CN108184664A - A kind of construction method of eleusine indica high-efficiency regeneration system - Google Patents
A kind of construction method of eleusine indica high-efficiency regeneration system Download PDFInfo
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- CN108184664A CN108184664A CN201711350046.6A CN201711350046A CN108184664A CN 108184664 A CN108184664 A CN 108184664A CN 201711350046 A CN201711350046 A CN 201711350046A CN 108184664 A CN108184664 A CN 108184664A
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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
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The present invention discloses a kind of construction method of eleusine indica high-efficiency regeneration system, belongs to herbicide resistance field.This method establishes its regenerating system using ripe responsive type Cowhells grass seed as explant；Induction and subculture including callus differential medium, are taken root, hardening, transplanting and other steps.The characteristics of this method, is that (1) pollution rate is low；Callus induction rate, differentiation rate are high.(2) callus water stainization degree is low.(3) regeneration period is short, and regeneration rate is high.
Description
Technical field
The invention belongs to herbicide resistance fields, are more particularly to a kind of construction method of eleusine indica high-efficiency regeneration system.
Background technology
Eleusine indica (Eleusineindica) is grass family annual grassy weeds, not only widely distributed, adaptable spy
Point, and it is strong with strong fertility, tillering ability, it has also become one of ten big malignant weed of the world.Due to the length of herbicide
Phase largely uses, and has 265 kinds of weeds at present and 15 class herbicides are produced with drug resistance, and eleusine indica produces 7 class herbicides
Multiple resistance is given birth to.In recent years, genetic transformation provides important technological means for the research of Resistant crop smothering.
It is that Genetic Transformation in Higher Plants is successfully basic to build high-frequency regenerating system.So far, eleusine indica regenerating system
Structure do not report that this greatly limits the development of genetic transformation also.So establish a kind of stabilization, efficient eleusine indica
Regenerating system can provide important method for the research of eleusine indica drug resistance.
Invention content
The shortcomings that in order to overcome the prior art, the purpose of the present invention is to provide a kind of eleusine indica highly efficient regeneration bodies with insufficient
The construction method of system.This method establishes its regenerating system using ripe responsive type Cowhells grass seed as explant.
The purpose of the present invention is achieved through the following technical solutions：
A kind of construction method of eleusine indica high-efficiency regeneration system, includes the following steps：
(1) induction of callus and subculture：Cowhells grass seed is removed the peel, after -20 DEG C of 20~30d of freezing, in 75% alcohol
It impregnates, with 0.05%~0.2%HgCl2After 10~20min of sterilization, aseptically, cleaned repeatedly with sterile water, wind
It is dry, in access inducing culture IC；After 30~40d, the callus of generation is transferred in subculture medium AC and is carried out after being commissioned to train
It supports, every 30~40d subcultures are primary, subculture 2~3 times；3~4d of preculture is carried out again, chooses faint yellow, compact-sized, graininess
Embryo callus as subsequent material；Condition of culture：25 ± 2 DEG C, it is dark for 24 hours.
(2) differential medium：The embryo callus that step (1) obtains is transferred in differential medium DC, is divided
Change culture；Condition of culture：25 ± 2 DEG C, 12h illumination cultivations.
(3) take root, hardening, transplanting：When differentiation seedling grows to 4~6cm, differentiation seedling is connected in root media RC,
25 ± 2 DEG C, 12h illumination cultivations, when root system is up to 4~6cm, after 3~4d of hardening, transplant survival.
The inducing culture IC：N6+ 0.5~2mgL-12,4-D+0.6~1gL-1Caseinhydrolysate+(30 ±
1)g·L-1Sucrose+7~8gL-1Agar, pH=5.8 ± 0.1；
Preferably, the inducing culture IC：N6+ 0.5~1mgL-12,4-D+0.6~1gL-1Caseinhydrolysate
+(30±1)g·L-1Sucrose+7~8gL-1Agar, pH=5.8 ± 0.1；
It is furthermore preferred that the inducing culture IC：N6+0.5mg·L-1 2,4-D+0.8g·L-1Caseinhydrolysate+
30g·L-1Sucrose+8gL-1Agar, pH=5.8 ± 0.1；
The subculture medium AC：N6+ 0.25~1mgL-12,4-D+3~4gL-1Plant gel+0.6~1g
L-1Caseinhydrolysate+(30 ± 1) gL-1Sucrose+7~8gL-1Agar, pH=5.8 ± 0.1.
Preferably, the subculture medium AC：N6+ 0.25~0.5mgL-12,4-D+3~4gL-1Plant gel+
0.6~1gL-1Caseinhydrolysate+(30 ± 1) gL-1Sucrose+7~8gL-1Agar, pH=5.8 ± 0.1.
It is furthermore preferred that the subculture medium AC：N6+0.25mg·L-1 2,4-D+3g·L-1Plant gel+0.8g
L-1Caseinhydrolysate+30gL-1Sucrose+7gL-1Agar, pH=5.8 ± 0.1.
The differential medium DC：MS+0.5~2mgL-16-BA+0.5~2mgL-1KT+0~0.2mgL- 1NAA+0~0.2mgL-1IAA+(30±1)g·L-1Sucrose+7~8gL-1Agar, pH=6.0 ± 0.1.
Preferably, the differential medium DC：MS+0.5mg·L-1 6-BA+0.5mg·L-1KT+0.05mg·L- 1NAA+0.05mg·L-1IAA+30g·L-1Sucrose+8gL-1Agar, pH=6.0 ± 0.1.
The root media RC：1/2MS+(30±1)g·L-1Sucrose+7~8gL-1Agar, pH=5.8 ±
0.1。
Preferably, the root media RC：1/2MS+30g·L-1Sucrose+8gL-1Agar, pH=5.8 ± 0.1.
Disinfection described in step (1) preferably uses 0.05%HgCl2Sterilization 10min.
Described MS, B5、N6Culture medium chemical composition is as follows：
The present invention is had the following advantages and effect relative to the prior art：
A kind of construction method of eleusine indica regenerating system, feature are that (1) pollution rate is low；Callus induction rate, differentiation rate
It is high.(2) callus water stainization degree is low.(3) regeneration period is short, and regeneration rate is high.
Description of the drawings
Fig. 1 is the distinct methods of embodiment breaking dormancy phase；A：The seed of (4 DEG C) storages of low temperature；B：- 20 DEG C of freezings 20~
Processing of basking seeds after 30d；C：Sterile water impregnates for 24 hours；D：30%NaOH (W/V) impregnates 15min, between lowercase letter processing
Level of difference (P<0.05).
Fig. 2 is the selection of embodiment seed disinfection sterilizing methods.
Fig. 3 is the selection of embodiment minimal medium.
Fig. 4 is the Callus morphology that embodiment generates；Wherein, a：I type structures, i.e. nutty structure and flocculence structure phase
The callus of separation；b：II type structures, the callus that nutty structure is wrapped up by spongy tissue, c：Type III structure, it is only extra large
The non-embryonic type callus of silk floss tissue.
Fig. 5 is the callus in embodiment subculture；Wherein, a, b, c, d, e are illustrated respectively in N6Culture medium (contains 1mgL-1 2,4-D+30g·L-1Sucrose) in be added to 7 (CK), 8,10,12gL-1Agar and 7.0gL-1Agar+3.0gL-1It plants
The processing of object gel；F represents to be added to 0.8gL on the basis of e-1The processing of caseinhydrolysate.
Fig. 6 is the bud that embodiment callus differentiates.
Fig. 7 is the culture of rootage of embodiment adventitious bud.
Fig. 8 is the transplant survival of embodiment regrowth.
Specific embodiment
With reference to embodiment and attached drawing, the present invention is described in further detail, but embodiments of the present invention are unlimited
In this.
The test method of specific experiment condition is not specified in the following example, usually according to conventional laboratory conditions or according to system
Make the experiment condition proposed by factory.
Embodiment 1
The best approach of seed breaking dormancy：The Cowhells grass seed of 4 DEG C of storages is removed into kind of a skin, is cleaned with tap water, wind
It is dry；Respectively at 4 DEG C of low-temperature storages, sterile water impregnate for 24 hours, the NaOH solution of 30% (W/V) impregnate 10min, -20 DEG C freezing 20~
30d；Later, on super-clean bench, by treated, Cowhells grass seed is soaked in 1min in 75% alcohol, is impregnated with 0.1% mercuric chloride
15min, uses sterile water wash 5 times afterwards, air-dries；The seed handled well is inoculated in culture medium (MS+1mgL-1 2,4-D+
30g·L-1Sucrose+8gL-1Agar, pH=5.8 ± 0.1) on, 25 DEG C, dark culturing for 24 hours.Each 5 repetitions of processing, one
Repeat 30 seeds.Callus quantity (Fig. 1) is recorded after 30d, -20 DEG C of 20~30d of freezing of Cowhells grass seed can be significantly improved
Callus induction rate, inductivity can inhibit callus water stainization up to 49%.
Embodiment 2
The best approach of eleusine indica seed disinfection sterilizing：The Cowhells grass seed of low-temperature storage (4 DEG C) removes kind of a skin, with certainly
Water cleaning air-dries；On super-clean bench, will treated Cowhells grass seed is soaked in 1min in 75% alcohol, then with a concentration of
0.05%th, 0.1%, 0.2% HgCl2(W/V) 10min, 15min, 20min are impregnated respectively；Finally use sterile water wash 5 times,
It air-dries；The Cowhells grass seed handled well is inoculated in culture medium (MS+1mgL-1 2,4-D+30g·L-1Sucrose+8gL-1Fine jade
Fat, pH=5.8 ± 0.1) on, 25 DEG C, dark culturing for 24 hours.Each 5 repetitions of processing, 30 seeds of a repetition.30d postscripts
Callus quantity (Fig. 2) and pollution rate are recorded, each processing is pollution-free, but 0.05%HgCl2Sterilization 10min, ox
The healing rate highest of muscle grass seed, up to 53.33%.
Embodiment 3
The selection of minimal medium：According to Examples 1 and 2, best method for treating seeds is selected, the Cowhells that will be handled well
Grass seed is inoculated on ICM, ICB and ICN inducing culture, 25 DEG C, dark culturing for 24 hours.Each processing repeats three times, a weight
Multiple 30 seeds.Every 7d record callus quantity (Fig. 3), N6Best minimal medium is should be, Fiber differentiation can be shortened
Period, and inductivity can be improved, inductivity 59%.
The ICM：MS+1mg·L-1 2,4-D+30g·L-1Sucrose+8gL-1Agar, pH=5.8 ± 0.1；
The ICN：N6+1mg·L-1 2,4-D+30g·L-1Sucrose+8gL-1Agar, pH=5.8 ± 0.1；
The ICB：B5+1mg·L-1 2,4-D+30g·L-1Sucrose+8gL-1Agar, pH=5.8 ± 0.1.
Embodiment 4
The concentration of 2,4-D is selected excellent in inducing culture：According to embodiment 1,2 and 3, best method for treating seeds is selected,
The Cowhells grass seed handled well is inoculated in best minimal medium, wherein 2,4-D concentration are respectively 0.5,1.0,2,
3mg·L-1, 25 DEG C, dark culturing for 24 hours.Each 5 repetitions of processing, 30 seeds of a repetition.Observe Callus morphology (figure
4), I types (Fig. 4 a), faint yellow embryo callus subculture are separated with flocculence milky spongy tissue, flocculence milky spongy tissue
The faint yellow embryo callus subculture of cell proliferation speed ratio is fast, and callus increases to certain phase, should remove flocculence milky sponge in time
Tissue；Faint yellow, compact-sized, granular embryonic type callus can be formed by a squamous subculture, callus proliferation is fast,
Differentiation capability is strong compared with II (Fig. 4 b) and III (Fig. 4 c).Callus number (table 1), 2,4-D 0.5mgL are recorded after 30d-1When,
Seed healing rate highest, up to 75%.
The selection of 2,4-D concentration in 1 inducing culture of table
Embodiment 5
Callus squamous subculture：The consistent callus subculture of water stainization degree in embodiment 4 is selected to containing 7.0 (CK)
(Fig. 5 a), 8.0 (Fig. 5 b), 10.0 (Fig. 5 c), 12.0gL-1Agar (Fig. 5 d) and 7.0gL-1Agar+3.0gL-1Plant coagulates
Subculture medium (the N of glue (Fig. 5 e)6+1mg·L-1 2,4-D+30g·L-1Sucrose, pH=5.8 ± 0.1) in, each processing 3
It repeats, 8 callus of a repetition.25 DEG C, dark culturing for 24 hours observe callus metamorphosis, find N6It is added in culture medium
0.8g·L-1Caseinhydrolysate, 7.0gL-1Agar and 3.0gL-1Plant gel can greatly reduce the water stain of callus
Change, callus is in faint yellow and nutty structure (Fig. 5 f).
Embodiment 6
Differential medium：Faint yellow, the granular embryo callus generated in embodiment 5 is transferred to hormon
Match culture medium (MS+30gL-1Sucrose+8gL-1Agar, pH=6.0 ± 0.1) in, carry out differentiation culture (Fig. 6).If one
Three repetitions of a processing, 8 callus of a repetition, 12h illumination cultivations；Differentiation rate (the table of callus is recorded after 10d
2), the collective effect of 6-BA and KT just can induce the differentiation of eleusine indica callus, add 0.05mgL again on this basis- 1NAA and 0.05mgL-1IAA can make eleusine indica callus differentiation rate reach 100%, and can shorten callus divergaence time.
The selection of hormone combination in 2 differential medium of table
| Hormone concentration and media | Differentiation rate (%) |
| 0.5mg·L-1 2,4-D+0.5mg·L-1 6-BA | 0 |
| 0.5mg·L-1 2,4-D+1mg·L-1 6-BA | 0 |
| 0.5mg·L-1 2,4-D+2mg·L-1 6-BA | 0 |
| 0.5mg·L-1 6-BA | 0 |
| 1mg·L-1 6-BA | 0 |
| 2mg·L-1 6-BA | 0 |
| 0.5mg·L-1 6-BA+0.5mg·L-1KT | 71.00±0.181a |
| 1mg·L-1 6-BA+1mg·L-1KT | 58.00±0.0844b |
| 2mg·L-1 6-BA+2mg·L-1KT | 50.00±0.144b |
Embodiment 7
Take root, hardening, transplanting：Adventitious bud in embodiment 6 is connected to root media RC (1/2MS+30gL-1Sucrose+
8g·L-1Agar, pH=5.8 ± 0.1) in, 25 DEG C, 12h illumination cultivations (Fig. 7), rooting rate 100%；When root system reaches 4cm
Remove sealed membrane, hardening 3d；Transplant survival (Fig. 8), survival rate 100%.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (9)
1. a kind of construction method of eleusine indica high-efficiency regeneration system, it is characterised in that include the following steps：
(1) induction of callus and subculture：Cowhells grass seed is removed the peel, and after -20 DEG C of 20~30d of freezing, is impregnated in 75% alcohol,
With 0.05%~0.2%HgCl2It after 10~20min of sterilization, aseptically, is cleaned repeatedly with sterile water, air-dries, connect
Enter in inducing culture IC；After 30~40d, the callus of generation is transferred in subculture medium AC and carries out squamous subculture, often
30~40d subcultures are primary, subculture 2~3 times；3~4d of preculture is carried out again, chooses faint yellow, compact-sized, granular embryo
Callus is as subsequent material；Condition of culture：25 ± 2 DEG C, it is dark for 24 hours；
(2) differential medium：The embryo callus that step (1) obtains is transferred in differential medium DC, carries out differentiation training
It supports；Condition of culture：25 ± 2 DEG C, 12h illumination cultivations；
(3) take root, hardening, transplanting：When differentiation seedling grows to 4~6cm, differentiation seedling is connected in root media RC, 25 ± 2
DEG C, 12h illumination cultivations, when root system is up to 4~6cm, after 3~4d of hardening, transplant survival.
2. the construction method of eleusine indica high-efficiency regeneration system according to claim 1, it is characterised in that：
The inducing culture IC：N6+ 0.5~2mgL-12,4-D+0.6~1gL-1Caseinhydrolysate+(30 ± 1) g
L-1Sucrose+7~8gL-1Agar, pH=5.8 ± 0.1.
3. the construction method of eleusine indica high-efficiency regeneration system according to claim 1 or 2, it is characterised in that：
The inducing culture IC：N6+ 0.5~1mgL-12,4-D+0.6~1gL-1Caseinhydrolysate+(30 ± 1) g
L-1Sucrose+7~8gL-1Agar, pH=5.8 ± 0.1.
4. the construction method of eleusine indica high-efficiency regeneration system according to claim 1, it is characterised in that：
The subculture medium AC：N6+ 0.25~1mgL-12,4-D+3~4gL-1Plant gel+0.6~1gL-1Water
Solve casein+(30 ± 1) gL-1Sucrose+7~8gL-1Agar, pH=5.8 ± 0.1.
5. the construction method of the eleusine indica high-efficiency regeneration system according to claim 1 or 4, it is characterised in that：
The subculture medium AC：N6+ 0.25~0.5mgL-12,4-D+3~4gL-1Plant gel+0.6~1gL-1
Caseinhydrolysate+(30 ± 1) gL-1Sucrose+7~8gL-1Agar, pH=5.8 ± 0.1.
6. the construction method of eleusine indica high-efficiency regeneration system according to claim 1, it is characterised in that：
The differential medium DC：MS+0.5~2mgL-16-BA+0.5~2mgL-1KT+0~0.2mgL-1NAA+0
~0.2mgL-1IAA+(30±1)g·L-1Sucrose+7~8gL-1Agar, pH=6.0 ± 0.1.
7. the construction method of the eleusine indica high-efficiency regeneration system according to claim 1 or 6, it is characterised in that：
The differential medium DC：MS+0.5mg·L-1 6-BA+0.5mg·L-1KT+0.05mg·L-1 NAA+0.05mg·
L-1IAA+30g·L-1Sucrose+8gL-1Agar, pH=6.0 ± 0.1.
8. the construction method of eleusine indica high-efficiency regeneration system according to claim 1, it is characterised in that：
The root media RC：1/2MS+(30±1)g·L-1Sucrose+7~8gL-1Agar, pH=5.8 ± 0.1.
9. the construction method of the eleusine indica high-efficiency regeneration system according to claim 1 or 8, it is characterised in that：
The root media RC：1/2MS+30g·L-1Sucrose+8gL-1Agar, pH=5.8 ± 0.1.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104620983A (en) * | 2015-01-28 | 2015-05-20 | 浙江省农业科学院 | Method for forming seedlings by carrying out tissue culture on echinochloacrus-galli |
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- 2017-12-15 CN CN201711350046.6A patent/CN108184664A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104620983A (en) * | 2015-01-28 | 2015-05-20 | 浙江省农业科学院 | Method for forming seedlings by carrying out tissue culture on echinochloacrus-galli |
Non-Patent Citations (1)
| Title |
|---|
| 魏吉平: "牛筋草遗传转化体系的建立及其抗百草枯的研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
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Application publication date: 20180622 |