CN104195171B - Fructus Fragariae Ananssae efficient fast and stable gene transformation method - Google Patents
Fructus Fragariae Ananssae efficient fast and stable gene transformation method Download PDFInfo
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Abstract
The invention discloses a kind of Fructus Fragariae Ananssae ' beauty ' efficiently fast and stable gene transformation method, prepare including actication of culture, vegetable material, infect, co-culture, screening and culturing, root culture step, solve Agrobacterium and the pollution problem of miscellaneous bacteria in tradition agrobacterium-mediated transformation；Solve the problem that Fructus Fragariae Ananssae ' beauty ' transformation efficiency is low；Solve Fructus Fragariae Ananssae ' beauty ' transgenic from transgenic, detection GMOs, the problem of field-transplanting cycle length, shortening cycle；Solving detection GMOs workload big, work heavy problem, simplification detection method, accelerates detection speed.On the basis of ' beauty ' Regeneration System of Strawberry leaf having built up, the every factor affecting Agrobacterium tumefaciens transformation ' beauty ' Strawberry Leaves is studied, optimized various parameter, simplify operating process, determine optimum conversion condition.
Description
Technical field
The invention belongs to gene engineering technology field field, particularly relate to the transgenic technology of a kind of Fructus Fragariae Ananssae.
Background technology
Fructus Fragariae Ananssae (Fragaria ananassa) it is the crop that a kind of economic worth is higher, it is of high nutritive value, in recent years Fructus Fragariae Ananssae
Industry develops rapidly in China's many areas, has become as the important component part of some area agriculture industrialization.The most in recent years
Carry out China's researcher in the research of Fructus Fragariae Ananssae, also put into substantial amounts of energy, especially with molecular biology in recent years and
Engineered developing rapidly, study hotspot turns to the degree of depth of strawberry germplasm excellent genes to excavate, and carries out molecular marker
Qualification with expressed sequence and mensuration.Simultaneously in the research of the aspects such as genetic improvement such as resistance, storage tolerance, flavor quality
Result has been reported, Strwberry Breeding just develops towards molecular breeding direction.And it is most important during Fructus Fragariae Ananssae molecular breeding
Means be exactly Fructus Fragariae Ananssae transgenic.From nineteen ninety James etc. have successfully been obtained agriculture bacillus mediated Transformation of Strawberry plant with
Coming, by the research of recent two decades, Fructus Fragariae Ananssae has become as after Semen Juglandis, Fructus Mali pumilae, the 3rd fruit tree obtaining transfer-gen plant.
So far, scholars utilizes agrobacterium-mediated transformation to be imported in Fructus Fragariae Ananssae genome by the multiple external source genes of interest having economic worth.
On the other hand, for fruit tree, Fructus Fragariae Ananssae is short to the result cycle from blooming, and can repeatedly become colored in 1 year, and inflorescence is many, Ke Yisi
Season result, plant is short and small, and plantation is convenient.Therefore, for fruit tree crop, Fructus Fragariae Ananssae research is used as a kind of pattern of fruit tree research
Research, especially in terms of the growth promoter of research fruit, the fruit development mechanism of research Fructus Fragariae Ananssae is easier to, and has efficiently
Quickly advantage.
The most conventional Fructus Fragariae Ananssae gene transformation method has agrobacterium-mediated transformation, particle bombardment and electric shocking method, wherein Agrobacterium
The leaf disk method of mediation is the main method of Transformation of Strawberry, is also to study maturation the most in plant transgene research up to now
The most frequently used a kind of transgenic method.Although the genetic transfoumation route of Fructus Fragariae Ananssae is ripe, but transformation efficiency is the lowest, it is thus achieved that turn
Gene plant very little, brings obstacle to the later stage appraisal of transfer-gen plant, and from transgenic, detection GMOs, big
It is longer that the whole cycle is transplanted in field, and detection GMOs workload is big, works heavy.Traditional agrobacterium-mediated transformation operating process is compared
Loaded down with trivial details, the selection of many problems, such as suitable carrier, the selection of Suitable strains, miscellaneous bacteria easily occur during transgenic
Pollution, Agrobacterium cannot suppress, bacterial concentration is too high or too low causes that transgenic is unsuccessful, the control of time of infection length
Etc., these problems all can affect efficiency and the speed of Fructus Fragariae Ananssae transgenic, the strawberry transgenic effect that traditional method obtains
Rate is less than one of percentage.The most how to obtain an efficiently quickly Fructus Fragariae Ananssae stable conversion system, be in Fructus Fragariae Ananssae transgenic urgently
The problem solved.
Strawberry cultivars ' beauty ' yielding ability is good, best in quality, is suitable to facility cultivation, current domestic these product of large-scale cultivation
Kind, also obtain bigger economic benefit, but to there is also many defects, such as resistance poor due to this kind, easily feels
Catch an illness insect pest and fruit not storage tolerance etc..
Summary of the invention
For solving above-mentioned technical problem, the present invention provides a kind of Fructus Fragariae Ananssae ' beauty ' efficiently fast and stable gene transformation method,
Not only solve Agrobacterium and the pollution problem of miscellaneous bacteria, the inefficient problem of Strawberry transformation in tradition agrobacterium-mediated transformation；But also
Solve Fructus Fragariae Ananssae transgenic from transgenic, detection GMOs, the problem of field-transplanting cycle length, detection GMOs workload big, work
Make heavy problem.
For reaching above-mentioned purpose, the present invention is achieved by the following technical solutions.
A kind of culture medium of Fructus Fragariae Ananssae ' beauty ' efficiently fast and stable gene transformation,
Strawberry subculture medium FMS1, including composition have: MS, sucrose 30g/L, agar 5.5 g/L, 6-BA0.5mg/
L, NAA0.1mg/L, and the pH value of culture medium is 5.8；
Fructus Fragariae Ananssae co-cultures culture medium FMS2, including composition have: MS, sucrose 30g/L, agar 5.5 g/L, TDZ 2mg/
L, 2,4-D 0.1mg/L, and the pH value of culture medium is 5.8；
Fructus Fragariae Ananssae screening culture medium FMS3, including composition have: MS, sucrose 30g/L, agar 5.5 g/L, TDZ 2mg/L,
2,4-D 0.1mg/L, kanamycin 0-50mg/L, Carbenicillin 100-400 mg/L, and the pH value of culture medium are 5.8；
Fructus Fragariae Ananssae takes root screening culture medium FMS4, including composition have: MS, sucrose 30g/L, agar 5.5 g/L, IBA
0.1mg/L, kanamycin 0-50mg/L, Carbenicillin 100-400 mg/L, and the pH value of culture medium are 5.8.
Fructus Fragariae Ananssae screening culture medium FMS3 described further, including composition have: MS, sucrose 30g/L, agar 5.5 g/L,
TDZ 2mg/L, 2,4-D 0.1mg/L, kanamycin 20mg/L, Carbenicillin 400 mg/L；Fructus Fragariae Ananssae takes root screening culture medium
FMS4, including composition have: MS, sucrose 30g/L, agar 5.5 g/L, IBA 0.1mg/L, kanamycin 20mg/L, carboxylic benzyl is blue or green
Mycin 300 mg/L.
In the every 1L of each culture medium described further, the consumption of MS culture medium powder is 4.4g；Fructus Fragariae Ananssae co-cultures culture medium FMS2
60 DEG C of subpackage flat boards it are cooled to standby after autoclaving；Fructus Fragariae Ananssae screening culture medium FMS3 and Fructus Fragariae Ananssae screening culture medium FMS4 of taking root is high
Being cooled to 60 DEG C after pressure sterilizing, to add kanamycin, Carbenicillin subpackage culture bottle standby.
A kind of Fructus Fragariae Ananssae ' beauty ' efficiently fast and stable gene transformation method, the method comprises the steps:
1) actication of culture: will contain plasmid pK7WG2D Agrobacterium EHA105 and rule on solid medium, cultivates 2 for 28 DEG C
My god, it is inoculated in fluid medium to shake in 28 DEG C of bacterium and cultivates to OD600 value 0.4～0.6, go to upper strata clear after centrifugal for bacterium solution
Liquid, collects bacterial sediment, and by resuspended for the precipitation MS fluid medium suspension collected, the resuspended rear bacterial concentration that measures is 0.2
～0.6, the bacterium solution after resuspended is put in refrigerator standby；
2) vegetable material prepares: is taken at the blade of subculture Fructus Fragariae Ananssae " beauty " tissue cultured seedling in strawberry subculture medium FMS1, cuts
Go to cut off blade tip and leaf margin, be cut into 0.4 cm2Leaf block,
3) infect: bacterium solution is poured in the blade sheared, slightly shakes up, contaminate 40～60 minutes, pour out after having infected
Bacterium solution, blots blade on filter paper；
4) co-culture: the blade inoculation after infecting in Fructus Fragariae Ananssae co-cultures base FMS2, dark culturing in 25 ± 2 DEG C of incubators
2～3 days；
5) screening and culturing: by the blade of the Fructus Fragariae Ananssae ' beauty ' that co-cultures 3 days, be seeded in Fructus Fragariae Ananssae screening culture medium FMS3,
In culturing room 25 ± 2 DEG C, illumination cultivation 20～30 days, Strawberry Leaves edge has callus to grow, and retains resistance after detection
Wound healing, removes non-resistance wound healing, by kanamycin-resistant callus tissue through the cultivation of 40～50 d, differentiates plant, and detection retains whole again
Plant leaf and petiole all resistant plants in resistance；
6) root culture: when resistant plant grows to 2-3 centimetre high, proceeds to resistant plant Fructus Fragariae Ananssae and takes root screening culture medium
Take root on FMS4, it is thus achieved that complete resistance Seedling, after detection, retain the blade of resistance Seedling, petiole and the root plant all in resistance.
In step 1) described further, solid medium is: LB solid medium 50 mL+ spectinomycin 50mg/L+ profit
Good fortune flat 100mg/ L；Wherein LB solid culture based formulas: comprising tryptone 10g in each liter of LB solid medium, yeast carries
Take thing 5g, sodium chloride 10g and Agar (agar) 15g；
Fluid medium is: LB fluid medium 50mL+ spectinomycin 50mg/L+ rifampicin 100mg/ L；Wherein LB liquid
Body culture medium prescription: comprise tryptone 10g, yeast extract 5g, sodium chloride 10g in each liter of LB fluid medium.
In step 1) described further, the resuspended rear bacterial concentration that measures is 0.2～0.3.
Described step 5), step 6) use the detection of body formula fluorescence microscope, it was observed that green glow is i.e. in resistance.
Beneficial effect: compared with prior art, it is an advantage of the current invention that:
1) improving Fructus Fragariae Ananssae transgene efficiency, conversion ratio can reach more than 30%；
2) being reduced by bacterial concentration, time of infection lengthens, and i.e. avoids Agrobacterium to pollute, improves again and infect efficiency；
3) traditional method is often added the medicament that the auxiliary such as acetosyringone infect, blade can be damaged, reduce leaf
Sheet regeneration capacity, and infect the drug medication infected in bacterium solution without any auxiliary, the injury to Strawberry Leaves can be reduced,
Improve survival rate and the regeneration rate of leaf culture；
4) carrier of strongly expressed reporter gene is carried in use, obtains transfer-gen plant by observing directly to screen, it is to avoid
Work loaded down with trivial details in traditional detection, shortens the cycle of transgenic.
Accompanying drawing explanation
Fig. 1 is the fluoroscopic examination picture of Transgenic Strawberry ' beauty ', wherein:
FA: callus fluorescence FB: blade fluorescence FC: petiole fluorescence
FD: tissue cultured seedling fluorescence FE: flower fluorescence FF: fruit fluorescence
Fig. 2 is the impact on Fructus Fragariae Ananssae regeneration rate of the kanamycin concentration；
Fig. 3 is the Carbenicillin impact on Fructus Fragariae Ananssae transgenic；
Fig. 4 is different bacterial concentration and time of infection pairEgfpThe impact of gene expression rate.
Detailed description of the invention
The present invention is described in further detail with embodiment below in conjunction with the accompanying drawings.
Embodiment 1:
One, material prepares
1, vegetable material
Fructus Fragariae Ananssae " beauty " tissue cultured seedling, in strawberry subculture medium, subculture is for experiment.
2, culture medium prescription and compound method:
Fructus Fragariae Ananssae ' beauty ' subculture medium FMS1:
MS4.4g/L, sucrose 30g/L, agar 5.5 g/L, 6-BA0.5mg/L, NAA0.1mg/L, pH5.8, high pressure goes out
Bacterium, standby；
Fructus Fragariae Ananssae ' beauty ' co-cultures culture medium FMS2:
MS4.4g/L, sucrose 30g/L, agar 5.5 g/L, TDZ 2mg/L, 2,4-D 0.1mg/L, pH5.8, high pressure goes out
Bacterium, is cooled to about 60 DEG C subpackage flat boards standby；
Fructus Fragariae Ananssae ' beauty ' screening culture medium FMS3:
MS4.4g/L, sucrose 30g/L, agar 5.5 g/L, TDZ 2mg/L, 2,4-D 0.1mg/L, pH5.8, high pressure goes out
Bacterium, is cooled to 60 DEG C and adds kanamycin 0-50mg/L, and Carbenicillin 100-400 mg/L subpackage culture bottle is standby；
Fructus Fragariae Ananssae ' beauty ' takes root screening culture medium FMS4:
MS4.4g/L, sucrose 30g/L, agar 5.5 g/L, IBA 0.1mg/L, pH5.8, autoclaving, it is cooled to 60
DEG C add kanamycin 0-50mg/L, Carbenicillin 100-400 mg/L subpackage culture bottle is standby.
MS:MS is MS culture medium, be Murashige and Skoog in 1962 is tobacco cell Training Design, its feature
Be inorganic salt and ion concentration higher, be more stable ionic equilibrium solution, its nitrate content is high, the quantity of its nutrient and
Ratio is suitable, can meet nutrition and the physiological need of plant cell, thus the scope of application is relatively wider, most plants tissue culture rapid
Speed breeding uses it as the minimal medium of culture medium.The present invention uses and is produced MS culture medium powder by sigma company of the U.S.
(Murashige and Skoog basal salt mixture), this MS culture medium powder requires that often one liter of culture medium of preparation adds
Adding 4.4 grams of powder, in the preparation of basal culture medium, often one liter of culture medium of preparation needs to add 4.4 grams of MS culture medium powder.
TDZ:TDZ is a kind of new plant growth regulator, has the strongest cytokine activity, and it can promote to plant
The regeneration of thing bud and breeding, that breaks bud stops eye, promotes seed germination, promote callus growth, delay plant senescence etc., and
And the effect of other phytohormone and biological active substances can be regulated the growth and development process of plant, it is an effect
The plant growth regulator that power is the strongest, be agriculturally widely used and promotional value in the tissue culture of Fructus Fragariae Ananssae the most normal
With, the TDZ adding 2.0mg/L in the present invention can induce ' beauty ' blade to form adventitious bud very well.TDZ is configured to 1mg/mL
Mother solution for preparation culture medium.
2,4-D:2,4-D are 2,4-dichlorphenoxyacetic acid, for the induction of callus with grow highly effective, are tissues
Plant growth regulator conventional in cultivation.The 2,4-D adding 0.1mg/L in the present invention just can be good at inducing ' beauty ' no
Normal bud is formed.2,4-D is configured to the mother solution of 1mg/mL for preparation culture medium.
6-BA:6-BA is 6-benzyl aminoadenine, is the material of a kind of cytokinin, has efficient, stable, honest and clean
Valency and the feature such as easy of use are the favorite basic elements of cell division of tissue culture person.The Main Function of 6-BA is the shape promoting bud
Become, in the present invention in Fructus Fragariae Ananssae ' beauty ' successive transfer culture.6-BA is configured to the mother solution of 1mg/mL for preparation culture medium.
IBA:IBA is indolebutyric acid, it is possible to promotes the growth of plant main root, improves germination percentage, survival rate.High concentration indole fourth
Acid also can promotion division packet seedlings cultivating propagation.In the present invention, IBA is mainly used in taking root of transfer-gen plant.It is configured to 1mg/mL's
Mother solution is for preparation culture medium.
Kanamycin: be biological antibiotic class medicament, buys from sigma company of the U.S., and medicine is white powder, is configured to
The concentration of 50mg/mL is for experiment；Kanamycin is the selectable plant marker in transgenic, by add in the medium card that
Mycin, can weed out a part of not genetically modified bud or plant.The anti-kanamycin of transfer-gen plant, therefore can add
The culture medium of kanamycin survives, and the common the most anti-kanamycin of plant, can death.
Rif: rifampicin is biological antibiotic class medicament, buys from sigma company of the U.S., and medicine is peony powder, joins
The concentration making 25mg/mL is for experiment.
Spectinomycin: be biological antibiotic class medicament, buys from sigma company of the U.S., and medicine is white powder, is configured to
The concentration of 50mg/mL is for experiment；Kanamycin is the selection markers of the carrier in transgenic, by adding in LB culture medium
Spectinomycin, it is possible to the accurately single bacterium colony of screening, reduces the probability of living contaminants.
Carbenicillin: Carbenicillin is for suppressing the growth of transgenic later stage Agrobacterium.
3, bacterial strain and plant expression vector
The bacterial strain used in the present invention is agrobacterium strains EHA105, and this bacterial strain is conventional bacterial strain in Fructus Fragariae Ananssae transgenic, invades
Dye ability is stronger.This bacterial strain is bought in American Type Culture collection warehousing ATCC (American type culture
collection)。
Plant expression vector selects pK7WG2D plant expression vector based on Gateway technology, Gateway technology
Significantly simplify the step of gene clone and subclone, Gateway make use of site-specific to recombinate, so building introduction
After carrier, it is no longer necessary to use restricted enzyme and ligase.In addition pK7WG2D plant expression vector carries superpower expression
Reporter gene Egfp, it is simple to the detection of transfer-gen plant.Carrier is bought in Invitrogen biotech company.
Two, Fructus Fragariae Ananssae ' beauty ' efficiently fast and stable gene transformation method
1, actication of culture
1) plasmid pK7WG2D Agrobacterium EHA105 will be contained rule on solid medium, cultivate 2 days for 28 DEG C；Wherein solid
Culture medium is: LB solid medium 50 mL+ spectinomycin 50mg/L+ rifampicin 100mg/ L；Wherein LB solid culture basigamy
Side: comprise Tryptone (tryptone) 10g, Yeast Extract (yeast extract) in each liter of LB solid medium
5g, NaCl (sodium chloride) 10g and Agar (agar) 15g..
2) long on picking flat board good single bacterium colony 2-3, is inoculated in 50 mL fluid mediums, 28 DEG C, 220 rpm
In shaking table, shaken cultivation is to OD600 value 0.4(about 12-16 h)；Wherein fluid medium is: LB fluid medium 50mL+ strengthens
Miromycin 50mg/L+ rifampicin 100mg/ L；Wherein LB liquid culture based formulas: comprise in each liter of LB fluid medium
Tryptone (tryptone) 10g, Yeast Extract (yeast extract) 5g, NaCl (sodium chloride) 10g.
3), under room temperature, 5000rmp is centrifuged 8 minutes, removes supernatant, back-off centrifuge tube on aseptic filter paper, as far as possible by upper
Clear liquid is removed, and with the 100 resuspended thalline of mLMS liquid, in shaking table, shaken cultivation measures OD600 value for about 1 hour is 0.2, this
Data are that most preferably (this OD600 value is critically important, it is impossible to less than 0.2, it is impossible to higher than 0.3, controls, between 0.2～0.3, connect
Nearly 0.2).The bacterium solution activated can be used to do next step vegetable material and infects.
2, vegetable material prepares
30 days leaf ages that the children that is taken in strawberry subculture medium FMS1 on subculture Fructus Fragariae Ananssae " beauty " tissue cultured seedling is tender, open and flat
Blade, cuts off blade tip and leaf margin, is cut into about 0.4 cm2Leaf block infect for bacterium solution.Make on Strawberry Leaves as far as possible
Surrounding has wound, so cuts easy long wound healing.
3, infect
The bacterium solution of suspension is poured in the blade sheared, slightly shakes up, contaminate 40 minutes, during every 5 minutes shake
Once, increase bacterium solution and the contact area of blade, pour out bacterium solution after having infected, blade is blotted on aseptic filter paper surface
Bacterium solution.
4, co-culture
Blade inoculation after infecting in Fructus Fragariae Ananssae co-cultures base FMS2, dark culturing 3 days in 25 ± 2 DEG C of incubators.
5, screening and culturing
Fructus Fragariae Ananssae ' beauty ' blade of 3 days will be co-cultured, and be seeded in Fructus Fragariae Ananssae screening culture medium FMS3, make wound and culture medium
It is fully contacted, in culturing room 25 ± 2 DEG C, illumination cultivation.Cultivating about 20 days, Strawberry Leaves edge has white wound healing group
Knit and grow, now observe under body formula fluorescence microscope, it can be clearly seen that a part of wound healing has the brightest green glow to send out
Going out, this part wound healing is kanamycin-resistant callus tissue, will retain, and a part does not has luminescence, and this part wound healing is non-resistance wound healing, removes
Fall, by the step for, non-resistance wound healing is removed, is greatly reduced further work amount.The kanamycin-resistant callus tissue retained passes through
After the cultivation of about about 40 d, resistant plant can be differentiated.Carry out first order fluorescence detection the most again, at body formula fluorescence microscope
Under carry out observing resistant plant, remaining of whole resistant plant blade and petiole all green light, glow gets rid of.
6, root culture
When resistant plant grows to about 2-3 centimetre high, resistant plant is proceeded to Fructus Fragariae Ananssae and takes root raw in screening culture medium FMS4
Root, it is thus achieved that complete resistance Seedling.Resistance Seedling now can't determine whether transgenic seedling completely, it is therefore desirable to carries out one again
Secondary fluoroscopic examination, the blade of resistance Seedling in fluoroscopic examination, the most shinny green glow of petiole may determine that as transfer-gen plant, such as Fig. 1
Middle FB, shown in FC, FD, the blade petiole of transfer-gen plant has fluorescence.
The present invention can also carry out the test of fruit to acquisition fruit: waits Fructus Fragariae Ananssae to yield positive results after transplanting, after yielding positive results is
Further determine that the stability of transgenic, can under fluorescence microscope detection flower and the fluorescence of fruit different developmental phases again
Such as FE in Fig. 1, shown in FF, the Fructus Fragariae Ananssae of transgenic and fruit have hyperfluorescence to send.
Three, beneficial effects of the present invention is expanded on further below by test.
1, the determination of selectable plant marker kanamycin concentration:
Outer implant is inoculated in respectively containing kanamycin be 0,10,15,20,25, in the FMS2 culture medium of 50mg/L, secretly
Cultivating 14 days, go to be further cultured under light 20 days, add up blade melting brown rate, wound healing formation rate and adventitious shoot regeneration rate determine the suitableeest
Kanamycin selects pressure.
Strawberry Leaves regeneration more sensitive to the concentration of kanamycin, along with the increase of kanamycin concentration in culture medium outside
Implant chlorosis, brownization, gauffer, downright bad phenomenon increase the weight of, and outer implant regeneration rate declines the most therewith, and albefaction bud increases.Accordingly, it is determined that
Suitably kanamycin concentration is the key being successfully obtained transfer-gen plant.In kanamycin concentration it is as can be seen from Figure 2
During 20mg/L, blade melting brown rate reaches 65.7%, has a number of Callus formation, but does not has adventitious bud to generate, therefore
The regeneration of Fructus Fragariae Ananssae adventitious bud has been completely inhibit when kanamycin concentration reaches 20mg/L.Can be as the choosing in adventitious buds differentiation stage
Select pressure (Fig. 2).
2, the selection of antibacterial antibiotic (Carbenicillin) concentration
After infecting blade after co-culturing be inoculated in respectively containing Carbenicillin 100,200,300,400mg/L
In FMS2 culture medium, through light culture 14 days, after screening and culturing to 40 day, add up blade melting brown rate, wound healing formation rate and adventitious bud
The bacteriostatic level of regeneration rate and Agrobacterium determines optimal antibacterial antibiotic concentration.
The most important effect of antibacterial antibiotic usage is the growth of Agrobacterium in suppression transgenic protocol, therefore, antibacterial
The concentration requirement of antibiotic can either suppress Agrobacterium to grow, and can not damage plant growing again.So determining antibacterial anti-
Raw element concentration is very important in whole transgenic experiments.In this test, antibacterial antibiotic concentration can when 300mg/L
Good suppression Agrobacterium growth, does not the most injure the normal growth of plant simultaneously, as it is shown on figure 3, do not have Agrobacterium to overflow in culture medium
Go out, and plant can normal growth.
3, bacterial concentration, time of infection optimization
By the bacterial concentration OD600=0.6 in traditional method, bacterial concentration OD600=in time of infection 8min, with this method
0.2, immerged time 40min compares, compare two kinds of distinct methods infect efficiency, infect with rear blade survival rate with
And the pollution level of later stage Agrobacterium determines optimal bacterial concentration and time of infection.
WithEgfpGene expression rate weighs the selection of time of infection and bacterial concentration.Method is to infect length after conversion
The blade going out wound healing is placed under body formula fluorescence microscope detection luciferase expression, and statistics has the brightest green glow and do not has the leaf of green glow
Sheet number.
EgfpGene expression rate=(having the outer implant number of the green light number of blade/total) × 100%
The optimization of bacterial concentration and time of infection is most important with part in the present invention, is different from traditional Fructus Fragariae Ananssae and turns base
Because of the bacterial concentration height time in method short infect method, the present invention uses low concentration bacterium solution, infects for a long time, through too much
Secondary test proves, low concentration infects for a long time and can increase substantially transformation efficiency, and late stage of culture is not easy to produce agriculture bar
Bacterium pollutes.Low concentration bacterium solution is used to infect for a long time and can significantly improve as shown in Figure 4EgfpGene expression rate, improves and converts effect
Rate.
Embodiment 2: substantially the same manner as Example 1, except that: Fructus Fragariae Ananssae ' beauty ' efficiently fast and stable gene transformation side
Method, specific as follows:
Second step in step 1 actication of culture, long on picking flat board good single bacterium colony 2-3, it is inoculated into 50 mL LB
In fluid medium, 28 DEG C, 220 rpm in shaking table shaken cultivation to OD600 value 0.6.
Step 3 infects operation: the bacterium solution of suspension is poured in the blade sheared, slightly shakes up, and contaminates 60 minutes, process
In every shake in 5 minutes once, increase bacterium solution and the contact area of blade, pour out bacterium solution after having infected, by blade aseptic
The bacterium solution on surface is blotted on filter paper.
Step 4 co-cultures operation: the blade inoculation after infecting is in Fructus Fragariae Ananssae co-cultures base FMS2, in 25 ± 2 DEG C of incubators
Dark culturing 2 days.
Step 5 screening and culturing operation:
Fructus Fragariae Ananssae ' beauty ' blade of 2 days will be co-cultured, and be seeded in Fructus Fragariae Ananssae screening culture medium FMS3, make wound and culture medium
It is fully contacted, in culturing room 25 ± 2 DEG C, illumination cultivation.Cultivating 30 days, it is long that Strawberry Leaves edge has white callus
Go out, now observe under body formula fluorescence microscope, it can be clearly seen that a part of wound healing has the brightest green glow to send, this
Part wound healing is kanamycin-resistant callus tissue, will retain, and a part does not has luminescence, and this part wound healing is non-resistance wound healing, gets rid of, logical
The step for of mistake, non-resistance wound healing is removed, is greatly reduced further work amount.The kanamycin-resistant callus tissue retained is through about 50 d
Cultivation after, resistant plant can be differentiated.Carry out first order fluorescence detection the most again, observe under body formula fluorescence microscope
Remaining of resistant plant, whole resistant plant blade and petiole all green light, glow gets rid of.
Claims (9)
1. the culture medium of a Fructus Fragariae Ananssae ' beauty ' efficiently fast and stable gene transformation, it is characterised in that include following composition:
Fructus Fragariae Ananssae ' beauty ' subculture medium FMS1, including composition have: MS, sucrose 30g/L, agar 5.5 g/L, 6-
BA0.5mg/L, NAA0.1mg/L, and the pH value of culture medium is 5.8；
Fructus Fragariae Ananssae ' beauty ' co-cultures culture medium FMS2, including composition have: MS, sucrose 30g/L, agar 5.5 g/L, TDZ
2mg/L, 2,4-D 0.1mg/L, and the pH value of culture medium is 5.8；
Fructus Fragariae Ananssae ' beauty ' screening culture medium FMS3, including composition have: MS, sucrose 30g/L, agar 5.5 g/L, TDZ 2mg/
L, 2,4-D 0.1mg/L, kanamycin 0-50mg/L, Carbenicillin 100-400 mg/L, and the pH value of culture medium are 5.8；
Fructus Fragariae Ananssae ' beauty ' takes root screening culture medium FMS4, including composition have: MS, sucrose 30g/L, agar 5.5 g/L, IBA
0.1mg/L, kanamycin 0-50mg/L, Carbenicillin 100-400 mg/L, and the pH value of culture medium are 5.8.
The culture medium of a kind of Fructus Fragariae Ananssae ' beauty ' efficiently fast and stable gene transformation the most according to claim 1, its feature exists
In described Fructus Fragariae Ananssae ' beauty ' screening culture medium FMS3, including composition have: MS, sucrose 30g/L, agar 5.5 g/L, TDZ
2mg/L, 2,4-D 0.1mg/L, kanamycin 20mg/L, Carbenicillin 400 mg/L；
Fructus Fragariae Ananssae takes root screening culture medium FMS4, including composition have: MS, sucrose 30g/L, agar 5.5 g/L, IBA 0.1mg/
L, kanamycin 20mg/L, Carbenicillin 300 mg/L.
The culture medium of a kind of Fructus Fragariae Ananssae ' beauty ' efficiently fast and stable gene transformation the most according to claim 1 and 2, its feature
It is: in the every 1L of each culture medium, the consumption of MS culture medium powder is 4.4g；Fructus Fragariae Ananssae is cold after co-culturing culture medium FMS2 autoclaving
But standby to 60 DEG C of subpackage flat boards；Fructus Fragariae Ananssae screening culture medium FMS3 and Fructus Fragariae Ananssae take root and cool down after screening culture medium FMS4 autoclaving
Kanamycin, Carbenicillin subpackage culture bottle is added standby to 60 DEG C.
4. utilize the culture medium described in claim 1 to carry out Fructus Fragariae Ananssae ' beauty ' efficiently fast and stable gene transformation method, its feature
Being, the method comprises the steps:
1) actication of culture: will contain plasmid pK7WG2D Agrobacterium EHA105 and rule on solid medium, cultivates 2 days, connects for 28 DEG C
Planting and shaking cultivation to OD600 value in 28 DEG C of bacterium in fluid medium is 0.4～0.6, goes the supernatant after centrifugal for bacterium solution,
Collecting bacterial sediment, by resuspended for the precipitation MS fluid medium suspension collected, the resuspended rear bacterium solution OD600 value that measures is 0.2
～0.6, the bacterium solution after resuspended is put in refrigerator standby；
2) vegetable material prepare: be taken at the blade of subculture Fructus Fragariae Ananssae ' beauty ' tissue cultured seedling subculture medium FMS1 on, cut off blade tip with
Leaf margin, is cut into 0.4 cm2Leaf block；
3) infect: bacterium solution is poured in the blade sheared, slightly shakes up, contaminate 40～60 minutes, after having infected, pour out bacterium
Liquid, blots blade on filter paper；
4) co-culture: the blade inoculation after infecting co-cultures in base FMS2 Fructus Fragariae Ananssae ' beauty ', dark training in 25 ± 2 DEG C of incubators
Support 2～3 days；
5) screening and culturing: by the blade of the Fructus Fragariae Ananssae ' beauty ' that co-cultures 2～3 days, be seeded in screening culture medium FMS3, is cultivating
In room 25 ± 2 DEG C, illumination cultivation 20～when 30 days, blade edge has callus to grow, and retains resistance more after detection
Wound, removes non-resistance wound healing, by kanamycin-resistant callus tissue through the cultivation of 40～50 days, differentiates plant, and detection retains whole planting again
Strain blade and petiole all resistant plants in resistance；
6) root culture: when resistant plant grows to 2-3 centimetre high, proceeds to Fructus Fragariae Ananssae by resistant plant and takes root in screening culture medium FMS4
Take root, it is thus achieved that complete resistance Seedling, after detection, retain the blade of resistance Seedling, petiole and the root plant all in resistance.
Fructus Fragariae Ananssae the most according to claim 4 ' beauty ' efficiently fast and stable gene transformation method, it is characterised in that described step
Rapid 1) in, solid medium is: LB solid medium 50 mL+ spectinomycin 50mg/L+ rifampicin 100mg/ L；Wherein LB
Solid culture based formulas: comprise tryptone 10g, yeast extract 5g, sodium chloride 10g in each liter of LB solid medium
And agar 15g；
Fluid medium is: LB fluid medium 50mL+ spectinomycin 50mg/L+ rifampicin 100mg/ L；Wherein LB liquid training
Support based formulas: each liter of LB fluid medium comprises tryptone 10g, yeast extract 5g, sodium chloride 10g.
Fructus Fragariae Ananssae the most according to claim 4 ' beauty ' efficiently fast and stable gene transformation method, it is characterised in that described step
Rapid 1) in, the resuspended rear bacterial concentration that measures is 0.2～0.3.
Fructus Fragariae Ananssae the most according to claim 4 ' beauty ' efficiently fast and stable gene transformation method, it is characterised in that described step
Rapid 2) blade in is the blade of young 30 days tender, open and flat leaf ages.
Fructus Fragariae Ananssae the most according to claim 4 ' beauty ' efficiently fast and stable gene transformation method, it is characterised in that described step
Rapid 3) during contaminating every shake in 5 minutes once.
Fructus Fragariae Ananssae the most according to claim 4 ' beauty ' efficiently fast and stable gene transformation method, it is characterised in that described step
Rapid 5), step 6) uses the detection of body formula fluorescence microscope, it was observed that green glow is i.e. in resistance.
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