CN105941162B - The method for building up of tillered-onion high-efficiency regeneration system - Google Patents
The method for building up of tillered-onion high-efficiency regeneration system Download PDFInfo
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- CN105941162B CN105941162B CN201610478188.XA CN201610478188A CN105941162B CN 105941162 B CN105941162 B CN 105941162B CN 201610478188 A CN201610478188 A CN 201610478188A CN 105941162 B CN105941162 B CN 105941162B
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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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- 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
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Abstract
The invention discloses a kind of method for building up of tillered-onion high-efficiency regeneration system, strips 1 2mm tillered-onion stem apexs of disinfection, is inoculated in callus inducing medium；Callus is first inoculated in MS+3.0mg L‑16‑BA+0.5mg•L‑1NAA culture mediums, in 15d follow-up generations, are in MS+1.0mg L‑16‑BA+1.0mg•L‑1NAA, differentiation rate is up to 98%；Root media is 1/2MS, and the test tube seedling after taking root passes through acclimatization and transplants, and transplanting survival rate is up to 100%.The method of the present invention uses conventional medium, and hormone prescription is simple, solves the technical barrier of the difficult differentiation of tillered-onion callus, and shoot regeneration frequency significantly improves.The tillered-onion stem tip tissue culture and regenerating system that the present invention establishes provide good acceptor material for follow-up transgenic research.
Description
Technical field
The invention belongs to agricultural biotechnology engineering field, and in particular to a kind of foundation of tillered-onion high-efficiency regeneration system
Method.
Background technology
Tillered-onion (Allium cepa L.var.multiplcans Bailey syn.var.Agrogatum Don)
A mao green onion, pearl green onion, multiplieronion or Egyptian onion etc. are commonly called as, is Liliaceae (Liliaceae) allium (Allium) annual cultivation
Herbaceous plant, is a mutation for onion, somatic chromosome number 2n=16.Originate in middle Asia and West Asia, 20th century
Just start large area plantation in the Northeast of China, also have small area plantation on Qinghai, Hebei, Henan, Hubei, Sichuan and other places,
And cultivated area has widened trend.Tillered-onion is adaptable, high yield, resistance to storage, supply the phase length, can adjust vegetables dull season confession
Should, it is generally acknowledged good preceding crop.
Tillered-onion generative propagation is difficult, and plant blossoms and has seeds compared with hard-pumped a kind of sedge, usually using bulb as organ of multiplication, so that
The virosis that single virus individually infects or several viral Combined Infections are triggered is extremely serious.Can be had using method for tissue culture
Effect ground Virusfree, improves the quality and quality of product.Tillered-onion bulb stem apex, plateau, bud, test tube seedling leaf at present
Piece, false stem etc. are selected as explant and carry out callus induction and plant regeneration research, but differentiation rate is not high.
The content of the invention
To solve the above problems, it is of the invention using the stronger tillered-onion stem apex of power of regeneration as explant, in disinfection, hormone
Concentration, induction, differentiation, the composition etc. of root media optimize, to obtain the stem apex callus group of high frequency regeneration rate
Induction and plant regeneration system are knitted, important basic material is provided 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 into bulb crust, excision top 1/2, while removes root.Flowing water rinses 40min, in ultra-clean
On workbench, 16min is soaked with 2% sodium hypochlorite after 75% ethanol disinfection 10 seconds, is during which swayed for several times, sterile water wash 3-5
Secondary, aseptic filter paper blots, and strips center 1-2mm stem apexs.
(2) callus induction of stem apex
1-2mm stem apexs are inoculated in callus inducing medium, 22~24 DEG C of light cultures of phjytotron, every 15
Its subculture is once；The 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 squamous subcultures, fine and close, light yellow callus is chosen, is cut into 3mm3Fritter, is inoculated in differentiation culture
Base, is placed in 22~24 DEG C, light intensity 2000lx of phjytotron, light application time 12~14h/ days；The differentiation culture
Base is：MS+30g/L sucrose+8g/L agar+3.0mg/L6-BA+0.5mg/L NAA culture mediums, in 15 days follow-up generations, are in MS+30g/L
Sucrose+8g/L agar+1.0mg/L6-BA+1.0mg/LNAA culture mediums, pH to 5.8 is adjusted before autoclaving.
(4) regeneration plant take root and rooting culture
It is vigorous to choose growing way, neat healthy and strong, the dark green Multiple Buds of color, are inoculated in root media, are placed in phjytotron 22
~24 DEG C, light intensity 2000lx, light application time 12~14h/ days；The root media is 1/2MS+30g/L sucrose+8g/L fine jades
Fat, pH to 5.8 is adjusted before autoclaving.
Culture of rootage treats that test tube seedling length to 8-10cm, root long 4-5cm, opens sealed membrane, a small amount of water moisturizing added, in room temperature
Lower astigmatism hardening 2-3d.After tillered-onion plant is taken out, clear water washes down the culture medium of root attachment, is transplanted to bactericidal nurishing soil
In.It is placed in 22~24 DEG C, light intensity 2000lx of phjytotron, light application time 12~14h/ days.
The beneficial effects of the invention are as follows：
The explant that the present invention uses is tillered-onion stem apex, and stem apex easily sterilizes success, and callus induction ability
By force.Component used in culture medium is cellar culture based component, and hormone prescription is simple, and cost is low.Tillered-onion callus is first
MS+30g/L sucrose+8g/L agar+3.0mg/L6-BA+0.5mg/L NAA culture mediums are inoculated in, 15d follow-up generations are in MS+30g/L
Sucrose+8g/L agar+1.0mg/L6-BA+1.0mg/L NAA culture mediums, differentiation rate solve tillered-onion up to 98%
The problem of differentiation rate is low.
Brief description of the drawings
Fig. 1 tillered-onion induction of callus；
Fig. 2 tillered-onion differentiation cultures；
Fig. 3 tillered-onion culture of rootage；
Fig. 4 tillered-onions regenerate transplantation of seedlings；
Embodiment
By combination attached drawing described further below it will be further appreciated that the features and advantages of the invention.The implementation provided
Example is only explanation to the method for the present invention, remaining content without limiting the invention in any way announcement.
Material and reagent：
Tillered-onion harvesting is in Harbin, Heilongjiang Province used in the present invention.
MS, 1/2MS powder：Qingdao GaoKeYuan Hai Bo Bioisystech Co., Ltd；
2,4-D, 6-BA, NAA, agar：Biosharp；
Sucrose：Sinopharm Chemical Reagent Co., Ltd.；
Nutrition Soil：Zhenjiang training flower bud matrix development in science and technology Co., Ltd.
【Embodiment 1】The acquisition of tillered-onion aseptic explant
Late August, bulb crust, excision top 1/2 are peelled off by the fresh tillered-onion of harvesting, while remove root.Stream
Water rinses 40min, on superclean bench, soaks 16min with 2% sodium hypochlorite after 75% ethanol disinfection 10s, during which sways number
Secondary, sterile water wash 3-5 times, aseptic filter paper blots, and strips 1-2mm stem apexs, spare.
【Embodiment 2】The callus induction of tillered-onion stem apex
The 1-2mm tillered-onions stem apex of sanitized is inoculated in callus inducing medium C respectively1-C6, they are common
With component be：MS+30g/L sucrose+8g/L agar, the component and concentration of hormone are shown in Table 1, pH are adjusted extremely before culture medium autoclaving
5.8.22~24 DEG C of light cultures of phjytotron, started to expand after 7 days, can induce out callus within 15 days, every 15 days subcultures
Once.By table 1 as it can be seen that hormon is handled, callus induction rate significant difference, in C after 30 days3：MS+30g/L sucrose+
Callus induction rate is up to 99.5% on 8g/L agar+2mg/L2,4-D culture medium, and callus quality is loose, bulky grain
Shape, color is faint yellow or light green, as shown in Figure 1.
Influences of the 1 various concentrations 2,4-D and KT of table to tillered-onion callus induction
Callus induction rate (%) represents average value ± standard error in table 1.Multiple range test uses Duncan methods, in same column
Significant difference (P between different letter expression processing<0.05).
【Embodiment 3】The differentiation of callus Multiple Buds
Fine and close, the light yellow callus after 2~3 squamous subcultures is chosen, is cut into 3mm3Fritter, is transferred to differentiation respectively
Culture medium Y1-Y12(being shown in Table 2), their common components are：MS+30g/L sucrose+8g/L agar, the concentration of respective 6-BA and NAA
2 are shown in Table, pH to 5.8 is adjusted before autoclaving.It is placed in 22~24 DEG C, light intensity 2000lx, 12~14h/ of light application time of phjytotron
My god.
Callus starts to break up green bud point after 15 days.Y8Culture medium callus breaks up green bud point rate
65.3%, differentiation degree is higher；Y3Though culture medium callus breaks up green bud point rate low 46.7%, Multiple Buds point
Change degree is high.With reference to Y8And Y3The advantages of culture medium, Y is first inoculated with by callus8Culture medium：MS+30g/L sucrose+8g/L agar
+ 3.0mg/L6-BA+0.5mg/LNAA, induces green bud point, and switching is in Y after 15 days3Culture medium：MS+30g/L sucrose+8g/L
Multiple Buds can be differentiated after agar+1.0mg/L6-BA+1.0mg/LNAA, 15d, differentiation rate is up to 98%, as shown in Figure 2.
Influences of the 2 various concentrations 6-BA and NAA of table to tillered-onion differentiation
Phenylacetic acid (%) represents average value ± standard error in table 2.Multiple range test uses Duncan methods, in same column
Significant difference (P between different letter expression processing<0.05).
【Embodiment 4】Regeneration plant take root and rooting culture
It is vigorous to choose growing way, neat healthy and strong, the dark green Multiple Buds of color, are inoculated in root media R respectively1-R3(being shown in Table 3),
Their common components are：1/2MS+30g/L sucrose+8g/L agar, the component and concentration of respective hormone are shown in Table 3, autoclaving
Preceding tune pH to 5.8.It is placed in 22~24 DEG C, light intensity 2000lx of phjytotron, light application time 12~14h/ days, culture of rootage 15 days
Afterwards, there are phenomenon of taking root, but rooting rate, root morphology significant difference in 3 kinds of culture mediums.R1Culture medium rooting rate highest, it is reachable
99.7%, root is elongated, number is more, plant growing way is vigorous, as shown in Figure 3.
Influences of the 3 various concentrations IBA and NAA of table to tillered-onion root induction
Rooting rate (%) represents average value ± standard error in table 3.Multiple range test uses Duncan methods, different letters in same column
Significant difference (P between expression processing<0.05).
In R1Middle culture of rootage, treats that test tube seedling length to 8-10cm, root long 4-5cm, opens sealed membrane, adds a small amount of water and protect
It is wet, astigmatism hardening 2-3 days at room temperature.After tillered-onion plant is taken out, clear water washes down the culture medium of root attachment, is transplanted to
In bactericidal nurishing soil.22~24 DEG C, light intensity 2000lx of phjytotron is placed in, light application time 12~14h/ days, transplants survival rate
100%, as shown in Figure 4.
Claims (1)
1. a kind of method for building up of tillered-onion high-efficiency regeneration system, it is characterised in that comprise the following steps：
（1）The acquisition of tillered-onion aseptic explant
Tillered-onion is peelled off into bulb crust, excision top 1/2, while removes root;Flowing water rinses 40min, in ultra-clean work
On platform, 16min is soaked with 2% sodium hypochlorite after 75% ethanol disinfection 10 seconds, is during which swayed for several times, sterile water wash 3-5 times is sterile
Filter paper blots, and strips center 1-2mm stem apexs；
（2）The callus induction of stem apex
1-2mm stem apexs are inoculated in callus inducing medium, 22 ~ 24 DEG C of light cultures of phjytotron, every 15 days after
In generation, is once；The callus inducing medium is：MS+30g/L sucrose+8g/L agar+2.0mg/L2,4-D, autoclaving
Preceding tune pH to 5.8；
（3）The differentiation of callus Multiple Buds
After 2 ~ 3 squamous subcultures, fine and close, light yellow callus is chosen, is cut into 3mm3Fritter, is inoculated in differential medium, is placed in
22 ~ 24 DEG C, light intensity 2000lx of phjytotron, light application time 12 ~ 14h/ days；The differentiation culture medium is：MS+
30g/L sucrose+8g/L agar+3.0mg/L6-BA+0.5mg/L NAA culture mediums, in 15 days follow-up generations, are in MS+30g/L sucrose+8g/
L agar+1.0mg/L6-BA+1.0mg/L NAA culture mediums, pH to 5.8 is adjusted before autoclaving；
（4）Regeneration plant take root and rooting culture
It is vigorous to choose growing way, neat healthy and strong, the dark green Multiple Buds of color, are inoculated in root media, are placed in phjytotron 22 ~ 24
DEG C, light intensity 2000lx, light application time 12 ~ 14h/ days；The root media is 1/2MS+30g/L sucrose+8g/L agar, high
PH to 5.8 is adjusted before pressure sterilizing；
Culture of rootage treats that test tube seedling length to 8-10cm, root long 4-5cm, opens sealed membrane, adds a small amount of water moisturizing, dissipate at room temperature
Light hardening 2-3d；After tillered-onion plant is taken out, clear water washes down the culture medium of root attachment, is transplanted in bactericidal nurishing soil,
It is placed in 22 ~ 24 DEG C, light intensity 2000lx of phjytotron, light application time 12 ~ 14h/ days.
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| 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 |
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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 |