CN104686341A - Tissue culture technique of aquilaria sinensis - Google Patents
Tissue culture technique of aquilaria sinensis Download PDFInfo
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- CN104686341A CN104686341A CN201510085931.0A CN201510085931A CN104686341A CN 104686341 A CN104686341 A CN 104686341A CN 201510085931 A CN201510085931 A CN 201510085931A CN 104686341 A CN104686341 A CN 104686341A
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Abstract
The invention discloses a tissue culture technique of aquilaria sinensis and relates to a seedling growing method for obtaining good-quality seedlings from the aquilaria sinensis by use of an in-vitro rapid propagation technique. According to the tissue culture technique, the tissue culture rapid propagation system of the aquilaria sinensis is established by taking the stem with buds of the aquilaria sinensis as the explant and by virtue of the processes of explant sterilization, multiple shoot induction, rooting culture, acclimatization and transplanting and the like; the tissue culture technique has important practical significance on acceleration of the popularization of the good-quality aquilaria sinensis variety and the development and utilization of resources.
Description
Technical field
The present invention relates to the method for Plant Tissue Breeding in Plant Biotechnology, specifically, relate to a kind of tissue culture technique of suspension culture of Aquilaria sinensis.
Background technology
Suspension culture of Aquilaria sinensis (
aquilaria sinensis) be the unique plant origin of China agalloch eaglewood, supply falls short of demand for a long time, adds that artificial predation formula felling causes suspension culture of Aquilaria sinensis wild resource exhausted, be therefore necessary to protect its resource.
In recent years, suspension culture of Aquilaria sinensis development of resources has had certain scale, and in Guangxi, Guangdong, Fujian etc. establishes GAP planting base, serves certain facilitation to suspension culture of Aquilaria sinensis wild resource protection work.Current suspension culture of Aquilaria sinensis carries out sapling multiplication mainly through seed, but is separated due to seminal propagation characters of progenies, is unfavorable for high-quality plasm resource protection, constrains the popularizing planting of the high quality white banksia rose.Therefore, be necessary the tissue culture technique setting up suspension culture of Aquilaria sinensis, for its breeding Fast-propagation provides technical support and guarantee, also for meet China to the demand of suspension culture of Aquilaria sinensis seedling, accelerate its resource and expand and have important practical significance.
Summary of the invention
The object of the present invention is to provide out a kind of tissue culture technique of suspension culture of Aquilaria sinensis, the present invention with suspension culture of Aquilaria sinensis stem with bud for explant, establish suspension culture of Aquilaria sinensis tissue-culturing quick-propagation system through processes such as explant sterilization, inducing clumping bud, culture of rootage, acclimatization and transplantses, thus achieve object of the present invention.
The tissue culture technique of a kind of suspension culture of Aquilaria sinensis of the present invention, comprises the following steps:
(1) inducing clumping bud: the stem with bud that the treelet choosing the anosis worm of robust growth gives birth to then, as explant, with liquid detergent aqueous solution soaking 5 ~ 10min, then uses tap water 10 ~ 30min, for subsequent use after drying surface moisture.With 70% ~ 80% alcohol immersion 5 ~ 10s in superclean bench, 0.1% mercuric chloride solution sterilization 8 ~ 20min, use sterile water wash again 3 ~ 5 times, being cut into length after drying is that the stem section of 0.5 ~ 1.2cm is inoculated on inducing culture, first full light culture 15 ~ 20 days under 25 ~ 28 DEG C of conditions, be then placed in illumination every day 10 ~ 12 hours, intensity of illumination is 1000 ~ 1500lx, cultivation temperature is cultivate under 25 ~ 28 DEG C of conditions, until induced synthesis Multiple Buds.
(2) Multiplying culture: the Multiple Buds of step (1) gained is proceeded on proliferated culture medium and carries out Multiplying culture, after inoculation first under 25 ~ 28 DEG C of conditions full light culture until axil place germinates a lateral bud, then illumination every day is placed in 12 ~ 15 hours, intensity of illumination is 1000 ~ 1500lx, and cultivation temperature is cultivate until each Multiple Buds is elongated to 1 ~ 2cm under the condition of 25 ~ 28 DEG C.
(3) culture of rootage: the Multiple Buds bud height that step (1) or (2) obtain being about 1 ~ 2cm cuts and is 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 10 ~ 12 hours, intensity of illumination is 2000 ~ 2500lx, and cultivation temperature is be cultured under the condition of 25 ~ 28 DEG C to take root.
(4) acclimatization and transplants: after taking root in whitewood's smelling bottle 20 ~ 30 days, was placed in natural lighting lower refining seedling after 5 ~ 7 days, cleaned root medium, transplanted by yellow soil: in the matrix that river sand=3:1 is mixed into.
Inducing culture described in above-mentioned steps (1) is: MS+2 ~ 4mg/L6-BA+0.5 ~ 1mg/L NAA+0.1 ~ 0.5mmol/L La (NO
3)
2+ 2.0% ~ 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 (2) is: MS+1 ~ 2mg/L6-BA+0.1 ~ 0.5mg/L NAA+2.0% ~ 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 (3) is: 1/2MS+1 ~ 3mg/L NAA+2.0% ~ 3.5% sucrose+0.35% ~ 0.5% agar+0.05% ~ 0.1% active carbon, pH value is 5.4 ~ 5.8.
Compared with prior art advantage of the present invention is: the present invention obtains the seedling-cultivating method of high quality seedling by rapid propagation in vitro technology.With suspension culture of Aquilaria sinensis stem with bud for explant, establish suspension culture of Aquilaria sinensis tissue-culturing quick-propagation system through processes such as explant sterilization, inducing clumping bud, culture of rootage, acclimatization and transplantses, the popularization and the resources development and utilization that accelerate suspension culture of Aquilaria sinensis breeding have important practical significance.
Embodiment
Following examples further illustrate of the present invention, is not limitation of the present invention.
Embodiment 1:
(1) inducing clumping bud: the stem with bud that the treelet choosing the anosis worm of robust growth gives birth to then, as explant, with liquid detergent aqueous solution soaking 5min, then uses tap water 10min, for subsequent use after drying surface moisture.With 75% alcohol immersion 5s in superclean bench, 0.1% mercuric chloride solution sterilization 8min, use sterile water wash again 3 times, being cut into length after drying is that the stem section of 0.5 ~ 1.2cm is inoculated on inducing culture, first full light culture 15 days under 25 DEG C of conditions, be then placed in illumination every day 10 hours, intensity of illumination is 1000x, cultivation temperature is cultivate under 25 DEG C of conditions to get final product induced synthesis Multiple Buds in 39 days, and inductivity is 68.5%.Described inducing culture is MS+2mg/L6-BA+0.5mg/L NAA+0.1mmol/L La (NO
3)
2+ 2.0% sucrose+0.35% agar+0.05% active carbon, pH value is 5.8.
(2) Multiplying culture: the Multiple Buds of step (1) gained is proceeded on proliferated culture medium and carries out Multiplying culture, after inoculation first under 25 DEG C of conditions full light culture until axil place germinates a lateral bud, then illumination every day is placed in 13 hours, intensity of illumination is 1000lx, cultivation temperature is cultivate 31 days each Multiple Buds under the condition of 25 DEG C can be elongated to 1 ~ 2cm, and the rate of increase is 4.09.Described proliferated culture medium is MS+1mg/L6-BA+0.1mg/L NAA+2.0% sucrose+0.35% agar+0.05% active carbon, and pH value is 5.8.
(3) culture of rootage: the Multiple Buds bud height that step (1) or (2) obtain being about 1 ~ 2cm cuts and is inoculated on root media and carries out culture of rootage, first full light culture 3 days under 25 DEG C of conditions after inoculation, then illumination every day is placed in 10 hours, intensity of illumination is 2000lx, cultivation temperature is cultivate under the condition of 25 DEG C can take root for 39 days, and rooting rate is 73.4%.
(4) acclimatization and transplants: after taking root in whitewood's smelling bottle 20 days, was placed in natural lighting lower refining seedling after 5 days, cleaned root medium, transplanted by yellow soil: in the matrix that river sand=3:1 is mixed into.Transplanting survival rate is 87%.
Embodiment 2:
(1) inducing clumping bud: the stem with bud that the treelet choosing the anosis worm of robust growth gives birth to then, as explant, with liquid detergent aqueous solution soaking 8min, then uses tap water 15min, for subsequent use after drying surface moisture.With 78% alcohol immersion 10s in superclean bench, 0.1% mercuric chloride solution sterilization 10min, use sterile water wash again 4 times, being cut into length after drying is that the stem section of 0.5 ~ 1.2cm is inoculated on inducing culture, first full light culture 18 days under 28 DEG C of conditions, be then placed in illumination every day 12 hours, intensity of illumination is 1500x, cultivation temperature is cultivate under 28 DEG C of conditions to get final product induced synthesis Multiple Buds in 36 days, and inductivity is 74.9%.Described inducing culture is MS+4mg/L6-BA+0.9mg/L NAA+0.3mmol/L La (NO
3)
2+ 2.0% sucrose+0.35% agar+0.05% active carbon, pH value is 5.8.
(2) Multiplying culture: the Multiple Buds of step (1) gained is proceeded on proliferated culture medium and carries out Multiplying culture, after inoculation first under 28 DEG C of conditions full light culture until axil place germinates a lateral bud, then illumination every day is placed in 14 hours, intensity of illumination is 1600lx, cultivation temperature is cultivate 30 days each Multiple Buds under the condition of 28 DEG C can be elongated to 1 ~ 2cm, and the rate of increase is 5.29.Described proliferated culture medium is MS+1.5mg/L6-BA+0.4mg/L NAA+2.0% sucrose+0.35% agar+0.1% active carbon, and pH value is 5.8.
(3) culture of rootage: the Multiple Buds bud height that step (1) or (2) obtain being about 1 ~ 2cm cuts and is inoculated on root media and carries out culture of rootage, first full light culture 5 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 can take root for 33 days, and rooting rate is 80.9%.
(4) acclimatization and transplants: after taking root in whitewood's smelling bottle 20 days, was placed in natural lighting lower refining seedling after 5 days, cleaned root medium, transplanted by yellow soil: in the matrix that river sand=3:1 is mixed into.Transplanting survival rate is 90%.
Claims (4)
1. a tissue culture technique for suspension culture of Aquilaria sinensis, is characterized in that comprising the following steps:
(1) inducing clumping bud: the stem with bud that the treelet choosing the anosis worm of robust growth gives birth to then is as explant, with liquid detergent aqueous solution soaking 5 ~ 10min, then tap water 10 ~ 30min is used, for subsequent use after drying surface moisture, with 70% ~ 80% alcohol immersion 5 ~ 10s in superclean bench, 0.1% mercuric chloride solution sterilization 8 ~ 20min, use sterile water wash again 3 ~ 5 times, being cut into length after drying is that the stem section of 0.5 ~ 1.2cm is inoculated on inducing culture, first full light culture 15 ~ 20 days under 25 ~ 28 DEG C of conditions, then illumination every day is placed in 10 ~ 12 hours, intensity of illumination is 1000 ~ 1500lx, cultivation temperature is cultivate under 25 ~ 28 DEG C of conditions, until induced synthesis Multiple Buds,
(2) Multiplying culture: the Multiple Buds of step (1) gained is proceeded on proliferated culture medium and carries out Multiplying culture, after inoculation first under 25 ~ 28 DEG C of conditions full light culture until axil place germinates a lateral bud, then illumination every day is placed in 12 ~ 15 hours, intensity of illumination is 1000 ~ 1500lx, and cultivation temperature is cultivate until each Multiple Buds is elongated to 1 ~ 2cm under the condition of 25 ~ 28 DEG C;
(3) culture of rootage: the Multiple Buds bud height that step (1) or (2) obtain being about 1 ~ 2cm cuts and is 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 10 ~ 12 hours, intensity of illumination is 2000 ~ 2500lx, and cultivation temperature is be cultured under the condition of 25 ~ 28 DEG C to take root;
(4) acclimatization and transplants: after taking root in whitewood's smelling bottle 20 ~ 30 days, was placed in natural lighting lower refining seedling after 5 ~ 7 days, cleaned root medium, transplanted by yellow soil: in the matrix that river sand=3:1 is mixed into.
2. the tissue culture technique of a kind of suspension culture of Aquilaria sinensis according to claim 1, is characterized in that the inducing culture described in step (1) is: MS+2 ~ 4mg/L6-BA+0.5 ~ 1mg/L NAA+0.1 ~ 0.5mmol/L La (NO
3)
2+ 2.0% ~ 3.5% sucrose+0.35% ~ 0.5% agar+0.05% ~ 0.1% active carbon, pH value is 5.4 ~ 5.8.
3. the tissue culture technique of a kind of suspension culture of Aquilaria sinensis according to claim 1, it is characterized in that the proliferated culture medium described in step (2) is: MS+1 ~ 2mg/L6-BA+0.1 ~ 0.5mg/L NAA+2.0% ~ 3.5% sucrose+0.35% ~ 0.5% agar+0.05% ~ 0.1% active carbon, pH value is 5.4 ~ 5.8.
4. the tissue culture technique of a kind of suspension culture of Aquilaria sinensis according to claim 1, it is characterized in that the root media described in step (3) is: 1/2MS+1 ~ 3mg/L NAA+2.0% ~ 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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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104938336A (en) * | 2015-06-11 | 2015-09-30 | 仲恺农业工程学院 | Rapid propagation method for promoting germination of aquilaria sinensis isolated bud |
| CN106591215A (en) * | 2016-12-29 | 2017-04-26 | 广东药科大学 | Culture method for aquilaria sinensis callus cell suspension system |
| CN106818489A (en) * | 2017-03-23 | 2017-06-13 | 江苏农林职业技术学院 | A kind of lilac daphne stem section explant primary method for tissue culture |
| CN107347637A (en) * | 2017-06-26 | 2017-11-17 | 浦江县美泽生物科技有限公司 | Suspension culture of Aquilaria sinensis method for tissue culture for large-scale plantation |
| CN108841856A (en) * | 2018-06-19 | 2018-11-20 | 中国医学科学院药用植物研究所 | The hairy root induction of suspension culture of Aquilaria sinensis and its genetic transforming method |
| CN109220812A (en) * | 2018-11-25 | 2019-01-18 | 林登淞 | A kind of tissue culture technique of Chinese knotweed |
| CN109258476A (en) * | 2018-11-30 | 2019-01-25 | 林登淞 | A kind of tissue culture technique of aizoon stonecrop |
| CN109258471A (en) * | 2018-11-22 | 2019-01-25 | 林登淞 | A kind of tissue culture technique of sweet wormwood |
| CN109258472A (en) * | 2018-11-22 | 2019-01-25 | 林登淞 | A kind of tissue culture technique of powder tetrandra root |
| CN109275571A (en) * | 2018-11-29 | 2019-01-29 | 广西玉林市千力农业科技有限公司 | A kind of tissue culture technique of dianthus superbus |
| CN109329062A (en) * | 2018-11-27 | 2019-02-15 | 广西玉林市华睿茶业有限公司 | A kind of tissue culture technique of cottonrose hibiscus leaf |
| CN109380083A (en) * | 2018-11-22 | 2019-02-26 | 张世燊 | A kind of tissue culture technique of bicolor |
| CN109380120A (en) * | 2018-11-29 | 2019-02-26 | 钟天路 | A kind of tissue culture technique of chestnut |
| CN109496852A (en) * | 2018-11-27 | 2019-03-22 | 钟天路 | A kind of tissue culture technique of mountain tortoise |
| CN116584392A (en) * | 2023-06-09 | 2023-08-15 | 中国林业科学研究院热带林业研究所 | Asexual rapid propagation method of agilawood-easy-to-form excellent strain golden silk oil |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104938336A (en) * | 2015-06-11 | 2015-09-30 | 仲恺农业工程学院 | Rapid propagation method for promoting germination of aquilaria sinensis isolated bud |
| CN106591215A (en) * | 2016-12-29 | 2017-04-26 | 广东药科大学 | Culture method for aquilaria sinensis callus cell suspension system |
| CN106818489A (en) * | 2017-03-23 | 2017-06-13 | 江苏农林职业技术学院 | A kind of lilac daphne stem section explant primary method for tissue culture |
| CN106818489B (en) * | 2017-03-23 | 2019-03-15 | 江苏农林职业技术学院 | A kind of lilac daphne stem section explant method for tissue culture primary |
| CN107347637A (en) * | 2017-06-26 | 2017-11-17 | 浦江县美泽生物科技有限公司 | Suspension culture of Aquilaria sinensis method for tissue culture for large-scale plantation |
| CN108841856A (en) * | 2018-06-19 | 2018-11-20 | 中国医学科学院药用植物研究所 | The hairy root induction of suspension culture of Aquilaria sinensis and its genetic transforming method |
| CN109258472A (en) * | 2018-11-22 | 2019-01-25 | 林登淞 | A kind of tissue culture technique of powder tetrandra root |
| CN109258471A (en) * | 2018-11-22 | 2019-01-25 | 林登淞 | A kind of tissue culture technique of sweet wormwood |
| CN109380083A (en) * | 2018-11-22 | 2019-02-26 | 张世燊 | A kind of tissue culture technique of bicolor |
| CN109220812A (en) * | 2018-11-25 | 2019-01-18 | 林登淞 | A kind of tissue culture technique of Chinese knotweed |
| CN109329062A (en) * | 2018-11-27 | 2019-02-15 | 广西玉林市华睿茶业有限公司 | A kind of tissue culture technique of cottonrose hibiscus leaf |
| CN109496852A (en) * | 2018-11-27 | 2019-03-22 | 钟天路 | A kind of tissue culture technique of mountain tortoise |
| CN109275571A (en) * | 2018-11-29 | 2019-01-29 | 广西玉林市千力农业科技有限公司 | A kind of tissue culture technique of dianthus superbus |
| CN109380120A (en) * | 2018-11-29 | 2019-02-26 | 钟天路 | A kind of tissue culture technique of chestnut |
| CN109258476A (en) * | 2018-11-30 | 2019-01-25 | 林登淞 | A kind of tissue culture technique of aizoon stonecrop |
| CN116584392A (en) * | 2023-06-09 | 2023-08-15 | 中国林业科学研究院热带林业研究所 | Asexual rapid propagation method of agilawood-easy-to-form excellent strain golden silk oil |
| CN116584392B (en) * | 2023-06-09 | 2023-12-08 | 中国林业科学研究院热带林业研究所 | Asexual rapid propagation method of agilawood-easy-to-form excellent strain golden silk oil |
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