CN104686328A - Method for establishing in-vitro regeneration system of sinningia speciosa - Google Patents
Method for establishing in-vitro regeneration system of sinningia speciosa Download PDFInfo
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- CN104686328A CN104686328A CN201510085884.XA CN201510085884A CN104686328A CN 104686328 A CN104686328 A CN 104686328A CN 201510085884 A CN201510085884 A CN 201510085884A CN 104686328 A CN104686328 A CN 104686328A
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Abstract
The invention discloses a method for establishing an in-vitro regeneration system of sinningia speciosa and relates to a method for obtaining lots of good-quality sinningia speciosa seedlings from the sinningia speciosa in short time by use of a plant tissue culture method. The tender leaves of the sinningia speciosa are taken as explants, and the method for establishing the in-vitro regeneration system of the sinningia speciosa comprises the steps of explant sterilization, adventitious bud induced culture, rooting culture, acclimatization and transplant and the like; the method has important practical significance on meeting of the requirements of good-quality sinningia speciosa seedlings and accelerating of the popularization of the good-quality sinningia speciosa variety.
Description
Technical field
The present invention relates to the method for Plant Tissue Breeding in gardening plant biotechnology, specifically, relate to a kind of gloxinia vitro Regeneration System method for building up.
Background technology
Gloxinia (
sinningia speciosa) have another name called six snow Buddhist nuns, fall slush, Brazilian lotus etc., is that Gesneriaceae rock paulownia belongs to perennial napiform root herbage flower, stem tuber oblate spheroid, leaf is to life, plump and large, close raw fine hair, flower mitriform, rich color, large and beautiful, is colored desirable kind on National Day.The cultivar of gloxinia is various, and pattern has indigo plant, pink, white, red, purple etc., also has white edge indigo plant flower, the double-colored and double flower of white edge safflower.Common kind has William emperor, challenge, the god of fire etc., all has cultivation all over the world.Gloxinia happiness is warm, not low temperature resistant, and less than 5 DEG C are difficult to survive the winter, and cultivate cold-resistant kind and are expected to realize non-winter survival in greenhouse, expands cultivated area, reduces production cost.
At present, gloxinia seedling is mainly bred by modes such as seed, napiform root sprouting, cuttages.Various ways has respective characteristic, and major defect is that reproduction coefficient is low, and this have impact on the requirement of its large-scale development and the marketization to a great extent.And plant tissue culture technique has and accelerates breeding process, shortens repoductive time, improvement plant quality, saves space, reduces and work, can produce all the year round, be not subject to the features such as natural conditions restriction, gloxinia sapling multiplication length consuming time, problem that reproduction coefficient is low effectively can be solved.Therefore, set up gloxinia Regeneration in Vitro technical system, to promote excellent gloxinia garden-variety China production and apply significant.
Summary of the invention
The object of the present invention is to provide out a kind of gloxinia vitro Regeneration System method for building up, the present invention with gloxinia young leaflet tablet for explant, establish a kind of gloxinia vitro Regeneration System method for building up through explant sterilization, adventitious bud induction culture, Multiplying culture, culture of rootage, hardening and transplanting and other steps, thus achieve object of the present invention.
A kind of gloxinia vitro Regeneration System method for building up of the present invention, comprises the following steps:
(1) explant sterilization: the young leaflet tablet from the eugonic gloxinia plant that box is planted is explant, first be placed in superclean bench with rinsing 1 ~ 3h under flowing water, again after with 75% ethanol disinfection 5 ~ 30s with aseptic washing 3 ~ 5 times, again with 0.1% mercuric chloride solution sterilization 1 ~ 10min, for subsequent use after drying the globule on surface again with aseptic filter paper 4 ~ 6 times with aseptic water washing.
(2) bud inducement is cultivated: the blade after step (1) being processed is cut into 1 × 1cm
2long fritter is also inoculated on inducing culture, first full light culture 1 ~ 3 day under 25 ~ 28 DEG C of conditions, and be then placed in illumination every day 10 ~ 12 hours, intensity of illumination is 1500 ~ 3000lx, until induced synthesis indefinite bud.
(3) Multiplying culture: cut when the indefinite bud on the callus that the blade edge wound of step (2) being cultivated produces grows to about 2.0 ~ 3.0cm, cut its stem apex and be seeded on proliferated culture medium and carry out Multiplying culture, first full light culture 3 ~ 5 days under 25 ~ 28 DEG C of conditions after inoculation, then illumination every day is placed in 10 ~ 13 hours, intensity of illumination is 1500 ~ 2500lx, being placed in cultivation temperature is cultivate after 2 ~ 3 weeks under the condition of 25 ~ 28 DEG C, its axil place will grow many budlets, again these budlets are cut repeatedly and carry out squamous subculture, to obtain more indefinite bud.
(4) culture of rootage: the budlet that the height that step (3) process obtains is about 3.5 ~ 4.5cm is divided to cut and be inoculated on root media and carries out culture of rootage, first full light culture 3 ~ 7 days under 25 ~ 28 DEG C of conditions after inoculation, then illumination every day is placed in 12 ~ 15 hours, intensity of illumination is 1500 ~ 2500lx, and cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C within about 20 days, namely to start to take root.
(5) acclimatization and transplants: height about 5 ~ 7cm step (4) obtained, robust growth, the rooting tube plantlet decap that root system is sturdy, leaf look dark green were placed in natural lighting lower refining seedling after 3 ~ 5 days, test-tube plantlet is taken out from blake bottle, wash root medium off and soak 8 ~ 10s in carbendazim solution, plant into by Nutrition Soil: the matrix that sandy soil=3:1 is mixed into, keep humidity and give 60% ~ 80% process of sheltering from heat or light.
Inducing culture described in above-mentioned steps (2) is: MS+0.5 ~ 2mg/L6-BA+0.1 ~ 1mg/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 (3) is: MS+0.1 ~ 1mg/L NAA+0.5 ~ 1mg/L 6-BA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
Root media described in above-mentioned steps (4) is: 1/2MS+0.1 ~ 2mg/L NAA+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: the present invention is by obtaining the method for a large amount of high-quality gloxinia seedling in the method for plant tissue culture short time.With gloxinia young leaflet tablet for explant, a kind of gloxinia vitro Regeneration System method for building up is established, for the popularization of the satisfied demand to gloxinia high quality seedling, acceleration gloxinia breeding has important practical significance through explant sterilization, adventitious bud induction culture, Multiplying culture, culture of rootage, hardening and transplanting and other steps.。
Embodiment
Following examples further illustrate of the present invention, is not limitation of the present invention.
Embodiment 1:
(1) explant sterilization: the young leaflet tablet from the eugonic gloxinia plant that box is planted is explant, first be placed in superclean bench with rinsing 1h under flowing water, again after with 75% ethanol disinfection 5s with aseptic washing 3 times, again with 0.1% mercuric chloride solution sterilization 1min, for subsequent use after drying the globule on surface again with aseptic filter paper 4 times with aseptic water washing.
(2) bud inducement is cultivated: the blade after step (1) being processed is cut into 1 × 1cm
2long fritter is also inoculated on inducing culture, first full light culture 1 day under 28 DEG C of conditions, and be then placed in illumination every day 10 hours, intensity of illumination is that 1500lx CMC model gets final product induced synthesis indefinite bud in 30 days, and inductivity is 96%.Described inducing culture is MS+0.8mg/L6-BA+0.3mg/L NAA+30g/L sucrose+3.5g/L agar, and pH is 5.8.
(3) Multiplying culture: cut when the indefinite bud on the callus that the blade edge wound of step (2) being cultivated produces grows to about 2.0 ~ 3.0cm, cut its stem apex and be seeded on proliferated culture medium and carry out Multiplying culture, first full light culture 3 days under 28 DEG C of conditions after inoculation, then illumination every day is placed in 10 hours, intensity of illumination is 1500lx, being placed in cultivation temperature is cultivate after 14 days under the condition of 28 DEG C, its axil place will grow many budlets, again these budlets are cut repeatedly and carry out squamous subculture, to obtain more indefinite bud.Described proliferated culture medium is MS+0.5mg/L NAA+1mg/L 6-BA+30g/L sucrose+6.0g/L agar, and pH is 5.8.
(4) culture of rootage: the budlet that the height that step (3) process obtains is about 3.5 ~ 4.5cm is divided to cut and be inoculated on root media and carries out culture of rootage, 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 1500lx, cultivation temperature is cultivate under the condition of 28 DEG C within 18 days, namely to start to take root, rooting rate 100%.
(5) acclimatization and transplants: height about 5 ~ 7cm step (4) obtained, robust growth, the rooting tube plantlet decap that root system is sturdy, leaf look dark green were placed in natural lighting lower refining seedling after 3 days, test-tube plantlet is taken out from blake bottle, wash root medium off and soak 8s in carbendazim solution, plant into by Nutrition Soil: the matrix that sandy soil=3:1 is mixed into, keep humidity and give 60% process of sheltering from heat or light, transplanting survival rate is more than 98%.Described root media is 1/2MS+0.5mg/L NAA+15g/L sucrose+3.5 ~ g/L agar, and pH is 5.8.
Embodiment 2:
(1) explant sterilization: the young leaflet tablet from the eugonic gloxinia plant that box is planted is explant, first be placed in superclean bench with rinsing 1h under flowing water, again after with 75% ethanol disinfection 5s with aseptic washing 3 times, again with 0.1% mercuric chloride solution sterilization 1min, for subsequent use after drying the globule on surface again with aseptic filter paper 4 times with aseptic water washing.
(2) bud inducement is cultivated: the blade after step (1) being processed is cut into 1 × 1cm
2long fritter is also inoculated on inducing culture, first full light culture 3 days under 28 DEG C of conditions, and be then placed in illumination every day 11 hours, intensity of illumination is that 1500lx CMC model gets final product induced synthesis indefinite bud in 28 days, and inductivity is 92%.Described inducing culture is MS+1mg/L6-BA+0.5mg/L NAA+30g/L sucrose+3.5g/L agar, and pH is 5.8.
(3) Multiplying culture: cut when the indefinite bud on the callus that the blade edge wound of step (2) being cultivated produces grows to about 2.0 ~ 3.0cm, cut its stem apex and be seeded on proliferated culture medium and carry out Multiplying culture, first full light culture 5 days under 28 DEG C of conditions after inoculation, then illumination every day is placed in 11 hours, intensity of illumination is 2000lx, being placed in cultivation temperature is cultivate after 15 days under the condition of 28 DEG C, its axil place will grow many budlets, again these budlets are cut repeatedly and carry out squamous subculture, to obtain more indefinite bud.Described proliferated culture medium is MS+0.3mg/L NAA+0.9mg/L 6-BA+30g/L sucrose+6.0g/L agar, and pH is 5.8.
(4) culture of rootage: the budlet that the height that step (3) process obtains is about 3.5 ~ 4.5cm is divided to cut and be inoculated on root media and carries out culture of rootage, 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 1500lx, cultivation temperature is cultivate under the condition of 28 DEG C within 18 days, namely to start to take root, rooting rate 100%.Described root media is 1/2MS+0.8mg/L NAA+15g/L sucrose+3.5 ~ g/L agar, and pH is 5.8.
(5) acclimatization and transplants: height about 5 ~ 7cm step (4) obtained, robust growth, the rooting tube plantlet decap that root system is sturdy, leaf look dark green were placed in natural lighting lower refining seedling after 3 days, test-tube plantlet is taken out from blake bottle, wash root medium off and soak 8s in carbendazim solution, plant into by Nutrition Soil: the matrix that sandy soil=3:1 is mixed into, keep humidity and give 60% process of sheltering from heat or light, transplanting survival rate is more than 96%.
Claims (4)
1. a gloxinia vitro Regeneration System method for building up, is characterized in that comprising the following steps:
(1) explant sterilization: the young leaflet tablet from the eugonic gloxinia plant that box is planted is explant, first be placed in superclean bench with rinsing 1 ~ 3h under flowing water, again after with 75% ethanol disinfection 5 ~ 30s with aseptic washing 3 ~ 5 times, again with 0.1% mercuric chloride solution sterilization 1 ~ 10min, for subsequent use after drying the globule on surface again with aseptic filter paper 4 ~ 6 times with aseptic water washing;
(2) bud inducement is cultivated: the blade after step (1) being processed is cut into 1 × 1cm
2long fritter is also inoculated on inducing culture, first full light culture 1 ~ 3 day under 25 ~ 28 DEG C of conditions, and be then placed in illumination every day 10 ~ 12 hours, intensity of illumination is 1500 ~ 3000lx, until induced synthesis indefinite bud;
(3) Multiplying culture: cut when the indefinite bud on the callus that the blade edge wound of step (2) being cultivated produces grows to about 2.0 ~ 3.0cm, cut its stem apex and be seeded on proliferated culture medium and carry out Multiplying culture, first full light culture 3 ~ 5 days under 25 ~ 28 DEG C of conditions after inoculation, then illumination every day is placed in 10 ~ 13 hours, intensity of illumination is 1500 ~ 2500lx, being placed in cultivation temperature is cultivate after 2 ~ 3 weeks under the condition of 25 ~ 28 DEG C, its axil place will grow many budlets, again these budlets are cut repeatedly and carry out squamous subculture, obtain indefinite bud;
(4) culture of rootage: the budlet that the height that step (3) process obtains is about 3.5 ~ 4.5cm is divided to cut and be inoculated on root media and carries out culture of rootage, first full light culture 3 ~ 7 days under 25 ~ 28 DEG C of conditions after inoculation, then illumination every day is placed in 12 ~ 15 hours, intensity of illumination is 1500 ~ 2500lx, and cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C within about 20 days, namely to start to take root;
(5) acclimatization and transplants: height about 5 ~ 7cm step (4) obtained, robust growth, the rooting tube plantlet decap that root system is sturdy, leaf look dark green were placed in natural lighting lower refining seedling after 3 ~ 5 days, test-tube plantlet is taken out from blake bottle, wash root medium off and soak 8 ~ 10s in carbendazim solution, plant into by Nutrition Soil: the matrix that sandy soil=3:1 is mixed into, keep humidity and give 60% ~ 80% process of sheltering from heat or light.
2. a kind of gloxinia vitro Regeneration System method for building up according to claim 1, it is characterized in that the inducing culture described in step (2) is: MS+0.5 ~ 2mg/L6-BA+0.1 ~ 1mg/L NAA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
3. a kind of gloxinia vitro Regeneration System method for building up according to claim 1, it is characterized in that the proliferated culture medium described in step (3) is: MS+0.1 ~ 1mg/L NAA+0.5 ~ 1mg/L 6-BA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
4. a kind of gloxinia vitro Regeneration System method for building up according to claim 1, is characterized in that the root media described in step (4) is: 1/2MS+0.1 ~ 2mg/L NAA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
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Cited By (3)
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| CN106332780A (en) * | 2016-08-31 | 2017-01-18 | 李军 | Construction method for in-vitro regeneration system of flos carthami |
| CN107873520A (en) * | 2017-12-21 | 2018-04-06 | 湖南人文科技学院 | The cultural method of Sinningia speciosa tissue |
| CN112841032A (en) * | 2021-02-01 | 2021-05-28 | 中国科学院华南植物园 | Infinite bud multiplication medium for mallotus maritima and method for in-vitro rapid propagation of mallotus maritima |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106332780A (en) * | 2016-08-31 | 2017-01-18 | 李军 | Construction method for in-vitro regeneration system of flos carthami |
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| CN107873520B (en) * | 2017-12-21 | 2020-08-25 | 湖南人文科技学院 | Culture method of tissue of erythrina bigelovii |
| CN112841032A (en) * | 2021-02-01 | 2021-05-28 | 中国科学院华南植物园 | Infinite bud multiplication medium for mallotus maritima and method for in-vitro rapid propagation of mallotus maritima |
| CN112841032B (en) * | 2021-02-01 | 2022-03-22 | 中国科学院华南植物园 | Infinite bud multiplication medium for mallotus maritima and method for in-vitro rapid propagation of mallotus maritima |
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