CN106258952B - The method formed using the tender lateral root induction Ammopiptanthus mongolicus callus of Ammopiptanthus mongolicus children - Google Patents
The method formed using the tender lateral root induction Ammopiptanthus mongolicus callus of Ammopiptanthus mongolicus children Download PDFInfo
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- CN106258952B CN106258952B CN201510872381.7A CN201510872381A CN106258952B CN 106258952 B CN106258952 B CN 106258952B CN 201510872381 A CN201510872381 A CN 201510872381A CN 106258952 B CN106258952 B CN 106258952B
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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 methods formed using the tender lateral root induction Ammopiptanthus mongolicus callus of Ammopiptanthus mongolicus children, include the following steps：A. sterile nursery is carried out using Ammopiptanthus mongolicus seed, is formed by 1/2MS culture mediums constantly lateral root of the induction with root hair；B. then the tender hairy lateral root of clip is as explant, in the dark in induced synthesis callus on CDB1 culture mediums, and under light or dark place is in subculture on CDB6-2 culture mediums and numerous the induced callus of expansion.Method using the present invention, can efficiently, low cost and steadily obtain Ammopiptanthus mongolicus callus.
Description
Technical field
The present invention relates to a kind of methods formed using the tender lateral root induction Ammopiptanthus mongolicus callus of Ammopiptanthus mongolicus children.
Background technology
Ammopiptanthus Genus can the various poles of suitable for desert as a kind of super non-irrigated raw desert evergreen shrubs in pulse family Ammopiptanthus Genus
End ring border (arid, high temperature, low temperature and wind erosion etc.), while being the Tertiary Period ancient deleted species again, belong to endangered species, so husky
Chinese ilex is to carry preferable degeneration-resistant desirable genes resource plant, and have gene and phytobiology character inheritance Study on Evolution simultaneously
Value.Forefathers have only carried out largely about researchs such as its conservation of nature, community distribution, biological characteristics and Analysis of Genetic Background
Work, until recent, the research report of part is gene cloning and the work of transcript profile sequencing analysis about Ammopiptanthus mongolicus, is ground
It is also the valuable adversity gene resource and its degeneration-resistant mechanism for being to excavate that Ammopiptanthus mongolicus is contained to study carefully target, to solve current agricultural life
Production and weather variation issue.
But since Ammopiptanthus mongolicus growth cycle is long, seed-setting rate and germination percentage are low, while being subject to insect pest, so Ammopiptanthus mongolicus
Fertility is weak；Its mature tissue is compared with aging, and the growth of seedling later stage after germination is also easy to aging and lignifying is unfavorable for DNA
With RNA extractions etc., and scientific research personnel can not plant breeding Ammopiptanthus mongolicus indoors, it is necessary to pick a large amount of new seed developments section every year
It grinds, which greatly limits application of the molecular biotechnology in terms of the gene cloning of Ammopiptanthus mongolicus and functional study.Mesh
The preceding patent report induced not yet about Ammopiptanthus mongolicus tissue cultures and callus, only part Chinese periodical have a small amount of related husky winter
The report of green callus induction, but induced efficiency is relatively low and unstable (3%~80%), and method repeatability is poor.
Invention content
It is a primary object of the present invention to overcome the deficiencies of the prior art and provide a kind of tender lateral root of utilization Ammopiptanthus mongolicus children to induce
The method that Ammopiptanthus mongolicus callus is formed, easy, efficient, low cost and steadily acquisition Ammopiptanthus mongolicus callus.
To achieve the above object, the present invention uses following technical scheme：
A method of it is formed, is included the following steps using the tender lateral root induction Ammopiptanthus mongolicus callus of Ammopiptanthus mongolicus children：A. sharp
Sterile nursery is carried out with Ammopiptanthus mongolicus seed, is formed by 1/2MS culture mediums constantly lateral root of the induction with root hair；B. then clip is young
Tender hairy lateral root is as explant, in the dark in induced synthesis callus on CDB1 culture mediums, and under light or dark place in
Subculture and numerous the induced callus of expansion on CDB6-2 culture mediums.
Further：
Make germination in the culture dish containing 1/2MS solid mediums, then goes to the nothing equipped with 1/2MS culture mediums
Seedling is induced in bacterium culture bottle, after seedling grows more branch roots, is transplanted to the new sterile culture flask equipped with 1/2MS culture mediums
In, so that part lateral root is exposed at media surface, induction lateral root forms a large amount of short side roots for carrying root hair.
The process for carrying out sterile nursery in step a using Ammopiptanthus mongolicus seed includes：
(1) select the Ammopiptanthus mongolicus seed that maturity is good, shape is intact, was not eaten by insect, be attached to sterilized, closed bottle
Or in pipe；
(2) in bottle or pipe, first with 75% ethanol disinfection Ammopiptanthus mongolicus seed 10min；
(3) ethyl alcohol is removed, then with 6% hypochlorite disinfectant's Ammopiptanthus mongolicus seed 30min；
(4) sodium hypochlorite is removed, with sterile water wash sterilizing seed 5~6 times；
(5) sterile water is removed, is slightly drained away the water, you can switching seed to the culture dish containing solid 1/2MS culture mediums
In；
(6) it is cultivated 2~5 days under the conditions of 22 DEG C~28 DEG C, makes germination.
Include by the 1/2MS culture mediums process that constantly lateral root of the induction with root hair is formed in step a：
(1) selecting germinating energy, good seed is transferred in the sterilizing culture bottle equipped with solid 1/2MS culture mediums, in favor of hair
The growth of bud seed；
(2) after Ammopiptanthus mongolicus seedling grows a plurality of lateral root, carry out second and transfer, by the seedling replanting of robust growth to newly
The sterilizing culture bottle equipped with solid 1/2MS culture mediums in, and exposed portion lateral root is in media surface；
(3) after growing 20~40 days, many short side roots for carrying root hair can be grown by being exposed at the lateral root of media surface, with this
Explant material as evoked callus.
Include in the process of induced synthesis callus on CDB1 culture mediums in the dark in step b：
Aseptically, clip part hairy lateral root is placed on CDB1 culture mediums, induces training under the conditions of 28 DEG C in the dark
It supports 20~35 days, wherein CDB1 medium components are：MS+1.0mg/L 2,4-D+0.1mg/L 6-BA, pH5.8~7.5.
In step b under light or the process of numerous induced callus in subculture on CDB6-2 culture mediums and is expanded in dark place
Including：
The callus induced is transferred on CDB6-2 culture mediums, has at 28 DEG C and is cultivated under striation part or dark place, often
Primary every 10~15 days subcultures, wherein CDB6-2 medium components are：MS+0.2mg/L IBA+0.1mg/L KT+0.5mg/L
GA3, pH5.8~7.5.
Beneficial effects of the present invention：
Ammopiptanthus mongolicus callus is formed using the present invention, Ammopiptanthus mongolicus DNA and RNA extraction can be carried out, carry out genome survey
Sequence or gene cloning and functional study equimolecular biological study, without additional seed collecting nursery again, thus can reduce and grind
Study carefully cost, improves Efficiency, while protecting the population of Proterozoic country endangered plants Ammopiptanthus mongolicus.
The advantage of the present invention embodies in the following areas：
(1) it after the sterile nursery of Ammopiptanthus mongolicus grows more branch roots, is transplanted again by aseptic condition, and part root system is stayed to be exposed at 1/
2MS sterile culture primary surfaces can induce the short side root for growing many band root hairs in its lateral root again, the tender hairy lateral root of clip
It can be used as the explant material of Ammopiptanthus mongolicus callus tissue culture.The method of drawing material does not interfere with the normal of the single plant in aseptic bottle
Growth, and tender explant material can be obtained for a long time, it is used for follow-up callus induction.
(2) tender hairy lateral root is easy to callus induction and forms (inductivity >=80%), callus induced stable, and is not necessarily to
It collects seed again nursery.
(3) it present invention only requires CDB1 culture medium callus inductions are used in the dark, returns again under light or dark place is trained with CBD6-2
Base is supported by callus subculture and the numerous culture of expansion, step is simple.
(4) a large amount of sterile, uniform, stable Ammopiptanthus mongolicus callus are can get, (such as Ammopiptanthus mongolicus molecular biology research
Gene cloning and functional study) it is used, no longer need to field seed collecting or nursery.
(5) Ammopiptanthus mongolicus callus can be used for DNA or RNA extractions, be used for desirable genes Resource exploitation.
(6) if callus can regenerate seedling, may be implemented the molecular genetic manipulation of Ammopiptanthus mongolicus, obtain reproductive capacity it is strong,
The strong Ammopiptanthus mongolicus of resistance, realizes the protection of Ammopiptanthus mongolicus germplasm, and for checking winds and fixing drifting sand, afforestation etc..
Description of the drawings
Fig. 1 is the experiment sample schematic diagram that Ammopiptanthus mongolicus sterilizing seed is seeded in 1/2MS culture mediums；
Fig. 2 is that Ammopiptanthus mongolicus seed germinates the experiment sample schematic diagrames of 5 days phenotypes on 1/2MS culture mediums；
Fig. 3 is the experiment sample schematic diagram of the band root hair short side root Ammopiptanthus mongolicus seedling (35 days) of sterile culture；
Fig. 4 is the experiment sample schematic diagram for inducing callus (20 days) on CDB1 culture mediums with root hair lateral root；
Fig. 5 is that Ammopiptanthus mongolicus callus on CDB6-2 culture mediums (train subculture with numerous experiment sample schematic diagram is expanded by illumination
It supports).
Fig. 6 is that Ammopiptanthus mongolicus callus subculture on CDB6-2 culture mediums (is secretly trained with numerous experiment sample schematic diagram is expanded
It supports).
Specific implementation mode
It elaborates below to embodiments of the present invention.It is emphasized that following the description is only exemplary,
The range being not intended to be limiting of the invention and its application.
In one embodiment, a method of formed using the tender lateral root induction Ammopiptanthus mongolicus callus of Ammopiptanthus mongolicus children, packet
Include following steps：A. sterile nursery is carried out using a small amount of Ammopiptanthus mongolicus seed, passes through 1/2MS culture mediums constantly side of the induction with root hair
Root is formed；B. then the tender hairy lateral root of clip is as explant, in the dark in induced synthesis callus group on CDB1 culture mediums
It knits, and under light or numerous induced callus in subculture on CDB6-2 culture mediums and is expanded in dark place.
In a preferred embodiment, step a includes：By sterile nursery, in the culture dish containing 1/2MS solid mediums
In make germination, then go in the sterile culture flask equipped with 1/2MS culture mediums and induce seedling, wait for that seedling grows more branch roots
Afterwards, it is transplanted in the new sterile culture flask equipped with 1/2MS culture mediums, part lateral root is made to be exposed at media surface, induce lateral root
Form the short side root for largely carrying root hair.In step b, using this band root hair short side root as the tender explant of children, in CDB1 culture mediums
Evoked callus and subculture and expand numerous on CDB6-2 culture mediums, to obtain a large amount of uniform Ammopiptanthus mongolicus callus, supplies
Carry out the follow-up correlative study of Ammopiptanthus mongolicus to be used.Preferably, in the dark use CDB1 (MS+1.0mg/L 2,4-D+0.1mg/L 6-BA,
PH5.8~7.5) culture medium evoked callus formed, then under light or CBD6-2 (the MS+0.2mg/L IBA+ of dark place
0.1mg/L KT+0.5mg/L GA3, pH5.8~7.5) culture medium carries out the subculture of callus and expands numerous culture.
In a preferred embodiment, the sterile seedling raising process of Ammopiptanthus mongolicus specifically includes following steps：
(1) select the Ammopiptanthus mongolicus seed that maturity is good, shape is intact, was not eaten by insect, be attached to sterilized, closed bottle
Or in pipe；
(2) in bottle or pipe, first with 75% ethanol disinfection Ammopiptanthus mongolicus seed 10min；
(3) 75% ethyl alcohol is outwelled, then with 6% hypochlorite disinfectant's Ammopiptanthus mongolicus seed 30min；
(4) 6% sodium hypochlorite is outwelled, with sterile water wash sterilizing seed 5~6 times；
(5) sterile water is outwelled, is slightly drained away the water, you can switching seed to the culture dish containing solid 1/2MS culture mediums
In；
(6) it is cultivated 2~5 days under the conditions of 22 DEG C~28 DEG C, makes germination.
In a preferred embodiment, Ammopiptanthus mongolicus hairy lateral root Induction Process specifically includes following steps：
(1) after Ammopiptanthus mongolicus germination, selecting germinating energy, good seed is transferred to the sterilizing equipped with solid 1/2MS culture mediums
In culture bottle, in favor of the growth of chitting piece；
(2) after Ammopiptanthus mongolicus seedling grows a plurality of lateral root, second of switching can be carried out, by the seedling replanting of robust growth
Into the new sterilizing culture bottle equipped with solid 1/2MS culture mediums, and exposed portion lateral root is in media surface；
(3) after growing 20~40 days, many short side roots for carrying root hair can be grown by being exposed at the lateral root of media surface, with this
Explant material as callus induction.
In a preferred embodiment, Ammopiptanthus mongolicus During Callus Induction specifically includes following steps：
Aseptically, clip part hairy lateral root be placed in CDB1 (MS+1.0mg/L 2,4-D+0.1mg/L 6-BA,
PH5.8~7.5) on calli induction media, Fiber differentiation 20~35 days under the conditions of 28 DEG C in the dark.
In a preferred embodiment, Ammopiptanthus mongolicus callus subculture specifically includes following steps with numerous process is expanded：
The callus induced is transferred to CDB6-2 (MS+0.2mg/L IBA+0.1mg/L KT+0.5mg/L again
GA3, pH5.8~7.5) callus subculture with expand on breeding culture medium, have at 28 DEG C and cultivated under light or dark conditions, every 10~
Subculture is primary within 15 days.
Example
(1) the Ammopiptanthus mongolicus seed that shape is intact, is not eaten by worm is selected, is attached to 50mL in sterile centrifugation tube；
(2) on aseptic operating platform, 75% ethanol disinfection Ammopiptanthus mongolicus seed 10min of 40mL is first added, during which shake frequently
It swings；
(3) 75% ethyl alcohol is outwelled, 40mL high Le Shi bleaching waters (containing 6.25% sodium hypochlorite activity) the disinfection husky winter is then used
Green seed 30min, during which vibrates frequently；
(4) high Le Shi bleaching waters are outwelled, with sterile water wash sterilizing seed 5~6 times；
(5) sterile water is outwelled, on aseptic operating platform, with the tweezers of sterilizing, taking-up seed is directly inoculated with from centrifuge tube
Into the culture dish (90 × 15mm) containing solid 1/2MS culture mediums (as shown in Figure 1)；
Note：1/2MS culture mediums, 2.22g/L MS (vitamins containing Gamborg's) (Sigma)；2g/100mL
Sucrose(Caisson)；8g/L phytoblend (Caisson), with 1M KOH tune pH to 5.7；
It is cultivated 3 days under the conditions of (6) 22 DEG C of illumination cultivations, makes germination；
(7) it after Ammopiptanthus mongolicus germination, selects the neat seed (as shown in Figure 2) that germinates and is transferred to equipped with solid 1/2MS
In the sterilizing culture bottle of culture medium, in favor of the growth of chitting piece；
(8) after Ammopiptanthus mongolicus seedling grows a plurality of lateral root, second of switching can be carried out, by the seedling replanting of robust growth
Into the new sterilizing culture bottle equipped with solid 1/2MS culture mediums, and exposed portion lateral root is in media surface；
After (9) 35 days, many short side roots (as shown in Figure 3) for carrying root hair can be grown by being exposed at the lateral root of media surface,
In this, as the explant material of callus induction；
(10) on aseptic operating platform, clip part hairy lateral root is in CDB1 (MS+1.0mg/L2,4-D+0.1mg/L 6-
BA, pH5.8~7.5) on calli induction media, Fiber differentiation 20 days, the callus induced under the conditions of 28 DEG C in the dark
As shown in Figure 4；
(11) callus induced is transferred to CDB6-2 (MS+0.2mg/L IBA+0.1mg/L KT+0.5mg/ again
L GA3, pH5.8~7.5) callus subculture with expand breeding culture medium on, have callus under striation part (or dark place) at 28 DEG C
Expand it is numerous, it is primary every 15 days subcultures, expand numerous callus such as Fig. 5 (illumination cultivation, 20 days, medium pH 5.8), Fig. 6 (dark trainings
Support, 40 days, left figure pH7.5, right figure pH5.8) shown in.
The above content is specific/preferred embodiment further description made for the present invention is combined, cannot recognize
The specific implementation of the fixed present invention is confined to these explanations.For those of ordinary skill in the art to which the present invention belongs,
Without departing from the inventive concept of the premise, some replacements or modification can also be made to the embodiment that these have been described,
And these are substituted or variant all shall be regarded as belonging to protection scope of the present invention.
Claims (6)
1. a kind of method formed using the tender lateral root induction Ammopiptanthus mongolicus callus of Ammopiptanthus mongolicus children, which is characterized in that including following
Step：A. sterile nursery is carried out using Ammopiptanthus mongolicus seed, is formed by 1/2MS culture mediums constantly lateral root of the induction with root hair,
It is middle to be transplanted again by aseptic condition after the sterile nursery of Ammopiptanthus mongolicus grows lateral root, and part lateral root is stayed to be exposed at the sterile trainings of 1/2MS
Primary surface is supported, induces and grows the short side root with root hair again in lateral root；B. then the tender hairy lateral root of clip as explant,
Dark place is in induced synthesis callus on CDB1 culture mediums, and under light or numerous institute in subculture on CDB6-2 culture mediums and is expanded in dark place
The callus induced；The ingredient of wherein CDB1 culture mediums is：MS+1.0mg/L 2,4-D+0.1mg/L 6-BA, pH5.8~
7.5；The ingredient of CDB6-2 culture mediums is：MS+0.2mg/L IBA+0.1mg/L KT+0.5mg/L GA3, pH5.8~7.5.
2. the method as described in claim 1, which is characterized in that step a includes：In the culture containing solid 1/2MS culture mediums
Make germination in ware, then go in the sterile culture flask equipped with 1/2MS culture mediums and induce seedling, waits for that seedling grows many sides
It after root, is transplanted in the new sterile culture flask equipped with 1/2MS culture mediums, part lateral root is made to be exposed at media surface, induce side
Root forms a large amount of short side roots for carrying root hair.
3. method as claimed in claim 1 or 2, which is characterized in that carry out sterile nursery using Ammopiptanthus mongolicus seed in step a
Process includes：
(1) select the Ammopiptanthus mongolicus seed that maturity is good, shape is intact, was not eaten by insect, be attached to sterilized, closed bottle or pipe
In；
(2) in bottle or pipe, first with 75% ethanol disinfection Ammopiptanthus mongolicus seed 10min；
(3) ethyl alcohol is removed, then with 6% hypochlorite disinfectant's Ammopiptanthus mongolicus seed 30min；
(4) sodium hypochlorite is removed, with sterile water wash sterilizing seed 5~6 times；
(5) sterile water is removed, is slightly drained away the water, you can in switching seed to the culture dish containing solid 1/2MS culture mediums；
(6) it is cultivated 2~5 days under the conditions of 22 DEG C~28 DEG C, makes germination.
4. method as claimed in claim 1 or 2, which is characterized in that constantly induce band root in step a by 1/2MS culture mediums
Hair lateral root formed process include：
(1) selecting germinating energy, good seed is transferred in the sterilizing culture bottle equipped with solid 1/2MS culture mediums, in favor of germination kind
The growth of son；
(2) after Ammopiptanthus mongolicus seedling grows a plurality of lateral root, carry out second and transfer, by the seedling replanting of robust growth to new dress
In the sterilizing culture bottle for having solid 1/2MS culture mediums, and exposed portion lateral root is in media surface；
(3) after growing 20~40 days, many short side roots for carrying root hair can be grown by being exposed at the lateral root of media surface, in this, as
The explant material of evoked callus.
5. method as claimed in claim 1 or 2, which is characterized in that in the dark in induced synthesis on CDB1 culture mediums in step b
The process of callus includes：
Aseptically, clip part hairy lateral root is placed on CDB1 culture mediums, in the dark Fiber differentiation 20 under the conditions of 28 DEG C
~35 days.
6. method as claimed in claim 1 or 2, which is characterized in that in step b under light or dark place is on CDB6-2 culture mediums
Subculture and the process for expanding numerous induced callus include：
The callus induced is transferred on CDB6-2 culture mediums, has at 28 DEG C and is cultivated under light or dark conditions, every 10
Subculture is primary within~15 days.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN104255457A (en) * | 2014-09-12 | 2015-01-07 | 南京通泽农业科技有限公司 | Rapid propagation method for tissue culture of ilex nitidissima |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN104255457A (en) * | 2014-09-12 | 2015-01-07 | 南京通泽农业科技有限公司 | Rapid propagation method for tissue culture of ilex nitidissima |
Non-Patent Citations (1)
| Title |
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| 珍稀濒危植物新疆沙冬青胚根愈伤组织诱导及植株再生;焦培培;《北方园艺》;20121231(第20期);第101-104页，尤其是摘要、第1.1-1.2.2节及表1。 * |
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