CN109371057A - A kind of method of tobacco high throughput genetic transformation - Google Patents
A kind of method of tobacco high throughput genetic transformation Download PDFInfo
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- CN109371057A CN109371057A CN201811308661.5A CN201811308661A CN109371057A CN 109371057 A CN109371057 A CN 109371057A CN 201811308661 A CN201811308661 A CN 201811308661A CN 109371057 A CN109371057 A CN 109371057A
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Abstract
The present invention relates to a kind of methods of tobacco high throughput genetic transformation, belong to Genetic Transformation in Higher Plants field.This method include tobacco cultivating aseptic seedling, infect Agrobacterium prepare, infect, co-culture, select and breaks up cultivate and culture of rootage.The present invention eliminates time-consuming, laborious tobacco leaf disc preculture; infect Agrobacterium activation, co-culture after clean and etc.; manpower and time cost are saved; it reduces in conversion process due to cumbersome manual operation bring pollution risk; genetic transformation efficiency further increases simultaneously, is suitble to the scale genetic transformation of tobacco.
Description
Technical field
The present invention relates to a kind of methods of genetic transformation, and in particular to a kind of method of tobacco high throughput genetic transformation belongs to
In Genetic Transformation in Higher Plants field.
Background technique
Nicotiana tabacum (Nicotiana tabacum) is Solanaceae model organism, in model organism basic research and national warp
It is all played an important role in Ji production.Meanwhile tobacco is also a kind of plant for carrying out tissue cultures earliest, is developed according to tobacco
MS minimal medium (MurashigeandSkoog, 1962) be used widely in the tissue cultures of hundreds of plant.
Although having first-strike advantage, tobacco gene research always exists the small and weak limitation of weak foundation, scale.
In recent years, the gene editing technology based on CRISPR/Cas9 (hereinafter referred to as gene editing technology) is announced to the world splendidly, it is former
Reason is simple, mutation is efficient, low in cost, is widely used, and starts a field technology revolution in life science, grinds to gene function
Study carefully and plant industry and constantly brings new breakthrough, such as: in the U.S., scientist formulates anti-browning mushroom by editor's PPO gene；In day
This, scientist is sprouted by editor's SSR2 gene initiative is but free of the potato new varieties of toxin；In China, scientist passes through respectively
Edit MLO and OsBADH2 gene initiative powdery-mildew-resistance wheat and fragrant rice new varieties.
The development of gene editing technology and mature also study to tobacco gene bring new opportunity.Recent years, with cigarette
The foundation of careless gene editing technology, research scale constantly upgrade, and scale genetic transfoumation also becomes new challenge.By several
Technological accumulation in 10 years, all in all, it includes Aseptic seedling culture, Agrobacterium scribing line, Agrobacterium Dan Ke that tobacco, which has been formed a set of,
Grand liquid activation culture leaf dish preculture, is infected, is co-cultured, co-culturing leaf dish cleaning, selection and differentiation culture, culture of rootage
Deng the stabilization genetic conversion system of nine steps, but due to past research scale is too small, during tobacco genetic transformation
Operating procedure and the disadvantages of kinds of culture medium is various, labor intensive, very long time, also always exist, and have increasing need for one kind at present
Simply, efficiently, low human resources, be suitble to scale operation tobacco genetic transfoumation.
Summary of the invention
To solve the above problems, adapting to the demand of scale operation, the present invention constructs a kind of high-throughput heredity turn of tobacco
The method of change.
Method it is an object of the invention to be mediated using agrobacterium tumefaciens, creates a kind of genetic transformation skill of high throughput
Art gets rid of the limitation that tobacco does not have scale genetic transforming method, accelerates the speed of tobacco gene research, improves tobacco basis and grinds
The integral level studied carefully.
The specific technical solution of the present invention is as follows:
A kind of method of tobacco high throughput genetic transformation, includes the following steps:
Step (1) tobacco cultivating aseptic seedling: full tobacco seed is taken, in superclean bench respectively through alcohol and sodium hypochlorite
It is inoculated into after disinfection in nonreactive MS culture medium, 30-40 days acquisition aseptic seedling plant is cultivated at 20-25 DEG C.
Step (2) infects Agrobacterium preparation: taking out the Agrobacterium frozen in advance, 50-200ul even spread is drawn after thawing
Into the YEB solid resistance culture base comprising corresponding antibiotic, takes out and move to from incubator after being cultivated 48-72 hours at 28 DEG C
Superclean bench draws nonreactive MS fluid nutrient medium with pipettor and blows and beats thallus repeatedly, the mixed liquor of culture medium and thallus is turned
It moves in centrifuge tube, shakes up, take nonreactive MS Liquid Culture keynote OD value to 0.5；
Step (3) infects: aseptic seedling obtained in step (1) being punched, is prepared with the ratio of 50-100/pipe and step (2)
Infect Agrobacterium mixing, leaf dish is moved on sterile blotting paper from taking out in infected liquid immediately after 10min, blots leaf dish surface
Infected liquid；
Step (4) co-cultures: step (3) finally obtained leaf dish being transferred in co-culture medium in superclean bench, 28
DEG C dark culturing 72 hours；
Step (5) selection and differentiation culture: step (4) finally obtained leaf dish is moved in Selective agar medium in superclean bench
Culture, every 7-10 days one subcultures of replacement；There is the callus obviously expanded in leaf dish edge after replacement 3 times, replaces 5 times
Callus bears 2cm or so Bud Differentiation afterwards；
Step (6) culture of rootage: step (5) finally obtained Bud Differentiation is cut into shifting along base portion with scalpel in superclean bench
It cultivates, takes root into root media within about 4-10 days, can transplant within 10-15 days after taking root and carry out earth culture into basin.
Further, in step (1) and (2), MS culture medium are as follows: MS minimal medium 4.43g/L, sucrose 30g/L, agar
8g/L, PH=5.75.
Further, in step (2), the Agrobacterium strain is LBA4404.
Further, in step (3), after punching, the size of tobacco explant leaf dish is 5mm × 5mm, and time of infection is
10min。
Further, in step (4), co-culture medium are as follows: MS minimal medium 4.43g/L, sucrose 30g/L, agar
8g/L, methyl α-naphthyl acetate (NAA) 0.5mg/L, 6-benzyl aminopurine (6-BA) 2.0mg/L, PH=5.75.
Further, in step (5), co-culture medium are as follows: culture medium are as follows: MS minimal medium 4.43g/L, sucrose
30g/L, agar 8g/L, methyl α-naphthyl acetate (NAA) 0.5mg/L, 6-benzyl aminopurine (6-BA) 2.0mg/L, carbenicillin 250mg/
L, kanamycins 50mg/L, PH=5.75.
Further, in step (5) and (6), condition of culture are as follows: 16 hours/25 DEG C of 28 DEG C of illumination dark 8 hours.
Further, in step (6), culture medium specifically: MS minimal medium 4.43g/L, sucrose 30g/L, agar 8g/
L, carbenicillin 250mg/L, kanamycins 100mg/L, PH=5.75.
If the prior art is compared, the invention has the following beneficial effects:
1, medium component is simple, convenient easily to match.
2, streamline operation substantially saves manpower, is characterized in particular in:
(1) it infects Agrobacterium directly to be obtained by solid medium, saves the Liquid Culture link for needing culture overnight.
(2) tests for sterility can directly infect, and save 48 hours preculture links of explant, avoid mass propgation basigamy
System reduces repeatedly explant and shifts, reduce mechanical loss, improves leaf dish survival ability.
(3) Selective agar medium is directly antibacterial under conditions of infecting Agrobacterium bacterium solution OD value equal to 0.5, avoids co-cultivation
Many and diverse sterile water wash afterwards.
3, explant transfer number of the invention is reduced, and mechanical damage reduces, and transformation efficiency gets a promotion.
4, the present invention entire transformation period shortens 7-10 days compared with traditional scheme, and productivity is promoted.
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail.
It will be understood to those of skill in the art that the following example is merely to illustrate the present invention, and it should not be regarded as limiting this hair
Bright range.In the examples where no specific technique or condition is specified, described technology or conditions according to the literature in the art
Or it is carried out according to product description.Reagents or instruments used without specified manufacturer is that can be obtained by purchase
Conventional products.
Embodiment 1
The high-throughput genetic transforming method of the tobacco bred Hongda tobacco of the present embodiment, includes the following steps:
The cultivating aseptic seedling of step (1) tobacco bred Hongda tobacco: full tobacco seed is taken, is passed through in superclean bench
75% ethanol postincubation impregnates 30s, and aseptic water washing 2 times, then with 10% hypochlorite disinfectant 10min, sterile water wash 5 times,
Autoclaved filter paper is inoculated into MS1 culture medium after blotting the surface of the seed moisture, and at 25 DEG C, culture acquisition in 35 days or so is sterile
Seedling plant, as shown in Figure 1.
MS1 culture medium concrete composition are as follows: MS minimal medium 4.43g/L, sucrose 30g/L, agar 8g/L, PH=5.75.
Step (2) infects Agrobacterium preparation: take out from -80 DEG C of refrigerators freeze in advance containing pbi121 plasmid
LBA4404 engineering Agrobacterium, draws 50 μ Lpbi121 engineering Agrobacterium even spreads to comprising 50mg/L rifampin after thawing,
In the YEB resistance culture base of 50mg/L streptomysin and 50mg/L kanamycins, 28 DEG C culture 48 hours after taken out from incubator
Superclean bench is moved to, thallus is transferred in sterile 50mL centrifuge tube with pipettor after MS2 fluid nutrient medium repeated flushing, is shaken
It is even, adjust OD value to 0.5 to get to infecting Agrobacterium.
MS2 culture medium concrete composition are as follows: MS minimal medium 4.43g/L, sucrose 30g/L, PH=5.75.
Step (3) infects: aseptic seedling plant obtained in step (1) is obtained with punch punching in superclean bench
5mm × 5mm size tobacco leaf disc, take 50 with step (2) prepare Agrobacterium of infecting mix, immediately by leaf after 10min
Disk moves on sterile blotting paper from taking-up in infected liquid, blots leaf dish surface infected liquid.
Step (4) co-cultures: in superclean bench, step (3) finally obtained leaf dish is transferred in MS3 culture medium,
28 DEG C dark culturing 72 hours, obtained leaf dish is as shown in Figure 2.
MS3 culture medium concrete composition are as follows: MS minimal medium 4.43g/L, sucrose 30g/L, agar 8g/L, methyl α-naphthyl acetate
(NAA) 0.5mg/L, 6-benzyl aminopurine (6-BA) 2.0mg/L, PH=5.75.
Step (5) selection and differentiation culture: in superclean bench, step (4) finally obtained leaf dish is moved into MS4 culture
It cultivates under the conditions of (28 DEG C illumination 16 hours) in base/(25 DEG C dark 8 hours), every 10 days one subcultures of replacement, obtains
Bud Differentiation is as shown in Figure 3.
MS4 culture medium concrete composition are as follows: MS minimal medium 4.43g/L, sucrose 30g/L, agar 8g/L, methyl α-naphthyl acetate
(NAA) 0.5mg/L, 6-benzyl aminopurine (6-BA) 2.0mg/L, carbenicillin 250mg/L, kanamycins 50mg/L, PH=
5.75。
Step (6) culture of rootage: in superclean bench, by the finally obtained Bud Differentiation scalpel of step (5) along base portion
It cuts and moves in MS5 culture medium and cultivate under the conditions of (28 DEG C illumination 16 hours)/(25 DEG C dark 8 hours), take root within about 4-10 days,
As shown in Figure 4.MS5 culture medium concrete composition are as follows: MS minimal medium 4.43g/L, sucrose 30g/L, agar 8g/L, carboxylic benzyl mould
Plain 250mg/L, kanamycins 100mg/L, PH=5.75.
Embodiment 2
The high-throughput genetic transforming method of the K326 kind of the present embodiment, includes the following steps:
The cultivating aseptic seedling of step (1) K326 kind: full tobacco seed is taken, in superclean bench through 75% ethanol postincubation
Impregnate 30s, aseptic water washing 2 times, then with 10% hypochlorite disinfectant 10min, sterile water wash 5 times, autoclaved filter
Paper is inoculated into MS1 culture medium after blotting the surface of the seed moisture, at 25 DEG C, cultivates 35 days or so acquisition aseptic seedling plant.
Step (2) infects Agrobacterium preparation: take out from -80 DEG C of refrigerators freeze in advance containing pbi121 plasmid
LBA4404 engineering Agrobacterium, draws 50 μ Lpbi121 engineering Agrobacterium even spreads to comprising 50mg/L rifampin after thawing,
In the YEB resistance culture base of 50mg/L streptomysin and 50mg/L kanamycins, 28 DEG C culture 60 hours after taken out from incubator
Superclean bench is moved to, thallus is transferred in sterile 50mL centrifuge tube with pipettor after MS2 fluid nutrient medium repeated flushing, is shaken
It is even, adjust OD value to 0.5 to get to infecting Agrobacterium.
Step (3) infects: aseptic seedling plant obtained in step (1) is obtained with punch punching in superclean bench
5mm × 5mm size tobacco leaf disc, take 70 with step (2) prepare Agrobacterium of infecting mix, immediately by leaf after 10min
Disk moves on sterile blotting paper from taking-up in infected liquid, blots leaf dish surface infected liquid.
Step (4) co-cultures: in superclean bench, step (3) finally obtained leaf dish is transferred in MS3 culture medium,
28 DEG C dark culturing 72 hours.
Step (5) selection and differentiation culture: in superclean bench, step (4) finally obtained leaf dish is moved into MS4 culture
It is cultivated under the conditions of (28 DEG C illumination 16 hours) in base/(25 DEG C dark 8 hours), every 8 days one subcultures of replacement.
Step (6) culture of rootage: in superclean bench, by the finally obtained Bud Differentiation scalpel of step (5) along base portion
It cuts and moves in MS5 culture medium and cultivate under the conditions of (28 DEG C illumination 16 hours)/(25 DEG C dark 8 hours), take root within about 4-10 days.
Embodiment 3
The high-throughput genetic transforming method of the tobacco bred Hongda tobacco of the present embodiment, includes the following steps:
The cultivating aseptic seedling of step (1) tobacco bred Hongda tobacco: full tobacco seed is taken, is passed through in superclean bench
75% ethanol postincubation impregnates 30s, and aseptic water washing 2 times, then with 10% hypochlorite disinfectant 10min, sterile water wash 5 times,
Autoclaved filter paper is inoculated into MS1 culture medium after blotting the surface of the seed moisture, at 20 DEG C, is cultivated 30 days acquisition aseptic seedlings and is planted
Strain.
Step (2) infects Agrobacterium preparation: take out from -80 DEG C of refrigerators freeze in advance containing pbi121 plasmid
LBA4404 engineering Agrobacterium, draws 50 μ Lpbi121 engineering Agrobacterium even spreads to comprising 50mg/L rifampin after thawing,
In the YEB resistance culture base of 50mg/L streptomysin and 50mg/L kanamycins, 28 DEG C culture 70 hours after taken out from incubator
Superclean bench is moved to, thallus is transferred in sterile 50mL centrifuge tube with pipettor after MS2 fluid nutrient medium repeated flushing, is shaken
It is even, adjust OD value to 0.5 to get to infecting Agrobacterium.
Step (3) infects: aseptic seedling plant obtained in step (1) is obtained with punch punching in superclean bench
5mm × 5mm size tobacco leaf disc, take 90 with step (2) prepare Agrobacterium of infecting mix, immediately by leaf after 10min
Disk moves on sterile blotting paper from taking-up in infected liquid, blots leaf dish surface infected liquid.
Step (4) co-cultures: in superclean bench, step (3) finally obtained leaf dish is transferred in MS3 culture medium,
28 DEG C dark culturing 72 hours.
Step (5) selection and differentiation culture: in superclean bench, step (4) finally obtained leaf dish is moved into MS4 culture
It is cultivated under the conditions of (28 DEG C illumination 16 hours) in base/(25 DEG C dark 8 hours), every 10 days one subcultures of replacement.
Step (6) culture of rootage: in superclean bench, by the finally obtained Bud Differentiation scalpel of step (5) along base portion
It cuts and moves in MS5 culture medium and cultivate under the conditions of (28 DEG C illumination 16 hours)/(25 DEG C dark 8 hours), take root within about 8 days.
The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Claims (8)
1. a kind of method of tobacco high throughput genetic transformation, characterized by the following steps:
Step (1) tobacco cultivating aseptic seedling: full tobacco seed is taken, in superclean bench respectively through alcohol and sodium hypochlorite
It is inoculated into after disinfection in nonreactive MS culture medium, 30-40 days acquisition aseptic seedling plant is cultivated at 20-25 DEG C；
Step (2) infects Agrobacterium preparation: taking out the Agrobacterium frozen in advance, 50-200ul even spread is drawn after thawing to packet
In YEB solid resistance culture base containing corresponding antibiotic, after being cultivated 48-72 hours at 28 DEG C from incubator take out move to it is ultra-clean
Workbench draws nonreactive MS fluid nutrient medium with pipettor and blows and beats thallus repeatedly, the mixed liquor of culture medium and thallus is transferred to
It in centrifuge tube, shakes up, takes nonreactive MS Liquid Culture keynote OD value to 0.5；
Step (3) infects: aseptic seedling obtained in step (1) being punched, is prepared with the ratio of 50-100/pipe and step (2)
Infect Agrobacterium mixing, leaf dish is moved on sterile blotting paper from taking out in infected liquid immediately after 10min, blots leaf dish surface
Infected liquid；
Step (4) co-cultures: step (3) finally obtained leaf dish being transferred in co-culture medium in superclean bench, 28
DEG C dark culturing 72 hours；
Step (5) selection and differentiation culture: step (4) finally obtained leaf dish is moved in Selective agar medium in superclean bench
Culture, every 7-10 days one subcultures of replacement；There is the callus obviously expanded in leaf dish edge after replacement 3 times, replaces 5 times
Callus bears 2cm or so Bud Differentiation afterwards；
Step (6) culture of rootage: step (5) finally obtained Bud Differentiation is cut into shifting along base portion with scalpel in superclean bench
It cultivates, takes root into root media within about 4-10 days, can transplant within 10-15 days after taking root and carry out earth culture into basin.
2. the method for tobacco high throughput genetic transformation according to claim 1, it is characterised in that: in step (1) and (2),
MS culture medium are as follows: MS minimal medium 4.43g/L, sucrose 30g/L, agar 8g/L, PH=5.75.
3. the method for tobacco high throughput genetic transformation according to claim 1, it is characterised in that: in step (2), the agriculture
Bacillus species are LBA4404.
4. the method for tobacco high throughput genetic transformation according to claim 1, it is characterised in that: in step (3), punching
Afterwards, the size of tobacco explant leaf dish is 5mm × 5mm, and time of infection is 10min.
5. the method for tobacco high throughput genetic transformation according to claim 1, it is characterised in that: in step (4), co-culture
Culture medium are as follows: MS minimal medium 4.43g/L, sucrose 30g/L, agar 8g/L, methyl α-naphthyl acetate (NAA) 0.5mg/L, 6- benzyl amino are fast
Purine (6-BA) 2.0mg/L, PH=5.75.
6. the method for tobacco high throughput genetic transformation according to claim 1, it is characterised in that: in step (5), co-culture
Culture medium are as follows: culture medium are as follows: MS minimal medium 4.43g/L, sucrose 30g/L, agar 8g/L, methyl α-naphthyl acetate (NAA) 0.5mg/L,
6-benzyl aminopurine (6-BA) 2.0mg/L, carbenicillin 250mg/L, kanamycins 50mg/L, PH=5.75.
7. the method for tobacco high throughput genetic transformation according to claim 1, it is characterised in that: in step (5) and (6),
Condition of culture are as follows: 16 hours/25 DEG C of 28 DEG C of illumination dark 8 hours.
8. the method for tobacco high throughput genetic transformation according to claim 1, it is characterised in that: in step (6), culture medium
Specifically: MS minimal medium 4.43g/L, sucrose 30g/L, agar 8g/L, carbenicillin 250mg/L, kanamycins
100mg/L, PH=5.75.
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Cited By (2)
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| CN113207694A (en) * | 2021-06-10 | 2021-08-06 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Culture medium for tobacco callus culture, preparation method thereof and tobacco callus culture method |
| CN114107376A (en) * | 2021-12-17 | 2022-03-01 | 临沂大学 | Agrobacterium genetic transformation method for tobacco |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113207694A (en) * | 2021-06-10 | 2021-08-06 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Culture medium for tobacco callus culture, preparation method thereof and tobacco callus culture method |
| CN114107376A (en) * | 2021-12-17 | 2022-03-01 | 临沂大学 | Agrobacterium genetic transformation method for tobacco |
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