CN106047921A - Growth media for genetic modification of diploid strawberries and genetic modification method adopting growth media - Google Patents
Growth media for genetic modification of diploid strawberries and genetic modification method adopting growth media Download PDFInfo
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Abstract
The invention discloses growth media for genetic modification of diploid strawberries and a genetic modification method adopting the growth media. The growth media for the genetic modification of the diploid strawberries comprise a diploid strawberry seed culture medium S1, a diploid strawberry co-culture medium S2 and a diploid strawberry selection medium S3. The genetic modification method comprises the following steps: (1) activation of cultures; (2) genetic modification of the diploid strawberries. An efficient, fast and stable diploid strawberry transformation system is established, blades and petioles are adopted as explants, materials are used to the greatest extent, the transformation efficiency of genetic modification of the diploid strawberries is increased and the transformation ratio can be 50% or higher; the problems of complex steps and proneness to pollution of the traditional agrobacterium-mediated method can be solved, practice foundation is provided for improvement of strawberry varieties through further application of a genetic transformation method, development of strawberry molecular breeding in future is facilitated, and the genetic modification method has the advantages of simple operation process, high transformation efficiency, short test period, less consumed manpower and fewer consumed material resources.
Description
Technical field
The present invention relates to plant genetic engineering, be specifically related to the transgenic culture medium of a kind of diploid Fructus Fragariae Ananssae and turn base
Because of method.
Background technology
Fructus Fragariae Ananssae is a kind of fruit having nutritive value and ornamental value concurrently, and cultivated strawberry is increasingly becoming farmer richness in recent years
First-selection.And selection-breeding is best in quality, the strawberry cultivars that yield is high is also a vital task of Fructus Fragariae Ananssae research worker.Along with closely
Molecular biology and engineered develop rapidly over Nian, study hotspot turns to the degree of depth of strawberry germplasm excellent genes to dig
Pick, and carry out molecular marker and the qualification of expressed sequence and mensuration.Lose at resistance, storage tolerance, flavor quality etc. simultaneously
The result of study passing the aspects such as improvement has been reported, Strwberry Breeding just develops towards molecular breeding direction.And molecular breeding
In most important means be the transgenic of Fructus Fragariae Ananssae.Generally strawberry cultivars is divided into two big classes by us, is the cultivation of octoploid respectively
Fructus Fragariae Ananssae (Fragaria × ananassa, 2n=8x=56) and diploid fraises des bois kind.The common kind of cultigen all belongs to
Octoploid, and octoploid kind is height heterozygosis, genetic background is complicated, and therefore in breeding, the cycle is long, and uncontrollable factor is relatively
Many.Wild diploid strawberry cultivars ' Rugen ' is an excellent material in breeding, because it does not has stolon, can directly broadcast
Planting and be used for breeding, and this kind has only to 6 months from being seeded into fruit maturation, growth cycle is short, it is easy to breeding, therefore
It it is the kind commonly used of current Fructus Fragariae Ananssae research worker.Realize molecular breeding, it is necessary first to set up the transformation system of efficient stable.
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 Strawberry transformation, is also that to study maturation the most in plant transgene research up to now the most normal
A kind of transgenic method.Although the transgenic technology route comparative maturity of Fructus Fragariae Ananssae, but traditional method is bothersome arduously, time-consumingly
Between long, complex operation and efficiency is the lowest, it is thus achieved that transfer-gen plant very little, bring to the later stage appraisal of transfer-gen plant
Obstacle.Traditional agrobacterium-mediated transformation operating process is comparatively laborious, easily occurs many problems during transgenic, than
As the selection of suitable carrier, the selection of Suitable strains, the pollution of miscellaneous bacteria, Agrobacterium cannot suppress, bacterial concentration is too high or mistake
Low cause that transgenic is unsuccessful, the control of time of infection length etc., these problems all can affect the efficiency of Fructus Fragariae Ananssae transgenic with
And speed, the diploid strawberry transgene efficiency that traditional method obtains is less than one of percentage.And the transgenic reported at present
System builds in octoploid Fructus Fragariae Ananssae more, and diploid Fructus Fragariae Ananssae transformation system is seldom had correlational study.Especially diploid
The transformation system of kind ' Rugen ' yet there are no relevant report.
Summary of the invention
Goal of the invention: for existing issue, the present invention provides the transgenic culture medium of a kind of diploid Fructus Fragariae Ananssae and turns base
Because of method, the present invention establish one efficiently, quickly, stablize diploid Strawberry transformation system, petiole and blade can be used simultaneously
Do outer implant and carry out transgenic, material is farthest applied, and Traditional Agricultural bacillus Jie can be solved directly, efficiently
Step in inducing defecation by enema and suppository is numerous and diverse, the problem easily polluted；Solve the inefficient problem of diploid Strawberry transformation.
Technical scheme: to achieve these goals, the invention provides the transgenic culture medium of a kind of diploid Fructus Fragariae Ananssae, bag
Include following composition: diploid Fructus Fragariae Ananssae 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 Fructus Fragariae Ananssae co-cultures 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 Fructus Fragariae Ananssae screening culture 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 kanamycin, 300-500mg/L
Carbenicillin, pH5.5-6.0.
As preferably, described diploid Fructus Fragariae Ananssae seed culture medium S1, diploid Fructus Fragariae Ananssae co-culture culture medium S2, diploid grass
Certain kind of berries screening culture medium S3 all carries out autoclaving, standby.
6-BA is 6-benzyl aminoadenine, is the material of a kind of cytokinin, has efficient, stable, cheap and easy
It is the favorite basic element of cell division of tissue culture person in features such as uses, can occur with callus induction.
NAA is naphthalene acetic acid, is a kind of organic compound, is a kind of colorless solid being soluble in organic solvent, is that plant is raw
Auxin analog in long regulator, is usually used in root of hair powder or the rooting agent of commercialization, when plant uses cuttage breeding
Use it can also be used to plant tissue culture.
IBA is indolebutyric acid, is plant growth regulator conventional in tissue culture, for induction and the life of callus
Long highly effective.
TDZ is a kind of new plant growth regulator, has the strongest cytokine activity, and can be to phytohormone
Effect with biological active substances regulates the growth and development process of plant, is the plant growth regulating that an active force is the strongest
Agent, is agriculturally being widely used and promotional value.
ZT is zeatin (Zeatin), and a kind of natural plant cells mitogen being present in higher plant not only promotes side
Blastogenesis is long, stimulates cell differentiation (side advantage), promotes callus and germination, moreover it is possible to prevent leaf senile, reverses bud
The toxic insult that portion is subject to and the excessive root of suppression are formed.The zeatin of high concentration can also produce adventitious buds differentiation.
The transgenic method of the transgenic culture medium that present invention also offers a kind of diploid Fructus Fragariae Ananssae comprises the steps:
(1) actication of culture:
Plasmid pK7WG2D Agrobacterium EHA105 will be contained rule on LB solid medium, cultivate 1-3 days for 25-30 DEG C；
Described agrobacterium strains EHA105 is conventional bacterial strain in diploid Fructus Fragariae Ananssae transgenic, and infection ability is relatively strong, this bacterial strain
Buy in American Type Culture collection warehousing ATCC (American type culture collection)；
Described plasmid pK7WG2D is carried the reporter gene Egfp of superpower expression by plant expression vector pK7WG2D, and use is taken
With the carrier of strongly expressed reporter gene Egfp, obtain transfer-gen plant by observing directly to screen, it is to avoid in traditional detection
Loaded down with trivial details work, shortens the cycle of transgenic；Plant expression vector pK7WG2D buys in Invitrogen biotechnology public
Department, the accession number on NCBI of the Egfp sequence containing gene is GenBank:KU721836.1.
Plant expression vector is selected based on GatewayTMThe pK7WG2D plant expression vector of technology, GatewayTMTechnology is big
The earth simplifies the step of gene clone and subclone, GatewayTMSite-specific is make use of to recombinate, so carrying building introduction
After body, it is no longer necessary to use restricted enzyme and ligase；
Described containing plasmid pK7WG2D Agrobacterium EHA105 be will carry Egfp expression vector pK7WG2D convert
In agrobacterium strains EHA105.
Long on picking flat board good single bacterium colony 2-3, is inoculated in 50mL LB fluid medium, 25-30 DEG C, 200-
250rpm in shaking table shaken cultivation to OD600 value 0.3-0.5；
Under room temperature, 4000-6000rmp is centrifuged 7-9 minute, removes supernatant, back-off centrifuge tube on aseptic filter paper, by upper
Clear liquid is removed, resuspended thalline, shaken cultivation in shaking table, and measuring OD600 value is 0.1-0.3, prepares the bacterium solution activated.
(2) diploid Fructus Fragariae Ananssae transgenic
Material processes: diploid Fructus Fragariae Ananssae seed is planted in S1 culture medium, grows after blade aseptically by blade
Cut completely, prolong vein with scalpel crosscutting；Fructus Fragariae Ananssae petiole is cut into 0.5-1.5 centimeter length section simultaneously, on petiole, uses hands
Art graduating with cutter is hindered；
Infect: by the blade cut with carve the petiole of wound and be directly placed into the bacterium solution activated and infect, during slightly shake up,
Contaminate 30～40 minutes；After having infected, blade and petiole are taken out, are placed directly in and co-culture in culture medium S2, make blade and
Petiole wound is fully contacted with culture medium, in culturing room 23-27 DEG C, and blade petiole is proceeded to screening and culturing by light culture 2-4 days
Cultivating 18-22 days on base S3, diploid Strawberry Leaves edge has white callus and grows, now at body formula fluorescence microscope
Under observe, a part of wound healing has the brightest green glow to send, and a part does not has luminescence.The kanamycin-resistant callus tissue of green light is retained
Get off, after the cultivation of about 35-45 days, differentiate resistant plant；Carry out first order fluorescence detection the most again, by whole resistance
Remaining of plant leaf and petiole all green light, glow gets rid of；Resistant plant growth is carried out once for 55-65 days again
Fluoroscopic examination, in fluoroscopic examination, the most shinny green glow of the blade of resistance Seedling, petiole and root may determine that as transfer-gen plant.
As preferably, in step (1), plasmid pK7WG2D carries the reporter gene Egfp of superpower expression.Strong table is carried in use
Reach the carrier of reporter gene Egfp, obtain transfer-gen plant by observing directly to screen, it is to avoid loaded down with trivial details in traditional detection
Work, shortens the cycle of transgenic.
As preferably, in step (1) LB solid medium and LB fluid medium all contain 50mg/L spectinomycin and
50mg/L rifampicin.
As preferably, in described step (1), resuspended thalline is resuspended with 100mLMS liquid.
As preferably, shook once every 5 minutes during described step (2) is contaminated 30-40 minute.So can increase
Add the contact area of bacterium solution and blade.
As preferably, in described step (2), a part of wound healing has the brightest green glow to send as kanamycin-resistant callus tissue, retain
Under；It is non-resistance wound healing that a part does not has luminous, gets rid of.By the step for, non-resistance wound healing is removed, is greatly reduced
Further work amount.
The most either which kind of culture medium prescription, is all easily caused the glass of regeneration bud in diploid Fructus Fragariae Ananssae
Change, and regeneration efficiency is the highest, and the S2 culture medium in the present invention, by TDZ and the ZT compounding application of low concentration, can either avoid
The vitrification of regeneration bud, can improve again regeneration efficiency, thus improve transgene efficiency；Blade is directly proceeded to by the present invention simultaneously
S2 culture medium, blots clean bacterium solution without with filter paper, reduces the probability polluted in operating process.
In the present invention, co-culture and need not washing blade afterwards, be transferred directly in S3 culture medium, it becomes possible to well press down
Bacterium solution processed grows.
In detection, the process present invention is the simplest, carries out PCR detection because no matter dyeing or sampling, is required for plant
Carry out destructive sampling, and the present invention directly can screen dress gene plant at fluorescence microscopy Microscopic observation leaf color.Letter
Also avoid plant while changing workflow to be destroyed.
Beneficial effect: compared with prior art, the transgenic culture medium of a kind of diploid Fructus Fragariae Ananssae of the present invention and turning
Genetic method has the advantage that
1, improving diploid Fructus Fragariae Ananssae transgene efficiency, conversion ratio can reach more than 50%；
2, being reduced by bacterial concentration, time of infection lengthens, and i.e. avoids Agrobacterium to pollute, improves again and infect efficiency；
3, infect the drug medication infected without any auxiliary in bacterium solution and (traditional method is often added acetosyringone
The medicament infected Deng auxiliary, can damage blade, reduces leaf regeneration ability), reduce the wound to diploid Strawberry Leaves
Evil, improves survival rate and the regeneration rate of leaf culture；
4, outer implant uses blade and petiole, is farthest applied by material, improves transformation efficiency；
5, the 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；
6, operating process is simple, it is only necessary to hinder blade and petiole with operation graduating with cutter, no longer as conventional transgenic method one
Sample, is cut into leaf block by blade, which reduces the workload of worker, the speed speeded operations, it is also possible to farthest keep away
Exempt to pollute；
7, co-culturing in culture medium and in screening culture medium diploid Fructus Fragariae Ananssae, first by TDZ and ZT compounding application,
And concentration all ratios are relatively low, this hormone combination the most largely reduces the vitrification of regeneration bud, and improves blade
The regeneration efficiency of petiole, thus improve transformation efficiency.
The present invention establish one efficiently, quickly, stablize diploid Strawberry transformation system, for applying genetic transformation further
Method improvement strawberry cultivars provides practical basis, is conducive to the development of Fructus Fragariae Ananssae molecular breeding work from now on, transgenic side of the present invention
Method operating process is simple, and transformation efficiency is high, and the test period is short, and labor intensive material resources are less.
Detailed description of the invention
Embodiment 1
Culture medium:
Diploid Fructus Fragariae Ananssae 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 Fructus Fragariae Ananssae co-culture 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 Fructus Fragariae Ananssae screening culture 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 kanamycin, 300mg/L Carbenicillin, pH5.5；
Diploid Fructus Fragariae Ananssae seed culture medium S1, diploid Fructus Fragariae Ananssae co-culture culture medium S2, diploid Fructus Fragariae Ananssae screening culture medium
S3 carries out autoclaving, standby.
Transgenic method:
(1) actication of culture:
Plasmid pK7WG2D Agrobacterium EHA105 will be contained training containing 50mg/L spectinomycin and 50mg/L rifampicin LB solid
Support and rule on base, cultivate 3 days for 25 DEG C；
Good single bacterium colony 2 long on picking flat board, is inoculated into containing 50mg/L spectinomycin and 50mg/L rifampicin
In 50mL LB fluid 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, and supernatant is gone by back-off centrifuge tube on aseptic filter paper
Removing, by the resuspended thalline of 100mLMS liquid, shaken cultivation in shaking table, measuring OD600 value is 0.1, prepares the bacterium solution activated；
(2) diploid Fructus Fragariae Ananssae transgenic
Material processes: diploid Fructus Fragariae Ananssae ' Rugen ' seed is planted in S1 culture medium, grows 3-5 sheet leaf after 35 days,
On superclean bench, blade is cut completely and be positioned on aseptic filter paper, prolong vein crosscutting 3-5 cutter with scalpel, but can not be by
Blade cuts off, and the blade cut is placed directly in the bacterium solution activated and infects, and also Fructus Fragariae Ananssae petiole is cut into 0.5 centimeter length simultaneously
Section, hinders at two with operation graduating with cutter on petiole, carves and can not cut off petiole when hindering, and wound was directly placed into after completing and activated quarter
Bacterium solution infect；
Infect: by the blade cut with carve the petiole of wound and be directly placed into the bacterium solution activated and infect, during slightly shake up,
Contaminate 30 minutes, during every 5 minutes shake once；After having infected, blade and petiole are taken out, be placed directly in common training
Supporting in culture medium S2, make blade and petiole wound be fully contacted with culture medium, in culturing room 23 DEG C, light culture 2 days, by blade
Petiole proceeds to cultivate 18 days in screening culture medium S3, and diploid Strawberry Leaves edge has white callus and grows, and now exists
Observing under body formula fluorescence microscope, a part of wound healing has the brightest green glow to send, and this part wound healing is kanamycin-resistant callus tissue,
Retain；And a part does not has luminescence, this part wound healing is non-resistance wound healing, gets rid of；By the step for, by non-resistance
Wound healing is removed, and is greatly reduced further work amount；The kanamycin-resistant callus tissue of green light is remained, through the cultivations of about 35 days with
After, differentiate resistant plant；Carry out first order fluorescence detection the most again, carry out observing resistant plant under body formula fluorescence microscope,
By remaining of whole resistant plant blade and petiole all green light, glow gets rid of；Resistant plant grows 55 days again
Carrying out first order fluorescence detection, in fluoroscopic examination, the most shinny green glow of the blade of resistance Seedling, petiole and root may determine that as turning base
Because of plant.
Embodiment 2
Culture medium:
Diploid Fructus Fragariae Ananssae 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 Fructus Fragariae Ananssae co-cultures 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 Fructus Fragariae Ananssae screening culture 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 kanamycin, 500mg/L Carbenicillin, pH6.0；
Diploid Fructus Fragariae Ananssae seed culture medium S1, diploid Fructus Fragariae Ananssae co-culture culture medium S2, diploid Fructus Fragariae Ananssae screening culture medium
S3 carries out autoclaving, standby.
Transgenic method:
(1) actication of culture:
Plasmid pK7WG2D Agrobacterium EHA105 will be contained training containing 50mg/L spectinomycin and 50mg/L rifampicin LB solid
Support and rule on base, cultivate 3 days for 30 DEG C；
Good single bacterium colony 3 long on picking flat board, is inoculated into containing 50mg/L spectinomycin and 50mg/L rifampicin
In 50mL LB fluid 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, and supernatant is gone by back-off centrifuge tube on aseptic filter paper
Removing, by the resuspended thalline of 100mLMS liquid, shaken cultivation in shaking table, measuring OD600 value is 0.3, prepares the bacterium solution activated；
(2) diploid Fructus Fragariae Ananssae transgenic
Material processes: diploid Fructus Fragariae Ananssae ' Rugen ' seed is planted in S1 culture medium, grows 3-5 sheet leaf after 35 days,
On superclean bench, blade is cut completely and be positioned on aseptic filter paper, prolong vein crosscutting 3-5 cutter with scalpel, but can not be by
Blade cuts off, and the blade cut is placed directly in the bacterium solution activated and infects, and also Fructus Fragariae Ananssae petiole is cut into 1.5 centimeter length simultaneously
Section, hinders at two with operation graduating with cutter on petiole, carves and can not cut off petiole when hindering, and wound was directly placed into after completing and activated quarter
Bacterium solution infect；
Infect: by the blade cut with carve the petiole of wound and be directly placed into the bacterium solution activated and infect, during slightly shake up,
Contaminate 40 minutes, during every 5 minutes shake once；After having infected, blade and petiole are taken out, be placed directly in common training
Supporting in culture medium S2, make blade and petiole wound be fully contacted with culture medium, in culturing room 27 DEG C, light culture 2 days, by blade
Petiole proceeds to cultivate 22 days in screening culture medium S3, and diploid Strawberry Leaves edge has white callus and grows, and now exists
Observing under body formula fluorescence microscope, a part of wound healing has the brightest green glow to send, and this part wound healing is kanamycin-resistant callus tissue,
Retain；And a part does not has luminescence, this part wound healing is non-resistance wound healing, gets rid of；By the step for, by non-resistance
Wound healing is removed, and is greatly reduced further work amount；The kanamycin-resistant callus tissue of green light is remained, through the cultivations of about 45 days with
After, differentiate resistant plant；Carry out first order fluorescence detection the most again, carry out observing resistant plant under body formula fluorescence microscope,
By remaining of whole resistant plant blade and petiole all green light, glow gets rid of；Resistant plant grows 65 days again
Carrying out first order fluorescence detection, in fluoroscopic examination, the most shinny green glow of the blade of resistance Seedling, petiole and root may determine that as turning base
Because of plant.
Embodiment 3
Culture medium:
Diploid Fructus Fragariae Ananssae 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 Fructus Fragariae Ananssae co-cultures 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 Fructus Fragariae Ananssae screening culture 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 kanamycin, 400mg/L Carbenicillin, pH5.8；
Diploid Fructus Fragariae Ananssae seed culture medium S1, diploid Fructus Fragariae Ananssae co-culture culture medium S2, diploid Fructus Fragariae Ananssae screening culture medium
S3 carries out autoclaving, standby.
Transgenic method:
(1) actication of culture:
Plasmid pK7WG2D Agrobacterium EHA105 will be contained training containing 50mg/L spectinomycin and 50mg/L rifampicin LB solid
Support and rule on base, cultivate 2 days for 28 DEG C；
Good single bacterium colony 3 long on picking flat board, is inoculated into containing 50mg/L spectinomycin and 50mg/L rifampicin
In 50mL LB fluid 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, and supernatant is gone by back-off centrifuge tube on aseptic filter paper
Removing, by the resuspended thalline of 100mLMS liquid, shaken cultivation in shaking table, measuring OD600 value is 0.2, prepares the bacterium solution activated；
(2) diploid Fructus Fragariae Ananssae transgenic
Material processes: diploid Fructus Fragariae Ananssae ' Rugen ' seed is planted in S1 culture medium, grows 3-5 sheet leaf after 35 days,
On superclean bench, blade is cut completely and be positioned on aseptic filter paper, prolong vein crosscutting 3-5 cutter with scalpel, but can not be by
Blade cuts off, and the blade cut is placed directly in the bacterium solution activated and infects, and also Fructus Fragariae Ananssae petiole is cut into 0.1 centimeter length simultaneously
Section, hinders at two with operation graduating with cutter on petiole, carves and can not cut off petiole when hindering, and wound was directly placed into after completing and activated quarter
Bacterium solution infect；
Infect: by the blade cut with carve the petiole of wound and be directly placed into the bacterium solution activated and infect, during slightly shake up,
Contaminate 35 minutes, during every 5 minutes shake once；After having infected, blade and petiole are taken out, be placed directly in common training
Supporting in culture medium S2, make blade and petiole wound be fully contacted with culture medium, in culturing room 24 DEG C, light culture 3 days, by blade
Petiole proceeds to cultivate 20 days in screening culture medium S3, and diploid Strawberry Leaves edge has white callus and grows, and now exists
Observing under body formula fluorescence microscope, a part of wound healing has the brightest green glow to send, and this part wound healing is kanamycin-resistant callus tissue,
Retain；And a part does not has luminescence, this part wound healing is non-resistance wound healing, gets rid of；By the step for, by non-resistance
Wound healing is removed, and is greatly reduced further work amount；The kanamycin-resistant callus tissue of green light is remained, through the cultivations of about 40 days with
After, differentiate resistant plant；Carry out first order fluorescence detection the most again, carry out observing resistant plant under body formula fluorescence microscope,
By remaining of whole resistant plant blade and petiole all green light, glow gets rid of；Resistant plant growth 50 is entered again
Row first order fluorescence detects, and in fluoroscopic examination, the most shinny green glow of the blade of resistance Seedling, petiole and root may determine that as transgenic
Plant.
Embodiment 4
Culture medium:
Diploid Fructus Fragariae Ananssae 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 Fructus Fragariae Ananssae co-cultures 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 Fructus Fragariae Ananssae screening culture 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 kanamycin, 400mg/L Carbenicillin, pH5.7；
Diploid Fructus Fragariae Ananssae seed culture medium S1, diploid Fructus Fragariae Ananssae co-culture culture medium S2, diploid Fructus Fragariae Ananssae screening culture medium
S3 carries out autoclaving, standby.
Transgenic method:
(1) actication of culture:
Plasmid pK7WG2D Agrobacterium EHA105 will be contained training containing 50mg/L spectinomycin and 50mg/L rifampicin LB solid
Support and rule on base, cultivate 2 days for 28 DEG C；
Good single bacterium colony 3 long on picking flat board, is inoculated into containing 50mg/L spectinomycin and 50mg/L rifampicin
In 50mL LB fluid 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, and supernatant is gone by back-off centrifuge tube on aseptic filter paper
Removing, by the resuspended thalline of 100mLMS liquid, shaken cultivation in shaking table, measuring OD600 value is 0.2, prepares the bacterium solution activated；
(2) diploid Fructus Fragariae Ananssae transgenic
Material processes: diploid Fructus Fragariae Ananssae ' Rugen ' seed is planted in S1 culture medium, grows 3-5 sheet leaf after 35 days,
On superclean bench, blade is cut completely and be positioned on aseptic filter paper, prolong vein crosscutting 3-5 cutter with scalpel, but can not be by
Blade cuts off, and the blade cut is placed directly in the bacterium solution activated and infects, and also Fructus Fragariae Ananssae petiole is cut into 0.1 centimeter length simultaneously
Section, hinders at two with operation graduating with cutter on petiole, carves and can not cut off petiole when hindering, and wound was directly placed into after completing and activated quarter
Bacterium solution infect；
Infect: by the blade cut with carve the petiole of wound and be directly placed into the bacterium solution activated and infect, during slightly shake up,
Contaminate 35 minutes, during every 5 minutes shake once；After having infected, blade and petiole are taken out, be placed directly in common training
Supporting in culture medium S2, make blade and petiole wound be fully contacted with culture medium, in culturing room 24 DEG C, light culture 3 days, by blade
Petiole proceeds to cultivate 20 days in screening culture medium S3, and diploid Strawberry Leaves edge has white callus and grows, and now exists
Observing under body formula fluorescence microscope, a part of wound healing has the brightest green glow to send, and this part wound healing is kanamycin-resistant callus tissue,
Retain；And a part does not has luminescence, this part wound healing is non-resistance wound healing, gets rid of；By the step for, by non-resistance
Wound healing is removed, and is greatly reduced further work amount；The kanamycin-resistant callus tissue of green light is remained, through the cultivations of about 40 days with
After, differentiate resistant plant；Carry out first order fluorescence detection the most again, carry out observing resistant plant under body formula fluorescence microscope,
By remaining of whole resistant plant blade and petiole all green light, glow gets rid of；Resistant plant growth 50 is entered again
Row first order fluorescence detects, and in fluoroscopic examination, the most shinny green glow of the blade of resistance Seedling, petiole and root may determine that as transgenic
Plant.
Embodiment 5
Culture medium
Diploid Fructus Fragariae Ananssae 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 Fructus Fragariae Ananssae co-cultures 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 Fructus Fragariae Ananssae screening culture 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 kanamycin, 400mg/L Carbenicillin, pH5.8；
Diploid Fructus Fragariae Ananssae seed culture medium S1, diploid Fructus Fragariae Ananssae co-culture culture medium S2, diploid Fructus Fragariae Ananssae screening culture medium
S3 carries out autoclaving, standby.
Transgenic method:
(1) actication of culture:
Plasmid pK7WG2D Agrobacterium EHA105 will be contained training containing 50mg/L spectinomycin and 50mg/L rifampicin LB solid
Support and rule on base, cultivate 2 days for 28 DEG C；
Good single bacterium colony 3 long on picking flat board, is inoculated into containing 50mg/L spectinomycin and 50mg/L rifampicin
In 50mL LB fluid 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, and supernatant is gone by back-off centrifuge tube on aseptic filter paper
Removing, by the resuspended thalline of 100mLMS liquid, shaken cultivation in shaking table, measuring OD600 value is 0.2, prepares the bacterium solution activated；
(2) diploid Fructus Fragariae Ananssae transgenic
Material processes: diploid Fructus Fragariae Ananssae ' Rugen ' seed is planted in S1 culture medium, grows 3-5 sheet leaf after 35 days,
On superclean bench, blade is cut completely and be positioned on aseptic filter paper, prolong vein crosscutting 3-5 cutter with scalpel, but can not be by
Blade cuts off, and the blade cut is placed directly in the bacterium solution activated and infects, and also Fructus Fragariae Ananssae petiole is cut into 0.1 centimeter length simultaneously
Section, hinders at two with operation graduating with cutter on petiole, carves and can not cut off petiole when hindering, and wound was directly placed into after completing and activated quarter
Bacterium solution infect；
Infect: by the blade cut with carve the petiole of wound and be directly placed into the bacterium solution activated and infect, during slightly shake up,
Contaminate 35 minutes, during every 5 minutes shake once；After having infected, blade and petiole are taken out, be placed directly in common training
Supporting in culture medium S2, make blade and petiole wound be fully contacted with culture medium, in culturing room 24 DEG C, light culture 3 days, by blade
Petiole proceeds to cultivate 20 days in screening culture medium S3, and diploid Strawberry Leaves edge has white callus and grows, and now exists
Observing under body formula fluorescence microscope, a part of wound healing has the brightest green glow to send, and this part wound healing is kanamycin-resistant callus tissue,
Retain；And a part does not has luminescence, this part wound healing is non-resistance wound healing, gets rid of；By the step for, by non-resistance
Wound healing is removed, and is greatly reduced further work amount；The kanamycin-resistant callus tissue of green light is remained, through the cultivations of about 40 days with
After, differentiate resistant plant；Carry out first order fluorescence detection the most again, carry out observing resistant plant under body formula fluorescence microscope,
By remaining of whole resistant plant blade and petiole all green light, glow gets rid of；Resistant plant growth 50 is entered again
Row first order fluorescence detects, and in fluoroscopic examination, the most shinny green glow of the blade of resistance Seedling, petiole and root may determine that as transgenic
Plant.
Embodiment 6
If needing to obtain fruit to carry out the test of fruit, embodiment can be obtained after transfer-gen plant continues to cultivate and moving
Plant entrance field to yield positive results.In order to further determine that the stability of transgenic after yielding positive results, can be under fluorescence microscope
Detection flower and the fluorescence of fruit different developmental phases again
Embodiment 7
The conversion ratio of diploid Fructus Fragariae Ananssae transgenic of the present invention, is shown in Table 1.
Comparative example 1 is the conversion ratio of octoploid Fructus Fragariae Ananssae transgenic in prior art；
Comparative example 2 is the conversion ratio of diploid Fructus Fragariae Ananssae transgenic in prior art；
The conversion ratio of table 1 diploid of the present invention Fructus Fragariae Ananssae transgenic
As shown in Table 1, in the embodiment of the present invention, the conversion ratio of diploid Fructus Fragariae Ananssae transgenic is attained by more than 50%, the highest
Conversion ratio in prior art.
Claims (8)
1. the transgenic culture medium of a diploid Fructus Fragariae Ananssae, it is characterised in that include following composition: diploid Fructus Fragariae Ananssae seed culture
Base 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, 5.0-6.0g/
L agar powder, pH5.5-6.0；
Diploid Fructus Fragariae Ananssae co-cultures 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 Fructus Fragariae Ananssae screening culture 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 kanamycin, 300-500mg/L
Carbenicillin, pH5.5-6.0.
The transgenic culture medium of diploid Fructus Fragariae Ananssae the most according to claim 1, it is characterised in that described diploid Fructus Fragariae Ananssae seed
Culture medium S1, diploid Fructus Fragariae Ananssae co-culture culture medium S2, diploid Fructus Fragariae Ananssae screening culture medium S3 all carries out autoclaving, standby.
3. the transgenic method of the transgenic culture medium utilizing diploid Fructus Fragariae Ananssae described in claim 1, it is characterised in that bag
Include following steps:
(1) actication of culture:
Plasmid pK7WG2D Agrobacterium EHA105 will be contained rule on LB solid medium, cultivate 1-3 days for 25-30 DEG C；
Long on picking flat board good single bacterium colony 2-3, is inoculated in 50mL LB fluid medium, 25-30 DEG C, 200-
250rpm in shaking table shaken cultivation to OD600 value 0.3-0.5；
Under room temperature, 4000-6000rmp is centrifuged 7-9 minute, removes supernatant, and back-off centrifuge tube on aseptic filter paper, by supernatant
Removing, resuspended thalline, shaken cultivation in shaking table, measuring OD600 value is 0.1-0.3, prepares the bacterium solution activated；
(2) diploid Fructus Fragariae Ananssae transgenic
Material processes: diploid Fructus Fragariae Ananssae seed is planted in S1 culture medium, aseptically that blade is complete after growing blade
Cut, prolong vein with scalpel crosscutting；Fructus Fragariae Ananssae petiole is cut into 0.5-1.5 centimeter length section simultaneously, on petiole, uses scalpel
Carve wound；
Infect: by the blade cut with carve the petiole of wound and be directly placed into bacterium solution activate and infect, during slightly shake up, dip-dye
30-40 minute；After having infected, blade and petiole are taken out, be placed directly in and co-culture in culture medium S2, make blade and petiole
Wound is fully contacted with culture medium, in culturing room 23-27 DEG C, and blade petiole is proceeded to screening culture medium S3 by light culture 2-4 days
Upper cultivation 18-22 days, diploid Strawberry Leaves edge has white callus and grows, now enters under body formula fluorescence microscope
Row is observed, and a part of wound healing has the brightest green glow to send, and a part does not has luminescence；The kanamycin-resistant callus tissue of green light is retained
Come, after the cultivation of about 35-45 days, differentiate resistant plant；Carry out first order fluorescence detection the most again, whole resistance is planted
Remaining of strain blade and petiole all green light, glow gets rid of；Resistant plant growth carries out the most glimmering in 55-65 days again
Light detects, and in fluoroscopic examination, the most shinny green glow of the blade of resistance Seedling, petiole and root may determine that as transfer-gen plant.
Transgenic method the most according to claim 3, it is characterised in that in described step (1), plasmid pK7WG2D carries super
The reporter gene Egfp of strongly expressed.
Transgenic method the most according to claim 3, it is characterised in that LB solid medium and LB in described step (1)
Fluid medium all contains 50mg/L spectinomycin and 50mg/L rifampicin.
Transgenic method the most according to claim 3, it is characterised in that in described step (1), resuspended thalline is to use
100mLMS liquid is resuspended.
Transgenic method the most according to claim 3, it is characterised in that contaminate 30-40 minute process in described step (2)
In every 5 minutes shake once.
Transgenic method the most according to claim 3, it is characterised in that in described step (2) there be very a part of wound healing
Bright green glow sends as kanamycin-resistant callus tissue, retain；It is non-resistance wound healing that a part does not has luminous, gets rid of.
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| CN109187315A (en) * | 2018-08-18 | 2019-01-11 | 杭州市农业科学研究院 | Doubling of strawberry chromosome method and ploidy rapid identification method used |
| CN109735538A (en) * | 2018-12-27 | 2019-05-10 | 南京农业大学 | A kind of carrier and its preparation method and application improving forest Strawberry Leaves regeneration efficiency |
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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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109187315A (en) * | 2018-08-18 | 2019-01-11 | 杭州市农业科学研究院 | Doubling of strawberry chromosome method and ploidy rapid identification method used |
| CN109735538A (en) * | 2018-12-27 | 2019-05-10 | 南京农业大学 | A kind of carrier and its preparation method and application improving forest Strawberry Leaves regeneration efficiency |
| CN109735538B (en) * | 2018-12-27 | 2022-06-21 | 南京农业大学 | Carrier for improving forest strawberry leaf regeneration efficiency and preparation method and application thereof |
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