CN102124953B - Method for quickly breeding Litsea coreana test tube plantlets by spores - Google Patents
Method for quickly breeding Litsea coreana test tube plantlets by spores Download PDFInfo
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- CN102124953B CN102124953B CN 201110027359 CN201110027359A CN102124953B CN 102124953 B CN102124953 B CN 102124953B CN 201110027359 CN201110027359 CN 201110027359 CN 201110027359 A CN201110027359 A CN 201110027359A CN 102124953 B CN102124953 B CN 102124953B
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- 238000000034 method Methods 0.000 title claims abstract description 17
- 238000009395 breeding Methods 0.000 title claims abstract description 8
- 230000001488 breeding effect Effects 0.000 title claims abstract description 8
- 241000587213 Litsea coreana Species 0.000 title abstract description 5
- 239000002609 medium Substances 0.000 claims description 28
- 241000726221 Gemma Species 0.000 claims description 23
- 235000009024 Ceanothus sanguineus Nutrition 0.000 claims description 14
- 240000003553 Leptospermum scoparium Species 0.000 claims description 14
- 235000015459 Lycium barbarum Nutrition 0.000 claims description 14
- 229920001817 Agar Polymers 0.000 claims description 13
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 13
- 229930006000 Sucrose Natural products 0.000 claims description 13
- 239000008272 agar Substances 0.000 claims description 13
- 239000005720 sucrose Substances 0.000 claims description 13
- 238000005286 illumination Methods 0.000 claims description 12
- 230000001954 sterilising effect Effects 0.000 claims description 12
- 238000004659 sterilization and disinfection Methods 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 210000003038 endothelium Anatomy 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 229960002523 mercuric chloride Drugs 0.000 claims description 3
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 230000035584 blastogenesis Effects 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- 244000269722 Thea sinensis Species 0.000 claims 3
- 238000012258 culturing Methods 0.000 abstract 1
- 230000035755 proliferation Effects 0.000 abstract 1
- 241001122767 Theaceae Species 0.000 description 7
- 241000196324 Embryophyta Species 0.000 description 6
- 241000723346 Cinnamomum camphora Species 0.000 description 3
- 241000282373 Panthera pardus Species 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 2
- 241000209507 Camellia Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000218195 Lauraceae Species 0.000 description 1
- 241001081179 Litsea Species 0.000 description 1
- 235000012854 Litsea cubeba Nutrition 0.000 description 1
- 240000003705 Senecio vulgaris Species 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 235000018597 common camellia Nutrition 0.000 description 1
- 238000012364 cultivation method Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000004345 fruit ripening Effects 0.000 description 1
- 239000002932 luster Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000011169 microbiological contamination Methods 0.000 description 1
- 239000006870 ms-medium Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for quickly breeding Litsea coreana test tube plantlets by spores, comprising the following steps: taking one spore of the Litsea coreana as an explant; starting to cultivate to induce cluster buds; and carrying out branch bud proliferation cultivation, branch cultivation and rooting cultivation to obtain the Litsea coreana test tube plantlets. The method has the advantage of high speed and can be industrially produced, is convenient to operate and is suitable for industrially culturing plantlets, and the reproduction coefficient is 3-5 times.
Description
Technical field
The present invention relates to the plant corpus vegetative propagation, specifically cultivate the method for breeding eagle tea tree test-tube plantlet fast through gemma.
Background technology
The eagle tea tree, formal name used at school: leopard camphor tree (Litsea coreana), be Lauraceae, Litsea aiphyllium plant, leaf alternate, the food value of leaf are very thick, color and luster is dark green, China in Sichuan, there is fragmentary distribution on ground such as Guizhou, Yunnan, Anhui.Tea so that the tender leaf of leopard camphor tree is made into is claimed eagle tea, and it is a kind of Special tea, and eagle tea and beverage series thereof owing to be rich in the various plants elite, have characteristics such as pollution-free, that local flavor is good, favored by consumers in general.The eagle tea tree breeds difficulty naturally as a kind of wild seeds, and artificial cultivation sapling multiplication rate is low at present, has seriously restricted the industrialized development of eagle tea.
Leopard camphor tree wood is formed by natural, in the bird abdomen, digests and falls wax coat through the bird seed of pecking at behind the fruit maturation, grows new tree after discharging into ground in the ight soil, and it is extremely low to breed survival rate, breeds very slow naturally; The cultivation method with ripe branch cutting breeding is attempted in some area at present, because the branch degree of lignification is high, this mode shoot survival percent is less than 10%.Lack in fact because of the eagle camellia is many in the seminal propagation, and the seed maturity cycle is long, reasons such as collection seed difficulty are difficult to carry out the artificial propagation of eagle tea tree.
The method that tissue culture is bred is meant and uses aseptic method, and isolated organ, tissue and the cell that makes plant corpus is the culture technique of g and D under the condition that provides the people, also method for tissue culture is not applied in the breeding of eagle tea tree at present.
Summary of the invention
The present invention cultivates the method for breeding eagle tea tree test-tube plantlet fast for avoiding the existing weak point of above-mentioned prior art to provide a kind of through gemma, in the hope of obtaining the quick breeding to the eagle tea tree.
Technical solution problem of the present invention adopts following technical scheme:
The method that the present invention breeds eagle tea tree test-tube plantlet fast through gemma is: the gemma of getting eagle tea branch is an explant; After surface sterilization, be inoculated into to start in the medium and induce the bud of growing thickly; Through twig enrichment culture, branch cultivation, culture of rootage, obtain eagle tea tree test-tube plantlet again.
The plucking time of the eagle tea branch among the present invention is between the beginning of autumn to the Frost's Descent, and the branch that at first will newly adopt is positioned in 4 ℃ the environment, gets gemma on the branch after 1-3 days with flowing water flushing 120 minutes, carries out the explant sterilization then.
Explant sterilization method according to the invention is 75% alcohol-pickled 30 seconds at first using volumetric concentration, and using volumetric concentration again is 0.1% mercuric chloride jolting 8 minutes, uses aseptic water washing at last 3 times.
The present invention starts before training induces in that the gemma explant is inoculated in, and under aseptic condition, peels off the outer skin of gemma, keeps the base portion and the endothelium of gemma, again the gemma that strips is inoculated in to start in the medium and cultivates.
The medium that the present invention starts cultivation is to be basic solid culture medium with MS; Add 2mg/L6-BA, 0.2mg/LNAA, 2.6g/L agar, 30mg/L sucrose; The pH value transfers to 5.8, and 121 ℃, the autoclave sterilization of 0.1Mpa 20 minutes, said startup condition of culture was 16 ± 2 ℃ of temperature; Light intensity 1500LX, illumination 10h/d.
The blastogenesis of growing thickly that the present invention induces through the startup cultivation reaches length and is not less than 1cm; The medium of twig enrichment culture is to be minimal medium with MS; Add 3mg/L 6-BA, 30mg/L sucrose, 2.6g/L agar, the pH value transfers to 5.8 and 121 ℃, the autoclave sterilization of 0.1Mpa 20 minutes; Twig enrichment culture condition is 18 ± 2 ℃ of temperature, light intensity 1500LX, illumination 10h/d.
The twig length of the present invention behind the twig enrichment culture is not less than 1.5cm; The medium that branch is cultivated is to be minimal medium with MS; Add 1mg/L 6-BA, 0.1mg/L NAA, 30mg/L sucrose, 2.6g/L agar, 5.8,121 ℃ of pH values, 0.1MPa autoclaving 20 minutes; Condition of culture is 18 ± 2 ℃ of temperature, light intensity 1500LX, illumination 12h/d.
The branch length that the present invention cultivates through branch is 3-5cm, and the medium of culture of rootage is to be minimal medium with 1/2MS, adds 0.1mg/L NAA, 0.1g/L AC; 30mg/L sucrose, 2.6g/L agar, pH value 5.8, condition of culture is: 18 ± 2 ℃ of temperature, light intensity 1500LX, illumination 12h/d.
Compare with existing propagation technique, beneficial effect of the present invention is embodied in:
1, can obtain the test-tube plantlet of taking root by gemma in the present invention 4 months, by hundred strain plant, further expand and can obtain young plants up to ten thousand in numerous 1 year, simple to operation, reproduction coefficient reaches 3-5 doubly, and speed is fast, is fit to factorial seedling growth, but commercialization production.
Embodiment:
Present embodiment comprises following steps:
Step 1 is taken from eagle tea branch (the branch endophyte weight in spring, the branch microbiological contamination weight in summer between the beginning of autumn to the Frost's Descent; Should not be as explant); Branch was placed earlier 4 ℃ in refrigerator after 1-3 days, and the gemma of getting on the branch washes 120min with flowing water, carries out the explant sterilization then; Processes for disinfecting surfaces is used 0.1% mercuric chloride jolting 8min, aseptic water washing 3 times again for handling 30sec with 75% alcohol earlier.
Step 2, with the gemma that disinfects, the outer skin of under aseptic condition, peelling off gemma keeps the base portion and the endothelium of gemma, the gemma that strips is inoculated in to start in the medium cultivates.Start medium and be MS and be minimal medium and add respectively again that 2mg/L6-BA, 0.2mg/L NAA, 30mg/L sucrose, 2.6g/L agar (promptly in every liter of MS medium, add 2mg6-BA, 0.2mgNAA, 2.6g agar, 30mg sucrose, down with), pH value transfer to 5.8,20min sterilizes under 121 ℃, 0.1Mpa high-temperature and high-pressure conditions; Starting condition of culture is 16 ± 2 ℃ of temperature, light intensity 1500LX, and illumination 10h/d cultivates, and directly induced bundle is sprouted.After start cultivating in 40-50 days, there is 50% gemma to germinate approximately and to grow thickly bud.
Step 3 is learnt from else's experience to start and is cultivated the above bud of growing thickly of back growth length 1cm, changes the twig enrichment culture over to.The twig proliferated culture medium is minimal medium with MS, adds 3mg/L 6-BA, 30mg/L sucrose, 2.6g/L agar, and the pH value transfers to 5.8; Twig enrichment culture condition is 16 ± 2 ℃ of temperature, and light intensity 1500LX, illumination 10h/d cultivate, the rate of increase at 3-5 doubly, about 4 weeks of incubation time.
Step 4; The above twig of the twig enrichment culture of learning from else's experience growth length 1.5cm; The branch medium culture is gone in switching: the branch medium is to be minimal medium with MS, adds 1mg/L 6-BA, 0.1mg/L NAA, 30mg/L sucrose, 2.6g/L agar then, and the pH value transfers to 5.8; Condition of culture is 18 ± 2 ℃ of temperature, light intensity 1500LX, and illumination 12h/d cultivates, about 3 weeks of incubation time.
Step 5, the branch incubation growth of learning from else's experience length reach the above branch of 3-5cm, and root media is gone in switching, and root media is minimal medium with 1/2MS, add 0.1mg/L NAA, 0.1g/LAC, 30mg/L sucrose, 2.6g/L agar, and the pH value transfers to 5.8; Condition of culture is 18 ± 2 ℃ of temperature, light intensity 1500LX, and illumination 12h/d cultivates, and cultivates rooting rate about 95% about 30-40 days.
Medium in the above step all should be under 121 ℃, 0.1Mpa high-temperature and high-pressure conditions sterilization 20min.
Can obtain the rooting tube plantlet of eagle tea tree through step 5, the rooting tube plantlet in the present embodiment promptly can be used for tame seedling after domestication.
Claims (2)
1. breed the method for eagle tea tree test-tube plantlet through gemma fast, it is characterized in that:
The gemma of getting eagle tea branch is an explant, after surface sterilization, is inoculated into to start in the medium to induce the bud of growing thickly, and through twig enrichment culture, branch cultivation, culture of rootage, obtains eagle tea tree test-tube plantlet again;
The plucking time of said eagle tea branch is between the beginning of autumn to the Frost's Descent, and the tea branch of newly adopting was positioned in 4 ℃ the environment 1-3 days;
Said explant was peelled off the outer skin of gemma earlier under aseptic condition before being inoculated in the startup medium, keep the base portion and the endothelium of gemma, again the gemma that strips was inoculated in to start in the medium and cultivated;
The medium that said startup is cultivated is to be basic solid culture medium with MS; Add 2mg/L6-BA, 0.2mg/LNAA, 2.6g/L agar, 30mg/L sucrose; The pH value transfers to 5.8, and 121 ℃, the autoclave sterilization of 0.1Mpa 20 minutes, said startup condition of culture was 16 ± 2 ℃ of temperature; Light intensity 1500LX, illumination 10h/d;
The blastogenesis of growing thickly that warp startup cultivation is induced reaches length and is not less than 1cm; The medium of twig enrichment culture is to be minimal medium with MS; Add 3mg/L 6-BA, 30mg/L sucrose, 2.6g/L agar, the pH value transfers to 5.8 and 121 ℃, the autoclave sterilization of 0.1Mpa 20 minutes; Twig enrichment culture condition is 18 ± 2 ℃ of temperature, light intensity 1500LX, illumination 10h/d;
Twig length behind the twig enrichment culture is not less than 1.5cm; The medium that branch is cultivated is to be minimal medium with MS; Add 1mg/L 6-BA, 0.1mg/L NAA, 30mg/L sucrose, 2.6g/L agar, 5.8,121 ℃ of pH values, 0.1Mpa autoclave sterilization 20 minutes; Condition of culture is 18 ± 2 ℃ of temperature, light intensity 1500LX, illumination 12h/d;
The branch length of cultivating through branch is 3-5cm; The medium of culture of rootage is to be minimal medium with 1/2MS; Add 0.1mg/L NAA, 0.1g/L AC, 30mg/L sucrose, 2.6g/L agar, pH value 5.8, condition of culture is 18 ± 2 ℃ of temperature, light intensity 1500LX, illumination 12h/d.
2. method of breeding eagle tea tree test-tube plantlet through gemma fast according to claim 1; It is characterized in that; Said explant sterilization method is 75% alcohol-pickled 30 seconds at first using volumetric concentration, and using volumetric concentration again is 0.1% mercuric chloride jolting 8 minutes, uses aseptic water washing at last 3 times.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110027359 CN102124953B (en) | 2011-01-25 | 2011-01-25 | Method for quickly breeding Litsea coreana test tube plantlets by spores |
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|---|---|---|---|
| CN 201110027359 CN102124953B (en) | 2011-01-25 | 2011-01-25 | Method for quickly breeding Litsea coreana test tube plantlets by spores |
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| CN102124953B true CN102124953B (en) | 2012-06-20 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102318501B (en) * | 2011-09-01 | 2013-02-27 | 贵州省生物研究所 | Cutting propagation method for glede tea raw material plant |
| CN103843552B (en) * | 2012-12-04 | 2016-08-17 | 四川津海素盾实业有限公司 | A kind of method utilizing the asexual cottage propagation nursery of eagle Camellia sinensis cutting |
| CN104041409B (en) * | 2014-06-04 | 2015-06-17 | 四川省林业科学研究院 | Tissue culturing method of Litsea mollis |
| CN110622861B (en) * | 2019-10-15 | 2021-04-13 | 重庆市林业科学研究院 | Sterilization method for improving tissue culture starting rate of cinnamomum camphora explants |
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Non-Patent Citations (3)
| Title |
|---|
| 李俊等."毛貂皮樟的研究进展".《四川农业大学学报》.2005,第23卷(第2期),第247-252页. |
| 李廷松等."老鹰茶人工栽培初探".《贵州茶叶》.1999,(第1期),第9-10页. |
| 邓健."人工繁殖和栽培技术".《种植技术》.2005,第28-29页. |
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