CN106258952A - The method utilizing Caulis et Folium Ammopiptanthi Mongolici children tender side root induction Caulis et Folium Ammopiptanthi Mongolici Callus formation - Google Patents
The method utilizing Caulis et Folium Ammopiptanthi Mongolici children tender side root induction Caulis et Folium Ammopiptanthi Mongolici Callus formation Download PDFInfo
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- CN106258952A CN106258952A CN201510872381.7A CN201510872381A CN106258952A CN 106258952 A CN106258952 A CN 106258952A CN 201510872381 A CN201510872381 A CN 201510872381A CN 106258952 A CN106258952 A CN 106258952A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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Abstract
The invention discloses a kind of method utilizing Caulis et Folium Ammopiptanthi Mongolici children tender side root induction Caulis et Folium Ammopiptanthi Mongolici Callus formation, comprise the following steps: a. utilizes Caulis et Folium Ammopiptanthi Mongolici seed to carry out aseptic nursery, constantly induced by 1/2MS culture medium the side root of band root hair to be formed；The most then the tender hairy side root of clip children is as outer implant, in the dark induced synthesis callus in CDB1 culture medium, and under light or the callus that subculture and expanding propagation are induced in CDB6-2 culture medium of dark place.Use method of the invention, it is possible to efficiently, low cost and stably obtain Caulis et Folium Ammopiptanthi Mongolici callus.
Description
Technical field
The present invention relates to a kind of side utilizing Caulis et Folium Ammopiptanthi Mongolici children tender side root induction Caulis et Folium Ammopiptanthi Mongolici Callus formation
Method.
Background technology
Ammopiptanthus Genus is in pulse family Ammopiptanthus Genus, and it, can be suitable as one super non-irrigated raw desert evergreen shrubs
The various extreme environment in desert (arid, high temperature, low temperature and wind erosion etc.), is again that the Tertiary Period is ancient simultaneously
Deleted species, belong to Endangered species, so Caulis et Folium Ammopiptanthi Mongolici is with preferable degeneration-resistant desirable genes resource plant,
And there is gene and Plant Physiology character inheritance Study on Evolution value simultaneously.Forefathers have simply carried out greatly
Measure about research work such as its conservation of nature, community distribution, biological characteristics and Analysis of Genetic Backgrounds,
Until in the recent period, the research report having part is that the gene about Caulis et Folium Ammopiptanthi Mongolici is cloned and transcript profile sequencing analysis
Work, its goal in research is also to be to excavate the valuable adversity gene resource contained of Caulis et Folium Ammopiptanthi Mongolici and anti-
Inverse mechanism, to solve current agricultural production and weather variation issue.
But owing to Caulis et Folium Ammopiptanthi Mongolici growth cycle is long, seed-setting rate and germination percentage are low, subject to insect pest simultaneously,
So Caulis et Folium Ammopiptanthi Mongolici fertility is weak；Its mature tissue is more aging, and the growth of seedling later stage after germination is the easiest
All it is unfavorable for DNA and RNA extraction etc. in aging and lignifying, and scientific research personnel cannot be in indoor kind
Plant and breed Caulis et Folium Ammopiptanthi Mongolici, it is necessary to a large amount of new seed of annual harvesting carry out scientific research, and this limits to a great extent
Molecular biotechnology the gene of Caulis et Folium Ammopiptanthi Mongolici clone with functional study in terms of application.There is presently no
The patent report induced about Caulis et Folium Ammopiptanthi Mongolici tissue culture and wound healing, only part Chinese periodical has has on a small quantity
The report of pass Caulis et Folium Ammopiptanthi Mongolici callus induction, but induced efficiency (3%～80%) on the low side and unstable, side
Method repeatability is poor.
Summary of the invention
Present invention is primarily targeted at and overcome the deficiencies in the prior art, it is provided that one utilizes Caulis et Folium Ammopiptanthi Mongolici children
The method of tender side root induction Caulis et Folium Ammopiptanthi Mongolici Callus formation, easy, efficiently, low cost and stably obtaining
Obtain Caulis et Folium Ammopiptanthi Mongolici callus.
For achieving the above object, the present invention is by the following technical solutions:
A kind of method utilizing Caulis et Folium Ammopiptanthi Mongolici children tender side root induction Caulis et Folium Ammopiptanthi Mongolici Callus formation, including following
Step: a. utilizes Caulis et Folium Ammopiptanthi Mongolici seed to carry out aseptic nursery, constantly induces band root by 1/2MS culture medium
The side root of hair is formed；The most then the hairy side root that clip children is tender is as outer implant, in the dark in CDB1
Induced synthesis callus in culture medium, and under light or dark place in CDB6-2 culture medium subculture and
The callus that expanding propagation is induced.
Further:
In the culture dish containing 1/2MS solid medium, make germination, then go to equipped with 1/2MS
The sterile culture flask of culture medium is induced seedling, after seedling grows more branch roots, is transplanted to new dress
Have in the sterile culture flask of 1/2MS culture medium, make part side root be exposed at media surface, induction side root
Form the short side root in a large number with root hair.
The process utilizing Caulis et Folium Ammopiptanthi Mongolici seed to carry out aseptic nursery in step a includes:
(1) select the Caulis et Folium Ammopiptanthi Mongolici seed that Maturity is good, profile is intact, do not eaten by insect, install to
In sterilizing, airtight bottle or pipe；
(2) in bottle or pipe, first with 75% ethanol disinfection Caulis et Folium Ammopiptanthi Mongolici seed 10min；
(3) ethanol is removed, then with 6% hypochlorite disinfectant Caulis et Folium Ammopiptanthi Mongolici seed 30min；
(4) sodium hypochlorite is removed, with sterile water wash sterilizing seed 5～6 times；
(5) removing sterilized water, somewhat drain away the water, the most switchable seed is trained to containing solid 1/2MS
Support in the culture dish of base；
(6) cultivate 2～5 days under the conditions of 22 DEG C～28 DEG C, make germination.
The process constantly inducing the side root of band root hair to be formed by 1/2MS culture medium in step a includes:
(1) select the good seed of germinating energy and be transferred to the sterilizing culture bottle equipped with solid 1/2MS culture medium
In, it is beneficial to the growth of chitting piece；
(2) after Caulis et Folium Ammopiptanthi Mongolici seedling grows a plurality of side root, second time switching is carried out, by robust growth
Seedling replanting is in the new sterilizing culture bottle equipped with solid 1/2MS culture medium, and side, exposed portion root
In media surface；
(3), after growing 20～40 days, the side root being exposed at media surface can grow many with root hair
Short side root, in this, as the outer implant material of callus induction.
In step b, in the dark in CDB1 culture medium, the process of induced synthesis callus includes:
Aseptically, clip part hairy side root is placed in CDB1 culture medium, in the dark 28 DEG C
Under the conditions of inducing culture 20～35 days, wherein CDB1 medium component is: MS+1.0mg/L 2,4-D
+ 0.1mg/L 6-BA, pH5.8～7.5.
In step b under light or dark place subculture and expanding propagation in CDB6-2 culture medium induced more
The process of injured tissue includes:
The callus induced is transferred in CDB6-2 culture medium, has optical condition or dark at 28 DEG C
Place is lower cultivates, and every 10～15 days subcultures once, wherein CDB6-2 medium component is: MS+0.2
Mg/L IBA+0.1mg/L KT+0.5mg/L GA3, pH5.8～7.5.
Beneficial effects of the present invention:
Utilize the present invention to form Caulis et Folium Ammopiptanthi Mongolici callus, Caulis et Folium Ammopiptanthi Mongolici DNA and RNA can be carried out and extract,
Carry out gene order-checking or gene clone and functional study equimolecular biological study, without volume again
Outer seed collecting nursery, thus research cost can be reduced, improve Efficiency, simultaneously protection Proterozoic state
The population of family's endangered plants Caulis et Folium Ammopiptanthi Mongolici.
The advantage of the present invention is embodied in following aspect:
(1), after the aseptic nursery of Caulis et Folium Ammopiptanthi Mongolici grows more branch roots, transplanted again by aseptic condition, and stay
Part root system is exposed at 1/2MS aseptic culture primary surface, can induce and grow many band roots on its side root again
The short side root of hair, the clip tender hairy side root of children can be used as the outer implant of Caulis et Folium Ammopiptanthi Mongolici callus culture
Material.This method of drawing material does not interferes with the normal growth of this individual plant in aseptic bottle, can obtain again children for a long time
Tender outer implant material, is used for follow-up callus induction.
(2) the tender hairy side root of children is prone to callus induction and forms (inductivity >=80%), and wound healing lures
Lead stable, and without nursery of collecting seed again.
(3) present invention only requires in the dark with CDB1 culture medium callus induction, return again under light or dark
Callus subculture is cultivated by place's CBD6-2 culture medium with expanding propagation, and step is simple.
(4) the most aseptic, homogeneous, stable Caulis et Folium Ammopiptanthi Mongolici callus can be obtained, divide for Caulis et Folium Ammopiptanthi Mongolici
Sub-biological study (such as gene clone and functional study) is used, it is not necessary to go to field to collect seed or nursery again.
(5) Caulis et Folium Ammopiptanthi Mongolici callus can be used for DNA or RNA extraction, sends out for desirable genes resource
Pick.
(6) if callus can regenerate seedling, it is possible to achieve the molecular genetic manipulation of Caulis et Folium Ammopiptanthi Mongolici,
Acquisition reproductive capacity is strong, the Caulis et Folium Ammopiptanthi Mongolici of strong stress resistance, it is achieved the protection of Caulis et Folium Ammopiptanthi Mongolici kind matter, and for Radix Saposhnikoviae
Fix the sand, afforestation etc..
Accompanying drawing explanation
Fig. 1 is the experiment sample schematic diagram that Caulis et Folium Ammopiptanthi Mongolici sterilizing seed is seeded in 1/2MS culture medium；
Fig. 2 is that Caulis et Folium Ammopiptanthi Mongolici seed germinates the experiment sample schematic diagram of 5 days phenotypes in 1/2MS culture medium；
Fig. 3 is the experiment sample signal of band root hair short side root Caulis et Folium Ammopiptanthi Mongolici seedling (35 days) of sterile culture
Figure；
Fig. 4 is the experiment that band root hair side root induces callus (20 days) in CDB1 culture medium
Sample schematic diagram；
Fig. 5 is the experiment sample signal of Caulis et Folium Ammopiptanthi Mongolici callus subculture and expanding propagation in CDB6-2 culture medium
Figure (illumination cultivation).
Fig. 6 is that Caulis et Folium Ammopiptanthi Mongolici callus experiment sample of subculture and expanding propagation in CDB6-2 culture medium shows
It is intended to (light culture).
Detailed description of the invention
Hereinafter embodiments of the present invention are elaborated.It is emphasized that the description below is only
It is exemplary rather than in order to limit the scope of the present invention and application thereof.
In one embodiment, one utilizes Caulis et Folium Ammopiptanthi Mongolici children tender side root induction Caulis et Folium Ammopiptanthi Mongolici Callus formation
Method, comprise the following steps: a. utilizes a small amount of Caulis et Folium Ammopiptanthi Mongolici seed to carry out aseptic nursery, passes through 1/2MS
Culture medium constantly induces the side root of band root hair to be formed；The most then the hairy side root that clip children is tender is as outer planting
Body, in the dark induced synthesis callus in CDB1 culture medium, and under light or dark place is in CDB6-2
The callus that in culture medium, subculture and expanding propagation are induced.
In a preferred embodiment, step a includes: by aseptic nursery, containing 1/2MS solid
The culture dish of culture medium makes germination, then goes to the sterile culture flask equipped with 1/2MS culture medium
Middle induction seedling, after seedling grows more branch roots, is transplanted to the new nothing equipped with 1/2MS culture medium
In bacterium culture bottle, making part side root be exposed at media surface, induction side root is formed a large amount of with root hair
Short side root.In step b, using this band root hair short side root as the tender outer implant of children, lure in CDB1 culture medium
Lead callus and in CDB6-2 culture medium subculture and expanding propagation, thus obtain the most homogeneous Caulis et Folium Ammopiptanthi Mongolici
Callus, is used for carrying out the follow-up correlational study of Caulis et Folium Ammopiptanthi Mongolici.Preferably, in the dark with CDB1 (MS
+ 1.0mg/L 2,4-D+0.1mg/L 6-BA, pH5.8～7.5) culture medium callus induction
Formed, then under light or CBD6-2 (the MS+0.2mg/L IBA+0.1mg/L KT of dark place
+ 0.5mg/L GA3, pH5.8～7.5) culture medium carries out the subculture of callus and cultivates with expanding propagation.
In a preferred embodiment, the aseptic seedling raising process of Caulis et Folium Ammopiptanthi Mongolici specifically includes following steps:
(1) select the Caulis et Folium Ammopiptanthi Mongolici seed that Maturity is good, profile is intact, do not eaten by insect, install to
In sterilizing, airtight bottle or pipe；
(2) in bottle or pipe, first with 75% ethanol disinfection Caulis et Folium Ammopiptanthi Mongolici seed 10min；
(3) 75% ethanol is outwelled, then with 6% hypochlorite disinfectant Caulis et Folium Ammopiptanthi Mongolici seed 30min；
(4) 6% sodium hypochlorite is outwelled, with sterile water wash sterilizing seed 5～6 times；
(5) outwelling sterilized water, somewhat drain away the water, the most switchable seed is trained to containing solid 1/2MS
Support in the culture dish of base；
(6) cultivate 2～5 days under the conditions of 22 DEG C～28 DEG C, make germination.
In a preferred embodiment, Caulis et Folium Ammopiptanthi Mongolici hairy side root induction process specifically includes following steps:
(1), after Caulis et Folium Ammopiptanthi Mongolici germination, select the good seed of germinating energy and be transferred to equipped with solid 1/2MS
In the sterilizing culture bottle of culture medium, it is beneficial to the growth of chitting piece；
(2) after Caulis et Folium Ammopiptanthi Mongolici seedling grows a plurality of side root, second time switching can be carried out, growth is good for
Strong seedling replanting is in the new sterilizing culture bottle equipped with solid 1/2MS culture medium, and exposed portion
Side root is in media surface；
(3), after growing 20～40 days, the side root being exposed at media surface can grow many with root hair
Short side root, in this, as the outer implant material of callus induction.
In a preferred embodiment, Caulis et Folium Ammopiptanthi Mongolici During Callus Induction specifically includes following steps:
Aseptically, clip part hairy side root is placed in CDB1 (MS+1.0mg/L 2,4-D+
0.1mg/L 6-BA, pH5.8～7.5) on calli induction media, in the dark lure under the conditions of 28 DEG C
Lead cultivation 20～35 days.
In a preferred embodiment, Caulis et Folium Ammopiptanthi Mongolici callus subculture and expanding propagation process specifically include following step
Rapid:
The callus induced is transferred to again CDB6-2 (MS+0.2mg/L IBA+0.1mg/L
KT+0.5mg/L GA3, pH5.8～7.5) in callus subculture and expanding propagation culture medium, at 28 DEG C
Have under light or dark conditions and cultivate, every 10～15 days subcultures once.
Example
(1) select profile intact, the Caulis et Folium Ammopiptanthi Mongolici seed do not eaten by worm, install to 50mL sterilizing from
In heart pipe；
(2) on aseptic operating platform, it is initially charged 40mL 75% ethanol disinfection Caulis et Folium Ammopiptanthi Mongolici seed 10min,
Period vibrates frequently；
(3) 75% ethanol is outwelled, then with 40mL height Le Shi bleaching water (containing 6.25% sodium hypochlorite
Activity) sterilize Caulis et Folium Ammopiptanthi Mongolici seed 30min, and period vibrates frequently；
(4) high Le Shi bleaching water is outwelled, with sterile water wash sterilizing seed 5～6 times；
(5) outwell sterilized water, on aseptic operating platform, with the tweezers of sterilizing, take out from centrifuge tube
Seed direct inoculation in the culture dish (90 × 15mm) containing solid 1/2MS culture medium (such as Fig. 1
Shown in)；
Note: 1/2MS culture medium, 2.22g/L MS (containing Gamborg's vitamins) (Sigma)；
2g/100mL Sucrose(Caisson)；8g/L phytoblend (Caisson), uses 1M KOH
Adjust pH to 5.7；
Cultivate 3 days under the conditions of (6) 22 DEG C of illumination cultivation, make germination；
(7), after Caulis et Folium Ammopiptanthi Mongolici germination, select the neat seed (as shown in Figure 2) of germination and be transferred to
In sterilizing culture bottle equipped with solid 1/2MS culture medium, it is beneficial to the growth of chitting piece；
(8) after Caulis et Folium Ammopiptanthi Mongolici seedling grows a plurality of side root, second time switching can be carried out, growth is good for
Strong seedling replanting is in the new sterilizing culture bottle equipped with solid 1/2MS culture medium, and exposed portion
Side root is in media surface；
After (9) 35 days, be exposed at the side root of media surface can grow many short side roots with root hair (as
Shown in Fig. 3), in this, as the outer implant material of wound healing induction；
(10) on aseptic operating platform, clip part hairy side root is in CDB1 (MS+1.0mg/L
2,4-D+0.1mg/L 6-BA, pH5.8～7.5) on calli induction media, in the dark 28 DEG C
Under the conditions of inducing culture 20 days, the callus induced is as shown in Figure 4；
(11) callus induced is transferred to again CDB6-2 (MS+0.2mg/L IBA+0.1
Mg/L KT+0.5mg/L GA3, pH5.8～7.5) in callus subculture and expanding propagation culture medium,
Callus expanding propagation under optical condition (or dark place) is had at 28 DEG C, every 15 days subcultures once, expanding propagation
Callus such as Fig. 5 (illumination cultivation, 20 days, medium pH 5.8), Fig. 6 (light culture, 40
My god, left figure pH7.5, right figure pH5.8) shown in.
Above content is that combination is concrete/the most made for the present invention the most specifically
Bright, it is impossible to assert the present invention be embodied as be confined to these explanations.For technology belonging to the present invention
For the those of ordinary skill in field, without departing from the inventive concept of the premise, it can also be to this
The embodiment having described that a bit makes some replacements or modification, and these substitute or variant all should
It is considered as belonging to protection scope of the present invention.
Claims (6)
1. the method utilizing Caulis et Folium Ammopiptanthi Mongolici children tender side root induction Caulis et Folium Ammopiptanthi Mongolici Callus formation, it is special
Levy and be, comprise the following steps: a. utilizes Caulis et Folium Ammopiptanthi Mongolici seed to carry out aseptic nursery, is trained by 1/2MS
Foster base constantly induces the side root of band root hair to be formed；The most then the hairy side root that clip children is tender is as outer planting
Body, in the dark induced synthesis callus in CDB1 culture medium, and under light or dark place is in CDB6-2
The callus that in culture medium, subculture and expanding propagation are induced.
2. the method for claim 1, it is characterised in that step a includes: containing solid
Making germination in the culture dish of 1/2MS culture medium, then go to equipped with 1/2MS culture medium is aseptic
Culture bottle is induced seedling, after seedling grows more branch roots, is transplanted to new cultivating equipped with 1/2MS
In the sterile culture flask of base, make part side root be exposed at media surface, induction side root formed in a large number with
The short side root of root hair.
3. method as claimed in claim 1 or 2, it is characterised in that utilize the husky winter in step a
The process that blue or green seed carries out aseptic nursery includes:
(1) select the Caulis et Folium Ammopiptanthi Mongolici seed that Maturity is good, profile is intact, do not eaten by insect, install to
In sterilizing, airtight bottle or pipe；
(2) in bottle or pipe, first with 75% ethanol disinfection Caulis et Folium Ammopiptanthi Mongolici seed 10min；
(3) ethanol is removed, then with 6% hypochlorite disinfectant Caulis et Folium Ammopiptanthi Mongolici seed 30min；
(4) sodium hypochlorite is removed, with sterile water wash sterilizing seed 5～6 times；
(5) removing sterilized water, somewhat drain away the water, the most switchable seed is trained to containing solid 1/2MS
Support in the culture dish of base；
(6) cultivate 2～5 days under the conditions of 22 DEG C～28 DEG C, make germination.
4. the method as described in any one of claims 1 to 3, it is characterised in that logical in step a
Cross the process that 1/2MS culture medium constantly induces the side root of band root hair to be formed to include:
(1) select the good seed of germinating energy and be transferred to the sterilizing culture bottle equipped with solid 1/2MS culture medium
In, it is beneficial to the growth of chitting piece；
(2) after Caulis et Folium Ammopiptanthi Mongolici seedling grows a plurality of side root, second time switching is carried out, by robust growth
Seedling replanting is in the new sterilizing culture bottle equipped with solid 1/2MS culture medium, and side, exposed portion root
In media surface；
(3), after growing 20～40 days, the side root being exposed at media surface can grow many with root hair
Short side root, in this, as the outer implant material of callus induction.
5. the method as described in any one of Claims 1-4, it is characterised in that in step b
Dark place process of induced synthesis callus in CDB1 culture medium includes:
Aseptically, clip part hairy side root is placed in CDB1 culture medium, in the dark 28 DEG C
Under the conditions of inducing culture 20～35 days, wherein the composition of CDB1 culture medium is: MS+1.0mg/L
2,4-D+0.1mg/L 6-BA, pH5.8～7.5.
6. the method as described in any one of Claims 1-4, it is characterised in that in step b
The process bag of under light or the dark place callus that subculture and expanding propagation are induced in CDB6-2 culture medium
Include:
The callus induced is transferred in CDB6-2 culture medium, has light or dark place bar at 28 DEG C
Cultivating under part, every 10～15 days subcultures once, wherein the composition of CDB6-2 culture medium is: MS+
0.2mg/L IBA+0.1mg/L KT+0.5mg/L GA3, pH5.8～7.5.
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| CN201510872381.7A CN106258952B (en) | 2015-12-02 | 2015-12-02 | The method formed using the tender lateral root induction Ammopiptanthus mongolicus callus of Ammopiptanthus mongolicus children |
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| CN201510872381.7A CN106258952B (en) | 2015-12-02 | 2015-12-02 | The method formed using the tender lateral root induction Ammopiptanthus mongolicus callus of Ammopiptanthus mongolicus children |
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Cited By (1)
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| CN111011215A (en) * | 2019-12-30 | 2020-04-17 | 内蒙古蒙草生态环境（集团）股份有限公司 | Culture medium for ammopiptanthus mongolicus tissue culture |
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| CN103563745A (en) * | 2013-10-12 | 2014-02-12 | 杭州市园林绿化股份有限公司 | Tissue culture method of ilex verticillata |
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| CN103563745A (en) * | 2013-10-12 | 2014-02-12 | 杭州市园林绿化股份有限公司 | Tissue culture method of ilex verticillata |
| CN103583173A (en) * | 2013-10-12 | 2014-02-19 | 杭州市园林绿化股份有限公司 | Cuttage propagation method for North American holly branches |
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| CN111011215A (en) * | 2019-12-30 | 2020-04-17 | 内蒙古蒙草生态环境（集团）股份有限公司 | Culture medium for ammopiptanthus mongolicus tissue culture |
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