CN103348918A - Efficient detoxification tissue cultivating method of strawberries - Google Patents
Efficient detoxification tissue cultivating method of strawberries Download PDFInfo
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- CN103348918A CN103348918A CN2013103067691A CN201310306769A CN103348918A CN 103348918 A CN103348918 A CN 103348918A CN 2013103067691 A CN2013103067691 A CN 2013103067691A CN 201310306769 A CN201310306769 A CN 201310306769A CN 103348918 A CN103348918 A CN 103348918A
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Abstract
The invention relates to an efficient detoxification tissue cultivating method of strawberries, which takes the micro stem tips at the top of strawberry stolons as explants to conduct tissue cultivating to obtain a strawberry plant. According to the method provided by the invention, the germination rate of the micro stem tips is above 95 percent, and rooting percentage is above 98 percent. The method conducts rapid propagation by using the micro stem tips, the propagation coefficient is higher, original variety character is well kept, and the application prospect of new superior varieties strawberry plant short-term expanding propagation and rapid propagation of plants saved in a strawberry genebank is wide; furthermore, the method can be used as a new transgenosis way.
Description
Technical field
The present invention relates to the Plant Tissue Breeding field, particularly the high-efficiency detoxicating tissue culture method of a kind of strawberry.
Background technology
The Fragaria herbaceos perennial, cultivation is is mainly nourished and generated by stolon in producing, and after for many years plantation, owing to infecting multiple virus, causes quality and yield to reduce, and economic benefit obviously descends.At present extensive use in agricultural production of strawberry detoxification tissue culture seedling.It has very important effect for the quality that reduces production costs, improves strawberry product, raising peasant economy income.Utilize group culturation rapid propagating technology to carry out Fast-propagation and popularization to the strawberry detoxic seedling, emerge neat, can carry out the anniversary and produce, be not subject to seasonal restrictions, realize factorial seedling growth and rapid popularization of new varieties.
In the existing research, the explants that adopt are more: although all obtained success, all there are various defectives in axillalry bud, stem apex, blade, petiole, root, flower pesticide, petal, stipule, ovary etc.For example cultivate take stem apex as explant, mainly exist virus elimination rate to hang down and the low problem of reproduction coefficient; And take blade, petiole, root, flower pesticide, petal, stipule, ovary etc. as explant, although virus elimination rate and reproduction coefficient are greatly improved, have the larger problem of variation, can not well keep the kind of original kind.
Summary of the invention
Technical problem solved by the invention provides the high strawberry method for tissue culture of a kind of reproduction coefficient, and its concrete technical scheme is:
A kind of strawberry high-efficiency detoxicating tissue culture method is to organize cultivation take little stem apex on strawberry stolon top as explant, obtains strawberry.
Method of the present invention, little stem apex on described strawberry stolon top be will be wrapped in the blade in the shoot apical meristem outside remove, obtain not with little stem apex of leaf primordium.
Method of the present invention may further comprise the steps:
(1) described explant is placed induce on little stem apex germination medium and cultivate, induce little stem apex to sprout the seedling that obtains sprouting;
(2) seedling is received on the proliferated culture medium cultivated, breed and Multiple Buds;
(3) Multiple Buds that above-mentioned steps (2) is obtained is transferred on the root media to be cultivated, and root induction obtains complete regenerated plant.
Method of the present invention, adopt inducing culture, proliferated culture medium, successively purposive cultivation of root media, can improve the growth rate of strawberry, the incubation time of each step is generally: the incubation time of step (1) is 20 days, the incubation time of step (2) is 30 days, and the incubation time of step (3) is 30 days.The strawberry height of seedling 5-10cm that obtains after step (3) culture of rootage has the thick root of 3-5 bar, 5-10 bar radicula; Cultivation cycle is from shortened to for 12 weeks in 18 weeks.
Method of the present invention, the medium in each step mainly provide the necessary nutriment of each growth period of strawberry, find through test, and the medium component of each step is preferably as follows:
In the step (1), described to induce little stem apex germination medium be to add the semisolid culturemedium that 6-BA, NAA, carbon source and gel obtain in the MS basic culture solution; Wherein the final concentration of 6-BA is 0.1-2.0mg/L, and the final concentration of NAA is 0.01-0.1mg/L; The final concentration of 6-BA 0.3mg/L preferably wherein, the final concentration of NAA is 0.01mg/L preferably.
In the step (2), described proliferated culture medium is to add the semisolid culturemedium that 6-BA, NAA, carbon source and gel obtain in the MS basic culture solution; Wherein the final concentration of 6-BA is 0.1-2.0mg/L, and the final concentration of NAA is 0.01-0.1mg/L; The final concentration of 6-BA 0.5mg/L preferably wherein, the final concentration of NAA is 0.03mg/L preferably.
In the step (3), described root media is to add the semisolid culturemedium that IBA, carbon source and gel obtain in the 1/2MS basic culture solution; Wherein the final concentration of IBA is 0.02mg/L.
Gel in the medium of described each step is agar, carragheen or Gelrite, is preferably agar, and the final concentration in medium is 7-8g/L; Carbon source is glucose, maltose or sucrose, is preferably sucrose, and the final concentration in medium is 30g/L.
Method of the present invention, the condition of culture in described each step is: cultivation temperature: 25 ± 2 ℃; Adopt led light source in incubation, to carry out illumination, light application time: 12-16h/d; Light intensity: 2000-3000LX.That wherein said led light source preferably adopts is red, blue, the mixed light photograph of white light, and wherein the ratio between red, blue, the white light is: 5-7:1-3:8-10; Most preferred ratio is: 7:2:10.
The present invention adopts the little stem apex detoxify group training of strawberry, and virus elimination rate is high, selects optimum LED illumination proportioning and hormone in medium proportioning, reduces cultivation cycle, to carry out the training of high-efficiency detoxicating group.Little stem apex germination rate all reaches more than 95%, and rooting rate reaches more than 98%.The present invention carries out Fast-propagation take little stem apex as the basis, not only reproduction coefficient is higher, and the characteristic of the original kind that well keeps, and especially numerous in the short-term expansion of new excellent strawberry cultivars plant, fast numerous the having broad application prospects of plant preserved in the strawberry germplasm storehouse; In addition, can also be used as a kind of new transgenosis approach.
Embodiment
For further specifying the present invention, specify with the following Examples:
Embodiment 1:
(1) preparation medium:
One, the solvent of MS basic culture solution is that water, solute are as shown in table 1.
The solute of table 1.MS basic culture solution
| Macroelement | Concentration in the medium (g/L) |
| NH4NO3 | 1.65 |
| KNO3 | 1.9 |
| CaCl2 | 0.33 |
| MgSO4·7H2O | 0.37 |
| KH2PO4 | 0.17 |
| MES | 0.5 |
| Trace element | Concentration in the medium (mg/L) |
| MnSO4·4H2O | 22.3 |
| ZnSO4·7H2O | 8.6 |
| H3BO3 | 6.2 |
| KI | 0.83 |
| Na2MoO4·2H2O | 0.25 |
| CuSO4·5H2O | 0.025 |
| CoCl2·6H2O | 0.025 |
| Molysite | Concentration in the medium (mg/L) |
| Na2-EDTA | 27.8 |
| FeSO4·7H2O | 37.3 |
| Organic principle | Concentration in the medium (mg/L) |
| Inositol | 100 |
| Glycine | 2.0 |
| VB1 | 0.1 |
| VB6 | 0.5 |
| Nicotinic acid | 0.5 |
Two, the solvent of 1/2MS basic culture solution is water, and solute is as shown in table 2.
The solute of table 2.1/2MS basic culture solution
| Macroelement | Concentration in the medium (g/L) |
| NH4NO3 | 0.825 |
| KNO3 | 0.95 |
| CaCl2 | 0.165 |
| MgSO4·7H2O | 0.185 |
| KH2PO4 | 0.085 |
| MES | 0.5 |
| Trace element | Concentration in the medium (mg/L) |
| MnSO4·4H2O | 22.3 |
| ZnSO4·7H2O | 8.6 |
| H3BO3 | 6.2 |
| KI | 0.83 |
| Na2MoO4·2H2O | 0.25 |
| CuSO4·5H2O | 0.025 |
| CoCl2·6H2O | 0.025 |
| Molysite | Concentration in the medium (mg/L) |
| Na2-EDTA | 27.8 |
| FeSO4·7H2O | 37.3 |
| Organic principle | Concentration in the medium (mg/L) |
| Inositol | 100 |
| Glycine | 2.0 |
| VB1 | 0.1 |
| VB6 | 0.5 |
| Nicotinic acid | 0.5 |
In the MS basic culture solution, add 6-BA, NAA, carbon source and gel and obtain semisolid culturemedium, as inducing little stem apex germination medium; Wherein the final concentration of 6-BA is 0.3mg/L, the final concentration 0.01mg/L of NAA.Carbon source is 30g/L sucrose, and gel is 7-8g/L agar.
In the MS basic culture solution, add 6-BA, NAA, carbon source and gel and obtain semisolid culturemedium, as proliferated culture medium; Wherein the final concentration of 6-BA is 0.5mg/L, the final concentration 0.03mg/L of NAA.Carbon source is 30g/L sucrose, and gel is 7-8g/L agar.
In the 1/2MS basic culture solution, add IBA, carbon source and gel and obtain semisolid culturemedium, as root media; Wherein the final concentration of IBA is 0.02mg/L.Carbon source is 30g/L sucrose, and gel is 7-8g/L agar.
(2) cultivate:
Remove being wrapped in the crawl blade in the shoot apical meristem outside of strawberry, not with little stem apex of leaf primordium.Organize cultivation take little stem apex as explant:
(1) described explant is placed induce on little stem apex germination medium and cultivated 20 days, induce little stem apex to sprout the seedling that obtains sprouting;
(2) seedling is received on the proliferated culture medium and to be cultivated 30 days, breed and Multiple Buds;
(3) Multiple Buds that above-mentioned steps (2) is obtained is transferred on the root media and cultivated 30 days, and root induction obtains complete regenerated plant.Plant height of seedling 5-10cm has the thick root of 3-5 bar and 5-10 bar radicula.
Wherein the condition of culture of each step is:
Light source: led light source, red: indigo plant: white=7:2:10
Light intensity: 2500LX
Light application time: 16h/d
Cultivation temperature: 25 ± 2 ℃
Little stem apex germination rate reaches more than 95%, and rooting rate reaches more than 98%.
Above-described embodiment is described preferred embodiment of the present invention; be not that scope of the present invention is limited; design under the prerequisite of spirit not breaking away from the present invention; various distortion and improvement that the common engineers and technicians in this area make technical scheme of the present invention all should fall in the definite protection domain of claims of the present invention.
Claims (10)
1. strawberry high-efficiency detoxicating tissue culture method, it is characterized in that: the little stem apex take strawberry stolon top is organized cultivation as explant, obtains strawberry.
2. method according to claim 1 is characterized in that: little stem apex on described strawberry stolon top is that the blade that will be wrapped in the shoot apical meristem outside is removed, obtain not with little stem apex of leaf primordium.
3. method according to claim 1 and 2 is characterized in that: said method comprising the steps of:
(1) described explant is placed induce on little stem apex germination medium and cultivate, induce little stem apex to sprout the seedling that obtains sprouting;
(2) seedling is received on the proliferated culture medium cultivated, breed and Multiple Buds;
(3) Multiple Buds that above-mentioned steps (2) is obtained is transferred on the root media to be cultivated, and root induction obtains complete regenerated plant.
4. method according to claim 3 is characterized in that: in the step (1), described to induce little stem apex germination medium be to add the semisolid culturemedium that 6-BA, NAA, carbon source and gel obtain in the MS basic culture solution; Wherein the final concentration of 6-BA is 0.1-2.0mg/L, and the final concentration of NAA is 0.01-0.1mg/L; The final concentration of 6-BA 0.3mg/L preferably wherein, the final concentration of NAA is 0.01mg/L preferably.
5. method according to claim 3, it is characterized in that: in the step (2), described proliferated culture medium is to add the semisolid culturemedium that 6-BA, NAA, carbon source and gel obtain in the MS basic culture solution; Wherein the final concentration of 6-BA is 0.1-2.0mg/L, and the final concentration of NAA is 0.01-0.1mg/L; The final concentration of 6-BA 0.5mg/L preferably wherein, the final concentration of NAA is 0.03mg/L preferably.
6. method according to claim 3, it is characterized in that: in the step (3), described root media is to add the semisolid culturemedium that IBA, carbon source and gel obtain in the 1/2MS basic culture solution; Wherein the final concentration of IBA is 0.02mg/L.
7. each described method according to claim 3-6, it is characterized in that: the incubation time of described step (1) is 20 days, and the incubation time of step (2) is 30 days, and the incubation time of step (3) is 30 days.
8. each described method according to claim 3-6, it is characterized in that: the condition of culture in described each step is: cultivation temperature: 25 ± 2 ℃; Adopt led light source in incubation, to carry out illumination, light application time: 12-16h/d; Light intensity: 2000-3000LX.
9. method according to claim 8 is characterized in that: described led light source adopts mixed light photograph red, blue, white light, and wherein the ratio between red, blue, the white light is: 5-7:1-3:8-10; Preferred proportion is: 7:2:10.
10. each described method according to claim 3-9, it is characterized in that: the gel in the medium of described each step is agar, carragheen or Gelrite, is preferably agar, the final concentration in medium is 7-8g/L; Carbon source is glucose, maltose or sucrose, is preferably sucrose, and the final concentration in medium is 30g/L.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103975854A (en) * | 2014-04-29 | 2014-08-13 | 卞佳林 | Culture medium for culturing strawberry stem tips and inducing plant differentiation and culture method thereof |
| CN104186351A (en) * | 2014-09-24 | 2014-12-10 | 江苏农林职业技术学院 | Tissue culture method of strawberries |
| CN105594596A (en) * | 2016-01-13 | 2016-05-25 | 石磊 | Tissue culture method for strawberry virus-free and rapid propagation for large-scale production |
| CN106069750A (en) * | 2016-06-13 | 2016-11-09 | 四川省苗源生态农业科技有限公司 | A kind of cultural method of virus-free Fructus Fragariae Ananssae |
| CN107821162A (en) * | 2017-11-07 | 2018-03-23 | 玉溪云星生物科技有限公司 | A kind of large-scale method for producing of babysbreath Plug seedling |
| CN108464240A (en) * | 2018-04-13 | 2018-08-31 | 河北富硕农业科技发展有限公司 | The method of Snow White's strawberry detoxifying fast breeding |
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| CN102422816A (en) * | 2011-10-25 | 2012-04-25 | 上海航育种子基地场 | Method for rapidly culturing shoot tips in vitro |
| CN102893869A (en) * | 2012-10-22 | 2013-01-30 | 浙江省农业科学院 | Root tip detoxification and rapid propagation technology of strawberries |
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| JPH0731308A (en) * | 1993-06-29 | 1995-02-03 | Kubota Corp | Plant reproduction method and liquid medium therefor |
| RU2302106C1 (en) * | 2006-02-08 | 2007-07-10 | Институт физиологии растений им. К.А. Тимирязева РАН | Method for preparation of in vitro propagated strawberry (fragraria l) plants to ex vitro cultivation |
| CN102106261A (en) * | 2010-12-02 | 2011-06-29 | 浙江省农业科学院 | Strawberry detoxification tissue culture method under LED condition |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103975854A (en) * | 2014-04-29 | 2014-08-13 | 卞佳林 | Culture medium for culturing strawberry stem tips and inducing plant differentiation and culture method thereof |
| CN104186351A (en) * | 2014-09-24 | 2014-12-10 | 江苏农林职业技术学院 | Tissue culture method of strawberries |
| CN105594596A (en) * | 2016-01-13 | 2016-05-25 | 石磊 | Tissue culture method for strawberry virus-free and rapid propagation for large-scale production |
| CN106069750A (en) * | 2016-06-13 | 2016-11-09 | 四川省苗源生态农业科技有限公司 | A kind of cultural method of virus-free Fructus Fragariae Ananssae |
| CN107821162A (en) * | 2017-11-07 | 2018-03-23 | 玉溪云星生物科技有限公司 | A kind of large-scale method for producing of babysbreath Plug seedling |
| CN108464240A (en) * | 2018-04-13 | 2018-08-31 | 河北富硕农业科技发展有限公司 | The method of Snow White's strawberry detoxifying fast breeding |
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Application publication date: 20131016 |