CN104686339A - Method for constructing regeneration system of Magnolia officinalissubsp. biloba(Rehd. et Wils.) Law. - Google Patents
Method for constructing regeneration system of Magnolia officinalissubsp. biloba(Rehd. et Wils.) Law. Download PDFInfo
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- CN104686339A CN104686339A CN201510085929.3A CN201510085929A CN104686339A CN 104686339 A CN104686339 A CN 104686339A CN 201510085929 A CN201510085929 A CN 201510085929A CN 104686339 A CN104686339 A CN 104686339A
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Abstract
The invention discloses a method for constructing a regeneration system of Magnolia officinalissubsp. biloba(Rehd. et Wils.) Law., and relates to a tissue culture and rapid propagation method of Magnolia officinalissubsp. biloba(Rehd. et Wils.) Law.. According to the method disclosed by the invention, Magnolia officinalissubsp. biloba(Rehd. et Wils.) Law. is taken as an initial explant, the regeneration system of the Magnolia officinalissubsp. biloba(Rehd. et Wils.) Law. is established through processes of sterile system establishment, primary culture, multi-shoot propagation culture, multi-shoot strong seedling culture, test tube seedling rooting, test tube acclimatizating and transplanting, and the like, and a production method is provided for rapid regeneration of excellent Magnolia officinalissubsp. biloba(Rehd. et Wils.) Law. seed sources.
Description
Technical field
The present invention relates to the method for Plant Tissue Breeding in agricultural biotechnologies, specifically, relate to a kind of method that Magnolia bilola regenerating system builds.
Background technology
Magnolia bilola (
magnolia officinalissubsp.
biloba(Rehd. et Wils.) Law.)) also known as the Mount Lushan bark of official magnolia, be subspecies of the bark of official magnolia.For the peculiar economic tree of China, be national secondary national key protected plant, integrate medicinal, material with, the rare or endangered species viewed and admired, mainly distribute the ground such as Hunan, Hubei, Guangxi.Magnolia bilola root skin, bark, flower, bud and seed all can be used as medicine, and having the effect such as antibacterial, antiulcer, muscular flaccidity, anti-spasm, antiallergy, is enterogastric diseases conventional Chinese medicine.
At present, Magnolia bilola mainly relies on seminal propagation mode to carry out seeling industry, there is breeding cycle long, the problem such as efficiency is low, reproduction coefficient is low, not yet forms sound industrialization production mechanism.In recent years, because Magnolia bilola demand increases gradually, wild resource reduces gradually, and its seed itself has again a deep-sleep characteristic, and natural propagation rate is general lower.In addition, Magnolia bilola is as the comparatively original woody plant of Magnoliaceae, and tissue cultures is comparatively difficult.Therefore, the Magnolia bilola tissue culture quick breeding system setting up complete set is necessary very much.
Summary of the invention
The object of the present invention is to provide a kind of method that Magnolia bilola regenerating system builds, the present invention is initial explant with Magnolia bilola, the regenerating system of Magnolia bilola is established by the process such as sterile system foundation, Initial culture, adventitious buds proliferation cultivation, Multiple Buds strong seedling culture, rooting of vitro seedling and test-tube plantlet rooting culture, for the Fast-propagation of Magnolia bilola high-quality provenance provides production method, and then achieve object of the present invention.
The method that a kind of Magnolia bilola regenerating system of the present invention builds, comprises following processing step:
(1) sterile system is set up: choose mature and plump, the complete kind Magnolia bilola seed without damage by disease and insect, tentatively clean up under running water.Seed soaks 2 ~ 5h with 0.1% ~ 0.5% liquor potassic permanganate, until the lighter of liquor potassic permanganate is to pink, sterilize 1 ~ 5min after tap water is clean in 75% ethanolic solution, aseptic water washing 4 ~ 5 times, aseptic filter paper drains, manually remove exosper, be inoculated on germination medium and carry out Primary culture.
(2) Initial culture: the aseptic seedling that the sprouting cotyledon that sterile system is cultivated does not come off is inoculated on Initial culture base and cultivates, first full light culture 7 ~ 14 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, cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C, and switching in 30 ~ 40 days once.
(3) adventitious buds proliferation is cultivated: the aseptic seedling of Initial culture be inoculated on proliferated culture medium without the stem section that terminal bud length is 1.5 ~ 2.0cm and carry out Multiplying culture, first full light culture 7 ~ 14 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, cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C, and switching in 30 ~ 40 days once.
(4) Multiple Buds strong seedling culture: the indefinite bud that adventitious buds proliferation is obtained, the a small amount of callus of excision base portion, be inoculated on strong seedling culture base after removing bottom blade and carry out strong seedling culture, first full light culture 7 ~ 14 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, and switching in 30 ~ 40 days once.
(5) rooting of vitro seedling: will grow to 1.5 ~ 2.0cm's, the number of blade is no less than 3, what plant was more healthy and stronger cut to be inoculated on root media without offspring carries out root induction, first full light culture 7 ~ 14 days 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.
(6) test-tube plantlet rooting culture: the blake bottle bottle cap growing to the rooting tube plantlet of 6 ~ 8cm 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, to plant in the matrix be mixed into by yellow sand and fiery carbon mud (1:1) and to be colonizated in large Tanaka.
Germination medium described in above-mentioned steps (1) is: 3/4B
5+ 0.1 ~ 0.5mg/L 6-BA+0.1 ~ 0.2mg/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.
Initial culture base described in above-mentioned steps (2) is: B
5+ 1 ~ 4mg/L 6-BA+1 ~ 1.5mg/L 2,4-D+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+1 ~ 2mg/L KT+1 ~ 1mg/L NAA+1 ~ 3mg/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.
On the strong seedling culture base be set forth in described in step (4) be MS+0.1 ~ 0.5mg/L NAA+0.5 ~ 1mg/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+0.1 ~ 0.5mg/L NAA+2.5% ~ 3.5% sucrose+0.35% ~ 0.5% agar+0.05% ~ 0.1% active carbon, and pH value is 5.4 ~ 5.8.
Compared with prior art advantage of the present invention is: instant invention overcomes the problems such as the pollution rate existed in Magnolia bilola tissue cultures is high, melting brown rate is high, difficulty of taking root, transplanting survival rate are low, construct Magnolia bilola regenerating system, thus facilitate the commercialization process of Magnolia bilola.
Embodiment
Following examples further illustrate of the present invention, is not limitation of the present invention.
Embodiment 1:
(1) sterile system is set up: choose mature and plump, the complete kind Magnolia bilola seed without damage by disease and insect, tentatively clean up under running water.Seed soaks 2h with 0.1% liquor potassic permanganate, until the lighter of liquor potassic permanganate is to pink, sterilize 5min after tap water is clean in 75% ethanolic solution, aseptic water washing 4 times, aseptic filter paper drains, and manually removes exosper, is inoculated on germination medium and carries out Primary culture.Described germination medium is 3/4B
5+ 0.3mg/L 6-BA+0.2mg/L NAA+3.5% sucrose+0.5% agar+0.1% active carbon, pH value is 5.8.
(2) Initial culture: the aseptic seedling that the sprouting cotyledon that sterile system is cultivated does not come off is inoculated on Initial culture base and cultivates, first full light culture 10 days under 28 DEG C of conditions after inoculation, then illumination every day is placed in 15 hours, intensity of illumination is 2500lx, cultivation temperature is cultivate under the condition of 28 DEG C, and switching in 30 days once.Described Initial culture base is B
5+ 3mg/L 6-BA+1.5mg/L 2,4-D+2.5% sucrose+0.5% agar+0.05% active carbon, pH value is 5.8.
(3) adventitious buds proliferation is cultivated: the aseptic seedling of Initial culture be inoculated on proliferated culture medium without the stem section that terminal bud length is 1.5 ~ 2.0cm and carry out Multiplying culture, first full light culture 14 days under 28 DEG C of conditions after inoculation, then illumination every day is placed in 15 hours, intensity of illumination is 2500lx, cultivation temperature is cultivate under the condition of 28 DEG C, and switching in 40 days once.Described proliferated culture medium is MS+1mg/L KT+1mg/L NAA+3mg/L 6-BA+3.5% sucrose+0.5% agar+0.05% active carbon, and pH value is 5.8.
(4) Multiple Buds strong seedling culture: the indefinite bud that adventitious buds proliferation is obtained, the a small amount of callus of excision base portion, be inoculated on strong seedling culture base after removing bottom blade and carry out strong seedling culture, first full light culture 14 days under 28 DEG C of conditions after inoculation, then illumination every day is placed in 12 hours, intensity of illumination is 1500 ~ 2500lx, and cultivation temperature is cultivate under the condition of 28 DEG C, and switching in 30 days once.Described strong seedling culture base is MS+0.5mg/L NAA+0.5mg/L 6-BA+3.5% sucrose+0.35% agar+0.1% active carbon, and pH value is 5.8.
(5) rooting of vitro seedling: will grow to 1.5 ~ 2.0cm's, the number of blade is no less than 3, what plant was more healthy and stronger cut to be inoculated on root media without offspring carries out root induction, first full light culture 14 days under 28 DEG C of conditions after inoculation, then illumination every day is placed in 13 hours, intensity of illumination is 2500lx, and cultivation temperature is be cultured under the condition of 28 DEG C to take root, and rooting rate reaches 89%.Described root media is 1/2MS+0.5mg/L NAA+2.5% sucrose+0.35% agar+0.1% active carbon, and pH value is 5.8.
(6) test-tube plantlet rooting culture: the blake bottle bottle cap growing to the rooting tube plantlet of 6 ~ 8cm is opened and is placed in natural lighting lower refining seedling after 7 days, test-tube plantlet is taken out from blake bottle, wash root medium off, to plant in the matrix be mixed into by yellow sand and fiery carbon mud (1:1) and to be colonizated in large Tanaka, survival rate more than 93%.
Embodiment 2:
(1) sterile system is set up: choose mature and plump, the complete kind Magnolia bilola seed without damage by disease and insect, tentatively clean up under running water.Seed soaks 4h with 0.3% liquor potassic permanganate, until the lighter of liquor potassic permanganate is to pink, sterilize 5min after tap water is clean in 75% ethanolic solution, aseptic water washing 5 times, aseptic filter paper drains, and manually removes exosper, is inoculated on germination medium and carries out Primary culture.Described germination medium is 3/4B
5+ 0.5mg/L 6-BA+0.4mg/L NAA+2.5% sucrose+0.5% agar+0.1% active carbon, pH value is 5.6.
(2) Initial culture: the aseptic seedling that the sprouting cotyledon that sterile system is cultivated does not come off is inoculated on Initial culture base and cultivates, first full light culture 10 days under 28 DEG C of conditions after inoculation, then illumination every day is placed in 13 hours, intensity of illumination is 2000lx, cultivation temperature is cultivate under the condition of 28 DEG C, and switching in 40 days once.Described Initial culture base is B
5+ 2mg/L 6-BA+0.5mg/L 2,4-D+3.5% sucrose+0.3% agar+0.1% active carbon, pH value is 5.6.
(3) adventitious buds proliferation is cultivated: the aseptic seedling of Initial culture be inoculated on proliferated culture medium without the stem section that terminal bud length is 1.5 ~ 2.0cm and carry out Multiplying culture, first full light culture 12 days under 28 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 28 DEG C, and switching in 40 days once.Described proliferated culture medium is MS+1.5mg/L KT+0.5mg/L NAA+1mg/L 6-BA+3.5% sucrose+0.5% agar+0.05% active carbon, and pH value is 5.6.
(4) Multiple Buds strong seedling culture: the indefinite bud that adventitious buds proliferation is obtained, the a small amount of callus of excision base portion, be inoculated on strong seedling culture base after removing bottom blade and carry out strong seedling culture, first full light culture 14 days under 28 DEG C of conditions after inoculation, then illumination every day is placed in 12 hours, intensity of illumination is 1500 ~ 2500lx, and cultivation temperature is cultivate under the condition of 28 DEG C, and switching in 30 days once.Described strong seedling culture base is MS+0.6mg/L NAA+0.4mg/L 6-BA+3.5% sucrose+0.35% agar+0.08% active carbon, and pH value is 5.6.
(5) rooting of vitro seedling: will grow to 1.5 ~ 2.0cm's, the number of blade is no less than 3, what plant was more healthy and stronger cut to be inoculated on root media without offspring carries out root induction, first full light culture 14 days under 28 DEG C of conditions after inoculation, then illumination every day is placed in 13 hours, intensity of illumination is 2500lx, and cultivation temperature is be cultured under the condition of 28 DEG C to take root, and rooting rate reaches 89%.Described root media is 1/2MS+1mg/L NAA+2.8% sucrose+0.35% agar+0.08% active carbon, and pH value is 5.6.
(6) test-tube plantlet rooting culture: the blake bottle bottle cap growing to the rooting tube plantlet of 6 ~ 8cm is opened and is placed in natural lighting lower refining seedling after 7 days, test-tube plantlet is taken out from blake bottle, wash root medium off, to plant in the matrix be mixed into by yellow sand and fiery carbon mud (1:1) and to be colonizated in large Tanaka, survival rate more than 91%.
Claims (6)
1. a method for Magnolia bilola regenerating system structure, is characterized in that comprising following processing step:
(1) sterile system is set up: choose mature and plump, the complete kind Magnolia bilola seed without damage by disease and insect, tentatively clean up under running water, seed soaks 2 ~ 5h with 0.1% ~ 0.5% liquor potassic permanganate, until the lighter of liquor potassic permanganate is to pink, sterilize 1 ~ 5min after tap water is clean in 75% ethanolic solution, aseptic water washing 4 ~ 5 times, and aseptic filter paper drains, manually remove exosper, be inoculated on germination medium and carry out Primary culture;
(2) Initial culture: the aseptic seedling that the sprouting cotyledon that sterile system is cultivated does not come off is inoculated on Initial culture base and cultivates, first full light culture 7 ~ 14 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, cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C, and switching in 30 ~ 40 days once;
(3) adventitious buds proliferation is cultivated: the aseptic seedling of Initial culture be inoculated on proliferated culture medium without the stem section that terminal bud length is 1.5 ~ 2.0cm and carry out Multiplying culture, first full light culture 7 ~ 14 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, cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C, and switching in 30 ~ 40 days once;
(4) Multiple Buds strong seedling culture: the indefinite bud that adventitious buds proliferation is obtained, the a small amount of callus of excision base portion, be inoculated on strong seedling culture base after removing bottom blade and carry out strong seedling culture, first full light culture 7 ~ 14 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, and switching in 30 ~ 40 days once;
(5) rooting of vitro seedling: will grow to 1.5 ~ 2.0cm's, the number of blade is no less than 3, what plant was more healthy and stronger cut to be inoculated on root media without offspring carries out root induction, first full light culture 7 ~ 14 days 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;
(6) test-tube plantlet rooting culture: the blake bottle bottle cap growing to the rooting tube plantlet of 6 ~ 8cm 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, 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. the method for a kind of Magnolia bilola regenerating system structure according to claim 1, is characterized in that the germination medium described in step (1) is: 3/4B
5+ 0.1 ~ 0.5mg/L 6-BA+0.1 ~ 0.2mg/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.
3. the method for a kind of Magnolia bilola regenerating system structure according to claim 1, is characterized in that the Initial culture base described in step (2) is: B
5+ 1 ~ 4mg/L 6-BA+1 ~ 1.5mg/L 2,4-D+2.5% ~ 3.5% sucrose+0.35% ~ 0.5% agar+0.05% ~ 0.1% active carbon, pH value is 5.4 ~ 5.8.
4. the method for a kind of Magnolia bilola regenerating system structure according to claim 1, it is characterized in that the proliferated culture medium described in step (3) is: MS+1 ~ 2mg/L KT+1 ~ 1mg/L NAA+1 ~ 3mg/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. the method for a kind of Magnolia bilola regenerating system structure according to claim 1, it is characterized in that the strong seedling culture base described in step (4) is MS+0.1 ~ 0.5mg/L NAA+0.5 ~ 1mg/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.
6. the method for a kind of Magnolia bilola regenerating system structure according to claim 1, it is characterized in that the root media described in step (5) is 1/2MS+0.1 ~ 0.5mg/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.
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| CN105613287A (en) * | 2015-12-24 | 2016-06-01 | 华南农业大学 | Tissue rapid propagation seedling cultivation method for manglietia fadouensis |
| CN107509635A (en) * | 2017-10-11 | 2017-12-26 | 陈金水 | A kind of in vitro tissue culture and rapid propagation method of Chinese anise |
| CN109247237A (en) * | 2018-11-22 | 2019-01-22 | 林登淞 | A kind of construction method of river Ciliatenerve Knotweed Root regenerating system |
| CN109496853A (en) * | 2018-11-27 | 2019-03-22 | 钟天路 | A kind of construction method of trigone regenerating system |
| CN109496845A (en) * | 2018-11-23 | 2019-03-22 | 韦宇 | A kind of construction method of ardisia crispa regenerating system |
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| CN105409748A (en) * | 2015-12-08 | 2016-03-23 | 上海市园林科学研究所 | Fast breeding method for scirpus mariqueter |
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| CN105613287B (en) * | 2015-12-24 | 2017-09-05 | 华南农业大学 | A kind of method bucket cotton rose tissue rapid propagation method for culturing seedlings |
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| CN109247237A (en) * | 2018-11-22 | 2019-01-22 | 林登淞 | A kind of construction method of river Ciliatenerve Knotweed Root regenerating system |
| CN109496845A (en) * | 2018-11-23 | 2019-03-22 | 韦宇 | A kind of construction method of ardisia crispa regenerating system |
| CN109496853A (en) * | 2018-11-27 | 2019-03-22 | 钟天路 | A kind of construction method of trigone regenerating system |
| CN111557240A (en) * | 2020-04-24 | 2020-08-21 | 中国科学院昆明植物研究所 | Method for rapidly propagating embryonic cells of mangnolia officinalis |
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Application publication date: 20150610 |