CN110521603A - The construction method of tomato genetic conversion system - Google Patents
The construction method of tomato genetic conversion system Download PDFInfo
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- CN110521603A CN110521603A CN201910888114.7A CN201910888114A CN110521603A CN 110521603 A CN110521603 A CN 110521603A CN 201910888114 A CN201910888114 A CN 201910888114A CN 110521603 A CN110521603 A CN 110521603A
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- tomato
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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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
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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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- 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 application discloses a kind of construction method of tomato genetic conversion system, comprising: obtain sterile tomato cotyledon；The tomato cotyledon is infected using Agrobacterium, wherein the first preculture 1d on the first culture medium of the tomato cotyledon；After Agrobacterium is infected, the tomato cotyledon is placed on first culture medium and co-cultures 1d；First culture medium further includes 2mg/L zeatin and 0.5mg/L auxin on the basis of 1/2MS culture medium；Tomato cotyledon after being infected using differential medium induction successively breaks up callus and adventitious bud, to obtain regrowth；The regeneration seedling rooting is induced using root media；The regrowth taken root is transplanted into soil, to obtain Transgenic Tomato Plants.The application passes through the screening to every factor and training method, especially by the culture medium prescription of adjustment preculture and co-cultivation, the process for shortening preculture and co-cultivation establishes a kind of tomato fast genetic transformation system, and the system growing-seedling period is short, conversion ratio is higher, easy to operate.
Description
Technical field
This application involves field of transgenic technology more particularly to a kind of construction methods of tomato genetic conversion system.
Background technique
Tomato genus tubular flower mesh Solanaceae (Solanaceae) tomato genus (Lycopersicon) 1 year or perennial plant are
A kind of very high greengrocery industrial crops of nutritive value, in China, the South and the North has extensive plantation.Because tomato has complete
The features such as with reference to genome sequence, by self-pollination, fruit is big, and growth cycle is short, is easy observation, it is also research berries
Fruit development and mature classical mode plant.
Tomato can be eaten raw, cook, being processed as catsup, juice or whole fruit tank as the vegetables for having well processed characteristic
Hiding, popularization is wide, in good taste, full of nutrition, is the ideal carrier of plant oral vaccine.Currently, raw using genetically modified plants
Oral vaccine is produced to be taken seriously further.Tomato produces edible vaccine as bioreactor with good application prospect.
Apply for content
The purpose of the application is, existing tomato genetic transformation there are aiming at the problem that, provide that a kind of growing-seedling period is short, operation
Process is simple, the construction method of the higher tomato genetic conversion system of conversion ratio.
To reach the above technical purpose, the application the technical solution adopted is as follows:
A kind of construction method of tomato genetic conversion system, the following steps are included:
Obtain sterile tomato cotyledon；
The tomato cotyledon is infected using Agrobacterium, wherein the first preculture 1d on the first culture medium of the tomato cotyledon；
After Agrobacterium is infected, the tomato cotyledon is placed on first culture medium and co-cultures 1d；First culture medium is 1/
It further include 2mg/L zeatin and 0.5mg/L auxin on the basis of 2MS culture medium；
Tomato cotyledon after being infected using differential medium induction successively breaks up callus and adventitious bud, to be regenerated
Seedling；
The regeneration seedling rooting is induced using root media；
The regrowth taken root is transplanted into soil, to obtain Transgenic Tomato Plants.
Preferably, described the step of obtaining sterile tomato cotyledon are as follows:
Tomato seeds are obtained, sterilize 15min under conditions of revolving speed is 120rpm using 4% sodium hypochlorite；
After tomato seeds after sterile water cleaning and disinfecting, continue to be incubated on shaking table to germination；
The tomato seeds of germination are seeded on 1/2MS culture medium, culture to tomato cotyledon is unfolded under light；
Harvest sterile tomato cotyledon.
Further, before infecting the tomato cotyledon, the tomato cotyledon is cut into 0.5cm2Fritter.
Preferably, when preculture, the tomato cotyledon face down is placed on first culture medium.
It is further preferred that the process of preculture and co-cultivation, carries out under conditions of 28 DEG C be protected from light respectively.
Preferably, in Agrobacterium infection processs, the concentration of the Agrobacterium corresponds to the Agrobacterium at 600nm wavelength
Light absorption value reaches in the range of 0.5~0.8；Time of infection is 15min.
Preferably, the differential medium further includes 2mg/L zeatin, 0.5mg/L life on the basis of 1/2MS culture medium
Long element, 250mg/L carbapen and 50mg/L kanamycins.
Selectively, the differential medium needs 10~15d replacement primary, until the tomato cotyledon differentiation and regeneration seedling.
It is further preferred that the root media further include on the basis of 1/2MS culture medium 0.1mg/L auxin,
250mg/L carbapen and 50mg/L kanamycins.
Preferably, the construction method of foregoing tomato genetic conversion system answering in building berry model plant
With, or the application in building fruit type bioreactor.
Compared with prior art, the application has the advantage that
1, the construction method of the tomato genetic conversion system of the application, matches by adjusting preculture and the culture medium of co-cultivation
Side, shortens the process of preculture and co-cultivation, can be shortened the period for obtaining Transgenic Tomato Plants.
2, the construction method of the tomato genetic conversion system of the application, is invaded by the pretreatment to tomato cotyledon, Agrobacterium
The screening for contaminating every factors such as cotyledon, differentiation culture, culture of rootage and training method, establishes a kind of tomato fast genetic transformation
System, the system have growing-seedling period is short, conversion ratio is higher, simple operation and other advantages, for research tomato dna function provide
Convenient means, and produce tomato oral vaccine for later use technique for gene engineering and provide experimental basis.
3, tomato cotyledon is cut into 0.5cm by the construction method of the tomato genetic conversion system of the application2Fritter and by it
Face down is placed on culture medium, and cotyledon is enable to come into full contact with culture medium, is accelerated its growth and is improved survival rate.
4, the construction method of the tomato genetic conversion system of the application, using evoked callus culture medium, take root
Culture medium prescription more targetedly, is easier to promote tomato cotyledon raw due to the synergistic effect of each component so that culture medium is more scientific
At callus and adventitious bud, it is proliferated and takes root, significant effect.
Detailed description of the invention
Fig. 1 is in the construction method of the application tomato genetic conversion system, and the tomato seeds for showing germination are seeded into 1/2MS
On culture medium.
Fig. 2 is to show after being infected using differential medium induction in the construction method of the application tomato genetic conversion system
Tomato cotyledon differentiate callus.
Fig. 3 is to show in the construction method of the application tomato genetic conversion system and utilize root media regeneration induction seedling
It takes root.
Fig. 4 is to show the regrowth transplanted and taken root into soil in the construction method of the application tomato genetic conversion system.
Specific embodiment
The application is described in further detail below in conjunction with the drawings and specific embodiments.
Embodiment
A kind of tomato genetic conversion system, specific steps are as follows:
(1) acquisition of aseptic cotyledon: selecting full tomato seeds, and with 4% hypochlorite disinfectant, conditions for sterilization is to turn
15min is shaken on the shaking table that speed is 120rpm.It is put on shaking table after with aseptic water washing 3-4 and shakes 2-3d to germinateing, be seeded into 1/
On 2MS culture medium (as shown in Figure 1), goes under light and cultivate 2-3d, cut cotyledon after cotyledon is fully deployed.
(2) it infects cotyledon: the aseptic tomato cotyledon being fully deployed is cut into 0.5cm2Fritter, face down is placed in pre- training
It supports on culture medium (culture medium prescription: 1/2MS+2mg/L zeatin+0.5mg/L auxin), 28 DEG C of dark culture 1d.Cotyledon is set
After on precultivation medium, the Agrobacterium for being used to infect cotyledon is placed on 28 DEG C of shaking tables and is shaken for 24 hours.Activate Agrobacterium, 20m
+ 10 μ l rifampin (25mg/ml) of LB+10 μ l kanamycins (100mg/ml)+200 μ l acetosyringone (1.96mg/ml)+1ml
Agrobacterium bacterium solution, after shaking about 6h, bacterium solution presents golden yellow.With MS culture medium by the OD of Agrobacterium bacterium solution600Value is adjusted to
Between 0.5-0.8, then the cotyledon after preculture is immersed in Agrobacterium bacterium solution and infects 15min.Cotyledon is gone into co-cultivation culture
On base (1/2MS+2mg/L zeatin, 0.5mg/L auxin), it is placed in 28 DEG C of dark culture 1d.
(3) tissue obtained by (2) induction differentiation callus and adventitious bud: is transferred on differential medium (culture medium
Formula: 1/2MS+2mg/L zeatin+0.5mg/L auxin+250mg/L carbapen+50mg/L kanamycins) it is put into
The lower 25 DEG C of cultures of light, culture 35-40d can be differentiated callus (as shown in Figure 2), the general primary differentiation culture of 10-15d replacement
Base.
(4) culture of rootage: cutting when the regrowth that callus differentiates has apparent growing point and be transferred to root media, light
Lower 25 DEG C of cultures.(prescription of rooting medium: 1/2MS+0.1mg/L auxin+250mg/L carbapen+50mg/L blocks that
Mycin).About 30d is cultivated on root media can grow suitable (as shown in figure 3,3-5 root), then can be transplanted to it
(as shown in Figure 4) is cultivated in soil.
Cotyledon is obtained to transplanting from sowing, and entire genetic transformation process used time about 78d induces the germination rooting rate point of cotyledon
Not Wei 90%, conversion positive rate be 35%.
Comparative example 1
A kind of tomato genetic conversion system, specific steps are as follows:
(1) acquisition of aseptic cotyledon: selecting full tomato seeds, 75% ethanol disinfection 2min, and aseptic water washing 2 times,
With 4% hypochlorite disinfectant, conditions for sterilization is that 15min is shaken on the shaking table that revolving speed is 120rpm.With aseptic water washing 3-4 times,
It is seeded on 1/2MS culture medium, grows 2-3d in the dark, gone to after germinateing under light and cultivate 5-6d, after cotyledon is fully deployed
Cut off cotyledon.
(2) it infects cotyledon: the aseptic tomato cotyledon being fully deployed is cut into 0.5cm2Fritter, face down is placed in pre- training
It supports on culture medium (culture medium prescription: 1/2MS+2mg/L zeatin+0.5mg/L auxin), 28 DEG C of dark culture 2d.Cotyledon is set
After on precultivation medium, the Agrobacterium for being used to infect cotyledon is placed on 28 DEG C of shaking tables and shakes 48h.Activate Agrobacterium, 20m
+ 10 μ l rifampin (25mg/ml) of LB+10 μ l kanamycins (100mg/ml)+200 μ l acetosyringone (1.96mg/ml)+1ml
Agrobacterium bacterium solution, after shaking about 6h, bacterium solution presents golden yellow.With MS culture medium by the OD of Agrobacterium bacterium solution600Value is adjusted to
Between 0.5-0.8, then the cotyledon after preculture is immersed in Agrobacterium bacterium solution and infects 15min.Cotyledon is gone into co-cultivation culture
On base (1/2MS+2mg/L zeatin, 0.5mg/L auxin), it is placed in 28 DEG C of dark culture 2d.
(3) tissue obtained by (2) induction differentiation callus and adventitious bud: is transferred on differential medium (culture medium
Formula: 1/2MS+2mg/L zeatin+0.5mg/L auxin+250mg/L carbapen+50mg/L kanamycins) it is put into
The lower 25 DEG C of cultures of light, culture 35-40d can differentiate callus, and general 10-15d replaces a differential medium.
(4) culture of rootage: cutting when the regrowth that callus differentiates has apparent growing point and be transferred to root media, light
Lower 25 DEG C of cultures.(prescription of rooting medium: 1/2MS+0.1mg/L auxin+250mg/L carbapen+50mg/L blocks that
Mycin).About 30d is cultivated on root media can grow suitable (3-5 root), and then it can be transplanted in soil and cultivated.
Cotyledon is obtained to transplanting from sowing, and entire genetic transformation process used time about 83d induces the germination rooting rate point of cotyledon
Not Wei 80%, conversion positive rate be 30%.
Comparative example 2
A kind of tomato genetic conversion system, specific steps are as follows:
(1) acquisition of aseptic cotyledon: selecting full tomato seeds, 75% ethanol disinfection 2min, and aseptic water washing 2 times,
With 4% hypochlorite disinfectant, conditions for sterilization is that 15min is shaken on the shaking table that revolving speed is 120rpm.With aseptic water washing 3-4 times,
It is seeded on 1/2MS culture medium, grows 2-3d in the dark, gone to after germinateing under light and cultivate 5-6d, after cotyledon is fully deployed
Cut off cotyledon.
(2) it infects cotyledon: the aseptic tomato cotyledon being fully deployed is cut into 0.5cm2Fritter, face down is placed in pre- training
It supports on culture medium (culture medium prescription: 1/2MS+2mg/L zeatin+0.5mg/L auxin), 28 DEG C of dark culture 1d.Cotyledon is set
After on precultivation medium, the Agrobacterium for being used to infect cotyledon is placed on 28 DEG C of shaking tables and is shaken for 24 hours.Activate Agrobacterium, 20m
+ 10 μ l rifampin (25mg/ml) of LB+10 μ l kanamycins (100mg/ml)+200 μ l acetosyringone (1.96mg/ml)+1ml
Agrobacterium bacterium solution, after shaking about 6h, bacterium solution presents golden yellow.With MS culture medium by the OD of Agrobacterium bacterium solution600Value is adjusted to
Between 0.5-0.8, then the cotyledon after preculture is immersed in Agrobacterium bacterium solution and infects 15min.Cotyledon is gone into co-cultivation culture
On base, it is placed in 28 DEG C of dark culture 1d.
(3) tissue obtained by (2) induction differentiation callus and adventitious bud: is transferred on differential medium (culture medium
Formula: 1/2MS+2mg/L zeatin+0.5mg/L auxin+250mg/L carbapen+50mg/L kanamycins) it is put into
The lower 25 DEG C of cultures of light, culture 35-40d can differentiate callus, and general 10-15d replaces a differential medium.
(4) culture of rootage: cutting when the regrowth that callus differentiates has apparent growing point and be transferred to root media, light
Lower 25 DEG C of cultures.(prescription of rooting medium: 1/2MS+0.1mg/L auxin+250mg/L carbapen+50mg/L blocks that
Mycin).About 30d is cultivated on root media can grow suitable (3-5 root), and then it can be transplanted in soil and cultivated.
Cotyledon is obtained to transplanting from sowing, and entire genetic transformation process used time about 81d induces the germination rooting rate point of cotyledon
Not Wei 90%, conversion positive rate be 35%.
In conclusion the construction method of the tomato genetic conversion system of the application passes through pretreatment, the agriculture to tomato cotyledon
Bacillus infects the screening of every factors such as cotyledon, differentiation culture, culture of rootage and training method, especially by adjustment preculture
With the culture medium prescription of co-cultivation, the process of preculture and co-cultivation is shortened, establishes a kind of tomato fast genetic transformation body
System, the system growing-seedling period is short, conversion ratio is higher, easy to operate.
Above-described embodiment is the preferable embodiment of the application, but is not merely restricted to the described embodiments, other
It is any without departing from spirit herein essence with principle under made changes, modifications, substitutions, combinations, simplifications, should be equivalent
Substitute mode is all contained within the protection scope of the application.
Claims (10)
1. a kind of construction method of tomato genetic conversion system, it is characterised in that, the following steps are included:
Obtain sterile tomato cotyledon；
The tomato cotyledon is infected using Agrobacterium, wherein the first preculture 1d on the first culture medium of the tomato cotyledon；Agriculture bar
After bacterium is infected, the tomato cotyledon is placed on first culture medium and co-cultures 1d；First culture medium is trained in 1/2MS
It further include 2mg/L zeatin and 0.5mg/L auxin on the basis of feeding base；
Tomato cotyledon after being infected using differential medium induction successively breaks up callus and adventitious bud, to obtain regrowth；
The regeneration seedling rooting is induced using root media；
The regrowth taken root is transplanted into soil, to obtain Transgenic Tomato Plants.
2. the construction method of tomato genetic conversion system as described in claim 1, which is characterized in that described to obtain sterile kind
The step of eggplant leaf are as follows:
Tomato seeds are obtained, sterilize 15min under conditions of revolving speed is 120rpm using 4% sodium hypochlorite；
After tomato seeds after sterile water cleaning and disinfecting, continue to be incubated on shaking table to germination；
The tomato seeds of germination are seeded on 1/2MS culture medium, culture to tomato cotyledon is unfolded under light；
Harvest sterile tomato cotyledon.
3. the construction method of tomato genetic conversion system as described in claim 1, which is characterized in that infect the tomato cotyledon
Before, the tomato cotyledon is cut into 0.5cm2Fritter.
4. the construction method of tomato genetic conversion system as described in claim 1, which is characterized in that when preculture, described kind
Eggplant leaf face down is placed on first culture medium.
5. the construction method of tomato genetic conversion system as claimed in claim 4, which is characterized in that preculture and co-cultivation
Process carries out under conditions of 28 DEG C be protected from light respectively.
6. the construction method of tomato genetic conversion system as claimed in claim 4, which is characterized in that Agrobacterium infection processs
In, in the range of the concentration of the Agrobacterium reaches 0.5~0.8 corresponding to light absorption value of the Agrobacterium at 600nm wavelength；It invades
The dye time is 15min.
7. the construction method of tomato genetic conversion system as described in claim 1, which is characterized in that the differential medium exists
It further include 2mg/L zeatin, 0.5mg/L auxin, 250mg/L carbapen and 50mg/ on the basis of 1/2MS culture medium
L kanamycins.
8. the construction method of tomato genetic conversion system as claimed in claim 7, which is characterized in that the differential medium needs
10~15d replacement is primary, until the tomato cotyledon differentiation and regeneration seedling.
9. the construction method of tomato genetic conversion system as described in claim 1, which is characterized in that the root media exists
It further include 0.1mg/L auxin, 250mg/L carbapen and 50mg/L kanamycins on the basis of 1/2MS culture medium.
10. application of the construction method of tomato genetic conversion system as described in claim 1 in building berry model plant,
Or the application in building fruit type bioreactor.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116411021A (en) * | 2023-06-07 | 2023-07-11 | 隆平生物技术(海南)有限公司 | Conversion method of tomato glyphosate screening system |
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| CN116411021A (en) * | 2023-06-07 | 2023-07-11 | 隆平生物技术(海南)有限公司 | Conversion method of tomato glyphosate screening system |
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Application publication date: 20191203 |