CN103563745A - Tissue culture method of ilex verticillata - Google Patents
Tissue culture method of ilex verticillata Download PDFInfo
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- CN103563745A CN103563745A CN201310476057.4A CN201310476057A CN103563745A CN 103563745 A CN103563745 A CN 103563745A CN 201310476057 A CN201310476057 A CN 201310476057A CN 103563745 A CN103563745 A CN 103563745A
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Abstract
The invention provides a tissue culture method of ilex verticillata. The tissue culture method comprises the following steps: (1) selecting an explant from a female plant; (2) processing the explant, namely sterilizing the explant; (3) performing inducing culturing on buds; (4) culturing the buds in enrichment; and (5) performing inducing culturing on roots. The tissue culture method disclosed by the invention is high in survival rate and high in reproduction speed, the growth coefficient achieves 6.1, the rooting percentage achieves 95.2%, and the ilex verticillata female young plant with pure quality is obtained. The method is free from season limitations, and can be used for rapidly propagating the ilex verticillata throughout the year, so that the large production of the ilex verticillata is realized, and an available method is provided for the rapid extension of the ilex verticillata.
Description
Technical field
The invention belongs to technical field of plant culture, specifically the method for tissue culture of a kind of applicable North America Chinese ilex.
Background technology
North America Chinese ilex (Ilex verticillata) has another name called verticillate Chinese ilex, white holly, is the perennial shrub of Aquifoliaceae Ilex (Ilexlinn.).Originate in the new introduced variety in Northeastern United States ，Wei Zhejiang Province, introducing and planting finds, this plant resistance to environment stress is strong, wide adaptability, and winter, haw did not prolongedly fall, and was to cut branch, landscaping and potted plant good ornamental tree species the autumn and winter.
North America Chinese ilex only adopts sowing and cottage propagation at present.Adopt seminal propagation, its characters of progenies separation is very large, can not keep maternal merit, and the virgin phase of the breeding of growing directly from seeds is longer, and the time of yielding positive results is more late; Adopt cutting propagation, survival rate is lower, has had a strong impact on the coefficient of breeding with merit plant, and the immature obstacle that becomes North America Chinese ilex extensive use of raising technology, has limited its spread to a certain extent.
Summary of the invention
The invention provides and be not a kind ofly subject to seasonal restrictions, can be throughout the year, Fast-propagation North America Chinese ilex, realize the method for tissue culture of North America Chinese ilex large-scale production and extensive use.Adopt the method breeding North America Chinese ilex, the rate of increase can reach 6.1, and rooting rate reaches 95.2%, has successfully solved the low problem of North America Chinese ilex prior art reproduction coefficient,
The method for tissue culture of North America Chinese ilex, is characterized in that comprising the following steps:
1) in the morning of fine day, from the female young plant of North America Chinese ilex, choose health, fancy points shows typical plant as explant material, this material is necessary for robust growth and blade is complete, bulk material uniformity;
2) on superclean bench, the explant cleaning up is cut into 2.5cm left and right stem-segment with single bud, with alcoholic solution, mercuric chloride solution soaking disinfection flushing, process successively;
3) after explant sterilization, with aseptic cutter, cut the thanatogenic tissue of the upper and lower two ends 0.2cm of stem section left and right, stem section after processing is inserted in the inductive differentiation medium configuring and induced cultivation by plant morphology lower end, in this process, the removal of thanatogenic tissue is to affect other health tissues for fear of this tissue, thereby suppresses the differentiation of bud;
4) after stem section access medium 15-25d, axillary bud sprouting growth, and grow tender stem, tender stem, from the cutting of axillalry bud base portion, is cut into the tender stem of being longer than 0.5cm to the stem section of the 0.5cm left and right that at least contains a blade, transfer and breed cultivation into proliferated culture medium, in this process, the cutting of tender stem must be followed the principle of leaf one bud, tender stem multiselect is got sturdy stem section, chooses less thin and delicate stem section, and the propagation that is beneficial to the later stage is cultivated;
5) stem section is cultivated after 20-30d in proliferated culture medium, stem section propagation forms indefinite bud, and the tender stem of pumping, the tender stem that the length that stem section differential growth is gone out is 2-3cm cuts, access root media carries out root induction cultivation, the tender stem that stem section differential growth goes out can not be long, the long induction that will affect root.
The explant of described collection is to give birth to then the branch of semi-lignified, wipes out blade after collection, retains the petiole of 1.0cm left and right simultaneously, and after running water is rinsed well, liquid detergent solution soaks 5-10min, then it is standby with running water, to rinse 1-2h.The sterilization method concrete operations of described explant are:, the alcohol that is first 70-75% by concentration, soaks 30-50s, after aseptic water washing 3-5 time, then adds 0.5-1 ‰ mercuric chloride solution, soaks 4-8min, and constantly rocks, and finally uses aseptic water washing 7-8 time.
The formula of described inductive differentiation medium is WPM+0.5-1.5mg.L
-16-BA+0.05-0.2mg.L
-1nAA+2-4% sucrose+0.7% carragheen, regulates medium pH value with the NaOH of 1mol/L or HCl, to medium pH value be 5.4-5.8.
Described proliferation culture medium formula is WPM+0.5-1.5mg.L
-16-BA+0.05-0.2mg.L
-1iBA+2-4% sucrose+0.7% carragheen, regulates medium pH value with the NaOH of 1mol/L or HCl, to medium pH value be 5.4-5.8, wherein in culturing room, cultivation temperature is 25 ± 2 ℃, light application time is 12-14h/d, light intensity is 1500-2000LX.
The formula of described root media is that 1/2WPM(macroelement reduces by half)+0-0.2m
g.L
-1iBA+0.1-0.3mg.L
-1nAA+2-4% sucrose+0.7% carragheen+0.2g.L
-1active carbon, regulates medium pH value with the NaOH of 1mol/L or HCl, to medium pH value be 5.4-5.8: after cultivating 30-40d, form the healthy and strong seedling of taking root.
Described WPM medium, the composition of 1/2WPM medium are all with reference to relevant criterion or file.
Described 6-BA is 6-benzylaminopurine, and IBA is indolebutyric acid, and NAA is methyl α-naphthyl acetate.
The percentage composition relating in the present invention unless otherwise indicated, all refers to quality percentage composition.
The present invention compared with prior art has the following advantages:
Inducing culture of the present invention is with strong points, applicability good, explant cultivates through inductive differentiation medium that axillary bud growth speed is fast and branch is healthy and strong, stem section value-added coefficient after proliferated culture medium is cultivated is large, can reach 6.1, after cultivating, root media takes root fast, root amount is many and sturdy, and rooting rate reaches 95.2%.
Method survival rate of the present invention is high, quality is pure, and reproduction speed is fast, is not subject to seasonal restrictions, and can breed North America Chinese ilex throughout the year, realizes the large-scale production of North America Chinese ilex.
Embodiment
Now, in conjunction with embodiments of the invention, the invention will be further described.
Embodiment 1:
The morning on June 13rd, 2012, from the female young plant of North America Chinese ilex, choose health, fancy points shows typical plant, gather the branch of raw semi-lignified then.After collection, wipe out blade, retain the petiole of 1.2cm simultaneously, running water is rinsed well, and liquid detergent solution soaks 6min, then it is standby with running water, to rinse 1h; On superclean bench, the branch cleaning up is cut to the stem-segment with single bud that is cut into 2.5cm, successively disinfection, alcoholic solution with 75% soaks 30s, aseptic water washing 5 times, and 1 ‰ mercuric chloride solutions soak 6min, and constantly rock, make sterilization completely, finally with bacterium water, rinse 8 times; Then with aseptic cutter, cut the thanatogenic tissue of the upper and lower two ends 0.2cm of stem section, the stem section plant morphology lower end after processing is inserted in the inductive differentiation medium configuring: the formula of inductive differentiation medium is WPM+1.0mg.L
-16-BA+0.1mg.L
-1nAA+2% sucrose+0.7% carragheen, regulates medium pH value with the NaOH of 1mol/L or HCl, to medium pH value be 5.6; After inoculation, enter culturing room and cultivate, in culturing room, cultivation temperature is 25 ℃, and light application time is 12h/d, after light intensity is 1500LX:30d, axillary bud sprouting growth, and grow tender stem, tender stem is cut down from axillalry bud base portion, the tender stem of being longer than 0.5cm is cut at least containing 0.5 of a blade
The stem section of cm left and right, continues in the proliferated culture medium of transferring to cultivate, and proliferation culture medium formula is WPM+1.0mg.L
-16-BA+0.05mg.L
-1iBA+3% sucrose+0.7% carragheen, regulates medium pH value with the NaOH of 1mol/L or HCl, to medium pH value be 5.6; Cultivate after 30d, stem section propagation forms indefinite bud, and pumping goes out tender stem, and the tender stem that the length that stem section differential growth is gone out surpasses 2cm cuts, and access root media is cultivated: prescription of rooting medium is that 1/2WPM(macroelement reduces by half)+0.2mg.L
-1iBA+0.3mg.L
-1nAA+2% sucrose+0.7% carragheen+0.2g.L
-1active carbon,
With NaOH or the HCl of 1mol/L, regulate medium PH
Value forms the healthy and strong seedling of taking root after medium pH value is 5.8:40d.
The present embodiment rooting rate reaches 94.7%, and the number of on average taking root reaches 5.7, and the method is not subject to seasonal restrictions, can be throughout the year, Fast-propagation North America Chinese ilex, realize the large-scale production of North America Chinese ilex.
Embodiment 2:
The morning on May 14th, 2013, from the female young plant of North America Chinese ilex, choose health, fancy points shows typical plant, gather the branch of raw semi-lignified then.After collection, wipe out blade, retain the petiole of 1.0cm simultaneously, running water is rinsed well, and liquid detergent solution soaks 8min, then it is standby with running water, to rinse 1h; On superclean bench, the branch cleaning up is cut to the stem-segment with single bud that is cut into 2.2cm, with 75% alcoholic solution, soak 30s successively, outwell after alcohol, with aseptic water washing 5 times, then add 1 ‰ mercuric chloride solutions to soak 6min, constantly rock therebetween, outwell after mercuric chloride solution aseptic water washing 8 times standby; Then with aseptic cutter, cut the thanatogenic tissue of the upper and lower two ends 0.25cm of stem section, stem section plant morphology lower end is inserted in the inductive differentiation medium configuring and cultivated, in culturing room, cultivation temperature is 25 ℃, light application time is 12h/d, and light intensity is 1500LX: the formula of inductive differentiation medium is WPM+0.5mg.L
-16-BA+0.1mg.L
-1nAA+2% sucrose+0.7% carragheen, regulates medium pH value with the NaOH of 1mol/L or HCl, to medium pH value be 5.6; Cultivate after 30d, axillary bud sprouting growth, and grow tender stem, tender stem is cut down from axillalry bud base portion, the tender stem of being longer than 0.5cm is cut into the stem section that at least contains the 0.5cm of a blade, transfer and cultivate into proliferated culture medium, proliferation culture medium formula is WPM+1.0mg.L
-16-BA+0.1mg.L
-1iBA+3% sucrose+0.7% carragheen, regulates medium pH value with the NaOH of 1mol/L or HCl, to medium pH value be 5.6; After 30d, stem section propagation forms indefinite bud, and pumping goes out tender stem, and the tender stem that the length that stem section differential growth is gone out surpasses 2cm cuts, access root media: prescription of rooting medium is that 1/2WPM(macroelement reduces by half)+0.2mg.L
-1iBA+0.3mg.L
-1nAA+2% sucrose+0.7% carragheen+0.2g.L
-1active carbon, regulates medium pH value with the NaOH of 1mol/L or HCl, to medium pH value be 5.8 only: after cultivating 40d, form the healthy and strong seedling of taking root: other steps are with example 1.
The North America Chinese ilex that the present embodiment method for tissue culture the obtains seedling of taking root, survival rate is high, reproduction speed is fast, and average growth coefficient reaches 6.1, and rooting rate reaches 95.8%.
The above; it is only preferred embodiment of the present invention; not the present invention is done to any restriction, everyly according to invention technical spirit, any simple modification of above embodiment, change and equivalent structure are changed, all belong in the protection domain of technical solution of the present invention.
Claims (6)
1. a method for tissue culture for North America Chinese ilex, is characterized in that comprising the following steps:
1), in the morning of fine day, from the female young plant of North America Chinese ilex, choose health, fancy points shows typical plant as explant material;
2) on superclean bench, the explant cleaning up is cut into 2.2-2.6cm stem-segment with single bud, with alcoholic solution, mercuric chloride solution soaking disinfection flushing, process successively;
3) after explant sterilization, with aseptic cutter, cut the thanatogenic tissue of the upper and lower two ends 0.15-0.25cm of stem section, the stem section after processing is inserted in the inductive differentiation medium configuring and induced cultivation by plant morphology lower end;
4) after stem section access medium 15-25d, axillary bud sprouting growth, and grow tender stem, and tender stem, from the cutting of axillalry bud base portion, is cut into the tender stem of being longer than 0.5cm to the stem section that at least contains the 0.4-0.6cm of a blade, transfer and breed cultivation into proliferated culture medium;
5) stem section is cultivated after 20-30d in proliferated culture medium, and stem section propagation forms indefinite bud, and the tender stem of pumping, and the tender stem that the length that stem section differential growth is gone out is 2-3cm cuts, and access root media carries out root induction cultivation.
2. the method for tissue culture of North America as claimed in claim 1 Chinese ilex, it is characterized in that in step 1), the explant gathering is to give birth to then the branch of semi-lignified, after collection, wipe out blade, the petiole that simultaneously retains 0.9-1.2cm, after running water is rinsed well, liquid detergent solution soaks 5-10min, then it is standby with running water, to rinse 1-2h.
3. the method for tissue culture of North America as claimed in claim 1 Chinese ilex, it is characterized in that step 2) in, the sterilization method concrete operations of explant are: the alcohol that is first 70-75% by concentration, soak 30-50s, after aseptic water washing 3-5 time, then add 0.5-1 ‰ mercuric chloride solution, soak 4-8min, and constantly rock, finally use aseptic water washing 7-8 time.
4. the method for tissue culture of North America as claimed in claim 1 Chinese ilex, is characterized in that in step 3), and the formula of described inductive differentiation medium is WPM+0.5-1.5mg.L
-16-BA+0.05-0.2mg.L
-1nAA+2-4% sucrose+0.7% carragheen, regulates medium pH value with the NaOH of 1mol/L or HCl, to medium pH value be 5.4-5.8.
5. the method for tissue culture of North America as claimed in claim 1 Chinese ilex, is characterized in that in step 4), and described proliferation culture medium formula is WPM+0.5-1.5mg.L
-16-BA+0.05-0.2mg.L
-1iBA+2-4% sucrose+0.7% carragheen, regulates medium pH value with the NaOH of 1mol/L or HCl, to medium pH value be 5.4-5.8, wherein in culturing room, cultivation temperature is 25 ± 2 ℃, light application time is 12-14h/d, light intensity is 1500-2000LX.
6. the method for tissue culture of North America as claimed in claim 1 Chinese ilex, is characterized in that in step 5), and the formula of described root media is 1/2WPM+0-0.2mg.L
-1iBA+0.1-0.3mg.L
-1nAA+2-4% sucrose+0.7% carragheen+0.2g.L
-1active carbon, regulates medium pH value with the NaOH of 1mol/L or HCl, to medium pH value be 5.4-5.8: after cultivating 30-40d, form the healthy and strong seedling of taking root.
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| CN104255491A (en) * | 2014-09-19 | 2015-01-07 | 江苏农林职业技术学院 | Ilex crenata golden gem tissue culture breeding method |
| CN104304007A (en) * | 2014-10-08 | 2015-01-28 | 广东药学院 | In-vitro culture method for berchemia lineata |
| CN106258952A (en) * | 2015-12-02 | 2017-01-04 | 香港中文大学深圳研究院 | The method utilizing Caulis et Folium Ammopiptanthi Mongolici children tender side root induction Caulis et Folium Ammopiptanthi Mongolici Callus formation |
| CN107173231A (en) * | 2017-07-03 | 2017-09-19 | 甘肃省科学院生物研究所 | A kind of method of the Mongolian Ammopiptanthus mongolicus rapid propagation in vitro of rare or endangered species |
| CN108029559A (en) * | 2017-12-29 | 2018-05-15 | 南京林业大学 | A kind of method of quickly breeding bearberry tissue-cultured seedling |
| CN109729973A (en) * | 2019-01-21 | 2019-05-10 | 重庆市林业科学研究院 | A kind of excellent strain tissue culture and rapid propagation method of purplefruit holly leaf |
| CN112493124A (en) * | 2020-11-09 | 2021-03-16 | 江苏农林职业技术学院 | Culture medium and culture method for promoting subculture bud differentiation of ilex chinensis |
| CN113875590A (en) * | 2021-10-20 | 2022-01-04 | 江苏省林业科学研究院 | Rapid cultivation method for clustered North American winter seedlings |
| CN114793655A (en) * | 2022-06-01 | 2022-07-29 | 江苏青好景观园艺有限公司 | Cutting method of sildenafil holly |
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| CN104255491A (en) * | 2014-09-19 | 2015-01-07 | 江苏农林职业技术学院 | Ilex crenata golden gem tissue culture breeding method |
| CN104255491B (en) * | 2014-09-19 | 2016-03-30 | 江苏农林职业技术学院 | A kind of gold leaf tortoise plastron Chinese ilex tissue culture propagation method |
| CN104304007A (en) * | 2014-10-08 | 2015-01-28 | 广东药学院 | In-vitro culture method for berchemia lineata |
| CN106258952B (en) * | 2015-12-02 | 2018-11-09 | 香港中文大学深圳研究院 | The method formed using the tender lateral root induction Ammopiptanthus mongolicus callus of Ammopiptanthus mongolicus children |
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| CN108029559A (en) * | 2017-12-29 | 2018-05-15 | 南京林业大学 | A kind of method of quickly breeding bearberry tissue-cultured seedling |
| CN109729973A (en) * | 2019-01-21 | 2019-05-10 | 重庆市林业科学研究院 | A kind of excellent strain tissue culture and rapid propagation method of purplefruit holly leaf |
| CN109729973B (en) * | 2019-01-21 | 2020-12-11 | 重庆市林业科学研究院 | Tissue culture and rapid propagation method for superior plants of red fruit holly |
| CN112493124A (en) * | 2020-11-09 | 2021-03-16 | 江苏农林职业技术学院 | Culture medium and culture method for promoting subculture bud differentiation of ilex chinensis |
| CN113875590A (en) * | 2021-10-20 | 2022-01-04 | 江苏省林业科学研究院 | Rapid cultivation method for clustered North American winter seedlings |
| CN114793655A (en) * | 2022-06-01 | 2022-07-29 | 江苏青好景观园艺有限公司 | Cutting method of sildenafil holly |
| CN114793655B (en) * | 2022-06-01 | 2024-05-17 | 江苏青好景观园艺有限公司 | Cutting method of S.tepa |
| CN117099686A (en) * | 2023-10-25 | 2023-11-24 | 西南林业大学 | North American ilex tissue culture rapid propagation method and application |
| CN117099686B (en) * | 2023-10-25 | 2024-01-05 | 西南林业大学 | North American ilex tissue culture rapid propagation method and application |
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