CN101816286B - Method for tissue culture and rapid propagation of narcissus pseudonarcissus by using ramentum - Google Patents
Method for tissue culture and rapid propagation of narcissus pseudonarcissus by using ramentum Download PDFInfo
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- CN101816286B CN101816286B CN 200910264416 CN200910264416A CN101816286B CN 101816286 B CN101816286 B CN 101816286B CN 200910264416 CN200910264416 CN 200910264416 CN 200910264416 A CN200910264416 A CN 200910264416A CN 101816286 B CN101816286 B CN 101816286B
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Abstract
The invention relates to a method for the tissue culture and rapid propagation of narcissus pseudonarcissus by using ramentum. The method comprises the following steps of: taking the ramentum of the narcissus pseudonarcissus which is 1 to 2 years old as an explant; performing the steps of induction, differentiation, secondary multiplication, sound seedling, rooting, transplanting and the like; and finally obtaining commercialized narcissus pseudonarcissus seedlings. The method of the invention has the advantages of high direction, high adaptability, simple and practical operation, achievement in up to 10 induced propagation multiple, over 95 percent of rooting rate and over 95 percent of survival rate of transplanting, and application in industrial production.
Description
Technical field
The present invention relates to a kind ofly with the scale tissue culture, breed the new method of Narcissus tazetta fast, belong to agricultural technology field.
Technical background
Narcissus tazetta (Narcissus pseudonarcissus L.) cry hyacinth again, is the general designation of introducing the narcissus new varieties of China from Europe, is Amaryllidaceae Narcissus herbaceos perennial.Narcissus tazetta is compared with Chinese narcissus, characteristics such as have large flower and brilliant color, be widely used, and its flower footpath is 3～5 times of Chinese narcissus, and pattern is bright-coloured, the utmost point is welcome by the domestic market.After the eighties in 20th century, carry out fairly large introducing, be mainly used in the flower show in spring, a small amount of potted plantly use as annual flower.Narcissus tazetta be good landscape ground by flowers, can potted plantly view and admire and cut-flower uses, the also considerable leaf of both considerable flower, application prospect is very wide, can be used as emerging industry of flowers and plants kind.
At present, the Narcissus tazetta kind ball of domestic cultivation is mainly introduced by external, and every ball 5-10 unit does not wait, and cost is higher.Narcissus tazetta generally breeds by the bulbec of tillering, each female bulb 2～4 bulbs of can tillering.But because virus infections causes narcissus bulb separation reproduction rate lower, and easily produces degeneration, bulb diminishes, the ability drop of blooming.For this reason, we mouse out the method for the tissue culture that can quicken its breeding, for the propagation production of narcissus provides certain Technical Reference.The at present domestic existing axillalry bud between the Narcissus tazetta scale leaf that utilizes, as the report of the tissue culture quick breeding of explant, but with scale do the tissue culture of explant and fast breeding do not appear in the newspapers as yet.The Narcissus tazetta bulb contains a large amount of scales, utilizes scale to do explant, and is both cost-saved, can improve reproduction coefficient greatly again, is to expand numerous a kind of fast breeding technique on a large scale on a kind of abridged edition, efficient, the suitable for producing.
Summary of the invention
Goal of the invention: it is fast to the purpose of this invention is to provide a kind of reproduction speed, is not subjected to seasonal effect, and abridged edition, efficient can realize planting Narcissus tazetta quick breeding method for tissue culture seedling industrialized and large-scale production.
Particular content: the invention provides a kind of new method of Narcissus tazetta tissue-culturing rapid propagation, may further comprise the steps.
(1), explant is selected and sterilization: the selection of explant is the key that can decision carry out the callus induction differentiation smoothly.The Narcissus tazetta bulb of selecting no damage by disease and insect, robust growth and growing 1～2 year, through 5～9 ℃ of 4～8 weeks of low temperature storage, remove the scale and the remaining old root of base portion of 2 layers of shriveling of bulb outermost, put into washing powder water routinely and soak 5min, behind the flowing water flushing 30min, place the alcohol of 70～75% (percents by volume) to soak 1～2min, behind aseptic water washing 3～5 times, remove top 1/3 scale leaf, put into 0.1～0.2% (mass percent) HgCl again
2Soak 10-15min in the solution, use aseptic water washing again 3～5 times, as the lay-by material of inducing culture.In superclean bench, with the bulb of handling well, remove plateau earlier, again scale leaf is cut into the bulk of (5～10) mm * (10～15) mm.
(2), callus induction is cultivated: under aseptic condition, the block scale that cuts is inoculated in the following inducing culture.
The MS basic culture solution
6-benzylaminopurine (6-BA) 0.5～2.5mgL
-1
2,4 dichlorophenoxyacetic acid (2,4-D) 0.1～1.0mgL
-1
Sucrose 25～35gL
-1
Agar 5.0～6.0gL
-1
pH 5.6～6.0
Under the condition of 25 ± 3 ℃ of temperature, illuminance 1000～1500lx, light application time 10～12h/d, behind cultivation 20～30d, begin to produce callus around the scale, inductivity reaches more than 90%.
(3), indefinite bud or clove differentiation culture: the callus that step (2) is obtained is cut into the bulk of identical size, is inoculated in the following differential medium.
The MS basic culture solution
6-benzylaminopurine (6-BA) 1.0～3.0mgL
-1
Methyl (NAA) 0.1～0.5mgL
-1
Sucrose 25～35gL
-1
Agar 5.0～6.0gL
-1
pH 5.6～6.0
Under the condition of 25 ± 3 ℃ of temperature, illuminance 1500～3000lx, light application time 10～12h/d, behind cultivation 25～45d, around callus, break up a large amount of indefinite buds or clove, induce differentiation rate to reach more than 90%.
(4), shoot proliferation cultivates: indefinite bud that step (3) is obtained or clove cut into simple bud or 2～3 one group, change in the following shoot proliferation medium.
The MS basic culture solution
6-benzylaminopurine (6-BA) 1.0～2.0mgL
-1
Methyl (NAA) 0.1～0.5mgL
-1
Sucrose 20～30gL
-1
Agar 5.0～6.0gL
-1
pH 5.6～6.0
Under the condition of 25 ± 3 ℃ of temperature, illuminance 1000～2000lx, light application time 10～12h/d, behind cultivation 30～50d, can make 3～8 times of indefinite bud or clove propagation.No leaf indefinite bud or clove that propagation is obtained carry out the repetition subculture again, obtain the propagating materials of a large amount of band leaves.
(5), strong seedling culture: for further improving the quality of seedling, improve rooting rate and hardening survival rate, need to increase the link step in strong sprout.The indefinite bud of the band blade that step (4) is obtained is divided into one group of 1～2 bud, changes in the following strong seedling culture base.
The MS basic culture solution
6-benzylaminopurine (6-BA) 0.5～1.5mgL
-1
Methyl (NAA) 0.3～1.0mgL
-1
Sucrose 20～30gL
-1
Agar 5.0～6.0gL
-1
pH 5.6～6.0
Under the condition of 25 ± 3 ℃ of temperature, illuminance 1000～2000lx, light application time 10～12h/d, behind cultivation 20～35d, the base portion clove is expanded fast, grow 1～3 leaf, the leaf look dark green, robust growth.
(6), culture of rootage: the unrooted clove with 1～2 leaf, plant height 1.0～2.0cm with step (5) obtains changes in the following root media.
The MS basic culture solution
6-benzylaminopurine (6-BA) 0.1～0.5mgL
-1
Methyl (NAA) 0.3～1.0mgL
-1
Sucrose 20～30gL
-1
Agar 5.0～6.0gL
-1
pH 5.6～6.0
Under the condition of 25 ± 3 ℃ of temperature, illuminance 1000～2000lx, light application time 10～12h/d, behind cultivation 20～35d, can make clove grow 2～6 white roots, rooting rate can reach more than 90%, the height of seedling 2.5～4.0cm of taking root, 2～4 leaves of tool, the leaf look dark green, robust growth.
(7), acclimatization and transplants: with the seedling of taking root that obtains in the step (6),, place room scattering light place, open bottle cap behind 2～3d, make the clove upper exposed in air through behind indoor hardening 3～5d.Behind 3～4d bulb is taken out from blake bottle, select the seedling that number reaches more than 3, root reaches 2.0cm, diameter 〉=6mm that takes root, the residual medium of water flushing root, be transplanted to vermiculite: peat soil: in the mixed-matrix of garden mould=1: 1: 2 (through autoclaving), after watering sufficient water, keep 20～25 ℃ of temperature, humidity 85～90%, be positioned over shady and cool place, water weekly 1 time.Behind 35～45d, clove hardening survival rate can reach more than 95%, and taking root reaches more than 5,3～5 in blade, and get final product transplant survival this moment, and transplanting survival rate can reach more than 95%.
Beneficial effect:
1, in a single day the present invention accomplishes scale production, and then will drive the development of Narcissus tazetta industry rapidly, will break the situation of the complete dependence on import of Narcissus tazetta kind ball, and the price that reduces Narcissus tazetta kind ball is had revolutionary effect.
2, choose the Narcissus tazetta scale as explant, both saved cost, improved the availability of bulb again.
3, by explant choose, induce, break up, propagation, strong sprout, take root, the research of key technology such as transplanting, successfully made up the tissue culturing system of Narcissus tazetta, make and induce the propagation multiple to be up to 10 times, rooting rate reaches more than 95%, transplanting survival rate reaches more than 95%, and the reproduction speed that improves Narcissus tazetta kind ball, the quality that shortens production cycle and raising kind of ball are had great facilitation.
4, the present invention can also be applied to the preservation of Narcissus tazetta germ plasm resource, realizes the preservation of a large amount of germ plasm resources under less space.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail, but embodiment only is exemplary, and the present invention is not constituted any limitation.What it should be appreciated by those skilled in the art is: under the situation that does not depart from spirit and scope of the invention, can make amendment or replace the details of technical solution of the present invention and form, and these modifications and replacing all fall within the scope of protection of the present invention.
The Narcissus tazetta that following examples adopted is horticultural gardening kind " Dutch Master ", the culture medium preparation method of used Plant Tissue Breeding, all method of Plant Tissue Breeding culture medium preparation routinely preparations, no specific (special) requirements.
Embodiment one
Narcissus tazetta tissue culture and quick-breeding method, several steps in the following order carries out:
1, the selection at explant position and sterilization: the Narcissus tazetta bulb of life in 2 years, preserve 40d down at 5 ℃.Select the bulb of outward appearance stalwartness for use, peel off withered scale of outermost layer and the remaining old root of base portion at normal temperatures, put into washing powder water routinely and soak 5min, flowing water flushing 30min is placed in the alcohol of 70% (percent by volume) and soaks 1min, with removing top 1/3 scale leaf behind the aseptic water washing 4 times, put into 0.1% (mass percent) HgCl again
2Soak 10min in the solution, use 4 lay-by materials of aseptic water washing again as initial culture.The bulb of handling well is removed plateau earlier, again scale is cut into the bulk of 10mm * 10mm.
2, callus induction is cultivated: the scale bottom is inoculated into callus inducing medium (MS+6-BA1.5mgL
-1+ 2,4-D 0.5mgL
-1+ sucrose 25g/L+ agar 5.6g/L, pH 5.8) in cultivate.25 ± 3 ℃ of cultivation temperature, illuminance 1000lx, light application time 12h/d.Produce ripe callus around cultivating the 30d scale.
3, clove is cultivated with differentiation adventitious buds: cultured callus is cut into the bulk of identical size, is inoculated into (MS+6-BA 2.5mgL on the differential medium
-1+ NAA0.3mgL
-1+ sucrose 30g/L+ agar 5.6g/L, pH 5.8) in cultivate.25 ± 3 ℃ of cultivation temperature, illuminance 2000lx, light application time 12h/d.After cultivating 30d, the differentiation rate of inducing of clove and indefinite bud reaches 95%, and increment can reach 6 times, differentiates a large amount of indefinite buds.
4, shoot proliferation is cultivated: will induce the clove of differentiation or indefinite bud to cut into 2～3 one group, and be transferred to shoot proliferation medium (MS+6-BA2.0mgL
-1+ NAA0.3mgL
-1+ sucrose 25g/L+ agar 5.6g/L, pH 5.8) in breed.25 ± 3 ℃ of cultivation temperature, illuminance 1500lx, light application time 12h/d.After cultivating 30d, clove and indefinite bud are all bred in a large number, propagation is up to 8 times.The indefinite bud that obtained of propagation or clove divided be cut to simple bud or 1～2 one group, insert in the identical shoot proliferation medium, cultivate in identical culture environment, can obtain a large amount of unrooted indefinite buds, the clove that subculture produces does not have leaf usually or 1 leaf is arranged.
5, strong seedling culture: with the band leaf clove that step 4 obtained, divide to be cut to 1～2 group, change strong seedling culture base (MS+6-BA1.5mgL over to
-1+ NAA0.5mgL
-1+ sucrose 20g/L+ agar 5.8g/L, pH 5.8) in cultivate.25 ± 3 ℃ of cultivation temperature, illuminance 1500lx, light application time 12h/d.After cultivating 30d, the base portion clove is expanded fast, clove diameter average out to 0.50cm has 1～3 blade, and the leaf look dark green, robust growth.
6, culture of rootage:, be inoculated into root media (MS+6-BA 0.2mgL with the unrooted clove that step 5 obtained
-1+ NAA0.3mgL
-1+ sucrose 20g/L+ agar 5.8g/L, pH 5.8) middle root induction, the rooting rate of clove reaches more than 95% behind the 30d, takes root several 2～5.
7, acclimatization and transplants: with step 6 seedling that obtains to take root,, place room scattering light place, open bottle cap behind the 3d, make the clove upper exposed in air through behind the indoor hardening 3d.Behind 3～4d bulb is taken out from blake bottle, selecting takes root counts the bulb that reaches more than 3, the residual medium of water flushing root, be transplanted to vermiculite: peat soil: in the mixed-matrix of garden mould=1: 1: 2 (through autoclaving), should avoid during transplanting clove is imbedded in the matrix fully, only need to get final product imbedding matrix below the rhizome dish.After watering sufficient water, keep 25 ℃ of temperature, humidity 85% is positioned over shady and cool place, waters weekly 1 time, and clove hardening survival rate can reach more than 95% behind the 35d, take root more than 4, and 3～5 in blade, get final product transplant survival this moment, and transplanting survival rate can reach 98%.
Embodiment two
This Narcissus tazetta tissue culture and quick-breeding method, outside following distinguishing characteristics, all the other steps are all identical with embodiment one with method.
The used explant of the 1st step is the Narcissus tazetta bulb of life in 2 years, through 9 ℃ of low temperature treatment 45d, selects the internal layer bottom scale of bulb for use.
The callus inducing medium of the 2nd step is MS+6-BA 2.0mgL
-1+ 2,4-D 0.3mgL
-1+ sucrose 30g/L+ agar 5.6g/L, pH5.8,25 ℃ of cultivation temperature, light application time 12h/d, intensity of illumination 1000lx cultivates 30d, and callus induction rate is 98%.
The indefinite bud of the 3rd step or clove differential medium are MS+6-BA 2.0mgL
-1+ NAA0.2mgL
-1+ sucrose 30g/L+ agar 5.6g/L, pH 5.8,25 ℃ of cultivation temperature, illuminance 2000lx, light application time 12h/d cultivates 35d, and inducing differentiation rate is 96%.
The shoot proliferation medium of the 4th step is MS+6-BA 1.5mgL
-1+ NAA0.3mgL
-1+ sucrose 25g/L+ agar 5.6g/L, pH 5.8, illuminance 1500lx, 25 ℃ of cultivation temperature, illuminance 1500lx, light application time 12h/d cultivates 30d, and the propagation multiple is 10 times.
The strong seedling culture base of the 5th step is MS+6-BA 1.0mgL
-1+ NAA0.5mgL
-1+ sucrose 20g/L+ agar 5.8g/L, pH 5.8,25 ℃ of cultivation temperature, illuminance 1500lx, light application time 12h/d cultivates 25d, and planting percent reaches more than 90%.
The root media of the 6th step is MS+6-BA 0.3mgL
-1+ NAA0.5mgL
-1+ sucrose 20g/L+ agar 5.8g/L, pH 5.8,25 ℃ of cultivation temperature, illuminance 1500lx, light application time 12h/d cultivates 30d, and rooting rate reaches 100%.
Claims (1)
1. Narcissus tazetta quick breeding method for tissue culture, this method comprises following 7 steps:
1. explant is selected and is handled: clean up alcohol and HgCl after the bulb low temperature treatment
2Sterilization, the strip off scale is cut into small pieces; Described bulb is selected 1～2 year living bulb for use; Described low temperature treatment is for handling 30-60d under 5～9 ℃ condition; Described alcohol and HgCl
2Disinfecting process is: bulb kind ball is with the alcohol-pickled 1～2min of 70～75% (percents by volume), removes about 1/3 scale leaf in top with behind the aseptic water washing 3～5 times, puts into 0.1～0.2% (mass percent) HgCl again
2Soak 10～15min in the solution, use aseptic water washing again 3～5 times; Describedly be cut into small pieces for scale being cut into the bulk of (5～10) mm * (10～15) mm;
2. callus induction: with step 1. in treated aseptic scale tissue be inoculated in the callus inducing medium and cultivate; Described callus culture base is MS+6-BA0.5～2.5mgL
-1+ 2,4-D 0.1～1.0mgL
-1+ sucrose 25～35g/L+ agar 5.0～6.0g/L, pH5.6～6.0,25 ± 3 ℃ of cultivation temperature, illuminance 1000～1500lx, light application time 10～12h/d cultivates 20～30d;
3. indefinite bud or clove differentiation: step is cultivated the bulk that the callus obtain is cut into suitable size in 2., be inoculated in the differential medium and cultivate; Described differential medium is MS+6-BA1.0～3.0mgL
-1+ NAA0.1～0.5mgL
-1+ sucrose 25～35g/L+ agar 5.0～6.0g/L, pH 5.6～6.0,25 ± 3 ℃ of cultivation temperature, illuminance 1500～3000lx, light application time 10～12h/d cultivates 25～45d;
4. shoot proliferation is cultivated: induce the clove of differentiation or indefinite bud to cut into 2～3 one group in 3. step, be transferred to and carry out enrichment culture in the shoot proliferation medium; Described shoot proliferation medium is MS+6-BA 1.0～2.0mgL
-1+ NAA 0.1～0.5mgL
-1+ sucrose 20～30g/L+ agar 5.0～6.0g/L, pH 5.6～6.0,25 ± 3 ℃ of cultivation temperature, illuminance 1000～2000lx, light application time 10～12h/d cultivates 30～50d;
5. strong seedling culture: the band leaf clove that step is obtained in is 4. divided and is cut to 1～2 one group, changes in the strong seedling culture base and cultivates; Described strong seedling culture base is MS+6-BA 0.5～1.5mgL
-1+ NAA0.3～1.0mgL
-1+ sucrose 20～30g/L+ agar 5.0～6.0g/L, pH 5.6～6.0, and cultivation temperature 25+3 ℃, illuminance 1000～2000lx, light application time 10～12h/d cultivates 20～35d;
6. culture of rootage: the unrooted clove that step is obtained in 5. is inoculated into and carries out root induction in the root media; Described root media is MS+6-BA0.1～0.5mgL
-1+ NAA0.3～1.0mgL
-1+ sucrose 20～30g/L+ agar 5.0～6.0g/L, pH 5.6～6.0,25 ± 3 ℃ of cultivation temperature, illuminance 1000～2000lx, light application time 10～12h/d cultivates 20～35d;
7. acclimatization and transplants: the seedling of taking root that step is obtained in 6. takes out from blake bottle through after the indoor hardening, be transplanted in the suitable matrix, behind the certain hour with the sprigging of transplant survival in the land for growing field crops; Described indoor hardening process is: behind the covered indoor placement 3～5d of tissue culture bottle, place room scattering light place, open bottle cap behind 2～3d, make and take root the seedling upper exposed in air, behind 3～4d seedling is taken out from blake bottle, water is transplanted to matrix after washing the residual medium of root; Described matrix formulations is a vermiculite: peat soil: garden mould=1: 1: 2, and matrix need be passed through autoclaving; Should avoid during transplanting clove is imbedded in the matrix fully, only need get final product imbedding matrix below the rhizome dish, water sufficient water after, keep 20～25 ℃ of temperature, humidity 85～90% is positioned over shady and cool place, water weekly 1 time, behind 35～45d with the sprigging of transplant survival in the land for growing field crops.
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| CN102177850B (en) * | 2011-04-01 | 2012-07-25 | 上海植物园 | Quick tissue culture breeding and seedling raising method for narcissus tazetta var. chinensis |
| CN103004590B (en) * | 2012-12-06 | 2013-12-18 | 福建农林大学 | Chinese narcissus callus preservation and tissue culture method |
| CN103238457B (en) * | 2013-05-31 | 2015-05-27 | 太仓优活生物技术有限公司 | Method for utilizing narcissus flakes to cultivate young narcissus balls |
| CN105265315A (en) * | 2015-10-20 | 2016-01-27 | 韦丽 | Quick propagation method for narcissus |
| CN107318408A (en) * | 2017-06-19 | 2017-11-07 | 莆田学院 | A kind of Do.Dick wilden daffodil propagation methods |
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| CN101238793A (en) * | 2008-03-04 | 2008-08-13 | 云南省农业科学院 | A set of culture medium of eucharis grandiflora tissue culture and standardization fast replication method thereof |
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| CN101238793A (en) * | 2008-03-04 | 2008-08-13 | 云南省农业科学院 | A set of culture medium of eucharis grandiflora tissue culture and standardization fast replication method thereof |
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| 朱洪武等.洋水仙离体组织培养快速繁育技术的研究.《洋水仙离体组织培养快速繁育技术的研究》.2007,第34卷(第1期), * |
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