CN106106188B - A kind of induction of wild barley mature embryo callus and Regeneration System method - Google Patents
A kind of induction of wild barley mature embryo callus and Regeneration System method Download PDFInfo
- Publication number
- CN106106188B CN106106188B CN201610731771.7A CN201610731771A CN106106188B CN 106106188 B CN106106188 B CN 106106188B CN 201610731771 A CN201610731771 A CN 201610731771A CN 106106188 B CN106106188 B CN 106106188B
- Authority
- CN
- China
- Prior art keywords
- culture
- differentiation
- wild barley
- root
- mature embryo
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
- A01H4/008—Methods for regeneration to complete plants
-
- 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
- A01H4/001—Culture apparatus for tissue culture
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
A kind of wild barley mature embryo callus induction of present invention offer and Regeneration System method, steps are as follows：1）Selection wild barley seed simultaneously sterilizes；2）The screening of inducing culture, differentiation and proliferation culture medium and root media；3）The processing and induction differentiation of mature embryo：Take step（1）The wild barley seed of middle disinfection, it is placed in inducing culture and cultivates, subculture is transferred in differentiation and proliferated culture medium afterwards several times to be continued to cultivate, and subculture is transferred in root media is cultivated several times afterwards, when breaking up 4 6cm of root long of seedling, a bottle hardening is closed, tap water, corkage hardening 3d are injected after 2d, then root is cleaned, sterilizing, with matrix culture, obtains complete regeneration plant.The present invention is the tissue culture method of wild barley callus induction, and easy to operate, inexpensive is not limited by materials material space-time；The wild barley Mature embryo culture plant regeneration method system of foundation provides reliable technical support to explore its cell engineering breeding and genetic improvement research.
Description
Technical field
The present invention relates to the plant regeneration fields belonged in agricultural cultivation by tissue culture technique, and in particular to Yi Zhongye
Barley mature embryo callus induces and Regeneration System method.
Background technology
Wild barley is also known as short covered barley, is grass family Hordeum herbaceos perennial, be a kind of short rhizome of tool for many years
Raw herbage, vegetative propagation power is stronger, often with strain clump formal distribution in haophitic vegetation, alkalized meadow of Songnen Plain can
Form the single dominantcommunity of large area.Wild barley has herbaceous stem good, and full of nutrition, protein and fat content are higher in hay, fits
Mouth property is good, anti-adverse environment, and the good characteristics such as resistance to trample, are the preferred breedings during artificial cultivation meadow and grassland improvement.
With the degeneration on grassland and the increasingly reduction of cultivated area, high-quality, high yield, mostly anti-herbage new varieties are constantly cultivated
As the hot issue of domestic and international Forage Research worker concern.For breeding, in addition to traditional breeding technique, in conjunction with gene
Engineering technology can accelerate breeding process to cultivate new varieties, more effectively select target variety.And wild barley tissue cultures are
The basis that improvement of genes is carried out using technique for gene engineering is only established stable, high frequency regenerating system, could built preferably
Genetic conversion system, improve transformation efficiency, obtain transfer-gen plant, and then obtain improvement new varieties.
Currently, domestic wild barley vitro Regeneration System is studied and is still not perfect, Zhu is into equal using the mature embryo of wild barley as explant
Body evoked callus, the Salt-tolerant Variantss screened on the MS culture mediums containing NaCl are with certain salt tolerance and relatively steady
It is fixed, and differentiated regeneration plant.
Invention content
In order to solve the problems in the existing technology, the present invention provides a kind of inductions of wild barley mature embryo callus
And Regeneration System method.The invention discloses one kind by wild barley mature embryo be explant, structure callus induction,
Plant regeneration obtains regenerating system, and reliable technical support is provided to explore its cell engineering breeding and genetic improvement research.
A kind of wild barley mature embryo callus induction of present invention offer and Regeneration System method, steps are as follows：
（1）Selection wild barley seed simultaneously sterilizes；
（2）The screening of inducing culture, differentiation and proliferation culture medium and root media：
Inducing culture：Basal medium+0.1-0.5 mgL-1 6-BA +1.0-2.0 mg·L-1 2,4-D；
Differentiation and proliferation culture medium：Basal medium+0.5-1.5mg/L 6-BA+0.2-0.4mg/L NAA,
Root media：1/2 basal medium；
The basal medium is：MS culture mediums+sucrose 30gL-1+ agar 10gL-1；
（3）The processing and induction differentiation of mature embryo：Take step（1）The wild barley seed of middle disinfection, is placed in inducing culture
Middle culture, subculture is transferred in differentiation and proliferated culture medium afterwards several times to be continued to cultivate, subculture be transferred to afterwards several times in root media into
Bottle seedling is removed culturing room, a bottle hardening is closed under natural light, is injected originally after 2d by row culture when breaking up the root long 4-6cm of seedling
Water opens hardening 3d under natural light, then cleans root, and sterilizing with matrix culture, obtains complete regeneration plant.
Preferably, step（2）In,
The inducing culture is：+ 0.3 mgL of basal medium-1 6-BA +1.5 mg·L-1 2,4-D；
The differentiation and proliferation culture medium is：Basal medium+0.5mg/L6-BA+0.2mg/L NAA；
The root media is：1/2 basal medium；
The basal medium is：MS culture mediums+sucrose 30gL-1+ agar 10gL-1。
Preferably, step（3）Described in the condition of Fiber differentiation be：The dark culturing under the conditions of 25 ± 2 DEG C of constant temperature
30d。
Preferably, step（3）Described in the condition of differentiation and proliferation culture be：Illumination cultivation, intensity of illumination 1500-
2000Lux, 16 hd of daily illumination-1, cultivation temperature is 25 DEG C.
Preferably, step（3）Described in the condition of culture of culture of rootage be：In 25 ± 2 DEG C, intensity of illumination 2000-
3000lx, light application time 16hd-1Under the conditions of cultivate.
Beneficial effects of the present invention are：
（1）The invention discloses a kind of wild barley mature embryo callus Fiber differentiation and the construction method of regenerating system,
Using the wild barley mature embryo for acquiring Linze as explant, pass through induction, differentiation, the life of different growth regulator confrontation callus
Root and etc. influence, carry out the screening of callus inducing medium, obtaining the higher culture medium of healing rate is：Basis culture
Base+0.3mg/L6-BA+1.5mg/L2,4-D, inductivity 50%；Differentiation and plant regeneration culture medium be basic culture medium+
0.5mg/L6-BA+0.2mg/L NAA, differentiation rate 80%, seedling of taking root culture medium are：1/2 basal medium, rooting rate reach
100%；Wherein, basal medium is that MS culture mediums add sucrose 30gL-1With agar 10gL-1.It is wild to provide a kind of efficiently effect
Barley mature embryo method for tissue culture.Compared with existing wild barley regenerating system is reported, shoot regeneration frequency of the invention is notable
It improves.
（2）It is explant that the present invention, which selects wild barley mature embryo, carries out regeneration culture, not by materials material space-time limit
System.The present invention is the tissue culture method of wild barley callus induction, easy to operate, inexpensive；The wild barley mature embryo of foundation
Plant regeneration method system is cultivated, reliable technical support is provided to explore its cell engineering breeding and genetic improvement research.
Description of the drawings
Attached drawing is used to provide further understanding of the present invention, and a part for constitution instruction, the reality with the present invention
It applies example to be used to explain the present invention together, not be construed as limiting the invention.In the accompanying drawings：
Fig. 1 is mature embryo evoked callus；
Fig. 2 is callus Differentiation From Calli；
Fig. 3 is the seedling to take root；
Fig. 4 is the regeneration plant after transplanting.
Specific implementation mode
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified city
It sells.
The method of the present invention is as follows：
（1）Vegetable material chooses and processing：Select color and luster, wild barley mature seed of uniform size, remove lemma after
It is rinsed under flowing water 30 minutes, then with 75% ethanol postincubation 5min, aseptic water washing 2-3 times, with 10% hypochlorite disinfectant
Seed is placed in aseptic filter paper by 10min, aseptic water washing 3-5 times, until seed moisture content is for use after blotting；The wild barley
Seed refers to：It is collected in ecosystem in Linze, Gansu, China wild barley wild seed.
（2）Induction and differentiation and proliferation Screening of Media：Basal medium is that MS culture mediums add 30 gL of sucrose-1And agar
10 g·L-1；
Inducing culture and differentiation and proliferation culture medium are that basic culture medium adds different disposal hormone.
Inducing culture is the addition 6-BA and 2,4-D on the basis of basal medium.6-BA a concentration of 0.1 is set
mg·L-1、0.3 mg·L-1、0.5 mg·L-1；A concentration of 1. 0 mgL of 2,4-D-1、1.5 mg·L-1、2.0 mg·L-1
（Table 1）；
Differentiation and proliferated culture medium are the addition 6-BA and NAA on the basis of basal medium.6-BA a concentration of 0.5 is set
mg·L-1、1.0 mg·L-1、1.5 mg·L-1；A concentration of 0.2 mgL of NAA-1、0.4 mg·L-1（Table 2）；
Root media is 1/2 basal medium.
The above medium pH is adjusted to 5.8~6.0, the high pressure moist heat sterilization 20min at 121 DEG C.
（3）The processing and induction differentiation of mature embryo：Selection is optimized to the condition of culture of each section in this step, with
Best culture effect can be obtained when lower condition of culture.Specific cultural method is as follows：Take step（1）In the kind disinfected
Son is placed in the inducing culture containing various concentration, the dark culturing 30d under the conditions of constant incubator (25 ± 2) DEG C, during which
It is unexpected that microbiological contamination is repeated to remove, counts healing rate and upgrowth situation.And callus is transferred to optimal inducing culture and is carried out
Shoot proliferation, subculture cycle 15d, subculture is transferred to the differentiation containing various concentration afterwards three times and proliferated culture medium carries out illumination training
It supports, intensity of illumination 1500-2000Lux, 16 hd of daily illumination-1, cultivation temperature is 25 DEG C, and subculture is primary every 2 weeks, subculture 3
It is secondary, phenylacetic acid and emergence rate are counted after 30d；
（4）When differentiation seedling is grown to 2-3cm, the differentiation seedling of the optimal differentiation of application and proliferated culture medium culture is transferred to and is taken root
Culture medium is 2000~3000lx, 16 hd of light application time in (25 ± 2) DEG C, intensity of illumination-1Under the conditions of cultivate.It waits breaking up
Bottle seedling is removed culturing room, a bottle hardening is closed under natural light, tap water is injected after 2d, is opened under natural light by root long 5cm of seedling or so
Rooting rate is counted after hardening 3d.After cleaning root, matrix after high pressure steam sterilization is moved into（Perlite：Vermiculite：Nutrition Soil（It is purchased from
Hangzhou auspicious wave Gardening Materials portion）=2:3:1）Culture, obtains complete regeneration plant.
Table 1 various concentration 6-BA and 2,4-D combine the influence formed to wild barley callus
As shown in Table 1, when adding 0.1mg/L6-BA+1.5mg/L2,4-D in culture medium, inductivity is cured up to 75%
Injured tissue quality loosely, is unfavorable for the formation of the differentiated tissue in later stage；Addition 0.3mg/L6-BA+1.5mg/L2,4-D are lured
Conductance is 49.72%, and callus is faint yellow, and quality is fine and close, and growth rate is fast.In general, the inductivity of callus exists
Between a concentration of 0.1-0.3mg/L of 6-BA, as the increase of 2,4-D concentration is in first to increase to decline afterwards, but otherness is not shown
It writes.Therefore, best inducing culture is：+ 0.3 mgL of basal medium-1 6-BA +1.5 mg·L-1 2,4-D。
Table 2 various concentration 6-BA and NAA combine the influence to wild barley callus differentiation capability
As shown in Table 2, hormon and its concentration combination are added in the medium, there are notable differences for differentiation rate.Work as NAA
When concentration is identical, with the raising of 6-BA concentration, in downward trend after first increasing, hormone is the differentiation rate of callus
1.0mg/L6-BA+0.4mg/L NAA and 1.5mg/L6-BA+0.2mg/L NAA combinations, the differentiation rate of callus is most
Height respectively reaches 93.33% and 86.66%, but it is not fine to break up seedling late growth situation, and the death rate is higher.When hormone is dense
When degree is 0.5mg/L6-BA+0.2mg/L NAA, the differentiation rate of callus is 80%, and differentiation seedling later stage survival rate is high, growth
It is more vigorous.Therefore, the optimal medium of wild barley callus differentiation is basis culture medium+0.5mg/L6-BA+0.2mg/L
NAA。
Embodiment 1
The method of the present invention is as follows：
（1）Vegetable material chooses and processing：Select color and luster, wild barley mature seed of uniform size, remove lemma after
It is rinsed under flowing water 30 minutes, then with 75% ethanol postincubation 5min, aseptic water washing 2-3 times, with 10% hypochlorite disinfectant
Seed is placed in aseptic filter paper by 10min, aseptic water washing 3-5 times, until seed moisture content is for use after blotting；The wild barley
Seed refers to：It is collected in the wild wild barley seed of ecosystem in Linze, Gansu, China.
（2）The formula of inducing culture, differentiation and proliferation culture medium and root media is as follows：
Basal medium is that MS culture mediums add sucrose 30g × L-1With 10 g of agar × L-1；
Callus inducing medium is：+ 0.3 mgL of basal medium-1 6-BA +1.5 mg·L-1 2,4-D；
Differentiation and proliferation culture medium is：Basal medium+0.5mg/L6-BA+0.2mg/L NAA；
Root media is：1/2 basal medium.
The above medium pH is adjusted to 5.8~6.0, the high pressure moist heat sterilization 20min at 121 DEG C；
（3）The processing and induction differentiation of mature embryo：Take step（1）In the seed disinfected, be placed in inducing culture
In, during which unexpected microbiological contamination is repeated to remove, then carries out subculture by the dark culturing 30d under the conditions of constant incubator (25 ± 2) DEG C
Proliferation, subculture cycle 15d, subculture three times after, measure inductivity be 50%；It is transferred to differentiation and proliferation culture medium and carries out illumination cultivation,
Intensity of illumination 1500-2000Lux, 16 hd of daily illumination-1, cultivation temperature is 25 DEG C, and subculture is primary every 2 weeks, subculture 3 times,
Phenylacetic acid and emergence rate, proliferation rate 80% are counted after 30d；
（4）When breaking up seedling and growing to 2-3cm, be transferred to root media, (25 ± 2) DEG C, intensity of illumination be 2000~
3000lx, 16 hd of light application time-1Under the conditions of cultivate, rooting rate 100%.Root long 5cm of seedling to be broken up or so, bottle seedling is moved
Go out culturing room, injects tap water after bottle hardening 2d is closed under natural light, rooting rate is counted after opening hardening 3d under natural light.Clean root
Behind portion, matrix after high pressure steam sterilization is moved into（Perlite：Vermiculite：Nutrition Soil（Purchased from Hangzhou auspicious wave Gardening Materials portion）=2:3:1）
Culture, obtains complete regeneration plant.
Finally it should be noted that：The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention,
Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used
With technical scheme described in the above embodiments is modified or equivalent replacement of some of the technical features.
All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in the present invention's
Within protection domain.
Claims (4)
1. a kind of wild barley mature embryo callus induction and Regeneration System method, it is characterised in that：Steps are as follows：
（1）Selection wild barley seed simultaneously sterilizes；
（2）The screening of inducing culture, differentiation and proliferation culture medium and root media：
The inducing culture is：+ 0.3 mgL of basal medium-1 6-BA +1.5 mg·L-1 2,4-D；
The differentiation and proliferation culture medium is：Basal medium+0.5mg/L6-BA+0.2mg/L NAA；
The root media is：1/2 basal medium；
The basal medium is：MS culture mediums+sucrose 30gL-1+ agar 10gL-1；
（3）The processing and induction differentiation of mature embryo：Take step（1）The wild barley seed of middle disinfection, is placed in inducing culture and trains
It supports, subculture is transferred in differentiation and proliferated culture medium afterwards three times to be continued to cultivate, and subculture is transferred in root media is trained three times afterwards
It supports, when breaking up the root long 4-6cm of seedling, bottle seedling is removed into culturing room, a bottle hardening is closed under natural light, injects tap water after 2d, from
Hardening 3d is opened under right light, then cleans root, sterilizing with matrix culture, obtains complete regeneration plant.
2. according to the method described in claim 1, it is characterized in that：Step（3）Described in the condition of Fiber differentiation be：In constant temperature
Dark culturing 30d under the conditions of 25 ± 2 DEG C.
3. according to the method described in claim 1, it is characterized in that：Step（3）Described in the condition of differentiation and proliferation culture be：Light
According to culture, intensity of illumination 1500-2000Lux, 16 hd of daily illumination-1, cultivation temperature is 25 DEG C.
4. according to the method described in claim 1, it is characterized in that：Step（3）Described in the condition of culture of culture of rootage be：
25 ± 2 DEG C, intensity of illumination 2000-3000lx, light application time 16hd-1Under the conditions of cultivate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610731771.7A CN106106188B (en) | 2016-08-26 | 2016-08-26 | A kind of induction of wild barley mature embryo callus and Regeneration System method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610731771.7A CN106106188B (en) | 2016-08-26 | 2016-08-26 | A kind of induction of wild barley mature embryo callus and Regeneration System method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106106188A CN106106188A (en) | 2016-11-16 |
| CN106106188B true CN106106188B (en) | 2018-08-17 |
Family
ID=57275494
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610731771.7A Active CN106106188B (en) | 2016-08-26 | 2016-08-26 | A kind of induction of wild barley mature embryo callus and Regeneration System method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106106188B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114902914A (en) * | 2022-05-09 | 2022-08-16 | 兰州大学 | Construction method of wild barley-endophytic fungi symbiont |
| CN115710590B (en) * | 2022-11-29 | 2024-02-02 | 中国科学院青岛生物能源与过程研究所 | Agrobacterium tumefaciens mediated callus infection method |
| CN118020639B (en) * | 2024-03-19 | 2024-07-23 | 兰州大学 | Method for establishing callus induction and regeneration system of embryo of Chinese sheep Mao Chengshou |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1502226A (en) * | 2002-11-20 | 2004-06-09 | 中国人民解放军军需大学 | Method for breeding salt-resisting short-arn barley |
| CN101619303B (en) * | 2009-08-03 | 2011-01-05 | 浙江大学 | Method for inducing barley mature embryo callus and used inducing culture medium thereof |
-
2016
- 2016-08-26 CN CN201610731771.7A patent/CN106106188B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN106106188A (en) | 2016-11-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104429952B (en) | It is a kind of to cultivate the method that cabbage Isolated microspore efficiently obtains regeneration plant | |
| CN106106188B (en) | A kind of induction of wild barley mature embryo callus and Regeneration System method | |
| CN101200704B (en) | Tissue culturing method for broussonetia papyrifera | |
| CN102613083A (en) | North American redwood tissue cultivation method | |
| CN101897297B (en) | Two-step tissue culture quick propagation method for hemerocallis | |
| CN105961201A (en) | Tissue culture method for strawberry transplant seedlings | |
| CN104855294B (en) | A kind of Caulis Akebiae rapid propagation method | |
| CN108464240A (en) | The method of Snow White's strawberry detoxifying fast breeding | |
| CN105532450A (en) | Hybrid paper mulberry industrial tissue culture and breeding method | |
| CN103155862B (en) | Sinocalamus latiflorus flower pesticide inductor embryo the method obtaining regeneration plant | |
| CN106538382A (en) | A kind of method that eremochloa ophiuroides high-efficiency regeneration system is set up as explant with young fringe | |
| CN104938335B (en) | The method that regeneration plant is obtained using oil tea hypocotyls | |
| CN108901858A (en) | A kind of phoenix head ginger selenium-rich original silkworm egg quick-breeding method | |
| CN106106178A (en) | A kind of method for tissue culture of confection Rhizoma Iridis Tectori | |
| CN103238519B (en) | Rapid seedling raising method of switchgrass tissue culture | |
| CN104813931A (en) | Tissue culture and rapid propagation method for Dendrobium officinale | |
| CN115606503B (en) | Tissue culture method of aster | |
| CN106332779A (en) | Tissue culture and fast propagation method for gynostemma pentaphylla | |
| CN103477976A (en) | Stem tissue culture seedling method of dendrobium candidum | |
| CN103039360A (en) | Method for quickly propagating leeka through tissue culture | |
| CN105918132A (en) | Rapid breeding method of clerodendrum trichotomum thunb | |
| CN109197594A (en) | A kind of tissue culture expanding propagation method of radix pseudostellariae virus-elimination seedlings | |
| CN105746344A (en) | Virus-free tissue culture and rapid propagation method of castor oil plants | |
| CN109392717A (en) | A kind of cortex cinnamomi tissue culture and rapid propagation method | |
| CN104304032A (en) | Method suitable for efficient induction and plant regeneration of multi-genotype watermelon somatic embryos |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |