
Described proliferated culture medium be take MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, 1gL
-1active carbon, Medium's PH Value is 5.8, and adds 0.1-2.0mgL
-16-benzyladenine, 0.01-1.0mgL
-1methyl α-naphthyl acetate, 0.1-0.5mgL
-1heteroauxin;
Described strong seedling culture base be take MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, 1gL
-1active carbon, Medium's PH Value is 5.8, and adds 0.1-1.5mgL
-16-benzyladenine, 0.1-2mgL
-1methyl α-naphthyl acetate;
Described root media be take 1/2MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, 1gL
-1active carbon, Medium's PH Value is 5.8, and adds 0.05-1.0mgL
-1indolebutyric acid, 0.5-2.0mgL
-1methyl α-naphthyl acetate.
2. the tissue culture propagation of short lobe China pink according to claim 1, it is characterized in that the sterilization in step (1) undertaken by following operation: first 2-3 is dripped to liquid detergent and splash in the beaker of dress 50ml running water, explant is put into beaker and stir gently 2min, with cotton, clean explant surface dirt again, the slight flowing water of running water rinses 8-10min; Dislocation superclean bench, with 75% alcohol immersion 30s, aseptic water washing one time, and then be 0.1% mercuric chloride sterilization 6-8min, aseptic water washing 3-5 time by the 50ml concentration of having added 1-2 and drip Tween-20, finally place the stainless steel plate disinfecting, cut into the long stem with bud of 1.0-1.5cm, obtain aseptic explant.
3. the tissue culture propagation of short lobe China pink according to claim 1, is characterized in that the condition of culture of step (2) to (5) is: temperature is 23-25 ℃, intensity of illumination 1500-2000lux, and light application time is 10-12h/d.
4. the tissue culture propagation of short lobe China pink according to claim 3, is characterized in that the incubation time of step (2) to (5) is respectively: 15-20d, 30d, 30d, 25d.
5. the tissue culture propagation of short lobe China pink according to claim 1, it is characterized in that the acclimatization and transplants in step (6) is undertaken by following operation: through step (5), obtain the whole plant with root, treat that seedling grows to 5-6cm, when root grows to 2-4cm, select well-grown, neat, healthy and strong bottle seedling, it in room temperature, is the indoor hardening of carrying out of 23-25 ℃, and in bottle, add a small amount of running water to flood medium, hardening 5-7d, with tweezers, take out test-tube plantlet, clean root medium, be transplanted to the 40-50d that grows in matrix, then transplant to land for growing field crops.
6. the tissue culture propagation of short lobe China pink according to claim 5, is characterized in that: described matrix is that perlite, sandstone and silt mix in the ratio of 1:2:1.
7. the medium that short lobe China pink tissue is cultivated, comprises inducing culture, propagating culture medium, strong seedling culture base, root media, it is characterized in that:
Described inducing culture be take MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, Medium's PH Value is 5.8, and adds 1.0mgL
-16-benzyladenine, 0.1mgL
-1methyl α-naphthyl acetate;
Described proliferated culture medium be take MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, 1gL
-1active carbon, Medium's PH Value is 5.8, and adds 0.1-2.0mgL
-16-benzyladenine, 0.01-1.0mgL
-1methyl α-naphthyl acetate, 0.1-0.5mgL
-1heteroauxin;
Described strong seedling culture base be take MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, 1gL
-1active carbon, Medium's PH Value is 5.8, and adds 0.1-1.5mgL
-16-benzyladenine, 0.1-2mgL
-1methyl α-naphthyl acetate;
Described root media be take 1/2MS as minimal medium, additional 4.5gL
-1agar, 30gL
-1sucrose, 1gL
-1active carbon, Medium's PH Value is 5.8, and adds 0.05-1.0mgL
-1indolebutyric acid, 0.5-2.0mgL
-1methyl α-naphthyl acetate.
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CN105265320A (en) * | 2015-11-18 | 2016-01-27 | 广西壮族自治区药用植物园 | Tissue culture propagation method for herba aristolochia mollissima |
CN106258294A (en) * | 2015-06-24 | 2017-01-04 | 黄保成 | A kind of implantation methods of Brachystemma calycina D. Don. |
CN107155604A (en) * | 2017-06-16 | 2017-09-15 | 内蒙古蒙草生态环境(集团)股份有限公司 | The cultivation management method of " girl in red carnation " |
CN116711639A (en) * | 2023-06-26 | 2023-09-08 | 安徽农业大学 | Culture medium combination for preventing tissue culture browning of stevia rebaudiana and application thereof |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106258294A (en) * | 2015-06-24 | 2017-01-04 | 黄保成 | A kind of implantation methods of Brachystemma calycina D. Don. |
CN105265320A (en) * | 2015-11-18 | 2016-01-27 | 广西壮族自治区药用植物园 | Tissue culture propagation method for herba aristolochia mollissima |
CN107155604A (en) * | 2017-06-16 | 2017-09-15 | 内蒙古蒙草生态环境(集团)股份有限公司 | The cultivation management method of " girl in red carnation " |
CN116711639A (en) * | 2023-06-26 | 2023-09-08 | 安徽农业大学 | Culture medium combination for preventing tissue culture browning of stevia rebaudiana and application thereof |
CN116711639B (en) * | 2023-06-26 | 2024-06-11 | 安徽农业大学 | Culture medium combination for preventing tissue culture browning of stevia rebaudiana and application thereof |
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