CN104686351A - In-vitro rapid propagation method of cercidiphyllum japonicum - Google Patents
In-vitro rapid propagation method of cercidiphyllum japonicum Download PDFInfo
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Abstract
The invention discloses an in-vitro rapid propagation method of cercidiphyllum japonicum. The cercidiphyllum japonicum is the valuable, rare and endangered plant of secondary protection of China, and has extremely high scientific research, and medicinal and ornamental values. At present, the cercidiphyllum japonicum is generally propagated by cottage and grafting and by use of seeds, but due to the propagation methods, the reproduction rate is low and the reproduction period is long, and far fail in meeting the requirements of seedlings for landscaping, scientific research and the like; according to the in-vitro rapid propagation method of the cercidiphyllum japonicum, the in-vitro rapid propagation system of the cercidiphyllum japonicum is established by taking the stem with axillary buds of the cercidiphyllum japonicum as explant and by virtue of processes of explant processing, induced culture, propagation, rooting, acclimatization and transplanting and the like; the propagation coefficient of the cercidiphyllum japonicum can be increased, and then the industrialized seedling production of the cercidiphyllum japonicum can be realized.
Description
Technical field
The present invention relates to the method for Plant Tissue Breeding in agricultural biotechnologies, specifically, relate to a kind of katsura tree method for in-vitro rapid propagation.
Background technology
Katsura tree (
cercidiphyllum japonicum) have another name called Wu Junshu, Chinese filbert, cercis Ye Mu etc.,
For few survivors's single plant in the Tertiary Period, Disjunct distribution, in China and Japan, is China second class protection rare or endangered species.Due to the taste fragrance of katsura tree, just can smell within hundred meters, if set greatly, then shake more fragrant and more fragrant, therefore, be referred to as " katsura tree ".The purposes of katsura tree is very extensive, and trunk can be used for papermaking, and root, stem, leaf and fruit all have higher medical value, and its bark is decocted water and taken, and can treat flu, dysentery, and leaf can be used as the raw material extracting spices.Katsura tree trunk is logical straight, tree-like grace, floral white, little and attractive in appearance, and has light perfume (or spice).Leaf peculiar, beautiful uniqueness, autumn, blade became yellow or red, was therefore the Landscape Trees and avenue tree planting that in town and country, courtyard greening and Landscape, ornamental value is very high.Katsura tree wood quality is excellent, texture is logical directly, structure is careful, quality is hard, is world's preciousness material and important coinage tree.Therefore, katsura tree have higher economy, view and admire, medicinal and scientific research value.
At present, the breeding of katsura tree mainly relies on conventional propagation method, and the breeding cycle is long and reproduction speed is slow, is difficult to meet scientific research and market to the demand of katsura tree nursery stock.Plant tissue culture fast breeding technique can be bred by rapid, high volume at short notice, and obtains the consistent excellent nursery stock of genotype, and greatly accelerate nursery process, a new approach is opened up in the preservation for rare and endangered tree species germ plasm resource.Research at present in katsura tree Plant Tissue Breeding rarely has report, and the present invention is by inducing clumping bud and the key technique such as propagation, vegetative seedling root induction, greatly improving katsura tree reproduction coefficient, providing technical support for realizing katsura tree tissue cultures factorial praluction.
Summary of the invention
The object of the present invention is to provide out a kind of katsura tree method for in-vitro rapid propagation, the present invention is with the stem section of axillalry bud for explant with katsura tree, through explant process, Fiber differentiation, breed, take root, the process such as hardening and transplanting establishes katsura tree Vitro Quick Reproduction system, improve the growth coefficient of katsura tree, thus achieve object of the present invention.
A kind of katsura tree method for in-vitro rapid propagation of the present invention, comprises the following steps:
(1) explant sampling and processing: the dust on surface was rinsed well by raw stem segment with axillary bud running water in 1 year that adopts back from field, again with 1% ~ 3% drip dew carry out spraying be placed on climatic cabinate with 25 ~ 28 DEG C of temperature, 70% ~ 90% humidity, illumination every day 12 ~ 16 hours, intensity of illumination is carry out water planting 3 ~ 5 days under 1500 ~ 3000lx condition, and during water planting, every day changes a water.
(2) Fiber differentiation: the big leaf's slice of explant after step (1) process is cut off and is cut into the long stem section of about 3 ~ 4cm, first be added with 2 ~ 3 drip dew washing powder solution immersion treatment 10 ~ 30min after running water 1 ~ 3h, in superclean bench with after 75% ethanol disinfection 30 ~ 60s with aseptic washing 3 ~ 5 times, again with 0.1% mercuric chloride solution sterilization 10 ~ 25min, carry out inducing clumping bud cultivation with being inoculated in inducing culture after sucking moisture with aseptic filter paper after aseptic water washing 4 ~ 6 times.First full light culture 1 ~ 3 day under 25 ~ 28 DEG C of conditions after inoculation, be then placed in illumination every day 12 ~ 16 hours, intensity of illumination is 2000 ~ 3000lx, and being placed in cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C until formation Multiple Buds.
(3) Multiplying culture: by step (2) cultivate obtain grow thickly grow to 2 ~ 3cm height time, cut bud from base portion and proceed to proliferated culture medium and carry out squamous subculture, 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, and being placed in cultivation temperature is cultivate until form more Multiple Buds under the condition of 25 ~ 28 DEG C.
(4) culture of rootage: when the Multiple Buds of step (3) is grown to 3 ~ 4cm height, cut into simple bud and be inoculated on root media and carry 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 14 ~ 16 hours, intensity of illumination is 3000 ~ 5000lx, and cultivation temperature is be cultured under the condition of 25 ~ 28 DEG C to take root.
(5) acclimatization and transplants: by half-open for the cultivation bottle cap of the rooting tube plantlet of height about 6 ~ 7cm hardening 3 ~ 5 days, standard-sized sheet bottle cap adds appropriate running water and is placed in natural lighting lower refining seedling 5 ~ 7 days again, test-tube plantlet is taken out from blake bottle, wash root medium off, to plant in the matrix be mixed into by peat soil and yellow sand mud and to be colonizated in large Tanaka.
Inducing culture described in above-mentioned steps (2) is: MS+0.1 ~ 1.0mg/L6-BA+1.0 ~ 3.0mg/L2,4-D+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+1.0 ~ 3.0mg/L6-BA+0.1 ~ 1.0mg/LNAA+1.0 ~ 5.0mg/LGA
3+ 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+0.5 ~ 1.5mg/LNAA+1.0 ~ 5.0mg/LIBA+0.1 ~ 0.5mg/L6-BA+0.5 ~ 1.0g/LAC+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
Advantage of the present invention is: katsura tree (
cercidiphyllum japonicum) be China second class protection rare or endangered species, there is very high scientific research, medicinal and ornamental value.At present, the more employing cuttage of katsura tree, grafting and seminal propagation, but these propagation method reproduction rates are low, the breeding cycle is long, far can not meet the nursery stock demand of afforestation and scientific research etc., and the present invention is with the stem section of axillalry bud for explant with katsura tree, through explant process, Fiber differentiation, breed, take root, the process such as hardening and transplanting establishes katsura tree Vitro Quick Reproduction system, improve the growth coefficient of katsura tree, thus realize its factorial seedling growth.
Embodiment
Following examples further illustrate of the present invention, is not limitation of the present invention.
embodiment 1:
(1) explant sampling and processing: the dust on surface was rinsed well by raw stem segment with axillary bud running water in 1 year that adopts back from field, again with 1% drip dew carry out spraying be placed on climatic cabinate with 25 DEG C of temperature, 70% humidity, illumination every day 12 hours, intensity of illumination is carry out water planting 3 days under 1500lx condition, and during water planting, every day changes a water.
(2) Fiber differentiation: the big leaf's slice of explant after step (1) process is cut off and is cut into the long stem section of about 3 ~ 4cm, first be added with 2 drip dew washing powder solution immersion treatment 10min after running water 1h, in superclean bench with after 75% ethanol disinfection 30s with aseptic washing 3 times, again with 0.1% mercuric chloride solution sterilization 10min, carry out inducing clumping bud cultivation with being inoculated in inducing culture after sucking moisture with aseptic filter paper after aseptic water washing 4 times.First full light culture 1 day under 25 DEG C of conditions after inoculation, be then placed in illumination every day 12 hours, intensity of illumination is 2000lx, and being placed in cultivation temperature is cultivate under the condition of 25 DEG C can form Multiple Buds in straight 28 days, and inductivity is 78.3%.Described inducing culture is: MS+0.1mg/L6-BA+1.0mg/L2,4-D+19g/L sucrose+3.7g/L agar, pH is 5.6.
(3) Multiplying culture: by step (2) cultivate obtain grow thickly grow to 2 ~ 3cm height time, cut bud from base portion and proceed to proliferated culture medium and carry out squamous subculture, first full light culture 1 day under 25 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 under the condition of 25 DEG C, and growth coefficient is 6.33.Described proliferated culture medium is: MS+1.0mg/L6-BA+0.1mg/LNAA+2.0mg/LGA
3+ 30g/L sucrose+3.8g/L agar, pH is 5.6.
(4) culture of rootage: when the Multiple Buds of step (3) is grown to 3 ~ 4cm height, cut into simple bud and be inoculated on root media and carry out culture of rootage, first full light culture 1 day under 25 DEG C of conditions after inoculation, then illumination every day is placed in 14 hours, intensity of illumination is 3000lx, cultivation temperature is cultivate 35 days rooting rates under the condition of 25 DEG C to reach 87.5%, and mean elements is 5.5, and average root is long is 6.21cm.Described root media is: 1/2MS+0.5mg/L NAA+2.5mg/L IBA+0.1mg/L 6-BA+0.5g/L AC+18g/L sucrose+3.5g/L agar, pH is 5.4.
(5) acclimatization and transplants: by half-open for the cultivation bottle cap of the rooting tube plantlet of height about 6 ~ 7cm hardening 3 days, standard-sized sheet bottle cap adds appropriate running water and is placed in natural lighting lower refining seedling 5 days again, test-tube plantlet is taken out from blake bottle, wash root medium off, planting in the matrix be mixed into by peat soil and yellow sand mud and to be colonizated in large Tanaka, transplanting survival rate is 92.6%.
Embodiment 2
(1) explant sampling and processing: the dust on surface was rinsed well by raw stem segment with axillary bud running water in 1 year that adopts back from field, again with 2% drip dew carry out spraying be placed on climatic cabinate with 26 DEG C of temperature, 70% humidity, illumination every day 13 hours, intensity of illumination is carry out water planting 5 days under 2000lx condition, and during water planting, every day changes a water.
(2) Fiber differentiation: the big leaf's slice of explant after step (1) process is cut off and is cut into the long stem section of about 3 ~ 4cm, first be added with 2 drip dew washing powder solution immersion treatment 15min after running water 3h, in superclean bench with after 75% ethanol disinfection 45s with aseptic washing 5 times, again with 0.1% mercuric chloride solution sterilization 15min, carry out inducing clumping bud cultivation with being inoculated in inducing culture after sucking moisture with aseptic filter paper after aseptic water washing 5 times.First full light culture 3 days under 26 DEG C of conditions after inoculation, be then placed in illumination every day 12 hours, intensity of illumination is 2000lx, and being placed in cultivation temperature is cultivate under the condition of 26 DEG C can form Multiple Buds in straight 30 days, and inductivity is 84.9%.Described inducing culture is: MS+0.2mg/L6-BA+3.0mg/L2,4-D+19g/L sucrose+4.5g/L agar, pH is 5.8.
(3) Multiplying culture: by step (2) cultivate obtain grow thickly grow to 2 ~ 3cm height time, cut bud from base portion and proceed to proliferated culture medium and carry out squamous subculture, first full light culture 3 days under 26 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 under the condition of 26 DEG C, and growth coefficient is 6.16.Described proliferated culture medium is: MS+1.5mg/L6-BA+0.1mg/LNAA+3.0mg/LGA
3+ 30g/L sucrose+3.8g/L agar, pH is 5.8.
(4) culture of rootage: when the Multiple Buds of step (3) is grown to 3 ~ 4cm height, cut into simple bud and be inoculated on root media and carry out culture of rootage, first full light culture 3 days under 26 DEG C of conditions after inoculation, then illumination every day is placed in 15 hours, intensity of illumination is 3500lx, cultivation temperature is cultivate 31 days rooting rates under the condition of 26 DEG C to reach 90.06%, and mean elements is 5.8, and average root is long is 5.97cm.Described root media is: 1/2MS+0.5mg/L NAA+3.5mg/L IBA+0.2mg/L 6-BA+0.8g/L AC+25g/L sucrose+3.9g/L agar, pH is 5.8.
(5) acclimatization and transplants: by half-open for the cultivation bottle cap of the rooting tube plantlet of height about 6 ~ 7cm hardening 3 days, standard-sized sheet bottle cap adds appropriate running water and is placed in natural lighting lower refining seedling 5 days again, test-tube plantlet is taken out from blake bottle, wash root medium off, planting in the matrix be mixed into by peat soil and yellow sand mud and to be colonizated in large Tanaka, transplanting survival rate is 90.34%.
Claims (4)
1. a katsura tree method for in-vitro rapid propagation, is characterized in that comprising the following steps:
(1) explant sampling and processing: the dust on surface was rinsed well by raw stem segment with axillary bud running water in 1 year that adopts back from field, again with 1% ~ 3% drip dew carry out spraying be placed on climatic cabinate with 25 ~ 28 DEG C of temperature, 70% ~ 90% humidity, illumination every day 12 ~ 16 hours, intensity of illumination is carry out water planting 3 ~ 5 days under 1500 ~ 3000lx condition, and during water planting, every day changes a water;
(2) Fiber differentiation: the big leaf's slice of explant after step (1) process is cut off and is cut into the long stem section of about 3 ~ 4cm, first be added with 2 ~ 3 drip dew washing powder solution immersion treatment 10 ~ 30min after running water 1 ~ 3h, in superclean bench with after 75% ethanol disinfection 30 ~ 60s with aseptic washing 3 ~ 5 times, again with 0.1% mercuric chloride solution sterilization 10 ~ 25min, inducing clumping bud cultivation is carried out with being inoculated in inducing culture after sucking moisture with aseptic filter paper after aseptic water washing 4 ~ 6 times, 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, being placed in cultivation temperature is cultivate until form Multiple Buds under the condition of 25 ~ 28 DEG C,
(3) Multiplying culture: by step (2) cultivate obtain grow thickly grow to 2 ~ 3cm height time, cut bud from base portion and proceed to proliferated culture medium and carry out squamous subculture, 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, and being placed in cultivation temperature is cultivate until form more Multiple Buds under the condition of 25 ~ 28 DEG C;
(4) culture of rootage: when the Multiple Buds of step (3) is grown to 3 ~ 4cm height, cut into simple bud and be inoculated on root media and carry 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 14 ~ 16 hours, intensity of illumination is 3000 ~ 5000lx, and cultivation temperature is be cultured under the condition of 25 ~ 28 DEG C to take root;
(5) acclimatization and transplants: by half-open for the cultivation bottle cap of the rooting tube plantlet of height about 6 ~ 7cm hardening 3 ~ 5 days, standard-sized sheet bottle cap adds appropriate running water and is placed in natural lighting lower refining seedling 5 ~ 7 days again, test-tube plantlet is taken out from blake bottle, wash root medium off, to plant in the matrix be mixed into by peat soil and yellow sand mud and to be colonizated in large Tanaka.
2. a kind of katsura tree method for in-vitro rapid propagation according to claim 1, it is characterized in that the inducing culture described in step (2) is: MS+0.1 ~ 1.0mg/L6-BA+1.0 ~ 3.0mg/L2,4-D+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
3. a kind of katsura tree method for in-vitro rapid propagation according to claim 1, is characterized in that the proliferated culture medium described in step (3) is: MS+1.0 ~ 3.0mg/L6-BA+0.1 ~ 1.0mg/LNAA+1.0 ~ 5.0mg/LGA
3+ 15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
4. a kind of katsura tree method for in-vitro rapid propagation according to claim 1, it is characterized in that the root media described in step (4) is: 1/2MS+0.5 ~ 1.5mg/LNAA+1.0 ~ 5.0mg/LIBA+0.1 ~ 0.5mg/L6-BA+0.5 ~ 1.0g/LAC+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105028082A (en) * | 2015-06-17 | 2015-11-11 | 中国林业科学研究院亚热带林业研究所 | Common sassafras seed treating and sowing method |
| CN106538385A (en) * | 2016-11-04 | 2017-03-29 | 三峡大学 | A kind of extracorporeal culturing method of katsura tree |
| CN108770695A (en) * | 2018-09-10 | 2018-11-09 | 西南林业大学 | A kind of method of the in vitro tissue culture sprouting and rooting of red heart dragon fruit |
| CN111202003A (en) * | 2020-02-22 | 2020-05-29 | 长江大学 | Culture medium and method for tissue culture and rapid propagation of Lianxiang |
| CN111264368A (en) * | 2020-02-22 | 2020-06-12 | 长江大学 | Cultivation method of Lianxiang seedlings |
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| CN103430657A (en) * | 2013-08-16 | 2013-12-11 | 苏州仁成生物科技有限公司 | Method for promoting seed germination of cercidiphyllum japonicum and improving germination ratio of seeds |
| CN103444396A (en) * | 2013-08-16 | 2013-12-18 | 苏州仁成生物科技有限公司 | Cultivation for promoting seed germination of cercidiphyllum japonicum and increasing seed germination rate |
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| USPP21711P3 (en) * | 2009-05-26 | 2011-02-15 | Biringer Nursery Lp | Katsura tree named ‘Biringer’ |
| CN103430657A (en) * | 2013-08-16 | 2013-12-11 | 苏州仁成生物科技有限公司 | Method for promoting seed germination of cercidiphyllum japonicum and improving germination ratio of seeds |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105028082A (en) * | 2015-06-17 | 2015-11-11 | 中国林业科学研究院亚热带林业研究所 | Common sassafras seed treating and sowing method |
| CN105028082B (en) * | 2015-06-17 | 2018-04-20 | 中国林业科学研究院亚热带林业研究所 | A kind of sassafrases seed treatment and type of seeding |
| CN106538385A (en) * | 2016-11-04 | 2017-03-29 | 三峡大学 | A kind of extracorporeal culturing method of katsura tree |
| CN106538385B (en) * | 2016-11-04 | 2018-06-05 | 三峡大学 | A kind of extracorporeal culturing method of katsura tree |
| CN108770695A (en) * | 2018-09-10 | 2018-11-09 | 西南林业大学 | A kind of method of the in vitro tissue culture sprouting and rooting of red heart dragon fruit |
| CN108770695B (en) * | 2018-09-10 | 2022-01-21 | 西南林业大学 | Method for in-vitro tissue culture and rapid seedling establishment of red-heart pitaya |
| CN111202003A (en) * | 2020-02-22 | 2020-05-29 | 长江大学 | Culture medium and method for tissue culture and rapid propagation of Lianxiang |
| CN111264368A (en) * | 2020-02-22 | 2020-06-12 | 长江大学 | Cultivation method of Lianxiang seedlings |
| CN111202003B (en) * | 2020-02-22 | 2021-09-21 | 长江大学 | Culture medium and method for tissue culture and rapid propagation of Lianxiang |
| CN111264368B (en) * | 2020-02-22 | 2022-03-25 | 长江大学 | Cultivation method of Lianxiang seedlings |
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Application publication date: 20150610 |