CN105145354A - Method for aseptically seeding ebony and establishing leaf-cutting rapid propagation system - Google Patents
Method for aseptically seeding ebony and establishing leaf-cutting rapid propagation system Download PDFInfo
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- CN105145354A CN105145354A CN201510563448.9A CN201510563448A CN105145354A CN 105145354 A CN105145354 A CN 105145354A CN 201510563448 A CN201510563448 A CN 201510563448A CN 105145354 A CN105145354 A CN 105145354A
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Abstract
The invention relates to a method for aseptically seeding ebony and establishing a leaf-cutting rapid propagation system. The method comprises the following steps: 1, preparing a culture medium, wherein the culture medium comprises a basic culture medium and components for tissue culturing the culture medium at various stages and specifically includes: (1) a basic culture medium: an MS culture medium; (2) a propagation culture medium; (3) a seedling strengthening and rooting culture medium; 2, sterilizing seeds and culturing the seeds; 3, carrying out the leaf-cutting propagation culture: inoculating a basic part of a mature leaf of a plant in the step 2 onto the propagation culture medium; 4, carrying out the rooting and seedling strengthening culture. The method has the beneficial effects that a plant tissue culture technology is used for seeding and carrying out the leaf-cutting rapid propagation of the ebony, and the propagation coefficient is extremely high; moreover, a greater advantage can be achieved compared with the traditional way of propagating buds by using buds, a great amount of high-quality seedlings consistent in inheritable character can be obtained in a short time, the weakness that the conventional propagation way is slow can be overcome, and positive significance on the mass production and preservation of germplasm resources can be realized.
Description
Technical field
The present invention relates to field of plant tissue culture, be specifically related to a kind of ebony aseptic seeding and leaf and insert the method that rapid propagation system sets up.
Background technology
Ebony (EcheveriaagavoidesEbony) is Crassulaceae plan encrinite platymiscium, and originate in Mexico, its form is unique, and blade beautiful end black surround, arrogance show outward, belong to rare and view and admire succulent.The natural propagation rate of ebony is very low and growth rate is very slow; Simultaneously international succulent research organization (TheInternationalOrganizationforSucculentPlantStudy) predatoryly gathers the threat of even becoming extinct to protect wild succulent to exempt from; formulate " Rules of Conduct of succulent cultivation knowledge and gatherer " in the seventies; significantly limit succulent and introduce a fine variety work, make current ebony negligible amounts both domestic and external, expensive.
But; the Fast-propagation utilizing plant tissue culture technique to carry out ebony can obtain a large amount of high quality seedling consistent with maternal plant genetic background in a short time, and this is for meeting the domestic and international market demand for ebony, protecting rare ebony Wild ornamental resources to have great importance.But, also do not set up the report of ebony quick breeding method for tissue culture before this, more there is no successful example.
Summary of the invention
Object of the present invention just in order to overcome the deficiency of above-mentioned technology, and provides a kind of ebony aseptic seeding and leaf to insert the method for rapid propagation system foundation.
The object of the invention is to have come by following technical solution.This ebony aseptic seeding and leaf insert the method that rapid propagation system is set up, and comprise the steps:
1), the preparation of medium, the component that medium comprises minimal medium and each stage medium of group training is specially:
(1) minimal medium: MS medium, wherein sucrose 20 ~ 30g/L, agar 5 ~ 9g/L, pH=5.8;
(2) proliferated culture medium: MS+6-BA0.1 ~ 0.5mg/L+NAA0.01 ~ 0.05mg/L;
(3) strengthening seedling and rooting medium: MS+6-BA0.005 ~ 0.01mg/L+NAA0.05 ~ 0.2mg/L;
2) sterilization of seed and sowing are cultivated: with ebony seed as explant material, be aseptically inoculated on minimal medium by the seed after disinfecting and cultivate, and grow up to plant to seed germination;
3) leaf inserts Multiplying culture: by step 2) in the base portion of mature leaf of plant be inoculated in the formation of evoking adventive bud clump on proliferated culture medium;
4) Rooting and hardening-off culture: by step 3) the indefinite bud clump that obtains goes on Rooting and hardening-off culture base after being divided into individual plant and carries out Rooting and hardening-off culture.
In described step 2) in, with ebony seed for explant material, be placed on disinfection in the PE centrifuge tube of 1.5ml or 2ml, clean with running water again after first soaking 20min with liquid detergent solution, then successively volume ratio be 70% alcoholic solution and effective chlorine density be soak 1min and 8min respectively in the liquor natrii hypochloritis of 1%, finally use aseptic water washing 3 ~ 5 times, often all over 1min (liquid-transfering gun operation).
In described step 2) in, seed continues cultivation 3 ~ 5 weeks after within 2 ~ 4 weeks, sprouting, and becomes the plant with mature leaf.
In described step 3) in, be inoculated on proliferated culture medium after the mature leaf of ebony plant is peeled off, make face of blade upward and base in contact medium, cultivate and form indefinite bud clump in 4 ~ 8 weeks.
In described step 3) in, go to after indefinite bud clump is divided into individual plant on minimal medium and carry out strengthening seedling and rooting cultivation, cultivate and become the strong sprout with root system after 3 ~ 5 weeks.
In described step 2), 3), 4) in, described condition of culture is, cultivation temperature is 23 ± 2 DEG C, intensity of illumination is 60 ~ 100 μm of olm
-2s
-1, light application time is 10 ~ 16 hours/day.
Beneficial effect of the present invention is: utilize plant tissue culture technique to sow ebony and leaf inserts fast numerous cultivation; its growth coefficient is high; and than in bud numerous bud mode, there is larger advantage; the high-quality strong sprout that a large amount of genetic character is consistent can be obtained within a short period of time; overcome the shortcoming that Sterile culture mode is slower; all have positive effect to its large-scale production and Germ-plasma resources protection, this technology is also for experiment basis has been established in the foundation of its genetic conversion system simultaneously.
Embodiment
Below by embodiment, the present invention is further elaborated, and help is understood the present invention by embodiment better, but the present invention is not limited only to following embodiment.
Embodiment 1:
The invention provides a kind of ebony aseptic seeding and leaf and insert the method that rapid propagation system sets up, the steps include:
1), the preparation of medium
(1) minimal medium: MS medium, wherein sucrose 30g/L, agar 7g/L, pH=5.8;
(2) proliferated culture medium: MS+6-BA0.1 ~ 0.5mg/L+NAA0.01 ~ 0.05mg/L;
(3) Rooting and hardening-off culture base: MS+6-BA0.005 ~ 0.01mg/L+NAA0.05 ~ 0.2mg/L;
2), seed sterilization and sowing cultivate
With the seed of ebony for explant material, be placed on disinfection in the PE centrifuge tube of 1.5ml or 2ml, clean with running water again after first soaking 20min with liquid detergent solution, then successively volume ratio be 70% alcoholic solution and effective chlorine density be soak 1min and 8min respectively in the liquor natrii hypochloritis of 1%, finally use aseptic water washing 3 ~ 5 times, often all over 1min (liquid-transfering gun operation); Be seeded in after seed disinfection on minimal medium and cultivate, cultivate seed after 2 ~ 4 weeks and sprout successively and continue the plant that cultivation makes seedling grow up in 3 ~ 5 weeks to have mature leaf; Cultivation temperature is 23 ± 2 DEG C, intensity of illumination is 30 ~ 80 μm of olm
-2s
-1, light application time is 10 ~ 16 hours/day;
3), leaf inserts Multiplying culture
Be inoculated on proliferated culture medium after the mature leaf of ebony plant is peeled, make face of blade upward and base in contact medium, cultivate and form indefinite bud clump in 4 ~ 8 weeks; Cultivation temperature is 23 ± 2 DEG C, intensity of illumination is 30 ~ 80 μm of olm
-2s
-1, light application time is 10 ~ 16 hours/day; Have 6 mature leafs, often sheet blade to produce 2 propagation buds with average every strain ebony seedling length to calculate, its growth coefficient up to 12, and can not increase the probability of offspring's seedling generation genetic variation.
4), strengthening seedling and rooting is cultivated
Be transferred to after indefinite bud clump is aseptically cut into individual plant on strengthening seedling and rooting medium and cultivate, cultivate and become the strong sprout with root system after 3 ~ 5 weeks; Cultivation temperature is 23 ± 2 DEG C, intensity of illumination is 30 ~ 80 μm of olm
-2s
-1, light application time is 10 ~ 16 hours/day.
Embodiment 2:
The invention provides a kind of ebony aseptic seeding and leaf and insert the method that rapid propagation system sets up, the steps include:
1), the preparation of medium
(1) minimal medium: MS medium, wherein sucrose 20 ~ 30g/L, agar 5 ~ 9g/L, pH=5.8;
(2) proliferated culture medium: MS+6-BA0.3mg/L+NAA0.02mg/L;
(3) Rooting and hardening-off culture base: MS+6-BA0.01mg/L+NAA0.1mg/L;
2), seed sterilization and sowing cultivate
With the seed of ebony for explant material, be placed on disinfection in the PE centrifuge tube of 1.5ml or 2ml, clean with running water again after first soaking 20min with liquid detergent solution, then successively volume ratio be 70% alcoholic solution and effective chlorine density be soak 1min and 8min respectively in the liquor natrii hypochloritis of 1%, finally use aseptic water washing 3 ~ 5 times, often all over 1min (liquid-transfering gun operation); Be seeded in after seed disinfection on minimal medium and cultivate, cultivate seed after 2 ~ 4 weeks and sprout successively and continue the plant that cultivation makes seedling grow up in 3 ~ 5 weeks to have mature leaf; Cultivation temperature is 23 ± 2 DEG C, intensity of illumination is 30 ~ 80 μm of olm
-2s
-1, light application time is 10 ~ 16 hours/day;
3), leaf inserts Multiplying culture
Be inoculated on proliferated culture medium after the mature leaf of ebony plant is peeled, make face of blade upward and base in contact medium, cultivate and form indefinite bud clump in 4 ~ 8 weeks; Cultivation temperature is 23 ± 2 DEG C, intensity of illumination is 30 ~ 80 μm of olm
-2s
-1, light application time is 10 ~ 16 hours/day;
4), strengthening seedling and rooting is cultivated
Be transferred to after indefinite bud clump is aseptically cut into individual plant on strengthening seedling and rooting medium and cultivate, cultivate and become the strong sprout with root system after 3 ~ 5 weeks; Cultivation temperature is 23 ± 2 DEG C, intensity of illumination is 30 ~ 80 μm of olm
-2s
-1, light application time is 10 ~ 16 hours/day.
Finally, it should be pointed out that above example is only the more representational example of the present invention.Obviously, technical scheme of the present invention is not limited to above-mentioned example, can also have many distortion, all distortion that those of ordinary skill in the art can directly derive from content disclosed by the invention or associate, and all should think protection scope of the present invention.
Claims (6)
1. ebony aseptic seeding and leaf insert the method that rapid propagation system is set up, and it is characterized in that: comprise the steps:
1), the preparation of medium, the component that medium comprises minimal medium and each stage medium of group training is specially:
(1) minimal medium: MS medium, wherein sucrose 20 ~ 30g/L, agar 5 ~ 9g/L, pH=5.8;
(2) proliferated culture medium: MS+6-BA0.1 ~ 0.5mg/L+NAA0.01 ~ 0.05mg/L;
(3) strengthening seedling and rooting medium: MS+6-BA0.005 ~ 0.01mg/L+NAA0.05 ~ 0.2mg/L;
2) sterilization of seed and sowing are cultivated: with ebony seed as explant material, be aseptically inoculated on minimal medium by the seed after disinfecting and cultivate, and grow up to plant to seed germination;
3) leaf inserts Multiplying culture: by step 2) in the base portion of mature leaf of plant be inoculated in the formation of evoking adventive bud clump on proliferated culture medium;
4) Rooting and hardening-off culture: by step 3) the indefinite bud clump that obtains goes on Rooting and hardening-off culture base after being divided into individual plant and carries out Rooting and hardening-off culture.
2. ebony aseptic seeding according to claim 1 and leaf insert the method that rapid propagation system is set up, it is characterized in that: in described step 2) in, with ebony seed for explant material, be placed on disinfection in the PE centrifuge tube of 1.5ml or 2ml, clean with running water again after first soaking 20min with liquid detergent solution, then successively volume ratio be 70% alcoholic solution and effective chlorine density be soak 1min and 8min respectively in the liquor natrii hypochloritis of 1%, finally use aseptic water washing 3 ~ 5 times, often all over 1min.
3. ebony aseptic seeding according to claim 1 and leaf insert the method that rapid propagation system is set up, and it is characterized in that: in described step 2) in, seed continues cultivation 3 ~ 5 weeks after within 2 ~ 4 weeks, sprouting, and becomes the plant with mature leaf.
4. ebony aseptic seeding according to claim 1 and leaf insert the method that rapid propagation system is set up, it is characterized in that: in described step 3) in, be inoculated on proliferated culture medium after the mature leaf of ebony plant is peeled off, make face of blade upward and base in contact medium, cultivate and form indefinite bud clump in 4 ~ 8 weeks.
5. ebony aseptic seeding according to claim 1 and leaf insert the method that rapid propagation system is set up, it is characterized in that: in described step 3) in, go to after indefinite bud clump is divided into individual plant on minimal medium and carry out strengthening seedling and rooting cultivation, cultivate and become the strong sprout with root system after 3 ~ 5 weeks.
6. ebony aseptic seeding according to claim 1 and leaf insert the method that rapid propagation system is set up, it is characterized in that: in described step 2), 3), 4) in, described condition of culture is, cultivation temperature is 23 ± 2 DEG C, intensity of illumination is 60 ~ 100 μm of olm
-2s
-1, light application time is 10 ~ 16 hours/day.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106613988A (en) * | 2016-06-30 | 2017-05-10 | 云南省农业科学院花卉研究所 | Method for quickly cultivating small potted Echeveria plant goods difficult to cut and propagate on basis of in-bottle molding |
| CN108925428A (en) * | 2018-07-31 | 2018-12-04 | 上海应用技术大学 | Succulent gold mountainous region rose quick breeding by group culture method |
| CN109275564A (en) * | 2018-10-18 | 2019-01-29 | 陕西理工大学 | A kind of Qinling Mountains purpleflower stonecrop herb test tube internal lobe inserts the method for building up of rapid propagation system |
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| CN104273027A (en) * | 2013-07-03 | 2015-01-14 | 中国科学院上海生命科学研究院 | Aseptic germination method of Crassulaceae plant seeds |
| CN104380943A (en) * | 2014-10-08 | 2015-03-04 | 云南集创园艺科技有限公司 | Mist cuttage and rapid propagation method for Sinocrassula leaves |
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2015
- 2015-09-08 CN CN201510563448.9A patent/CN105145354A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104273027A (en) * | 2013-07-03 | 2015-01-14 | 中国科学院上海生命科学研究院 | Aseptic germination method of Crassulaceae plant seeds |
| CN104380943A (en) * | 2014-10-08 | 2015-03-04 | 云南集创园艺科技有限公司 | Mist cuttage and rapid propagation method for Sinocrassula leaves |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106613988A (en) * | 2016-06-30 | 2017-05-10 | 云南省农业科学院花卉研究所 | Method for quickly cultivating small potted Echeveria plant goods difficult to cut and propagate on basis of in-bottle molding |
| CN108925428A (en) * | 2018-07-31 | 2018-12-04 | 上海应用技术大学 | Succulent gold mountainous region rose quick breeding by group culture method |
| CN109275564A (en) * | 2018-10-18 | 2019-01-29 | 陕西理工大学 | A kind of Qinling Mountains purpleflower stonecrop herb test tube internal lobe inserts the method for building up of rapid propagation system |
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