CN106047921B - A kind of the transgenosis culture medium and its transgenic method of diploid strawberry - Google Patents
A kind of the transgenosis culture medium and its transgenic method of diploid strawberry Download PDFInfo
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Abstract
The invention discloses a kind of transgenosis culture medium of diploid strawberry and its transgenic method, which includes: diploid strawberry seed culture medium S1, diploid strawberry co-culture medium S2, diploid strawberry screening and culturing medium S3；The transgenic method includes: (1) actication of culture, (2) diploid strawberry transgenosis；The present invention establishes an efficient, quick, stable diploid Strawberry transformation system, and explant uses blade and petiole, material is applied to the greatest extent, improves and improve diploid strawberry transgenosis transformation efficiency, and conversion ratio can reach 50% or more；And the step being able to solve in traditional agrobacterium-mediated transformation is many and diverse, the problem of being easy pollution, practical basis is provided further to apply genetic transforming method to improve strawberry cultivars, be conducive to the development of strawberry molecular breeding work from now on, transgenic method operating process of the present invention is simple, high conversion efficiency, the test period is short, spends human and material resources smaller.
Description
Technical field
The present invention relates to plant genetic engineerings, and in particular to a kind of diploid strawberry transgenosis culture medium and its turn base
Because of method.
Background technique
Strawberry is a kind of fruit for having both nutritive value and ornamental value, and cultivated strawberry is increasingly becoming farmer richness in recent years
First choice.And breeding is best in quality, the high strawberry cultivars of yield are also the vital task of strawberry research worker.With close
The rapid development of molecular biology and genetic engineering over year, the depth that research hotspot turns to strawberry germplasm excellent genes are dug
Pick, and carry out the identification and measurement of molecular labeling and expressed sequence.It is lost simultaneously in resistance, storage tolerance, flavor quality etc.
The result of study for passing improvement etc. is reported that Strwberry Breeding just develops towards molecular breeding direction in succession.And molecular breeding
In most important means be strawberry transgenosis.Usually strawberry cultivars are divided into two major classes by us, are the cultivation of octoploid respectively
Strawberry (Fragaria × ananassa, 2n=8x=56) and diploid fraises des bois kind.The common kind of cultivar all belongs to
Octoploid, and octoploid kind is height heterozygosis, genetic background is complicated, therefore the period is long in breeding, uncontrollable factor compared with
It is more.Wild diploid strawberry cultivars ' Rugen ' are that an excellent material in breeding can directly be broadcast because it does not have stolon
Kind is for breeding, and the kind only needs 6 months from fruit maturation is seeded into, and growth cycle is short, is easy to breed, therefore
It is the current common kind of strawberry research worker.Realize molecular breeding, it is necessary first to establish the transformation system of efficient stable.
Currently used strawberry 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 Strawberry transformation, and is studied in plant transgene research so far the most mature most normal
A kind of transgenic method.Although the transgenic technology route comparative maturity of strawberry, conventional method is bothersome laborious, time-consuming
Between it is long, cumbersome and efficiency is very low, and the transgenic plant of acquisition is very little, brings to the later period identification work of transgenic plant
Obstacle.Traditional agrobacterium-mediated transformation operating process is comparatively laborious, and many problems are easy to appear during transgenosis, than
Such as the selection of suitable carrier, the selection of Suitable strains, the pollution of miscellaneous bacteria, Agrobacterium can not inhibit, bacterial concentration is excessively high or mistake
It is low to lead to that transgenosis is unsuccessful, the control of time of infection length etc., these problems can all influence the efficiency of strawberry transgenosis with
And speed, the diploid strawberry transgene efficiency that conventional method obtains is less than 1 percent.And the transgenosis reported at present
System is built in octoploid strawberry more, and to the few correlative studys of diploid strawberry transformation system.Especially diploid
The transformation system of kind ' Rugen ' yet there are no relevant report.
Summary of the invention
Goal of the invention: being directed to existing issue, and the present invention provides a kind of transgenosis culture medium of diploid strawberry and its turns base
Because of method, the present invention establishes an efficient, quick, stable diploid Strawberry transformation system, can use petiole and blade simultaneously
Transgenosis is carried out to do explant, material is applied to the greatest extent, and can directly, efficiently solve traditional Agrobacterium and be situated between
The problem of step in inducing defecation by enema and suppository is many and diverse, is easy pollution；Solve the problems, such as diploid Strawberry transformation low efficiency.
Technical solution: to achieve the goals above, the present invention provides a kind of transgenosis culture medium of diploid strawberry, packets
Include following ingredient: diploid strawberry seed culture medium S1:2.0-2.5g/L MS, 15-25g/L sucrose, 0.2-0.5mg/L 6-
BA, 0.05-0.15mg/L NAA, 5.0-6.0g/L agar powder, pH5.5-6.0；
Diploid strawberry co-culture medium S2:4.2-4.6g/LMS, 25-35g/L sucrose, 0.2-0.4mg/L ZT,
0.4-0.6mg/L TDZ, 0.05-0.15mg/L IBA, 5.0-6.0g/L agar powder, pH5.5-6.0；
Diploid strawberry screening and culturing medium S3:4.2-4.6g/L MS, 25-35g/L sucrose, 0.2-0.4mg/L ZT, 0.4-
0.6mg/L TDZ, 0.05-0.15mg/L IBA, 5.0-6.0g/L agar powder, 10-30mg/L kanamycins, 300-500mg/L
Carbenicillin, pH5.5-6.0.
Preferably, the diploid strawberry seed culture medium S1, diploid strawberry co-culture medium S2, diploid grass
Certain kind of berries screening and culturing medium S3 carries out high pressure sterilization, spare.
6-BA is 6- benzyl aminoadenine, is a kind of substance of cytokinin, is had efficient, stable, cheap and easy
The favorite basic element of cell division of person that is tissue cultures in use the features such as, can be occurred with evoked callus.
NAA is methyl α-naphthyl acetate, is a kind of organic compound, is a kind of colorless solid for being soluble in organic solvent, is that plant is raw
Auxin analog in long regulator is usually used in commercial root of hair powder or rooting agent, when plant uses cuttage breeding
It uses, it can also be used to Plant Tissue Breeding.
IBA is indolebutyric acid, is common plant growth regulator in tissue cultures, induction and life for callus
Length is highly effective.
TDZ is a kind of new plant growth regulator, has very strong cytokine activity, and can be to plant hormone
Effect with physiological activator adjusts the growth and development process of plant, is the very strong plant growth regulating of active force
Agent is agriculturally being widely used and promotional value.
ZT is zeatin (Zeatin), is to be present in a kind of natural plant cells mitogen of higher plant not only to promote side
Bud growth, stimulates cell differentiation (side advantage), promotes callus and germination, moreover it is possible to prevent leaf senile, reverse bud
The toxic insult and excessive root is inhibited to be formed that portion is subject to.The zeatin of high concentration can also generate adventitious buds differentiation.
The present invention also provides a kind of transgenic methods of the transgenosis culture medium of diploid strawberry to include the following steps:
(1) actication of culture:
Plasmid pK7WG2D Agrobacterium EHA105 will be contained to cross on LB solid medium, 25-30 DEG C culture 1-3 days；
The agrobacterium strains EHA105 is that bacterial strain is commonly used in diploid strawberry transgenosis, and infection ability is stronger, the bacterial strain
It buys in American Type Culture collection warehousing ATCC (American type culture collection)；
The plasmid pK7WG2D is carried the reporter gene Egfp of superpower expression by plant expression vector pK7WG2D, using taking
Carrier with strongly expressed reporter gene Egfp can be screened directly to obtain transgenic plant, avoided in traditional detection by observation
Cumbersome work shortens the period of transgenosis；Plant expression vector pK7WG2D purchase is public in Invitrogen biotechnology
Department, accession number of the sequence of Egfp containing gene on NCBI are GenBank:KU721836.1.
Plant expression vector, which is selected, is based on GatewayTMThe pK7WG2D plant expression vector of technology, GatewayTMTechnology is big
The earth simplifies the step of gene cloning and subclone, GatewayTMSite-specific recombination is utilized, so carrying in building introduction
After body, it is no longer necessary to use restriction enzyme and ligase；
The Agrobacterium of pK7WG2D containing the plasmid EHA105's is the expression vector pK7WG2D conversion that will carry Egfp
Into agrobacterium strains EHA105.
Long good single colonie 2-3, is inoculated into 50mL LB liquid medium, 25-30 DEG C, 200- on picking plate
250rpm in shaking table shaken cultivation to OD600 value 0.3-0.5；
Under room temperature, 4000-6000rmp is centrifuged 7-9 minutes, removes supernatant, centrifuge tube is buckled on aseptic filter paper, will be upper
Thallus, the shaken cultivation in shaking table is resuspended in clear liquid removal, and measurement OD600 value is 0.1-0.3, and activated bacterium solution is made.
(2) diploid strawberry transgenosis
Material processing: the plantation of diploid strawberry seed is after on S1 culture medium, growing blade aseptically by blade
It cuts completely, it is crosscutting to prolong vein with scalpel；Strawberry petiole is cut into 0.5-1.5 centimeter length section simultaneously, hand is used on petiole
Art graduating with cutter wound；
It infects: the blade cut and the petiole for carving wound being directly placed into activated bacterium solution and infected, is slightly shaken up in the process,
Dip dyeing 30~40 minutes；Blade and petiole are taken out after the completion of infecting, are placed directly on co-culture medium S2, make blade and
Petiole wound comes into full contact with culture medium, and 23-27 DEG C in culturing room, dark culture 2-4 days, blade petiole is transferred to screening and culturing
It is cultivated 18-22 days on base S3, diploid Strawberry Leaves edge has white callus and grows, at this time in body formula fluorescence microscope
Under observed, a part of callus has the non-green light that is always on to issue, and a part is without shining.The kanamycin-resistant callus tissue of green light is retained
Get off, after culture in about 35-45 days, differentiates resistant plant；First order fluorescence detection is carried out again at this time, by entire resistance
Plant leaf and petiole all green lights remain, and what is glowed gets rid of；Resistant plant is grown to carry out once for 55-65 days again
Fluorescence detection, the blade of resistance seedling in fluorescence detection, petiole and all shinny green light of root can be determined as transgenic plant.
Preferably, plasmid pK7WG2D carries the reporter gene Egfp of superpower expression in step (1).Use the strong table of carrying
Up to the carrier of reporter gene Egfp, it can directly screen to obtain transgenic plant by observation, avoid cumbersome in traditional detection
Work, shortens the period of transgenosis.
Preferably, in step (1) LB solid medium and LB liquid medium all contain 50mg/L spectinomycin and
50mg/L rifampin.
Preferably, thallus is resuspended in the step (1) to be resuspended with 100mLMS liquid.
Preferably, being shaken once during dip dyeing 30-40 minutes every 5 minutes in the step (2).It can increase in this way
Add the contact area of bacterium solution and blade.
Preferably, it is kanamycin-resistant callus tissue that a part of callus, which has the non-green light being always on to issue, in the step (2), to retain
Under；A part does not shine as non-resistance callus, gets rid of.By the step, non-resistance callus is removed, is greatly reduced
Further work amount.
Either which kind of culture medium prescription in the prior art, is all easy to cause the glass of regeneration bud in diploid strawberry
Change, and regeneration efficiency is not high, and the S2 culture medium in the present invention can either avoid TDZ the and ZT compounding application of low concentration
The vitrifying of regeneration bud, and can be improved regeneration efficiency, to improve transgene efficiency；Blade is directly transferred to by the present invention simultaneously
S2 culture medium reduces a possibility that polluting in operating process without blotting net bacterium solution with filter paper.
In the present invention, washing blade is not needed after co-culturing, is transferred directly on S3 culture medium, it will be able to suppression well
Bacterium solution growth processed.
In detection process, the present invention is more simple, carries out PCR detection because no matter dyeing or sampling, requires to plant
Carry out destructive sampling, and the present invention directly can screen dress gene plant in fluorescence microscopy microscopic observation leaf color.Letter
It is also destroyed from plant while changing workflow.
The utility model has the advantages that compared with prior art, a kind of transgenosis culture medium of diploid strawberry of the present invention and its turn
Genetic method has the advantages that
1, diploid strawberry transgene efficiency is improved, conversion ratio can reach 50% or more；
2, bacterial concentration is reduced, time of infection lengthens, that is, avoids Agrobacterium from polluting, and improve infect efficiency；
3, it infects and does not add the drug medication that any auxiliary infects in bacterium solution and (often add acetosyringone in conventional method
Etc. the medicament that infects of auxiliary, blade can be damaged, reduce leaf regeneration ability), reduce the wound to diploid Strawberry Leaves
Evil, improves the survival rate and regeneration rate of leaf culture；
4, explant uses blade and petiole, and material is applied to the greatest extent, improves transformation efficiency；
5, using the carrier for carrying strongly expressed reporter gene, it can directly screen to obtain transgenic plant by observation, avoid
Cumbersome work in traditional detection, shortens the period of transgenosis；
6, operating process is simple, it is only necessary to hurt blade and petiole with operation graduating with cutter, no longer as conventional transgenic method one
Blade is cut into leaf block, reduces the workload of worker in this way by sample, and the speed to speed operations can also be kept away to the greatest extent
Exempt to pollute；
7, in the co-culture medium of diploid strawberry and in screening and culturing medium, for the first time by TDZ and ZT compounding application,
And concentration is all relatively low, this hormone combination not only largely reduces the vitrifying of regeneration bud, but also improves blade
The regeneration efficiency of petiole, to improve transformation efficiency.
The present invention establishes an efficient, quick, stable diploid Strawberry transformation system, further to apply genetic transformation
Method improvement strawberry cultivars provide practical basis, are conducive to the development of strawberry molecular breeding work from now on, transgenosis side of the present invention
Method operating process is simple, and high conversion efficiency, the test period is short, spends human and material resources smaller.
Specific embodiment
Embodiment 1
Culture medium:
Diploid strawberry seed culture medium S1:2.0g/L MS, 15g/L sucrose, 0.2mg/L 6-BA, 0.0.5mg/L
NAA, 5.0g/L agar powder, pH5.5；
Diploid strawberry co-culture medium S2:4.2g/LMS, 25g/L sucrose, 0.2mg/L ZT, 0.4mg/L TDZ,
0.05mg/L IBA, 5.0g/L agar powder, pH5.5；
Diploid strawberry screening and culturing medium S3:4.2g/L MS, 25g/L sucrose, 0.2mg/L ZT, 0.4mg/L TDZ,
0.05mg/L IBA, 5.0g/L agar powder, 10mg/L kanamycins, 300mg/L carbenicillin, pH5.5；
Diploid strawberry seed culture medium S1, diploid strawberry co-culture medium S2, diploid strawberry screening and culturing medium
S3 carries out high pressure sterilization, spare.
Transgenic method:
(1) actication of culture:
Plasmid pK7WG2D Agrobacterium EHA105 will be contained to train containing 50mg/L spectinomycin and 50mg/L rifampin LB solid
It supports and crosses on base, 25 DEG C are cultivated 3 days；
Long good single colonie 2, is inoculated into containing 50mg/L spectinomycin and 50mg/L rifampin on picking plate
In 50mL LB liquid medium, 25 DEG C, 250rpm in shaking table shaken cultivation to OD600 value 0.3；
Under room temperature, 4000rmp is centrifuged 9 minutes, removes supernatant, centrifuge tube is buckled on aseptic filter paper, supernatant is gone
It removes, thallus, the shaken cultivation in shaking table is resuspended with 100mLMS liquid, measurement OD600 value is 0.1, and activated bacterium solution is made；
(2) diploid strawberry transgenosis
Material processing: the plantation of diploid strawberry ' Rugen ' seed grows 3-5 piece leaf, In on S1 culture medium after 35 days
Blade is cut completely on superclean bench and is placed on sterile filter paper, prolongs the crosscutting 3-5 knife of vein with scalpel, but cannot incite somebody to action
Blade cutting, the blade cut is placed directly in activated bacterium solution and infects, while strawberry petiole is also cut into 0.5 centimeter length
Section, it is used at operation graduating with cutter wound two on petiole, petiole can not be cut off quarter when hurting, be directly placed into and activate after the completion of hurting at quarter
Bacterium solution infect；
It infects: the blade cut and the petiole for carving wound being directly placed into activated bacterium solution and infected, is slightly shaken up in the process,
Dip dyeing 30 minutes was shaken every 5 minutes primary in the process；Blade and petiole are taken out after the completion of infecting, are placed directly in total training
It supports on culture medium S2, comes into full contact with blade and petiole wound with culture medium, 23 DEG C in culturing room, dark culture 2 days, by blade
Petiole is transferred on screening and culturing medium S3 and cultivates 18 days, and diploid Strawberry Leaves edge has white callus and grows, and exists at this time
It is observed under body formula fluorescence microscope, a part of callus has the non-green light being always on to issue, this part callus is kanamycin-resistant callus tissue,
It retains；And a part is no luminous, this part callus is non-resistance callus, is got rid of；It, will be non-resistance by the step
Callus removal, greatly reduces further work amount；The kanamycin-resistant callus tissue of green light is remained, by culture in about 35 days with
Afterwards, resistant plant is differentiated；It carries out first order fluorescence detection again at this time, carries out observation resistant plant under body formula fluorescence microscope,
By remaining for entire resistant plant blade and petiole all green lights, what is glowed is got rid of；Resistant plant grows 55 days again
First order fluorescence detection is carried out, the blade of resistance seedling in fluorescence detection, being determined as of petiole and all shinny green light of root turns base
Because of plant.
Embodiment 2
Culture medium:
Diploid strawberry seed culture medium S1:2.5g/L MS, 25g/L sucrose, 0.5mg/L6-BA, 0.15mg/L NAA,
6.0g/L agar powder, pH6.0；
Diploid strawberry co-culture medium S2:4.6g/LMS, 35g/L sucrose, 0.4mg/L ZT, 0.6mg/L TDZ,
0.15mg/L IBA, 6.0g/L agar powder, pH6.0；
Diploid strawberry screening and culturing medium S3:4.6g/L MS, 35g/L sucrose, 0.4mg/L ZT, 0.6mg/L TDZ,
0.15mg/L IBA, 6.0g/L agar powder, 30mg/L kanamycins, 500mg/L carbenicillin, pH6.0；
Diploid strawberry seed culture medium S1, diploid strawberry co-culture medium S2, diploid strawberry screening and culturing medium
S3 carries out high pressure sterilization, spare.
Transgenic method:
(1) actication of culture:
Plasmid pK7WG2D Agrobacterium EHA105 will be contained to train containing 50mg/L spectinomycin and 50mg/L rifampin LB solid
It supports and crosses on base, 30 DEG C are cultivated 3 days；
Long good single colonie 3, is inoculated into containing 50mg/L spectinomycin and 50mg/L rifampin on picking plate
In 50mL LB liquid medium, 30 DEG C, 200rpm in shaking table shaken cultivation to OD600 value 0.5；
Under room temperature, 6000rmp is centrifuged 7 minutes, removes supernatant, centrifuge tube is buckled on aseptic filter paper, supernatant is gone
It removes, thallus, the shaken cultivation in shaking table is resuspended with 100mLMS liquid, measurement OD600 value is 0.3, and activated bacterium solution is made；
(2) diploid strawberry transgenosis
Material processing: the plantation of diploid strawberry ' Rugen ' seed grows 3-5 piece leaf, In on S1 culture medium after 35 days
Blade is cut completely on superclean bench and is placed on sterile filter paper, prolongs the crosscutting 3-5 knife of vein with scalpel, but cannot incite somebody to action
Blade cutting, the blade cut is placed directly in activated bacterium solution and infects, while strawberry petiole is also cut into 1.5 centimeter lengths
Section, it is used at operation graduating with cutter wound two on petiole, petiole can not be cut off quarter when hurting, be directly placed into and activate after the completion of hurting at quarter
Bacterium solution infect；
It infects: the blade cut and the petiole for carving wound being directly placed into activated bacterium solution and infected, is slightly shaken up in the process,
Dip dyeing 40 minutes was shaken every 5 minutes primary in the process；Blade and petiole are taken out after the completion of infecting, are placed directly in total training
It supports on culture medium S2, comes into full contact with blade and petiole wound with culture medium, 27 DEG C in culturing room, dark culture 2 days, by blade
Petiole is transferred on screening and culturing medium S3 and cultivates 22 days, and diploid Strawberry Leaves edge has white callus and grows, and exists at this time
It is observed under body formula fluorescence microscope, a part of callus has the non-green light being always on to issue, this part callus is kanamycin-resistant callus tissue,
It retains；And a part is no luminous, this part callus is non-resistance callus, is got rid of；It, will be non-resistance by the step
Callus removal, greatly reduces further work amount；The kanamycin-resistant callus tissue of green light is remained, by culture in about 45 days with
Afterwards, resistant plant is differentiated；It carries out first order fluorescence detection again at this time, carries out observation resistant plant under body formula fluorescence microscope,
By remaining for entire resistant plant blade and petiole all green lights, what is glowed is got rid of；Resistant plant grows 65 days again
First order fluorescence detection is carried out, the blade of resistance seedling in fluorescence detection, being determined as of petiole and all shinny green light of root turns base
Because of plant.
Embodiment 3
Culture medium:
Diploid strawberry seed culture medium S1:2.2g/L MS, 20g/L sucrose, 0.3mg/L 6-BA, 0.1mg/L NAA,
5.5g/L agar powder, pH 5.8；
Diploid strawberry co-culture medium S2:4.4g/LMS, 30g/L sucrose, 0.3mg/L ZT, 0.5mg/L TDZ,
0.1mg/L IBA, 5.5g/L agar powder, pH5.8；
Diploid strawberry screening and culturing medium S3:4.4g/L MS, 30g/L sucrose, 0.3mg/L ZT, 0.5mg/L TDZ,
0.1mg/L IBA, 5.5g/L agar powder, 20mg/L kanamycins, 400mg/L carbenicillin, pH5.8；
Diploid strawberry seed culture medium S1, diploid strawberry co-culture medium S2, diploid strawberry screening and culturing medium
S3 carries out high pressure sterilization, spare.
Transgenic method:
(1) actication of culture:
Plasmid pK7WG2D Agrobacterium EHA105 will be contained to train containing 50mg/L spectinomycin and 50mg/L rifampin LB solid
It supports and crosses on base, 28 DEG C are cultivated 2 days；
Long good single colonie 3, is inoculated into containing 50mg/L spectinomycin and 50mg/L rifampin on picking plate
In 50mL LB liquid medium, 28 DEG C, 220rpm in shaking table shaken cultivation to OD600 value 0.4；
Under room temperature, 5000rmp is centrifuged 8 minutes, removes supernatant, centrifuge tube is buckled on aseptic filter paper, supernatant is gone
It removes, thallus, the shaken cultivation in shaking table is resuspended with 100mLMS liquid, measurement OD600 value is 0.2, and activated bacterium solution is made；
(2) diploid strawberry transgenosis
Material processing: the plantation of diploid strawberry ' Rugen ' seed grows 3-5 piece leaf, In on S1 culture medium after 35 days
Blade is cut completely on superclean bench and is placed on sterile filter paper, prolongs the crosscutting 3-5 knife of vein with scalpel, but cannot incite somebody to action
Blade cutting, the blade cut is placed directly in activated bacterium solution and infects, while strawberry petiole is also cut into 0.1 centimeter length
Section, it is used at operation graduating with cutter wound two on petiole, petiole can not be cut off quarter when hurting, be directly placed into and activate after the completion of hurting at quarter
Bacterium solution infect；
It infects: the blade cut and the petiole for carving wound being directly placed into activated bacterium solution and infected, is slightly shaken up in the process,
Dip dyeing 35 minutes was shaken every 5 minutes primary in the process；Blade and petiole are taken out after the completion of infecting, are placed directly in total training
It supports on culture medium S2, comes into full contact with blade and petiole wound with culture medium, 24 DEG C in culturing room, dark culture 3 days, by blade
Petiole is transferred on screening and culturing medium S3 and cultivates 20 days, and diploid Strawberry Leaves edge has white callus and grows, and exists at this time
It is observed under body formula fluorescence microscope, a part of callus has the non-green light being always on to issue, this part callus is kanamycin-resistant callus tissue,
It retains；And a part is no luminous, this part callus is non-resistance callus, is got rid of；It, will be non-resistance by the step
Callus removal, greatly reduces further work amount；The kanamycin-resistant callus tissue of green light is remained, by culture in about 40 days with
Afterwards, resistant plant is differentiated；It carries out first order fluorescence detection again at this time, carries out observation resistant plant under body formula fluorescence microscope,
By remaining for entire resistant plant blade and petiole all green lights, what is glowed is got rid of；Resistant plant growth 50 again into
The detection of row first order fluorescence, the blade of resistance seedling in fluorescence detection, petiole and all shinny green light of root can be determined as transgenosis
Plant.
Embodiment 4
Culture medium:
Diploid strawberry seed culture medium S1:2.0g/L MS, 25g/L sucrose, 0.5mg/L 6-BA, 0.15mg/L NAA,
5.0g/L agar powder, pH5.7；
Diploid strawberry co-culture medium S2:4.6g/LMS, 35g/L sucrose, 0.2mg/L ZT, 0.4TDZ,
0.15mg/L IBA, 6.0g/L agar powder, pH5.7；
Diploid strawberry screening and culturing medium S3:4.2g/L MS, 25g/L sucrose, 0.4mg/L ZT, 0.6mg/L TDZ,
0.05mg/L IBA, 6.0g/L agar powder, 20mg/L kanamycins, 400mg/L carbenicillin, pH5.7；
Diploid strawberry seed culture medium S1, diploid strawberry co-culture medium S2, diploid strawberry screening and culturing medium
S3 carries out high pressure sterilization, spare.
Transgenic method:
(1) actication of culture:
Plasmid pK7WG2D Agrobacterium EHA105 will be contained to train containing 50mg/L spectinomycin and 50mg/L rifampin LB solid
It supports and crosses on base, 28 DEG C are cultivated 2 days；
Long good single colonie 3, is inoculated into containing 50mg/L spectinomycin and 50mg/L rifampin on picking plate
In 50mL LB liquid medium, 28 DEG C, 220rpm in shaking table shaken cultivation to OD600 value 0.4；
Under room temperature, 5000rmp is centrifuged 8 minutes, removes supernatant, centrifuge tube is buckled on aseptic filter paper, supernatant is gone
It removes, thallus, the shaken cultivation in shaking table is resuspended with 100mLMS liquid, measurement OD600 value is 0.2, and activated bacterium solution is made；
(2) diploid strawberry transgenosis
Material processing: the plantation of diploid strawberry ' Rugen ' seed grows 3-5 piece leaf, In on S1 culture medium after 35 days
Blade is cut completely on superclean bench and is placed on sterile filter paper, prolongs the crosscutting 3-5 knife of vein with scalpel, but cannot incite somebody to action
Blade cutting, the blade cut is placed directly in activated bacterium solution and infects, while strawberry petiole is also cut into 0.1 centimeter length
Section, it is used at operation graduating with cutter wound two on petiole, petiole can not be cut off quarter when hurting, be directly placed into and activate after the completion of hurting at quarter
Bacterium solution infect；
It infects: the blade cut and the petiole for carving wound being directly placed into activated bacterium solution and infected, is slightly shaken up in the process,
Dip dyeing 35 minutes was shaken every 5 minutes primary in the process；Blade and petiole are taken out after the completion of infecting, are placed directly in total training
It supports on culture medium S2, comes into full contact with blade and petiole wound with culture medium, 24 DEG C in culturing room, dark culture 3 days, by blade
Petiole is transferred on screening and culturing medium S3 and cultivates 20 days, and diploid Strawberry Leaves edge has white callus and grows, and exists at this time
It is observed under body formula fluorescence microscope, a part of callus has the non-green light being always on to issue, this part callus is kanamycin-resistant callus tissue,
It retains；And a part is no luminous, this part callus is non-resistance callus, is got rid of；It, will be non-resistance by the step
Callus removal, greatly reduces further work amount；The kanamycin-resistant callus tissue of green light is remained, by culture in about 40 days with
Afterwards, resistant plant is differentiated；It carries out first order fluorescence detection again at this time, carries out observation resistant plant under body formula fluorescence microscope,
By remaining for entire resistant plant blade and petiole all green lights, what is glowed is got rid of；Resistant plant growth 50 again into
The detection of row first order fluorescence, the blade of resistance seedling in fluorescence detection, petiole and all shinny green light of root can be determined as transgenosis
Plant.
Embodiment 5
Culture medium
Diploid strawberry seed culture medium S1:2.5g/L MS, 25g/L sucrose, 0.2mg/L 6-BA, 0.05mg/L NAA,
6.0g/L agar powder, pH5.8；
Diploid strawberry co-culture medium S2:4.6g/LMS, 35g/L sucrose, 0.4mg/L ZT, 0.6mg/L TDZ,
0.05mg/L IBA, 6.0g/L agar powder, pH5.8；
Diploid strawberry screening and culturing medium S3:4.6g/L MS, 35g/L sucrose, 0.2mg/L ZT, 0.6mg/L TDZ,
0.05mg/L IBA, 5.0g/L agar powder, 20mg/L kanamycins, 400mg/L carbenicillin, pH5.8；
Diploid strawberry seed culture medium S1, diploid strawberry co-culture medium S2, diploid strawberry screening and culturing medium
S3 carries out high pressure sterilization, spare.
Transgenic method:
(1) actication of culture:
Plasmid pK7WG2D Agrobacterium EHA105 will be contained to train containing 50mg/L spectinomycin and 50mg/L rifampin LB solid
It supports and crosses on base, 28 DEG C are cultivated 2 days；
Long good single colonie 3, is inoculated into containing 50mg/L spectinomycin and 50mg/L rifampin on picking plate
In 50mL LB liquid medium, 28 DEG C, 220rpm in shaking table shaken cultivation to OD600 value 0.4；
Under room temperature, 5000rmp is centrifuged 8 minutes, removes supernatant, centrifuge tube is buckled on aseptic filter paper, supernatant is gone
It removes, thallus, the shaken cultivation in shaking table is resuspended with 100mLMS liquid, measurement OD600 value is 0.2, and activated bacterium solution is made；
(2) diploid strawberry transgenosis
Material processing: the plantation of diploid strawberry ' Rugen ' seed grows 3-5 piece leaf, In on S1 culture medium after 35 days
Blade is cut completely on superclean bench and is placed on sterile filter paper, prolongs the crosscutting 3-5 knife of vein with scalpel, but cannot incite somebody to action
Blade cutting, the blade cut is placed directly in activated bacterium solution and infects, while strawberry petiole is also cut into 0.1 centimeter length
Section, it is used at operation graduating with cutter wound two on petiole, petiole can not be cut off quarter when hurting, be directly placed into and activate after the completion of hurting at quarter
Bacterium solution infect；
It infects: the blade cut and the petiole for carving wound being directly placed into activated bacterium solution and infected, is slightly shaken up in the process,
Dip dyeing 35 minutes was shaken every 5 minutes primary in the process；Blade and petiole are taken out after the completion of infecting, are placed directly in total training
It supports on culture medium S2, comes into full contact with blade and petiole wound with culture medium, 24 DEG C in culturing room, dark culture 3 days, by blade
Petiole is transferred on screening and culturing medium S3 and cultivates 20 days, and diploid Strawberry Leaves edge has white callus and grows, and exists at this time
It is observed under body formula fluorescence microscope, a part of callus has the non-green light being always on to issue, this part callus is kanamycin-resistant callus tissue,
It retains；And a part is no luminous, this part callus is non-resistance callus, is got rid of；It, will be non-resistance by the step
Callus removal, greatly reduces further work amount；The kanamycin-resistant callus tissue of green light is remained, by culture in about 40 days with
Afterwards, resistant plant is differentiated；It carries out first order fluorescence detection again at this time, carries out observation resistant plant under body formula fluorescence microscope,
By remaining for entire resistant plant blade and petiole all green lights, what is glowed is got rid of；Resistant plant growth 50 again into
The detection of row first order fluorescence, the blade of resistance seedling in fluorescence detection, petiole and all shinny green light of root can be determined as transgenosis
Plant.
Embodiment 6
The test of fruit is carried out if necessary to obtain fruit, embodiment acquisition transgenic plant can be continued into culture and moved back
Cultivation is yielded positive results into field.It, can be under fluorescence microscope in order to further determine the stability of transgenosis after yielding positive results
The colored fluorescence with fruit different developmental phases of detection again
Embodiment 7
The conversion ratio of diploid strawberry transgenosis of the present invention, is shown in Table 1.
Comparative example 1 is the conversion ratio of octoploid strawberry transgenosis in the prior art；
Comparative example 2 is the conversion ratio of diploid strawberry transgenosis in the prior art；
The conversion ratio of the diploid strawberry transgenosis of the present invention of table 1
As shown in Table 1, the conversion ratio of diploid strawberry transgenosis is attained by 50% or more in the embodiment of the present invention, remote high
In the conversion ratio of the prior art.
Claims (7)
1. a kind of transgenic method of the transgenosis culture medium of diploid strawberry, which comprises the steps of:
(1) actication of culture:
Plasmid pK7WG2D Agrobacterium EHA105 will be contained to cross on LB solid medium, 25-30 DEG C culture 1-3 days；
Long good single colonie 2-3, is inoculated into 50 mL LB liquid mediums, 25-30 DEG C, 200-250 on picking plate
Rpm in shaking table shaken cultivation to OD600 value 0.3-0.5；
Under room temperature, 4000-6000rmp is centrifuged 7-9 minutes, removes supernatant, centrifuge tube is buckled on aseptic filter paper, by supernatant
Thallus, the shaken cultivation in shaking table is resuspended in liquid removal, and measurement OD600 value is 0.1-0.3, and activated bacterium solution is made；
(2) diploid strawberry transgenosis
Material processing: the plantation of diploid strawberry seed is aseptically complete by blade after on S1 culture medium, growing blade
It cuts, it is crosscutting to prolong vein with scalpel；Strawberry petiole is cut into 0.5-1.5 centimeter length section simultaneously, scalpel is used on petiole
Carve wound；
It infects: the blade cut and the petiole for carving wound being directly placed into activated bacterium solution and infected, is slightly shaken up in the process, is disseminated
30-40 minutes；Blade and petiole are taken out after the completion of infecting, is placed directly on co-culture medium S2, makes blade and petiole
Wound comes into full contact with culture medium, and 23-27 DEG C in culturing room, dark culture 2-4 days, blade petiole is transferred to screening and culturing medium
It is cultivated 18-22 days on S3, diploid Strawberry Leaves edge has white callus and grows, at this time under body formula fluorescence microscope
It is observed, a part of callus has the non-green light being always on to issue, and a part is without shining；The kanamycin-resistant callus tissue of green light is retained
Come, after culture in 35-45 days, differentiates resistant plant；First order fluorescence detection is carried out again at this time, by entire resistant plant
Blade and petiole all green lights remain, and what is glowed gets rid of；Resistant plant is grown 55-65 days carries out first order fluorescence again
Detection, the blade of resistance seedling in fluorescence detection, petiole and all shinny green light of root can be determined as transgenic plant；
Diploid strawberry seed culture medium S1:2.0-2.5g/L MS, 15-25g/L sucrose, 0.2-0.5mg/L 6-BA, 0.0.5-
0.15mg/L NAA, 5.0-6.0 g/L agar powder, pH5.5-6.0；
Diploid strawberry co-culture medium S2:4.2-4.6 g/LMS, 25-35g/L sucrose, 0.2-0.4mg/L ZT, 0.4-
0.6mg/L TDZ, 0.05-0.15mg/L IBA, 5.0-6.0 g/L agar powder, pH5.5-6.0；
Diploid strawberry screening and culturing medium S3:4.2-4.6 g/L MS, 25-35g/L sucrose, 0.2-0.4mg/L ZT, 0.4-
0.6mg/L TDZ, 0.05-0.15mg/L IBA, 5.0-6.0 g/L agar powder, 10-30mg/L kanamycins, 300-500
Mg/L carbenicillin, pH5.5-6.0.
2. the transgenic method of the transgenosis culture medium of diploid strawberry according to claim 1, which is characterized in that described two
Times body strawberry seed culture medium S1, diploid strawberry co-culture medium S2, diploid strawberry screening and culturing medium S3 carry out height
Pressure sterilizing, it is spare.
3. transgenic method according to claim 1, which is characterized in that plasmid pK7WG2D carries super in the step (1)
The reporter gene of strongly expressedEgfp。
4. transgenic method according to claim 1, which is characterized in that LB solid medium and LB in the step (1)
Fluid nutrient medium all contains 50 mg/L spectinomycins and 50 mg/L rifampins.
5. transgenic method according to claim 1, which is characterized in that it is with 100 that thallus is resuspended in the step (1)
MLMS liquid is resuspended.
6. transgenic method according to claim 1, which is characterized in that disseminate 30-40 minutes mistakes in the step (2)
It was shaken once in journey every 5 minutes.
7. transgenic method according to claim 1, which is characterized in that there is very a part of callus in the step (2)
It is kanamycin-resistant callus tissue that bright green light, which issues, to be retained；A part does not shine as non-resistance callus, gets rid of.
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| CN105200080A (en) * | 2015-09-24 | 2015-12-30 | 江苏农林职业技术学院 | Method for efficiently and rapidly stabilizing gene transformation for tomatoes |
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| WO2012069539A1 (en) * | 2010-11-24 | 2012-05-31 | Nunhems B. V. | Dual purpose pollenizer watermelons |
| CN104195171A (en) * | 2014-09-16 | 2014-12-10 | 江苏农林职业技术学院 | Efficient, rapid and stable genetic transformation method for strawberries |
| CN105200080A (en) * | 2015-09-24 | 2015-12-30 | 江苏农林职业技术学院 | Method for efficiently and rapidly stabilizing gene transformation for tomatoes |
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