CN104686346A - Tissue culture and rapid propagation method for divaricate saposhnikovia root - Google Patents
Tissue culture and rapid propagation method for divaricate saposhnikovia root Download PDFInfo
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- CN104686346A CN104686346A CN201510086064.2A CN201510086064A CN104686346A CN 104686346 A CN104686346 A CN 104686346A CN 201510086064 A CN201510086064 A CN 201510086064A CN 104686346 A CN104686346 A CN 104686346A
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Abstract
The invention discloses a tissue culture and rapid propagation method for divaricate saposhnikovia root, i.e., an in-vitro rapid propagation system of the divaricate saposhnikovia root is established by taking divaricate saposhnikovia root seeds as an explant to adapt to large-scale and standard production needs. According to the tissue culture and rapid propagation method disclosed by the invention, the seeds are taken as the explant, the tissue culture and rapid propagation of the divaricate saposhnikovia root is realized through processes of callus induction, propagation culture, adventitious bud induction, root and shoot induction as well as test tube seedling acclimatizating and transplanting and the like, and technical support is provided for the industrial production of divaricate saposhnikovia seedlings.
Description
Technical field
The present invention relates to the method for Plant Tissue Breeding in agricultural biotechnologies, specifically, relate to a kind of windproof tissue cultivation rapid breeding method.
Background technology
Windproof (
saposhnikovia divaricata) be Umbelliferae medicinal plant, its dry root is used as medicine, and containing multiple biochemical composition, has antipyretic, analgesia, anti-inflammatory, the effect such as antibacterial, antitumor, antiviral.Traditional windproof acquisition methods excavates wild plant resource, owing to blindly excavating, not only makes wild resource day by day reduce but also the natural ecological balance of heavy damage.In addition, the kind sexual involution of cultivar, yield and quality declines, and can not adapt to the needs of scale and standardized production.
Mainly concentrate in plant resources, chemical composition analysis, Pharmacognosy Studies, pharmacological action and clinical practice etc. about windproof research at present, certain basis has been established in these clinical practices studied as with windproof being base source preparation.Along with the development of biotechnology, tissue culture technique is that windproof large-scale production opens new approach.Up to now, there is not yet the institute road of RADIX SAPOSHINOKOVIAE from Northeast vitro Regeneration System, this has had a strong impact on the development of windproof sapling multiplication.Therefore, be necessary very much the windproof rapid propagation in vitro system of system of setting up, for windproof seedling suitability for industrialized production provides technical support.
Summary of the invention
The object of the present invention is to provide a kind of windproof tissue cultivation rapid breeding method, the present invention take seed as explant, by the windproof tissue-culturing quick-propagation of callus induction, Multiplying culture, adventitious bud inducing, offspring induction and the process implementation such as test-tube plantlet rooting culture, thus realize object of the present invention.
A kind of windproof tissue cultivation rapid breeding method of the present invention, comprises following processing step:
(1) process of explant: choose and receive full windproof seed then, scrub gently with liquid detergent water, be placed in running water flushing 10 ~ 30min and be placed on clear water immersion 8 ~ 12h, proceed to again in the Gibberellins solution of 50 ~ 120mg/mL and soak 10 ~ 15h, then in superclean bench with 75 ~ 80% alcohol solution dipping 10 ~ 30s, aseptic water washing 3 ~ 5 times, again with 0.1 ~ 0.5% mercuric chloride solution sterilization 5 ~ 10min containing 0.01 ~ 0.05% Tween-20, for subsequent use blot the moisture on surface after aseptic water washing 3 ~ 5 times with aseptic filter paper after.
(2) callus induction: cut to be inoculated on inducing culture after 1 ~ 3 cutter causes wound with sterile scalpel on the seed after step (1) process and carry out induction of callus, carry out full light culture after inoculation under 25 ~ 28 DEG C of conditions until form callus.
(3) Multiplying culture: behind callus growth to 20 ~ 30 day, the callus grown fine is forwarded in proliferated culture medium and carries out Multiplying culture, first full light culture 1 ~ 3 day under 25 ~ 28 DEG C of conditions after inoculation, then illumination every day is placed in 12 ~ 16 hours, intensity of illumination is 2000 ~ 3000lx, cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C, within about 20 days, transfers once.
(4) adventitious bud inducing: the callus grown fine being cultured to 15 ~ 20 days is forwarded in differential medium and carries out adventitious bud inducing, first full light culture 2 ~ 5 days under 25 ~ 28 DEG C of conditions after inoculation, then illumination every day is placed in 12 ~ 16 hours, intensity of illumination is 2000 ~ 3000lx, and cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C until form indefinite bud.
(5) culture of rootage: when Multiple Buds grows to 2.5 ~ 3.5cm height, tufted seedling is cut into single seedling to be seeded in root media and to carry out culture of rootage, first full light culture 1 ~ 4 day under 25 ~ 28 DEG C of conditions after inoculation, then illumination every day is placed in 12 ~ 16 hours, intensity of illumination is 2000 ~ 3000lx, and cultivation temperature is cultivate until send out roots under the condition of 25 ~ 28 DEG C.
(6) test-tube seedling transplanting: the new root system grown when seedling reaches 2 ~ 3.5cm, and grow side root, during more than height of seedling 5cm, blake bottle bottle cap is opened and is placed in natural lighting lower refining seedling after 5 ~ 7 days, test-tube plantlet is taken out from blake bottle, wash root medium off, do not injure root as far as possible, to plant in the matrix be mixed into by yellow sand and fiery carbon mud (1:1) and to be colonizated in large Tanaka.
Inducing culture described in above-mentioned steps (2) is: MS+ 1 ~ 5mg/L 2,4-D+ 0.5 ~ 2mg/L 6-BA+ 0.1 ~ 1mg/L KT+2.5% ~ 3.5% sucrose+0.35% ~ 0.5% agar+0.05% ~ 0.1% active carbon, pH value is 5.4 ~ 5.8.
Proliferated culture medium described in above-mentioned steps (3) is: MS+0.5 ~ 3.0mg/L 6-BA+0.1 ~ 1.0mg/L NAA+2.5% ~ 3.5% sucrose+0.35% ~ 0.5% agar+0.05% ~ 0.1% active carbon, pH value is 5.4 ~ 5.8.
Differential medium described in above-mentioned steps (4) is: MS+0.1 ~ 0.5mg/L NAA+1.0 ~ 2.0mg/L 6-BA+2.5% ~ 3.5% sucrose+0.35% ~ 0.5% agar+0.05% ~ 0.1% active carbon, pH value is 5.4 ~ 5.8.
Root media described in above-mentioned steps (5) is: 1/2MS+0.05 ~ 0.5mg/L 6-BA+0.1 ~ 1.0mg/L NAA+2.0% ~ 2.5% sucrose+0.4% ~ 0.6% agar+0.05% ~ 0.1% active carbon, pH value is 5.4 ~ 5.8.
Advantage of the present invention is: with windproof seed for explant establishes windproof rapid propagation in vitro system, to adapt to the needs of scale and standardized production.The present invention take seed as explant, by the windproof tissue-culturing quick-propagation of callus induction, Multiplying culture, adventitious bud inducing, offspring induction and the process implementation such as test-tube plantlet rooting culture, for windproof seedling suitability for industrialized production provides technical support.
Embodiment
Following examples further illustrate of the present invention, is not limitation of the present invention.
Embodiment 1:
(1) process of explant: choose and receive full windproof seed then, scrub gently with liquid detergent water, be placed in running water to rinse 10min and be placed on clear water and soak 8h, proceed to again in the Gibberellins solution of 80mg/mL and soak 10h, then in superclean bench with 75% alcohol solution dipping 10s, aseptic water washing 3 times, then with the 0.1% mercuric chloride solution sterilization 5min containing 0.01% Tween-20, for subsequent use blot the moisture on surface after aseptic water washing 3 times with aseptic filter paper after.
(2) callus induction: cut to be inoculated on inducing culture after 1 cutter causes wound with sterile scalpel on the seed after step (1) process and carry out induction of callus, under 28 DEG C of conditions, carry out full light culture after inoculation can form callus in 25 days, inductivity is up to 96.8%.Described inducing culture is: MS+ 3mg/L 2,4-D+ 0.5mg/L 6-BA+ 0.3mg/L KT+3.5% sucrose+0.35% agar+0.08% active carbon, pH value is 5.8.
(3) Multiplying culture: after callus growth to 23 day, the callus grown fine is forwarded in proliferated culture medium and carries out Multiplying culture, first full light culture 2 days under 28 DEG C of conditions after inoculation, be then placed in illumination every day 13 hours, intensity of illumination is 2000lx, cultivation temperature is cultivate under the condition of 28 DEG C, once, callus growth is rapid, in yellow green in switching in 23 days, quality is tight, and callus proliferation times reaches 5.8.Described proliferated culture medium is MS+1.5mg/L 6-BA+0.3mg/L NAA+2.5% sucrose+0.35% agar+0.1% active carbon, and pH value is 5.8.
(4) adventitious bud inducing: the callus grown fine being cultured to 15 days is forwarded in differential medium and carries out adventitious bud inducing, first full light culture 3 days 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 callus after 15 days under the condition of 28 DEG C to turn green gradually, and form a lot of projection, and then form Multi bud body, differentiation rate is up to 86.3%.Described differential medium is: MS+0.3mg/L NAA+1.5mg/L 6-BA+2.5% sucrose+0.5% agar+0.1% active carbon, pH value is 5.8.
(5) culture of rootage: when Multiple Buds grows to 2.5 ~ 3.5cm height, tufted seedling is cut into single seedling to be seeded in root media and to carry out culture of rootage, first full light culture 2 days under 28 DEG C of conditions after inoculation, then illumination every day is placed in 14 hours, intensity of illumination is 2000lx, cultivation temperature is cultivate under the condition of 28 DEG C after 15 days to start to take root, and rooting rate reaches 88.4%, and mean elements is 17.1.Described root media is: 1/2MS+0.1mg/L 6-BA+0.6mg/L NAA+2.0% sucrose+0.4% agar+0.1% active carbon, pH value is 5.8.
(6) test-tube seedling transplanting: the new root system grown when seedling reaches 2 ~ 3.5cm, and grow side root, during more than height of seedling 5cm, blake bottle bottle cap is opened and is placed in natural lighting lower refining seedling after 5 days, test-tube plantlet is taken out from blake bottle, washes root medium off, do not injure root as far as possible, planting in the matrix be mixed into by yellow sand and fiery carbon mud (1:1) and to be colonizated in large Tanaka, transplanting survival rate reaches more than 93%.
Embodiment 2:
(1) process of explant: choose and receive full windproof seed then, scrub gently with liquid detergent water, be placed in running water to rinse 15min and be placed on clear water and soak 8h, proceed to again in the Gibberellins solution of 110mg/mL and soak 12h, then in superclean bench with 75% alcohol solution dipping 15s, aseptic water washing 5 times, then with the 0.1% mercuric chloride solution sterilization 8min containing 0.01% Tween-20, for subsequent use blot the moisture on surface after aseptic water washing 5 times with aseptic filter paper after.
(2) callus induction: cut to be inoculated on inducing culture after 3 cuttves cause wound with sterile scalpel on the seed after step (1) process and carry out induction of callus, under 28 DEG C of conditions, carry out full light culture after inoculation can form callus in 22 days, inductivity is up to 89.8%.Described inducing culture is: MS+ 5mg/L 2,4-D+ 0.6mg/L 6-BA+ 0.1mg/L KT+3.5% sucrose+0.35% agar+0.1% active carbon, pH value is 5.8.
(3) Multiplying culture: after callus growth to 22 day, the callus grown fine is forwarded in proliferated culture medium and carries out Multiplying culture, first full light culture 3 days under 28 DEG C of conditions after inoculation, be then placed in illumination every day 13 hours, intensity of illumination is 2500lx, cultivation temperature is cultivate under the condition of 28 DEG C, once, callus growth is rapid, in yellow green in switching in 23 days, quality is tight, and callus proliferation times reaches 6.3.Described proliferated culture medium is MS+2.0mg/L 6-BA+0.5mg/L NAA+2.5% sucrose+0.35% agar+0.1% active carbon, and pH value is 5.8.
(4) adventitious bud inducing: the callus grown fine being cultured to 15 days is forwarded in differential medium and carries out adventitious bud inducing, first full light culture 3 days 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 callus after 15 days under the condition of 28 DEG C to turn green gradually, and form a lot of projection, and then form Multi bud body, differentiation rate is up to 88.9%.Described differential medium is: MS+0.5mg/L NAA+1.8mg/L 6-BA+2.5% sucrose+0.5% agar+0.1% active carbon, pH value is 5.8.
(5) culture of rootage: when Multiple Buds grows to 2.5 ~ 3.5cm height, tufted seedling is cut into single seedling to be seeded in root media and to carry out culture of rootage, first full light culture 2 days under 28 DEG C of conditions after inoculation, then illumination every day is placed in 14 hours, intensity of illumination is 2000lx, cultivation temperature is cultivate under the condition of 28 DEG C after 15 days to start to take root, and rooting rate reaches 92%, and mean elements is 15.8.Described root media is: 1/2MS+0.05mg/L 6-BA+0.8mg/L NAA+2.0% sucrose+0.4% agar+0.1% active carbon, pH value is 5.8.
(6) test-tube seedling transplanting: the new root system grown when seedling reaches 2 ~ 3.5cm, and grow side root, during more than height of seedling 5cm, blake bottle bottle cap is opened and is placed in natural lighting lower refining seedling after 7 days, test-tube plantlet is taken out from blake bottle, washes root medium off, do not injure root as far as possible, planting in the matrix be mixed into by yellow sand and fiery carbon mud (1:1) and to be colonizated in large Tanaka, transplanting survival rate reaches more than 90.5%.
Claims (5)
1. a windproof tissue cultivation rapid breeding method, is characterized in that comprising following processing step:
(1) process of explant: choose and receive full windproof seed then; scrub with liquid detergent water; be placed in running water flushing 10 ~ 30min and be placed on clear water immersion 8 ~ 12h; proceed to again in the Gibberellins solution of 50 ~ 120mg/mL and soak 10 ~ 15h; then in superclean bench with 75 ~ 80% alcohol solution dipping 10 ~ 30s; aseptic water washing 3 ~ 5 times; again with 0.1 ~ 0.5% mercuric chloride solution sterilization 5 ~ 10min containing 0.01 ~ 0.05% Tween-20, for subsequent use blot the moisture on surface after aseptic water washing 3 ~ 5 times with aseptic filter paper after;
(2) callus induction: cut to be inoculated on inducing culture after 1 ~ 3 cutter causes wound with sterile scalpel on the seed after step (1) process and carry out induction of callus, carry out full light culture after inoculation under 25 ~ 28 DEG C of conditions until form callus;
(3) Multiplying culture: behind callus growth to 20 ~ 30 day; the callus grown fine is forwarded in proliferated culture medium and carries out Multiplying culture; first full light culture 1 ~ 3 day under 25 ~ 28 DEG C of conditions after inoculation; then illumination every day is placed in 12 ~ 16 hours; intensity of illumination is 2000 ~ 3000lx; cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C, within about 20 days, transfers once;
(4) adventitious bud inducing: the callus grown fine being cultured to 15 ~ 20 days is forwarded in differential medium and carries out adventitious bud inducing; first full light culture 2 ~ 5 days under 25 ~ 28 DEG C of conditions after inoculation; then illumination every day is placed in 12 ~ 16 hours; intensity of illumination is 2000 ~ 3000lx, and cultivation temperature is cultivate until form indefinite bud under the condition of 25 ~ 28 DEG C;
(5) culture of rootage: when Multiple Buds grows to 2.5 ~ 3.5cm height; tufted seedling is cut into single seedling to be seeded in root media and to carry out culture of rootage; first full light culture 1 ~ 4 day under 25 ~ 28 DEG C of conditions after inoculation; then illumination every day is placed in 12 ~ 16 hours; intensity of illumination is 2000 ~ 3000lx, and cultivation temperature is cultivate until send out roots under the condition of 25 ~ 28 DEG C;
(6) test-tube seedling transplanting: the new root system grown when seedling reaches 2 ~ 3.5cm, and grow side root, during more than height of seedling 5cm, blake bottle bottle cap is opened and is placed in natural lighting lower refining seedling after 5 ~ 7 days, test-tube plantlet is taken out from blake bottle, wash root medium off, do not injure root as far as possible, to plant in the matrix be mixed into by yellow sand and fiery carbon mud (1:1) and to be colonizated in large Tanaka.
2. a kind of windproof tissue cultivation rapid breeding method according to claim 1, it is characterized in that the inducing culture described in step (2) is: MS+ 1 ~ 5mg/L 2,4-D+ 0.5 ~ 2mg/L 6-BA+ 0.1 ~ 1mg/L KT+2.5% ~ 3.5% sucrose+0.35% ~ 0.5% agar+0.05% ~ 0.1% active carbon, pH value is 5.4 ~ 5.8.
3. a kind of windproof tissue cultivation rapid breeding method according to claim 1, it is characterized in that the proliferated culture medium described in step (3) is: MS+0.5 ~ 3.0mg/L 6-BA+0.1 ~ 1.0mg/L NAA+2.5% ~ 3.5% sucrose+0.35% ~ 0.5% agar+0.05% ~ 0.1% active carbon, pH value is 5.4 ~ 5.8.
4. a kind of windproof tissue cultivation rapid breeding method according to claim 1, it is characterized in that the differential medium described in step (4) is: MS+0.1 ~ 0.5mg/L NAA+1.0 ~ 2.0mg/L 6-BA+2.5% ~ 3.5% sucrose+0.35% ~ 0.5% agar+0.05% ~ 0.1% active carbon, pH value is 5.4 ~ 5.8.
5. a kind of windproof tissue cultivation rapid breeding method according to claim 1, it is characterized in that the root media described in step (5) is: 1/2MS+0.05 ~ 0.5mg/L 6-BA+0.1 ~ 1.0mg/L NAA+2.0% ~ 2.5% sucrose+0.4% ~ 0.6% agar+0.05% ~ 0.1% active carbon, pH value is 5.4 ~ 5.8.
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Cited By (2)
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| CN111616054A (en) * | 2020-06-16 | 2020-09-04 | 河南科技学院 | Method for obtaining regenerated plants by using Ledebouriella seseloides leaves |
| CN115005096A (en) * | 2022-05-26 | 2022-09-06 | 桂林医学院 | Ethnic medicine alangium platanifolium seedling breeding method |
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Application publication date: 20150610 |