CN105941162A - Establishment method of high-efficiency regeneration system for allium cepa - Google Patents
Establishment method of high-efficiency regeneration system for allium cepa Download PDFInfo
- Publication number
- CN105941162A CN105941162A CN201610478188.XA CN201610478188A CN105941162A CN 105941162 A CN105941162 A CN 105941162A CN 201610478188 A CN201610478188 A CN 201610478188A CN 105941162 A CN105941162 A CN 105941162A
- Authority
- CN
- China
- Prior art keywords
- culture medium
- root
- tillered
- onion
- culture
- 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.)
- Granted
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
The invention discloses an establishment method of a high-efficiency regeneration system for allium cepa. Stem apexes, with lengths of 1-2mm, of sterilized allium cepa are stripped and taken, and the stem apexes are inoculated in a callus inducing culture medium; calluses are first inoculated in an MS+3.0mg L<-1>6-BA+0.5mg L<-1> NAA (naphthylacetic acid) culture medium, and are sub-cultured in an MS+1.0mg L<-1>6-BA+1.0mg L<-1> NAA culture medium after 15d, and a differentiation ratio of cluster buds can reach 98%; a rooting culture medium is 1/2MS, test-tube plantlets after rooting are subjected to acclimatization and transplant, and a transplant survival rate can reach 100%. The method provided by the invention uses a conventional culture medium, is simple in hormone prescription, solves a technical problem that the calluses of the allium cepa are difficult to differentiate, and obviously increases the regeneration rate of plants. A tissue culture and regeneration system for the stem apexes of the allium cepa, which is established by the establishment method of the high-efficiency regeneration system for the allium cepa, provides a favorable acceptor material for subsequent transgenic research.
Description
Technical field
The invention belongs to agricultural biotechnology engineering field, be specifically related to the foundation of a kind of tillered-onion high-efficiency regeneration system
Method.
Background technology
Tillered-onion (Allium cepa L.var.multiplcans Bailey syn.var.Agrogatum Don)
It is commonly called as a mao Herba Alii fistulosi, pearl Herba Alii fistulosi, multiplieronion or Egyptian Bulbus Allii Cepae etc., for Liliaceae (Liliaceae) Allium (Allium) annual cultivation
Herbaceous plant, is a mutation of Bulbus Allii Cepae, and somatic chromosome number is 2n=16.Originate in middle Asia and West Asia, 20th century
Just start establishing in large scale in the Northeast of China, in Qinghai, Hebei, Henan, Hubei, the ground such as Sichuan also have little area to plant,
And cultivated area has the trend of expansion.Tillered-onion strong adaptability, high yield, resistance to storage, the supply phase is long, and vegetable of can adjusting supplies dull season
Should, the good preceding crop being well recognized as.
Tillered-onion sexual propagation difficulty, plant relatively hard-pumped a kind of sedge is blossomed and had seeds, generally using bulb as organ of multiplication, so that
The virosis that single virus individually infects or several virus Combined Infection is caused is extremely serious.Method for tissue culture is used to have
Effect ground Virusfree, improves quality and the quality of product.Tillered-onion bulb stem apex, plateau, alabastrum, test tube Seedling leaf at present
Sheet, cauloid etc. are selected as outer implant and carry out callus induction and plant regeneration research, but differentiation rate is the highest.
Summary of the invention
For solving the problems referred to above, the present invention is outer implant with the tillered-onion stem apex that regeneration capacity is stronger, at sterilization, hormone
Concentration, induce, break up, the aspect such as the composition of root media is optimized, to obtaining the stem apex wound healing group of high frequency regeneration rate
Knit induction and plant regeneration system, provide important basic material for tillered-onion genetic transformation.
The method for building up of tillered-onion high-efficiency regeneration system of the present invention, comprises the following steps:
(1) acquisition of tillered-onion aseptic explant
Tillered-onion is peelled off bulb crust, excises top 1/2, remove root simultaneously.Flowing water rinses 40min, in ultra-clean
On workbench, soaking 16min with 2% sodium hypochlorite after 75% ethanol disinfection 10 seconds, period sways for several times, sterile water wash 3-5
Secondary, aseptic filter paper blots, and strips center 1-2mm stem apex.
(2) callus induction of stem apex
Being inoculated in callus inducing medium by 1-2mm stem apex, phjytotron 22～24 DEG C of light culture, every 15
It subculture is once；Described callus inducing medium is: MS+30g/L sucrose+8g/L agar+2.0mg/L2,4-D, high pressure
PH to 5.8 is adjusted before sterilizing.
(3) differentiation of callus Multiple Buds
After 2～3 successive transfer culture, choose densification, light yellow wound healing, be cut into 3mm3Fritter, is inoculated in differentiation culture
Base, is placed in phjytotron 22～24 DEG C, light intensity 2000lx, light application time 12～14h/ days；Described differentiation is cultivated
Base is: MS+30g/L sucrose+8g/L agar+3.0mg/L6-BA+0.5mg/L NAA culture medium, 15 days follow-up generations are in MS+30g/L
Sucrose+8g/L agar+1.0mg/L6-BA+1.0mg/LNAA culture medium, adjusts pH to 5.8 before autoclaving.
(4) the taking root and rooting culture of regeneration plant
Choose growing way vigorous, neatly healthy and strong, the dark green Multiple Buds of color, it is inoculated in root media, is placed in phjytotron 22
～24 DEG C, light intensity 2000lx, light application time 12～14h/ days；Described root media is 1/2MS+30g/L sucrose+8g/L fine jade
Fat, adjusts pH to 5.8 before autoclaving.
Root culture treats that test tube Seedling length, to 8-10cm, root length 4-5cm, opens sealed membrane, adds a small amount of water moisturizing, in room temperature
Lower astigmatism seedling exercising 2-3d.After being taken out by tillered-onion plant, clear water washes down the culture medium of root attachment, is transplanted to bactericidal nurishing soil
In.It is placed in phjytotron 22～24 DEG C, light intensity 2000lx, light application time 12～14h/ days.
The invention has the beneficial effects as follows:
The outer implant that the present invention uses is tillered-onion stem apex, and stem apex is easily sterilized successfully, and callus induction ability
By force.Composition used by culture medium is cellar culture based component, and hormone prescription is simple, low cost.By tillered-onion callus first
Being inoculated in MS+30g/L sucrose+8g/L agar+3.0mg/L6-BA+0.5mg/L NAA culture medium, in 15d follow-up generation, is in MS+30g/L
Sucrose+8g/L agar+1.0mg/L6-BA+1.0mg/L NAA culture medium, differentiation rate, up to 98%, solves tillered-onion
The problem that differentiation rate is low.
Accompanying drawing explanation
Fig. 1 tillered-onion induction of callus；
Fig. 2 tillered-onion differentiation is cultivated；
Fig. 3 tillered-onion root culture；
Fig. 4 tillered-onion regrowth is transplanted；
Detailed description of the invention
By combination accompanying drawing described further below it will be further appreciated that the features and advantages of the invention.The enforcement provided
Example is only the explanation to the inventive method, and limits remaining content that the present invention discloses never in any form.
Material and reagent:
Used by the present invention, tillered-onion is gathered in Harbin, Heilongjiang Province.
MS, 1/2MS powder: Qingdao GaoKeYuan Hai Bo Bioisystech Co., Ltd；
2,4-D, 6-BA, NAA, agar: Biosharp；
Sucrose: Chemical Reagent Co., Ltd., Sinopharm Group；
Nutrition Soil: Zhenjiang training flower bud substrate development in science and technology company limited.
The acquisition of [embodiment 1] tillered-onion aseptic explant
Late August, the fresh tillered-onion gathered is peelled off bulb crust, excise top 1/2, remove root simultaneously.Stream
Water rinses 40min, on superclean bench, soaks 16min with 2% sodium hypochlorite after 75% ethanol disinfection 10s, and period sways number
Secondary, sterile water wash 3-5 time, aseptic filter paper blots, and strips 1-2mm stem apex, standby.
The callus induction of [embodiment 2] tillered-onion stem apex
By sterilizing, complete 1-2mm tillered-onion stem apex is inoculated in callus inducing medium C respectively1-C6, they are altogether
Same composition is: MS+30g/L sucrose+8g/L agar, and the composition of hormone and concentration are shown in Table 1, adjust pH extremely before culture medium autoclaving
5.8.Phjytotron 22～24 DEG C of light culture, started to expand after 7 days, within 15 days, can induce callus, every 15 days subcultures
Once.From table 1, hormon processes, and callus induction rate significant difference, at C after 30 days3: MS+30g/L sucrose+
8g/L agar+2mg/L2, in 4-D culture medium, callus induction rate is up to 99.5%, and callus quality is loosened, bulky grain
Shape, color is faint yellow or light green, as shown in Figure 1.
Table 1 variable concentrations 2,4-D and the KT impact on tillered-onion callus induction
In table 1, callus induction rate (%) represents meansigma methods ± standard error.Multiple comparisons uses Duncan method, in same column
Significant difference (P < 0.05) between different letter representations process.
The differentiation of [embodiment 3] callus Multiple Buds
Choose the densification after 2～3 successive transfer culture, light yellow wound healing, be cut into 3mm3Fritter, is transferred to differentiation respectively
Culture medium Y1-Y12(being shown in Table 2), they common compositions are: MS+30g/L sucrose+8g/L agar, the concentration of respective 6-BA and NAA
It is shown in Table 2, before autoclaving, adjusts pH to 5.8.It is placed in phjytotron 22～24 DEG C, light intensity 2000lx, light application time 12～14h/
My god.
After 15 days, callus starts to break up green bud point.Y8Culture medium callus differentiation green bud point rate is up to
65.3%, differentiation degree is higher；Y3Though culture medium callus differentiation green bud point rate is low by 46.7%, but Multiple Buds divides
Change degree is high.In conjunction with Y8And Y3The advantage of culture medium, first inoculates Y by callus8Culture medium: MS+30g/L sucrose+8g/L agar
+ 3.0mg/L6-BA+0.5mg/LNAA, induces green bud point, transfers in Y after 15 days3Culture medium: MS+30g/L sucrose+8g/L
Agar+1.0mg/L6-BA+1.0mg/LNAA, can differentiate Multiple Buds after 15d, differentiation rate reaches 98%, as shown in Figure 2.
Table 2 variable concentrations 6-BA and the NAA impact on tillered-onion differentiation
In table 2, phenylacetic acid (%) represents meansigma methods ± standard error.Multiple comparisons uses Duncan method, in same column
Significant difference (P < 0.05) between different letter representations process.
Taking root and rooting culture of [embodiment 4] regeneration plant
Choose growing way vigorous, neatly healthy and strong, the dark green Multiple Buds of color, it is inoculated in root media R respectively1-R3(being shown in Table 3),
They common compositions are: 1/2MS+30g/L sucrose+8g/L agar, and the composition of respective hormone and concentration are shown in Table 3, autoclaving
Front tune pH to 5.8.It is placed in phjytotron 22～24 DEG C, light intensity 2000lx, light application time 12～14h/ days, root culture 15 days
After, all have, 3 kinds of culture medium, a phenomenon of taking root, but rooting rate, root morphology significant difference.R1Culture medium rooting rate is the highest, up to
99.7%, root is elongated, number is many, plant growing way is vigorous, as shown in Figure 3.
Table 3 variable concentrations IBA and the NAA impact on tillered-onion root induction
In table 3, rooting rate (%) represents meansigma methods ± standard error.Multiple comparisons uses Duncan method, different letters in same column
Significant difference (P < 0.05) between expression process.
At R1Middle root culture, treats that test tube Seedling length, to 8-10cm, root length 4-5cm, opens sealed membrane, adds a small amount of water and protects
Wet, at room temperature astigmatism seedling exercising 2-3 days.After being taken out by tillered-onion plant, clear water washes down the culture medium of root attachment, is transplanted to
In bactericidal nurishing soil.It is placed in phjytotron 22～24 DEG C, light intensity 2000lx, light application time 12～14h/ days, transplants survival rate
100%, as shown in Figure 4.
Claims (1)
1. the method for building up of a tillered-onion high-efficiency regeneration system, it is characterised in that comprise the following steps:
(1) acquisition of tillered-onion aseptic explant
Tillered-onion is peelled off bulb crust, excises top 1/2, remove root simultaneously;Flowing water rinses 40min, in ultra-clean work
On platform, soaking 16min with 2% sodium hypochlorite after 75% ethanol disinfection 10 seconds, period sways for several times, and sterile water wash 3-5 time is aseptic
Filter paper blots, and strips center 1-2mm stem apex；
(2) callus induction of stem apex
1-2mm stem apex is inoculated in callus inducing medium, 22 ~ 24 DEG C of light culture of phjytotron, continued every 15 days
In generation, is once；Described callus inducing medium is: MS+30g/L sucrose+8g/L agar+2.0mg/L2,4-D, autoclaving
Front tune pH to 5.8；
(3) differentiation of callus Multiple Buds
After 2 ~ 3 successive transfer culture, choose densification, light yellow wound healing, be cut into 3mm3Fritter, is inoculated in division culture medium, is placed in
Phjytotron 22 ~ 24 DEG C, light intensity 2000lx, light application time 12 ~ 14h/ days；Described differentiation culture medium is: MS+
30g/L sucrose+8g/L agar+3.0mg/L6-BA+0.5mg/L NAA culture medium, in 15 days follow-up generations, are in MS+30g/L sucrose+8g/
L agar+1.0mg/L6-BA+1.0mg/L NAA culture medium, adjusts pH to 5.8 before autoclaving；
(4) the taking root and rooting culture of regeneration plant
Choose growing way vigorous, neatly healthy and strong, the dark green Multiple Buds of color, it is inoculated in root media, is placed in phjytotron 22 ~ 24
DEG C, light intensity 2000lx, light application time 12 ~ 14h/ days；Described root media is 1/2MS+30g/L sucrose+8g/L agar, high
PH to 5.8 is adjusted before pressure sterilizing；
Root culture treats that test tube Seedling length, to 8-10cm, root length 4-5cm, opens sealed membrane, adds a small amount of water moisturizing, at room temperature dissipates
Light seedling exercising 2-3d；After being taken out by tillered-onion plant, clear water washes down the culture medium of root attachment, is transplanted in bactericidal nurishing soil,
It is placed in phjytotron 22 ~ 24 DEG C, light intensity 2000lx, light application time 12 ~ 14h/ days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610478188.XA CN105941162B (en) | 2016-06-27 | 2016-06-27 | The method for building up of tillered-onion high-efficiency regeneration system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610478188.XA CN105941162B (en) | 2016-06-27 | 2016-06-27 | The method for building up of tillered-onion high-efficiency regeneration system |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105941162A true CN105941162A (en) | 2016-09-21 |
| CN105941162B CN105941162B (en) | 2018-04-24 |
Family
ID=56905108
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610478188.XA Active CN105941162B (en) | 2016-06-27 | 2016-06-27 | The method for building up of tillered-onion high-efficiency regeneration system |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105941162B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107027633A (en) * | 2017-06-19 | 2017-08-11 | 东北林业大学 | A kind of purple skin onion adventitious root suspension culture method for improving prostaglandin A content |
| CN110607323A (en) * | 2019-09-24 | 2019-12-24 | 四川育良生物科技有限公司 | Agrobacterium tumefaciens-mediated rice genetic transformation method |
| CN113106121A (en) * | 2021-05-25 | 2021-07-13 | 赣南师范大学 | Method for establishing genetic transformation system of tillered onion |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101960991A (en) * | 2010-11-15 | 2011-02-02 | 南京农业大学 | Onion callus induction method and special culture medium thereof |
-
2016
- 2016-06-27 CN CN201610478188.XA patent/CN105941162B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101960991A (en) * | 2010-11-15 | 2011-02-02 | 南京农业大学 | Onion callus induction method and special culture medium thereof |
Non-Patent Citations (1)
| Title |
|---|
| 陈典等: "分蘖洋葱茎尖愈伤组织诱导及植株再生", 《园艺学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107027633A (en) * | 2017-06-19 | 2017-08-11 | 东北林业大学 | A kind of purple skin onion adventitious root suspension culture method for improving prostaglandin A content |
| CN110607323A (en) * | 2019-09-24 | 2019-12-24 | 四川育良生物科技有限公司 | Agrobacterium tumefaciens-mediated rice genetic transformation method |
| CN113106121A (en) * | 2021-05-25 | 2021-07-13 | 赣南师范大学 | Method for establishing genetic transformation system of tillered onion |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105941162B (en) | 2018-04-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jun-jie et al. | An efficient micropropagation protocol for direct organogenesis from leaf explants of an economically valuable plant, drumstick (Moringa oleifera Lam.) | |
| Guo et al. | Establishment and plant regeneration of somatic embryogenic cell suspension cultures of the Zingiber officinale Rosc. | |
| CN107155898B (en) | A kind of dendrobium candidum carries out expanding numerous method using stem section thin slice | |
| CN104813939A (en) | Method for constructing lotus regeneration system | |
| WO2019134331A1 (en) | A method for quick breeding of abies beshanzuensis employing embryo rescue technology | |
| CN113331059B (en) | Method for establishing efficient regeneration system by taking bird king tea tree hypocotyls as explants | |
| CN104396742B (en) | The method that aseptic seedling is differentiated again with five-step approach induction Lilium sulphureum Baker bulbil calluss | |
| CN102657092A (en) | Tissue culture method of dioscorea opposita stem with axillary buds | |
| CN105684901A (en) | Quick propagation method for medicinal plant lycium ruthenicum in deserts | |
| Ju et al. | High frequency somatic embryogenesis and plant regeneration from hypocotyl and leaf explants of gherkin (Cucumis anguria L.) | |
| CN105145365B (en) | A kind of tissue culture propagation method of China Caulis cyatheae spinulosae green spheroids approach | |
| CN105941162A (en) | Establishment method of high-efficiency regeneration system for allium cepa | |
| EP3485726A1 (en) | Culturing method for cultured leguminous roots having increased coumestrol content | |
| CN106538382B (en) | Method for establishing efficient eremochloa ophiuroides regeneration system by taking young ears as explants | |
| CN108739396B (en) | Rapid seedling method for stem segments of red honeysuckle | |
| CN112753582B (en) | Method for sterilizing and rapidly proliferating stem segments of aleurites montana | |
| CN102239801A (en) | Method for pollination and fructification of orchids in test tubes | |
| WO2021189544A1 (en) | Method for efficiently detoxifying lilies | |
| CN108064699A (en) | A kind of tissue Propagation In Vitro method of pueraria lobata plant | |
| CN105638468B (en) | Efficient inducing soybean cotyledonary node explant produces the method and culture medium of Multiple Buds | |
| CN104604390B (en) | A kind of method of podocarpus nagi seed sprouting and rooting | |
| CN109220809B (en) | Koelreuteria paniculata somatic embryogenesis and plant regeneration culture method | |
| CN103814823A (en) | Culture method for inducing angelica dahurica anther to seedlings | |
| CN107439211B (en) | Method for shortening eggplant germination time | |
| Jesmin et al. | Callus induction and efficient plant regeneration in Cucumber (Cucumis sativus L.) |
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 | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20191216 Address after: Han Shi Lu 430415 Hubei province Wuhan city Xinzhou District of Yangluo Economic Development Zone No. 1 Co-patentee after: Wuhan Changhe Agricultural Technology Development Co., Ltd Patentee after: Wuhan Bioengineering Institute Address before: Han Shi Lu 430415 Hubei province Wuhan Yangluo City Economic Development Zone No. 1 Patentee before: Wuhan Bioengineering Institute |