CN104663446A - In-vitro rapid propagation method of penstemon barbatus - Google Patents
In-vitro rapid propagation method of penstemon barbatus Download PDFInfo
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- CN104663446A CN104663446A CN201510087223.0A CN201510087223A CN104663446A CN 104663446 A CN104663446 A CN 104663446A CN 201510087223 A CN201510087223 A CN 201510087223A CN 104663446 A CN104663446 A CN 104663446A
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Abstract
The invention discloses an in-vitro rapid propagation method of penstemon barbatus and relates to an in-vitro rapidly propagation method of the penstemon barbatus through a plant tissue culture technology for overcoming the problems of long period and low efficiency of the traditional propagation technology. The tissue culture and rapid propagation system of the penstemon barbatus can be built by taking the bud stem segment of the penstemon barbatus as an explant, performing induced cultivation, successive transfer culture and root induction, seedling and transplanting. A technical support can be provided for the large-scale production, and a foundation can be laid for increasing the content of the echinacoside in the penstemon barbatus through a gene engineering solution.
Description
Technical field
The present invention relates to the method for Plant Tissue Breeding in Plant Biotechnology, specifically, relate to a kind of method for in-vitro rapid propagation of safflower beard-tongue.
Background technology
Beard-tongue is being commonly called as of Scrophulariaceae penstemon, and majority is perennial herb, and minority is annual herb.Beard-tongue pattern enriches, and flower pattern is changeable, has the ornamental quality of its uniqueness, and they are so that adaptability, abundant natural colour and changeable colored shape are famous widely.Therefore, penstemon is widely used in afforestation, is outstanding ornamental plants in garden.Now extensively introduced a fine variety in many areas such as China Beijing, Shanghai, Handan, Hangzhou, Zhangjiagangs.Beard-tongue almost complete stool is all pharmaceutically acceptable, the multiple compounds such as ring rare ether terpene glycosides, benzyl carbinol glycosides, alkaloid are rich in, wherein, echinacoside be current people find in beard-tongue there is one of compound of higher medical value, it has anti-inflammation, the effect such as anti-oxidant.
Safflower beard-tongue have another name called draft ivory red, five stamens flower.Due to beautiful in colour, flower pattern is changeable, has high ornamental value, is outstanding landscape flower material, is extensively introduced a fine variety in each big city of China.Echinacoside contained by safflower beard-tongue has antitumor, anti-oxidant, neuroprotective and liver, improves the pharmacological action such as memory and immunity, and in treatment senile dementia and cardiovascular and cerebrovascular disease etc., curative effect is given prominence to.Safflower beard-tongue is bred by modes such as sowing, plant division, cuttages.The multiplex seminal propagation of safflower beard-tongue original species.Seed is tiny, and has dormant trait, and sowing is germinateed slowly.Tissue culture propagation research at present about beard-tongue is less, about the Study on tissue culture of safflower beard-tongue does not almost have.The tissue culture technique of maturation can not only provide an a large amount of excellent nursery stock quickly and efficiently, also can be the later stage to introduce genes of interest by gene engineering in section, increases safflower beard-tongue its secondary metabolites content and lays the foundation.
Summary of the invention
The object of the present invention is to provide out a kind of method for in-vitro rapid propagation of safflower beard-tongue, the present invention with safflower beard-tongue stem with bud for explant, establish safflower beard-tongue tissue-culturing rapid propagation system through processes such as Fiber differentiation, squamous subculture, root induction, hardening and transplantings, thus achieve object of the present invention.
The method for in-vitro rapid propagation of a kind of safflower beard-tongue of the present invention, comprises the following steps:
(1) inducing clumping bud: choose the annual lignification stem with bud of healthy and strong safflower beard-tongue plant as explant, cut off blade, be cut into the long sections with 2 ~ 3 axillalry buds of 3.0 ~ 5.0cm and first soak 1 ~ 3h with 0.2% liquor potassic permanganate, scrub explant surface gently with banister brush again the dust on its surface and part thalline are removed, then be placed in superclean bench with tap water 1 ~ 3h, first use after 75% ethanol disinfection 5 ~ 30s with aseptic washing 3 ~ 5 times, again with 0.1% mercuric chloride solution sterilization 5 ~ 15min, be inoculated into Primary culture base after drying the globule on surface again with aseptic filter paper 4 ~ 6 times with aseptic water washing and carry out Fiber differentiation, first full light culture 1 ~ 5 day under 25 ~ 28 DEG C of conditions after inoculation, then illumination every day is placed in 10 ~ 12 hours, intensity of illumination is 1500 ~ 3000lx, until induced synthesis Multiple Buds.
(2) squamous subculture: the Multiple Buds obtained by Fiber differentiation cuts into single bud and vertically carries out squamous subculture to being inoculated on proliferated culture medium, first full light culture 1 ~ 3 day under 25 ~ 28 DEG C of conditions after inoculation, then illumination every day is placed in 12 ~ 14 hours, intensity of illumination is 2000 ~ 3000lx, being placed in cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C, repeatedly cutting carries out squamous subculture, to obtain more Multiple Buds repeatedly.
(3) root induction: the Multiple Buds bud height that step (1) or (2) process obtain being about 1.5 ~ 2.5cm is cut to be inoculated on root media and carried out culture of rootage, first full light culture 1 ~ 3 day under 25 ~ 28 DEG C of conditions after inoculation, then illumination every day is placed in 12 ~ 15 hours, intensity of illumination is 2000 ~ 3000lx, and cultivation temperature is be cultured under the condition of 25 ~ 28 DEG C to take root.
(4) acclimatization and transplants: the test-tube plantlet blake bottle of the well developed root system of high more than 4cm step (3) obtained shifts out culturing room, bottle cap is opened after 3 ~ 5 days gradually in natural lighting lower refining seedling after 5 ~ 7 days in indoor placement, test-tube plantlet is taken out from blake bottle, wash root medium off, plant and fill in the nutrition cup of field soil, throw off after 5 ~ 10 days with covered rearing with plastic film, after 30 days, add up survival rate.
Inducing culture described in above-mentioned steps (1) is: MS+1 ~ 3mg/L6-BA+0.01 ~ 0.5mg/L NAA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
Proliferated culture medium described in above-mentioned steps (2) is MS+0.01 ~ 0.5mg/LNAA+1 ~ 3mg/L 6-BA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, and pH is 5.4 ~ 5.8.
Root media described in above-mentioned steps (3) is: 1/2MS+0.1 ~ 1mg/L NAA+0.1 ~ 1mg/L IBA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
Compared with prior art advantage of the present invention is: by plant tissue culture technique, quick in-vitro propagate safflower beard-tongue (
penstemon barbatus(Can.) Roth), with problems such as the cycle overcoming the existence of traditional propagation technique are long, efficiency is low.The present invention with safflower beard-tongue stem with bud for explant; safflower beard-tongue tissue-culturing rapid propagation system is established through processes such as Fiber differentiation, squamous subculture, root induction, hardening and transplantings; for its large-scale production provides technical support, and lay the foundation for improving echinacoside content in safflower beard-tongue further by genetic engineering means.
Embodiment
Following examples further illustrate of the present invention, is not limitation of the present invention.
Embodiment 1:
(1) inducing clumping bud: choose the annual lignification stem with bud of healthy and strong safflower beard-tongue plant as explant, cut off blade, be cut into the long sections with 2 ~ 3 axillalry buds of 3.0 ~ 5.0cm and first soak 1h with 0.2% liquor potassic permanganate, scrub explant surface gently with banister brush again the dust on its surface and part thalline are removed, then be placed in superclean bench with tap water 1h, first use after 75% ethanol disinfection 15s with aseptic washing 3 times, again with 0.1% mercuric chloride solution sterilization 5min, be inoculated into Primary culture base after drying the globule on surface again with aseptic filter paper 4 times with aseptic water washing and carry out Fiber differentiation, first full light culture 1 day under 28 DEG C of conditions after inoculation, then illumination every day is placed in 10 hours, intensity of illumination is cultivate under 2500lx condition can form Multiple Buds in 30 days, inductivity 100%, reproduction coefficient reaches 9.8.Described inducing culture is: MS+1mg/L6-BA+0.01mg/L NAA+15g/L sucrose+3.5g/L agar, pH is 5.4.
(2) squamous subculture: the Multiple Buds obtained by Fiber differentiation cuts into single bud and vertically carries out squamous subculture to being inoculated on proliferated culture medium, first full light culture 1 day under 28 DEG C of conditions after inoculation, then illumination every day is placed in 12 hours, intensity of illumination is 2000lx, being placed in cultivation temperature is cultivate 30 days under the condition of 28 DEG C, growth coefficient is 6.7, Multiple Buds well-grown.Repeatedly cutting carries out squamous subculture, to obtain more Multiple Buds repeatedly.Described proliferated culture medium is MS+0.01mg/LNAA+2mg/L 6-BA+15g/L sucrose+3.5g/L agar, and pH is 5.4.
(3) root induction: the Multiple Buds bud height that step (1) or (2) process obtain being about 1.5 ~ 2.5cm is cut to be inoculated on root media and carried out culture of rootage, first full light culture 1 day under 28 DEG C of conditions after inoculation, then illumination every day is placed in 12 hours, intensity of illumination is 2000lx, cultivation temperature is cultivate under the condition of 28 DEG C can take root for 26 days, the adventive root produced has more side root, well developed root system, and test-tube plantlet growth is healthy and strong.Described root media is: 1/2MS+0.1mg/L NAA+0.1mg/L IBA+15g/L sucrose+3.5g/L agar, pH is 5.4.
(4) acclimatization and transplants: the test-tube plantlet blake bottle of the well developed root system of high more than 4cm step (3) obtained shifts out culturing room, bottle cap is opened after 3 days gradually in natural lighting lower refining seedling after 5 days in indoor placement, test-tube plantlet is taken out from blake bottle, wash root medium off, plant and fill in the nutrition cup of field soil, throw off after 5 days with covered rearing with plastic film, after 30 days, test-tube plantlet survival rate is higher, reaches 94.6%.
Embodiment 2:
(1) inducing clumping bud: choose the annual lignification stem with bud of healthy and strong safflower beard-tongue plant as explant, cut off blade, be cut into the long sections with 2 ~ 3 axillalry buds of 3.0 ~ 5.0cm and first soak 2h with 0.2% liquor potassic permanganate, scrub explant surface gently with banister brush again the dust on its surface and part thalline are removed, then be placed in superclean bench with tap water 2h, first use after 75% ethanol disinfection 20s with aseptic washing 3 times, again with 0.1% mercuric chloride solution sterilization 8min, be inoculated into Primary culture base after drying the globule on surface again with aseptic filter paper 5 times with aseptic water washing and carry out Fiber differentiation, first full light culture 3 days under 25 DEG C of conditions after inoculation, then illumination every day is placed in 13 hours, intensity of illumination is cultivate under 2500lx condition can form Multiple Buds in 26 days, inductivity 96%, reproduction coefficient reaches 10.0.Described inducing culture is: MS+3mg/L6-BA+0.5mg/L NAA+25g/L sucrose+4.0g/L agar, pH is 5.8.
(2) squamous subculture: the Multiple Buds obtained by Fiber differentiation cuts into single bud and vertically carries out squamous subculture to being inoculated on proliferated culture medium, first full light culture 2 days under 25 DEG C of conditions after inoculation, then illumination every day is placed in 13 hours, intensity of illumination is 2500lx, being placed in cultivation temperature is cultivate 27 days under the condition of 25 DEG C, growth coefficient is 7.4, Multiple Buds well-grown.Repeatedly cutting carries out squamous subculture, to obtain more Multiple Buds repeatedly.Described proliferated culture medium is MS+0.03mg/LNAA+3mg/L 6-BA+20g/L sucrose+3.5g/L agar, and pH is 5.8.
(3) root induction: the Multiple Buds bud height that step (1) or (2) process obtain being about 1.5 ~ 2.5cm is cut to be inoculated on root media and carried out culture of rootage, first full light culture 2 days under 25 DEG C of conditions after inoculation, then illumination every day is placed in 13 hours, intensity of illumination is 2500lx, cultivation temperature is cultivate under the condition of 25 DEG C can take root for 29 days, the adventive root produced has more side root, well developed root system, and test-tube plantlet growth is healthy and strong.Described root media is: 1/2MS+0.5mg/L NAA+1mg/L IBA+15g/L sucrose+3.5g/L agar, pH is 5.8.
(4) acclimatization and transplants: the test-tube plantlet blake bottle of the well developed root system of high more than 4cm step (3) obtained shifts out culturing room, bottle cap is opened after 3 days gradually in natural lighting lower refining seedling after 7 days in indoor placement, test-tube plantlet is taken out from blake bottle, wash root medium off, plant and fill in the nutrition cup of field soil, throw off after 5 days with covered rearing with plastic film, after 30 days, test-tube plantlet survival rate is higher, reaches 96.3%.
Claims (4)
1. a method for in-vitro rapid propagation for safflower beard-tongue, is characterized in that comprising the following steps:
(1) inducing clumping bud: choose the annual lignification stem with bud of healthy and strong safflower beard-tongue plant as explant, cut off blade, be cut into the long sections with 2 ~ 3 axillalry buds of 3.0 ~ 5.0cm and first soak 1 ~ 3h with 0.2% liquor potassic permanganate, scrub explant surface gently with banister brush again the dust on its surface and part thalline are removed, then be placed in superclean bench with tap water 1 ~ 3h, first use after 75% ethanol disinfection 5 ~ 30s with aseptic washing 3 ~ 5 times, again with 0.1% mercuric chloride solution sterilization 5 ~ 15min, be inoculated into Primary culture base after drying the globule on surface again with aseptic filter paper 4 ~ 6 times with aseptic water washing and carry out Fiber differentiation, first full light culture 1 ~ 5 day under 25 ~ 28 DEG C of conditions after inoculation, then illumination every day is placed in 10 ~ 12 hours, intensity of illumination is 1500 ~ 3000lx, until induced synthesis Multiple Buds,
(2) squamous subculture: the Multiple Buds obtained by Fiber differentiation cuts into single bud and vertically carries out squamous subculture to being inoculated on proliferated culture medium; first full light culture 1 ~ 3 day under 25 ~ 28 DEG C of conditions after inoculation; then illumination every day is placed in 12 ~ 14 hours; intensity of illumination is 2000 ~ 3000lx; being placed in cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C; repeatedly cutting carries out squamous subculture, to obtain more Multiple Buds repeatedly;
(3) root induction: the Multiple Buds bud height that step (1) or (2) process obtain being about 1.5 ~ 2.5cm is cut to be inoculated on root media and carried out culture of rootage; first full light culture 1 ~ 3 day under 25 ~ 28 DEG C of conditions after inoculation; then illumination every day is placed in 12 ~ 15 hours; intensity of illumination is 2000 ~ 3000lx, and cultivation temperature is be cultured under the condition of 25 ~ 28 DEG C to take root;
(4) acclimatization and transplants: the test-tube plantlet blake bottle of the well developed root system of high more than 4cm step (3) obtained shifts out culturing room, bottle cap is opened after 3 ~ 5 days gradually in natural lighting lower refining seedling after 5 ~ 7 days in indoor placement, test-tube plantlet is taken out from blake bottle, wash root medium off, plant and fill in the nutrition cup of field soil, throw off after 5 ~ 10 days with covered rearing with plastic film, after 30 days, add up survival rate.
2. the method for in-vitro rapid propagation of a kind of safflower beard-tongue according to claim 1, it is characterized in that the inducing culture described in step (1) is: MS+1 ~ 3mg/L6-BA+0.01 ~ 0.5mg/L NAA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
3. the method for in-vitro rapid propagation of a kind of safflower beard-tongue according to claim 1, it is characterized in that the proliferated culture medium described in step (2) is MS+0.01 ~ 0.5mg/LNAA+1 ~ 3mg/L 6-BA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
4. the method for in-vitro rapid propagation of a kind of safflower beard-tongue according to claim 1, it is characterized in that the root media described in step (3) is: 1/2MS+0.1 ~ 1mg/L NAA+0.1 ~ 1mg/L IBA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105830926A (en) * | 2016-04-13 | 2016-08-10 | 广东金作农业科技有限公司 | Tissue culture and rapid propagation method of striga asiatica |
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Non-Patent Citations (3)
| Title |
|---|
| DALE T. LINDGREN: "Multiplication of Four Penstemon Species in Vitro", 《HORTSCIENCE》 * |
| 汤绍虎等: "卵叶钓钟柳的离体再生及特殊中药成分的HPLC检测", 《西南大学学报（自然科学版）》 * |
| 莫秀媚等: "红花钓钟柳的组织培养及离体快速繁殖", 《西南大学学报（自然科学版）》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105830926A (en) * | 2016-04-13 | 2016-08-10 | 广东金作农业科技有限公司 | Tissue culture and rapid propagation method of striga asiatica |
| CN105830926B (en) * | 2016-04-13 | 2017-10-24 | 广东金作农业科技有限公司 | A kind of quick breeding method for tissue culture of witchweed |
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Application publication date: 20150603 |