CN1285259C - Breeding method of Wedelia trilobata - Google Patents
Breeding method of Wedelia trilobata Download PDFInfo
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- CN1285259C CN1285259C CN 200410077597 CN200410077597A CN1285259C CN 1285259 C CN1285259 C CN 1285259C CN 200410077597 CN200410077597 CN 200410077597 CN 200410077597 A CN200410077597 A CN 200410077597A CN 1285259 C CN1285259 C CN 1285259C
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Abstract
The present invention relates to a propagating method for African wedelia trilobata. The propagating method comprises: (1) explants are sterilized by ethanol of 65 to 75% and mercuric chloride of 0.1%; (2) aseptic seedlings are obtained in an MS culture medium, the cultivation is carried out for 15 to 30 days, the temperature is from 23 to 27 DEG C, the illumination intensity is 1500 Lux, and the illumination time is 12 h.d<-1>; (3) the multiple shoots are generated by the induction in the culture medium which comprises MS, 6-BA of 0.5 to 5 mg/L and NAA of 0 to 0.5 mg/L, the cultivation is carried out for 15 to 35 days, and other conditions such as the temperature, the illumination intensity and the illumination time are same as the temperature, the illumination intensity and the illumination time during the process of obtaining of the aseptic seedlings; (4) the root induction is carried out in the culture medium which comprises 1/2 MS, IBA or NAA of 0 to 0.5 mg/L, the cultivation is carried out for 5 to 20 days, and other conditions such as the temperature, the illumination intensity and the illumination time are same as the temperature, the illumination intensity and the illumination time during the process of obtaining of the aseptic seedlings; (5) the regenerated plants are transplanted after the seedlings of regenerated plants are cultivated. The fast separated propagation of African wedelia trilobata can be realized by the method, and the operation is easy.
Description
(1) technical field
The present invention relates to a kind of method for plant tissue culture, specifically, be meant a kind of method that adopts tissue culture technique that the Wedelia trilobata cultured in vitro is bred fast.
(2) background technology
Plant Tissue Breeding is meant any organ, tissue or the cell with plant, carries out the process that aseptic culture is grown under the manual control condition.This technology is drawn materials few, and it is low to cultivate the vegetable material cost, and growth cycle is short, convenient management.Utilize this quick propagating technology of Plant Tissue Breeding, can multiply the plant of maternal biological nature of a large amount of maintenances and genetic character at short notice.It is reported that China plant of breeding fast has nearly thousand kinds.Normally used minimal medium has several, as MS, B5, White, N6 etc.In tissue culture, plant explants will be under the effect of plant growth substance, through inducing dedifferentiation, recover fissional ability, influencing processes such as breaking up, promote organ formation again.The size of plant explants reproduction coefficient directly affects the quality and quantity of producing the offspring plant.
Wedelia trilobata (Wedelia prostrata) has another name called single flower wedelia chinensis, another name Cat tongue chrysanthemum, bog starwort herb uncle (mother's brother), ?flower, prostrate wedelia herb, be the amphibious crab Chrysanthemum perennial herb of composite family.Be widely distributed in the general torrid zone to warm band seashore, Taiwan, south China, Southeast Asia, Japan's one band all have growth.Wedelia trilobata can be done flower bed, bank protection ground quilt, is the good fixed husky plant of seashore; Leaf is pharmaceutically acceptable; It is the nectar source of middle-size and small-size butterfly.The breeding of Wedelia trilobata is mainly by cottage propagation, but that natural propagation exists reproduction coefficient is low, problem such as is subject to seasonal restrictions.Can reach the purpose of breeding fast in the short time by tissue culture, improve output, and can overcome the influence of season breeding.Carrying out the scale micropropagation with tissue culture technique, is the effective way that obtains a large amount of high quality seedlings.We are intended to utilize method for tissue culture at such situation, improve the reproduction rate of Wedelia trilobata by the vegetative propagation means, provide technical scheme for producing in its anniversary.The method for tissue culture of relevant Wedelia trilobata vegetative propagation seedling at home as yet the someone report, do not appear in the newspapers in the world yet.
The basic process of Plant Tissue Breeding is: the organ, tissue or the cell that utilize plant corpus to exsomatize, (as root, stem, leaf, flower, fruit, seed, embryo, ovule, ovary, flower pesticide, pollen and storage organ's parenchyma, vascular bundle tissue etc.) is under conditions such as aseptic and suitable synthetic medium and illumination, temperature, induce callus and be differentiated to form indefinite bud again, perhaps directly directly differentiate the bud of growing thickly by plant in vitro tissue or organ, root induction then forms complete plant at last.Aftergrowth is transplanted after hardening, finally forms the commodity plant.
(3) Fa Ming content
The object of the present invention is to provide a kind of propagation method of Wedelia trilobata, it can obviously improve the vegetative propagation coefficient of Wedelia trilobata, and reproduction speed is fast and easy and simple to handle.
Specifically, the inventive method comprises the steps:
(1) explant sterilization: place 65-75% ethanol to soak 20-60s (representing " second ", down together) Wedelia trilobata terminal bud or lateral bud, the 4-12min that sterilizes in 0.1% mercuric chloride (expression " minute ", down together), use aseptic water washing 3-6 time again;
(2) acquisition of aseptic seedling: Wedelia trilobata terminal bud after will sterilizing or lateral bud are inoculated into to cultivate in the MS medium and obtained aseptic seedling in 15～30 days, and condition of culture is 25 ± 2 ℃ of temperature, intensity of illumination 1500Lux, light application time 12hd
-1(expression " hour/day ", down together);
(3) the induced bundle generation of sprouting: the stem apex of aseptic seedling or stem section placed cultivate the bud that obtained in 15～35 days to grow thickly on the medium of prescription for MS+0.5-5mg/L6-benzylaminopurine (being called for short 6-BA)+0-0.5mg/L methyl (being called for short NAA) and (highly be generally 1～2cm), the same step of condition of culture (2);
(4) root induction: the resulting bud of growing thickly of step (3) is cut into individual plant, on 1/2MS+0-5mg/L indolebutyric acid (being called for short IBA) or NAA medium, cultivate and obtained regeneration plant in 5～20 days, the same step of condition of culture (2).
(5) regeneration plant is transplanted: the regeneration plant that step (4) is obtained is transplanted in vermiculite soil or the flower cultivating soil, with running water or the pouring of 1/4MS culture fluid after hardening.
In the said method, in step (3), the preferred version of medium is MS+1-5.0mg/L6-BA+0.1-0.5mg/L NAA, preferably MS+2.0mg/L6-BA+0.5mg/LNAA.The optimization formula of medium is to adopt 1/2MS+0.5-1mg/L IBA or NAA prescription in the step (4), wherein the concentration of IBA or NAA 1.0mg/L preferably.
The present invention has following advantage and effect:
1. the experiment proved that, the inductivity of indefinite bud reaches as high as 100% in the inventive method step (3), each explant can differentiate a plurality of indefinite buds, wherein maximum with the indefinite bud in the medium of 2.0mg/L6-BA+0.5mg/L NAA, each explant has 8～10 buds, in step (4), adopt IBA, NAA to induce, rooting rate 100%, and grow fine, test-tube plantlet adventive root quality guaranteed.Compare with existing propagation technique, this method reproductive efficiency height, the anniversary can produce in a large number, has overcome prior art and has been subjected to the influence of season to breeding.
2. the inventive method need not task equipment, and is easy and simple to handle.
(4) concrete embodiment
Embodiment 1:
(1) sterilization of explant: get Wedelia trilobata terminal bud or lateral bud and place 70% ethanol to soak 30s, the 6min that sterilizes in 0.1% mercuric chloride uses aseptic water washing 5 times again;
(2) acquisition of aseptic bacterium: the Wedelia trilobata explant that will sterilize is inoculated on the MS medium and cultivated 15 days, obtains aseptic seedling, and condition of culture is: 25 ± 2 ℃ of temperature, intensity of illumination 1500Lux, light application time 12hd
-1(expression " hour/day ", down together);
(3) differentiation of bud: being inoculated into after one month to add has on the MS medium of 0.5mg/L 6-BA, cultivated 15 days, the differentiation adventitious buds rate is 87.5%, each explant that can break up has 1～2 indefinite bud, cultivated 35 days, the differentiation adventitious buds rate is 100%, and each explant that can break up has 2 indefinite buds.
(4) formation of root: the seedling of formation, switching goes into to add on the 1/2MS medium of 1.0mg/L IBA, when cultivating 5 days, rooting rate is 100%, and each explant all has 10～11 to induce root, induce root to become radial, cultivate after 10 days, the root of inducing of each explant increases to 15～16, cultivates after 20 days, induce root growth elongated, climb the full bottle end.The upgrowth situation of seedling is good.
(5) test-tube seedling transplanting: after inducing the root experiment to cultivate 20 days, will change vermiculite soil after the test-tube plantlet hardening over to, with the pouring of 1/4MS culture fluid, seedling well-grown after 15 days, survival rate 100%.
Embodiment 2:
Other operation is with embodiment 1, and different is: in the step (1), get Wedelia trilobata terminal bud or lateral bud and place 65% ethanol to soak 20s, the 4min that sterilizes in 0.1% mercuric chloride uses aseptic water washing 6 times again; In the step (2), incubation time is 20 days; In the step (3), culture medium prescription is MS+0.5mg/L 6-BA+0.1mg/LNAA, and incubation time is 15 days, and the differentiation adventitious buds rate is 100%, and each explant has 2～3 differentiation buds, and when cultivating 35 days, each explant on average has 3 differentiation buds; In the step (4), medium changes 1/2MS+1.0mg/L NAA into, when cultivating 5 days, rooting rate also is 100%, the root of inducing of each explant is 8～9, induce root to become radial and bear, and situation of growth is better, cultivate after 10 days, the root of inducing of each explant increases to 13～14, cultivates after 20 days, induces root growth elongated, climb the full bottle end, the upgrowth situation of seedling is good; In the step (5), the vermiculite land reform is a flower cultivating soil, with the running water pouring, and after 15 days, the seedling well-grown, survival rate is 100%.
Embodiment 3:
Other operation is with embodiment 1, and different is: in the step (1), get Wedelia trilobata terminal bud or lateral bud and place 75% ethanol to soak 60s, the 12min that sterilizes in 0.1% mercuric chloride uses aseptic water washing 3 times again; In the step (2), incubation time is 30 days; In the step (3), culture medium prescription is MS+0.5mg/L 6-BA+0.2mg/L NAA, when incubation time is 15 days, the differentiation adventitious buds rate is 75%, each explant that can break up has 2～3 differentiation buds, when incubation time was 35 days, the differentiation adventitious buds rate was 100%, and each explant has 3～5 differentiation buds; In the step (4), medium changes 1/2MS+0.5mg/L NAA into, when cultivating 5 days, induce root to become radial and bear, rooting rate also is 100%, and the root of inducing of each explant is 7～8, cultivate that the root of inducing of each explant increases to 9～10 after 10 days, cultivate after 20 days, induce root growth elongated, climb the full bottle end.The upgrowth situation of seedling is good.
Embodiment 4:
Other operation is with embodiment 1, different is: in the step (3), culture medium prescription is MS+0.5mg/L 6-BA+0.5mg/L NAA, when incubation time was 15 days, the differentiation adventitious buds rate was 66.7%, and each explant that can break up has 1～2 differentiation bud, incubation time is 35 days, the differentiation adventitious buds rate is 100%, and each explant has 3～5 differentiation buds, and the part explant has the root generation; In the step (4), medium changes 1/2MS+0.5mg/L IBA into, when cultivating 5 days, root becomes radial and bears, and rooting rate also is 100%, the root of inducing of each explant is 9～10, and situation of growth is better, cultivates that the root of inducing of each explant increases to 13～14 after 10 days, cultivates after 20 days, root is long long, at the bottom of being covered with bottle.The upgrowth situation of seedling is good.
Embodiment 5:
Other operation is with embodiment 2, and different is: in the step (3), culture medium prescription is MS+1mg/L 6-BA, and incubation time is 20 days, and the differentiation adventitious buds rate is 100%, and each explant has 3～4 differentiation buds; In the step (4), medium changes 1/2MS into, in the time of 5 days, rooting rate is 75%, and the root of inducing of each explant is 2, rooting rate is 95.8% after 10 days, the root of inducing of each explant is 2～3, cultivates after 20 days, and rooting rate is 100%, the root of inducing of each explant is 2～3, induces root growth elongated.The upgrowth situation of seedling is good.
Embodiment 6:
Other operation is with embodiment 2, different is: in the step (3), culture medium prescription is MS ten 1mg/L 6-BA+0.1mg/L NAA, when incubation time is 20 days, the differentiation adventitious buds rate is 100%, each explant has 4～5 differentiation buds, and when incubation time was 30 days, the differentiation bud of each explant increased to 6～8; In the step (4), medium changes 1/2MS+2mg/L NAA into, cultivates after 5 days, explant sends out roots a little, does not still have obviously and takes root, and callus is arranged at the bottom, the seedling well-grown was cultivated after 8 days, and root begins to be shaped, it is intensive to grow, and each explant is induced 9～10 of roots, and root is comparatively short, have only 1～2mm, rooting rate is 100%, cultivates after 15 days, each explant has 19～20 roots, and root increases, but the longlyest has only about 1cm.Seedling grows tall, and grows fine.
Embodiment 7:
Other operation is with embodiment 2, different is: in the step (3), culture medium prescription is MS+1mg/L 6-BA+0.2mg/L NAA, when incubation time is 20 days, the differentiation adventitious buds rate is 100%, each explant has 3～5 differentiation buds, and when incubation time was 30 days, the differentiation bud of each explant increased to 8～9; In the step (4), medium changes 1/2MS+5mg/L NAA into, cultivates after 5 days, callus is arranged at the explant bottom, and root point is not obvious, cultivates after 10 days, it is big that callus is formed on the bottom, all has root to grow on the callus, and the root point of bottom is intensive, cultivate after 15 days, each explant has 19～20 roots, and rooting rate is 100%, and root increases, but major part only is 2～3mm, and the longest is 2cm.Seedling grows tall, and grows fine.
Embodiment 8:
Other operation is with embodiment 2, different is: in the step (3), culture medium prescription is MS+1mg/L 6-BA+0.5mg/L NAA, incubation time is 20 days, the differentiation adventitious buds rate is 100%, each explant has 3～4 differentiation buds, and when incubation time was 30 days, the indefinite bud of each explant increased to 6～9; In the step (4), medium changes 1/2MS+2mg/L IBA into, cultivates after 5 days, callus is arranged at the explant bottom, sends out roots a little, does not still have obviously and takes root, the seedling well-grown was cultivated after 8 days, and root growth is intensive, each explant is induced 14～15 of roots, but root is comparatively short, has only 1～2mm, rooting rate is 100%, cultivates after 15 days, and each explant has 22～23 roots, root increases, but on average has only about 1cm.Seedling grows tall, and grows fine.
Embodiment 9:
Other operation is with embodiment 1, and different is: in the step (3), culture medium prescription is MS+2mg/L 6-BA, and incubation time is 20 days, and the differentiation adventitious buds rate is 100%, and each explant has 3～5 differentiation buds; In the step (4), medium changes 1/2MS+5mg/L IBA into, cultivates after 5 days, callus is arranged at the explant bottom, and root point is not obvious, cultivates after 10 days, it is big that callus is formed on the bottom, all has root to grow on the callus, and the root point of bottom is intensive, cultivate after 15 days, each explant has 17～18 roots, and rooting rate is 100%, root increases, but on average have only 4～5mm, seedling grows tall, and grows fine.
Embodiment 10:
Other operation is with embodiment 1, different is: in the step (3), culture medium prescription is MS+2mg/L 6-BA+0.1mg/L NAA, incubation time is 20 days, the differentiation adventitious buds rate is 100%, each explant has 3～4 differentiation buds, and when incubation time was 30 days, the indefinite bud of each explant increased to 5～7.
Embodiment 11:
Other operation is with embodiment 1, different is: in the step (3), culture medium prescription is MS+2mg/L 6-BA+0.2mg/L NAA, incubation time is 20 days, the differentiation adventitious buds rate is 100%, each explant has 4～5 differentiation buds, and when incubation time was 30 days, the indefinite bud of each explant increased to 5～8.
Embodiment 12:
Other operation is with embodiment 1, different is: in the step (3), culture medium prescription is MS+2mg/L 6-BA+0.5mg/L NAA, incubation time is 20 days, the differentiation adventitious buds rate is 100%, each explant has 4～6 differentiation buds, and when incubation time was 30 days, the indefinite bud of each explant increased to 8～10.
Embodiment 13:
Other operation is with embodiment 2, different is: in the step (3), culture medium prescription is MS+5mg/L 6-BA, incubation time is 20 days, the differentiation adventitious buds rate is 30%, and each explant that can break up has 1～2 differentiation bud, when incubation time is 30 days, the differentiation adventitious buds rate is 50%, and the differentiation bud of each explant increases to 2.
Embodiment 14:
Other operation is with embodiment 2, different is: in the step (3), culture medium prescription is MS+5mg/L 6-BA+0.1mg/L NAA, incubation time is 20 days, and the differentiation adventitious buds rate is 100%, and each explant has 3～5 differentiation buds, when incubation time is 30 days, the indefinite bud of each explant increases to 5～6, and when incubation time was 35 days, the indefinite bud of each explant increased to 7～8.Differentiation bud color is more shallow.
Embodiment 15:
Other operation is with embodiment 2, different is: in the step (3), culture medium prescription is MS+5mg/L 6-BA+0.2mg/L NAA, incubation time is 20 days, and the differentiation adventitious buds rate is 100%, and each explant has 3～5 differentiation buds, when incubation time is 30 days, the indefinite bud of each explant increases to 5～8, and when incubation time was 35 days, the indefinite bud of each explant was 5～9.Differentiation bud color is more shallow.
Embodiment 16:
Other operation is with embodiment 2, different is: in the step (3), culture medium prescription is MS+5mg/L 6-BA+0.2mg/L NAA, incubation time is 20 days, and the differentiation adventitious buds rate is 100%, and each explant has 3～5 differentiation buds, when incubation time is 30 days, the indefinite bud of each explant increases to 5～8, and when incubation time was 35 days, the indefinite bud of each explant was 5～9.Differentiation bud color is more shallow.
Embodiment 17:
Other operation is with embodiment 2, different is: in the step (3), culture medium prescription is MS+5mg/L 6-BA+0.5mg/L NAA, incubation time is 20 days, and the differentiation adventitious buds rate is 100%, and each explant has 4～6 differentiation buds, when incubation time is 30 days, the indefinite bud of each explant increases to 7～9, and when incubation time was 35 days, the indefinite bud of each explant was 8～10.Differentiation bud color is more shallow.
Claims (5)
1, a kind of propagation method of Wedelia trilobata is characterized in that comprising the steps:
(1) explant sterilization: place 65-75% ethanol to soak 20-60s Wedelia trilobata terminal bud or lateral bud, the 4-12min that sterilizes in 0.1% mercuric chloride uses aseptic water washing 3-6 time again;
(2) acquisition of aseptic seedling: Wedelia trilobata terminal bud after will sterilizing or lateral bud are inoculated into to cultivate in the MS medium and obtained aseptic seedling in 15～30 days, and condition of culture is 25 ± 2 ℃ of temperature, intensity of illumination 1500Lux, light application time 12hd
-1
(3) the induced bundle generation of sprouting: place prescription to obtain to grow thickly bud, the same step of condition of culture (2) in 15～35 days the stem apex or the stem section of aseptic seedling for cultivating on the medium of MS+0.5-5mg/L 6-BA+0-0.5mg/L NAA;
(4) root induction: the resulting bud of growing thickly of step (3) is cut into individual plant, on the medium of 1/2MS+0-5mg/L IBA or NAA, cultivate and obtained regeneration plant in 5～20 days, the same step of condition of culture (2);
(5) regeneration plant is transplanted: after the regeneration plant hardening that step (4) is obtained, be transplanted in vermiculite soil or the flower cultivating soil, with running water or the pouring of 1/4MS culture fluid.
2, the method for claim 1 is characterized in that: in step (3), culture medium prescription is MS+1-5.0mg/L6-BA+0.1-0.5mg/L NAA.
3, method as claimed in claim 1 or 2 is characterized in that: the prescription of medium is in the step (3): MS+2.0mg/L6-BA+0.5mg/L NAA.
4, the method for claim 1 is characterized in that: in the step (4), the prescription of medium is 1/2MS+0.5-1mg/L IBA or NAA.
5, want 1 or 4 described methods as right, it is characterized in that: the prescription of medium is in the step (4): 1/2MS+1.0mg/L IBA or NAA.
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| CN 200410077597 CN1285259C (en) | 2004-12-27 | 2004-12-27 | Breeding method of Wedelia trilobata |
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| CN 200410077597 CN1285259C (en) | 2004-12-27 | 2004-12-27 | Breeding method of Wedelia trilobata |
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| CN1631106A CN1631106A (en) | 2005-06-29 |
| CN1285259C true CN1285259C (en) | 2006-11-22 |
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| CN100553446C (en) * | 2007-03-29 | 2009-10-28 | 浙江省农业科学院 | A kind of method of European wild black cherry mahogany tissue-culturing rapid propagation |
| CN103081806B (en) * | 2013-01-15 | 2014-04-09 | 江苏大学 | In vitro micropropagation two-step method of trilobate wedelia |
| CN103340152B (en) * | 2013-06-28 | 2015-03-25 | 新疆凤凰远山农业发展有限公司 | Chrysanthemum screening, detoxification and rapid propagation method |
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