CN103766220A - Method for establishing efficient regeneration system of Chahu date - Google Patents
Method for establishing efficient regeneration system of Chahu date Download PDFInfo
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- CN103766220A CN103766220A CN201410043975.2A CN201410043975A CN103766220A CN 103766220 A CN103766220 A CN 103766220A CN 201410043975 A CN201410043975 A CN 201410043975A CN 103766220 A CN103766220 A CN 103766220A
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Abstract
The invention relates to a method for establishing an efficient regeneration system of Chahu date, belonging to the field of biotechnologies. The method comprises the following steps: by adopting a tender leaf of a Chahu date aseptic seedling as an explant of a regeneration system, inducing on an MS (Murashige and Skoog) culture medium containing thidiazuron and 6-benzylaminopurine to form an adventitious bud, inoculating the formed adventitious bud to an MS culture medium containing 6-benzylaminopurine and indolebutyric acid, strengthening the seedling, and rooting, hardening and transplanting to obtain a Chahu date regeneration plant. According to the method, a high-frequency regeneration system is established aiming at the Chahu date, a theoretical basis and a testing basis are provided for the breeding work of the Chahu date biotechnology, and a technical reference is provided for breeding the Chahu excellent variety in future.
Description
Technical field
Biological technical field under the present invention, is specifically related to a kind of method that obtains teapot jujube regeneration plant take aseptic seedling young leaflet tablet as explant.
Background technology
Jujube (Zizyphus.jujuba Mill.) is Rhamnaceae (Rhamnaceae) zizyphus (Zizyphus.Mill.) plant, to originate in Chinese characteristic advantage economic tree, its delicious flavour, fruit shape is attractive in appearance, be rich in several amino acids and the vitamin of needed by human body, also containing the medicinal ingredients such as multiple polysaccharide, trace element and cancer-resisting substance cyclic adenosine monophosphate, is medicine-food two-purpose invigorant.
Teapot jujube (' Chahuzao ') originates in Shandong, and quantity is few.Have meat protrusion at fruit body shoulder or nearly shoulder, size, shape, like Hu Zui, kettle handle, join together with oval fruits main body, likeness in form teapot and gaining the name.Teapot jujube pulp is soft, many in juice, the sweet slightly acid of taste, and the level and smooth gloss of the more excellent fruit face in addition of fresh food quality is bright-coloured, and likeness in form teapot and lovable is the high good jujube cultivars of a kind of ornamental value.In recent years, the ornamental use of teapot jujube is valued by the people gradually, or for afforestation, make scape, or view and admire etc. for go sightseeing harvesting and garden, potted landscape, have a extensive future.
Jujube cultivars and the position difference of drawing materials, the medium of suitable regeneration is just different from plant growth regulator concentration proportioning.At present, although the jujube cultivars such as existing winter jujube, grey jujube, wild jujube have been set up regenerating system, its renovation process can not be applicable to teapot jujube.
Summary of the invention
The invention provides a kind of method of setting up teapot jujube high-efficiency regeneration system, set up regenerating system with the indirect adventitious organogenesis of teapot jujube aseptic seedling young leaflet tablet callus, to realizing a large amount of Fast-propagations of these seeds, for genetic transformation, the breed improvement of teapot jujube lay the foundation.
Set up a method for teapot jujube high-efficiency regeneration system, comprise the steps: the young leaflet tablet of (1) clip teapot jujube aseptic seedling, be inoculated in the MS inducing culture that contain plant growth regulator after wound quarter, cultivates 5-6 week; (2) indefinite bud leaf regeneration being gone out is peeled off from blade, is transferred in the MS strong seedling culture base that contains plant growth regulator, cultivates 3 weeks; (3) indefinite bud of robust growth is transferred in the improvement MS root media that contains plant growth regulator, cultivates 4 weeks; (4) treat that indefinite bud bears the root of 4-5cm, transplants after hardening.
In a preferred embodiment of the present invention, the Thidiazuron that the plant growth regulator in described MS inducing culture is 0.5mg/L and the 6-benzyl aminoadenine of 1.0mg/L.
In a preferred embodiment of the present invention, the 6-benzyl aminoadenine that the plant growth regulator in described strong seedling culture base is 2.0mg/L and the indolebutyric acid of 0.4mg/L, and add the polyvinylpyrrolidone of 1g/L.
In a preferred embodiment of the present invention, the methyl α-naphthyl acetate that the plant growth regulator in described root media is 0.2mg/L and the indolebutyric acid of 0.4mg/L.
In a preferred embodiment of the present invention, the MS medium in described induction, strong sprout is the common MS medium in this area (Murashige and Skoog, 1962), wherein, sucrose 30g/L, ph=5.8-6.2, it is filled a prescription as following table:
Wherein, the modified MS medium formula of described root media is, sucrose 20g/L, and ph=5.8-6.2, its formula is as following table:
In a preferred embodiment of the present invention, described young leaflet tablet be teapot jujube aseptic seedling plant forms learn upper end under 3-5 piece of blade.
In a preferred embodiment of the present invention, described young leaflet tablet is carved and is hindered method for 2-3 wound of arteries and veins vertical direction crosscut in edge, does not injure leaf margin, does not cut off blade and is advisable.
In a preferred embodiment of the present invention, the inoculation direction of described blade be axle head far away upwards.
In a preferred embodiment of the present invention, 5-6 week is cultivated in described blade induction, within first 3 weeks, is dark cultivation.
In a preferred embodiment of the present invention, described manually operated illumination, temperature condition are intensity of illumination 2000lx ± 100lx, light application time 14 ± 2h/d, 25 ± 2 ℃ of temperature.
The present invention adopts the young leaflet tablet of teapot jujube aseptic seedling as the explant of regenerating system, on the MS medium that contains Thidiazuron and 6-benzyl aminoadenine, induction forms indefinite bud, the indefinite bud of formation is transferred to strong sprout on the MS medium that contains 6-benzyl aminoadenine and indolebutyric acid, through taking root, hardening, transplanting, finally obtain teapot jujube regeneration plant.Set up high frequency regenerating system for teapot jujube first by the present invention, survival rate and the breeding efficiency of teapot jujube regeneration plant are effectively improved, for the work of teapot jujube biotechnology breeding provides theoretical foundation and test basis, simultaneously also for seed selection teapot jujube improved seeds from now on provide Technical Reference.
Accompanying drawing explanation
Fig. 1 is that the aseptic young leaflet tablet of teapot jujube in the embodiment of the present invention is cultivated the photo after 20 days in inducing culture;
Fig. 2 is that the aseptic young leaflet tablet of teapot jujube in the embodiment of the present invention is cultivated the photo after 40 days in inducing culture;
Fig. 3 is the teapot jujube blade regenerated adventitious bud micrograph in the embodiment of the present invention;
Fig. 4 is the photo after the teapot jujube blade regenerated adventitious bud strong seedling culture 20d in the embodiment of the present invention;
Fig. 5 is teapot jujube blade regenerated adventitious bud acclimatization and transplants in the embodiment of the present invention photo after 10 days.
Embodiment
For making the technical problem to be solved in the present invention, technical scheme and advantage clearer, will be described in detail at specific embodiment below.If do not specialized, in embodiment, technological means used is the conventional means that those of ordinary skills know, the raw materials used commercial goods that is.
Below in conjunction with embodiment, the present invention is described in further detail.
Medium and condition of culture:
Adventitious bud induction culture base is the Thidiazuron that contains 0.5mg/L, the 6-benzyl aminoadenine of 1.0mg/L, and the sucrose of 30g/L, the MS medium of the agar of 7g/L;
Strong seedling culture base is the 6-benzyl aminoadenine of 2.0mg/L, the indolebutyric acid of 0.4mg/L, and the polyvinylpyrrolidone of 1g/L, the sucrose of 30g/L, the MS medium of the agar of 7g/L;
Root media is the methyl α-naphthyl acetate of 0.2mg/L, the indolebutyric acid of 0.4mg/L, and the sucrose of 20g/L, the modified MS medium of the agar of 7g/L.
Medium pH in experiment is all 5.8-6.2.
In each stage of cultivating, manually operated illumination, temperature condition are intensity of illumination 2000lx, light application time 14h/d, 25 ℃ of temperature.
The MS medium in induction, strong sprout is the common MS medium in this area (Murashige and Skoog, 1962):
The modified MS medium formula of root media is:
1, the acquisition of aseptic explant: get the teapot jujube group training seedling growing fine, cut down several 3-5 piece of blades of morphology upper end, cut 2-3 wound perpendicular to vein, but do not cut off.
2, blade evoking adventive bud: will shear the young leaflet tablet of wound, proximal ends is seeded to downwards on the MS medium of indolebutyric acid of the methyl α-naphthyl acetate that contains 0.2mg/L and 0.4mg/L, inoculates altogether 160 pieces of blades.Dark cultivation after 20d, petiole and wound have all grown white callus.Move under illumination and cultivate after 20d, have 134 pieces of blades and induce indefinite bud, average every piece of leaf regeneration bud number is 2.36.Adventitious shoot regeneration rate is 83.75%, wherein adventitious shoot regeneration rate=induce indefinite bud blade number × 100%/inoculation explant number.
3, the strong seedling culture of indefinite bud: the indefinite bud that grows to 2cm is carefully peeled off from blade, be inoculated on the MS medium of indolebutyric acid of the 6-benzyl aminoadenine that contains 2.0mg/L and 0.4mg/L, inoculate altogether 229 indefinite buds.Cultivate after 30d, have 107 indefinite buds to grow up to the regrowth of 4cm left and right, indefinite bud survival rate is 46.72%.
4, the culture of rootage of indefinite bud: by through the regenerated adventitious bud of strong seedling culture, be inoculated in the modified MS medium of indolebutyric acid of the methyl α-naphthyl acetate that contains 0.2mg/L and 0.4mg/L.Cultivate after 35d, all indefinite buds are all taken root, and rooting rate is 100%, and root length is at 3-5cm.
5, indefinite bud hardening and transplanting: the indefinite bud after taking root is opened to tissue culture bottle lid, hardening 3d.Dry for preventing medium, can in medium, add a small amount of running water.Then by the medium of root and callus wash clean, transplant to thin river sand: the matrix of vermiculite: perlite=1:1:1 is watered sufficient water, keep 70% extremely group training seedling seedling of humidity, after 40 days, group training seedling has grown a lot of capillary root systems, long 8cm left and right, is transferred to continued growth in nutritive cube, waits for out garden.
The present invention adopts the young leaflet tablet of teapot jujube aseptic seedling as the explant of regenerating system, on the MS medium that contains Thidiazuron and 6-benzyl aminoadenine, induction forms indefinite bud, the indefinite bud of formation is transferred to strong sprout on the MS medium that contains 6-benzyl aminoadenine and indolebutyric acid, through taking root, hardening, transplanting, finally obtain teapot jujube regeneration plant.Set up high frequency regenerating system for teapot jujube first by the present invention, survival rate and the breeding efficiency of teapot jujube regeneration plant are effectively improved, for the work of teapot jujube biotechnology breeding provides theoretical foundation and test basis, simultaneously also for seed selection teapot jujube improved seeds from now on provide Technical Reference.
The above is the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of principle of the present invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a method of setting up teapot jujube high-efficiency regeneration system, is characterized in that, comprises the steps:
(1) induction is cultivated: the young leaflet tablet of clip teapot jujube aseptic seedling, and be inoculated in the MS inducing culture that contain plant growth regulator after wound quarter, cultivates 35-45d;
(2) strong seedling culture: the indefinite bud that leaf regeneration is gone out is peeled off from blade, is transferred in the MS strong seedling culture base that contains plant growth regulator, cultivates 20d;
(3) culture of rootage: the indefinite bud of robust growth is transferred in the improvement MS root media that contains plant growth regulator, cultivates 4 weeks;
(4) treat that indefinite bud bears the root of 4-5cm, acclimatization and transplants.
2. the method for setting up teapot jujube high-efficiency regeneration system according to claim 1, is characterized in that, the 6-benzyl aminoadenine of the Thidiazuron that the plant growth regulator in described inducing culture is 0.5mg/L and 1.0mg/L.
3. the method for setting up teapot jujube high-efficiency regeneration system according to claim 1, it is characterized in that, the 6-benzyl aminoadenine that plant growth regulator in described strong seedling culture base is 2.0mg/L and the indolebutyric acid of 0.4mg/L, and add the polyvinylpyrrolidone of 1g/L.
4. the method for setting up teapot jujube high-efficiency regeneration system according to claim 1, is characterized in that, the methyl α-naphthyl acetate that the plant growth regulator in described root media is 0.2mg/L and the indolebutyric acid of 0.4mg/L.
5. the method for setting up teapot jujube high-efficiency regeneration system according to claim 1, is characterized in that, the MS medium in described induction, strong sprout is this area common MS medium (Murashige and Skoog, 1962), wherein, containing sucrose 30g/L, ph=5.8-6.2, filling a prescription is:
6. the method for setting up teapot jujube high-efficiency regeneration system according to claim 1, is characterized in that, the modified MS medium formula of described root media is sucrose 20g/L, ph=5.8-6.2, and other composition is:
7. the method for setting up teapot jujube high-efficiency regeneration system according to claim 1, is characterized in that, described young leaflet tablet be teapot jujube aseptic seedling plant forms learn upper end under 3-5 piece of blade.
8. the method for setting up teapot jujube high-efficiency regeneration system according to claim 1, is characterized in that, described young leaflet tablet is carved and hindered method for 2-3 wound of arteries and veins vertical direction crosscut in edge, does not injure leaf margin, does not cut off blade.
9. the method for setting up teapot jujube high-efficiency regeneration system according to claim 1, is characterized in that, the inoculation direction of described blade be axle head far away upwards.
10. the method for setting up teapot jujube high-efficiency regeneration system according to claim 1, is characterized in that, 5-6 week is cultivated in described blade induction, within first 3 weeks, is dark cultivation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104082146A (en) * | 2014-07-14 | 2014-10-08 | 北京林业大学 | Method for inducing polyploid through wild jujube adventitious buds |
| CN106561460A (en) * | 2016-11-15 | 2017-04-19 | 广西大学 | Tissue culture method of early fruiting green dates |
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| CN1934932A (en) * | 2006-10-09 | 2007-03-28 | 河北农业大学 | Method for in vitro induction of winter jujube anther callus |
| CN103190347A (en) * | 2013-04-26 | 2013-07-10 | 北京林业大学 | Teapot dates tissue culturing method |
| CN103461124A (en) * | 2013-09-15 | 2013-12-25 | 德州市农业科学研究院 | Tissue culture and rapid propagation process for seedless golden silk jujube |
-
2014
- 2014-01-30 CN CN201410043975.2A patent/CN103766220A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1934932A (en) * | 2006-10-09 | 2007-03-28 | 河北农业大学 | Method for in vitro induction of winter jujube anther callus |
| CN103190347A (en) * | 2013-04-26 | 2013-07-10 | 北京林业大学 | Teapot dates tissue culturing method |
| CN103461124A (en) * | 2013-09-15 | 2013-12-25 | 德州市农业科学研究院 | Tissue culture and rapid propagation process for seedless golden silk jujube |
Non-Patent Citations (2)
| Title |
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| NISHI MATHUR ET.AL.,: "Rapid in vitro multiplication of jujube through mature stem explants", 《PLANT CELL, TISSUE AND ORGAN CULTURE》, vol. 43, 31 December 1995 (1995-12-31), pages 75 - 77 * |
| 周瑞金等: "枣离体叶片高效再生植株的研究", 《园艺学报》, vol. 33, no. 3, 31 December 2006 (2006-12-31) * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104082146A (en) * | 2014-07-14 | 2014-10-08 | 北京林业大学 | Method for inducing polyploid through wild jujube adventitious buds |
| CN104082146B (en) * | 2014-07-14 | 2016-04-27 | 北京林业大学 | A kind of method of wild jujube indefinite bud approach induction polyploid |
| CN106561460A (en) * | 2016-11-15 | 2017-04-19 | 广西大学 | Tissue culture method of early fruiting green dates |
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