CN101502239B - Method for rapid propagation and cultivation of carnation seedling by tissue culture - Google Patents
Method for rapid propagation and cultivation of carnation seedling by tissue culture Download PDFInfo
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- CN101502239B CN101502239B CN 200910094281 CN200910094281A CN101502239B CN 101502239 B CN101502239 B CN 101502239B CN 200910094281 CN200910094281 CN 200910094281 CN 200910094281 A CN200910094281 A CN 200910094281A CN 101502239 B CN101502239 B CN 101502239B
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Abstract
The present invention provides a rapid propagation culture method of carnation tissue culture seedling, and the method comprises the steps of cutting stock and sterilizing the explant, enrichment culturing and rooting culturing. Namely in the rooting period of seedling, the traditional method that the non-root stem segment is executed with in-bottle rooting is replaced. The non-root stem segment is directly inserted into a hybrid culture medium for primary rooting and seedling formation. The technical operation instruction of rooting culture technical system is simplified. The shearing of tissue cultured non-root seedling is executed in big shelter. A program of rooting culturing in bottle is saved. The material which is sheared for micro-sticking can include stem tip, stem segment and stem base. The material which can be used for rooting according to traditional in-bottle rooting culture program only includes stem tip. The rooting material is more than 3-4 times compared with the traditional method. The cultured rooting seedling can become finished seedling after outdoor transition management without secondary replanting. The produced seedlings has the advantages of strongness, orderliness and excellent quality under the precondition that the cost is saved. The standard of excellent-quality seeding is obtained. The rapid propagation culture method of carnation tissue culture seedling is suitable for the large-scale production of flower seedling.
Description
Technical field
The present invention relates to carnation group training quick reproduction technique in conjunction with the disposable one-tenth seedling of outside sprout-cultivating-bottle technology, especially the carnation group is cultivated the large-scale production technology of seedling.
Background technology
Carnation (Dianthus caryophyllus L) has another name called carnation, is Caryophyllaceae Caryophyllaceae, Carnation perennial herb perennial plant.Originate in Northern Europe and southern Europe.This genus has 80 plant species approximately, is the local products plant in Ireland British, Northern Europe and southern Europe, likes shady and cool, dry, sunny and draughty ecotope.The cold resistance of carnation is good, but warm tolerance is relatively poor, and optimum growth temperature is 14-21 ℃, and temperature surpasses 27 ℃ or when being lower than 14 ℃, plant strain growth is slow.Suitable being planted in is rich in humus, well-drained lime soil, and happiness is fertile.
Tissue culture about carnation; from the eighties domestic and international research person just at the explant position of carnation; medium component; condition of culture; detoxification seed preservation etc. has launched number of research projects; but these all are confined to the research to certain link in the tissue culture technology, though research of carnation seedling tissue culture technology and large-scale production also have report at home, systematically do not relate to the little disposable one-tenth seedling technology of taking root of inserting of the outer stem section of carnation bottle.
It is to carry out culture of rootage with stem apex on the basis of subculture bottle seedling that traditional group is cultivated seedling, its coefficient of taking root is not high, move on to after taking root and carry out the previous stage transition in the pearlite interstitial substance, be transplanted to again after waiting to survive and carry out the secondary transition in the mixed-matrix, the seedling bar that cultivate this moment is thin and individual high, thereby is difficult for surviving.Traditional in addition tissue cultivating seedling yields poorly, cultivation program complexity, and the cost height has been difficult to meet the need of market.Therefore, must be improved prior art.
Summary of the invention
For simplifying incubation, improve kind of a shoot survival percent, reduce cost, the invention provides a kind of method for tissue culture, rapid propagation and cultivation of carnation seedling.
The present invention finishes by following technical proposal: a kind of method for tissue culture, rapid propagation and cultivation of carnation seedling, comprise explant segment and sterilization, and explant induction cultivation, enrichment culture and culture of rootage is characterized in that culture of rootage carries out under following condition:
Enrichment culture is cut into stem section with 1～2 pair of leaf to growing 4 pairs of propagation seedlings more than the leaf, after sterile-processed, basal part of stem dips in the water of taking root that contains 300～500mg/L hormone, insert in the medium of following mass ratio: the peat composed of rotten mosses: humus soil: perlite=2.5～3: 0.5～1: 0.8～1, water permeable, at one deck plastic foil and one deck shading rate is 75% shade net, humidity under the tight and air tight condition more than 95%, culture of rootage 1～5 day; Be one deck plastic foil in the morning again, be that one deck plastic foil and one deck shading rate are 75% shade net afternoon, humidity under the condition more than 95%, culture of rootage 5～14 days; Removing plastic foil afterwards, only is 75% shade net at one deck shading rate, and temperature is 18～26 ℃, and humidity is under the condition of 85-90%, and being cultured to seedling, uprightly can to remove shading rate be 75% shade net; Afterwards weekly to 1-2 1/4MS nutrient solution of foliage-spray, and take the circumstances into consideration in Liriomyza, the control of noctuid spraying pesticide in multiple season, pesticide concentration is a 800-1000 times of liquid, until growing up to the seedling of taking root.
Described disinfecting is: at thimerosal is thiophanate methyl solution, perhaps carbendazim solution, and perhaps one or more in the tpn solution, the concentration of thimerosal is under 800～1000 times the condition, soaked 20～60 seconds, and thimerosal to be the commercially available prod.
Described agricultural chemicals is an avermectin, or formidable opponent 315, or spot dives only, or in the Avermectin one or more during use are made into agricultural chemicals concentration and are 800～2000 times liquid, and agricultural chemicals is the commercially available prod.
Described explant segment and sterilization are conventional method of the prior art.
Described explant induction is cultivated the medium that adopts: the agar of sucrose+7000mg/L of methyl+30000mg/L of 6-benzylaminopurine+0.1～0.5mg/L of MS+1.0～1.5mg/L, condition of culture is: cultivation temperature: 25 ± 2 ℃, light application time: 10～12 hours/day, intensity of illumination: 1500～2000Lx.
The medium that described enrichment culture adopts is: the agar of sucrose+7000mg/L of methyl+30000mg/L of 6 chaff aminopurine+0.1～0.5mg/L of 6-benzylaminopurine+0.1～0.5mg/L of MS+0.1～0.5mg/L, condition of culture is: cultivation temperature: 25 ± 2 ℃, light application time: 10～12 hours/day, intensity of illumination: 1500～2000Lx.
Method for tissue culture, rapid propagation and cultivation of carnation seedling concrete steps provided by the invention are as follows:
A, draw materials and sterilize: choosing flower type is normal in the plant that has bloomed, pattern is pure, branch is straight and upright, no damage by disease and insect, individual plant that strain shape is full, cleans, after the sterilization, carries out the sterile working segment and inoculate;
B, inducing culture: the segment of A step gained explant is inoculated in the following inducing culture, that is: the agar of sucrose+7000mg/L of methyl+30000mg/L of 6-benzylaminopurine+0.1～0.5mg/L of MS+1.0～1.5mg/L, in temperature is 25 ± 2 ℃, light application time is 10～12 hours/day, intensity of illumination is under the condition of culture of 1500～2000Lx, carries out inducing culture to terminal bud and grows tall and differentiate lateral bud;
C, enrichment culture: change in the following proliferated culture medium after terminal bud that the B step is induced and indefinite bud are cut, that is: the agar of sucrose+7000mg/L of methyl+30000mg/L of 6 chaff aminopurine+0.1～0.5mg/L of 6-benzylaminopurine+0.1～0.5mg/L of MS+0.1～0.5mg/L, in temperature is 25 ± 2 ℃, light application time is 10～12 hours/day, intensity of illumination is under the condition of 1500～2000Lx, be cultured to terminal bud and grow tall and merogenesis, adventitious bud proliferation grows tall and extracts blade out;
D, culture of rootage: enrichment culture is cut into stem section with 1 to 2 pair of leaf to growing 4 pairs of propagation seedlings more than the leaf, after in 800～1000 times thimerosal, disinfecting 20～60 seconds, basal part of stem dips in the water of taking root that contains 300～500mg/L hormone, insert in the medium of following mass ratio: the peat composed of rotten mosses: humus soil: perlite=2.5～3: 0.5～1: 0.8～1, water permeable, it at one deck plastic foil and one deck shading rate 75% shade net, humidity under the tight and air tight condition more than 95%, culture of rootage 1～5 day; Be one deck plastic foil in the morning again, be that one deck plastic foil and one deck shading rate are 75% shade net afternoon, humidity under the condition more than 95%, culture of rootage 5～14 days; Removing plastic foil afterwards, only is 75% shade net at one deck shading rate, and temperature is 18～26 ℃, and humidity is under the condition of 85-90%, and being cultured to seedling, uprightly can to remove shading rate be 75% shade net; Afterwards weekly to 1-2 1/4MS nutrient solution of foliage-spray, and take the circumstances into consideration in Liriomyza, the control of noctuid spraying pesticide in multiple season, pesticide concentration is a 800-1000 times of liquid, until growing up to the seedling of taking root.
The present invention has following advantage and effect: adopt such scheme; promptly at first the carnation seedling is carried out tissue-culturing rapid propagation propagation; carrying out the outer stem section of the carnation bottle disposable one-tenth seedling of taking root then cultivates; i.e. taking root the stage at seedling; changing takes root in traditional bottle is no rhizome section; and will not have the rhizome section and directly insert a secondary root Cheng Miao in the mixed culture medium; the regulations for technical operation of culture of rootage technical system have been simplified; the clip of the no offspring of group training carries out (need not superclean bench) in booth; saved a bottle interior culture of rootage program; clip carries out little slotting material can comprise stem apex; the stem section; basal part of stem; and the material that the culture of rootage program can be used for taking root in the conventional bottles is only got stem apex; the material of taking root that the present invention can use on this link manys 3-4 doubly than conventional method; the traditional secondary of the outdoor disposable Cheng Miaoyu of the present invention becomes the seedling technology to compare; on the cultivation program, also done important improvement; the seedling of taking root through turning out does not need secondary to transplant; but once-seedling forming after outdoor transition management; under the prerequisite of saving cost; the seedling stalwartness of output; neatly; quality better; reach the standard of high quality seedling, be suitable for the large-scale production of flower seed plantlet.The present invention can effectively avoid the generation of polluting in incubation, reduce the number of times of transplanting seedlings, and has simplified the cultivation program of tissue culture technology in the production of industrialized tissue culture seedling, once-seedling forming, and the planting percent height, amount is big.Because the seedling of producing has remarkable advantages on quantity and production cost, in the large-scale production of flower seed plantlet, have broad application prospects simultaneously.
Embodiment
Below in conjunction with embodiment the present invention is described further.
Embodiment 1
A, draw materials and sterilize: choosing flower type is normal in the plant that has bloomed, pattern is pure, branch is straight and upright, no damage by disease and insect, individual plant that strain shape is full, getting the healthy and strong and vegetative bud that do not carry out flower bud differentiation of base portion is explant, peel off blade at branch terminal bud 2CM place, stay and do not open up 2-3 to young leaves and cut and stay 1.5CM, handled 20 minutes with 0.15% mercuric chloride with the clean back of washing powder washing, handled 20 minutes with 2% clorox, rinsed with sterile water 2 times gets the explant segment again;
B, inducing culture: the segment of A step gained explant is inoculated in the following inducing culture, that is: the agar of sucrose+7000mg/L of methyl+30000mg/L of 6-benzylaminopurine+0.1mg/L of MS+1.0mg/L, in temperature is 25 ± 2 ℃, light application time is 10 hours/day, intensity of illumination is under the condition of culture of 1500Lx, carries out inducing culture to terminal bud and grows tall and differentiate lateral bud;
C, enrichment culture: change in the following proliferated culture medium after terminal bud that the B step is induced and indefinite bud are cut, that is: the agar of sucrose+7000mg/L of methyl+30000mg/L of 6 chaff aminopurine+0.1mg/L of 6-benzylaminopurine+0.1mg/L of MS+0.1mg/L, in temperature is 25 ± 2 ℃, light application time is 10 hours/day, intensity of illumination is under the condition of 1500Lx, be cultured to terminal bud and grow tall and merogenesis, adventitious bud proliferation grows tall and extracts blade out;
D, culture of rootage: enrichment culture is cut into stem section with 1 to 2 pair of leaf to growing 4 pairs of propagation seedlings more than the leaf, after in 1000 times thiophanate methyl solution, disinfecting 20 seconds, basal part of stem dips in the water of taking root that contains the 500mg/L hormone, insert the medium of following mass ratio: the peat composed of rotten mosses: humus soil: perlite=in 3: 1: 1, water permeable, at one deck plastic foil and one deck shading rate is 75% shade net, humidity under the tight and air tight condition more than 95%, culture of rootage 5 days; Be one deck plastic foil in the morning again, be that one deck plastic foil and one deck shading rate are 75% shade net afternoon, and humidity is under the condition more than 95%, and culture of rootage 14 days is sprayed water therebetween, and the blade face is moistening, here withering does not appear in the blade face to keep, matrix keeps moistening being advisable; Removing plastic foil afterwards, only is 75% shade net at one deck shading rate, and temperature is 25 ℃, and humidity is under 90% the condition, be cultured to seedling upright after, can remove shading rate and be 75% shade net; Spray the control of Ai Fuding solution multiple season afterwards weekly to 1-2 1/4MS nutrient solution of foliage-spray, and Liriomyza, noctuid, the avermectin solution concentration is 1500 times, until growing up to the seedling of taking root, can transplant the land for growing field crops.
Embodiment 2
A, draw materials and sterilize: choosing flower type is normal in the plant that has bloomed, pattern is pure, branch is straight and upright, no damage by disease and insect, individual plant that strain shape is full, getting the healthy and strong and vegetative bud that do not carry out flower bud differentiation of base portion is explant, peel off blade at branch terminal bud 2CM place, stay and do not open up 2-3 to young leaves and cut and stay 1.5CM, handled 20 minutes with 0.15% mercuric chloride with the clean back of washing powder washing, handled 20 minutes with 2% clorox, rinsed with sterile water 2 times gets the explant segment again;
B, inducing culture: the segment of A step gained explant is inoculated in the following inducing culture, that is: the agar of sucrose+7000mg/L of methyl+30000mg/L of 6-benzylaminopurine+0.5mg/L of MS+1.5mg/L, in temperature is 25 ± 2 ℃, light application time is 12 hours/day, intensity of illumination is under the condition of culture of 2000Lx, carries out inducing culture to terminal bud and grows tall and differentiate lateral bud;
C, enrichment culture: change in the following proliferated culture medium after terminal bud that the B step is induced and indefinite bud are cut, that is: the agar of sucrose+7000mg/L of methyl+30000mg/L of 6 chaff aminopurine+0.5mg/L of 6-benzylaminopurine+0.5mg/L of MS+0.5mg/L, in temperature is 25 ± 2 ℃, light application time is 12 hours/day, intensity of illumination is under the condition of 2000Lx, be cultured to terminal bud and grow tall and merogenesis, adventitious bud proliferation grows tall and extracts blade out;
D, culture of rootage: enrichment culture is cut into stem section with 1 to 2 pair of leaf to growing 4 pairs of propagation seedlings more than the leaf, after in 800 times tpn solution, disinfecting 50 seconds, basal part of stem dips in the water of taking root that contains the 400mg/L hormone, insert the medium of following mass ratio: the peat composed of rotten mosses: humus soil: perlite=in 2.5: 0.5: 0.8, water permeable, at one deck plastic foil and one deck shading rate is 75% shade net, humidity under the tight and air tight condition more than 95%, culture of rootage 1 day; Be one deck plastic foil in the morning again, be that one deck plastic foil and one deck shading rate are 75% shade net afternoon, and humidity is under the condition more than 95%, and culture of rootage 5 days is sprayed water therebetween, and the blade face is moistening, here withering does not appear in the blade face to keep, matrix keeps moistening being advisable; Removing plastic foil afterwards, only is 75% shade net at one deck shading rate, and temperature is 25 ℃, and humidity is under 90% the condition, be cultured to seedling upright after, can remove shading rate and be 75% shade net; Spray the control of formidable opponent's 315 solution multiple season afterwards weekly to 1-2 1/4MS nutrient solution of foliage-spray, and Liriomyza, noctuid, formidable opponent's 315 solution concentrations are 800 times, until growing up to the seedling of taking root, can transplant the land for growing field crops.
Claims (4)
1. method for tissue culture, rapid propagation and cultivation of carnation seedling, comprise explant segment and sterilization, explant induction cultivation, enrichment culture and culture of rootage, described explant induction is cultivated the medium that adopts: the agar of sucrose+7000mg/L of methyl+30000mg/L of 6-benzylaminopurine+0.1～0.5mg/L of MS+1.0～1.5mg/L, condition of culture is: cultivation temperature: 25 ± 2 ℃, light application time: 10～12 hours/day, intensity of illumination: 1500～2000Lx; The medium that described enrichment culture adopts is: the agar of sucrose+7000mg/L of methyl+30000mg/L of 6 chaff aminopurine+0.1～0.5mg/L of 6-benzylaminopurine+0.1～0.5mg/L of MS+0.1～0.5mg/L, condition of culture is: cultivation temperature: 25 ± 2 ℃, light application time: 10～12 hours/day, intensity of illumination: 1500～2000Lx is characterized in that culture of rootage carries out under following condition:
Enrichment culture to the propagation seedling that grows 4 pairs of leaves is cut into stem section with 1～2 pair of leaf, after sterile-processed, basal part of stem dips in the water of taking root that contains 300～500mg/L hormone, insert in the medium of following mass ratio: the peat composed of rotten mosses: humus soil: perlite=2.5～3: 0.5～1: 0.8～1, water permeable, at one deck plastic foil and one deck shading rate is 75% shade net, humidity under the tight and air tight condition more than 95%, culture of rootage 1～5 day; Be one deck plastic foil in the morning again, be that one deck plastic foil and one deck shading rate are 75% shade net afternoon, humidity under the condition more than 95%, culture of rootage 5～14 days; Removing plastic foil afterwards, only is 75% shade net at one deck shading rate, and temperature is 18～26 ℃, and humidity is under the condition of 85-90%, and being cultured to seedling, uprightly can to remove shading rate be 75% shade net; Afterwards weekly to 1-2 1/4MS nutrient solution of foliage-spray, and take the circumstances into consideration in Liriomyza, the control of noctuid spraying pesticide in multiple season, pesticide concentration is a 800-1000 times of liquid, until growing up to the seedling of taking root.
2. method for tissue culture, rapid propagation and cultivation of carnation seedling according to claim 1, it is characterized in that described disinfecting is: at thimerosal is thiophanate methyl solution, perhaps carbendazim solution, perhaps one or more in the tpn solution, the concentration of thimerosal is under 800～1000 times the condition, soaked 20～60 seconds, and thimerosal is the commercially available prod.
3. method for tissue culture, rapid propagation and cultivation of carnation seedling according to claim 1 is characterized in that described agricultural chemicals is an avermectin, or formidable opponent 315, or spot is dived clean, or in the Avermectin one or more, during use agricultural chemicals being made into concentration and being 800～2000 times liquid, agricultural chemicals is the commercially available prod.
4. method for tissue culture, rapid propagation and cultivation of carnation seedling according to claim 1 is characterized in that concrete steps are as follows:
A, draw materials and sterilize: choosing flower type is normal in the plant that has bloomed, pattern is pure, branch is straight and upright, no damage by disease and insect, individual plant that strain shape is full, cleans, after the sterilization, carries out the sterile working segment and inoculate;
B, inducing culture: the segment of A step gained explant is inoculated in the following inducing culture, that is: the agar of sucrose+7000mg/L of methyl+30000mg/L of 6-benzylaminopurine+0.1～0.5mg/L of MS+1.0～1.5mg/L, in temperature is 25 ± 2 ℃, light application time is 10～12 hours/day, intensity of illumination is under the condition of culture of 1500～2000Lx, carries out inducing culture to terminal bud and grows tall and differentiate lateral bud;
C, enrichment culture: change in the following proliferated culture medium after terminal bud that the B step is induced and indefinite bud are cut, that is: the agar of sucrose+7000mg/L of methyl+30000mg/L of 6 chaff aminopurine+0.1～0.5mg/L of 6-benzylaminopurine+0.1～0.5mg/L of MS+0.1～0.5mg/L, in temperature is 25 ± 2 ℃, light application time is 10～12 hours/day, intensity of illumination is under the condition of 1500～2000Lx, be cultured to terminal bud and grow tall and merogenesis, adventitious bud proliferation grows tall and extracts blade out.
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| CN 200910094281 CN101502239B (en) | 2009-03-31 | 2009-03-31 | Method for rapid propagation and cultivation of carnation seedling by tissue culture |
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| CN 200910094281 CN101502239B (en) | 2009-03-31 | 2009-03-31 | Method for rapid propagation and cultivation of carnation seedling by tissue culture |
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| CN101502239B true CN101502239B (en) | 2011-09-28 |
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102090308A (en) * | 2010-11-29 | 2011-06-15 | 天津滨海国际花卉科技园区股份有限公司 | Test tube seedling transplanting substrate for carnation |
| CN102217545B (en) * | 2011-05-27 | 2013-04-10 | 云南省农业科学院花卉研究所 | Method for cultivating polyploid dianthus caryophyllus germplasm |
| CN102318485A (en) * | 2011-07-27 | 2012-01-18 | 金华职业技术学院 | Cuttage propagation method based on nutrient solution spraying |
| CN103636493B (en) * | 2013-11-21 | 2016-06-01 | 青岛佰众化工技术有限公司 | A kind of Dianthus caryophyllus L. detoxication and tissue culture rapid propagation method |
| CN103718962B (en) * | 2013-12-13 | 2017-01-18 | 内蒙古和信园蒙草抗旱绿化股份有限公司 | Culture mediums for tissue culturing of maiden pink |
| CN104186315B (en) * | 2014-08-06 | 2016-06-29 | 云南集创园艺科技有限公司 | Rapidly and efficiently carnation Regeneration System method |
| CN111990259B (en) * | 2020-09-11 | 2021-11-30 | 上海辰山植物园 | High-fidelity seedling breeding method for carnation |
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